Welcome to Porto and BioIron 2009

We express our appreciation to both Portos: the city and our local chair Graça Porto, who contributed so much to the preparation of the meeting. On behalf of the organizers we also thank you for coming to Porto 200 years after it became a battleground of Europe divided by men who sought to establish their power by war. Science has counteracted that notion of power, by promoting shared knowledge as a unifying force that transcends these artificial divisions and now encompasses women as equals.

Let us make Bioiron 2009 a successful forum for scientific exchange and collegial friendship between scientists from all over the globe!

Pierre Brissot, IBIS President Tomas Ganz, IBIS Elect-President Maria de Sousa, Co-Chairman of the Local Organizing Committee

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Table of Contents

Schedule at a Glance ...... Inside Front Cover IBIS Board of Directors & Committees...... 4 General Meeting Information...... 5 Social Activities ...... 8 Program Schedule ...... 10 Invited Speaker Abstracts and Bios ...... 50 Podium Abstracts ...... 64 Poster Abstracts...... 95 Alphabetical Index of Authors by Presentation...... 188 Acknowledgements...... Inside Back Cover

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2009 IBIS Board of Directors & Committees

OFFICERS Pierre Brissot, MD, President DIRECTORS Tomas Ganz, MD, PhD, President-Elect Robert Fleming, MD Paolo Arosio, PhD, Secretary Prem Ponka, MD, PhD Caroline Philpott, MD, Treasurer Graça Porto, MD, PhD Grant A. Ramm, PhD EXECUTIVE OFFICE Kathryn Robson, PhD Wendy J. Weiser, Executive Director Guenter Weiss, MD Sue O’Sullivan, Associate Director

International Scientific Committee Local Organizing Committee

PRESIDENT CHAIRS Pierre Brissot, MD Graça Porto, MD, PhD Maria de Sousa, MD, PhD MEMBERS Paolo Arosio, PhD MEMBERS Tomas Ganz, MD, PhD Sérgio de Almeida, PhD Robert Fleming, MD Idalina Beirão, MD Jerry Kaplan, PhD Carla Sofia Cardoso, PhD Caroline Philpott, MD Ana Paula Correia, MD Prem Ponka, MD, PhD Eugénia Cruz, MD, PhD Graça Porto, MD, PhD Hal Drakesmith, PhD Grant Ramm, PhD Maria Salomé Gomes, PhD Kathryn Robson, PhD Pedro Moradas-Ferreira, PhD Guenter Weiss, MD José Fraga, MD Guenther Winkelmann, PhD José Moura, MD Joana Palha, PhD Jorge Pinto, PhD Local Organizations Alexandre Quintanilha, PhD FCT, Portuguese Science and Technology Conceição Rangel, PhD Foundation Pedro Rodrigues, PhD IBMC, Institute for Molecular and Cellular Ana Tomás, PhD Biology Jorge Vieira, PhD

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2009 IBIS General Meeting Information

Venue Porto Congress Center Alfândega Rua Nova da Alfândega Edificio da Alfândega 4050-430 Porto, Portugal Phone: +351 223 403 000 Fax: +351 223 403 099 Website: www.amtc.pt/congresso

Dates of the Congress June 7 – 11, 2009 On Sunday, June 7, registration will open at 14:00 and the Welcome Reception (Get-Together Party) will begin at 19:00. The congress will begin with the Opening Ceremony at the Porto Congress Center Alfândega on Monday, June 8, at 09:00 and concludes with the Closing Ceremony at the Congress Center on Thursday, June 11, at 17:00.

Language The official language of the congress is English.

Map of Porto

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2009 IBIS General Meeting Information

Money The local currency is the Euro (€). Banks in Portugal are open Monday – Friday, 08:30 – 15:00 (Banks at the airports are open 08:00 – 20:00). Portugal also has an extensive network of cash dispensers (labeled Multibanco) that operate 24 hours a day.

Shopping The most traditional shopping area is Rua Santa Catarina, along with the narrow streets going up and down from the city center till Ribeira, by the Douro riverside. Several shopping centers, in and around Porto, are open daily until 23:00. Besides the major international franchising and brands, national textiles, leather, pottery, shoes, crystal, wines and gold handcraft can be found in the shops.

Visas All non-EU citizens should check if it is necessary to obtain a visa, which can be obtained at the Portuguese Embassy or Consulate in their own country. A list of countries that require a visa to Portugal is given on the following website: www.wordtravels.com/Travelguide/Countries/Portugal/Visa

Local Time The local time in Portugal is the Greenwich Mean Time, which is the same as in London. GMT + 1 Hour (Western Europe Daylight/Summer Time)

Electricity Supply: 220 V

Emergency Number 112

Registration / Information Desk The registration / information desk is located in the Porto Congress Center Alfândega. Sunday, June 7th 14:00 – 20:00 Monday, June 8th 08:00 – 18:30 Tuesday, June 9th 08:00 – 14:30 Wednesday, June 10th 08:00 – 18:15 Thursday, June 11th 08:00 – 18:15

Registration fee for participant includes: • Admission to Opening Ceremony and all scientific sessions • Admission to the exhibition area • Congress materials (congress bag, badge, certificate of attendance) • Abstract Book • Coffee breaks, lunch, and afternoon refreshments • Welcome Reception on Sunday, June 7, 2009 • Closing Ceremony and scientific sessions on Thursday, June 11, 2009

Registration fee for accompanying person includes: • Admission to the exhibition area • Coffee breaks, lunch, and afternoon refreshments • Welcome Reception on Sunday, June 7, 2009 • Closing Ceremony on Thursday, June 11, 2009

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2009 IBIS General Meeting Information

Name Badges Attendees are requested to wear their name badges while at the meeting.

Presenters – Slide Review Room The Slide Review Room is in Miragaia in the Porto Congress Center Alfândega. Presentations must be delivered the day prior to your presentation. Those presenting on Monday, June 8th should submit their presentation on Sunday. Sunday, June 7th 15:00 – 20:00 Monday, June 8th 08:00 – 18:30 Tuesday, June 9th 08:00 – 14:30 Wednesday, June 10th 08:00 – 18:15 Thursday, June 11th 08:00 – 18:15

Poster Sessions Posters will be available for viewing throughout the entire meeting; however, presentations will occur during the following times/sessions: Poster Session I – June 8th 14:00 – 15:15 Poster Session II – June 10th 14:00 – 15:15 Poster Session III – June 11th 14:00 – 15:15

Tourist Information Tourist information is available from your hotel concierge or at the Porto Congress Center Alfândega information desk.

Lunch Lunch will be served Monday though Thursday in Noble Hall.

Cancellations and Refunds Cancellations must be made in writing to the BioIron 2009 Office. Cancellations received after May 1, 2009 will be considered at the discretion of the Organizing Committee at the conclusion of the meeting.

Dress The meeting dress is business casual throughout the conference, except for the Gala Dinner, which is formal dress.

First Aid Medical doctors can be called by your hotel in case of emergencies or by the Porto Congress Center Alfândega.

Special Needs (including dietary needs / allergies) If you have specific needs to facilitate your comfort during the conference (i.e. wheelchair access, dietary, auditory or other assistance), all concerns should be addressed at the conference registration / information desk. In general, soups in Portugal are very good and are not prepared with milk. Fish is generally served with potatoes and vegetables; however, some codfish dishes (popular bacalhau dishes) are prepared with milk, so please beware.

Liability BioIron will not be liable for illness, accidents or thefts suffered by participants or accompanying persons during the conference or their stay in Portugal.

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2009 IBIS Social Activities

Welcome Reception (Get-Together Party) Venue: Alfândega Mooring by the Douro Date: Sunday, June 7, 2009 Time: 19:00 – 21:00 Cost: Registration fee covers cost of participant and guest. Attire: Casual Dress The Congress Center is located at “Alfândega”, an imposing former Customs House standing on an artificial platform and a remarkable example of nineteenth century engineering and iron architecture. The Welcome Reception will be held at the “Salão Nobre” or noble room, which extends to the dock thus offering, in favorable weather conditions, a splendid view to the Douro River.

Gala Dinner Venue: Palácio da Bolsa Date: Thursday, June 11, 2009 Departure: 19:30 from all official congress hotels Time: 20:30 Cost: 90 USD Attire: Formal Dress The BioIron 2009 Award Ceremony will be held during the Gala Dinner. The “Palácio da Bolsa” is considered one of the most beautiful buildings in Porto and one of the richest in Portugal, as well as a favorite place for most relevant social, cultural and political events linked to the life of the city. It was built as a trade center in middle nineteenth century with the great investment and dedication of Porto merchandisers. The Gala Dinner and Award Ceremony of Bioiron 2009 is organized in this beautiful environment and is certainly a program not to miss.

Sightseeing For sightseeing tours you can contact Skyros-Congressos (Phone: +351 22 616 54 50; Email: [email protected])

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2009 IBIS Social Activities

Optional Tours The Porto tours have been arranged through Skyros-Congressos. You may contact them direct by phone at +351 22 616 54 50 or email at [email protected] if you have any questions. (Minimum of 25 participants per tour, excludes river tour.) The IBIS office is not involved in the logistics or registration for the optional tours.

Optional Dinner at Port Wine Cellars with River Cruise Venue: Port Wine Cellar (to be specified) Date: Monday, June 8, 2009 Time: Departure from Alfândega (Venue) at 19:00 for a river cruise; arrival to Port Wine Cellar for visit and dinner at 20:00; arrival (end of dinner) to the official hotels at 23:30 by bus Cost: 70 Euros per person Starting at the Alfândega mooring, a small cruise will take you along the Douro River, passing the traditional Porto iron bridges, to visit a port wine house and enjoy dinner at the cellars.

Option 1: Porto City Tour Full Day, 09:30 – 17:30 Price: 65 Euros (Includes: lunch in Portuguese restaurant, one-hour river cruise, visit to the port wine cellars, port wine taste) This tour will give you a general idea of Porto as a city, with its historical and modern quarters linking the past to the future, shown in its social and cultural aspects. For a city tour including the Old Town (with World Heritage status): Infante Square and the Stock Exchange Palace is the choice for you. We will also visit the St. Francis Church, built in 13th Century Roman and Gothic styles, and famous for its superb Baroque-style interior, with the gilt carvings covering the granite. “Sé Catedral”, the second oldest Cathedral in Portugal (12th and 13th Centuries) is known for its Goth Cloister with charming ornamentation in blue and white tile panels of 18th Century by Nicolau Nasoni. Afterwards, we will visit a port wine cellar, where we will taste different types of port wine.

There will be time for lunch in a typical Portuguese restaurant, savoring our authentic gastronomy. After lunch, we will have a one-hour river cruise, during which we will enjoy the beautiful landscape with its old riverine quarters and its remarkable iron bridges over the Douro River and “Serra do Pilar Belvedere”. After the cruise we will continue to “Avenida da Boavista”, then to the 17th Century fortress “Castelo do Queijo” and finally returning to the congress venue.

Option 2: Minho Tour Full Day, 09:30 – 18:00 Price: 90 Euros (Includes: lunch in Portuguese restaurant, auto Pullman, English speaking tourist guide)

On this tour we will visit Minho, the northernmost province of Portugal, known for being one of the most beautiful and historical regions in the entire country. It is a captivating region, where the symbiosis between nature, man and tradition is so perfect that simply looking at it is a source of pure happiness. Over the centuries, this mountain region, where the sound of murmuring water is everywhere, has become a garden in which every inch of land is cultivated, creating a curtain of green along the roadsides. We will pass through Vila do Conde and Póvoa do Varzim, poetic and picturesque fishing towns, while driving along the coastal road. The tour then continues on to Ponte de Lima, with its imposing Roman Bridge that gave name to the Villa, remembering the ancient Roman influence on this land. Lunch will be provided in a traditional restaurant. After, we will visit Viana do Castelo, the capital of Portuguese folklore, known for its colorful embroidery customs, where women’s beauty is set off by its filigree jewelry. Return to Porto.

Option 3: Post Congress Tour – Douro World Heritage See the Skyros-Congressos website for details: Website: www.skyroscongressos.com 9

2009 International BioIron Society Program Schedule

SUNDAY, JUNE 7th, 2009

14:00 – 20:00 Registration / Information Desk Open Location: Porto Congress Center Alfândega

19:00 – 21:00 Welcome Reception (Get-Together Party) Location: Alfândega Mooring by the Douro

MONDAY, JUNE 8th, 2009

08:00 – 18:30 Registration / Information Desk Open Location: Porto Congress Center Alfândega

09:00 – 09:30 Opening Ceremony Pierre Brissot, President of IBIS Graça Porto, Chair of BioIron 2009

09:30 – 10:30 Plenary Session I: The Global Iron Cycle & Evolution Location: Arquivo Chair: Antonello Pietrangelo, Modena, Italy

09:30 – 10:00 Keynote Lecture 1: Global Iron Connections Between Desert Dust, Ocean Biogeochemistry and Climate Tim Jickells, University of East Anglia, Norwich, UK

10:00 – 10:30 Keynote Lecture 2: Iron and Oxygen Sensing Peter Ratcliffe, University of Oxford, UK

10:30 – 11:00 Coffee Break Location: Noble Hall

11:00 – 12:30 Plenary Session II: Erythropoiesis and Iron Recycling Location: Arquivo Chairs: Mario Cazzola, Pavia, Italy and François Canonne-Hergaux, Paris, France

11:00 – 11:30 Lecture 1: Erythropoiesis Olivier Hermine, Institut Pasteur, Paris, France (Presented By: Geneviève Courtois)

11:30 – 12:00 Lecture 2: Ineffective Erythropoiesis in Beta-Thalassemia Stefano Rivella, Cornell University, New York, USA

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12:00 – 12:30 Podium Presentations

12:00 1 IDENTIFYING GENES ESSENTIAL FOR HEME BIOSYNTHESIS THROUGH LARGE-SCALE GENE EXPRESSION SCREENING Iman Schultz, PhD¹, Roland Nilsson, PhD², Eric Pierce³, Kathleen Soltis³, Amorrnrat Naranuntarat, MSc4, Prasad Paradkar, PhD5, Joshua Baughman², Paul Kinsley, PhD6, Valeria Culotta, PhD4, Jerry Kaplan, PhD5, James Palis, MD6, Vamsi Mootha, MD² and Barry Paw, MD, PhD³ ¹Brigham and Women’s Hospital, Harvard Medical School; ²Department of Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ³Department of Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA; 4Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD; 5Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; 6Department of Pediatrics, University of Rochester School of Medicine & Dentistry, Rochester, NY (Presented By: Iman Schultz, PhD)

12:15 2 FPN1B, A NOVEL FERROPORTIN TRANSCRIPT LACKING AN IRON-RESPONSIVE ELEMENT, ENABLES DUODENAL AND ERYTHROID PRECURSOR CELLS TO EVADE TRANSLATIONAL REPRESSION Deliang Zhang, PhD, Robert Hughes, Hayden Ollivierre-Wilson, Manik Ghosh, PhD and Tracey Rouault, MD NICHD, NIH (Presented By: Tracey Rouault, MD)

12:30 – 14:00 Lunch Location: Noble Hall

14:00 – 15:15 Poster Session I Presentations0 Location: Arquivo

Poster# 1 A NEW FLVCR ISOFORM ABLE TO SUPPORT ERYTHROPOIESIS Deborah Chiabrando, PhD Student, Samuele Marro, Erika Messana, Sonia Mercurio, Emilia Turco, Lorenzo Silengo, Fiorella Altruda and Emanuela Tolosano, PhD Molecular Biotechnology Center, Torino (Presented By: Emanuela Tolosano, PhD)

Poster# 2 ABSENCE OF THE HEMOCHROMATOSIS GENE HFE CONFERS PROTECTION UNDER CONDITIONS OF STRESS ERYTHROPOIESIS Pedro Ramos¹, Ella Guy1, Robert W Grady, PhD1, Maria de Sousa, MD, PhD² and Stefano Rivella, PhD1 ¹Weill Cornell Medical College, New York; ²IBMC, Porto (Presented By: Pedro Ramos)

Poster# 3 A CLOSER LOOK AT CELLULAR IRON METABOLISM IN IRP2 DEFICIENT ERYTHROBLASTS Matthias Schranzhofer, PhD¹, Manfred Schifrer, MSc², Bruno Galy, PhD³, Matthias Hentze, MD³, Muellner Ernst, PhD² and Prem Ponka, MD, PhD4 ¹McGill University; ²Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Austria; ³European Molecular Biology Laboratories, Heidelberg, Germany; 4Lady Davis Institute for Medical Research, McGill University, Montreal, Canada (Presented By: Matthias Schranzhofer, PhD)

Poster# 4 DOES HEME OXYGENASE 1 PLAY A ROLE IN ERYTHROID DIFFERENTIATION? Daniel Garcia dos Santos, Msc¹,³, Matthias Schranzhofer, Msc, Phd¹,4, Jesse Eisenberg¹, Jose Artur Bogo Chies, Msc, PhD² and Prem Ponka, MDd, PhD¹,5 ¹Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada; ²Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pos Graduacao em Genetica e Biologia Molecular (PPGBM), Departamento de Genetica, Porto Alegre, RS, Brazil; ³Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pos Graduacao em Genetica e Biologia Molecular (PPGBM), Departamento de Genetica, Brazil; 4Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pos Graduacao em Genetica e Biologia Molecular, Departamento de Genetica, Porto Alegre, RS, Brazil; 5Department of Physiology, McGgill University, Montreal, QC, Canada (Presented By: Daniel Garcia dos Santos, Msc)

Poster# 5 LOW HEPCIDIN AND ELEVATED GDF15 LEVELS IN PATIENTS WITH CONGENITAL DYSERYTHROPOIETIC ANEMIA (CDA) Guillem Casanovas¹, Judit Kiss², Sandro Altamura², Hermann Heimpel³ and Martina U. Muckenthaler² ¹European Molecular Biology Laboratory; ²Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg, Germany; ³Department for Internal Medicine III (Hematology/Oncology), University Hospital of Ulm, Germany (Presented By: Guillem Casanovas)

Poster# 6 WITHDRAWN TRANSFERRIN RECEPTOR ACTIVATION BY POLYMERIC IGA1 MODULATES CYTOKINE THRESHOLD OF EARLY ERYTHROBLASTS Séverine Coulon¹, Céline Callens¹, Pamella Huey Mei Wang², Julie Vandekerckhove¹, Damien Grapton², Houda Tamouza², Yael Zermati¹, Jean-Antoine Ribeil¹, Bertrand Arnulf³, Marie-Alexandra Alyanakian4, Marc Benhamou², Renato C. Monteiro², Olivier Hermine¹ and Ivan Cruz Moura² ¹CNRS UMR 8147; ²INSERM U699; ³EA3963; 4INSERM U580

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Poster# 7 STUDYING IRON METABOLISM IN ERYTHROID CELLS UNDER DIFFERENT OXYGEN CONDITONS Manfred Schifrer, MSc¹, Matthias Schranzhofer, PhD² and Ernst Muellner, PhD¹ ¹Medical University of Vienna/MFPL; ²McGill University/Lady Davis Institute (Presented By: Manfred Schifrer, MSc)

Poster# 8 FRATAXIN EXPRESSION LEVELS DURING INCREASED HEME SYNTHESIS IN K562 CELLS Laura Neumann, Hannes Steinkellner, MScsc, Barbara Scheiber-Mojdehkar, PhD, Hans Goldenberg, PhD and Brigitte Sturm, PhD Medical University of Vienna (Presented By: Laura Neumann)

Poster# 9 CHARACTERIZATION OF IRON METABOLISM AND ANEMIA IN A MOUSE MODEL OF CONGENITAL ERYTHROPOIETIC PORPHYRIA Said Lyoumi, PhD¹, Constance Delaby, PhD², Sarah Millot, MD¹, Zoubida Karim, PhD¹, David Haile, PhD³, Hubert de Verneuil, MD, PhD4, Herve Puy, MD, PhD5 and Carole Beaumont¹ ¹INSERM U773; ²University Paris Diderot; ³University of Texas; 4University of Bordeaux; 5Versailles-Saint Quentin University (Presented By: Carole Beaumont)

Poster# 10 REGULATION OF IRON HOMEOSTASIS IN ANEMIA OF CHRONIC DISEASES AND IRON DEFICIEANCY ANEMIA: DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS Igor Theurl², Elmar Aigner³, Milan Theurl4, Manfred Nairz², Markus Seifert², Andrea Schroll², Thomas Sonnweber², Lukas Eberwein², Derrick R. Witcher5, Anthony T. Murphy5, Victor J. Wroblewski5, Eva Wurz², Christian Datz³ and Günter Weiss¹ ¹Internal Medicine I, Clinical Immunolgy and Infectious Diseases; ²Department of Internal Medicine I, Clinical Immunology and Infectious Diseases, Medical University of Innsbruck, Austria; ³General Hospital Oberndorf, Department of Internal Medicine, Oberndorf, Austria; 4Departments of Ophthalmology and Internal Medicine I, Clinical Immunology and Infectious Diseases, Medical University of Innsbruck, Austria; 5Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA (Presented By: Günter Weiss)

Poster# 11 COPPER-DEFICIENT ANEMIC RATS, BUT NOT MICE, UP-REGULATE FERROPORTIN EXPRESSION AND DOWN- REGULATE HEPCIDIN Supak Jenkitkasemwong¹, Joseph Prohaska, PhD² and Mitchell Knutson¹ ¹University of Florida; ²University of Minnesota (Presented By: Mitchell Knutson)

Poster# 12 HIGH PHOSPHATE CONCENTRATIONS DISRUPT IRON LOADING INTO FERRITIN FORMING IRON (III)- PHOSPHATE COLLOID PARTICLES: CORRELATIONS TO NON-TRANSFERRIIN BOUND IRON IN CHRONIC KIDNEY DISEASE Richard Watt, PhD, Robert Hilton, BS, Nathan Andros, BSc and Zachary Kenealey, Undergraduate Brigham Young University (Presented By: Richard Watt, PhD)

Poster# 13 FERRIC PYROPHOSPHATE: BIOCHEMICAL CHARACTERISATION AND METABOLIC FATE IN HEPG2 CELLS Brigitte Sturm, PhD, Nina Ternes, PhD, Hans Goldenberg, PhD and Barbara Scheiber-Mojdehkar, PhD Medical University of Vienna (Presented By: Barbara Scheiber-Mojdehkar, PhD)

Poster# 14 ERYTHROPOIETIC AND IRON FEATURES IN THE DIFFERENT CLINICAL FORMS OF HEREDITARY SPHEROCYTOSIS – A PRELIMINARY STUDY Susana Rocha¹, Petronila Rocha-Pereira, PhD², Fátima Ferreira, MD³, Esmeralda Cleto, MD4, Marika Antunes, MD4, José Barbot, MD5, Alexandre Quintanilha, PhD6, Luís Belo, PhD¹ and Alice Santos-Silva, PhD¹ ¹Faculdade de Farmácia/Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto; ²Centro de Investigação em Ciências da Saúde (CICS), Universidade da Beira Interior/Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto; ³Hospital de S. João, Porto; 4Hospital de Santo António, Centro Hospitalar do Porto; 5Hospital Maria Pia, Centro Hospitalar do Porto; 6Instituto de Ciências Biomédicas Abel Salazar (ICBAS)/Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto (Presented By: Susana Rocha)

Poster# 15 PATHOGENESIS OF ANEMIA IN PREGNANCY AND ANEMIA OF INFLAMMATION ARE SIMILAR Valeriy Demikhov, MD, ELena Morshchakova, MD and Anatole Pavlov, MD Federal Research Center for Pediatric Hematology (Presented By: Valeriy Demikhov, MD)

Poster# 16 EFFECTS OF RECOMBINANT HUMAN ERYTHROPOIETIN THERAPY ON MODERATE STAGES OF CARDIO-RENAL ANAEMIA SYNDROME – AN EXPERIMENTAL MODEL Patrícia Garrido, MSci², Flávio Reis, PhD¹, Elísio Costa, MSci³, Belmiro Parada, MD¹, Edite Teixeira de Lemos, PhD¹, Nuno Piloto, Biochem D¹, José Sereno, Biochem D¹, Carlos Alberto Tavares, MD4, Carlos Ferrer Antunes, MD4, Arnaldo Figueiredo, PhD5, Lina Carvalho, PhD6, Petronila Rocha Pereira, PhD7, Luis Belo, PhD³, Alice Santos Silva, PhD³ and Frederico Teixeira, PhD¹ ¹Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University; ²Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University, Portugal; ³Biochemistry Service, Pharmacy Faculty & IBMC, University of Porto; 4Laboratory of Haematology, Coimbra University Hospital; 5Service of Urology and Renal Transplantation, Coimbra University Hospital; 6Institute of Anatomic Pathology, Medicine Faculty, Coimbra University; 7Research Centre for Health Sciences, Beira Interior University, Covilhã (Presented By: Flávio Reis, PhD)

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Poster# 17 ATYPICAL IRON DEFICIENCY ANAEMIA – ASSOCIATION OF TWO NEW MUTATIONS IN FERROPORTIN AND TMPRSS6 GENES Gonçalo Caetano, MD², Luis Relvas, BScsC², Celeste Bento, MsCSc², Maria Pedro Silveira, MD² and Leticia Ribeiro, MD¹ ¹Centro Hospitalar Coimbra; ²Serv. Hematologia, CHC (Presented By: Leticia Ribeiro, MD)

Poster# 18 BIOMINERALIZATION IN MAGNETOTACTIC BACTERIA PROCEEDS WITHOUT MINERAL PRECURSOR AND MAGNETOSOMES MOVE DURING CHAIN ASSEMBLY Berthold F. Matzanke, PhD, DiplChem¹, D. Faivre, PhD², L.H. Boettger, PhD³, N. Menguy, PhD4, G. Ona-Nguema, PhD4, M. Pósfai, PhD5, H.P. Gunnlaugson, Prof, PhD6 and D. Schueler, Prof, PhD7 ¹University of Luebeck, Isotopes Laboratory; ²Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424 Potsdam, Germany; ³Institute of Physics, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; 4Universités Paris 6 and 7 and Institut de Physique du Globe de Paris, 140 rue de Lourmel, 75015 Paris, France; 5Department of Earth and Environmental Sciences, University of Pannonia, POB 158, 8200 Veszprém, Hungary; 6Institute of Physics and Astronomy, Århus University, Ny Munkegade, 8000 Århus C, Denmark; 7Department of Biology, LMU München, Biozentrum, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany (Presented By: Berthold F. Matzanke, PhD, DiplChem)

Poster# 19 IRON-SULFUR PROTEINS AND THE NITRIC OXIDE CYTOTOXICITY IN BACTERIA Huangen Ding, PhD, Binbin Ren, BSc¹, Xuewu Duan, PhD, Aaron Landry, BSc and Juanjuan Yang, MSc Louisiana State University (Presented By: Huangen Ding, PhD)

Poster# 20 SERCA PUMP EXPRESSION AND IRON UPTAKE IN A DROSOPHILA CELL LINE Wenlin Wu, PhD and Maria Lind Karlberg Comparative Physiology, Uppsala University, Sweden (Presented By: Maria Lind Karlberg)

Poster# 21 THE IMPACT OF DIETARY COPPER AND ZINC ON DROSOPHILA FERRITIN DISTRIBUTION AND IRON METABOLISM Fanis Missirlis, PhD Queen Mary University of London (Presented By: Fanis Missirlis, PhD)

Poster# 22 OVEREXPRESSION OF THE YEAST FRATAXIN HOMOLOG (YFH1): CONTRASTING EFFECTS ON IRON-SULFUR CLUSTER ASSEMBLY, HEME SYNTHESIS AND RESISTANCE TO OXIDATIVE STRESS Alexandra Seguin, Aurélien Bayot, Andrew Dancis, Adelina Rogowska-Wrzesinska, Françoise Auchère, Jean-Michel Camadro, Anne- Laure Bulteau and Emmanuel Lesuisse Institut Jacques Monod, Université Paris Diderot and UPMC (Presented By: Alexandra Seguin)

Poster# 23 KLAFT, THE KLUYVEROMYCES LACTIS ORTHOLOG OF AFT1 AND AFT2, MEDIATES IRON-RESPONSIVE GENE EXPRESSION THROUGH AN AFT-TYPE ELEMENT Natalia Conde e Silva, PhD, Isabelle R. Gonçalves, PhD, Marc Lemaire, PhD, Emmanuel Lesuisse, PhD, Jean Michel Camadro, PhD and Pierre Louis Blaiseau, PhD Institut Jacques Monod (Presented By: Natalia Conde e Silva, PhD)

Poster# 24 HAPTOGLOBIN AND HAPTOGLOBIN-RELATED PROTEIN ARE HEMOGLOBIN-BINDING PROTEINS WITH DIVERSE FUNCTIONS Marianne Jensby Nielsen, PhD, Benoit Vanhollebeke, Christian Jacobsen, Etienne Pays and Søren Kragh Moestrup University of Aarhus (Presented By: Marianne Jensby Nielsen, PhD)

Poster# 25 IRON AND VIRUSES – INTERACTIONS OF HIV-1 WITH CELLULAR IRON METABOLISM Lucy Eddowes, Andrew Armitage, Alain Townsend and Hal Drakesmith University of Oxford (Presented By: Lucy Eddowes)

Poster# 26 MILD INCREASES IN SERUM HEPCIDIN AND INTERLEUKIN-6 CONCENTRATIONS IMPAIR IRON INCORPORATION IN HEMOGLOBIN DURING EXPERIMENTAL HUMAN MALARIA INFECTION Quirijn de Mast, MD, Edmee Dongen-van Lases, PhD, Dorine Swinkels, PhD, An-Emmie Nieman, MD, Meta Roestenberg, MD, Pierre Druilhe, PhD, Theo Arens, MSc, Adrian Luty, PhD, Cornelis Hermsen, PhD, Robert Sauerwein, PhD and Andre van der Ven Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands (Presented By: Andre van der Ven)

Poster# 27 INTRACELLULAR PARASITE LEISHMANIA DONOVANI MANIPULATES IRON HOMEOSTASIS OF HOST MACROPHAGE BY DEPLETING INTRACELLULAR LABILE IRON POOL Chinmay Mukhopadhyay, PhD and Nupurkanti Das, MBBSc Jawaharlal Nehru University (Presented By: Chinmay Mukhopadhyay, PhD)

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Poster# 28 IDENTIFICATION OF A NEW HEXADENTATE IRON CHELATOR CAPABLE OF RESTRICTING THE INTRAMACROPHAGIC GROWTH OF MYCOBACTERIUM AVIUM Sofia Sousa Fernandes, PhD², Ana Nunes, PhD³, Ana Rita Gomes, BSsc4, Baltazar de Castro, PhD³, Robert C. Hider, PhD5, Maria Rangel, PhD6, Rui Appelberg, PhD, MD7 and Maria Salomé Gomes, PhD¹ ¹IBMC and ICBAS- Universidade do Porto; ²IBMC-Universidade do Porto; ³REQUIMTE-Faculdade de Ciências, U.P.; 4IBMC-U.P.; 5Division of Pharmaceutical Sciences, King’s College London; 6REQUIMTE-Faculdade de Ciências and ICBAS-U.P.; 7IBMC and ICBAS-Universidade do Porto (Presented By: Maria Salomé Gomes, PhD)

Poster# 29 ZIP TRANSPORTERS IN LEISHMANIA INFANTUM Sandra Carvalho, Tânia Cruz, Licenciatura, Rosa Silva, Licenciatura, Vítor Costa, PhD and Ana M. Tomás, PhD Institute for Molecular and Cell Biology, IBMC, Porto (Presented By: Sandra Carvalho)

Poster# 30 EVALUATION OF IRON STATUS IN PATIENTS WITH GASTRITIS AND HELICOBACTER PYLORI INFECTION Helena Grotto, MD, PhD and Eliana Alvarenga, postgraduate student State University of Campinas, Brasil (Presented By: Helena Grotto, MD, PhD)

Poster# 31 IRON-HEME METABOLISM IN LEISHMANIA INFANTUM INTRACELLULAR STAGE Tânia Cruz, Sandra Carvalho and Ana Tomás Instituto de Biologia Molecular e Celular, IBMC, Universidade do Porto (Presented By: Tânia Cruz)

Poster# 32 SYNTHESIS AND CHARACTERIZATION OF A NOVEL TRIPODAL CATECHOLATE HEXADENTATE CHELATOR Carla Queiroz, MScsC¹, Ana M.G. Silva, PhD², Mariana Andrade, BScsC², Paula Gameiro, PhD², Baltazar Castro, Agregação² and Maria Rangel, PhD³ ¹REQUIMTE, ICBAS, Universidade do Porto; ²REQUIMTE, Departamento de Quìmica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal; ³REQUIMTE, Instituto de Ciências Biomédicas de Abel Salazar, 4099-003 Porto, Portugal (Presented By: Carla Queiroz, MScsC)

Poster# 33 DIAGNOSTIC ACCURACY OF SERUM HEPCIDIN FOR IRON DEFICIENCY IN CRITICALLY ILL PATIENTS WITH ANEMIA Sigismond Lasocki, Isabelle Boutron, PhD, Fathi Driss, MD, PhD, Mark Westerman, PhD, Herve Puy, MD, PhD, Sarah Millot, MD, Philippe Montravers, MD, PhD and Carole Beaumont, PhD INSERM U773 (Presented By: Sigismond Lasocki)

Poster# 34 MHC CLASS I ANTIGEN PRESENTATION: EFFECT OF HFE, A PROTEIN INVOLVED IN IRON METABOLISM Alexandre Reuben, Manuela M. Santos, PhD and Réjean Lapointe, PhD University of Montreal/CRCHUM/Montreal Cancer Institute (ICM) (Presented By: Alexandre Reuben)

Poster# 35 HEPATIC MCP-1 GENE EXPRESSION IS SUPPRESSED IN IRON-LOADED MALE C282Y HOMOZYGOTES AND CORRELATES WITH HEPCIDIN EXPRESSION John D. Ryan, MBBCh¹, Eleanor Ryan, PhD², Matthew Lawless, PhD², Jens Stolte³, Martina U. Muckenthaler, PhD³ and John Crowe, PhD² ¹Centre for Liver Disease; ²Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin, Ireland; ³Department of Pediatric Haematology, Oncology and Immunology, University of Heidelberg, Germany (Presented By: John D. Ryan, MBBCh)

Poster# 36 INFLAMMATION-INDUCED HEPCIDIN IS ASSOCIATED WITH THE DEVELOPMENT OF ANEMIA IN SEPTIC PATIENTS Lucas van Eijk, MD¹, Joyce Kroot, MSc², Mirjam Tromp, MA, RN³, Peter Pickkers, MD, PhD4 and Dorine Swinkels, MD, PhD² ¹Radboud University Nijmegen Medical Centre; ²Department of Clinical Chemistry, Radboud University Nijmegen Medical Centre; ³Nijmegen Institute for Infection, Inflammation, and Immunity (N4i), Department of Internal Medicine, Radboud University Nijmegen Medical Centre; 4Department of Intensive Care Medicine, Radboud University Nijmegen Medical Centre (Presented By: Lucas van Eijk)

Poster# 37 INFLAMMATION UPREGULATES NON-TRANSFERRIN BOUND IRON UPTAKE BY HEPATOCYTES Debbie Trinder, BSc, PhD, Carly Herbison, BSc (Hons), Roheeth Delima, BSc (Hons), Anita Chua, BSc (Hons) PhD, Ross Graham, BSc (Hons), PhD and John Olynyk, MBBS, MD University of Western Australia (Presented By: Debbie Trinder, BSc, PhD)

Poster# 38 HIGH NUMBERS OF THE MOST MATURE CD8+ EFFECTOR MEMORY T CELLS ARE ASSOCIATED WITH THE NEW GGG HAPLOTYPE, A MARKER OF LESS SEVERE EXPRESSION OF HEREDITARY HEMOCHROMATOSIS M. Fátima Macedo, PhD¹, Monica Costa², Graça Porto, MD, PhD³,4 and Eugénia Cruz, MD, PhD²,4 ¹IBMC; ²IRIS, IBMC, Porto University; ³IRIS, IBMC, ICBAS, Porto University; 4Hematology S. António Hospital, Porto (Presented By: M. Fátima Macedo, PhD)

Poster# 39 INCREASED FREQUENCY OF CD4+CD28+ T-LYMPHOCYTES IN MICE WITH HFE-DEFICIENCY Maja Vujic Spasic, PhD¹, Alexis Perez Gonzalez, PhD², Andy Riddell, PhD², Matthias Hentze, Prof MD² and Martina Muckenthaler, Prof PhD¹ ¹University Hospital of Heidelberg; ²EMBL, Heidelberg, Germany (Presented By: Maja Vujic Spasic, PhD) 14

Monday, June 8, 2009

Poster# 40 ELEVATED ICAM-1 AND SUPPRESSION OF VCAM-1 SERUM PROTEIN IN HEREDITARY HEMOCHROMATOSIS PATIENTS Matthew Lawless, PhD, MSc¹, Suzanne Norris, MD, PhD² and John Crowe, MD, PhD³ ¹Mater Misericordiae Hospital; ²Trinity College Dublin; ³Centre for Liver Disease, Mater Misericordiae Hospital, University Hospital, Dublin (Presented By: Matthew Lawless, PhD, MSc)

Poster# 41 THE MULTICOPPER OXIDASE CERULOPLASMIN AND THE IRON EXPORTER FERROPORTIN ARE BOTH PRESENT AT THE CELL SURFACE OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES Liliana Marques, Rui Malhó, François Canonne-Hergaux and Luciana Costa Instituto Nacional de Saúde Dr. Ricardo Jorge (Presented By: Liliana Marques)

Poster# 42 LEUKOCYTE APOPTOSIS AND INFLAMMATION IN IRON-OVERLOADED PATIENTS WITH SICKLE CELL DISEASE OR BETA-THALASSEMIA: A MECHANISM FOR INCREASED STROKE AND DISEASE SEVERITY IN SICKLE CELL DISEASE Patrick Walter, PhD¹, Killilea David, PhD², Fung Ellen, PhD¹, Anne Higa, BScs¹, Jacqueline Madden, BScs, RN¹, John Porter, MD², Pat Evans, PhD², Bruce Ames, PhD¹, Elliott Vichinsky, MD¹ and Paul Harmatz, MD¹ ¹Children’s Hospital & Research Center Oakland; ²University College London (Presented By: Patrick Walter, PhD)

Poster# 43 MOVED TO NOVEL TECHNOLOGIES SYMPOSIUM, WEDNESDAY, JUNE 10, 17:20 VASCULAR INFLAMMATORY RESPONSE AND SPATIAL ARRANGEMENT OF CELL ADHESION MOLECULES ASSOCIATED WITH REDOX CYCLING OF IRON IN PULMONARY BLAST INJURY Nikolai Gorbunov, PhD The Henry M. Jackson Foundation for the Advancement of Military Medicine Inc. (Presented By: Nikolai Gorbunov, PhD)

Poster# 44 ENDOSOMAL TRANSPORT OF TRANSFERRIN TO MITOCHONDRIA IS ESSENTIAL FOR EFFICIENT UTILIZATION OF IRON FOR HEME SYNTHESIS Alex D. Sheftel, PhD, An-Sheng Zhang, MD, Tanya Kahawita, MSc, Orian S. Shirihai, MD, PhD and Prem Ponka, MD, PhD Institut für Zytobiologie, Philipps-Universität-Marburg (Presented By: Alex D. Sheftel, PhD)

Poster# 45 POTENTIAL INVOLVEMENT OF DMT1 AND ZIP8 IN IRON RELEASE FROM LYSOSOMES Chevaun Morrison, BS¹, Ningning Zhao², Annie Nguyen¹, Angelica Gonzalez¹, Eric Sauble¹, Maria C Linder¹ and Mitchell Knutson² ¹California State University, Fullerton; ²University of Florida, Gainesville (Presented By: Chevaun Morrison, BS)

Poster# 46 EVIDENCE FOR AN ENDOPLASMIC RETICULUM IRON TRANSPORT SYSTEM Marie-Berengere Troadec, PhD, Diane M. Ward, PhD and Jerry Kaplan, PhD University of Utah (Presented By: Marie-Berengere Troadec)

Poster# 47 DELIVERY OF IRON FROM TRANSFERRIN TO MITOCHONDRIA VIA PATHWAYS INVOLVING LABILE AND NON- CHELATABLE FORMS OF IRON Maya Shvartsman, MSc¹, Eugenia Prus, PhD², Eitan Fibach, Prof² and Ioav Cabantchik, MD, PhD Prof³ ¹Hebrew University of Jerusalem; ²Hadassah Medical Center, Jerusalem, Israel; ³Hebrew University of Jerusalem, Jerusalem, Israel (Presented By: Maya Shvartsman, MSc)

Poster# 48 HEME AND IRON TRANSPORT SYSTEMS IN NON-POLARIZED AND POLARIZED CELLS Izumi Yanatori¹, Mitsuaki Tabuchi, PhD, Reiko Akagi, PhD² and Fumio Kishi, MD, PhD ¹Kawasaki Medical School; ²Yasuda Women’s University (Presented By: Izumi Yanatori)

Poster# 49 IRON SUPPLY DETERMINES APICAL/BASOLATERAL MEMBRANE DISTRIBUTION OF INTESTINAL IRON TRANSPORTERS DMT1 AND FERROPORTIN 1 Marco T. Nunez, PhD¹, Victoria Tapia, BSc¹, Alejandro Rojas, PhD¹, Pabla Aguirre, PhD¹, Francisco Gomez, BSc¹, Daniela Sandoval, Engineer in Biotecnology¹ and Francisco Nualart, PhD² ¹Universidad de Chile; ²Universidad de Concepcion (Presented By: Marco T. Nunez, PhD)

Poster# 50 SUBCELLULAR DISTRIBUTIONS OF ZIP14 AND DMT1 IN HEPG2 CELLS: IMPLICATIONS FOR CELLULAR IRON IMPORT AND ENDOSOMAL IRON TRANSPORT Ningning Zhao and Mitchell Knutson University of Florida (Presented By: Mitchell Knutson)

Poster# 51 FERRITIN DOES NOT DONATE ITS IRON FOR HEME SYNTHESIS IN CULTIVATED MACROPHAGES Marc R. Mikhael, BSc¹, Alex D. Sheftel, BSc, PhD² and Prem Ponka, MD, PhD³ ¹McGill University; ²Institut für Zytobiologie, Philipps-Universität-Marburg, Marburg, Germany; ³Lady Davis Institute and Department of Physiology, McGill University, Montreal, QC, Canada (Presented By: Marc R. Mikhael, BSc)

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Poster# 52 MOVED TO NOVEL TECHNOLOGIES SYMPOSIUM, WEDNESDAY, JUNE 10, 17:40 PROBING THE INTERRELATIONSHIPS OF THE CELLULAR LABILE IRON POOLS (LIP) IN NORMAL AND PATHOLOGICAL CONDITIONS: AN OVERVIEW William Brwuer, PhD², Yan-Sung Sohn, MSc, Maya Shvartsman, MSc, Or Kakhlon, PhD and Ioav Cabantchik, MD, PhD¹ ¹Hebrew University of Jerusalem; ²Institute of Life Sciences, Hebrew University of Jerusalem (Presented By: Ioav Cabantchik, MD, PhD)

Poster# 53 CHARACTERIZATION OF A POTENTIAL IRON SHUTTLE WITHIN THE CYTOPLASM OF MAMMALIAN CELLS Natasha Hill, BS¹, Ben Rojas¹, Vireak Thon¹, Robert Malanga¹, Lena Ton-nu¹, Lina Planutyte¹, Maria C. Linder¹, Andrew Z. Mason² and Ashraf Elamin² ¹California State University, Fullerton; ²California State University, Long Beach (Presented By: Natasha Hill, BS)

Poster# 54 DIFFERENTIAL PROTEOMIC APPROACH TO INVESTIGATE IRON RESPONSE IN MURINE MACROPHAGES Annalisa Castagna, PhD¹, Ivana De Domenico², Rita Polati³, Alessandra Bossi³, Natascia Campostrini4, Federica Zaninotto4, Fabiana Busti4, Lello Zolla5, AnnaMaria Timperio5, Oliviero Olivieri4, Jerry Kaplan6 and Domenico Girelli4 ¹University of Verona, department of Clinical and Experimental Medicine; ²University of Utah, School of Medicine, Department of Internal Medicine, Salt Lake City UT, US; ³University of Verona, Dip. di Biotecnologie, Verona, Italy; 4University of Verona, Dip. di Medicina Clinica e Sperimentale, Verona, Italy; 5University of Tuscia, Department of Environmental Sciences, Italy; 6University of Utah, School of Medicine, Department of Pathology, Salt Lake City UT, US (Presented By: Annalisa Castagna, PhD)

Poster# 55 MITOFERRIN 1 AND 2 ACT AS MITOCHONDRIAL IRON IMPORTERS IN HELA CELLS Sara Luscieti, Patrizia Cavadini, PhD, Maura Poli, PhD, Federica Maccarinelli, PhD, Dario Finazzi, PhD and Paolo Arosio, PhD Dipartimento Materno Infantile e Ttecnologie Biomediche, Università di Brescia, Italy (Presented By: Sara Luscieti)

Poster# 56 THE ASSEMBLY OF RESPIRATORY COMPLEX I REQUIRES THE IRON-SULFUR CLUTER PROTEIN HUIND1 Alex D. Sheftel, PhD, Oliver Stehling, PhD, Antonio J. Pierik, PhD, Daili J.A. Netz, PhD, Stefan Kerscher, PhD, Hans-Peter Elsässer, PhD, Ilka Wittig, PhD, Ulrich Brandt, PhD, Janneke Balk, PhD and Roland Lill, PhD Institut für Zytobiologie, Philipps-Universität-Marburg (Presented By: Alex D. Sheftel, PhD)

Poster# 57 DOWN-REGULATION OF COMPONENTS OF THE IRON-SULFUR CLUSTER ASSEMBLY MACHINERIES IN INFLAMMATORY MACROPHAGES Cédric Picot, PhD, Frédéric Canal, PhD, Kahina Abbas, PhD student, Cendrine Seguin, research assistant, Valérie Quesniaux, PhD, Jean- Claude Drapier, PhD and Cecile Bouton ICSN-CNRS avenue de la Terrasse, 91190 Gif-sur-Yvette, France (Presented By: Cecile Bouton)

Poster# 58 MITOCHONDRIAL FERRITIN ATTENUATES DOXORUBICIN EFFECTS ON IRON HOMEOSTASIS Maura Poli, Dr, Federica Maccarinelli, Dr, Vanessa Porrini, Dr, Emiliano Cocco, Dr, Derosas Manuela, Dr, Sara Luscieti, Dr, Paolo Arosio, PhD, Prof and Isabella Zanella, Dr Dipartimento Materno Infantile e Tecnologie Biomediche Università Brescia (Presented By: Maura Poli, Dr)

Poster# 59 A HIGH-THROUGHPUT SIRNA SCREEN FOR HEPCIDIN REGULATORS REVEALS SMAD7 AS A NOVEL NEGATIVE REGULATOR OF HEPCIDIN EXPRESSION Katarzyna Mleczko-Sanecka¹, Anan Ragab, Dr², Guillem Casanovas, BSc³, Michael Boutros, Prof², Matthias W. Hentze, Prof4 and Martina U. Muckenthaler, Prof¹ ¹Molecular Medicine Partnership Unit, Heidelberg University; ²German Cancer Research Center (DKFZ); ³Molecular Medicine Partnership Unit; 4European Molecular Biology Laboratory (Presented By: Katarzyna Mleczko-Sanecka)

Poster# 60 BONE MORPHOGENETIC PROTEIN (BMP)-RESPONSIVE ELEMENTS LOCATED IN THE PROXIMAL AND DISTAL HEPCIDIN PROMOTER ARE CRITICAL FOR ITS RESPONSE TO HJV/BMP/SMAD Guillem Casanovas¹, Katarzyna Mleczko-Sanecka¹, Sandro Altamura², Matthias W. Hentze¹ and Martina U. Muckenthaler² ¹European Molecular Biology Laboratory, Heidelberg, Germany; ²Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg (Presented By: Guillem Casanovas)

Poster# 61 RENAL HANDLING OF HEPCIDIN Hilde Peters, MD², Coby Laarakkers, BSc, Jack Wetzels and Dorine Swinkels, MD, PhD¹ ¹Radboud Department of Clinical Chemistry 441, University Nijmegen Medical Centre; ²Departments of Clinical Chemistry and Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands (Presented By: Dorine Swinkels, MD, PhD)

Poster# 62 FURIN AND MAPK (ERK1/2) PARTICIPATE IN THE REGULATION OF HEPCIDIN EXPRESSION IN HEPG2 CELLS Maura Poli, PhD, Sara Luscieti, PhD, Federica Maccarinelli, PhD, Dario Finazzi, PhD and Paolo Arosio, PhD Department MITB, University of Brescia (Presented By: Maura Poli, PhD)

Poster# 63 PHYSIOLOGIC AND PATHOPHYSIOLOGIC REGULATION OF HEPCIDIN EXPRESSION Tom Bartnikas, MD, PhD and Mark Fleming, MD, DPhil Children’s Hospital Boston (Presented By: Tom Bartnikas, MD, PhD) 16

Monday, June 8, 2009

Poster# 64 SERUM HEPCIDIN LEVELS IN PATIENTS WITH THALASSEMIA OR SICKLE CELL DISEASE: A CONFOUNDING RELATIONSHIP WITH TRANSFUSION CYCLE Patrick Walter, PhD¹, Zahra Pakbaz, MD¹, Elizabeta Nemeth, PhD², Roland Fischer, PhD³, Tomas Ganz, MD, PhD², Elliott Vichinsky, MD¹, Ward Hagar, MD¹, John Porter, MD4, Pat Evans, PhD4, Matt Hertz, BScs¹, Lynne Neumayr, MD¹ and Paul Harmatz, MD¹ ¹Children’s Hospital & Research Center Oakland; ²University of California Los Angeles; ³University Medical Center Hamburg-Eppendorf; 4University College London (Presented By: Patrick Walter)

Poster# 65 HAEM METABOLISM INHIBITORS AFFECT HEPCIDIN EXPRESSION IN MICE A.H. Laftah, MSc; PhD¹, A.T. McKie, Prof², S.K. Srai, Prof³ and R.J. Simpson, Dr² ¹King’s College London; ²Division of Nutrition, School of Biomedical & Health Sciences, King’s College London, London, UK; ³Department of Strutural & Molecular Biology, University College London, London, UK (Presented By: A.H. Laftah, MSc, PhD)

Poster# 66 CHARACTERISATION OF THE DIVALENT METAL ION BINDING PROPERTIES OF HEPCIDIN BY FTICR MASS SPECTROMETRY Chris Tselepis, Cleidiane Zampronio, Tariq Iqbal and Douglas Ward University of Birmingham (Presented By: Chris Tselepis)

Poster# 67 PREPARATION AND INVESTIGATION OF FLUORESCENT HEPCIDIN IN MDCK CELLS AND MACROPHAGES Dareen Jaiash, BSc (Hons), MSc, A. Parmar, V. Marshal, G.O. Latunde-Dada, R.C. Hider and S. Bansal King’s College London (Presented By: Dareen Jaiash, BSc (Hons))

Poster# 68 HEPCIDIN DEFICIENCY IN PATIENTS WITH CHRONIC HEPATITIS C Domenico Girelli, MD¹, Michela Pasino²,6, Julia B. Goodnough³, Elizabeta Nemeth³, Maria Guido4, Annalisa Castagna², Fabiana Busti², Natascia Campostrini², Federica Zaninotto², Nicola Martinelli², Italo Vantini5, Roberto Corrocher², Tomas Ganz³ and Giovanna Fattovich6 ¹University of Verona; ²Department of Clinical and Experimental Medicine, University of Verona, Verona, Italy; ³Department of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, California, USA; 4Department of Diagnostic Sciences & Special Therapies, University of Padova, Padova, Italy; 5Department of Biomedical and Surgical Sciences, University of Verona, Verona, Italy; 6Department of Surgical and Gastroenterological Sciences, University of Verona, Verona, Italy (Presented By: Domenico Girelli, MD)

Poster# 69 EFFECTS OF RECOMBINANT HEPCIDIN, PROHEPCIDIN AND THEIR FLUORESCENT DERIVATIVES ON FERROPORTIN DEGRADATION Marie-Agnes Sari, PhD¹, Bruno Gagliardo¹, Nicole Kubat², Nicolas Desbenoit¹, Maryse Jaouen¹, Jean-Christophe Deschemin², Francois Canonne-Hergaux³, Isabelle Artaud¹ and Sophie Vaulont² ¹Université Paris Descartes, CNRS (UMR 8601), Paris, France; ²Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Inserm U 567Paris, France; ³CNRS, Institut de Chimie des Substances Naturelles, Gif-Sur-Yvette, France (Presented By: Marie-Agnes Sari, PhD)

Poster# 70 HIERARCHY BETWEEN ERYTHROPOIESIS AND IRON AS HEPCIDIN REGULATORS Jan Krijt, PhD¹, Martin Vokurka², Ludek Sefc², Tereza Hlobenova² and Emanuel Necas² ¹Charles University in Prague, First Faculty of Medicine, Institute of Pathophysiology; ²Charles University in Prague, First Faculty of Medicine, Institute of Pathophysiology and Center of Experimental Hematology (Presented By: Jan Krijt, PhD)

Poster# 71 HEPCIDIN EFFECT ON TRACE ELEMENTS IN SERUM OF WT AND HEPCIDIN1 KNOCKOUT MICE A.H. Laftah, MSc; PhD¹, A.H. Laftah, PhD², P. Masaratana, PhD², A.T. McKie, PhD, Prof², R.J. Simpson, PhD² and K. Raja, PhD³ ¹King’s College London; ²Division of Nutrition, School of Biomedical & Health Sciences, King’s College London, London, UK; ³Department of Clinical Biochemistry, King’s College Hospital, London, UK (Presented By: A.H. Laftah, MSc; PhD)

Poster# 72 METABOLIC STEATOSIS AND ALCOHOL-LOADING REGULATE THE EXPRESSION OF TRANSFERRIN RECEPTOR 1 AND HEPCIDIN IN MICE LIVER Takaaki Ohtake, MD, Katsuya Ikuta, MD, PhD, Koji Sawada, MD, Masami Abe, MD, Takaaki Hosoki, MD, Shigeki Miyoshi, MD, Yasuaki Suzuki, MD, Katsunori Sasaki, PhD, Yoshihiro Torimoto, MD, PhD and Yutaka Kohgo, MD, PhD Asahikawa Medical College (Presented By: Takaaki Ohtake, MD)

Poster# 73 HEPCIDIN MEASUREMENT BY QUANTITATIVE SELDI-TOF-MS IN HEMODIALYSIS PATIENTS: EVALUATION OF IRON STATUS DURING ERYTHROPOIESIS-STIMULATING AGENTS THERAPY Natascia Campostrini, PhD¹, Annalisa Castagna², Federica Zaninotto², Fabiana Busti², Nicola Tessitore³, Albino Poli4, Valeria Bedogna³, E. Melilli³, Oliviero Olivieri², Roberto Corrocher², Antonio Lupo³ and Domenico Girelli² ¹University of Verona; ²Department of Clinical and Experimental Medicine, University of Verona, Verona, Italy; ³Nephrology Division, University of Verona, Verona, Italy; 4Public Health Department, University of Verona, Verona, Italy (Presented By: Natascia Campostrini, PhD)

Poster# 74 RADIOIMMUNOASSAY FOR HUMAN SERUM HEPCIDIN Nicolai Grebenchtikov, MSc, Anneke Geurts-Moespot, BSc, Joyce Kroot, MSc, Martin den Heijer, MD, PhD, Harold Tjalsma, PhD, Fred Sweep, PhD and Dorine Swinkels, MD, PhD Radboud Department of Clinical Chemistry 441, University Nijmegen Medical Centre (Presented By: Dorine Swinkels, MD, PhD)

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Poster# 75 QUANTITATION OF HEPCIDIN Sukhi Bansal, PhD¹, John Halket, PhD¹, Adrian Bomford, MBBS, PhD², Robert Simpson, PhD², Nisha Vasavda, PhD³, Swee Lay Thein, MBBS PhD4 and Robert Hider, PhD5 ¹King’s College London, Pharmaceutical Sciences Division; ²King’s College London, Nutritional Sciences Division; ³King’s College London, Division of Gene and Cell Based Therapy; 4King’s College London, Department of Hematological Medicine; 5King’s College London Pharmaceutical Sciences Division (Presented By: Sukhi Bansal, PhD)

Poster# 76 CONTRIBUTION OF STAT3 AND SMAD4 PATHWAYS TO THE REGULATION OF HEPCIDIN BY OPPOSING STIMULI Hua Huang, Marco Constante, MSc, Antonio Layoun, BSc and Manuela M. Santos, PhD CRCHUM, University of Montreal (Presented By: Hua Huang)

Poster# 77 REGULATION OF HEPCIDIN EXPRESSION DURING PHENYLHYDRAZINE-INDUCED HAEMOLYTIC ANAEMIA IN MICE Yemisi Latunde-Dada, PhD, Robert Simpson, PhD and Andrew McKie, PhD Kings College London (Presented By: Yemisi Latunde-Dada, PhD)

Poster# 78 HFE MUTATIONS MODULATE THE EFFECT OF IRON STORES AND INFLAMMATION ON SERUM HEPCIDIN-25 IN CHRONIC HEMODIALYSIS PATIENTS Luca Valenti, MD, Domenico Girelli, Giovanni Francesco Valenti, Annalisa Castagna, Giovanna Como, Natascia Campostrini, Raffaela Rametta, Paola Dongiovanni, Piergiorgio Messa and Silvia Fargion University of Milano (Presented By: Luca Valenti, MD)

Poster# 79 HEPCIDIN RESPONSE TO ACUTE ORAL IRON AND CHRONIC IRON OVERLOAD IN DYSMETABOLIC HEPATIC IRON OVERLOAD SYNDROME Paola Trombini, MD¹, Valentina Paolini, Sara Pelucchi², Raffaella Mariani, Alessandra Salvioni, Domenico Girelli³, Clara Camaschella4, Elizabeta Nemeth5, Tomas Ganz5 and Alberto Piperno ¹Clinical Medicine, San Gerardo Hospital; ²University of Milano-Bicocca, Monza, Italy; ³University of Verona, Italy; 4San Raffaele University, Milano, Italy; 5Departments of Medicine and Pathology, UCLA, USA (Presented By: Paola Trombini, MD)

Poster# 80 THE IRON-REGULATING HORMONE HEPCIDIN IS EXPRESSED IN BLADDER CANCER TISSUE AND CORRELATES TO POOR PATIENT SURVIVAL Maciej Bogdan Maniecki, MHSc¹, Benedicte Parm Ulhøi, MD², Henrik Schmidt, MD, DMSc³, Lars Dyrskjøt, MSc, PhD4, Torben Falck Ørntoft, MD, DMSc4, Søren Kragh Moestrup, MD, DMSc5 and Holger Jon Møller, MD, PhD¹ ¹Department of Clinical Biochemistry, Århus Sygehus, Aarhus University Hospital; ²Institute of Pathology, Århus Sygehus, Aarhus University Hospital; ³Cancer Immunotherapy Group, Department of Oncology, Aarhus University Hospital; 4Molecular Diagnostic Laboratory, Department of Molecular Medicine, Aarhus University Hospital, Skejby; 5Institute of Medical Biochemistry, Aarhus University (Presented By: Maciej Bogdan Maniecki, MHSc)

Poster# 81 FIRST DUTCH PATIENT WITH MAPTRIPTASE-2 MUTATION LEADING TO IRON-REFRACTORY IRON DEFICIENT ANEMIA Marloes Cuijpers, MD, Erwin Wiegerinck, Theo de Witte, Prof and Dorine Swinkels, MD, PhD, Prof Radboud University Nijmegen Medical Centre (Presented By: Marloes Cuijpers, MD)

Poster# 82 COMBINED DELETION OF HFE AND TRANSFERRIN RECEPTOR 2 IN MICE LEADS TO MARKED DYSREGULATION OF HEPCIDIN AND IRON OVERLOAD Nathan Subramaniam, PhD, Lesa Summerville, Emily Crampton, David Frazer, Greg Anderson and Daniel Wallace Queensland Institute of Medical Research (Presented By: Nathan Subramaniam, PhD)

Poster# 83 DISRUPTION OF BOTH HFE AND TFR2 CAUSES MORE SEVERE HEPATIC IRON OVERLOAD IN HEREDITARY HAEMOCHROMATOSIS Roheeth Delima, BSc (Hons), Anita Chua, Carly Herbison, Ross M. Graham, John Olynyk and Debbie Trinder University of Western Australia (Presented By: Roheeth Delima, BSc (Hons))

Poster# 84 EXTRA-HEPATIC HFE FUNCTIONS MAY BE RESPONSIBLE FOR IRON OVERLOAD IN THE HEART AND ALTERATION WITHIN THE ERYTHRON Maja Vujic Spasic, PhD¹, Matthias Hentze, Prof MD² and Martina Muckenthaler, Prof PhD¹ ¹University Hospital of Heidelberg; ²EMBL, Heidelberg, Germany (Presented By: Maja Vujic Spasic, PhD)

Poster# 85 HFE POLYMORPHISMS AFFECT CHOLESTEROL METABOLISM: INSIGHTS INTO NEURODEGENERATIVE DISEASES Fatima Ali-Rahmani, Sang Lee, James Connor and Cara-Lynn Schengrund Pennsylvania State University (Presented By: James Connor)

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Poster# 86 HFE POLYMORPHISMS AND DRUG RESISTANCE IN CANCER Sang Lee, PhD, Siying Liu, Becky Slagle-Webb, Elana Farace, PhD, Jonas Sheehan, MD and James Connor, PhD Penn State College of Medicine (Presented By: Sang Lee, PhD)

Poster# 87 DIFFERENT EFFECTS ON IRON CONTENT IN THE LIVER AND SPLEEN OF HFE-KO MICE AFTER HEPATIC INJECTION OF A LENTIVIRAL VECTOR BEARING THE HFE GENE Pedro Ramos¹, Sara Gardenghi, PhD1, Ella Guy1, Nan Chan1, Antonia Follenzi, PhD², Robert W Grady, PhD1, Maria de Sousa, MD, PhD3 and Stefano Rivella, PhD1 ¹Weill Cornell Medical College, New York; ²Albert Einstein, New York; 3IBMC, Porto (Presented By: Pedro Ramos)

Poster# 88 HFE IS ESSENTIAL TO INDUCE HEPCIDIN AND TO DEVELOP HYPOFERREMIA IN RESPONSE TO LOW DOSES OF LPS Maja Vujic Spasic, PhD, Richard Sparla, Dipl, Judit Kiss, PhD, Jens Stolte, Dipl, Birgit Rathkolb, PhD, Matthias Hentze, PhD and Martina Muckenthaler, PhD University Hospital of Heidelberg (Presented By: Maja Vujic Spasic, PhD)

Poster# 89 REDUCED SERUM TRANSFERRIN LEVELS CHARACTERISE IRON-LOADED C282Y HAEMOCHROMATOSIS DESPITE UPREGULATED HEPATIC TRANSFERRIN TRANSCRIPTION John D. Ryan, MBBCh¹, Eleanor Ryan, PhD², Matthew Lawless, PhD², Jennifer Russell, PhD², Jens Stolte³, Martina U. Muckenthaler, PhD³, T. Barry Kelleher, MD² and John Crowe, PhD² ¹Centre for Liver Disease; ²Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin, Ireland; ³Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg, Germany (Presented By: John D. Ryan, MBBCh)

Poster# 90 HEMOJUVELIN IN MOUSE TISSUES Yuzo Fujikura, PhD, Jan Krijt, PhD and Emanuel Necas, MD, PhD Charles University in Prague (Presented By: Yuzo Fujikura, PhD)

Poster# 91 HOMOZYGOUS DELETION OF HFE AS A CAUSE OF HEMOCHROMATOSIS IN SARDINIA Sara Pelucchi¹, Raffaella Mariani, Matteo Pozzi, Francesca Bertola², Sabina Coletti, Cristina Arosio² and Alberto Piperno ¹University of Milano-Bicocca; ²Consortium of Human Molecular Genetics (Presented By: Alberto Piperno)

Poster# 92 HFE PROTEIN PRODUCED IN EUKARYOTIC CELLS BINDS THE CATION INDEPENDENT MANNOSE-6-PHOSPHATE RECEPTOR IN VITRO Lisa Schimanski, BSc, MSc, Hal Drakesmith, Emma Sweetland, Mariola Edelmann, Dellel Razgui, Chandran Ka, Alison Merryweather- Clarke, Kathryn Robson, Benedikt Kessler and Alain Townsend Oxford University (Presented By: Lisa Schimanski, BSc, MSc)

Poster# 93 E277K AND V295A HFE MUTATIONS – A MATTER OF α3-DOMAIN LOCATION? B. Silva, R. Martins, D. Proença, R. Fleming and P. Faustino Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal (Presented By: B. Silva)

Poster# 94 DIFFERENTIAL EXPRESSION OF HFE SPLICE VARIANTS D. Proença², R. Martins¹, B. Silva and P. Faustino ¹Instituto Nacional de Saude Dr. Ricardo Jorge; ²Departamento de Genética, Instituto Nacional de Saude Dr. Ricardo Jorge, Lisboa, Portugal (Presented By: R. Martins)

15:15 – 16:45 Concurrent Sessions: I – III

15:15 – 16:45 Concurrent Session I: Intracellular Iron Trafficking Location: D. Maria Chairs: Fumio Kishi, Kurashiki, Okayama, Japan and Prem Ponka, Montreal, PC, Canada

15:15 3 LYSOSOMAL PROTEOLYSIS IS THE PRIMARY DEGRADATION PATHWAY FOR CYTOSOLIC FERRITIN AND IS NECESSARY FOR IRON EXIT FROM FERRITIN Yinghui Zhang, MS², Marc Mikhael, BS³, Yiye Li, PhD4, Shan Soe-Lin, MS³, Bo Ning, MS², Guangjun Nie¹, Yuliang Zhao, PhD² and Prem Ponka, PhD, MD5 ¹National Center for Nanoscience and Technology; ²CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology of China, Beijing 100190; ³Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and Departments of Physiology and Medicine, McGill University, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada; 41CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology of China, Beijing 100190; 5Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and Departments of Physiology and Medicine, McGill University (Presented By: Guangjun Nie)

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Monday, June 8, 2009

15:30 4 LIVE IMAGING OF METAL-ION TRANSPORT IN OOCYTES EXPRESSING HUMAN DIVALENT METAL-ION TRANSPORTER-1 (DMT1): SUBSTRATE PROFILE AND SELECTIVITY Bryan Mackenzie, PhD, Anthony C. Illing, Christopher L. Cunningham and Ali Shawki University of Cincinnati College of Medicine (Presented By: Bryan Mackenzie, PhD)

15:45 5 IMAGING OF IRON ENTRY AND IRON-MEDIATED TOXICITY IN PRIMARY NEURONS AND ASTROCYTES Franca Codazzi, PhD¹, Ilaria Pelizzoni, Dr², Romina Macco, Dr¹, Marco Francesco Morini¹, Ilaria Vitali¹, Federico Maria Rossi¹, Alessandra Consonni, Dr¹, Barbara Bettegazzi, Dr¹, Daniele Zacchetti, PhD4 and Fabio Grohovaz, Professor4 ¹S. Raffele Scientific Institute; 2Italian Institute of Technology (IIT) (Presented By: Franca Codazzi, PhD)

16:00 6 CHARACTERIZATION OF HOLO-TF-REGULATED TFR2 TRAFFICKING Juxing Chen, PhD, Jinzhi Wang, MS, Kathrin Meyers, BS and Caroline Enns, PhD Oregon Health & Science University, Portland, OR (Presented By: Juxing Chen, PhD)

16:15 7 ABCB10 INTERACTS WITH MITOFERRIN-1 (SLC25A37) TO ENHANCE ITS PROTEIN STABILITY AND FUNCTION TO IMPORT MITOCHONDRIAL IRON IN ERYTHROBLASTS Wen Chen, PhD¹, Prasad Paradkar, PhD², Liangtao Li, MD², Nathaniel Langer, BA¹, Eric Pierce, BA¹, Iman Schultz, PhD¹, Brigham Hyde, PhD³, Orian Shirihai, MD, PhD³, Jerry Kaplan, PhD² and Barry Paw, MD, PhD¹ ¹Harvard Medical School; ²University of Utah; ³Boston University (Presented By: Barry Paw, MD, PhD)

16:30 8 EXPRESSION OF MITOCHONDRIAL FERRITIN AFFECTS JAK2/STAT5 PATHWAY IN K562 CELLS Sonia Levi, PhD, Alessandro Campanella, PhD, Elisabetta Rovelli, PhD, Benedetta Erba, Student, Anna Cozzi, PhD and Paolo Santambrogio, PhD Vita-Salute San Raffaele University (Presented By: Sonia Levi, PhD)

15:15 – 16:45 Concurrent Session II: Iron and Inflammation Location: D. Luis Chairs: Manuela Santos, Montreal, PQ, Canada and Guenter Weiss, Innsbruck, Austria

15:15 9 STRESS ERYTHROPOIESIS AND IRON MOBILIZATION IN A MOUSE MODEL MIMICKING ANEMIA OF CHRONIC DISEASE; RESPONSE TO ERYTHROPOIETIN INJECTIONS Sarah Millot¹, Valerie Andrieu, Dr², Philippe Letteron, Dr¹, Olivier Thibaudeau³, Ivana de Domenico, Dr4, Sigismond Lasocki, Dr5 and Carole Beaumont, Dr¹ ¹INSERM U773; ²Department of Hematology, Hopital Bichat; ³Department of Anatomopathology, Hopital Bichat; 4University of Utah, Salt lLake City; 5ICU, Hopital Bichat (Presented By: Sarah Millot)

15:30 10 DIFFERENTIAL EXPRESSION OF IRON METABOLISM GENES AND IRON HANDLING IN DISTINCT POPULATIONS OF HUMAN INFLAMMATORY MACROPHAGES Stefania Recalcati, MD, PhD¹, Massimo Locati, MD¹, Agnese Marini, PhD¹, Paolo Santambrogio, PhD², Annalisa Castagna, PhD³, Domenico Girelli, MD³ and Gaetano Cairo, PhD¹ ¹University of Milan; ²DIBIT HS S. Raffaele; ³University of Verona (Presented By: Gaetano Cairo, PhD)

15:45 11 THE ACTIVATION OF IRON REGULATORY PROTEIN 1 DOMINATES IRON HOMEOSTASIS IN INFLAMED INTESTINAL EPITHELIUM Esther Meyron-Holtz, Shirly Moshe, Orly Savion, Adi Kammer, Yoram Bujanover, MD, Batia Weiss, MD and Ram Reifen, MD, PhD Technion-Israel Institute of Technology (Presented By: Esther Meyron-Holtz)

16:00 12 ROLE OF TOLL-LIKE RECEPTOR / ADAPTOR PROTEIN MYD88 SIGNALING IN HYPOFERREMIA ASSOCIATED WITH INFLAMMATION Antonio Layoun, Hua Huang and Manuela M. Santos Institut du Cancer de Montréal, CRCHUM, University of Montréal (Presented By: Antonio Layoun)

16:15 13 ENDOTOXIN-MEDIATED INDUCTION OF HEPCIDIN EXPRESSION IS ATTENUATED IN TFR2 MUTANT MICE DESPITE NORMAL ACTIVATION OF STAT3 Robert Fleming, MD, Robert Britton, PhD, Mary Migas, MS, William Sly, MD and Abdul Waheed, PhD Saint Louis University School of Medicine (Presented By: Robert Fleming, MD)

16:30 14 FUNCTION OF THE IRE/IRP REGULATORY NETWORK IN MACROPHAGES Bruno Galy, PhD, Dunja Ferring-Appel, Lydie Viatte, Klaus Schuemann and Matthias Hentze European Molecular Biology Laboratory (Presented By: Bruno Galy, PhD)

20

Monday, June 8, 2009

15:15 – 16:45 Concurrent Session III: IRE / IRP Location: Arquivo Chairs: Robert Crichton, Wavre, Belgium and Lucas Kühn, Epalinges, Switzerland

15:15 15 FUNCTION OF THE IRE/IRP REGULATORY NETWORK IN SYSTEMIC IRON METABOLISM AND CENTRAL ORGANS FOR IRON HOMEOSTASIS Bruno Galy, PhD, Dunja Ferring-Appel, Sylvia Kaden, Hermann-Josef Groene and Matthias Hentze European Molecular Biology Laboratory (Presented By: Bruno Galy, PhD) 15:30 16 A NOVEL IRP2-/- MOUSE MODEL DISPLAYS LOCOMOTOR DYSFUNCTION AND NEURONAL IRON ACCUMULATION Kimberly Zumbrennen, PhD, Sabine Holter, PhD, Lore Becker, PhD, Brigit Rathkolb, PhD, Eva Rodansky, BSc and Elizabeth Leibold, PhD University of Utah (Presented By: Kimberly Zumbrennen, PhD)

15:45 17 GENOME-WIDE MRNA IDENTIFICATION AND PROTEOMIC ANALYSIS OF THE IRE/IRP REGULATORY NETWORK Matthias Hentze, Prof Dr¹, Mayka Sanchez, Dr¹, Bjoern Schwanhaeusser, Dr², Jonathon Blake, Dr³, Bruno Galy, Dr³, Yehven Vainshtein³, Tomi Bähr-Ivacevic³, Vladimir Benes, Dr³, Matthias Selbach, Dr² and Martina U. Muckenthaler, Dr4 ¹Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 153, 69120 Heidelberg, Germany and European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; ²Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin, Germany; ³European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; 4Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 153, 69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany (Presented By: Matthias Hentze, Prof Dr)

16:00 18 DEGENERATION OF MOTOR NEURONS IN THE SPINAL CORD OF IRP NULL MICE Suh Young Jeong, PhD¹, Bernard S. Jortner, VMD², Hayden Ollivierre³, Manik Ghosh, PhD³, Sharon Cooperman, MD, PhD³, Rachid Sougrat, PhD³ and Tracey A. Rouault, MD³ ¹NIH; ²Virginia Tech; ³NICHD, NIH (Presented By: Suh Young Jeong, PhD)

16:15 19 FERROCHELATASE DEFICIENCY IN ERYTHROPOIETIC TISSUES OF MICE LACKING IRON REGULATORY PROTEIN 2 Daniel Crooks, MS¹, Manik Ghosh, PhD², Hayden Olivierre-Wilson² and Tracey Rouault, MD² ¹Georgetown University; ²NICHD (Presented By: Daniel Crooks, MS)

16:30 20 IRON-INDEPENDENT PHOSPHORYLATION OF IRP2 REGULATES G2/M TRANSITION DURING CELL CYCLE PROGRESSION Elizabeth Leibold, PhD, Michelle Wallander, PhD, Kimberly Zumbrennen, PhD, Joshua Romney, PhD and Eva Rodansky, BS University of Utah (Presented By: Elizabeth Leibold, PhD)

17:00 – 18:30 Concurrent Sessions: IV – V

17:00 – 18:30 Concurrent Session IV: Iron Homeostasis and HFE Revisited Location: Arquivo Chairs: Martina Muckenthaler, Hiedelberg, Germany and Nathan Subramaniam, Brisbane, QLD, Australia

17:00 21 HIF-2 ALPHA BUT NOT HIF-1 ALPHA IS A KEY REGULATOR OF IRON ABSORPTION Maria Mastrogiannaki¹, Pavle Matak, PhD², Brian Keith, PhD³, Celeste Simon, PhD³, Sophie Vaulont, PhD² and Carole Peyssonnaux, PhD² ¹Institut Cochin; ²Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Inserm U567, Paris 75014, France; ³Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA (Presented By: Maria Mastrogiannaki)

17:15 22 BMP-SMAD SIGNALING IS IMPAIRED IN AN HFE KO MOUSE MODEL Elena Corradini, MD¹,4, Cinzia Garuti, PhD², Giuliana Montosi, PhD², Billy Andriopoulos Jr, PhD³, Yin Xia, PhD³, Herbert Y. Lin, MD, PhD³, Antonello Pietrangelo, MD, PhD² and Jodie L. Babitt, MD³ ¹Program in Membrane Biology, Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ²Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena, Italy; ³Program in Membrane Biology, Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 4Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena, Italy (Presented By: Elena Corradini, MD)

21

Monday, June 8, 2009

17:30 23 PHOSPHORYLATION OF RECEPTOR-ACTIVATED SMAD1/5/8 IS NOT ENHANCED IN HFE-DEFICIENT MICE DESPITE INCREASED BMP6 EXPRESSION Leon Kautz, PhD student¹, Delphine Meynard, PhD², Valerie Darnaud², Helene Coppin, PhD² and Marie-Paule Roth, MD, PhD² ¹INSERM; ²INSERM U563 (Presented By: Leon Kautz, PhD)

17:45 24 INTERACTION OF THE HEREDITARY HEMOCHROMATOSIS PROTEIN, HFE, WITH TRANSFERRIN RECEPTOR 2 IS REQUIRED FOR TRANSFERRIN-INDUCED HEPCIDIN EXPRESSION Junwei Gao, PhD¹, Juxing Chen, PhD¹, Maxwell Kramer, BA¹, Hidekazu Tsukamoto, MD², An-Sheng Zhang, MD¹ and Caroline Enns¹ ¹Oregon Health & Science University; ²University of Southern California (Presented By: Caroline Enns, PhD)

18:00 25 REGULATION OF HEPCIDIN BY DIETARY IRON IN MICE WITH FUNCTIONAL LOSS OF HFE AND TRANSFERRIN RECEPTOR 2 Robert Fleming, MD, Robert Britton, PhD, Mary Migas, MS, Margaret Rozier, BSc, Abdul Waheed, PhD and William Sly, MD Saint Louis University School of Medicine (Presented By: Robert Fleming, MD)

18:15 26 INVESTIGATION OF HFE-DEPENDENT REGULATION OF HEPCIDIN EXPRESSION Paul Schmidt, PhD¹, Mark Fleming, MD, DPhil¹ and Nancy Andrews, MD, PhD² ¹Children's Hospital Boston; ²Duke University School of Medicine (Presented By: Paul Schmidt)

17:00 – 18:30 Concurrent Session V: Iron, Proliferation and Cancer Location: D. Luis Chairs: D.R. Richardson, Wales, Australia and Suzy Torti, Winston-Salem, NC, USA

17:00 27 IRON DEPENDENT REGULATION OF MDM2 INFLUENCES P53 ACTIVITY AND HEPATIC CARCINOGENESIS Paola Dongiovanni, PhD², Anna Ludovica Fracanzani, MD², Gaetano Cairo, PhD³, Chiara Paola Megazzini, PhD², Stefano Gatti, MD4, Raffaela Rametta, PhD², Silvia Fargion, MD² and Luca Valenti, MD¹ ¹University of Milano; ²Internal Medicine, University of Milano; ³General Pathology, University of Milano; 4Surgery, Policlinico Hospital Milano (Presented By: Luca Valenti)

17:15 28 IRON CHELATORS FOR THE TREATMENT OF PANCREATIC CANCER: MECHANISM OF ACTION INVOLVING THE IRON-REGULATED GROWTH AND METASTASIS SUPPRESSOR, NDRG-1 Zaklina Kovacevic and Des R. Richardson, Professor of Cell Biology University of Sydney (Presented By: Zaklina Kovacevic)

17:30 29 FERRITIN H INDUCTION BY HISTONE DEACETYLASE INHIBITORS Wei Wang, PhD, Xiumin Di, MSc, Suzy Torti, PhD and Frank M. Torti, MD Wake Forest University School of Medicine (Presented By: Suzy Torti, PhD)

17:45 30 IRON TRANSPORT PROTEINS ARE INFLUENCED BY WNT SIGNALLING Matthew Brookes, Samuel Ford, Tariq Iqbal and Chris Tselepis University of Birmingham (Presented By: Chris Tselepis)

18:00 31 DEFERASIROX IS THE ONLY IRON CHELATOR INDUCING NF-KB INHIBITION IN MDS PATIENTS AND IN LEUKEMIC CELL LINES AND ACTS INDEPENDENTLY FROM REACTIVE OXYGEN SPECIES REDUCTION Emanuela Messa, MD, Sonia Carturan, PhD, Chiara Maffè, PhD, Marisa Pautasso, MD, Enrico Bracco, PhD, Antonella Roetto, PhD, Francesca Messa, PhD, Francesca Arruga, PhD, Ilaria Defilippi, PhD, Valentina Rosso, PhD, Chiara Zanone, PhD, Daniela Cilloni, MD, PhD and Giuseppe Saglio, MD Dept. of Clinical and Biological Sciences, University of Turin (Presented By: Emanuela Messa, MD)

18:15 32 CHARACTERIZATION OF THE PROMOTER REGION OF THE HAMP GENE IN THE BLACK SOUTH AFRICAN POPULATION AND ITS POSSIBLE ASSOCIATION WITH OESOPHAGEAL CANCER Nathaniel McGregor, BSc (Hon), Ann Louw, PhD, Louise Warnich, PhD and Monique Zaahl, PhD University of Stellenbosch (Presented By: Nathaniel McGregor, BSc (Hon))

22

Tuesday, June 9, 2009

17:00 – 18:30 Symposium: Iron Overload and Hematological Disorders Location: D. Maria Chairs: Maria Cappellini, Milan, Italy and Letícia Ribeiro, Coimbra, Portugal

17:00 – 17:20 Iron Overload in Myelodysplastic Syndromes Mario Cazzola, Pavia, Italy

17:20 – 17:40 MRI for Cardiac Iron Evaluation John Wood, Los Angeles, USA

17:40 – 18:00 State of Art of Iron Chelation Therapy Maria Cappellini, Milan, Italy

18:00 – 18:20 Future Therapeutic Approaches to Target Imbalances in Iron Homeostasis Antonello Pietrangelo, Modena, Italy

18:20 – 18:30 General Discussion

TUESDAY, JUNE 9th, 2009

08:00 – 14:30 Registration / Information Desk Open Location: Porto Congress Center Alfândega

09:00 – 10:30 Plenary Session III: Microbial Iron Metabolism Location: Arquivo Chairs: Guenther Winkelmann, Tübingen, Germany and Maria Salomé Gomes, Porto, Portugal

09:00 – 09:30 Lecture 3: Iron Metabolism in Yeast Caroline Philpott, University of UTA, USA

09:30 – 10:00 Lecture 4: Siderophores and the Battle for Iron by Aspergillus Hubertus Haas, Innsbruck Medical University, Austria

10:00 – 10:30 Podium Presentations

10:00 33 IRON UPTAKE IN GRAM-NEGATIVE BACTERIA: THE ROLE OF PERIPLASMIC PROTEINS IN SIDEROPHORE AND HEME TRANSPORT Hans Vogel, Byron Chu, MSc, Cheryl Lau, BSc, Zoya Slavinskaya, BSc, Alicia Garcia-Herrero, PhD, Renee Otten, MSc and Frans Mulder, PhD University of Calgary (Presented By: Hans Vogel)

10:15 34 THE ADAPTATION TO DIFFERENT DEGREES OF IRON DEFICIENCY IN SACCHAROMYCES CEREVISIAE DEPENDS ON THE AUTO- AND CROSS-REGULATION OF THE MRNA-DESTABILIZING PROTEINS CTH1 AND CTH2 Sandra Vergara, BSc and Dennis Thiele, PhD Duke University (Presented By: Sandra Vergara, BSc)

10:30 – 11:00 Coffee Break Location: Noble Hall

23

Tuesday, June 9, 2009

11:00 – 12:30 Plenary Session IV: Iron, Immunity and Inflammation Location: Arquivo Chairs: Guenter Weiss, Innsbruck, Austria and Hal Drakesmith, Oxford, UK

11:00 – 11:30 Lecture 5: The Immune System at the Crossroads of Iron Metabolism Maria de Sousa, ICBAS, University of Porto, Portugal

11:30 – 12:00 Lecture 6: Iron and Host-Pathogen Interactions Andrew Prentice, London School of Hygiene and Tropical Medicine, UK

12:00 – 12:30 Podium Presentations

12:00 35 HEPCIDIN: A CLUE FOR THE ROLE OF LYMPHOCYTES IN IRON HOMEOSTASIS Jorge Pinto, PhD¹, Vera Dias, MsC², Heinz Zoller, MD, PhD³, Pedro Rodrigues, PhD², Pedro Ramos², Graça Porto, MD, PhD² and Maria de Sousa, MD, PhD² ¹Institute for Molecular and Cell Biology – IBMC; ²Iron Genes and Immune System, Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal; ³Gastroenterology and Hepatology, Medical University, Innsbruck, Austria (Presented By: Jorge Pinto, PhD)

12:15 36 INCREASED PHOSPHORYLATION OF SMAD1/5/8 AND HEPCIDIN EXPRESSION IN RESPONSE TO LPS IN BMP6- DEFICIENT MICE Leon Kautz, PhD student, Delphine Meynard, PhD, Valerie Darnaud, Celine Fournier, Marie-Paule Roth, MD, PhD and Helene Coppin, PhD INSERM (Presented By: Leon Kautz, PhD student)

12:30 – 13:30 IBIS Business Meeting Location: Arquivo

13:30 – 14:30 Lunch Location: Noble Hall

Afternoon Free

24

Wednesday, June 10, 2009

WEDNESDAY, JUNE 10th, 2009

08:00 – 18:15 Registration / Information Desk Open Location: Porto Congress Center Alfândega

09:00 – 10:30 Plenary Session V: Cellular Iron Metabolism Location: Arquivo Chairs: Paolo Arosio, Milan, Italy and Ivana De Domenico, Salt Lake City, USA

09:00 – 09:30 Lecture 7: Role of Mitochondria in Iron-Sulfur and Iron Metabolism Roland Lill, Philipps Universität Marburg, Marburg, Germany

09:30 – 10:00 Lecture 8: Hepcidin Regulation and Processing Sophie Vaulont, Université Paris Descartes, CNRS, Paris, France

10:00 – 10:30 Podium Presentations

10:00 37 CELLULAR TRAFFICKING OF IRON AND IRON SULFUR CLUSTERS: THE ROLE OF MITONEET, A NOVEL OUTER MITOCHONDRIAL MEMBRANE PROTEIN WITH A LABILE IRON SULFUR CLUSTER Yael Harir, MSc², A. Conlan, MSc³, D. Michael, MSc, M. Shvartsman, MSc, D. Roisman-Sade, MSc, M. Paddock, PhD, P. Jennings, PhD, R. Mittler, PhD, I. Cabantchik, MD, PhD and Rachel Nechushtai, PhD¹ ¹Institute of Life Sciences/Hebrew University of Jerusalem; ²Hebrew University of Jerusalem; ³University of California San Diego (Presented By: Rachel Nechushtai, PhD)

10:15 38 HUMAN TRANSFERRIN RECEPTOR-1 IS AN ENDOCYTOSING RECEPTOR FOR H-FERRITIN Li Li, PhD¹, Mary C. Nakamura, MD¹, Celia J. Fang, MD¹, James C. Ryan, MD¹, Erene C. Niemi, BA¹, Hisashi Arase, MD, PhD², Suzy V. Torti, PhD³, Frank M. Torti, MD³ and William E. Seaman, MD¹ ¹University of California San Francisco; ²Osaka Univeristy; ³Wake Forest University (Presented By: William E. Seaman, MD)

10:30 – 11:00 Coffee Break Location: Noble Hall

11:00 – 12:30 Plenary Session VI: Systemic Iron Homeostasis – An Evolutionary Perspective Location: Arquivo Chairs: Tomas Ganz, LA, USA and Jerry Kaplan, Salt Lake City, USA

11:00 – 11:20 Lecture 9: Iron Metabolism in Plants Mary Lou Guerinot, Dartmouth College, Hanover USA

11:20 – 11:40 Lecture 10: Iron Metabolism in C. elegans Iqbal Hamza, University of Maryland, USA

11:40 – 12:00 Lecture 11: Iron Metabolism in Zebrafish Barry Paw, Harvard Medical School, USA

12:00 – 12:20 Lecture 12: Iron Metabolism in Mammals Marie-Paule Roth, Toulouse University, France

12:20 – 12:30 General Discussion

12:30 – 14:00 Lunch Location: Noble Hall

25

Wednesday, June 10, 2009

14:00 – 15:15 Poster Session II Presentations Location: Arquivo

Poster# 95 TFR2 BETA ISOFORMS ARE DIFFERENTIALLY LOCALIZED IN CELLS AND RESPOND DIFFERENTLY TO IRON TREATMENT Rosa Maria Pellegrino¹, Ilaria Defilippi, Dr², Enrico Bracco, PhD², Sonia Carturan, Dr², Antonietta Palmieri², Daniela Cilloni, MD², Giuseppe Saglio, MD² and Antonella Roetto, PhD² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Science, Az Osp San Luigi Gonzaga, Orbassano Torino Italy (Presented By: Rosa Maria Pellegrino)

Poster# 96 CHARACTERIZATION OF MOUSE SERUM FERRITIN Lyora A. Cohen, MSc, Avigail Morgenstern and Esther G. Meyron-Holtz Israel Institute of Technology, Technion (Presented By: Lyora A. Cohen, MSc)

Poster# 97 POSTNATAL INCREASES IN HEPCIDIN EXPRESSION AT BIRTH AND AT WEANING ARE DEPENDENT ON TRANSFERRIN RECEPTOR 2 Heather Morgan, MD, Robert Britton, PhD, Mary Migas, MS, Gail Palmer, BS, Abdul Waheed, PhD and Robert Fleming, MD Saint Louis University School of Medicine (Presented By: Robert Fleming, MD)

Poster# 98 THE ROLE OF DCYTB IN INTESTINAL IRON ABSORPTION Jeehyea Choi, Abas Laftah, Gladys Oluyemisi Latunde-Dada, Andrew McKie and Robert Simpson King’s College London (Presented By: Jeehyea Choi)

Poster# 99 ERYTHROPOIETIN INCREASES IRON ABSORPTION BY HUMAN INTESTINAL EPITHELIAL CELLS Bomee Chung¹, Timothy Chaston, PhD¹, Chiara Rapisarda, BSc¹, Katayoun Pourvali, BSc¹, Carin Da Silva, Bsc¹, Surjit Kaila Srai, PhD² and Paul Sharp, PhD¹ ¹King’s College London; ²University College London (Presented By: Bomee Chung)

Poster# 100 ALTERNATIVE FERRITIN MRNA TRANSLATION UNDER STRESS CONDITIONS VIA INTERNAL INITIATION Alina Daba¹, Anja Hausmann, PhD¹, Antonis Koromilas, PhD² and Kostas Pantopoulos, PhD² ¹Lady Davis Institute for Medical Research; ²Lady Davis Institute for Medical Research and McGill University (Presented By: Alina Daba)

Poster# 101 IN-CELL-WESTERN ANALYSIS OF ENDOGENOUS FERROPORTIN REGULATION IN PRIMARY MACROPHAGES Anne Auriac, Master, Alexandra Willemetz, Master and François Canonne-Hergaux, PhD UPR2301, CNRS, Centre de recherche de GIF-sur-Yvette, France (Presented By: Anne Auriac, Master)

Poster# 102 ROLE OF MUCIN 13 IN IRON METABOLISM Marizela Saric, Dr, Abas Laftah, Dr, Oluyemisi Latunde-Dada, Dr, Patarabutr Masaratana, MD, Robert Simpson, Dr and Andrew T. McKie, Prof Nutritional Sciences Division, King’s College London (Presented By: Marizela Saric, Dr)

Poster# 103 A NOVEL MUTATION IN INTESTINAL ISOFORM OF DIVALENT METAL TRANSPORTER 1 (DMT 1A) Luigia De Falco, Francesca Totaro, Mrs, Maria Nunzia Scoppettuolo, Mrs, Immacolata Andolfo, Mrs, David Beneitez Pastor, MD and Achille Iolascon, MD, PhD Ceinge Advanced Biotechnologies (Presented By: Luigia De Falco)

Poster# 104 UPSTREAM STIMULATORY FACTORS (USF-1/USF-2) REGULATE HUMAN HEMOJUVELIN GENE EXPRESSION Mohamed Salama, Henry Bayele, PhD and Kaila Srai, PhD University College London (Presented By: Mohamed Salama)

Poster# 105 REDOX CONTROL OF IRON REGULATORY PROTEIN 2 STABILITY Anja Hausmann¹, Julie Lee, MSc¹ and Kostas Pantopoulos, PhD² ¹Lady Davis Institute for Medical Research; ²Lady Davis Institute for Medical Research and McGill University (Presented By: Anja Hausmann)

Poster# 106 STRUCTURAL STUDIES OF THE SOUL/HBP FAMILY OF HEME BINDING PROTEINS Brian Goodfellow, PhD, Filipe Freire, BSc, Susana Aveiro, BSc, Maria João Romão, PhD, Ana Luisa Carvalho, PhD, Vitor Felix, PhD, Nuno Micaelo, PhD, Gloria Ferreira, PhD and Anjos Macedo, PhD Universidade de Aveiro (Presented By: Brian Goodfellow, PhD)

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Poster# 107 STRUCTURAL ANALYSIS OF HEMIN DEMETALLATION BY L-CHAIN APOFERRITINS Robert Crichton, BSc, PhD, FRSC, Natalia De Val, BSc, PhD, Changkee Lim, BSc, PhD and Jean-Paul Declercq, BSc, PhD University of Louvain (Presented By: Robert Crichton, BSc, PhD, FRSC)

Poster# 108 DEFINITION OF THE RESIDUES REQUIRED FOR THE INTERACTION BETWEEN GLYCINE-EXTENDED GASTRIN AND TRANSFERRIN IN VITRO Suzana Kovac, PhD², Audrey Ferrand, PhD², Jean-Pierre Estève, PhD³, Anne Mason, PhD4 and Graham Baldwin, PhD, DSc¹ ¹University of Melbourne; ²University of Melbourne Department of Surgery, Austin Health, Melbourne, Australia; ³INSERM U.858, Plateforme d’interaction moléculaire, I2MR, Institut Louis Bugnard, IFR31, Toulouse, France; 4University of Vermont, College of Medicine, Department of Biochemistry, Vermont, USA (Presented By: Graham Baldwin)

Poster# 109 DMT1 EXPRESSION CAN LEAD TO ACCUMULATION OF METALS IN CELLS Michael Garrick, PhD¹, Lin Zhao, MD¹, Saied Ghadersohi¹, Jackie Stonehuerner, BS², Andy Ghio, MD² and Laura Garrick, PhD¹ ¹SUNY at Buffalo; ²EPA (Presented By: Michael Garrick, PhD)

Poster# 110 AGGREGATION AND DISSOCIATION OF PEA SEED FERRITIN REGULATED BY PH AND FERRIC ION Guanghua Zhao, PhD, Chaorui Li, PhD, Xiaoping Fu, PhD, Fang Chen, PhD and Xiaosong Hu, PhD China Agricultural University (Presented By: Guanghua Zhao, PhD)

Poster# 111 THE EFFECT OF COPPER LOADING AND DEPLETION ON IRON TRANSPORTER EXPRESSION IN CACO-2 CELLS Katayoun Pourvali and Paul Sharp, PhD King’s College London (Presented By: Katayoun Pourvali)

Poster# 112 ACTIVATION OF AN UNFOLDED PROTEIN RESPONSE MODULATES THE EXPRESSION OF IRON-RELATED GENES Susana Oliveira, PhD student, S.F. de Almeida, PhD, G. Picarote, undergraduate student, V.M. Costa, PhD student, F. Carvalho, PhD, J.E. Azevedo, PhD, J.P. Pinto, PhD and M. de Sousa, MD, PhD Instituto de Biologia Molecular e Celular, Universidade do Porto (Presented By: Susana Oliveira, PhD student)

Poster# 113 BMP6 IS A KEY ENDOGENOUS REGULATOR OF HEPCIDIN EXPRESSION AND IRON METABOLISM Billy Andriopoulos, Jr.², Elena Corradini²,5, Yin Xia², Sarah A. Faasse², Shanzhuo Chen², Lovorka Grgurevic³, Mitchell D. Knutson4, Antonello Pietrangelo5, Slobodan Vukicevic³, Herbert Y. Lin² and Jodie L. Babitt, MD¹ ¹Massachusetts General Hospital, Harvard Medical School; ²Program in Membrane Biology, Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ³Laboratory of Mineralized Tissues, School of Medicine, University of Zagreb, Zagreb, Croatia; 4Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida; 5Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena Italy (Presented By: Jodie L. Babitt, MD)

Poster# 114 FUNCTION OF THE IRP/IRE REGULATORY NETWORK IN HEPATOCYTES Bruno Galy, PhD, Dunja Ferring-Appel, Lydie Viatte, Sylvia Kaden, Hermann-Josef Groene and Matthias Hentze European Molecular Biology Laboratory (Presented By: Bruno Galy, PhD)

Poster# 115 FUNCTINAL RNAI SCREEN IDENTIFIES CCL2 (MCP-1) AS A NOVEL REGULATOR OF TRANSFERRIN UPTAKE Martina Muckenthaler¹, Mingang Zhu, PhD²,³, Fabian Bartz³,4, Till Meinhof4, Holger Erfle5, Matthias Hentze, MD³,6, Rainer Pepperkok, PhD³,5 and Heiko Runz, MD³,4 ¹Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg; ²Department of Pediatrics, University of Heidelberg; ³Molecular Medicine Partnership Unit (MMPU), University of Heidelberg/EMBL Heidelberg; 4Institute of Human Genetics, University of Heidelberg; 5Cell Biology/Biophysics Unit, European Molecular Biological Laboratories (EMBL); 6Gene Expression Unit, European Molecular Biological Laboratories (EMBL) (Presented By: Martina Muckenthaler)

Poster# 116 HEPCIDIN TRIGGERS REDUCTION IN DMT1 PROTEIN IN INTESTINAL CELLS Carole Lagnel, Dr¹, Andre Bado, Dr¹, Philippe Letteron, Dr¹, Corinne Nazaret¹, Dimitri Tchernitchko, Dr¹, Soumeya Bekri, Dr² and Carole Beaumont, Dr¹ ¹INSERM U773; ²University of Rouen, Rouen (Presented By: Carole Lagnel, Dr)

Poster# 117 INTESTINAL IRON ABSORPTION IN NEONATAL RATS DOES NOT CORRELATE WITH CHANGES IN THE EXPRESSION OF THE IRON REGULATORY HORMONE HEPCIDIN Deepak Darshan, MBBS, PhD, David M. Frazer, PhD, Sarah J. Wilkins, BSc (Hons) and Greg J. Anderson, PhD Queensland Institute of Medical Research, PO Royal Brisbane & Women’s Hospital, Brisbane, QLD 4029, Australia (Presented By: Deepak Darshan, MBBS, PhD)

Poster# 118 PHLEBOTOMY-INDUCED IRON MOBILISATION FROM TISSUES OF HJV-/- MICE Jan Krijt, PhD, Ludek Sefc, Martin Vokurka, Tereza Hlobenova and Emanuel Necas Charles University in Prague, First Faculty of Medicine, Institute of Pathophysiology and Center of Experimental Hematology (Presented By: Jan Krijt, PhD)

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Poster# 119 THE ROLES OF IRON AND FERRITIN IN ISCHEMIC PRECONDITIONING OF THE NON-DIABETIC AND DIABETIC HEART Mottie Chevion, BSc, MSc, PhD, Vladimir Vinokur, MSc, Baruch Bulvik, MSc, Abraham Konijn, PhD and Eduard Berenshtein, MD, PhD Hebrew University of Jerusalem (Presented By: Mottie Chevion, BSc, MSc, PhD)

Poster# 120 IMMUNOASSAY FOR HUMAN HEPCIDIN IN BLOOD AND DEVELOPMENT OF A PROTOTYPE ELISA KIT Mark Westerman, BS, MS, PhD¹, Gordana Olbina, BS, MS, PhD¹, Siong Wie, BS, PhD², Vaughn Ostland, BS, MS, PhD¹, Domenico Girelli, MD, PhD³, Kimberley O'Brien, BS, PhD4, Elizabeta Nemeth, BS, MS, PhD¹ and Tomas Ganz, BS, PhD, MD¹ ¹Intrinsic LifeSciences; ²Immunodiagnostic Reagents Inc; ³University of Verona; 4Cornell University (Presented By: Mark Westerman, BS, MS, PhD)

Poster# 121 ALTERATIONS IN IRON STATUS ARE ASSOCIATED WITH CHANGES IN THE CHOLESTEROL BIOSYNTHESIS PATHWAY Ross M. Graham, BSc (Hons) PhD¹, Anita C.G. Chua, BSc (Hons) PhD¹, Kim Carter, BSc (Hons) PhD², Roheeth D. Delima, BSc (Hons)¹, Daniel Johnstone, BSc (Hons)³, Carly E. Herbison, BSc (Hons)¹, Martin J. Firth, BSc (Hons) PhD², Rebecca O’Leary, BSc (Hons) PhD², Elizabeth A. Milward, BSc (Hons) PhD³, John K. Olynyk, MBBS, MD¹ and Debbie Trinder, BSc (Hons) PhD¹ ¹University of Western Australia and Western Australian Institute for Medical Research; ²University of Western Australia and Telethon Institute for Child Health Research, Western Australia; ³University of Newcastle, New South Wales, Australia (Presented By: Ross M. Graham, BSc (Hons) PhD)

Poster# 122 DIFFERENCES IN GENE EXPRESSION OF DUODENAL IRON TRANSPORTERS IN HEMOCHROMATOSIS SUBJECTS WITH AND WITHOUT IRON OVERLOAD James Nelson, PhD¹, Stuart Raaka, Virginia Mugford, Ellen Kilcourse and Kris Kowdley, MD² ¹Benaroya Research Institute; ²Benaroya Research Institute and Virginia Mason Medical Center (Presented By: James Nelson)

Poster# 123 IDENTIFICATION OF AN IRON-REGULATED E3 UBIQUITIN LIGASE AND ITS ROLE IN IRON REGULATORY PROTEIN 2 STABILITY Ameen Salahudeen, BS, Joel Thompson, BS and Richard Bruick, PhD UTSW Medical Center (Presented By: Richard Bruick, PhD)

Poster# 124 59FE-DISTRIBUTION IN CONDITIONAL FERRITIN H KNOCKOUT MICE Klaus Schuemann, Dr¹, Anna Even¹, Stefan Wagner¹, Heike Weinheimer, MD¹, Depak Darshan, PhD², Liviu Vanoaica² and Lukas Kuehn, PhD² ¹Technical University Munich; ²ISREC (Presented By: Klaus Schuemann, Dr)

Poster# 125 GENOME-WIDE SIRNA SCREEN TO IDENTIFY REGULATORS OF THE HEPCIDIN/FERROPORTIN REGULATORY SYSTEM Sandro Altamura, PhD¹, Wolfgang Gilles, PhD², Heiko Runz, PhD², Rainer Pepperkok, PhD³, Matthias Hentze, Prof4 and Martina Muckenthaler, Prof² ¹MMPU – Molecular Medicine Partnership Unit; ²University of Heidelberg / MMPU; ³EMBL – European Molecular Biology Laboratory; 4EMBL / MMPU (Presented By: Sandro Altamura, PhD)

Poster# 126 DAILY REGULATION OF SERUM AND URINARY HEPCIDIN IS NOT INFLUENCED BY SUBMAXIMAL CYCLING- BASED EXERCISE IN HUMANS WITH NORMAL IRON METABOLISM Marie-Bérengère Troadec, PhD¹,¹°, Fabrice Lainé, MD², Vincent Daniel, MD³, Pierre Rochcongar, MD, Professor³, Martine Ropert, MD4, Florian Cabillic, MD5, Michèle Perrin², Jeff Morcet², Olivier Loréal, MD, PhD6, Gordana Olbina, PhD7, Mark Westerman, MD, PhD7, Elizabeta Nemeth, MD, PhD8, Tomas Ganz, MD, PhD, Professor8 and Pierre Brissot, MD, Professor1, 9 ¹Inserm U-522, Inserm CIC 203, Laboratory of Physiology and Sportsmedicine, Liver Disease Unit, University Hospital Pontchaillou; ²Center For Clinical Investigation, Inserm 0203,University Hospital Pontchaillou, Rennes, France; ³Laboratory of Physiology and Sportsmedicine, University Hospital Pontchaillou, Rennes, France; 4Laboratory of Biochemistry, University Hospital Pontchaillou, Rennes, France; 5URU biothérapies innovantes, and Laboratoire de cytogénétique et biologie cellulaire, University Hospital Pontchaillou, Rennes, France; 6Inserm U-522, IFR 140, University Hospital Pontchaillou, Rennes, France; 7Intrinsic LifeSciences, LLC, La Jolla, California, USA; 8David Geffen School of Medicine, University of California, Los Angeles, USA; 9Liver Disease Unit, Inserm U-522, IFR 140, University Hospital Pontchaillou, Rennes, France; ¹°Intrinsic Life Sciences, La Jolla, California, David Geffen School of Medicine, UCLA, California, USA (Presented By: Marie-Bérengère Troadec, PhD)

Poster# 127 ARABIDOPSIS NRAMP1 IS REQUIRED FOR HIGH-AFFINITY MANGANESE UPTAKE BY THE ROOT Rémy Cailliatte, PhD, Jean-François Briat, PhD, Stéphane Mari, PhD and Catherine Curie BPMP-IBIP (Presented By: Catherine Curie)

Poster# 128 THE STRUCTURE OF THE HEPCIDIN-FERROPORTIN BINDING INTERFACE Elizabeta Nemeth, PhD, Gloria Preza, MS, Alan Waring, PhD, Richard Clark, PhD, David Craik, PhD and Tomas Ganz, PhD, MD University of California, Los Angeles (Presented By: Elizabeta Nemeth, PhD)

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Poster# 129 TWO TFR2 MICE MODELS REPRODUCE HEMOCHORMATOSIS TYPE 3 AND CAN GIVE INSIGHT INTO MOLECULAR MECHANISMS OF TFR2 PROTEIN Antonella Roetto, PhD¹, Rosa Maria Pellegrino, Dr², Ferdinando Di Cunto, Prof³, Emilio Hirsch, Prof³, Ornella Azzolino, Dr³, Sonia Carturan, Dr², Fiorella Altruda, Prof³, Clara Camaschella, Prof4 and Giuseppe Saglio, Prof² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Science, Az Osp San Luigi Gonzaga, Orbassano Torino Italy; ³University of Torino Department of Genetics, Biology and Biochemistry Molecular Biotecnology Center, Torino, Italy; 4Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milan, Italy (Presented By: Antonella Roetto, PhD)

Poster# 130 DEGRADATION PATHWAYS OF HUMAN IRON REGULATORY PROTEIN 2 Camille Dycke², Peggy Charbonnier, Catherine Bougault, Kostas Pantopoulos³ and Jean-Marc Moulis, PhD¹ ¹CEA-Grenoble/IRTSV-LCBM; ²CEA-CNRS-Université Grenoble, France; ³Lady Davis Institute for Medical Research, Department of Medicine, McGill University, Montreal, Quebec, Canada (Presented By: Jean-Marc Moulis, PhD)

Poster# 131 WITHDRAWN METABOLIC REGULATION OF IRON HOMEOSTASIS IN HEREDITARY LEIOMYOMATOSIS AND RENAL CELL CARCINOMA Wing-Hang Tong, Suh Young Jeong, PhD, Gennadiy Kovtunovych, PhD, Youfeng Yang, PhD, Marston Linehan, MD and Tracey A. Rouault, MD National Institute of Child Health and Human Development, USA

Poster# 132 HEPATIC COPPER CONTENT IS REDUCED IN WILSON DISEASE KNOCKOUT MICE FED AN IRON-ENRICHED DIET Uta Merle, PD, Dr, Sabine Tuma, Valer Muntean, Martin Volkmann, Sven Gehrke and Wolfgang Stremmel, Prof Dr University of Heidelberg (Presented By: Wolfgang Stremmel, Prof Dr)

Poster# 133 FPN1 INTERACTS WITH HEPHAESTIN AND HEME OXYGENASE 1 BUT NOT ABCG2 IN INTESTINAL IRON ABSORPTION Kwo-yih Yeh, PhD, Mary Yeh, MS, Laura Mims, BS and Jonathan Glass, MD LSUHSC, Shreveport, LA (Presented By: Kwo-yih Yeh, PhD)

Poster# 134 HEREDITARY HAEMOCHROMATOSIS PROTEIN (HFE)-DEPENDENT REGULATION OF IRON TRANSFER ACROSS PLACENTA Rumeza Hanif, PhD student¹, Sara Balesaria, PhD¹, Harry J. McArdle, PhD² and Kaila Srai, PhD¹ ¹University College London; ²University of Aberdeen (Presented By: Rumeza Hanif, PhD student)

Poster# 135 GENERATION OF A MOUSE STRAIN WITH INDUCIBLE IRP1 OVEREXPRESSION: A NEW MODEL FOR UNDERSTANDING IRON METABOLISM DISORDERS D. Casarrubea¹, L. Viatte, R. Eisenstein², B. Galy1 and M.W. Hentze1 ¹European Molecular Biology Laboratory; ²University of Wisconsin (Presented By: D. Casarrubea)

Poster# 136 EXPRESSION OF GENES INVOLVED IN IRON IMPORT/EXPORT IN HUMAN INTESTINAL AND LIVER CELLS: EFFECT OF IRON AVAILABILITY AND MODULATION BY ETHANOL Jan Kovar, DSc, Kamila Balusikova, Jitka Neubauerova and Marketa Dostalikova-Cimburova Third Faculty of Medicine, Charles University, Prague (Presented By: Jan Kovar, DSc)

Poster# 137 INTERRELATIONS BETWEEN IRON METABOLISM AND ETHANOL EFFECT ON CELL PROLIFERATION IN THE HUMAN HEPATOMA HEPARG CELL LINE Thi Hong Tuoi Do², François Gaboriau², Romain Moirand²,³, Caroline Le Lan²,³, Isabelle Cannie², Lucie Gouffier², Olivier Loréal², Pierre Brissot²,³ and Gérard Lescoat¹ ¹INSERM U522; ²Inserm U522 – EA « MDC », Université de Rennes 1, IFR 140, Rennes; ³Service des maladies du Foie, CHU Pontchaillou, Rennes (Presented By: Gérard Lescoat)

Poster# 138 HIGHLY SENSITIVE HISTOCHEMICAL STAINING OF IRON COMBINED TO GENETIC ANALYSIS OF ARABIDOPSIS EMBRYOS REVEAL THAT IRON SPECIFICALLY ACCUMULATES IN THE ENDODERMAL CELL LAYER Hannetz Roschzttardtz, PhD, Genevieve Conejero, PhD, Stéphane Mari, PhD and Catherine Curie BPMP-IBIP (Presented By: Catherine Curie)

Poster# 139 CHARACTERIZATION OF FELINE SERUM FERRITIN-BINDING POTEINS: THE PRESENCE OF A NOVEL FERRITIN- BINDING PROTEIN AS AN INHIBITORY FACTOR IN FERRITIN IMMUNOASSAY Koichi Orino, PhD, DVM and Kiyotaka Watanabe, PhD, DVM Kitasato University (Presented By: Koichi Orino, PhD, DVM)

Poster# 140 REGULATORY EFFECTS OF FERRITIN ON H-KININOGEN: IMPLICATIONS FOR ANGIOGENESIS Lan Coffman, PhD, Yufeng Song, BA, Frank Torti, MD and Suzy Torti, PhD Wake Forest University School of Medicine (Presented By: Suzy Torti, PhD)

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Poster# 141 OVEREXPRESSION OF IRP2 INCREASES THE GROWTH OF TUMOR XENOGRAFTS IN NUDE MICE Carmen Maffettone¹ and Kostas Pantopoulos, PhD² ¹Lady Davis Institute for Medical Research; ²Lady Davis Institute for Medical Research and McGill University (Presented By: Carmen Maffettone)

Poster# 142 C282Y AND H63D MUTATIONS IN HFE GENE IMPACT ON EPITHELIAL OVARIAN CANCER RISK AND SURVIVAL Christine M. Maugard, MD, PhD, Sanae Medelci, Beaulieu Martin, Philipe Gannon, Diane M. Provencher, Anne-Marie Mes-Masson and Manuela M. Santos Montreal Cancer Institute / CRCHUM / University of Montréal (Presented By: Christine M. Maugard, MD, PhD)

Poster# 143 GENOME-WIDE MICROARRAY ANALYSIS OF MELANOMA REVEALS UNEXPECTED ANOMALIES IN IRON- RELATED GENE EXPRESSION Daniel Johnstone, Martin Ravetti, PhD, Carlos Riveros, PhD, Pablo Moscato, PhD, Peter Hersey, MD, Rodney Scott, PhD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Poster# 144 A ROLE FOR LUMINAL IRON IN COLORECTAL CARCINOGENESIS Chris Tselepis, Sorina Radulescu, Matthew Brookes, Tariq Iqbal and Owen Sansom University of Birmingham (Presented By: Chris Tselepis)

Poster# 145 MOLECULAR-GENETIC ANALYSIS OF CERULOPLASMIN IN OESOPHAGEAL CANCER Natalie Strickland, MSc Genetics¹, Tandi Matsha and Monique Zaahl, PhD Genetics² ¹University of Stellenbosch; ²University of Genetics (Presented By: Natalie Strickland, MSc Genetics)

Poster# 146 MUTATION ANALYSIS OF THE SLC40A1 PROMOTER IN BLACK SOUTH AFRICAN OESOPHAGEAL CANCER PATIENTS Jessica Vervalle, BSc (Hons), Ann Louw, PhD, Louise Warnich, PhD and Monique Zaahl, PhD Stellenbosch University (Presented By: Jessica Vervalle, BSc (Hons))

Poster# 147 MOLECULAR CHARACTERIZATION OF THE 5’ UNTRANSLATED REGION (UTR) OF THE CYTOCHROME B REDUCTASE 1 (CYBRD1) GENE OF OESOPHAGEAL CANCER PATIENTS Veronique Human, MSc¹, Thandi Matsha, PhD², Louise Warnich, PhD¹ and Monique Zaahl, PhD¹ ¹Stellenbosch University; ²Cape Peninsula University of Technology (Presented By: Veronique Human, MSc)

Poster# 148 HEPHAESTIN IS INVOLVED IN CONTROLLING IRON EFFLUX FROM OLIGODENDROCYTES IN THE CENTRAL NERVOUS SYSTEM Katrin Schulz¹, Chris D. Vulpe, PhD² and Samuel David, PhD³ ¹Center for Research in Neuroscience; ²Department of Nutritional Sciences and Toxicology, University of California; ³Center for Research in Neuroscience, The Research Institute of the McGill University Health Center (Presented By: Katrin Schulz)

Poster# 149 EXTRACELLULAR H-FERRITIN IS AN ALTERNATIVE IRON IMPORT MECHANISM IN OLIGODENDROCYTES, AND ITS RECEPTOR IS TIM-2 James R. Connor, PhD, Bozho Todorich, BS, Xuesheng Zhang, PhD and Becky Slagle-Webb, BS Penn State College of Medicine (Presented By: James R. Connor, PhD)

Poster# 150 THE COMPARTAMENTALIZED DISTRIBUTION OF FERROPORTIN IN THE MOUSE CENTRAL NERVOUS SYSTEM IS INDICATIVE OF A DELICATE FUNCTION FOR NEURONAL IRON METABOLISM M. Boserup², J. Lichota², D. Haile³ and Torben Moos¹ ¹Aalborg University; ²Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; ³Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA (Presented By: Torben Moos)

Poster# 151 IRON UPTAKE IN THE RETINA OF THE RAT: IMPLICATIONS FOR AGE-INDEPENT AND CONTINOUS IRON UPTAKE, PLUS ANTEROGRADE AXONAL IRON TRANSPORT BY RETINAL GANGLION CELLS Torben Moos¹, N. Bernth², Y. Courtois³ and E.H. Morgan4 ¹Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; ²Danish Technological Institute, Taastrup, Denmark; ³INSERM 598, University Pierre and Marie Curie, Paris, France; 4Department of Physiology, University of Western Australia, Crawley, Australia (Presented By: Torben Moos)

Poster# 152 RELATIONSHIP BETWEEN BRAIN R2 AND LIVER AND SERUM IRON CONCENTRATIONS IN ELDERLY MEN Michael House, PhD¹, Timothy St. Pierre¹, Elizabeth Milward², David Bruce¹ and John Olynyk¹ ¹The University of Western Australia; ²The University of Newcastle (Presented By: Michael House, PhD)

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Poster# 153 THE HEPCIDIN MRNA AND FPN1 PROTEIN EXPRESSION IN THE CP-/- MOUSE BRAIN Shu-Min Wang, Pei Guo, Zhen-Ling Yang, Xiang-Lin Duan and Yan-Zhong Chang, PhD Hebei Normal University (Presented By: Yan-Zhong Chang, PhD)

Poster# 154 THE INCREASING IRON ACCUMULATION IN THE AGING BRAIN IS REFLECTED BY AN INCREASE IN NEURONAL FERRITIN RATHER THAN OF FERROPORTIN M. Boserup¹, L. Lichota¹, D. Haile² and Torben Moos¹ ¹Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.; ²Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA (Presented By: Torben Moos)

Poster# 155 INHIBITION OF PROLIFERATION OF OLN 93 CELLS BY IRON CHELATORS: USE AS TEST SYSTEM TO STUDY THE BIOAVAILABILITY OF IRON IN IRON OXIDE NANOPARTICLES Michaela Hohnholt, Mark Geppert and Ralf Dringen, Dr University Bremen (Presented By: Michaela Hohnholt)

Poster# 156 THE IONOTROPIC GLUTAMATE RECEPTOR AGONIST N-METHYL D-ASPARTATE (NMDA) AND SPATIAL MEMORY TRAINING ENHANCE THE EXPRESSION OF THE IRON TRANSPORTER DMT1 IN HIPPOCAMPAL NEURONS Paola Haeger, PhD², Pablo Munoz, PhD³, Alexis Humeres, MSc², M. Angelica Carrasco, PhD4, Marco T. Nunez, PhD5 and Cecilia Hidalgo, PhD¹ ¹F. Medicine, U. Chile; ²CEMC, F. Medicine, U. de Chile, Santiago, Chile; ³CIBR, U. de Valparaíso, Valparaíso, Chile; 4ICBM & CEMC, F. Medicine, U. de Chile, Santiago, Chile; 5MI CDB, F. Sciences, U. de Chile, Santiago, Chile (Presented By: Cecilia Hidalgo, PhD)

Poster# 157 BRAIN IRON MANAGEMENT PROTEIN GENE EXPRESSION VARIES ACROSS THE DIURNAL CYCLE AND WITH DIETARY IRON DEPRIVATION Erica Unger, PhD, John Beard, PhD, Lei Hao, Narasimha Hegde, PhD, Byron Jones, PhD and James Connor, PhD Pennsylvania State University (Presented By: Erica Unger, PhD)

Poster# 158 GENOME-WIDE MICROARRAY ANALYSIS OF BRAIN FROM A HEMOCHROMATOSIS HFE KNOCKOUT MOUSE MODEL SHOWS FEW CHANGES IN IRON-RELATED GENE EXPRESSION Daniel Johnstone, Ross Graham, PhD, Deborah Trinder, PhD, Rodney Scott, PhD, John Olynyk, MD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Poster# 159 QUANTIFICATION OF IRON IN HUMAN BRAIN IN VIVO USING APPARENT TRANSVERSE RELAXATION RATE OBTAINED AT HIGH FIELD MRI Fumiyuki Mitsumori, PhD, Hidehiro Watanabe, PhD and Nobuhiro Takaya National Institute for Environmental Studies (Presented By: Fumiyuki Mitsumori, PhD)

Poster# 160 CHARACTERIZATION OF THE IRON MANAGEMENT PROTEIN PROFILES IN THE BRAINS OF MICE CARRYING THE H67D MUTATION IN HFE GENE James Connor, PhD, Wint Nandar, BS and Elizabeth Neely, BS Penn State University, M.S. Hershey Medical Center (Presented By: James Connor, PhD)

Poster# 161 FORWARD GENETIC ANALYSIS OF BRAIN IRON MANAGEMENT DURING IRON DEFICIENCY Byron C. Jones, PhD, Leslie C. Jellen, BS, Erica L. Unger, PhD and John L. Beard, PhD The Pennsylvania State University (Presented By: Byron C. Jones, PhD)

Poster# 162 CELLULAR IMPACT OF H63D HFE MUTANT PROTEIN ON ALS Sang Lee, PhD¹, Yiting Liu, PhD¹, Zachary Simmons, MD² and James Connor, PhD¹ ¹Penn State College of Medicine; ²Penn State MS Hershey Medical Center (Presented By: Sang Lee, PhD)

Poster# 163 CERULOPLASMIN DIFFERENTIAL EXPRESSION IN THE CEREBROSPINAL FLUID OF PARKINSON’S DISEASE REFLECTS OXIDATION AND FUNCTIONAL IMPAIRMENT FAVOURING INTRACELLULAR IRON OVERLOAD Stefano Olivieri¹, Sandro Iannaccone, Doctor², Antonio Conti, Doctor², Carlo Cannistraci, Doctor², Diego Franciotta, Doctor³, Laura Piccio, Doctror4, Stefano Cappa, Professor5 and Massimo Alessio, Doctor² ¹San Raffaele Scientific Institute; ²San Raffaele Scientific Institute, Milan; ³Istituto Neurologico Mondino, Pavia; 4Washington University School of Medicine, St. Louis; 5San Raffaele University, Milan (Presented By: Stefano Olivieri)

Poster# 164 ASSOCIATION STUDIES IN ALZHEIMER’S DISEASE EXCLUDE CAT53 AS A NEW SUSCEPTIBILITY GENE BUT SUPPORT A PUTATIVE PROTECTIVE ROLE OF THE C282Y HFE MUTATION Ana Paula Correia, MD¹,³, Jorge Pinto, PhD², Vera Dias, Technician², Cláudia Mascarenhas, Technician², Susana Almeida, Technician² and Graça Porto, MD, PhD²,³,4 ¹Hospital de Magalhães Lemos, Porto; ²IBMC – Institute for Molecular and Cellular Biology, Porto; ³Centro Hospitalar do Porto, Hospital de Santo António, Porto; 4ICBAS – Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal (Presented By: Ana Paula Correia, MD)

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Poster# 165 HFE POLYMORPHISM, H63D, IMPACTS TAU PHOSPHORYLATION James Connor, PhD¹, Eric Hall, II, BS¹, Sang Lee, PhD¹, Noot Mairuae, MS² and Zach Simmons, MD¹ ¹Penn State University; ²Chulalongkorn University (Presented By: James Connor, PhD)

Poster# 166 REACTIVE OXYGEN SPECIES REGULATE CERULOPLASMIN BY A NOVEL MRNA DECAY MECHANISM INVOLVING ITS 3’-UNTRANSLATED REGION: IMPLICATIONS IN IRON DEPOSITION IN NEURODEGENERATIVE DISEASES Nisha Tapryal, MSc and Chinmay Mukhopadhyay, PhD Jawaharlal Nehru University (Presented By: Nisha Tapryal, MSc)

Poster# 167 LONG TERM DEFERIPRONE INDUCES IRON DEFICIENCY IN FRIEDREICH ATAXIA PATIENTS Filomena Longo, MD², Simona Roggero, MD², Chiara Compagni, MD² and Antonio Piga, Prof¹ ¹Department of Biological and Clinical Sciences; ²University of Turin (Presented By: Antonio Piga, Prof)

Poster# 168 ABNORMAL METAL DISTRIBUTION IN THE WHITE MATTER OF THE BRAIN IN FRIEDREICH ATAXIA Bogdan Popescu, MD¹, Deborah Renaud, MD², Joseph Parisi, MD² and Helen Nichol, PhD¹ ¹University of Saskatchewan; ²Mayo Clinic Foundation (Presented By: Bogdan Popescu, MD)

Poster# 169 THE EXPRESSION AND ROLE OF LIPOCALIN 2 AND ITS RECEPTOR AFTER SPINAL CORD INJURY Khizr Rathore, Adriana Redensek, MSc, Ruben Lopez-Vales, PhD, Manuela Santos, PhD, Shizuo Akira, PhD, Alan Aderem, PhD and Samuel David, PhD Centre for Research in Neuroscience, McGill University (Presented By: Khizr Rathore)

Poster# 170 HO-1-MEDIATED MACROAUTOPHAGY: A MECHANISM FOR UNREGULATED BRAIN IRON DEPOSITION Hillel Zukor¹, Wei Song, MD, PhD¹, Adrienne Liberman, BSc¹, Jeannie Mui, BSc¹, Hojatollah Vali, PhD¹, Carine Fillebeen, PhD¹, Kostas Pantopoulos, PhD¹, Ting-Di Wu, PhD², Jean-Luc Guerquin-Kern, PhD² and Hyman Morris Schipper, MD, PhD¹ ¹McGill University; ²Institut Curie (Presented By: Hillel Zukor)

Poster# 171 GENE EXPRESSION CHANGES RELATED TO ALZHEIMER’S DISEASE AND OTHER NEURODEGENERATIVE DISORDERS IN A HEMOCHROMATOSIS HFE KNOCKOUT MOUSE MODEL Daniel Johnstone, Ross Graham, PhD, Deborah Trinder, PhD, Rodney Scott, PhD, John Olynyk, MD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Poster# 172 THE ROLE OF MITOCHONDRIAL FERRITIN ON HYDROGEN PEROXIDE INDUCED SH-SY5Y CELL DAMAGE Nan Zhang, Zhen-Hua Shi, Yan-Zhong Chang, PhD and Xiang-Lin Duan Hebei Normal University (Presented By: Xiang-Lin Duan)

Poster# 173 IRON, FRATAXIN AND FRIEDREICH’S ATAXIA NEURODEGENERATION: DEFECTIVE FOLDING AND FUNCTION IN COMPOUND HETEROZYGOTES Cláudio M. Gomes, PhD and Ana R. Correia ¹Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-756 Oeiras, Portugal (Presented By: Cláudio M. Gomes, PhD)

Poster# 174 ANALYSIS OF IRON HOMEOSTASIS IN SKIN FIBROBLASTS FROM PANTOTHENATE KINASE ASSOCIATED NEURODEGENERATION PATIENTS Alessandro Campanella¹, Elisabetta Rovellli², Barbara Garavaglia³ and Sonia Levi4 ¹University Vita-Salute San Raffaele; ²San Raffaele Scientific Institute, DIBIT, Milan, Italy; ³Fondazione IRCCS Istituto Neurologico “C. Besta”, Milan, Italy; 4University Vita-Salute San Raffaele and San Raffaele Scientific Institute, DIBIT, Milan, Italy (Presented By: Alessandro Campanella)

Poster# 175 SYNCHROTRON X-RAY FLUORESCENCE REVEALS ABNORMAL DISTRIBUTION OF METALS IN BRAIN AND SPINAL CORD IN SPINOCEREBELLAR ATAXIA: A CASE REPORT Bogdan Popescu, MD, Christopher Robinson, MD, L. Dean Chapman, PhD and Helen Nichol, PhD University of Saskatchewan (Presented By: Bogdan Popescu, MD)

Poster# 176 SERUM FERRITIN IS NOT RELATED TO COGNITIVE STATUS – RESULTS FROM A PERSPECTIVE COMMUNITY STUDY OF OLDER AUSTRALIANS Liz Milward, David Bruce, MD, Michelle Cole, NA, Matthew Knuiman, PhD, Mark Divitini, PhD, Helen Bartholomew, GradDipComp, Graham Maier, GradDipComp and John Olynyk, MD University of Newcastle (Presented By: Liz Milward)

Poster# 177 THE ROLE OF EXTERNAL LOOPS OF CERULOPLASMIN IN STABILIZING FERROPORTIN ON CELL MEMBRANE Maria Carmela Bonaccorsi di Patti, Dr, Nunziata Maio, Fabio Polticelli and Giovanni Musci University La Sapienza (Presented By: Maria Carmela Bonaccorsi di Patti, Dr)

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Poster# 178 CYSTEINE OXIDATION REGULATES THE RNA-BINDING ACTIVITY OF IRON REGULATORY PROTEIN 2 DURING OXIDATIVE STRESS Kimberly Zumbrennen, PhD, Michelle Wallander, PhD, Joshua Romney, PhD and Elizabeth Leibold, PhD University of Utah (Presented By: Kimberly Zumbrennen, PhD)

Poster# 179 PROTECTION OF CARDIAC CELLS AGAINST OXIDATIVE INJURY BY NOVEL IRON CHELATORS – KETONE ANALOGS OF SALICYLALDEHYDE ISONICOTINOYL HYDRAZONE (SIH) Tomas Simunek, PhD, Petra Bendova, Katerina Hruskova, Pavlina Haskova, Petra Kovarikova and Katerina Vavrova Charles University in Prague, Faculty of Pharmacy (Presented By: Tomas Simunek, PhD)

Poster# 180 EFFECTS OF OXIDATIVE STRESS ON IRON REGULATORY GENES IN TWO DIFFERENT MICE MODELS FOR NAFLD Rolf Hultcrantz, MD, C. Söderberg, P. Stål, K. Eckes and M. Sjöström Department of ’Gastroenterology, Karolinska University Hospital, Stockholm, Sweden (Presented By: Rolf Hultcrantz, MD)

Poster# 181 THE CRITICAL ROLE OF INTRACELLULAR LABILE IRON AND FERRITIN IN THE RESISTANCE OF CELLS TO HYDROGEN PEROXIDE Charareh Pourzand, PhD¹, Abdullah Al-Qenaei, PhD², Anthie Yiakouvaki, PhD², Paolo Santambrogio, PhD³, Sonia Levi, PhD4, Rex Tyrrell, PhD² and Nick Hall, PhD5 ¹Department of Pharmacyand Pharmacology/University of Bath; ²Department of Pharmacy and Pharmacology, Bath University, Bath, UK; ³San Raffaele Scientific Institute, Milano, Italy; 4Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milano, Italy; 5Bath Institute for Rheumatic Diseases, Bath, UK (Presented By: Charareh Pourzand, PhD)

Poster# 182 PROTECTION OF CARDIOMYOCYTES AGAINST CATECHOLAMINE INJURY BY IRON CHELATOR SALICYLALDEHYDE ISONICOTINOYL HYDRAZONE (SIH) Pavlina Haskova, MSc¹, Petra Kovarikova¹, Anna Vavrova¹, Prem Ponka² and Tomas Simunek¹ ¹Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Czech Republic; ²Lady Davis Institute for Medical Research and Department of Physiology, McGill University, Montreal, QC, Canada (Presented By: Pavlina Haskova, MSc)

Poster# 183 PARAQUAT DOWN-REGULATES IRON REGULATORY PROTEIN 1 ACTIVITIES AND EXPRESSION IN MURINE RAW 264.7 CELLS AND BONE MARROW-DERIVED MACROPHAGES Rafal Starzynski, PhD¹, Agnieszka Grzelak, PhD², Agnieszka Stys, MSc¹, Grzegorz Bartosz, PhD² and Pawel Lipinski, PhD¹ ¹Institute of Genetics and Animal Breeding; ²University of Lodz (Presented By: Rafal Starzynski, PhD)

Poster# 184 IRON METABOLISM UNDER DIQUAT-INDUCED OXIDATIVE STRESS IN FISCHER-344 RATS Masashi Higuchi, graduate student, Junnichi Oshida, Koichi Orino and Kiyotaka Watanabe Kitasato University (Presented By: Masashi Higuchi, graduate student)

Poster# 185 HEMIN IS TOXIC TO ASTROCYTES: ARE IRON OR OXIDATIVE STRESS INVOLVED? Stephen Robinson, PhD¹, Theresa Dang², Ralf Dringen² and Glenda Bishop² ¹Monash University; ²SPPPM, Monash University (Presented By: Stephen Robinson, PhD)

Poster# 186 IRON CHELATION BY SYNTHETIC CHELATORS AND FLAVONOIDS DEPENDS ON PH Katerina Macakova, Premysl Mladenka, PhD, Libuse Zatloukalova, MSc, Katerina Vavrova, PhD, Lubomir Opletal, Asst Prof, Radomir Hrdina, Asst Prof and Ludek Jahodar, Prof Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Czech Republic (Presented By: Katerina Macakova)

Poster# 187 POLYANHYDROGLUCURONIC ACID DERIVATIVE OF LACTOFERRIN DOES NOT IMPROVE ITS PROTECTIVE EFFECTS ON CATECHOLAMINE CARDIOTOXICITY Premysl Mladenka, PhD¹, Vladimir Semecky, Asst Prof¹, Libuse Zatloukalova, MSc¹, Jiri Briestensky² and Radomir Hrdina, Asst Prof¹ ¹Faculty of Pharmacy, Charles University; ²Alltracel Pharma CZ (Presented By: Premysl Mladenka, PhD)

15:15 – 16:45 Concurrent Sessions VI – VIII

15:15 – 16:45 Concurrent Session VI: Hepcidin Location: Arquivo Chairs: Sophie Vaulont, Paris, France and Dorine Swinkels, Nijmegen, the Netherlands

15:15 39 PRODUCTION OF CORRECTLY-FOLDED, BIOLOGICALLY-ACTIVE HEPCIDIN ENABLED REVISION OF THE PUBLISHED HEPCIDIN STRUCTURE AND GENERATION OF NEUTRALIZING ANTIBODIES Tara Arvedson, PhD, J. Brad Jordan, PhD, Keegan Cooke, Aaron Winters, Leszek Poppe, PhD, Mitsuru Haniu, Greg Dyas, Jacob Corcoran, Helen Kim, PhD, Hiko Kohno, PhD, Vivian Li, Ching Chen, PhD, Paul Schnier, PhD, Rashid Syed, PhD, Les P. Miranda, PhD, Janet Cheetham, PhD and Barbra J. Sasu, PhD Amgen (Presented By: Tara Arvedson, PhD) 33

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15:30 40 HEPCIDIN INDUCED INTERNALIZATION OF FERROPORTIN REQUIRES BINDING AND COOPERATIVE INTERACTION WITH JAK2 Ivana De Domenico, PhD, Diane Ward, PhD and Jerry Kaplan, PhD University of Utah (Presented By: Ivana De Domenico, PhD)

15:45 41 MECHANISMS OF HEPCIDIN INHIBITION BY NOVEL TMPRSS6 (MATRIPTASE-2) MUTATIONS ASSOCIATED WITH IRIDA Laura Silvestri, PhD¹, Flavia Guillem², Alessia Pagani³, Antonella Nai³, Luigia De Falco4, Achille Iolascon4,5, Carole Beaumont², Bernard Grandchamp6,7 and Clara Camaschella³ ¹San Raffaele Scientific Institute; ²INSERM UMR773, University Paris Diderot, site Bichat, Paris, France; ³Vita-Salute San Raffaele University, Istituto Scientifico San Raffaele, Milan, Italy; 4Centre of Genetics Engineering (CEINGE) Advanced Biotechnologies, Naples, Italy; 5Department of Biochemistry and Medical Biotechnologies, University Federico II, Naples; 6INSERM UMR773, University Paris Diderot, site Bichat, Paris; 7APHP, Hôpital Xavier Bichat, Service de Biochimie Hormonale et Génétique, Paris, France (Presented By: Laura Silvestri, PhD)

16:00 42 HEMOJUVELIN/NEOGENIN INTERACTION IS REQUIRED FOR BMP4-INDUCED HEPCIDIN EXPRESSION An-Sheng Zhang¹, Fan Yang², Jiaohong Wang³, Hidekazu Tsukamoto³ and Caroline Enns¹ ¹Oregon Health & Science University; ²California Institute of Technology; ³University of Southern California (Presented By: An-Sheng Zhang)

16:15 43 RESULTS OF THE FIRST INTERNATIONAL ROUND ROBIN FOR THE QUANTIFICATION OF URINARY AND PLASMA HEPCIDIN ASSAYS: NEED FOR STANDARDISATION Joyce Kroot¹, Erwin Kemna, PhD², Sukhvinder Bansal, PhD³, Mark Busbridge, PhD4, Natascia Campostrini, MSc5, Domenico Girelli, MD, PhD5, Robert Hider, PhD³, Vasiliki Koliaraki, MSc6, Avgi Mamalaki, PhD6, Gordana Olbina, PhD7, Naohisa Tomosugi, MD8, Chris Tselepis, PhD9,¹°, Douglas Ward, PhD9,¹°, Tomas Ganz, MD, PhD¹¹, Jan Hendriks, PhD¹² and Dorine Swinkels, MD, PhD² ¹Radboud University Nijmegen Medical Center – Dept. of Clinical Chemistry; ²Radboud University Medical Center – Dept. of Clinical Chemistry; ³King’s College London – Pharmaceutical Sciences Division; 4Imperial College HealthCare Trust, Hammersmith Hospital – Dept. of Clinical Chemistry; 5University of Verona – Dept. of Clinical and Experimental Medicine, Section of Internal Medicine; 6Hellenic Pasteur Institute – Laboratory of Molecular Biology & Immunobiotechnology, Dept. of Biochemistry; 7Intrinsic Life Sciences; 8Kanazawa Medical University – 9Division of Advanced Medicine, Medical Research Institute/Division of Nephrology; ¹°University of Birmingham – CRUK Institute for Cancer Studies; ¹¹Intrinsic Life Sciences/David Geffen School of Medicine UCLA – Dept. of Medicine; ¹²Radboud University Nijmegen Medical Center – Dept. of Epidemiology and Biostatistics (Presented By: Joyce Kroot)

16:30 44 ANTI-HEPCIDIN THERAPY IS AN EFFECTIVE TREATMENT FOR THE ANEMIA OF INFLAMMATION IN MICE Keegan Cooke, Jeanne Sloan, Adrienne Augustic, Tara Arvedson, Cherylene Plewa, Aaron Ellison, Jackie Sheng, Aaron Winters, Hongyan Li, C. Glenn Begley, Graham Molineux and Barbra J. Sasu Amgen (Presented By: Tara Arvedson, PhD)

15:15 – 16:45 Concurrent Session VII: Novel Aspects of Iron and Infection Location: D. Maria Chairs: Ana Tomás, Porto, Portugal and Chinmay Mukhopadhyay, New Delhi, India

15:15 45 CHARACTERIZATION AND EXPRESSION ANALYSIS OF IMMUNE-RELATED IRON GENES DURING EXPERIMENTAL IRON OVERLOAD AND INFECTION IN FISH João Neves, MSc¹, Jonathan Wilson, PhD² and Pedro Rodrigues, PhD³ ¹Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal; ²CIIMAR; ³IBMC and ICBAS (Presented By: João Neves, MSc)

15:30 46 THE HEMOCHROMATOSIS-ASSOCIATED HFE MUTATION PROTECTS MICE FROM SALMONELLA TYPHIMURIUM INFECTION VIA INDUCTION OF LIPOCALIN 2 Manfred Nairz¹, Igor Theurl², Andrea Schroll², Milan Theurl², Sabine Mair², Thomas Sonnweber², Gernot Fritsche², Ewald Lindner², Klaus Hantke³, Shizuo Akira4,5 and Günter Weiss² ¹Internal Medicine I; ²Department of Internal Medicine I, Clinical Immunology and Infectious Diseases, Medical University of Innsbruck, 6020 Innsbruck, Austria; ³Department of Microbiology/Membrane Physiology, University of Tübingen, 72076 Tübingen, Germany; 4Department of Host Defense, Osaka University, and Exploratory Research for Advanced Technology (ERATO), Japan; 5Science and Technology Corporation, Osaka 565-0871, Japan (Presented By: Manfred Nairz)

15:45 47 MYCOBACTERIA-INDUCED ANAEMIA REVISITED: A MOLECULAR APPROACH REVEALS THE INVOLVEMENT OF NRAMP-1 AND LIPOCALIN-2, BUT NOT OF HEPCIDIN Pedro Rodrigues, PhD², Sandro Gomes, BsSc³, João Neves, BsMSc³, Sandra Gomes-Pereira, PhD³, Margarida Correia-Neves, PhD, VetD4, Cláudio Nunes-Alves, BSsc4, Rui Appelberg, PhD, MD5, Martina Muckenthaler, PhD6 and Maria Salomé Gomes, PhD¹ ¹IBMC and ICBAS – Universidade do Porto; ²IBMC and ICBAS – Universidade do Porto, Porto, Portugal; ³IBMC – Universidade do Porto, Porto, Portugal; 4Life and Health Sciences Reasearch Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.; 5IBMC and ICBAS – Universidade do Porto; 6Department of Pediatric Oncology, Haematology and Immunology, University Hospital of Heidelberg, Heidelberg, Germany (Presented By: Maria Salomé Gomes, PhD)

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16:00 48 HEME SENSITIZATION TO TNF-MEDIATED PROGRAMMED CELL DEATH DICTATES THE OUTCOME OF PLASMODIUM INFECTION IN MICE Raffaella Gozzelino, PhD¹, Elsa Seixas, PhD, Ângelo Chora, PhD, Ana Ferreira, PhD, Gabriela Silva, PhD, Rasmus Larsen, PhD, Sofia Rebelo, PhD, Carme Penido, PhD, Neal Smith, MD, PhD², Antonio Coutinho, MD, PhD and Miguel Soares, PhD ¹Instituto Gulbenkian de Ciência; ²Dept. of Pathology, Massachusetts General Hospital, Boston MA, USA (Presented By: Raffaella Gozzelino, PhD)

16:15 49 THE AVIAN LIPOCALIN EX-FABP IS A SIDEROPHORE BINDING ANTIBACTERIAL PROTEIN Fiorella Descalzi, PhD¹, Matthew C. Clifton, PhD², Ranieri Cancedda, MD³, Tricia M. Hoette, PhD4, Kennet N. Raymond, PhD4 and Roland K. Strong, PhD² ¹IBFM & Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; ²Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; ³Istituto Nazionale per la Ricerca sul Cancro, University of Genova,Genova, Italy; 4Department of Chemistry, University of California, Berkeley, California , USA (Presented By: Fiorella Descalzi, PhD)

16:30 50 HEPCIDIN INDUCTION BY PATHOGEN-ASSOCIATED STIMULI Andrew Armitage², Lucy Eddowes², Lisa Schimanski², Alain Townsend² and Hal Drakesmith¹ ¹Oxford University; ²Molecular Immunology Group, WIMM, Oxford University (Presented By: Hal Drakesmith, PhD)

15:15 – 16:45 Concurrent Session VIII: Iron Around the Body Location: D. Luis Chairs: Alberto Piperno, Monza, Italy and Martin Vokurka, Prague, Czaech Republic

15:15 51 LIVER-SPECIFIC DELETION OF FRATAXIN IN THE MOUSE AS A NEW MODEL TO UNDERSTAND IRON DYSREGULATION IN FRIEDREICH ATAXIA Alain Martelli, PhD¹, Stéphane Schmucker, student², Marie Wattenhofer-Donzé, PhD², Laurence Reutenauer, technician², Hervé Puy, Professor³, Nadia Messaddeq, Engineer² and Hélène Puccio, PhD² ¹IGBMC; ²IGBMC Illkirch; ³Centre Français des Porphyries (Presented By: Alain Martelli, PhD)

15:30 52 IRON STORES AND IRON ABSORPTION IN HEREDITARY HEMOCHROMATOSIS Roland Fischer, PhD¹, Hannah Seligmann², Rainer Engelhardt, PhD², Rosemarie Kongi², Gritta E. Janka, MD² and Peter Nielsen, MD, PhD² ¹University Medical Center Hamburg-Eppendorf, Germany & Children’s Hospital & Research Center Oakland, USA; ²University Medical Center Hamburg-Eppendorf, Germany (Presented By: Roland Fischer, PhD)

15:45 53 HYPERGLYCEMIA AND IMPAIRED BETA-CELL FUNCTION CAUSED BY IRON REGULATORY PROTEIN 2 DEFICIENCY Kimberly Zumbrennen, PhD, Susanne Neschen, PhD, Brigit Rathkolb, PhD, Wolfgang Hans, PhD and Elizabeth Leibold, PhD University of Utah (Presented By: Kimberly Zumbrennen, PhD)

16:00 54 ALTERATIONS OF SYSTEMIC AND MUSCLE IRON METABOLISM IN HUMAN SUBJECTS TREATED WITH LOW DOSE RECOMBINANT ERYTHROPOIETIN Gaetano Cairo, PhD¹, Stefania Recalcati, MD, PhD¹, Domenico Girelli, MD³, Cecilia Gelfi, PhD¹, Niels Aachmann-Andersen, PhD4, Jonas Thomsen, PhD4, Anne Norgaard, PhD4, Alessandra Alberghini, PhD¹, Natascia Campostrini, PhD³, Annalisa Castagna, PhD³, Agnese Viganò, PhD¹, Paolo Santambrogio, PhD5, Tibor Kempf, MD6, Kay Wollert, PhD6, Stephane Moutereau, PhD7, Carsten Lundby, PhD4 and Paul Robach, PhD² ¹University of Milan; ²ENSA, France; ³University of Verona; 4University of Copenhagen; 5DIBIT, HS S. Raffaele; 6Hannover Medical School; 7Hopital H. Mondor (Presented By: Gaetano Cairo, PhD)

16:15 55 ANALYSIS OF CONDITIONAL FERRITIN H DELETION IN MOUSE ADULT HEART Liviu Vanoaica², Deepak Darshan, PhD², Larry Richman², Corinne Berthonneche³, Thierry Pedrazzini, MD4 and Lukas Kühn, PhD¹ ¹Ecole Polytechnique Federale de Lausanne (EPFL) – Swiss Institute for Experimental Cancer Research (ISREC); ²Ecole Polytechnique Fédérale de Lausanne (EPFL) – Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland; ³Cardiovascular Assessment Facility, University of Lausanne Medical School, Lausanne, Switzerland; 4Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, Lausanne, Switzerland (Presented By: Lukas Kühn, PhD)

16:30 56 EFFICACY AND SAFETY OF DEFERASIROX (EXJADE®) IN REDUCING AND PREVENTING MYOCARDIAL SIDEROSIS IN PATIENTS WITH BETA-THALASSEMIA Dudley Pennell, MD, FRCP, FACC, FESC¹, John B. Porter², Maria Domenica Cappellini³, Amal El-Beshlawy4, Lee Lee Chan5, Yesim Aydinok6, Mohsen Saleh Elalfy7, Pranee Sutcharitchan8, Chi-Kong Li9, Hishamshah Ibrahim¹°, Vip Viprakasit¹¹, Antonis Kattamis¹², Gillian Smith¹³, Dany Habr¹4, Gabor Domokos¹5, Abdel Hmissi¹5 and Ali Taher¹6 ¹Royal Brompton Hospital; ²University College London, London, UK; ³Universitá di Milano, Policlinico Foundation IRCCS, Milan, Italy; 4Cairo University, Cairo, Egypt; 5University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia; 6Ege University Medical Faculty, Izmir, Turkey; 7Ain Shams University, Cairo, Egypt; 8Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 9Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong; ¹°Hospital Kuala Lumpur, Malaysia; ¹¹Faculty of Medicine Siriraj Hospital/Dept of Pediatrics/Division of Haematology-Oncology, Prannok, Bangkoknoi, Bangkok, Thailand; ¹²First Dept of Pediatrics, University of Athens, Athens, Greece; ¹³Royal Brompton Hospital, London, UK; ¹4Novartis Pharmaceuticals, New Jersey, USA; ¹5Novartis, Basel, Switzerland; ¹6American University Beirut, Beirut, Lebanon (Presented By: Dudley Pennell, MD, FRCP, FACC, FESC) 35

Wednesday, June 10, 2009

17:00 – 18:30 Concurrent Sessions IX – X

17:00 – 18:30 Concurrent Session IX: Anemia Revisited Location: D. Maria Chairs: Carole Beaumont, Paris, France and Sylvia Bottomley, Oklahoma City, OK, USA

17:00 57 NRAMP1 PROMOTES EFFICIENT IRON RECYCLING FOLLOWING ERYTHROPHAGOCYTOSIS IN VIVO Shan Soe-Lin, BSc¹, Bill Andriopoulos, Jr., BSc, PhD², Sameer Apte, BSc³, Marc Andrews, BSc4, Matthias Schranzhofer, BSc, PhD³, Tanya Kahawita, BSc, MSc5, Daniel Garcia-Santos, BSc³ and Prem Ponka, MD, PhD6 ¹McGill University; ²Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; ³Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada; 4Department of Molecular Genetics and Development, Institut de Recherches Cliniques de Montreal, Quebec, Canada; 5Department of Physiology, McGill University, Montreal, Quebec, Canada; 6Department of Physiology, McGill University, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada (Presented By: Shan Soe-Lin, BSc)

17:15 58 INCREASED HEPCIDIN EXPRESSION IN MICE AFFECTED BY BETA-THALASSEMIA REDUCES IRON OVERLOAD WITH NO EFFECT ON ANEMIA Sara Gardenghi, PhD¹, Maria Franca Marongiu, PhD², Kristen Muirhead², Pedro Ramos³, Cindy N. Roy, PhD4, Nancy C. Andrews, MD, PhD5, Elizabeta Nemeth, PhD6, Eliezer A. Rachmilewitz, MD7, Patricia J. Giardina, MD², Robert W. Grady, PhD² and Stefano Rivella, PhD² ¹Weill Cornell Medical College; ²Weill Cornell Medical College, New York; ³Weill Cornell Medical College, New York - IBMC, Portugal; 4John Hopkins University, Baltimore; 5Duke University, Durham; 6UCLA, Los Angeles; 7Wolfson Medical Center, Israel (Presented By: Sara Gardenghi, PhD)

17:30 59 THE ROLES OF HEPCIDIN AND INFLAMMATION IN ANEMIA OF CANCER Airie Kim, MD, Tomas Ganz, MD, PhD and Seth Rivera, MD, PhD UCLA Medical Center (Presented By: Airie Kim, MD)

17:45 60 MUTATIONS IN A NOVEL MITOCHONDRIAL TRANSPORTER GENE CAUSE AUTOSOMAL RECESSIVE CONGENITAL SIDEROBLASTIC ANEMIA Sylvia S. Bottomley, MD¹, Duane L. Guernsey, PhD²,8, Paul J. Schmidt, PhD³, Andrew Orr, MD4,8, Mark Ludman, MD5, 8, Conrad V. Fernandez, MD6, 8, Mark E. Samuels, PhD7,8 and Mark D. Fleming, MD, DPhil³ 1Department of Medicine, Hematology-Oncology Section, University of Oklahoma College of Medicine, Oklahoma City, USA; 2Department of Pathology, Dalhousie University, Halifax, Canada; 3Departments of Pathology, Children’s Hospital and Harvard Medical School, Boston, USA; 4Department of Ophthalmology and Visual Sciences, Dalhousie University, Halifax, Canada; 5Department of Pediatrics, Division of Medical Genetics, IWK Health Centre and Dalhousie University, Halifax, Canada; 6Department of Pediatrics, Division of Hematology and Oncology, IWK Health Centre and Dalhousie University, Halifax, Canada; 7Centre de Recherche de Ste- Justine, Université de Montréal, Montréal, Canada; 8Atlantic Medical Genetics and Genomics Initiative and Genome Canada, Dalhousie University, Halifax, Canada (Presented By: Sylvia S. Bottomley, MD)

18:00 61 ALLELIC HETEROGENEITY OF TMPRSS6 MUTATIONS IN IRIDA Caroline Kannengiesser, PhD², Flavia Guillem, Laura Silvestri, Claire Oudin, Anne Marfaing, Leïla Chaiba-Berrouche, Jean Donadieu, Fabienne Toutain, Murielle Da Silva, Bertrand Isidor, Geneviève Marguerite, Patricia Aguila-Martinez, Clara Camaschella, Carole Beaumont and Bernard Grandchamp, MD, PhD¹,³,4 ¹AP-HP, Hopital Bichat; ²APHP, INSERM U773, University Paris Diderot; ³INSERM CRB3; 4University Paris Diderot (Presented By: Bernard Grandchamp, MD, PhD)

18:15 62 THE ROLE OF ERYTHROPOIETIN IN FAMILIAL AMYLOIDOSIS TTR V30M Idalina Beirão¹, Luisa Lobato, PhD²,³, Paulo PM Costa, PhD³, Luciana Moreira, MSc³ and Graça Porto, PhD4,5 ¹Santo António General Hospital; ²Nephrology, Santo António General Hospital; ³Aamyloidosis Research Centre; 4Hematology, Santo António General Hospital; 5IBMC (Presented By: Idalina Beirão, MD)

17:00 – 18:30 Concurrent Session X: Other Significant Proteins, Other Metals Location: Arquivo Chairs: Esther Meyron-Holtz, Haifa, Israel and Alain Townsend, Oxford, UK

17:00 63 LIPID RAFTS BREAKDOWN DECREASES THE ACTION OF HEPCIDIN ON MACROPHAGE FERROPORTIN Anne Auriac, Master², Alexandra Willemetz, Master² and François Canonne-Hergaux, PhD¹ ¹INSERM-CNRS; ²UPR2301, CNRS, Centre de recherche de GIF-sur-Yvette, France (Presented By: François Canonne-Hergaux, PhD)

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17:15 64 HEPCIDIN, THE HORMONE OF IRON METABOLISM, IS BOUND SPECIFICALLY TO Alpha2-MACROGLOBULIN IN BLOOD D.R. Richardson¹, G. Peslova², J. Petrak²,7, K. Kuzelova², I. Hrdy³, P. Halada4, P.W. Kuchel5, S. Soe-Lin6, P. Ponka6, R. Sutak¹, E. Becker¹, M. Huang¹, Y.S. Rahmanto¹ and D. Vyoral¹,² ¹Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia; ²Institute of Hematology and Blood Transfusion U Nemocnice 1, Prague 2, 128 20, Czech Republic; ³Department of Parasitology, Faculty of Science, Charles University, 12844 Prague, Czech Republic; 4Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídenská 1083, CZ-142 20 Prague 4, Czech Republic; 5School of Molecular and Microbial Biosciences, Biochemistry and Microbiology Building (G08), University of Sydney, Sydney, New South Wales, 2006 Australia; 6Lady Davis Institute, 3755 Cote Ste. Catherine Road, Montréal, Québec, Canada H3T 1E2; 7Institute of Pathophysiology, U Nemocnice 5, Prague 2, 128 53, Charles University in Prague, First Faculty of Medicine (Presented By: D.R. Richardson)

17:30 65 IRON-DEPENDENT REGULATION OF FERRITIN BY HIF-1 IN C. ELEGANS Elizabeth Leibold, PhD, Joshua Romney, BS and Ben Newman, BS University of Utah (Presented By: Elizabeth Leibold, PhD)

17:45 66 CHARACTERISATION OF THE INTERACTION BETWEEN CERULOPLASMIN AND LACTOFERRIN Robert Evans, BA, PhD¹, Celia Conesa², Maryam Amini¹, Lourdes Sanchez² and Sebastien Farnaud³ ¹Brunel University; ²University of Zaragoza; ³Dr Hadwen Trust & University of Westminster (Presented By: Robert Evans, BA, PhD)

18:00 67 MAGNETIC DYNAMIC SUSCEPTIBILITY MEASUREMENTS AS A PRE-SCREENING TOOL TO INVESTIGATE THE PRESENCE OF FERRITIN IN DIFFERENT ORGANISMS Lucia Gutierrez and Francisco J. Lazaro Universidad de Zaragoza (Presented By: Lucia Gutierrez)

18:15 68 RELEVANCE OF COMMON POST-TRANSLATION MODIFICATIONS OF HUMAN SERUM ALBUMIN FOR NON- TRANSFERRIN-BOUND IRON SPECIATION Andre Silva, BSc and Robert Hider, PhD King’s College London (Presented By: Andre Silva, BSc)

17:00 – 18:30 Symposium: Novel Technologies Location: D. Luis Chairs: Maria de Sousa, Porto, Portugal and José Moura, Lisbon, Portugal

17:00 – 17:20 Imaging the Distribution of Iron-Ferritin-Hemosiderin at the Subcellular Level Carmen Quintana, Instituto Microelectronica de Madrid, Spain

17:20 – 17:40 Spacial Coordination of Cell-Adhesion Molecules and Redox Cycling of Iron in Pulmonary Blast Injury Nikolai Gorbunov, Silver Spring, Maryland, USA

17:40 – 18:00 Shuttling Iron Between Cellular Organelles Ioav Cabantchik, Hebrew University of Jerusalem, Israel

18:00 – 18:20 Drug Targeting to the Brain Using the Transferrin Receptor Ruben Boado, UCLA, USA

18:20 – 18:30 General Discussion

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THURSDAY, JUNE 11th, 2009

08:00 – 18:15 Registration / Information Desk Open Location: Porto Congress Center Alfândega

09:00 – 10:30 Plenary Session VII: Tissue Iron Metabolism Location: Arquivo Chairs: Olivier Loréal, Rennes, France and Joana Palha, Braga, Portugal

09:00 – 09:30 Lecture 13: Iron and the Brain Torben Moos, Aalborg University, Denmark

09:30 – 10:00 Lecture 14: Iron-Sulfur Cluster Biogenesis and Human Disease Tracey Rouault, National Institute of Neurological Disorders and Stroke, Bethesda, USA

10:00 – 10:30 Podium Presentations

10:00 69 RETINAL DEGENERATION IN CP, HEPH, FPN AND HEPC MUTANT MOUSE MODELS AMELIORATED BY IRON CHELATION Joshua Dunaief, MD, PhD¹, Majda Hadziahmetovic, MD¹, Jared Iacovelli, MS¹, Paul Hahn, MD, PhD¹, Ying Song, MD¹, Allan Hunter, MD¹, Steven Grieco, BA¹, Adriana Donovan, PhD², Nancy Andrews, PhD³ and Sophie Vaulont, PhD4 ¹University of Pennsylvania; ²Novartis; ³Duke; 4Cochin Institute (Presented By: Joshua Dunaief, MD, PhD)

10:15 70 THE LIVER-SPECIFIC MICRORNA 122A REGULATES THE EXPRESSION OF HFE, HFE2 AND HEPCIDIN AND CONTROLS SYSTEMIC IRON HOMEOSTASIS Mirco Castoldi, PhD¹,4, Maja Vujic-Spasic, PhD¹,4, Jens Stolte², Vladimir Benes, PhD², Joachim Elmen, PhD³, Morton Lindow, PhD³, Sakari Kauppinen, PhD³, Matthias Hentze, MD²,4 and Martina Muckenthaler¹ ¹Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg; ²European Molecular Biology Laboratory, Heidelberg, Germany; ³Santaris Pharma, Hørsholm, Denmark; 4Molecular Medicine Partnership Unit, University of Heidelberg, Germany (Presented By: Martina Muckenthaler)

10:30 – 11:00 Coffee Break Location: Noble Hall

11:00 – 12:30 Plenary Session VIII: Hemochromatosis Location: Arquivo Chairs: Pierre Brissot, Rennes, France and Grant Ramm, Brisbane, Australia

11:00 – 11:30 Lecture 15: Hemochromatosis – Screening and Penetrance Katie Allen, University of Melbourne, Australia

11:30 – 12:00 Podium Presentations

11:30 71 -153 C/T MUTATION IN HEPCIDIN PROMOTER MAY IMPACT PRESENTATION OF HFE RELATED GENETIC IRON OVERLOAD Marie-Laure Island, PhD¹, Anne-Marie Jouanolle, PhD², Annick Mosser, PhD², Yves Deugnier, MD³, Véronique David, PhD², Pierre Brissot, MD4 and Olivier Loréal, MD, PhD5,6,7 ¹INSERM U522, University of Rennes 1, Rennes, France; ²Molecular Genetic Department, Hopital Pontchaillou, Rennes, France; ³Liver Diseases Unit and INSERM U522, Rennes, France; 4Liver Diseases Unit anddn INSERM U522, Rrennes; 5Inserm U522; 6IFR140; 7University of Rennes 1, Rennes, France (Presented By: Marie-Laure Island, PhD)

11:45 72 THE PATHOGENESIS OF HEREDITARY HEMOCHROMATOSIS Emilio Ramos, Y. Phung, V. Gabayan, T. Ganz and E. Nemeth Departments of Medicine and Chemistry and Biochemistry, David Geffen School of Medicine at UCLA (Presented By: Emilio Ramos)

12:00 – 12:30 Lecture 16: Hereditary Hemochromatosis as a Conformational Disorder Matthew W. Lawless, Trinity College Dublin, Ireland 38

Thursday, June 11, 2009

12:30 – 14:00 Lunch Location: Noble Hall

14:00 – 15:15 Poster Session III Presentations Location: Arquivo

Poster# 188 IRONING OUT FRIEDREICH’S ATAXIA: THE MOLECULAR CHANGES BEHIND MITOCHONDRIAL IRON-LOADING AND EXPLORATION OF IRON-CHELATION THERAPY FOR TREATMENT D.R. Richardson, M. Whitnall, Y. Suryo Rahmanto, R. Sutak, X. Xu, E. Becker, M.R. Mikhael and P. Ponka Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia (Presented By: D.R. Richardson)

Poster# 189 FURIN EXPRESSION IS DECREASED IN THE LIVER OF HFE-HEMOCHROMATOSIS PATIENTS Donatella Barisani, MD², Sara Pelucchi, PhD¹, Alessandra Pansa, PhD³, Raffaella Meneveri, PhD³, Paola Trombini, MD¹, Alessandra Salvioni, MD¹, Raffaella Mariani, MD¹ and Alberto Piperno, MD¹ ¹Department of Clinical Medicine, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy; ²Faculty of Medicine, University of Milano-Bicocca, Monza, Italy; ³Department of Experimental Medicine, University of Milano-Bicocca, Monza, Italy (Presented By: Alberto Piperno, MD) (Presented By: Alberto Piperno, MD)

Poster# 190 LIVER TRANSCRIPTOME OF THE F2 PROGENY OF B6 AND D2 HFE-DEFICIENT MICE: ROLE OF IRON LOADING AND GENETIC BACKGROUND. Annabelle Monnier¹, Léon Kautz, PhD student², Régis Bouvet, Technical support³, Valérie Darnaud, Technical support², Hélène Coppin, DR², Marie-Paule Roth, MD² and Jean Mosser, PU-PH4 ¹RTO Team UMRCNRS6061; ²Centre de Physiopathology of Toulouse Purpan, INSERM U563 Département de génétique- Toulouse- France; ³platform OUEST-genopole®-Rennes-France; 4UMRCNRS6061/platform OUEST-genopole® /Hospital Medical Genomics Laboratory (Presented By: Annabelle Monnier)

Poster# 191 INTERACTION OF DEFERASIROX (ICL670) WITH METAL IONS USED IN CONTRAST AGENTS Nadine Jane Koch¹, Stefan Stucky, Dipl Chem¹, Kaspar Hegetschweiler, Prof Dr¹ and Hanspeter Nick² ¹Saarland University; ²Novartis Pharma AG Research, Basel, Switzerland (Presented By: Stefan Stucky, Dipl Chem)

Poster# 192 A NOVEL SIMULTANEOUS QUANTITATIVE METHOD FOR HEPCIDIN ISOFORMS USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY Katsuya Ikuta, MD, PhD¹, Yasushi Shimonaka², Takaaki Hosoki, MD³, Yusuke Sasaki², Hideyuki Yasuno², Takaaki Ohtake, MD, PhD³, Katsunori Sasaki, PhD4, Yoshihiro Torimoto, MD, PhD5, Keiji Saito, PhD² and Yutaka Kohgo, MD, PhD³ ¹Asahikawa Medical College; ²Kamakura Research Labs, Chugai Pharmaceutical Co., Ltd.; ³Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical College; 4Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical College; 5Oncology Center, Asahikawa Medical College Hospital (Presented By: Katsuya Ikuta, MD, PhD)

Poster# 193 CARDIAC GENE EXPRESSION IN MOUSE MODELS OF IRON LOADING DISORDERS Sharon Hollins, N/A, Daniel Johnstone, Ross Graham, PhD, Dirk van Helden, PhD, Karen Kerr, PhD, Derek Laver, PhD, Kirsty Metelerkamp, N/A, Deborah Trinder, PhD, Robert Fleming, MD, Rodney Scott, PhD, John Olynyk, MD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Poster# 194 PROPERTIES OF THE METAL-ION TRANSPORTER ZIP14 SUGGEST A ROLE IN CELLULAR UPTAKE OF NONTRANSFERRIN-BOUND IRON (NTBI) CHARACTERISTIC OF IRON-OVERLOAD CONDITIONS Jorge J. Pinilla Tenas¹, Brian K. Sparkman¹, Anthony C. Illing¹, Ali Shawki¹, Juan P. Liuzzi², Robert J. Cousins², Mitchell D. Knutson² and Bryan Mackenzie¹ ¹University of Cincinnati College of Medicine; ²University of Florida (Presented By: Jorge J. Pinilla Tenas)

Poster# 195 MORTALITY AMONG PATIENTS WITH PHENOTYPIC HEREDITARY HEMOCHROMATOSIS AND AMONG THEIR FAMILY MEMBERS: ARE FIRST-DEGREE RELATIVES AT INCREASED RISK? Maria Elmberg, MD, Rolf Hultcrantz, MD, Anders Ekbom, MD, Stefan Lindgren, MD and Johan Askling, MD Karolinska (Presented By: Rolf Hultcrantz, MD)

Poster# 196 BONE STATUS IN A MOUSE MODEL OF GENETIC HEMOCHROMATOSIS Pascal Guggenbuhl, MD, PhD¹, Patricia Fergelot, MD, PhD², Hélène Libouban, PhD³, Marie-Paule Roth, MD, PhD4, Yves Gallois, PhD5, Olivier Loréal, MD, PhD6, Gerard Chales, MD7 and Daniel Chappard, MA, PhHD3 ¹Rheumatology Department University Hospital, Rennes, France; ²CNRS UMR 6061, Rennes; ³INSERM, EMI 00335, Angers, France; 4INSERM, U563, IFR 130, Toulouse; 5INSERM, EMI 00335, Angers; 6INSERM U522, IFR 140, Rennes 1, France; 7Rheumatology Department, University Hospital, Rennes (Presented By: Pascal Guggenbuhl, MD, PhD)

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Poster# 197 COORDINATION CHEMISTRY OF TWO METABOLITES OF DEFERASIROX (ICL670) AND THE CRYSTAL STRUCTURE OF AN IRON-DEFERASIROX-COMPLEX Stefan Stucky, Dipl Chem¹, Kaspar Hegetschweiler¹ and Hanspeter Nick² ¹Saarland University; ²Novartis Pharma AG Research (Basel, Switzerland) (Presented By: Stefan Stucky, Dipl Chem)

Poster# 198 MUTATION ANALYSIS OF GENES INVOLVED IN IRON REGULATION IN AN INDIAN FAMILY PRESENTING WITH IRON OVERLOAD Michelle-Angelique Hallendorff, MSc¹, Nathaniel McGregor, HonsBSc¹, Richard Hift, MD² and Monique Zaahl, PhD¹ ¹Stellenbosch University; ²University of KwaZulu-Natal (Presented By: Monique Zaahl, PhD)

Poster# 199 WITHDRAWN IRON ABSORPTION IN DYSMETABOLIC IRON OVERLOAD SYNDROME IS DECREASED CORRELATING WITH INCREASED PLASMA HEPCIDIN Fabrice Lainé, MD¹, Marc Ruivard, MD², Tomas Ganz, MD, PhD³, Gordana Olbina, PhD³, Mark Westerman, PhD³, Mathieu Rambeau, PhD4, André Mazur, PhD4, Laurent Gerbaud, PhD², Valérie Tournilhac, MD², Armand Abergel, MD, PhD², Pierre Philippe, MD², Charles Coudray, PhD4 and Yves Deugnier, MD5 ¹CIC INSERM 0203; ²CHU, Clermont-Ferrand, France; ³University of California, Los Angeles, USA; 4INRA, Clermont-Ferrand, France; 5INSERM CIC 203 and U 522, Rennes, France

Poster# 200 MULTIPLE PREGNANCIES DO NOT REDUCE BODY IRON STORES IN THE HFE-/-MOUSE MODEL OF HEMOCHROMATOSIS Pedro Rodrigues, PhD, João Neves, Ellin Weber, Graça Porto, PhD and Anna Olsson, PhD IBMC, University of Porto (Presented By: Pedro Rodrigues, PhD)

Poster# 201 HAEMOCHROMATOSIS-ASSOCIATED GENES IN THE ZEBRAFISH Chun Yu Lok, A.T. Merryweather-Clarke, P. Pinheiro, R. Patient and K.J.H. Robson Weatherall Institute of Molecular Medicine (Presented By: Chun Yu Lok)

Poster# 202 THE EFFECT OF DIETARY IRON ON TISSUE IRON LEVELS IN INTACT AND SPLENECTOMIZED MICE AFFECTED BY Beta-THALASSEMIA Maria Marongiu, PhD, Kristen Muirhead, BS, Sara Gardenghi, PhD, Ella Guy, Stefano Rivella, PhD and Robert Grady, PhD Weill Cornell Medical College (Presented By: Robert Grady, PhD)

Poster# 203 SYNTHESIS AND COMPLEX FORMATION OF WATER SOLUBLE DERIVATIVES OF THE IRON-CHELATING LIGAND DEFERASIROX (ICL670) Nadine Jane Koch, Dipl Chem, Frank Teucke and Kaspar Hegetschweiler, Prof Dr Saarland University (Presented By: Nadine Jane Koch)

Poster# 204 DISTRIBUTION AND CHEMICAL SPECIATION OF IRON DEPOSITS IN HEARTS FROM TRANSFUSION DEPENDENT PATIENTS Tim St. Pierre, PhD¹, Dudley Pennell², Adam Fleming¹, Mike House¹, Greg Black¹, Paul Kirk², John Paul Carpenter², Lisa Anderson² and Taigang He² ¹The University of Western Australia; ²Royal Brompton Hospital and Imperial College, London (Presented By: Tim St. Pierre)

Poster# 205 MUTATION ANALYSIS OF HEPCIDIN AND FERROPORTIN GENES: POSSIBLE RELATIONSHIP WITH IRON OVERLOAD IN ITALIAN PROSPECTIVE BLOOD-DONORS Lorena Duca, Biology, Paola Delbini, Biotechnology, Isabella Nava, Biology, Valentina Vaja, Biotechnology, Gemino Fiorelli, Medicine and Maria Domenica Cappellini, Medicine ¹Policlinico, Mangiagalli, and Regina Elena Hospital IRCCS Foundation, University of Milan (Presented By: Lorena Duca, Biology)

Poster# 206 PRELIMINARY STUDY ON GENETIC MODIFIERS THAT MAY INFLUENCE THE IRON OVERLOAD IN SUBJECTS WITH H63D MUTATION Isabella Nava, Biology, Paola Delbini, Biotechnology, Lorena Duca, Biology, Gemino Fiorelli, Medicine and Maria Domenica Cappellini, Medicine Policlinico, Mangiagalli, and Regina Elena Hospital IRCCS Foundation, University of Milan (Presented By: Lorena Duca)

Poster# 207 IRON-CHELATOR-THERAPY RESTORES MAJOR SYMPTOMS OF A FEMALE PATIENT SUFFERING FROM MICROCYTIC ANEMIA AND SEVERE IRON-OVERLOAD OF THE BONE-MARROW AND THE LIVER Wolfgang Gilles, PhD¹, Sandro Altamura, PhD¹, Mayka Sanchez, PhD², Christa Flechtenmacher, MD¹, Thomas Herrmann, MD¹, Martina U. Muckenthaler, Prof¹ and Wolfgang Stremmel, Prof¹ ¹University of Heidelberg; ²EMBL Heidelberg (Presented By: Wolfgang Gilles, PhD)

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Poster# 208 IS IRON OVERLOAD IN ALCOHOL-RELATED CIRRHOSIS MEDIATED BY HEPCIDIN? Tariq Iqbal¹, Azzam Diab, MRCP¹, Douglas Ward, BSc, PhD², Matthew Brookes, PhD, MRCP² and Chris Tselepis, BSc, PhD² ¹University Hospital Birmingham; ²University of Birmingham (Presented By: Tariq Iqbal)

Poster# 209 USEFULNESS OF MAGNETIC RESONANCE IMAGING FOR NON-INVASIVE TISSUE AND HISTOLOGICAL CHARACTERIZATION OF IRON OVERLOAD DEPOSITS IN MICE Eric Hitti, PhD5,6, Pierre-Antoine Eliat, PhD², Emmanuelle Abgueguen, PhD5,7, Bruno Turlin, MD,¨PhD5,7, Hervé Saint-Jalmes, MD, PhD³, Pierre Brissot, MD5,7, Yves Gandon, MD4 and Olivier Loréal, MD, PhD¹ ¹INSERM U522; ²Prism IFR 140 GFAS, University of Rennes 1, Rennes, France; ³,5Departement d'imagerie, Centre Eugène Marquis; 4Imaging Departement, University Hhospital, Rennes, France; 5IFR140; 6University of Rennes 1, Rennes, France; 7University of Rennes 1, Rennes (Presented By: Olivier Loréal, MD, PhD)

Poster# 210 THE EFFECT OF ACCESS TO NON-INVASIVE LIVER IRON CONCENTRATION MEASUREMENTS ON PATIENTS AT RISK OF IRON OVERLOAD FROM MULTIPLE BLOOD TRANSFUSIONS: AN AUDIT AND RETROSPECTIVE STUDY Tim St. Pierre, PhD¹, Greg Brown², Nigel Patton³, Heather Tapp4 and James Taylor² ¹The University of Western Australia; ²Royal Adelaide Hospital; ³Auckland City Hospital; 4Children, Youth, and Womens Health Service, South Australia (Presented By: Tim St. Pierre, PhD)

Poster# 211 LIVER IRON OVERLOAD IN NON HFE RELATED HYPERFERRITINEMIC PATIENTS Gian Luca Forni, MD, Manuela Balocco, MD, Roberto Villa, MD, Cinzia Bruzzone, Phisician, Andrea Macco, Phisician and Barbara Gianesin, Phisician Centro della Microcitemia e delle Anemie Congenite, Ospedale Galliera, Genova (Presented By: Gian Luca Forni, MD)

Poster# 212 INTESTINAL IRON ABSORPTION DURING PHLEBOTOMY THERAPY IN PATIENTS AFFECTED BY HEMOCHROMATOSIS TYPE 1 Gian Luca Forni, MD, Roberto Villa, MD, Manuela Balocco, MD, Andrea Macco, Physician, Barbara Gianesin, Physician and Cinzia Bruzzone, Physician Centro della Microcitemia e delle Anemie Congenite, Ospedale Galliera, Genova (Presented By: Gian Luca Forni, MD)

Poster# 213 ASSOCIATION BETWEEN HFE GENOTYPE AND IRON OVERLOAD IN PATIENTS WITH LIVER DISEASES FROM WESTERN ROMANIA Adriana Maria Neghina¹, Ketil Thorstensen, PhD³, Andrei Anghel, PhD², Iosif Marincu, MD, PhD² and Raul Neghina, MD² ¹Victor Babes University of Medicine and Pharmacy; ²Victor Babes University of Medicine and Pharmacy, Timisoara, Romania; ³Department of Medical Biochemistry, St. Olavs Hospital, Trondheim, Norway (Presented By: Adriana Maria Neghina)

Poster# 214 FREQUENCY OF C282Y AND H63D MUTATIONS OF THE HEMOCHROMATOSIS GENE IN A NORTHEASTERN BRAZILIAN POPULATION Luiz Silva, Jr., MD¹, Andre Silva, MD², Adavio Silva, MD³ and Jairo C. Cavalcante, Statiscal4 ¹Souza Junior Laboratorio Medico; ²Hospital A. C. Camargo; ³Hospital da Benficiencia Portguesa; 4PMM (Presented By: Luiz Silva, Jr., MD)

Poster# 215 IRON CHELATION IN PATIENTS WITH THALASSEMIA INTERMEDIA AND OTHER NON TRANSFUSION DEPENDENT CONGENITAL HEMOLYTIC ANEMIAS Alexandra Pereira, MD¹, Maria Pedro Silveira, MD² and Maria Leticia Ribeiro, MD, PhD³ ¹Departamento de Hematologia, Centro Hospitalar de Coimbra; ²Serviço de Hematologia, Centro Hospitalar de Lisboa Ocidental, E.P.E, Portugal; ³hematologia, Centro Hospitalar de Coimbra, E.P.E., Portugal (Presented By: Alexandra Pereira, MD)

Poster# 216 HYPERFERRITINEMIA CATARACT SYNDROME, IS THERE A RENAL COMPONENT? Samuel Krikler, MBChB, FRCPC, Chris Whittington, MBBS, Anthony Vivian, MBBS and Daniel Schwartz, MD University of British Columbia (Presented By: Samuel Krikler, MBChB, FRCPC)

Poster# 217 HEREDITARY HEMOCROMATOSIS AND DIABETES MELLITUS (DM), CAUSE OR COINCIDENCE? A NORTHEASTERN BRAZILIAN EXPERIENCE Luiz Silva, Jr., MD¹, Andre Silva, MD², Adavio Silva, MD³ and Jairo Cavalcante, Statistical4 ¹Souza Junior Laboratorio Medico; ²Hospital A.C. Camargo; ³Hospital da Beneficiencia Portuguesa; 4PMM (Presented By: Luiz Silva, Jr., MD)

Poster# 218 PILOT PHARMACOKINETIC STUDY IN PATIENTS WITH INADEQUATE RESPONSE TO DEFERASIROX (EXJADE) S. Deborah Chirnomas, MD¹, Amber Smith, BA², Luis Pereira, PhD³, Jennifer Braunstein, PNP4, Carole Paley, MD5 and Ellis Neufeld, MD, PhD² ¹Children’s Hospital Boston; ²Children’s Hospital Boston/Hematology/Oncology; ³Children’s Hospital, Boston/Clinical Pharmacology Unit; 4Children’s Hospital Boston/Hematology/Oncology; 5Novartis Pharmaceuticals (Presented By: S. Deborah Chirnomas, MD)

Poster# 219 MITOCHONDRIAL DYSFUNCTION MAY EXPLAIN THE CARDIOMYOPATHY OF CHRONIC IRON OVERLOAD John Eaton, PhD, MDhc University of Louisville (Presented By: John Eaton, PhD, MDhc) 41

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Poster# 220 THE IRON CHELATOR DEXRAZOXANE PROTECTS CARDIOMYOCYTES FROM DOXORUBICIN-MEDIATED CELL DEATH: ROLE OF HIF-1 Lorenza Tacchini, PhD, Stefania Recalcati, MD, PhD, Rosalin Spagnuolo, PhD and Gaetano Cairo, PhD University of Milan (Presented By: Gaetano Cairo, PhD)

Poster# 221 INCREASED IRON ABSORPTION IS ASSOCIATED WITH UPREGULATION OF DUODENAL DMT1, DCYTB AND HEPHAESTIN EXPRESSION IN PATIENTS WITH NONALCOHOLIC STEATOHEPATITIS Masayoshi Kobune, MD, and PhD, Kohichi Takada, MD, PhD, Yutaka Kawano, MD, PhD, Koji Miyanishi, MD, PhD, Hiroyuki Nagahima, MD, PhD, Rishu Takimoto, MD, PhD, Shohei Kikuchi, MD, Tsutomu Sato, MD, PhD, Yasushi Sato, MD, PhD, Yoshiro Niitsu, MD, PhD and Junji Kato, MD, PhD Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine (Presented By: Masayoshi Kobune, MD)

Poster# 222 THE COMPARATIVE ANALYSES OF SINGLE NUCLEOTIDE POLYMORPHISM OF OXIDATIVE DNA REPAIR GENES IN PATIENTS WITH HCV-RELATED LIVER DISEASE Koji Miyanishi, MD, and PhD, Masayoshi Kobune, MD, PhD, Yutaka Kawano, MD, PhD, Hiroyuki Nagahima, MD, PhD, Shohei Kikuchi, MD, Kohichi Takada, MD, PhD, Rishu Takimoto, MD, PhD, Tsutomu Sato, MD, PhD, Yasushi Sato, MD, PhD, Yoshiro Niitsu, MD, PhD and Junji Kato, MD, PhD Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine (Presented By: Koji Miyanishi, MD)

Poster# 223 THE CLINICAL AND GENETIC SPECTRUM OF TMPRSS6 MUTATIONS LEADING TO INAPPROPRIATE HEPCIDIN EXPRESSION AND IRON REFRACTORY IRON DEFICIENCY ANEMIA (IRIDA) Matthew Heeney, MD, Dean Campagna and Mark Fleming, MD, DPhil Children’s Hospital Boston (Presented By: Matthew Heeney, MD)

Poster# 224 GENOME-WIDE ASSOCIATION STUDY OF IRON DEFICIENCY C.E. McLaren, PhD¹, C.P. Garner, PhD¹, C.C. Constantine, PhD², S. Masle, PhD4, C.D. Vulpe, MD, PhD4, B.M. Snively, PhD¹³, D.A. Nickerson, PhD¹¹, J.D. Cook, MD¹², C. Leiendecker-Foster, MS5, K.B. Beckman, MS5, J.H. Eckfeldt, MD5, V.R. Gordeuk, MD³, L.F. Barcellos, PhD4, A. Killeen, MD5, J.A. Murray, MD6, P.C. Adams, MD7, R.T. Acton, PhD8, D.M. Reboussin, PhD¹³, K. Goddard, PhD9 and G.D. McLaren, MD¹° ¹University of California, Irvine; ²University of Melbourne, Australia; ³Howard University; 4University of California, Berkeley; 5University of Minnesota; 6Mayo Clinic; 7London Health Sciences Centre, London, Ontario; 8University of Alabama at Birmingham; 9Kaiser Permanente Northwest; ¹°Univ. of California, Irvine, and Department of Veterans Affairs Medical Center, Long Beach, CA; ¹¹University of Washington; ¹²University of Kansas; ¹³Wake Forest University School of Medicine (Presented By: C.E. McLaren, PhD)

Poster# 225 IRON DEFICIENCY DETECTED BY ESTIMATION OF BODY IRON IN A PRIMARY CARE POPULATION: PREVALENCE AND ETIOLOGY Gordon D. McLaren, MD¹, Joanna A. Kolodney, MD¹, Naveen Doki, MD¹, Wen-Pin Chen, MS¹, Sakineh Khalaghizadeh, BS¹, Mahmood Novin, MD¹, James D. Cook, MD², Mazen Jamal, MD, MPH¹ and Christine E. McLaren, PhD¹ ¹Dept. of Veterans Affairs Long Beach Healthcare System, Long Beach, and University of California, Irvine, CA; ²University of Kansas Medical Center, Kansas City, KS, USA (Presented By: Gordon D. McLaren, MD)

Poster# 226 IRON UTILISATION FROM ORAL IRON TREATMENT IS DEPENDENTD FROM THE INDIVIDUAL BLEEDING RATE IN PATIENTS WITH HYPERMENORRHEA OR GASTROINTESTINAL HEMORRHAGE Peter Nielsen, Rosemarie Kongi and Roland Fischer, PhD University Hospital, Hamburg (Presented By: Peter Nielsen)

Poster# 227 THE MENKES COPPER ATPASE (ATP7A) GENE ENCODES TWO NUCLEAR PROTEIN VARIANTS THAT INTERACT WITH DNA AND ARE INDUCED DURING IRON DEFICIENCY James Collins, PhD, Yan Lu, BS and P.N. Ranganathan, PhD University of Florida (Presented By: James Collins, PhD)

Poster# 228 POTENTIAL ABNORMALITIES IN IRON PROCESSING BY MUCOLIPIDOSIS TYPE IV PATIENT FIBROBLASTS Chevaun Morrison, BS¹, Eric N. Sauble, MS Chemistry¹, Annie Nguyen, undergraduate¹, Maria C Linder, PhD¹ and Gideon Bach, MD² ¹California State University, Fullerton; ²Hadassah Medical School, Jerusalem (Presented By: Chevaun Morrison, BS)

Poster# 229 WITHDRAWN ISOTHERMAL TITRATION CALORIMETRY OF HUMAN SERUM TRANSFERRIN INTERACTION WITH THE SOLUBLE PORTION OF THE RECEPTOR Banu Kandemir, undergraduate¹, Shaina Byrne, PhD student², Anne Mason, PhD², N. Dennis Chasteen, PhD³ and Fadi Bou-Abdallah, PhD¹ ¹State University of New York at Potsdam, NY, USA; ²University of Vermont, VT, USA; ³University of New Hampshire, NH, USA

Poster# 230 “IN SILICO” MAPPING OF LIVER IRON LEVELS IN INBRED MICE Stela Masle, Seung-Min Lee and Chris Vulpe UC Berkeley (Presented By: Stela Masle)

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Poster# 231 A HIGH PREVALENCE OF SUB-CLINICAL IRON DEFICIENCY IN BLOOD DONORS THAT PASSED THE SCREENING TEST FOR HEMOGLOBIN Mireille Baart, MSc, Carine Doggen, PhD, Wim de Kort, PhD and Paulus van Noord, PhD Sanquin Blood Bank (Presented By: Mireille Baart, MSc)

Poster# 232 ROLE OF IRON REPLACEMENT THERAPY IN THE ROUTINE MANAGEMENT OF BLOOD DONORS Barbara Bryant, MD¹, Yu Ying Yau, RN², Sarah Arceo, RN², Julie Hopkins, RN² and Susan Leitman, MD² ¹University of Texas Medical Branch; ²National Institutes of Health (Presented By: Barbara Bryant, MD)

Poster# 233 VALIDATING A SELF-REPORTED PROTOCOL FOR MENSTRUAL BLOOD LOSS IN PATIENTS WITH IRON DEFICIENCY ANAEMIA Peter Nielsen, Thomas Jansen, MD, Rosemarie Kongi and Roland Fischer, PhD University Hospital, Hamburg (Presented By: Peter Nielsen)

Poster# 234 PREVALENCE AND TRANSFORMATION’S PROBABILITY OF IRON DEPLETION TO ANEMIA IN CHILDREN Elena Morshchakova, MD, Valeriy Demikhov, MD, Nataliya Inyakova, Oksana Isakova and Nadezhda Kravtsova Russian Federal Research Center for Pediatric Hematology (Presented By: Valeriy Demikhov, MD)

Poster# 235 HFE-RELATED HEMOCHROMATOSIS PHENOTYPIC SCREENING IN 53119 SUBJECTS: THE BRITTANY EXPERIENCE Hervé Goarin, MD², Jeff Morcet³, Pascale Gérault4, Yves Trégouët4, Michèle Bourrien4, Véronique David5, Yves Deugnier, MD6, Anne- Marie Jouanolle5, Hervé Seignard, MD4 and Pierre Brissot, MD¹ ¹Liver Disease Unit, Inserm U-522, University Hospital Pontchaillou, Rennes, France; ²Mutualité Sociale Agricole,Inserm CIC 203, Laboratory of Molecular Genetics, Liver Disease Unit, Inserm U-522, Pontchaillou University Hospital, Rennes, France; ³Inserm CIC 203, Rennes, France; 4Mutualité Sociale Agricole, Rennes, France; 5Laboratory of Molecular Genetics, University Hospital Pontchaillou, Rennes, France; 6Inserm CIC 203, Liver Disease Unit, University Hospital Pontchaillou, Rennes, France (Presented By: Pierre Brissot, MD)

Poster# 236 CLINICAL EXPRESSION OF HFE-ASSOCIATED HAEMOCHROMATOSIS IN SUBJECTS UNDER 40 YEARS OF AGE Jeannette Dixon, BA, MPH, Elizabeth Watt, BComm, MD, Grant Ramm, BSc, PhD, Nathan Subramaniam, BSc, PhD, Lawrie Powell, MD, PhD and Greg Anderson, BSc, MSc, PhD Queensland Institute of Medical Research (Presented By: Jeannette Dixon, BA)

Poster# 237 SCREENING FOR IRON OVERLOAD: LESSONS FROM THE HEIRS STUDY Paul Adams, MD¹, James Barton, MD², Gordon McLaren, MD³, Ronald Acton, PhD4, Mark Speechley, PhD5, Christine McLaren, PhD6, David Reboussin, PhD7, Cathie Leiendecker-Foster, MS8, Beverly Snively, PhD7, Emily Harris, PhD9, Tom Vogt, MD¹°, Phyliss Sholinsky, MSPH¹¹, Elizabeth Thomson, DNSc¹², Fitzroy Dawkins, MD¹³, Victor Gordeuk, MD¹³ and John Eckfeldt, MD, PhD8 ¹University Hospital; ²Southern Iron Disorders Center, Birmingham, AL; ³VA Long Beach Healthcare System, Irvine, CA; 4Dept. of Microbiology, Birmingham, AL; 5Dept. of Epidemiology, University of Western Ontario; 6Dept. of Epidemiology, UCI, Irvine, CA; 7Division of Public Health Sciences, Wake Forest University, Winston-Salem, NC; 8Department of Laboratory Medicine, University of Minnesota, Minneapolis, MN; 9National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD; ¹°Kaiser Permanente Center for Health Research, Honolulu, HI; ¹¹Dept of Epidemiology, NHLBI, NIH, Bethesda, MD; ¹²National Genomics Institute, NIH, Bethesda, MD; ¹³Dept. of Hematology, Howard University, Washington, DC (Presented By: Paul Adams)

Poster# 238 HFE-C282Y HOMOZYGOTES WITH ELEVATED SF LESS THAN 1000 µG/L ARE NOT AT INCREASED RISK OF HAEMOCHROMATOSIS-ASSOCIATED DISEASE FEATURES K.J. Allen¹,²,³, L.C. Gurrin, N.A. Bertalli, N.J. Osborne, C.C. Constantine, M.B. Delatycki, A.E. Nisselle, A.J. Nicoll, D.M. Gertig, C.E. McLaren, G.G. Giles, D.R. English, J.L. Hopper, G.J. Anderson, J.K. Olynyk and L.W. Powell ¹Murdoch Childrens Research Institute; ²Dept of Paediatrics The University of Melbourne; ³Dept of Gastroenterology Royal Children’s Hospital, Australia (Presented By: K.J. Allen)

Poster# 239 NOMOGRAM TO DETECT THE RISK FOR IRON OVERLOAD-ATTRIBUTED DISEASE IN SIBLINGS OF PROBANDS WITH HFE-RELATED SYMPTOMATIC HEMOCHROMATOSIS: RESULTS OF THE HEMOCHROMATOSIS FAMILY STUDY Esther Jacobs, MD, PhD², Jan Hendriks, Herman Kreeftenberg, Richard de Vries, Joannes Marx, Cees van Deursen, Anton Stalenhoef, Andre Verbeek and Dorine Swinkels, MD, PhD¹ ¹Radboud Department of Clinical Chemistry 441, University Nijmegen Medical Centre; ²Department of Clinical Chemistry, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands (Presented By: Dorine Swinkels, MD, PhD)

Poster# 240 HFE COMPOUND HETEROZYGOTES ARE AT LOW RISK OF HEMOCHROMATOSIS-RELATED MORBIDITY L.C. Gurrin, N.A. Bertalli, G.W. Dalton, N.J. Osborne, C.C. Constantine, C.E. McLaren, D.R. English, D.M. Gertig, M.B. Delatycki, A.J. Nicoll, M.C. Southey, J.L. Hopper, G.G. Giles, G.J. Anderson, J.K. Olynyk, L.W. Powell and K.J. Allen Centre for MEGA Epidemiology, The University of Melbourne. Australia (Presented By: L.C. Gurrin)

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Poster# 241 WITHDRAWN JAPANESE PATIENTS WITH HEMOJUVELIN-HEMOCHROMATOSIS WHO SURVIVED OVER 50 YEARS Hisao Hayashi, MD¹, Yasuaki Tatsumi, PhD², Ai Hattori, BS², Motoyoshi Yano, MD³, Ryuichi Kidokoro, MD4, Shinya Wakusawa, PhD5, Kazuhiko Hayashi, MD5, Yoshiaki Katano, MD5 and Hidemi Goto, MD5 ¹Aichi Gakuin University; ²Aichi Gakuin University School of Pharmacy; ³Yokkaichi City Hospital; 4Kidokoro Clinic; 5Nagoya University School of Medicine

Poster# 242 ADRENERGIC OVERDRIVE IN IRON OVERLOADED PATIENTS WITH HEMOCHROMATOSIS Raffaella Mariani, MD¹, Alberto Piperno, Paola Trombini², Valentina Paolini, Matteo Pozzi, Gino Seravalle³, Fosca Quarti-Trevano, Guido Grassi and Giuseppe Mancia ¹University of Milano-Bicocca; ²Department of Clinical Medicine, San Gerardo Hospital, Monza, Italy; ³Istituto Auxologico Italiano, Milano, Italy (Presented By: Paola Trombini)

Poster# 243 EXTRINSIC FACTORS INFLUENCING THE EXPRESSIVITY OF THE HFE VARIANT C282Y, H63D, S65C PHENOTYPES IN DANISH MEN AGED 30-53 YEARS Palle Pedersen, PhD² and Nils Milman, MD¹ ¹Næstved Hospital, Department of Clinical Biochemistry; ²Department of Clinical Biochemistry, Næstved Hospital, Næstved, Denmark (Presented By: Nils Milman, MD)

Poster# 244 DOES HLA-TYPE OR T-LYMPHOCYTE SUBSETS HAVE ANY RELEVANCE FOR PLANNING THE FOLLOWUP OF PERSONS WITH SCREENING-DETECTED HEMOCHROMATOSIS? PRELIMINARY DATA FROM NORTH TRØNDELAG COUNTY, NORWAY Ketil Thorstensen¹, Wenche Irgens¹, Arne Åsberg¹, Berit Borch-Iohnsen², Torolf Moen³ and Kristian Hveem4 ¹Dept. of Medical Biochemistry, St. Olav Hospital, Trondheim, Norway.; ²Inst. of Basic Medical Sciences, Department of Nutrition, University of Oslo, Norway.; ³Dept. of Immunology and Transfusion Medicine, St. Olav Hospital, Trondheim, Norway.; 4Medical Department, Levanger Hospital, Levanger, Norway (Presented By: Ketil Thorstensen)

Poster# 245 SELDI-TOF MS DETECTION OF URINARY HEPCIDIN Sandro Altamura, PhD, Judith Kiss, Claudia Blattmann, Wolfgang Gilles and Martina Muckenthaler MMPU – Molecular Medicine Partnership Unit (Presented By: Sandro Altamura, PhD)

Poster# 246 PHENOTYPIC PENETRANCE OF THE HFE GENE C282Y, H63D AND S65C VARIANTS IN DANISH MEN AGED 30-53 YEARS Nils Milman, MD and Palle Pedersen, PhD Næstved Hospital, Næstved, Denmark (Presented By: Nils Milman, MD)

Poster# 247 DELINEATING THE EXPERIENCES OF HEREDITARY HEMOCHROMATOSIS PATIENTS ALONG THE PATIENT PATHWAY: AN INTERNATIONAL QUANTITATIVE SURVEY OF 210 PATIENTS Pierre Brissot, MD¹, Vivian Wu Jin², Simon Ball³ and Helena Cannon³ ¹Liver Disease Unit Inserm U-522, University Hospital Pontchaillou, Rennes, France; ²Novartis Pharmaceuticals Corporation, New Jersey, USA; ³Adelphi International Research, Bollington, UK (Presented By: Pierre Brissot, MD)

Poster# 248 A PORTUGUESE PATIENT HOMOZYGOUS FOR THE HAMP-25G→ A MUTATION-30 YEARS FOLLOW UP Rita Fleming, MD Hospital Santa Maria (Presented By: Rita Fleming, MD)

Poster# 249 EVALUATION OF PATIENT SATISFACTION WITH PHLEBOTOMY FOR THE TREATMENT OF HEREDITARY HEMOCHROMATOSIS: AN INTERNATIONAL QUANTITATIVE SURVEY OF 210 PATIENTS Pierre Brissot, MD¹, Jean Francis Baladi², Simon Ball³ and Helena Cannon³ ¹Liver Disease Unit Inserm U-522, University Hospital Pontchaillou, Rennes, France; ²Novartis Pharmaceuticals Corporation, New Jersey, USA; ³Adelphi International Research, Bollington, UK (Presented By: Pierre Brissot, MD)

Poster# 250 INFECTIOUS DISEASES IN DIAGNOSED PATIENTS OF GENETIC HEMOCHROMATOSIS Alejandro Del Castillo, MD, Susana Gordo, Teresa Aldámiz, Fernando De la Calle and Luis A. Álvarez-Sala Hospital G. U. Gregorio Marañón (Presented By: Alejandro Del Castillo, MD)

Poster# 251 ARTERIAL HYPERTENSION IN PATIENTS WITH GENETIC HEMOCHROMATOSIS Alejandro Del Castillo, MD, Susana Gordo, Teresa Aldámiz, Fernando De la Calle and Luis A. Álvarez-Sala Hospital G. U. Gregorio Marañón (Presented By: Alejandro Del Castillo, MD)

Poster# 252 SNPS ASSOCIATED WITH IRON INDICES IN A CANDIDATE GENE STUDY OF HEREDITARY HAEMOCHROMATOSIS C.C. Constantine, PhD², C.D. Vulpe, G.J. Anderson, C.E. McLaren, M. Bahlo, H.L. Yeap, K.J. Allen, N.J. Osborne, N.A. Bertalli, D.M. Gertig, M.B. Delatycki, J.K. Olynyk, K.B. Beckman, V. Chen, P. Matak, A.T. McKie, L.C. Gurrin¹ and HealthIron Investigators ¹Centre for MEGA Epidemiology, The University of Melbourne; ²Centre for MEGA Epidemiology, School of Population Health, University of Melbourne (Presented By: L.C. Gurrin)

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Poster# 253 CELLULAR CHARACTERISATION OF MAMMALIAN MON1A: A PUTATIVE TRAFFICKING PROTEIN AND MODIFIER OF THE HAEMOCHROMATOSIS PHENOTYPE Daniel Wallace, PhD, Lesa Summerville, Nick Barker and Nathan Subramaniam Queensland Institute of Medical Research (Presented By: Daniel Wallace, PhD)

Poster# 254 HFE, SLC40A1, HAMP, HJV, TFR2, AND FTL MUTATONS DETECTED BY DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AFTER IRON PHENOTYPING AND HFE C282Y AND H63D GENOTYPING IN 785 HEIRS STUDY PARTICIPANTS James Barton, MD¹, Susie DelRio-LaFreniere, MS², Catherine Leiendecker-Foster, MS², Honggui Li, MS², Ronald Acton, PhD³, Richard Press, MD, PhD4 and John Eckfeldt, MD, PhD² ¹Southern Iron Disorders Center; ²University of Minnesota, Minneapolis, MN; ³University of Alabama at Birmingham, Birmingham, AL; 4Oregon Health & Science University, Portland, OR (Presented By: James Barton, MD)

Poster# 255 MUTATION ANALYSIS OF SEVEN GENES INVOLVED IN IRON METABOLISM IN A MENNONITE POPULATION Samuel Krikler, MBChB¹, Chris Whittington, MBChB¹, Michelle-Angelique Hallendorff, MSc², Natalie Strickland, MSc², Veronique Human, MSc², Nathaniel McGregor, HonsBSc², Jessica Vervalle, HonsBSc² and Monique Zaahl, PhD² ¹University of British Columbia; ²Stellenbosch University (Presented By: Samuel Krikler, MBChB)

Poster# 256 SYNTHESIZING THE EVIDENCE FOR THE ASSOCIATION BETWEEN THE G277S SUBSTITUTION IN THE TRANSFERRIN GENE AND IRON DEFICIENCY BY COMBINING DATA FROM POPULATION STUDIES L.C. Gurrin, N.A. Bertalli and C.C. Constantine Centre for MEGA Epidemiology, The University of Melbourne (Presented By: L.C. Gurrin)

Poster# 257 CHANGES OF THE EXPRESSIONS OF THE GENES INVOLVED IN IRON METABOLISM BY THE IRON CHELATION THERAPY AND PHLEBOTOMY IN THE IRON OVERLOADED MOUSE MODEL Takaaki Hosoki, MD, PhD, Katsuya Ikuta, MD, PhD, Naoka Okamura, MD, Motohiro Shindo, MD, PhD, Kazuya Sato, MD, PhD, Takaaki Ohtake, MD, PhD, Katsunori Sasaki, PhD, Yoshihiro Torimoto, MD, PhD and Yutaka Kohgo, MD, PhD Asahikawa Medical College (Presented By: Takaaki Hosoki, MD, PhD)

Poster# 258 COMPARATIVE ABILITY OF IRON CHELATORS TO REMOVE IRON FROM ALBUMIN-IRON (III) COMPLEXES Jay Parshotam, BSc (Hons)¹, André Silva², Xiao Kong³ and Robert Hider4 ¹KCL/ PhD student; ²KCL/PhD student; ³KCL/PhD; 4KCL/Professor (Presented By: Jay Parshotam, BSc (Hons))

Poster# 259 H-FERRITIN SIRNA DELIVERED BY CATIONIC LIPOSOMES INCREASES GLIOMA CELL SENSITIVITY TO RADIATION AND CHEMOTHERAPEUTIC AGENTS Xiaoli Liu, MS, A.B. Madhankumar, PhD, Jonas Sheehan, MD, Becky Slagle-Webb, BSc and James Connor, PhD Penn State Hershey Medical center (Presented By: James Connor, PhD)

Poster# 260 A COMPARATIVE STUDY OF THE ANTIPROLIFRATIVE ACTIVITY OF IRON CHELATORS PIH, SIH AND THEIR PHOTOLABILE CAGED-DERIVATIVES IN SKIN CELLS Charareh Pourzand, PhD, Asma Aroun, PhD² and Olivier Reelfs, PhD Department of Pharmacy and Pharmacology, Bath University, Bath, UK (Presented By: Charareh Pourzand, PhD)

Poster# 261 L-TYPE CALCIUM CHANNEL BLOCKER, VERAPAMIL, IMPROVES SURVIVAL AND REDUCES IRON LOADING IN HYPOTRANSFERRINAEMIC MICE Patarabutr Masaratana, MD, Abas Laftah, PhD, Gladys Oluyemisi Latunde-Dada, PhD, Robert Simpson, PhD and Andrew McKie, PhD King’s College London (Presented By: Patarabutr Masaratana, MD)

Poster# 262 IRON DEPLETION THERAPY FOR PATIENTS WITH METABOLIC SYNDROME AND NON-ALCOHOLIC FATTY LIVER DISEASE: PRELIMINARY FINDINGS James Nelson, PhD¹, Kristina Utzschneider, MD², Bryan Maliken, BS¹, Jacob Alexander, MD² and Kris Kowdley, MD³ ¹Benaroya Research Institute; ²University of Washington Medical Center; ³Benaroya Research Institute and Virginia Mason Medical Center (Presented By: James Nelson, PhD)

Poster# 263 HIGH THROUGH-PUT SCREENING OF CHEMICALS THAT STIMULATE IRON UPTAKE – A NOVEL APPROACH TO DISCOVERY OF ANTI-CANCER DRUGS Zhen Li, PhD, Hiroki Tanaka, PhD, Hong Yin, PhD, Kwo-Yih Yeh, PhD, Mary Yeh, PhD and Jonathan Glass, MD Feist-Weiller Cancer Center, LSUHSC (Presented By: Jonathan Glass, MD)

Poster# 264 FRIEDREICH’S ATAXIA: PROTEIN TRANSDUCTION OF TAT-CONJUGATED FRATAXIN MAY BE A NOVEL THERAPY Hannes Steinkellner, Barbara Scheiber-Mojdehkar, Dr, Franco Laccone, Dr, Hans Goldenberg, Dr and Brigitte Sturm, Dr Medical University of Vienna (Presented By: Hannes Steinkellner)

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Poster# 265 POTENT IRON CHELATORS AS ANTI-TUMOR AGENTS: THE NOVEL 2-ACETYLPYRIDINE THIOSEMICARBAZONE SERIES Danuta Kalinowski, BSc, PhD, V. Richardson, P.C. Sharpe, D.B. Lovejoy, M. Islam, P.V. Bernhardt and D.R. Richardson University of Sydney (Presented By: Danuta Kalinowski, BSc, PhD)

Poster# 266 FRATAXIN-EXPRESSION IS INCREASED BY RECOMBINANT HUMAN ERYTHROPOIETIN INDEPENDENT OF EXPRESSION OF THE CLASSICAL ERYTHROPOIETIN RECEPTOR Melissa Helminger¹, Hannes Steinkellner, Msc², Hans Goldenberg, PhD², Brigitte Nina Sturm, PhD² and Barbara Scheiber-Mojdehkar, PhD² ¹Medical University of Vienna; ²Department of Medical Chemistry, Medical University of Vienna, Austria (Presented By: Melissa Helminger)

Poster# 267 MOLECULAR IMPACT OF HUMAN DIVALENT METAL-ION TRANSPORTER-1 (DMT1) MUTATIONS ASSOCIATED WITH DISEASE PHENOTYPES Ali Shawki, Anthony C. Illing and Bryan Mackenzie University of Cincinnati College of Medicine (Presented By: Ali Shawki)

Poster# 268 THE FIRST CELLULAR MODELS FOR FRIEDREICH ATAXIA BASED ON FRATAXIN MISSENSE MUTATIONS THAT REPRODUCE SPONTANEOUSLY FE-S CLUSTERS DEFECT AND INTRAMITOCHONDRIAL IRON DEPOSITS Stéphane Schmucker¹, Nadège Carelle-Calmels, PhD, Marie Wattenhofer-Donzé, PhD, Nadège Vaucamps, technician, Laurence Reutenauer, Technician, Nadia Messaddeq, Engineer, Koenig Michel, Professor and Hélène Puccio, PhD² ¹IGBMC; ²IGBMC Illkirch (Presented By: Stéphane Schmucker)

Poster# 269 RETROMER IS REQUIRED FOR PROPER ENDOSOMAL SORTING OF MAMMALIAN IRON TRANSPORTER, DMT1 Mitsuaki Tabuchi, PhD, Izumi Yanatori, Bachelor and Fumio Kishi, MD, PhD Kawasaki Medical School (Presented By: Fumio Kishi, MD, PhD)

Poster# 270 EVENTS IN THE ENDOSOME LINK HEME AND IRON HOMEOSTASIS WITH THAT OF COPPER IN THE HEMOPEXIN SYSTEM Ann Smith, BSc, PhD, Kimberley Rish, MS, Rachel Lovelace, BS, Jennifer Hackney, PhD and Rachel Helston, PhD University of Missouri-KC (Presented By: Ann Smith, BSc, PhD)

Poster# 271 MECHANISMS UNDERLYING HEPATIC IRON ACCUMULATION IN A DIETARY EXPERIMENTAL MODEL OF NAFLD Paola Dongiovanni, PhD, S. Gatti, R Rametta, A.L. Fracanzani, S. Fargion and L. Valenti Department of Internal Medicine, University of Milan (Presented By: Paola Dongiovanni, PhD)

Poster# 272 HEPATIC IRON DEPOSITION IN RETICULOENDOTHELIAL CELLS BUT NOT HEPATOCYTES IS ASSOCIATED WITH MORE SEVERE NASH: RESULTS FROM THE NASH CLINICAL RESEARCH NETWORK James Nelson, PhD¹, Laura Wilson, MS², Elizabeth Brunt, MD³, Matthew Yeh, MD, PhD4, David Kleiner, MD, PhD5, Aynur Unalp-Arida, MD, PhD² and Kris Kowdley, MD6 ¹Benaroya Research Institute; ²Johns Hopkins University; ³Washington University, Department of Pathology; 4University of Washington, Department of Pathology; 5National Cancer Institute; 6Benaroya Research Institute and Virginia Mason Medical Center (Presented By: James Nelson, PhD)

Poster# 273 OSYSL15 IS AN IRON-REGULATED IRON (III)-PHYTOSIDEROPHORE TRANSPORTER AND IS ESSENTIAL FOR IRON UPTAKE IN RICE PLANTS N.K. Nishizawa, PhD, T. Kobayashi, T. Nozoye, M. Takahashi, Y. Kakei, H. Nakanishi, S. Mori University of Tokyo (Presented By: Naoko Nishizawa, PhD)

Poster# 274 PORPHYRIN DISTORTION AND METAL ION SELECTIVITY IN FERROCHELATASE Neil McIntyre, PhD², Ricardo Franco, PhD³, John Shelnutt, PhD4 and Gloria Ferreira¹ ¹Univ South Florida; ²University of South Florida; ³Universidade Nova de Lisboa; 4Sandia National Laboratories (Presented By: Gloria Ferreira)

Poster# 275 SERUM FERRITIN AND HEPCIDIN LEVELS PREDICT VASCULAR DAMAGE IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE Luca Valenti, MD, Dorine W. Swinkels, Larry Burdick, Harold Tjalsma, Cristina Bertelli, Erika Fatta, Daniela Bignamini, Paola Dongiovanni, Raffaela Rametta, Benedetta M. Motta, Silvia Fargion and Anna Ludovica Fracanzani University of Milano (Presented By: Luca Valenti, MD)

Poster# 276 RECOMBINANT HUMAN ERYTHROPOIETIN DOPING IN AN EXPERIMENTAL MODEL OF CHRONIC AEROBIC EXERCISE TRAINING Nuno Piloto, Biochem D², Flávio Reis, PhD¹, Helena M. Teixeira, PhD, Edite Teixeira de Lemos, PhD, Patrícia Garrido, MSci, Elísio Costa, MSCi, José Sereno, Biochem D, Margarida Lourenço, MD, Carlos A. Tavares, MD, Carlos A. Ferrer Antunes, MD, Petronila Rocha Pereira, PhD, Luis Belo, PhD, Alice Santos Silva, PhD and Frederico Teixeira, PhD ¹Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University; ²Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University, Portugal (Presented By: Flávio Reis, PhD) 46

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Poster# 277 IRON UPTAKE BY CHROMERA VELIA Robert Sutak, Emmanuel Lesuisse and Jean-Michel Camadro Institut Jacques Monod (Presented By: Robert Sutak)

Poster# 278 SMALL MOLECULE INHIBITORS OF HYPOXIA INDUCIBLE FACTOR 2A (HIF2A) TRANSLATION LINK ITS 5’- UNTRANSLATED REGION (5’-UTR) IRON RESPONSIVE ELEMENT (IRE) TO IRP-1 MEDIATED OXYGEN SENSING Michael Zimmer, PhD², Christoher Neil, BSc² and Othon Iliopoulos, MD¹ ¹Massachusetts General Hospital Cancer Center; ²Massachusetts General Hospital Cancer Center and Harvard Medical School (Presented By: Othon Iliopoulos, MD)

15:15 – 16:45 Concurrent Sessions XI – XIII

15:15 – 16:45 Concurrent Session XI: Iron, Brain and Neurodegeneration Location: Arquivo Chairs: James Connor, Hershey, PA, USA and Roberta Ward, Louvain-la-Neu, Belgium

15:15 73 A NOVEL TRANSFERRIN/TFR2-MEDIATED MITOCHONDRIAL IRON TRANSPORT SYSTEM IS DISRUPTED IN PARKINSON’S DISEASE Pier Mastroberardino, PhD¹, Maxx Horowitz¹, Ranjita Betarbet, PhD², Claire-Anne Gutekunst, PhD², Marla Gearing, PhD², John Q. Trojanowski, PhD³, Marjorie Anderson, PhD4, Charleen T. Chu, MD, PhD¹, Junmin Peng, PhD² and John T Greenamyre, MD, PhD¹ ¹University of Pittsburgh; ²Emory University; ³University of Pennsylvania; 4University of Washington (Presented By: Pier Mastroberardino, PhD)

15:30 74 DOMINANT MUTANTS OF CERULOPLASMIN INDUCE GOLGI FRAGMENTATION AND IMPAIRMENT OF THE COPPER LOADING MACHINERY IN ACERULOPLASMINEMIA Giovanni Musci, Prof, Nunziata Maio, Gianluca Rizzo, Giovanni De Francesco, Tiziana Persichini, Marco Colasanti, Fabio Polticelli and Maria Carmela Bonaccorsi di Patti University Roma Tre (Presented By: Giovanni Musci, Prof)

15:45 75 HUMAN AMYLOID-B BINDS HEME TIGHTLY: WHY HUMANS BUT NOT RODENTS DEVELOP ALZHEIMER’S DISEASE Hani Atamna The Commonwealth Medical College, Pennsylvania, USA (Presented By: Hani Atamna)

16:00 76 REGULATION OF IRON TRANSPORT ACROSS THE BLOOD BRAIN BARRIER James Connor, PhD¹, Padmavathi Ponnuru, PhD², Becky Webb, BS³, David Antonetti, PhD4, Anthony Carruthers, PhD5 and Ian Simpson, PhD6 ¹Penn State University College of Medicine; ²Dept. of Neurosurgery, Penn State University College of Medicine, Hershey, PA; ³Dept. of Neurosurgery, Penn State University College of Medicine, Hershey, PA; 4Dept. of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA; 5Department of Biochemistry, UMMS, Worcester, MA; 6Department of Neural and Behavioral Sciences Penn State University College of Medicine, Hershey, PA (Presented By: James Connor, PhD)

16:15 77 THE CHOROID PLEXUS IS A SITE FOR REGULATING BRAIN IRON HOMEOSTASIS Fernanda Marques, Ana Mendanha Falcao, BSc, Joao Carlos Sousa, PhD, Giovanni Coppola, MD, PhD, Daniel Geschwind, MD, PhD, Nuno Sousa, MD, PhD, Margarida Correia-Neves, PhD and Joana Almeida Palha, PhD University of Minho, Life and Health Sciences Research Institute (ICVS), Portugal (Presented By: Fernanda Marques)

16:30 78 DESFERRIOXAMINE AND DEFERIRSIROX TRAVERSE THE BLOOD BRAIN BARRIER AND PREVENT DEGENERATION OF DOPAMINE NEURONS IN THE 6-HYDROXY DOPAMINE MODEL OF PARKINSON’S DISEASE David Dexter, BSc, PhD¹, Hugh Chan, BSc¹, Robert Crichton, BSc, PhD, FRSC² and Roberta Ward, BSc, PhD² ¹Imperial College London; ²University of Louvain, Belgium (Presented By: David Dexter, BSc, PhD)

15:15 – 16:45 Concurrent Session XII: Novelties – From Approaches to Therapies Location: D. Maria Chairs: Sonia Levi, Milano, Italy and Elisabeta Nemeth, Los Angeles, CA, USA

15:15 79 USE OF JAK2 INHIBITORS TO LIMIT INEFFECTIVE ERYTHROPOIESIS AND IRON ABSORPTION IN MICE AFFECTED BY BETA-THALASSEMIA MAJOR, N°79 Luca Melchiori, graduate student¹, Sara Gardenghi, PhD², Ella Guy, BaS², Nan Chen, BaS², Eliezer A. Rachmilewitz, MD³, Patricia J. Giardina, MD², Robert W. Grady, PhD² and Stefano Rivella, PhD² ¹Weill Cornell Medical College; ²Weill Cornell Medical College, New York; ³Wolfson Medical Center, Israel (Presented By: Luca Melchiori, graduate student)

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15:30 80 EXOGENOUS TRANSFERRIN AMELIORATES DISEASE IN BETA-THALASSEMIC MICE Yelena Ginzburg, MD¹, Huihui Li, MS¹, Anne Rybicki, PhD², Sandra Suzuka, MS³, Leni von Bonsdorff, PhD4, William Breuer, PhD5, Z. Ioav Cabantchik, MD, PhD5, Eric Bouhassira, PhD³ and Mary Fabry, PhD³ ¹New York Blood Center; ²Montefiore Medical Center; ³Albert Einstein College of Medicine; 4Sanquin; 5Hebrew University of Jerusalem (Presented By: Yelena Ginzburg, MD)

15:45 81 DEVELOPMENT OF BMP ANTAGONISTS FOR EXPERIMENTAL AND THERAPEUTIC CONTROL OF HEPCIDIN EXPRESSION Randall Peterson, PhD, Paul Yu, MD, PhD, Chetana Sachidanandan, PhD, Greg Cuny, PhD, Charles Hong, MD, PhD and Kenneth Bloch, MD Harvard Medical School (Presented By: Randall Peterson, PhD)

16:00 82 NATURAL AND SYNTHETIC STAT3 INHIBITORS REDUCE SIGNIFICANTLY HEPCIDIN EXPRESSION IN DIFFERENTIATED MOUSE HEPATOCYTES EXPRESSING THE ACTIVE PHOSPHORYLATED FORM OF STAT3 Nadia Fatih¹, Emilie Camberlein, PhD², Marie-Laure Island, PhD², Anne Corlu, PhD², Emmanuelle Abgueguen, PhD², Patricia Leroyer², Pierre Brissot, MD² and Olivier Loréal, MD, PhD² ¹INSERM U522, University of Rennes1, Rennes France; ²INSERM U522, University of Rennes 1, Rennes (Presented By: Nadia Fatih)

16:15 83 GENE REGULATION THERAPY UTILIZING FERRITIN HEAVY CHAIN Robert H. Broyles, BSc, PhD¹, Visar Belegu, PhD², Marie Trudel, DSc³, Sonia Levi, PhD4 and Paoloa Arosio, PhD5 ¹Univ of Oklahoma Health Sciences Ctr.; ²Johns Hopkins University; ³Institut de Recherches Cliniques de Montréal; 4DIBIT-IRCCS, H.San Raffaele, Milano; 5Materno Infantile e Tecnologie Biomedicine, University of Brescia (Presented By: Robert H. Broyles, BSc, PhD)

16:30 84 MINIHEPCIDINS: SMALL PEPTIDES INVOLVED IN DISULFIDE EXCHANGE WITH FERROPORTIN ACT AS AGONISTS Tomas Ganz, PhD, MD, Gloria Preza, MS, Peter Ruchala, PhD and Elizabeta Nemeth, PhD University of California, Los Angeles (Presented By: Tomas Ganz, PhD, MD)

15:15 – 16:45 Concurrent Session XIII: Iron Overload Around the World Location: D. Luis Chairs: John Crowe, Dublin, Ireland and Nils Milman, Naestved, Denmark

15:15 85 HFE MUTATIONAL ANALYSIS HAS REDUCED THE NEED FOR LIVER BIOPSY AT DIAGNOSIS BUT NOT IMPACTED ON INITIAL DISEASE BURDEN IN HEREDITARY HAEMOCHROMATOSIS PROBANDS John D. Ryan, MBBCh, Mary T. O’Neill, MBBCh, Eleanor Ryan, PhD, T. Barry Kelleher, MD and John Crowe, PhD Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin, Ireland (Presented By: John D. Ryan, MBBCh)

15:30 86 HERITABILITY OF SERUM IRON MEASURES IN THE HEMOCHROMATOSIS AND IRON OVERLOAD SCREENING (HEIRS) FAMILY STUDY C.E. McLaren, PhD¹, J.C. Barton², G.D. McLaren³, R.T. Acton4, P.C. Adams5, L.F. Henkin6, V.R. Gordeuk7, J.H. Eckfeldt8, C.D. Vulpe9, B.W. Harrison¹°, J.A. Reiss¹¹ and B.M. Snively6 ¹University of California, Irvine; ²Southern Iron Disorders Center, Birmingham, AL; ³Dept. of Veterans Affairs Long Beach Healthcare System, Long Beach, CA; 4Univ. of Alabama at Birmingham, AL; 5London Health Sciences Centre, London, Ontario, Canada; 6Wake Forest Univ. School of Medicine, Winston-Salem, NC; 7Howard Univ.; 8Univ. of Minnesota, Minneapolis, MN; 9Univ. of California, Berkeley, CA; ¹°Howard Univ., Washington, DC; ¹¹Kaiser Permanente NW, Portland, OR (Presented By: C.E. McLaren, PhD)

15:45 87 TFR2-RELATED HEREDITARY HEMOCHROMATOSIS IN JAPAN Chisaki Mizumoto, MD¹, Junya Kanda, MD², Hiroshi Kawabata, MD², Takashi Uchiyama, MD², Katsutoshi Kojima, MD³, Tatsuya Hayashi, MD³, Masakazu Hirata, MD³, Kazuma Nakao, MD³, Ken-ichi Tsuchida, MD4, Miwa Kawanaka, MD5, Gotaro Yamada, MD5, Naohisa Tomosugi, MD6, Yasuaki Tatsumi, PhD7, Aya Hattori, MSc7 and Hisao Hayashi, MD7 ¹Kyoto University; ²Dept. Hematology/Oncology, Kyoto University; ³Dept. Medicine/Clinical Science, Kyoto University; 4Manda Memorial Hospital Diabtes Center; 5Center of Liver Diseases, Kawasaki Hospital, Kawasaki Medical School; 6Proteomics Research Unit, Division of Advanced Medicine, Medical Research Institute, Kanazawa Medical University; 7Dept. Medicine, Aichi Gakuin University School of Pharmacy (Presented By: Chisaki Mizumoto, MD)

16:00 88 IRON OVERLOAD IN THE ASIAN COMMUNITY Chun Yu Lok, A.T. Merryweather-Clarke, D.J. Weatherall and K.J.H. Robson Weatherall Institute of Molecular Medicine, Oxford, UK (Presented By: Chun Yu Lok)

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Thursday, June 11, 2009

16:15 89 DELETION OF THE HFE GENE IS PRESENT AT THE POPULATION LEVEL IN SARDINIA Gerald Le Gac, Dr¹,6, Antonio Cao, Pr², Rita Congiu, Dr³, Isabelle Gourlaouen4, Claude Férec, Pr4 and Maria Antonietta Melis, Pr5 ¹Inserm U613, Etablissement Français du Sang, Brest, F-29200 FRANCE; ²Istituto di Neurogenetica e Neurofarmacologia CNR, Cagliari, Italy; ³Ospedale microcitemico ASL8, Cagliari, Italy; 4Inserm U613, Etablissement Français du Sang, Centre Hospitalier Universitaire, Brest, F-29200 France; 5Dipartimento Scienze biomediche e biotecnologia, Università di Cagliari, Cagliari, Italy; 6Istituto di Neurogenetica e Neurofarmacologia CNR, Ospedale microcitemico ASL8, Dipartimento Scienze biomediche e biotecnologia, Università di Cagliari, Cagliari, Italy (Presented By: Gerald Le Gac, Dr)

16:30 90 DO ALL HEMOCHROMATOSIS PATIENTS ORIGINATE FROM THE SAME ANCESTRAL ORIGIN BY MITOCHONDRIAL DNA ANALYSIS? Paul Adams, MD University Hospital (Presented By: Paul Adams, MD)

17:00 – 18:00 Closing Ceremony Location: Arquivo

Ernest Beutler Lecture Chair: Maria de Sousa, Porto, Portugal

Iron as Engine of Basic Biological Processes and Disease: The Future of BioIron Nancy Andrews, Duke University, USA

18:00 Meeting Adjourns

19:30 Departure for Gala Dinner and Awards Ceremony from all official congress hotels 20:30 Gala Dinner and Awards Ceremony Begins Location: Palácio da Bolsa

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Invited Speaker Abstracts and Bios

Keynote Lecture 1: Global Iron Connections Between Desert Dust, Ocean Biogeochemistry and Climate Tim Jickells, University of East Anglia, Norwich, UK Ocean primary production by microscopic algae (phytoplankton) is a central part of the global carbon cycle which profoundly influences global climate as well as food supply. Iron is a key component of several phytoplankton enzymes. The high pH and oxygen rich conditions of the oceans lead to very low dissolved iron concentrations and to iron limitation of phytoplankton productivity in many parts of the oceans. Iron limitation can be relieved by inputs of iron, and the atmospheric supply of iron as a component of desert dust is of particular importance. This creates a complex feedback system between climate, dust supply and ocean productivity which I will explore in this talk.

Tim Jickells graduated with a BSc in Chemistry from Reading in the UK and an MSc in Oceanography from Southampton UK. He then worked in Glasgow and Bermuda and gained a PhD in Oceanography from Southampton at the same time. He took up a lectureship at University of East Anglia in 1985 where he am now a professor. He currently also works part time for the UK Natural research Council as Theme Leader for Earth System Science. His research interests focus on the biogeochemistry of coastal waters and the impact of atmospheric deposition of iron, nitrogen and phosphorus on the oceans.

Keynote Lecture 2: Iron and Oxygen Sensing Peter Ratcliffe, University of Oxford, UK Analysis of the oxygen sensitive pathways that regulate the transcription factor HIF has revealed a novel mode of signaling through hydroxylation of specific prolyl and asparaginyl residues. HIF prolyl hydroxylation promotes destruction of HIF-alpha sub-units by promoting association with the von Hippel-Lindau ubiquitin E3 ligase; HIF asparaginyl hydroxylation inhibits transactivation. These processes link the HIF transcriptional response (which includes the regulation of a wide range of proteins involved in iron metabolism) to the availability of oxygen. The HIF hydroxylations are all catalysed by members of the Fe(II) and 2-oxoglutarate dioxygenase superfamily. During catalysis, the splitting of molecular oxygen is coupled to the hydroxylation of HIF and the oxidative decarboxylation of 2-oxoglutarate, in a radical reaction that most probably involves the generation of a highly reactive ferryl (FeIV=O) ion at the catalytic centre. Ascorbate is required for full catalytic activity, either through reducing the catalytic Fe centre in the event of uncoupled turnover, or by enhancing the availability of cellular Fe(II) more generally. The absolute requirement for molecular oxygen as co-substrate conveys oxygen sensitivity. However emerging evidence suggests that limiting levels of co-factors including Fe(II), ascorbate and 2-oxoglutarate may under certain circumstances reduce hydroxylase activity and thus modulate the signalling of hypoxia. At the catalytic centre Fe(II) is co-ordinated by a 2-histidine-1-carboxylate facial triad. Binding of Fe(II) is labile and recent work indicates that changes in cellular iron status occurring under physiological and patho-physiological conditions can have significant effects on the operation of the HIF system both in cells and in humans. This supports the existence of a physiologically important overlap between oxygen and iron sensing in the regulation of HIF. Recent work has also revealed that at least one of HIF hydroxylases, the HIF asparaginyl hydroxylases FIH has many other substrates. Together with the recognition that the human genome likely encodes as many as 60 members of the Fe(II) and 2-oxoglutarate dependent dioxygenase superfamily, many of which are predicted to catalyse protein oxidations, these findings suggest the existence of a wide range of previously unforeseen mechanisms by which iron status may regulate cellular functions.

Peter Ratcliffe is Nuffield Professor and Head of the Department of Clinical Medicine at the University of Oxford. He trained as a Nephrologist and was Academic Director of Renal Medicine prior to appointment to the current position in 2004. He heads the hypoxia biology laboratory at the Henry Wellcome Building for Molecular Physiology and has published over 200 peer-reviewed manuscripts in hypoxic signalling and related fields. In 2002 he was elected to the Fellowship of the Royal Society, and in 2009 was awarded the Louis- Jeantet Prize for Medicine for his work on mechanisms in cellular oxygen sensing.

Lecture 1: Erythropoiesis Olivier Hermine, Institut Pasteur, Paris, France Erythropoiesis is a multistep process that occurs in the bone marrow and involves the sequential formation of proerythroblasts - basophilic, polychromatic and orthochromatic -erythroblasts and enucleated reticulocytes. Terminal erythroid differentiation is characterized by hemoglobin synthesis and major morphological changes including chromatin condensation, cell size reduction and finally, nuclei extrusion to generate reticulocytes that enter in the blood circulation. This differentiation process involves on one hand the transcription factor GATA-1, which positively regulates promoters of erythroid genes such as Glycophorin A, erythropoietin receptor (EPO-R) and hemoglobin synthesis and on the other hand, major nuclear and cytoskeleton reorganization. Stem cell factor (SCF) and erythropoietin (EPO) are the two main cytokines that are required for erythropoiesis respectively at early and late stage of differentiation. The amount of the production of red cells is regulated by apoptosis of late eythroid precursors that depends on the level of circulating Epo. Low level of Epo induces caspases activation and cell death and maturation arrest. Mechanisms underlying Epo sensitivity of erythroid precursors are still not fully understood and may involve both intrinsic and extrinsic factors.

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Interestingly, the late differentiation of erythroid cells shares similarities with features occurring during apoptosis such as chromatin condensation, organelle loss and membrane remodeling. We have previously demonstrated that transient activation of effector caspases at the basophilic stage is required for terminal erythroid maturation. Caspases induce cleavage of several proteins involved in nuclear structural changes such as lamin B, acinus or PARP. In contrast, GATA-1, a target of caspases during apoptosis, is not cleaved during differentiation, and cell death does not occur. This phenomenon is explained by the translocation into the nucleus at the onset of caspase activation of the chaperone protein HSP70 and its interaction with GATA-1. Mechanisms by which SCF and Epo regulate caspase activation and HSP70 nuclear translocation during differentiation are under evaluation. Systematic identification of each caspase substrate might be helpful to understand mechanisms that determine the fate -differentiation versus apoptosis- of erythroid cells upon different physiological or pathological conditions. Terminal maturation from polychromatophilic erythroblasts to reticulocytes occurs independently of cytokine signaling. This cytokine independence for late maturation of erythroid precursors is a peculiar phenomenon that may be in vivo dependant on integrins signaling for erythroid progenitors proliferation. However, it remains to be understood by which mechanisms orthochromatic erythroblasts can exhibit cytoskeleton reorganization, contractile actin ring formation for extrusion of pycnotic nucleus, a process highly dependent on kinases signaling. In this process, caspases could play an indirect role through proteins cleavage and activation. The understanding of all these processes will shed light in the pathophysiology of anemia associated with various erythroid disorders. All these mechanisms may be translated beyond erythropoiesis and may be involved in neoplastic transformation of cells and in degenerative disorders.

Olivier Hermine is professor of clinical haematology and head of a research group in CNRS at Necker Hospital in Paris France. He received his medical degree after a residency training in Paris University Hospital of Assistance Publique. He is board certified in clinical haematology. He received his PhD in hematopoiesis from Paris University based on his work on normal and pathological erythroid differentiation that has been performed both in the Pr Goldwasser’s laboratory at the University of Chicago and in the Dr Vainchenker’s laboratory at the INSERM unit in Villejuif, France. Currently, his clinical practice focuses on haematological malignancies with a special emphasis on lymphoma, and non malignant haematological disorders including primary immune deficiencies, autoimmune disorders, and mastocytosis. His clinical research interests include physiopathology and treatment of mantle cell lymphoma, virus associated lymphoproliferation particularly those associated with HTLV-1 and HCV, and mastocytosis. He has participated in numerous clinical studies. His research activity focuses on erythropoiesis and pathophysiology and targeted therapy of malignant lymphoproliferative diseases and mastocytosis. He has co authored more than 200 papers in leading scientific journals including Nature, New England Journal of Medicine, Lancet, Blood, Journal of clinical oncology, Journal of experimental medicine, PNAS, and Nature immunology, and serves as a reviewer for many of these journals. He has delivered several lectures at local, national and international conferences and symposia as invited speaker.

Lecture 2: Ineffective Erythropoiesis in Beta-Thalassemia Stefano Rivella, Cornell University, New York, USA In thalassemia, ineffective erythropoiesis is characterized by apoptosis of the maturing nucleated erythroid cells. New studies also suggest that limited erythroid cell differentiation plays a role in the development of ineffective erythropoiesis. Some of the major consequences of ineffective erythropoiesis are extra−medullary hematopoiesis (EMH), splenomegaly and systemic iron overload mediated by transfusion therapy and down−regulation of hepcidin. We hypothesized that the protein kinase Jak2 plays a major role in ineffective erythropoiesis. Use of Jak2 inhibitors may limit the overproduction of immature erythroid cells in thalassemia, with the potential of reversing extramedullary hematopoiesis and preventing splenectomy. For this reason, we administered a Jak2 inhibitor to mice affected by beta−thalassemia intermedia, showing that this treatment was associated with a marked decrease in ineffective erythropoiesis, and a more moderate decrease in hemoglobin levels (~1 g/dL). This last observation indicates that the use of Jak2 inhibitors might exacerbate anemia in thalassemia. Recall, however, that blood transfusion is required, not only by thalassemia−major patients, but also by those with thalassemia−intermedia who develop splenomegaly. Therefore, administration of a Jak2 inhibitor, together with blood transfusions, might be a sensible way to limit splenomegaly while preventing further anemia. This strategy might even complement our efforts to decrease iron accumulation in thalassemia. Therefore, we are conducting an analysis of erythropoiesis and iron metabolism in animals affected by beta−thalassemia major, which require blood transfusion for survival. In addition, we showed that hepcidin is expressed at a relatively low level compared to iron load in mice with beta−thalassemia. For this reason we hypothesized that thalassemic mice absorb more iron than required for erythropoiesis and that reducing their iron intake by increasing hepcidin levels could limit iron overload without affecting their erythropoiesis. The experiments to generate pre-clinical data with the use of a Jak2 inhibitor and hepcidin are in progress and will be discussed.

Stefano Rivella is an associate professor at Weill Cornell Medical College (WCMC) in New York City. He earned his BS and PhD in genetics at the University of Pavia, Italy. He then completed a post-doctoral fellowship in molecular biology from the Memorial Sloan- Kettering Cancer Center in New York. Dr. Rivella has received numerous honors, most recently Travel Grant Awards from the American Society of Gene Therapy (ASGT) in 2001 and the International Society of Experimental Hematology (ISEH) in 2002. Also in 2002, he received first prize New Investigator Award for the ISEH.

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Iron Overload in Myelodysplastic Syndromes Mario Cazzola, Pavia, Italy

Abstract not received in time for the final program.

MRI for Cardiac Iron Evaluation John Wood, Los Angeles, USA Iron cardiomyopathy remains the leading cause of death in thalassemia major. Serum ferritin and liver iron measurements have previously been used to infer cardiac risk. While these studies have demonstrated that noncompliance with iron chelation represents the leading cause of cardiac iron overload, iron cardiomyopathy develops in patients who otherwise appear well chelated. Cardiac MRI can be used to detect preclinical myocardial iron and stratify cardiac risk according. Tissue iron shortens T2 and T2*, the time constants for transverse relaxation, causing MRI images to darken more quickly with echo time. These techniques have been validated in human and animal models. We will introduce the methodology for cardiac T2* measurements, as well as some of the insights that these techniques have provided regarding the pathophysiology of cardiac iron overload. Lastly, we will discuss the role of cardiac T2* in the management of patients with transfusional and nontransfusional siderosis.

John Wood has been on the medical staff at both Children’s Hospital Los Angeles (CHLA) and Los Angeles County Hospital since 1999. He is also an assistant professor of pediatrics with a dual appointment in the Department of Radiology at CHLA, along with a joint appointment in the Department of Bioengineering at the University of Southern California. Dr. Wood’s eduational background consists of a BS from the University of California, Davis, postgraduate work at Norges Tekniske Hogskole, Norwary and the University of Michigan Medical Scientist Training Program where he completed a PhD in bioengineering and his MD. He served a residency in the Department of Pediatrics at Children’s Hospital at Yale-New Haven and a fellowship in the Division of Pediatric Cardiology, Department of Pediatrics at Yale University School of Medicine.

State of Art of Iron Chelation Therapy Maria Cappellini, Milan, Italy

Abstract not received in time for the final program.

Future Therapeutic Approaches to Target Imbalances in Iron Homeostasis Antonello Pietrangelo, Modena, Italy There is a distinct risk of developing iron overload in humans. While iron is essential for many vital functions, there is no regulated means by which excess iron can be disposed. Therefore, whenever body iron exceeds its needs and storage capabilities are saturated, toxicity due to iron overload may arise. Classic causes of severe iron overload states in humans include transfusion-dependent iron overload due genetic or acquired anaemias and hereditary haemochromatosis. However, recently, a number of pathologic states have been identified where even marginal iron excess or iron misdistribution may exert a pathogenic cofactorial activity: e.g. viral hepatitis, the metabolic syndrome, and certain neurodegenerative disorders. Traditional iron removal strategies have been phlebotomy , restricted mainly to hemochromatosis, or pharmacological iron chelation, to treat post-transfusion iron overload. It appears now that iron chelation therapy might be also beneficial in patients with non-transfusion-related forms of iron overload. Moreover, recent, exciting discoveries in iron metabolism pave the way for the development of future novel therapies aimed at modulating the activity of the iron hormone hepcidin.

Antonello Pietrangelo, MD, PhD, graduated in medicine at the University of Modena in 1981. He held research associate positions at the Universities of Modena and Milan, and from, 1989 , at the Liver Research Center, AEC0M, New York. In 1993, he returned to the University of Modena, where he has been Professor of Medicine since 2001. During his career, he has received a number of awards, such as the EASL Investigator Award, the Fogarty Advanced Fellowship Award, and the Marcel Simon Award for excellence in Iron Metabolism. In 1999, he organized and chaired the First International Consensus Conference on Hemochromatosis supported by NIH, CDC and WHO, and in 2003, he chaired the founding committee of the International BioIron Society (IBIS) of which he was elected as the first president. The fields of hemochromatosis, molecular and cell biology of iron-related oxidant stress and fibrogenesis, and liver gene expression are Professor Pietrangelo’s special research interests. He has served in the editorial board of renowned international journals and published more than 150 peer-reviewed artiche, and contributed chapters to numerous books in iron metabolism and hemochromatosis.

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Lecture 3: Iron Metabolism in Yeast Caroline Philpott, University of UTA, USA In determining the cellular response to iron deficiency and iron overload, we and others have focused on changes in the levels of transcripts and proteins involved in iron homeostasis. Yet alterations in transcript or proteins levels do not always result in the predicted changes in transport activity or flux through a metabolic pathway. For example, despite an increase in the expression of reductases and iron transporters during growth in iron-poor medium, yeast cells accumulate much less iron in this setting. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine the extent to which flux through metabolic pathways is maintained or altered in the face of falling iron availability. We find that in yeast, although several iron-dependent enzymes are required for amino acid biosynthesis, amino acid homeostatic mechanisms are robust and levels of individual amino acids do not fall in the face of iron deficiency. Carbon source utilization and energy metabolism are dramatically altered during iron-limited growth, as cells shift exclusively to fermentation. Lipid biosynthesis is affected by iron deficiency. We found specific alterations in the flux through the ergosterol and sphingolipid biosynthetic pathways at points where heme and diiron enzymes function.

Caroline Philpott received her bachelor and medical degrees from Duke University. She completed her internship and residency training in internal medicine at the Johns Hopkins Hospital. Following her clinical training, Philpott began a research fellowship in the laboratory of Richard Klausner in the National Institute of Child Health and Human Development at the National Institutes of Health. In 1998, she joined the intramural program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH and in 2004, became a tenured Senior Investigator. Dr. Philpott now serves as the Chief of the Genetics and Metabolism Section, Liver Diseases Branch, NIDDK, NIH. Her laboratory focuses on the mechanisms by which eukaryotic cells take up and use iron.

Lecture 4: Siderophores and the Battle for Iron by Aspergillus Hubertus Haas, Innsbruck Medical University, Austria To acquire iron, all species have to overcome the problems of iron insolubility and toxicity. In recent years it became clear that siderophores (low molecular mass iron chelators) constitute a central element in the iron homeostasis of most fungi. The saprobic fungus Aspergillus fumigatus, which is the most common airborne fungal pathogen, produces four different hydroxamate type siderophores: extracellular fusarinine C and triacetylfusarinine C, hyphal ferricrocin and conidial hydroxyferricrocin. Siderophore biosynthesis is repressed by iron via the GATA-transcription factor SreA and induced via interaction of HapX with the CCAAT binding complex. Genome-wide transcriptional profiling combined with gene deletion analysis identified siderophore biosynthetic genes. Analysis of generated A. fumigatus siderophore mutant strains demonstrated that the extracellular siderophores are involved in iron uptake, whereas the intracellular siderophores are involved in iron storage, and intracellular iron transport. Consistent with these roles, siderophores affect germination, hyphal growth, sporulation, oxidative stress resistance, and, in the close relative Aspergillus nidulans, also homothallic sexual development. Comparison of wild type and siderophore mutant strains revealed that both extra- and intracellular siderophores are important for virulence in a mouse model of pulmonary aspergillosis. Moreover, siderophore biosynthesis has also been found to be crucial for virulence of many plant-pathogenic fungi. The fungal requirement for iron could potentially open up perspectives towards the development of novel antifungal treatments, e.g., iron chelation therapy, blocking of high-affinity iron acquisition, or development of siderophore-drug conjugates for "Trojan Horse"-inhibition of fungal growth.

Hubertus Haas is vice-director and associate professor in the Division of Molecular Biology/Biocenter at Innsbruck Medical University (IMU) in Austria. His educational background includes a BS/MS in microbiology/biochemistry and a PhD in molecular mycology/genetics from the Leopold-Franzens University (LFU) Innsbruck and Sandoz GmbH, Kundl, Austria. His research interests include Characterization of the the fungal metabolism (e.g. Aspergillus spp.) for improvement of (i) treatment and diagnosis of fungal infections and (ii) biotechnological exploitation of fungi. The current research focus is the fungal iron metabolism.

Lecture 5: The Immune System at the Crossroads of Iron Metabolism Maria de Sousa, ICBAS, University of Porto, Portugal The one characteristic that perhaps better distinguishes the immune system from iron metabolism is the nature of their interaction with the external environment. The immune system has its roots in the realization that some people can become immune to certain infections coming from the outside world, a phenomenon first observed spontaneously and later provoked by immunization. Iron metabolism on the other hand represents the ultimate paradigm of freedom from the external environment. As fields of knowledge, however, their youth approximates them. Both the complexity of immunological function and the complexity of iron biology are major scientific developments of our time. In the case of immunology the complexity started to be perceived in the last 50 years. In the case of iron metabolism, that process is even younger. People contributing to that youth are largely participating in this conference. The weight of history and the availability of tools, impose the questions we ask. But when two fields are so young there is room to question whether they only do what we know today. Infection as a first reason for the very existence of the immune system has naturally dominated the crossroads between the immune system and iron metabolism. Because bacteria utilize haem and non-haem iron for their own metabolism, an iron chelation function has been sought for the immune system focusing in particular on the association with neutrophils of iron chelating proteins like lactoferrin and the siderocalins. Reciprocally, iron loaded macrophages and neutrophils are thought to be havens for bacterial and parasite growth and a threat to the infected host. Gene Weinberg is acknowledged as the prophetical voice that propagated the message for iron withholding as a defense strategy. Hal Drakesmith the one who brought iron to its most contemporary crossroad with HIV. But the path has been crossed in the reciprocal direction where iron deficiency is thought to diminish a host immunological response to infection. The intricate role of 53 cytokines in these reciprocal crossings has been extensively studied by Gunter Weiss. The role of iron genes expressed in macrophages responsible for iron recycling has been the focus of the work by the groups of Cannone-Hergaux and Ponka. The less expected is the growing evidence that the immune system as a physiological system can have critical regulatory functions of other systems. The system that perhaps has received the greater attention is the central nervous system, giving rise to a discipline known as Neuroimmunology. But the one that in my view deserves equal if not greater attention is the one I have in several instances described as Hemmunology, to encompass the fantastic frontier continuously crossed by immune system cells, red blood cells and iron, securing iron homeostasis and immunological function in general. We know that crossing involves iron overload associated to a non-classical MHC class I molecule, HFE, abnormally low lymphocyte numbers in situations of deregulation of iron absorption and iron overload in humans and in mice, molecules liberated by red blood cells that signal trafficking for lymphocytes, and abnormal erythropoiesis in mice knock out for HFE. To conclude I will present recent work from my lab demonstrating that iron gene expression in lymphocytes starts to offer a molecular basis for some of those least expected findings.

Maria de Sousa is professor of Immunology at the Abel Salazar Institute for the Biomedical Sciences and Head of the Division of Human Genetics and Genetic Disorders at the IBMC , both at the University of Oporto. Her work has focused on dissecting a role for the immune system in iron homeostasis describing the interface between the two systems as Hemmunology. She is a member of EMBO, a corresponding member of the Portuguese Academy of Sciences and, a Fellow of the Royal College of Pathologists.

Lecture 6: Iron and Host-Pathogen Interactions Andrew Prentice, London School of Hygiene and Tropical Medicine, UK Evidence from a wide variety of sources, ranging from genomic analysis of micro-organisms to large-scale human intervention studies, confirms that iron lies at the centre of a battleground for nutritional resource between mammals and their invading pathogens. Iron appears to be more critical in this role than any other micronutrient, and can modulate the pathogenicity of bacterial, viral and protozoal infections. These battles have raged for countless generations leading to the evolution in both host and pathogen of complex iron- responsive adaptive mechanisms that favour survival. By definition these mechanisms are finely balanced (otherwise one side would be extinct) and hence are difficult to study in human clinical medicine. They are also niche specific; for instance, E coli strains capable of colonising the low iron environment of the urinary tract are characterised by their iron acquisition capabilities. Iron may also have paradoxical effects in certain micro-organisms; for instance, whilst an abundant iron supply may favour pathogen growth, a restricted supply may have even more harmful consequences by triggering toxin production (eg by Vibrio cholerae) designed to ‘leak’ iron and other nutrients from the host. These insights suggest a need for caution in the therapeutic administration of iron in human populations that still suffer a high burden of infectious diseases. A number of clinical trials and epidemiological surveys have confirmed these theoretical concerns by showing enhanced morbidity and mortality in groups receiving iron or characterised by a high iron status. This poses a serious barrier to global efforts to harness the positive benefits of iron (in relation to cognition and child development) in the world’ poorest nations, and hence represents a crucial area for further research at both basic science and clinical/epidemiological levels.

Andrew Prentice leads the MRC International Nutrition Group at the London School of Hygiene and Tropical Medicine, and at the group’s rural fieldstation in Keneba, The Gambia where he has worked for over 30 years. His interest in iron and host-pathogen interactions is relatively recent and was first prompted by a revelatory moment when reading of the heavy genomic investment in iron acquisition systems in the plague organism, Yersinia pestis, and of their crucial role in its niche jump from being an enteric to a systemic pathogen.

Lecture 7: Role of Mitochondria in Iron-Sulfur and Iron Metabolism Roland Lill, Philipps Universität Marburg, Marburg, Germany Research over the past few years has established the crucial role of mitochondria in both the biogenesis of iron-sulfur (Fe/S) proteins and the regulation of cellular iron homeostasis. The two processes are coupled mechanistically in that defects in Fe/S protein biogenesis elicit increases in both cellular and mitochondrial iron import. The molecular basis of Fe/S cluster synthesis and assembly into apoproteins in a living cell has been subject to intense research activities (see Review). Biogenesis is accomplished by three complex proteinaceous machineries (Figure). Mitochondrial Fe/S proteins require the iron-sulfur cluster (ISC) assembly machinery which was inherited from bacteria during evolution. Cytosolic/nuclear Fe/S protein assembly also requires the function of this machinery, yet additionally depends on the mitochondrial ISC export apparatus and the cytosolic iron-sulfur protein assembly (CIA) machinery. The components of all three systems (more than 25 proteins) are highly conserved in eukaryotes from yeast to man suggesting similar mechanisms of Fe/S protein assembly. We are currently attempting to define the molecular basis of the assembly processes, in particular the contribution of mitochondria to cytosolic/nuclear Fe/S protein biogenesis. In fact, the latter process is the major reason why mitochondria (or mitochondria-derived organelles such as mitosomes and hydrogenosomes) are indispensable in virtually all eukaryotic cells. Essential cytosolic/nuclear Fe/S proteins depending on mitochondrial ISC function are involved in DNA replication and repair as well as ribosome assembly and translation, thus linking the function of mitochondria to basic processes of gene expression in a eukaryotic cell. The process is of importance for human disease in that more than ten diseases are associated with ISC, CIA or Fe/S protein defects. For instance, depletion of the ISC assembly component frataxin leads to the neurodegenerative disease Friedreich’s ataxia, and a defect in the ISC export protein ABCB7 is associated with X-linked sideroblastic anemia and ataxia (XLSA/A). Fe/S protein defects in the nucleus impairing DNA replication and repair link Fe/S protein biogenesis to numerous diseases including various forms of cancer. 54

Reviews: Lill, R., and Mühlenhoff, U. (2008). Maturation of iron-sulfur proteins in eukaryotes: Mechanisms, connected processes, and diseases. Annu Rev Biochem 77, 669-700. Sheftel, A. D., and Lill, R. (2009). The power plant of the cell is also a smithy: The emerging role of mitochondria in cellular iron homeostasis. Ann Med 41, 82-99.

Figure: Simplified model for Fe/S protein biogenesis in eukaryotes. For details see reviews.

Roland Lill is a full professor (W3) at the Universität Marburg in Germany. He is also currently an advisory board member of the German Society for Biochemistry and Molecular Biology (GBM). His undergraduate studies in chemistry were completed at the Universitäten Ulm and München and received a PhD in biochemistry from the Universität München. He has received many awards and honors and has organized several scientific meetings, most recently (2009) the FEBS Advanced Lecture Course on “Mitochondria in Life, Death and Disease”, Aussois, France. Dr. Lill’s research interests include function and biogenesis of mitochondria; post-translational modification; iron-sulfur protein biogenesis in mitochondria, cytosol and nucleus; structure and function of eukaryotic iron-sulfur proteins; transport across the mitochondrial membranes; intracellular iron homeostasis; transcriptional and post-transcriptional control of iron uptake and regulation; structure and function of ABC transporters, mitochondrial carriers, Isu scaffold proteins, ferredoxins, Hsp70 chaperones, glutaredoxins, P-loop ATPases, WD40 proteins, and hydrogenase-like proteins; intracellular redox control; Friedreich’s ataxia; iron storage diseases.

Lecture 8: Hepcidin Regulation and Processing Sophie Vaulont, Université Paris Descartes, CNRS, Paris, France

Abstract not received in time for the final program.

Lecture 9: Iron Metabolism in Plants Mary Lou Guerinot, Dartmouth College, Hanover USA Although the mechanisms controlling iron uptake from the soil are well understood, relatively little is known subcellular trafficking or iron deficiency signaling in plants. Studies in Arabidopsis identified FIT as a transcription factor essential for uptake of iron from the soil. FIT controls expression of many but not all iron-regulated genes, including the IRT1 gene encoding a high affinity Fe(II) transporter that transports Fe into root cells and the FRO2 gene encoding the ferric chelate reductase that reduces Fe at the root-soil interface. Although IRT1 mRNA abundance is somewhat decreased in roots of Fe deficient fit plants, IRT1 protein is not detectable in fit. We decided to carry out a genetic screen for other genes that control IRT1 expression. One recessive mutant [uri (upstream regulator of IRT1 does not induce IRT1 expression in response to Fe deficiency. As might be expected of a mutant that does not express the essential IRT1 protein, the uri mutant dies after germinating in soil unless supplied with high levels of soluble Fe. The mutant also does not induce ferric chelate reductase activity, consistent with a lack of induction of FRO2 mRNA. F1 plants from a cross between a fit loss of function line and the uri mutant were phenotypically wild type, indicating the uri line does not have a mutation in FIT. Interestingly, FIT expression is not induced under Fe deficiency in the uri mutant, suggesting that URI lies upstream of FIT in the Fe deficiency signaling pathway. We have also been continuing our studies on localization of metals using synchrotron X-ray fluorescence spectroscopy and can now image at the subcellular level. In addition, we have been using high throughput elemental profiling via inductively coupled plasma spectroscopy – mass spectroscopy (ICP-MS) to define a plant’s ionome and have used this information for functional characterization of various mutants, for gene discovery, for developing ionomic signatures of different physiological states and for exploring natural variation. For example, we have identified several promising lines that accumulate more Fe in their seed. Our long-term goal is to understand the network of genes responsible for integrating information about iron status and orchestrating a coordinated response. Such an understanding will transform efforts towards sustainable improvements of crop yields in terms of plant productivity and nutrient content.

Mary Lou Guerinot is a professor in the Department of Biological Sciences. She earned her bachelor’s degree in biology at Cornell University in 1975 and her PhD in biology from Dalhousie University in 1979. After completing postdoctoral studies at the University of Maryland and at the DOE–MSU Plant Research Laboratory, she came to Dartmouth as an assistant professor in 1985. She was promoted to an associate professor with tenure in 1991 and to full professor in 1997. She was chair of the Department of Biological Sciences from 1994 to 1998, served as the associate dean of the Faculty for the Sciences from 1998 to 2001 and as Vice Provost from 2001 to 2004. Guerinot is a molecular geneticist whose principal expertise and research interests are in the area of metal transport and regulation of 55 gene expression by metals. For most of the world, plants are the major point of entry for essential metals into the food chain, so her work is laying the foundation for crops that offer sustainable solutions for malnutrition. Guerinot has published over 90 articles and her work is funded by major grants from the National Science Foundation, the National Institutes of Health and the Department of Energy. In her lab at Dartmouth, she has trained 13 Ph.D. students and over 60 undergraduates. Guerinot has taught genetics and microbiology lecture and lab courses, as well as upper-level and graduate courses in molecular genetics and biochemistry. She has served on numerous competitive grant panels and as a member of the Advisory Committee for Biological Sciences at NSF. She has been a member of the editorial boards of the Journal of Bacteriology and Applied and Environmental Microbiology and is an associate editor of Plant Molecular Biology and Plant, Cell and Environment. She is a Past President of the American Society of Plant Biologists, a 6000-member organization that promotes the growth and development of plant biology as well as publishes research in plant biology.

Lecture 10: Iron Metabolism in C. elegans Iqbal Hamza, University of Maryland, USA Hemes are metalloporphyrins that serve as prosthetic groups for diverse biological processes including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control, metabolic reprogramming, and microRNA processing. With a few exceptions, heme is synthesized via a multistep biosynthetic pathway comprising defined intermediates that are highly conserved throughout evolution. Despite our extensive knowledge of heme biosynthesis and degradation, the cellular pathways and molecules which mediate intracellular heme trafficking are unknown. The experimental setback in identifying heme trafficking pathways has been the inability to dissociate the highly regulated cellular synthesis and degradation of heme from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural heme auxotrophs that acquire environmental heme for incorporation into hemoproteins, which have vertebrate orthologs. We exploited this auxotrophy to identify HRG-1 proteins in C. elegans and show that they are essential for heme homeostasis and normal development in worms and vertebrates. Depletion of either hrg-1, or its paralog hrg-4, in worms results in disruption of organismal heme sensing and abnormal response to heme analogs. HRG-1 and HRG-4 are novel transmembrane proteins, which reside in distinct intracellular compartments. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations, and, most strikingly, profound defects in erythropoiesis - phenotypes that are fully rescued by worm HRG-1. Human and worm proteins co-localize, and bind and transport heme, thus establishing an evolutionarily conserved function for HRG-1. These findings reveal unanticipated and conserved pathways for cellular heme trafficking in animals that define the paradigm for eukaryotic heme transport. Thus, uncovering the mechanisms of heme transport in C. elegans may provide novel insights into human disorders of heme metabolism and reveal new drug targets for developing anthelmintics to combat worm infestations.

Iqbal Hamza received his BSc and MSc in Biochemistry from the University of Bombay. He completed his PhD in Biochemistry in 1997 from the State University of New York at Buffalo under the mentorship of Dr. Mark O'Brian. Dr. Hamza conducted his postdoctoral fellowship in the laboratory of Dr. Jonathan Gitlin at Washington University School of Medicine till 2002 when he joined the University of Maryland as an Assistant Professor. He is currently an Associate Professor at the UM and the main thrust of his research group is to identify the genes and elucidate the mechanisms of heme trafficking and transport in eukaryotes.

Lecture 11: Iron Metabolism in Zebrafish Barry Paw, Harvard Medical School, USA The zebrafish (Danio rerio) has emerged as a powerful model system to study vertebrate development and heritable disorders in humans, in particular disorders of iron and heme metabolism (1). The combined genetic and embryological advantages of the zebrafish are ideal for studying the complex processes of organogenesis, and in particular, the hematopoietic system. Zebrafish have the same primitive and definitive waves of erythropoiesis as higher vertebrates (2). The optical transparency and size of the zebrafish embryos allow for the direct visualization of blood cells using a simple dissecting microscope. The external fertilization and rapid development of the embryos allow the study of early events in hematopoiesis, which are not feasible in mammalian systems. The relatively short generation time to sexual maturity, prolific fecundity, and large brood size allow one to do large-scale classical genetic screens. These forward genetic screens have the distinct advantage of allowing one to assign function to genes, based on the observed phenotype, in an unbiased manner. Genetic screens performed in the zebrafish have uncovered novel genes identified from anemic mutants, and these mutants that have given insights into iron, heme and Fe/S cluster metabolism: ferroportin1 (slc40a1, weißherbst) (3); glutaredoxin5 (glrx5, shiraz) (4); mitoferrin1 (slc25a37, frascati) (5). Transient knockdown technology using antisense morpholino oligomers has allowed investigators to evaluate candidate genes and their loss-of-function phenotype in iron and heme metabolism (6). References: 1. Shafizadeh E, Paw BH. (2004) Curr. Opinion Hematol. 11:255-261. 2. Orkin SH, Zon LI. (2008) Cell 132:631-644. 3. Donovan A, et al. (2000) Nature 403:776-781. 4. Wingert RA, et al. (2005) Nature 436:1035-1039. 5. Shaw GC, et al. (2006) Nature 440:96-100. 6. Nilsson R*, Schultz IJ*, et al. (2009) Cell Metabolism, revision in review.

Barry Paw is currently an assistant professor of medicine at Harvard Medical School, Brigham & Women’s Hospital and Children’s Hospital Boston. He earned an AB in biochemistry from the University of California, Berkeley and an MD and PhD in biological chemistry, both from the University of California Los Angeles School of Medicine. Dr. Paw completed all of his postdoctoral training at the Children’s Hospital Boston, including a residency in pediatrics, a pediatric hematology/oncology clinical fellowship and a postdoctoral fellowship.

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Lecture 12: Iron Metabolism in Mammals Marie-Paule Roth, Toulouse University, France An important breakthrough in understanding the regulation of iron metabolism has been the discovery of hepcidin in 2001. Hepcidin is produced primarily by hepatocytes and secreted into the blood circulation. It interacts with the cellular iron exporter ferroportin, causing its degradation and preventing release of iron from macrophages or intestinal cells into the plasma. The consequent increase in splenic iron and decrease in dietary iron absorption lead to decreased circulating iron levels. As expected, a feedback relationship exists between body iron status and hepcidin expression: hepcidin is upregulated in response to iron loading and decreased in response to iron deficiency. A second breakthrough has been the discovery of the role of the BMP signaling pathway in hepcidin regulation in 2006. BMPs are members of the TGF- superfamily of ligands and initiate an intracellular signaling cascade by binding to two type I and two type II serine-threonine kinase receptors. The activated receptor complex phosphorylates cytoplasmic effectors, the Smad1, 5 and 8 proteins, which form heteromeric complexes with Smad4 and translocate to the nucleus to modulate gene expression. Mutations in the BMP coreceptor, hemojuvelin, are associated with inappropriately low hepcidin expression and massive iron overload in both humans and mice. Suppression of hepatic BMP signaling by a liver-specific conditional knockout of Smad4 has similar consequences. BMP signaling can be inhibited in vivo with soluble hemojuvelin which binds to endogenously secreted BMP ligands and prevents their interaction with cell surface receptors, or with dorsomorphin which blocks the ability of activated BMP type I receptors to phosphorylate Smad1/5/8. The inhibition of BMP signaling reduces hepcidin expression and increases serum iron. Conversely, BMP administration in vivo increases hepcidin expression and reduces serum iron. In vitro, numerous BMP ligands, including BMP2, BMP4, BMP5, BMP6, BMP7 and BMP9, can regulate hepcidin when added exogenously but the observations made in cell culture do not always translate to the situation in vivo. A systemic iron challenge induces hepatic Smad1/5/8 phosphorylation, which indicates that BMP signaling is involved in the feedback regulation of hepcidin transcription by iron. Interestingly, the analysis of the liver transcriptomes of mice kept on a low- or high-iron diet showed that the levels of Bmp6 mRNA, but not that of other Bmps, were concordant with changes in Hamp mRNA concentrations, suggesting that BMP6 could be the endogenous regulator of iron metabolism. Our group and that of J. Babitt recently confirmed this possibility. Indeed, targeted disruption of Bmp6 in mice causes a rapid and massive iron accumulation in the liver, pancreas, heart and kidney. Despite their severe iron overload, these mice have low levels of hepatic Smad1/5/8 phosphorylation and markedly reduced hepcidin synthesis, indicating that BMP6 is critical for iron homeostasis and functionally nonredundant with other members of the BMP subfamily of ligands. The mechanisms that allow the liver to sense iron levels in order to maintain iron homeostasis still need to be clarified. Physiological modulation of hepcidin by circulating holotransferrin not only requires BMP6 and hemojuvelin but also HFE and TfR2. High holotransferrin levels alter the interaction of HFE with TfR1 and favor the formation of a Tf/TfR2/HFE complex that probably facilitates BMP6 signal transduction. Humans and mice with mutated HFE or TfR2 have insufficient hepcidin production, increased intestinal iron absorption, and hepatic iron overload. Noticeably, this nonphysiological iron accumulation in the liver of Hfe-deficient mice leads to a secondary increase in Bmp6 production and an elevation of hepcidin expression to approximately ‘normal’ levels. Modulation of BMP6 levels by iron could thus be important in pathological situations like genetic hemochromatosis.

Marie-Paule Roth has been the director of research for the National Institute of Health and Medical Research (Inserm), France, since 1996. She is leader of the group “Abnormalities of iron homeostasis: functional genomics and physiopathology” at the Centre of Physiopathology of Toulouse Purpan (CPTP), and co-director of the Department “Genetics: Human Diseases and Animal Models” (Inserm Research Unit 563, Toulouse). Dr. Roth completed her MD at Strasbourg Medical School in 1981, board certification in medical genetics. She then received an MSc in anthropology and genetic epidemiology from the University Paris VII in 1983. She was a postdoctoral fellow, Department of Medical Genetics, UCLA School of Medicine, Los Angeles, 1986-1988 and HDR (accreditation to supervise research), University of Toulouse, 1993.

Imaging the Distribution of Iron-Ferritin-Hemosiderin at the Subcellular Level Carmen Quintana, Instituto Microelectronica de Madrid, Spain Ferritin (Ft) and hemosiderin (Hm) are the main proteins that store the excess of iron in the human organism. Ft, a spherical protein, can store until 5000 atoms of iron as hydrated Fe 3+ iron oxide nanocrystals in their internal cavity (the core). Hemosiderin, considered to be a partial proteolytic product of Ft is principally present in pathological iron overload (hemochromatosis and siderosis). These proteins can be identified by histochemical and biochemical approaches. Classical physical techniques for metals studies can be also used for iron identification in Ft and Hm. Isolated Ft molecules and Hm clusters can be directly and specifically visualized at subcellular level in unstained or lightly stained TEM preparations. Chemical microanalysis can be performed using X-ray and EELS associated to TEM and by secondary ion mass spectrometry (SIMS). X-ray and EELS methods allow the identification of the compositional elements of the nanocrystals; oxygen and iron in the case of Ft and Hm cores. EELS also permit us to know the valence state of the iron oxide that composes the Ft and Hm cores. Using Analytical TEM methods as Electron Nanodiffraction (END) or High resolution TEM (HRTEM) the mineral phase of physiological and pathological Ft individual cores can be identified and compared. 2D-chemical mapping, obtained using STEM/EDX, EFTEM/ESI or the latest generation of SIMS instruments works in scanning mode, allows obtaining the distribution of Iron-Ferritin-Hemosiderin at Subcellular Level. The instrumental progress of modern high-resolution electron microscopes has led to development of 3D-chemical mapping associating electron tomography and chemical information (EFTEM tomography). Consequently, 3D mapping of distribution of Iron-Ferritin- Hemosiderin at Subcellular Level is now possible.

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Carmen Quintana started her scientific career with postgraduate degrees in the Physical Sciences and Biophysics (Madrid, 1973, Paris, 1975). Through positions at the Medical Faculty of Creteil et Bobigny (France), INSERM-France and the CSIC-Spain she has made several significant contributions in bringing to two fields closer. In addition, she participates in “CNRS-formation permanent” (France) and postgraduate teaching at the Complutense University in Madrid. Using Analytical TEM methods as Electron Nanodiffraction (END) or High resolution TEM (HRTEM) she and her group have identified the mineral phase of physiological and pathological Ferritin individual cores. The distinguishing feature of Quintana’s contribution is her pioneering work across Physics and Biology, being among the first physicists to apply physics to resolve biological questions.

Spacial Coordination of Cell-Adhesion Molecules and Redox Cycling of Iron in Pulmonary Blast Injury Nikolai Gorbunov, Silver Spring, Maryland, USA The inflammatory response to lung trauma is characterized by the emigration of blood-borne phagocytic leukocytes (PLC), across the microvascular wall into the alveolar lumen of injured lung. This event requires a conversion of the microvascular endothelial cells (EC) to the pro-inflammatory phenotypes and spatiotemporal interplay of different types of cell adhesion molecules (CAM) on PLC and endothelium. The current report is focused on iron-dependent signaling mechanisms mediating spatial coordination of lung CAM in models simulating blast-induced pulmonary trauma. Impact of blast shock wave (SW) in animals was produced using experimental shock- tube described previously [Gorbunov et al., Exp Mol Pathol. 80(1):11, 2006]. Impact of SW caused instant rupture of alveolar microvessels and blood extravasation. The following activation of pulmonary redox cycling of iron was assessed with electron spin resonance spectroscopy and histochemical techniques. Pro-inflammatory remodeling in vascular endothelium and increase in PLC-EC interaction in the injured lung was investigated with immunofluorescence confocal imaging and biochemical techniques. Effect of catalytically reactive iron in the injured lung was simulated by endotracheal administration of iron complexes [Gorbunov et al., Antioxid Redox Signal. 9(4):483, 2007]. The gathered data demonstrated that the presence of catalytically-active iron and induction of the oxidative stress in the lung injury models were accompanied by (1) down-regulation of VE-cadherin adhesion junction, (2) up-regulation of endothelial intercellular adhesion molecules (ICAM-1) and the PLC integrins, and (3) endothelial cell polarization, formation of cuplike rafts abundant with pro-inflammatory ICAM-1, and increase in vascular permeability. The observed vascular endothelial response to catalytically reactive iron in lung was simulated in a culture of rat pulmonary artery endothelial cells (RPAECs). Cellular iron influx and cell shape profile were monitored with time-lapse imaging techniques. Iron-induced oxidative stress was estimated by depletion of cellular glutathione and by response of redox-sensitive transcriptional factor Nrf2 (NF-E2-related factor 2). Spatial rearrangement of pro- inflammatory ICAM-1 and cell structural protein F-actin and caveolin-1 was assessed with confocal immunofluorescence imaging techniques. For assessment of alterations of RPAEC barrier integrity we determined relative electrical resistance of endothelial monolayer (REREM) using ENDOHM-12 chamber and EVOM Voltohmmeter. The iron-induced alterations in RPAECs were characterized by cell polarization and formation of membrane cuplike and microvilli-like projections abundant with ICAM-1, caveolin-1, and F-actin. The iron-induced re-arrangements in cytoskeleton, alterations in focal cell-cell interactions, and cell buckling were accompanied by decrease in REREM. The iron-induced effects observed in in vivo and in vitro experiments were partially eliminated in the presence of N,N'-bis (2- hydroxybenzyl) ethylenediamine-N,N'-diacetic acid, an iron chelator. We suggest that iron-induced structural alterations in endothelial cells occur in redox-dependent manner and can contribute to vascular inflammation. Based on gathered data we suggest that deposition of hemoglobin and activation of iron cycling in lung hemorrhagic lesions can extend inflammation phase in pulmonary trauma and can cause abscopal (e.g., distant bystander) vascular effects.

Dr. Nikolai V. Gorbunov is currently Research Associate Professor with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (Bethesda, Maryland). Dr. Gorbunov have earned his PhD degree from the Russian Academy Sciences in St. Petersburg, Russia. He then completed a two-year postdoctoral training in Biomedicine at the Research Center of the Consorzio “Mario Negri” Sud (Italy) and a two-year DOE postdoctoral training program in Radiation Sciences at the University of Pittsburgh, Graduate School of Public Health. Pittsburgh, Pennsylvania. In 1994 and 2000 Dr. Gorbunov was a recipient of the National Research Council Fellowship Awards to pursue the NRC Senior Research Associateship at the Walter Reed Army Institute of Research (Silver Spring, Maryland). Working on models of blast-related injury he and co-authors have introduced a mechanism of the inflammatory response to shock wave induced pulmonary trauma and described the role of iron and cell redox signaling in the related pulmonary microvascular remodeling. In the recently published book “Explosions and Blast Related Injuries” Dr. Gorbunov and Dr. Elsayed have proposed the redox metabolic machinery balanced by antioxidants as an “interface module” which mediates systemic response to the blast-induced oxidative stress.

Shuttling Iron Between Cellular Organelles Ioav Cabantchik, Hebrew University of Jerusalem, Israel Cells maintain steady pools of labile iron (LIP) in their various organelles. The labile iron in these pools is characterized by its redox activity (II <-> III convertibility), its propensity to generate noxious radicals when challenged with pro-oxidants and by its amenability to chelation with membrane permeant agents that can gain access to the various cell compartments. However, for analytical biochemists, tracing LIP levels and its dynamics in living systems has traditionally posed a technical challenge and therefore has limited its potential role in health and disease. The advent of fluorescence-responsive metal-sensors (FMS) and redox-sensors (RS) and their coupling to cell targeting devices have opened the road for transmitting real-time information about iron localization and dynamics in biological compartments (reviewed in 1). The information gained with FMS and RS has revealed that the intracellular LIPs are interrelated, chemically (by oxidation-reduction) depending on the reductive capacity of a given compartment and topographically, in that changes in a given cell compartment are often accompanied by a concomitant change in another. Thus for example, cellular import of iron from transferrin or organic salts into non-erythroid cells leads to a sequential change in the cytosolic and mitochondrial LIPs or vice versa when 58 chelators act at the mitochondrial level (2). In iron overload disorders there is a pathological rise in labile plasma iron that increases the cellular LIPs in both cytosol and mitochondria. As LIPs might attain clinically toxic levels in metabolic, cardiac, skin and neurological diseases, they have been evaluated with FMS as diagnostic parameters (3) and as therapeutic targets of chelators, namely by agents that are classically used for reducing systemic iron burden. However, in some pathophysiological conditions, a change in the expression of particular genes (frataxin, abcb7, mitochondrial ferritin or ala-synthase) or a pharmacological treatment (blockage of heme synthesis) that cause accumulation of iron in mitochondria, lead to a concomitant iron depletion in the cytosol or vice versa. In those cases of misdistribution of iron, siderophoric chelators have proven useful as vehicles for redistributing iron from sites of accumulation to sites of deprivation (4) and thereby correcting an aberrant the phenotype (5). Such results strengthen the rationale for treating diseases of iron miesdistibution as NBIA and ACD (6) with siderophores . Supported in part by the Israel Science Foundation (ISF) and by the EEC F6 (LSHM-CT-2006-037296 Euroiron1). 1. Breuer, W., et al (2008) Intracellular labile iron. Int J Biochem Cell Biol. 40: 350-4; 2. Shvartsman M et al. (2007). Iron accesses mitochondria from a cytosolic pool of non-labile iron. Am J Physiol Physiol 293: C1383-94; 3. Sung, Y.S. et al. (2008) Redistribution of accumulated cell iron. A modality of chelation with therapeutic implications. Blood 111:1690-9.; 4. Kakhlon, O. et al. (2009). Cell functions impaired by frataxin deficiency are restored by iron relocation. Blood. 112:5219-27; 5. Prus E, Fibach E.(2008) Flow cytometry measurement of labile iron pools in human hematopoietic cells. Cytometry 73:22-27; 6. Breuer W. and Cabantchik, Z.I. (2009).Disorders affecting iron distribution. BloodMed http://www.bloodmed.com/800000/mini-reviews1.asp?id=253&p=1&v=1

Ioav Cabantchik is Professor of Biochemistry and Biophysics and the Della Pergola Chair in Life Sciences at the Hebrew University of Jerusalem, Israel. He studied undergraduate Chemistry and Biology and graduate Biochemistry at the Hebrew University of Jerusalem (Israel) (1961–1966), and an MD-PhD program in Medical Biophysics at the University of Rochester School of Medicine, NY, USA, (1970–1974). He later trained as Research Fellow at the Medical Research Institute of the Hospital for Sick Children (Toronto, Canada) (1975–1976) and worked on membrane physiology and biophysics at the National Institute of Heath (Laboratory of Kidney and Electrolyte Metabolism, NHLBI) as a visiting scientist (1976–1978) and as CF-NIH Scholar (1987). In 1979, Ioav Cabantchik joined the Hebrew University of Jerusalem Institute of Life Sciences as a senior lecturer. He was promoted to Associate Professor of Biochemistry and Biophysics in 1984 and was appointed as Full Professor in 1987. He holds the Adelina and Massimo DellaPergola Chair in Life Sciences Professor Cabantchik has served on the editorial board of several scientific journals as an expert in the area of membrane transport research. His main scientific research, in the area of ion transport mechanisms in healthy and diseased tissues, led to the identification, isolation and reconstitution of the first mammalian membrane transporter — the anion exchange protein (band 3) of red blood cells — and to the description of its physiological role in the removal of CO2 from tissue to the lungs. He pursued research in malaria, choosing the unique features of haemoglobin handling by parasites as a therapeutic target for novel anti-malarial drugs. In the last 10 years, he has focused on novel approaches for tracing the status (levels and dynamics) of labile iron in biological samples (living cells and fluids), its involvement in iron overload of organs (as seen in Thalassaemia and, haemochromatosis) and in recent years in neurodegenerative disorders such as Friedreich’s ataxia, and the promotion of oxidative stress and its susceptibility to iron chelators in preclinical and clinical use. His research was supported over the years by NIH, WHO, AID, ISF, US-Israel BSF, EEC, AFM and AFIRN and by pharmaceutical companies such as Aferrix, Novartis and Apotex.

Drug Targeting to the Brain Using the Transferrin Receptor Ruben Boado, UCLA, USA The progress in the development of biotherapeutics and non-viral gene therapies for the treatment of disorders of the central nervous system is impaired by the presence of the blood-brain barrier (BBB), which is only permeable to lipophilic molecules of <400 Da. Neuronal growth factors, i.e. BDNF and GDNF, are potential neuroprotective agents for the treatment of stroke, Parkinson's and Alzheimer's disease, respectively. However, these proteins do not cross the BBB, and they have poor brain penetration when injected directly into the brain parenchyma. However, it is possible to reengineer proteins to cross the BBB. Monoclonal antibodies (MAb) directed to exofacial epitopes of endogenous BBB transporters, like transferrin receptor (TfR) or insulin receptor (IR), are used as Molecular Trojan horses to carry peptides and proteins of pharmacological interest into the brain via receptor-mediated transcytosis. Conjugates of anti-TfR MAb and BDNF or FGF2 were shown to be effective neuroprotectants in various experimental models of stroke and ischemia. More recently, conjugates of anti-TfR and streptavidin were able to deliver biotinylated-siRNA to brain tumors in vivo and to produce silencing of a target transcript within the brain tumor. The potential of this technology is also extended to enzymes (i.e. iduronidase for Hurler's disease), to bispecific antibodies and to pegylated immunoliposomes carrying plasmid DNA. The latter has been reduced to practice in gene therapy and RNAi protocols, including experimental models of Parkinson’s disease and brain tumors. The recent development in this area will be discussed.

Ruben J. Boado is Professor of Medicine and member of the Brain Research Institute at the University of California at Los Angeles (UCLA). He is also Vice-President of ArmaGen Technologies, Inc. of Santa Monica, CA. Dr. Boado received a Ph.D. Degree in Biochemistry from the School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), Argentina in 1982. He completed a postdoctoral fellowship and conducted research as Established Investigator at the UBA Center for Nuclear Medicine. In 1985, Dr. Boado joined the UCLA School of Medicine with a grant from the Fogarty International Center, National Institutes of Health (NIH). He was appointed Visiting Research Endocrinologist in the UCLA Department of Medicine in 1988, Assistant Professor in 1991, Associate Professor in 1997, and Professor of Medicine in 2001. At UCLA, Dr. Boado's research program examined BBB genomics, and the genetic engineering of plasmid DNA for non-viral gene therapy for the brain. At ArmaGen, Dr. Boado leads the molecular biology division, which is responsible for the genetic engineering, expression, and validation of novel recombinant fusion proteins, which cross the human 59 blood-brain barrier (BBB). He is a member of The Society for Neuroscience, and has authored more than 150 scientific journal articles and book chapters in his field. Dr. Boado has served on numerous scientific review committees including the US Department of Energy, the Alzheimer’s Association and the NIH. Dr. Boado is co-inventor of U.S. and European patents related to BBB drug delivery.

Lecture 13: Iron and the Brain Torben Moos, Aalborg University, Denmark This presentation will cover the putative mechanism underlying uptake and transport of iron by the various cell types of the brain, and discuss their involvement for maintaining a sufficient iron homeostasis within the brain microenvironment. A section of the presentation will cover the implications of altered iron levels in the brain for contributing to psychiatric and neurological disease. The demonstration of transferrin receptors on brain capillary endothelial cells (BCECs) more than 20 years ago provided evidence for the accepted view that the first step in blood to brain transport of iron is receptor-mediated endocytosis of transferrin. Subsequent steps are less clear. However, recent investigations provide new evidence on how iron is released from transferrin on the abluminal surface of BCECs, including the role of astrocytes in this process, how iron is transported in brain extracellular fluid, and how iron is taken up by neurons and glia. We propose that the divalent metal transporter 1 (DMT1) is not involved in iron transport through the BCECs. Instead, iron is probably released from transferrin on the abluminal surface of these cells by the action of citrate and ATP that are released by astrocytes, which form a very close relationship with BCECs. Complexes of iron with citrate and ATP can then circulate in brain extracellular fluid and may be taken up in these low-molecular weight forms by all types of brain cells. Some iron most likely also circulates bound to transferrin, since neurons contain both transferrin receptors and DMT1 and can take up transferrin-bound iron. The most likely source for transferrin in the brain interstitium derives from diffusion from the ventricles. Neurons express the iron exporting carrier, ferroportin, which probably allows them to excrete unneeded iron. The process of iron export mainly occurs where neurons are devoid of myelin. Astrocytes lack transferrin receptors and do not contain ferroportin at detectable levels. Their source of iron is probably that released from transferrin on the abluminal surface of BCECs. The astrocytes are further thought to export iron by a mechanism involving a membrane-bound form of the ferroxidase, ceruloplasmin. Oligodendrocytes also lack transferrin receptors, both they do contain ferroportin; they probably take up non- transferrin bound iron that gets incorporated in newly synthesized transferrin, which in turn may play an important role for intracellular iron transport.

Lecture 14: Iron-Sulfur Cluster Biogenesis and Human Disease Tracey Rouault, National Institute of Neurological Disorders and Stroke, Bethesda, USA Iron sulfur clusters prosthetic groups are crucial to mitochondrial respiratory chain function, where they facilitate electron transfer. There are twelve iron-sulfur clusters in mammalian respiratory chain complexes, with the majority found in complex I. Iron-sulfur cluster assembly is a complex process that involves cysteine desulfurase activity to provide sulfur, chaperone proteins, scaffolds, and reductases. In mammalian cells, a cytosolic protein important in iron homeostasis, iron regulatory protein 1 (IRP2), contains a [4Fe-4S] cluster, whereas a related protein, IRP2 is not regulated by iron-sulfur cluster status. To understand how iron-sulfur clusters are synthesized in mammalian cells, we have cloned and characterized many of the assembly proteins. We find that the cysteine desulfurase, IscS, and the primary scaffold, IscU, encode both mitochondrial and cytosolic isoforms through alternative splicing and start codon utilization. Interestingly, when mitochondrial iron-sulfur cluster assembly is disrupted, mitochondria develop iron overload. We suggest that mitochondrial iron overload results from a regulatory response to disruption of mitochondrial iron-sulfur cluster assembly, which cells interpret as mitochondrial iron depletion. Abnormal iron-sulfur cluster assembly is important in the human disease, Friedreich ataxia, and we have recently shown that defective splicing of one of the iron-sulfur cluster assembly proteins, ISCU, causes an inherited human muscle disease. In addition, treatment of IRP2-/- animals with the stable nitroxide, Tempol, prevents neurodegenerative disease by disassembling the iron-sulfur cluster of IRP1 and restoring normal iron homeostasis in the central nervous system. References: Li K, Besse EK, Ha D, Kovtunovych G, Rouault TA (2008) Iron-dependent regulation of frataxin expression: implications for treatment of Friedreich ataxia. Hum Mol Genet 17:2265-73. Mochel F, Knight MA, Tong WH, Hernandez D, Ayyad K, Taivassalo T, Andersen PM, Singleton A, Rouault TA, Fischbeck KH, Haller RG (2008) Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance. Am J Hum Genet 82:652-60. Rouault TA, Tong WH (2008) Iron-sulfur cluster biogenesis and human disease. Trends Genet 24:398-407. Ghosh, M. C., Tong, W. H., Zhang, D., Ollivierre-Wilson, H., Singh, A., Krishna, M. C., Mitchell, J. B., and Rouault, T. A. (2008). Tempol-mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice. Proc Natl Acad Sci U S A. 105, 12028-12033.

Tracey Rouault graduated from Yale College and obtained her M.D. at Duke University. Following residency in internal medicine, she completed a genetics research fellowship at the National Institutes of Health in Bethesda, Maryland. There, she worked to understand regulation of mammalian iron metabolism, cloning iron regulatory proteins 1 and 2 (IRPs), and characterizing their roles in physiology using mouse targeted gene deletion studies. Her work led to recognition of the role of an iron-sulfur cluster in regulation of IRP1 and to the dominant role played by IRP2 in regulation of cytosolic iron metabolism in most tissues. Studies on mammalian iron-sulfur cluster biogenesis have revealed that iron-sulfur cluster biogenesis occurs not only in mitochondria, but also in the cytosolic/nuclear compartment of mammalian cells. Several human diseases result from mutations in proteins involved in iron-sulfur cluster biogenesis, and the pathophysiology of these diseases likely results not only from impaired function of iron-sulfur proteins, but also impaired regulation of mitochondrial iron homeostasis. Recent work reveals that adult-onset neurodegenerative disease in IRP2-/- mice can be prevented with a pharmacologic reagent that destabilizes the iron-sulfur cluster of latent IRP1, thereby converting IRP1 to an active IRE- 60 binding form that compensates for loss of IRP2. Elucidation of the molecular basis of mouse and human diseases opens the way to discovery of new targeted treatment strategies in a phenotypically disparate group of diseases caused by misregulation of cytosolic or mitochondrial iron homeostasis.

Lecture 15: Hemochromatosis – Screening and Penetrance Katie Allen, University of Melbourne, Australia The recent completion of the Human Genome Project has offered great promise that medical genetics will have a population-based impact on the prevention and treatment of genetically inherited conditions. Hereditary haemochromatosis (HH), was initially touted as a “poster child” for population genetic screening (1,2,3). Most cases of hereditary haemochromatosis are due to homozygosity for a single mutation leading to iron overload and it is considered an ideal candidate for population genetic screening because genetic susceptibility is common, testing is inexpensive, and iron studies can detect early stages of disease (4). Most importantly, venesection is a simple and effective way to both prevent and manage the potential sequelae of iron overload (5). However even for a genetically inherited condition as apparently straightforward as HH, justification for the implementation of population screening have proven more complicated than initially expected. Population estimates of the prevalence of both non-specific signs and symptoms of HH (such as arthritis and fatigue) and disease due to documented iron overload (such as cirrhosis) in C282Y homozygotes have been hindered by either the failure to clinically assess individuals prior to knowledge of their genetic status or by an inability to account for the long lead time of preclinical iron overload status. In the largest longitudinal prospective study to date, we assessed 203 homozygotes amongst a healthy population of 31,192 followed over 12 years (6). We assessed the proportion of C282Y homozygotes who had disease that could be directly attributable to iron overload (iron-overload-related disease) using the combined definition of documented iron-overload (as defined by Whitlock et al (7)) and objective measures of disease with physicians and participants blinded to genotype. We found evidence of iron overload- related disease in 28% of male but only 1% of female C282Y homozygotes. These results clearly have implications for a cost-effective analysis for genetic-based population screening for HH. Other criticisms leveled at population screening for HH (8,9) including concerns over insurance implications and creating a cohort of “worried well’ amongst those at genetic risk of HH have proved unfounded (10,11) and it appears that cost remains the last barrier to screening. The questions that now remain with regards to population screening for HH include whether it would be more cost-effective to simply offer screening to men, what age is the most cost-effective to screen and what would be the most pragmatic way to access a population prior to an age when disease is likely to develop. Hereditary haemochromotosis may yet offer a prototype for population- based genetic screening programs but the journey of justification has offered unexpected challenges. References 1. Allen K, Williamson R. Screening for hereditary haemochromatosis should be implemented now. BMJ 2000: 320: 183-184. 2. Stuhrmann M, Graf N, Dörk T, Schmidtke J. Mutation screening for prenatal and presymptomatic diagnosis: cystic fibrosis and haemochromatosis. Eur J Pediatr. 2000 Dec;159 Suppl 3:S186-91. 3. Yapp TR, Eijkelkamp EJ, Powell LW. Population screening for HFE-associated haemochromatosis: should we have to pay for our genes? Int Med J 2001: 31:48-52. 4. Allen KJ, Williamson R. Should we genetically test everyone for haemochromatosis? J Med Ethics 1999: 25: 209-214. 5. Niederau C, Fischer R, Purschel A, Stremmel W, Haussinger D, Strohmeyer G. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996;110(4):1107-19. 6. Allen KJ, Gurrin LC, Osborne NJ, et al. Iron-overload-related disease in HFE hereditary hemochromatosis. New England Journal of Medicine 2008;358:221-30. 7. Whitlock EP, Garlitz BA, Harris EL, Beil TL, Smith PR. Screening for hereditary hemochromatosis: a systematic review for the U.S. Preventive Services Task Force. Ann Intern Med 2006;145(3):209-23. 8. Burke W, Thomson E, Khoury MJ et al. Hereditary hemochromatosis: gene discovery and its implications for population- based screening. JAMA 1998: 280: 172-178. 9. Haddow JE, Bradley LA. Hereditary haemochromatosis: to screen or not. Conditions for screening are not yet fulfilled. BMJ 1999: 319: 531-532. 10. Delatycki MB, Allen KJ, Nisselle AE, et al. Use of community genetic screening to prevent HFE-associated hereditary haemochromatosis. Lancet 2005;366(9482):314-6. 11. Delatycki M, Allen K, Williamson R. Insurance agreement to facilitate genetic testing. Lancet 2002: 359: 1433.

A/Prof Katrina (Katie) Allen, MBBS (Hons), B Med Sc (Hons), FRACP, PhD is a Pediatric Gastroenterologist and Allergist with research interests in common, preventable gastrointestinal diseases that present a significant public health burden. Her clinical interests include childhood food allergy and haemochromatosis. Prof Allen instigated and was co-prinicipal investigator of HaemScreen, a population genetic screening study of hereditary haemochromatosis. Prof Allen is also co-principal investigator for an NHMRC and NIH-funded study (HealthIron) that is the largest and most comprehensive longitudinal study of disease penetrance of hereditary haemochromatosis.

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Lecture 16: Hereditary Hemochromatosis as a Conformational Disorder Matthew W. Lawless, Trinity College Dublin, Ireland

Hereditary hemochroamtosis (HH) refers to a unique clinicopathologic subset of iron overload syndromes that includes the disorder related to C282Y homozygous mutation of the hemochromatosis protein (HFE), the most common form of hereditary hemochromatosis. Individuals homozygous for the mutant C282Y HFE associated allele are at risk of developing liver disease, diabetes and arthritis. HFE C282Y is a mutant protein that does not fold correctly forming aggregates and is retained in the Endoplasmic Reticulum (ER). Consequently, HH associated with the C282Y HFE mutation has been proposed to be included in the medical grouping of disorders known as conformational disorders. Conformational diseases are a class of disorders associated with the expression of misfolded protein and examples include conditions such as Alzheimer's, Parkinson's, Z alpha 1-antitrypsin deficiency and Huntington's diseases. In conformational disorders, accumulation of unfolded or misfolded proteins in the ER can activate a complex cascade linked to the regulation of diverse physiologic processes, variable disease onset and progression. Recent reports have highlighted C282Y HFE analogies with this class of disorders, whereby HFE C282Y mutant protein forms aggregates and is subsequently retained in the ER. To- Date, investigations of HFE C282Y HH has been largely dealt with the end-stage consequence of this disorder (iron overload). This plenary lecture focuses on mechanisms of cell injury in diseases associated with aggregation-prone proteins, pathways by which cells respond to protein aggregation and mislocatization, and relating these specific events to HFE C282Y HH. Viewing HFE C282Y HH in the context of the conformational disorders may give rise to novel therapeutic strategies and greater insights into the onset and progression of this disorder.

Matthew Lawless graduated from NUI Maynooth with a degree in Biology and obtained a PhD in Medicine from the Department of Medicine, Royal College of Surgeons in Ireland in 2004, with Professor Gerry McElvaney. He was awarded the Sheppard’s prize research medal, from the Royal College of Surgeons hospital and a Health Research Board Ireland project grant in 2004. During this time he was involved with a large-scale clinical trial with the Alpha One foundation and University of Florida. He was appointed as senior postdoctoral researcher and lecturer in liver immunology at the Institute of Molecular Medicine at Trinity College Dublin and St. James Hospital, with Professor Suzanne Norris. He developed his own research group focused on the regulation of endoplasmic reticulum signalling and epigenetics in liver disease. In 2008, Dr. Lawless was appointed to the Mater Misericordiae Hospital and University College Dublin School of Medicine with Professor John Crowe, and has expanded these research areas into several chronic liver diseases and altered pathogenic immune states in patients. The group at the centre for liver disease investigates the molecular basis and the immunology behind chronic liver disease. His principal achievements to date include the discovery that the compound Tauroursodeoxycholic acid may be used to treat the childhood liver disease in Z A1AT patients and suggesting for the first time hereditary hemochromatosis should be consider a Conformational Disorder. Dr. Lawless’s academic activities include the supervision and co-supervised of several students to competition of MSc, MD and PhD degree. He has published in a number of high impact journals including Hepatology, Journal of Immunology and the Journal of Cellular and Molecular Medicine. He serves as an invited reviewer for several international journals including Cytokine and the British Journal of Haematology, and his editor of his own forthcoming book. He has presented his research findings both nationally and internationally at several diverse internationally well-recognised conferences.

Iron as Engine of Basic Biological Processes and Disease: The Future of BioIron Nancy Andrews, Duke University, USA Over the past 15 years, our understanding of iron biology has increased enormously. Advances have come from parallel and complementary studies in unicellular organisms, cultured cells, animal models and human patients. In this talk I will focus on what we have learned about mammalian iron homeostasis, its perturbations in disease, and what new directions should be explored in the next decade. We now know that genetic disorders of iron homeostasis result from dysregulated production of a key iron regulatory hormone, hepcidin, or Hepcidin mutation of its physiological target, the iron transporter ferroportin. producing Hepcidin expression is meticulously controlled in response to body iron adenoma needs and available stores. Under normal circumstances, hepcidin IRIDA expression increases as the body’s iron endowment increases, creating a inflammation TMPRSS6 se regulatory mechanism that controls both dietary iron absorption and iron pon es l r recycling. Disorders of iron balance result from perturbations that change a hemochromatosis rm the “set point” for hepcidin expression (Figure). no hepcidin Genes have been identified to account for most or all iron disorders that HFE, TFR2, FPN show Mendelian inheritance. Over the coming years, there will likely be increased focus on how mammalian iron homeostasis interacts with other Juvenile hemochromatosis normal and pathologic homeostatic processes, including metabolism, host HAMP, HJV defense, carcinogenesis, metastasis, neurodegeneration and co-existence with our natural microbiome. iron endowment

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Nancy Andrews is Vice Chancellor for Academic Affairs and Dean of the Duke University School of Medicine. She is also a Professor in the Departments of Pediatrics and Pharmacology & Cancer Biology. Dr. Andrews received her B.S. and M.S. degrees in Molecular Biophysics and Biochemistry from Yale University, her Ph.D. in Biology from MIT and her M.D. from Harvard Medical School. She completed her internship and residency in Pediatrics at Children’s Hospital Boston, and her Hematology/Oncology fellowship at Children’s Hospital and the Dana-Farber Cancer Institute. After her training Dr. Andrews stayed on at Harvard and Children’s Hospital, rising through the academic ranks to become the George Richards Minot Professor of Pediatrics at Harvard, Senior Associate in Medicine at the Children’s Hospital Boston, and a Distinguished Physician of the Dana-Farber Cancer Institute. She served as an attending physician in hematology and oncology at Children’s Hospital until 2003. She was director of the Harvard-M.I.T. M.D.-Ph.D. Program from 1999 to 2003 and Dean for Basic Sciences and Graduate Studies at Harvard Medical School from 2003 to 2007. Dr. Andrews has maintained an active NIH-funded research laboratory studying mouse models of human diseases. She was an investigator of the Howard Hughes Medical Institute at Harvard for 13 years. She has authored over 150 articles and book chapters, and has received many awards and honors for her research, including election to the Institute of Medicine of the National Academies and to the American Academy of Arts and Sciences. She was the 2009 President of the American Society of Clinical Investigation.

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Podium Abstracts

1 IDENTIFYING GENES ESSENTIAL FOR HEME BIOSYNTHESIS THROUGH LARGE-SCALE GENE EXPRESSION SCREENING Iman Schultz, PhD¹, Roland Nilsson, PhD², Eric Pierce³, Kathleen Soltis³, Amorrnrat Naranuntarat, MSc4, Prasad Paradkar, PhD5, Joshua Baughman², Paul Kinsley, PhD6, Valeria Culotta, PhD4, Jerry Kaplan, PhD5, James Palis, MD6, Vamsi Mootha, MD² and Barry Paw, MD, PhD³ ¹Brigham and Women’s Hospital, Harvard Medical School; ²Department of Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ³Department of Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA; 4Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD; 5Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; 6Department of Pediatrics, University of Rochester School of Medicine & Dentistry, Rochester, NY (Presented By: Iman Schultz, PhD)

Heme biosynthesis is central to red blood cell physiology, and defects in this pathway cause hematological disorders. The eight ‘core’ heme biosynthesis enzymes are distributed evenly between the mitochondrial and cytosolic compartments and are well characterized, but additional transporters and regulatory factors are likely present. To discover such missing components, we used large-scale computational screening to identify novel mitochondrial transcripts that co-express tightly with the eight heme biosynthesis enzymes. Our screen predicted SLC25A39, SLC22A4 and TMEM14C as putative mitochondrial transporters and also implicated the iron-sulfur cluster proteins C1orf69 and ISCA1. These genes showed erythropoiesis related regulation in vitro, and were expressed at moderate to high levels in the zebrafish intermediate cell mass, the yolk-sac blood island equivalent in teleosts. Gene-specific knockdowns in zebrafish using antisense morpholinos resulted in profound anemia for all five genes. Normal expression of globin and myeloperoxidase in the morpholino-injected embryos showed that erythroid and myeloid lineage specification remained unaffected, indicating the genes are red blood cell specific and function late in erythroid maturation. A further focus on the mitochondrial solute carrier SLC25A39 showed high expression of this gene in mouse embryonic blood islands, and in the hematopoietic organs, fetal liver and adult bone marrow. Mitochondrial iron flux studies in mouse erythroleukemia cells showed that silencing of Slc25a39 by siRNA did not affect total mitochondrial iron, while silencing of Slc25a37 (Mitoferrin-1)1, greatly reduced such levels. However, Slc25a39 silencing resulted in a significant reduction in heme levels, similar to those obtained for Slc25a37 silencing. Also, zebrafish embryos injected with the slc25a39 specific morpholinos did not show accumulation of heme intermediates, suggesting the gene functions upstream of ALA synthase (ALAS2). Mutants of the yeast SLC25A39 ortholog, MTM1, show mitochondrial iron accumulation and reduced activity of SOD2, phenotypes that were complemented by both zebrafish and mouse SLC25A39. Our data, and unpublished data by Naranuntarat et al2. that shows up-regulation of the iron sulphur cluster scaffold proteins Isu1/2 in yeast mtm1D mutants, strongly suggest SLC25A39 is involved in iron suplhur cluster assembly, and may affect heme synthesis in an analogous way as previously described for GLRX53. Taken together, these results warrant further studies of the genes identified by our large-scale gene expression screen, and may shed light on the nature of hematological disorders. 1. Shaw GC et al. (2006). Mitoferrin is essential for erythroid iron assimilation. Nature 440, 96-100. 2. Naranuntarat A et al. (2009). XXXX, Proc. Natl. Acad. Sci. USA, in review. 3. Wingert RA et al. (2005). Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis. Nature 436, 1035-1039.

2 FPN1B, A NOVEL FERROPORTIN TRANSCRIPT LACKING AN IRON-RESPONSIVE ELEMENT, ENABLES DUODENAL AND ERYTHROID PRECURSOR CELLS TO EVADE TRANSLATIONAL REPRESSION Deliang Zhang, PhD, Robert Hughes, Hayden Ollivierre-Wilson, Manik Ghosh, PhD and Tracey Rouault, MD NICHD, NIH (Presented By: Tracey Rouault, MD)

Background & Aims: Ferroportin (FPN1), the sole characterized mammalian iron exporter, has an iron responsive element (IRE) in the 5’-untranslated region of its transcript, which ensures that its translation is repressed by iron regulatory proteins (IRPs) to maintain cellular iron availability in iron-deficient cells. However, enterocytes differ from other cells because they must be able to export iron even when cells throughout the body are iron-deficient, as occurs in animals on iron-deficient diet. We hypothesized that a non-IRE containing FPN1 isoform might exist in enterocytes that would enable enterocytes to export sufficient iron to satisfy systemic demands, even when enterocytes were iron-depleted. Methods: Using 5’-rapid amplification of cDNA end assay, a new FPN1 isoforms were identified. The regulation, distribution, expression pattern of the new FPN1 isoforms were characterized by luciferase assays, northern blot, real-time PCR and in situ hybridization. The erythroid cells and macrophages of mouse bone marrow were sorted by cell-type specific antibodies. The changes of FPN1 transcripts in the differentiating erythroid cells were investigated in G1E-ER4 cells. Results: A novel FPN1 isoform, FPN1B, which lacked the IRE, was identified. FPN1B contained a new exon located 845bp 5’prime of the first exon of the old isoform (FPN1A), was generated from an alternative promoter, and shared the same open reading frame as FPN1A. Luciferase assays proved that FPN1B expression was not regulated by the IRE/IRP machinery. FPN1B was expressed specifically in duodenum and bone marrow. In duodenum, FPN1B was expressed highly in the enterocytes, and its expression increased in animals maintained on a low-iron diet. In bone marrow, FPN1B was specifically expressed in erythroid precursor cells. The FPN1B transcript, which encodes ferroportin with an identical open reading frame, contributes significantly to total expression of FPN1 protein in erythroid precursors, and likely also in the duodenum of iron-starved animals. The promoter analysis showed that FPN1B promoter was conserved between mouse and human, and GATA and KLF transcriptional elements were essential for its promoter activity. Conclusions and Hypothesis: The identification of FPN1B reveals how FPN1 expression can bypass IRPdependent repression in intestinal iron uptake, even when cells throughout the body are iron-deficient. High expression of FPN1B in erythroid precursor cells probably enhances real-time sensing of systemic iron status and restriction of erythropoiesis in response to low systemic iron. It also provides the possibility that hepcidin, the small peptide produced by liver, could regulate the iron status, and maybe even further, the proliferation and differentiation of erythroid precursor cells.

3 LYSOSOMAL PROTEOLYSIS IS THE PRIMARY DEGRADATION PATHWAY FOR CYTOSOLIC FERRITIN AND IS NECESSARY FOR IRON EXIT FROM FERRITIN Yinghui Zhang, MS², Marc Mikhael, BS³, Yiye Li, PhD4, Shan Soe-Lin, MS³, Bo Ning, MS², Guangjun Nie¹, Yuliang Zhao, PhD² and Prem Ponka, PhD, MD5 ¹National Center for Nanoscience and Technology; ²CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology of China, Beijing 100190; ³Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and Departments of Physiology and Medicine, McGill University, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada; 41CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology of China, Beijing 100190; 5Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and Departments of Physiology and Medicine, McGill University (Presented By: Guangjun Nie)

Ferritin sequesters and stores iron and, consequently, protect cells against iron-mediated free radical damage. However, the mechanism of iron exit from the ferritin nanocage and its reutilization within cells are largely unknown. A recent study showed that ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasomal pathway (De Domenico et al. EMBO J. 25:5396-5404, 2006). Our previous study found that mitochondrial ferritin (MtFt) expression led to a decrease in cytosolic ferritin (Nie et al. Blood 108:2428-2342, 2005). To further examine the mechanism of cytosolic ferritin degradation and iron exit, we studied the mechanism of ferritin degradation under basal cell culture conditions and following the overexpression of mouse MtFt under the control of a tetracycline-responsive promoter. We found that treatment with the lysosomal inhibitors, leupeptin and E-64D, but not the proteasomal inhibitor lactacystin, largely blocked ferritin degradation in H1299 and Huh7 cells under normal cell culture conditions. Moreover, the ferritin in the cells treated with lysosomal inhibitors, leupeptin and E-64D, was found to store more radio-labeled

64 iron in the protein nanocage than the ferritin in the cells without the protease inhibitor treatment. Furthermore, we found lysosomal inhibitors also prevented ferritin degradation in the cells when MtFt expression was induced. The prevention of cytosolic ferritin degradation in the MtFt expressing cells significantly blocked radio-iron mobilization from the protein nanocage induced by MtFt expression. Moreover, the induction of MtFt resulted in the mobilization of iron from other intracellular iron sources, but not from cytosolic ferritin in the presence of lysosomal inhibitors. Our studies also showed that blockage of ferritin degradation by leupeptin resulted in decreased ferritin synthesis and prolonged ferritin stability, indicative of intracellular iron depletion. Lastly, despite evidence suggesting a lack of proteasomal involvement in basal ferritin turnover in log-phase cultured cells, we found proteasomes were responsible for ferritin degradation in prolonged-culture cells and cells pretreated with ferric ammonium citrate. In conclusion, the current studies suggest that ferritin protein degradation precedes the release of stored iron and that basal ferritin turnover may be a necessary step for iron release and utilization. MtFt induced cytosolic ferritin decrease was found to be preventable by lysosomal protease inhibitors, and both lysosomal and proteasomal pathways may be involved in ferritin degradation under different growth conditions. De Domenico, I. D., Vaughn, M. B. et al. (2006). "Ferroportin- mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome." EMBO J. 25(22): 5396-5404. Nie, G., Sheftel, A. D. et al. (2005). "Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis." Blood 105(5): 2161-2167.

4 LIVE IMAGING OF METAL-ION TRANSPORT IN OOCYTES EXPRESSING HUMAN DIVALENT METAL-ION TRANSPORTER-1 (DMT1): SUBSTRATE PROFILE AND SELECTIVITY Bryan Mackenzie, PhD, Anthony C. Illing, Christopher L. Cunningham and Ali Shawki University of Cincinnati College of Medicine (Presented By: Bryan Mackenzie, PhD)

DMT1 is essential for intestinal iron absorption and erythroid iron utilization. A broad range of transition metal ions, including Cd2+, are capable of inducing currents in Xenopus oocytes expressing DMT1; however—like metal-ion inhibition of radiotracer transport—evoked currents demonstrate DMT1 reactivity with these metal ions but do not demonstrate that each is actually transported. We used a fluorescence-based approach for live imaging of metal-ion transport in Xenopus oocytes and provide here a comprehensive substrate-profile analysis for human DMT1. We confirmed the reactivity of the fluorophore PhenGreen SK (PGSK) with a range of metal ions (which cause fluorescence quenching) in a cell-free system. We injected control oocytes and oocytes expressing DMT1 with PGSK and used the confocal laser-scanning microscope (excitation at 514 nm and detection in the band 530-600 nm) to monitor fluorescence changes during 10-min superfusion with metal ions (typically at pH 5.5). We also measured radiotracer metal-ion uptake and metal-ion-evoked currents using the voltage clamp. We took the first-order rate constant of quenching as an index of metal-ion uptake and validated our fluorescence approach by comparing our findings with radiotracer data: (1) The half-maximal concentration (K0.5) for Fe2+ (4.2 ± 2.2 µM, SEE) determined from PGSK quenching at pH 5.5 matched that determined from 55Fe2+ uptake (5.9 ± 0.9 µM); (2) Fe2+-induced PGSK-quenching rates were pH-dependent in the same manner as 55Fe2+ uptake; (3) Cr (in the presence or absence of Lascorbic acid) did not quench PGSK fluorescence, consistent with 51Cr data revealing that Cr is excluded by DMT1. Cd2+, Co2+, Mn2+, Ni2+, and VO2+ resulted in more rapid quenching in DMT1 oocytes than in control oocytes. Sn2+ and VO+ did not induce fluorescence changes. We observed no fluorescence changes when oocytes were superfused with CuCl2 in the presence of L-histidine with or without L-ascorbic acid. In the presence of L-ascorbic acid but in the absence of histidine, 100 mM CuCl2 induced in DMT1 oocytes only delayed fluorescence changes that did not differ from control oocytes and which may be attributable to membrane damage. Our data therefore suggest that Cu is not a DMT1 substrate. Whereas Zn2+ evoked large currents, 65Zn2+ was very poorly transported and Zn2+ only weakly inhibited 55Fe2+ transport (Ki ≈ 50 µM). 55Fe2+ transport was inhibited by Cd2+, Mn2+ and Co2+ in a competitive manner. We established the order of selectivity for transported substrates of DMT1 using the ratio Imax/K0.5 obtained from evoked currents (I): Fe2+, Cd2+ > Co2+, Mn2+ > Ni2+, VO2+, Zn2+ (where “>” ≈ 0.5 log10 units). We conclude that DMT1 is a ferrous iron-preferring transporter that efficiently transports Cd2+ but that Cu is not transported by DMT1. We speculate that DMT1 will serve as a route of entry for the toxic heavy metal Cd — especially in iron deficiency — and contribute to the absorption of Co and Mn (and trace metals Ni and V), but that DMT1 is unlikely to be physiologically relevant to the absorption of Zn or Cu, both of which are served by other transport systems. We expect that our PGSK fluorescence-based metal-ion transport assay will have considerable utility in the live imaging of metalion transport in intact tissues.

5 IMAGING OF IRON ENTRY AND IRON-MEDIATED TOXICITY IN PRIMARY NEURONS AND ASTROCYTES Franca Codazzi, PhD¹, Ilaria Pelizzoni, Dr², Romina Macco, Dr¹, Marco Francesco Morini¹, Ilaria Vitali¹, Federico Maria Rossi¹, Alessandra Consonni, Dr¹, Barbara Bettegazzi, Dr¹, Daniele Zacchetti, PhD4 and Fabio Grohovaz, Professor4 ¹S. Raffele Scientific Institute; 2Italian Institute of Technology (IIT) (Presented By: Franca Codazzi, PhD)

Introduction: Iron is essential for many cellular functions but it is also a key player in oxidative stress. Accordingly, when in excess, it has toxic effects that can contribute to the pathogenesis of several neurodegenerative disorders. Methods: In order to investigate the effects of intracellular iron misregulation in cultured primary neurons and astrocytes, we combined calcium imaging and Total Internal Reflection Microscopy with approaches of “high content cell based screening”. A protocol was set up to concomitantly monitor iron influx, iron oxidative status, ROS formation and cell death at the single cell level. Results: Rapid iron entry into the cell (mediated by an iron ionophore and monitored by the quenching of Fura-2) impaired cellular reducing potential and promoted ROS production (monitored by increase of DCFDA fluorescence), ultimately leading to cell death (monitored by the loss of the cytosolic dye and the entry of a dead-cell indicator). Cell death was preceded by the oxidation of Fe2+ to Fe3+, which occurred during the Fenton reaction and was revealed by the de-quenching of Fura-2. Neuronal and astrocytic death was speeded up by glutathione depletion. Moreover, the analysis of mitochondrial membrane potential and the assessment of mitochondrial integrity (monitored by fluorescent probes), along with the use of mitochondrial scavenger molecules, point to a crucial role of these organelles in iron-mediated cell death. In a parallel line of experiments, we found that the process known as “astrocyte activation” (induced in vitro by treatment with pro-inflammatory cytokines) was able to protect cultured astrocytes from the effects of iron-induced oxidative stress. We are currently investigating at the molecular level the differences between resting and “activated” astrocytes in terms of their capability to detoxify iron and ROS. Finally, we have studied the possible iron influx pathways in neurons, by evaluating the role of the Divalent Metal Transporter 1 (DMT1), as well as other potential routes such as Ca2+ channels and NMDA receptors. In this respect, we are also investigating how calcium elevations (upon depolarization or NMDA receptor stimulation) influence iron entry thereby ensuing neuronal toxicity. Discussion and Conclusions: Acute iron over-load promoted the death of cortical astrocytes, as well as hippocampal primary neurons. In contrast, hippocampal astrocytes appeared less vulnerable to iron-mediated oxidative stress, unless depleted of glutathione. However, even cortical astrocytes, after activation, appeared to be protected from the effects of iron. Further analysis is in progress to characterize this mechanism of acquired protection as well as its possible implication in neuroprotection under oxidative stress conditions. We propose that routes other than DMT1 can be involved in iron entry and that a strict interplay between iron and calcium control might play a central role in neurodegenerative processes.

6 CHARACTERIZATION OF HOLO-TF-REGULATED TFR2 TRAFFICKING Juxing Chen, PhD, Jinzhi Wang, MS, Kathrin Meyers, BS and Caroline Enns, PhD Oregon Health & Science University, Portland, OR (Presented By: Juxing Chen, PhD)

Transferrin receptor 2 (TfR2) is a homolog of TfR1 but has distinct functions from TfR1 in iron homeostasis. In contrast to TfR1, the trafficking of TfR2 is largely unexplored. Previous studies have shown that TfR2 is degraded in the lysosome, and that holo-Tf stabilizes TfR2 and increases its half-life by redirecting it from a degradative pathway to a recycling pathway. In this study, we characterized the endocytosis, recycling and degradation of TfR2. Since TfR2 and TfR1 have similar endocytic motifs (YXXØ), we tested whether TfR2 internalization was also mediated by AP-2. We found that knockdown of m2 subunit of AP-2 blocked internalization of TfR2 by examining surface TfR2 level and holo-Tf-488 uptake using flow cytometry analysis and 125I-holo-Tf uptake. Thus like TfR1, TfR2 endocytosis was also AP-2- 65 dependent. This prompted us to investigate whether TfR1 and TfR2 internalize through similar or different pathways. Flow cytometry analysis showed that TfR1 and TfR2 competed for endocytosis only in the presence of holo-Tf, indicating that holo-Tf redirects TfR2 to internalize through the same pathway as TfR1. TfR2 but not TfR1 interacted with PACS-1 through the acidic cluster motif in the cytoplasmic domain of TfR2. PACS-1 is a sorting protein implicated in the trafficking of proteins containing an acidic cluster sorting motif between endosomes and the TGN and between endosomes and the plasma membrane. Knockdown of PACS-1 resulted in less TfR2 on the cell surface by flow cytometry analysis, but did not alter degradation and internalization of TfR2. These results indicate that knockdown of PACS-1 slowed TfR2 recycling and that TfR2 recycled to the cell surface by interacting with PACS-1. Finally, we examined the mechanism by which TfR is degraded. Many membrane proteins are degraded by a ubiquitin-mediated TSG101 pathway whereby the protein is targeted to multivesicular bodies that eventually fuse with lysosomes. Depletion of TSG101 by siRNA blocked degradation of TfR2, suggesting that TfR2 degradation occurs through a multivesicular body (MVB) pathway. TfR2 possesses a single lysine residue (K31) in its cytoplasmic domain. Degradation of K31A-TfR2 was similar to wild type TfR2, demonstrating that TfR2 degradation is not mediated through ubiquitination on lysine 31. Using HA-ubiquitin no detectable ubiquitination of TfR2 could be detected. Therefore, TfR2 degradation is either mediated through a ubiquitination-independent but TSG101-mediated MVB pathway or by interaction with a ubiquitinated protein. This work was supported by National Institute of Health Grants RO1-DK072166 (to C.A.E.). J.C. was partially supported by National Institutes of Health Grant T32 HD049309.

7 ABCB10 INTERACTS WITH MITOFERRIN-1 (SLC25A37) TO ENHANCE ITS PROTEIN STABILITY AND FUNCTION TO IMPORT MITOCHONDRIAL IRON IN ERYTHROBLASTS Wen Chen, PhD¹, Prasad Paradkar, PhD², Liangtao Li, MD², Nathaniel Langer, BA¹, Eric Pierce, BA¹, Iman Schultz, PhD¹, Brigham Hyde, PhD³, Orian Shirihai, MD, PhD³, Jerry Kaplan, PhD² and Barry Paw, MD, PhD¹ ¹Harvard Medical School; ²University of Utah; ³Boston University (Presented By: Barry Paw, MD, PhD)

Mitoferrin-1 (Mfrn1, Slc25a37), a member of the solute carrier family located in the mitochondrial inner membrane, functions as an essential iron-importer for the synthesis of mitochondrial heme and iron-sulfur clusters in erythroblasts1. Mfrn1 mRNA is expressed highly in hematopoietic organs of developing zebrafish and mouse. The biochemistry of Mfrn1-mediated iron transport into the mitochondria, however, is poorly understood. Here, we employed the strategy of in vivo epitope-tagging affinity purification and mass spectrometry to investigate Mfrn1-mediated mitochondrial iron homeostasis. Abcb10, an inner mitochondrial ATP-binding cassette transporter highly induced during erythroid maturation in hematopoietic tissues2, was found as one key protein that physically interacts with Mfrn1 during mouse erythroleukemia (MEL) cell differentiation. Mfrn1 was previously shown to have a longer protein half-life in differentiated MEL cells compared to undifferentiated cells3. In this study, Abcb10 was found to enhance the stabilization of Mfrn1 protein in MEL cells and transfected heterologous COS7 cells. In undifferentiated MEL cells, co-transfected Abcb10 specifically interacts with Mfrn1 to enhance its protein stability and promote Mfrn1-dependent mitochondrial iron importation. The structural stabilization of the Mfrn1:Abcb10 complex is independent of the transport activity of Abcb10, thereby demonstrating a novel function for Abcb10 in the mitochondria. These results suggest the tight regulation of mitochondrial iron acquisition and heme synthesis in erythroblasts is mediated by both transcriptional and post-translational mechanisms, whereby high levels of Mfrn1 is stabilized by oligomeric protein complexes. 1. Shaw GC, et al. (2006) Nature 440:96-100. 2. Shirihai OS, et al. (2000) EMBO J. 19:2492-2502. 3. Paradkar PN, et al. (2009) Mol. Cell. Biol. 29:1007-1016.

8 EXPRESSION OF MITOCHONDRIAL FERRITIN AFFECTS JAK2/STAT5 PATHWAY IN K562 CELLS Sonia Levi, PhD, Alessandro Campanella, PhD, Elisabetta Rovelli, PhD, Benedetta Erba, Student, Anna Cozzi, PhD and Paolo Santambrogio, PhD Vita-Salute San Raffaele University (Presented By: Sonia Levi, PhD)

Mitochondrial ferritin (FtMt) is a nuclear encoded iron-storage protein localized in mitochondria and whose physiological role is only partly characterized. It is a 24-homo- polymeric shell protein, with structure and function similar to those of cytosolic H-ferritin. Differently from cytosolic ferritins, FtMt does not show ubiquitous distribution and its expression is not irondependent. Studies on mice tissues revealed that the protein has a tight tissue-specific expression pattern: it is preferentially expressed in cells characterized by high-energy consumption and it is not present in iron storage tissues. In human, the protein is also highly expressed in sideroblasts of patients affected by sideroblastic anemia (SA) and by refractory anemia with ringed sideroblasts (RARS), where it was demonstrated to be a specific marker of these myelodysplastic syndromes. Previous studies on HeLa cells showed that FtMt expression affected cellular iron homeostasis: it drew iron into mitochondria, induced cytosolic iron starvation and, depending on cellular growth conditions, improved or limited mitochondrial Fe/S enzymes activity. However, FtMt maintained ROS buffering ability, even in enhanced respiratory condition, indicating that its primary function consisted in the control of ROS formation, through the regulation of mitochondrial iron availability. To verify if the FtMt-induced ROS reduction could affect the cellular redox state and, consequently, influence the regulation of ROS-dependent pathways, like JAK2/STAT5, we analysed the effect of the overexpression of FtMt in K562 erythroleukemic cell lines. We performed transient transfections with vectors containing the cDNA of FtMt and its ferroxidase activity deleted mutant (FtMt222) and, using lentiviral vectors, we developed K562 stable clones overexpressing different amount of FtMt. Cells were analysed for iron homeostasis parameters, ROS production, total and phosphorilated STAT5 and apoptosis. Transient transfections revealed that the FtMt had dose dependent effect on JAK2/STAT5 signal transduction pathway, while the FtMt222 and uncorrelated protein expression had no effect. Analysis of stable clones showed different results depending on FtMt amount. Low levels of FtMt expression resulted in: a reduction of basal ROS production of about 15%; a decrease of about 30% of phosphorilated STAT5; a decrease of about 50% of IRP1 and 2 and of 95% of TfR1 proteins amount, compared to control cells. High levels of FtMt expression, although maintained low ROS level and the phosphorilation of STAT5 diminished of 70%, induced IRP2 and TfR1 increase of 2.5 and 1.6 fold respectively, compared to control cells, suggesting that the transcriptional regulation of STAT5 pathway was overcome by the strong cytosolic iron starvation status. Thus, FtMt appeared to determine cellular iron deprivation and to limit the phosphorilation of STAT5, with consequence on the transcription of iron homeostasis controlling proteins. Furthermore, the expression of FtMt resulted also in enhanced apoptosis, as indicated by the increase of PARP85 peptide. Altogether the results indicate that FtMt could interfere on the ROS-mediated regulatory pathway of erythropoiesis and it might be involved in the pathogenesis of some myelodysplastic syndromes.

9 STRESS ERYTHROPOIESIS AND IRON MOBILIZATION IN A MOUSE MODEL MIMICKING ANEMIA OF CHRONIC DISEASE; RESPONSE TO ERYTHROPOIETIN INJECTIONS Sarah Millot¹, Valerie Andrieu, Dr², Philippe Letteron, Dr¹, Olivier Thibaudeau³, Ivana de Domenico, Dr4, Sigismond Lasocki, Dr5 and Carole Beaumont, Dr¹ ¹INSERM U773; ²Department of Hematology, Hopital Bichat; ³Department of Anatomopathology, Hopital Bichat; 4University of Utah, Salt Lake City; 5ICU, Hopital Bichat (Presented By: Sarah Millot)

Introduction: The anemia of chronic disease (ACD) is the most frequent anemia in hospitalized patients. Several factors contribute to the pathology of ACD: development of hypoferremia with subsequent limitation of iron availability for erythroid progenitor cells, impaired proliferation of erythroid progenitors, and blunted erythropoietin response (Epo). Hypoferremia results form a strong activation of hepcidin expression and subsequent internalization and degradation of ferroportin in macrophages. In mouse, whereas erythropoiesis in the bone marrow is primarily homeostatic, stress erythropoiesis occurs in the spleen in response to acute anemia. BMP4/Smad5-dependent signalling initiates the expansion of BMP4-responsive cells and their differentiation into stress BFU-e responsive to high levels of Epo. The aim of our work was to compare spleen and bone marrow erythropoiesis in a mouse model of chronic inflammation, and to follow the effects of Epo injections on erythropoiesis and mobilization of iron stores in this ACD model.

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Methods: We developed an animal model of chronic inflammation with multiple organ dysfunctions in C57BL/6 mice, by intraperitoneal injections of Zymosan (Z), combined or not with Epo injections (Z+Epo). Z is injected at day 1, then EPO is injected on four consecutive days starting at day 5 (D5). Sacrifices are performed 1, 4 or 9 days after the final injection. Bone marrow and spleen cell composition as well as maturation of erythroblast were investigated by flow cytometry using Ter119/CD 71 double labeling. The expression of BMP4 in the spleen was followed by RT-PCR and immunohistochemistry Mobilization of iron stores in Z and Z+Epo conditions was assessed by ferroportin expression in spleen (western blot and immunochemistry), measurement of serum hepcidin levels by a functional assay and of liver and spleen iron concentrations. Results: Inflammation suppressed bone marrow erythropoiesis whereas spleen erythropoiesis was moderately activated and anemia persisted over time (Hb: 10.2g/l at D12). BMP4 was not expressed in this condition. The increase in serum hepcidin concentration following Zymosan injection correlated with the down regulation of ferroportin expression in liver and spleen. In mice receiving both Z and Epo, bone marrow erythroblast production remained low, whereas in the spleen, Epo injections induced a significant 10-fold increase in immature erythroblasts at D9 of inflammation, followed at D12 by an increase in mature erythroblasts. Almost complete recovery of the anemia was obtained by D17. Epo injections also induced a significant increase in spleen BMP4 mRNA in Z+EPO as compared to basal state and BMP4 protein was found in macrophages of the red pulp. The injection of Epo in the presence of inflammation also up regulated ferroportin expression, liver iron concentrations decreased and serum hepcidin concentrations returned to basal level. Conclusion: In mice, spleen “stress erythropoiesis” is responsive to Epo injections even in the presence of inflammation, on the contrary to bone marrow erythropoiesis. This response is due to the Epo-mediated activation of BMP4 expression by spleen macrophages. Efficient erythropoiesis is also possible due to repression of hepcidin and increased ferroportin expression in macrophages.

10 DIFFERENTIAL EXPRESSION OF IRON METABOLISM GENES AND IRON HANDLING IN DISTINCT POPULATIONS OF HUMAN INFLAMMATORY MACROPHAGES Stefania Recalcati, MD, PhD¹, Massimo Locati, MD¹, Agnese Marini, PhD¹, Paolo Santambrogio, PhD², Annalisa Castagna, PhD³, Domenico Girelli, MD³ and Gaetano Cairo, PhD¹ ¹University of Milan; ²DIBIT HS S. Raffaele; ³University of Verona (Presented By: Gaetano Cairo, PhD)

Most of the information available on macrophage iron metabolism has been provided by studies in which macrophages have been challenged with LPS/IFN gamma, a model system mimicking exposure to microrganisms and/or Th1 pro-inflammatory cytokines (M1 polarization). However, macrophagescould also follow a different activation pathway after stimulationwith the Th2 cytokines IL-4 or IL-13 (M2 polarization) acquiring a role in regulatingadaptive immunity and in the control of cell growth and tissue repair. Importantly, tumour infiltrating macrophages that are relevant for tumour growth, progression and invasion acquire a M2 phenotype. In the present study, we characterized the differential expression of genes of iron metabolism and iron trafficking in M1 and M2 polarized activation processes. Three different cell types were generated from human monocytes obtained from blood donor buffy coats: fully differentiated macrophages (7 days of culture in RPMI plus 20% FCS and 100 ng/ml M- CSF), M1 (differentiated macrophages incubated for additional 3 days in the presence of LPS/IFN gamma), M2 (differentiated macrophages incubated for additional 3 days in the presence of IL-4). Microarray analysis of the transcriptional profile associated with M1 or M2 polarization focusing on genes of iron metabolism revealed differences in the expression of genes involved in iron storage (ferritin), traffic (TfR1, ferroportin, ceruloplasmin) and regulation (IRP2, heme oxygenase). RT-PCR analysis and immunoassays confirmed these data at the mRNA and protein levels. The molecular signature of M1 macrophages was in line with previous data showing changes in the expression of genes (e.g. ferroportin repression and ferritin induction) that could favour iron sequestration in reticuloendothelial cells, a hallmark of inflammatory disorders. Conversely, we found in M2 macrophages an expression profile of iron genes (upregulation of ferroportin and heme oxygenase and down-regulation of ceruloplasmin and H ferritin) which was opposite to that of M1 macrophages and could greatly enhance iron release. This different expression was accompanied by functional effects on iron availability because 55Fe release experiments showed a three fold higher iron export from M2 than from M1 cells. To gain insight into the biological implications of the different iron release and storage capacity shown by the two macrophage populations we analyzed the growth promoting activity of their conditioned media. Various cell lines showed a growth rate twice as fast when incubated in the presence of the conditioned medium obtained from M2 cells, as compared to that of M1 macrophages. Importantly, the difference in cell growth was blunted if macrophages were incubated in the presence of a cellimpermeant form of the iron chelator DFO or were purified from a patient with ferroportin disease characterized by a ferroportin mutation that prevents iron release. Overall, the present data provide a better understanding of the modifications of iron metabolism under inflammatory conditions, in particular those characterized by the presence of M2 macrophages. In fact, they suggest pathophysiological implications related to: 1) a role for iron in influencing the distinct phenotypic heterogeneity of the two macrophage populations as it regards cytokine production; and 2) an effect on neighbour cells in the microenvironment.

11 THE ACTIVATION OF IRON REGULATORY PROTEIN 1 DOMINATES IRON HOMEOSTASIS IN INFLAMED INTESTINAL EPITHELIUM Esther Meyron-Holtz, Shirly Moshe, Orly Savion, Adi Kammer, Yoram Bujanover, MD, Batia Weiss, MD and Ram Reifen, MD, PhD Technion-Israel Institute of Technology (Presented By: Esther Meyron-Holtz)

Crohn’s disease (CD) is an inflammatory bowel disease that causes transmural inflammation of the gastrointestinal mucosa and is often accompanied by microcytic anemia. Often the cause of this anemia is a combination of iron deficiency due to intestinal bleeding and decreased iron absorption and anemia of chronic inflammation that causes iron retention in macrophages and intestinal epithelial cells. Treating this anemia with oral iron supplementation may contribute to the iron accumulation and elevated oxidative stress markers found in inflammatory lesions of CD patients. In addition, in animal models of inflammatory bowel disease, iron supplements have aggravated the inflammation. Iron regulatory proteins (IRPs) modulate the expression of proteins involved in the transport and storage of iron, through binding to their target mRNA templates during iron deficiency. IRP binding generates upregulation of the iron importer transferrin receptor1 (TfR1) and downregulation of the iron storage protein ferritin and possibly the iron exporter ferroportin. At physiologic O2 conditions, IRP2 is the dominant regulator as IRP1 sensing depends on the stability of its iron sulfur cluster and physiologic O2 concentration is too low for this cluster to act as a sensor. We hypothesized that IRP1 will be activated by reactive oxygen or nitrogen species in inflamed tissue and will play a dominant role in the regulation of iron homeostasis in these lesions. We therefore evaluated IRP activity, downstream target proteins and iron accumulation in CD patient biopsies, a mouse model for inflammatory bowel disease and an intestinal epithelial cell-line (Caco-2 cells). IRP1-RNA binding activity was found elevated in CD patients compared to non-inflamed controls. In agreement, levels of ferritin were decreased. We were able to mimic this IRP1 mediated mis-regulation of iron homeostasis in Caco-2 cells and found that inflammation induced IRP-1 activation caused elevated TfR1 and decreased ferroportin levels suggesting iron accumulation in the inflamed cells. Interestingly the effect of IRP1 activation on IRP target proteins dominated over the IRP2 deactivation in these models. In addition, preliminary results in a mouse model of inflammatory bowel disease show iron1 accumulation in the inflamed lesions, suggesting that perturbation of iron homeostasis mediated by IRP1 activation leads to this iron accumulation. As ferritin levels were decreased, safe iron storage may not be optimal which can enhance generation of reactive oxygen species and further worsen the inflammation. We conclude that IRP1 activation in inflammatory lesions can perturb local iron homeostasis and aggravate the inflammation. Interfering with this process could be beneficial for the patient.

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12 ROLE OF TOLL-LIKE RECEPTOR / ADAPTOR PROTEIN MYD88 SIGNALING IN HYPOFERREMIA ASSOCIATED WITH INFLAMMATION Antonio Layoun, Hua Huang and Manuela M. Santos Institut du Cancer de Montréal, CRCHUM, University of Montréal (Presented By: Antonio Layoun)

The interaction between the immune system and iron metabolism is well exemplified in the anemia of chronic disease (ACD), which is frequently encountered in chronic infections, inflammation and cancer. The major changes in iron parameters observed in ACD ultimately reflect modifications in iron trafficking, with relocation of the metal from both the circulation and sites of erythropoiesis to the storage compartment in macrophages. Mechanisms in the hypoferremic response involve cytokines, including TNF-alpha and IL-6. These pro-inflammatory cytokines regulate the levels of several iron metabolism genes, including iron transporters and hepcidin, resulting in the inhibition of iron export from the intestine and macrophages. Previous studies showed that IL-6 upregulates hepcidin in hepatocytes, but there are very few data regarding how hepcidin expression is regulated in macrophages. More recently, we found that hepcidin induction in the liver by lipopolysaccharide (LPS) is dependent on the signaling pathway mediated by toll-like receptor 4 (TLR4). TLRs are an essential component of signaling receptors expressed on the cell surface that recognize pathogen- associated molecular patterns or ligands that are produced by microorganisms. In the present study, we investigated the regulation of hepcidin expression in macrophages through TLR signaling after stimulation with distinct ligands and their requirement for the development of hypoferremia. To identify TLR ligands capable of inducing hepcidin in macrophages, we treated Raw 264.7 macrophages and thioglycollatestimulated mouse peritoneal macrophages (MPMs) with various TLR ligands and measured hepcidin expression by real-time RT-PCR. We observed that Pam3CSK4 (Pam), a TLR1/2 ligand; LPS, a TLR-4 ligand; and FSL1 a TLR6/2 ligand, but not polyinosinic:polycytidylic acid (poly I:C), a TLR3 ligand, upregulate hepcidin expression in both Raw 264.7 cells and MPMs. To further define the requirement for the identified TLRs, we used TLR-4-deficient and TLR-2 knockout mice and established that upregulation of macrophage hepcidin expression by LPS, Pam or FSL1 is TLR4 or TLR2 dependent, respectively. In agreement with the in vitro experiments, when tested in vivo polyI:C failed to induce hepatic hepcidin expression but was effective in the triggering of hypoferremia. We next investigated whether MyD88, the primary but not exclusive intracellular signal transduction pathway for TLR-4, is necessary for hepcidin induction in macrophages and for the occurrence of hypoferremia in response to LPS. Using MyD88 knockout mice, we found that the absence of MyD88 signaling abolishes hepcidin induction triggered by Pam, LPS and FSL1 in MPMs as well as in the liver of LPS treated mice. However, despite severely depressed circulating IL-6 and TNF-alpha levels found in the knockout mice, absence of MyD88 signaling did not completely prevent LPS from causing hypoferremia within the first phase of the LPS response. These observations indicate that hepcidin expression in macrophages can be regulated through multiple TLRs, suggesting that macrophage hepcidin production is part of an immune response activated by the TLR-mediated recognition of molecules associated with pathogens. The suppression of hepcidin expression by LPS is associated with cytokine production through the MyD88 signaling. In contrast, the development of hypoferremia during the first phase of the LPS response seems to occur via a MyD88- independent mechanism that may be disassociated from its effect both on peripheral cytokine production and hepatic hepcidin induction.

13 ENDOTOXIN-MEDIATED INDUCTION OF HEPCIDIN EXPRESSION IS ATTENUATED IN TFR2 MUTANT MICE DESPITE NORMAL ACTIVATION OF STAT3 Robert Fleming, MD, Robert Britton, PhD, Mary Migas, MS, William Sly, MD and Abdul Waheed, PhD Saint Louis University School of Medicine (Presented By: Robert Fleming, MD)

Introduction: Functional loss of transferrin receptor 2 (TfR2) in humans and mice results in decreased hepcidin expression and hemochromatosis. Hepcidin expression is regulated by several factors, including iron status, erythropoietic activity, and inflammation. The effect of inflammation on hepcidin expression is primarily mediated via the induction of STAT3 phosphorylation by interleukin 6. Studies in other murine models of hereditary hemochromatosis (HH) have suggested that the induction of hepcidin expression during inflammation is attenuated. We compared the effect of endotoxin on the activation of STAT3 and on liver hepcidin expression in Tfr2(Y245X) homozygous and wild-type mice. Methods: Tfr2(Y245X) mice were bred to homogeneity on an FVB background. Homozygous wild-type and Tfr2(Y245X) colonies were maintained on chow ad libitum. Five-week-old wild-type and Tfr2(Y245X) mice on a standard iron diet (N=6-7 each group) were treated with 1 µg/g of endotoxin (LPS E. coli 055:B5) or carrier intraperitoneally and sacrificed six hours later. Endosomes and microsomes were prepared from liver homogenates by sucrose gradient centrifugation. Cytosolic and nuclear proteins were prepared as described (NE-Per, Pierce). Equivalent amounts of protein were subjected to SDS-PAGE and immunoblotted using antibodies to STAT3 and phospho-STAT3 (Cell Signaling). Signals obtained by chemiluminescence were quantified and blots re-probed with antibodies to clathrin (BD Biosciences) and/or ß-actin (Sigma) for normalization. RNA analysis was performed using a 32P-labeled antisense RNA probe which detects mHamp1 and 2 mRNAs, and by realtime RT-PCR for each transcript individually. RNA blot signals were quantified by phosphorimagery and normalized to the 28S rRNA signal from ethidium bromide-stained gels. Results: Tfr2 mutant mice demonstrated decreased basal hepcidin expression compared with wild-type mice. The expression of hepcidin was increased in response to endotoxin in both wild-type and Tfr2 mutant mice. The induction of hepcidin expression in response to endotoxin over baseline was similar in wild-type and Tfr2 mutant mice; however, the magnitude of expression in the Tfr2 mutant mice remained lower than in wild-type mice. By contrast, the induced level of expression of ceruloplasmin (another acute-phase liver gene) was even greater in Tfr2 mutant mice compared with wild-type mice. Endotoxin administration resulted in similar increases in phospho- STAT3 in endosomal, cytosolic, and nuclear compartments of both wild-type and Tfr2 mutant mice. Conclusions: These observations indicate that while the inflammatory activation of STAT3 is intact in the Tfr2 mutant mice, this signal tranduction mechanism does not overcome the supression of hepcidin expression caused by functional loss of Tfr2. We suggest that the relatively low hepcidin expression during inflammation in HH may have important consequences on iron distribution during the host-defense response.

14 FUNCTION OF THE IRE/IRP REGULATORY NETWORK IN MACROPHAGES Bruno Galy, PhD, Dunja Ferring-Appel, Lydie Viatte, Klaus Schuemann and Matthias Hentze European Molecular Biology Laboratory (Presented By: Bruno Galy, PhD)

Tissue macrophages clear effete erythrocytes, free iron from hemoglobin, and release it back into the circulation via ferroportin; macrophages also use the redox properties of iron to generate radicals and to eliminate invading pathogens. Systemically, the iron hormone hepcidin exerts a key control on macrophage iron fluxes by directly inhibiting ferroportinmediated iron export; cellularly, translational control of ferritin and ferroportin expression by the IRPs is anticipated to play a major role in macrophage iron management from studies using cultured cells. To study the function of the IRE/IRP regulatory network in macrophage iron metabolism in vivo, we have generated conditional alleles of the murine Irp1 and Irp2 genes, respectively, allowing total as well as cell-specific ablation of IRP1 and/or IRP2 based on Cre/Lox technology. While mice with total and constitutive IRP1 deficiency present with no steady-state abnormality, IRP2-deficient animals display relative iron deficiency in splenic macrophages associated with ferritin and ferroportin downregulation, a surprising finding following removal of a translational repressor protein. Selective ablation of IRP2 in macrophages using the LysozymeM-Cre deletor strain does not phenocopy the abnormalities of macrophages from systemically IRP2-deficient animals, indicating that these require IRP2 disruption in other cell type(s). The relatively mild impact of IRP1 or IRP2 deficiency, respectively, likely reflects reciprocal compensation between the two IRPs. Since disruption of both Irp genes results in early embryonic lethality, we used the LysozymeM-Cre line to simultaneously ablate both IRPs selectively in macrophages (Irp1,Irp2LysMCre mice). While bone marrow derived macrophages (BMDM) lacking either IRP1 or IRP2, respectively, display normal expression of IRP- target genes or slight ferritin upregulation, BMDM lacking both IRPs exhibit a dramatic increase in ferritin and ferroportin levels associated with transferrin receptor 1 (TfR1) downregulation, indicating strong functional redundancy between the two IRPs ex vivo; of note, expression of DMT1 mRNA variants and mitochondrial aconitase remains unchanged in these cells. Exposure of doubly-deficient BMDM to synthetic hepcidin, desferoxamine or hemin, respectively, reveals IRP-dependent and - independent mechanisms of ferritin, ferroportin, and TfR1 regulation. Similar to primary BMDM cultures, Kupffer cells in the liver of Irp1,Irp2LysMCre mice exhibit 68 ferritin and ferroportin upregulation; surprisingly, the spleen displays increased ferritin expression but unchanged ferroportin levels. The lack of ferroportin protein upregulation in the spleen cannot be explained by compensatory downregulation of the corresponding mRNA, nor by increased splenic or hepatic expression of hepcidin mRNA. This suggests an additional, currently unidentified mechanism to control ferroportin expression. At the systemic level, plasma ferritin levels in Irp1,Irp2LysMCre mice are markedly increased, likely due to secretion from ferritin-overexpressing macrophages; however, plasma iron levels, transferrin saturation values, and body 59Fe distribution remain normal. This study shows that the IRPs control ferritin and ferroportin expression in macrophages in vivo; nevertheless, iron metabolism perturbation in IRP-deficient macrophages does not detectably impact systemic iron homeostasis, at least under standard laboratory conditions. Mice with double IRP deficiency in macrophages are currently being subjected to inflammatory and infectious stimuli to unveil the role of the IRE/IRP system in macrophage iron management and immune function.

15 FUNCTION OF THE IRE/IRP REGULATORY NETWORK IN SYSTEMIC IRON METABOLISM AND CENTRAL ORGANS FOR IRON HOMEOSTASIS Bruno Galy, PhD, Dunja Ferring-Appel, Sylvia Kaden, Hermann-Josef Groene and Matthias Hentze European Molecular Biology Laboratory (Presented By: Bruno Galy, PhD)

Because both lack and excess of iron are detrimental, iron homeostasis must be tightly controlled on both the systemic and cellular levels to provide just the right amounts of iron at all times. While hepcidin plays a central role in systemic iron homeostasis, extensive work with cultured cells has shown that cellular iron levels are predominantly balanced by the IRE/IRP system that coordinately regulates the posttranscriptional expression of genes involved in iron uptake, utilization, storage, and export. The in vivo roles of the IRE/IRP system have recently become a focus of attention with the creation of various mouse lines with altered IRP function. We used homologous recombination in ES cells and Cre/Lox technology to generate mouse models with conditional loss of IRP function (1,2). These mouse lines enable disruption of the Irp1 and/or Irp2 genes, either in the entire organism or in a time- and/or tissue-specific manner. While mice lacking IRP1 expression are aphenotypic under standard laboratory conditions, total inactivation of the Irp2 locus shows that IRP2 is required for normal erythropoiesis and to secure physiological iron distribution between the duodenum, the liver, and splenic macrophages (3); tissue-specific ablation of IRP2 reveals that the manifestations of systemic IRP2 deficiency are largely explained by cell autonomous functions (4). Disruption of both Irp genes results in early embryonic lethality (5), showing that the IRE/IRP system is essential and that the two IRPs can largely replace each other. To bypass the embryonic lethality of doubly deficient mice, we simultaneously inactivated both Irp genes in a cell-specific manner and created the first living organisms with complete IRP deficiency in one of their organs, namely the macrophages, the intestine, or the liver, respectively. Mice lacking IRP expression in macrophages display ferritin and ferroportin upregulation notably in Kupffer cells, associated with elevated serum ferritin levels, but develop normally. By contrast, complete IRP deficiency in enterocytes (5) or in hepatocytes, respectively, results in lethality during the early postnatal period, showing that the IRE/IRP system is essential for intestinal and hepatic function and organismal survival. We are now using Cre-deletor strains with, respectively, intestinal- or hepatic-specific expression of a ligand-inducible Cre recombinase in order to study IRP function in, normally developed, adult mice. The versatility of mouse lines with conditional loss of IRP function offer the possibility to finely dissect the tissue-specific function of the IRE/IRP regulatory system in the control of cellular, tissular and organismic iron fluxes. In combination with treatments known to challenge the iron homeostasis machinery, they will help to unveil the overlaping a well as specific functions of IRP1 and IRP2. Furthermore, investigation of these mouse lines begins to reveal interesting aspects of the interplay between the IRE/IRP and the hepcidin/ferroportin regulatory systems. References: (1) Galy B, et al. (2004) RNA 10:1019-25. (2) Galy B, et al. (2005) Genesis 43:181-88. (3) Galy B, et al. (2005) Blood 106:2580-89. (4) Ferring-Appel D, et al. (2009) Blood 113:679-687. (5) Galy B, et al. (2008) Cell Metab. 7:79-85.

16 A NOVEL IRP2-/- MOUSE MODEL DISPLAYS LOCOMOTOR DYSFUNCTION AND NEURONAL IRON ACCUMULATION Kimberly Zumbrennen, PhD, Sabine Holter, PhD, Lore Becker, PhD, Brigit Rathkolb, PhD, Eva Rodansky, BSc and Elizabeth Leibold, PhD University of Utah (Presented By: Kimberly Zumbrennen, PhD)

Iron regulatory protein 2 (Irp2) is a RNA-binding protein that regulates the expression of proteins required for iron import (transferrin receptor-1 (TfR1)), export (ferroportin), and storage (ferritin). Two distinct Irp2 knock-out mouse models have been generated by independent laboratories (LaVaute et al., Nat Genet 2001; Galy et al., Genesis 2005). Both Irp2-/- mouse strains were shown to have altered expression of TfR1 and ferritin in multiple tissues which resulted in increased hepatic and duodenal iron loading, as well as microcytic anemia (Cooperman et al., Blood 2005; Galy et al., Blood 2005). The Irp2-/- mouse strain generated by Lavaute et al. was also described to have kiphosis, vestibular dysfunction, ataxia, tremors, unsteady gait, and bradykinesia (LaVaute et al., Nat. Genet. 2001). These severe locomotor abnormalities were attributed to neuronal iron accumulation and neuronal degeneration characterized by increased amino cupric silver staining in white matter tracts, activation of microglia, loss of Purkinje cells, and vacuolization in the substantia nigra (Smith et al., Ann NY Acad Sci 2004). In contrast, Galy et al. found no evidence of neuronal iron accumulation or degeneration, and only a mild alteration in locomotor function in their Irp2-/- mouse strain (Galy et al., Nat Genet 2006). The discrepancies between these two Irp2-/- mouse models have caused the role of Irp2 in brain iron homeostasis to be controversial. We have used gene targeting to generate a novel Irp2-/- mouse strain. Similar to the previously characterized Irp2-/- mouse models, our mice show altered expression of TfR1 and ferritin in numerous tissues, increased hepatic and duodenal iron, and a hypochromic microcytic anemia. While we did not observe postural, vestibular, or gait defects, neurological and behavioral tests revealed significant decreases in vertical and horizontal exploratory activities in our Irp2-/- mice compared to Wt controls. Perls-DAB staining on perfused brains revealed iron accumulation in the frontal cortex, mid-brain, and cerebellum of Irp2-/- mice. Increased iron staining in the forebrain of Irp2-/- mice was localized to areas required for motor coordination. Analysis of neuronal degeneration using Flouro-Jade staining revealed no differences between Irp2-/- mice and Wt controls. Microglial analysis using IBA-1 (microglial specific) and Reactive Microglial staining showed no changes in morphology or activation of microglia in Irp2-/- mice compared to Wt controls. Our studies indicate that loss of Irp2 causes significant locomotor dysfunction, which is associated with the accumulation of iron in regions of the brain required to coordinate muscle function.

17 GENOME-WIDE MRNA IDENTIFICATION AND PROTEOMIC ANALYSIS OF THE IRE/IRP REGULATORY NETWORK Matthias Hentze, Prof Dr¹, Mayka Sanchez, Dr¹, Bjoern Schwanhaeusser, Dr², Jonathon Blake, Dr³, Bruno Galy, Dr³, Yehven Vainshtein³, Tomi Bähr-Ivacevic³, Vladimir Benes, Dr³, Matthias Selbach, Dr² and Martina U. Muckenthaler, Dr4 ¹Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 153, 69120 Heidelberg, Germany and European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; ²Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin, Germany; ³European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; 4Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 153, 69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany (Presented By: Matthias Hentze, Prof Dr)

Iron regulatory proteins (IRP) 1 and 2 post-transcriptionally control cellular iron metabolism by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5’ or 3’ untranslated regions (UTRs). Over the past 20 years, IREs have been identified in less than a dozen mRNAs. To define the IRE/IRP regulatory network systematically on a genome-wide scale,IRP1/IRE and IRP2/IRE mRNP complexes were immunoselected using total RNA from five different mouse tissues (liver, spleen, duodenum, bone marrow and brain). For each tissue, the mRNA composition of the immunoprecipitated IRP/IRE mRNPs was determined using genome-wide Affymetrix arrays and validated by qPCR. We also developed a software package to identify IRE-like motifs in the entire mouse and human transcriptomic database (ENSEMBL and NCBI) and correlated ‘hits’ with the experimentally identified IRE candidate mRNAs. In addition, murine hepatocytic Hepa1-6 cells are used for mass-spec proteomic analysis of iron-regulated proteins by pulsed SILAC.

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Microarray analysis identified 43 mRNAs that bind both IRP1 and IRP2 in at least one tissue. This list includes all known murine IRE-containing mRNAs (Fth1, Ftl1, Tfrc, Alas2, Aco2, Slc40a1, Slc11a2, Hif2alpha). Approximately half of these 43 mRNAs (24 out of 43, 56%) harbour an IRE motif (8 known IREs and 16 novel IRE-like motifs), while the other half displays specific IRP binding without an identifiable IRE. In addition, we found 101 mRNAs that exclusively bind IRP1 (37% with a predicted IRE-like motif) and 113 IRP2-specific mRNAs (35% with a predicted IRE-like motif) that were consistently detected in at least one tissues. This work explores the IRE/IRP regulatory network in unprecedented depth and uncovers previously unrecognized mRNAs that specifically interact with IRP1 and/or IRP2, potentially connecting iron metabolism to other metabolic pathways. Ongoing proteomic analysis further helps to define iron-regulated polypeptides and assignment to IRE- controlled or non-IRE regulatory pathways. This work was supported by the Young Investigator Award of the Medical Faculty, University of Heidelberg (Germany) and the post-doctoral Fellowship Beatriu de Pinos, Generalitat de Catalunya (Spain) to M.S and by grants from the Forschungsschwerpunktprogramm des Landes Baden-Württemberg (RNA and disease) to M.W.H. and M.U.M.

18 DEGENERATION OF MOTOR NEURONS IN THE SPINAL CORD OF IRP NULL MICE Suh Young Jeong, PhD¹, Bernard S. Jortner, VMD², Hayden Ollivierre³, Manik Ghosh, PhD³, Sharon Cooperman, MD, PhD³, Rachid Sougrat, PhD³ and Tracey A. Rouault, MD³ ¹NIH; ²Virginia Tech; ³NICHD, NIH (Presented By: Suh Young Jeong, PhD)

Iron Regulatory Proteins (IRPs) are the key regulators for maintaining cellular iron homeostasis. These proteins regulate expression of various proteins essential for iron homeostasis by binding to their transcripts. Previously, we have reported neurodegenerative symptoms of mice models lacking IRP1 and/or IRP2. Here we examined spinal cord of IRP1+/+;IRP2-/- and IRP1+/-;IRP2-/- mice especially focusing on the degeneration of motor neurons. Light microscopic analysis of 0.7um sections from IRP1+/- ;IRP2-/- mice spinal cord showed that there is massive increase of myelin dense bodies in the ventral white matter (65-fold compared to the wildtype). These myelin dense bodies (MDBs) are a histological hallmark of axonal degeneration and collapse of myelin sheath. There was also significant increase in IRP1+/+;IRP2-/-mice compared to the control (24-fold). These MDBs are only found in the ventral and lateral funiculars of the spinal cord where nerve tracts involved in motor functions are localized, suggesting motor function problems in these mice. Therefore we also examined dorsal and ventral root nerves of these animals and confirmed that the most of affected neurons are in the ventral root nerve. Longitudinal sections of ventral nerve root showed axonal swelling and degeneration of cytoskeleton in the IRP null mice. Losing IRPs in a cell is expected to generate a false signal for iron overload therefore increasing ferritin expression and decrease of transferring receptor1. This misregulation was found in the most of the cell types throughout spinal cord, but the most prominent change was found in the motor neuronal cell bodies using immunohistochemistry. Therefore, these two big changes will cause functional iron deprivation in the motor neurons that will diminish the usable iron pool. Since iron is an essential cofactor for the mitochondrial respiratory chain, we measured activity of Complex I. Complex I activity was decreased 30% in the IRP null mice while the amount of a 43 peptide complex that contains eight iron-sulfur cluster subunits did not change significantly. Therefore this data suggest that there might be a problem in iron-sulfur cluster assembly in these mice possibly due to iron deprivation. EM analysis also showed that there are number of swollen mitochondria in the axons of IRP1+/-;IRP2-/- mice with occasional demyelination and aggregation of neurofilaments. Oral treatment with a free radical scavenger, Tempol, spared around 50% of axons from degeneration in IRP1+/+;IRP2-/- while there was no effect on IRP1+/-;IRP2-/- mice. We have previously shown that Tempol increases IRP1 activity. These data demonstrate that iron homeostasis disrupted by lack of IRP cause motor neuronal degeneration in these mice and also these neurons are more vulnerable than other cells. Currently there is little data how neurons manage iron homeostasis along their axons, but many neurodegenerative disorders in human show disrupted iron homeostasis. Therefore, understanding the function of IRP in the central nervous system will help us explore these diseases in the future.

19 FERROCHELATASE DEFICIENCY IN ERYTHROPOIETIC TISSUES OF MICE LACKING IRON REGULATORY PROTEIN 2 Daniel Crooks, MS¹, Manik Ghosh, PhD², Hayden Olivierre-Wilson² and Tracey Rouault, MD² ¹Georgetown University; ²NICHD (Presented By: Daniel Crooks, MS)

Iron-regulatory proteins (IRP) 1 and 2 post-transcriptionally regulate protein expression by binding to iron-responsive elements present in the mRNA transcripts of genes important for iron metabolism such as ferritin and transferrin receptor. We previously demonstrated that mice lacking IRP2 (IRP2-/-) exhibit an atypical anemia, characterized by a low hematocrit and microcytosis, but with normal serum transferrin saturation and markedly increased serum ferritin levels. Free protoporphyrin IX levels in the erythrocytes of IRP2-/- mice are also dramatically increased, a symptom often observed in human patients suffering from erythropoietic protoporphyria, a disease typically caused by deficiency of the heme biosynthetic enzyme known as ferrochelatase. To further investigate the cause of elevated protoporphyrin IX levels in the blood of IRP2-/- mice, we measured ferrochelatase activity and protein abundance in tissues of IRP2-/- mice. Ferrochelatase activity in bone marrow and spleens of IRP2-/- mice was significantly decreased to 59% and 50% of control, respectively. This activity decrease was accompanied by a significant decrease in ferrochelatase protein levels, as measured by western blot. In tissues with a lower demand for iron such as heart and brain, ferrochelatase activity was unchanged, suggesting a specific defect in hematopoietic tissues of IRP2-/- mice. Despite the significantly decreased ferrochelatase protein abundance in the spleens of IRP2-/- mice, ferrochelatase mRNA transcript levels were increased as measured by qRT-PCR and northern blot. These findings suggest that decreased ferrochelatase abundance in the spleens of IRP2-/- mice is a result of a post-transcriptional regulatory mechanism involving either diminished protein synthesis or enhanced protein degradation. Current in-vitro studies in our lab using mouse erythroleukemia cells as a model for early erythropoiesis are directed at further characterizing the molecular mechanism of post-transcriptional ferrochelatase regulation during iron-deficient erythropoiesis, with special consideration being given to the role of the [2Fe-2S] cluster of ferrochelatase in this process. In summary, these data suggest a mechanism by which elevated erythrocyte protoporphyrin IX might arise in IRP2-/- mice. In developing erythrocytes of IRP2-/- mice, protoporphyrin IX is synthesized in abundance by ALAS2, the rate-limiting enzyme of heme biosynthesis which we previously found to be upregulated in IRP2-/- mice. However, due to intracellular iron deficiency caused by decreased transferrin receptor expression, and markedly decreased ferrochelatase activity, erythrocytes are unable to incorporate iron into protoporphyrin IX in sufficient quantities leading to erythrocyte protoporphyrin IX over-accumulation and anemia.

20 IRON-INDEPENDENT PHOSPHORYLATION OF IRP2 REGULATES G2/M TRANSITION DURING CELL CYCLE PROGRESSION Elizabeth Leibold, PhD, Michelle Wallander, PhD, Kimberly Zumbrennen, PhD, Joshua Romney, PhD and Eva Rodansky, BS University of Utah (Presented By: Elizabeth Leibold, PhD)

Iron regulatory protein 2 (IRP2) is a key iron sensor that regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) located in the untranslated regions of mRNAs encoding proteins involved in iron import and sequestration, such as transferrin receptor 1 (TfR1) and ferritin, respectively. During iron deficiency, IRP2-IRE binding represses ferritin mRNA translation and stabilizes TfR1 mRNA, whereas during iron sufficiency IRP2 is degraded by the proteasome, leading to derepression of ferritin synthesis and destabilization of TfR1 mRNA. Here, we identify an iron-independent IRP2 phosphorylation site at S157 that is regulated by the cell cycle. IRP2 S157 is phosphorylated by Cdk1/cyclin B1 during G2/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Phosphorylation of S157 reduces the RNA-binding activity of IRP2 resulting in increased ferritin synthesis. S157 dephosphorylation restores IRP2 RNA binding, causing repression of ferritin synthesis and increased TfR1 mRNA abundance. Expression of a non-phosphorylatable S157A mutant in Irp2-/- mouse embryonic fibroblasts (MEFs) causes a delay in cell cycle progression from S phase to G2/M phase and reduces the rate of cell proliferation compared to Irp2-/- MEFs reconstituted with wild type IRP2. Conversely, expression of a phosphomimetic S157D mutant in Irp2-/- MEFs causes a slight increase in G2/M progression and increased cellular proliferation. These data indicate that reversible phosphorylation of IRP2 during G2/M modulates the iron-independent expression of ferritin and TfR1 during the cell cycle, and contributes to the regulation of the G2/M transition during cell cycle progression. 70

21 HIF-2 ALPHA BUT NOT HIF-1 ALPHA IS A KEY REGULATOR OF IRON ABSORPTION Maria Mastrogiannaki¹, Pavle Matak, PhD², Brian Keith, PhD³, Celeste Simon, PhD³, Sophie Vaulont, PhD² and Carole Peyssonnaux, PhD² ¹Institut Cochin; ²Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Inserm U567, Paris 75014, France; ³Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA (Presented By: Maria Mastrogiannaki)

Maintenance of iron homeostasis is critical, as iron deficiency can lead to anemia while iron overload is toxic and detrimental to human health. Since humans do not possess a regulated iron excretion pathway, iron homeostasis must be tightly regulated at the level of absorption. Because of its juxtaposition with the anoxic lumen, the gastrointestinal mucosa has a uniquely steep oxygen gradient ranging from the richly vascularized sub-epithelial mucosa to the constant hypoxic state of the epithelium. Hypoxia Inducible Factors (HIFs), HIF-1 and HIF-2, are central mediators of cellular adaptation to hypoxia and consist of two helix-loop-helix proteins: a regulatory subunit HIF-1α or HIF-2α, which is the oxygen-responsive component, and HIF-1β which is constitutively expressed. We hypothesized that the HIF transcription factors, stabilized in the hypoxic epithelial layers of the intestine, could play a central role in maintaining iron balance in the body by regulating iron absorption. Divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), and ferroportin (FPN1), are the major genes involved in iron uptake and export and have only recently been characterized, but the molecular mechanisms of their basal, iron-dependant or hypoxic regulation are still largely unknown. We generated mice with targeted deletion of HIF-1α or HIF-2α specifically in the intestinal epithelium including the proximal part of the duodenum, which is the main site of dietary iron uptake. Here, we report a specific regulation of murine DMT1, Dcytb and FPN mRNA levels in vivo by HIF-2α and not by HIF-1α. Promoter mutagenesis and Chromatin Immunoprecipitation (CHIP) in vitro showed the requirement of a highly conserved Hypoxia Responsive Element (HRE) element, crucial for the trans- activation of human DMT1 promoter by HIF-2. In vivo, diminished DMT1 expression due to disrupted HIF-2 signaling in the duodenum correlated with a decrease in serum and liver iron, and a marked decrease in hepcidin expression. Interestingly, the decrease in hepcidin expression was not sufficient to induce the expected increase in ferroportin stabilization in the duodenum. The phenotype observed at the basal level could be further exacerbated by iron deficiency. After a 2-months iron deficient diet, while WT mice showed strong induction of DMT1 and Dcytb mRNA levels, the response was decreased in the HIF-2α knock-out duodenum further demonstrating that HIF-2α in the intestine is essential for sensing the iron deficiency and the regulation of the adaptive response. Our findings suggest that HIF-2α, stabilized in the hypoxic epithelial layers of the intestine could be important in maintaining the iron balance in the body by counteracting any transient changes in tissue oxygenation or iron deficiency that could happen daily under a regular diet. In addition, upon an iron deficient diet, not only would hepcidin be low to prevent ferroportin degradation, but HIF-2α, further stabilized by local iron deficiency in enterocytes would boost iron absorption by increasing the expression of apical transporters. In conclusion, HIF-2α is a central local iron sensor and iron absorption regulator in the duodenum. Therefore, monitoring the levels of HIF-2α could benefit patients with iron disorders, its activation allowing iron mobilization and its reduction favoring decreased iron absorption.

22 BMP-SMAD SIGNALING IS IMPAIRED IN AN HFE KO MOUSE MODEL Elena Corradini, MD¹,4, Cinzia Garuti, PhD², Giuliana Montosi, PhD², Billy Andriopoulos Jr, PhD³, Yin Xia, PhD³, Herbert Y. Lin, MD, PhD³, Antonello Pietrangelo, MD, PhD² and Jodie L. Babitt, MD³ ¹Program in Membrane Biology, Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ²Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena, Italy; ³Program in Membrane Biology, Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 4Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena, Italy (Presented By: Elena Corradini, MD)

Hemochromatosis (HC) is a genetically heterogeneous hereditary disorder of iron metabolism caused by a failure to prevent unneeded iron from entering the circulatory pool, and characterized by progressive iron overload with potential for multi-organ damage and disease. Mutations in the gene encoding HFE are the first discovered and most common cause of HC, affecting approximately 5/1000 individuals of northern European descent. While the discovery of the HFE gene dates back more than 12 years, and our understanding of disease prevalence and expressivity has increased appreciably, the pathophysiology of HFE-HC is still elusive. One clue to understanding the pathophysiology of HFE-HC comes from the observation that the main iron regulatory hormone hepcidin is inappropriately low and/or not adequate for the iron burden in mouse models and human patients with HFE-HC. Secreted by the liver, hepcidin controls the extent of iron entering the bloodstream from macrophages and enterocytes, by degrading the iron exporter ferroportin. Although impaired hepcidin expression and unchecked ferroportin activity are therefore thought to play a role in the iron overload phenotype of HFE-HC, the mechanism by which HFE affects hepcidin expression is still unknown. We and others have recently shown that BMP-SMAD signaling is a main regulatory pathway acting in hepcidin regulation and iron metabolism. BMP signals positively regulate hepcidin expression at the transcriptional level both in vivo and in vitro. Impaired hepatic BMP signaling, through mutations in genes encoding the BMP co-receptor hemojuvelin, the common BMP/TGF-b intracellular mediator Smad4, or the ligand Bmp6, leads to low hepcidin levels and iron overload. Additionally, iron increases SMAD signaling in the liver and Bmp6 mRNA levels correlate with iron burden in mice. Based on these premises, the aim of this project was to dissect the BMP-SMAD signaling pathway in the Hfe knockout (KO) mouse model. On iron balanced diet, liver Bmp6 mRNA levels in Hfe KO mice were higher than in wild-type (WT) mice and appropriate to increased body iron levels seen in Hfe KO mice. However, phospho-Smad 1/5/8 proteins (intracellular mediators of BMP signaling) and Id1 mRNA (a target gene of BMPs), in the liver of Hfe KO mice were similar to those in WT mice, therefore inappropriate to iron burden and Bmp6 mRNA levels. Hepcidin mRNA (Hamp) expression was lower in Hfe KO mice than in WT mice, consistent with previous reports. To further explore the BMP6 pathway in mice lacking Hfe, WT and Hfe KO mice were challenged with an iron-enriched diet. While Bmp6 mRNA, transferrin saturation and liver iron were increased in iron-fed WT mice to levels comparable to those of Hfe KO mice, phopsho-Smad 1/5/8 proteins, Id1 mRNA, and Hamp mRNA expression levels were significantly lower in Hfe KO than in WT mice. Collectively, our data indicate that HFE is involved in the downstream signals of BMP6 that are triggered by iron.

23 PHOSPHORYLATION OF RECEPTOR-ACTIVATED SMAD1/5/8 IS NOT ENHANCED IN HFE-DEFICIENT MICE DESPITE INCREASED BMP6 EXPRESSION Leon Kautz, PhD student¹, Delphine Meynard, PhD², Valerie Darnaud², Helene Coppin, PhD² and Marie-Paule Roth, MD, PhD² ¹INSERM; ²INSERM U563 (Presented By: Leon Kautz, PhD)

Although the site of HFE regulatory function is the hepatocyte, the exact mechanisms by which HFE regulates iron homeostasis remain elusive. Because the expected up- regulation of hepcidin expression in response to iron loading is impaired in hemochromatosis patients, HFE is thought to be involved in the regulation of hepcidin expression. The role of the BMPSmad signaling pathway in directing hepcidin expression is now well established and we recently showed that BMP6, whose mRNA expression is regulated by iron in vivo, is critical to activate this signaling cascade. However, whether HFE has a role in this pathway is still unknown. In this study, we used a mouse model of hemochromatosis to determine how Hfe-deficiency affects the Bmp6/Smad signaling pathway in vivo. Iron-loaded Hfe-deficient mice retain the ability to increase Bmp6 in response to body iron excess, as do mice with secondary iron overload or mice with genetic iron overload due to the inactivation of the Smad4 or the Hamp1 gene. However, Smad1/5/8 phosphorylation (an indicator of BMP signaling), translocation of the receptor-activated Smads to the nuclei of hepatocytes, and hepcidin synthesis are all inappropriately low in these mice, given their liver iron burden. This is the first demonstration that lack of HFE impairs propagation of the signaling cascade induced by the BMP6 ligand and suggests that HFE and the BMP type I and II serine/threonine kinase receptors are associated at the hepatocyte cell membrane and that this association is required to ensure proper signal transduction. We also showed by immunochemistry that BMP6 localizes to the hepatocyte basolateral membrane domain, as do hemojuvelin and TFR2, which suggests a functional interaction of these molecules in the context of iron metabolism regulation. HFE, TFR2 and other proteins like BMP6, its receptors and hemojuvelin would then form in this functional membrane domain an iron signaling complex that induces hepcidin transcription via Smad proteins. 71

24 INTERACTION OF THE HEREDITARY HEMOCHROMATOSIS PROTEIN, HFE, WITH TRANSFERRIN RECEPTOR 2 IS REQUIRED FOR TRANSFERRIN-INDUCED HEPCIDIN EXPRESSION Junwei Gao, PhD¹, Juxing Chen, PhD¹, Maxwell Kramer, BA¹, Hidekazu Tsukamoto, MD², An-Sheng Zhang, MD¹ and Caroline Enns¹ ¹Oregon Health & Science University; ²University of Southern California (Presented By: Caroline Enns, PhD)

The mechanisms that allow the body to sense iron levels in order to maintain iron homeostasis are unknown. Patients with the most common form of hereditary iron overload have mutations in the hereditary hemochromatosis protein HFE. They have lower levels of hepcidin, than unaffected individuals. Hepcidin, a hepatic peptide hormone, negatively regulates iron efflux from the intestines into the blood. We report two hepatic cell lines, WIF-B cells and HepG2 cells transfected with HFE, where hepcidin expression responded to iron-loaded transferrin. The response was abolished when endogenous transferrin receptor 2 (TfR2) was suppressed or in primary hepatocytes lacking either functional TfR2 or HFE. Furthermore, transferrin-treated HepG2 cells transfected with HFE chimeras containing only the α3 and cytoplasmic domains upregulated hepcidin expression. Since the HFE α3 domain interacts with TfR2, these results support our finding that TfR2/HFE complex is required for transcriptional regulation of hepcidin by holo-Tf. Supported by NIH DK54488.

25 REGULATION OF HEPCIDIN BY DIETARY IRON IN MICE WITH FUNCTIONAL LOSS OF HFE AND TRANSFERRIN RECEPTOR 2 Robert Fleming, MD, Robert Britton, PhD, Mary Migas, MS, Margaret Rozier, BSc, Abdul Waheed, PhD and William Sly, MD Saint Louis University School of Medicine (Presented By: Robert Fleming, MD)

Introduction: Functional loss of either HFE or transferrin receptor 2 (TfR2) leads to decreased liver hepcidin (HAMP) expression and hereditary hemochromatosis. Whether these molecules regulate hepcidin by a common pathway or by separate signal transduction pathways is unknown. The aim of this study was to compare the regulation of hepcidin by dietary iron in mice in which each of these genes had been disrupted individually or together. Methods: We bred Hfe knockout and Tfr2(Y245X) mice to homogeneity on an FVB background and generated lines homozygous for each gene disruption individually or together. Mice with each of the four genotypes were weaned at 21 days onto one of four different diets: <20 ppm iron, 60 ppm iron, 250 ppm iron, or 25,000 ppm iron. Mice were sacrificed at 28 days and analysis of tissue non-heme iron content, transferrin saturation, hematocrit was performed on male mice (N=3-5 per each of 16 groups). Expression of Hamp1 mRNA was quantified by real-time RT-PCR and normalized to ß-actin. Results: Increases in dietary iron were associated with increases in both splenic iron and hepatic iron concentrations in each of the four mouse lines. Hamp1 mRNA levels varied with dietary iron content. Disruption of either Hfe or Tfr2 led to increased hepatic iron and decreased splenic iron compared with wild-type mice on each of the four diets. The magnitude of hepatic iron loading and splenic iron sparing was overall greater in the Tfr2(Y245X) mice compared with the Hfe knockout mice. Tissue iron concentrations were not significantly different in the combined Hfe/Tfr2 mice compared with Tfr2 mutant mice. On the 60 ppm or 250 ppm iron diets, hematocrits were higher in the Tfr2(Y245X) mice, Hfe knockout mice or combined mutant mice (m=48) compared with wild type mice (m=44). While Hamp1 mRNA levels were lower in the Hfe knockout mice compared with wild-type mice on each diet, hepcidin expression was nonetheless regulated by dietary iron. Similar findings were observed in Tfr2(Y245X) mice. The combined Hfe/Tfr2 mice demonstrated markedly lower Hamp1 expression than did disruption of either gene individually. Regulation of hepcidin by dietary iron was not completely ablated, however, despite functional loss of both Hfe and Tfr2. Conclusions: Hepcidin expression at any given level of dietary iron is diminished in mice with functional loss of Hfe or Tfr2. However, Hamp1 mRNA expression increases as dietary iron is increased, despite loss of either of these molecules. Combined loss of Hfe and Tfr2 causes a marked decrease in hepcidin expression at any given level of dietary iron. These results raise the possibility that Hfe and Tfr2 each independently augment a separate iron signaling pathway.

26 INVESTIGATION OF HFE-DEPENDENT REGULATION OF HEPCIDIN EXPRESSION Paul Schmidt, PhD¹, Mark Fleming, MD, DPhil¹ and Nancy Andrews, MD, PhD² ¹Children's Hospital Boston; ²Duke University School of Medicine (Presented By: Paul Schmidt)

Hereditary hemochromatosis (HH) is caused by mutations in several genes, including the classic, type I HH gene (HFE). All forms of HH share the common pathophysiology of inappropriately increased dietary iron uptake as compared to total body iron burden. We and others previously demonstrated that transferrin receptor 1 (TFR1) normally acts to sequester HFE in a HFE/TFR1 complex that can be displaced by iron bound transferrin (TF-Fe), and have proposed that this competition is a crucial part of a liver-centered, systemic iron regulatory pathway. In particular, we have previously showed that expression of full length Hfe under the control of the liver-specific transthyretin (Ttr) promoter not only corrects the Hfe-/- iron overloaded phenotype, but also leads to iron deficiency and increased hepcidin expression. Here we refined this model by employing several additional transgenic mouse lines in which variant forms ofHfe are expressed under the control of the Ttr promoter. First, to assess the effect of the common human HFE C282Y disease-causing mutation in our transgenic system, we introduced the analogous murine C294Y missense mutation into our Ttr-Hfe transgene. As was true in previously published work, we found that the C294Y allele is not a complete null allele and can partially rescue the Hfe-/- phenotype. Secondly, we examined whether the cytoplasmic tail of Hfe was required for the Hfe-modulated regulation of hepcidin expression in the liver by expressing Hfe lacking its cytoplasmic domain in Hfe-/- mice. We found that truncated Hfe lacking its cytoplasmic tail was equally effective in inducing an iron deficient state in Hfe-/- mice, indicating that the cytoplasmic tail is not essential for function. Finally, it has been well documented that mutations in hemojuvelin (HJV) lead to a severe form of juvenile hemochromatosis and that HJV plays a key role in the regulation of hepcidin through its stimulation of the bone morphogenetic protein (BMP) signaling pathway. It has been postulated that HFE is upstream in this iron regulatory signaling cascade. Thus, we bred a full length Ttr-Hfe transgene onto an Hjv-/- mouse background in order to determine if Hjv is necessary for the Hfe-dependent modulation of hepcidin. We found that this transgene did not complement the high Fe-Tf saturation and non-heme liver iron of Hjv-/- animals. Taken together, our data suggests that the Ttr-Hfe transgenic mouse is an excellent tool for elucidating the Hfe-dependent modulation of hepcidin expression. This work demonstrates that the cytoplasmic domain of HFE is not essential for its role in the regulation of hepcidin expression, and that HFE is upstream of HJV in the hepcidin signaling pathway. Future work will help to clarify the role of HFE and other HH genes in hepcidin regulation.

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27 IRON DEPENDENT REGULATION OF MDM2 INFLUENCES P53 ACTIVITY AND HEPATIC CARCINOGENESIS Paola Dongiovanni, PhD², Anna Ludovica Fracanzani, MD², Gaetano Cairo, PhD³, Chiara Paola Megazzini, PhD², Stefano Gatti, MD4, Raffaela Rametta, PhD², Silvia Fargion, MD² and Luca Valenti, MD¹ ¹University of Milano; ²Internal Medicine, University of Milano; ³General Pathology, University of Milano; 4Surgery, Policlinico Hospital Milano (Presented By: Luca Valenti)

Iron overload is a risk factor for hepatocarcinoma, whereas iron chelators are being evaluated as anti-neoplastic agents, but the biological pathways involved are poorly characterized. Microarrays analysis of gene expression profiles in immortalized mouse hepatocytes exposed to iron or the iron chelator deferoxamine showed that iron addition downregulated, whereas iron chelation upregulated mRNA levels of MDM2, an ubiquitin ligase involved in the degradation of p53, transcription factor that regulates antioxidant response and apoptosis and is frequently mutated in hepatocarcinoma. Down-regulation of MDM2 by iron was confirmed at protein levels, and was associated with upregulation of p53. Conversely, iron deficiency induced MDM2 and repressed p53 in mouse hepatocytes and rat livers. Antioxidants inhibited iron- dependent increase in p53. Ironpoor monocytes isolated from patients with hereditary hemochromatosis showed increased MDM2 expression associated with reduced expression of p53 and its target genes with antioxidant and proapoptotic activity. Consistently with ubiquitinationcontrolled modulation of p53 levels, iron status influenced p53 ubiquitination and degradation rate in human monocytes, and the MDM2 inhibitor Nutlin-3 increased p53 levels in iron-depleted cells. In line with experimental data, the MDM2 –309T>G promoter polymorphism, previously linked with increased MDM2 and lower p53 activity, was associated with increased hepatocarcinoma risk in cirrhotic patients with hereditary hemochromatosis (n=72, OR 2.1; P=0.0085), and with hemochromatosis mutations in patients with hepatocarcinoma from the general population (n=143, p=0.05), suggesting an interaction between genetic factors predisposing to iron accumulation and lower antioxidant response in the pathogenesis of hepatocarcinoma. Conclusion: Iron status influences p53 activity and antioxidant response by modulating MDM2 expression. This regulatory mechanism possibly protects from iron dependent cancerogenesis. MDM2 inhibitors may enhance the antineoplastic activity of iron chelators.

28 IRON CHELATORS FOR THE TREATMENT OF PANCREATIC CANCER: MECHANISM OF ACTION INVOLVING THE IRON-REGULATED GROWTH AND METASTASIS SUPPRESSOR, NDRG-1 Zaklina Kovacevic and Des R. Richardson, Professor of Cell Biology University of Sydney (Presented By: Zaklina Kovacevic)

Introduction: Pancreatic cancer (PaCa) is a belligerent disease with a 95% mortality rate primarily due to limited treatment options. The aim of this study was to examine the efficacy of novel iron (Fe) chelators, Bp4eT and Dp44mT, against PaCa. These new compounds, which have been specifically developed as anti-cancer agents in our laboratory, have previously been shown to exhibit potent anti-proliferative activity against a range of cancers both in vitro and in vivo (Richardson, DR et al. PNAS USA 2006,103:14901-6). Their mechanism of action is partly mediated by their ability to affect a range of proteins involved in tumorigenesis. One such protein, the iron- regulated N-myc downstream regulated gene-1 (Ndrg-1), a growth and metastasis suppressor, was found to be significantly up-regulated by Fe chelators in PaCa cells (Kovacevic, Z et al. BBA 2008, 1783:1981-92; Richardson, DR et al. Blood 2004, 104:2967-75). Ndrg-1 has been shown to inhibit growth and metastasis of PaCa although the mechanisms behind this are unclear. Hence, another aim of this study was to further investigate the molecular effects of Ndrg-1 up-regulation in PaCa and its potential therapeutic benefits. Methods: The anti-proliferative effects of novel Fe chelators Bp4et and Dp44mT, were examined in vitro using MTT assays with 6 PaCa cell lines and compared to currently used treatments gemcitabine and 5-fluorouracil. Potential molecular targets of Ndrg-1 were identified using micro-array analysis, RT-PCR and western blots on MIAPaCa-2 cells transfected with Ndrg-1. Results and Discussion: Our MTT analysis revealed that the Fe chelators Bp4et and Dp44mT were able to significantly inhibit proliferation of PaCa. Moreover, these agents were at least 100-fold more effective than the gold-standard treatment for PaCa, gemcitabine. These results demonstrate, for the first time, that Fe chelators may be an effective new treatment strategy against PaCa. To understand the effect of Fe chelators on PaCa, we further investigated one of its target genes, the metastasis suppressor Ndrg-1, and its molecular function in PaCa. Ndrg-1 was found to be up-regulated by Bp4eT and Dp44mT in each of the 6 PaCa cell lines, and may be one of the key mechanisms by which these agents function to inhibit growth and metastasis. Our study has identified two novel Ndrg-1 protein targets, Nedd4L and Glis3, both of which are up-regulated in PaCa cells. Nedd4L is involved in inhibiting the TGF-β pathway that is often oncogenic in PaCa. Glis3 is a transcription factor with a critical role in pancreatic development. Furthermore, the p53 regulatory gene, MDM2 has also been identified as another Ndrg-1 target. Interestingly, we found that Ndrg-1 up-regulates the p60MDM2 isoform, which functions to protect p53 by preventing the full length p90MDM2 isoform from mediating the degradation of p53. These data provide the first evidence that Ndrg-1 may protect the tumor-suppressor p53, leading to G1/S arrest and inhibition of proliferation. In conclusion, our study has identified novel Fe chelators as a promising new strategy against PaCa, showing considerably more activity than the best current treatments. We have also identified three novel Ndrg-1 protein targets in PaCa including Nedd4L, Glis3 and p60MDM2 which have revealed new insights into the function of Ndrg-1.

29 FERRITIN H INDUCTION BY HISTONE DEACETYLASE INHIBITORS Wei Wang, PhD, Xiumin Di, MSc, Suzy Torti, PhD and Frank M. Torti, MD Wake Forest University School of Medicine (Presented By: Suzy Torti, PhD)

Ferritin, an iron storage protein, plays a key role in maintenance of iron homeostasis. Over-expression of ferritin H has been shown to deplete intracellular iron and inhibit cell growth. Here we demonstrate that ferritin H is induced by histone deacetylase inhibitors (HDACIs), a promising new class of anti-cancer drugs. Cultured cancer cells were treated with HDACI and the effect on ferritin H mRNA measured by real-time RT-PCR. Levels of ferritin H mRNA were induced in a dose and time dependent manner. Consistent with this result, levels of ferritin H protein were also elevated. Deletion analysis and EMSA assays revealed that the induction of ferritin H occurred at a transcriptional level via Sp1 and NF-Y binding sites near the transcriptional start site of the human ferritin H promoter. Classically, HDACIs modulate gene expression by increasing histone acetylation in the gene promoter region. However, chromatin immunoprecipitation assays demonstrate that HDACImediated increases in ferritin H occur via a different mechanism, increasing NF-Y binding to the ferritin H promoter without directly increasing the association of acetylated histone to the ferritin H promoter. To assess the contribution of ferritin H induction to the cellular response to HDACI, ferritin H induction was blocked by knockdown with siRNA. Knockdown of ferritin H attenuated the HDACI-mediated apoptotic response. These results suggest that ferritin H induction may contribute to the apoptotic function of HDACIs.

30 IRON TRANSPORT PROTEINS ARE INFLUENCED BY WNT SIGNALLING Matthew Brookes, Samuel Ford, Tariq Iqbal and Chris Tselepis University of Birmingham (Presented By: Chris Tselepis)

Introduction: Emerging evidence suggests that iron is implicated in colorectal carcinogenesis. We have previously demonstrated that the expression of the cellular iron import proteins (transferrin receptor 1 and DMT-1) is increased in expression in colorectal cancer, whilst there a loss of expression and functionality of the cellular iron export proteins. What causes this modulation remains undetermined. With more iron being acquired and less being exported this results in an accumulation of iron in cancer cells. However, according to IRP/IRE signalling increased cellular iron loading should result in decreased cellular import and increased cellular export which is not observed in tumour tissue. This would suggest that there is another competing pathway which modulates the expression of these cellular iron transport proteins in colorectal carcinogenesis. We have previously shown that increased cellular iron deposition can amplify the Wnt signalling pathway; the major cancer causing pathway in the colon. 73

Wnt signalling is activated commonly by mutations in the tumour suppressor gene APC (adenomatous polyposis coli) which culminates in Wnt targets. These include c-myc an oncogene which has previously been shown to modulate the expression of iron transport proteins. Thus our hypothesis is that iron triggers c-myc expression by amplifying Wnt signalling and c-myc can increase the expression of TfR1, DMT-1 and decrease the expression of ferritin despite high cellular iron levels. Methods: Colorectal cell lines RKO (wild-type APC) and SW480 (mutated APC) were cultured in the presence of either ferrous sulphate (100uM) or hemin (50uM) for up to 48hrs. Cells were then lysed and expression of iron transport proteins (TfR1, DMT-1, ferritin and ferroportin) assessed by qRT-PCR and western blotting. Additionally, cells were transiently transfected with either c-myc or IRP-2 and cellular iron concentration determined using a ferrozine assay. Colonic adenomatous tissue from patients with i) no evidence of colorectal cancer (n=8) and ii) familial adenomatous polyposis coli (a condition associated with a germline mutation in APC; n=4), were comparably analysed for expression of iron transport proteins and c-myc. Results: Challenging wild type APC containing RKO cells with iron either as inorganic iron or hemin resulted in decreased TfR1, DMT-1 and increased ferritin expression. However, overexpression of either c-myc or IRP-2 caused increased expression of TfR1, DMT-1 and decreased ferritin expression. These changes were associated with increased cellular iron. Challenging APC mutant SW480 cells with iron resulted in increased TfR1, DMT-1 and decreased ferritin expression which could be reversed when reconstituting with wild type APC. mRNA expression analyses for adenomatous tissue showed statistical fold increases in TfR1, DMT1 and c-myc expression in 5/12, 7/12 and 9/12 tissue samples respectively. Furthermore, correlation analyses suggested that both TfR1 and DMT1 were tightly correlated with c-myc expression (R2>0.94 and 0.81 respectively). Discussions: This study suggests that c-myc which is induced by iron-mediated Wnt signalling is crucial in increasing TfR1 and DMT-1 and repressing ferritin expression in the face of high cellular iron. This suggests that the expression of iron transport proteins is influenced not only by the well characterised IRE/IRP pathway but in addition Wnt signalling.

31 DEFERASIROX IS THE ONLY IRON CHELATOR INDUCING NF-KB INHIBITION IN MDS PATIENTS AND IN LEUKEMIC CELL LINES AND ACTS INDEPENDENTLY FROM REACTIVE OXYGEN SPECIES REDUCTION Emanuela Messa, MD, Sonia Carturan, PhD, Chiara Maffè, PhD, Marisa Pautasso, MD, Enrico Bracco, PhD, Antonella Roetto, PhD, Francesca Messa, PhD, Francesca Arruga, PhD, Ilaria Defilippi, PhD, Valentina Rosso, PhD, Chiara Zanone, PhD, Daniela Cilloni, MD, PhD and Giuseppe Saglio, MD Dept. of Clinical and Biological Sciences, University of Turin (Presented By: Emanuela Messa, MD)

Iron overload is a critical issue for low risk myelodisplastic syndrome (MDS) patients with a long tranfusional history. Iron chelation is a proven tool improving survival in MDS but can also increase haemoglobin level in some cases with different drugs and modalities. Recently a once daily oral chelator deferasirox became available for MDS patients and it has been described an haemoglobin improvement in many patients within a few months of treatment. Moreover it was demonstrated that the transcriptional factor NF-kB is abnormally activated in MDS blast cells. Its pathway can be mediated by a broad variety of stimuli, among them also reactive oxygen species (ROS) but it is activated even in the absence of iron overload. Aim of our study was to compare the effects of the 3 commercially available iron chelators on NF-kB activity in MDS and to identify a possible mechanism responsible for the observed reduced transfusion requirement during iron chelation therapy. 40 PB samples from MDS patients with different diagnosis were collected. 21 out of 40 presented iron overload. Mononuclear cells were incubated with 50 microM deferasirox for 18 hrs. K562 and HL60 cell lines as controls were incubated with deferasirox 50 microM for 18 hrs, deferiprone 0,5 mM and deferioxamine 0,5 mM (both for 30 mins) and with the DLDithiothreitol 100 mM for 18 hours. NF-kB activity was evaluated using both EMSA and ELISA methods. Apoptosis was evaluated by FACS for annexin V expresion. ROS assay was performed by FACS analysis after incubation with 2’-7’- dichlorofluorescin diacetate. Chemosensitivity test was performed by FACS analysis for annexin V expression of K562 cells incubated with deferasirox 50 microM for 18 hours and etoposide 10 microM for 72 hrs. Deferasirox incubation induced a significant decrease of NF-kB activity both in HL60 and K562 cells lines. We detected an increased activation of NF-kB in 26 out of 40 patients as compared to healthy subjects. No significant difference was detected in NF-kB activity by comparing patients with or without iron overload (p=0,5). Among patients with increased NF-kB activity the incubation with deferasirox induced a significant reduction of it (p=0,0002). Apoptosis was not significantly triggered by incubation with any of the different chelators. HL60 and K562 cells incubation with the deferiprone and deferioxamine failed to induce a significant reduction of NF-kB activity even if ROS level reduction was comparable after incubation with all the three drugs. K562 cells incubation with both etoposide and deferasirox induce a significant increase in apopotosis rate. NF-kB is abnormally activated in MDS patients without a strong relation to iron overload status of the patients. Only deferasirox induces NF-kB inhibition while deferioxamine and deferiprone do not despite a similar ROS scavenging effect. Due to its peculiar effect, deferasirox is able to potentiate chemoterapy activity on blast cells. Our in vitro observation could be an explanation for the early improvement of hemoglobin levels observed in some patients under deferasirox chelation that seems not related to a sharp decrease in iron overload.

32 CHARACTERIZATION OF THE PROMOTER REGION OF THE HAMP GENE IN THE BLACK SOUTH AFRICAN POPULATION AND ITS POSSIBLE ASSOCIATION WITH OESOPHAGEAL CANCER Nathaniel McGregor, BSc (Hon), Ann Louw, PhD, Louise Warnich, PhD and Monique Zaahl, PhD University of Stellenbosch (Presented By: Nathaniel McGregor, BSc (Hon))

Objectives: The HAMP gene; encoding for the 25 amino acid hepcidin protein, is paramount in the regulation of the bodily iron status. Mutations disrupting the regular expression of this gene may therefore result in an irregular bodily iron standing, i.e. iron overload. An iron overload status is a previously described risk factor in the development of numerous cancers, oesophageal cancer (OC) included, and therefore the dysregulation of the HAMP gene may be implicated. Methods: Mutational analysis of approximately 1500 bp of the 5’UTR of the HAMP gene was performed to determine the effect of promoter variants identified on gene expression. Initially, 50 Black, Xhosa-speaking South African individuals were screened for variants against a cohort of 50 population matched controls. Variants were identified by means of HEX-SSCP analysis and subsequent bi-directional sequencing analysis. These variants were cloned into a vector and transfected into HepG2 cells, to assess HAMP gene expression levels in relation to the specific variants. Exogenous stimuli, namely ferric ammonium citrate (FAC), interferon gamma (INF-γ) and lipopolysaccharides (LPS) were used to stimulate gene expression by replicating hypoxia within the cells. Results: Variants identified within the promoter region of the HAMP gene comprise: -153 C/T, -188 C/T, -429 G/T and -582 A/G; with the -429 G/T variant being of particular interest. The -429 G/T variant was found to occur at a low frequency within the general Mixed Ancestry, Caucasian and Black South African populations contradictory to the high frequency identified in the Black South African OC patient cohort. This variant is found to disrupt a previously identified p53 response element required for the regulation of HAMP gene expression. The expression profile for the -429 G/T variant showed a definite decrease in the transcriptional activity of the HAMP gene in the presence of the variant. Treatment with exogenous stimuli showed transcriptional up-regulation, as expected under wild-type conditions, however, in the presence of the variant even with this up-regulation, an expression level above that of the wild-type was rarely achieved. Conclusions: Variants found within regions crucial to the transcriptional regulation of the gene are able to alter gene expression, possibly contributing to disease pathogenesis. Dysregulation of the HAMP gene results in an increased uptake of dietary iron, which in turn could feed the proliferation of carcinogenic cells lines. The presence of the -429 G/T and -582 A/G variants indicate a trend in the alteration of HAMP gene expression. Whether this alteration is causative or merely accessory to the cancer phenotype exhibition is a hypothesis that warrants further investigation.

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33 IRON UPTAKE IN GRAM-NEGATIVE BACTERIA: THE ROLE OF PERIPLASMIC PROTEINS IN SIDEROPHORE AND HEME TRANSPORT Hans Vogel, Byron Chu, MSc, Cheryl Lau, BSc, Zoya Slavinskaya, BSc, Alicia Garcia-Herrero, PhD, Renee Otten, MSc and Frans Mulder, PhD University of Calgary (Presented By: Hans Vogel)

All bacteria, with the possible exception of the lactobacilli, have an absolute requirement for iron to promote regular growth and proliferation. In microorganisms and in all multi-cellular organisms iron is an essential component of the electrontransport chain and in some of the enzymes involved in nucleotide biosynthesis. Some bacteria such as Helicobacter pylori can survive by utilizing ferrous iron (Fe II), which is prevalent under anaerobic conditions and at low pH. However under the regular aerobic conditions and at normal pH-values iron is exclusively present in the ferric form (Fe III). Ferric iron is extremely insoluble and hence many bacteria secrete specific iron chelators, called siderophores, for solubilization. Some pathogenic bacteria can also scavenge for heme, or other host proteins, as an iron source inside the host. These bacteria possess intricate pathways for the acquisition of the siderophore or heme complexes. In Gram-negative bacteria the Fe III uptake systems are comprised of four components: an outer membrane receptor, a periplasmic binding protein, a cytoplasmic membrane transporter (CMT) and an intracellular ATPase protein that binds to the intracellular side of the CMT and energizes the transport into the cytoplasm. The siderophore binding protein collects the ferric-siderophore complex in the periplasmic space, the area in between the outer and the cytoplasmic membrane, which comprises about 30% of the overall cell volume. Subsequently the siderophore binding protein docks onto the periplasmic surface of the CMT to mediate transport. Transport of ferric-siderophore or -heme complexes through the outer membrane transporters is not a spontaneous event and the energy for this part of the uptake is provided by the TonB-system. The latter is comprised of three proteins TonB, ExbB and ExbD that are anchored in the cytoplasmic membrane. TonB can span the entire periplasmic space and it interacts directly with the outer membrane receptors. Here we will review our structural studies of the TonB and ExbD proteins. Moreover, we have studied the proteins FhuD and FepB from Escherichia coli, which mediate the transport of the hydroxamate and catecholate classes of siderophores, respectively. TonB has an unusual unfolded N-terminal region that is followed by a globular Cterminal region that has a marked three- or four-stranded beta-sheet structure. The folded TonB domain binds along the edge of its beta sheet to a specific region in the outer membrane receptors, where it can dislodge the ‘cork’ domain, which prevents the entry of the siderophore, from the inside of the beta-barrel structure of the receptor. The periplasmic domain of the ExbD protein has a well-defined globular structure that can bind to the TonB domain. The siderophore-binding proteins have a unique bilobal structure that is distinct from other periplasmic binding proteins that are involved in the transport of other nutrients, such as amino acids. Our recent NMR results indicate that the binding of the ferric- siderophore enterobactin requires the unfolding of part of the C-terminal lobe of the siderophore-binding protein FepB. This mode of action is again distinct from the regular periplasmic binding proteins, which undergo typical large-scale domain reorientation movements. Clearly the acquisition of iron in Gram-negative bacteria relies on periplasmic proteins that have unique properties and characteristics.

34 THE ADAPTATION TO DIFFERENT DEGREES OF IRON DEFICIENCY IN SACCHAROMYCES CEREVISIAE DEPENDS ON THE AUTO- AND CROSS- REGULATION OF THE MRNA-DESTABILIZING PROTEINS CTH1 AND CTH2 Sandra Vergara, BSc and Dennis Thiele, PhD Duke University (Presented By: Sandra Vergara, BSc)

Under conditions of limited environmental Fe, the Saccharomyces cerevisiae transcription factors Aft1 and Aft2 induce the expression of genes whose protein products are involved in the acquisition and mobilization of Fe. These transcription factors also induce the expression of two genes encoding the paralogous Cth1 and Cth2 proteins, which complementarily mediate a genome-wide remodeling of metabolism facilitating the utilization of limited cellular Fe. Cth1 and Cth2 belong to the wellconserved family of CCCH-tandem zinc finger family of mRNA-binding proteins. Under conditions of limited Fe, Cth1 and Cth2 bind to AU-rich elements (AREs) within the 3’unstranslated regions (3’UTRs) of select mRNAs, thus accelerating their rate of decay. Messenger RNAs bound by Cth1 and Cth2 include those encoding proteins involved in highly Fe-demanding processes such mitochondrial respiration, the TCA cycle, amino acid biosynthesis, and heme biosynthesis. Moreover, while Cth1 predominantly targets ~20 mRNAs that encode proteins with mitochondrial functions, Cth2 targets both these mRNAs and ~80 additional mRNAs with a broader spectrum of Fe-dependent or Fe-storage functions. The physiological consequence of Cth1- and Cth2-dependent mRNA degradation is the metabolic adaptation to conditions of low Fe, where Fe utilization is prioritized away from non-essential processes and storage. Our recent data indicate that the pattern of expression of Cth1 is different from Cth2 during Fe-deficiency. Immunoblotting experiments reveal that while Cth2 expression gradually increases during Fe-deficiency over time, Cth1 is more rapidly and only transiently expressed soon after the Fe-deficient conditions are imposed. In the absence of the paralogous protein, however, both proteins reach maximum expression levels more rapidly, and these levels are significantly higher than in wild type cells grown under the same conditions. Furthermore, visual inspection of the CTH1 and CTH2 3’UTRs reveal putative ARE sequences, raising the possibility that both mRNAs may themselves be under the regulation of the Cth1 and Cth2 proteins. Yeast three-hybrid assays, Lac-Z reporter assays, RNA blotting experiments, as well as other biochemical and genetic assays indicate that the CTH1 and CTH2 genes are not only activated at the transcriptional level in response to Fe-limitation by Aft1 and Aft2, but also that the Cth1 and Cth2 proteins auto- and trans-regulate their expression via the modulation of their mRNA stability. This form of tight regulation may be a cellular strategy to adjust to either mild or transient versus severe or sustained forms Fe- deficiency.

35 HEPCIDIN: A CLUE FOR THE ROLE OF LYMPHOCYTES IN IRON HOMEOSTASIS Jorge Pinto, PhD¹, Vera Dias, MsC², Heinz Zoller, MD, PhD³, Pedro Rodrigues, PhD², Pedro Ramos², Graça Porto, MD, PhD² and Maria de Sousa, MD, PhD² ¹Institute for Molecular and Cell Biology – IBMC; ²Iron Genes and Immune System, Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal; ³Gastroenterology and Hepatology, Medical University, Innsbruck, Austria (Presented By: Jorge Pinto, PhD)

Introduction: Experimental and clinical evidence suggest a role for lymphocytes in iron homeostasis, with increased severity of iron overload observed in Hereditary Hemochromatosis patients and in animal models of iron overload with deficiencies in lymphocyte numbers. However, no mechanism(s) has been suggested to explain these observations. Aim: To study hepcidin expression by lymphocytes and assess its contribution to the role of lymphocytes in iron homeostasis. Methods: To determine how lymphocytes may influence iron metabolism, we studied the expression of hepcidin in Peripheral Blood Lymphocytes and in lymphocyte populations in steady-state conditions and in response to fully saturated holotransferrin by Real-time PCR. The effect of hepcidin in Ferroportin expression was analyzed by Real-Time PCR and analysis of Ferroportin-GFP localization performed by confocal immunofluorescence. Intracellular iron levels were quantified by measurement of 55Fe and silencing of gene expression in lymphocytes performed with siRNAs. Results: Hepcidin is expressed in lymphocytes and is up-regulated in response to holo-transferrin. Hepcidin levels induced by holo-transferrin are sufficient to reduce ferroportin 1 expression and cytoplasmic membrane localization in lymphocytes. We found that hepcidin expression increases with T-lymphocyte activation. siRNA- mediated silencing of hepcidin in activated T lymphocytes impaired cell proliferation and was associated with a reduction of intracellular iron levels. Analysis of the role of activation-related cytokines in the regulation of hepcidin by holo-transferrin in lymphocytes showed that Tumor Necrosis Factor-α expression increased with holo- transferrin treatments. siRNA-mediated silencing of TNF-α abrogated hepcidin up-regulation by holo-transferrin, suggesting the involvement of this cytokine in this process. Discussion: By increasing hepcidin expression in response to holo-transferrin, peripheral blood lymphocytes can be seen to exercise a function of first line of response to changes in transferrin saturation, as sensitive sensors of early changes in serum iron levels. The present findings that hepcidin modulates ferroportin expression, intracellular iron levels and activationinduced cell proliferation in lymphocytes suggests autocrine modulation of intracellular iron levels as the mechanism for control of lymphocyte proliferation by hepcidin. These results extend the role of hepcidin in the regulation of iron homeostasis. It is possible that, under the control of hepcidin, lymphocytes could be included in the iron-chelation machinery, which introduces “site-directed mobility” as a new asset in the overall role of this protein as the main effector in the 75 maintenance of systemic and localized iron homeostasis. Conclusion: With the demonstration, for the first time, of hepcidin synthesis by peripheral blood lymphocytes, of its regulation by holo-transferrin and of its involvement in T cell activation and proliferation, the present results put forward a molecular mechanism for the described role of lymphocytes in iron homeostasis.

36 INCREASED PHOSPHORYLATION OF SMAD1/5/8 AND HEPCIDIN EXPRESSION IN RESPONSE TO LPS IN BMP6-DEFICIENT MICE Leon Kautz, PhD student, Delphine Meynard, PhD, Valerie Darnaud, Celine Fournier, Marie-Paule Roth, MD, PhD and Helene Coppin, PhD INSERM (Presented By: Leon Kautz, PhD student)

Hepcidin is enhanced by dietary or parenteral iron loading and by inflammation. It normally provides a feed-back mechanism to limit intestinal absorption. However, its up- regulation in chronic inflammatory diseases results in inefficient macrophage iron release and subnormal intestinaliron absorption, limiting the availabilityof iron to red blood cell precursors. This is thought to play a major role in anemia of chronic disease, a prevalent condition that afflicts patients with a wide variety of diseases,including infections, malignancies, and rheumatologic disorders.It is not clear yet how inflammation stimulates hepcidin transcription. We recently showed that BMP6 is crucial to regulate hepcidin expression. Indeed, Bmp6-deficient mice have low levels of phosphorylated Smad proteins 1/5/8 in the liver, markedly reduced hepcidin synthesis, and massive iron overload. However, despite the lack of Bmp6, these mice are able to induce hepcidin expression in response to LPS- mediated inflammation. Because iron is unable to activate the Smad1/5/8 signaling pathway in the Bmp6-deficient mice, they offer a unique opportunity to analyse the in vivo response to inflammation. In a first step, we examined nuclear translocation of phosphorylated Smad proteins in liver extracts of Bmp6-deficient mice and wild-type controls treated with LPS or saline solution. Interestingly, we found that both phospho-Smad1/5/8 and phospho- Smad2/3 were strongly induced in the hepatocyte nuclei of Bmp6-deficient mice. We next examined expression of the Bmp 2, 4, 6, 7 and 9 transcripts in the liver of these mice by quantitative PCR and found no statistically significant difference between LPS and saline-treated animals, which suggests that BMP molecules are not the ligands that activate the BMP/Smad1/5/8 signaling pathway in response to LPS. We conclude from these experiments that the pathway that leads to the phosphorylation of the Smad proteins 1/5/8 is induced by a ligand other than BMPs. One candidate is TGF-beta, a cytokine induced by inflammation and known to classically activate the phosphorylation of the Smad proteins 2/3, a phenomenon also observed in Bmp6- deficient mice treated with LPS. Interestingly, TGF-beta was recently shown to use the same type II receptor but different type I receptors to induce the phosphorylation of both Smad2/3 and Smad1/5/8 in endothelial cells. To check the possibility that TGF-beta is the ligand that activates the signaling cascade directing hepcidin synthesis in response to LPS, we are currently using neutralizing antibodies and test whether these block hepcidin induction following treatment with LPS.

37 CELLULAR TRAFFICKING OF IRON AND IRON SULFUR CLUSTERS: THE ROLE OF MITONEET, A NOVEL OUTER MITOCHONDRIAL MEMBRANE PROTEIN WITH A LABILE IRON SULFUR CLUSTER Yael Harir, MSc², A. Conlan, MSc³, D. Michael, MSc, M. Shvartsman, MSc, D. Roisman-Sade, MSc, M. Paddock, PhD, P. Jennings, PhD, R. Mittler, PhD, I. Cabantchik, MD, PhD and Rachel Nechushtai, PhD¹ ¹Institute of Life Sciences/Hebrew University of Jerusalem; ²Hebrew University of Jerusalem; ³University of California San Diego (Presented By: Rachel Nechushtai, PhD)

MitoNEET, an outer mitochondrial membrane 2Fe-2S protein (ISP) that binds the insulin-sensitizing drug pioglitazone used as anti-type-2 diabetes (1), shows by X-ray- analysis a structure composed of two protomers intertwined forming a unique dimeric structure (among the ~650 known ISPs) (2). The presence of His instead of Cys in the iron sulfur cluster (ISC)-binding site of mitoNEET is unique and believed to confer ISC lability. However, the stability of ISC is increased by binding of pioglitazone (2) and by His87 to Cys87 mutation ( 2). Here we assessed MitoNEET's possible involvement in ISC trafficking by examining its ability to donate ISC and/or Fe to putative acceptor proteins and to mitochondria. Soluble recombinant mitoNEET (33-108 aa) incubated with apo-ferredoxin (apo-mFd) under reducing conditions evoked a decrease in 458nm and an increase in 423nm absorbance, indicating ISC transfer from mitoNEET to apo-mFd. This was confirmed by native PAGE as colorless apo-mFd got converted to red holo-mFd (containing ISC) after incubation with mitoNEET. The interaction of soluble recombinant mitoNEET (33-108 aa sequence) with apo-mFd followed by real time Surface Plasmon Resonance revealed an associating with apo-mFd but not holo-mFD (KA of 2.3x106 M and KD of 4.3x10- 7M-1) as predicted from structural analysis. mitoNEET’s ability to donate ISC's or iron to mitochondria was assessed in digitonin- permeabilized HEK293 cells double-labeled with fluorescent labile iron sensors rhodamine-phenanthroline in mitochondria and calcein-green in cytosol (4). Fluorescence imaging microscopy of permeabilized cells showed that wt mitoNEET caused a time dependent quenching in mitochondrial RPA fluorescence, indicating the transfer of reduced iron to the mitochondrial matrix residing probe. In solution, transfer of iron from wt mitoNEET to o-phenanthroline occurred only in the presence of exogenously added reducing agents. No transfer or protein-protein interactions were observed with the H87C mutant. These studies reveal that mitoNEET has a unique, labile ISC transferable in its entirety to cytosolic apo-ISP acceptor(s) such as ferredoxin or prone to release chelatable iron to mitochondria. The transfer in solution of ISC from wt-mitoNEET to other ISP's and of iron to iron(II) chelators, was obtained only in reducing environments, whereas transferred to mitochondria it occured spontaneously. Based on these and other observations we suggest that mitoNEET might play a functional role in the cellular trafficking of iron/ISC. Supported in part by ISF, NIH and EEC F6(LSHM-CT-2006-037296 Euroiron1). 1. Colca et al. (2004) Am J Physiol (Endocrinol Metab) 286:E252- 60; 2. Paddock et al. (2007) Proc Natl Acad Sci USA 104:14342-47; 3. Wiley et al. (2007) J Biol Chem 282:23745-49; Shvartsman et al. (2007) Am J Physiol (Cell) 293:C1383-94.

38 HUMAN TRANSFERRIN RECEPTOR-1 IS AN ENDOCYTOSING RECEPTOR FOR H-FERRITIN Li Li, PhD¹, Mary C. Nakamura, MD¹, Celia J. Fang, MD¹, James C. Ryan, MD¹, Erene C. Niemi, BA¹, Hisashi Arase, MD, PhD², Suzy V. Torti, PhD³, Frank M. Torti, MD³ and William E. Seaman, MD¹ ¹University of California San Francisco; ²Osaka Univeristy; ³Wake Forest University (Presented By: William E. Seaman, MD)

H-ferritin (HFt) binds specifically and saturably to a variety of cell types. We previously identified Tim-2 in mice as a receptor for HFt, but Tim-2 is not expressed in humans. The human B cell line 721.221 binds HFt. We therefore used a cDNA library from these cells for expression cloning of receptors for human HFt, using fluorescent HFt to screen for binding by flow cytometry. Eight of >100 clones were sequenced, and all eight encoded transferrin receptor-1 (TfR1). Expression of TfR1 in mouse Ba/F3 B cells confirmed that TfR1 binds HFt but not LFt, with binding detectable at ~10 nM and saturating at ~300nM. MOLT4 T cells were used to examine the fate of human HFt following binding to TfR1. As assessed by fluorescence microscopy, HFt was transported to endosomes. Unlike Tf, however, HFt subsequently entered lysosomes. We identified a mAb against TfR1 (M-A712) that blocked binding of HFt to TfR1. This mAb, however, did not block binding of transferrin (Tf) to TfR1, suggesting that the TfR1 binding sites for HFt and Tf differ. In accord with this, HFt did not block binding of Tf, and Tf only partially blocked binding by HFt, even in large molar excess. At no concentration did Tf facilitate binding by HFt to TfR1, demonstrating that Tf is not required for the binding of HFt. Using M-A712 anti-TfR1 mAb to block binding of HFt to TfR1, we assessed the role of TfR1 in the binding of HFt to cell lines and to fresh human peripheral blood cells, using flow cytometry. For cell lines that bound HFt, most binding was blocked by M-A712, indicating that binding was mediated through TfR1. In addition to 721.221 B cells and MOLT4 T cells, cell lines that bound HFt through TfR1 included K-562 erythroleukemia cells, U937 monocytes, HeLa cervical cancer cells, and 293T kidney epithelial cells. T cell blasts induced from the blood of normal volunteers by phytohemagglutinin, as well as B cell blasts from the same cultures, showed upregulation of TfR1, as previously reported by others, and these cells showed increased binding of HFt that was blocked by anti-TfR1. The peripheral blood cells from one volunteer included erythrocyte precursors, as identified by morphology and by the surface expression of glycophorin A but not CD45. These cells bound HFt at high levels, and binding was again blocked by anti-TfR1. Konijn and his colleagues have previously shown that human erythrocyte precursors bind 76 and endocytose HFt and they can use HFt as a source of iron for the synthesis of hemoglobin (J Biol Chem 103:1211, 2008). Our studies suggest that this utilization of HFt by erythrocyte precursors is mediated by TfR1. This is the first characterization of a human receptor for HFt. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and utilization of iron by these iron-binding molecules.

39 PRODUCTION OF CORRECTLY-FOLDED, BIOLOGICALLY-ACTIVE HEPCIDIN ENABLED REVISION OF THE PUBLISHED HEPCIDIN STRUCTURE AND GENERATION OF NEUTRALIZING ANTIBODIES Tara Arvedson, PhD, J. Brad Jordan, PhD, Keegan Cooke, Aaron Winters, Leszek Poppe, PhD, Mitsuru Haniu, Greg Dyas, Jacob Corcoran, Helen Kim, PhD, Hiko Kohno, PhD, Vivian Li, Ching Chen, PhD, Paul Schnier, PhD, Rashid Syed, PhD, Les P. Miranda, PhD, Janet Cheetham, PhD and Barbra J. Sasu, PhD Amgen (Presented By: Tara Arvedson, PhD)

Hepcidin, a 25 amino acid peptide with four disulfide bonds, has been shown to be a key component in the maintenance of iron homeostasis. Hepcidin elevation is proposed to be a contributing factor in the development of anemia in inflammatory patients and may be involved in progression of other diseases with an iron maldistribution component such as atherosclerosis and diabetes. Hepcidin neutralization may therefore have significant therapeutic potential. The aim of this study was to produce correctly- folded, biologically-active hepcidin and to use this material to generate high affinity, neutralizing antibodies. Successful hepcidin production was considered challenging due to the small size and highly disulfide bonded nature of the peptide. Several types of mature hepcidin were generated (E. coli-produced, Chinese hamster ovary (CHO) cell-expressed and synthetic) and the quality of these preparations was assessed through structural and biological comparison to purified urinary hepcidin. Structural equivalence was confirmed by comparing the 1D NMR spectra and disulfide bonding pattern of each sample. The disulfide connectivity was determined by partial reduction alkylation and all preparations had an equivalent bonding pattern (C1-C8, C2-C4, C3-C6 and C5-C7) which was distinct from the published connectivity. This connectivity was confirmed through determination of a full NMR solution structure of CHO-expressed hepcidin at high temperature (standard 2D NMR methods at 325K) and determination of a crystal structure of hepcidin associated with an anti-hepcidin Fab (1.9 Å resolution). All hepcidin samples had similar biological activity as indicated by comparable activity (20-60nM) observed in a cell based intracellular iron retention assay. This assay was based on an established assay with the addition of a β-lactamase reporter gene controlled by an iron-sensitive t ranslation control element. High affinity antibodies were generated using KLH-conjugated, E. coli-expressed human hepcidin as the immunogen and rank-ordered according to binding (1pM to 40nM affinity determined by Biacore and KinexA experiments) and neutralization activity in the cell based intracellular iron retention assay (IC50 Ab 2.7 = 14nM in the presence of EC50 hepcidin concentration). These antibodies were also able to neutralize 25µg hepcidin in vivo as measured in a previously reported hepcidin injection model. Characterization of hepcidin/Ab binding by Biacore analysis showed that all antibodies tested recognized a non linear epitope. Analysis of the hepcidin/anti-hepcidin Fab co-crystal structure indicated that the binding epitope consisted of non-contiguous amino acids and required intact disulfide bonds. In conclusion, structural characterization as a means of quality control for hepcidin preparations enabled determination of a complete hepcidin structure and correction of the previously proposed disulfide connectivity. In addition, use of these biologically-active hepcidin preparations made possible the production of high affinity neutralizing antibodies which can be employed to evaluate the therapeutic role of hepcidin neutralization in disease.

40 HEPCIDIN INDUCED INTERNALIZATION OF FERROPORTIN REQUIRES BINDING AND COOPERATIVE INTERACTION WITH JAK2 Ivana De Domenico, PhD, Diane Ward, PhD and Jerry Kaplan, PhD University of Utah (Presented By: Ivana De Domenico, PhD)

In vertebrates, iron homeostasis is regulated by the binding of hepcidin to the cells surface iron transporter ferroportin (Fpn) resulting in Fpn internalization and degradation. Hepcidin is a hormone secreted in response to iron loading and inflammation. The removal of Fpn from the surface of the cell leads to an increase in cellular ferritin and consequently cellular iron retention. We determined that hepcidin binding to Fpn results in the binding and activation of the protein kinase Jak2 which is required for the phosphorylation of Fpn. This step is necessary for Fpn internalization by clathrin-coated pits. Binding of Jak2 to Fpn is highly cooperative and requires hepcidin binding to both Fpn monomers. Jak2 will not bind to the Fpn dimer in the absence of one hepcidin binding site. Once Jak2 has bound to the Fpn dimer, both monomers must be capable of activating bound Jak2 in order for Fpn to be phosphorylated. These findings suggest that cooperation between the Fpn monomers is required for both hepcidin- mediated Jak2 activation and Fpn downregulation. This highly cooperative nature of Jak2 activation explains the dominant transmission of hepcidin-resistant Fpn disease. Furthermore, binding of Jak2 to Fpn leads to a transcriptional response that modulates inflammation in vivo and in vitro. This transcriptional response adds a third function to hepcidin as a signal transducer in addition to its antibacterial activity and its ability to downregulation Fpn.

41 MECHANISMS OF HEPCIDIN INHIBITION BY NOVEL TMPRSS6 (MATRIPTASE-2) MUTATIONS ASSOCIATED WITH IRIDA Laura Silvestri, PhD¹, Flavia Guillem², Alessia Pagani³, Antonella Nai³, Luigia De Falco4, Achille Iolascon4,5, Carole Beaumont², Bernard Grandchamp6,7 and Clara Camaschella³ ¹San Raffaele Scientific Institute; ²INSERM UMR773, University Paris Diderot, site Bichat, Paris, France; ³Vita-Salute San Raffaele University, Istituto Scientifico San Raffaele, Milan, Italy; 4Centre of Genetics Engineering (CEINGE) Advanced Biotechnologies, Naples, Italy; 5Department of Biochemistry and Medical Biotechnologies, University Federico II, Naples; 6INSERM UMR773, University Paris Diderot, site Bichat, Paris; 7APHP, Hôpital Xavier Bichat, Service de Biochimie Hormonale et Génétique, Paris, France (Presented By: Laura Silvestri, PhD)

Hepcidin is the master regulator of iron homeostasis since it controls the surface expression of the cellular iron exporter ferroportin on the duodenal enterocytes and macrophages. Hepcidin expression is increased in iron overload and inflammation and decreased in iron deficiency, hypoxia and erythropoiesis expansion. TMPRSS6, which encodes the hepatic serine-protease matriptase-2, is a negative regulator of hepcidin in vivo. TMPRSS6 mutations are associated with autosomal recessive iron deficiency iron refractory anemia (IRIDA) in patients and with a similar iron deficiency and hair abnormalities in Tmprss6-deficient mice and in Mask mouse, carrier of a truncated Tmprss6. We have recently shown that membrane associated hemojuvelin (m-HJV), which has a pivotal role in hepcidin activation acting as a BMP coreceptor, is the substrate of matriptase-2 (Silvestri et al, Cell Met 2008), that the MASK human homologue is unable to cleave m-HJV and that the serineprotease domain mutant identified in patients, R774W, has partial cleavage activity. Here we extend our studies on new TMPRSS6 mutations identified in IRIDA patients. Wild type and TMPRSS6 mutants affecting different domains of the protease (I212T and R271Q [N-terminal], G442R [CUB], D521N and E522K [LDLRA]) were analyzed in the presence of HJV and their interaction with HJV studied by coimmunoprecipitation in transfected HeLa cells. The mutant ability to inhibit hepcidin was assessed in Hep3B cells transfected with a hepcidin promoter-luciferase construct. When coexpressed with HJV, all matriptase-2 variants have partial inhibitory activity on hepcidin activity, except R271Q that shows an inhibitory activity comparable to wild type matriptase-2. All mutants retain only partial ability to cleave HJV from plasma membrane, except LDLRA mutants that are fully inactive. m-HJV decreases in a dose dependent manner in cells expressing increased concentration of wild type matriptase-2, does not change when MASK is expressed and is variably decreased in the presence of the different variants. Independently of their HJV cleavage/hepcidin inhibitory effect, all matriptase-2 variants interact with HJV. Our studies confirm that HJV is a substrate of matriptase-2. The inability to fully cleave HJV from plasma membrane might explain the clinical features of IRIDA, since the persistence of hemojuvelin on plasma membrane favors BMPdependent hepcidin activation, with consequent decreased serum iron and iron refractoriness. Acknowledgements: this work was supported by EU Contract N° LSHM-CT-2006-037296, Italian Telethon Foundation grant GGP08089.

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42 HEMOJUVELIN/NEOGENIN INTERACTION IS REQUIRED FOR BMP4-INDUCED HEPCIDIN EXPRESSION An-Sheng Zhang¹, Fan Yang², Jiaohong Wang³, Hidekazu Tsukamoto³ and Caroline Enns¹ ¹Oregon Health & Science University; ²California Institute of Technology; ³University of Southern California (Presented By: An-Sheng Zhang)

Hemojuvelin (HJV) is a GPI-linked protein, which binds both bone morphogenic proteins (BMPs) and neogenin. Cellular HJV acts as a BMP co-receptor to enhance the transcription of hepcidin, a key iron regulatory hormone secreted predominantly from liver hepatocytes. In this study we characterized the role of neogenin in HJV-regulated hepcidin expression. Both HJV and neogenin were expressed in liver hepatocytes. Knockdown of neogenin decreased BMP4-induced hepcidin mRNA levels by 15-fold in HJV-expressing HepG2 cells, but only by about 2-fold in cells transfected with either empty vector or the G99V mutant HJV that does not bind BMPs. Further studies indicated that disruption of HJV/neogenin interaction is responsible for the marked suppression of hepcidin expression. Moreover, in vivo studies showed that a soluble portion of neogenin that interacts with HJV, could significantly suppress hepatic hepcidin mRNA in mice. Together, these results suggest that the HJV/neogenin interaction is required for the BMP-mediated induction of hepcidin expression when HJV is expressed. Combined with our previous studies, our results support that hepatic neogenin possesses two functions, to mediate cellular HJV release and to guide the HJV-enhanced hepcidin expression.

43 RESULTS OF THE FIRST INTERNATIONAL ROUND ROBIN FOR THE QUANTIFICATION OF URINARY AND PLASMA HEPCIDIN ASSAYS: NEED FOR STANDARDISATION Joyce Kroot¹, Erwin Kemna, PhD², Sukhvinder Bansal, PhD³, Mark Busbridge, PhD4, Natascia Campostrini, MSc5, Domenico Girelli, MD, PhD5, Robert Hider, PhD³, Vasiliki Koliaraki, MSc6, Avgi Mamalaki, PhD6, Gordana Olbina, PhD7, Naohisa Tomosugi, MD8, Chris Tselepis, PhD9,¹°, Douglas Ward, PhD9,¹°, Tomas Ganz, MD, PhD¹¹, Jan Hendriks, PhD¹² and Dorine Swinkels, MD, PhD² ¹Radboud University Nijmegen Medical Center – Dept. of Clinical Chemistry; ²Radboud University Medical Center – Dept. of Clinical Chemistry; ³King’s College London – Pharmaceutical Sciences Division; 4Imperial College HealthCare Trust, Hammersmith Hospital – Dept. of Clinical Chemistry; 5University of Verona – Dept. of Clinical and Experimental Medicine, Section of Internal Medicine; 6Hellenic Pasteur Institute – Laboratory of Molecular Biology & Immunobiotechnology, Dept. of Biochemistry; 7Intrinsic Life Sciences; 8Kanazawa Medical University – 9Division of Advanced Medicine, Medical Research Institute/Division of Nephrology; ¹°University of Birmingham – CRUK Institute for Cancer Studies; ¹¹Intrinsic Life Sciences/David Geffen School of Medicine UCLA – Dept. of Medicine; ¹²Radboud University Nijmegen Medical Center – Dept. of Epidemiology and Biostatistics (Presented By: Joyce Kroot)

The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Quantification of hepcidin concentrations in urine and plasma holds promise as a biomarker for diagnosis and monitoring of disorders of iron metabolism. To date, various mass spectrometry (MS) and immunochemical (IC) methods, exploiting an internal or external standard, have been developed for hepcidin quantification. Only a few of them have been published. Differences in methodology and analytical performance, however, hinder the comparability of data. In this study we compared the analytical characteristics of several available urinary and plasma hepcidin assays as a first step towards standardization of hepcidin quantification methods. Six and 8 laboratories participated in an international interlaboratory evaluation of hepcidin levels from a common human urinary (n=8) and plasma (n=7) sample set, respectively, exploiting MS and IC based methods. Participating laboratories were requested to perform 3 hepcidin measurements for each sample on 4 different days, making a total of 96 urine and 84 plasma measurements per laboratory. The methods differed considerably in mean urine and plasma hepcidin level, i.e. range 2.9 – 427.1 nmol/mmol creatinine and 11.4 – 124.6 nM, respectively. The between- sample variation (distinctive ability) and the analytical variation are similar for both urine and plasma. Importantly, the contribution of the analytical variation to the total % of variance is low for all methods, i.e. < 15% and < 20% for urine and plasma, respectively, which indicates the suitability of all methods to distinguish hepcidin levels of different samples. Accordingly, mutual Spearman correlations were generally high. In summary, all methods have sufficient concordance to be used for research studies. Further harmonization between the various hepcidin assays would be facilitated by 1) introducing an internal standard for all MS-based methods; 2) reaching optimal antibody specificity for the IC-methods; 3) reaching consensus on level assignment and level adjustment of the calibrators used in every procedure; and 4) by regular testing of shared samples for quality control. Finally, clinical studies should provide information on which analytical characteristics are most pertinent for mechanistic, diagnostic and therapeutic applications.

44 ANTI-HEPCIDIN THERAPY IS AN EFFECTIVE TREATMENT FOR THE ANEMIA OF INFLAMMATION IN MICE Keegan Cooke, Jeanne Sloan, Adrienne Augustic, Tara Arvedson, Cherylene Plewa, Aaron Ellison, Jackie Sheng, Aaron Winters, Hongyan Li, C. Glenn Begley, Graham Molineux and Barbra J. Sasu Amgen (Presented By: Tara Arvedson, PhD)

Hepcidin is a 25aa peptide shown to be the central mediator of iron homeostasis. Hepcidin can be induced by inflammation, leading to speculation that it causes iron sequestration and limits response to erythropoietic stimulating agents (ESAs) in patients with inflammation. For this reason, the role of hepcidin in ESA resistance and the anemia of inflammation (AI) was examined. Adeno-associated virus (AAV) was used to induce over-expression of hepcidin in mice. Over-expression of mouse hepcidin 1 (mHepc1) for 4 months resulted in stunted growth and iron deficiency anemia (Hb 10.7 ± 0.8 g/dL compared to 13.1 ± 0.5 g/dL in control group; p<0.05). Over-expression of human hepcidin (hHepc) produced a similar phenotype. Forced expression of 30-100 ng/mL hHepc for as little as 2 weeks decreased serum iron and induced resistance to high dose ESA treatment (Hb response one week after ESA of only 0.7 ± 0.1 g/dL compared to 3.6 ± 0.3 g/dL in control animals; p<0.0001). This illustrated that hepcidin over-expression alone could limit response to ESA. To create an animal model of AI, mice were treated with Brucella abortus (BA). This led to IL-6 induction and hepcidin upregulation within 6 hours and anemia within 7 days (decrease in Hb varied between 2-5 g/dL in different experiments). Mice treated with BA did not respond to high dose ESA (Hb response of -1.0 ± 0.2 g/dL compared to 3.5 ± 0.05 g/dL in control animals; p<0.0001). shRNAs were identified which specifically down-regulated mHepc1 in vitro and in vivo. ShRNAmediated reduction of hepcidin to pre-inflammatory levels in BA-treated animals led to increased serum iron (315 ± 61 µg/dL compared to 84 ± 7 µg/dL for control shRNA treatment; p<0.01) and restored response to ESA treatment (Hb response of 2.2 ± 0.4 g/dL compared to 0.4 ± 0.2 g/dL for control shRNA group; p<0.005). These data indicated that hepcidin played a causative role in the development of AI and that neutralization of hepcidin constituted an effective anemia treatment in this model. High affinity antibodies against hHepc were generated and shown to be neutralizing in vitro and in vivo. Use of a representative antibody (Ab2.7; 110pM affinity) restored ESA response in AAV-hHepc animals (Hb response of 3.0 ± 0.4 g/dL compared to 0.2 ± 0.2 g/dL for control Ab treated group; p<0.001). Ab2.7 therapy also worked in combination with ESA to prevent progression of anemia after BA treatment (Hb change one week after treatment of -0.8 ± 0.8 g/dL compared to -4.0 ± 0.1 g/dL in control Ab treated animals; p<0.05). These data indicated that antibodies capable of suppressing hepcidin may be an effective treatment for AI. Iron maldistribution has been implicated in many diseases in addition to anemia including atherosclerosis, diabetes and neurodegenerative disorders. The availability of a reagent such as an anti-hepcidin antibody to manipulate iron uptake and redistribution in the body may allow exploration of the role of iron metabolism in these conditions.

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45 CHARACTERIZATION AND EXPRESSION ANALYSIS OF IMMUNE-RELATED IRON GENES DURING EXPERIMENTAL IRON OVERLOAD AND INFECTION IN FISH João Neves, MSc¹, Jonathan Wilson, PhD² and Pedro Rodrigues, PhD³ ¹Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal; ²CIIMAR; ³IBMC and ICBAS (Presented By: João Neves, MSc)

Iron is an essential element required for the growth and survival of most organisms. However, free iron is also toxic to cells, when in excess. Since many organisms, including fish, lack a regulated pathway for iron excretion, iron balance is maintained by a tight regulation of absorption in the intestine, responding to the level of body iron stores and to the amount needed for erythropoiesis and other functions. This tight regulation of iron metabolism keeps a balance between its benefits and toxic effects, accomplished by the interactions of several genes, many of which are also known to be involved in the response to infection, such as the iron carrier transferrin and the iron storage protein ferritin. The aims of this study were to identify and characterize transferrin and ferritin in sea bass (Dicentrarchus labrax), a marine fish that has acquired a great importance in European aquaculture, thus providing a better understanding of the role of immune-related iron genes in response to iron modulation and bacterial infection. Sea bass transferrin and ferritin H cDNAs were isolated from the liver by RT-PCR, cloned and characterized, and constitutive expression determined. Transferrin mRNA was also detected by in situ hybridization in the liver and brain. Fish were subjected to either experimental iron modulation (overload/deficiency) or bacterial infection with the pathogenic bacteria Photobacterium damselae, and transferrin and ferritin expression levels in the liver and brain were analyzed by real-time PCR, along with several other erythroid (RBC, hemoglobin, mean corpuscular volume) and iron related (transferrin saturation, serum and tissue iron) parameters. Transferrin constitutive expression was found to be highest in the liver, among all tested organs. Significant expression was also detected in the brain, particularly in a highly vascularized region between the hypothalamus and saccus vasculosus. Ferritin was expressed in all tested organs, although significantly higher levels were found in the liver. In the liver, transferring expression was found to decrease in response to infection and iron overload, and to increase in conditions of iron deficiency, whereas in the brain, expression was found to increase significantly during the first hours of infection, recovering to constitutive levels during its course. Ferritin, on the other hand, inversely reflected transferrin liver expression, increasing in infection and iron overload and decreasing in iron deficiency. In the brain, ferritin expression significantly increased during infection. This study demonstrates that transferrin and ferritin, besides their role in iron metabolism, have important functions during bacterial infection in fish. In addition to the liver, a surprising transcriptional response to infection was detected in the brain, both for transferrin and ferritin. Although the overall importance of the brain’s locally synthesized transferrin is uncertain, it may have a significant role in the interaction between brain iron metabolism and defense mechanisms.

46 THE HEMOCHROMATOSIS-ASSOCIATED HFE MUTATION PROTECTS MICE FROM SALMONELLA TYPHIMURIUM INFECTION VIA INDUCTION OF LIPOCALIN 2 Manfred Nairz¹, Igor Theurl², Andrea Schroll², Milan Theurl², Sabine Mair², Thomas Sonnweber², Gernot Fritsche², Ewald Lindner², Klaus Hantke³, Shizuo Akira4,5 and Günter Weiss² ¹Internal Medicine I; ²Department of Internal Medicine I, Clinical Immunology and Infectious Diseases, Medical University of Innsbruck, 6020 Innsbruck, Austria; ³Department of Microbiology/Membrane Physiology, University of Tübingen, 72076 Tübingen, Germany; 4Department of Host Defense, Osaka University, and Exploratory Research for Advanced Technology (ERATO), Japan; 5Science and Technology Corporation, Osaka 565-0871, Japan (Presented By: Manfred Nairz)

Mutations in HFE predispose to hereditary hemochromatosis type I, a frequent genetic disorder characterized by progressive parenchymal iron deposition and eventual organ failure. Since HFE mutations are associated with reduced iron levels within mononuclear phagocytes, we hypothesized that Hfe deficiency may be beneficial in infections with intramacrophage pathogens. Using Hfe+/+, Hfe+/- and Hfe-/- mice in a model of typhoid fever, we found that animals lacking one or both Hfe alleles are protected from systemic infection with Salmonella typhimurium, displaying prolonged survival and improved bacterial control. This increased resistance can be referred to an enhanced production of the siderophore-binding peptide lipocalin 2 and the reduced availability of iron for Salmonella engulfed by Hfe deficient macrophages. This effect is mediated via stimulation of lipocalin 2-dependent iron export from infected cells since Hfe-/- macrophages concurrently knocked out for lipocalin 2 are unable to efficiently control the infection or to withhold iron from intracellular Salmonella. Correspondingly, infection of Hfe+/+ and Hfe-/- mice with siderophore deficient Salmonella abolishes the protection conferred by the Hfe defect. Thus, by inducing the formation of the iron-capturing peptide lipocalin 2, the Hfe mutation harbors a genetically determined immunological advantage towards infections with intracellular pathogens such as Salmonella.

47 MYCOBACTERIA-INDUCED ANAEMIA REVISITED: A MOLECULAR APPROACH REVEALS THE INVOLVEMENT OF NRAMP-1 AND LIPOCALIN- 2, BUT NOT OF HEPCIDIN Pedro Rodrigues, PhD², Sandro Gomes, BsSc³, João Neves, BsMSc³, Sandra Gomes-Pereira, PhD³, Margarida Correia-Neves, PhD, VetD4, Cláudio Nunes-Alves, BSsc4, Rui Appelberg, PhD, MD5, Martina Muckenthaler, PhD6 and Maria Salomé Gomes, PhD¹ ¹IBMC and ICBAS – Universidade do Porto; ²IBMC and ICBAS – Universidade do Porto, Porto, Portugal; ³IBMC – Universidade do Porto, Porto, Portugal; 4Life and Health Sciences Reasearch Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.; 5IBMC and ICBAS – Universidade do Porto; 6Department of Pediatric Oncology, Haematology and Immunology, University Hospital of Heidelberg, Heidelberg, Germany (Presented By: Maria Salomé Gomes, PhD)

Anaemia is a frequent complication of chronic infectious diseases. The deficient production of red blood cells under these conditions is thought to result from a decrease in iron availability, due to the efforts of the host to sequester the iron away from the invading microbes. The exact mechanisms underlying this observation remain to be clarified. Hepcidin is considered a key regulator of iron metabolism. The fact that it is produced in response to microbial products and that it can block iron release to the circulation, through the inactivation of Ferroportin, make it a good candidate to explain anaemia of chronic infection. So far, the involvement of hepcidin in the development of anaemia of inflammation has been analyzed only in short-course studies examining mainly the effect of LPS. Overall, in vivo studies of iron metabolism in animal models of chronic infections are clearly missing. In the present work, we used a mouse model of mycobacterial infection to study the molecular alterations on iron metabolism induced by this kind of pathogen. We have confirmed that BALB/c mice infected with Mycobacterium avium for four weeks exhibited a moderate anaemia, with significant reductions in red blood cells, haemoglobin and haematocrit. Interestingly, this effect was not paralleled by increased hepatic Hepcidin mRNA expression. Microarray analysis of approx. 400 iron-related genes in the liver of M. avium infected mice revealed increased mRNA expression of Ferroportin, Ceruloplasmin, Hemopexin and Heme-oxygenase-1. Conversely, the expression of Transferrin Receptor-1 and Aminolevulinic acid synthase 1 was decreased. The gene with the highest level of differential mRNA expression was Lipocalin-2. Using immunocytochemistry, we validated these data showing that Lipocalin-2 was present in various hepatic cell types, namely neutrophils and hepatocytes. BALB/c mice carry the mutant (susceptibility-associated) allele of the Nramp1 gene. In order to investigate a possible role for Nramp1 in anaemia of chronic infection, we compared the response of BALB/c mice to that of a congenic mouse strain- C.D2-that bears the wild-type (resistance-associated) allele of this gene. C.D2 mice did not show any alterations on haematological parameters, four weeks after infection with M. avium. Concordantly, only a few iron-related genes are regulated in the liver, in response to infection. The attenuated gene expression response of C.D2 mice were maintained even when the bacterial loads in their organs were similar to the ones in BALB/c mice. These data suggest that a functional NRAMP1 protein protects mice from the anaemia as a consequence of mycobacterial infection. In conclusion, we show that the anaemia that develops in BALB/c mice infected for four weeks with Mycobacterium avium is not associated with increased levels of Hepcidin or downregulation of Ferroportin mRNA and protein levels in the liver. Conversely, increased mRNA and protein levels of Lipocalin-2 may play a role in the development of anaemia, as has been recently suggested. Furthermore, Nramp1 is required to maintain systemic iron homeostasis in M. avium infected mice. 79

48 HEME SENSITIZATION TO TNF-MEDIATED PROGRAMMED CELL DEATH DICTATES THE OUTCOME OF PLASMODIUM INFECTION IN MICE Raffaella Gozzelino, PhD¹, Elsa Seixas, PhD, Ângelo Chora, PhD, Ana Ferreira, PhD, Gabriela Silva, PhD, Rasmus Larsen, PhD, Sofia Rebelo, PhD, Carme Penido, PhD, Neal Smith, MD, PhD², Antonio Coutinho, MD, PhD and Miguel Soares, PhD ¹Instituto Gulbenkian de Ciência; ²Dept. of Pathology, Massachusetts General Hospital, Boston MA, USA (Presented By: Raffaella Gozzelino, PhD)

Under homeostasis, heme acts as a prosthetic group in hemoproteins. Under inflammatory conditions however, non-covalently bound heme can be released and generate cytotoxic free heme. We have previously shown that free heme released from hemoglobin (an hemoprotein) can dictate the lethal outcome of Plasmodium infection (malaria) in mice (Pamplona A. et al., Nat. Med. 2007, 13, 703). We now provide a molecular mechanism underlying this pathogenic effect. Namely, free heme sensitizes cells to undergo TNF-mediated programmed cell death. This cytotoxic effect, which occurs independently of newly gene transcription and/or protein synthesis, relies on the unfettered generation of free radicals in response to TNF, as demonstrated in hepatocytes in vitro. When exposed to free heme in vitro, hepatocytes respond to TNF by sustaining the activation of the c-jun N-terminal kinase (JNK), which leads to further accumulation of free radicals and to apoptosis, i.e. caspase-8 and -3 activation, DNA condensation. Inhibition of free radical accumulation by N acetylcysteine (NAC) or Butylated hydroxyanisole (BHA), inhibition of JNK activation (pharmacologic) or JNK expression (shRNA) as well as inhibition of caspase -8 and 3 activation (pharmacologic) suppress the cytotoxic effects of free heme plus TNF in hepatocytes. Expression of the heme catabolyzing enzyme heme oxygenase 1 (HO-1) or the iron sequestering protein H Ferritin in hepatocytes acts in an anti-oxidant manner to afford cytoprotection against heme plus TNF in vitro as well as in vivo, providing complete protection against Plasmodium infection in mice. In conclusion, this data reveals a novel mechanism via which free heme sensitizes hepatocytes to TNF mediated programmed cell death, an effect countered by the expression of cytoprotective genes that prevent the lethal outcome of Plasmodium infection.

49 THE AVIAN LIPOCALIN EX-FABP IS A SIDEROPHORE BINDING ANTIBACTERIAL PROTEIN Fiorella Descalzi, PhD¹, Matthew C. Clifton, PhD², Ranieri Cancedda, MD³, Tricia M. Hoette, PhD4, Kennet N. Raymond, PhD4 and Roland K. Strong, PhD² ¹IBFM & Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; ²Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; ³Istituto Nazionale per la Ricerca sul Cancro, University of Genova,Genova, Italy; 4Department of Chemistry, University of California, Berkeley, California , USA (Presented By: Fiorella Descalzi, PhD)

Ex-FABP is a chicken lipocalin, expressed in several tissues including: embryonic hypertrophic cartilage, newly formed muscle fibers, myocardium in developing heart, liver, and granulocytes. Ex-FABP suppression by gene targeting resulted in extensive cell death and in a strong inhibition of cell proliferation and differentiation in cultures of both myoblasts and chondrocytes. Ex-FABP behaves as an acute phase protein. In “in vitro” cultures of differentiating hypertrophic chondrocytes, of myoblasts leading to the formation of myotubes, of cardiomyocytes and of fragments of embryonic liver, the expression of Ex-FABP is enhanced by inflammatory agents, such as bacterial endotoxin LPS and Interleukin 6. In adult animals, Ex-FABP is expressed in articular cartilage of osteoarthritic chickens and in the lesion areas of dyschondroplastic chicken bones. We proposed that Ex-FABP is a stress protein, expressed both during development in tissues where active remodeling is taking place and in adult animal tissues as part of an acute phase response to different pathological conditions. We have previously demonstrated that Ex-FABP binds long chain unsaturated fatty acids. Interestingly the protein is also present in the egg white where antibacterial proteins, such as avidin and ovotransferrin, are also present. The mammalian counterpart of Ex- FABP, Siderocalin (Lcn2), which is also involved in differentiation in several tissues, has been described as an antibacterial protein able to inhibit bacterial growth both by binding the bacterial siderophore enterobactin and competing with bacteria for iron binding. Siderocalin’s effects on differentiation and tumorigenesis are thought to be the result of endogenous iron transport. In this study we investigated the possible iron binding capability and antibacterial activity of Ex-FABP. 1) By labeling cartilage cells with 55FeCl3 in the presence and in the absence of siderophore we demonstrated that the released protein can bind iron, but only in the presence of added siderophore. 2) Siderophore-free recombinant protein was obtained from a strain of E. coli unable to make siderophore; using fluorescence quenching, we showed that this molecule can bind different, catecholate-type siderophores. 3) Growth inhibition experiments indicated that the recombinant protein is able to inhibit bacterial growth in agreement with its siderophore binding property and specificity. In conclusion Ex-FABP is an iron binding lipocalin with antibacterial function. This property can be related to its expression in egg white and in tissues in inflammatory conditions. The function in tissues undergoing differentiation is under investigation.

50 HEPCIDIN INDUCTION BY PATHOGEN-ASSOCIATED STIMULI Andrew Armitage², Lucy Eddowes², Lisa Schimanski², Alain Townsend² and Hal Drakesmith¹ ¹Oxford University; ²Molecular Immunology Group, WIMM, Oxford University (Presented By: Hal Drakesmith, PhD)

Disturbances in iron homeostasis occur during infection with viral, bacterial and protozoan pathogens. Iron overload and/or redistribution of iron are linked to poor prognosis and increased mortality with HCV, HIV, Salmonella typhimurium and Plasmodium infections. Because hepcidin regulates iron transport, fluctuations in hepcidin synthesis during infection may influence the outcome of disease. The host recognizes components of invading pathogens using the Toll-like receptor (TLR) and Nod-like receptor (NLR) families of proteins. Ligation of TLRs provokes synthesis of inflammatory mediators including IL-6, which induces hepcidin synthesis by hepatocytes. Previous work has shown that the TLR4 ligand LPS increases IL-6 synthesis but also directly stimulates hepcidin production by myeloid cells. We have tested a panel of purified TLR and NLR agonists for their ability to induce hepcidin or IL-6 mRNA in human hepatocyte cell lines, blood mononuclear cells and monocyte-derived macrophages. TLR and NLR agonists failed to induce hepcidin synthesis by hepatocyte lines or macrophages, although TLR ligands stimulated IL-6 synthesis by macrophages. Blood mononuclear cells responded to several TLR ligands (including LPS) by making hepcidin, and the most potent of these agonists was the TLR5 ligand flagellin. Flagellin is a component of flagella, which are used by motile bacteria to acquire nutrients. S. typhimurium and Bacillus subtilis flagellin both induced hepcidin mRNA within 3 hours of exposure to blood mononuclear cells or enriched blood monocytes. Flagellin induction of hepcidin was not blocked by dorsomorphin, which inhibits the BMP/SMAD route of hepcidin stimulation described for hepatocytes. The rapid production of hepcidin by blood cells in response to flagellin may represent an attempt to reduce local serum iron availability to swimming pathogens. Flagellin-induced hepcidin is probably independent of hepatocyte-derived hepcidin and so may not influence systemic iron transport. However if chronic TLR ligation as a result of established infections (of viral, bacterial or protozoan origin) leads to persistently increased hepcidin, this could conceivably contribute to anaemia.

51 LIVER-SPECIFIC DELETION OF FRATAXIN IN THE MOUSE AS A NEW MODEL TO UNDERSTAND IRON DYSREGULATION IN FRIEDREICH ATAXIA Alain Martelli, PhD¹, Stéphane Schmucker, student², Marie Wattenhofer-Donzé, PhD², Laurence Reutenauer, technician², Hervé Puy, Professor³, Nadia Messaddeq, Engineer² and Hélène Puccio, PhD² ¹IGBMC; ²IGBMC Illkirch; ³Centre Français des Porphyries (Presented By: Alain Martelli, PhD)

Friedreich ataxia, a neurodegenerative disease associating cardiomyopathy, is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur (Fe-S) biosynthesis and/or heme synthesis. We have previously generated conditional mouse models which reproduce important pathophysiological and biochemical features of the human disease: progressive cardiac hypertrophy, progressive sensory and cerebellar ataxia, multiple mitochondrial Fe-S enzyme deficiency, time-dependent intramitochondrial iron accumulation. Particularly, we showed that Fe-S deficit precedes the iron aggregate formation in mitochondria of the cardiac mouse model (MCK), suggesting that the deregulation of Fe-S proteins is a primary event of the disease.

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To further elucidate the relationship between frataxin deficiency, Fe-S biosynthesis and iron metabolism dysregulation, we generated a liver-specific frataxin deleted mouse model. Liver is a central organ for systemic iron homeostasis, notably through its iron storage activity and its iron sensing property that modulates hepcidin transcription. The liver-specific frataxin knock-out shows a severe phenotype with a drastic decrease in lifespan. Histological studies revealed a lipid accumulation in the liver of mutant mice with a clear mitochondrial dysfunction. The primary Fe-S biosynthesis deficit was confirmed while no deficit in the heme pathway was observed. Furthermore, the expression of various genes involved in iron metabolism as well as the IRPs activities were assessed. The results clearly indicate that iron dysregulation appears after the primary Fe-S biosynthesis deficit. Together, the data enables to define a molecular mechanism to explain the events leading to iron dysregulation in Friedreich ataxia.

52 IRON STORES AND IRON ABSORPTION IN HEREDITARY HEMOCHROMATOSIS Roland Fischer, PhD¹, Hannah Seligmann², Rainer Engelhardt, PhD², Rosemarie Kongi², Gritta E. Janka, MD² and Peter Nielsen, MD, PhD² ¹University Medical Center Hamburg-Eppendorf, Germany & Children’s Hospital & Research Center Oakland, USA; ²University Medical Center Hamburg-Eppendorf, Germany (Presented By: Roland Fischer, PhD)

Total body iron (TBI) stores in patients with hereditary hemochromatosis (HH) can be calculated from the blood volumes drawn during a complete phlebotomy series. In a 2nd step, the phlebotomized iron is corrected for hemoglobin synthesis, i.e. changes in the circulating iron, which leads to mobilized iron [Haskins et al, 1952]. However, elevated iron absorption in HH competes with iron mobilization from TBI, especially at the end of a phlebotomy series. Liver iron concentration is also used to estimate TBI. Although, a linear relationship between LIC and TBI was shown [Fischer et al, Bioiron 2007], this relationship in patients with HH is not well known in contrast to ex- thalassemia patients after bone marrow transplantation [Angelucci et al, 2000]. In 1989-2008, we followed 441 homozygous patients with HH by SQUID biomagnetic liver susceptometry (BLS). In 72 patients with at least 2 measurements by BLS, the change in LIC was related to the change in mobilized iron (see Fig 1). Fig 1. Relationship between changes in mobilized iron per body weight (TBI/BW), TBI/BW after subtraction of a daily iron absorption rate of 4.5 mg and liver iron concentration (LIC). During the total phlebotomy treatment intervals of 100 to 1100 days, 10 – 200 mg iron per week (mean: 115 ± 47 mg/w) was mobilized corresponding with changes in LIC of 5 to 75 µg/gliver. In Fig 1, in vivo LIC measurements converted by a factor c of 6 into dry-weight values correlated with mobilized iron (R2 = 0.74), but with a significant intercept, which indicates average iron absorption of 15.8 mg/kg. Subtraction of a daily iron absorption rate of 4.5 mg/d leads to a negligible intercept with a regression coefficient of m = 5.75 mg/kg per 1 mg/gdry wgt of LIC. This slope is only half of that found in ex-thalassemia patients after bone marrow transplantation [Angelucci et al, 2000]. From the slope m, the liver iron fraction (LIF) can be calculated as LIF = Vliver / (c · m). From liver volume measurements in HH, a specific liver volume Vliver = 23.7 ml/kg was found (R2 = 0.4). These results indicate that 69 % of total body storage iron is concentrated in the liver of HH patients. However in a few patients, iron stores in other organs than the liver significantly contribute to TBI. In addition, iron absorption rates seem to be very different in some patients (0.5 to 10 mg/d). Further efforts will be undertaken to investigate these cases and also gender differences.

53 HYPERGLYCEMIA AND IMPAIRED BETA-CELL FUNCTION CAUSED BY IRON REGULATORY PROTEIN 2 DEFICIENCY Kimberly Zumbrennen, PhD, Susanne Neschen, PhD, Brigit Rathkolb, PhD, Wolfgang Hans, PhD and Elizabeth Leibold, PhD University of Utah (Presented By: Kimberly Zumbrennen, PhD)

Excessive iron in humans is associated with a number of pathologies including neurodegeneration, cardiomyopathy, and diabetes. For example, type 2 diabetes is part of the classical presentation in patients with hereditary hemochromatosis; a disease characterized by increased iron loading in liver, heart, and pancreas, as well as increased transferrin saturation and serum ferritin. In a mouse model of hemochromatosis (Hfe-/-), iron accumulates in pancreatic islets and is associated with impaired insulin secretion and increased apoptosis of beta-cells. Iron regulatory protein 2 (Irp2) is a key regulator of iron homeostasis in mammalian cells. Irp2 functions as a RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as transferrin receptor 1 (TfR1) and ferritin. We have generated a mouse model with a targeted deletion of Irp2 and have found altered expression of TfR1 and ferritin in numerous tissues resulting in increased hepatic, duodenal, and neuronal iron accumulation, as well as increased serum ferritin levels. We hypothesized that disruptions in iron homeostasis in our Irp2-/- mouse strain may affect glucose homeostasis similar to Hfe-/- mice. Analysis of whole body bone, fat, and lean mass revealed a significant decrease in lean mass and an increase in fat mass in Irp2-/- mice compared to Wt controls suggesting impaired energy metabolism. Blood glucose analysis showed a mild hyperglycemia in Irp2-/- mice compared to Wt controls in both fasted and random-fed animals. Intraperitoneal (ip) glucose tolerance tests revealed decreased glucose tolerance in Irp2-/- mice characterized by increased glucose excursions and decreased glucose clearance. Insulin tolerance tests as well as euglycemic-hyperinsulinemic clamp studies showed no difference in glucose disposal rates between Irp2-/- mice and Wt controls. In contrast, insulin analysis revealed a mild reduction in serum insulin levels in Irp2-/- mice after an overnight fast and 30 min after an ip-glucose injection. HOMA-beta cell function analysis (insulin/glucose ratio) showed a significant reduction in insulin secretion in Irp2-/- mice. These data indicate that Irp2 deficiency causes impaired beta-cell function resulting in reduced glucose-stimulated insulin secretion and subsequently glucose intolerance. Interestingly, we have also found that, in contrast to Hfe-/- mice, Irp2-/- mice have decreased levels of pancreatic iron. These data suggest that efficient beta-cell function is dependent on an optimal range of iron, and that extreme increases or decreases in pancreatic iron may result in beta-cell dysfunction.

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54 ALTERATIONS OF SYSTEMIC AND MUSCLE IRON METABOLISM IN HUMAN SUBJECTS TREATED WITH LOW DOSE RECOMBINANT ERYTHROPOIETIN Gaetano Cairo, PhD¹, Stefania Recalcati, MD, PhD¹, Domenico Girelli, MD³, Cecilia Gelfi, PhD¹, Niels Aachmann-Andersen, PhD4, Jonas Thomsen, PhD4, Anne Norgaard, PhD4, Alessandra Alberghini, PhD¹, Natascia Campostrini, PhD³, Annalisa Castagna, PhD³, Agnese Viganò, PhD¹, Paolo Santambrogio, PhD5, Tibor Kempf, MD6, Kay Wollert, PhD6, Stephane Moutereau, PhD7, Carsten Lundby, PhD4 and Paul Robach, PhD² ¹University of Milan; ²ENSA, France; ³University of Verona; 4University of Copenhagen; 5DIBIT, HS S. Raffaele; 6Hannover Medical School; 7Hopital H. Mondor (Presented By: Gaetano Cairo, PhD)

The increased red cell production that occurs in physiological and clinical situations threatening tissue oxygen availability is mediated by erythropoietin (Epo), and requires an increased iron supply to erythropoietic tissue. The high iron demand associated with enhanced erythropoiesis during high-altitude hypoxia leads to skeletal muscle iron mobilisation and decrease in myoglobin levels (Robach P et al. Blood 109:4724-31, 2007). In order to investigate the effect of enhanced erythropoiesis on systemic and muscle iron metabolism under non-hypoxic conditions, eight healthy volunteers were treated with Epo for one month and blood, urine (for hepcidin determination) and muscle biopsies (for evaluation of mRNAs and proteins of iron metabolism) were taken at various time points. As expected, the treatment efficiently increased erythropoiesis and stimulated bone marrow iron use. It was also associated with a prompt (day 2) and considerable decrease in urinary hepcidin, a slight transient increase in GDF-15 (day 4) and a decrease in transferrin saturation (day 6) The increased iron use and reduced hepcidin levels suggested increased iron mobilisation, but the treatment was associated with increased muscle iron and L ferritin levels. The muscle expression of transferrin receptor and ferroportin was up-regulated by Epo administration, without appreciable change in myoglobin levels, which suggests unaltered muscle oxygen homeostasis. In conclusion, our results provide new insights into the interactions between erythropoiesis and iron metabolism in humans by showing that Epo administration induces a strong and early suppression of hepcidin, that may be mediated directly by Epo, as also suggested by complementary experiments in the mouse showing that an Epo derivative devoid of erythropoietic stimulating activity was still able to downregulate liver hepcidin mRNA levels. Secondly, the demonstration of iron accumulation in skeletal muscle tissue despite hepcidin suppression highlights the role of hepcidin-independent mechanisms in regulating iron trafficking in this tissue. Our data may have clinical implications for optimising the use of erythropoiesis-stimulating agents and/or iron therapy for treatment of anemia.

55 ANALYSIS OF CONDITIONAL FERRITIN H DELETION IN MOUSE ADULT HEART Liviu Vanoaica², Deepak Darshan, PhD², Larry Richman², Corinne Berthonneche³, Thierry Pedrazzini, MD4 and Lukas Kühn, PhD¹ ¹Ecole Polytechnique Federale de Lausanne (EPFL) – Swiss Institute for Experimental Cancer Research (ISREC); ²Ecole Polytechnique Fédérale de Lausanne (EPFL) – Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland; ³Cardiovascular Assessment Facility, University of Lausanne Medical School, Lausanne, Switzerland; 4Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, Lausanne, Switzerland (Presented By: Lukas Kühn, PhD)

Ferritin is thought to play a defensive role against oxidative damage. Since ferritin H is expressed in high proportion in heart muscle, we have analyzed the effects of a specific conditional ferritin H gene deletion in this tissue. Our established Fthlox/lox mice were crossed to Myh6-CreERT mice expressing Cre recombinase with a tamoxifen-inducible estrogen receptor domain only in heart muscle due to the cardiac-specific α-myosin heavy chain (Myh6) promoter. Mice were analyzed 15 days post deletion under normal iron regimen. Heart ferritin H mRNA was reduced to 80% and hemeoxygenase-1 mRNA increased 1.5- fold. Cardiac failure and hypertrophy is known to induce genes characteristic of embryonic heart. Here, in ferritin H-deleted mice, we observed such a shift in expression from cardiac to skeletal actin and from myosin α to myosin β heavy chain. Moreover, tissue sections showed a significant increase in fibrosis revealed by Masson’s trichrome method. The fibrotic regions were quite evenly distributed in the heart muscle. In absence of additional stress mice showed no further increase in fibrosis. Moreover, under normal iron regimen, we found only weak transient changes in heart physiology and mice lived normally for 10 months. However, when mice were loaded with a single dose of iron dextran (1 mg/g body weight) at 2 weeks prior to the ferritin H deletion, effects were dramatically aggravated and the experiment had to be terminated between day 8 and 15. Hemeoxygenase-1 mRNA was induced 45-fold, and other oxidative stress induced genes, like superoxide dismutase 1, glutathione peroxidase and catalase were significantly upregulated. Fibrosis was clearly visible. The TUNEL assay measuring strand breaks in DNA, a sign of apoptosis and necrosis, revealed regions of prominent staining in deleted but not control mice. Most importantly, mice showed strong physiological changes in echocardiography. The cardiac output was only about 40% of that in control animals as measured by the left ventricular fractional shortening (%FS) and ejection fraction (%EF). Our results provide evidence for the protective role of ferritin H against iron toxicity linked to oxidative stress in the heart. This conditional ferritin H knock-out mouse may provide a model system to evaluate the role of iron overload in heart failure.

56 EFFICACY AND SAFETY OF DEFERASIROX (EXJADE®) IN REDUCING AND PREVENTING MYOCARDIAL SIDEROSIS IN PATIENTS WITH BETA- THALASSEMIA Dudley Pennell, MD, FRCP, FACC, FESC¹, John B. Porter², Maria Domenica Cappellini³, Amal El-Beshlawy4, Lee Lee Chan5, Yesim Aydinok6, Mohsen Saleh Elalfy7, Pranee Sutcharitchan8, Chi-Kong Li9, Hishamshah Ibrahim¹°, Vip Viprakasit¹¹, Antonis Kattamis¹², Gillian Smith¹³, Dany Habr¹4, Gabor Domokos¹5, Abdel Hmissi¹5 and Ali Taher¹6 ¹Royal Brompton Hospital; ²University College London, London, UK; ³Universitá di Milano, Policlinico Foundation IRCCS, Milan, Italy; 4Cairo University, Cairo, Egypt; 5University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia; 6Ege University Medical Faculty, Izmir, Turkey; 7Ain Shams University, Cairo, Egypt; 8Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 9Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong; ¹°Hospital Kuala Lumpur, Malaysia; ¹¹Faculty of Medicine Siriraj Hospital/Dept of Pediatrics/Division of Haematology-Oncology, Prannok, Bangkoknoi, Bangkok, Thailand; ¹²First Dept of Pediatrics, University of Athens, Athens, Greece; ¹³Royal Brompton Hospital, London, UK; ¹4Novartis Pharmaceuticals, New Jersey, USA; ¹5Novartis, Basel, Switzerland; ¹6American University Beirut, Beirut, Lebanon (Presented By: Dudley Pennell, MD, FRCP, FACC, FESC)

Heart failure due to myocardial siderosis is the main cause of death in patients (pts) with β-thalassemia undergoing regular blood transfusions. The efficacy of deferasirox (Exjade®) in reducing or preventing myocardial siderosis was assessed in 192 β-thalassemia pts in a 1-year, prospective, multicenter study. The treatment arm (n=114) included pts aged ≥10 years with cardiovascular magnetic resonance (CMR) imaging of myocardial T2* >5 to <20ms (indicating cardiac siderosis), left ventricular ejection fraction (LVEF) ≥56%, serum ferritin (SF) >2500ng/mL, liver iron concentration (LIC) >10mg Fe/g dry weight (dw), and a lifetime minimum of 50 transfused blood units. The prevention arm (n=78) included otherwise eligible pts but with normal myocardial iron content (T2* ≥20ms). Deferasirox was initiated at 30mg/kg/day (treatment arm) or 20– 30mg/kg/day (prevention arm) with subsequent dose adjustments of 5 or 10mg/kg/day based on SF trends, month-6 myocardial T2*, and safety parameters. The primary endpoint was change in myocardial T2* at 12 months. In the treatment arm (54 M:60 F; mean age 20.9±7.3 years) the mean actual deferasirox dose over 1 year was 32.6±4.0mg/kg/day. Myocardial T2* improved significantly from a baseline of 11.2ms ±40.5% to 12.9ms ±49.5% (geometric mean ±CV; P<0.0001; +16%). LVEF was unchanged: 67.4 ±5.7% to 67.1 ±6.0% (mean ±SD; P=0.53). Both mean LIC and absolute change of median SF were reduced significantly from baseline by –6.6±9.9mg Fe/g dw and –1257ng/mL, respectively (both P<0.0001). Investigator-assessed drug-related adverse events (AEs) were reported in 56 patients (49.1%); rash was the most common (n=15; 13.2%). Most AEs were mild-to-moderate. Two drug-related serious AEs (one nephritis leading to acute renal failure, one renal tubular disorder) were reported that resolved following drug discontinuation. Five pts (4.4%) had a non-progressive increase in serum creatinine >33% above baseline and the upper limit of normal (ULN) on two consecutive visits. Two (1.8%) pts had an increase in alanine aminotransferase (ALT) >10xULN on two consecutive visits; levels were already elevated at baseline in both pts. In the prevention arm (35 M:43 F; mean age 20.2±7.5 years) the mean deferasirox dose over 1 year was 27.6±6.0mg/kg/day. Baseline myocardial T2* was unchanged from a baseline of 32.0 ±25.6% to 32.5ms ±25.1% (+2.0%; P=0.56). LVEF increased from baseline 67.7 ±4.7% to 69.6 ±4.5% (mean absolute change +1.9; P<0.0001). Mean LIC and absolute change of median SF were also significantly reduced from baseline by –7.2±10.5mg Fe/g dw and –1048ng/mL, respectively (both P<0.0001). Drug-related AEs were mostly mild-to-moderate and were reported in 31/78 (39.7%) patients, including diarrhea (n=8; 10%) and rash (n=7; 9%). One pt (1.3%) had a non-progressive increase in serum creatinine >33% above baseline and the ULN on two consecutive visits. No pts had an increase in ALT 82

>10xULN on two consecutive visits. No pts died in either group. In conclusion, deferasirox treatment over 1 year in heavily transfused β-thalassemia removed iron from the heart in pts with myocardial siderosis and prevented myocardial iron accumulation in pts with normal baseline myocardial iron levels. Concomitantly, SF and LIC were significantly reduced in both pt groups and treatment was generally well tolerated.

57 NRAMP1 PROMOTES EFFICIENT IRON RECYCLING FOLLOWING ERYTHROPHAGOCYTOSIS IN VIVO Shan Soe-Lin, BSc¹, Bill Andriopoulos, Jr., BSc, PhD², Sameer Apte, BSc³, Marc Andrews, BSc4, Matthias Schranzhofer, BSc, PhD³, Tanya Kahawita, BSc, MSc5, Daniel Garcia-Santos, BSc³ and Prem Ponka, MD, PhD6 ¹McGill University; ²Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; ³Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada; 4Department of Molecular Genetics and Development, Institut de Recherches Cliniques de Montreal, Quebec, Canada; 5Department of Physiology, McGill University, Montreal, Quebec, Canada; 6Department of Physiology, McGill University, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada (Presented By: Shan Soe-Lin, BSc)

Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells such as macrophages and neutrophils. Based on our earlier in vitro study (Soe-Lin et al. Exp Hematol. 2008;36:929-937), we hypothesize that Nramp1 may participate in the recycling of iron acquired through the phagocytosis of senescent red blood cells by macrophages in vivo. In preliminary characterization experiments of the iron parameters of wild-type (Nramp1+/+) and Nramp1 knockout mice (Nramp1-/-), we found that untreated knockout mice exhibited greater serum transferrin saturation and splenic iron content, with higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression. Furthermore, hepatocyte iron content and hepcidin mRNA levels were dramatically lower in knockout mice, indicating that hepcidin levels can be regulated by low hepatocyte iron stores despite increased transferrin saturation. In addition, we observed significant iron loading of the reticuloendothelial organs of knockout mice that increased with age. After injection of 59Fe-labeled heat damaged reticulocytes, knockout animals accumulated erythrophagocytosed 59Fe within their liver and spleen, whereas wild-type animals were able to efficiently recycle the phagocytosed 59Fe to the marrow for incorporation into newly formed erythrocytes. In order to further examine the effect of Nramp1 on iron recycling in vivo, accelerated erythrophagocytosis was induced in wild-type and knockout mice by administration of the hemolytic agent phenylhydrazine. Following acute phenylhydrazine treatment, Nramp1-/- mice experienced a significant decrease in serum iron levels and hematocrit, while their Nramp1+/+ counterparts were relatively unaffected. Following a month-long phenylhydrazine regimen, Nramp1-/- mice retained markedly increased quantities of iron within the liver and spleen, and exhibited greater splenomegaly and reticulocytosis than wild-type mice. The data presented in this report suggest that in the absence of Nramp1, iron accumulates to a greater degree within ironrecycling macrophages in the liver and spleen following erythrophagocytosis. We hypothesize that hepatocytes iron stores are released to compensate for the lack of iron release from macrophages, resulting in a lower production of hepcidin mRNA. Our observation of increased DMT1 and ferroportin within the duodenums of the Nramp1-/- animals imply that the increase in transferrin saturation despite the impaired iron release from erythrophagocytosing macrophages occurs due to a compensatory increase in iron absorption from the diet. These findings are consistent with our hypothesis that Nramp1 promotes the efficient recycling of iron in erythrophagocytosing macrophages.

58 INCREASED HEPCIDIN EXPRESSION IN MICE AFFECTED BY BETA-THALASSEMIA REDUCES IRON OVERLOAD WITH NO EFFECT ON ANEMIA Sara Gardenghi, PhD¹, Maria Franca Marongiu, PhD², Kristen Muirhead², Pedro Ramos³, Cindy N. Roy, PhD4, Nancy C. Andrews, MD, PhD5, Elizabeta Nemeth, PhD6, Eliezer A. Rachmilewitz, MD7, Patricia J. Giardina, MD², Robert W. Grady, PhD² and Stefano Rivella, PhD² ¹Weill Cornell Medical College; ²Weill Cornell Medical College, New York; ³Weill Cornell Medical College, New York - IBMC, Portugal; 4John Hopkins University, Baltimore; 5Duke University, Durham; 6UCLA, Los Angeles; 7Wolfson Medical Center, Israel (Presented By: Sara Gardenghi, PhD)

The hepatic peptide hepcidin (Hamp) triggers the degradation of ferroportin, the iron-exporter localized on absorptive enterocytes and tissue macrophages. We showed that iron absorption increases with time in mice with beta-thalassemia intermedia (th3/+), while Hamp is expressed at a relatively low level compared to iron load. We hypothesized that th3/+ mice absorb more iron than required for erythropoiesis and that reducing their iron intake by increasing Hamp levels could limit iron overload. However, this might also impair erythropoiesis by preventing release of iron from macrophages. Accordingly, we fed wt and th3/+ mice with diets containing 2.5- and 35- ppm iron (low iron and iron sufficient, respectively) and compared them to transgenic mice over expressing Hamp (designated Tg-Hamp and Tg-Hamp/th3, respectively) fed the 35-ppm diet. After 1 month, the hemoglobin values of wt and th3/+ mice on the 2.5-ppm diet did not change significantly compared to those of mice on the 35-ppm diet. Moreover, the 2.5-ppm diet did not affect the iron content of the liver and spleen in wt mice, or expression of Hamp in the liver. However, in th3/+ mice, the same diet reduced the mean amount of iron in the liver from 284±43 to 85±48 ug, and that in the spleen from 370±50 to 115±13 ug. It also caused a 9-fold reduction of Hamp expression in the liver. After 5 months, wt mice fed the 2.5-ppm diet became anemic, their mean hemoglobin levels being 8.7±3.0 g/dl vs 14.6±0.7 g/dL at 1 month. They also showed a reduced amount of iron in the liver (35±1 ug vs 112±10 ug) and spleen (12±5 ug vs 21±12 ug), as well as a 28-fold reduction of Hamp expression in the liver. Interestingly, th3/+ mice fed the 2.5-ppm diet were not more anemic (8.2±1.7 g/dl vs 8.2±1.7 g/dL at 1 month), despite having a reduced amount of iron in the liver (52±14 ug vs 665±81 ug) and spleen (37±19 ug vs 483±161 ug), and a 5-fold reduction of Hamp expression compared to animals on the 35-ppm diet. In transgenic mice, Tg-Hamp and Tg-Hamp/th3, the iron content of the liver was reduced in both groups after 1 month on the 35-ppm diet (59±18 ug in Tg-Hamp vs 152±22 ug in controls; 147±91 ug in Tg-Hamp/th3 vs 264±18 ug in controls). Only the Tg-Hamp/th3 mice exhibited a decrease in splenic iron (132±96 ug vs 249±8 ug in controls). However, while Tg-Hamp mice showed a slight anemia, the hematological parameters of Tg-Hamp/th3 mice did not change significantly. Analysis at 5 months is in progress. Overall, these data indicate that more iron is absorbed in thalassemia than required for erythropoiesis, since a reduced iron intake in th3/+ mice did not worsen their anemia. Moreover, over- expression of Hamp in thalassemic mice could reduce tissue iron overload with no effect on anemia. In conclusion, these studies are expected to contribute to the development of new pharmacological approaches to the treatment of abnormal iron absorption in beta-thalassemia and related disorders.

59 THE ROLES OF HEPCIDIN AND INFLAMMATION IN ANEMIA OF CANCER Airie Kim, MD, Tomas Ganz, MD, PhD and Seth Rivera, MD, PhD UCLA Medical Center (Presented By: Airie Kim, MD)

Introduction: Anemia is a common complication of many cancers, and is associated with increased morbidity and shortened survival. Anemia of cancer (AC) likely has mechanistic similarities to the anemia of inflammation, but the roles of inflammation and hepcidin in the development of AC have yet to be elucidated. We used animal models of lung cancer to investigate the role of hepcidin in the development of AC. Methods: We created two mouse models of lung cancer using the syngeneic lung cancer cell lines TC-1 and LLC. C57/Bl6 mice were injected intraperitoneally with TC-1, LLC, or PBS, and sacrificed on days 7 and 14. Results: By day 14, the TC-1 and LLC mice were more anemic than the control mice (control hgb 15.5 g/dL, TC-1 13.4, LLC 7.7; p=0.004, <0.001, respectively). Zinc protoporphyrin (ZPP), a measure of iron restricted erythropoiesis, was significantly elevated in the tumor mice compared to controls (control ZPP 86, TC-1 142, LLC 212; p<0.001, p=0.007, respectively), which reflects that the anemia was due to iron restriction. The tumor mice had a higher MCV at day 14 which may have been due to a vigorous reticulocytosis. Liver median hepcidin measurements were not statistically significantly different in the LLC, TC-1, and control mice at day 7 (0.92, 0.76, 1.39, respectively), but they were significantly different by day 14. The TC-1 mice had higher hepcidin levels than the control mice with a trend towards significance (TC-1 7.14 arbitrary units vs PBS 3.28, p=0.052), but the LLC mice had lower hepcidin levels than both the control and TC-1 mice (0.075, p<0.001). Preliminary data also indicate that the TC-1 mice mount a hepatic acute phase response as measured by SAA-1 expression (0.146 vs 0.003, p=0.034) while the LLC mice do not.

83

Discussion: We were able to create mouse models of anemia of cancer. By day 14, sizable tumors were present, and there was significant anemia. This occurred in conjunction with higher ZPP measurements, which points to iron restriction as an important contributor to the anemia. The TC-1 mice have inappropriately elevated hepcidin at day 14 despite the presence of significant anemia, indicating that hepcidin likely causes the iron restriction. This is likely mediated by inflammation since there is a hepatic acute phase response. LLC mice, by contrast, have suppressed hepcidin production. The differing hepcidin production seen in the two models may indicate a hepcidin independent mechanism for iron sequestration in the LLC mice. Alternatively, it may be that the LLC mice represent a later, more severe, form of AC where anemia suppression of hepcidin outstrips the inflammatory stimulation. Conclusions: Our mouse models demonstrate that there may be varying mechanisms for the development of anemia of cancer. Despite the development of anemia and iron- restriction in both models, there are large differences in hepcidin measurements and inflammatory markers. Further studies are pending using hepcidin knockout mice for further elucidation of the mechanisms of anemia of cancer.

60 MUTATIONS IN A NOVEL MITOCHONDRIAL TRANSPORTER GENE CAUSE AUTOSOMAL RECESSIVE CONGENITAL SIDEROBLASTIC ANEMIA Sylvia S. Bottomley, MD¹, Duane L. Guernsey, PhD²,8, Paul J. Schmidt, PhD³, Andrew Orr, MD4,8, Mark Ludman, MD5, 8, Conrad V. Fernandez, MD6, 8, Mark E. Samuels, PhD7,8 and Mark D. Fleming, MD, DPhil³ 1Department of Medicine, Hematology-Oncology Section, University of Oklahoma College of Medicine, Oklahoma City, USA; 2Department of Pathology, Dalhousie University, Halifax, Canada; 3Departments of Pathology, Children’s Hospital and Harvard Medical School, Boston, USA; 4Department of Ophthalmology and Visual Sciences, Dalhousie University, Halifax, Canada; 5Department of Pediatrics, Division of Medical Genetics, IWK Health Centre and Dalhousie University, Halifax, Canada; 6Department of Pediatrics, Division of Hematology and Oncology, IWK Health Centre and Dalhousie University, Halifax, Canada; 7Centre de Recherche de Ste-Justine, Université de Montréal, Montréal, Canada; 8Atlantic Medical Genetics and Genomics Initiative and Genome Canada, Dalhousie University, Halifax, Canada (Presented By: Sylvia S. Bottomley, MD)

Numerous unexplained cases of congenital sideroblastic anemia (CSA) with hypochromic microcytic erythrocytes have occurred sporadically or in siblings of both sexes, suggesting autosomal recessive inheritance. In this study, a dense SNPbased genome-wide scan was performed in three CSA patients from a population isolate from the Canadian Maritime provinces and demonstrated a segment of chromosome 3 that was apparently homozygous by descent in the three affected individuals. After sequencing 15 annotated genes in the linked region, a homozygous p.117R>X (c.726 C>T) stop codon in exon 4 of a novel gene was detected in all three patients. Subsequently, multiple additional biallelic mutations in the gene were identified among three separate families and 9 unrelated individuals from a cohort of 41 probands with familial or sporadic CSA from different ethnic backgrounds. In total, eleven distinct variants were found, including 3 stop codons, 2 frameshifts, 1 splice acceptor site mutation, 4 missense mutations, and one involving the stop codon and extending the C-terminus. The gene product is a member of a family of inner mitochondrial membrane transporters, which is preferentially expressed in erythroid cells and is predicted to be an amino acid carrier. Injection of orthologous antisense morpholinos into zebrafish embryos yielded an anemic phenotype, confirming a role for the transporter in erythropoiesis. To explore the possibility that the CSA results from a heme biosynthetic defect, the phenotype of S. cerevisiae with a germline deletion of its putative orthologue of the human gene was examined. Yeast lacking the mitochondrial carrier grew poorly aerobically and were unable to reduce sodium nitroprusside that could be rescued by glycine or 5-aminolevulinic acid (ALA). Cellular ALA was reduced 6.7-fold in the deletion strain, suggesting that loss of the carrier impairs heme biosynthesis in vivo. In conclusion, our data indicate that the mitochondrial transporter is essential in erythroid cells to meet their high heme synthesis requirement. We hypothesize that it facilitates ALA production by importing glycine into mitochondria or by exchanging glycine for ALA across the mitochondrial inner membrane. Loss of the transporter likely results in recessive CSA due to ALA deficiency as occurs with 5-aminolevulinate synthase 2 (ALAS2) mutations. Mutations in the gene appear to be a relatively common cause of CSA, accounting for 29% of 41 previously undefined probands with the disorder and 17% of a complete cohort of 72 probands with CSA, which includes 31 with ALAS2 mutations.

61 ALLELIC HETEROGENEITY OF TMPRSS6 MUTATIONS IN IRIDA Caroline Kannengiesser, PhD², Flavia Guillem, Laura Silvestri, Claire Oudin, Anne Marfaing, Leïla Chaiba-Berrouche, Jean Donadieu, Fabienne Toutain, Murielle Da Silva, Bertrand Isidor, Geneviève Marguerite, Patricia Aguila-Martinez, Clara Camaschella, Carole Beaumont and Bernard Grandchamp, MD, PhD¹,³,4 ¹AP-HP, Hopital Bichat; ²APHP, INSERM U773, University Paris Diderot; ³INSERM CRB3; 4University Paris Diderot (Presented By: Bernard Grandchamp, MD, PhD)

Matriptase-2 is a transmembrane serine protease encoded by the TMPRSS6 gene that negatively regulates hepcidin expression by cleaving membrane-bound hemojuvelin. Matriptase-2 mutations have been reported in several patients with iron-refractory iron deficiency anemia (IRIDA), an autosomal recessive disease characterised by high levels of plasma hepcidin and iron deficient erythropoiesis. Here we describe 8 novel cases from 6 families. The entire coding region of TMPRSS6 and exonintron borders were sequenced and serum hepcidin was measured using an Elisa assay. Four patients were compound heterozygotes for 2 missense mutations targeting different subdomains of the ectodomain (families I,II,III) and affected the function of matriptase-2. Two patients were homozygous for a frameshift deletion. Finally, in two patients the mutation was found in only one allele suggesting that some mutations may be located outside the explored parts of the gene (exons and intron-exon junctions). A large range of serum hepcidin concentrations was found in different patients. All patients but two brothers (family II) were not sensitive to oral iron supplementation but had received IV iron which resulted in an increase in serum ferritin and almost complete correction of the anemia. After a course of IV iron, the response was usually sustained, with hemoglobin levels reaching normal values and remaining near normal for several months. This observation supports the idea that in the presence of abnormally elevated hepcidin levels, a part of infused iron is sequestered in macrophages then is subsequently slowly released and utilized by the erythropoietic cells. Our results further document the allelic heterogeneity of TMPRSS6 mutations and show the importance of different modules of the ectodomain. Clinical data suggest that IRIDA is a relatively benign genetic disease provided appropriate therapy is given to the patients

$ cDNA sequence reference AY055384 * normal range : 28-244 ng/ml § current values for patients under iron therapy (oral or IV) excepted for patient 7, indicated by #. Hb of patient 7 reached 11.5 in 2001 after IV iron therapy. abreviations : IRIDA : iron-refractory iron deficiency anemia, LDLRA: low density lipoprotein receptor class A domain, CUB : complement factor C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein, SEA : sea urchin sperm protein, enteropeptidase, agrin, SP: serine protease 84

62 THE ROLE OF ERYTHROPOIETIN IN FAMILIAL AMYLOIDOSIS TTR V30M Idalina Beirão¹, Luisa Lobato, PhD²,³, Paulo PM Costa, PhD³, Luciana Moreira, MSc³ and Graça Porto, PhD4,5 ¹Santo António General Hospital; ²Nephrology, Santo António General Hospital; ³Aamyloidosis Research Centre; 4Hematology, Santo António General Hospital; 5IBMC (Presented By: Idalina Beirão, MD)

Familial amyloidosis (FAP) TTR V30M is caused by a mutated transthyretin (TTR V30M). A sensorimotor and autonomic neuropathy is the main clinical feature. Anemia affects 24.8% of symptomatic FAP Portuguese patients associated to low serum levels of erythropoietin (EPO). This cytokine is essential for erythropoiesis and neuroprotection. To evaluate the serum EPO levels in FAP and its relation with anemia we studied a group of 140 patients (65 M; 75 F) with TTR V30M mutation confirmed by DNA analysis, 124 of them symptomatic with an evolution time of the disease of 6±5 (1-26) years. In order to classify the individual EPO serum levels as appropriate or not for the degree of anemia, the observed values were systematically compared with the expected values, estimated according to the logarithmic regression equation of EPO against hemoglobin in healthy adult controls. Anemia was considered for hemoglobin levels lower than 13 g/dl in men and lower than 12g/dl in women. Anemia was present in 58/124 (46.8%) symptomatic patients and affected equally males and females. Anemic patients had 9±5 (2-26) years of symptomatic disease. The serum hemoglobin levels ranged from 6.3 to 12.9 g/dl (10.5±1.6). The mean transferrin saturation was 21±12.7% and the mean serum ferritin was 145.1±139.3 ng/ml. No abnormalities in serum folic acid or serum vitamin B12 were found. An inappropriate serum EPO level, lower than 80% of the expected level, was observed in 43/58 (74.1%) anemic patients. Twenty patients with severe anemia were successfully treated with recombinant human erythropoietin. The group of non anemic patients presented with a mean evolution time of the disease of 4±4 (0-17) years. Interestingly, 39/82 (45.6%) of these patients also presented a serum EPO level lower than 80% of the expected level. Low serum EPO levels were observed independently of the presence of clinical renal disease, proteinuria or renal insufficiency. This group included a subgroup of 16 asymptomatic carriers of the TTR V30M mutation. Low serum EPO levels were also observed in 44% of these asymptomatic carriers. In summary, anemia in FAP is a common feature and affects 46.8% of the symptomatic patients. A defective endogenous EPO production is the cause of anemia in FAP and can be corrected by treatment with recombinant human erythropoietin. Moreover, low serum EPO levels are observed early in the course of the disease, before anemia and even in the pre- symptomatic phase. It is possible that the low EPO production has other implications in the disease, besides anemia. FAP is a neurodegenerative disease of the peripheral nervous system and there is an increasing evidence of the neuroprotective role of EPO in several neurological disorders. The contribution of the defective EPO production to the neuropathy needs to be clarified as well as the potential benefit of EPO in the treatment of FAP.

63 LIPID RAFTS BREAKDOWN DECREASES THE ACTION OF HEPCIDIN ON MACROPHAGE FERROPORTIN Anne Auriac, Master², Alexandra Willemetz, Master² and François Canonne-Hergaux, PhD¹ ¹INSERM-CNRS; ²UPR2301, CNRS, Centre de recherche de GIF-sur-Yvette, France (Presented By: François Canonne-Hergaux, PhD)

Ferroportin (Fpn), the only mammalian iron exporter identified to date, is highly expressed at the basolateral cell surface of duodenal enterocytes and in tissues macrophages. In these cells, Fpn traffics from intracellular vesicular compartments to the cell surface. At that site, the presence of Fpn is strongly increased by iron loading and decreased by the action of the iron hormone hepcidin (Hepc). Hepc induces rapid endocytosis and subsequent degradation of the iron transporter. We previously showed that, in macrophages, Fpn defines specific cell surface domains suggestive of lipid rafts. In addition, in these cells, Fpn colocalises with CD11b, a plasma membrane marker also used as a raft associated antigen. Lipid rafts are known to be involved in signal transduction and intracellular trafficking. We therefore clarified the subcellular localization of Fpn in macrophage membranes and tested if rafts mediated endocytosis could play a role in hepcidin effect. Lipid rafts from untreated and iron (Fe-NTA) treated macrophages (murine bone marrow derived macrophages BMDM and J774a1) were isolated as detergent (TritonX- 100) -resistant membrane (DRM) fractions using ultracentrifugation on discontinuous iodixanol gradients. Fpn, raft (caveolin1, flotillin) and non-raft (TfR) markers were analyzed on these fractions by western blotting. The effect of Hepc on Fpn expression in the presence of drugs inhibiting lipid rafts- (filipin and methyl-ßcyclodextrin: MßCD) or clathrin- (chlorpromazin) endocytosis was study by classical and “In cell” western blotting. After treatment, localization of macrophage Fpn was also studied by immunofluorescence. In both BMDM and J774a1, Fpn was strongly detected in the raft/DRM fractions. Iron treatment increased the presence of Fpn in the lightest raft/DMR fractions highly enriched in caveolin1 and flotillin. Interestingly, lipid rafts breakdown by cholesterol sequestration (filipin) or depletion (MßCD) impaired the hepcidin mediated Fpn degradation. The presence of Fpn at the cell surface after disruption of lipid rafts suggest that the process of hepc mediated endocytosis and subsequent degradation are affected. On the other hand, inhibition of clathrin endocytosis did not significantly interfere with hepcidin effect in these cells. Further investigations are needed to clarify the role of lipid rafts in Hepc mediated effects as well as in iron transport activity of Fpn at the cell surface of macrophages.

64 HEPCIDIN, THE HORMONE OF IRON METABOLISM, IS BOUND SPECIFICALLY TO Alpha2-MACROGLOBULIN IN BLOOD D.R. Richardson¹, G. Peslova², J. Petrak²,7, K. Kuzelova², I. Hrdy³, P. Halada4, P.W. Kuchel5, S. Soe-Lin6, P. Ponka6, R. Sutak¹, E. Becker¹, M. Huang¹, Y.S. Rahmanto¹ and D. Vyoral¹,² ¹Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia; ²Institute of Hematology and Blood Transfusion U Nemocnice 1, Prague 2, 128 20, Czech Republic; ³Department of Parasitology, Faculty of Science, Charles University, 12844 Prague, Czech Republic; 4Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídenská 1083, CZ-142 20 Prague 4, Czech Republic; 5School of Molecular and Microbial Biosciences, Biochemistry and Microbiology Building (G08), University of Sydney, Sydney, New South Wales, 2006 Australia; 6Lady Davis Institute, 3755 Cote Ste. Catherine Road, Montréal, Québec, Canada H3T 1E2; 7Institute of Pathophysiology, U Nemocnice 5, Prague 2, 128 53, Charles University in Prague, First Faculty of Medicine (Presented By: D.R. Richardson) D.V. and D.R.R. contributed equally to this investigation

Introduction: Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify α2-macroglobulin (α2-M) as the specific hepcidin-binding molecule in blood. Methods: Interaction of 125I-hepcidin with α2-M was identified using fractionation of plasma proteins followed by native gradient PAGE and mass spectrometry. Results and Discussion: Hepcidin-binding to non-activated α2-M displays high affinity (Kd 177 ± 27 nM), whereas hepcidin binding to albumin was non-specific and displayed non-saturable kinetics. Surprisingly, the interaction of hepcidin with activated α2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high affinity (Kd 0.3 µM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Since α2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to α2-M may influence its functions. In fact, the α2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. Conclusions: The demonstration that α2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

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65 IRON-DEPENDENT REGULATION OF FERRITIN BY HIF-1 IN C. ELEGANS Elizabeth Leibold, PhD, Joshua Romney, BS and Ben Newman, BS University of Utah (Presented By: Elizabeth Leibold, PhD)

Ferritin is the principal iron sequestration protein in organisms. C. elegans express two ferritin proteins (FTN-1 and FTN-2) that are highly expressed in intestine. ftn-1 and ftn-2 mRNA abundance are increased by iron and decreased by iron chelators. In transgenic worms, the expression of GFP transcriptional reporters ftn-1::gfp and ftn-2::gfp are also increased by iron and decreased by iron chelators. We identified a conserved 63-bp sequence in the 5' upstream promoter regions of ftn-1 and ftn-2, which we term the iron-dependent element (IDE). The IDE consists of two GATA and three canonical hypoxia-inducible factor-1 (HIF-1) binding sites. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNAi blocks ftn-1::gfp reporter expression in vivo. Furthermore, deletion of the IDE eliminates expression of ftn-1::gfp and ftn-2::gfp reporters, while insertion of the IDE into the promoter region of a heterologous gene activates iron-dependent transcription in intestine. These data indicate that the IDE is necessary and sufficient for iron regulation of ferritin in intestine. The presence of three HIF-1 binding sequences in the IDE suggests a role for HIF-1 in ftn-1 and ftn-2 transcription. HIF-1 protein stability is controlled by the prolyl hydroxylase EGL-9 and von-Hippel-Lindau VHL-I tumor suppressor protein, which mediate HIF-1 degradation in the presence of oxygen and iron. Depletion of HIF-1 by RNAi in ftn-1:gfp and ftn- 2:gfp reporter strains grown in the presence of the iron chelator bipyridyl do not show the expected reduction in GFP expression. Similarly, ftn-1 and ftn-2 mRNAs were upregulated in a HIF-1 knockout strain (KO) or in wildtype N2 worms depleted of HIF-1 by RNAi. Conversely, ftn-1 and ftn-2 mRNAs are downregulated in a strain carrying a loss-of function mutation in vhl-l, which constitutively expresses HIF-1. We also show that HIF-1 binds to the IDE by chromatin immunoprecipitation. These data indicate that HIF-1 binding to the IDE represses ftn-1 and ftn-2 transcription under iron deficient conditions. Because HIF-1 KO worms constitutively express ferritin, we predicted that they may be sensitive to iron deficiency. To test this, HIF-1 KO and N2 worms were made iron deficient by growth under different concentrations of bipyridyl. N2 worms develop normally under these conditions, whereas HIF-1 KO worms display a profound developmental delay. Depletion of FTN-1 and FTN-2 by RNAi in HIF-1 KO worms restores normal development. These data indicate that HIF-1 regulates the iron-dependent transcription of ferritin genes in C. elegans.

66 CHARACTERISATION OF THE INTERACTION BETWEEN CERULOPLASMIN AND LACTOFERRIN Robert Evans, BA, PhD¹, Celia Conesa², Maryam Amini¹, Lourdes Sanchez² and Sebastien Farnaud³ ¹Brunel University; ²University of Zaragoza; ³Dr Hadwen Trust & University of Westminster (Presented By: Robert Evans, BA, PhD)

Lactoferrin is a basic iron-binding glycoprotein present in milk and other secretory fluids. Ceruloplasmin is an acidic coppercontaining glycoprotein with ferroxidase activity. The selective binding of human lactoferrin to human ceruloplasmin has been observed both in vitro and in vivo, in blood plasma and milk. Such an interaction could be physiologically relevant as both proteins are known to be involved in acute inflammation processes. Ceruloplasmin maintains low concentrations of potentially toxic ferrous iron by oxidising it to ferric iron, while lactoferrin present in the foci of inflammation is thought to sequester ferric iron, thus preventing its utilisation by pathogenic bacteria and avoiding the formation of free radicals. The precise identity of the interacting regions on human lactoferrin and human ceruloplasmin are unknown. The aim of the present study was to investigate the interaction further using overlapping peptide libraries that are used routinely for the identification of immunological epitopes. An overlapping peptide library for human ceruloplasmin, comprising 13-amino acid linear peptides with a 6-amino acid overlap, was immobilised onto a cellulose membrane by N-terminal acetylation. The membrane was incubated with human milk lactoferrin conjugated with horseradish peroxidase (HRP). After removal of the excess, unbound, lactoferrin the bound lactoferrin was detected using a peroxidase substrate. After regeneration, the membrane was subsequently probed with HRPlabelled samples of bovine milk lactoferrin, human serum transferrin and camel milk lactoferrin. When the human ceruloplasmin library was probed with HRP-labelled human lactoferrin, the protein was observed to bind to three peptides corresponding to residues 204- 216 (VVDENFSWYLEDN), 715-727 (YYIAAVEVEWDYS) and 904-916 (FDENESWYLDDNI) in the amino acid sequence of human ceruloplasmin. The same three, predominantly acidic, peptides were also identified when the library was probed with HRP-labelled bovine lactoferrin, however, no binding was observed with HRP- labelled human serum transferrin or HRP-labelled camel milk lactoferrin. The solved crystal structure of human ceruloplasmin has revealed that the protein is formed of six domains arranged in a triangular array, with the catalytic ferroxidase centre located between domains 1 and 6. When the three lactoferrin-binding peptides, VVDENFSWYLEDN, YYIAAVEVEWDYS and FDENESWYLDDNI, were mapped onto the solved crystal structure of human ceruloplasmin they were found to be located in domains 2, 5 and 6, respectively. The third peptide is thus located close to the catalytic centre and provides further support for the proposal that in the lactoferrin:ceruloplasmin complex, oxidised iron can be made directly available to lactoferrin during the inflammation process. Such an arrangement would not only prevent the formation of potentially toxic hydroxyl radicals but also sequester iron and deprive pathogenic bacteria of a source of essential iron. As ceruloplasmin and lactoferrin have been shown to form 1:2 and 1:1 complexes in vitro but only 1:1 complexes in vivo it is possible that the lactoferrin-binding peptide residing in domain 2, corresponding to residues 204-216, could represent a secondary, non-physiologically relevant binding region. Further work is now in progress to identify ceruloplasmin-binding regions on lactoferrin using an overlapping peptide library for lactoferrin.

67 MAGNETIC DYNAMIC SUSCEPTIBILITY MEASUREMENTS AS A PRE-SCREENING TOOL TO INVESTIGATE THE PRESENCE OF FERRITIN IN DIFFERENT ORGANISMS Lucia Gutierrez and Francisco J. Lazaro Universidad de Zaragoza (Presented By: Lucia Gutierrez)

Iron storage is an important ability in the life of many organisms. This capacity, based on the presence of ferritin, is currently being investigated in many species. In particular, the genes that encode ferritin are now being studied among different phytoplankton species, as the mechanisms of iron storage may play an important role in photosynthetic algae blooms, with their concomitant environmental relevance in terms of atmospheric carbon fixation. Recent research has revealed the presence of the genes that encode ferritin in the diatom Pseudo-nitzschia multiseries (Marchetti 2008) while these genes have not been observed in other diatoms. As the iron stored inside the ferritin protein shell is usually in mineral form, its magnetic properties differ from those of other iron-containing proteins allowing the magnetic detection of ferritin-iron without the need of a previous isolation procedure. The use of this technique, whose first attempts started some time ago (Allen 2000), has been intensified in recent years. In particular isolated ferritins from horse spleen and rat liver present a low temperature out-of-phase susceptibility maximum around 10 K. This finding has allowed the systematic monitoring of the protein in rat whole tissues (see, e.g., Gutiérrez 2006) as result of different iron metabolism processes, and clearly opened the possibility of investigating the presence of ferritin in other organisms. In this presentation, a comparative overview of the systematic research that is currently taking place in the detection of ferritin iron from the out-of-phase susceptibility vs temperature profiles in different organisms is shown. Several mice tissues together with mutants of the fruit fly, Drosophila melanogaster, and some marine phytoplankton species have been characterized showing the presence ferritin-iron. Differences in the location in temperature of the out-of-phase susceptibility maxima (see figure) for some of these organisms (three of them shown in the graph: isolated horse spleen ferritin, mouse liver tissue and Isochrysis galbana (Haptophyta) cells have been observed, suggesting slight differences in their iron biomineralisation in terms of either different crystallinity of the iron compounds and/or different sizes of the crystallites.

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Although the presence of the iron-storage protein is not directly detected through this method, the systematic and greatly reproducible results undoubtedly indicate a storage mechanism in some species justifying its interest as a pre-screening tool in the search of ferritin in different organisms and possibly in the frame of biological evolutionary studies. References - P.D. Allen et al., Biochimica et Biophysica Acta 1500, 186-196 (2000) - L. Gutiérrez et al, Journal of Inorganic Biochemistry, 100 (11), 1790-1799 (2006) - A. Marchetti et al, Nature 457, 467-470 (2008)

68 RELEVANCE OF COMMON POST-TRANSLATION MODIFICATIONS OF HUMAN SERUM ALBUMIN FOR NON-TRANSFERRIN-BOUND IRON SPECIATION Andre Silva, BSc and Robert Hider, PhD King’s College London (Presented By: Andre Silva, BSc)

Non-transferrin-bound iron (NTBI) is present in the serum of patients suffering from thalassemia, hemochromatosis and other iron-overloading diseases. Despite the central role of NTBI for the understanding of these pathologies its exact biochemical nature is yet to be determined. Previous studies indicate that citrate is involved in binding and solubilizing this iron pool and is thought to be its primary low molecular weight ligand. However, Human serum albumin (HSA) has been shown to behave as a low affinity iron binding protein and as it constitutes almost two thirds of the total serum protein content, HSA is bound to influence the speciation of NTBI. Further, HSA is subject to several post-translation modifications, such as glycation, oxidation and formation of mix-disulfides at residue 34, the extent of which varies with disease state. Aiming at clarifying the role of HSA in the speciation of NTBI and understanding the effect of post-translation modifications on modulating the iron binding ability of albumin, a set of experiments was design to compete different albumin preparations with citrate for iron binding under serum like conditions of pH, ionic strength and concentration. Recombinant human albumin (Novozymes Biopharma UK) was utilized in this study in order to overcome the transferring contamination present in commerciallly available albumin purified from human serum. Supplementary, recombinant albumin is a more uniform sample presenting a smaller extent of modification, thus proving to be the ideal material to study the independent effect of common post-translation modifications. Different HSA samples were prepared in vitro according to previously described methods and the extent of modification was found to fall within relevant biological ranges. The fraction of albumin-bound iron was determined by gel filtration chromatography linked to inductively coupled plasma mass spectrometry (GF-ICP-MS). HSA was found to bind between 60 and 20% of the available iron when the iron concentration was varied over the common clinical range of NTBI concentrations (1 to 10 microM), thus proving to be a relevant ligand for NTBI speciation. Analysis of modified albumins showed that both glycation and oxidation increase the albumin iron binding capacity while cysteinilation or lipid loading had no significant effect. Glycated albumin was able to bind the total available iron under the adopted experimental conditions, proving to be a high affinity iron ligand. These two modifications are prevalent in diabetic patients, but serum markers for oxidative damage are also increased in iron overload patients. The results obtained in the course of this study indicate that non-enzymatic modifications of albumin may have a fundamental role in NTBI speciation and scavenging of modified HSA – iron complex for protein degradation may prove to be relevant in explaining the differences in tissue iron deposition observed in different iron overloading diseases. We also suggest that the low NTBI levels so far measured in diabetic sera may be understated and that more robust analytical techniques are required to determine NTBI levels in diabetic patients. Acknowledgement: A. Silva would like to thank “Fundacao para a Ciencia e Tecnologia”, Lisbon, Portugal for his PhD grant [SFRH/BD/22633/2005].The authors would also like to thank Novozymes Biopharma UK, Ltd. (www.novozymes.com) for the offer of the research grade recombinant human albumin utilized in this study.

69 RETINAL DEGENERATION IN CP, HEPH, FPN AND HEPC MUTANT MOUSE MODELS AMELIORATED BY IRON CHELATION Joshua Dunaief, MD, PhD¹, Majda Hadziahmetovic, MD¹, Jared Iacovelli, MS¹, Paul Hahn, MD, PhD¹, Ying Song, MD¹, Allan Hunter, MD¹, Steven Grieco, BA¹, Adriana Donovan, PhD², Nancy Andrews, PhD³ and Sophie Vaulont, PhD4 ¹University of Pennsylvania; ²Novartis; ³Duke; 4Cochin Institute (Presented By: Joshua Dunaief, MD, PhD)

Introduction: Iron dysregulation contributes to the retinal degeneration in several diseases, including age-related macular degeneration, aceruloplasminemia, Friedreich's Ataxia, and Pantothenate Kinase Associated Neurodegeneration. This is not surprising, as the retina has high metabolic activity, high oxygen concentration, and a high concentration of easily oxidized polyunsaturated fatty acids. We have sought to increase understanding of retinal iron homeostasis, create models of retinal degeneration, and test chelation therapy by studying mice harboring mutations in iron homeostasis genes. Methods: Iron overload and retinal degeneration has been studied in Cp/Heph knockout, Hepc knockout, and conditional Fpn knockout mice using histology, electron microscopy, Perls' staining, atomic absorption spectrophotometry. Labile iron levels are measured indirectly through qPCR measurement of TfR mRNA. Oxidative stress is measured using mass spectroscopy to quantify isoprostane F2-alpha. Iron chelators are administered PO, IP, and by eye drops. Results: All of the mutant mice listed above have an age-dependent retinal degeneration. In the Cp/Heph and the Hepc knockout mice, the degeneration is morphologically similar: the photoreceptors die over time, while the retinal pigment epithelial cells become laden with iron-containing lysosomes, autofluorescent and hypertrophic. Ferroportin conditional knockouts have an age-dependent retinal pigment epithelial cell atrophy. Chelator administration can decrease retinal labile iron levels and markedly protect against retinal degeneration. Discussion: These results demonstrate the importance of Cp, Heph, Fpn and Hepc in retinal iron regulation and maintenance of retinal health. Chelator studies indicate the possibility of therapeutic efficacy for protection against retinal degeneration. Conclusions: Iron homeostasis is important for retinal health, and many of the genes involved in systemic iron regulation are expressed by the retina and play a local role in control of retinal iron levels. In situations where iron dysregulation may occur, as with gene mutation, chronic inflammation, or retinal bleeding, iron chelation may help restore healthy iron levels.

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70 THE LIVER-SPECIFIC MICRORNA 122A REGULATES THE EXPRESSION OF HFE, HFE2 AND HEPCIDIN AND CONTROLS SYSTEMIC IRON HOMEOSTASIS Mirco Castoldi, PhD¹,4, Maja Vujic-Spasic, PhD¹,4, Jens Stolte², Vladimir Benes, PhD², Joachim Elmen, PhD³, Morton Lindow, PhD³, Sakari Kauppinen, PhD³, Matthias Hentze, MD²,4 and Martina Muckenthaler¹ ¹Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg; ²European Molecular Biology Laboratory, Heidelberg, Germany; ³Santaris Pharma, Hørsholm, Denmark; 4Molecular Medicine Partnership Unit, University of Heidelberg, Germany (Presented By: Martina Muckenthaler)

Hepcidin mRNA expression is fine tuned by positive (e.g. HFE, TfR2 or HJV) and negative regulators (e.g. TMPRSS6) to maintain iron homeostasis. In search for hemochromatosis protein-dependent changes in gene expression which may contribute to the development of HH we have analyzed microRNA expression in the liver of HFE-deficient mice applying a microarray platform that sensitively and accurately detects changes in microRNA expression (miChip). MicroRNAs have emerged as important regulators of metabolic processes, immunity and cancer. Differential expression of several microRNAs was detected in mice with HFE-deficiency and secondary iron overload, suggesting that microRNAs are regulated by iron availability. MiR122a, which is abundantly expressed specifically in the liver, caught our particular attention: similar to hepcidin mRNA, it showed decreased expression in HFE-deficient mice which did not increase following iron-dextran injection. To investigate the functional consequences of miR122a deficiency, wild-type mice were injected with a locked nucleic acid (LNA)-modified oligonucleotide complementary to miR122a (antimiR122a). AntimiR122a mice show reduced detectability of miR122a in the liver, reduced serum iron levels and signs of microcytic anemia. In addition, depletion of miR122a causes iron redistribution with a decrease in liver and an increase in spleen iron levels. Analysis of mRNA expression of iron-related genes in the liver reveals that miR122a depletion correlates with increased HFE, hemojuvelin and hepcidin mRNA expression suggesting a potential role for miR122a in the regulation of these HH associated proteins. In conclusion, our data show for the first time that microRNA levels are regulated by iron and vice versa that microRNA 122a is involved in the control of HH-protein dependent hepcidin expression and systemic iron homeostasis.

71 -153 C/T MUTATION IN HEPCIDIN PROMOTER MAY IMPACT PRESENTATION OF HFE RELATED GENETIC IRON OVERLOAD Marie-Laure Island, PhD¹, Anne-Marie Jouanolle, PhD², Annick Mosser, PhD², Yves Deugnier, MD³, Véronique David, PhD², Pierre Brissot, MD4 and Olivier Loréal, MD, PhD5,6,7 ¹INSERM U522, University of Rennes 1, Rennes, France; ²Molecular Genetic Department, Hopital Pontchaillou, Rennes, France; ³Liver Diseases Unit and INSERM U522, Rennes, France; 4Liver Diseases Unit anddn INSERM U522, Rrennes; 5Inserm U522; 6IFR140; 7University of Rennes 1, Rennes, France (Presented By: Marie-Laure Island, PhD)

Hepcidin plays a major role in maintaining body iron homeostasis. Abnormally low levels of hepcidin regarding iron status are involved in the development of genetic iron overload, as found in genetic hemochromatosis related to pC282Y mutation in HFE. It can be hypothesized that every mutation favouring a low expression of hepcidin gene could contribute to the development of a severe phenotype during HFE related hemochromatosis. Based on this hypothesis, we identified a heterozygous mutation (nc.-153 C>T) in hepcidin gene promoter in one patient with homozygous C282Y mutation presenting an extremely severe iron overload resisting to well conduct iron depletive treatment. This mutation was not found in 100 healthy volunteers not presenting abnormal iron parameters. There was no other mutation found in the coding sequence of hepcidin gene as well as in transferrin receptor 2, hemojuvelin, and ferroportine sequence. Functional impact of this mutation, located within the proximal BMP-RE of the hepcidin promoter, was evaluated by analysing activities of wild-type and mutated hepcidin promoter gene reporter construct, containing 970 bp upstream of the initiation transcription site of hepcidin gene, which were transfected in HepG2 cells stimulated or not by BMP4 or 9, Furthermore, we characterized the SMAD protein complex binding on wild-type or mutated corresponding oligonucleotides by Electrophoretic Mobility Shift Assay (EMSA). Our results demonstrate that this mutation strongly decreases basal hepcidin promoter transcriptional activity. The expected responsiveness of hepcidin promoter to BMP-4 and 9 was totally abolished, whatever the dose of BMPs, thus demonstrating the severity of the mutation. In addition, despite the fact that transcriptional activity of the promoter was not affected by the mutation in term of fold of induction, the total transcriptional activity remains lower with mutated forms compared to wild type form of the promoter. EMSA and coupled with supershift analysis demonstrate that the mutation prevented the binding of SMAD protein complex (1/5/8 and 4) to the BPM-RE. In conclusion, our results demonstrates that the nc.-153 C>T affects transcriptional activity of the hepcidin promoter by inhibiting the transduction signal related to the BMP/SMAD pathway. Such mutation in hepcidin promoter may have a cumulative effect which impact the phenotype of patient presenting with genetic HFE related hemochromatosis. This work is supported by EEC FP6 Euroiron1, LSHM−CT−2006−037296.

72 THE PATHOGENESIS OF HEREDITARY HEMOCHROMATOSIS Emilio Ramos, Y. Phung, V. Gabayan, T. Ganz and E. Nemeth Departments of Medicine and Chemistry and Biochemistry, David Geffen School of Medicine at UCLA (Presented By: Emilio Ramos)

Hereditary hemochromatosis (HH) is an iron overload disease which can result from mutations in genes encoding HFE, transferrin receptor 2 (TfR2), hemojuvelin (HJV) or hepcidin. The unifying consequence of all these mutations is inadequate synthesis of the hormone hepcidin, the principal regulator of iron absorption and tissue iron distribution. Hepcidin deficiency in HH results in excessive iron absorption and deposition of iron in liver and other organs, leading to their dysfunction. The genetics of HH suggests that HFE, TfR2 and hemojuvelin are regulators of hepcidin expression, but their mechanism of action is still unclear. In normal homeostasis, hepcidin production is increased by iron loading. In HH, however, hepcidin is inappropriately low for the degree of iron load suggesting that HFE, TfR2 and HJV may be involved in hepcidin regulation by iron. We explored the regulation of hepcidin by iron in vivo in three mouse models of HH: TfR2 mutants (TfR2m/m), HFE knockouts (HFE-/-) and HJV knockouts (HJV-/-), as well as in vitro, in primary hepatocytes from WT and mutant mice. Mice were fed low iron diet (4 ppm) for 2 weeks, then switched to standard chow (336ppm iron) for 1, 7 or 21 days. Similar to human disease, HFE-/- mice had the least severe, and HJV-/- the most severe iron overload, based on their liver iron content. Baseline hepcidin mRNA levels, when compared to the wt counterparts, were most decreased in HJV-/- and least in HFE-/-. TfR2 had an intermediate hepcidin decrease and liver iron load. Importantly, all three mutant mouse strains had impaired hepcidin response to iron feeding. HFE-/- and TfR2m/m mice had a dramatically blunted response to 1 day iron feeding, but a different response to chronic iron challenge. While HFE-/- failed to increase hepcidin even after 21 days on iron diet, TfR2m/m mice hepcidin mRNA increase comparable to wt mice after 21 days on iron diet. The results suggest that that there are two types of iron signals regulating hepcidin, one likely involving circulating iron and depending on both HFE and TfR2 (acute signal) and the other involving HFE but not TfR2 (chronic signal). HJV-/- mice, which had extremely low hepcidin mRNA expression, were not able to upregulate hepcidin mRNA in response to either acute (1 day) or chronic (21 days) iron feeding, indicating that apart from setting hepcidin baseline, HJV may also be necessary for hepcidin regulation by iron. Our studies show that hepcidin deficiency, and consequently hereditary hemochromatosis, can result from both decreased hepcidin baseline expression and the loss of hepcidin response to acute or chronic changes in iron levels.

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73 A NOVEL TRANSFERRIN/TFR2-MEDIATED MITOCHONDRIAL IRON TRANSPORT SYSTEM IS DISRUPTED IN PARKINSON’S DISEASE Pier Mastroberardino, PhD¹, Maxx Horowitz¹, Ranjita Betarbet, PhD², Claire-Anne Gutekunst, PhD², Marla Gearing, PhD², John Q. Trojanowski, PhD³, Marjorie Anderson, PhD4, Charleen T. Chu, MD, PhD¹, Junmin Peng, PhD² and John T Greenamyre, MD, PhD¹ ¹University of Pittsburgh; ²Emory University; ³University of Pennsylvania; 4University of Washington (Presented By: Pier Mastroberardino, PhD)

More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson’s disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell - mitochondria and the respiratory complexes - has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention.

74 DOMINANT MUTANTS OF CERULOPLASMIN INDUCE GOLGI FRAGMENTATION AND IMPAIRMENT OF THE COPPER LOADING MACHINERY IN ACERULOPLASMINEMIA Giovanni Musci, Prof, Nunziata Maio, Gianluca Rizzo, Giovanni De Francesco, Tiziana Persichini, Marco Colasanti, Fabio Polticelli and Maria Carmela Bonaccorsi di Patti University Roma Tre (Presented By: Giovanni Musci, Prof)

Aceruloplasminemia is a rare autosomal recessive iron overload disease caused by mutations in the gene of ceruloplasmin (Cp), a multicopper oxidase playing a key role in iron homeostasis. Cp is mainly secreted by hepatocytes, where the P-type ATPase ATP7B incorporates copper into apo-Cp. An alternatively spliced GPI-anchored isoform of Cp has been identified in brain astrocytes and in other cell types. Recent data have demonstrated that the ferroxidase activity of Cp is required for the stability of cell surface ferroportin (Fpn), the only known mammalian iron exporter, and that Fpn is rapidly internalized and degraded in the absence of Cp. In this study, the funcionality of missense mutations causing aceruloplasminemia was investigated by testing the ability of the mutants to prevent ferroportin degradation. Since brain iron overload is a hallmark of aceruloplasminemia, we chose the rat C6 glioma cell line as our model system. Cells were previously silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin), and we found that this was due to the irreversible loss of copper binding ability. Mutant R701W, which is found in a heterozygous very young patient with severe neurological problems, was unable to complement per se, but did so in the presence of copper-glutathione, or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and induced extensive Golgi fragmentation and subcellular relocalization of ATP7B. From a structural point of view, a disulphide bridge close to arginine 701 was found to be involved in these effects, as both C699S and C618S mutants behaved like R701W. It should be noted that fragmentation of the Golgi apparatus appears to be a general hallmark of neurodegenerative disease. Earlier examinations of human brain tissues and animal models have shown that fragmentation of the Golgi apparatus is found in Alzheimer’s disease, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, multiple system atrophy, Parkinson’s disease, spinocerebellar ataxia type 2, and Niemann-Pick disease type C. This has led to the hypothesis that the morphological status of the Golgi apparatus may be a reliable index of activity of degeneration. Our results constitute the first evidence of ‘functional silencing’ of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.

75 HUMAN AMYLOID-B BINDS HEME TIGHTLY: WHY HUMANS BUT NOT RODENTS DEVELOP ALZHEIMER’S DISEASE Hani Atamna The Commonwealth Medical College, Pennsylvania, USA (Presented By: Hani Atamna)

Amyloid-b (Ab) peptide, which accumulates in the brains of Alzheimer’s disease (AD) patients, is neurotoxic. Although Ab aggregates are proposed to play key role in AD, the mechanism of Ab neurotoxicity is not known. We investigated how AD affect the neuronal heme metabolism and found that heme metabolism is abnormal in AD brains. We then searched for a molecular link between heme and Ab. We found that Ab binds and sequesters heme (i.e., regulatory heme), forming an Abheme complex. We further provided experimental evidence that sequestration of heme by Ab plays key role in Ab’s neurotoxicity. Because heme binding with Ab is fundamental for this mechanism, we searched for additional evidence for the relevance of Ab-heme to AD. It is known that species that develop AD-like neuropathology share an identical amino acid sequence of Ab. Rodents, on the other hand, lack AD-like neuropathology. The amino acid sequence of rodent Ab (roAb) is identical to human Ab (huAb), except for three amino acids within the 16 hydrophilic amino acids that make the N-terminal region of Ab (Site-H). We previously proposed that site-H in Ab binds with heme. The lipophilic amino acids 17-42 of Ab (Site-L) are identical among all the species regardless of the presence or absence of AD-like neuropathology. The lipophilic residues in site-L are responsible for Ab aggregation and fibrillation. We hypothesized that roAb has low affinity for heme compared to huAb, however it is as efficient in forming aggregates as huAb. We determined and compared the binding constants of roAb and huAb with heme, the resulting peroxidase activity, and the tendency to form aggregates. We found that unlike roAb, huAb binds heme tightly (Kd=140±60 nM, Hyperbola binding) and forms a peroxidase. The peroxidase efficiency increases as the huAb/heme ratio increases. roAb, on the other hand binds heme with affinity 18 fold less than huAb with sigmoidal binding kinetics. Interestingly, we found that both roAb and huAb form aggregates equally. These results suggest that heme depletion and Ab-heme peroxidase formation contribute to huAb’s neurotoxicity and oxidative stress in the early stages of AD. We conclude that phylogenic variations in the amino acid sequence of Ab result in tight heme-binding to huAb and increased human susceptibility to AD.

76 REGULATION OF IRON TRANSPORT ACROSS THE BLOOD BRAIN BARRIER James Connor, PhD¹, Padmavathi Ponnuru, PhD², Becky Webb, BS³, David Antonetti, PhD4, Anthony Carruthers, PhD5 and Ian Simpson, PhD6 ¹Penn State University College of Medicine; ²Dept. of Neurosurgery, Penn State University College of Medicine, Hershey, PA; ³Dept. of Neurosurgery, Penn State University College of Medicine, Hershey, PA; 4Dept. of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA; 5Department of Biochemistry, UMMS, Worcester, MA; 6Department of Neural and Behavioral Sciences Penn State University College of Medicine, Hershey, PA (Presented By: James Connor, PhD)

Iron is essential for all cell types in the body including those of the brain. The endothelial cells of brain vasculature, however, are linked by tight junctions to form a barrier between the blood and the brain known as the blood-brain-barrier (BBB) that limits access of the brain to iron. The existing paradigm for brain iron acquisition teaches that iron bound to transferrin is taken up from the blood via the interaction of transferrin (Tf) and the transferrin receptor (TfR) on endothelial cells. This complex is then transcytosed across the endothelial cells and the transferrin-bound iron in released into the brain. This model identifies the function of endotheliaI cells of the BBB as simple conduits for iron transport and fails to account for the iron requirements of the endothelial cells or provide any sense of regulation of brain iron uptake. Our hypothesis is that 1) endothelial cells of the BBB regulate brain iron acquisition and are not simply a conduit but also serve as a reservoir for brain iron. To establish that iron regulatory 89 components exist in brain microvasculature, we performed quantitative real time PCR which indicated the presence of Iron Regulatory Protein 1, ferroportin and transferrin in the human brain microvasculature. The presence of IRP1 indicates that endothelial cells have the mechanism for regulating TfR and ferritin expression in response to their labile iron pool. These results suggest that the amount of TfR expression and thus brain iron uptake is controlled by the amount of iron in the endothelial cells. The presence of ferroportin suggests a mechanism for iron export independent of transferrin. To address the second part of our hypothesis we used an in vitro BBB model system using a monolayer of bovine retinal endothelial cells (BRECs). 59Fe-NTA was added to the upper chamber of a BREC monolayer and incubated overnight. The cells were rinsed the following morning and apo-Tf, holo-Tf, hepcidin or erythropoietin (EPO) were placed into the lower chamber. 59Fe flux was measured into the lower chamber. There was a significant increase in iron flux into the lower chamber when apo-Tf was present whereas the presence of holo-Tf had significantly decreased the basal rate of iron release. While EPO had no effect, hepcidin decreased the rate of iron release from the BRECs. These results strongly suggest that endothelial cells forming the BBB act as reservoirs of iron and that there is a feedback system to the basal surface of endothelial cells that alters the basal rate of iron release. The release of iron from the endothelial cells is an exciting finding and our next step is to determine if signals regarding iron status are sent from cells in the brain and from the cerebrospinal fluid to the endothelial cells in the BBB and how the endothelial cells respond to these signals. A computer model that simulates brain iron acquisition and regulation will be presented.

77 THE CHOROID PLEXUS IS A SITE FOR REGULATING BRAIN IRON HOMEOSTASIS Fernanda Marques, Ana Mendanha Falcao, BSc, Joao Carlos Sousa, PhD, Giovanni Coppola, MD, PhD, Daniel Geschwind, MD, PhD, Nuno Sousa, MD, PhD, Margarida Correia-Neves, PhD and Joana Almeida Palha, PhD University of Minho, Life and Health Sciences Research Institute (ICVS), Portugal (Presented By: Fernanda Marques)

Iron is an essential element to the body homeostasis. Hepcidin (HAMP), a peptide hormone mainly synthesized in the liver, is the principal regulator of systemic iron homeostasis. HAMP is also one of the major proteins induced during the innate immune response, inhibiting iron efflux from the tissues to the serum. Because the choroid plexus responds to an inflammatory stimulus we studied Hamp expression in the choroid plexus upon lipopolysaccharide (LPS) peripheral administration. As early as 3h after LPS injection, Hamp mRNA levels are up-regulated in the choroid plexus returning to basal levels after 24h. HAMP expression is stimulated by Interleukin 6, which reaches choroid plexus epithelial cells from the bloodstream, and activates the signal transducer and activator of transcription 3 that in turn induces the transcription of HAMP. An additional mechanism through the transferrin receptor 2 is also proposed in the choroid plexus after LPS stimulation. This study shows that inflammation triggers the choroid plexus to respond by producing important iron-related proteins that, being secreted into the cerebrospinal fluid, may modulate brain iron metabolism. This novel function of the choroid plexus may be of relevance not only with respect to the brain innate immune response against infection, but also in neurodegenerative disorders in which iron accumulation has been reported.

78 DESFERRIOXAMINE AND DEFERIRSIROX TRAVERSE THE BLOOD BRAIN BARRIER AND PREVENT DEGENERATION OF DOPAMINE NEURONS IN THE 6-HYDROXY DOPAMINE MODEL OF PARKINSON’S DISEASE David Dexter, BSc, PhD¹, Hugh Chan, BSc¹, Robert Crichton, BSc, PhD, FRSC² and Roberta Ward, BSc, PhD² ¹Imperial College London; ²University of Louvain, Belgium (Presented By: David Dexter, BSc, PhD)

The deposition of iron in specific brain regions is associated with specific neurodegenerative diseases, e.g. the substantia nigra in Parksinson’s disease (PD) and the hippocampus, amygdala and neocortex in Alzheimer’s disease. Such iron may exacerbate the progression of each disease such that its removal by iron chelators may be of clinical benefit. In our earlier studies we clearly showed that deferriprone and desferrioxamine were able to cross the blood brain barrier (BBB) and remove iron from various brain regions in the ferrocene loaded rat. (Ward et al., 1995). There has been controversy as to whether other chelators, namely deferasirox is also able to traverse the BBB. In the present studies, the 6-hydroxy dopamine (6-OHDA) animal model of PD has been utilised to investigate whether these chelators can traverse the BBB and are neuroprotective. Such a model replicates the loss of nigrostriatal dopaminergic neurons, depletion of striatal dopamine content, activation of the innate inflammatory response, iron accumulation and oxidative stress as observed in PD. Rats received either desferrioxamine, 30 mg/kg, or deferirsirox, 20 mg/kg, for 1 day prior to the stereotaxic induction of the 6-OHDA lesion to the left medial forebrain bundle. After a further 4 days of daily chelator administration, the rats were killed, the brains removed and striatum dissected from the forebrain whilst the hindbrain containing the substantia nigra was placed in 4% paraformaldehyde fixative. The integrity of the nigrostriatal tract was assessed utilising HPLC analysis of the striatal dopamine content and its metabolites; while tyrosine hydroxylase immunohistochemistry was used to assess the numbers of dopaminergic neurons in the substantia nigra (SNc –principle area affected in PD). 6-OHDA lesioning produced a 43% loss of TH+ve dopaminergic neurons in the SNc and a 68% depletion of striatal dopamine content. The loss of TH+ve neurons in the SNc was significantly lower in both groups of rats which had received the chelators by comparison to the 6-OHDA treated controls (desferrioxamine 32% loss, deferirsirox 25% loss). In addition, deferirsirox and desferrioxamine administration attenuated the 6-OHDA induced depletion of striatal dopamine levels (desferirsirox 24% loss and desferrioxamine 35% loss compared to 6-OHDA alone 68% loss). The results of the current study indicate that both of the chelators are able to traverse the blood brain barrier and neuroprotect against damage induced by the 6-OH dopamine. R.J.Ward , D. Dexter , A. Florence, F. Aouad , R. Hider, P.Jenner and R.R. Crichton (1995). Biochemical Pharmacology 49 1821-1826 Acknowledgements to COST D34 for supporting our international cooperation

79 USE OF JAK2 INHIBITORS TO LIMIT INEFFECTIVE ERYTHROPOIESIS AND IRON ABSORPTION IN MICE AFFECTED BY BETA-THALASSEMIA MAJOR, N°79 Luca Melchiori, graduate student¹, Sara Gardenghi, PhD², Ella Guy, BaS², Nan Chen, BaS², Eliezer A. Rachmilewitz, MD³, Patricia J. Giardina, MD², Robert W. Grady, PhD² and Stefano Rivella, PhD² ¹Weill Cornell Medical College; ²Weill Cornell Medical College, New York; ³Wolfson Medical Center, Israel (Presented By: Luca Melchiori, graduate student)

Beta-thalassemia is characterized by ineffective erythropoiesis (IE). Some of the major consequences of IE are extra-medullary hematopoiesis (EMH), splenomegaly and systemic iron overload mediated by transfusion therapy and down-regulation of hepcidin. We hypothesized that the protein kinase Jak2 plays a major role in IE. For this reason, we administered a Jak2 inhibitor (TG101209) to mice affected by beta-thalassemia intermedia (th3/+), showing that this treatment was associated with a marked decrease in IE, and a more moderate decrease in hemoglobin levels (~1 g/dL). This last observation indicates that the use of Jak2 inhibitors might exacerbate anemia in thalassemia. Recall, however, that blood transfusion is required, not only by thalassemia-major patients, but also by those with thalassemia-intermedia who develop splenomegaly. Therefore, administration of a Jak2 inhibitor, together with blood transfusions, might be a sensible way to limit splenomegaly while preventing further anemia. This strategy might even complement our efforts to decrease iron accumulation in thalassemia. Therefore, we conducted an analysis of erythropoiesis and iron metabolism in animals affected by beta-thalassemia major (th3/th3), which require blood transfusion for survival. We first established the proper conditions for administering TG101209 to transfused th3/th3 mice. After 10 days, use of TG101209 not only reduced the spleen size dramatically (0.19±0.10 g vs. 0.42±0.15 g, P= 0.007) in TG101209- (N=8) versus placebo-treated (N=4) mice, but also resulted in higher hemoglobin levels (9.3±1.2g/dl vs. 7.3±1.1g/dl, P=0.019), perhaps because the reduced spleen size limited red cell sequestration. Unlike the situation in the placebo-treated mice, no foci of extra-medullary hematopoiesis were detectable in the parenchema of mice treated with TG101209. We observed a trend toward lower spleen weights with higher hepcidin expression, possibly indicating that suppression of IE (due both to blood transfusion and TG101209 administration) had a positive effect on hepcidin expression. Hemoglobin levels directly correlated with hepcidin expression in the same animals. In this case, however, transfusion undoubtedly played the major role in increasing hepcidin expression, 90

TG101209 perhaps having a positive effect by reducing the spleen size and thereby indirectly increasing hemoglobin levels. It has been proposed that Stat5, a transcriptional factor activated by Jak2, controls the level of transcription the transferring receptor-1 (TfR1). For this reason, we analyzed the mean fluorescence of CD71-positive cells, a marker of TfR1, in bone marrow and spleen, observing that it was reduced (P<0.01) in the animals treated with TG101209, indirectly supporting the notion that inhibition of Jak2 phosphorylation decreases TfR1 expression. These observations suggest that the use of Jak2 inhibitors may reduce the amount of blood per transfusion and/or the rate of transfusion necessary to properly manage thalassemia patients. Since the increased iron absorption in thalassemic mice is a direct consequence of IE, treatment with TG101209 could also be beneficial in ameliorating this process in patients. These studies were performed utilizing only transfused animals. Since blood transfusions repress erythropoiesis, they may mask the effect of the Jak2 inhibitor, thereby limiting our interpretations. To better evaluate this situation, we are now conducting studies in th3/+ mice treated with either TG101209 or placebo in the presence or absence of blood transfusions.

80 EXOGENOUS TRANSFERRIN AMELIORATES DISEASE IN BETA-THALASSEMIC MICE Yelena Ginzburg, MD¹, Huihui Li, MS¹, Anne Rybicki, PhD², Sandra Suzuka, MS³, Leni von Bonsdorff, PhD4, William Breuer, PhD5, Z. Ioav Cabantchik, MD, PhD5, Eric Bouhassira, PhD³ and Mary Fabry, PhD³ ¹New York Blood Center; ²Montefiore Medical Center; ³Albert Einstein College of Medicine; 4Sanquin; 5Hebrew University of Jerusalem (Presented By: Yelena Ginzburg, MD)

ß-thalassemia patients develop iron overload, resulting in major morbidity and mortality. These complications are caused by labile plasma iron which is taken up by parenchymal cells in a dysregulated manner. Because normal erythropoiesis requires transferrin-bound iron uptake via transferrin receptor, we hypothesized that anemia in ß-thalassemia results, in part, from insufficient circulating transferrin, which leads to expanded, but ineffective, erythropoiesis. We determined the capacity of exogenous transferrin to modulate iron metabolism and erythropoiesis in mice with ß-thalassemia. Providing exogenous transferrin to increase the efficiency of iron transport has several potential advantages: 1) Transferrin is able to sequester unbound iron, 2) iron is maintained in a redox-inactive form, and 3) iron is prevented from precipitating in tissues. Using human transferrin administered by daily intraperitoneal injections in ß-thalassemic mice, we compared parameters in untreated and age and gender matched mice treated for 10 days with apotransferrin or holotransferrin. Additionally, several mice were treated for 60 days. We analyzed changes in circulating red blood cell parameters (including hemoglobin and reticulocyte count); survival of red blood cells; non-heme iron distribution in different organs; the degree of extramedullary erythropoiesis; erythropoietin levels and apoptosis of erythroid precursors; circulating indicators of iron overload (such as labile plasma iron and transferrin saturation); and hepcidin expression in the liver. Exogenous transferrin reversed or significantly improved pathological characteristics of ß-thalassemia. Specifically, transferrin injections 1) normalized red cell survival, 2) increased hemoglobin production with concomitantly decreased reticulocytosis, erythropoietin levels, splenomegaly, and extramedullary erythropoiesis in the liver, 3) normalized levels of labile plasma iron, and 4) increased hepcidin expression. No appreciable difference was observed between apotransferrin and holotransferrin injected mice. Furthermore, transferrin improved the ability of erythroid precursors to mature, indicating its role in increasing the effectiveness of erythropoiesis in ß-thalassemia. In addition, although the spleen has the dual role as the site for extramedullary erythropoiesis and the site for erythroid precursor apoptosis, reversal of splenomegaly in transferrin-treated mice – despite an increase in both the number of mature erythroid precursors and in apoptosis of immature erythroid precursors – indicates that splenomegaly in ß-thalassemia serves as a reservoir for quiescent immature erythroid precursors that do not mature, yet, as a consequence of elevated erythropoietin levels, cannot undergo apoptosis. Lastly, although some of the details of these findings need clarification, the observation that hepcidin expression increased despite low transferrin saturation in mice with decreased extramedullary erythropoiesis provides further evidence for the existence of an “erythroid regulator” of hepcidin. Together, these results indicate that transferrin is a limiting factor contributing to anemia in these mice. We speculate that transferrin purified from human donors may have several potential uses not previously considered. The most obvious would be for treatment of patients with diseases of concurrent anemia and iron overload. In these circumstances, additional transferrin could be used to abrogate ineffective erythropoiesis by redirecting iron from storage and parenchymal deposition to erythropoietic machinery for hemoglobin synthesis. Examples of such diseases include ß-thalassemia, sickle cell anemia, and myelodysplastic syndromes. A novel approach would greatly benefit this patient population for whom standard management has consisted of transfusion followed by chelation therapy for the last half-century.

81 DEVELOPMENT OF BMP ANTAGONISTS FOR EXPERIMENTAL AND THERAPEUTIC CONTROL OF HEPCIDIN EXPRESSION Randall Peterson, PhD, Paul Yu, MD, PhD, Chetana Sachidanandan, PhD, Greg Cuny, PhD, Charles Hong, MD, PhD and Kenneth Bloch, MD Harvard Medical School (Presented By: Randall Peterson, PhD)

The hormone hepcidin is a central regulator of iron homeostasis. By controlling the turnover of the iron transporter ferroportin, hepcidin regulates the rate of iron release from intracellular stores. Recently, Bone Morphogenetic Protein (BMP) signaling has emerged as a critical regulator of the expression of hepcidin in the liver. Therefore, small molecule antagonist of BMP signaling may be useful for studying hepcidin regulation and may have therapeutic utility for diseases characterized by hepcidin overexpression, including the anemia of inflammation (AI). Using an in vivo small molecule screening approach conducted in developing zebrafish embryos, we discovered dorsomorphin, the first small molecule antagonist of BMP signaling. Dorsomorphin antagonizes BMP signaling by inhibiting the kinase activity of the Type I BMP receptors Alk2 and Alk3. Extensive medicinal chemistry has yielded derivatives of dorsomorphin with improved properties, including LDN-193189, which exhibits high-potency and selectivity for the BMP receptors and favorable pharmacokinetics for in vivo studies. In zebrafish studies, we have found that exogenous iron and the inflammatory cytokine IL-6 both induce hepcidin expression, and that BMP antagonists block these effects. The BMP antagonists also reduce hepcidin expression in mice, with a concomitant increase in serum iron concentration. These findings point to the hypothesis that BMP antagonism could be used to treat anemia of inflammation and provide powerful chemical tools for testing this hypothesis in animal models of the disease.

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82 NATURAL AND SYNTHETIC STAT3 INHIBITORS REDUCE SIGNIFICANTLY HEPCIDIN EXPRESSION IN DIFFERENTIATED MOUSE HEPATOCYTES EXPRESSING THE ACTIVE PHOSPHORYLATED FORM OF STAT3 Nadia Fatih¹, Emilie Camberlein, PhD², Marie-Laure Island, PhD², Anne Corlu, PhD², Emmanuelle Abgueguen, PhD², Patricia Leroyer², Pierre Brissot, MD² and Olivier Loréal, MD, PhD² ¹INSERM U522, University of Rennes1, Rennes France; ²INSERM U522, University of Rennes 1, Rennes (Presented By: Nadia Fatih)

During the inflammatory process of chronic diseases, hepcidin expression increases. Anaemia of chronic diseases is the second most common form of anaemia worldwide after nutritional iron deficiency. Therefore, the identification of therapeutic agents decreasing hepcidin expression is an important goal. Our aim was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. Material and Methods: Hepatocytes isolated from C57Bl/6 mice were maintained in coculture with Rat Liver Epithelial Cells (RLEC) during 2 to 6 days. mRNA levels of genes of interest were evaluated using quantitative RT-PCR using mouse specific primers. Hepatocyte differentiation was evaluated by quantification of albumin, aldolase B and Cyp3A4 mRNA levels. Expression of genes involved in the control of iron metabolism, including hepcidin1 (mouse equivalent of human hepcidin), HFE, hemojuvelin, transferrin receptor 2 were quantified by measuring their respective mRNAs steady state levels. STAT3 expression was evaluated by measuring its mRNA level and by western-blot analysis of either its non-phosphorylated or its active phosphorylated form. STAT3 impact on hepcidin expression was studied by using specific siRNAs. The effect of specific inhibitors of STAT3 (curcumin, AG490 and a synthetic peptide) was evaluated on hepcidin1 expression. Results: Coculture model allowed maintenance of hepatocyte differentiation in contrast to pure primary hepatocyte culture. Hepcidin1 mRNA was detected in coculture at very high levels compared to hepatocytes plated in pure culture. Moreover, hepcidin1 mRNA levels at D2 and D6 were 2 and 4 folds higher than those obtained with freshly isolated hepatocytes. This sustained hepcidin1 expressionwas associated with maintained mRNA levels of other iron regulatory genes, contrasting with the drop observed in pure primary culture. Furthermore, we found in coculture that STAT3 expression was characterized by the presence of the active phosphorylated form of STAT3 protein and that STAT3 siRNAs, targeted toward hepatocytes, strongly decreased hepcidin1 expression. Screening the efficiency of STAT3 natural and synthetic inhibitors when added to the culture medium, we found a significant decrease of both the STAT3 phosphorylated form and hepcidin1 expression, without significant toxicity. Conclusions: Our hepatocyte coculture model permits: i) the maintenance of hepatocyte differentiation and ii) a sustained hepcidin1 expression associated to preservation of the transduction pathways which control its expression. In addition, we found that STAT3 inhibitors, tested in a differentiated hepatocyte model, are potential tools for the control of hepatocyte hepcidin overexpression during the inflammatory process. Globally, these results suggest that the model that we developed is useful to test in vitro new compounds supposed to target the STAT3 pathway. This work was supported by EEC FP6 Euroiron1, LSHM-CT-2006-037296.

83 GENE REGULATION THERAPY UTILIZING FERRITIN HEAVY CHAIN Robert H. Broyles, BSc, PhD¹, Visar Belegu, PhD², Marie Trudel, DSc³, Sonia Levi, PhD4 and Paoloa Arosio, PhD5 ¹Univ of Oklahoma Health Sciences Ctr.; ²Johns Hopkins University; ³Institut de Recherches Cliniques de Montréal; 4DIBIT-IRCCS, H.San Raffaele, Milano; 5Materno Infantile e Tecnologie Biomedicine, University of Brescia (Presented By: Robert H. Broyles, BSc, PhD)

Ferritin heavy chain (FtH) is an embryonically-expressed protein, prominently expressed in the erythroid lineage and in developing brain. In adults, FtH is highly expressed in CNS neurons, in heart and in kidney. In the adult erythroid lineage, FtH expression is low and disappears as erythroid differentiation and hemoglobin (Hb) synthesis begins. High FtH expression in embryonic erythroid cells versus low-to-nil expression in adult erythroid cells is a key developmental difference in the mechanism of developmental Hb switching (Broyles et al., PNAS 98: 9145, 2001). FtH represses adult ß-globin expression and activates gamma (fetal)-globin gene expression in embryonic/K562 erythroid cells. These findings have led us to propose utilizing FtH as a therapeutic agent in sickle cell disease (SCD) and ß-thalassemias, since it is well known that high expression of gamma (fetal)-globin markedly alleviates both disorders. Our results with K562 cells that express only embryonic and fetal Hbs have supported the theoretical basis for FtH as an Hb switching factor. FtH localizes to the nucleus in K562 cells and represses the human adult ß-globin promoter in transient expression assays. Chromatin immunoprecipitation (ChIP) assays using anti-FtH polyclonal antisera show that FtH is bound to the -150 promoter repression site in vivo in K562 cells in which the ß-globin gene is repressed. An Alexa488-tagged antisense oligonucleotide to FtH transfected into K562 cells enters the nucleus and derepresses the ß-globin gene. Strickingly, the anti-FtH oligo knocks down both FtH and gamma (fetal)-globin gene expression by over 90 percent, confirming FtH’s role as an activator of fetal Hb. Competitive EMSA assays have revealed that the mouse ßMajor-globin promoter has an analogous CAGTGC motif at –160 bp from the cap site that competes specifically with the human CAGTGC site for K562 nuclear FtH binding. However, the mouse ßMinor-globin promoter lacks the –150/-160 CAGTGC motif and, therefore, the FtH binding site. Thus, a human FtH transgenic mouse, in which the FtH gene is driven by a truncated ß-promoter lacking the CAGTGC motif, expresses human FtH in definitive erythroid cells which results in repression of ßMajor-globin but not ßMinor-globin. Thus, these TgFtH mice are born with a reduced ratio of ßMajor/ßMinor globins, resulting in α-globin excess and a mild ß-thalassemia. FtH-tg mice with high copy numbers of the transgene have greater numbers of target cells, due to inclusions of excess a-globin. In applying this therapy to humans, thalassemia would not be expected since we have found that FtH represses ßS-globin and activates gamma (fetal)- globin gene expression in maturing human erythroid precursor cells from pediatric sickle cell patients. FITC-labeled FtH enters these human erythroid precursor cells, presumably via a FtH-specific receptor that has been found on the surface of these cells. FtH receptors have also been found on liver cells and in the blood brain barrier, suggesting that FtH might be used as a therapeutic in a number of highly prevalent human diseases.

84 MINIHEPCIDINS: SMALL PEPTIDES INVOLVED IN DISULFIDE EXCHANGE WITH FERROPORTIN ACT AS AGONISTS Tomas Ganz, PhD, MD, Gloria Preza, MS, Peter Ruchala, PhD and Elizabeta Nemeth, PhD University of California, Los Angeles (Presented By: Tomas Ganz, PhD, MD)

The hepcidin-ferroportin interaction is the central regulatory event in systemic iron homeostasis. To facilitate the understanding of hepcidin structure and to aid the development of useful hepcidin agonists we searched for the minimal structure that would display hepcidin agonist activity. The bioactivity of candidate peptides was assayed by flow cytometry measuring the degradation of GFP-ferroportin in a cellular model. We previously showed that the five N-terminal residues, DTHFP, were essential but not sufficient for bioactivity. Our ferroportin studies identified C326 cysteine thiol as an essential feature of the binding site for hepcidin suggesting that disulfide exchange may be necessary for hepcidin agonist activity. We therefore synthesized a series of minihepcidins that contained the N-terminus but also included the first or second cysteine in a thiol form. These peptides exerted substantial bioactivity. Substitution of the cysteine with isosteric serine not capable of disulfide exchange ablated minihepcidin activity as did blocking of the cysteine thiol. Substitution with unnatural amino acids capable of disulfide exchange retained activity and confirmed the importance of ligand-receptor disulfide formation. Additional substitutions were devised to increase the resistance of hepcidin to proteolysis and its chemical stability. Small hepcidin agonists should provide the benefit of decreased cost of production, favorable pharmacokinetics and possibly oral bioavailability, which should make them useful as therapeutics for iron overload disorders.

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85 HFE MUTATIONAL ANALYSIS HAS REDUCED THE NEED FOR LIVER BIOPSY AT DIAGNOSIS BUT NOT IMPACTED ON INITIAL DISEASE BURDEN IN HEREDITARY HAEMOCHROMATOSIS PROBANDS John D. Ryan, MBBCh, Mary T. O’Neill, MBBCh, Eleanor Ryan, PhD, T. Barry Kelleher, MD and John Crowe, PhD Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin, Ireland (Presented By: John D. Ryan, MBBCh)

Hereditary Haemochromatosis (HH) is the commonest inherited disorder in Ireland. Homozygosity for the C282Y substitution is responsible for HH in 93% of Irish HH patients and occurs in 1% of the Irish population. Disease penetrance is variable due to genetic and environmental factors. Genetic testing for HFE mutations became routine in this institution in 1997, prior to which liver biopsy was mandatory to establish a diagnosis of HH. Liver biopsy is since reserved for those with serum ferritin >1000 µg/L, age >40yrs or abnormal liver function tests. Objective: The objective of this study was to retrospectively analyse the impact of genetic testing on the severity of disease burden at presentation in patients without a family history of HH and having evidence of iron overload (i.e. probands). Methods: Approximately 300 C282Y homozygotes were diagnosed at this centre prior to August 2008. Of these, 159 were HH probands with biochemical and/or histological data available. Demographic information, iron parameters, serum ALT, and liver siderosis and fibrosis (METAVIR) grading were obtained from patient medical records, the hospital information system and the HH genotyping database. Patients were grouped according to gender and time of diagnosis, i.e. pre- and post- 1997. Data was analysed using SPPS for Windows (13.0). Results:Table 1 demonstrates the comparison of disease severity at presentation pre- and post- 1997. HH probands did not have statistically significant differences in serum ferritin, transferrin saturation, ALT, degree of hepatic fibrosis or siderosis upon presentation before and after 1997. Histological differences remained non-significant when post-1997 criteria for liver biopsy (i.e. ferritin >1000 µg/L etc.) were applied to the pre-1997 cohort. Significant correlations were found between serum ferritin and fibrosis grade (p<0.001), liver iron staining (p=0.001), and serum ALT (p<0.001). A 61% reduction in liver biopsy at presentation was observed overall, and a significantly lower frequency of HCC at diagnosis in males. Conclusion: Since the advent of HFE mutational analysis there has been a 61% reduction in liver biopsy at diagnosis but no alteration in severity of disease at presentation in HH probands.

86 HERITABILITY OF SERUM IRON MEASURES IN THE HEMOCHROMATOSIS AND IRON OVERLOAD SCREENING (HEIRS) FAMILY STUDY C.E. McLaren, PhD¹, J.C. Barton², G.D. McLaren³, R.T. Acton4, P.C. Adams5, L.F. Henkin6, V.R. Gordeuk7, J.H. Eckfeldt8, C.D. Vulpe9, B.W. Harrison¹°, J.A. Reiss¹¹ and B.M. Snively6 ¹University of California, Irvine; ²Southern Iron Disorders Center, Birmingham, AL; ³Dept. of Veterans Affairs Long Beach Healthcare System, Long Beach, CA; 4Univ. of Alabama at Birmingham, AL; 5London Health Sciences Centre, London, Ontario, Canada; 6Wake Forest Univ. School of Medicine, Winston-Salem, NC; 7Howard Univ.; 8Univ. of Minnesota, Minneapolis, MN; 9Univ. of California, Berkeley, CA; ¹°Howard Univ., Washington, DC; ¹¹Kaiser Permanente NW, Portland, OR (Presented By: C.E. McLaren, PhD)

Hereditary hemochromatosis (HH) is a common iron overload disorder of people of western European descent. Most whites with HH have common mutations (C282Y and H63D) in HFE. The spectrum of HH phenotypes is broad, and mutations in known iron-related genes do not account for most phenotype variability. Putative genetic factors may influence dietary choices or modulate iron absorption or loss via mechanisms presently undefined. The Hemochromatosis and Iron Overload Screening (HEIRS) Study is a multi-center, multi-ethnic study in which transferrin saturation (TS), serum ferritin (SF), and C282Y and H63D mutations were determined in 101,168 adults. We examined familial aggregation and genetic contributions to ironrelated phenotypes in the HEIRS Family Study and hypothesized that both genetic and environmental factors influence serum iron measures after adjustment for gender, age, HFE C282Y and H63D genotype, and other clinical and demographic characteristics. Heritability (h2) measures the fraction of variation between individuals in a population attributable to additive effects of their genotypes. We estimated heritability of TS, SF, and unbound iron-binding capacity (UIBC) in participants from the HEIRS Family Study (N=180 families, mean size 5.5). Eligible probands (aged >24 y) had both TS and SF values above gender-specific thresholds (TS>50% and SF >300 µg/L in men; TS >45% and SF >200 µg/L in women), or were C282Y homozygotes. Family members, 19 years of age or older, were eligible. There were 77% Caucasians, 3% African Americans, 8% Hispanics, and 10% Asians; average age (SD) was 49 (16) y; 56% were female. The distribution of HFE genotypes was 22% C282Y/C282Y, 7% C282Y/H63D, 2% H63D/H63D, 34% C282Y/+, 8% H63D/+, and 26% +/+. A variance component approach using SOLAR software estimated residual heritability, adjusting for age, gender, age x gender interaction, race/ethnicity, and HFE genotype (model 1). In another model, study site, body mass index, menopausal status, phlebotomy treatment, hepatitis, average daily intake of alcohol, and level of C-reactive protein were added to the core set of covariates (model 2). Natural log transformation of serum ferritin was performed. In model 1 (N=938), h2 was 0.25 (SE 0.056) for TS, 0.26 (0.055) for ln SF, and 0.40 (0.06) for UIBC; P < 0.000001 for each test of h2=0. Age, gender, race/ethnicity, and HFE genotype accounted for 36%, 38%, and 38% of the variability in TS, ln SF, and UIBC, respectively. In model 2 (N=828), adjusted for the full set of covariates, h2 was 0.16 (0.061) for TS, 0.27 (0.067) for ln SF, and 0.32 (0.067) for UIBC; P < 0.0009 for each. The percentage of variance due to covariates was 41% (TS), 43% (ln SF), and 43% (UIBC). Residual heritability estimates for data from probands and families ascertained by C282Y homozyosity were slightly higher than for all families. We conclude that quantitative serum iron measures in HEIRS Family Study participants have significant heritability components in addition to the effects of HFE C282Y and H63D genotypes, suggesting that other genetic variants contribute to iron phenotype variability. Gene discovery studies could provide insight into clinical disorders that are characterized by perturbations of iron metabolism.

87 TFR2-RELATED HEREDITARY HEMOCHROMATOSIS IN JAPAN Chisaki Mizumoto, MD¹, Junya Kanda, MD², Hiroshi Kawabata, MD², Takashi Uchiyama, MD², Katsutoshi Kojima, MD³, Tatsuya Hayashi, MD³, Masakazu Hirata, MD³, Kazuma Nakao, MD³, Ken-ichi Tsuchida, MD4, Miwa Kawanaka, MD5, Gotaro Yamada, MD5, Naohisa Tomosugi, MD6, Yasuaki Tatsumi, PhD7, Aya Hattori, MSc7 and Hisao Hayashi, MD7 ¹Kyoto University; ²Dept. Hematology/Oncology, Kyoto University; ³Dept. Medicine/Clinical Science, Kyoto University; 4Manda Memorial Hospital Diabtes Center; 5Center of Liver Diseases, Kawasaki Hospital, Kawasaki Medical School; 6Proteomics Research Unit, Division of Advanced Medicine, Medical Research Institute, Kanazawa Medical University; 7Dept. Medicine, Aichi Gakuin University School of Pharmacy (Presented By: Chisaki Mizumoto, MD)

Hereditary hemochromatosis (HH) is an inherited systemic iron overload syndrome that causes various clinical symptoms such as skin pigmentation, diabetes mellitus, hepatic dysfunction, cardiac failure, hypothyroidism, and hypogonadism. In Caucasians, mutations in the HFE gene, such as C282Y, are the common etiologies of HH. In

93 contrast, HFE-related HH is extremely rare in Japan. To determine the genetic etiology of Japanese HH, we have been analyzing mutations in known Hhrelated genes, including HFE, TFR2, HJV, and HAMP, in suspected cases. Here, we present a new case of TFR2-related HH in a Japanese patient and summarize our genetic study on Japanese HH. A Japanese woman was diagnosed with hemochromatosis at 56 years of age. She was taking oral iron supplement since she was 48 years old. At the time of the diagnosis, diabetes, hepatic dysfunction, and hypothyroidism were found, and her serum ferritin level was 5390 mg/dl. Hepatic iron accumulation was documented by liver biopsy and magnetic resonance imaging. After the diagnosis, iron supplement was discontinued and insulin therapy and infrequent phlebotomy were initiated. Her serum alanine aminotransferase level was gradually normalized. When she was 64 years old, a genetic analysis for HH was performed. At the time of this analysis, her serum ferritin level was 216 ng/ml, transferrin saturation was 93%, and serum hepcidin-25 level was 3.3 ng/ml (normal range is 2.3-37 ng/ml). We found a homozygous missense mutation in exon 8 of the TFR2 gene resulting in an amino acid substitution of A364T. This mutation has not been reported previously. No mutation was detected in the HFE, HJV, or HAMP gene. Her parents were second cousins. Thus far, we have analyzed 27 suspected cases of HH in Japan, and have identified 7 TFR2-related HH cases including the present case, 3 HJV-related cases, and no HFE-related case. In all the 7 TFR2-related cases, the patients had liver diseases and in 4 cases, the patients had diabetes. Transferrin saturation was above 88% in all TFR2-related cases. Our results indicate that mutations in the TFR2 gene may be the major cause of HH in the Japanese population. Further study is required to elucidate the frequency and significance of TFR2-related HH in patients, especially with diabetes and liver dysfunction in Japan.

88 IRON OVERLOAD IN THE ASIAN COMMUNITY Chun Yu Lok, A.T. Merryweather-Clarke, D.J. Weatherall and K.J.H. Robson Weatherall Institute of Molecular Medicine, Oxford, UK (Presented By: Chun Yu Lok)

Hereditary haemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 haemochromatosis is the most common form of the disease and due to mutations in the HFE gene. Juvenile haemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary haemochromatosis is commonly found in populations of European origin. By contrast, haemochromatosis in Asia is rare and less well understood, and can be masked by the presence of iron deficiency and secondary iron overload from thalassaemia. Here, we provide a comprehensive report of haemochromatosis in a group of patients of Asian origin. Through DHPLC and sequence analysis, we have identified novel mutations in HJV, HAMP and SLC40A1 in countries not normally associated with hereditary haemochromatosis (Pakistan, Bangladesh, Sri Lanka and Thailand). This will have consequences for the treatment of thalassaemia in these populations. Our family studies also show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.

89 DELETION OF THE HFE GENE IS PRESENT AT THE POPULATION LEVEL IN SARDINIA Gerald Le Gac, Dr¹,6, Antonio Cao, Pr², Rita Congiu, Dr³, Isabelle Gourlaouen4, Claude Férec, Pr4 and Maria Antonietta Melis, Pr5 ¹Inserm U613, Etablissement Français du Sang, Brest, F-29200 FRANCE; ²Istituto di Neurogenetica e Neurofarmacologia CNR, Cagliari, Italy; ³Ospedale microcitemico ASL8, Cagliari, Italy; 4Inserm U613, Etablissement Français du Sang, Centre Hospitalier Universitaire, Brest, F-29200 France; 5Dipartimento Scienze biomediche e biotecnologia, Università di Cagliari, Cagliari, Italy; 6Istituto di Neurogenetica e Neurofarmacologia CNR, Ospedale microcitemico ASL8, Dipartimento Scienze biomediche e biotecnologia, Università di Cagliari, Cagliari, Italy (Presented By: Gerald Le Gac, Dr)

Introduction: Very recently, we reported the case of a woman of Sardinian descent who had a major structural alteration in the HFE gene. Molecular characterization revealed an Alu-mediated recombination causing the loss of the complete HFE gene sequence. Although homozygous for the HFE deleted allele, the woman had a phenotype similar to that seen in most women homozygous for the common p.C282Y mutation. The deletion was not detected in a cohort of iron overload patients of Northern European descent. Here, we focused on DNA from Sardinia patients. Methods: We looked for the HFE deletion by using a rearrangement specific PCR. Positive results were confirmed by QFM-PCR and sequencing. Results: The HFE deleted allele was detected in two of 24 unrelated patients. Both patients were previously viewed as homozygous for the common p.H63D variation. At diagnosis, they presented with moderate iron overloads. Conclusion / Discussion: Deletion of the complete HFE gene sequence is not private, but present at the population level in Sardinia. Additional studies have been started to ascertain the assumption of a founder effect and precisely investigate frequency of the HFE deleted allele. In future, the recognition of several individuals with the HFE deletion will provides another opportunity to better understand penetrance of the common p.C282Y/p.C282Y genotype, which remains a matter of debates.

90 DO ALL HEMOCHROMATOSIS PATIENTS ORIGINATE FROM THE SAME ANCESTRAL ORIGIN BY MITOCHONDRIAL DNA ANALYSIS? Paul Adams, MD University Hospital (Presented By: Paul Adams, MD)

Background: Mitochondrial and y-DNA analysis have been widely used to predict ancestral origin. Genetic anthropologists have predicted that human civilizations may have originated in central Africa 1-2 million years ago. The Out of Africa hypothesis suggests that humans migrated to Europe from Africa. Primary iron overload is not a common diagnosis in northern Africa amongst indigenous people but hereditary hemochromatosis is seen in approximately 1 in 200 in Northern Europe. Mitochondrial DNA analysis has the potential to determine if today’s hemochromatosis patients have an ancient ancestral linkage. Methods: DNA was obtained from buccal smears for mitochrondrial DNA (mt-DNA) and y-DNA analysis. Y-DNA analysis included 20 STR markers on the Y chromosome. Mt-DNA analysis included genetic sequencing of the HVR-1 sequence (16001-16520) and compared to the Cambridge Reference Sequence (CRS). Mt-DNA ancestral haplotypes were predicted from the HVR-1 analysis. Results: Thirteen male C282Y homozygotes were studied. This included participants with ancestors from the United Kingdom, Portugal, Netherlands, and Belgium. There were 35 polymorphisms in HVR-1 seen in the 13 particpants. The most common polymorphism was seen in position 16519 in 7 participants and at position 16513 in 5 participants. There were 10 different ancestral haplotypes predicted by mt-DNA analysis with the H haplotype (Near East/Caucasus 20,000 years ago) being seen in 3 participants. By Y-DNA analysis there were 5 different haplotypes, with R1b (Cro-Magnons, 35,000 years ago) being found in 9 of 13 participants. Conclusions: The study of mt-DNA and Y-DNA in 13 hemochromatosis patients has suggested that they did not all originate from the same original tribe in ancestral Africa. These findings are consistent with the theory that the original hemochromatosis mutation occurred after migration of these ancestral people to central Europe possibly 4,000 years ago.

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Poster Abstracts

Poster# 1 A NEW FLVCR ISOFORM ABLE TO SUPPORT ERYTHROPOIESIS Deborah Chiabrando, PhD Student, Samuele Marro, Erika Messana, Sonia Mercurio, Emilia Turco, Lorenzo Silengo, Fiorella Altruda and Emanuela Tolosano, PhD Molecular Biotechnology Center, Torino (Presented By: Emanuela Tolosano, PhD)

Feline Leukemia Virus, subgroup C, Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukaemic virus, subgroup C causing in cats pure red-blood-cell aplasia. Recent works have demonstrated that FLVCR is a heme exporter essential for erythropoiesis. The heme efflux via FLVCR was shown to be essential for erythroid differentiation in K562 cells as well as in CD34+ precursors cells1. Moreover, Keel and co-authors have recently reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis2. We have recently identified a novel FLVCR isoform, called FLVCRb, coding for a putative 7 transmembrane domain-containing protein. Both FLVCRb and the canonical FLVCR (that, henceforth we will name FLVCRa), were expressed in all tissues: FLVCRa mRNA level was significantly higher than FLVCRb in liver, kidney, colon and duodenum, whereas it was the opposite in heart and skeletal muscle. In bone marrow, spleen and brain FLVCRa and FLVCRb mRNA levels were comparable. To gain insights into the specific roles of the two isoforms, we have generated Flvcr mutant mice different from those previously reported2, as they do not express the canonical FLVCRa, but an allele coding for FLVCRb. Flvcr-a heterozygous mice were grossly normal, fertile and indistinguishable from their wild-type littermates. When Flvcr-a+/– mice were intercrossed, no Flvcr-a homozygous knock-out newborns were obtained, and analysis of the embryos from timed Flvcr-a+/– intercrosses showed that the Flvcr-a–/– genotype was lethal between E14.5-E16.5. Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, and subcutaneous edema. Alcian blue-alizarin red staining demonstrated skeletal abnormalities in limbs and head. Flow cytometric analyses of E14.5fetal liver cells double-stained for Ter119 (erythroid-specificantigen) and CD71 (transferrin receptor) demonstrated normal erythropoiesis in Flvcr-a-null embryos: all populations corresponding to different maturational stages of differentiating erythroblastswere present and indistinguishable from those in wildtype littermates. Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis, but not to prevent endothelial ruptures responsible for hemorrhages, thus suggesting that FLVCRa is needed for detoxifying heme excess at these sites. References 1. Quigley JG, Yang Z, Worthington MT, Phillips JD, Sabo KM, Sabath DE, Berg CL, Sassa S, Wood BL, Abkowitz JL. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004;118:757-66. 2. Keel SB, Doty RT, Yang Z, Quigley JG, Chen J, Knoblaugh S, Kingsley PD, De Domenico I, Vaughn MB, Kaplan J, Palis J, Abkowitz JL. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319:825-8.

Poster# 2 ABSENCE OF THE HEMOCHROMATOSIS GENE HFE CONFERS PROTECTION UNDER CONDITIONS OF STRESS ERYTHROPOIESIS Pedro Ramos¹, Ella Guy1, Robert W Grady, PhD1, Maria de Sousa, MD, PhD² and Stefano Rivella, PhD1 ¹Weill Cornell Medical College, New York; ²IBMC, Porto (Presented By: Pedro Ramos)

Hereditary hemochromatosis type-I (HH) is a disease associated mainly with the C282Y-HFE mutation and characterized by iron overload. Some studies have reported altered erythropoietic values in HH patients. Our goal was to study erythropoiesis in the Hfe-KO mice, discriminating between the contribution of iron overload and an intrinsic role of Hfe. Hfe-KO and wt mice were compared. Complete blood counts, flow cytometry profiles and organ iron contents were determined. Lentiviral vectors were used to overexpress Hfe in the bone marrow (BM) of Hfe-KO animals. Compared to wt animals, Hfe-KO mice had increased hemoglobins, MCHs, MCVs and higher proportions of immature erythroid cells in the BM and spleen (p≤0.05). They also recovered faster from anemia induced by either phlebotomy or phenylhydrazine. To minimize the contribution of iron overload to these alterations, we transplanted Hfe-KO or wt hematopoietic stem cells (HSCs) into wt recipients, these mice being designated Hfe→wt and wt→wt, respectively. At steady state we observed that Hfe→wt animals had decreased RBC counts and slightly increased MCHs compared to wt→wt mice. Recovery from induced anemia was faster in Hfe→wt than wt→wt. To extend our analysis to a naturally occurring anemia, Hfe-KO mice were crossed with animals affected by thalassemia intermedia (th3/+). HSCs from the th3/+-Hfe-KO mice were transplanted into wt or Hfe-KO recipients. Th3/+ HSCs were utilized as controls. Our preliminary data indicates that lack of Hfe expression in either the liver or the BM prevented worsening of anemia in th3/+ mice, unlike the situation in wt mice that received th3/+ HSCs. To compare the maturation of erythroid cells while minimizing differences in microenvironment, Hfe-KO and wt animals were phlebotomized and immature erythroid cells isolated and cultured in vitro for 48 hours. First, we detected low expression of Hfe in wt progenitor cells. Second, the proliferation of Hfe-KO cells was 25% greater than that of wt cells (p≤0.01). This result was confirmed by mixing the same number of cultured cells from the two genotypes, after labeling them with different dyes. We observed that the percentage of Hfe-KO cells was consistently higher than that of wt cells (figure). Finally we generated lentiviral vectors to express Hfe in the BM. High levels of Hfe expression led to anemia. Low/normal expression led to reversion of steady state erythropoietic alterations compared to control animals, without affecting tissue iron distribution/content. From our results we can conclude that iron overload contributes to the increased erythropoiesis in Hfe-KO mice. Additionally, absence of Hfe has a beneficial role in erythroid cells under conditions of stress erythropoiesis. Expression of Hfe may control iron uptake in erythroid progenitors so as to avoid excessive iron intake and the toxicity associated with it. However, in conditions of acute anemia, perhaps induced by infection, lack of Hfe might be protective, leading to faster recovery. This may provide and alternative explanation for the high frequency of the C282Y mutation.

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Poster# 3 A CLOSER LOOK AT CELLULAR IRON METABOLISM IN IRP2 DEFICIENT ERYTHROBLASTS Matthias Schranzhofer, PhD¹, Manfred Schifrer, MSc², Bruno Galy, PhD³, Matthias Hentze, MD³, Muellner Ernst, PhD² and Prem Ponka, MD, PhD4 ¹McGill University; ²Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Austria; ³European Molecular Biology Laboratories, Heidelberg, Germany; 4Lady Davis Institute for Medical Research, McGill University, Montreal, Canada (Presented By: Matthias Schranzhofer, PhD)

Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post- transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia. For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice. This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.

Poster# 4 DOES HEME OXYGENASE 1 PLAY A ROLE IN ERYTHROID DIFFERENTIATION? Daniel Garcia dos Santos, Msc¹,³, Matthias Schranzhofer, Msc, Phd¹,4, Jesse Eisenberg¹, Jose Artur Bogo Chies, Msc, PhD² and Prem Ponka, MDd, PhD¹,5 ¹Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada; ²Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pos Graduacao em Genetica e Biologia Molecular (PPGBM), Departamento de Genetica, Porto Alegre, RS, Brazil; ³Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pos Graduacao em Genetica e Biologia Molecular (PPGBM), Departamento de Genetica, Brazil; 4Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pos Graduacao em Genetica e Biologia Molecular, Departamento de Genetica, Porto Alegre, RS, Brazil; 5Department of Physiology, McGgill University, MontrealQC, Canada (Presented By: Daniel Garcia dos Santos, Msc)

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Poster# 5 LOW HEPCIDIN AND ELEVATED GDF15 LEVELS IN PATIENTS WITH CONGENITAL DYSERYTHROPOIETIC ANEMIA (CDA) Guillem Casanovas¹, Judit Kiss², Sandro Altamura², Hermann Heimpel³ and Martina U. Muckenthaler² ¹European Molecular Biology Laboratory; ²Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg, Germany; ³Department for Internal Medicine III (Hematology/Oncology), University Hospital of Ulm, Germany (Presented By: Guillem Casanovas)

The congenital dyserythropoietic anemias (CDAs) encompass a group of rare hereditary disorders of erythropoiesis that is hallmarked by ineffective erythropoiesis and increased iron absorption. Previous studies suggest that growth factor 15 (GDF15), a member of the transforming growth factor-ß superfamily of cytokines is highly over expressed in thalassemia patients and contributes to the iron overload by inhibiting hepcidin expression. In this study we analysed the serum of 2 patients diagnosed with CDA I and 12 patients with CDA II for GDF-15, transferrin, ferritin, haemoglobin, reticulocytes and bilirubin levels. We show that GDF-15 levels are significantly increased in the serum of CDA patients (with the highest values detected in CDA I patients), in addition to elevated ferritin, reticulocyte and bilirubin and diminished transferrin and haemoglobin values. GDF-15 levels significantly correlate with reticulocyte numbers and bilirubin levels. Additionally, we measured urinary hepcidin expression by a SELDI based assay in 3 CDA I and 4 CDA II patients compared to 24 healthy controls. We show that hepcidin levels are significantly lower in the CDA patients suggesting that high serum GDF15 levels may contribute to inappropriately low hepcidin expression and iron overload observed in CDA patients.

Poster# 6 – WITHDRAWN TRANSFERRIN RECEPTOR ACTIVATION BY POLYMERIC IGA1 MODULATES CYTOKINE THRESHOLD OF EARLY ERYTHROBLASTS Séverine Coulon¹, Céline Callens¹, Pamella Huey Mei Wang², Julie Vandekerckhove¹, Damien Grapton², Houda Tamouza², Yael Zermati¹, Jean-Antoine Ribeil¹, Bertrand Arnulf³, Marie-Alexandra Alyanakian4, Marc Benhamou², Renato C. Monteiro², Olivier Hermine¹ and Ivan Cruz Moura² ¹CNRS UMR 8147; ²INSERM U699; ³EA3963; 4INSERM U580

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Poster# 7 STUDYING IRON METABOLISM IN ERYTHROID CELLS UNDER DIFFERENT OXYGEN CONDITONS Manfred Schifrer, MSc¹, Matthias Schranzhofer, PhD² and Ernst Muellner, PhD¹ ¹Medical University of Vienna/MFPL; ²McGill University/Lady Davis Institute (Presented By: Manfred Schifrer, MSc)

Adult erythropoiesis takes place in the bone marrow, where the local oxygen concentration corresponds to the maturity of hematopoietic cells: less oxygen coincides with immature cells and higher oxygen concentration with more differentiated cells. Moreover, oxygen also influences the regulation of cellular iron metabolism by affecting the iron regulatory system, and thus influences hemoglobinization, which is the major task of differentiating erythroid cells. In the work shown here we evaluated the influence of oxygen concentration on both, the regulation of iron metabolism as well as the terminal differentiation of erythroblasts. We cultivated primary erythroid progenitors as well as immortalized erythroblasts at different oxygen concentrations ranging from 3 to 20%. After expanding the erythroblasts under self-renewal conditions, synchronous terminal differentiation including enucleation and hemoglobin synthesis was induced by administration of high erythropoietin and insulin. The progression of differentiation was analyzed by monitoring parameters like heme content and size decrease. The effect of different oxygen concentrations on the regulation of iron metabolism was analyzed by determining the binding activity of iron regulatory proteins as well as the expression of the transferrin- receptor. Expansion of erythroid progenitors from fetal livers was significantly modulated by oxygen concentration. Reduced oxygen resulted in lower proliferation rates and less spontaneous differentiation compared to cells that were cultivated at 20% oxygen. A similar reduction in the proliferation rate under low oxygen levels was observed when we shifted erythroblasts to differentiating conditions. Furthermore, differentiation parameters like cell size decrease and hemoglobinization were reduced under hypoxic conditions. At the level of cellular iron metabolism iron regulatory protein binding affinities and transferrin-receptor expression not only changed with different oxygen concentration, but in addition depended on the differentiation status. Oxygen influences mechanisms like the binding activity of IRPs as well as the maturation of red blood cells. The standard oxygen concentration in tissue culture incubators is often not suitable for the analysis of these mechanisms. Our results show that a simple adjustment of the oxygen concentration to values resembling those in corresponding tissues is not sufficient to establish in vivo like conditions. Based on our experiments we conclude that the high metabolic activity of differentiating erythroblasts reduces the oxygen concentration in the direct vicinity of the cells resulting in reduced proliferation and perturbed terminal differentiation, which in turn interfere with the analysis of direct effects of oxygen on the iron regulatory system. Therefore, oxygen levels have to be individually adjusted to the specific needs of different cell types and culture conditions in order to prevent secondary effects on the iron regulatory system. Supported by the Austrian Science Funds (FWF) and the Herzfelder Family Foundation (to EWM).

Poster# 8 FRATAXIN EXPRESSION LEVELS DURING INCREASED HEME SYNTHESIS IN K562 CELLS Laura Neumann, Hannes Steinkellner, MScsc, Barbara Scheiber-Mojdehkar, PhD, Hans Goldenberg, PhD and Brigitte Sturm, PhD Medical University of Vienna (Presented By: Laura Neumann)

Introduction: Friedreich’s Ataxia (FRDA) is a neurodegenerative hereditary disease with an incidence of 1:50.000 people. The disease manifests in individuals carrying a GAA repeat expansion on frataxin’s first intron which results in reduced protein expression levels down to 5-30% of normal. The exact function of frataxin is still unclear, but it may be involved in mitochondrial iron homeostasis, assembly of iron-sulfur (Fe/S) proteins and heme synthesis. [1] In this work I focus on heme synthesis in regard to the expression of frataxin during differentiation of K562 erythroleukaemia cells. Materials and Methods: K562 cells were differentiated into erythrocyte – like cells using 25µM hemin. Frataxin protein and mRNA levels as well as haemoglobin concentrations were measured over a time period of 4 days. Frataxin expression was measured by electrochemiluminescent immunoassay, mRNA levels by PCR and haemoglobin concentration by a photometric assay using Drabkins Reagent. Results: Differentiation of K562 cells into erythrocyte-like cells with 25µM hemin increases haemoglobin levels. The measurement of frataxin mRNA levels and the frataxin protein concentration showed no changes during the 4 day incubation. Discussion / Conclusion: We found no changes in frataxin expression during heme synthesis in differentiated K562 cells. Future studies using erythroid progenitor cells as a more appropriate cell line will possibly give a better opportunity to approach this open question. [1] NAPIER J., PONKA P., RICHARDSON DR., Iron trafficking in the mitochondrion: novel pathways revealed by disease. Blood. 2005 Mar 1;105 (5):1867-74

Poster# 9 CHARACTERIZATION OF IRON METABOLISM AND ANEMIA IN A MOUSE MODEL OF CONGENITAL ERYTHROPOIETIC PORPHYRIA Said Lyoumi, PhD¹, Constance Delaby, PhD², Sarah Millot, MD¹, Zoubida Karim, PhD¹, David Haile, PhD³, Hubert de Verneuil, MD, PhD4, Herve Puy, MD, PhD5 and Carole Beaumont¹ ¹INSERM U773; ²University Paris Diderot; ³University of Texas; 4University of Bordeaux; 5Versailles-Saint Quentin University (Presented By: Carole Beaumont)

Introduction: Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder due to a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. This deficiency leads to excessive synthesis and accumulation of type I isomers of porphyrins in the bone marrow, followed by their massive excretion. CEP is characterized by severe cutaneous photosensitivity with mutilating involvement and chronic hemolysis Methods: In a mouse model of CEP (Urosmut248), we studied tissue iron, expression of proteins of iron metabolism (by Western blot, immunohistochemistry and RT- qPCR) and bone marrow and spleen erythropoiesis by FACS. Renal injury was examined by studying electrolyte homeostasis of mice separately placed in metabolic cages for 24h. Results: Hematological data confirmed the presence of regenerative anemia with reduced number of RBCs (6 x 106/µL vs 10 x 106/µL) and important reticulocytosis (20% vs 3%). Accumulation of Uro I in RBCs leads to increased osmotic fragility, severe hemolysis as shown by 1) increased of plasma LDH (2500 UI/L vs 500 UI/L) 2) the presence of large amount of iron in urines (220 µmol/L vs 9 µmol/L) with iron overload of kidney tubular cells (650 µg/g-tissue vs 100 µg/g-tissue) 3) increased expression of heme oxygenase 1 (HO-1) in liver and kidney and reduced expression of CD163 (Hbhaptoglobin receptor). In the liver, there was an increase in iron content (1600 µg/g-tissue vs 200 µg/g-tissue) but Perls staining showed increased iron deposits in macrophages but not in hepatocytes, despite reduced liver hepcidin mRNA expression. Spleen iron was only moderately induced (1700 µg/g-tissue vs 1200 µg/gtissue), with no activation of HO-1, suggesting that erythrophagocytosis is not increased. Ferroportin was strongly increased in liver and kidney but not in spleen. Stress erythropoiesis was observed in the spleen in response to activation of BMP4 expression, with an increase in immature erythroblasts, reduced apoptosis and accelerated maturation. GD15 mRNAs were also increased. Metabolic cages studies and blood sampling analysis showed relevant renal phosphate retention with hyperphosphatemia. Perspectives: In this CEP model, intravascular hemolysis induces a strong erythropoietic response in the spleen, characteristics of « stress » erythropoiesis. Hepcidin repression probably facilitates intestinal absorption but the rapid maturation of erythroblasts and reticulocytosis prevent iron overload, at odds with what is usually observed in dyserythropoietic disorders. Iron excretion in urine is probably responsible for kidney iron deposits and alteration of renal functions.

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Poster# 10 REGULATION OF IRON HOMEOSTASIS IN ANEMIA OF CHRONIC DISEASES AND IRON DEFICIEANCY ANEMIA: DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS Igor Theurl², Elmar Aigner³, Milan Theurl4, Manfred Nairz², Markus Seifert², Andrea Schroll², Thomas Sonnweber², Lukas Eberwein², Derrick R. Witcher5, Anthony T. Murphy5, Victor J. Wroblewski5, Eva Wurz², Christian Datz³ and Günter Weiss¹ ¹Internal Medicine I, Clinical Immunolgy and Infectious Diseases; ²Department of Internal Medicine I, Clinical Immunology and Infectious Diseases, Medical University of Innsbruck, Austria; ³General Hospital Oberndorf, Department of Internal Medicine, Oberndorf, Austria; 4Departments of Ophthalmology and Internal Medicine I, Clinical Immunology and Infectious Diseases, Medical University of Innsbruck, Austria; 5Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA (Presented By: Günter Weiss)

The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and the master regulator hepcidin. Hepcidin controls cellular iron efflux upon binding to the iron export protein ferroportin. Many patients, however, present with both, ACD and iron deficiency anemia (ACD/IDA), the later due to chronic blood loss. We used a rat model of ACD due to chronic arthritis and mimicked ACD/IDA by additional phlebotomy in order to define differing iron regulatory pathways. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In rats and humans suffering from ACD, serum hepcidin concentrations are elevated, which is paralleled by reduced duodenal and macrophage expression of ferroportin. Individuals suffering from ACD/IDA have significantly lower hepcidin levels than ACD subjects, and ACD/IDA individuals, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Circulating hepcidin levels affect iron traffic in ACD and ACD/ IDA and are more responsive to the erythropoietic demands for iron than to inflammation. Hepcidin determination may aid to differentiate between ACD and ACD/IDA and in selecting appropriate therapy for these patients.

Poster# 11 COPPER-DEFICIENT ANEMIC RATS, BUT NOT MICE, UP-REGULATE FERROPORTIN EXPRESSION AND DOWN-REGULATE HEPCIDIN Supak Jenkitkasemwong¹, Joseph Prohaska, PhD² and Mitchell Knutson¹ ¹University of Florida; ²University of Minnesota (Presented By: Mitchell Knutson)

Ferroportin is a transmembrane iron-export protein that plays an important role in dietary iron absorption and iron recycling from senescent red blood cells. Ferroportin expression increases with tissue iron loading but decreases in response to hepcidin, the circulating iron-regulatory hormone that binds to ferroportin, causing its internalization and degradation. Hepcidin expression increases with iron loading but decreases during anemia. A previous study found that ferroportin levels did not change in copper-deficient (CuD) mice, despite hepatic iron loading and anemia (Chung et al. J Nutr 2004;134:517-21). More recent studies report that CuD rats, but not mice, have decreased plasma iron, thus highlighting species-specific differences in iron/copper metabolism (Pyatskowit and Prohaska, Comp Biochem Physiol C Toxicol Pharmacol. 2008 Apr;147(3):316-23). We therefore examined ferroportin expression in rats, comparing CuD and copper-adequate (CuA) animals (n=4/group) after one month of treatment postweaning. Hemoglobin levels were lower in CuD than in CuA rats (101 vs. 159 g/L). Western blot analysis revealed that ferroportin levels increased in CuD rats relative to CuA rats by 4- and 1.6-fold in liver and spleen, respectively (P<0.02). Increased ferroportin expression in CuD rat tissues was associated with increased ferroportin transcript abundance, as measured by qRT-PCR. Hepatic hepcidin mRNA levels were dramatically lower in CuD than CuA rats despite a doubling of liver iron. Analyses of copper-deficient mouse liver indicated no change in ferroportin expression, similar to the previous studies in mice. Furthermore, we found no change in hepatic hepcidin mRNA levels in copper-deficient mice, despite their anemia. We conclude that tissue ferroportin levels increase in anemic CuD rats, but not mice, because only rats display low serum iron concentrations and diminished hepcidin expression. These data suggest that serum iron, rather than anemia per se, is the signal for hepcidin during copper deficiency.

Poster# 12 HIGH PHOSPHATE CONCENTRATIONS DISRUPT IRON LOADING INTO FERRITIN FORMING IRON (III)-PHOSPHATE COLLOID PARTICLES: CORRELATIONS TO NON-TRANSFERRIIN BOUND IRON IN CHRONIC KIDNEY DISEASE Richard Watt, PhD, Robert Hilton, BS, Nathan Andros, BSc and Zachary Kenealey, Undergraduate Brigham Young University (Presented By: Richard Watt, PhD)

Introduction: Chronic kidney disease (CKD) shows symptoms of abnormal iron processing. Symptoms include anemia, oxidative damage, elevated serum ferritin, low transferrin saturation levels, and non-transferrin bound iron (NTBI). NTBI is observed even before iron supplementation. Free iron is a catalyst for the formation of reactive oxygen species (ROS) and oxidative damage. Another consequence of kidney failure is the elevation of serum metabolites normally removed by healthy kidneys. Phosphate reaches concentrations more than twice the healthy concentration in the serum of CKD patients. This work examines the hypothesis that elevated phosphate concentrations disrupt iron binding by ferritin. Methods: Iron loading into ferritin was monitored in the presence of physiological serum phosphate concentrations (1 mM) and elevated concentrations of phosphate (2-10 mM) by a spectrophotometric assay and oximetry. Experiments used horse spleen ferritin as a serum ferritin mimic because of the high L to H ferritin subunit ratio (~90% L). Recombinant human H and L ferritin were also used. A method to separate ferritin from the iron phosphate complex was developed. Elemental analysis was performed on ferritin to determine the amount of iron and phosphate loaded into the protein shell. Results: Control reactions without ferritin showed that phosphate stimulated the rate of Fe2+ oxidation. When ferritin and phosphate were present, iron loading appears to occur faster in both spectrophotometric and oximetry assays. Unfortunately these assays are compromised by the formation of a soluble iron(III)-phosphate complex. Elemental analysis shows that the formation of the iron(III)-phosphate complex side reaction competes with ferritin and results in ferritin samples loaded to a much lower extent than expected. In the presence of phosphate, the ferritin mineral core has a much higher iron/phosphate ratio (~ 1:1) than ferritin purified from tissue (~ 10:1). Similar iron loading experiments were performed using homopolymers of H or L ferritin. H ferritin showed significantly better iron loading in the presence of elevated phosphate due to the ferroxidase center that allows ferritin to rapidly bind and oxidize iron. Iron loading into L ferritin, lacking the ferroxidase center, was significantly inhibited by phosphate. Electron microscope EM analysis of the purified iron(III)-phosphate complex shows spherical nanoparticles between 20-200 nm. Discussion / Conclusions: We conclude normal serum phosphate concentrations (1 mM) cause a slight inhibition of iron loading into ferritin. However, phosphate concentrations observed in CKD patients (2.5 mM) inhibited iron loading into ferritin by ~ 50%. Phosphate inhibits iron loading into ferritin by forming a soluble iron(III)- phosphate complex in solution. This complex is very stable and forms a colloidal solution with nanoparticles 20-200 nm in size. We propose that this iron(III)-phosphate complex represents a fraction of the NTBI found in CKD patients and may act as a catalyst for oxidative damage. Experiments with H and L ferritin showed that homopolymers of H ferritin were more efficient at sequestering iron than L ferritin because of the ferroxidase center. This observation provides yet another role for the ferroxidase allowing ferritin to bind and sequester iron in the presence of phosphate.

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Poster# 13 FERRIC PYROPHOSPHATE: BIOCHEMICAL CHARACTERISATION AND METABOLIC FATE IN HEPG2 CELLS Brigitte Sturm, PhD, Nina Ternes, PhD, Hans Goldenberg, PhD and Barbara Scheiber-Mojdehkar, PhD Medical University of Vienna (Presented By: Barbara Scheiber-Mojdehkar, PhD)

Background and Aims: Intestinal iron absorption is insufficient to meet the enhanced iron demand in recombinant human erythropoietin (rHuEPO) treated hemodialysis (HD)-patients. Parenteral iron preparations are widely used to correct iron deficiency in anaemic HD-patients. There is still a search for an optimal iron source. So far soluble iron salts have hitherto been considered too toxic for parenteral administration because free iron catalyzes formation of oxygen free radicals. However it was suggested by Gupta et.al. to use ferric-pyrophosphate as iron source which can be added directly to the dialysate solution for hemodialysis and peritoneal dialysis patients. This approach is currently tested in Phase II clinical studies. In this study we investigated important biochemical properties of ferric pyrophosphate and its metabolic fate in liver cells. Methods: HepG2 cells were used to monitor changes in the total amount of cellular iron by atomic absorption spectroscopy and in the intracellular labile iron pool by using the metal-sensitive fluorescent probe calcein-AM during exposure to ferric pyrophosphate. Binding of iron from ferric pyrophosphate to apotransferrin and the influence of ascorbic acid on binding to transferrin was assayed with fluorescent apotransferrin. In addition cytotoxicity of ferric pyrophosphate was assayed by a neutral red cytotoxicity assay. Generation of redox-active iron during exposure to ferric pyrophosphate was assayed by a dichlorofluorescein assay. Results: Exposure of HepG2 cells to ferric pyrophosphate for 24 hours led to a rapid, but transient increase in the intracellular labile iron pool within two hours which returned to the baseline level after 6 hours. Total cellular iron content of the cells increased with time. After 24 hours the efficiency to increase total intracellular iron was comparable to that obtained with the same concentration of iron sucrose. Ferric pyrophosphate could donate iron to transferring in vitro. Iron transfer to transferrin was higher in buffer than in transferrin depleted serum, indicating that serum components beside transferrin can efficiently chelate iron and reduce therfore iron binding to transferrin. Addition of increasing concentration of ascorbic acid to ferric pyrophosphate reduced the amount of iron which was bound to transferrin, suggesting chelation of iron by ascorbic acid. Exposure of HepG2 cells to ferric pyrophosphate was only cytotoxic in the neutral red assay above concentrations of 150µM of ferric pyrophosphate. Redox-active iron was present when ferric pyrophosphate was diluted in buffer, but disappeared in the presence of transferrin depleted serum (obtained by ultrafiltration with a 50kD MWCO filter device), indicating that serum components form redox-inactive chelates with iron. Conclusion: In this study we show that ferric pyrophosphate can donate iron very efficiently to HepG2 cells. However there was a rapid, but transient increase in the intracellular labile iron pool within the first 2 hours which may generate intracellular oxidative stress. Vitamin C, which is currently tested as an adiuvant therapy in HD- patients reduced transfer of iron from ferric-pyrophosphate to transferrin indicating that ascorbic acid acts as a low molecular weight iron chelator in the plasma. In addition to ascorbic acid other serum components chelate iron and reduce the amount of transferrin bound iron which is critical for iron delivery to the erythron.

Poster# 14 ERYTHROPOIETIC AND IRON FEATURES IN THE DIFFERENT CLINICAL FORMS OF HEREDITARY SPHEROCYTOSIS – A PRELIMINARY STUDY Susana Rocha¹, Petronila Rocha-Pereira, PhD², Fátima Ferreira, MD³, Esmeralda Cleto, MD4, Marika Antunes, MD4, José Barbot, MD5, Alexandre Quintanilha, PhD6, Luís Belo, PhD¹ and Alice Santos-Silva, PhD¹ ¹Faculdade de Farmácia/Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto; ²Centro de Investigação em Ciências da Saúde (CICS), Universidade da Beira Interior/Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto; ³Hospital de S. João, Porto; 4Hospital de Santo António, Centro Hospitalar do Porto; 5Hospital Maria Pia, Centro Hospitalar do Porto; 6Instituto de Ciências Biomédicas Abel Salazar (ICBAS)/Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto (Presented By: Susana Rocha)

Hereditary Spherocytosis (HS) is the most common non-immune hemolytic anemia in individuals of northern European ancestry, ranging from an asymptomatic condition to a severe life-threatening anemia. HS is usually classified as mild, typical or severe according to the severity of the symptoms and analytical presentation. Erythrocyte membrane protein defects underlie HS and are responsible for RBC membrane destabilization that leads to their premature removal in the spleen. Splenectomy, when performed, corrects the anemic state. The aim of our work was to evaluate the erythropoietic and iron features in the different clinical forms of HS and to search for a relationship with HS severity/erythrocyte destruction. We studied 17 splenectomized and 41 unsplenectomized HS patients from the north of Portugal. The latter patients were classified as having mild (n=26), moderate (n=11) and severe (n=4) HS, according to Guidelines for the diagnosis and management of Hereditary Spherocytosis (Bolton-Maggs et al., 2004, Br. J. Haematol.126:455). We performed the basic hematological and iron status studies by standard routine methods, including the reticulocyte count; we also evaluated the levels of erythropoietin (EPO) and soluble transferrin receptor (sTfR) by ELISA; the reticulocyte production index (RPI) and the iron transferrin saturation (TS) were determined. Data was analysed for unsplenectomized HS patients according to HS severity and for splenectomized patients. For correlation analysis patients with moderate and severe HS were grouped together. We found that in splenectomized HS patients, reticulocytes, RPI, EPO and sTfR values were statistically and significantly lower than in unsplenectomized patients; the opposite was observed for iron, ferritin and transferirin levels and TS. A significant increase in reticulocyte count, RPI, EPO, sTfR and ferritin, as well as a significant decrease in transferrin, were observed in unsplenectomised HS patients according to clinical severity; iron and TS presented a trend of decreasing values. In mild HS patients, RPI was found to be statistically and positively correlated with reticulocytes, sTfR and ferritin and to be statistically and negatively correlated with transferrin; in patients with more severe forms (moderate and severe HS) these correlations were lower and only a statistically and positively correlation between RPI and reticulocytes was found. Our results show an inadequate erythropoietic response in the more severe cases of HS, given that, the erythropoietic stimuli (shown by EPO and indirectly by sTfR, iron, ferritin and transferrin levels) was not able to trigger an adequate erythropoietic response as shown by the reticulocyte number and RPI, as occured in mild HS patients. We wonder if this insufficient response could be due to the organism’s restricted capacity in producing more RBCs. In conclusion, HS seems to be related to an increase in erythropoietic stimuli, according to the severity of the disease; however, the response to that stimulus appears to be impaired in the more severe cases of HS. Acknowledgements: This study was supported by a PhD grant (SFRH/BD/22442/2005) attributed to S. Rocha by FCT and FSE.

Poster# 15 PATHOGENESIS OF ANEMIA IN PREGNANCY AND ANEMIA OF INFLAMMATION ARE SIMILAR Valeriy Demikhov, MD, ELena Morshchakova, MD and Anatole Pavlov, MD Federal Research Center for Pediatric Hematology (Presented By: Valeriy Demikhov, MD)

Obviously iron deficiency is one of the main causes of an anemia in pregnancy. But we suppose that anemia in pregnancy pathogenesis is more complex, than ineffective erythropoiesis, caused by iron or folate deficiency alone. To show an anemia in pregnancy has a more complex pathogenesis we investigated the adequacy of the erythropoietin (EPO) production for the degree of anemia in pregnant women. A total 172 anemic pregnant women were tested. Control group consisted of 19 non-pregnant women with iron-deficiency anemia (IDA). We determined Hb concentration, reticulocyte count, erythrocyte indexes, serum iron, total iron binding capacity and serum ferritin concentrations. EPO values were measured immunoenzymometrically by using ELISA-EPO (IBL, Germany) kits. To evaluate serum levels of some inflammatory cytokines in anemic pregnants we determined serum levels of IL-6, IL-8, INF-γ in 60 pregnant women and in 13 healthy non-pregnant women immunoenzymometrically by using commercial ELISA kits. All anemic women were divided into 4 groups on the basis of key laboratory tests: 1 - IDA, n=55; 2 - IDA + infection, n=17; anemia of chronic diseases (ACD), n=13 and anemia with normal iron status (ACD-like), n = 87. Mean EPO level was similar only in pregnant women with IDA as compared with control group. All other groups showed significantly lower EPO levels, than non-pregnant women with IDA (20.1±2.8 U/l, 27.2±4.7 and 17.3±1.7 versus 58.7±9.1 U/l in 99

IDA + infections, ACD, ACD-like and control groups, respectively). As compared with anemic controls, the mean O/E (log EPO) ratio was significantly lower in all groups of anemic pregnant women. The significant elevated serum levels of IL-8 and INF-γ observed at all pregnant women groups versus healthy non-pregnant women. Serum INF-γ concentration in IDA pregnant women was significant higher than in anemic pregnants with normal iron status: 240.2±80.37 ng/L and 84.2±30.59 ng/L respectively (p<0.05). Serum levels of IL-8 in IDA pregnants and in anemic pregnants with normal iron status were higher, than in pregnant females with normal Hb levels and in healthy non-pregnant women: 85.7±43.18 ng/L, 81.2±34.59 ng/L and 27.4±5.99 ng/L, 10.2±0.98 ng/L respectively. Inadequately low production of EPO for the degree of the anemia is important mechanism in pathogenesis of anemia in pregnancy. Blunted erythropoiesis in anemia during pregnancy is obviously related to increased production of inflammatory cytokines. We suppose some causes of proinflammatory cytokines increased production in anemic pregnant women. First, infections during pregnancy may lead to elevated serum levels of inflammatory cytokines. Second, it is known that reduced O2 stimulates placental production of inflammatory cytokines. Third, there is increased serum level of estrogens during pregnancy. It is well known that estrogens probably increasing production of INF-γ inhibit the erythropoiesis. Earlier we have shown that anemic pregnants females had higher serum levels of INF-γ and estrogens as compared pregnants women with normal Hb levels. The own and other investigators data about high efficacy of a recombinant human EPO therapy of anemic pregnant women are well confirmation of such comprehension of an anemia in pregnancy pathogenesis. Therefore anemia in pregnancy pathogenesis is multifactorial. The anemia has some typical features of anemia of inflammation.

Poster# 16 EFFECTS OF RECOMBINANT HUMAN ERYTHROPOIETIN THERAPY ON MODERATE STAGES OF CARDIO-RENAL ANAEMIA SYNDROME – AN EXPERIMENTAL MODEL Patrícia Garrido, MSci², Flávio Reis, PhD¹, Elísio Costa, MSci³, Belmiro Parada, MD¹, Edite Teixeira de Lemos, PhD¹, Nuno Piloto, Biochem D¹, José Sereno, Biochem D¹, Carlos Alberto Tavares, MD4, Carlos Ferrer Antunes, MD4, Arnaldo Figueiredo, PhD5, Lina Carvalho, PhD6, Petronila Rocha Pereira, PhD7, Luis Belo, PhD³, Alice Santos Silva, PhD³ and Frederico Teixeira, PhD¹ ¹Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University; ²Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University, Portugal; ³Biochemistry Service, Pharmacy Faculty & IBMC, University of Porto; 4Laboratory of Haematology, Coimbra University Hospital; 5Service of Urology and Renal Transplantation, Coimbra University Hospital; 6Institute of Anatomic Pathology, Medicine Faculty, Coimbra University; 7Research Centre for Health Sciences, Beira Interior University, Covilhã (Presented By: Flávio Reis, PhD)

Chronic renal failure (CRF) patients develop anaemia which further promotes cardiovascular insufficiency. This triad of dysfunctions, already known as cardio-renal anaemia syndrome, seems to be favoured by the low production of erythropoietin (EPO) by the kidney. The introduction of recombinant human EPO (rhEPO) therapy has marked a significant advance in the management of anaemia associated with CRF. However, in advanced stages rhEPO therapy becomes progressively ineffective and, despite the increment in doses, treatment usually fails. Since EPO exhibits beneficial effects on non-hematopoietic tissues, we hypothesize if rhEPO therapy given in moderate CRF will have a better performance. The aim of this work was, thus, to access the effects of rhEPO treatment in moderate anaemia/CRF stages, using an animal model. Four groups (n=8 each) of male Wistar rats (~280 g) were evaluated during a follow up period of 12 weeks: control, without drugs and surgery; rhEPO, treated with 50 IU/Kg/week Recormon® (Roche Pharm.); CRF, induced by two-stage partial (3/4) nephrectomy; CRF+rhEPO, treated with rhEPO after the 3rd week of surgery. Blood samples were collected at 0, 3, 9 and 15 weeks and the following profiles were evaluated: renal function, haematology, iron metabolism, blood lipids, oxidative stress and inflammation. Results are means ± sem (ANOVA/Fisher’s PLSD; p<0.05 was considered as significant). The experimental model of CRF showed an increase in creatinine (0.91±0.06 mg/dl; p<0.01) vs control (0.45±0.02), development of moderate, but quickly corrected, anaemia, with iron metabolism markers deregulation (iron: 124.5±11.9 µg/dL; ferrritin: 24.7±5.3 ng/dL, p<0.05; transferrin: 81.6±11.0 ng/dL, p<0.001) vs control (iron: 154.2±26.9 µg/dL; ferrritin: 12.9±3.7 ng/dL; transferrin: 120.0±8.0 ng/dL). CRF rats exhibits hypertension and dyslipidaemia, unchanged serum markers of inflammation, contrasting with the increment of 3-nitrotyrosine (50.5±3.2 nM; p<0.001), a marker of peroxynitrite, suggesting the increment of oxidative stress, vs control (15.7±1.4 nM). rhEPO treatment in the CRF animals (CRF+rhEPO group) has increased haematological values (RBC, Hct and Hb), attenuated dyslipidaemia, as well as corrected oxidative stress (3-NT: 17.1±4.3 nM; p<0.01). Furthermore, iron metabolism markers were unchanged, except serum ferritin, which showed increased values (119.1±3.5 ng/dL; p<0.001). In highly dysfunctional kidneys (2 rats), rhEPO treatment aggravates all the evaluated biochemical profiles, as well as the renal histomorphological modifications (lesions). In conclusion, our study suggests that rhEPO therapy might be more efficient if recommended in moderate CRF stage in order to properly correct not only the underlying anaemia but also the cardio-renal complications.

Poster# 17 ATYPICAL IRON DEFICIENCY ANAEMIA – ASSOCIATION OF TWO NEW MUTATIONS IN FERROPORTIN AND TMPRSS6 GENES Gonçalo Caetano, MD², Luis Relvas, BScsC², Celeste Bento, MsCSc², Maria Pedro Silveira, MD² and Leticia Ribeiro, MD¹ ¹Centro Hospitalar Coimbra; ²Serv. Hematologia, CHC (Presented By: Leticia Ribeiro, MD)

Background: Hereditary iron deficiency is a rare condition, involving some iron metabolism and regulation proteins: Hereditary Atransferrinaemia, Aceruloplasminaemia, DMT1 Disease, Ferroportin Disease, Iron-Refractory Iron Deficiency Anaemia (IRIDA) and many others. IRIDA, an autosomal recessive condition, has been associated with mutations in TMPRSS6 (22q), resulting in inappropriate high Hepcidin synthesis with increased Ferroportin degradation and decreased cellular iron export. IRIDA presents as microcytic hypochromic anaemia, with low transferring saturation, refractory to oral iron and partially responsive to intravenous (iv) iron. Ferroportin disease, caused by mutation in gene SLC40A1, is an autosomal dominant iron overload disease, due to reduced ferroportin iron transport with iron accumulation in reticuloendothelial cells and a heterogenous clinical presentation often associated with mild anaemia, increased ferritin and normal/low transferrin saturation. Aims: Describe two cases of familial iron deficiency anaemia, refractory to iron supplementation. Methods: Hematological data according to Dacie & Lewis Practical Haematology. HBA, HFE, TMPRSS6, DMT1 and SLC40A1 gene studies by standard procedures. Results: We presente a56 years old woman, with a long history of iron deficiency anaemia (Hb 7-8g/dL) and menorrhagia, with poor response to iv iron. In the last few years she had mild anaemia (Hb 11-12g/dL) with no hepatosplenomegaly or other relevant findings. Hb 12,2g/dL, MCV 79,8fL, MCH 26,8g/dL, Retic 1,36%, Leuc 8,52x109/L, Plt 353x109/L. Ferritin 65,1ng/mL, Transferrin saturation 5,6%. GI tract study: no bleeding lesions, no Celiac Disease, Helicobacter pylori was positive but eradication had no influence in haematological parameters. Her 32 years old daughter, presented with asthenia and tiredness, long history of iron deficiency and poor response to multiple oral iron treatments. Physical exam: no hepatosplenomegaly or other relevant findings. Hb 12,1g/dL, MCV 77,5fL, MCH 25,4g/dL, Retic 1,19%, Leuc 11,9x109/L, Plt 546x109/L. Ferritin 31,4ng/mL, Transferrin saturation 5,6%. GI tract study: no Celiac Disease, Helicobacter pylori negative. She was treated with iv iron infusion but maintained the levels of Hb, ferritin and transferrin saturation.There is no consanguinity in the family. Both presented mild liver iron overload (MRI), mother: 80 µmol/g ± 30; daughter: 70 µmol/g ± 30 (N <36 µmol/g).Both had normal Hemoglobin studies (HPLC) and negative screening for -α4.2 and -α3.7 thalassemia deletions. Gene study revealed: Gene HFE: C282Y, H63D, S65C – no mutations in mother, H63D heterozygozity in the daughter; Normal DMT1 gene. Mother and daughter heterozygous SLC40A1 CD232 GCTàGAT (AlaàAsp) and TMPRSS6: IVS 11+4 (aàt). Conclusions: We present 2 cases of hereditary iron deficiency anemia refractory to iv iron, with low transferrin saturation, normal ferritin and mild liver iron overload. Both of them are heterozygous for two mutations not describe before:a splicing mutation IVS 11+4 (aàt) in TMPRSS6 and a missense mutation CD232 GCTàGAT (AlaàAsp) in SLC40A1. The SLC40A1 CD232 GCTàGAT mutation results in an Ferroportin localization defect and a loss of iron export function. The normalization of ferritin levels may reflect the association between the mutations found in the SLC40A1 and TMPRSS6 genes.

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Poster# 18 BIOMINERALIZATION IN MAGNETOTACTIC BACTERIA PROCEEDS WITHOUT MINERAL PRECURSOR AND MAGNETOSOMES MOVE DURING CHAIN ASSEMBLY Berthold F. Matzanke, PhD, DiplChem¹, D. Faivre, PhD², L.H. Boettger, PhD³, N. Menguy, PhD4, G. Ona-Nguema, PhD4, M. Pósfai, PhD5, H.P. Gunnlaugson, Prof, PhD6 and D. Schueler, Prof, PhD7 ¹University of Luebeck, Isotopes Laboratory; ²Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424 Potsdam, Germany; ³Institute of Physics, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; 4Universités Paris 6 and 7 and Institut de Physique du Globe de Paris, 140 rue de Lourmel, 75015 Paris, France; 5Department of Earth and Environmental Sciences, University of Pannonia, POB 158, 8200 Veszprém, Hungary; 6Institute of Physics and Astronomy, Århus University, Ny Munkegade, 8000 Århus C, Denmark; 7Department of Biology, LMU München, Biozentrum, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany (Presented By: Berthold F. Matzanke, PhD, DiplChem)

Magnetotactic bacteria form intracellular magnetic nanoparticles in organelles called magnetosomes that are arranged in one or several chains. One of the most intriguing questions in magnetosome biomineralization is how the synthesis of the nano-sized crystals is controlled, and specifically, whether iron-based precursors of magnetite biomineralization are present. Further aspects that require clarification are the characteristic features of the assembly processes, particularly whether or not magnetite nucleates in adjacent organelles. We performed time-resolved studies of magnetite biomineralization in magnetotactic bacteria after induction of magnetosome formation in iron-starved resting cells under highly controlled conditions. Our analyses of the biogenic mineral were performed by in situ Mössbauer spectroscopy, high-resolution transmission electron microscopy, electron diffraction, and X-ray absorption spectroscopy. Our results are in striking contrast to those recently published by Staniland et al. (Proc Natl Acad Sci USA (2007), 104, 19524- 19528), who reported a 3 - 4 nm surface layer of hematite in ~ 20 nm magnetosome crystallites and a very rapid chain formation. Here, we show that no mineral precursor of magnetite was present in the magnetosomes, hematite never occurred intracellularily, and chain formation was a slow and directed process.

Poster# 19 IRON-SULFUR PROTEINS AND THE NITRIC OXIDE CYTOTOXICITY IN BACTERIA Huangen Ding, PhD, Binbin Ren, BSc¹, Xuewu Duan, PhD, Aaron Landry, BSc and Juanjuan Yang, MSc Louisiana State University (Presented By: Huangen Ding, PhD)

The nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions, and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (IlvD), an iron-sulfur enzyme essential for the branched-chain amino acids biosynthesis, is completely inactivated in E. coli cells by NO with the concomitant formation of the IlvD-bound dinitrosyl iron complex. Combining a sensitive NO electrode with electron paramagnetic resonance (EPR) spectroscopy and an enzyme activity assay, we further demonstrate that NO can rapidly react with the IlvD [4Fe-4S] cluster and inactivate the enzyme activity under both aerobic and anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0±2.0) x106 M-2 x s-1 at 25 oC, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. When E. coli cells containing recombinant IlvD are exposed to NO using the Silastic tubing NO delivery system under both aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD are efficiently modified forming the IlvD-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD than with O2 in vivo. Fractionation of the cell extracts prepared from the NO-exposed E. coli cells reveals that a large number of protein-bound DNICs are formed by NO. While the IlvD-bound DNIC and other protein-bound DNICs are stable in E. coli cells under anaerobic growth conditions, they are efficiently repaired under aerobic growth conditions even without new protein synthesis. Additional studies indicate that L-cysteine and oxygen have important roles in repairing the NO-modified iron-sulfur proteins in E. coli cells. These results suggest that iron-sulfur clusters in cells are the primary targets of the NO cytotoxicity and that cellular deficiency to repair the NO-modified iron-sulfur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions.

Poster# 20 SERCA PUMP EXPRESSION AND IRON UPTAKE IN A DROSOPHILA CELL LINE Wenlin Wu, PhD and Maria Lind Karlberg Comparative Physiology, Uppsala University, Sweden (Presented By: Maria Lind Karlberg)

Background: Sarco/endoplasmic reticulum (SER) Ca2+ ATPases (SERCA) represent a highly conserved family of Ca2+ pumps that transport Ca2+ from the cytosol into SER. All animals have multiple isoforms, which are expressed in a developmental and tissue specific manner. We have investigated the iron uptake in the Drosophila melanogaster cell line I(2)mbn in response to the activity and expression level of the SERCA. Methods: SERCA overexpressing (stable transfected) and knockdown cells (RNAi) were produced and compared with control cells. The cells were incubated in normal medium or medium containing ferric ammonium citrate (FAC) or FeCl2. Furthermore, cells were also treated by SERCA pump inhibitor, thapsigarin, and by Ni2+ to investigate the effect on iron uptake. Cellular iron concentration was quantified by an improved ferrizine based assay. The expression of the ferritin heavy chain homologue, Fer1HCH and SERCA was measured by real time RT-PCR or by Western blot analysis. Results: To determine if SERCA effected the iron uptake, we investigated cellular iron concentration in SERCA overexpressing, knockdown and control cells after incubation in iron-rich medium for 24 hrs. Compared with cells cultured in normal medium, the intracellular iron concentration was highly increased in response to the iron concentration in the cell medium: 15-fold for normal cells, over 20-fold for SERCA-overexpressing cells and only 10-fold in SERCA knockdown cells. Independently of the treatment, cells overexpressing SERCA always showed highest intracellular iron concentration. The mRNA abundance of the Fer1HCH was in agreement with these results showing that ferritin is more common in SERCA-overexpressing cells. Furthermore, the expression of SERCA was not significant changed in response to cellular iron level. Treatment of cells with the irreversible inhibitor of the SERCA pump showed the same results as the knockdown cells. Addition of Ni2+ to cell medium abolished the increased iron uptake in SERCA overexpressin cells. Conclusion: These data show that there is a correlation between the amount of the SERCA and intracellular iron level suggest that Ca2+ pumps serve as unspecific iron transporters in Drosophila melanogaster.

Poster# 21 THE IMPACT OF DIETARY COPPER AND ZINC ON DROSOPHILA FERRITIN DISTRIBUTION AND IRON METABOLISM Fanis Missirlis, PhD Queen Mary University of London (Presented By: Fanis Missirlis, PhD)

Drosophila is an emerging model organism for the investigation of metal metabolism. The insect intestine contains cells that are responsive to dietary iron levels (ferritin regulation is typical), “copper cells” that sequester excess copper to metallothionein and a specialized group of “iron cells” that function as body iron stores. Ferritin regulation in the iron cells is atypical, as iron deficiency does not lead to ferritin degradation (despite iron mobilization from ferritin). A recent observation (Yepiskoposyan et al, 2006) that Drosophila Metal Transcription Factor-1 (MTF-1) induces ferritin RNA transcripts in response to dietary zinc, and possibly copper, levels has prompted a closer investigation into the crosstalk between heavy metal metabolisms in the fly. I will present a cell-specific, zinc and MTF-1 dependent induction of ferritin in a fourth group of intestinal cells that are usually devoid of detectable iron. Curiously, despite ferritin induction, cellular iron content remains below levels of histochemical detection suggesting that induced ferritin is low in iron content and therefore its precise 101 function remains unknown. Zinc feeding did not alter total iron or copper levels, but resulted in a strong depletion of manganese, by an unknown mechanism. We have also studied the unique fly homolog of the human disease gene pantothenate kinase (PANK) because in human patients iron depositions occur in the globus pallidus and substantia nigra regions of their brains. We discovered that fly loss-of-function mutants for PANK have a three-fold induction of their total zinc content. A metabolomics analysis is underway in our efforts to discover a possible link between coenzyme A biosynthesis and metal metabolism. We are also testing if the above mentioned ferritin induction via MTF-1 plays a role in the mutant flies. Copper feeding, on the other hand, did not result in any induction of ferritin protein. Therefore, post-transcriptional control mechanisms (possibly mediated by the iron regulatory protein) may compensate for ferritin mRNA upregulation reported under these conditions (Yepiskoposyan et al, 2006). Intriguingly, copper feeding leads to very substantial iron mobilization from ferritin in the “iron cells”. I am currently testing whether iron mobilization from stores is mediated by multicopper ferroxidase activity (ceruloplasmin-like) in the fly hemolymph. The results suggest that copper dependence for iron mobilization from body iron stores, which has also been documented in mice (Nose et al, 2006), is evolutionarily conserved. References: Nose Y, Kim BE and Thiele DJ (2006). Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function. Cell Metabolism 4, 235-244 Yepiskoposyan H, Egli D, Fergestad T, Selvaraj A, Treiber C, Multhaup G, Georgiev O and Schaffner W (2006). Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc. Nucleic Acids Research 34, 4866-4877

Poster# 22 OVEREXPRESSION OF THE YEAST FRATAXIN HOMOLOG (YFH1): CONTRASTING EFFECTS ON IRON-SULFUR CLUSTER ASSEMBLY, HEME SYNTHESIS AND RESISTANCE TO OXIDATIVE STRESS Alexandra Seguin, Aurélien Bayot, Andrew Dancis, Adelina Rogowska-Wrzesinska, Françoise Auchère, Jean-Michel Camadro, Anne-Laure Bulteau and Emmanuel Lesuisse Institut Jacques Monod, Université Paris Diderot and UPMC (Presented By: Alexandra Seguin)

Friedreich’s ataxia is generally associated with defects in [Fe–S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model Friedreich’s ataxia to study the physiological consequences of modulating the expression of the frataxin gene (YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2µYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe–S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2µYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2µYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2µYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe–S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different.

Poster# 23 KLAFT, THE KLUYVEROMYCES LACTIS ORTHOLOG OF AFT1 AND AFT2, MEDIATES IRON-RESPONSIVE GENE EXPRESSION THROUGH AN AFT-TYPE ELEMENT Natalia Conde e Silva, PhD, Isabelle R. Gonçalves, PhD, Marc Lemaire, PhD, Emmanuel Lesuisse, PhD, Jean Michel Camadro, PhD and Pierre Louis Blaiseau, PhD Institut Jacques Monod (Presented By: Natalia Conde e Silva, PhD)

Iron is an essential nutrient and different mechanisms of iron homeostasis regulation have been described in fungi. This study explores iron regulation of iron homeostasis in the respiratory yeast Kluyveromyces lactis that diverged from Saccharomyces cerevisiae before the whole genome duplication. The K. lactis genome contains an ortholog of the S. cerevisiae iron-responsive transcription activators genes AFT1/AFT2, designated KlAFT. Deletion of KlAFT led to the inability to grow under iron limitation. Quantitative real-time PCR analyses demonstrated that KlAft activates the transcription of most homologs of the Aft1 regulated genes but not the homologs of genes specifically regulated by Aft2. Computer analysis identified an Aft-type element PuCACCC in the promoters of most KlAft regulated genes. We used gel shift experiments to demonstrate that KlAft mediates iron-responsive regulation through its direct binding to this element. K. lactis is the first yeast to be shown to exhibit a positive iron- transcriptional control mediated by one sole copy of the Aft-type regulator. This suggests that this iron-regulatory function was acquired before the whole genome duplication.

Poster# 24 HAPTOGLOBIN AND HAPTOGLOBIN-RELATED PROTEIN ARE HEMOGLOBIN-BINDING PROTEINS WITH DIVERSE FUNCTIONS Marianne Jensby Nielsen, PhD, Benoit Vanhollebeke, Christian Jacobsen, Etienne Pays and Søren Kragh Moestrup University of Aarhus (Presented By: Marianne Jensby Nielsen, PhD)

Haptoglobin (Hp) and haptoglobin-related protein (Hpr) are plasma proteins sharing 91% sequence identity. Despite this high degree of identity, the proteins perform completely different biological functions. Hp primarily functions as a high-affinity hemoglobin-binding protein capturing ‘free’ hemoglobin released into the circulation during intravascular hemolysis. The interaction of Hp with the liberated hemoglobin protects tissues from hemecatalyzed oxidative stress and directs hemoglobin to the monocyte/macrophage scavenger receptor CD163 which specifically recognizes the Hphemoglobin complex and removes it from the circulation by receptor-mediated endocytosis. The hpr gene arose by a triplication of the hp gene in early primate evolution. The Hpr protein is associated with a minor subfraction of high density lipoproteins termed Trypanosome Lytic Factor (TLF) with an important role in the primate-specific innate immune system against the African parasite Trypanosoma brucei brucei (T. b. brucei). The latter is transmitted to the bloodstream of mammals through the bite of infected tsetse flies and is the causative agent of the lethal disease sleeping sickness in cattle and other animals. Humans and a number of other primates are, however, resistant to this particular trypanosome subspecies because their Hprcontaining TLF particles have the ability to induce lysis of T. b. brucei. Despite earlier assumptions, that Hpr is inactive in terms of binding hemoglobin, we have demonstrated, by means of surface plasmon resonance (SPR) studies, that Hpr in fact binds hemoglobin with an affinity similar to that of Hp. This finding paved the way for the discovery of a novel trypanosomal receptor for Hp/Hpr-hemoglobin complexes using Hp-hemoglobin affinity chromatography (Vanhollebeke et al., Science 2008 320:677-81). Apparently, this parasite receptor has evolved to take up Hp- hemoglobin from the host bloodstream as a means to supply heme to the parasite which itself is unable to synthesize the heme necessary for incorporation into hemo proteins. Due to the high degree of resemblance between Hp and Hpr, the toxic hemoglobin-associated Hpr-containing TLF particles are, however, also taken up, inadvertently, by this receptor, thus leading to subsequent lysis of the parasite. In contrast, SPR studies demonstrate that the mammalian Hp-hemoglobin receptor, CD163, has the ability to effectively discriminate between Hp-hemoglobin and Hpr-hemoglobin complexes and therefore does not mediate uptake of the toxic TLF particles. In conclusion, like Hp, Hpr is a hemoglobin-binding protein and thus complies with the designation ‘haptoglobin’(Greek: haptein, to bind + (hemo)globin). Upon hemoglobin- and receptor interaction these two homologous proteins contribute to distinct biological events.

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Poster# 25 IRON AND VIRUSES – INTERACTIONS OF HIV-1 WITH CELLULAR IRON METABOLISM Lucy Eddowes, Andrew Armitage, Alain Townsend and Hal Drakesmith University of Oxford (Presented By: Lucy Eddowes)

There is clinical evidence that elevated iron status correlates with a poor prognosis in HIV-1 infection. In vitro the HIV protein Nef downregulates the haemochromatosis protein HFE, and work with iron chelators has also demonstrated an iron dependency for HIV replication, but these studies are few. Four steps of the HIV-1 life cycle are thought to be iron dependent: reverse transcription, transcription, nuclear export of mRNA and virion assembly. Given the clinical correlation between prognosis and iron and the in vitro data we investigated the dependency of HIV-1 replication on iron in monocytederived macrophages (MDMs) and monocytic cell lines. MDMs from both HFE wild type and C282Y-haemochromatosis patients were infected with R5 HIV-1 strains. U937 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) then infected with VSV-G pseudotyped HIV-1. Cultures were supplemented with iron salts or iron chelators. Infections were monitored by p24 ELISA and, where GFP- linked HIV-1 was used, by flow cytometry. Our data from multiple patients suggests that the extent of HIV-1 replication is independent of HFE genotype in MDMs. Infecting differentiated U937 cells gave inconsistent results when different viral strains and experimental conditions were used. It may therefore not be a good model for investigating the iron dependency of HIV-1 replication. As well as investigating whether iron has a direct effect on HIV-1 replication we have also studied whether the virus causes or effects changes in iron handling. Pathogen- associated molecular patterns (PAMPs) are recognised by a family of proteins including the Toll-like receptors (TLRs). These interactions stimulate an antigen-nonspecific response that includes the production of proinflammatory cytokines within hours of recognising the pathogen. The master regulator of iron, hepcidin, is produced as part of the innate immune response to bacteria and bacterial products and is structurally related to beta-defensins, some of which inhibit HIV-1 infection. HIV ssRNA has been found to stimulate the production of proinflammatory cytokines such as TNF-alpha and IL-6 through TLR7/8. We therefore investigated whether expression of hepcidin mRNA is induced by the presence of whole or inactivated HIV virions and whether hepcidin can act similarly to defensins and directly inhibit HIV-1 infection. Hepcidin mRNA expression was measured by quantitative real time PCR (qRT-PCR) after incubating fresh PBMCs with whole or inactivated viruses. HIV-1 infection inhibition was measured by incubating hepcidin with HIV-1 and infecting the TZM-bl reporter cell line. The data shows that hepcidin mRNA expression is unchanged by the presence of whole or inactivated HIV-1 virions after 3 hours in bulk PBMCs, although virus-associated TLR agonists do induce hepcidin. Our work also shows that hepcidin does not directly inhibit HIV-1 infection of reporter cell lines. Further work will address whether changes in hepcidin mRNA expression are observed at later time points and whether a response is limited to a particular PBMC subset.

Poster# 26 MILD INCREASES IN SERUM HEPCIDIN AND INTERLEUKIN-6 CONCENTRATIONS IMPAIR IRON INCORPORATION IN HEMOGLOBIN DURING EXPERIMENTAL HUMAN MALARIA INFECTION Quirijn de Mast, MD, Edmee Dongen-van Lases, PhD, Dorine Swinkels, PhD, An-Emmie Nieman, MD, Meta Roestenberg, MD, Pierre Druilhe, PhD, Theo Arens, MSc, Adrian Luty, PhD, Cornelis Hermsen, PhD, Robert Sauerwein, PhD and Andre van der Ven Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands (Presented By: Andre van der Ven)

Background and Objective: Iron deficiency is common in children in malaria-endemic regions and associated with significant adverse effects on child health. Iron supplementation to iron-replete children may however increase malaria-associated morbidity and mortality. Proper selection of those children who may benefit from iron supplementation requires improved insight in the effects of malaria on host iron homeostasis and new and reliable biomarkers. Recently, we found strongly elevated concentrations of urine hepcidin and serum interleukin (IL)-6 concentrations in anemic children with high-density Plasmodium falciparum malaria infections in Tanzania. To study whether disturbances of iron homeostasis also occur in low-density P. falciparum malaria infections, we assessed changes in serum hepcidin and interleukin (IL)-6 concentrations in volunteers during an experimental human P. falciparum malaria infection; a prospective malaria infection model that offers unique possibilities to study these processes in detail. We also related changes in hepcidin and IL-6 concentrations to changes in iron parameters and the incorporation of iron in hemoglobin. Methods: Serial concentrations of serum hepcidin, traditional biochemical iron status indicators and IL-6 were determined in healthy volunteers, who were experimentally infected with P. falciparum. Samples were taken at various time points, i.e. before, during and after the malaria blood infection. The hemoglobin content of reticulocytes (Ret-He) and mature red blood cells (RBC-He) and the Delta He (Ret-He minus RBC-He) were determined as measures for iron incorporation in hemoglobin. Results: Serum hepcidin and IL-6 concentrations increased only mildly during low-density parasitemia (maximum concentrations 12.7 nmol/L and 70 pg/mL, respectively). However, despite these only minimal changes, a marked hypoferremia with a strong increase in serum ferritin concentrations developed and this was associated with a sharp fall in Ret-He and Delta-He, while RBC-He remained unchanged. The ratio of soluble transferrin receptor to the log ferritin concentrations (sTfR/log ferritin index) decreased to an average nadir of 63% of the baseline value. Discussion and Conclusions: The mild increases in serum hepcidin and IL-6 concentrations, as are seen in low-density P. falciparum malaria infections, may already cause a marked disturbance in host iron homeostasis, including impaired iron incorporation in hemoglobin. Iron supplements should therefore be withheld from patients with malaria until after antimalarial treatment has been completed. Serum hepcidin, Ret-He and Delta- He are promising biomarkers to select those individuals who will benefit from iron supplements in malaria endemic regions, while the sTfR/log ferritin ratio should be used with caution to assess iron status during malaria.

Poster# 27 INTRACELLULAR PARASITE LEISHMANIA DONOVANI MANIPULATES IRON HOMEOSTASIS OF HOST MACROPHAGE BY DEPLETING INTRACELLULAR LABILE IRON POOL Chinmay Mukhopadhyay, PhD and Nupurkanti Das, MBBSc Jawaharlal Nehru University (Presented By: Chinmay Mukhopadhyay, PhD)

Leishmania donovani (LD) is a digenetic trypanosomatid parasite causes fatal visceral leishmaniasis in mammalian hosts. After successful entry into macrophages, the promastigote form of parasite proliferates within the mature phagolysosome compartment as amastigote, multiplies within and finally bursts the host to infect neighboring macrophages. Like other intracellular pathogens one of the major challenges leishmania faces is to acquire iron from the harsh environment of host macrophages for its intracellular survival and growth. While, intracellular pathogens employ several strategies for iron acquisition from host, macrophage resists infection by actively sequestering iron. Thus, strategy of iron acquisition from host macrophage is very important for leishmania, which has not been explored much except the recent identification of ZIP family iron transporter LIT1 in leishmania amazonensis expressing only in its amastigote form. However, the source(s) of available iron as well as effect of leishmania infection on iron homeostasis of host macrophage remained largely unexplored. In this study, we demonstrate that instead of allowing macrophages to sequester iron, virulent LD directly scavenges iron from labile iron pool (LIP) of macrophages. As a result of LIP depletion iron sensors iron regulatory proteins IRP1 and IRP2 are activated in the host. IRPs then bind to iron responsive elements (IREs) present in the 3’ UTR of iron uptake gene transferrin receptor 1 (TfR1) and modulate its expression by a post-transcriptional mRNA stability mechanism. Increased IRE-IRP interaction and TfR1 expressions in spleen derived macrophages from LD infected mice confirm LD employs similar mechanism to acquire iron during infection into mammalian hosts. To our knowledge this is the first demonstration of increased IREIRP interaction for any infection in vivo. Increased intracellular LD growth by holo-transferrin supplementation and inhibited growth by iron chelator treatment confirm the significance of this modulated iron uptake pathway of host in favour of the parasite. In summary, we detected an active mechanism of intracellular LD by which it directly scavenges iron from LIP to activate iron-sensing proteins to influence TfR1 mediated host iron uptake so that LD could use the iron for its intracellular growth. This work is supported by the Wellcome Trust grant to CKM.

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Poster# 28 IDENTIFICATION OF A NEW HEXADENTATE IRON CHELATOR CAPABLE OF RESTRICTING THE INTRAMACROPHAGIC GROWTH OF MYCOBACTERIUM AVIUM Sofia Sousa Fernandes, PhD², Ana Nunes, PhD³, Ana Rita Gomes, BSsc4, Baltazar de Castro, PhD³, Robert C. Hider, PhD5, Maria Rangel, PhD6, Rui Appelberg, PhD, MD7 and Maria Salomé Gomes, PhD¹ ¹IBMC and ICBAS- Universidade do Porto; ²IBMC-Universidade do Porto; ³REQUIMTE-Faculdade de Ciências, U.P.; 4IBMC-U.P.; 5Division of Pharmaceutical Sciences, King’s College London; 6REQUIMTE-Faculdade de Ciências and ICBAS-U.P.; 7IBMC and ICBAS-Universidade do Porto (Presented By: Maria Salomé Gomes, PhD)

Iron accumulation has been suggested to contribute to an increase of the susceptibility to mycobacterial infections. In this study we tested the effect of an array of iron chelating ligands of the 3-hydroxy-4-pyridinone family, in the intramacrophagic growth of M. avium. We found that bidentate chelators, namely N-alkyl-3-hydroxy-4- pyridinones and N-aryl-3-hydroxy-4-pyridinones, did not affect the growth of M. avium inside mouse macrophages. In the case of the hexadentate chelators, those synthesized using an alkylamine (HCP25) or a benzene ring (CP252) to link the three bidentate units, did not have an inhibitory effect on intramacrophagic growth of M. avium while those synthesized from a tripodal structure to anchor the bidentate units were capable of inhibiting the intramacrophagic growth of M. avium. The molecule we designated CP777 had the strongest inhibitory activity. The growthreducing activity of CP777 was abrogated when this molecule was saturated with iron. This result confirms that iron deprivation, by the use of iron chelating compounds, restricts M. avium growth and that new iron chelators offer an approach to controlling mycobacterial infections.

Poster# 29 ZIP TRANSPORTERS IN LEISHMANIA INFANTUM Sandra Carvalho, Tânia Cruz, Licenciatura, Rosa Silva, Licenciatura, Vítor Costa, PhD and Ana M. Tomás, PhD Institute for Molecular and Cell Biology, IBMC, Porto (Presented By: Sandra Carvalho)

Leishmania are the causative agents of the neglected disease leishmaniasis. These protozoan parasites are transmitted to mammals during the blood meal of the female sand- fly. Once in the vertebrate host, parasites are phagocytosed by macrophages and undergo a set of changes that lead to the mammalian stage, the amastigote. In order to survive and replicate in the phagolysosome harsh environment, the amastigote must acquire iron. We have shown that Leishmania infantum axenic amastigotes are able to use heme- and non-heme-bound iron to replicate (Carvalho et al, 2009). The identification and characterization of the proteins involved in iron and heme internalization in the mammalian stage of L. infantum is important as its inhibition, by chemotherapeutic or immunological approaches, could help in the control of leishmaniasis. Mining the L. infantum genome for iron transporters, three candidates were selected. These genes have features of the ZIP (ZRT/IRT-like Protein) family, whose members are divalent metal transporters. The characterization of the encoded proteins involves several aspects: localization in the parasite, function and essentiality for infection. By immunofluorescence, LiIZT1 (Iron/Zinc Transporter #1 of L. infantum) was observed near the mitochondrial DNA, probably in the flagellar pocket, in both stages of the parasite. In the case of the vector stage, the promastigote, LiIZT1 was also localized in the cellular membrane. Data collected from western blots, shows that this protein is mainly expressed in the early stages of promastigote growth and seems to be processed at a post-translational level. Confirmation will be achieved by mass spectrometry. LiIZT2 is expressed both in amastigotes and promastigotes but no localization could be determined. In what concerns to LiIZT3, neither localization nor expression was observed yet. However, this was the only protein able to rescue yeast deficient in the high- and low-affinity systems for the transport of zinc, suggesting that LiIZT3 is a zinc transporter. Radioactive metal (55Fe and 65Zn) uptake assays will be performed using transgenic parasites (LiIZT-knockout and LiIZT-overexpressing) to determine the exact function of the proteins. The essentiality of LiIZT to infection will be evaluated in animal models. Project funded by POCTI/ESP/41982/2001 from Fundação para a Ciência e Tecnologia. SC is recipient of doctoral fellowship SFRH/BD/28712/2006.

Poster# 30 EVALUATION OF IRON STATUS IN PATIENTS WITH GASTRITIS AND HELICOBACTER PYLORI INFECTION Helena Grotto, MD, PhD and Eliana Alvarenga, postgraduate student State University of Campinas, Brasil (Presented By: Helena Grotto, MD, PhD)

Background: The role of H.pylori (HP) infection as a possible cause of iron deficiency anemia (IDA) is not completely established. Some mechanisms are invoked to explain the relationship between HP gastritis and IDA: occult gastrointestinal bleeding, competition for dietary iron by the bacteria, and an abnormality of the gastric juice composition with consequent impaired iron absorption. Moreover, HP causes continuous gastric inflammation which may contribute to anemia. Objective: to evaluate the possible association between HP infection and anemia in a group of Brazilian adult patients with gastritis. Patients: 126 patients submitted to endoscopy and gastric biopsy requested for gastritis investigation. Methods: CBC including reticulocyte hemoglobin content (Ret-He) ( Sysmex XE 2100, Sysmex, Japan), serum iron and total iron binding capacity (Roche/ Hitachi Modular Analyzer), serum ferritin (Roche/ Elecsys 2010). C-reactive protein (CRP) was measured as an indicator of inflammatory activity (Roche/ Hitachi Modular Analyzer). Serum gastrin-chemiluminescent test (gastrin IMMULITE/IMMULITE 2000, Siemens). Gastritis was diagnosed according to histologic findings (Sidney classification) and HP presence was detected by H-E staining. Detection of IgG antibodies to HP: chemiluminescent assay kit (IMMULITE 1000, Siemens). Results: Endoscopic investigation -110/126 patients presented gastritis, classified as: mild (n= 64), moderate (n=33), nonspecific (n=9) and intense chronic gastritis (n=4). HP detection by histological examination was positive in 93 patients (84.55%). Anemia investigation: 14/110 (12.7%) patients with gastritis presented anemia, 12 were HP+ and 2 HP-. Six patients showed alterations in iron parameters compatible with iron deficiency (3 with IDA and 3 with reduced storage but without microcytosis) and all of them were HP+; 2 patients had hemoglobinopathies and in 6 patients was not possible to define anemia diagnosis using the present protocol. Ret-He values were reduced (< 24.0 pg) only in those 3 patients with IDA. There were no significant differences between groups HP+ (n= 93) and HP- (n= 17) concerning hematologic and iron status parameters, CRP and serum gastrin levels. Serum IgG titles were higher in HP + group when compared with HP - (p= 0.0026). The same results were observed when the patients were divided according to gastritis intensity. In order to evaluate the possible contribution of gastritis inflammatory activity on erythropoiesis, CRP and gastrin values were correlated with hematologic parameters. In the total group there was a a weak but significant correlation between CRP and Ret-He (r= - 0.20125, p= 0.0348), gastrin X Hb (r= -0.28644, p= 0.0024), gastrin x Ret-He (r= - 0.25240, p= 0.0078) and gastrin x ferritin (r= - 0.24320, p= 0.0105). When the group with anemia (n= 14) was evaluate separately the correlation between gastrin and Ret-He was stronger (r= -0.5473, p= 0.0428). Conclusions: IDA incidence was not higher in patients with gastritis or HP+ than in normal population, differently from epidemiological studies conducted over diverse geographic areas, but especially in children and using HP serology result as indicator of infection. Our data in a group of Brazilian patients are in agreement with other results who found that the HP infection was not associated with low hemoglobin level or iron deficiency. The inverse correlation between serum gastrin levels and Ret-He values reinforces the possibility of hypo or achlorhydria influence on iron absorption and, consequently in reticulocyte hemoglobin synthesis. Financial Support: FAEPEX/UNICAMP nº 230/07.

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Poster# 31 IRON-HEME METABOLISM IN LEISHMANIA INFANTUM INTRACELLULAR STAGE Tânia Cruz, Sandra Carvalho and Ana Tomás Instituto de Biologia Molecular e Celular, IBMC, Universidade do Porto (Presented By: Tânia Cruz)

Leishmania, a protozoan parasite transmitted to mammals by Phlebotomine sand flies, must obtain iron in order to survive. Being responsible for visceral leishmaniasis, Leishmania infantum shows a tropism for macrophages of both the spleen and the liver. These phagocytes, which are involved in the catabolism of senescent erythrocytes, are likely to have high amounts of iron and/or heme. In order to survive inside the host cell, L. infantum must have evolved tightly regulated processes to acquire iron/heme. We have previously shown that, indeed, axenic amastigotes, the intracellular stage of L. infantum, can use both heme and non-heme bound iron for nutritional purposes [1]. Furthermore, the growth of amastigotes in DFO-treated medium supplemented with Fe-NTA, indicates that these cells, as in the vector stage, use a ferric reductase for iron internalization. Here, we present data that elucidates about the localization and regulation of this protein (Li Fred) in both stages of the parasite. Residing inside the parasitophorous vacuole of spleen and liver macrophages, L. infantum amastigotes may be influenced by the host cell iron/heme levels. To understand if the infection is modulated by the presence of different iron/heme concentrations, we are studying the impact of erythrophagocytosis on L. infantum survival and replication in vitro. More recently, we have also started to investigate the effect of the parasite on the iron metabolism of the host. To address this issue we made use of RT-qPCR and started by measuring HAMP gene expression levels in L. infantum infected macrophages. So far, we have indication that HAMP is upregulated early in the infection and decreases to basal levels in later stages. This work was financed by FCT (project POCTI/ESP/41982/2001 and doctoral fellowships SFRH/BD/23352/2005 and SFRH/BD/28712/2006). [1] Carvalho S., Cruz T., Santarém T., Castro H., Costa V., Tomás AM. (2009) Heme as a source of iron to Leishmania infantum amastigotes. Acta Tropica, 109, (2):131-5.

Poster# 32 SYNTHESIS AND CHARACTERIZATION OF A NOVEL TRIPODAL CATECHOLATE HEXADENTATE CHELATOR Carla Queiroz, MScsC¹, Ana M.G. Silva, PhD², Mariana Andrade, BScsC², Paula Gameiro, PhD², Baltazar Castro, Agregação² and Maria Rangel, PhD³ ¹REQUIMTE, ICBAS, Universidade do Porto; ²REQUIMTE, Departamento de Quìmica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal; ³REQUIMTE, Instituto de Ciências Biomédicas de Abel Salazar, 4099-003 Porto, Portugal (Presented By: Carla Queiroz, MScsC)

A systematic investigation of the potential anti-microbial activity of a set of iron chelators revealed that a novel tripodal hydroxypyridinone hexadentate ligand is effective in inhibiting the growth of Mycobacterium avium in mouse bone marrow derived macrophages.1 Taking into account the chemical structure of natural siderophores, we are currently exploring the potential anti-microbial activity of other tripodal hexadentate molecules which are prepared with chelating units of the catechol and hydroxamate type. In the present work we report the synthesis and physico-chemical properties of a novel tripodal catecholate hexadentate chelator (L1, Figure 1).

Figure 1 The chelator synthesis is based on a tetrahedral structure to which three bidentate catecholate units are linked via amide bonds.2 The synthetic approach developed to synthesize L1 involves the preparation of 2,3-dibenzyloxy-1-aminomethylbenzene, which is the key compound for the amide coupling reaction with a triacid anchor. Different coupling reagents were employed in order to achieve the best reaction conditions, including DCCI/ HOBT, DCCI/ N-hydroxysuccinimide, EDAC/ N- hydroxysuccinimide and pentafluorophenyl trifluoroacetate/ Py. Subsequent removal of the protecting benzyl groups was carried out using BCl3 in CH2Cl2. All compounds synthesised were fully characterized by elemental analysis, mass spectrometry and NMR spectroscopy (1D and 2D techniques including 1H, 13C, COSY, HSQC and HMBC). The dissociation constants of the ligand and the affinity constant towards iron(III) were determined using spectrophotometric methods. The values obtained allowed the plot of the corresponding distribution diagrams as a function of pH. The synthetic route for the preparation and futher functionalization of novel tripodal catecholates has been established. The synthesis and physicochemical characterization of the first compound prepared is presented in this work. References: 1. "Identification of a new hexadentate iron chelator capable of restricting the intramacrophagic growth of Mycobacterium avium" Sofia Sousa Fernandes, Ana Nunes, Ana Rita Gomes, Baltazar de Castro, Robert C. Hider, Maria Rangel, Rui Appelberg, Maria Salomé Gomes, manuscript in preparation. 2. "Iron Binding Dendrimers: A Novel Approach for the Treatment of Haemochromatosis", Tao Zhou, Hendrik Neubert, Ding Yong Liu, Zu Dong Liu, Yong Min Ma, Xiao Le Kong, Wei Luo, Sykes Mark, and Robert C. Hider, J. Med. Chem. 2006, 49, 4171-4182.

Poster# 33 DIAGNOSTIC ACCURACY OF SERUM HEPCIDIN FOR IRON DEFICIENCY IN CRITICALLY ILL PATIENTS WITH ANEMIA Sigismond Lasocki, Isabelle Boutron, PhD, Fathi Driss, MD, PhD, Mark Westerman, PhD, Herve Puy, MD, PhD, Sarah Millot, MD, Philippe Montravers, MD, PhD and Carole Beaumont, PhD INSERM U773 (Presented By: Sigismond Lasocki)

Context: Anemia is frequent in critically ill patients and is due to inflammation and blood losses. The latter induces iron deficiency (ID), which is difficult to diagnose using usual biological parameters. Serum hepcidin may be a good clinical indicator of ID. We designed a prospective observational study to determine the diagnostic accuracy of hepcidin for detecting ID in critically ill patients with anemia. Patients and Methods: Between June 2006 and February 2007, 51 patients suffering from anemia (hemoglobin<100g/L) and expected to stay for more than 7 days in our surgical intensive care unit were enrolled. Weekly determinations of hematological and iron parameters were performed (iron, transferrin saturation, ferritin, soluble transferrin receptor, zinc protoporphyrin, and C-reactive protein). The status (normal, inflammatory, true ID, functional ID (ie ID despite inflammation) or unknown) was defined for each set of measures by the consensus of three experts (reference standard), blinded to hepcidin levels determined using a competitive ELISA test. For the analysis, true and functional ID were pooled together and compared to no ID (normal or inflammatory status), while unknown status were not included.

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Results: At study inclusion, of 51 patients (36 male/15 female), 43 had inflammation (86%), 2 had normal iron status (4%), 2 had true ID (4%), and 3 had functional ID (6%). One patient had an “unknown” profile. The iron profile did not change during the ICU stay in 39 patients (32 inflammation, 2 true ID, 2 functional ID, 2 normal, and one unknown). For the remaining 12 patients having an inflammatory profile at first assessment, 8 developed functional ID while in the ICU, iron profiles normalized in 3, and one patient with functional ID developed an inflammatory profile. Altogether, 25 % of screenings were classified as ID. A total of 128 iron profiles from 47 patients was available for analysis of hepcidin. Median hepcidin levels were 80.5 [0.05-548.3] and 526.6[246.7-891.4] µg/L for ID and non-ID profiles, respectively. The optimal threshold for serum hepcidin for diagnosis of ID was assessed by building 100 ROC curves using a bootstrap method and found at 129.5 µg/L (95%CI=[115.5-143.4]). Development of an iron deficiency profile during the ICU stay was accompanied by a decrease in hepcidin levels, whereas patients with a persistent inflammatory profile maintained high plasma hepcidin concentrations. Overall, there was a good negative correlation between serum hepcidin levels and soluble transferrin receptor or zinc protoporphyrin, and a positive correlation between hepcidin and CRP. Conclusions: In these critically ill patients, ID was present in 10 % of them at study inclusion and evolved to 25 % during ICU stay. Serum hepcidin level of 129.5 µg/L was the most accurate threshold for ID diagnosis in critically ill patients with anemia. Furthermore, undetectable hepcidin levels are observed in some of these patients despite inflammation, suggesting that this assay might be useful to identify patients that could benefit from iron treatment.

Poster# 34 MHC CLASS I ANTIGEN PRESENTATION: EFFECT OF HFE, A PROTEIN INVOLVED IN IRON METABOLISM Alexandre Reuben, Manuela M. Santos, PhD and Réjean Lapointe, PhD University of Montreal/CRCHUM/Montreal Cancer Institute (ICM) (Presented By: Alexandre Reuben)

HFE is a β2-microglobulin (β2m)-dependent protein involved in the regulation of iron absorption in the intestine. Mutations in this gene which are highly prevalent in the population of European descent may lead to the development of hereditary hemochromatosis (HH), a disease which causes increased iron absorption in the intestine and its accumulation in several organs. HFE is a structural homolog of major histocompatibility complex class I (MHC I) molecules that are involved in presentation of cellular antigens to the immune system, therefore contributing to the elimination of infected or tumor cells. Several lines of evidence suggest that the cellular immune system may contribute to HH. For example, immunological defects have been observed in patients with HH and Hfe-deficient mice lacking lymphocytes develop a more severe form of iron overloading. Furthermore, it has been shown that HFE can be recognized by T lymphocytes and that this interaction influences the development of the T-cell repertoire. We therefore hypothesized that HFE could play a role in cellular immunity by impacting on MHC I antigen presentation. In turn, this influence could contribute to the survival and persistence of cancer cells due to reduced immune recognition. We used a system which has been well established in our laboratory. We first co-transfected HEK-293 or MelS melanoma cell lines with HFE and plasmids encoding a model melanoma tumor antigen (TA). We then co-cultured transfected cells with CD8+ T lymphocytes which were generated in our laboratory to specifically recognize processed TA originating from the various transfected melanoma tumor plasmids. To assess the magnitude of T-cell recognition of processed melanoma TA, we quantified interferon-γ (IFN-γ) secretion by CD8+ T lymphocytes in the supernatant of co-cultures by ELISA. We found that the presence of HFE in HEK-293 and MelS cells leads to decreased IFN-γ secretion by melanoma-TA-specific CD8+ T lymphocytes. This could be explained in part by a decrease in expression levels of MHC I molecules at the cell surface, which would result in decreased recognition. However, the partial decrease in MHC I surface expression in the presence of HFE did not completely account for the difference in T-cell recognition. In fact, although MHC I expression decreased to similar levels in cells transfected with melanoma tumor antigens, HFE-transfected cells which were pulsed with tumor-specific peptides showed no differences in recognition levels when co-cultured with specific CD8+ T lymphocytes . Further experiments with co-transfections of β2m-encoding plasmids showed that competition for β2m, which is shared between HFE and the MHC I molecules involved in the presentation of the selected melanoma TA, also could not entirely explain this diminished immune recognition of HFE-expressing tumor cells by CD8+ T lymphocytes. Finally, experiments with MelS cells permitted us to discriminate between an effect of HFE on newly synthesized or recycled peptide- MHC I complexes and confirmed that HFE targets newly synthesized complexes. Taken together, our results suggest that HFE may interfere with antigen processing in the endoplasmic reticulum. HFE interference with antigen processing and recognition may lead to decreased efficiency in eliminating infected and malignant cells. Further experiments are underway to establish the mechanism by which HFE interferes with antigen processing.

Poster# 35 HEPATIC MCP-1 GENE EXPRESSION IS SUPPRESSED IN IRON-LOADED MALE C282Y HOMOZYGOTES AND CORRELATES WITH HEPCIDIN EXPRESSION John D. Ryan, MBBCh¹, Eleanor Ryan, PhD², Matthew Lawless, PhD², Jens Stolte³, Martina U. Muckenthaler, PhD³ and John Crowe, PhD² ¹Centre for Liver Disease; ²Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin, Ireland; ³Department of Pediatric Haematology, Oncology and Immunology, University of Heidelberg, Germany (Presented By: John D. Ryan, MBBCh)

Hereditary Haemochromatosis (HH) is an autosomal recessive disorder characterised by defective iron metabolism. The C282Y mutation in the HFE gene underlies the majority of cases. Systemic iron overload results from deficient hepcidin production, and excessive iron absorption and release from duodenal enterocytes and macrophages. Reduced intra-macrophagic iron levels may result in impaired cytokine secretion and altered immune response in HH. Hepcidin expression is stimulated by inflammation through cytokines, especially IL-6. MCP-1 (Monocyte chemoattractant protein-1) is a prolific inflammatory chemokine, recruiting monocytes and T lymphocytes to areas of tissue injury and inflammation. MCP-1 mRNA expression is responsive to pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF-α), and increased serum levels are found in alcoholic hepatitis and non-alcoholic steatohepatitis (NASH). MCP-1 has been implicated in hepatic fibrogenesis and steatosis, as well as various inflammatory conditions including atherosclerosis, and malignancy. A previous study found significantly reduced serum MCP-1 levels in C282Y homozygotes, compared with compound C282Y/H63D heterozygotes, H63D homozygotes and normal controls. Objective: The aim of this study was to examine the hepatic expression of MCP-1 and hepcidin mRNA in untreated C282Y HH patients, and to correlate expression with clinical disease. Methods: Gene microarray (using the human Ironchip®) performed on liver tissue from 10 C282Y HH patients revealed reduced hepatic MCP-1 and hepcidin expression when compared to controls. This was validated by RT-PCR on 25 untreated male patients homozygous for the C282Y mutation with evidence of iron overload; mean age 48yrs, ferritin 1569ug/l, and median transferrin saturation 89%. Mean grade of hepatocellular iron staining was 3+ (out of 4). Control liver tissue was obtained from male donor transplant livers (n=4) negative for HFE gene mutations. Results: Both MCP-1 and hepcidin mRNA transcription were significantly lower in the HH cohort when compared to controls (p<0.002 and p<0.015 respectively). Furthermore, MCP-1 expression correlated significantly with that of hepcidin (p<0.004). Expression of these genes was not found to significantly reflect iron status, or degree of inflammation or fibrosis at histology. Discussion / Conclusion: This study demonstrates suppression of hepatic MCP-1 transcription occurs in iron-loaded patients with C282Y HH and complements a previous report of reduced serum MCP-1 levels in a similar HH cohort. Taken together, these data suggest an inherent reduction of MCP-1 expression in C282Y HH. This suppression correlates significantly with diminished hepcidin expression and may reflect the defective immune response described in these patients. While reduced recruitment of mononuclear cells may convey protective effects through attenuated hepatic inflammation and fibrogenesis, this was not evident clinically. Further studies are warranted to elucidate this interesting finding.

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Poster# 36 INFLAMMATION-INDUCED HEPCIDIN IS ASSOCIATED WITH THE DEVELOPMENT OF ANEMIA IN SEPTIC PATIENTS Lucas van Eijk, MD¹, Joyce Kroot, MSc², Mirjam Tromp, MA, RN³, Peter Pickkers, MD, PhD4 and Dorine Swinkels, MD, PhD² ¹Radboud University Nijmegen Medical Centre; ²Department of Clinical Chemistry, Radboud University Nijmegen Medical Centre; ³Nijmegen Institute for Infection, Inflammation, and Immunity (N4i), Department of Internal Medicine, Radboud University Nijmegen Medical Centre; 4Department of Intensive Care Medicine, Radboud University Nijmegen Medical Centre (Presented By: Lucas van Eijk)

Introduction: Infection and inflammation lead to anemia. During systemic inflammation, cytokines stimulate iron sequestration by inducing hepcidin, leading to the internalization and degradation of ferroportin in intestinal epithelial cells and macrophages1, thereby abrogating the iron supply to the bone marrow, and inducing ‘anemia of inflammation’. The knowledge concerning these pathophysiological mechanisms is mainly based on in vitro and animal experiments. The role of hepcidin in the development of acute anemia of inflammation observed in the first two weeks of human sepsis has not yet been identified. Therefore, we explored the association between hepcidin and sepsis−associated anemia. Methods: 92 consecutive patients were enrolled after presentation on the emergency ward of a university hospital with sepsis, indicated by the presence of >= 2 SIRS criteria, and a proven or suspected infection. Blood was drawn at day 0, 1 and 2 after admission for the measurement of IL−6 and hepcidin−25. Hepcidin−25 was measured by quantitative surface enhanced laser desorption/ionization time−of−flight mass spectrometer (SELDI−TOF MS) as described previously2. IL−6 levels were correlated with hepcidin concentrations. Hemoglobin levels and data of blood transfusions during 14 days after hospital admission were retrieved and decreases in hemoglobin compared to admission levels were correlated to hepcidin levels. Patients who received a blood transfusion were excluded for correlation analysis. Data are expressed as mean±SEM, median[range], or Pearson’s correlation coefficient. Results: 53 men and 39 women with a mean age of 53.3±1.8 were included. Hepcidin levels were highest at admission (19.0[1.2−72.7]nmol/l) and decreased to normal levels in most patients within 3 days (9.6[0.8−40.5]nmol/l). Hepcidin levels were higher in patients with more SIRS criteria (p=0.049). Highest IL−6 levels were measured at admission (288.3±45.0pg/ml) and significantly correlated with hepcidin levels at admission (r=0.38, p=0.007) and on day 1 (r=0.32, p=0.029). Twelve patients received one or more blood transfusions during the first 2 weeks of admission, not related to active bleeding. These patients had borderline significant higher hepcidin level at admission (preceding their blood transfusion) compared to non−transfused patients (32.2[8.9−65.3] vs 22.1[1.2−72.7]nmol/l, p=0.052). IL−6 concentrations did not differ between both groups. Correlation analyses showed significant associations between hepcidin levels on day 1 and 2 and decreases in hemoglobin from day 7−14, with strongest correlation between hepcidin on day 2 and decreases in hemoglobin from day 9−11 (r ranging from −0.70, p=0.001 to −0.86, p<0.001, respectively). Discussion: Up to now, effects of inflammation and hepcidin release on the development of anemia have been obtained in in vitro and animal experiments, while human in vivo data is lacking. Our study demonstrates that increased IL−6 concentrations in septic patients are associated with hepcidin concentrations. Moreover, the increased hepcidin concentrations observed in early sepsis negatively correlated with hemoglobin levels during the hospital stay of these patients. These human in vivo correlations suggest that hepcidin release is an important modulator of anemia in septic patients with systemic inflammation. References: 1. Nemeth E, Tuttle MS, Powelson J et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090−2093. 2. Swinkels DW, Girelli D, Laarakkers C et al. Advances in quantitative hepcidin measurements by time−of−flight mass spectrometry. PLoS.ONE. 2008;3:e2706.

Poster# 37 INFLAMMATION UPREGULATES NON-TRANSFERRIN BOUND IRON UPTAKE BY HEPATOCYTES Debbie Trinder, BSc, PhD, Carly Herbison, BSc (Hons), Roheeth Delima, BSc (Hons), Anita Chua, BSc (Hons) PhD, Ross Graham, BSc (Hons), PhD and John Olynyk, MBBS, MD University of Western Australia (Presented By: Debbie Trinder, BSc, PhD)

Inflammation modifies iron metabolism causing anaemia of chronic disease, a condition in which iron accumulates in the liver and spleen, decreasing blood iron levels and thereby reducing the availability of iron for erythropoiesis. It has been shown that the inflammatory cytokine interleukin-6 (IL-6) upregulates hepcidin, inducing ferroportin degradation and reducing iron recycling which is likely to contribute to the dysregulation of iron homeostasis. In addition, studies have shown that IL-6 increases transferrin-bound iron uptake by hepatocytes, with a concomitant increase in hepatic iron levels but whether IL-6 regulates non-transferrin bound iron (NTBI) uptake as well is not known. Several candidate NTBI transporters have been identified including the zinc transporter Zip-14 and divalent metal transporter 1 (DMT1) which are both regulated by inflammation. The aim of this study was to investigate the effects of IL-6 on NTBI uptake by mouse hepatocytes and the hepatocyte cell line AML-12 and to identify if Zip-14 plays a role in this process. AML-12 cells were treated with 20-100 ng/ml IL-6 for 24 hours. Hepatocytes were isolated from female AKR mice (10 weeks of age) and treated with 20 ng/ml IL-6 for 24 hours. mRNA expression of iron metabolism genes was measured by real-time PCR and protein expression by immunoblot. NTBI uptake was measured by incubating cells with 59Fe-citrate (10 µM) for 60 min at 37oC. Zip14 was over-expressed or knocked down in AML12 cells by transient transfection of pCMV-Sport6 containing mZip14A cDNA or mZip14 SMARTpool siRNA. Cells were analysed for gene expression and NTBI uptake at 48 hours post- transfection. IL-6 increased NTBI uptake by AML-12 cells in a concentration dependent manner with uptake increasing up to 1.9-fold at a concentration of 50 ng/ml IL-6 compared with untreated cells. mRNA expression of Zip14A and Zip14B isoforms and Hamp1 was also increased in a concentration dependent manner with a maximum increase of 1.5, 1.7 and 2.3-fold, respectively, at 50 ng/ml IL-6. DMT1 and ferroportin mRNA expression was not significantly altered by IL-6. Similarly, IL-6 increased NTBI uptake by hepatocytes by 30% while Zip14A, Zip14B and Hamp1 but not DMT1 mRNA expression was upregulated significantly. Transfection of AML-12 cells with a mZip-14 plasmid upregulated Zip14 protein levels markedly and increased NTBI uptake by approximately 75% compared with the control cells. Suppression of endogenous Zip-14 expression in AML-12 cells using Zip14 siRNA inhibited NTBI uptake by 35% while treatment of the Zip-14 over-expressing cells with siRNA downregulated NTBI uptake by 55% suggesting that Zip14 mediates the uptake of NTBI in hepatocytes. Furthermore, IL-6 induced upregulation of NTBI uptake in AML-12 cells was inhibited by Zip14 siRNA to the level observed in the absence of IL-6 treatment. In conclusion these findings suggest that the inflammatory cytokine IL-6 upregulates NTBI transport in hepatocytes, which is likely to be due to the upregulation of Zip-14, and that this process may contribute to hepatic iron overload in anaemia of chronic disease.

Poster# 38 HIGH NUMBERS OF THE MOST MATURE CD8+ EFFECTOR MEMORY T CELLS ARE ASSOCIATED WITH THE NEW GGG HAPLOTYPE, A MARKER OF LESS SEVERE EXPRESSION OF HEREDITARY HEMOCHROMATOSIS M. Fátima Macedo, PhD¹, Monica Costa², Graça Porto, MD, PhD³,4 and Eugénia Cruz, MD, PhD²,4 ¹IBMC; ²IRIS, IBMC, Porto University; ³IRIS, IBMC, ICBAS, Porto University; 4Hematology S. António Hospital, Porto (Presented By: M. Fátima Macedo, PhD)

Hereditary hemochromatosis (HH) is a common genetic disorder characterized by a variable penetrance. Recently we have described two 500 Kilobases (Kb) haplotypes in the 6p21 region predicting CD8+ T lymphocyte numbers and the severity of disease in HH patients. The AAT haplotype was found associated with low CD8+ T lymphocyte numbers and a more severe expression of HH. In contrast, the GGG haplotype was found in a minority of cases and was associated with high CD8+ T lymphocyte numbers and a less severe disease. These data associate this region with the regulation of CD8+ T cell numbers and clinical heterogeneity observed in HH. The CD8+ T cell population is highly heterogeneous but can be grouped into three major subpopulations: naïve (CCR7+ CD45RA+), central memory (CM) (CCR7+ CD45RA-) and effector memory (EM) (CCR7-). The EM subpopulation can be further subdivided into four groups identified by the expression of the costimulatory molecules CD27 and CD28: CD27+CD28+ (DP), CD27-CD28+ (CD28SP), CD27+CD28- (CD27SP) and CD27-CD28- (DN). As each of these groups display an homogeneous expression of effector molecules known to be important for CD8+ T cell function, a distinctive degree of differentiation from the most immature (resembling the naïve phenotype) to the most differentiated (resembling effector cells) can be drawn: DP to CD28SP to CD27SP and finally to the most differentiated DN. Therefore we 107 investigated the CD8+ T cell subpopulations in HH patients in order to explore the type(s) of CD8+ T cell subpopulation(s) that is(are) being hypothetically regulated by the conserved haplotypes AAT and GGG. Forty-six HH patients, all homozygous for the C282Y mutation of the HFE gene, were included in this study. Patients were regularly followed up at the Hemochromatosis Outpatient Clinic of Santo António Hospital, Porto, Portugal. The CD8+ T lymphocytes subpopulations were analyzed by 5-colors flow cytometry. The AAT and GGG haplotypes were identified by genetic analyses as described previously (Cruz et al 2008). As previously described, patients carrying the GGG haplotype (n=7) have higher numbers of CD8+ T cells than patients homozygous for the AAT haplotype (n=39) (0.55±0.21, 0.33±0.18 x106/ml respectively, p=0.004). For CD8+ naïve T cells there is no statistically significant differences between GGG and AAT patients. The difference observed in the number of CD8+ T cells reflects the difference in the memory pool. Both CM and EM CD8+ T cell numbers are increased in GGG versus AAT (CM 0.051±0.025, 0.024±0.015 respectively p=0.0004; EM 0.358±0.195, 0.206±0.119 respectively p=0.008). Considering the CD8+ EM subpopulations (see table) it is clearly observed that the CD8+ EM DN is the population with a higher contribution for the differences observed between GGG and AAT EM numbers (0.246±0.202, 0.066±0.068 respectively p=0.0002).

Poster# 39 INCREASED FREQUENCY OF CD4+CD28+ T-LYMPHOCYTES IN MICE WITH HFE-DEFICIENCY Maja Vujic Spasic, PhD¹, Alexis Perez Gonzalez, PhD², Andy Riddell, PhD², Matthias Hentze, Prof MD² and Martina Muckenthaler, Prof PhD¹ ¹University Hospital of Heidelberg; ²EMBL, Heidelberg, Germany (Presented By: Maja Vujic Spasic, PhD)

Hereditary hemochromatosis (HH) is a common disorder of iron metabolism. It is most frequently caused by mutations in the Hfe gene, a MHC class I like molecule. The clinical picture of this disease is complex and includes abnormalities in T lymphocyte numbers. In particular, low CD8+ T cell counts were observed in HH patients that correlate with the severity of iron overload. Hfe knock-out mice recapitulate many aspects of the HH phenotype, including liver iron accumulation, reduced splenic iron content, increased transferrin saturation and hyperferremia. In this study we have analyzed aged, male Hfe-deficient mice and show that they present severe liver iron overload and reduced duodenal iron content while the spleen iron content was unchanged compared to wild-type mice of the same age. Analysis of the T-lymphocyte cell surface molecules CD3, CD4, CD8 and CD28 revealed no major differences in the relative proportion of CD4+ and CD8+ T cells in the blood, spleen, thymus and lymph nodes between Hfe-deficient and WT mice, contrasting previous data in patients. Interestingly, however, within the CD3+CD4+ T cell population an increased percentage of CD28+ cells was detected in Hfe-deficient mice while the percentage of CD28+ cells in the CD3+CD8+ T cell compartment revealed no differences between control and Hfe-deficient mice. The CD28 co-receptor is proposed to aid antigendependent T-cell activation by promoting T-cell proliferation, cytokine production and cell survival. Further investigations will clarify whether the shift within the CD4+ T-cell population in Hfe-deficient mice is genetically determined or, whether increased systemic iron levels are responsible for this observation.

Poster# 40 ELEVATED ICAM-1 AND SUPPRESSION OF VCAM-1 SERUM PROTEIN IN HEREDITARY HEMOCHROMATOSIS PATIENTS Matthew Lawless, PhD, MSc¹, Suzanne Norris, MD, PhD² and John Crowe, MD, PhD³ ¹Mater Misericordiae Hospital; ²Trinity College Dublin; ³Centre for Liver Disease, Mater Misericordiae Hospital, University Hospital, Dublin (Presented By: Matthew Lawless, PhD, MSc)

Introduction: Hereditary Hemochroamtosis (HH) is a disorder of iron metabolism associated with accumulation of iron in the body. HH is an autosomal recessive disorder resulting from mutations in the HFE (hemochromatosis) gene, with clinical consequences, which can range from cirrhosis of the liver, diabetes, heart failure, arthritis to liver cancer. HFE, a Major Histocompatability Complex class I (MHC class I) gene located on chromosome 6 resembles other MHC I proteins that have a key role in immunology. HFE gene’s potential role on the immune system remains poorly understood. Several abnormalities in the immune status of patients with HH have been reported, suggesting an imbalance in their immune function. Abnormally low proportions of lymphocytes with more severe clinical expression of hemochromatosis have been consistently reported (not correlated to phlebotomy treatment). Intercellular adhesion molecule−1 (ICAM−1) and vascular cell adhesion molecule−1 (VCAM−1) are involved in immune processes and altered levels of these proteins have been found in pathological states including cardiovascular disease, arthritis and liver cancer. We sought to examine weather soluble ICAM−1 and VCAM−1 protein expression is altered in the serum of HFE HH patients. Method: One hundred and thirty−nine serums from subjects diagnosed with HH (C2828Y homozygotes = 86, C282Y/H63D = 26 heterozygotes, H63D homozygotes = 26), 27 healthy control subjects with no HFE mutation (N/N), and 18 control subjects heterozygous for the H63D mutation served as age− and sex matched controls were analysed. Ferritin and transferrin saturation and the presence of HFE mutation status were correlated with ICAM−1 and VCAM−1 levels. Full white blood cell count analysis was also performed. Biochip Array Technology using the adhesion molecules array was used to simultaneously and quantitatively detect soluble ICAM−1 and VCAM−1 in each patient sample. Results Obtained: ICAM−1 levels were significantly elevated in HH patients compared to controls (p < 0.0059). This was associated with individual HFE genotypes (YY vs. NN, p < 0.01) (YD vs. NN, p < 0.02) (DD vs. NN, p < 0.0314) respectively. ICAM−1 protein expression did not correlate with iron indexes. Marked suspension of VCAM−1 protein expression negatively correlated in patients with raised ferritin level over 500mg/L and transferrin saturation > 75% (p < 0.002). VCAM−1 showed no significant association with HFE genetic component. Discussion: Elevated soluble ICAM−1 has been suggested to hinder lymphocyte activation thus exerting direct immune suppressive action. Increased ICAM−1 levels are reported in the pathogeneses of inflammatory liver disease, diabetes and arthritis. VCAM−1 expression exhibits known chemotactic activity for immune cells and has a role in monocyte recruitment. Conclusion: This study demonstrates that immunological differences in ICAM−1 and VCAM−1 expression in HH patients are associated with HFE genotype and iron overload effect respectively. ICAM−1 and VCAM−1 expression are essential for the generation of effective inflammatory responses. This study favours the hypothesis that abnormalities in the immune status of patients with HH can be both intrinsic due to HFE genetic defects and as a consequence of progressive iron overload. Identifying abnormalities in the immune states of HH patients provides a possible insight into the variable clinical spectrum of this multifactorial disorder.

Poster# 41 THE MULTICOPPER OXIDASE CERULOPLASMIN AND THE IRON EXPORTER FERROPORTIN ARE BOTH PRESENT AT THE CELL SURFACE OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES Liliana Marques, Rui Malhó, François Canonne-Hergaux and Luciana Costa Instituto Nacional de Saúde Dr. Ricardo Jorge (Presented By: Liliana Marques)

Iron (Fe) metabolism and its close relationship with immunity, particularly inflammation, are extremely complex and still needs major research. Because Fe is an indispensable but toxic element when present in excess, numerous proteins are involved in the maintenance of Fe homeostasis through highly regulated mechanisms. In particular, ceruloplasmin (Cp), a multicopper ferroxidase, and ferroportin (Fpn), the only Fe exporter identified in mammals, work in concert to drive Fe out of the cells. In addition, Cp is an acute phase protein that presents both anti- and pro-oxidant activities playing a role in both host defense against oxidative stress and in oxidative damage, especially in inflammatory conditions. 108

Our previous mRNA studies suggest that human peripheral blood lymphocytes (PBL) express Fpn and glycosylphosphatidylinositol (GPI) membraneanchored Cp in addition to the secreted Cp isoform. Herein, we investigate the protein expression and the localization of Cp and Fpn in these cells. Human mononuclear cells were isolated from peripheral blood by density gradient centrifugation using Lymphoprep™. PBL were then separated from monocytes by cell culture adherence and analyzed for Cp and Fpn expression by western blot and immunofluorescence. Western blot analysis of PBL subcellular fractions showed that membrane-associated Cp is the main isoform expressed in these cells, likely corresponding to GPI-Cp. Indeed, treatment with PI-PLC, an enzyme that cleaves specifically GPI-anchored proteins, induced a significant decrease in Cp staining at PBL surface confirming this hypothesis. Both immunofluorescence and western blot results also supports Fpn expression in PBL membrane. Importantly, confocal studies suggest that Fpn and GPI-Cp colocalize at the surface of these cells. In conclusion, PBL express Fpn and both Cp isoforms: secreted Cp and GPI-anchored Cp. This latter one seems to be the major Cp isoform in human PBL, being specially enriched at cell surface where it colocalizes with Fpn. Expression of such proteins in PBL supports the postulate that immune cells could be directly involved in the maintenance of Fe homeostasis. Moreover, getting a better insight into Cp and Fpn physiologic role in circulating PBL may also contribute to understand the underlying mechanisms of oxidative protection/damage in inflammation. Particularly, we are now investigating the putative shedding of GPI-Cp under the effect of specific inflammatory stimulus as a rapid mechanism to deliver a protein to a particular site of action. The physiologic relevance of PBL Cp/Fpn interaction in the regulation of Fe metabolism associated with particular pathological conditions is discussed.

Poster# 42 LEUKOCYTE APOPTOSIS AND INFLAMMATION IN IRON-OVERLOADED PATIENTS WITH SICKLE CELL DISEASE OR BETA-THALASSEMIA: A MECHANISM FOR INCREASED STROKE AND DISEASE SEVERITY IN SICKLE CELL DISEASE Patrick Walter, PhD¹, Killilea David, PhD², Fung Ellen, PhD¹, Anne Higa, BScs¹, Jacqueline Madden, BScs, RN¹, John Porter, MD², Pat Evans, PhD², Bruce Ames, PhD¹, Elliott Vichinsky, MD¹ and Paul Harmatz, MD¹ ¹Children’s Hospital & Research Center Oakland; ²University College London (Presented By: Patrick Walter, PhD)

Introduction: Sickle cell disease (SCD) is a hemoglobinopathy characterized by micro-vascular hypoxia-reperfusion, inflammation and leukocytosis. Studies in SCD have shown that leukocytosis is a strong predictor of stroke and disease severity. It is known that leukocytosis and inflammation contribute to increased leukocyte-endothelial adhesion and vasoocclusive events. Leukocytosis or increased leukocyte number is determined by the balance between cell death programs (apoptosis) and proliferation. In this study, we examine markers of apoptosis and proliferation in SCD as compared to thalassemia (a hemoglobinopathy that is vasculitis negative) and a control group. Methods: Markers of leukocyte turnover, inflammation and free iron (NTBI, non-transferrin bound iron) were compared in 11 patients with SCD (7M, 13±4 yrs), 18 with thalassemia (7M, 24±9 yrs) and 10 disease-free controls (5M, 27±12 yrs). All SCD and thalassemia patients were healthy and event free in the previous 4 months. Blood was obtained fasting and prior to RBC transfusion; and plasma, serum and cells were separated by centrifugation. The pro-apoptotic markers nucleosome protein (DNA laddering) and Bax (initiator of mitochondrial permeability) and inflammatory marker, high-sensitivity C Reactive Protein (hsCRP) were determined by ELISA. NTBI (DNA damage inducing, pro-apoptotic) was measured by HPLC. Plasma levels of cytokines (anti-apoptotic, proliferation stimulating) were determined by using a multiplex bead-based immunoassay. Plasma lactate dehydrogenase (LDH), a marker of hemolysis, was also measured because hemolysis products have been shown to inhibit apoptosis. Results: Leukocyte number and absolute neutrophil count were 1.8 and 2.2 fold higher in SCD compared to thalassemia (P<0.01). Evidence for decreased apoptosis in SCD included reduced nuleosome protein as compared to thalassemia and reduced Bax as compared to controls. In addition, LDH was significantly increased 1.9 fold in SCD relative to thalassemia (p=0.012). In contrast, NTBI was 2 fold higher in thalassemia than SCD and correlated with nucleosome protein (R=0.45; p=0.013). Finally, high levels of the cytokines IL-6 and IL-10 in SCD relative to thalassemia may be stimulating leukocyte proliferation and survival. These cytokines and hsCRP are also positively correlated with leukocyte number. Discussion and Conclusions: These preliminary findings demonstrate that the balance of apoptosis vs. proliferation favors delayed cell death and enhanced cell proliferation in SCD, leading to increased circulating leukocytes. These changes may be related to hemolysis, increased levels of inflammatory cytokines, and lower amounts of NTBI in SCD. This leukocytosis increases the potential for vasoocclusive events that contribute to stroke, acute chest syndrome or renal tubule damage.

Poster# 43 – MOVED TO NOVEL TECHNOLOGIES SYMPOSIUM, WEDNESDAY, JUNE 10, 17:20 VASCULAR INFLAMMATORY RESPONSE AND SPATIAL ARRANGEMENT OF CELL ADHESION MOLECULES ASSOCIATED WITH REDOX CYCLING OF IRON IN PULMONARY BLAST INJURY Nikolai Gorbunov, PhD The Henry M. Jackson Foundation for the Advancement of Military Medicine Inc. (Presented By: Nikolai Gorbunov, PhD)

Poster# 44 ENDOSOMAL TRANSPORT OF TRANSFERRIN TO MITOCHONDRIA IS ESSENTIAL FOR EFFICIENT UTILIZATION OF IRON FOR HEME SYNTHESIS Alex D. Sheftel, PhD, An-Sheng Zhang, MD, Tanya Kahawita, MSc, Orian S. Shirihai, MD, PhD and Prem Ponka, MD, PhD Institut für Zytobiologie, Philipps-Universität-Marburg (Presented By: Alex D. Sheftel, PhD)

An exquisite relationship between iron and heme in haemoglobin (Hb)-synthesizing cells makes blood red. Erythroid cells are the most avid consumers of iron (Fe) in the organism and synthesize heme at a breakneck rate. Developing red blood cells (RBC) can take up Fe only from the plasma glycoprotein transferrin (Tf). Delivery of iron to these cells occurs following the binding of Tf to its cognate receptors on the cell membrane. The Tfreceptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification. Iron, following its reduction to Fe2+ by Steap3, is then transported across the endosomal membrane by the divalent metal transporter, DMT1. However, the post-endosomal path of Fe in the developing RBC remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones Fe in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron binding intermediate has never been identified. In erythroid cells, more than 90% of iron must enter mitochondria since ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides in the inner mitochondrial membrane, facing the matrix. In fact, in erythroid cells, strong evidence exists for a specific targeting of Fe toward mitochondria. This targeting is demonstrated in Hb-synthesizing cells in which Fe acquired from Tf continues to flow into mitochondria even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated a hypothesis that in erythroid cells a transient mitochondrion- endosome interaction is involved in iron translocation to its final destination. Recently, we have collected strong experimental evidence supporting this hypothesis: We have shown that Fe, delivered to mitochondria via the Tf pathway, is unavailable to cytoplasmic chelators. Moreover, we have demonstrated that Tf-containing endosomes move and contact mitochondria in erythroid cells, that vesicular movement is required for iron delivery to mitochondria and that “free” cytoplasmic Fe is not efficiently used for heme biosynthesis. Additonally, performing flow cytometry on cell lysates from reticulocytes incubated with two different fluorescent markers for endosomes and mitochondria, we have identified three distinct populations: endosomes, mitochondria, and a population of particles labelled with both fluorescent markers. The size of the double-labelled population increases with the incubation time and plateaus in ~30 min. Reticulocyte re-incubation with unlabelled Fe2-Tf leads to a timedependent decrease, and ultimate disappearance, of the double-labelled population, indicating a reversible nature of mitochondria-endosome interactions. As mentioned above, the substrate for the endosomal transporter DMT1 is Fe2+, the redox form of iron which is also the substrate for ferrochelatase. These facts make the above hypothesis quite attractive, since the “chaperone”-like function of endosomes may be one of the mechanisms that keeps the concentrations of reactive Fe2+ at extremely low levels in the oxygen-rich cytosol of erythroblasts, preventing ferrous ion’s participation in a dangerous Fenton reaction.

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Poster# 45 POTENTIAL INVOLVEMENT OF DMT1 AND ZIP8 IN IRON RELEASE FROM LYSOSOMES Chevaun Morrison, BS¹, Ningning Zhao², Annie Nguyen¹, Angelica Gonzalez¹, Eric Sauble¹, Maria C Linder¹ and Mitchell Knutson² ¹California State University, Fullerton; ²University of Florida, Gainesville (Presented By: Chevaun Morrison, BS)

We and others have shown in different cell types that to release iron from ferritin, its protein “shell” must first be degraded. This occurs primarily in lysosomes, since inhibitors of lysosomal proteases prevent ferritin iron release (Kidane et al. Am. J. Physiol. Cell Physiol. 291: C445-55, 2006). Iron deprivation stimulates entry of cytoplasmic ferritin into lysosomes (presumably by autophagy), and after removal of the protein shell, the exposed ferrihydrite crystallites in the ferritin core are dissolved and the iron returns to the cytoplasm for further distribution. How (and in what form) iron is returned to the cytoplasm is unclear; but two distinct transporters could be involved. DMT1 has been reported to associate with lysosomes, which we confirmed with confocal microscopy. Zip 8 is also associated with lysosomes, and we established that its transfection into HEK cells enhanced uptake of iron, given as 59Fe-labeled ferric citrate. In first attempts to determine whether one or both of these transporters were important for release of iron from lysosomes, we examined movement of DMT1 to lysosomes in response to iron deprivation, using confocal microscopy, and attempted to knock down expression of Zip 8 to determine whether that resulted in accumulation of lysosomal iron. In HepG2 cells, exposure to DFO (to induce iron deprivation) resulted in a rapid movement of ferritin from the cytoplasm to the lysosomes, as determined by density gradient fractionation and confocal microscopy. This was accompanied by a rapid 2-3-fold increase in colocalization of DMT1 with LAMP2 (a lysosomal membrane marker), the maximum effect being evident already after 2h, and implicating involvement of DMT1 in lysosomal iron metabolism. Expression of Zip 8 mRNA was knocked down 80-90% with siRNA in rat hepatoma cells (H4-IIE-C3) over 72-96h, compared with control cells treated with scrambled siRNA. Accumulation of iron in lysosomes was tracked after labeling ferritin with 59Fe during 24h treatment with 59Fe-ferric ammonium citrate and inducing its movement into lysosomes with DFO, as well as by assays of iron with bathophenanthroline. Although Zip8 mRNA was markedly knocked down, there was no major accumulation of iron in the lysosomes. However, there were difficulties with the immunoblotting required to determine whether Zip 8 protein expression had also been reduced. We conclude that DMTI is likely to be involved in the transfer of iron released from lysosomal ferritin back into the cytoplasm. It seems less likely that Zip 8 is important, but further studies are needed. Supported in part by PHS Grants RO1 HD 46949 (MCL) and RO1DK080706 (MK).

Poster# 46 EVIDENCE FOR AN ENDOPLASMIC RETICULUM IRON TRANSPORT SYSTEM Marie-Berengere Troadec, PhD, Diane M. Ward, PhD and Jerry Kaplan, PhD University of Utah (Presented By: Marie-Berengere Troadec)

Many enzymes require transition metals as co-factors for activity. For enzymes that reside within the vesicular apparatus (endoplasmic reticulum (E.R.), Golgi, lysosomes) metal transporters must exist to supply the required transition metal for the luminal apoenzyme. The presence of iron-containing enzymes with the secretory apparatus indicates a need for a vesicular iron transporter. The iron-containing enzyme prolyl-4-hydroxylase is a resident E.R. suggesting that the E.R. might contain an iron transporter. To identify this transporter we took advantage of the easily assayed Tartrate Resistant Acid Phosphatase (TRAP). TRAP is a conserved oxo-diiron containing phosphatase present in plants and mammals. The expression of a recombinant TRAP in mammalian cells led to a fully active enzyme suggesting that iron is incorporated in this protein in the E.R. Expression of TRAP in the budding yeast Saccharomyces cerevisiae also led to an active enzyme within the secretory apparatus. We generated a chimeric TRAP construct with a canonical KDEL endoplasmic reticulum retention signal. Expression of this construct in mammalian cells resulted in an active enzyme restricted to the early secretory apparatus. We are using this system in conjunction with RNAi silencing in mammalian cells to determine if TRAP gains its iron solely from the E.R. We are also testing the effect of deletions in specific candidate transition metal transporter genes to determine their effect on the activity of E.R. localized TRAP in yeast. These studies will permit us to determine the mechanism and regulation of vesicular iron transport. (This work is funded from an DK30534 grant to JK from the NIH).

Poster# 47 DELIVERY OF IRON FROM TRANSFERRIN TO MITOCHONDRIA VIA PATHWAYS INVOLVING LABILE AND NON-CHELATABLE FORMS OF IRON Maya Shvartsman, MSc¹, Eugenia Prus, PhD², Eitan Fibach, Prof² and Ioav Cabantchik, MD, PhD Prof³ ¹Hebrew University of Jerusalem; ²Hadassah Medical Center, Jerusalem, Israel; ³Hebrew University of Jerusalem, Jerusalem, Israel (Presented By: Maya Shvartsman, MSc)

Since the major portion of iron acquired by cells is ultimately delivered to mitochondria for incorporation into heme and iron sulfur clusters, the transfer of iron from endocytic vesicles to mitochondria entails a principal cell iron traffic pathway. Mitochondrial iron import has been associated with the iron carrier mitoferrin (1), yet little is known about the steps preceding it. In reticulocytes, transferrin-delivered iron might be acquired by mitochondria via direct physical contact between endosomes and mitochondria (2). Hitherto, it is unclear whether: a. an inter-organellar transfer of iron is mechanistically easible and whether or not it is applicable to all erythroid and non- erythroid cells and b. transfer of iron from TfFe or non-transferrinic sources to mitochondria is mediated by specific cytosolic factors. We used human K562 erythroleukemic cells and in-vitro differentiated erythroid cells (3) labeled with fluorescent iron-sensors for tracing iron delivery to mitochondria (4). Methods: Iron transit into cytosol and mitochondria was monitored by double labeling cells with two fluorescent metal-sensors of labile iron, the cytosolic calcein green (CALG (5) and the mitochondrial red fluorescent RPA (rhodamine B-phenanthroline (6)). On line fluorescence measurements were done (by spectrofluorimetry or flow cytometry), following addition of 1-5 mM TfFe or ferrous ammonium sulfate (FAS) Cytosolic chelators DMBAM) 5,5’-dimethyl BAPTA-AM (or bipyridyl carboxylate - AM (BIPC-AM) were loaded as described (5). Results and Discussion: Addition of TfFe to K562 or primary erythroid cells evoked time-dependent quenching of both cytosolic (CALG) and mitochondrial (RPA) metal- sensors, indicating a parallel entry of labile iron into these compartments. The cytosolic iron chelators DMB and BIPC reduced iron ingress from TfFe into cytosol; but while BIPC completely inhibited iron ingress into mitochondria, DMB did not. A similar pattern of chelator effects was observed with FAS uptake into K562 cells. Chloroquine, which prevents endosome acidification and thereby the release of iron from Tf within the endosome, curbed iron delivery into the cytosol and into mitochondria. On the other hand, wortmannin, an inhibitor of vesicle mobility, failed to reduce iron ingress from Tf into mitochondria. Based on the differential inhibition of iron ingress into mitochondria by cytosol-localized chelators and by inhibitors of vesicle trafficking, we deduce that iron delivery to mitochondria is likely to be mediated by more than one pathway, involving cytosolic passage of chelatable iron as well as parallel pathways that are inaccessible to chelators. Supported by ISF and EEC-F6 (LSHM-CT-2006-037296-Euroiron1. (1) Paradkar, PN et al. (2009). Molec Cell Biol. 29: 1007-1016. (2) Sheftel, AD et al. (2007) Blood 110: 125-132. (3) Prus, E; Fibach, E. (2008). Br J Hematol. 142: 301-307. (4) Breuer, W., et al (2008). Int J Biochem Cell Biol. 40: 350-4. (5) Shvartsman M et al. (2007). Am J Physiol Physiol 293: C1383-94. (6) Petrat, F. et al. (2002). Biochem J 362: 137-147.

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Poster# 48 HEME AND IRON TRANSPORT SYSTEMS IN NON-POLARIZED AND POLARIZED CELLS Izumi Yanatori¹, Mitsuaki Tabuchi, PhD, Reiko Akagi, PhD² and Fumio Kishi, MD, PhD ¹Kawasaki Medical School; ²Yasuda Women’s University (Presented By: Izumi Yanatori)

Human is able to utilize two types of iron, heme and non-heme iron. Heme is an important nutritional source of iron to synthesize iron-containing molecules and it is thought to be more readily absorbed than non-heme iron. Once heme is incorporated into cells, it is degraded by heme oxygenases (HOs) into iron, carbon monoxide and biliverdin. The non-heme iron is transported by divalent metal transporter 1 (DMT1), which is located at apical membrane and recycling endosome in enterocyte or lysosome in macrophage. To clarify the process of heme uptake and its degradation and the reutilization of iron, we analyzed the subcellular localizations of two isoenzymes of HOs, four isoforms of DMT1 and two candidates of the heme transporter, heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1), in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1 and DMT1A-I are located at plasma membrane and DMT1B-I at lysosome. In polarized cells, they show distinct localizations; HCP1 and DMT1A-I are located at apical membrane and HRG-1 at basolateral membrane and lysosome. HOs are located at smooth endoplasmic reticulum and colocalized with NADPH-cytochrome P-450 reductase. The incorporated heme in cytosol is degraded by HOs and the resulting iron is exported into tissue fluid via iron transporter ferroportin 1 expressed on basolateral membrane in enterocyte or on plasma membrane in macrophage. The liberated iron is transported by transferrin and reutilized for the hemoglobin synthesis in the erythroid system. These findings suggest that HCP1 can function on apical membrane of enterocyte to uptake dietary heme, and HRG-1 on lysosomal membrane to transport the endocytosed heme into cytosol. Recently, it has been reported that HCP1is able to transport folate more efficientlythan heme and thus it is speculated that folate is the more physiologically relevant target of HCP1. However our localization study suggests that HCP1 may play a role in dietary heme uptake, and HRG-1 may transport heme from body fluid into enterocyte and sense the heme concentration in the blood stream.

Poster# 49 IRON SUPPLY DETERMINES APICAL/BASOLATERAL MEMBRANE DISTRIBUTION OF INTESTINAL IRON TRANSPORTERS DMT1 AND FERROPORTIN 1 Marco T. Nunez, PhD¹, Victoria Tapia, BSc¹, Alejandro Rojas, PhD¹, Pabla Aguirre, PhD¹, Francisco Gomez, BSc¹, Daniela Sandoval, Engineer in Biotecnology¹ and Francisco Nualart, PhD² ¹Universidad de Chile; ²Universidad de Concepcion (Presented By: Marco T. Nunez, PhD)

Introduction: Intestinal iron absorption comprises the coordinated activity of the influx transporter DMT1 and the efflux transporter ferroportin 1 (FP1). In this work, we studied the movement of DMT1 and FP1 between cellular compartments as a function of the iron supply. Methods: Membrane localization of DMT1 and FP1 was assessed by selective biotinylation. Movement of transporters was determined by time-lapsed fluorescence microscopy. DMT1 and FP1 knock-down was done using antisense technology. Iron fluxes were determined using 55Fe as a tracer. Results: In rat duodenum, iron starvation resulted in the preferential distribution of DMT1 to the apical membrane and FP1 to the basolateral membrane, although considerable FP1 was also found in apical domains. Iron gavage resulted in the re-location of DMT1 to intracellular basal domains and the internalization of basolateral FP1. In Caco-2 cells, relocalization of CFP-tagged DMT1 was complete 90 min after addition of iron. Membrane localization of DMT1 and FP1, studied by selective biotinylation, revealed that iron induced a striking re-distribution of DMT1 to the basolateral membrane, and a modest re-localization of FP1 to the apical membrane. In agreement with the membrane distribution of the transporters, 55Fe flux experiments revealed inward and outward iron fluxes at both membrane domains. Antisenses targeted to DMT1 or FP1 inhibited basolateral iron uptake and apical iron efflux, respectively, indicating the participation of DMT1 and FP1 in these fluxes. The fluxes were regulated by the iron supply: increased iron produced decreased apical uptake and increased basolateral uptake. Discussion: Movement of transporters between plasma membrane and intracellular domains is an important mechanism by which influx of ions and metabolites is regulated. Examples of this process are found in the TRP family of calcium channels, the Glut4 transporter, neurotransmitter receptors and aquaporin channels. The results reported here show that intestinal iron absorption is also regulated by the movement of iron transporters. Conclusions: Our findings reveal a novel cell mechanism by which intestinal cells regulate intestinal iron absorption through the re-positioning of DMT1 and FP between apical and basolateral membranes. Financed by FONDECYT grant 1070840 and ICM grant P-05-001-F.

Poster# 50 SUBCELLULAR DISTRIBUTIONS OF ZIP14 AND DMT1 IN HEPG2 CELLS: IMPLICATIONS FOR CELLULAR IRON IMPORT AND ENDOSOMAL IRON TRANSPORT Ningning Zhao and Mitchell Knutson University of Florida (Presented By: Mitchell Knutson)

The liver is the main storage site for body iron. This organ normally takes up plasma transferrin-bound iron (TBI), but can also acquire non-transferrinbound iron (NTBI), which appears in plasma during iron overload. Uptake of TBI involves binding to the transferrin receptor at the cell surface, followed by internalization into recycling endosomes. Upon endosomal acidification, iron is released from transferrin and translocated into the cytosol by a transmembrane protein. Despite the central role of the liver in iron metabolism, the transmembrane proteins that participate in the uptake of NTBI and TBI are not firmly established. We used confocal laser-scanning microscopy to examine the subcellular distribution of the metal-ion transporters DMT1 (Divalent Metal Transporter 1) and Zip14 in human HepG2 hepatoma cells. For the localization studies, cells were transfected to express green fluorescent protein (GFP) fusion proteins. We found that Zip14-GFP was readily detectable at the hepatocyte plasma membrane whereas DMT1-GFP was not. Both fusion proteins displayed abundant punctuate intracellular staining that partially colocalized with internalized Texas-red labeled transferrin, a marker of recycling endosomes. Localization of Zip14-GFP to the plasma membrane is consistent with its ability to take up NTBI at the cell surface. Detection of Zip14-GFP in recycling endosomes further implicates this protein in the assimilation of TBI. We conclude that Zip14 may therefore play a role in normal uptake of iron by the liver.

Poster# 51 FERRITIN DOES NOT DONATE ITS IRON FOR HEME SYNTHESIS IN CULTIVATED MACROPHAGES Marc R. Mikhael, BSc¹, Alex D. Sheftel, BSc, PhD² and Prem Ponka, MD, PhD³ ¹McGill University; ²Institut für Zytobiologie, Philipps-Universität-Marburg, Marburg, Germany; ³Lady Davis Institute and Department of Physiology, McGill University, Montreal, QC, Canada (Presented By: Marc R. Mikhael, BSc)

Iron is essential for all life yet can be dangerous under certain conditions. Iron storage by the 24-subunit protein, ferritin, renders excess amounts of the metal non-reactive and, consequentially, is crucial for life. While the mechanism detailing the storage of iron in ferritin has been well characterized, little is known about the fate of ferritin- stored iron and whether it can be released and reutilized by cells. Although it has been shown that ferritin-derived iron cannot be used for heme synthesis in developing red blood cells, virtually nothing is known about the use of ferritin-derived iron in non-erythroid cells. We therefore attempted to answer the question of whether iron from ferritin can be used for heme synthesis in the murine macrophage cell line, RAW 264.7 cells. Cells treated with 5-aminolevulinic acid (ALA; a precursor of heme synthesis) show increased heme production as determined by enhanced incorporation of transferrin-bound 59Fe into heme. However, our study shows that, upon the addition of ALA, 59Fe from ferritin cannot be incorporated into heme. Additionally, little 59Fe is liberated from ferritin when heme synthesis is increased. In conclusion, ferritin in cultivated macrophages is not a significant source of iron for the cell’s own metabolic functions. 111

Poster# 52 – MOVED TO NOVEL TECHNOLOGIES SYMPOSIUM, WEDNESDAY, JUNE 10, 17:40 PROBING THE INTERRELATIONSHIPS OF THE CELLULAR LABILE IRON POOLS (LIP) IN NORMAL AND PATHOLOGICAL CONDITIONS: AN OVERVIEW William Brwuer, PhD², Yan-Sung Sohn, MSc, Maya Shvartsman, MSc, Or Kakhlon, PhD and Ioav Cabantchik, MD, PhD¹ ¹Hebrew University of Jerusalem; ²Institute of Life Sciences, Hebrew University of Jerusalem (Presented By: Ioav Cabantchik, MD, PhD)

Poster# 53 CHARACTERIZATION OF A POTENTIAL IRON SHUTTLE WITHIN THE CYTOPLASM OF MAMMALIAN CELLS Natasha Hill, BS¹, Ben Rojas¹, Vireak Thon¹, Robert Malanga¹, Lena Ton-nu¹, Lina Planutyte¹, Maria C. Linder¹, Andrew Z. Mason² and Ashraf Elamin² ¹California State University, Fullerton; ²California State University, Long Beach (Presented By: Natasha Hill, BS)

In mammalian cells, iron shuttling in and out of cellular proteins and compartments involves a labile iron pool (LIP) in which the iron, loosely bound to uncharacterized proteins or other factors, is available to desferrioxamine (DFO) and other high affinity chelators. Following 3h labeling of cultured human cell lines with tracer 59Fe as Fe(II)-ascorbate or ferric ammonium citrate, Biogel P2 size exclusion chromatography and native PAGE were used to isolate two labeled cytoplasmic fractions, one being ferritin, the second being a smaller unknown moiety. The latter, collected after ultrafiltration of lysates, had an apparent molecular weight of 1600 Da and degraded to 600 Da if not separated from the bulk of the cellular protein. This component could be labeled directly by treatment of fresh lysates with 59Fe(III)-nitrilotriacetate (NTA), and conversion to the 600 Da fragment was induced by incubation with proteinase K, but not trypsin. The 1600 and 600 Da components were negatively charged at pH 7, migrating with an Rf of about 0.9 in native PAGE with 5% acrylamide. 59Fe in the 1600 Da moiety was completely chelated by DFO in fresh lysates but became unextractable over time. The 1600 Da component had a UV absorption spectrum with peaks at 220 and 280 nm. MALDI analyses of the 1600 Da entity resulted in a fragmentation pattern having approximately thirteen fragments decreasing in mass by 44 Da units down to a core component of about 850 Da, implying a polymeric structure. 59Fe in the unknown versus in ferritin varied inversely with total cellular Fe. However, analysis of Centricon 10 ultrafiltrates from human HepG2 and Caco2 cells showed that the Fe content of this fraction did not vary appreciably with cellular Fe status in control cells or cells pretreated by 24h exposure to 180 uM Fe or 30 uM DFO. Fe concentrations in the <10,000 Da pool were 90- 200 ng/mg cell protein, with total iron contents ranging from 200 to1600 ng/mg. We conclude that the concentration of Fe in the LIP stays relatively constant despite changes in Fe status and that the LIP is likely to consist mainly of a complex of Fe with a small peptide, possibly having a polymeric modification. Supported in part by PHS Grant No. RO1 HD 46949 (MCL) as well as CSUPERB and the W.M. Keck Foundation (AZM).

Poster# 54 DIFFERENTIAL PROTEOMIC APPROACH TO INVESTIGATE IRON RESPONSE IN MURINE MACROPHAGES Annalisa Castagna, PhD¹, Ivana De Domenico², Rita Polati³, Alessandra Bossi³, Natascia Campostrini4, Federica Zaninotto4, Fabiana Busti4, Lello Zolla5, AnnaMaria Timperio5, Oliviero Olivieri4, Jerry Kaplan6 and Domenico Girelli4 ¹University of Verona, department of Clinical and Experimental Medicine; ²University of Utah, School of Medicine, Department of Internal Medicine, Salt Lake City UT, US; ³University of Verona, Dip. di Biotecnologie, Verona, Italy; 4University of Verona, Dip. di Medicina Clinica e Sperimentale, Verona, Italy; 5University of Tuscia, Department of Environmental Sciences, Italy; 6University of Utah, School of Medicine, Department of Pathology, Salt Lake City UT, US (Presented By: Annalisa Castagna, PhD)

Macrophages cells are specialized for iron storage and recycling from senescent erythrocytes, so they play a central role in the fine tuning of iron balance and distribution. Thus, they are ideal candidates to elucidate the complex responses associated to iron levels. Murine macrophages are a suitable model for a comprehensive evaluation of all the proteins interested in up- and down-regulation due to a stimulus (iron-rich culture medium). In this study we performed a 2-DE differential analysis to investigate the molecular bases of iron response in macrophages. Mouse Bone Marrow macrophages were isolated from C3H WT mice and maintained in culture for 7 days. Macrophages were incubated with ferric ammonium citrate (FAC) (10mM Fe) for 24h in serum-free media. Protein extraction was obtained using an innovative protocol [1] and 2D maps were performed and analyzed accordingly. Differentially expressed spots were identified by nanoRP-HPLC-ESI-MS/MS. Many proteins resulted to be differentially expressed in FAC treated cells, as compared to untreated cells. Among the cytosolic proteins, as expected FAC induced the over-expression of ferritins, a family of iron storage proteins. FAC treatment also determined down-regulation of several proteins of the cytoskeleton, e.g. beta-actin (↓ 3.44), gamma-actin-like protein (↓ 2.88), A-X actin (↓ 2.88), actin (↓ 2.52). Down-regulation was also observed for proteins involved in control of signaling pathways and the formation of vesicles, e.g. the glia maturation factor gamma-isoform 1 (↓ 2.42), the vesicles mediated transport mKIAA3012 protein (↓ 1.54) and Rho GDP dissociation inhibitor (GDI) beta. In general, the presence of high iron level appeared to induce de-structuration and/or de-polymerisation of the actin filaments of the cytoskeleton. Of note, the membrane proteins up- regulated by Fe included the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (↑2.36), which is normally localized within the cytoplasm and is associated with the glycolysis. However, it has been reported that the GAPDH could also locate on macrophage cell surfaces, and that its expression correlated with the availability of iron in the medium [2]. Indeed, membrane GAPDH has been proposed as an alternate route of iron acquisition through interaction with transferrin and subsequent internalization of the GAPDH-transferrin complex into the early endosomes. Finally, it is interesting to note that FAC up-regulated vimentin (↑ 4.54) and galectin-3 (↑ 1.97), both known to be involved in phagocytosis/internalization processes, and localized on phagocytic structures, replacing actin. This proteomic study represents an example of the potential role of this methodology in the field of iron biology, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions. [1] Polati R, Castagna A, Bossi A, Campostrini N, Zaninotto F, Timperio AM, Zolla L, Olivieri O, Corrocher R, Girelli D. 2009 Proteome Science (in press). [2] Raje CI, Kumar S, Harle A, Nanda JS, Raje M. 2007 J Biol Chem. 282:3252-3261.

Poster# 55 MITOFERRIN 1 AND 2 ACT AS MITOCHONDRIAL IRON IMPORTERS IN HELA CELLS Sara Luscieti, Patrizia Cavadini, PhD, Maura Poli, PhD, Federica Maccarinelli, PhD, Dario Finazzi, PhD and Paolo Arosio, PhD Dipartimento Materno Infantile e Ttecnologie Biomediche, Università di Brescia, Italy (Presented By: Sara Luscieti)

The mitochondrion is the place of the biosynthesis of heme and iron sulphur clusters, and it processes most of cellular iron for the synthesis of iron proteins. Mitochondrial iron must be tightly regulated to minimise reactions with reactive oxygen species produced by respiration, which would lead to the production of toxic free radicals. This involves a balance between iron import, usage and export with mechanisms that have not being clarified. In yeast iron is mainly imported by MRS3/4 and exported as Fe/S complexes by ATMp1. Their mammalian homologs seem to be mitoferrin1 and 2 (MFRN1 and 2) and ABCB7, respectively. The silencing of ABCB7 in HeLa cells was previously shown to reduce cell proliferation and cause iron accumulation in the mitochondria in a form not easily available to heme synthesis and mitochondrial ferritin. This work was aimed to analyse the effects of the silencing of mitoferrin 1 and 2 on cell proliferation, iron status and mitochondrial enzymes. The levels of MFRN1 and 2 transcripts, quantified by qRT-PCR, were comparable in HeLa cells. They were not affected by iron supplementation or oxidative stress, while iron chelation by desferrioxamine or deferiprone caused a reduction of MFRN2 mRNA but not of MFRN1. Transfection with specific siRNAs reduced the level of their transcripts to 20-30% of the control. This was accompanied by about a 50% reduction of cell proliferation, measured by MTT assay, without evident signs of apoptosis. Iron status was modified: transferrin receptor 1 level was increased, while ferritin level was altered differently by the two types of siRNAs. The activity of cytosolic and of total aconitase was reduced, that of mitochondrial citrate synthase and succinate dehydrogenase was not affected, while that of Mnsuperoxide dismutase was increased, together with its protein level. The silencing of both MFRN1 and 2 caused also an increase of free protoporphyrin, which further increased when the two were silenced together. The phenotype was not modified by the expression of mitochondrial ferritin, which incorporated slightly less iron than in control cells.

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Finally we found that the silencing of MFRN2 partially rescued some of the defects caused by the deficiency of ABCB7, a Fe/S exporter: it increased cell proliferation, the levels of ferritin and aconitase activity. In contrast, the silencing of MFRN1 had only marginal effects. In conclusion our data confirm that MFRN1 and 2 act as mitochondrial iron importers in HeLa cells. Their functionality is similar, and the expression of MFRN1 is not erythroid specific as previously suggested. MFRN2 is modulated by iron chelation and its silencing caused stronger effects on cellular iron status then that of MFRN1, suggesting functional specificities.

Poster# 56 THE ASSEMBLY OF RESPIRATORY COMPLEX I REQUIRES THE IRON-SULFUR CLUTER PROTEIN HUIND1 Alex D. Sheftel, PhD, Oliver Stehling, PhD, Antonio J. Pierik, PhD, Daili J.A. Netz, PhD, Stefan Kerscher, PhD, Hans-Peter Elsässer, PhD, Ilka Wittig, PhD, Ulrich Brandt, PhD, Janneke Balk, PhD and Roland Lill, PhD Institut für Zytobiologie, Philipps-Universität-Marburg (Presented By: Alex D. Sheftel, PhD)

Respiratory complex I (NADH:ubiquinone oxidoreductase) is a large mitochondrial inner membrane enzyme consisting of 45 subunits and eight ironsulfur (Fe/S) clusters. While complex I dysfunction is the most common reason for mitochondrial diseases, the assembly of complex I and its Fe/S cofactors remains elusive. Here, we identify the human mitochondrial P-loop NTPase, designated huInd1, that is specifically required for the assembly of complex I. huInd1 can bind an Fe/S cluster via a conserved CXXC motif in a labile fashion. Knockdown of huInd1 in HeLa cells by RNAi technology led to highly specific decreases in complex I protein and activity levels, that could not be rescued by over-production of a mutated huInd1 with alanine instead of cysteine residues in the CXXC motif. The activities of other respiratory complexes were hardly altered upon depletion of huInd1, even though the respiratory super-complexes were massively remodeled showing the dynamic character of respiratory chain assemblages. By a novel radiolabelling technique, the amount of iron associated with complex I also reflected the dependence of this enzyme on huInd1 for assembly. Depletion of huInd1 resulted in strongly decreased amounts of Fe/S cluster-containing complex I subunits and the concomitant appearance of a 450 kDa sub-complex. As a consequence of the respiratory defect in huInd1-deficient cells the mitochondrial morphology was altered. Together, these data suggest that huInd1 is a bona fide Fe/S cluster assembly factor performing a specific role in the delivery of one or more Fe/S clusters to complex I subunits.

Poster# 57 DOWN-REGULATION OF COMPONENTS OF THE IRON-SULFUR CLUSTER ASSEMBLY MACHINERIES IN INFLAMMATORY MACROPHAGES Cédric Picot, PhD, Frédéric Canal, PhD, Kahina Abbas, PhD student, Cendrine Seguin, research assistant, Valérie Quesniaux, PhD, Jean-Claude Drapier, PhD and Cecile Bouton ICSN-CNRS avenue de la Terrasse, 91190 Gif-sur-Yvette, France (Presented By: Cecile Bouton)

Biogenesis of Fe-S clusters in eukaryotes is a complex process involving numerous mitochondrial and cytosolic proteins divided into three multicomponent systems (ISC, export and CIA). We have previously shown that Nfs1 and IscU, the two main components of the ISC machinery, are down-regulated in macrophages physiologically stimulated with interferon-gamma (IFN-g) and lipopolysaccharide (LPS), two key inducers of nitric oxide (NO) synthesis (1). In this study, we have investigated whether immunostimulated macrophages orchestrate a common regulation of the genes homologous to the yeast ISC, export and CIA machineries. Using the TaqMan® Low Density Array Applied Biosystems microfluidic card technology, we have observed that most genes involved in Fe-S biogenesis are down-regulated in IFN-g/LPS-stimulated macrophages, in particular frataxin, glutaredoxin 5 and the mitochondrial transporter ABCb7. As reported before for Nfs1 and IscU, regulation does not rely on NO production because decrease in transcript levels occurred to the same extent both in macrophages from wild type and from NO synthase 2 knock-out mice. By exposing cells to IFN-g or LPS separately, we have shown that frataxin and ABCb7 genes were the most sensitive to LPS (~50% decrease). We have then explored the signaling events that repress these two genes in LPS-stimulated macrophages. As expected the effect of LPS on frataxin and ABCb7 mRNA downregulation was mediated by the Toll-like receptor 4 (TLR4). TLR4 signals through two adaptor pathways, namely MyD88/TIRAP and TRIF/TRAM, which in turn trigger signaling cascades leading to activation of NF-kB and MAPKs. By using bone marrow-derived macrophages from MyD88-/- or TRIF-/- mice, we have shown that ABCb7 down-regulation by LPS was dependent on the TRIF branch of TLR4 signaling pathway while frataxin required both MyD88 and TRIF for its regulation. Moreover, by exposing macrophages to a synthetic ligand of TRL3 which solely interacts with the TRIF adapter, both frataxin and ABCb7 mRNA levels were significantly decreased. We have then assessed the role of MAPKs in LPS-dependent reduction of frataxin and ABCb7 gene expression. Addition of the specific JNK inhibitor SP600125 or the ERK-specific MEK1/2 inhibitor U0126 to LPS- stimulated macrophages markedly blocked frataxin and ABCb7 down-regulation while the p38 inhibitor SB203580 was not effective. In conclusion, our results show that LPS alone down-regulates the expression of frataxin and ABCb7 in macrophages through a common TRIF-dependent activation of MAPK pathways. By limiting the production of Fe-S proteins which can contribute to oxidative injury (2), macrophages may resist damage due to their own production of reactive oxygen species during inflammatory stress. (1) Canal, F., Fosset, C., Chauveau, MJ., Drapier, JC, Bouton C, Arch. Biochem. Biophys., 465 (2007) 282-292 (2) Strain, J., Lorenz, C.R., Bode, J., Garland, S., Smolen, G.A., Ta, D.T., Vickery, L.E. Culotta V.C, J. Biol. Chem., 273 (1998) 31138-144

Poster# 58 MITOCHONDRIAL FERRITIN ATTENUATES DOXORUBICIN EFFECTS ON IRON HOMEOSTASIS Maura Poli, Dr, Federica Maccarinelli, Dr, Vanessa Porrini, Dr, Emiliano Cocco, Dr, Derosas Manuela, Dr, Sara Luscieti, Dr, Paolo Arosio, PhD, Prof and Isabella Zanella, Dr Dipartimento Materno Infantile e Tecnologie Biomediche Università Brescia (Presented By: Maura Poli, Dr)

Doxorubicin (DOX) is an anthracyclin widely used in cancer treatment, however with dangerous side effects on heart. A relationship with iron metabolism is recognised: iron chelators are used in therapy to reduce its cardiotoxicity while iron-loading potentiates its effects. The role of iron can be related to oxidative damage, however alterations of iron homeostasis are described in cellular/animal models. ROS production and iron trafficking are intense in mitochondria, the major site of iron-anthracycline interaction in heart. Mitochondrial ferritin (FtMt) has been recently identified and shown to be expressed in heart. Its exact role is unknown, however it incorporates iron, protects from oxidative damage and from defects caused by frataxin deficiency. This work was aimed to analyse the effect of DOX in mouse and cell models on iron homeostasis and to verify if FtMt can attenuate defects. Acute DOX treatment of mice induced in heart: 1) cytosolic ferritin increase and TfR1 decrease, measured as mRNA by RT-PCR and protein level by blotting, possibly due to down-modulation of IRP2 as previously reported; 2) ferritin iron saturation increase analysed by native PAGE and Perls’-DAB staining; 3) up-regulation of hepcidin mRNA, possibly linked to ferritin upregulation. IRP1/cytosolic aconitase, little expressed in heart, remained unchanged as measured by blotting. In heart mitochondria DOX induced: 1) interestingly up- regulation of FtMt mRNA; 2) a slight but significative decrease of SOD2 activity measured by native-PAGE and NBT staining, without modification of protein level measured by blotting, suggesting a partial inactivation of the enzyme. Iron sulfur cluster machinery seemed not impaired since mitochondrial aconitase and SDH activity, measured spectrophotometrically, and frataxin protein level remained unchanged. DOX combined with iron overload exhacerbated only some indices. Cytosolic ferritin and its iron content increased with iron, as expected, but DOX+iron had additive effects although mRNA decreased. In mitochondria SOD2 activity decreased also with iron and DOX+iron effects were again additive, with protein level unchanged. FtMt mRNA was upregulated also by iron but DOX+iron did not further increased its level; DOX induced citotoxicity in HeLa cells, that was exhacerbated by H2O2 and FAC treatment. Ferritins and ferritin-bound iron increased, TfR1 decreased, as in mice. While mitochondrial aconitase and SDH activity were unchanged as in mice, cytosolic aconitase activity decreased, with a parallel protein content decrease unlike in mice heart. SOD2 activity was defective, despite an increase in protein level. FtMt overexpression in DOX treated cells induced a greater viability and resistance to H2O2 and FAC. Ferritin levels were enhanced but in a minor way. TfR1 was substantially unchanged. Despite ferritin increase, bounded iron was unchanged but interestingly FtMt was not overloaded. FtMt was however not able to rescue cytosolic aconitase activity decrease, despite a minor decrease of protein levels. Interestingly FtMt recovered and even enhanced SOD2 activity, also with a parallel increase of protein content.

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In conclusion, the prevalent effects of DOX acute treatment on iron metabolism in mice were more evident in cytosol than in mitochondrion and partially exhacerbated by iron-overloading. HeLa cell model resembled mouse model in most effects. Interestingly the overexpression of FtMt recovered some iron-related defects induced by DOX, thus possibly protecting heart from its effects.

Poster# 59 A HIGH-THROUGHPUT SIRNA SCREEN FOR HEPCIDIN REGULATORS REVEALS SMAD7 AS A NOVEL NEGATIVE REGULATOR OF HEPCIDIN EXPRESSION Katarzyna Mleczko-Sanecka¹, Anan Ragab, Dr², Guillem Casanovas, BSc³, Michael Boutros, Prof², Matthias W. Hentze, Prof4 and Martina U. Muckenthaler, Prof¹ ¹Molecular Medicine Partnership Unit, Heidelberg University; ²German Cancer Research Center (DKFZ); ³Molecular Medicine Partnership Unit; 4European Molecular Biology Laboratory (Presented By: Katarzyna Mleczko-Sanecka)

Hepcidin is a hepatic hormone that acts as a key regulator of iron homeostasis in humans. Several signalling pathways and transcription factors have been identified to activate hepcidin transcription including the Bone morphogenetic protein (BMP) pathway involving the hemochromatosis protein HFE2 and SMAD transcription factors as well as the inflammatory JAK-STAT pathway. By contrast, hepcidin inhibition by negative regulators is still poorly understood. Suppressors of hepcidin mRNA expression include the serine protease TMPRSS6 and GDF15. By applying a cell-based high-throughput siRNA screen we identified the SMAD7 gene as a novel suppressor of hepcidin expression. This gene is an inhibitory SMAD protein (I-SMAD), activated via the TGFß pathway and involved in negative feedback loops that suppress both TGFß and BMP signalling. Here, we show that siRNA- mediated knockdown of the SMAD7 gene increases hepcidin mRNA expression. Using luciferase reporter constructs containing the human hepcidin promoter we demonstrate that the effect of the SMAD7 knockdown is only partially dependent on the proximal and distal BMP-responsive element within the hepcidin promoter. In agreement with previous reports, we observe that SMAD7 expression on the mRNA level requires the SMAD4 transcription factor. Interestingly, SMAD7 expression is increased in response to BMP-6 suggesting that this regulatory mechanism may be linked to the iron-mediated hepcidin response. In summary, we show that the I-SMAD protein SMAD7 is a potent hepcidin suppressor in vitro that is co-regulated with hepcidin by the BMP-signalling pathway.

Poster# 60 BONE MORPHOGENETIC PROTEIN (BMP)-RESPONSIVE ELEMENTS LOCATED IN THE PROXIMAL AND DISTAL HEPCIDIN PROMOTER ARE CRITICAL FOR ITS RESPONSE TO HJV/BMP/SMAD Guillem Casanovas¹, Katarzyna Mleczko-Sanecka¹, Sandro Altamura², Matthias W. Hentze¹ and Martina U. Muckenthaler² ¹European Molecular Biology Laboratory, Heidelberg, Germany; ²Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg (Presented By: Guillem Casanovas)

The hemochromatosis proteins HFE, transferrin receptor 2 (TfR2) and hemojuvelin (HJV, HFE2) positively control expression of the major iron regulatory hormone hepcidin. HJV is a bone morphogenetic protein (BMP) co-receptor that enhances the cellular response to BMP cytokines via the phosphorylation of SMAD proteins. In search for transcription factor binding motifs that activate or repress hepcidin promoter activity we analyzed luciferase reporter constructs containing the human hepcidin promoter (2.7kb) as well as derivatives with mutated motifs to analyse their role in hepcidin transcription. We show that two highly conserved and sequence-identical BMP- responsive elements located at positions -84/-79 (BMP-RE1) and -2255/- 2250 (BMP-RE2) of the human hepcidin promoter are critical for both the basal hepcidin mRNA expression and the hepcidin response to BMP-2 and BMP-6. While BMP-RE1 and BMP-RE2 show additive effects in responding to HJV-mediated BMP signals, only BMP-RE1 that is located in close proximity to a previously identified STAT-binding site is important for the hepcidin response to IL-6. These data identify a missing link between the HJV/BMP signaling pathways and hepcidin transcription, and further define the connection between inflammation and BMP-dependent hepcidin promoter activation. As such, they provide important new information furthering our understanding of disorders of iron metabolism and the anemia of inflammation.

Poster# 61 RENAL HANDLING OF HEPCIDIN Hilde Peters, MD², Coby Laarakkers, BSc, Jack Wetzels and Dorine Swinkels, MD, PhD¹ ¹Radboud Department of Clinical Chemistry 441, University Nijmegen Medical Centre; ²Departments of Clinical Chemistry and Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands (Presented By: Dorine Swinkels, MD, PhD)

Introduction and Aims: Hepcidin is a 25-amino acid peptide and a key regulator of iron homeostasis. Currently the renal handling of hepcidin is unclear. In order to evaluate the influence of estimated glomerular filtration rate (eGFR) on serum hepcidin levels, we determined serum levels of both hepcidin-25 and its isoforms (hepcidin- 20 and 22), in patients with various degrees of chronic kidney disease (CKD) and in dialysis patients. Furthermore, we assessed whether hepcidin was reabsorbed by the renal tubules. Methods: We obtained blood samples of patients with CKD who visited our outpatient clinic. Relevant clinical and biochemical data were retrieved from patients' records. In haemodialysis patients, blood samples were collected at the start and end of dialysis. To determine whether the artificial kidney removed hepcidin, blood samples were drawn from both the arterial and the venous line. Gelofusine®, an inhibitor of tubular protein reabsorption, was administered intravenously to three healthy volunteers. Blood samples were drawn at the start and the end (60 min) of the infusion and urine samples were collected after 0, 30, 60 and 180 min. The following parameters were measured: hemoglobin (Hb), serum iron, iron binding capacity, ferritin, C-reactive protein (CRP), serum and urinary creatinine and hepcidin and urinary β2-microglobulin (β2m). β2m was measured with ELISA. Serum and urine hepcidin measurements were performed by the use of Time-Of-Flight mass spectrometry after enrichment for hepcidin by weak cation exchange chromatography. Results: 83 Predialysis patients were included. 25% Were treated with erythropoetin, mean age was 55 years, median eGFR 36 ml/min/1.73m2, median ferritin 111 µ g/L, mean Hb 7.6 mmol/L, median serum hepcidin-25 5.1 nM, median serum hepcidin-20 1.8 nM and median serum hepcidin-22 1.1 nM. Serum hepcidin-25 correlated strongly with ferritin (r= 0.74, p <0.01). Multiple regression analysis showed ferritin to be the only significant predictor of hepcidin-25 levels. The ratio hepcidin-25/total hepcidin was influenced by eGFR: we observed a significant decrease in the ratio of hepcidin-25/total hepcidin in patients with more severe renal failure. 34 Haemodialysis patients were included; median hepcidin-25 levels were higher than in CKD patients (9.5 nM), but were again strongly correlated with ferritin (r= 0.76, P <0.01). Arterial and venous samples differed significantly (15.7 nM versus 9.2 nM, p =0.04), but no significant decrease in serum hepcidin occured during dialysis. After the infusion of Gelofusine® a rapid increase in absolute and fractional urinary excretion of β2m (150-300 fold) and hepcidin (3-10 fold) was observed. Conclusions: Our study indicates that hepcidin is filtered by the glomerulus and reabsorbed by the renal tubules. However, eGFR is not a major determinant of serum hepcidin levels. Moreover, serum levels of hepcidin remain unchanged during dialysis, despite removal of hepcidin by the artificial kidney. Taken together, this suggests a tight and rapid regulation of serum hepcidin-25 levels in patients with CKD.

Poster# 62 FURIN AND MAPK (ERK1/2) PARTICIPATE IN THE REGULATION OF HEPCIDIN EXPRESSION IN HEPG2 CELLS Maura Poli, PhD, Sara Luscieti, PhD, Federica Maccarinelli, PhD, Dario Finazzi, PhD and Paolo Arosio, PhD Department MITB, University of Brescia (Presented By: Maura Poli, PhD)

The BMP/SMAD signalling pathway is central to regulation of hepcidin expression in the liver. It involves various receptors, co-receptors and proteins, some of which may be involved in the HFE/TfR2 and iron-dependent regulation. Furin is particularly interesting since it is able to act on various proteins of the pathway, by promoting the maturation of hepcidin (Valore & Ganz, 2008), of the BMPs 2, 4 and 6 (Constam &, Robertson, 1999), and the production of soluble HJV (Silvestri et al. 2008). Furin also 114 activates TGF-beta, which in turn upregulates furin in a regulatory loop which involves the phosphorylation of SMAD2/3 and ERK (Blanchette et al. 2001). Interestingly, transferrin binding to TfR2 in K562 and HepG2 cells was found to activate MAP kinases with the phosphorylation of Erk (Calzolari, et al. 2006), and Erk phosphorylation is induced also by activation of the BMP/SMAD pathway. To verify the importance of furin and MAP kinases in hepcidin expression HepG2 cells were treated with a furin inhibitor, with BMP2 or with specific siRNAs, and hepcidin mRNA and signalling pathways were analysed. The level of furin protein, the phosphorylation of SMAD1/5/8 and Erk1/2 and the expression of hepcidin and other genes were analysed by western blotting and qRT-PCR, respectively. HepG2 cells were treated with BMP2, the furin inhibitor CMK (Decanoyl-Arg-Val-Lys-Arg-Chloromethylketone), or with siRNAs for HFE, TfR2, Neogenin and HJV. Moreover, the cells were treated for iron supplementation (FAC, FeTf), chelation (DFO, DFP) and oxidative stress (H2O2). The furin inhibitor CMK caused a strong reduction of hepcidin mRNA in dose and time dependent manner, probably due to reduction in BMP2/4/6 maturation. BMP2 stimulation upregulated hepcidin expression via SMAD1/5/8 phosphorylation, as expected, but also increased up to 2-4 folds the level of HJV and Furin in dose and time dependent manner. The specific inhibitor of SMAD phosphorylation, Dorsomorphyn (5uM), inhibited the constitutive and BMP2-dependent expression of Hepcidin and HJV, while it reduced the induction of furin by BMP2.. Moreover, we found that the silencing of Neogenin and of HJV inhibited SMAD phosphorylation and hepcidin transcription , while that of TfR2 and HFE, alone and together, strongly reduced Erk1/2 phosphorylation and the level of furin mRNA and protein, without major effects on SMAD phosphorylation and hepcidin expression. In conclusion. our data indicate a possible new pathway for hepcidin regulation that involves furin and MAP kinases. Iron supplementation and chelation, and also TfR2 / HFE modulate Erk1/2 phosphorylation . This regulates furin expression and activity, which, in turn regulates the maturation of major effectors of hepcidin expression, such as BMP2/4/6. Moreover, MAPK activation was shown assist the BMP/SMAD pathway to support hepcidin expression. Supported by EEC program Euroiron1 and by MIUR-PRIN to PA

Poster# 63 PHYSIOLOGIC AND PATHOPHYSIOLOGIC REGULATION OF HEPCIDIN EXPRESSION Tom Bartnikas, MD, PhD and Mark Fleming, MD, DPhil Children’s Hospital Boston (Presented By: Tom Bartnikas, MD, PhD)

A central regulator of iron metabolism, the hepatic peptide hepcidin inhibits dietary iron absorption and macrophage iron recycling. To investigate the physiologic and pathophysiologic regulation of hepcidin expression, we have chosen the hypotransferrinemic (hpx) mouse as an experimental system. Hpx mice carry a splice site mutation in the transferrin gene that leads to extremely low levels of the serum iron-binding protein transferrin. While transferrin deficiency results in profound anemia, the paradoxically low level of hepcidin expression in these mice results in severe iron overload. We investigated the regulation of hepcidin expression by transferrin, erythropoietic activity and iron by treating these mice with a variety of pharamacologic modulators and then following parameters of iron metabolism. Repeated injections of transferrin into hpx mice normalized parameters of iron metabolism and led to increased hepcidin levels; cessation of transferrin injections led to a reversion to abnormal parameters of iron metabolism and low hepcidin levels. Treatment of hpx mice with the chemotherapeutic agent doxorubicin led to inhibition of erythropoietic activity yet did not result in increased hepcidin levels despite severe tissue iron overload. Taken together, these results suggest that the regulation of hepcidin expression is a dynamic process that responds rapidly to in vivo perturbations and that transferrin is central to such regulation.

Poster# 64 SERUM HEPCIDIN LEVELS IN PATIENTS WITH THALASSEMIA OR SICKLE CELL DISEASE: A CONFOUNDING RELATIONSHIP WITH TRANSFUSION CYCLE Patrick Walter, PhD¹, Zahra Pakbaz, MD¹, Elizabeta Nemeth, PhD², Roland Fischer, PhD³, Tomas Ganz, MD, PhD², Elliott Vichinsky, MD¹, Ward Hagar, MD¹, John Porter, MD4, Pat Evans, PhD4, Matt Hertz, BScs¹, Lynne Neumayr, MD¹ and Paul Harmatz, MD¹ ¹Children’s Hospital & Research Center Oakland; ²University of California Los Angeles; ³University Medical Center Hamburg-Eppendorf; 4University College London (Presented By: Patrick Walter)

Introduction: Hepcidin is a key regulator of systemic iron metabolism and is implicated in the pathogenesis of iron disorders. In normal homeostasis, iron loading and inflammation increase hepcidin levels, and erythropoietic activity and hypoxia decrease hepcidin. In the present study, we report the effect of transfusion cycle on hepcidin, as well as measurements of comparative bio-markers of erythropoietic drive in transfused and non-transfused beta-thalassemia and sickle cell disease (SCD) patients. Methods: Sixty-eight transfused (16 thalassemia, 7M, 19 yrs, Hb 10.4 g/dl and 16 SCD, 10M, 17 yrs, Hb 9.6 g/dl) and non-transfused (11 thalassemia, 9M, 22 yrs, Hb 9.0 g/dl and 16 SCD, 9M, 34 yrs, Hb 10.1 g/dl) thalassemia and sickle cell patients and 56 normal controls were enrolled after approval by IRB and informed consent. Liver iron concentration was measured by a SQUID Ferritometer® on thalassemia and SCD patients. Serum hepcidin was determined by competitive ELISA (Intrinsic LifeSciences, La Jolla, CA) which detects the mature bioactive hepcidin peptide. Results: Transfused patients had higher levels of serum hepcidin compared to controls (median 221 vs. 88 ng/ml respectively, p<0.001) with no significant difference between the two transfused groups (median serum hepcidin: thalassemia 168 vs. SCD 203 ng/ml). Serum hepcidin decreased significantly over the period of the transfusion cycle (r=-0.5, p=0.004). Serum hepcidin for non-transfused patients was significantly lower than controls (median 53 vs. 88 ng/ml respectively, p<0.001). In Spearman Rank correlations, serum hepcidin was negatively correlated with soluble transferrin receptor (Rs=-0.63, p=0.00001) and positively correlated with serum ferritin (Rs=0.56, p=0.0001), hemoglobin (Rs=0.45, p=0.001) and liver iron concentration (Rs=0.38, p=0.008). However, multivariate analysis that included these variables in a single model showed that only serum ferritin (p=0.003) and soluble transferrin receptor (p=0.024) were significant predictors of hepcidin. Discussion and Conclusions: Significantly higher levels of hepcidin occur in transfused compared to non-transfused patients with SCD or thalassemia. These elevated levels of hepcidin appear to be related to iron burden and erythropoietic drive. It is important that investigations of hepcidin in transfused patients take the stage of transfusion cycle into account. We suggest that the increased hepcidin levels in transfused patients may protect by decreasing dietary iron absorption and favoring retention of iron in the reticuloendothelial system.

Poster# 65 HAEM METABOLISM INHIBITORS AFFECT HEPCIDIN EXPRESSION IN MICE A.H. Laftah, MSc; PhD¹, A.T. McKie, Prof², S.K. Srai, Prof³ and R.J. Simpson, Dr² ¹King’s College London; ²Division of Nutrition, School of Biomedical & Health Sciences, King’s College London, London, UK; ³Department of Strutural & Molecular Biology, University College London, London, UK (Presented By: A.H. Laftah, MSc, PhD)

Our recent work has implicated 5-aminolaevulinic acid (ALA) as a possible regulator of intestinal iron absorption in conditions where haem metabolism is altered. As a step towards understanding how ALA could lead to altered iron absorption rates, we set out to measure the effect of haem metabolism inhibitors and ALA on hepcidin expression in mice in vivo. Modulators of haem metabolism had significant effects on hepcidin mRNA levels in livers of mice consistent with previously reported effects on iron absorption. Mice injected with 5-aminolaevulinic acid (ALA) and ferrochelatase inhibitorsgriseofulvin (GF) had a significant increase in hepatic hepcidin mRNA levels. Treatment of mice with ALA dehydratase inhibitor (succinylacetone (SA)) and haem Oxygenase-1 inhibitor (tin mesoporphyrin (SnMP)) decreased liver hepcidin mRNA levels. The phosphorylation of SMAD 1/5/8 and STAT3 in liver lysate expressed by western blot was increased in Gf- and decreased in SnMP-treated mice relative to SMAD1 and STAT3 as compared to control groups. Significant decreases of BMP6 & 9 mRNA expression levels were observed in mice injected with SnMP. However, mice fed Gf diet resulted in an increased mRNA expression levels of BMP6 and 9 as compared to controls. We hypothesized that changes in hepcidin expression level in mice treated with haem biosynthesis inhibitors was mediated by the signal mechanism through the bone morphogenetic protein (BMP) pathway, ultimately activating by phosphorylation the SMAD/ STAT3 complexes to alter hepcidin transcription.

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Poster# 66 CHARACTERISATION OF THE DIVALENT METAL ION BINDING PROPERTIES OF HEPCIDIN BY FTICR MASS SPECTROMETRY Chris Tselepis, Cleidiane Zampronio, Tariq Iqbal and Douglas Ward University of Birmingham (Presented By: Chris Tselepis)

Introduction: Hepcidin is now widely accepted to be the master regulator of iron metabolism and has rigorously been shown to mediate its biological effect through the relocalisation and degradation of the iron efflux protein ferroportin. Although it has previously been shown that copper can bind to hepcidin, most likely via the histidine at position 3, the binding has not been thoroughly characterised and it is not known if this influences biological activity. Furthermore, a separate study has suggested that iron can also bind to hepcidin. Thus the aims of this study were to determine if hepcidin-metal binding occurs and if this interaction is of relevance to biological activity. Methods: Electospray ionisation under non-denaturing conditions and fourier transform ion cyclotron resonance mass spectroscopy were employed to characterise complex formation between human hepcidin and the divalent ions of a range of transition metals (Cu2+, Ag2+, Zn2+, Ni2+, Co2+, Fe2+, at a range of concentrations (0-200mM) Human hepcidin-25 was synthesised and folded as previously described by Ward et al 2008. Results: Synthetic human hepcidin-25 binds one Cu2+ or Ag2+ ion with very high-affinity and up to 2 more ions with much lower affinity. The high affinity binding is not seen when the experiments are repeated with hepcidin-20 implying that the metal ion binding site is formed by the N-terminal 5 residues of hepcidin-25. Hepcidin-25 was also able to bind Zn2+ and Ni2+ with lower affinity but not Co2+, Fe2+ or Fe3+ ions. Interestingly, in hepcidin with the histidine at position 3 switched to asparagine; (the residue found at position 3 in rodents) copper binding was retained. Discussions: Our results suggest that the very high-affinity of Cu2+ for the N-terminal region of hepcidin might allow an interplay between copper and iron homeostasis. We are currently generating novel hepcidin-25 mutants with amino acid substitutions in the N-terminal copper/ferroportin binding region in an attempt test this hypothesis.

Poster# 67 PREPARATION AND INVESTIGATION OF FLUORESCENT HEPCIDIN IN MDCK CELLS AND MACROPHAGES Dareen Jaiash, BSc (Hons), MSc, A. Parmar, V. Marshal, G.O. Latunde-Dada, R.C. Hider and S. Bansal King’s College London (Presented By: Dareen Jaiash, BSc (Hons))

Introduction: Iron is an essential element for almost all living organisms. Both iron deficiency and accumulation in the body lead to serious diseases such as anaemia and haemachromatosis. Recently, the peptide, hepcidin has been demonstrated to be an essential regulator in iron metabolism and in the immune system. Hepcidin regulates plasma iron concentrations by suppression of intestinal iron absorption and recycling of iron from macrophages. Hepcidin binds to the sole iron exporter ferroportin which is located on the membrane of enterocytes and macrophages. This in turn leads to internalization and degradation of ferroportin. However, the mechanism of action of hepcidin is not fully understood. Aims: To synthesize a fluorescent hepcidin analog to investigate its function on ferroportin in vitro in two cell types; MDCK cells and J774 macrophages. Results / Discussion: Peptide synthesis was successfully carried out using Fmoc amino acids pre-activated using HBTU. Fmoc deprotection was carried out using piperidine in dimethylformamide. On completion of the synthesis the peptide was selectively labelled with the fluorophore. The fluorescent linear peptide was removed from the resin using trifluoroacetic acid and scavengers and then purified by HPLC. The linear fluorescent peptide was folded in redox buffers containing GSSG, GSH, EDTA and ABC at a concentration of 10 _g/mL over 60 h. The folded peptide was fractionated by HPLC. Ferroportin transfected kidney cells (MDCK) and mouse macrophages (J774) were treated with the peptide for various hours to determine its inhibitory role. Consequently, ferroportin of these cells was partly internalized from the membrane into the cytoplasm in comparison to ferroportin of cells incubated with serum free medium only. Conclusion: Hepcidin has been selectively labelled with both carboxyfluorescein and tetramethylrhodamine. The labelled peptides are biologically active and led to a promising internalization of ferroportin in both MDCK and J774 cells. These results suggest the successful synthesis and folding of fluorescent hepcidin which might be further developed as a molecular probe. This is the first report of a fluorescent hepcidin derivative.

Poster# 68 HEPCIDIN DEFICIENCY IN PATIENTS WITH CHRONIC HEPATITIS C Domenico Girelli, MD¹, Michela Pasino²,6, Julia B. Goodnough³, Elizabeta Nemeth³, Maria Guido4, Annalisa Castagna², Fabiana Busti², Natascia Campostrini², Federica Zaninotto², Nicola Martinelli², Italo Vantini5, Roberto Corrocher², Tomas Ganz³ and Giovanna Fattovich6 ¹University of Verona; ²Department of Clinical and Experimental Medicine, University of Verona, Verona, Italy; ³Department of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, California, USA; 4Department of Diagnostic Sciences & Special Therapies, University of Padova, Padova, Italy; 5Department of Biomedical and Surgical Sciences, University of Verona, Verona, Italy; 6Department of Surgical and Gastroenterological Sciences, University of Verona, Verona, Italy (Presented By: Domenico Girelli, MD)

Patients with chronic hepatitis C (CHC) often have increased liver iron, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is a major regulator of iron metabolism that inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (shepcidin). In this study we used a recently validated quantitative immunoassay for serum hepcidin [1] to evaluate correlations of hepcidin with disease status in CHC patients. We measured s-hepcidin levels in 81 untreated CHC patients and 57 sex- matched healthy controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score [2]. S-hepcidin was significantly lower in CHC than in controls (geometric means with 95% CIs: 33.7, 21.5-52.9 versus 90.9, 76.1-108.4 ng/ml, respectively; P<0.001). Correlation analyses showed strong positive associations of hepcidin levels with serum ferritin, both in controls and in CHC patients (r=0.741, P<0.001; and r = 0.718, P<0.001; respectively), and with total iron score (TIS) at histology (r=0.488, P<0.001). Similar significant correlations were found among hepcidin levels and either parenchymal (r=0.428, P<0.001) or mesenchymal (r=0.423, P<0.001) hepatic iron. Conversely, no significant correlation was found between hepcidin and either viral loading or s-interleukin-6 (IL-6) levels. Ferritin and TIS were the only independent significant predictors of hepcidin levels at a Multiple Linear Regression model (R2= 0.56, P<0.001; standardized b coefficients: 0.74 for ferritin, 0.64 for TIS, P<0.001 for both). After stratification for ferritin quartiles, s-hepcidin increased significantly across quartiles in both controls and CHC patients (chi for trend, P<0.001). However, in the latter group s-hepcidin was significantly lower than in controls for each corresponding quartile (ANOVA, P<0.001). These results, together with very recent studies in animal and cellular models [3,4], indicate that though hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by HCV is likely an important factor in liver iron accumulation in this condition. [1] Ganz T, et al. Immunoassay for Human Serum Hepcidin. Blood 2008; 112:4292-7. [2] Deugnier YM, et al. Liver pathology in genetic hemochromatosis: a review of 135 homozygous cases and their bioclinical correlations. Gastroenterology 1992;102:2050–2059. [3] Nishina S, et al. Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription. Gastroenterology 2008;134:226-238. [4] Miura K, et al. Hepatitis C virus-induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. Hepatology 2008; 48:1420-9.

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Poster# 69 EFFECTS OF RECOMBINANT HEPCIDIN, PROHEPCIDIN AND THEIR FLUORESCENT DERIVATIVES ON FERROPORTIN DEGRADATION Marie-Agnes Sari, PhD¹, Bruno Gagliardo¹, Nicole Kubat², Nicolas Desbenoit¹, Maryse Jaouen¹, Jean-Christophe Deschemin², Francois Canonne-Hergaux³, Isabelle Artaud¹ and Sophie Vaulont² ¹Université Paris Descartes, CNRS (UMR 8601), Paris, France; ²Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Inserm U 567Paris, France; ³CNRS, Institut de Chimie des Substances Naturelles, Gif-Sur-Yvette, France (Presented By: Marie-Agnes Sari, PhD)

Background / Aims: The iron-regulatory peptide hepcidin is synthesized in the liver in the form of a pre-pro-hormone of 84-aa maturated by proteolysis through a consensus furin cleavage site to generate the bioactive 25-aa peptide secreted in the circulation. This peptide was shown to regulate iron export from enterocytes and macrophages by binding to the membrane iron exporter, ferroportin, leading to its degradation. Whether pro-hepcidin could be secreted and reflect hepcidin levels remains an open question. The aim of this work was to develop methods to produce recombinant mouse and human hepcidin and prohepcidin and to measure their biological activity towards ferroportin. Methods: Human and mouse hepcidins were produced in E. coli as thioredoxin fusion proteins. Upon cleavage, peptides were purified by HPLC and characterized. Following the same strategy, mouse pro-hepcidin, either in the wild type form or a version mutated at the furin cleavage site, was produced and purified from E. coli. Hepcidins and pro-hepcidin were also modified using fluorescent reactive. The activity of these recombinant proteins was tested for their ability to decrease ferroportin levels in macrophage J774 cells. Results: Human and mouse hepcidins, purified after cleavage from thioredoxin, were properly folded and contained the expected 4 disulfide bridges without any need for renaturation or oxidation steps. Hepcidins were able to promote ferroportin degradation. Inactive aggregated forms of hepcidin, unrelated to disulfide bridge formation, were observed depending on purification and storage conditions. We showed that recombinant pro-hepcidin was able to induce ferroportin degradation, but only when processed by furin to generate the mature hepcidin-25 form. Indeed, pro-hepcidin activity was abolished in presence of furin inhibitor. Furthermore, the mutated version of pro- hepcidin (R35H) was completely inefficient in degrading cell membrane-associated ferroportin in macrophages. Finally, fluorescent hepcidin and pro-hepcidin derivatives (both recombinant and synthetic) were prepared and preliminary results on their biological activity indicate that the fluorescent hepcidins are also active. Conclusions: Our results demonstrate that recombinant hepcidins are capable to induce ferroportin degradation and that pro-hepcidin is devoid of biological activity, unless fully maturated by a furin-dependent process to yield the bioactive 25-aa peptide. The fluorescent derivatives are also able to promote ferroportin degradation, such molecules ought to be valuable tools to study biological activity of hepcidin and its derivatives.

Poster# 70 HIERARCHY BETWEEN ERYTHROPOIESIS AND IRON AS HEPCIDIN REGULATORS Jan Krijt, PhD¹, Martin Vokurka², Ludek Sefc², Tereza Hlobenova² and Emanuel Necas² ¹Charles University in Prague, First Faculty of Medicine, Institute of Pathophysiology; ²Charles University in Prague, First Faculty of Medicine, Institute of Pathophysiology and Center of Experimental Hematology (Presented By: Jan Krijt, PhD)

Introduction: Erythropoiesis and body iron status are known to control the expression of hepcidin. We have previously shown that the downregulation of murine Hamp1 expression by erythropoietin requires functional erythropoiesis. In the presented study we tried to further explore the functional hierarchy between erythropoiesis and iron overload as hepcidin gene regulators. Methods: To downregulate hepcidin expression, C57Bl/6N mice were fed an iron-deficient diet and bled (0.6 ml of blood) once weekly for 4 weeks. To suppress erythropoiesis, mice were hypertransfused with 0.7 ml of 70% red blood cell suspension in saline on 2 consecutive days before sacrifice. In experiments combining radiation and erythropoietin treatment, mice were irradiated (6Gy) on day 1; administered three daily doses of erythropoietin at 50 U/mouse on days 2, 3 and 4, and sacrificed on day 5. Iron was administered once at 300 mg/kg one week prior to bleeding or erythropoietin administration. Results: As previously demonstrated, administration of erythropoietin significantly decreased liver Hamp1 mRNA levels, while mice subjected to irradiation before erythropoietin treatment actually displayed increased Hamp1 mRNA content. The effect of erythropoietin on Hamp1 mRNA levels was also observed in hemojuvelin- knockout mice, indicating that functional hemojuvelin protein is not necessary for the erythropoietin-dependent downregulation of hepcidin expression. Repeated phlebotomies decreased liver Hamp1 mRNA content by two orders of magnitude. After irradiation or hypertransfusion of phlebotomised mice, Hamp1 expression returned to near normal levels, again demonstrating the crucial role of erythropoiesis in hepcidin gene downregulation. Although phlebotomy resulted in a very significant decrease of Hamp1 mRNA content, pretreatment with a high dose of iron prevented the phlebotomymediated downregulation of Hamp1 expression. The same results were obtained when iron was administered prior to erythropoietin treatment – iron administration prevented the expected erythropoietin-mediated decrease of Hamp1 mRNA levels. Discussion: We have previously demonstrated the importance of functional erythropoiesis for hepcidin downregulation by erythropoietin. In the presented study, we confirm the previous results in an alternative experimental setting using repeated phlebotomies and suppression of erythropoiesis by hypertransfusion. The obtained data again demonstrate a very significant effect of erythropoiesis on hepcidin downregulation. Interestingly, pretreatment with a high dose of iron prevented the downregulation of hepcidin by phlebotomies or erythropoietin. This suggests that macrophage iron stores play an important role in the control of hepcidin expression. When macrophage iron was present in excess, increased erythropoietic drive did not result in the expected hepcidin downregulation. In contrast, when excess iron was stored in hepatocytes, as is the case in animals with disrupted hemojuvelin gene, erythropoietin treatment or bleeding dramatically decreased hepcidin expression. Conclusion: In conclusion, the presented results show that repeated bleeding or erythropoietin administration lead to significant downregulation of hepcidin expression, which can be prevented by suppression of erythropoiesis or by pretreatment with a high dose of iron. In addition, the data also suggest that the iron-mediated and erythropoiesis-mediated pathways of hepcidin regulation are probably partially independent of each other. Supported by grants MSM 0021620806 and LC06044 from the Ministry of Education of the Czech Republic.

Poster# 71 HEPCIDIN EFFECT ON TRACE ELEMENTS IN SERUM OF WT AND HEPCIDIN1 KNOCKOUT MICE A.H. Laftah, MSc; PhD¹, A.H. Laftah, PhD², P. Masaratana, PhD², A.T. McKie, PhD, Prof², R.J. Simpson, PhD² and K. Raja, PhD³ ¹King’s College London; ²Division of Nutrition, School of Biomedical & Health Sciences, King’s College London, London, UK; ³Department of Clinical Biochemistry, King’s College Hospital, London, UK (Presented By: A.H. Laftah, MSc; PhD)

Hepcidin, a liver produced peptide, has recently been found not only to regulate intestinal iron absorption (via inverse alterations in duodenal iron transporter, Ireg1) (1) but also to influence reticuloendothelial iron release: these effects account for the marked hypoferraemia seen in inflammatory conditions (2,3) . As the acute phase response is associated with alterations in elements other then iron, it was of interest to ascertain whether these are associated with changes in hepcidin level. In this study, we therefore investigated changes in circulating levels of some important trace elements following hepcidin administration in mice. Male wild type (WT) and Hepcidin1 knockout (KO) mice were injected (i.p) with hepcidin (10µg/mouse) dissolved in sterile saline. Controls received an equivalent volume of saline. 4h post injection, serum and tissues were collected from the animals in metal-free tubes and analysed for iron (Fe), Copper (Cu), Zinc (Zn), and Selenium (Se) using flame/furnace atomic absorption spectrophotometry (Perkin-Elmer Model 3100/4100ZL) or Thermo XII ICP-MS. Our results showed that serum Fe and Zn were significantly reduced (p<0.05) with no change in Cu, and Se serum level in WT mice after hepcidin injection. Hepcidin1 KO mice showed no change in serum Fe and Cu level as compared to saline injected control group: however, Zn and Se level showed non-significant decreases. Conclusion: Absence of reduction in serum Fe level in hepcidin1 KO post hepcidin injection might be attributable to high iron level in the tissues and high transferrin saturation, and warrants further studies. The trend in the changes in trace element levels is similar to acute phase responses. The effects are however, less marked as compared to those seen after endotoxin treatment (4, 5). 1. Nemeth E, et al (2005) Science. 306:2090-3. 2. Frazer et al (2001) Gastroenterology. 123, 835-44 3. Nemeth et al (2004) J Clin Invest. 113, 1271-6 4. Park et al (2001) J Biol Chem. 276, 7806-10 5. Rofe et al (1996) Biochem J. 314, 793-7 117

Poster# 72 METABOLIC STEATOSIS AND ALCOHOL-LOADING REGULATE THE EXPRESSION OF TRANSFERRIN RECEPTOR 1 AND HEPCIDIN IN MICE LIVER Takaaki Ohtake, MD, Katsuya Ikuta, MD, PhD, Koji Sawada, MD, Masami Abe, MD, Takaaki Hosoki, MD, Shigeki Miyoshi, MD, Yasuaki Suzuki, MD, Katsunori Sasaki, PhD, Yoshihiro Torimoto, MD, PhD and Yutaka Kohgo, MD, PhD Asahikawa Medical College (Presented By: Takaaki Ohtake, MD)

Introduction: Both obesity and chronic alcohol intake frequently cause to hepatic iron overload and may contribute to hepatic carcinogenesis through production of free radicals. Previously, we reported that the expression of transferrin receptor 1 (TfR1), which mediates cellular transferrin-bound iron uptake, was increased in hepatocytes in patients with alcoholic liver disease (ALD). Recently it has been reported that alcohol metabolism dawnregulates the expression of hepcidin (Hepc) both in vivo mouse model and in vitro hepatoma cell lines. However the mechanism of iron overload in metabolic steatosis due to obesity, frequently merges with ALD, is still unclear. In this study, we investigated the gene expressions of TfR1 and Hepc in the liver and the serum concentration of mature Hepc peptide in obesity and/or alcohol-loaded mouse model. Methods: C57BL/6 and ob/ob mice were fed on a regular rodent chow diet. 10% ethanol was added in the drinking water to alcohol-loaded group for 7 days. Hematoxylin– eosin and oil red O stains were performed for histopathologic evaluation. Hepatic iron concentration was estimated by atomic absorption spectrophotometry method. The gene expressions of TfR1 and Hepc in the mice liver were evaluated by quantitative real-time RT-PCR method. The serum concentration of mature Hepc peptide was evaluated by Liquid chromatography/electrospray ionization tandem mass spectrometric technology (LC/ESI-MS/MS) (Murao, Rapid Commun. Mass Spectrom. 2007). Results: Total iron content in the whole liver in ob/ob mice was significantly higher than in wild type (p<0.005). The expression of TfR1 mRNA in the liver was significantly higher in ob/ob mice and/or alcohol-loaded mice than in control (p<0.05). The expression of Hepc mRNA in the liver was significantly lower in ob/ob mice and/or alcohol-loaded mice than in control (p<0.05). Furthermore, the serum concentrations of mature Hepc in ob/ob mice with water and with alcohol-loaded were significantly lower than in control (p<0.01). Conclusions: Our data suggest that both metabolic steatosis and alcohol load might up-regulate TfR1 expression and down-regulate Hepc expression respectively, following iron absorption from the small intestine and increased iron uptake into hepatocytes.

Poster# 73 HEPCIDIN MEASUREMENT BY QUANTITATIVE SELDI-TOF-MS IN HEMODIALYSIS PATIENTS: EVALUATION OF IRON STATUS DURING ERYTHROPOIESIS-STIMULATING AGENTS THERAPY Natascia Campostrini, PhD¹, Annalisa Castagna², Federica Zaninotto², Fabiana Busti², Nicola Tessitore³, Albino Poli4, Valeria Bedogna³, E. Melilli³, Oliviero Olivieri², Roberto Corrocher², Antonio Lupo³ and Domenico Girelli² ¹University of Verona; ²Department of Clinical and Experimental Medicine, University of Verona, Verona, Italy; ³Nephrology Division, University of Verona, Verona, Italy; 4Public Health Department, University of Verona, Verona, Italy (Presented By: Natascia Campostrini, PhD)

It has been postulated that Hepcidin (Hep), an iron-regulatory peptide linked to anemia and inflammation, may be a novel biomarker of iron status in patients with chronic kidney disease, based on its association with established indices of iron status. The aim of our study was to evaluate determinants of serum Hep and its role in detecting iron- deficient (ID) erythropoiesis in hemodialysis (HD) patients (pts) on maintenance erythropoiesis-stimulating agents (ESAs). In 45 stable, thrice-weekly (w) HD pts (19 females, 26 males, aged 62 ± 14 years) on maintenance i.v. ESAs (26 on Epoetin α 11103±6862 IU/w, 19 on Darbepoetin α 28.0±24.3 µg/w) we measured predialysis serum 25-Hep by a quantitative mass spectrometry-based assay, SELDI-TOF MS, using synthetic 24-hep as an internal standard [1], hemoglobin (Hb), reticulocyte count (ret), C-reactive protein (CRP), ferritin, Transferrin saturation (TSat), transferrin receptor (TfR), TfR Index (TfR/log ferritin), % of hypochromic RBC (%Hypo), CHr. All pts underwent an i.v. iron load (1 g NaFerric gluconate at the end of HD over 6 w period) to identify ID (Hb increase >1 g/dl) and iron-repleted (IR) pts (Hb increase <1 mg/dl). At the Generalized Linear Model analysis (including gender, age, Hb, ret, CRP, TfR, %Hypo, ferritin), 25-Hep was independently predicted by ferritin (β=0.55, p<0.001), %Hypo (β=0.36, p=0.008), TfR (β= -0.32, p=0.013) and ret (β=0.24, p=0.044) (R 0.781, p<0.001). Following the iron load, 15 pts were considered ID (Hb increase 1.8±0.6 g/dl) and 30 IR (Hb increase 0.4±0.3 g/dl). At ROC curve analysis, %Hypo (AUC 0.83, 95% CI 0.70-0.97, p<0.001), TfR (AUC 0.74, 95% CI 0.59-0.89, p=0.008) and TfR Index (AUC 0.73, 95% CI 0.57-0.89, p=0.01) were significant predictor of ID, while CHr (AUC 0.60, 95% CI 0.41-0.79), Ferritin (AUC 0.60, 95% CI 0.42-0.67), TSat (AUC 0.60, 95% CI 0.41-0.76) and Hep (AUC 0.54, 95% CI 0.34-0.72) were not. Our study confirms that Hep levels are predicted by indices of iron status and erythroid activity, suggesting that, in HD pts on maintenance ESAs, Hep is primarily determined by the iron stores, and also by the erythroid iron supply and utilization, and response to ESAs. Hep, however, could not predict Hb response to i.v. iron, indicating that it is not a first-line biomarker to monitor iron needs in clinical practice. [1] Swinkels DW, Girelli D, Laarakkers C, Kroot J, Campostrini N, Kemna EH, Tjalsma H. Advances in quantitative hepcidin measurements by time-offlight mass spectrometry. PLoS ONE. 2008 Jul 16;3(7):e2706.

Poster# 74 RADIOIMMUNOASSAY FOR HUMAN SERUM HEPCIDIN Nicolai Grebenchtikov, MSc, Anneke Geurts-Moespot, BSc, Joyce Kroot, MSc, Martin den Heijer, MD, PhD, Harold Tjalsma, PhD, Fred Sweep, PhD and Dorine Swinkels, MD, PhD Radboud Department of Clinical Chemistry 441, University Nijmegen Medical Centre (Presented By: Dorine Swinkels, MD, PhD)

The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Despite its promise as a biomarker, the availability of highthroughput hepcidin assays is still limited. Therefore, we developed and validated a RadioImmunoAssay (RIA) to measure hepcidin quantitatively in human serum. The hepcidin RIA exhibited a relatively low detection limit (0.02 µg/L), low imprecision (CV range 4.4-6.2%) and good parallelism between serial dilutions and a mean recovery of 95 % (range 81- 105%). Hepcidin levels of 18 patient samples showed an excellent correlation with our previous described quantitative time of flight mass spectrometry assay (TOF-MS, range 2.5 to 266.8 µg/L, r=0.92, P<0.0001). The RIA moreover, showed to be able to detect: i) differences in mean hepcidin levels between men (n=29) and women (n=35), ii) differences between individuals of different HFE-genotypes (n=60), and iii) daily increases in hepcidin levels (n=64). The assay is easy to perform, requires standard state-of-the-art laboratory equipment and many samples can be processed within one assay-run. In conclusion: we developed a RIA method for the measurement of hepcidin in human serum with high sensitivity and specificity that is useful in clinical studies to assess hepcidin levels in large cohorts of patients with iron metabolism disorders in a relatively cheap and high throughput manner.

Poster# 75 QUANTITATION OF HEPCIDIN Sukhi Bansal, PhD¹, John Halket, PhD¹, Adrian Bomford, MBBS, PhD², Robert Simpson, PhD², Nisha Vasavda, PhD³, Swee Lay Thein, MBBS PhD4 and Robert Hider, PhD5 ¹King’s College London, Pharmaceutical Sciences Division; ²King’s College London, Nutritional Sciences Division; ³King’s College London, Division of Gene and Cell Based Therapy; 4King’s College London, Department of Hematological Medicine; 5King’s College London Pharmaceutical Sciences Division (Presented By: Sukhi Bansal, PhD)

The reliable quantitation of hepcidin in biological matrices is widely sought after. There are a range of immunoassays available and recently mass spectrometric methods have been reported. With some immunoassays there is confussion as to whether the immunoglobin binds pro-hepcidin as well as hepcidin. Different mass spectrometric 118 methods have been utilised for hepcidin quantitation. The methods employ either external calibration or stable isotope labelled or truncated hepcidin internal standards. In this presentation we will critically review the currently available methods for the determination of hepcidin concentration in biological matrices. We will also present details relating to a new mass spectrometric method for the quantitation of hepcidin which utalises a stable isotope labelled internal standard and sample preparation using magnetic nanoparticles. The validation and the robustness of the method will be described. The application of the method for hepcidin quantitation in a range of clinical samples will be discussed. Acknowledgement: SB,AB and RS thank the Guys and St Thomas' Trust and RCH and RS thank the MRC for fanincial support

Poster# 76 CONTRIBUTION OF STAT3 AND SMAD4 PATHWAYS TO THE REGULATION OF HEPCIDIN BY OPPOSING STIMULI Hua Huang, Marco Constante, MSc, Antonio Layoun, BSc and Manuela M. Santos, PhD CRCHUM, University of Montreal (Presented By: Hua Huang)

Hepcidin, a key regulator of iron metabolism, is a small antimicrobial peptide produced by the liver that regulates intestinal iron absorption and iron recycling by macrophages. Several factors are known to regulate hepcidin expression, including iron levels (store regulator), inflammation (inflammatory regulator), anemia (erythroid regulator) and hypoxia(hypoxia regulator). More precisely, hepcidin is stimulated when iron stores increase and during inflammation, and conversely, is inhibited by anemia. Suppression of hepcidin during anemia may be mediated by tissue hypoxia or erythropoietin (EPO). In many pathological situations, such as in the anemia of chronic disease (ACD) and iron-loading anemias, several of these factors may be present concomitantly and may generate opposing signaling to regulate hepcidin expression. In the present study we addressed the question of dominance among the regulators of hepcidin expression. To assess whether EPO could affect the induction of hepcidin during inflammation, mice were first treated with EPO, and then with lipopolysaccharide (LPS) to induce systemic inflammation. Similarly, we tested whether EPO treatment could influence the induction of hepcidin by iron. For this purpose, mice were placed on an iron- supplemented diet containing 2.5% of carbonyl iron (CI), and then treated with EPO. We found that EPO treatment was able to significantly block hepcidin mRNA induction by both, LPS or dietary iron supplementation. However, EPO treatment did not affect IL-6 production in LPS injected mice, indicating that, in vivo, an IL-6-deficit is unlikely to explain EPO inhibition of hepcidin expression induced by LPS. Next, to test whether hypoxia could similarly block LPS or iron-mediated hepcidin induction, mice were injected with LPS or were kept on the ironsupplemented diet while exposed to normobaric hypoxia (10% O2). In contrast to what was observed with EPO, hypoxia was manifestly insufficient to block hepcidin induction by LPS or dietary iron supplementation. To understand the molecular mechanisms through which EPO achieves its effects, we assessed the activation level of two major signaling pathways known to be important for the transcriptional regulation of hepcidin. We found that EPO partially inhibited both the LPS-activated STAT3 pathway and the iron-mediated SMAD pathway. To determine whether the apparent dominance of the erythroid over the immune and stores regulator depends on the extent of erythropoietic activity, we treated mice with increasing amounts of EPO, alone or in combination with LPS injections or with the CI-supplemented diet. We found that the capacity of EPO to block hepcidin induction by LPS or CI was strongly dependent on the EPO dosage, with lower EPO dosages being significantly less effective in suppressing hepcidin expression compared to higher dosages. Finally, when both LPS and dietary iron treatments were combined to antagonize EPO-mediated hepcidin suppression, an additive effect of LPS and dietary iron counteracting EPO treatment was observed. In conclusion, hepcidin expression levels in the presence of opposing signaling are determined by the strength of the individual stimuli, rather than by an absolute hierarchy among signaling pathways. Our findings also suggest that, in vivo, erythropoietic drive can inhibit both inflammatory and ironsensing pathways, at least in part, via the suppression of STAT3 and SMAD4 signaling.

Poster# 77 REGULATION OF HEPCIDIN EXPRESSION DURING PHENYLHYDRAZINE-INDUCED HAEMOLYTIC ANAEMIA IN MICE Yemisi Latunde-Dada, PhD, Robert Simpson, PhD and Andrew McKie, PhD Kings College London (Presented By: Yemisi Latunde-Dada, PhD)

Haemolytic anaemia induced by phenylhydrazine (PHZ) is characterised by increased erythropoiesis, erythrophagocytosis, hepatic iron loading and enhanced intestinal absorption of iron, manifesting an overriding dominant effect of the erythroid over the stores regulation of iron homeostasis. Although hepcidin expression is reduced under this condition, the molecular mechanisms of the response remain unresolved. This current study was conducted to investigate the expression of genes involved in the transcriptional regulation of hepcidin expression in mice administered with PHZ. Genes of the TGFβ and the BMP/SMAD4 signal transduction cascades were analysed by quantitative PCR and Western blot. The emerging results show a trend towards reduced mRNA expression levels of hfe, tfr2, hjv and the BMPs. Expression of the mRNA of GDF15 and the transmembrane serine protease matriptase-2 (TMPRSS6) in mice treated with PHZ were reduced only slightly. In conclusion, erythropoietic inhibition of hepcidin synthesis during haemolytic anaemia induced by PHZ appears to be due to the suppression of the BMP/SMAD4 signal pathway.

Poster# 78 HFE MUTATIONS MODULATE THE EFFECT OF IRON STORES AND INFLAMMATION ON SERUM HEPCIDIN-25 IN CHRONIC HEMODIALYSIS PATIENTS Luca Valenti, MD, Domenico Girelli, Giovanni Francesco Valenti, Annalisa Castagna, Giovanna Como, Natascia Campostrini, Raffaela Rametta, Paola Dongiovanni, Piergiorgio Messa and Silvia Fargion University of Milano (Presented By: Luca Valenti, MD)

Increased serum hepcidin has been reported in patients receiving chronic hemodialysis treatment, and hypothesized to contribute to the alterations of iron metabolism of end stage renal disease. However, no quantitative assessment is available to date, the clinical determinants are still under definition, and the role of genetic factors, namely HFE gene mutations, has not yet been evaluated. Aim of this study was to quantitatively assess serum hepcidin-25 in hemodialysis patients vs. controls, and analyze the relationship between hepcidin, iron indices, HFE genotype, and erythropoietic parameters. We considered 65 hemodialysis patients and 57 healthy controls. Hepcidin-25 was evaluated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, HFE genotype by restriction analysis. Serum hepcidin-25 was higher in hemodialysis patients compared to controls (p=0.0021). In patients, hepcidin-25 correlated positively with ferritin (p=0.0001), and C reactive protein (p=0.029), and negatively with serum iron after adjustment for confounders (p=0.014). Hepcidin/ferritin ratio was lower in patients with (n=25) than in those without (n=40) HFE mutations (p=0.045). At multivariate analysis, hepcidin-25 was independently associated with ferritin (p<0.0001), and HFE status (p=0.018). In a subgroup of 22 “stable” patients, i.e. with Hb levels on target, normal CRP levels, and absence of complications for at least 1-year, hepcidin-25 was negatively correlated with Hb levels independently of confounders (p=0.0011). In conclusion, serum hepcidin-25 is increased in hemodialysis patients, regulated by iron stores and inflammation, and relatively reduced in subjects carrying frequent HFE mutations. Hepcidin-25 may contribute to the pathogenesis of anemia by decreasing iron availability.

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Poster# 79 HEPCIDIN RESPONSE TO ACUTE ORAL IRON AND CHRONIC IRON OVERLOAD IN DYSMETABOLIC HEPATIC IRON OVERLOAD SYNDROME Paola Trombini, MD¹, Valentina Paolini, Sara Pelucchi², Raffaella Mariani, Alessandra Salvioni, Domenico Girelli³, Clara Camaschella4, Elizabeta Nemeth5, Tomas Ganz5 and Alberto Piperno ¹Clinical Medicine, San Gerardo Hospital; ²University of Milano-Bicocca, Monza, Italy; ³University of Verona, Italy; 4San Raffaele University, Milano, Italy; 5Departments of Medicine and Pathology, UCLA, USA (Presented By: Paola Trombini, MD)

Dysmetabolic Hepatic Iron Overload Syndrome (DHIOS) is the most frequent iron disorder but its pathogenesis is still unclear. Hepcidin is the key inhibitory regulator of iron homeostasis. A blunted response of urinary hepcidin to oral iron has been recently described in HFE-hemochromatosis (HH). Our aim was to explore hepcidin response to oral iron and the effect of iron overload and depletion on hepcidin levels in patients with DHIOS. Presence of iron overload was established by liver biopsy in patients with DHIOS. We analyzed urinary hepcidin at baseline and 24 hours after a single 65mg dose of oral iron in 24 DHIOS patients at diagnosis. Fifteen of them were also studied after iron depletion. Data were compared to those of 34 HFE-HH patients (C282Y/C282Y and C282Y/H63D) at diagnosis, 62 HFE-HH at iron depletion and 23 healthy controls [Blood 2007;110:4096-4100]. At iron depletion, oral iron challenge was done at a minimal time delay of 30 days from the last phlebotomy. Urinary hepcidin was measured according to Nemeth [J Clin Invest 2004;113:1271-76]. At diagnosis, basal hepcidin in DHIOS was markedly higher than in healthy control and C282Y/C282Y (both p<0.0001), and only slightly increased than in C282Y/H63D (p=0.04). At iron depletion, basal hepcidin in DHIOS significantly decreased, but was still higher than in HFE-HH (especially compared to C282Y/C282Y). It did not differ to healthy controls who had comparable iron indices. One day after iron challenge, a significant hepcidin increase (10ng/mg creatinine) was observed in 55% DHIOS vs 74% healthy controls (p=ns), vs 32% C282Y/H63D (p=ns), and vs 15% C282Y/C282Y (p=0.02), independently to iron status. Basal hepcidin and its response to oral iron did not correlate with any parameter of the metabolic syndrome, hepatic steatosis, liver tests, PCR, age and sex. In DHIOS, hepcidin response to chronic iron overload is more adequate than in HFE-HH. The normalization of hepcidin levels at iron depletion suggests that hepcidin is not innately low in DHIOS, differently to HFE-HH. The hepcidin response at 24 hours suggests that acute iron sensing is not significantly altered in DHIOS and that other mechanisms are involved in the pathogenesis of iron overload. Since the maximal urinary response to acute oral iron may occur earlier than 24 hours [Blood 2007;110:2182- 2189], this result should be taken with caution and a more detailed hepcidin time course is needed.

Poster# 80 THE IRON-REGULATING HORMONE HEPCIDIN IS EXPRESSED IN BLADDER CANCER TISSUE AND CORRELATES TO POOR PATIENT SURVIVAL Maciej Bogdan Maniecki, MHSc¹, Benedicte Parm Ulhøi, MD², Henrik Schmidt, MD, DMSc³, Lars Dyrskjøt, MSc, PhD4, Torben Falck Ørntoft, MD, DMSc4, Søren Kragh Moestrup, MD, DMSc5 and Holger Jon Møller, MD, PhD¹ ¹Department of Clinical Biochemistry, Århus Sygehus, Aarhus University Hospital; ²Institute of Pathology, Århus Sygehus, Aarhus University Hospital; ³Cancer Immunotherapy Group, Department of Oncology, Aarhus University Hospital; 4Molecular Diagnostic Laboratory, Department of Molecular Medicine, Aarhus University Hospital, Skejby; 5Institute of Medical Biochemistry, Aarhus University (Presented By: Maciej Bogdan Maniecki, MHSc)

Introduction: Hepcidin is a recently discovered liver-produced peptide hormone which is responsible for the maintenance of iron homeostasis. Hepcidin controls extracellular iron levels by regulating the absorption in the duodenum, recycling by macrophages, and release from stores. Hepcidin binds to the cell membrane iron exporter ferroportin-1 and induces internalization and degradation resulting in a decrease of serum iron. The expression of hepcidin is downregulated in response to low body iron stores, anemia or hypoxia. Conversely, hepcidin expression is induced by iron overload or during inflammation by the pro-inflammatory cytokine IL-6. The upregulation of hepcidin expression by inflammatory signals has been strongly linked to the “anemia of chronic disease” (ACD). Recently, hepcidin expression in macrophages has been described, but the function is still unclear. Anemia is a frequent manifestation of almost all cancers. However, at present there are no studies addressing whether hepcidin expression induces anemia in bladder cancer or whether hepcidin plays a role in cancer pathogenesis. Materials and Methods: Hepcidin (HAMP), ferroportin-1 (FPN1), and IL-6 mRNA expression levels were measured using quantitative real-time polymerase chain reaction in bladder cancer biopsy samples obtained from 88 patients followed for a median of 39 months (range, 1-116 months). The levels of mRNA were normalized against the β-actin (ACTB) mRNA content. Results: Hepcidin and ferroportin-1 mRNA was expressed in all bladder cancer biopsies. In invasive tumors (T2-T4), the expression of hepcidin was significantly higher compared with superficial tumors (Ta; p<0.01). Furthermore, hepcidin expression positively correlated with tumor aggressiveness (p<0.01), whereas ferroportin-1 negatively correlated with tumor aggressiveness (p<0.001). No correlation between hepcidin and ferroportin-1 expression was observed and the expression of IL-6 was not elevated in advanced disease nor correlated with hepcidin expression levels. Hepcidin mRNA expression in anemic bladder cancer patients (median: 1.560; range: 0.185- 9.747) was significantly higher compared with non-anemic patients (median: 0.627; range: 0.0337-31.980), p<0.05. Notably, we found that high hepcidin expression was significantly associated with poor survival in bladder cancer patients (p<0.05). Additionally, a highly significant correlation was observed between combined high expression of hepcidin/low expression of ferroportin-1 and poor patient survival rate (p<0.005). Conclusion: We demonstrate for the first time that hepcidin is expressed in bladder cancer tumors, probably due to infiltrating macrophages. Furthermore, we show that high hepcidin expression is associated with poor patient survival. Our data indicate that hepcidin is up-regulated in bladder cancer tissue by an IL-6 independent pathway. In addition, our data show that hepcidin expression in bladder cancer tissue is associated with anemia. The triggering factor for the elevated expression of hepcidin in bladder cancer tissue is unclear, however, hepcidin might represent a novel pro-oncogenic signaling mechanism.

Poster# 81 FIRST DUTCH PATIENT WITH MAPTRIPTASE-2 MUTATION LEADING TO IRON-REFRACTORY IRON DEFICIENT ANEMIA Marloes Cuijpers, MD, Erwin Wiegerinck, Theo de Witte, Prof and Dorine Swinkels, MD, PhD, Prof Radboud University Nijmegen Medical Centre (Presented By: Marloes Cuijpers, MD)

Introduction: Several regulation mechanisms for hepcidin synthesis have been discovered the last few years, including matriptase-2, a serine protease, which inhibits hepatocyte hepcidin synthesis by cleaving hemojuvelin, a regulator of hepcidin, on the plasma membrane. These new insights may contribute to resolve unexplained cases of anemia. Case: A female patient, born from non-consanguineous parents in 1968, attended our outpatient clinic during her first pregnancy at 30 years of age because of a microcytic anemia (Hb 4.8 mmol/L; MCV 72 fL) in the presence of iron deficiency. From her childhood onward, she was diagnosed and treated with oral iron supplements without any result. After her second pregnancy in 2002, the iron deficient anemia persisted (Hb 5.8 mmol/L; MCV 66 fL; serum iron 2 µmol/L; transferrin saturation 4%; serum ferritin 34 µmol/L). Subsequently, an extended analysis was made, excluding causes of iron deficiency, including blood loss of the gastrointestinal tract, decreased iron absorption due to celiac disease, and bone marrow pathology. Finally, she was treated with intravenous iron followed by a partial response: the anemia was corrected (Hb 7.6 mmol/L; MCV 83 fL), and serum ferritin level increased (428 µmol/L), but the serum iron level and transferrin saturation remained low (8 µmol/L and 19% respectively). Iron kinetic studies, using oral and intravenous administered 59Fe, showed a decreased absorption and a fast distribution, with normal erythrocyte-iron incorporation, respectively, suggesting an iron absorption disorder. Hepcidin concentration was quantified in the urine by SELDI-TOF mass spectrometry. In contrary to what is expected in iron deficient anemia, the hepcidin level in the urine was in high-normal range, indicative of a primary defect in the regulation of hepcidin. Upon DNA-sequencing, mutations in the genes coding for hepcidin-regulating proteins (HFE, TfR2, hemojuvelin, hepcidin) were absent, but a homozygous p.Cys702Phe mutation in the recently discovered TMPRSS6-gene, encoding for matriptase-2, was found. Modeling studies reveal that this novel mutation leads to the loss of a disulfide bond and thereby is expected to change the local structure, disturbing the intermolecular contacts with domains of other proteins.

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Conclusion: We describe a patient with an iron deficient anemia refractory to oral iron treatment caused by a homozygous mutation in the recently discovered TMPRSS6- encoded protein matriptase-2, leading to inadequately elevated hepcidin levels. This defect might be responsible for unsolved cases of ironrefractory iron deficient anemia. The optimal treatment strategy of these patients appears to consist of intravenous administration of iron.

Poster# 82 COMBINED DELETION OF HFE AND TRANSFERRIN RECEPTOR 2 IN MICE LEADS TO MARKED DYSREGULATION OF HEPCIDIN AND IRON OVERLOAD Nathan Subramaniam, PhD, Lesa Summerville, Emily Crampton, David Frazer, Greg Anderson and Daniel Wallace Queensland Institute of Medical Research (Presented By: Nathan Subramaniam, PhD)

Hepcidin is a central regulator of body iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary haemochromatosis and regulate the hepatic expression of hepcidin in response to iron. However, whether they act through the same or parallel pathways is unclear. Mutations in hepcidin (HAMP) and hemojuvelin (HJV) lead to a severe form of early onset HH termed juvenile haemochromatosis (JH). There has been one report of two siblings who displayed clinical symptoms characteristic of JH but did not carry mutations in either HAMP or HJV but instead had a combination of mutations in both the HFE and TFR2 genes. This observation suggests that HFE and TFR2 are complementary regulators of hepcidin expression. While loss of either HFE or TFR2 results in an appreciable iron overload, with onset normally in adulthood, the loss of both would be predicted to lead to a combined loss of regulation, akin to that observed in JH. To understand how Hfe and Tfr2 regulate hepcidin expression and in turn iron homeostasis, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe-null(Hfe-/-) and Tfr2-null (Tfr2-/-)mice on a genetically identical C57BL/6 background. Tissue and serum from 5-week old wild-type, single and double null male mice were analysed. Serum transferrin saturation and hepatic iron concentrations were determined. Iron was detected in formalin-fixed liver sections using the Perls’ Prussian blue staining. The expression of iron-related mRNA transcripts in liver and duodenum was analysed by real-time PCR. Levels of the iron-related proteins Tfr1, Tfr2, ferritin and prohepcidin in the liver were analysed by immunoblotting. Hfe-/-/Tfr2-/- mice had more severe iron loading than mice lacking either Hfe or Tfr2 at the same age; Tfr2-/- mice had a greater iron burden than Hfe-/- mice. Iron loading in the livers of Hfe-/- mice had a predominantly periportal distribution, the extent of iron loading expanding to include more pericentral regions in the Tfr2-/- and Hfe-/- /Tfr2-/- mice. Expression of iron transporters was increased in the duodenum of Hfe-/-/Tfr2-/- mice. Hepcidin expression in relation to iron stores was reduced in the Hfe-/- mice, with significantly lower values in the Tfr2-/- mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. The phenotypic comparison of Hfe-/-, Tfr2-/- and Hfe-/-/Tfr2-/- mice on identical genetic backgrounds has demonstrated for the first time that loss of Tfr2 results in more severe iron loading than loss of Hfe. When extrapolated to humans our results suggest that TFR2-HH is a more severe iron loading disorder than HFE-HH. Iron loading is enhanced further by the loss of both Hfe and Tfr2 resulting in a more severe early onset haemochromatosis, similar in phenotype to JH caused by mutations in HJV or HAMP. Analysis of Hfe-/-/Tfr2-/-mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways, and contributes to our understanding of iron metabolism and the pathogenesis of iron loading disorders.

Poster# 83 DISRUPTION OF BOTH HFE AND TFR2 CAUSES MORE SEVERE HEPATIC IRON OVERLOAD IN HEREDITARY HAEMOCHROMATOSIS Roheeth Delima, BSc (Hons), Anita Chua, Carly Herbison, Ross M. Graham, John Olynyk and Debbie Trinder University of Western Australia (Presented By: Roheeth Delima, BSc (Hons))

Hereditary haemochromatosis (HH) is an autosomal recessive disorder of iron metabolism that causes iron overload. HH type 1 is usually caused by a homozygous C282Y mutation in the HFE gene and HH type 3, a rare form of HH, is caused by mutations in the transferrin receptor (TFR) 2 gene. HFE and TFR2 are hypothesized to control iron homeostasis by regulating synthesis of the liver peptide hepcidin (HAMP), and its receptor ferroportin (FPN) to regulate iron absorption, recycling and storage. The aim of this study was to examine the effects of disruptions in both Hfe and Tfr2 genes on iron homeostasis using mouse models of HH. Tissues and plasma were collected from 10 week old female Hfe knockout (Hfe-KO), Tfr2 Y245X mutant (Tfr2mut) and double mutant (Hfe- KOxTfr2mut) mice, as well as wild-type mice and iron-loaded wild-type mice. All mice were on a homogeneous AKR genetic background. They were all fed a normal diet except the iron-loaded mice which were fed a diet supplemented with 2% iron for 3 weeks prior to sacrifice. Iron status was assessed by plasma iron and transferrin saturation, as well as liver and spleen non-haem iron levels. Liver iron transporters (Tfr1, Tfr2, Fpn, Zip14) and regulator (Hamp1) gene expression was measured by real-time PCR. Hfe-KOxTfr2mut mice had elevated plasma iron levels and transferrin saturation compared with Hfe-KO, Tfr2mut and iron loaded wild-type mice. Liver non-haem iron levels in Hfe-KOxTfr2mut mice were significantly increased by 50% compared with Hfe-KO, TfR2mut, and iron-loaded wild-type mice (p<0.01), which in turn were 3-fold higher than non-iron loaded wild-type mice (p<0.0001) (Hfe-KOxTfr2mut, 27±1; Hfe-KO, 17±1; Tfr2mut, 18±2; iron-loaded wild-type, 19±1; non-iron loaded wild-type, 6±1 µmol iron/g wet wt liver; mean ± SEM; n=6-15). Splenic non-haem iron levels in Hfe- KOxTfr2mut, Hfe-KO and Tfr2mut mice were similar and these were significantly lower by approximately 50% and 60% compared with non-iron loaded and iron-loaded wild-type mice, respectively. Liver Tfr1 mRNA expression in Hfe- KOxTfr2mut, Hfe-KO and Tfr2mut mice was similar and reduced by approximately 40% compared with wild-type mice. Tfr2 expression in Hfe-KOxTfr2mut and Tfr2mutmice was reduced significantly compared with all other types of mice. Fpn expression was similar in all types of mice. Hamp1 expression in Hfe-KOxTfr2mut mice was reduced markedly and was 3% of that found in Hfe-KO and Tfr2mut and 1% of that in wild-type mice. Zip14 (isoform A) was decreased by 60% in the livers from Hfe- KOxTfr2mut mice and by 40% in Hfe-KO, Tfr2mut and iron-loaded wild-type mice compared to non-loaded wild-type mice. We conclude that disruption of both Hfe and Tfr2 in mice causes a more severe form of HH with increased plasma iron parameters and enhanced liver iron levels when compared with mice with impaired Hfe or Tfr2 alone. Hepcidin expression is negligible in the absence of both Hfe and Tfr2 suggesting that Hfe and Tfr2 are acting through parallel signalling pathways to regulate hepcidin expression and iron homeostasis.

Poster# 84 EXTRA-HEPATIC HFE FUNCTIONS MAY BE RESPONSIBLE FOR IRON OVERLOAD IN THE HEART AND ALTERATION WITHIN THE ERYTHRON Maja Vujic Spasic, PhD¹, Matthias Hentze, Prof MD² and Martina Muckenthaler, Prof PhD¹ ¹University Hospital of Heidelberg; ²EMBL, Heidelberg, Germany (Presented By: Maja Vujic Spasic, PhD)

Systemic iron homeostasis is disrupted in the common iron overload disorder hereditary hemochromatosis (HH), which is hallmarked by increased intestinal iron absorption, hyperferremia and tissue iron overload. Clinical manifestations include liver cirrhosis and carcinoma, diabetes, heart failure, hypermelanotic pigmentation of the skin, arthritis and hypogonadism. Most frequently, HH is caused by mutations in the ubiquitously expressed Hfe gene, which codes for a MHC class I-like molecule. We recently generated and analyzed conditional Hfe knock-out mice, where Hfe is deleted in duodenal enterocytes, macrophages or hepatocytes, respectively. Analysis of systemic and cellular changes in iron parameters in response to Hfe deficiency unambiguously revealed that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis and hepcidin expression. To investigate whether extra-hepatic functions of the broadly expressed Hfe are responsible to maintain iron-related and non-related physiological functions we have systematically investigated (i) iron levels in extra-hepatic tissues (spleen, heart, kidney, lungs, duodenum, brain) and (ii) haematological parameters in constitutive and tissue-specific Hfe-deficient mice. Our results show that in addition to the liver, Hfe-deficient mice accumulate iron in the heart, kidney and brain, while the duodenum and the lungs are relatively iron spared. Iron accumulation in the kidneys and brain is explained by the excess of iron absorbed as a consequence of hepatic Hfe-deficiency. Unexpectedly, iron-loading of the heart is not observed in the mouse line deficient for hepatic Hfe suggesting that Hfe in other tissues than the liver, possibly the heart, is required to maintain tissue iron homeostasis.

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We further observe diminished red blood cell counts (RBC), as well as macrocytic and hyperchromic erythr cytes in Hfe-deficient mice. The alterations in erythrocyte size (MCV) and haemoglobin content (MCH) can be rescued by hepatic Hfe expression suggesting that iron overload and possibly hepcidin expression may contribute to these observations. Interestingly, diminished RBC counts are only observed in mice with global Hfe-deficiency and not in mice with hepatocyte-specific Hfe ablation, suggestive of an extra-hepatic function of Hfe possibly in erythroid precursor cells. In summary our results suggest that, in addition to the liver, Hfe exerts functions in other tissues like the heart and the erythron and that extra-hepatic functions of Hfe may play a role in some of the clinical manifestations associated with hereditary hemochromatosis.

Poster# 85 HFE POLYMORPHISMS AFFECT CHOLESTEROL METABOLISM: INSIGHTS INTO NEURODEGENERATIVE DISEASES Fatima Ali-Rahmani, Sang Lee, James Connor and Cara-Lynn Schengrund Pennsylvania State University (Presented By: James Connor)

Disruption of iron homeostasis and of cholesterol metabolism has been implicated in several neurodegenerative diseases including Alzheimer disease (AD) and motor neuron disease. The appearance of HFE gene variants, especially the H63D variant, has been under investigation as a risk factor for neurodegenerative diseases and mutations in the APOE gene (cholesterol transporter) have also been linked to these diseases. The goal of this study is to determine whether the presence of the HFE polymorphisms affects cholesterol metabolism. Because iron is a required cofactor for 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR) -- the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis -- the hypothesis that changes in iron levels could affect cholesterol metabolism is logical. To begin to explore the link between iron and cholesterol and neurodegenerative diseases, we examined a cellular model that expressed various polymorphisms of the HFE gene. To test the effect of increased iron accumulation, as seen in AD brains, on membrane associated cholesterol, we are using SH-SY5Y human neuroblastoma cells that stably express either wild type (WT) or the H63D or C282Y variant HFE protein. Analysis of cholesterol content indicated that cells expressing H63D variant had <50% of the cholesterol content found in WT HFE expressing cells. In contrast, C282Y HFE variant cells had ~10% more cholesterol than WT cells. Targeted gene array (lipoprotein signaling and cholesterol metabolism) analysis was used to determine whether alterations in cholesterol reflected changes in the expression of genes involved in either its transport or metabolism. Interestingly, expression of a number of genes encoding proteins involved in cholesterol transport was altered in cells expressing HFE variants. To determine whether the observed changes affect localization of HFE within the plasma membrane, studies to determine whether HFE associates with lipid rafts (lipid domains enriched in cholesterol), are in progress. While a direct connection between HFE and cholesterol has not been shown, the results indicate that specific mutations in HFE which are highly prevalent in AD and are known to cause increased iron accumulation, are also related to changes in cholesterol metabolism.

Poster# 86 HFE POLYMORPHISMS AND DRUG RESISTANCE IN CANCER Sang Lee, PhD, Siying Liu, Becky Slagle-Webb, Elana Farace, PhD, Jonas Sheehan, MD and James Connor, PhD Penn State College of Medicine (Presented By: Sang Lee, PhD)

HFE polymorphic alleles have been associated with a variety of cancers such as hepatocellular carcinoma (HCC), breast cancer, colorectal cancer, childhood acute lymphoblastic leukemia, and gliomas. Given the differences in cell phenotypes when expressing the different HFE gene variants we hypothesized that there are differences in sensitivity to treatment based on HFE genotype. Therefore, we determined the association of drug resistance and HFE polymorphisms using a human neuroblastoma cell line (SH-SY5Y) and a number of human glioma cell lines. In the HFE variants stably transfected neuroblastoma cells, cells carrying the C282Y allelic variant were significantly more resistant to the chemotherapeutic agent temozolomide (currently the standard of care for brain tumors) than the same cell line carrying wildtype or H63D variant of HFE. The resistance to temozolomide was also found in commercially available human astrocytoma cell lines with the C282Y allele. In addition to temozolomide, the C282Y allele expressing glioma cells were also resistant to geldanamycin. Geldanamycin was chosen for analysis because it has a different mechanism of action on the tumor cells than temozolomide. Resistance to chemotoxins was also observed in the H63D allele expressing astrocytoma cells compared to wildtype HFE cells, but the resistance is less than seen in the C282Y cells. To begin to discover the drug resistance mechanism of the C282Y HFE variant cells, we performed functional gene arrays in HFE stably transfected neuroblastoma cells. Based on the gene array analysis, we identified p16ink4a (cyclin dependent kinase inhibitor 2A) gene for further analysis because the difference in expression of this gene was significantly increased in the C282Y cells compared to the wildtype cell lines. Consistent with the gene expression, p16INK4A protein expression was elevated in association with the C282Y allele relative to wildtype HFE. C282Y expressing astrocytoma cells also expressed high levels of p16INK4A protein. Therefore, we further determined the role of p16INK4A in temozolomide resistant and C282Y expressing cells using p16ink4a siRNA. Decreasing the expression of p16INK4A protein following transfection with the siRNA is associated with increased sensitivity of the C282Y expressing cells to temozolomide. At present, we are expanding other possible drug resistance mechanisms from the gene array data in tetracycline inducible cells. The clinical relevance of these observations was examined by performing a survival analysis on brain tumor patients. The survival analysis of brain tumor patients indicated that C282Y polymorphism showed a trend (p<.07) towards being an independent predictor of shorter length of survival even after age, grade, and baseline functional status were accounted for. The clinical sample size continues to be expanded. In conclusion, these data suggest the sensitivity to available treatment strategies can be affected by the HFE genotype and the mechanism of resistance appears to be via expression of levels of p16INK4A. (Temozolomide used in this study was a gift from Schering Plough)

Poster# 87 DIFFERENT EFFECTS ON IRON CONTENT IN THE LIVER AND SPLEEN OF HFE-KO MICE AFTER HEPATIC INJECTION OF A LENTIVIRAL VECTOR BEARING THE HFE GENE Pedro Ramos¹, Sara Gardenghi, PhD1, Ella Guy1, Nan Chan1, Antonia Follenzi, PhD², Robert W Grady, PhD1, Maria de Sousa, MD, PhD3 and Stefano Rivella, PhD1 ¹Weill Cornell Medical College, New York; ²Albert Einstein, New York; 3IBMC, Porto (Presented By: Pedro Ramos)

Subjects homozygous for a mutation (C282Y) in the HFE gene often suffer from type-1 hereditary hemochromatosis (HH), showing low levels of urinary hepcidin and increased iron absorption. Studies in vitro suggest that HFE controls cellular iron intake and is involved in regulation of hepcidin expression. Our goal was to study changes in iron metabolism, organ iron distribution and gene expression in an HH mouse model (Hfe-KO) after incorporation of Hfe into the liver using a lentiviral system. To validate our approach, livers of 3-day-old pups were infected with vectors bearing green fluorescent protein (GFP). Analysis 90 days after injection revealed long-term expression of GFP in hepatocytes. We then generated lentiviral vectors expressing Hfe under control of the human HFE promoter (HFE-Hfe) or the hepatocyte-specific transthyretin (Ttr) promoter (Ttr-Hfe). These promoters lead to expression of the transgene at different levels in hepatic cell lines, the Ttr promoter being an order of magnitude stronger. Hfe-KO pups (N=5) were infected with the HFE-Hfe vector, the mice being designated Hfe-HHW. These animals were analyzed for organ and serum iron content and gene expression. Mice injected with the Ttr-Hfe vector are currently being studied. Two months post-injection, the level of Hfe expression in the liver of Hfe-HHW animals was approximately one-third that of wt mice, expression in the spleen being negligible. The vector copy number was 1.3±0.4. Hepcidin expression was increased approximately 1.5 fold compared to Hfe-KO controls. Hfe-HHW mice exhibited a marked decrease in liver iron content and transferrin saturation compared to Hfe-KO mice, but did not reach the levels observed in wt animals. Their respective liver irons were 238±24, 367±51 and 108±5 ug and the transferrin saturations 71±5%, 86±4% and 69±1%. In contrast, iron levels in the spleen of Hfe-HHW mice were increased compared to both Hfe-KO and wt control mice (95±9, 36±3 and 36±12 ug of iron, respectively). A lentiviral vector bearing an Hfe-GFP fusion protein is being utilized to evaluate the distribution of iron in the liver relative to Hfe expression.

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Comparing Hfe-HHW mice with Hfe-KO and wt control animals, we observed that the amount of splenic iron more than doubled. This could be due to retention of iron in macrophages as a consequence of increased hepcidin expression. Our data are also compatible with the notion that lack of Hfe increases hepatic iron intake, while its expression limits this process. Since iron absorption was still elevated in Hfe-HHW mice, shunting of iron to the spleen could also be explained by decreased hepatic iron intake. In summary, we developed a new tool for studying the role of Hfe in iron metabolism in vivo. This tool fosters increased expression of hepcidin in the liver, partially reversing the hemochromatosis phenotype. This approach might further help us to dissect the role of Hfe in iron metabolism.

Poster# 88 HFE IS ESSENTIAL TO INDUCE HEPCIDIN AND TO DEVELOP HYPOFERREMIA IN RESPONSE TO LOW DOSES OF LPS Maja Vujic Spasic, PhD, Richard Sparla, Dipl, Judit Kiss, PhD, Jens Stolte, Dipl, Birgit Rathkolb, PhD, Matthias Hentze, PhD and Martina Muckenthaler, PhD University Hospital of Heidelberg (Presented By: Maja Vujic Spasic, PhD)

Hereditary hemochromatosis (HH) is mainly caused by mutations in the HFE/Hfe gene, which codes for a MHC class I-like molecule. HH patients and Hfe-deficient mice accumulate iron because they fail to express adequate levels of the hepatic peptide hepcidin, which negatively regulates duodenal iron absorption and iron release from macrophages. Hepcidin is also induced during the acute phase response and is thought to represent a causative factor in the pathogenesis of the “Anemias of Chronic Disease (ACD)”. Our previous findings demonstrated that Hfe-deficient mice fail to express increased hepcidin and decreased serum iron levels after LPS injection (Roy et al., Nature Genetics, 36:481-485, 2004), a finding that was not supported by several other investigators using different stimuli and time points of analysis. We have now systematically analyzed WT and Hfe-/- mice 1,5h, 8h and 24h after applying 5µg or 500µg of LPS. We show a lack of hypoferremia in Hfe-/- mice injected with 5µg of LPS at all time points studied which coincides with a lack of hepcidin induction. By contrast, in Hfe-/- mice injected with 500µg of LPS hepcidin is induced and hypoferremia develops. These data suggest that Hfe is critical for the hepcidin response to a low dose of LPS, while high, sublethal doses of LPS override the contribution of Hfe to the hepcidin response. We further report that the observed increase in hepatic iron levels in response to low and high doses of LPS is independent of altered hepcidin mRNA expression but may be explained by diminished hepatic ferroportin mRNA levels. Additionally, LPS injection reduces expression of Hfe2 and TfR2 in WT and Hfe-/- mice suggesting that decreased expression of these HH associated proteins may contribute to the lack of hepcidin activation in the case Hfe is also lacking. Studies are ongoing to analyse the activity of signalling mediated by the HH proteins (BMP/SMAD) or IL6 (JAK/STAT) to understand how hepcidin is differentially regulated in response to LPS in Hfe-/- and WT mice providing insight into the crosstalk of inflammation and HH protein associated hepcidin activation.

Poster# 89 REDUCED SERUM TRANSFERRIN LEVELS CHARACTERISE IRON-LOADED C282Y HAEMOCHROMATOSIS DESPITE UPREGULATED HEPATIC TRANSFERRIN TRANSCRIPTION John D. Ryan, MBBCh¹, Eleanor Ryan, PhD², Matthew Lawless, PhD², Jennifer Russell, PhD², Jens Stolte³, Martina U. Muckenthaler, PhD³, T. Barry Kelleher, MD² and John Crowe, PhD² ¹Centre for Liver Disease; ²Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin, Ireland; ³Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg, Germany (Presented By: John D. Ryan, MBBCh)

C282Y Hereditary Haemochromatosis (HH) is characterised by a defective HFE protein and deficient hepcidin synthesis. Failure to degrade ferroportin is hypothesised to cause duodenal iron deficiency, resulting in excessive dietary iron absorption and iron overload. Transferrin (Tf), the main distributor of plasma iron, is produced mainly by the liver. Tf levels are increased in iron deficiency and reduced in iron overload. The Tf gene is regulated by iron at a translational level in animal models and upregulated by iron deficiency. Hypotransferrinaemic humans and mice exhibit very low serum Tf, deficient hepcidin production, and severe iron overload. Low serum Tf is a known feature of HH, while hepatic Tf gene expression has not been described in a C282Y HH cohort. Objective: The aim of this study was to examine hepatic Tf gene expression in iron-loaded male C282Y homozygotes and correlate this with serum Tf. Methods: Using the human Ironchip®, gene expression profiling was performed on liver tissue from 10 untreated male C282Y homozygotes and 4 male liver donors negative for HFE mutations. Results were validated by QRT-PCR on liver biopsies from 22 iron-loaded C282Y HH males and 4 controls. In addition, serum Tf was measured in a cohort of C282Y homozygotes compared to age and sex-matched controls negative for HFE mutations. With regard to the 22 HH males, the mean age at diagnosis was 48yrs(+/-9), mean Tf saturation was 88%(+/-6.7), serum iron 43umol/L(+/-6), serum Tf 1.97g/L(+/-0.26), and median serum ferritin 1573µg/L and ALT 85IU/L. Mean grade of hepatocellular iron staining was 3+. Transferrin expression was examined in HFE, TfR2 and HJV knockdown mice. Results: Microarray analysis demonstrated a mean >5 fold increase in hepatic Tf mRNA expression. QRT-PCR confirmed significantly elevated levels of Tf mRNA compared to controls(p=0.009). Tf mRNA levels correlated significantly with serum Tf levels(p=0.001) and ferroportin gene expression (P<0.0001). Surprisingly, suppression of serum Tf was observed in untreated C282Y HH compared to controls(p=0.001). Discussion / Conclusion: Untreated C282Y Haemochromatosis is characterised by depressed serum Tf levels, while hepatic Tf gene expression is significantly elevated. This finding was not substantiated in HFE-deficient mice, possibly suggesting a role for the C282Y mutated HFE protein. We hypothesize that an iron deficient duodenum may signal to increase Tf transcription, while excess hepatocyte iron may regulate Tf translation in these patients. Further studies are needed to clarify these findings.

Poster# 90 HEMOJUVELIN IN MOUSE TISSUES Yuzo Fujikura, PhD, Jan Krijt, PhD and Emanuel Necas, MD, PhD Charles University in Prague (Presented By: Yuzo Fujikura, PhD)

Expression and processing of hemojuvelin (HJV) have been studied mostly using tissue specific cell cultures but direct studies of animal tissue are rare. In order to investigate possible involvement of HJV in hepcidin regulation we have been analyzing HJV in mouse tissues. Extracts of liver (L), muscle (M), and IgG/albumin depleted- plasma (P) were analyzed by reducing (R) and non-reducing (NR) SDS-PAGE , followed by western blot (WB), using a commercial antibody (Ab) against mouse HJV. Firstly, extracts from wild-type (WT) and HJV knock-out (KO) mice (kind gifts by Prof. S. Arber, Switzerland) were analyzed by R-SDS-PAGE/WB to check the detection by the Ab. In L samples, distinct 35 and 20 kDa positive bands were detected in WT but not in KO. In M samples, a faint 35 kDa and a clear and thick 18 kDa positive bands were observed in WT but not in KO. In P samples, no WT specific positive band was detected, which may indicate quick degradation of extracellular HJV. By NR- SDS-PAGE, a 50 kDa band was detected as a predominant positive band in both L and M samples, indicating that the 35 and 20/18 kDa proteins may form a 50 kDa hetero dimer. The extracts were treated with phosphatidylinositol-specific phospholipase C (PI-PLC), which releases glycosyl-phosphatidyl-inositol (GPI)-anchored proteins from cell membrane, then analyzed by both NR- and R- SDS-PAGE. In L samples, PI-PLC treatment produced the similar results to those of the untreated samples, indicating the hetero dimer may already be released from the membrane in the extract. On the other hand, in M samples, much stronger intensity of the 35 kDa band and less intensity of 18 kDa band than those of the untreated sample was detected by R-SDS-PAGE, indicating that a significant amount of 35 kDa peptides was exposed by PI-PLC treatment. This was another distinct difference found between L and M samples. A difference in the lipid composition of plasma membranes might be the reason. Next, PI-PLC treated L and M samples were digested by N-glycosidase. In both L and M samples, a slight reduction of 35 kDa bands to about 33 kDa was detected. The smaller bands, 20 kDa of L or 18 kDa of M, almost disappeared after the treatment, indicating their instability, even in the presence of protease inhibitors.

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Our results demonstrated that HJV hetero dimer consists of a 35 and a 20 kDa peptides in L and a 35 and a 18 kDa in M and that a 35 kDa peptide in L and M may be identical and similarly glycosylated but its attachment to the plasma membranes may be in different manners Also instability of the 20/18 kDa peptides was detected. Mature mouse HJV consists of 361 amino acids which make up a 39 kDa protein. Considering the glycosidase results, one of furin cleavage products, a 32 kDa peptide, may be a primary candidate for the detected 35 kDa peptide. Since the other furin cleavage product is a 7 kDa peptide, it is interesting to speculate the origin of the 20/18 kDa peptides. This project is supported by the grants, MSM 0021620806 and LC 06044, from the Ministry of Education of Czech Republic.

Poster# 91 HOMOZYGOUS DELETION OF HFE AS A CAUSE OF HEMOCHROMATOSIS IN SARDINIA Sara Pelucchi¹, Raffaella Mariani, Matteo Pozzi, Francesca Bertola², Sabina Coletti, Cristina Arosio² and Alberto Piperno ¹University of Milano-Bicocca; ²Consortium of Human Molecular Genetics (Presented By: Alberto Piperno)

Type 1 hemochromatosis is generally due to homozygous p.C282Y mutation in HFE. We report the case of a young woman with a classical hemochromatosis phenotype due to a homozygous deletion in the 6p chromosome region containing HFE. The proband is a 29-year-old woman of Sardinian origin (Italy). At the age of 27, serum iron was 186 µg/dL, and serum ferritin 1400 µg/L. There were no causes of secondary iron overload. A test for HFE mutations (NLM, Settala, Italy) gave no signal. She began phlebotomies every two weeks and after 3.5 g of iron removal serum ferritin decreased to 188 µg/L, but serum iron did not change (201 µg/dL), indicating she was not iron depleted, yet. After 3 months without therapy, serum ferritin raised again to 547 µg/L. She came to our attention in June 2008. She denied family consanguinity up to the fifth generation. Her grandparents originated from different Sardinia regions. After informed consent, we analysed her DNA sample for p.C282Y and p.H63D by Real-Time PCR (Celbio, Milano, Italy), confirming the absence of signal and suggesting a deletion in HFE-containing region. By analysing several polymorphic markers, centromeric and telomeric to HFE, we roughly define the upstream and downstream limits at 26.181.749 and 26.225.235, respectively. By using specific primers, we confirmed that the proband was homozygous for a gross deletion [Chr6 g.(26,175,442)_g.(26,208,186)del] that corresponds to that recently reported by Le Gac et al [Blood 2008;112:5238-5240] in another woman originating from Sardinia, too. The absence of close consanguinity in the present family and with the case reported by LeGac et al, suggests that this deletion is present at the population level in Sardinia and may derive from a common ancestor. However, considering the mechanism ofdeletion, we cannot exclude that it might result from multiple mutational events and explain other hemochromatosis cases.

Poster# 92 HFE PROTEIN PRODUCED IN EUKARYOTIC CELLS BINDS THE CATION INDEPENDENT MANNOSE-6-PHOSPHATE RECEPTOR IN VITRO Lisa Schimanski, BSc, MSc, Hal Drakesmith, Emma Sweetland, Mariola Edelmann, Dellel Razgui, Chandran Ka, Alison Merryweather-Clarke, Kathryn Robson, Benedikt Kessler and Alain Townsend Oxford University (Presented By: Lisa Schimanski, BSc, MSc)

Since its association with Haemochromatosis was discovered in 1996, the function of HFE, an MHC class-1b molecule, has remained elusive. The binding of HFE to transferrin receptor-1 (TfR1) in competition with Holo-transferrin (Fe-Tf) is generally believed to be part of a mechanism to sense serum Fe-Tf concentrations. HFE freed from TfR1 by Fe-Tf may induce the peptide hormone hepcidin, which inhibits the iron exporter ferroportin. How HFE induces hepcidin in this context is not known, but it could involve an interaction with as yet undetermined ligands. We searched for new HFE ligands that might be involved in iron sensing. Plasmid constructs were made encoding class-1 heavy chain (HFE, HLA-A2 or CD1d), β2- microglobulin and a tag containing 6 x histidine for purification, a biotinylation motif and c-myc for detecting with monoclonal antibodies. These were expressed in 293T cells and soluble protein purified from the culture media. Fluorescent tetramers of biotinylated HFE protein bound to the surface of cell lines tested by flow cytometry. In cells such as HL60 and Hep3b, binding could be abolished by addition of Fe-Tf, indicating binding only to TfR1. However THP-1, K562 and Jurkat cells retained HFE binding even in the presence of excess Fe-Tf, indicating a non-TfR1 ligand. To isolate this ligand we added soluble HFE protein and anti c-myc to lysate from I125 labelled K562 cells and precipitated HFE associated proteins. As expected TfR1 co-precipitated with HFE protein. However we also observed a 250kDa co-immunoprecipitated band, which unlike TfR1, was retained in the presence of Fe-Tf. This protein was identified by mass spectroscopy as the cation independent mannose-6-phosphate receptor (ci-MPR, also known as IGF2R). Transfection of 293T cells with a plasmid encoding ci-MPR increased binding by HFE tetramer, and full-length HFE protein co-immunoprecipitated with ci-MPR. HFE tetramer binding to ci-MPR transfected cells was blocked by mannose-6-phosphate (M6P), and likewise, binding of HFE tetramer to K562 cells was blocked by M6P and by Endoglycosidase F pre-treatement of HFE tetramer. BIACORE experiments also showed HFE binding to ci- MPR, and blocking by M6P. We therefore concluded that HFE expressed in 293T cells was modified with a M6P causing it to bind ci-MPR. Moreover, binding of HFE to ci-MPR or to TfR1 was found to be mutually exclusive in solution and influenced by Fe-Tf concentrations, consistent with an iron sensing mechanism. Control protein CD1d did not bind ci-MPR but there was a small amount of binding of HLA-A2 to cells highly expressing ci-MPR and this low level binding was confirmed in BIACORE experiments. We are concerned that over-expressing proteins in 293T cells could lead to inappropriate M6P modification. Experiments are underway to isolate endogenous HFE and investigate its binding to ci-MPR, to establish whether this is a functional interaction in vivo or an artefact of our protein expression system. It may be important to be aware of this potential artefact when experimenting with exogenously produced proteins.

Poster# 93 E277K AND V295A HFE MUTATIONS – A MATTER OF α3-DOMAIN LOCATION? B. Silva, R. Martins, D. Proença, R. Fleming and P. Faustino Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal (Presented By: B. Silva)

HFE is a major histocompatibility class I-like (MHC) molecule that has a function in cell and body iron metabolism. Usually, HFE is firstly associated with its light chain, β2-microglobulin (β2-M), and then presented to the cell surface where interacts with the transferrin receptor pool (TfR-1 and -2). When these interactions are impaired they may lead to a hereditary hemochromatosis (HH) phenotype characterized by an excessive iron loading and deposition at some vital tissues, as liver and heart. Since HFE gene discovery, two mutations have majorly been associated with this disease. One, results from a histidine to aspartic acid substitution at the position 63 (H63D) and other, from a cysteine to tyrosine substitution at the position 282 (C282Y) of the unprocessed protein. The former is located at the α1-domain and it seems to reduce the affinity of the HFE protein to TfR-1, while the latter, located at α3-domain, disrupts a disulphide bond at this level, impairing protein association with β2-M and subsequent transport to cell surface. Other rare HFE mutations have been found in association with HH, but their impact in the structure and function of the protein remains to be clarified. Between them, there are E277K and V295A, both located at α3-domain, and found in portuguese patients with a HH phenotype. Since these two non-classical HFE mutations are located at the same domain as C282Y, we aimed to analyse if they also affect the protein association with β2-M and its transport to cell surface. To achieve this, we made constructs in pEGFP-N1 vector that allowed the expression of both wild-type and mutated HFE proteins tagged to GFP at the C-terminus. The constructs were transfected in HeLa and HepG2 cells and protein expression confirmed by immunoblotting. Transfected cells were submitted to an immunofluorescence protocol, where antibodies were raised against β2-M, TfR-1 and calnexin (endoplasmic reticulum marker), followed by confocal microscopy analysis. Results revealed that E277K and V295A mutated proteins became partially retained in endoplasmic reticulum, what is shown by the partial co-Iocalization between calnexin and HFE-GFP protein in about 60 and 30% of the cells, respectively. In the same proportions they also failed the association to β2-M, compromising their binding to TfR1 and subsequent transport to cytoplasmic membrane. Immunoprecipitation assays are undergoing to confirm if these interactions are physically impaired. This is a phenotype that resembles the one obtained with C282Y mutation. Thus, we may conclude that the mutation E277K, more than V295A, has a deleterious effect in the HFE protein. 124

Furthermore, in silico analysis predicted that the G to A substitution in nucleotide 829, leading to E277K, increases the number or exonic splicing enhancer consensus sequences in the affected area. To test the hypothesis that this mutation may lead to aberrant HFE splicing transcripts, a construct including E277K and its flanking introns was made, and transfected in HepG2 cells. RNA has been extracted and Real-Time PCR analysis is currently undergoing. Thus, besides impairing the correct processing of HFE protein to the cell surface, E277K mutation may also be inducing splicing alterations. Both effects might contribute to the HH development in these patients. Partially funded by FCT: Programa de Financiamento Plurianual do CIGMH

Poster# 94 DIFFERENTIAL EXPRESSION OF HFE SPLICE VARIANTS D. Proença², R. Martins¹, B. Silva and P. Faustino ¹Instituto Nacional de Saude Dr. Ricardo Jorge; ²Departamento de Genética, Instituto Nacional de Saude Dr. Ricardo Jorge, Lisboa, Portugal (Presented By: R. Martins)

Hereditary hemochromatosis is an autosomal recessive disorder characterized by excessive iron stores, generally caused by a mutated HFE protein. Nevertheless, the mechanism by which HFE acts to regulate iron metabolism remains elusive. It has been suggested that its role on iron absorption depends on the complex relationship between the HFE protein and the transferrin receptor (TfR) but its precise function is still unknown. HFE transcripts are widely expressed and the predominant mRNA identified has ~4.2 kb in length. Even so, several additional transcripts have been reported, which seem to differ in both expression level and tissue- or cellular-specificity. However, their structural characterization, relative abundance and tissue specificity have been poorly studied. Therefore, to clarify this matter, we carried out cDNA synthesis using RNA obtained from different human tissues: liver, duodenum, heart, testis, ovary, spleen, small intestine and kidney. RT-PCR followed by nested PCR were performed and the amplified products were cloned into the TOPO® XL plasmid (Invitrogen) and sequenced. Eight different HFE transcripts were identified as an outcome of alternative splicing events. Raising the hypothesis that some of these variants might have a biological role, we determined the relative abundance of those resulting from exon 2 skipping or the inclusion of intron 4. Quantification was performed by Real-time PCR, relatively to the total amount of HFE mRNA present in the different tissues. Furthermore, to study the intracellular trafficking of the corresponding protein variants, GFP was tagged to their C-terminus as well as to the HFE wild-type. HeLa cells were transfected with these constructs and the intracellular location of the proteins was analyzed by immunofluorescence, using several antibodies. Our results show that both splice variants are present in all the tissues analysed. The relative amount of exon 2 skipping transcript ranges between 3 and 26% of the total HFE transcripts, whereas the intron 4 variant has a relative abundance between 6 and 35%. Preliminary results show that in cells transfected with the HFE cDNA where the exon 2 is skipped, the correspondent protein does not co-localize with beta2-microglobulin or TfR1 as it happens in the wt, which may represent a misprocessing of the mutated protein. Consequently, by abrogating these interactions, the altered protein is unable to bind and present itself at the cellular membrane. Immunoprecipitation assays are undergoing to clarify if these interactions are disrupted. Amongst the analysed tissues, the liver presents the lowest amount of both transcripts, possibly reflecting the central role for the HFE protein in this organ. On the other hand, since it remains unknown how HFE regulates iron homeostasis in the body, the complex interaction of HFE with its ligands may be uncovered by some of its splicing variants. Alternatively spliced isoforms with significant biological function are known to exist for MHC class I genes (HLA-A, HLA-B and HLA-G) as for related genes (MR1). Since HFE belongs to this family of genes, we forecast that some HFE transcripts may play a specific role on iron homeostasis. Partially funded by FCT: PTDC/SAU-GMG/64494/2006; Programa de Financiamento Plurianual do CIGMH and SFRH/BD/21340/2005

Poster# 95 TFR2 BETA ISOFORMS ARE DIFFERENTIALLY LOCALIZED IN CELLS AND RESPOND DIFFERENTLY TO IRON TREATMENT Rosa Maria Pellegrino¹, Ilaria Defilippi, Dr², Enrico Bracco, PhD², Sonia Carturan, Dr², Antonietta Palmieri², Daniela Cilloni, MD², Giuseppe Saglio, MD² and Antonella Roetto, PhD² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Science, Az Osp San Luigi Gonzaga, Orbassano Torino Italy (Presented By: Rosa Maria Pellegrino)

TFR2 gene, responsible of Hemochromatosis type 3, codifies for one of the HH sensing protein (Tfr2) involved in body iron amount sensing and in signalling devoted to hepcidin transcription modulation. Two alternative transcripts of TFR2 have been reported, named alfa and beta. TFR2 alfa mRNA is highly expressed in the hepatocytes while TFR2 beta has a low ubiquitous expression (Kawabata et al, 1999). The beta isoform is identical to alfa but it lacks the transmembrane and cytoplasmic domains; it has been considered an alternative splicing form and its role is still poorly understood. Nevertheless, clinical observations support the hypothesis that TFR2 beta has an important functional role since HFE3 patients with mutations not compromising its production have a milder phenotype compared to those unable to synthesize both alfa and beta TFR2 isoforms (Roetto et al,2002; Le Gac et al, 2004). In order to elucidate whether TFR2 beta has some function, we cloned Tfr2 alfa and beta isoforms in fluorescent proteins (red and green respectively) expressing vectors and transfected the two clones in Tfr2 negative HEK293 and in COS cell lines. Since TFR2 beta protein has never been demonstrated to exist before, beta-Tfr2 transfected cells lysates have been decorated with Tfr2 Ab and visualized on Western Blot. A 65 kDa band has been obtained from the transfection, consistent with the aminoacid sequence predicted molecular weight. We investigate Tfr2 alfa and beta localization in basal iron condition and after iron modulation treatments (50_M olo-Tf and apo-Tf) for increasing periods of time. At the same experimental points we analyzed the other HH genes expression pattern. We observed a transmembrane localization of Tfr2 alfa isoform and a cytoplasmic diffuse localization of the beta both in separate transfection and in alfa/beta co transfection in basal conditions. Starting from 15 min after treatment with oloTf, Tfr2 beta isoform localizes in well defined spots under the plasma membrane while Tfr2 alfa remains on the cell surface. Thirty minutes after olo-Tf treatment we notice a stabilization of the beta isoform and a partial colocalization of the two isoforms both on the plasmatic membrane and in cytoplasmic vesicles. No apparent modification of Tfr2 isoforms has been observed in apo-Tf treated cells. Quantitative transcription analysis revealed that only co-transfected cells responds to iron treatment increasing Hepc and HJV transcription 5 min after olo-Tf adding. On the whole, these results suggest a functional role for the TFR2 beta isoform in iron sensing, since this isoform changes its cellular localization as a consequence of iron treatment and increased transferrin saturation. Furthermore, partial colocalization of Tfr2 known isoforms strengthens the hypothesis they could collaborate in iron sensing pathway. Finally Hepc Tf-dependent regulation seems to be restored in vitro only when both Tfr2 isoforms are produced.

Poster# 96 CHARACTERIZATION OF MOUSE SERUM FERRITIN Lyora A. Cohen, MSc, Avigail Morgenstern and Esther G. Meyron-Holtz Israel Institute of Technology, Technion (Presented By: Lyora A. Cohen, MSc)

Ferritin is a soluble protein that stores iron in a spherical protein cage, constituted of 24 subunits of two types, H and L. In mammals, ferritin is mostly cytosolic. A secreted form of ferritin is found in serum and levels of serum ferritin usually correlate with levels of body iron stores. Exceptions to this correlation are pathologic states such as chronic inflammation and several kinds of cancer, where serum ferritin levels are elevated. Recently two different receptors for ferritin have been identified (TIM2 and Scara5) and in addition to iron transport, a role for extracellular ferritin in angiogenesis has been demonstrated. Therefore understanding the mechanism and regulation of ferritin secretion has become an important question. Human serum ferritin is reported to contain little iron, mostly L-subunits and binds to the lectin Concanavalin A. It is therefore considered glycosylated but the exact nature and location of the glycosylation is not known.

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We characterized mouse serum ferritin in respect to amino-acid and subunit composition, iron content and nature of glycosylation. Serum ferritin is mostly a 24-mer but some very high molecular weight complexes are found to contain ferritin in serum. Immunoprecipitation followed by Western blot with H- and L-specific antibodies and mass spectrometry of the main ferritin peak from a Sephacryl S-300 26/60 column fractionation verified the presence of both H- and L-ferritin subunits. Mass spectrometry also suggests an amino acid composition identical with intracellular ferritin. To better understand the origin and secretion pathway of serum ferritin we characterized the subunit ratio. This ratio suggests that many ferritin molecules in serum contain only L-subunit and therefore most likely no iron, and only about 25 percent of serum ferritin molecules contain one Hsubunit. Average iron determination was done by the ferrozine assay and showed that serum ferritin contains significantly less iron than liver ferritin (700 and 2100 Fe atoms/ ferritin molecule respectively). Analysis of mouse serum ferritin binding to Sepharose-Concanavalin A beads, detected very low ferritin binding to this resin and similar amounts of ferritin purified from liver or heart bound to these beads as well. We therefore analyzed the nature of serum ferritin N-glycosylation further. Comparison of serum ferritin before and after deglycosylation with PNGase F showed that only a very small fraction of serum ferritin is N-glycosylated and that after deglycosylation the 22kDa L-subunit band slightly increased. This small fraction does not correlate with the published 30-50% glycosylation of human serum ferritin. In summary, our results suggest that mouse serum ferritins are 24-mers, composed mostly of L-subunit homo-polymers and of some hetero-polymers containing little H- subunit. Secreted ferritin structurally resembles intracellular ferritin but differs in its subunit ratio and iron content, suggesting that serum ferritin subunits are transcribed from the same genes as cytosolic ferritin, but that assembly of serum ferritin takes place in a compartment other than the cytosol of the secreting cells. In addition only a minor fraction of serum ferritin is N-glycosylated, suggesting a non-classical secretory pathway for serum ferritin, that is now elucidated in detail.

Poster# 97 POSTNATAL INCREASES IN HEPCIDIN EXPRESSION AT BIRTH AND AT WEANING ARE DEPENDENT ON TRANSFERRIN RECEPTOR 2 Heather Morgan, MD, Robert Britton, PhD, Mary Migas, MS, Gail Palmer, BS, Abdul Waheed, PhD and Robert Fleming, MD Saint Louis University School of Medicine (Presented By: Robert Fleming, MD)

Introduction: The ontogeny of liver hepcidin expression demonstrates several phases. While prenatal hepcidin expression is very low, a transient surge has been observed in the immediate newborn timeframe, with a persistent increase noted after several weeks. The aim of these studies was to characterize the events associated with these marked changes in hepcidin expression and the potential role of transferrin receptor 2 (TfR2). Methods: Timed pregnant wild-type FVB and Tfr2(Y245X) mice (N=5-10) were analyzed at multiple ages from 19 days gestation to day-of-life 42. To determine the effect of time of weaning, we alternated four lactating dams per litter from a feeding cage with chow to a nursing cage with the pups. To determine the effect of dietary iron intake, mice were weaned at 21 days onto chow with <20 ppm iron or 250 ppm iron. Hepcidin mRNA expression was determined by RNA blot analysis, and Hamp1 transcripts were measured by real-time RT-PCR normalized to ß-actin. Liver and spleen non-heme iron concentrations were also measured. Hematocrits were determined by packed cell volumes from cervical bleeds. Results: A postnatal surge in hepcidin expression was observed at day 1 in wild-type but not Tfr2 mutant mice. The higher hepcidin expression after 1 day in the wild-type mice was associated with lower liver iron and higher splenic iron concentrations than seen in the Tfr2 mutant mice. By day 10, hepcidin levels had markedly decreased in wild-type mice and remained low until weaning. The Tfr2 mutant mice demonstrated higher hematocrit levels (m=40) at days 6-10 than the wild-type mice (m=37). Mice weaned onto a standard iron diet (250 ppm) demonstrated a marked increase in hepcidin expression within 4 days; however, mice weaned onto a low-iron diet (< 20 ppm) continued to have very low hepcidin expression. Tfr2 mutant mice did not demonstrate the marked weaning-associated increase in hepcidin expression observed in the wild- type mice. Conclusions: The immediate postnatal increase in liver hepcidin expression is dependent upon Tfr2. We speculate that this increase is due to the increase in serum transferrin saturation reported during this timeframe. The increase in hepcidin expression observed at several weeks of age is related to weaning from a low-iron diet (milk) to the relatively high-iron diet provided by chow, and is likewise influenced by Tfr2. The higher hematocrits observed in the Tfr2 mutant mice suggest that the postnatal surge in hepcidin may contribute to iron-restrictive erythropoiesis during this period.

Poster# 98 THE ROLE OF DCYTB IN INTESTINAL IRON ABSORPTION Jeehyea Choi, Abas Laftah, Gladys Oluyemisi Latunde-Dada, Andrew McKie and Robert Simpson King’s College London (Presented By: Jeehyea Choi)

Absorption of dietary iron is a highly regulated process and is the major determinant of body iron stores. Non haem iron absorption involves at least two proteins present on the duodenal brush border membrane, a divalent metal transporter (DMT1) and a ferric reductase (DcytB) and at least two proteins located on the basolateral membrane, a ferrous iron exporter (Ferroportin or Ireg1) and a multicopper oxidase (Hephaestin). Ferric reduction is essential for transport of the metal via DMT1 which exclusively transports ferrous iron. DcytB mRNA and protein levels in the duodenal enterocytes are highly upregulated by iron deficiency and in iron overload diseases, such as haemochromatosis, where iron absorption is increased. The aim of the project is to fully characterise the impact of loss of DcytB in DcytB KO mice on intestinal iron absorption. Reductase activity was measured in DcytB Knock-out mice after treatment with hypoxia, iron deficiency and pregnancy, all of which increase the body iron demand. In contrast to wild type mice which show three to four fold increase in all cases, there is no change in reductase activity in the KO mice (p<0.0005, n=4 to 9), suggesting DcytB is the primary iron regulated ferric reductase in the gut. Non-haem iron levels were measured in KO and WT mice treated with 3-day hypoxia or six-week iron deficient diet. After hypoxic treatment, 50% reduction in spleen non- haem iron was measured in KO mice (p<0.05, n=3) but no change was observed in liver, compared to WT. However there was a significant drop in both liver and spleen, 30% (p<0.005, n=9-10) and 50% (p<0.005, n=6-8) respectively, in KO mice compared with WT after six-week iron deficient diet feeding. The rate of iron absorption was measured in vivo in hypoxic KO mice using radioactive 59Fe. Under hypoxic condition, DcytB KO mice showed significantly lower mucosal retention (measures how much 59Fe is retained in the duodenum) but higher percentage mucosal transfer (measures how much 59Fe is exported out of the duodenum into the body). Overall this data suggests that iron uptake into the mucosa from the diet is lower in Dcytb KO mice under increased iron demand. Expression and localization of other genes such as DMT1and Ferroportin are currently under investigation.

Poster# 99 ERYTHROPOIETIN INCREASES IRON ABSORPTION BY HUMAN INTESTINAL EPITHELIAL CELLS Bomee Chung¹, Timothy Chaston, PhD¹, Chiara Rapisarda, BSc¹, Katayoun Pourvali, BSc¹, Carin Da Silva, Bsc¹, Surjit Kaila Srai, PhD² and Paul Sharp, PhD¹ ¹King’s College London; ²University College London (Presented By: Bomee Chung)

Enhanced erythropoiesis in iron deficiency anaemia (IDA) increases the body’s demand for iron. Erythropoietin (Epo) levels are increased in IDA and are thought to act as the main homonal stimulus for red blood cell production. The presumed mode of action of Epo is via inhibition of hepatic hepcidin production which in turn increases iron absorption by intestinal enterocytes and permits iron release from reticuloendothelial macrophages. However, it is also possible that Epo signals the body’s erythroid requirements directly to the enterocytes. To investigate this possibility, we have studied the effects of Epo on intestinal iron absorption in human intestinal Caco-2 cells. Caco-2 cells were grown on Transwell inserts for 17 days prior to experimentation. Epo (1U/ml) was added to the basolateral medium and iron uptake was measured 24 h later. Parallel plates of Epo-treated cells were processed to provide membrane protein and total RNA for the measurement of iron transporter expression.

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Iron uptake across the apical membrane of Caco-2 cell monolayers and efflux across the basolateral membrane were both significantly enhanced in Epotreated cells. These transport effects were associated with significant increases in protein and mRNA expression of DMT1, ferroportin and hephaestin in Caco-2 cell exposed to Epo. However, there was no significant change in Dcytb expression. Our data suggest that Epo can act directly on intestinal epithelial cells to increase dietary iron absorption for subsequent utilisation in the bone marrow. These effects are mediated by Epo receptors, which are expressed on both Caco-2 cells and rat duodenal enterocytes. These data provide a novel insight into the mechanisms by which the body senses and responds to changes in the erythroid requirements for iron. This work was funded by BBSRC (BB/D015456). Bomee Chung was part-funded by an ORS award.

Poster# 100 ALTERNATIVE FERRITIN MRNA TRANSLATION UNDER STRESS CONDITIONS VIA INTERNAL INITIATION Alina Daba¹, Anja Hausmann, PhD¹, Antonis Koromilas, PhD² and Kostas Pantopoulos, PhD² ¹Lady Davis Institute for Medical Research; ²Lady Davis Institute for Medical Research and McGill University (Presented By: Alina Daba)

Ferritin stores and detoxifies excess of intracellular iron and thereby plays an important role in metabolism of this metal. As unshielded iron promotes oxidative stress, ferritin is crucial in maintaining cellular redox balance and may also modulate cell growth, survival and apoptosis. The expression of ferritin is controlled by transcriptional and post-transcriptional mechanisms. In light of the well-established transcriptional induction of ferritin by inflammatory signals, we examined how ferritin mRNA translation responds to stress conditions. We first employed HT1080 fibrosarcoma cells engineered for coumermycin-inducible expression of PKR, a stress kinase that inhibits protein synthesis in virus-infected cells by phosphorylating eIF2α. In this setting, iron triggered ferritin mRNA translation despite a PKR-induced global shutdown in protein synthesis. Moreover, iron-mediated ferritin synthesis efficiently proceeded in cells infected with an attenuated strain of poliovirus, under conditions where eIF4GI was cleaved, eIF2α was phosphorylated, and host protein synthesis was inhibited. Further experiments with a positive feedback bicistronic vector to screen for sequences that enhance translation, revealed that the 5’ untranslated region of ferritin mRNA contains a bona fide internal ribosomal entry site (IRES). Our data are consistent with an alternative, cap-independent mechanism for ferritin mRNA translation under stress conditions, which may be crucial for cell viability.

Poster# 101 IN-CELL-WESTERN ANALYSIS OF ENDOGENOUS FERROPORTIN REGULATION IN PRIMARY MACROPHAGES Anne Auriac, Master, Alexandra Willemetz, Master and François Canonne-Hergaux, PhD UPR2301, CNRS, Centre de recherche de GIF-sur-Yvette, France (Presented By: Anne Auriac, Master)

Being the sole identified cellular iron exporter, Ferroportin (Fpn) was shown to play a key role in mammals iron homeostasis. Fpn is involved in the export of iron from duodenal enterocytes (iron absorption) and from tissues macrophages (iron recycling) into the plasma. The regulation of Fpn is complex relying on several transcriptional and post-transcriptional regulations. In macrophages, Fpn protein expression is strongly increased by heme or iron and is downregulated by hepcidin (Hepc), a small peptide expressed in liver in response to inflammation and iron loading. Some observations suggest that Fpn regulation is likely cell type and tissue specific and highlight the need to study Fpn regulation in its physiological cellular context. For this purpose, we developed an “In-cell-western” assay to directly quantify the expression of Fpn in fixed primary macrophage cultures using the Odyssey infrared imaging system (LI-COR Biosciences). In this assay, mouse bone marrow derived macrophages (BMDM) cultured in microplate were treated with different doses of human hemin (Normosang®, Orphan Europe, heme arginate), iron (Fe-NTA) and human hepcidin (Peptide International). After cells fixation and permeabilisation, Fpn was detected using a high affinity purified primary antibody and a near infrared-labelled secondary antibody. As a control of iron loading, the expression of the iron storage protein ferritin (Ft) was also studied. To correct for variations in cell number from well to well, quantification and normalization of the specific fluorescent signal (Ft or Fpn; Channel 1) was performed against fluorescent detection of a second protein target or DNA (Channel 2). We found that an overnight heme or iron treatment strongly increased Fpn and Ft expression in a dose dependant manner. Maximum effect on Fpn (up to 10 fold increase) was observed with 100µM of Fe-NTA or human hemin. After iron loading treatments, the presence of Hepc in the culture medium (300nM for 6 hours) decreased by 70 % the amount of Fpn. A low concentration of 10 nM of the Hepc peptide was strongly efficient in Fpn downregulation. Using an In-cell-western microplate-based assay, we were able to specifically reproduce the physiological effect of iron and hepcidin on macrophage Fpn expression. In addition the assay developed was shown to be sensitive with accurate quantification of Fpn expression changes after treatments. Such a model will be useful to test new regulators of Fpn that could have an effect on expression and/or stability of the iron exporter in macrophages.

Poster# 102 ROLE OF MUCIN 13 IN IRON METABOLISM Marizela Saric, Dr, Abas Laftah, Dr, Oluyemisi Latunde-Dada, Dr, Patarabutr Masaratana, MD, Robert Simpson, Dr and Andrew T. McKie, Prof Nutritional Sciences Division, King’s College London (Presented By: Marizela Saric, Dr)

Mucin 13 (MUC13) belongs to the family of transmembrane mucins. MUC13 was identified in a screen for iron regulated genes in mouse duodenum. It has been shown recently that the TIM-2 protein, which contains a similar mucin-domain to MUC13, is a receptor for H-ferritin. The aim of this study was to characterize the expression of MUC13 in different tissues of mouse gastrointestinal tract, to test if expression of MUC13 is regulated by iron and if MUC13 can function as a ferritin-binding protein. Semi-quantitative PCR, Northern blot analysis, Western blot analysis and immunhistochemical staining for MUC13 was performed using iron deficient (ID), iron loaded and control CD1 mice as well as Caco-2 cells treated with ferritin, Fe+3, Fe+2 and heme. Results show that MUC13 is expressed throughout the whole length of the mouse gut. MUC13 expression was regulated by iron at the protein level in stomach and colon, and at the mRNA and protein level in duodenum. MUC13 protein level was increased in Caco-2 cells treated with ferritin. Immunoprecipitation suggested that ferritin and MUC13 co-precipitated together. We hypothesize that MUC13 acts as a ferritin-binding protein. Mass spectroscopy will be used in order to confirm this hypothesis.

Poster# 103 A NOVEL MUTATION IN INTESTINAL ISOFORM OF DIVALENT METAL TRANSPORTER 1 (DMT 1A) Luigia De Falco, Francesca Totaro, Mrs, Maria Nunzia Scoppettuolo, Mrs, Immacolata Andolfo, Mrs, David Beneitez Pastor, MD and Achille Iolascon, MD, PhD Ceinge Advanced Biotechnologies (Presented By: Luigia De Falco)

Divalent metal transporter 1 (DMT1) mediates apical iron uptake into duodenal enterocytes and also transfers iron from the endosome into the cytosol after cellular uptake via the transferrin receptor. Mutations in DMT1 cause systemic iron deficiency and microcytic anemia. The three patients with DMT1 mutations described so far developed highly transferrin saturation and mildly elevated serum ferritin, despite liver iron overload. Anemia is present from the birth. The molecular defect is common to intestinal and erythroid isoforms, which could explain the elevated transferrin saturation and the microcytosis. Four DMT1 isoforms exist resulting from mRNA transcripts that vary both at their 5’ ends (starting in exon 1A or exon 1B) and at their 3’ ends giving rise to mRNAs containing (+) or lacking (−) the 3’ IRE (iron-responsive element). Exon 1A is located 1.9 kb upstream of the first exon (“1B”) of the previously characterized human DMT1 and is followed by a consensus splice sequence. The exon 1A is spliced directly to exon 2. The 1B isoform is ubiquitous, whereas the 1A isoform is tissue-specific, predominantly in the duodenum and kidney. Here, we report a novel DMT1 variant with a mutation in 1A isoform due to A>T transition at nucleotide position 652. This 55-years-old Caucasian female with unrelated parents was admitted to our hospital for mild anemia and microcytosis. We observed a mild hypochromic microcytic anemia with a hemoglobin level of 9 g/dL, a mean cell volume (MCV) of 56 fL, and a mean hemoglobin concentration (MCH) of 18.1 pg, the reticulocyte count was 69 x 103/µL. Transferrin saturation was elevated (129%) as well as ferritin levels and soluble transferrin receptor values. Haptoglobin and bilirubin levels were normal, and there were no alterations in hemoglobin electrophoresis and there was no 127 enzymatic defect. Sequencing of the exons and exon-intron boundaries of the DMT1 gene revealed the proband compound heterozygote for c.GGC723>GTC, also described in literature, and a novel A>T substitution in intron 1A at position 1 following the acceptor splice site. The latter could lead to splicing alterations as verify by in silico tools and functional assay. The DMT1 1A mutation has an homozygote expression in enterocytes, otherwise there is no expression of this isoform in hepatic or erythroid cells. Compound heterozygote condition is present only in enterocytes, where we hypothesize that the exon 8 G>T transition causes a functional alteration while mutation in DMT1 1A causes a reduction of protein expression. All those subjects who are compound heterozygote in peripheral cells show a decrease in MCV , so untill now it has been believed that the distribution of iron to peripheral tissues could have a foundamental role in MCV value. Even our case has a decrease in MCV, but she is heterozygote in erythroid cells (double heterozygote in intestinal cells) and we know that heterozygote subjects are asymptomatic. So our conclusions are that MCV could depend on intestinal iron absorption, and not on distribution of iron to peripheral tissues.

Poster# 104 UPSTREAM STIMULATORY FACTORS (USF-1/USF-2) REGULATE HUMAN HEMOJUVELIN GENE EXPRESSION Mohamed Salama, Henry Bayele, PhD and Kaila Srai, PhD University College London (Presented By: Mohamed Salama)

Hepcidin is presumed to be the master regulator of systemic iron homeostasis; its gene expression is affected by inflammation, hypoxia, iron levels, the rate of erythropoiesis, and oxidative stress. Hemojuvelin (HJV), a recently identified protein expressed in muscle, heart, and liver, has been shown to be an upstream regulator of hepcidin expression by acting as a bone morphogenetic protein (BMP) co-receptor. Consequently, HJV genetic mutations lead to the severe iron overload disorder juvenile hemochromatosis. There are two HJV isoforms: a membrane-bound and a soluble form, both of which are proposed to regulate hepcidin expression in response to changes in iron levels in vitro and in vivo. However, there is no information on how HJV is regulated. In this study, we identified E-box elements in the human HJV promoter and investigated the role of upstream stimulatory factors (USF) in the regulation of HJV expression through the E-boxes. By co-transfection experiments, we demonstrated that over-expression of USF trans-activated the HJV promoter. This was confirmed with electrophoretic mobility shift assays which showed that recombinant USF1 and USF2 bound specifically to the HJV E-box. Our data suggests that USF transcription factors are important regulators of HJV expression, and strengthens the link between USF and iron metabolism.

Poster# 105 REDOX CONTROL OF IRON REGULATORY PROTEIN 2 STABILITY Anja Hausmann¹, Julie Lee, MSc¹ and Kostas Pantopoulos, PhD² ¹Lady Davis Institute for Medical Research; ²Lady Davis Institute for Medical Research and McGill University (Presented By: Anja Hausmann)

Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron- responsive elements (IREs) and regulates their expression. Iron promotes proteasomal IRP2 degradation by an incompletely characterized mechanism. Previous work provided evidence that IRP2 degradation involves post-translational modification by 2-oxoglutarate-dependent dioxygenase(s). These enzymes require oxygen, ferrous iron and ascorbate (to maintain iron reduced), and are subjected to redox regulation. We explored the effects of oxygen and cellular redox status on IRP2 stability in H1299 human lung cancer cells. Iron-dependent decay of tetracyclineinducible IRP2 proceeded efficiently under mild hypoxic conditions (3% oxygen) but was compromised in severe hypoxia (0.1% oxygen). The administration of exogenous H2O2 to the cells stabilized IRP2, resulting in elevated IRE-binding activity, stimulation of transferrin receptor 1 expression and decrease in ferritin levels. IRP2 was also stabilized in response to menadione-induced oxidative stress. Ascorbate dramatically inhibited IRP2 stabilization by the hypoxia mimetic Co2+, but did not significantly affect IRP2 stabilization by menadione, the iron chelator desferrioxamine or hypoxia. These data demonstrate that the degradation of IRP2 in iron-replete cells is oxygen-dependent and sensitive to redox perturbations, consistently with a mechanism involving 2- oxoglutarate-dependent dioxygenases.

Poster# 106 STRUCTURAL STUDIES OF THE SOUL/HBP FAMILY OF HEME BINDING PROTEINS Brian Goodfellow, PhD, Filipe Freire, BSc, Susana Aveiro, BSc, Maria João Romão, PhD, Ana Luisa Carvalho, PhD, Vitor Felix, PhD, Nuno Micaelo, PhD, Gloria Ferreira, PhD and Anjos Macedo, PhD Universidade de Aveiro (Presented By: Brian Goodfellow, PhD)

Both members of the SOUL/HBP family of heme-binding proteins were discovered around 1999. The first member, p22HBP, was found to be a monomeric 22 kDa cytoplasmic heme or porphyrin binding protein isolated from mouse liver cell extracts. The solution structure has been determined and chemical shift mapping located the tetrapyrrole-binding site and binding constants were found to be in the nM range [1]. p22HBP is thought to be involved in heme regulation or heme synthesis. The second member of the family, SOUL, was isolated from retinal and pineal gland tissues [2], and is a cytoplasmic 23kDa monomeric protein that upon heme binding becomes hexameric [3]. No structural or functional information is available although SOUL has been found to induce necrotic cell death via mitochondrial membrane permeability in the presence of calcium. No structure of the bound form of HBP exists therefore modeling has been used to probe the interaction between HBP and various tetrapyrroles. A flexible docking protocol was carried out using Autodock4 centered on the binding site identified by NMR. Subsequent molecular dynamics and comparison with calculated and experimental ring current shifts indicated that both hemin and protoporphyrin-IX are stabilized by strong electrostatic interactions via their propionate groups to K64, K177, and R56 located at the edges of the binding pocket. Subtle differences between calculated and experimental ring current shifts suggest that the protein, especially around the D173-G180 loop, changes conformation on binding. Initial structural studies of hSOUL by NMR and X-ray diffraction in conjunction with fluorescence quenching studies suggest that hSOUL binds tetrapyrroles but with a mM Kd suggesting a different binding mode to HBP. Also, the single His residue, thought to bind heme, probably plays no part as Kds were found to be similar for hemin and PPIX. No evidence for hexamer formation was found. An initial low resolution X-ray structure solved by molecular replacement using the HBP structure has been obtained [4]. [1] J.S. Dias, A.L. Macedo, G.C. Ferreira, N. Jeanty, F.C. Peterson, B.F. Volkman, B.J. Goodfellow Journal of Biologial Chemistry 2006 281(42); 31553 [2] M.J. Zylka, S.M. Reppert Brain Res Mol Brain Res 1999 74(1-2); 175-181 [3] E. Sato, I. Sagami, T. Uchida, A. Sato, T. Kitagawa, J. Igarashi, T. Shimizu Biochemistry 2004 43(44); 14189-98 [4] F. Freire, M.J. Romão, A.L. Macedo, S.S. Aveiro, B.J. Goodfellow, A.L. Carvalho Journal of Structural Biology (submitted, 2009) Work supported by Fundação para a Ciência e Tecnologia, FEDER, MCTES (SFRH/BD/30239/2006, and PTDC/QUI/64203/2006)

Poster# 107 STRUCTURAL ANALYSIS OF HEMIN DEMETALLATION BY L-CHAIN APOFERRITINS Robert Crichton, BSc, PhD, FRSC, Natalia De Val, BSc, PhD, Changkee Lim, BSc, PhD and Jean-Paul Declercq, BSc, PhD University of Louvain (Presented By: Robert Crichton, BSc, PhD, FRSC)

There are extensive structural similarities between eukaryotic ferritins and prokaryotic ferritins. However, there is one essential difference between these two types of ferritins: bacterioferritins (BFRs) contain haem (located in a hydrophobic pocket along the 2-fold symmetry axes and liganded by two axial Met residues), whereas eukaryotic ferritins are considered to be non-haem proteins. Previous studies had shown that horse spleen apoferritin, isolated by classic procedures, contains a cofactor with many of the characteristics of a porphyrin, while in a series of in-vitro experiments, it was shown that when horse spleen apoferritin or a number of recombinant horse L 128 chain apoferritins are co-crystallised with haemin, the metalloporphyrin is demetallated, and a demetallated porphyrin is found within the same hydrophobic pocket as in BFRs. In the present study the cofactor has been isolated directly from horse spleen apoferritin and from crystals of the mutant horse L chain apoferritin E53,56,57,60Q + R59M which had been co-crystallised with haemin. In both cases the HPLC/ESI-MS results confirm that the cofactor is the N-alkyl porphyrin, N-ethylprotoporphyrin IX (m/Z 591). Crystal structures of wild type L chain horse apoferritin and its three mutants E53,56,57,60Q / R59M and E53,56,57,60Q + R59M co-crystallised with haemin have been determined to high resolution and in all cases a metal-free molecule derived from haemin was found in the hydrophobic pocket, close to the two-fold axis. The X-ray structure of the E53,56,57,60Q + R59M recombinant horse L-chain apoferritin has been obtained at a higher resolution (1.16 Å) than previously reported for any mammalian apoferritins. Similar evidence for a metal-free molecule derived from haemin was found in the electron density map of horse spleen apoferritin (at a resolution of 1.5 Å) which had been prepared by a method which retains the cofactor. The out-of-plane distortion of the observed porphyrin is clearly compatible with an N-alkyl porphyrin, but because of local disorder it has proved difficult to entirely fit the electron density.The mechanism by which the demetallation and alkylation of the metalloporphyrin might take place, together with the wider implications of this phenomenon in a physiological context are briefly discussed

Poster# 108 DEFINITION OF THE RESIDUES REQUIRED FOR THE INTERACTION BETWEEN GLYCINE-EXTENDED GASTRIN AND TRANSFERRIN IN VITRO Suzana Kovac, PhD², Audrey Ferrand, PhD², Jean-Pierre Estève, PhD³, Anne Mason, PhD4 and Graham Baldwin, PhD, DSc¹ ¹University of Melbourne; ²University of Melbourne Department of Surgery, Austin Health, Melbourne, Australia; ³INSERM U.858, Plateforme d’interaction moléculaire, I2MR, Institut Louis Bugnard, IFR31, Toulouse, France; 4University of Vermont, College of Medicine, Department of Biochemistry, Vermont, USA (Presented By: Graham Baldwin)

Introduction: Transferrin is the main iron transport protein found in the circulation. The level of transferrin saturation in the blood is an important indicator of iron status since it modulates the concentration of hepcidin, the protein responsible for regulation of iron release from cells which store iron. The peptides amidated gastrin17 (Gamide) and glycine-extended gastrin17 (Ggly) are well known for their roles in controlling gastric acid secretion, as well as for their effects as growth factors in the gastrointestinal tract.An interaction between gastrin and transferrin was first demonstrated by covalent crosslinking techniques1. Several recent lines of evidence, including the facts that gastrins bind ferric ions and that the level of expression of gastrins positively correlates with transferrin saturation, suggest the potential involvement of the gastrin/transferrin interaction in iron homeostasis2. Aim: To investigate, using surface plasmon resonance and crosslinking, the residues required for the interaction between gastrins and transferrin. Results: Firstly, an interaction between iron-free apo-transferrin and Gamide or Ggly (Kd = 39 ± 1 mM) was observed. The fact that the interaction was not observed in the presence of the chelator EDTA suggests that the gastrin-ferric ion complex is the interacting species. Moreover, removal of ferric ions with EDTA reduced the stability of the complex between apo-transferrin and gastrins. No interaction was observed between Gamide or Ggly and diferric-transferrin. Secondly, some or all of glutamates 8-10 of Ggly, together with the C-terminal domain of the Ggly molecule, were shown to be involved in the interaction with apo-transferrin. Thirdly, transferrin mutants unable to bind iron in either the N- or C-terminal lobes still bound Ggly. Conclusion: These findings are consistent with the hypothesis that gastrin peptides are involved in the loading of iron onto transferrin in vivo. References: 1Baldwin, G.S., Chandler, R., and Weinstock, J. (1986). FEBS Lett 205, 147-149. 2Kovac, S., Smith, K., Anderson, G.J., Burgess, J.R., Shulkes, A., and Baldwin, G.S. (2008). Am J Physiol Gastrointest Liver Physiol 295, G855-861. This work was supported by USPHS grant R01-DK-21739 (ABM) and NIH grant 5RO1-GM65926-07 (GSB).

Poster# 109 DMT1 EXPRESSION CAN LEAD TO ACCUMULATION OF METALS IN CELLS Michael Garrick, PhD¹, Lin Zhao, MD¹, Saied Ghadersohi¹, Jackie Stonehuerner, BS², Andy Ghio, MD² and Laura Garrick, PhD¹ ¹SUNY at Buffalo; ²EPA (Presented By: Michael Garrick, PhD)

DMT1 (Divalent Metal Transporter 1) has two isoforms starting at the N-termini in exon 1A or 2; and two isoforms having C-termini from exon 16A or 17. As the corresponding mRNAs have or do not have an IRE in the 3’ UTR, the 4 protein isoforms are 1A/+IRE; 1A/-IRE; 2/+IRE; and 2/-IRE. To learn if over-expression of DMT1 in HEK293 cells causes accumulation of metals in the cells, we cloned rat 1A/+IRE and mouse 2/-IRE DMT1 into HEK293 cells subject to doxycycline regulation. Both cell lines incorporated more 59Fe or 54Mn into cells when DMT1 levels were higher (Garrick et al 2006 Biochem. J. 398:539). Because cells exhibited stress at pH 5.5-6.0 (optimal for DMT1), we had to incubate the cells at neutral pH for periods longer than one hour to detect net accumulation of metal substrates. After incubation, cells were separated from the medium and washed; then the supernatant and cell digest were assayed for metals by inductively coupled plasma - atomic emission spectroscopy or mass spectrometry (ICP-AES or - MS). Mn accumulation was time and DMT1 dependent. Other metals that accumulated in a DMT1 dependent fashion included Fe, Cd, Ni, Co and Cu1+. Vanadyl, Tl, Ga, Cu2+, and Ca did not accumulate; while Pb, Zn, Mg and Ti yielded ambivalent data. Some other metals like Pt (as chloroplatin) are still under study. We conclude that one can use accumulation of metals as a way to detect which metals are DMT1 substrates, but that there are factors that limit interpretation of data for some metals. The metal binding properties of DMT1 may also allow one to detect the transporter.

Poster# 110 AGGREGATION AND DISSOCIATION OF PEA SEED FERRITIN REGULATED BY PH AND FERRIC ION Guanghua Zhao, PhD, Chaorui Li, PhD, Xiaoping Fu, PhD, Fang Chen, PhD and Xiaosong Hu, PhD China Agricultural University (Presented By: Guanghua Zhao, PhD)

Iron stored in phytoferritin plays an important role in the germination and early growth of seedlings. The protein is located in the amyloplast where Fe2+ was oxidized and incorporated into its shell-like structure. In the present work, soluble and insoluble forms of pea seed ferritin (PSF) were isolated from dried seeds, and were found to be identical 24mer ferritins composed of 28.0 and 26.5-kDa subunits in a ~2:1 ratio with the insoluble form that has aggregated into large assemblies of ~ 100 – 400 ferritin molecules. The two forms reversibly interconvert in the pH range 6.0 to 7.8 with an apparent pKa ~ 6.8, the insoluble form being favored at low pH. The oxidative addition of Fe2+ to the apoprotein to form Fe3+ or the direct addition of Fe3+ to apoferritin also causes protein aggregation when the binding capacity of the ferroxidase centers (48 Fe3+/shell) is exceeded. Experiments show that excess Fe2+ binding and oxidation occurs on the exterior of the protein at sites involving the N-terminus extension peptide, a structural component unique to plant ferritins, causing the protein to associate into large multimeric species. The oxidized iron eventually migrates to the interior of the protein cavity where it is deposited in the iron core by a process representing a new pathway for iron mineralization of the protein. Concurrently, the protein dissociates back into ferritin monomers. These results point toward a possible role for PSF association/dissociation reactions in the management of iron within the seed.

Poster# 111 THE EFFECT OF COPPER LOADING AND DEPLETION ON IRON TRANSPORTER EXPRESSION IN CACO-2 CELLS Katayoun Pourvali and Paul Sharp, PhD King’s College London (Presented By: Katayoun Pourvali)

Copper is an essential trace metal in the body required for many physiological functions. The intimate relationship between copper and iron metabolism has been known for many years. In in vitro intestinal epithelial cell models, experimental evidence suggests that copper and iron may compete for uptake by divalent metal transporter1 (DMT1). Furthermore, in the same cells hephaestin, a copper-containing ferrioxidase, is required for iron efflux.

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The aim of this study was to investigate the effects of copper on the protein and mRNA expression of the iron transporters DMT1 and Ferroportin, and hephaestin in an intestinal epithelial cell line (Caco-2). Fully-differentiated Caco-2 cells were treated with 50 µM copper chloride, for 4, 8, and 24 hours. In addition, the effect of treating cells with 0.5 mM of Triethylenetetramine dihydrochloride (TETA), a copper chelator, over the same time scale was investigated. We examined the changes in the whole cell levels of transport proteins by western blotting and mRNA expression by Real Time PCR. Western blotting data were semi-quantified using Scion Image software for were analysed by one- way ANOVA and Tukey’s post hoc test (significant at p<0.05). Following exposure to copper for 24h there was a significant decrease in the mRNA expression of DMT1 (p< 0.001) compared with control. Following treatment with TETA for 24h, DMT1 mRNA expression was significantly increased (p<0.006). TETA treatment also resulted in a significant increase in hephaestin (p<0.04) mRNA expression. At the protein level, DMT1 and hephaestin were unaffected by copper or TETA treatment; however, Ferroportin protein expression was transiently and significantly increased by TETA treatment for 4 and 8 hours compared to controls (p< 0.01). These data show that the expression of iron transporters is altered by modulating copper levels. The amount of copper used in these experiments is similar to that used in previous studies; however, it is considerably higher than normal dietary levels. As most of the changes in transporter expression were only evident after 24 hour treatment, the next stage of this study will be to investigate the effects of chronic treatment (up to 7 days) with nutritionally relevant levels of copper (10µM Cu:histidine) or with TETA on transporter expression.

Poster# 112 ACTIVATION OF AN UNFOLDED PROTEIN RESPONSE MODULATES THE EXPRESSION OF IRON-RELATED GENES Susana Oliveira, PhD student, S.F. de Almeida, PhD, G. Picarote, undergraduate student, V.M. Costa, PhD student, F. Carvalho, PhD, J.E. Azevedo, PhD, J.P. Pinto, PhD and M. de Sousa, MD, PhD Instituto de Biologia Molecular e Celular, Universidade do Porto (Presented By: Susana Oliveira, PhD student)

The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) lumen compromises the homeostatic balance within this organelle and in the entire cell. Aiming at relieving such stress, a signaling program known as Unfolded Protein Response (UPR) that includes global repression of translation, induction of ER chaperones and foldases and improvement of ER-associated degradation (ERAD) is elicited [1]. There is growing recognition that the scope of the UPR-derived transcriptional signals is broader than initially anticipated. Transferrin (TF) was identified as a downstream target of CHOP, a stress-inducible transcription factor that can heterodimerize with other members of the same family. Acting as dominant negative inhibitor of C/EBPα, CHOP was reported to down-modulate TF gene expression [2]. Although not formally confirmed, an identical mechanism was evoked to justify the impaired hepcidin expression in two models of hepatic iron overload, induced by HCV [3] and alcohol [4]. The crosstalk between the UPR and cellular iron metabolism remains unclear, however. In the present work we examine whether an UPR affects the expression of relevant iron- related genes. An UPR was pharmacologically elicited in human HepG2 cells by dithiothreitol (DTT) which, preventing the formation of disulphide bonds, burdens the ER lumen with misfolded proteins. The mRNA and/or protein content of GRP94, BiP (two ER-resident chaperones) and CHOP was monitored as hallmark of UPR activation. Gene profiling of HAMP, SLC40A1 and FTH1 (coding for hepcidin, ferroportin and ferritin H, respectively) was performed by qRT-PCR during the DTT-induced UPR. The results show that the expression pattern of the analyzed iron-related genes is modulated in the context of an active UPR. SLC40A1 and FTH1 were both up-regulated in a time- and dose-dependent manner. The temporal expression of HAMP was characterized by an early repression followed by a marked increase after 24 h of DTT-triggered UPR. To provide further insights into the molecular mechanisms underlying such a biphasic regulation, C/EBPα was assessed. A CHOP-dependent decline of C/EBPα protein levels was detected upon 5 h of DTT incubation and might account for the early decrease of hepcidin transcripts. Conversely, an enhanced binding activity of this nuclear factor to HAMP promoter rather than quantitative protein changes, was found to be implicated in the stimulation of hepcidinexpression observed thereafter. Our data provide valuable clues when extrapolated to a physiological scenario. Considering the recently established link between the HFE C282Y mutant protein and the activation of an UPR [5], it is tempting to speculate that a CHOP-C/EBPα interconnection might contribute to the reported poor induction of hepcidin in response to iron load in Hereditary Hemochromatosis. 1. Schroder et al. (2005). Annu Rev Biochem 74: 739-89. 2. You et al. (2003). Hepatology 38: 745-5. 3. Nishina et al. (2008). Gastroenterology 134: 226-38. 4. Harrison- Findik et al. (2007). Hepatology 46: 1979-85. 5. de Almeida et al. (2007). J Immunol 178: 3612-9. Acknowledgments: We thank the Fundação para a Ciência e Tecnologia (FCT) for the financial support through the research project PTDC/BIABCM/66818/2006 and the PhD grant SFRH/BD/19682/2004.

Poster# 113 BMP6 IS A KEY ENDOGENOUS REGULATOR OF HEPCIDIN EXPRESSION AND IRON METABOLISM Billy Andriopoulos, Jr.², Elena Corradini²,5, Yin Xia², Sarah A. Faasse², Shanzhuo Chen², Lovorka Grgurevic³, Mitchell D. Knutson4, Antonello Pietrangelo5, Slobodan Vukicevic³, Herbert Y. Lin² and Jodie L. Babitt, MD¹ ¹Massachusetts General Hospital, Harvard Medical School; ²Program in Membrane Biology, Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ³Laboratory of Mineralized Tissues, School of Medicine, University of Zagreb, Zagreb, Croatia; 4Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida; 5Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena Italy (Presented By: Jodie L. Babitt, MD)

Hepcidin is a key regulator of systemic iron balance. By decreasing cell-surface expression of the iron exporter ferroportin, hepcidin decreases iron absorption from the intestine and iron release from reticuloendothelial cells. Hepcidin deficiency and unchecked ferroportin activity are the common pathogenic mechanisms underlying hereditary hemochromatosis due to mutations in hepcidin itself, hemojuvelin, transferrin receptor 2 or HFE. On the other end of the spectrum, hepcidin is upregulated by inflammatory cytokines and hepcidin excess is implicated in the pathogenesis of anemia of inflammation. We have previously shown that hemojuvelin, the gene mutated in most cases of juvenile hemochromatosis, is a bone morphogenetic protein (BMP) co-receptor and that hemojuvelin-mediated BMP signals regulate hepcidin expression and iron metabolism. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here, we show that compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc is a more potent inhibitor of hepcidin promoter activation by BMP2 and BMP4, but a less potent inhibitor of BMP6 in vitro. In vivo, DRAGON.Fc has no effect on hepcidin expression and iron metabolism. In contrast, HJV.Fc or a neutralizing BMP6 antibody inhibits hepcidin expression and increase serum iron. Furthermore, Bmp6 null mice have a phenotype that resembles hereditary hemochromatosis with reduced hepcidin expression, increased ferroportin expression, increased serum iron and transferrin saturation, reduced spleen iron stores, and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP6 protein by pull-down assay, and we show that BMP6 administration in mice increases hepcidin expression and reduces serum iron. Together, these data support a key role for BMP6 as a ligand for HJV and an endogenous regulator of hepcidin expression and iron metabolism in vivo. Our data suggest that selective BMP6 inhibitors may be effective agents for treating anemia of inflammation due to hepcidin excess. Additionally, BMP6-like agonists may be an alternative treatment strategy for managing iron overload disorders in patients resistant to current therapies. Although no human patients with BMP6 mutations have yet been described, our data also suggests that BMP6 mutations or BMP6 gene variants may function as another cause of hereditary hemochromatosis or a modifier of disease penetrance.

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Poster# 114 FUNCTION OF THE IRP/IRE REGULATORY NETWORK IN HEPATOCYTES Bruno Galy, PhD, Dunja Ferring-Appel, Lydie Viatte, Sylvia Kaden, Hermann-Josef Groene and Matthias Hentze European Molecular Biology Laboratory (Presented By: Bruno Galy, PhD)

The liver plays a key role in iron homeostasis. It constitutes the major storage depot for excess iron and secretes important effectors of iron metabolism (e.g. transferrin and ceruloplasmin); importantly, it expresses the iron hormone hepcidin and molecules that sense systemic cues (e.g. HFE, hemojuvelin, TfR2) to adjust its synthesis. It is also equipped with the IRP/IRE regulatory system that coordinately regulates the posttranscriptional expression of genes involved in cellular iron uptake, utilization, storage, and export. To dissect the hepatocytic function(s) of the IRE/IRP regulatory network in vivo, we have generated conditional alleles of the murine Irp1 and Irp2 genes, respectively, which enable total as well as cell-specific ablation of IRP1 and/or IRP2 using Cre/Lox technology. Mice with total and constitutive IRP2 deficiency display increased iron levels and ferritin upregulation in the liver, showing that IRP2 is important for hepatic iron metabolism; furthermore, IRP2 appears to be dispensable for adequate hepcidin regulation in mice subjected to dietary iron manipulation, to acute sterile inflammation upon turpentine oil injection, or to phenylhydrazine-induced hemolytic anemia. Mice with selective IRP2 deficiency in the liver were obtained by crossing mice harbouring conditional Irp2 alleles with a transgenic strain expressing the Cre recombinase under the control of both mouse Albumin regulatory elements and the Alpha fetoprotein enhancers (Alfp-Cre line). Interestingly, these animals display liver iron loading associated with ferritin upregulation, showing that the hepatic manifestation of systemic IRP2 deficiency can be explained by cell autonomous functions of IRP2. Importantly, hepatic iron loading in mice with systemic or hepatocytic IRP2 deficiency does not affect basal hepcidin mRNA expression. In contrast to animals with IRP2 insufficiency, mice lacking IRP1 expression exhibit no phenotypic abnormality under standard laboratory conditions, likely due to functional redundancy between IRP1 and IRP2. Hence, we used the Alfp-Cre line to bypass the embryonic lethality of mice with systemic deficiency for IRP1 and IRP2 and selectively ablate both IRPs in hepatocytes, shortly after the appearance of the liver bud (~E9.5-E10.5). While mice with complete IRP deficiency in hepatocytes are macroscopically indistinguishable from wild-type littermates at birth, they die between 8 and 12 days of age, showing that the IRE/IRP system is essential for hepatic function and organismal survival. To overcome the early lethality in animals with embryonic IRP deficiency and disrupt both Irp genes in the liver of adult mice, we are now using a Cre-deletor strain with hepatocyte-specific expression of a ligandinducible Cre recombinase.

Poster# 115 FUNCTINAL RNAI SCREEN IDENTIFIES CCL2 (MCP-1) AS A NOVEL REGULATOR OF TRANSFERRIN UPTAKE Martina Muckenthaler¹, Mingang Zhu, PhD²,³, Fabian Bartz³,4, Till Meinhof4, Holger Erfle5, Matthias Hentze, MD³,6, Rainer Pepperkok, PhD³,5 and Heiko Runz, MD³,4 ¹Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg; ²Department of Pediatrics, University of Heidelberg; ³Molecular Medicine Partnership Unit (MMPU), University of Heidelberg/EMBL Heidelberg; 4Institute of Human Genetics, University of Heidelberg; 5Cell Biology/Biophysics Unit, European Molecular Biological Laboratories (EMBL); 6Gene Expression Unit, European Molecular Biological Laboratories (EMBL) (Presented By: Martina Muckenthaler)

Cellular iron homeostasis must be precisely maintained to avoid cell damage due to iron excess or deficiency. Most cell types control iron levels by regulating uptake of iron-bound transferrin via the ubiquitously expressed transferrin receptor 1 (TfR1). To identify novel regulators of transferrin uptake we selected candidate genes by gene expression profiling of cells and tissues with altered iron levels. By applying targeted siRNA knock-down using RNAi arrays and automated high throughput microscopy we assessed their role in regulating fluorescent labelled transferrin-uptake. We identified 14 novel genes that either enhance or diminish the uptake of transferrin. Selected genes were then further characterized for their role in regulating TfR1 mRNA and protein expression. These data functionally link PTP4A1, TGFBI, ATP6V1B2, HSPA1A, PROM1, CTH and KPNB1 among others to the regulation of Tf uptake. Interestingly, the iron-regulated chemokine CCL2 (MCP-1), a member of the proinflammatory CC chemokine superfamily that was recently shown to be expressed at low levels in patients with hereditary hemochromatosis (HH) was identified as a repressor of Tf uptake. The siRNA-mediated knock-down of CCL2 increases TfR1 mRNA and protein expression mediated in parts by increased activity of IRP1, the central cellular regulator of iron homeostasis. Our data thus functionally link the regulation of iron uptake by CCL2 to the recruitment of monocytes and lymphocytes during inflammation, a mechanism that may be relevant in hereditary hemochromatosis.

Poster# 116 HEPCIDIN TRIGGERS REDUCTION IN DMT1 PROTEIN IN INTESTINAL CELLS Carole Lagnel, Dr¹, Andre Bado, Dr¹, Philippe Letteron, Dr¹, Corinne Nazaret¹, Dimitri Tchernitchko, Dr¹, Soumeya Bekri, Dr² and Carole Beaumont, Dr¹ ¹INSERM U773; ²University of Rouen, Rouen (Presented By: Carole Lagnel, Dr)

Hepcidin is the key regulatory hormone that mediates iron homeostasis. It is a small peptide produced by liver and secreted into the circulation that acts at two critical sites of iron homeostasis: tissue macrophages and small intestin. Recently, the mechanism by wich hepcidin regulates iron export from macrophage has been well established. However, in the small intestin the effect of hepcidin is unclear. In the duodenum, following the reduction of iron from the ferric to the ferrous form by a duodenal ferric reductase, iron crosses to the cytoplasm via the (+IRE) isoform I of the divalent metal transporter-1 (DMT1), an apical iron transporter. Iron is either stored by ferritin or exported out of the enterocytes into the bloodstream by ferroportin, a basolateral transporter. In this study, the influence of hepcidin on intestinal iron absorption was investigated. The action of hepcidin (Peptide International) was studied on cultured epithelial intestinal cell line, CaCO-2/TC7 and on ex vivo isolated mouse duodenal segments. DMT1 and ferroportin expression were analyzed by RT-qPCR at the mRNA levels and by Western blotting at the protein level. A construct expressing the isoform I of DMT1 fused to eGFP was transfected into CaC02/TC7 cells and the effect of hepcidin was followed in real time by in vivo confocal imaging. The effect of hepcidin on transepithelial iron transport was studied using 55FeCl3 in CaCO2 cells and ex vivo isolated duodenal segments. Expression of DMT1 and ferroportin is strongly activated during polarization of CaCO2 cells grown on filters and reaches a maximum at 16 days of culture. Addition of hepcidin at the baso-lateral side of polarized cells induces a progressive reduction in DMT1 and ferroportin mRNAs. The effect is maximum at 2 hours, and dose-response curves show that the maximum effect is obtained at 200 nM hepcidin, with a half maximal effective concentration of 75 nM. This corresponds to the physiological concentration of hepcidin in plasma. Surprisingly, when we incubated the polarized cells for 2hrs in the presence of 200 nM hepcidin at the baso-lateral side, we only observed a two-fold drop in the amount of DMT1 protein, without changes in ferroportin, on the contrary to what is found in macrophages. This reduction in DMT1 protein is blocked when the cells are pre-incubated with MG132, a proteasome inhibitor. This result was confirmed by confocal imaging on live CaCO2 cells transfected with a DMT1 (+IRE)-GFP encoding vector. Incubation of the transfected cells 2 hrs with 200 nM hepcidin showed progressive internalization and reduction in DMT1 protein. The same concentration of hepcidin was also able to inhibit by 50 % the apical uptake of 55Fe by polarized CaCO2 cells and by ex vivo isolated duodenal segments. In conclusion, these results show that hepcidin could lead to an inhibition of intestinal iron absorption through an increase of proteasome-dependent degradation of DMT1. We propose that minor fluctuations in hepcidin concentrations within the physiological range inhibits intestinal iron absorption, by a mechanism not entirely dependent on ferroportin, whereas more important increases in hepcidin levels trigger ferroportin internalization and degradation in macrophages.

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Poster# 117 INTESTINAL IRON ABSORPTION IN NEONATAL RATS DOES NOT CORRELATE WITH CHANGES IN THE EXPRESSION OF THE IRON REGULATORY HORMONE HEPCIDIN Deepak Darshan, MBBS, PhD, David M. Frazer, PhD, Sarah J. Wilkins, BSc (Hons) and Greg J. Anderson, PhD Queensland Institute of Medical Research, PO Royal Brisbane & Women’s Hospital, Brisbane, QLD 4029, Australia (Presented By: Deepak Darshan, MBBS, PhD)

Intestinal iron absorption is extremely high in neonatal mammals to cater for the high iron requirements associated with rapid growth and development, and is thought to reflect intrinsic characteristics of the neonatal intestine. We have previously shown that increased expression of iron transport molecules, particularly ferroportin (Fpn), in the distal regions of the GI tract correlates well with the increased absorption. The aim of this study was to investigate whether changes in systemic iron homeostasis (and in particular the iron regulatory hormone hepcidin, the principal regulator of Fpn function) also affect iron absorption in neonates as they do in adults. Sprague Dawley rats were used for all experiments and animals were studied at 15 (suckling) and 49 days post partum. At each time point animals were injected i.p. with iron dextran (300mg/kg) or bacterial lipopolysaccharide (LPS)(0.1mg/kg). Blood, liver and proximal small intestinal samples were collected 4 days after iron dextran injection and 6 hrs after the injection for LPS. Total RNA was extracted from liver and gut to study the expression of Hamp (the gene that encodes hepcidin) and intestinal iron transporters by ribonuclease protection assay. In a separate group of animals at the same time points treated in the same way, the absorption of an oral dose of 59Fe was measured by whole body counting. Intestinal iron absorption was very high in 15 day old neonates and declined significantly on weaning (93.1% vs 39.1%). Treatment with iron dextran led to a small decrease in absorption in 15 day old rats (93.1% vs 79.9%), but a much larger change in the weaned animals (39.1% vs 18.4%). In both groups of animals, iron dexran treatment greatly increased hepatic iron levels (120 fold in neonates and 10 fold in adults). LPS administration did not alter iron absorption in the neonates (93.1% vs 88.2%), but led to a 2.6 fold decrease in weaned rats (39.1% vs 14.9%). The expression of intestinal Fpn mRNA paralleled the absorption responses, and was much higher in the distal GI tract in neonates than in the adult animals. The limited response of neonatal iron absorption to systemic stimuli was not reflected in the expression of Hamp. Hamp mRNA expression was negligible in untreated neonates but increased substantially following both iron dextran and LPS administration. A similar pattern was seen in the weaned animals, although the effect of iron dextran administration was less pronounced. These data suggest that iron absorption in the neonate is independent of changes in the expression of the systemic iron regulator hepcidin. They suggest that either the link between hepcidin and its major target FPN is uncoupled in neonates, or that ferroportin does not play a critical role in intestinal iron transport in the neonatal intestine.

Poster# 118 PHLEBOTOMY-INDUCED IRON MOBILISATION FROM TISSUES OF HJV-/- MICE Jan Krijt, PhD, Ludek Sefc, Martin Vokurka, Tereza Hlobenova and Emanuel Necas Charles University in Prague, First Faculty of Medicine, Institute of Pathophysiology and Center of Experimental Hematology (Presented By: Jan Krijt, PhD)

Introduction: Phlebotomy is the standard treatment for patients with hemochromatosis, including juvenile hemochromatosis. It has been demonstrated that repeated phlebotomies decrease liver hepcidin expression, thus increasing ferroportin levels and iron export from tissues. Interestingly, phlebotomy treatment is also effective in patients with juvenile hemochromatosis, which already have very low hepcidin levels. The purpose of the presented study was to examine the effect of repeated phlebotomies on tissue iron mobilisation from Hjv-/- mice. Methods: Hjv-/- mice were a generous gift from prof. Silvia Arber, Basel, Switzerland. Six months old male and female Hjv-/- animals were bled once weekly for 7 weeks. mRNA levels were determined by real-time PCR. Results: During the seven weeks of phlebotomy treatment, approximately 1.8 mg of iron was removed from the animals. In Hjv-/-males, liver iron content decreased from 1000 µg/g wet wt. to 150 µg/g, pancreas iron content from 2100 µg/g to 1500 µg/g and myocardium iron content from 480 µg/g to 300 µg/g. In Hjv-/- females of the same age, liver iron decreased from 1390 µg/g to 130 µg/g and pancreatic iron content from 690 µg/g to 450 µg/g. Compared to the liver, iron mobilisation from pancreas and myocardium was several times slower. Particularly, the high levels of pancreatic iron in male Hjv-/- mice decreased only by 30 % during phlebotomy treatment. The calculated approximate daily iron loss from the liver was 25 µg/g. Hamp1 mRNA content in Hjv-/- males was by an order of magnitude lower when compared to Hjv+/+ mice; phlebotomies resulted in a further decrease by up to 4 PCR cycles. No effect on hepatic ferroportin mRNA or ceruloplasmin mRNA was observed. To establish the rate of iron accumulation in Hjv-/- mice, an additional experiment was set up, in which Hjv-/- mice were fed on low-iron diet since birth, and switched to normal diet at six weeks of age. After 17 days on normal diet, the animals were sacrificed. During normal diet feeding, liver iron content in Hjv-/- animals rose from 20 to 430 µg/g wet weight, representing liver iron accumulation at a rate of approximately 20 µg/day. Discussion: Repeated phlebotomies are known to effectively decrease liver iron content in hemochromatosis patients. The effective mobilisation of hepatocyte iron in HFE hemochromatosis can be possibly explained by the phlebotomy-induced decrease of hepcidin expression, and the resulting increase in ferroportin protein levels. However, such a mechanism of hepatocyte iron mobilisation is probably not of much importance in juvenile hemochromatosis, where ferroportin-mediated hepatocyte iron export must be very high even in untreated patients, due to low hepcidin levels. A possible explanation for the efficiency of phlebotomy treatment in juvenile hemochromatosis is the lowering of plasma iron, and the consequent decrease of iron import into the hepatocyte. This model postulates a relatively high rate of hepatocyte iron turnover, which, in Hjv-/- mice, reaches values comparable with the daily iron requirement of erythropoiesis (about 25 µg/day). Supported by grants MSM 0021620806 and LC06044 from the Ministry of Education of the Czech Republic.

Poster# 119 THE ROLES OF IRON AND FERRITIN IN ISCHEMIC PRECONDITIONING OF THE NON-DIABETIC AND DIABETIC HEART Mottie Chevion, BSc, MSc, PhD, Vladimir Vinokur, MSc, Baruch Bulvik, MSc, Abraham Konijn, PhD and Eduard Berenshtein, MD, PhD Hebrew University of Jerusalem (Presented By: Mottie Chevion, BSc, MSc, PhD)

Objectives: Ischemic preconditioning (IPC) is a well-known procedure transiently protecting the heart against injury associated with prolonged ischemia and 'reperfusion injury'. The mechanism/s proposed are only partly understood. Myocardial IPC protection is impaired in hearts of animals with experimentally-induced diabetes. The aim of this study was to examine the involvement of an iron-based mechanism in IPC protection - generation of an iron signal during IPC, which is followed by accumulation of ferritin (Ft), in non-diabetic (control) and diabetic hearts. Methods: We Used isolated rat hearts (Langendorff configuration), from control and streptozotocin-induced diabetic animals. Heart function and biochemical parameters, including Ft levels, Ft-iron content, and mRNA levels of Ft subunits were monitored. The biochemical parameters were measured at four pre-determined time points along the experimental proteocol: at the end of the stabilization phase (baseline), completion of the IPC procedure (IPC – composed of three cycles of 2min ischemia followed by 3min perfusion), end of ischemia (I) (35min), and after 60min of reperfusion (R). Results: In control hearts - IPC caused rapid accumulation of Ft, reaching 3.6-fold of the baseline value. This was accompanied by a parallel decline in Ft-bound iron content: from 2200±550 down to 760±30 Fe atoms/ferritin molecule, p<0.05. Ft levels remained high during the subsequent prolonged ischemia phase, and returned to nearly the baseline value during the reperfusion phase. Selective iron chelators, which infiltrate into cells (acetyl hydroxamate or Zn-desferrioxamine), abrogated the functional protection and suppressed Ft accumulation. These demonstrated the essentiality of the IPC-induced generation of an iron signal, in the protective mechanism. Also, the correlation between high Ft and functional protection indicated that Ft plays an important role in IPC protection. Moreover, introducing, into the perfusate, an iron- containing ternary complex known to import iron into cells, caused a 3-fold accumulation of Ft and simulated the functional protection against prolonged ischemia. Following IPC, the relative levels of mRNAs of H- and L-Ft subunits remained unchanged, versus baseline values, indicating an IPC-mediated activated Ft-translation. In contrast, when hearts were subjected to (IPC+I) - the increase in Ft levels correlated well with the accumulation of the L-subunit mRNA, versus the baseline, suggesting that transcriptional control of L-subunit synthesis had been activated. This change in the relative level of mRNAs of the subunits, giving rise to a marked change in Ft protein composition, could explain the rapid dissipation of Ft during reperfusion. In diabetic hearts – analogous correlations were obtained: increased resistance of these hearts to 132

I/R and their inability to respond to IPC went together with changes in Ft levels. Conclusions: The results from both control and diabetic hearts could be explained by the iron-based mechanism of IPC protection, and further substantiated the pivotal role of iron homeostasis in adequate heart function. IPC initiates de novo synthesis of Ft in the control heart, but not in the diabetic myocardium; the extra Ft is proposed to serve as a 'sink' for labile and redox-active iron, thus protecting the heart against the 'reperfusion injury'. The results substantiate the validity of the iron-based protective mechanism of heart IPC, and could pave the way for the development of new modalities of heart protection.

Poster# 120 IMMUNOASSAY FOR HUMAN HEPCIDIN IN BLOOD AND DEVELOPMENT OF A PROTOTYPE ELISA KIT Mark Westerman, BS, MS, PhD¹, Gordana Olbina, BS, MS, PhD¹, Siong Wie, BS, PhD², Vaughn Ostland, BS, MS, PhD¹, Domenico Girelli, MD, PhD³, Kimberley O'Brien, BS, PhD4, Elizabeta Nemeth, BS, MS, PhD¹ and Tomas Ganz, BS, PhD, MD¹ ¹Intrinsic LifeSciences; ²Immunodiagnostic Reagents Inc; ³University of Verona; 4Cornell University (Presented By: Mark Westerman, BS, MS, PhD)

We developed and validated the first ELISA for measurement of hepcidin is serum, plasma, and urine. Hepcidin is the principal iron-regulatory hormone in humans and has been a very difficult antigen to raise antibodies against due to it’s compact structure and conserved sequence in mammals. The competitive ELISA uses a biotin-tagged hepcidin that retains bioactivity against ferroportin as a tracer (competitor) in the assay with bioactive hepcidin as reference material for construction of duplicate 12 point standard curves. In healthy volunteers, the 5-95% range of serum hepcidin was 29-254 ng/ml in men (n=65) and 17-286 ng/ml in women (n=49), with median concentrations 112 vs. 65, p<0.001. The lower limit of detection was 5 ng/ml serum. Serum, EDTA plasma, and heparin plasma hepcidin measurements in matched samples were strongly correlated (r> 0.98). Matched serum and urinary hepcidin concentrations in 24 healthy subjects correlated well (r=0.82) and serum hepcidin was positively correlated with serum ferritin (r=0.63) in healthy subjects, reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8 pm compared to 8 am, and transient rise of serum and urine hepcidin in response to ingestion of iron sulfate in a volunteer. In 18 young healthy women, serum hepcidin correlated well (p<0.05) with intestinal iron absorption both from a food (r=0.49) and a supplemental iron source (r=0.51) when determined by stable iron isotope techniques. The assay also correlates closely (r> 0.97) with a rigorously validated isotope dilution micro-HPLC tandem MS (MS/MS) assay for hepcidin that allows quantification at nanomole/L levels. In a variety of clinical conditions associated with iron disturbances we observed the appropriate changes in hepcidin concentrations. Hepcidin was undetectable or low in patients with iron deficiency anemia (ferritin<10 ng/ml), and treated HFE hemochromatosis and juvenile hemochromatosis patients. Conversely, hepcidin was abnormally high in patients with inflammation (CRP>10 mg/dl), multiple myeloma, or chronic kidney disease. The new blood hepcidin ELISA yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic and genetic background, and is informative about etiology of iron disorders. To expand the availability of this competitive ELISA assay to investigators worldwide, we have produced the first prototype 96-well microtiter plates that will be the major component of a kit. A lot of five microtiter plates were coated with rabbit anti-hepcidin antibodies, blocked with a protein solution and dried under vacuum. The plates were packaged in a vacuum sealed package with a desiccant until testing. Evaluation of these first prototype hepcidin ELISA plates was performed by measuring 6 replicates from two plasma and one urine sample independently on four plates. The samples had mean hepcidin concentrations of 22.93, 54.25, and 1156.85 ng/ml, respectively. In the preliminary experiments, intra-assay coefficient of variability (CV) ranged from 3.9-13.7% and inter-assay CV for four experiments ranged from 5.74-19.1%. The variability decreased with increasing hepcidin concentration in the sample and was similar to results generated with freshly coated and blocked plates used for routine analysis. These data provide strong proof-of-principle that transfer of the hepcidin ELISA to 96-well microtiter kit is feasible.

Poster# 121 ALTERATIONS IN IRON STATUS ARE ASSOCIATED WITH CHANGES IN THE CHOLESTEROL BIOSYNTHESIS PATHWAY Ross M. Graham, BSc (Hons) PhD¹, Anita C.G. Chua, BSc (Hons) PhD¹, Kim Carter, BSc (Hons) PhD², Roheeth D. Delima, BSc (Hons)¹, Daniel Johnstone, BSc (Hons)³, Carly E. Herbison, BSc (Hons)¹, Martin J. Firth, BSc (Hons) PhD², Rebecca O’Leary, BSc (Hons) PhD², Elizabeth A. Milward, BSc (Hons) PhD³, John K. Olynyk, MBBS, MD¹ and Debbie Trinder, BSc (Hons) PhD¹ ¹University of Western Australia and Western Australian Institute for Medical Research; ²University of Western Australia and Telethon Institute for Child Health Research, Western Australia; ³University of Newcastle, New South Wales, Australia (Presented By: Ross M. Graham, BSc (Hons) PhD)

Iron is an essential trace element and an important structural or functional component of many biochemical systems. The liver is the major storage site for iron and is important in the regulation of iron metabolism being the site of production of the hormone hepcidin which regulates iron absorption. Iron metabolism is tightly regulated; nevertheless, changes in body iron status can occur and may have serious clinical consequences ranging from fatigue to liver cirrhosis and hepatocellular carcinoma. The biochemical changes leading to these consequences are difficult to predict due to the wide variety of processes in which iron is involved. However, iron has been linked to changes in many metabolic processes including those of glucose and insulin, leading to diabetes, and to disorders of lipid metabolism, such as non-alcoholic fatty liver disease. Iron deficient, iron loaded and control mice were used to investigate changes in gene expression associated with altered iron status. To investigate the effects of mutations which cause iron overload, two mouse models of haemochromatosis (Hfe-/- and TfR2Y245X) were also examined. Mice were fed a diet containing 2% carbonyl iron (iron loaded), 0.001% iron (iron deficient) or 0.01% iron (control, Hfe-/- and TfR2Y245X). Mice were sacrificed at 10 weeks, perfused with isotonic saline and organs harvested. Iron status was assessed by measurement of liver non-haem iron. Total RNA was extracted from the liver and subjected to whole-genome microarray analysis using Illumina Sentrix Mouse-6 (v1.1) Beadarray chips. Data were normalised using the Lumi Bioconductor package. Isotonic regression was used to rank changes in gene expression with increasing iron burden and gene set enrichment analysis was conducted using GSEA 2.0 to identify pathways enriched with these genes. Liver non-haem iron concentration of control mice was 5.5±0.5 (mean ± SE) µmolFe gwet wt liver -1, whilst that of iron deficient mice was 2.4±0.2 and iron loaded mice was -/- Y245X 19±1. Liver non-haem iron concentrations of Hfe and TfR2 mice were 16.6±0.8 and 17±1 µmolFe gwet wt liver -1, respectively, not significantly different from iron loaded mice. The transcripts of many enzymes in the cholesterol synthesis pathway were identified as being upregulated with increasing iron burden. Transcript levels of HMG- CoA reductase, the enzyme catalysing the rate-limiting step in cholesterol biosynthesis, correlated positively with liver non-haem iron (P<0.01), as did the transcript levels of sterol-14 demethylase (P<0.02). Transcript levels of ∆24-sterol reductase, which catalyses, inter alia, the reaction that produces cholesterol, also correlated positively with liver non-haem iron (P<0.001). Interestingly, genes in the cholesterol synthesis pathway were not identified as being significantly dysregulated in mice with mutations associated with haemochromatosis compared to iron loaded mice These results suggest that cholesterol metabolism may be altered in response to changes in iron status and that the alterations in the cholesterol pathway are independent of the genetic changes which cause haemochromatosis.

Poster# 122 DIFFERENCES IN GENE EXPRESSION OF DUODENAL IRON TRANSPORTERS IN HEMOCHROMATOSIS SUBJECTS WITH AND WITHOUT IRON OVERLOAD James Nelson, PhD¹, Stuart Raaka, Virginia Mugford, Ellen Kilcourse and Kris Kowdley, MD² ¹Benaroya Research Institute; ²Benaroya Research Institute and Virginia Mason Medical Center (Presented By: James Nelson)

Background: Several large-scale population screening studies have shown that a substantial proportion of C282Y homozygotes may not have phenotypic expression of iron overload. Differences in duodenal iron absorption could explain the variable penetrance of the HFE C282Y homozygous genotype. The goal of this study was to test the following hypotheses: A) the incomplete penetrance of the C282Y mutation is the result of differences in duodenal iron transporter gene expression; B) iron depletion therapy would increase expression of duodenal iron transporter genes and; C) there exists a positive coordinated regulation of DMT1 and FPN1 expression and a negative regulation of these genes in response to body iron stores. 133

Methods: Duodenal biopsy samples were analyzed using RT-PCR for expression of DMT1, FPN1, DCYTB and HEPH relative to GAPDH from 23 C282Y homozygotes (HH), including 5 “non-expressors” (serum ferritinResults: We did not observe decreased expression of any of these genes between the HH non-expressor and untreated HH patients with elevated ferritin or normal controls. In contrast, iron-depleted HH patients showed significantly increased expression of DMT1 compared to untreated patients (p= 0.048) or controls (p= 0.033). DMT1(IRE) expression was also significantly increased in iron depleted HH patients compared to both non-expressors and untreated patients (p= 0.024). DMT1(IRE) expression was increased in non-expressors compared to untreated HH patients (p=0.026). There was no significant difference between HH groups in the expression of FPN1, HEPH or DCYTB. A positive relationship was observed among all HH patients regardless of phenotype or treatment status between relative expression of FPN1 and DMT1 (r=0.6948, p=0.0001); between FPN1 and DCYTB (r=0.5377, p=0.0056); between FPN1 and HEPH (r=0.5868, p=0.0020); and between DCYTB and HEPH (r=0.6692, p=0.0003). Untreated HH patients appeared to cluster into two separate subgroups according to their serum ferritin level; high ferritin (n=5; 2085.6±1248.2 µg/L or low ferritin (n=4; 845.8±364.0 µg/L, p=0.05). The untreated high ferritin subgroup had significantly higher gene expression of DMT1 (8.4 fold, p=0.0002) and FPN1 (5.7 fold, p=0.013) compared to the untreated low ferritin subgroup. An inverse relationship between ferritin and DMT1 (r=-0.892) or FPN1 (r=-0.591) which did not achieve statistical significance was observed in the low ferritin subgroup. Conclusions: These data suggest that the incomplete penetrance of the C282Y homozygous mutation cannot be explained by differences in duodenal expression of iron transport genes. Phlebotomy therapy is associated with upregulation of DMT1 expression in HH patients. There is coordinated regulation of FPN expression to the other iron transport genes DMT1, DCYTB and HEPH and also between DCYTB and HEPH. Finally our data suggest that variability in iron overload in HH patients may be due to a compensatory response to downregulate iron transport genes.

Poster# 123 IDENTIFICATION OF AN IRON-REGULATED E3 UBIQUITIN LIGASE AND ITS ROLE IN IRON REGULATORY PROTEIN 2 STABILITY Ameen Salahudeen, BS, Joel Thompson, BS and Richard Bruick, PhD UTSW Medical Center (Presented By: Richard Bruick, PhD)

While iron is an important cofactor for a number of proteins, the same chemical properties that facilitate its biological roles lead to toxic side reactions that damage macromolecules when it is present in excess. For this reason the availability of intracellular iron must be tightly regulated. Mammalian cellular iron homeostasis is maintained in part by the coordinate posttranscriptional regulation of a number of gene products responsible for iron uptake, release, utilization, and storage. Expression of these genes is regulated at the level of translation or mRNA stability through the actions of Iron Regulatory Proteins 1 and 2 (IRPs) that bind to Iron Responsive Elements within their mRNAs. While it is known that the IRP2 protein is preferentially degraded under iron replete conditions, the underlying mechanism of how cells sense iron levels and subsequently regulate IRP2 degradation is incompletely understood. Using a siRNA screen we have recently identified an E3 ubiquitin ligase whose RNAi- mediated knockdown results in stabilization of IRP2 under conditions of excess iron. Additional data suggests that IRP2 may be a direct substrate for ubiquitination by this E3 ligase. Interestingly, the E3 ligase itself responds to changes in cellular iron availability, suggesting a model by which the IRP arm of the cellular iron homeostasis pathway is regulated.

Poster# 124 59FE-DISTRIBUTION IN CONDITIONAL FERRITIN H KNOCKOUT MICE Klaus Schuemann, Dr¹, Anna Even¹, Stefan Wagner¹, Heike Weinheimer, MD¹, Depak Darshan, PhD², Liviu Vanoaica² and Lukas Kuehn, PhD² ¹Technical University Munich; ²ISREC (Presented By: Klaus Schuemann, Dr)

Background and Objectives: Ferritin H knock-out (KO) should abolish ferritin iron storage due to lack of redox capacity. Here we analyse 59Fe-storage and release Fe from the tissues of MX-Cre inducible ferritin H KO mice. Material and Methods: Iron adequately fed male adult C57BL6 mice with lox-Cre inducible ferritin H knock-out as described earlier (Kühn et al., Abstract IBIS 2007, Kyoto) and their Cre-negative litter mates were i.v. injected with trace amounts of 59Fe to analyse iron distribution kinetics (Schümann et al., 2007, Toxicology 241: 19-32). Ferritin H KO was induced by i.p. poly-IC injection to compare 59Fe-distribution in 11 tissues between induced MX-Cre mice and Cre-negative controls 12h, 24h and 7d after injection (n=8-10). KO was also induced under steady-state conditions 28d after 59Fe-injection to check 59Fe-mobilisation from all 11 tissues on days 2-8 after KO induction (n=4 for each day). Hb, hk and non-haem iron in liver and plasma were determined by standard procedures. Results: Mx-Cre and Cre-negative mice showed no significant differences in hb, hk, or plasma- and liver non-haem iron content. In contrast to Crenegative controls, MX- Cre-mice with fully induced ferritin H KO showed a transitory increase in 59Fe plasma concentrations 12h after injection. In contrast to Cre-negative controls no transient 59Fe-increments after 24h in tissues like e.g. heart, muscle or stomach. 59Fe-content in spleen and liver, in contrast, was markedly lower in induced MX-Cre mice 24h and 7 days after 59Fe-injection. In compensation, 59Fe content e.g. in heart and kidney increased steadily over 7 days and 59Fe associated more rapidly with circulating erythrocytes to reach similar levels 7 after days, as 59Fe-incorporation in blood catches up in Cre-negative controls. When ferritin H KO was induced 28d after 59Fe injection, 59Fe-retention was markedly lower in liver, bone marrow, and spleen from MX-Cre mice as compared to Cre-negative litter mates, starting on days 6, 3 and 2 after KO-induction, respectively. No significant 59Fe-mobilisation from other tissues or increased 59Fe-losses from the body were observed. Discussion and Conclusions: Analysis of 59Fe-distribution kinetics after ferritin H KO shows lower standard deviations and is more sensitive than analysis of 59Fe- mobilsation from the tissues in 59Fe-preloaded mice and subsequent KO-induction, which may be due to high 59Fe-background radiation in tissues from these mice. Still, both methods agreed in their results. Ferritin H KO mice released sizable 59Fe quantities only from liver, spleen and bone marrow – these organs had shown most marked reduction of ferritin H mRNA before. However, organs with no predominant function in iron homoeostasis (e.g. heart, kidney, stomach) had lost the capacity for transient storage of 59Fe from bolus-doses observed in Cre-negative controls.

Poster# 125 GENOME-WIDE SIRNA SCREEN TO IDENTIFY REGULATORS OF THE HEPCIDIN/FERROPORTIN REGULATORY SYSTEM Sandro Altamura, PhD¹, Wolfgang Gilles, PhD², Heiko Runz, PhD², Rainer Pepperkok, PhD³, Matthias Hentze, Prof4 and Martina Muckenthaler, Prof² ¹MMPU – Molecular Medicine Partnership Unit; ²University of Heidelberg / MMPU; ³EMBL – European Molecular Biology Laboratory; 4EMBL / MMPU (Presented By: Sandro Altamura, PhD)

Ferroportin (Fpn, SLC40A1) is a transmembrane protein mainly expressed in hepatocytes, duodenal enterocytes, macrophages, and placental trophoblasts. To date, it is the only known cellular iron exporter in vertebrates. The hepatic peptide Hepcidin binds to Fpn, induces its phosphorylation at Tyr 302 and 303 and its subsequent internalization and degradation. Hence the hepcidin/Fpn regulatory system controls dietary iron absorption, iron release from macrophages, mobilization of iron from hepatic stores and iron transfer across the placenta. Dysregulation of the hormonal hepcidinferroportin-system causes frequent iron-related diseases like hereditary hemochromatosis, secondary iron overload in iron-loading-anemias, iron refractory iron deficiency anemia (IRIDA) as well as the anemias of chronic inflammation (ACD). To further our understanding of the hepcidin/ferroportin regulatory system we established robust experimental conditions to conduct a genome-wide siRNA screen to identify regulators of hepcidin-mediated ferroportin-internalisation and degradation. We established a stable cell line that expresses the ferroportin/EGFP fusion protein in an inducible manner. In this cellular model the fluorescent signal indicative of ferroportin protein expression is visualized on the cell surface by microscopy. In a time- dependent manner, Hepcidin treatment shifts the fluorescence from the outer membrane to intracellular vesicles until the signal disappears. With this cell-based assay in hand we currently apply a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. In principle, synthetic siRNAs that represent a subset of 27.306 genes, the druggable genome (Ambion Inc) are arrayed in chambered slides, incubated with cells and Fpn expression in response to hepcidin treatment is probed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. This innovative assay combined with our expertise in studying disorders of iron metabolism will be a powerful approach to identify druggable regulators of hepcidin controlled iron export. 134

Poster# 126 DAILY REGULATION OF SERUM AND URINARY HEPCIDIN IS NOT INFLUENCED BY SUBMAXIMAL CYCLING-BASED EXERCISE IN HUMANS WITH NORMAL IRON METABOLISM Marie-Bérengère Troadec, PhD¹,¹°, Fabrice Lainé, MD², Vincent Daniel, MD³, Pierre Rochcongar, MD, Professor³, Martine Ropert, MD4, Florian Cabillic, MD5, Michèle Perrin², Jeff Morcet², Olivier Loréal, MD, PhD6, Gordana Olbina, PhD7, Mark Westerman, MD, PhD7, Elizabeta Nemeth, MD, PhD8, Tomas Ganz, MD, PhD, Professor8 and Pierre Brissot, MD, Professor1, 9 ¹Inserm U-522, Inserm CIC 203, Laboratory of Physiology and Sportsmedicine, Liver Disease Unit, University Hospital Pontchaillou; ²Center For Clinical Investigation, Inserm 0203,University Hospital Pontchaillou, Rennes, France; ³Laboratory of Physiology and Sportsmedicine, University Hospital Pontchaillou, Rennes, France; 4Laboratory of Biochemistry, University Hospital Pontchaillou, Rennes, France; 5URU biothérapies innovantes, and Laboratoire de cytogénétique et biologie cellulaire, University Hospital Pontchaillou, Rennes, France; 6Inserm U-522, IFR 140, University Hospital Pontchaillou, Rennes, France; 7Intrinsic LifeSciences, LLC, La Jolla, California, USA; 8David Geffen School of Medicine, University of California, Los Angeles, USA; 9Liver Disease Unit, Inserm U-522, IFR 140, University Hospital Pontchaillou, Rennes, France; ¹°Intrinsic Life Sciences, La Jolla, California, David Geffen School of Medicine, UCLA, California, USA (Presented By: Marie-Bérengère Troadec, PhD)

Background: Hepcidin and hemojuvelin are two critical regulators of iron metabolism as indicated by the development of major iron overload associated to mutations in hepcidin and hemojuvelin genes. Hepcidin and hemojuvelin are respectively highly expressed in the liver and muscles. Intensive muscular exercise has been reported to modify serum iron parameters and to increase hepcidinuria. AIM. The present study aimed at evaluating the potential impact of low intensity muscle exercise on iron metabolism and on its key hormone regulator, hepcidin. Subjects and Methods: Fourteen normal male volunteers, aged 18-40, underwent a 45 min submaximal cycling-based muscle exercise at 60% of their heart rate reserve in a prospective, controlled, randomized and crossover study. Iron parameters were recorded including serum iron, serum transferrin, serum ferritin, serum and urinary hepcidin. Hepcidin was assayed using a validated competitive ELISA which detects the 25 amino-acid form of hepcidin, the biological active form of the peptide. Serum liver and muscle enzymes (alanine aminotransferase (ALT) and creatine kinase (CK), respectively), and inflammatory tests (serum C-Reactive Protein (CRP) and interleukin-6 (IL-6)) were also assayed. All these parameters were serially studied from the beginning of the exercise and no exercise periods up to 24 h after. ANCOVA analyses, Neuman-Keuls tests and Duncan’s multiple range tests were used for statistical studies. Results: The results demonstrated that submaximal ergocycle exercise did not modulate serum or urinary hepcidin. ALT, CRP and IL-6 were also not modified by exercise. In contrast, submaximal exercise increased significantly, but slightly, serum iron, serum transferrin, serum ferritin, and CK, just after the end of the exercise. This study also indicated that hepcidinuria did not show any daily variation whereas serum hepcidin did. Conclusion: A submaximal endurance ergocycle exercise, performed in volunteers with normal basal iron metabolism, did not modify serum hepcidin concentrations, which maintained their diurnal variation. The data suggests that, under this setting, muscle iron needs may not be increased, therefore not eliciting significant changes in the metabolic cascade of iron regulation. These results indicate that diurnal variation, but not routine exercise, may impact on the interpretation of serum hepcidin concentrations in clinical practice.

Poster# 127 ARABIDOPSIS NRAMP1 IS REQUIRED FOR HIGH-AFFINITY MANGANESE UPTAKE BY THE ROOT Rémy Cailliatte, PhD, Jean-François Briat, PhD, Stéphane Mari, PhD and Catherine Curie BPMP-IBIP (Presented By: Catherine Curie)

Among the 6 NRAMP homologs in Arabidopsis thaliana, only the two members AtNRAMP3 and AtNRAMP4 have been assigned a function. These two proteins are located on the vacuolar membrane and cooperate to remobilize iron stores upon germination (Lanquar et al., 2005). We previously reported that expression of the AtNRAMP1 member in yeast restores growth of the Fe and Mn uptake mutants fet3∆fet4∆ and smf1∆ (curie et al., 2000). We have isolated an Arabidopsis loss-of-function mutant of AtNRAMP1, named nramp1-1, and shown that it is defective in high-affinity manganese uptake. In planta short-time uptake experiments enabled to calculate a Km for the Mn transport activity in the range of tens of nM. We show using transgenic plants expressing AtNRAMP1 promoter fused to the GUS reporter gene, that AtNRAMP1 is expressed in outer layers of the root and upregulated by Mn deficiency. Despite its predicted localization in the chloroplast thylakoids, we demonstrate that an AtNRAMP1::GFP protein fusion, shown to be functional by complementation of the nramp1-1 mutant phenotype, is addressed to the plasma membrane. Altogether, these results point to role of AtNRAMP1 in Mn uptake from the soil in conditions of Mn limitation.

Poster# 128 THE STRUCTURE OF THE HEPCIDIN-FERROPORTIN BINDING INTERFACE Elizabeta Nemeth, PhD, Gloria Preza, MS, Alan Waring, PhD, Richard Clark, PhD, David Craik, PhD and Tomas Ganz, PhD, MD University of California, Los Angeles (Presented By: Elizabeta Nemeth, PhD)

Hepcidin-ferroportin interaction is the central regulatory event in systemic iron homeostasis. To determine the structural basis of this interaction, we performed site-directed mutagenesis of the putative interface between the receptor ferroportin and its ligand hepcidin. Mutations in ferroportin C326 residue are known to cause an early onset form of hereditary hemochromatosis. We showed that substitutions in C326 ablate the binding of hepcidin to ferroportin and that a thiol form of C326 was required for hepcidin binding. C326 is located in a short extracellular loop and we carried out site-directed mutagenesis on each of its 20 residues. We found that several additional mutated sites ablated hepcidin binding but not iron export, confirming that the loop was a part of the hepcidin binding site. RosettaDock computer analysis of the interaction of this loop with hepcidin converged on a single low energy state in which the key contacts included aromatic side chains in pi-stacking interactions, charged side chains in ionic pairing and a potential ligand-receptor disulfide exchange involving the C326 residue. Additional mutations in the contact region of hepcidin altered binding in agreement with the computer model. Understanding the structural basis of the hepcidin-ferroportin interaction will facilitate the rational design of hepcidin agonists and antagonists for the treatment of iron disorders.

Poster# 129 TWO TFR2 MICE MODELS REPRODUCE HEMOCHORMATOSIS TYPE 3 AND CAN GIVE INSIGHT INTO MOLECULAR MECHANISMS OF TFR2 PROTEIN Antonella Roetto, PhD¹, Rosa Maria Pellegrino, Dr², Ferdinando Di Cunto, Prof³, Emilio Hirsch, Prof³, Ornella Azzolino, Dr³, Sonia Carturan, Dr², Fiorella Altruda, Prof³, Clara Camaschella, Prof4 and Giuseppe Saglio, Prof² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Science, Az Osp San Luigi Gonzaga, Orbassano Torino Italy; ³University of Torino Department of Genetics, Biology and Biochemistry Molecular Biotecnology Center, Torino, Italy; 4Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milan, Italy (Presented By: Antonella Roetto, PhD)

The transferrin receptor 2 gene (TFR2) encodes for a transmembrane protein that plays a pivotal role in iron metabolism, since TFR2 mutations are responsible of type 3 Hereditary Hemochromatosis (HH). Together with other HH proteins (Hfe and Rgmc) it participates to modulation of Hepcidin (Hepc) production, a small peptide able to regulate body iron availability through its negative effect on cellular iron exporter Ferroportin (Fpn1).

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Due to the patophisiology of HH, the necessity to create animal models is mandatory in order to study in vivo the molecular mechanisms of iron proteins. Here we present the creation of two animal models in which Tfr2 gene was inactivated both germinally (Tfr2 KO) and specifically in liver (Tfr2 LCKO) utilizing a liver specific cre/lox recombination system. Left and right arms were cloned in Bluescript-LoxP modified plasmid and the multifunctional construct obtained was electroporated in C14 ES cells. Homologous recombination was verified through Southern blot. Floxed and null Tfr2 alleles positive clones were microinjected in a C57BL/6 blastocyst and chimeras were obtained. Chimeras descendents were genotypized using two couples PCR protocols. Floxed mice were crossed with mice expressing cre gene in the liver to obtain LCKO. Phenotype analysis of Tfr2 targeted adult mice was done at ten weeks of age. Blood cell counts and erythrocyte parameters were determined, together with Serum Iron and Total Iron Binding Capacity. For histological studies liver and spleen sections were stained with Perls method. HH genes expression was measured by quantitative real time PCR (qRT-PCR). Results were analyzed using the ∆∆Ct method. Homozygous Tfr2KO mice revealed elevated transferrin saturation since 4 weeks of age while Tfr2 LCKO present a lower increase vs germinal KO. Iron deposit was found in KO mice 10 weeks old further increasing with mice age and becoming massive at 40 weeks. On the contrary, KO mice spleens are not overloaded at the same age. This data is consistent with the higher splenic Fpn1 expression in germinal KO mice compared to controls. HH genes transcripts quantitative analysis in the two targeted mice groups revealed some differences. While Hepc gene does not seem to increase in Tfr2 KO mice, it appears to be significantly down modulated in LCKO mice since 10 week of age. HFE expression remains constant in both Tfr2 targeted mice together with liver Fpn1. Rgmc gene transcription is significantly reduced both in Tfr2 KO and in LCKO mice. Protein analysis essentially confirms the transcriptional data. From phenotype analysis of germinal and liver conditional Tfr2 KO mice we can conclude that they represent good animal models to study iron metabolism proteins. Furthermore, the Rgmc protein decrease in Tfr2 null mice supports the hypothesis that this two proteins interact directly to modulate hepcidin transcription. From the significative decrease of Hepc transcription in LCKO compared to KO we can argue that Tfr2 extra hepatic expression could exert a negative effect on Hepc production.

Poster# 130 DEGRADATION PATHWAYS OF HUMAN IRON REGULATORY PROTEIN 2 Camille Dycke², Peggy Charbonnier, Catherine Bougault, Kostas Pantopoulos³ and Jean-Marc Moulis, PhD¹ ¹CEA-Grenoble/IRTSV-LCBM; ²CEA-CNRS-Université Grenoble, France; ³Lady Davis Institute for Medical Research, Department of Medicine, McGill University, Montreal, Quebec, Canada (Presented By: Jean-Marc Moulis, PhD)

The mammalian Iron Regulatory Proteins, IRP1 and IRP2, are post-transcriptional regulators of cellular iron homeostasis. They control the translation of proteins involved in storage, transport, and utilization of iron by binding to their mRNA. Whereas IRP1 protein levels remain nearly constant, IRP2 is rapidly degraded by the proteasome in iron-replete cells. Disappearance of transfected hemagglutinin-tagged IRP2 in H1299 human lung cancer cells has been monitored in the presence of different proteolytic inhibitors [1]. The contribution of the specific IRP2 domain of 73 amino acids (Ins5) that is absent from IRP1 has been probed by use of constructions devoid of the corresponding exon and by spectroscopic analysis of purified IRP2 protein and Ins5 peptide [2]. Without iron overload, the half-lives of both IRP2 and IRP2-Ins5 significantly increased by addition of not only proteasomal, but also lysosomal inhibitors. These data uncover an alternative, iron independent, mechanism of IRP2 degradation via the lysosomal pathway. Experiments with IRP2 variants indicated that the C-terminus of IRP2 is responsible for lysosomal targeting. In addition, recombinant IRP2 and Ins5 are both sensitive to proteolysis at a lysine-rich site located in this fragment. IRP2 cleavage at the same site also occurs in H1299 cells. Binding of heme to Ins5 was suggested as the initial iron-sensing mechanism of IRP2. Heme interacts with a cysteine residue only in truncated forms of Ins5. The full-length peptide or intact IRP2 do not provide an axial ligand to heme. IRP2 is degraded by both lysosomes and the proteasome under normal growth conditions, but excess iron enhances the proteasomal pathway. The involvement of heme as a signal molecule for degradation of IRP2 via the Heme Regulatory Motif-like in Ins5 is doubtful since it requires previous cleavage of the protein for efficient binding. [1] Dycke et al. The International Journal of Biochemistry & Cell Biology (2008), 40, 2826-2832. [2] Dycke et al. Biochem. J. (2007), 408, 429–439

Poster# 131 – WITHDRAWN METABOLIC REGULATION OF IRON HOMEOSTASIS IN HEREDITARY LEIOMYOMATOSIS AND RENAL CELL CARCINOMA Wing-Hang Tong, Suh Young Jeong, PhD, Gennadiy Kovtunovych, PhD, Youfeng Yang, PhD, Marston Linehan, MD and Tracey A. Rouault, MD National Institute of Child Health and Human Development, USA

Defects in mitochondrial enzymes predispose to severe developmental defects as well as tumorigenesis, and heterozygous germline mutations in the nuclear gene encoding tricarboxylic acid (TCA) cycle enyzme fumarate hydratase (FH) are associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Loss of FH in HLRCC shunts the TCA cycle and leads to overaccumulation of intracellular fumarate. Fumarate competitively inhibits hypoxia-inducible factor (HIF) prolyl hydroxylase and prevents proteosomal degradation of HIF, resulting in an increase in HIF targets such as VEGF and glucose transporter Glut-1 in HLRCC tumors (Pollack et al 2005; Isaac et al 2005). It has now been recognized that HIF is upregulated across a broad range of cancers and increased levels of HIF can facilitate tumorigenesis through dysregulation of angiogenesis, glucose metabolism, and growth stimulation. In mammalian cells, iron regulatory proteins IRP1 and IRP2 are key regulators of cellular iron homeostasis through their regulation of specific mRNAs encoding proteins for iron trafficking and usage. In this study, we showed that altered glucose metabolism in response to the defects in TCA cycle in HLRCC is accompanied by changes in IRP1 and IRP2, as well as the iron transport proteins TfR1 and DMT1. Moreover, the changes in iron metabolism genes can be attenuated by inhibition of mTOR which is a key point of convergence for a number of signaling pathways that regulate cell metabolism and proliferation. Given the critical role that iron plays in mitochondrial oxidative phosphorylation, our results suggest a reprogramming of cellular iron metabolism in response to the transition from oxidative phosphorylation to aerobic glycolysis in HLRCC. Furthermore, the reprogramming of iron metabolism in HLRCC can also further favor the stabilization and activation of HIF and potentially fuels tumorigenesis through the action of HIF on angiogenesis, energy production and growth stimulation.

Poster# 132 HEPATIC COPPER CONTENT IS REDUCED IN WILSON DISEASE KNOCKOUT MICE FED AN IRON-ENRICHED DIET Uta Merle, PD, Dr, Sabine Tuma, Valer Muntean, Martin Volkmann, Sven Gehrke and Wolfgang Stremmel, Prof Dr University of Heidelberg (Presented By: Wolfgang Stremmel, Prof Dr)

Background & Aims: The interaction of copper and iron metabolism has been recognized for many years. However, the effect of dietary iron on copperoverload in Wilson disease is unclear. The aim of the present study was to assess the effect of dietary iron on hepatic copper and iron content and gene expression of selected hepatic and intestinal copper- and iron- related genes in Wilson disease gene knockout mice (Atp7b(-/-) mice). Methods: Atp7b-/- mice and wildtype mice aged 5 weeks were allocated to a standard rodent diet or the same diet enriched with 20 g/kg carbonyl iron. After 8 weeks the animals were killed and sections of duodenum and liver removed. Quantitatively mRNA expression levels of iron- and copper-related genes in the liver and the duodenum, hepatic iron and copper concentration, and serum AST and ALT levels of Atp7b(-/-) mice (n=10) and wildtype mice (n=10) were analyzed. Results: Hepatic iron content of Atp7b(-/-) mice and wildtype mice was significantly higher in the group fed the iron-enriched diet compared to standard diet. Hepatic copper content of Atp7b(-/-) mice fed standard diet was ~50-fold higher than that of wildtype mice. Atp7b(-/-) mice fed the iron-enriched diet displayed a 50%-decreased hepatic copper content (paralleled by a decreased metallothionein-1 and metallothionein-2 mRNA expression) compared to Atp7b(-/-) mice fed the standard chow. This effect was not seen in wildtype mice. Atp7b(-/-) mice fed the iron-enriched diet, but not wildtype mice, showed a significantly increased serum AST and ALT level. Hepatic and duodenal mRNA expression levels of Dmt1+IRE were decreased, hepatic hepcidin expression increased, and duodenal hephaestin expression decreased in mice fed the iron-enriched diet compared to mice fed the standard chow. This was true for both, Atp7b(-/-) mice and wildtype mice. The duodenal expression of Zip14, a transporter of

136 non-transferrin-bound iron and putatively copper, was significantly decreased in animals (Atp7b(-/-) and wildytpe mice) fed the iron-enriched diet compared to standard diet. Conclusion: Our data clearly show a decrease in hepatic copper content and a decreased duodenal mRNA expression level of Dmt1 and Zip 14 in Atp7b (-/-) mice on an iron-enriched diet compared to mice fed a standard chow. These results indicate that dietary iron might trigger a reduced copper absorption by downregulation of the iron transporters Dmt1 and Zip14, and suggest an additional role role of these genes in copper transport.

Poster# 133 FPN1 INTERACTS WITH HEPHAESTIN AND HEME OXYGENASE 1 BUT NOT ABCG2 IN INTESTINAL IRON ABSORPTION Kwo-yih Yeh, PhD, Mary Yeh, MS, Laura Mims, BS and Jonathan Glass, MD LSUHSC, Shreveport, LA (Presented By: Kwo-yih Yeh, PhD)

Ferroportin (FPN1, MTP1, or Ireg1) is a multi-spanning membrane protein of enterocytes and is essential for iron export in to the systemic circulation. Iron export by FPN1 requires hephaestin (Heph) to reduce Fe(II) to Fe(III) before release to systemic circulation. Heme, a major source of dietary iron, is absorbed by the heme carrier protein HCP1 (PCFT or Slc46a1) and catabolized by heme oxygenase 1 or 2 (HO1 or HO2) with release of Fe(II) for export by ferroportin. The interaction of FPN1 and Heph has been previously described (Am J Physiol Gastintest Liver Physiol 296: G55, 2009) but whether FPN1 and/or Heph cooperates with HO1 for the export of Fe released from heme is unclear. In addition, unexplored is whether the apical heme export protein Abcg2 interacts with the proteins involved in basolateral Fe export to modulate iron transport from heme. Expression of all of the above mentioned genes (FPN1, Heph, H01, Abcg2) and the apical iron transporter DMT1 are increased in the intestinal mucosa of iron deficient rats. Hence, it is possible that the protein products of these genes might cooperate to increase iron absorption. To explore this possibility we generated DsRed (mono) and EmGFP fusion genes and examined the immunolocalization of gene products in HEK cells and in isolated duodenal epithelial cell sheets by two-dimensional blue native (BN)/SDS PAGE to identify possible interaction of these proteins. Following transfection into HEK cells DsRed-FPN1 colocalized with EmGFP-Heph, EmGPF-HO1 and EmGFP-Abcg2 and DsRed-HO1 colocalized with EmGFP-Abcg2. Western blot analysis of BN/SDS PAGE, demonstrated that a complex migrating together in the 1st dimension could be identified as containing in the 2nd dimension FPN1, Heph, and H01 as all three proteins appeared in the same lane. However, these interactions are complex with FPN1 exhibiting 155 kDa dimers, even in the presence of SDS, and 75 and 55 kDa monomers, Heph migrating at 155 kDa, and H01presenting 55 and 50 kDa bands. In contrast, Abcg2 comigrated with the apical iron transporter DMT1 in an insoluable mass and a 90 kDa band that was not associated with H01 as suggested by the transfection studies. The FPN1 and Heph structures in the complexes with H01 remain to be solved.

Poster# 134 HEREDITARY HAEMOCHROMATOSIS PROTEIN (HFE)-DEPENDENT REGULATION OF IRON TRANSFER ACROSS PLACENTA Rumeza Hanif, PhD student¹, Sara Balesaria, PhD¹, Harry J. McArdle, PhD² and Kaila Srai, PhD¹ ¹University College London; ²University of Aberdeen (Presented By: Rumeza Hanif, PhD student)

Background and Aims: HFE functions as an upstream regulator of liver hepcidin which has been demonstrated to be negative regulator of intestinal absorption of dietary iron and macrophage efflux of recycled iron. Hepcidin has also been proposed to be a negative regulator of iron efflux from placenta, however it is not known if this of maternal or foetal origin. HFE has been demonstrated to be present in the placenta, but its role in the dynamics of maternal foetal in iron transfer independent of hepcidin is unknown. In this study we investigated the effects of HFE and dietary iron levels on transfer of iron from mother to foetus in order to determine the importance of maternal and foetal HFE status on iron transfer Materials and Methods:HFE knockout (KO), wild type (WT), and heterozygote (HET) dams were fed 50 and 200ppm iron diets and mated with HET males to produce pups of all genotypes. Dams and pups were sacrificed and pup liver iron levels were determined. mRNA levels of iron transporter genes (Ferroportin1, TfR1, DMT1+IRE, and DMT1-IRE) were determined in placental tissue by real-time PCR. Results: Liver iron levels of HET pups from KO dams fed 50ppm iron diet were considerably higher (137.42 + 9.19 µg/g dryweight, n = 10) than those from WT dams (112.90 + 3.71 µg/g dryweight, n = 10). This difference was also apparent in the pup liver iron levels when the dams were fed 200ppm iron diet. The gene expression was higher in HET pups from KO dams compared to those from WT dams fed 50ppm iron diet but this difference was not significant in HET pups from dams fed 200ppm iron diet. There was no difference in liver iron levels of KO (115.9 + 7.09 µg/g dryweight, n = 10) and WT pups (113.47 + 3.61 µg/g dryweight, n = 10) from HET dams fed 50ppm iron diet. This was mirrored in the gene expression data, which showed no significant difference between all of the placental genes tested. However liver iron levels of KO and WT pups from HET dams fed 200ppm iron diet were higher in KO pups (146.95 + 9.34 µg/g dryweight, n = 10) than WT (122.08 + 4.74 µg/g dryweight, n = 10). In addition, expression of placental genes was also found to be significantly elevated in KO pups than WT. Conclusions: Maternal genotype plays a role in regulating placental iron transfer independent of dietary iron content. However, foetal genotype seems to affect liver iron accumulation and certain genes expression only with adequate iron intake.

Poster# 135 GENERATION OF A MOUSE STRAIN WITH INDUCIBLE IRP1 OVEREXPRESSION: A NEW MODEL FOR UNDERSTANDING IRON METABOLISM DISORDERS D. Casarrubea¹, L. Viatte, R. Eisenstein², B. Galy1 and M.W. Hentze1 ¹European Molecular Biology Laboratory; ²University of Wisconsin (Presented By: D. Casarrubea)

Iron Regulatory Proteins (IRP) -1 and -2 coordinately secure cellular iron homeostasis by binding to cis-regulatory iron-responsive elements (IRE) on mRNAs encoding proteins involved in iron uptake, handling, storage and export. In turn, the cellular labile iron pool regulates the IRE-binding activity of the two IRPs through distinct mechanisms. To explore the in vivo functions of the IRP/IRE system and to complement investigations with loss-of-function lines, we have developed a mouse model allowing conditional expression of a gain-of-function IRP1 mutant using Cre/Lox technology. We first generated a cDNA encoding a flag-tagged mutant version of IRP1 that binds IREs in a constitutive manner due to the lack of critical residues required for Fe-S cluster assembly. This cDNA was inserted into the permissive Rosa26 locus by homologous recombination in murine embryonic stem (ES) cells together with a floxed ß-geo stop cassette placed between the ubiquitous Rosa26 promoter and the IRP1 cDNA. This floxed ß-geo stop cassette prevents transcription of the IRP1 transgene; its excision upon Cre-mediated recombination enables expression of the IRP1 mutant in a conditional manner. The functionality of the targeted Rosa26 allele was validated by transfection of ES clones with a plasmid encoding Cre recombinase, showing the expected deletion of the ß-geo stop sequence as assessed by PCR, and the ensuing induction of IRP1 synthesis as shown by western blotting. Mouse lines harbouring the targeted Rosa26 locus have been obtained after injection of ES clones into blastocysts. Rosa26-IRP knock-in mice will be crossed with various Cre-deletor strains to achieve pan- as well as tissue and/or stage-specific expression of the IRP mutant; of particular interest will be to analyze the consequences of forced IRP overexpression on iron absorption, utilization, storage, and recycling, respectively, by selectively triggering expression of the IRP mutant in enterocytes, erytrocytes, hepatocytes, or macrophages. This study will establish the physiological importance of appropriate IRP regulation in different organs specialized in the control of body iron fluxes. Our new mouse line should also provide a valuable model to study diseases associated with abnormally high IRP activity as observed e.g. in the substantia nigra of patients with Parkinson’s disease or in patients suffering from sideroblastic anemia linked to glutaredoxin 5 deficiency.

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Poster# 136 EXPRESSION OF GENES INVOLVED IN IRON IMPORT/EXPORT IN HUMAN INTESTINAL AND LIVER CELLS: EFFECT OF IRON AVAILABILITY AND MODULATION BY ETHANOL Jan Kovar, DSc, Kamila Balusikova, Jitka Neubauerova and Marketa Dostalikova-Cimburova Third Faculty of Medicine, Charles University, Prague (Presented By: Jan Kovar, DSc)

Introduction: We studied the effect of iron availability (iron deficiency vs. iron excess) on the expression of genes involved in iron transport across plasma membrane in human intestinal cells and hepatocytes. We also tested modulatory effect of short-term and long-term exposure to ethanol on the expression of studied genes. Methods: The expression of studied genes (DMT1, Dcytb, ferroportin, hephaestin, ceruloplasmin, transferrin receptor 1, transferrin receptor 2, HFE, hepcidin) was assessed in Caco-2 cells (human colorectal carcinoma) and HEP-G2 cells (human hepatocellular carcinoma) after 24-hour preincubation in defined iron-free medium (iron deficiency) or in defined medium containing 500 µM ferric citrate (iron excess). The effect of ethanol (2%) was tested as short-term exposure (24 h) or long-term exposure (6 months). The expression was assessed on mRNA level by quantitative real-time PCR and on protein level by western blot analysis. Results: Concerning the effect of iron deficiency, increased mRNA level of DMT1 in Caco-2 cells was confirmed by increased level of DMT1 protein. As to the effect of iron excess, only the increase of ferroportin mRNA level in Caco-2 cells and the decrease of ceruloplasmin mRNA level in HEP-G2 cells correlated with a relevant change of protein level. Long-term exposure to ethanol, but not short-term exposure, resulted in a dramatical decrease (approximately 10-fold) of mRNA level of Dcytb in Caco-2 cells. On the other hand, in HEP-G2 cells long-term exposure to ethanol led to a dramatical increase (approximately 30-fold) and short-term exposure to some increase (approximately 3-fold) of the level of Dcytb mRNA. However, these changes in Dcytb mRNA level were not followed by relevant changes in Dcytb protein level. Long- term exposure to ethanol also led to a significant increase (approximately 2-fold) of hepcidin mRNA level in HEP-G2 cells. It seems, that long-term exposure to acetaldehyde affected mRNA level of the genes similarly like ethanol but to a lesser degree. Long-term exposure to ethanol did not affect ROS production in both cell types. On the other hand, it resulted in significant upregulation of hypoxia inducible factor 1α (HIF-1α) in HEP-G2 cells but not in Caco-2 cells. Furthermore, long-term exposure to ethanol significantly potentiated the effect of iron deficiency on the increase of ferroportin mRNA level in Caco-2 cells. Long-term exposure to ethanol, together with iron excess, also synergistically affected the decrease of DMT1 mRNA level in HEP-G2 cells. Discussion: We demonstrate that long-term exposure to ethanol can modulate the effect of iron availability on the expression of genes involved in iron import/export. It seems that acetaldehyde does not mediate the modulatory effect of ethanol. The effect of ethanol, at least in HEP-G2 cells, could be related to the regulation involving HIF- 1α. Conclusion: Both iron availability and ethanol affect the expression of genes involved in iron transport across plasma membrane in human intestinal cells and hepatocytes. The effects are cell type-specific. Changes in the expression found on the mRNA level do not often correlate with relevant alterations of the expression on the protein level. This study was supported by research program MSM 0021620814 of the Third Faculty of Medicine of Charles University in Prague.

Poster# 137 INTERRELATIONS BETWEEN IRON METABOLISM AND ETHANOL EFFECT ON CELL PROLIFERATION IN THE HUMAN HEPATOMA HEPARG CELL LINE Thi Hong Tuoi Do², François Gaboriau², Romain Moirand²,³, Caroline Le Lan²,³, Isabelle Cannie², Lucie Gouffier², Olivier Loréal², Pierre Brissot²,³ and Gérard Lescoat¹ ¹INSERM U522; ²Inserm U522 – EA « MDC », Université de Rennes 1, IFR 140, Rennes; ³Service des maladies du Foie, CHU Pontchaillou, Rennes (Presented By: Gérard Lescoat)

Introduction: Recent studies concern the role of ethanol on the modulation of iron metabolism, particularly in relation to cell growth including hepatocyte proliferation. Excessive ethanol consumption can result in a slow, progressive accumulation of excess hepatic iron and increases the risk of fibrosis, cirrhosis and/or hepatocellular carcinoma. Individually, ethanol and iron modulate cell proliferation. Their synergy could intensify the deleterious effects on hepatocyte proliferation and the risk of liver disease. The purpose of the present study was: 1) to evaluate the effect of ethanol alone, or ethanol plus iron on cell proliferation; 2) to correlate the ethanol-induced effect to a modulation of iron metabolism. Methods: We used the human hepatoma HepaRG cell line. Cells were seeded in culture plates using William’s Medium E supplemented with 10% foetal calf serum and exposed to different concentrations of ethanol (0, 50, 100, 200 and 300 mM) for 24, 48 or 72h. Cellular viability was evaluated by MTT test, DNA synthesis by thymidine incorporation, protein level by Bradford method, cytotoxicity by extracellular LDH/total LDH ratio. Apoptosis was studied by measuring caspase 3/7 activity. The mRNA levels of L-ferritin, transferrin, transferrin receptor 1 and DMT1 were analyzed by quantitative RT-PCR in real time. Results: Ethanol decreased all the parameters associated with cell proliferation (cellular viability, DNA and protein syntheses) in a dose-and timedependent manner. This phenomenon was correlated to apoptosis and necrosis. In ethanol-exposed cells, reduction of cell viability, thymidine incorporation and protein level was intensified by the presence of iron-citrate or iron-NTA (20 µM) in the culture medium. These decreases were associated with a considerable increase in extracellular LDH/ total LDH ratio. Ethanol increased significantly the expression of iron metabolism genes (L-ferritin, transferrin, transferrin receptor 1 and DMT 1). Discussion: In this study, ethanol effect on HepaRG cells was in agreement with already published data in which ethanol reduced the proliferation of hepatocytes. Indeed, ethanol, by activation of cytochrome P450 2E1 or mitochondrial dysfunction, is known to produce free radicals via the Fenton reaction and to increase cell susceptibility to signals of apoptosis or necrosis.The intensification of ethanol toxic effects by iron-citrate or iron-NTA could be explained by the catalytic role of iron in the free radical synthesis induced by ethanol. Moreover, the observed increase in iron metabolism gene expression may correspond to enhanced hepatocyte iron uptake, therefore increasing ethanol toxicity. In conclusion, our results put forward a clear relationship between ethanol and iron metabolism: ethanol-induced inhibition on cell proliferation is closely related to an increase in iron metabolism markers expression. The results suggest that iron metabolism dysfunction could play a mechanistic role in the ethanol effect on HepaRG cell proliferation. Acknowledgements: This work was supported by « Institut de Recherches Scientifiques sur les Boissons » (IREB, Paris).

Poster# 138 HIGHLY SENSITIVE HISTOCHEMICAL STAINING OF IRON COMBINED TO GENETIC ANALYSIS OF ARABIDOPSIS EMBRYOS REVEAL THAT IRON SPECIFICALLY ACCUMULATES IN THE ENDODERMAL CELL LAYER Hannetz Roschzttardtz, PhD, Genevieve Conejero, PhD, Stéphane Mari, PhD and Catherine Curie BPMP-IBIP (Presented By: Catherine Curie)

The development of simple techniques of in vivo metal imaging is of potential great interest to better understand of metal homeostasis in plants. The well-known Perls histochemical method for iron staining is hardly usable in plants due to its lack of sensitivity, unless plants are overloaded due to either an excessive iron supply or a genetic disorder. We have adapted the Perls intensification method with diaminobenzidine (DAB), used in animals to stain organs by perfusion, to detect iron in Arabidopsis embryos. Comparison of the distribution of Perls/DAB staining in embryos of wild type and vit1, a mutant with altered Fe distribution, combined to in vitro tests of staining specificity, have demonstrated that in plants too, the method is specific for iron and further established that it detects both FeII and FeIII. During embryogenesis, Perls/DAB staining showed a rather diffuse iron distribution in early stages of the development but, as the embryonic provascular system differentiates, staining concentrated to the vascular region. Thin cross-sections of stained embryos established that Fe accumulates specifically in a single cell layer surrounding the provascular cells and corresponding to the endodermis. In vit1 however, we found that the subepidermal localization previously detected by X-ray tomography corresponded in fact to cortical cells. Finally, in embryos of the short root mutant, that lack endodermis, Fe accumulated in the central cylinder suggesting that endodermis functions as a barrier for the radial movement of Fe in the root. Taken together, these data establish that the Perls/DAB method, highly sensitive and straightforward, represents a powerful tool to detect iron in plant tissues, and enabled us to uncover a new role of the endodermis in iron storage in the embryo.

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Poster# 139 CHARACTERIZATION OF FELINE SERUM FERRITIN-BINDING POTEINS: THE PRESENCE OF A NOVEL FERRITIN-BINDING PROTEIN AS AN INHIBITORY FACTOR IN FERRITIN IMMUNOASSAY Koichi Orino, PhD, DVM and Kiyotaka Watanabe, PhD, DVM Kitasato University (Presented By: Koichi Orino, PhD, DVM)

Introduction: Ferritin-binding proteins (FBPs) are known to interact with circulating ferritin to eliminate it from circulation. Mammalian FBP were identified as follows; H-kininogen, apolipoprotein B, alpha-2-macroglobulin, fibrinogen, and anti-ferritin autoantibody. However, we found that feline serum strongly inhibited the detection of canine and feline liver ferritins by immunoassay; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. The purpose of this study is to characterize and purify feline serum FBPs. Methods: Feline FBP was partially purified from feline serum using canine liver ferritin coupled-Sepharose 4B affinity column. After complex formation between affinity- purified FBPs and canine liver ferritin, the complex was co-immunoprecipitated by anti-bovine spleen ferritin antibody. Coimmunoprecipitates obtained were subjected to immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. In addition, a novel feline FBP was partially purified from feline serum by (NH4)2SO4 fractionation (33-50%) followed by gel filtration chromatography and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the novel FBP was separated and purified from complexes formed in a native-PAGE gel. Results and Discussion: Immunoblotting analysis showed that affinity-purified FBPs contain autoantibodies (IgG, IgM, and IgA) to ferritin. On the other hand, affinity- purified FBPs did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has a novel FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. Purified novel FBP found to be a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds on SDS-PAGE analysis, indicating that the FBP has different molecular property with other FBPs previously described. Detection of feline liver ferritin by immunoassay was dose-dependently inhibited by FBP. These results demonstrate that feline serum has a novel FBP in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.

Poster# 140 REGULATORY EFFECTS OF FERRITIN ON H-KININOGEN: IMPLICATIONS FOR ANGIOGENESIS Lan Coffman, PhD, Yufeng Song, BA, Frank Torti, MD and Suzy Torti, PhD Wake Forest University School of Medicine (Presented By: Suzy Torti, PhD)

Angiogenesis is an essential component of physiologic and pathologic processes ranging from wound healing to tumor growth. Angiogenesis is regulated by a balance between pro- and anti-angiogenic factors. One of the major cleavage products of high molecular weight kininogen (HK), termed HKa, binds endothelial cells through specific receptors and inhibits angiogenesis. We have previously demonstrated that ferritin is a binding partner of HK and may be an important overall regulator of HK activity. We explored ferritin’s potential to regulate the anti-angiogenic activity of HKa. Using cultured primary endothelial cells, we demonstrate that ferritin successfully antagonizes the anti-angiogenic effects of HKa: ferritin blocked the ability of HKa to reduce endothelial cell viability and induce endothelial cell apoptosis. Ferritin also antagonized the anti-migratory effects of HKa and opposed the ability of HKa to inhibit tube formation in vitro, resulting in enhanced migration, assembly, and survival of endothelial cells treated with HKa. Effects of ferritin on modulation of angiogenesis by HKa were independent of the iron content of ferritin. Regulatory effects of ferritin on Hka anti-angiogenic activity were also observed in cancer angiogenesis: ferritin blocked the anti-angiogenic effect of HKa in human prostate cancer xenografts, restoring tumor-dependent assembly of blood vessels to control levels. Regulation of angiogenesis represents a new role for ferritin in cell biology. We propose that ferritin, which is elevated during inflammation and malignancy, may play a role in regulating the levels of angiogenesis during these conditions.

Poster# 141 OVEREXPRESSION OF IRP2 INCREASES THE GROWTH OF TUMOR XENOGRAFTS IN NUDE MICE Carmen Maffettone¹ and Kostas Pantopoulos, PhD² ¹Lady Davis Institute for Medical Research; ²Lady Davis Institute for Medical Research and McGill University (Presented By: Carmen Maffettone)

Iron is essential for cell growth and proliferation, as cofactor of ribonucleotide reductase, the rate-limiting enzyme for de novo DNA synthesis. Nevertheless iron overload may be associated with fibrosis and carcinogenesis, as shown in hemochromatosis patients. The mechanisms by which iron metabolism interferes with carcinogenesis are not well defined. We hypothesized that misregulation of cellular iron homeostasis by altering the expression of Iron Regulatory Proteins, IRP1 or IRP2, may affect tumor growth. IRP1 and IRP2 post-transcriptionally control cellular iron uptake, storage and efflux by binding to Iron-Responsive Elements (IREs) within the mRNAs encoding transferrin receptor 1 (TfR1), ferritin and ferroportin. We previously showed that the overexpression of IRP1 inhibits the growth of tumor xenografts in nude mice (1). Here we investigated the effects of IRP2 in a similar setting. We utilized human H1299 lung cancer cells and clones engineered to overexpress wild type (IRP2WT ) and mutant (IRP2∆73) IRP2 by a tetracycline-inducible promoter; the IRP2∆73 mutant lacks an IRP2-specific stretch of 73 amino acids. Parent H1299 cells, IRP2WT or IRP2∆73 transfectants were injected subcutaneously into the flanks of BALB/c nude mice and evaluated for their capacity to promote tumor growth. The xenografts derived from IRP2WT and IRP2∆73-expressing cells were growing faster compared to those derived from parent H1299 cells. Therefore, the IRP2WT and IRP2∆73 tumors were bigger in mass and in size. Western blot analysis of the tumor extracts confirmed the expression of hemagglutinin (HA) epitope-tagged IRP2WT or IRP2∆73. As expected, increased expression of TfR1 was observed in the tumor xenografts derived from IRP2WT and IRP2∆73 overexpressing cells, while there was no difference in the ferritin and ferroportin content. These preliminary data suggest that IRP2WT and IRP2∆73 exhibits pro-oncogenic properties. The molecular basis underlying this phenotype is being investigated.

Poster# 142 C282Y AND H63D MUTATIONS IN HFE GENE IMPACT ON EPITHELIAL OVARIAN CANCER RISK AND SURVIVAL Christine M. Maugard, MD, PhD, Sanae Medelci, Beaulieu Martin, Philipe Gannon, Diane M. Provencher, Anne-Marie Mes-Masson and Manuela M. Santos Montreal Cancer Institute / CRCHUM / University of Montréal (Presented By: Christine M. Maugard, MD, PhD)

Epithelial ovarian carcinoma is one of the most aggressive gynecologic cancers with the highest mortality rate. Characterized by rapid and localized expansion of tumor cells in the peritoneal cavity, the growth of epithelial ovarian tumor cells is limited by environmental supply of nutrients, of which one is, indisputably, iron. Because of their increased requirement for iron, cancer cells express high levels of transferrin receptor and internalize iron from transferrin at a very high rate. Transferrin receptor function is regulated by HFE, a major histocompatibility class I-like molecule that forms complexes with the transferrin receptor and modulates the transferrin cycle. Mutations in the HFE gene are associated with hemochromatosis type I, an autosomal recessive inherited iron overload disease which is frequently encountered in Caucasians. We determined the prevalence of the H63D and C282Y HFE mutations in a cohort of 526 French-Canadian women. 79 of them, which underwent surgery for non-cancerous pathologies, were recruited as controls. Cases included 102 women with epithelial ovarian cancers of low malignant potential (LMP), 253 with invasive epithelial ovarian cancer (EOC) grades 1 to 3 (G1-G3), and 92 with endometrial cancer. Genotyping was performed using a polymerase chain reaction (PCR) amplification followed by restriction enzyme digestion and by multiplex quantitative real-time PCR. We conducted a survival analysis in a subgroup of 188 serous malignant ovarian tumors, including 140 invasive EOC (G1-G3) with at least 18 months of follow-up. Kaplan-Meier survival curves were determined and compared using a log-rank test. We computed a multivariate Cox model to define the prognosis value of 3 variables (age, grade and presence of a C282Y allele) for overall survival.

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No significant differences were found between the HFE allele distribution in the entire cohort compared to the reported frequency in the province of Québec neonates cohort [1], i.e. 4.6% vs. 4.4% for the C282Y mutant allele and 16.5% vs. 18% for the H63D mutant allele. While no significant differences were observed for the H63D allele frequency, the C282Y allele frequency in LMP together with invasive EOC (G1 to G3) was higher than in our control population (5.9% vs. 1.3%, P < 0.02). Moreover, it was also higher in ovarian tumors than in endometrial cancer patients (5.9% vs. 2.2%, P < 0.04). Survival curve analysis did not find any significant association between the H63D allele and patient outcome. However, patients with at least one C282Y allele have a decreased survival rate compared to those with no C282Y allele, irrespective of the grade: P < 0.01 when all tumors were considered together (LMP and EOC); P < 0.01 for G1-G3 EOC tumors; and P < 0.005 for G3 EOC tumors (hazard ratio of 3.3 with a 95% confidence interval [CI] = 1.55 to 6.25; P < 0.005). These preliminary findings indicate for the first time that the C282Y allele could be associated with an increased risk of ovarian cancer and a poor outcome in EOC patients.

Poster# 143 GENOME-WIDE MICROARRAY ANALYSIS OF MELANOMA REVEALS UNEXPECTED ANOMALIES IN IRON-RELATED GENE EXPRESSION Daniel Johnstone, Martin Ravetti, PhD, Carlos Riveros, PhD, Pablo Moscato, PhD, Peter Hersey, MD, Rodney Scott, PhD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

It has been known for many years that cancer cells take up large amounts of iron, exhibiting increased expression of transferrin receptor 1 and some other iron-related molecules. Here we report microarray data from human melanoma biopsy samples revealing large alterations in expression of a range of genes important in iron storage and metabolism. These differences discriminated melanoma cells from controls extremely effectively and raise intriguing questions about the regulation of iron in melanoma. Total RNA was extracted from melanoma tissues from 62 patients and seven control melanocyte cell lines and hybridized to Illumina Sentrix HumanRef-8 Expression BeadChips (v2.0). Following normalization (Cubic Spline and median) an in-house algorithm using a Fayyad-Irani-based entropy filter was applied to discretize the data. Feature set modeling (Max Cover (α,β)-κ-Feature Set) identified probes discriminating melanomas from controls. We observed increases in transcripts for transferrin receptor 1 (TFRC), consistent with previous observations in cancer cells, transferrin (TF), hepcidin (HAMP), ferroportin (SLC40A1) and the two major hemoglobins (HBA1, HBB). There were decreases in transcripts for ferritin light (FTL) and heavy chain (FTH1), iron-regulatory protein 2 (IREB2) and melanotransferrin (MFI2). There were no changes in HFE transcripts and HFE2 (hemojuvelin) gene expression, while decreasing, was expressed at extremely low levels. These alterations are difficult to explain but suggest considerable dysregulation of iron metabolism. Possibly iron is rapidly taken up by melanomas and utilized without being stored but this would not fully account for the parallel increases in hepcidin and ferroportin transcripts or parallel transcriptional down-regulation of IREB2 and ferritin transcripts. Gene transcription does not directly measure protein levels, post-transcriptional changes are frequently important, and it will be valuable to investigate changes at the protein level. However there is unlikely to be increases in, for example, IRP-2 activity or ferritin levels in the face of decreased gene expression, suggesting protein discrepancies are also occurring. The average decrease in melanotransferrin expression in melanomas relative to controls was unexpected as increased melanotransferrin expression is reported in melanomas however melanotransferrin gene expression varied considerably between biopsies, some having very high expression, others very low. Increases in hemoglobin gene expression are also intriguing but require studies using laser capture microdissection to determine if this reflects melanoma expression or immature blood cells within the biopsies. These results demonstrate considerable dysregulation of iron-related gene expression in melanomas and have potential therapeutic implications.

Poster# 144 A ROLE FOR LUMINAL IRON IN COLORECTAL CARCINOGENESIS Chris Tselepis, Sorina Radulescu, Matthew Brookes, Tariq Iqbal and Owen Sansom University of Birmingham (Presented By: Chris Tselepis)

Introduction: Excessive iron is detrimental to health, as exemplified by the clinical consequences of hereditary haemochromatosis. Alarmingly there is a growing body of epidemiological, animal and cellular evidence implicating excessive iron in carcinogenesis in particular colorectal cancer. However, what remains unclear is how iron mediates carcinogenesis and whether it is high iron in the lumen of the colon or high circulating iron levels in the blood which poses the greatest cancer risk. Thus the aims of this study were to delineate the importance of luminal and systemic iron in a murine model of colorectal cancer. Methods: One of the best established models of intestinal tumourigenesis is the ApcMin/+ mouse. These mice carry a point mutation at codon 850 (which disrupts the ability of the Apc tumour suppressor to bind and turnover b-catenin) and develop 100’s of benign adenomas on the loss of the wild type Apc allele. This model therefore represents an excellent model to test potential chemopreventive agents and the role of iron in colorectal carcinogenesis. ApcMin/+ mice post weaning were fed either a standard control diet (n=9) or an iron deficient diet (Harlan UK) (n=11). In addition a third group of ApcMin/+ mice (n=8) were fed the standard control diet but upon reaching 50 days of age were injected sub-cutaneously with iron dextran (50mgs). All mice were then culled at 85 days of age and their gastrointestinal tract assessed for tumour burden. Results: Total intestinal tumour number was significantly decreased by 3.5 fold in mice fed an iron deficient diet compared to mice on a control iron diet (p=0.0002). Mice injected with iron dextran did not show a statistical difference in tumour number compared to control mice. The average size of the tumours in mice fed an iron deficient diet were also decreased by an average of 1.4 fold compared to mice on a control diet (p=0.005). Analysing total tumour burden revealed an overall 4 fold decrease in mice fed an iron deficient diet compared to mice fed a control diet (p=0.0002). Discussions: This study adds further weight to a growing body of evidence suggesting that iron is involved in colorectal carcinogenesis. Furthermore our data suggests that decreasing dietary iron levels may diminish the risk of developing colorectal cancer.

Poster# 145 MOLECULAR-GENETIC ANALYSIS OF CERULOPLASMIN IN OESOPHAGEAL CANCER Natalie Strickland, MSc Genetics¹, Tandi Matsha and Monique Zaahl, PhD Genetics² ¹University of Stellenbosch; ²University of Genetics (Presented By: Natalie Strickland, MSc Genetics)

Oesophageal Cancer (OC) is a disease characterised by the development of malignant tumours in the epithelial cells lining the oesophagus. It demonstrates marked ethnic variation, with squamous cell carcinoma (SCC) being more prevalent in the Black population and adenocarcinoma (ADC) occurring more often in Caucasians. OC is one of the leading causes of cancer-related deaths worldwide; it is the 15th most common cancer in developed nations and the 4th most common in developing countries such as South Africa. The Transkei region of South Africa is thought to be the centre of the disease in Africa, with an age standardised incidence rate (ASIR) of 46.7/100 000 for males and 19.2/100 000 for females previously being reported. OC shows clear geographic variation and occurs at a high incidence in certain areas of the world, which are termed “oesophageal cancer belts”.The aetiology of this complex disease has been attributed to a variety of factors, including an excess of iron (resulting in increased tumourigenesis), oesophageal injury and inflammation (due in part to Barrett's oesophagus and smoking, amongst others). Ceruloplasmin (CP) is a ferroxidase enzyme synthesized in the liver which catalyses the oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+), thereby creating an ion gradient favouring iron export from the cells. Therefore, CP which is located primarily in the plasma is responsible for driving iron transport from stores in various tissues In this study, we aim to demonstrate the relationship between genes involved in iron metabolism (specifically CP) and the development of OC, by identifying gene variations that could potentially contribute toward iron dysregulation and subsequent disease pathogenesis. It is anticipated that the results obtained from this study will lead to a greater understanding of the role that iron homeostasis plays in the aetiology of OC.

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The study cohort consisted of 96 unrelated OC patients from the Black Xhosa-speaking South African population and 88 population-matched control individuals. The promoter and coding regions of the CP gene were analysed for DNA sequence variation using the polymerase chain reaction (PCR), heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis and semiautomated bidirectional DNA sequencing analysis. Allele and genotype frequencies were estimated by allele counting and statistical differences between patient and control groups were tested for significance by chi-squared (X2) analysis. A probability value (P) smaller than 0.05 was regarded as statistically significant. The Hardy-Weinberg equilibrium (HWE) test was performed to determine equilibrium for the genetic traits investigated in the respective populations. Fourteen previously described (5’UTR-567C→G, 5’UTR-563T→C, 5’UTR-439C→T, 5’UTR-364delT, 5’UTR-354T→C, 5’UTR-350C→T, 5’UTR- 282A→G, V223, Y425, R367C, D544E, IVS4-14C→T, IVS7+9T→C and IVS15-12T→C) and four novel (5’UTR-308G→A, T83, V246A and G633) variants were identified. Statistical analysis revealed that two of the novel variants identified in the patient cohort were significantly associated with OC in this study; the promoter variant 5'UTR-308G→A (P=0.012) and the exonic variant G633 (P=0.0003). This is the first study to examine CP with respect to OC in the Black South African population. As such, these findings should serve to further our understanding of the relationship between iron metabolism and disease pathogenesis, specifically OC.

Poster# 146 MUTATION ANALYSIS OF THE SLC40A1 PROMOTER IN BLACK SOUTH AFRICAN OESOPHAGEAL CANCER PATIENTS Jessica Vervalle, BSc (Hons), Ann Louw, PhD, Louise Warnich, PhD and Monique Zaahl, PhD Stellenbosch University (Presented By: Jessica Vervalle, BSc (Hons))

Solute carrier family 40 member 1 (SLC40A1) is a protein responsible for the iron transport across the apical membrane of enterocytes as well as iron export from tissue cells. Iron regulation is crucial in the human body as iron is required for enzyme synthesis. However excess iron promotes the formation of reactive oxygen species, linking iron overload to cancer. We investigated the promoter region of the SLC40A1 gene to identify promoter variants that could potentially be involved in gene expression regulation. A Black South African Oesophageal cancer patient cohort (n=45), and a population matched control cohort (n=50) was subjected to PCR amplification of overlapping fragments of the promoter region of the SLC40A1 gene. Subsequently, PCR products were subjected to heteroduplex single-stranded conformational polymorphism (HEX- SSCP) analysis for mutation detection. Variation was confirmed with bi-directional semi-automated DNA sequencing analysis. Genotyping of these variants was extended to a larger patient cohort (n=90) using restriction fragment length polymorphism (RFLP) analysis. The significance of variants was determined by estimating allele and genotype frequencies and determining probability values using chi-squared analysis. Several variants were identified following mutation analysis, including -1461T/C, -1470C/T, -1399G/A, -1355G/C, -1098G/A, -1087G/C, -750G/A and -23A/G. Allele and genotype frequencies did not show any statistically significant associations. Bioinformatic results, using JASPAR, showed creation and abolishment of various transcription factor binding sites involved in iron regulation, including the transcription factors Yin Yang1 (YY1), GATA2 and forkhead transcription factor (FOXL2) respectively. Both YY1 and GATA2 activity have been linked to tumorigenesis. Research has demonstrated that YY1 plays a pivotal role in biological processes such as replication, differentiation and cellular proliferation, although the variety of functions of YY1 can only be inferred from the genes that it regulates. YY1 is a multifunctional transcription factor that can act as a transcriptional activator, repressor or as an initiator element binding protein. YY1 has been found to act as a negative regulator of p53, a tumor suppressor protein. GATA2 belongs to the family of GATA transcription factors, which bind to the consensus DNA sequence GATA, and acts as transcriptional activators. GATA2 is required for proliferation of hematopoietic cells. Studies demonstrated that p53 counteracts the defects of GATA2 deficient cells to some extent, suggesting a potential link between the functions of GATA2 and p53. FOXL2 is part of the family of forkhead proteins, which is thought to promote gene activation directly by opening chromatin. Although no statistically significant associations were identified, allele and genotype frequencies for the variants identified in the Black South African population have been documented. Further investigation of the SLC40A1 promoter should be pursued. Bioinformatic results are in the process of being confirmed by luciferase reporter gene constructs. These results would add to better understanding of SLC40A1 promoter regulation and its involvement in iron regulation.

Poster# 147 MOLECULAR CHARACTERIZATION OF THE 5’ UNTRANSLATED REGION (UTR) OF THE CYTOCHROME B REDUCTASE 1 (CYBRD1) GENE OF OESOPHAGEAL CANCER PATIENTS Veronique Human, MSc¹, Thandi Matsha, PhD², Louise Warnich, PhD¹ and Monique Zaahl, PhD¹ ¹Stellenbosch University; ²Cape Peninsula University of Technology (Presented By: Veronique Human, MSc)

The promoter region of a gene may contain DNA polymorphisms exerting influences on the transcriptional activity of the downstream gene. Identifying and characterizing these polymorphisms are thus important as they may influence gene expression in various disease states. CYBRD1, the membrane protein responsible for the reduction of ferric to ferrous iron, enables the absorption of dietary iron into the enterocytes. The precise mechanisms involved here are still unknown and limited information is available regarding the regulation of CYBRD1 transcription and expression. Promoter variants identified in this study may create or disrupt transcription factor binding sites (TFBS), affecting the feedback regulatory circuit existing between iron levels and CYBRD1 expression. To identify if a correlation exists between CYBRD1 promoter variants and gene expression, the 5’ regulatory region of CYBRD1 of oesophageal cancer (OC) patients will be screened for novel and/or known variants possibly affecting gene expression. The study cohort comprised 110 Black Xhosa-speaking OC patients and 100 population-matched control individuals. Genomic DNA was amplified by PCR and approximately 2100 bp of the 5’ regulatory region of 50 OC patients were characterized by semi-automated DNA sequencing. The remaining cohort was further genotyped by HEX-SSCP analysis and restriction enzyme analysis, where appropriate. Variants identified within the 5’UTR of the various genes under investigation were subjected to in silico analysis to determine whether they created or disrupted any transcription factor binding sites (TFBS). Statistically significant differences were determined by the Fisher exact test and/or chi squared analysis, with a P<0.05 regarded as statistically significant. Variants identified in the 5’ UTR of the CYBRD1 gene included -1844C/G, -1834G/A, -1459T/C, -1346T/C, -624G/A and the G(T)8G(T)nG(T)nG(T)9 repeat. These variants presented in both the homozygous and heterozygous states in patients as well as the controls. Predictive functional analyses using bioinformatic tools indicated that the presence of a number of these variants affected TFBS crucial in processes such as iron homeostasis, haematopoiesis and liver regulation. It is therefore suggested that these variants may have a potential role in the regulation of CYBRD1 expression. In silico analysis is merely a predictive tool used to identify putative binding motifs. As the transcriptional regulation of the gene has not yet been clarified, we can only speculate that these TFs have a role in CYBRD1 regulation. Further studies are required to identify key regulatory elements in promoter regions, analyze the functional effects of polymorphisms identified by employing luciferase reporter gene assays and also investigate the possibility of cell specific differences in gene expression.

Poster# 148 HEPHAESTIN IS INVOLVED IN CONTROLLING IRON EFFLUX FROM OLIGODENDROCYTES IN THE CENTRAL NERVOUS SYSTEM Katrin Schulz¹, Chris D. Vulpe, PhD² and Samuel David, PhD³ ¹Center for Research in Neuroscience; ²Department of Nutritional Sciences and Toxicology, University of California; ³Center for Research in Neuroscience, The Research Institute of the McGill University Health Center (Presented By: Katrin Schulz)

Iron is essential for many biological processes. However, because of its redox activity, its levels must be tightly regulated. Ferroxidases, which convert toxic ferrous iron into its non-toxic ferric form, are involved in maintaining iron homeostasis. One of these ferroxidases is Hephaestin (Heph), a transmembrane copper-dependent enzyme that is required for the export of iron from intestinal enterocytes. Heph is thought to facilitate iron efflux from enterocytes by oxidizing the ferrous iron transported across the 141 cell membrane via the iron exporter ferroportin (Fpn). Here we show by double immunofluorescence that Heph and Fpn are expressed in oligodendrocytes in purified primary cell cultures from rat cortex and in spinal cord sections of C57BL/6 mice.Furthermore, Heph expression in oligodendrocytes is restricted to mature cells, while Fpn is expressed in mature as well as immature oligodendrocytes. We also show that Heph is required for iron efflux from oligodendrocytes in primary cell culture. In addition, immunofluorescence and Perl´s histochemistry of spinal cord and brain sections of ‘sex-linked anaemia’ (sla) mice show iron accumulation in grey matter oligodendrocytes. These mice carry a mutation in the heph gene resulting in the expression of a truncated form of Heph with only partial ferroxidase activity. Furthermore, the increased iron accumulation was also accompanied by decreased motor coordination on a rotorod. These data show that Heph is expressed by oligodendrocytes and that it might be involved in exporting iron from these glial cells.

Poster# 149 EXTRACELLULAR H-FERRITIN IS AN ALTERNATIVE IRON IMPORT MECHANISM IN OLIGODENDROCYTES, AND ITS RECEPTOR IS TIM-2 James R. Connor, PhD, Bozho Todorich, BS, Xuesheng Zhang, PhD and Becky Slagle-Webb, BS Penn State College of Medicine (Presented By: James R. Connor, PhD)

Oligodendrocytes, the only myelin-producing cells in CNS, contain more iron than other cells in CNS, and peak iron import into oligodendrocytes coincides with peak myelinogenesis. Because iron deficiency leads to hypomyelination in humans and animal model, characterizing mechanisms of iron import into oligodendrocytes is important. Even though traditionally transferrin has been considered essential for survival of oligodendrocyte progenitors (OPCs), during differentiation these cells lose expression of transferrin receptor, but continue to uptake iron, which suggested alternative mechanism of iron import. Previous studies demonstrated saturable and specific receptors for H-ferritin in OPCs, yet the identity of that protein has been unknown. In the current study we evaluated hypothesis that extracellular H-ferritin is important in iron delivery and myelination of oligodendrocytes and that the receptor for H-ferritin in oligodendroglia is Tim-2. In support for our hypothesis we show that incubating primary rat OPCs with recombinant H-ferritin increases iron content of these cells and that the loss of cell viability of OPCs due to transferrin deprivation in media can be completely reversed by administration of recombinant H-ferritin. In complete media, however, 6 day incubation with H-ferritin results in structurally more differentiated oligodendrocytes, which immunocytochemically and by Western blot expressed significantly more myelin basic protein and olig2. Previously we reported that ferritin is taken into the cells via a specific receptor which was recently identified as Tim-2 (T cell immunoglobulin domain 2 protein) by Chen et al.(2005) in lymphocytes. We further demonstrated that pre-treating rat primary OPCs with blocking antibody raised to extracellular domain of Tim-2 completely abated the specific H-ferritin binding to these cells, Thus, we conclude that extracellular H-ferritin is an alternative iron-transport mechanism and a pro-myelinating agent to developing OPCs and its uptake in oligodendrocytes is mediated via Tim-2 receptors.

Poster# 150 THE COMPARTAMENTALIZED DISTRIBUTION OF FERROPORTIN IN THE MOUSE CENTRAL NERVOUS SYSTEM IS INDICATIVE OF A DELICATE FUNCTION FOR NEURONAL IRON METABOLISM M. Boserup², J. Lichota², D. Haile³ and Torben Moos¹ ¹Aalborg University; ²Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; ³Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA (Presented By: Torben Moos)

Introduction: Iron is essential for a variety of cellular functions in neuronal cells. Accordingly, neurons must maintain a delicate balance between uptake and excretion of iron to simultaneously ensure proper uptake and prevention of iron intoxicating the neurons. Neuronal uptake of iron is reflected by a robust and consistent expression of proteins like transferrin receptors and divalent metal transporter I (DMTI). Some neuronal pools express ferritin indicating that they scavenge iron by storage. Conversely, the capability of neurons to neutralize and possibly excrete iron is much lesser studied. Concerning the possibility of neurons excreting iron, there are clear indications from few studies in rodents that neurons express ferroportin, which probably reflect a function for neuronal iron export. In the present study, we attempted to obtain a complete distribution of ferroportin in the adult mouse central nervous system using an antibody specifically raised against mouse ferroportin. Methods: The distribution of ferroportin was studied in adult male 8 weeks old 129/SvJ mice using immunohistochemistry. Specific binding of the antibody was detected using the avidin-biotin complex method added with tyramide signal amplification. DAB was used as chromogen. The specificity of the antibody against ferroportin in the mouse brain was verified in immunoblots of dissected preparations of the dorsal portion of the rostral mesencephalon that contained the habenular nucleic region. As negative controls homogenates of cultured mouse brain endothelial cells b.End.3 were used. Results Obtained: In brain sections, neuronal ferroportin was strikingly confined to certain pathways, i.e. neurons of the pyramidal tract projecting from the cerebral cortex to cranial motor nerve nuclei and motor neurons of the spinal cord, neurons of the hippocampus in particular those projecting to the entorhinal cortex, neurons of the habenular-interpeduncular tract, and neurons of the cerebellum projecting to the red nucleus of the brain stem. In other regions of the brain, e.g. striatum, globus pallidus, hypothalamus, and molecular layer of the cerebellum ferroportin-expressing neurons were hardly identified. Among non-neuronal cells, ferroportin was observed in cells with morphology corresponding to oligodendrocytes. Interestingly this finding was only made in oligodendrocytes of the grey matter. Ferroportin was not observed in cell types denoting the blood-brain- or blood-cerebrospinal (CSF) barriers. Discussion: Prior studies have shown that many of these neuronal pools are also highly transferrin receptor-expressing. Hence, these data together suggest that neuronal iron homeostasis relies on a balance between transferrin receptor-mediated uptake of iron-transferrin and excretion of iron based on a mechanism involving ferroportin. The consistent expression of both proteins in some regions clearly suggests a particular high turnover of iron in these neuronal pools. Conclusions: Neuronal iron metabolism seems to rely on a delicate balance between uptake mediated by transferrin receptors and excretion involving ferroportin.

Poster# 151 IRON UPTAKE IN THE RETINA OF THE RAT: IMPLICATIONS FOR AGE-INDEPENT AND CONTINOUS IRON UPTAKE, PLUS ANTEROGRADE AXONAL IRON TRANSPORT BY RETINAL GANGLION CELLS Torben Moos¹, N. Bernth², Y. Courtois³ and E.H. Morgan4 ¹Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; ²Danish Technological Institute, Taastrup, Denmark; ³INSERM 598, University Pierre and Marie Curie, Paris, France; 4Department of Physiology, University of Western Australia, Crawley, Australia (Presented By: Torben Moos)

Introduction: Iron uptake has been quite extensively studied in the brain. The retina is developed from the diencephalic portion of the brain and carries the same barrier- properties to circulating proteins found in the brain. Methods: Differently aged postnatal rats were studied for [59Fe-125I]transferrin and [131I]albumin uptake in eyeballs with a main emphasis on iron uptake in the retina. The rats were injected intravenously with a mixture of [59Fe-125I]transferrin and [131I]albumin. Blood was sampled from the ventral tail vein into microcapillary tubes 5 min after the injection to estimate the hematocrit and plasma volume, and thereafter at various time intervals up to 60 min, to estimate a plasma concentration for [59Fe-125I]transferrin and [131I]albumin. Additionally, P1 and P16 rats were injected intraperitoneally with [59Fe-125I]transferrin and [131I]albumin. Eyeballs and retinas were carefully dissected and counted for radioactivity. The volume of distribution, VD, for [59Fe-125I]transferrin in total eyeballs and retina were estimated by dividing the radioactivity per sample of ocular tissue with the terminal plasma radioactivity. P16 rats were also studied for their distribution of [55Fe]transferrin using autoradiography. Results Obtained: In the adult rat eyeball and retina, the uptake of 59Fe was significantly higher than that of [125I]transferrin from 5 onwards. In P16 rat retinas, the uptake of 59Fe was significantly higher than that of [125I]transferrin from 5 min onwards. Comparing P1 and P16 rats, the uptake of [125I] transferrin and [131I]albumin was higher at P1 in both eyeball and retina, whereas the uptake of 59Fe remained at the same level. Autoradiography showed a marked uptake of [55Fe] in the developing photoreceptor cells and retinal ganglion cells. Studies of the dissected optic pathway revealed a significant anterograde axonal transport of 59Fe into the optic nerve, optic tract, thalamus, and superior colliculus. Iron in the nervous tissue was precipitable with an antibody raised against ferritin indicative of detachment of iron from transferrin within retinal ganglion neurons. 142

Discussion: The data demonstrates that iron is specifically taken up by transferrin receptor expressing capillaries of the retina. Iron is continuously taken up by the eye including the retina at adult age, which is coherent with data from similar experiments in the developing and adult brain. The sustained iron uptake implies that photoreceptor cells are devoid a significant capability of reuse of iron released from degenerating photoreceptor disks. Iron also enters ganglion neurons and undergoes anterograde axonal transport into the brain. Conclusions: Iron is continuously taken up by the eye including the retina at adult age. Iron is taken up by photoreceptor cells and retinal ganglion neurons, the latter projecting and transporting iron deeper into the brain.

Poster# 152 RELATIONSHIP BETWEEN BRAIN R2 AND LIVER AND SERUM IRON CONCENTRATIONS IN ELDERLY MEN Michael House, PhD¹, Timothy St. Pierre¹, Elizabeth Milward², David Bruce¹ and John Olynyk¹ ¹The University of Western Australia; ²The University of Newcastle (Presented By: Michael House, PhD)

Introduction: Studies of iron overload in humans and animals suggest that brain iron concentrations may be related in a regionally specific way to body iron status. However, few quantitative studies have investigated the associations between body iron stores and regional brain iron in a normal elderly cohort. To examine these relationships, we used magnetic resonance imaging (MRI) to measure the proton transverse relaxation rate (R2) in 13 brain regions in 18 elderly men (average age 75.5 years) with normal cognition and compared it to liver iron concentrations (LIC) and serum iron indices. Methods: FerriScan® (1) MRI data were acquired for the measurement of LIC concurrently with the brain scan. Brain and liver data acquisitions used a multislice, single spin-echo pulse sequence. R2, was calculated by fitting the signal intensity for all echo times to monoexponential (brain) or biexponential (liver) equations. Brain R2 values were compared to contemporaneous LIC and serum iron measurements (iron, ferritin, transferrin saturation) made at the time of the MRI scan (time point 2) and 3 years before the current study (baseline). Results: Post-mortem iron concentrations from the literature (2) were strongly correlated with mean R2 values from five brain regions (r = 0.99, p = 0.0008). R2 values in the medial globus pallidus were significantly correlated with LIC (r = 0.61, p = 0.007), baseline serum iron concentrations (r = 0.66, p = 0.003), and transferrin saturation (baseline: r = 0.69, p = 0.002; time point 2: r = 0.54, p = 0.019). R2 values in the ventral pallidum were significantly correlated with LIC (r = 0.60, p = 0.015). The caudate nucleus and putamen also showed significant positive correlations with serum iron concentrations and transferrin saturation. In contrast, there were significant negative correlations between brain R2 values in the corpus callosum and serum iron concentrations (r = -0.58, p = 0.012) and transferrin saturation (r = -0.60, p = 0.009) measured at time point 2. There were no significant correlations between brain R2 values and serum ferritin concentrations. Discussion: This exploratory study suggests that, in humans, higher R2 values in basal ganglia grey matter reflect liver iron stores. This interpretation is consistent with rat studies that show brain iron can be affected by dietary intake of iron and that changes are not uniform across all brain regions. R2 in white matter brain regions showed the opposite dependency on serum iron indices compared to grey matter. Conclusions: Our results suggest that iron levels in specific grey matter brain regions are influenced by systemic iron status in elderly men and that the response of the elderly male brain to higher body iron stores and serum iron measures is not uniform and is tissue and region specific. Serum ferritin levels cannot be used as an indicator of brain iron status. References: 1) St Pierre TG, et al. Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance. Blood 2005;105(2):855-861. 2) Hallgren B, Sourander P. The effect of age on the non-haemin iron in the human body. J Neurochem 1958;3:41-51.

Poster# 153 THE HEPCIDIN MRNA AND FPN1 PROTEIN EXPRESSION IN THE CP-/- MOUSE BRAIN Shu-Min Wang, Pei Guo, Zhen-Ling Yang, Xiang-Lin Duan and Yan-Zhong Chang, PhD Hebei Normal University (Presented By: Yan-Zhong Chang, PhD)

Hepcidin is a peptide hormone secreted by the liver that plays a central role in the regulation of iron homeostasis, through binding to the membranous ferroportin1 (Fpn1) following internalization and degradation. Recently, accumulating evidences have revealed that high concentrations of iron in the brain are the primary cause of neuronal death in some age-related neurodegenerative disorders. Lack of Ceruloplasmin (Cp) results in iron accumulation in the brain and neurodegeneration. The previous studies support that hepcidin is widely expressed in brain. Therefore, we want to explore the role of hepcidin on the iron accumulation in the Cp gene deficient mice brain. We firstly compare ferritin levels in the three gene types (Cp+/+, Cp+/- and Cp-/-; 17 month) mice cerebral cortex, hippocampus and striatum. The ferritin level was evidently high in the three regions of Cp-null mice. We found that the hepcidin mRNA was significantly increased in the cerebral cortex and hippocampus but not in striatum of Cp-/- mice by real-time PCR method. We used the western bolt method to detect Fpn1 and TfR1 protein expression in the brain of Cp-null mice. The data show that Fpn1 and TfR1 protein decreased significantly in the Cp-null mice, including the cerebral cortex (CC) and hippocampus (hippo). However, there is slightly increased in the striatum. In order to clarify the role of hepcidin on Fpn1 and iron level in the brain, we injected the hepcidin into the lateral cerebral ventricle and found that the Fpn1 significant decreased in cerebral cortex, hippocampus and striatum and ferritin increased significant in cortex and striatum slight in hippocampus. Our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism. The interactions of hepcidin and Fpn1 may control the iron efflux from cells, and disruption of this balance could lead to iron accumulation in the central nervous system.

Poster# 154 THE INCREASING IRON ACCUMULATION IN THE AGING BRAIN IS REFLECTED BY AN INCREASE IN NEURONAL FERRITIN RATHER THAN OF FERROPORTIN M. Boserup¹, L. Lichota¹, D. Haile² and Torben Moos¹ ¹Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.; ²Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA (Presented By: Torben Moos)

Introduction: Iron in the brain accumulates with increasing age. This increase is reflected by an increase in neuronal ferritin and indicates that neurons store and neutralize excess iron. Another principle mechanism for scavenge of iron excess would be to excrete it via induction of ferroportin that is present in many neuronal pools of the brain. Methods: In this study we examined the expression at the mRNA and protein level in the aging (8 months) brain and correlated with that of ferroportin in young (2 weeks) and normal adult (8 weeks) old rats using PCR, Western-blotting and immunohistochemistry. Results Obtained: In contrast to a prominent increase in neuronal ferritin throughout in the aging brain, ferroportin remained at unaltered levels. The ferroportin- immunoreactivity was particularly high in neurons of the neocortex, hippocampus, habenular nuclei, red nucleus, interpeduncular nucleus, cerebellar cortex, and locus ceruleus, but the immunoreactivity was not higher than seen in the normal adult brain. The study did not find evidence that ferroportin was induced in glial cells of the aging brain. This also applied to endothelial cells of the blood-brain barrier and epithelial cells of the choroids plexuses. Discussion: Ferroportin is supposed to provide a route for export of iron from cells. In the aging brain, iron increases gradually with increasing age, but this was not reflected in a higher expression of ferroportin. The ferroportin protein could be of significance for immediate changes in neuronal iron concentrations and as such operate to maintain iron levels within a given steady state. By contrast, ferritin could operate to neutralize excess iron occurring from a decreased capability to reuse iron with increasing age of the neurons leading to a need for continuously iron-uptake in spite of a prominent cellular iron level. Following this hypothesis, it might be the case that iron is preferentially directed towards neutralization in ferritin rather than directed to ferroportin for export. More research is needed to delineate the conditions for neuronal iron levels that might lead to ferroportinmediated release of iron. Ferroportin appears to distribute to somata, dendrites and axons, but given the fact that many neurons have their axons covered by myelin neurons may mainly release their iron from the soma.

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Conclusions: The data indicate that the rise in brain iron with increasing age does not trigger neurons to export iron by raising ferroportin-expression, but rather leads to increased storage in ferritin.

Poster# 155 INHIBITION OF PROLIFERATION OF OLN 93 CELLS BY IRON CHELATORS: USE AS TEST SYSTEM TO STUDY THE BIOAVAILABILITY OF IRON IN IRON OXIDE NANOPARTICLES Michaela Hohnholt, Mark Geppert and Ralf Dringen, Dr University Bremen (Presented By: Michaela Hohnholt)

Iron is an essential metal for the cellular metabolism due to its redox function in various enzymes. Iron is especially important for ATP production by the respiratory chain and for DNA synthesis during proliferation. Deferoxamine (DFX), a well known bacterial ferric iron chelator, has been reported to inhibit cell proliferation in tumor cells. We have used OLN 93 cells, an oligodendroglial cell line derived from rat brain cell cultures, as model system to investigate the consequences of iron deprivation on cell proliferation. During incubation in culture medium containing 10% fetal calf serum in the absence of iron chelators, OLN cells proliferate strongly as demonstrated by substantial increases in cell density, in the protein content per well and in the activity of the cytosolic enzyme lactate dehydrogenase (LDH). Compared to initial values (0 h), the protein content per well and the LDH activity per well increased within 48 h by factors of eight and three, respectively. Presence of DFX in concentrations of 100 or 1000 µM completely prevented the proliferation of OLN cells. For an incubation period of 48 h half maximal effect on cell proliferation were obtained at a DFX concentration of about 50 µM. Presence of DFX in concentrations of up to 1000 µM was not toxic to the cells as indicated by the lack of an increase in extracellular LDH activity. In addition to DFX, also the presence of the membrane impermeable iron chelator bathophenanthroline disulfonate or of the membrane permeable iron chelators 1,10-phenanthroline and 2,2´-dipyridyl resulted in complete inhibition of cell proliferation. The inhibitory effect of DFX on the proliferation of OLN cells was prevented by application of ferric ammonium citrate (FAC), a soluble ferric iron salt. Presence of 30 µM FAC was partly able to overcome the DFX-induced inhibition of proliferation of OLN cells that were incubated for 48 h with 100 µM DFX, whereas presence of FAC in a concentration of 100 µM allowed maximal proliferation of the cells. Analysis of the cellular iron contents revealed that presence of DFX significantly lowered the cellular iron content compared to controls, if the chelator was present in excess compared to FAC. To study the bioavailability of iron in iron oxide nanoparticles, we tested whether application of such particles was able to prevent the DFX-mediated inhibition of cell proliferation. Application of 100 µM iron in form of iron oxide nanoparticles in the presence of 100 µM DFX allowed partial proliferation of OLN cells. In addition, the cells accumulated more iron from iron oxide nanoparticles than from equal concentrations of FAC within 48 h and the increase of the specific amount of cellular iron after exposure to iron oxide nanoparticles was not affected by the presence of DFX. These data demonstrate that OLN cells are able to accumulate iron after exposure to iron oxide nanoparticles and that iron from these particles is used by the cells to support proliferation.

Poster# 156 THE IONOTROPIC GLUTAMATE RECEPTOR AGONIST N-METHYL D-ASPARTATE (NMDA) AND SPATIAL MEMORY TRAINING ENHANCE THE EXPRESSION OF THE IRON TRANSPORTER DMT1 IN HIPPOCAMPAL NEURONS Paola Haeger, PhD², Pablo Munoz, PhD³, Alexis Humeres, MSc², M. Angelica Carrasco, PhD4, Marco T. Nunez, PhD5 and Cecilia Hidalgo, PhD¹ ¹F. Medicine, U. Chile; ²CEMC, F. Medicine, U. de Chile, Santiago, Chile; ³CIBR, U. de Valparaíso, Valparaíso, Chile; 4ICBM & CEMC, F. Medicine, U. de Chile, Santiago, Chile; 5MI CDB, F. Sciences, U. de Chile, Santiago, Chile (Presented By: Cecilia Hidalgo, PhD)

Introduction: Activation of hippocampal postsynaptic NMDA receptors triggers calcium-dependent intracellular signaling cascades, which in turn induce the expression of proteins necessary for sustained synaptic plasticity, memory, and learning. Here, we investigated a) the role of iron in the generation of hippocampal calcium signals, and b) the possible effects of NMDA and spatial memory training on the expression of the iron transporter DMT1. Methods and Results: NMDA receptor activation, produced by 5 min incubation of hippocampal cells in primary culture with NMDA (50 µM), caused an intracellular Ca2+ concentration increase, which through Ca2+-induced Ca2+ release (CICR) stimulated ryanodine receptor (RyR)-mediated Ca2+ release from intracellular stores. The iron chelator desferrioxamine abolished this increase, indicating that RyR-mediated CICR requires iron. Furthermore, addition of iron to primary hippocampal cultures increased the labile iron pool, stimulated reactive oxygen species generation (ROS) and via ROS generation elicited Ca2+ release through the highly redox-sensitive intracellular RyR channels. In view of iron’s requirement for RyR-mediated CICR, which is a necessary intermediate step of sustained long-term potentiation in the hippocampus, we investigated the effects of agonist-induced NMDA receptor activation and spatial memory training on the expression of the iron transporter DMT1, which mediates iron uptake into neurons. We found that 5 min incubation with NMDA (25-50 µM) enhanced >2-fold DMT1(+IRE) mRNA levels (measured 24-48 h later) without changing DMT1(-IRE) mRNA expression. In these conditions, NMDA also produced a significant enhancement of total DMT1 protein expression, which was significantly inhibited by RyR inhibition with ryanodine or a ROS scavenger. Next, we tested if spatial memory training of male rats in a Morris water maze increased DMT1 expression. For these experiments, the hippocampus from male rats was dissected 6 h after the last behavioral task (5d, 2d rest, 1d platform free) and tissue samples were prepared for Western blot and RT-PCR analysis. We found that spatial memory training increased > 5-fold DMT1(+IRE) mRNA and 2-fold total DMT1 protein expression. Discussion and Conclusions: Our results correlate for the first time agonist-induced activation of the NMDA receptor and spatial memory acquisition/consolidation with enhanced in vitro and in vivo expression, respectively, of the iron transporter DMT1. We propose that iron-induced ROS production facilitates RyR-mediated CICR, which promotes Ca2+-dependent DMT1 expression after NMDA-induced neuronal activation or during the spatial memory task. Supported by FONDECYT postdoctoral project 3070035, CEMC-FONDAP grant 15010006, Millennium Scientific Initiative grant P05-001F and FONDECYT grants1070840 and 1060177.

Poster# 157 BRAIN IRON MANAGEMENT PROTEIN GENE EXPRESSION VARIES ACROSS THE DIURNAL CYCLE AND WITH DIETARY IRON DEPRIVATION Erica Unger, PhD, John Beard, PhD, Lei Hao, Narasimha Hegde, PhD, Byron Jones, PhD and James Connor, PhD Pennsylvania State University (Presented By: Erica Unger, PhD)

In our previous work using BXD/Ty recombinant inbred mouse strains we showed significant, genetically-based variations both systemic iron status and brain iron and iron management proteins (Jones et al 2003, 2007, 2008). Restless Legs syndrome patients appear to have low brain iron levels while maintaining a low-normal systemic iron status. Several candidate strains of mice from the RI panel have been examined for diurnal variation in both brain and systemic iron status but also for gene expression of iron management proteins. We have chosen strains that have a high, medium, low ratio of ventral midbrain iron/liver iron as investigational models to describe iron flux across diurnal cycles and with co-existing iron deficiency. Unger et al note that BXD 40 is a strain with low ventral midbrain iron content but with a modest reduction in hemoglobin and liver iron when fed a low iron diet (see Jones et al). Mice from this strain had 40% greater liver iron in the dark versus the light and liver ferritin protein, and ferritin and hepcidin mRNA levels also varied accordingly. As might be expected, dietary iron deficiency eliminated this exchangeable pool of iron and there were no longer differences between night and day. At the same time that liver iron and hepcidin increase, ventral midbrain and nucleus accumbens iron are reduced by 15-25% suggesting an exchange of iron across the blood-brain-barrier is possible as part of the normal diurnal flux of iron between organs. qRT-PCR of ventral midbrain showed strains of mice with varying levels of ventral midbrain iron also had relative differences in hepcidin mRNA. With dietary iron restriction in Strain 40 mice, there was a 16% reduction in gene expression for hepcidin, a 34% reduction in ferroportin, and a 40% reduction in ferritin H. It is not clear what particular cell types in the ventral midbrain, or other brain regions, contain the message, though microglia is a candidate cell type. In situ hybridization studies should reveal this information shortly. In addition, the source of the signal for this diurnal variation is also not immediately known, but a number of candidate regulators of hepcidin gene expression are regulated by CLOCK and PER genes. The periodicity of symptoms in Restless Legs Syndrome and the new suggestions of periodicity in brain iron content make it tempting to make a connection between variations in brain iron and motor movement. There is already published data that show correction of brain iron with iron treatment is associated with a diminution 144 in RLS symptoms. Future studies, in connection with Dr. Connor on blood brain barrier iron biology and Dr. Jones on genetic networks affecting brain iron homeostasis will provide more clarity. Supported in part by USPHS Grants PO1 AG 021190.

Poster# 158 GENOME-WIDE MICROARRAY ANALYSIS OF BRAIN FROM A HEMOCHROMATOSIS HFE KNOCKOUT MOUSE MODEL SHOWS FEW CHANGES IN IRON-RELATED GENE EXPRESSION Daniel Johnstone, Ross Graham, PhD, Deborah Trinder, PhD, Rodney Scott, PhD, John Olynyk, MD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Iron dysregulation and tissue damage are relatively common in people with hereditary hemochromatosis, a disorder usually attributable to dual polymorphism in the HFE gene. Historically, the brain is not considered at risk in hemochromatosis to the same extent as other organs, as the bloodbrain barrier appears to provide some protection against excessive iron uptake. However genetic conditions that alter proteins important in iron metabolism may affect internal brain iron homeostasis even in the absence of abnormal brain iron uptake. To examine genetic changes that might influence brain iron homeostasis, we have performed genome-wide microarray analysis to investigate differential gene expression in brains from an Hfe knockout mouse model of hemochromatosis and wildtype AKR mice. Total RNA was extracted from brain hemispheres of 10 week-old biological replicates of Hfe knockout mice and wildtype AKR mice (n≥4 per group). Differential expression analysis by microarray was performed using Illumina Sentrix MouseRef-8 (v1.1) BeadChip arrays. Data were normalized and analyzed using BeadStudio v.3 (Illumina) and GeneSpring GX 7.3 (Agilent Technologies). Relative to wildtype mice, there were few changes in expression of genes usually associated with iron homeostasis. There was a small but significant increase of 1.4-fold in expression of the gene encoding the iron storage protein ferritin light chain (p=0.010), accompanied by a small but significant decrease of 1.13-fold in transcripts for transferrin receptor 1 (p=0.046). There were no significant expression changes for genes encoding ferritin heavy chain, transferrin, iron regulatory proteins 1 and 2, ferroportin, hepcidin, divalent metal transporter (Dmt1), hephestin, ceruloplasmin or beta-2 microglobulin (all p>0.05). We did, however, observe significant increases in transcripts for transferrin receptor 2 (1.6-fold; p=0.036) and hemojuvelin (2.7-fold; p<0.001). Loss-offunction mutations in both these proteins cause forms of hemochromatosis and, while their interrelationship with Hfe in regulating brain iron homeostasis is unknown, it is possible that the increases observed here may result in gain-of-function that compensates at least in part for the Hfe defect, reducing rather than increasing pathology and maintaining expression of most iron-related genes at control levels. There was little evidence of any substantial effects on genes involved in oxidative stress, inflammatory responses or apoptosis. The overall lack of marked changes in expression of key iron-related genes suggests there may be relatively few changes in brain iron homeostasis in hemochromatosis. However several possibilities remain to be considered. The brain may rely on different mechanisms of regulating iron homeostasis compared to peripheral tissues. Expression of several proteins related to iron regulation is also controlled post-transcriptionally by intracellular iron levels through factors such as iron regulatory proteins and iron responsive elements, so the lack of changes observed in mRNA transcripts may not correspond to what occurs at the protein level. Different cell types and regions in the brain may also regulate iron differently, so it will be valuable to study specific cell types and regions. In conclusion, while disruption of the Hfe gene may influence other brain systems, these findings suggest that it does not strongly influence the expression of genes known to be involved in iron homeostasis or in pathways central to neural damage and death.

Poster# 159 QUANTIFICATION OF IRON IN HUMAN BRAIN IN VIVO USING APPARENT TRANSVERSE RELAXATION RATE OBTAINED AT HIGH FIELD MRI Fumiyuki Mitsumori, PhD, Hidehiro Watanabe, PhD and Nobuhiro Takaya National Institute for Environmental Studies (Presented By: Fumiyuki Mitsumori, PhD)

Iron is an essential metal to human brain, but its overabundance causes neurodegenerative diseases like aceruloplasminemia [1]. Recently it has been reported that the occurrence of Alzheimer’s and Parkinson’s diseases are closely related to the presence of regional iron [2]. Thus, estimation of the brain iron concentration in vivo is relevant to assess the risk of these diseases. We reported that the apparent transverse relaxation rate (R2† = 1/T2†) of the tissue water in human brain obtained at 4.7T shows a high linear correlation with the published level of non-hemin iron concentration ([Fe]) (R2† = 0.551[Fe] + 14.1, r = 0.97) [3]. In the present paper we propose a method to quantify [Fe] in vivo based on the above linear relationship. We collected T2† maps of the brain in 54 healthy subjects using a multi-echo adiabatic spin echo (MASE) sequence on a high field MRI at 4.7T (Varian, Palo Alto, USA) [3]. A simple application of the linear equation between R2† and [Fe] to the observed R2† values at five grey matter (GM) regions in each subject revealed clear age-dependent changes in [Fe]. The trend is consistent with the change reported by Hallgren and Sourander [4] with increases with the age at frontal cortex, caudate, putamen, and globus pallidus, while a linear decrease at thalamus. The limitation with this simple estimation is that [Fe] at thalamus was 43% overestimated, while the estimate in other GM regions remained within ±20% of the reported value. Another limitation is that [Fe] in the white matter (WM) region cannot be estimated. To overcome above limitations a refinement was attempted by considering a contribution of macromolecules to R2†. When the observed R2† was analyzed by a multi- regression scheme with [Fe] and the macromolecular fraction (fM), R2† in both GM and WM regions were better fitted. The result gives an equation of R2† = 0.47[Fe] + 24.9fM + 9.54. The recalculated [Fe] in thalamus and frontal WM were 4.9 ± 2.0 (vs. reported value of 4.76 ±1.16), and 4.0 ± 1.7 (vs. reported value of 4.24 ± 0.88) mg/100g fresh weight, respectively. It is possible to quantify the iron concentration as low as 2 mg/100g fr. wt. (0.36 µmol Fe/g fr. wt.). References [1] Miyajima H et al, Neurology, 37, 761 (1987)., [2] Smith DG et al, BBA, 1768, 1976 (2007)., [3] Mitsumori F et al, Magn. Reson. Med., 58, 1054 (2007)., [4] Hallgren B, Sourander P, J. Neurochem., 3, 41 (1958).

Poster# 160 CHARACTERIZATION OF THE IRON MANAGEMENT PROTEIN PROFILES IN THE BRAINS OF MICE CARRYING THE H67D MUTATION IN HFE GENE James Connor, PhD, Wint Nandar, BS and Elizabeth Neely, BS Penn State University, M.S. Hershey Medical Center (Presented By: James Connor, PhD)

Iron accumulation is associated with the pathogenesis and the progression of neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). One of the mechanisms that can lead to iron overloading is a mutation in hemochromatosis gene (HFE), which encodes a nonclassical MHC class-1 protein. The most common HFE mutation, H63D, has been has been identified as a potential risk factor in a number of neurodegenerative disorders. This mutation has been shown to influence the iron homeostasis, increase oxidative stress, neuroinflammation and neuroexcitotoxicity in SH-SY5Y neuroblastoma cell lines. However, the role of the H63D mutation in brain iron homeostasis and its effect on neurological disorders in vivo has not been investigated. Since both environmental and genetic factors are involved in iron homeostasis and the role of H63D mutation in iron accumulation has been controversial, in vivo model is necessary to evaluate the neurological consequences of H63D mutation. The mouse model for H63D mutation used in the present study was established by intercrossing between HfeH67D/+ mice that carry wild type and H67D alleles (mice homologous to H63D in humans; homozygous H67D mutants) or crossing HfeH67D/+ mice with HFE knock out mice (heterozygous H67D mutants). Although hepatic iron loading has been reported in mice carrying the H67D mutation, the neurological characterization of H67D homozygous mice have not been reported yet. Thus, we evaluated the expression of the brain iron management protein in heterozygous (+/-) and homozygous (-/-) H67D mice. At 6 months of age, the brains from +/- or -/- H67D mice were harvested and transferrin receptor expression (TfR), one of the proteins involved in iron uptake, was detected by immunohistochemistry. The brains of -/- and +/- H67D mice were not different macroscopically from each other. However, TfR expression was downregulated in the striatum and the hippocampus of -/- H67D mice compared to +/- H67D mice. Since TfR expression is influenced by the cellular iron level, downregulation of TfR expression in -/- H67D mice suggested that -/- H67D mice 145 would have increased brain iron level compared to +/- H67D mice. Although evaluation of other iron management proteins in H67D knock in mice compared to both heterozygous H67D and wild type mice is still in process, H67D knock in mice appear to be a potential animal model to investigate the role of H63D mutation in iron homeostasis and its effect on the pathogenesis of neurodegenerative diseases.

Poster# 161 FORWARD GENETIC ANALYSIS OF BRAIN IRON MANAGEMENT DURING IRON DEFICIENCY Byron C. Jones, PhD, Leslie C. Jellen, BS, Erica L. Unger, PhD and John L. Beard, PhD The Pennsylvania State University (Presented By: Byron C. Jones, PhD)

Previously, we have shown that iron concentrations in brain, to show wide, genetically-based regional differences among inbred mouse strains. Our animal model consists of the 20+ recombinant inbred strains of the BXD/Ty panel, derived from the C57BL/6 and DBA/2 inbred strains. Recently, we subjected male and female mice from these strains to an iron-deficient diet (~4 ppm Fe) beginning at weaning at 21 days and continuing for 100 days. At sacrifice, we measured iron concentrations in several tissues, including the liver, spleen, and brain; in the brain, we focused on the ventral midbrain, as a key player in central dopamine neurobiology. Upon analysis of iron concentration in the ventral midbrain, we observed large differences in iron loss, compared to control. In some strains, the difference was minimal and not statistically significant, i.e., these animals were completely resistant to treatment. In other strains, however losses to near 40% were observed and the extent of loss was not at all correlated with iron concentrations in the control animals. Genetic mapping revealed several suggestive polymorphic sites on the mouse genome (QTL) harboring possible candidate genes. One such QTL on mouse chromosome 7 identified in mice fed an iron-adequate diet was amplified by iron deficiency and points to a Riken Institute theoretical gene as possible candidate. We are presently conducting gene expression studies of iron management (and other) proteins to identify those genes and gene networks that underlie individual differences in brain iron loss. Similar to iron losses observed in the ventral midbrain, spleen and liver iron losses in response to an iron-deficient diet also show wide, genetically-based variability. Interestingly, the losses in these peripheral tissues are not correlated nor are the losses from either of these tissues correlated with iron loss in the brain. It is evident from our work that the management of iron in brain is largely independent from peripheral iron management and may have important implications for understanding diseases related to brain iron such as restless legs syndrome and Parkinson’s disease. Supported in part by USPHS Grants PO1 AG 021190 and F31 NS060393 to LCJ.

Poster# 162 CELLULAR IMPACT OF H63D HFE MUTANT PROTEIN ON ALS Sang Lee, PhD¹, Yiting Liu, PhD¹, Zachary Simmons, MD² and James Connor, PhD¹ ¹Penn State College of Medicine; ²Penn State MS Hershey Medical Center (Presented By: Sang Lee, PhD)

Iron imbalance in the brain has been implicated in the mechanisms that underlie many neurodegenerative diseases. In 2004, we were the first to report that a specific polymorphism in the HFE gene, H63D, was over-represented in ALS. The prevalence (30%) of the H63D allele that we and the other groups reported was the second most frequent genetic variation reported in ALS. However, how H63D HFE might increase the vulnerability to ALS is as yet unknown. We developed a cell model by introducing tetracycline inducible expression system in human neuroblastoma SH-SY5Y cell line to determine the cellular effects of HFE H63D allele. The expression of H63D HFE mutant protein initially triggered the unfolded protein response (UPR), as revealed by the elevated level of the major UPR sensor, BiP/GRP78. This response was followed by down-regulation or cleavage of some UPR sensors, such as IRE1α and ATF4, and the activation of Caspase 3, indicating apoptosis was being promoted as a result of persistent ER stress. In addition, H63D HFE protein expression was associated with a higher level of protein oxidation, compared to wild-type protein. In the tetracycline inducible HFE expressing SH-SY5Y cell line, the presence of H63D HFE also resulted in the decreased expression and activity of Cu/Zn superoxide dismutase 1 (SOD1). In consistence with this result in the cell model, we found that in ALS patients, the individuals carrying H63D HFE allele have 40% less SOD1 expression in the muscle tissue compared to those with wild-type HFE. The effect of H63D HFE on SOD1 expression could be mimicked by stimulating iron over-load with ferrous ammonium citrate (FAC). In the mouse neuroblastoma N2a cell lines stably expressing wild-type or ALS –linked mutant human SOD1, FAC treatment reduced wild-type SOD1 expression, but did not alter the expression of mutant proteins. Together, our data suggest that iron-overload caused by the H63D HFE gene variant activates UPR, promotes apoptosis and increases oxidative stress. Iron accumulation has different regulatory effects on wild-type SOD1 and ALS mutants. These studies shed light on the molecular mechanisms on how iron accumulation and the H63D HFE polymorphism contribute to ALS. Supported by the Paul and Harriett Campbell Fund for ALS Research, the Zimmerman Family Love Fund, and the Judith & Jean Pape Adams Charitable Foundation

Poster# 163 CERULOPLASMIN DIFFERENTIAL EXPRESSION IN THE CEREBROSPINAL FLUID OF PARKINSON’S DISEASE REFLECTS OXIDATION AND FUNCTIONAL IMPAIRMENT FAVOURING INTRACELLULAR IRON OVERLOAD Stefano Olivieri¹, Sandro Iannaccone, Doctor², Antonio Conti, Doctor², Carlo Cannistraci, Doctor², Diego Franciotta, Doctor³, Laura Piccio, Doctror4, Stefano Cappa, Professor5 and Massimo Alessio, Doctor² ¹San Raffaele Scientific Institute; ²San Raffaele Scientific Institute, Milan; ³Istituto Neurologico Mondino, Pavia; 4Washington University School of Medicine, St. Louis; 5San Raffaele University, Milan (Presented By: Stefano Olivieri)

Ceruloplasmin (Cp) is a ferroxidase protein that oxidises toxic ferrous iron to non-toxic ferric form, thus regulates cellular iron loading and protects tissues from iron deposition, especially in central nervous system (CNS). Using 2-Dimensional electrophoresis analysis we investigated Cp profile in the cerebrospinal fluid (CSF) of neurodegenerative patients (Parkinson’s disease, PD; Amyotrophic Lateral Sclerosis, ALS) compared to control subjects (CN). We found that differential expression of Cp isoforms is able to statistically discriminate among the groups. In particular, PD showed a more acidic Cp pattern not present in CN and ALS patients. Automatic image analysis performed with machine learning algorithm is able to identify the two CN and PD clusters. In vitro CSF oxidation generates a Cp shift to acid region similar to that observed in neurodegenerative patients. The CSF proteins oxidation correlates with the increase in carbonylation, a modification that induces protein acidification. The analysis of CSF from PD patients confirms the carbonylation increase on specific protein-targets, included Cp. Interestingly, the acidic Cp isoform present in PD patients resulted to be the carbonylated Cp isoforms. Moreover, in vitro oxidation of purified Cp resulted in a decrease of its ferroxidase activity. Functionally inactive oxidized-Cp, promote intracellular iron overload in the SH-SY5Y neuroblastoma cell line, indicating a role of Cp ferroxidase impairment in the pathological mechanisms. In conclusion, these results showed that a specific isoform of Cp is generated in the CSF of PD patients as consequence of the increase of the oxidative level in the CNS. The presence of oxidized Cp might be used as biomarker useful for early diagnosis and oxidative damage quantitation, further providing insight into disease pathogenesis.

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Poster# 164 ASSOCIATION STUDIES IN ALZHEIMER’S DISEASE EXCLUDE CAT53 AS A NEW SUSCEPTIBILITY GENE BUT SUPPORT A PUTATIVE PROTECTIVE ROLE OF THE C282Y HFE MUTATION Ana Paula Correia, MD¹,³, Jorge Pinto, PhD², Vera Dias, Technician², Cláudia Mascarenhas, Technician², Susana Almeida, Technician² and Graça Porto, MD, PhD²,³,4 ¹Hospital de Magalhães Lemos, Porto; ²IBMC – Institute for Molecular and Cellular Biology, Porto; ³Centro Hospitalar do Porto, Hospital de Santo António, Porto; 4ICBAS – Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal (Presented By: Ana Paula Correia, MD)

Alzheimer´s disease (AD) is a complex disorder, resulting from an interaction between environmental and genetic factors. Several studies addressed the association of AD with MHC class-I polymorphisms without definite conclusions. Considering the remarkable linkage disequilibrium at the MHC region, it is not possible to assume if the reported associations result from a direct effect of the respective genes or result from associations with other closely linked genes transmitted in an extended conserved haplotype. Recent evidence pointed to CAT53, a newly described gene located at the MHC class-I region in the vicinity of HLA-C, as a candidate modifier gene in AD. CAT53 encodes a phosphatase 1 nuclear inhibitor protein and is strongly expressed in brain regions involved in memory and AD. Here we tested the potential association of CAT53 with the risk of developing AD and searched for potential haplotypic associations of CAT53 with two common mutations (H63D, C282Y) in the HFE gene, also located at chromosome 6p21.3. The allele frequencies of these mutations in AD patients were compared to the expected frequencies previously established in the normal Portuguese population. We detected only one polymorphism (G>A) in CAT53,at position 8232, in intron 17. Screening of this polymorphism in 113 AD patients and 82 controls did not show any evidence of association, therefore excluding the hypothetical role of the CAT53 polymorphism as modifier in AD. In contrast, we found a global highly significant negative association in AD patients for the presence of the C282Y mutation (a.f.=0,013 vs 0,058 in controls; P=0,0022) supporting a putative protective role of this protein variant in neurodegeneration. This result was confirmed when the analysis was restricted to the subgroup of late-onset AD patients (a.f.=0,010 vs 0,058 in controls; P=0.0025). The mechanism how the HFE C282Y mutation could confer protection to neurodegeneration is still speculative. It has been recently described that the presence of the mutant C282Y protein stimulates an Unfolded Protein Response (UPR) with consequent increased expression of other chaperone proteins implicated both in the response to cellular iron overload and in protection from oxidative stress (Lawless 2007, de Almeida 2007, 2008). One of these proteins, calreticulin, was shown to have a protective role in the clinical expression of Hereditary Hemochromatosis (Pinto 2007).It is plausible to consider that other proteins may be also involved in the HFE- induced UPR that could explain the protective response associated with an up-regulation of the signal transduction pathway in response to stress. Lawless et al. BMC Cell Biol. (2007) 8:30 De Almeida et al. J Immunol (2007) 178:3612-9 De Almeida and De Sousa J Cell Mol Med (2008) 12:421-34 Pinto et al. Free Radic. Biol. Med. 44 (2008) 99-108

Poster# 165 HFE POLYMORPHISM, H63D, IMPACTS TAU PHOSPHORYLATION James Connor, PhD¹, Eric Hall, II, BS¹, Sang Lee, PhD¹, Noot Mairuae, MS² and Zach Simmons, MD¹ ¹Penn State University; ²Chulalongkorn University (Presented By: James Connor, PhD)

Numerous studies have examined the association between HFE gene polymorphisms and neurodegenerative disease, including Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Results from these studies are highly variable, strongly implying a gene-environment interaction. The HFE protein is associated with regulating iron homeostasis where iron dyshomeostasis is represented in numerous neurological diseases. HFE protein is expressed on blood vessels, choroid plexus, and ependymal cells representing the cerebral vasculature, placing HFE in position to impact iron uptake into the brain. Thus, it is important to determine the cellular effects, if any, of HFE polymorphisms in contributing to neurological pathogenesis. In the present study, we hypothesized that our stably transfected H63D-expressing SH-SY5Y neuroblastoma cell culture model would have increased tau phosphorylation compared to the wild-type polymorphism. The hyperphosphorylation of tau leads to neurofibrillary tangle formation, a hallmark characteristic of Alzheimer’s disease, and a disruption in the neuronal cytoskeleton leads to neurodegeneration. We have previously shown expression of the H63D allele to result in a higher baseline of oxidative stress and altered glutamate homeostasis, two factors that indirectly contribute to increased tau phosphorylation. In testing our hypothesis, we have found an increase in total tau and increased phosphorylation at the serine 199, serine 202, serine 396, and serine 404 amino acid sites in the presence of the H63D allele using ELISA and semi-quantitative western blot methods. Through evaluating the direct mechanisms responsible for increased tau phosphorylation, we have found increased kinase expression/activity of GSK-3β (glycogen synthase kinase). Protein phosphatase (PP-1 and PP-2A) expression was not altered under the expression of H63D HFE. Additionally, we have found that cellular iron levels can directly influence GSK-3β activity by exposing the neuroblastoma cells to exogenous iron (ferric ammonium citrate) and removing iron with a chelator (desferrioxamine); increased iron leads to increased GSK-3β activity consistent with increased iron levels in the presence of the H63D allele. Furthermore, oxidative stress is associated with increased tau phosphorylation at the same epitopes elevated in H63D cells and treatment with Trolox, an anti-oxidant, lowered tau phosphorylation. Our data showing the association of the H63D HFE variant with increased tau phosphorylation and up-regulated GSK-3β activity is compelling evidence that the H63D allele is associated with an altered intracellular environment that would be consistent with its putative role as a risk factor for neurodegenerative disorders. These data begin to provide a framework as to how allelic variants of the HFE gene can influence neurological diseases. The data suggests that the H63D allele can increase tau phosphorylation through iron-mediated oxidative stress and GSK-3β activity.

Poster# 166 REACTIVE OXYGEN SPECIES REGULATE CERULOPLASMIN BY A NOVEL MRNA DECAY MECHANISM INVOLVING ITS 3’-UNTRANSLATED REGION: IMPLICATIONS IN IRON DEPOSITION IN NEURODEGENERATIVE DISEASES Nisha Tapryal, MSc and Chinmay Mukhopadhyay, PhD Jawaharlal Nehru University (Presented By: Nisha Tapryal, MSc)

Ceruloplasmin (Cp), a copper containing protein, plays a significant role in body iron homeostasis as aceruloplasminemia patients and Cp knock out mice exhibit iron overload in several tissues including liver and brain. Several other functions as oxidant, antioxidant and in nitric oxide metabolism are also attributed to Cp. Despite its role in iron oxidation and other biological oxidation reactions the regulation of Cp by reactive oxygen species (ROS) remains unexplored. In this study we explored the possibility of Cp regulation by ROS. Since, Cp is synthesized in liver as a secretory protein and predominantly as a glycosylphosphatidylinositol (GPI) -anchored membrane bound form in astroglia, human hepatic cell HepG2, rat glial cell C6 or human astroglial cell U373MG are subjected to ROS treatment either by addition of H2O2 or by mitochondrial electron transport chain blockers i.e., rotenone or antimycin A. In response to ROS, Cp synthesis is decreased as detected by western analysis. We further demonstrated that Cp expression is decreased by an mRNA decay mechanism in response to extracellular (H2O2) or intracellular oxidative stress in both hepatic and astroglial cells. To understand the mechanism a chimera of chloramphenicol acetyl transferase (CAT) with Cp 3’UTR was constructed and transfected in to HepG2 or U373MG cells. In response to either H2O2, rotenone or antimycin A, the expression of CAT transcript is decreased, whereas, expression of 3’UTR less CAT transcript remains unaffected suggesting role of Cp 3’UTR in this Cp mRNA decay mechanism. RNA gel shift assay showed significant reduction in 3’UTR binding protein complex by ROS in both the cell types and is reversed by antioxidant N-acetyl cysteine suggesting that ROS affects RNA protein complex formation to promote Cp mRNA decay. This is the first demonstration of regulation of Cp by ROS by a novel post-transcriptional mechanism. In summary, we have identified a novel post transcriptional regulation of Cp in response to ROS, which could explain the mechanism of iron accumulation and related free radical injury in neurodegenerative diseases like Parkinson’s.

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Poster# 167 LONG TERM DEFERIPRONE INDUCES IRON DEFICIENCY IN FRIEDREICH ATAXIA PATIENTS Filomena Longo, MD², Simona Roggero, MD², Chiara Compagni, MD² and Antonio Piga, Prof¹ ¹Department of Biological and Clinical Sciences; ²University of Turin (Presented By: Antonio Piga, Prof)

Background: In patients with Friedreich ataxia (FRDA), deficiency in the mitochondrial iron chaperone frataxin, results in a defective use of iron and its misdistribution between mitochondria and cytosol. Previous studies indicated that at relatively low concentrations, the orally active membrane-permeant iron chelator deferiprone (DFP), can efficiently mobilize labile iron from cells and tissues. In a preliminary open trial, DFP has been shown to reduce iron accumulation in the dentate nuclei of cerebella of young FRDA patients and to improve their neurological condition with no significant change in hemoglobin or plasma iron levels (Boddaert N, 2007). Methods: We conducted an open label study on iron chelation therapy with DFP in FRDA patients. Compliance was assessed crosschecking patient’ referral, pharmacy records and medical interview. Hematological follow up included weekly blood count and quarterly dosage of iron markers. Liver iron concentration (LIC) was assessed by Magnetic Biosusceptometry SQUID. Neurologic assessment was performed every 3 months with appropriate scales. Results: We enrolled 17 FRDA patients (10 males, 7 females) with a mean age of 23.7 years (range:10.8-39.8 years) The mean follow-up duration was 15.5 months (range: 4-26 months). All patients received oral iron chelation with DFP at a dosage of 20-30 mg/kg/d. The compliance with treatment was optimal (mean 99%; range 96-100%). Two patients dropped out (1 agranulocitosis; 1 subjective discomfort;). Neurologic follow-up showed a mild improvement not only in cerebellar and postural performances but also in sensibility after one year of therapy in most of the patients. No patient showed a significant decrease of baseline LIC levels compared to those at the end of follow-up ( 277 vs 263 mcg/gliver). Also not significant was the reduction observed in serum ferritin (77 ng/mL vs 39 ng/mL), serum iron (87mcg/dL vs 58 mcg/dL), transferrin (264 mg/dL vs 307 mg/dL), transferrin saturation (25% vs 17%) and in reticulocytes count (1.29% vs 1.25%). Eight patients required an iron supplementation. The worst hemoglobin delta was observed in a male boy at the longest treatment duration. The hemoglobin changes were not related with gender, deferiprone doses or duration. Conclusions:Long term DFP treatment, even at low dosage, does not reduce total body iron but induces iron deficient erythropoiesis in FRDA patients. The risk of anemia is not limited to women in childbearing age. Temporarily interruption, withdrawal or iron supplementation may be considered.

Poster# 168 ABNORMAL METAL DISTRIBUTION IN THE WHITE MATTER OF THE BRAIN IN FRIEDREICH ATAXIA Bogdan Popescu, MD¹, Deborah Renaud, MD², Joseph Parisi, MD² and Helen Nichol, PhD¹ ¹University of Saskatchewan; ²Mayo Clinic Foundation (Presented By: Bogdan Popescu, MD)

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease inherited as an autosomal recessive trait that affects 1:50,000 Caucasians. It is caused by deficiency of frataxin, a mitochondrial protein that functions both as an iron chaperone and storage protein. Although iron levels may be elevated in mitochondria in some tissues, direct evidence of iron accumulation in the central nervous system of FRDA patients is scarce and controversial and no data is available on global distribution of multiple metals in the CNS. We simultaneously mapped iron, copper and zinc in a formalin fixed coronal brain slice of a case of FRDA using rapid scanning X-ray fluorescence (RS-XRF). We compared the relative metal distributions to another case of FRDA and previously published controls. In the normal brain, white matter has low levels of iron and high levels of zinc relative to the adjacent grey matter. In the two FRDA brains some of these distinctions are lost. In particular, iron was elevated and zinc reduced in the white matter relative to the adjacent grey matter, compared to normal brain. In the normal brain the U fibers are richest in copper but in FRDA, copper was also elevated throughout the white matter. These changes were particularly pronounced in the basal ganglia. The internal capsule and frontal and temporal white matter were strikingly high in iron, approaching levels in the adjacent putamen and caudate. In zinc maps of the normal brain, the basal ganglia stand out because zinc is low relative to surrounding zinc-rich white matter. In FRDA, the basal ganglia are not readily discernible in the zinc map because the adjacent white matter is very low in zinc. An increased content of iron and copper paralleled by a decrease in “protective” zinc could trigger oxidative damage. However, our data cannot clarify if these metal abnormalities are the cause or the result of such an increase in the formation of reactive oxygen species.

Poster# 169 THE EXPRESSION AND ROLE OF LIPOCALIN 2 AND ITS RECEPTOR AFTER SPINAL CORD INJURY Khizr Rathore, Adriana Redensek, MSc, Ruben Lopez-Vales, PhD, Manuela Santos, PhD, Shizuo Akira, PhD, Alan Aderem, PhD and Samuel David, PhD Centre for Research in Neuroscience, McGill University (Presented By: Khizr Rathore)

Lipocalin 2 (Lcn2) is an acute phase protein involved in the innate immune response and in the apoptosis of cells. Lcn2 sequesters iron-loaded siderophores and chelates iron, which underlies its ability to induce and promote apoptosis in certain mammalian cells. It has been shown that Lcn2 binds the cell surface receptor, 24p3R, and that this interaction is required for its pro-apoptotic effects. Our previous work in spinal cord contusion injury (SCI) has shown that iron-mediated oxidative mechanisms can promote significant secondary damage after SCI. Based on these results we hypothesized that Lcn2 may play a role in SCI. To assess this role for Lcn2, we conducted spinal cord contusion injuries in wildtype and Lcn2-/- mice using the IH impactor. Functional recovery was assessed in these mice for a period of 28 days using the Basso Mouse Scale, an open-field locomotor test. Spinal cord tissue was harvested for mRNA expression studies, western blotting and histological assessment at 1, 3, 7, 14, 21 and 28 days post injury. Quantitative Real-Time PCR analysis showed that there is a ~16-fold induction of Lcn2 mRNA expression in the injured spinal cord at 1 dpi, which drops to ~ 2-fold by day 7. By 21dpi, however, Lcn2 mRNA levels are increased ~8-fold compared to uninjured controls. Western blot analysis revealed that Lcn2 is robustly induced in the injured spinal cord after SCI. Lcn2 protein peaked at 1dpi and followed a similar pattern as the mRNA levels for the time points examined. Immunofluorescence staining of frozen sections at 1dpi showed that Lcn2 localized mainly to astrocytes in the gray and white matter, motor neurons in the ventral gray matter and endothelial cells. We also stained for the Lcn2 receptor, 24p3R. Interestingly, neurons and infiltrating leukocytes were the most intensely stained cells at 1dpi in addition to some astrocytes. Finally, our assessment of locomotor behavior after SCI showed that Lcn2-/- mice performed significantly better than wildtype controls, suggesting a negative role for Lcn2 after SCI

Poster# 170 HO-1-MEDIATED MACROAUTOPHAGY: A MECHANISM FOR UNREGULATED BRAIN IRON DEPOSITION Hillel Zukor¹, Wei Song, MD, PhD¹, Adrienne Liberman, BSc¹, Jeannie Mui, BSc¹, Hojatollah Vali, PhD¹, Carine Fillebeen, PhD¹, Kostas Pantopoulos, PhD¹, Ting-Di Wu, PhD², Jean-Luc Guerquin-Kern, PhD² and Hyman Morris Schipper, MD, PhD¹ ¹McGill University; ²Institut Curie (Presented By: Hillel Zukor)

Background: Oxidative stress, augmented iron deposition, and mitochondrial insufficiency have been amply documented in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). The transferrin pathway of iron delivery appears to play little or no role in the aberrant iron mobilization characteristic of the senescent and degenerating CNS. We previously demonstrated that heme oxygenase-1 (HO-1) (i) is up-regulated in AD and PD brain and (ii) promotes the accumulation of non- transferrin iron in astroglial mitochondria. In the present study we set out to: 1) determine whether human HO-1 (hHO-1) over-expression impacts astroglial mitochondrial morphology in vitro, 2) map the topography of aberrant iron sequestration in astrocytes overexpressing HO-1, and 3) to delineate a possible role of iron regulatory proteins in ho-1 mediated iron deposition.

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Methods: Light and transmission electron microscopy and diaminobenzidine histochemistry were employed to evaluate mitochondrial morphology and iron deposition in control and HO-1 transfected primary rat astrocytes. Nano secondary ion mass spectrometry (SIMS) was used to generate topographical maps of iron sequestration in relation to accruing cytopathology. Immunoblotting was used to evaluate flag-tagged HO-1 (transgene), transferrin receptor 1 (TfR1), ferritin, ferroportin, and iron regulatory protein-2 (IRP2) expression. Electrophoretic mobility shift assay was used to monitor IRP1 activation. Results:Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial swelling, membrane damage and macroautophagy, and the accumulation of redox-active iron and sulphur within these cytopathological profiles. HO-1 upregulation had no significant effects on ferroportin, TfR1, ferritin, and IRP 2 protein levels or IRP1 activity. Conclusions: In cultured astroglia, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD and other human CNS conditions characterized by the unregulated deposition of brain iron.

Poster# 171 GENE EXPRESSION CHANGES RELATED TO ALZHEIMER’S DISEASE AND OTHER NEURODEGENERATIVE DISORDERS IN A HEMOCHROMATOSIS HFE KNOCKOUT MOUSE MODEL Daniel Johnstone, Ross Graham, PhD, Deborah Trinder, PhD, Rodney Scott, PhD, John Olynyk, MD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Several neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease, can be associated with abnormalities in brain iron status but whether abnormal body iron status predisposes to neurodegeneration remains controversial. The most common form of the genetic disorder hereditary hemochromatosis occurs as a result of mutations in the HFE gene. This disorder is often typified by tissue iron loading, usually in the liver but other organs can also be affected. Magnetic resonance imaging suggests iron can accumulate in the brains of hemochromatosis patients and mutations of the HFE gene have been reported to be a risk factor for AD. We have performed a microarray study of genome-wide changes in brain gene expression in an Hfe knockout mouse model of hemochromatosis. In particular we have analyzed genes involved in various important neurodegenerative disorders, including AD, frontotemporal dementia, Parkinson’s disease, Huntington’s disease and Creutzfeld-Jacob disease and other prion-related conditions. RNA was isolated from brain hemispheres of biological replicates (n≥4) of wildtype AKR mice and Hfe knockout mice at 10 weeks of age. Differential expression analysis was performed using Illumina Sentrix MouseRef-8 (v1.1) BeadChip microarrays. One-way ANOVA determined differential gene expression between the two groups (p < 0.05). Gene sets were analyzed for enriched pathways and ontologies using commercial bioinformatics applications and public domain tools. Select changes were further investigated by real-time reverse-transcription PCR (RT-PCR). Pathway classification of the differentially expressed gene set determined that genes in the “Alzheimer’s disease-amyloid secretase pathway”, as defined by the Panther classification system, were significantly overrepresented in comparisons of brain tissue from Hfe knockout and wildtype mice (p = 0.001). Notably, there was a 2-fold decrease in expression of the amyloid precursor protein gene (App), a decrease of up to 8-fold in expression of the presenilin 1 gene (Psen1) and a decrease of up to 2.4-fold in expression of the tau (Mapt) gene (all p<0.05). Real-time RT-PCR confirmed significant decreases in both App and Psen1 transcripts. The expression of apolipoprotein E and various important inflammatory response genes (e.g. tumor necrosis factor alpha and most interleukins) were not significantly altered (all p>0.05). We observed small but significant increases in transcripts for some related proteins, including lipoprotein receptorrelated protein 1 (Lrp1) and apolipoprotein A-1 binding protein (Apoa1bp). While some genes related to other neurodegenerative disorders show altered expression, notably a 1.7-fold increase in transcripts for Niemann Pick type C2 (Npc2), a gene associated with frontotemporal dementia, and a 1.3-fold increase in transcripts for Park7, involved in Parkinson’s disease, there was, in general, little evidence of substantial changes in transcripts for the main genes known to be involved in these diseases or other neurodegenerative diseases such as Huntington’s disease or Creutzfeld-Jacob disease. These results reveal considerable alterations in transcripts for important AD genes in the Hfe knockout mouse brain. However the directions of some of these changes were opposite to what would be expected in AD, making it difficult to predict whether the sum of these changes would be more likely to promote AD, protect against AD or not change AD risk.

Poster# 172 THE ROLE OF MITOCHONDRIAL FERRITIN ON HYDROGEN PEROXIDE INDUCED SH-SY5Y CELL DAMAGE Nan Zhang, Zhen-Hua Shi, Yan-Zhong Chang, PhD and Xiang-Lin Duan Hebei Normal University (Presented By: Xiang-Lin Duan)

Mitochondrial ferritin (MtFt) is a newly identified mitochondria protein encoded by an intronless gene on chromosome 5q23.1 in humans. MtFt is structurally and functionally similar to the cytosolic H-ferritin, but different to H-ferritin. MtFt expression is restricted mainly to the testis, neuronal cells and islets of Langherans, which require high mitochondria metabolic activity and oxygen consumption. In addition, it is highly expressed in erythroblast mitochondria of patients with sideroblasts anemia. Previous studies suggested that overexpression of MtFt caused a redistribution of iron from cytosol to mitochondria，thus high levels of MtFt result in an iron deficient phenotype in cytosol, it expression also inhibited the in vivo tumor growth due to cytosolic iron deprivation, and MtFt over-expression in HeLa cells increases resistance to oxidative stress. These results indicate that MtFt plays a role in cellular iron uptake, distribution, and mobilization, additionally, in protecting mitochondria from iron- induced damage. In addition, the study of MtFt may be useful for revealing mitochondria iron metabolism and finding possible therapeutic applications in neurological disorders involving increased iron deposition. In this study, we chose MtFt overexpressed SH-SY5Y cells as experimental model, P10-3-SY5Y (blank vector) and SH-SY5Y cells as control, Hydrogen Peroxide (H2O2) as oxidative stress-induced cell damage drug, to examine the role of MtFt in H2O2 induced neuroblastoma cell damage. Cells were grown in DMEM medium supplemented with 10% fatal bovine serum,100IU penicillin and streptomycin, at 37℃ and 5% CO2, 95% air. Then, cells were exposed for 24hrs to H2O2 (100µM). MTT, western blotting, and flow cytometry were used for cell viability assay, protein analysis, cell cycle analysis, labile iron pool(LIP) and cytosolic ROS measurement, mitochondrial membrane potential monitoring, and cell apoptosis detection, respectively. The results indicated that overexpression of MtFt in SH-SY5Y cells inhibited the cell growth, it maybe caused by depletion of iron in intracytoplasm; overexpression of MtFt in SH-SY5Y cells restrained the changes of iron metabolism related proteins in expression, which induced by H2O2; overexpression of MtFt in SH-SY5Y cells restrained the increase of ROS and the decrease of mitochondrial membrane potential, maintained the level of anti-apoptotic protein Bcl-2 expression, inhibited the activation of pro-apoptotic protein caspase3, thus inhibited the apoptosis of SH-SY5Y cells induced by H2O2. Our results showed that MtFt played an important role in oxidative damage to mitochondrial induced by H2O2.The mechanism perhaps is that overexpression of MtFt decreased the free iron level by regulating the iron metabolism related proteins, and then inhibited the Fenton reaction, decreased the production of ROS, maintained the mitochondrial membrane potential, inhibited the activation of pro-apoptotic proteins, maintained the level of antiapoptotic proteins, thus inhibited the apoptosis induced by H2O2.

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Poster# 173 IRON, FRATAXIN AND FRIEDREICH’S ATAXIA NEURODEGENERATION: DEFECTIVE FOLDING AND FUNCTION IN COMPOUND HETEROZYGOTES Cláudio M. Gomes, PhD and Ana R. Correia ¹Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-756 Oeiras, Portugal (Presented By: Cláudio M. Gomes, PhD)

Human frataxin is a mitochondrial protein whose deficiency is associated with the neurodegenerative disease Friedreich's Ataxia (FRDA, OMIM: 229300) [1]. The majority of FRDA patients (>95%) are homozygous for a GAA repeat expansion within the first intron of the frataxin gene that affects frataxin transcription impairing its expression. A small but significant number of FRDA patients are compound heterozygotes containing a GAA expansion in one allele and a deleterious point mutation on the other [2]. At the molecular level, this disease is characterized by mitochondrial iron accumulation, impairment of the biosynthesis of iron-sulfur (Fe-S) proteins and increased oxidative stress. Frataxin function is not yet completely understood but experimental evidence suggests that frataxin is an iron chaperone binding iron in vivo, protecting cells from iron-induced oxidative stress, and delivering the metal ion to other proteins involved in heme and Fe-S cluster biosynthesis, as well as Fe-S clusters repair. Through biophysical and biochemical methodologies, we have analysed in vitro the consequences of different disease-related point mutations (D122Y, G130V, I154F and W155R) on the folding, stability, dynamics and function of human frataxin [3]. Aiming at a better molecular and structural understanding of FRDA mechanism, we have focused our study on clinical mutations associated to both mild (D122Y and G130V) and aggressive (I154F and W155R) clinical phenotypes. Our results show that, at 25ºC, the mutations do not change significantly the protein fold or its flexibility, and that the clinical mutants retain a compact and relatively rigid globular core. However, the mutants have a decreased thermodynamic stability, an increased propensity towards aggregation and compromised function as iron-chaperones. Whereas I154F and W155R are unable to bind iron, which destabilizes the proteins inducing precipitation, the mutants D122Y and G130V show a lower binding stoichiometry but no iron induced precipitation is noted [4]. At 37ºC, the mutants show an increased susceptibility towards proteolytic degradation which is indicative of an enhanced flexibility of the polypeptide chain. Framing our results with the available in vivo data, suggests that the clinical effects in heterozygous patients may arise from a combination of effects, such as reduced efficiency of protein folding (resulting in increased aggregation rates), accelerated degradation in vivo and misfolding and conformational destabilization, that together contribute to a decrease in the levels of functional frataxin. In this scenario, FRDA in heterozygous patients, although intrinsically different from other neurodegenerative disorders involving toxic aggregation, could also be linked to a process of defective protein folding due to specific destabilization of frataxin, and in this respect be considered a conformational disease. [1] Campuzano, V. et al. (1996) Science 271, 1423-7. [2] Cossee, M. et al. (1999) Ann Neurol 45, 200-6. [3] Correia, A.R., et al. (2008) FEBS J 275, 3680-90. [4] Correia, A.R., et al. (2006) Biochem J 398, 605-11.

Poster# 174 ANALYSIS OF IRON HOMEOSTASIS IN SKIN FIBROBLASTS FROM PANTOTHENATE KINASE ASSOCIATED NEURODEGENERATION PATIENTS Alessandro Campanella¹, Elisabetta Rovellli², Barbara Garavaglia³ and Sonia Levi4 ¹University Vita-Salute San Raffaele; ²San Raffaele Scientific Institute, DIBIT, Milan, Italy; ³Fondazione IRCCS Istituto Neurologico “C. Besta”, Milan, Italy; 4University Vita-Salute San Raffaele and San Raffaele Scientific Institute, DIBIT, Milan, Italy (Presented By: Alessandro Campanella)

Pantothenate Kinase Associated Neurodegeneration (PKAN) is the most prevalent form of Neurodegenerations with Brain Iron Accumulation, a heterogeneous group of inherited disorders characterized by progressive impairment of movement, speech and cognition. PKAN is characterized by lateral degeneration of the globus pallidus and substantia nigra pars reticulata with deposition of iron in the affected region that results in the distinctive "eye of the tiger" signal in T2 weighted MRI brain pictures. The disease is associated with several mutations in the Pantothenate Kinase 2 (PANK2) gene that encodes for a mitochondrial protein involved in the first step of coenzymes A synthesis. To investigate the link between the gene defect and iron accumulation we analyzed skin fibroblasts from four PKAN patients (homozygous for single mutations: R286C, Y190X and two F419fsX472) compared with three healthy controls. It was reported that R286C and Y190X mutations produced functional and un-functional Pank2, respectively. Patients’ cells did not show statistical significant differences in cytosolic H and L ferritins or in Transferrin Receptor 1 (TfR1) levels. No differences were observed also during high iron supplementation, as 300 µM FAC for 18 hours, a treatment that strongly modulated ferritins and TfR1 levels. Despite this, cells from three patients, representative of R286C and F419fsX472 mutations, showed statistical significant differences in iron manipulation during prolonged mild iron supplementation as 20 µM FAC for 72 hours. Although comparable total iron incorporation, these patients’ fibroblasts stored in ferritins a minor amount of iron than controls. This was not justified by defective iron storage capacity of ferritins, which was similar to controls, but resulted from a minor ferritin up-regulation in response to iron supplementation. Ferritins up-regulation was measured 2,9 and 1,8 times for controls and patients respectively. Moreover preliminary data indicated that also the down- regulation of TfR1 levels occurred in minor extent in patients than in controls. Defective regulation of these proteins reflected abnormalities in cytosolic Labile Iron Pool (cytLIP) dynamics. Although cytLIP increased in all the cells after short time iron supplementation as 20 µM FAC for 4 hours, in the controls, after 72 hours treatment cytLIP resulted lower than in the untreated, probably due to ferritins up-regulation. On the contrary, patient cells were not able to reduce cytLIP after prolonged iron supplementation. This did not occur in patients’ cells representative of Y190X mutation. They correctly modulated cytLIP and up-regulated ferritins after prolonged mild iron supplementation, however they incorporated more iron than controls and preliminary results showed that they still maintained the defect in TfR1 reduction. The relevance of Iron Regulatory Proteins on this miss-regulation and the potential damages induced by increased cytLIP are under investigation. In conclusion, our data proposed that Pank2 protein could be involved in cellular iron homeostasis and utilization with a mechanism that probably did not rely on its enzymatic activity. Interestingly, in the cases analyzed, the iron homeostasis defects appeared more relevant in patient cells with the longest residual Pank2 proteins, suggesting an involvement of some protein interaction or aggregation defects.

Poster# 175 SYNCHROTRON X-RAY FLUORESCENCE REVEALS ABNORMAL DISTRIBUTION OF METALS IN BRAIN AND SPINAL CORD IN SPINOCEREBELLAR ATAXIA: A CASE REPORT Bogdan Popescu, MD, Christopher Robinson, MD, L. Dean Chapman, PhD and Helen Nichol, PhD University of Saskatchewan (Presented By: Bogdan Popescu, MD)

Synchrotron rapid-scanning X-ray fluorescence (RS-XRF) was used for the first time to simultaneously localize iron, copper and zinc in situ in a case of spinocerebellar ataxia (SCA). The relative levels of each metal were quantified in order to compare SCA with control tissue. Formalin fixed SCA and age- and sex-matched control human brain and spinal cord slices were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, Maryland. The neuropathology and clinical report provided by the brain bank combined with our histopathology confirmed a diagnosis of SCA. The dorsal columns of the spinal cord, spinocerebellar tracts, amiculum olivae and cerebellar white matter showed loss of myelinated fibres. Neuronal loss and gliosis were evident in the inferior olivary nucleus, dentate nucleus and cerebellar cortex. RS-XRF analysis showed that iron, copper and zinc were all decreased in the SCA regions characterized by myelin degeneration or neuronal loss and gliosis compared to the control. In the normal medulla a previously undescribed copper enrichment was seen associated with spinocerebellar fibres and amiculum olivae. This region was virtually devoid of all metals in the SCA case. The ventral columns of the SCA spinal cord that exhibited only moderate myelin pallor had increased metal levels. SCA cerebral blood vessels also had elevated metal levels. Both iron and zinc, but not copper were elevated in the globus pallidus pars externa relative to the control. We hypothesize that metals increase as part of the initial neurodegenerative process but once degeneration is advanced the metal levels drop. This implies a role for multiple metals in SCA neurodegeneration but further study is required to establish a causative role. We suggest that if these findings are generally true of at least some cases of SCA, not only iron but also copper and zinc should be considered as possible therapeutic targets.

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Poster# 176 SERUM FERRITIN IS NOT RELATED TO COGNITIVE STATUS – RESULTS FROM A PERSPECTIVE COMMUNITY STUDY OF OLDER AUSTRALIANS Liz Milward, David Bruce, MD, Michelle Cole, NA, Matthew Knuiman, PhD, Mark Divitini, PhD, Helen Bartholomew, GradDipComp, Graham Maier, GradDipComp and John Olynyk, MD University of Newcastle (Presented By: Liz Milward)

There has been considerable interest in the relationship of abnormal iron status with cognitive impairment and risks of neurodegenerative conditions such as Alzheimer’s disease. While this has been investigated in various small clinical studies, it has not been examined in depth in larger, epidemiological studies of older people in the community, particularly in conjunction with clinical examinations of participants. In 2003-4, we examined cognition and dementia in 800 community-dwelling Australians aged 60 years or older and investigated the relationships with serum ferritin and other blood iron measures determined at the time of testing in 2003-4 and from archival data obtained in an earlier survey of the same participants in 1994-5. Serum ferritin in 1994-5 or 2003-4 was categorized by gender-specific reference ranges as low, normal-low, normal-high and high. Cognition was assessed in 2003-4 by the Cambridge Cognitive Examination (CAMCOG), which has been validated in the elderly. The CAMCOG interrogates the domains of memory, orientation, language, attention/calculation, praxis, abstract thinking and perception. It also includes a separate measure of executive function. Participants with possible cognitive impairment (CAMCOG score <84) also attended a clinical examination with a senior geriatrician, which included Clinical Dementia Rating (CDR). We evaluated the relationships between serum iron measures and cognitive scores by ANOVA F-test on unadjusted values and ANCOVA F-test after adjustment for potential confounders (age, gender, National Adult Reading Test as a measure of premorbid ability and history of stroke) with post-hoc pairwise testing. In participants without dementia as gauged by the CAMCOG and by clinical evaluation and CDR (n=749), there was no evidence for any significant relationships, with or without adjustment for confounders, between the total CAMCOG scores and either the serum ferritin level in 1994-5 or 2003-4 or the change in serum ferritin level between these two times (all p>0.05). There was also no evidence for any significant relationships with executive function scores. In addition we did not detect any correlations with serum ferritin levels for the group of participants with possible or probable dementia gauged by CAMCOG, clinical examination and CDR (n=51). Similarly we found no effect for other serum iron measures (serum iron, transferring saturation). The findings from this community epidemiological study of elderly Australians found no evidence for clinically significant effects of either low or high abnormal body iron stores, as gauged by serum ferritin, on cognitive status or dementia risk in older people within the community. Larger studies are required to investigate the possibility that effects may influence dementia severity or progress rate or be restricted to some particular subset of dementia etiologies. The results do not rule out effects in a few people with very low or high body iron levels. In addition, serum ferritin cannot be assumed to be a reliable gauge of abnormal brain iron status. However in general, the results suggest most elderly individuals in the community with abnormal serum ferritin levels do not have increased risks of clinically significant cognitive impairment or dementia.

Poster# 177 THE ROLE OF EXTERNAL LOOPS OF CERULOPLASMIN IN STABILIZING FERROPORTIN ON CELL MEMBRANE Maria Carmela Bonaccorsi di Patti, Dr, Nunziata Maio, Fabio Polticelli and Giovanni Musci University La Sapienza (Presented By: Maria Carmela Bonaccorsi di Patti, Dr)

Aceruloplasminemia arises from mutations of ceruloplasmin (Cp), a multicopper ferroxidase present both as a soluble form, and as a GPI-anchored isoform first identified in brain astrocytes. A study concerning the functionality of aceruloplasminemia missense Cp mutants has recently shown that some mutants do not load copper in vivo, due to impairment of the copper ATPase ATP7B following Golgi fragmentation. Cp is a multidomain protein made up of six plastocyanin-like domains. Interaction between domains 1-2, 3-4, and 5-6 are through extensive hydrophobic interfaces, while polar interactions connect domains 2-3 and 4-5, with the interface between domains 6 and 1 hosting the catalytically essential trinuclear copper cluster. We here report a structural and functional analysis showing that homologous flexible loops found in Cp are essential for proper functioning of the protein in a cellular model. For this purpose, several mutants on the loops were analyzed for their ability to prevent the internalization and the rapid degradation of cell surface ferroportin (Fpn), which occurs when a functional Cp is missing, in rat glioma C6 cells previously silenced for endogenous Cp. The starting point was the observation (reported in a separate poster) that mutation R701W is highly critical for Cp activity in vivo, generating a dominant negative mutant unable to rescue Fpn even in the presence of wild type Cp, and able to induce Golgi fragmentation and relocalization of ATP7B. R701 is located in a large solvent exposed loop connecting domains 4 and 5 and comprising residues 700-708; a corresponding loop which connects domains 2 and 3 is found at positions 339-350. Domains held together by hydrophobic interactions are also connected by surface loops. Despite a low degree of sequence homology, all these loops start with a C-X-R/K motif, with the cysteine residue stabilizing the loop by forming a disulphide bridge. The role of the 700-708 loop was therefore investigated. Residue R701 was either deleted or substituted with glutamine (R701Q), glutamic acid (R701E) or phenylalanine (R701F). All these mutants did not rescue Fpn, yet they were not dominant and did not induce relocalization of ATP7B. Mutants Q702M, S703A, E704M, D705M were functional and rescued Fpn in Cp-GPI silenced cells, while substitutions which were definitely non-conservative and structurally more challenging (R700W, Q702W, E704W, D705P) yielded proteins unable to complement the loss of endogenous Cp; all proteins were localized at the plasma membrane and the inactive mutants were not dominant. Disruption of the disulphide bridge C618-C699 preceding R701 by replacement of C699 with serine generates a dominant Cp mutant, highlighting the importance of this loop. The possible role of homologous loops was tested by mutating K340 and R883, the basic residues corresponding to R701 and located on the loops connecting domains 2 and 3 and 5 and 6. Mutants K340M, K340W, R883M and R883W were non-functional but not dominant and they did not alter the localization of ATP7B. Mutants R701W, C699S, K340W and R883W were enzymatically active when produced in P. pastoris. In mammalian C6 cells transfected with these mutants, co- transfection of the yeast copper ATPase Ccc2p, or addition of Cu(I)-GSH to the culture medium significantly rescued Fpn, confirming that the loops are critical for proper copper incorporation in mammalian cells.

Poster# 178 CYSTEINE OXIDATION REGULATES THE RNA-BINDING ACTIVITY OF IRON REGULATORY PROTEIN 2 DURING OXIDATIVE STRESS Kimberly Zumbrennen, PhD, Michelle Wallander, PhD, Joshua Romney, PhD and Elizabeth Leibold, PhD University of Utah (Presented By: Kimberly Zumbrennen, PhD)

Iron regulatory protein 2 (IRP2) is a RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as ferritin and transferrin receptor-1 (TfR1). IRP2 is primarily regulated by iron-mediated proteasomal degradation, but studies have suggested that the RNA-binding activity of IRP2 can also be regulated by thiol-oxidation. To characterize the role of cysteines in regulating IRP2 iron degradation and/or RNA binding, we mutated all 18 cysteines to serine either individually or in combination. Using an iron-dependent degradation assay, we found that all IRP2-cysteine mutants were sensitive to iron-dependent protein turnover indicating that cysteines do not function in regulating IRP2 iron-mediated degradation. We next generated a model of IRP2 bound to RNA and found that two cysteines, C512 and C516, were predicted to lie within the RNAbinding cleft suggesting that these cysteines may regulate the RNA-binding activity of IRP2. Mutagenesis of C512, but not C516, reduced IRP2 RNA binding while chemical-oxidation or modification of either cysteine eliminated IRP2 RNA-binding activity. These data indicate that C512 and C516 are located in close proximity to the RNA but that neither cysteine directly interacts with the RNA. These data also show that both C512 and C516 must be unmodified and reduced for IRP2 to interact with RNA. To determine if oxidation of either C512 or C516 may affect IRP2 RNA binding in cells, we treated human embryonic kidney 293 cells with glucose deprivation, which induces oxidative stress, and assayed IRP2 RNA-binding activity over time. We found that the RNA-binding activity of IRP2 was reduced by 40% after only 15 min of glucose deprivation and a maximal decrease of 70% was observed after 2 hours. The loss of IRP2 RNA binding was due to protein oxidation and not degradation as immunoblotting showed no change in IRP2 protein levels and the RNA-binding activity of IRP2 could be restored in glucose-deprived extracts with the addition of a reducing agent. In addition, the RNA-binding activity of IRP2 could be rapidly restored in cells when glucose was 151 reintroduced to the medium. To determine which cysteines were oxidized during oxidative stress, IRP2 cysteine mutants were tested for their sensitivity to glucose deprivation. We found that IRP2 RNA binding was unchanged during glucose deprivation when either C512 or C516 were mutated. These data indicate that both C512 and C516 must be oxidized to inhibit IRP2 RNA binding during glucose deprivation and suggest that during oxidative stress a disulfide bond is formed between these two cysteines that precludes IRP2-RNA interactions. We also found that the loss of IRP2 RNA-binding activity during glucose deprivation correlated with reduced TfR1 mRNA abundance. These studies provide insight into the structural basis for IRP2-RNA interactions and reveal an iron-independent mechanism for regulating iron homeostasis through the redox regulation of IRP2 cysteines.

Poster# 179 PROTECTION OF CARDIAC CELLS AGAINST OXIDATIVE INJURY BY NOVEL IRON CHELATORS – KETONE ANALOGS OF SALICYLALDEHYDE ISONICOTINOYL HYDRAZONE (SIH) Tomas Simunek, PhD, Petra Bendova, Katerina Hruskova, Pavlina Haskova, Petra Kovarikova and Katerina Vavrova Charles University in Prague, Faculty of Pharmacy (Presented By: Tomas Simunek, PhD)

Recent evidence suggests that oxidative stress is a common denominator in many aspects of cardiovascular pathogenesis. Its important role is now established in a number of cardiovascular disorders, such as ischemia/reperfusion injury, cardiac arrhythmias, congestive heart failure, or myocarditis. Free cellular iron (Fe) plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the contribution of oxidative stress to cardiovascular disease. Therefore, shielding of free or loosely-bound iron via its chelation may be an effective therapeutic approach. Salicylaldehyde isonicotinoyl hydrazone (SIH) is able to quickly penetrate cellular membranes and efficiently chelate labile intracellular Fe pool. SIH has been previously demonstrated as extremely effective in protection of cardiac cells against oxidative stress. The main drawback of SIH is its rapid hydrolysis due to the labile hydrazone bond resulting in a short half life in biological environment. We therefore hypothesized that chemical modification aimed at increasing its stability may result in further improvement of its antioxidant action. A series of nine novel SIH analogs was synthesized by substitution of salicylaldehyde with various hydroxyketones in order to sterically shield the hydrazone bond and change its electron density. Using Fe-calcein dequenching assay, all the novel agents have shown very high and rapid Fe(III) chelation activity (97 - 103 %), comparable to that of SIH (100 %). HPLC analysis revealed that all these chelators possessed significantly lower rate of hydrolytic breakdown in aqueous solution compared to SIH. Neutral red uptake assay was then used to examine the agents for their ability to protect rat embryonic cardiomyoblast cell line H9c2 against the model oxidative injury induced by 24-hour exposure to 200 uM tert-butyl-hydroperoxide (t-BHP). In addition, own toxicity of chelators towards the H9c2 cells was assayed following 24h and 72h incubations. Simple replacement of salicylaldehyde in SIH with a corresponding methylketone, resulted in higher maximal protection by this agent - 1-(2-hydroxyphenyl) ethylidene isonicotinoyl hydrazone - 2HAP-INH (90% protection with 100 uM 2HAP-INH vs. 66% protection by equimolar SIH; n.s.). This was apparently related to either higher stability or lower acute (24h) toxicity of 2HAP-INH than that of SIH (0% vs. 45% of cellular viability reduction; P<0.05). However, following the 72h cellular exposure, the toxicity of 2HAP-INH was higher than of SIH (88% vs. 68% viability reduction; n.s.). It is possible that the increased stability resulted in higher Fe depletion by removing or withholding Fe from critical Fe-containing proteins. Of the various structure modifications tested, the best ratio of cytoprotective to toxic properties was achieved with 1- (2- hydroxy- 7-oxo- 7,8- dihydronaphthalen- 1-yl) ethylidene isonicotinoyl hydrazone (AHC-INH), where the relative decrease in antioxidant action (as related to SIH) was accompanied by even more pronounced decrease in toxicity. In conclusion, our study confirms a key role of free cellular Fe in peroxidative cell damage and points to significant protective ability of iron chelation. The cardioprotective potential of novel ketone aroylhydrazone agents warrants further investigation. Study supported by the Charles University in Prague (grant 124307/C/2007) and the Ministry of Education of the Czech Republic (project MSM0021620822).

Poster# 180 EFFECTS OF OXIDATIVE STRESS ON IRON REGULATORY GENES IN TWO DIFFERENT MICE MODELS FOR NAFLD Rolf Hultcrantz, MD, C. Söderberg, P. Stål, K. Eckes and M. Sjöström Department of ’Gastroenterology, Karolinska University Hospital, Stockholm, Sweden (Presented By: Rolf Hultcrantz, MD)

Introduction: Non-alcoholic steato-hepatitis (NASH) can develop from non-alcoholic fatty liver (NAFLD) and progress to cirrhosis. Iron is suspected to enhance hepatic damage associated with non-alcoholic fatty liver disease. Hyperferritinemia, is frequently observed in (NAFLD). Hepatic iron overload in of NAFLD models is associated with lipid peroxidation products and increased necro-inflammation. Oxidative stress leads to increased lipid peroxidation, elevated markers of oxidative stress in vivo have been shown in patients with NAFLD. Aims: To show how oxidative stress and different cytokine stimulation affected iron regulatory gene expression in various degree of fatty liver. Methods: Liver tissue samples and primary hepatocytes from Ob/Ob-mice, MTTP-knockout mice and their respective controls were used. Hepatocytes were isolated, cultured and subjected to Tert-butyl-hydroperoxide (TBH), between 0.05 and 0.5 mM, TNF-α (10ng/ml) or IL-6 (20ng/ml). LDH and MDA were measured to evaluate cell death and lipid peroxidation. Iron was measured with atomic absorbtion spectophotometry.. For gene expression Quantitative Real-Time PCR,was used. Results were normalized to house-keeping genes HPRT or GAPDH. Primer/probe mixture was obtained commercially for Hepcidin (HAMP1), Ferroportin (FPN), Transferrinreceptor 1 and 2 (TfR1, TfR2), Transferrin Trf, DMT1, Ferritin H (FtH), HFE and Serum Amyloid P component (SAPc) Results: Total iron content in liver tissue was significantly lower in Ob/Ob mice than in controls. In liver tissue samples, the expression was lower in Ob/Ob mice than in controls for TfR2, FtH, HFE and DMT1, and the expression was higher for HAMP and TfR1 than controls. SAPc mRNA expression was strongly upregulated by TBH but no significant differences were seen between models. No clear alterations were seen for the MTTP mouse which contained less fat. Fatty liver mice hepatocytes were more sensitive to induced oxidative stress, measured with LDH and MDA than their controls. Incubation with TBH gave decreased expression of HAMP1, TfR1, TfR2, HFE and DMT1. FtH differed from the other genes by having decreased expression in Ob/Ob with higher oxidative stress, while expression in controls increased. No significant difference was seen in Trf and FPN expression but FPN was slightly increased in controls. Incubation of IL6 on the two types of hepatocytes leads to increase in HAMP and TfR2, the same for the controls. Conclusion: In hepatocytes from mouse models of fatty liver (Ob/Ob mouse) exposed to oxidative stress and inflammation, iron regulating genes showed alterations in expression, compared to controls cells, indicating a decreased defence against intracellular iron. Ob/Ob mice had a higher expression of HAMP which may cause the lower iron content in the liver. Furthermore the increased HAMP expression may explain the low response to proinflammatry cytokines.

Poster# 181 THE CRITICAL ROLE OF INTRACELLULAR LABILE IRON AND FERRITIN IN THE RESISTANCE OF CELLS TO HYDROGEN PEROXIDE Charareh Pourzand, PhD¹, Abdullah Al-Qenaei, PhD², Anthie Yiakouvaki, PhD², Paolo Santambrogio, PhD³, Sonia Levi, PhD4, Rex Tyrrell, PhD² and Nick Hall, PhD5 ¹Department of Pharmacyand Pharmacology/University of Bath; ²Department of Pharmacy and Pharmacology, Bath University, Bath, UK; ³San Raffaele Scientific Institute, Milano, Italy; 4Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milano, Italy; 5Bath Institute for Rheumatic Diseases, Bath, UK (Presented By: Charareh Pourzand, PhD)

Acute exposure of skin cells to UVA or H2O2 provokes an immediate increase in the level of Labile Iron Pool (LIP) and a related increase in the extent of necrosis. To investigate this phenomenon in chronic oxidative stress conditions, a new Jurkat T cell line, HJ16 (H2O2-resistant) was developed by gradual adaptation of parental J16 cells to high doses of H2O2. Flow cytometric analysis revealed that HJ16 cells were resistant to H2O2- but not to UVA-induced oxidative damage and necrosis. UVA radiation triggered an immediate and dose-dependent increase in the level of LIP of both cell lines (calcein assay). However following H2O2 treatment, LIP level was only increased in J16 but not in HJ16 cells. These results suggested a link between the lack of induction of labile iron release and cellular resistance of HJ16 to H2O2-induced necrosis. To further understand the correlation between the level of H2O2-induced labile iron release and the extent of necrosis, both cell lines were treated with either desferrioxamime (DFO) or hemin prior to exposure to a range of doses of H2O2. The DFO treatment abolished both the basal and H2O2-mediated increase in LIP in cells 152 and protected both cells against necrosis. Hemin treatment dramatically decreased the H2O2-induced level of labile iron release in J16 cells and provided considerable protection against H2O2-induced necrosis. In contrast, in HJ16 cells, hemin treatment increased dramatically the basal level of LIP (up to 8-fold) and following H2O2 treatment increased further the level of labile iron release. Accordingly, the percentage of H2O2-induced necrosis increased dramatically in the hemin-treated HJ16 cells. The differential response of hemin-treated parental and H2O2-resistant cells highlighted the crucial importance of oxidantinduced labile iron release in the increased susceptibility of cells to oxidative cell death. To further demonstrate the strict dependence of H2O2-induced necrosis to LIP levels present in cells, hemin treatment was followed by an additional treatment with DFO. This significantly decreased the level of H2O2-induced necrosis in both cell lines when compared to hemin-treated cells. ELISA analysis using monoclonal H-ferritin (H-Ft) and L-Ferritin (L-Ft) antibodies revealed that the intracellular level of H-Ft (but not L-Ft) was 7-fold lower in HJ16 cells when compared to J16 cells. The lower H-Ft levels in HJ16 cells might be part of the adaptive response developed during the gradual adaptation of this cell line to H2O2. Furthermore, the higher accumulation of labile iron in hemin-treated HJ16 cells could be due to the substantial difference in the level of intracellular H-Ft in this cell line as this level might not be sufficient to sequester the excess labile iron load by hemin. In addition, the level of heme-oxygenase 1 protein in HJ16 cells was two-fold higher than in J16 cells. H2O2 treatment substantially decreased the level of H-Ft in J16 cells (up to 70% of the control value) but did not affect significantly the L-Ft level. In contrast in HJ16 cells, H-Ft level was not affected by H2O2 treatment. The latter results highlight a further link between the lack of induction of LIP, the lack of modulation of H-Ft and cellular resistance to H2O2-induced necrosis.

Poster# 182 PROTECTION OF CARDIOMYOCYTES AGAINST CATECHOLAMINE INJURY BY IRON CHELATOR SALICYLALDEHYDE ISONICOTINOYL HYDRAZONE (SIH) Pavlina Haskova, MSc¹, Petra Kovarikova¹, Anna Vavrova¹, Prem Ponka² and Tomas Simunek¹ ¹Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Czech Republic; ²Lady Davis Institute for Medical Research and Department of Physiology, McGill University, Montreal, QC, Canada (Presented By: Pavlina Haskova, MSc)

Catecholamines are important regulators of myocardial contractility and metabolism. However, an excessive release or administration of catecholamines may induce cardiotoxic effects such as myocardial necrosis, myocarditis and manifestations of ischemic heart disease, including acute myocardial infarction and development of heart failure. Catecholamine cardiotoxicity has traditionally been attributed to continuous excessive β-receptor stimulation and consequent development of calcium overload. More recently, focus has shifted to the ability of catecholamines to undergo redox cycling and form aminochromes and reactive oxygen species (ROS), which are both potentially toxic for the cell. Free intracellular iron (Fe) serves as a key catalyst of the Haber-Weiss reaction; free iron may participate in aminochrome formation and induction of oxidative stress. This suggests that catecholamine cardiotoxicity could be prevented by iron chelation. The aims of this study were to determine time- and dose-dependence of catecholamine toxicity towards cardiac cells and to examine the potential for protection by iron chelation. In this study cardiotoxic properties of one synthetic and two naturally occurring catecholamines – isoprenaline, noradrenaline and adrenaline, were studied. The strong lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was evaluated for its protective potential. Using spectrophotometry and HPLC analysis, the rate and extent of catecholamine oxidation in solution were determined in various times. Cellular toxicity following 24h cell exposure was assessed by neutral red uptake assay, and the H9c2 cell line, derived from rat embryonic cardiomyoblasts, was used as a cellular model for cardiotoxicity determination. Initial experiments revealed that all three catecholamines undergo spontaneous oxidation to a number of products – changes in absorption spectra were observed after 6-12h incubation attributed to the formation rose-carmine coloured oxidative products. At the same times, HPLC analysis revealed a gradual decrease in concentrations of parent catecholamines. This oxidation was markedly potentiated by the addition of exogenous Fe-citrate and reduced by Fe chelation with SIH. Subsequent cellular experiments showed that these oxidation products (formed in catecholamine-containing medium during 24h preincubation period prior addition to cells) were markedly more toxic than parent (non-preincubated) drugs. Both toxicities of parent and oxidized catecholamines were time- and dose-dependent and while the IC50 values of parent catecholamines were in the range of 200-300 µM, the IC50 values of oxidized catecholamines were 40-50 µM (all following 24h cell exposure). Significant reductions of toxicities of all catecholamines and their oxididation products were achieved by co-incubations with SIH (10-100 µM) using three various experimental protocols: 1) when SIH was added to fresh catecholamines immediately before cellular experiment; 2) when SIH was added immediately before cellular experiments to medium preincubated for 24h with catecholamines; and 3) when SIH was preincubated for 24h together with catecholamines and then added to cells. All these results support the hypothesis that redox-active iron is significantly involved in catecholamine oxidation as well as subsequent cardiotoxicity. The iron chelator SIH has shown considerable potential to protect cardiac cells by both inhibition of catecholamine conversion and prevention of ROSmediated cellular injury. Study funded by a Charles University grant No. 51308/C/2008.

Poster# 183 PARAQUAT DOWN-REGULATES IRON REGULATORY PROTEIN 1 ACTIVITIES AND EXPRESSION IN MURINE RAW 264.7 CELLS AND BONE MARROW-DERIVED MACROPHAGES Rafal Starzynski, PhD¹, Agnieszka Grzelak, PhD², Agnieszka Stys, MSc¹, Grzegorz Bartosz, PhD² and Pawel Lipinski, PhD¹ ¹Institute of Genetics and Animal Breeding; ²University of Lodz (Presented By: Rafal Starzynski, PhD)

Iron regulatory protein 1 (IRP1) post-transcriptionally regulates expression of iron metabolism genes to optimize cellular iron availability and minimize iron toxicity. The two activities of IRP1 have been shown to be modulated by fluctuations in cellular iron content, nitric oxide, phosphorylation. IRP1 serves also as sensor of reactive oxygen species (ROS), inevitable byproducts of aerobic metabolism. As iron and oxygen are essential but potentially toxic constituents of most organisms, ROS-mediated modulation of IRP1 activity is an important regulatory element in dissecting iron homeostasis and oxidative stress. Previously, we have shown that expression of Irp1 gene is markedly decreased at both mRNA and protein levels in livers of superoxide dismutase 1 (SOD1) knockout mice (KO SOD1), lacking Cu,Zn-SOD, an enzyme that acts to reduce the concentration of superoxide anion (O2•-) in cytosol. Here, we show that strong down-regulation of hepatic IRP1 expression in KO SOD1 mice starts on day 1 after birth and is not observed in the liver of KO SOD1 embryos. Moreover, developmental changes in hepatic IRP1 abundance in normal (wild-type SOD1) mice are not related to moderate fluctuations in SOD1 activity. Results of comparative analysis of both IRP1 protein level and SOD1 activity in the liver of Sod1+/+, Sod1+/-, and Sod1- /- mice clearly demonstrate that complete lack of SOD1 activity observed in Sod1-/- mice is absolutely necessary for hepatic IRP1 down-regulation. The biological mechanisms underlying the decrease in IRP1 expression under conditions of SOD1 deficiency (supposed to increase the O2 •- steady-state level) are unknown, although it has been suggested that O2.-–mediated oxidative stress may affect IRP1 protein stability. Unfortunately, bone marrowderived macrophages (BMDM) obtained from KO SOD1 mice are not viable in in vitro culture even at low (3%) O2 concentration, and thus are useless as a cellular model. Therefore, we have attempted to study mechanisms underlying O2•-—mediated regulation of IRP1 using mouse RAW 264.7 macrophages and Sod1+/- BMDM (displaying decreased activity of SOD1) treated with paraquat (PQ; 1,1’-dimethyl-4,4’-bipyridinium dichloride), a redox cycling agent, which is widely used to stimulate O2.- production in cells. Exposure of macrophages to PQ for 3 hrs resulted in a marked decrease in both aconitase and IRE-binding activity of IRP1 as well as in down-regulation of IRP1 protein level 12 hrs after the treatment. Macrophages were recovering IRP1 level (but not cytosolic aconitase activity) 36-48 hrs after PQ treatment. We showed also that lysosomal inhibitors do not prevent decrease in IRP1 protein level in PQ-treated macrophages, which suggests that under our experimental conditions IRP1 is not degraded via the lysosomal pathway. Importantly, we showed that mRNA IRP1 level is considerably decreased in PQ-treated RAW 264.7 macrophages, and that the time-course pattern of this decrease largely overlaps changes in IRP1 protein level.

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Poster# 184 IRON METABOLISM UNDER DIQUAT-INDUCED OXIDATIVE STRESS IN FISCHER-344 RATS Masashi Higuchi, graduate student, Junnichi Oshida, Koichi Orino and Kiyotaka Watanabe Kitasato University (Presented By: Masashi Higuchi, graduate student)

Introduction: Diquat (DQ) is a bipyridyl herbicide that induces hepatotoxicity in vivo by the iron-mediated oxidative stress. The higher levels of stored iron in the body are associated with the more severe DQ toxicity in Fischer-344 rats and free iron is known to participate in producing highly reactive hydroxyl radicals via Fenton reaction. In the present study, we aimed to investigate hepatic nonheme iron, free iron and ferritin, and plasma iron and ferritin to examine an iron metabolism under DQ-induced oxidative stress in Fischer-344 rats. Methods: Ten-week-old male Fischer-344 rats fed the purified diet containing 0.15 ppm Se and 320 ppm Fe were injected subcutaneously with DQ (0.1 mmol/kg body weight) in 0.9% NaCl. Control rats were injected with an equivalent volume of 0.9% NaCl. One, three, six, or twenty hr after DQ treatment, the rats were anesthetized with pentobarbital, and blood samples were collected by cardiac puncture. The rats were then sacrificed and the livers were removed. Free iron was measured as nitrilotriacetate (NTA)-chelatable iron. Results: One hr after DQ injection, the level of hepatic free iron was about 4-fold higher than that of control and its high level was maintained until 6 hr. Six hr after DQ injection, plasma alanine aminotransferase (ALT) activities were markedly increased. DQ administration had no effect on hepatic nonheme iron concentration until 20 hr. Although no changes in hepatic ferritin concentration was observed until 6 hr after DQ treatment, it was markedly increased at 20 hr as compared with the control rats. At 20 hr, the iron content of liver ferritin was significantly lower in DQ-treated rats than in control rats, although there was no significant difference in the H/L subunit ratio of liver ferritin between the two groups. The plasma iron level in DQ-treated rats was gradually decreased, and it was less than 10% of that in control rats at 20 hr. The plasma ferritin level was significantly increased after DQ administration. Discussion: These findings suggest that the free iron concentration in the liver is markedly increased in early stages after DQ treatment, and then hepatotoxicity is induced by iron-mediated oxidative stress. The free iron appears to induce ferritin in the liver and then to be incorporated into ferritin synthesized. The increase of plasma ferritin is considered to be due to liver injury. The mechanism that reduces the plasma iron remains to be clarified.

Poster# 185 HEMIN IS TOXIC TO ASTROCYTES: ARE IRON OR OXIDATIVE STRESS INVOLVED? Stephen Robinson, PhD¹, Theresa Dang², Ralf Dringen² and Glenda Bishop² ¹Monash University; ²SPPPM, Monash University (Presented By: Stephen Robinson, PhD)

Background: During haemorrhagic stroke, lysed red blood cells release hemin, an iron-containing protoporphyrin. We have shown that cultured astrocytes rapidly accumulate heme-iron in a linear and concentration-dependent manner, similar to that observed for non-heme iron from ferric ammonium citrate (FAC). The accumulation of hemin kills astrocytes, and it is widely believed that the degradation of hemin by heme oxygenase releases iron, which is then free to produce toxic hydroxyl radicals via Fenton chemistry. The present study has investigated this proposal. Methods: Primary cultures of astrocytes derived from neonatal rats were cultured in 24-well plates for 2 weeks prior to experimentation. Cells were incubated with 0 or 30µM hemin or FAC in serum-free growth media (Dulbecco’s modified Eagle medium). After 2h, 6h or 24h, the cellular accumulation of iron was determined using a ferrozine-based colorimetric assay. Cell viability was assessed with the lactate dehydrogenase assay, and the intracellular contents of hemin and bilirubin were estimated from the absorbance spectra of cell lysates. To neutralise any free radicals produced from heme-iron, additional cultures were treated with 500µM Trolox, 1mM N-acetyl-L- cysteine, 1mM phenyl-α-tert-butyl-nitrone or 100µM 1,10-phenanthroline. To slow the clearance of intracellular hydrogen peroxide, cultures were treated with the glutathione synthesis inhibitor, buthionine sulfoximine (1mM), which decreases cellular glutathione content. Each condition was performed in triplicate on three independent cultures. Results: Hemin treatment increased LDH release by 6h, reaching 40% by 24h, whereas uptake of an equivalent amount of free iron resulted in approximately 10% LDH release, which is not different to that from untreated control cultures. Neither the chelator nor the antioxidants were able to reduce the toxicity of hemin. By contrast, inhibition of glutathione synthesis increased the LDH released following incubation with FAC for 24h to 45%, whereas it did not exacerbate the toxicity of hemin. Spectrophotometric measurements showed that no more than 25% of the hemin accumulated by cells had been metabolised to bilirubin after 24h. Discussion: 30µM of free iron is not toxic to astrocytes unless glutathione synthesis is inhibited, indicating that the cytotoxicity of iron is associated with its interaction with endogeneous hydrogen peroxide. Astrocytes respond very differently to equivalent amounts of hemin: 30µM of hemin is toxic yet the level of toxicity does not increase when glutathione synthesis is inhibited nor does it decrease when iron chelators are applied. It should be noted that the small amounts of iron which are released by the metabolism of hemin are far below the threshold for toxicity to astrocytes. Conclusion: The present results show that the toxicity of hemin to astrocytes is not due to the release of redox-active iron when hemin is metabolised to bilirubin.

Poster# 186 IRON CHELATION BY SYNTHETIC CHELATORS AND FLAVONOIDS DEPENDS ON PH Katerina Macakova, Premysl Mladenka, PhD, Libuse Zatloukalova, MSc, Katerina Vavrova, PhD, Lubomir Opletal, Asst Prof, Radomir Hrdina, Asst Prof and Ludek Jahodar, Prof Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Czech Republic (Presented By: Katerina Macakova)

Iron chelators are investigated as drugs in many pathological states. Their use in the prevention of various conditions associated with ischaemiareperfusion injury depends on their ability to inhibit Fenton reaction by binding redox-active iron. Moreover, ischaemia-injury is associated with cellular acidosis, therefore this pilot study aimed to test iron chelation activity from neutral to acidic conditions. A simple spectrophotometric approach by means of ferrozine was used in this pilot study for assessment of ferrous and total iron binding capacity of deferoxamine, three synthetic iron chelators (dexrazoxane, deferasirox and salicylaldehyde isonicotinoyl hydrazone – SIH) and three flavonoids (kaempferol, rutin and apigenin). Ferrous chelation was established at pH 4.5, 5.5, 6.8 and 7.5 by means of acetate and HEPES buffers, respectively. Moreover, total iron binding capacity was tested at pH 4.5. Ferrous chelation of all tested drugs rose from pH 4.5 to more neutral pH. All tested compounds were significantly more active ferric chelators than ferrous at pH 4.5. Dexrazoxane is considered as a pro-drug and, in fact, its iron chelation activity was low, measurable only in neutral conditions. The most active drug in acidic conditions was deferoxamine. It was slightly more efficient than deferasirox followed by SIH. The differences at neutral pH were much lower. The efficacy of flavonoids was generally lower than that of deferoxamine although the efficacies of kaempferol and even that of rutin were comparable to synthetic chelators (deferasirox and SIH) in some cases. Notwithstanding, flavonoids tested in this study were able to chelate iron, their iron chelation potencies seems to be lower in acidic conditions when compared to deferoxamine, deferasirox and SIH. This work was supported by grant 53707 B of Charles University in Prague and by the Ministry of Education of the Czech Republic (MSM0021620822).

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Poster# 187 POLYANHYDROGLUCURONIC ACID DERIVATIVE OF LACTOFERRIN DOES NOT IMPROVE ITS PROTECTIVE EFFECTS ON CATECHOLAMINE CARDIOTOXICITY Premysl Mladenka, PhD¹, Vladimir Semecky, Asst Prof¹, Libuse Zatloukalova, MSc¹, Jiri Briestensky² and Radomir Hrdina, Asst Prof¹ ¹Faculty of Pharmacy, Charles University; ²Alltracel Pharma CZ (Presented By: Premysl Mladenka, PhD)

Large doses of catecholamines are cardiotoxic and exogenous application of synthetic catecholamine isoprenaline (ISO) evokes a pathological state with many similarities to acute myocardial infarction where reactive oxygen species generated especially by free iron play a role. Recently, it has been shown that some iron chelators act protectively on cardiac injury caused by catecholamines. Clear bovine lactoferrin (La) was only moderately active. One hypothesis was that low efficacy of La is caused by its very short half-life (t1/2). A non-toxic derivate of La with polyanhydroglucuronic acid (LaD) with preserved iron chelating properties was synthetized in order to prolong La t1/2. Male Wistar: Han rats were randomly divided in 6 groups: the controls (saline only), ISO (100 mg/kg s.c.), La in a dose of 50 mg/kg i.v., LaD (10 mg/kg i.v.) and combination groups of ISO with La or LaD in the same doses as above. Analysis of haemodynamic and selected biochemical parameters was performed 24 hours after drug application. Application of ISO led to 31% mortality and marked myocardial damage. La had no effect on mortality although it reduced cardiac index drop and peripheral resistance increase caused by ISO. Administration of La to healthy animals was associated with some tissue disturbances (an increase in wet ventricles weight index and a tendency to increase diastolic blood pressure). LaD had diminished influence on these parameters on healthy animals as compared to La, but its co-administration with ISO was not more efficient in reduction of myocardial damage and moreover, it even increased mortality to 50%. Conclusively, prolongation of La t1/2 by derivatization with polyanhydroglucuronic acid seems not to potentiate La beneficial effects on catecholamine cardiotoxicity. This work was supported by grants 94/2006/C/FaF and 39207 C of Charles University in Prague.

Poster# 188 IRONING OUT FRIEDREICH’S ATAXIA: THE MOLECULAR CHANGES BEHIND MITOCHONDRIAL IRON-LOADING AND EXPLORATION OF IRON-CHELATION THERAPY FOR TREATMENT D.R. Richardson, M. Whitnall, Y. Suryo Rahmanto, R. Sutak, X. Xu, E. Becker, M.R. Mikhael and P. Ponka Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia (Presented By: D.R. Richardson)

There is no effective treatment for the severe cardiomyopathy that occurs in Friedreich’s ataxia (FA). The identification of significant mitochondrial iron (Fe) deposits in the heart of FA patients suggests Fe may play a role in its pathogenesis. However, the molecular pathways responsible for this toxic accumulation of Fe are unknown. Hence, we used the MCK frataxin knockout mouse model to examine the molecular mechanisms responsible for the mitochondrial Fe-loading associated with FA cardiac pathogenesis. In addition, we explored the use of Fe-chelation therapy as a possible therapeutic regime. We observed significant changes in the trafficking of cardiac Fe in wild-type (WT) compared to mutant mice1. Iron uptake studies using intravenously injected 59Fe- transferrin demonstrated that in mutants, there is increased cellular uptake of 59Fe. This was ascribed to up-regulation of the transferring receptor-1 (TfR1) that is responsible for Fe uptake from transferrin. There was also significant disruption to the intracellular distribution of 59Fe in mutant mice, with a marked decrease in the proportion of cytosolic 59Fe and an increase in mitochondrial 59Fe in mutants compared to WT mice. This suggested that Fe was being targeted to the mitochondrion and away from the cytosol to effect cytosolic Fe-deficiency. Indeed, there was significant down-regulation of the cytosolic Fe-storage molecule, ferritin, in mutant mice. Mediating the regulation of TfR1 and ferritin expression are the Feregulatory proteins (IRPs), sensors of cytosolic Fe levels. Gel retardation analysis showed that IRP2 activity was increased in mutant mice due to the cytosolic Fe-deficit. For the first time, these data elucidate the mechanisms responsible for mitochondrial Fe-loading observed in FA. We also showed that the mitochondrial-permeable chelator PIH, used in combination with the hydrophilic chelator DFO, prevented cardiac Fe-loading and limited hypertrophy in mutants with no observable toxicity1, suggesting Fe-chelation may be useful in the treatment of FA. 1Whitnall M, Rahmanto YS, Xiangcong X, Becker M, Mikhael MR, Ponka P and Richardson DR (2008) The MCK mouse heart model of Friedreich's ataxia: Alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation. PNAS USA 105(28):9757-62.

Poster# 189 FURIN EXPRESSION IS DECREASED IN THE LIVER OF HFE-HEMOCHROMATOSIS PATIENTS Donatella Barisani, MD², Sara Pelucchi, PhD¹, Alessandra Pansa, PhD³, Raffaella Meneveri, PhD³, Paola Trombini, MD¹, Alessandra Salvioni, MD¹, Raffaella Mariani, MD¹ and Alberto Piperno, MD¹ ¹Department of Clinical Medicine, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy; ²Faculty of Medicine, University of Milano-Bicocca, Monza, Italy; ³Department of Experimental Medicine, University of Milano-Bicocca, Monza, Italy (Presented By: Alberto Piperno, MD)

Various studies have recently demonstrated the ability of different molecules to inhibit the production of hepcidin by hepatocytes. They include soluble hemojuvelin, generated by furin cleavage at either muscle skeletal cells and hepatocytes and the protease TMPRSS6, which has a tissue-restricted strong liver expression. Currently there are no data on the expression of these genes in the liver of patients with different iron overload conditions. We previously reported a decreased hepcidin expression, compared to the iron burden, in liver biopsies of patients with HFE hemochromatosis and dismetabolic hepatic iron overload syndrome (DHIOS) [1]. We thus analyzed liver mRNA levels of furin and TMPRSS6 in patients with iron overload of different ethiologies to detect possible variation in expression and to correlate them with hepcidin expression, iron indexes and other clinical and histological data. Liver biopsies were obtained from patients with DHIOS (no. 25), HFE hemochromatosis (no. 10) and secondary iron overload due to beta-thalassemia (no. 6). Patients with non alcoholic fatty liver disease (NAFLD) without iron overload or inflammation served as controls (no. 8). mRNA levels were determined by quantitative PCR using Taqman probes and normalized over HPRT expression. Differences between groups were compared by ANOVA. Furin mRNA expression was significantly downregulated in HFE hemochromatotic patients as compared to the NAFLD group, and a reduction in expression, although not significant, was observed also in DHIOS and thalassemic subjects. No variation in mRNA expression of TMPRSS6 was observed in any of the groups. In the HFE group, furin expression was positively correlated to hepcidin mRNA levels (p<0.001), whereas it was inversely related to transferrin saturation and the Total Iron Score determined by histology. On the other hand, in DHIOS and NAFLD groups no correlation with hepcidin expression or iron stores could be detected. Furin affects iron metabolism via the generation of soluble hemojuvelin, that should block the BMP pathway which increases hepcidin expression [2]. On the other hand, furin cleaves prohepcidin generating the mature hepcidin peptide, activity shared by other proprotein convertases [3]. Furin expression has been reported to increase in iron deficiency [2], and the data here presented suggest that, in iron overload due to HFE hemochromatosis, furin mRNA expression is downregulated by iron burden. This might generate two distinct and opposite effects, since a decrease in the production of soluble hemojuvelin could partially counteract the hepcidin inhibition by defective HFE, whereas a reduced prohepcidin conversion could further decrease the amount of active hepcidin. This regulation by iron seems to be present, although to a lesser extent, also in DHIOS. On the contrary, iron overload does not affect expression of TMPRSS6, at least at mRNA level. 1) Barisani D et al. J Hepatol. 2008;49(1):123-133. 2) Silvestri L et al. Blood 2008;57(11):1573-82. 3) Scamuffa N et al. Gut 2008;57(11):1573-82.

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Poster# 190 LIVER TRANSCRIPTOME OF THE F2 PROGENY OF B6 AND D2 HFE-DEFICIENT MICE: ROLE OF IRON LOADING AND GENETIC BACKGROUND. Annabelle Monnier¹, Léon Kautz, PhD student², Régis Bouvet, Technical support³, Valérie Darnaud, Technical support², Hélène Coppin, DR², Marie-Paule Roth, MD² and Jean Mosser, PU-PH4 ¹RTO Team UMRCNRS6061; ²Centre de Physiopathology of Toulouse Purpan, INSERM U563 Département de génétique- Toulouse-France; ³platform OUEST- genopole®-Rennes-France; 4UMRCNRS6061/platform OUEST-genopole® /Hospital Medical Genomics Laboratory (Presented By: Annabelle Monnier)

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron homeostasis characterized by increased dietary iron absorption. Progressive iron accumulation in parenchymal cells may result in severe tissue damage. In Northern Europe, most patients with HH are homozygous for a single mutation (C282Y) in the HFE gene. However, the disease penetrance is very low. To understand why the severity of iron loading is variable in HH, we took advantage of the two strains of Hfe-deficient mice we recently derived. Whereas Hfe-/- mice of the DBA/2 (D2) genetic background are heavily iron loaded, the iron burden of Hfe-/- mice of the C57BL/6 (B6) background is much milder. We produced a F2 progeny, phenotyped and genotyped the F2 mice, and identified five genomic intervals that control hepatic iron loading (QTL) on chromosomes 3, 7, 8, 11, and 12. We then used a transcriptomic approach to target modifier genes in these regions. Here we report preliminary data obtained by analyzing expression profiles of different groups of mice: 1) 15 B6 and 15 D2 wild-type mice fed a diet with low, normal or high iron content (5 mice/group); 2) 60 Hfe-/- males from the F2 progeny of D2 and B6 Hfe-/- mice. Liver RNA from these mice was hybridized on Agilent 4x44K whole- genome expression arrays. We applied a multistep gene screening strategy. First, we selected genes differentially expressed between the two groups (n=10) of F2 mice with extreme (low and high) liver iron content (unpaired two-tailed Student’s t-test; raw p-values ≤ 0.1). We then selected the subset of genes whose expression in the F2 males was correlated with liver iron loading (GLM; raw p-values ≤ 0.1) and applied a correction for multiple testing (Benjamini & Hochberg correction adjusted at p-values ≤ 0.05). 709 genes emerged from this selection, of which 85 genes also had their expression influenced by the dietary iron content (Benjamini-Hochberg adjusted p-values ≤ 0.05). Interestingly, 34 map within 3 of the 5 QTL intervals. We considered more carefully two of these, mapping to chromosome 7, because their expression was positively associated with dietary iron loading and negatively associated with iron loading induced by Hfe-deficiency in the F2 progeny. This suggests that they may be causally involved in the pathogenesis of hemochromatosis. Furthermore, genome-wide mapping of the loci controlling their expression in the F2 progeny (eQTL) shows that these genes are regulated in cis. These preliminary results pinpoint several genes that could possibly be the modifiers of iron loading in our candidate regions. A similar study will be performed on the female sample of F2 mice as well as on the total sample after correction for sex-related differences in gene expression.

Poster# 191 INTERACTION OF DEFERASIROX (ICL670) WITH METAL IONS USED IN CONTRAST AGENTS Nadine Jane Koch¹, Stefan Stucky, Dipl Chem¹, Kaspar Hegetschweiler, Prof Dr¹ and Hanspeter Nick² ¹Saarland University; ²Novartis Pharma AG Research, Basel, Switzerland (Presented By: Stefan Stucky, Dipl Chem)

a Deferasirox ( LH3, ICL670, figure 1) is a new, orally active iron chelator, which is currently used for the treatment of iron overload. Complexformation of deferasirox with FeIII has been investigated comprehensively;[1] however, nothing is known about its interaction with other trivalent metal cations such as GaIII and GdIII. Such information is, however, crucial, since GaIII and GdIII complexes are widely used as contrast agents. The knowledge of the binding ability of deferasirox for GaIII and GdIII is thus a pre- requisite to answer the question, whether an iron overloaded patient treated with deferasirox, may or may not receive Ga or Gd containing formulations for an imaging study. a A series of titration experiments was performed at T = 25 °C, I = 0.1 M (KCl) to determine formation constants of the metal complexes. LH3 is poorly soluble in H2O and III metal complex formation was therefore studied in a DMSO/H2O mixture (xDMSO = 0.2). The investigation of the Gd complexes proved to be particularly difficult since a solid was generally formed. Consequently, only titration data with pH > 8 and pH < 4 were taken into account. The formation constants are: logβ(GdaLH2) = 25.1(2), a a a b logβ(Gd LH) = 18.6(1), logβ(Gd L) = 12.9(1) and logβ(Gd aL2) = 20.6(1). The sulfonic acid derivative LH3 showed better solubility in H2O and precipitation of any solid b b b during the titrations was never noted. The titrations were all carried out in pure H2O: logβ(Gd L) = 10.2(1), logβ(Gd L2H2) = 32.5(1), logβ(Gd L2H) = 25.5(1) and b a b III logβ(Gd L2) = 18.7(1). The remarkably low affinity of LH3 and LH3 for Gd probably follows from the preference of these two ligands for small metal ions.[1] Hence in terms of thermodynamic considerations, a metal transfer from [Gd(dota)]- or [Gd(dtpa)]2- (dota = tetra-aza-cyclododecane-tetra-acetic acid, dtpa = diethylene-triamine- pentaacetic acid) to aL3- is not to be expected, and the simultaneous administration of deferasirox and a Gd-containing contrast agent should not lead to any interferences between the two pharmaceuticals. As expected, stability constants of the smaller GaIII with both ligands are markedly higher.Complexformation of [GaaL] was completed at the beginning of the titration. - a Based on the formation of the well known [Ga(OH)4] in basic solution, logβ(Ga L2) = 33.8(1) could be measured in pure H2O by an acidimetric titration. The determination a b of further stability constants was, however, not possible due to the poor solubility of LH3 in acidic media. Stability constants with LH3 could well be determined in H2O: b b b b III logβ(Ga LH) = 23.1(2), logβ(Ga L) = 20.5(2), logβ(Ga L2H) = 39.3 (2), logβ(Ga L2) = 34.1(1). In contrast to Gd , administration of a Ga containing medicine to a patient treated with deferasirox at the same time may be problematic, since a transfer of GaIII to deferasirox cannot be ruled out. On the contrary, the high affinity of deferasirox for GaIII, would even allow to consider its Ga complex as a radiopharmaceutical itself. [1]S. Steinhauser, U. Heinz, M. Bartholomä, T. Weyhermüller, H. Nick, K. Hegetschweiler, Eur. J. Inorg. Chem. 2004, 4177-4192.

Poster# 192 A NOVEL SIMULTANEOUS QUANTITATIVE METHOD FOR HEPCIDIN ISOFORMS USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY Katsuya Ikuta, MD, PhD¹, Yasushi Shimonaka², Takaaki Hosoki, MD³, Yusuke Sasaki², Hideyuki Yasuno², Takaaki Ohtake, MD, PhD³, Katsunori Sasaki, PhD4, Yoshihiro Torimoto, MD, PhD5, Keiji Saito, PhD² and Yutaka Kohgo, MD, PhD³ ¹Asahikawa Medical College; ²Kamakura Research Labs, Chugai Pharmaceutical Co., Ltd.; ³Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical College; 4Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical College; 5Oncology Center, Asahikawa Medical College Hospital (Presented By: Katsuya Ikuta, MD, PhD)

Introduction: Hepcidin is known to have 3 isoforms, hepcidin-20, -22, and -25. Hepcidin-25 is thought to be the major isoform and involved in iron metabolism, but the physiological roles of other isoforms are poorly understood. The regulation of hepcidin expression is complex so that the experiments in vitro would be valuable, but most studies have been performed only at the transcriptional level because of the difficulty of quantification of hepcidin in cell culture media. We therefore aimed to develop a sensitive new method for measuring hepcidin that can simultaneously measure hepcidin isoforms in culture media.

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Methods: Human hepcidin-20, -22, -25 was synthesized, and dissolved in cell culture media supplemented with 10% FBS. Trichloroacetic acid precipitations were performed to clean up the media, and supernatants were applied for quantification of hepcidin isoforms. Quantification was performed using liquid chromatography -tandem mass spectrometry. Selected reaction monitoring (SRM) transitions and the collision energies were settled for each isoform respectively. Quantification of hepcidin isoforms in HepG2 culture medium was also performed. Results:Upon optimization of SRM conditions, the most intense precursor ions were selected in each mass spectrum to detect hepcidin isoforms. Product ions were selected to maximize sensitivity and selectivity. Despite using culture media including 10% FBS as matrix, isoform peaks were not interfered with by a blank matrix, indicating the method has good selectivity.Calibration curves were constructed over the range 2-1,000 ng/mL, and linearity of the calibration curves by weighted (1/x2) linear regression was excellent (correlation coefficient: r=0.9974 for hepcidin-20, r=0.9937 for hepcidin-22, r=0.9950 for hepcidin-25). Accuracies for back-corrected concentrations were 99.7-122.1% for hepcidin-20, 102.6-132.5% for hepcidin-22, and 99.1-141.2% for hepcidin-25. These results indicate that the method is adequate for quantifying hepcidin isoforms in culture media. In the HepG2 medium, peaks corresponding to the retention time of synthetic hepcidin-22 and hepcidin-25, but not hepcidin-20, were detected. Conclusions: We have devised a method for simultaneous quantification of hepcidin isoforms in culture media. Quantification of hepcidin isoforms was also possible in the culture medium with HepG2 cells. This new method can contribute to in vitro research on the regulation of hepcidin expression.

Poster# 193 CARDIAC GENE EXPRESSION IN MOUSE MODELS OF IRON LOADING DISORDERS Sharon Hollins, N/A, Daniel Johnstone, Ross Graham, PhD, Dirk van Helden, PhD, Karen Kerr, PhD, Derek Laver, PhD, Kirsty Metelerkamp, N/A, Deborah Trinder, PhD, Robert Fleming, MD, Rodney Scott, PhD, John Olynyk, MD and Liz Milward, PhD University of Newcastle (Presented By: Daniel Johnstone)

Perturbations in iron homeostasis can lead to arrhythmia, cardiomyopathy and infarction and contribute substantially to premature mortality in thalassemia and other diseases involving iron overload. Despite extensive research, the mechanisms causing cardiac dysfunction in primary or secondary iron overload remain largely unknown. We performed genome-wide microarray analysis of mouse cardiac gene expression in 10 week-old biological replicates (n≥3 per group) of AKR mice on regular chow or a short-term high iron diet, and in two models of hemochromatosis, Hfe knockout mice and transferrin receptor 2 (TfR2) mutant mice. Differential expression analysis by microarray was performed using Illumina Sentrix MouseRef-8 (v1.1) BeadChip arrays. One-way ANOVA compared treatment groups to wildtype. Array data were analysed using commercial bioinformatics software (e.g. Agilent GeneSpring GX, Pathway Studio) and various public domain online tools (Panther, Gather, KEGG) for pathway and ontology analysis. Standard biostatistical methods in combination with inhouse programs using heuristic feature set modeling and other approaches were applied to identify additional genes undergoing changes of potential interest. Select changes were further investigated by real-time reverse-transcription PCR. Analysis using the GeneSpring program identified 1488, 867 and 1832 gene transcripts as changing significantly (p<0.05) in cardiac tissue from the high iron diet, Hfe knockout and TfR2 mutant models respectively compared to wildtype AKR controls. Of these transcripts, 167 were altered in all three models, with most overlap occurring between the high iron diet and TfR2 mutant models (611 common transcripts). Just under half (41%) of the transcripts altered in the high iron diet model were also altered in the TfR2 mutant model, whereas only 17% of transcripts altered in the high iron diet model were also altered in the Hfe knockout model. Over half the transcripts with altered expression in each of the two genetic models were altered only in that particular model. There were changes in expression of various genes linked to cardiac function. Notably there was a significant increase in expression of two potassium channel genes, Kcne1 and Kcnq1. Mutations in these genes cause both Long QT and Short QT syndromes, characterized by alterations in the QT interval of the cardiac action potential leading to alterations in heart rate and arrhythmias. Compared to wildtype control mice, cardiac transcripts for the Kcnq1 gene were increased by 1.2-1.4 fold in all iron overload models and transcripts for the Kcne1 were increased 2-2.4 fold in the two genetic models. There was also increased expression of the serum response factor (Srf) gene, linked to QT prolongation. Heart rate assessed from sinoatrial node preparations was slower in Hfe knockout mice compared to wildtype (242.7±21.7 vs 293.3±7.6; n=9, P<0.05) without apparent differences in rhythmicity or responses to noradrenaline or carbachol. Greater effects might occur with intense exercise or other cardiac stress. We conclude that even mild abnormalities in systemic iron cause alterations in cardiac gene expression which may contribute to cardiac morbidity. While effects appear relatively small, such changes may be important in patients with pre-existing cardiovascular disease or severe iron overload and may help identify early biomarkers and treatment strategies.

Poster# 194 PROPERTIES OF THE METAL-ION TRANSPORTER ZIP14 SUGGEST A ROLE IN CELLULAR UPTAKE OF NONTRANSFERRIN-BOUND IRON (NTBI) CHARACTERISTIC OF IRON-OVERLOAD CONDITIONS Jorge J. Pinilla Tenas¹, Brian K. Sparkman¹, Anthony C. Illing¹, Ali Shawki¹, Juan P. Liuzzi², Robert J. Cousins², Mitchell D. Knutson² and Bryan Mackenzie¹ ¹University of Cincinnati College of Medicine; ²University of Florida (Presented By: Jorge J. Pinilla Tenas)

Iron-overload conditions (e.g., thalassemia, hereditary hemochromatosis) are characterized by the appearance in plasma of nontransferrin-bound iron (NTBI), and result in cardiomyopathy, diabetes, hepatic cancer and cirrhosis. Since the metal-ion transporter ZIP14 (SLC39A14) is strongly expressed in liver, heart, and pancreas — major sites of organ damage in iron overload — we tested the hypothesis that ZIP14 transports free iron. We expressed murine ZIP14 in Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. ZIP14 stimulated the uptake of 55Fe2+, 54Mn2+, 65Zn2+ and 109Cd2+ by 50- to 1,000-fold over that in control oocytes. ZIP14- 55 2+ 2+ −1 2+ 2+ mediated Fe uptake was saturable (K0.5 for Fe of 2.6 ± 0.6 µM), temperature dependent (activation energy, Ea = 14 ± 2 kcal.mol ), and inhibited by Co , Mn , and 2+ 2+ 55 2+ Zn . Ca and HCO3 − stimulated Fe transport. These data agree well with those obtained from NTBI uptake measurements in perfused rat liver [Wright et al (1986) J Biol Chem 261, 10909], and suggest that ZIP14- mediated iron transport may be of pathophysiological relevance in iron overload. We observed no currents associated with metal-ion transport in oocytes expressing ZIP14 and conclude that ZIP14 is not rheogenic (i.e., net charge movement is zero). The phorbol ester PMA (a PKC activator, 100 nM) decreased by 48% ± 5% the uptake of 65Zn2+ compared with that in untreated oocytes. This was associated with a 28% ± 3% decrease in membrane surface area (measured by capacitance) suggesting that PKC activation results in the derecruitment of ZIP14 from the plasma membrane. Western blotting of whole-cell extracts revealed significant loss of ZIP14 protein in oocytes treated with PMA. Our data reveal that ZIP14 is a complex, broad-scope metal-ion transporter capable of mediating cellular NTBI uptake, and that PKC activation results in the internalization and degradation of ZIP14 expressed in oocytes.

Poster# 195 MORTALITY AMONG PATIENTS WITH PHENOTYPIC HEREDITARY HEMOCHROMATOSIS AND AMONG THEIR FAMILY MEMBERS: ARE FIRST-DEGREE RELATIVES AT INCREASED RISK? Maria Elmberg, MD, Rolf Hultcrantz, MD, Anders Ekbom, MD, Stefan Lindgren, MD and Johan Askling, MD Karolinska (Presented By: Rolf Hultcrantz, MD)

Background: Hereditary Hemochromatosis (HH) is an autosomal recessive disorder characterized by iron overload and an excess mortality. Through family screening in HH, a substantial pool of close relatives, heterozygous for the C282Y mutation, with mild to moderately increased iron load is identified. The mortality pattern among such individuals is largely unknown, and so is their optimal management. We therefore assessed the mortality among Swedish patients with clinical HH and among their first- degree relatives using nation-wide, population-based, high-quality health- and census registers. Methods: We performed a matched population-based cohort study of 3,832 patients with HH and their 14,496 first-degree relatives using nationwide, population based health and census registers. The cohort of patients were assembled using data from four overlapping sources.

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(i) The Swedish inpatient Register, that covers all inpatient care in Sweden since 1964. (ii) The Swedish Outpatient Register that covers Swedish non-GP outpatient care since 2001 (iii) A Regional register of individuals diagnosed with HH (as inpatients and/or as outpatients) at either of eleven Swedish hospitals during 1970-2004, together serving a population-based catchment area of around 3 million inhabitants. (iii) A Regional register of individuals diagnosed with HH (as inpatients and/or as outpatients) at either of eleven Swedish hospitals during 1970-2004, together serving a population-based catchment area of around 3 million inhabitants (iv) The Swedish Cause of Death register (that contain information on all deaths of Swedish residents from 1961). For each patient identified in the registers described above, 10 controls matched for sex, date of birth, county of residence, and marital status were randomly selected from the Register of population maintained by Statistics Sweden. The cohort of first degree relatives were identified by linking patients and controls to the nation-wide Multi- Generation Register, which holds information on Swedish residents, born 1932 or later, and alive in 1961 or later. All unique individuals in either of the above cohorts were linked to the Swedish cause of Death Register and to the Swedish Population Register. Through these linkages, information on cause of death, vital status and emigrations throughout the follow-up period (1990 through November 30th 2007) was assessed. The mortality among patients and their first-degree relatives was compared to that of general population controls using Cox regression. Results: Patients identified on the basis of hospitalisation with HH were at increased risk of death (RR=2.45, 95% CI 2.27-2.64, 857 deaths). Patients identified through other means had a lower, yet elevated risk of death (RR=1.15, 95% CI 1.00-1.33, 216 deaths). By contrast, their first-degree relatives were only at a marginally increased risk of death (RR=1.05, 95%CI 1.01-1.10), which was similar to the mortality among patients' spouses (RR=1.09, 95% CI 0.86-1.38, 82 deaths). Patients with HH who also had a family history of HH were not at any additional increased risk of death (RR=1.05, 95% CI 0.67-1.62, 21 deaths). Conclusions: The mortality among contemporary patients with HH is only modestly increased. The mortality among their closest relatives is marginally increased and similar for biological and non-biological relatives.

Poster# 196 BONE STATUS IN A MOUSE MODEL OF GENETIC HEMOCHROMATOSIS Pascal Guggenbuhl, MD, PhD¹, Patricia Fergelot, MD, PhD², Hélène Libouban, PhD³, Marie-Paule Roth, MD, PhD4, Yves Gallois, PhD5, Olivier Loréal, MD, PhD6, Gerard Chales, MD7 and Daniel Chappard, MA, PhHD3 ¹Rheumatology Department University Hospital, Rennes, France; ²CNRS UMR 6061, Rennes; ³INSERM, EMI 00335, Angers, France; 4INSERM, U563, IFR 130, Toulouse; 5INSERM, EMI 00335, Angers; 6INSERM U522, IFR 140, Rennes 1, France; 7Rheumatology Department, University Hospital, Rennes (Presented By: Pascal Guggenbuhl, MD, PhD)

Background: Genetic hemochromatosis (GH) is an autosomal recessive disease mainly due to HFE protein loss of function (homozygous C282Y mutation). This mutation leads to inappropriate iron absorption and visceral accumulation in liver and other organs. We found Osteopenia in 78.9% and osteoporosis in 34.2% in males with GH (OI 2005;16:1809). However, the mechanism of bone loss is not well understood and is not explained by hypogonadism in the most recent series. Material and Methods: We studied 6 and 12 mo (H6 and H12) old male HFE-/- C57BL/6 mice susceptible to iron loading and 6 and 12 mo wild-type controls (C6 and C12); there were 8-10 mice in each group. After sacrifice, they were assessed for liver iron overload by measuring the hepatic iron concentration (HIC, in µmol iron/liver g dry weight). Bone microarchitecture was assessed by MicroCT and by histomorphometry. Results: In HFE-/-mice there was a significantly higher HIC, 17.13±7.2 (H6) vs 4.5±0.8 (C6), p<0.0001 and 14.4±4.9 (H12) vs 6.6±2.1 (C12), p=0.001 and a significant decrease in bone volume fraction (BV/TV). The increase in Tb.Pf and SMI indicated a loss of connectivity and an evolution to a rod-like trabeculae model. Trabeculae were fewer and thinner but Tb. Sp did not change. The osteoclast number N.Oc/B.ar was dramatically increased in HFE-/- mice; there was no difference in serum testosterone levels measured in the 6 mo mice between H and C. Iron was found only in HFE-/- bone trabeculae; Ir.S/BS (iron trabeculae content) was 54.6±34.2% in H6 and 51.8±28.8% in H12. A strong correlation was found respectively in 6 and 12 mo mice between Ir.S/BS and HIC (r=0.895, p<0.0001 and r=0.783, p<0.0001), N.Oc/B.Ar (r=0.59, p=0.01 and r=0.673, p=0.001) and BV/TV (r=-0.647, p=0.004 and r=-0.643, p=0.002). Conclusion: hemochromatosis in HFE-/- mice leads to a significant decreased bone mass and deterioration of microarchitecture. There was a strong correlation between iron trabeculae content, HIC and osteoclast number, which dramatically increased despite the absence of hypogonadism. This suggests a direct role of iron in bone loss in hemochromatosis.

Poster# 197 COORDINATION CHEMISTRY OF TWO METABOLITES OF DEFERASIROX (ICL670) AND THE CRYSTAL STRUCTURE OF AN IRON- DEFERASIROX-COMPLEX Stefan Stucky, Dipl Chem¹, Kaspar Hegetschweiler¹ and Hanspeter Nick² ¹Saarland University; ²Novartis Pharma AG Research (Basel, Switzerland) (Presented By: Stefan Stucky, Dipl Chem)

x 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (deferasirox, ICL670, H3L ) is an orally active iron chelator which is used for the treatment of iron overload. It x III II [1, 2] is currently on the market under the name Exjade. Complex formation of H3L with Fe and Fe is well known. Deferasirox is metabolized in the human body. Some of the main metabolites have been isolated. The structural characterization and synthesis was based on the previously published protocol [1] with some minor modifications. One of the metabolites (M3) was identified as a derivative of deferasirox with the benzoic acid-group bound to a glucuronic acid residue via an ester linkage. Two additional metabolites (M1 and M2) carry an additional OH-group in para- (M1) or ortho-position (M2) to one of the phenolic OH-groups (Fig. 1). In view of the coordination- behavior and redox properties, the latter two are of particular interest: a catecholate-type binding of Fe(III) by M2 and formation of quinones in a redox cycle must be considered. x For the unmodified deferasirox, it was possible to grow single crystals of an Fe(III) complex of composition [Fe(L )2]Li3·MeOH·9H2O. These experiments confirmed the proposed meridional coordination of the O,N,O donor set on the Fe(III) center, yielding a distorted octahedral FeO4N2 coordination sphere. The pKa-values of M1 and M2 and the stability-constants of their iron(III) complexes were determined by potentiometric titrations (25°C, I = 0.1M KCl) in a DMSO/H2O medium (xDMSO = 0.2): M1: pKa1 = 4.64(5), pKa2 = 10.08(3), pKa3 = 11.34(4), pKa4 = 13.7(4), log ß110 = 21.6(1), log ß120 = 36.3(1); M2: pKa1 = 4.60(2), pKa2 = 9.98(1), pKa3 = 11.02(1), pKa4 = 14.4(1), log ß110 = 22.04(6), log ß120 = 36.38(7). In addition to the simple 1:1 and 1:2 complexes, formation of protonated and deprotonated species were also observed. Binding of a third M2-ligand to Fe(III) via the catecholate moiety, i.e. formation of a 1:3-complex with a possible bidentate O,O-coordination mode was only observed for an excess of M2 in strong alkaline solution. The evaluated formation constants show that complexation with the two metabolites M1 and M2 is very similar to that of Lx. The redox potential of [Fe(M1)2]3-/4- in alkaline solution could be determined by cyclic voltammetry using a Hg (hanging drop) electrode. The potential of -0.54V vs. NHE corresponds x 3-/4- closely to the value of -0.58V, observed for [Fe(L )2] , indicating that undesired side effects caused by oxidative stress (redox cycling, generation of hydroxyl radicals by Fenton reaction) are not to be expected. It is noteworthy that the biological iron excretion of M1 and M2 is substantially lower than that achieved with deferasirox.[3]

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However, the results of the solution study presented here clearly evidence that the low in vivo efficacy of M1 and M2 cannot be explained by means of thermodynamic parameters such as stability constants or redox potentials. [1] S. Steinhauser, U. Heinz, M. Bartholomä, T. Weyhermüller, H. Nick, K. Hegetschweiler, Eur. J. Inorg. Chem., 2004, 4177. [2] U. Heinz, K. Hegetschweiler, P. Acklin, B. Faller, R. Lattmann, H.P. Schnebli, Angew. Chem. Int. Ed., 1999, 38, 2568. [3] G.J.M. Bruin, T. Faller, H. Wiegand, A. Schweitzer, H. Nick, J. Schneider, K.-O. Boernsen, F. Waldmeier, Drug Metabolism and Disposition, 2008, 2523.

Poster# 198 MUTATION ANALYSIS OF GENES INVOLVED IN IRON REGULATION IN AN INDIAN FAMILY PRESENTING WITH IRON OVERLOAD Michelle-Angelique Hallendorff, MSc¹, Nathaniel McGregor, HonsBSc¹, Richard Hift, MD² and Monique Zaahl, PhD¹ ¹Stellenbosch University; ²University of KwaZulu-Natal (Presented By: Monique Zaahl, PhD)

Iron metabolism disorders comprise the most common disorders in humans. Hereditary haemochromatosis (HH) is a common condition resulting from inappropriate iron absorption. The most common form of the disease (Type 1) is associated with mutations in the HFE gene. The C282Y homozygous genotype accounts for approximately 80% of all reported cases of HH within the Caucasian population. A second HFE mutation, H63D, is associated with less severe disease expression. The C282Y mutation is extremely rare in Asian and African populations. The H63D mutation is more prevalent and has been observed in almost all populations. Iron overload resulting from haemochromatosis is predicted to be rare in Asian Indian populations and is not associated with common HFE mutations that are responsible for HH in the Caucasian population. The aberrant genes associated with HH in India have not yet been identified. The present study attempted to identify variants in six iron regulatory genes that were resulting in the Type 1 HH phenotype observed in two Asian Indian probands from a highly consanguineous family. The promoter and coding regions of the HMOX1, HFE, HAMP, SLC40A1, CYBRD1 and HJV genes were subjected to mutation analysis. Gene fragments were amplified employing the polymerase chain reaction (PCR) and subsequently subjected to heteroduplex single-strand conformational polymorphism (HEX-SSCP) analysis. Samples displaying aberrations were then analysed using bi-directional semi-automated DNA sequencing analysis to identify any known or novel variants within the six genes. Variants disrupting restriction enzyme recognition sites were genotyped employing restriction fragment length polymorphism (RFLP) analysis. Mutation analysis of the six genes revealed 24 previously identified variants, five novel variants (HFE: 5’UTR-840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-1272T→C; HJV: 5’UTR-534G→T, 5’UTR-530G→T), one previously described microsatellite and two novel repeats. Variants identified within the SLC40A1, CYBRD1 and HJV genes do not seem to be associated with the iron overload phenotype. A previously described HAMP variant (5’UTR-335G→T) was observed in the homozygous state in both probands. This variant seems to be the genetic aberration responsible for iron overload in this Indian family. The severe juvenile haemochromatosis phenotype usually associated with HAMP mutations, was not exhibited by the two Indian probands. Their symptoms resembled those observed in classic Type 1 HH. It is suggested that variants identified in the HMOX1 and HFE genes are modifying the effect of the HAMP variant and resulting in the less severe disease phenotype. Although this variant has only been identified in one Indian family, it could shed some light in the hunt for the iron-loading gene in India.

Poster# 199 – WITHDRAWN IRON ABSORPTION IN DYSMETABOLIC IRON OVERLOAD SYNDROME IS DECREASED CORRELATING WITH INCREASED PLASMA HEPCIDIN Fabrice Lainé, MD¹, Marc Ruivard, MD², Tomas Ganz, MD, PhD³, Gordana Olbina, PhD³, Mark Westerman, PhD³, Mathieu Rambeau, PhD4, André Mazur, PhD4, Laurent Gerbaud, PhD², Valérie Tournilhac, MD², Armand Abergel, MD, PhD², Pierre Philippe, MD², Charles Coudray, PhD4 and Yves Deugnier, MD5 ¹CIC INSERM 0203; ²CHU, Clermont-Ferrand, France; ³University of California, Los Angeles, USA; 4INRA, Clermont-Ferrand, France; 5INSERM CIC 203 and U 522, Rennes, France

Background: The dysmetabolic iron overload syndrome (DIOS) is a common disorder whose pathophysiology remains unclear, especially with respect to intestinal iron absorption. Method: A case-control study was conducted to compare intestinal absorption of iron in 16 men with DIOS (age 53 ± 11 years, body mass index 31.4 ± 4.3 kg/m², serum ferritin 750 ± 372 µg/l and hepatic iron 78 ± 25 µmol/g) and in 32 age-matched male controls with normal body iron stores (16 overweight subjects matched for body mass index and 16 lean subjects with body mass index 23.6 ± 1.2 kg/m²). Intestinal absorption was calculated as the area under the curve (AUC) of 0.8 mg of 58Fe given by oral route and correlated with plasma hepcidin measurements and with insulin resistance parameters including homeostasis model assessment (HOMA). Results: Intestinal iron absorption was significantly lower in DIOS (AUC = 22.4 ± 15.9 µg/l/h) compared to both overweight controls (AUC = 40.5 ± 29.4 µg/l/h, p = 0.04) and to lean controls (AUC = 102.5 ± 113.5µg/l/h, p < 0.01). There was a significant inverse correlation between intestinal iron absorption and plasma hepcidin (r = - 0.61, p < 0.001), HOMA (r = -0.35, p = 0.01) and C reactive protein (r = - 0.52, p < 0.001). Conclusion: In overweight subjects with normal iron stores, iron absorption is decreased through hepcidin upregulation secondary to a low-grade inflammatory state. In overweight and iron-overloaded subjects (DIOS), this decrease is more pronounced due to an additional stimulatory effect of iron excess on circulating hepcidin levels.

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Poster# 200 MULTIPLE PREGNANCIES DO NOT REDUCE BODY IRON STORES IN THE HFE-/-MOUSE MODEL OF HEMOCHROMATOSIS Pedro Rodrigues, PhD, João Neves, Ellin Weber, Graça Porto, PhD and Anna Olsson, PhD IBMC, University of Porto (Presented By: Pedro Rodrigues, PhD)

Hereditary Hemochromatosis (HH), a widespread hereditary iron metabolism disorder, is characterized by an excessive absorption of dietary iron which results in increased body iron stores. Since the discovery of the HFE gene underlying the disease (Feder et al. 1996 NatGenet 13, 399-418), a vast number of epidemiological and experimental studies have been conducted. Some of these studies indicate a gender difference in disease expression, in that women show a slower disease progression and a less severe clinical profile. This is usually attributed to iron loss during menstruation and pregnancy (Ryan et al. 2002 Gut 51, 108-112). However, this link has not been clearly demonstrated. The Hfe-/- mouse recapitulates key aspects of HH, including an iron overload phenotype similar to that observed in human patients, and has therefore been extensively used as a model. In this study, we used this animal model to test whether multiple pregnancies would have an impact on iron stores. One year old nulliparous and pluriparous (on average 29 weaned pups per female) B6 and Hfe-/- female mice (n=6) were sacrificed and blood and tissues were collected. From the blood, several erythroid (hematocrit, hemoglobin, mean corpuscular volume and red blood cells) and iron related (serum iron and transferrin saturation) parameters were determined. Liver, spleen, heart and pancreas non-heme iron was measured by the bathophenanthroline method. Additionally, histological sections of liver, spleen and heart were stained for iron with the Pearl’s method. There was no significant difference between pluriparous and nulliparous mice in either of the genotypes as regards levels of serum iron (Hfe-/-: 293±29 vs 347±52 µg/dl; B6 208±67 vs 196±10 µg/dl) and levels of hemoglobin (Hfe-/-: 14,8±0,4 vs 15,1±0,4 mmol/L; B6 13,9±0,6 vs 14,4±0,6 mmol/L). However, in pluriparous Hfe-/- females, we observed a significantly lower transferrin saturation, when compared with Hfe-/- nulliparous females (63,0±6,9 vs 82,2±2,1 %), a difference not statistically different in B6 mice (37,3±7,9 vs 43,7±3,3 %). In both Hfe-/- and B6 mice, liver iron concentration did not differ significantly between pluriparous and nulliparous females (Hfe-/- 855±202 vs 813±206 µgFe/g dry wt; B6 265±33 vs 310±52 µgFe/g dry wt). However, the combined iron stores (liver + spleen + pancreas + heart total iron content) from pluriparous females was significantly higher than those of nulliparous females, both in Hfe-/- and B6 mice (Hfe-/- 592±141 vs 400±72 µg Fe; B6 244±38 vs 159±25 µg Fe) Our results show that multiple pregnancies do not reduce body iron stores in mice. With the exception of transferrin saturation, all relevant clinical parameters of HH were either not significantly affected or even increased in pluriparous females compared to nulliparous females. These results may suggest that in spite of abundant iron stores, female mice increase their iron uptake in response to the increased demands of pregnancy and lactation. Consequently, the iron overload phenotype of Hfe-/- females remains largely unaffected by multiple pregnancies and subsequent lactations. While a confounding effect of physiological and behavioural differences between humans and mice cannot be ruled out, these results nevertheless challenge the view that reproductive iron loss explains gender differences in the expression of Hereditary Hemochromatosis, opening for the possibility of differences between males and females in the regulation of iron metabolism.

Poster# 201 HAEMOCHROMATOSIS-ASSOCIATED GENES IN THE ZEBRAFISH Chun Yu Lok, A.T. Merryweather-Clarke, P. Pinheiro, R. Patient and K.J.H. Robson Weatherall Institute of Molecular Medicine (Presented By: Chun Yu Lok)

Hereditary haemochromatosis is an iron overload disorder caused by mutations in several genes involved in iron metabolism. The common form, type I, is caused by mutations in the HFE gene. Mutations disrupting either hemojuvelin (HJV) or hepcidin (HAMP) cause juvenile haemochromatosis, type II, while mutations in transferrin receptor 2 (TFR2) cause haemochromatosis type III. An autosomal dominant form of the disease, type IV, is caused by mutations in ferroportin (FPN), a transmembrane iron exporter which can be inhibited by the peptide hormone hepcidin, thereby restricting iron uptake and release.Hepcidin itself is upregulated by hemojuvelin via the bone morphogenetic protein (BMP) signalling pathway. Many of these haemochromatosis-associated genes are highly conserved among species including the zebrafish. The transparency and rate of development of the zebrafish makes it a convenient model system for studying the interactions of these genes. Morpholino anti-sense knock-down of HJV expression resulted in abnormal development and heart oedema of zebrafish embryos. Whole mount RNA in situ hybridisation revealed a marked decrease in HAMP expression. DAB-enhanced Perl’s staining showed a systemic increase in iron levels including in the heart, when compared to wildtype uninjected embryos. Our results suggest that the abnormal phenotype is a direct result of the increased iron accumulation in the zebrafish. Rescue of the phenotype was achieved by microinjection of wild-type HJV RNA transcripts into embryos, while injection of HJV RNA containing different disease-causing mutations failed to rescue the phenotype. Morpholino knock-down of HAMP in wild-type embryos produced a similar phenotype to that observed in HJV morphants. To study the effects of the different FPN mutations, RNA over-expression was analysed in zebrafish embryos. Microinjection of wild-type FPN transcripts resulted in the abnormal development of wild-type embryos. Injected fish appeared truncated, with reduced anterior development and slight heart oedema. These embryos also showed strong HAMP expression. The abnormal phenotype was also observed in embryos injected with loss-ofregulation FPN mutants that are resistant to hepcidin inhibition. In contrast, embryos injected with loss-of-function FPN mutant transcripts were indistinguishable from uninjected wild-type fish. GFP analysis revealed expression of FPN in circulation, most probably in macrophages. These results show that an imbalance in gene expression can result in defective iron metabolism. The zebrafish model can be applied to the study of phenotypic characteristics of the different mutant proteins involved in iron homeostasis.

Poster# 202 THE EFFECT OF DIETARY IRON ON TISSUE IRON LEVELS IN INTACT AND SPLENECTOMIZED MICE AFFECTED BY Beta-THALASSEMIA Maria Marongiu, PhD, Kristen Muirhead, BS, Sara Gardenghi, PhD, Ella Guy, Stefano Rivella, PhD and Robert Grady, PhD Weill Cornell Medical College (Presented By: Robert Grady, PhD)

Patients with β-thalassemia hyper absorb dietary iron, most of which is stored in the liver. They also suffer from ineffective erythropoiesis (IE) which leads to hepatosplenomegaly, often requiring a splenectomy. We have been conducting a series of studies utilizing the th3/+ mouse model of thalassemia intermedia to investigate the absorption, distribution and erythroid utilization of iron in the presence and absence of splenectomy. Here we focus on changes in the iron content of liver and spleen resulting from diets containing low (2.5 ppm), sufficient (35 ppm) and high (200 ppm) levels of iron, and assess the impact of splenectomy on its distribution. Th3/+ mice were either bred or generated by transplantation of th3/+ hematopoietic stem cells into lethally irradiated wild type (+/+) recipients. Wild type controls were similarly obtained. Splenectomy of bred and recipient mice was performed at 5 weeks of age and bone marrow transplantation at 8 weeks. Non-transplanted mice were placed on the test diets at 8 weeks of age, and transplanted mice at 11 weeks. All animals were sacrificed after 4 weeks on the test diets, and livers and spleens harvested for determination of their iron content by atomic absorption. Group sizes ranged from 3 to 10 mice (median 7). In general, transplanted and non-transplanted +/+ and th3/+ animals showed comparable differences in organ iron content at the end of the treatments. We saw no major differences between mice fed the high iron diet with those fed the iron sufficient diet. Animals fed the low iron diet had reduced levels of tissue iron. Therefore, for simplicity, we focused on transplanted animals, comparing animals kept on the 35- and 2.5-ppm diets. Wt animals analyzed just before being placed on the test diets exhibited 76.9 and 19.1 ug of iron in their livers and spleens, respectively. Splenectomized wt mice had 98.7 ug of iron in their livers. After 4 weeks, the mean iron contents of the livers and spleens from +/+ animals fed the 35-ppm diet were increased 64% and 186%, respectively. The corresponding values on the 2.5-ppm diet were 27% and 72%. In th3/+ animals fed the 35-ppm diet, the mean iron contents of the livers and spleens were increased 269% and 1,836%, while in animals fed the 2.5-ppm diet the increases were 11% and 712%, showing preferential diversion of iron to the spleen.

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Splenectomizing the animals resulted in further increasing the liver iron, more that 2.5-fold in those fed the low iron diet. The hemoglobin levels of all mice evaluated were unchanged as a result of the dietary changes, except for a 20% decrease seen in bred +/+ mice fed the low iron diet, there being no differences between splenectomized and non-splenectomized animals. We are currently studying splenectomized and non-splenectomized mice after 5- months on the different diets. In summary, a low iron diet has marked effects on the iron levels of liver and spleen, which are accentuated under conditions of IE. Secondly, more iron is absorbed under conditions of IE than is needed for erythropoiesis, the excess being shunted to the liver for storage.

Poster# 203 SYNTHESIS AND COMPLEX FORMATION OF WATER SOLUBLE DERIVATIVES OF THE IRON-CHELATING LIGAND DEFERASIROX (ICL670) Nadine Jane Koch, Dipl Chem, Frank Teucke and Kaspar Hegetschweiler, Prof Dr Saarland University (Presented By: Nadine Jane Koch)

a The iron chelator deferasirox (ICL670, LH3, Figure 1) was developed by Novartis Pharma AG to treat iron overload caused by blood transfusions which are necessary in the treatment of hematological defects such as β-Thalassaemia. The distinctive features of deferasirox are its oral application and long plasma half-life. The pharmaceutical is known by the trade name Exjade which is meanwhile available all over the world. Deferasirox is only sparingly soluble in water which renders the elucidation of its solution properties (in particular the investigation of metal complex formation) difficult.[1] b We therefore prepared a corresponding monosulfonic acid derivative LH3 (Figure 1). Although somewhat more soluble in water, comprehensive investigation of metal complex formation in pure water was sometimes still hampered by the formation of insoluble components.[3] Subsequently, we prepared a new disulfonate derivative (2,5- c [3,5-Bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzodisulfonic acid, LH4,) which finally was sufficiently hydrophilic to keep the various metal complexes in solution. In particular, the problems with precipitation during the titration experiments with CuII and ZnII reported previously for deferasirox[3] could be completely eliminated. c LH4 was prepared from simple building blocks such as salicylic acid, salicylamide and aniline-2,5-disulfonic acid in a four step reaction, following a protocol described first by Ryabukhin[2]. The intermediate phenylhydrazin-2,5-disulfonic acid was prepared from aniline-2,5-disulfonic acid by diazotisation followed by reduction to the c hydrazine with Na2SO3. Singlecrystals of LH4 which could be used for an X-ray structural analysis were grown from methanol / ethyl acetate solution. cLH4 crystallizes as a zwitterion with one of the triazol nitrogen atoms (N4) protonated and one sulfonate group deprotonated. The pKa values of the free cLH4 and the stability constants of c III II II -3 LH4 complexes with Fe and some other relevant biometal cations such as Cu and Zn were determined (H2O, T = 25°C, 0.1 mol dm KCl). The pKa,1 is < 0 and could not be measured. The pKa,2 = 1.4(1) and pKa,3 = 9.32(3), pKa,4 = 11.24(3) were determined by spectrophotometric and potentiometric titrations, respectively. The latter two pKa can be assigned to the two phenolic hydroxy groups. Based on the crystal structure, where a zwitterionic form was observed, it appears probable that the pKa,2 can be assigned to a deprotonation of the protonated triazol unit rather than a sulfonic acid group. The determination of the stability constant logβ(FecL) = 23.50(3) was carried out c c c c c c by spectrophotometric titration; logβ(Fe L2) = 39.4(1), logβ(FeH L2)= 43.7(1), logβ(Zn L) = 10.89(4), logβ(Zn L2) = 15.2(1), logβ(Cu L) = 18.19(6) and logβ(Cu L2) = a c 22.7(1) were obtained by potentiometric titrations. The formation constants of the ligand LH3 (deferasirox, DMSO/H2O) and the new LH4 (H2O) are correlated in terms of a c a c simple linear free energy relations: logβ(M L)=4.6+0.79xlogβ(M L) and logβ(M L2)=4.2+0.87xlogβ (M L2). The two correlations can be used to estimate unknown formation constants of deferasirox complexes, which cannot be measured directly due to the above mentioned solubility problems.[3] [1]S. Steinhauser, U. Heinz, M. Bartholomä, T. Weyhermüller, H. Nick, K. Hegetschweiler, Eur. J. Inorg. Chem. 2004, 4177-4192. [2]Yu.I. Ryabukhin, L.N. Faleeva, V.G. Korobkova, Chem. Heter. Com. 1983, 19, 332-336. [3]U. Heinz, K. Hegetschweiler, P. Acklin, B. Faller, R. Lattmann, H.P. Schnebli, Angew. Chem. Int. Ed. 1999, 38, 2568-2570.

Figure 1 Poster# 204 DISTRIBUTION AND CHEMICAL SPECIATION OF IRON DEPOSITS IN HEARTS FROM TRANSFUSION DEPENDENT PATIENTS Tim St. Pierre, PhD¹, Dudley Pennell², Adam Fleming¹, Mike House¹, Greg Black¹, Paul Kirk², John Paul Carpenter², Lisa Anderson² and Taigang He² ¹The University of Western Australia; ²Royal Brompton Hospital and Imperial College, London (Presented By: Tim St. Pierre)

Background: Iron chelation therapy removes iron from the liver more readily than from the heart. The chemical speciation of the iron in the heart is a factor that is likely to influence iron availability to chelators. Methods: Hearts were obtained post mortem from 10 transfusion dependent patients then stored in formalin. The left ventricle of each heart was dissected into pieces (mean mass ± SD = 1001±620 mg) according to a geometrical plan that comprised 5 axial slices, each slice being divided into 6 sectors, each sector being divided into an inner, middle, and outer part of the left ventricular wall. Samples were subsequently freeze-dried. Mössbauer spectroscopy measurements were made on the dried septum samples from the mid axial slices. Total iron content measurements were made on all samples. Results: The mean wet (dry) tissue iron concentrations for the left ventricles ranged from 0.08 (0.39) to 5.0 (25) mg Fe/g. The coefficients of variation of dry tissue iron concentration ranged from 14% to 40%. The proportion of non-septal sectors with greater wet weight iron concentration in the outer wall than the inner wall (154 of 184) was significantly greater (p<0.0001) than the proportion with greater iron concentration in the inner wall (30 of 184). There was no corresponding significant trend in iron concentration across the septal sectors (n=92). Wet weight iron concentrations of the mid axial slice of the septum (target of T2* measurements) ranged from being 30% less to 26% more (mean deviation 2% less) than the mean iron concentration for the entire left ventricle. Mössbauer spectra indicated that some of the hearts contained iron(III) oxyhydroxide in a more crystalline form than that found in ferritin. In vitro studies have previously shown that this more crystalline form is less readily available to chelators than that found in ferritin [1]. Conclusions: Iron deposits in left ventricular heart tissue have a greater concentration in the outer wall except in the septal region where no significant gradient is detected. Measurements of iron concentration in the septa are a reasonable indicator of overall left ventricular iron loading. The inorganic form of the iron deposits are likely to be less soluble and less available to chelators than the form of iron in ferritin. [1] Chua-anusorn, W. and St. Pierre, T.G. (2006) Pathological biomineralization of iron. In: Biomineralization – Medical Aspects of Solubility. (Eds: Königsberger, E. and Königsberger, L-C.) Wiley and Sons Ltd, pp 219-276.

Poster# 205 MUTATION ANALYSIS OF HEPCIDIN AND FERROPORTIN GENES: POSSIBLE RELATIONSHIP WITH IRON OVERLOAD IN ITALIAN PROSPECTIVE BLOOD-DONORS Lorena Duca, Biology, Paola Delbini, Biotechnology, Isabella Nava, Biology, Valentina Vaja, Biotechnology, Gemino Fiorelli, Medicine and Maria Domenica Cappellini, Medicine ¹Policlinico, Mangiagalli, and Regina Elena Hospital IRCCS Foundation, University of Milan (Presented By: Lorena Duca, Biology)

Hepcidin and ferroportin dysregulation resulting from mutations in its encoding genes is implicated in the pathogenesis of several iron disorders. In this study mutations and polymorphisms of hepcidin and ferroportin genes in Italian prospective blood donors that showed iron overload not due to HFE genotype or to other causes (inflammation, liver disease and alcohol abuse) were examined. Hematological and iron parameters were obtained from routine laboratory procedures. Hepcidin and ferroportin genes were analyzed by PCR amplifications of single exons 161 ollowed by mutations screening with restriction analysis or direct sequencing. From two-hundred blood-donors originating from different areas of Italy, thirty subjects (20 males and 10 females, 18-57 years) with increased transferrin saturation (%TS=51±23%; normal values <45%) and/or serum ferritin (SF=558±437 ng/mL; normal values <250 ng/mL) were identified. Control cohort (n=50; 30 males and 20 females, 20-60 years) included individuals with the same geographical origin of iron-loaded carriers, normal haematological and iron parameters, and negative viral and autoimmune screening. No mutations in hepcidin gene were found in both groups. A polymorphism in intron 2 (IVS2+7G>A) has been identified in two blood donors: one had SF=488 ng/mL and normal %TS, while the other had SF=617 ng/mL and TS=93%. The last also resulted heterozygous for two common ferroportin mutations (Q248H and R561G). Five common polymorphisms and one microsatellite in the ferroportin gene promoter region were identified: 5’UTR(-98)G>C, 5’UTR(-8)C>G, (CGG)n 5’UTR microsatellites, IVS1-24C>G and 977 T>C (V221V). The -98C, -8G and (CGG)7 genotypes occurred at an higher frequency in blood donors with iron overload (0.43, 0.35 and 0.32 respectively) than in control group (0.06, 0.05 and 0.20 respectively). The frequency of (CGG)9 allele was 0.22 in iron-overload carriers and 0.00 in control group. The (CGG) 9 allele was in partial linkage disequilibrium with the –98C and the –8G alleles. The intronic polymorphism IVS1(-24) and the T>C substitution in exon 6 at nt 663 (V221V) were common polymorphisms that exhibited allelic frequencies significantly higher in subjects with alterated iron parameters (0.50 and 0.70, respectively) compared to control group (0.25 and 0.08, respectively). R561G mutation was detected in subjects affected by iron overload at polymorphic frequencies (0.10). Heterozygosity was found in 4 individuals with moderate iron load (SF=617-779 ng/mL and TS=48-69%), while homozygosity was present in 1 out of 30 subjects and associated with SF=1724 ng/mL and TS=63%. One Val162del mutation was detected in a subject with high serum ferritin (2750 ng/mL) and transferrin saturation (70%). This mutation was associated to microsatellite (CGG) 7, IVSI-24G and V221V. No mutations or polymorphisms in hepcidin gene that might be responsible for iron overload were found, while an higher genetic variability of ferroportin gene has been observed in blood donors with increased TS% and/or SF values. The polymorphic ferroportin variants could have a significant effect on iron metabolism and explain the remarkable phenotypic variability that exists in non-HFE-linked iron overload. The significance of these observations are uncertain and further investigations are needed to determine if hepcidin and ferroportin genes polymorphisms affect the parameters of iron overload in subjects with normal HFE genotype.

Poster# 206 PRELIMINARY STUDY ON GENETIC MODIFIERS THAT MAY INFLUENCE THE IRON OVERLOAD IN SUBJECTS WITH H63D MUTATION Isabella Nava, Biology, Paola Delbini, Biotechnology, Lorena Duca, Biology, Gemino Fiorelli, Medicine and Maria Domenica Cappellini, Medicine Policlinico, Mangiagalli, and Regina Elena Hospital IRCCS Foundation, University of Milan (Presented By: Lorena Duca)

Hereditary hemochromatosis (HH) is mainly due to C282Y mutation in HFE gene. In the pathogenesis of HH the role of H63D, a second known mutation of HFE, is still unclear. Although the majority of homozygotes for H63D has normal iron parameters, sometimes H63D homozygosity is associated with increased transferrin saturation (TS) and serum ferritin (SF). These observations raise the question whether H63D mutation itself results in changes of iron parameters, or other associated risk factors are required. Mutations in hepcidin and ferroportin genes are other known causes of iron storage disease. The aim of this study was to determine if mutations and polymorphisms in hepcidin and ferroportin genes may influence iron homeostasis and promote the progression of iron overload-associated disease in H63D homozygotes. Forty patients (35 males and 5 females, 20-65 years) with high transferring saturation (%TS=59±10%; normal values <45%) and/or ferritin (SF=550±520 ng/mL; normal values <250 ng/mL) with H63D homozygosity genotype were included. Other factors responsible for iron overload (C282Y genotype, inflammation, liver disease and alcohol abuse) were excluded. Fifty healthy individuals (30 males and 20 females, 20-60 years) with normal haematological and iron parameters, and negative viral and autoimmune screening were included as controls. C282Y and H63D mutations were identified using allele- specific oligonucleotide hybridization. Hepcidin and ferroportin genes were amplified by PCR and submitted to sequence analysis. In hepcidin gene a T31M mutation was identified in homozygosity in one H63D subject with SF=506 ng/mL and TS=73%. The mutation does not affect any amino acid and its significance is elusive. Frequency of this mutation in controls was 0.002. In H63D-carriers 3 polymorphisms of ferroportin gene were identified: a microsatellite (CGG)7 repeat in the TATA box of the promoter region (0.20), a polymorphism IVSI-24 G→C within the first intron (0.75), a silent coding region mutation 663 T→C (V221V) (0.55). None of these polymorphisms has significant allelic frequency in controls (~0.001). The haplotype resulting from (CGG)7, IVS1-24C and V221V showed SF=617±244 ng/mL and TS=61±8% (n.12), while without or with one or two of these variants SF was 408±92 ng/mL and TS was 58±10% (n. 26). Two ferroportin polymorphisms in the 5’-UTR, a G→C at nt.98 and a C→G at nt.8 relative to the start of translation, occurred at an higher frequency (0.25 and 0.35, respectively) in H63D subjects with SF=372±92 ng/mL and TS=58±12% than in controls (~0.001). The R561G was associated with H63D-carriers at an allele frequency of 0.06 (n=5 alleles) and absent in controls. This mutation was associated with high iron load (SF=907±129 ng/mL and TS=61±4%). Val162del mutation was detected in one H63D subject with very high SF (3528 ng/mL) and TS (74%). This subject showed the ferroportin haplotype (CGG)8, IVSI-24C, 663C and -98C associated to Val162del mutation. In conclusion polymorphic variants of ferroportin investigated might be implicated in the variable phenotypic expression of H63D homozygotes. Further studies in a larger series of H63D homozygotes with and without iron overload are needed to reach definitive conclusions. Additionally, mutations in other genes different from those screened for and the involvement of non-genetic factors cannot be discounted.

Poster# 207 IRON-CHELATOR-THERAPY RESTORES MAJOR SYMPTOMS OF A FEMALE PATIENT SUFFERING FROM MICROCYTIC ANEMIA AND SEVERE IRON-OVERLOAD OF THE BONE-MARROW AND THE LIVER Wolfgang Gilles, PhD¹, Sandro Altamura, PhD¹, Mayka Sanchez, PhD², Christa Flechtenmacher, MD¹, Thomas Herrmann, MD¹, Martina U. Muckenthaler, Prof¹ and Wolfgang Stremmel, Prof¹ ¹University of Heidelberg; ²EMBL Heidelberg (Presented By: Wolfgang Gilles, PhD)

Microcytic anemia is commonly caused by iron deficiency but can also occur as a consequence of abnormalities of iron metabolism. The most common forms of microcytic anemia include the thalassemias, the X-linked sideroblastic anemia, sickle-cell-anemia and pyruvate-kinase-deficiency. Beside these inherited forms of microcytic anemias several acquired forms exist which are mostly due to monoclonal changes in the red-blood-cell-line. Microcytic anemias which are due to iron-metabolism-disorders frequently belong to the group of iron loading anemias. In this case, the anemia ought to be compensated by increased erythropoesis and a simultaneous increase in duodenal iron-uptake. The precise relationship between increased erythropoesis and iron-uptake is only poorly understood. Here we describe the case of a 44-years old woman, who presented with an anemia, severe microcytosis and hyperferritinemia of unclear etiology, which ameliorated during the treatment with Deferasirox. A Caucasian woman (age 44) was noted to suffer from a microcytic anemia (Hemoglobin: 11.6 g/dl, MCV: 70 fl) since several years. The patient suffered from arthralgia of the subtalar joints, occasionally occurring edemas of the lower legs and fatigue. Further examinations of the blood revealed highly elevated ferritin (1038 µg/l), decreased transferrin (1.8 g/l) and increased transferrin-saturation (86%), which lead to the suspicion for systemic iron-overload. Systemic iron-overload could be confirmed by biopsy of the liver and the bone-marrow where high iron-concentrations could be detected. In the liver-biopsy iron-accumulation was detected in the surrounding of the portal fields in addition to hepatic steatosis and beginning fibrosis. The biopsy of the bone-marrow revealed impaired erythropoesis and dispersed iron-overload probably due to an iron- utilization-disorder. We performed genetic analysis of the whole coding sequence without finding any mutation in ALAS2, TMPRSS6 and GLRX5. Also in HFE, Ferroportin, HAMP and DMT1 none of the common mutations could be found. Also spherocytosis, pyruvate-kinase-deficiency, porphyria and hemoglobinopathy could be excluded. Esophagogastoduodenoskopy revealed esophageal varices. In order to avoid further damage of liver, bone-marrow and other organs we decided to begin an iron-depletion-therapy. As phlebotomy seemed not to be reasonable because of the low hemoglobin we started the administration of Deferasirox (20 mg/kg). Undergoing this therapy Ferritin-levels dropped from 1038 to 250 µg/l and hemoglobin rose from 11.6 to 14 g/dl within 6 month. In the meantime the mean corpuscular volume rose from 70 to 75 fl. Apart from these laboratory findings all clinical symptoms of the patient disappeared during the therapy. As iron-depletion solved the major symptoms and and reverted the laboratory findings of the patient to normal we conclude that the disease of the patient was due to the toxic effect of free iron particularly in the bone marrow which led to an impairment of erythropoesis. The causality of the disease has not been revealed until now. 162

Poster# 208 IS IRON OVERLOAD IN ALCOHOL-RELATED CIRRHOSIS MEDIATED BY HEPCIDIN? Tariq Iqbal¹, Azzam Diab, MRCP¹, Douglas Ward, BSc, PhD², Matthew Brookes, PhD, MRCP² and Chris Tselepis, BSc, PhD² ¹University Hospital Birmingham; ²University of Birmingham (Presented By: Tariq Iqbal)

Introduction: Spur cell anaemia (SCA) is a condition in which both erythropoeisis and tissue iron stores are altered by excessive alcohol consumption such that sufferers develop severe tissue iron overload in the face of ongoing haemolysis. The haemolysis, due to a structural abnormality of red cell membranes, results in spiculated erythrocytes (acanthocytes) which undergo early splenic destruction. Treatment of SCA has been disappointing and it is commonly viewed as an indicator of end-stage liver disease. We postulated that reduced hepcidin production in the cirrhotic liver would be a significant factor contributing to the body iron excess in patients with end stage alcohol-induced cirrhosis and SCA who underwent orthotopic liver transplantation(OLT). Method: We measured hepcidin production pre, peri and post liver transplantation in a 55 year old female patients with cirrhosis. The patient gave consent for blood and urine samples to be collected for study (LREC 08/MRE09/2). Samples were taken immediately pre and at 12, 36, 48 hrs and 2 months post transplant. Blood was analysed for ferritin, haemoglobin, erythropoietin, IL-6 and CRP as markers of haemolysis inflammation. Hepcidin was measured on urine and serum samples by assessment of peak intensity at m/z 2792 in MALDI-TOF-mass spectra of urine or SELDI-TOF spectra of serum using stable isoptope labelled hepcidin as an internal standard. Urine samples were normalised with respect to protein concentration (20 ug/ml, Bradford assay) and desalted with C8 magnetic beads. Cu2+ loaded IMAC SELDI chips were used to enrich hepcidin from serum (2 ul serum per analysis). A sample of the explant liver was analysed for hepcidin mRNA by rtPCR and this was compared to samples taken from 8 normal livers. Results: Hepcidin levels pre-transplant were very low compared to the elevated ferritin. Following transplant hepcidin production rose with falling ferritin. At 2 months post transplant the hepcidin production had fallen in the context of the now normal serum ferritin. Hepcidin/ferritin ratio rose progressively following transplantation. Hepcidin mRNA expression in the explant liver from our patient was significantly lower than that seen in normal liver (data not shown). Conclusion: This case illustrates “inappropriate” low hepcidin production in an iron-over loaded cirrhotic patient with alcohol-related SCA. Following successful transplantation hepcidin production by the new liver increased accompanied by falling serum ferritin. “Appropriate” hepcidin production by the new liver would act to sequester iron in reticulo-endothelial stores and to repress duodenal iron uptake. At 2 months post-OLT both hepcidin and ferritin have fallen to low levels.

Poster# 209 USEFULNESS OF MAGNETIC RESONANCE IMAGING FOR NON-INVASIVE TISSUE AND HISTOLOGICAL CHARACTERIZATION OF IRON OVERLOAD DEPOSITS IN MICE Eric Hitti, PhD5,6, Pierre-Antoine Eliat, PhD², Emmanuelle Abgueguen, PhD5,7, Bruno Turlin, MD,¨PhD5,7, Hervé Saint-Jalmes, MD, PhD³, Pierre Brissot, MD5,7, Yves Gandon, MD4 and Olivier Loréal, MD, PhD¹ ¹INSERM U522; ²Prism IFR 140 GFAS, University of Rennes 1, Rennes, France; ³,5Departement d'imagerie, Centre Eugène Marquis; 4Imaging Departement, University Hhospital, Rennes, France; 5IFR140; 6University of Rennes 1, Rennes, France; 7University of Rennes 1, Rennes (Presented By: Olivier Loréal, MD, PhD)

Iron deposits repartition and histological picture of iron overload disease vary regarding the disease aetiology. Obtaining, by non invasive method, relevant information concerning iron localisation within the body, i.e. liver on spleen iron concentration ratio, and nature of the loaded cells, i.e. hepatocytes versus Kupffer cells, could be useful to guide diagnostic procedure. Our aim was to study the usefulness of MRI in the determination of tissue and histological iron parameters. Material and Methods: Iron-dextran (0; 0,25 or 0,5 mg/kg) loaded mice investigated shortly after the iron injection (2 months), with iron deposits localized predominantly in macrophages, or later (5months) iron being then mainly located within hepatocytes. MR images were acquired at 4.7 T using a Bruker Biospec spectrometer. For each mouse a multi echo spin echo sequence (eight echoes) was acquired (TR=2000ms, TE=6.5ms matrix 256x 256, FOV = 3.2x3.2cm, 2 Nex, slice thickness=1.5mm). After MRI examinations, mice were sacrificed; liver and spleen were excised for the biochemical and histological determination of liver and splenic iron content (LIC and SIC respectively) and repartition.T2 was calculated using the mean signal in a region of interest (ROI) over the 8 echoes. We also studied spleen/muscle (S/M) and liver/muscle (L/M) signal intensity ratios. A texture analysis was performed on a square ROI 20x20 pixels using Mazda 4.5 software through the use of Fisher test to select best discriminating parameters between the different studied groups. A linear discriminating analysis (LDA) was then performed. Histological iron quantification, including quantification of iron excess within hepatocytes, sinusoidal cells including hepatic macrophages was performed on Perl’s stained liver slice. Results: The best correlation between LIC and MRI parameters was obtained using liver/muscle signal intensity ratio in the second echo (r²=0.91), accordingly to previous reports. The spleen/muscle signal intensity was also well correlated (r²=0.89) with the SIC. Histological scores demonstrate that i) two months after injection the iron was mainly located within the sinusoidal cell, and at five months the HIS increased and the SIS decreased significantly whereas the LIC was not modified. The Fisher test isolated the 10 best discrimant parameters and The LDA performed using these parameters showed that the two groups (2 and 5 months) groups were well discriminated. Conclusion: Our results show that MRI is useful for iron quantification in spleen, in addition to the liver, and could be used to evaluate therapeutic efficiency. In addition, texture analysis allowed us to classify images according to cellular iron repartition in the liver and could be a guide for diagnostic procedure.

Poster# 210 THE EFFECT OF ACCESS TO NON-INVASIVE LIVER IRON CONCENTRATION MEASUREMENTS ON PATIENTS AT RISK OF IRON OVERLOAD FROM MULTIPLE BLOOD TRANSFUSIONS: AN AUDIT AND RETROSPECTIVE STUDY Tim St. Pierre, PhD¹, Greg Brown², Nigel Patton³, Heather Tapp4 and James Taylor² ¹The University of Western Australia; ²Royal Adelaide Hospital; ³Auckland City Hospital; 4Children, Youth, and Womens Health Service, South Australia (Presented By: Tim St. Pierre, PhD)

Background: The study aims were to determine whether the body iron burden in a cohort of subjects with hemolytic anemias or ineffective hematopoiesis improved after the introduction of non-invasive monitoring of liver iron concentrations (LIC). Design and Methods: A retrospective audit was conducted on clinical data from 40 consecutive subjects with hemolytic anemias or ineffective hematopoiesis who had been monitored non-invasively for LIC over a period of at least one year. LIC was measured with spin-density-projectionassisted R2-magnetic resonance imaging (FerriScan® R2-MRI). The median number of R2-MRI measurements made on each subject was 5 (range 2 to 9). The median period between the first and last R2-MRI measurements for each subject was 3.4 years (range 1.0 to 6.1 years). 163

Results: The geometric mean LIC for the cohort dropped significantly (p = 0.008) from 6.8 mg Fe/g dry tissue at initial measurement to 4.8 mg Fe/g dry tissue at final measurement. The proportion of subjects with LIC in the range associated with greatly increased risk of cardiac disease and death ( > 15 mg Fe/g dry tissue) dropped significantly (p = 0.01) from 14 of 40 subjects at initial measurement to 5 of 40 subjects at final measurement. A total of 19 clinical decisions based of LIC results were documented in the case notes. Decisions comprised initiation of chelation therapy, increasing chelator dose, decreasing chelator dose, and change of mode of delivery of deferioxamine from subcutaneous to intravenous. Conclusions: The data support the hypothesis that introduction of non-invasive monitoring of LIC can lead to a decreased body iron burden through improved clinical decision making and improved feedback to patients and hence improved adherence to chelation therapy.

Poster# 211 LIVER IRON OVERLOAD IN NON HFE RELATED HYPERFERRITINEMIC PATIENTS Gian Luca Forni, MD, Manuela Balocco, MD, Roberto Villa, MD, Cinzia Bruzzone, Phisician, Andrea Macco, Phisician and Barbara Gianesin, Phisician Centro della Microcitemia e delle Anemie Congenite, Ospedale Galliera, Genova (Presented By: Gian Luca Forni, MD)

Between 2003 and 2008, we analyzed the suspected hepatic iron overload in sixty-eight subjects with unexplaited persistent hyperferritinemia with normal value of transferrin saturation. Ferritin is a major iron storage protein essential to iron homeostasis, involved in a wide range of physiologic and pathologic processes. It is also usually utilized as a serum marker of total body iron stores. None of the patients had a clear etiology of hyperferritinemia such as HFE mutations, viral hepatitis or liver disease, haematological or malignant disorders, inflammatory causes or non alcoholic fatty liver disease. None had a familial history of hyperferritin. There were 51 males and 17 females with a median age of 51 years (range 30-78). The median duration of hyperferritinemia until the time that patients arrived to our observation was 2 years (range 1-11). The normal range value of ferritin is 13-150 ng/ml in females and 30-400 ng/ml in males. In the subjects that we have analyzed, the median serum ferritin level was 615 ng/ml for females (range 239-3532) and 687 mg/ml for males (401-2144). The median transferrin saturation was 35% (15-48). The median values of transaminases were respectively 25 U/L (SGOT: range 12-143) and 30 U/L (range 12-84 U/L) and GGT 37 U/L (range 16-641). Twenty-three subjects had an heterozygosity H63D. Thirtyseven subjects underwent liver elastography (FibroScan®) and the median stiffness values were classified in Metavir score (F0-F1: 24 patients; F2: 9 patients and F3: 4 patients). All subjects were measured with Magnetic Iron Detector (MID),a room temperature biosusceptometer, which evaluates iron overload in the liver region. The error in the measurement of iron overload by MID is estimated to be equal to ±0.8 g (1 SD). Forty-eight patients had hepatic iron overload lower than the sensibility of the MID. Seventeen patients had a mild hepatic iron overload (LIC 1200-2400 mcg/gr/ww) and three patients with high iron overload (LIC 2700-5000 mcg/gr/ww). The hepatic iron overload was significantly correlated with hyperferritin (p< 0.0001) and hepatic fibrosis (p< 0,0001).The median duration of hyperferritinemia, in patients with liver iron overload, until our observation was 2 years (range 1-11). Of twenty subjects with hepatic iron overload, only four showed an increase of transaminases (2-5 times the normal value) with a significant correlation (p=0,02). Our results show an unlike increase of LIC greater than 4 times the upper limit of normal in hyperferritinemic subjects who do not have HFE-related hereditary hemochromatosis or causes of secondary iron overload. On the basis of these results, we can so argue that a chronical increasing in ferritin values, even in absence of clear primary or secondary causes of hyperferritinemia, can lead to a detectable liver iron overload responsible of progressive fibrotic degeneration. In our opinion, a non-invasive liver assessment for iron overload should be performed in every subjects with persistent hyperferritinemia.

Poster# 212 INTESTINAL IRON ABSORPTION DURING PHLEBOTOMY THERAPY IN PATIENTS AFFECTED BY HEMOCHROMATOSIS TYPE 1 Gian Luca Forni, MD, Roberto Villa, MD, Manuela Balocco, MD, Andrea Macco, Physician, Barbara Gianesin, Physician and Cinzia Bruzzone, Physician Centro della Microcitemia e delle Anemie Congenite, Ospedale Galliera, Genova (Presented By: Gian Luca Forni, MD)

Hemochromatosis type 1 is the most important congenital cause of iron overload in caucasian population. This genetic trait causes iron overload leading to progressive organ failure, mainly in liver, spleen, pancreas, heart, brain. To avoid this kind of complications the therapeutic approach usually used consists in phlebotomy. We assessed iron overload in the liver region in 15 patients (14 male and 1 female in menopause) with C282Y homozygous, serum ferritin > 1000 ng/ml and transferrin saturation > 90% undergoing phlebotomy therapy. Measurements were performed before and during phlebotomy therapy by a room temperature biosusceptometer, named Magnetic Iron Detector (M.I.D.), which evaluates iron overload in the liver region. Phlebotomies (300 – 450 mlblood ) containing an average iron amount of 150–225 mg, were made at weekly intervals until the serum ferritin level was < 50 ng/ml and transferring saturation < 30%. Complete blood count, serum transferrin saturation and serum ferritin were scheduled at the beginning, during and at the end of the treatment. Most patients underwent several iron overload measurements within a period from 1 to 40 months depending on the therapy regimen. A total of 41 valid measurements were performed. The error in the measurement of iron overload is estimated to be equal to ±0.8 g (1 SD), but for two or more measurements of the same patient it becomes better than 0.5g. All fifteen patients had at least two MID measurements with a result in iron liver value reduction related to the rate of reduction expected on the basis of the iron removed by phlebothomy. The slope of the linear fit is 0.5 (R2=0.72), showing we really measure approximately half a gram for every expected gram of iron reduction. To our knowledge this difference is to be referred to increased iron absorption from diet. The rate of iron absorption in treated patients was found to be 4.9 mg/day (R2=0.58) more than the normal iron balance. Furthermore we observed a very weak statistical correlation between the values obtained by M.I.D. and ferritin (R2= 0.46) and transferrin saturation (R2=0.05) if next to normal ranges. Indeed, we observed that even if ferritin and transferrin saturation values are low M.I.D. detects a residual liver iron overload. However, values of iron overload above 3g, correlate significantly with ferritin values (R2=0.73), but they do not with transferrin saturation ones. These results suggest M.I.D. iron assessment can quantify liver iron overload also in patients presenting biochemical iron markers values next to normal ranges. On the basis of these evidences we think the interruption of phlebotomic therapy has to be relat ed not only to biomarkers, but also to direct non invasivemeasurement.

Poster# 213 ASSOCIATION BETWEEN HFE GENOTYPE AND IRON OVERLOAD IN PATIENTS WITH LIVER DISEASES FROM WESTERN ROMANIA Adriana Maria Neghina¹, Ketil Thorstensen, PhD³, Andrei Anghel, PhD², Iosif Marincu, MD, PhD² and Raul Neghina, MD² ¹Victor Babes University of Medicine and Pharmacy; ²Victor Babes University of Medicine and Pharmacy, Timisoara, Romania; ³Department of Medical Biochemistry, St. Olavs Hospital, Trondheim, Norway (Presented By: Adriana Maria Neghina)

Hereditary haemochromatosis (HH) is an inherited autosomal recessive disorder characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The clinical penetrance and frequency of complications of HH is a subject of controversy. The present study aimed at assessing the HFE mutations (C282Y, H63D and S65C) in western Romanian patients with liver disease of diverse aetiologies suspected of iron overload.

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A total of 21 patients, all Romanian residents hospitalized with clinical suspicion of iron overload and liver disease, were assayed for C282Y, H63D and S65C mutations and serum ferritin. The characteristics of the study group are summarized in Table 1. Of these 21 patients, 4 (19%) were C282Y homozygotes, 1 (4.8%) was C282Y/H63D heterozygote, 1 (4.8%) was C282Y heterozygote, 2 (9.5%) were H63D heterozygotes, 1 (4.8%) was S65C heterozygote and 12 (57.1%) had none of these 3 mutations. Out of 10 cases with hyperferritinemia and/or HIS ≥ 1, without hepatocellular carcinoma, 5 (50%) patients had mutations in the HFE gene in accordance with iron overload (4 C282Y homozygotes, 1 C282Y/H63D heterozygote). They had significantly increased ferritin levels compared to wild-type cases (P = 0.029). The inclusion of iron studies during routine clinical visits coupled with the availability of HFE genotyping for family and population studies will facilitate the early detection of HH in Romania.

Poster# 214 FREQUENCY OF C282Y AND H63D MUTATIONS OF THE HEMOCHROMATOSIS GENE IN A NORTHEASTERN BRAZILIAN POPULATION Luiz Silva, Jr., MD¹, Andre Silva, MD², Adavio Silva, MD³ and Jairo C. Cavalcante, Statiscal4 ¹Souza Junior Laboratorio Medico; ²Hospital A. C. Camargo; ³Hospital da Benficiencia Portguesa; 4PMM (Presented By: Luiz Silva, Jr., MD)

This study aimed at determining mutations in the hemochromatosis gene in a Northeastern Brazilian population. At first, six hundred patients with high serum ferritin or high transferrin saturation, all of them examined by the same physician, were studied in a longitudinal investigation developed from December 1996 to December 2008. All patients went under laboratory screening: Hemogram, Platelets count, Glicose, Glicosilated Haemoglobin, Frutosamin, C peptid, anti nuclear anti body test, LE cell, Erythrocyte sedimentation rate, Alanine Aminotransferase, Aspartate Amino Transferase, Alkaline Phosphatase, Gamma-glutamyl transferase, Lactic acid dehydrogenase, Billirrubin, Total Protein and Albumin, liver sonography, screening for B and C hepatites, and schistosomiases, serum iron level, transferrin saturation and ferritin and genetic test to identify mutation for the haemochromatosis gene both C282 and H63D. Of the six hundred initials patients, 65 (10,83%) went out of the survey according the following causes: 29 C hepatite, 11 got anemia with the phlebotomies,11 schistosomiasis, 4 Sistemic Eritematous Lupus, 2 B hepatite, 2 alcoolism,and aplastic anemia, mielofibrosis, thalassemia, sickle cell disease, mielodisplasia and on chemotherapy one of each, then we became with 535 patients. In the next step of the research, 74,39% (398/535) were evaluated for the presence of C282 and H63D mutation of the Hemochromatosis gene. This analysis procedure showed that 52,9% (208/393) of the selected sample didn´t present any mutation, while 47,1% (185/393) were reported with one or both of the above mentioned mutations. Among the latter subjects who presented mutations, the following results were documented: 27,03% (50 out of 185) were positive for the C282Y, whilst 62% (151 out of 185) showed positivity for the H63D mutation. In the positive C282Y group, 86% (43 out of 50) were identified as heterozigotic and 14% (7 out of 50) as homozigotic. The subjects who showed positivity for the H63D mutation were mainly heterozigotic, 86.09% (130 out of 151), while the homozigotic group answered for 13,91 % (21 out of 151) of the sample. From the one hundred and eighty-five patients analysed, sixteen of them were identified as compound heterozigous. When we compare our results with some other publications (see tabel 1), we didn’t find important diferences, and them probably are because diferents genetical constitution of the populations studied, ours being formed with great influence of African descendents. (Tabel 1, yellow columm)

Table 01 – Prevalence of mutations of the hemochromatosis gene observed by differents authors in different Countries.

Poster# 215 IRON CHELATION IN PATIENTS WITH THALASSEMIA INTERMEDIA AND OTHER NON TRANSFUSION DEPENDENT CONGENITAL HEMOLYTIC ANEMIAS Alexandra Pereira, MD¹, Maria Pedro Silveira, MD² and Maria Leticia Ribeiro, MD, PhD³ ¹Departamento de Hematologia, Centro Hospitalar de Coimbra; ²Serviço de Hematologia, Centro Hospitalar de Lisboa Ocidental, E.P.E, Portugal; ³hematologia, Centro Hospitalar de Coimbra, E.P.E., Portugal (Presented By: Alexandra Pereira, MD)

Iron overload is one of the main causes of morbimortality in patients with congenital anaemias. Erythroid hyperplasia and chronic tissue hypoxia increase iron absorption in non transfusion dependent anaemic patients, most of them requiring iron overload monitoring and, eventually, iron chelation. Cardiac disease and pulmonary hypertension are the leading cause of death among these patients, nevertheless myocardial iron is not usually significantly increased. The aim of this study was to analyse iron chelation data on 22 non transfusion dependent patients with congenital anaemias.

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In a group of 22 patients (18 β-thalassemia intermedia, 2 piruvate kinase deficiency, 1 pyrimidine 5’ nucleotidase deficiency and 1 hereditary xerocytosis), ALT, AST, serum ferritin level and liver iron concentration (LIC) by MRI were determined before and 42 ± 5 weeks after iron chelation therapy. Cardiac iron overload was evaluated by MRI T2* only at the end of the observational study (Table I). All the patients had normal liver enzymes, except one with active hepatitis C, and most of them presented high initial serum ferritin levels and significant hepatic iron overload. Patients with good therapeutical compliance had a very significant serum ferritin decrease, with a less significant decrease on LIC. There was no consistent correlation between serum ferritin levels and LIC especially after iron chelation. Myocardial T2* was above 20 ms (normal) in all the patients. In conclusion: according to the present data, iron overload in patients with non transfusion dependent congenital anaemias may be underestimated by serum ferritin levels alone. Assessment of LIC in these patients may establish the need for iron chelation to prevent liver dysfunction, and to monitor the treatment.

Poster# 216 HYPERFERRITINEMIA CATARACT SYNDROME, IS THERE A RENAL COMPONENT? Samuel Krikler, MBChB, FRCPC, Chris Whittington, MBBS, Anthony Vivian, MBBS and Daniel Schwartz, MD University of British Columbia (Presented By: Samuel Krikler, MBChB, FRCPC)

Introduction: Hyperferritinemia cataract syndrome is a rare autosomal dominant disorder due to mutations of the iron responsive element of the L-ferritin mRNA. These patients have not been previously reported as having any renal compromise. Methods: A 57 year old adopted, non drinking white male presented for investigation of right upper quadrant pain. The patient had a BMI of 32, slightly increased cholesterol level and mild hypertension. On presentation his ferritin level was 1854 ug/L and his transferrin saturation (TS) 26%. The patient had a normal complete blood count with a hemoglobin of 158 g/L. He was investigated for iron overload and phlebotomized 5 times. DNA was analyzed for hyperferritinemia cataract syndrome after the patient reported bilateral cataract extraction at age 42 years. Results Obtained: The patient had a 2 finger breadth palpable liver and a fatty liver was shown on ultrasound. Liver function tests were normal. Hepatitis serology was negative and colonoscopy was normal. He was wild type/wild type for the HFE mutation and had normal urinary porphyrin excretion. At initial investigation he was shown to have a borderline creatinine level of 102 umol/L (normal 45 – 110 umol/L). Differential diagnoses of dysmetabolic hepatosiderosis or ferroportin disease were made. Two phlebotomies initially decreased the ferritin level to 1497 ug/L, but the ferritin level rose to 1749 ug/L after five phlebotomies and the TS fell to 9% and hemoglobin to 134 g/L. At the same time the creatinine increased to 127 umol/L. Urinalysis was negative for blood and protein but microscopy showed unusual deeply pigmented casts. Subsequent testing of the urine was positive for hemosiderin. Calcium/phosphate profile and myeloma screen were negative. One year after the last phlebotomy the patient’s creatinine level had dropped to 106 umol/L with no hemosiderin detected in the urine. The TS had risen to 28% and the ferritin level had risen to over 2000 ug/L. DNA analysis showed the patient to have the G43A variant of hyperferritinemia cataract syndrome. Discussion: Hyperferritinemia cataract syndrome is frequently initially misdiagnosed and patients may progress to liver biopsy or marrow aspiration before the diagnosis is considered. Phlebotomy may cause severe anemia. Here the patient presented as a possible dysmetabolic hepatosiderosis, though the ferritin level seemed too high. Ferroportin disease was also a possibility. The clinical scenario of anemia with a rising ferritin level suggested another diagnosis. The renal deterioration was pursued in view of the initial “borderline” serum creatinine. Referral to a nephrologist elicited the unidentified heavily pigmented casts. Urine testing that was initially positive for hemosiderin (and subsequently cleared with cessation of phlebotomies) suggested a possible renal excretion of abnormal ferritin elements. Renal biopsy was deferred as the patient’s renal function improved after cessation of phlebotomies. Conclusions: Hyperferritinemia cataract syndrome may have a renal component.

Poster# 217 HEREDITARY HEMOCROMATOSIS AND DIABETES MELLITUS (DM), CAUSE OR COINCIDENCE? A NORTHEASTERN BRAZILIAN EXPERIENCE Luiz Silva, Jr., MD¹, Andre Silva, MD², Adavio Silva, MD³ and Jairo Cavalcante, Statistical4 ¹Souza Junior Laboratorio Medico; ²Hospital A.C. Camargo; ³Hospital da Beneficiencia Portuguesa; 4PMM (Presented By: Luiz Silva, Jr., MD)

This study aimed at avaluate the incidence of Diabetes Mellitus among hemochromatosis patients in a Northeastern Brazilian population. At first, six hundred patients with high serum ferritin or high transferrin saturation, all of them examined by the same physician, were studied in a longitudinal investigation developed from December 1996 to December 2008. All patients went under laboratory screening: Hemogram, Platelets count, Glicose, Glicosilated Haemoglobin, Frutosamin, C peptid, anti nuclear anti body test, LE cell, Erythrocyte sedimentation rate, Alanine Aminotransferase, Aspartate Amino Transferase, Alkaline Phosphatase , Gamma-glutamyl transferase, Lactic acid dehydrogenase, Billirrubin, Total Protein and Albumin, liver sonography, screening for B and C hepatites, and schistosomiases, serum iron level, transferrin saturation and ferritin and genetic test to identify mutation for the haemochromatosis gene both C282 and H63D. Of the six hundred initials patients, 65 (10,83%) went out of the survey according the following causes: 29 C hepatite, 11 got anemia with the phlebotomies,11 schistosomiasis, 4 Sistemic Eritematous Lupus, 2 B hepatite, 2 alcoolism,and aplastic anemia, mielofibrosis, thalassemia, sickle cell disease, mielodisplasia and on chemotherapy one of each, then we became with 535 patients. In the next step of the research, 74,39% (398/535) were evaluated for the presence of C282 and H63D mutation of the Hemochromatosis gene. This analysis procedure showed that 52,9% (208/393) of the selected sample didn´t present any mutation, while 47,1% (185/393) were reported with one or both of the above mentioned mutations. Among the latter subjects who presented mutations, the following results were documented: 27,03% (50 out of 185) were positive for the C282Y, whilst 62% (151 out of 185) showed positivity for the H63D mutation. In the positive C282Y group, 86% (43 out of 50) were identified as heterozigotic and 14% (7 out of 50) as homozigotic. The subjects who showed positivity for the H63D mutation were mainly heterozigotic, 86.09% (130 out of 151), while the homozigotic group answered for 13,91 % (21 out of 151) of the sample. From the one hundred and eighty-five patients analysed, sixteen of them were identified as compound heterozigous. The patients who showed fast Glicemy above 95 mg/dl, were submitted to Intolerance test for glicose, and then classified according the criterias for diabetes mellitus of the American Diabetes Association. When we stratified our patients into: Heterozigous ou Homozigous for the C282Y mutation, Hetero or Homozigous for the H63D mutation, and compound heterozigous, no difference of incidence of DM in the different groups. When we compare the levels of ferritin, Transferrin Saturation (TS), we coludn´t find differences of incidence in Diabetics and Non Diabetics patients. In the same way, no differences among patients related to sex or age. In conclusion: in our studied population there is not elevated incidence of Diabetes Mellitus. 166

Poster# 218 PILOT PHARMACOKINETIC STUDY IN PATIENTS WITH INADEQUATE RESPONSE TO DEFERASIROX (EXJADE) S. Deborah Chirnomas, MD¹, Amber Smith, BA², Luis Pereira, PhD³, Jennifer Braunstein, PNP4, Carole Paley, MD5 and Ellis Neufeld, MD, PhD² ¹Children’s Hospital Boston; ²Children’s Hospital Boston/Hematology/Oncology; ³Children’s Hospital, Boston/Clinical Pharmacology Unit; 4Children’s Hospital Boston/Hematology/Oncology; 5Novartis Pharmaceuticals (Presented By: S. Deborah Chirnomas, MD)

Background: Iron chelation is essential for patients with transfusion-dependent anemias to prevent fatal iron overload. The oral chelator, deferasirox (DFX), available since 2005, is potentially a major advance for patients, but a large hurdle is the variability in response. On DFX as many as 30% of patients have rising serum ferritin and iron burden assessed by liver biopsy or R2 MRI (Ferriscan) measurement. This is a serious problem for those patients with few or no alternatives for therapy. Objective: To investigate the mechanism of variable DFX response by obtaining pharmacokinetic (PK) data in patients with good versus poor response. Design / Methods: A prospective study of ten patients with poor response to DFX and five with good response was approved by the IRB. After a 48-hour washout period, patients were admitted to the Children response to DFX is distinguishable by measuring exposure. As this is not a function of Vd and elimination, this is likely due to bioavailability. The UGT1A1 analysis was not statistically significant but suggests a trend that should be studied further along with pharmacogenomic differences in absorption of DFX. In addition, effective dosing options for poorly responding patients must be determined.

Poster# 219 MITOCHONDRIAL DYSFUNCTION MAY EXPLAIN THE CARDIOMYOPATHY OF CHRONIC IRON OVERLOAD John Eaton, PhD, MDhc University of Louisville (Presented By: John Eaton, PhD, MDhc)

Chronic iron overload, whether congenital or acquired, frequently leads to progressive damage of organs such as liver, pancreas and heart. In the specific case of iron- mediated cardiomyopathy, cardiac dysfunction may appear years after patients have achieved a state of iron overload. We earlier suggested (Gao et al., J. Biol. Chem. 284:4767, 2009) that this slow progression implies some sort of biologic ‘memory’, perhaps in the form of progressive mitochondrial dysfunction. The heart has highly active mitochondria that incidentally generate reactive oxygen species capable of causing synergistic toxicity with intracellular iron. In the present investigations, we have tested in vivo the idea that progressive iron-catalyzed oxidant damage to mitochondrial DNA (mtDNA) might explain the often lethal cardiac dysfunction in chronic iron overload. Mice (male B6D2F1, 6 weeks of age) were given repetitive intraperitoneal injections of iron dextran (10 mg elemental Fe/day/mouse) 5 days per week for a total of 4 weeks. At the end of this time, the iron loaded mice had modest cardiac hypertrophy and cardiac dysfunction. qPCR amplification of near-full-length (15.9 kb) mtDNA revealed >50% loss of full length product whereas amounts of a qPCR product of a nuclear gene (13 kb region of beta globin) were unaffected. Quantitative rtPCR analyses revealed 60-70% loss of mRNA for proteins encoded by mtDNA - 12S rRNA, cytochrome c oxidase subunit 1, and NADH dehydrogenase subunits 4 and 6 (light chain) in both heart and liver - with no change in mRNA abundance for nuclear encoded respiratory subunits. These changes coincided with decreased respiration of isolated cardiac mitochondria. We conclude that chronic iron overload leads to cumulative iron-mediated damage to mtDNA and impaired synthesis of mitochondrial respiratory chain subunits. The resulting respiratory dysfunction may explain the slow development of cardiomyopathy in chronic iron overload and similar accumulation of damage to mtDNA may also explain the mitochondrial dysfunction observed in slowly progressing disorders such as neurodegenerative diseases.

Poster# 220 THE IRON CHELATOR DEXRAZOXANE PROTECTS CARDIOMYOCYTES FROM DOXORUBICIN-MEDIATED CELL DEATH: ROLE OF HIF-1 Lorenza Tacchini, PhD, Stefania Recalcati, MD, PhD, Rosalin Spagnuolo, PhD and Gaetano Cairo, PhD University of Milan (Presented By: Gaetano Cairo, PhD)

Iron aggravates the cardiotoxicity of doxorubicin (DOX), a widely used anticancer anthracycline. Indeed, dexrazoxane(DRZ) is the only agent clearly able to protectthe myocardium from anthracycline-induced toxicity both in experimentaland clinical settings. Iron has been proposed to catalyse ROS formation in reactions primed by DOX. However, the concept of the oxidative nature of the role of iron in cardiotoxicity is challenged by results showing that antioxidants do not always protect laboratory animals against cardiotoxicity, or mitigate cardiotoxicity in patients. It clearly remains to be established what the precise mechanism of DOX-mediated cardiotoxicity is, and how it may be prevented by the use of iron chelators, such as DRZ. The hypoxia inducible factor (HIF-1) is a transcription factor activated in response to reduced oxygen levels, which regulates the expression of a variety of genes mediating adaptive responses to lack of oxygen. HIF-1 is a heterodimer of the regulated HIF-1 alpha subunit and the constitutively expressed HIF-1 beta subunit. Iron is required for HIF-1 degradation and therefore decreased iron availability activates HIF-1 in normoxic cells. In consideration of the antiapoptotic role of some HIF-1 regulated genes and of the general protective role of HIF-1 activation against a number of different insults, in this study we tested the hypothesis that iron chelation by DRZ may result in HIF-1 activation and play a role in the protective effect of DRZ from DOX cardiotoxicity. We found a strong dose-dependent induction of HIF-1 protein levels in the H9c2 cardiomyocyte cell line exposed to DRZ. Treatment with DRZ also increased the capacity of HIF-1 to activate the transcription of a reporter luciferase gene under the control of hypoxia responsive elements (HRE). The transactivation capacity of HIF-1 was almost completely abolished by the transfection of a dominant negative of the constitutive beta subunit (DARNT). DRZ also significantly prevented the induction of cell death and apoptosis caused by the exposure of H9c2 cells to concentrations of DOX within the range of plasma levels found in patients undergoing chemotherapy. Genetic suppression of HIF-1 activity by means of transfection of DARNT showed that the protective effect of DRZ was strongly dependent on HIF-1 activity. Conversely, HIF-1 activation provided cardioprotection from DOX also in cells not exposed to iron chelators. In fact, transfection with an expression vector coding for HIF-1 resulted in HIF-1 overexpression and great stimulation of HRE-dependent transcription, and was able to provide significant cytoprotection from DOX-mediated cell death and apoptosis. Finally, we examined the expression of HIF-1 target genes with a possible role in cell survival in DRZ-treated H9c2 cells and we found a strong increase in the levels of Mcl1, an antiapoptotic member of the BCL2 gene family. In summary, our results showing HIF-1-dependent prevention of DOX toxicity in DRZ-treated H9c2 cardiomyocytes suggest that HIF-1 activation may be a mechanism contributing to the protective effect of DRZ against anthracycline cardiotoxicity.

Poster# 221 INCREASED IRON ABSORPTION IS ASSOCIATED WITH UPREGULATION OF DUODENAL DMT1, DCYTB AND HEPHAESTIN EXPRESSION IN PATIENTS WITH NONALCOHOLIC STEATOHEPATITIS Masayoshi Kobune, MD, and PhD, Kohichi Takada, MD, PhD, Yutaka Kawano, MD, PhD, Koji Miyanishi, MD, PhD, Hiroyuki Nagahima, MD, PhD, Rishu Takimoto, MD, PhD, Shohei Kikuchi, MD, Tsutomu Sato, MD, PhD, Yasushi Sato, MD, PhD, Yoshiro Niitsu, MD, PhD and Junji Kato, MD, PhD Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine (Presented By: Masayoshi Kobune, MD)

It has been recently revealed that iron accumulation is attributable to liver injury and hepatic fibrosis in nonalcoholic steatohepatitis (NASH). However, the precise mechanism of iron accumulation in the NASH liver remains to be clarified. In this study, we evaluated iron absorption from the gastrointestinal tract in NASH cases, and further examined expressions of a panel of molecules in association with iron absorption in the duodenum and hepatic hepcidin to gain insight into the mechanism for iron accumulation in the NASH liver. In addition, oxidative DNA stress due to excessive iron was examined. First, it was revealed that absorption of iron from the gastrointestinal tract significantly increased in NASH patients (n = 22), as compared with healthy subjects (p = 0.034). Next, a significant increase in mRNA of DMT1 (patients vs. healthy subjects; 25.33 [0.372 - 492.06] vs. 0.78 [0.54 - 2.67]), Dcytb (16.51 [1.62 - 182.22] vs. 0.71 [0.38 - 2.64]), and hephaestin (2.99 [0.45 - 9.90] vs. 1.02 [0.82 - 3.25]) was demonstrated (p = 0.004, 0.001, and 0.004, respectively). In addition, hepatic hepcidin mRNA expression increased in NASH patients (p = 0.043). Moreover, hepatic 8-OH-dG levels significantly increased (3.26 [1.23 - 5.99] vs. 1.02 [0.99 - 1.12], p = 0.001), and showed a positive correlation with the hepatic iron score 167

(r = 0.736 and p = 0.001). Furthermore, one year after iron depletion therapy by phlebotomy and low iron diet, elevated hepatic 8-OH-dG levels in NASH significantly decreased to nearly normal levels. In conclusion, an increase in the expressions of DMT1, Dcytb, and hephaestin in the duodenum could stimulate iron absorption from the gastrointestinal tract, leading to induce excessive iron accumulation in the liver.

Poster# 222 THE COMPARATIVE ANALYSES OF SINGLE NUCLEOTIDE POLYMORPHISM OF OXIDATIVE DNA REPAIR GENES IN PATIENTS WITH HCV- RELATED LIVER DISEASE Koji Miyanishi, MD, and PhD, Masayoshi Kobune, MD, PhD, Yutaka Kawano, MD, PhD, Hiroyuki Nagahima, MD, PhD, Shohei Kikuchi, MD, Kohichi Takada, MD, PhD, Rishu Takimoto, MD, PhD, Tsutomu Sato, MD, PhD, Yasushi Sato, MD, PhD, Yoshiro Niitsu, MD, PhD and Junji Kato, MD, PhD Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine (Presented By: Koji Miyanishi, MD)

Recent studies have shown that excess hepatic iron accumulation contributes to liver injury in chronic hepatitis C (CHC) patients. Free iron in the liver is believed to facilitate the formation of reactive oxygen species (ROS), including hydroxyl radicals, which cause oxidative damage of numerous cellular components such as lipids, proteins, and nucleic acids, and also upregulate collagen synthesis. Further, the hydroxyl radical is known to generate promutagenic bases such as 8-hydroxy-2- deoxyguanosine (8-OHdG), which has been implicated in DNA mutagenesis and carcinogenesis. In general, 8- OHdG lesion can be promptly repaired by human 8-oxo- guanine DNA glycosylase (hOGG1) and human MutY homolog (MutYH). Therefore, it is plausible that the development of hepatocellular carcinoma (HCC) from CHC is determined by the balance between DNA damage and repair. In this study, we conducted comparative analyses of single nucleotide polymorphisms (SNPs) in hOGG1 and MutYH genes on patient of CHC with or without development of HCC. Genomic DNA and total RNA were extracted from peripheral-blood mononuclear cells obtained from 57 patients of CHC without HCC (non-HCC group) and 27 patients with HCC (HCC group). We analyzed the several SNPs and mRNA of hOGG1 and MutYH by direct sequence and RT-PCR. The frequency of SNPs in hOGG1 did not differ between HCC group and non-HCC group. Interestingly, in one of MutYH SNPs (under patent application), the frequency of minor homo or hetero allele carrier in the HCC group significantly higher than that in HCC group. In addition, the expression of MutYH mRNA in minor homo allele tended to be lower than that in major homo allele. These results indicated that the minor hetero allele of this SNP in MutYH could be a significant risk factor of liver carcinogenesis from CHC and could be a predictive marker of HCC development in CHC patients.

Poster# 223 THE CLINICAL AND GENETIC SPECTRUM OF TMPRSS6 MUTATIONS LEADING TO INAPPROPRIATE HEPCIDIN EXPRESSION AND IRON REFRACTORY IRON DEFICIENCY ANEMIA (IRIDA) Matthew Heeney, MD, Dean Campagna and Mark Fleming, MD, DPhil Children’s Hospital Boston (Presented By: Matthew Heeney, MD)

Introduction: The peptide hormone hepcidin is a negative regulator of iron efflux from cells. In iron deficiency anemia (IDA) hepcidin levels are ordinarily undetectable, thereby stimulating the release of iron from macrophages, hepatocytes and duodenal enterocytes, promoting iron availability for erythropoieisis and increasing iron stores. Rare individuals and kindreds with lifelong hypoferremia, microcytic red blood cells and IDA unresponsive to enteral iron therapy and only an incomplete, transient response to parenteral iron supplementation — so-called iron refractory iron deficiency anemia — have been described. We, and others, previously reported that mutations in the hepatocyte-specific transmembrane serine protease TMPRSS6 cause IRIDA and that the physiology of the phenotype can be attributed to inappropriately elevated hepcidin levels. Here, we extend our description of the homozygous TMPRSS6-mutated IRIDA phenotype, identify disease-causing mutations in an additional probands, including the original family reported by Buchanan and Sheehan (J Pediatr, 1981, 98:72) and describe the phenotype of heterozygous TMPRSS6 deficiency. Methods: Individuals and kindreds with IRIDA were recruited in an IRB approved protocol and assessed for hematologic and biochemical markers of iron status, inflammation, urinary and plasma hepcidin and TMPRSS6 sequencing by previously described methods. Given that the transferrin saturation (TfSat) appears to have a central role in hepcidin regulation, an index relating the hepcidin to transferrin saturation, TfSat/log10hepcidin, was evaluated to attempt to distinguish inappropriate from appropriate hepcidin expression in probands and their family members. Results: Five additional probands that carried 7 novel and 2 recurrent mutations in TMPRSS6 were identified. Each of these patients had elevated absolute urinary and plasma hepcidin concentrations that were profoundly elevated compared to familial wild type controls when normalized for serum transferrin saturation. Family members heterozygous for TMPRSS6 mutations had normal hematological and biochemical iron studies, but had an intermediate TfSat/log10hepcidin, indicative of a codominant phenotype. Several other individuals referred for a clinically milder IRIDA phenotype were found to have heterozygous mutations, suggesting that some heterozygotes may manifest clinically apparent disease. In addition, several individuals with a severe IRIDA phenotype were found to have high hepcidins in the absence of evidence of inflammation and TMPRSS6 mutations, raising the possibility that IRIDA is a heterogeneous disorder. Discussion / Conclusions: TMPRSS6 mutations leading to inappropriately elevated hepcidin levels explain many, but not all, patients with the IRIDA phenotype. We add seven new mutations to the previous sixteen described mutations in TMPRSS6. The TfSat/log10hepcidin can assist in distinguishing those with iron deficiency from those with TMPRSS6 mutations.

Poster# 224 GENOME-WIDE ASSOCIATION STUDY OF IRON DEFICIENCY C.E. McLaren, PhD¹, C.P. Garner, PhD¹, C.C. Constantine, PhD², S. Masle, PhD4, C.D. Vulpe, MD, PhD4, B.M. Snively, PhD¹³, D.A. Nickerson, PhD¹¹, J.D. Cook, MD¹², C. Leiendecker-Foster, MS5, K.B. Beckman, MS5, J.H. Eckfeldt, MD5, V.R. Gordeuk, MD³, L.F. Barcellos, PhD4, A. Killeen, MD5, J.A. Murray, MD6, P.C. Adams, MD7, R.T. Acton, PhD8, D.M. Reboussin, PhD¹³, K. Goddard, PhD9 and G.D. McLaren, MD¹° ¹University of California, Irvine; ²University of Melbourne, Australia; ³Howard University; 4University of California, Berkeley; 5University of Minnesota; 6Mayo Clinic; 7London Health Sciences Centre, London, Ontario; 8University of Alabama at Birmingham; 9Kaiser Permanente Northwest; ¹°Univ. of California, Irvine, and Department of Veterans Affairs Medical Center, Long Beach, CA; ¹¹University of Washington; ¹²University of Kansas; ¹³Wake Forest University School of Medicine (Presented By: C.E. McLaren, PhD)

Iron deficiency is the most common nutritional disorder in the world with an estimated 4−5 billion affected persons. Although commonly considered environmental in origin, the existence of multiple genetic disorders of iron metabolism in man, rodents and other vertebrates suggest a genetic contribution to iron deficiency. The Hemochromatosis and Iron Overload Screening (HEIRS) Study is a multi−center, multi−ethnic study in which transferrin saturation (TS), serum ferritin (SF), and HFE mutations were determined in 101,168 adults. To identify genomic locations associated with iron deficiency, we performed a genome−wide association study (GWAS) using DNA collected from Caucasian HEIRS Study participants who had SF < 12 µg/L (cases) and an equal number of Caucasian controls (SF>100 µg/L in men, SF>50 µg/L in women. Men aged > 25 y and women > 50 y were included in both groups. Controls were frequency−matched to cases by sex and geographic location. Body iron, an index of iron deficiency, was estimated from serum transferrin receptor (sTfR) and SF. Genotyping was performed with the Illumina HumanCNV370K Beadchip platform. Quality control filters excluded single nucleotide polymorphisms (SNPs) or samples with >5% missing genotypes, SNPs showing heterozygosity or Hardy- Weinberg deviations (P<10−7), and SNPs with minor allele frequency < 0.02. Concordance between genotypes in quality control duplicate samples exceeded 99%. Population admixture/structure was assessed using principal component analysis. Average identity-by-state was computed for all sample pairs to identify duplicate samples or related individuals. The genomic control parameter was not significantly different from 1.0. There were 392 cases (96 men) and 390 controls (96 men) with average age (SD) of 59 (10) y and 61 (11) y, respectively. Geometric mean SF (minimum, maximum), and mean (SD) for sTfR and body iron index were 7.5 (1.2, 12) µg/L, 6.4 (3.77) mg/kg and −2.0 (2.50) for cases and 141 (51, 881) µg/L, 3.0 (0.98) mg/kg and 10.8 (2.5) for controls. After quality control tests, GWAS analysis included genotype data for 331,060 SNPs in 736 individuals (365 cases, 371 controls). Logistic regression was used for the association analysis of case−control status with each SNP genotype variable; covariates included age, sex, and geographic location. Twenty−nine SNPs distributed across 21 unique regions showed evidence for association at P<0.0001. Two SNPs on chromosome 22q11.22 and two SNPs on chromosome 8q24.12 were significant at P < 0.00001. The SNP rs987710 on chromosome 22q11.22 showed the most 168 significant association (P=6.4x10-08) with a Bonferroni−corrected genome−wide P−value of 0.02. Iron deficient cases showed a reduced minor allele frequency at rs987710, compared to the controls (0.25 vs. 0.38; odds ratio = 0.53); the frequency is 0.33 in HapMap samples of European ancestry. The most significant SNP in a recent GWAS of transferrin was rs3811647 in the TF gene. Secondary analyses of our GWAS data identified this SNP as the most significantly associated with total iron binding capacity (P=1.6x10-08). From this initial GWAS, we have identified genetic loci associated with iron deficiency and iron phenotype variability. Replication studies are underway.

Poster# 225 IRON DEFICIENCY DETECTED BY ESTIMATION OF BODY IRON IN A PRIMARY CARE POPULATION: PREVALENCE AND ETIOLOGY Gordon D. McLaren, MD¹, Joanna A. Kolodney, MD¹, Naveen Doki, MD¹, Wen-Pin Chen, MS¹, Sakineh Khalaghizadeh, BS¹, Mahmood Novin, MD¹, James D. Cook, MD², Mazen Jamal, MD, MPH¹ and Christine E. McLaren, PhD¹ ¹Dept. of Veterans Affairs Long Beach Healthcare System, Long Beach, and University of California, Irvine, CA; ²University of Kansas Medical Center, Kansas City, KS, USA (Presented By: Gordon D. McLaren, MD)

Iron deficiency (ID), although prevalent in women, is considered a relatively uncommon disorder among men and usually is associated with blood loss. We screened veterans attending primary care clinics at a VA medical center by measuring serum transferrin receptor (TfR) and serum ferritin (SF) concentrations. Body iron (Fe) was assessed from the TfR/SF ratio calibrated by quantitative phlebotomy. In this approach, body Fe is expressed as a positive value when stores are present and negatively with tissue Fe deficiency. A body Fe < -4 mg/kg body weight represents a deficit severe enough to produce anemia. A total of 2,225 participants, 2,099 male (M) and 126 female (F) were enrolled. ID was detected in 93 participants (4.2%; 77 M and 16 F, ages 35-88 yr, median 65.3 yr) based either on a negative body iron value or SF < 12 µg/L, indicating total depletion of iron stores. Of these, 79 (3.6%) had a negative body iron value (68 M and 11 F, ages 38-88, median 65.9 yr). The diagnosis of ID was not previously suspected in the majority of cases (N=62, 78%). A likely etiology of ID was apparent in only 15 (19%), including 10 with blood loss and 5 with probable malabsorption. The average tissue Fe deficit in men was -2.2 mg/kg (range –7.9 mg/kg to –0.01 mg/kg). Only 30 (44%) of the men with a negative body iron were anemic, based on blood hemoglobin (Hb) concentration. Participants not having an apparent etiology underwent further evaluation, including upper and lower gastrointestinal (GI) endoscopy and testing for Helicobacter pylori antibody (ab), anti-gliadin ab, and anti-parietal cell ab. Participants whose ID had spontaneously resolved or who refused to undergo further testing, or who subsequently had died or were lost to follow-up, were excluded. After completion of evaluation, 44% of the remaining cases had an established etiology for ID. Of these, 38% had a GI source, the most prevalent etiologies being blood loss or malabsorption associated with H. pylori (24%); other diagnoses included ulcerative colitis, colon cancer, Barrett’s esophagus, and bleeding arterio-venous malformations. In a few cases (7%), there were other forms of blood loss, such as bladder cancer, radiation proctitis, or a history of phlebotomy for polycythemia vera. Among the small number of women in this cohort, other causes of ID included post- menopausal bleeding or dysfunctional uterine bleeding. The results indicate an unexpectedly high prevalence of ID in this predominantly adult male population and demonstrate that measurement of Hb fails to detect many cases. Further evaluation often identifies serious GI lesions. Thus, assessment of iron status is important to identify persons with ID who require diagnostic evaluation to determine the etiology.

Poster# 226 IRON UTILISATION FROM ORAL IRON TREATMENT IS DEPENDENTD FROM THE INDIVIDUAL BLEEDING RATE IN PATIENTS WITH HYPERMENORRHEA OR GASTROINTESTINAL HEMORRHAGE Peter Nielsen, Rosemarie Kongi and Roland Fischer, PhD University Hospital, Hamburg (Presented By: Peter Nielsen)

Iron deficiency (ID) affects more than 3.5 billion people in the developing world, stealing health and vitality from the young and old and impairing the cognitive development of children. In developed countries, iron deficiency anaemia (IDA) is found mainly in certain risk groups with increased demand for iron (growing children, menstruating women) or is a clinical symptom for pathological blood loss (hypermenorrhea or gastrointestinal haemorrhage). In our diagnostic centre we use routinely 59Fe and whole body counting for diagnosing the individual cause of iron deficiency in patients with unclear IDA. Parallel to the diagnostic workup, oral iron therapy with several commercial available iron drugs were introduced. In a series of 108 patients with IDA, the daily iron utilisation, (dIU), was calculated from the daily iron loss (IL), measured by whole body counting, corrected for the haemoglobin change DHb in a treatment interval dt of 6-10 weeks by dIU=IL + dHb/ Dt • FeHb • f • BV, (FeHb=3.47 mg-Fe/g-Hb, f= correction factor venous blood/total blood = 0.913). Most of the patients (95/108) suffered from ongoing blood loss due to hypermenorrhea (n=39) or gastrointestinal haemorrhage (n=56). In non-bleeding patients, a “basal iron utilisation” of 5-10 mg is provided (see figure). With increasing blood loos, more iron can be used and the oral iron treatment with a daily single dose of 100 mg could clearly compensate for the blood loss in almost all patients. This study documents a self-regulation of the oral iron therapy in subjects with chronic blood loss according to the demand of iron. Using a good iron drug, oral iron therapy is effective and safe and should remain the first line treatment in iron deficiency. Figure1: Daily iron utilisation from oral iron medication (100 mg/d) in bleeding patients with iron deficiency anaemia (crosses, gastrointestinal haemorrhage; circles, hypermenorrhea; line, linear regression). Three different iron preparations with known high bioavailability were used.

Poster# 227 THE MENKES COPPER ATPASE (ATP7A) GENE ENCODES TWO NUCLEAR PROTEIN VARIANTS THAT INTERACT WITH DNA AND ARE INDUCED DURING IRON DEFICIENCY James Collins, PhD, Yan Lu, BS and P.N. Ranganathan, PhD University of Florida (Presented By: James Collins, PhD)

The Menkes Copper Atpase (Atp7a) is strongly induced in the intestine of iron deficient rats. We surmised that this induction was part of the transcriptional response to iron deprivation, intended to increase copper absorption which in turn could function to enhance absorption of dietary iron. During our efforts to determine the molecular mechanism of Atp7a induction, we discovered alternative splicing at 5’ end of the mRNA molecule, which would produce 2 protein variants; two transcripts would encode the full length protein and one transcript would encode a truncated protein missing the first copper-binding domain. qRT-PCR experiments showed that all splice variants were induced ~7-fold in the duodenum of iron deficient rats. We then developed antibodies against the N-terminus of the protein (which would be present in one protein variant and missing in the other) to test the hypothesis that different Atp7a protein variants existed. The antibodies were validated and utilized in combination with a 169 previously validated Atp7a-specific antiserum to demonstrate that indeed different protein variants existed, but unexpectedly, we discovered 2 variants that localized to the nucleus in rat (IEC-6) and human intestinal epithelial cells (Caco-2), and in rat intestine. The purpose of the current studies was thus to determine if the nuclear protein variants interacted with DNA and to biochemically characterize such interactions, if detected. By immunoblotting, the nuclear protein variants were ~97 and 64 kDa, as compared to the predicted full length protein of >180 kDa. As Atp7a is known to be a membrane protein, we performed experiments to address this issue. Our data suggested that the nuclear protein variants however were soluble proteins. Both nuclear proteins could be pulled down with a cellulose resin containing calf thymus DNA. The specificity of this reaction was assessed by competing the binding reaction with single stranded, random 25mers, calf thymus and salmon sperm DNA, restriction digested calf thymus and salmon sperm DNA, plasmid DNA, and RNA. Competition was observed only with calf thymus DNA (uncut and cut), suggesting that the interaction was specific and perhaps also sequence specific as salmon sperm DNA and bacterial plasmid DNA did not compete. We further sought to determine the biochemical nature of the DNA protein interaction so as to be able to predict the possible function of the proteins. We thus eluted the DNA resin-protein complex with various chemical compounds/molecules which would interfere with electrostatic interactions (high salt), hydrophobic forces (detergents), and hydrogen binding (urea). We consistently found that only urea was able to disrupt the DNA resin-protein interaction suggesting that hydrogen bonding played a predominant role. Interestingly, hydrogen bonding forces are what give most transcription factors their DNA sequencespecific binding properties. We have several lines of experimental evidence suggesting that these proteins are indeed Atp7a variants (some of which have not been mentioned here). These observations thus lead us to predict that these novel nuclear proteins function as trans-acting factors that could be involved in the genetic response to iron deprivation. Future studies will be designed to explore this possibility and to determine which genes may be regulated, and also to determine whether these proteins could additionally play a role in copper homeostasis.

Poster# 228 POTENTIAL ABNORMALITIES IN IRON PROCESSING BY MUCOLIPIDOSIS TYPE IV PATIENT FIBROBLASTS Chevaun Morrison, BS¹, Eric N. Sauble, MS Chemistry¹, Annie Nguyen, undergraduate¹, Maria C Linder, PhD¹ and Gideon Bach, MD² ¹California State University, Fullerton; ²Hadassah Medical School, Jerusalem (Presented By: Chevaun Morrison, BS)

Mucolipidosis type IV (MLIV) is an autosomal recessive neurodegenerative disorder that results from a mutation in mucolipin 1, a 580 amino acid nonselective cation channel present on lysosomal membranes. This mutation disrupts sorting, transport and/or fusion of endosomes and lysosomes, and subjects suffer from iron deficiency anemia. To establish whether lysosomal turnover of endogenous ferritin was disrupted, fibroblasts from normal and MLIV subjects were pretreated with 59Fe-labeled ferric ammonium citrate (180 uM) for 24 h to produce ferritin and then with desferrioxamine to induce ferritin turnover in lysosomes (Kidane et al, Am J Physiol 291: C445, 2006). Movement of 59Fe-ferritin to lysosomes was followed by separating it from cytoplasmic ferritin on iodixanol gradients, after bomb cavitation disruption of the cells. Ferritin was tracked by ELISA; release of its iron was tracked by radioactivity of 59Fe and by quantitation of total iron using bathophenanthroline. No defects in turnover of endogenous ferritin and its iron were observed. MLIV fibroblasts (and normal fibroblasts) did not take up significant quantities of native, exogenous horse spleen or rat liver ferritin. They did, however, take up exogenous ferritin that had been cationized. Turnover of iron administered through cationized horse spleen ferritin was slowed compared with what occurred in fibroblasts of normal subjects. Also, more iron from exogenous ferritin was held in lysosomes and endosomes in cells from diseased versus normal subjects. Accumulation of cytoplasmic ferritin protein in response to the incoming iron was also reduced. We conclude that a reduced rate of processing of iron entering by endocytosis may slow recycling of red cell iron and contribute to the development of an iron deficiencylike anemia. Supported in part by PHS Grant No. RO1 HD 46949.

Poster# 229 – WITHDRAWN ISOTHERMAL TITRATION CALORIMETRY OF HUMAN SERUM TRANSFERRIN INTERACTION WITH THE SOLUBLE PORTION OF THE RECEPTOR Banu Kandemir, undergraduate¹, Shaina Byrne, PhD student², Anne Mason, PhD², N. Dennis Chasteen, PhD³ and Fadi Bou-Abdallah, PhD¹ ¹State University of New York at Potsdam, NY, USA; ²University of Vermont, VT, USA; ³University of New Hampshire, NH, USA

The transferrins are the major iron binding and transport proteins in vertebrates. They consist of a single polypeptide chain of ~700 amino acid residues organized into two lobes, termed the N- and C-lobe. Each lobe is divided into two subdomains (N1,N2, C1 and C2) forming a cleft in which a single Fe3+ ion binds with an association constant of approximately 1022 M-1. In mammals, serum-transferrins transport iron from the neutral environment of the blood to the cytoplasm by receptor-mediated endocytosis. Diferric transferrin preferentially binds to its specific receptor residing on the membrane of iron requiring cells. Transferrin delivers its iron within the cell stimulated by the acidic environment of the endosome (pH ~ 5.6) and with the participation of the receptor. The transferrin (lacking iron) remains bound to the receptor and is recycled to the cell surface where it is released to repeat the cycle. Much data indicates that both lobes of transferrin are required for high affinity binding to the receptor. Using ITC, we have measured the interaction of different constructs of human serum-transferrin (recombinant apo, monoferric, and diferric forms) with the soluble portion of the receptor at pH 7.4 and at pH 5.6 in order to obtain the complete thermodynamic profile of their interaction. The ITC data indicate that each monoferric transferrin (Fe-loaded N-lobe or Fe-loaded C-lobe) binds to the transferrin receptor with a 1:1 stoichiometry (one transferrin per receptor monomer) and with similar high binding affinity (Kassoc ~ 3 x 107 M-1) confirming that each lobe participates in the association. The fully loaded diferric transferrin also binds to the receptor with a 1:1 stoichiometry but with a 10-fold higher affinity than either monoferric transferrin (Kassoc = 3 x 108 M-1). As expected, although apotransferrin binds with high affinity to the receptor at the acidic pH of the endosome (Kassoc = 1 x 108 M-1) this iron-free protein does not bind to the receptor at physiological pH. ITC data for several constructs of diferric transferrin containing a single mutation in the N2 subdomain (P142A, R143A, K144A, or P145A) indicates that at pH 7.4 all of these residues are involved in the high affinity binding of the N-lobe to the transferrin receptor, a result in accord with a previous suggestion. In all samples, binding of transferrin to the transferrin receptor is both enthalpically and entropically driven resulting in a Gibbs energy of binding (- 42 kJ/mol < ∆Go < - 48 kJ/mol). The large positive change in entropy (∆So = 37 – 85 J/K-mol) suggests a significant loss of water of hydration upon docking of the two proteins.

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Poster# 230 “IN SILICO” MAPPING OF LIVER IRON LEVELS IN INBRED MICE Stela Masle, Seung-Min Lee and Chris Vulpe UC Berkeley (Presented By: Stela Masle)

Disorders of iron metabolism are the most common chronic disorders. Among them, iron deficiency represents the biggest problem. There is a substantial environmental influence in the development of iron deficiency. However, studies in humans and mice provide strong evidence for a genetic contribution to the iron status. Identification of loci involved in iron deficiency in mice will facilitate understanding of iron deficiency in humans and will have important implications for numerous clinical disorders associated with disturbances of iron homeostasis. Large sets of publicly available genotyping data on number of inbred strains made performance of direct association studies on measured phenotypes by “in silico” mapping possible. Recent genome wide association study demonstrated the robustness of this method comparing the results with previously detected quantitative trait loci (QTL) from classical linkage analyses. We determined the liver iron levels in179 mice of 14 inbred strains (~13 animals per strain) on defined iron diets. To identify candidate QTL associated with liver iron levels we carried out an association study with 68,657 informative single nucleotide polymorphisms (SNPs) using efficient Mixed-Model Association (EMMA) method and 137,871 informative SNPs using Haplotype Association Mapping (HAM) method. Both methods correct for population structure present in inbred mouse strains. EMMA corrects for multiple hypothesis testing using conservative Bonferroni approach, while HAM uses weighted permutation. Results from both methods were compared. We found dramatic differences in liver iron levels between strains, varying from 233 ug iron/g dried tissue for NZB/BINJ to 660 ug iron/g dried tissue for AKR/J. QTL analysis identified several regions associated with liver iron levels. The most significant results (-log10 p-value > 2.5 by both methods) include 35 SNPs on seven different chromosomes. However identified regions do not contain genes apparently related to iron metabolism and represent possible “novel” QTL regions influencing iron homeostasis. Further refinement of all identified regions is necessary in order to identify possible candidate genes for further functional analysis.

Poster# 231 A HIGH PREVALENCE OF SUB-CLINICAL IRON DEFICIENCY IN BLOOD DONORS THAT PASSED THE SCREENING TEST FOR HEMOGLOBIN Mireille Baart, MSc, Carine Doggen, PhD, Wim de Kort, PhD and Paulus van Noord, PhD Sanquin Blood Bank (Presented By: Mireille Baart, MSc)

Introduction: Donors that meet the hemoglobin (Hb) cutoff criteria for blood donation (≥8.4 mmol/L for men and ≥7.8 mmol/L for women in The Netherlands) may have depleted iron stores or even iron deficient erythropoiesis. The latter stage is called sub-clinical iron deficiency: iron has become deficient but this has not yet led to overt anemia. Under this condition blood donation may result in anemia and deferral at the next visit to the blood bank. The golden standard for measuring iron deficiency is a bone marrow biopsy. This method can also detect sub-clinical iron deficiency. However, this test is invasive and expensive and is inappropriate for donor screening. Zinc protoporphyrin (ZPP) measurement is an alternative test for diagnosing (sub-clinical) iron deficiency. Under conditions of iron deficiency, a zinc atom instead of iron is incorporated in the last step of heme synthesis, resulting in the formation of ZPP instead of heme. ZPP measurement can be easily applied and is cheap. The aim of this study was to estimate the prevalence of sub-clinical iron deficiency in blood donors that have Hb levels above cutoff levels by measuring ZPP levels. Methods: A random sample of 515 whole blood donors from the Southeast Region of The Netherlands who met the criteria for blood donation participated in the study. Venous samples were collected in EDTA at donations between July and October 2008. Hb and ZPP measurements were performed on the day after blood collection. Hb levels were analyzed on a hematology analyzer (Sysmex XT-1800i). ZPP was measured with a hematofluorometer (AVIV, 206D). Before ZPP measurement erythrocytes were washed with phosphate buffered saline. Results: Mean Hb levels were 9.0 (SD 0.5) mmol/L for men and 8.3 (SD 0.4) mmol/L for women. Mean ZPP levels for men were 58 (SD 22) µmol/mol heme and for women 59 (SD 17) µmol/mol heme. A negative correlation (r -0.336) was found between ZPP and Hb in men. With a ZPP cutoff of 60 µmol/mol heme, sub-clinical iron deficiency was present in 35.5 % of male donors and in 44.0 % of female donors. With a ZPP cutoff of 80 µmol/mol heme, this percentage was 12.9 for male donors and 9.6 for female donors. Discussion: Sub-clinical iron deficiency is highly prevalent among blood donors that meet the Hb cutoff criteria and are allowed to donate. With ZPP measurement iron deficiency can be detected in the sub-clinical state. Normal Hb levels with high ZPP levels may be an indication of future anemia. After detection of sub-clinical iron deficiency, interventions such as dietary advice and prolonged donation intervals may prevent the development of clinical iron deficiency.

Poster# 232 ROLE OF IRON REPLACEMENT THERAPY IN THE ROUTINE MANAGEMENT OF BLOOD DONORS Barbara Bryant, MD¹, Yu Ying Yau, RN², Sarah Arceo, RN², Julie Hopkins, RN² and Susan Leitman, MD² ¹University of Texas Medical Branch; ²National Institutes of Health (Presented By: Barbara Bryant, MD)

Introduction: Iron depletion and deficiency in blood donors frequently results in deferrals for low hemoglobin values, yet blood centers remain reluctant to dispense oral iron replacement therapy. The purpose of this study was to prospectively assess the prevalence of iron depletion/deficiency in blood donors, study the effects of long-term blood donation on donors’ hemoglobin values and iron stores, and to evaluate the safety and efficacy of routine oral iron replacement therapy. Methods: The study was conducted at a hospital-based donor center with a registry of approximately 4500 donors. During a 36-month period, 1506 consecutive blood donors deferred for fingerstick hemoglobin values <12.5 g/dL (“low hemoglobin” group) and 413 nondeferred “control” donors underwent health history screening and laboratory testing including CBC, ferritin, iron, and transferrin. The presence of pica (persistent craving and compulsive consumption of nonnutritive substances) was specifically assessed. Iron deficiency and depletion were defined as ferritin levels of <18 mcg/L and 18-29 mcg/L in males, respectively (normal range 18-370 mcg/L) and <9 mcg/L and 9-19 mcg/L in females (normal range 9-120 mcg/L). Deferred donors and iron-deficient control donors were given a 60-pack of ferrous sulfate 325 mg tablets and instructed to take one tablet daily. Donors intolerant to ferrous sulfate were given ferrous gluconate 325 mg tablets. Reassessments were performed and another 60-pack of iron tablets dispensed at all subsequent donation visits. Results: In the low hemoglobin group, 31% and 22% of females and 9% and 51% of males had iron depletion or deficiency, respectively, compared with 29% and 9% of females and 18% and 22% of males in the control group. Pica was reported at study entry in 10% of subjects with iron depletion/deficiency, compared with 3% who were not iron depleted/deficient. Pagophagia (ice pica) was the most commonly reported craving, followed by geophagia (dirt), amylophagia (laundry starch) and cravings for raw pasta, chalk, and coal. Iron depleted/deficient donors taking iron showed normalization of iron-related laboratory parameters, even as they continued to donate blood. Donors presenting with pagophagia and placed on oral iron replacement therapy reported a profound reduction in the need to eat ice by day 5 to 8 of therapy, with complete disappearance of symptoms by day 10-14. Treatment-related iron overload did not occur even in donors with normal iron stores. Compliance with iron therapy was 73%. Adverse effects occurred in 18% of donors given ferrous sulfate and 18% of the subgroup of donors intolerant to sulfate who were given gluconate. The most commonly reported adverse effect of iron therapy was constipation. The study screening algorithm identified nine donors with serious medical conditions: five with GI bleeding, three with vitamin B12 deficiency, one with thyrotoxicosis. No donors were found to have cancer or hemochromatosis. Discussion: Iron depletion or deficiency was found in 54% of female and 60% of male donors with low fingerstick hemoglobin values, and in 38% of female and 40% of male control donors. Low iron stores are endemic in blood donors. Conclusion: The routine administration of iron replacement therapy by the donor center is safe and effective, preserves donor well-being, provides a community service, and may decrease donor deferral rates.

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Poster# 233 VALIDATING A SELF-REPORTED PROTOCOL FOR MENSTRUAL BLOOD LOSS IN PATIENTS WITH IRON DEFICIENCY ANAEMIA Peter Nielsen, Thomas Jansen, MD, Rosemarie Kongi and Roland Fischer, PhD University Hospital, Hamburg (Presented By: Peter Nielsen)

Hypermenorrhoe is one of the major causes of severe iron deficiency in developed countries. In female patients the differentiation between gastrointestinal and/or increased menstrual blood loss as the pathological finding for the clinical symptom iron deficiency anaemia (IDA) is sometimes difficult. We developed a simple protocol for the self-diagnosis of increased menstrual blood loss (www.mens-test.de), in which the number and the degree of filling of tampons or sanitary pads during individual menstruations were registered. This test gives back to the respective women a degree of risk for having an up normal menstrual blood loss and in positive cases the recommendation to see her doctor. In a series of 17 patients with 28 menstrual periods we compared the calculated results of the menstruation test with individually measured values of menstrual blood loss using a 59Fe-based blood loss quantification technique based on whole body counting. Although there was a large variation of the inter-individual results, we found overall a significant linear relation between calculated and measured values. The variation is related mainly to different capacities of the menstrual pads used. However, we think this menstruation protocol offers a simple and valuable tool both for the patients and the doctors in order to find the correct diagnosis of a given unclear severe iron deficiency. Figure 1: Relation between calculated (score points) and measured blood loss during menstruation in 17 patients. Crosses, tamp ons; circles sanitary pads used. Blood loss was measured by 59Fe whole body counting in patients with IDA following the oral intake of 10 µmol 59Fe at least 2 weeks before the start of retention measurements.

Poster# 234 PREVALENCE AND TRANSFORMATION’S PROBABILITY OF IRON DEPLETION TO ANEMIA IN CHILDREN Elena Morshchakova, MD, Valeriy Demikhov, MD, Nataliya Inyakova, Oksana Isakova and Nadezhda Kravtsova Russian Federal Research Center for Pediatric Hematology (Presented By: Valeriy Demikhov, MD)

It is estimated that iron depletion's (ID) prevalence in Russian children is high. That is why program’s development of routine iron supplementation for children (actually preschooler aged) are advocated widely. We have studied the prevalence of ID and anemia in school boys and girls aged 7 to 17 years from rural areas and Ryazan city. We observed 264 children, including 124 (35 boys and 89 girls) from Ryazan city and 140 (61 boys and 79 girls) from rural areas. Serum ferritin and Hb levels has been determined. Children who found ID and hasn’t received iron therapy were observed repeatedly in 15 month from first observation (n=39). ID was found in 47 (17.8%) and anemia in 15 (5.7%) of 264 children (Table). The prevalence of ID in children from rural area (23.6%) was significantly higher, than in children from Ryazan city (11.3%). The value of anemic schoolboys from Ryazan city was higher, than ones in boys from rural area: 11.4% vs 3.3% respectively (p<0.05).Of 6 anemic children from rural area 5 (83.3%) had iron deficiency anemia (IDA). On the contrary only 3 (53.3%) of 9 anemic urban children were IDA (p < 0.05). Of 47 ID children 39 have been examined in 15 month repeatedly. Anemia was found in 9 (23%) of 39 children. In 18 (46.2%) children iron status was normal. And 12 (10.8%) children was ID as before. Thus the transformation’s probability of ID to anemia (without treatment) in 15 month in children was only 23%. The present study showed that prevalence and transformation’s probability of ID to anemia in children aren’t high. There were significantly more rural children suffering from ID and IDA than their ur ban counterparts, while there were more urban children with non-IDA. Rural children has higher risk of ID, than urban children. Higher value of ID and IDA in rural children is obviously related to lower socioeconomic status of their families. More prevalent non-IDA in urban children may be related to their increased infectious morbidity as compared rural children. Routine iron supplementation for all children school aged is inexpediently.

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Poster# 235 HFE-RELATED HEMOCHROMATOSIS PHENOTYPIC SCREENING IN 53119 SUBJECTS: THE BRITTANY EXPERIENCE Hervé Goarin, MD², Jeff Morcet³, Pascale Gérault4, Yves Trégouët4, Michèle Bourrien4, Véronique David5, Yves Deugnier, MD6, Anne-Marie Jouanolle5, Hervé Seignard, MD4 and Pierre Brissot, MD¹ ¹Liver Disease Unit, Inserm U-522, University Hospital Pontchaillou, Rennes, France; ²Mutualité Sociale Agricole,Inserm CIC 203, Laboratory of Molecular Genetics, Liver Disease Unit, Inserm U-522, Pontchaillou University Hospital, Rennes, France; ³Inserm CIC 203, Rennes, France; 4Mutualité Sociale Agricole, Rennes, France; 5Laboratory of Molecular Genetics, University Hospital Pontchaillou, Rennes, France; 6Inserm CIC 203, Liver Disease Unit, University Hospital Pontchaillou, Rennes, France (Presented By: Pierre Brissot, MD)

Background: HFE(C282Y/C282Y)-related hemochromatosis is a recessive iron overload disorder, especially observed in Caucasian populations. Brittany (France), due to its celtic roots, is particularly exposed to hemochromatosis hazard. Using the genetic approach for population screening is hampered by partial penetrance of C282Y homozygosity (especially in women) and by ethical issues. The interest of using plasma transferrin saturation (TfSat) as a phenotypic screening test remains controversial, essentially due to its lack of specificity. However, on one hand, it is the earliest biochemical abnormality of iron metabolism related to the expression of C282Y homozygosity (through hepcidin deficiency), being present before the development of tissue iron deposition ; on the other hand, the absence of increased TfSat at the adult age excludes, at the time of determination, any indication for iron depletive treatment and renders highly improbable the future development of hemochromatosis disease. AIM. The present study aimed at evaluating, in a large population of individuals attending MSA (Mutualité Sociale Agricole) health appraisal centers all over Brittany, the efficacy of a screening strategy restricting genotyping to individuals with increased plasma transferrin saturation. Subjects and Methods: 53119 subjects, 55.8% men-44.2% women, aged 35-65 (mean age ± SD: 49.2±8.6 for men, 49.9±8.7 for women), were checked for TfSat, during a systematic clinical and biochemical work-up [age, weight, height, BMI (body mass index), glycemia, cholesterol, triglycerides, red cell corpuscular volume, GGT]. TfSat was calculated from the data obtained by separate assays for plasma iron and plasma transferrin. Whenever transferrin saturation was >50% in men and > 45% in women, the search for C282Y mutation was performed after having obtained a written informed consent. Statistical analysis consisted of a logistic regression model in order to determine the most discriminant TfSat values, among those >50 (45)%, for detecting C282Y homozygotes. Men and women data were studied separately in a two-step process: 80% of the population served for determining the best model, and the remainder 20% for testing the model. Results: 1476 men and 1272 women, according to selection criteria, had a genetic test. C282Y homozygosity with increased TfSat, corresponding to (at least) grade 1 hemochromatosis, was detected in 70 men (4.7%) and 72 women (5.7%). When applying the determined statistical models: i) in men: TfSat>68%, or Tfsat 45-68% plus transferrin<2g/L, permitted to detect 93% of homozygotes, avoiding to realize the genetic test in 983 subjects (over 1476) ; ii) in women: by excluding subjects with TfSat 45-58% plus plasma iron≥24µmol/L plus BMI≤25 (i.e. normal), 98.6% of homozygotes (71/72) remained detected, avoiding to perform the genetic test in 542 subjects (over 1272). Conclusion: This study of a large population in Brittany: i) points out the limitation of using classical TfSat threshold values for detecting expressing C282Y homozygotes ; ii) permits to define new discriminant TfSat-based models, upgrading significantly this phenotypic screening procedure.

Poster# 236 CLINICAL EXPRESSION OF HFE-ASSOCIATED HAEMOCHROMATOSIS IN SUBJECTS UNDER 40 YEARS OF AGE Jeannette Dixon, BA, MPH, Elizabeth Watt, BComm, MD, Grant Ramm, BSc, PhD, Nathan Subramaniam, BSc, PhD, Lawrie Powell, MD, PhD and Greg Anderson, BSc, MSc, PhD Queensland Institute of Medical Research (Presented By: Jeannette Dixon, BA)

HFE-associated haemochromatosis (HH) is the most common iron overload disorder in persons of northern European descent. Whilst it is widely recognised that clinical expression most frequently occurs after the age of 40 years, there have been very limited data on HH in younger subjects. The goal of this study was to investigate disease expression and factors affecting expression in a large cohort of younger HH subjects. This information will improve our understanding of the natural history of the disorder and help inform the debate on population screening for HH and factors affecting clinical expression. A retrospective analysis of 234 C282Y homozygous HH subjects under the age of 40 years was performed. These subjects were selected from the large HH database at the QIMR. Parameters analysed included age, serum ferritin (SF; µg/L), transferrin saturation (TS; %), liver function tests (LFTs), hepatic iron concentration (HIC; µmol/g dry weight), alcohol consumption (g/day), body mass index (BMI), body weight, stage of hepatic fibrosis, and the presence or absence of cirrhosis. For most analyses, subjects were divided into three groups based on levels of SF - low (males < 300 µg/L; females < 200 µg/L), moderate (< 1000 µg/L but > 200/300 µg/L) and high (≥ 1000 µg/L). Of the 234 subjects (M 149; F 85), 44 (M 23; F 21) were in the low SF group, 124 (M 68; F 56) were in the moderate SF group and 66 (M 58; F 8) in the high SF group. Thus 81% had SF levels above the normal range and 28% had SFs at a level strongly associated with hepatic damage (≥1000 µg/L). Significant positive relationships were present between the level of SF and increasing age, TS, HIC, abnormal LFTs, alcohol consumption, body weight, hepatic fibrosis stage and the presence of cirrhosis (P = 0.03 for weight; P < 0.0001 for all other relationships). Male subjects were also more likely to have elevated SF (P < 0.0001), as were probands (P < 0.0001). Those with high SF were more likely to present with HH-related symptoms, such as lethargy and arthralgia, and iron overload-related disease (SF ≥ 1000 µg/L and abnormal LFTs) was found in 21% of subjects. Subjects with hepatic fibrosis were more than twice as likely to have one or more cofactors (diabetes, excess alcohol, steatosis) than subjects with no fibrosis (52% vs 24%). No significant correlation was found between SF and BMI, but BMI data were available for only a small number of subjects. The results of this study indicate that persons aged less than 40 years with HFE-associated haemochromatosis frequently exhibit biochemical expression of iron loading. Many subjects also showed signs of hepatic injury and fibrosis, and 21% of subjects had iron overload-related disease. Key modifiable environmental factors associated with increased disease expression included increased alcohol consumption and increased body weight. On this basis, young C282Y homozygotes should be encouraged to limit their alcohol consumption and to maintain an ideal body weight in order to decrease the risk of early clinical expression.

Poster# 237 SCREENING FOR IRON OVERLOAD: LESSONS FROM THE HEIRS STUDY Paul Adams, MD¹, James Barton, MD², Gordon McLaren, MD³, Ronald Acton, PhD4, Mark Speechley, PhD5, Christine McLaren, PhD6, David Reboussin, PhD7, Cathie Leiendecker-Foster, MS8, Beverly Snively, PhD7, Emily Harris, PhD9, Tom Vogt, MD¹°, Phyliss Sholinsky, MSPH¹¹, Elizabeth Thomson, DNSc¹², Fitzroy Dawkins, MD¹³, Victor Gordeuk, MD¹³ and John Eckfeldt, MD, PhD8 ¹University Hospital; ²Southern Iron Disorders Center, Birmingham, AL; ³VA Long Beach Healthcare System, Irvine, CA; 4Dept. of Microbiology, Birmingham, AL; 5Dept. of Epidemiology, University of Western Ontario; 6Dept. of Epidemiology, UCI, Irvine, CA; 7Division of Public Health Sciences, Wake Forest University, Winston-Salem, NC; 8Department of Laboratory Medicine, University of Minnesota, Minneapolis, MN; 9National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD; ¹°Kaiser Permanente Center for Health Research, Honolulu, HI; ¹¹Dept of Epidemiology, NHLBI, NIH, Bethesda, MD; ¹²National Genomics Institute, NIH, Bethesda, MD; ¹³Dept. of Hematology, Howard University, Washington, DC (Presented By: Paul Adams)

The HEmochromatosis and IRon Overload Screening (HEIRS) study provided data on a racially, ethnically and geographically diverse cohort of participants in North America screened from primary care populations. There were 101,168 participants screening by testing for HFE C282Y and H63D mutations and measuring serum ferritin and transferrin saturation. In this overview, lessons from the HEIRS study are highlighted in the context of the principles of screening for a medical disease as outlined previously by the World Heatlh Organization.

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1.Generalized population screening in a primary care population as performed in the HEIRS study is not recommended. 2. Genetic testing for hemochromatosis is well accepted with minimal risk of discrimination. 3. An elevated serum ferritin is very common, particularly in Asians, Pacific Islanders, and African Americans and may not be an indication for phlebotomy. In the absence of HFE C282Y homozygosity, this finding usually does not represent an increase of iron stores of > 4 g. 4. Transferrin saturation has high biological variability and relatively low sensitivity to detect HFE C282Y homzygotes which limits its role as a screening test. 5. Symptoms attributable to HFE C282Y homozygosity are no more common in individuals identified by population screening than in control subjects. 6. Mild increases in body iron stores in the range of 2-3 g were common in non-HFE C282Y homozygotes, but iron overload defined as iron stores > 4 g was most common in Caucasian men who were C282Y homozygotes. 7. There may be a role for focused screening in Caucasian men with some debate about genotyping followed by phenotyping or phenotyping followed by genotyping.

Poster# 238 HFE-C282Y HOMOZYGOTES WITH ELEVATED SF LESS THAN 1000 µG/L ARE NOT AT INCREASED RISK OF HAEMOCHROMATOSIS- ASSOCIATED DISEASE FEATURES K.J. Allen¹,²,³, L.C. Gurrin, N.A. Bertalli, N.J. Osborne, C.C. Constantine, M.B. Delatycki, A.E. Nisselle, A.J. Nicoll, D.M. Gertig, C.E. McLaren, G.G. Giles, D.R. English, J.L. Hopper, G.J. Anderson, J.K. Olynyk and L.W. Powell ¹Murdoch Childrens Research Institute; ²Dept of Paediatrics The University of Melbourne; ³Dept of Gastroenterology Royal Children’s Hospital, Australia (Presented By: K.J. Allen)

Introduction: Although serum ferritin (SF) >1000 µg/L remains the strongest indicator of disease expression amongst individuals who are homozygous for the C282Y mutation in the HFE gene, it is not known whether moderately elevated SF that is less than 1000 µg/L is also associated with an increased risk of haemochromatosis (HH)- associated disease compared to either HFE wild-type controls or C282Y homozygotes with normal SF. We aimed to assess the prevalence of HH-associated disease in C282Y homozygotes with SF<1000 µg/L in a prospective population-based cohort. Methods: 41, 528 people aged 40–69 years were recruited to the Melbourne Collaborative Cohort Study (MCCS) between 1990-94 and followed for an average of 12 years. All C282Y homozygotes plus a random sample stratified by HFE-genotype (C282Y and H63D) mutations were invited to clinical follow-up (n=1,438). Medical, dietary and lifestyle information were collected and iron studies performed at baseline and follow-up. Liver aminotransferases (AST/ALT) were measured at follow-up. Clinical examination at follow-up was performed by doctors blinded to HFE-genotype. All C282Y homozygotes who were undiagnosed at baseline and HFE wild-types (in both groups with SF<1000 µg/L at both baseline and follow-up) were included in the analysis. HH-associated disease features examined were abnormal 2nd/3rd metacarpophalangeal joints (MCP 2/3), self-reported fatigue and raised AST/ALT. Results: Male and female C282Y homozygotes had a similar prevalence of disease features compared to HFE wild-types (see Table) despite that fact that C282Y homozygotes had a significantly greater prevalence of elevated SF compared to their sex-specific wild-type groups. For those with elevated SF, there was no significant difference in prevalence of disease features between the genotype groups of either sex, the one exception being an increased prevalence of abnormal MCP 2/3 for male C282Y homozygotes (5/14 (36%)) compared to male HFE wild-types (3/32 (9%)) (p=0.030). C282Y homozygotes of both sexes with elevated SF had a comparable prevalence of disease features compared to C282Y homozygotes with normal SF. Conclusion: C282Y homozygotes with SF<1000 µg/L do not appear to be at increased risk of HH-associated disease compared to wild-type controls with the exception of arthritis in males. Nor does moderately elevated SF (that is less than 1000 µg/L) appear to be associated with an increased prevalence of HH-associated disease features compared to homozygotes with normal iron studies.

Poster# 239 NOMOGRAM TO DETECT THE RISK FOR IRON OVERLOAD-ATTRIBUTED DISEASE IN SIBLINGS OF PROBANDS WITH HFE-RELATED SYMPTOMATIC HEMOCHROMATOSIS: RESULTS OF THE HEMOCHROMATOSIS FAMILY STUDY Esther Jacobs, MD, PhD², Jan Hendriks, Herman Kreeftenberg, Richard de Vries, Joannes Marx, Cees van Deursen, Anton Stalenhoef, Andre Verbeek and Dorine Swinkels, MD, PhD¹ ¹Radboud Department of Clinical Chemistry 441, University Nijmegen Medical Centre; ²Department of Clinical Chemistry, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands (Presented By: Dorine Swinkels, MD, PhD)

The clinical expression of HFE-associated hereditary hemochromatosis (HH) is highly variable, complicating early screening options to prevent ironoverload related disease. The aim of this study was to verify the existence of HH-related disease in C282Y homozygous siblings of C282Y homozygous probands with clinically detected HFE- related HH and to identify factors predictive for the iron-related disease in these siblings. To this end, C282Y homozygous (n=110, males n=53) and non-homozygous siblings (n=318, males n=145) of 224 probands were compared for levels of serum iron parameters, self-reported environmental and lifestyle factors and HH-related diseases. Compared to non-homozygous C282Y siblings, C282Y homozygous siblings more often mentioned to have been diagnosed with arthropathy (Odds Ratio [OR] 2.76, 95% Confidence Interval [CI] 1.71-4.46) and liver disease (OR 2.90, 95%CI 1.27-6.62). Using multivariate logistic regression modelling, age (OR 1.09, 95% CI 1.05-1.12) and age in the presence of elevated ferritin levels gender (OR 0.94, 95%CI 0.90-0.99) were independent predictors for the development of iron-associated organ disease next to C282Y homozygosity (OR 2.11, 95%CI 1.12-3.96). In contrast, gender, BMI and alcohol intake were not. A nomogram including these significant predictors is provided to simply and accurately calculate the probability of being affected by HH-related diseases. We additionally calculated the benefit of using this model in the decision to treat or not treat by phlebotomies. In conclusion, HH related disease risk is best predicted by C282Y homozygosity, age and ferritin levels. These findings will help to define a high-risk group for iron overload-related disease among siblings of clinically detected C282Y homozygous probands and may contribute to the evaluation of the cost-effectiveness of screening programs.

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Poster# 240 HFE COMPOUND HETEROZYGOTES ARE AT LOW RISK OF HEMOCHROMATOSIS-RELATED MORBIDITY L.C. Gurrin, N.A. Bertalli, G.W. Dalton, N.J. Osborne, C.C. Constantine, C.E. McLaren, D.R. English, D.M. Gertig, M.B. Delatycki, A.J. Nicoll, M.C. Southey, J.L. Hopper, G.G. Giles, G.J. Anderson, J.K. Olynyk, L.W. Powell and K.J. Allen Centre for MEGA Epidemiology, The University of Melbourne. Australia (Presented By: L.C. Gurrin)

Background: Most clinical cases of HFE-associated hemochromatosis (HH) are homozygous for the C282Y mutation. Although C282Y/H63D compound heterozygosity is associated with some iron loading, the risk of HH-related morbidity in this genotype group is unknown at the population level. Methods: We used a prospective population-based cohort study to estimate the prevalence of elevated iron indices and HH-related morbidity for compound heterozygotes. 31,192 subjects of northern European descent were genotyped for HFE C282Y and H63D. A genotype stratified random sample of 1,438 subjects, when followed an average of 12 years since baseline to a mean age 65 years, completed questionnaires and gave blood for measurement of iron indices (serum ferritin (SF) and transferrin saturation (TS)) and liver aminotransferases (AST/ALT). Clinical examinations were blinded to HFE genotype. 180 (84 males) C282Y/H63D participants were compared with 330 (149 males) controls with neither HFE mutation. 132 (65 males) and 270 (122 males) respectively had serum iron measures at both time-points. Results: The prevalence of elevated SF and TS was greater in compound heterozygotes than subjects with neither HFE mutation. For subjects with serum measures from both baseline and follow-up, mean SF and TS levels did not change significantly for men or for post-menopausal women, but for pre-menopausal women SF levels increased from 43 to 109 µg/L for compound heterozygotes and from 35 to 64 µg/L for wild-types (both p<0.001). The prevalence of HH-related disease features (abnormal 2nd/3rd metacarpophalangeal joints (MCP 2/3), self-reported fatigue and raised AST/ALT) was similar in the two genotype groups (see Table). 1/82 males and 0/95 females had documented iron overload-related disease (Allen et al. NEJM 358 (2008). Conclusion: For male compound heterozygotes mean iron indices do not change during middle age but for female compound heterozygotes menopause results in increased mean SF. Although compound heterozygotes might maintain elevated iron indices during middle age, documented iron overloadrelated disease is rare.

Poster# 241 – WITHDRAWN JAPANESE PATIENTS WITH HEMOJUVELIN-HEMOCHROMATOSIS WHO SURVIVED OVER 50 YEARS Hisao Hayashi, MD¹, Yasuaki Tatsumi, PhD², Ai Hattori, BS², Motoyoshi Yano, MD³, Ryuichi Kidokoro, MD4, Shinya Wakusawa, PhD5, Kazuhiko Hayashi, MD5, Yoshiaki Katano, MD5 and Hidemi Goto, MD5 ¹Aichi Gakuin University; ²Aichi Gakuin University School of Pharmacy; ³Yokkaichi City Hospital; 4Kidokoro Clinic; 5Nagoya University School of Medicine

Aims: Hemojuvelin (HJV) was originally cloned as an iron transport protein responsible for a juvenile type of hereditary hemochromatosis (HH). We report a dissociation of the phenotype from genotype in three Japanese patients with HJV-HH. Methods: Two unrelated families were enrolled in a 15-year followed-up study. The first family included a 48-year-old male patient with D249H/D249H in HJV, and the second family included a 51-year-old male patient and his sister with Q312X/Q312X in HJV (1). These male patients had fathered one and two children, respectively. Results: One male patient survived to the age of 62-years with insulin therapy combined with venesection. He was positive for anti-Helicobactor pylori antibody but free from gastrointestinal complications. His social activity had been preserved 60 min. before cardiopulmonary arrest. The other male patient had received intensive treatment for 6 months due to cardiac failure and DM. He was finally admitted with a hypoglycemic episode, and died from multiple organ failure on the 4th hospital day. His sister currently remains alive at the age of 58 years. Discussion: Early diagnosis and appropriate treatment are always important. One male patient underwent complete venesection, which was associated with prolonged survival, while the other male patient did not have the opportunity to receive this treatment. Regardless of treatment effect, the phenotype of three patients appeared to be classic rather than juvenile HH. Since each of the male patients had become a father, hypogonadism was not a main feature of these patients, but cardiac disorder was a common final event. Reason our patients showed less severe organ damage due to iron overload remained unclear, but Helicobactor pylori infection may be a candidate for the cause of impaired iron absorption or iron depletion. As far as Q312X mutation is concerned, phenotype and genotype correlation might be unlikely, since the same mutation was involved in a Japanese family with juvenile HH (2). Conclusion: Japanese patients HJV-HH showed a wider range of clinical features than expected previously. References: 1. Koyama C, Hayashi H, Wakusawa S, et al. Three patients with middle-age-onset hemochromatosis caused by novel mutations in the hemojuvelin gene. J Hepatol 43:740-742, 2005. 2. Nagayoshi Y, Nakayama M, Suzuki S, et al. A Q312X mutation in the hemojuvelin gene is associated with cardiomyopathy due to juvenile haemochromatosis. Eur J Heart Fail 10:1001-1006, 2008.

Poster# 242 ADRENERGIC OVERDRIVE IN IRON OVERLOADED PATIENTS WITH HEMOCHROMATOSIS Raffaella Mariani, MD¹, Alberto Piperno, Paola Trombini², Valentina Paolini, Matteo Pozzi, Gino Seravalle³, Fosca Quarti-Trevano, Guido Grassi and Giuseppe Mancia ¹University of Milano-Bicocca; ²Department of Clinical Medicine, San Gerardo Hospital, Monza, Italy; ³Istituto Auxologico Italiano, Milano, Italy (Presented By: Paola Trombini)

Adrenergic vascular hyper-responsiveness is a hallmark of different cardiometabolic disease, including essential hypertension, diabetes, obesity, metabolic syndrome, heart failure and chronic kidney disease. Previous studies have shown that sympathetic activation and insulin resistance are closely related each other, but the cause-and-effect relationship remains undefined. Iron overload impairs glucose metabolism in hemochromatosis patients by either insulin resistance or decreased insulin secretion. There are no data on the effect of iron overload on the adrenergic overdrive. The study groups consisted of 15 iron-loaded hemochromatosis male patients without iron-related organ damage, at diagnosis, and 10 age-matched healthy male controls. We measured, during a 30-minute resting period, beat-to-beat blood pressure (Finapres), heart rate (ECG) and postganglionic muscle sympathetic nerve traffic (MSNA) via microneurography into the peroneal nerve. Seven hemochromatosis patients were evaluated also after iron depletion. All patients underwent Magnetic Resonance for the assessment of hepatic iron concentration (HIC). Metabolic syndrome (defined according to the ATPIII criteria), essential hypertension, diabetes, obesity, heart and kidney

175 failure, and cirrhosis were considered metabolic confounders and they represented the exclusion criteria. Insulin resistance was estimated by HOMA index. In hemochromatosis patients at diagnosis, mean serum ferritin was 825.26+406.83 µg/L, HIC 206+57.17 µmol/g and HOMA index was normal (1.7+0.82). MSNA values were significantly higher than in controls (63.53+10.93 vs 35.36+10.85 bursts/100 heart beats, p=0.0001). After iron depletion sympathetic activation showed 41% mean reduction and significantly decreased in the seven patients studied before and after therapy (68.24+12.26 vs 40.39+11.04 bursts/100 heart beats, p=0.0156) reaching the normal range. The present study indicates that iron overloaded male patients with hemochromatosis are characterized by a hyperadrenergic state. Iron overload contributes to adrenergic hyper-responsiveness and could be directly involved in the overactivity of the autonomic nervous system also in the absence of an insulin resistance condition. Iron- dependent generation of reactive oxygen species might be involved in the pathogenesis of the adrenergic overdrive and more generally in cardiovascular damage.

Poster# 243 EXTRINSIC FACTORS INFLUENCING THE EXPRESSIVITY OF THE HFE VARIANT C282Y, H63D, S65C PHENOTYPES IN DANISH MEN AGED 30-53 YEARS Palle Pedersen, PhD² and Nils Milman, MD¹ ¹Næstved Hospital, Department of Clinical Biochemistry; ²Department of Clinical Biochemistry, Næstved Hospital, Næstved, Denmark (Presented By: Nils Milman, MD)

This study analysed the influence of extrinsic factors on the phenotypic expression of HFE gene variants in ethnic Danish men. A cohort of 6,020 men aged 30-53 years was screened for HFE C282Y, H63D and S65C variants. Serum iron, serum transferrin, transferrin saturation, and serum ferritin were analysed in 1,452 men and 1,294 men completed a questionnaire on factors, which could influence iron balance. The C282Y allele was present in 5.6%, H63D in 12.8% and S65C in 1.8% of the men. In the entire series, 3% had elevated iron status markers (transferrin saturation ≥50%, ferritin ³300 µg/L). Self-reported liver disease had an elevating effect and peptic ulcer a lowering effect on iron status markers. Age increased the fraction of men with elevated ferritin from 8.3% at 32-38 years to 16.2% at 46-53 years of age (p=0.002). Blood donation had a lowering effect on iron status markers (p=0.0001).Alcohol consumption elevated serum iron and serum ferritin (p=0.001). Meat consumption had an elevating effect (p=0.02) and milk consumption a lowering effect (p=0.03) on serum ferritin. There was no influence of vitamin-mineral tablets on iron status markers. In adjusted logistic regression analysis, the HFE genotype had the highest impact on iron status markers; high alcohol consumption was significantly associated with elevated transferrin saturation. High age and high alcohol consumption were significantly associated with elevated ferritin and high egg consumption and blood donation was significantly associated with normal ferritin levels. In conclusion,the expressivity of HFE variant phenotypes in Danish men was enhanced by alcohol and meat consumption and decreased by milk and egg consumption and blood donation.

Poster# 244 DOES HLA-TYPE OR T-LYMPHOCYTE SUBSETS HAVE ANY RELEVANCE FOR PLANNING THE FOLLOWUP OF PERSONS WITH SCREENING- DETECTED HEMOCHROMATOSIS? PRELIMINARY DATA FROM NORTH TRØNDELAG COUNTY, NORWAY Ketil Thorstensen¹, Wenche Irgens¹, Arne Åsberg¹, Berit Borch-Iohnsen², Torolf Moen³ and Kristian Hveem4 ¹Dept. of Medical Biochemistry, St. Olav Hospital, Trondheim, Norway.; ²Inst. of Basic Medical Sciences, Department of Nutrition, University of Oslo, Norway.; ³Dept. of Immunology and Transfusion Medicine, St. Olav Hospital, Trondheim, Norway.; 4Medical Department, Levanger Hospital, Levanger, Norway (Presented By: Ketil Thorstensen)

Background: In a previous screening study we identified a large number of patients with genotypic and phenotypic hemochromatosis (Scand. J. Gastroenterol. 2001; 36, 1108). A common feature of these patients is the relatively low iron load as determined by their serum ferritin levels. The proportion of C282Y homozygotes that will progress to life-threatening complications of the disease is still unknown, and it is unclear which patients that need the closest followup. The finding of additional, modifying factors that could help predict the course of the disease may lead to a more efficient use of health resources. Previous work on T-lymphocyte subsets (J. Hepatol. 1998; 28, 1) has indicated that abnormally high CD4+/CD8+ ratios due to low peripheral blood CD8+ counts are associated with more severe forms of iron overload and with a faster re-entry of iron after treatment. This association is supposedly persistent. In addition, HLA-type has been reported to modify the degree of iron accumulation (Eur. J. Haematol. 2007; 79, 429). For that reason we undertook a study examining these factors in a cohort of patients found through a hemochromatosis screening program as part of a general population helath survey. Methods: A total of 529 individuals found through screening were invited to participate in the study, and 333 subjects responded positively. Blood samples were analysed for HLA-type on the Luminex system. T-lymphocyte subsets (CD4+ , CD8+, total T-lymphocytes) were determined by flowcytometry. The newly obtained data were compared with data obtained in the original screening study. Results: All results are for C282Y homozygous probands. Ferritin data are from the time of diagnosis. Data for T-lymphocyte subsets are from recent analyses. Females had significantly higher CD4+ count (P=0.0014) and total T-lymphocyte count (P=0.0079) than men. No difference was seen in CD8+ count or CD4+/CD8+ ratio. The age distribution was identical for women and men. Univariate models showed negative correlations between age and CD4+ count (P=0.008), CD8+ count (P=0.049) and total T- lymphocyte count (P=0.004), and a positive correlation between age and CD4+/CD8+ ratio (P=0.003). In females, but not in males, there was a significant positive correlation (P=0.007) between age and ferritin levels, independent of genotype. In males a positive correlation was observed between ferritin levels and the CD4+ count (P=0.002), as well as total T-lymphocyte count (P=0.033). The strong correlation between age and T-lymphocyte subsets, as well as with ferritin in women, complicates data analysis and interpretation of results even after correcting for age by using age-adjusted parameters (e.g. ferritin/age or CD4+/age). In a multiple regression model age, CD4+, CD8+ and total Tlymphocyte counts were significant predictors of ferritin levels for females (overall P=0.001). For males this association was much weaker (overall P=0.025). We also observed a trend in the data that increasing number of HLA-A3 alleles were associated with higher ferritin levels. However, this association was not statistically significant. The analyses of HLA-type are not completed (88 samples typed, approx. 200 remaining), and it is possible that upon completion this trend may become significant. Conclusions: Our data show that there are correlations between the CD4+, CD8+ and total lymphocyte counts and the iron load, as well as a trend towards higher ferritin levels with increasing numbers of HLA-A3 alleles. However, these differences may be too subtle to be of any relevance for planning the followup of persons with screening-detected hemochromatosis.

Poster# 245 SELDI-TOF MS DETECTION OF URINARY HEPCIDIN Sandro Altamura, PhD, Judith Kiss, Claudia Blattmann, Wolfgang Gilles and Martina Muckenthaler MMPU – Molecular Medicine Partnership Unit (Presented By: Sandro Altamura, PhD)

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.

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Poster# 246 PHENOTYPIC PENETRANCE OF THE HFE GENE C282Y, H63D AND S65C VARIANTS IN DANISH MEN AGED 30-53 YEARS Nils Milman, MD and Palle Pedersen, PhD Næstved Hospital, Næstved, Denmark (Presented By: Nils Milman, MD)

The aim of this epidemiologic population survey was to assess the phenotypic penetrance of the most frequent haemochromatosis (HFE) gene variants in ethnic Danish men. A cohort of 6,020 men aged 30-53 years was screened for HFE C282Y, H63D and S65C variants by Restriction Fragment Length Polymorphism analysis. Subsequently, iron status markers (serum transferrin saturation, serum ferritin) were analysed in 1,452 men. The C282Y allele was present in 5.6%, H63D in 12.8% and S65C in 1.8% of the men. We found 23/6,020 (0.38%) C282Y homozygotes, of whom 2 had been treated with phlebotomy.Among untreated C282Y homozygotes (n=21) with available iron status markers (transferrin saturation n=18, ferritin n=16), 89% had elevated transferrin saturation ³50%, 94% had elevated ferritin ³300 µg/L and 88% had elevation of both iron status markers; 7/16 (44%) had ferritin values >800 µg/L. One C282Y homozygote had normal iron status markers possibly due to non-expressivity. Among C282Y/H63D compound heterozygotes (n=66), 23% had elevated transferrin saturation, 27% elevated ferritin and 9% elevation of both iron status markers. Among H63D/H63D homozygotes (n=74), 15% had elevated transferrin saturation, 19% elevated ferritin and 5.4% elevation of both iron status markers. Among C282Y/wild type (wt) heterozygotes (n=255), 9% had elevated transferrin saturation, 9% elevated ferritin and 1.2% elevation of both iron status markers. Among H63D/wt heterozygotes (n=600), 8% had elevated transferrin saturation, 12% elevated ferritin and 2% elevation of both iron status markers. None of the men with the S65C variant displayed elevation of both iron status markers. In conclusion, this study demonstrates a high penetrance of the C282Y variant in Danish men followed by the H63D variant while the S65D variant had no significant impact on iron status markers in this study.

Poster# 247 DELINEATING THE EXPERIENCES OF HEREDITARY HEMOCHROMATOSIS PATIENTS ALONG THE PATIENT PATHWAY: AN INTERNATIONAL QUANTITATIVE SURVEY OF 210 PATIENTS Pierre Brissot, MD¹, Vivian Wu Jin², Simon Ball³ and Helena Cannon³ ¹Liver Disease Unit Inserm U-522, University Hospital Pontchaillou, Rennes, France; ²Novartis Pharmaceuticals Corporation, New Jersey, USA; ³Adelphi International Research, Bollington, UK (Presented By: Pierre Brissot, MD)

Background & Aims: In order to improve the treatment and management of Hereditary Hemochromatosis (HH) it is important to gain insight into patients’ experiences: from diagnosis, through treatment and beyond. This study quantified the routes to diagnosis, patient self-reported involvement in treatment decisions, treatment experiences, the emotional impact of HH, and sources of information considered most useful in learning about their condition. Method: Following a preliminary qualitative study, which explored the correct language to use in the quantitative survey, 210 patients diagnosed with HH completed an on- line survey across four countries: US, France, Ireland and UK. Patients were recruited into three groups: (1) currently receiving induction phlebotomy (n=62), (2) currently receiving maintenance phlebotomy having finished their induction phase within the last year (n=102) and (3) never received phlebotomy but likely to do so in the next 6 months (n=46). Induction and maintenance patients were required to have received phlebotomy for at least 6 months. Patients were recruited via patient associations, media advertisements, healthcare professionals, and family members/ friends with HH. Results: 88% of patients claimed to have been experiencing HH symptoms at diagnosis: most commonly fatigue/anemia-like symptoms and joint pain (74% and 55% of patients respectively). However, only 33% of patients visited their physician because of the symptoms and therefore received diagnosis. They were most often diagnosed by chance (42%), through routine check ups or other investigations. Even though 61% of patients claimed to be involved or highly involved in decisions about their HH treatment, 27% were unaware of their serum ferritin level at diagnosis and 35% were unaware of their target level. 55% of patients reported that phlebotomy is a weekly or more frequent procedure during the induction phase, with a further 26% claiming it is done fortnightly. For 44% of maintenance phase patients it took 9 months or longer for their serum ferritin to reach ‘normal’ levels. Many patients continued to experience HH symptoms even after receiving phlebotomy; 60% reported currently experiencing fatigue/ anemia-like symptoms and 50% experiencing joint pain. 38% of patients agree/ totally agree that they are afraid of the effect HH is having on their long term health. Hemochromatosis support groups/societies and the internet were considered the most useful information sources by patients (rated useful or extremely useful by 70% and 69% respectively). Conclusions: The findings provide insight into the burden HH and its treatment places on patients. There is room to improve recognition of HH symptoms within the at risk population, which can facilitate early diagnosis of the condition. Phlebotomy represents a burden on patients in the induction phase, in terms of the frequency of attending treatment and the length of the induction phase. A substantial number of patients continue to experience HH symptoms after receiving phlebotomy, highlighting an unmet treatment need. Support groups/societies are considered valuable to patients and can play a key role in educating and alleviating patient concerns. As most patients consider themselves highly involved in treatment decisions, sources like the internet also provide useful supplements to communication with doctors/nurses.

Poster# 248 A PORTUGUESE PATIENT HOMOZYGOUS FOR THE HAMP-25→ A MUTATION-30 YEARS FOLLOW UP Rita Fleming, MD Hospital Santa Maria (Presented By: Rita Fleming, MD)

Juvenile or type 2 Hereditary Hemochromatosis (JH) is an autosomal recessive disorder linked to mutated HJV or HAMP genes. HAMP gene encodes hepcidin, the liver- derived iron regulatory hormone, which maintains iron homeostasis by tight regulation of intestinal iron absorption, as well as macrophage and hepatocyte iron release. JH is associated with the most severe phenotype of hereditary hemochromatosis. Usually, the onset of the hipogonadism and cardiomyopathy appears before the age of 30 and before cirrhosis. Here we present a 54 year-old man, presented, at the age of 24, signals of hipogonadism (loss of hair, sexual dysfunction) and low levels of testosterone and luteinizing hormone. One year after, the liver function tests were abnormal, the ultrasonography showed diffuse hepatomegaly and liver biopsy showed intense hepatic siderosis without cirrhosis. Serum iron: 200µg/dl; transferrine saturation: 70%; normal glycemia. Three years later, he started a severe heart failure, left ventricular (LV) ejection fraction of 17% and arrhythmias. In the last 30 years, after two years weekly phlebotomies he became asymptomatic, having dismissed cardiologic therapy and continued maintenance therapy to keep serum ferritin level below 50ng/ml. Liver functional tests and glycemia were normal since then. Since 1997, complains of joint pain involving knees and ankles. He was negative for C282Y and H63D mutations in HFE. In 2005, screening for other genes in association with hemochromatosis revealed a homozygosity for the 5´UTR- 25→A mutation in the HAMP gene. This mutation creates a new iniciation codon at position + 14 related to the cap site, which induces a shift of the open reading frame. Homozygosity for this mutation was previously reported as the cause of JH in two other individuals of Portuguese origin. Four years ago, although with no cardiac symptoms and normal standard echocardiografic parameters, using Tissue Doppler Imaging, it was able to detect LV dysfunction. Early identification of LV dysfunction before onset of symptoms is very important for the monitoring of these patients.

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Poster# 249 EVALUATION OF PATIENT SATISFACTION WITH PHLEBOTOMY FOR THE TREATMENT OF HEREDITARY HEMOCHROMATOSIS: AN INTERNATIONAL QUANTITATIVE SURVEY OF 210 PATIENTS Pierre Brissot, MD¹, Jean Francis Baladi², Simon Ball³ and Helena Cannon³ ¹Liver Disease Unit Inserm U-522, University Hospital Pontchaillou, Rennes, France; ²Novartis Pharmaceuticals Corporation, New Jersey, USA; ³Adelphi International Research, Bollington, UK (Presented By: Pierre Brissot, MD)

Background and Aims: Currently, phlebotomy is the only approved treatment for hereditary hemochromatosis (HH). This study aims to assess patient satisfaction with phlebotomy and its impact on patients’ lives. Method: Following a preliminary qualitative study with HH patients, which explored the correct language to use in the quantitative survey, 210 patients diagnosed with HH completed an on-line survey across four countries: US, France, Ireland and UK. The survey examined patient perceptions of ‘the worthiness’ of phlebotomy, tolerability of treatment, convenience, impact on work and leisure and their preference towards another form of treatment. Patients were recruited into three groups: (1) currently receiving induction phlebotomy (n=62), (2) currently receiving maintenance phlebotomy having finished their induction phase within the last year (n=102) and (3) never received phlebotomy but likely to do so in the next 6 months (n=46). Induction and maintenance patients were required to have received phlebotomy for at least 6 months. Patients were recruited via patient associations, media advertisements, healthcare professionals, and family members/friends with HH. Results: 87% of those patients currently receiving phlebotomy, regarded phlebotomy as “quite” or “very” worthwhile. 43% frequently experienced sideeffects (e.g. tiredness, fainting and loss of appetite) after phlebotomy and 25% of patients “frequently” felt upset about tolerability issues associated with phlebotomy. 40% of patients reported that attending phlebotomy appointments frequently affects their work/daily routine. 45% of patients said that their appointments take 2-5 hours out of their day, resulting in 39% of patients considering attending phlebotomy appointments to be “quite” or “very inconvenient”. 16% of patients receiving phlebotomy “definitely” or “probably” would prefer to receive an alternative therapy, and a further 29% of patients were undecided as to whether an alternative would be preferable to phlebotomy. Conclusions: The majority of HH patients surveyed claim to be relatively satisfied with most aspects of phlebotomy and consider it worthwhile. However, data suggests that a group of patients have concerns with phlebotomy, in terms of tolerability and impact on their lives. These concerns represent unmet treatment needs for some HH patients.

Poster# 250 INFECTIOUS DISEASES IN DIAGNOSED PATIENTS OF GENETIC HEMOCHROMATOSIS Alejandro Del Castillo, MD, Susana Gordo, Teresa Aldámiz, Fernando De la Calle and Luis A. Álvarez-Sala Hospital G. U. Gregorio Marañón (Presented By: Alejandro Del Castillo, MD)

Introduction and Objectives: The relationship between iron and infection (bacterial, viral or fungal) has been the object of study for many years. We aimed to determine the incidence of infectious diseases in patients diagnosed with genetic hemochromatosis (GH) in a unit caring for patients with iron metabolism disorders. Methods: The study population was composed of patients diagnosed with GH between 2002 and 2008. We compared the phenotype and genotype of all patients with GH with the subgroup of patients who at the time of diagnosis had a history of infectious disease. Results: Of a total of 200 patients with GH (71 % men and 29 % women, mean age 49 years [range 13 to 81 and standard deviation (SD) 15.84]) 10 had a history of infectious diseases (5 % of the total, 5 men [50 %] and 5 women [50 %], mean age 54 years [range 35-73 and SD 13,6]). The patients with infections (5 hepatitis B virus, 3 hepatitis C virus, 1 Helicobacter pylori and 1 Klebsiella pneumoniae) had lower ferritin levels (443 +/-308 ng/ml vs 545 +/-440 ng/ml) than the general group. The most frequent genotypes in the general group were the C282Y/H63D compound heterozygote (26 %) and the C282Y homozygote (22 %), whereas in the infected patients, the most frequent genotype was the H63D homozygote (40 %), of which 75 % were associated with infection by the hepatitis C virus. All patients infected by the hepatitis C virus had the H63D homozygotic genotype whereas those infected by the hepatitis B virus had no typical genotype. All the infected patients tolerated and benefited from bleeding as treatment. Discussion: Patients with a history of infectious disease make up 5% of the population of patients with GH and, like the others patients, tolerated bleedings as part of their treatment. There were no differences between the groups in terms of age, sex, or iron overload, although the most frequent genotype was the H63D homozygote and the most frequent infection was by hepatitis C virus. Conclusions: There were no differences in phenotype between GH patients with a history of infection. The most frequent etiologic agent was hepatitis C virus, which was associated with the H63D homozygotic genotype.

Poster# 251 ARTERIAL HYPERTENSION IN PATIENTS WITH GENETIC HEMOCHROMATOSIS Alejandro Del Castillo, MD, Susana Gordo, Teresa Aldámiz, Fernando De la Calle and Luis A. Álvarez-Sala Hospital G. U. Gregorio Marañón (Presented By: Alejandro Del Castillo, MD)

Introduction and Objectives: Iron has a deleterious effect on arteriosclerotic disease by mechanisms of oxidative stress and inflammation . It is directly associated with blood pressure levels, and iron deposits contribute to high blood pressure. We aimed to determine the incidence of arterial hypertension in patients diagnosed with Genetic Hemochromatosis (GH) in a unit that cares for patients with iron metabolism disorders. Methods: The study population was composed of patients diagnosed with GH between 2002 and 2008. We compared the phenotype and the genotype of all patients with GH (general group) with those of patients who had a history of arterial hypertension and were receiving medical treatment at the time of the diagnosis. Results: Of a total of 200 patients diagnosed with GH (71 % men and 29 % women, mean age 49 years [range 13 to 81 and standard deviation (SD) 15.84]), 14 had hypertension (7 % of the total, 10 men [71 %] and 4 women [29 %], mean age 61 years [range 38-81 and sd 12,22]). The hypertensive patients presented higher ferritin levels (660 +/-364 ng/ml vs 545 +/-440 ng/ml) than the general group. The most frequent genotypes in the general group were the C282Y/H63D compound heterozygote (26 %) and the C282Y homozygote (22 %), whereas in the hypertensive patients the most dominant genotypes were the C282Y/H63D compound heterozygote (42 %) and cases with no HFE-associated mutations (35 %). No hypertensive patients had the classic C282Y homozygotic genotype. All the hypertensive patients tolerated and benefited from bleeding as treatment to control their blood pressure. Discussion: Hypertensive patients made up 7 % of the general group and, like the others, tolerated bleeding as part of their treatment.The basis characteristics of this subgroup are older age, greater iron overload, and lack of the classic mutation of the HFE gene (C282Y homozygote). Therefore, iron overload can destabilize the compensating mechanisms of blood pressure. Conclusions: Hypertension affects 7 % of the population with GH.These patients are older than other patients and have a greater iron overload, although this is not associated with the classic gh genotype.

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Poster# 252 SNPS ASSOCIATED WITH IRON INDICES IN A CANDIDATE GENE STUDY OF HEREDITARY HAEMOCHROMATOSIS C.C. Constantine, PhD², C.D. Vulpe, G.J. Anderson, C.E. McLaren, M. Bahlo, H.L. Yeap, K.J. Allen, N.J. Osborne, N.A. Bertalli, D.M. Gertig, M.B. Delatycki, J.K. Olynyk, K.B. Beckman, V. Chen, P. Matak, A.T. McKie, L.C. Gurrin¹ and HealthIron Investigators ¹Centre for MEGA Epidemiology, The University of Melbourne; ²Centre for MEGA Epidemiology, School of Population Health, University of Melbourne (Presented By: L.C. Gurrin)

Background: Haemochromatosis is a disease characterised by excessive absorption of iron. Although more than 80% of clinically diagnosed cases of iron overload are homozygous for the mutation that causes the C282Y substitution in the HFE protein (about 1:200 northern Europeans), many C282Y homozygotes do not develop clinical symptoms of the disease. There is recent evidence from family and twin studies, candidate gene studies and genome wide association studies that common genetic variants influence iron indices. Our aim was to identify single nucleotide polymorphisms (SNPs) that are associated with iron indices and which potentially influence the progression to disease in a prospectively-recruited population-based cohort study. Subjects: The HealthIron study, as part of the Melbourne Collaborative Cohort Study (MCCS), genotyped the C282Y and H63D mutations in 31,192 participants of northern European descent who were recruited from 1990-1994 when aged 40–69 years. An HFE genotype stratified random sample of 1438 people was invited for follow- up between 2004-2006. Participants gave a blood sample to measure iron indices, completed questionnaires, underwent clinical examination by physicians blinded to HFE genotype and provided a cheekbrush sample to confirm HFE genotype. Genotyping: We genotyped (in 863 participants) 476 SNPs from 44 genes encoding proteins involved in iron metabolism. The selection of SNPs for genotyping was based on data available from four sources (i) The International HapMap Project (ii) SeattleSNPs (iii) The NHLBI Resequencing and Genotyping Service and (iv) Resequencing from 94 C282Y homozyogotes and 94 randomly chosen individuals from our own study. Statistical Analysis: Single SNP association testing using a linear regression model adjusted for sex, menopause and HFE genotype (C282Y and H63D) was conducted for four continuous valued outcomes: serum ferritin (natural log transformed), transferrin saturation, serum transferrin, and serum iron. Results: A novel modifier specific to HFE C282Y homozygotes (rs884409) was found in the CYBRD1 gene. Median unadjusted serum ferritin dropped from 1194 (N=27) to 387 µg/L (N=16) in male C282Y homozygotes heterozygous for rs884409, compared to those with no copies, and from 357 (N=42) to 69 µg/L (N=12) in female C282Y homozygotes. After adjusting for sex, menopause and HFE-genotype in the whole sample, the average effect on SF levels of one and two copies of was a reduction of 21% (95% CI (6%,34%), p = 0.008) and 33% (95% CI (-5%,57%), p = 0.082) respectively. Functional testing of this promoter polymorphism in a heterologous expression assay demonstrated significantly decreased promoter activity relative to the more common genotype. Replication: Our results also replicate two of the main findings from a 300K GWAS by Benyamin et al. 2009 AJHG 84:1-6: rs4820268 in TMPRSS6 is associated with lower serum iron and transferrin saturation; and rs3811647 and rs1358024, both TF, r2=0.6, are associated with increased serum transferrin. Conclusion: We report that rs884409 in CYBRD1 is a novel genetic modifier of serum ferritin levels, particularly in C282Y homozygotes, and replicate two findings from a 300K GWAS by Benyamin et al. 2009.

Poster# 253 CELLULAR CHARACTERISATION OF MAMMALIAN MON1A: A PUTATIVE TRAFFICKING PROTEIN AND MODIFIER OF THE HAEMOCHROMATOSIS PHENOTYPE Daniel Wallace, PhD, Lesa Summerville, Nick Barker and Nathan Subramaniam Queensland Institute of Medical Research (Presented By: Daniel Wallace, PhD)

The iron overload disorder hereditary haemochromatosis is a heterogeneous disease caused by mutations in a number of genes involved in either regulation or transport of iron. A marked variation in disease phenotype has been observed between patients with identical mutations, suggesting the presence of genetic and other modifiers of disease. Monensin sensitive protein 1 (Mon1) was first discovered in Saccharomyces cerevisiae in a hypersensitivity screen to drugs that interfere with intracellular trafficking, Brefeldin A and Monensin. Recently, a polymorphism in mouse Mon1A was shown to influence the cell surface levels of the iron exporter ferroportin, thus modifying splenic iron levels in C57BL mice. The mammalian orthologs of Mon1 have yet to be fully characterised. To further understand the role of mammalian Mon1A in protein trafficking and as a modifier of disease we performed a molecular and cellular characterisation of mammalian Mon1A. A specific antibody was generated and used to characterise the expression of Mon1a in mammalian tissues and cell lines. In order to study the expression of Mon1a in mammalian tissues, and human and mouse derived cells lines, western blotting, real-time RTPCR and immunofluorescence were employed. Western blotting of Mon1A in mouse tissues, and in human and mouse cell lines showed similar expression levels across these sample types. Real-time RT-PCR showed that Mon1A RNA expression was ubiquitous in all human cell lines and tissues studied. Immunofluorescence and confocal microscopy analysis of an epitope-tagged Mon1A construct transfected into Huh7 liver cells and endogenous Mon1a in a number of cell lines showed a perinuclear localisation with some cell surface expression. Studies with antibodies against markers of intracellular organelles showed significant co-localisation with endoplasmic reticulum markers. The ubiquitous expression of Mon1A suggests that it plays a fundamental role that is required in most cell and tissue types. The cellular localization studies indicate Mon1A is a protein localised intracellularly and on the cell surface, and may itself be involved in regulating the trafficking of proteins to the cell surface. Mon1a may play a role as a potential modifier by affecting protein trafficking of molecules involved in iron regulation and transport and thus influence the haemochromatosis phenotype.

Poster# 254 HFE, SLC40A1, HAMP, HJV, TFR2, AND FTL MUTATONS DETECTED BY DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AFTER IRON PHENOTYPING AND HFE C282Y AND H63D GENOTYPING IN 785 HEIRS STUDY PARTICIPANTS James Barton, MD¹, Susie DelRio-LaFreniere, MS², Catherine Leiendecker-Foster, MS², Honggui Li, MS², Ronald Acton, PhD³, Richard Press, MD, PhD4 and John Eckfeldt, MD, PhD² ¹Southern Iron Disorders Center; ²University of Minnesota, Minneapolis, MN; ³University of Alabama at Birmingham, Birmingham, AL; 4Oregon Health & Science University, Portland, OR (Presented By: James Barton, MD)

Introduction: HFE mutations other than C282Y and H63D or mutations in other iron-related genes could explain iron phenotype heterogeneity in adults. Participants and Methods: We converted initial screening transferrin saturation (TS) and serum ferritin (SF) levels of HEIRS Study participants into gender-specific and HFE C282Y and H63D genotype-specific percentile values. From the 333 available HFE C282Y homozygotes, we selected 75 with the highest TS and SF (high TS/SF) and another 75 with the lowest TS and SF (low TS/SF) using aforementioned criteria. We selected a control group of 76 C282Y homozygotes randomly with no constraints on gender, race/ethnicity, or Field Center. We did not exclude those chosen for either the high or low TS/SF groups from also being selected for the control group. We also selected participants from each racial/ethnic group who had the highest percentile for either TS or SF (high TS/SF), because we had no a priori reason to know whether mutations discovered by denaturing high-performance liquid chromatography (DHPLC) would influence TS or SF more strongly (74 whites, 75 Hispanics, 74 blacks, 72 Asians). For controls, we selected other participants from the same racial/ethnic groups without regard for initial screening TS and SF values (75 whites, 74 Hispanics, 75 blacks, 75 Asians). Using DHPLC, we screened 20 regions of HFE, SLC40A1, HAMP, HJV, TFR2, and FTL in each of 791 participants. The regions we screened were: HFE exons 2, 3, and 4; SLC40A1 exons 3, 5, and 6; HAMP promoter and exons 1, 2, and 3; HJV exons 1, 2, 3, and 4; TFR2 exons 2, 4, 5, and 6; and FTL exons 1 and 2. Results: DHPLC analyses were successful in 785 participants (99.3%) and detected 117 different mutations. In C282Y homozygotes, 4.0% of high TS/SF participants had SLC40A1 Q248H, HAMP -72C>T, or HAMP R59G heterozygosity (0% controls; p = 0.1200). In whites, 4.1% with high TS/SF and 1.3% of controls had HFE S65C or E168Q (p = 0.3049). HJV c.-6C>G and FTL L55L frequencies were greater in whites with high TS/SF than controls (0.0811 vs. 0.0200, p = 0.0144; 0.5743 vs. 0.4400, p = 0.0204, respectively). One Hispanic with high TS/SF (1.3%) had HAMP G71D heterozygosity. In blacks, SLC40A1 Q248H frequencies did not differ significantly between high TS/SF and control participants. Among Asians, 2.8% with high TS/SF were HFE V295A heterozygotes.

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Conclusions: Mutations other than HFE C282Y and H63D reported to be pathogenic were infrequently detected in high TS/SF participants. Genetic regions in linkage disequilibrium with HJV c.-6C>G and FTL L55L could partly explain high TS/SF phenotypes in whites.

Poster# 255 MUTATION ANALYSIS OF SEVEN GENES INVOLVED IN IRON METABOLISM IN A MENNONITE POPULATION Samuel Krikler, MBChB¹, Chris Whittington, MBChB¹, Michelle-Angelique Hallendorff, MSc², Natalie Strickland, MSc², Veronique Human, MSc², Nathaniel McGregor, HonsBSc², Jessica Vervalle, HonsBSc² and Monique Zaahl, PhD² ¹University of British Columbia; ²Stellenbosch University (Presented By: Samuel Krikler, MBChB)

Background: Hereditary hemochromatosis (HH) is a common autosomal recessive disease and is characterized by increased iron absorption leading to progressive iron accumulation in organs such as the liver, heart and pancreas. The disease has been well-characterized in the Caucasian population from Northern European descent, with homozygosity for the HFE gene C282Y mutation being associated with the iron overload observed in the majority of cases. The Mennonites are an Anabaptist group who originated from northern Europe approximately 400 years ago. They are mainly of Dutch/North German origin, with less than 400 family names. Iron overload observed in these patients are not all explained by the presence of the C282Y mutation or compound heterozygosity (C282Y/H63D) and other modifier genes have previously been implicated. The aim of this study was to identify modifier loci in Mennonites with significant iron overload which could not be accounted for by C282Y mutations alone. Methods: Polymerase chain reaction (PCR) amplification of the promoter and coding region of the genes HFE, HMOX1, SLC40A1, HAMP, CYBRD1, CP and HJV was performed. This was followed by mutation analysis by means of heteroduplex single-stranded conformation polymorphism (HEX-SSCP) analysis and subsequent bi- directional semi-automated DNA sequencing analysis. DNA analysis was performed on samples from 14 individuals, including 3 unrelated families with 7, 4 and 2 family members respectively (including probands), and one other unrelated individual. All individuals who participated in the study tested either negative or heterozygous for C282Y. Results: Fourteen previously described and six novel variants were identified. The novel variants include the following: HFE: -970G/T, IVS1+9G/A; SLC40A1: -750G/A; HAMP: E36K; and CP: IVS2+20C/T, H977. These variants were identified in both a heterozygous and homozygous state. Further analysis of the three families demonstrated common haplotypes in the HFE and SLC40A1 genes that inherits with the iron overload phenotype in these families. The majority of variants comprising these haplotypes are located within the promoter region of these genes. Conclusion: Transcriptional regulation of gene expression has previously been shown to be influenced by variants in the promoter region of a gene. The promoter variants comprising the haplotypes identified in the families investigated were shown, by means of in silico analysis, to disrupt putative transcription binding sites. This would, in turn, lead to differential expression of the respective genes, therefore possibly playing a role in the iron overload observed in these families. Screening of the variants identified in general Mennonite population as well as functional analyses of the promoter variants should lead to improved diagnosis and treatment of Mennonites presenting with iron overload.

Poster# 256 SYNTHESIZING THE EVIDENCE FOR THE ASSOCIATION BETWEEN THE G277S SUBSTITUTION IN THE TRANSFERRIN GENE AND IRON DEFICIENCY BY COMBINING DATA FROM POPULATION STUDIES L.C. Gurrin, N.A. Bertalli and C.C. Constantine Centre for MEGA Epidemiology, The University of Melbourne (Presented By: L.C. Gurrin)

Background: There is evidence from one large cross-sectional study that the G277S substitution in the Transferrin (TF) gene is a risk factor for iron deficiency anaemia in white, pre-menopausal women. Two further studies examined the effect of G277S on iron indices but the results were equivocal due to the small sample sizes. Data are also available on this association from the HealthIron study of genetic and environmental modifiers of hereditary haemochromatosis which is on-going in Melbourne, Australia. This project aimed to use the statistical techniques of meta-analysis to combine data from the HealthIron study with existing published results on the association between mean levels of serum ferritin (SF) and the G277S substitution in TF to provide greater precision in determining the magnitude of the association. Data: Data are available from three published studies on genotype-specific mean SF levels: Lee et al. (2001, Brit.J.Haem. 115, 329-333, n = 1987), Delanghe et al. (2003, Brit.J.Haem. 132, 244-250, n = 91) and Sarria et al. (2007, Eur.J Nut., 46, 57–60, n = 25). In one study these means are not presented separately for pre- and post- menopausal women although the proportion of women below a fixed serum iron threshold is available for premenopausal women. In another study data are presented only for women below a similar fixed serum iron threshold. The HealthIron study as part of the Melbourne Collaborative Cohort Study (MCCS) genotyped C282Y and H63D in 31,192 participants of northern European descent who were recruited from 1990-1994 when aged 40–69 years. An HFE genotype stratified random sample of 1,438 people were selected for invitation to follow-up between 2004- 2006. Participants gave a blood sample to measure iron indices, completed questionnaires, underwent clinical examination by physicians blinded to HFE genotype. Data on baseline and follow-up SF and genotype for the G277S substitution were available for n = 450 women. Statistical Analysis: Linear regression models were fitted to data from the HealthIron study to estimate differences in mean SF between G277S genotype groups while including HFE-genotype and menopausal status as covariates. Probit regression assuming an underlying log-normal distribution for SF was used to model published data on the proportion of women with SF measurements below a fixed threshold, results of which were also used to estimate differences in mean SF between G277S genotype groups. These results and published data on genotype-specific mean SF levels were combined in a meta-analysis using the WinBUGS statistical software to generate overall estimates of the difference in mean SF between genotype groups defined by the G277S substitution. Results: Women with one copy of the G277S substitution have serum iron levels that are on average 11.8% lower (s.e. 5.50%, p=0.032) than women who are wild-type for the G277S substitution. There is some evidence that the effect is greater in pre-menopausal women (13.0% lower, s.e. 6.90% p=0.060) that in post-menopausal women (7.50% lower, s.e. 15.8%, p=0.64). Conclusion: The G277S substitution is associated with lower mean levels of serum ferritin in pre-menopausal women, but the magnitude of this effect is mild and unlikely to be of clinical relevance.

Poster# 257 CHANGES OF THE EXPRESSIONS OF THE GENES INVOLVED IN IRON METABOLISM BY THE IRON CHELATION THERAPY AND PHLEBOTOMY IN THE IRON OVERLOADED MOUSE MODEL Takaaki Hosoki, MD, PhD, Katsuya Ikuta, MD, PhD, Naoka Okamura, MD, Motohiro Shindo, MD, PhD, Kazuya Sato, MD, PhD, Takaaki Ohtake, MD, PhD, Katsunori Sasaki, PhD, Yoshihiro Torimoto, MD, PhD and Yutaka Kohgo, MD, PhD Asahikawa Medical College (Presented By: Takaaki Hosoki, MD, PhD)

Introduction: Iron chelation therapy and phlebotomy have been widely applied for iron overload. On the other hand, many new molecules involved in iron metabolism have been discovered over a decade, and the understanding of the molecular mechanisms of iron metabolism has progressed. However, the changes of the genes involved in iron metabolism in iron chelation therapy and phlebotomy have not been fully investigated. Therefore, the aim of this study is to clarify the responses of the body and the mechanisms of iron removal by iron chelator DFO and phlebotomy. We investigated the changes of genes involved in iron metabolism in the important organs, such as the liver and the gastrointestinal tract, at the early stage of iron removal by treating the iron overloaded mouse with DFO and phlebotomy. Methods: Iron overloaded mouse model was made by giving iron dextran interperitonealy (ironloaded group). DFO was given for the iron overloaded mice regularly for four weeks interperitonealy (iron-loaded + DFO group). Phlebotomy (100 µL/ week) was performed for four weeks (iron-loaded + phlebotomy group). Mice without any treatment were participated as the control. After 4 weeks, mice were sacrificed, and the liver, small intestine, spleen, and blood were collected from all mice. Total RNA was then purified, and quantitative RTPCR (qRT-PCR) was performed for genes involved in iron metabolism. The investigated genes were HFE, transferrin receptor 1 (TfR1), 180 transferring receptor 2 (TfR2), hepcidin-1 (hamp1), hepcidin-2 (hamp2), ferroportin 1 (FPN1), divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), hephaestin and ferritin. Results: There was no significant difference in the physique, the weight of the liver and spleen, serum iron concentration among all groups of mice. Hemoglobin was decreased in the phlebotomy group. The serum ferritin was remarkably increased in iron-loaded group, and there was no decline of serum ferritin in the iron-loaded + DFO and +phlebotomy group. Although the expressions of HFE, TfR2, hamp1, hamp2 in the liver didn’t show significant difference among each of the groups by qRT-PCR, the ferritin mRNA was decreased in the iron-loaded + DFO and +phlebotomy group, and FPN1 mRNA was decreased significantly in the iron-loaded + DFO group compared to the iron-loaded group. In the duodenum, there was no difference in the expressions of DMT1, Dcytb, hephaestin, but the expressions of ferritin mRNA was decreased in the iron-loaded + DFO and +phlebotomy group, and FPN1 mRNA was decreased significantly in the iron-loaded + DFO group. Conclusion: The decreases of ferritin mRNA at both of the liver and the duodenum might imply that intracellular free iron was removed by iron chelator DFO and phlebotomy. Concerning the decline of FPN1 mRNA, there might be a possibility that intracellular iron depletion by DFO cause the inhibition of translation of FPN1 mRNA via iron responsive element which exists in 5’-untranslated region of FPN1 mRNA. Iron chelation would lead to decreased iron uptake from the duodenum, and this would be preferable effect of DFO in the view of iron chelation therapy for iron overload.

Poster# 258 COMPARATIVE ABILITY OF IRON CHELATORS TO REMOVE IRON FROM ALBUMIN-IRON (III) COMPLEXES Jay Parshotam, BSc (Hons)¹, André Silva², Xiao Kong³ and Robert Hider4 ¹KCL/ PhD student; ²KCL/PhD student; ³KCL/PhD; 4KCL/Professor (Presented By: Jay Parshotam, BSc (Hons))

The number of patients suffering from iron-overloaded conditions remains at a high level. Deferiprone, Desferrioxamine and Exjade, all clinically approved drugs, have been shown to restore iron balance. In normal conditions, serum iron will be mainly bound to transferrin but in patients suffering from iron-overloading disorders such as thalasseamia, iron will also be found in the non-transferrin form which is toxic, leading to an inappropriate distribution of iron and organ failure. The main low molecular weight ligand for iron (III) is citrate. Albumin is the most prevalent protein in serum and will also bind iron (III). Previous studies have shown that some modified forms of serum albumin possess an increased affinity for iron (III). In the present study, iron citrate was incubated with non-modified, glycated and oxidized forms of albumin. These solutions were then incubated with the chelators mentioned above, at different concentrations to verify to what extent iron could be removed from the albumin. Spectrophotometric determinations and LC coupled ICP-MS were used to assess the rate of iron transfer.

Poster# 259 H-FERRITIN SIRNA DELIVERED BY CATIONIC LIPOSOMES INCREASES GLIOMA CELL SENSITIVITY TO RADIATION AND CHEMOTHERAPEUTIC AGENTS Xiaoli Liu, MS, A.B. Madhankumar, PhD, Jonas Sheehan, MD, Becky Slagle-Webb, BSc and James Connor, PhD Penn State Hershey Medical center (Presented By: James Connor, PhD)

Approximately fifty percent of gliomas are resistant to radiation and chemotherapy. Therefore, efforts into the development of new mechanisms to enhance efficacy of treatment strategies are clearly warranted. We hypothesized that potential targets that will disrupt iron homeostasis in tumor cells could prove effective at disrupting tumor growth because cancer cells require high levels of iron to meet the increased energy requirements associated with rapid growth. Ferritin, is considered an intracellular iron storage protein, but also has been found in the nucleus of tumor cells where it appears to protect DNA from damage and promote transcription. Consequently, we hypothesized that silencing H-ferritin gene expression could increase the sensitivity of gliomas to radiation and chemotoxins. To test this hypothesis, H-ferritin siRNA was delivered to U251 human astrocytoma cells via cationic liposomes. The siRNA delivered in this manner decreased H-ferritin protein expression by 50 % within 24 hours. The decrease in H-ferritin was associated with a decrease in the LD50 for carmustine (BCNU) from greater than 100 µM to 38 µM in the U251 cells. In addition, the presence of Hferritin siRNA was associated with a 50% increase in cell death of U251 cells at 20 Gy of radiation. The in vivo efficacy of siRNA delivered by cationicliposomes was tested in the athymic nude mouse subcutaneous glioma tumor model. Intratumorally injections of liposomes containing H-ferritin siRNA reduced the required effective dose of BCNU for tumor suppression by more than 50%. Thus our data demonstrated that silencing the H-ferritin gene appears to be an effective way to sensitize tumors to radiation and chemotherapy. As importantly, we have demonstrated that siRNA can be delivered effectively by cationic liposomes in an in vivo tumor model.

Poster# 260 A COMPARATIVE STUDY OF THE ANTIPROLIFRATIVE ACTIVITY OF IRON CHELATORS PIH, SIH AND THEIR PHOTOLABILE CAGED- DERIVATIVES IN SKIN CELLS Charareh Pourzand, PhD, Asma Aroun, PhD² and Olivier Reelfs, PhD Department of Pharmacy and Pharmacology, Bath University, Bath, UK (Presented By: Charareh Pourzand, PhD)

Because of the crucial role of iron in cell proliferation, iron chelation therapy has become an attractive alternative for the treatment of hyper-proliferative disease, notably cancer. The antiproliferative activity of the pyridoxal isonicotinoyl hydrazone (PIH) and salicaldehyde isonicotinoyl hydrazone (SIH) is well documented; however prolonged systemic administration of iron chelators (ICs) causes severe side effects, which currently limits its applicability. To overcome this problem, smart ICs are needed that may be selectively switched on in the diseased tissues. For this purpose, we have recently synthesized simple prototypical compounds derived from SIH and PIH elaborated with ortho-nitrobenzyl caging group (2NPE). Initial in vitro (skin culture) and in vivo (animal) studies in the field of skin-photoprotection have revealed that unlike the parent compounds, the caged-ICs (i.e. 2NPE-SIH and –PIH) do not diminish the normal labile iron pool in cells. However, exposure of caged-ICs-treated skin to a physiologically relevant dose of UVA subsequently provides promising levels of protection against UVA-mediated iron-induced oxidative damage and necrotic cell death. In the present study, we have investigated the potential of these caged-ICs as powerful alternative antiproliferative agents for skin cancer. The spontaneously immortalised human keratinocyte cell line, HaCaT has proved a useful and reliable in vitro model of human skin cell carcinoma. This cell line is hyperproliferative and shows a significantly higher proliferation rate compared to normal human skin keratinocytes and fibroblasts. Here we characterised HaCaT cell responses to SIH, PIH and their caged-derivates 2NPE-PIH and 2NPE-SIH followed or not by exposure to extremely low doses of UVA: The MTT assay demonstrated that SIH has a more pronounced growth inhibitory effect than PIH. In the absence of UVA, 2NPE-PIH and –SIH did not alter the growth of HaCaT cells, but following UVA irradiation provided a significant decrease in growth rate of cells that was comparable to the effect observed with the parental chelators alone. The UVA-irradiated controls did not alter the growth of cells, indicating that the effect observed in caged-treated irradiated cells is unrelated to radiation but rather related to uncaging of the 2NPE-caging group and release of active antiproliferative ICs. In an attempt to relate these findings to an in vivo setting, the colony forming ability assay was performed in parallel and the results showed that in agreement with MTT data, both PIH and SIH have strong antiprolifrative activity. Interestingly, the colony forming assay revealed that the antiproliferative action of UVA- irradiated caged-ICs was much superior to that of their parental compounds. We also analysed the impact of the chelators on the cell cycle by flow cytometry, using bromodeoxyuridine incorporation. The results showed that both parental PIH and SIH and their UVA-irradiated caged-derivatives have effective impact on cell cycle. Taken together, these results suggest that our newly developed caged-ICs which remain inactive inside the cells until their iron-binding capacity is activated by exposure to an external light source are effective antiproliferative agents. This novel family of compounds offers an alternative treatment for hyperproliferative skin disorders such as skin cancer, and could be used in protocols involving straightforward topical application on skin and selective activation by low doses of UVA.

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Poster# 261 L-TYPE CALCIUM CHANNEL BLOCKER, VERAPAMIL, IMPROVES SURVIVAL AND REDUCES IRON LOADING IN HYPOTRANSFERRINAEMIC MICE Patarabutr Masaratana, MD, Abas Laftah, PhD, Gladys Oluyemisi Latunde-Dada, PhD, Robert Simpson, PhD and Andrew McKie, PhD King’s College London (Presented By: Patarabutr Masaratana, MD)

Background: Parenchymal iron loading through nontransferrin bound iron (NTBI) uptake can lead to tissue injury and organ dysfunction. L-type calcium channels have been shown to transport NTBI into cardiomyocytes. Recent studies showed that L-type calcium channel blockers administration to iron loaded mice lessened cardiac iron content and improved cardiac function. This study aimed to explore the effect of an L-type calcium blocker, verapamil, in genetic iron loading models, hypotransferrinaemic (HPX) and Hepc1 knockout mice. Methods: HPX mice and normal littermates were treated with either 0.1 mg/ml verapamil in 2.5% dextrose as drinking water (treatment) or 2.5% dextrose alone (control). After 4 weeks treatment, tissue nonhaem iron content was measured. Prussian blue stain (for iron) was performed on paraffinembedded tissues. Similar experiments were performed in Hepc1 knockout mice and littermates for 8- or 12-week duration. Results: The survival rate in treated HPX was higher than control HPX mice. Verapamil treatment in HPX & normal littermates was associated with significantly reduced nonhaem iron in the heart, spleen and lung in normal and decreased nonhaem iron in the pancreas of HPX mice. Prussian blue staining suggested decreased iron accumulaion in many viscera of treated HPX mice compared to control counterpart. In contrast, the treatment had no effect in Hepc1 knockout and littermates. Conclusion: This study showed beneficial effects of verapamil in severe iron loading as demonstrated by increased survival and decreased iron loading in HPX mice. However, the effects appeared to be strain specific as no response was found in Hepc1 knockout mice strain.

Poster# 262 IRON DEPLETION THERAPY FOR PATIENTS WITH METABOLIC SYNDROME AND NON-ALCOHOLIC FATTY LIVER DISEASE: PRELIMINARY FINDINGS James Nelson, PhD¹, Kristina Utzschneider, MD², Bryan Maliken, BS¹, Jacob Alexander, MD² and Kris Kowdley, MD³ ¹Benaroya Research Institute; ²University of Washington Medical Center; ³Benaroya Research Institute and Virginia Mason Medical Center (Presented By: James Nelson, PhD)

Background: Insulin resistance is a prominent feature of a number of conditions including diabetes, metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). While iron and insulin metabolic pathways share a complex and reciprocal relationship, the role of iron in the progression of these conditions remains unclear. The goal of this pilot study is to determine the effect of iron depletion on insulin sensitivity in patients with the metabolic syndrome and NAFLD. Methods: Participants with NAFLD and the metabolic syndrome, defined by the World Health Organization criteria (at least 3 of the following; elevated waist circumference, elevated triglycerides, reduced HDL cholesterol, hypertension and elevated fasting blood glucose), who were enrolled into the NASH Clinical Research Network Database Study were eligible to participate. Hepatic and peripheral insulin sensitivity were measured using a 6,6 2-d glucose labeled two-step hyperinsulinemic euglycemic clamp. Additional study procedures included a 2 hr oral glucose tolerance test (OGTT), a CT scan for intrahepatic fat (liver/spleen ratio) and a DEXA scan for body fat composition. Fasting blood and urine samples were taken for analysis of lipids, aminotransferases, cytokines and oxidative stress markers. After completing baseline study procedures, subjects underwent repeated bimonthly phlebotomies until iron depleted (i.e., serum transferrin saturation ≤10% and/or serum ferritin ≤50µg/L). Study procedures were repeated following iron depletion and again 6 months later. Results: To date seven subjects are enrolled, four subjects have completed the first two visits and three subjects have completed all three study visits. The following treatment outcomes were improved in 3 out of 4 subjects after phlebotomy treatment: ALT, AST, total cholesterol, serum triglycerides, and LDL cholesterol. Improvement from baseline values for ALT, AST and total cholesterol remained for at least 6 months following iron depletion in two out of three subjects, while improvements in serum triglycerides and LDL cholesterol persisted for 6 months in one subject each. Insulin resistance as determined by the Homeostasis Model Assessment (HOMA-IR = fasting insulin x fasting glucose/22.5) was improved in 3 out of 4 subjects after phlebotomy. Based on clamp data (n=3), hepatic insulin sensitivity, determined as basal endogenous glucose production/fasting insulin, and peripheral insulin sensitivity, determined as the glucose infusion rate during the high dose insulin infusion, improved in 2 of 3 subjects following phlebotomy. Phlebotomy treatment improved β-cell function calculated from the OGTT using several different methods including A) incremental area under the curve insulin response from 0-120 minutes (incAUCinsulin/incAUCglucose) in all four subjects post-phlebotomy and 2/3 after 6 months; B) early insulin response from 0-30 minutes (∆Ins0-30/∆Glu0-30) in 3/4 subjects postphlebotomy and 2/3 after 6 months; C) oral disposition index (∆Ins0-30/∆Glu0-30 x 1/HOMA-IR) in all four subjects post-phlebotomy and 1/3 after 6 months. There were no changes among subjects in intrahepatic fat, body fat composition or levels of the oxidative stress markers, malondialdehyde and F2-isoprostane. Conclusions: Our preliminary findings suggest that iron depletion via phlebotomy therapy may improve both hepatic and peripheral insulin sensitivity, β-cell function, lipids and aminotransferases in patients with metabolic syndrome and NAFLD.

Poster# 263 HIGH THROUGH-PUT SCREENING OF CHEMICALS THAT STIMULATE IRON UPTAKE – A NOVEL APPROACH TO DISCOVERY OF ANTI- CANCER DRUGS Zhen Li, PhD, Hiroki Tanaka, PhD, Hong Yin, PhD, Kwo-Yih Yeh, PhD, Mary Yeh, PhD and Jonathan Glass, MD Feist-Weiller Cancer Center, LSUHSC (Presented By: Jonathan Glass, MD)

Introduction: Actively growing cells, including cancer cells, appear to have a greater need for iron as measured by increased expression of the transferring receptor (TfR). Attempts have been made to use iron chelators to deplete cells of iron as a means of decreasing the growth of cancer cells. We hypothesized that a contrary approach of enhancing iron entry would allow for generation of increased reactive oxygen species which could potentiate radiation and radiomimetic drugs and accelerate hydroxylation of HIF1α and HIF2α decreasing HIF activity, modifying a driving force for cancer cell proliferation. Methods:To identify small molecules that would accelerate iron uptake we used a high through-put screening system in conjunction with a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. Small molecules that stimulate Fe uptake were defined as causing an increase of calcein fluorescence quenching compared to Fe alone. Results: An in-house chemical library of ~10,000 compounds was screened and 30 chemicals identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose response curves and the ability to increase ferritin levels when the sole source of iron was FeTf. One of the chemicals, LS-0108076, effectively increased iron uptake into the prostate cancer line, PC3, and increased ROS as measured with DCFDA by 150±18%. Cell growth of K562, PC3, and the breast cancer cell line MDA-MB-231 cells decreased when incubated with iron facilitators and Fe2Tf. Following radiation LS-0108076 and other facilitators induced increased DNA strand breaks in PC3 cells as measured by comet assay. LS-0108076 also enhanced bleomycin cell killing both in PC3 and DU145 prostate cancer cell lines but not normal prostate cells, and potentiated radiation induced cell death. Finally, LS-0108076 markedly decreased levels of HIF1α and HIF2α in K562 and PC3 cells. Similar effects including increased bleomycin and radiation sensitivity and decreased HIF1α and HIF2α, were seen with the MDA-MB-231 cells but not normal breast cell lines. Discussion: In summary, we have developed a high through-put screening technique that identified small molecules that stimulate iron uptake both from ferriTf and non-Tf bound iron and demonstrated that compounds which facilitate iron uptake increase cell sensitivity to radiation therapy and chemotherapy and decrease HIF1α and HIF2α expression. These effects were apparent in both prostate and breast cancer malignant cell lines but not in the non-malignant counterparts. As the effects were seen with Fe2Tf these iron facilitators must be acting on some aspect of receptor mediated endocytosis. Conclusion: The results support the concept of increased levels of ROS in cancer cells and that further increases in ROS may lead to cell death. Hence, these iron facilitator chemicals may serve as anti-cancer agents.

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Poster# 264 FRIEDREICH’S ATAXIA: PROTEIN TRANSDUCTION OF TAT-CONJUGATED FRATAXIN MAY BE A NOVEL THERAPY Hannes Steinkellner, Barbara Scheiber-Mojdehkar, Dr, Franco Laccone, Dr, Hans Goldenberg, Dr and Brigitte Sturm, Dr Medical University of Vienna (Presented By: Hannes Steinkellner)

Introduction: Friedreich’s ataxia (FA) is an autosomal recessive neurodegenerative disease. It is caused by a GAA trinucleotide expansion in the frataxin gene located on chromosome 9q13, resulting in a reduced expression of frataxin. However, the exact physiological function of this small protein is still unknown. Nevertheless, frataxin deficiency is involved in several aspects of intracellular iron metabolism, low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression. Laccone and co-workers have used a Transactivator of Transcription (TAT) from HIV to develop a frataxin fusion protein, which is able to deliver frataxin into mitochondria. Methods: Fibroblast cell lines from Friedreich’s ataxia patients and age and sex-matched controls were used for all experiments. Aconitase activity was determined by an adaption of the indirect method using the reduction of nicotinamide adenine dinucleotide phosphate (NADPH) by isocitric dehydrogenase. SDH activity was measured spectrophotometrically. Frataxin-expression was measured byelectrochemiluminescence — immunoassay (ECLIA). Results: We showed that TAT-Fx can compensate Fx-deficiency in a concentration dependent manner. Turnover of TAT-frataxin was time-dependent. We also examined the influence of TAT-Fx on aconitase and SDH−activity. Discussion: The aim of this study was to investigate functional and stability properties of exogenously applied TAT-frataxin. Our study shows that TAT-frataxin can be delivered into mitochondria of fibroblasts from FA patients using the protein transduction domain TAT-frataxin and can take over functions of endogenous frataxin at least partially. Conclusion: Our results indicate that TAT-frataxin should be further investigated to develope a novel therapy for the treatment of FA using protein transduction.

Poster# 265 POTENT IRON CHELATORS AS ANTI-TUMOR AGENTS: THE NOVEL 2-ACETYLPYRIDINE THIOSEMICARBAZONE SERIES Danuta Kalinowski, BSc, PhD, V. Richardson, P.C. Sharpe, D.B. Lovejoy, M. Islam, P.V. Bernhardt and D.R. Richardson University of Sydney (Presented By: Danuta Kalinowski, BSc, PhD)

Introduction: Utilizing iron (Fe) chelators as a means of inhibiting DNA synthesis represents a novel chemotherapy treatment strategy (Richardson, DR et al. PNAS USA 2006, 103, 14901-6). Chelators act by inhibiting the activity of Fe-containing enzymes, while some Fe chelator complexes demonstrate redox cycling ability, generating damaging reactive oxygen species (ROS). The previously developed di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) series of Fe chelators illustrate potent in vivo anti-proliferative activity through redox cycling. In an attempt to examine the effect of substituents at the imine carbon on anti-tumor and redox activity, we developed novel DpT analogues, the 2-acetylpyridine thiosemicarbazone (ApT) series. Methods: Through this strategy, the electron-withdrawing pyridine ring of the DpT series was replaced by an inductively-donating methyl group in the ApT chelators by the Schiff-base condensation of 2-acetylpyridine with thiosemicarbazides. The ability of the ApT Fe complexes to redox cycle was measured through cyclic voltammetry and the oxidation of ascorbate. Additionally, the anti-proliferative activity of these novel chelators was assessed via the MTT assay and their ability to mobilize intracellular Fe was examined through 59Fe efflux and uptake studies in the SK-N-MC neuroepithelioma cell line. Results and Discussion: Four of the six ApT chelators had potent anti-tumor activity (IC50: 0.001-0.002 µM) and demonstrated potent Fe chelation efficacy that was similar to the most effective BpT and DpT ligands. The ApT Fe complexes had the lowest Fe(III/II) redox potentials of any thiosemicarbazone series generated by our group and maintained the ability to oxidize ascorbate, having activity comparable to the DpT series, but less than the BpT analogues. This suggests that redox cycling and generation of ROS may play a role in the anti-proliferative activity of the ApT series. Conclusion: These results demonstrate that the ApT series is able to act via a “double punch” mechanism, where the ligand is able to induce anti-proliferative activity through (1) efficient Fe-depletion and (2) the generation of damaging ROS. This property makes the ApT series one of the most potent anti-cancer agents developed by our group that are suitable for further clinical development.

Poster# 266 FRATAXIN-EXPRESSION IS INCREASED BY RECOMBINANT HUMAN ERYTHROPOIETIN INDEPENDENT OF EXPRESSION OF THE CLASSICAL ERYTHROPOIETIN RECEPTOR Melissa Helminger¹, Hannes Steinkellner, Msc², Hans Goldenberg, PhD², Brigitte Nina Sturm, PhD² and Barbara Scheiber-Mojdehkar, PhD² ¹Medical University of Vienna; ²Department of Medical Chemistry, Medical University of Vienna, Austria (Presented By: Melissa Helminger)

Background: Friedreich’s ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the mitochondrial protein frataxin, described to be an iron chaperone for the assembly of iron-sulfur clusters in the mitrochondria causing iron accumulation in mitochondria, oxidative stress and cell damage. Recently we showed that recombinant human erythropoietin (rhuEPO) significantly increases frataxin expression by a still unknown mechanism. In this study we investigate the role of the classical erythropoietin receptor (EPO-R) in the frataxin- increasing effect of rhuEPO. Materials and Methods: Expression of the EPO-R in human erythroleukaemic K562 cells and human monocytes THP-1 cells was detected by western blot. In the experiments K562-cells, THP-1-cells as well as primary lymphocytes from control and FRDA patients were incubated with different concentrations of rhuEPO. Frataxin expression was detected by an electrochemical luminescence assay (ECLIA) and real-time RT-PCR for frataxin-mRNA. Results: Western blot analysis confirmed expression of EPO-R in K562 cells, but complete absence of EPO-R expression in THP-1 cells. However the increase in frataxin protein expression following treatment with rhuEPO correlated with the concentration of rhuEPO used in both cell lines, comparable to the effect of rhuEPO on frataxin expression in primary lymphocytes from control and FRDA patients. RhuEPO increased frataxin expression without increasing frataxin-mRNA. Discussion: We investigated if the frataxin increasing effect of rhuEPO is mediated via the classical EPO-R. To test if EPO binding to the classical EPO-R is an essential step to mediate EPO`s effect on frataxin-expression we used two cell culture models: one expressing the classical EPO-R, the erythroleukaemic K562 cells and a cell line not expressing this receptor, human monocyte THP-1 cells. We found that rhuEPO increased frataxin expression in both cell lines, which indicates that the effect of rhuEPO on frataxin expression is not limited to cells expressing the classical EPO-R. RhuEPO had no effect on frataxin mRNA suggesting that the observed increase in frataxin protein is attributable to a posttranslational mechanism. Conclusion: RhuEPO increases frataxin expression by a mechanism without involvement of the classical EPO-R. The results of this study provide a scientific basis to further examine the effectiveness of non-erythropoietic EPO-derivatives which do not bind to the classical EPO-R as a possible treatment option for FRDA patients. 183

Poster# 267 MOLECULAR IMPACT OF HUMAN DIVALENT METAL-ION TRANSPORTER-1 (DMT1) MUTATIONS ASSOCIATED WITH DISEASE PHENOTYPES Ali Shawki, Anthony C. Illing and Bryan Mackenzie University of Cincinnati College of Medicine (Presented By: Ali Shawki)

DMT1 is a H+-coupled divalent metal-ion transporter critical for intestinal iron (Fe) absorption and erythroid Fe utilization. One of our core objectives is to understand the structure-function of DMT1, and identify critical residues and structural elements responsible for metal-ion binding and H+ coupling. Our present approach is to study the molecular impact of human DMT1 mutations associated with disease phenotypes. Recent reports describe three unrelated cases in which DMT1 mutations were associated with severe microcytic anemia and hepatic iron overload. We expressed mutant and wildtype (wt) DMT1 in Xenopus oocytes and used voltage-clamp and radiotracer assays to test the hypothesis that the identified mutations disrupt iron transport. Proband 1 [Mims et al (2005) Blood 105, 1337] is homozygous for a G>C mutation that results in a Glu-to-Asp substitution (E399D) and increased skipping of exon 12. We found that E399D-DMT1 functioned like wtDMT1 except for a modest reduction in apparent 2+ affinity for Fe (K0.5 = 2.1 ± 0.3 µM; wtDMT1, 1.0 ± 0.4 µM). The phenotype of the patient homozygous for this mutation likely arises from the increased frequency of an aberrant splice variant lacking exon 12, since the exon-12-deleted construct lost 55Fe2+ transport activity in oocytes. Proband 2 [Iolascon et al (2006) Blood 107, 349] is compound heterozygous for a 3-bp deletion in intron 4 and a substitution (R416C) at a conserved amino acid residue. R416C significantly lowered Fe2+ transport activity in 2+ + -1 oocytes without a loss of affinity for Fe or H . The reduced Vmax resulted largely from a reduction in the turnover rate of the transport cycle (7 s ) compared with wtDMT1 (23 s-1, 30 °C); however, 30-35% of the mRNA in this patient also arises from abnormal splicing that deletes 40 amino acids in exon 5, and we found that exon-5 deletion abolished Fe2+ transport activity in oocytes. Proband 3 [Beaumont et al (2006) Blood 107, 4168] is compound heterozygous for a single amino acid deletion (V114) and a substitution (G212V). The proband’s sibling and parents each possess one mutated allele but were asymptomatic. V114 deletion abolished 55Fe2+ transport activity whereas 55Fe2+ transport mediated by G212V-DMT1 did not differ from wtDMT1. G212V had no effect on the pH dependence of 55Fe2+ transport, presteady-state kinetics, or the affinity for Fe2+ (K0.5 = 1.7 ± 1.1 µM cf. 1.3 ± 0.5 µM in wtDMT1). To our surprise, however, G212V-DMT1 mediated much smaller Fe2+-evoked currents than did wtDMT1. From simultaneous measurement of currents and 55Fe2+ fluxes, we arrived at a wildtype H+/Fe2+ ratio of 17 ± 1 which greatly exceeds that expected for strict stoichiometric transport, i.e. most of the wtDMT1-mediated current arises from H+ slippage. Such slippage is normally thought of in terms of the energetic penalty to the cell; however, indications that H+ slippage, but not Fe2+ transport, is disrupted in the G212V mutant raise the possibility that H+ slippage serve a physiological role. Disruption of iron transport activity in most of the identified human DMT1 mutations therefore explains most of the patients’ phenotypes; however, analysis of some mutations (e.g. G212V) is revealing novel aspects of the molecular physiology of DMT1.

Poster# 268 THE FIRST CELLULAR MODELS FOR FRIEDREICH ATAXIA BASED ON FRATAXIN MISSENSE MUTATIONS THAT REPRODUCE SPONTANEOUSLY FE-S CLUSTERS DEFECT AND INTRAMITOCHONDRIAL IRON DEPOSITS Stéphane Schmucker¹, Nadège Carelle-Calmels, PhD, Marie Wattenhofer-Donzé, PhD, Nadège Vaucamps, technician, Laurence Reutenauer, Technician, Nadia Messaddeq, Engineer, Koenig Michel, Professor and Hélène Puccio, PhD² ¹IGBMC; ²IGBMC Illkirch (Presented By: Stéphane Schmucker)

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial ironchaperone involved in iron- sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)n expansion within the first intron of the frataxin gene. A few patients either with typical or atypical clinical presentation are compound heterozygous for the GAA expansion and a micromutation. We have developed a new strategy, that can be brought to general use, to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin, notably the G130V and I154F mutations which cause mild and severe phenotypes in patients, respectively. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXNG130V and hFXNI154F) frataxin. Nevertheless, both mutations affect mitochondrial ISC enzymes activities and mitochondria ultrastructure with intramitochondrial iron aggregates. Furthermore, in agreement with the severity observed in patients, the hFXNI154F mutant has more severe consequences, with an extramitochondrial ISC enzumes deficiency, an increased sensitivity to oxidative stress and an iron dysregulation with an increased IRP1 activity. These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotype associated with FRDA, are important tools to gain new insights into the in vivo role of frataxin and the consequences of pathological mutations as well as for large-scale pharmacological screening.

Poster# 269 RETROMER IS REQUIRED FOR PROPER ENDOSOMAL SORTING OF MAMMALIAN IRON TRANSPORTER, DMT1 Mitsuaki Tabuchi, PhD, Izumi Yanatori, Bachelor and Fumio Kishi, MD, PhD Kawasaki Medical School (Presented By: Fumio Kishi, MD, PhD)

Divalent metal transporter 1 (DMT1) is indispensable molecule for both the dietary-iron absorption from apical plasma membrane in the duodenum and the iron acquisition from the transferrin-cycle endosomes in peripheral tissues. Previously we showed that two isoforms of DMT1, differing at their Ctermini (DMT1-I and -II) resulted from the alternative splicing of 3' exons, target to the distinct endosomal compartments and identified the Y555XLXsequence in the C-terminus of DMT1-II as the sorting signal for delivery to the transferrin receptor (TfR)-positive endosome. To examine the structural requirement for the appropriate sorting signal of DMT1-II, we performed a detailed mutational analysis of this sequence. As a result, we have found that the sequence motif "TM-(X)≥15FX(L/M)" (where X can be any residues, and F can be any hydrophobic residues) is structurally sufficient for the function of this sorting signal. Through the search for the known sorting motifs, we found the structurally similar sequence motif WLM, which has been recently identified in the cation-independent mannose-6-phoshate receptor tail as a retromer-mediated endosome-to-trans-Golgi network (TGN) retrieval. To investigate the potential role of retromer complex on DMT1-II-recycling endosome targeting, we have examined the effect of depletion of retromer function on DMT1-II localization. Depletion of one of the retromer subunits, VPS35, by RNAi missorts the DMT1-II from recycling endosomes to late endosome and lysosome. On the other hand, TfR, which normally colocalizes with DMT1-II, appears to be sorted to recycling endosome. It indicates that DMT1-II uses the proper sorting pathway to get to the recycling endosome, which is distinct from that of TfR. We also found that expression of RNAi-resistant GFP-Vps35 in VPS35-depleted cells significantly rescued the mislocalization of DMT1-II to late endosome/lysosome, but not that of nonfunctional-mutant VPS35 or GFP alone. These data directly show that retromer is required for sorting of DMT1-II to recycling endosome probably via the TGN. This sorting event must be of especial importance for iron acquisition from transferrin-cycle endosome during the iron recycling and the red blood cell maturation.

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Poster# 270 EVENTS IN THE ENDOSOME LINK HEME AND IRON HOMEOSTASIS WITH THAT OF COPPER IN THE HEMOPEXIN SYSTEM Ann Smith, BSc, PhD, Kimberley Rish, MS, Rachel Lovelace, BS, Jennifer Hackney, PhD and Rachel Helston, PhD University of Missouri-KC (Presented By: Ann Smith, BSc, PhD)

Hemopexin (HPX) binds heme tightly, protecting cells from heme toxicity, and also targets heme to the liver for catabolism. Thus, HPX helps to conserve and recycle iron (1,2). Heme is catabolized by heme oxygenase−1 (HMOX1) after endocytosis of heme−HPX. This raises regulatory iron pools thus linking heme metabolism with that of iron. Normal iron homeostasis requires copper−replete cells, and copper−proteins are needed to move iron across membranes. The copper and zinc storing metallothioneins (MT) are induced simultaneously with HMOX1 by heme−HPX. CCS1, the copper chaperone that responds to changes in intracellular copper, acts together with MTs to minimize levels of redox−active copper in the cytosol(3). As part of our ongoing investigations to define how heme and iron homeostasis in the HPX system are linked with copper, we focus here on events in the endosome since heme moves through this organelle before reaching HMOXs for degradation and iron release. Our findings that hmox1 regulation requires heme passage through the endosome(4) and the copper chelator bathocuproinedisulfonate (BCDS) prevents the simultaneous regulation of hmox1 and mt1 provides the rationale to investigate two hypotheses: 1) that copper is needed for heme−HPX endocytosis, which we tested by determining whether copper deficiency decreased or prevented endocytosis of heme−HPX; and 2) whether copper acts at the levels of the endosome to release heme from heme−HPX, which we tested by determining whether copper prevented the re−association of heme with HPX at acidic pH. Significantly, heme−HPX endocytosis decreases the CCS1 levels and CCS1 decreases as intracellular copper rises. Heme−HPX endocytosis is needed to regulate CCS1 since the signaling ligand cobalt protoporphyrin (CoPP)−HPX, which does not induce HMOX1 but does co−localize with heme−HPX in endosomes, also decreases CCS1. These observations support that heme−HPX endocytosis mobilizes copper into cells. The regulation of both hmox1 and mt1 is prevented by BCDS, but not uptake of heme−AlexaFluor−labeled HPX into endosomes. Copper is needed for HMOX1 regulation by heme−HPX, since nutritional copper deficiency prevented HMOX1 induction. Using conditions that mimic maturing endosomes, we found that copper prevents rebinding of heme to apo−HPX. We conclude that heme uptake by HPX is linked with copper at the level of the endosome before heme reaches HOs in the smooth endoplasmic reticulum and subsequent iron release into the cytosol. Supported by NIH (R21DK 64363). 1. Davies, D. M., Smith, A., Muller-Eberhard, U., and Morgan W.T. (1979) Biochem.Biophys.Res.Commun. 91, 1504-1511 2. Smith, A., and Morgan, W.T. (1979) Biochem.J. 182, 47-54 3. Tapia, L., Gonzalez-Aguero, M., Cisternas, M.F., Suazo, M., Cambiazo, V., Uauy, R., and Gonzalez, M. (2004) Biochem J378, 617-624 4. Flaherty, M.M., Rish, K.R., Smith, A., and Crumbliss, A.L. (2008) Biometals 21, 239-248

Poster# 271 MECHANISMS UNDERLYING HEPATIC IRON ACCUMULATION IN A DIETARY EXPERIMENTAL MODEL OF NAFLD Paola Dongiovanni, PhD, S. Gatti, R Rametta, A.L. Fracanzani, S. Fargion and L. Valenti Department of Internal Medicine, University of Milan (Presented By: Paola Dongiovanni, PhD)

Background & Aims: Increased iron stores are frequently observed in NAFLD, and associated with more advanced hepatic and vascular damage. Controversial data have been reported as to the mechanisms underlying iron accumulation based on gene expression studies in humans. Aim was to investigate the effect of high fat diet (HFD) on iron metabolism in rats. Methods: Six weeks male Sprague-Dawley rats were fed for 12 weeks with standard chow (control, n=6), or HFD (n=6), or HFD plus s.c. iron administration (250 mg iron sulfate, n=6), hepatic iron concentration (HIC) evaluated by atomic absorption spectrometry, mRNA by qRT-PCR, proteins by Western blotting. Results: HFD resulted in mild steatosis and increased glucose and insulin resistance compared to standard chow. Dietary iron intake was lower in HFD treated rats vs. controls (.74±.3 vs. 8.5±1.5 mcg/week, p<0.0001), but HIC (16±9 controls, 33±10 HFD, 122±40 mcg/100mg HFD+iron, p=0.01), H and L ferritins mRNA and proteins were higher in HFD vs. standard chow fed rats, whereas serum iron was not influenced by diet. mRNA of hepcidin, the hormone regulating iron absorption, was higher in HFD fed vs. control rats (1.2±.3 controls, 1.9±.4 HFD, 2.2±.6 HFD+iron, p=0.01), but hepcidin/ferritin ratio was not affected. mRNA levels of the hepcidin regulators HFE, TfR-2, hemojuvelin, neogenin, Tmprss6, and IL-6 were regulated by HFD consensually to iron stores, whereas transcription of TNFalpha, neogenin and BMP-2,-4,-6 was not affected. Expression of the iron exporter ferroportin-1, but not that of ceruloplasmin, was decreased relative to iron stores by HFD (p=0.01). mRNA and protein levels of Transferrin recepetor-1 (TfR-1), the physiological mediator of iron uptake from plasma usually downregulated by iron, were upregulated by HFD (2.6-fold, p<0.05), and normalized, albeit to a degree not proportional to iron stores, by iron supplementation. Conclusions: HFD affects the expression of iron genes and favors hepatic iron accumulation. As previously report in patients with NAFLD, HFD decreased the expression of ferroportin-1 relative to iron. However, HFD was not associated with impairment in hepcidin transcription and with that of its main regulators, but with up-regulation of TfR-1 reversed by iron supplementation. We are currently evaluating transcriptional mechanisms underlying these findings in the in vivo and a cellular model and confirming data in human samples. TfR-1 up-regulation may contribute to early phases of iron accumulation associated with HFD.

Poster# 272 HEPATIC IRON DEPOSITION IN RETICULOENDOTHELIAL CELLS BUT NOT HEPATOCYTES IS ASSOCIATED WITH MORE SEVERE NASH: RESULTS FROM THE NASH CLINICAL RESEARCH NETWORK James Nelson, PhD¹, Laura Wilson, MS², Elizabeth Brunt, MD³, Matthew Yeh, MD, PhD4, David Kleiner, MD, PhD5, Aynur Unalp-Arida, MD, PhD² and Kris Kowdley, MD6 ¹Benaroya Research Institute; ²Johns Hopkins University; ³Washington University, Department of Pathology; 4University of Washington, Department of Pathology; 5National Cancer Institute; 6Benaroya Research Institute and Virginia Mason Medical Center (Presented By: James Nelson, PhD)

Background: Previous studies have found conflicting results regarding the prevalence and pattern of iron deposition and relationship to histologic severity in nonalcoholic fatty liver disease (NAFLD). Most of these previous studies were limited by small sample sizes and did not distinguish between parenchymal and non-parenchymal iron deposition in the analyses. The goal of this study was to examine both the prevalence and pattern(s) of iron deposition in NAFLD, and to evaluate any relationships between iron deposition and NAFLD severity in a large cohort of well-characterized US patients in a multicenter network. Methods: All subjects were enrolled in the NASH Clinical Research Network (NASH CRN). The following clinical and laboratory variables were obtained for each subject: age, gender, ethnicity, BMI, history of diabetes, history of iron overload, history of GI bleeding, menstrual history, HFE genotype (when available), serum aminotransferases, fasting glucose, fasting insulin and lipids, serum iron studies and ferritin. All liver biopsies were evaluated by the Pathology Committee of the NASH CRN according to the method of Kleiner et al (Hepatology 2005; 41:1313- 1321; in addition to a NAFLD Activity Score (NAS) and fibrosis scores, diagnoses of NASH, not NASH and borderline were also assigned. Iron stains, performed in a single central lab, were scored for amount, cellular and parenchymal localization. Results: 768 adult NASH CRN subjects’ biopsies were reviewed. One third (253/768, 32.9%) had stainable hepatic iron. Stainable iron was present in three histologic patterns; parenchymal hepatocytes only (HC; 59/768, 7.7%), cells in the reticuloendothelial system only (RES; 77/768, 10%), or a mixed pattern of hepatocellular and reticuloendothelial cells (HC/RES; 117/768, 15.2%). Subjects with iron staining were more likely to be male (59.3% vs. 26.8%; p<0.0001), have a lower BMI (33.2 ± 6.5 vs. 35.0 ± 5.9; p<0.0001), and lower cholesterol (186.6 ± 41.8 vs. 204.2 ± 43.2; p=0.001). Compared with hepatocellular iron staining only, the presence of iron exclusively in RES was most frequently associated with increased grade of steatosis (p=0.07 vs. HC only), lobular inflammation (p=0.005 vs. HC only), portal inflammation (p=0.0003 vs. HC only), advanced (stage 3 or 4) fibrosis (p=0.002 vs. HC only) and ballooned hepatocytes (p=0.00009 vs. HC only). Subjects with HC iron only less often had a definite NASH diagnosis compared to either the no iron (p=0.001) or RES only groups (p=0.0002).

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Conclusions: The following relationship between disease severity (i.e., increased NAS, advanced fibrosis, and cases of definitive NASH) and hepatic iron deposition was shown: from most severe to least severe: RES only>no iron≈HC/RES mixed>HC only (see figure). We speculate that the increased histologic activity and fibrosis in subjects with RES only iron is due to the combined effects of an increased inflammatory response and ROS production, causing greater necroinflammation and Kupffer cell- mediated hepatic stellate cell activation. We further speculate that the apparent protective effects of HC iron alone may be related to the observed decreased steatosis and inflammation in these patients by an as yet undefined mechanism such as an increased anti-inflammatory response or decreased lipogenesis.

Poster# 273 OSYSL15 IS AN IRON-REGULATED IRON (III)-PHYTOSIDEROPHORE TRANSPORTER AND IS ESSENTIAL FOR IRON UPTAKE IN RICE PLANTS N.K. Nishizawa, PhD, T. Kobayashi, T. Nozoye, M. Takahashi, Y. Kakei, H. Nakanishi, S. Mori University of Tokyo (Presented By: Naoko Nishizawa, PhD)

Graminaceous plants take up iron through yellow stripe 1 (YS1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in rice plants. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid, the primary phytosiderophore, and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-b-glucuronidase (GUS) analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron (III)-deoxymugineic acid transporter responsible for iron uptake from the soil and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth.

Poster# 274 PORPHYRIN DISTORTION AND METAL ION SELECTIVITY IN FERROCHELATASE Neil McIntyre, PhD², Ricardo Franco, PhD³, John Shelnutt, PhD4 and Gloria Ferreira¹ ¹Univ South Florida; ²University of South Florida; ³Universidade Nova de Lisboa; 4Sandia National Laboratories (Presented By: Gloria Ferreira)

Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX (1, 2). Non-planar deformation of the porphyrin substrate has been proposed to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to yield heme (1-3). In fact, previously, we demonstrated that the degree of saddling, a specific non-planar porphyrin deformation, contributes to the catalytic efficiency of ferrochelatase towards the porphyrin substrate (4). To examine the role of the enzyme-induced porphyrin on metal ion substrate selectivity and ultimately product release, ferrochelatase variants were directly evolved to acquire increased nickel-chelatase activity. Using resonance Raman (RR) spectroscopy, the structural properties of the chelatase-bound porphyrins were examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. While the extent of the induced porphryin saddling decreased for all variants, the steady-state kinetic parameters for both Ni(II) and Fe(II) chelatase activities increased relative to those of wild-type ferrochelatase. The decrease in the porphyrin saddling mode appeared to relax metal ion preference for ferrochelatase through activation of another non-planar porphyrin mode, ruffling. Supported by grant GM080270 (to G.C.F.) from the National Institute of General Medical Sciences. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-AC04-94AL85000. (1) Dailey, H. A., and Dailey, T. A. (2003) Ferrochelatase, in The Porphyrin Handbook (Kadish, K. M., Smith, K. M., and Guillard, R., Eds.) pp 93-121, Elsevier Science, California, USA. (2) Ferreira, G. C. (1999) Ferrochelatase. Int J Biochem Cell Biol 31, 995-1000. (3) Al-Karadaghi, S., Franco, R., Hansson, M., Shelnutt, J. A., Isaya, G., and Ferreira, G. C. (2006) Chelatases: distort to select? Trends in Biochem. Sci. 31, 135-142. (4) Shi, Z., Haddad, R., Franco, R., Shelnutt, J. A., and Ferreira, G. C. (2006) The Conserved Active-Site Loop Residues of Ferrochelatase Induce Porphyrin Conformational Changes Necessary for Catalysis. Biochemistry 45, 2904-2912.

Poster# 275 SERUM FERRITIN AND HEPCIDIN LEVELS PREDICT VASCULAR DAMAGE IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE Luca Valenti, MD, Dorine W. Swinkels, Larry Burdick, Harold Tjalsma, Cristina Bertelli, Erika Fatta, Daniela Bignamini, Paola Dongiovanni, Raffaela Rametta, Benedetta M. Motta, Silvia Fargion and Anna Ludovica Fracanzani University of Milano (Presented By: Luca Valenti, MD)

Background and Aims: Increased ferritin levels and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with increased susceptibility to vascular damage. Excess iron has been hypothesized to induce vascular damage by increasing levels of the hormone hepcidin, determining iron trapping in macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD. Patients and Methods: 506 consecutive patients with NAFLD, without hereditary hemochromatosis, whose alcohol intake was <210/180 g/week in M/F. Vascular damage was evaluated by common carotid intimamedia thickness (CC-IMT) measurement, and plaque detection by ecocolor-doppler ultrasonography. HFE mutations were detected by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. Results: Mean CC-IMT was 0.87±0.23, 27% had plaques. At multivariate analysis CC-IMT was associated with systolic blood pressure (p=0.001), glucose (p=0.002), LDL cholesterol (p=0.002), abdominal circumference (p=0.006), age (p=0.01), and ferritin (p=0.048). Carotid plaques were independently associated with age (p<0.0001), ferritin (p=0.0004), glucose (p=0.02), and hypertension (p=0.05). Ferritin reflected indices of iron stores and of metabolic syndrome, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores (p<0.0001), and hepcidin-25 was independently associated with carotid plaques (p=0.05). Conclusions: Ferritin and hepcidin are independent predictors of vascular damage in NAFLD, suggesting that iron may promote atherogenesis. The mechanism may be related to increased hepcidin determining iron trapping into macrophages.

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Poster# 276 RECOMBINANT HUMAN ERYTHROPOIETIN DOPING IN AN EXPERIMENTAL MODEL OF CHRONIC AEROBIC EXERCISE TRAINING Nuno Piloto, Biochem D², Flávio Reis, PhD¹, Helena M. Teixeira, PhD, Edite Teixeira de Lemos, PhD, Patrícia Garrido, MSci, Elísio Costa, MSCi, José Sereno, Biochem D, Margarida Lourenço, MD, Carlos A. Tavares, MD, Carlos A. Ferrer Antunes, MD, Petronila Rocha Pereira, PhD, Luis Belo, PhD, Alice Santos Silva, PhD and Frederico Teixeira, PhD ¹Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University; ²Institute of Pharmacology & Experimental Therapeutics, Medicine Faculty, Coimbra University, Portugal (Presented By: Flávio Reis, PhD)

Recombinant human EPO (rhEPO) doping remains a huge concern, not only because of distortion of sport truth, but particularly because of the risks for athletes under doping administration. Besides the well known increase of hematocrit and blood viscosity, other disturbances could occur. The aim of this study was to evaluate the effects of rhEPO treatment on rats under chronic exercise. Four groups (n=7 each) of male Wistar rats (~280 g) were evaluated: a) control – sedentary (SED); b) rhEPO – 50 IU/Kg/wk Recormon®, Roche Pharmaceuticals (EPO); c) Exercised (EX) – swimming training (1 hr, 3 times/wk); d) Exercised+rhEPO (EX+EPO). After the 10-weeks period of treatment, blood and tissue samples were collected in order to assess the following profiles: haematology, iron metabolism, biochemistry, peripheral and central catecholamine and 5-HT contents (by HPLC-ECD), lipid peroxidation (through MDA content), 3-nitrotyrosine (3-NT) levels, total antioxidant status (TAS) and inflammation (serum TGF-ß, TNF-alpha, PCR, IL-1ß and IL-2), through immunoassay kits. Results are means ± sem (ANOVA/Fisher’s PLSD; p<0.05 was considered as significant). RBC count was increased in the EX+EPO group (8.2±0.1) when compared with EPO or EX groups (7.7±0.1 and 7.6±1.5; p<0.05), as well as Hct and Hb. Iron metabolism was almost unaffected by treatments: EX (iron: 170,86±5,63 µg/dL; ferritin: 25,30±2,00 ng/dL; transferrin: 126,86±5,52 ng/dL) and EX+EPO (iron: 182,00±12,99 µg/dL; ferrritin: 22,90±0,87 ng/dL; transferrin: 129,86±4,62 ng/dL). Plasma NA, AD and 5-HT (9.3±1.4; 1.9±0.2; 30.1±4.4 ng/ml; p<0.01) were higher in the EX+EPO group when compared with the EPO (4.8±0.4; 1.5±0.1; 5.8±0.6) and EX (5.1±1.0; 1.0±0.1; 11.1±0.6) groups. While serum MDA/TAS was unchanged, muscle MDA/TAS ratio was increased in the EX+EPO group (3.6±0.3) vs EPO (3.1±0.2) and EX (2.9±0.2; p<0.05). Concerning the inflammatory markers, there was an increment in IL-2 in the EX+EPO group vs EPO (p<0.05). In conclusion, rhEPO doping in rats under chronic aerobic exercise training promotes not only blood viscosity, but also sympathetic activation and oxidative stress, suggesting an increment in thromboembolic and cardiometabolic complications and risk of cardio/cerebrovascular events.

Poster# 277 IRON UPTAKE BY CHROMERA VELIA Robert Sutak, Emmanuel Lesuisse and Jean-Michel Camadro Institut Jacques Monod (Presented By: Robert Sutak)

The newly discovered marine species called Chromera velia is the closest known photosynthetic relative to apicomplexan parasites including Plasmodium and Toxoplasma. This alveolate is easily maintained in culture, providing a suitable model to study apicomplexan metabolism and evolution. In the marine environment, iron is believed to be one of the most limiting nutrients. Therefore, in this study, we investigated the mechanisms of iron uptake by C. velia. Cells were incubated with different iron (Fe55) sources (ferric citrate, ferrous ascorbate and various siderophores) under different conditions (temperature, light, copper and iron abundance in the growth medium) and the rate of iron uptake was determined. Our results indicate a two-step mechanism of iron uptake consisting of strong, fast specific binding of iron by cell surface proteins followed by slower incorporation into the cells. The cells showed no detectable ferric-reductase activity and were able to take up iron from different sources (ferric and ferrous iron). Iron binding/uptake was dependent on iron abundance in the growth medium. We hypothesise that the presence of a highly effective uptake system for different iron sources, different than the well-known reductive uptake system, is involved in iron assimilation by C. velia, an organism living in constant iron-deficient conditions.

Poster# 278 SMALL MOLECULE INHIBITORS OF HYPOXIA INDUCIBLE FACTOR 2A (HIF2A) TRANSLATION LINK ITS 5’-UNTRANSLATED REGION (5’-UTR) IRON RESPONSIVE ELEMENT (IRE) TO IRP-1 MEDIATED OXYGEN SENSING Michael Zimmer, PhD², Christoher Neil, BSc² and Othon Iliopoulos, MD¹ ¹Massachusetts General Hospital Cancer Center; ²Massachusetts General Hospital Cancer Center and Harvard Medical School (Presented By: Othon Iliopoulos, MD)

Cells transiently adapt to hypoxia by globally decreasing protein translation by inhibiting mTOR activity. However, specific proteins needed to respond to hypoxia evade this translational repression. The mechanisms of this phenomenon remain unclear. We screened for and identified small molecules that selectively decrease HIF-2a translation in an mTOR independent manner. The HIF2a 5’-UTR is necessary and sufficient for the response to inhibitors. Structure-function analysis and electrophoretic mobility shift assays indicated that the inhibitors enhance the binding of Iron Regulatory Protein 1 (IRP1) to an Iron-Responsive Element (IRE) within the 5’-UTR of the HIF-2a message by a non-iron chelating mechanism. We noticed no detectable binding of endogenous IRP2 in HIF2a IRE, at least in the cell lines tested. Knocking down the expression of IRP1 by shRNA abolished the effect of the compounds. Exposure of cultured cells to hypoxia results in disruption of the IRP1- HIF-2a IRE interaction and rapid de-repression of HIF-2a translation. Thus, this chemical genetic analysis describes a molecular mechanism by which translation of the HIF-2a message is maintained during conditions of cellular hypoxia through inhibition of IRP-1 dependent repression. It also provides the chemical tools that can help investigating how hypoxia modifies IRP1-IRE interaction.

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Alphabetical Index of Authors by Presentation (Presenting Authors Only Include Email Address)

Aachmann-Andersen, N. Alessio, M. 06/10/09 16:00 Podium# 54 06/10/09 14:00 Poster# 163

Abbas, K. Alexander, J. 06/08/09 14:00 Poster# 57 06/11/09 14:00 Poster# 262

Abe, M. Ali-Rahmani, F. [ [email protected] ] 06/11/09 14:00 Poster# 72 06/08/09 14:00 Poster# 85

Abergel, A. Allen, K. [ [email protected] ] 06/11/09 14:00 Poster# 199 (WITHDRAWN) 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 240 Abgueguen, E. 06/11/09 14:00 Poster# 252 06/11/09 14:00 Poster# 209 06/11/09 16:00 Podium# 82 Almeida, S. 06/10/09 14:00 Poster# 164 Acton, R. 06/11/09 14:00 Poster# 224 Al-Qenaei, A. 06/11/09 14:00 Poster# 237 06/10/09 14:00 Poster# 181 06/11/09 14:00 Poster# 254 06/11/09 15:30 Podium# 86 Altamura, S. [ [email protected] ] 06/08/09 14:00 Poster# 5 Adams, P. [ [email protected] ] 06/08/09 14:00 Poster# 60 06/11/09 14:00 Poster# 224 06/10/09 14:00 Poster# 125 06/11/09 14:00 Poster# 237 06/11/09 14:00 Poster# 207 06/11/09 15:30 Podium# 86 06/11/09 14:00 Poster# 245 06/11/09 16:30 Podium# 90 Altruda, F. Aderem, A. 06/08/09 14:00 Poster# 1 06/10/09 14:00 Poster# 169 06/10/09 14:00 Poster# 129

Aguila-Martinez, P. Alvarenga, E. 06/10/09 18:00 Podium# 61 06/08/09 14:00 Poster# 30

Aguirre, P. Álvarez-Sala, L. 06/08/09 14:00 Poster# 49 06/11/09 14:00 Poster# 250 06/11/09 14:00 Poster# 251 Aigner, E. 06/08/09 14:00 Poster# 10 Alyanakian, M. 06/08/09 14:00 Poster# 6 (WITHDRAWN) Akagi, R. 06/08/09 14:00 Poster# 48 Ames, B. 06/08/09 14:00 Poster# 42 Akira, S. 06/10/09 14:00 Poster# 169 Amini, M. 06/10/09 15:30 Podium# 46 06/10/09 17:45 Podium# 66

Alberghini, A. Anderson, G. 06/10/09 16:00 Podium# 54 06/08/09 14:00 Poster# 82 06/10/09 14:00 Poster# 117 Aldámiz, T. 06/11/09 14:00 Poster# 236 06/11/09 14:00 Poster# 250 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 251 06/11/09 14:00 Poster# 240 06/11/09 14:00 Poster# 252 188

Anderson, L. Armitage, A. 06/11/09 14:00 Poster# 204 06/08/09 14:00 Poster# 25 06/10/09 16:30 Podium# 50 Anderson, M. 06/11/09 15:15 Podium# 73 Arnulf, B. 06/08/09 14:00 Poster# 6 (WITHDRAWN) Andolfo, I. 06/10/09 14:00 Poster# 103 Arosio, C. 06/08/09 14:00 Poster# 91 Andrade, M. 06/08/09 14:00 Poster# 32 Arosio, P. 06/08/09 14:00 Poster# 55 Andrews, M. 06/08/09 14:00 Poster# 58 06/10/09 17:00 Podium# 57 06/08/09 14:00 Poster# 62 06/11/09 16:15 Podium# 83 Andrews, N. 06/08/09 18:15 Podium# 26 Aroun, A. 06/10/09 17:15 Podium# 58 06/11/09 14:00 Poster# 260 06/11/09 10:00 Podium# 69 Arruga, F. Andrieu, V. 06/08/09 18:00 Podium# 31 06/08/09 15:15 Podium# 9 Artaud, I. Andriopoulos, Jr., B. 06/08/09 14:00 Poster# 69 06/08/09 17:15 Podium# 22 06/10/09 14:00 Poster# 113 Arvedson, T. 06/10/09 17:00 Podium# 57 06/10/09 15:15 Podium# 39 06/10/09 16:30 Podium# 44 Andros, N. 06/08/09 14:00 Poster# 12 Åsberg, A. 06/11/09 14:00 Poster# 244 Anghel, A. 06/11/09 14:00 Poster# 213 Askling, J. 06/11/09 14:00 Poster# 195 Antonetti, D. 06/11/09 16:00 Podium# 76 Atamna, H. [ [email protected] ] 06/11/09 15:45 Podium# 75 Antunes, M. 06/08/09 14:00 Poster# 14 Auchère, F. 06/08/09 14:00 Poster# 22 Appelberg, R. 06/08/09 14:00 Poster# 28 Augustic, A. 06/10/09 15:45 Podium# 47 06/10/09 16:30 Podium# 44

Apte, S. Auriac, A. [ [email protected] ] 06/10/09 17:00 Podium# 57 06/10/09 14:00 Poster# 101 06/10/09 17:00 Podium# 63 Arase, H. 06/10/09 10:15 Podium# 38 Aveiro, S. 06/10/09 14:00 Poster# 106 Arceo, S. 06/11/09 14:00 Poster# 232 Aydinok, Y. 06/10/09 16:30 Podium# 56 Arens, T. 06/08/09 14:00 Poster# 26 Azevedo, J. 06/10/09 14:00 Poster# 112

189

Azzolino, O. Barcellos, L. 06/10/09 14:00 Poster# 129 06/11/09 14:00 Poster# 224

Baart, M. [ [email protected] ] Barisani, D. 06/11/09 14:00 Poster# 231 06/11/09 14:00 Poster# 189

Babitt, J. [ [email protected] ] Barker, N. 06/08/09 17:15 Podium# 22 06/11/09 14:00 Poster# 253 06/10/09 14:00 Poster# 113 Bartholomew, H. Bach, G. 06/10/09 14:00 Poster# 176 06/11/09 14:00 Poster# 228 Bartnikas, T. [ [email protected] ] Bado, A. 06/08/09 14:00 Poster# 63 06/10/09 14:00 Poster# 116 Barton, J. Bahlo, M. 06/11/09 15:30 Podium# 86 06/11/09 14:00 Poster# 252 Barton, J. [ [email protected] ] Bähr-Ivacevic, T. 06/11/09 14:00 Poster# 237 06/08/09 15:45 Podium# 17 06/11/09 14:00 Poster# 254

Baladi, J. Bartosz, G. 06/11/09 14:00 Poster# 249 06/10/09 14:00 Poster# 183

Baldwin, G. [ [email protected] ] Bartz, F. 06/10/09 14:00 Poster# 108 06/10/09 14:00 Poster# 115

Balesaria, S. Baughman, J. 06/10/09 14:00 Poster# 134 06/08/09 12:00 Podium# 1

Balk, J. Bayele, H. 06/08/09 14:00 Poster# 56 06/10/09 14:00 Poster# 104

Ball, S. Bayot, A. 06/11/09 14:00 Poster# 247 06/08/09 14:00 Poster# 22 06/11/09 14:00 Poster# 249 Beard, J. Balocco, M. 06/10/09 14:00 Poster# 157 06/11/09 14:00 Poster# 211 06/10/09 14:00 Poster# 161 06/11/09 14:00 Poster# 212 Beaumont, C. Balusikova, K. 06/08/09 14:00 Poster# 9 06/10/09 14:00 Poster# 136 06/08/09 14:00 Poster# 33 06/08/09 15:15 Podium# 9 Bansal, S. 06/10/09 14:00 Poster# 116 06/08/09 14:00 Poster# 67 06/10/09 15:45 Podium# 41 06/10/09 18:00 Podium# 61 Bansal, Sukhi [ [email protected] ] 06/08/09 14:00 Poster# 75 Becker, E. 06/10/09 17:15 Podium# 64 Bansal, Sukhvinder 06/11/09 14:00 Poster# 188 06/10/09 16:15 Podium# 43 Becker, L. Barbot, J. 06/08/09 15:30 Podium# 16 06/08/09 14:00 Poster# 14

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Beckman, K. Berthonneche, C. 06/11/09 14:00 Poster# 224 06/10/09 16:15 Podium# 55 06/11/09 14:00 Poster# 252 Bertola, F. Bedogna, V. 06/08/09 14:00 Poster# 91 06/08/09 14:00 Poster# 73 Betarbet, R. Begley, C. 06/11/09 15:15 Podium# 73 06/10/09 16:30 Podium# 44 Bettegazzi, B. Beirao, I. [ [email protected] ] 06/08/09 15:45 Podium# 5 06/10/09 18:15 Podium# 62 Bignamini, D. Bekri, S. 06/11/09 14:00 Poster# 275 06/10/09 14:00 Poster# 116 Bishop, G. Belegu, V. 06/10/09 14:00 Poster# 185 06/11/09 16:15 Podium# 83 Black, G. Belo, L. 06/11/09 14:00 Poster# 204 06/08/09 14:00 Poster# 14 06/08/09 14:00 Poster# 16 Blaiseau, P. 06/11/09 14:00 Poster# 276 06/08/09 14:00 Poster# 23

Bendova, P. Blake, J. 06/10/09 14:00 Poster# 179 06/08/09 15:45 Podium# 17

Beneitez Pastor, D. Blattmann, C. 06/10/09 14:00 Poster# 103 06/11/09 14:00 Poster# 245

Benes, V. Bloch, K. 06/08/09 15:45 Podium# 17 06/11/09 15:45 Podium# 81 06/11/09 10:15 Podium# 70 Boettger, L. Benhamou, M. 06/08/09 14:00 Poster# 18 06/08/09 14:00 Poster# 6 (WITHDRAWN) Bogo Chies, J. Bento, C. 06/08/09 14:00 Poster# 4 06/08/09 14:00 Poster# 17 Bomford, A. Berenshtein, E. 06/08/09 14:00 Poster# 75 06/10/09 14:00 Poster# 119 Bonaccorsi di Patti, M. Bernhardt, P. 06/10/09 14:00 Poster# 177 06/11/09 14:00 Poster# 265 06/11/09 15:30 Podium# 74

Bernth, N. Borch-Iohnsen, B. 06/10/09 14:00 Poster# 151 06/11/09 14:00 Poster# 244

Bertalli, N. Boserup, M. 06/11/09 14:00 Poster# 238 06/10/09 14:00 Poster# 150 06/11/09 14:00 Poster# 240 06/10/09 14:00 Poster# 154 06/11/09 14:00 Poster# 252 06/11/09 14:00 Poster# 256 Bossi, A. 06/08/09 14:00 Poster# 54 Bertelli, C. 06/11/09 14:00 Poster# 275

191

Bottomley, S. [ [email protected] ] Britton, R. 06/10/09 17:45 Podium# 60 06/08/09 16:15 Podium# 13 06/08/09 18:00 Podium# 25 Bou-Abdallah, F. 06/10/09 14:00 Poster# 97 06/11/09 14:00 Poster# 229 (WITHDRAWN) Brookes, M. Bougault, C. 06/08/09 17:45 Podium# 30 06/10/09 14:00 Poster# 130 06/10/09 14:00 Poster# 144 06/11/09 14:00 Poster# 208 Bouhassira, E. 06/11/09 15:30 Podium# 80 Brown, G. 06/11/09 14:00 Poster# 210 Bourrien, M. 06/11/09 14:00 Poster# 235 Broyles, R. [ [email protected] ] 06/11/09 16:15 Podium# 83 Bouton, C. [ [email protected] ] 06/08/09 14:00 Poster# 57 Bruce, D. 06/10/09 14:00 Poster# 152 Boutron, I. 06/10/09 14:00 Poster# 176 06/08/09 14:00 Poster# 33 Bruick, R. [ [email protected] ] Boutros, M. 06/10/09 14:00 Poster# 123 06/08/09 14:00 Poster# 59 Brunt, E. Bouvet, R. 06/11/09 14:00 Poster# 272 06/11/09 14:00 Poster# 190 Bruzzone, C. Bracco, E. 06/11/09 14:00 Poster# 211 06/08/09 18:00 Podium# 31 06/11/09 14:00 Poster# 212 06/10/09 14:00 Poster# 95 Brwuer, W. Brandt, U. 06/08/09 14:00 Poster# 52 (Moved to June 10, 17:40) 06/08/09 14:00 Poster# 56 Bryant, B. [ [email protected] ] Braunstein, J. 06/11/09 14:00 Poster# 232 06/11/09 14:00 Poster# 218 Bujanover, Y. Breuer, W. 06/08/09 15:45 Podium# 11 06/11/09 15:30 Podium# 80 Bulteau, A. Briat, J. 06/08/09 14:00 Poster# 22 06/10/09 14:00 Poster# 127 Bulvik, B. Briestensky, J. 06/10/09 14:00 Poster# 119 06/10/09 14:00 Poster# 187 Burdick, L. Brissot, P. [ [email protected] ] 06/11/09 14:00 Poster# 275 06/10/09 14:00 Poster# 126 06/10/09 14:00 Poster# 137 Busbridge, M. 06/11/09 11:30 Podium# 71 06/10/09 16:15 Podium# 43 06/11/09 14:00 Poster# 209 06/11/09 14:00 Poster# 235 Busti, F. 06/11/09 14:00 Poster# 247 06/08/09 14:00 Poster# 54 06/11/09 14:00 Poster# 249 06/08/09 14:00 Poster# 68 06/11/09 16:00 Podium# 82 06/08/09 14:00 Poster# 73

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Byrne, S. Canal, F. 06/11/09 14:00 Poster# 229 (WITHDRAWN) 06/08/09 14:00 Poster# 57

Cabantchik, I. [ [email protected] ] Cancedda, R. 06/08/09 14:00 Poster# 47 06/10/09 16:15 Podium# 49 06/08/09 14:00 Poster# 52 (Moved to June 10, 17:40) 06/10/09 10:00 Podium# 37 Cannie, I. 06/11/09 15:30 Podium# 80 06/10/09 14:00 Poster# 137

Cabillic, F. Cannistraci, C. 06/10/09 14:00 Poster# 126 06/10/09 14:00 Poster# 163

Caetano, G. Cannon, H. 06/08/09 14:00 Poster# 17 06/11/09 14:00 Poster# 247 06/11/09 14:00 Poster# 249 Cailliatte, R. 06/10/09 14:00 Poster# 127 Canonne-Hergaux, F. [ [email protected] ] 06/08/09 14:00 Poster# 41 Cairo, G. 06/08/09 14:00 Poster# 69 06/08/09 15:30 Podium# 10 06/10/09 14:00 Poster# 101 06/08/09 17:00 Podium# 27 06/10/09 17:00 Podium# 63 06/10/09 16:00 Podium# 54 06/11/09 14:00 Poster# 220 Cao, A. 06/11/09 16:15 Podium# 89 Callens, C. 06/08/09 14:00 Poster# 6 (WITHDRAWN) Cappa, S. 06/10/09 14:00 Poster# 163 Camadro, J. 06/08/09 14:00 Poster# 22 Cappellini, M. 06/08/09 14:00 Poster# 23 06/10/09 16:30 Podium# 56 06/11/09 14:00 Poster# 277 06/11/09 14:00 Poster# 205 06/11/09 14:00 Poster# 206 Camaschella, C. 06/08/09 14:00 Poster# 79 Carelle-Calmels, N. 06/10/09 14:00 Poster# 129 06/11/09 14:00 Poster# 268 06/10/09 15:45 Podium# 41 06/10/09 18:00 Podium# 61 Carpenter, J. 06/11/09 14:00 Poster# 204 Camberlein, E. 06/11/09 16:00 Podium# 82 Carrasco, M. 06/10/09 14:00 Poster# 156 Campagna, D. 06/11/09 14:00 Poster# 223 Carruthers, A. 06/11/09 16:00 Podium# 76 Campanella, A. [ [email protected] ] 06/08/09 16:30 Podium# 8 Carter, K. 06/10/09 14:00 Poster# 174 06/10/09 14:00 Poster# 121

Campostrini, N. [ [email protected] ] Carturan, S. 06/08/09 14:00 Poster# 54 06/08/09 18:00 Podium# 31 06/08/09 14:00 Poster# 68 06/10/09 14:00 Poster# 95 06/08/09 14:00 Poster# 73 06/10/09 14:00 Poster# 129 06/08/09 14:00 Poster# 78 06/10/09 16:00 Podium# 54 Carvalho, A. 06/10/09 16:15 Podium# 43 06/10/09 14:00 Poster# 106

193

Carvalho, F. Chapman, L. 06/10/09 14:00 Poster# 112 06/10/09 14:00 Poster# 175

Carvalho, L. Chappard, D. 06/08/09 14:00 Poster# 16 06/11/09 14:00 Poster# 196

Carvalho, S. Charbonnier, P. 06/08/09 14:00 Poster# 29 06/10/09 14:00 Poster# 130 06/08/09 14:00 Poster# 31 Chasteen, N. Casanovas, G. [ [email protected] ] 06/11/09 14:00 Poster# 229 (WITHDRAWN) 06/08/09 14:00 Poster# 5 06/08/09 14:00 Poster# 59 Chaston, T. 06/08/09 14:00 Poster# 60 06/10/09 14:00 Poster# 99

Casarrubea, D. [ [email protected] ] Cheetham, J. 06/10/09 14:00 Poster# 135 06/10/09 15:15 Podium# 39

Castagna, A. Chen, C. 06/08/09 14:00 Poster# 54 06/10/09 15:15 Podium# 39 06/08/09 14:00 Poster# 68 06/08/09 14:00 Poster# 73 Chen, F. 06/08/09 14:00 Poster# 78 06/10/09 14:00 Poster# 110 06/08/09 15:30 Podium# 10 06/10/09 16:00 Podium# 54 Chen, J. [ [email protected] ] 06/08/09 16:00 Podium# 6 Castoldi, M. 06/08/09 17:45 Podium# 24 06/11/09 10:15 Podium# 70 Chen, N. Castro, B. 06/11/09 15:15 Podium# 79 06/08/09 14:00 Poster# 32 Chen, S. Cavadini, P. 06/10/09 14:00 Poster# 113 06/08/09 14:00 Poster# 55 Chen, V. Cavalcante, J. 06/11/09 14:00 Poster# 252 06/11/09 14:00 Poster# 214 06/11/09 14:00 Poster# 217 Chen, W. 06/08/09 16:15 Podium# 7 Chaiba-Berrouche, L. 06/11/09 14:00 Poster# 225 06/10/09 18:00 Podium# 61 Chevion, M. [ [email protected] ] Chales, G. 06/10/09 14:00 Poster# 119 06/11/09 14:00 Poster# 196 Chiabrando, D. Chan, H. 06/08/09 14:00 Poster# 1 06/11/09 16:30 Podium# 78 Chirnomas, S. [ [email protected] ] Chan, L. 06/11/09 14:00 Poster# 218 06/10/09 16:30 Podium# 56 Choi, J. [ [email protected] ] Chan, N. 06/10/09 14:00 Poster# 98 06/08/09 14:00 Poster# 87 Chora, A. Chang, Y. [ [email protected] ] 06/10/09 16:00 Podium# 48 06/10/09 14:00 Poster# 153 06/10/09 14:00 Poster# 172

194

Chu, B. Conde e Silva, N. [ [email protected] ] 06/09/09 10:00 Podium# 33 06/08/09 14:00 Poster# 23

Chu, C. Conejero, G. 06/11/09 15:15 Podium# 73 06/10/09 14:00 Poster# 138

Chua, A. Conesa, C. 06/08/09 14:00 Poster# 37 06/10/09 17:45 Podium# 66 06/08/09 14:00 Poster# 83 06/10/09 14:00 Poster# 121 Congiu, R. 06/11/09 16:15 Podium# 89 Chung, B. [ [email protected] ] 06/10/09 14:00 Poster# 99 Conlan, A. 06/10/09 10:00 Podium# 37 Cilloni, D. 06/08/09 18:00 Podium# 31 Connor, J. 06/10/09 14:00 Poster# 95 06/08/09 14:00 Poster# 85 06/08/09 14:00 Poster# 86 Clark, R. 06/10/09 14:00 Poster# 149 06/10/09 14:00 Poster# 128 06/10/09 14:00 Poster# 157 06/10/09 14:00 Poster# 160 Cleto, E. 06/10/09 14:00 Poster# 162 06/08/09 14:00 Poster# 14 06/10/09 14:00 Poster# 165 06/11/09 14:00 Poster# 259 Clifton, M. 06/11/09 16:00 Podium# 76 06/10/09 16:15 Podium# 49 Consonni, A. Cocco, E. 06/08/09 15:45 Podium# 5 06/08/09 14:00 Poster# 58 Constante, M. Codazzi, F. [ [email protected] ] 06/08/09 14:00 Poster# 76 06/08/09 15:45 Podium# 5 Constantine, C. Coffman, L. 06/11/09 14:00 Poster# 224 06/10/09 14:00 Poster# 140 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 240 Cohen, L. [ [email protected] ] 06/11/09 14:00 Poster# 252 06/10/09 14:00 Poster# 96 06/11/09 14:00 Poster# 256

Colasanti, M. Conti, A. 06/11/09 15:30 Podium# 74 06/10/09 14:00 Poster# 163

Cole, M. Cook, J. 06/10/09 14:00 Poster# 176 06/11/09 14:00 Poster# 224 06/11/09 14:00 Poster# 225 Coletti, S. 06/08/09 14:00 Poster# 91 Cooke, K. [ [email protected] ] 06/10/09 15:15 Podium# 39 Collins, J. [ [email protected] ] 06/10/09 16:30 Podium# 44 06/11/09 14:00 Poster# 227 Cooperman, S. Como, G. 06/08/09 16:00 Podium# 18 06/08/09 14:00 Poster# 78 Coppin, H. Compagni, C. 06/08/09 17:30 Podium# 23 06/10/09 14:00 Poster# 167 06/09/09 12:15 Podium# 36 06/11/09 14:00 Poster# 190

195

Coppola, G. Craik, D. 06/11/09 16:15 Podium# 77 06/10/09 14:00 Poster# 128

Corcoran, J. Crampton, E. 06/10/09 15:15 Podium# 39 06/08/09 14:00 Poster# 82

Corlu, A. Crichton, R. [ [email protected] ] 06/11/09 16:00 Podium# 82 06/10/09 14:00 Poster# 107 06/11/09 16:30 Podium# 78 Corradini, E. [ [email protected] ] 06/08/09 17:15 Podium# 22 Crooks, D. [ [email protected] ] 06/10/09 14:00 Poster# 113 06/08/09 16:15 Podium# 19

Correia, A.P. [ [email protected] ] Crowe, J. 06/10/09 14:00 Poster# 164 06/08/09 14:00 Poster# 35 06/08/09 14:00 Poster# 40 Correia, A.R. 06/08/09 14:00 Poster# 89 06/10/09 14:00 Poster# 173 06/11/09 15:15 Podium# 85

Correia-Neves, M. Cruz Moura, I. 06/10/09 15:45 Podium# 47 06/08/09 14:00 Poster# 6 (WITHDRAWN) 06/11/09 16:15 Podium# 77 Cruz, E. Corrocher, R. 06/08/09 14:00 Poster# 38 06/08/09 14:00 Poster# 68 06/08/09 14:00 Poster# 73 Cruz, T. [ [email protected] ] 06/08/09 14:00 Poster# 29 Costa, E. 06/08/09 14:00 Poster# 31 06/08/09 14:00 Poster# 16 06/11/09 14:00 Poster# 276 Cuijpers, M. [ [email protected] ] 06/08/09 14:00 Poster# 81 Costa, L. 06/08/09 14:00 Poster# 41 Culotta, V. 06/08/09 12:00 Podium# 1 Costa, M. 06/08/09 14:00 Poster# 38 Cunningham, C. 06/08/09 15:30 Podium# 4 Costa, V. 06/08/09 14:00 Poster# 29 Cuny, G. 06/10/09 14:00 Poster# 112 06/11/09 15:45 Podium# 81

Coudray, C. Curie, C. [ [email protected] ] 06/11/09 14:00 Poster# 199 (WITHDRAWN) 06/10/09 14:00 Poster# 127 06/10/09 14:00 Poster# 138 Coulon, S. [ [email protected] ] 06/08/09 14:00 Poster# 6 (WITHDRAWN) Da Silva, C. 06/10/09 14:00 Poster# 99 Courtois, Y. 06/10/09 14:00 Poster# 151 Da Silva, M. 06/10/09 18:00 Podium# 61 Cousins, R. 06/11/09 14:00 Poster# 194 Daba, A. [ [email protected] ] 06/10/09 14:00 Poster# 100 Coutinho, A. 06/10/09 16:00 Podium# 48 Dalton, G. 06/11/09 14:00 Poster# 240 Cozzi, A. 06/08/09 16:30 Podium# 8 196

Dancis, A. de Kort, W. 06/08/09 14:00 Poster# 22 06/11/09 14:00 Poster# 231

Dang, T. De la Calle, F. 06/10/09 14:00 Poster# 185 06/11/09 14:00 Poster# 250 06/11/09 14:00 Poster# 251 Daniel, V. 06/10/09 14:00 Poster# 126 de Mast, Q. 06/08/09 14:00 Poster# 26 Darnaud, V. 06/08/09 17:30 Podium# 23 de Sousa, M. 06/09/09 12:15 Podium# 36 06/08/09 14:00 Poster# 2 06/11/09 14:00 Poster# 190 06/08/09 14:00 Poster# 87 06/09/09 12:00 Podium# 35 Darshan, D. [ [email protected] ] 06/10/09 14:00 Poster# 112 06/10/09 14:00 Poster# 117 06/10/09 14:00 Poster# 124 De Val, N. 06/10/09 16:15 Podium# 55 06/10/09 14:00 Poster# 107

Das, N. de Verneuil, H. 06/08/09 14:00 Poster# 27 06/08/09 14:00 Poster# 9

Datz, C. de Vries, R. 06/08/09 14:00 Poster# 10 06/11/09 14:00 Poster# 239

David, K. de Witte, T. 06/08/09 14:00 Poster# 42 06/08/09 14:00 Poster# 81

David, S. Declercq, J. 06/10/09 14:00 Poster# 148 06/10/09 14:00 Poster# 107 06/10/09 14:00 Poster# 169 Defilippi, I. David, V. 06/08/09 18:00 Podium# 31 06/11/09 11:30 Podium# 71 06/10/09 14:00 Poster# 95 06/11/09 14:00 Poster# 235 Del Castillo, A. [ [email protected] ] Dawkins, F. 06/11/09 14:00 Poster# 250 06/11/09 14:00 Poster# 237 06/11/09 14:00 Poster# 251 de Almeida, S. Delaby, C. 06/10/09 14:00 Poster# 112 06/08/09 14:00 Poster# 9 de Castro, B. Delatycki, M. 06/08/09 14:00 Poster# 28 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 240 De Domenico, I. [ [email protected] ] 06/11/09 14:00 Poster# 252 06/08/09 14:00 Poster# 54 06/08/09 15:15 Podium# 9 Delbini, P. 06/10/09 15:30 Podium# 40 06/11/09 14:00 Poster# 205 06/11/09 14:00 Poster# 206 De Falco, L. 06/10/09 14:00 Poster# 103 Delima, R. 06/10/09 15:45 Podium# 41 06/08/09 14:00 Poster# 37 06/08/09 14:00 Poster# 83 De Francesco, G. 06/10/09 14:00 Poster# 121 06/11/09 15:30 Podium# 74

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DelRio-LaFreniere, S. Doki, N. 06/11/09 14:00 Poster# 254 06/11/09 14:00 Poster# 225

Demikhov, V. [ [email protected] ] Domokos, G. 06/08/09 14:00 Poster# 15 06/10/09 16:30 Podium# 56 06/11/09 14:00 Poster# 234 Donadieu, J. den Heijer, M. 06/10/09 18:00 Podium# 61 06/08/09 14:00 Poster# 74 Dongen-van Lases, E. Derosas, M. 06/08/09 14:00 Poster# 26 06/08/09 14:00 Poster# 58 Dongiovanni, P. Desbenoit, N. 06/08/09 14:00 Poster# 78 06/08/09 14:00 Poster# 69 06/08/09 17:00 Podium# 27 06/11/09 14:00 Poster# 271 Descalzi, F. [ [email protected] ] 06/11/09 14:00 Poster# 275 06/10/09 16:15 Podium# 49 Donovan, A. Deschemin, J. 06/11/09 10:00 Podium# 69 06/08/09 14:00 Poster# 69 Dostalikova-Cimburova, M. Deugnier, Y. 06/10/09 14:00 Poster# 136 06/11/09 11:30 Podium# 71 06/11/09 14:00 Poster# 199 (WITHDRAWN) Drakesmith, H. 06/11/09 14:00 Poster# 235 06/08/09 14:00 Poster# 25 06/08/09 14:00 Poster# 92 Dexter, D. [ [email protected] ] 06/10/09 16:30 Podium# 50 06/11/09 16:30 Podium# 78 Drapier, J. Di Cunto, F. 06/08/09 14:00 Poster# 57 06/10/09 14:00 Poster# 129 Dringen, R. Di, X. 06/10/09 14:00 Poster# 155 06/08/09 17:30 Podium# 29 06/10/09 14:00 Poster# 185

Diab, A. Driss, F. 06/11/09 14:00 Poster# 208 06/08/09 14:00 Poster# 33

Dias, V. Druilhe, P. 06/09/09 12:00 Podium# 35 06/08/09 14:00 Poster# 26 06/10/09 14:00 Poster# 164 Duan, X.L. [ [email protected] ] Ding, H. [ [email protected] ] 06/10/09 14:00 Poster# 153 06/08/09 14:00 Poster# 19 06/10/09 14:00 Poster# 172

Divitini, M. Duan, X. 06/10/09 14:00 Poster# 176 06/08/09 14:00 Poster# 19

Dixon, J. [ [email protected] ] Duca, L. [ [email protected] ] 06/11/09 14:00 Poster# 236 06/11/09 14:00 Poster# 205 06/11/09 14:00 Poster# 206 Do, T. 06/10/09 14:00 Poster# 137 Dunaief, J. [ [email protected] ] 06/11/09 10:00 Podium# 69 Doggen, C. 06/11/09 14:00 Poster# 231

198

Dyas, G. Elmberg, M. 06/10/09 15:15 Podium# 39 06/11/09 14:00 Poster# 195

Dycke, C. Elmen, J. 06/10/09 14:00 Poster# 130 06/11/09 10:15 Podium# 70

Dyrskjøt, L. Elsässer, H. 06/08/09 14:00 Poster# 80 06/08/09 14:00 Poster# 56

Eaton, J. [ [email protected] ] Engelhardt, R. 06/11/09 14:00 Poster# 219 06/10/09 15:30 Podium# 52

Eberwein, L. English, D. 06/08/09 14:00 Poster# 10 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 240 Eckes, K. 06/10/09 14:00 Poster# 180 Enns, C. 06/08/09 16:00 Podium# 6 Eckfeldt, J. 06/08/09 17:45 Podium# 24 06/11/09 14:00 Poster# 224 06/10/09 16:00 Podium# 42 06/11/09 14:00 Poster# 237 06/11/09 14:00 Poster# 254 Erba, B. 06/11/09 15:30 Podium# 86 06/08/09 16:30 Podium# 8

Eddowes, L. [ [email protected] ] Erfle, H. 06/08/09 14:00 Poster# 25 06/10/09 14:00 Poster# 115 06/10/09 16:30 Podium# 50 Ernst, M. Edelmann, M. 06/08/09 14:00 Poster# 3 06/08/09 14:00 Poster# 92 Estève, J. Eisenberg, J. 06/10/09 14:00 Poster# 108 06/08/09 14:00 Poster# 4 Evans, P. Eisenstein, R. 06/08/09 14:00 Poster# 42 06/10/09 14:00 Poster# 135 06/08/09 14:00 Poster# 64

Ekbom, A. Evans, R. [ [email protected] ] 06/11/09 14:00 Poster# 195 06/10/09 17:45 Podium# 66

Elalfy, M. Even, A. 06/10/09 16:30 Podium# 56 06/10/09 14:00 Poster# 124

Elamin, A. Faasse, S. 06/08/09 14:00 Poster# 53 06/10/09 14:00 Poster# 113

El-Beshlawy, A. Fabry, M. 06/10/09 16:30 Podium# 56 06/11/09 15:30 Podium# 80

Eliat, P. Faivre, D. 06/11/09 14:00 Poster# 209 06/08/09 14:00 Poster# 18

Ellen, F. Falcao, A. 06/08/09 14:00 Poster# 42 06/11/09 16:15 Podium# 77

Ellison, A. Fang, C. 06/10/09 16:30 Podium# 44 06/10/09 10:15 Podium# 38

199

Farace, E. Fibach, E. 06/08/09 14:00 Poster# 86 06/08/09 14:00 Poster# 47

Fargion, S. Figueiredo, A. 06/08/09 17:00 Podium# 27 06/08/09 14:00 Poster# 16 06/08/09 14:00 Poster# 78 06/11/09 14:00 Poster# 271 Fillebeen, C. 06/11/09 14:00 Poster# 275 06/10/09 14:00 Poster# 170

Farnaud, S. Finazzi, D. 06/10/09 17:45 Podium# 66 06/08/09 14:00 Poster# 55 06/08/09 14:00 Poster# 62 Fatih, N. [ [email protected] ] 06/11/09 16:00 Podium# 82 Fiorelli, G. 06/11/09 14:00 Poster# 205 Fatta, E. 06/11/09 14:00 Poster# 206 06/11/09 14:00 Poster# 275 Firth, M. Fattovich, G. 06/10/09 14:00 Poster# 121 06/08/09 14:00 Poster# 68 Fischer, R. Faustino, P. 06/08/09 14:00 Poster# 64 06/08/09 14:00 Poster# 93 06/10/09 15:30 Podium# 52 06/08/09 14:00 Poster# 94 06/11/09 14:00 Poster# 226 06/11/09 14:00 Poster# 233 Felix, V. 06/10/09 14:00 Poster# 106 Flechtenmacher, C. 06/11/09 14:00 Poster# 207 Férec, C. 06/11/09 16:15 Podium# 89 Fleming, A. 06/11/09 14:00 Poster# 204 Fergelot, P. 06/11/09 14:00 Poster# 196 Fleming, M. 06/08/09 14:00 Poster# 63 Fernandez, C. 06/08/09 18:15 Podium# 26 06/10/09 17:45 Podium# 60 06/10/09 17:45 Podium# 60 06/11/09 14:00 Poster# 223 Ferrand, A. 06/10/09 14:00 Poster# 108 Fleming, Rita [ [email protected] ] 06/08/09 14:00 Poster# 93 Ferreira, A. 06/11/09 14:00 Poster# 248 06/10/09 16:00 Podium# 48 Fleming, Robert [ [email protected] ] Ferreira, F. 06/08/09 16:15 Podium# 13 06/08/09 14:00 Poster# 14 06/08/09 18:00 Podium# 25 06/10/09 14:00 Poster# 97 Ferreira, G. [ [email protected] ] 06/11/09 14:00 Poster# 193 06/10/09 14:00 Poster# 106 06/11/09 14:00 Poster# 274 Follenzi, A. 06/08/09 14:00 Poster# 87 Ferrer Antunes, C. 06/08/09 14:00 Poster# 16 Ford, S. 06/11/09 14:00 Poster# 276 06/08/09 17:45 Podium# 30

Ferring-Appel, D. Forni, G. [ [email protected] ] 06/08/09 16:30 Podium# 14 06/11/09 14:00 Poster# 211 06/08/09 15:15 Podium# 15 06/11/09 14:00 Poster# 212 06/10/09 14:00 Poster# 114 200

Fournier, C. Gannon, P. 06/09/09 12:15 Podium# 36 06/10/09 14:00 Poster# 142

Fracanzani, A. Ganz, T. [ [email protected] ] 06/08/09 17:00 Podium# 27 06/08/09 14:00 Poster# 64 06/11/09 14:00 Poster# 271 06/08/09 14:00 Poster# 68 06/11/09 14:00 Poster# 275 06/08/09 14:00 Poster# 79 06/10/09 14:00 Poster# 120 Franciotta, D. 06/10/09 14:00 Poster# 126 06/10/09 14:00 Poster# 163 06/10/09 14:00 Poster# 128 06/10/09 16:15 Podium# 43 Franco, R. 06/10/09 17:30 Podium# 59 06/11/09 14:00 Poster# 274 06/11/09 11:45 Podium# 72 06/11/09 14:00 Poster# 199 (WITHDRAWN) Frazer, D. 06/11/09 16:30 Podium# 84 06/08/09 14:00 Poster# 82 06/10/09 14:00 Poster# 117 Gao, J. 06/08/09 17:45 Podium# 24 Freire, F. 06/10/09 14:00 Poster# 106 Garavaglia, B. 06/10/09 14:00 Poster# 174 Fritsche, G. 06/10/09 15:30 Podium# 46 Garcia dos Santos, D. [ [email protected] ] 06/08/09 14:00 Poster# 4 Fu, X. 06/10/09 14:00 Poster# 110 Garcia-Herrero, A. 06/09/09 10:00 Podium# 33 Fujikura, Y. [ [email protected] ] 06/08/09 14:00 Poster# 90 Garcia-Santos, D. 06/10/09 17:00 Podium# 57 Gabayan, V. 06/11/09 11:45 Podium# 72 Gardenghi, S. 06/08/09 14:00 Poster# 87 Gaboriau, F. 06/10/09 17:15 Podium# 58 06/10/09 14:00 Poster# 137 06/11/09 14:00 Poster# 202 06/11/09 15:15 Podium# 79 Gagliardo, B. 06/08/09 14:00 Poster# 69 Garner, C. 06/11/09 14:00 Poster# 224 Gallois, Y. 06/11/09 14:00 Poster# 196 Garrick, L. 06/10/09 14:00 Poster# 109 Galy, B. [ [email protected] ] 06/08/09 14:00 Poster# 3 Garrick, M. [ [email protected] ] 06/08/09 15:15 Podium# 15 06/10/09 14:00 Poster# 109 06/08/09 15:45 Podium# 17 06/08/09 16:30 Podium# 14 Garrido, P. 06/10/09 14:00 Poster# 114 06/08/09 14:00 Poster# 16 06/10/09 14:00 Poster# 135 06/11/09 14:00 Poster# 276

Gameiro, P. Garuti, C. 06/08/09 14:00 Poster# 32 06/08/09 17:15 Podium# 22

Gandon, Y. Gatti, S. 06/11/09 14:00 Poster# 209 06/08/09 17:00 Podium# 27 06/11/09 14:00 Poster# 271

201

Gearing, M. Ginzburg, Y. [ [email protected] ] 06/11/09 15:15 Podium# 73 06/11/09 15:30 Podium# 80

Gehrke, S. Girelli, D. 06/10/09 14:00 Poster# 132 06/08/09 14:00 Poster# 54 06/08/09 14:00 Poster# 68 Gelfi, C. 06/08/09 14:00 Poster# 73 06/10/09 16:00 Podium# 54 06/08/09 14:00 Poster# 78 06/08/09 14:00 Poster# 79 Geppert, M. 06/08/09 15:30 Podium# 10 06/10/09 14:00 Poster# 155 06/10/09 14:00 Poster# 120 06/10/09 16:00 Podium# 54 Gérault, P. 06/10/09 16:15 Podium# 43 06/11/09 14:00 Poster# 235 Glass, J. [ [email protected] ] Gerbaud, L. 06/10/09 14:00 Poster# 133 06/11/09 14:00 Poster# 199 (WITHDRAWN) 06/11/09 14:00 Poster# 263

Gertig, D. Goarin, H. 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 235 06/11/09 14:00 Poster# 240 06/11/09 14:00 Poster# 252 Goddard, K. 06/11/09 14:00 Poster# 224 Geschwind, D. 06/11/09 16:15 Podium# 77 Goldenberg, H. 06/08/09 14:00 Poster# 8 Geurts-Moespot, A. 06/08/09 14:00 Poster# 13 06/08/09 14:00 Poster# 74 06/11/09 14:00 Poster# 264 06/11/09 14:00 Poster# 266 Ghadersohi, S. 06/10/09 14:00 Poster# 109 Gomes, A. 06/08/09 14:00 Poster# 28 Ghio, A. 06/10/09 14:00 Poster# 109 Gomes, C. [ [email protected] ] 06/10/09 14:00 Poster# 173 Ghosh, M. 06/08/09 12:15 Podium# 2 Gomes, M. [ [email protected] ] 06/08/09 16:00 Podium# 18 06/08/09 14:00 Poster# 28 06/08/09 16:15 Podium# 19 06/10/09 15:45 Podium# 47

Gianesin, B. Gomes, S. 06/11/09 14:00 Poster# 211 06/10/09 15:45 Podium# 47 06/11/09 14:00 Poster# 212 Gomes-Pereira, S. Giardina, P. 06/10/09 15:45 Podium# 47 06/10/09 17:15 Podium# 58 06/11/09 15:15 Podium# 79 Gomez, F. 06/08/09 14:00 Poster# 49 Giles, G. 06/11/09 14:00 Poster# 238 Gonçalves, I. 06/11/09 14:00 Poster# 240 06/08/09 14:00 Poster# 23

Gilles, W. Gonzalez, A. 06/10/09 14:00 Poster# 125 06/08/09 14:00 Poster# 45 06/11/09 14:00 Poster# 207 06/11/09 14:00 Poster# 245 Goodfellow, B. [ [email protected] ] 06/10/09 14:00 Poster# 106

202

Goodnough, J. Grgurevic, L. 06/08/09 14:00 Poster# 68 06/10/09 14:00 Poster# 113

Gorbunov, N. [ [email protected] ] Grieco, S. 06/08/09 14:00 Poster# 43 (Moved to June 10, 17:20) 06/11/09 10:00 Podium# 69

Gordeuk, V. Groene, H. 06/11/09 14:00 Poster# 224 06/08/09 15:15 Podium# 15 06/11/09 14:00 Poster# 237 06/10/09 14:00 Poster# 114 06/11/09 15:30 Podium# 86 Grohovaz, F. Gordo, S. 06/08/09 15:45 Podium# 5 06/11/09 14:00 Poster# 250 06/11/09 14:00 Poster# 251 Grotto, H. [ [email protected] ] 06/08/09 14:00 Poster# 30 Goto, H. 06/11/09 14:00 Poster# 241 (WITHDRAWN) Grzelak, A. 06/10/09 14:00 Poster# 183 Gouffier, L. 06/10/09 14:00 Poster# 137 Guernsey, D. 06/10/09 17:45 Podium# 60 Gourlaouen, I. 06/11/09 16:15 Podium# 89 Guerquin-Kern, J. 06/10/09 14:00 Poster# 170 Gozzelino, R. [ [email protected] ] 06/10/09 16:00 Podium# 48 Guggenbuhl, P. [ [email protected] ] 06/11/09 14:00 Poster# 196 Grady, R. [ [email protected] ] 06/08/09 14:00 Poster# 2 Guido, M. 06/08/09 14:00 Poster# 87 06/08/09 14:00 Poster# 68 06/10/09 17:15 Podium# 58 06/11/09 14:00 Poster# 202 Guillem, F. 06/11/09 15:15 Podium# 79 06/10/09 15:45 Podium# 41 06/10/09 18:00 Podium# 61 Graham, R. 06/08/09 14:00 Poster# 37 Gunnlaugson, H. 06/08/09 14:00 Poster# 83 06/08/09 14:00 Poster# 18 06/10/09 14:00 Poster# 121 06/10/09 14:00 Poster# 158 Guo, P. 06/10/09 14:00 Poster# 171 06/10/09 14:00 Poster# 153 06/11/09 14:00 Poster# 193 Gurrin, L. [ [email protected] ] Grandchamp, B. [ [email protected] ] 06/11/09 14:00 Poster# 238 06/10/09 15:45 Podium# 41 06/11/09 14:00 Poster# 240 06/10/09 18:00 Podium# 61 06/11/09 14:00 Poster# 252 06/11/09 14:00 Poster# 256 Grapton, D. 06/08/09 14:00 Poster# 6 (WITHDRAWN) Gutekunst, C. 06/11/09 15:15 Podium# 73 Grassi, G. 06/11/09 14:00 Poster# 242 Gutierrez, L. [ [email protected] ] 06/10/09 18:00 Podium# 67 Grebenchtikov, N. 06/08/09 14:00 Poster# 74

Greenamyre, J. 06/11/09 15:15 Podium# 73

203

Guy, E. Hao, L. 06/08/09 14:00 Poster# 2 06/10/09 14:00 Poster# 157 06/08/09 14:00 Poster# 87 06/11/09 14:00 Poster# 202 Harir, Y. 06/11/09 15:15 Podium# 79 06/10/09 10:00 Podium# 37

Habr, D. Harmatz, P. 06/10/09 16:30 Podium# 56 06/08/09 14:00 Poster# 42 06/08/09 14:00 Poster# 64 Hackney, J. 06/11/09 14:00 Poster# 270 Harris, E. 06/11/09 14:00 Poster# 237 Hadziahmetovic, M. 06/11/09 10:00 Podium# 69 Harrison, B. 06/11/09 15:30 Podium# 86 Haeger, P. 06/10/09 14:00 Poster# 156 Haskova, P. 06/10/09 14:00 Poster# 179 Hagar, W. 06/10/09 14:00 Poster# 182 06/08/09 14:00 Poster# 64 Hattori, A. Hahn, P. 06/11/09 14:00 Poster# 241 (WITHDRAWN) 06/11/09 10:00 Podium# 69 06/11/09 15:45 Podium# 87

Haile, D. Hausmann, A. [ [email protected] ] 06/08/09 14:00 Poster# 9 06/10/09 14:00 Poster# 100 06/10/09 14:00 Poster# 150 06/10/09 14:00 Poster# 105 06/10/09 14:00 Poster# 154 Hayashi, H. Halada, P. 06/11/09 14:00 Poster# 241 (WITHDRAWN) 06/10/09 17:15 Podium# 64 06/11/09 15:45 Podium# 87

Halket, J. Hayashi, K. 06/08/09 14:00 Poster# 75 06/11/09 14:00 Poster# 241 (WITHDRAWN)

Hall, E. Hayashi, T. 06/10/09 14:00 Poster# 165 06/11/09 15:45 Podium# 87

Hall, N. He, T. 06/10/09 14:00 Poster# 181 06/11/09 14:00 Poster# 204

Hallendorff, M. Heeney, M. [ [email protected] ] 06/11/09 14:00 Poster# 198 06/11/09 14:00 Poster# 223 06/11/09 14:00 Poster# 255 Hegde, N. Hanif, R. [ [email protected] ] 06/10/09 14:00 Poster# 157 06/10/09 14:00 Poster# 134 Hegetschweiler, K. Haniu, M. 06/11/09 14:00 Poster# 191 06/10/09 15:15 Podium# 39 06/11/09 14:00 Poster# 197 06/11/09 14:00 Poster# 203 Hans, W. 06/10/09 15:45 Podium# 53 Heimpel, H. 06/08/09 14:00 Poster# 5 Hantke, K. 06/10/09 15:30 Podium# 46 Helminger, M. [ [email protected] ] 06/11/09 14:00 Poster# 266

204

Helston, R. Hift, R. 06/11/09 14:00 Poster# 270 06/11/09 14:00 Poster# 198

Hendriks, J. Higa, A. 06/10/09 16:15 Podium# 43 06/08/09 14:00 Poster# 42 06/11/09 14:00 Poster# 239 Higuchi, M. [ [email protected] ] Henkin, L. 06/10/09 14:00 Poster# 184 06/11/09 15:30 Podium# 86 Hill, N. [ [email protected] ] Hentze, M. 06/08/09 14:00 Poster# 53 06/08/09 14:00 Poster# 3 06/08/09 14:00 Poster# 39 Hilton, R. 06/08/09 14:00 Poster# 59 06/08/09 14:00 Poster# 12 06/08/09 14:00 Poster# 60 06/08/09 14:00 Poster# 84 Hirata, M. 06/08/09 14:00 Poster# 88 06/11/09 15:45 Podium# 87 06/08/09 15:15 Podium# 15 06/08/09 15:45 Podium# 17 Hirsch, E. 06/08/09 16:30 Podium# 14 06/10/09 14:00 Poster# 129 06/10/09 14:00 Poster# 114 06/10/09 14:00 Poster# 115 Hitti, E. 06/10/09 14:00 Poster# 125 06/11/09 14:00 Poster# 209 06/10/09 14:00 Poster# 135 06/11/09 10:15 Podium# 70 Hlobenova, T. 06/08/09 14:00 Poster# 70 Herbison, C. 06/10/09 14:00 Poster# 118 06/08/09 14:00 Poster# 37 06/08/09 14:00 Poster# 83 Hmissi, A. 06/10/09 14:00 Poster# 121 06/10/09 16:30 Podium# 56

Hermine, O. Hoette, T. 06/08/09 14:00 Poster# 6 (WITHDRAWN) 06/10/09 16:15 Podium# 49

Hermsen, C. Hohnholt, M. [ [email protected] ] 06/08/09 14:00 Poster# 26 06/10/09 14:00 Poster# 155

Herrmann, T. Hollins, S. 06/11/09 14:00 Poster# 207 06/11/09 14:00 Poster# 193

Hersey, P. Holter, S. 06/10/09 14:00 Poster# 143 06/08/09 15:30 Podium# 16

Hertz, M. Hong, C. 06/08/09 14:00 Poster# 64 06/11/09 15:45 Podium# 81

Hidalgo, C. [ [email protected] ] Hopkins, J. 06/10/09 14:00 Poster# 156 06/11/09 14:00 Poster# 232

Hider, R. Hopper, J. 06/08/09 14:00 Poster# 28 06/11/09 14:00 Poster# 238 06/08/09 14:00 Poster# 67 06/11/09 14:00 Poster# 240 06/08/09 14:00 Poster# 75 06/10/09 16:15 Podium# 43 Horowitz, M. 06/10/09 18:15 Podium# 68 06/11/09 15:15 Podium# 73 06/11/09 14:00 Poster# 258

205

Hosoki, T. Iannaccone, S. 06/08/09 14:00 Poster# 72 06/10/09 14:00 Poster# 163 06/11/09 14:00 Poster# 192 06/11/09 14:00 Poster# 257 Ibrahim, H. 06/10/09 16:30 Podium# 56 House, M. [ [email protected] ] 06/10/09 14:00 Poster# 152 Ikuta, K. 06/11/09 14:00 Poster# 204 06/08/09 14:00 Poster# 72 06/11/09 14:00 Poster# 192 Hrdina, R. 06/11/09 14:00 Poster# 257 06/10/09 14:00 Poster# 186 06/10/09 14:00 Poster# 187 Iliopoulos, O. [ [email protected] ] 06/11/09 14:00 Poster# 278 Hrdy, I. 06/10/09 17:15 Podium# 64 Illing, A. 06/08/09 15:30 Podium# 4 Hruskova, K. 06/11/09 14:00 Poster# 194 06/10/09 14:00 Poster# 179 06/11/09 14:00 Poster# 267

Hu, X. Inyakova, N. 06/10/09 14:00 Poster# 110 06/11/09 14:00 Poster# 234

Huang, H. [ [email protected] ] Iolascon, A. 06/08/09 14:00 Poster# 76 06/10/09 14:00 Poster# 103 06/08/09 16:00 Podium# 12 06/10/09 15:45 Podium# 41

Huang, M. Iqbal, T. 06/10/09 17:15 Podium# 64 06/08/09 14:00 Poster# 66 06/08/09 17:45 Podium# 30 Huey Mei Wang, P. 06/10/09 14:00 Poster# 144 06/08/09 14:00 Poster# 6 (WITHDRAWN) 06/11/09 14:00 Poster# 208

Hughes, R. Irgens, W. 06/08/09 12:15 Podium# 2 06/11/09 14:00 Poster# 244

Hultcrantz, R. [ [email protected] ] Isakova, O. 06/10/09 14:00 Poster# 180 06/11/09 14:00 Poster# 234 06/11/09 14:00 Poster# 195 Isidor, B. Human, V. 06/10/09 18:00 Podium# 61 06/10/09 14:00 Poster# 147 06/11/09 14:00 Poster# 255 Islam, M. 06/11/09 14:00 Poster# 265 Humeres, A. 06/10/09 14:00 Poster# 156 Island, M. 06/11/09 11:30 Podium# 71 Hunter, A. 06/11/09 16:00 Podium# 82 06/11/09 10:00 Podium# 69 Jacobs, E. Hveem, K. 06/11/09 14:00 Poster# 239 06/11/09 14:00 Poster# 244 Jacobsen, C. Hyde, B. 06/08/09 14:00 Poster# 24 06/08/09 16:15 Podium# 7 Jahodar, L. Iacovelli, J. 06/10/09 14:00 Poster# 186 06/11/09 10:00 Podium# 69

206

Jaiash, D. [ [email protected] ] Kahawita, T. 06/08/09 14:00 Poster# 67 06/08/09 14:00 Poster# 44 06/10/09 17:00 Podium# 57 Jamal, M. 06/11/09 14:00 Poster# 225 Kakei, Y. 06/11/09 14:00 Poster# 273 Janka, G. 06/10/09 15:30 Podium# 52 Kakhlon, O. 06/08/09 14:00 Poster# 52 (Moved to June 10, 17:40) Jansen, T. 06/11/09 14:00 Poster# 233 Kalinowski, D. [ [email protected] ] 06/11/09 14:00 Poster# 265 Jaouen, M. 06/08/09 14:00 Poster# 69 Kammer, A. 06/08/09 15:45 Podium# 11 Jellen, L. 06/10/09 14:00 Poster# 161 Kanda, J. 06/11/09 15:45 Podium# 87 Jenkitkasemwong, S. 06/08/09 14:00 Poster# 11 Kandemir, B. [ [email protected] ] 06/11/09 14:00 Poster# 229 (WITHDRAWN) Jennings, P. 06/10/09 10:00 Podium# 37 Kannengiesser, C. 06/10/09 18:00 Podium# 61 Jeong, S. 06/08/09 16:00 Podium# 18 Kaplan, J. 06/10/09 14:00 Poster# 131 (WITHDRAWN) 06/08/09 12:00 Podium# 1 06/08/09 14:00 Poster# 46 Jin, V. 06/08/09 14:00 Poster# 54 06/11/09 14:00 Poster# 247 06/08/09 16:15 Podium# 7 06/10/09 15:30 Podium# 40 Johnstone, D. [ [email protected] ] 06/10/09 14:00 Poster# 121 Karim, Z. 06/10/09 14:00 Poster# 143 06/08/09 14:00 Poster# 9 06/10/09 14:00 Poster# 158 06/10/09 14:00 Poster# 171 Katano, Y. 06/11/09 14:00 Poster# 193 06/11/09 14:00 Poster# 241 (WITHDRAWN)

Jones, B. [ [email protected] ] Kato, J. 06/10/09 14:00 Poster# 157 06/11/09 14:00 Poster# 221 06/10/09 14:00 Poster# 161 06/11/09 14:00 Poster# 222

Jordan, J. Kattamis, A. 06/10/09 15:15 Podium# 39 06/10/09 16:30 Podium# 56

Jortner, B. Kauppinen, S. 06/08/09 16:00 Podium# 18 06/11/09 10:15 Podium# 70

Jouanolle, A. Kautz, L. [ [email protected] ] 06/11/09 11:30 Podium# 71 06/08/09 17:30 Podium# 23 06/11/09 14:00 Poster# 235 06/09/09 12:15 Podium# 36 06/11/09 14:00 Poster# 190 Ka, C. 06/08/09 14:00 Poster# 92 Kawabata, H. 06/11/09 15:45 Podium# 87 Kaden, S. 06/08/09 15:15 Podium# 15 06/10/09 14:00 Poster# 114 207

Kawanaka, M. Kirk, P. 06/11/09 15:45 Podium# 87 06/11/09 14:00 Poster# 204

Kawano, Y. Kishi, F. [ [email protected] ] 06/11/09 14:00 Poster# 221 06/08/09 14:00 Poster# 48 06/11/09 14:00 Poster# 222 06/11/09 14:00 Poster# 269

Keith, B. Kiss, J. 06/08/09 17:00 Podium# 21 06/08/09 14:00 Poster# 5 06/08/09 14:00 Poster# 88 Kelleher, T. 06/11/09 14:00 Poster# 245 06/08/09 14:00 Poster# 89 06/11/09 15:15 Podium# 85 Kleiner, D. 06/11/09 14:00 Poster# 272 Kemna, E. 06/10/09 16:15 Podium# 43 Knuiman, M. 06/10/09 14:00 Poster# 176 Kempf, T. 06/10/09 16:00 Podium# 54 Knutson, M. 06/08/09 14:00 Poster# 11 Kenealey, Z. 06/08/09 14:00 Poster# 45 06/08/09 14:00 Poster# 12 06/08/09 14:00 Poster# 50 06/10/09 14:00 Poster# 113 Kerr, K. 06/11/09 14:00 Poster# 194 06/11/09 14:00 Poster# 193 Kobayashi, T. Kerscher, S. 06/11/09 14:00 Poster# 273 06/08/09 14:00 Poster# 56 Kobune, M. Kessler, B. 06/11/09 14:00 Poster# 221 06/08/09 14:00 Poster# 92 06/11/09 14:00 Poster# 222

Khalaghizadeh, S. Koch, N. [ [email protected] ] 06/11/09 14:00 Poster# 225 06/11/09 14:00 Poster# 191 06/11/09 14:00 Poster# 203 Kidokoro, R. 06/11/09 14:00 Poster# 241 (WITHDRAWN) Kohgo, Y. 06/08/09 14:00 Poster# 72 Kikuchi, S. 06/11/09 14:00 Poster# 192 06/11/09 14:00 Poster# 221 06/11/09 14:00 Poster# 257 06/11/09 14:00 Poster# 222 Kohno, H. Kilcourse, E. 06/10/09 15:15 Podium# 39 06/10/09 14:00 Poster# 122 Kojima, K. Killeen, A. 06/11/09 15:45 Podium# 87 06/11/09 14:00 Poster# 224 Koliaraki, V. Kim, A. [ [email protected] ] 06/10/09 16:15 Podium# 43 06/10/09 17:30 Podium# 59 Kolodney, J. Kim, H. 06/11/09 14:00 Poster# 225 06/10/09 15:15 Podium# 39 Kong, X. Kinsley, P. 06/11/09 14:00 Poster# 258 06/08/09 12:00 Podium# 1

208

Kongi, R. Kuchel, P. 06/10/09 15:30 Podium# 52 06/10/09 17:15 Podium# 64 06/11/09 14:00 Poster# 226 06/11/09 14:00 Poster# 233 Kuhn, L. [ [email protected] ] 06/10/09 14:00 Poster# 124 Konijn, A. 06/10/09 16:15 Podium# 55 06/10/09 14:00 Poster# 119 Kuzelova, K. Koromilas, A. 06/10/09 17:15 Podium# 64 06/10/09 14:00 Poster# 100 Laarakkers, C. Kovac, S. 06/08/09 14:00 Poster# 61 06/10/09 14:00 Poster# 108 Laccone, F. Kovacevic, Z. [ [email protected] ] 06/11/09 14:00 Poster# 264 06/08/09 17:15 Podium# 28 Laftah, A. [ [email protected] ] Kovar, J. [ [email protected] ] 06/08/09 14:00 Poster# 65 06/10/09 14:00 Poster# 136 06/08/09 14:00 Poster# 71 06/10/09 14:00 Poster# 98 Kovarikova, P. 06/10/09 14:00 Poster# 102 06/10/09 14:00 Poster# 179 06/11/09 14:00 Poster# 261 06/10/09 14:00 Poster# 182 Lagnel, C. [ [email protected] ] Kovtunovych, G. 06/10/09 14:00 Poster# 116 06/10/09 14:00 Poster# 131 (WITHDRAWN) Lainé, F. Kowdley, K. 06/10/09 14:00 Poster# 126 06/10/09 14:00 Poster# 122 06/11/09 14:00 Poster# 199 (WITHDRAWN) 06/11/09 14:00 Poster# 262 06/11/09 14:00 Poster# 272 Landry, A. 06/08/09 14:00 Poster# 19 Kramer, M. 06/08/09 17:45 Podium# 24 Langer, N. 06/08/09 16:15 Podium# 7 Kravtsova, N. 06/11/09 14:00 Poster# 234 Lapointe, R. 06/08/09 14:00 Poster# 34 Kreeftenberg, H. 06/11/09 14:00 Poster# 239 Larsen, R. 06/10/09 16:00 Podium# 48 Krijt, J. [ [email protected] ] 06/08/09 14:00 Poster# 70 Lasocki, S. 06/08/09 14:00 Poster# 90 06/08/09 14:00 Poster# 33 06/10/09 14:00 Poster# 118 06/08/09 15:15 Podium# 9

Krikler, S. [ [email protected] ] Latunde-Dada, G. [ [email protected] ] 06/11/09 14:00 Poster# 216 06/08/09 14:00 Poster# 67 06/11/09 14:00 Poster# 255 06/08/09 14:00 Poster# 77 06/10/09 14:00 Poster# 98 Kroot, J. [ [email protected] ] 06/10/09 14:00 Poster# 102 06/08/09 14:00 Poster# 36 06/11/09 14:00 Poster# 261 06/08/09 14:00 Poster# 74 06/10/09 16:15 Podium# 43 Lau, C. 06/09/09 10:00 Podium# 33 Kubat, N. 06/08/09 14:00 Poster# 69

209

Laver, D. Lesuisse, E. 06/11/09 14:00 Poster# 193 06/08/09 14:00 Poster# 22 06/08/09 14:00 Poster# 23 Lawless, M. [ [email protected] ] 06/11/09 14:00 Poster# 277 06/08/09 14:00 Poster# 35 06/08/09 14:00 Poster# 40 Letteron, P. 06/08/09 14:00 Poster# 89 06/08/09 15:15 Podium# 9 06/10/09 14:00 Poster# 116 Layoun, A. [ [email protected] ] 06/08/09 14:00 Poster# 76 Levi, S. 06/08/09 16:00 Podium# 12 06/08/09 16:30 Podium# 8 06/10/09 14:00 Poster# 174 Lazaro, F. 06/10/09 14:00 Poster# 181 06/10/09 18:00 Podium# 67 06/11/09 16:15 Podium# 83

Le Gac, G. [ [email protected] ] Li, C. 06/11/09 16:15 Podium# 89 06/10/09 14:00 Poster# 110

Le Lan, C. Li, C.K. 06/10/09 14:00 Poster# 137 06/10/09 16:30 Podium# 56

Lee, J. Li, Honggui 06/10/09 14:00 Poster# 105 06/11/09 14:00 Poster# 254

Lee, S. [ [email protected] ] Li, Hongyan 06/08/09 14:00 Poster# 85 06/10/09 16:30 Podium# 44 06/08/09 14:00 Poster# 86 06/10/09 14:00 Poster# 162 Li, Huihui 06/10/09 14:00 Poster# 165 06/11/09 15:30 Podium# 80

Lee, S.M. Li, Li 06/11/09 14:00 Poster# 230 06/10/09 10:15 Podium# 38

Leibold, E. [ [email protected] ] Li, Liangtao 06/08/09 15:30 Podium# 16 06/08/09 16:15 Podium# 7 06/08/09 16:30 Podium# 20 06/10/09 14:00 Poster# 178 Li, V. 06/10/09 15:45 Podium# 53 06/10/09 15:15 Podium# 39 06/10/09 17:30 Podium# 65 Li, Y. Leiendecker-Foster, C. 06/08/09 15:15 Podium# 3 06/11/09 14:00 Poster# 224 06/11/09 14:00 Poster# 237 Li, Z. 06/11/09 14:00 Poster# 254 06/11/09 14:00 Poster# 263

Leitman, S. Liberman, A. 06/11/09 14:00 Poster# 232 06/10/09 14:00 Poster# 170

Lemaire, M. Libouban, H. 06/08/09 14:00 Poster# 23 06/11/09 14:00 Poster# 196

Leroyer, P. Lichota, J. 06/11/09 16:00 Podium# 82 06/10/09 14:00 Poster# 150

Lescoat, G. [ [email protected] ] Lichota, L. 06/10/09 14:00 Poster# 137 06/10/09 14:00 Poster# 154

210

Lill, R. Lopez-Vales, R. 06/08/09 14:00 Poster# 56 06/10/09 14:00 Poster# 169

Lim, C. Loréal, O. [ [email protected] ] 06/10/09 14:00 Poster# 107 06/10/09 14:00 Poster# 126 06/10/09 14:00 Poster# 137 Lin, H. 06/11/09 11:30 Podium# 71 06/08/09 17:15 Podium# 22 06/11/09 14:00 Poster# 196 06/10/09 14:00 Poster# 113 06/11/09 14:00 Poster# 209 06/11/09 16:00 Podium# 82 Linder, M. 06/08/09 14:00 Poster# 45 Lourenço, M. 06/08/09 14:00 Poster# 53 06/11/09 14:00 Poster# 276 06/11/09 14:00 Poster# 228 Louw, A. Lindgren, S. 06/08/09 18:15 Podium# 32 06/11/09 14:00 Poster# 195 06/10/09 14:00 Poster# 146

Lind-Karlberg, M. [ [email protected] ] Lovejoy, D. 06/08/09 14:00 Poster# 20 06/11/09 14:00 Poster# 265

Lindner, E. Lovelace, R. 06/10/09 15:30 Podium# 46 06/11/09 14:00 Poster# 270

Lindow, M. Lu, Y. 06/11/09 10:15 Podium# 70 06/11/09 14:00 Poster# 227

Linehan, M. Ludman, M. 06/10/09 14:00 Poster# 131 (WITHDRAWN) 06/10/09 17:45 Podium# 60

Lipinski, P. Lundby, C. 06/10/09 14:00 Poster# 183 06/10/09 16:00 Podium# 54

Liu, S. Lupo, A. 06/08/09 14:00 Poster# 86 06/08/09 14:00 Poster# 73

Liu, X. Luscieti, S. [ [email protected] ] 06/11/09 14:00 Poster# 259 06/08/09 14:00 Poster# 55 06/08/09 14:00 Poster# 58 Liu, Y. 06/08/09 14:00 Poster# 62 06/10/09 14:00 Poster# 162 Luty, A. Liuzzi, J. 06/08/09 14:00 Poster# 26 06/11/09 14:00 Poster# 194 Lyoumi, S. Lobato, L. 06/08/09 14:00 Poster# 9 06/10/09 18:15 Podium# 62 Macakova, K. [ [email protected] ] Locati, M. 06/10/09 14:00 Poster# 186 06/08/09 15:30 Podium# 10 Maccarinelli, F. Lok, C. [ [email protected] ] 06/08/09 14:00 Poster# 55 06/11/09 14:00 Poster# 201 06/08/09 14:00 Poster# 58 06/11/09 16:00 Podium# 88 06/08/09 14:00 Poster# 62

Longo, F. 06/10/09 14:00 Poster# 167

211

Macco, A. Maniecki, M. [ [email protected] ] 06/11/09 14:00 Poster# 211 06/08/09 14:00 Poster# 80 06/11/09 14:00 Poster# 212 Marfaing, A. Macco, R. 06/10/09 18:00 Podium# 61 06/08/09 15:45 Podium# 5 Marguerite, G. Macedo, A. 06/10/09 18:00 Podium# 61 06/10/09 14:00 Poster# 106 Mari, S. Macedo, F. [ [email protected] ] 06/10/09 14:00 Poster# 127 06/08/09 14:00 Poster# 38 06/10/09 14:00 Poster# 138

Mackenzie, B. [ [email protected] ] Mariani, R. 06/08/09 15:30 Podium# 4 06/08/09 14:00 Poster# 79 06/11/09 14:00 Poster# 194 06/08/09 14:00 Poster# 91 06/11/09 14:00 Poster# 267 06/11/09 14:00 Poster# 189 06/11/09 14:00 Poster# 242 Madden, J. 06/08/09 14:00 Poster# 42 Marincu, I. 06/11/09 14:00 Poster# 213 Madhankumar, A. 06/11/09 14:00 Poster# 259 Marini, A. 06/08/09 15:30 Podium# 10 Maffè, C. 06/08/09 18:00 Podium# 31 Marongiu, M. 06/10/09 17:15 Podium# 58 Maffettone, C. [ [email protected] ] 06/11/09 14:00 Poster# 202 06/10/09 14:00 Poster# 141 Marques, F. [ [email protected] ] Maier, G. 06/11/09 16:15 Podium# 77 06/10/09 14:00 Poster# 176 Marques, L. [ [email protected] ] Maio, N. 06/08/09 14:00 Poster# 41 06/10/09 14:00 Poster# 177 06/11/09 15:30 Podium# 74 Marro, S. 06/08/09 14:00 Poster# 1 Mair, S. 06/10/09 15:30 Podium# 46 Marshal, V. 06/08/09 14:00 Poster# 67 Mairuae, N. 06/10/09 14:00 Poster# 165 Martelli, A. [ [email protected] ] 06/10/09 15:15 Podium# 51 Malanga, R. 06/08/09 14:00 Poster# 53 Martin, B. 06/10/09 14:00 Poster# 142 Malhó, R. 06/08/09 14:00 Poster# 41 Martinelli, N. 06/08/09 14:00 Poster# 68 Maliken, B. 06/11/09 14:00 Poster# 262 Martins, R. [ [email protected] ] 06/08/09 14:00 Poster# 93 Mamalaki, A. 06/08/09 14:00 Poster# 94 06/10/09 16:15 Podium# 43 Marx, J. Mancia, G. 06/11/09 14:00 Poster# 239 06/11/09 14:00 Poster# 242

212

Masaratana, P. McKie, A. 06/08/09 14:00 Poster# 71 06/08/09 14:00 Poster# 65 06/10/09 14:00 Poster# 102 06/08/09 14:00 Poster# 71 06/11/09 14:00 Poster# 261 06/08/09 14:00 Poster# 77 06/10/09 14:00 Poster# 98 Mascarenhas, C. 06/10/09 14:00 Poster# 102 06/10/09 14:00 Poster# 164 06/11/09 14:00 Poster# 252 06/11/09 14:00 Poster# 261 Masle, S. [ [email protected] ] 06/11/09 14:00 Poster# 224 McLaren, C. 06/11/09 14:00 Poster# 230 06/11/09 14:00 Poster# 224 06/11/09 14:00 Poster# 225 Mason, Andrew 06/11/09 14:00 Poster# 237 06/08/09 14:00 Poster# 53 06/11/09 14:00 Poster# 238 06/11/09 14:00 Poster# 240 Mason, Anne 06/11/09 14:00 Poster# 252 06/10/09 14:00 Poster# 108 06/11/09 15:30 Podium# 86 06/11/09 14:00 Poster# 229 (WITHDRAWN) McLaren, G. [ [email protected] ] Mastroberardino, P. [ [email protected] ] 06/11/09 14:00 Poster# 224 06/11/09 15:15 Podium# 73 06/11/09 14:00 Poster# 225 06/11/09 14:00 Poster# 237 Mastrogiannaki, M. [ [email protected] ] 06/11/09 14:00 Poster# 263 06/08/09 17:00 Podium# 21 06/11/09 15:30 Podium# 86

Matak, P. Medelci, S. 06/08/09 17:00 Podium# 21 06/10/09 14:00 Poster# 142 06/11/09 14:00 Poster# 252 Megazzini, C. Matsha, T. 06/08/09 17:00 Podium# 27 06/10/09 14:00 Poster# 145 06/10/09 14:00 Poster# 147 Meinhof, T. 06/10/09 14:00 Poster# 115 Matzanke, B. [ [email protected] ] 06/08/09 14:00 Poster# 18 Melchiori, L. [ [email protected] ] 06/11/09 15:15 Podium# 79 Maugard, C. [ [email protected] ] 06/10/09 14:00 Poster# 142 Melilli, E. 06/08/09 14:00 Poster# 73 Mazur, A. 06/11/09 14:00 Poster# 199 (WITHDRAWN) Melis, M. 06/11/09 16:15 Podium# 89 McArdle, H. 06/10/09 14:00 Poster# 134 Meneveri, R. 06/11/09 14:00 Poster# 189 McGregor, N. 06/08/09 18:15 Podium# 32 Menguy, N. 06/11/09 14:00 Poster# 198 06/08/09 14:00 Poster# 18 06/11/09 14:00 Poster# 255 Mercurio, S. McIntyre, N. 06/08/09 14:00 Poster# 1 06/11/09 14:00 Poster# 274 Merle, U. 06/10/09 14:00 Poster# 132

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Merryweather-Clarke, A. Millot, S. 06/08/09 14:00 Poster# 92 06/08/09 14:00 Poster# 9 06/11/09 14:00 Poster# 201 06/08/09 14:00 Poster# 33 06/11/09 16:00 Podium# 88 06/08/09 15:15 Podium# 9

Mes-Masson, A. Milman, N. [ [email protected] ] 06/10/09 14:00 Poster# 142 06/11/09 14:00 Poster# 243 06/11/09 14:00 Poster# 246 Messa, E. [ [email protected] ] 06/08/09 18:00 Podium# 31 Milward, A. [ [email protected] ] 06/10/09 14:00 Poster# 121 Messa, F. 06/10/09 14:00 Poster# 143 06/08/09 18:00 Podium# 31 06/10/09 14:00 Poster# 152 06/10/09 14:00 Poster# 158 Messa, P. 06/10/09 14:00 Poster# 171 06/08/09 14:00 Poster# 78 06/10/09 14:00 Poster# 176 06/11/09 14:00 Poster# 193 Messaddeq, N. 06/10/09 15:15 Podium# 51 Mims, L. 06/11/09 14:00 Poster# 268 06/10/09 14:00 Poster# 133

Messana, E. Miranda, L. 06/08/09 14:00 Poster# 1 06/10/09 15:15 Podium# 39

Metelerkamp, K. Missirlis, F. [ [email protected] ] 06/11/09 14:00 Poster# 193 06/08/09 14:00 Poster# 21

Meyers, K. Mitsumori, F. [ [email protected] ] 06/08/09 16:00 Podium# 6 06/10/09 14:00 Poster# 159

Meynard, D. Mittler, R. 06/08/09 17:30 Podium# 23 06/10/09 10:00 Podium# 37 06/09/09 12:15 Podium# 36 Miyanishi, K. Meyron-Holtz, E. [ [email protected] ] 06/11/09 14:00 Poster# 221 06/08/09 15:45 Podium# 11 06/11/09 14:00 Poster# 222 06/10/09 14:00 Poster# 96 Miyoshi, S. Micaelo, N. 06/11/09 14:00 Poster# 72 06/10/09 14:00 Poster# 106 Mizumoto, C. [ [email protected] ] Michael, D. 06/11/09 15:45 Podium# 87 06/10/09 10:00 Podium# 37 Mladenka, P. Michel, K. 06/10/09 14:00 Poster# 186 06/11/09 14:00 Poster# 268 06/10/09 14:00 Poster# 187

Migas, M. Mleczko-Sanecka, K. 06/08/09 16:15 Podium# 13 06/08/09 14:00 Poster# 59 06/08/09 18:00 Podium# 25 06/08/09 14:00 Poster# 60 06/10/09 14:00 Poster# 97 Moen, T. Mikhael, M. [ [email protected] ] 06/11/09 14:00 Poster# 244 06/08/09 14:00 Poster# 51 06/08/09 15:15 Podium# 3 Moestrup, S. 06/11/09 14:00 Poster# 188 06/08/09 14:00 Poster# 24 06/08/09 14:00 Poster# 80

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Moirand, R. Moscato, P. 06/10/09 14:00 Poster# 137 06/10/09 14:00 Poster# 143

Molineux, G. Moshe, S. 06/10/09 16:30 Podium# 44 06/08/09 15:45 Podium# 11

Møller, H. Mosser, A. 06/08/09 14:00 Poster# 80 06/11/09 11:30 Podium# 71

Monnier, A. [ [email protected] ] Mosser, J. 06/11/09 14:00 Poster# 190 06/11/09 14:00 Poster# 190

Monteiro, R. Motta, B. 06/08/09 14:00 Poster# 6 (WITHDRAWN) 06/11/09 14:00 Poster# 275

Montosi, G. Moulis, J. [ [email protected] ] 06/08/09 17:15 Podium# 22 06/10/09 14:00 Poster# 130

Montravers, P. Moutereau, S. 06/08/09 14:00 Poster# 33 06/10/09 16:00 Podium# 54

Moos, T. [ [email protected] ] Muckenthaler, M. 06/10/09 14:00 Poster# 150 06/08/09 14:00 Poster# 5 06/10/09 14:00 Poster# 151 06/08/09 14:00 Poster# 35 06/10/09 14:00 Poster# 154 06/08/09 14:00 Poster# 39 06/08/09 14:00 Poster# 59 Mootha, V. 06/08/09 14:00 Poster# 60 06/08/09 12:00 Podium# 1 06/08/09 14:00 Poster# 84 06/08/09 14:00 Poster# 88 Morcet, J. 06/08/09 14:00 Poster# 89 06/10/09 14:00 Poster# 126 06/08/09 15:45 Podium# 17 06/11/09 14:00 Poster# 235 06/10/09 14:00 Poster# 115 06/10/09 14:00 Poster# 125 Moreira, L. 06/10/09 15:45 Podium# 47 06/10/09 18:15 Podium# 62 06/11/09 10:15 Podium# 70 06/11/09 14:00 Poster# 207 Morgan, E. 06/11/09 14:00 Poster# 245 06/10/09 14:00 Poster# 151 Muellner, E. Morgan, H. 06/08/09 14:00 Poster# 7 06/10/09 14:00 Poster# 97 Mugford, V. Morgenstern, A. 06/10/09 14:00 Poster# 122 06/10/09 14:00 Poster# 96 Mui, J. Mori, S. 06/10/09 14:00 Poster# 170 06/11/09 14:00 Poster# 273 Muirhead, K. Morini, M. 06/10/09 17:15 Podium# 58 06/08/09 15:45 Podium# 5 06/11/09 14:00 Poster# 202

Morrison, C. [ [email protected] ] Mukhopadhyay, C. 06/08/09 14:00 Poster# 45 06/08/09 14:00 Poster# 27 06/11/09 14:00 Poster# 228 06/10/09 14:00 Poster# 166

Morshchakova, E. Mulder, F. 06/08/09 14:00 Poster# 15 06/09/09 10:00 Podium# 33 06/11/09 14:00 Poster# 234 215

Munoz, P. Neely, E. 06/10/09 14:00 Poster# 156 06/10/09 14:00 Poster# 160

Muntean, V. Neghina, A. [ [email protected] ] 06/10/09 14:00 Poster# 132 06/11/09 14:00 Poster# 213

Murphy, A. Neghina, R. 06/08/09 14:00 Poster# 10 06/11/09 14:00 Poster# 213

Murray, J. Neil, C. 06/11/09 14:00 Poster# 224 06/11/09 14:00 Poster# 278

Musci, G. Nelson, J. [ [email protected] ] 06/10/09 14:00 Poster# 177 06/10/09 14:00 Poster# 122 06/11/09 15:30 Podium# 74 06/11/09 14:00 Poster# 262 06/11/09 14:00 Poster# 272 Nagahima, H. 06/11/09 14:00 Poster# 221 Nemeth, E. 06/11/09 14:00 Poster# 222 06/08/09 14:00 Poster# 64 06/08/09 14:00 Poster# 68 Nai, A. 06/08/09 14:00 Poster# 79 06/10/09 15:45 Podium# 41 06/10/09 14:00 Poster# 120 06/10/09 14:00 Poster# 126 Nairz, M. [ [email protected] ] 06/10/09 14:00 Poster# 128 06/08/09 14:00 Poster# 10 06/10/09 17:15 Podium# 58 06/10/09 15:30 Podium# 46 06/11/09 11:45 Podium# 72 06/11/09 16:30 Podium# 84 Nakamura, M. 06/10/09 10:15 Podium# 38 Neschen, S. 06/10/09 15:45 Podium# 53 Nakanishi, H. 06/11/09 14:00 Poster# 273 Netz, D. 06/08/09 14:00 Poster# 56 Nakao, K. 06/11/09 15:45 Podium# 87 Neubauerova, J. 06/10/09 14:00 Poster# 136 Nandar, W. 06/10/09 14:00 Poster# 160 Neufeld, E. 06/11/09 14:00 Poster# 218 Naranuntarat, A. 06/08/09 12:00 Podium# 1 Neumann, L. [ [email protected] ] 06/08/09 14:00 Poster# 8 Nava, I. 06/11/09 14:00 Poster# 205 Neumayr, L. 06/11/09 14:00 Poster# 206 06/08/09 14:00 Poster# 64

Nazaret, C. Neves, J. [ [email protected] ] 06/10/09 14:00 Poster# 116 06/10/09 15:15 Podium# 45 06/10/09 15:45 Podium# 47 Necas, E. 06/11/09 14:00 Poster# 200 06/08/09 14:00 Poster# 70 06/08/09 14:00 Poster# 90 Newman, B. 06/10/09 14:00 Poster# 118 06/10/09 17:30 Podium# 65

Nechushtai, R. [ [email protected] ] Nguyen, A. 06/10/09 10:00 Podium# 37 06/08/09 14:00 Poster# 45 06/11/09 14:00 Poster# 228

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Nichol, H. Nozoye, T. 06/10/09 14:00 Poster# 168 06/11/09 14:00 Poster# 273 06/10/09 14:00 Poster# 175 Nualart, F. Nick, H. 06/08/09 14:00 Poster# 49 06/11/09 14:00 Poster# 191 06/11/09 14:00 Poster# 197 Nunes, A. 06/08/09 14:00 Poster# 28 Nickerson, L. 06/11/09 14:00 Poster# 224 Nunes-Alves, C. 06/10/09 15:45 Podium# 47 Nicoll, A. 06/11/09 14:00 Poster# 238 Nunez, M. [ [email protected] ] 06/11/09 14:00 Poster# 240 06/08/09 14:00 Poster# 49 06/10/09 14:00 Poster# 156 Nie, G. [ [email protected] ] 06/08/09 15:15 Podium# 3 O’ Neill, M. 06/11/09 15:15 Podium# 85 Nielsen, M. [ [email protected] ] 06/08/09 14:00 Poster# 24 O’Brien, K. 06/10/09 14:00 Poster# 120 Nielsen, P. [ [email protected] ] 06/10/09 15:30 Podium# 52 Ohtake, T. [ [email protected] ] 06/11/09 14:00 Poster# 226 06/08/09 14:00 Poster# 72 06/11/09 14:00 Poster# 233 06/11/09 14:00 Poster# 192 06/11/09 14:00 Poster# 257 Nieman, A. 06/08/09 14:00 Poster# 26 Okamura, N. 06/11/09 14:00 Poster# 257 Niemi, E. 06/10/09 10:15 Podium# 38 Olbina, G. 06/10/09 14:00 Poster# 120 Niitsu, Y. 06/10/09 14:00 Poster# 126 06/11/09 14:00 Poster# 221 06/10/09 16:15 Podium# 43 06/11/09 14:00 Poster# 222 06/11/09 14:00 Poster# 199 (WITHDRAWN)

Nilsson, R. O’Leary, R. 06/08/09 12:00 Podium# 1 06/10/09 14:00 Poster# 121

Ning, B. Oliveira, S. [ [email protected] ] 06/08/09 15:15 Podium# 3 06/10/09 14:00 Poster# 112

Nishizawa, N. [ [email protected] ] Olivieri, O. 06/11/09 14:00 Poster# 273 06/08/09 14:00 Poster# 54 06/08/09 14:00 Poster# 73 Nisselle, A. 06/11/09 14:00 Poster# 238 Olivieri, S. [ [email protected] ] 06/10/09 14:00 Poster# 163 Norgaard, A. 06/10/09 16:00 Podium# 54 Ollivierre-Wilson, H. 06/08/09 12:15 Podium# 2 Norris, S. 06/08/09 16:00 Podium# 18 06/08/09 14:00 Poster# 40 06/08/09 16:15 Podium# 19

Novin, M. Olsson, A. 06/11/09 14:00 Poster# 225 06/11/09 14:00 Poster# 200

217

Olynyk, J. Palha, J. 06/08/09 14:00 Poster# 37 06/11/09 16:15 Podium# 77 06/08/09 14:00 Poster# 83 06/10/09 14:00 Poster# 121 Palis, J. 06/10/09 14:00 Poster# 152 06/08/09 12:00 Podium# 1 06/10/09 14:00 Poster# 158 06/10/09 14:00 Poster# 171 Palmer, G. 06/10/09 14:00 Poster# 176 06/10/09 14:00 Poster# 97 06/11/09 14:00 Poster# 193 06/11/09 14:00 Poster# 238 Palmieri, A. 06/11/09 14:00 Poster# 240 06/10/09 14:00 Poster# 95 06/11/09 14:00 Poster# 252 Pansa, A. Ona-Nguema, G. 06/11/09 14:00 Poster# 189 06/08/09 14:00 Poster# 18 Pantopoulos, K. Opletal, L. 06/10/09 14:00 Poster# 100 06/10/09 14:00 Poster# 186 06/10/09 14:00 Poster# 105 06/10/09 14:00 Poster# 130 Orino, K. 06/10/09 14:00 Poster# 141 06/10/09 14:00 Poster# 139 06/10/09 14:00 Poster# 170 06/10/09 14:00 Poster# 184 Paolini, V. Ørntoft, T. 06/08/09 14:00 Poster# 79 06/08/09 14:00 Poster# 80 06/11/09 14:00 Poster# 242

Orr, A. Parada, B. 06/10/09 17:45 Podium# 60 06/08/09 14:00 Poster# 16

Osborne, N. Paradkar, P. 06/11/09 14:00 Poster# 238 06/08/09 12:00 Podium# 1 06/11/09 14:00 Poster# 240 06/08/09 16:15 Podium# 7 06/11/09 14:00 Poster# 252 Parisi, J. Oshida, J. 06/10/09 14:00 Poster# 168 06/10/09 14:00 Poster# 184 Parmar, A. Ostland, V. 06/08/09 14:00 Poster# 67 06/10/09 14:00 Poster# 120 Parshotam, J. [ [email protected] ] Otten, R. 06/11/09 14:00 Poster# 258 06/09/09 10:00 Podium# 33 Pasino, M. Oudin, C. 06/08/09 14:00 Poster# 68 06/10/09 18:00 Podium# 61 Patient, R. Paddock, M. 06/11/09 14:00 Poster# 201 06/10/09 10:00 Podium# 37 Patton, N. Pagani, A. 06/11/09 14:00 Poster# 210 06/10/09 15:45 Podium# 41 Pautasso, M. Pakbaz, Z. 06/08/09 18:00 Podium# 31 06/08/09 14:00 Poster# 64 Pavlov, A. Paley, C. 06/08/09 14:00 Poster# 15 06/11/09 14:00 Poster# 218

218

Paw, B. [ [email protected] ] Peters, H. 06/08/09 12:00 Podium# 1 06/08/09 14:00 Poster# 61 06/08/09 16:15 Podium# 7 Peterson, R. [ [email protected] ] Pays, E. 06/11/09 15:45 Podium# 81 06/08/09 14:00 Poster# 24 Petrak, J. Pedersen, P. 06/10/09 17:15 Podium# 64 06/11/09 14:00 Poster# 243 06/11/09 14:00 Poster# 246 Peyssonnaux, C. 06/08/09 17:00 Podium# 21 Pedrazzini, T. 06/10/09 16:15 Podium# 55 Philippe, P. 06/11/09 14:00 Poster# 199 (WITHDRAWN) Pelizzoni, I. 06/08/09 15:45 Podium# 5 Phung, Y. 06/11/09 11:45 Podium# 72 Pellegrino, R. 06/10/09 14:00 Poster# 95 Picarote, G. 06/10/09 14:00 Poster# 129 06/10/09 14:00 Poster# 112

Pelucchi, S. [ [email protected] ] Piccio, L. 06/08/09 14:00 Poster# 79 06/10/09 14:00 Poster# 163 06/08/09 14:00 Poster# 91 06/11/09 14:00 Poster# 189 Pickkers, P. 06/08/09 14:00 Poster# 36 Peng, J. 06/11/09 15:15 Podium# 73 Picot, C. 06/08/09 14:00 Poster# 57 Penido, C. 06/10/09 16:00 Podium# 48 Pierce, E. 06/08/09 12:00 Podium# 1 Pennell, D. 06/08/09 16:15 Podium# 7 06/10/09 16:30 Podium# 56 06/11/09 14:00 Poster# 204 Pierik, A. 06/08/09 14:00 Poster# 56 Pepperkok, R. 06/10/09 14:00 Poster# 115 Pietrangelo, A. 06/10/09 14:00 Poster# 125 06/08/09 17:15 Podium# 22 06/10/09 14:00 Poster# 113 Pereira, A. [ [email protected] ] 06/11/09 14:00 Poster# 215 Piga, A. [ [email protected] ] 06/10/09 14:00 Poster# 167 Pereira, L. 06/11/09 14:00 Poster# 218 Piloto, N. 06/08/09 14:00 Poster# 16 Perez Gonzalez, A. 06/11/09 14:00 Poster# 276 06/08/09 14:00 Poster# 39 Pinheiro, P. Perrin, M. 06/11/09 14:00 Poster# 201 06/10/09 14:00 Poster# 126 Pinilla Tenas, J. [ [email protected] ] Persichini, T. 06/11/09 14:00 Poster# 194 06/11/09 15:30 Podium# 74

Peslova, G. 06/10/09 17:15 Podium# 64

219

Pinto, J. Porter, J. 06/09/09 12:00 Podium# 35 06/08/09 14:00 Poster# 42 06/10/09 14:00 Poster# 112 06/08/09 14:00 Poster# 64 06/10/09 14:00 Poster# 164 06/10/09 16:30 Podium# 56

Piperno, A. Porto, G. 06/08/09 14:00 Poster# 79 06/08/09 14:00 Poster# 38 06/08/09 14:00 Poster# 91 06/09/09 12:00 Podium# 35 06/11/09 14:00 Poster# 189 06/10/09 14:00 Poster# 164 06/11/09 14:00 Poster# 242 06/10/09 18:15 Podium# 62 06/11/09 14:00 Poster# 200 Planutyte, L. 06/08/09 14:00 Poster# 53 Pósfai, M. 06/08/09 14:00 Poster# 18 Plewa, C. 06/10/09 16:30 Podium# 44 Pourvali, K. [ [email protected] ] 06/10/09 14:00 Poster# 99 PM Costa, P. 06/10/09 14:00 Poster# 111 06/10/09 18:15 Podium# 62 Pourzand, C. [ [email protected] ] Polati, R. 06/10/09 14:00 Poster# 181 06/08/09 14:00 Poster# 54 06/11/09 14:00 Poster# 260

Poli, A. Powell, L. 06/08/09 14:00 Poster# 73 06/11/09 14:00 Poster# 236 06/11/09 14:00 Poster# 238 Poli, M. [ [email protected] ] 06/11/09 14:00 Poster# 240 06/08/09 14:00 Poster# 55 06/08/09 14:00 Poster# 58 Pozzi, M. 06/08/09 14:00 Poster# 62 06/08/09 14:00 Poster# 91 06/11/09 14:00 Poster# 242 Polticelli, F. 06/10/09 14:00 Poster# 177 Press, R. 06/11/09 15:30 Podium# 74 06/11/09 14:00 Poster# 254

Ponka, P. Preza, G. 06/08/09 14:00 Poster# 3 06/10/09 14:00 Poster# 128 06/08/09 14:00 Poster# 4 06/11/09 16:30 Podium# 84 06/08/09 14:00 Poster# 44 06/08/09 14:00 Poster# 51 Proença, D. 06/08/09 15:15 Podium# 3 06/08/09 14:00 Poster# 93 06/10/09 14:00 Poster# 182 06/08/09 14:00 Poster# 94 06/10/09 17:00 Podium# 57 06/10/09 17:15 Podium# 64 Prohaska, J. 06/11/09 14:00 Poster# 188 06/08/09 14:00 Poster# 11

Ponnuru, P. Provencher, D. 06/11/09 16:00 Podium# 76 06/10/09 14:00 Poster# 142

Popescu, B. [ [email protected] ] Prus, E. 06/10/09 14:00 Poster# 168 06/08/09 14:00 Poster# 47 06/10/09 14:00 Poster# 175 Puccio, H. Poppe, L. 06/10/09 15:15 Podium# 51 06/10/09 15:15 Podium# 39 06/11/09 14:00 Poster# 268

Porrini, V. 06/08/09 14:00 Poster# 58 220

Puy, H. Ranganathan, P. 06/08/09 14:00 Poster# 9 06/11/09 14:00 Poster# 227 06/08/09 14:00 Poster# 33 06/10/09 15:15 Podium# 51 Rangel, M. 06/08/09 14:00 Poster# 28 Quarti-Trevano, F. 06/08/09 14:00 Poster# 32 06/11/09 14:00 Poster# 242 Rapisarda, C. Quesniaux, V. 06/10/09 14:00 Poster# 99 06/08/09 14:00 Poster# 57 Rathkolb, B. Quintanilha, A. 06/08/09 14:00 Poster# 88 06/08/09 14:00 Poster# 14 06/08/09 15:30 Podium# 16 06/10/09 15:45 Podium# 53 Queiroz, C. [ [email protected] ] 06/08/09 14:00 Poster# 32 Rathore, K. [ [email protected] ] 06/10/09 14:00 Poster# 169 Raaka, S. 06/10/09 14:00 Poster# 122 Ravetti, M. 06/10/09 14:00 Poster# 143 Rachmilewitz, E. 06/10/09 17:15 Podium# 58 Raymond, K. 06/11/09 15:15 Podium# 79 06/10/09 16:15 Podium# 49

Radulescu, S. Razgui, D. 06/10/09 14:00 Poster# 144 06/08/09 14:00 Poster# 92

Ragab, A. Rebelo, S. 06/08/09 14:00 Poster# 59 06/10/09 16:00 Podium# 48

Rahmanto, Y. Reboussin, D. 06/10/09 17:15 Podium# 64 06/11/09 14:00 Poster# 224 06/11/09 14:00 Poster# 188 06/11/09 14:00 Poster# 237

Raja, K. Recalcati, S. 06/08/09 14:00 Poster# 71 06/08/09 15:30 Podium# 10 06/10/09 16:00 Podium# 54 Rambeau, M. 06/11/09 14:00 Poster# 220 06/11/09 14:00 Poster# 199 (WITHDRAWN) Redensek, A. Rametta, R. 06/10/09 14:00 Poster# 169 06/08/09 14:00 Poster# 78 06/08/09 17:00 Podium# 27 Reelfs, O. 06/11/09 14:00 Poster# 271 06/11/09 14:00 Poster# 260 06/11/09 14:00 Poster# 275 Reifen, R. Ramm, G. 06/08/09 15:45 Podium# 11 06/11/09 14:00 Poster# 236 Reis, F. [ [email protected] ] Ramos, E. [ [email protected] ] 06/08/09 14:00 Poster# 16 06/11/09 11:45 Podium# 72 06/11/09 14:00 Poster# 276

Ramos, P. Reiss, J. 06/08/09 14:00 Poster# 2 06/11/09 15:30 Podium# 86 06/08/09 14:00 Poster# 87 06/09/09 12:00 Podium# 35 Relvas, L. 06/10/09 17:15 Podium# 58 06/08/09 14:00 Poster# 17

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Ren, B. Robinson, C. 06/08/09 14:00 Poster# 19 06/10/09 14:00 Poster# 175

Renaud, D. Robinson, S. [ [email protected] ] 06/10/09 14:00 Poster# 168 06/10/09 14:00 Poster# 185

Reuben, A. [ [email protected] ] Robson, K. 06/08/09 14:00 Poster# 34 06/08/09 14:00 Poster# 92 06/11/09 14:00 Poster# 201 Reutenauer, L. 06/11/09 16:00 Podium# 88 06/10/09 15:15 Podium# 51 06/11/09 14:00 Poster# 268 Rocha, S. [ [email protected] ] 06/08/09 14:00 Poster# 14 Ribeil, J. 06/08/09 14:00 Poster# 6 (WITHDRAWN) Rocha-Pereira, P. 06/08/09 14:00 Poster# 14 Ribeiro, L. [ [email protected] ] 06/08/09 14:00 Poster# 16 06/08/09 14:00 Poster# 17 06/11/09 14:00 Poster# 276 06/11/09 14:00 Poster# 215 Rochcongar, P. Richardson, D. [ [email protected] ] 06/10/09 14:00 Poster# 126 06/08/09 17:15 Podium# 28 06/10/09 17:15 Podium# 64 Rodansky, E. 06/11/09 14:00 Poster# 188 06/08/09 15:30 Podium# 16 06/11/09 14:00 Poster# 265 06/08/09 16:30 Podium# 20

Richardson, V. Rodrigues, P. 06/11/09 14:00 Poster# 265 06/09/09 12:00 Podium# 35 06/10/09 15:15 Podium# 45 Richman, L. 06/10/09 15:45 Podium# 47 06/10/09 16:15 Podium# 55 06/11/09 14:00 Poster# 200

Riddell, A. Roestenberg, M. 06/08/09 14:00 Poster# 39 06/08/09 14:00 Poster# 26

Rish, K. Roetto, A. [ [email protected] ] 06/11/09 14:00 Poster# 270 06/08/09 18:00 Podium# 31 06/10/09 14:00 Poster# 95 Rivella, S. 06/10/09 14:00 Poster# 129 06/08/09 14:00 Poster# 2 06/08/09 14:00 Poster# 87 Roggero, S. 06/10/09 17:15 Podium# 58 06/10/09 14:00 Poster# 167 06/11/09 14:00 Poster# 202 06/11/09 15:15 Podium# 79 Rogowska-Wrzesinska, A. 06/08/09 14:00 Poster# 22 Rivera, S. 06/10/09 17:30 Podium# 59 Roisman-Sade, D. 06/10/09 10:00 Podium# 37 Riveros, C. 06/10/09 14:00 Poster# 143 Rojas, A. 06/08/09 14:00 Poster# 49 Rizzo, G. 06/11/09 15:30 Podium# 74 Rojas, B. 06/08/09 14:00 Poster# 53 Robach, P. 06/10/09 16:00 Podium# 54 Romão, M. 06/10/09 14:00 Poster# 106

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Romney, J. Ryan, J.C. 06/08/09 16:30 Podium# 20 06/10/09 10:15 Podium# 38 06/10/09 14:00 Poster# 178 06/10/09 17:30 Podium# 65 Ryan, J.D. [ [email protected] ] 06/08/09 14:00 Poster# 35 Ropert, M. 06/08/09 14:00 Poster# 89 06/10/09 14:00 Poster# 126 06/11/09 15:15 Podium# 85

Roschzttardtz, H. Rybicki, A. 06/10/09 14:00 Poster# 138 06/11/09 15:30 Podium# 80

Rossi, F. Sachidanandan, C. 06/08/09 15:45 Podium# 5 06/11/09 15:45 Podium# 81

Rosso, V. Saglio, G. 06/08/09 18:00 Podium# 31 06/08/09 18:00 Podium# 31 06/10/09 14:00 Poster# 95 Roth, M. 06/10/09 14:00 Poster# 129 06/08/09 17:30 Podium# 23 06/09/09 12:15 Podium# 36 Saint-Jalmes, H. 06/11/09 14:00 Poster# 190 06/11/09 14:00 Poster# 209 06/11/09 14:00 Poster# 196 Saito, K. Rouault, T. [[email protected] ] 06/11/09 14:00 Poster# 192 06/08/09 12:15 Podium# 2 06/08/09 16:00 Podium# 18 Salahudeen, A. 06/08/09 16:15 Podium# 19 06/10/09 14:00 Poster# 123 06/10/09 14:00 Poster# 131 (WITHDRAWN) Salama, M. [ [email protected] ] Rovelli, E. 06/10/09 14:00 Poster# 104 06/08/09 16:30 Podium# 8 06/10/09 14:00 Poster# 174 Salvioni, A. 06/08/09 14:00 Poster# 79 Roy, C. 06/11/09 14:00 Poster# 189 06/10/09 17:15 Podium# 58 Samuels, M. Rozier, M. 06/10/09 17:45 Podium# 60 06/08/09 18:00 Podium# 25 Sanchez, L. Ruchala, P. 06/10/09 17:45 Podium# 66 06/11/09 16:30 Podium# 84 Sanchez, M. Ruivard, M. 06/08/09 15:45 Podium# 17 06/11/09 14:00 Poster# 199 (WITHDRAWN) 06/11/09 14:00 Poster# 207

Runz, H. Sandoval, D. 06/10/09 14:00 Poster# 115 06/08/09 14:00 Poster# 49 06/10/09 14:00 Poster# 125 Sansom, O. Russell, J. 06/10/09 14:00 Poster# 144 06/08/09 14:00 Poster# 89 Santambrogio, P. Ryan, E. 06/08/09 15:30 Podium# 10 06/08/09 14:00 Poster# 35 06/08/09 16:30 Podium# 8 06/08/09 14:00 Poster# 89 06/10/09 14:00 Poster# 181 06/11/09 15:15 Podium# 85 06/10/09 16:00 Podium# 54

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Santos Silva, A. Scheiber-Mojdehkar, B. 06/08/09 14:00 Poster# 16 06/08/09 14:00 Poster# 8 06/11/09 14:00 Poster# 276 06/08/09 14:00 Poster# 13 06/11/09 14:00 Poster# 264 Santos, M. 06/11/09 14:00 Poster# 266 06/08/09 14:00 Poster# 34 06/08/09 14:00 Poster# 76 Schengrund, C. 06/08/09 16:00 Podium# 12 06/08/09 14:00 Poster# 85 06/10/09 14:00 Poster# 142 06/10/09 14:00 Poster# 169 Schifrer, M. [ [email protected] ] 06/08/09 14:00 Poster# 3 Santos-Silva, A. 06/08/09 14:00 Poster# 7 06/08/09 14:00 Poster# 14 Schimanski, L. [ [email protected] ] Sari, M. [ [email protected] ] 06/08/09 14:00 Poster# 92 06/08/09 14:00 Poster# 69 06/10/09 16:30 Podium# 50

Saric, M. [ [email protected] ] Schipper, H. 06/10/09 14:00 Poster# 102 06/10/09 14:00 Poster# 170

Sasaki, K. Schmidt, H. 06/08/09 14:00 Poster# 72 06/08/09 14:00 Poster# 80 06/11/09 14:00 Poster# 192 06/11/09 14:00 Poster# 257 Schmidt, P. [ [email protected] ] 06/08/09 18:15 Podium# 26 Sasaki, Y. 06/10/09 17:45 Podium# 60 06/11/09 14:00 Poster# 192 Schmucker, S. [ [email protected] ] Sasu, B. 06/10/09 15:15 Podium# 51 06/10/09 15:15 Podium# 39 06/11/09 14:00 Poster# 268 06/10/09 16:30 Podium# 44 Schnier, P. Sato, K. 06/10/09 15:15 Podium# 39 06/11/09 14:00 Poster# 257 Schranzhofer, M. Sato, T. 06/08/09 14:00 Poster# 3 06/11/09 14:00 Poster# 221 06/08/09 14:00 Poster# 4 06/11/09 14:00 Poster# 222 06/08/09 14:00 Poster# 7 06/10/09 17:00 Podium# 57 Sato, Y. 06/11/09 14:00 Poster# 221 Schroll, A. 06/11/09 14:00 Poster# 222 06/08/09 14:00 Poster# 10 06/10/09 15:30 Podium# 46 Sauble, E. 06/08/09 14:00 Poster# 45 Schueler, D. 06/11/09 14:00 Poster# 228 06/08/09 14:00 Poster# 18

Sauerwein, R. Schuemann, K. [ [email protected] ] 06/08/09 14:00 Poster# 26 06/08/09 16:30 Podium# 14 06/10/09 14:00 Poster# 124 Savion, O. 06/08/09 15:45 Podium# 11 Schultz, I. [ [email protected] ] 06/08/09 12:00 Podium# 1 Sawada, K. 06/08/09 16:15 Podium# 7 06/11/09 14:00 Poster# 72 Schulz, K. [ [email protected] ] 06/10/09 14:00 Poster# 148

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Schwanhaeusser, B. Shawki, A. 06/08/09 15:45 Podium# 17 06/08/09 15:30 Podium# 4 06/11/09 14:00 Poster# 194 Schwartz, D. 06/11/09 14:00 Poster# 267 06/11/09 14:00 Poster# 216 Sheehan, J. Scoppettuolo, M. 06/08/09 14:00 Poster# 86 06/10/09 14:00 Poster# 103 06/11/09 14:00 Poster# 259

Scott, R. Sheftel, A. [ [email protected] ] 06/10/09 14:00 Poster# 143 06/08/09 14:00 Poster# 44 06/10/09 14:00 Poster# 158 06/08/09 14:00 Poster# 51 06/10/09 14:00 Poster# 171 06/08/09 14:00 Poster# 56 06/11/09 14:00 Poster# 193 Shelnutt, J. Seaman, W. [ [email protected] ] 06/11/09 14:00 Poster# 274 06/10/09 10:15 Podium# 38 Sheng, J. Sefc, L. 06/10/09 16:30 Podium# 44 06/08/09 14:00 Poster# 70 06/10/09 14:00 Poster# 118 Shi, Z. 06/10/09 14:00 Poster# 172 Seguin, A. [ [email protected] ] 06/08/09 14:00 Poster# 22 Shimonaka, Y. 06/11/09 14:00 Poster# 192 Seguin, C. 06/08/09 14:00 Poster# 57 Shindo, M. 06/11/09 14:00 Poster# 257 Seifert, M. 06/08/09 14:00 Poster# 10 Shirihai, O. 06/08/09 14:00 Poster# 44 Seignard, H. 06/08/09 16:15 Podium# 7 06/11/09 14:00 Poster# 235 Sholinsky, P. Seixas, E. 06/11/09 14:00 Poster# 237 06/10/09 16:00 Podium# 48 Shvartsman, M. [ [email protected] ] Selbach, M. 06/08/09 14:00 Poster# 47 06/08/09 15:45 Podium# 17 06/08/09 14:00 Poster# 52 (Moved to June 10, 17:40) 06/10/09 10:00 Podium# 37 Seligmann, H. 06/10/09 15:30 Podium# 52 Silengo, L. 06/08/09 14:00 Poster# 1 Semecky, V. 06/10/09 14:00 Poster# 187 Silva, L. [ [email protected] ] 06/11/09 14:00 Poster# 214 Seravalle, G. 06/11/09 14:00 Poster# 217 06/11/09 14:00 Poster# 242 Silva, Adavio Sereno, J. 06/11/09 14:00 Poster# 214 06/08/09 14:00 Poster# 16 06/11/09 14:00 Poster# 217 06/11/09 14:00 Poster# 276 Silva, Ana M. G. Sharp, P. 06/08/09 14:00 Poster# 32 06/10/09 14:00 Poster# 99 06/10/09 14:00 Poster# 111 06/11/09 14:00 Poster# 265

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Silva, André [ [email protected] ] Sloan, J. 06/10/09 18:15 Podium# 68 06/10/09 16:30 Podium# 44 06/11/09 14:00 Poster# 214 06/11/09 14:00 Poster# 217 Sly, W. 06/11/09 14:00 Poster# 258 06/08/09 16:15 Podium# 13 06/08/09 18:00 Podium# 25 Silva, B. [ [email protected] ] 06/08/09 14:00 Poster# 93 Smith, Amber 06/08/09 14:00 Poster# 94 06/11/09 14:00 Poster# 218

Silva, G. Smith, Ann [ [email protected] ] 06/10/09 16:00 Podium# 48 06/11/09 14:00 Poster# 270

Silva, R. Smith, G. 06/08/09 14:00 Poster# 29 06/10/09 16:30 Podium# 56

Silveira, M. Smith, N. 06/08/09 14:00 Poster# 17 06/10/09 16:00 Podium# 48 06/11/09 14:00 Poster# 215 Snively, B. Silvestri, L. [ [email protected] ] 06/11/09 14:00 Poster# 224 06/10/09 15:45 Podium# 41 06/11/09 14:00 Poster# 237 06/10/09 18:00 Podium# 61 06/11/09 15:30 Podium# 86

Simmons, Z. Soares, M. 06/10/09 14:00 Poster# 162 06/10/09 16:00 Podium# 48 06/10/09 14:00 Poster# 165 Söderberg, C. Simon, C. 06/10/09 14:00 Poster# 180 06/08/09 17:00 Podium# 21 Soe-Lin, S. Simpson, I. 06/08/09 15:15 Podium# 3 06/11/09 16:00 Podium# 76 06/10/09 17:00 Podium# 57 06/10/09 17:15 Podium# 64 Simpson, R. 06/08/09 14:00 Poster# 65 Sohn, Y. 06/08/09 14:00 Poster# 71 06/08/09 14:00 Poster# 52 (Moved to June 10, 17:40) 06/08/09 14:00 Poster# 75 06/08/09 14:00 Poster# 77 Soltis, K. 06/10/09 14:00 Poster# 98 06/08/09 12:00 Podium# 1 06/10/09 14:00 Poster# 102 06/11/09 14:00 Poster# 261 Song, W. 06/10/09 14:00 Poster# 170 Simunek, T. [ [email protected] ] 06/10/09 14:00 Poster# 179 Song, Ying 06/10/09 14:00 Poster# 182 06/11/09 10:00 Podium# 69

Sjöström, M. Song, Yufeng 06/10/09 14:00 Poster# 180 06/10/09 14:00 Poster# 140

Slagle-Webb, B. Sonnweber, T. 06/08/09 14:00 Poster# 86 06/08/09 14:00 Poster# 10 06/10/09 14:00 Poster# 149 06/10/09 15:30 Podium# 46 06/11/09 14:00 Poster# 259 Sougrat, R. Slavinskaya, Z. 06/08/09 16:00 Podium# 18 06/09/09 10:00 Podium# 33

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Sousa Fernandes, S. Stonehuerner, J. 06/08/09 14:00 Poster# 28 06/10/09 14:00 Poster# 109

Sousa, J. Stremmel, W. [ [email protected] ] 06/11/09 16:15 Podium# 77 06/10/09 14:00 Poster# 132 06/11/09 14:00 Poster# 207 Sousa, N. 06/11/09 16:15 Podium# 77 Strickland, N. [ [email protected] ] 06/10/09 14:00 Poster# 145 Southey, M. 06/11/09 14:00 Poster# 255 06/11/09 14:00 Poster# 240 Strong, R. Spagnuolo, R. 06/10/09 16:15 Podium# 49 06/11/09 14:00 Poster# 220 Stucky, S. [ [email protected] ] Sparkman, B. 06/11/09 14:00 Poster# 191 06/11/09 14:00 Poster# 194 06/11/09 14:00 Poster# 197

Sparla, R. Sturm, B. 06/08/09 14:00 Poster# 88 06/08/09 14:00 Poster# 8 06/08/09 14:00 Poster# 13 Speechley, M. 06/11/09 14:00 Poster# 264 06/11/09 14:00 Poster# 237 06/11/09 14:00 Poster# 266

Srai, S. Stys, A. 06/08/09 14:00 Poster# 65 06/10/09 14:00 Poster# 183 06/10/09 14:00 Poster# 99 06/10/09 14:00 Poster# 104 Subramaniam, N. [ [email protected] ] 06/10/09 14:00 Poster# 134 06/08/09 14:00 Poster# 82 06/11/09 14:00 Poster# 236 St. Pierre, T. [ [email protected] ] 06/11/09 14:00 Poster# 253 06/10/09 14:00 Poster# 152 06/11/09 14:00 Poster# 204 Summerville, L. 06/11/09 14:00 Poster# 210 06/08/09 14:00 Poster# 82 06/11/09 14:00 Poster# 253 Stål, P. 06/10/09 14:00 Poster# 180 Sutak, R. [ [email protected] ] 06/10/09 17:15 Podium# 64 Stalenhoef, A. 06/11/09 14:00 Poster# 188 06/11/09 14:00 Poster# 239 06/11/09 14:00 Poster# 277

Starzynski, R. Sutcharitchan, P. 06/10/09 14:00 Poster# 183 06/10/09 16:30 Podium# 56

Stehling, O. Suzuka, S. 06/08/09 14:00 Poster# 56 06/11/09 15:30 Podium# 80

Steinkellner, H. [ [email protected] ] Suzuki, Y. 06/08/09 14:00 Poster# 8 06/11/09 14:00 Poster# 72 06/11/09 14:00 Poster# 264 06/11/09 14:00 Poster# 266 Sweep, F. 06/08/09 14:00 Poster# 74 Stolte, J. 06/08/09 14:00 Poster# 35 Sweetland, E. 06/08/09 14:00 Poster# 88 06/08/09 14:00 Poster# 92 06/08/09 14:00 Poster# 89 06/11/09 10:15 Podium# 70

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Swinkels, D. [ [email protected] ] Tavares, C. 06/08/09 14:00 Poster# 26 06/08/09 14:00 Poster# 16 06/08/09 14:00 Poster# 36 06/11/09 14:00 Poster# 276 06/08/09 14:00 Poster# 61 06/08/09 14:00 Poster# 74 Taylor, J. 06/08/09 14:00 Poster# 81 06/11/09 14:00 Poster# 210 06/10/09 16:15 Podium# 43 06/11/09 14:00 Poster# 239 Tchernitchko, D. 06/11/09 14:00 Poster# 275 06/10/09 14:00 Poster# 116

Syed, R. Teixeira de Lemos, E. 06/10/09 15:15 Podium# 39 06/08/09 14:00 Poster# 16 06/11/09 14:00 Poster# 276 Tabuchi, M. 06/08/09 14:00 Poster# 48 Teixeira, F. 06/11/09 14:00 Poster# 269 06/08/09 14:00 Poster# 16 06/11/09 14:00 Poster# 276 Tacchini, L. 06/11/09 14:00 Poster# 220 Teixeira, H. 06/11/09 14:00 Poster# 276 Taher, A. 06/10/09 16:30 Podium# 56 Ternes, N. 06/08/09 14:00 Poster# 13 Takada, K. 06/11/09 14:00 Poster# 221 Tessitore, N. 06/11/09 14:00 Poster# 222 06/08/09 14:00 Poster# 73

Takahashi, M. Teucke, F. 06/11/09 14:00 Poster# 273 06/11/09 14:00 Poster# 203

Takaya, N. Thein, S. 06/10/09 14:00 Poster# 159 06/08/09 14:00 Poster# 75

Takimoto, R. Theurl, I. 06/11/09 14:00 Poster# 221 06/08/09 14:00 Poster# 10 06/11/09 14:00 Poster# 222 06/10/09 15:30 Podium# 46

Tamouza, H. Theurl, M. 06/08/09 14:00 Poster# 6 (WITHDRAWN) 06/08/09 14:00 Poster# 10 06/10/09 15:30 Podium# 46 Tanaka, H. 06/11/09 14:00 Poster# 263 Thibaudeau, O. 06/08/09 15:15 Podium# 9 Tapia, V. 06/08/09 14:00 Poster# 49 Thiele, D. 06/09/09 10:15 Podium# 34 Tapp, H. 06/11/09 14:00 Poster# 210 Thompson, J. 06/10/09 14:00 Poster# 123 Tapryal, N. [ [email protected] ] 06/10/09 14:00 Poster# 166 Thomsen, J. 06/10/09 16:00 Podium# 54 Tatsumi, Y. 06/11/09 14:00 Poster# 241 (WITHDRAWN) Thomson, E. 06/11/09 15:45 Podium# 87 06/11/09 14:00 Poster# 237

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Thon, V. Townsend, A. 06/08/09 14:00 Poster# 53 06/08/09 14:00 Poster# 25 06/08/09 14:00 Poster# 92 Thorstensen, K. 06/10/09 16:30 Podium# 50 06/11/09 14:00 Poster# 213 06/11/09 14:00 Poster# 244 Trégouët, Y. 06/11/09 14:00 Poster# 235 Timperio, A. 06/08/09 14:00 Poster# 54 Trinder, D. 06/08/09 14:00 Poster# 37 Tjalsma, H. 06/08/09 14:00 Poster# 83 06/08/09 14:00 Poster# 74 06/10/09 14:00 Poster# 121 06/11/09 14:00 Poster# 275 06/10/09 14:00 Poster# 158 06/10/09 14:00 Poster# 171 Todorich, B. 06/11/09 14:00 Poster# 193 06/10/09 14:00 Poster# 149 Troadec, M. [ [email protected] ] Tolosano, E. [ [email protected] ] 06/08/09 14:00 Poster# 46 06/08/09 14:00 Poster# 1 06/10/09 14:00 Poster# 126

Tomás, A. Trojanowski, J. 06/08/09 14:00 Poster# 29 06/11/09 15:15 Podium# 73 06/08/09 14:00 Poster# 31 Trombini, P. Tomosugi, N. 06/08/09 14:00 Poster# 79 06/10/09 16:15 Podium# 43 06/11/09 14:00 Poster# 189 06/11/09 15:45 Podium# 87 06/11/09 14:00 Poster# 242

Tong, W. [ [email protected] ] Tromp, M. 06/10/09 14:00 Poster# 131 (WITHDRAWN) 06/08/09 14:00 Poster# 36

Ton-nu, L. Trudel, M. 06/08/09 14:00 Poster# 53 06/11/09 16:15 Podium# 83

Torimoto, Y. Tselepis, C. [ [email protected] ] 06/08/09 14:00 Poster# 72 06/08/09 14:00 Poster# 66 06/11/09 14:00 Poster# 192 06/08/09 17:45 Podium# 30 06/11/09 14:00 Poster# 257 06/10/09 14:00 Poster# 144 06/10/09 16:15 Podium# 43 Torti, F. 06/11/09 14:00 Poster# 208 06/08/09 17:30 Podium# 29 06/10/09 10:15 Podium# 38 Tsuchida, K. 06/10/09 14:00 Poster# 140 06/11/09 15:45 Podium# 87

Torti, S. [ [email protected] ] Tsukamoto, H. 06/08/09 17:30 Podium# 29 06/08/09 17:45 Podium# 24 06/10/09 10:15 Podium# 38 06/10/09 16:00 Podium# 42 06/10/09 14:00 Poster# 140 Tuma, S. Totaro, F. 06/10/09 14:00 Poster# 132 06/10/09 14:00 Poster# 103 Turco, E. Tournilhac, V. 06/08/09 14:00 Poster# 1 06/11/09 14:00 Poster# 199 (WITHDRAWN) Turlin, B. Toutain, F. 06/11/09 14:00 Poster# 209 06/10/09 18:00 Podium# 61

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Tyrrell, R. Vanoaica, L. 06/10/09 14:00 Poster# 181 06/10/09 14:00 Poster# 124 06/10/09 16:15 Podium# 55 Uchiyama, T. 06/11/09 15:45 Podium# 87 Vantini, I. 06/08/09 14:00 Poster# 68 Ulhøi, B. 06/08/09 14:00 Poster# 80 Vasavda, N. 06/08/09 14:00 Poster# 75 Unalp-Arida, A. 06/11/09 14:00 Poster# 272 Vaucamps, N. 06/11/09 14:00 Poster# 268 Unger, E. [ [email protected] ] 06/10/09 14:00 Poster# 157 Vaulont, S. 06/10/09 14:00 Poster# 161 06/08/09 14:00 Poster# 69 06/08/09 17:00 Podium# 21 Utzschneider, K. 06/11/09 10:00 Podium# 69 06/11/09 14:00 Poster# 262 Vavrova, A. Vainshtein, Y. 06/10/09 14:00 Poster# 182 06/08/09 15:45 Podium# 17 Vavrova, K. Vaja, V. 06/10/09 14:00 Poster# 179 06/11/09 14:00 Poster# 205 06/10/09 14:00 Poster# 186

Valenti, G. Verbeek, A. 06/08/09 14:00 Poster# 78 06/11/09 14:00 Poster# 239

Valenti, L. [ [email protected] ] Vergara, S. [ [email protected] ] 06/08/09 14:00 Poster# 78 06/09/09 10:15 Podium# 34 06/08/09 17:00 Podium# 27 06/11/09 14:00 Poster# 271 Vervalle, J. [ [email protected] ] 06/11/09 14:00 Poster# 275 06/10/09 14:00 Poster# 146 06/11/09 14:00 Poster# 255 Vali, H. 06/10/09 14:00 Poster# 170 Viatte, L. 06/08/09 16:30 Podium# 14 Van der Ven, A. [ [email protected] ] 06/10/09 14:00 Poster# 114 06/08/09 14:00 Poster# 26 06/10/09 14:00 Poster# 135 van Deursen, C. Vichinsky, E. 06/11/09 14:00 Poster# 239 06/08/09 14:00 Poster# 42 06/08/09 14:00 Poster# 64 van Eijk, L. [ [email protected] ] 06/08/09 14:00 Poster# 36 Viganò, A. 06/10/09 16:00 Podium# 54 van Helden, D. 06/11/09 14:00 Poster# 193 Villa, R. 06/11/09 14:00 Poster# 211 van Noord, P. 06/11/09 14:00 Poster# 212 06/11/09 14:00 Poster# 231 Vinokur, V. Vandekerckhove, J. 06/10/09 14:00 Poster# 119 06/08/09 14:00 Poster# 6 (WITHDRAWN) Viprakasit, V. Vanhollebeke, B. 06/10/09 16:30 Podium# 56 06/08/09 14:00 Poster# 24

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Vitali, I. Wallander, M. 06/08/09 15:45 Podium# 5 06/08/09 16:30 Podium# 20 06/10/09 14:00 Poster# 178 Vivian, A. 06/11/09 14:00 Poster# 216 Walter, P. [ [email protected] ] 06/08/09 14:00 Poster# 42 Vogel, H. [ [email protected] ] 06/08/09 14:00 Poster# 64 06/09/09 10:00 Podium# 33 Wang, Jiaohong Vogt, T. 06/10/09 16:00 Podium# 42 06/11/09 14:00 Poster# 237 Wang, Jinzhi Vokurka, M. 06/08/09 16:00 Podium# 6 06/08/09 14:00 Poster# 70 06/10/09 14:00 Poster# 118 Wang, S. 06/10/09 14:00 Poster# 153 Volkmann, M. 06/10/09 14:00 Poster# 132 Wang, W. 06/08/09 17:30 Podium# 29 von Bonsdorff, L. 06/11/09 15:30 Podium# 80 Ward, Diane 06/08/09 14:00 Poster# 46 Vujic Spasic, M. [ [email protected] ] 06/10/09 15:30 Podium# 40 06/08/09 14:00 Poster# 39 06/08/09 14:00 Poster# 84 Ward, Douglas 06/08/09 14:00 Poster# 88 06/08/09 14:00 Poster# 66 06/11/09 10:15 Podium# 70 06/10/09 16:15 Podium# 43 06/11/09 14:00 Poster# 208 Vukicevic, S. 06/10/09 14:00 Poster# 113 Ward, R. 06/11/09 16:30 Podium# 78 Vulpe, C. 06/10/09 14:00 Poster# 148 Waring, A. 06/11/09 14:00 Poster# 224 06/10/09 14:00 Poster# 128 06/11/09 14:00 Poster# 230 06/11/09 14:00 Poster# 252 Warnich, L. 06/11/09 15:30 Podium# 86 06/08/09 18:15 Podium# 32 06/10/09 14:00 Poster# 146 Vyoral, D. 06/10/09 14:00 Poster# 147 06/10/09 17:15 Podium# 64 Watanabe, H. Wagner, S. 06/10/09 14:00 Poster# 159 06/10/09 14:00 Poster# 124 Watanabe, K. Waheed, A. 06/10/09 14:00 Poster# 139 06/08/09 16:15 Podium# 13 06/10/09 14:00 Poster# 184 06/08/09 18:00 Podium# 25 06/10/09 14:00 Poster# 97 Watt, E. 06/11/09 14:00 Poster# 236 Wakusawa, S. 06/11/09 14:00 Poster# 241 (WITHDRAWN) Watt, R. [ [email protected] ] 06/08/09 14:00 Poster# 12 Wallace, D. [ [email protected] ] 06/08/09 14:00 Poster# 82 Wattenhofer-Donzé, M. 06/11/09 14:00 Poster# 253 06/10/09 15:15 Podium# 51 06/11/09 14:00 Poster# 268

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Weatherall, D. Witcher, D. 06/11/09 16:00 Podium# 88 06/08/09 14:00 Poster# 10

Webb, B. Wittig, I. 06/11/09 16:00 Podium# 76 06/08/09 14:00 Poster# 56

Weber, E. Wollert, K. 06/11/09 14:00 Poster# 200 06/10/09 16:00 Podium# 54

Weinheimer, H. Wroblewski, V. 06/10/09 14:00 Poster# 124 06/08/09 14:00 Poster# 10

Weiss, G. [ [email protected] ] Wu, T. 06/08/09 14:00 Poster# 10 06/10/09 14:00 Poster# 170 06/10/09 15:30 Podium# 46 Wu, W. Weiss, B. 06/08/09 14:00 Poster# 20 06/08/09 15:45 Podium# 11 Wurz, E. Westerman, M. [ [email protected] ] 06/08/09 14:00 Poster# 10 06/08/09 14:00 Poster# 33 06/10/09 14:00 Poster# 120 Xia, Y. 06/10/09 14:00 Poster# 126 06/08/09 17:15 Podium# 22 06/11/09 14:00 Poster# 199 (WITHDRAWN) 06/10/09 14:00 Poster# 113

Wetzels, J. Xu, X. 06/08/09 14:00 Poster# 61 06/11/09 14:00 Poster# 188

Whitnall, M. Yamada, G. 06/11/09 14:00 Poster# 188 06/11/09 15:45 Podium# 87

Whittington, C. Yanatori, I. 06/11/09 14:00 Poster# 216 06/08/09 14:00 Poster# 48 06/11/09 14:00 Poster# 255 06/11/09 14:00 Poster# 269

Wie, S. Yang, F. 06/10/09 14:00 Poster# 120 06/10/09 16:00 Podium# 42

Wiegerinck, E. Yang, J. 06/08/09 14:00 Poster# 81 06/08/09 14:00 Poster# 19

Wilkins, S. Yang, Y. 06/10/09 14:00 Poster# 117 06/10/09 14:00 Poster# 131 (WITHDRAWN)

Willemetz, A. Yang, Z. 06/10/09 14:00 Poster# 101 06/10/09 14:00 Poster# 153 06/10/09 17:00 Podium# 63 Yano, M. Wilson, J. 06/11/09 14:00 Poster# 241 (WITHDRAWN) 06/10/09 15:15 Podium# 45 Yasuno, H. Wilson, L. 06/11/09 14:00 Poster# 192 06/11/09 14:00 Poster# 272 Yau, Y. Winters, A. 06/11/09 14:00 Poster# 232 06/10/09 15:15 Podium# 39 06/10/09 16:30 Podium# 44

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Yeap, H. Zhang, A. 06/11/09 14:00 Poster# 252 06/08/09 14:00 Poster# 44 06/08/09 17:45 Podium# 24 Yeh, K. 06/10/09 16:00 Podium# 42 06/10/09 14:00 Poster# 133 06/11/09 14:00 Poster# 263 Zhang, D. [ [email protected] ] 06/08/09 12:15 Podium# 2 Yeh, Mary 06/10/09 14:00 Poster# 133 Zhang, N. 06/11/09 14:00 Poster# 263 06/10/09 14:00 Poster# 172

Yeh, Matthew Zhang, X. 06/11/09 14:00 Poster# 272 06/10/09 14:00 Poster# 149

Yiakouvaki, A. Zhang, Y. 06/10/09 14:00 Poster# 181 06/08/09 15:15 Podium# 3

Yin, H. Zhao, G. [ [email protected] ] 06/11/09 14:00 Poster# 263 06/10/09 14:00 Poster# 110

Yu, P. Zhao, L. 06/11/09 15:45 Podium# 81 06/10/09 14:00 Poster# 109

Zaahl, M. Zhao, N. 06/08/09 18:15 Podium# 32 06/08/09 14:00 Poster# 45 06/10/09 14:00 Poster# 145 06/08/09 14:00 Poster# 50 06/10/09 14:00 Poster# 146 06/10/09 14:00 Poster# 147 Zhao, Y. 06/11/09 14:00 Poster# 198 06/08/09 15:15 Podium# 3 06/11/09 14:00 Poster# 255 Zhu, M. Zacchetti, D. 06/10/09 14:00 Poster# 115 06/08/09 15:45 Podium# 5 Zimmer, M. Zampronio, C. 06/11/09 14:00 Poster# 278 06/08/09 14:00 Poster# 66 Zolla, L. Zanella, I. 06/08/09 14:00 Poster# 54 06/08/09 14:00 Poster# 58 Zoller, H. Zaninotto, F. 06/09/09 12:00 Podium# 35 06/08/09 14:00 Poster# 54 06/08/09 14:00 Poster# 68 Zukor, H. [ [email protected] ] 06/08/09 14:00 Poster# 73 06/10/09 14:00 Poster# 170

Zanone, C. Zumbrennen, K. [ [email protected] ] 06/08/09 18:00 Podium# 31 06/08/09 15:30 Podium# 16 06/08/09 16:30 Podium# 20 Zatloukalova, L. 06/10/09 14:00 Poster# 178 06/10/09 14:00 Poster# 186 06/10/09 15:45 Podium# 53 06/10/09 14:00 Poster# 187

Zermati, Y. 06/08/09 14:00 Poster# 6 (WITHDRAWN)

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