Seventh Congress of the International BioIron Society (IBIS) Biennial World Meeting (BioIron 2017) May 7 – 11, 2017 UCLA Meyer & Renee Luskin Conference Center | Los Angeles, USA
Featuring Special Events: Introductory Course – May 7, 2017 “Essentials of BioIron for Clinicians and Scientists”
“Meet the Expert” Sessions for Trainees May 10 – 11, 2017
INTERNATIONAL BIOIRON SOCIETY
PROGRAM BOOK Acknowledgments IBIS
This conference has received support from the NIH NCATS UCLA CTSI Grant Number UL1TR0001881.
This conference has received support from the NIH/NIDDK R-13 Grant.
This conference has received support from the NIXHNHLBI-R-13 Grant.
Seventh Congress of the International BioIron Society Page 2 Table of Contents IBIS
Seventh Congress of the Internationl BioIron Society (IBIS) Biennial World Meeting (BioIron 2017) May 7 - 11, 2017 UCLA Meyer & Renee Luskin Conference Center | Los Angeles, USA
Welcome Message ...... Page 4
Board of Directors ...... Page 5
General Meeting Information ...... Page 6
Special Events ...... Page 8
Local Activities ...... Page 9
Acknowledgments ...... Page 10
Program Schedule ...... Page 11
Keynote Speaker ...... Page 51
Abstracts ...... Page 52
Featured Podium/Poster Abstracts ...... Page 52
Podium Abstracts ...... Page 61
Poster Abstracts ...... Page 116
Late-Breaking Abstracts ...... Page 325
Alphabetical Index of Presenters ...... Page 378
Meeting Title Page 3 Welcome IBIS
Dear Friends and Colleagues,
Welcome to the Seventh Congress of the International BioIron Society (IBIS) being held at the University of California, Los Angeles. In keeping with the character of the organization, the BioIron 2017 Meeting is held is a different continent every biennial cycle, returning to North America for the first time since Vancouver in 2011.
This continues to be an exciting time in the field of iron research. Molecular discoveries over the past two decades have led to a rapid expansion in the number of potential biomarkers and targets for treatment for iron-related diseases. Indeed many of these discoveries hold promise to make major contributions to the well-being of a large number of people suffering from iron-related disorders across the globe. The program planned by the IBIS Board and Local Organizing Committee reflects this broadening character of the most compelling questions in iron research. This year, we are including two celebratory sessions: The 30-year anniversary of the discovery of the iron responsive elements, which laid the foundation for understanding cellular iron regulation, and the 50-year anniversary of therapeutic iron chelation which improved and prolonged the lives of so many patients with iron overload. These sessions feature outstanding presentations highlighting not only why these events were so seminal, but where they provide opportunities for future application.
We especially welcome participants new to iron research. In keeping with past several meetings, the first day will be an introductory course to provide a helpful framework for critical understanding of the research to be presented during the main meeting. The course is intended for an audience of diverse backgrounds, and provides ample time for questions. We strongly encourage trainees in the audience to contribute to this discussion. In addition to scheduled opportunities for trainees to meet-the-experts, we have added two special “Interviews with the Masters” to share the wisdom provided by long and highly successful careers in iron research.
The name of our organization serves as an acknowledgement that science is a social as well as investigative process. We hope you will take time to enjoy the wonderful setting the Southern California provides. We will have a communal outing to the beautiful and unique Getty Museum on Tuesday afternoon, and a fun, entertaining Gala and Awards Dinner to close the meeting.
We wish to express our gratitude to all our sponsors for their generous contributions to this event. It is impossible to hold a meeting of this scope and caliber without the contributions from the biotechnology and pharmaceutical companies that ultimately translate scientific discovery into practical applications. We equally appreciate the support from National Institutes of Health in providing travel bursaries for trainees to attend this meeting. We would also like to express our thanks for the highly professional organizational work performed by Heather Swanson and her staff at W.J. Weiser.
Most of all, we are grateful to each of you for contributing your time and effort to participate in this global sharing of ideas and discoveries. Thank you again for being part of BioIron 2017.
Robert E Fleming, MD, IBIS President
Elizabeta Nemeth, PhD and Tomas Ganz, MD, PhD, Co-Chairs of the Local Organizing Committee
Ioav Cabantchik PhD, IBIS President-Elect
Seventh Congress of the International BioIron Society Page 4 Board of Directors IBIS
OFFICERS President Stefano Rivella, PhD Robert E. Fleming, MD Philadelphia, PA, USA St. Louis, MO, USA Laura Silvestri, PhD President-Elect Milan, Italy Ioav Cabantchik, MD, PhD Jerusalem, Israel Program Committee Robert E. Fleming, MD Secretary St. Louis, MO, USA Helene Coppin, PhD Toulouse Cedex, France Tomas Ganz, MD, PhD Los Angeles, CA, USA Treasurer Domenico Girelli, MD, PhD Elizabeta Nemeth, PhD Verona, Italy Los Angeles, CA, USA
Past President Local Organizing Committee Greg Anderson, PhD Tomas Ganz, MD, PhD Brisbane, Australia Los Angeles, CA, USA
Board Of Directors Elizabeta Nemeth, PhD Director Los Angeles, CA, USA Robert W. Evans, BA, PhD Uxbridge Middlesex, United Kingdom Headquarter Office WJ Weiser & Associates, Inc. Yelena Ginzburg, MD 1100 E. Woodfield Road, Suite 350 New York, NY, USA Schaumburg, IL 60173
Mitchell Knutson, PhD Executive Director Gainesville, FL, USA Heather Swanson Schaumburg, IL Elizabeta Nemeth, PhD Los Angeles, CA, USA
ABSTRACT REVIEWERS Hossein Ardehali, MD, PhD Sonia Levi, PhD Paulo Arosio, PhD Sijin Liu, PhD Ioav Cabantchik, MD, PhD Bryan Mackenzie, PhD Clara Camaschella, MD Esther Meyron-Holtz, PhD Nica Cappellini, MD Elizabeta Nemeth, PhD Hal Drakesmith, PhD Guangjun Nie, PhD Caroline Enns, PhD Kostas Pantopoulos, PhD Mark D. Fleming, MD, DPhil Sant-Rayn Pasricha, MBBS, MPH, PhD Robert E. Fleming, MD John B. Porter, MA, MD, FRCP DRCPath Lucia De Franceschi, MD Grant Ramm, PhD Tomas Ganz, MD, PhD Marie-Paule Roth, MD, PhD Iqbal Hamza, PhD Tracey Rouault, MD Zoubida Karim, PhD Yatrik Shah, PhD Mitchell Knutson, PhD Laura Silvestri, PhD Jan Krijt, PhD V. Nathan Subramaniam, PhD Samira Lakhal-Littleton, PhD Dorine Swinkels, PhD
Seventh Congress of the International BioIron Society Page 5 General Meeting Information IBIS
Dates of the Conference May 7 – 11, 2017 On Sunday, May 7, 2017, registration will open at 9:00 and the Welcome Reception will begin at 18:30. The BioIron 2017 Meeting will begin with the Opening Ceremony on Monday, May 8, 2017 at 8:00 and will conclude on Thursday, May 11, 2017 at 19:00 with the Gala Dinner and Award Ceremony.
Venue Meyer & Renee Luskin Convention Center 425 Westwood Plaza Los Angeles, California, 90095 (855) 522-8252 luskinconferencecenter.ucla.edu
Language The official language of the BioIron 2017 Conference will be English.
Weather Los Angeles averages a daily maximum temperature for May that's between 72 and 77 degrees Fahrenheit (22 to 25 degrees Celsius). The minimum temperature usually falls between 55 and 59 °F (13 to 15 °C). This time of year may also garner some rain, so waterproof wear is recommended.
Money The official currency of Los Angeles, California, USA is the United States Dollar (USD).
Notes: $100, $50, $20, $10, $5, $1 Coinage: quarter (25 cents), dime (10 cents), nickel (5 cents), penny (1 cent)
Cards: Access, MasterCard, American Express, Diners Club, VISA are widely accepted in shops, restaurants, etc. Retailers now have the option to charge more for goods and services bought by credit card, although they are obliged to display a clear indication that differentiated pricing applies.
Visiting Los Angeles For information on documentation required for entrance into the United States, please visit the U.S. Customs and Border Protection website at: www.cbp.gov
Local Time All times listed observe the 24-hour clock in Greenwich Mean Time (GMT).
Electricity Supply: 110 volts (International travelers should prepare to bring an adapter and a converter)
Emergency Number For ambulance, fire or police, dial 911.
Seventh Congress of the International BioIron Society Page 6 General Meeting Information IBIS
Registration/Information Desk Hours Exhibit Hall Hours Location: Centennial Hall Prefunction, Level One Location: Centennial Hall Prefunction, Level One Sunday, May 7, 2017 9:00 – 20:00 Monday, May 8, 2017 9:00 – 15:00 Monday, May 8, 2017 7:00 – 18:30 Tuesday, May 9, 2017 8:00 – 13:00 Tuesday, May 9, 2017 7:00 – 14:00 Wednesday, May 10, 2017 7:30 – 15:00 Wednesday, May 10, 2017 7:00 – 18:15 Thursday, May 11, 2017 7:00 – 18:15
Registration Fee Includes: o Entrance to Scientific Sessions o Coffee breaks and afternoon refreshments o Lunch on full-day sessions o One ticket to the Welcome Reception o Program Materials IBIS has a green initiative, so instead of cutting down trees to make paper program books, we are cutting down on the use of paper and going electronic, which also cuts costs for the society as a whole. A link to the electronic version of the program book will be available on the website one week before the meeting.
Accompanying Person Registration Fee Includes: o One ticket to the Welcome Reception
Name Badges Attendees are requested to wear their name badges while at the BioIron 2017 Meeting.
Poster Sessions Poster Session I: Monday, May 8, 2017 16:45 – 19:00 Location: South Meeting Rooms, Level Two
Poster Session II: Wednesday, May 10, 2017 17:15 – 19:00 Location: South Meeting Rooms, Level Two
Lunch Location: Centennial Terrace, Level Three Monday, May 8, 2017 11:45 – 13:30 Wednesday, May 10, 2017 12:30 – 14:00 Thursday, May 11, 2017 12:30 – 14:00
Dress The meeting dress is business casual throughout the conference, except for the Gala Dinner and Award Ceremony, which is all L.A. wear – anything from Hollywood Glam to L.A. cool streetwear.
Special Needs 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.
Liability BioIron will not be liable for illness, accidents or thefts suffered by participants or accompanying person during the BioIron 2017 Meeting or their stay in Los Angeles, California, USA.
Seventh Congress of the International BioIron Society Page 7 Special Events IBIS
Welcome Reception Date: Sunday, May 7, 2017 Time: 18:30 Location: Centennial Terrace, Level Three Attire: Casual Cost: One ticket included in Attendee & Accompanying Person registration fees. Additional tickets can be purchased for $50.00 each.
Gala Dinner & “Academy Awards” Ceremony Date: Thursday, May 11, 2017 Time: 19:00 Location: Centennial Hall Attire: Hollywood Glam to L.A. Cool Cost: $90.00
Enjoy tributes and award presentations to rival the Oscars, followed by dancing into the night! Dress up for the occasion: anything from Hollywood Glam to L.A. cool! The gala will kick off with cocktails on the terrace of Centennial Hall at 19:00 and move inside for dinner, awards, and dancing. Set amid the iconic and historic campus buildings, this year's Gala Dinner and International BioIron Society "Academy Awards" Ceremony will surely be an event to remember.
The conference registration fee does NOT include the Gala Dinner & Award Ceremony. Tickets must be purchased separately.
Prior to dinner, cocktail hour, from 19:00 to 20:00, will offer an open bar. Complimentary wine service will be available during dinner service. The bar will remain open at the conclusion of dinner, but beverage orders will be at individual’s expense (payments accepted through credit card only).
Afternoon Activity Guided Tour at The Getty Center* Date: Tuesday, May 9, 2017 Time: 15:00 – 16:30 Cost: $25.00 per person
Bus Pick-up to The Getty Center: 14:00 Bus Departure to UCLA Meyer & Renee Luskin Conference Center: 18:00
* Space is limited – event will take place with a first come, first serve basis.
Seventh Congress of the International BioIron Society Page 8 Local Activities IBIS
Los Angeles’ Griffith Park Griffith Park is the tenth-largest municipally owned park in the United States and one of the largest urban parks in North America, spanning over 4,300 acres of land. Originally a part of the Spanish land grant, Rancho Los Feliz, the park was named for its former owner, “Colonel” Griffith J. Griffith, a Welsh industrialist and philanthropist. In 1896, Griffith bequeathed five square miles of his Rancho Los Feliz estate to the people of Los Angeles.
Los Angeles Zoo and Botanical Gardens Founded in 1966, the Los Angeles Zoo and Botanical Gardens is a 133-acre zoo that is home to 1,100 animals from over 250 species, including 29 that are endangered. The botanical collection features several planted gardens and over 800 different plant species with over 7,400 individual plants. Highlights include the LAIR (Living Amphibians, Invertebrates and Reptiles), Elephants of Asia, Campo Gorilla Reserve and the Tom Mankiewicz Conservation Carousel. General parking is free, and preferred parking is available for a fee on weekends. Your visit supports conservation! Just by purchasing a ticket, you’re helping protect endangered and vulnerable species.
General Admission Fees: Adult (ages 13-61) - $20.00 Senior (ages 62+) - $17.00 Child (ages 2-12) - $15.00 Hours of Operation: Monday – Sunday: 10am – 5pm Address: 5333 Zoo Dr. Los Angeles, CA 90027 Phone: (323) 644-4200
Griffith Observatory Since its opening in 1935, the Griffith Observatory has become one of L.A.’s great cultural icons. The Observatory is located above Los Feliz on the southern slope of Mount Hollywood, and offers some of the best views of L.A., from the Pacific Ocean to downtown. In addition to its fascinating exhibits, the Observatory’s features include the Samuel Oschin Planetarium, Leonard Nimoy Event Horizon, Zeiss Telescope and Gottlieb Transit Corridor. The Griffith Observatory is also one of L.A.’s most famous movie locations.
Hours of Operation: Wednesday – Friday: 12:00 p.m. – 10:00 p.m. Saturday – Sunday: 10:00 a.m. – 10:00 p.m. Address: 2800 E. Observatory Rd. Los Angeles, CA 90027 Phone: (213) 473-0800 Cost: Free Entry
Hollywood Sign The world-famous Hollywood Sign, which recently celebrated its 90th anniversary, is an instantly recognized symbol of Los Angeles. The 45-foot-tall, 350-foot-long sign was recently restored by the nonprofit Hollywood Sign Trust. In 2010, thanks to a $900,000 donation by Hugh Hefner, a $12.5 million fundraising goal was met and the area around the Hollywood Sign was saved from developers.
The Greek Theatre The Greek Theatre is one of L.A.’s premier outdoor venues, nestled in a picturesque, tree-enclosed setting within Griffith Park. Opened in 1929, the historic theatre has hosted some of the biggest names in entertainment, from pop and rock to classical and reggae. The 5,900-seat venue offers an intimate concert setting with state-of-the-art acoustics combined with excellent sight lines. The Greek Theatre has been named North America’s “Best Small Outdoor Venue” multiple times by Pollstar magazine.
Hours of Operation: Monday – Friday: 12:00 p.m. – 6:00 p.m. Saturday – Sunday: 10:00 a.m. – 4:00 p.m. Address: 2700 N. Vermont Ave. Los Angeles, CA 90027 Phone: (323) 665-5857
For additional sightseeing information along with shopping and dining options, please visit: discoverlosangeles.com
Seventh Congress of the International BioIron Society Page 9 Acknowledgments IBIS
Thank You to Our 2017 Promotional Partners (alphabetical as of 5/2/2017)
Platinum Level ApoPharma Inc. Keryx Biopharmaceuticals, Inc. Vifor Pharma Ltd
Gold Level American Regent, Inc. La Jolla Pharmaceutical Novartis
Silver Level DRG International Inc PharmaNutra Spa
Bronze Level Akebia Therapeutics, Inc AMAG Pharmaceuticals Ionis Pharmaceuticals Pfizer, Inc. Rockwell Medical Silarus Therapeutics Sysmex Tolero Pharmaceuticals, Inc
Thank You to Our 2017 Contributors (alphabetical as of 5/2/2017)
Akebia Therapeutics, Inc Intrinsic Lifesciences Kymab Ltd Protagonist Therapeutics, Inc
Thank You To Our 2017 Educational Grant Supporters (alphabetical as of 5/2/2017)
BioMarin Pharmaceutical, Inc.
Seventh Congress of the International BioIron Society Page 10 Program Schedule IBIS
May 8 – 11, 2017
All sessions will be located at the UCLA Meyer & Renee Luskin Conference Center, Centennial Hall, Level One, unless otherwise noted. Speakers and times are subject to change. MONDAY, MAY 8, 2017
OVERVIEW
7:00 – 18:30 Registration/Information Desk Open Location: Centennial Hall Prefunction, Level One 9:00 – 15:00 Exhibit Hall Open Location: Centennial Hall Prefunction, Level One GENERAL SESSION
8:00 – 8:15 Opening Ceremony/Welcome Address 8:15 – 10:20 Plenary Session I: 30-Year Anniversary of IRE Discovery Chairs: Lukas Kuhn, PhD Ecole Polytechnique Federale de Lausanne (EPFL) Switzerland William Walden, PhD University of Illinois at Chicago USA 8:15 – 8:40 The IRP– IRE System – Historical Perspective Speaker: Elizabeth A. Leibold, PhD University of Utah USA 8:40 – 9:05 The Role of the IRE– IRP System in Regulation of Iron Homeostasis in Cells and Tissues Speaker: Tracey Rouault, MD National Institutes of Health USA 9:05 – 9:30 Organismal Functions of the IRP/IRE Regulatory Network Speaker: Bruno Galy, PhD German Cancer Research Center Heidelberg Germany 9:30 – 9:55 New Insights into the Regulation and Function of IRP1 Speaker: Richard Eisenstein, PhD University of Wisconsin USA 9:55 – 10:20 The IRE, Metabolism and RNA Biology: The Next 10 Years Speaker: Matthias Hentze, MD EMBL, Heidelberg Germany 10:20 – 10:45 Coffee Break 10:45 – 11:45 Keynote Lecture Chair: Robert E. Fleming, MD St. Louis University School of Medicine USA Microbial Iron Piracy and Evolution Speaker: Nels C. Elde, PhD The University of Utah School of Medicine USA 11:45 – 13:30 Lunch – Interviews with the Masters Location: Centennial Terrace, Level Three Interviewer: Esther Meyron-Holtz, PhD Interviewee: Chaim Hershko, MD
Seventh Congress of the International BioIron Society Page 11 Program Schedule IBIS
13:30 – 15:00 Concurrent Session I: Iron Sensing in the Liver Location: Centennial Hall AB, Level One Chairs: Leon Kautz, PhD Université Toulouse Paul Sabatier France Laura Silvestri, PhD San Raffaele Scientific Institute & Vita– Salute University Italy 13:30 – 13:45 #1 HEPATOCYTE TRANSFERRIN RECEPTOR 1 OPERATES AS A SENSOR OF SERUM IRON AND IS DISPENSABLE FOR HEPATOCELLULAR IRON SUPPLY John Wagner, MSc, Carine Fillebeen, PhD and Kostas Pantopoulos, PhD Lady Davis Institute for Medical Research/McGill University Presented By: Kostas Pantopoulos 13:45 – 14:00 #2 NRF2 CONTROLS SYSTEMIC IRON HOMEOSTASIS VIA BMP6 AND HEPCIDIN AND ALLEVIATES HEMOCHROMATOSIS Pei Jin Lim, Tiago L. Duarte, Sant-Rayn Pasricha, Andrew E. Armitage, Ana G. Santos, Alireza Morovat, Elizabeth Soilleux, Chia-Yu Wang, Jodie L. Babitt, Graça Porto and Hal Drakesmith MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Pei Jin Lim 14:00 – 14:15 #3 FUNCTIONAL CONSEQUENCES OF N– LOBE SPECIFIC MUTATIONS IN THE TRANSFERRIN BINDING SITE Nermi Parrow¹, Yihang Li, PhD¹, Princy Prasad¹, Yelena Ginzburg, MD², Stefano Rivella, PhD³ and Robert Fleming, MD¹ ¹Saint Louis University School of Medicine; ²Icahn School of Medicine at Mount Sinai; ³Children’s Hospital of Philadelphia Presented By: Nermi Parrow 14:15 – 14:30 #4 ENDOTHELIAL CELLS PRODUCE BMP6 REQUIRED FOR IRON HOMEOSTASIS IN MICE AND PROVIDE A MODEL FOR STUDYING IRON– MEDIATED REGULATION OF THE BMP6– HEPCIDIN AXIS Susanna Canali, PhD, Chia– Yu Wang, PhD, Kimberly B. Zumbrennen– Bullough, PhD, Amanda B. Core, PhD, Manfred Nairz, MD, PhD, Richard Bouley, PhD, Filip K. Swirski, PhD and Jodie L. Babitt, MD Program in Anemia Signaling Research, Division of Nephrology, Program in Membrane Biology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA Presented By: Susanna Canali 14:30 – 14:45 #5 NEOGENIN FACILITATES THE INDUCTION OF HEPCIDIN EXPRESSION BY HEMOJUVELIN IN THE LIVER Ningning Zhao², Julia Maxson¹, Richard Zhang¹, Mastura Wahedi¹, Caroline Enns¹ and An– Sheng Zhang¹ ¹ Oregon Health & Science University; ²University of Arizona Presented By: An– Sheng Zhang 14:45 – 15:00 #6 HEPATIC HEPARAN SULFATE REGULATES HEPCIDIN EXPRESSION BY MODULATING BMP6/SMAD SIGNALING Maura Poli, PhD¹, MF Anower– E– Khuda, PhD², Michela Asperti, PhD¹, Paola Ruzzenenti, PhD student¹, Philip Gordts, PhD², Jeffrey D. Esko, PhD² and Paolo Arosio, PhD¹ ¹University of Brescia; ²UCSD Presented By: Maura Poli
13:30 – 15:00 Concurrent Session II: Brain and Iron Location: Centennial Hall CD, Level One Chairs: James Connor, PhD Pennsylvania State University USA Daniel Kosman, PhD University of Buffalo USA 13:30 – 13:45 #7 IPSC–DERIVED NEURONS OF PANTOTHENATE KINASE–ASSOCIATED NEURODEGENERATION SHOW MITOCHONDRIAL IRON/CALCIUM MISHANDLING Paolo Santambrogio, PhD², Daniel I. Orellana, PhD², Alicia Rubio, PhD², Anna Cozzi, PhD², Vania Broccoli, PhD³ and Sonia Levi, PhD¹ ¹Vita– Salute San Raffaele University; ²San Raffaele Scientific Institute Milano, Italy; ³CNR– Institute of Neuroscience, Milano, Italy Presented By: Sonia Levi
Seventh Congress of the International BioIron Society Page 12 Program Schedule IBIS
13:45 – 14:00 #8 RESTORATION OF ENERGY METABOLISM AND CYTOSOLIC IRON BURDEN USING A COA SYNTHESIS PATHWAY INTERMEDIATE IN PKAN CELLS Suh Young Jeong, PhD, Rachel Fox, BA, Jeffrey Hamada, BA, Martina Ralle, PhD and Susan Hayflick, MD Dept. of Molecular & Medical Genetics, Oregon Health & Science University Presented By: Suh Young Jeong 14:00 – 14:15 #9 STUDY OF BRAIN IRON ACCUMULATION AND DEPOSITION OF AMYLOID PLAQUES IN A MOUSE MODEL THAT COMBINES BRAIN IRON LOADING WITH ALZHEIMER’S DISEASE AMYLOID PATHOLOGY Ritambhara Aryal¹, Jason Woods¹, Kristy Martin¹, Seak– Lin Ly¹, Daniel Johnstone, PhD² and Elizabeth Milward, PhD¹ ¹The University of Newcastle; ²University of Sydney Presented By: Ritambhara Aryal 14:15 – 14:30 #10 SINGLE CELL RNASEQ ANALYSIS PROVIDES EVIDENCE FOR RELATIONSHIPS BETWEEN IRON–RELATED GENE EXPRESSION AND BRAIN OLIGODENDROGLIAL MYELINATING CAPABILITY Seak Lin Ly¹, Carlos Riveros, PhD², Moones Heidari, PhD³, Daniel Johnstone, PhD4 and Elizabeth Milward, PhD² ¹The University of Newcastle; ²The University of Newcastle, Australia; ³University of Wisconsin, USA; 4University of Sydney, Australia Presented By: Seak Lin Ly 14:30 – 14:45 #11 MODULATING ANTIBODY AFFINITY TOWARDS THE TRANSFERRIN RECEPTOR TO INCREASE BRAIN UPTAKE OF ANTI–TRANSFERRIN RECEPTOR ANTIBODY TARGETED GOLD NANOPARTICLES Kasper Bendix Johnsen, MSci², Martin Bak, Civil Ing³, Fredrik Melander, Civil Ing, PhD3, Annette Burkhart, MSci, PhD², Paul Kempen, MSci, PhD³, Thomas Lars Andresen, Civil Ing, PhD³ and Torben Moos, MD, PhD, DMSci¹ ¹Aalborg University; ²Laboratory for Neurobiology, Institute of Health Science and Technology, Aalborg University, Denmark; ³Center for Nanomedicine and Theranostics, Department of Micro–and Nanotechnology, Technical University of Denmark, Denmark Presented By: Torben Moos 14:45 – 15:00 #12 CONDITIONAL MULLER CELL ABLATION LEADS TO RETINAL IRON ACCUMULATION Bailey Baumann, BA, Ying Song, MD, Delu Song, MD, PhD, Jacob Sterling, BS, MS, Weiyong Shen, MD, PhD, Mark Gillies, PhD and Joshua Dunaief, MD, PhD FM Kirby Center for Molecular Ophthalmology, School of Veterinary Medicine, University of Pennsylvania Presented By: Bailey Baumann
15:00 – 15:15 Break
15:15 – 16:45 Concurrent Session III: Intermediary Metabolism as a Regulator of Erythropoiesis and Iron Location: Centennial Hall AB, Level One Chairs: Max Gassmann, DVM University of Zurich Switzerland
Carole Peyssonnaux, PhD Institut Cochin France 15:15 – 15:40 Acetate Switch Regulates Erythropoiesis Speaker: Joseph Garcia, MD, PhD University of Texas Southwestern Medical Center USA 15:40 – 16:05 Isocitrate and Aconitase in Regulation of Erythropoiesis Speaker: Adam Goldfarb, MD University of Virginia School of Medicine USA 16:05 – 16:30 New Pathways Controlling Cellular Response to Iron Deficiency Speaker: Hossein Ardehali, MD, PhD Northwestern University USA 16:30 – 16:45 #13 ACONITASE: AN IRON SENSING REGULATOR OF MITOCHONDRIAL OXIDATIVE METABOLISM AND ERYTHROPOIESIS Grant Bullock, MD, PhD, Naomi Gunawardena, BS, Valerie Miller, MS, Chante Richardson, PhD, Teague Cole, BS, Catherine Corey, MPH, Yinna Wang, MD, PhD and Sruti Shiva, PhD University of Pittsburgh Presented By: Grant Bullock
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15:15 – 16:45 Concurrent Session IV: The Role of Iron in Metabolic and Cardiovascular Disorders Location: Centennial Hall CD, Level One Chairs: Martina Muckenthaler, PhD University Hospital Heidelberg Germany Gaetano Cairo, PhD University of Milan School of Medicine Italy 15:15 – 15:30 #14 FUNCTIONAL IRON DEFICIENCY CAUSED BY LOSS OF IRON REGULATORY PROTEIN 2 LEADS TO PANCREATIC ß CELL DYSFUNCTION Elizabeth Leibold, PhD, Cole P. Anderson, PhD and Steven J. Romney, BS University of Utah, USA Presented By: Elizabeth Leibold 15:30 – 15:45 #15 DISREGULATED IRON AND LIPID METABOLISM IN MICE WITH LIVER–SPECIFIC KNOCKOUT OF IRON CHAPERONE PCBP1 Olga Protchenko, PhD¹, Fengmin Li, PhD¹, Minoo Shakoury-Elizeh, MS¹, Manik C. Ghosh, PhD², Oksana Gavrilova, PhD¹ and Caroline C. Philpott, MD¹ ¹National Institutes of Health, NIDDK; ²National Institutes of Health, NICHD Presented By: Olga Protchenko 15:45 – 16:00 #16 AN ESSENTIAL ROLE FOR THE BMP–SMAD PATHWAY IN HEPCIDIN INDUCTION DURING ER STRESS AND NAFLD/NASH Audrey Belot, PhD student², Ophélie Gourbeyre, Aude Rubio, Céline besson-Fournier, Chloé Latour, Inka Gallitz, Alexandra Montagner, Arnaud Polizzi, Marion Régnier, Andrea U. Steinbicker, Hervé Guillou, Marie-Paule Roth, Hélène Coppin and Delphine Meynard, PhD¹ ¹INSERM; ²IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, France Presented By: Delphine Meynard 16:00 – 16:15 #17 LOW IRON DIET AND CHELATION THERAPY PREVENT IRON–AGGRAVATED ATHEROSCLEROSIS IN A MOUSE MODEL OF HEREDITARY HEMOCHROMATOSIS Francesca Vinchi¹, Andreas Simmelbauer, MD student¹, Sandro Altamura, PhD¹, Richard Sparla, BTA¹, Graca Porto, MD², Sebastian Speich, MD³, Bruno Galy, PhD4, Matthias W. Hentze, MD5 and Martina U. Muckenthaler, PhD¹ ¹Molecular Medicine Partnership Unit (MMPU), University of Heidelberg & EMBL, Heidelberg, Germany; ²University of Porto, Porto, Portugal; ³University of Heidelberg, Germany; 4German Cancer Research Center (DKFZ), Germany; 5European Molecular Biology Laboratory (EMBL), MMPU, Heidelberg, Germany Presented By: Francesca Vinchi 16:15 – 16:30 #18 GENETIC LOSS OF TMPRSS6, THE GENE MUTATED IN IRON–REFRACTORY IRON DEFICIENCY ANEMIA, DISRUPTS PHOSPHATE HOMEOSTASIS IN MICE Xiuqi Li, BS, Larisa Lozovatsky, MS, Abitha Sukumaran, PhD, Steven Tommasini, PhD, Jackie Fretz, PhD and Karin Finberg, MD, PhD Yale School of Medicine Presented By: Xiuqi Li 16:30 – 16:45 #19 TYPE 2 DIABETES RISK AND NORMATIVE VALUES FOR IRON BIOMARKERS Leo Zacharski¹, Galina Shamayeva, MS², Bruce Chow, MS² and Ralph DePalma, MD³ ¹VA Hospital & Dartmouth Hitchcock Medical Center; ²Palo Alto VA Hospital; ³VA Cebtral Office Presented By: Leo Zacharski 16:45 – 19:00 Poster Session I Location: South Meeting Rooms, Level Two Refreshments available. Beer and wine service to begin at 17:00. Poster #10 EXPLORING THE EFFECTS OF IRON DEFICIENCY ANAEMIA AND REPLETION WITH INTRAVENOUS IRON ON EXERCISE PERFORMANCE IN C57 WILD TYPE MICE Anna Butcher, MBBS, PhD¹, Marija Sajic, MBBS, PhD², Kenneth Smith, PhD² and Toby Richards, MD, FRCS³ ¹UCL; ²Department of Neuroinflammation, Institute of Neurology, UCL, London, United Kingdom; ³Division of Surgery and Interventional Medicine, UCL, London, United Kingdom Presented By: Anna Butcher, MBBS, PhD Poster #11 NCOA4 INCREASES IN CONDITION OF MODERATE IRON OVERLOAD BUT NOT IN HIGLY IRON OVERLOADED MICE AND IN TFR2 KO ANIMALS Rosa Maria Pellegrino, PhD², Martina Boero, Dr², Antonietta Palmieri, Dr², Mariarosa Mezzanotte, Dr², Giuseppe Saglio, MD² and Antonella Roetto, PhD¹ ¹University of Torino; ²University of Torino, Department of Clinical and Biological Sciences, AOU San Luigi Gonzaga, 10043 Orbassano, Torino, Italy Presented By: Antonella Roetto, PhD
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Poster #12 BASIDIOCHROME: MAIN SIDEROPHORE OF MYCORRHIZAL FUNGAL SYMBIONTS OF ORCHIDS WITH INTERESTING CHEMICAL AND BIOLOGICAL PROPERTIES Kurt Haselwandter, Dr¹, D. Pretsch, Dr² and J.M. Rollinger, Dr² ¹University of Innsbruck; ²University of Vienna Presented By: Kurt Haselwandter Poster #13 DISRUPTION OF THE HEPCIDIN/FERROPORTIN REGULATORY CIRCUITRY CAUSES PULMONARY IRON OVERLOAD AND RESTRICTIVE LUNG DISEASE Joana Neves¹,⁴, Dominik Leit ², Christina Brandenberger³, Christian Mühlfeld³, Raman Agrawal², Marcus A. Mall²,⁵, Sandro Altamura¹,⁴,⁵ and Martina U. Muckenthaler¹,⁴,⁵ ¹Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg, Germany; ²Translational Lung Research Center Heidelberg , German Center for Lung Research (DZL), University of Heidelberg, Germany; ³Institute of Functional and Applied Anatomy, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover Medical School, Germany; ⁴Molecular Medicine Partnership Unit, Heidelberg, Germany; ⁵ ual contribution Presented By: Joana Neves Poster #14 GENETIC DISRUPTION OF A REDOX ENZYME NCB5OR IN MICE RESULTS IN IRON DYSHOMEOSTASIS, NEUROLOGICAL DEFECTS AND HEARING LOSS IN ADDITION TO EARLY–ONSET LEAN DIABETES Hao Zhu, PhD, WenFang Wang, PhD, Matthew Stroh, PhD, Marcello Peppi, PhD, Haping Wang, MD, Ming Xu, PhD, Zhi– nong Yin, PhD and Jie Dai, MD University of Kansas Medical Center Presented By: Hao Zhu, PhD Poster #15 METHODOLOGICAL CONSIDERATIONS IN IRON STAINING OF SELECT NON–HEPATIC TISSUES OF MOUSE MODELS OF HEMOCHROMATOSIS Kristy Martin¹, William Myles¹, Daniel Johnstone², Ali Shahandeh¹ and Elizabeth Milward¹ ¹The University of Newcastle; ²University of Sydney Presented By: Kristy Martin Poster #16 HAEMOCHROMATOSIS INTERNATIONAL–AN ALLIANCE OF HEREDITARY HAEMOCHROMATOSIS GROUPS AROUND THE WORLD Desley White, Pierre Brissot, Barbara Butzeck, Howard Don, Robert Evans, Paulo Santos, Emerencia Teixeira and Ben Marris Plymouth University Presented By: Desley White, PhD, BSc Hons Poster #17 HEPCIDIN DETERMINATION IN NON–HFE HEMOCHROMATOSIS. A CASE–CONTROL STUDY Ana López Aparicio¹, Maria Dolores Pulfer, Resident², Alejandra García, Resident², Maria Josefa Moran, PH², Maria Isabel Moreno, PH² and Alejandro Del Castillo, Staff² ¹Gregorio Marañon university hospital; ²Gregorio Marañón Universitary Hospital Presented By: Ana Aparicio, MD Poster #18 FUNCTIONAL STUDIES OF A SINGLE NUCLEOTIDE POLYMORPHISM IN DUODENAL CYTOCHROME B Fabian Schlottmann, BSc, M. Vera– Aviles, and G.O. Latunde– Dada, PhD Faculty of Life Sciences, Diabetes and Nutritional Sciences Division, King s College London, United Kingdom Presented By: Gladys Latunde– Dada, PhD Poster #19 TYPE 4 HEREDITARY HEMOCHROMATOSIS: CHARACTERIZATION OF PATIENTS REFERRING TO THE REGIONAL CENTER FOR IRON DISORDERS IN VERONA (ITALY) Annalisa Castagna¹,², Chiara Piubelli, PhD, Giacomo Marchi, MD, Monica Rizzi, MD, Fabiana Busti, MD, Alice Vianello, MD, Paola Capelli, MD, Roberto Pozzi– Mucelli, MD, Luciano Xumerle, BSc, Massimo Delledonne, PhD, Oliviero Olivieri, MD and Domenico Girelli, MD, PhD ¹Veneto Region Referral Center for Iron Disorders, Azienda Ospedaliera Universitaria Integrata; ²Department of Medicine, Section of Internal Medicine, University of Verona, Verona, Italy Presented By: Annalisa Castagna, PhD Poster #20 NCOA4 IS WELL EXPRESSED IN MURINE AND HUMAN PERYPHERAL BLOOD AND CORRELATES WITH AGE: A NEW MARKER OF IRON AMOUNT IN FRAIL ELDERLY PEOPLE? Rosa Maria Pellegrino, PhD¹, Antonietta Palmieri, Dr², Martina Boero, Dr², Gianluca Isaia, MD³, Mariarosa Mezzanotte, Dr², Giuseppe Saglio, MD², Angelo Guerrasio, MD² and Antonella Roetto, PhD² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Sciences, AOU San Luigi Gonzaga, Torino, Italy; ³SCDU Geriatrics, AOU San Luigi Gonzaga Hospital, Orbassano, Turin, Italy Presented By: Rosa Maria Pellegrino
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Poster #21 IRON–RICH DIET DECREASES MYCOBACTERIAL (M. Bovis BCG) BURDEN AND CORRELATES WITH HEPCIDIN UPREGULATION, LOWER PRO–INFLAMMATORY MEDIATORS AND HIGHER T CELL RECRUITMENT Rafiou Agoro, PhD², Stephanie Rose, MSc³, Irene Garcia, PhD⁴, Bernhard Ryffel, MD, PhD², Valerie Quesniau , PhD² and Catherine Mura, PhD¹ ¹CNRS/University Orleans; ²CNRS University Orleans, France; ³CNRS, France; ⁴University of Geneva, Switzerland Presented By: Catherine Mura, PhD Poster #22 CELL IRON STATUS ATTENUATES PRO–INFLAMMATORY MACROPHAGE POLARIZATION Rafiou Agoro, PhD² and Catherine Mura, PhD¹ ¹CNRS/University Orleans; ²CNRS/University Orleans, France Presented By: Catherine Mura, PhD Poster #23 FERROPORTIN DOWNREGULATION IN THE LIVER OF LEISHMANIA INFANTUM–INFECTED MICE THROUGH A HEPCIDIN–INDEPENDENT MECHANISM Tania Cruz, PhD¹, Maria Vieira, Bachelor², Joana Passos, Master³, Helena Castro, PhD², Margarida Duarte, PhD², François Cannone– Hergau , PhD⁴, Jorge Pinto, PhD³ and Ana Tom s, PhD² ¹I3S – Instituto de Investiga o e Inova o em Sa de, Universidade do Porto; ²I S; ³IBMC; ⁴CNRS Presented By: Tania Cruz, PhD Poster #24 THE ROLE OF HFE GENOTYPE IN MACROPHAGE PHENOTYPE Anne Nixon, Elizabeth Neely and James Connor Penn State College of Medicine, Hershey Medical Center, Hershey, PA Presented By: Anne Nixon, BS Poster #25 ELUCIDATING THE ROLE OF IL6 AND REACTIVE OXYGEN SPECIES (ROS) IN BONE MARROW ERYTHROPOIESIS AND IN THE DEVELOPMENT OF ANEMIA UNDER INFLAMMATORY CONDITIONS Ritama Gupta¹, Roberta Chessa, PhD², Sara Gardenghi, PhD³, Ping La, PhD² and Stefano Rivella, PhD² ¹Children's Hospital of Philadelphia, University of Pennsylvania; ²Children's Hospital of Philadelphia; ³Weill Cornell Medical College Presented By: Ritama Gupta, MSc Poster #26 H–FERRITIN IS A MACROPHAGE HOST DEFENSE FACTOR AGAINST SALMONELLA INFECTION David Haschka, Dr¹, Piotr Tymoszuk, PhD¹, Verena Petzer, Dr¹, Stefanie Dichtl, Mag¹, Dirk Bumann, Prof², Markus Seifert¹, Sylvia Berger¹, Lukas Kühn, Prof³, Igor Theurl, Prof¹ and Günter Weiss, Prof¹ ¹Medical University of Innsbruck, Internal Medicine VI; ²University of Basel, Focal Area Infection Biology; ³Swiss Institute for Experimental Cancer Research Presented By: David Haschka Poster #27 RESTRICTION OF FE–S CLUSTER BIOGENESIS UNDERPINS THE METABOLIC AND EPIGENETIC REPROGRAMMING IN TOLL–LIKE RECEPTORS–STIMULATED MACROPHAGES Wing– Hang Tong, PhD, Nunziata Maio, PhD, De– liang Zhang, PhD and Tracey Rouault, MD Eunice Kennedy Shriver National Institute of Child Health and Human Development, USA Presented By: Wing– Hang Tong, PhD Poster #28 NOVEL SPLICING SITE AND MISSENSE MUTATIONS CAUSE ACERULOPLASMINEMIA Marina Dorigatti Borges, BSc, Dulcineia Martins Albuquerque, BSc, PhD, Carolina Lanaro, BSc, PhD, Fernando Ferreira Costa, MD, PhD and Kleber Yotsumoto Fertrin, MD, PhD University of Campinas Presented By: Marina Dorigatti Borges, BSc Poster #29 THE INFLUENCE OF IRON HOMEOSTASIS ON MICROGLIA FUNCTION Andreas Popp, Dr¹, Thomas Möller, Dr², Jessica Abele³ and Bernhard K. Mueller, PD Dr⁴ ¹Abbvie Deutschland GmbH & Co. KG; ²AbbVie Inc., Neuroscience Foundational Discovery, Cambridge, USA; ³AbbVie Deutschland GmbH Co. KG, Preclinical Safety, Lud igshafen, Germany; ⁴AbbVie Deutschland GmbH & Co. KG, Neuroscience Discovery, Ludwigshafen, Germany Presented By: Andreas Popp, Med. Vet Poster #30 A ROLE FOR SEX AND A COMMON HFE GENE VARIANT IN BRAIN IRON UPTAKE James Connor, PhD, Kari Duck, PhD, Ian Simpson, PhD and Elizabeth Neely Penn State Hershey Medical Center Presented By: James Connor, PhD Poster #31 THE ROLE OF THE BLOOD–BRAIN BARRIER IN MAINTAINING BRAIN IRON STATUS James Connor, PhD, Kari Duck, PhD and Ian Simpson, PhD Penn State Hershey Medical Center Presented By: James Connor, PhD
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Poster #32 FERRITIN UPTAKE INTO THE DEVELOPING MOUSE BRAIN Brian Chiou, Elizabeth Neely and James Connor Penn State Hershey College of Medicine Presented By: Brian Chiou Poster #33 EXOGENOUS α–SYNUCLEIN INDUCES INFLAMMATION AND ALTERS NON–TRANSFERRIN BOUND IRON UPTAKE GENES AND PROTEINS IN CELLS OF THE CENTRAL NERVOUS SYSTEM Veronika Kallo, BSc MRes, Vernon Skinner, BSc PhD, David Dexter, PhD and Surjit Kaila Singh Srai, PhD University College London Presented By: Veronika Kallo, BSc, MRes Poster #34 COMPUTATIONAL MODELLING OF IRON TRANSPORT BETWEEN BLOOD CIRCULATION, BRAIN, AND DOPAMINERGIC NEURONS OF THE SUBSTANTIA NIGRA Vindy Tjendana Tjhin, MSc¹, Simon Mitchell, PhD², Elizabeth A. Milward, PhD³, Charles E. Hutchinson, MD¹, Michael J. Chappell, PhD¹ and Joanna F. Collingwood, PhD¹ ¹University of Warwick; ²UCLA; ³University of Newcastle Presented By: Vindy Tjendana Tjhin, MSc Poster #35 ADVANCES IN X–RAY MICROSCOPY FOR THE ANALYSIS OF TRANSITION METALS IN THE BRAIN Joanna Collingwood, PhD¹, James Everett, PhD¹, Neil Telling, PhD² and Freddy Adams, PhD³ ¹University of Warwick; ²Keele University; ³University of Antwerp Presented By: Joanna Collingwood, MPhys, PhD Poster #36 THE DISTRIBUTION OF IRON–RELATED PROTEINS IN INTRACEREBRAL HEMORRHAGE Lisa Juul Routhe, MSc¹, Torben Moos, Prof¹ and Guohua Xi, MD² ¹Aalborg University; ²University of Michigan Presented By: Lisa Juul Routhe, MSc Poster #37 LIVER IRON CONTENT BY MAGNETIC RESONANCE IN CHRONIC KIDNEY DISEASE AT THE START OF MAINTENANCE HEMODIALYSIS Patricia Carrilho, MD¹, In s Santiago, MD², Marta Alves, MSc³, Pedro Fidalgo, MD⁴, lsa Rodrigues, MD² and Bruno Rodrigues, MD⁴ ¹Hospital Fernando Fonseca; ²Hospital Fernando Fonseca, Dept Radiology; ³3Centro Hospitalar de Lisboa Central, pidemiology and Statistics Office of the Research Unit; ⁴Hospital Fernando Fonseca, Dept Nephrology Presented By: Patricia Carrilho, MD Poster #38 PROTECTIVE EFFECT OF HISTIDINE AGAINST IRON–INDUCED TOXICITY IN HEK293 CELLS F. Schlottmann, MSc, M. Vera– Aviles, MSc and G.O. Latunde– Dada, PhD Faculty of Life Sciences, Diabetes and Nutritional Sciences Division, King s College London, United Kingdom Presented By: Mayra Vera– Aviles, MSc Poster #39 HEMOGLOBIN UPTAKE AND INJURY IN RENAL DISTAL TUBULES Rachel van Swelm, PhD¹, Frank Verhoeven¹, Tom Gielkens¹, Frank Thévenod, PhD² and Dorine Swinkels, PhD¹ ¹Radboud University Medical Center, Nijmegen, The Netherlands; ²University of Witten/Herdecke, Witten, Germany Presented By: Rachel Van Swelm, PhD Poster #40 INTRAVENOUS IRON OXIDE INJECTION WITH HIGH POTENCY AND SAFETY PROFILES IN IRON DEFICIENCY ANEMIA RAT MODEL IN VIVO Chih–Lung Chen, PhD, Wen–Yuan Hsieh, PhD, Yuan–Hung Hsu, PhD, Nina Wang, PhD and Shian–Jy Wang, PhD MegaPro Biotech Company Presented By: Chih–Lung Chen, PhD Poster #41 HOW CAN RECENT INSIGHTS INTO IRON METABOLISM AND INFECTION HELP US OVERCOME THE GLOBAL BURDEN OF ANEMIA? Sant-Rayn Pasricha, MBBS, MPH, PhD, Andrew Armitage, Andrew Prentice and Hal Drakesmith MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Sant-Rayn Pasricha, MBBS, MPH, PhD Poster #42 IMPACT OF IRON DEFICIENCY DIAGNOSIS USING HEPCIDIN MS DOSAGE METHODS ON HOSPITAL STAY AND COSTS AFTER A PROLONGED ICU STAY: A MULTICENTER, RANDOMIZED, SINGLE–BLINDED TRIAL Sigismond Lasocki, MD, PhD¹, Herve Puy, MD,PhD², Thibaud Lefevre, MD, PhD², Gregoire Mercier, MD³, Constance Delaby, PhD³ and Sylvain Lehmann, MD, PhD³ ¹CHU Angers; ²INSERM U1149; ³CHU de Montpellier Presented By: Sigismond Lasocki, MD, PhD
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Poster #43 DEVELOPMENT OF HUMAN SOLUBLE TRANSFERRIN RECEPTOR ELISA ASSAY Liubov Litvinkova, MS¹, Ale ey Kravtsov, PhD², Matthias Herkert, PhD³, Ian Cushman, PhD⁴, Wilhelm Sänger, MBA⁵ and Cyril Geacintov, PhD⁶ ¹DRG International, Inc.; ²Senior Research Scientist, DRG International, Inc.; ³Director R&D, DRG Instruments GMBH; ⁴Director R D, DRG International, Inc.; ⁵General Manager, DRG Instruments GMBH; ⁶C O, DRG International, Inc. Presented By: Liubov Litvinkova Poster #44 COMPARISON OF THE THERAPEUTICAL POTENTIAL OF ORAL SUCROSOMIAL® IRON AND PARENTERAL IRON DEXTRAN SUPPLEMENTATIONS IN NEONATAL IRON DEFICIENCY ANEMIA IN PIGS Rafa Star y ski, PhD¹, Mateus S ud ik, MSc², Robert Staro , MSc², Aneta Jo c y, MSc², a Smuda, MSc², Marek Pies ka, PhD³, Marian Kamyc ek, PhD⁴ and Pa e Lipi ski, PhD² ¹IGAB PAS; ²Institute of Genetics and Animal Breeding PAS, Jastr biec, Poland; ³Department of Animal Nutrition & Feed Science, National Research Institute of Animal Production, Kraków, Poland; ⁴Pig Hybridi ation Centre, National Research Institute of Animal Production, Pa o ice, Poland Presented By: Pawel Lipinski, PhD, DSc Poster #45 NEONATAL IRON DEFICIENCY ANEMIA IN PIGS: CAUSES, REGULATION OF IRON METABOLISM, AND STRATEGIES FOR IRON SUPPLEMENTATION THERAPY Pa e Lipi ski, PhD¹, Rafa Star y ski, PhD², Robert Staro , MSc², Anna Gajo iak, MSc², a Smuda, MSc², Marek Pies ka, PhD³ and Ma gor ata Lenarto ic , PhD⁴ ¹IGAB PAS; ²Institute of Genetics and Animal Breeding PAS, Jastr biec, Poland; ³Department of Animal Nutrition & Feed Science, National Research Institute of Animal Production, Kraków, Poland; ⁴Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, Kraków, Poland Presented By: Pawel Lipinski, PhD, DSc Poster #46 PLACENTAL EXPRESSION OF IRON HOMEOSTASIS GENES IN IRON DEFICIENCY ANEMIA OF PREGNANCY Sreenithi Santhakumar, MTech¹, Rekha Athiyarath, PhD¹, Anne George Cherian, MD², Vinod Joseph Abraham, MD² and Eunice Sindhuvi Edison, PhD¹ ¹Department of Haematology, Christian Medical College, Vellore, India; ²Department of Community Health, Christian Medical College, Vellore, India Presented By: Eunice Sindhuvi Edison, PhD Poster #47 DEFERASIROX AND PHLEBOTOMIES IN A PATIENT WITH EXTREME IRON OVERLOAD BY HEREDITARY HEMOCHROMATOSIS AND BETA–THALASSEMIA MINOR AFTER DIAGNOSIS OF LISTERIOSIS Alejandro Del Castillo-Rueda, Dolores Pulfer, Alejandra García-García and Ana López-Aparicio Hospital Gregorio Marañón Presented By: Alejandro Del Castillo-Rueda, MD, PhD Poster #48 EVALUATING IRON METABOLISM AND SUPPLEMENTATION IN GAMBIAN TODDLERS USING A NOVEL, HIGH–PRECISION METHOD BASED ON THE DILUTION OF LABELLED BODY IRON Diego Moretti, MSc, PhD¹, Rita Wegmüller, MSc, PhD²,³, Cornelia Speich, MSc¹, Christophe Zeder, MSc¹, Magdalena Cichucka, MSc¹, Colin Ivano Cercamondi, MSc, PhD¹, Andrew Prentice, MD, Professor²,³, Gary Brittenham, MD, Professor and Michael Bruce Zimmermann, MD, Professor¹ ¹ETH Zürich; ²MRC Unit, The Gambia; ³LSHTM, London, UK. Presented By: Diego Moretti, PhD Poster #49 EFFECT OF SUCROSOMIAL® IRON IN A IRON DEFICIENCY ANEMIA MOUSE MODEL lisa Brilli, PhD¹, Maura Poli, PhD², Michela Asperti, PhD², Angela Fabiano, PhD³, Annalisa Castagna, PhD⁴, Domenico Girelli, MD⁴, Ylenia Zambito, PhD³, Paolo Arosio, PhD² and Germano Tarantino¹ ¹Pharmanutra S.p.A.; ²DMMT, University of Brescia; ³Pharmacological Sciences, University of Pisa; ⁴Internal Medicine, University of Verona Presented By: Germano Tarantino Poster #50 EFFECTS OF BONE MARROW IRON ON BONE MINERAL DENSITY ASSESSMENTS: A PHANTOM STUDY Marcela Weyhmiller, PhD¹, Neil Buac², Ellen Fung, PhD RD¹, Ashutosh Lal, MD¹ and Roland Fischer, PhD³ ¹UCSF Benioff Children's Hospital Oakland; ²California State University, East Bay; ³UCSF Benioff Children's Hospital Oakland/UKE – Pediatrische Hämatologie/Onkologie Presented By: Marcela G. Weyhmiller, PhD Poster #51 HFE GENOTYPE ALTERS EXOSOME PROFILES IN CANCER Oliver Mrowczynski, A.B. Madhankumar, PhD, Brad Zacharia, MD, MS, Becky Slagle-Webb, BS and James Connor, PhD Penn State Hershey Presented By: Oliver Mrowczynski Poster #52 ANALYSIS OF SINGLE NUCLEOTIDE VARIATION OF HFE GENE FOR CANCER RISK AND ASSOCIATION TO SURVIVAL IN THE CANCER GENOME ATLAS (TCGA) GBM DATA Sang Lee, Junjia Zhu, Anna Salzberg, Bo Zhang, Dajiang Liu and James Connor Penn State University College of Medicine Presented By: Sang Lee, PhD Seventh Congress of the International BioIron Society Page 18 Program Schedule IBIS
Poster #53 FERROPORTIN INDUCES IRON DEPLETION, AUTOPHAGY, AND CELL CYCLE ARREST IN PROSTATE CANCER CELLS OF DIFFERENT HISTOPATHOLOGICAL SUBTYPES Suzy Torti, PhD, Zhiyong Deng, PhD, David Manz, BS and Frank Torti, MD University of Connecticut Health Center Presented By: Suzy Torti, PhD Poster #54 DCYTB IS A PREDICTOR OF OUTCOME IN BREAST CANCER THAT FUNCTIONS VIA IRON– INDEPENDENT MECHANISMS Frank Torti, MD¹, David Lemler, PhD², Miranda Lynch, PhD³, Lia Tesfay, MS¹, Zhiyong Deng, PhD¹, Bibbin Paul, PhD¹, Poornima Hegde, MD¹ and Suzy Torti, PhD¹ ¹University of Connecticut Health Center; ²Univesity of Connecticut Health Center; ³Los Alamos National Laboratory Presented By: Frank Torti, MD Poster #55 APOPTOTIC EFFECT OF FENUGREEK ON MCF–7 CELLS IS INHIBITED BY FERRITIN Kholoud Khoja¹ MSc, PhD, Paul Sharp² PhD and Yemisi Latunde-Dada² PhD ¹King's College London; ²King’s College London Presented By: Kholoud Khoja, PhD Poster #56 ADMINISTRATION OF MINIHEPCIDINS TO ANIMALS AFFECTED BY ß–THALASSEMIA MAJOR REDUCES ANEMIA AND SPLENOMEGALY Roberta Chessa, PhD¹, Ritama Gupta², Carla Casu, PhD², Robert Fleming, MD³, Yelena Gin burg, MD⁴, Brian MacDonald, MD⁵ and Stefano Rivella, PhD² ¹The Children s Hospital of Philadelphia; ²The Children s Hospital of Philadelphia; ³Saint Louis University; ⁴Icahn School of Medicine at Mount Sinai; ⁵Merganser Biotech Presented By: Roberta Chessa, PhD Poster #57 THRESHOLD FERRITIN VALUES TO PREDICT CONTROL OF LIVER IRON BURDEN IN THALASSEMIA Ashutosh Lal, MD, Marcela Weyhmiller, PhD and Elliott Vichinsky, MD UCSF Benioff Children's Hospital Oakland Presented By: Ashutosh Lal, MD Poster #58 DIFFERENTIAL OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION RESPONSES INDUCED BY BRAND AND GENERIC SODIUM FERRIC GLUCONATE COMPLEX FORMULATIONS Amy Pai, PharmD¹, Paul Neumann, BS² and Teresa Regis, BS² ¹University of Michigan College of Pharmacy; ²Albany College of Pharmacy and Health Sciences Presented By: Amy Pai, PharmD Poster #59 BIOEQUIVALENCE EVALUATION OF GENERIC INTRAVENOUS IRON PRODUCTS– IMPLICATIONS OF LABILE IRON RELEASE Amy Pai, PharmD¹, Dan Meyer, PhD², Brian Bales, PhD², Victoria Cotero, PhD² and Manjunath Pai, PharmD¹ ¹University of Michigan College of Pharmacy; ²General Electric Global Research Presented By: Amy Pai, PharmD Poster #60 EXERCISE–INDUCED DECREASE IN HEPATOCELLULAR IRON OVERLOAD IMPROVES INSULIN SENSITIVITY IN AGED RATS yuqian liu¹, Haitao Wang, PhD², Yingjun Zhang² and Yanzhong Chang, PhD³ ¹College of Physical Education; ²College of Physical Education, Hebei Normal University; ³Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University Presented By: Yuqian Liu, PhD Poster #61 HEPCIDIN– FERRITIN RATIO IS DECREASED IN DIABETES MELLITUS Molly Jacob, MBBS, MD, PhD¹, Padmanaban Venkatesan, MBBS², Joe Varghese, MBBS, MD, DNB², Jithu James, MSc² and Jasmin Prasad, MBBS, MD² ¹Christian Medical College, Vellore, India; ²Christian Medical College, Velllore, India Presented By: Molly Jacob, MD, PhD, MBBS Poster #62 DIET–INDUCED INSULIN RESISTANCE IN MICE IS ASSOCIATED WITH DECREASED LEVELS OF LIVER IRON AND SERUM HEPCIDIN Joe Varghese, MBBS, MD, Jithu Varghese James, MSc and Molly Jacob, MBBS, MD, PhD Department of Biochemistry, Christian Medical College, Vellore, India Presented By: Joe Varghese, MD Poster #63 HYPERFERRITINEMIA INCREASES THE RISK OF HYPERURICEMIA IN PATIENTS WITH GENETIC HEMOCHROMATOSIS RELATED TO P.CYS282TYR MUTATION IN THE HFE GENE Jeremy Flais, MD², Edouard Bardou– Jacquet, MD, PhD³, Yves Deugnier, MD³, Guillaume Coiffier, MD², Aleth Perdriger, MD, PhD², G rard Chal s, MD², Martine Ropert, MD⁴, Olivier Loreal, MD, PhD¹ and Pascal Guggenbuhl, MD, PhD² ¹INS RM UMR ; ²Department of Rheumatology; ³Liver Disease Unit; ⁴Department of Biochemistry, Rennes University Hospital, France Presented By: Olivier Loreal, MD, PhD
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Poster #64 HEPCIDIN KNOCK–OUT MICE DEVELOP LESS INSULIN RESISTANCE THAN WILD–TYPE MICE WHEN FED A HIGH–FAT DIET Jithu James, MSc, Joe Varghese, Sophie Vaulont and Molly Jacob Christian Medical College, Vellore, India Presented By: Jithu James, MSc Poster #65 PROTEIN LEVELS OF IRON HOMEOSTASIS RELATED MOLECULES IN SKELETAL MUSCLE TISSUE IN VEGAN POPULATION Kamila Balusikova, Jan Gojda, David Albrecht, Jana Patockova, Jan Kovar and Michal Andel 3rd Faculty of Medicine, Charles University Presented By: Kamila Balusikova Poster #66 THE DIVERSE ROLES OF SIDEROCALIN / NGAL / LCN2 IN ANTI–BACTERIAL IRON SEQUESTRATION, CANCER, AND CLINICAL APPLICATION Colin Correnti, PhD, Peter Rupert, PhD and Roland Strong, PhD Fred Hutchinson Cancer Research Center Presented By: Roland Strong, PhD Poster #67 THE ROLES OF THE THREE IRON–STORAGE PROTEINS OF ESCHERICHIA COLI IN SUPPORTING LOW–IRON GROWTH AND REDOX STRESS RESISTANCE Afrah Salman, PhD student and Simon Andrews, Professor The University of Reading Presented By: Ahmad Salman, MBBS, MCOS Poster #68 A STUDY OF NCOA4 EXPRESSION AND BINDING TO FERRITINS Magdalena Gryzik, PhD student, Fernando Carmona, PhD, Maura Poli, PhD and Paolo Arosio, PhD Department of Molecular and Translational Medicine, University of Brescia, Italy Presented By: Magdalena Gryzik Poster #69 THE HYDROPHOBIC IRON CHELATOR DESFERRIOXAMINE–CAFFEINE, DFCAF Jesus Antonio Alvarado Huayhuaz, Hector Aguilar Vitorino, Othon Souto Campos, Silvia Helena Pires Serrano, Telma Mary Kaneko and Breno Esposito University of Sao Paulo Presented By: Breno P. Esposito Poster #70 PHARMACOKINETICS (PK) OF FERRIC PYROPHOSPHATE CITRATE (TRIFERIC): MODELING DIURNAL IRON AND ADDITIVITY OF TRIFERIC IN HEALTHY VOLUNTEERS IRON Raymond Pratt, MD¹, Mark Bush, PhD², Scott Brantley, PhD² and Ajay Gupta, MD¹ ¹Rockwell Medical Inc; ²Nuventra Inc. Presented By: Raymond D. Pratt, MD Poster #71 CRISPR–CAS9 GENOME–WIDE KNOCKOUT SCREENING IDENTIFIES MOLECULAR COMPONENTS INVOLVED IN CELLULAR IRON UPTAKE Amin Sobh, Alex Loguinov, PhD and Chris Vulpe, MD, PhD University of California, Berkeley Presented By: Amin Sobh Poster #72 DNA METHYLATION REGULATES THE EXPRESSION OF TRANSFERRIN RECEPTOR 2 Paul Sharp¹, Robert Weeks² and Ian Morison² ¹King's College London; ²University of Otago, New Zealand Presented By: Paul A. Sharp Poster #73 LC– ICP– MS CHARACTERIZATION OF NON–TRANSFERRIN–BOUND IRON Nathanial Dziuba, BS, Joanne Hardy, PhD, DVM and Paul Lindahl, PhD Texas A&M University Presented By: Paul Lindahl, PhD Poster #74 ERYTHROID CELL MITOCHONDRIA RECEIVE ENDOSOMAL IRON BY A “KISS–AND–RUN” MECHANISM Amel Hamdi, PhD¹, Daniel Garcia-Santos, PhD¹, Tariq Roshan, MD¹, Alex Sheftel, PhD² and Prem Ponka, MD, PhD¹ ¹McGill University, Lady Davis Institute, Montreal, Canada; ²Spartan Bioscience Inc, Ottawa, Canada Presented By: Amel Hamdi, PhD Poster #75 SPECIATION OF NON–TRANSFERRIN BOUND IRON IN BLOOD PLASMA OF IRON DEFICIENT SWINE Nathaniel Dziuba, Joanne Hardy and Paul A. Lindahl Texas A&M University Presented By: Nathaniel Dziuba, BA
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Poster #76 TISSUE–AUTONOMOUS REGULATION OF RETINAL IRON TRANSPORT Joshua Dunaief, MD, PhD, Ying Song, MD, Delu Song, MD, PhD, Bailey Baumann, BA, Milan Theurl, MD, Paul Hahn, MD, PhD, Majda Hadziahmetovic, MD and Natalie Wolkow, MD, PhD FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Perelman School of Medicine, University of Pennsylvania Presented By: Joshua L. Dunaief, MD, PhD Poster #77 REVERSAL OF IRON OVERLOAD IN TRANSFERRIN–DEFICIENT MICE Thomas Bartnikas, MD PhD, Courtney Mercadante, Michael Pettiglio, Carolina Herrera, Miriam Dash, Julia Bu and Edward Stopa, MD Brown University Presented By: Thomas Bartnikas, MD, PhD Poster #78 THE FATE OF TRANSFERRIN BOUND TO IRON OXIDE NANOPARTICLES IN VITRO AND IN VIVO Denise Bargheer, PhD, Julius Nielsen, Artur Giemsa, PhD, Barbara Freund, PhD and Peter Nielsen, MD, PhD University Medical Centre Hamburg, Dept. of Biochemistry and Molecular Cell Biology Presented By: Peter Nielsen, MD, PhD Poster #79 A DOMINANT MUTATION IN MITOCHONDRIAL UNFOLDASE CLPX RESULTS IN ERYTHROPOIETIC PROTOPORPHYRIA Barry Paw, MD, PhD¹, Yvette Yien, PhD¹, Gael Nicolas, PhD², Lisa van der Vorm, MS¹, Laurent Gouya, PhD², Hector Bergonia, PhD³, Martin Kafina, MS¹, Julia Kardon, PhD⁴, Tania Baker, PhD⁴, John Phillips, PhD³ and Herve Puy, MD, PhD² ¹Harvard Medical School; ²INS RM U , Paris; ³Univ. Utah; ⁴MIT Presented By: Barry H Paw, MD, PhD Poster #80 ERYTHROPOIETIN SIGNALING REGULATES HEME BIOSYNTHESIS Barry Paw, MD, PhD¹, Jacky Chung, PhD¹, Johannes Wittig, MS¹, Alireza Ghamari, PhD¹, Manami Maeda, MD, PhD⁵, Tamara Dailey², Hector Bergonia³, Martin Kafina, MS¹, Liangtao Li, MD³, Jerry Kaplan, PhD³, Harvey Lodish, PhD⁴, Daniel Bauer, MD, PhD¹, Stuart Orkin, MD¹, Alan Cantor, MD, PhD¹, Takahiro Maeda, MD, PhD⁵, John Phillips, PhD³, Joshua Coon, PhD⁶, David Pagliarini, PhD⁶ and Harry Dailey, PhD² ¹Harvard Medical School; ²Univ. Georgia; ³Univ. Utah; ⁴MIT; ⁵Fukuoka, Japan; ⁶Univ. Wisconsin, Madison Presented By: Barry H. Paw, MD, PhD Poster #81 FUNCTIONAL ANALYSIS OF HETEROZYGOUS GATA1 DELETERIOUS MUTATIONS IN TWO CHINESE FEMALE PATIENTS AFFECTED BY DYSERYTHROPOIETIC ANEMIA, THROMBOCYTOPENIA AND PORPHYRIA, RESPECTIVELY Yong ei Wang, BS¹, Gang Liu, PhD¹,⁴, Shanshan Guo, BS¹, Bing Han, MD², Yan hong Chang, PhD³ and Guangjun Nie, PhD¹ ¹CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China; ²Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; ³Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei Province, China; ⁴Section on Human Iron Metabolism, NIH, Bethesda, MD Presented By: Yongwei Wang, BS Poster #82 PHARMACOLOGICAL ACTIVATION OF IRE–BINDING OF IRON REGULATORY PROTEIN1 BY TEMPOL PROTECTS VhlR200W MUTANT MICE FROM POLYCYTHEMIA THROUGH TRANSLATIONAL REPRESSION OF Hif2a Manik Ghosh, PhD, De-Liang Zhang, PhD, Wade Ollivierre and Tracey Rouault, MD Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH Presented By: Manik Ghosh, PhD Poster #83 CARBON NANOTUBES DISRUPT IRON HOMEOSTASIS AND INDUCE ANEMIA OF INFLAMMATION THROUGH INFLAMMATORY PATHWAY AS A SECONDARY EFFECT DISTANT TO THEIR PORTAL–OF–ENTRY Juan Ma, PhD and Sijin Liu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Presented By: Juan Ma Poster #84 GENERATION OF INDUCED PLURIPOTENT STEM CELL–DERIVED ERYTHROBLASTS FROM A PATIENT WITH X–LINKED SIDEROBLASTIC ANEMIA Shunsuke Hatta, MD, PhD¹, Tohru Fujiwara¹, Takako Yamamoto, MD, PhD², Mayumi Kamata, MD, PhD¹, Yoshiko Tamai, MD, PhD³, Yukio Nakamura, MD, PhD⁴, Shin Ka amata, MD, PhD² and Hideo Harigae, MD, PhD¹ ¹Tohoku University Graduate School of Medicine; ²Foundation of Biomedical Research and Innovation; ³Hirosaki University Graduate School of Medicine; ⁴RIK N BioResource Center Presented By: Tohru Fujiwara, MD, PhD
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Poster #85 THE INTERPLAY OF GATA1 WITH HEME REGULATES AN ERYTHROID CELL’S DIFFERENTIATION Raymond Doty, PhD¹, Xiaowei Yan, PhD², Christopher Lausted, MS², Zhantao Yang, MD¹, Li Liu, PhD¹, Neda Jabbari, PhD², Sioban Keel, MD¹, Qiang Tian, MD, PhD² and Janis Abkowitz, MD¹ ¹University of Washington; ²Institute for Systems Biology Presented By: Raymond Doty, PhD Poster #86 GLUTATHIONE AND THE CYTOSOLIC HEME POOL Robert Hider, BSc, PhD¹, Yu-Lin Chen, PhD², Rosemary Rawlinson, BSc² and Yemisi Latunde-Dada, PhD² ¹King's College London; ²KCL Presented By: Robert Hider, BSc, PhD Poster #87 ERYTHROPOIETIN INCREASES BONE AND CIRCULATING FGF23 LEVELS Mark Hanudel, MD, MS¹, Maxime Rappaport, BS², Kristine Chua, BS², Erica Clinkenbeard, PhD³, Victoria Gabayan, BS², Tomas Ganz, MD, PhD², Kenneth White, PhD³, Isidro Salusky, MD² and Elizabeta Nemeth, PhD² ¹UCLA Pediatric Nephrology; ²David Geffen School of Medicine at UCLA; ³Indiana University School of Medicine Presented By: Mark Hanudel, MD, MS Poster #88 DYSREGULATED IRON METABOLISM IN BONE MARROW FROM A MOUSE MODEL OF CHRONIC KIDNEY DISEASE (CKD): HEPCIDIN AND ERYTHROPOIESIS Takeshi Nakanishi, MD, PhD, Tomoko Kimura, MD, Kiyoko Yamamoto, MD, Masayoshi Nanami, MD, PhD and Takahiro Kuragano, MD, PhD Hyogo College of Medicine Presented By: Takeshi Nakanishi, MD, PhD Poster #89 INVESTIGATING THE ROLE OF HRG1 IN HEME TRANSPORT Rini Pek¹, Jianbing Zhang, M Sc¹, Nicole Rietzschel, PhD¹, John Phillips, PhD², David Bodine, PhD³ and Iqbal Hamza, PhD¹ ¹University of Maryland; ²University of Utah; ³National Human Genome Research Institute Presented By: Rini Pek, BA Poster #90 THE HEME EXPORTER FLVCR1a IS CRUCIAL FOR BRAIN DEVELOPMENT Deborah Chiabrando¹, Tullio Genova², Francesca Bertino¹, Lorenzo Silengo¹, Fiorella Altruda¹ and Emanuela Tolosano¹ ¹Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy.; ²Department of Surgical Sciences, CIR Dental School, University of Turin, Presented By: Deborah Chiabrando, PhD Poster #91 EXPLORING POTENTIAL MEDIATORS OF EARLY HEPCIDIN DOWNREGULATION BY ERYTHROPOIETIN IN VIVO Irene Artuso, Antonella Nai, Mariateresa Pettinato, Silvia Colucci, Clara Camaschella and Laura Silvestri San Raffaele Scientific Institute Presented By: Irene Artuso Poster #92 MOUSE STRAIN DIFFERENCES IN THE RESPONSE TO TREATMENT WITH A HEMOJUVELIN NEUTRALIZING MONOCLONAL ANTIBODY Andreas Popp, Dr¹, Melissa Matzelle, Dr² and Bernhard K. Mueller, PD Dr³ ¹Abbvie Deutschland GmbH & Co. KG; ²AbbVie Inc., Foundational Immunology; ³AbbVie Deutschland GmbH & Co. KG, Neuroscience Discovery Presented By: Andreas Popp, Med. Vet Poster #93 VALPROIC ACID AND SHORT CHAIN FATTY ACIDS BUTYRATE AND PROPIONATE MAY INDUCE HEPCIDIN MRNA EXPRESSION IN HEPATIC CELLS Marie-Laure Island, PhD², Amandine Gaudin, MsC, Thibault Cavey, MD, Patricia Leroyer, Martine Ropert, MD, Pierre Brissot, MD and Olivier Loréal, MD, PhD¹ ¹INSERM UMR991; ²INSERM UMR991, Rennes, France Presented By: Olivier Loreal, MD, PhD Poster #94 SUPPRESSION OF ERYTHROPOIESIS IN THE MOUSE MODEL MIMICKING REPEATED– TRANSFUSION INDUCED IRON OVERLOAD Masayo Yamamoto, MD, PhD¹, Hiroki Tanaka, PhD², Masatomo Ishioh, MD¹, Takeshi Saito, MD¹, Yasumichi Toki, MD, PhD¹, Mayumi Hatayama, MD¹, Motohiro Shindo, MD, PhD¹, Katsuya Ikuta, MD, PhD¹, Yoshihiro Torimoto, MD, PhD³ and Toshikatsu Okumura, MD, PhD¹ ¹Division of Gastroenterology & Hematology/Oncology, Asahikawa Medical University; ²Division of Legal Medicine, Asahikawa Medical University; ³Oncology Center, Asahikawa Medical University Hospital Presented By: Masayo Yamamoto, MD, PhD Poster #95 PREVENTION OF IRON TOXICITY FROM HEME CATABOLISM IS LINKED TO COPPER– MEDIATED REGULATORY EVENTS Ann Smith, PhD¹, Kim Rish, BS MS², LokMan Sung, BS MD², Rachel Helston, BS PhD³ and Roberto Vanacore, BS PhD⁴ ¹SBS-UMKC; ²University of Missouri-Kansas City; ³Stowers Institute for Medical Research; ⁴Vanderbilt University Presented By: Ann Smith, BSc, PhD
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Poster #96 TRIPHENYLPHOSPHONIUM–DESFERRIOXAMINE AS A CANDIDATE MITOCHONDRIAL IRON CHELATOR Roxana Yesenia Pastrana Alta, Hector Vitorino, Simon Wisnovsky, Shana Kelley, M. Teresa Machini and Breno Esposito University of Sao Paulo Presented By: Breno P. Esposito Poster #97 WITHDRAWN Poster #98 CHANGES OF “IRON GENE” SIGNATURE RESPONSIBLE FOR EMBRYONIC STEM CELL STEMNESS AND DIFFERENTIATION Jing Liu and Sijin Liu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Presented By: Jing Liu Poster #99 A TRIPEPTIDE MOTIF– LEU– TYR– ARG (LYR) PLAYS A SIGNIFICANT ROLE IN Fe–S CLUSTER ACQUISITION BY CELLULAR PROTEINS Anshika Jain, PhD¹, Anamika Singh, MS², Nunziata Maio, PhD² and Tracey Rouault, MD² ¹NIH; ²NICHD/NIH Presented By: Anshika Jain, PhD Poster #100 SHORT– ACTING MUCOSAL BLOCK AND FERRITIN–INDEPENDENT ANTIOXIDATIVE SYSTEM WORKS IN THE IRON–DEFICIENT RATS DUODENUM Shoko Shinoda, PhD, Machiko Yamamoto, MS and Anna Arita, MS Tokyo Metropolitan University Presented By: Shoko Shinoda, PhD Poster #101 GENETIC ANALYSIS OF SURVIVAL OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN EGG WHITE; IMPACT TO IRON RESTRICTION AND LYSOZYME Abdulameer Ghareeb, PhD student¹, Pauline Sabrou², Sophie Jan², Florence Baron² and Simon Andrews, Professor ¹The University of Reading; ²Agrocampus Ouest Presented By: Abdulameer Ghareeb Poster #102 INCREASED DUODENAL IRON ABSORPTION IN PATIENTS WITH CHRONIC HEPATITIS C AND NONALCOHOLIC STEATOHEPATITIS Koji Miyanishi, Masanori Sato, Shingo Tanaka, Akira Sakurada, Hiroki Sakamoto, Yutaka Kjawano, Toshifumi Hoki, Masayoshi Kobune and Junji Kato Department of Medical Oncology, Sapporo Medical University. School of Medicine Presented By: Koji Miyanishi, MD, PhD Poster #103 THE NOVEL FINDING OF ELTROMBOPAG (ELT) IMPROVING PANCREATIC CELL FUNCTION BY INCREASING INSULIN PRODUCTION, CHELATING IRON AND SCAVENGING ROS Evangelia Vlachodimitropoulou, MBBS, BSc, P. Koonyosying, N. Cooper, B. Psaila, M. Sola-Visner and J. Porter UCl Presented By: Evangelia Vlachodimitropoulou, MBBS, BSc Poster #104 NEW FILM–COATED TABLET FORMULATION OF DEFERASIROX IS WELL TOLERATED IN PATIENTS WITH THALASSEMIA OR MYELODYSPLASTIC SYNDROMES: RESULTS OF THE RANDOMIZED, PHASE II ECLIPSE STUDY Ali T. Taher¹, Raffaella Origa², Silverio Perrotta³, Ale andra Kouraklis⁴, Giovan Battista Ruffo⁵, Antonis Kattamis⁶, Ai– Sim Goh⁷, Annelore Cortoos⁸, Vicky Huang⁸, Marine Weill⁹, Ra uel Merino Herran ⁹ and John B. Porter¹ ¹American University of Beirut Medical Center; ²University of Cagliari; ³Second University of Naples; ⁴University of Patras Medical School; ⁵U.O.C. matolog. Con Talassemia; ⁶University of Athens; ⁷Hospital Pulau Pinang; ⁸Novartis Pharmaceuticals Corporation; ⁹Novartis Pharma AG; ¹ University College London Presented By: John B. Porter, MA, MD, FRCP DRCPath Poster #105 RENAL SAFETY AFTER MORE THAN A DECADE OF DEFERASIROX USE IN PATIENTS WITH TRANSFUSIONAL HEMOSIDEROSIS Raffaella Origa¹, Antonio Piga², Immacolata Tartaglione³, Giuseppina Della Corte⁴, Andreas Bruederle⁵, Jackie Han⁶, Chiara Castiglioni⁷ and Gian Luca Forni⁸ ¹University of Cagliari; ²University of Turin; ³Second University of Naples; ⁴Universit di Milano; ⁵Novartis Pharma AG; ⁶Novartis Pharmaceuticals Corporation; ⁷Novartis Farma SpA; ⁸Ospedale Galliera Presented By: Antonio Piga, MD Poster #106 MEASUREMENT OF HEPCIDIN ISOFORMS IN HUMAN SERUM BY LIQUID CHROMATOGRAPHY WITH HIGH RESOLUTION MASS SPECTROMETRY (LC– HR– MS) Simon Handley¹, Lewis Couchman, MSc¹, Paul Sharp, PhD², Iain Macdougall, PhD³ and Caje Moni , PhD⁴ ¹Toxicology Unit, Viapath Analytics; ²Diabetes and Nutritional Sciences Division, King's College London; ³Department of Renal Medicine, King s College Hospital; ⁴Department of Clinical Biochemistry, King's College Hospital Presented By: Simon Handley
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Poster #107 HEPCIDIN ISOFORM CONCENTRATIONS IN SERUM SAMPLES FROM PATIENTS WITH CHRONIC KIDNEY DISEASE Simon Handley¹, Paul Sharp, PhD², Caje Moni , PhD³ and Iain Macdougall, PhD⁴ ¹Toxicology Unit, Viapath Analytics; ²Diabetes and Nutritional Sciences Division, King’s College London; ³Department of Clinical Biochemistry, King s College Hospital; ⁴Department of Renal Medicine, King s College Hospital Presented By: Simon Handley Poster #108 HEPCIDIN IS AN HDAC3 REGULATED GENE AND ITS EXPRESSION IS DETERMINED BY PROMOTER– ASSOCIATED HISTONE ACETYLATION Sant-Rayn Pasricha, MBBS, MPH, PhD, Pei Jin Lim, Tiago Duarte, Carla Casu, Kasia Mleczko-Sanecka, Maria Suciu, Joao Areas, Ana Rita Da Silva, Joe Frost, Kirsty McHugh, Graca Porto, Simon Draper, Sarah Gooding, Peter Olinga, Martina Muckenthaler, Stefano Rivella, Jim Hughes, Tom Milne, Andrew Armitage and Hal Drakesmith MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Sant-Rayn Pasricha, MBBS, MPH, PhD Poster #109 THE ROLE OF HEPCIDIN IN RENAL FAILURE Alejandra García¹, Ana Lopez, Resident², Maria Dolores Pulfer, Resident², Maria Josefa Moran, Ph², Maria Isabel Moreno, Ph² and Alejandro Del Castillo, Satff² ¹University Hospital Gregorio Marañon; ²Gregorio Marañon University Hospital Presented By: Alejandra Garcia Poster #110 IS HEPCIDIN AN INFECTION MARKER? Maria Dolores Pulfer¹, Ana López, Resident², Alejandra García, Resident², Maria Josefa Moran, Ph², Maria Isabel Moreno, PH² and Alejandro Del Castillo, staff² ¹Gregorio Marañón Universitary Hospital; ²University Hospital Gregorio Marañon Presented By: Maria Dolores Pulfer Poster #111 FERROPORTIN Q248H MUTATION INDUCES AUTOPHAGY AND FERRITINOPHAGY Guelaguetza Vazquez– Ortiz, PhD, Andrey Ivanov, MD, PhD, Xionghao Lin, PhD, Namita Kumari, PhD and Sergei Nekhai, PhD Howard University Presented By: Sergei Nekhai, PhD Poster #112 TP– 0184 IS AN INHIBITOR OF ALK2 AND A POTENTIAL THERAPEUTIC FOR THE TREATMENT OF ANEMIA OF CHRONIC DISEASE Peter Peterson, PhD, Wontak Kim, BS, Hillary Haws, MS, Clifford Whatcott, PhD, Adam Siddiqui-Jain, PhD, David Bearss, PhD and Steven Warner, PhD Tolero Pharmaceuticals, Inc. Presented By: Clifford Whatcott, PhD Poster #113 MATERNAL HEPCIDIN DETERMINES FETAL IRON HOMEOSTASIS DURING PREGNANCY V. Sangkhae, A.L. Fisher, K. Chua, V. Gabayan, E. Valore, T. Ganz and E. Nemeth University of California, Los Angeles Presented By: Veena Sangkhae, PhD Poster #114 DEVELOPMENTAL ROLES FOR THE IRP1- IF AXIS AND IRP1 ALONE IN THE CONTROL OF ERYTHROPOIESIS AND IRON METABOLISM Gretchen Seim, James Votava, Chris Nizzi and Rick Eisenstein University of Wisconsin-Madison Presented By: Richard Eisenstein, PhD Poster #133 EFFECTS OF BRAIN TISSUE PROCESSING ON SENSITIVITY OF DAB–ENHANCED PERL’S METHOD FOR IRON STAINING Moones Heidari, PhD, Kristy Martin, Daniel M. Johnstone, Ritambhara Aryal, Shanmathi Parameswaran, Linh Lim, Roberto Cappai, Samantha Richardson and Elizabeth A. Milward University of Newcastle Presented By: Moones Heidari, MSc Poster #142 IRIDA DUE TO HETEROZYGOUS MUTATIONS OF TMPRSS6 AND ACVR1A ENCODING THE BMP RECEPTOR ALK2 Alessia Pagani, Silvia Colucci, Renata Bocciardi, Roberto Ravazzolo, Laura Silvestri and Clara Camaschella Vita Salute University and Division of Genetics and Cell Biology, San Raffaele Scientific Institute Milano, Italy Presented By: Alessia Pagani, PhD Poster #LB1 MORE HARM THAN HELP: DOES EARLY–LIFE IRON EXPOSURE INCREASE THE RISK OF AGE–RELATED NEURODEGENERATION? Dominic Hare, PhD The Florey Institute of Neuroscience and Mental Health Presented By: Dominic Hare, PhD
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Poster #LB2 NANOSTRUCTURED IRON COMPOUNDS FOR NUTRITION – IN VITRO AND IN VIVO ASSESSMENT OF SAFETY AND ABSORPTIVE PATHWAYS Hans Christian Winkler, DVM, PhD, Lea M. von Moos, PhD, Julian Kornprobst, MSc, Mirjam Schneider, Florentine M. Hilty, PhD, Monika Hilbe, DVM, Myrtha Arnold, Nathalie Ziegler, Diogo Sales Mato, Elisabeth M. Schraner, Peter Wick, PhD, Barbara Bathke, Shana J. Sturla, PhD, Ioannis A. Trantakis, PhD, Hubertus Hochrein, PhD, Wolfgang Langhans, DVM, Mark Suter, DVM, Hanspeter Naegeli, DVM and Michael B. Zimmermann, MD, MSc Swiss Federal Institute of Technology ETH, Laboratory of Human Nutrition Presented By: Hans Winkler Poster #LB3 WITHDRAWN Poster #LB4 EFFECT OF A NOVEL ORALLY ACTIVE IRON CHELATOR: 1– (N– ACETYL– 6– AMINOHEXYL)– 3– HYDROXY– 2– METHYLPYRIN– 4– ONE (CM1) IN IRON– OVERLOADED AND NON– OVERLOADED MICE Somdet Srichairatanakool, PhD¹, Nittaya Chansiw, PhD², Kanjana Pangjit, PhD³, Chada Phisalaphong, PhD⁴, John Porter, FRCP, FRCPath, MD⁵, Patricia vans, PhD⁵, Robert Hider, PhD⁶ and Suthat Fucharoen, MD⁷ ¹Chiang Mai University; ²Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Thailand; ³College of Medicine and Public Health, Ubon Ratchathani University, Thailand; ⁴Government Pharmaceutical Organi ation, Thailand; ⁵Department of Haematology, University College London, United Kingdom; ⁶Institute of Pharmaceutical Science, King's College London, United Kingdom; ⁷Institute of Molecular Bioscience, Mahidol University, Thailand Presented By: Somdet Srichairatanakool, PhD Poster #LB5 ROLE OF EFEUOB IN IRON ACQUISITION AND PATHOGENICITY OF ESCHERICHIA COLI O157:H7 Arvindkumar Salunkhe and Simon Andrews University of Reading, UK Presented By: Arvindkumar Salunkhe Poster #LB6 IRON INDUCES INSULIN RESISTANCE IN CARDIOMYOCYTES VIA REGULATION OF OXIDATIVE STRESS Hyekyoung Sung and Gary Sweeney, PhD York University Presented By: Hyekyoung Sung Poster #LB7 MITOCHONDRIAL FERRITIN DELETION EXACERBATES ß–AMYLOID–INDUCED NEUROTOXICITY IN MICE Peina Wang, Qiong Wu, Wenyue Wu, Haiyan Li, Yuetong Guo, Peng Yu, Guofen Gao, Zhenhua Shi, Baolu Zhao and Yan-Zhong Chang Hebei Normal University Presented By: Peina Wang Poster #LB8 THE PROTECTIVE ROLE OF MITOCHONDRIAL FERRITIN ON ERASTIN–INDUCED FERROPTOSIS Yueqi Wang, Shiyang Chang, Qiong Wu, Yujing Gou, Linpei Jia, Yanmei Cui, Peng Yu, Zhenhua Shi, Wenshuang Wu, Guofen Gao and Yan-Zhong Chang Hebei Normal University Presented By: Shiyang Chang Poster #LB9 CHARACTERIZATION OF MITOCHONDRIAL FERRITIN–OVEREXPRESSION MICE Peina Wang, Xin Li, Shiyang Chang, Weina Kong and Yan-Zhong Chang Hebei Normal University Presented By: Peina Wang Poster #LB10 MITOCHONDRIAL FERRITIN PROTECTS HYDROGEN PEROXIDE–INDUCED NEURONAL CELL DAMAGE Guofen Gao, Nan Zhang, Yueqi Wang, Zhen Li, Peng Yu, Zhenhua Shi, Baolu Zhao, Wenshuang Wu, Yan-Zhong Chang and Shiyang Chang Hebei Normal University Presented By: Shiyang Chang Poster #LB11 WITHDRAWN Poster #LB12 FERRIC CARBOXYMALTOSE VERSUS IRON SUCROSE COMPLEX IN WOMEN WITH IRON DEFICIENCY ANEMIA – A RANDOMISED CONTROLLED TRIAL Garima Chaudhry Obstetrics & Gynaecology Presented By: Garima Chaudhry
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Poster #LB13 INTRA– AND INTER–MOLECULAR SIGNAL TRANSDUCTION MECHANISMS OF HEME SENSING SYSTEMS Hitomi Sawai, PhD¹, Gareth S. A. Wright, PhD², Akane Saeki, MSc¹, Takaaki Hikima, PhD³, Masaki Yamamoto, PhD³, Svetlana Antonyuk, PhD², S. Samar Hasnain, PhD² and Yoshitsugu Shiro, PhD¹,³ ¹Graduate School of Life Science, University of Hyogo; ²Institute of Integrative Biology, University of Liverpool; ³RIKEN SPring– 8 Center Presented By: Hitomi Sawai, PhD Poster #LB14 STRUCTURAL INSIGHTS INTO ASCORBATE–DEPENDENT FERRIREDUCTASE, DCYTB, IN HUMAN Menega Ganasen¹, Hitomi Sawai, PhD², Hiromi Togashi, PhD³, Hanae Takeda, MSc³, Yoshitsugu Shiro, PhD²,³ and Hiroshi Sugimoto, PhD³ ¹Graduate School of Life Science, University of Hyogo; ²Grad. Sch. of Life Sci., Univ. Hyogo; ³RIKEN SPring– 8 Center Presented By: Menega Ganasen, MS Poster #LB15 NASAL DELIVERY OF NANOLIPOSOME–ENCAPSULATED FERRIC AMMONIUM CITRATE CAN INCREASE THE IRON CONTENT OF RAT BRAIN ueling Guo, MS², Hong Zheng, MS³, Yuetong Guo, MS², Yan Wang, MS², Gregory J. Anderson, Ph D⁴, Yun he Ci, MS², Peng Yu, Ph D¹, Lina Geng, Ph D³ and Yanzhong Chang, Ph D² ¹Laboratory of Molecular Iron Metabolism, Key laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University; ²College of Life Sciences, Hebei Normal University; ³College of Chemistry and Material Science, Hebei Normal University; ⁴Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute Presented By: Peng Yu, PhD Poster #LB16 FERRITIN IN THE CEREBROSPINAL FLUID PREDICTS RATE OF AMYLOID BETA ACCUMULATION IN BIOMARKER–CONFIRMED ALZHEIMER’S DISEASE Scott Ayton, PhD¹, Ibrahima Diouf, PhD² and Ashley Bush, MB BS, PhD³,⁴,⁵ ¹Florey Institute of Neuroscience and Mental Health; ²CSIRO Health and Biosecurity/Australian E– Health Research Centre, Brisbane, Australia; ³The Florey Institute for Neuroscience and Mental Health; ⁴The University of Melbourne, Australia; ⁵Cooperative Research Center for Mental Health, Victoria, Australia Presented By: Scott Ayton, PhD Poster #LB17 HAEMOCHROMATOSIS AUSTRALIA – A POTENTIAL RESEARCH PARTNER? Dianne Prince Haemochromatosis Australia Presented By: Dianne Prince Poster #LB18 FERRIC CITRATE AND FERRIC EDTA DRIVE PROLIFERATION OF GUT EPITHELIAL CANCER CELLS VIA AMPHIREGULIN MEDIATED ACTIVATION OF THE MAP KINASE PATHWAY Nathalie Scheers¹,², Dora I.A. Pereira²,³, Nuno Faria²,⁴ and Jonathan J.J. Po ell²,⁴ ¹Chalmers University of Technology; ²Elsie Widdowson Laboratory, Medical Research Council, Cambridge, UK; ³Department of Pathology, University of Cambridge; ⁴Department of Veterinary Medicine, University of Cambridge Presented By: Natalie Scheers, PhD Poster #LB19 SHORT FORM HEALTH SURVEY (SF–36) DIFFERS BETWEEN GENOTYPIC GROUPS OF PATIENTS WITH SUSPECTED HEREDITARY HEMOCHROMATOSIS Paula Fernanda, MSc², Rodolfo Cançado, MD, PhD³, Fl vio Naoum, MD, PhD⁴, Carla Dinardo, MD, PhD⁵,⁹, Guilherme Fonseca, MD, MSc⁶, Sandra Gualandro, MD, PhD⁷, Jose Krieger, MD, PhD², Ale andre Pereira, MD, PhD², Pierre Brissot, MD, PhD⁸ and Paulo Santos, PhD¹ ¹Federal University of Sao Paulo; ²Laboratory of Genetics and Molecular Cardiology, Heart Institute , University of São Paulo Medical School, São Paulo, Brazil; ³Hematology and Hemotherapy Section, Santa Casa Medical School, S o Paulo, Bra il; ⁴Academia de Ci ncia e Tecnologia, S o Jos do Rio Preto, Bra il; ⁵Funda o Pr – Sangue, Hemocentro de S o Paulo, S o Paulo, SP, Bra il; ⁶Hematology Service, Hospital das Clinicas, Medical School, University of S o Paulo, S o Paulo, Bra il; ⁷Hematology and Hemotherapy Discipline, Hospital das Clinicas, Medical School, University of S o Paulo, S o Paulo, Bra il; ⁸Liver Disease Unit, Pontchaillou University Hospital, University of Rennes, and National Reference Centre for Rare Iron Overload Diseases of Genetic Origin, Rennes, France; ⁹Universidade de S o Paulo (USP), São Paulo, SP, Brazil Presented By: Paulo Caleb J L Santos, PhD Poster #LB20 HDAC INHIBITION AND GAIN–OF–FUNCTION P53 MUTATION DICTATES ONCOGENIC PHENOTYPE AND INFLUENCES IRON METABOLISM IN THE SW13 CELL LINE McKale Montgomery¹ and Elizabeth Hull, PhD² ¹Midwestern University; ²Midwestern Universty Presented By: McKale Montgomery Poster #LB21 FERROPORTIN INHIBITORS PREVENT IRON LOADING IN A MOUSE MODEL OF HEREDITARY HEMOCHROMATOSIS Naja Nyffenegger, PhD, Anna Flace, Camillo Canclini, Franz Duerrenberger, PhD and Vania Manolova, PhD Vifor Pharma Ltd. Presented By: Naja Nyffenegger, PhD
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Poster #LB22 GINGER NANOPARTICLE–DERIVED LIPID VECTOR SIRNA DELIVERY SYSTEM TARGETING INTESTINAL DIVALENT METAL–ION TRANSPORTER 1 (DMT1) AS A NOVEL TREATMENT FOR HEREDITARY HEMOCHROMATOSIS Xiaoyu Wang¹, Mingzhen Zhang, PhD², Didier Merlin, PhD² and James Collins, PhD¹ ¹University of Florida; ²Georgia State University Presented By: Xiaoyu Wang Poster #LB23 LOSS OF LIVER SPECIFIC IRON CHAPERONE PCBP1 LEADS TO STEATOSIS AND HYPERLIPIDEMIA Shyamalagauri Jadhav, Olga Protchenko, Fengmin Li, Oksana Gavrilova, James Cox, Howard Smith, Minoo Shakoury– Elizeh and Caroline Philpott National Institute of Diabetes and Digestive and Kidney Diseases Presented By: Shyamalagauri Jadhav Poster #LB24 THE HEPCIDIN KNOCKOUT RAT: A NOVEL MODEL OF JUVENILE HEREDITARY HEMOCHROMATOSIS IN HUMANS Shireen Flores, MS, Xiaoyu Wang, MS, Tao Wang, Jungheun Ha, PhD, Caglar Doguer, PhD and James Collins, PhD University of Florida Presented By: Shireen Flores, MS Poster #LB25 PLUTONIUM–TRANSFERRIN BINDING MONITORED BY CE– ICP– MS Robert Evans, BA, PhD¹, Sandrine Sauge– Merle²,³,⁴, David Lemaire²,³,⁴, Catherine Berthomieu²,³,⁴ and Jean Aupiais²,⁵,⁶ ¹Brunel University; ²C A; ³CNRS; ⁴Ai – Marseille University; ⁵DAM; ⁶DIF Presented By: Robert William Evans, BA, PhD
TUESDAY, MAY 9, 2017
OVERVIEW
7:00 – 14:00 Registration/Information Desk Open Location: Centennial Hall Prefunction, Level One 8:00 – 13:00 Exhibit Hall Open Location: Centennial Hall Prefunction, Level One GENERAL SESSION
8:00 – 9:30 Plenary Session II: Unmet Clinical Needs in Iron Deficiency Chairs: Sigismond Lasocki, MD, PhD CHU Angers France Igor Theurl, MD University of Innsbruck Austria 8:00 – 8:30 Patient Blood Management Programs Speaker: Axel Hofmann, ME University Hospital Zurich Switzerland 8:30 – 9:00 Anemia of the Elderly Speaker: Domenico Girelli, MD, PhD University of Verona Italy 9:00 – 9:30 Iron Deficiency in Heart Failure Speaker: Lawrence T. Goodnough, MD The Stanford University Medical Center USA 9:30 – 10:00 Panel Discussion: Controversies in Iron Supplementation Chairs: Chaim Hershko, MD The Hebrew University of Jerusalem Israel John Wood, MD, PhD Children's Hospital Los Angeles, Keck School of Medicine USA Panelists: Hossein Ardehali, MD, PhD Northwestern University USA
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Domenico Girelli, MD, PhD University of Verona Italy Lawrence T. Goodnough, MD The Stanford University Medical Center USA Axel Hofmann, ME University Hospital Zurich Switzerland Iain C. Macdougall, MD, FRCP King's College Hospital United Kingdom 10:00 – 10:30 Coffee Break 10:30 – 11:00 Paul Erlich Lecture Chair: Robert E. Fleming, MD St. Louis University School of Medicine USA Iron Chelation: Past, Present and Future Speaker: Robert Hider, BSc, PhD King's College London United Kingdom 11:00 – 12:30 Plenary Session III: 50-Year Anniversary of Iron Chelation Chairs: Ioav Cabantchik, MD, PhD The Hebrew University of Jerusalem Israel John B. Porter, MD, FRCP University College London United Kingdom 11:00 – 11:30 Iron Metabolism in the CNS: Implication for Neurodegenerative Diseases Speaker: Tracey Rouault, MD National Institutes of Health USA 11:30 – 12:00 Molecular Mechanisms of Chelation Therapy Speaker: Ashley Bush, MD, PhD The University of Melbourne Florey Institute of Neuroscience and Mental Health Australia 12:00 – 12:30 Chelation Therapy in Neurodegenerative Disease: Clinical Evidence Speaker: David Devos, MD, PhD Lille Nord De France University France 12:30 – 13:00 Business Meeting (IBIS Members) WEDNESDAY, MAY 10, 2017
OVERVIEW
7:00 – 18:15 Registration/Information Desk Open Location: Centennial Hall Prefunction, Level One 7:30 – 15:00 Exhibit Hall Open Location: Centennial Hall Prefunction, Level One GENERAL SESSION
8:00 – 9:00 Meet the Experts I: Animal Models in Iron Research Location: Exploration, Level Two Moderator: Laura Silvestri, PhD San Raffaele Scientific Institute & Vita– Salute University Italy *Students/Trainees ONLY – Space is limited
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9:00 – 10:30 Plenary Session IV: Iron and Anemia in Chronic Kidney Disease Chairs: Jodie Babitt, MD Massachusetts General Hospital USA Marie-Paule Roth, MD, PhD Université Toulouse Paul Sabatier France 9:00 – 9:30 Pathogenesis of Anemia in CKD Speaker: Iain C. Macdougall, MD, FRCP King's College Hospital United Kingdom 9:30 – 10:00 Emerging Therapies for CKD Anemia Speaker: Volker H. Haase, MD Vanderbilt University USA 10:00 – 10:30 FGF23 in CKD: The Role of Iron and Anemia Speaker: Myles S. Wolf, MD, MMSc Duke University School of Medicine USA 10:30 – 11:00 Coffee Break 11:00 – 12:30 Plenary Session V: Iron Sensor and Chaperones Chairs: Caroline Enns, PhD Oregon Health Sciences University USA Francesca Carlomagno, PhD University of Naples Federico II Italy 11:00 – 11:30 Ferritinophagy: The Mechanism and Biological Role Speaker: Joseph D. Mancias, MD, PhD Dana-Farber Cancer Institute, Harvard Medical School USA 11:30 – 12:00 FBXL5: Sensing Intracellular Iron and Oxygen Speaker: Richard K. Bruick, PhD UTSW Medical Center USA 12:00 – 12:30 Biological Role of PCBPs, the Intracellular Iron Chaperones Speaker: Caroline Philpott, MD Liver Disease Section, NIDDK USA 12:30 – 14:00 Lunch Location: Centennial Terrace, Level Three 14:00 – 15:30 Concurrent Session V: Iron, Infection and Inflammation Location: Centennial Hall AB, Level One Chairs: Zoubida Karim, PhD INSERM France Guenter Weiss, MD Medical University of Innsbruck Austria 14:00 – 14:25 Iron and Hepcidin in Infectious Disease and Adaptive Immunity: Emerging Principles Speaker: Alexander H. Drakesmith, PhD Weatherall Institute of Molecular Medicine, Univesity of Oxford United Kingdom 14:25 – 14:50 TfR1 Mutation Causes Combined Immunodeficiency Speaker: Janet Chou, MD Boston Children's Hospital, Harvard Medical School USA
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14:50 – 15:05 #20 HEPCIDIN MEDIATES HOST DEFENSE AGAINST GRAM–NEGATIVE PATHOGENS BY CONTROLLING NON–TRANSFERRIN–BOUND IRON Deborah Stefanova1, Antoan Raychev1, Joao Arezes2, Kathryn Michels3, Barbara Dillon1, Marcus Horwitz1, Borna Mehrad3, Tomas Ganz1, Elizabeta Nemeth1 and Yonca Bulut1 1University of California, Los Angeles, 2University of Oxford, 3University of Virginia Presented By: D. Stefanova 15:05 – 15:20 #21 EPIDERMAL HEPCIDIN IS PROTECTIVE AGAINST GROUP A STREPTOCOCCUS INFECTION AND SYSTEMIC BACTERIAL SPREAD Mariangela Malerba, PhD student, Jacques Mathieu, Sabine Louis, Sylvain Cuvellier and Carole Peyssonnaux INSERM, U1016, CNRS UMR8104, Université Paris Descartes, Sorbonne Paris Cité, Institut Cochin Presented By: Mariangela Malerba 15:20 – 15:30 #22 A NOVEL IRON–MEDIATED MECHANISM FOR DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE Shirly Moshe-Belisowski, PhD, Lulu Fahoum, MSc, Lena Lifshitz, PhD, Abraham Nyska, PhD, Avi Zuckerman, PhD, Matti Waterman, MD, Roni Weisshof, MD, Orly Savion, PhD, Fabio Cominelli, MD PhD and Esther G. Meyron-Holtz Technion -Israel Institute of Technology Presented By: Esther G. Meyron-Holtz
14:00 – 15:30 Concurrent Session VI: Iron and Cancer Location: Centennial Hall CD, Level One Chairs: Sijin Liu, PhD Chinese Academy of Sciences China Suzy Torti, PhD University of Connecticut Health Center USA 14:00 – 14:15 #23 BREAST CANCER TUMORIGENICITY IS DEPENDENT ON HIGH EXPRESSION LEVELS OF NAF– 1 AND THE LABILITY OF ITS FE– S CLUSTERS Rachel Nechushtai¹, Merav Darash-Yahana, PhD¹, Ola Karmi¹, Y– S Sohn, PhD¹, Eli Pikarsky, MD/PhD², Ioav Cabantchik, MD/PhD¹ and Ron Mittler, PhD³ ¹The Hebrew University of Jerusalem; ²Hadassah; ³University of North Texas Presented By: Rachel Nechushtai 14:15 – 14:30 #24 LOSS OF LOCAL HEPCIDIN DECREASES TUMORIGENESIS IN COLORECTAL CANCER Andrew Schwartz, MS¹, Samira Lakhal-Littleton, DPhil², Peter Robbins, DPhil² and Yatrik Shah, PhD¹ ¹Molecular and Integrative Physiology, University of Michigan; ²Department of Physiology, Anatomy and Genetics, University of Oxford Presented By: Andrew Schwartz 14:30 – 14:45 #25 DYSREGULATION OF IRON METABOLISM IN CHOLANGIOCARCINOMA STEM– LIKE CELLS Stefania Recalcati, MD/PhD¹, Chiara Raggi, PhD², Elena Gammella, PhD¹, Margherita Correnti, PhD², Paolo Buratti, PhD¹, Elisa Forti, PhD², Jasper Andersen, PhD³, Gianfranco Alpini, MD/PhD4, Domenico Alvaro, MD/PhD5, Pietro Invernizzi, MD/PhD6 and Gaetano Cairo, PhD¹ ¹University of Milan; ²Humanitas Clinical and Research Center, Rozzano, Italy; ³University of Copenhagen, Denmark; 4A&M Health Science Center, Texas; 5Sapienza University of Rome, Italy; 6University of Milan Bicocca Presented By: Gaetano Cairo 14:45 – 15:00 #26 A NOVEL GALLIUM COMPOUND DISRUPTS TUMOR IRON HOMEOSTASIS AND RETARDS THE GROWTH OF GLIOBLASTOMA IN VIVO BY INHIBITING THE IRON– DEPENDENT M2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE (RRM2) Christopher Chitambar, MD, Mona Al-Gizawiy, PhD, Andrew Lozen, MD, Kimberly Pechman, PhD, Janine Wereley, BS, Robert Wujek, BS, Paul Clark, PhD, John Kuo, MD, PhD, Howard Elford, PhD, Bo Hedlund, PhD, Devashish Joshi, BS, William Antholine, PhD and Kathleen Schmainda, PhD Medical College of Wisconsin Presented By: Christopher Chitambar 15:00 – 15:15 #27 FERROPTOSIS: A NEW POTENTIAL THERAPEUTIC TARGET FOR AGGRESSIVE TUMORS. A PRELIMINARY STUDY ON HELA, HEPG2 AND RHABDOMYOSARCOMA CELL LINES Michela Asperti, PhD, Silvia Codenotti, PhD Student, Magdalena Gryzik, PhD Student, Paolo Crotti, Student, Maria Regoni, PhD Student, Paolo Arosio, PhD, Alessandro Fanzani, PhD and Maura Poli, PhD University Of Brescia, Italy Presented By: Michela Asperti
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15:15 – 15:30 #28 IMPACT OF HEMOCHROMATOSIS ON COLON: INCREASED SUSCEPTIBILITY TO INFLAMMATION AND CARCINOGENESIS Bojana Ristic, BS in Chemistry, Sathish Sivaprakasam, PhD, Sabarish Ramachandran, PhD and Vadivel Ganapathy, PhD Texas Tech University Health Sciences Center Presented By: Bojana Ristic 15:30 – 15:45 Break 15:45 – 17:15 Featured Podium Presentations Location: Centennial Hall AB, Level One Chair: Robert E. Fleming, MD St. Louis University School of Medicine USA Podium #1 SMAD1/5 IS REQUIRED FOR ERYTHROPOIETIN SUPPRESSION OF HEPCIDIN IN MICE (15:45 – 15:55) Chia-Yu Wang, Amanda B. Core, Kimberly B. Zumbrennen– Bullough, Susanna Canali, Sinan Ozer, Lieve Umans, An Zwijsen and Jodie L. Babitt Massachusetts General Hospital, Harvard Medical School Presented By: Chia-Yu Wang Podium #2 LIVER HFE PROTEIN CONTENT IN IRON DEFICIENT MICE (15:55 – 16:05) Jan Krijt¹, Jana Frýdlová², Iuliia Gurieva² and Martin Vokurka² ¹Institute of Pathological Physiology, First Faculty of Medicine, Charles University; ²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic Presented By: Jan Krijt Podium #3 EVIDENCE THAT HEPCIDIN OCCLUDES FERROPORTIN TO INHIBIT IRON EXPORT (16:05 – 16:15) Sharraya Aschemeyer, Bo Qiao, Erika Valore, Tomas Ganz and Elizabeta Nemeth University of California, Los Angeles Presented By: S. Aschemeyer Podium #4 CALCIUM ACTIVATES FERROPORTIN– MEDIATED CELLULAR IRON EFFLUX (16:15 – 16:25) Bryan Mackenzie¹, T. Alex Ruwe¹, Chandrika N. Deshpande², Reiya Taniguchi³, Vicky Xin², Ali Shawki¹, Kyle R. Vieth¹, Bo Qiao4, Erika V. Valore4, Osamu Nureki³, Tomas Ganz4, Elizabeta Nemeth4 and Mika Jormakka² ¹University of Cincinnati College of Medicine; ²Centenary Institute-Sydney; ³RIKEN– Saitama; 4David Geffen School of Medicine at UCLA Presented By: Bryan Mackenzie Podium #5 AGE RELATED IRON ACCUMULATION: ACCUMULATING WHAT EXACTLY? (16:25 – 16:35) Simon James, PhD¹, Dominic Hare, PhD¹, Martin de Jonge, PhD² and Gawain McColl, PhD¹ ¹Florey Institute of Neuroscience and Mental Health; ²Australian Synchrotron, Australia Presented By: Simon James Podium #6 RECOVERY OF HEME OXYGENASE 1– DEFICIENT MICE THROUGH REPOPULATION OF WT (16:35 – 16:45) MACROPHAGES IN RETICULOENDOTHEILAL SYSTEM Ki Soon Kim and Tracey A Rouault, PhD NIH Presented By: Ki Soon Kim Podium #7 DEVELOPING A GALNAC– CONJUGATED TMPRSS6 ANTISENSE THERAPY FOR THE (16:45 – 16:55) TREATMENT OF ß–THALASSEMIA Shuling Guo, PhD¹, Mariam Aghajan, PhD¹, Tom Zanardi, PhD¹, Rudy Gunawan, PhD¹, Carla Casu, PhD², Vania Presti², Sheri Booten¹, Stefano Rivella, PhD² and Brett Monia, PhD¹ ¹Ionis Pharmaceuticals; ²Children’s Hospital of Philadelphia Presented By: Shuling Guo Podium #8 HEME ACCUMULATION IN ENDOTHELIAL CELLS IMPAIRS ANGIOGENESIS BY TRIGGERING (16:55 – 17:05) PARAPTOSIS Sara Petrillo¹, Deborah Chiabrando, PhD¹, Tullio Genova, PhD¹, Veronica Fiorito, PhD¹, Giada Ingoglia, PhD¹, Francesca Vinchi, PhD², Federico Mussano³, Stefano Carossa³, Lorenzo Silengo¹, Fiorella Altruda¹, Giorgio Merlo¹, Luca Munaron¹ and Emanuela Tolosano¹ ¹University of Turin; ²Univerity of Heidelberg; ³University of Turin, Dental School Presented By: Sara Petrillo Podium #9 TWO SIDES OF THE SAME COIN: A FRESH PERSPECTIVE ON THE ROLE OF TNFα IN (17:05 – 17:15) INFLAMMATION, IRON METABOLISM AND HEMATOPOIESIS Vania Lo Presti, MSc, Carla Casu, PhD, Rea Paraskevi Oikonomidou, MD, Emir O'Hara, BSc, Sara Gardenghi, PhD and Stefano Rivella, PhD Children's Hospital of Philadelphia Presented By: Vania Lo Presti
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17:15 – 19:00 Poster Session II Location: South Meeting Rooms, Level Two Refreshments available. Beer and wine service to begin at 17:30. Poster #1 SMAD1/5 IS REQUIRED FOR ERYTHROPOIETIN SUPPRESSION OF HEPCIDIN IN MICE Chia– Yu Wang, Amanda B. Core, Kimberly B. Zumbrennen– Bullough, Susanna Canali, Sinan Ozer, Lieve Umans, An Zwijsen and Jodie L. Babitt Massachusetts General Hospital, Harvard Medical School Presented By: Chia– Yu Wang Poster #2 LIVER HFE PROTEIN CONTENT IN IRON DEFICIENT MICE Jan Krijt¹, Jana Frýdlová², Iuliia Gurieva² and Martin Vokurka² ¹Institute of Pathological Physiology, First Faculty of Medicine, Charles University; ²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic Presented By: Jan Krijt Poster #3 EVIDENCE THAT HEPCIDIN OCCLUDES FERROPORTIN TO INHIBIT IRON EXPORT S. Aschemeyer, B. Qiao, E. Valore, T. Ganz and E. Nemeth University of California, Los Angeles Presented By: S. Aschemeyer Poster #4 CALCIUM ACTIVATES FERROPORTIN–MEDIATED CELLULAR IRON EFFLUX Bryan Mackenzie¹, T. Alex Ruwe¹, Chandrika N. Deshpande², Reiya Taniguchi³, Vicky Xin², Ali Shawki¹, Kyle R. Vieth¹, Bo Qiao4, Erika V. Valore4, Osamu Nureki³, Tomas Ganz4, Elizabeta Nemeth4 and Mika Jormakka² ¹University of Cincinnati College of Medicine; ²Centenary Institute– Sydney; ³RIKEN– Saitama; 4David Geffen School of Medicine at UCLA Presented By: Bryan Mackenzie Poster #5 AGE RELATED IRON ACCUMULATION: ACCUMULATING WHAT EXACTLY? Simon James, PhD¹, Dominic Hare, PhD¹, Martin de Jonge, PhD² and Gawain McColl, PhD¹ ¹Florey Institute of Neuroscience and Mental Health; ²Australian Synchrotron Presented By: Simon James Poster #6 RECOVERY OF HEME OXYGENASE 1– DEFICIENT MICE THROUGH REPOPULATION OF WT MACROPHAGES IN RETICULOENDOTHEILAL SYSTEM Ki Soon Kim and Tracey A Rouault, PhD NIH Presented By: Ki Soon Kim Poster #7 DEVELOPING A GALNAC–CONJUGATED TMPRSS6 ANTISENSE THERAPY FOR THE TREATMENT OF ß–THALASSEMIA Shuling Guo, PhD¹, Mariam Aghajan, PhD¹, Tom Zanardi, PhD¹, Rudy Gunawan, PhD¹, Carla Casu, PhD², Vania Presti², Sheri Booten¹, Stefano Rivella, PhD² and Brett Monia, PhD¹ ¹Ionis Pharmaceuticals; ²Children’s Hospital of Philadelphia Presented By: Shuling Guo Poster #8 HEME ACCUMULATION IN ENDOTHELIAL CELLS IMPAIRS ANGIOGENESIS BY TRIGGERING PARAPTOSIS Sara Petrillo¹, Deborah Chiabrando, PhD¹, Tullio Genova, PhD¹, Veronica Fiorito, PhD¹, Giada Ingoglia, PhD¹, Francesca Vinchi, PhD², Federico Mussano³, Stefano Carossa³, Lorenzo Silengo¹, Fiorella Altruda¹, Giorgio Merlo¹, Luca Munaron¹ and Emanuela Tolosano¹ ¹University of Turin; ²Univerity of Heidelberg; ³University of Turin, Dental School Presented By: Sara Petrillo Poster #9 TWO SIDES OF THE SAME COIN: A FRESH PERSPECTIVE ON THE ROLE OF TNFα IN INFLAMMATION, IRON METABOLISM AND HEMATOPOIESIS Vania Lo Presti, MSc, Carla Casu, PhD, Rea Paraskevi Oikonomidou, MD, Emir O'Hara, BSc, Sara Gardenghi, PhD and Stefano Rivella, PhD Children's Hospital of Philadelphia Presented By: Vania Lo Presti Poster #115 LOW HEPCIDIN LEVELS CAN COMPENSATE FOR THE LACK OF A FERROPORTIN ALLELE Sandro Altamura, PhD¹,⁴, Julia Schmidt¹,⁴, Katja Muedder¹,⁴, Bruno Galy², Matthias Hent e³,⁴ and Martina Muckenthaler¹,⁴ ¹Department of Pediatric Hematology, Oncology and Immunology – University of Heidelberg, Heidelberg, Germany; ²Division of Virus– associated Carcinogenesis, German Cancer Research Centre , Heidelberg, Germany; ³European Molecular Biology Laboratory, Heidelberg, Germany; ⁴Molecular Medicine Partnership Unit, Heidelberg, Germany Presented By: Sandro Altamura, PhD
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Poster #116 LIPOSOME ENCAPSULATED DEFEROXAMINE (LDFO) HAS A HIGH MOLAR EFFICIENCY IN REMOVING IRON IN AN IRON–OVERLOADED MOUSE MODEL Mark Hayes, PhD¹, David Tran, PhD², Max Petersen, BSc², Charles Noble, PhD², Zhipeng Dai, PhD², Peter Working, PhD² and Francis Szoka, PhD³ ¹ZoneOne Pharma Inc; ²ZoneOne Pharma, Inc; ³ZoneOne Pharma, Inc and UCSF Dept. Bioengineering and Therapeutics Presented By: Charles Noble, BSc, PhD Poster #117 HUMAN MACROPHAGE FERROPORTIN BIOLOGY AND THE BASIS FOR THE FERROPORTIN DISEASE Manuela Sabelli, Giuliana Montosi, Cinzia Garuti, Angela Caleffi, Stefania Oliveto, Stefano Biffo and Antonello Pietrangelo Division of Internal Medicine 2 and Center for Hemochromatosis, University Hospital of Modena, Italy. Presented By: Antonello Pietrangelo, MD, PhD Poster #118 ACERULOPLASMINEMIA: EXPERIENCES AT THE VENETO REGION REFERRAL CENTER FOR IRON DISORDERS Chiara Piubelli, PhD¹,², Annalisa Castagna, PhD, Giacomo Marchi, MD, Monica Rizzi, MD, Fabiana Busti, MD, Paola Capelli, MD, Roberto Pozzi-Mucelli, MD, Massimiliano Calabrese, MD, Alice Vianello, MD, Luciano Xumerle, BSc, Massimo Delledonne, PhD, Oliviero Olivieri, MD and Domenico Girelli, MD, PhD ¹Veneto Region Referral Center for Iron Disorders, Azienda Ospedaliera Universitaria Integrata; ²Department of Medicine, Section of Internal Medicine, University of Verona Verona Italy Presented By: Chiara Piubelli, PhD Poster #119 NON– HFE HAEMOCHROMATOSIS: A PHENOTYPIC AND CLINICAL ANALYSIS OF PUBLISHED CASES AND COMPARISON WITH HFE– HAEMOCHROMATOSIS Daniel Wallace, BSc, PhD¹, Kam Sandhu², Kaledas Flintoff², Jeannette Dixon², Louise Ramm², Grant Ramm², Lawrie Powell² and V. Nathan Subramaniam¹ ¹Queensland University of Technology; ²QIMR Berghofer Medical Research Institute Presented By: Daniel Frederick Wallace, PhD, BSc (hons) Poster #120 INDEPENDENT ASSOCIATION OF GNPAT p.D519G WITH MARKEDLY INCREASED IRON STORES IN HFE p.C282Y HOMOZYGOTES James Barton, MD², Wen– Pin Chen, MS³, Mary mond, PhD⁴, Pradyumna Phatak, MD⁵, V. Nathan Subramaniam, PhD⁶, Paul Adams, MD⁷, Lyle Gurrin, PhD⁸, Gregory Anderson, PhD⁹, Grant Ramm, PhD⁹, La rie Po ell, MD, PhD⁹, Katrina Allen, FRACP, PhD¹ , John Phillips, PhD¹¹, Charles Parker, MD¹², Gordon McLaren, MD¹ and Christine McLaren, PhD³ ¹VA Long Beach Healthcare System and University of California, Irvine; ²Southern Iron Disorders Center; ³University of California, Irvine; ⁴University of Washington; ⁵Rochester General Hospital; ⁶QIMR Berghofer Medical Research Institute and The University of Queensland; ⁷London Health Sciences Centre; ⁸The University of Melbourne; ⁹QIMR Berghofer Medical Research Institute; ¹ Murdoch Childrens Research Institute; ¹¹University of Utah; ¹²University of Utah School of Medicine Presented By: Gordon McLaren, MD Poster #121 ANY GAIN OF 300 ALTITUDE METERS INCREASES HEMOGLOBIN LEVELS IN YOUNG MEN LIVING AT LOW ALTITUDE IN SWITZERLAND Kaspar Staub, Dr¹, Martin Häusler, Dr¹, Martina Muckenthaler, Prof Dr², Frank Rühli, Prof Dr¹ and Max Gassmann¹ ¹University of Zurich; ²University of Heidelberg Presented By: Max Gassmann, DVM Poster #122 IRON OVERLOAD ENHANCED CELL DEATH INDUCED BY CARCINOGENIC FIBROUS MATERIALS Fumiya Ito, Master¹, Lei Shi, MD² and Shinya Toyokuni, MD, PhD² ¹Nagoya University Graduate School of Medicine; ²Nagoya University Graduate School of Medicine Department of Pathology and Biological Responses Presented By: Fumiya Ito, MA Poster #123 TOLL–LIKE RECEPTORS AND REGULATION OF SYSTEMIC IRON HOMEOSTASIS Oriana Marques, MSc, PhD Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg Presented By: Oriana Marques, MSc, PhD Poster #124 HEMOJUVELIN IS CRITICAL FOR INFLAMMATORY SIGNALING TO HEPCIDIN Nicole Wilkinson, PhD, Carine Fillebeen, PhD, John Wagner, MSc and Kostas Pantopoulos, PhD Lady Davis Institute for Medical Research/McGill University Presented By: Kostas Pantopoulos, PhD Poster #125 INSIGHT INTO THE MECHANISM OF HEME EXTRACTION FROM HUMAN HEMOGLOBIN BY THE ISDH RECEPTOR FROM STAPHYLOCOCCUS AUREUS Ramsay Macdonald¹, Megan Sjodt, PhD¹, Joanna Marshall, BS¹, John Olson, PhD², David Gell, PhD³ and Robert Clubb, PhD¹ ¹UCLA; ²Rice University; ³University of Tasmania Presented By: Ramsay Macdonald
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Poster #126 IRON SUPPLEMENTS MODULATE COLON MICROBIOTA COMPOSITION AND DEXTRAN SODIUM SULFATE–INDUCED COLITIS Marco Constante, Gabriela Fragoso, PhD, Joseph Lupien-Meilleur, MSc, Annie Calve, MSc and Manuela M. Santos, PhD CRCHUM Presented By: Marco Constante, BSc Poster #127 HEMIN AND ALTERED IRON METABOLISM IN SICKLE CELL DISEASE MODULATE HIV– 1 RESTRICTION FACTORS Namita Kumari¹, Miguel Mulder, PhD², Douglas Nixon, PhD² and Sergei Nekhai, PhD¹ ¹Howard University; ²George Washington University Presented By: Namita Kumari Poster #128 ROLE OF EPIGENETIC REGULATION OF IRON HOMEOSTASIS GENES AND NEURODEGENERATION Steinunn Sara Helgudottir¹, Jacek Lichota, PhD² and Torben Moos, Professor, PhD, MD² ¹Health Science and Technology, Aalborg University; ²Aalborg University, Denmark Presented By: Steinunn Sara Helgudottir Poster #129 EFFECTS OF PARENTAL IRON THERAPY TO PREGNANT RATS WITH SYSTEMIC IRON DEFICIENCY WITHOUT ANEMIA ON CEREBRAL IRON STATUS AT BIRTH AND IN ADULTHOOD Jacek Lichota¹, Annette Burkhart, PhD², Tina Skjørringe, PhD², Lisa Juul Routhe, MSc², Lars Lykke Thomsen, PhD, MD³ and Torben Moos, Professor, PhD, MD² ¹Health Science and Technology, Aalborg University; ²Aalborg University, Denmark; ³Pharmacosmos, Denmark Presented By: Jacek Lichota Poster #130 INFLAMMATION– DRIVEN DISRUPTION OF IRON HOMEOSTASIS IN PARKINSON’S DISEASE Ana Catarina Vaz Carreto Martins, MSc², Susana Ramos, PhD³, Miguel P. Soares, PhD³ and Raffaella Gozzelino, PhD¹ ¹Chronic Diseases Research Center /NOVA Medical School; ²CEDOC/NOVA Medical School; ³Instituto Gulbenkian de Ciência Presented By: Raffaella Gozzelino, PhD Poster #131 RESTING STATE MRI BRAIN DIFFERENCES IN HFE AND TRANSFERRIN MUTATION CARRIERS Mark Meadowcroft, PhD¹, Jianli Wang, MD, PhD², Carson Purnell, BS², Paul Eslinger, PhD², Elizabeth Neely, BS², Qing Yang, PhD² and James Connor, PhD² ¹The Pennsylvania State University – College of Medicine; ²The Pennsylvania State University – College of Medicine Presented By: Mark Meadowcroft, PhD Poster #132 IRON PROMOTES SENESCENCE IN FIBROBLASTS AND IPS–DERIVED NEURONS FROM NEUROFERRITINOPATHY PATIENTS Anna Cozzi, PhD², Daniel I. Orellana, PhD³, Paolo Santambrogio, PhD³, Alicia Rubio, PhD³, Angelo Iannielli, PhD³, Chiara Fiorillo, MD⁴, Gian Luca Forni, MD⁵, Vania Broccoli, PhD⁶ and Sonia Levi, PhD¹ ¹Vita–Salute San Raffaele University; ²San Raffaele Scientific Institute, Milano, Italy.; ³San Raffaele Scientific Institute Milano, Italy.; ⁴University of Genoa, Genoa, Italy.; ⁵ .O. Ospedali Galliera, Genova, Italy.; ⁶CNR–Institute of Neuroscience, Milano, Italy Presented By: Sonia Levi, PhD Poster #134 PATHOLOGICAL RELATIONSHIPS INVOLVING IRON AND MYELIN MAY CONSTITUTE A SHARED MECHANISM LINKING VARIOUS RARE AND COMMON BRAIN DISEASES Moones Heidari, PhD², Daniel Johnstone, Brianna Bassett, Conceição Bettencourt, Joanna Collingwood, Sam Gerami, Michael House, Kristy Martin, Anita Chua, Mina Ryten, Henry Houlden, John Olynyk, Debbie Trinder and Elizabeth Milward¹ ¹The University of Newcastle; ²University of Newcastle Presented By: Elizabeth Milward Poster #135 TFR2 KI MICE PRESENT AN ENRICHMENT IN CARDIAC FERRITIN L THAT CONFERS CARDIOPROTECTION AGAINST I/R INJURIES Martina Boero, Biologist¹, Rosa Maria Pellegrino, PhD², Mariarosa Mezzanotte, Dr², Antonietta Palmieri, Dr², Giuseppe Saglio, MD² and Antonella Roetto, PhD² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Sciences, AOU San Luigi Gonzaga, Torino, Italy Presented By: Martina Boero Poster #136 DMT1, ZIP14, AND ZIP8 ARE DISPENSIBLE FOR IRON LOADING OF THE HEART Chia-Yu Wang, PhD, Supak Jenkitkasemwong, PhD and Mitchell Knutson, PhD University of Florida, USA Presented By: Mitchell Knutson, PhD
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Poster #137 CARDIAC IRON BURDEN IS CORRELATED WITH INNATE IMMUNE MARKERS, BODY MASS INDEX AND LABILE PLASMA IRON IN ßETA–THALASSEMIA MAJOR PATIENTS UNDERGOING COMBINED CHELATION THERAPY Nicholas Slater, BSc², Marcela Weyhmiller, PhD¹, Patricia Evans, PhD³, John Porter, MD³, Annie Higa, BS¹, Vivian Ng, BS¹, Nancy Sweeters, PNP¹, Jackson Price, BSc², Alisha Manji, BSc², David Killilea, PhD¹, Lynne Neumayer, MD¹, Ashutosh Lal, MD¹, Paul Harmatz, MD¹, Elliott Vichinsky, MD¹ and Patrick Walter, PhD¹ ¹UCSF Benioff Children's Hospital Oakland; ²Biology, University of Victoria; ³University College London Presented By: Patrick Walter, PhD Poster #138 RENAL HANDLING OF NON–TRANSFERRIN BOUDN IRON AND ASSOCIATED INJURY IN PROXIMAL TUBULAR EPITHELIAL CELLS Sanne van Raaij¹, Rosalinde Masereeuw, PhD², Dorine Swinkels, PhD¹ and Rachel van Swelm, PhD¹ ¹Radboud University Medical Center, Nijmegen, The Netherlands; ²Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands Presented By: Sanne van Raaij Poster #139 INCREASED IRON ACCUMULATION AND UPREGULATED HEPCIDIN EXPRESSION IN KIDNEYS OF SICKLE CELL DISEASE MICE Marina Jerebtsova, PhD¹, Guelaguetza Vazquez– Meves, PhD¹, Namita Kumari, PhD¹, Nowah Afangbedji, BS¹, Alfia Khaibullina, PhD², Zena Quezado, MD² and Sergei Nekhai, PhD¹ ¹Howard University; ²Sheikh Zayed Institute, Children’s National Health System Presented By: Sergei Nekhai, PhD Poster #140 ENHANCED MEDULLARY IRON TRAFFICKING MAY EXPLAIN RESISTANCE OF KIDNEY TO IRON OVERLOAD INJURY Avital Weiss, PhD, Lior Spector, MSc, Inbar Gold, MSc, Lena Lifshitz, PhD, Abraham Nyska, PhD, Sefi Addadi, PhD, Diana Goldberg, Yael Haimovich, MSc, Tracey A. Rouault, MD and Esther G. Meyron-Holtz Technion –Israel Institute of Technology Presented By: Esther Meyron-Holtz, PhD Poster #141 ELEVATIONS IN FGF23 PRECEDE DISRUPTIONS IN EITHER PHOSPHATE OR IRON HOMEOSTASIS IN THE EBF1– KO MODEL OF RENAL INSUFFICIENCY Sarah Jenkins¹, Xuiqi Li, BS¹, Tracy Nelson², Karin Finberg, MD, PhD¹ and Jackie Fretz, PhD¹ ¹Yale School of Medicine; ²Yale School of Medicne Presented By: Jackie Fretz, PhD Poster #143 COMPARISON OF MATERNAL AND FETAL IRON REGULATORS IN IRON DEFICIENCY ANEMIA OF PREGNANCY Sreenithi Santhakumar, MTech¹, Rekha Athiyarath, PhD¹, Anne George Cherian, MD², Vinod Joseph Abraham, MD² and Eunice Sindhuvi Edison, PhD¹ ¹Department of Haematology, Christian Medical College, Vellore, India; ²Department of Community Health, Christian Medical College, Vellore, India Presented By: Eunice Sindhuvi Edison, PhD Poster #144 A NOVEL IN VITRO MODEL OF SIMULATED TWICE A DAY IRON SUPPLEMENTATION FOR ASSESSMENT OF IRON UPTAKE AND CELLULAR DAMAGE Petia Apostolova¹, Voni Blesia, MSc², Christelle Tossou, MPharm², Derek Renshaw, MSc, PhD³, Sebastien Farnaud, MSc, PhD³, Robert vans, BA, PhD⁴ and M.Gulrez Zariwala, BPharm, MSc, PhD² ¹University of Westminster; ²Faculty of Science & Technology / University of Westminster; ³Centre for Applied Biological and ercise Sciences Coventry University; ⁴Centre For lectronic Systems Research Brunel University Presented By: Petia Apostolova Poster #145 IRONWOMAN TRIAL: THE IMPACT OF INTRAVENOUS IRON ON EXERCISE PERFORMANCE IN IRON DEFICIENT, EXERCISING WOMEN Georgie Bruinvels⁵, Charles Pedlar, PhD¹, Richard Burden, PhD², Nicola Bro n, PhD³, Anna Butcher, MD⁴, Marisa Chau, BSc⁴ and Toby Richards, MD⁴ ¹Massachusetts General Hospital, St Mary's University, Orreco Ltd; ²St Mary's University, Orreco Ltd, English Institute of Sport; ³St Mary s University; ⁴University College London; ⁵UCL Presented By: Georgie Bruinvels Poster #146 THE IMPACT OF HEAVY MENSTRUAL BLEEDING AND IRON STATUS ON FATIGUE IN MENSTRUATING WOMEN Georgie Bruinvels¹, Charles Pedlar, PhD², Richard Burden, PhD³, Anna Butcher, MD⁴, Marisa Chau, BSc⁴, Timothy Cush ay⁵ and Toby Richards, MD⁴ ¹UCL; ²Massachusetts General Hospital, St Mary's University, Orreco Ltd.; ³St Mary's University, Orreco Ltd., nglish Institute of Sport; ⁴University College London; ⁵Vifor Pharma Presented By: Georgie Bruinvels
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Poster #147 EFFECTS OF THE IRON CHELATOR CURCUMIN IN THE DSS–INDUCED COLITIS MOUSE MODEL Macha Samba Mondonga, MSc, Marco Constante, PhD, Gabriela Fragoso, PhD and Manuela M. Santos, PhD CRCHUM, Universite de Montreal Presented By: Macha Samba Mondonga, MSc Poster #148 OSTEOMALACIA AFTER TREATMENT WITH INTRAVENOUS IRON Benedikt Schaefer, MD¹, André Viveiros, MD¹, Irina Nardin², Bernhard Glodny, MD² and Heinz Zoller, MD¹ ¹Medical University of Innsbruck, Department of Medicine II, Gastroenterology and Hepatology; ²Medical University of Innsbruck, Department of Radiology Presented By: Benedikt Schaefer, MD Poster #149 PREDICTORS OF IRON ABSORPTION FROM A NOVEL NANO IRON SUPPLEMENT (IHAT) IN GAMBIAN WOMEN IN THE ABSENCE OF INFLAMMATION Dora Pereira, PhD, MEng¹, Rita Wegmuller, PhD², Carla Cerami, PhD, MD² and Andrew Prentice, PhD² ¹University of Cambridge; ²MRC Unit, The Gambia Presented By: Dora Pereira, PhD, MEng Poster #150 ENTERAL IRON SUPPLEMENTATION INCREASES ERYTHROPOIESIS IN PREWEANLING WILD TYPE AND TMPRSS6 KNOCKOUT MICE Rebecca Verhaeghe, Ramya Bharathi, Nermi Parrow, Princy Prasad and Robert Fleming Saint Louis University School of Medicine Presented By: Rebecca Verhaeghe, DO Poster #151 PANCREATIC IRON IN PATIENTS WITH HEMOCHROMATOSIS: NOT AS RARE AS CARDIAC IRON Jin Yamamura, MD², Roland Fischer, PhD³, Björn Schönnagel, MD², Regine Grosse, MD⁴ and Peter Nielsen, MD, PhD¹ ¹University Medical Centre Hamburg, Dept. of Biochemistry and Molecular Cell Biology; ²University Medical Centre Hamburg, Dept. of Diagnostic and Interventional Radiology; ³UCSF Benioff Children's Hospital and Research Centre Oakland, Dept. of Hematology Oncology; ⁴University Medical Centre Hamburg, Pediatric Hematology/Oncology Presented By: Peter Nielsen, MD, PhD Poster #152 WITHDRAWN Poster #153 HEME AND IRON SHAPE THE PHENOTYPE OF MACROPHAGES IN THE TUMOR MICROENVIRONMENT OF NON–SMALL CELL LUNG CANCER Milene Costa da Silva¹,⁵, Carl Ma imilian Thielmann¹,⁵, Margareta P. Correia², Michael O. Breck oldt³, Michael Meister⁴, Thomas Muley⁴, Adelheid Cer enka² and Martina U. Muckenthaler¹,⁵ ¹Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Germany; ²Innate Immunity Group, German Cancer Research Center , Heidelberg, Germany; ³Department of Neuroradiology, University Hospital Heidelberg, Heidelberg, Germany; ⁴Translational Research Unit, Thora klinik at University Hospital Heidelberg, Heidelberg, Germany; ⁵Molecular Medicine Partnership Unit MMPU , Heidelberg University & EMBL, Heidelberg, Germany Presented By: Milene Costa Da Silva, PhD Poster #154 THE ROLE OF IRON IN THE PROGRESSION OF SOFT TISSUE SARCOMAS Michela Asperti, PhD, Silvia Codenotti, PhD student, Paolo Crotti, student, Paolo Arosio, PhD, Alessandro Fanzani, PhD and Maura Poli, PhD University of Brescia Presented By: Maura Poli, PhD Poster #156 INDUCTION OF IRON EXCESS IN PROSTATE CANCER: A NOVEL POTENTIAL THERAPEUTIC APPROACH Federica Morisi, Jessica Bordini, Gianluca Venturi, Alessia Fulli, Matteo Bellone, Francesco Montorsi, Alberto Briganti, Clara Camaschella and Alessandro Campanella IRCCS Ospedale San Raffaele Presented By: Alessandro Campanella, PhD Poster #157 IRON ADMINISTRATION INCREASES BORTEZOMIB EFFICACY IN MULTIPLE MYELOMA MURINE MODELS Jessica Bordini, Federica Morisi, Alessia Fulli, Gianluca Venturi, Clara Camaschella and Alessandro Campanella Vita–Salute San Raffaele University Presented By: Jessica Bordini
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Poster #158 ELEVATED SYSTEMIC HEME AND IRON LEVELS AS RISK FACTORS FOR VASCULOPATHY AND ATHEROSCLEROSIS: EVIDENCE FROM A ß–THALASSEMIA COHORT STUDY Francesca Vinchi¹, Graca Porto, MD², Gregory M Vercellotti, MD³, John Belcher, PhD³, Richard Sparla, BTA¹, itan Fibach, PhD⁴, Hala Zreid, MD⁵, Rushdy Rasras, MD⁶, Hussam Ghoti, MD⁷, Martina U. Muckenthaler, PhD¹ and lie er A. Rachmile it , MD⁸ ¹Molecular Medicine Partnership Unit , University of Heidelberg & EMBL, Heidelberg, Germany; ²University of Porto, Porto, Portugal; ³University of Minnesota, Minneapolis, USA; ⁴The Hebre University Hadassah Medical Center, Jerusalem, Israel; ⁵AlShifa Hospital, Ga a, Palestine; ⁶ uropean Hospital of Gaza, Gaza, Palestine; ⁷Assuta Medical Center, Tel Aviv, Israel; ⁸The dith Wolfson Medical Center, Holon, Israel Presented By: Francesca Vinchi, PhD Poster #159 IN FIFTY YEARS: A UNIQUE WINDOW ON HEME AND IRON METABOLISM FROM THE HEMOPEXIN SYSTEM Ann Smith, PhD SBS–UMKC Presented By: Ann Smith, BSc, PhD Poster #160 HEPCIDIN AS A PREDICTOR TO TREAT PREOPERATIVE ANEMIA EFFECTIVELY WITH INTRAVENOUS IRON Christina Wittkamp², Lisa Traeger², Ines Ellermann³, Maria Eveslage, Dipl-Stat⁴ and Andrea Steinbicker, MD MPH¹ ¹University Hospital Muenster, Department of Anesthesiology, Intensive Care and Pain Medicine; ²Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Muenster, University of Muenster, Muenster, Germany; ³Department of Pharmacy, University Hospital Muenster, University of Muenster, Muenster, Germany; ⁴Institute of Biostatistics and Clinical Research, University of Muenster, Germany Presented By: Andrea Steinbicker, MD, MPH Poster #161 INVESTIGATION OF MITOCHONDRIAL FUNCTION WITH IRON AND LIPID LOADING IN AML12 CELLS Abhishek Singh¹, Cyril Mamotte, BSc , PhD², Leon Adams, MB BS, PhD WAust, FRACP³ and Ross Graham, PhD, CBiol² ¹Curtin University/ Biomedical Science; ²Curtin University/ School of Biomedical Science and Curtin Health Innovation Research Institute; ³University of Western Australia/ School of Medicine and Pharmacology Presented By: Abhishek Singh Poster #162 UNCOUPLED IRON HOMEOSTASIS IN TYPE 2 DIABETES MELLITUS Sandro Altamura, PhD¹,³, Stefan Kopf², Julia Schmidt¹,³, Ana Rita da Silva¹,³, Peter Nawroth² and Martina Muckenthaler¹,³ ¹Department of Pediatric Hematology, Oncology and Immunology – University of Heidelberg, Heidelberg, Germany; ²Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany; ³Molecular Medicine Partnership Unit, Heidelberg, Germany Presented By: Sandro Altamura, PhD Poster #163 CROSSTALK BETWEEN ACIDOSIS AND IRON METABOLISM: DATA ON HEPCIDIN AND H+/K+ ATPASE Raed Daher¹, Thibaud Lefebvre²,⁵, Nicolas Ducrot², Philippe Lett ron³, Anne Couvelard⁴, Herv Puy²,⁵ and Zoubida Karim² ¹INSERM U1149, Université Paris Diderot, Laboratory of Excellence, GR– Ex; ²INSERM U1149, Université Paris Diderot, Laboratory of Excellence, GR– Ex, Paris, France; ³INSERM U1149, Université Paris Diderot, Paris, France; ⁴INS RM U , Universit Paris Diderot, AP– HP, D partement d anatomo– pathologie, Hôpital Bichât, Paris, France; ⁵AP– HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France Presented By: Raed Daher Poster #164 TRANSFERRIN: A PREDICTOR OF SURVIVAL IN EARLY STAGE LIVER CIRRHOSIS André Viveiros, MD, Armin Finkenstedt, MD, Benedikt Schaefer, MD, Konrad Lehner and Heinz Zoller, MD Medical University Innsbruck Presented By: Andre Viveiros, MD Poster #165 ERYTHROFERRONE DOES NOT CONTRIBUTE TO GLUCOSE HOMEOSTASIS AND DOES NOT AFFECT INSULIN SENSITIVITY IN RESPONSE TO CHRONIC ERYTHROPOIETIN TREATMENT R. Coffey, V. Gabayan, E. Nemeth and T. Ganz University of California, Los Angeles, USA Presented By: Richard Coffey Poster #166 GENOME WIDE ASSOCIATION STUDY (GWAS) OF STEATOSIS DUE TO DIETARY IRON OVERLOAD IN INBRED STRAINS OF MICE Dongyi Lu, Brie Fuqua, Simon Hui, Frode Norheim, Calvin Pan, Sarada Charugundla, Nam Che, Brian Parks, Eleazar Eskin, Chris Vulpe and Aldons Lusis University of California, Los Angeles, USA Presented By: Dongyi Lu
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Poster #167 GENETIC LOSS OF TMPRSS6 MODIFIES THE PHYSIOLOGICAL RESPONSE TO A HIGH FAT DIET Abitha Sukumaran, PhD, Larisa Lozovatsky, MS, Xiuqi Li, BS, Luis Gonzalez, BS and Karin Finberg, MD, PhD Yale School of Medicine, USA Presented By: Karin Finberg, MD, PhD Poster #168 STUDY OF FERRITIN SELF– ASSEMBLY AND HETEROPOLYMER FORMATION BY FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) Fernando Carmona, PhD, Maura Poli, PhD, Magdalena Gryzik, PhD student, Andrea Denardo, Student and Paolo Arosio, PhD Department of Molecular and Translational Medicine, University of Brescia, Italy Presented By: Andrea Denardo Poster #169 HEPARIN BINDING DOMAINS IN BMP6: STUDY ON THE SYNTHETIC PEPTIDES AND THE RECOMBINANT PROTEIN Paola Ruzzenenti, PhD student¹, Andrea Denardo, Student, Fernando Carmona, PhD, Stefania Federici, PhD², Michela Asperti, PhD, Paolo Bergese, PhD, Maura Poli, PhD and Paolo Arosio, PhD ¹Department of Molecular and Translational Medicine, University of Brescia, Italy; ²INSTM and Department of Mechanical and Industrial Engineering, University of Brescia, via Branze 38 25123 Brescia, Italy Presented By: Paola Ruzzenenti Poster #170 COMPARATIVE CHARACTERISATION OF THE FERROUS IRON TRANSPORT SYSTEMS EFEUOB AND FEOABC IN E. COLI Salem Al– Aidy and Simon Andrews, Professor University of Reading Presented By: Salem Al– Aidy Poster #171 HEME OVERLOAD IN MICE PROMOTES IRON EXPORT FROM MACROPHAGES VIA ROS– MEDIATED FERROPORTIN ACTIVATION Maja Vujic Spasic, PhD¹, Naveen Kumar Tangudu, MSc², Betuel Alan, MSc², Dilay Lai², Francesca Vinchi, PhD³, Katharina Woehrle, MSc⁴, Sabine Vettora i, PhD² and Kerstin Leopold, PhD⁴ ¹Institute of Comparative Molecular Endocrinology; ²Institute of Comparative Molecular Endocrinology, Ulm, Germany; ³Molecular Medicine Partnership Unit, Heidelberg, Germany; ⁴Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany Presented By: Maja Vujic Spasic, PhD Poster #172 INTRACELLULAR SYSTEMS OF IRON COFACTOR DELIVERY: ANALYSIS OF MURINE MODELS OF IRON CHAPERONE DEFICIENCY Caroline Philpott, MD, Moon– Suhn Ryu, PhD, Fengmin Li, PhD, Avery Frey, PhD, Olga Protchenko, PhD and Minoo Shakoury– Elizeh, MS NIDDK, NIH Presented By: Caroline Philpott, MD Poster #173 THE MAIN PHYSIOLOGIC SUBSTRATE FOR ZIP14 IS NEITHER IRON NOR ZINC Supak Jenkitkasemwong, PhD and Mitchell Knutson, PhD University of Florida, USA Presented By: Mitchell Knutson, PhD Poster #174 INTESTINAL HEPHAESTIN IS NECESSARY FOR OPTIMAL IRON ABSORPTION IN PREGNANT MICE, BUT IS DISPENSABLE DURING LACTATION Caglar Doguer, PhD¹, Jung-Heun Ha, PhD¹, Shireen R.L. Flores, MS¹, Tao Wang, MS¹,² and James F. Collins, PhD¹ ¹University of Florida; ²Sichuan University Presented By: Caglar Doguer, PhD Poster #175 TRANSCRIPTIONAL REGULATION OF TRANSFERRIN RECEPTOR BY HEME IN ERYTHROID CELLS Daniel Garcia dos Santos, PhD¹, Amel Hamdi, PhD², Matthias Schranzhofer, PhD¹, Nam-Chun Lok, PhD³ and Prem Ponka, MD, PhD² ¹Lady Davis Institute, Jewish General Hospital; ²Lady Davis Institute, Jewish General Hospital/ Physiology Department, McGill University; ³Department of Chemistry, The University of Hong Kong Presented By: Daniel Garcia-Santos, PhD Poster #176 FURTHER EVIDENCE THAT INTESTINAL COPPER ABSORPTION DOES NOT OCCUR THROUGH DMT1 BUT THROUGH CTR1 AND A CHLORIDE DEPENDENT UPTAKE TRANSPORTER, USING THE CACO2 CELL MODEL Adrian Ricarte, BS, Aleksander Wertz, undergraduate, Yuseinis Gutierrez, undergraduate, Angelica Valadez, undergraduate, Theodros Kidane, MS and Maria Linder, PhD California State University Fullerton, USA Presented By: Adrian Ricarte, BS
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Poster #177 STEPS IN THE MOBILIZATION AND RELEASE OF FERRITIN IRON DURING STIMULATION OF HEMATOPOIESIS Maria Linder, PhD¹, Alice La, BS Biochem¹, Trinity Nguyen, BS Biochem¹, Ketson Tran, BS Biochem¹, Eric Sauble, MS¹, David Tu, BS Biochem¹, Angelica Gonzalez, MS¹, Theodros Kidane, MS¹, Cesar Soriano, BS Biochem¹, Jessica Morgan, BS Biochem¹, Michael Doan, BS Biochem¹, Diane Jun, Undergraduate¹, Cia– Yu Wang, PhD² and Mitchell Knutson, PhD² ¹California State University Fullerton; ²University of Florida, USA Presented By: Maria C. Linder, PhD Poster #178 A MATHEMATICAL MODEL OF IRON DYNAMICS IN A MOUSE Jignesh Parmar, PhD and Pedro Mendes, PhD Center for Quantitative Medicine, UCONN Health Presented By: Jigneshkumar Parmar, PhD Poster #179 A GLUTAREDOXIN– BOLA COMPLEX SERVES AS AN IRON–SULFUR CLUSTER CHAPERONE FOR THE CYTOSOLIC CLUSTER ASSEMBLY MACHINERY Avery Frey, PhD¹, Dan Palenchar¹, Kym Zumbrennen¹, Toshiki Yabe¹, Ajay Vashisht², Justin Wildemann¹, James Wohlschlegel² and Caroline Philpott¹ ¹National Institutes of Health– NIDDK; ²Department of Biological Chemistry, UCLA Presented By: Avery Frey, PhD Poster #180 NOVEL SENSORS FOR SIMULTANEOUS DETECTION OF LABILE Fe2+ AND Fe3+ BASED ON IN VITRO– SELECTED DNAZYMES Ryan Lake, BA¹, Seyed– Fakhreddi Torabi, PhD², Peiwen Wu, PhD² and Yi Lu, PhD¹ ¹Department of Chemistry, University of Illinois Urbana– Champaign; ²Department of Biochemistry, University of Illinois Urbana– Champaign Presented By: Ryan Lake, BA Poster #181 ERYTHROFERRONE EXPRESSION LEVELS IN MYELODYSPLATIC SYNDROMES SHOW CLINICAL RELEVANCE Augusta Di Savino, Arianna Savi, Student, Daniela Gallo, MD, Valentina Gaidano, MD, Patrizia Scaravaglio, BSc, Mauro Mezzabotta, MD, Giuseppe Saglio, MD, PhD, Daniela Cilloni, MD, PhD and De Gobbi Marco, MD, PhD Department of Clinical and Biological Sciences Presented By: Augusta Di Savino, PhD Poster #182 EFFECT OF ERYTHROPOIETIN ON IRON HOMEOSTASIS IN TMPRSS6– MUTATED MASK MICE Jana Frydlova², Zuzana Rychtarcikova³, Iuliia Gurieva², Martin Vokurka², Jaroslav Truksa¹ and Jan Krijt² ¹Czech Academy of Sciences, Institute of Biotechnology; ²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic; ³Institute of Biotechnology, BIOCEV Research Center, Czech Academy of Sciences, Vestec, Czech Republic Presented By: Jaroslav Truksa, PhD Poster #183 THE ROLE OF GDF11 IN THE TREATMENT OF ß–THALASSEMIA Rea Oikonomidou, MD¹, Amaliris Gonzalez, PhD, Ping La, PhD, Vania Lo Presti, Ritama Gupta, Carla Casu, PhD, Laura Breda, PhD, Rajasekhar NVS Suragani, PhD², Ravi Kumar, PhD and Stefano Rivella, PhD¹ ¹Children's Hospital of Philadelphia; ²Acceleron Presented By: Stefano Rivella, PhD Poster #184 THE HIGH–RESOLUTION CRYSTAL STRUCTURE OF THE GLOBULAR DOMAIN OF MOUSE ERFE Kirsty McHugh, DPhil Clinical Medicine, Jing Jin, Joao Arezes, Reema Jasuja, Alexander Drakesmith, Simon Draper and Matt Higgins Oxford University, U.K. Presented By: Kirsty McHugh, PhD Poster #185 IDENTIFICATION OF RNAi TECHNOLOGY TO TARGET TRANSFERRIN RECEPTOR 1 FOR REDUCTION OF HEMICHROME FORMATION IN ß–THALASSEMIA Amaliris Gonzalez, PhD¹, Valentina Ghiaccio², Laura Breda, PhD, Yelena Gin burg, MD³, Robert Fleming, MD⁴ and Stefano Rivella, PhD ¹Children's Hospital of Philadelphia; ²Universit degli studi di Cagliari; ³Mount Sinai Medical Center, NY; ⁴Saint Louis University Presented By: Amaliris Gonzalez, PhD Poster #186 EX VIVO GENE THERAPY APPROACH BY TARGT TECHNOLOGY FOR THE TREATMENT OF ß–THALASSEMIA INTERMEDIA Carla Casu, Vania Lo Presti, Emir O'Hara, Garry Neil, Reem Miari, Nir Shapir and Stefano Rivella The Children’s Hospital of Philadelphia, USA Presented By: Carla Casu, PhD Poster #187 A NOVEL ROLE FOR ZIP8 IN ERYTHROPOIESIS Supak Jenkitkasemwong, PhD, Wei Zhang, PhD and Mitchell Knutson, PhD University of Florida, USA Presented By: Supak Jenkitkasemwong, PhD
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Poster #188 GLYCINE TRANSPORTER 1 PLAYS A CRUCIAL ROLE IN HEMOGLOBINIZATION Daniel Garcia dos Santos, PhD¹, Matthias Schranzhofer, PhD¹, Richard Bergeron, MD², Alex D Sheftel, PhD³ and Prem Ponka, MD, PhD⁴ ¹Lady Davis Institute, Jewish General Hospital; ²Ottawa Hospital Research Institute, University of Ottawa; ³Spartan Bioscience Inc High Impact diting; ⁴Lady Davis Institute for Medical Research, Je ish General Hospital/Physiology Department, McGill University Presented By: Daniel Garcia-Santos, PhD Poster #189 HEME OXYGENASE 1 DIRECTS STRESS–INDUCED REMODELING OF IRON– RECYCLING MACROPHAGES Carine Fillebeen, PD¹, Daniel Garcia-Santos, PhD¹, Konstantinos Gkouvatsos, MD¹, Saljoughian Noushin, PhD², Louis– Philippe Leroux, PhD², Maritza Jaramillo, PhD², Tina Haliotis, MD¹, Marzel Buffler, PhD³, Christiane Becker, PhD³, Klaus Schümann, MD³, Prem Ponka, MD¹ and Kostas Pantopoulos, PhD¹ ¹Lady Davis Institute for Medical Research/McGill University; ²INRS Institut Armand Frappier Research Centre; ³Science Center Weihenstephan, Technical University Munich Presented By: Kostas Pantopoulos, PhD Poster #190 IDENTIFICATION OF NOVEL LOCI ASSOCIATED WITH HEMATOLOGICAL TRAITS IN MICE Brie Fuqua, Stela McLachlan, Richard C. Davis, Simon T. Hui, Nam Che, Zhiqiang Zhou, Carmen Ng, Claire Yukhtman, Raffi Gharakhanian, Hannah Qi, Milagros C. Romay, Calvin Pan, Yehudit Hasin– Brumshtein, Dongyi Lu, Brian W. Parks, Eleazar Eskin, Aldons J. Lusis and Chris D. Vulpe University of Florida and University of California, Los Angeles Presented By: Brie Fuqua, BS, PhD Poster #191 IMATINIB AND SPIRONOLACTONE SUPPRESS HEPCIDIN EXPRESSION Katarzyna Mleczko– Sanecka¹, Debora Call², Ana Rita da Silva², Nikolai Schmeer, Melanie Kiessig, Georg Damm and Martina Muckenthaler ¹International Institute of Molecular and Cell Biology, Warsaw, Poland; ²Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Germany Presented By: Katarzyna Mleczko-Sanecka, PhD Poster #192 FASTING INCREASES HEPATIC HEPCIDIN EXPRESSION AND CAN OVERCOME THE EFFECT OF HFE DELETION IN MICE Cornel Mirciov, BSc, Sarah Wilkins, BSc, Greg Anderson, PhD and David Frazer, PhD Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: David Frazer, PhD Poster #193 EFFECT OF DIETARY VITAMIN D SUPPLEMENTATION ON SERUM HEPCIDIN LEVELS IN MICE WITH ACUTE INFLAMMATION Abitha Sukumaran, Larisa Lozovatsky, MS and Karin E Finberg, MD, PhD Yale University School of Medicine, USA Presented By: Abitha Sukumaran, PhD Poster #194 THE PROTECTIVE ROLE OF HAPTOGLOBIN/HEMOPEXIN AXIS IN ANKYLOSING SPONDYLITIS– DRIVEN SPINAL CORD INJURY Ana Catarina Vaz Carreto Martins, MSc², Illyane Sofia Martins Lima, MSc² and Raffaella Gozzelino, PhD¹ ¹Chronic Diseases Research Center /NOVA Medical School; ²CEDOC/NOVA Medical School Presented By: Raffaella Gozzelino, PhD Poster #195 INCREASING DIETARY IRON INTAKE ANTAGONIZES COPPER METABOLISM IN A DOSE– DEPENDENT FASHION IN WEANLING RATS Jung– Heun Ha, PhD¹, Caglar Doguer, PhD¹, Shireen R. L. Flores, MS¹, Tao Wang, MS¹,², Sun Young Jeong, MS¹ and James F. Collins, PhD¹ ¹University of Florida; ²Sichuan University Presented By: Jung– Heun Ha, PhD Poster #196 A SINGLE ADAPTABLE COCHAPERONE–SCAFFOLD COMPLEX DELIVERS NASCENT IRON– SULFUR CUSTERS TO MAMMALIAN RESPIRATORY CHAIN COMPLEXES I–III Nunziata Maio, PhD¹, Ki Soon Kim, PhD², Anamika Singh² and Tracey Rouault, MD² ¹National Institutes of Health; ²NIH/NICHD Presented By: Nunziata Maio, PhD Poster #197 ENTRY OF IRON AND OTHER METAL IONS INTO MITOCHONDRIA MichaeL Garrick, PhD¹, Natascha Wolff, PhD², Lin Zhao, MD¹, Laura Garrick, PhD¹ and Frank Thévenod, MD, PhD² ¹UB; ²Witten/Herdecke University Presented By: Michael Garrick, PhD Poster #198 A NOVEL PLAYER IN THE REGULATION OF HEPCIDIN REVEALED BY THE MTOR INHIBITOR RAPAMYCIN Laura Silvestri, PhD, Silvia Colucci, BSc, Alessia Pagani, PhD, Irene Artuso, BSc, Mariateresa Pettinato, BSc, Antonella Nai, PhD and Clara Camaschella, MD IRCCS San Raffaele Scientific Institute & San Raffaele Vita– Salute University Presented By: Laura Silvestri, PhD
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Poster #199 WORLDWIDE STANDARDIZATION OF SERUM HEPCIDIN ASSAYS Dorine Swinkels, MD, PhD Radboudumc Presented By: Dorine W. Swinkels, MD, PhD Poster #200 MARINE SPONGE EXTRACTS AS A PROMISING SOURCE OF IRON CHELATORS Hector Aguilar Vitorino, PhD¹ and Breno Pannia Espósito, PhD² ¹University of São Paulo – USP; ²University of São Paulo Presented By: Hector Aguilar Vitorino, PhD Poster #201 NUCLEAR HORMONE RECEPTOR– 14 INTEGRATES IRON UPTAKE AND INNATE IMMUNITY IN CAENORHABDITIS ELEGANS THROUGH PQM–1 SIGNALING Elizabeth Leibold, PhD, Cole P. Anderson, PhD, Steven J. Romney, BS and Paul Rindler, PhD University of Utah, USA Presented By: Elizabeth A Leibold, PhD Poster #202 IMPROVED PATIENT–REPORTED OUTCOMES WITH A FILM–COATED VERSUS DISPERSIBLE TABLET FORMULATION OF DEFERASIROX: RESULTS FROM THE RANDOMIZED, PHASE II ECLIPSE STUDY Ali T Taher¹, Raffaella Origa², Silverio Perrotta³, Ale andra Kouraklis⁴, Giovan Battista Ruffo⁵, Antonis Kattamis⁶, Ai– Sim Goh⁷, Annelore Cortoos⁸, Vicky Huang⁸, Marine Weill⁹, Ra uel Merino Herran ⁹ and John B Porter¹ ¹American University of Beirut Medical Center; ²University of Cagliari; ³Second University of Naples; ⁴University of Patras Medical School; ⁵U.O.C. matolog. Con Talassemia; ⁶University of Athens; ⁷Hospital Pulau Pinang; ⁸Novartis Pharmaceuticals Corporation; ⁹Novartis Pharma AG; ¹ University College London Presented By: John B. Porter, MA, MD, FRCP DRCPath Poster #203 LONG–TERM IRON CHELATION THERAPY WITH DEFERASIROX IN PEDIATRIC PATIENTS WITH TRANSFUSIONAL HEMOSIDEROSIS (ENTRUST) Elliott Vichinsky¹, Amal El–Beshla y², A am Al oebie³, Annie Kamdem⁴, Su anne Koussa⁵, Thirachit Chotsampancharoen⁶, Andreas Bruederle⁷, Jackie Han⁸ and Mohsen lalfy⁹ ¹Children s Hospital Research Center; ²Cairo University; ³Sheikh Khalifa Medical City; ⁴Centre Hospitalier Intercommunal; ⁵Chronic Care Center; ⁶Songklanagarind Hospital; ⁷Novartis Pharma AG; ⁸Novartis Pharmaceuticals; ⁹Ain Shams University Presented By: Elliott Vichinsky, MD Poster #204 LABILE PLASMA IRON PREDICTS FOR SURVIVAL IN PATIENTS UNDERGOING ALLOGENIC– STEM CELL TRANSPLANTATION – RESULTS FROM THE PROSPECTIVE MULTICENTER GERMAN–AUSTRIAN ALLIVE TRIAL Martin Wermke, Julia Eckoldt, Katharina Goetze, Stefan Klein, Gesine Bug, Friedrich Stoelzel, Malte von Bonin, Johannes Schetelig, Michael Laniado, Verena Plodeck, Wolf– Karsten Hofmann, Gerhard Ehninger, Martin Bornhaeuser, Dominik Wolf, Igor Theurl and Uwe Platzbecker TU Dresden, Medical Faculty, Department of Medicine I Presented By: Martin Wermke Poster #205 EXPANDING THE CLINICAL AND MOLECULAR SPECTRUM OF FERROPORTIN DISEASE André Viveiros, MD, Benedikt Schaefer, MD, Armin Finkenstedt, MD, Benjamin Henninger, MD, Karin Nachbaur, MD, Herbert Tilg, MD and Heinz Zoller, MD Medical University Innsbruck Presented By: Andre Viveiros, MD Poster #206 HEPCIDIN IN HEALTHY AND ANEMIC PREGNANT WOMEN Valerii Demikhov, MD, Manel Ayari, Igor Mirov, MD, Prof, Elena Zinovieva, Elena Demikhova and Olga Zhurina, PhD Ryazan State Medical University, Russia Presented By: Valerii Demikhov, MD Poster #207 HOMOMERIC AND HETEROMERIC RECEPTOR COMPLEX FORMATION OF BONE MORPHOGENETIC TYPE I RECEPTORS ALK2 AND ALK3 Lisa Traeger¹, Inka Gallitz, PhD¹, Rohit Sekhri¹, Nicole Bäumer, PhD², Tanja Kuhlmann, MD³, Claudia Kemming³, Thomas B. Bartnikas, MD, PhD⁴ and Andrea U. Steinbicker, MD, MPH¹ ¹Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Muenster, Muenster, Germany; ²Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany; ³Institute for Neuropathology, University Hospital Muenster, Muenster, Germany; ⁴Department of Pathology and Laboratory Medicine, Brown University, Providence, USA Presented By: Lisa Traeger Poster #208 THE STRUCTURAL AND FUNCTIONAL RELATIONSHIPS OF TRANSFERRIN RECEPTOR 2– MEDIATED HEPCIDIN SIGNALING Mark Kleven and Caroline Enns, PhD Oregon Health & Science University, USA Presented By: Mark Kleven, PhD
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Poster #209 HAMP1 mRNA AND PLASMA HEPCIDIN LEVELS ARE INFLUENCED BY SEX AND STRAIN BUT DON’T PREDICT TISSUE IRON LEVELS IN INBRED MICE Stela McLachlan, PhD¹, Kathryn Page, PhD², Seung– Min Lee, PhD³, rika Valore, Ms⁴, Simon Hui, PhD⁴, Brie Fu ua, PhD⁵, Aldons Lusis, PhD⁴, Tomas Gan , PhD⁴, li abeta Nemeth, PhD⁴ and Chris Vulpe, MD PhD⁶ ¹University of dinburgh; ²University of California, Berkeley; ³Yonsei University, Seoul, Korea; ⁴University of California at Los Angeles; ⁵University of California at Los Angeles and University of Florida; ⁶University of Florida Presented By: Brie Fuqua, BS, PhD Poster #210 DEVELOPMENT OF A NOVEL ASSAY FOR STUDYING THE HEPCIDIN–FERROPORTIN AXIS AND ITS APPLICATION TO CHARACTERIZATION OF A NEW FERROPORTIN DISEASE– CAUSING MUTATION Nathan Subramaniam, PhD, Cameron McDonald, PhD, Lesa Ostini, BSc, Diploma, Gautam Rishi, PhD, David Iser, MD, Annabel Iser, MD and Daniel Wallace, PhD Institute of Health and Biomedical Innovation, Queensland University of Technology and QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: Daniel Wallace, PhD Poster #211 INAPPROPRIATE EXPRESSION OF HEPCIDIN IN THE LIVER OF PROFILIN–2 KNOCK OUT MICE Sara Lucieti², Bruno Galy, PhD³, Lucia Gutierre , PhD⁴, Michael Reinke⁵, Jorge Couso², Maya Shvartsman, PhD⁶, Antonio Di Pascale, PhD⁷, Walter Witke, PhD⁵, Matthias W. Hent e, PhD, MD⁸, Pietro Pilo Boyl, PhD⁵ and Mayka Sanchez, Dr¹ ¹Josep Carreras Leukaemia Research Institute ; ²Josep Carreras Leukaemia Research Institute , Iron Metabolism: Regulation and Disease group; ³Division of Virus– associated Carcinogenesis, German Cancer Research Centre DKFZ , Heidelberg, Germany; ⁴Department of Biomaterials and Bioinspired Materials, Instituto de Ciencia de Materiales de Madrid (ICMM– CSIC , Madrid, Spain; ⁵Institute of Genetics, University of Bonn, Bonn, Germany; ⁶ uropean Molecular Biology Laboratory MBL , Monterotondo, Italy; ⁷Department of Pharmacy, University of Naples Federico II, Naples, Italy; ⁸ uropean Molecular Biology Laboratory (EMBL), Heidelberg, Germany Presented By: Mayka Sanchez Fernandez, PhD Poster #LB26 IMMUNOASSAY FOR HUMAN SERUM ERYTHROFERRONE Tomas Ganz, PhD, MD, Grace Jung, MS, Arash Naeim, MD, Yelena Ginzburg, MD, Zahra Pakbaz, MD, Patrick Walter, PhD, Leon Kautz, PhD and Elizabeta Nemeth, PhD UCLA, USA Presented By: Tomas Ganz, PhD, MD Poster #LB27 ANTI– INFLAMMATORY EFFECT OF FGF– 23 INHIBITION PREVENTS LIVER HEPCIDIN UPREGULATION IN A CHRONIC KIDNEY DISEASE MOUSE MODEL Rafiou Agoro, PhD¹, Anna Montagna, PhD¹, Onyedikachi Aligbe, BA¹, Regina Goetz, PhD¹, Moosa Mohammadi, PhD¹, Stefano Rivella, PhD² and Despina Sitara, PhD¹ ¹New York University; ²Children's Hospital of Philadelphia Presented By: Rafiou Agoro, MSc Poster #LB28 ROLE OF C– JUN NH(2)– TERMINAL KINASE AND IRON IN CELLULAR MODEL OF ACUTE PANCREATITIS Zbigniew Sled i ski, MD PhD¹, Michal Wo niak, MD PhD², Maciej Sled inski, MD² and Jedr ej Antosie ic , PhD³ ¹Medical University of Gda sk; ²Medical University of Gdansk, Poland; ³Gdansk University of Physical ducation and Sport, Gdansk, Poland Presented By: Zbigniew Sledzinski, MD, PhD Poster #LB29 EXERCISE INDUCED–INCREASE IN BLOOD HEPCIDIN IS THE SAME IN WT AND HETEROZYGOTES FOR HFE H63D Jedrzej Antosiewicz, PhD¹, Jakub Kortas, PhD², Maja Tomczyk, MS², Krzysztof Prusik,, PhD², Katarzyna Prusik, PhD², Joanna Jaworska, MS², Barbara Kaczorowska, MD² and Ewa Ziemann, PhD² ¹Gdansk University of Physical ducation and Sport, Ka imier a Go rskiego ,; ²Gdansk University of Physical Education and Sport, Gdansk, Poland Presented By: Jedrzej Antosiewicz, PhD Poster #LB30 BACTERIAL DYSBIOSIS IN COLON AND BREAKDOWN OF MUCOSAL PERMEABILITY BARRIER IN A MOUSE MODEL OF HEMOCHROMATOSIS SATHISH SIVAPRAKASAM¹, BOJANA RISTIC, BS¹, ABDUL N HAMOOD2, PhD¹, NITHYA S MUDALIAR, MS¹, JANE COLMER HAMOOD, PhD¹, KAMESWARA RAO KOTTAPALLI, PhD² and VADIVEL GANAPATHY, PhD¹ ¹Texas Tech University Health Sciences Center; ²Texas Tech University Presented By: Sathish Sivaprakasam, PhD Poster #LB31 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL NA+– COUPLED, CITRATE– DEPENDENT TRANSPORT SYSTEM FOR NON–TRANSFERRIN–BOUND IRON Jiro Ogura, PhD¹, Ellappan Babu, PhD¹, Seiji Miyauchi, PhD² and Vadivel Ganapathy, PhD¹ ¹Texas Tech University Health Sciences Center; ²Toho University Presented By: Jiro Ogura, PhD
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Poster #LB32 UNCOMMON IRON CHELATORS AURINTRICARBOXYLIC ACID (ATA) AND LIPOIC ACID (LA) AS ANTICANCER AGENTS Michal Wozniak¹, Alicja Kuban– Jankowska, PhD², Magdalena Gorska– Ponikowska, PhD², Kamlesh Sahu, PhD³ and Jack Tus ynski, PhD⁴ ¹Medical Univeristy of Gdansk, Poland; ²Medical Chemistry Department, Medical University of Gdansk, Gdansk, Poland; ³Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada; ⁴Department of Oncology, University of Alberta, dmonton, Canada Presented By: Matt Wozniak Poster #LB33 SMART IRON DELOCALIZATORS IN COMBINATION WITH NEW GPRX4 PROTECTOR PREVENT DAMAGE OF TRANSPLANTED KIDNEY FROM FERROPTOTIC INSULT Narcyz Knap¹, Michal Wozniak, MD PhD², Magdalena Gorska– Ponikowska, PhD², Alicja Kuban– Jankowska, PhD², Ryszard Smolenski, MD PhD³, Michal Zmije ski, PhD⁴, Jedr ej Antosie ic , PhD⁵ and Zbignie Sled inski, MD PhD⁶ ¹Medical Univeristy of Gdansk, Poland; ²Department of Medical Chemistry, Medical University of Gdansk; ³Department of Biochemistry, Medical University of Gdansk; ⁴Department of Histology, Medical University of Gdansk; ⁵Department of Bioenergetics and Physiology of ercise Medical University of Gdansk; ⁶Department of General, Endocrine Surgery and Transplantation, Medical University of Gdansk Presented By: Narcyz Knap Poster #LB34 INDEPENDENT RISK FACTORS FOR BIOPSY– PROVEN CIRRHOSIS IN HEMOCHROMATOSIS: A STUDY OF 110 HFE p.C282Y HOMOZYGOTES WITH GNPAT p.D519G GENOPTYING James Barton, MD²,¹³, Christine McLaren, PhD³, Wen– pin Chen, MS⁴, Grant Ramm, PhD⁵, Gregory Anderson, PhD⁶, La rie Po ell, MD, PhD⁶, V. Nathan Subramaniam, PhD⁷, Paul Adams, MD⁸, Pradyumna Phatak, MD⁹, Lyle Gurrin, PhD¹ , John D. Phillips, MD¹¹, Charles J. Parker, PhD¹¹, Mary J. dmond, PhD¹² and Gordon McLaren, MD¹,³ ¹Tibor Rubin VA Medical Center; ²Southern Iron Disorders Center; ³University of California, Irvine; ⁴Chao Family Comprehensive Cancer Center; ⁵QIMR Berghofer Medical Research Institute; ⁶The University of Queensland; ⁷Queensland University of Technology; ⁸London Health Sciences Centre; ⁹Rochester General Hospital; ¹ The University of Melbourne; ¹¹University of Utah School of Medicine; ¹²University of Washington; ¹³University of Alabama at Birmingham Presented By: Gordon McLaren, MD Poster #LB35 IDENTIFICATION OF THE NUCLEATION SITE FOR IRON BIOMINERALIZATION IN L– FERRITIN BY X– RAY CRYSTALLOGRAPHY Silvia Ciambellotti¹,³, Cecilia Pozzi², Caterina Bernacchioni¹,³, Flavio Di Pisa², Stefano Mangani² and Paola Turano¹,³ ¹Center for Magnetic Resonance , University of Florence; ²Department of Biotechnology, Chemistry, and Pharmacy, University of Siena; ³Department of Chemistry, University of Florence Presented By: Silvia Ciambellotti Poster #LB36 IDENTIFICATION OF STAPHYLOCOCCUS AUREUS IRON ACQUISITION LOCI REQUIRED FOR REPLICATION WITHIN MACROPHAGES David Heinrichs and Ron Flannagan, PhD University of Western Ontario, London, Ontario, Canada Presented By: David Heinrichs, PhD Poster #LB37 THE CORE COMPONENT OF THE MITOCHONDRIAL CALCIUM UNIPORTER (MCU) PROMOTES ACETAMINOPHEN HEPATOXICITY AND IS REQUIRED FOR MITOCHONDRIAL Fe2+ UPTAKE Jiangting Hu, MD, PhD², James Weemhoff, PhD³, Andaleb Kholmukhamedov, MD², Hartmut Jaeschke, PhD³, Anna– Liisa Nieminen, PhD² and John Lemasters, MD, PhD¹ ¹Medical University of South Carolina; ²Medical Univeristy of South Carolina; ³University of Kansas Medical Center Presented By: John Lemasters, MD, PhD Poster #LB38 PRETREATMENT OF RICE SEEDS WITH IRON AND ZINC DECREASES CADMIUM TRANSPORT IN RICE DURING GROWTH Di Guan Hunan Academy of Agricultural Sciences, China Presented By: Di Guan Poster #LB39 ERFE– INDEPENDENT REGULATION OF HEPCIDIN IN A MOUSE MODEL OF INFLAMMATORY BOWEL DISEASE Ugo Sardo, Benjamin Billoré, BSc, Perrine Rousset, MSc and Leon Kautz, PhD Inserm U1220/IRSD Presented By: Ugo Sardo Poster #LB40 RESTORED IRON TRANSPORT BY A SMALL MOLECULE PROMOTES GUT IRON ABSORPTION AND HEMOGLOBINIZATION IN MICE, RATS, AND ZEBRAFISH Alexander Cioffi, Anthony Grillo, Anna SantaMaria and Martin Burke, MD/PhD University of Illinois at Urbana– Champaign, USA Presented By: Alexander Cioffi
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Poster #LB41 RESTORED IRON TRANSPORT BY A SMALL MOLECULE PROMOTES GUT IRON ABSORPTION AND HEMOGLOBINIZATION IN MICE, RATS, AND ZEBRAFISH Anna SantaMaria, Anthony Grillo, Alex Cioffi and Martin Burke, MD/PhD University of Illinois at Urbana– Champaign, USA Presented By: Anna SantaMaria Poster #LB42 TREATING DARK CIRCLE UNDER THE EYE BY ADDRESSING IRON IN FERRITIN AND HEMOGLOBIN Xi Huang, PhD Marivan Skincare Inc. Presented By: Xi Huang, PhD Poster #LB43 RETINAL IRON OVERLOAD ACCELERATES THE PROGRESSION OF DIABETIC RETINOPATHY Kapil Chaudhary, MD, PhD², Wan isa Promsote, PhD³, Sudha Ananth, MSc⁴, Rajalakshmi Veeranan– Karmegam, MSc⁴, Amany Ta fik, PhD⁴, Pamela Martin, PhD⁴, Sylvia Smith, PhD⁴, Vadivel Ganapathy, PhD⁵ and Jaya P. Gnana– Prakasam, PhD¹ ¹Saint Louis University, St Louis, MO; ²Washington University, St Louis, MO; ³National Institute of Health, Bethesda, MD; ⁴Augusta University, Augusta, GA; ⁵Te as Tech Health Sciences University, Lubbock, T Presented By: Jaya P. Gnana– Prakasam, PhD Poster #LB44 MITOFERRIN 2 ASSOCIATES WITH MCU, THE CORE PROTEIN OF THE MITOCHONDRIAL CALCIUM UNIPORTER Anna– Liisa Nieminen, Monika Gooz, MD, PhD and John Lemasters, MD, PhD Medical University of South Carolina, USA Presented By: Anna– Liisa Nieminen, PhD Poster #LB45 DISTAL TUBULAR IRON LOADING IN EXPERIMENTAL FOCAL SEGMENTAL GLOMERULOSCLEROSIS Rachel van Swelm, Henry Dijkman, Jack Wetzels, Erwin Wiegerinck, Rian Roelofs, Johan van der Vlag, Bart Smeets and Dorine Swinkels Radboud University Medical Center, Netherlands Presented By: Rachel Van Swelm, PhD Poster #LB46 IRON REGULATORY PROTEINS REGULATE FRATAXIN AND ISCU TO SECURE MITOCHONDRIAL FUNCTION Huihui Li, BS and Kuanyu Li Nanjing University, China Presented By: Kuanyu Li Poster #LB47 CHARACTERIZATION OF IRON HANDLING IN HEALTHY AND DISEASED HUMAN RENAL BIOPSIES Sanne van Raaij¹, Rachel van Swelm, PhD¹, Karlijn Bouman¹, Maaike Bouman¹, Paul Bass, MD², Robert Unwin, PhD², Jeanne Pertijs¹, Dorine Swinkels, PhD¹ and Kaila Srai, PhD³ ¹Radboud University Medical Center, Nijmegen, the Netherlands; ²Royal Free Hospital, London, United Kingdom; ³University College London, London, United Kingdom Presented By: Sanne van Raaij Poster #LB48 ZIP14 DEFICIENCY PREVENTS IRON LOADING OF THE ANTERIOR PITUITARY AND ADRENAL GLAND Joseph Olivera, Master of Science in Food Science and Human Nutrition, Supak Jenkitkasemwong, PhD in Nutritional Sciences and Mitchell Knutson, PhD in Nutrition University of Florida, USA Presented By: Joseph Olivera Poster #LB49 DELETION OF BMP6 WORSENS THE PHENOTYPE OF HJV–ASSOCIATED HEMOCHROMATOSIS Chloé Latour, Céline Besson-Fournier, Ophélie Gourbeyre, Delphine Meynard, PhD, Marie-Paule Roth, MD and Hélène Coppin, PhD IRSD Inserm Presented By: Marie-Paule Roth, MD, PhD Poster #LB50 HUMAN HEPATIC HEPARAN SULFATE REGULATES HEPCIDIN EXPRESSION Ferdous Anower– E– Khuda, PhD¹, Maura Poli, PhD², Philip Gordts, PhD³, Paolo Arosio, PhD² and Jeffrey Esko, PhD⁴ ¹University of California, San Diego; ²Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, Brescia, Italy; ³Department of Medicine, University of California, San Diego, La Jolla, CA; ⁴Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA Presented By: Ferdous Anower– E– Khuda, PhD
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Poster #LB51 URATE OFFSETS MANGANESE–INDUCED IRON DYSHOMEOSTASIS AND NEURONAL DEGENERATION Rachit Bakshi, PhD², Vivek Venkataramani, MD³,⁹, Sanghamitra Bandyopadhyay, PhD⁴, Yadong Huang, PhD¹, Yanyan Liu, PhD¹, Xudong Huang, PhD¹, Ashley Bush, MD¹, Catherine Cahill, PhD¹, Debomoy Lahiri, PhD⁵, Hong Jiang, MD⁶, Fudi Wang, MD⁷, Ann Smith, PhD⁸ and Jack Rogers, PhD¹ ¹MGH; ²Neurochemistry lab, Depts. Neurology and Psychiatry MGH; ³Department of Hematology and Medical Oncology, University Medicine Göttingen; ⁴Developmental Toxicology Division, India; ⁵Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, USA; ⁶Dept. Physiology, Qingdao University, China; ⁷Department of Nutrition Zhejiang University, China; ⁸School of Biological Sciences, University of Missouri– K.C, Kansas City, MO, USA,; ⁹Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA Presented By: Jack Rogers, PhD Poster #LB52 HEPCIDIN DETERMINATION IN BETA THALASSAEMIA MAJOR CHILDREN BY ELISA AND QPCR Yvonne Elliott, PhD¹, Shradda Bhange, BSc¹, Rob Evans, PhD² and Sebastien Farnaud, PhD¹ ¹Coventry University; ²Brunel University Presented By: Sebastien Farnaud, PhD Poster #LB53 HFE mRNA EXPRESSION IS NOT DIRECTLY RELATED TO IRON SENSING BUT IS RELATED TO TFRC EXPRESSION Kosha Mehta, PhD², Robert Evans, PhD³, Vinood Patel, PhD² and Sebastien Farnaud, PhD¹ ¹Coventry University; ²University of Westminster; ³Brunel University Presented By: Sebastien Farnaud, PhD Poster #LB54 PHARMACOKINETICS AND PHARMACODYNAMICS MODEL OF SYNTHETIC HUMAN HEPCIDIN LJPC– 401 FOLLOWING SINGLE DOSE ADMINISTRATION IN DOGS Hong Jin, PhD¹, Wojciech Krzyzanski, PhD², Sima Patel, MS¹, Robin Marsden, BS¹, James Rolke, BS¹ and Andrew Seacat, PhD¹ ¹La Jolla Pharmaceutical Company; ²University at Buffalo Presented By: Hong Jin, PhD
Poster# LB55 INHIBITION OF INTESTINAL NON–HAEM IRON ABSORPTION BY QUERCETIN IS MEDIATED BY BOTH LUMINAL AND SYSTEMIC MECHANISMS M. Lesjak1, 2, S. Balesaria1, V. Skinner1, E. S. Debnam3, S. K. S. Srai1 1Research Department of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom; 2Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića, Serbia; 3Research Department of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, Royal Free Campus, London, United Kingdom Presented By: M. Lesjak THURSDAY, MAY 11, 2017
OVERVIEW
7:00 – 18:15 Registration/Information Desk Open Location: Centennial Hall Prefunction, Level One 19:00 – 23:30 Gala Dinner/Award Ceremony Location: Centennial Hall, Level One GENERAL SESSION
8:00 – 9:00 Meet the Experts II: Measurement of Iron Transport Location: Exploration, Level Two Moderators: Mitchell Knutson, PhD University of Florida USA
Bryan Mackenzie, PhD University of Cincinnati USA Students/Trainees ONLY – Space is limited 9:00 – 10:30 Plenary Session VI: Heme Physiology and Pathology Chairs: Janis L. Abkowitz, MD University of Washington USA Ann Smith, PhD University of Missouri– Kansas City USA
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9:00 – 9:30 Heme Trafficking Speaker: Iqbal Hamza, PhD University of Maryland USA 9:30 – 10:00 Heme Exporter FLVCR1: Biological Role in Different Organ Systems Speaker: Deborah Chiabrando, PhD University of Torino Italy 10:00 – 10:30 Inflammation, Heme and Parkinson's Disease Speaker: Raffaella Gozzelino, PhD Chronic Diseases Research Center (CEDOC) – Faculty of Medicine of NOVA Portugal 10:30 – 11:00 Coffee Break
11:00 – 12:25 Concurrent Session VII: Iron, Heme and Mitochondria Location: Optimist, Level Two Chairs: Hideo Harigae, MD, PhD Tohoku University Japan Prem Ponka, MD, PhD, FCMA Lady Davis Institute for Medical Research Canada 11:00 – 11:25 Congential Sideroblastic Anemia Speaker: Mark D. Fleming, MD, DPhil Harvard Medical School USA 11:25 – 11:40 #29 TARGET OF ERYTHROPOIETIN, FAM210B, REGULATES ERYTHROID HEMOGLOBINIZATION Barry Paw, MD, PhD¹, Yvette Yien, PhD¹, Jiahai Shi, PhD², Caiyong Chen, PhD¹, Anthony Grillo4, Liangtao Li, MD³, Jacky Chung, PhD¹, Martin Kafina, MS¹, Paul Kingsley, PhD5, Julien Ablain, PhD¹, Leonard Zon, MD¹, James Palis, MD¹, Martin Burke, MD, PhD4, Daniel Bauer, MD, PhD¹, Stuart Orkin, MD¹, Carla Koehler, PhD6, John Phillips³, Jerry Kaplan³, Diane Ward, PhD³ and Harvey Lodish, PhD² ¹Harvard Medical School; ²MIT; ³Univ. Utah; 4Univ. Illinois, Urbana; 5Univ. Rochester; 6UCLA Presented By: Barry Paw 11:40 – 11:55 #30 LOSS OF IRON UPREGULATES MITOCHONDRIA FERRITIN AND TRIGGERS MITOPHAGY THROUGH THE BINDING OF MITOCHONDRIA FERRITIN TO NUCLEAR RECEPTOR COACTIVATOR 4 Yuichi Hara¹, Izumi Yanatori, MD, PhD², Kyo Sasaki, MD², Sohji Nishina, MD, PhD², Fumio Kishi, MD, PhD² and Keisuke Hino, MD, PhD² ¹Kawasaki Medical School; ²Kawasaki Medical Shool, Japan Presented By: Yuichi Hara 11:55 – 12:10 #31 CD13 (ALANYL AMINOPEPTIDASE OR ANPEP) IS AN ENDOCYTIC HEME–AND CLATHRIN–BINDING INTESTINAL BRUSH BORDER MEMBRANE PROTEIN WHOSE DOWN–REGULATION BLOCKS HEME–INDUCED INDUCTION OF HEME OXYGENASE Glenn Gerhard, MD, Fan Chun, PhD, Qunyan Jin, MD, Hui Chen, PhD and Shiming Zhang, PhD Temple University, Pennsylvania Presented By: Glenn Gerhard 12:10 – 12:25 #32 TARGETED DELETION OF THE MOUSE MITOFERRIN2 GENE SLC25A28: MFRN1 AND MFRN2 ARE NECESSARY FOR CELL PROLIFERATION Diane Ward¹, Alexandra Seguin, PhD1, Xuan Jia1, John Phillips, PhD1, Barry Paw, MD, PhD2, Jonathan Barasch, MD3 and Jerry Kaplan, PhD1 ¹University of Utah; 2Harvard; 3Columbia Presented By: Diane Ward
11:00 – 12:30 Concurrent Session VIII: Novel Targets and Therapies in Iron Overload Location: Legacy, Level Two Chairs: Yelena Ginzburg, MD Icahn School of Medicine at Mount Sinai USA Stefano Rivella, PhD Children's Hospital of Philadelphia USA
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11:00 – 11:15 #33 TFR2: A NOVEL POTENTIAL THERAPEUTIC TARGET FOR ß–THALASSEMIA Antonella Nai, PhD, Maria Rosa Lidonnici, PhD, Irene Artuso, MSc, Giacomo Mandelli, PhD, Mariateresa Pettinato, MSc, Laura Silvestri, PhD, Giuliana Ferrari, PhD and Clara Camaschella, MD Division of Genetics and Cell Biology, Ospedale San Raffaele, Italy Presented By: Antonella Nai 11:15 – 11:30 #34 POTENTIAL THERAPEUTIC APPLICATIONS OF JAK2 INHIBITORS AND HIF2α–ASO FOR THE TREATMENT OF ß–THALASSEMIA INTERMEDIA AND MAJOR Carla Casu, Paraskevi Rea Oikonomidou, Vania Lo Presti, Mariam Aghajan, Shuling Guo, Abdulmalik Osheiza, Luca Melchiori, Pedro Ramos and Stefano Rivella The Children’s Hospital of Philadelphia, USA Presented By: Carla Casu 11:30 – 11:45 #35 HEME OXYGENASE 1 INHIBITION REVERSES ANEMIA IN ß–THALASSEMIA MICE Daniel Garcia dos Santos, PhD¹, Amel Hamdi, PhD², Zuzana Zidova, PhD³, Monika Horvathova, PhD³ and Prem Ponka, MD, PhD² ¹Lady Davis Institute, Jewish General Hospital; ²Lady Davis Institute, Jewish General Hospital/Physiology Department, McGill University; ³Faculty of Medicine and Dentistry, Palacky University Presented By: Daniel Garcia dos Santos 11:45 – 12:00 #36 HEPCIDIN MIMETIC PTG–300 FOR TREATMENT OF INEFFECTIVE ERYTHROPOIESIS AND IRON OVERLOAD Gregory Bourne, PhD¹,², Li Zhao, PhD¹, Ashok Bhandari, PhD¹, Brian Frederick, MS¹, Jaimee McMahon, BS¹,², Vinh Tran, BS¹, Jenny Zhang, PhD¹,², Adam Stephenson, PhD¹,², James Tovera, BS¹, Lu Bai, BS¹, Thamil Annamalai, MSc, M Phil¹, Dinesh Patel, PhD¹, Mark Smythe, PhD¹,² and David Liu, PhD¹ ¹Protagonist Therapeutics, Milpitas, CA; ²Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia Presented By: Gregory Bourne 12:00 – 12:15 #37 FERROPORTIN INHIBITORS IMPROVE INEFFECTIVE ERYTHROPOIESIS AND PREVENT IRON LOADING IN A BETA–THALASSEMIA DISEASE MODEL Vania Manolova, PhD, Anna Flace, Patrick Altermatt, MSc, Hanna Sundström, PhD, Naja Nyffenegger, PhD and Franz Duerrenberger, PhD Vifor (International) AG Presented By: Vania Manolova 12:15 – 12:30 #38 IDENTIFICATION OF GUANOSINE 5ʹ– DIPHOPSHATE AS POTENTIALLY IRON MOBILIZER: PREVENTING THE HEPCIDIN– FERROPORTIN INTERACTION AND MODULATING THE INTERLEUKIN– 6/STAT– 3 PATHWAY Stanzin Angmo and Nitin Kumar Singhal, PhD National Agri– Food Biotechnology Institute, India Presented By: Stanzin Angmo 12:30 – 14:00 Lunch – Interviews with the Masters Location: Centennial Terrace, Level Three Interviewer: Tomas Ganz, PhD, MD Interviewee: Prem Ponka, MD, PhD, FCMA
14:00 – 15:30 Concurrent Session IX: Iron Deficiency and Inflammatory Iron Restriction Location: Optimist, Level Two Chairs: James Collins, PhD University of Florida USA Andrea Steinbicker, MD, MPH University Hospital Munster Germany
14:00 – 14:15 #39 EFFECTS OF MATERNAL IRON STATUS ON PLACENTAL IRON TRANSFER Veena Sangkhae, Allison L Fisher, Victoria Gabayan, Erika Valore, Tomas Ganz and Elizabeta Nemeth University of California Los Angeles, USA Presented By: Veena Sangkhae 14:15 – 14:30 #40 ALTERNATE DAY DOSING, AND SINGLE MORNING DOSES RATHER THAN B.I.D. DOSING, MAXIMIZE TOTAL AND FRACTIONAL IRON ABSORPTION FROM ORAL IRON SUPPLEMENTS: STABLE IRON ISOTOPE STUDIES IN IRON–DEPLETED WOMEN Nicole Stoffel¹, Diego Moretti¹, Colin Cercamondi¹, Christophe Zeder¹, Dorine Swinkels², Gary Brittenham³ and Michael Bruce Zimmermann¹ ¹ETH Zurich, Switzerland; ²Radboud University Medical Centre, Nijmegen, The Netherlands; ³Columbia University, New York, USA Presented By: Nicole Stoffel
Seventh Congress of the International BioIron Society Page 47 Program Schedule IBIS
14:30 – 14:45 #41 IMPROVING SAFETY AND EFFICACY OF IRON FORTIFICATION IN AFRICA BY COMBINING IRON AND PREBIOTICS: A STABLE ISOTOPE ABSORPTION STUDY AND A RANDOMIZED CONTROLLED TRIAL IN KENYAN INFANTS Michael Bruce Zimmermann¹, Daniela Paganini¹, Mary Uyoga², Diego Moretti¹, Colin Cercamondi¹, Francis Mutuku³, Edith Mwasi³, Clarissa Schwab¹, Christophe Lacroix¹ and Simon Karanja² ¹ETH Zurich, Switzerland; ²JKUAT, Nairobi, Kenya; ³Msambweni Hospital, Kenya Presented By: Michael Bruce Zimmermann 14:45 – 15:00 #42 RISK–BENEFIT AND COST–EFFECTIVENESS OF UNIVERSAL DISTRIBUTION OF IRON SUPPLEMENTATION AND IRON–CONTAINING MICRONUTRIENT POWDERS TO YOUNG CHILDREN IN 79 LOW AND MIDDLE INCOME COUNTRIES: A MICROSIMULATION STUDY Sant-Rayn Pasricha, MBBS MPH PhD, Michael Bode, Fayrouz Ashour, Amrita Arcot, Laura Murray-Kolb, Parminder Suchdev and Adrian Gheorghe MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Sant-Rayn Pasricha 15:00 – 15:15 #43 TRIFERIC (FERRIC PYROPHOSPHATE CITRATE), A NOVEL THERAPY THAT TREATS ANEMIA OF INFLAMMATION AND OVERCOMES FUNCTIONAL IRON DEFICIENCY Ajay Gupta, MD Rockwell Medical, USA Presented By: Ajay Gupta 15:15 – 15:30 #44 IRON–FORTIFIED VS LOW–IRON INFANT FORMULA: COGNITIVE OUTCOMES AT 10 AND 16 YEARS Sheila Gahagan1, Erin Delker1, Marcella Castillo2, Betsy Lozoff3 1University of California, San Diego; 2Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile; 3University of Michigan Presented By: Sheila Gahagan
14:00 – 15:25 Concurrent Session X: Hereditary and Acquired Hemochromatosis and Its Treatment Location: Legacy, Level Two Chairs: Pierre Brissot, MD Faculté De Médecine-Unversité De Rennes1 France David Frazer, PhD QIMR Berghofer Medical Research Institute Australia 14:00 – 14:25 NTBI Transporters Speaker: Mitchell Knutson, PhD University of Florida USA 14:25 – 14:40 #45 A NOVEL H–FERRITIN SIGNALING RECEPTOR THAT ACTIVATES NLRP3 INFLAMMASOME–INDUCED INFLAMMATION IN HEPATIC STELLATE CELLS: IMPLICATIONS FOR HEPATIC FIBROGENESIS IN HAEMOCHROMATOSIS Manuel Fernandez-Rojo, PhD, Anita Burgess, PhD, Diem Hoang-Le, BSc, Amber Glanfield, PhD, Sujeevi Nawaratna, PhD, Geoffrey Gobert, PhD and Grant Ramm, PhD QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: Grant Ramm 14:40 – 14:55 #46 A RANDOMIZED PATIENT–BLINDED STUDY OF TRUE VERSUS SHAM REDUCTION OF BODY IRON IN HFE RELATED HEMOCHROMATOSIS WITH MODERATE IRON OVERLOAD REVEALS BENEFIT OF TREATMENT Martin Delatycki, Sim Ong, Lyle Gurrin, Lara Dolling, Jeanette Dixon, Amanda Nicoll, Michelle Wolthuizen, Erica Wood, Greg Anderson, Grant Ramm, Katie Allen, John Olynyk, Darrell Crawford, Louise Ramm, Kevin Croft, Paul Gow, Simon Durrant and Lawrie Powell Murdoch Children’s Research Institute, Victoria, Australia Presented By: Martin Delatycki 14:55 – 15:10 #47 THIZOLIDINONE COMPOUNDS PREVENT IRON OVERLOAD IN HFE MURINE MODEL THROUGH PROMOTING HEPCIDIN EXPRESSION Jing Liu and Sijin Liu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Presented By: Jing Liu
Seventh Congress of the International BioIron Society Page 48 Program Schedule IBIS
15:10 – 15:25 #48 IMPROVING THE EFFICIENCY OF IRON CHELATORS USING NANOTECHNOLOGY Shanshan Guo¹, Gang Liu, PhD¹, Tianqing Liu, PhD², David Frazer, PhD², Guangjun Nie, PhD¹ and Greg Anderson, PhD² ¹CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, China; ²Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: Shanshan Guo
15:25 – 15:45 Coffee Break
15:45 – 17:20 Concurrent Session XI: Tissue– Specific Iron Regulation Location: Optimist, Level Two Chairs: Dorine W. Swinkels, MD, PhD Radboud University Nijmegen Medical Center Netherlands Fudi Wang, PhD Zhejiang University, Department of Nutrition, School of Public Health China 15:45 – 16:10 Iron Homeostasis in the Heart Speaker: Samira Lakhal-Littleton, PhD University of Oxford United Kingdom 16:10 – 16:35 Ironing Out Smoke–Induced Inflammation in the Lung Speaker: Suzanne Cloonan, PhD Weill Cornell Medical College USA 16:35 – 16:50 #49 IRON–RELATED PROTEINS IN THE KIDNEY AND THEIR REGULATION BY RENAL IRON OVERLOAD Laura Diez Ricote and Mitchell D. Knutson, PhD University of Florida, Gainesville, USA Presented By: Laura Diez Ricote 16:50 – 17:05 #50 FERROPORTIN PROTECTS RED BLOOD CELLS FROM OXIDATIVE STRESS AND MALARIA INFECTION BY EXPORTING FREE INTRACELLULAR IRON De-Liang Zhang, PhD¹, Jian Wang, PhD², Binal Shah, PhD³, Manik Ghosh, PhD4, Hayden Ollivierre, BSc4, Xin-zhuan Su, PhD², Philip Thuma, PhD5, Victor Gordeuk, PhD³ and Tracey Rouault, PhD4 ¹Section on Human Iron Metabolism, NICHD, NIH; ²National Institute of Allergy and Infectious Diseases; ³University of Illinois at Chicago; 4Eunice Kennedy Shriver National Institute of Child Health and Human Development; 5Malaria Research Trust, Choma, Zambia Presented By: De-Liang Zhang 17:05 – 17:20 #51 AN UNEXPECTED ROLE OF HFE IN MACROPHAGES Maja Vujic Spasic, PhD, Naveen Kumar Tangudu and Dilay Lai Institute of Comparative Molecular Endocrinology, Ulm University, Germany Presented By: Maja Vujic Spasic
15:45 – 17:10 Concurrent Session XII: Iron: The Good, The Bad, and The Ugly Location: Legacy, Level Two Chair: Sandro Altamura, PhD University of Heidelberg Germany Thomas Bartnikas, MD, PhD Brown University USA 15:45 – 16:10 Speciation of Iron in Tissues and Plasma During Development and in Disease Conditions Speaker: Paul Lindahl, PhD Texas A&M University USA 16:10 – 16:25 #52 RESTORED IRON TRANSPORT BY A SMALL MOLECULE PROMOTES GUT IRON ABSORPTION AND HEMOGLOBINIZATION IN MICE, RATS, AND ZEBRAFISH Anthony Grillo, Anna SantaMaria, Alexander Cioffi and Martin Burke University of Illinois, USA Presented By: Anthony Grillo
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16:25 – 16:40 #53 ASTROCYTE HEPCIDIN IS A KEY FACTOR IN LPS–INDUCED NEURONAL APOPTOSIS LH You², Caizhen Yan, Bingjie Zheng, Yunzhe Ci, Shiyang Chang, Peng Yu, Guofen Gao and Yan– Zhong Chang¹ ¹Hebei Normal Unviersity; ²Hebei Normal University Presented By: Yan-Zhong Chang 16:40 – 16:55 #54 EFFECTS OF FERRIC CITRATE ADMINISTRATION IN A MURINE MODEL OF CHRONIC KIDNEY DISEASE Connor Francis¹, Samantha Neuburg², Claire Gerber², Xueyan Wang², Corey Dussold², Lixin Qi², Aline Martin², Myles Wolf² and Valentin David² ¹Division of Nephrology and Hypertension, Department of Medicine, and Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine; ²Division of Nephrology and Hypertension, Department of Medicine, and Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Presented By: Connor Francis 16:55 – 17:10 #55 CROSSTALK BETWEEN HYPOXIC AND AUTOPHAGIC PATHWAYS IN INTESTINAL IRON METABOLISM Nupur Das, MBBS, PhD¹, Amanda Sankar, MD², Andrew Schwartz, MS¹ and Yatrik Shah, PhD¹,³,4 ¹Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI; ²Department of Pediatrics and Communicable Diseases, Division of Pediatric Hematology– Oncology University of Michigan, Ann Arbor, MI; ³Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, MI; 4Department of Surgery, University of Michigan, Ann Arbor, MI Presented By: Nupur Das
19:00 – 23:30 Gala Dinner/Award Ceremony Location: Centennial Hall, Level One
Seventh Congress of the International BioIron Society Page 50 Keynote Speaker IBIS
Keynote Lecture Monday, May 8, 2017 10:45 - 11:45
Microbial Iron Piracy and Evolution Nels C. Elde, PhD The University of Utah School of Medicine USA
My research program investigates host-pathogen interfaces and the evolutionary impact of these interactions on genomic and cellular complexity. Protein surfaces at these interfaces often evolve in a manner resembling molecular arms races, providing a conspicuous means to investigate mechanisms underlying the process of evolution. A major focus of our efforts is to understand how even highly conserved host functions, such as iron transport and storage, might be modified to block exploitation by pathogens. We use integrated phylogenetic and experimental approaches to identify new sources of genetic resistance to infectious diseases.
Seventh Congress of the International BioIron Society Page 51 Featured Podium/Poster Abstracts IBIS
Podium/Poster #1
SMAD1/5 IS REQUIRED FOR ERYTHROPOIETIN SUPPRESSION OF HEPCIDIN IN MICE Chia-Yu Wang, Amanda B. Core, Kimberly B. Zumbrennen-Bullough, Susanna Canali, Sinan Ozer, Lieve Umans, An Zwijsen and Jodie L. Babitt Massachusetts General Hospital, Harvard Medical School Presented By: Chia-Yu Wang, PhD
Background: Anemia and other conditions that increase erythropoietic drive suppress liver expression of the main iron regulatory hormone hepcidin to supply adequate iron for red blood cell production. Erythroferrone was recently proposed as one such mediator of hepcidin suppression by anemia; however, its mechanism of action remains uncertain. The BMP signaling pathway is a central regulator of hepcidin transcription in response to iron. BMPs act by inducing phosphorylation of intracellular receptor-activated SMAD (R-SMAD) transcription factors: SMAD1, SMAD5, and SMAD9 (also known as SMAD8). Although R-SMADs have been shown to have redundant, dose-dependent functions in many biological contexts, accumulating data suggest that they do not always have overlapping functions. Moreover, the role of the BMP-SMAD signaling in hepcidin suppression by anemia is not known. Methods: We used R-SMAD siRNA knockdown in vitro and generated mice with a hepatocyte-specific knockout of R- SMADs in vivo to determine the relative contribution of individual R-SMADs to hepcidin regulation. We also used these mice to elucidate the role of SMAD signaling in hepcidin suppression by erythropoietin and erythroferrone. Results: Knockdown studies in human hepatoma Hep3B cells demonstrated that SMAD5 siRNA robustly inhibited hepcidin expression, whereas SMAD1 siRNA had a more modest inhibitory effect and SMAD9 siRNA did not inhibit hepcidin expression. At 8 weeks of age, double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice developed massive serum and liver iron overload, whereas single-knockout Smad5fl/fl;Cre+ or Smad1fl/fl;Cre+ mice had minimal to no iron loading. Liver hepcidin (Hamp) mRNA was reduced in double-knockout mice, but not in single-knockouts, at 8 weeks of age. However, Hamp mRNA was reduced in isolated primary hepatocytes from single-knockout mice at the baseline with a similar fold increase when stimulated by Bmp6, indicating the inducibility of Hamp in these animals was well-preserved. Similarly, Hamp mRNA was reduced in liver tissue from single-knockout mice at 12-day-old, suggesting some degree of hepcidin deficiency that was compensated for after exposure to the high iron content of the standard rodent diet. In Smad1fl/fl;Smad5fl/wt;Cre+ mice and Smad1fl/wt;Smad5fl/fl;Cre+ females containing one functional allele of Smad5 and Smad1, respectively, modestly increased liver iron loading with elevated transferrin saturation was observed, suggesting a gene dosage effect. Consistent with prior studies, epoetin alfa (EPO) treatment led to a robust induction in bone marrow erythroferrone (Fam123b) mRNA and reduction in liver Hamp mRNA levels in Smad1fl/fl;Smad5fl/fl;Cre- control mice. However, EPO did not suppress Hamp in double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice. Similarly, overexpression of Fam132b decreased Hamp and Id1 mRNA levels in primary hepatocytes from Smad1fl/fl;Smad5fl/fl;Cre- mice; however, neither Hamp nor Id1 mRNA was reduced in Smad1fl/fl;Smad5fl/fl;Cre+ primary hepatocytes. Hamp mRNA was further suppressible in double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice under other conditions such as treatment with a low iron diet. Conclusions: These data demonstrate that hepatocyte Smad5 and Smad1 have overlapping functions and work collaboratively to govern hepcidin transcription and that EPO and erythroferrone require an intact SMAD signaling pathway to suppress hepcidin expression.
Seventh Congress of the International BioIron Society Page 52 Featured Podium/Poster Abstracts IBIS
Podium/Poster #2
LIVER HFE PROTEIN CONTENT IN IRON DEFICIENT MICE Jan Krijt¹, Jana Frýdlová², Iuliia Gurieva² and Martin Vokurka² ¹Institute of Pathological Physiology, First Faculty of Medicine, Charles University; ²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic Presented By: Jan Krijt, PhD
HFE gene was described in 1996; its mutations cause hereditary hemochromatosis. In 2008, it was shown that the disruption of Hfe in mouse hepatocytes decreases liver hepcidin expression. However; up to now, there is no information on liver HFE expression at the protein level. In the presented study, we examined the effect of iron deficiency on liver HFE protein content. Iron deficiency was induced in adult mice by feeding of an iron-deficient diet for 10 weeks; liver HFE protein content was determined in plasma membrane-enriched fraction of liver homogenates by immunoblotting using a commercial antibody (SC-18810). Feeding of iron-deficient diet to adult male C57BL/6 mice for 10 weeks did not result in an increase in splenic Fam132b expression; liver non-heme iron content was decreased to approximately 30% of controls. Liver Hamp mRNA content was decreased to approximately 20% of controls. There was no change in liver Hfe mRNA content; however, liver HFE protein content was significantly decreased (Fig.1). The results indicate a strong effect of iron deficiency on HFE protein content. Since functional liver HFE protein is necessary for adequate hepcidin expression, it can be speculated that the observed decrease in HFE protein content could contribute to attenuated hepcidin expression in iron deficiency. In contrast to iron deficiency anemia models based on administration of iron deficient diets to weaned mice, feeding of iron deficient diet to adult mice did not result in an increase of splenic Fam132b expression. It is therefore suggested that the observed decrease of liver HFE protein content could contribute to Hamp gene downregulation in states of relatively mild iron depletion, which does not yet result in anemia, hypoxia and increased expression of erythroferrone. Supported by Czech Science Foundation grant 15-16803S. Fig.1. HFE protein content in plasma-membrane enriched fraction of liver homogenate. C: mice fed control diet; ID: mice fed iron deficient diet for 10 weeks. KO indicates a sample from Hfe-deficient mouse (negative control), E- cadherin is included as loading control.
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Podium/Poster #3
EVIDENCE THAT HEPCIDIN OCCLUDES FERROPORTIN TO INHIBIT IRON EXPORT S. Aschemeyer, B. Qiao, E. Valore, T. Ganz and E. Nemeth University of California, Los Angeles Presented By: Sharraya Aschemeyer
The hepcidin-ferroportin (FPN) axis controls intestinal absorption of iron, as well as its internal recycling and tissue distribution. FPN is the only known cellular iron exporter in vertebrates and is the conduit through which iron is supplied into plasma. Hepcidin is a systemically acting iron-regulatory peptide hormone and the only known natural FPN ligand. We have previously shown that hepcidin binding to FPN causes the ubiquitination, endocytosis, and degradation of the ligand-receptor complex thereby decreasing iron supply to plasma. Based on its similarity to the major facilitator superfamily of alternating access transporters, of which FPN is a member, we generated a computational structural model of human FPN. To test our model, we mutated residues that are candidates for hepcidin binding or are mutated in the gain of function form of human ferroportin disease. Our FPN structural model implies that when hepcidin binds FPN in the open-out conformation, iron export should be inhibited even if FPN is not internalized and degraded. To test this conjecture, we prevented the hepcidin-dependent ubiquitination of FPN by generating K8R, a FPN mutant in which 8 lysines in or near the long intracellular loop connecting the two 6-helix lobes of FPN were mutated to arginines. When overexpressed in HEK293 cells, K8R localized to the cell membrane and exported iron normally, similarly to wild type FPN (WT). In the presence of hepcidin, the WT FPN was ubiquitinated, internalized, and degraded, but the K8R mutant and C326S mutant, a mutant that cannot bind hepcidin, were not ubiquitinated and remained localized to the membrane as determined by microscopy. However, hepcidin treatment inhibited iron efflux from the WT, and at higher concentrations, K8R, but not C326S. Cellular ferritin, indicative of cellular iron retention, was also elevated after hepcidin addition for K8R and WT, but not C326S. Our results are consistent with a model in which hepcidin binding occludes FPN, thus interfering with iron export and promoting iron storage in ferritin. PR73, a 9 amino acid hepcidin analog, also inhibited iron export by K8R. Engineered for high affinity binding to FPN, PR73, at higher concentrations, degraded even C326S, causing iron retention in the cell. We propose that hepcidin and its analogs can inhibit iron export through FPN either by inducing its endocytosis or, at higher concentrations, by occluding its open-out conformation. The two mechanisms may be differentially active depending on the cell type and its endocytic machinery, explaining the reported differences in tissue sensitivity to hepcidin and discrepancies between the effect of hepcidin on iron export as compared to endocytosis.
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Podium/Poster #4
CALCIUM ACTIVATES FERROPORTIN-MEDIATED CELLULAR IRON EFFLUX Bryan Mackenzie¹, T. Alex Ruwe¹, Chandrika N. Deshpande², Reiya Taniguchi³, Vicky Xin², Ali Shawki¹, Kyle R. Vieth¹, Bo Qiao⁴, Erika V. Valore⁴, Osamu Nureki³, Tomas Ganz⁴, Elizabeta Nemeth⁴ and Mika Jormakka² ¹University of Cincinnati College of Medicine; ²Centenary Institute–Sydney; ³RIKEN–Saitama; ⁴David Geffen School of Medicine at UCLA Presented By: Bryan Mackenzie, PhD
Ferroportin—the only known cellular iron exporter—is responsible for iron efflux from enterocytes and macrophages to the blood plasma. This transporter, under the control of hepcidin, serves as a key site of regulation in human iron homeostasis. The thermodynamic mechanism driving ferroportin-mediated iron export is unknown. As a first step in exploring potential thermodynamic mechanisms, we have used biophysical analyses, site-directed mutagenesis, and functional assays to test the hypothesis that ferroportin is a secondary-active transporter that functions in iron/cation antiport. We expressed human ferroportin in RNA-injected Xenopus oocytes and measured efflux of radiotracer metal (microinjected into the oocyte). Expression of ferroportin stimulated the first-order rate constants describing the efflux of 55Fe. Ferroportin-mediated 55Fe efflux activity was unaffected by replacement of extracellular Na+ by choline. Removal of extracellular calcium (i.e. 0 Ca2+ plus 1 mM EGTA) abolished the ferroportin-mediated efflux of 55Fe or 57Co. Mg2+ could 2+ 2+ 55 not substitute for Ca . Whereas extracellular Ca stimulated the ferroportin-mediated Fe efflux (K0.5 for Ca of 0.8 ± [SE] 0.1 mM), we found that (1) pharmacologically raising intracellular Ca2+ concentration had no effect on 55Fe efflux, (2) expression of ferroportin did not stimulate the uptake of 45Ca2+ in oocytes with or without microinjection of iron, and (3) expression of ferroportin did not stimulate 45Ca2+ efflux. We also tested the calcium dependence of ferroportin-mediated iron efflux in a human expression system, and found that removal of extracellular calcium (i.e. 0 Ca2+ plus 1 mM EGTA) inhibited 55Fe efflux from HEK cells stably expressing ferroportin. We have used X-ray crystallography to solve the structure of a Ca2+-bound BbFPN protein (a prokaryotic ferroportin ortholog) and identified four strictly conserved residues that coordinate Ca2+. By using isothermal titration calorimetry and purified protein, we confirmed Ca2+ binding in wildtype 2+ BbFPN (Kd for a of 1 0.2 ) and mammalian ferroportin (Kd 1 ), whereas we could detect no a binding in mutants in which we had mutated any one of the four putative Ca2+ coordinating residues. BbFPN exhibited no detectable affinity for Mg2+ or K+ under the experimental conditions. Mutagenesis of any one of the putative Ca2+ coordination residues in human ferroportin abolished iron-transport activity in our vertebrate expression systems, with one exception; one mutant exhibited modest residual activity that was accelerated by our increasing the extracellular calcium concentration. This mutant exhibited decreased apparent affinity for extracellular calcium (K0.5 = 2.4 ± 0.4 mM, n = 9) compared with wildtype ferroportin (K0.5 = 0.9 ± 0.1 mM; n = 8) (P = 0.004). In conclusion, (1) we have identified the site of calcium coordination in BbFPN and human ferroportin; (2) we conclude that calcium is a required cofactor of ferroportin but find no evidence that calcium is transported; and (3) we dismiss the notion that ferroportin is an iron/cation antiporter. We speculate that Ca2+ triggers the outward-facing to inward-facing conformational change, exposing the iron-binding site towards the cytoplasm. Our findings raise the possibility that ferroportin-mediated cellular iron export may be restricted in conditions of hypocalcemia. Support: NIH–NIDDK grant R01 DK107309
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Podium/Poster #5
AGE RELATED IRON ACCUMULATION: ACCUMULATING WHAT EXACTLY? Simon James, PhD¹, Dominic Hare, PhD¹, Martin de Jonge, PhD² and Gawain McColl, PhD¹ ¹Florey Institute of Neuroscience and Mental Health; ²Australian Synchrotron Presented By: Simon James, PhD
Iron is a fundamentally important biochemical catalytic cofactor thanks to a capacity to accommodate a range of electronic configurations throughout its valence shell. Coordination chemistry controls the reactivity and availability of iron across cellular environments but characterisation of specific iron-ligand species requires isolation of the complex, necessitating disruption of biological systems despite the attendant risk of mismetallation. The tools available to study biological coordination chemistry in situ have remained limited. The synergy of synchrotron-based X-ray fluorescence microscopy (XFM) and X-ray absorption near edge structure (XANES) spectroscopy represents a powerful analytical approach for studying iron biochemistry at the micro-scale. This permits both quantitative mapping of metal distribution and profiling of the native coordination environment without the need for exogenous molecular probes. We combined these two measurement strategies using the same synchrotron beamline to develop an imaging approach we have called fluorescence imaging (‘fi’, or for the reek phi ) AN . We demonstrate the development of non-destructive AN imaging under standard laboratory conditions, validated in a Caenorhabditis elegans model of disrupted metal metabolism. Further we utilise this technique to explore the changes in iron metabolism that accompany ageing in C. elegans and show how and where iron homeostasis is lost during ageing, and its relationship to the age-related elevation of damaging reactive oxygen species. We find that ferritin is used to sustain longevity, buffering against exogenous iron and ablation of ferritin induces rapid ageing. We propose that loss of iron homeostasis may be fundamental to and inescapable consequence of ageing and may represent a potent target for therapeutic strategies aimed at maintaining health at advancing age.
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Podium/Poster #6
RECOVERY OF HEME OXYGENASE 1-DEFICIENT MICE THROUGH REPOPULATION OF WT MACROPHAGES IN RETICULOENDOTHEILAL SYSTEM Ki Soon Kim and Tracey A Rouault, PhD NIH Presented By: Ki Soon Kim, PhD
Heme oxygenase 1 (Hmox1) is an inducible enzyme that catalyzes the degradation of heme into biliverdin, carbon monoxide and ferric iron. Loss-of-function mutations in the Hmox1 gene cause a rare and lethal disease in children, characterized by severe anemia, intravascular hemolysis and tissue damage. Macrophages of the reticuloendothelial system play a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes. Macrophages were depleted from the liver and spleen of Hmox1 knockout (KO) mice, which resulted in intravascular hemolysis and severe damage to the endothelial system and organs (1). Previously, we have shown that bone marrow transplantation rescues the phenotype by providing Wild-type (WT) reticuloendothelial cells in Hmox1 KO mice (2). To investigate whether the macrophages in the reticuloendothelial system can repopulate and divide in tissue, we injected macrophages into the tail vein of KO mice. We isolated the bone marrows from GFP-expressing mice to enable us to detect them in recipient animals. Donor bone marrow cells were extracted from femur and tibial bones of 2 month-old mice, and cells were differentiated with L-cell conditioned medium for 1 week. Subsequently, the differentiated GFP expressing macrophages were injected into recipient KO mice. The introduction of WT macrophages reversed abnormal blood parameters of KO mice. Mean cell volume and hematocrits were significantly improved in transplanted KO mouse, compared to KO mouse. Elevated plasma concentrations for lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in KO mice normalized after macrophage transplantation. Furthermore, the white blood cell counts (WBC) returned normal. The renal iron accumulation was shown in kidney of KO mice through Perls’ blue staining. WT macrophage transduction revealed normalization of the iron-staining pattern, and significant numbers of Kupffer cells were identified in the livers of transplanted KO mice through the F4/80 staining. These were sufficient to support red blood cell recycling. Our results suggest there is no barrier to repopulating the reticuloendothelial system, and the macrophages in the reticuloendothelial system may divide in tissues as needed. Our results suggest that Hmox1-/- patients could be potentially be treated by repair of their mutations in macrophages, followed by reinfusion, without resorting to bone marrow transplantation. Our preliminary results will form the basis of further experiments. References 1) Dysfunction of the heme recycling system in heme oxygenase 1-deficient mice : effects on macrophage viability and tissue iron distribution, Blood, 2010, V116:6054- 6062 2) Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice, Blood, 2014, V124 : 1522-1530
Seventh Congress of the International BioIron Society Page 57 Featured Podium/Poster Abstracts IBIS
Podium/Poster #7
DEVELOPING A GALNAC-CONJUGATED TMPRSS6 ANTISENSE THERAPY FOR THE TREATMENT OF ß- THALASSEMIA Shuling Guo, PhD¹, Mariam Aghajan, PhD¹, Tom Zanardi, PhD¹, Rudy Gunawan, PhD¹, Carla Casu, PhD², Vania Presti², Sheri Booten¹, Stefano Rivella, PhD² and Brett Monia, PhD¹ ¹Ionis Pharmaceuticals; ²Children’s Hospital of Philadelphia Presented By: Shuling Guo, PhD
Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H1–mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Recently, we developed a ligand conjugated antisense (LICA) strategy to deliver antisense oligonucleotide (ASO) to hepatocytes which significantly improves the potency (Prakash et al. Nucleic Acids Res. 2014; 42(13):8796-807). This LICA approach has been validated in both rodents and in humans. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previously we have shown that TMPRSS6 ASO treatment can up-regulate hepcidin expression and ameliorate the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (Hbbth3/+ mice), which recapitulates beta-thalassemia intermedia in humans (Guo et al. J Clin Invest. 2013; 123(4):1531-41). This ASO can be combined efficiently with iron chelators for the management of iron overload and anemia in non-transfusion-dependent thalassemia (Casu et al. Haematologica. 2016; 101(1):e8-e11). Using LICA strategy, we demonstrated that similar pharmacology can be achieved with a 10-fold lower dose. A LICA human TMPRSS6 clinical candidate (Ionis-TMPRSS6-LRx) has been identified. Similar to the mouse TMPRSS6 ASO, Ionis-TMPRSS6-LRx showed superior potency in human primary hepatocyte culture and in human TMPRSS6 transgenic mice compared with the parent ASO. Ionis-TMPRSS6-LRx has been evaluated in a 13-week mouse study and a 9-month monkey study and demonstrated a favorable tolerability profile. Ionis-TMPRSS6-LRx was rapidly absorbed to the systemic circulation and the plasma exposure increased greater than dose proportionally. The nonlinearity was due to a dose- dependent reduction in apparent plasma clearance. After attaining the maximum plasma concentration, it decreased in an apparent multi-exponential fashion with time. ASO concentrations in liver increased slightly less than dose-proportionally to dose. Pharmacokinetic profile of this compound is similar to what have been observed with other LICA ASOs. After treatment with Ionis-TMPRSS6-LRx, we observed a dose-dependent reduction of monkey TMPRSS6 up to >90%. Serum iron and transferrin saturation decreased in a dose-dependent and time-dependent manner as expected. Collectively, our data demonstrate that GalNAc-conjugated TMPRSS6 ASO could be an effective therapeutic for patients with beta- thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2017.
Seventh Congress of the International BioIron Society Page 58 Featured Podium/Poster Abstracts IBIS
Podium/Poster #8
HEME ACCUMULATION IN ENDOTHELIAL CELLS IMPAIRS ANGIOGENESIS BY TRIGGERING PARAPTOSIS Sara Petrillo¹, Deborah Chiabrando, PhD¹, Tullio Genova, PhD¹, Veronica Fiorito, PhD¹, Giada Ingoglia, PhD¹, Francesca Vinchi, PhD², Federico Mussano³, Stefano Carossa³, Lorenzo Silengo¹, Fiorella Altruda¹, Giorgio Merlo¹, Luca Munaron¹ and Emanuela Tolosano¹ ¹University of Turin; ²Univerity of Heidelberg; ³University of Turin, Dental School Presented By: Sara Petrillo, PhD
Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. In the future, a deeper understanding of the molecular actors involved in heme-triggered paraptosis might provide new tools to modulate angiogenesis in pathophysiologic conditions.Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. In the future, a deeper understanding of the molecular actors involved in heme-triggered paraptosis might provide new tools to modulate angiogenesis in pathophysiologic conditions.
Seventh Congress of the International BioIron Society Page 59 Featured Podium/Poster Abstracts IBIS
Podium/Poster #9
TWO SIDES OF THE SAME COIN: A FRESH PERSPECTIVE ON THE ROLE OF TNFa IN INFLAMMATION, IRON METABOLISM AND HEMATOPOIESIS Vania Lo Presti, MSc, Carla Casu, PhD, Rea Paraskevi Oikonomidou, MD, Emir O'Hara, BSc, Sara Gardenghi, PhD and Stefano Rivella, PhD Children's Hospital of Philadelphia Presented By: Vania Lo Presti, MSc
The anemia of inflammation (AI) is a well-recognized clinical condition, commonly observed in patients with chronic infections, malignancy, trauma, or inflammatory diseases; however, its etiology remains unclear. In AI, inflammation is associated with abnormal levels of cytokines produced by the innate immune system. Some cytokines are considered to be pro-inflammatory while others anti-inflammatory. The role of pro-inflammatory cytokines, such as IL6 and IL1, has been well documented in AI. However, the precise contribution to AI of these and additional pro-inflammatory cytokines has not been elucidated yet. In particular, abnormally elevated levels of TNFα have been associated with many inflammatory conditions and a symptom of disease progression. For this reason, pharmacological inhibition of TNFα represents a major focus of drug development. It has been shown that administration of Heat Killed Brucella Abortus (HKBA) in C57BL/6 (WT) mice triggers expression of several inflammatory cytokines, including TNFα. Furthermore, animals treated with a single injection of HKBA develop a severe but reversible anemia. In this study, we investigate the role of TNFα in the development of anemia by administration of HKBA to WT and Tnfα-/- mice. Our results demonstrate that Tnfα-/- mice, following a single injection of HKBA, develop an irreversible macrocytic, hyperchromic anemia, characterized by complete failure in bone marrow erythropoiesis and extramedullary erythropoiesis in the spleen. We also demonstrate that HKBA- treated Tnfα-/- mice present low serum levels of hepcidin while that of erythropoietin and iron is elevated, excluding that these factors contributes to the development of AI. Interestingly, compared to WT mice, Tnfα-/- animals treated with HKBA exhibit a chronic anemia associated with sustained production of cytokines such as IFNγ, IL12p40 and ΙL1β. In addition, we observe a quantitative and qualitative difference in T cells and macrophages, likely contributing to the anemia. Moreover, HKBA-treated Tnfα-/- mice show increased Reactive Oxygen Species (ROS) production in progenitor and terminally differentiated erythroid cells, which exhibit a reduced lifespan. In conclusion, our results suggest a possible dual role of TNFα during AI (pro- and anti-inflammatory), highlighting the extreme importance of this cytokine in the context of the immune function and anemia resolution.
Seventh Congress of the International BioIron Society Page 60 Podium Abstracts IBIS
Podium #1
HEPATOCYTE TRANSFERRIN RECEPTOR 1 OPERATES AS A SENSOR OF SERUM IRON AND IS DISPENSABLE FOR HEPATOCELLULAR IRON SUPPLY John Wagner, MSc, Carine Fillebeen, PhD and Kostas Pantopoulos, PhD Lady Davis Institute for Medical Research/McGill University Presented By: Kostas Pantopoulos, PhD
Transferrin receptor 1 (TfR1) mediates uptake of circulating transferrin-bound iron to developing erythroid cells and other cell types. Complete disruption of the murine Tfrc gene, encoding TfR1, is associated with early embryonic lethality, while tissue-specific ablation of Tfrc in neurons, skeletal muscle cells, cardiomyocytes or intestinal enterocytes, leads to several pathologies. We generated floxed Tfrcfl/fl mice and crossed them to Alb-Cre transgenics for hepatocyte-specific disruption of Tfrc. TfrcAlb-Cre offspring are viable and lack any detectable Tfr1 expression in hepatocytes. The animals do not show any apparent phenotypic abnormalities and have similar hepatic iron content compared to Tfrcfl/fl controls. Moreover, they develop a similar degree of hepatic iron overload following feeding with a high iron diet, and lack any apparent liver pathology following feeding with an iron-deficient diet. TfrcAlb-Cre mice exhibit a ~10-fold increase in liver hepcidin mRNA levels. This is most likely mediated by Hfe, since TfR1 is known to physically interact with Hfe and thereby inhibit its hepcidin-inducing activity. In addition, the TfrcAlb-Cre mice readily induce hepcidin in response to the high-iron diet. However, while Tfrcfl/fl controls appropriately suppressed hepcidin mRNA expression following switch from a high-iron to an iron-deficient diet, TfrcAlb-Cre mice failed to do so and maintained inappropriately elevated hepcidin mRNA levels. Finally, we observed that TfrcAlb-Cre mice failed to upregulate hepcidin mRNA in response to oral gavage with inorganic iron, contrary to Tfrcfl/fl controls. Considering that hepcidin responds to serum and hepatic iron by distinct pathways, our data suggest an involvement of hepatocellular TfR1 in the sensing serum iron levels. Thus, TfR1 appears to be dispensable for iron delivery to hepatocytes, and rather operates in these cells as an upstream regulator of iron-signaling to hepcidin.
Seventh Congress of the International BioIron Society Page 61 Podium Abstracts IBIS
Podium #2
NRF2 CONTROLS SYSTEMIC IRON HOMEOSTASIS VIA BMP6 AND HEPCIDIN AND ALLEVIATES HEMOCHROMATOSIS Pei Jin Lim, Tiago L. Duarte, Sant-Rayn Pasricha, Andrew E. Armitage, Ana G. Santos, Alireza Morovat, Elizabeth Soilleux, Chia-Yu Wang, Jodie L. Babitt, Graça Porto and Hal Drakesmith MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Pei Jin Lim
Iron is essential but toxic in excess because it catalyzes the formation of reactive oxygen species (ROS). To maintain systemic iron homeostasis, iron drives the synthesis of the iron-regulatory hormone hepcidin in part via increasing hepatic Bmp6; however the sensing mechanism linking iron to Bmp6 is unknown. We sought to determine the molecular mechanism for how iron upregulates Bmp6, a critical step in maintaining iron homeostasis. We hypothesized the involvement of Nrf2, the key transcription factor that responds to and detoxifies oxidative stress factors. Our data demonstrate parallel activation of Nrf2 and enhancement of Bmp6 expression by iron or excess heme in cell lines, purified liver sinusoidal endothelial cells and in vivo in the liver. Knockdown of Nrf2 in vitro inhibits induction of Bmp6 by iron. Nrf2- knockout mice have impaired Bmp6 and hepcidin induction in response to chronic high iron diet, injection of iron-dextran and oral iron gavage, and are more susceptible to liver iron overloading and iron toxicity. The free radical scavenger mitoTEMPO quenches ROS and prevents iron-mediated Bmp6 upregulation in vitro and in vivo, indicating that iron- induced oxidative stress rather than iron per se drives Bmp6 upregulation. Hfe/Nrf2 double knockout mice have suppressed Bmp6 expression relative to Hfe-knockout mice, and accumulate iron in heart and pancreas as well as in liver. Pharmacological activation of Nrf2 by CDDO-Imidazole increases Bmp6 in liver sinusoidal endothelial cells in vitro, enhances expression of hepatic Bmp6 in vivo, and rescues Hfe-knockout hemochromatosis mice from iron overload. In conclusion, Nrf2 is a transcription factor activated by iron-associated oxidative stress that is required for appropriate regulation of Bmp6 and hepcidin by iron. As such, Nrf2 links cellular sensing mechanisms to the control of systemic iron homeostasis, and serves as a target to ameliorate iron overload disorders.
Seventh Congress of the International BioIron Society Page 62 Podium Abstracts IBIS
Podium #3
FUNCTIONAL CONSEQUENCES OF N-LOBE SPECIFIC MUTATIONS IN THE TRANSFERRIN BINDING SITE Nermi Parrow¹, Yihang Li, PhD¹, Princy Prasad¹, Yelena Ginzburg, MD², Stefano Rivella, PhD³ and Robert Fleming, MD¹ ¹Saint Louis University School of Medicine; ²Icahn School of Medicine at Mount Sinai; ³Children’s Hospital of Philadelphia Presented By: Nermi Parrow, PhD
Introduction: Transferrin (Tf) plays a role in iron homeostasis as both an iron cargo molecule and as a signaling molecule. Tf is comprised of two homologous lobes (N and C), each of which can bind one iron molecule. As such Tf circulates as four forms: diferric, monoferric C, monoferric N and apo. The most abundant molecules in the circulation at normal transferrin saturations are the monoferric forms. The N and C lobes differ in several properties, including binding affinities for iron. Whether the lobes are functionally distinct in iron delivery has been a subject of controversy; however some data suggest that iron is released initially from the C-lobe during delivery to the erythron. Moreover, the relative abundance of monoferric N in healthy individuals is greater than C, and increased further with increased iron status. These observations led early investigators of the properties of Tf to suggest the possibility that iron occupancy of the N lobe of Tf served as means of signaling iron sufficiency and decreasing iron absorption. We thus hypothesized that loss of iron-binding capability from the N lobe would lead to hemochromatosis. Methods: In order to test this hypothesis, we generated mice producing exclusively monoferric C Tf by mutating two essential tyrosine residues, Y114 and Y207, to phenylalanine in the iron binding site of the N-lobe (N-bl Tf). Heterozygous mice were crossed and offspring analyzed for hematological parameters, tissue iron concentrations, and expression by RT-PCR of selected genes relevant to iron homeostasis. Results: At two months of age, N-bl Tf mutant mice demonstrated hepatic iron overload (1349 +/- 95.5 ug Fe/g dry weight) compared to wild-type littermates (357.1 +/- 40.9 ug Fe/g dry weight; p < 0.0001) concomitant with an approximate 2-fold decrease in Hamp1 expression (p = 0.0196). They also demonstrated a mild microcytic anemia, with hemoglobin concentrations of 12.2 +/- 0.19 g/dL vs 15.2 +/- 0.26 g/dL and MCV values of 42.55 +/- 0.21 fL vs 53.06 +/- 0.24 fL for homozygous and wild-type mice, respectively (p < 0.001 for each). Bone marrow Erfe expression is increased ~ 4-fold relative to controls (p < 0.0001), whereas expression of the Bmp/Smad target, Id1, is decreased ~ 2-fold in homozygous mice relative to controls (p = 0.03). Conclusions: Mice in which iron binding to the N-lobe of Tf is abrogated demonstrate a hemochromatosis phenotype with a mild microcytic anemia. These observations are qualitatively similar but quantitatively milder than the phenotype observed in hypotransferrinemic mice. The decreased hepcidin expression in these mice may be in part mediated by increased Erfe; however the observed decreased expression of Id1 suggests an additional Erfe-indepedent component. The observations support a model whereby loss of iron binding in the N-lobe of transferrin attenuates a signal (e.g. via transferrin receptor 2) that would normally serve to upregulate hepcidin through the Bmp/Smad pathway. Parallel investigations of the effects of exclusive production of N-lobe transferrin in C-bl Tf mutant mice are underway, with the aim of determining whether the individual lobes of transferrin are functionally heterogeneous in terms of either iron delivery or signaling.
Seventh Congress of the International BioIron Society Page 63 Podium Abstracts IBIS
Podium #4
ENDOTHELIAL CELLS PRODUCE BMP6 REQUIRED FOR IRON HOMEOSTASIS IN MICE AND PROVIDE A MODEL FOR STUDYING IRON-MEDIATED REGULATION OF THE BMP6-HEPCIDIN AXIS Susanna Canali, PhD, Chia-Yu ang, PhD, imberly umbrennen ullough, PhD, Amanda ore, PhD, Manfred Nairz, MD, PhD, Richard Bouley, PhD, Filip K. Swirski, PhD and Jodie L. Babitt, MD Program in Anemia Signaling Research, Division of Nephrology, Program in Membrane Biology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA Presented By: Susanna Canali, PhD
Background: Bone morphogenetic protein (BMP)6 signaling in hepatocytes is a central transcriptional regulator of the iron hormone hepcidin to control systemic iron balance. How iron levels are sensed to regulate hepcidin production is not known, but local induction of liver BMP6 expression by iron is proposed to have a critical role. Methods: To identify the cellular source of BMP6 responsible for hepcidin and iron homeostasis regulation, we generated global Bmp6 knockout mice and mice with a tissue specific ablation of Bmp6 in different liver cell populations, and we evaluated their iron phenotype. Efficiency and specificity of Cre-mediated recombination was assessed using Cre reporter mice, PCR of genomic DNA, and quantitation of Bmp6 mRNA expression from isolated liver cell populations. Localization of the BMP co-receptor hemojuvelin was visualized by immunofluorescence microscopy. The ability of iron to regulate Bmp6 mRNA was tested in isolated liver cell populations from mice fed diets of different iron content and in primary mouse liver cell cultures. Results: Analysis of the Bmp6 conditional knockout mice revealed that liver endothelial cells expressed Bmp6, whereas resident liver macrophages (Kupffer cells) and hepatocytes did not. Loss of Bmp6 in endothelial cells recapitulated the hemochromatosis phenotype of global Bmp6 knockout mice, whereas hepatocyte and macrophage Bmp6 conditional knockout mice exhibited no iron phenotype. Hemojuvelin was localized on the hepatocyte sinusoidal membrane immediately adjacent to Bmp6-producing sinusoidal endothelial cells. Bmp6 mRNA was regulated by dietary iron in isolated liver endothelial cells from mice and in primary liver endothelial cell cultures. Conclusions: Together, these data demonstrate that endothelial cells are the predominant source of BMP6, and support a model where endothelial cell BMP6 has paracrine actions on hepatocyte hemojuvelin to regulate hepcidin transcription and maintain systemic iron homeostasis. Analysis of isolated liver endothelial cells and primary liver endothelial cell cultures provides a foundation to elucidate the molecular mechanisms by which iron regulates BMP6 expression.
Seventh Congress of the International BioIron Society Page 64 Podium Abstracts IBIS
Podium #5
NEOGENIN FACILITATES THE INDUCTION OF HEPCIDIN EXPRESSION BY HEMOJUVELIN IN THE LIVER Ningning Zhao², Julia Maxson¹, Richard Zhang¹, Mastura Wahedi¹, Caroline Enns¹ and An-Sheng Zhang¹ ¹OHSU; ²University of Arizona Presented By: An-Sheng Zhang, MD
Hemojuvelin (HJV) regulates iron homeostasis by direct interaction with bone morphogenetic protein (BMP) ligands to induce hepcidin expression through the BMP signaling pathway in the liver. Crystallography studies indicate that HJV can simultaneously bind to both BMP2 and the ubiquitously expressed cell surface receptor neogenin. However, the role of the neogenin-HJV interaction in the function of HJV is unknown. Here we identify a mutation in HJV that specifically blocks its interaction with neogenin. Expression of this mutant Hjv in the liver of Hjv-/- mice dramatically attenuated its induction of BMP signaling and hepcidin mRNA, suggesting that interaction with neogenin is critical for the iron regulatory function of HJV. Further studies revealed that neogenin co-immunoprecipitated with ALK3, an essential type-I BMP receptor for hepatic hepcidin expression. Neogenin has also been shown to facilitate the cleavage of HJV by furin in transfected cells. Surprisingly, although cleavage of HJV by furin has been implicated in the regulation of HJV function in cell culture models and furin-cleaved soluble Hjv is detectable in the serum of mice, mutating the furin cleavage site did not alter the stimulation of hepcidin expression by HJV in mice. Additionally, in vivo studies also validated the important role of HJV-BMP interaction for HJV stimulation of BMP signaling and hepcidin expression. Together these data support a model in which neogenin acts as a scaffold to facilitate the assembly of HJV-BMP-BMP receptor complex to induce hepcidin expression.
Seventh Congress of the International BioIron Society Page 65 Podium Abstracts IBIS
Podium #6
HEPATIC HEPARAN SULFATE REGULATES HEPCIDIN EXPRESSION BY MODULATING BMP6/SMAD SIGNALING Maura Poli, PhD¹, MF Anower-E-Khuda, PhD², Michela Asperti, PhD¹, Paola Ruzzenenti, PhD student¹, Philip Gordts, PhD², Jeffrey D. Esko, PhD² and Paolo Arosio, PhD¹ ¹University of Brescia; ²UCSD Presented By: Maura Poli, PhD
BMP6/SMAD signaling regulates the expression of hepcidin, a liver peptide hormone that controls systemic iron availability by altering iron transport in gut enterocytes and macrophages. In the liver, BMP6 activates hepcidin expression through BMP receptors Type I and II and various ancillary membrane proteins, including HJV and TMPRSS6. Our previous studies showed that exogenous heparin and heparanase strongly inhibit this pathway, suggesting that cell surface heparan sulfate (HS) expressed by hepatocytes interacts and modulates BMP6 and/or its receptors. To obtain genetic evidence supporting this hypothesis, we analyzed hepcidin expression in cell and animal models bearing defects in enzymes involved in HS biosynthesis. Human hepatoma cell lines were treated with agents to inhibit HS biosynthesis (sodium chlorate), siRNAs targeting EXT1 and EXT2 (enzymes responsible for polymerization of HS) and CRISPR/Cas9 technology to knock-out key HS sulfotransferases involved in sulfation of HS (NDST1, HS6ST1 and HS6ST2). Altering HS assembly resulted in significantly reduced expression of hepcidin, in the presence and absence of BMP6 stimulation. Hepcidin expression was reduced to the greatest extent in NDST1-null cells, which express undersulfated HS. Inactivation of HS6ST1 and HS6ST2 impacted hecidin expression to a lesser extent, in agreement with previous data showing that 6- O- and 2-O-desulfation reduces the inhibitory effect of heparin on hepcidin expression. Mice with hepatocyte-specific deletion of Ndst1 (Ndst1f/fAlbCre+) have reduced expression of hepatic hepcidin and serum hepcidin levels. The reduction in hepcidin was associated with significant liver iron accumulation, which persisted on an iron-rich diet and after LPS-induced systemic inflammation. These observations provide the first genetic evidence that hepatic HS plays a key role in hepcidin expression and iron homeostasis in mice. The results suggest HS assembly as a potential therapeutic target for treatment of disorders of iron metabolism.
Seventh Congress of the International BioIron Society Page 66 Podium Abstracts IBIS
Podium #7 IPSC-DERIVED NEURONS OF PANTOTHENATE KINASE-ASSOCIATED NEURODEGENERATION SHOW MITOCHONDRIAL IRON/CALCIUM MISHANDLING Paolo Santambrogio, PhD², Daniel I. Orellana, PhD², Alicia Rubio, PhD², Anna Cozzi, PhD², Vania Broccoli, PhD³ and Sonia Levi, PhD¹ ¹Vita-Salute San Raffaele University; ²San Raffaele Scientific Institute Milano, Italy.; ³CNR- Institute of Neuroscience, Milano, Italy. Milano, Italy Presented By: Sonia Levi, PhD
Panthothenate kinase-associated neurodegeneration (PKAN) is caused by mutations in PANK2, which encodes for the mitochondrial enzyme pantothenate kinase 2. Its function is to catalyse the first limiting step of Coenzyme A (CoA) biosynthesis. PKAN accounts for about 50% of the cases of neurodegeneration with brain iron accumulation (NBIA) disorders, which are characterized by progressive disabling neurodegeneration and excessive iron deposition in the brain and for which no therapy is available. Despite iron accumulation hallmarks PKAN, its link with CoA dysfunction is not clear. In a previous work, we generated induced pluripotent stem cells (iPSC) from fibroblasts derived by three PKAN patients and three healthy subjects. Their derived neurons exhibited premature death, increased ROS production, mitochondrial dysfunctions, including impairment of mitochondrial iron-dependent biosynthesis and major membrane excitability defects respect to the control ones. CoA supplementation prevented some of these phenotypes, restoring mitochondrial and neuronal functionality. To investigate in deeper detail the link between iron dys-regulation and CoA- deficency derived mitochondrial impairment, we performed ultrastructural analysis and evaluated mitophagy and lysosome enzymatic activity. Electron microscopy analysis revealed the presence of many electron dense granules in mitochondria of PKAN neurons, which are largely prevented by the Pank2 re-expression. Electron spectroscopic imaging identified calcium phophate as component of these granules while no iron was detected. Immunofluorescence co- localization of Lamp1 and Tim44 revealed an enhanced mitophagy in fibroblasts and iPSC-derived neurons from PKAN patients respect the control ones. Moreover, PKAN iPSC-derived neurons resulted positive to the staining of b-gal activity, suggesting that also lysosomal activity is altered in PKAN neurons. These data on PKAN human neuronal model indicate that impairment of mitochondrial iron-dependent biosynthesis coexists with a large accumulation of calcium in the matrix of mitochondria, suggesting deregulation of mitochondrial calcium homeostasis. Alteration of mitochondrial iron and calcium metabolism seems to promote organelles impairment that may trigger neuronal death. The financial support of Telethon (Grant n°: GGP11088 and GGP16234) and AISNAF is gratefully acknowledged.
Seventh Congress of the International BioIron Society Page 67 Podium Abstracts IBIS
Podium #8
RESTORATION OF ENERGY METABOLISM AND CYTOSOLIC IRON BURDEN USING A COA SYNTHESIS PATHWAY INTERMEDIATE IN PKAN CELLS Suh Young Jeong, PhD, Rachel Fox, BA, Jeffrey Hamada, BA, Martina Ralle, PhD and Susan Hayflick, MD Dept. of Molecular & Medical Genetics, Oregon Health & Science University Presented By: Suh Young Jeong, PhD
Neurodegeneration with brain iron accumulation (NBIA) is a collective term for various diseases with abnormal brain iron accumulation. Among ten subtypes, PKAN (pantothenate kinase-associated neurodegeneration) is the most common type and is caused by mutations in a mitochondrial protein PANK2 (pantothenate kinase 2). Although PKAN patients show prominent iron accumulation in their GP (globus pallidus) area of the brain, PANK2 is not directly involved in iron homeostasis, yet it is a rate limiting enzyme in the CoA (coenzyme A) synthesis pathway. Moreover, another subtype of NBIA, called CoPAN (COASY protein-associated neurodegeneration) is also caused by a disease gene involved the CoA synthesis pathway. CoA is an essential substrate for numerous cellular processes including energy metabolism. Using primary fibroblasts raised from PKAN patients and controls, we found that PKAN cells generate about 4 fold more ATP compared to the control cells. However, we also found that expression of genes involved in the electron transport chain in mitochondria were all significantly decreased (about 30% compared to control). Therefore, we hypothesized that mutations in PANK2 caused ‘glycolytic shift’ in these cells. Using a Seahorse extracellular flux analyzer, we measured both OCR (oxygen consumption rate) and ECAR (extracellular acidification rate). PKAN cells showed decreased oxygen consumption and increased lactate formation in vitro. In addition to the decreased mitochondrial activity, PKAN cells also demonstrated increased cytosolic iron concentration assessed by both decreased TfR1 mRNA and ICP-MS (inductively coupled plasma mass spectroscopy). As mentioned above, PANK2 is involved in the first step of the CoA synthesis pathway converting pantothenate to the phosphorylated form. Therefore, we decided to bypass the mutation and provide cells with a CoA synthesis pathway intermediate and test whether this treatment can recover changes in energy metabolism and iron homeostasis. This treatment restored about 50% of the increased ECAR and fully increased TfR1 mRNA expression to the control level in PKAN cells after three days. These data demonstrate that mutations in PANK2 cause deranged energy metabolism in mitochondria and iron accumulation in cytosol. Using a CoA synthesis pathway intermediate, we were able to partially restore these phenotypes, suggesting these intermediates might hold potential as a drug to increase cell viability in both PKAN and CoPAN patients.
Seventh Congress of the International BioIron Society Page 68 Podium Abstracts IBIS
Podium #9
STUDY OF BRAIN IRON ACCUMULATION AND DEPOSITION OF AMYLOID PLAQUES IN A MOUSE MODEL THAT COMBINES BRAIN IRON LOADING WITH ALZHEIMER’S DISEASE AMYLOID PATHOLOGY Ritambhara Aryal¹, Jason Woods¹, Kristy Martin¹, Seak-Lin Ly¹, Daniel Johnstone, PhD² and Elizabeth Milward, PhD¹ ¹The University of Newcastle; ²University of Sydney Presented By: Ritambhara Aryal
Introduction: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that has been proposed by some researchers to be associated with brain iron accumulation. However it is unclear whether excess brain iron is a causal factor for AD or whether it is a secondary consequence of AD. By cross- din d f AD, possessing mutant versions of human APP and PS1 on a C57BL6/C3H background, with a Hfe-/-xTfr2mut mouse model of hemochromatosis, and backcrossing onto the AKR strain, which supports higher iron loading, we have generated a novel mouse model (Fe+APP/PS1) which displays chronic brain iron accumulation as well as neuropathological features of AD. Objectives: To examine brain iron levels and distribution in the Fe+APP/PS1 mouse model and assess the effect of chronic iron loading on AD-related neuropathology. Methods: Randomized fields of hippocampus and cortex between Bregma 2.45 mm and 3.87 mm from fixed-frozen brains of Fe+APP/PS1 mice (6 months age; 2 males, 2 females) and age-matched and gender-matched control APP/PS1 mice (n=4) were stained for amyloid plaques using Congo Red staining (modified Puchtler method). Simultaneously, iron was visualized in either the same section or adjacent serial sections using 3,3’-diaminobenzidine (DAB)-enhanced Perls’ stain. Relative quantification of amyloid and iron was performed using a semi-automated protocol in the Fiji distribution of ImageJ. Results a i in n i f ainin f i n a i nifi an in a d in PS1 mice compared to control APP/PS1 mice (p=0.005, n=4 mice/group), consistent with increased brain iron loading in the new model, as expected. Iron was primarily localized to myelinated structures and myelin-associated cells, as is also commonly observed in control APP/PS1 mice, normal wildtype AKR or C57BL6/C3H mice and human brain. There was no apparent redistribution of iron from myelin-associated cells with oligodendroglial morphologies to cells with neuronal morphologies, with regions of densely pack d n na a in and i a a a in i a f f aina i n a possible trend toward increased plaque load was observed for Fe+APP/PS1 mice compared to APP/PS1 mice, this was not significant (p=0.16, n=4 mice/group). By either fluorescence or polarized light microscopy, 100% of detected amyloid a a ia d i aina i n in i n a in d a d in sections (p=0.03, n=4 mice/group). Conclusion: The ini ia findin f i n d f d in a i n i n i n and i di a a not consistent with substantially increased neuronal iron levels. Instead our observations suggest that even with chronically elevated brain iron levels, myelin can provide a reservoir for brain iron storage in neuropathological conditions a i di a a a in n a ain i a ain ain in na n na i n adin a physiological levels despite extra-neuronal increases and protect against neurodegeneration. It remains possible that neuronal damage may instead occur as a secondary consequence of iron loading in non-neuronal cells or increased interactions between extracellular iron and amyloid. Further analyses to determine whether iron deposition precedes or is secondary to plaque formation and behavioral studies at different ages are underway.
Seventh Congress of the International BioIron Society Page 69 Podium Abstracts IBIS
Podium #10
SINGLE CELL RNASEQ ANALYSIS PROVIDES EVIDENCE FOR RELATIONSHIPS BETWEEN IRON-RELATED GENE EXPRESSION AND BRAIN OLIGODENDROGLIAL MYELINATING CAPABILITY Seak in y¹, arlos iveros, PhD , Moones eidari, PhD³, Daniel ohnstone, PhD⁴ and lizabeth Milward, PhD ¹The University of Newcastle; ²The University of Newcastle, Australia; ³University of Wisconsin, USA; ⁴University of Sydney, Australia Presented By: Seak Lin Ly
Introduction: Iron in the brain is stored primarily in myelin and myelin-forming cells called oligodendrocytes, which may be vulnerable to damage if iron levels become excessive. Iron dyshomeostasis could therefore contribute to myelin damage in brain white matter lesions in conditions such as multiple sclerosis, stroke and Alzheimer’s disease. Yet intriguingly only a subset of oligodendrocytes appear to contain substantial amounts of iron, even when brain iron is considerably elevated.1 This suggests there may be subgroups of oligodendrocytes that differ in regard to regulation and storage of iron. A recent Science paper by Marques and colleages2 has described subgroups of brain oligodendrocytes with different transcriptomic profiles depending on maturity, expression of key genes responsible for myelin formation and location within the brain. The objective of our study was to determine the relationships between expression of genes encoding iron-regulatory proteins and the subgroups reported by Marques and colleagues.2 Methods: We analyzed expression of 82 iron-related genes (GRCm38.85 Mus Musculus annotation) across the 13 cell compartments identified by Marques and colleagues, which included newly-formed oligodendrocytes as well as myelin- forming oligodendrocytes.2 Original RNA-Seq count data aggregated at the gene level was analyzed for differential expression of iron-related genes between pairwise combinations of compartments, without distinction of cell localization. Contrast groups were chosen according to maturation and myelinating status. The analysis was performed with a generalized linear model and normalization across cell localizations. We also used post hoc t-testing to directly compare a restricted set of compartments within the corpus callosum, a highly myelinated region. Results: Oligodendroglial compartments distinguished on the basis of maturity and myelinating capability showed differences in transcript levels for various iron-related proteins. Mature and myelinating oligodendroglial compartments had elevated levels of transcripts for transferrin (TFR), a recognized oligodendrocyte marker that was used by Marques and colleagues2 to identify mature oligodendrocytes, and for ferritin heavy chain (FTH1). For example, for cells groups dissected from the corpus callosum, the relative levels of transcripts were significantly higher for the ‘myelin-forming oligodendrocyte group’ (expressing key genes responsible for myelin formation) compared to newly-formed oligodendrocytes for both TFR (fold-change 5.6, p=0.0068, n=10-65 cells/group) and FTH1 (fold-change 6.0, p=0.0197, n=10-65 cells/group). In contrast, transcripts for ferritin light chain (FTL1) and both transferrin receptors (TFRC, TFR2) were detected at much lower apparent levels in the myelinating group and did not differ significantly from the newly- formed oligodendrocyte group (all p>0.05, n=10-65 cells/group). Of note, our preliminary analysis suggests fatty acid 2 hydroxylase (FA2H), an enzyme with recognized roles in myelin synthesis genetically implicated in the disease ‘neurodegeneration with brain iron accumulation (NBIA)’, showed similar patterns of transcript expression to TFR and FTH1 across the various cell groups, with greatest expression in mature and myelinating oligodendrocytes, although apparent transcript levels were lower. Conclusions: Alterations in iron-related gene expression may distinguish myelinating from newly-formed oligodendrocyte compartments and reveal relationships between iron homeostasis and brain oligodendroglial myelinating capability. This gives new perspectives both on the importance of iron in normal myelinogenesis and on how iron dysregulation may contribute to a variety of brain diseases. 1 2 References: Heidari et al. Molecular Psychiatry, 2016;11:1599-1607; Marques et al. Science, 2016;352:1326-1329.
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Podium #11
MODULATING ANTIBODY AFFINITY TOWARDS THE TRANSFERRIN RECEPTOR TO INCREASE BRAIN UPTAKE OF ANTI-TRANSFERRIN RECEPTOR ANTIBODY TARGETED GOLD NANOPARTICLES asper endi ohnsen, MSci , Martin ak, ivil Ing³, redrik Melander, ivil Ing, PhD⁴, Annette urkhart, MSci, PhD , Paul Kempen, MSci, PhD³, Thomas Lars Andresen, Civil Ing, PhD³ and Torben Moos, MD, PhD, DMSci¹ ¹Aalborg University; ²Laboratory for Neurobiology, Institute of Health Science and Technology, Aalborg University, Denmark; ³Center for Nanomedicine and Theranostics, Department of Micro- and Nanotechnology, Technical University of Denmark, Denmark; ⁴ enter for anomedicine and heranostics, Department of Micro- and Nanotechnology, Technical University of Denmark, Denmark Presented By: Torben Moos, MD, PhD, DMSc
Drug delivery to the brain is hampered by the presence of the blood-brain barrier (BBB) that under physiological conditions precludes entrance of most substances contained in the systemic circulation. Thus, this barrier must be overcome to deliver medicines into the brain parenchyma. The transferrin receptor is exclusively expressed on capillaries of the brain, which makes it an interesting target for transport of drugs towards the brain. However, the current evidence on the receptor movement in brain capillaries does not suggest transcytosis, and delivering medicines or nanoparticles using antibodies towards this receptor has largely been without success. We investigated the impact of antibody affinity on the transport of gold nanoparticles into the brain parenchyma. PEGylated gold nanoparticles were conjugated to either a high or low affinity antibodies towards the transferrin receptor, an isotype IgG control, or no antibodies, and injected into mice. Brain capillary depletion, ICP-MS, and various microscopy techniques were employed to analyse the resulting tissue. For the transferrin receptor-targeted groups, gold nanoparticles could be detected along vessel structures as revealed by silver enhancement and light microscopy. Electron microscopy showed that the particles had been efficiently endocytosed into the endothelial cells of the BBB. A small fraction of particles could also be detected in the brain parenchyma, which was underscored by measuring the gold content in brain parenchyma after capillary depletion. Furthermore, the uptake of gold nanoparticles into both the brain capillaries and brain parenchyma were significantly affected by the affinity of the attached antibodies.
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Podium #12
CONDITIONAL MULLER CELL ABLATION LEADS TO RETINAL IRON ACCUMULATION Bailey Baumann, BA, Ying Song, MD, Delu Song, MD,PhD, Jacob Sterling, BS,MS, Weiyong Shen, MD,PhD, Mark Gillies, PhD and Joshua Dunaief, MD,PhD FM Kirby Center for Molecular Ophthalmology, School of Veterinary Medicine, University of Pennsylvania Presented By: Bailey Baumann, BA
Purpose: For largely unknown reasons, iron homeostasis becomes dysregulated and retinal iron accumulation is observed in retinal degenerative diseases, including age-related macular degeneration (AMD). Müller cells, the major glial cells of the retina, may be important mediators of retinal iron transport, distribution, or regulation due to their role in blood- retinal barrier formation, their expression of proteins involved in cellular iron regulation, and their trans-retinal position. A novel transgenic model of Müller cell loss recently demonstrated that primary Müller cell ablation leads to blood-retinal barrier leakage and photoreceptor degeneration and recapitulates clinical features observed in Macular Telangectasia type 2 (MacTel2), a rare human disease that features perifoveal Müller cell loss. In this study, we used this mouse model to determine the effect of Müller cell loss on retinal iron homeostasis. Methods: Changes in total retinal iron levels after Müller cell ablation were directly assessed using inductively coupled mass spectrometry. Corresponding changes in the expression of proteins involved in the control of iron flux and storage were also characterized using qPCR and Western analysis. Results: We found that Müller cell loss leads to increased iron levels throughout the neurosensory retina. Among the mechanisms that we explored, the most compelling mechanism for retinal iron accumulation after Müller cell loss is excessive influx of iron into the retina related to the loss of blood-retinal barrier integrity. We also found increased iron levels within the photoreceptors of a retinal section from a patient with MacTel2, further indicating Müller cell loss leads to retinal iron dysregulation. Conclusions: This study shows that Müller cells play a pivotal role in the regulation of retinal iron homeostasis; an important finding as retinal iron dysregulation following Müller cell loss may contribute to retinal degeneration in this model and in diseases such as MacTel2.
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Podium #13
ACONITASE: AN IRON SENSING REGULATOR OF MITOCHONDRIAL OXIDATIVE METABOLISM AND ERYTHROPOIESIS Grant Bullock, MD, PhD, Naomi Gunawardena, BS, Valerie Miller, MS, Chante Richardson, PhD, Teague Cole, BS, Catherine Corey, MPH, Yinna Wang, MD, PhD and Sruti Shiva, PhD University of Pittsburgh Presented By: Grant Bullock, MD, PhD
Anemia is a global health problem that decreases quality of life for billions of people. Previous studies have concluded that there is an iron-regulated checkpoint in erythropoiesis that suppresses red blood cell production. This results in anemia and the conservation of iron for use in other vital processes. We have shown that the aconitase enzymes are key to this pathway and that inhibition of aconitase enzyme activity by iron restriction or pharmacologic inhibitors blocks erythropoiesis in primary human hematopoietic progenitor cells (HPCs). Mitochondrial aconitase (ACO2) functions as an isomerase within the tricarboxylic acid (TCA) cycle to convert citrate into isocitrate, which contributes to ATP and heme synthesis. During iron restriction, there is a significant decrease of intracellular isocitrate with only a slight increase in intracellular citrate. A corresponding increase in ATP-citrate lyase activity suggests that excess citrate is shunted into acetyl-CoA production. The addition of exogenous isocitrate to iron-deprived HPCs abrogates the block in erythropoiesis and protects iron-deprived mice and chronically-inflamed rats from anemia. These results suggest that ACO2 regulates mitochondrial metabolism and erythropoiesis. Recent unpublished data shows that ACO2 inhibition by iron deprivation or by treatment with an ACO2 inhibitor decreases mitochondrial respiratory rates (RR) and increases mitochondrial reactive oxygen species (mito-ROS). Isocitrate normalizes RR and mito-ROS and restores erythropoiesis. Importantly, disruption of ROS generation with a variety of anti-oxidants blocks erythropoiesis, while surprisingly, treatment of iron restricted HPCs with oxidant generators or ROS promotes erythropoiesis. These data inform our overarching hypothesis that iron- restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. We have recently extended these studies to ACO2 knock down (ACO2-KD) K562 cell lines which provide more material for biochemical assessment of mitochondrial function. New pilot data shows that a 70% decrease in ACO2 expression significantly reduces the induction of erythroid specific genes during hydroxyurea or hemin treatment. This confirms that ACO2 plays a direct role in erythropoiesis. Extracellular flux (XF, Seahorse Bioscience) experiments show a decreased RR in ACO2-KD cells. Mitochondrial complex activity assays show no differences in complex IV or citrate synthase activity between control and ACO2-KD cell lines. These studies also demonstrate that the shRNA-ACO2 lentiviral constructs are effectively targeting ACO2 and can be used in our HPC model system. We now have evidence in two different human cell culture models of erythropoiesis that mitochondrial aconitase is an iron-sensing regulator of both mitochondrial respiration and erythropoiesis. Our long term goals are to identify novel therapeutic targets in this iron dependent metabolic regulatory pathway that enhance or suppress erythropoiesis and have potential clinical application in the treatment of anemia or polycythemia. We are also investigating the role of the mitochondrion in the differentiation of other hematopoietic cell lineages.
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Podium #14
FUNCTIONAL IRON DEFICIENCY CAUSED BY LOSS OF IRON REGULATORY PROTEIN 2 LEADS TO PANCREATIC ß CELL DYSFUNCTION Elizabeth Leibold, PhD, Cole P. Anderson, PhD and Steven J. Romney, BS University of Utah Presented By: Elizabeth A Leibold, PhD
Iron regulatory protein 2 (Irp2) is a key regulator of cellular iron metabolism in vertebrates. Irp2 controls the post- transcriptional expression of the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin. Previous studies have shown that mice with a targeted deletion of the Irp2 gene develop microcytic anemia, erythropoietic protoporphyria and neurobehavioral impairments. Here, we show that Irp2-/- mice develop fasting hyperglycemia, glucose intolerance and impaired glucose- i a d in in i n f an a i -cells, all of which have been implicated in the pathogenesis of diabetes mellitus. Impaired glucose-stimulated insulin secretion in Irp2-/- -cells is not caused by d d a a d d in in n n d d f i in in i n i n a i i dysregulated in Irp2-d fi i n a id n d by decreased TfR1 expression and iron content, and increased ferritin expression and iron sequestration causing functional iron deficiency. Disruption of cellular iron homeostasis is associated with reduced activity/levels Fe-S proteins, reduced ATP production and endoplasmic reticulum stress. Increasing cellular iron in Irp2-d fi i n n a i in in n n and i -stimulated insulin secretion. These studies reveal a previously unidentified link between insulin biosynthesis and cellular iron homeostasis, and show that Irp2 is i d f n a f n i n and d a i
Seventh Congress of the International BioIron Society Page 74 Podium Abstracts IBIS
Podium #15
DISREGULATED IRON AND LIPID METABOLISM IN MICE WITH LIVER-SPECIFIC KNOCKOUT OF IRON CHAPERONE PCBP1 Olga Protchenko, PhD¹, Fengmin Li, PhD¹, Minoo Shakoury-Elizeh, MS¹, Manik C. Ghosh, PhD², Oksana Gavrilova, PhD¹ and Caroline C. Philpott, MD¹ ¹National Institutes of Health, NIDDK; ²National Institutes of Health, NICHD Presented By: Olga Protchenko, PhD
Complex regulation of cellular iron requires proteins of diverse molecular function, such as transcription factors, ubiquitin ligases, chaperones, etc. PCBP1-4 proteins have been initially identified as RNA-binding, scaffold proteins engaged in different protein- and RNA-protein complexes. PCBP1 and PCBP2 are abundant cellular proteins that share 83% identity. PCBPs can bind iron and work as cytosolic iron chaperones for ferritin, prolyl hydroxylase 2, and deoxyhypusine hydroxylase. In the present study, we addressed how the depletion of PCBP1 in the liver, the main iron storage organ, affected iron homeostasis and liver physiological function. We have bred PCBP1flox/flox mice with hepatocyte-specific expression of Cre recombinase. Alb-Cre PCBP1flox/flox mice exhibited near complete depletion of PCBP1 in the livers. Alb- Cre PCBP1flox/flox mice did not appear sick or different from wild type littermates. However, livers of Alb-Cre PCBP1flox/flox mice had ~ two-fold reduced non-heme iron content on normal iron diet (50 ppm). Transferrin receptor 1 mRNA was increased two fold in knockout mice when compared to the PCBP1flox/flox mice. There was no significant difference for other iron-related transcripts: BMP6, DMT1, HO-1, ferroportin. Ferritin and IRP2 protein levels also did not differ significantly between Alb-Cre PCBP1flox/flox and PCBP1flox/flox mice. Surprisingly, measurement IRP1 activity by bandshift assay showed lower IRE binding activity in the samples from PCBP1 knockout livers. Overall these data suggest that depletion of PCBP1 resulted in dis-regulated iron homeostasis and minor iron-deficiency phenotype. Liver-specific knockout of PCBP1 affected lipid homeostasis in this organ. Young Alb-Cre PCBP1flox/flox mice had increased triglycerides (TG) in the liver. Lipidomics analysis revealed that livers depleted of PCBP1 had also increased free fatty acids, diacylglycerides and phospholipids. Preliminary data from RNA sequencing indicated up-regulation of genes activated by sterol-regulatory element binding proteins, SREBP1 and 2, in knockout livers. Increased lipid accumulation was associated with steatohepatitis and hypercholesterolemia in aged PCBP1 knockout mice (10 month). Dietary iron supplementation did not affect liver TG in Alb-Cre PCBP1flox/flox mice, while PCBP1flox/flox mice had increased liver TG under these conditions. However, lowering iron in the diet might have effect and these experiments are in progress. Taken together our data indicate that liver-specific depletion of PCBP1 resulted in altered iron regulation and lipid homeostasis.
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Podium #16
AN ESSENTIAL ROLE FOR THE BMP-SMAD PATHWAY IN HEPCIDIN INDUCTION DURING ER STRESS AND NAFLD/NASH Audrey Belot, PhD student², Ophélie Gourbeyre, Aude Rubio, Céline besson-Fournier, Chloé Latour, Inka Gallitz, Alexandra Montagner, Arnaud Polizzi, Marion Régnier, Andrea U Steinbicker, Hervé Guillou, Marie-Paule Roth, Hélène Coppin and Delphine Meynard, PhD¹ ¹INSERM; ²IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, France Presented By: Delphine Meynard, PhD
There is mounting evidence that both endoplasmic reticulum (ER) stress and hepatic iron deposition contribute to nonalcoholic steatohepatitis (NASH). Interestingly, ER stress is known to upregulate hepcidin, which could prevent iron efflux from macrophages and hepatocytes and cause hepatic iron deposition. The objective of this study was to characterize in vivo the mechanisms responsible for increased hepcidin expression during ER stress and NASH. To address this question, ER stress was induced in mice with tunicamycin (Tm). In wild-type mice (WT), Tm injection triggers ER-stress in the liver and leads to an increase in hepcidin mRNA expression. Crebh, a liver-specific transcription factor, was previously suggested to play essential role in hepcidin regulation in response to ER stress. However, in our hands, hepcidin mRNA was increased to similar levels in Tm-injected Crebh-/- and wild-type (WT) mice, which seriously questions this hypothesis. Rather, we observed a rise in Smad5 phosphorylation and in Id1 mRNA expression in the liver in Tm-injected mice, indicating that Bmp-Smad signaling is activated in response to ER stress. An essential role for this pathway was provided using Bmpr1a-/- mice. Indeed, lack of Bmpr1a, the BMP type I receptor Alk3, prevents the induction of hepcidin mRNA expression in response to Tm treatment. To find out what activates Bmp-Smad signaling in a in d i n f diff n i and f fa i H n n f ligands was induced in response to ER stress. Notably, activin B-deficient mice injected with Tm induce hepcidin as much as do WT mice, which eliminates the possibility that activin B plays a role in ER stress. BMP-Smad signaling in ER stress could also be activated independently of the induction of a ligand. Interestingly, we found a dramatic repression of Tmprss6 gene expression in Tm-injected mice, which very likely explains the increase in Smad5 phosphorylation and the induction of Id1 and hepcidin mRNA expression seen in these mice. To determine whether a similar mechanism explains the induction of hepcidin during NASH, we fed WT mice with a Methionine Choline Deficient (MCD) diet for 10 and 24 days. As expected, MCD diet leads to accumulation of lipid droplets and inflammatory foci in the liver of these mice, and a n ind i n f if2α a i n an di a i a idin expression and promotes liver iron deposition in hepatocytes and Kupffer cells. Remarkably, hepcidin upregulation in these mice coincides with an increase in Smad5 phosphorylation, an induction of Id1 and a repression of Tmprss6 expression, which suggests that the molecular mechanism of hepcidin upregulation in NASH is similar to the mechanism highlighted previously in Tm-injected mice and very likely reflects ER stress caused by lipid accumulation. Altogether, this study shows that hepcidin up-regulation in response to ER stress and NAFLD/NASH is promoted by the activation of Bmp-Smad signaling. Inhibiting the BMP-SMAD pathway could be a promising therapeutic option for NASH patients to reduce hepcidin expression, avoid liver iron deposition and thus prevent disease progression.
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Podium #17
LOW IRON DIET AND CHELATION THERAPY PREVENT IRON-AGGRAVATED ATHEROSCLEROSIS IN A MOUSE MODEL OF HEREDITARY HEMOCHROMATOSIS Francesca Vinchi¹, Andreas Simmelbauer, MD student¹, Sandro Altamura, PhD¹, Richard Sparla, BTA¹, Graca Porto, MD , Sebastian Speich, MD³, runo Galy, PhD⁴, Matthias entze, MD and Martina U Muckenthaler, PhD¹ ¹Molecular Medicine Partnership Unit , University of Heidelberg & EMBL, Heidelberg, Germany; ²University of Porto, Porto, Portugal; ³University of eidelberg,Germany; ⁴German ancer esearch enter D , Germany; uropean Molecular Biology Laboratory (EMBL), MMPU, Heidelberg, Germany Presented By: Francesca Vinchi, PhD
In 1981, Sullivan proposed the “iron hypothesis”, which states that iron is detrimental for the cardiovascular system, promoting atherosclerosis progression. Iron levels are increased in hereditary hemochromatosis as well as in iron-loading anemias, such as thalassemia, sickle cell disease and myelodysplastic syndromes. In the latter iron levels may be further increased due to chronic red blood cell transfusions. To date it is unclear whether iron overload in these disorders promotes atherosclerosis. Conflicting evidence is provided by epidemiological data and studies in disease models. To assess whether iron overload affects atherosclerosis progression, we analyzed ApoE-deficient mice crossbred with a mouse model of hereditary hemochromatosis type IV, due to a point mutation in the iron exporter ferroportin, which prevents hepcidin binding. We show that at 6 and 12 months of age hemochromatotic ApoE-null mice show a strong increase in lesion size and numbers compared to ApoE-null mice. The atherosclerotic phenotype positively correlates with increased levels of serum iron and transferrin saturation, as well as with iron deposition in the vascular smooth muscle cells, which cause vascular oxidative stress and vessel stiffness. High circulating iron levels promote circulating LDL oxidation, vascular endothelium activation and permeabilization, nitric oxide consumption and inflammation (increased MCP1 and VEGF). In hemochromatotic ApoE-null mice atherosclerotic plaques show reduced collagen deposition and elevated macrophage numbers as well as increased lipid content and calcification, suggesting enhanced plaque vulnerability and accelerated disease progression. Consistently, these mice develop compensatory left ventricular hypertrophy, associated with increased left ventricle diastolic volume and area. To reduce iron levels we maintained hemochromatotic ApoE-null mice either on a low iron diet or on iron chelation therapy (Deferasirox). Both, prolonged maintenance on a low iron diet or iron chelator treatment rescued the severe atherosclerotic phenotype in 6 and 10 month-old hemochromatotic ApoE-null mice. Importantly, these treatments significantly lowered serum iron levels and transferrin saturation as well as arterial iron deposition. As a consequence, endothelial activation, pro-inflammatory molecule production and LDL oxidation are strongly reduced, limiting atherosclerosis progression in these mice. Interestingly, the analysis of serum samples from a cohort of hemochromatotic patients (HFE C282Y mutation) shows positive correlation between iron indices and markers of endothelial dysfunction, inflammation and lipid oxidation. Taken together our data suggest that systemic iron overload aggravates the severity of atherosclerosis, thus indicating that high circulating iron levels are a risk factor for cardiovascular disease. Furthermore our results demonstrate the beneficial effects of iron restriction, achieved either via dietary iron limitation or iron chelation therapy, in counteracting iron-induced atherosclerosis progression. Importantly, these observations highlight the pro-atherosclerotic implications of iron for pathological conditions associated with iron overload and underscore the importance of lowering systemic iron levels in these patients.
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Podium #18
GENETIC LOSS OF TMPRSS6, THE GENE MUTATED IN IRON-REFRACTORY IRON DEFICIENCY ANEMIA, DISRUPTS PHOSPHATE HOMEOSTASIS IN MICE Xiuqi Li, BS, Larisa Lozovatsky, MS, Abitha Sukumaran, PhD, Steven Tommasini, PhD, Jackie Fretz, PhD and Karin Finberg, MD, PhD Yale School of Medicine Presented By: Xiuqi Li, BS
The hepatic transmembrane protease TMPRSS6 inhibits the expression of hepcidin, a circulating hormone that decreases intestinal iron absorption and macrophage iron recycling. Accordingly, TMPRSS6 mutations result in elevated hepcidin levels, impaired dietary iron absorption, and systemic iron deficiency in both human and mice. Recent studies suggest a link between systemic iron homeostasis and production of FGF23, a hormone secreted by osteocytes that regulates systemic phosphate homeostasis. Here we ask if mice with genetic loss of Tmprss6 exhibit alterations in Fgf23 expression and phosphate homeostasis. By intercrossing Tmprss6+/- mice, we generated Tmprss6-/- mice and littermate controls, which were raised on a standard rodent diet (Teklad 2018S containing 200 ppm iron and 0.7% phosphorous) prior to analysis at 8 weeks of age. Compared to littermate controls, Tmprss6-/- mice displayed hepcidin elevation, markedly reduced liver non-heme iron concentration, and microcytic anemia, consistent with prior studies. Analysis of RNA isolated from flushed femurs revealed that Fgf23 mRNA levels were significantly elevated in Tmprss6-/- mice compared to littermate controls. Additionally, compared to littermate controls, Tmprss6-/- mice showed significantly higher circulating levels of the Fgf23 hormone, as assessed by an enzyme-linked immunosorbent assay (ELISA) that detects the biologically-active (“intact”) form of the Fgf23 hormone, and also as assessed by an ELISA that detects the active form as well as C-terminal products of Fgf23 cleavage. Because Fgf23 is known to inhibit renal tubular phosphate reabsorption, we also examined if the Fgf23 elevation in Tmprss6-/- mice was associated with alterations in serum and/or urine phosphate parameters. Indeed, Tmprss6-/- mice displayed a trend toward lower serum phosphate concentration, as well as a significant elevation in urinary phosphate concentration when normalized to urine creatinine. Because phosphate is required for bone mineralization, we also analyzed the tibiae of Tmprss6-/- mice and littermate controls, and we discovered that the tibiae of Tmprss6-/- mice showed alterations in biomechanical properties. Fgf23 has been proposed as the basis for a bone-kidney axis that coordinates bone health with renal handling of phosphate. Together, our findings suggest that elevation of Fgf23 in Tmprss6-/- mice with iron deficiency may be the cause of phosphaturia and altered bone function. These results may have relevance to the skeletal health of patients with Iron Refractory Iron Deficiency Anemia due to TMPRSS6 mutation and those with other forms of iron restricted anemia resulting from hepcidin elevation. Additionally, as Tmprss6-/- mice are known to exhibit growth retardation, our results raise the possibility that disrupted phosphate homeostasis may be a potential mechanism that contributes to the commonly observed association of childhood iron deficiency with growth impairment.
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Podium #19
TYPE 2 DIABETES RISK AND NORMATIVE VALUES FOR IRON BIOMARKERS Leo Zacharski¹, Galina Shamayeva, MS², Bruce Chow, MS² and Ralph DePalma, MD³ ¹VA Hospital & Dartmouth Hitchcock Medical Center; ²Palo Alto VA Hospital; ³VA Cebtral Office Presented By: Leo Zacharski, MD
Type 2 diabetes (T2D) risk is associated with altered iron metabolism but risk assessment using common iron biomarkers, ferritin and percent transferrin saturation (%TS), is uncertain. We examined normative levels of ferritin, %TS and hemoglobin (representing physiologic iron) to estimate their utility for T2D risk prediction. Ferritin and %TS values predicting T2D reported epidemiologically were compared to ferritin, %TS and hemoglobin levels from 1,277 primarily males with peripheral arterial disease (PAD; 37% diabetic) entered to a randomized trial of iron reduction by calibrated phlebotomy. Iron biomarker relationships based on over 8,300 paired samples were displayed by Loess Locally-Weighted Regression curves. Increasing hemoglobin corresponded to increasing ferritin up to about 80-100 ng/mL (left figure); further ferritin increase lacked physiologic correspondence. Rising hemoglobin and %TS up to about 25% TS corresponded to physiologic iron transport for erythropoiesis. Loess plots in the diabetic cohort were similar to non- diabetics but displaced by lower hemoglobin values. Proportionately increasing ferritin up to about 80-100 ng/mL and %TS up to about 25% corresponded to epidemiologic and clinical trial outcome measures associated with minimal T2D and PAD risk reflecting compensation for excess iron by iron-scavenging transferrin. T2D and PAD outcome data suggest further clinical risk limitation with %TS levels up to about 50% (right figure). Ferritin levels above an upper threshold of about 80-100 ng/mL and %TS above about 25-50% in PAD corresponded to epidemiologic measures of iron biomarkers associated with increased T2D and PAD risk. These levels may reflect normative values useful for monitoring interventions to reduce risk of T2D and other diseases of excess iron.
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Podium #20
HEPCIDIN MEDIATES HOST DEFENSE AGAINST GRAM-NEGATIVE PATHOGENS BY CONTROLLING NON- TRANSFERRIN-BOUND IRON D. Stefanova, A. Raychev, J. Arezes, K. Michels, B. Dillon, M. Horwitz, B. Mehrad, T. Ganz, E. Nemeth and Y. Bulut University of California, Los Angeles Presented By: Debora Stefanova
Hepcidin, the iron-regulatory hormone, is strongly induced by inflammation causing rapid hypoferremia, a mechanism proposed to limit bacterial replication. Indeed, iron-overloaded patients are more susceptible to characteristic infections, and iron supplementation in some human trials increased life-threatening infections. However, the mechanism by which hepcidin and hypoferremia mediate resistance to infection are poorly understood. Conversely, hepcidin causes iron accumulation in macrophages which could enhance pathogenesis by intracellular microbes. To study the effect of hepcidin and iron on microbial pathogenesis we infected hepcidin knockout mice (HKO), a model of hereditary hemochromatosis, with: 1) Extracellular siderophilic pathogens reported to cause infections in iron-overloaded patients: Gram- Yersinia enterocolitica and Vibrio vulnificus; 2) Extracellular non-siderophilic bacteria that are clinically common: Gram- Klebsiella pneumoniae and Gram+ Staphylococcus aureus; 3) Intracellular, macrophage-tropic Mycobacterium tuberculosis. Hepcidin ablation had the greatest effect on infections with siderophilic pathogens Y. enterocolitica O9 and V. vulnificus. At inocula where WT mice were resistant and had 0% mortality, iron-loaded HKO mice suffered 100% mortality. Compared to WT mice, iron-loaded HKO were also more susceptible to infection with K. pneumoniae, and displayed more rapid and higher mortality. In all these models, treatment of infected iron-loaded HKO mice with hepcidin analogs prevented or decreased mortality. Short-term administration of hepcidin analogs altered serum iron concentrations but not iron stores, indicating that only extracellular iron availability modulated infection with these pathogens. In contrast, non-siderophilic Y.enterocolitica serotype O8 infected WT and iron-loaded HKO with similar virulence (50% mortality). Despite having greater hosts range, Y.enterocolitica O8 was significantly less pathogenic in iron overload conditions than O9, suggesting that its more effective iron uptake system comes at the cost of decreased virulence when iron is abundant. For two other pathogens, catheter-associated Gram+ S. aureus and the macrophage- tropic M. tuberculosis there was no difference in the outcome of infection between HKO and WT mice. We next examined the mechanism by which hepcidin deficiency promotes susceptibility to systemic infection and mortality. Extracellular iron is bound by transferrin but in iron-loaded conditions the binding capacity of transferrin can be exceeded leading to the formation of non-transferrin-bound iron (NTBI), which may be more readily utilizable by microbes. Indeed, total serum iron concentration in different groups of mice did not correlate with their susceptibility to infection. In contrast, NTBI was detectable only in the plasma of animals that developed systemic infection and/or suffered mortality. To test the role of NTBI, we developed an in vitro model of iron-dependent bacterial growth. Human plasma was supplemented with increasing concentrations of ferric iron to produce agar plates with increasing transferrin saturation, and when transferrin was saturated, increasing NTBI concentrations. V.vulnificus, Y.enterocolitica O9 and K.pneumoniae only grew when NTBI was present. Thus, hepcidin is essential for resistance to infection with certain gram-negative pathogens, but plays no role in the susceptibility to intracellular M. tuberculosis or gram-positive S. aureus. We further provide evidence that the important host defense function of hepcidin is the elimination of NTBI, and that resistance to iron-enhanced infections can be potentiated by the use of synthetic hepcidin analogues to prevent fulminant infection and associated mortality.
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Podium #21
EPIDERMAL HEPCIDIN IS PROTECTIVE AGAINST GROUP A STREPTOCOCCUS INFECTION AND SYSTEMIC BACTERIAL SPREAD Mariangela Malerba, PhD student, Jacques Mathieu, Sabine Louis, Sylvain Cuvellier and Carole Peyssonnaux INSERM, U1016, CNRS UMR8104, Université Paris Descartes, Sorbonne Paris Cité, Institut Cochin Presented By: Mariangela Malerba
Hepcidin, demonstrated to be the key iron regulatory hormone, is mainly produced by the hepatocytes. However, an increasing number of studies showed that hepcidin is also expressed by other tissues. Thanks to the generation of conditional knockout of hepcidin, we previously demonstrated that hepatic hepcidin is sufficient to ensure systemic iron homeostasis in physiological conditions suggesting that production of hepcidin by extra-hepatic tissues may have local roles. Hepcidin was originally identified as a cationic antimicrobial peptide (AMP) by its close similarity to the beta defensins. AMPs are known to play essential roles in maintaining epithelia integrity but also in innate immunity and host defense against infection. So far hepcidin expression in the epithelia, a major source of AMP, and its role as a local AMP in vivo has been poorly investigated. We develop new mouse models in order to investigate the role of hepcidin in the skin d in inf i n i -hemolytic streptococci (GAS). These gram-positive bacteria are responsible for a wide range of both invasive and non-invasive infections causing more than 500,000 deaths per year. We first found, by immunohistochemistry, that hepcidin was highly induced in the skin of WT mice subcutaneously (SC) infected with GAS. Particularly, we detected hepcidin expression in keratinocytes and in myeloid cells recruited to the site of infection. To discriminate the contribution of these two sources of hepcidin production during infection, we specifically deleted hepcidin in keratinocytes and in myeloid cells, by breeding Hepclox/lox mice with a transgenic strain expressing the Cre recombinase respectively under the control of the murine K14 promoter (Hepc KOkerat) or under the control of the murine Lys M promoter (Hepc KOmyeloid). Four days after the SC infection by GAS, we found that the Hepc KOmyeloid mice and WT littermates showed the same number of bacteria at the local and systemic level. In contrast, the Hepc KOkerat mice had a significantly higher number of bacteria than the WT littermates at the lesion site (2,5 .106 vs 8. 104 CFU/mg) but also in the blood (2,7.104 vs 2.103 CFU/ml) and in peripheral organs such as the spleen (103 vs 30 CFU/mg). We next investigated the mechanisms by which epidermal hepcidin restrains the infection. We showed that hepcidin had no direct bacteriostatic effect against GAS in vitro and that primary keratinocytes derived from WT and Hepc KOkerat mice displayed the same bactericidal activity against this pathogen. However, we found that hepcidin had immuno-modulatory function on keratinocytes in vitro and that the number of neutrophils recruited at the site of infection was lower in the Hepc KOkerat mice compared to WT littermates. Our results suggest that hepcidin production by keratinocytes but not by myeloid cells is important in controlling bacterial infection through a new immunoregulatory role of hepcidin, that we are currently investigating. In conclusion, hepcidin production by keratinocytes mice restricts local and systemic spread of infection from an initial tissue focus, through regulation of immune cells. Hepcidin agonists may represent a novel approach for adjunctive therapy of complicated infections due to antibiotic-resistant pathogens or compromised host immunity.
Seventh Congress of the International BioIron Society Page 81 Podium Abstracts IBIS
Podium #22
A NOVEL IRON-MEDIATED MECHANISM FOR DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE Shirly Moshe-Belisowski, PhD, Lulu Fahoum, MSc, Lena Lifshitz, PhD, Abraham Nyska, PhD, Avi Zuckerman, PhD, Matti Waterman, MD, Roni Weisshof, MD, Orly Savion, PhD, Fabio Cominelli, MD PhD and Esther G. Meyron-Holtz Technion - Israel Institute of Technology Presented By: Esther Meyron-Holtz, PhD
Background: Iron accumulation in inflammatory lesions has been described in many settings but a molecular mechanism for this accumulation was not completely elucidated. We hypothesized that an iron independent activation of RNA-binding activity of the Iron Regulatory Protein (IRP)1, via reactive oxygen and nitrogen species is the driving force for this iron accumulation and for the exacerbation of inflammation. Methods: To test this hypothesis, we examined changes in iron homeostasis during inflammation in human biopsies from Crohn’s disease patients, a mouse-model of inflammatory bowel disease - α in mouse (TNF ) - and an epithelial cell model. We analyzed the effect of IRP1 and 2 deletions on the course of the inflammation and performed differential analyses of different cell-types within the inflamed tissue. Results: We found that in human biopsies the ferritin content of epithelial cells was significantly reduced, compared to non-inflamed sections of the same patient. The terminal ileitis, which developed in the TNF mouse, was accompanied not only by a local iron accumulation in the lamina propria but also by profound iron redistribution within the inflamed tissue. While the local and infiltrating macrophages accumulated iron, the inflamed epithelial cells showed a state of significant iron deficiency and elevated iron flux, which was mimicked in the epithelial cell model of inflamed Caco-2 cells. The significant epithelial iron depletion was accompanied by elevated levels of the Hypoxia inducible transcription fa H 2α a d -binding activity of both IRP1 and IRP2 in the epithelial layer, but a decrease in IRP2 activity in the lamina propria. Accordingly, levels of proteins involved in the cellular iron homeostasis were altered. These new dynamics of iron homeostasis were completely reversed by the deletion of IRP1 in the TNF mouse and the n i i f inf a a i n f mouse was not detectable in the TNF x IRP1-/- mice. In contrast, TNF x IRP2-/- mice showed severe transmural ileitis. Discussion: The non-iron mediated activation of IRP1 during inflammation triggered an impaired iron homeostasis within the inflammatory lesion that led to iron depletion of the epithelial barrier and the accumulation of iron in the local immune cells. The iron accumulation in these cells and the iron depletion of the epithelial cells both support the recruitment of a systemic immune response and the propagation of an initially local and close to physiologic inflammation of the gut to a severe inflammation with massive infiltration of systemic immune cells. Conclusions: The finding that targeted deletion of IRP1 but not IRP2 completely abolished the inflammation in the a a a a f ifi in i i i n a inf a a diseases.
Seventh Congress of the International BioIron Society Page 82 Podium Abstracts IBIS
Podium #23
BREAST CANCER TUMORIGENICITY IS DEPENDENT ON HIGH EXPRESSION LEVELS OF NAF-1 AND THE LABILITY OF ITS Fe-S CLUSTERS Rachel Nechushtai¹, Merav Darash-Yahana, PhD¹, Ola Karmi¹, Y-S Sohn, PhD¹, Eli Pikarsky, MD/PhD², Ioav Cabantchik, MD/PhD¹ and Ron Mittler, PhD³ ¹HUJI; ²Hadassah; ³UNT Presented By: Rachel Nechushtai, PhD DSc
NEET proteins comprise a unique class of iron-sulfur (2Fe-2S) proteins. Their clusters are coordinated by a 3Cys:1His structure which allows it to be both relatively stable, as well as to transfer to apo-acceptor protein(s). This unique feature of NEET proteins is likely controlled by the NEET's 2Fe-2S's coordinating His that is positioned at the protein surface and can undergo protonation that destabilizes the cluster and enables it's transfer (1). Recent studies implicated NEET proteins in a diverse array of biological processes and diseases. Localized to the ER, outer mitochondria and the membranes that connect them, the NEET protein NAF-1 (encoded by CISD2) is implicated in the regulation of autophagy, apoptosis, ER-calcium signaling, and iron and reactive oxygen species (ROS) homeostasis. NAF-1 was also linked to neurodegenerative diseases, skeletal muscle maintenance, cancer and aging. Enhanced expression of NAF-1 is associated with the progression of different cancers including breast, prostate, gastric, cervical, liver, and laryngeal cancer (2). Silencing of NAF-1 expression in breast, or gastric, cancer cells significantly inhibited cellular proliferation and tumorigenicity, whereas overexpression of NAF-1 in gastric or breast cancer cells significantly enhanced cellular proliferation (2). Moreover, gastric and liver cancer patients with high NAF-1 expression displayed a shorter survival and higher recurrence rate compared to patients with low NAF-1 expression. Taken together, these studies point to a potential key role for the NEET protein NAF-1 in promoting the proliferation of different epithelial cancers. Nevertheless, it is unknown what role the 2Fe-2S clusters of NAF-1 play in promoting cancer cell proliferation and tumorigenicity. Using a variety of molecular, cell biology, tissue culture and mouse model techniques, we studied the role of NAF-1 in the development and proliferation of human epithelial breast cancer. Here we report that overexpression of NAF-1 in triple- negative xenograft breast cancer tumors (MDA-MB-231) results in a dramatic augmentation in tumor size and aggressiveness. Furthermore, NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF- 1(H114C), in xenograft breast cancer tumors, results in a dramatic decrease in tumor size that was accompanied by enhanced mitochondrial iron and ROS accumulation and reduced cellular tolerance to oxidative stress. Moreover, treating breast cancer cells with pioglitazone that stabilizes the cluster of NAF-1, results in a similar effect on mitochondrial iron and ROS accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. We conclude that cluster transfer reactions mediated by the overexpressed NAF-1 protein are critical for inducing oxidative stress tolerance in cancer cells leading to rapid tumor growth. In addition, we propose that drugs which stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression (2). 1. Tamir et al., (2014) Biochim Biophys Acta. 1853(6):1294-315. 2. Darash-Yahanaa et al., (2016) Proc Natl Acad Sci U S A. 113(39):10890-10895.
Seventh Congress of the International BioIron Society Page 83 Podium Abstracts IBIS
Podium #24
LOSS OF LOCAL HEPCIDIN DECREASES TUMORIGENESIS IN COLORECTAL CANCER Andrew Schwartz, MS¹, Samira Lakhal-Littleton, DPhil², Peter Robbins, DPhil² and Yatrik Shah, PhD¹ ¹Molecular and Integrative Physiology, University of Michigan; ²Department of Physiology, Anatomy and Genetics, University of Oxford Presented By: Andrew Schwartz, MS
The metabolism of macronutrients such as lipids, amino acids, and glucose are altered in cancers for efficient growth. These metabolic alterations in cancers are currently being exploited for novel therapies. However, very little is known about micronutrient metabolism in cancer. In many cancers, iron transport genes are dysregulated when compared to normal epithelium, particularly in colorectal cancer (CRC). Iron becomes sequestered in colorectal tumor tissue, leading to massive intratumoral iron stores. We have shown that colon tumors are addicted to high iron levels for growth. The master regulator of systemic iron metabolism is hepcidin, a small peptide hormone that is predominately synthesized and released by the liver. To control iron efflux and systemic iron load, hepcidin binds to the only known mammalian iron exporter, ferroportin, resulting in rapid internalization and degradation of ferroportin. Human epidemiological studies have shown a direct correlation between dietary iron intake and/or systemic iron levels and CRC risk. Despite this knowledge, the precise molecular role of iron metabolism in the pathogenesis of CRC remains largely unknown. The goal of this project is to investigate cellular iron metabolism in CRC in order to better understand how changes to iron metabolism drive the progression of CRC. An RT-qPCR analysis for iron metabolic genes in 10-paired human colorectal adenocarcinoma and normal epithelium samples revealed that the hepcidin transcript is one of the most robustly activated iron-related genes in human colorectal tumors. Kaplan-Meier survival analysis generated from 530 human CRC biopsies showed that high levels of intratumoral hepcidin expression portend a significant decrease in overall patient survival. An intestinal epithelial enrichment strategy demonstrated that tumor epithelial cells are a source for intratumoral hepcidin, data that aligns with a previous report. However, we have data to suggest that there are other intratumoral sources of hepcidin as well. The hepcidin target, ferroportin, was significantly decreased in tumor compared to adjacent normal tissues as assessed by IHC. To address the functional significance of hepcidin produced by tumor epithelium in CRC, mice deficient for the hepcidin gene specifically in colon epithelium were challenged in a sporadic model of CRC, which resulted in a significant decrease in tumor number, burden, and size compared to mice wild-type littermates. Hypoxia is a hallmark of the tumor microenvironment and a luciferase-based reporter construct of the hepcidin promoter demonstrated that hypoxia and its downstream transcription factor hypoxia inducible factors (HIFs) are sufficient to increase hepcidin promoter activity in colon-derived cell lines. HIFs regulate gene transcription by binding to HIF response elements (HRE) in the promoter of genes. Truncation of the hepcidin reporter construct to remove the HREs significantly dampened the responsiveness of the hepcidin promoter to hypoxia. Furthermore, a mouse model of sporadic CRC revealed that colonic H 2α i n ia f in a a idin i n in i e data suggest that the hypoxia-mediated induction of hepcidin in tumor epithelium may be one of the iron-dependent processes that are selected for by CRC cells, whereby intratumoral hepcidin might establish a paracrine and/or autocrine axis to degrade colonic ferroportin and thus sequester iron in colorectal tumors.
Seventh Congress of the International BioIron Society Page 84 Podium Abstracts IBIS
Podium #25
DYSREGULATION OF IRON METABOLISM IN CHOLANGIOCARCINOMA STEM-LIKE CELLS Stefania Recalcati, MD/PhD¹, Chiara Raggi, PhD², Elena Gammella, PhD¹, Margherita Correnti, PhD², Paolo Buratti, PhD¹, lisa orti, PhD , asper Andersen, PhD³, Gianfranco Alpini, MD PhD⁴, Domenico Alvaro, MD PhD , Pietro Invernizzi, MD PhD and Gaetano airo, PhD¹ ¹University of Milan; ²Humanitas Clinical and Research Center, Rozzano, Italy; ³University of Copenhagen, Denmark; ⁴A M ealth Science enter, e as; Sapienza University of ome, Italy; University of Milan icocca Presented By: Gaetano Cairo, PhD
Cholangiocarcinoma (CCA), a severe and mostly intractable adenocarcinoma arising from malignant transformation of bile duct epithelial cells, is the second most common liver tumor. Cancer Stem Cells (CSC), a subset of cells within a tumor endowed with stem-like properties and higher resistance to chemotherapy, are involved in tumor initiation, recurrence and metastasis. By using the functional tool of 3D sphere (SPH) formation, we recently highlighted the presence of a stem-like compartment in human CCA. Since recent evidence showed that the regulation of iron homeostasis is altered in cancer, we investigated the role of iron in CCA, focusing on the stem-cells compartment. We analyzed the expression of iron- related proteins in established (CCLP1) and primary (CCA4) human intrahepatic CCA-derived cell lines, cultured both as adherent monolayers (MON) and in 3D SPH conditions. The expression of the major proteins of intracellular iron traffic was opposite between MON [transferrin receptor (TfR1)high, ferritinlow, ferroportinhigh and IRPhigh, indicative of low labile iron pool] and SPH, which have a phenotype symptomatic of elevated intracellular iron availability [TfR1low, IRPlow, ferroportinlow, ferritinhigh]. The different iron content was accompanied by changes in ROS levels and heme oxygenase expression, which were remarkably higher in SPH than in MON. These alterations in cellular iron availability had functional consequences, as exposure to ferric ammonium citrate (FAC) or desferrioxamine (DFO) affected SPH forming efficiency as representation of tumor stem compartment; in particular, we observed decreased SPH formation in the presence of DFO. Molecular characterization of both CCA SPH and MON exposed to DFO or FAC showed that CSC- related markers and stem-like genes specifically involved in pluripotency and self-renewal, epithelial mesenchymal transition and survival are induced by FAC and down-modulated by DFO, thus suggesting an involvement of iron in the control of the tumor stem-like features of CCA. To assess the prognostic relevance of the expression of iron-related genes in CCA, we interrogated a published transcriptome of 104 patient database. H ferritin, hepcidin and ferroportin transcripts were significantly decreased in tumors respect to surrounding liver samples, whereas TfR1 mRNA levels were remarkably up-regulated. Moreover, there was a trend toward poor prognosis in patients with higher hepcidin expression (p= 0.059). The same type of analysis in tumor samples after separation of epithelial and stromal cells by laser capture microdissection in a dataset of 23 CCA patients showed a trend toward shorter survival (p= 0.09) in patients with high levels of H ferritin mRNA expression in epithelial cells. The current model of altered iron homeostasis in cancer cells can be summarized in a framework in which decreased iron storage, increased iron uptake and decreased iron export contribute to enhancing the levels of iron to sustain the high requirement of growing tumor cells. A different picture emerges for iron metabolism in CSC, as the concerted expression of the major proteins of cellular iron homeostasis in CCA SPH, an accepted model of CCS, is indicative of elevated iron storage. This is accompanied by substantial changes in the expression of CSC markers and is mirrored by data indicating a trend toward shorter survival in CCA patients with higher H ferritin expression.
Seventh Congress of the International BioIron Society Page 85 Podium Abstracts IBIS
Podium #26
A NOVEL GALLIUM COMPOUND DISRUPTS TUMOR IRON HOMEOSTASIS AND RETARDS THE GROWTH OF GLIOBLASTOMA IN VIVO BY INHIBITING THE IRON-DEPENDENT M2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE (RRM2) Christopher Chitambar, MD, Mona Al-Gizawiy, PhD, Andrew Lozen, MD, Kimberly Pechman, PhD, Janine Wereley, BS, Robert Wujek, BS, Paul Clark, PhD, John Kuo, MD, PhD, Howard Elford, PhD, Bo Hedlund, PhD, Devashish Joshi, BS, William Antholine, PhD and Kathleen Schmainda, PhD Medical College of Wisconsin Presented By: Christopher Chitambar, MD
Background and Hypotheses: Glioblastoma is a malignant brain tumor with a dismal prognosis and a median patient survival of only 14.6 months with standard therapy. Most chemotherapeutic drugs lack efficacy against brain tumors because they are unable to cross the blood brain barrier (BBB) and penetrate the tumor. New drugs with mechanisms of action different from existing therapies are thus needed for this disease. Tumor iron homeostasis is an attractive target for cancer therapy since malignant cells have a greater requirement for iron than normal cells. Previously we showed that gallium, a trivalent metal that shares properties with iron, enters lymphoma cells by transferrin (Tf)-Tf receptor (TfR) endocytosis similar to iron. Within tumor cells, gallium disrupts iron-dependent processes resulting in cell death. We hypothesized that TfRs on brain microvascular endothelial cells (BMECs) of the BBB would facilitate Tf-Ga entry into the brain while TfRs on glioblastoma would facilitate gallium’s entry into this tumor and inhibit its growth. Methods: The antiproliferative effects of gallium maltolate (GaM) in human U87 and D54 glioblastoma cell lines and human glioblastoma stem cells (GSCs) were measured in vitro by the MTT cytotoxicity assay. TfR, ferritin, and RRM2 proteins were measured by immunohistochemistry (IHC) or immunoblotting; cellular Tf-TfR binding and internalization was studied using 125I-Tf. The iron-dependent RRM2 tyrosyl radical signal was measured by EPR spectroscopy. GaM’s antineoplastic efficacy in vivo was examined in a U87 glioblastoma xenograft brain tumor rat model and tumor response to treatment was measured by magnetic resonance imaging of the brain in live animals. Results: GaM formed complexes with Tf in vitro and inhibited the growth and invasiveness of glioblastoma cells at an IC50 relevant to Ga concentrations theoretically attainable in vivo. Although D54 and U87 cells had greater numbers of TfRs than GSCs, the latter cells displayed rapid internalization of TfR-bound 125I-Tf indicative of highly functional TfRs. Within 24 h of incubation with U87 cells, GaM reduced the iron-dependent RRM2 EPR signal by 65% relative to controls but did not reduce RRM2 protein content. On IHC analysis, TfR and RRM2 expression was seen in glioblastoma but not in normal brain. H- and L-ferritin expression was greater in glioblastoma cells than in normal brain. GaM administered intravenously for 10 days to glioblastoma-bearing rats significantly retarded the growth of established U87 xenograft brain tumors compared with untreated control animals (p= 0.0159). A trend toward a reduction in relative cerebral blood volume in GaM-treated tumors was also noted. Tumors from GaM-treated animals displayed a significant reduction in their mitotic figures and mitotic indices compared with untreated controls (p=0.045). GaM-treated tumors had a marked increase in TfR expression suggesting that GaM produced tumor iron deprivation in vivo. In other studies, iron chelation was shown to potentiate the growth-inhibitory action of the RRM2 inhibitor Didox, suggesting that combination therapies against iron- dependent RR activity in glioblastoma may have therapeutic potential. Conclusions: Our studies are the first to show that GaM, a novel iron homeostasis-targeting agent, may have potential as a therapeutic metallodrug for brain tumors. Further investigation into the ferrobiology of glioblastoma and the development of agents that can target and disrupt critical iron-dependent processes in brain tumors is warranted.
Seventh Congress of the International BioIron Society Page 86 Podium Abstracts IBIS
Podium #27
FERROPTOSIS: A NEW POTENTIAL THERAPEUTIC TARGET FOR AGGRESSIVE TUMORS. A PRELIMINARY STUDY ON HELA, HEPG2 AND RHABDOMYOSARCOMA CELL LINES Michela Asperti, PhD, Silvia Codenotti, PhD Student, Magdalena Gryzik, PhD Student, Paolo Crotti, Student, Maria Regoni, PhD Student, Paolo Arosio, PhD, Alessandro Fanzani, PhD And Maura Poli, PhD University Of Brescia Presented By: Michela Asperti, PhD
Iron is essential for many vital physiological functions in mammalian cells but its excess is associated with tumor initiation, growth and metastasis. High levels of iron sustain tumor growth but even increase oxidative stress, rendering cancer cells more prone to Ferroptosis, a recently discovered form of iron-dependent cell death caused by accumulation of lipid peroxides. Ferroptosis can be stimulated pharmacologically by administration of Erastin, which inhibiting the cystine- glutamate antiporter (System Xc-) is responsible of the intracellular depletion of glutathione (GSH), the major antioxidant system in cells. Also, inhibiting the activity of Glutathione Peroxidase 4 (GPX4) and of other enzymes involved in the cell detoxification from ROS can result in a ferroptotic cell death. The aim of this study was to verify if Erastin may be exploited to selectively target aggressive tumor cells. For this purpose, we employed three cell lines,namely HeLa, HepG2 and RMS cells, which represent well-known in vitro models of cervical cancer, hepatocarcinoma and rhabdomyosarcoma, respectively. After treating the tumor cell lines with Erastin in a dose- and time-dependent manner, we evaluated the cell death by performing cell viability (MTT or Neutral red) and cell density assays (Crystal violet) as well as by measuring the cytosolic ROS levels by Cell ROX Orange dye. Our data showed that: (1) Erastin increased ROS production in the three tumor lines in a dose-dependent manner, promoting ferroptosis especially in RMS cells. (2) Proliferating but not differentiated RMS cells were sensitive to erastin-induced ferroptosis , suggesting that the effect of erastin, being cell- cycle-dependent, could be especially confined to the more proliferating and aggressive cancer cells. (3) Iron supplementation by means of Ferric ammonium citrate or Fe-Transferrin administration enhanced the Erastin-induced- cell-death, while iron chelators (BPS, DFO) or Ferrostatin-I prevented ferroptosis.. Since ferritin regulates iron availability in cells , we analyzed whether modulation of ferritin levels, as obtained via H ferritin or NCOA4 (the protein responsible for ferritinophagy) overexpression and knock-out using the CRISPR/Cas9 technology, was able to impact the erastin-induced ferroptosis in HeLa cells. Our results showed that high ferritin (upon NCOA4 knock out or FTH overexpression) and low ferritin (NCOA4 overexpression or FTH knock out) levels correlated with an increased or decreased Ferroptosis in response to Erastin, respectively. Overall, our data suggest that Erastin effects are dominant on proliferating and thus more aggressive tumor cells; moreover, ferroptosis is critically enhanced by higher iron levels thanks to a ferritin dependent-mechanism. In conclusion, the modulation of intracellular iron content could offer a new potential approach to eradicate aggressive tumor cells.
Seventh Congress of the International BioIron Society Page 87 Podium Abstracts IBIS
Podium #28
IMPACT OF HEMOCHROMATOSIS ON COLON: INCREASED SUSCEPTIBILITY TO INFLAMMATION AND CARCINOGENESIS Bojana Ristic, BS in Chemistry, Sathish Sivaprakasam, PhD, Sabarish Ramachandran, PhD and Vadivel Ganapathy, PhD Texas Tech University Health Sciences Center Presented By: Bojana Ristic, BS
Hemochromatosis, an inherited iron-overload disease, causes tissue damage due to deposition of iron to toxic levels in most tissues including the liver, heart, kidney, pancreas and colon. Free iron, both in the inorganic form (i.e, non- transferrin-bound and non-ferritin-bound) and organic form (heme), facilitates Fenton reaction to generate hydroxyl radicals and induces oxidative damage in cells. However, the extent of its participation in a variety of other cellular processes has not been well explored. Although hemochromatosis is well known for it association with the development of liver cirrhosis and liver cancer, the magnitude of its impact on carcinogenesis in tissues other than liver remains unexplored. Considering that colon tissues obtained from hemochromatosis patients exhibit biochemical features correlated with inflammation, we postulate that hemochromatosis will enhance the susceptibility to colonic inflammation (colitis) and colon cancer, and that the molecular mechanisms of this effect would involve iron-associated manipulation of p53, a major tumor and immunosuppressor, and NF-kB, a pro-inflammatory transcription factor. To test this postulate, we performed in vivo and in vitro studies. In vivo studies were performed to examine the influence of hemochromatosis on experimental colitis, colitis-associated colon cancer, and genetically driven colon cancer. Experimental colitis was induced in Hfe-/- and wild type mice by administration of dextran sulfate sodium (DSS) in drinking water. Colitis-associated colon cancer was induced in Hfe-/- and wild type mice with the colon-specific carcinogen azoxymethane that was injected into mice intraperitoneally, in tandem with administration of dextran sulfate sodium in drinking water as a cancer promoter. ApcMin+ mice were generated on wild type and Hfe-/- backgrounds and were monitored for the development and progression of colon and intestinal polyps. In vitro studies included chronic treatment (several passages) of normal human colonic epithelial cells, CCD841, with ferric ammonium citrate (FAC). They were used to examine the influence that iron had on the p53 and NF-kB. When compared to the control group, DSS in drinking water instigated a severe decrease in body weight and an increase in the diarrhea and bleeding scores in the Hfe-/- group, indicating increased susceptibility to experimental colitis in hemochromatosis mice. Similarly, in the colitis-associated colon cancer model (DSS/AOM), there were more and larger size polyps in the colon in hemochromatosis mice than in wild type mice. This increased susceptibility to carcinogenesis was also seen in the genetically driven colon cancer. ApcMin/+ mice had increased number and larger size of polyps in the small intestine and colon when present on hemochromatosis background. CCD841 cells treated with FAC showed epithelial-to-mesenchymal transition, which is known to be associated with carcinogenesis. In addition, their heme content was significantly higher than in untreated control cells. Furthermore, p53 was depleted from the nucleus in the FAC-treated cells, while NF-kB moved to the nucleus. Thus, our in vivo data show that hemochromatosis increases the susceptibility to colitis and colon cancer in mice. Our in vitro data show potential involvement of excess iron/heme-induced changes in p53 and NF-kB signaling in this phenomenon. We conclude that the iron-overload disease is a risk factor for colitis and colon cancer. These findings may also be relevant to excess intake dietary iron/heme and its known association with colon cancer.
Seventh Congress of the International BioIron Society Page 88 Podium Abstracts IBIS
Podium #29
TARGET OF ERYTHROPOIETIN, FAM210B, REGULATES ERYTHROID HEMOGLOBINIZATION arry Paw, MD, PhD¹, Yvette Yien, PhD¹, iahai Shi, PhD , aiyong hen, PhD¹, Anthony Grillo⁴, iangtao i, MD³, acky hung, PhD¹, Martin afina, MS¹, Paul ingsley, PhD , ulien Ablain, PhD¹, eonard on, MD¹, ames Palis, MD¹, Martin urke, MD, PhD⁴, Daniel auer, MD, PhD¹, Stuart rkin, MD¹, arla oehler, PhD , ohn Phillips³, erry aplan³, Diane Ward, PhD³ and Harvey Lodish, PhD² ¹ arvard Medical School; MI ; ³Univ Utah; ⁴Univ Illinois, Urbana; Univ ochester; U A Presented By: Barry H. Paw, MD, PhD
Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well understood. To better understand these regulatory mechanisms, we profiled gene expression in EPO-treated fetal liver cells to identify novel iron regulatory genes. We determined that FAM210B, a mitochondrial inner membrane protein, was essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects are corrected with a lipid-soluble small molecule iron transporter in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that Fam210b is not a mitochondrial iron transporter per se. FAM210B mediates mitochondrial iron import and heme synthesis by interacting with terminal hemesynthesis enzymes. We propose that FAM210B functions as an adaptor protein to facilitate the formation of a mitochondrial iron transport oligomeric complex, which is required for the increase in iron acquisition for heme and hemoglobin synthesis during terminal erythropoiesis. Collectively, our data reveal a novel mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.
Seventh Congress of the International BioIron Society Page 89 Podium Abstracts IBIS
Podium #30
LOSS OF IRON UPREGULATES MITOCHONDRIA FERRITIN AND TRIGGERS MITOPHAGY THROUGH THE BINDING OF MITOCHONDRIA FERRITIN TO NUCLEAR RECEPTOR COACTIVATOR 4 Yuichi Hara¹, Izumi Yanatori, MD, PhD², Kyo Sasaki, MD², Sohji Nishina, MD, PhD², Fumio Kishi, MD, PhD² and Keisuke Hino, MD, PhD² ¹Kawasaki Medical School; ²Kawasaki Medical Shool Presented By: Yuichi Hara
Background and aim: Mitochondria selective autophagy (mitophagy) plays a critical role in mitochondrial quality control. Although PINK1/Parkin signaling is a well characterized mechanism underlying mitophagy, iron chelation has recently been shown to induce mitophagy independent of PINK1/Parkin signaling. The aim of this study was to elucidate the mechanisms by which loss of iron triggers mitophagy. Methods: Huh7 cells were treated with iron chelator, deferiprone (DFP), deferasirox (DFX), or deferoxamine (DFO). STAM® mice that develop liver cancer preceded by steatohepatitis were administered 0.075g of DFP dissolved in water through gastric tube for 3 months. The effect of iron chelators on mitophagy and mitochondrial function was examined in vitro and in vivo. Results: We measured mitochondrial ferrous iron content in Huh7 cells, using mitochondrial iron-specific fluorescent probe (Ac-MT-FluNox1) and calorimetric ferrozine-based assay. Mitochondrial ferrous iron content decreased in the order of DFP, DFX and DFO. DFP most induced mitophagy among the three iron chelators, as evidenced by increased expression of LC3-II, decreased expression of p62, and increased number of mitophagosomes, but did not make Parkin translocate to the mitochondria. DFP decreased reactive oxygen species (ROS) production, but did not affect mitochondrial electron transport activity and ATP production. DFP increased the expression of mitochondria ferritin (FtMt) in the mitochondrial fraction in vitro and in vivo in a dose dependent manner. Silencing FtMt suppressed DFP-induced mitophagy, suggesting that FtMt may have a functional role in iron chelator-induced mitophagy. Of note, immunofluorescence analysis and pull-down assay revealed that FtMt was bound to nuclear receptor coactivator 4 (NCOA4), a cargo receptor for autophagy, in mitochondria. In addition, the binding site of both proteins was identified, using recombinant proteins of FtMt and NCOA4, both of which have site-directed amino acid substitution. Because FtMt is targeted to mitochondria by leader sequence, the deletion of leader sequence abolished colocalization of FtMt with NCOA4 in mitochondria, even though it retained the ability of FtMt to bind to NCOA4. Finally, DFP suppressed ROS production, steatosis and fibrosis in the liver, and liver tumor development in STAM® mice. The inhibitory effect of DFP on disease progression and liver tumor development was cancelled by knockdown of Atg5, autophagy related gene, or FtMt. Conclusions: These results indicate that iron chelation upregulates FtMt and triggers mitophagy through the binding of FtMt to NCOA4, which consequently suppresses hepatocarcinogenesis in STAM® mice.
Seventh Congress of the International BioIron Society Page 90 Podium Abstracts IBIS
Podium #31
CD13 (ALANYL AMINOPEPTIDASE OR ANPEP) IS AN ENDOCYTIC HEME- AND CLATHRIN-BINDING INTESTINAL BRUSH BORDER MEMBRANE PROTEIN WHOSE DOWN-REGULATION BLOCKS HEME-INDUCED INDUCTION OF HEME OXYGENASE Glenn Gerhard, MD, Fan Chun, PhD, Qunyan Jin, MD, Hui Chen, PhD and Shiming Zhang, PhD Temple University Presented By: Glenn Gerhard, MD
The molecular pathways involved in the cellular uptake of free heme are poorly defined. In the enterocyte, two primary mechanisms have been hypothesized, transport through “channel” proteins and receptor-mediated endocytosis (RME), or both. Over a decade ago, Heme Carrier Protein 1 (HCP1) was reported to serve as the intestinal heme transporter. However, this protein was subsequently shown to transport folate and not heme, except perhaps through a non-physiological low affinity process. Another transporter, SLC48A1 (HRG-1), is a bona fide heme transport protein but appears to be primarily an endosomal protein. Most data to date, based on in vivo and in situ studies, as well as by in vitro and our preliminary studies, support RME as the primary mechanism by which free heme can be acquired, analogous to the endocytic cellular uptake of inorganic iron carried by transferrin, as well as uptake of hemoglobin- haptoglobin complexes by CD163 and heme-hemopexin complexes by CD91. Compartmentalization of heme, similar to inorganic iron, may be necessary due to its inherent chemical reactivity. An evolutionary, proteomic approach was used to identify CD13 (alanyl aminopeptidase or ANPEP) as a candidate for RME of heme. Heme binding membrane proteins obtained from the brush border of intact duodenal fragments from a domestic cat, selected as a carnivorous heme utilizing species, were separated using high performance liquid chromatography and then subject to Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry. CD13 was selected as a candidate because it is a multifunctional plasma membrane peptidase that functions on the small intestinal brush-border in the terminal steps of protein digestion by hydrolyzing oligopeptides to single amino acids. Native and bacterially expressed CD13 can bind heme assayed via in-gel staining, and by pull down of bacterially expressed CD13 protein using a heme-biotin conjugate and streptavidin. Site-directed mutagenesis of two key zinc-binding histidines in CD13 greatly diminished heme binding. Reduction of CD13 expression by RNA interference abrogated heme induced induction of heme oxygenase 1 (HMOX1) in vitro. Co-immunoprecipitation studies indicated that CD13 bound clathrin. Chemical inhibition of clathrin- mediated endocytosis blocked induction of heme-induced HMOX1 expression. These data suggest that CD13 binds extracellular heme for RME of heme.
Seventh Congress of the International BioIron Society Page 91 Podium Abstracts IBIS
Podium #32
TARGETED DELETION OF THE MOUSE MITOFERRIN2 GENE SLC25A28: MFRN1 AND MFRN2 ARE NECESSARY FOR CELL PROLIFERATION Diane ard¹, Ale andra Seguin, PhD , uan ia , ohn Phillips, PhD , arry Paw, MD, PhD³, onathan arasch, MD⁴ and Jerry Kaplan, PhD² ¹University of Utah; Univ of Utah; ³ arvard; ⁴ olumbia Presented By: Diane Ward, PhD
Mitochondrial iron import is essential for the biosynthesis of Fe-S clusters and heme. Dysregulation of mitochondrial iron import results in human diseases ranging from anemia to cardiac dysfunction to Freidrich’s ataxia. Two nuclear encoded mitochondrial iron importers Mitoferrin1 and Mitoferrin2 have been identified. Here we report mice with a complete deletion of Mitoferrin2 (Slc25a28) are viable and are born at Mendelian ratios. Mitoferrin2-/- mice show decreased male fertility and increased hepatic steatosis. Tissues deleted for Mitoferrin2 do not show evidence of compensatory increases in Mitoferrin1 mRNA levels except in kidney. Deletion of both Mitoferrin1 in heart, liver or adipocytes of Mitoferrin2-/- mice does not affect viability and animals are born at Mendelian ratios. Loss of Mitoferrin1 and Mitoferrin2 in skin fibroblasts, however, resulted the inability of cells to proliferate. A defect in cell proliferation was confirmed in other tissues; liver regeneration was severely compromised in Mitoferrin2-/- hepatocyte-specific Mitoferrin1-/- mice. Further, cultured bone marrow-derived macrophages fail to proliferate and die in the absence of Mitoferrin1 and Mitoferrin2. Our results suggest that there must be alternative mitochondrial iron transporters that can support the iron needs of developing organs and non-multiplying cells in the absence of Mitoferrin1 and Mitoferrin2.
Seventh Congress of the International BioIron Society Page 92 Podium Abstracts IBIS
Podium #33
TFR2: A NOVEL POTENTIAL THERAPEUTIC TARGET FOR ß-THALASSEMIA Antonella Nai, PhD, Maria Rosa Lidonnici, PhD, Irene Artuso, MSc, Giacomo Mandelli, PhD, Mariateresa Pettinato, MSc, Laura Silvestri, PhD, Giuliana Ferrari, PhD and Clara Camaschella, MD Division of Genetics and Cell Biology, Ospedale San Raffaele Presented By: Antonella Nai, PhD
Transferrin receptor 2 (TFR2), a liver activator of the iron hormone hepcidin mutated in hemochromatosis type 3, is an erythropoietin (EPO) receptor partner in erythroid cells. We have recently shown that the loss of bone marrow (BM) Tfr2 increases erythroblast Epo sensitivity and mimics a condition of mild iron-d fi i n -thalassemias are iron-loading an ia d i a i n in -globin gene, characterized by severe ineffective erythropoiesis. Since several studies demon a d a i n i i n a i a -thalassemia phenotype, we generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) transplanting BM cells (BMT) from Hbbth3/+ and Tfr2-/-/Hbbth3/+ mice into lethally irradiated wt recipients. Nine weeks after BMT Tfr2BMKO/Hbbth3/+ mice have RBCs count and Hb levels higher than Hbbth3/+, lower circulating reticulocytes and serum Epo levels, comparable liver (LIC) and spleen (SIC) iron content and hepcidin levels and increased Epo-target genes (Epor, Bcl-xl, Erfe) expression in BM and spleen relative to Epo levels. The phenotype amelioration last until 22 weeks after BMT, when the hematological phenotype of Tfr2BMKO/Hbbth3/+ starts to worsen. At this time point Tfr2BMKO/Hbbth3/+ animals have lower serum Epo, LIC, SIC and Hamp levels than Hbbth3/+, but higher Erfe expression. In another cohort Tfr2BMKO/Hbbth3/+ animals underwent a more dramatic worsening of anemia, with overproduction of Erfe and reduction of Hamp and consequent increased LIC and reduced SIC. Our results suggest that deleting Tfr2 in thalassemic erythroblasts increases their Epo sensitivity as occurs in normal ones. This effect is positive during BM repopulation and for a long period of time, making Tfr2 a good therapeutic target andida f -thalassemia. However, likely due to excessive Epo stimulation and continuous Erfe production iron- overload worsens on long-term, further damaging the thalassemic erythropoiesis. In order to test this assumption we fed a cohort of animals an iron-deficient diet starting at 9 weeks after BMT to prevent iron-overload: surprisingly Tfr2BMKO/Hbbth3/+ underwent a worsening of the hematological phenotype as occurred in iron-balance conditions, despite LIC reached the level of wt animals. SIC was reduced in iron-deficient Tfr2BMKO/Hbbth3/+ mice as compared to Hbbth3/+: since low SIC always paralleled the worsening of the hematological phenotype, our present hypothesis is that in long-term SIC reduction may impair the compensatory spleen erythropoiesis. In order to verify this assumption we are challenging transplanted mice with iron-dextran injection to increase SIC and hopefully prevent the development of the severe phenotype. Also germ-line deletion of Tfr2 ameliorates the hematological phenotype of Hbbth3/+ mice with a gene dosage effect, further confirming Tfr2 as a potential therapeutic target. Follow up analysis will assess the phenotype of Tfr2-/- /Hbbth3/+ mice in aging animals. Finally erythroid cells at different stages of maturation sorted from all the models described will be subjected to RNAseq analysis with the aim of defining the pathway/s altered by the loss of Tfr2 n i f a i a i n f n and id n if in n ia a f a i in - thalassemia.
Seventh Congress of the International BioIron Society Page 93 Podium Abstracts IBIS
Podium #34
POTENTIAL THERAPEUTIC APPLICATIONS OF JAK2 INHIBITORS AND HIF2a-ASO FOR THE TREATMENT OF ß - THALASSEMIA INTERMEDIA AND MAJOR Carla Casu, Paraskevi Rea Oikonomidou, Vania Lo Presti, Mariam Aghajan, Shuling Guo, Abdulmalik Osheiza, Luca Melchiori, Pedro Ramos and Stefano Rivella The Children’s Hospital of Philadelphia Presented By: Carla Casu, PhD
ß-Thalassemia is a genetic red blood cell (RBC) disorder characterized by reduced (such as in non-transfusion dependent thalassemia or NTDT) or absent (such as in transfusion dependent thalassemia or TDT) production of ß-globin chains. Ineffective erythropoiesis (IE) with consequent anemia leads to extra-medullary hematopoiesis (EMH), splenomegaly and i n ad dia d f idin i d n a d a in -thalassemia is associated with increased proliferation and reduced differentiation of erythroid progenitors. This is mediated by increased production of erythropoietin (EPO), which activates the downstream JAK2 kinase in erythroid progenitors. As a consequence, hepatosplenomegaly may result, often requiring splenectomy to prevent serious morbidities and mortality. The increased synthesis of EPO in thalassemia is the result of hypoxia. Hypoxia Inducible Factor-2a (HIF2a) is a central mediator of cellular adaptation to hypoxia and stimulates renal and hepatic EPO synthesis. Furthermore, splenomegaly in thalassemia also exacerbates the anemia, as a large proportion of the circulating RBCs are engulfed and eliminated by an enlarged spleen. We hypothesized that targeting the EPO/JAK2 pathway would limit the number of erythroid progenitor cells and reduce the splenomegaly, serving as an alternative to splenectomy. We utilized JAK2 inhibitors or HIF2a antisense oligonucleotides (Hif2a-ASO) to target the EPO/JAK2 pathway. Moreover, we hypothesized that combination of these drugs with blood transfusion therapy will further reduce the splenomegaly. We tested two commercially available JAK2 inhibitors [INCB018424 (Ruxolitinib) and TG101348 (Fedratinib, SAR302503)] and the antisense oligonucleotide Hif2a- ASO in mice affected by NTDT (Hbbth3/+). Both JAK2 inhibitors were administered for 10 days, twice daily at a dose of 180 and 120 mg/kg respectively, while Hif2a-ASO was given at a dose of 25 mg/kg twice weekly for 10 days. A mild reduction in hemoglobin levels (in the range of 9%) was observed in animals treated with both inhibitors while a more pronounced reduction (about 29%) was observed after Hif2a-ASO administration when compared to vehicle-treated mice. Splenomegaly was significantly reduced with all three compounds (up to 58%). Flow cytometry studies on spleen cells revealed that all treated animals exhibited a reduction in the number of erythroid progenitors compared to the controls. We then combined these drugs with blood transfusion. Blood transfusion reduced splenomegaly 49% and 53% when compared, respectively, to non-transfused controls and animals treated with JAK2 inhibitors alone. When transfusion was combined with the administration of JAK2 inhibitors or Hif2a-ASO, the spleen size was further reduced (up to 72%). We then tested these drugs in mice affected by TDT. These mice show dramatic splenomegaly and require chronic blood transfusion for survival. Preliminary studies suggest that administration of JAK2 inhibitors or Hif2a-ASO for 10 days, together with blood transfusion, further reduces spleen weight by 71%. We are now finalizing our study by analyzing ferroportin expression in the duodenum, serum Erythroferrone levels, tissue iron concentration and distribution. In summary, JAK2 inhibitors and Hif2a-ASO reduce splenomegaly by targeting the EPO/JAK2 pathway and limiting the excessive proliferation of erythroid cells. Therefore, these drugs could be effective in reversing the splenomegaly in thalassemia patients and may offer an important alternative to splenectomy.
Seventh Congress of the International BioIron Society Page 94 Podium Abstracts IBIS
Podium #35
HEME OXYGENASE 1 INHIBITION REVERSES ANEMIA IN ß-THALASSEMIA MICE Daniel Garcia dos Santos, PhD¹, Amel Hamdi, PhD², Zuzana Zidova, PhD³, Monika Horvathova, PhD³ and Prem Ponka, MD, PhD² ¹Lady Davis Institute, Jewish General Hospital; ²Lady Davis Institute, Jewish General Hospital/Physiology Department, McGill University; ³Faculty of Medicine and Dentistry, Palacky University Presented By: Daniel Garcia-Santos, PhD
Thalassemias are a heterogeneous group of red blood cell (RBC) disorders ranging from a clinically severe phenotype requiring lifesaving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffectiv i i n - a a ia -globin synthesis is diminished a in α-globin accumulation. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. in -thalassemia an erythroid-specific protease destroy α-globin chains, likely leading to the generation of a pool of “free” heme in erythroblasts. Physiologically, heme can be degraded only viahemeoxygenases (HO). Circulating erythrocytes contain the majority of heme destined for catabolism; this process takes place primarily in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. Recently, we have provided unequivocal evidence that this enzyme is present in erythroid progenitors as well as their differentiated progenies.1 “Unshielded” heme is toxic, but this toxicity will likely be augmented, if HO1 releases iron from heme. We hy i a in -thalassemic erythroblasts HO1-mediated release of iron from heme is the major culprit responsible for cellular damage. Additionally, it has been shown that prevention of heme-derived iron release from splenic and hepatic macrophages i -thalassemia phenotype2. Therefore, suppression of HO1-mediated heme catabolism from senescent RBC could be beneficial in reversing thalassemic phenotype. To test this hypothesis, we exploited the mouse d f -thalassemia known as th3/th3; we obtained these mice from Dr. Stefano Rivella. Our data indicates that HO1 i n i in a d in i f -thalassemic mice as compared to wildtype mice. To investigate the contribution of HO a a ia d i -thalassemia, wild type and thalassemic (Th3/+) mice were injected intraperitoneally with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO inhibitor) during a 4-weeks, 3-times a week. Our a -thalassemic mice injected with SnPP have increased hemoglobin levels and red blood cell counts, and display a decrease in the spleen index, reticulocyte counts and liver iron content when compared to PBS-in d - thalassemic mice. Furthermore, hepcidin levels increase and liver ferroportin expression decreases in SnPP-in d - a a i i n ad ini a i n d a d α-globin precipitation and increased RBC half life in thalassemic mice. HO-1 inhibition Ter119+ cells of the bone marrow, leading to a decrease in heme regulatory pool in these cells. Our indi a a H in i i i n an ia in -thalassemia intermedia mice model. Further research is needed to determine whether iron liberated from heme by HO i di n i f da a f -thalassemic erythroblasts. 1GarciaSantos D, et al. Hemeoxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 226977, 2014. 2Nai A, et al. Deletion of TMPRSS6 attenuates the phenotype in a d f -thalassemia. Blood 119 (21): 5021, 2012.
Seventh Congress of the International BioIron Society Page 95 Podium Abstracts IBIS
Podium #36 HEPCIDIN MIMETIC PTG-300 FOR TREATMENT OF INEFFECTIVE ERYTHROPOIESIS AND IRON OVERLOAD Gregory Bourne, PhD¹,², Li Zhao, PhD¹, Ashok Bhandari, PhD¹, Brian Frederick, MS¹, Jaimee McMahon, BS¹,², Vinh Tran, BS¹, Jenny Zhang, PhD¹,², Adam Stephenson, PhD¹,², James Tovera, BS¹, Lu Bai, BS¹, Thamil Annamalai, MSc, M Phil¹, Dinesh Patel, PhD¹, Mark Smythe, PhD¹,² and David Liu, PhD¹ ¹Protagonist Therapeutics, Milpitas, CA; ²Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia Presented By: Gregory Thomas Bourne, PhD
Hepcidin is a 25–amino acid peptide synthesized by hepatocytes and is involved in iron homeostasis. The known molecular target of hepcidin is the ferroportin receptor, which functions as a trans-membrane channel for the transport of cellular iron. Hepcidin induces the endocytosis and proteolysis of ferroportin and thereby decreases the delivery of iron to plasma. Hepcidin is prone to aggregation and difficult to synthesize, characteristics that could hinder its development as a drug. The hormone is a highly structured 4-disulfide bonded beta-hairpin that is highly conserved. Protagonist used a proprietary ‘scaffold hopping’ approach, VectrixTM to identify a novel peptidic scaffold that matches key pharmacophores of hepcidin. Early hits identified compounds with micromolar affinity within the HEK cell based GFP assay. After further optimization PTG-300 was selected as a clinical development candidate. It was selected based on its drug-like characteristics, such as human plasma stability t1/2>24h and aqueous solubility >100 mg/mL. It reduces cell surface expression of ferroportin with an EC50 of 5 nM and causes >80% reduction in serum iron with significant sustained reduction for 48 hr after a single subcutaneous dose of 1 mg/kg in healthy cynomolgus monkeys. To show erythropoietic efficacy, 1 mg/kg of PTG-300 was injected subcutaneously Q5D for 4 weeks in six week old Hbbth3+ mice. This resulted in a significant hemoglobin increase of 1.75 g/dL. Reticulocyte count and spleen size were also reduced, and an increase in peripheral red cell number was observed. In separate studies in Hbbth3+ mice, flow cytometry analysis of bone marrow and spleen erythroid populations from PTG-300 treated animals demonstrated an increase in the relative proportion of mature erythroid cells. Accumulation of tissue iron was also reduced in the liver and spleen. In this model there was no disease- related iron accumulation in the heart or duodenum. This data support the development of PTG-300 as a novel therapy for the potential treatment of anemia and iron overload disorders associated with hemoglobinopathies. This work was supported by Protagonist Therapeutics and NIH SBIR grant under the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R43HL132702.
Seventh Congress of the International BioIron Society Page 96 Podium Abstracts IBIS
Podium #37
FERROPORTIN INHIBITORS IMPROVE INEFFECTIVE ERYTHROPOIESIS AND PREVENT IRON LOADING IN A BETA-THALASSEMIA DISEASE MODEL Vania Manolova, PhD, Anna Flace, Patrick Altermatt, MSc, Hanna Sundström, PhD, Naja Nyffenegger, PhD and Franz Duerrenberger, PhD Vifor AG Presented By: Vania Manolova, PhD
Imbalanced synthesis of alpha- and beta-globin chains of hemoglobin in beta-thalassemia causes ineffective erythropoiesis, anemia and iron overload. Iron restriction by activation of endogenous hepcidin1 or repeated dosing of minihepcidins2 has been shown to improve erythropoiesis and reduce iron overload in a mouse model of beta- thalassemia intermedia. We have identified orally bioavailable small molecules Ferroportin Inhibitors with nanomolar potency and a similar mode of action as the physiological ferroportin ligand hepcidin. These Ferroportin Inhibitors triggered ferroportin ubiquitination and degradation in a mouse macrophage cell line with similar kinetics as hepcidin. Accordingly, Ferroportin Inhibitors blocked iron efflux in a human cell line expressing endogenous ferroportin. Oral administration of Ferroportin Inhibitors to wild-type mice rapidly lowered serum iron levels thereby demonstrating efficient iron restriction in vivo. Strikingly, Ferroportin Inhibitors dose-dependently corrected anemia, ameliorated ineffective erythropoiesis, decreased formation of insoluble hemichromes, reduced splenomegaly and prevented liver iron loading in a mouse model of beta-thalassemia intermedia. Therefore, iron restriction by pharmacologically targeting the ferroportin- hepcidin axis with oral Ferroportin Inhibitors provides a novel therapeutic opportunity in beta-thalassemia. 1 Schmidt, 2013, Blood; Guo, 2013, JClinInvest; 2 Casu, 2016, Blood
Seventh Congress of the International BioIron Society Page 97 Podium Abstracts IBIS
Podium #38
IDENTIFICATION OF GUANOSINE 5'-DIPHOPSHATE AS POTENTIALLY IRON MOBILIZER: PREVENTING THE HEPCIDIN-FERROPORTIN INTERACTION AND MODULATING THE INTERLEUKIN-6/STAT-3 PATHWAY Stanzin Angmo and Nitin Kumar Singhal, PhD National Agri-Food Biotechnology Institute Presented By: Stanzin Angmo
Introduction: Anemia of inflammation (AI) is one of the most common manifestations of iron deficiency in the patients with inflammatory conditions. AI is responsible for hypoferremia, with consequent iron-restricted erythropoiesis. In this work, using virtual screening and molecular modelling, a natural compound guanosine- -diphosphate (GDP) was identified to show good binding affinity with hepcidin and thus prevents its binding to ferroportin (FPN). Further in vitro and in vivo studies confirmed the role of GDP in preventing hepcidin-mediated FPN degradation, reversing iron restrictive effect of inflammation. Methods: A systematic approach involving in silico, in vitro and in vivo studies was employed to identify hepcidin inhibiting agents. To identify a potent hepcidin-binding agent, natural compounds were screened using molecular docking and dynamics simulations and further investigated on cell lines (GFP-FPN, Caco-2, HepG2) using flow cytometry and western blotting. Normal or turpentine induced anemic mice were used in the associated studies. Results: The virtual screening via molecular modelling showed that GDP as a potent hepcidin-binding agent as shown in the (Figure 1A). In vitro studies revealed that GDP significantly increased ferroportin stabilization in GFP-FPN cell lines (Figure 1C) and In vivo results showed that co-administration of GDP and ferrous sulphate (FeSO4) significantly improved the turpentine-induced anemic state with increase in haemoglobin level (Figure 1B). Discussion: AI is a normocytic anemia, common among patients with chronic infection and inflammatory disorders. We found that GDP molecule have higher contribution to the stability of hepcidin-GDP complex and thus blocks its interaction with FPN. The results support the novel hypothesis that GDP along with iron supplement regime can overcome the binding of hepcidin from interaction with FPN that would be an effective treatment for AI. Conclusions These results suggest that GDP may be a potent novel natural compound that can be incorporated with iron supplement regimens to ameliorate AI.
Seventh Congress of the International BioIron Society Page 98 Podium Abstracts IBIS
Podium #39
EFFECTS OF MATERNAL IRON STATUS ON PLACENTAL IRON TRANSFER V. Sangkhae, A.L. Fisher, V. Gabayan, E. Valore, T. Ganz and E. Nemeth University of California, Los Angeles Presented By: Veena Sangkhae, PhD
Pregnancy profoundly affects maternal metabolism to provide optimal conditions for fetal development. A specific example is the massive movement of iron from the mother across the placenta to satisfy the iron requirements of the developing fetus. Iron-deficiency during pregnancy has negative effects on both mother and fetus, including increased maternal morbidity and mortality, preterm birth, low birth weight, impaired immune function, and cognitive defects in newborns and infants. Although iron transfer from mother to fetus is entirely dependent on transport through the placenta, our understanding of this process and its relationship to maternal iron status remains limited. In this study, maternal iron status of wild-type C57BL/6 mice was altered using diet or iron dextran injections. Mothers were fed either a standard chow diet (185ppm iron), a low iron diet (4ppm iron) 2 weeks prior to and throughout the pregnancy, or were injected with 20mg iron dextran at time of mating. In iron-replete pregnancies (standard chow), maternal hepcidin is robustly suppressed with advancing pregnancy, allowing for increased dietary iron absorption and release from stores for transfer to the embryo. Iron deficiency (low-iron diet) resulted in development of anemia and hypoferremia in both the mother and embryos. In iron dextran injected mothers, maternal iron stores were high; however, fetal iron status of the embryos did not differ from those of iron-replete mothers. This was in part due to an increase in maternal hepcidin, which prevented an increase in maternal serum iron. Among the known placental iron transporters and regulators, ferroportin (FPN) showed the most dramatic changes with gestational age and maternal iron status. FPN mRNA and protein increased with gestational age, presumably to meet the increasing iron requirement of the developing embryo. With maternal iron deficiency, placental iron content decreased, and transferrin receptor 1 (TFR1) expression increased as expected. However, FPN protein levels were surprisingly decreased, suggesting existence of placental iron-sensing mechanisms that ensure maintenance of placental iron homeostasis at the expense of iron transfer to the fetus. Conversely, in iron- loaded mothers, higher placental iron content was accompanied by decreased TFR1 expression and increased FPN protein levels. Electrophoretic mobility shift assays (EMSAs) for measurement of iron regulatory element (IRE)-binding activity of iron regulatory proteins (IRPs) demonstrated iron-dependent changes in the IRP regulatory system within the placenta. These results confirm that iron transfer to the embryo is dependent on maternal iron status. We demonstrate existence of placental iron sensing mechanisms, including the IRE/IRP regulatory system, which modulate TFR1 and FPN levels to ensure placental iron homeostasis. Maternal iron deficiency resulted in embryo iron deficiency, but maternal and placental iron regulatory mechanisms prevented iron overload of embryos when mothers were iron loaded.
Seventh Congress of the International BioIron Society Page 99 Podium Abstracts IBIS
Podium #40
ALTERNATE DAY DOSING, AND SINGLE MORNING DOSES RATHER THAN B.I.D. DOSING, MAXIMIZE TOTAL AND FRACTIONAL IRON ABSORPTION FROM ORAL IRON SUPPLEMENTS: STABLE IRON ISOTOPE STUDIES IN IRON-DEPLETED WOMEN Nicole Stoffel¹, Diego Moretti¹, Colin Cercamondi¹, Christophe Zeder¹, Dorine Swinkels², Gary Brittenham³ and Michael Bruce Zimmermann¹ ¹ETH Zurich, Switzerland; ²Radboud University Medical Centre, Nijmegen, The Netherlands; ³Columbia University, New York, USA Presented By: Nicole Stoffel, Msc
Background: Oral iron supplementation is a primary approach to treat iron deficiency anemia, but there is no clear consensus on the optimal regimen, and compliance is often poor. Oral iron supplements acutely increase plasma hepcidin (PHep); the duration and magnitude of this increase and its dose dependence could be used to optimize iron supplementation to maximize absorption. We have previously tested this in short term studies; we have now extended these findings in longer-term studies. Objectives: In short term studies, we: (study 1) defined the duration and magnitude of the PHep increase and iron absorption after 40, 80, 160 and 240 mg of oral iron, comparing single day versus two consecutive daily doses; and (study 2) compared iron absorption from 60 mg oral iron given single day versus two consecutive daily doses and (study 3) single versus b.i.d. 60 mg doses. In long-term studies (study 4), we compared iron absorption (60 mg) from consecutive day dosing for 14 days versus alternate day dosing for 28 days (equal total iron dose) and (study 5) from 120 mg given in daily doses versus split b.i.d. 60 mg over 3 days. Methods: In the studies (study 1: n=25; 2: n=16; 3: n=13; 4: n=40; 5: n=20) labeled Fe [54Fe]-, [57Fe]- or [58Fe]-FeSO4 was given to fasting iron-depleted women (plasma ferritin <25µg/L). Iron absorption was measured as erythrocyte incorporation of iron stable isotopes 14 days later. Results: In study 1, there was an increase in PHep at 24 h after the doses of 60, 80, 160 and 240mg (P<.05), but not at 40mg; PHep was not elevated 48 h after any of the doses. Fractional iron absorption (FIA) of the second day’s dose decreased (35-45% lower) with increasing dose (P<.01). In study 2, FIA decreased by 36% when 60 mg was administered on the second day compared with the first day (P<.001). In study 3, total iron absorption (TIA) was 23.6 mg if 3 doses were administered within 24 hours (b.i.d.) compared with 22.6 mg iron when only the 2 morning doses were given (P=.79); b.i.d. dosing increased PHep on the following day compared with single dosing (P<.01). In study 4, TIA from consecutive day dosing was increased compared to alternate day dosing: geometric mean (-SD,+SD) 131.0 (71.4,240.5) versus 175.3 (110.3,278.5) mg (P=.001). Mean PHep was lower in the consecutive versus alternate day dosing group (P<.05), but was higher in the first two weeks of supplementation (P<.01). In study 5, there was no difference in TIA over 3 days between single dosing and b.i.d: geometric mean (-SD,+SD) 40.7 (40.9,40.6) versus 44.7 (44.8,44.5) mg, respectively; b.i.d. dosing increased PHep over the 3 days compared with single day dosing (P<.05) Conclusions: In both short- and long-term studies in iron-depleted women: 1) FIA and TIA are higher from 60 mg doses given on alternate days versus consecutive days; and 2) an iron dose (120 mg) split b.i.d. does not increase TIA compared with a single morning dose. These studies provide new evidence to optimize oral iron supplementation regimens.
Seventh Congress of the International BioIron Society Page 100 Podium Abstracts IBIS
Podium #41
IMPROVING SAFETY AND EFFICACY OF IRON FORTIFICATION IN AFRICA BY COMBINING IRON AND PREBIOTICS: A STABLE ISOTOPE ABSORPTION STUDY AND A RANDOMIZED CONTROLLED TRIAL IN KENYAN INFANTS Michael Bruce Zimmermann¹, Daniela Paganini¹, Mary Uyoga², Diego Moretti¹, Colin Cercamondi¹, Francis Mutuku³, Edith Mwasi³, Clarissa Schwab¹, Christophe Lacroix¹ and Simon Karanja² ¹ETH Zurich, Switzerland; ²JKUAT, Nairobi, Kenya; ³Msambweni Hospital, Kenya Presented By: Michael Zimmermann, MD
Background: Most African infants are anemic, and iron deficiency anemia (IDA) impairs neurocognitive development; thus, effective and safe interventions are urgently needed. Iron-containing micronutrient powders (MNPs) added to complementary foods at home can reduce IDA in infants, but the high iron dose increases the enterobacteria/bifidobacteria ratio, numbers of enteropathogenic E. coli, gut inflammation and diarrhea. Iron absorption from MNPs in African infants is low (4-9%), due to inhibitory complementary foods and common infections. We have developed a safer and more efficacious approach by: 1) reducing the iron dose in MNPs while maximizing absorption by providing iron as NaFeEDTA and ferrous fumarate (FeFumEDTA); and 2) including a prebiotic galacto-oligosacchariode (GOS) that may mitigate the adverse effects on on the gut by promoting growth of the beneficial barrier bacteria. Objectives: The objective of this study was to determine if a MNP combining GOS and a low dose (5 mg iron) as FeFumEDTA would be efficacious and safe in African infants. We hypothesized that this MNP formula would not only reduce anemia but also protect against the adverse effects of iron on the gut. Methods: To test absorption from the new formulation, Kenyan infants (n=50; age 6–14 months; 64% iron deficient anemic) consumed daily for 3 wks maize porridge fortified with a MNP containing FeFumEDTA and 7.5 g GOS) or the MNP without GOS. Then we fed isotopically-labelled maize porridge and MNP test meals containing 5 mg iron as 57FeFum+Na58FeEDTA or ferrous sulfate (54FeSO4) to all infants. Iron absorption was measured as erythrocyte incorporation of stable isotopes after 14 d and was compared between groups. We then perfomed a 4 month randomized controlled trial (RCT) in infants (n=150, age 6-8 months) where infants received: 1) MNP without Fe (control); 2) MNP with 5 mg Fe as FeFumEDTA (Fe); or 3) MNP with the same iron dose and with 7.5 g GOS (FeGOS). Microbiome analysis was with 16S RNA sequencing and targeted qPCR for pathogens. Results: In the absorption study, prior GOS consumption lowered fecal pH and increased iron absorption by 62% (11.6 to 18.8%, P<0.001) from FeFumEDTA, possibly reflecting greater colonic iron absorption; iron absorption was indirectly correlated with fecal pH and directly correlated with fecal bifidobacteria (for both p<0.05). In the RCT, in both Fe and FeGOS, there were increases in hemoglobin (+6-9 g/L) and decreases in anemia (>50%) compared to control (for both, p<0.01). Comparing Fe to control, there were significantly higher plasma intestinal fatty acid binding protein (enterocyte damage) (+26%), and fecal calprotectin (gut inflammation) (+11%) and a dysbiosis with increased enteropathogenic E.coli (all p<0.05). Comparing FeGOS to control, there was no significant increase in any of the above variables, higher abundances of bifidobacteria and lactobacilli (both, p<0.02) and decreased morbidty from diarrhea and respiratory tract infections (p<0.05). Conclusions: In anemic African infants, a low iron dose MNP is efficacious against IDA, and the addition of a prebiotic to the MNP increases iron absorption, mitigates adverse effects of iron on the gut microbiome and inflammation, and reduces infectious morbidity.
Seventh Congress of the International BioIron Society Page 101 Podium Abstracts IBIS
Podium #42
RISK-BENEFIT AND COST-EFFECTIVENESS OF UNIVERSAL DISTRIBUTION OF IRON SUPPLEMENTATION AND IRON-CONTAINING MICRONUTRIENT POWDERS TO YOUNG CHILDREN IN 79 LOW AND MIDDLE INCOME COUNTRIES: A MICROSIMULATION STUDY Sant-Rayn Pasricha, MBBS MPH PhD, Michael Bode, Fayrouz Ashour, Amrita Arcot, Laura Murray-Kolb, Parminder Suchdev and Adrian Gheorghe MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Sant-Rayn Pasricha, MBBS, MPH, PhD
The World Health Organization (WHO) recommends universal distribution of iron supplementation or multiple micronutrient powders to all children under-2 years in settings where anaemia is prevalent. However, there is evidence these interventions may also promote infections including malaria and diarrhoea. Thus, a decision to implement anaemia control interventions must incorporate a holistic analysis of both expected benefits and risks along with associated costs or economic gains. Differing epidemiology and infectious disease burdens likely mean the effects of interventions are different in each country-context. In each country where the prevalence of anaemia is such that universal iron interventions are recommended, or where interventions are presently being piloted, we modeled the risk-benefit and cost effectiveness of the iron interventions to under-2 children. We developed a microsimulation model that generated individual patient histories over a lifetime horizon. We populated the model with country-specific epidemiologic, health service utilization and cost data, and simulated three cohorts of 2,500 infants receiving different anaemia-control interventions between the ages of 6 and 12 months: 1) MNPs, 2) iron supplementation or 3) no intervention. We estimated country-specific, lifetime net differences in health outcomes (measured as disability-adjusted life years [DALYs] averted) and costs between MNPs and iron supplementation when compared with no intervention. Effects sizes were based on updated systematic reviews of MNPs or iron supplements versus control which have been used to inform WHO anaemia control recommendations, and modulated at the country level depending on malaria endemicity and individual level depending on access to malaria prevention. Cost data were based on estimates of unit-costs of interventions, programmatic costs, probability of health seeking, and costs per visit of health attendances (Figure 1). There was marked variation in overall benefit in net benefit-risk from iron interventions between different regions, and in different countries within regions, from overall beneficial to overall harmful. The burden of disease attributable to anaemia was chiefly due to moderate and severe, but not mild, anaemia. Increases in diarrhoea from anaemia control interventions contribute considerable to DALYs in this model. Data on long term effects of iron interventions on cognitive performance were sparse but indicate no effect; this precluded incorporation of an effect from intervention on earning potential. There were differing costs per DALYs averted and different affordability of programmes between countries, with costs in some countries exceeding US$5000 per DALY averted. Benefits from iron would be entirely negated if iron raises mortality risk in children by 1 per 10000. Iron interventions generally have a positive but modest effect on child health and are moderately cost-effective. Data from randomised controlled trials reporting on effects of iron interventions on functional health outcomes would inform and improve precision of the model.
Seventh Congress of the International BioIron Society Page 102 Podium Abstracts IBIS
Podium #43
FERRIC PYROPHOSPHATE CITRATE (TRIFERIC®), A NOVEL THERAPY THAT TREATS ANEMIA OF INFLAMMATION AND OVERCOMES FUNCTIONAL IRON DEFICIENCY Raymond Pratt, MD and Ajay Gupta, MD Rockwell Medical Inc Presented By: Raymond D. Pratt, MD
Ferric Pyrophosphate Citrate (FPC) is the only iron salt for parenteral administration, indicated to replace iron and maintain hemoglobin in adult hemodialysis patients. FPC comprises Fe(III) complexed to 1 pyrophosphate (PPi) and 2 citrate molecules by coordinate covalent bonds (MW 1300 Da). PPi promotes rapid direct binding of FPC iron to plasma transferrin. Co-crystallization of FPC with human apo-transferrin in a 2:1 molar ratio produced pink crystals indicating presence of iron bound to transferrin. Crystal structure demonstrates a N-lobe fully closed around iron with carbonate as the synergistic anion, while C-lobe has pyrophosphate as the synergistic anion. The iron in FPC is highly inert and does not promote oxidant stress or inflammation. The Pharmacokinetics of FPC are dose proportional up to 100% TIBC. FPC is rapidly cleared from the blood (t½=~1.2 hr) without non-transferrin bound iron or labile plasma iron present. FPC’s unique properties make it an ideal candidate to overcome iron sequestration, functional iron deficiency and anemia of inflammation. Citrate makes FPC highly water-soluble and therefore suitable for infusion via the dialysate in hemodialysis (HD) patients. A series of randomized, placebo-controlled clinical trials have demonstrated that FPC delivered via hemodialysis can maintain iron balance by replacing obligatory iron loss, sparing ESA use [PRIME Study, Kidney Int. 2015] and maintaining hemoglobin (Hgb) [CRUISE studies, Nephrol. Dial Transplant 2015]. The PRIME study was a double blind RCT in 104 iron-replete (baseline ferritin 200-1000 µg/L) HD patients randomized to FPC or placebo for up to 36 weeks. ESA was titrated to maintain Hgb in the target range. IV iron was administered for serum ferritin < 200 µg/L. At the end of treatment, prescribed ESA doses in the FPC group were reduced by 35% (p=0.045) and prescribed IV iron by 45% (p=0.028) versus placebo. FPC did not increase serum hepcidin levels or markers of oxidative stress/inflammation. In the CRUISE studies iron-replete HD patients were randomized to FPC (N=299) or placebo (N=300) for up to 48 weeks. ESA dose changes and IV/oral iron were prohibited. FPC maintained Hgb at baseline in both studies, while placebo treated subjects developed iron restricted erythropoiesis. The primary end point, the mean change in hemoglobin from baseline to end of treatment, was 3.6 g/L lower in the placebo group (p-value = 0.011). The safety profile of FPC administered via dialysate was similar to placebo in all studies, with no anaphylaxis. There were no increases in intradialytic hypotension, cardiovascular events or infections compared to placebo. Intraperitoneal administration of FPC to rabbits and rats has demonstrated good bioavailability without peritoneal toxicity. Studies are ongoing to examine the short term safety and pharmacokinetics of delivering FPC via the peritoneal dialysate in CKD-PD patients. Unlike intravenous iron-carbohydrate complexes which are known to destabilize lipid emulsions, in vitro studies have demonstrated excellent compatibility of FPC with lipid containing parenteral nutrition admixtures, without destabilizing the emulsion. Conclusions: FPC is a novel, highly soluble iron salt suitable for parenteral administration. FPC donates iron immediately to transferrin, avoids iron sequestration in the RES, and appears effective in treating functional iron deficiency in states of inflammation.
Seventh Congress of the International BioIron Society Page 103 Podium Abstracts IBIS
Podium #44
IRON-FORTIFIED VS LOW-IRON INFANT FORMULA: COGNITIVE OUTCOMES AT 10 AND 16 YEARS Sheila Gahagan1, Erin Delker1, Marcella Castillo2, Betsy Lozoff3 1University of California, San Diego; 2Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile; 3University of Michigan Presented By: Sheila Gahagan
Introduction: Infancy iron deficiency anemia (IDA) is a global public health problem. Even in developed countries, infancy IDA is associated with negative health outcomes including poorer cognitive, motor and socio-emotional development. Expert organizations worldwide differ on the recommended dose of prophylactic iron for infants. Despite routine iron fortification of infant formulas, there is limited research assessing the optimal dose of iron fortification and its long-term effects on the developing brain. We report here on long-term outcomes from a study of long-term outcomes of infants who received iron fortified formula or low-iron formula in a double-blind, randomized-controlled trial of iron from 6 to 12 months. Methods: The trial took place in Chile when iron deficiency anemia was widespread and there was no national program of iron fortification. We recruited non-anemic, singleton, full- infan i i i g, who had routine vaginal delivery, no congenital anomalies, perinatal complications, phototherapy, or chronic illness. Infants (n = were randomized to iron fortified formula (12.7 mg/L) or low-iron formula (2.3 mg/L) (1991 – 1994). (In the last years of participant enrollment (1994 – 1996), low-iron formula was no longer used and infants were randomized to iron-fortified formula or a no-added-iron condition.) At 12 and 18 months, 835 infants randomized to iron-fortified formula or low-iron formula, had venous blood assessed for hemoglobin, mean cell volume, erythrocyte protoporphyrin, red blood cells and serum ferritin. The infants were followed up at 10 and 16 years with comprehensive testing of development and health. At 10 years, 473 of the infancy sample were assessed (56.6%). Outcome measures of interest at 10 years included IQ, spatial memory, arithmetic achievement, visual-motor integration, visual perception, and motor functioning. Those randomized to iron- fortified formula scored lower than those who received low iron formula on all tests. The differences were significant for spatial memory and visual-motor integration with effects varying by 6-month hemoglobin level. Specifically, those with high 6-month hemoglobin levels (>12.8g/dL) performed worse when supplemented with iron-fortified compared to low-iron formula (2.3 mg/L) whereas those with low 6-month hemoglobin levels (<10.5g/dL) performed better in the iron-fortified condition. We report here on neurocognitive and socioemotional outcomes at 16 years. Results: At 16 years, 49% of the infancy sample were assessed (n=405). Of the 9 cognitive tests administered, 4 showed statistically significant lower scores in the iron-fortified vs. low-iron group (Rey-Osterrieth visual memory, WRAT-R arithmetic achievement, Reading comprehension achievement, WISC-IV Verbal Similarities), adjusting for background characteristics. We found no evidence of a significant interaction between 6-month hemoglobin level and formula group for any of the outcomes at 16 years. Discussion: At 10 and 16 years, those randomized to high-iron formula in infancy scored lower on some neurocognitive tests compared to those randomized to low-iron formula. These findings raise important questions about the optimal dose of iron to prevent IDA in infancy. Nonetheless, they do not negate the robust evidence demonstrating the importance of utilizing iron supplementation to prevent ID and IDA.
*Please note that an earlier version of the 16-year findings were presented at a poster session at APHA 2016.
Seventh Congress of the International BioIron Society Page 104 Podium Abstracts IBIS
Podium #45
A NOVEL H-FERRITIN SIGNALING RECEPTOR THAT ACTIVATES NLRP3 INFLAMMASOME-INDUCED INFLAMMATION IN HEPATIC STELLATE CELLS: IMPLICATIONS FOR HEPATIC FIBROGENESIS IN HAEMOCHROMATOSIS Manuel Fernandez-Rojo, PhD, Anita Burgess, PhD, Diem Hoang-Le, BSc, Amber Glanfield, PhD, Sujeevi Nawaratna, PhD, Geoffrey Gobert, PhD and Grant Ramm, PhD QIMR Berghofer MRI Presented By: Grant A. Ramm, PhD, BSc (Hons)
Introduction: Hepatic stellate cells (HSCs) are responsible for collagen deposition leading to fibrosis following liver injury/inflammation. Serum ferritin levels reflect body iron stores in haemochromatosis and are elevated in inflammation where they act as an indicator of disease severity in chronic liver disease. We have previously demonstrated that H- subunit ferritin (FTH1) actually contributes to this process as a pro-inflammatory mediator in HSC biology via an iron- independent, NFkappaB-regulated signalling pathway, inducing the expression of cytokines, e.g., IL- i na in receptor responsible for the effect of FTH1 on HSC-induced inflammation remains unknown. Aims: To characterize the FTH1 receptor and underlying molecular mechanisms responsible for proinflammatory cytokine stimulation in HSCs. o o Methods: In order to identify the FTH1 receptor, primary rat HSCs were exposed to 10nM FTH1 for 2hrs at 4 C vs 37 C and cell extracts were subjected to immunoprecipitation of FTH1-binding proteins with subsequent analysis by mass spectrometry. This identified numerous potential candidate molecules. Validation of potential FTH1 receptor candidates included analysis of IL- in i n n f in i ain- and loss-of function experiments; and ii) disruption of plasma membrane-endocytic signaling/trafficking pathways using specific inhibitors (i.e., clathrin-coated pit endocytosis via PitStop). In addition, we examined iii) the reciprocal regulation between FTH1- signalling and components of the inflammasome by western-blotting, immunofluorescence, qRT-PCR and ELISA; and iv) determination of the potential for FTH1 to stimulate proinflammatory cytokine synthesis in whole liver using an ex vivo precision-cut liver slice tissue culture model. Results: This study revealed QIMR1 (de-identified, as subject of a pending patent application) as a novel H-Ferritin signaling receptor candidate in HSCs. Accordingly, transfection of primary rat HSCs with QIMR1 siRNA (which achieved 77% QIMR1 knock-down, p=0.023), significantly inhibited FTH1-induced IL- i n p=0.00021), while over-expression of QIMR1 exacerbated the FTH1-induced IL- an i p=0.028). Maturation of active IL- in i dia d -associated proteins within the inflammasome. Western-blot analysis demonstrated that FTH1 stimulates NLRP3, but not NLRP1, protein levels in HSCs and that this is prevented via inhibition of clathrin-coated pit endocytosis. We also showed that FTH1 induced the expression of active Caspase-1 protein and active IL- in secretion. FTH1-induced expression of NLRP3 was prevented by knock-down of QIMR1. Finally, as a proof of concept that FTH1 has the ability to influence liver tissue inflammation, we have demonstrated that FTH1 stimulates IL- expression (~3 fold; p=0.033) in precision-cut (ex-vivo) liver slices. Conclusions: We have characterized a novel FTH1 receptor (QIMR1) that is responsible for the transduction of FTH1- induced proinflammatory signalling, resulting in NLRP3-dependent inflammasome activation and IL- i n in H This process is dependent on clathrin-coated pit endocytosis. Therefore, this study provides the first evidence of a role for FTH1-induced signaling via QIMR1 in HSCs as a unique molecular mechanism for the promotion of hepatic inflammation in chronic liver disease.
Seventh Congress of the International BioIron Society Page 105 Podium Abstracts IBIS
Podium #46
A RANDOMIZED PATIENT-BLINDED STUDY OF TRUE VERSUS SHAM REDUCTION OF BODY IRON IN HFE RELATED HEMOCHROMATOSIS WITH MODERATE IRON OVERLOAD REVEALS BENEFIT OF TREATMENT Martin Delatycki, Sim Ong, Lyle Gurrin, Lara Dolling, Jeanette Dixon, Amanda Nicoll, Michelle Wolthuizen, Erica Wood, Greg Anderson, Grant Ramm, Katie Allen, John Olynyk, Darrell Crawford, Louise Ramm, Kevin Croft, Paul Gow, Simon Durrant and Lawrie Powell Murdoch Childrens Research Institute Presented By: Martin Delatycki, MBBS, FRACP, PhD
Background: Hereditary haemochromatosis (HH), an iron overload disorder, is most commonly caused by HFE p.C282Y homozygosity. There is insufficient evidence to determine if individuals with HH and moderately elevated serum ferritin (SF), defined as SF 300-1000µg/L, should undergo iron reduction treatment. The aim of this study was to determine if treatment in this cohort improves symptoms and biochemical markers. Methods: Ninety-four HFE p.C282Y homozygotes with SF 300-1000µg/L were randomized to either iron reduction by erythrocytapharesis, or to sham treatment by plasmapheresis. Patient reported outcomes and biochemical markers were a d i a n and i n indin a ifi d a in 600µg/L), gender and site. Results: There was a significantly greater improvement in Modified Fatigue Impact Scale score (primary outcome measure) in the treatment group compared to the control group, particularly in the cognitive subcomponent. The higher pretreatment SF group showed greater improvement in the cognitive aspect of MFIS and in the mental component of the Medical Outcomes Study Health Survey Version 2. Hepascore and serum F2-isoprostanes had greater improvement with treatment in the lower pretreatment SF group and urine F2-isorpstanes in the higher pretreatment SF group. There was no parameter that showed greater improvement in the control group Conclusions: This randomized blinded study identified significantly greater improvement in various patient reported outcome measures and biochemical markers in the treatment group when compared to the control group. This suggests iron removal should be instigated for all individuals with HH and elevated iron levels. MB Delatycki, SY Ong, LC Gurrin, L Dolling, J Dixon, AJ Nicoll, M Wolthuizen, EM Wood, GJ Anderson, GA Ramm, KJ Allen, J Olynyk, D Crawford, L Ramm, K Croft, P Gow, S Durant, LW Powell. Murdoch Childrens Research Institute. Background and Aims: Hereditary haemochromatosis (HH), an iron overload disorder, is most commonly caused by HFE p.C282Y homozygosity. There is insufficient evidence to determine if individuals with HH and moderately elevated serum ferritin (SF), defined as SF 300- 1000µg/L, should undergo iron reduction treatment. The aim of this study was to determine if treatment in this cohort improves symptoms and biochemical markers. Methods: Ninety-four HFE p.C282Y homozygotes with SF 300-1000µg/L were randomized to either iron reduction by erythrocytapharesis, or to sham treatment by plasmapheresis. Patient reported outcomes and biochemical markers were measured prior to treatment and prior to unblinding. Subjects were a ifi d a in 600µg/L), gender and site. Results: There was a significantly greater improvement in Modified Fatigue Impact Scale score (primary outcome measure) in the treatment group compared to the control group, particularly in the cognitive subcomponent. The higher pretreatment SF group showed greater improvement in the cognitive aspect of MFIS and in the mental component of the Medical Outcomes Study Health Survey Version 2. Hepascore and serum F2-isoprostanes had greater improvement with treatment in the lower pretreatment SF group and urine F2-isorpstanes in the higher pretreatment SF group. There was no parameter that showed greater improvement in the control group Conclusion: This randomized blinded study identified significantly greater improvement in various patient reported outcome measures and biochemical markers in the treatment group when compared to the control group. This suggests iron removal should be instigated for all individuals with HH and elevated iron levels.
Seventh Congress of the International BioIron Society Page 106 Podium Abstracts IBIS
Podium #47
THIZOLIDINONE COMPOUNDS PREVENT IRON OVERLOAD IN HFE MURINE MODEL THROUGH PROMOTING HEPCIDIN EXPRESSION Jing Liu and Sijin Liu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Presented By: Jing Liu
Iron overload disorders, such as hereditary hemochromatosis and thalassemia diseases, affect a huge number of population worldwide. The occurrence of iron overload is due to repressed hepcidin expression in these diseases. Thus, induction of hepcidin is a therapeutic target to relieve iron overload in these diseases. In the current study, more than 200 compounds were screened through the hepcidin luciferase reporter assay and qRT-PCR assay. Of them, 10 compounds were identified to upregulate hepcidin mRNA expression in hepatocytes. Afterwards. compounds 93, 156 and 165 were verified to elevate hepatic hepcidin expression, associated with according changes of splenic and serum iron concentrations. Furthermore, these 3 compounds were subjected to assessment in hemochromatosis mouse model. Our results manifested that compounds 93, 156 and 165 robustly elevated hepcidin expression in Hfe-/- mice relative to untreated control, resulting in diminished serum and liver iron content. Further mechanistic investigations revealed that Smad1/5/8 phosphorylation and Stat3 phosphorylation were activated to enhance hepcidin transcription in vitro and in vivo in response to these compounds. Further, reduction of Erk phosphorylation was demonstrated to contribute to the increase of Smad1/5/8 and Stat3 phosphorylation upon these compounds. Together, our combined data suggested that thiazolidonine derivatives may offer novel therapeutics for the treatment of iron overload disorders.Iron overload disorders, such as hereditary hemochromatosis and thalassemia diseases, affect a huge number of population worldwide. The occurrence of iron overload is due to repressed hepcidin expression in these diseases. Thus, induction of hepcidin is a therapeutic target to relieve iron overload in these diseases. In the current study, more than 200 compounds were screened through the hepcidin luciferase reporter assay and qRT-PCR assay. Of them, 10 compounds were identified to upregulate hepcidin mRNA expression in hepatocytes. Afterwards. compounds 93, 156 and 165 were verified to elevate hepatic hepcidin expression, associated with according changes of splenic and serum iron concentrations. Furthermore, these 3 compounds were subjected to assessment in hemochromatosis mouse model. Our results manifested that compounds 93, 156 and 165 robustly elevated hepcidin expression in Hfe-/- mice relative to untreated control, resulting in diminished serum and liver iron content. Further mechanistic investigations revealed that Smad1/5/8 phosphorylation and Stat3 phosphorylation were activated to enhance hepcidin transcription in vitro and in vivo in response to these compounds. Further, reduction of Erk phosphorylation was demonstrated to contribute to the increase of Smad1/5/8 and Stat3 phosphorylation upon these compounds. Together, our combined data suggested that thiazolidonine derivatives may offer novel therapeutics for the treatment of iron overload disorders.
Seventh Congress of the International BioIron Society Page 107 Podium Abstracts IBIS
Podium #48
IMPROVING THE EFFICIENCY OF IRON CHELATORS USING NANOTECHNOLOGY Shanshan Guo¹, Gang Liu, PhD¹, Tianqing Liu, PhD², David Frazer, PhD², Guangjun Nie, PhD¹ and Greg Anderson, PhD² ¹CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, China; ²Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: Shanshan Guo, BE
Introduction: Significant pathology accompanies body iron accumulation in both primary and secondary iron loading di d i n a i a a i a n i n adin a in a i a -thalassaemia, iron is removed chemically using iron chelators. Of the three chelators in clinical use, Desferrioxamine (DFO) is the most effective iron binding compound with the most favourable safety profile, but an onerous parenteral administration regimen means that patient compliance is low. If DFO could be delivered with greater efficiency, its clinical utility could be improved. Nanotechnology approaches have greatly improved the delivery of a broad range of therapeutic agents. The aims of this study were to determine whether amphiphilic copolymer nanoparticles (NPs) can be used to deliver DFO efficiently and whether they are effective at depleting body iron. Methods: PEGylated PLGA NPs containing DFO (DFO-NP) were generated by the double emulsion method and characterized by transmission electron microscopy and dynamic light scattering. Macrophage (RAW264.7, J774) and hepatoma (HepG2, HuH7) cell lines were used to study toxicity and efficacy of iron removal in vitro. In vivo iron removal was examined by administering NPs to wild-type mice that had previously been loaded using iron dextran (0.3mg/kg, every second day, twice), as well as Hfe knockout mice and Hbbth3/+ mice. In all studies, DFO-NPs were compared to free DFO and NPs without encapsulated chelator. Results: Physical characterisation showed a uniform preparation of NPs with an average diameter of 113nm. They were stable in the pH range 4.4-7.4. When applied to the macrophage and hepatoma cell lines, DFO-NPs were much more effective at depleting cellular iron levels than free DFO, particularly at higher levels of chelators (up to 80 µM). Mice previously iron loaded with iron dextran, as well as Hfe knockout mice and Hbbth3/+ mice were treated with either saline, free DFO, empty NPs or DFO-NPs (40 mg/kg DFO) on alternate days for 4 weeks. DFO-NPs were more efficient at reducing liver and spleen iron levels than free DFO in each of the models of iron loading studied (e.g. liver: iron dextran loaded mice, 6.15, 5.12, 6.03 and 3.88 mg/g dry weight; Hbbth3/+ mice: 2.28, 2.17, 2.20 and 1.05 mg/g dry weight in the saline, free DFO, empty NPs and DFO-NP groups respectively). Western blotting for ferritin in various organs and staining for tissue iron confirmed these findings. In iron dextran loaded mice, DFO-NPs proved more efficient at promoting urinary iron excretion that free DFO (e.g. 16.0, 20.9, 13.9 and 28.9 µg of iron in 24 h for the treatment groups as specified above). In vitro, DFO-NPs were less toxic to several cell lines than free DFO, and in vivo they did not elicit any specific inflammatory response. Conclusions: Taken together, our results suggest that using a nanoformulation of DFO is potentially a highly valuable strategy for increasing its efficiency as an iron chelating agent and that it could greatly improve the therapeutic potential of this chelator in human iron overloading disorders.
Seventh Congress of the International BioIron Society Page 108 Podium Abstracts IBIS
Podium #49
IRON-RELATED PROTEINS IN THE KIDNEY AND THEIR REGULATION BY RENAL IRON OVERLOAD Laura Diez Ricote and Mitchell D Knutson, PhD University of Florida Presented By: Laura Diez Ricote, MSc
In iron overload conditions, the kidney is exposed to high levels of iron from transferrin-bound iron as well as non- transferrin-bound iron. However, how the kidney handles iron is poorly understood. Historically, kidney iron metabolism has received comparatively little attention because this organ does not commonly display iron overload-related pathology as do other tissues such as the liver, pancreas, and heart. The lack of clear iron-related pathology in iron overload may also be due to the fact that the kidney does not load as much iron as do other tissues. While studying the role of ZIP14 in tissue iron loading, we observed that hemojuvelin-knockout (Hfe2-/-) mice, when crossed with ZIP14 knockout (Slc39a14-/-) mice, failed to load iron in the liver but loaded a large amount of iron in the kidney (Jenkitkasemwong et al. Cell Metab. 2015). We therefore sought to use this animal model of kidney iron overload to investigate how iron loading affects the expression and localization of iron-related proteins in the kidney. We investigated kidneys from four groups of mice: wild- type (WT), Slc39a14-/-, Hfe2-/-, and Hfe2-/-;Slc39a14-/-. At 6 weeks of age kidney non-heme iron levels were 49.4, 31.0, 147.8, and 506.3 µg/g, respectively. Perls’ Prussian blue stain revealed that iron loaded primarily in the proximal straight tubules (PST) in Hfe2-/- mice, consistent with what has been reported previously (B. Moulouel et al., Kidney International, 2013; Young et al. Antioxidants & Redox Signaling, 2014). In contrast, Hfe2-/-;Slc39a14-/- mice loaded iron mainly in the thick ascending loop (TAL). Both Hfe2-/- and Hfe2-/-;Slc39a14-/- mice also showed iron accumulation in the distal tubules (DT). Western blotting and immunohistochemistry were used to investigate the expression and localization of ferritin, transferrin receptor (TfR1), DMT1, ferroportin, and ZIP8 (a homologue of ZIP14). As expected, iron loading of Hfe2-/- and Hfe2-/-;Slc39a14-/- kidneys was associated with higher levels of L-ferritin and lower levels of TfR1. Ferroportin levels were also elevated in the Hfe2-/- and Hfe2-/-;Slc39a14-/- kidneys, likely because these mice lack hepcidin. By contrast, levels of DMT1 and ZIP8 did not differ among groups. Similar to previous reports, we detected DMT1 on the apical side of tubular epithelial cells in the proximal tubules (PT) and PST and ferroportin mainly on the basolateral side of PT, PST and DT. ZIP8 localized to the apical side of PT and PST. Notably, ZIP8 expression in the PST clearly co-localized with Perls’ positive iron. Collectively these results suggest that lack of ZIP14 alters the iron loading pattern in the kidney under iron overload and that ZIP8 and/or DMT1 participate in iron reabsorption, mainly in PST. Lastly, the observation that iron loads in the kidney despite elevated ferroportin levels suggests that the iron may be unavailable for export (e.g., bound to ferritin) or that the rate of cellular iron uptake exceeds the capacity for cellular iron efflux.
Seventh Congress of the International BioIron Society Page 109 Podium Abstracts IBIS
Podium #50
FERROPORTIN PROTECTS RED BLOOD CELLS FROM OXIDATIVE STRESS AND MALARIA INFECTION BY EXPORTING FREE INTRACELLULAR IRON De-Liang Zhang, PhD¹, ian ang, PhD , inal Shah, PhD³, Manik Ghosh, PhD⁴, ayden llivierre, Sc⁴, in-zhuan Su, PhD , Philip huma, PhD , ictor Gordeuk, PhD³ and racey ouault, PhD⁴ ¹Section on Human Iron Metabolism, NICHD, NIH; ²National Institute of Allergy and Infectious Diseases; ³University of Illinois at hicago; ⁴ unice ennedy Shriver ational Institute of hild ealth and uman Development; Malaria Research Trust, Choma, Zambia Presented By: Deliang Zhang, PhD
All vertebrates use red blood cells (RBC) to transport O2 from the lungs to tissues in vivo for ATP production. Each human RBC is packed with about 280 million hemoglobin molecules, equivalent to 1.12 billion iron atoms that may be released during hemoglobin autoxidation. However, how RBCs manage to avoid iron-mediated damage in such a ROS- rich microenvironment is not known yet. RBCs also host the erythrocytic stage of malaria, a life-threatening parasite causing about half a million deaths annually. Iron supplementation enhances the severity of malarial infection, but the mechanisms by which RBCs regulate intracellular iron homeostasis are incompletely understood. Recently we found that ferroportin (FPN), the only known mammalian iron exporter, is highly expressed in erythroblasts. To figure out its function in erythroblasts, we created erythroblast-specific Fpn knockout mice (Fpn EryKO) and found that these mice developed hemolytic anemia compared to their wild type (WT) littermates. Our results showed that mature RBCs highly expressed FPN at about 54,000 copies per cell. Fpn deficiency in RBCs upregulated labile iron concentrations by 80%, which promoted ROS production, increased osmotic fragility, and induced intravascular hemolysis, which subsequently led to relative anemia of Fpn EryKO mice. The RBC life span of Fpn EryKO mice was reduced by 50%, and the small percentage of Fpn-expressing RBCs that escaped from Cre-mediated recombination in the blood of Fpn EryKO mice became dominant in 4-week age of RBCs, suggesting that RBCs are under selective pressure to express Fpn in vivo. Fpn EryKO mice had tissue iron overload and high serum ferritin, but their serum iron levels were significantly lower than those of WT mice, suggesting that iron exported by Fpn from erythroid cells also contributes to systemic iron homeostasis. Moreover, Fpn deficiency in RBCs also promoted parasitemia, and the Fpn EryKO mice died earlier than WT littermates upon malaria infection, suggesting that Fpn protects RBCs from malaria infection. In contrast, a FPN mutation prevalent in Africa, Q248H, which inhibits hepcidin-induced degradation, protected humans from developing severe malarial anemia in newly analyzed samples from a population study in Africa. Our results suggest that FPN exports free intracellular iron to protect RBCs from iron overload and malarial infection, helping to explain why iron supplementation promotes malarial infection and why the FPN Q248H mutation apparently underwent positive selection in African populations.
Seventh Congress of the International BioIron Society Page 110 Podium Abstracts IBIS
Podium #51
AN UNEXPECTED ROLE OF HFE IN MACROPHAGES Maja Vujic Spasic, PhD, Naveen Kumar Tangudu and Dilay Lai Institute of Comparative Molecular Endocrinology Presented By: Maja Vujic Spasic, PhD
Systemic iron homeostasis is disrupted in prevalent genetic iron overload disorder caused by the mutations in the HFE/Hfe gene. Our earlier work revealed that selective Hfe deficiency in macrophages (HfeLysMCre mutant mice) does not impair systemic and cellular iron homeostasis. By contrast, Hfe actions in hepatocytes were shown to be crucial to maintain appropriate hepcidin expression and to prevent iron overload. These data suggest that actions of Hfe in extra- hepatocytic cells are dispensable for the maintenance of physiological iron homeostasis. Intriguingly, this phenotype develops in age-dependent manner since young HfeLysMCre mutant mice display no overt changes in iron homeostasis. Here we present an unexpected finding: we demonstrate that mice with selective Hfe deficiency in macrophage develop iron-deficient phenotype in an age-dependent manner. We show that isolated Hfe-deficient macrophages display significantly reduced intracellular iron levels. On systemic levels, iron deficiency was measured in the liver, spleen and duodenum of these mice. In line with this, the expression of hepcidin and several other iron-responsive genes is decreased. Likewise, the activity of hepatic Bmp/Smad signaling is accordingly affected in the liver of mutant mice. Given these observations we investigated whether macrophage-Hfe act to maintain cellular iron balance by controlling iron uptake of iron release. To this end we induced acute iron overload. We show that acute iron-dextran injections lead to normalization of iron levels in HfeLysMcre mutant mice. These data suggest that other, Hfe-independent, signaling pathways suffice to level the iron-uptake in mutant mice. We further demonstrate that dysbalanced cellular and systemic iron homeostasis in HfeLysMcre mice is due to enhanced iron export mediated by increased expression of ferroportin. Over-expression of Hfe in macrophages prevented ferroportin induction and normalized cellular iron levels. We next investigated the role of macrophage-Hfe in pathogenic septic conditions. During the septic shock conditions, a significantly increased mortality rate was scored in hepatocyte-specific mutant mice (HfeAlfpCre), a model which phenocopies the constitutive Hfe knock-out mice. By contrast, HfeLysMCre mutant mice show improved survival suggesting that mild iron deficiency may be beneficial under these conditions. Our study provides for the fist time evidences for the role of Hfe in macrophages and innate immunity.
Seventh Congress of the International BioIron Society Page 111 Podium Abstracts IBIS
Podium #52
RESTORED IRON TRANSPORT BY A SMALL MOLECULE PROMOTES GUT IRON ABSORPTION AND HEMOGLOBINIZATION IN MICE, RATS, AND ZEBRAFISH Anthony Grillo, Anna SantaMaria, Alexander Cioffi and Martin Burke University of Illinois Presented By: Anthony Grillo
Deficiencies and/or dysfunction of proteins involved in iron transport, homeostasis, or metabolism often impede the movement of iron into, within, and/or out of cells, and are associated with more than twenty-five Mendelian diseases. Because other iron-transport proteins remain active, site- and direction-selective iron gradients likely build up across the corresponding protein-deficient membranes. We questioned whether a small molecule iron transporter could leverage such gradients to restore the movement of iron into, within, and/or out of cells, and thereby enable its use in endogenous iron-dependent physiological processes (Fig. 1A). In this vein, we discovered the small molecule natural product, hinokitiol, can autonomously perform transmembrane iron transport and thereby restore growth to iron-deficient yeast missing the iron transporting complex Fet3Ftr1. Hinokitiol promotes the movement of iron into, within, and/or out of cells deficient in DMT1, Mfrn1, or FPN1 to restore transepithelial iron transport across DMT1-deficient gut epithelia (Caco-2 monolayers), hemoglobinization in DMT1- and Mfrn1-deficient erythroid progenitors (MEL cells), and iron release from FPN1-deficient gut epithelia (Caco-2 monolayers) and reticuloendothelial macrophages (J774 macrophages). Further, we linked the remarkable capacity for this small molecule to restore iron-dependent physiological processes to the site- and direction-selective build-up of iron gradients. Using spatiotemporal imaging with iron-sensitive fluorescent dyes, we observed increased endosomal iron and reduced cytosolic and mitochondrial iron in DMT-1 deficient MEL cells. We discovered hinokitiol leverages these gradients to release iron from endosomes and increase cytosolic and mitochondrial iron levels. We also observed a build-up of iron in FPN1-deficient J774 macrophages. We further provide evidence for collaboration between the small molecule and endogenous IRE- and Hif2α-mediated regulatory networks, with levels of ferritin, IRP2, TfR2, and FPN1 responding to changes in the dynamic iron status upon hinokitiol treatment. Administration of 1.5 mg/kg hinokitiol via oral gavage promotes gut iron absorption in DMT1-deficient belgrade (b/b) rats and FPN1- deficient flatiron (ffe/+) mice (Fig. 1B, C). Further, hinokitiol treatment restores hemoglobinization and reverses anemia in DMT1- and Mfrn1-deficient chardonnay (cdy/cdy) and frascati (frs/frs) embryos (Fig. 1D, E) and in morpholino-injected DMT1- and FPN1-deficient zebrafish. Thus, a small molecule restores site- and direction-selective iron transport in cells deficient in three distinct iron-transport proteins, and the same compound promotes gut iron absorption or peripheral hemoglobinization in corresponding animal models. Mechanistic studies support the role of transmembrane ion gradients that build up in the setting of missing iron transporters, enabling hinokitiol to restore site- and direction-selective transmembrane iron transport. Further, endogenous protein-based homeostatic mechanisms interface with this imperfect small molecule to promote iron-related physiological processes without disrupting other cellular processes. These findings suggest small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases.
Seventh Congress of the International BioIron Society Page 112 Podium Abstracts IBIS
Podium #53
ASTROCYTE HEPCIDIN IS A KEY FACTOR IN LPS-INDUCED NEURONAL APOPTOSIS LH You², Caizhen Yan, Bingjie Zheng, Yunzhe Ci, Shiyang Chang, Peng Yu, Guofen Gao and Yan-Zhong Chang¹ ¹Hebei Normal Unviersity; ²Hebei Normal University Presented By: Yan-Zhong Chang, PhD
Inflammatory responses involving microglia and astrocytes contribute to the pathogenesis of neurodegenerative diseases (NDs). In addition, inflammation is tightly linked to iron metabolism dysregulation. However, it is not clear whether the brain inflammation induced iron metabolism dysregulation contributes to the NDs pathogenesis. Herein, we demonstrate that the expression of the systemic iron regulatory hormone, hepcidin, is induced by lipopolysaccharide (LPS) through the IL-6/STAT3 pathway in the cortex and hippocampus. In this paradigm, activated glial cells are the source of IL-6 which was essential in the iron overload-activated apoptosis of neurons. Disrupting astrocyte hepcidin expression prevented the apoptosis of neurons, which were able to maintain levels of FPN1 adequate to avoid iron accumulation. Together, our data are consistent with a model whereby inflammation initiates an intercellular signaling cascade in which activated microglia, through IL-6 signaling, stimulate astrocytes to release hepcidin which, in turn, signals to neurons, via hepcidin, to prevent their iron release. Such a pathway is relevant to NDs in that it links inflammation, microglia and astrocytes to neuronal damage. Acknowledgments This work was supported by National Natural Sciences Foundation of China (31471035, 30871260) and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (SKLN-201405).
Seventh Congress of the International BioIron Society Page 113 Podium Abstracts IBIS
Podium #54
EFFECTS OF FERRIC CITRATE ADMINISTRATION IN A MURINE MODEL OF CHRONIC KIDNEY DISEASE Connor Francis¹, Samantha Neuburg², Claire Gerber², Xueyan Wang², Corey Dussold², Lixin Qi², Aline Martin², Myles Wolf² and Valentin David² ¹Division of Nephrology and Hypertension, Department of Medicine, and Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine; ²Division of Nephrology and Hypertension, Department of Medicine, and Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Presented By: Connor Francis
Background: Elevated levels of fibroblast growth factor 23 (FGF23) are strongly associated with cardiovascular disease, mortality, and progression of chronic kidney disease (CKD). Hyperphosphatemia and iron deficiency are powerful stimuli of FGF23 production. This suggests that reducing dietary phosphate intake or absorption and increasing serum iron may lower FGF23 levels and improve clinical outcomes in CKD, but to date, this concept has not been tested in detail. Methods: We tested the hypotheses that ferric citrate treatment will simultaneously correct iron deficiency and also bind to dietary phosphate in the Col4a3ko mouse model of progressive CKD. We fed 4 week-old wild-type (WT) and Col4a3ko (CKD) mice, a control (Ctr) or a 5% Ferric Citrate enriched (FC) diet for 6 weeks and performed biochemical, molecular and histological analysis of iron and mineral metabolism status. Results and Discussion: At ten weeks, Ctr-CKD animals displayed signs of iron deficiency anemia as evidenced by low serum iron (99±12 vs 128±5 mg/dL), hemoglobin (Hb) (15±1 vs 19±1 g/dL) and hematocrit (Hct) (50±3 vs 62±1 %) (p<0.05 vs WT). Ctr-CKD mice also displayed a decline in renal function as shown by a 8-fold increase in Blood Urea Nitrogen (BUN) and urinary albumin compared to WT. Renal histology analyses showed advanced glomerulosclerosis, tubular atrophy and interstitial fibrosis in Ctr-CKD compared to Ctr-WT animals. This was concomitant with a marked increase in both total (tFGF23, which includes intact and cleaved proteins) and intact FGF23 (iFGF23) serum levels, compared to WT (11426±2623 vs. 433±32 pg/mL and 7312±1749 vs. 207±57 pg/mL respectively, p<0.05). In addition, serum PTH levels were also markedly increased (3676±755 vs 375±53 pg/mL) and 1,25Vitamin D levels were low (34±7 vs 143±45 pg/mL) in Ctr-CKD animal (p<0.05, vs. Ctr-WT). Ferric citrate supplementation increased serum iron and ferritin levels by 1.5 and 17-fold respectively in WT and by 1.6 and 7-fold in CKD. Interestingly, FC diet also increased fecal phosphate excretion by 9 fold in CKD mice, resulting in a 1.5-and 4-fold serum phosphate and urinary phosphate reduction (p<0.05, vs. Ctr-CKD). The correction of iron deficiency and serum phosphate levels, dramatically and significantly reduced tFGF23 by 4-fold and iFGF23 by 3-fold (p<0.05 vs. Ctr-CKD). Consistent with decreased iFGF23, serum 1,25Vitamin D levels increased by 2-fold in FC-CKD mice (p<0.05 vs. Ctr-CKD). Interestingly, the FC diet also decreased BUN (127±21 vs. 218±24 mg/dL), 24h urine albumin (101±69 vs. 586±91 µg) and reduced mean blood pressure by 11% (p<0.05, vs. Ctr-CKD). Reductions in interstitial fibrosis and tubular dystrophy were also evident by histology in FC-CKD animals compared to Ctr-CKD group. Conclusions: Our data show that ferric citrate administration in CKD mice reduces the magnitude of FGF23 increase and slows disease progression. This suggests that ferric citrate might mitigate renal injury and possibly improve survival in CKD.
Seventh Congress of the International BioIron Society Page 114 Podium Abstracts IBIS
Podium #55
CROSSTALK BETWEEN HYPOXIC AND AUTOPHAGIC PATHWAYS IN INTESTINAL IRON METABOLISM Nupur Das, MBBS, PhD¹, Amanda Sankar, MD², Andrew Schwartz, MS¹ and Yatrik Shah, PhD¹,³,⁴ ¹Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI; ²Department of Pediatrics and Communicable Diseases, Division of Pediatric Hematology-Oncology University of Michigan, Ann Arbor, MI.; ³Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, MI; ⁴Department of Surgery, University of Michigan, Ann Arbor, MI Presented By: Nupurkanti Das, MBBS, PhD
Iron is critical for many vital processes including oxygen transport and erythropoiesis. As both deficiency and excess of iron can lead to disorders such as anemia and iron overload, iron homeostasis is tightly regulated. Our work has shown a critical role for the transcription factor hypoxia-inducible factor-2a (HIF-2a) in intestinal iron absorption. Transcriptomic analysis demonstrates that HIF-2a regulates over 90% of all transcripts induced following iron deficiency in the intestine. However, beyond divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1) and duodenal cytochrome b (Dcytb), no other genes/pathways have been critically assessed with respects to their importance in intestinal iron absorption. Ferritinophagy is associated with cargo specific autophagic breakdown of ferritin and subsequent release of iron. Our work is the first to show that intestinal ferritinophagy is integrated to systemic iron demand via HIF-2a. Nuclear receptor co-activator 4 (NCOA4) is the cargo receptor to initiate ferritinophagy. We show that duodenal NCOA4 expression is regulated by HIF-2a during high systemic iron demands. Moreover, overexpression of intestinal HIF-2a is sufficient to activate NCOA4 and promote lysosomal degradation of ferritin. Promoter analysis revealed NCOA4 as a direct HIF-2a target. To demonstrate the importance of intestinal HIF-2a/ferritinophagy axis in systemic iron homeostasis, the role of autophagy inhibitors or a novel NCOA4-null mouse line was assessed. These analyses demonstrate an iron sequestration in the enterocytes, and significantly high tissue ferritin levels during low iron diets. Moreover, in a hemolytic anemia model, altering ferritinophagy significantly disrupted RBC homeostasis. Together, our data suggests efficient ferritinophagy is critical for intestinal iron absorption and systemic iron homeostasis. This work provides novel mechanisms and targets that regulate systemic iron metabolism in iron-related disorders.
Seventh Congress of the International BioIron Society Page 115 Poster Abstracts IBIS
Poster #1
SMAD1/5 IS REQUIRED FOR ERYTHROPOIETIN SUPPRESSION OF HEPCIDIN IN MICE Chia-Yu Wang, Amanda B. Core, Kimberly B. Zumbrennen-Bullough, Susanna Canali, Sinan Ozer, Lieve Umans, An Zwijsen and Jodie L. Babitt Massachusetts General Hospital, Harvard Medical School Presented By: Chia-Yu Wang, PhD
Background: Anemia and other conditions that increase erythropoietic drive suppress liver expression of the main iron regulatory hormone hepcidin to supply adequate iron for red blood cell production. Erythroferrone was recently proposed as one such mediator of hepcidin suppression by anemia; however, its mechanism of action remains uncertain. The BMP signaling pathway is a central regulator of hepcidin transcription in response to iron. BMPs act by inducing phosphorylation of intracellular receptor-activated SMAD (R-SMAD) transcription factors: SMAD1, SMAD5, and SMAD9 (also known as SMAD8). Although R-SMADs have been shown to have redundant, dose-dependent functions in many biological contexts, accumulating data suggest that they do not always have overlapping functions. Moreover, the role of the BMP-SMAD signaling in hepcidin suppression by anemia is not known. Methods: We used R-SMAD siRNA knockdown in vitro and generated mice with a hepatocyte-specific knockout of R- SMADs in vivo to determine the relative contribution of individual R-SMADs to hepcidin regulation. We also used these mice to elucidate the role of SMAD signaling in hepcidin suppression by erythropoietin and erythroferrone. Results: Knockdown studies in human hepatoma Hep3B cells demonstrated that SMAD5 siRNA robustly inhibited hepcidin expression, whereas SMAD1 siRNA had a more modest inhibitory effect and SMAD9 siRNA did not inhibit hepcidin expression. At 8 weeks of age, double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice developed massive serum and liver iron overload, whereas single-knockout Smad5fl/fl;Cre+ or Smad1fl/fl;Cre+ mice had minimal to no iron loading. Liver hepcidin (Hamp) mRNA was reduced in double-knockout mice, but not in single-knockouts, at 8 weeks of age. However, Hamp mRNA was reduced in isolated primary hepatocytes from single-knockout mice at the baseline with a similar fold increase when stimulated by Bmp6, indicating the inducibility of Hamp in these animals was well-preserved. Similarly, Hamp mRNA was reduced in liver tissue from single-knockout mice at 12-day-old, suggesting some degree of hepcidin deficiency that was compensated for after exposure to the high iron content of the standard rodent diet. In Smad1fl/fl;Smad5fl/wt;Cre+ mice and Smad1fl/wt;Smad5fl/fl;Cre+ females containing one functional allele of Smad5 and Smad1, respectively, modestly increased liver iron loading with elevated transferrin saturation was observed, suggesting a gene dosage effect. Consistent with prior studies, epoetin alfa (EPO) treatment led to a robust induction in bone marrow erythroferrone (Fam123b) mRNA and reduction in liver Hamp mRNA levels in Smad1fl/fl;Smad5fl/fl;Cre- control mice. However, EPO did not suppress Hamp in double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice. Similarly, overexpression of Fam132b decreased Hamp and Id1 mRNA levels in primary hepatocytes from Smad1fl/fl;Smad5fl/fl;Cre- mice; however, neither Hamp nor Id1 mRNA was reduced in Smad1fl/fl;Smad5fl/fl;Cre+ primary hepatocytes. Hamp mRNA was further suppressible in double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice under other conditions such as treatment with a low iron diet. Conclusions: These data demonstrate that hepatocyte Smad5 and Smad1 have overlapping functions and work collaboratively to govern hepcidin transcription and that EPO and erythroferrone require an intact SMAD signaling pathway to suppress hepcidin expression.
Seventh Congress of the International BioIron Society Page 116 Poster Abstracts IBIS
Poster #2
LIVER HFE PROTEIN CONTENT IN IRON DEFICIENT MICE Jan Krijt¹, Jana Frýdlová², Iuliia Gurieva² and Martin Vokurka² ¹Institute of Pathological Physiology, First Faculty of Medicine, Charles University; ²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic Presented By: Jan Krijt, PhD
HFE gene was described in 1996; its mutations cause hereditary hemochromatosis. In 2008, it was shown that the disruption of Hfe in mouse hepatocytes decreases liver hepcidin expression. However; up to now, there is no information on liver HFE expression at the protein level. In the presented study, we examined the effect of iron deficiency on liver HFE protein content. Iron deficiency was induced in adult mice by feeding of an iron-deficient diet for 10 weeks; liver HFE protein content was determined in plasma membrane-enriched fraction of liver homogenates by immunoblotting using a commercial antibody (SC-18810). Feeding of iron-deficient diet to adult male C57BL/6 mice for 10 weeks did not result in an increase in splenic Fam132b expression; liver non-heme iron content was decreased to approximately 30% of controls. Liver Hamp mRNA content was decreased to approximately 20% of controls. There was no change in liver Hfe mRNA content; however, liver HFE protein content was significantly decreased (Fig.1). The results indicate a strong effect of iron deficiency on HFE protein content. Since functional liver HFE protein is necessary for adequate hepcidin expression, it can be speculated that the observed decrease in HFE protein content could contribute to attenuated hepcidin expression in iron deficiency. In contrast to iron deficiency anemia models based on administration of iron deficient diets to weaned mice, feeding of iron deficient diet to adult mice did not result in an increase of splenic Fam132b expression. It is therefore suggested that the observed decrease of liver HFE protein content could contribute to Hamp gene downregulation in states of relatively mild iron depletion, which does not yet result in anemia, hypoxia and increased expression of erythroferrone. Supported by Czech Science Foundation grant 15-16803S.
Fig.1. HFE protein content in plasma-membrane enriched fraction of liver homogenate. C: mice fed control diet; ID: mice fed iron deficient diet for 10 weeks. KO indicates a sample from Hfe-deficient mouse (negative control), E- cadherin is included as loading control.
Seventh Congress of the International BioIron Society Page 117 Poster Abstracts IBIS
Poster #3
EVIDENCE THAT HEPCIDIN OCCLUDES FERROPORTIN TO INHIBIT IRON EXPORT S. Aschemeyer, B. Qiao, E. Valore, T. Ganz and E. Nemeth University of California, Los Angeles Presented By: Sharraya Aschemeyer
The hepcidin-ferroportin (FPN) axis controls intestinal absorption of iron, as well as its internal recycling and tissue distribution. FPN is the only known cellular iron exporter in vertebrates and is the conduit through which iron is supplied into plasma. Hepcidin is a systemically acting iron-regulatory peptide hormone and the only known natural FPN ligand. We have previously shown that hepcidin binding to FPN causes the ubiquitination, endocytosis, and degradation of the ligand-receptor complex thereby decreasing iron supply to plasma. Based on its similarity to the major facilitator superfamily of alternating access transporters, of which FPN is a member, we generated a computational structural model of human FPN. To test our model, we mutated residues that are candidates for hepcidin binding or are mutated in the gain of function form of human ferroportin disease. Our FPN structural model implies that when hepcidin binds FPN in the open-out conformation, iron export should be inhibited even if FPN is not internalized and degraded. To test this conjecture, we prevented the hepcidin-dependent ubiquitination of FPN by generating K8R, a FPN mutant in which 8 lysines in or near the long intracellular loop connecting the two 6-helix lobes of FPN were mutated to arginines. When overexpressed in HEK293 cells, K8R localized to the cell membrane and exported iron normally, similarly to wild type FPN (WT). In the presence of hepcidin, the WT FPN was ubiquitinated, internalized, and degraded, but the K8R mutant and C326S mutant, a mutant that cannot bind hepcidin, were not ubiquitinated and remained localized to the membrane as determined by microscopy. However, hepcidin treatment inhibited iron efflux from the WT, and at higher concentrations, K8R, but not C326S. Cellular ferritin, indicative of cellular iron retention, was also elevated after hepcidin addition for K8R and WT, but not C326S. Our results are consistent with a model in which hepcidin binding occludes FPN, thus interfering with iron export and promoting iron storage in ferritin. PR73, a 9 amino acid hepcidin analog, also inhibited iron export by K8R. Engineered for high affinity binding to FPN, PR73, at higher concentrations, degraded even C326S, causing iron retention in the cell. We propose that hepcidin and its analogs can inhibit iron export through FPN either by inducing its endocytosis or, at higher concentrations, by occluding its open-out conformation. The two mechanisms may be differentially active depending on the cell type and its endocytic machinery, explaining the reported differences in tissue sensitivity to hepcidin and discrepancies between the effect of hepcidin on iron export as compared to endocytosis.
Seventh Congress of the International BioIron Society Page 118 Poster Abstracts IBIS
Poster #4
CALCIUM ACTIVATES FERROPORTIN-MEDIATED CELLULAR IRON EFFLUX Bryan Mackenzie¹, T. Alex Ruwe¹, Chandrika N. Deshpande², Reiya Taniguchi³, Vicky Xin², Ali Shawki¹, Kyle R. Vieth¹, Bo iao⁴, Erika . alore⁴, samu Nureki³, omas Ganz⁴, Elizabeta Nemeth⁴ and Mika Jormakka² ¹University of Cincinnati College of Medicine; ²Centenary Institute–Sydney; ³RIKEN–Saitama; ⁴David Geffen School of Medicine at UCLA Presented By: Bryan Mackenzie, PhD
Ferroportin—the only known cellular iron exporter—is responsible for iron efflux from enterocytes and macrophages to the blood plasma. This transporter, under the control of hepcidin, serves as a key site of regulation in human iron homeostasis. The thermodynamic mechanism driving ferroportin-mediated iron export is unknown. As a first step in exploring potential thermodynamic mechanisms, we have used biophysical analyses, site-directed mutagenesis, and functional assays to test the hypothesis that ferroportin is a secondary-active transporter that functions in iron/cation antiport. We expressed human ferroportin in RNA-injected Xenopus oocytes and measured efflux of radiotracer metal (microinjected into the oocyte). Expression of ferroportin stimulated the first-order rate constants describing the efflux of 55Fe. Ferroportin-mediated 55Fe efflux activity was unaffected by replacement of extracellular Na+ by choline. Removal of extracellular calcium (i.e. 0 Ca2+ plus 1 mM EGTA) abolished the ferroportin-mediated efflux of 55Fe or 57Co. Mg2+ could 2+ 2+ 55 not substitute for Ca . Whereas extracellular Ca stimulated the ferroportin-mediated Fe efflux (K0.5 for Ca of 0.8 ± [SE] 0.1 mM), we found that (1) pharmacologically raising intracellular Ca2+ concentration had no effect on 55Fe efflux, (2) expression of ferroportin did not stimulate the uptake of 45Ca2+ in oocytes with or without microinjection of iron, and (3) expression of ferroportin did not stimulate 45Ca2+ efflux. We also tested the calcium dependence of ferroportin-mediated iron efflux in a human expression system, and found that removal of extracellular calcium (i.e. 0 Ca2+ plus 1 mM EGTA) inhibited 55Fe efflux from HEK cells stably expressing ferroportin. We have used X-ray crystallography to solve the structure of a Ca2+-bound BbFPN protein (a prokaryotic ferroportin ortholog) and identified four strictly conserved residues that coordinate Ca2+. By using isothermal titration calorimetry and purified protein, we confirmed Ca2+ binding in wildtype 2+ BbFPN (Kd for Ca of 13 0.2 μM) and mammalian ferroportin (Kd 17 6 μM), whereas we could detect no Ca binding in mutants in which we had mutated any one of the four putative Ca2+ coordinating residues. BbFPN exhibited no detectable affinity for Mg2+ or K+ under the experimental conditions. Mutagenesis of any one of the putative Ca2+ coordination residues in human ferroportin abolished iron-transport activity in our vertebrate expression systems, with one exception; one mutant exhibited modest residual activity that was accelerated by our increasing the extracellular calcium concentration. This mutant exhibited decreased apparent affinity for extracellular calcium (K0.5 = 2.4 ± 0.4 mM, n = 9) compared with wildtype ferroportin (K0.5 = 0.9 ± 0.1 mM; n = 8) (P = 0.004). In conclusion, (1) we have identified the site of calcium coordination in BbFPN and human ferroportin; (2) we conclude that calcium is a required cofactor of ferroportin but find no evidence that calcium is transported; and (3) we dismiss the notion that ferroportin is an iron/cation antiporter. We speculate that Ca2+ triggers the outward-facing to inward-facing conformational change, exposing the iron-binding site towards the cytoplasm. Our findings raise the possibility that ferroportin-mediated cellular iron export may be restricted in conditions of hypocalcemia. Support: NIH–NIDDK grant R01 DK107309
Seventh Congress of the International BioIron Society Page 119 Poster Abstracts IBIS
Poster #5
AGE RELATED IRON ACCUMULATION: ACCUMULATING WHAT EXACTLY? Simon James, PhD¹, Dominic Hare, PhD¹, Martin de Jonge, PhD² and Gawain McColl, PhD¹ ¹Florey Institute of Neuroscience and Mental Health; ²Australian Synchrotron Presented By: Simon James, PhD
Iron is a fundamentally important biochemical catalytic cofactor thanks to a capacity to accommodate a range of electronic configurations throughout its valence shell. Coordination chemistry controls the reactivity and availability of iron across cellular environments but characterisation of specific iron-ligand species requires isolation of the complex, necessitating disruption of biological systems despite the attendant risk of mismetallation. The tools available to study biological coordination chemistry in situ have remained limited. The synergy of synchrotron-based X-ray fluorescence microscopy (XFM) and X-ray absorption near edge structure (XANES) spectroscopy represents a powerful analytical approach for studying iron biochemistry at the micro-scale. This permits both quantitative mapping of metal distribution and profiling of the native coordination environment without the need for exogenous molecular probes. We combined these two measurement strategies using the same synchrotron beamline to develop an imaging approach we have called fluorescence imaging (‘fi’, or for the reek ‘phi’) A ES. e demonstrate the development of non-destructive A ES imaging under standard laboratory conditions, validated in a Caenorhabditis elegans model of disrupted metal metabolism. Further we utilise this technique to explore the changes in iron metabolism that accompany ageing in C. elegans and show how and where iron homeostasis is lost during aging and its relationship to the age-related elevation of damaging reactive oxygen species. We find that ferritin is used to sustain longevity, buffering against exogenous iron and ablation of ferritin induces rapid ageing. We propose that loss of iron homeostasis may be fundamental to and inescapable consequence of ageing and may represent a potent target for therapeutic strategies aimed at maintaining health at advancing age.
Seventh Congress of the International BioIron Society Page 120 Poster Abstracts IBIS
Poster #6
RECOVERY OF HEME OXYGENASE 1-DEFICIENT MICE THROUGH REPOPULATION OF WT MACROPHAGES IN RETICULOENDOTHEILAL SYSTEM Ki Soon Kim and Tracey A Rouault, PhD NIH Presented By: Ki Soon Kim, PhD
Heme oxygenase 1 (Hmox1) is an inducible enzyme that catalyzes the degradation of heme into biliverdin, carbon monoxide and ferric iron. Loss-of-function mutations in the Hmox1 gene cause a rare and lethal disease in children, characterized by severe anemia, intravascular hemolysis and tissue damage. Macrophages of the reticuloendothelial system play a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes. Macrophages were depleted from the liver and spleen of Hmox1 knockout (KO) mice, which resulted in intravascular hemolysis and severe damage to the endothelial system and organs (1). Previously, we have shown that bone marrow transplantation rescues the phenotype by providing Wild-type (WT) reticuloendothelial cells in Hmox1 KO mice (2). To investigate whether the macrophages in the reticuloendothelial system can repopulate and divide in tissue, we injected macrophages into the tail vein of KO mice. We isolated the bone marrows from GFP-expressing mice to enable us to detect them in recipient animals. Donor bone marrow cells were extracted from femur and tibial bones of 2 month-old mice, and cells were differentiated with L-cell conditioned medium for 1 week. Subsequently, the differentiated GFP expressing macrophages were injected into recipient KO mice. The introduction of WT macrophages reversed abnormal blood parameters of KO mice. Mean cell volume and hematocrits were significantly improved in transplanted KO mouse, compared to KO mouse. Elevated plasma concentrations for lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in KO mice normalized after macrophage transplantation. Furthermore, the white blood cell counts (WBC) returned normal. The renal iron accumulation was shown in kidney of KO mice through Perls’ blue staining. WT macrophage transduction revealed normalization of the iron-staining pattern, and significant numbers of Kupffer cells were identified in the livers of transplanted KO mice through the F4/80 staining. These were sufficient to support red blood cell recycling. Our results suggest there is no barrier to repopulating the reticuloendothelial system, and the macrophages in the reticuloendothelial system may divide in tissues as needed. Our results suggest that Hmox1-/- patients could be potentially be treated by repair of their mutations in macrophages, followed by reinfusion, without resorting to bone marrow transplantation. Our preliminary results will form the basis of further experiments. References 1) Dysfunction of the heme recycling system in heme oxygenase 1-deficient mice : effects on macrophage viability and tissue iron distribution, Blood, 2010, V116:6054- 6062 2) Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice, Blood, 2014, V124 : 1522-1530
Seventh Congress of the International BioIron Society Page 121 Poster Abstracts IBIS
Poster #7
DEVELOPING A GALNAC-CONJUGATED TMPRSS6 ANTISENSE THERAPY FOR THE TREATMENT OF ß- THALASSEMIA Shuling Guo, PhD¹, Mariam Aghajan, PhD¹, Tom Zanardi, PhD¹, Rudy Gunawan, PhD¹, Carla Casu, PhD², Vania Presti², Sheri Booten¹, Stefano Rivella, PhD² and Brett Monia, PhD¹ ¹Ionis Pharmaceuticals; ²Children’s Hospital of Philadelphia Presented By: Shuling Guo, PhD
Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H1–mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Recently, we developed a ligand conjugated antisense (LICA) strategy to deliver antisense oligonucleotide (ASO) to hepatocytes which significantly improves the potency (Prakash et al. Nucleic Acids Res. 2014; 42(13):8796-807). This LICA approach has been validated in both rodents and in humans. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previously we have shown that TMPRSS6 ASO treatment can up-regulate hepcidin expression and ameliorate the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (Hbbth3/+ mice), which recapitulates beta-thalassemia intermedia in humans (Guo et al. J Clin Invest. 2013; 123(4):1531-41). This ASO can be combined efficiently with iron chelators for the management of iron overload and anemia in non-transfusion-dependent thalassemia (Casu et al. Haematologica. 2016; 101(1):e8-e11). Using LICA strategy, we demonstrated that similar pharmacology can be achieved with a 10-fold lower dose. A LICA human TMPRSS6 clinical candidate (Ionis-TMPRSS6-LRx) has been identified. Similar to the mouse TMPRSS6 ASO, Ionis-TMPRSS6-LRx showed superior potency in human primary hepatocyte culture and in human TMPRSS6 transgenic mice compared with the parent ASO. Ionis-TMPRSS6-LRx has been evaluated in a 13-week mouse study and a 9-month monkey study and demonstrated a favorable tolerability profile. Ionis-TMPRSS6-LRx was rapidly absorbed to the systemic circulation and the plasma exposure increased greater than dose proportionally. The nonlinearity was due to a dose- dependent reduction in apparent plasma clearance. After attaining the maximum plasma concentration, it decreased in an apparent multi-exponential fashion with time. ASO concentrations in liver increased slightly less than dose-proportionally to dose. Pharmacokinetic profile of this compound is similar to what have been observed with other LICA ASOs. After treatment with Ionis-TMPRSS6-LRx, we observed a dose-dependent reduction of monkey TMPRSS6 up to >90%. Serum iron and transferrin saturation decreased in a dose-dependent and time-dependent manner as expected. Collectively, our data demonstrate that GalNAc-conjugated TMPRSS6 ASO could be an effective therapeutic for patients with beta- thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2017.
Seventh Congress of the International BioIron Society Page 122 Poster Abstracts IBIS
Poster #8
HEME ACCUMULATION IN ENDOTHELIAL CELLS IMPAIRS ANGIOGENESIS BY TRIGGERING PARAPTOSIS Sara Petrillo¹, Deborah Chiabrando, PhD¹, Tullio Genova, PhD¹, Veronica Fiorito, PhD¹, Giada Ingoglia, PhD¹, Francesca Vinchi, PhD², Federico Mussano³, Stefano Carossa³, Lorenzo Silengo¹, Fiorella Altruda¹, Giorgio Merlo¹, Luca Munaron¹ and Emanuela Tolosano¹ ¹University of Turin; ²Univerity of Heidelberg; ³University of Turin, Dental School Presented By: Sara Petrillo, PhD
Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. In the future, a deeper understanding of the molecular actors involved in heme-triggered paraptosis might provide new tools to modulate angiogenesis in pathophysiologic conditions.Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. In the future, a deeper understanding of the molecular actors involved in heme-triggered paraptosis might provide new tools to modulate angiogenesis in pathophysiologic conditions.
Seventh Congress of the International BioIron Society Page 123 Poster Abstracts IBIS
Poster #9
TWO SIDES OF THE SAME COIN: A FRESH PERSPECTIVE ON THE ROLE OF TNFa IN INFLAMMATION, IRON METABOLISM AND HEMATOPOIESIS Vania Lo Presti, MSc, Carla Casu, PhD, Rea Paraskevi Oikonomidou, MD, Emir O'Hara, BSc, Sara Gardenghi, PhD and Stefano Rivella, PhD Children's Hospital of Philadelphia Presented By: Vania Lo Presti, MSc
The anemia of inflammation (AI) is a well-recognized clinical condition, commonly observed in patients with chronic infections, malignancy, trauma, or inflammatory diseases; however, its etiology remains unclear. In AI, inflammation is associated with abnormal levels of cytokines produced by the innate immune system. Some cytokines are considered to be pro-inflammatory while others anti-inflammatory. The role of pro-inflammatory cytokines, such as IL6 and IL1, has been well documented in AI. However, the precise contribution to AI of these and additional pro-inflammatory cytokines has not been elucidated yet. n particular, abnormally elevated levels of T have been associated with many inflammatory conditions and a symptom of disease progression. For this reason, pharmacological inhibition of T represents a major focus of drug development. It has been shown that administration of Heat Killed Brucella Abortus (HKBA) in C57BL/6 ( T) mice triggers expression of several inflammatory cytokines, including T . urthermore, animals treated with a single injection of BA develop a severe but reversible anemia. n this study, we investigate the role of T in the development of anemia by administration of BA to T and Tnf -/- mice. Our results demonstrate that Tnf -/- mice, following a single injection of HKBA, develop an irreversible macrocytic, hyperchromic anemia, characterized by complete failure in bone marrow erythropoiesis and extramedullary erythropoiesis in the spleen. We also demonstrate that HKBA- treated Tnf -/- mice present low serum levels of hepcidin while that of erythropoietin and iron is elevated, excluding that these factors contributes to the development of A . nterestingly, compared to T mice, Tnf -/- animals treated with HKBA exhibit a chronic anemia associated with sustained production of cytokines such as , L12p40 and L1β. n addition, we observe a quantitative and qualitative difference in T cells and macrophages, likely contributing to the anemia. Moreover, HKBA-treated Tnf -/- mice show increased Reactive Oxygen Species (ROS) production in progenitor and terminally differentiated erythroid cells, which exhibit a reduced lifespan. In conclusion, our results suggest a possible dual role of T during A (pro- and anti-inflammatory), highlighting the extreme importance of this cytokine in the context of the immune function and anemia resolution.
Seventh Congress of the International BioIron Society Page 124 Poster Abstracts IBIS
Poster #10
EXPLORING THE EFFECTS OF IRON DEFICIENCY ANAEMIA AND REPLETION WITH INTRAVENOUS IRON ON EXERCISE PERFORMANCE IN C57 WILD TYPE MICE Anna Butcher, MBBS, PhD¹, Marija Sajic, MBBS, PhD², Kenneth Smith, PhD² and Toby Richards, MD, FRCS³ ¹UCL; ²Department of Neuroinflammation, Institute of Neurology, UCL, London, United Kingdom; ³Division of Surgery and Interventional Medicine, UCL, London, United Kingdom Presented By: Anna Butcher, MBBS, PhD
Background: Anaemia is common and regarded by the World Health Organisation (WHO) as one of the top 10 causes of disability. The most common cause of anaemia is iron deficiency (ID). Previous research in both animals and humans have demonstrated the effects of ID, contributing to impaired energy production, physical work and exercise capacity. The aim of this pilot study was to establish a murinic model of diet controlled iron-deficiency anaemia (IDA) and a timeframe for analysing functional changes in terms of exercise endurance following treatment with intravenous iron. Methods: Thirty C57/Bl6 male (6-week old weaned) mice were divided into three groups, normal diet (n = 10; 50-58ppm total Iron), ID diet (n = 20; 2-6ppm total Iron), and iron replete (n = 10; ID mice treated with IV iron) for six-weeks duration. During week two of the diet, mice commenced endurance training on a treadmill (set at 5-degree incline, and starting speed of 13m/min, increasing by approximately 2m/min every 5min), until exhaustion. Mice underwent twice- weekly weights and weekly haemoglobin (Hb) measurements via tail-vein sampling and analyzing on a HemoCue 201 (Radiometer Inc., Denmark) until iron-deficient mice became anaemic (Hb <136g/L). At the end of the diet, ten mice from the ID group were administered IV FCM (15 mg/kg) and exercised daily for up to 72 hours after dosing. Mice were sacrificed at the end of the treadmill training, and plasma samples were collected for analysis of ferritin levels. Results: During the six-week course of experiments there was no difference in the average body weight of animals across groups (21.70 ± 0.97g for control vs. 21.14 ± 1.15g for ID). Three weeks after commencement of the diet, ID mice demonstrated a significant reduction in running times (11.03 ± 1.08 mins) compared to the control diet group (21.56 ± 1.76 mins; p <0.05). At the same time, Hb remained unchanged and similar between the control and ID groups (153.3 ± 6.5 g/L vs. 148.4 ± 2.9 g/L, p >0.05). After six weeks ID mice developed anaemia with recorded a Hb of 130.4 ± 2.6 g/L, compared to control (151.3 ± 4.1 g/L; p <0.05). The progressive difference in running times between groups remained significant between ID and control (10.43 ± 4.02 mins vs. 24.20± 3.89 mins, p <0.05). Seventy-two hours following injection of IV FCM to ten ID mice, a 48% increase from 11.26 ± 0.83 mins to 21.56 ± 1.81 mins in running time was observed. Iron replete mice were running on average 10 minutes longer than mice who remained iron deficient (21.56 ± 1.81 mins vs. 11.56 ± 2.21 mins, p <0.05). No recorded change to Hb was observed in iron replete mice after FCM compared to those who remained ID (132.2 ± 4.3 g/L vs. 128.5 ± 1.4 g/L, p >0.05). A significant increase in ferritin levels was also observed post FCM dosing in iron replete mice (161.20 ± 21.28 ng/ml protein) compared to ID mice (53.47 ± 31.66 ng/ml protein). Conclusions: ID negatively impacts on exercise performance before the onset of anaemia. Treatment with IV FCM resulted in a rapid improvement in exercise performance.
Seventh Congress of the International BioIron Society Page 125 Poster Abstracts IBIS
Poster #11
NCOA4 INCREASES IN CONDITION OF MODERATE IRON OVERLOAD BUT NOT IN HIGLY IRON OVERLOADED MICE AND IN TFR2 KO ANIMALS Rosa Maria Pellegrino, PhD², Martina Boero, Dr², Antonietta Palmieri, Dr², Mariarosa Mezzanotte, Dr², Giuseppe Saglio, MD² and Antonella Roetto, PhD¹ ¹University of Torino; ²University of Torino, Department of Clinical and Biological Sciences, AOU San Luigi Gonzaga, Orbassano, Torino, Italy Presented By: Antonella Roetto, PhD
Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently demonstrated to be involved in iron metabolism as a cargo protein appointed to ferritin autophagy (Dowdle et al, 2014; Mancias et al, 2014). The NCOA4 KO mouse has an iron overload phenotype with microcytic anemia (Bellelli et al, 2016). Furthermore, NCOA4 seems to be involved in a programmed cell death induced by iron and reactive oxygen species (ROS) named ferroptosis. Its ferritin degradation would lead to an increases of the cellular labile iron pool (LIP) promoting in this way ROS increase and ferroptosis (Gao et al, 2016). Transferrin Receptor 2 (Tfr2) is one of the several proteins involved in iron homeostasis through iron hormone Hepcidin regulation and Tfr2 KO animals present an inappropriately low amount of Hepcidin leading to a systemic iron overload. To investigate if Tfr2 could have some role in NCOA4 dependent ferroptosis we decided to analyze NCOA4 in liver of our iron overloaded Tfr2 KO mouse in comparison to WT sib pairs feeded with an iron enriched diet (WT IED). Since Tfr2 KO animals had an higher LIC compared to WT IED mice (4x vs 2x respectively), we decided to analyze two other groups: Tfr2 KO mice treated with an iron chelator (Tfr2 KO Exj), that decreased animals LIC to levels comparable to that of WT IED, and WT animals submitted to parenteral iron overload (WT PIO), that reached 20x LIC values. NCOA4 has been evaluated in these four animals groups vs WT strain, age and sex matched sib pairs. We found that in WT IED animals LIC and ferritin increased as expected. Surprisingly, also NCOA4 transcript and protein significantly increased in these moderately iron overloaded animals while they were very low in the severely iron overloaded WT PIO animals. NCOA4 protein amount did not increase in Tfr2 KO mice and in Tfr2 KO Exj mice livers, although these presented LIC and liver ferritin similar to WT IED animals. Furthermore, despite comparable LIC values, ferritin amount was significantly higher in Tfr2 KO Exj than in WT IED mice. ROS resulted to be increased in WT IED compared to Tfr2 KO animals liver. The same was true for Tfr1, a marker demonstrated to be selectively increased in ferroptosis sensible cells (Xie et al, 2016). On the contrary, apoptosis markers (p53 and Cleaved Caspase-3) resulted to be increased only in Tfr2 KO animals liver. As a consequence of these results we can conclude that: a) under moderate iron overload condition (WT IED mice), NCOA4 exerts is ferritin cargo protein function causing an increase of ROS that leads to ferroptosis; b) when iron overload significantly increases (WT PIO mice) NCOA4 signals to ferroptosis is overwhelmed by the necessity to have as much ferritin as possible to chelate the metal; c) NCOA4 amount does not change in Tfr2 KO mice independently from stored iron amount. So, we can hypothesize that Tfr2 is in some way involved in NCOA4 regulation and that its lack causes an apoptotic cell death that severely compromise organ function.
Seventh Congress of the International BioIron Society Page 126 Poster Abstracts IBIS
Poster #12
BASIDIOCHROME: MAIN SIDEROPHORE OF MYCORRHIZAL FUNGAL SYMBIONTS OF ORCHIDS WITH INTERESTING CHEMICAL AND BIOLOGICAL PROPERTIES Kurt Haselwandter, Dr¹, D. Pretsch, Dr² and J.M. Rollinger, Dr² ¹University of Innsbruck; ²University of Vienna Presented By: Kurt Haselwandter
Almost all plant species live in symbiosis with fungi and form root structures known as mycorrhizae. A screening of siderophores released by mycorrhizal fungi has revealed that most of them synthesize hydroxamate siderophores of known structures. However, with basidiochrome orchidaceous mycorrhizal fungi (Ceratobasidium and Rhizoctonia spp.) produce a hydroxamate siderphore with a structure unknown until 2006. Basidiochrome represents a linear tripeptide consisting of three L-N5-hydroxyornithines each linked to 3-methyl-2-cis-pentenedioic acid residues (Figure 1). With a log ß110 of 27.8 ± 0.1 and a pFe of 25.0 the iron binding characteristics of basidiochrome are similar to those of the hexapeptides ferrichrome and ferrichrysin. This suggests that the backbone of the molecule has little influence on complex formation with ferric iron. Chelation of ferric iron is certainly the main function of siderophores. However, there is more and more evidence that siderophores mediate a number of important interactions between microorganisms and their hosts. In general, the metal binding properties of siderophores make them promising agents for medical applications. The structure of basidiochrome is rather unique as it represents a linear trishydroxamate siderophore with carboxy- and methyl-groups on each of its acid side chains (see Figure 1). Basidiochrome is currently being tested for evaluating its effect on neurodegenerative processes using Caenorhabditis elegans as model organism. Based on the metal hypothesis of neurodegenerative disorders increased levels of iron are the main cause of dopaminergic cell death in Parkinson´s disease. Thus, the influence of basidiochrome on -synuclein toxicity is currently under investigation in the transgenic Parkinson strain NL5901 as well as in transgenic C. elegans strains of Alzheimer´s disease. Figure 1. Structure of ferric basidiochrome: The linear trishydroxamate siderophore contains a tripeptide sequence of L- N5-hydroxyornithine; each of the amino acid derivatives is linked to one cis-methylglutaconic acid residue.
Seventh Congress of the International BioIron Society Page 127 Poster Abstracts IBIS
Poster #13
DISRUPTION OF THE HEPCIDIN/FERROPORTIN REGULATORY CIRCUITRY CAUSES PULMONARY IRON OVERLOAD AND RESTRICTIVE LUNG DISEASE Joana Neves¹,⁴, Dominik Leitz², Christina Brandenberger³, Christian M hlfeld³, Raman Agra al², Marcus A. Mall²,⁵, Sandro Altamura¹,⁴,⁵ and Martina U. Muckenthaler¹,⁴,⁵ ¹Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg, Germany; ²Translational Lung Research Center Heidelberg , German Center for Lung Research (DZL), University of Heidelberg, Germany; ³Institute of Functional and Applied Anatomy, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (D L), Hannover Medical School, Germany; ⁴Molecular Medicine Partnership Unit, Heidelberg, Germany; ⁵E ual contribution Presented By: Joana Neves
Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in organ pathologies that are so far not considered classical iron-related disorders.
Seventh Congress of the International BioIron Society Page 128 Poster Abstracts IBIS
Poster #14
GENETIC DISRUPTION OF A REDOX ENZYME NCB5OR IN MICE RESULTS IN IRON DYSHOMEOSTASIS, NEUROLOGICAL DEFECTS AND HEARING LOSS IN ADDITION TO EARLY-ONSET LEAN DIABETES Hao Zhu, PhD, WenFang Wang, PhD, Matthew Stroh, PhD, Marcello Peppi, PhD, Haping Wang, MD, Ming Xu, PhD, Zhi- nong Yin, PhD and Jie Dai, MD University of Kansas Medical Center Presented By: Hao Zhu, PhD
Cytosolic NADH cytochrome b5 oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues and capable of reducing ferric iron in vitro. We previously reported that global gene ablation (KO) of NCB5OR in mice resulted in early- onset diabetes in association with elevated mitochondrial proliferation, oxidative stress and endoplasmic reticulum (ER) stress in cells. Mild anemia and iron deficiency have also been observed in prediabetic KO mice. To further investigate the effects of NCB5OR deficiency on iron homeostasis, we subjected these mice to dietary iron modulation. While an iron deficient diet (< 5 ppm) worsens all the phenotypes, an iron sufficient diet (300 ppm) alleviates only anemia, not the others. 59Fe pulse-feeding experiments revealed increased iron flux among major tissues, especially in the brain, consistent with increased transcript and protein expression of genes involved in iron uptake and export, such as transferrin receptor 1 and ferroportin 1, in parallel to decreased ferritin protein levels. Prediabetic KO mice also exhibit hyperactivity in association with elevated dopamine levels in the cortex, as well as hearing loss through analysis of auditory brainstem responses (ABE) and distortion product otoacoustic emissions (DPOAE). The latter phenotype was associated with increased expression of genes involved in oxidative stress response and iron uptake in the cochlea. The roles of NCB5OR deficiency in diabetes and neurologic function were concluded to be cell autonomous by studies of NCB5OR conditional knockout mice of the pancreatic beta-cells (PKO) and the cerebellum / midbrain (CKO), respectively. Early-onset diabetes was observed in PKO mice, and the islets in the prediabetic animals displayed reduced activity of mitochondrial Complex I that is rich in iron sulfur clusters. CKO mice are diabetes free but hypersensitive to harmaline- induced tremor and diet-induced neurologic changes, including reduced locomotor activity, altered complex motor task execution, and gait abnormalities. Assessment of molecular markers in the cerebellum of CKO mice revealed changes in pathways associated with cellular and mitochondrial iron homeostasis. These observations show that iron dyshomeostasis caused by NCB5OR deficiency leads to major cellular defects and conditions that are commonly associated with mitochondrial dysfunction and oxidative stress. Studies are ongoing to characterize how NCB5OR may be involved in the cellular processing of iron.
Seventh Congress of the International BioIron Society Page 129 Poster Abstracts IBIS
Poster #15
METHODOLOGICAL CONSIDERATIONS IN IRON STAINING OF SELECT NON-HEPATIC TISSUES OF MOUSE MODELS OF HEMOCHROMATOSIS Kristy Martin¹, William Myles¹, Daniel Johnstone², Ali Shahandeh¹ and Elizabeth Milward¹ ¹The University of Newcastle; ²University of Sydney Presented By: Kristy Martin
Introduction: While investigating retinal distributions of iron and ferritin in a Hfe-/-xTfr2mut mouse model of hemochromatosis on an albino AKR background, discrepancies were noticed between distributions of iron and ferritin when iron staining was performed in tandem with immunofluorescent labeling for ferritin. Although initially postulated to reflect artefactual staining losses due to the simultaneous double-labeling procedure, similar discrepancies occurred for separate iron and ferritin labeling of adjacent sections. Objective: To identify reasons for discrepancies and improve knowledge of retinal iron and ferritin localization in normal and iron-loaded retina. Methods: Retina were collected from a Hfe-/-xTfr2mut mouse model of hemochromatosis on an albino AKR background at three and nine months and age- and gender-matched control AKR mice following transcardial perfusion with 10% formalin alone or with 1% HCl, 1% potassium ferricyanide in 0.1M PBS, pH 1.0. Tissue was embedded in paraffin for microtome sectioning or in O.C.T. compound for cryostat sectioning. Iron was detected by 3,3’-diaminobenzidine (DAB)- enhanced Perls’ staining or DAB-enhanced Turnbull’s staining and ferritin light or heavy chains were detected by immunohistochemical labeling. Some sections were pre-incubated overnight at room temperature in 3% hydrogen peroxide (H2O2), used to bleach retinal pigments such as melanin that mask iron staining. Results: Iron staining was increased in fixed-frozen Hfe-/-xTfr2mut retinal sections compared to wildtype retina, with increases mainly localized to the choroid and retinal pigment epithelial layers. According to the literature, detectable iron should also be present in the photoreceptor layer, and ferritin immunoreactivity was present in this layer. Yet surprisingly, although prior bleaching should not be required since no pigment is visible in this layer in albino AKR strain mice, photoreceptor iron staining was only present after overnight pre-incubation with 3% H2O2. We postulated that H2O2 pre- incubation may partly influence staining patterns by oxidizing Fe2+ to Fe3+. We were initially unable to detect retinal Fe2+ using standard Turnbull’s staining so this hypothesis was first validated in heart, which has high levels of Fe2+ and Fe3+. Pre-incubation of heart sections overnight in 3% H2O2 increased Perls’ and decreased Turnbull’s staining intensity. Similar results were subsequently observed in retina using a modified Turnbull’s transcardial perfusion fixation technique to improve retention and detectability of retinal Fe2+, which tends to leach out using traditional Turnbulls staining. Conclusions: Although there is long-standing literature addressing various limitations of iron staining, modern authors often fail to consider these, compromising interpretation. Effects of pre-bleaching are not fully understood but may include valence conversions that may be difficult to assess with standard staining, particular as Fe2+ tends to be ‘free’ or bound to small non-proteinaceous species and may leach out of fixed tissue. This may be partly prevented by transcardial perfusion of Turnbull’s ferricyanide solution concurrent with fixation. We still cannot completely explain discrepancies between photoreceptor iron and ferritin distributions. Further studies, including modified Perls’ transcardial perfusion fixation, are needed to assess more fully issues around tissue fixation and processing. Detection may also be influenced by mouse pigmentation or tissue type. For example, disproportionate losses of a particular iron species may occur in tissues where levels are relatively low.
Seventh Congress of the International BioIron Society Page 130 Poster Abstracts IBIS
Poster #16
HAEMOCHROMATOSIS INTERNATIONAL – AN ALLIANCE OF HEREDITARY HAEMOCHROMATOSIS GROUPS AROUND THE WORLD Desley White, Pierre Brissot, Barbara Butzeck, Howard Don, Robert Evans, Paulo Santos, Emerencia Teixeira and Ben Marris Plymouth University Presented By: Desley White, PhD, BSc Hons
HFE-related haemochromatosis (HH) is an iron-overloading disease which, in people with Northern European ancestry, is one of the commonest genetic conditions, with an estimated one in 150 to 200 individuals having p.Cys282Tyr homozygosity. Excess iron cannot be excreted by the body, and is taken up by the liver, pancreas, and heart. The disease is polymorphic, and up to 50% of p.Cys282Tyr homozygotes may need venesection therapy. Diagnosed early, patients, treated by venesections, have a normal life expectancy. However, early symptoms such as tiredness and arthralgia, typically developing in mid-life, are non-specific, and frequently overlooked. Patients often present too late to prevent shortened life-expectancy, typically presenting with liver cirrhosis or cancer, diabetes, or cardiac disease. Whilst treatment guidelines have been developed for use in a number of countries, they are inconsistent in detail. Many countries have no specific HH recommendations, for example, at which serum ferritin level venesections should start and stop, and the use of values based on treating iron overload in the beta thalassemia population is common, despite differences in ferritin-iron loading between these two clinical groups. Haemochromatosis International (HI) is an alliance of haemochromatosis patients’ groups around the world. It seeks to bring together the numerous national HH groups, to advance the health of people with haemochromatosis everywhere, via the following objectives: To improve awareness of haemochromatosis throughout the world. To develop internationally recognised therapeutic recommendations for HFE haemochromatosis. HI is run by a committee made up of clinicians, academics, and patients, meeting on a monthly basis via video-audio calls, with committee members attending from Australia, Brazil, France, Germany, Portugal, and the UK. We maintain a website, http://haemochromatosis-international.org/, where more information about HI can be found, with links to member organisations. HI spectrum also covers the rare non-HFE related forms of haemochromatosis. HI is grateful to Novartis for a grant which has enabled it to develop, becoming formally established as a Charitable Incorporated Organisation in March 2016.
Seventh Congress of the International BioIron Society Page 131 Poster Abstracts IBIS
Poster #17
HEPCIDIN DETERMINATION IN NON-HFE HEMOCHROMATOSIS. A CASE-CONTROL STUDY Ana López Aparicio¹, Maria Dolores Pulfer, Resident², Alejandra García, Resident², Maria Josefa Moran, PH², Maria Isabel Moreno, PH² and Alejandro Del Castillo, Staff² ¹Gregorio Marañon university hospital; ²Gregorio Marañón Universitary Hospital Presented By: Ana Aparicio, MD
Unfrequent hemochromatosis forms are those that are not associated to HFE gene mutation. We selected a group of patients with non- HFE hemochromatosis. Hepcidin levels were determined to analyze the iron overload’ phenotype. We conducted a case-control study in which we compared hepcidin levels in patients with non-HFE hemochromatosis and healthy controls. Patients were recruited from a specialized iron disorders unit in the Internal Medicine department in a tertiary hospital. Serum levels of hepcidin were determined in batches using a commercial en yme linked immunosorbent kit (DRG Instruments, GmbH, Marburg, Germany). The statistical analysis was made with SPSS version 21.0. The correlation between both groups was compared using the U-Mann-Withney test. We considered statistical significancy p< 0,05. Eight patients were included in the non-HFE hemochromatosis group, and fifteen persons were included in the healthy control group. In the first group, 6 patients were men, and there were two women. The mean age was 55 years. Regarding to their comorbidities, 25 percent of patients were ex- smokers and another 25 percent were smokers. 63 percent suffered from metabolic syndrome. Hepcidin mean level in the hemochromatosis group was 25.88, while in the controls group was 8.53 (p=0.034). Even though our study has few patients, a wider cohort of patients in further studies could demonstrate that hepcidin defines the disease phenotype in patients with no HFE-hemochromatosis, so that high levels of hepcidin would confirm the absence of iron overload.
Seventh Congress of the International BioIron Society Page 132 Poster Abstracts IBIS
Poster #18
FUNCTIONAL STUDIES OF A SINGLE NUCLEOTIDE POLYMORPHISM IN DUODENAL CYTOCHROME B Fabian Schlottmann, BSc, M. Vera-Aviles, and G.O. Latunde-Dada, PhD Faculty of Life Sciences, Diabetes and Nutritional Sciences Division, King’s College London, United Kingdom Presented By: Gladys Latunde-Dada, PhD
Dietary ferric non-heme iron is reduced by ferric reductase enzyme (1), duodenal cytochrome b (Dcytb) before absorption by the divalent metal transporter 1 (DMT1). A single nucleotide (SNP rs884409) that is located in the promoter region of Dcytb in HFE subjects exhibited a phenotype of lower serum ferritin than the control group (2). Moreover, functional luciferase reporter assays of the SNP revealed a 30% reduction in basal promoter activity. Interestingly, Dcytb rs14055 SNP was also identified in HFE subjects in the same study. The present work therefore investigated the phenotype of Dcytb rs14055 mutation in Chinese hamster ovary (CHO) cells. CHO cells were transfected with wild type (WT) or the SNP vector plasmids of Dcytb. Ferric reductase assays were performed in Dcytb transgenic CHO cells using the ferrozine spectrophometric assay protocol. Furthermore, expression of the protein was confirmed by Western blot analysis. The Dcytb SNP showed a gain-of-function since ferric reductase activity significantly (P<0.01) increased in the cells. Increased ferric reductase activity was consistent when CHO cells were pretreated with modulators of Dcytb protein expression. While ferric reductase in endogenous CHO cells increased by desferoxamine and CoCl2, iron loading with ferrous ascorbate in contrast, had the opposite effect. The SNP rs14055 in Dcytb is a putative modifier that may contribute to variance of penetrance in HFE C282Y homozygotes. The mechanisms by which the polymorphism enhanced ferric reductase activity remain to be fully elucidated. References: (1) McKie AT. et al (2001), 291(5509):1755-9. (2) Constantine CC. et al, (2009), Br J Haematol. 147(1):140-9.
Seventh Congress of the International BioIron Society Page 133 Poster Abstracts IBIS
Poster #19
TYPE 4 HEREDITARY HEMOCHROMATOSIS: CHARACTERIZATION OF PATIENTS REFERRING TO THE REGIONAL CENTER FOR IRON DISORDERS IN VERONA (ITALY) Annalisa Castagna¹,², Chiara Piubelli, PhD, Giacomo Marchi, MD, Monica Rizzi, MD, Fabiana Busti, MD, Alice Vianello, MD, Paola Capelli, MD, Roberto Pozzi-Mucelli, MD, Luciano Xumerle, BSc, Massimo Delledonne, PhD, Oliviero Olivieri, MD and Domenico Girelli, MD, PhD ¹Veneto Region Referral Center for Iron Disorders, Azienda Ospedaliera Universitaria Integrata; ²Department of Medicine, Section of Internal Medicine, University of Verona Verona Italy Presented By: Annalisa Castagna, PhD
Type 4 Hereditary Hemochromatosis (HH) is a genetic disorder due to SLC40A1 mutations, and it accounts for most of atypical non-HFE HH forms (Wallace DF, Genet Med 2015). Recently we developed a next generation sequencing 2nd level genetic test for HH at our Referral Center for Iron Disorders (Badar S, Am J Hematol 2016), being able to detect new cases of type 4 HH. We have characterized so far 9 patients from 7 families; among them 8 carried loss-of-function mutations (typical of the so called “Ferroportin Disease”-FD) and one a gain-of-function mutation. The 3bp-deletion p.Val162del was found in 3 families, according to literature reporting this as the most frequent type 4 HH-associated variant. We recently detected a new variant (p.Leu233Gln) in a patient showing mixed biochemical/pathological features between Type 4A/4B HH, i.e. marked hyperferritinemia (6,923 µg/L), high TSAT (85% at diagnosis), iron deposition in both liver and spleen at MRI, and iron overload in both Kupffer cells and hepatocytes at liver biopsy. Of note, the mutation affected the same residue involving a different mutation (p.Leu233Pro) previously described by us in a patient with a quite similar phenotype (Girelli D, J Hepatol 2008). All 4 patients with p.Val162del (all females, age range 30-69) had marked hyperferritinemia (range 1,101-3,020 µg/L) and normal transferrin saturation. All patients were clinically asymptomatic, with no overt biochemical signs of liver disease, notwithstanding in some cases hyperferritinemia was present for several years without treatment before the correct diagnosis. Our single center experience confirm Type 4 HH disease as the most frequent atypical HH, and call for multicentre registries to better define the clinical features of this heterogeneous condition.
Seventh Congress of the International BioIron Society Page 134 Poster Abstracts IBIS
Poster #20
NCOA4 IS WELL EXPRESSED IN MURINE AND HUMAN PERYPHERAL BLOOD AND CORRELATES WITH AGE: A NEW MARKER OF IRON AMOUNT IN FRAIL ELDERLY PEOPLE? Rosa Maria Pellegrino, PhD¹, Antonietta Palmieri, Dr², Martina Boero, Dr², Gianluca Isaia, MD³, Mariarosa Mezzanotte, Dr², Giuseppe Saglio, MD², Angelo Guerrasio, MD² and Antonella Roetto, PhD² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Sciences, AOU San Luigi Gonzaga, Orbassano, Torino, Italy; ³SCDU Geriatrics, AOU San Luigi Gonzaga Hospital, Orbassano, Turin, Italy Presented By: Rosa Maria Pellegrino
With the rise of age in Western populations, physicians have to face condition, comorbidities, and syndromes demanding different diagnostic and therapeutic approaches compared to those employed for younger patients. The frailty syndrome is an age-associated psychosocial and clinical conditions characterized by unintentional weight loss, self-reported exhaustion, and chronic conditions including anemia (Röhring et al, 2016). Anemia of the elderly in particular is a common finding that, in some cases, could be caused by continuous exposure to subclinical inflammation leading to hepcidin increase (Nemeth et al, 2014) and to a consequent tissue iron deposition. This correlates with higher levels of serum ferritin (Ft), the main marker of tissues iron availability. Also, MRI studies in young and old population clearly confirm iron deposition during aging (Acosta-Cabronero et al, 2016). Furthermore, large number of in vitro and in vivo evidences suggest that uncontrolled iron increase causes oxidative stress with ROS accumulation, that has been considered an important factor in frailty (Kawanishi S, Oikawa S. 2004). Thus, the disruption of iron homeostasis and its accumulation during aging may contribute to the senescence process. Aim of this work was to verify if NCOA4, a protein that regulates cellular ferritin amount and whose degradation is subjected to an iron-dependent control (Dowdle et al, 2014; Mancias et al, 2014), could be involved in iron bound ferritin increase during normal aging. Since NCOA4 promotes ferritin degradation and its deficiency in a mouse model causes increased serum ferritin and systemic iron overload (Bellelli R et al., 2016), we expected a decrease of NCOA4 amount during ageing in normal population. To perform this analysis a total of 22 human healthy subjects and 20 wild type mice were recruited and classified by age ranges accordingly to mouse vs human life phases equivalence (https://www.jax.org). Human white blood cells obtained from buffy coat fraction and murine total peripheral blood samples were used for NCOA4 RNA and proteins study through quantitative real time PCR and WB analysis respectively. Human NCOA4 resulted to be well transcribed and produced in peripheral blood and, after an increase from children until young adults age it started to decrease during the middle age and fall down to very low levels in old people group (> 70 y). Murine NCOA4 expression started to decrease from 2 months old animals and was very low in 17 months old mice. From these preliminary data from a limited number of subjects we can argue that NCOA4 may be involved in cellular ferritin increase during ageing and could represent an easily available and informative marker of ageing in mouse and humans that could be utilized to evaluate iron metabolism abnormality in frailty patients compared to normal elderly population
Seventh Congress of the International BioIron Society Page 135 Poster Abstracts IBIS
Poster #21
IRON-RICH DIET DECREASES MYCOBACTERIAL (M. Bovis BCG) BURDEN AND CORRELATES WITH HEPCIDIN UPREGULATION, LOWER PRO-INFLAMMATORY MEDIATORS AND HIGHER T CELL RECRUITMENT Rafiou Agoro, PhD², Stephanie Rose, MSc³, Irene Garcia, PhD⁴, Bernhard Ryffel, MD, PhD², alerie uesniau , PhD² and Catherine Mura, PhD¹ ¹CNRS University rleans; ²CNRS University rleans, France; ³CNRS, France; ⁴University of Geneva, S itzerland Presented By: Catherine Mura, PhD
Introduction: A competition between host and mycobacteria for iron acquisition occurs during infection. Pathogens have developed iron acquisition systems, which are essential for their growth and pathogenicity, while in mammals cell iron redistribution is triggered by infection. Mycobacterium tuberculosis complex (which include M. bovis and M. tuberculosis), can cause tuberculosis in humans or other organisms. After inhalation, mycobacteria are phagocytosed by macrophages which move to deeper tissue. The myeloid and lymphoid cell recruitment at the site of infection contributes to the formation of granuloma which is formed by a core of infected macrophages surrounded by T lymphocytes. Granuloma contain the infection but support bacterial survival over long periods of time. Coordinated innate and adaptive immune responses are required for the efficient control of mycobacteria infection. In this study, we revisited the effects of experimental iron-enriched diet on Mycobacterium bovis BCG infection. Methods: Mice fed with standard diet (280 mg/kg) or diet moderately enriched with iron (2500 mg/kg) were infected with Mycobacterium bovis BCG-GFP. Liver, spleen, lung and lynph nodes were harvested at day 7, 28 and 63 post-infection. Bacterial load (CFU), bacterial uptake, host myeloid cells, cell recruitment, cytokine production and iron gene expression were determined at different stages of infection by using flow cytometry, ELISA and quantitative PCR. Results: A decrease of bacilli levels (CFU) in liver, lung and spleen was observed in infected mice under iron-rich diet as compared to infected animals fed with standard diet .This decrease correlated with the level of mycobacteria uptake by host myeloid cells. Enhanced bacterial clearance correlated with reduced pro-inflammatory cytokines expression. Furthermore, although the number of granuloma was similar in M. bovis BCG infected mice fed standard or iron rich diet, liver histology from M. bovis BCG infected mice under iron rich diet was characterized by decrease neutrophil and macrophage recruitment and enhanced T cell recruitment in granulomas, as compared with infected mice fed with standard diet. Hepcidin and ferroportin mRNA expression was induced in liver and lung following M. bovis BCG infection, iron rich diet strongly exacerbated hepcidin upregulation in infected mice, while it reduced ferroportin mRNA overexpression in the liver. Discussion: In mice fed with a standard diet, we observed a decrease in bacterial burden in the liver, correlating with an early increase in hepcidin gene expression. Iron supplementation induced hepcidin expression, and infected mice exhibited a sharper decline in bacterial burden. We thus could assume a role for hepcidin activity in M. bovis BCG clearance as this peptide has proved anti-microbial properties, altering Mycobacterium structure. We also show higher T cell levels in infected organs, with granuloma displaying higher lymphocyte population, in M. bovis BCG infected mice fed with iron-rich diet, as compared to standard diet. These results suggest that iron supplementation which promotes T cell response in infected mice, with higher T cells able to recognize infected macrophages and activate them, would promote M. bovis BCG clearance. Conclusions: On the whole, moderate iron increase diminished inflammation and growth of Mycobacterium bovis BCG via enhanced immune cell activation and local hepcidin expression.
Seventh Congress of the International BioIron Society Page 136 Poster Abstracts IBIS
Poster #22
CELL IRON STATUS ATTENUATES PRO-INFLAMMATORY MACROPHAGE POLARIZATION Rafiou Agoro, PhD² and Catherine Mura, PhD¹ ¹CNRS/University Orleans; ²CNRS/University Orleans, France Presented By: Catherine Mura, PhD
Introduction: Macrophages play a central role in both immunity and tissue homeostasis, protecting the organism from infection as well as completing essential tissue-specific functions. Indeed, macrophages are key modulator and effector cells in the immune response, their activation influences and responds to other arms of the immune system, coordinating both initiation and resolution of immune responses. They are involved in pathogen recognition, antigen processing, inflammation, phagocytic clearance, immune regulation as well as tissue repair. Macrophages represent a heterogeneous cell population of immune cells, a dynamic spectrum of functional states whose extremes are pro-inflammatory M1 and anti-inflammatory M2 macrophages, exhibiting distinct phenotypes with marked signatures of gene expression and effector functions. Systemic iron balance is maintained through the couple hepcidin hormone and ferroportin cellular iron exporter, and the expression of hepcidin and ferroportin genes is altered upon inflammation. Here, we addressed the influence of iron on immune marker expression and macrophage polarization, and its impact during LPS-induced inflammation. Methods: Mice were fed with standard diet (280 mg/kg), iron-rich diet (2500 mg/kg), or iron-deficient diet (< 20 mg/kg). Acute inflammation was induced by LPS, liver and peritoneal exudate cells were harvested after 4 hours. Murine bone marrow cells were isolated from femurs, differentiated into macrophages and cultured with/without ferric ammonium citrate. Membranes protein, NO, gene expression were analyzed by flow cytometry, Griess assay, ELISA and quantitative PCR. NF-kB nuclear translocation was analyzed in cells fixed, and incubated with anti-NF-kB p65 antibody and Alexa Fluor 488-conjugated secondary antibody. Results: Iron-enriched diet increased M2 marker Arg1 and Ym1 expression in liver and peritoneal macrophages, while iron deficiency decreased Arg1 expression. Under LPS-induced inflammatory conditions while low iron diet exacerbated the proinflammatory response, IL-12/IL-10 balance decreased with iron-rich diet thus polarizing to type 2 response. In addition, macrophage iron loading reduced the basal percentage of cell expressing M1 co-stimulatory CD86 and MHC-II molecules. Further in vitro, iron loading macrophages prevented the pro-inflammatory response induced by LPS by reducing NF-kB p65 nuclear translocation as well as the expression of iNos and IL-1b, IL-6, IL-12 and TNF-a at the mRNA and protein or product levels. The increase of intracellular iron reduced LPS-induced hepcidin gene expression and abolished ferroportin down-regulation in macrophages, in line with macrophage polarization. Discussion: Our data show that high iron levels influence monocyte polarisation towards a M2-like phenotype and dampen pro-inflammatory immune responses, while the iron deficiency had the opposite effect. Indeed, in vivo iron-rich diet stimulated expression of M2-associated markers and cytokines, and repressed some type 1 immune response to TLR4 stimulation. On the other hand, iron deficiency promoted type 1 pro-inflammatory cytokine expression in response to TLR4 stimulation. In vitro, iron-loading of macrophages repressed the expression of M1-associated markers while promoting M2-associated markers, and decreased NF-kB p65 nuclear translocation and pro-inflammatory cytokines release and NO formation in response to TLR4 stimulation. Conclusions: Therefore changes in cell iron concentration can modulate macrophage phenotype and function with clear implications for the immune responses.
Seventh Congress of the International BioIron Society Page 137 Poster Abstracts IBIS
Poster #23
FERROPORTIN DOWNREGULATION IN THE LIVER OF LEISHMANIA INFANTUM-INFECTED MICE THROUGH A HEPCIDIN-INDEPENDENT MECHANISM Tania Cruz, PhD¹, Maria Vieira, Bachelor², Joana Passos, Master³, Helena Castro, PhD², Margarida Duarte, PhD², François Cannone-Hergau , PhD⁴, Jorge Pinto, PhD³ and Ana om s, PhD² ¹I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto; ²I S; ³IBMC; ⁴CNRS Presented By: Tania Cruz, PhD
Iron is crucial for the survival of both parasites and their hosts, yet it can be highly toxic if in excess. As a means to control infections, hosts often take advantage of the dual properties of iron engaging a diversity of microbicidal mechanisms such as deprivation of this essential metal or intoxication of the microorganisms with this micronutrient. In this work our ultimate goal is to understand the importance of iron for the development of in vivo infections by Leishmania infantum, a causative agent of canine and human visceral leishmaniasis. Thus, as a starting point, we investigated whether ferroportin-1 (FPN- 1), the sole iron exporter of mammalian cells, and hepcidin, the main FPN-1 regulator, where altered in the liver of L. infantum-infected mice. For that, we performed a set of in vivo infections and, at different time-points, the liver of both L. infantum-infected and control mice were collected. Immunofluorescence assays were used to assess the expression and localization of FPN-1 and iron deposition was identified through Perls-DAB staining. To assess hepcidin relevance for this parasitic infection the hamp KO mouse model was used. So far, we have uncovered that i) in L. infantum-infected kupffer cells FPN-1 expression is altered, this protein being either internalized or highly downregulated. This is probably important for the accumulation of iron observed in those cells. ii) Strikingly, FPN-1 alterations in infected macrophages occur through a hepcidin-independent mechanism which likely requires the contact between the parasite and its host cell. Finally, we iii) have data suggesting that iron deposition is an important factor for the containment of L. infantum in mice. In conclusion, our work provides evidence that L. infantum infection impacts on the host iron metabolism. More importantly, we report for the first time that upon infection with L. infantum, FPN-1 is downregulated by a hepcidin- independent mechanism. In the future we will investigate whether iron is indeed used by the host as a microbicidal strategy and we will also dissect the mechanism leading to FPN-1 decrease. This work was financed by FEDER funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), and by FCT - Fundação para a Ciência e a Tecnologia in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-000012) and project PTDC/QEQ- MED/7097/2014.
Seventh Congress of the International BioIron Society Page 138 Poster Abstracts IBIS
Poster #24
THE ROLE OF HFE GENOTYPE IN MACROPHAGE PHENOTYPE Anne Nixon, Elizabeth Neely and James Connor Penn State College of Medicine, Hershey Medical Center, Hershey, PA Presented By: Anne Nixon, BS
The HFE gene was discovered to encode the HFE protein, an essential protein involved in the iron homeostasis through the regulation of cellular iron uptake. HFE is thought to contribute to the management of iron through its ability to bind to the transferrin receptor on the cellular membrane. In addition, it is speculated that HFE functions to regulate hepcidin levels through bone morphogenetic protein 6 pathway (BMP6). Mutations of the HFE gene, specifically C282Y and H63D, are commonly associated with iron dysregulation and iron related diseases. Our laboratory and others have reported an increased prevalence of the H63D mutation in neurodegenerative diseases such as ALS and Alzheimer’s disease. Importantly, HFE gene polymorphisms of reticuloendothelial cells, primarily macrophages, have been found to be iron deficient despite overall high levels of iron within the body and parenchymal cells. However, elucidation of the underlying mechanism and the precise role of HFE mutations in macrophages remains unsatisfied. We hypothesize that the HFE/H63D gene variant is the source of the altered macrophage phenotype, and aim to characterize the role of HFE in macrophages. To test this hypothesis, we isolated macrophages from the bone marrow of mice harboring the H67D mutation, a homolog for the human H63D mutation. We demonstrate that H67D macrophages have a similar capacity to iron load compared to wildtype bone marrow macrophages. Furthermore, there was no difference in expression of transferrin receptor and ferroportin; suggesting iron uptake and export are similar to wildtype. However, we found the H67D macrophages stored significantly more iron in the form of L-ferritin compared to the wildtype macrophages, suggesting an alternative mechanism for iron processing in the cell. H67D macrophages were also found to have higher levels of BMP6 and IL-1ra, indicating hepcidin regulation may be altered. The H67D also exhibited increased phagocytic activity and perturbed migratory ability, as well as a cytokine profile corresponding to altered chemotaxis. Together, these results suggest H67D macrophages have an altered phenotype relating to iron processing and motility. These changes in macrophage function are likely related to alterations in inflammatory responses and the role of macrophages in disease progression.
Seventh Congress of the International BioIron Society Page 139 Poster Abstracts IBIS
Poster #25
ELUCIDATING THE ROLE OF IL6 AND REACTIVE OXYGEN SPECIES (ROS) IN BONE MARROW ERYTHROPOIESIS AND IN THE DEVELOPMENT OF ANEMIA UNDER INFLAMMATORY CONDITIONS Ritama Gupta¹, Roberta Chessa, PhD², Sara Gardenghi, PhD³, Ping La, PhD² and Stefano Rivella, PhD² ¹Children's Hospital of Philadelphia, University of Pennsylvania; ²Children's Hospital of Philadelphia; ³Weill Cornell Medical College Presented By: Ritama Gupta, MSc
Inflammatory states seen in infection and other chronic disorders are often characterized by a condition called anemia of inflammation (AI). The iron deficiency in AI is predominantly due to an altered balance of the cytokine, interleukin-6 (IL6) and the hormone hepcidin (Hamp). We have previously shown that lack of IL6 or Hamp in knockout mouse models (IL6- KO, Hamp-KO) injected with the heat-killed pathogen Brucella abortus (BA) results in improved recovery from anemia. This recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. To formally demonstrate the independent roles of the two genes in AI, we generated a double knock out of IL6 and Hamp (DKO) and hypothesized that the DKO would show the best recovery from anemia, superior to the single KO models. However, after an initial phase of accelerated recovery, the DKO showed a worsening of erythropoiesis in the BM, similar to WT-BA and Hamp-KO-BA and unlike what observed in the IL6-KO treated with BA. The DKO-BA exclusively showed a biphasic effect of reticulocyte count, which was elevated in the first week and then reduced in the second week. In the first week, this effect was mirrored in improved BM-erythroid recovery and an accelerated production of new RBCs in the DKO-BA. However, BM-erythropoiesis and RBC production declined on the second week, while the spleen showed increased erythropoiesis, similar to Hamp-KO-BA. Because the IL6-KO-BA showed increased erythroid progenitors in the BM in contrast to the DKO-BA and Hamp-KO-BA, we hypothesized that lack of IL6 protects erythroid progenitors from inflammatory insult in the BM, but this protection is lost in presence of high iron levels or in absence of Hamp. As Hamp- KO mice show high levels of Tf-sat and, potentially NTBI, we investigated total protein oxidation and reactive oxygen species (ROS) levels in erythroid progenitors, in the BM and spleen. Whole BM and spleen cells showed differing changes in total protein oxidation after BA treatment, as seen by immunoblot. Furthermore, unlike the IL6-KO, at 2 weeks from BA administration, WT, Hamp-KO and DKO showed upregulation of ROS in erythroid progenitors in the BM. Based on these observations, we further hypothesized that presence of high iron levels are positively correlated with BM ROS and negatively correlated with BM erythroid recovery under AI. In IL6-KO-BA mice injected with iron dextran to raise iron levels, we found an upregulation of BM-ROS and a concurrent decline in BM-erythroid recovery. We hereby conclude that upregulated ROS potentially contributes to impaired BM-erythroid recovery under AI. Lack of IL6 inhibits this effect, but this inhibition is reversible in the presence of high iron levels and, possibly, generation of NTBI. We speculate that high calcium levels in the mitochondria contribute to the upregulation of ROS and this in turn triggers apoptosis in erythroid progenitors, thereby preventing recovery under AI. We are currently investigating calcium levels as well as superoxide radicals within the mitochondria, under AI and how this may differ in the BM versus spleen. We are also exploring recovery under AI in the presence of antioxidants or other reagents that may decrease NTBI.
Seventh Congress of the International BioIron Society Page 140 Poster Abstracts IBIS
Poster #26
H-FERRITIN IS A MACROPHAGE HOST DEFENSE FACTOR AGAINST SALMONELLA INFECTION David Haschka, Dr¹, Piotr Tymoszuk, PhD¹, Verena Petzer, Dr¹, Stefanie Dichtl, Mag¹, Dirk Bumann, Prof², Markus Seifert¹, Sylvia Berger¹, Lukas Kühn, Prof³, Igor Theurl, Prof¹ and Günter Weiss, Prof¹ ¹Medical University of Innsbruck, Internal Medicine VI; ²University of Basel, Focal Area Infection Biology; ³Swiss Institute for Experimental Cancer Research Presented By: David Haschka
Background: Iron is a vital microelement for both the host and pathogen and iron with-holding from bacteria by host macrophages constitutes an important defense mechanism. The iron storage protein ferritin is thought to be a crucial player in this process. Functional ferritin consists of 24 subunits of heavy (H) and light (L) chains, where H-Ferritin facilitates iron uptake into the complex. In this study we investigated the role of H-ferritin in regulation of inflammation and anti-bacterial response under normal and iron loading conditions. Methods: To specifically delete H-Ferritin in the myeloid cell line of mice, we used a strain expressing Cre recombinase under the promotor of the lysozyme 2 gene. Infection assays were performed with Salmonella Typhimurium (S.tm.) expressing the green fluorescent protein (GFP), which enabled us to determine infection rate of a particular leukocyte population on a single-cell level. Results: Infection of FtH knockout mice with Salmonella-GFP revealed no significant difference in bacterial load in organs after 96 hours as compared with WT animals. However, survival of iron loaded knockout mice after Salmonella challenge was drastically reduced in comparison to WT mice. Similar effects were observed after LPS treatment. Whole-genome expression analysis of the spleens of infected animals indicate that this phenotype can be attributed to unrestricted reactive oxygen species ( OS) production and B activation. Conclusions: Lack of H-Ferritin in macrophages/monocytes promotes bacterial growth or survival of Salmonella in iron challenged mice, presumably due to immune cell and tissue damage as a consequence of enhanced oxidative stress and inflammation.
Seventh Congress of the International BioIron Society Page 141 Poster Abstracts IBIS
Poster #27
RESTRICTION OF FE-S CLUSTER BIOGENESIS UNDERPINS THE METABOLIC AND EPIGENETIC REPROGRAMMING IN TOLL-LIKE RECEPTORS-STIMULATED MACROPHAGES Wing-Hang Tong, PhD, Nunziata Maio, PhD, De-liang Zhang, PhD and Tracey Rouault, MD Eunice Kennedy Shriver National Institute of Child Health and Human Development, USA Presented By: Wing-Hang Tong, PhD
Iron-sulfur clusters are essential cofactors for many pathways involved in cellular metabolism, mediating electron transfer in the mitochondrial respiratory chain, enzymatic catalysis in aconitase, and lipoylation of the pyruvate dehydrogenase complex. Previous studies have shown that mitochondrial respiratory function decreases in myeloid cells that are activated with the bacterial lipopolysaccharide, LPS, and the cytokine, , mainly due to the induction of inducible nitric oxide synthase (iNOS), which produces nitric oxide that damages the Fe-S cluster cofactors that are essential for mitochondrial electron transport, TCA cycle activities and heme biosynthesis. Paradoxically, the expression levels of two key Fe-S cluster biogenesis factors NFS1 and ISCU were repressed at both mRNA and protein levels in M1 macrophages (Canal et al. Arch. Biochem. Biophys. 2007) (Recalcati et al. Eur. J. Immunol. 2007). To understand the role of Fe-S cluster biogenesis in macrophage activation, we examined the factors that are involved in Fe-S cluster biogenesis in macrophages that are activated with LPS and IFNg as well as several other macrophage activating agents. Our studies indicated that at least eight members of the Fe-S cluster biogenesis machinery were down-regulated in M1 macrophages, which inhibited the recovery of damaged Fe-S clusters in respiratory complex I, succinate dehydrogenase, aconitases, and ferrochelatase, resulting in prolonged fragmentation of the TCA cycle and reduced OXPHOS. Furthermore, restriction of Fe-S cluster biogenesis resulted in chromatin modifications in the late phase of the LPS response. Recent studies in immunometabolism have shown that profound transcriptional, metabolic and epigenetic reprogramming govern the immune cell response to danger signals and cytokines, and the metabolism shift from OXPHOS to glycolysis is critical for M1 macrophage function. Our results suggested that repression of Fe-S cluster biogenesis/repair plays an integral role in driving metabolic and epigenetic changes needed for the immune response in inflammatory macrophages. Importantly, the link between Fe-S cluster biogenesis with metabolic and epigenetic reprogramming has implications in both immune and non-immune cells. Our studies have shown that iron deprivation not only limited the availability of iron for Fe-S cluster biogenesis, but also caused a decrease in the levels of multiple Fe-S cluster biogenesis factors. Previous studies have shown that macrophage iron depletion in iron deficiency and in hereditary haemochromatosis may have pro-inflammatory effects (Pagani et al. Blood 2011), and iron deprivation further reduced the levels of FXN transcripts in cells from Friedreich’s ataxia patients (Li et al. Hum. Mol. Gen 2008). The implications of the link between the regulation of Fe-S cluster biogenesis with iron homeostasis, intermediary metabolism, and chromatin dynamics on immune cell function and in riedreich’s ataxia will be discussed.
Seventh Congress of the International BioIron Society Page 142 Poster Abstracts IBIS
Poster #28
NOVEL SPLICING SITE AND MISSENSE MUTATIONS CAUSE ACERULOPLASMINEMIA Marina Dorigatti Borges, BSc, Dulcineia Martins Albuquerque, BSc, PhD, Carolina Lanaro, BSc, PhD, Fernando Ferreira Costa, MD, PhD and Kleber Yotsumoto Fertrin, MD, PhD University of Campinas Presented By: Marina Dorigatti Borges, BSc
Aceruloplasminemia is a rare form of cerebral iron overload of autossomal recessive inheritance that results from mutations in the sequence of the CP gene, which encodes the iron oxidase ceruloplasmin. It causes progressive neurodegenerative disease associated with anemia and diabetes, with only 56 described cases. No cases have been characterized molecularly in the Brazilian population, so we aimed at determining the molecular basis of aceruloplasminemia in two patients with undetectable serum ceruloplasmin and brain iron overload confirmed by magnetic resonance imaging. Peripheral blood samples were collected for total leukocyte DNA extraction. Automated sequencing on an ABI 3500 Genetic Analyzer platform (Applied Biosystems, USA) was performed for the regions of the CP gene in which most mutations have been described and for the whole sequence of the pseudogene CPP. Data from NCBI were used as reference. In addition, in silico analyses of the mutations identified were performed to establish the probable consequences to the tertiary structure of the protein with the aid of RaptorX web server (see Figure). No mutations were identified in the CPP pseudogene sequences. Both patients were homozygous for CP mutations not previously described in the literature. The first patient was diagnosed at the age of 38, and was found to bear a c.2879-1 G>T splicing site mutation in the region corresponding to the donor site of intron 16. With the help of fruitfly.org, it was verified that this mutation leads to the loss of exon 17, with significant change in the tertiary structure of the predicted mutated protein. The second patient was 46 years old at diagnosis, and was homozygous for a c.2756 T>C missense mutation in exon 16, which causes the exchange of a leucine residue for a proline at codon 919. These novel mutations corroborate the idea that, in aceruloplasminemia, mutations tend to be unique to specific families and mostly appear in homozygosity, although there was no confirmed history of consanguinity in the families we studied. As in the majority of the cases described in the literature, the clinical features did not vary significantly in relation to the mutation found, with comparable ages at diagnosis, degrees of anemia, diabetes, and brain iron overload in both cases. Both mutations are associated with undetectable levels of serum ceruloplasmin, although conventional nephelometry assays used to measure serum ceruloplasmin may be unable to recognize mutated versions of ceruloplasmin secreted. Comparing with other similar mutations previously described, CP c.2879-1 G>T splicing site mutation may possibly lead to disturbed trafficking of the protein to the endoplasmic reticulum due to its severe effect on protein structure, while it also remains to be determined if CP c.2756 T>C is secreted, retains the ability to bind copper or has impaired ferroxidase activity to stabilize ferroportin. Further studies to elucidate the effect of these mutations on protein synthesis and ferroportin expression are ongoing. Our study is the first to characterize two novel mutations in Brazilian patients with aceruloplasminemia, which now allows for genetic counseling of these families, and may elucidate the importance and function of the region of the protein encoded by CP exons 16 and 17 in the physiology of ceruloplasmin production.
Seventh Congress of the International BioIron Society Page 143 Poster Abstracts IBIS
Poster #29
THE INFLUENCE OF IRON HOMEOSTASIS ON MICROGLIA FUNCTION Andreas Popp, Dr¹, Thomas Möller, Dr², Jessica Abele³ and Bernhard K. Mueller, PD Dr⁴ ¹Abbvie Deutschland GmbH & Co. KG; ²AbbVie Inc., Neuroscience Foundational Discovery, Cambridge, USA; ³AbbVie Deutschland GmbH Co. G, Preclinical Safety, Lud igshafen, Germany; ⁴Abb ie Deutschland GmbH Co. G, Neuroscience Discovery, Ludwigshafen, Germany Presented By: Andreas Popp, Med. Vet
Microglia as resident defense cell in the brain has properties which are in general comparable to the macrophage population in the peripheral organ systems, but differs significantly in origin, specific marker expression pattern and morphologic characteristics. In the past, the role of microglia was mainly understood as a first line defense cell acting during inflammation in the brain. But its role in physiological processes like brain plasticity and synaptic pruning on the one and pathological changes during aging and neurodegeneration (e.g. Alzheimer and Multiple Sclerosis) on the other side needs more intensive evaluation. Of specific interest is to evaluate the function of different polarization states of microglia subpopulations under these conditions, and to identify mechanisms which can be used to influence its polarization as a novel specific therapeutic concept. Microglia seems to have the similar function and reactivity in the regulation cascade of iron homeostasis compared to peripheral macrophages. Since it is known that the polarization of macrophages is dependent on the iron related microenvironment and a high iron load of macrophages stabilizes the pro- inflammatory properties of these cells, we evaluated whether microglia shows a similar reaction pattern. The presentation will give an overview on the current knowledge of microglia function in comparison to macrophages with special focus on iron regulation and the possible role of iron in neurodegeneration. Results of own experiments with microglia cultivated and then stimulated under different conditions followed by treatment with hemin and/or hepcidin will be presented. Treatment of microglia with hemin and hepcidin led to an upregulation of iNOS, IL-6 and IL-12. In addition it could be shown that treatment of microglia with increasing concentrations of hemin and hepcidin impaired cell viability and showed cell toxicity. These results demonstrate that microglia polarization can be influenced by iron microenvironment. Targeting iron metabolism of microglia in the context of disease modification in neurodegenerative diseases is therefore proposed as an alternative treatment strategy. Disclosure statement: All authors are or have been employees of AbbVie at the time of data generation. The design, study conduct, and financial support for the research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Seventh Congress of the International BioIron Society Page 144 Poster Abstracts IBIS
Poster #30
A ROLE FOR SEX AND A COMMON HFE GENE VARIANT IN BRAIN IRON UPTAKE James Connor, PhD, Kari Duck, PhD, Ian Simpson, PhD and Elizabeth Neely Penn State Hershey Medical Center Presented By: James Connor, PhD
HFE (high iron protein) is an essential protein for regulating iron transport into cells. Two common mutations of the HFE gene, C282Y and H63D, result in loss of this regulation and are associated with accumulation of iron within the cell. The cellular iron accumulation can reach toxic levels; and in its most clinically aggressive form is known as hemochromatosis. HFE is a key iron management protein that is responsible for limiting cellular iron uptake. The mutated protein has been found increasingly in a range of neurodegenerative disorders. Additionally, recent evidence that these mutations are associated with elevated brain iron challenges the paradigm that the brain is protected by the blood-brain barrier (BBB). While much has been studied regarding the role of HFE in cellular iron uptake, it has remained unclear what role the protein plays in the transport of iron into the brain. Here, we investigated the regulation of iron transport into the brain using a mouse model with a mutation in the HFE protein. We demonstrated that the rate of uptake of 59Fe was similar between the two genotypes despite there being higher brain iron concentrations in the mutant. However, there were significant differences in iron uptake between males and females regardless of genotype. These data suggest that brain iron status could be set during development while the brain is growing and that systemic iron changes or even mutations designed to favor iron uptake do not change brain iron levels once the level is set.
Seventh Congress of the International BioIron Society Page 145 Poster Abstracts IBIS
Poster #31
THE ROLE OF THE BLOOD-BRAIN BARRIER IN MAINTAINING BRAIN IRON STATUS James Connor, PhD, Kari Duck, PhD and Ian Simpson, PhD Penn State Hershey Medical Center Presented By: James Connor, PhD
Normal metabolic functions in the brain such as myelination and neurotransmitter synthesis require adequate and timely delivery of iron. Many common neurological disorders and cognitive performance levels are associated with too much or too little iron. Therefore, it is surprising that regulation of brain iron uptake mechanisms are poorly understood. Our recent studies and the data presented herein establish the blood-brain-barrier (BBB) as the key site for regulation of brain iron uptake. The endothelial cells store iron and that iron can be made available to cellular functions within endothelial cells or released to the brain. In this study, we demonstrate that both iron-poor transferrin (apo-Tf) and an iron chelator, deferoxamine (DFO), stimulate release of iron from iron-loaded endothelial cells in an in vitro BBB model. Despite the presence of the iron exporter, ferroportin, on the endothelial cells, the release of iron from these cells is not affected by hepcidin. One mechanism by which iron becomes available to the endothelial cells and subsequently to the brain, if released, is through the divalent metal transporter 1 (DMT-1). Here we show that blocking DMT-1 results in decreased iron and transferrin release from endothelial cells, but not complete inhibition. These data support models of iron transport that invoke movement of transferrin bound iron across the endothelial cells. This study in conjunction with our previous findings identify potential mechanisms for local regional regulation of brain iron uptake and provide insights to the limited success associated with using transferrin mediated drug delivery to the brain.
Seventh Congress of the International BioIron Society Page 146 Poster Abstracts IBIS
Poster #32
FERRITIN UPTAKE INTO THE DEVELOPING MOUSE BRAIN Brian Chiou, Elizabeth Neely and James Connor Penn State Hershey College of Medicine Presented By: Brian Chiou
Iron is a critical factor that is essential for all facets of life, especially important for all cell types in the developing and adult brain. Altered delivery of iron into the brain during development may have long-standing effects that may result in altered brain biology later in development or in the adult brain. Currently, the accepted paradigm for iron uptake involves transferrin as the sole provider of iron to the brain during development. In this study, we propose that an alternative iron carrier protein, ferritin, is heavily involved in delivering iron to the brain during development. As such, we looked at the ability for ferritin loaded with the radionuclide Fe-59 to be delivered to the brain. We compared this uptake to the established transferrin uptake as a control. Our results show a high level of iron uptake through both transferrin and ferritin during the period of increased brain development (1-2 weeks) with a marked decline in uptake at 3 weeks. These data suggest a major role for ferritin in the delivery of iron to the developing brain; an observation consistent with our previous reports that ferritin in the major iron delivery protein for oligodendrocytes that are producing myelin in the brain. We also compared the uptake of both transferrin and ferritin between males and females in both the wild-type and H67D genotypes. The H67D mutation is the mouse homolog of the H63D mutation in humans, resulting in a mutation in HFE, the hemochromatosis protein. There were no differences between the two genotypes in rates of uptake of ferritin. However, there were differences in the levels of iron uptake via ferritin between sexes in the H67D group in the early and late time points. We are currently in the process of evaluating the iron status between each of these mice at the various time points; a difference between genotypes may suggest significant differences in the level of efflux of iron into the brain. Overall, the study of ferritin uptake into the developing brain establishes a non-transferrin bound mechanism for brain iron uptake.
Seventh Congress of the International BioIron Society Page 147 Poster Abstracts IBIS
Poster #33
EXOGENOUS -SYNUCLEIN INDUCES INFLAMMATION AND ALTERS NON-TRANSFERRIN BOUND IRON UPTAKE GENES AND PROTEINS IN CELLS OF THE CENTRAL NERVOUS SYSTEM Veronika Kallo, BSc MRes, Vernon Skinner, BSc PhD, David Dexter, PhD and Surjit Kaila Singh Srai, PhD University College London Presented By: Veronika Kallo, BSc, MRes
Introduction: Inflammation is a key pathological feature of sporadic and hereditary neurodegenerative disorders characterized by resident activated microglial cells. Furthermore, iron accumulation is often jointly present with inflammation in brain areas where neuronal cell death is evident. This study indicates involvement of microglial activation, with -synuclein (a major constituent of Lewy bodies) induced-inflammation as a potential mediator. Here we assess the impact of the innate inflammatory response induced by purified recombinant human -synuclein on iron metabolism in microglial cells in vitro, and the effect of activated microglial cells on neurons in a co–culture system. Methods: Microglial cells (N9 cell line) were treated with -synuclein directly or a-synuclein-treated microglial cells were co-cultured with neurons (N27 cell line) for various times, up to 24h. Key inflammatory markers, nitric oxide (NO), interleukin 6 (IL-6) and tumor necrosis alpha (TNF- ) measured by the Griess assay, quantitative real-time PCR (qPCR), enzyme-linked immunosorbent assay (ELISA) respectively. The expression of the iron importers DMT1, Zip14, and the iron exporter, ferroportin, were also detected with qPCR. Influx of non-transferrin bound iron (NTBI) was assessed using the radioactive isotope of iron (55Fe). Results: -synuclein treated microglia confirmed activation by inducing the up-regulation of NO and TNF-a levels significantly at 12 and 24h (p<0.0001). IL-6 mRNA levels were significantly up-regulated at all time points; 2h (22.78x)(p<0.001), 4h (63.52x), 6h (88.68x), 12h (50.74x)(p<0.0001) and at 24h (15.04x)(p<0.05). DMT1 mRNA levels displayed significant up-regulation at 2h (3.34x)(p<0.0001), 4h (3.08x)(p<0.001), 6h (5.51x)(p<0.0001), 12h (4.76x)(p<0.0001) and at 24h (2.12x)(p<0.05). Zip14 transcripts also showed significant up-regulatory responses at 2h (8.71x) (p<0.0001), 4h (4.12x) (p<0.0001), 12h (1.16x) (p<0.05). This was mirrored by a significant increase of NTBI uptake at 6, 12h (p<0.001) and 24h (p<0.0001). Ferroportin transcripts were down regulated significantly at all time points during the 24h -synuclein treatment. N27 neurons co-cultured with -synuclein activated N9 cells displayed significant up-regulation of DMT1 transcripts at 6h (4.31x) (p<0.0001), Zip14 mRNA levels showed up-regulation at 12h (2.52x) (p<0.001) and 24h (3.42x) (p<0.0001). NTBI uptake studies support the involvement of these genes in importing iron under inflammatory conditions at 24h (p<0.0001). Ferroportin mRNA levels were up-regulated by 24h (2.26x) (p<0.0001) in response to inflammatory stimuli. Conclusions: -synuclein not only induces an inflammatory response in microglia with the production of diverse pro- inflammatory species but also changes the gene expression profile of iron importer and exporter proteins. Neuronal and microglial co-culture studies further support the effect of inflammation on iron regulating proteins that show increased influx of iron in both neurons and microglia. These results highlight that the inflammatory process can influence the iron content of brain cells, and provide further grounds for investigating the role of iron in neurodegeneration.
Seventh Congress of the International BioIron Society Page 148 Poster Abstracts IBIS
Poster #34
COMPUTATIONAL MODELLING OF IRON TRANSPORT BETWEEN BLOOD CIRCULATION, BRAIN, AND DOPAMINERGIC NEURONS OF THE SUBSTANTIA NIGRA Vindy Tjendana Tjhin, MSc¹, Simon Mitchell, PhD², Elizabeth A. Milward, PhD³, Charles E. Hutchinson, MD¹, Michael J. Chappell, PhD¹ and Joanna F. Collingwood, PhD¹ ¹University of Warwick; ²UCLA; ³University of Newcastle Presented By: Vindy Tjendana Tjhin, MSc
Objective: to create a mechanistic computational model of iron transport between the blood circulation and the brain, permitting in silico investigation of homeostatic regulation of iron at the human blood-brain barrier (BBB) and blood- cerebrospinal fluid barrier (BCB), and to extend this with compartments that facilitate analysis of altered iron loading in neurodegenerative disorders. Introduction: Iron trafficking in the human brain is tightly regulated, helping to minimize damage catalyzed by redox- active iron. Uptake of iron across the BBB utilizes the iron transport protein transferrin, and is demonstrably dependent on transferrin receptor (TfR) expression at the BBB. Iron uptake and efflux from neurons is also tightly regulated, with uptake primarily dependent on TfRs. In healthy dopaminergic neurons of the substantia nigra (SN), the expression level of TfRs is lower than for many other brain regions, and average tissue iron concentration in the SN exceeds other regions excepting globus pallidus and red nucleus. Within dopaminergic neurons of the SN, ferritin concentration is atypically low compared to other neurons; much of the intracellular iron is complexed with neuromelanin which may serve as an intracellular iron store and perform a protective function in isolating iron from the cytosol. Disrupted brain iron homeostasis is evidenced in certain neurodegenerative disorders; elevated iron in dopaminergic neurons of the SN pars compacta is a particular feature of Parkinson’s disease (PD). Iron dysregulation in PD is not fully understood, and while treatment of early-stage PD patients with deferiprone appeared to delay deterioration and modify iron levels in the SN, the mechanism is not confirmed. A computational model that accurately describes brain iron homeostasis at a level that replicates experimental observations, and allows prediction of the impact of perturbations (e.g. with iron-modifying drugs) is therefore highly desirable. Methods: A compartmental in silico model of brain iron trafficking between the blood and the brain, and between the interstitial fluid (ISF) and the dopaminergic neurons, has been created using COPASI: Biochemical System Simulator software. Nonlinear ordinary differential equations characterized the kinetics of each chemical species involved in the transport of iron between the compartments. Experimentally determined model parameter values for compartmental volumes, initial species concentrations, and rate constants for each species, were derived from the literature. Results: Model simulations accurately replicate the kinetics of iron uptake across the BBB with independently determined data from various experimental systems. In response to simulated iron overload, the model predicts that regulatory activity at the BBB/BCB protects against excessive iron loading in the brain, evidenced in the response of the ISF iron levels to varying blood iron levels. Within the dopaminergic neurons, steady state simulations of iron concentration (including ferritin- and neuromelanin-bound iron), and concentrations of iron response proteins and TfRs compare well with independently determined experimental data from the literature. Conclusions: This new model provides an in silico resource to explore the impact of iron dysregulation and iron modifying drugs on the primary compartments of the human brain, to improve understanding of the mechanism of action of existing therapeutics, and to predict effective targets for novel therapeutics.
Seventh Congress of the International BioIron Society Page 149 Poster Abstracts IBIS
Poster #35
ADVANCES IN X-RAY MICROSCOPY FOR THE ANALYSIS OF TRANSITION METALS IN THE BRAIN Joanna Collingwood, PhD¹, James Everett, PhD¹, Neil Telling, PhD² and Freddy Adams, PhD³ ¹University of Warwick; ²Keele University; ³University of Antwerp Presented By: Joanna Collingwood, MPhys, PhD
Introduction: X-rays have the advantage that they have a short wavelength and can penetrate through a thick biological sample. It was the need to ‘see inside’ opaque objects, especially biological tissues, and to resolve features too small for optical microscopes or too thick for electron microscopes, that spurred the development of X-ray microscopes. Their much shorter wavelength means they are less hindered by the diffraction limit which has historically limited spatial observation to micro dimensions for visible or UV light. Many of the X-ray microscopy techniques that provide the greatest sensitivity and specificity for transition metals in biological materials are emerging at synchrotron X-ray facilities. Here, the extremely high flux available across a wide range of soft and hard X-rays, combined with state-of-the-art focussing techniques and ultra-sensitive detectors, makes it viable to undertake direct imaging, chemical and mineral speciation of trace element concentrations in biological materials. Methods: Synchrotron X-ray methods for characterization of trace metal accumulations and metal-binding species are compared and placed in context with other techniques for trace metal analysis in tissues, taking into account the sample preparation requirements and the relative advantages and disadvantages of each technique for analysis at regional, cellular, and sub-cellular spatial resolution. Conclusions: X-ray microscopy meets the criteria of complementing data from existing modalities while providing totally unique views of cells and tissues. In particular, it allows the semi-quantitative or even quantitative imaging of numerous elements and elemental compounds within intact biological cells down to the level of nanometre-sized objects and structures. The combination of the superior penetration power of X-rays, and their capacity for high sensitivity quantitative elemental analysis at high spatial resolution, creates a unique tool with capabilities that other microscopy techniques based on electron or light microscopy cannot provide. While X-ray techniques need synchrotron radiation sources for optimum performance, they can also be implemented with micro-focus laboratory sources at reduced spatial resolution and sensitivity. With synchrotron X-ray microscopy, comparatively non-destructive elemental imaging and speciation of iron and other transition metals in brain tissue is possible with sub-cellular spatial resolution, specificity and sensitivity unmatched by any other imaging techniques.
Seventh Congress of the International BioIron Society Page 150 Poster Abstracts IBIS
Poster #36
THE DISTRIBUTION OF IRON-RELATED PROTEINS IN INTRACEREBRAL HEMORRHAGE Lisa Juul Routhe, MSc¹, Torben Moos, Prof¹ and Guohua Xi, MD² ¹Aalborg University; ²University of Michigan Presented By: Lisa Juul Routhe, MSc
Background: Hemorrhagic stroke as occurring in intracerebral hemorrhage (ICH) is associated with increased level of non-transferrin bound iron (NTBI) leading to increased concentration of total iron in brain. NTBI represents a potentially toxic form of iron that could play a detrimental role in promoting Fenton chemistry. The transporter ZRT/IRT-like protein 14 (Zip14) directly transports NTBI across the cell membrane with maximal transport near physiological pH, which occurs contrary to the pumping activity of divalent metal transporter 1 (DMT1) that is most efficient at pH 5.5 and only plays a minor role in NTBI uptake. In spite all cell types in the brain are capable of taking up NTBI, the primary cell type responsible for scavenging NTBI is astrocytes, since they have a high capacity for uptake. Recent studies indicate that minocycline reduces iron-induced toxicity through chelation and significantly improves the outcome after ICH. In this study, we characterized the occurrence of Zip14 in different brain cells and the expression of Zip14 in rats after ICH and ICH with minocycline treatment. Methods: The current study was performed on adult male rats and divided in three parts:1) Rats were stereotactically injected with whole blood (ICH) and sacrificed after different time points (1, 7, 14, and 28 days), 2) Rats with ICH were treated with minocycline or vehicle and sacrificed after 7 days, 3) Rats were injected into the caudate nucleus with saline, iron, iron plus minocycline, or iron plus macrophage/microglia inhibitory factor. All experimental setups were used for immunohistochemistry in order to count the number of Zip14 immunoreactive (+) cells after ICH and iron accumulation in the brain. Results: Zip14 co-localized with GFAP, but not with NeuN or Iba1, after ICH or iron injection. After ICH, a delayed upregulation of Zip14 was observed with the number of Zip14+ cells increasing 7 days after ICH compared to both contralateral and 1 day ipsilateral (p<0.01), and peaking after 14 days (p<0.01). Minocycline treatment did not affect the number of Zip14+ cells. Injection of iron into the caudate nucleus did not upregulate Zip14 compared to the sham, but a tendency towards an increased number of Zip14+ cells was observed after 3 days, suggesting that astrocytes increased Zip14 expression. Conclusions: Zip14 increased ipsilaterally after ICH compared to the contralateral side. Minocycline is known to reduce iron overload and brain injury after ICH according to the literature, and thus the increased number of Zip14+ cells in astrocytes could reflect the brains attempt to recover after ICH through iron-chelation.
Seventh Congress of the International BioIron Society Page 151 Poster Abstracts IBIS
Poster #37
LIVER IRON CONTENT BY MAGNETIC RESONANCE IN CHRONIC KIDNEY DISEASE AT THE START OF MAINTENANCE HEMODIALYSIS Patricia Carrilho, MD¹, In s Santiago, MD², Marta Alves, MSc³, Pedro Fidalgo, MD⁴, Elsa Rodrigues, MD² and Bruno Rodrigues, MD⁴ ¹Hospital Fernando Fonseca; ²Hospital Fernando Fonseca, Dept Radiology; ³3Centro Hospitalar de Lisboa Central, Epidemiology and Statistics ffice of the Research Unit; ⁴Hospital Fernando Fonseca, Dept Nephrology Presented By: Patricia Carrilho, MD
Background: Intravenous iron therapy is increasingly used as part of the treatment of anemia in chronic kidney disease (CKD) and hemodialysis patients. Hepatic iron overload assessed by hepatic magnetic resonance imaging (MRI) has been described in the majority of patients on chronic hemodialysis receiving intravenous iron therapy. Whether iron overload is present at the time of dialysis initiation is unknown. We aimed to describe the liver iron content (LIC) assessed by MRI among a population of incident hemodialysis patients and explore the association with clinical, biochemical markers of iron metabolism and prior anemia therapy. Methods: After approval by the Hospital’s Ethical Committee, a prospective, observational study was conducted at Hospital Fernando Fonseca between March 2014 and February 2016 that included adult patients starting maintenance hemodialysis. MRI without gadolinium was performed to calculate LIC based on the signal intensity ratio method developed and validated by Gandon at Rennes University. Results: Of the 23 patients included, only 6 (26%) had normal LIC, evaluated by MRI (<40µmol/g). 14 (61%) patients showed mild overload (41-100 µmol/g) and 3 (13%) patients already had moderate overload (101-200 µmol/g) at the start of hemodialysis. None had severe overload, as found in prevalent hemodialysis patients. Previous anemia therapy, clinical and biochemical parameters were not associated with hepatic iron status. Discussion: Unexpectedly, the majority of CKD patients already showed some degree of hepatic iron overload, assessed by MRI, at the time of dialysis start. Even those patients who didn’t receive significant amounts of iron or any iron at all had some iron overload, suggesting that CKD patients have a propensity to store iron in the liver instead of using it efficiently in erythropoiesis. Conclusions: This study provides for the first time evidence of the value of verifying LIC before HD is started. These findings suggest that iron administration to CKD patients should be cautious since the majority of patients already have hepatic iron overload at the time of dialysis start.
Seventh Congress of the International BioIron Society Page 152 Poster Abstracts IBIS
Poster #38
PROTECTIVE EFFECT OF HISTIDINE AGAINST IRON-INDUCED TOXICITY IN HEK293 CELLS F. Schlottmann, MSc, M. Vera-Aviles, MSc and G.O. Latunde-Dada, PhD Faculty of Life Sciences, Diabetes and Nutritional Sciences Division, King’s College London, United Kingdom Presented By: Mayra Vera-Aviles, MSc
The prevalence of anaemia of chronic kidney disease (ACKD) is high in most countries of the world. Kidney damage implies negative balance of iron and haemoglobin, transferrin, and other markers of iron levels, as a result, patients with chronic kidney disease and anaemia are in need of iron supplementation and higher amounts of iron have been related to stress that leads to tissue damage. Histidine has been postulated to protect against damage produced by excess iron. The study therefore evaluated the protective function of histidine in human embryonic kidney cells (HEK-293) that were challenged with a potent iron substrate. HEK-293 cells were treated with different concentrations of histidine before being subjected to 20 μmol/L of 8-hydroxy uinoline and 50 μmol/L of ferric ammonium citrate for 2 hours. Cell viability was tested with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, (MTT assay). Ferric reducing and antioxidant potentials of histidine were determined respectively by ferrozine and H2DCFDA fluorescent antioxidant assays. Histidine (100-750 µM) significantly (P<0.05) exerted a protective effect on HEK293 cell viability against iron-induced stress. N- Acetylcysteine (NAC) at 100-500 µM, used as a positive control, was equally cytoprotective against iron-induced cell damage. Histidine did not exhibit ferric reducing potential when compared with ascorbic acid. Furthermore, the antioxidant capacity of histidine was not significant compared to NAC. The antioxidant potential of NAC was significant (P<0.01) at 250 or 500 µM. Histidine and NAC seem to modulate cytosolic ferrous iron content using a probe-based immunofluorescence detection by microscopy. Histidine seems to protect HEK 293 cells against iron-induced cytotoxicity in a concentration dependent manner from 100 to 750 µM. Higher concentrations of histidine however, could have a cytotoxic effect. Although the mechanism by which histidine confers cytoprotection against excess iron damage seems not to be related to its antioxidant capability, histidine, however, may not accentuate ferric redox cycling as ascorbate in the generation of reactive oxygen species. Histidine could be important in reducing the toxic effects of excess iron in the treatment of ACKD.
Seventh Congress of the International BioIron Society Page 153 Poster Abstracts IBIS
Poster #39
HEMOGLOBIN UPTAKE AND INJURY IN RENAL DISTAL TUBULES Rachel van Swelm, PhD¹, Frank Verhoeven¹, Tom Gielkens¹, Frank Thévenod, PhD² and Dorine Swinkels, PhD¹ ¹Radboud university medical center, Nijmegen, the Netherlands; ²University of Witten/Herdecke, Witten, Germany Presented By: Rachel Van Swelm, PhD
Postoperative acute kidney injury (AKI) is an increasingly common life-threatening complication in up to 30% of the patients undergoing major surgery with cardiopulmonary bypass. Patients with postoperative AKI are at increased risk for mortality and morbidity, including chronic kidney disease. Currently, adequate preventive measures or specific treatment modalities are lacking. It is now well-established that hemolysis is an essential mechanism that contributes to postoperative AKI. During hemolysis, levels of circulating iron-containing hemoglobin increase, leading to accumulation in the kidney. Interestingly, the exact molecular mechanisms that lead to cellular injury in case of tubular hemoglobin overload have not been fully elucidated, but oxidative stress may play a role. In addition, molecular interactions of intra- tubular hemoglobin have only been described for proximal tubular cells. However, since hemoglobin casts have been observed in the distal nephron after hemolysis, tubular hemoglobin excess is also likely to affect the distal nephron. Recently, it has been demonstrated that proteins may also be reabsorbed in the distal tubules via the lipocalin-2 receptor in case the proximal tubules are overwhelmed. To investigate hemoglobin uptake by the lipocalin-2 receptor, mouse cortical collecting duct cells (mCCDcl1) were treated with scrmbl or lipocalin-2 receptor siRNA followed by incubation with fluorescently labeled hemoglobin. Expression of the lipocalin-2 receptor was reduced to 40% (p<0.001) and resulted in an 80% reduction of hemoglobin uptake (p<0.001). Moreover, incubation of mCCDcl1 cells with hemoglobin for 24, 48 or 72h resulted in a dose-dependent increase in mRNA expression of Ho-1 (p<0.001), suggesting intracellular hemoglobin catabolism. Interestingly, exposure with hemoglobin concentrations up to 10 uM did not lead to cell death in the mCCDcl1 cells, although the elevated mRNA expression levels of H-and L-ferritin (p<0.01 and p<0.001, respectively) after 24h incubation suggests increased intracellular iron levels. Hemoglobin-induced cell stress may be indicated by the observed increased secretion of lipocalin-2by the mCCDcl1 cells. Moreover, we observed increased Hamp1 mRNA expression (p<0.05) and hepcidin immunostaining (p<0.05) in mCCDcl1 cells after hemoglobin exposure. In conclusion, hemoglobin is reabsorbed in the mCCDcl1 cells via the lipocalin-2 receptor and catabolized by HO-1. Hemoglobin exposure does not induce cell death, but may cause transient cell stress in the mCCDcl1 cells. The mCCDcl1 cells may be protected from hemoglobin-induced injury by endogenous increased expression of hepcidin. In the near future we will evaluate the protective effect of hepcidin against hemoglobin exposure in mCCDcl1 cells by hepcidin siRNA silencing followed by hemoglobin incubation. Moreover, (transient) cell stress will be assessed after shorter hemoglobin incubation times to evaluate acute responses. Ultimately, enhanced insights in distal tubular hemoglobin handling will indicate whether there is a need and opportunity to prevent or treat deleterious effects of hemoglobin exposure in the distal nephron during hemolysis.
Seventh Congress of the International BioIron Society Page 154 Poster Abstracts IBIS
Poster #40
INTRAVENOUS IRON OXIDE INJECTION WITH HIGH POTENCY AND SAFETY PROFILES IN IRON DEFICIENCY ANEMIA RAT MODEL IN VIVO Chih-Lung Chen, PhD, Wen-Yuan Hsieh, PhD, Yuan-Hung Hsu, PhD, Nina Wang, PhD and Shian-Jy Wang, PhD MegaPro Biotech Company Presented By: Chih-Lung Chen, PhD
Abstract: Iron therapy is required for hemodialysis patients receiving erythropoietic stimulators in order to achieve the target hemoglobin (Hb) level in the most efficient way. Although parenteral iron drugs have been widely used, significant incidence of severe adverse effects including hypersensitivity and iron overload were observed. Despite many concerns remained, better efficacy and higher safety parenteral iron drug was still needed for hemodialysis patients. MegaPro newly developed the IOP injectionTM product, which is a superparamagnetic iron oxide nanoparticle conjugated with polyethylene glycol with an overall hydrodynamic colloidal particle size of 45 – 85 nm in diameter, containing 20 mg/mL elemental iron. The product has the potential for treating iron deficient anemia (IDA) in hemodialysis patients. In this study, an iron deficiency animal model was established by feeding (AIG-93) 30 weanling Sprague-Dawley rats for 21 days until low Hb level achieved 5 g/dL. We have compared the efficacy and safety results between IOP injection and iron- dextran based product for the treatment of IDA rats. Hb and ferritin level were measured on the day three after parenteral iron drug was injected into IDA rat, which were considered as efficacy markers. Serum iron and reactive oxidative stress (ROS) enzyme (malonyldialdehyde) were also detected which represented as safety markers. The results showed that IOP injection performed better efficacy and safety in the IDA rat. The detailed results will be presented at the meeting.
Seventh Congress of the International BioIron Society Page 155 Poster Abstracts IBIS
Poster #41
HOW CAN RECENT INSIGHTS INTO IRON METABOLISM AND INFECTION HELP US OVERCOME THE GLOBAL BURDEN OF ANEMIA? Sant-Rayn Pasricha, MBBS MPH PhD, Andrew Armitage, Andrew Prentice and Hal Drakesmith MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Sant-Rayn Pasricha, MBBS, MPH, PhD
Anemia affects ~273 million children and ~529 million women. In 2012, the World Health Assembly adopted a goal of 50% reduction in the prevalence of anemia as one of the key 2025 global nutrition targets. However, the 2016 Global Nutrition Report estimated that despite longstanding recommendations from WHO and UNICEF, progress to meet this goal is about 100 years behind schedule. Safe and effective control of the enormous unresolved burden of anemia worldwide requires translation of new scientific insights into the biology of iron homeostasis, interactions between iron and infection, and the epidemiology of anemia, into public health policy and programs. Ten years ago a randomized controlled trial from Pemba Island, Tanzania evaluating the effects of iron-folate in over 24,000 children aged 6-36 months was terminated prematurely because of increased mortality and hospitalization attributable to malaria and other infections were identified in children receiving iron. This study provoked suspension of WHO recommendations for universal iron for young children in malaria-endemic areas, and an ongoing considerable research effort into understanding iron-infection interactions. We present the six key messages from this research to be as follows: 1) Hepcidin –mediated hypoferremia is protective against some bacterial infections but can be overcome by iron supplementation that can lead to transient generation of harmful NTBI; 2) Iron deficient children are protected from malaria but are at elevated risk when given iron; 3) Infections besides malaria, and intestinal inflammation, can be exacerbated by iron supplementation; 4) asymptomatic and clinical malaria may cause iron deficiency; 5) Iron supplementation to pregnant women in malaria-endemic areas is safe and improves child outcomes; and 6) The proportion of anemia that is iron-responsive is lower than previously estimated. The implications of these findings for global health policy are significant. The World Health Assembly targets for anemia control in women and children are unlikely to be attained via iron interventions alone. In many low-income settings with high infection prevalence, the majority of children have high hepcidin concentrations and thus high dose iron interventions run counter to physiology in most cases. Furthermore, controlling malaria (and other infections) should be seen as an important intervention for anemia control, both by directly preventing malaria-related anemia and simultaneously improving iron absorption, thereby alleviating iron deficiency. Policy makers should evaluate the benefit-risk for iron interventions in children; these should be considered unsafe until infection control measures are robustly implemented. In contrast, iron appears safe and beneficial in pregnant women even without malaria control, although further investigations of effects on non-malarial morbidities (e.g. diarrhea) are warranted. Efforts should ensure women receive iron during (and perhaps before) pregnancy, to safeguard a healthy pregnancy, improved chances of a term birth, optimal birthweight and iron endowment.
Seventh Congress of the International BioIron Society Page 156 Poster Abstracts IBIS
Poster #42
IMPACT OF IRON DEFICIENCY DIAGNOSIS USING HEPCIDIN MS DOSAGE METHODS ON HOSPITAL STAY AND COSTS AFTER A PROLONGED ICU STAY: A MULTICENTER, RANDOMIZED, SINGLE-BLINDED TRIAL Sigismond Lasocki, MD, PhD¹, Herve Puy, MD,PhD², Thibaud Lefevre, MD, PhD², Gregoire Mercier, MD³, Constance Delaby, PhD³ and Sylvain Lehmann, MD, PhD³ ¹CHU Angers; ²INSERM U1149; ³CHU de Montpellier Presented By: Sigismond Lasocki, MD, PhD
Background: Iron deficiency (ID) is present in 25-40% of critically ill patients on intensive care unit (ICU) admission. Even higher ID is expected on ICU discharge due to bleedings, blood samplings, surgeries etc. ID may be responsible for prolonged post-ICU hospital stays, since it results in fatigue, muscle weakness and anemia. However, ID is difficult to diagnose in ICU using the usual biomarkers (i.e feritin or transferrin saturation). Hepcidin, the key iron metabolism hormone, may be a good marker of ID in these patients. The aim of this study is to determine whether using Mass Spectrometry hepcidin determination to diagnose (and treat) ID after prolonged ICU stays may reduce patients’ subsequent hospital stays and costs in comparison with conventional (ferritin and transferrin saturation) methods. Methods: This is a randomized, controlled, single blinded (patients and non-ICU physicians) multicenter study. Blood samples will be collected for hepcidin quantification from 400 anemic critically ill adult patients about to be discharged from the C , after a stay ≥5 days. n the intervention arm ( epcidin) results will be given to the C -physicians, whereas hepcidin dosages will be done at the end of the study on patients in the control arm. The following treatment will be recommended for D in intervention arm iron when hepcidin is 20 μg/l; iron erythropoietin when hepcidin ranges between 20-41 μg/l; and for patients in the control arm iron when ferritin is 300 μg/l and Transferrin saturation is <20%. ID diagnosis will be repeated every week on patients whose stay at the ICU has been extended. The primary endpoint will be the number of days spent in hospital 90 days after leaving the ICU and the direct hospital costs. Secondary endpoints will be anemia and iron deficiency on D15, fatigue on D30 and D90, and the proportion of patients alive and at home on D90. Discussion: The recrutement for the study is closed, with 400 patients included as scheduled. The results of this study will show whether diagnosing iron deficiency using MS hepcidin determination methods is liable to reduce patients’ post- ICU hospital stay and costs, as well as their anemia and fatigue. Trial Registration: Clinical Trial registration number: Clinicaltrials.gov NCT02276690 (27 October 2014)
Seventh Congress of the International BioIron Society Page 157 Poster Abstracts IBIS
Poster #43
DEVELOPMENT OF HUMAN SOLUBLE TRANSFERRIN RECEPTOR ELISA ASSAY Liubov Litvinkova, MS¹, Alexey Kravtsov, PhD², Matthias Herkert, PhD³, Ian Cushman, PhD⁴, Wilhelm S nger, MBA⁵ and Cyril Geacintov, PhD⁶ ¹DRG International, Inc.; ²Senior Research Scientist, DRG International, Inc.; ³Director R&D, DRG Instruments GMBH; ⁴Director R D, DRG International, Inc.; ⁵General Manager, DRG Instruments GMBH; ⁶CE , DRG International, Inc. Presented By: Liubov Litvinkova
Soluble Transferrin receptor (sTfR) is a circulating, cleaved form of a membrane receptor protein. The serum concentration of sTfR reflects the amount of the cellular receptor (1,2). As Transferrin expression increases with decrease in iron concentrations, measurement of its soluble form can be a valuable indicator of iron deficiency. sTfR level is elevated in iron deficiency, but not in anemia associated with chronic disease (3-5). Therefore, measurement of sTfR is particularly useful, when trying to distinguish between iron deficient individuals and anemic individuals with chronic disease (6). DRG International, Inc. developed a sandwich ELISA assay, to measure sTfR levels. With Correlation Coefficient = 0.938, our test shows excellent correlation to the reference Soluble Transferrin Receptor ELISA test. Our standard curve is set to World Health Organization reference material, providing for more accurate sTfR concentration measurements. sTfR DRG ELISA is a rapid test. Assay procedure: 30 minutes at room T for Standard, sample or Control incubation (sample volume required - 6 µl); 30 minutes at room T for Conjugate incubation, 15 minutes at room T for Substrate incubation. To assess the linearity of the assay three samples containing high, medium and low concentrations were evaluated. Average % recovery between samples was 93 – 105%. Recovery of sTfR spiked to 4 different levels in 3 samples with high, medium and low concentrations was evaluated. Average % recovery between spiked samples was 95 – 108%. Analytical Sensitivity of the assay was determined as 0.01µg/ml. Mean Intra Assay CV = 5.39%. This assay was validated and shows very good performance. Currently available as RUO in the USA. References: Cook, J.D., Baynes, R.D., Skikne, B.S. (1994) The physiological significance of circulating transferrin receptors, Adv Exp Med Biol. 352:119- 26. Luck, A.N., Mason, A. B. (2013) Structure and dynamics of drug carriers and their interaction with cellular receptors: focus on serum transferrin, Adv Drug Deliv Rev. 65(8):1012-1019. Ferguson, B.J, Skikne, B.S., Simpson, K.M., Baynes, R.D., Cook, J.D. (1992) Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia, J Lab Clin Med. 119(4):385-90. Skikne, B.S., Flowers, C.H., Cook, JD. (1990) Serum transferrin receptor: a quantitative measure of tissue iron deficiency, Blood. 1:75(9):1870-6. Braga, F., Infusino I., Dolci, A., Panteghini, M. (2014) Soluble transferrin receptor in complicated anemia, Clinica Chimica Acta. 431: 143-147 Harms, K., Kaiser, T., (2015) Beyond soluble transferrin receptor: old challenges and new horizons, Best Practice & Research Clinical Endocrinology & Metabolism. 29:799-810
Seventh Congress of the International BioIron Society Page 158 Poster Abstracts IBIS
Poster #44
COMPARISON OF THE THERAPEUTICAL POTENTIAL OF ORAL SUCROSOMIAL® IRON AND PARENTERAL IRON DEXTRAN SUPPLEMENTATIONS IN NEONATAL IRON DEFICIENCY ANEMIA IN PIGS Rafa Starzy ski, PhD¹, Mateusz Szudzik, MSc², Robert Staro , MSc², Aneta Jo czy, MSc², Ewa Smuda, MSc², Marek Pieszka, PhD³, Marian amyczek, PhD⁴ and Pa e Lipi ski, PhD² ¹IGAB PAS; ²Institute of Genetics and Animal Breeding PAS, Jastrz biec, Poland; ³Department of Animal Nutrition Feed Science, National Research Institute of Animal Production, rak , Poland; ⁴Pig Hybridization Centre, National Research Institute of Animal Production, Pa o ice, Poland Presented By: Pawel Lipinski, PhD, DSc
Background: Early postnatal iron deficiency is a widespread phenomenon in various mammalian species. Iron deficiency anemia (IDA) has long been recognized as a serious iron disorder in suckling piglets of all breeds. In consequence of a negative iron balance resulting from insufficient iron stores and dietary intake, the supply of iron for erythropoiesis is reduced in pig neonates. To counteract the development of IDA, a supplementation with an exogenous iron must be routinely applied to piglets, which most frequently consists on the intramuscular injection of large amounts of iron dextran (FeDex). Although this treatment has been proven to rectify the hematological status of piglets, there is evidence from our studies that it induces iron-dependent DNA oxidative damage in the liver. Sucrosomial® Iron (SI) has been shown to be directly transported into the bloodstream across gastric and enteric mucosa, presumably bypassing the pathways involved in heme and non-heme iron absorption in the duodenum. In this context, SI, a next generation oral iron supplement displaying high bioavailability and low incidence of side effects, seems to be a promising compound for delineating new strategies for curing iron deficiency in the neonatal period. Objectives: (i) evaluation of the efficacy and tolerance of Sucrosomial® Iron in the oral treatment of IDA in newborn piglets; (ii) comparison of the therapeutical potential of this new procedure with FeDex parenteral treatment, largely used in pig industry. Methods: The experiment was performed on Polish Landrace piglets in the age from 3 to 28 days (i.e. up to the day of weaning). During this period piglets were given per os a daily dose of 6 mg Fe in the form of SI (SiderAL®, PharmaNatura, Italy, liposomial iron pyrophosphate III, ultradispersed in sucrester) suspended in 2 ml of milk replacer (application with a sterile syringe). As a positive control group we used piglets supplemented intramuscularly with 150 and 40 mg Fe/kg b.w. in the form of FeDex (a complex of ferric ions with low molecular weight dextran; Ferran 100, Vet-Agro, Lublin, Poland) on days 3 and 21 postpartum, respectively (routine supplementation). Piglets receiving no iron supplementation were used as a negative control group. Results: All piglets tolerated well supplementation with SI. Oral SI application to piglets efficiently prevented them from IDA as evidenced by high values of all analyzed red blood cell indices as well as biochemical iron parameters in the plasma (iron concentration, TIBC, transferrin saturation). The iron status of SI-supplemented piglets was similar to that of FeDex-supplemented animals. However, in contrast to FeDex-supplemented piglets, animals receiving SI show no excessive iron accumulation in liver, spleen, brain, heart and kidney. Importantly, hepatic hepcidin mRNA level in SI- treated animals was not elevated. Conclusions: The results of our pilot study show for the first time that oral application of Sucrosomial® Iron to newborn iron-deficient piglets may be an effective and reliable option for IDA treatment. Further studies including the analysis of gut microbiom as well toxicological analysis are needed to confirm the usefulness of SI for curing iron-deficient piglets on a large-scale. In a long-term perspective, our results may be important to optimize strategies for preventing neonatal iron deficiency in humans, especially in preterm human neonates showing similarly to newborn piglets - low level of hepatic iron stores.
Seventh Congress of the International BioIron Society Page 159 Poster Abstracts IBIS
Poster #45
NEONATAL IRON DEFICIENCY ANEMIA IN PIGS: CAUSES, REGULATION OF IRON METABOLISM, AND STRATEGIES FOR IRON SUPPLEMENTATION THERAPY Pa e Lipi ski, PhD¹, Rafa Starzy ski, PhD², Robert Staro , MSc², Anna Ga o iak, MSc², E a Smuda, MSc², Marek Pieszka, PhD³ and Ma gorzata Lenarto icz, PhD⁴ ¹IGAB PAS; ²Institute of Genetics and Animal Breeding PAS, Jastrz biec, Poland; ³Department of Animal Nutrition & Feed Science, National Research Institute of Animal Production, rak , Poland; ⁴Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, Kraków, Poland Presented By: Pawel Lipinski, PhD, DSc
Neonatal iron deficiency anemia (IDA) commonly occurs in pigs to a greater or lesser extent, regardless of the breed and the system of piglet rearing. The major etiological factors in pig IDA include low hepatic iron stores, the high intensity of piglet growth, increasing litter size, inadequate levels of iron in sow’s milk and the immaturity of molecular mechanisms of iron absorption. Indeed, our studies show that in 3-day-old piglets iron reserves in the liver are completely depleted. We have also demonstrated that the expression of divalent metal transporter 1 and ferroportin, two iron transporters critical for duodenal iron absorption, was barely detectable during the first days of life. In consequence, dietary iron supplements show limited efficacy in preventing and curing pig IDA. In contrast, the use of intramuscular injection of large amounts of iron dextran (FeDex) has been proved to be beneficial in correcting IDA in suckling piglets and is obligatory in pig breeding. However, the potential toxicity of such supplemental iron implies the necessity for caution when applying this treatment. Indeed, we show that supplementation of piglets with a single high dose of FeDex resulted in a high increase in the hepatic oxidative DNA damage. Moreover, this routine treatment of piglets with FeDex induces a sustained hepatic hepcidin expression at both the mRNA (liver) and protein (plasma and urine) levels, which in turn may impair both the utilization of supplemental iron deposited in reticuloendothelial macrophages and the absorption of dietary iron. We therefore established an optimized protocol of FeDex administration to pig neonates (by both reducing the amount of supplemented iron and modifying the timing of its dosage), which better meets the iron demand for erythropoiesis, attenuates the risks of excessive induction of hepcidin expression, and minimizes the toxicity of supplemental iron. Finally, considering that heme is an efficient source of iron in the diet, we employed young iron-deficient piglets as a convenient animal model to determine the efficacy of oral heme iron supplementation and investigate the pathways of heme iron absorption. The use of bovine hemoglobin as a dietary source of heme iron was found to efficiently counteract the development of iron deficiency anemia in piglets. Furthermore, our results revealed a concerted increase in the expression of genes responsible for heme breakdown and for apical and basolateral heme transport in the duodenum of piglets fed a heme-enriched diet. In addition, we demonstrate that the hepcidin-duodenal ferroportin axis acts to enhance the basolateral transport of elemental iron released from the protoporphyrin ring of heme in enterocytes. We hypothesize that the well-known high bioavailability of heme iron may rely on the presence of two independent pathways mediating the transport of intact heme and heme-derived elemental iron across the duodenal enterocytes into the circulation.
Seventh Congress of the International BioIron Society Page 160 Poster Abstracts IBIS
Poster #46
PLACENTAL EXPRESSION OF IRON HOMEOSTASIS GENES IN IRON DEFICIENCY ANEMIA OF PREGNANCY Sreenithi Santhakumar, MTech¹, Rekha Athiyarath, PhD¹, Anne George Cherian, MD², Vinod Joseph Abraham, MD² and Eunice Sindhuvi Edison, PhD¹ ¹Department of Haematology, Christian Medical College, Vellore, India; ²Department of Community Health, Christian Medical College, Vellore, India Presented By: Eunice Sindhuvi Edison, PhD
Iron Deficiency Anaemia (IDA) is a major public health concern with frequency greater than 50% being reported in pregnant women in India. IDA in pregnancy is known to be a risk factor for preterm delivery and low birth weight. The placenta plays a dynamic regulatory function in maintaining the iron stores for foetal survival. The materno-foetal iron regulation is very complex and less understood. The present study aims to understand the regulation of iron transport across the placenta by analysing the expression of iron transporters and regulators in subjects with IDA. Subjects who came for child birth to the Department of Community health were screened and consenting patients were included in the study. Peripheral blood samples were collected and a cut placental tissue (~0.5cm) was obtained at the time of delivery.The tissue was washed in sterile PBS and immediately stored in RNA stabilising agent. Haematological and biochemical parameters were analysed according to standard methods.Serum hepcidin and GDF15 levels were measured using ELISA (DRG Diagnostics and Ray Biotech, respectively). RNA was extracted using PARIS kit (Ambion) and cDNA was synthesized using RT2 first strand Kit (QIAGEN). The relative quantification of twenty two genes involved in iron uptake, export, transport and regulation was done using Custom RT2 PCR Array (SA Biosciences, QIAGEN). Statistical analysis was done using the software SPSS. Of the 138 subjects recruited in the study, 18 subjects with anaemia and 13 subjects without anaemia (control) were randomly selected. Among these 18 anaemic subjects, 8 were iron deficient. The haematological and biochemical parameters of the different groups are tabulated (Table.1). A significant negative correlation was observed between the GDF15 and hepcidin levels in the IDA cohort (r= -0.821, p=0.023). Several genes in iron transport, uptake and regulation (DMT1, FPN, TFRC, and TWSG1) were differentially expressed between the anaemic and the control group. A significant difference in expression of genes involved in heme transport (SLC46A1 and LRP1) was also observed. In subjects with IDA, significant differential expression of ACO1 (Aconitase1), SLC11A2, FTL, SLC46A1, HIF1A and LRP1 genes were noted. These findings also need to be validated at the protein level. The expression of specificity Factor-1 (Sp1, a transcription factor required for the transcriptional response to iron deprivation) had a significant positive correlation with the expression of DMT1 (r=0.964, p=0.00), FPN (r=0.857, p=0.014) and TFRC (r=0.893, p=0.007) in the iron deficient group alone. Several of these genes involved in placental iron transport and regulation have significant changes in iron deficiency suggesting that they play a crucial role in maternal and foetal iron regulation.
Table.1 Haematological and Biochemical parameters of the study cohort (Values of Hb is represented as Mean±S.D, Ferritin, Hepcidin, Hep:Ferr ratio and GDF15 are represented as Median, Range)
Seventh Congress of the International BioIron Society Page 161 Poster Abstracts IBIS
Poster #47
DEFERASIROX AND PHLEBOTOMIES IN A PATIENT WITH EXTREME IRON OVERLOAD BY HEREDITARY HEMOCHROMATOSIS AND BETA-THALASSEMIA MINOR AFTER DIAGNOSIS OF LISTERIOSIS Alejandro Del Castillo-Rueda, Dolores Pulfer, Alejandra García-García and Ana López-Aparicio Hospital Gregorio Marañón Presented By: Alejandro Del Castillo-Rueda, MD, PhD
The two most important treatment options for iron overload are phlebotomy and chelation. Phlebotomy is the initial treatment of choice in hemochromatosis, while chelation with deferasirox could be a safe alternative in selected patients. Patients with hemochromatosis have increased susceptibility to infections such as L. monocytogenes due to hepcidin deficiency and iron overload. We present a patient who was diagnosed after an episode of bacteremia by Listeria monocytogenes of classical hemochromatosis and beta thalassemia minor with iron overload and who was treated with phlebotomies and deferasirox. A 45-year-old male patient was referred to our iron disorders unit due to extreme hyperferritinemia, thrombocytopenia and hypertransaminasemia after discharge from the hospital due to listeria infection with primary bacteremia. The results of his laboratory test were: 1) Iron panel: serum ferritin (sF) 11.080 ng/mL, transferrin iron saturation (TS) 43% and soluble transferrin receptor (sTfR) 6.18 mg/L. 2) Blood count: red blood count (RBC) 4.9 million/uL, hemoglobin (Hgb) 10.1 g/dl, hematocrit (hct) 0,31, mean corpuscular volume (MCV) 63.5 and platelet count 64.000. 3) Liver test Aspartate transaminase 89 /L, alanine transaminase 95 /L, alkaline phosphatase 45 /L and - glutamyltransferase 62 IU/L. 4) Serum hepcidin was undetectable. 5) DNA mutation analysis showed that the patient was homozygosis for C282Y mutation. 6) Hb electrophoresis show the following pattern: 81.2% Hb A, 5.4% Hb A2 and Hb fetal 2.8%, whitout abnormal Hb and whit beta defect that determines hetero ygous (β0) thalassemia. Liver magnetic resonance imaging measurement resulted in >350 µmol/g (normal value <36µmol/g), with extreme iron overload. Cardiac magnetic resonance methods resulted in normal value, without iron overload. The patient had bi-weekly therapeutic phlebotomies of 250 ml. However, there was only a slight reduction of the serum ferritin values and was necessary to introduce deferasirox at starting doses of 10 mg / kg/day for 10 months with good effectiveness and tolerance without anemization with ferritin of 500 ng/mL and normalization of the tests of liver function, continuing with phlebotomies monthly. The decreased and normalization of iron parameters using deferasirox adjunct to venesections during the initial treatment phase were observed. While waiting for large studies, it is possible to raise the need for therapy combined with deferasirox and phletomies in the diagnosis of severe iron overload.
Seventh Congress of the International BioIron Society Page 162 Poster Abstracts IBIS
Poster #48
EVALUATING IRON METABOLISM AND SUPPLEMENTATION IN GAMBIAN TODDLERS USING A NOVEL, HIGH- PRECISION METHOD BASED ON THE DILUTION OF LABELLED BODY IRON Diego Moretti, Msc; PhD¹, Rita Wegmüller, Msc; PhD²,³, Cornelia Speich, Msc¹, Christophe Zeder, Msc¹, Magdalena Cichucka, Msc¹, Colin Ivano Cercamondi, Msc; PhD¹, Andrew Prentice, MD; Professor²,³, Gary Brittenham, MD; Professor and Michael Bruce Zimmermann, MD; Professor¹ ¹ETH Zürich; ²MRC Unit, The Gambia; ³LSHTM, London, UK Presented By: Diego Moretti, PhD
Global efforts to reduce iron deficiency anemia are hindered by the unreliability of current iron status biomarkers in the presence of infection and inflammation. We developed and evaluated a novel stable iron isotope method based on the dilution of labelled body iron to measure and quantify the efficacy of an iron intervention and to quantify iron balance in 57 rural Gambian toddlers. To label total body iron, we orally administered 2 mg Fe as FeSO4 to anemic infants (mean (SD) age: 16.7(1.6) months; Hb<11.0 g/dl) and waited 41(4.9) weeks for equilibration of the 57Fe in body iron. We then randomly allocated the toddlers to 12 weeks of directly supervised feeding with: A) a micronutrient powder (MNP) containing 12 mg Fe given every day; B) a MNP containing 12 mg Fe given for seven days when hepdicin <5.5 ng/ml at weekly screening; C) a MNP containing 6 mg Fe given for seven days when hepdicin <5.5ng/ml at weekly screening. We uantified the rate of change of body iron ( C e), iron absorption ( eAbs), as well as the change in b ( b), soluble transferrin receptor ( sTf ) and plasma ferritin ( P ). These markers were assessed in all toddlers in blood samples on day 49 and 84 of the intervention, and after a 12-week control period post-intervention. Complete data from 41 subjects was available for analysis. Time elapsed from isotopic administration to intervention start was not correlated with RCFe, suggesting complete isotopic label equilibration. RCFe significantly differed between the intervention and control period (P 0.01), as did b (P 0.01) and sTf (P 0.01) but not P (P 0.17). Compared to conventional iron biomarkers, RCFe had the strongest association with total administered iron dose (R2=0.44, P<0.01). RCFe was also associated with b, sTf and P (R2=0.37, P <0.01; R2 =0.49, P<0.01 and R2 = 0.11, P<0.05, respectively) but not with the CRP levels at the end of each period. In contrast, and as expected, PF was associated with CRP (R2=0.14; P<0.01). Across the three groups, FeAbs during the intervention (0.36±0.13 mg Fe/d) was significantly higher than during the control period (0.13±0.07 mg Fe/d, P <0.01). During the intervention, FeAbs was significantly higher in group A (0.47±0.18 mg Fe/d) than in groups B or C (0.30±0.10 mg Fe/d and 0.33±0.12 mg Fe/d, P=0.01). Daily iron losses were significantly higher during the intervention (0.25±0.11 mg Fe/d) than during the control period (0.11±0.12mg Fe/d, P<0.01); there were no significant differences in daily iron losses between the three groups. Conclusions: RCFe measured with this technique is superior to conventional iron biomarkers in prediction of the administered iron dose. It may allow to quantify iron absorption, losses and overall iron balance over the long term, and is a promising tool to evaluate iron interventions with high precision entirely free from confounding by inflammation. Listed first and second author contributed equally to this paper.
Seventh Congress of the International BioIron Society Page 163 Poster Abstracts IBIS
Poster #49
EFFECT OF SUCROSOMIAL® IRON IN A IRON DEFICIENCY ANEMIA MOUSE MODEL Elisa Brilli, PhD¹, Maura Poli, PhD², Michela Asperti, PhD², Angela Fabiano, PhD³, Annalisa Castagna, PhD⁴, Domenico Girelli, MD⁴, lenia ambito, PhD³, Paolo Arosio, PhD² and Germano arantino¹ ¹Pharmanutra S.p.A.; ²DMM , University of Brescia; ³Pharmacological Sciences, University of Pisa; ⁴Internal Medicine, University of Verona Presented By: Germano Tarantino
Iron, an essential element for organisms, plays a role in vital processes. Proteins such as Ferritin (Ft), Hemoglobin (Hb) and Transferrin Receptor (TfR1) are involved in tissue iron distribution while systemic iron metabolism is regulated by Hepcidin. Iron deficiency anemia (IDA) is a condition where the body iron store are too low to support normal red blood cells production. Oral iron delivery is especially attractive due to the ease of administration, but commonly used iron salts are poorly absorbed and tolerated. Recently, some reports showed that phospholipid-like nano-carriers are able to cross intestinal barrier thanks to the presence of specialized lymphatic cells named M cells. Up to date, there are no studies on the effects of oral iron administration in anemic murine models. We have developed a new oral Iron formulation named Sucrosomial® Iron, preparation of ferric pyrophosphate, covered by a phospholipids plus sucrester matrix, with high bioavailability and tolerability, which promotes ferric iron absorption thanks to gastro-resistant and matrix composition properties and showed Hb concentration improvement not inferior to intravenous iron in chronic kidney disease and myelodisplastic patients. We have developed a fluorescin-labelled Sucrosomial® Iron in order to perform a fate-mapping analysis in ex-vivo experiments using rats isolate intestine. To study the effects of oral supplementation on Iron, Hb, Ft, TfR1 and Hepcidin concentration, we have supplemented wild-type (wt) and iron deficient (ID) mice, maintained in iron- free diet for 8 weeks, with 1mg/kg/die of Sucrosomial® Iron or Iron Sulphate or Placebo by oral gavage, for 2 weeks. Finally, animals were sacrificed and blood, liver and spleen were collected. Fate-mapping study showed that Sucrosomial® Iron is able to cross the intestinal barrier over time. Results from mice showed that both groups of animals respond to Sucrosomial® Iron treatment increasing serum and tissue iron concentration. In ID mice, Hb level mean increase was 4,9 g/dl and liver TfR1 and Ft protein expressions were significantly higher compared to placebo and control mice, while Ferroportin concentration did not differ from control mice. Hepcidin mRNA expression was not induced during Sucrosomial® Iron treatment in both ID and wt mice while was up-regulated in mice treated with Iron Sulphate. In summary, the results showed that the murine model responds to Sucrosomial® Iron treatment regulating iron homeostasis through an increase in Hb concentration but without an induction of hepcidin mRNA expression. These evidences may suggest that this new oral iron formulation carried by phospholipids and sucrester matrix behaves differently than other oral iron salts, perhaps due to different absorption pathways.
Seventh Congress of the International BioIron Society Page 164 Poster Abstracts IBIS
Poster #50
EFFECTS OF BONE MARROW IRON ON BONE MINERAL DENSITY ASSESSMENTS: A PHANTOM STUDY Marcela Weyhmiller, PhD¹, Neil Buac², Ellen Fung, PhD RD¹, Ashutosh Lal, MD¹ and Roland Fischer, PhD³ ¹UCSF Benioff Children's Hospital Oakland; ²California State University, East Bay; ³UCSF Benioff Children's Hospital Oakland/UKE - Pediatrische Hämatologie/Onkologie Presented By: Marcela G. Weyhmiller, PhD
Chronically transfused patients are at risk for iron overload in various organs. Recent MRI studies have shown that the vertebral bone marrow (VBM) of these patients can be highly iron overloaded. In addition, many of these patients are at risk for endocrine disorders with can result in low bone density. Dual Energy Xray Absorptiometry (DXA) analysis is used clinically to assess bone mineral density (BMD) of the lumbar vertebrae. Iron is an electronic-dense element (Z=26) that has been shown to artificially increase bone density assessment by DXA in patients with highly iron overloaded livers. Additionally, liver iron has been qualitatively assessed with whole body scan DXA. This phantom study was designed to investigate whether VBM iron can effect BMD assessment by DXA. Spine phantoms of high, normal and low BMD (made from beef shin bone, DXA spine phantom and cuttlefish bone, respectively) were overlaid with bone marrow phantoms of various amounts of iron and measured by DXA. Bone marrow phantoms were made from mixtures of mayonnaise, water and various concentrations of iron dextran (R2* up to 600 s-1). Mayonnaise was used to resemble the fat/water structure of VBM. BMD scans were acquired on a Hologic Horizon A (Hologic Inc., Bedford, MA) software version Apex 5.6.1. Raw data of phantom BMD were measured using lumbar spine fast array and forearm scan acquisition. Net average bone mineral content (BMC) was abstracted from subregional analysis for each scan. R2* and fat quantification were assessed from MRI scans acquired at 1.5 T (Phillips Intera software version 3.2.3.2) using a multiple gradient-echo sequence. Region of interest (ROI) analysis was performed with CMRtools 2016. Values were fit for iron concentration (R2*) and fat content were determined by water-fat chemical shift relaxometry from signal intensities using a Levenberg-Marquardt fit algorithm (SlideWrite Plus V 7.0). This will be the first study to compare iron proportional R2* rates with BMD measurements by DXA. BMD did increase with R2*, however this was most pronounced in the low BMD phantoms. Since many chronically transfused patients are at risk for low bone density/mass, this study indicates that iron may interfere with bone density assessments. Further work is needed, but we expect that VBM iron analysis by MRI may be used in conjunction with DXA to better assess bone health for iron overloaded patients.
Seventh Congress of the International BioIron Society Page 165 Poster Abstracts IBIS
Poster #51
HFE GENOTYPE ALTERS EXOSOME PROFILES IN CANCER Oliver Mrowczynski, A.B. Madhankumar, PhD, Brad Zacharia, MD, MS, Becky Slagle-Webb, BS and James Connor, PhD Penn State Hershey Presented By: Oliver Mrowczynski
Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. A major focus in cancer biology is the impact of exosomes on metastasis and the microenvironment. The major goal of this study was to determine if changes in HFE genotype, associated with aggressive cancer phenotype, have significant effects on exosome profile. HFE, the most common autosomal recessive polymorphism in the Caucasian population (affecting 1:200 Caucasians), originally associated with hemochromatosis, is also associated with increased tumor burden, therapeutic resistance boost, and negative impact on survival. The outcome of this study will improve our limited knowledge of neuroblastoma exosomes and provide direct insights into the impact of HFE gene variants on exosomes. This is a novel concept because the effect of germline mutations on exosome profile has not been avidly studied. We utilized immunoblots to analyze protein expression differences between exosomes. We also used functional assays, such as invasion, angiogenesis, and cytotoxicity assays, to determine functional changes caused by HFE mutant cell derived exosomes. We demonstrate that HFE mutant derived exosomes have increased expression of proteins relating to the ferrome, invasion, angiogenesis, and cancer therapeutic resistance. These exosomes can also transfer their cargo to recipient cells causing an increased functionality with regards to oncogenesis. These are novel findings because the contents within exosomes have been proven to cause an enormous impact in the tumor microenvironment by heightening the tumorigenic potential of neighboring cells. Our study has demonstrated that HFE mutant derived exosomes have increased expression of proteins related to oncogenesis, which has major implications on not only HFE variant patients with neuroblastoma, but all types of cancer.
Seventh Congress of the International BioIron Society Page 166 Poster Abstracts IBIS
Poster #52
ANALYSIS OF SINGLE NUCLEOTIDE VARIATION OF HFE GENE FOR CANCER RISK AND ASSOCIATION TO SURVIVAL IN THE CANCER GENOME ATLAS (TCGA) GBM DATA Sang Lee, Junjia Zhu, Anna Salzberg, Bo Zhang, Dajiang Liu and James Connor Penn State University College of Medicine Presented By: Sang Lee, PhD
It has been known that H63D and C282Y HFE polymorphisms have been associated with an increased risk of cancers. Recently, we reported gender affects in overall survival based on two common HFE polymorphisms (H63D or C282Y) in glioblastoma (GBM) patients at the Hershey Medical Center. However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. In this study, we analyzed SNV of HFE gene using The Cancer Genome Atlas (TCGA) GBM database to find cancer risk in human GBM and also to expand our finding in a larger sample size for the survival relationship with HFE polymorphisms. We used only Caucasian’s data in this study, because HFE polymorphisms are more common in Caucasian than other races. We compared the frequency of SNV in HFE gene between primary tumor samples of TCGA GBM and 1000Genome (genome sequence data of at least 1000 anonymous participants) as well as blood normal samples (same patient of tumor tissue). According to ExAC database, there are 142 SNVs in the HFE gene. We found total 9 SNVs have increased frequency in tumor tissues compared to the 1000Genome. Among 9 SNVs, 7 SNVs locate in intron and 2 SNVs (i.e., H63D, C282Y) are in exon of HFE gene. The statistical analysis indicates that tumor samples of TCGA GBM have more H63D (p=0.0003, 95% Confidence interval (CI): 0.2088-0.317) or C282Y (p=0.0153, 95% CI: 0.046-0.1125) HFE polymorphisms than 1000Genome. The frequency of other HFE gene mutations such as S65C and Q277K in tumor tissue is not different with 1000Genome. Interestingly, one intron position SNV (near exon 2), which has been known a benign risk factor for hemochromatosis, also shows increased frequency in tumor samples than the frequency of 1000Genome (p<0.0001, 95% CI: 0.4553-0.5778). In addition, there is an increased ratio at 5 SNVs (all intron position of HFE gene) in tumor samples compared to the blood normal samples. Although the incidence for H63D and C282Y is increased in tumor samples, our results indicate that there is no difference at the Kaplan-Meier survival curve between HFE polymorphisms (H63D, C282Y) patients and patients without a mutation. The contradictory findings between TCGA GBM and our previous work for the association between HFE polymorphisms and patient’s survival may be due to different ratio of gender and other unidentified reasons. In other preliminary animal study, however, we observed H67D (mouse homolog of the human H63D) homozygote mouse dying early than control wildtype mouse in orthotopic mouse glioma tumor model. In conclusion, the current results suggest that two common HFE polymorphisms (H63D, C282Y) and several SNVs in intron of HFE are risk factors for GBM and need further study for the role of cancer progression and development.
Seventh Congress of the International BioIron Society Page 167 Poster Abstracts IBIS
Poster #53
FERROPORTIN INDUCES IRON DEPLETION, AUTOPHAGY, AND CELL CYCLE ARREST IN PROSTATE CANCER CELLS OF DIFFERENT HISTOPATHOLOGICAL SUBTYPES Suzy Torti, PhD, Zhiyong Deng, PhD, David Manz, BS and Frank Torti, MD University of Connecticut Health Center Presented By: Suzy Torti, PhD
Prostate cancer is the most common malignancy and the second most frequent cause of cancer death in men (American Cancer Society, 2016). The vast majority of prostate cancer is classified as adenocarcinoma, and is characterized by uncontrolled proliferation of cells with characteristics of luminal differentiation, including glandular formation and expression of the androgen receptor (AR). Targeting of the AR is a highly successful frontline therapy for prostatic adenocarcinoma that causes clinical remission for prolonged periods of time; however, most tumors ultimately become resistant to such therapies (referred to as development of castrate-resistant disease), which eventually leads to disease progression. A smaller fraction of prostatic epithelial malignancies are of other histological types, including small cell (neuroendocrine) carcinoma (SCNC), which occurs in approximately 0.5-2 % of men with prostate cancer. SCNC do not express AR, are clinically more aggressive than adenocarcinoma, and are treated with different therapeutic regimens. Ferroportin is an iron efflux pump that is best known for its role in the maintenance of systemic iron homeostasis. Apart from its expression in tissues that regulate systemic iron balance, ferroportin is also expressed in peripheral tissues and tumors. Our laboratory and others have reported that ferroportin is decreased in prostate tumors at the transcript and protein level when compared to normal prostate tissue. Induction of hepcidin, a negative regulator of FPN, also contributes to the decrease in ferroportin in prostate cancer. The consequence of decreased ferroportin is increased tumoral retention of iron, a necessary nutrient for tumor growth. Here, we used cell culture models of prostate adenocarcinoma, castrate-resistant adenocarcinoma, and neuroendocrime carcinoma to probe the mechanism(s) by which iron depletion mediated by FPN affects these disparate types of prostate cancer. We show that enhanced iron efflux mediated by overexpression of FPN is sufficient to trigger substantial iron depletion and induce profound effects in all prostate cancer cells by activating different but converging pathways. These include activation of the transcription factors p53 and KLF6 and their common downstream target, p21; induction of growth arrest and DNA-damage inducible genes; and induction of autophagy. Effects of FPN are attributable to its effects on intracellular iron and are similar to those seen using iron chelators. Importantly, high levels of FPN inhibit proliferation of all prostate cancer cells in vitro and reduce growth of castrate-resistant adenocarcinoma C4-2 cells in vivo. These activities may explain why FPN is suppressed in cancer cells, and suggest that FPN agonists may be beneficial in treatment of prostate cancer.
Seventh Congress of the International BioIron Society Page 168 Poster Abstracts IBIS
Poster #54
DCYTB IS A PREDICTOR OF OUTCOME IN BREAST CANCER THAT FUNCTIONS VIA IRON-INDEPENDENT MECHANISMS Frank Torti, MD¹, David Lemler, PhD², Miranda Lynch, PhD³, Lia Tesfay, MS¹, Zhiyong Deng, PhD¹, Bibbin Paul, PhD¹, Poornima Hegde, MD¹ and Suzy Torti, PhD¹ ¹University of Connecticut Health Center; ²Univesity of Connecticut Health Center; ³Los Alamos National Laboratory Presented By: Frank Torti, MD
Background: Duodenal cytochrome b (DCYTB) is a ferrireductase that functions together with divalent metal transporter 1 (DMT1) to mediate dietary iron reduction and uptake in the duodenum. DCYTB is a member of a 16 gene Iron Regulatory Gene Signature (IRGS) that predicts metastasis-free survival in breast cancer patients. To better understand the relationship between DCYTB and breast cancer, we explored in detail the prognostic significance and molecular function of DCYTB in breast cancer. Methods: The prognostic and predictive significance of DCYTB expression was evaluated using publicly available microarray data. Signaling Pathway Impact Analysis (SPIA) of microarray data was used to identify potential novel functions of DCYTB. The role of DCYTB was assessed using immunohistochemistry and analysis of the effect of DCYTB on iron uptake, iron metabolism and FAK signaling. Results: High DCYTB expression was associated with prolonged survival in two large independent cohorts, together totaling 1610 patients (cohort #1, p= 1.6e-11, n= 741; cohort #2, p= 1.2e-05, n= 869; log-rank test). DCYTB also predicted response to tamoxifen and chemotherapy in patients treated with these agents. Immunohistochemistry revealed that DCYTB is localized on the plasma membrane of breast epithelial cells, particularly on the surface of the ductal lumen, and that expression is dramatically reduced in high grade tumors. DMT1 was also expressed in breast cancer tissue, but did not co-localize with DCYTB. Surprisingly, neither overexpression nor knockdown of DCYTB affected levels of ferritin H, transferrin receptor, labile iron or total cellular iron in breast cancer cells. Because SPIA analysis of patient microarray data revealed an association between DCYTB and the focal adhesion pathway, we examined the influence of DCYTB on FAK activation in breast cancer cells. These experiments reveal that DCYTB reduces activation of focal adhesion kinase (FAK). Conclusions: DCYTB is an important predictor of outcome and response to therapy in breast cancer patients. DCYTB does not affect intracellular iron in breast cancer cells. Instead, DCYTB may retard cancer progression by reducing activation of FAK, a kinase that plays a central role in tumor cell migration and metastasis.
Seventh Congress of the International BioIron Society Page 169 Poster Abstracts IBIS
Poster #55
APOPTOTIC EFFECT OF FENUGREEK ON MCF-7 CELLS IS INHIBITED BY FERRITIN Kholoud Khoja¹, MSc, PhD, Paul Sharp², PhD and Yemisi Latunde-Dada², PhD ¹King's College London; ²King’s College London Presented By: Kholoud Khoja, MSc, PhD
There is evidence that fenugreek (Trigonella foenum graecum) could be used to treat cancer because of its ability to enhance the expression of some apoptotic genes. The mechanisms and the signally pathways that induced are however not certain known [1]. Ferritin is the main protein which stores iron in the body and it is therefore important in maintaining iron homeostasis. Furthermore, ferritin has impact on many cancer cells, but the mechanisms of some of these effects are not known [2]. The study explored the effect of fenugreek plant methanol extract (FPME), fenugreek seeds methanol extract (FSME), and ferritin (human liver ferritin), in the regulation of cancer progression using MCF7 as a breast cancer cell line. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and reactive oxygen species (ROS) was estimated by H2DCFDA fluorescent assays. Ferritin treatment enhanced MCF-7 cell viability in a time-dependent manner, both (FPME) and (FSME), by contrast, caused cell death. The cytotoxic influence of both (FPME) and (FSME) were significantly reduced by average of 13% for (FPME) and 16.50% (FSME) when MCF7 cells were treated with 50 nM of ferritin for 24 h. Treatment of MCF-7 cells with ferritin resulted in decreased ROS production while both FPME and FSME at their respective IC50 significantly (P<0.0013) and (P<0.0001) increased intracellular ROS levels. Fenugreek induces apoptosis possibly by the generating ROS, while ferritin decreases ROS as an antioxidant. Thus, ferritin may be antagonising the apoptotic effect of fenugreek as an anti-cancer agent. References: 1. Khoja KK, Shaf G, Hasan TN, Syed NA, Al-Khalifa AS, Al-Assaf AH, Alshatwi AA., 2011. Asian Pac J Cancer Prev.;12(12):3299-304 2. Buranrat, B. and Connor, J.R., 2015. Oncology reports, 34(5), pp.2790-2796.
Seventh Congress of the International BioIron Society Page 170 Poster Abstracts IBIS
Poster #56
ADMINISTRATION OF MINIHEPCIDINS TO ANIMALS AFFECTED BY ß-THALASSEMIA MAJOR REDUCES ANEMIA AND SPLENOMEGALY Roberta Chessa, PhD¹, Ritama Gupta², Carla Casu, PhD², Robert Fleming, MD³, elena Ginzburg, MD⁴, Brian MacDonald, MD⁵ and Stefano Rivella, PhD² ¹ he Children s Hospital of Philadelphia; ² he Children s Hospital of Philadelphia; ³Saint Louis University; ⁴Icahn School of Medicine at Mount Sinai; ⁵Merganser Biotech Presented By: Roberta Chessa, PhD
ß-thalassemia intermedia (BTI) and major (BTM) are characterized by formation of hemichromes in erythroid cells, impairing their survival and the lifespan of red blood cells (RBC). Minihepcidins (MH) are novel compounds that function as hepcidin agonists and reduce iron absorption and transferrin saturation. Hbbth3/+ mice show features of BTI, such as ineffective erythropoiesis (IE), anemia and reduced hepcidin synthesis, but do not require blood transfusion (Tx) for survival (non-transfusion dependent thalassemia or NTDT). Administration of MH in these animals decreased transferrin saturation, erythroid iron intake, heme synthesis and hemichrome formation, with a significant beneficial effect on RBC quality, lifespan and anemia. In order to test if this approach could also benefit animals affected by BTM we focused on generating a model that exhibited a low production of RBC, severe anemia and Tx requirement for survival, as in patients affected by transfusion dependent thalassemia (TDT). The engraftment of Hbbth3/th3 fetal liver cells (FLCs) into GFP+ transgenic mice leads to a very severe anemia that requires Tx for survival. Hbbth3/th3 FLCs do not contain any adult or fetal-globin genes and are unable to make hemoglobin in the transplanted animals, in contrast to human ß-thalassemia. Therefore, animals cannot benefit from therapies that decrease hemichrome formation and target IE. We crossed models of NTDT Hbbth1/th1 with Hbbth2/+ that harbor alternative mutations to decrease the synthesis of the mouse ß-globin genes to mimic TDT. Hbbth1/th2 were alive at birth, but unable to survive even with the support of Tx. However, recipient transgenic animals expressing GFP and engrafted with Hbbth1/th2 FLCs showed the desired phenotype 2 month post-transplant including low production of GFP+-RBC, and anemia. In the long term, despite production of thalassemic RBC, these animals showed enlarging splenomegaly and require Tx for survival. We have performed two experiments 2 month post engraftment with Hbbth1/th2 FLCs. Two groups were treated for six weeks with two different doses of MH (5.25mg/kg, 2.625mg/kg administered every other day) in absence of Tx. A third group was treated with high dose of MH (5.25mg/kg) and Tx. The animals treated with vehicle showed severe IE and worsening anemia. Compared to these animals, mice treated with MH showed improvement of anemia and RBC morphology. MH significantly increased hemoglobin levels in animals treated with Tx compared to mice treated with Tx alone. At 6 weeks, splenomegaly was significantly reduced by the treatment of MH. In particular, combination of MH and Tx decreased the spleen weight to levels similar to that of wild type mice. This suggests that administration of MH has additive effects in presence of Tx in limiting the splenomegaly. This limits the sequestration of transfused RBC and increases the hemoglobin in circulation. The treatment with MH results in a decrease of serum EPO concentration with a concurrent increase in serum hepcidin compared to controls and animals treated with Tx alone. We are now finalizing our study by analyzing RBC lifespan, hemichrome formation, serum Erythroferrone levels, tissue iron concentration and distribution. In conclusion, these models can be utilized to characterize severe thalassemia phenotypes and new drugs, like MH, that have the potential to ameliorate IE and improve RBC generation.
Seventh Congress of the International BioIron Society Page 171 Poster Abstracts IBIS
Poster #57
THRESHOLD FERRITIN VALUES TO PREDICT CONTROL OF LIVER IRON BURDEN IN THALASSEMIA Ashutosh Lal, MD, Marcela Weyhmiller, PhD and Elliott Vichinsky, MD UCSF Benioff Children's Hospital Oakland Presented By: Ashutosh Lal, MD
Transfusion-dependent (TD) and non-transfusion dependent (NTD) forms of thalassemia are associated with progressive iron overload reflected by an increase in serum ferritin and liver iron concentration (LIC). Despite the adoption of non- invasive methods, measurement of LIC is only available to a minority of patients or limited to annual assessment. Maintenance of low systemic iron usually depends on frequent determination of serum ferritin, a less reliable marker whose level can vary among individuals with similar LIC. We evaluated the relationship between LIC and ferritin in TD, and NTD (including un-transfused (NT) or intermittently transfused (TI)) to determine the validity of average annual ferritin to predict adequate control of liver iron (defined as LIC <5 mg/g dry-liver-weight) as determined by SQUID- biosusceptometry. After excluding individuals with active hepatitis C virus infection, the LIC and ferritin values were available from 115 TD (729 observations), 36 TI (139 observations) and 14 NT (28 observations) individual patients. The median (range) ferritin and LIC were 1,684 (91-14,155) ng/mL and 10.2 (0.14- 59.4) mg/g for TD, 469 (50-4,536) ng/mL and 8.8 (2.4-33.1) mg/g for TI, and 407 (30-1,061) ng/mL and 5.7 (1.3-22.8) mg/g for NT groups, respectively. The slope of the regression line between ferritin and LIC was significantly different between the 3 groups (p=0.019), being lowest in TD, and highest in NT. Receiver operating characteristics analysis showed that in NTD group (NT+TI), a ferritin threshold of 300 and 500 ng/mL failed to correctly classify 21% and 48% of individuals with LIC >5 mg/g, respectively. In contrast, in the TD group, a ferritin of 300 and 500 ng/mL were associated with false negative rate of 0.7% and 3.8%, respectively. In conclusion, transfusions significantly alter the relationship between serum ferritin and LIC, with the slope being steepest in non-transfused individuals. Consequently, LIC can be 50% higher in NTD than in TD for the same serum ferritin level. These relationships should be factored into making decisions about screening or treatment of iron overload in un- transfused or intermittently transfused individuals with thalassemia.
Seventh Congress of the International BioIron Society Page 172 Poster Abstracts IBIS
Poster #58
DIFFERENTIAL OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION RESPONSES INDUCED BY BRAND AND GENERIC SODIUM FERRIC GLUCONATE COMPLEX FORMULATIONS Amy Pai, PharmD¹, Paul Neumann, BS² and Teresa Regis, BS² ¹University of Michigan College of Pharmacy; ²Albany College of Pharmacy and Health Sciences Presented By: Amy Pai, PharmD
Intravenous (IV) iron products are widely used in management of anemia in a wide spectrum of disease state including anemia of chronic kidney disease. IV iron formulations are colloidal suspensions of iron-carbohydrate nanoparticles which makes production of bioequivalent generics challenging. Differences in oxidative stress induction with several generic iron sucrose products compared to the reference listed drug (RLD) have been shown in markets outside the United States. There are no published studies comparing oxidative stress induction and endothelial dysfunction associated with the RLD Ferrlecit® and the only FDA approved generic, sodium ferric gluconate complex (SFGC). Human umbilical vein endothelial cells (HUVECS) were incubated with Ferrlecit® and SFGC in dose response (0, 10 25, 50, 100, 200 mcg/mL for 24 h) and time (0, 0.5, 1, 3, 6, 24 h) studies. Induction of the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of detoxification and heme-oxygenase 1 (HO-1) a Nrf2-regulated cytoprotective enzyme were determined in cell lysate by immunochemistry. Intracellular labile iron (ILI) was determined using the fluorescent probe Phen Green FL. To determine endothelial permeability, rat aortic endothelial cell (RAEC) monolayers were treated with each product at 50 µg/ml for 24 h. Permeability was measured by the clearance rate of Evans Blue-labeled albumin between the luminal and abluminal compartments. Both formulations significantly increased ILI dose dependently. Ferrlecit® treated cells had higher ILI than SFGC at all studied doses except 50 µg/ml (p<0.01). Stabilization and translocation of Nrf2 by Ferrlecit® was greater than SFGC at all doses and was statistically significant at the 100 mcg/mL dose (p<0.05). Similarly, Ferrlecit®-induced upregulation of HO-1 was greater than SFGC at all doses, and was statistically significant at the 100 and 200µg/mL doses (p<0.001). At 24 hours, HO-1 was upregulated by Ferrlecit® and was significantly higher than SFGC (p<0.0004). Incubation of RAEC with both IV iron formulations for 24h at 50µg/ml increased endothelial barrier permeability, which was statistically significant only for Ferrlecit® (p<0.05), compared to untreated controls. Differences in oxidative stress response and endothelial barrier dysfunction between the RLD Ferrlecit® and the generic SFGC were observed which infers limitations may exist in current bioequivalence evaluation methods. These differences may be due, in part, to differences in labile iron release from the iron-carbohydrate nanoparticle. These differences may impact safety and efficacy of these formulations and warrant further exploration.
Seventh Congress of the International BioIron Society Page 173 Poster Abstracts IBIS
Poster #59
BIOEQUIVALENCE EVALUATION OF GENERIC INTRAVENOUS IRON PRODUCTS-IMPLICATIONS OF LABILE IRON RELEASE Amy Pai, PharmD¹, Dan Meyer, PhD², Brian Bales, PhD², Victoria Cotero, PhD² and Manjunath Pai, PharmD¹ ¹University of Michigan College of Pharmacy; ²General Electric Global Research Presented By: Amy Pai, PharmD
Commercially available intravenous (IV) iron formulations are colloidal suspensions of iron oxide nanoparticles (NPs). Small changes in the NP may allow more labile (“free”) iron to be released after injection causing adverse side effects. Thus, bioequivalence (BE) evaluation of generics remains a challenge for non-biologic complex drugs (NBCDs). We evaluated physicochemical characteristics (PCC) and labile iron release in vitro and in vivo to develop a relational model to support BE. We studied Venofer®, Ferrlecit®, generic sodium ferric gluconate complex (NDC 00591-0149-87, Watson Laboratories, Inc), InFeD®, Feraheme® and a pre-clinical investigational formulation GE121333. Sodium ferric gluconate complex (SFGC) is the only FDA approved generic for the reference listed drug (RLD) Ferrlecit®. PCC included; filterable free iron, dynamic light scattering, field flow fractionation/quasi-elastic light scattering (FFF/QELS) and zeta potential. Five published in vivo assay methods for labile iron (LI) determination evaluated for suitability for in vitro application. LI release profiles were studied in vitro in saline and a biorelevant matrix (rat serum) at 0.952 mg/mL simulating Cmax after a 40 mg/kg dose. In vivo plasma LI concentration time profiles (t0-240 min) were then studied in rats after a 40mg/kg IV dose. Several challenges were encountered during PCC that included lack of formulation stability to FFF and non-specific membrane binding during free filterable iron analyses. In vitro LI release in rat serum was similar to in vivo release between the RLD Ferrlecit® and the generic. In vitro LI release in saline was much greater than in serum, especially for InFeD® which has been shown to have low LI release patients receiving this agent. [Pai et al. Pharmacotherapy 2007;27:343-350] This suggests stability of the formulation is reduced, likely due to ionic shielding by sodium chloride, reducing electrostatic repulsion. A modest correlation was observed between the concentration of labile iron released in vitro and Cmax in vivo which suggests an in vitro to in vivo correlation model is possible to develop for application to bioequivalence evaluation. These data indicate bioequivalence evaluation for intravenous iron formulations requires additional studies of best practice methods for physicochemical characterization methods and iron nanoparticle disposition in vitro and in vivo. Funding for this presentation was made possible, in part, by the Food and Drug Administration through grant 1U01FD004889-01 (Pai AB). Views expressed in written materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services, nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government
Seventh Congress of the International BioIron Society Page 174 Poster Abstracts IBIS
Poster #60
EXERCISE-INDUCED DECREASE IN HEPATOCELLULAR IRON OVERLOAD IMPROVES INSULIN SENSITIVITY IN AGED RATS Yuqian Liu¹, Haitao Wang, PhD², Yingjun Zhang² and Yanzhong Chang, PhD³ ¹College of Physical Education; ²College of Physical Education, Hebei Normal University; ³Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University Presented By: Yuqian Liu, PhD
Iron overload is a risk factor for diabetes. Serum Ferritin (SF) concentration，an important index of iron stores, was positively correlated with serum insulin, blood glucose, obesity and other metabolic syndrome. Aerobic exercise is widely used to improve glycometabolism in clinical. Liver is one of the main iron storage organs, and hepatic glycogen plays a key role in glycometabolism. It is unclear that iron overload in the hepatocyte could be decreased by aerobic exercise and as a result, the glycometabolism could be improved. The research aimed to illuminate effects of aerobic exercise on improving insulin sensitivity via decrease iron overload in hepatocytes. Methods: 8 male SD rats, 15-month old, were the control group (CG). 16 male SD rats, 15-month old, with decreased insulin sensitivity and high plasma glucose (induced by High-sugar-lipid fodder for 6 weeks). The HOMA insulin sensitivity index (HOMA-ISI) was 0.0016±0.00015 VS 0.0055±0.00023 in CG. Plasma glucose of rats were 13.42±0.80 mmol/L VS 5.83±0.27 mmol/L in CG. These 16 aged rats were randomly divided into two groups, the high plasma glucose control group (HC) and the high plasma glucose with aerobic exercise Group (HE, run on treadmill at 19 m/min (75%~80%Vo2max), 45 min/d, 6 d/w, 5 w). The content of SF and insulin were examined by ELISA kits. Serum iron, hepatic iron, plasma glucose and the activity of SOD in the liver were examined by reagent kits. The expression of iron transporter hepatic ferroportin 1 (FPN1) was examined by Western Blot. Results and discussions: (1) Compared with HG, the Insulin Sensitivity was increased of HE (HG: 0.0014±0.00028, HE: 0.0041±0.00022, P<0.01), the content of hepatic glycogen was increased in HE (HC:10.64±0.97mg/g, HE:12.49±0.63mg/g, P<0.01). (2) Compared with HC, the content of SF in HE was decreased (HC: 337.43±28.14 ng/ml, HE: 210.95±16.79 ng/ml, P<0.01). The iron store in hepatocytes were decreased in HE than that of HC (HC:48.38±3.76 μmol/g, E 38.58 2.69 μmol/g). (3) The activity of hepatic SOD of E was significantly increased compared with C ( C 30.28±5.31 U/mg, HE: 43.26±3.84 U/mg, P<0.01). (4) The expression of hepatic FPN1 of HE was significantly increased than that of HC (P<0.01). Conclusions: (1) The body iron store was decreased after the aerobic exercise, and the activity of antioxidant was increased, and as a result the oxidative stress and insulin resistance were relieved, and insulin sensitivity was improved. (2) The increased hepatocellular FPN1 after aerobic exercise might play an important role in exporting iron from hepatocyte into circulation, which led to reduced iron overload and improving insulin sensitivity.
Seventh Congress of the International BioIron Society Page 175 Poster Abstracts IBIS
Poster #61
HEPCIDIN-FERRITIN RATIO IS DECREASED IN DIABETES MELLITUS Molly Jacob, MBBS, MD, PhD¹, Padmanaban Venkatesan, MBBS², Joe Varghese, MBBS, MD, DNB², Jithu James, MSc² and Jasmin Prasad, MBBS, MD² ¹Christian Medical College, Vellore, India; ²Christian Medical College, Velllore, India Presented By: Molly Jacob, MD, PhD, MBBS
Background: Increases in body iron stores have been reported to be associated with diabetes mellitus (DM). The mechanisms that link these two conditions are unclear. Hepcidin is the central regulator of iron homeostasis. It is, therefore, conceivable that changes in hepcidin may underlie the association between these conditions. This study was designed to determine if this was so. Methods: Adult males, not previously known to be diabetic, who were referred for medically-indicated estimations of fasting and post-prandial blood glucose levels, were recruited after obtaining informed consent. Anthropometric measurements were also done for each subject. The results of the fasting plasma glucose level were used to categorize subjects as diabetics (diagnosed for the first time) and non-diabetics based on criteria recommended by the American Diabetic Association. The blood sample obtained was used to estimate haematological parameters, glucose, insulin, C- reactive protein (CRP), ferritin, iron, total iron-binding capacity (TIBC) and hepcidin. Homeostatic model assessment (insulin resistance [HOMA-IR]), an indirect measure of insulin resistance, and HOMA-B (an index of pancreatic beta cell function) were calculated (using values for fasting glucose and insulin). Appropriate tests were used for statistical analyses, depending on the distribution of the data obtained. Results: A preliminary analysis was done for 42 subjects in whom biochemical estimations have been completed. The diabetic (n=21) and non-diabetic subjects (n=21) were similar in age and anthropometric measurements. Fasting plasma glucose levels, post-prandial plasma glucose and HOMA-IR values were significantly higher and HOMA-B values significantly lower in the diabetic group. Serum ferritin levels were significantly higher in those with DM (median 147 [IQR 114-251.5] ng/ml) than in non-diabetics (median 79 [IQR 46.65-155.5] ng/ml). Serum hepcidin levels were similar in the 2 groups (controls: median 12.28 [IQR 9.86 - 15.37] vs diabetics: 13.15 (IQR 8.4 1-25.54]). Hepcidin-ferritin ratio was significantly lower in those with DM (0.15 [IQR 0.08 - 0.24]) than in control subjects (0.08 (IQR 0.06-0.12). Other parameters measured (haemoglobin, serum iron, TIBC, transferrin saturation, CRP and insulin levels) were similar in the two groups. On univariate analysis, serum ferritin showed known significant correlations with other parameters of iron metabolism. Fasting plasma glucose levels correlated significantly and positively with serum ferritin and negatively with hepcidin-ferritin ratios. Multivariate analyses showed correlations between serum ferritin and other iron-related parameters (as mentioned above) and a trend towards a negative correlation between fasting plasma glucose levels and hepcidin- ferritin ratio (p = 0.07). Conclusions: Serum ferritin, often used as a marker of body iron stores, was significantly higher in subjects with diabetes mellitus, but hepcidin levels were not. Lower hepcidin-ferritin ratios seen in the diabetics suggest that serum hepcidin levels were inappropriately low for the increased iron stores in these subjects, as indicated by elevated serum ferritin levels. The significance of these observations requires elucidation. This study is ongoing with analyses pending on more patients and also in those with pre-diabetes.
Seventh Congress of the International BioIron Society Page 176 Poster Abstracts IBIS
Poster #62
DIET-INDUCED INSULIN RESISTANCE IN MICE IS ASSOCIATED WITH DECREASED LEVELS OF LIVER IRON AND SERUM HEPCIDIN Joe Varghese, MBBS, MD, Jithu Varghese James, MSc and Molly Jacob, MBBS, MD, PhD Department of Biochemistry, Christian Medical College, Vellore, India Presented By: Joe Varghese, MD
Background: Increased body iron stores have been reported to be associated with increased incidence of type 2 diabetes mellitus (T2DM). The mechanisms that link insulin resistance (IR), the hallmark of T2DM, and iron homeostasis are not clearly understood. It is not known whether increased body iron stores are a cause or consequence of IR. There is very little information on the effect of IR on hepcidin, the central iron-regulatory hormone. In addition, it is not known whether IR affects iron distribution in the body. In view of this, a time-course study was carried out to determine the interplay between IR and iron homeostasis, using a mouse model of diet-induced obesity as a model of T2DM. Methods: Male C57Bl/6 mice were fed a high-fat diet (HFD) or control diet for various time periods (up to 24 weeks). Glucose and insulin tolerance tests were done to monitor development of glucose intolerance and IR. Subsets of mice were euthanized after 4, 8, 12, 16, 20 or 24 weeks of feeding. Western blot analyses and qPCR were done to determine expression levels of various iron-related parameters in the liver. Iron content of various tissues was visualized, using perfusion Perls’ Prussian blue staining. Iron content was also measured in the liver, spleen, gonadal white adipose tissue (gWAT) and skeletal muscle. Serum levels of hepcidin, insulin and interleukin-6 (IL6) were estimated. Maturation of erythroid precursors (Ter119+ cells) in the bone marrow was assessed by flow cytometry and expression of erythroid regulators of hepcidin in TER119+ cells was determined by qPCR. Results: HFD induced obesity, hepato-steatosis and IR in mice; these changes were seen from 8 weeks of HFD feeding and were maximal at 20 and 24 weeks. Serum hepcidin levels and hepatic Hamp1 mRNA expression were significantly lower at 20 and 24 weeks of HFD. There was a progressive decrease in hepatic iron levels from 12 weeks of HFD, as indicated by decreased tissue iron content, decreased iron staining after in situ Perls’ perfusion, increased hepatic mRNA expression of transferrin receptor and decreased protein levels of ferritin. Serum hepcidin levels and liver hepcidin mRNA expression were positively correlated with liver iron levels. There were no significant differences between HFD and control diet-fed mice, with regard to serum iron and IL-6 levels, and iron content of adipose tissue, skeletal muscle and spleen at any of the time points studied. HFD also did not significantly affect maturation of erythroid precursors in the bone marrow or gene expression of putative bone marrow-derived erythroid regulators of hepcidin (erythroferrone, growth differentiation factor 15 and twisted gastrulation factor 1). Conclusions: High-fat feeding induced IR and dysregulation of iron homeostasis in a mouse model of T2DM. Onset of IR (at 8 weeks) appeared to precede the onset of iron dysregulation (at 12 weeks). HFD resulted in decreased liver iron levels, which may be responsible for observed suppression of hepatic hepcidin. There was no evidence to suggest re-distribution of iron from the liver to extra-hepatic sites. The mechanisms that underlie decreased liver iron stores in this model warrant further investigations.
Seventh Congress of the International BioIron Society Page 177 Poster Abstracts IBIS
Poster #63
HYPERFERRITINEMIA INCREASES THE RISK OF HYPERURICEMIA IN PATIENTS WITH GENETIC HEMOCHROMATOSIS RELATED TO P.CYS282TYR MUTATION IN THE HFE GENE Jeremy Flais, MD², Edouard Bardou-Jacquet, MD, PhD³, Yves Deugnier, MD³, Guillaume Coiffier, MD², Aleth Perdriger, MD, PhD², G rard Chal s, MD², Martine Ropert, MD⁴, livier Loreal, MD, PhD¹ and Pascal Guggenbuhl, MD, PhD² ¹INSERM UMR ; ²Department of Rheumatology; ³Liver Disease Unit; ⁴Department of Biochemistry, Rennes University Hospital, France Presented By: Olivier Loreal, MD, PhD
Background: Hyperuricemia is becoming increasingly frequent in the population, and is known to favor the occurrence of gout. The iron metabolism may interact with the uric acid metabolism. Aim: Our objective was to investigate the relationships between the serum uric acid and serum ferritin levels in a cohort of hemochromatosis patients who were homozygous for the HFE p.Cys282Tyr mutation. Methods: 738 patients exhibiting an homozygous p.Cys282Tyr mutation in the HFE gene, identified during the diagnosis procedure or familial screening, were included in this retrospective study. Collected bioclinical variables included sex, age, body mass index (BMI), alcohol intake, CRP, glycaemia, transaminases, GGT, ferritinemia, serum iron and transferrin saturation levels, and hepatic iron concentration (biochemical quantification or MRI). The bioclinical variables measured during the initial evaluation were compared in univariate analysis by Student’s t test. In multivariate analysis, linear stepwise regression was used. Results: Two groups of patients, with (n=158) and without (n=574) hyperuricemia were identified. In the group of hyperuricemic patients, the serum ferritinemia level was significantly higher than in the group of non-hyperuricemic patients (1576.7 ± 1387.4 ng/l vs. 1095.63 ± 1319.24 ng/l, p< 0.005). With multivariate analysis, only ferritin and BMI independently explained the uricemia (R2= 0.258) after adjustment for age, glycaemia and CRP levels. The correlation between uricemia and log (ferritin) with partial regression correlation coefficients was 0.307 (P < 0.01). Discussion: In a population of patients homozygous for the HFE gene mutation, an increase in uricemia was associated with the increase in ferritin level. This was observed independently of factors commonly associated with hyperuricemia. Knowing the antioxidant potentiality of uric acid, its increase associated with hyperferritinemia, could participate to a physiological response against visceral toxicity of iron linked to oxidative stress. Conclusions: Our study suggests that an increase of uricemia is associated with the ferritin increase in patients homozygous for the HFE p.Cys282Tyr gene mutation. The evaluation of mechanisms involved in this association and the impact of uricemia increase will require further studies. References: Flais J. et al. Joint Bone spine, 2016
Seventh Congress of the International BioIron Society Page 178 Poster Abstracts IBIS
Poster #64
HEPCIDIN KNOCK-OUT MICE DEVELOP LESS INSULIN RESISTANCE THAN WILD-TYPE MICE WHEN FED A HIGH-FAT DIET Jithu James, MSc, Joe Varghese, Sophie Vaulont and Molly Jacob Christian Medical College, Vellore, India Presented By: Jithu James, MSc
Introduction: Increased body iron stores and inflammation in adipose tissue are known to play key roles in the pathogenesis of insulin resistance (IR). It is not clearly known how these factors interplay in the development of IR. Hepcidin is the central regulator of iron homeostasis. Mice in which hepcidin is knocked out develop iron over-load. We hypothesized that such mice would be more susceptible to development of IR and inflammation in adipose tissue than wild-type mice when fed a high-fat diet (HFD). Methods: Male hepcidin knockout (Hamp1-/-) and wild-type (WT) mice, on a C57BL/6 background, were maintained on a HFD (60% of calories from fat) for 24 weeks. Development of IR was assessed by insulin tolerance test (ITT) and calculation of homeostatic model assessment–insulin resistance (HOMA-IR) index. Stromal vascular fractions (SVF) were isolated from epididymal adipose tissue from the mice and used to study macrophage infiltration in adipose tissue and their M1/M2 phenotype, using flow cytometric analysis. Adipose tissue iron content and ferritin protein levels were also determined. Results: Hamp1-/- mice on HFD gained less weight (41.5 + 1.4 g) than WT on HFD (46.3 + 1.4 g) (p=0.045). They demonstrated less insulin resistance (area-under-the-curve for ITT 44.1 ± 2.4 in Hamp1-/- vs. 54.2 ± 2.7 in WT, p=0.054) than HFD-fed WT mice. These mice also had lower serum insulin levels (0.59 + 0.07 µg/L in Hamp1-/- vs 1.01 + 0.58 µg/L in WT, p=0.006) and values for HOMA-IR index (10.5 + 2.0 in Hamp1-/- vs. 27.4 + 3.4 in WT, p=0.006) than their WT counterparts. Hamp1-/- mice on HFD showed higher adipose tissue iron content (1.03 + 0.1 µg/mg tissue in Hamp1-/- vs 0.58 + 0.1 µg/mg in WT, p=0.002) and ferritin protein levels than WT counterparts on HFD. Flow cytometric analyses showed significantly fewer macrophages (F4/80+ CD11b+ cells) in adipose tissue of Hamp1-/- HFD mice than in WT-HFD mice. Furthermore, these macrophages from Hamp1-/--HFD mice demonstrated a higher M2 (CD11c- CD301+) phenotype than macrophages from WT mice. Discussion and Conclusions: In response to high-fat feeding, Hamp1-/- mice were found to develop less insulin resistance than WT mice. Macrophage infiltration of adipose tissue in the mice was less than in WT mice, with the infiltrating cells being of M2 macrophage phenotype, which is anti-inflammatory in nature. This suggests decreased adipose tissue inflammation. This observation may underlie the decreased insulin resistance seen in these mice. However, further investigations are required to delineate the molecular and cellular mechanisms involved.
Seventh Congress of the International BioIron Society Page 179 Poster Abstracts IBIS
Poster #65
PROTEIN LEVELS OF IRON HOMEOSTASIS RELATED MOLECULES IN SKELETAL MUSCLE TISSUE IN VEGAN POPULATION Kamila Balusikova, Jan Gojda, David Albrecht, Jana Patockova, Jan Kovar and Michal Andel 3rd Faculty of Medicine, Charles University Presented By: Kamila Balusikova
Food consumed without any limitation leads to higher body iron stores when compared with non-animal (vegan) food. Since body iron stores positively correlates with the intake of especially red meat together with the fact that higher iron stores positively correlate with insulin resistance it can be assumed that the lower iron uptake in vegans is one of the factors increasing insulin sensitivity. Therefore, we tested whether iron homeostasis and overall cell conditions (e.g. level of oxidative and endoplasmic reticulum stress) are affected in skeletal muscle tissue (important for glucose metabolism) in vegan population when compared with non-vegan population. Our study included 24 vegans and 25 corresponding controls. Exclusion criteria were age under 18 years and the presence of any chronic disease with an impact on the intermediary metabolism. Each subject underwent a collection of peripheral blood under basal conditions and skeletal muscle biopsy. Following parameters of iron metabolism and glucose homeostasis were determined in serum: hepcidin, prohepcidin, ferritin, iron binding capacity, transferrin, serum iron level, blood glucose, insulin and C-peptide. Tissue samples were processed for total protein isolation and protein levels were analyzed using western blot analysis. A significant difference was found between the group of vegans and controls in the levels of hepcidin, prohepcidin and glucose. In vegan group, measured levels were decreased for all parameters. Other iron metabolism and glucose homeostasis parameters differences were off significance. On protein level, significant differences were found for molecules of iron homeostasis (ferroportin, ferritin, transferrin receptors, iron regulatory proteins) as well as for both oxidative stress (superoxid dismutases, glutathion reductase) and endoplasmic reticulum stress (calnexin, PDI, BiP, CHOP) markers in muscle cells. Again, protein levels of tested molecules were decreased in vegans when compared to control group. Our results shows that vegans compared with the control group present lower serum hepcidin and prohepcidin. This corresponds to an effort of the organism to increase the intake of iron through enterocytes, but also increased release of iron into circulation at the level of individual cells. This is with agreement with our results of protein analysis of iron homeostasis molecules in muscle cells. Probably as a result of lower iron levels, muscle cells in vegans manifest also lower level of oxidative and endoplasmic reticulum stress. These facts, together with the known negative association between vegan diet and insulin resistance may indicate that lower dietary intake of iron assists to the reduction of cell injury and insulin resistance development. This study was funded by research programs of Charles University, PRVOUK-P31 and UNCE 204015, and was supported by a Grant of the Ministry of Health of the Czech Republic, number NT/14416.
Seventh Congress of the International BioIron Society Page 180 Poster Abstracts IBIS
Poster #66
THE DIVERSE ROLES OF SIDEROCALIN / NGAL / LCN2 IN ANTI-BACTERIAL IRON SEQUESTRATION, CANCER, AND CLINICAL APPLICATION Colin Correnti, PhD, Peter Rupert, PhD and Roland Strong, PhD Fred Hutchinson Cancer Research Center Presented By: Roland Strong, PhD
Siderocalin, the archetype of a family of highly-conserved mammalian proteins, acts to slow bacterial infections by binding to and sequestering iron as ferric complexes with bacterial siderophores. Siderocalin’s recognition mechanism, detailed through exhaustive structural studies, enables binding of a wide range of siderophores, though successful pathogens use modified siderophores to evade Siderocalin binding. Avian analogues of Siderocalin demonstrate the general effectiveness of siderophore-depletion as an antibacterial defense. Siderocalin is also implicated in a wide range of physiological responses, including normal iron homeostasis, apoptosis, tumorigenesis, and metastasis, though confirming proposed mechanisms and identifying affected molecular pathways has remained challenging. Many proposed activities, candidate endogenous siderophores, and possible cell-surface receptors have been subsequently disproved, though copious indirect evidence continues to build supporting many pleiotropic roles likely involving alternate receptors and endogenous siderophore-like molecules. Siderocalin’s interactions with its only confirmed endogenous receptor, Megalin, are now being detailed at the molecular level. Siderocalin also possesses many unique biochemical properties that enable its use as a molecular tool, for generally improving target protein expression and simplifying purification, and for the delivery and transport of a variety of elements, including actinides, enabling a variety of diagnostic and therapeutic applications.
Seventh Congress of the International BioIron Society Page 181 Poster Abstracts IBIS
Poster #67
THE ROLES OF THE THREE IRON-STORAGE PROTEINS OF ESCHERICHIA COLI IN SUPPORTING LOW-IRON GROWTH AND REDOX STRESS RESISTANCE Afrah Salman, PhD student and Simon Andrews, Professor University of Reading Presented By: Ahmad Salman, MBBS, MCOS
Organisms must balance their iron requirement to achieve iron homeostasis. This occurs through several mechanisms, including sequestration of excess cellular iron by iron-storage proteins. Such proteins (‘ferritins’) act as an iron source under iron restriction; they also counter redox stress imposed by excess cellular iron that might contribute to Fenton chemistry. In Escherichia coli, there are three iron-storage proteins: ferritin A (FtnA), bacterioferritin (Bfr) and the ‘DNA- protection during starvation’ (Dps). Experiments were conducted to further investigate their respective functions and to determine whether these proteins are mutually interchangeable with each other, i.e. can one of these iron-storage proteins be used in place of any of the other two. This aim was progressed by first generating a triple mutant of the corresponding iron-storage genes (B 25113 ftnA dps bfr); this mutant was then complemented separately with dps, ftnA and bfr, each under the control of a rhamnose-inducible promoter in order to divorce the physiological function of these proteins from any expression-related (e.g. iron, redox stress and growth phase) influences. Triple mutants of the corresponding genes were constructed by P1 transduction and confirmed by colony PCR. The phenotypic status of FtnA, Bfr and Dps production was further confirmed by Western blot. The gene fragments of interest were amplified using PCR from wild type chromosomal DNA; complementation was confirmed by Western blot. The complemented triple mutant was pre-cultured in minimal medium (M9) in the presence of excess iron (20 µM) to allow iron stores to be deposited, with/without the inducer (rhamnose), and were then subsequently grown in M9 under iron restriction (0.5 µM of the chelator, DTPA [diethylenetriamin pentaacetic acid]) or redox-stress conditions (200 µM of hydrogen peroxide). Growth under each condition was monitored over 24 h using a Bioscreen. The results showed that Dps and Bfr pre-induction enhances growth (6.5- and 8-fold, respectively) of the triple mutant in the presence of the hydrogen peroxide, indicating that both these iron-storage proteins have capacity to provide resistance against peroxide-induced stress. However, FtnA pre-induction had virtually no effect on growth with hydrogen peroxide and so, in contract, does not appear to provide resistance to redox stress. Pre-induction of Bfr increased growth (~2.5-fold) under iron restriction with DTPA; however, neither Dps nor FtnA pre-induction caused any notable growth advantage under Fe-restriction. Thus, somewhat surprisingly, only Bfr acted to provide an apparent iron source that could promote iron-restricted growth. However, the expression levels achieved for FtnA were far less than those for Bfr and Dps, despite all three corresponding genes being under control of identical promoters. Subsequent studies showed that this weak FtnA expression effect appears to be due to rapid turnover of the protein through ‘N-end rule dependent’ degradation. Indeed, replacement of the second FtnA amino acid (L A/ ) resulted in a 10-fold increase in FtnA levels. Thus, FtnA function as an iron-storage protein might be partly related to its susceptibility to rapid turnover by the ClpS-AP system through mediation of iron release. Current work is focusing on the role of ClpS-AP, and other proteases, in FtnA function as an iron source, and on the ability of ferritins/bacterioferritins/Dps-proteins from diverse sources to complement iron-storage defects in E. coli.
Seventh Congress of the International BioIron Society Page 182 Poster Abstracts IBIS
Poster #68
A STUDY OF NCOA4 EXPRESSION AND BINDING TO FERRITINS Magdalena Gryzik, PhD student, Fernando Carmona, PhD, Maura Poli, PhD and Paolo Arosio, PhD Department of Molecular and Translational Medicine, University of Brescia, Italy Presented By: Magdalena Gryzik
Ferritin stores and detoxifies cellular iron by sequestering up to 4000 Fe atoms in its large cavity. The recycling of this iron is crucial and it involves the Nuclear Receptor Coactivator 4 (NCOA4) that acts as a selective cargo receptor mediating ferritin autophagy (ferritinophagy). NCOA4 is a 70 kDa protein of 614 amino acids, predominantly localized in the cytoplasm, involved in regulation of nuclear hormone receptors, microtubule activity, transcription and ferritinophagy. The aim of the study is to analyze NCOA4 expression in HeLa cells under different cellular conditions, and to characterize NCOA4-ferritin interaction in vitro. HeLa cells were treated with 100µM of the chelator desferoxamine (DFO) or 100µM of ferric ammonium citrate (FAC) for 24h and the effect of treatments was confirmed by change of FTL and TFR1 levels in western blot. We observed significant 20% decrease of NCOA4 transcript level after both the DFO and FAC treatments. Oxidative stress was induced in HeLa cells by treatment with 50µM and 100µM of hydrogen peroxide (H2O2) for 24h. NCOA4 mRNA expression decreased to 75% after treatment with 100µM H2O2 in HeLa cells. On the other hand, treatments with 5µM of tamoxifen and with 10mM of ammonium chloride for 24h to induce autophagy and to inhibit lysosome activity respectively, did not affect NCOA4 level. Next, we cloned and expressed in E. coli the recombinant NCOA4 domain (383-522aa), known to be involved in NCOA4-FTH interaction. When added to H-ferritin, it strongly retarded its mobility in nondenaturing-PAGE, while did not affect L-ferritin mobility. The affinity binding was confirmed by an ELISA assay in which the his-tagged NCOA4 domain was incubated with solid phase ferritins. The recombinant NCOA4 domain bounds the H-ferritin with a characteristic saturation curve, but did not bind L-ferritin. Next, we found that Arg23 to Ala mutagenesis of FTH resulted in complete suppression of NCOA4-ferritin interaction both in Native-PAGE and ELISA analyses, confirming Arg23 to be a key residue for NCOA4 binding. We also found that the NCOA4-FTH interation resisted up to 6M urea. The ELISA assay was used to analyze the impact of various factors on the NCOA4 (383-522)- FTH interaction in vitro. We found that iron(II) prevents the interaction in a dose-dependent manner and that the effect is counteracted by the addition of iron chelator. Other divalent metals (Cu(II), Mn(II), Zn(II), Mg(II), Ca(II)) and Fe(III) did not inhibit the interaction. We also found that NCOA4-FTH binding affinity is pH dependent in the range 5-7, with the highest association at pH 6, whereas at pH 3-4 the binding was abolished. In conclusion, we found that NCOA4 expression is regulated by iron and H2O2 in HeLa cells, and that NCOA4 domain (383-522aa) has a strong H-, but not L-ferritin binding activity, that is iron (II) and pH dependent and involves ferritin Arg23. Our results confirm NCOA4 interaction with H-ferritin and allow us to propose new model of ferritinophagy regulation by iron level and oxidative stress at NCOA4 trancript level, as well as iron-mediated inhibition of NCOA4-ferritin binding.
Seventh Congress of the International BioIron Society Page 183 Poster Abstracts IBIS
Poster #69
THE HYDROPHOBIC IRON CHELATOR DESFERRIOXAMINE-CAFFEINE, DFCAF Jesus Antonio Alvarado Huayhuaz, Hector Aguilar Vitorino, Othon Souto Campos, Silvia Helena Pires Serrano, Telma Mary Kaneko and Breno Esposito University of Sao Paulo Presented By: Breno P. Esposito
The extreme aqueous solubility of desferrioxamine (DFO) results in high clearance rates and the need for prolonged, intravenous application rendering its application troublesome for the patient. In recent years we developed DFCAF, a caffeine derivative of DFO, as a means to improve cell permeability in our search to repurpose approved drugs by minimal modifications with nontoxic conjugating agents. The application of DFCAF as a carrier of toxic metals to target microrganisms (fungus and bacteria) and the docking of Fe(DFCAF) to the ferrichrome transporter FhuA will be presented.
Seventh Congress of the International BioIron Society Page 184 Poster Abstracts IBIS
Poster #70
PHARMACOKINETICS (PK) OF FERRIC PYROPHOSPHATE CITRATE (TRIFERIC): MODELING DIURNAL IRON AND ADDITIVITY OF TRIFERIC IN HEALTHY VOLUNTEERS IRON Raymond Pratt, MD¹, Mark Bush, PhD², Scott Brantley, PhD² and Ajay Gupta, MD¹ ¹Rockwell Medical Inc; ²Nuventra Inc. Presented By: Raymond D. Pratt, MD
Ferric pyrophosphate citrate (FPC, Triferic) is a soluble carbohydrate-free complex iron salt approved as an iron replacement product. FPC is added to hemodialysate to maintain hemoglobin in patients receiving chronic hemodialysis. In healthy subjects, endogenous serum iron concentrations undergo a profound diurnal variation that complicates estimation of PK parameters. The objective of this study was to evaluate the PK of FPC by simultaneously modeling the diurnal profile of endogenous serum iron and the contributions of FPC iron administered by slow IV infusion or by bolus IV injection. A secondary objective was direct measurement of serum non-transferrin bound iron and labile plasma iron. Healthy volunteers were administered no treatment (Basal), FPC 6 mg iron IV over 3 hours, or FPC 35 mg Fe/kg IV bolus over 1 minute. All observations started at 08:00 hours (time 0). FPC administered IV was well tolerated. The diurnal variation of basal serum iron concentrations was modeled with a circadian cosine-driven function. The endogenous iron pattern was well-characterized by the model with the highest serum iron concentrations at ~8AM and the lowest at ~8PM (Figure 1). FPC by IV infusion or IV bolus was assumed to be additive with the basal circadian iron profile. By accounting for the diurnal pattern, the compartmental model provides better estimates of Clearance (CL) and Volume of Distribution (Vd) than non-compartmental methods. The resulting clearance (CL) and distribution volume (Vd) estimates were 0.92 L/hr. and 5.3 L, respectively. No circulating non transferrin bound iron (NTBI) or labile serum iron (LPI) was detected at any time. Conclusions: FPC iron concentrations in healthy volunteers can be modeled as additive to the circadian basal iron concentrations. The distribution of Triferic iron administered IV was slightly greater than plasma volume. Together with total unsaturated iron binding capacity, these results allow estimation of total body exogenous iron-binding capacity. This further allows determination of the maximal IV bolus dose of FPC iron that will not exceed the total iron-binding capacity. This approach may permit bolus administration of FPC iron as an alternate dosing regimen when addition of FPC to the dialysate is not feasible.
Figure 1: Baseline Serum Iron Values Fit to the Model
Seventh Congress of the International BioIron Society Page 185 Poster Abstracts IBIS
Poster #71
CRISPR-CAS9 GENOME-WIDE KNOCKOUT SCREENING IDENTIFIES MOLECULAR COMPONENTS INVOLVED IN CELLULAR IRON UPTAKE Amin Sobh, Alex Loguinov, PhD and Chris Vulpe, MD, PhD University of California, Berkeley Presented By: Amin Sobh
Iron uptake by erythrocyte precursors is an indispensable process for erythropoiesis. Under normal physiological conditions, erythroid cells acquire iron mainly through the uptake of transferrin-bound iron (TBI), a well-characterized process that involves the transferrin receptor (TfR). Alternatively, erythroid cells have been shown to take up non- transferrin bound iron (NTBI) which exists in the circulation under conditions of iron overload and can be accumulated as a redox-active labile iron. ncontrolled TB uptake by erythroid precursors in β-thalassemia is suggested to underlie premature destruction of erythroid precursors resulting in ineffective erythropoiesis (IE). Comprehensive elucidation of molecular pathways governing and regulating the cellular acquisition of either TBI or NTBI is required to better understand physiological as well as pathological processes that interfere with cellular iron homeostasis and would shed light on novel therapies that can be used in iron-related disorders. In this study, we employed a functional approach to identify cellular components involved in the uptake of TBI and NTBI by human erythroid cells. We performed a genome-wide loss of function screen using the CRISPR-Cas9 system in human K562 erythroleukemic cells, which can utilize either TBI or NTBI as an essential iron source for proliferation. We generated a pooled genome-wide library of mutant K562 cells by global gene disruption using the CRISPR-Cas9 knockout tool. The mutant cell library was screened to identify mutants that exhibit a growth disadvantage when either TBI or NTBI is the sole iron source in the culture medium. We identified known genes involved in cellular iron uptake, including TFRC in TBI uptake, which suggested that our screening protocol was effective. In addition, we identified multiple candidate genes that are potentially involved in either TBI or NTBI cellular acquisition. The top candidates included gene products involved in intracellular trafficking, cell surface molecules, metal transporters and signaling factors that have not been previously linked to cellular iron uptake. Our work established novel links between cellular iron homeostasis and multiple cellular processes.
Seventh Congress of the International BioIron Society Page 186 Poster Abstracts IBIS
Poster #72
DNA METHYLATION REGULATES THE EXPRESSION OF TRANSFERRIN RECEPTOR 2 Paul Sharp¹, Robert Weeks² and Ian Morison² ¹King's College London; ²University of Otago, New Zealand Presented By: Paul A Sharp
Transferrin receptor 2 (Tfr2) is a key iron sensing gene in hepatocytes, and also plays and important role in controlling haematopoiesis. Two Tfr2 variants have been identified, and are differentially expressed in various tissues. Tfr2 is the predominant isoform in liver and mononuclear cells, whereas Tfr2β, a truncated isoform lacking the membrane spanning domain is highly expressed in spleen and T-cell leukaemia cells (Kawabata et al J. Biol. Chem. 1999, 274:20826-32). The aim of our study was to understand the basis for the tissue-specific expression of Tfr2 isoforms. An inspection of the 5’ promoter region of both gene variants revealed a number of CpG dinucleotides. We therefore hypothesized that DNA methylation regulates the expression of Tfr2 in different tissues. Experiments were performed on HepG2 and Huh7 human hepatoma cells, K562 human erythro-leukaemia cells, and Jurkat T-cell leukaemia cells. DNA was isolated from each cell line and subjected to bisulphite conversion. ragments of the 5’ promoter regions of both Tfr2 variants were PCR amplified using bisulphite specific primers. Each amplicon contained a methylation-specific restriction digestion site which would be cut if the sequence was methylated. Aliquots of amplicons from each cell line were further amplified in E. coli using a TOPO cloning vector. DNA was isolated and sent for sequencing. To determine whether methylation had influenced Tfr2 mRNA expression, cells were treated with 5-aza-2’-deoxycitidine (AZA, 5 µM) for 72 hours; medium containing AZA was replaced every 24 hours. Tfr2 isoform mRNA was measured by Q-PCR. or Tfr2 , agarose gel electrophoresis following restriction digestion showed only a single product for HepG2 and K562 cell DNA indicating that the gene promoter was unmethylated; two distinct products were identified in Huh7 and Jurkat cells indicating (partial) methylation of the promoter. Sequencing data confirmed these finding. In contrast, electrophoresis indicated that there was methylation of the Tfr2β promoter in 562 and uh7 cells. nterestingly, these data indicate that there was a differential pattern of Tfr2 methylation in the two human hepatoma cell lines. We therefore assessed whether this influenced Tfr2 mRNA expression in the two cell lines, and whether treatment with the demethylating agent AZA altered m A expression. Basal expression of Tfr2 was 3.4-fold higher in HepG2 cells compared with Huh7 cells (P<0.02); however, there was no significant difference in Tfr2β levels. A A increased expression of both Tfr2 (3.2-fold; P<0.01) and Tfr2β (5.1-fold; P 0.001) in uh7 cells, but only modified Tfr2β expression (2.6-fold; P<0.001) in HepG2 cells. These findings are consistent with our DNA sequencing data and support our hypothesis that methylation, at least in part, regulates the expression of Tfr2 gene variants. Acknowledgement: This work was supported by funding from the Dunedin School of Medicine, University of Otago.
Seventh Congress of the International BioIron Society Page 187 Poster Abstracts IBIS
Poster #73
LC-ICP-MS CHARACTERIZATION OF NON-TRANSFERRIN-BOUND IRON Nathanial Dziuba, BS, Joanne Hardy, PhD, DVM and Paul Lindahl, PhD Texas A&M University Presented By: Paul Lindahl, PhD
After nutrient iron passes through enterocytes in the duodenum and into the blood, the vast majority binds to transferrin. Non-transferrin-bound iron (NTBI) is thought to be composed of one or more low-molecular-mass non-proteinaceous Fe complexes in blood plasma. The collective concentration of NTBI is very low in healthy individuals but higher in those with Fe-overload diseases. NTBI is toxic and accumulates in the liver. Despite major advances in characterizing NTBI, the number of NTBI species, their chemical identities, and exact physiological properties remain largely unknown. NTBI is typically detected using a chelator-based assay, but this destroys the NBTI species as an inherent part of detection. The goal of this project is to develop a chromatography-based assay for NTBI that leaves the metal complex(es) intact to allow downstream characterization. Toward this end we have built a liquid chromatography system within a refrigerated inert- atmosphere glove box and interfaced it on-line to an inductively coupled plasma mass spectrometer. This LC-ICP-MS system allows low-mass metal complexes to be speciated according to molecular mass, and quantified in terms of absolute concentration in the plasma. Using this system, we have detected numerous NTBI candidate species that we are further characterizing. In a more advanced experiment, catheters are surgically implanted in the portal vein and caudal/cranial vena cava of Fe-deficient pigs, and a feeding tube is installed into the stomach. This setup allows us to characterize the trafficking of nutrient iron up to the point at which it binds transferrin. At the beginning of the experiment, aqueous 57Fe(II) is injected into the stomach, and blood samples are removed from both catheters at increasing times. Plasma samples are examined using the LC-ICP-MS system. Approximately 94% of the Fe in the pig is 56Fe, such that this experiment allows the pathway of nutrient 57Fe absorption to be traced. Using this approach, we have detected a number of low-molecular-mass Fe species as well as 57Fe-labeled transferrin. These species are currently being characterized. The role of the liver in NTBI trafficking, under the conditions of the experiment, has also been assessed. Our approach has a rich capacity to explore mammalian Fe trafficking involving NTBI, transferrin and the liver at an unprecedented level of detail.
Seventh Congress of the International BioIron Society Page 188 Poster Abstracts IBIS
Poster #74
ERYTHROID CELL MITOCHONDRIA RECEIVE ENDOSOMAL IRON BY A “KISS-AND-RUN” MECHANISM Amel Hamdi, PhD¹, Daniel Garcia-Santos, PhD¹, Tariq Roshan, MD¹, Alex Sheftel, PhD² and Prem Ponka, MD, PhD¹ ¹McGill University, Lady Davis Institute, Montreal, Canada; ²Spartan Bioscience Inc, Ottawa, Canada Presented By: Amel Hamdi, PhD
In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. An opposing view is that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (“kiss-and-run” hypothesis; Ponka Blood 89:1, 1997). Despite the longevity of the prevailing opinion, experimental evidence (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007) only supports the latter hypothesis. Using 3D live confocal imaging of reticulocytes following their incubation with MitoTracker Deep Red (MTDR) and Alexa Green Transferrin (AGTf), we have demonstrated transient endosome-mitochondria interactions. We have also documented these interactions by a novel method exploiting flow sub-cytometry to analyze reticulocyte lysates labeled with MTDR and AGTf. We have thus identified a population of particles labeled with both fluorescent markers, representing endosomes interacting with mitochondria. FACS followed by 2D confocal microscopy confirmed the association of both organelles in the double-labeled population (Hamdi A et al. BBA Molecular Cell Research 2859:2867, 2016). In the current study, we examined whether reticulocyte mitochondria interact with transferrin (Tf) in a cell-free system. Lysates of reticulocytes previously labeled with MTDR were incubated with AGTf for various time intervals. Examination of lysates by 2D confocal microscopy revealed a time-dependent increase in the number of mitochondria in contact with fluorescent Tf. This can be prevented by the presence of excess, unlabeled Fe2-Tf, but not by albumin. In addition, we demonstrate that endosomes containing mutated recombinant holotransferrin, which cannot release iron, remain associated with mitochondria, while endosomes containing mutated recombinant apotransferrin, which cannot bind iron, are not associated with mitochondria. Our findings indicate that endosomes containing holo-Tf promote their attachment to, and drive the detachment of apo-Tf-endosomes from, mitochondria, respectively. By co- immunoprecipitation assay (from murine eryhroleukemia [MEL] cells and reticulocytes lysates), we purified the voltage- dependent anion channel 2 (VDAC2), which is located at the outer membrane of the mitochondrion with DMT1. We confirmed the colocalization of VDAC2 and DMT1 in MEL cells and reticulocytes by both immunofluorescence and confocal microscopy. Moreover, we found a significant decrease in the number of mitochondria in contact with Tf- endosomes after depletion of VDAC2 in MEL cells or after treatment of reticulocyte lysates with the mitochondrial uncoupler CCCP, further supporting the concept of a physical interaction between endosomes and mitochondria. To examine a possible role of VDAC2 in iron trafficking, we depleted MEL cells of VDAC2 or inhibited VADC2 using erastin (a specific VDAC2 inhibitor that alters its gating) followed by the measurement of 59Fe incorporation from 59Fe-Tf into heme. Our finding of decreased 59Fe incorporation into heme of MEL cells with silenced or inhibited VDAC2 supports the idea that this outer-membrane mitochondrial protein is involved in the interaction of endosomes with mitochondria. We are currently continuing to delineate the molecular mechanisms involved in endosome-mitochondria interactions focusing on the “signal(s)” that direct iron-carrying endosomes towards mitochondria, the players involved in the docking of endosomes to mitochondria and the “signal(s)” that determine the detachment of iron-free endosomes from mitochondria.
Seventh Congress of the International BioIron Society Page 189 Poster Abstracts IBIS
Poster #75
SPECIATION OF NON-TRANSFERRIN BOUND IRON IN BLOOD PLASMA OF IRON DEFICIENT SWINE Nathaniel Dziuba, Joanne Hardy and Paul A. Lindahl Texas A&M University Presented By: Nathaniel Dziuba, BA
Non-transferrin bound iron (NTBI) can be operationally defined as iron-containing species in blood plasma that have molecular masses less than 30 kDa. NTBI is implicated in damaging the liver and other tissues throughout the body, and is primarily relevant in iron-overload disorders. NTBI was first discovered over 35 years ago, however, much remains to be learned with regard to the composition of this pool in vivo. Additionally, little is known as to the pathway of NTBI formation or the species generated therefrom. The current model of iron transport through enterocytes presumes that iron is released into the portal vein as an NTBI species. The liver removes NTBI from the portal vein supply, reducing the levels observed in the caudal vena cava. We have developed an experimental approach using isotopically-labeled dietary iron to probe the trafficking of such iron in the blood plasma, as well as the role of the liver in this process. We surgically implant catheters in the portal vein and caudal vena cava (or cranial vein) in iron deficient swine. A feeding tube is implanted in the stomach, and is used to administer a reduced 57Fe solution. Blood samples from each catheter are removed periodically. Blood plasma from each sample is filtered through a 10 kDa molecular cutoff membrane or through a 0.45 uM syringe filter and then run through our novel anaerobic and chilled liquid chromatography system that is interfaced to an inductively coupled mass spectrophotometer system. Filtered plasma solutions are passed down a size- exclusion column and sent to the ICP-MS system in real time. This approach allows us to determine the extent of transferrin loading dietary iron, as well as detect NTBI species and quantify their concentrations in the blood. This research may be the first of its kind to speciate endogenously-generated NTBI using a non-destructive method while determining the approximate molecular mass and individual specie concentration. The role of the liver in NTBI absorption can be assess by comparing results obtained from samples collected from the portal vein catheter vs. the caudal vena cava catheter. We are using this approach to test the current mechanism of how nutrient iron is trafficked by enterocytes to transferrin, and will present our latest results.
Seventh Congress of the International BioIron Society Page 190 Poster Abstracts IBIS
Poster #76
TISSUE-AUTONOMOUS REGULATION OF RETINAL IRON TRANSPORT Joshua Dunaief, MD, PhD, Ying Song, MD, Delu Song, MD, PhD, Bailey Baumann, BA, Milan Theurl, MD, Paul Hahn, MD, PhD, Majda Hadziahmetovic, MD and Natalie Wolkow, MD, PhD FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Perelman School of Medicine, University of Pennsylvania Presented By: Joshua L. Dunaief, MD, PhD
Introduction: ron dysregulation contributes to retinal degeneration in several diseases, including age related macular degeneration (AMD), aceruloplasminemia, Friedreich's Ataxia, and Pantothenate Kinase Associated Neurodegeneration. This is not surprising, as the retina has high metabolic activity, oxygen concentration, and easily oxidized polyunsaturated fatty acid. These findings prompted investigation of retinal iron regulation at the organismal and tissue levels. Methods: Retinal iron distribution and levels were assessed by ICP-MS, VIP-enhanced Perls’ stain and indirectly using immunolabeling for Ft, Fpn, and TfR and quantitative PCR for Tfrc, Ftn, and Hamp1 in the following systemic and conditional knockout mice: Cp-/-Heph-/-, Hamp1-/-, Bmp6-/-, Fpn C326S/C326S, BEST1-cre;Cp-/-HephFlox/Flox and Rx- cre;Cp-/-HephFlox/Flox. The effect of increased Hepc levels was evaluated using retinal gene transfer of AAV-Hepc. Systemic iron levels were increased by intravenous injection of iron-sucrose. Results: Mice with genotypes Cp-/-Heph-/-, Hamp1-/-, Bmp6-/-, and FpnC326S/C326S all had retinal iron accumulation inducing retinal degeneration. Conditional knockout mice with retina-specific knockout of Heph on a Cp-/- background had retinal iron accumulation, demonstrating that these genes function locally within the retina. Fpn localized to the blood retinal barriers (the retinal pigment epithelial cells and the vascular endothelial cells, suggesting it promotes iron flux across these barriers. Iron-loaded retinas from Cp-/-Heph-/- mice increased their production of Hepc, which may represent an effort to limit retinal iron influx. This hypothesis is supported by the finding of decreased retinal vascular endothelial cell Fpn and increased Ft following retinal injection with AAV-Hepc. IV iron treatment led to increased iron levels in the retinal pigment epithelial cells (one of the blood-retinal barriers) but only minimally in the neural retina. Discussion: These results suggest that the retina can use Hepc to regulate its iron levels in a tissue-autonomous manner. Fpn/Cp/Heph exports iron from cells of the blood-retinal barrier either into, or out of, the retina, and Hepc can regulate Fpn. Therefore, dysregulation of any of these processes could lead to retinal iron dysregulation and degeneration. Conclusions: Local iron regulation within the retina represents a microcosm of systemic iron regulation, and plays a critical role in retinal health.
Seventh Congress of the International BioIron Society Page 191 Poster Abstracts IBIS
Poster #77
REVERSAL OF IRON OVERLOAD IN TRANSFERRIN-DEFICIENT MICE Thomas Bartnikas, MD PhD, Courtney Mercadante, Michael Pettiglio, Carolina Herrera, Miriam Dash, Julia Bu and Edward Stopa, MD Brown University Presented By: Thomas Bartnikas, MD, PhD
Trfhpx/hpx mice are a model of inherited transferrin deficiency characterized by severe anemia and iron excess. Anemia reflects the essential role for transferrin in iron delivery to erythroid precursors. Iron excess is attributed to secondary deficiency in hepcidin, a hormone that inhibits dietary iron absorption and macrophage iron export. Trfhpx/hpx mice develop hepcidin deficiency because transferrin is essential for hepcidin expression and because anemia suppresses hepcidin expression. We have shown previously that short-term transferrin treatment of adult Trfhpx/hpx mice not only corrects anemia and hepcidin deficiency but also decreases liver iron levels. To explore this decrease in liver iron levels in more detail, we determined if a two-week transferrin treatment of two-month-old Trfhpx/hpx mice alters iron levels in organs other than liver. Transferrin treatment of mutant mice decreased iron levels in liver, pancreas, and kidney, indicating that treatment induces mobilization of iron from several organs in Trfhpx/hpx mice. To explore the fate of mobilized iron in treated Trfhpx/hpx mice, we interrogated total iron levels in mice before and after transferrin treatment. We postulated that total body iron levels would not change if mobilized iron was utilized largely for erythropoiesis. However, a decrease in total body iron levels would indicate that a prominent fraction of mobilized iron was excreted from the body. To test this, we first established baseline total body iron levels in Trf+/+ and Trfhpx/hpx mice without transferrin treatment. Total body iron levels were measured by inductively coupled plasma emission spectroscopy in mouse carcasses, defined as a mouse body from which the skin and small and large intestines had been removed. Total body iron levels were stable in Trf+/+ mice and increased progressively in untreated Trfhpx/hpx mice. Transferrin treatment of mutant mice beginning at two months resulted in normalization of total body iron levels to wild-type levels by six months. This indicates that Trfhpx/hpx mice can excrete a significant iron load. To our knowledge, complete correction of iron overload in an animal model has not been reported previously. To explore the correction of iron excess in transferrin-treated mutant mice in more detail, we measured rates of 59Fe absorption and excretion. Gavage of mice with 59Fe followed one hour later by harvest of gastrointestinal tracts and carcasses indicated that untreated Trfhpx/hpx mice have increased absorption rates and that transferrin treatment normalizes absorption rates in mutant mice to wild-type levels. Analysis of 59Fe excretion is underway, which will determine if rates of iron excretion are affected by the hpx/hpx genotype and by transferrin treatment and if excretion occurs via urine and/or feces.
Seventh Congress of the International BioIron Society Page 192 Poster Abstracts IBIS
Poster #78
THE FATE OF TRANSFERRIN BOUND TO IRON OXIDE NANOPARTICLES IN VITRO AND IN VIVO Denise Bargheer, PhD, Julius Nielsen, Artur Giemsa, PhD, Barbara Freund, PhD and Peter Nielsen, MD, PhD University Medical Centre Hamburg, Dept. of Biochemistry and Molecular Cell Biology Presented By: Peter Nielsen, MD, PhD
Nanoparticles (NPs) have unique capabilities to interact with cells and organs which mark them as attractive working material in nanobioscience and nanomedicine. In order to make full use of their potential it is essential to understand what controls at the molecular level recognition by cells, cell entering and intracellular processing. One important implication is that most NP upon contact with biological matrices such as the blood, are immediately coated by a layer of proteins, resulting in a so called protein corona. We used 125I labelled transferrin to study the fate of a designed protein corona in vitro and in vivo. A monodisperse superparamagnetic iron oxide nanoparticle (SPIO, 11 nm core) was encapsulated with a amphiphilic polymer, poly (maleic anhydride-alt-1-octadecene), forming carboxyl groups at the surface. From this negatively charged model particle, a platform of SPIOs with different surface characteristics was performed by controlled PEGylation. The SPIOs were first incubated with 125I-transferrin in vitro. After removal of free unbound protein by filtration, a excess of bovine albumin was added. FPLC analysis followed a 2 hours incubation at room temperature. The same experiment was performed in which 125I-transferrin was first covalently bound to the particles by EDC-coupling. A clear leakage of protein from the corona was observed when 125I-transferrin was initially only adsorbed but not when covalently bound. It shows also in this setup that covalently bound transferrin prevents the binding of 125I-albumin. The remaining transferrin on the SPIOs is still functional and can bind 59Fe when a stable precursor (Fe-NTA) is administered to the reaction mixture. For an in vivo experiment, two batches of SPIOs, double-labelled with 59Fe in the core and 125I- transferrin at the surface, were prepared in with transferrin was either adsorbed or covalently bound. Following the intravenous injection in mice, covalently bound or in-vitro adsorbed 125I-labeled mouse-transferrin was not separated from the 59Fe-label in blood and was transported into the liver, where, however, a complex metabolism and re-distribution of this physiological relevant plasma protein occurred (Figure). This study shows the complexity of nanoparticles distribution and degradation in vivo. The use of radiolabelled compounds is a powerful technique to follow the fate of nanoparticles in vivo in detail. Fig. Removal of radiolabels from blood after injection of 59Fe-core labelled SPIOs and 125I labelled transferrin covalently bound to the surface polymer in mice. Most of the radioactivity was incorporated into liver and spleen after two hours.
Seventh Congress of the International BioIron Society Page 193 Poster Abstracts IBIS
Poster #79
A DOMINANT MUTATION IN MITOCHONDRIAL UNFOLDASE CLPX RESULTS IN ERYTHROPOIETIC PROTOPORPHYRIA Barry Paw, MD, PhD¹, Yvette Yien, PhD¹, Gael Nicolas, PhD², Lisa van der Vorm, MS¹, Laurent Gouya, PhD², Hector Bergonia, PhD³, Martin afina, MS¹, Julia ardon, PhD⁴, ania Baker, PhD⁴, John Phillips, PhD³ and Herve Puy, MD, PhD² ¹Harvard Medical School; ²INSERM U , Paris; ³Univ. Utah; ⁴MI Presented By: Barry H Paw, MD, PhD
Porphyrias are a group of disorders caused by accumulation of heme intermediates in tissues, usually caused by loss of function mutations in the heme synthetic enzymes. However, their genetic pentrance is variable, underscoring the role of modifier genes and our incomplete understanding of the heme synthesis pathway. Here, we report a p.Gly298Asp dominant mutation in the ATP binding Walker A domain of the mitochondrial ATP-dependent unfoldase CLPX, which is causative for a novel variant of erythropoietic protoporphyria (EPP), characterized by protoporphyrin IX (PPIX) accumulation in erythroid cells. Mutant CLPX (CLPXGD) has no ATPase activity and interacts with the wild-type enzyme, suppressing its ATPase. Expression of CLPXGD stabili es -aminolevulinate synthase, causing downstream accumulation of PPIX, accounting for the development of protoporphyria. Our results identify a new variant of EPP which has a clinical phenotype similar to X-linked protoporphyria. The control of ALAS enzymatic activity and protein stability by CLPX unveils a novel cause of protoporphyria and insights revealing the ways in which mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with a key step in heme synthesis and provides an important genetic tool for understanding the pathology of porphyrias.
Seventh Congress of the International BioIron Society Page 194 Poster Abstracts IBIS
Poster #80
ERYTHROPOIETIN SIGNALING REGULATES HEME BIOSYNTHESIS Barry Pa , MD, PhD¹, Jacky Chung, PhD¹, Johannes Wittig, MS¹, Alireza Ghamari, PhD¹, Manami Maeda, MD, PhD⁵, Tamara Dailey², Hector Bergonia³, Martin afina, MS¹, Liangtao Li, MD³, Jerry aplan, PhD³, Harvey Lodish, PhD⁴, Daniel Bauer, MD, PhD¹, Stuart rkin, MD¹, Alan Cantor, MD, PhD¹, akahiro Maeda, MD, PhD⁵, John Phillips, PhD³, Joshua Coon, PhD⁶, David Pagliarini, PhD⁶ and Harry Dailey, PhD² ¹Harvard Medical School; ²Univ. Georgia; ³Univ. Utah; ⁴MI ; ⁵Fukuoka, Japan; ⁶Univ. Wisconsin, Madison Presented By: Barry H Paw, MD, PhD
Heme is required for the survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for a plethora of cellular processes, how it is coupled to cellular differentiation is unknown. Here, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH, EC 4.99.1.1) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway in vivo strikingly results in a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post- translational mechanisms to regulate heme metabolism during normal development.
Seventh Congress of the International BioIron Society Page 195 Poster Abstracts IBIS
Poster #81
FUNCTIONAL ANALYSIS OF HETEROZYGOUS GATA1 DELETERIOUS MUTATIONS IN TWO CHINESE FEMALE PATIENTS AFFECTED BY DYSERYTHROPOIETIC ANEMIA, THROMBOCYTOPENIA AND PORPHYRIA, RESPECTIVELY ong ei Wang, BS¹, Gang Liu, PhD¹,⁴, Shanshan Guo, BS¹, Bing Han, MD², Yanzhong Chang, PhD³ and Guangjun Nie, PhD¹ ¹CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China; ²Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; ³Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei Province, China; ⁴Section on Human Iron Metabolism, NIH, Bethesda, MD Presented By: Yongwei Wang, BS
GATA binding protein 1 (GATA1) is one of six members of GATA gene family which is located on Xp11.23 and contains six exons. GATA1, as a transcription factor, encodes a 413 amino acid protein and plays essential role in erythrocyte and megakaryocyte development and differentiation. GATA1 mutations may cause red blood cells and platelet disorders. Four functional domains (N-terminal and C-terminal transactivation domains and two zinc fingers) have been identified in previous study and seven missense mutations in the N-terminal zinc finger have been demonstrated on the basis of HGMD (Human Gene Mutation Database) which cause dyserythropoietic anaemia, thrombocytopenia, and congenital erythropoietic porphyria, respectively. Functional analysis of these missense mutations in erythroid precursors (G1E) and megakaryocyte-erythroid progenitors (G1ME) cell lines revealed that the defect of the FOG1 binding or TAL1 complex interaction with N-terminal zinc finger of GATA1 protein caused a severe hematological disease. In the current study, we identified a novel GATA1 heterozygous deletion in 46-year-old Chinese female, who presented with severe anemia (HGB 30-90 g/L, normal range 110-150 g/L) and thrombocytopaenia (PLT 50-80 10^9/L normal range 100-350 10^9/L). We also sequenced the cDNA of GATA1 gene, extracted from bone marrow of the patient and the results showed this deleted mutation. In addition, we also demonstrated another novel GATA1 heterozygous missense mutation in another 30-year- old Chinese female who carried a heterozygous small deletion in ALAS2 (c. 1706-1709 del AGTG) gene and manifested severe X-linked dominant protoporphyria while the plasma fluorescence emission spectroscopy test showed an emission peak at 625-628 nm. Mechanistic investigation with GATA1 knockout K562 and TF-1 cell lines via CRISPR-Cas9 technique provides novel understanding of these identified pathogenic mutations in Chinese patients and further highlights the multifaceted nature of GATA1 involved in human diseases.
Seventh Congress of the International BioIron Society Page 196 Poster Abstracts IBIS
Poster #82
PHARMACOLOGICAL ACTIVATION OF IRE-BINDING OF IRON REGULATORY PROTEIN1 BY TEMPOL PROTECTS VhlR200W MUTANT MICE FROM POLYCYTHEMIA THROUGH TRANSLATIONAL REPRESSION OF Hif2a Manik Ghosh, PhD, De-Liang Zhang, PhD, Wade Ollivierre and Tracey Rouault, MD Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH Presented By: Manik Ghosh, PhD
Chuvash polycythemia is an inherited disease that is endemic, but not limited to the Chuvash region of Russia (Miasnikova, et al. 2011). Chuvash polycythemia patients die prematurely, and the reported major causes of death are thrombosis and cerebral hemorrhage (Sergeyeva et al. 1997). In Chuvash polycythemia, a homozygous germ-line VHLR200W mutation leads to impaired degradation of 2 , and an elevated level of serum erythropoietin, the hormone that promotes production of red blood cells (RBC) (Sergueeva et al, 2015). The mouse model generated with homozygous R200W mutation in the Vhl gene also developed polycythemia similar to human disease. Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are two homologous mammalian proteins that regulate cytosolic iron level by binding to RNA stem-loops known as iron responsive elements ( Es) located in the 5’ or 3’-untranslated regions (UTR) of mRNAs encoding proteins involved in iron import, utilization, storage and export. IRP1 exists in a dynamic equilibrium between two forms; a cytosolic aconitase form with a 4Fe-4S cluster in its active site, and an apoprotein form that binds to IREs. When cellular iron levels are low, IRP1 exists predominantly in IRE-binding form. When cellular iron levels are high, IRP1 regains its iron-sulfur cluster, and exists mainly in cytosolic aconitase form. The cytosolic aconitase form is also stabilized at low oxygen concentrations (Rouault, 2006). We and others recently discovered that mice with targeted deletion of Irp1 developed polycythemia through translational de-repression of if2 ( hosh et al, 2013; Anderson et al, 2013; ilkinson et al, 2013). In another study, we showed that Irp1 can be pharmacologically activated in cells and mice by Tempol, a small molecule stable nitroxide (Ghosh et al, 2008). Therefore, we hypothesized that Tempol would activate IRE binding activity of rp1, which would thereby inhibit if2 translation and diminish polycythemia in VHLR200W mice. In this study, we fed VHLR200W mutation mice with a diet supplemented with Tempol, and observed that increased hematocrit levels of VhlR200W mice diminished to WT levels when these mice were treated with Tempol. RBC and hemoglobin levels were also significantly lowered in Tempol fed VhlR200W mice. Elevated serum erythropoietin levels in the mutant mice were reduced to T levels upon Tempol treatment. Our results showed that if2 protein levels in VhlR200W mice decreased significantly when the mice were fed with Tempol-supplemented diet. Tempol treatment remarkably increased IRE-binding activity of Irp1. However, Irp1 protein levels remained unchanged. Our results demonstrated that increased binding of Irp1 with if2 - E in its 5’ T in Tempol treated VhlR200W mice led to translational repression of if2 , resulting in down- regulation of erythropoietin, thus preventing VhlR200W mice from developing polycythemia. These results suggest that Chuvash polycythemia patients, who do not get better with phlebotomy therapy, could be treated with Tempol, to activate if2 - E binding activity of P1, thereby translationally repress 2 expression, and thus offer an effective therapy to Chuvash polycythemia patients.
Seventh Congress of the International BioIron Society Page 197 Poster Abstracts IBIS
Poster #83
CARBON NANOTUBES DISRUPT IRON HOMEOSTASIS AND INDUCE ANEMIA OF INFLAMMATION THROUGH INFLAMMATORY PATHWAY AS A SECONDARY EFFECT DISTANT TO THEIR PORTAL-OF-ENTRY Juan Ma, PhD and Sijin Liu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Presented By: Juan Ma
Although numerous toxicological studies have been performed on carbon nanotubes (CNTs), few studies investigated their secondary and indirect effects beyond the primary target tissues/organs. Here, we looked into the cascade of events: the initiating event and the subsequent key events necessary for the development of phenotypes, namely CNT-induced pro-inflammatory effects on iron homeostasis and red blood cell (RBC) formation, which are linked to anemia of inflammation (AI). A panel of CNTs were prepared including pristine multi-wall CNTs (P-MWCNTs), aminated (MWCNTs- NH2), polyethylene glycol (MWCNTs-PEG), polyethyleneimine (MWCNTs-PEI) and carboxylated MWCNTs (MWCNTs- COOH). We demonstrated that all CNT materials provoked inflammatory cytokine interleukin-6 (IL-6) production and stimulated hepcidin induction, associated with disordered iron homeostasis, irrespective of exposure routes including intratracheal (I.T.), intravenous (I.V.) and intraperitoneal (I.P.) administration. Meanwhile, PEG and COOH modifications could ameliorate the activation of IL-6-hepcidin signaling. Long-term exposure of MWCNTs resulted in AI and extramedullary erythropoiesis. Thus, we identified an adverse outcome pathway (AOP): MWCNT exposure led to inflammation, hepatic hepcidin induction and disordered iron metabolism. Together, our combined data depicted the hazardous secondary toxicity of CNTs in incurring anemia through inflammatory pathway. This study would also open a new avenue for future investigations on CNT-induced indirect and secondary adverse effects.
Seventh Congress of the International BioIron Society Page 198 Poster Abstracts IBIS
Poster #84
GENERATION OF INDUCED PLURIPOTENT STEM CELL-DERIVED ERYTHROBLASTS FROM A PATIENT WITH X- LINKED SIDEROBLASTIC ANEMIA Shunsuke Hatta, MD, PhD¹, Tohru Fujiwara¹, Takako Yamamoto, MD, PhD², Mayumi Kamata, MD, PhD¹, Yoshiko Tamai, MD, PhD³, ukio Nakamura, MD, PhD⁴, Shin a amata, MD, PhD² and Hideo Harigae, MD, PhD¹ ¹Tohoku University Graduate School of Medicine; ²Foundation of Biomedical Research and Innovation; ³Hirosaki University Graduate School of Medicine; ⁴RIKEN BioResource Center Presented By: Tohru Fujiwara, MD, PhD
Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow ring sideroblasts (RSs). To clarify the epidemiology and pathophysiology of CSA, a nationwide survey was performed in Japan. As of December 2016, 25 cases with CSA have been recruited. Seventeen cases (68%) were diagnosed as X- linked sideroblastic anemia (XLSA), which was attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 mutations included missense mutation (n = 12), mutations at intron 1 enhancer involving GATA- binding domain (n = 4), and a partial duplication of the coding region among ALAS2 exon 11 (n = 1). Remaining CSA cases included SLC25A38 mutation (n = 1), Pearson marrow-pancreas syndrome (n = 2), and yet genetically undefined cases (n = 5). Furthermore, whereas vitamin B6, a cofactor of ALAS2, has been used for the treatment of XLSA, more than half of the cases were refractory to the treatment. Thus, there is a need to establish a model of XLSA to reveal the detailed molecular mechanism contributing to RS formation as well as to explore novel therapeutic strategy for XLSA. Here, we established induced pluripotent stem (iPS) cells from a patient with XLSA harboring mutation of ALAS2 (c.T1737C, p.V562A). Based on serum-free monolayer culture, we obtained erythroblasts from normal and XLSA-derived iPS cells. Whereas the erythroblasts expressed predominantly embryonic globins and did not enucleate, XLSA-derived iPS cells harbored the phenotype reflecting decreased enzymatic activity of ALAS2. Interestingly, when the XLSA-derived was induced to undergo erythroid differentiation by co-culture with OP9 cells, aberrant mitochondrial iron deposition was detected. We are conducting biological analyses to characterize established RSs. Thus, the iPS can be used as an important tool for clarifying the molecular etiology and exploring novel therapeutic strategy.
Seventh Congress of the International BioIron Society Page 199 Poster Abstracts IBIS
Poster #85
THE INTERPLAY OF GATA1 WITH HEME REGULATES AN ERYTHROID CELL’S DIFFERENTIATION Raymond Doty, PhD¹, Xiaowei Yan, PhD², Christopher Lausted, MS², Zhantao Yang, MD¹, Li Liu, PhD¹, Neda Jabbari, PhD², Sioban Keel, MD¹, Qiang Tian, MD, PhD² and Janis Abkowitz, MD¹ ¹University of Washington; ²Institute for Systems Biology Presented By: Raymond Doty, PhD
GATA1 promotes the transcription of ALAS2, the first and rate limiting step of heme synthesis, and the transcription of many other erythroid-specific genes. It also increases its own transcription while silencing proliferation genes active in early progenitors and thus assures that erythroid differentiation correctly initiates. Heme then transcriptionally and translationally upregulates globin to guarantee adequate hemoglobin production in each cell as it matures. Mice lacking the heme exporter, FLVCR1, develop severe macrocytic anemia (HGB 4.4±0.97 vs 14.8±0.57 g/dL; MCV 66.9±6.2 vs 48.4±0.65 fL) with excess erythroblast heme and increased ROS. Since Flvcr1-deleted mice have no mutations in metabolic or erythroid genes, we can use this mouse to address the physiological role of heme in erythropoiesis. Erythroid differentiation proceeds normally until cells synthesize heme and cannot export it. Thus they need to deal with its consequences. To determine how excess heme causes ineffective erythropoiesis and how excess heme results in failed erythroid differentiation, we performed RNA sequencing of single early erythroid cells (BFU-E to basophilic erythroblasts) from wildtype control and Flvcr1-deleted mice and linked this transcription data to the total quantity of Ter119, CD71, and CD44 on that cell. Principal component analysis identified 4 transcriptionally unique clusters A, B, C, & D, which contained cells with negative, low, intermediate, and high Ter119 levels respectively. - and β-globin transcripts were expressed in all cells and were highly correlated (r=0.975). Gene set enrichment analysis (GSEA) comparing control cells to Flvcr1- deleted cells revealed excess heme results in significant downregulation of Gata1 with a 67% reduction in Flvcr1-deleted cells. Coupled two-way clustering analysis identified 150 genes co-regulated with Gata1 including 106 known GATA1 target genes which were poorly upregulated in Flvcr1-deleted cells in clusters B-D. Independent microarray analysis of mRNA from control and Flvcr1-deleted erythroid cells confirmed low Gata1 mRNA and low GATA1-dependent gene expression in the Flvcr1-deleted cells. To determine if excess heme was directly responsible for Gata1 downregulation, we treated primary mouse and human erythroid marrow cells and K562 cells with aminolevulinic acid (ALA) and iron to bypass the rate limiting step and increase endogenous heme synthesis. In the human cells, GATA1 protein decreased by 30-43% (p=0.03) within 15 minutes and 66% by 90 minutes suggesting that heme disrupts GATA1 protein function resulting in the loss of autoregulation and reduced GATA1 mRNA. Additionally, of the 86 genes in the ribosome pathway, 73 were significantly upregulated in Flvcr1-deleted cells, including 16 of the 17 ribosomal protein genes linked to Diamond-Blackfan anemia (DBA) or del(5q) myelodysplastic syndrome (MDS). When heme synthesis was induced in primary human erythroid marrow cells with ALA and iron, the transcript levels of ribosome protein genes such as Rps19, Rps14, and Rpl35 increased rapidly, supporting the concept that heme assures sufficient ribosome production for globin protein synthesis. Thus, GATA1 turns on heme synthesis and initiates the erythroid differentiation program. GATA1 with heme assure each cell’s appropriate progression. Then heme increases at the orthochromatic erythroblast stage when FLVCR expression is lowest and reduces GATA1, allowing terminal erythroid differentiation. By linking excess heme to prematurely low GATA1, our data may also help explain the ineffective (early termination of) erythropoiesis in DBA.
Seventh Congress of the International BioIron Society Page 200 Poster Abstracts IBIS
Poster #86
GLUTATHIONE AND THE CYTOSOLIC HEME POOL Robert Hider, PhD¹, Yu-Lin Chen, PhD², Rosemary Rawlinson, BSc² and Yemisi Latunde-Dada, PhD² ¹King's College London; ²KCL Presented By: Robert Hider, BSc, PhD
Introdution: Recently glutathione (GSH) has been proposed as a key component of the cytosolic iron pool.1,2 GSH possesses a buffering role for cytosolic iron(II) protecting it from autoxidation. However the chemical nature of the cytosolic heme pool is unknown. We have investigated whether or not GSH also binds to heme; if it does, it would be expected to increase the water solubility of this extremely hydrophobic molecule and thereby reduce its rate of partitioning into membranes. Methods: The affinity constant for the 1:1 complex of GSH and hemin has been determined by absorption spectroscopy. Standard bioassays were adopted to assess the influence of GSH on heme oxidase activity and the partitioning of hemin into liposomes of varying lipid composition. Monolayers of CaCo-2 cells have been investigated in order to establish the possible involvement of GSH in heme transport. Results: GSH does bind hemin to form a 1:1 complex with a log affinity constant of 4.7. As a result of the high cytosolic GSH level (2-10 mM) the predominant form of ‘heme’ in cytosol is S emin, where iron is in the 3+ redox state. S eme (with iron (II) in the complex) is rapidly autoxidised to S emin. The presence of S has a profound effect on the partitioning of hemin into bilayers, effectively inhibiting the partition completely. Data will be presented indicating the involvement of GSH in heme transport in gastrointestinal epithilical cells (CaCo-2 cells). Discussion: The formation of the hemin GSH complex markedly increases the water solubility of hemin and stabilises the redox state of iron as iron (III). The complex is relatively stable in the prescence of physiological levels of ascorbic acid, hydrogen peroxide and oxygen. Furthermore, in the presence of GSH, hemin does not rapidly partition into membranes, which is one of the main causes of heme toxicity. We also provide evidence for the involvement of GSH in heme transport. Conclusions: Glutathione is the main ligand to bind both iron (II) and hemin in the cytosol of mammalian cells. In both cases a 1:1 complex is formed. With hemin iron (III) is the most stable redox state. References: Hider RC and Kong XL (2011) Biometals 24 1179-1187. Hider RC and Kong XL (2013) Dalton Trans. 42 3220-3229.
Seventh Congress of the International BioIron Society Page 201 Poster Abstracts IBIS
Poster #87
ERYTHROPOIETIN INCREASES BONE AND CIRCULATING FGF23 LEVELS Mark Hanudel, MD, MS¹, Maxime Rappaport, BS², Kristine Chua, BS², Erica Clinkenbeard, PhD³, Victoria Gabayan, BS², Tomas Ganz, MD, PhD², Kenneth White, PhD³, Isidro Salusky, MD² and Elizabeta Nemeth, PhD² ¹UCLA Pediatric Nephrology; ²David Geffen School of Medicine at UCLA; ³Indiana University School of Medicine Presented By: Mark Hanudel, MD, MS
Introduction: In chronic kidney disease (CKD), circulating levels of fibroblast growth factor 23 (FGF23), a bone-derived phosphaturic hormone, increase dramatically. Although elevated FGF23 levels help to maintain normophosphatemia until late in the CKD course, FGF23 is associated with CKD progression, the development of left ventricular hypertrophy, and increased cardiovascular morbidity and mortality. Iron deficiency anemia increases FGF23 production. We hypothesized that other anemia-related factors, specifically erythropoietin (EPO), also affect FGF23 levels. Methods: First, to assess the effects of high endogenous EPO levels on FGF23 parameters, we characterized transgenic (TG) EPO overexpressing mice. Next, to assess the acute effects of exogenous EPO on FGF23, we assessed wild type (WT) C57BL/6 mice after a single intraperitoneal dose of EPO. To determine whether the acute effects of EPO on FGF23 are dependent on iron status and/or erythroferrone, we repeated the experiment with iron dextran co-administration and in erythroferrone knockout mice, respectively. Lastly, to assess the acute effects of exogenous EPO on FGF23 in humans, we assessed three anemic adults with normal kidney function who received a single dose of subcutaneous EPO. Results: Compared to their WT littermates, the TG EPO mice had similar kidney function, similar phosphate, almost double the hemoglobin and hematocrit, and markedly increased bone Fgf23 mRNA expression and circulating FGF23 levels. However, secondary to constant erythropoietic demand, the TG EPO mice were also relatively iron deficient, which also may increase FGF23 levels. However, iron dextran administration normalized serum iron but did not attenuate the increased FGF23 levels. In the WT mice treated with a single intraperitoneal dose of EPO, six hours after injection of 70 units/gram recombinant human EPO, bone Fgf23 mRNA expression and circulating FGF23 levels markedly increased; by 24 hours post-injection, the effects of EPO on FGF23 dissipated. Neither iron dextran co-administration nor erythroferrone knockout attenuated the EPO-induced increases in bone and circulating FGF23. In three human subjects, a single subcutaneous dose of 20,000-40,000 units EPO increased circulating FGF23 levels hours after injection. Discussion: Cardiovascular disease is the leading cause of death for CKD patients. Elevated FGF23 levels, which directly induce cardiac hypertrophy, likely contribute to CKD-associated cardiovascular morbidity and mortality. Regulation of FGF23 is incompletely understood, and there are currently no CKD treatment paradigms focused on lowering pathologically high FGF23 levels. Iron deficiency and EPO administration represent novel, potentially modifiable stimuli of FGF23 production. Moreover, increased FGF23 levels may account for some of the unexplained adverse cardiovascular effects associated with iron deficiency anemia and EPO therapy. Conclusions: TG EPO mice have high FGF23 levels that persist despite iron repletion. FGF23 concentrations increase after EPO administration in WT mice, an effect not attenuated by iron dextran co-administration or erythroferrone knockout. EPO administration increases FGF23 levels in anemic adults with normal kidney function
Seventh Congress of the International BioIron Society Page 202 Poster Abstracts IBIS
Poster #88
DYSREGULATED IRON METABOLISM IN BONE MARROW FROM A MOUSE MODEL OF CHRONIC KIDNEY DISEASE (CKD): HEPCIDIN AND ERYTHROPOIESIS Takeshi Nakanishi, MD, PhD, Tomoko Kimura, MD, Kiyoko Yamamoto, MD, Masayoshi Nanami, MD, PhD and Takahiro Kuragano, MD, PhD Hyogo College of Medicine Presented By: Takeshi Nakanishi, MD, PhD
Background: Regarding the causes of anemia in CKD, in addition to inadequate synthesis of erythropoietin (EPO), shortened erythrocyte life span and failure in bone marrow due to chronic inflammation have been postulated. However, the principal mechanism linked between CKD and erythropoiesis has not been fully established. In the present study, we examined the differentiation pattern of erythroid in the hematopoietic system of bone marrow and spleen using flow cytometry, EPO production in kidney and hepcidin expression in liver using mouse model of adenine-induced renal failure (RF). Methods: RF was induced by the administration of 0.15 % to 0.3 % adenine-containing chow for 8 weeks to male C57BL/6J mice. Liver, kidney, bone marrow, and blood were obtained from control (C) C57BL/6J mice and RF mice. Then Hb, serum iron and ferritin levels were measured. Serum hepcidin levels were quantified using LC-MS/MS methods. Hepatic hepcidin (Hamp) mRNA, renal EPO mRNA and bone marrow Fam132b (erythroferrone) mRNA from C and RF were semi-quantified using RT-PCR. For the purpose of evaluating the maturation of erythron, bone marrow erythroid precursors were analyzed using CD71 and Ter119 markers by FACS. Results: We confirmed that Hb levels were significantly lower (8.8±0.3 vs 13.4±0.1 g/dL) and serum ferritin levels were higher (2.6 0.6 vs 1.1 0.1μg/mL) in than C. enal EPO m A expression in was increased compared with C (2.5 : 1). FACS analysis showed the percentages of Pro erythroblast (CD71+/Ter119-) and Baso erythroblast (CD71+/Ter119+) in the BM of femur were decreased in RF compared with C. Hepatic Hamp mRNA expression was increased (2.4 : 1) and serum hepcidin levels were also higher (397.6±79.4 vs 99.6±6.9 ng/dL) in RF compared with C. Serum iron (105.4 5.1 vs 121.4 3.8μg/dL) and transferrin saturation (37.2 1.1 % vs 43.0 1.2%) were lower in than C. Further, BM Fam132b mRNA expression was significantly decreased (0.6 : 1) in RF compared with C. Conclusions: Erythroblast maturation was affected in the steps of late differentiation, those are the decrease in CD71+ (transferrin receptor expressing) cells. In RF, erythroferrone might be decreased, which could cause the increase in hepcidin although renal EPO expression was increased. Finally we presumed that the increase in hepcidin expression could be associated with the dysregulated erythroblast differentiation in RF model.
Seventh Congress of the International BioIron Society Page 203 Poster Abstracts IBIS
Poster #89
INVESTIGATING THE ROLE OF HRG1 IN HEME TRANSPORT Rini Pek¹, Jianbing Zhang, M Sc¹, Nicole Rietzschel, PhD¹, John Phillips, PhD², David Bodine, PhD³ and Iqbal Hamza, PhD¹ ¹University of Maryland; ²University of Utah; ³National Human Genome Research Institute Presented By: Rini Pek, BA
Heme is a significant source of iron for both eukaryotes and prokaryotes. However, free heme is a toxic, hydrophobic macrocycle. Thus, organisms have to maintain heme homeostasis at the subcellular, cellular, and organismal levels, a concept heretofore poorly understood. To identify these presumptive pathways, our lab established the roundworm Caenorhabditis elegans as a genetic animal model because worms lack the highly conserved heme biosynthetic pathway but absolutely require heme for sustenance. We identified heme-responsive gene-1 (hrg-1), which encodes the first metazoan heme importer/transporter, using a genomic screen in C. elegans. CeHRG-1 is a permease with four transmembrane domains and is conserved in vertebrates. Depletion of hrg-1 in worms resulted in disruption of organismal heme sensing and abnormal responses to heme analogs. Zebrafish with lower levels of zfhrg1 show anemia; severe depletion of zfhrg1 causes hydrocephalus, yolk tube malformations, and profound defects in erythropoiesis - phenotypes that were fully rescued by CeHRG-1. In cellula experiments using bone marrow derived macrophages revealed that HRG1 is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during phagocytosis of senescent RBCs – a process called erythrophagocytosis (EP). HRG1 specifically localizes to the phagolysosomal membranes during EP and depletion of HRG1 in macrophages causes attenuation of heme transport from the phagolysosomal compartment. These cell biological studies showed that HRG1 transports heme for heme-iron recycling in macrophages. To further elucidate the role of HRG1 in vivo, we generated a mouse model of Hrg1 deficiency. Our genetic and nutritional studies with mutant mice are ongoing and will be discussed.
Seventh Congress of the International BioIron Society Page 204 Poster Abstracts IBIS
Poster #90
THE HEME EXPORTER FLVCR1a IS CRUCIAL FOR BRAIN DEVELOPMENT Deborah Chiabrando¹, Tullio Genova², Francesca Bertino¹, Lorenzo Silengo¹, Fiorella Altruda¹ and Emanuela Tolosano¹ ¹Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy.; ²Department of Surgical Sciences, CIR Dental School, University of Turin, Italy Presented By: Deborah Chiabrando, PhD
Heme is an essential co-factor involved in multiple biological processes. However, excess free-heme is highly toxic due to its ability to promote oxidative stress, proteasome inhibition and mitochondrial dysfunction that ultimately lead to cell death. In the nervous system, heme is required for neuronal survival and differentiation. On the other hand, the toxic properties of heme in the brain have been extensively studied in the context on intracerebral hemorrhages but little is known about the physio-pathological relevance to control intracellular heme levels in the nervous system. FLVCR1 (Feline Leukaemia Virus subgroup C Receptor 1) is a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in the FLVCR1 gene have been reported in individuals affected by Posterior Column Ataxia and Retinitis Pigmentosa (PCARP), Retinitis Pigmentosa (RP) and Hereditary Sensory and Autonomic Neuropathy (HSAN). These findings suggest that FLVCR1 is required for the survival of specific neuronal populations. However, the precise role of FLVCR1 in the nervous system still remain to be investigated. We previously showed that the silencing of the plasma membrane isoform FLVCR1a induces oxidative stress and programmed cell death in neuroblastoma SHSY5Y cells. To investigate the consequences of Flvcr1a loss in the nervous system in vivo, we generated neuronal-specific Flvcr1a-/- mice. To this end, we took advantage of transgenic mice expressing the Cre recombinase under the control of the rat nestin-promoter. Nestin is an intermediate filament protein, highly expressed in neuronal progenitors cells starting from embryonic day 11. Neuronal-specific Flvcr1a- /- newborns were delivered in normal mendelian ratios but died immediately after birth. Histological analysis showed that their pulmonary alveoli did not expand at birth. Thus, apnea could well explain the newborn lethality. At E18,5 neuronal- specific Flvcr1a-/- embryos appears comparable to wild-type embryos. X-ray computed micro-tomography (micro-CT) analysis showed severe alterations of brain morphology. Neuronal-specific Flvcr1a-/- embryos showed microcephaly and an enlargement of ventricles, subarachnoid and perivascular spaces (Figure). These defects are likely secondary to the reduction of cerebral parenchyma volume. We hypothesize that loss of FLVCR1a may affect the proliferation, differentiation and/or survival of neuronal progenitors. The ability of heme to regulate specific pathways required for neuronal survival is still under investigation. Taken together these data indicate that FLVCR1a-mediated heme export plays a crucial role for proper brain development.
Seventh Congress of the International BioIron Society Page 205 Poster Abstracts IBIS
Poster #91
EXPLORING POTENTIAL MEDIATORS OF EARLY HEPCIDIN DOWNREGULATION BY ERYTHROPOIETIN IN VIVO Irene Artuso, Antonella Nai, Mariateresa Pettinato, Silvia Colucci, Clara Camaschella and Laura Silvestri San Raffaele Scientific Institute Presented By: Irene Artuso
Introduction: Erythroid cells release erythroferrone (ERFE) to signal their iron needs to the liver through hepcidin inhibition. We demonstrated that high BMP-SMAD pathway activation contrasts the Erfe-dependent hepcidin inhibition (Nai, Rubio et al., 2016). However, how ERFE inhibits hepcidin and its crosstalk with the BMP-SMAD pathway remain unknown. The major source of iron for erythroid cells is diferric-transferrin (Holo-TF). We hypothesized that the overexpression of TFR1 in conditions of expanded erythropoiesis increases iron uptake and decreases holo-TF, reducing the BMP-pathway activation and favoring ERFE-dependent hepcidin inhibition. To investigate whether the EPO-induced erythropoiesis expansion may contribute to hepcidin inhibition through holo-TF and independently of Erfe, we analyzed ERFE, hepcidin expression and circulating iron in wild-type mice challenged with a single EPO injection and sacrificed at early time points post-treatment. Methods: C57BL/6 male mice (7-8 week-old) were treated with EPO and sacrificed 1.5-3-6-9-15 hrs later. Erfe, iron- and erythroid-related genes were analyzed by qRT-PCR. Percent transferrin saturation (%TS, an indirect measure of holo-TF) and LIC were assayed using standard procedures. Hep3B cells were transfected with the ERFE expressing vector and treated with the ERK-MAPK inhibitor U0126 or the mTOR inhibitor rapamycin. After 15 hrs cells were lysed and processed for RNA isolation and gene expression analyses. Results: To investigate how erythropoiesis expansion influences circulating iron at short time points, we measure %TS in EPO-treated mice. Percent TS is decreased by EPO at 6 hrs post-injection. The expression of the BMP-SMAD target gene Id1 is decreased starting from 6 hrs post-EPO and remains low until 15 hrs. Erfe expression is significantly upregulated by EPO in bone marrow and spleen at 1.5 hrs post injection. Interestingly, although Erfe expression is increased, Hamp remains high until the BMP-SMAD signaling is attenuated, confirming our previous hypothesis (Nai, Rubio et al., 2016). To investigate the signaling pathway/s involved in ERFE-dependent hepcidin inhibition, ERFE- transfected cells were treated with U0126 and rapamycin. Hepcidin is strongly increased in U0126-treated cells, confirming that the ERK-MAPK pathway has an inhibitory effect, as suggested (Chen H. et al., 2016). Nonetheless, U0126 treatment does not interfere with the ERFE-effect on hepcidin. On the contrary, rapamycin partially inhibits the ERFE- dependent hepcidin inhibition, suggesting a role for the mTOR pathway in ERFE-mediated hepcidin downregulation. Conclusions: Holo-TF is decreased at 6 hrs post-EPO treatment in a hepcidin-independent way, suggesting that high erythroid Tfr1 expression increases iron consumption for Hb and RBCs production. Liver BMP-SMAD pathway is downregulated at 6 hrs and hepcidin fully suppressed at 15 hrs, although Erfe is upregulated at shorter time points. Overall these data suggest that Holo-TF-dependent BMP-SMAD-pathway inhibition is a prerequisite for hepcidin suppression by Erfe. Studies are in progress to confirm preliminary data suggesting that mTOR might play a role in ERFE- dependent hepcidin inhibition. Acknowledgements Supported by Telethon Grant GGP15064 to L
Seventh Congress of the International BioIron Society Page 206 Poster Abstracts IBIS
Poster #92
MOUSE STRAIN DIFFERENCES IN THE RESPONSE TO TREATMENT WITH A HEMOJUVELIN NEUTRALIZING MONOCLONAL ANTIBODY Andreas Popp, Dr¹, Melissa Matzelle, Dr² and Bernhard K. Mueller, PD Dr³ ¹Abbvie Deutschland GmbH & Co. KG; ²AbbVie Inc., Foundational Immunology; ³AbbVie Deutschland GmbH & Co. KG, Neuroscience Discovery Presented By: Andreas Popp, Med. Vet
Physiological normal values of serum iron are known to be different between species and strains. In addition, species show different susceptibility to iron deficiency or excessive supply of iron via the diet. We used a hemojuvelin neutralizing monoclonal antibody which has been shown previously to decrease serum hepcidin level and iron binding capacity and to increase iron level in the serum to evaluate the influence of this treatment in three different mouse strains (Rag KO, C57Bl/6 and Balb/c). The animals were treated with a single dose of 20 mg/kg intraperitoneally and blood was taken after 4, 24, 48 and 96 hours post application. In the serum of the animals, drug exposure, hepcidin, iron level and the unsaturated iron binding capacity were measured. There was a clear correlation between antibody exposure with the other parameters measured in the three strains. Rag KO and C57Bl/6 had low baseline iron of approximately 140 µg/dl compared to 300 µg/dl in Balb/c mice. However, RAG KO and C57Bl/6 mice responded to the hemojuvelin neutralizing antibody with a 100-150% increase in iron compared to 30% in Balb/c 24 hours after antibody treatment. We concluded that there are strain specific differences in normal serum iron and iron binding capacity level in mice and corresponding differences in the response on treatment with compounds having an influence on iron homeostasis. These strain differences have to be considered in future experiments when selection of a proper mouse strain is required. Disclosure statement: All authors are employees of AbbVie. The design, study conduct, and financial support for the research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Seventh Congress of the International BioIron Society Page 207 Poster Abstracts IBIS
Poster #93
VALPROIC ACID AND SHORT CHAIN FATTY ACIDS BUTYRATE AND PROPIONATE MAY INDUCE HEPCIDIN MRNA EXPRESSION IN HEPATIC CELLS Marie-Laure Island, PhD², Amandine Gaudin, MsC, Thibault Cavey, MD, Patricia Leroyer, Martine Ropert, MD, Pierre Brissot, MD and Olivier Loréal, MD, PhD¹ ¹INSERM UMR991; ²INSERM UMR991, Rennes, France Presented By: Olivier Loreal, MD, PhD
Background: A relative deficiency in hepatic hepcidin expression is involved in iron overload development during genetic hemochromatosis related to mutations in genes controlling hepcidin expression (HFE, TFR2, HJV), and in b-thalassemia, in which both anemia and the erythropoietic process decrease hepatic hepcidin expression. Hepcidin deficiency induces high levels of serum iron, transferrin saturation and non-transferrin-bound iron that, in turn, induce parenchymal iron overload. Increasing hepcidin levels, either by hepcidin supplementation or by promoting higher levels of hepcidin expression, represents a potential therapeutic approach. Aim. To investigate whether valproic acid, an antiepileptic drug and mood stabilizer derived from valerate, a short chain fatty acid (SCFA), could be an inducer of hepcidin expression. Methods: HepG2 cells and/or pure cultures of freshly isolated human hepatocytes were exposed to valproic acid from 125µM to 1mM. Hepcidin mRNA levels were quantified by RT-PCR in cell cultures. In vitro, the impact of VPA on the transcription of hepcidin gene was analyzed through gene reporter strategy and luciferase activity. The impact of natural SCFA, butyrate, propionate and acetate, on hepcidin mRNA level expression was analysed. BMP/SMAD pathway was explored by performing SMAD1/5/8 western-blot. Results: Exposure of HepG2 cells and cultured human hepatocytes to VPA induced a dose dependent increase of hepcidin mRNA levels. In HepG2 cells, the gene reporter strategy identified the BMP-Responsive elements as a potential mediator of VPA activity on hepcidin transcription level. However, phosphorylation of SMAD1/5/8 proteins was not significantly modulated by VPA exposure. Regarding the impact of natural SCFA, butyrate (0.5mM, 1mM) induced a dose- dependent increase of hepcidin mRNA level. Propionate (1mM) was also able to induce hepcidin expression. Conversely, acetate and valerate did not exert significant effects on hepcidin expression. Discussion: Our data indicates that: i) VPA induces hepcidin expression in human hepatocytes; ii) This induction involves BPM-Responsive elements in the hepcidin promoter, but the precise mechanism remains to be dissected, iii) Butyrate and propionate may induce hepcidin expression in contrast with acetate and valerate,the precursor of VPA. Conclusions: The present data suggests that hepatocyte hepcidin expression can be induced by valproic acid, and by natural SCFA that may be produced by gut bacteria, therefore suggesting that this track could be followed for designing innovative therapeutic actions in the field of hepcidin deficiency-related diseases.
Seventh Congress of the International BioIron Society Page 208 Poster Abstracts IBIS
Poster #94
SUPPRESSION OF ERYTHROPOIESIS IN THE MOUSE MODEL MIMICKING REPEATED-TRANSFUSION INDUCED IRON OVERLOAD Masayo Yamamoto, MD, PhD¹, Hiroki Tanaka, PhD², Masatomo Ishioh, MD¹, Takeshi Saito, MD¹, Yasumichi Toki, MD, PhD¹, Mayumi Hatayama, MD¹, Motohiro Shindo, MD, PhD¹, Katsuya Ikuta, MD, PhD¹, Yoshihiro Torimoto, MD, PhD³ and Toshikatsu Okumura, MD, PhD¹ ¹Division of Gastroenterology & Hematology/Oncology, Asahikawa Medical University; ²Division of Legal Medicine, Asahikawa Medical University; ³Oncology Center, Asahikawa Medical University Hospital Presented By: Masayo Yamamoto, MD, PhD
Patients with bone marrow failure such as myelodysplastic syndrome, aplastic anemia and myelofibrosis require transfusion repeatedly, but transfusion-induced iron overload threatens their life. Therefore, iron chelation therapy (ICT) is widely used in the patients. Recent reports have suggested that ICT could reduce transfusion dependency indicating that iron itself directly causes anemia. However, it has not been known whether iron itself directly affects hematopoiesis and the function of bone marrow. We therefore tried to clarify the above hypothesis in an iron-overload (IO) mice model. In order to mimic repeated-transfusion, male C57BL/6 mice were randomly assigned into following 2 separate treatment groups at age 4 weeks. In a control group, PBS (100 µL/head/day) was injected, and iron dextran (10 mg/head/day) was injected in rats (IO). These injections were performed twice per a week for 8 weeks. After each treatment, peripheral blood and bone marrow cells (contained in thighbones) were collected. Additionally, because the kidney in IO mice seemed to be atrophic, the kidney was examined pathologically. In the complete blood count of peripheral blood, the red blood cells were significantly reduced and the platelets were slightly reduced, while the white blood cells were significantly increased in IO mice. The total numbers of bone marrow cells per thighbone were significantly lower in IO mice than control mice. In Prussian blue staining of bone marrow cells, the IO mice showed iron deposits in the macrophages and blast cells while control mice did not show any iron deposits. These iron deposits were considered to be a cause of myelosuppression in IO mice. Additionally, the histopathological analysis demonstrated that iron deposit was localized to renal peritubular interstitial fibroblasts where erythropoietin (EPO) is produced in IO mice. The serum analysis by ELISA revealed EPO levels were significantly lower in IO mice. Our results indicate that repeated-transfusion causes iron deposit in bone marrow cells and renal peritubular interstitial fibroblasts. We would suggest that iron deposit in the bone marrow cells could be a cause of reduction of erythroblasts and that iron deposit in renal peritubular interstitial fibroblasts might impair the production of EPO in kidney, followed by renal anemia.
Seventh Congress of the International BioIron Society Page 209 Poster Abstracts IBIS
Poster #95
PREVENTION OF IRON TOXICITY FROM HEME CATABOLISM IS LINKED TO COPPER-MEDIATED REGULATORY EVENTS Ann Smith, PhD¹, im Rish, BS MS², LokMan Sung, BS MD², Rachel Helston, BS PhD³ and Roberto anacore, BS PhD⁴ ¹SBS-UMKC; ²University of Missouri- Kansas City; ³Stowers Institute for Medical Research; ⁴ anderbilt University Presented By: Ann Smith, BSc, PhD
Intracellular heme toxicity develops rapidly from methemoglobin- and methemalbumin-derived heme in several different cell types including neurons, hepatocytes, endothelial and immune systems cells. Heme toxicity occurs from the rapid, unregulated uptake of heme that does not involve endocytosis and leads to extensive amounts of intracellular heme. Given sufficient O2 and coenzymes for heme oxygenase activities and for their obligatory reductases, heme catabolism then generates high levels of redox-active ferrous iron for which the cells is unprepared in terms of storage, biochemical or regulatory uses or export. Consequently, as shown in human primary hepatocytes, heme generates reactive oxygen species (ROS) and is toxic releasing DAMPS such as the chromatin associated protein high mobility group box 1. In contrast, after heme uptake by heme-hemopexin endocytosis, ROS are not produced, several signaling pathways are activated and, uniquely, copper levels rise as part of the cytoprotective reprogramming of cells. The rise in copper levels is not due to the activation of signaling pathways but likely requires co-endocytosis of copper with heme-hemopexin. Copper binding to hemopexin prevents the re-association of heme at the acidic pH of endosomes, thus aiding in heme release for intracellular trafficking to HOs in the SER, to the nucleus for gene regulation, and also for the coordinated induction of HO1 and metallothioneins (MTs). MTs are cytosolic proteins that bind copper minimizing intracellular levels to protect cells against oxidative stress as does the tripeptide glutathione and several copper chaperones that traffic copper for cupro- enzyme synthesis (e.g. CuZnSOD). Intracellular antioxidant defenses rely upon the enzymatic removal of ROS before they can react with any available redox-active metals such as heme, iron and copper. Both SOD and CCS1 have been shown to play a significant role in protecting cells against oxidative damage. Intriguingly, MT is also needed for induction by copper of the genes for CuZnSOD and CCS1. Cells replete with HO1, CuZnSOD, MTs and GSH would be able to survive the oxidative stress in situations where hemopexin is protective such as the inflammation that accompanies hemolysis, trauma, reperfusion injury as well as infection. Importantly, “free” heme does not regulate CCS1, MTs or MTF1. Thus, this copper-mediated response to heme delivery by hemopexin is one key for cells to survive raised abnormally high iron levels and the increased intracellular redox state. Understanding the mode of action of the endogenous protective hemopexin system will continue to provide new insights into the mechanisms involved for safe transport of heme to the hepatocytes and neuronal cells for its catabolism and for iron conservation. This will in turn help guide new diagnostics, treatments and therapies for patients when methemoglobin and heme levels in biological fluids such as plasma and cerebral spinal fluid start to generate heme toxicity that is due predominantly to increased intracellular iron.
Seventh Congress of the International BioIron Society Page 210 Poster Abstracts IBIS
Poster #96
TRIPHENYLPHOSPHONIUM-DESFERRIOXAMINE AS A CANDIDATE MITOCHONDRIAL IRON CHELATOR Roxana Yesenia Pastrana Alta, Hector Vitorino, Simon Wisnovsky, Shana Kelley, M. Teresa Machini and Breno Esposito University of Sao Paulo Presented By: Breno P. Esposito
Cell-impermeant desferrioxamine (DFO) can have access to organelles if appended to suitable vectors. Triphenylphosphonium (TPP) is a delocalized lipophilic cation used to ferry molecules to mitochondria. Here we report the synthesis and characterization of TPP-DFO as a mitochondrial iron chelator. TPP-DFO retains significant iron chelation ability (logKFe(TPP-DFO) ~33) with the same antioxidant activity against reactive oxygen species in the iron-ascorbate system. TPP-DFO is less toxic than TPP alone to A2780 cells (IC50 = 135.60±1.08 µM and 4.34±1.06 µM, respectively). Its native fluorescence was used to assess mitochondrial localization (Rr = +0.20). Altogether these properties suggest that TPP- DFO could be an interesting alternative for the treatment of mitochondrial iron overload e.g. in Friedreich’s ataxia.
Seventh Congress of the International BioIron Society Page 211 Poster Abstracts IBIS
Poster #97
IRONING OUT SMOKE-INDUCED INFLAMMATION IN LUNG DISEASE Suzanne Cloonan, PhD Weill Cornell Medical College, NY, USA Presented By: Suzanne Cloonan, PhD
Chronic obstructive pulmonary disease (COPD) is as a complex debilitating lung disease that encompasses three main clinical and pathologic phenotypes: airway inflammation (chronic bronchitis), destruction of lung tissue (emphysema) and small-airway remodeling. The pathogenesis of COPD involves aberrant inflammatory and dysregulated cellular responses of the lung to cigarette smoke (CS) exposure. CS exposure remains the greatest environmental risk factor for COPD; however, multiple studies have demonstrated that genetic factors influence COPD susceptibility. Iron-responsive element binding protein 2 (IRP2, also known as IREB2) is a well-replicated identified COPD susceptibility gene based on genome- wide association studies (GWAS), with IRP2 protein increased in the lungs of COPD subjects. In this study we sought to delineate the function of the COPD susceptibility gene IRP2 in the pathogenesis of CS-induced COPD. Using novel experimental approaches, integrating unbiased IRP2-target identification in cells and mouse lungs with human COPD expression data, we demonstrate that IRP2 regulates mitochondrial-related pathways in the lung. We validate this critical regulation in two well-established human COPD cohorts, showing a strong association between the differential-expression of mitochondrial genes and IRP2 expression in COPD subjects. Our studies are the first to characterize a functional role for IRP2 in the lung wherein IRP2 promotes mitochondrial dysfunction in experimental COPD by regulating mitochondrial iron loading and cytochrome c oxidase. Furthermore, we demonstrate that IRP2 promotes mitochondrial dysfunction in experimental COPD, which may ultimately lead to epithelial cell death and emphysematous destruction associated with COPD. These data are consistent with earlier observations that increasing IRP2 is detrimental to cells and mice and that COPD itself is associated with mitochondrial dysfunction and altered mitochondrial dynamics. Our data that IRP2 promotes mitochondrial iron overload in experimental COPD is also consistent with previous observations that CS increases iron deposition systemically and in alveolar macrophages from human subjects with COPD. Such iron deposition may also be consistent with recent reports of anemia being associated with poor clinical and functional outcomes in COPD and with reports that systemic inflammation in COPD up-regulates serum hepcidin. Importantly, we also show that mitochondrial iron chelation using the siderophore deferiprone alleviates established disease in a model of CS-induced pulmonary inflammation and injury (experimental COPD), highlighting a potential novel therapeutic approach for selective mitochondrial iron chelation in COPD. References: Cloonan S.M, Choi A.M.K et al. The iron regulatory protein Irp2 mediates cigarette smoke–induced bronchitis and emphysema via mitochondrial iron loading and cytochrome c oxidase. Nature Medicine 2016 Feb; 22(2): 163-74.
Seventh Congress of the International BioIron Society Page 212 Poster Abstracts IBIS
Poster #98
CHANGES OF “IRON GENE” SIGNATURE RESPONSIBLE FOR EMBRYONIC STEM CELL STEMNESS AND DIFFERENTIATION Jing Liu and Sijin Liu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Presented By: Jing Liu
The importance of iron-related genes in embryonic stemness mantainance and differentiation is nearly unknown. In the current study, we evaluated the alterations and physiological roles of 22 iron-related genes in murine embryonic stem (ES) CCE cells under self-renewal and during differentiation. Among these genes, 8 genes were found to be significantly increased by more than 5 fold during ES cells differentiation, and another 8 genes were demonstrated to be elevated by >2 fold upon differentiation compared to undifferentiated ES cells. Meanwhile, other genes revealed slight changes during differentiation. To look into the contribution of these differentially expressed genes to regulating ES cell differentiation through altering cellular iron status, we deliberately compared the cellular iron metabolism of ES cells to mouse hepatocytes, Hepa1-6 cells. Knocking-down (KD) the expression of Slc25A28, Steap3 and Sfxn1 did not affect ES cell self-renewal, as reflected by the unchanged levels of Oct4 and Nanog; however, cell proliferation was restrained under retinoic acid (RA) differentiation, associated with cell death upon KD these genes. Meanwhile, Steap3 KD and Slc25A28 KD cells exhibited lower ATP and SDH activity after RA induction. To look for the molecular mechanisms underlying the changes of iron-related genes, we further determined the molecular regulation of these genes. Mechanistic investigations uncovered that the induction of Slc25A28, Steap3 and Sfxn1 was accounted for by the enhanced demethylation of their promoter regions due to differential regulation by DNA methylation and demethylation processes. This study unveiled an important contribution of iron metabolism in modulating ES cell self-renewal and differentiation.
Seventh Congress of the International BioIron Society Page 213 Poster Abstracts IBIS
Poster #99
A TRIPEPTIDE MOTIF- LEU-TYR-ARG (LYR) PLAYS A SIGNIFICANT ROLE IN Fe-S CLUSTER ACQUISITION BY CELLULAR PROTEINS Anshika Jain, PhD¹, Anamika Singh, MS², Nunziata Maio, PhD² and Tracey Rouault, MD² ¹NIH; ²NICHD/NIH Presented By: Anshika Jain, PhD
Fe-S clusters are important cofactors in cellular biochemistry. The ability of Fe to readily accept or donate single electrons makes them an efficient redox center for various proteins. While several Fe-S cluster containing proteins, including DNA polymerases, primases, respiratory chain enzymes have been reported in the past; many more that harbor the cofactor are still unknown. My project focuses on the identification of a common consensus that facilitates a protein to accept the cofactor and function as a Fe-S cluster containing protein. Fe-S cluster biogenesis is a well elucidated pathway. Recently, a co-chaperone, HSC20 (exclusively involved in Fe-S cluster pathway) was discovered, which accepts electrons from the Fe-S cluster scaffold protein, ISCU and transfers it to the recipient protein, SDHB. HSC20, recognizes a tripeptide motif, Leu-Tyr-Arg (LYR) on SDHB and transfers the cluster to the target. This study provides an important insight into the Fe-S acquisition clusters by recipient proteins. Based on this study, we hypothesized that the presence of LYR motif serves as a recognition site for HSC20 and this interaction allows the delivery of Fe-S cluster from the co-chaperone to the target protein. In this study, we identified approximately 1000 proteins in human proteome containing the LYR-tripeptide motif and four cysteine residues to house the cofactor. The study was initiated with the series of proteins involved in kidney cancer pathway (PTEN, mTOR, CUL2, TSC1). Our results from co-immunoprecipitation suggest a positive interaction between HSC20 and these proteins. Subsequently, these proteins were anaerobically purified from E.coli; and currently, are being investigated for the presence of Fe-S clusters using spectrophotometric and radioactive assays. Identification of Fe-S clusters in these proteins will yield new mechanistic insight into the signal transduction cascades and divulge a novel signaling molecule in cellular biochemistry. Further on, a structural analysis on the purified proteins and the binding pocket will enable us to extrapolate and simulate of the features of unknown Fe-S containing protein. Additionally, ISD11, an accessory factor to NSF1 (cysteine desulfurase1) was identified in the insilico LYR-protein search. NSF1 in association with ISD11 provides the sulfide radical to the Fe-S cluster. In our studies, an LYR mediated interaction was demonstrated between ISD11 and HSC20. However, the absence of any cys-residues incapacitates ISD11 to house the Fe-S cluster. This suggests a role of HSC20 in not only the delivery but also the biogenesis of Fe-S clusters. Currently, we are investigating the physiological relevance of this interaction to conclusively establish the role of HSC20 in Fe-S cluster biogenesis pathway.
Seventh Congress of the International BioIron Society Page 214 Poster Abstracts IBIS
Poster #100
SHORT-ACTING MUCOSAL BLOCK AND FERRITIN-INDEPENDENT ANTIOXIDATIVE SYSTEM WORKS IN THE IRON-DEFICIENT RATS DUODENUM Shoko Shinoda, PhD, Machiko Yamamoto, MS and Anna Arita, MS Tokyo Metropolitan University Presented By: Shoko Shinoda, PhD
Excess iron loading suppresses intestinal iron absorption, a phenomenon known as “mucosal block” which is characterized by the down-regulation of mRNA and proteins. Regardless of whether mucosal block occurs only in iron deficient animals, the iron concentration required to induce this phenomenon and the time to onset remain unclear. We administered 10 mg of iron to the ligated duodenum of iron-deficient and iron-sufficient rats. One hour later, arterial serum iron concentration was higher in iron-deficient than in iron-sufficient rats, suggesting increased iron absorption in the former, but duodenal iron uptake did not differ significantly. Based on these results, we hypothesized that short-acting mucosal block occurs prior to the regular mucosal block. Excessive iron generate the hydroxyl radicals (•OH) from the Fenton reaction, but this reaction is usually inhibited by ferritin in the mucosal cells. In iron deficiency, ferritin expression is downregulated, therefore it is possible that labile iron pool and oxidative stress are increased in the duodenal mucosa. We found glutathione (GSH) in iron deficient duodenum is maintained higher than iron sufficient duodenum; it is possible to work ferritin-independent antioxidative system. We also examined the effects of iron deficiency on the antioxidative enzyme activities and antioxidant concentration related to the Fenton reaction. Weaned Wistar male rats were fed an AIN- 93G or an iron-free diet for 3weeks. We assessed the effects of 30–2,000 μg iron load on iron uptake in the duodenal loop of iron-deficient and iron-sufficient rats under anesthesia. The duodenums were removed from rats and mucosal cells were scraped using a slide glass. Mucosal GSH and hydrogen peroxide concentrations, activities and mRNA expressions of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were measured. As lipid peroxide marker, malondialdehyde (MDA) was also examined in the presence and absence of GSH. We found that short-acting mucosal block could be induced by much lower iron concentration and much shorter induction time than previously reported, without affecting levels of gene expression. Compared with iron-sufficient rats, GSH concentration was higher, and H2O2 concentration, SOD and CAT activities were lower in iron-deficient rats. MDA was lower in iron deficient than iron-sufficient rats at iron loading of 1,000 μg, it remained lower even at low level of S concentration. These observations indicate that short-acting mucosal block and ferritin-independent antioxidative system are working to suppress acute iron oxidative stress in the iron-deficient duodenum with high absorption capacity of iron.
Seventh Congress of the International BioIron Society Page 215 Poster Abstracts IBIS
Poster #101
GENETIC ANALYSIS OF SURVIVAL OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN EGG WHITE; IMPACT TO IRON RESTRICTION AND LYSOZYME Abdulameer Ghareeb, PhD student¹, Pauline Sabrou², Sophie Jan², Florence Baron² and Simon Andrews, Professor ¹The University of Reading; ²Agrocampus Ouest Presented By: Abdulameer Ghareeb
Salmonella enterica serovar Enteritidis (SE) is an important disease causing serovar in many animal species as well as humans [1]. Eggs and egg products are the major sources of human infection with SE [2]. Egg white is highly resistant to bacterial infection partly because of the presence of ovotransferrin that acts to restrict iron availability. Identifying SE genes that confer resistance to egg white might explain the specific ability of SE to resist the harsh conditions of egg white. Global expression analysis of SE exposed to egg-white medium showed that three distinct gene clusters, likely representing a novel regulon, are strongly induced: the dgoRKADT operon, the uxuAB-uxaC operon and the SEN1433-6 genes. These genes are related to in utilisation of hexonates and hexuronates, and have not been previously reported to possess any role in egg-white survival. The aim of this work is to determine the role of these genes in the survival of SE in egg white and low iron conditions, and to further explore the mechanism of egg white iron restriction in combating infection. Nine of the putative promoter regions of interest (ybhC, SEN1435, SEN1436, SEN1432, dgoR, dgoT, SEN2977, SEN2978 and SEN2979) were tested for expression by generation of lacZ fusions. Activities were variable, with highest observed for SEN1436 (encoding for putative dehydratase) and SEN2977. The SEN1436-lacZ fusion was selected for subsequent work for the study of environmental regulation. Upon exposure to egg white (EW), SEN1436 expression was induced up to 60 fold. However, egg-white filtrate (EWF; lacking proteins >10 kDa) and iron had no marked effect on expression. This result suggests that EW proteins are responsible for the induction of the hexonate genes during EW exposure. Experiments involving exposure to isolated EW proteins showed that that major EW protein affecting SEN1436 expression is lysozyme (14 fold) with little effect of ovotransferrin. Interestingly, low levels of EW (2.5%) completely inhibited growth of SE, and this effect was reversed by addition of iron suggesting that iron restriction is the dominant anti-microbial activity of EW with respect to SE.
Seventh Congress of the International BioIron Society Page 216 Poster Abstracts IBIS
Poster #102
INCREASED DUODENAL IRON ABSORPTION IN PATIENTS WITH CHRONIC HEPATITIS C AND NONALCOHOLIC STEATOHEPATITIS Koji Miyanishi, Masanori Sato, Shingo Tanaka, Akira Sakurada, Hiroki Sakamoto, Yutaka Kjawano, Toshifumi Hoki, Masayoshi Kobune and Junji Kato Department of Medical Oncology, Sapporo Medical University. School of Medicine Presented By: Koji Miyanishi, MD, PhD
Background: Hepatic iron accumulation is generally increased in chronic hepatitis C (CHC) and nonalcoholic steatohepatitis (NASH) liver. However, the precise mechanism of iron accumulation in the liver remains unclear. Hepcidin is a 25-amino acid peptide hormone primarily synthesized by hepatocytes. Hepcidin regulates serum iron level. In the duodenum, dietary iron is transported to enterocytes by the divalent metal transporter 1(DMT1). After entering enterocytes, iron is stored in ferritin or transferred to the portal blood by the basolateral exporter ferroportin 1 (FPN). FPN transports iron from the duodenum to the serum. No study has investigated the dynamics of iron absorption in patients with CHC. In this study, we evaluated iron absorption from the gastrointestinal tract in patients with CHC or NASH. We measured the expression of a panel of molecules associated with iron absorption in the duodenum and serum hepcidin level to analyze the mechanism of iron accumulation in the CHC or NASH liver. Methods: We recruited 24 patients with CHC and 25 patients with NASH. An oral iron absorption test (OIAT) was used, in which 100 mg of sodium ferrous citrate was administered to each individual. The serum level of hepcidin-25 was measured by liquid chromatography-tandem mass spectrometry. DMT1 and FPN mRNA was measured by RT-PCR. DMT1 and FPN protein was analyzed by Western blot. The samples were obtained duodenum biopsy tissues from each CHC patients and NASH. Caco-2/TC7 cells were incubated on Costar transwell (0.4μm pores). Results: The OIAT showed that absorption of iron from the gastrointestinal tract was increased significantly in CHC patients and NASH patients, compared to control subjects (p<0.001). Serum level of hepcidin-25 was significantly low in the CHC group and significantly high in the NASH group. Compared to the controls, duodenal FPN mRNA expression levels in patients with CHC were significantly up-regulated and median DMT1 mRNA expression levels in CHC groups showed no significant difference. Compared to the controls, duodenal DMT1 mRNA expression levels in patients with NASH were significantly upregulated and median FPN mRNA expression levels in NASH groups showed no significant difference. The FPN mRNA levels and protein levels increased significantly in Caco-2/TC7 cell monolayers cultured in transwells with hepcidin. Conclusions: These results indicated that lower serum hepcidin-25 level might up-regulate not only FPN protein expression but also mRNA expression in the duodenum and cause iron accumulation in patients with CHC. In spite of elevation of serum hepcidin, iron absorption from the gastrointestinal tract in patients with NASH increased through upregulation of DMT1.
Seventh Congress of the International BioIron Society Page 217 Poster Abstracts IBIS
Poster #103
THE NOVEL FINDING OF ELTROMBOPAG (ELT) IMPROVING PANCREATIC CELL FUNCTION BY INCREASING INSULIN PRODUCTION, CHELATING IRON AND SCAVENGING ROS Evangelia Vlachodimitropoulou, MBBS, BSc, P. Koonyosying, N. Cooper, B. Psaila, M. Sola-Visner and J. Porter UCl Presented By: Evangelia Vlachodimitropoulou, MBBS, BSc
Introduction: Eltrombopag (ELT) is an oral, nonpeptide, small-molecule thrombopoietin receptor (TPO-R) agonist known to efficiently treat chronic immune thrombocytopenic purpura (ITP) and thrombocytopenia in the presence of Hepatitis C. We have previously confirmed its ability to mobilise iron, and act as an iron shuttle when combined with current chelation therapy (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21), to improve current treatment for iron overload which is often slow and suboptimal. We here model the ability of ELT to improve pancreatic cell function by improving insulin production shown to be reduced by iron overload, chelate iron and reduce ROS. The above findings further support the concurrent use of ELT in the management of iron overload, particularly in patients who are diabetic or likely to have impaired glycaemic control secondary to iron overload. Methods: Cell line: RINm5F insulin-producing rat pancreatic cell line (LGC ATCC Sales, UK). Intracellular Iron: Cellular iron loading and iron mobilisation were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al. 2015, British Journal of Haematology). A four-fold increase in intracellular iron compared to control was obtained by serially treating cells with 10% FBS RPMI media in pancreatic cells over two ten hour periods (Figure 1A). The cells were then exposed to iron chelators/ELT, lysed and intracellular iron concentration determined via the ferrozine assay, normalised against protein content. Reactive oxygen species (ROS) estimation: To monitor the intracellular ROS, we utilised a cell-permeable oxidation-sensitive fluorescent probe 5,6-carboxy-2’,7’- dichlorofluorescein diacetate (DCFH-DA); (Molecular Probes, Leiden, Netherlands). Following iron loading, cells were pre-incubated with 6 mM H2DCF-DA for 30 minutes at 37°C. Chelators were added and the fluorescence of control and treated cells was read throughout the treatment period in the plate reader (excitation at 504 nm, emission at 526 nm). Results: Pancreatic cell iron loading with serial changes of media containing 10% FBS reduced cellular insulin production in a concentration-dependent manner (Figure 1B). Two changes of 10% FBS containing media, cellular iron is increased by 4 fold (Figure 1A) and insulin production reduced by 2.6 fold (Figure 1B). Iron loading with media containing 10% FBS (two ten-hour treatments) was comparable to treating with FAC for 24 hours (Figure 1A).When cells are treated with ELT at just 1μ , insulin production is restored back to normal levels, similar to D 1μ ( igure 1C). Monotherapy with ELT induced a dose-dependent iron removal from the m5 pancreatic cell line. At 10μ following 8 hours of treatment, ELT was able to mobilise approximately 40% of total cellular iron (Figure 2). In Figure 3 A, B and C we can see the comparison of iron mobilisation between ELT and commercially available chelators. The effect when ELT is used in combination with all three commercially available iron chelators is substantial. or example, when 10μ D P is combined with 3μ ELT, iron mobilisation increases by a further 17% when compared to DFP treatment alone (Figure 3B). Finally, ELT reduces ROS in iron-loaded cells in a concentration-dependent manner, in a similar fashion to DFO, DFX and DFP (Figure 4). Conclusions: To our knowledge this is the first demonstration of ELT improving pancreatic cell function by increasing insulin production, removing iron and reducing ROS. ELT concentrations used to achieve the above results are clinically achievable and unlikely to increase platelet counts in patients without thrombocytopaenia.
Seventh Congress of the International BioIron Society Page 218 Poster Abstracts IBIS
Poster #104
NEW FILM-COATED TABLET FORMULATION OF DEFERASIROX IS WELL TOLERATED IN PATIENTS WITH THALASSEMIA OR MYELODYSPLASTIC SYNDROMES: RESULTS OF THE RANDOMIZED, PHASE II ECLIPSE STUDY Ali T. aher¹, Raffaella riga², Silverio Perrotta³, Ale andra ouraklis⁴, Giovan Battista Ruffo⁵, Antonis attamis⁶, Ai-Sim Goh⁷, Annelore Cortoos⁸, icky Huang⁸, Marine Weill , Ra uel Merino Herranz and John B. Porter¹ ¹American University of Beirut Medical Center; ²University of Cagliari; ³Second University of Naples; ⁴University of Patras Medical School; ⁵U. .C. Ematolog. Con alassemia; ⁶University of Athens; ⁷Hospital Pulau Pinang; ⁸Novartis Pharmaceuticals Corporation; Novartis Pharma AG; ¹ University College London Presented By: John B. Porter, MA, MD, FRCP DRCPath
Background: The once-daily oral iron chelator, deferasirox (DFX) dispersible tablet (DT) has a well-defined safety and efficacy profile, and offers an alternative choice to parenteral deferoxamine. However, barriers exist to patient adherence to DFX DT, including gastrointestinal tolerability and palatability, resulting in the development of a film-coated tablet (FCT) formulation. The FCT contains the same active substance as DT but has different excipients, and can be taken with or without a light meal. The objective of the randomized, open-label, phase II ECLIPSE study was to evaluate safety of DFX FCT and DT formulations in patients with transfusion-dependent thalassemia (TDT) or IPSS-R very low-, low- or intermediate-risk myelodysplastic syndromes (MDS). Methods: Iron chelation therapy (ICT)- naïve or pre-treated patients aged ≥10 years, re uiring CT at D DT ≥30 mg/kg/day (TDT) or ≥20 mg/kg/day (MDS), with serum ferritin (S ) 1000 ng/mL, were eligible for enrollment. CT- naïve patients were requested to start on DFX DT 20 mg/kg/day or DFX FCT 14 mg/kg/day and ICT pre-treated patients were requested to start on DT or FCT dose equivalent to their pre-washout dose. FCT doses are 30% lower compared to DT doses due to improved bioavailability. Primary endpoint was overall safety over 24 weeks. Results: Of the 173 transfusion-dependent patients (TDT, n=140; MDS, n=32; missing, n=1) enrolled, 86 received DT (mean actual dose±SD, 27.5±7.7 mg/kg/day) and 87 received FCT (20.8±5.4 mg/kg/day). Adverse events (AEs) regardless of relationship with study drug were reported in similar proportions of patients for each formulation (Table 1). 61.6% (95% CI, 50.5–71.9%) of DT patients and 58.6% (95% CI, 47.6–69.1%) of FCT patients had at least one gastrointestinal AE on study. Constipation and vomiting were numerically lower in FCT patients compared to DT patients. FCT patients also experienced fewer severe gastrointestinal AEs (4.6%, FCT; 12.8%, DT) than DT patients. In patients receiving DT prior to study entry, fewer FCT patients had gastrointestinal related AEs (53.5%, FCT; 60.3%, DT), particularly diarrhea, vomiting and constipation. Notable laboratory evaluation frequencies were similar with both formulations (Table 2). Increased urine protein/creatinine ratio was reported in more FCT pts (12.8%, DT; 20.7%, FCT), which could be due to more FCT pts receiving a higher than recommended starting dose. Renal event frequency was similar when patients were started on a correct starting dose (30.9%, DT; 33.3%, FCT). 10/23 patients discontinued due to AEs (n=6, DT; n=4, FCT). SF was not a study endpoint and was assessed as supportive non-safety outcome. Conclusions: DFX FCT and DT safety profiles were consistent with the known DFX profile. DFX FCT may improve patient experience with ICT resulting in greater compliance, and reduced frequency and severity of iron overload related complications.
Seventh Congress of the International BioIron Society Page 219 Poster Abstracts IBIS
Poster #105
RENAL SAFETY AFTER MORE THAN A DECADE OF DEFERASIROX USE IN PATIENTS WITH TRANSFUSIONAL HEMOSIDEROSIS Raffaella Origa¹, Antonio Piga², Immacolata artaglione³, Giuseppina Della Corte⁴, Andreas Bruederle⁵, Jackie Han⁶, Chiara Castiglioni⁷ and Gian Luca Forni⁸ ¹University of Cagliari; ²University of urin; ³Second University of Naples; ⁴Universit di Milano; ⁵Novartis Pharma AG; ⁶Novartis Pharmaceuticals Corporation; ⁷Novartis Farma SpA; ⁸ spedale Galliera Presented By: Antonio Piga, MD
Background: Patients with transfusion-dependent anemias often require lifelong iron chelation therapy (ICT). Longer drug exposure increases the chance of complications, such as changes in renal function, which may be age-, disease- or drug-related. This study assessed long-term renal safety of deferasirox in Italian patients with transfusional hemosiderosis who were enrolled in the deferasirox registration studies and continued treatment for up to 10 or more years. Methods: Baseline and worst values were recorded during the prospective studies; values were collected retrospectively in 3-month periods from registration trial end until the latest patient assessment for serum creatinine (SCr), urinary protein/creatinine ratio (UPCR) and creatinine clearance (CrCl, if available). Primary objective was SCr trend over time. Secondary objectives included evaluation of notable renal function values (Figure) and renal AEs from registration study end to retrospective period end. Results: Of the 366 talian patients in the registration studies, 282 patients (mean SD age, 26.0 12.0 years) received ≥1 dose of deferasirox (mean±SD, duration of deferasirox exposure, 3.5±1.6 years; mean deferasirox dose, 1032.2±438.8 mg/day). In the retrospective period (215 [76.2%]), mean±SD duration of deferasirox exposure was 6.1±2.8 years and mean deferasirox dose was 1385.6±500.0 mg/day. 63.5% of patients received other ICT (deferoxamine, 158 [56.0%]; deferiprone, 136 [48.2%]; FBS0701, 11 [3.9%]; deferitrin, 2 [0.7%] during the retrospective period either as combination therapy or alone. In the overall population, mean SCr and UPCR values were within normal limits and stable over time during the retrospective period (Figure). In the retrospective period, regardless of ICT received, renal AEs were reported in 86 (30.5%) pts, severe renal AEs in 7 (2.5%) pts. 54.2% of pts with a renal AE took ≥1 concomitant medication associated with potential nephrotoxicity. The most common renal AEs were nephrolithiasis (n=31, 11.0%), renal colic (n=28, 9.9%), increased/abnormal UPCR (n=16, 5.7%), increased blood creatinine (n=12, 4.3%) and proteinuria (n=13, 4.6%). 8 (2.8%) pts had 10 serious renal AEs: nephrolithiasis, renal colic (n=2 each), increased blood creatinine, acute kidney injury, hematuria, hydronephrosis, renal failure, urethral stenosis (n=1 each); at the time of serious AE pts were receiving deferasirox (n=4), deferoxamine or deferiprone (n=3) or no ICT (n=1). Of 86 pts who had renal AEs, 33 (11.7%) were suspected to be deferasirox-related, most commonly increased UPCR (n=14, 5.0%), proteinuria (n=11, 3.9%) and increased blood creatinine (n=8, 2.8%). 5 (1.8%) pts discontinued because of renal AEs: increased blood creatinine, glycosuria (n=2 each), proteinuria (n=1). Conclusions: This is the longest follow-up analysis in patients with transfusion-dependent anemias available today and indicates stable renal function and a lack of any progressive worsening of renal function during long-term deferasirox treatment (3.5 years registration studies; 6.1 years retrospective period). Results support a favorable long-term risk:benefit renal safety profile for deferasirox in the treatment of transfusional iron overload.
Seventh Congress of the International BioIron Society Page 220 Poster Abstracts IBIS
Poster #106
MEASUREMENT OF HEPCIDIN ISOFORMS IN HUMAN SERUM BY LIQUID CHROMATOGRAPHY WITH HIGH RESOLUTION MASS SPECTROMETRY (LC-HR-MS) Simon Handley¹, Lewis Couchman, MSc¹, Paul Sharp, PhD², Iain Macdougall, PhD³ and Caje Moniz, PhD⁴ ¹Toxicology Unit, Viapath Analytics; ²Diabetes and Nutritional Sciences Division, King's College London; ³Department of Renal Medicine, ing s College Hospital; ⁴Department of Clinical Biochemistry, ing s College Hospital Presented By: Simon Handley
Hepcidin-25 acts as the master regulator of iron homeostasis by binding to and degrading ferroportin. A number of N- truncated isoforms of hepcidin-25 have been identified (hepcidin-20, -22, and -24), although data on the concentrations of these isoforms encountered in patient samples is scant. Furthermore, a validated method for the measurement of these isoforms has not been published to date. We present a fully validated LC-HR-MS method for the measurement of hepcidin-25 and truncated isoforms in human serum, which has been applied to 47 paired human plasma (BD Vacutainer dipotassium EDTA) and serum samples (BD Vacutainer SST II Advance) from hospitalised patients. Samples (200 µL) 13 15 were mixed with internal standard [( C8, N3)-hepcidin-25, 10 µL], and 0.1 % (v/v) aqueous formic acid (600 µL). Following vortex mixing (5 min), and centrifugation (5 min, 16,060 x g), the supernatants were loaded into wells of a Waters Oasis HLB µElution 96-well SPE plate under vacuum. Each well was then washed sequentially with (i) deionised water (200 µL), and (ii) 0.1 % (v/v) aqueous formic acid (200 µL). Analytes were eluted under vacuum with 70 % (v/v) acetonitrile in deionised water containing 1 % (v/v) TFA (60 µL). After elution, 0.1 % (v/v) aqueous formic acid (70 µL) was added to each extract. Prepared samples (100 µL) were injected onto an ACE C18 analytical column (100 x 1.0 mm, 3 µm). Detection was by positive heated-electrospray-ionisation, in full-scan mode (400–1000 m/z, ThermoFisher Scientific Q Exactive). Summed ion counts for the [M+3H]3+, [M+4H]4+, and [M+5H]5+ charge states were extracted for assay calibration and quantitation. Calibration (hepcidin-20, -22, -24; 1–100 µg/L, hepcidin-25; 1–200 µg/L), and internal quality control solutions (hepcidin-20, -22, -24; 4, 8, and 25 µg/L, hepcidin-25; 8, 75, and 150 µg/L) were prepared in charcoal- stripped human serum. Calibration was linear over the range studied, and the lower limit of quantitation was 1 µg/L for all analytes. Intra- and inter-assay accuracy were ± 20 % of the nominal concentration, and imprecision was < 20 % at each IQC concentration. There was a good correlation (R2 > 0.90) for all analytes between human plasma and serum. Hepcidin-20, -22, -24, and -25 were detected in 77, 47, 53, and 89 % of all samples. The median (range) serum hepcidin- 20, -22, -24, and-25 concentrations measured in these samples were; 4 (1–40), 8 (2–20), 8 (1–50), and 39 (1–334) µg/L, respectively. There was a good correlation between hepcidin-25 and hepcidin-24 in human serum (r = 0.88, p = < 0.05), but less so between hepcidin-25, and hepcidin-20 (r = 0.70, p = < 0.05), and hepcidin-22 (r = 0.57, p = 0.05). In 12 samples where hepcidin-25 was detected (median 98, range 15–133 µg/L), all other isoforms were also detected, median (range), hepcidin-20; 10 [2–34], hepcidin-22; 6 [2–21], and hepcidin-24; 16 [3–26] µg/L. In no sample was hepcidin-20, - 22, or -24 detected in the absence of hepcidin-25 A precise, accurate, and sensitive LC-HR-MS assay has been developed for all available hepcidin isoforms in human serum. There was a good correlation for all analytes between human plasma and serum. Further studies are required to ascertain the clinical and diagnostic relevance of hepcidin-20, - 22, and-24.
Seventh Congress of the International BioIron Society Page 221 Poster Abstracts IBIS
Poster #107
HEPCIDIN ISOFORM CONCENTRATIONS IN SERUM SAMPLES FROM PATIENTS WITH CHRONIC KIDNEY DISEASE Simon Handley¹, Paul Sharp, PhD², Ca e Moniz, PhD³ and Iain Macdougall, PhD⁴ ¹Toxicology Unit, Viapath Analytics; ²Diabetes and Nutritional Sciences Division, King’s College London; ³Department of Clinical Biochemistry, ing s College Hospital; ⁴Department of Renal Medicine, ing s College Hospital Presented By: Simon Handley
Hepcidin is a 25-amino acid polypeptide considered to be the critical regulator of iron homeostasis. Plasma hepcidin-25 is raised in patients with chronic kidney disease (CKD), and in patients undergoing haemodialysis (HD). N-truncated isoforms of hepcidin-25 have been identified (hepcidin-20, -22, and -24); however data on the concentrations of these isoforms in patients with CKD not requiring haemodialysis, and those undergoing haemodialysis is scant. We used a validated liquid chromatography-high resolution-mass spectrometry based assay to investigate hepcidin isoform concentrations in serum from 72 patients with CKD not requiring haemodialysis, and in serum from 34 CKD patients taken pre-, and post-haemodialysis. Hepcidin isoforms were also measured in 47 serum samples from healthy volunteers. Ferritin, iron, TIBC, and creatinine were measured on every sample. The median (range) hepcidin-25 concentration in healthy volunteers was: 8 (< 1–31) µg/L, hepcidin-20 -22, or -24 were not detected (LLoQ: 1 µg/L) in any sample. In samples from patients with CKD not requiring dialysis [median (range) creatinine: 144 (54–333) µmol/L], the median (range) hepcidin-25 concentration was 14 (2–134) µg/L. Hepcidin-20 and -24 were detected in 75, and 19 % of these samples respectively. Hepcidin-22 was not detected in any sample from patients with CKD. Median (range) hepcidin-20, and 24 concentrations measured in CKD patients not requiring dialysis were 4 (1–31) µg/L, and 3 (2–12) µg/L, respectively. There was a positive correlation between hepcidin-25 and ferritin (r = 0.40, p = < 0.05), and a negative correlation with TIBC (r = -0.61, p = < 0.05). There was no correlation between hepcidin-25 and creatinine (r = -0.25, p = 0.07), and serum iron (r = -0.25, p = 0.05). In pre-dialysis samples the median (range) hepcidin-25 concentration was 50 (11–234) µg/L, post-dialysis this had declined to 35 (4–143) µg/L. Hepcidin-20, -22, and-24 were detected in 72, 8, and 25 % of samples taken pre-dialysis, and the median (range) concentrations measured were; 17 (4–129), 19 (9–32), and 9 (5– 51) µg/L, respectively. Post-dialysis all hepcidin isoforms had declined by some 40 %. In both patient groups, hepcidin-25 was raised as compared to healthy volunteers. N-truncated isoforms of hepcidin-25 were identified in patient samples, and serum concentrations of all hepcidin isoforms were significantly lower post-dialysis. The clinical importance of N- truncated isoforms of hepcidin-25 is not clear and requires further investigation.
Seventh Congress of the International BioIron Society Page 222 Poster Abstracts IBIS
Poster #108
HEPCIDIN IS AN HDAC3 REGULATED GENE AND ITS EXPRESSION IS DETERMINED BY PROMOTER- ASSOCIATED HISTONE ACETYLATION Sant-Rayn Pasricha, MBBS MPH PhD, Pei Jin Lim, Tiago Duarte, Carla Casu, Kasia Mleczko-Sanecka, Maria Suciu, Joao Areas, Ana Rita Da Silva, Joe Frost, Kirsty McHugh, Graca Porto, Simon Draper, Sarah Gooding, Peter Olinga, Martina Muckenthaler, Stefano Rivella, Jim Hughes, Tom Milne, Andrew Armitage and Hal Drakesmith MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented By: Sant-Rayn Pasricha, MBBS, MPH, PhD
Systemic iron homeostasis is coordinated by the hepatic hormone hepcidin, which is primarily transcriptionally regulated. Epigenetic regulation of hepcidin transcription has not been studied in detail. We now demonstrate that epigenetic events at the hepcidin locus regulate its expression. In mice, increased erythropoietic drive and iron deficiency suppress hepcidin via distinct erythroferrone-dependent and -independent mechanisms respectively, but both states involve reversible loss of the activation-associated histone marks H3K9ac and H3K4me3 at the hepcidin locus. The pan-histone deacetylase (HDAC) inhibitor panobinostat maintains H3K9ac at hepcidin-associated chromatin and prevents hepcidin suppression by erythropoietin and iron deficiency in vivo, demonstrating that histone deacetylation is required for gene suppression. Panobinostat raises hepcidin expression and potentiates effects of BMP in both Huh7 cells and primary liver slices. Panobinostat also rescues hepcidin expression in murine models of hemochromatosis and thalassemia that exhibit pathologic hepcidin suppression. Pharmacologic screens of HDAC-specific inhibitors along with re-analysis and validation of a genome-wide siRNA knockdown screen in Huh7 cells demonstrate implicate HDAC3 in hepcidin regulation. The HDAC3 specific inhibitor RGFP966 and knockdown of HDAC3 both increase hepcidin expression in vitro, along with the recognised HDAC3 canonically-regulated gene CDKN1A. HDAC3 knockdown ameliorates hepcidin suppression caused by erythroferrone or BMP signaling blockade in vitro. Double knockdown of HDAC3 and its co-factor NCOR1 further enhances hepcidin expression. In iron-deficient mice, treatment with RGFP966 rapidly increased hepcidin without affecting Bmp6 or Bmp target genes, suggesting a direct effect of HDAC3 on hepcidin expression in vivo. HDAC3 has been previously implicated in regulation hepatic lipid and energy metabolism. Collectively, our data indicate that epigenetic modifications at the hepcidin locus control its pathophysiological expression, HDAC3 appears to directly regulate hepcidin expression, and manipulation of epigenetic modifiers may offer therapeutic opportunities.
Seventh Congress of the International BioIron Society Page 223 Poster Abstracts IBIS
Poster #109
THE ROLE OF HEPCIDIN IN RENAL FAILURE Alejandra García¹, Ana Lopez, Resident², Maria Dolores Pulfer, Resident², Maria Josefa moran, Ph², Maria Isabel Moreno, Ph² and Alejandro Del Castillo, Satff² ¹University hospital Gregorio Marañon; ²Gregorio Marañon University Hospital, Spain Presented By: Alejandra Garcia
Introduction: Hepcidin (HEPC) is a peptide hormone produced by the liver in response to iron concentrations. It is physiologically increased by high levels of iron and decreased in erythropoiesis to ensure iron supply, so that it regulates iron homeosthasis. It is pathologically increased in inflammatory states, so it can be considered as an acute phase reactant. In this study we try to stablish a correlation between hepcidin levels and renal function. Methods: We compared ferritin and hepcidin levels in a group of patients who were interned in the internal medicine department due to different causes. We stablished different groups depending on the renal function (Glomerular filtration rate (GFR) measured by MDRD-4). Two groups were determined according to GFR: patients with normal renal function (GFR> 60 ml/min) belonged to the first group and patients with mild to moderate kidney disease (GFR 30-60 ml/min) to the second group. No patients with severe chronic kidney disease were included in our study. Serum levels of hepcidin were determined in batches using a commercial en yme linked immunosorbent kit (D nstruments, mb , Marburg, Germany). The statistical analysis was made with SPSS version 21.0 software. The correlation between both groups was compared using the U-Mann-Withney test. We considered statistical significancy p< 0,05. Results: 83 patients were included in our study. 64 patients (77%) were included in the first group (normal renal function) and 19 patients were included in the second one. Regarding to the ferritin levels, two determinations had to be excluded because of their extremely high values. In the first group the median ferritin level was 151 µg/l, and in the second one was 90.50 µg/l (p= 0.376). In relation to the hepcidin measurements, in the normal renal function group the median hepcidin level was 9 vs 10.5 in the mild-to moderate kidney disease group (p= 0.57). No statistically significant differences were found in our study. Conclusions: In this study no statistically significant differences were found comparing hepcidin levels between normal renal function vs mild to moderate kidney disease. Additional comorbidities and the limited sample could be possible explanations for this result. However, further studies with a wider sample could clarify this possible relationship more accurately.
Seventh Congress of the International BioIron Society Page 224 Poster Abstracts IBIS
Poster #110
IS HEPCIDIN AN INFECTION MARKER? Maria Dolores Pulfer¹, Ana López, Resident², Alejandra García, Resident², Maria Josefa Moran, Ph², Maria Isabel Moreno, PH² and Alejandro Del Castillo, staff² ¹Gregorio Marañón Universitary Hospital; ²University hospital Gregorio Marañon, Spain Presented By: Maria Dolores Pulfer
Introduction: Hepcidin is a peptide hormone produced by the liver in response to iron concentrations. It is physiologically increased by high levels of iron and decreased in erythropoiesis to ensure iron supply, so that it regulates iron homeosthasis. It is pathologically increased in inflammatory states in order to limit extracellular iron availability to microbes. In this study we compared hepcidin levels in infected patients vs non-infected patients recruited from an internal medicine unit to test its usefulness as an infection marker. An infectious state was considered if the patient had positive cultures, classic infectious symptoms and positive classic infection markers. The most common localizations were urine infection and pneumonia (about 80% of the patients). Methods: We compared ferritin and hepcidin levels in a group of infected patients with non-infected ones (patients who were interned in the internal medicine department of a tertiary hospital due to other causes). Serum levels of hepcidin were determined in batches using a commercial en yme linked immunosorbent kit (D G Instruments, GmbH, Marburg, Germany). The statistical analysis was made with SPSS version 21.0 software. The correlation between both groups was compared using the U-Mann-Withney test. We considered statistical significancy p< 0,05. Results: 81 patients were included in our study, 28 patients in the infected group and 53 in the non-infected one. Regarding to the ferritin levels, two determinations had to be excluded because of their extremely high values. In the first group the median ferritin level was 233.00 µg/l, and in the second one was 325.00 µg/l (p=0,768). In relation to the hepcidin comparison study, in the infected group the mean hepcidin level was 21.00 vs 29.49 in non-infected ones (p= 388). No statistical significancy was observed in any of the analysis. Conclusions: In this study we can conclude that hepcidin is not a good infection marker as no statistically significant differences were found between both groups. One possible explanation is that our patients had a lot of comorbidities that could interfere in hepcidin levels (anemia, chronic liver disease, etc). However further studies with a wider sample of patients could clarify role of hepcidin in infection.
Seventh Congress of the International BioIron Society Page 225 Poster Abstracts IBIS
Poster #111
FERROPORTIN Q248H MUTATION INDUCES AUTOPHAGY AND FERRITINOPHAGY Guelaguetza Vazquez-Ortiz, PhD, Andrey Ivanov, MD PhD, Xionghao Lin, PhD, Namita Kumari, PhD and Sergei Nekhai, PhD Howard University Presented By: Sergei Nekhai, PhD
Introduction: Ferroportin (FPN) Q248H mutant is the only variant present in African and African American populations with a prevalence of 2.2% to 13.4%. We showed that this mutation has a hepcidin resistant phenotype, but the effect of this mutation on iron homeostasis and cellular metabolism is not fully understood. Thus analysis of FPN Q248H is of great interest not only as fundamental for iron physiology in Africans and African Americans but also for possible drug development. Methods: To determine the effect of FPN Q248H on intracellular iron, we expressed FPN-EGFP and FPN-Q248H-EGFP in HEK293 cells and measured cellular ferritin and labile iron. To determine whether Q248H mutation affects FPN ubiquitination, we immunoprecipitated EGFP-fused FPN with anti-EGFP antibodies and analyzed its ubiquitination using MS/MS analysis. To visualize the effect of FPN-Q248H-GFP mutation in the cells, we used confocal microscopy to detect FPN and lysosomes stained with LysoTracker (Invitrogen). We tested the effect of FPN Q248H mutation on the autophagy by analyzing the expression of GABARAP1, LC3, p62, TRIM5 and TRIM22 in the presence and absence of the proteasome inhibitor MG-132 and the autophagy inhibitor Bafylomicin. Finally, we measured distribution of Cathepsin D, a lysosome contained enzyme, between lysosomes and cytosol. Finally, we measured cytochrome C expression to check mitochondrial integrity. Results: As previously shown, FPN Q248H demonstrated delayed kinetics of degradation in hepcidin treated cells. Moreover, hepcidin treatment led to no changes in the intracellular iron in FPN Q248H cells in contrast to the WT FPN expressing cells that showed an increase in labile iron levels. There was not increased of ferritin in FPN-Q248H cells compared to WT FPN cells. FPN was found to be ubiquitinated on K240, and, unexpectedly, ubiquitination of FPN Q248H was increased, even in the absence of hepcidin treatment. Cells expressing FPN Q248H showed about twice more lysosomes than the cells expressing WT FPN. After hepcidin treatment, the number of lysosomes in FPN Q248H cells was further increased. This observation was supported by the increased expression of a lysosme membrane-incorporated protein, LAMP1. These results indicated that FPN Q248H might induce autophagy. We found autophagy to be significantly upregulated in the cells expressing FPN Q248H comparing to the WT FPN. Hepcidin treatment further induced autophagy. Similar upregulation of NCOA4 protein was observed in FPN Q248H. The autophagy related proteins were expressed to higher levels when proteasome was blocked and the levels of ferritin were further reduced. Finally, we found more Cathepsin D the cytoplasm of FPN-Q248H cells and also detected increased cytochrome C expression. Conclusions: Our results indicated that FPN Q248H induces autophagy and promotes ferritinophagy, based on the upregulation NCOA4, a selective cargo receptor for ferritin. Our results also indicated that mitochondria are getting damaged in FPN Q248H cells which could be due to the leakage of Cathepsin D from lysosomes. Increased autophagy can be triggered by ubiquitination of FPN Q248H and function as an “emergency pathway” to eliminate iron by selective ferritinophagy, bypassing proteasomes. As autophagy is a pro-survival mechanism that provides intermittent energy source and continuously disposes damaged organelles, this pathway could also increase cell survival and prevents iron toxicity. Acknowledgments: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.
Seventh Congress of the International BioIron Society Page 226 Poster Abstracts IBIS
Poster #112
TP-0184 IS AN INHIBITOR OF ALK2 AND A POTENTIAL THERAPEUTIC FOR THE TREATMENT OF ANEMIA OF CHRONIC DISEASE Peter Peterson, PhD, Wontak Kim, BS, Hillary Haws, MS, Clifford Whatcott, PhD, Adam Siddiqui-Jain, PhD, David Bearss, PhD and Steven Warner, PhD Tolero Pharmaceuticals, Inc. Presented By: Clifford Whatcott, PhD
Chronic inflammatory diseases, such as cancer or rheumatoid arthritis, commonly lead to a debilitating functional anemia, often referred to as anemia of chronic disease (ACD). Persistent T β/BMP receptor family signaling, or more specifically activin receptor-like kinase 2 (ALK2) signaling, is thought to play a significant role in the development of this anemia. Activation of ALK2 leads to SMAD family transcription factor-mediated gene expression, which includes induction of the liver peptide hormone, hepcidin. Increased hepcidin promotes the degradation of the iron transporter, ferroportin, which then leads to reduced iron bioavailability and subsequent anemia. Treatment of ACD requires transfusions, intravenous iron, or erythropoietin-based therapies. Unfortunately, these approaches often do not address the underlying pathology. We hypothesized that lowering hepcidin levels via inhibition of the ALK2 kinase would be a potential therapeutic approach for the treatment of ACD. We have developed a novel small-molecule, selective ALK2 inhibitor, TP-0184, which achieves an IC50 of 5 nM in biochemical assays. In this report, we demonstrated robust in vitro and in vivo activity of TP-0184 in multiple models. In cultured cells, we saw robust inhibition of SMAD signaling and hepcidin expression at concentrations of 100-300 nM. In the turpentine oil mouse model for inflammation-induced ACD, we observed a 14-fold induction of liver hepcidin with the administration of turpentine oil. TP-0184 pre-treatment largely abrogated this induction, resulting in a 92% reduction in turpentine oil-mediated stimulation. In the TC-1 model for cancer-induced ACD, injection of TC-1 lung cancer cells into nude mice results in anemia, manifested in a 2.6-fold increase in liver hepcidin. TP-0184 treatment, however, completely abrogated the induction of hepcidin at doses as low as 25 mg/kg. Finally, we also interrogated TP- 0184 in the heat-killed Brucella abortus (HKBA) model for ACD. Following intraperitoneal injection with HKBA, mice experienced a transient increase in plasma hepcidin levels that resulted in anemia manifested in significant serum iron and hemoglobin (HGB) loss. Treatment with TP-0184, even following onset of HKBA-induced anemia, results in a 66.5% reduction in the HKBA-mediated HGB loss. Plasma and liver pharmacokinetic studies in mice revealed that TP-0184 has a high distribution to the liver. In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Taken together, our data support the targeting of ALK2 as a potential approach for the inhibition of ACD, as well as the development of TP-0184 as a therapeutic option for patients with ACD.
Seventh Congress of the International BioIron Society Page 227 Poster Abstracts IBIS
Poster #113
MATERNAL HEPCIDIN DETERMINES FETAL IRON HOMEOSTASIS DURING PREGNANCY V. Sangkhae, A.L. Fisher, K. Chua, V. Gabayan, E. Valore, T. Ganz and E. Nemeth University of California, Los Angeles Presented By: Veena Sangkhae, PhD
Disorders of iron homeostasis, including iron deficiency and iron deficiency anemia, are very common during pregnancy and are associated with adverse outcomes. Despite its importance, little is known about the basic physiology of maternal and fetal iron regulation during pregnancy. In human pregnancy, maternal hepcidin levels are suppressed, presumably to allow for greater iron absorption and mobilization from stores, and thus increased iron supply to the placenta and the fetus. Of note, ferroportin (FPN) is also expressed on placental trophoblasts where it transports iron into the fetal circulation, but because it is localized facing fetal circulation, placental FPN is thought to be under the direct control of fetal but not maternal hepcidin. We confirmed that similarly to humans, maternal hepcidin concentrations in the mouse model progressively decrease during pregnancy. In mice fed standard chow (185ppm iron), time-course analysis between E12.5 and birth indicated that maternal hepcidin suppression occurs prior to the changes in other iron parameters. Maternal hepcidin suppression was followed by the mobilization of liver iron stores which allowed maintenance of stable serum iron levels throughout the pregnancy. We then examined the relative contribution of maternal vs embryo hepcidin to the control of placental iron transport. Hamp1-/- (KO) or Hamp1+/- (HET) females were crossed with Hamp1+/- males to generate combinations of mothers and offspring that lack hepcidin or not. Neonatal liver iron concentrations were measured as an indicator of the amount of iron transferred across placenta. We found that maternal, but not embryonic, hepcidin determined iron transfer across placenta: pups from iron-overloaded KO mothers had higher liver iron concentrations than pups from HET mothers. The effect of maternal genotype was highly significant (p<0.001 by two-way ANOVA), with no significant effect of pup genotype. Staining of E18.5 embryo livers also showed greater iron loading of pups from KO mothers than HET mothers, irrespective of embryo genotype. Similarly, placental iron at E18.5 was significantly higher in hepcidin KO mothers than HET mothers. The lack of effect of embryo-derived hepcidin may be due to the low expression levels of embryonic hepcidin, too low to exert an observable effect on iron transport. To determine how essential maternal hepcidin suppression is for fetal iron homeostasis, we generated a model with high maternal hepcidin activity. We administered a bioactive hepcidin peptide mimetic to pregnant mice during the second and third trimester. Compared to control mothers injected with solvent, the hepcidin mimetic caused severe maternal iron restriction resulting in anemia in mothers and pups, low birth weight, and even increased mortality of mouse embryos. Our studies confirm that appropriate maternal hepcidin levels during pregnancy are essential for fetal iron homeostasis.
Seventh Congress of the International BioIron Society Page 228 Poster Abstracts IBIS
Poster #114
DEVELOPMENTAL ROLES FOR THE IRP1-HIF2Α AXIS AND IRP1 ALONE IN THE CONTROL OF ERYTHROPOIESIS AND IRON METABOLISM Gretchen Seim, James Votava, Chris Nizzi and Rick Eisenstein University of Wisconsin - Madison Presented By: Richard Eisenstein, PhD
Previous studies by our lab and others has shown that IRP1 is critical for coordinating erythropoiesis and iron metabolism. In Irp1-/- mice fed a normal diet, HIF-2 synthesis is derepressed leading to inappropriate induction of its prototypical gene target erythropoietin (Epo). The enhanced Epo expression leads to polycythemia in young mice (4-10 wks old) that subsequently resolves. To define the developmental signals acting through IRP1 during the early peri- and post-natal period a detailed kinetic characterization of the Irp1-/- phenotype was undertaken. Erythropoiesis, as examined by flow cytometry, was inappropriately activated in IRP1 deficient mice as early as 3 weeks of age with the strongest effect noted in spleen. These changes in erythroblast differentiation were similar to that of wild-type mice acutely treated with erythropoietin (Epo). The increase in erythropoiesis was preceded with what appears to be a pulse of Epo gene expression that was strongest at one week of age (~100-fold increase in renal Epo mRNA) indicating early postnatal HIF- 2 hyperactivity. The increase in renal Epo mRNA at this time in Irp1-/- mice was coincident with a 2-fold activation of IRP1 binding activity in wildtype kidney. Strikingly, dysregulation of liver Fe stores and the Fe regulator hepcidin was observed at d1 in Irp1-/- mice, before clear evidence of enhanced erythropoiesis. Irp1-/- d1 pups had significantly lower total body iron and liver non-heme Fe and a 17-fold reduction in liver hepcidin mRNA level when compared to age matched Irp1+/+ neonates. Our data suggests that in addition to its role in controlling renal and hepatic HIF-2 synthesis, IRP1 is critical for proper distribution of Fe in the neonate, a role that appears to be independent of the IRP1-HIF-2 axis. (Supported by NIH DK66600)
Seventh Congress of the International BioIron Society Page 229 Poster Abstracts IBIS
Poster #115
LOW HEPCIDIN LEVELS CAN COMPENSATE FOR THE LACK OF A FERROPORTIN ALLELE Sandro Altamura, PhD¹,⁴, Julia Schmidt¹,⁴, at a Muedder¹,⁴, Bruno Galy², Matthias Hentze³,⁴ and Martina Muckenthaler¹,⁴ ¹Department of Pediatric Hematology, Oncology and Immunology - University of Heidelberg, Heidelberg, Germany; ²Division of Virus-associated Carcinogenesis, German Cancer Research Centre , Heidelberg, Germany; ³European Molecular Biology Laboratory, Heidelberg, Germany; ⁴Molecular Medicine Partnership Unit, Heidelberg, Germany Presented By: Sandro Altamura, PhD
The hepcidin-ferroportin regulatory circuitry is crucial to maintain physiological systemic iron levels. Mutations that confer ferroportin resistance to hepcidin binding cause uncontrolled iron export and systemic iron overload. Conversely, mutations that affect ferroportin protein stability or its targeting to the cell surface cause iron retention in iron exporting cells and reduced systemic iron levels. Although having a functional ferroportin protein is clearly critical for keeping the body iron balance, nonsense mutations or translocations into the ferroportin locus have not been identified so far. Here we analyzed mice with a heterozygous disruption of the ferroportin allele (Slc40a1wt/trp). In this model hematological parameters and plasma iron levels are unaffected, while plasma hepcidin levels are dramatically decreased. Low hepcidin expression is explained by hepatic iron deficiency and decreased activity of the BMP/SMAD signaling pathway. The decrease in hepatic iron levels is already manifest at the fetal stage and could possibly reflect inadequate maternofetal iron exchange when one functional ferroportin allele is missing. We further demonstrate that decreased hepcidin levels in adult Slc40a1wt/trp mice allow for ferroportin protein expression in duodenal enterocytes and splenic macrophages that is similar to wild-type mice. As a consequence sufficient plasma iron is available to satisfy the demand for erythropoiesis. Taken together, our results show that in Slc40a1wt/trp mice hepcidin production is controlled by the hepatic iron content rather than systemic iron levels and that low hepcidin expression can compensate for the lack of one ferroportin allele. This possibly explains why patients with heterozygous ferroportin null mutations have not been reported.
Seventh Congress of the International BioIron Society Page 230 Poster Abstracts IBIS
Poster #116
LIPOSOME ENCAPSULATED DEFEROXAMINE (LDFO) HAS A HIGH MOLAR EFFICIENCY IN REMOVING IRON IN AN IRON-OVERLOADED MOUSE MODEL Mark Hayes, PhD¹, David Tran, PhD², Max Petersen, BSc², Charles Noble, PhD², Zhipeng Dai, PhD², Peter Working, PhD² and Francis Szoka, PhD³ ¹ZoneOne Pharma Inc; ²ZoneOne Pharma, Inc; ³ZoneOne Pharma, Inc and UCSF Dept. Bioengineering and Therapeutics Presented By: Charles Noble, BSc, PhD
Introduction: To manage the toxic effects of iron-overload, β-thalassemia patients are prescribed iron chelators, administered either orally or infused on a daily basis. One strategy to more effectively deliver chelators to the sites of iron accumulation is to encapsulate them in liposomes. Liposomes also accumulate in the liver, spleen and bone marrow; therefore LDFO targets the chelator directly to the iron stores. We tested the hypothesis that the LDFO approach provides highly efficient (moles chelator administered/moles iron removed) iron chelation in an iron-overloaded mouse model. Methods: A comparison of various iron-overloading protocols was performed. We prepared two LDFO formulations composed of (hydrogenated soy phosphatidylcholine, HSPC)/cholesterol (60/40) or (palmitoyloleoylphosphatidylcholine, POPC)/cholesterol (55/45). Efficacy was evaluated in CF-1 mice overloaded with iron dextran. The LDFO formulations were administered IV three times at 200 mg/kg at 14-day intervals. A control group (n=10) of non-encapsulated DFO was administered by SC infusion from an implantable minipump at 20 mg/kg/day for a total dose of 280 mg/kg DFO over 14 days. The iron concentration in urine and feces was measured by a modified ferrozine-based spectroscopic assay. Results: The HSPC/cholesterol and POPC/cholesterol LDFO liposomes had 88 nm and 119 nm diameters and encapsulated 354 and 266 g DFO/mole phospholipid respectively. At 6 and 24h post IV injection, there is 67.0% and 27.2% ID DFO in plasma for HSPC liposomes and 54.2% and 18.1% ID DFO in plasma for POPC liposomes. In treating iron-overloaded mice, IV administered LDFO removed 2.3-2.8 times more iron than deferoxamine mesylate (DFO) given by SC continuous infusion. After LDFO treatment, iron was continuously eliminated for 14 days post dosing. At day 14 LDFO-HSPC and LDFO-POPC cumulatively had a 3.1 and 3.5-fold higher iron elimination in urine compared to SC infused DFO and 1.8 and 1.3-fold higher in feces respectively, corrected for background iron using the saline control group. The second and third dose of LDFO at 14-day intervals showed similar iron elimination patterns to the first dose. The iron removal efficiencies were 68% for LDFO-HSPC, 55% for LDFO-POPC and 24% for Free DFO. The liposome composition shifted the relative iron elimination profiles within the liposome groups. Of the two formulations, LDFO-HSPC produced more fecal iron removal while the LDFO-POPC group gave higher urinary iron removal. The iron elimination curves for both liposome formulations were statistically different than the infused DFO curve (p<0.0001) when analyzed by a regression and covariance analysis. At study end, liver and renal function markers were normal. Of the iron-overloading protocols examined, iron dextran dosed IV provided the most reproducible and consistent loading. However, the iron- dextran mouse model of iron overload presented some challenges, including delayed iron bio-assimilation times and high levels of background iron eliminated in feces of control animals. Conclusions: In the iron-dextran overload mouse model, a single injection of LDFO greatly accelerates iron elimination compared to two weeks of continuous SC infused DFO at similar cumulative dosages. If validated clinically, the high molar efficiency of iron removal of LDFO could lead to an improved treatment for patients suffering from iron overload conditions.
Seventh Congress of the International BioIron Society Page 231 Poster Abstracts IBIS
Poster #117
HUMAN MACROPHAGE FERROPORTIN BIOLOGY AND THE BASIS FOR THE FERROPORTIN DISEASE Manuela Sabelli, Giuliana Montosi, Cinzia Garuti, Angela Caleffi, Stefania Oliveto, Stefano Biffo and Antonello Pietrangelo Division of Internal Medicine and Center for Hemochromatosis, University Hospital of Modena, Italy Presented By: Antonello Pietrangelo, MD, PhD
Ferroportin (FPN1) is the sole iron-exporter in mammals but its cell–specific function and regulation are still elusive. The Ferroportin Disease (FD), an autosomal dominant disorder due to lack-of-function mutations of FPN, is characterized by marked iron accumulation in tissue macrophages, preserved intestinal iron transfer, and subclinical anemia. The molecular basis and mechanisms of the FD have not been clarified so far. We aimed at studying FPN1 expression in human macrophages, the cells that mostly contribute on a daily basis to plasma iron turnover and are central in the pathogenesis of the disease due to lack-of-function FPN1 mutations, the Ferroportin Disease (FD). We characterized FPN1 protein expression and traffic by confocal microscopy, western blot, gel-filtration and immunoprecipitation studies in macrophages from blood donors and patients with either lack-of-function p.A77D, p.G80S and p.Val162del or p.A69T gain-of-function FPN1 mutations. In gel-filtrations studies FPN1 partitioned in the expected ~ 65 kDa MW form and in a larger complex, ≥ 250 kDa, e.g. solubili ed microsomal membranes. Other M forms detected by western-blot were likely artifactual post-lysis aggregates and disappeared upon heating or in the presence of reducing agents. In donor macrophages, we found that FPN1 cycles in the early endocytic compartment, and is promptly degraded by hepcidin, its physiologic inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for higher accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines and similarly to donor macrophages, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to hepcidin. On the other hand, when FD macrophages were exposed to large amount of heme iron, differently from donor and p.A69T macrophages, FPN1 could no longer reach the cell surface leading to intracellular iron retention. Even during strong induction of protein traffic to the cell surface, FPN1 did not multimerize. We conclude that native FPN1 is a multiple-pass transmembrane protein that may aggregate ex vivo, but it exists, in vivo, in two stable forms, a monomeric one, and one associated with high molecular weight structures/microsomal membranes. FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiologic inhibitor, hepcidin, in both donor and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover, likely due to faulty protein degradation and/or endocytic cycling pathways. Our findings do not support a classic dominant- negative model proposed by some in vitro studies where the mutant allele product is retained intracellularly and precludes traffic and membrane targeting of the wild-type allele product. On the other hand, it provides a model for the FD, which is characterized by a preserved iron transfer in the enterocytes (i.e. cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages.
Seventh Congress of the International BioIron Society Page 232 Poster Abstracts IBIS
Poster #118
ACERULOPLASMINEMIA: EXPERIENCES AT THE VENETO REGION REFERRAL CENTER FOR IRON DISORDERS Chiara Piubelli, PhD¹,², Annalisa Castagna, PhD, Giacomo Marchi, MD, Monica Rizzi, MD, Fabiana Busti, MD, Paola Capelli, MD, Roberto Pozzi-Mucelli, MD, Massimiliano Calabrese, MD, Alice Vianello, MD, Luciano Xumerle, BSc, Massimo Delledonne, PhD, Oliviero Olivieri, MD and Domenico Girelli, MD, PhD ¹Veneto Region Referral Center for Iron Disorders, Azienda Ospedaliera Universitaria Integrata; ²Department of Medicine, Section of Internal Medicine, University of Verona Verona Italy Presented By: Chiara Piubelli, PhD
Hereditary aceruloplasminemia (HA) is a rare genetic neurodegenerative disorder characterized by peculiar iron deposition in the brain and other organs, such as liver and pancreas. HA represents an atypical iron overload disease, leading to neurological symptoms, along with retinitis, diabetes, liver disease, and microcytic anemia. HA is caused by mutations in the Ceruloplasmin (CP) gene, which results in the absence or malfunctioning of CP, a copper-containing ferroxidase, involved in the oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+), necessary for the coupling with plasma transferrin. At our referral centre for iron disorder, a 46 year old male patient with unexplained hyperferritinemia (2,100 µg/l), low TSAT and mild anemia has been characterized. He showed iron deposition in liver at MRI and iron overload in hepatocytes at liver biopsy, but a negative 1st level molecular test for HFE-Hemochromatosis (HH). We then performed our next generation sequencing 2nd level genetic test (broad panel) for suspected non-classical HH. The patient resulted compound heterozygous for two novel mutations in the CP gene (p.Cys338Ser and p.Ile991Thr). Both mutations had a low frequency in ExAC, and a possible damaging prediction by bioinformatics analysis, indicating a likely diagnosis of HA. In silico modeling based on the available 3D structure of CP showed that the Cys338 residue is directly involved in the Cu binding, while the Ile991 residue is closed to a second Cu-binding site. Thus, both mutations are predicted to impair copper incorporation, which could results in protein instability. The diagnosis was confirmed by undetectable levels of serum CP. Of note, the patient was asymptomatic at neurological examination. Brain MRI was negative for iron accumulation, and no cognitive impairment was detected at functional tests. Chelation therapy with deferasirox was started, but soon after suspended because of the development of renal insufficiency even a lower dose. The patient is now treated with deferoxamine. A second patient (male, 62 year old), with a previous diagnosis in the third decade of life of fully penetrant HFE-related HH (ferritin 3,650 µg/l, TSAT 88%, p.Cys282Tyr homozygosity in the HFE gene), was recently referred from another province at our Center. Search for variants that could have contributed to unusually full and early clinical expression surprisingly revealed a compound heterozygosity for two possibly damaging variants in the CP gene (the novel p.Cys534Trp, and p.Gly895Ala, already associated to HA in ClinVar). In silico modeling showed that the Cys534 residue is involved in a disulphide bridge, with amino acid substitution at this level being predicted to create a massive conformational change of the protein. Change at Gly895 residue could impair the local dynamic properties of the protein. Of note, being this patient also affected by mild anemia due to ß-thalassemia trait, he was treated for a long time (30 years) by chelation, which could have likely prevented the development of neurologic damage. Further analyses in the patient and relatives are ongoing. Considering the rarity of HA, a multicenter registry seems to be fundamental to better define the molecular and clinical features of this highly invalidating disease.
Seventh Congress of the International BioIron Society Page 233 Poster Abstracts IBIS
Poster #119
NON-HFE HAEMOCHROMATOSIS: A PHENOTYPIC AND CLINICAL ANALYSIS OF PUBLISHED CASES AND COMPARISON WITH HFE-HAEMOCHROMATOSIS Daniel Wallace, BSc, PhD¹, Kam Sandhu², Kaledas Flintoff², Jeannette Dixon², Louise Ramm², Grant Ramm², Lawrie Powell² and V Nathan Subramaniam¹ ¹Queensland University of Technology; ²QIMR Berghofer Medical Research Institute Presented By: Daniel Frederick Wallace, PhD, BSc (hons)
Background: Hereditary haemochromatosis (HH) is normally caused by homozygosity for the HFE-C282Y mutation (HFE-HH). Other less common genetic causes include homozygosity for mutations in the genes hemojuvelin (HFE2), hepcidin (HAMP) and transferrin receptor 2 (TFR2). While these causes of non-HFE HH are known to be more severe than HFE-HH, no formal analysis has thus far been performed to compare their phenotypic and clinical features. Methods: We performed a systematic literature review and identified all published cases of autosomal recessive non-HFE HH up until July 2015. Genetic, phenotypic and clinical information was extracted from the journal articles where available. Data from HFE-C282Y homozygous individuals from the QIMR Berghofer Haemochromatosis Database was used as a comparison group in the analyses. Statistical comparisons between groups were performed using 1-way-ANOVA or Fisher’s exact test. Results: Age at diagnosis was significantly lower in individuals with non-HFE HH compared with HFE-HH (Age (years): HFE: 43, HFE2: 24, HAMP: 26, TFR2: 32, p<0.0001). Serum ferritin (SF) and transferrin saturation (TS) at diagnosis were significantly higher in individuals with non-HFE HH compared with HFE-HH (SF (µg/L): HFE 1086, HFE2: 3644, HAMP: 3806, TFR2: 2612, p<0.0001; TS: HFE: 74%, HFE2: 94%, HAMP: 92%, TFR2: 91%, p<0.0001). Clinical features that were more prevalent in non-HFE HH compared with HFE-HH at diagnosis were cardiac involvement, hypogonadism, hepatic fibrosis, diabetes and skin hyperpigmentation. There was no difference in the prevalence of arthropathy between groups. Conclusions: These results confirm that HH due to mutations in HFE2 and HAMP has an earlier age of onset, with more severe iron overload and clinical course compared with HFE-HH. TFR2-HH also has an earlier age of onset but is more intermediate in its onset and disease severity. This analysis provides a reference framework that will be useful for clinicians for the differential diagnosis and management of patients with different forms of HH.
Seventh Congress of the International BioIron Society Page 234 Poster Abstracts IBIS
Poster #120
INDEPENDENT ASSOCIATION OF GNPAT p.D519G WITH MARKEDLY INCREASED IRON STORES IN HFE p.C282Y HOMOZYGOTES James Barton, MD², Wen-Pin Chen, MS³, Mary Emond, PhD⁴, Pradyumna Phatak, MD⁵, . Nathan Subramaniam, PhD⁶, Paul Adams, MD⁷, Lyle Gurrin, PhD⁸, Gregory Anderson, PhD , Grant Ramm, PhD , La rie Po ell, MD, PhD , atrina Allen, FRACP, PhD¹ , John Phillips, PhD¹¹, Charles Parker, MD¹², Gordon McLaren, MD¹ and Christine McLaren, PhD³ ¹VA Long Beach Healthcare System and University of California, Irvine; ²Southern Iron Disorders Center; ³University of California, Irvine; ⁴University of Washington; ⁵Rochester General Hospital; ⁶ IMR Berghofer Medical Research Institute and he University of ueensland; ⁷London Health Sciences Centre; ⁸ he University of Melbourne; IMR Berghofer Medical Research Institute; ¹ Murdoch Childrens Research Institute; ¹¹University of Utah; ¹²University of Utah School of Medicine Presented By: Gordon McLaren, MD
Introduction: GNPAT (chromosome 1q42.2) encodes the peroxisomal enzyme glyceronephosphate O-acyltransferase. In a previous study, DNA of men with hemochromatosis and HFE p.C282Y homozygosity and either markedly increased iron stores or normal or mildly increased iron stores were evaluated with exome sequencing. Positivity for the polymorphism p.D519G (rs11558492) was significantly greater in men with markedly increased iron stores (McLaren CE et al., Hepatology 2015;62:429-39). This result suggests that the p.D519G is a candidate modifier of iron phenotypes in p.C282Y homozygotes. To learn more, we examined associations of p.D519G, age, iron-related variables, and daily alcohol consumption with iron stores in p.C282Y homozygotes classified by extremes of iron overload phenotypes. Methods: We defined markedly increased iron stores as serum ferritin >1000 µg/L and either hepatic iron >236 µmol/g dry weight or mobilizable iron >10 g by induction phlebotomy (men and women). Normal or mildly elevated iron stores were defined as serum ferritin <300 µg/L and either age ≥40 y with ≤2.5 g iron removed by induction phlebotomy or age ≥50 y with ≤3.0 g iron removed by induction phlebotomy (men only). e first compared general characteristics of participant subgroups using univariate methods. Then, using multivariable logistic regression, we evaluated associations of markedly increased iron stores with the following six variables observed at diagnosis of hemochromatosis that might account for markedly increased iron stores: age; iron supplement use (dichotomous); number of whole blood units donated; number of erythrocyte units received as transfusion; daily alcohol consumption, g; and p.D519G positivity (heterozygosity or homozygosity). Results: There were 56 participants (53 men, 3 women), of whom 41 (38 men, 3 women) had markedly increased iron stores and 15 others had normal or mildly increased iron stores (all men). The mean age of the 56 participants was 55 ± 10 (SD) y. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated serum levels of aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis proven by biopsy. Odds ratios of having markedly increased iron stores for each of the six variables, as determined by univariate logistic regression, are displayed in Table 1. In the multivariable analysis, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p = 0.0126). Area under the curve for the multivariable logistic regression analysis was 0.82. Conclusions: We conclude that GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and daily alcohol consumption. It remains unknown whether p.D519G directly enhances iron transport into the blood by absorptive enterocytes, indirectly augments iron absorption by suppressing hepcidin, or is linked to a putative iron absorption promoter on chromosome 1q.
Seventh Congress of the International BioIron Society Page 235 Poster Abstracts IBIS
Poster #121
ANY GAIN OF 300 ALTITUDE METERS INCREASES HEMOGLOBIN LEVELS IN YOUNG MEN LIVING AT LOW ALTITUDE IN SWITZERLAND Kaspar Staub, Dr¹, Martin Häusler, Dr¹, Martina Muckenthaler, Prof Dr², Frank Rühli, Prof Dr¹ and Max Gassmann¹ ¹University of Zurich; ²University of Heidelberg Presented By: Max Gassmann, DVM
Background: It is well known that living at high altitude elevates hemoglobin (Hb) levels in humans but scarce information exists on the effect of low altitude on erythrocyte production. Most attempts to define the latter are hampered by the heterogeneity of the tested population especially due variations in ethnicity and socio-economical aspects. Nevertheless, the altitude an individual resides in most probably defines the Hb threshold that in is required to define anemia at the individual and population level. Of note, the WHO is currently revising these thresholds for the diagnosis of anemia and its severity. Methods and Results: To circumvent the limitations caused by the population’s heterogeneity we compared Hb and ferritin levels, body-mass index (BMI) and C-reactive protein (CRP) values to the altitude of residence of 71,798 Swiss men aged 18-22 years. The anonymized individual conscription data (covering >90% of birth cohorts) were collected by the Swiss Army in the years 2010-12 and included the place of residence thus allowing assessment of the altitude the conscripts live at. Blood samples of volunteers (ca. 65% of all conscripts) were all analyzed within 24 hours by the same certified Swiss laboratory. Interestingly, we observed a significant increase of Hb values for every 300 meters of augmented altitude the young Swiss men live at. Indeed, Hb values significantly increased by 2.5% from 300 to 1800 m. While increasing altitude did not affect BMI and CRP, blood ferritin levels that represent a marker for tissue iron stores raised with elevation. Conclusions: Using this unique set of data of a very homogenous population we show - to our knowledge - for the first time that even a modest increase in the residence altitude significantly elevates Hb values. Apart from gender, age, ethnicity and socio-economical effects, altitude should be considered when defining the Hb threshold for a given population even when residing at altitudes below 1000 m above sea level.
Seventh Congress of the International BioIron Society Page 236 Poster Abstracts IBIS
Poster #122
IRON OVERLOAD ENHANCED CELL DEATH INDUCED BY CARCINOGENIC FIBROUS MATERIALS Fumiya Ito, Master¹, Lei Shi, MD² and Shinya Toyokuni, MD, PhD² ¹Nagoya University Graduate School of Medicine; ²Nagoya University Graduate School of Medicine Department of Pathology and Biological Responses Presented By: Fumiya Ito, MA
Introduction: Asbestos inhalation can lead to neoplastic disease, lung cancer and malignant mesothelioma. But, how cell death from asbestos contributes to carcinogenesis is unclear. Ferroptosis is associated with iron-dependent lipid peroxidation as a recently recognized form of cell death. Because iron overload is involved in the carcinogenesis of malignant mesothelioma, we speculated ferroptosis is associated with asbestos-induced disease. Here we investigated the association between iron and asbestos-induced cell death. Methods: We used two types of fibrous materials in this experiment, carcinogenetic fiber (crocidolite, MWCNT-50), and non-carcinogenetic one (anthophyllite, MWCNT-tngl). We established mesothelioma model in which fibrous materials are (0.5-5.0 mg) i.p. injected to rats. We collected cell and fluid samples by centrifugation of the peritoneal lavage (PL) and used the cell samples for western blot and the fluid samples for iron and hydrogen peroxide (H2O2) detection respectively. Catalytic Fe(II) was detected by RhoNox-1. H2O2 detection was assessed by using Amplex® Red. Mesothelial (Met5A) and macrophage (RAW 264.7) cell lines were exposed to two kinds of asbestos (crocidolite, anthophyllite) (1-25 µg/cm2). Ferric ammonium citrate (FAC; 3.3-66.0 µg /ml) was used to load iron, deferoxamine (DFO; 100 µM) to chelate iron and hydrogen peroxide (1-50 µM) to induce Fenton reaction. We evaluated necrosis by LDH assay and proliferation by MTT assay. Results: In in vivo experiments, cell sample from carcinogenic PL showed higher DMT-1 (divalent metal transporter-1) expression than non-carcinogenic one. Catalytic Fe(II) increased in both of cellular and fluid samples of carcinogenic PL. In in vitro experiments, the necrosis of mesothelial cell was induced dose-dependently by crocidolite, but not by anthophyllite. FAC increased intracellular catalytic Fe(II) and DFO canceled the effect. Treatment of FAC significantly increased the necrotic effect of crocidolite on mesothelial cells. FAC treatment without crocidolite suppressed proliferation in macrophage, and DFO canceled the effect. (Discussion) In in vivo experiments, although inflammatory cells collected extracellular iron with DMT-1 overexpression, extra-cellular catalytic Fe(II) still increased in carcinogenic inflammation because the inflammatory cells were saturated with iron. In in vitro experiments, asbestos induced necrosis in mesothelial cells, which was enhanced by iron overload. Anti-proliferation induced by iron and inhibited by DFO in macrophage suggests that the phenomenon is ferroptosis. These necrotic cells may contribute to form a vicious cycle due to a release of intracellular catalytic Fe(II) and hydrogen peroxide which accelerates Fenton reaction. Conclusions: Carcinogenic fibers induce necrosis in mesothelial cells and necrotic cells release catalytic iron, which enhances death of the surrounding cells, forming a vicious cycle.
Seventh Congress of the International BioIron Society Page 237 Poster Abstracts IBIS
Poster #123
TOLL-LIKE RECEPTORS AND REGULATION OF SYSTEMIC IRON HOMEOSTASIS Oriana Marques, MSc, PhD Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg Presented By: Oriana Marques, MSc, PhD
Toll-like receptors (TLRs) constitute key sensors of the innate immune system that trigger an acute phase response by recognizing pathogen-associated molecular patterns and danger signals. We have recently shown that stimulation of TLR2/6 decreases ferroportin messenger RNA and protein expression, in bone marrow-derived macrophages, liver and spleen of mice, without changing hepcidin expression. C326S ferroportin mutant mice, with a disrupted hepcidin/ferroportin regulatory circuitry, respond to injection of the TLR2/6 ligands FSL1 or Pam3CSK4 by ferroportin downregulation and a reduction of serum iron levels. These findings challenged the prevailing role of hepcidin in hypoferremia in response to inflammatory signals. We now asked whether TLR2/6 control ferroportin expression and systemic iron homeostasis under steady-state conditions. We show a mild increase in red blood cell counts (RBC) in TLR6 knock-out (KO) mice, while the mean corpuscular volume (MCV) is decreased in both TLR2KO and TLR6KO mice. The lack of TLR6 causes increased ferroportin levels in the liver despite unaltered non-heme iron content and hepcidin levels. By contrast, splenic iron levels are reduced in TLR2KO and TLR6KO mice. Preliminary findings suggest that erythrophagocytosis is affected in TLR6KO mice, suggesting that TLRs play a role in maintaining systemic iron homeostasis under steady state conditions.
Seventh Congress of the International BioIron Society Page 238 Poster Abstracts IBIS
Poster #124
HEMOJUVELIN IS CRITICAL FOR INFLAMMATORY SIGNALING TO HEPCIDIN Nicole Wilkinson, PhD, Carine Fillebeen, PhD, John Wagner, MSc and Kostas Pantopoulos, PhD Lady Davis Institute for Medical Research/McGill University Presented By: Kostas Pantopoulos, PhD
Hereditary hemochromatosis is a disorder of systemic iron overload that is caused by genetic inactivation of genes that regulate iron signaling to hepcidin, the iron regulatory hormone. The most common form is associated with mutations in the hemochromatosis protein HFE, an atypical MHC class 1 molecule, and is characterized by mild suppression of hepcidin. Mutations in hemojuvelin (HJV), a bone morphogenetic protein (BMP) co-receptor, lead to juvenile hemochromatosis due to severe suppression of hepcidin. These phenotypes are recapitulated in Hfe-/- and Hjv-/- mice. We recently showed that Hjv-/- and double Hjv-/-Hfe-/- mice manifest the same degree of iron overload, suggesting that Hjv and Hfe operate in the same pathway for iron signaling to hepcidin (Kent et al, J. Mol. Med., 2015). Considering that hepcidin is also induced by inflammatory stimuli, we next assessed the responses of these animals to acute inflammation. To this end, wild type, Hfe-/-, Hjv-/- and double Hjv-/-Hfe-/- mice were injected with LPS. Serum iron levels decreased profoundly in LPS-treated wild type and Hfe-/- mice, and this was associated with induction of hepatic hepcidin mRNA in both genotypes. However, the hypoferremic response was blunted in LPS-treated Hjv-/- and Hjv-/-Hfe-/- mice, which maintained negligible hepcidin mRNA levels. Consistently, the LPS treatment decreased ferroportin expression in Kupffer cells and splenic macrophages of wild type and Hfe-/-, but not Hjv-/- and Hjv-/-Hfe-/- mice. These findings suggest that Hjv is essential for inflammatory signaling to hepcidin. Biochemical experiments are underway to dissect the underlying mechanism.
Seventh Congress of the International BioIron Society Page 239 Poster Abstracts IBIS
Poster #125
INSIGHT INTO THE MECHANISM OF HEME EXTRACTION FROM HUMAN HEMOGLOBIN BY THE ISDH RECEPTOR FROM STAPHYLOCOCCUS AUREUS Ramsay Macdonald¹, Megan Sjodt, PhD¹, Joanna Marshall, BS¹, John Olson, PhD², David Gell, PhD³ and Robert Clubb, PhD¹ ¹UCLA; ²Rice University; ³University of Tasmania Presented By: Ramsay Macdonald
The Gram-positive bacterium Staphylococcus aureus (S. aureus) is the causative agent of a wide range of infections that are frequently lethal. Antibiotic-resistant strains of S. aureus are on the rise and necessitate the development of novel therapeutics. One attractive target is the iron acquisition system of S. aureus, as the microbe must acquire iron from its host in order to mount an infection. Hemoglobin (Hb), located within erythrocytes, contains ~70% of the body’s iron tightly bound to heme (protoporphyrin IX + iron) and is preferentially used by S. aureus as an iron source. S. aureus acquires iron using the Iron-regulated Surface Determinant (Isd) system, which extracts heme from Hb, transports it across the cell membrane into the bacterial cytoplasm, and degrades the pyrrole ring to yield iron. The focus of our work is on the first step in iron acquisition by the Isd system, the capture of Hb and the extraction of its heme molecule by the IsdH receptor. Heme extraction from Hb is mediated by a conserved tri-domain unit within IsdH consisting of two NEAr iron Transporter (NEAT) domains separated by an alpha-helical linker (IsdHN2N3). In this conserved unit, the second NEAT domain (N2) and the third NEAT domain (N3) bind Hb and heme respectively. Recent structural work on the Hb:IsdHN2N3 complex has revealed that the linker and N3 domains (LN3) in IsdHN2N3 engage Hb and perturb its heme binding pocket presumably weakening its affinity for heme. Here we use biochemical and biophysical experiments to probe the kinetics and thermodynamics of the heme extraction mechanism from Hb by IsdHN2N3. Isothermal Titration Calorimetry (ITC) and nuclear magnetic resonance (NMR) data have demonstrated that the N2 domain binds Hb with high affinity while the LN3 domains engage Hb with weak affinity. The ITC data has shown that there is an enthalpic penalty associated with the LN3:Hb binding event. We hypothesize that this enthalpic penalty is a result of the perturbation of the heme binding pocket of Hb which consists of the unwinding of the final turn of Hb’s F-helix. The crystal structure has revealed that two residues (Y495 and K499) in the linker make contact with the perturbed F-helix. Stopped-flow kinetics data has indicated that these residues are critical for accelerated heme release from Hb by IsdHN2N3. Current efforts are devoted to obtaining structural information of IsdHN2N3 mutants in complex with Hb via X-ray crystallography in order to gain insight into the extraction process. Collectively, this data will contribute to a deeper understanding of the mechanism by which IsdHN2N3 rapidly extracts heme from Hb.
Seventh Congress of the International BioIron Society Page 240 Poster Abstracts IBIS
Poster #126
IRON SUPPLEMENTS MODULATE COLON MICROBIOTA COMPOSITION AND DEXTRAN SODIUM SULFATE- INDUCED COLITIS Marco Constante, Gabriela Fragoso, PhD, Joseph Lupien-Meilleur, MSc, Annie Calve, MSc and Manuela M. Santos, PhD CRCHUM Presented By: Marco Constante, BSc
Introduction: Iron is an important nutrient for both the host and the bacteria that inhabit it. Since most of the iron ingested by the host remains largely unabsorbed, oral iron supplementation effectively leads to an accumulation of iron in the gastrointestinal tract. Excess iron in the gut may potentially impact the composition of the microbiota, and may be particularly damaging to susceptible individuals, such as patients suffering from inflammatory bowel disease (IBD) in which gut dysbiosis has been reported. However, IBD patients may require oral iron supplementation to treat their iron- deficiency anemia. Methods: We fed mice diets supplemented with ferrous sulfate (FS) at different doses (5, 50 and 500 mg of iron/Kg chow) and with different iron formulations (FS, ferrous bisglycinate (FBG) and ferric ethylenediaminetetraacetic acid (FEDTA)), then analyzed the effects on the composition of the gut microbiota via 16S ribosomal RNA gene sequencing. Using the Dextran Sodium Sulfate (DSS)-induced colitis mouse model, we investigated the effects of iron supplementation in colitis severity. Results: Iron supplementation with different doses of FS induced shifts in the gut microbial communities and several inferred metabolic pathways. In addition, FS iron supplementation had a modest, but significant protective effect on DSS- induced colitis. However, depending on the iron formulation used in the diets, the impact of iron supplementation on colitis ranged from beneficial (FBG) to highly detrimental (FEDTA). Discussion and Conclusions: Our data shows that the form of the iron used to treat iron deficiency may differentially impact the gut microbiota and colitis in mice, and as such suggests that iron forms may be of particular relevance to IBD patients.
Seventh Congress of the International BioIron Society Page 241 Poster Abstracts IBIS
Poster #127
HEMIN AND ALTERED IRON METABOLISM IN SICKLE CELL DISEASE MODULATE HIV-1 RESTRICTION FACTORS Namita Kumari¹, Miguel Mulder, PhD², Douglas Nixon, PhD² and Sergei Nekhai, PhD¹ ¹Howard University; ²George Washington University Presented By: Namita Kumari
Introduction: Numerous previous studies pointed to the possibility that SCD patients might restrict HIV-1 infection. We recently described a mechanism of HIV-1 restriction in PBMC from SCD patient infected with HIV-1 ex vivo. We showed that ferroportin acts as an upstream regulator of SAMHD1 and links a reduction in iron levels, inhibition of CDK2 activity and a decrease in SAMHD1 phosphorylation to the inhibition of HIV-1 infection in SCD. Here, we looked for additional restriction factors induced in SCD PBMC and PBMCs treated with hemin and iron chelator. Methods: A customized array was utilized to determine the expression of restriction factors in SCD PBMCs. Real-time q- PCR was used to validate the expression of restriction factors. The shRNA-mediated knockdowns of the identified genes were used to further validate the role of these factors in HIV-1 replication in cultured and primary cells. VSVG- pseudotyped pNL4-3.Luc.R-E-virus was used to analyze HIV-1 replication. Results: Restriction factor SAMHD1 was upregulated in hemin treated PBMC and shRNA mediated knockdown of SAMHD1 reduced the effect of HIV-1 inhibition by hemin. Customized array for human restriction factors showed upregulation of 19 genes in SCD PBMC. APOBEC3A, APOBEC3C, T M5 , T M22, M 2 and RSAD2 were most significantly upregulated and were further validated using real-time q-PCR. ABOBEC3A, RASD2 and MX2 showed upregulation in PBMCs treated with hemin, whereas T M5 was upregulated in PBMC treated with hemin and iron chelators. Knockdowns using sh A against APOBEC3A, APOBEC3C, T M5 , T M22 showed upregulation of viral replication in the cells with APOBEC3A, T M5 and T M22 knockdowns. Conclusions: n addition to SAM D1, other restriction factors such as APOBEC3A, APOBEC3C, T M5 , TRIM22, MX2 and RSAD2 are upregulated in SCD PBMC. These additional factors might contribute to HIV-1 restriction observed in hemin and iron chelators treated cells and also PBMCs obtained from SCD patients. Acknowledgments: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, 5G12MD007597 and 1P30AI117970. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIAID, or NIMHD, NIH.
Seventh Congress of the International BioIron Society Page 242 Poster Abstracts IBIS
Poster #128
ROLE OF EPIGENETIC REGULATION OF IRON HOMEOSTASIS GENES AND NEURODEGENERATION Steinunn Sara Helgudottir¹, Jacek Lichota, PhD² and Torben Moos, Professor, PhD, MD² ¹Health Science and Technology, Aalborg University; ²Aalborg University, Denmark Presented By: Steinunn Sara Helgudottir
Introduction: It is well established that iron is one of the key factors involved in the progression of different neurodegenerative diseases, but further studies are needed to fully elucidate its role. Iron is the most abundant metal in the human body and is essential for all mammalian cells, including neurons, in order to maintain important cellular functions. However, the level of intracellular iron has to be tightly regulated as excessive amounts of iron are potentially toxic for the cells through the formation of ROS. The cellular mechanisms for regulating the iron homeostasis are complex and involves various regulatory proteins. The expression of the genes involved in iron homeostasis and their epigenetic regulation as well as function of these proteins and how they are affected by iron accumulation, is still not clear. Methods: Differentiated by neuron growth factor (NGF) neuron-like PC12 were used as an in vitro model for the study of effect of excessive iron concentrations or iron depletion. Light and fluorescent microscopy images were taken in order to follow the progression of PC12 cell differentiation by morphology changes and detection of neuronal markers (IHC). WST- 1 cytotoxicity assay was employed in order to investigate the effect of iron load and depletion on cell viability. Bisulfite sequencing and qPCR-analysis were done to investigate the epigenetic status of ferroportin promoter and expression of genes involved in iron homeostasis (ferroportin, ferritin, hepcidin) and epigenetic regulation (HDAC1, KDM5B). Results: Light microscopy images showed the extensive growth of neurites and ceasing of proliferation in PC12 treated with 50 ng/ml NGF over a period of 6 days. This was further shown by immunocytochemistry images. WST-1 cytotoxicity assay showed stepwise decrease in cell viability when cells were treated with 10-200µM FAC qPCR analysis showed and markedly upregulation of both Ferritin-H and Ferritin-L as well as Ferroportin in cells treated with FAC. DNA methylation changes of Ferroportin gene promoter were observed in different brain regions dependent on the iron concentration. Conclusions: The findings showed that PC12 cells easily differentiate into neuron-like cells, and as such are simple and convenient to use as in vitro model for studying neurotoxicity and neurodegeneration. Increasing concentrations of FAC had a decreasing effect on cellular viability confirming that even though iron is essential for normal cellular function, excessive amounts can be toxic for the cell. Additionally, the gene expression analyses confirmed an inducing effect of iron in the expression of the iron regulatory proteins Ferritin and Ferroportin, which is also regulated epigenetically as shown by changes in DNA methylation.
Seventh Congress of the International BioIron Society Page 243 Poster Abstracts IBIS
Poster #129
EFFECTS OF PARENTAL IRON THERAPY TO PREGNANT RATS WITH SYSTEMIC IRON DEFICIENCY WITHOUT ANEMIA ON CEREBRAL IRON STATUS AT BIRTH AND IN ADULTHOOD Jacek Lichota¹, Annette Burkhart, PhD², Tina Skjørringe, PhD², Lisa Juul Routhe, MSc², Lars Lykke Thomsen, PhD, MD³ and Torben Moos, Professor, PhD, MD² ¹Health Science and Technology, Aalborg University; ²Aalborg University, Denmark; ³Pharmacosmos, Denmark Presented By: Jacek Lichota
Background: Iron (Fe)-deficiency (ID) affects fertile females worldwide. Fe is co-factor for many enzymes in the developing brain. Therefore, strategies to ensure sufficient cerebral fetal Fe supply are crucial. Objectives: We investigated the effects of administering Iron Isomaltoside 1000 (IIM) subcutaneously to ID pregnant rats for offspring viability, and cerebral concentration of Fe, copper (Cu), and zinc (Zn). We also examined offspring cerebral and hepatic fetal gene expression of hepcidin, plus gene and protein expression of ferroportin, and ferritin H and L subunits. Methods: Fertilized females subjected to ID without anemia were injected with IIM at the day of mating (E0), 14 days into pregnancy (E14), or at the day of birth (Postnatal (P) 0). Brain and liver in the offspring were examined on P0 or in adulthood on postnatal day P70. Results: Females fed a Fe-low diet (5.2 mg/kg Fe) had offspring with significantly lower cerebral and hepatic Fe compared to a control group fed a standard diet (178.6 mg/kg Fe). Injection of IIM to pregnant ID females on E0 or E14 yielded normalization of Fe in the PO brain. On P70, fetal cerebral ID was accompanied by a decrease in cerebral expression of ferritin H+L gene and protein expression, and unaltered ferroportin gene expression with lower protein expression. Hepcidin gene expression was absent both from the normal and the ID brain. In spite diets were identical with respect to Cu and Zn, liver Cu was much higher on P70, and cerebral and hepatic Zn lower on both P0 and P70. Conclusions: IIM administrated parentally to ID pregnant mothers can normalize cerebral Fe in the fetal brain. ID prior to and during pregnancy can chronically affect metals and affect gene expression related to metal homeostasis in brain and liver in the offspring.
Seventh Congress of the International BioIron Society Page 244 Poster Abstracts IBIS
Poster #130
INFLAMMATION-DRIVEN DISRUPTION OF IRON HOMEOSTASIS IN PARKINSON’S DISEASE Ana Catarina Vaz Carreto Martins, MSc², Susana Ramos, PhD³, Miguel P. Soares, PhD³ and Raffaella Gozzelino, PhD¹ ¹Chronic Diseases Research Center /NOVA Medical School; ²CEDOC/NOVA Medical School; ³Instituto Gulbenkian de Ciência Presented By: Raffaella Gozzelino, PhD
Parkinson’s disease (PD) is a complex neurodegenerative disorder caused by the progressive loss of dopaminergic neurons (DNs) in the substantia nigra of the brain. Although several causes seem to contribute to PD progression, the etiology of this disease remains still poorly understood. In the recent years, immunity and inflammation were shown to play major roles in the pathogenesis of PD, a notion supported by the detection of infiltrated immune cells in post-mortem brains. Whether inflammation might act as a risk factor for the development of PD, it seems to be the case. By using an animal model of pharmacologic PD, we observed that the severity of PD is significantly enhanced when mice are previously exposed to immune-mediated inflammatory diseases (IMID). Exacerbated locomotor dysfunction and DNs loss are associated with the development of PD upon inflammatory priming, a phenomenon accompanied by IMID-mediated disruption of iron (Fe) homeostasis in peripheral immune cells. Since this circulating compartment was shown capable to store Fe upon IMID induction, it is reasonable to hypothesize that the infiltration of Fe-loaded immune cells into the brain might act as a predisposing factor for the development of PD. To support this notion, our results demonstrate the existence of an immune-mediated neuronal interaction that, fueled by Fe, is crucially involved in the pathogenesis of PD.
Seventh Congress of the International BioIron Society Page 245 Poster Abstracts IBIS
Poster #131
RESTING STATE MRI BRAIN DIFFERENCES IN HFE AND TRANSFERRIN MUTATION CARRIERS Mark Meadowcroft, PhD¹, Jianli Wang, MD, PhD², Carson Purnell, BS², Paul Eslinger, PhD², Elizabeth Neely, BS², Qing Yang, PhD² and James Connor, PhD² ¹The Pennsylvania State University - College of Medicine; ²The Pennsylvania State University – College of Medicine Presented By: Mark Meadowcroft, PhD
Introduction: HFE and transferrin (Tf) mutations have often been implicated in neurodegenerative disease etiology, however their effect remains uncertain. Our previous work illustrated reduced white matter integrity in mutation carriers. To determine if these white matter differences manifest brain connection alterations, resting state functional MRI was utilized to detect if subtle differences in brain temporal connectivity are present in cognitively normal HFE and Tf mutations carriers. Methods: Thirty-four cognitively normal subjects were genotyped for HFE and transferrin SNPs. Twenty-one participants were determined to have at least one HFE or Tf SNP, while thirteen were wild-type carriers. Rest periods between task events in a previous functional block design paradigm were separated from the dataset and used for resting-state analysis, for a total concatenated resting period of 186 repetitions (6m12s). Data was slice-timing adjusted, motion corrected, co-registered, normalized to a template brain, and then smoothed with SPM8. Using the MNET toolbox, subject-level resting-state correlation matrices were generated using a 116-region AAL template. Statistical analyses with false discovery rate detection were conducted with MATLAB. Results: Resting-state functional connectivity patterns were highly conserved between the two groups, where large-scale connectivity was very similar (Fig. 1a and b). However, differences were observed between multiple frontal, cerebellar, temporal, occipital, and midbrain structures, as well as differences in cingulate connections to parietal and occipital regions (Fig. 1c). Several of these areas correspond to components of the default mode network. Discussion Brain connectivity and the default mode network have been demonstrated to be asynchronous in patients with neurodegenerative disease. The differences observed in this work demonstrate that HFE and transferrin carriers have modified baseline neural connectivity compared to wild-types. Differences in resting-state activity may refer to a nascent breakdown of neural networks; however it remains uncertain if these changes represent early network disruption, prior to any noticeable behavioral symptoms. As detailed tests of intelligence and executive function were not performed, only cognitive testing to discern dementia-type cognitive disruption, it remains unknown if the outlined neuronal changes have a behavioral effect. Conclusions: HFE and transferrin SNP carriers present with altered resting-state functional connectivity compared to wild-type subjects, despite all indications of being cognitively normal. Finer tests of cognitive capabilities are necessary to discover if these refer to a difference in behavior or cognition. Furthermore, long-term studies would be required to determine in these mutations confer on influence on the development of neurodegeneration and dementia.
Seventh Congress of the International BioIron Society Page 246 Poster Abstracts IBIS
Poster #132
IRON PROMOTES SENESCENCE IN FIBROBLASTS AND IPS-DERIVED NEURONS FROM NEUROFERRITINOPATHY PATIENTS Anna Cozzi, PhD², Daniel I. Orellana, PhD³, Paolo Santambrogio, PhD³, Alicia Rubio, PhD³, Angelo Iannielli, PhD³, Chiara Fiorillo, MD⁴, Gian Luca Forni, MD⁵, ania Broccoli, PhD⁶ and Sonia Levi, PhD¹ ¹Vita-Salute San Raffaele University; ²San Raffaele Scientific Institute, Milano, Italy.; ³San Raffaele Scientific Institute Milano, Italy.; ⁴University of Genoa, Genoa, Italy.; ⁵E. . spedali Galliera, Genova, Italy.; ⁶CNR-Institute of Neuroscience, Milano, Italy Presented By: Sonia Levi, PhD
Neuroferritinopathy (NF) is a rare autosomal dominant inherited movement disorder, caused by alterations of the L-ferritin gene (FTL). It belongs to the group of disorders named neurodegeneration with brain iron accumulation (NBIA), which are characterized by iron deposition in the basal ganglia and neurodegenerative process. Patients present abundant spherical inclusions in the brain positive for iron, ferritin and ubiquitin staining. Fibroblasts from two Italian patients affected by NF (c469_484dup and c352 delG_insTTT), were characterized and reprogrammed in human induced pluripotent stem cell (hiPSC) to develop a new human NF neuronal model. We established multiple lines of hiPSCs from patients and from three normal subjects by Sendai virus-mediated expression of the four Yamanaka’s factors. Next, we differentiated these lines by overexpressing the neurogenin-2 (Ngn2) neurogenic factor to generate a cellular population of enriched glutamatergic neurons. In addition, to generate FTL-corrected iPSCs, we performed a gene knockin of the wt allele by combining CRISPR/Cas9 technology with a single-stranded oligodeoxynucleotide donor template and a sgRNA directed specifically to the mutated allele. Fibroblasts, neuronal precursors cells (NPC) and hiPSC-derived neurons were then analyzed for morphology, iron metabolism, cell oxidative status and senescence. All these types of NF cells showed ferritin aggregates detected by immunofluorescence. Perl’s staining and electron spectroscopic imaging confirmed the presence of iron in the aggregates. NF cells showed reduced 55Fe-ferritin incorporation ability, decreased TfR1 expression (~10 fold), increased amount of oxidized proteins and ROS production respect to the control cells. Moreover, reduced proliferation, together with the induction of SA-b-GAL activity suggested a senescence phenotype in NF cells. The SA- b -GAL activity was increased after iron addition and this was prevented by co-treatment with an antioxidant agent. FTL-corrected iPSCs prevented the SA- b -GAL increased signal. The fibroblasts morphology alteration and the increased autophagosome formation, confirmed the senescence status of this cell type. In addition, the expression of cytosolic NCOA4 appeared strongly reduced in NF fibroblasts and NPC, supporting a major cytosolic iron availability. We report for the first time in human NF primary cells and in a new neuronal model that the poor intracellular ferritin iron buffering triggers oxidative stress and senescence in the cells, involving alteration of NCOA4 expression. The financial support of Telethon–Italy (Grant n°GGP10099) and AISNAF is gratefully acknowledged.
Seventh Congress of the International BioIron Society Page 247 Poster Abstracts IBIS
Poster #133
EFFECTS OF BRAIN TISSUE PROCESSING ON SENSITIVITY OF DAB-ENHANCED PERL’S METHOD FOR IRON STAINING Moones Heidari, PhD, Kristy Martin, Daniel M Johnstone, Ritambhara Aryal, Shanmathi Parameswaran, Linh Lim, Roberto Cappai, Samantha Richardson and Elizabeth A. Milward University of Newcastle Presented By: Moones Heidari, MSc
Introduction: In a normal healthy brain most of the iron is mainly stored in myelin and oligodendrocytes. There has been increasing evidence suggesting pathological roles for excess iron in the development of various neurodegenerative diseases including Alzheimer’s disease. Traditionally the 3, 3’- diaminobenzidine tetrahydrochloride (DAB) -enhanced Perls’ staining method has been widely used as an easy and cost-effective method to detect iron accumulation in different tissues. However various studies have suggested that although DAB-enhanced Perls’ method stains iron in oligodendrocytes and myelin, it lacks enough sensitivity required to stain subtle amount of iron in neurons. Here we reported that tissue processing reagents and embedding mediums are a potential cause affecting DAB-enhanced Perls’ staining sensitivity, leading to inconsistent neuronal iron staining. Methods: Male Hfe-/-xTfr2mut mice at three months of age were anesthetized using either 50 mg/kg ketamine and 10 mg/kg xylazine or 60 mg/kg sodium pentobarbital. Brains were fixed in 4% paraformaldehyde and embedding was done using two different methods including the sucrose cryoprotection followed by freezing and embedding in O.C.T. compound or tissues were processed and embedded in a paraffin block. All brain sections were stained using 1% potassium ferrocyanide, pH 1 followed by incubation in methanol containing 0.01M NaN3 and 0.3% H2O2 and finally staining was enhanced with DAB. Results: The density of staining and cells which were detected to accumulate iron were different in paraffin compared with O.C.T. compound embedded brain sections. Iron was stained in myelinated structures and oligodendrocytes in both paraffin and O.C.T. compound embedded brain sections but the density of staining is higher in O.C.T. compound embedded brain sections comparing with paraffin embedded sections. Little if any iron staining was detected in cortical or hippocampal neurons in O.C.T. compound embedded sections while clear iron staining was visible in neurons in both cortex and hippocampus in paraffin embedded brain sections. Iron staining in choroid plexus was very strong in both paraffin embedded and frozen section. However, the density of staining was higher in frozen brain section. Conclusions: The exact effect of various tissue processing reagent and embedding medium on Prussian blue reaction is not clear but these findings suggest that brain tissue processing protocols routinely used for histochemichal studies, particularly iron staining based on Perls’ method, could remarkably affect sensitivity and accuracy of the technique. The exact reasons and involved reactions leading to the iron leaching from paraffin embedded brain sections need to be investigated further but minimizing incubation time required prior paraffin tissue processing could simply prevent iron loss from tissue sections. However extensive lipid clearing due to tissue processing prior paraffin embedding improves staining of the subtle amount of iron in neurons. Therefore it is suggested to consider applying both tissue processing and embedding techniques in parallel to improve sensitivity and accuracy of iron staining in brain tissue.
Seventh Congress of the International BioIron Society Page 248 Poster Abstracts IBIS
Poster #134
PATHOLOGICAL RELATIONSHIPS INVOLVING IRON AND MYELIN MAY CONSTITUTE A SHARED MECHANISM LINKING VARIOUS RARE AND COMMON BRAIN DISEASES Moones Heidari, PhD², Daniel Johnstone, Brianna Bassett, Conceição Bettencourt, Joanna Collingwood, Sam Gerami, Michael House, Kristy Martin, Anita Chua, Mina Ryten, Henry Houlden, John Olynyk, Debbie Trinder and Elizabeth Milward¹ ¹The University of Newcastle; ²University of Newcastle Presented By: Elizabeth Milward
Brain iron abnormalities may contribute to psychiatric disorders, including major depression, bipolar disorder and autism, and to diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease, that can have psychiatric features. Psychiatric symptoms are seen in the rare, debilitating and sometimes fatal neurogenetic disease family termed ‘neurodegeneration with brain iron accumulation' (NBIA). Patients often have movement disorders such as spasticity and parkinsonism. Neuropsychological features can include delusions, personality changes, depression, violent outbursts, impulsivity, hyperactivity, poor attention or cognitive impairment. Neuropsychiatric symptoms are not recognized features of the more common and typically less severe iron disease hemochromatosis. However case reports document conditions such as bipolar, major depressive and psychotic disorders in some patients, including instances where patients resistant to conventional psychiatric treatments experienced partial or complete recovery following phlebotomy or chelation treatment. To understand how iron loading affects the brain, we have studied mice with mutations in the hemochromatosis (Hfe) and transferrin receptor 2 (Tfr2) genes. Inductively coupled plasma atomic emission spectroscopy demonstrated increased iron in the Hfe-/-×Tfr2mut brain (P 0.002, n ≥5/group), primarily locali ed by Perls staining to myelinated structures and myelin-associated cells. Immunoblotting showed increases of ferritin light polypeptide, associated with the NBIA disease neuroferritinopathy. Microarray and real-time reverse transcription-PCR revealed decreased transcript levels (P 0.04, n ≥5/group) for five other NBIA genes - phospholipase A2 group VI, fatty acid 2-hydroxylase, ceruloplasmin, chromosome 19 open reading frame 12 and ATPase type 13A2. Apart from the ferroxidase ceruloplasmin, all are involved in myelin homeostasis. Using expanded data mining and ontological analyses, we have now identified in total 40 myelin-related genes showing altered transcript levels, significantly more than predicted by chance alone (Chi square P<0.0001). Of these, 36 had reduced expression, although gross myelin structure and integrity appeared unaffected (P>0.05). These analyses implicated, among others, genes linked to various rare central hypomyelinating leukodystrophies and peripheral neuropathies including Pelizaeus-Merzbacher-like disease and Charcot- Marie-Tooth disease as well as to other rare neurological diseases such as Niemann-Pick disease. Overlap (P<0.0001) of differentially expressed genes in Hfe-/-×Tfr2mut brain with human gene co-expression networks suggests iron loading influences expression of NBIA-related and myelin-related genes co-expressed in normal human basal ganglia. There was overlap (P<0.0001) between genes that were differentially expressed in Hfe-/-×Tfr2mut brain and post-mortem NBIA basal ganglia, a brain region with high iron levels. Concordance between mouse transcriptome changes and human myelin- related gene expression networks in normal and NBIA brain testifies to potential clinical relevance. Besides showing molecular changes, Hfe-/-×Tfr2mut mice were hyperactive (P<0.0112) but without apparent cognitive impairment by IntelliCage testing (P>0.05), although effects of factors such as stress on performance were not examined. These results implicate myelin-related systems involved in NBIA neuropathogenesis in early responses to iron loading. This may contribute to behavioral symptoms in NBIA and hemochromatosis and is relevant to patients with abnormal iron status and psychiatric disorders involving myelin abnormalities or resistant to conventional treatments. These findings may help understand interrelationships of iron and myelin in more common conditions such as hemochromatosis, multiple sclerosis and various psychiatric disorders.
Seventh Congress of the International BioIron Society Page 249 Poster Abstracts IBIS
Poster #135
TFR2 KI MICE PRESENT AN ENRICHMENT IN CARDIAC FERRITIN L THAT CONFERS CARDIOPROTECTION AGAINST I/R INJURIES Martina Boero, Biologist¹, Rosa Maria Pellegrino, PhD², Mariarosa Mezzanotte, Dr², Antonietta Palmieri, Dr², Giuseppe Saglio, MD² and Antonella Roetto, PhD² ¹University of Torino; ²University of Torino, Department of Clinical and Biological Sciences, AOU San Luigi Gonzaga, Orbassano, Torino, Italy Presented By: Martina Boero
Beta isoform of Tfr2 gene is involved in iron efflux from splenic reticuloendothelial cells via iron exporter Ferroportin 1 (Fpn1) since it was demonstrated that Tfr2 beta lack causes a decrease of Fpn1 and a consequent iron retention in splenic macrophages of Tfr2 beta null mouse model (Tfr2 KI) (Roetto et al, 2010). It was previously documented that Tfr2 beta is highly transcribed in heart (Kawabata et al, 1999) and its loss is sufficient to have a protective effects on cardiac Ischemia Reperfusion (I/R) induced damages (Boero et al, 2015). To investigate what was the molecular changes that induces cardioprotection in these hearts, we analyze Ferritins (Ft), Transferrin Receptor 1 (Tfr1) as well as markers of oxidative stress (SOD1 and Catalase) in Tfr2 KI whole hearts. Furthermore, due to the evident iron retention previously found in Tfr2 beta splenic macrophages, we decided to analyze iron amount in cardiac macrophages. Hearts from Tfr2 KI and WT mice, were explanted and immediately frozen in liquid nitrogen for molecular analysis of whole hearts, or digested with 0.1% collagenase type A at 37oC for 1h for macrophages isolation. Cardiac macrophages were isolated using cd11b MicroBeads (Miltenyi Biotec). Protein extracts were electrophoresed in 10-12 % SDS polyacrylamide gel and immunoblotted according to standard protocols. Antibody against FtL, FtH, Tfr1, SOD1, and Catalase were used. To evaluate oxidative stress in whole hearts OxyBlot Protein Oxidation Detection Kit (Millipore) was utilized. No significant alteration of the analyzed proteins and of oxidative stress could be observed in Tfr2 KI whole hearts compared to strain, age and sex matched WT controls hearts. On the contrary, significant differences were evidenced in the two Ferritin subunits amount in cardiac macrophages and in macrophage-negative cardiac cells fraction. Tfr2 KI cardiac macrophages present an increased production of FtL vs WT macrophages while FtH is comparable in these two experimental groups. On the contrary, macrophage-negative cardiac cells fraction of Tfr2 KI hearts presents a significant decrease of FtH amount vs WT cells, with equal FtL amount in these two experimental groups. In other words, an enrichment of FtL was evidenced both in Tfr2 KI cardiac macrophages and in macrophage-negative cardiac cells fraction compared to WT cells. In both cases this FtL subunits enrichment could lead to the production of L ferritin-rich heteropolymers, that are typical of iron deposits Ferritins (Koorts 2007) and supports the hypothesis that Tfr2 KI mice are cardio-protected because iron is entrapped in L-rich ferritins and less available for I/R injuries.
Seventh Congress of the International BioIron Society Page 250 Poster Abstracts IBIS
Poster #136
DMT1, ZIP14, AND ZIP8 ARE DISPENSIBLE FOR IRON LOADING OF THE HEART Chia-Yu Wang, PhD, Supak Jenkitkasemwong, PhD and Mitchell Knutson, PhD University of Florida Presented By: Mitchell Knutson, PhD
In iron overload conditions such as thalassemia major and hereditary hemochromatosis, the iron-carrying capacity of plasma transferrin is exceeded, giving rise to non-transferrin-bound iron (NTBI). NTBI is taken up preferentially by the liver, and to a lesser extent, the kidney, pancreas, and heart. How NTBI is taken up by various tissues has been elusive. We recently demonstrated that the plasma membrane metal-ion transporter SLC39A14 (ZIP14) mediates NTBI uptake and iron loading of the liver and pancreas, but not the kidney, heart or most other tissues (Jenkitkasemwong et al., Cell Metab., 2015). Given that the heart is particularly susceptible to iron-related toxicity, we are currently investigating the contribution of other iron transporters to iron loading of this organ. Possible alternative cardiac iron importers include L- type and T-type calcium channels, divalent metal transporter 1 (DMT1), and SLC39A8 (ZIP8). To examine the role of DMT1 and ZIP8 in cardiac iron metabolism, we generated mice with cardiomyocyte-specific disruption of DMT1 (Dmt1heart/heart) or ZIP8 (Zip8heart/heart). The mice were then crossed with hemojuvelin knockout (Hfe2-/-) mice, a model of juvenile hemochromatosis characterized by high circulating levels of NTBI. Dmt1heart/heart mice were found to have cardiac non-heme iron concentrations that were 30% lower (P<0.01) than those of wild-type littermate controls at 6 weeks of age. Interestingly, however, double mutant Hfe2-/-;Dmt1heart/heart mice accumulated more cardiac non-heme iron (3.9X control) than did single-mutant Hfe2-/- mice (2.3X control) at 6 weeks of age. Cardiac-specific disruption of Zip8 did not affect cardiac non-heme iron concentrations under basal conditions or when mice were crossed with Hfe2-/- mice. Collectively, these data indicate that DMT1 and ZIP8 (as well as ZIP14) are dispensable for iron loading of the heart in a mouse model of hemochromatosis. Our data additionally suggest that DMT1 may play a role in normal cardiac iron metabolism.
Seventh Congress of the International BioIron Society Page 251 Poster Abstracts IBIS
Poster #137
CARDIAC IRON BURDEN IS CORRELATED WITH INNATE IMMUNE MARKERS, BODY MASS INDEX AND LABILE PLASMA IRON IN ßETA-THALASSEMIA MAJOR PATIENTS UNDERGOING COMBINED CHELATION THERAPY Nicholas Slater, BSc², Marcela Weyhmiller, PhD¹, Patricia Evans, PhD³, John Porter, MD³, Annie Higa, BS¹, Vivian Ng, BS¹, Nancy Sweeters, PNP¹, Jackson Price, BSc², Alisha Manji, BSc², David Killilea, PhD¹, Lynne Neumayer, MD¹, Ashutosh Lal, MD¹, Paul Harmatz, MD¹, Elliott Vichinsky, MD¹ and Patrick Walter, PhD¹ ¹UCSF Benioff Children's Hospital Oakland; ²Biology, University of Victoria; ³University College London Presented By: Patrick Walter, PhD
ntroduction β-thalassemia major (TM) is an inherited anemia that is characterized by severe transfusion dependence which can lead to hepatic and extra-hepatic cardiac iron overload, causing cardiomyopathy, the leading cause of death. Despite improved chelation therapies, TM patients still endure cardiomyopathy as well as chronic inflammation, organ failure and infection. Innate immune cells are central to iron trafficking and response to organ injury. To travel to areas of iron-induced tissue injury, innate immune cells must extravasate from the vasculature using surface presentation of ligands such as LewisX (aka CD15). LewisX is a trisaccharide adhesion molecule, used by granulocytes to mediate endothelial attachment and phagocytosis. In this study, TM granulocyte LewisX expression was compared to iron-burden markers (such as cardiac T2*) and body mass index when undergoing combined chelation therapy with deferoxamine and deferasirox. A comparative healthy control group of subjects were also studied. LewisX surface presentation was also quantified on cultured THP-1 cells exposed to exogenous iron. Patients and Methods: TM patients were investigated at baseline and after 6 months and a year of combined chelation therapy with deferasirox and deferoxamine and analyzed for the expression of LewisX on peripheral-blood granulocytes. Eighteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox and deferoxamine (Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Fasting blood samples were obtained i) at baseline after a 72 hr. washout of chelator, and ii) at 6 and 12 months on study. Fourteen healthy controls (10 - 35 yrs old) were also enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and granulocytes were separated from dilute blood using ficoll. Granulocytes were purified using antibody- linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of LewisX+ (CD15+) granulocytes were verified by flow cytometry. The expression level of LewisX on purified granulocytes was determined and reported as the median fluorescent intensity (MFI). Results: A 5% decrease in LewisX positive granulocytes was observed in TM compared to healthy age and ethnically matched controls (TM = 90.19 ± 7.14, control = 96.6 ± 1.90, p = 0.034). LewisX expression was also decreased on THP-1 monocytic cells exposed to iron-citrate for greater than six weeks in vitro (p=0.04). In TM, LewisX expression correlated with labile plasma iron (r = -0.47 and p=0.015), T2* (myocardial iron, r= 0.69 and p=0.001), left ventricular ejection fraction (r = 0.49 and p=0.030) and body mass-index (r = -0.78 and p=0.002). Multiple regression analysis revealed that myocardial iron was best predicted by LewisX independently (r = 0.66, p = 0.001), whereas, LewisX expression was best predicted by BMI (r = 0.76, p = 0.002). 52 weeks of combined chelation therapy successfully restored TM expression of LewisX to the level of healthy controls (baseline = 90.19±7.14, 26 weeks = 96.10±3.44, 52 weeks = 96.61±3.10, p= 0.011). Conclusions: We found significant evidence that granulocyte LewisX surface presentation may be decreased in response to LPI and is predicted by BMI. Furthermore, LewisX and BMI appear to be predictive of cardiac iron loading and function, providing potential novel indicators for the leading cause of mortality in TM.
Seventh Congress of the International BioIron Society Page 252 Poster Abstracts IBIS
Poster #138
RENAL HANDLING OF NON-TRANSFERRIN BOUDN IRON AND ASSOCIATED INJURY IN PROXIMAL TUBULAR EPITHELIAL CELLS Sanne van Raaij¹, Rosalinde Masereeuw, PhD², Dorine Swinkels, PhD¹ and Rachel van Swelm, PhD¹ ¹Radboud University Medical Center, Nijmegen, The Netherlands; ²Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands Presented By: Sanne van Raaij
n systemic iron overload disorders, such as β-thalassemia, systemic iron regulation is disturbed. Patients with these disorders have increased circulating levels of iron bound to the iron transport protein transferrin as well as increased levels of non-transferrin bound iron (NTBI). These increased amounts of iron may be filtered by the kidney, leading to increased exposure of the proximal tubular epithelial cells to potentially harmful iron. NTBI may have toxic effects on the proximal tubular cells by means of catalyzing the production of reactive oxygen species (ROS). Indeed, an association between increased renal iron exposure and renal injury is supported by the fact that proximal tubular dysfunction as well as iron deposition have been observed in patients with β-thalassemia. However, little is known about the molecular mechanisms involved in renal NTBI handling or the subsequent potential harmful effects. Current knowledge suggests the potential involvement of the divalent metal transporters ZIP8 and ZIP14, but not DMT-1. Human conditionally immortalized proximal tubular epithelial cells (ciPTECs) were used to characterize the role of ZIP8 and ZIP14 in renal NTBI handling and to evaluate the potential of NTBI-mediated renal toxicity. ZIP8, ZIP14 and DMT-1 all co-localized with markers for early (EEA1) and late (RAB7) endosomed as assessed by immunocytochemistry. However, cell surface biotinylation showed that only ZIP8 and ZIP14, but not DMT-1, were located on the ciPTEC plasma membrane. This suggest that ZIP8 and/or ZIP14 may be involved in NTBI reabsorption. Incubation with 0-500 µM iron citrate (FeC) up to 24h resulted in a time-dependent increase in intracellular ciPTEC iron levels (p<0.001), associated with increased total ferritin and reduced transferrin receptor-1 protein levels as demonstrated by Western blot. Moreover, using the H2-DCFDA fluorescent probe we showed that incubation of FeC resulted in a dose-dependent oxidative stress response. Despite the increased oxidative stress, no cell death was observed in the ciPTECs after incubation with 0-500 µM FeC up to 72h. Using a QPCR-based tox-array, we discovered that the absence of FeC-induced cell death might be attributable to a NRF-2 mediated antioxidant response. Indeed, additional QPCR experiments showed significant dose-dependent increases in NQO1 and TXNRD1 mRNA expression levels after 48 and 72h of FeC incubation and increased GCLC and GCLM mRNA expression after 72h. At these time points also significant increases in heme oxygenase-1 (HO-1) mRNA expression were observed after FeC exposure. Our data suggests that ZIP8 and/or ZIP14 might be involved in increased iron uptake in proximal tubular epithelial cells during systemic iron overload. Additional experiments using ZIP8 and ZIP14 silencing are conducted to further elucidated their roles in NTBI uptake in ciPTEC cells. Moreover, incubation of ciPTECs with FeC up to 72h resulted in an antioxidant response that might protect the cells from cell death via the NRF2-pathway. This protective system might become exhausted during chronic NTBI exposure as would be the case in ß-thalassemia, resulting in iron-mediated damage to the proximal tubular epithelial cells. Future experiments will focus on the NRF2- mediated defense system against FeC exposure in ciPTECs.
Seventh Congress of the International BioIron Society Page 253 Poster Abstracts IBIS
Poster #139
INCREASED IRON ACCUMULATION AND UPREGULATED HEPCIDIN EXPRESSION IN KIDNEYS OF SICKLE CELL DISEASE MICE Marina Jerebtsova, PhD¹, Guelaguetza Vazquez-Meves, PhD¹, Namita Kumari, PhD¹, Nowah Afangbedji, BS¹, Alfia Khaibullina, PhD², Zena Quezado, MD² and Sergei Nekhai, PhD¹ ¹Howard University; ²Sheikh Zayed Institute, Children’s National Health System Presented By: Sergei Nekhai, PhD
Introduction: Hemolysis and frequent blood transfusions lead to iron overload and organ iron accumulation in patients with red blood cells disorders. The pattern of iron accumulation within different organs is disease specific. Abnormalities of renal iron metabolism and cortical iron deposition is characteristic for sickle cell disease (SCD) but not for β-thalassemia. Renal iron deposition does not correlate with iron overload and blood transfusion. Iron is reabsorbed from primary urine in the renal proximal epithelial cells and released into the renal intersitium by ferroportin. Iron-regulating hormone, hepcidin controls ferroportin expression. Binding of hepcidin to the ferroportin induces ferroportin degradation and intracellular iron accumulation. Low concentrations of circulating hepcidin are common in SCD patients and do not explain paradoxical renal iron accumulation. SCD mice accumulate iron in the epithelial cells of proximal tubules and may be a suitable model to study iron metabolism in SCD. Objective: To characterize proteins of the renal iron metabolism in SCD mouse model. Methods: The SCD (Townes) mice do not express mouse - or β-globin alleles, but carry two copies of a human 1- globin gene and two copies of a human A -globin and βS-globin genes. These animals synthesize approximately 94% human sickle (HbS) and 6% human fetal hemoglobin (HbF), and no murine hemoglobin. Control animals carry two copies of the human 1-globin gene and two copies of the human hemoglobin gamma (A ) gene and the human wildtype hemoglobin beta (βA) gene. idneys were collected from 5 months old SCD and control mice. Renal cortex was used for RNA and protein isolation. Levels of renal hepcidin, ferroportin, transferrin receptor (TFR1), divalent cation receptor (DMT1), ferritin and hepheastin were determined by q-RT-PCR, WB and ELISA. Paraffin-embedded sections were used for immunostaining. Perl’s Prussian blue staining was used for detection of renal iron accumulation. Results: We detected significant accumulation of iron in the epithelial cells of proximal tubules in SCD mice. Expression of renal hepcidin was increased in SCD mice compared to controls. Surprisingly mRNA levels of all other proteins involved in renal iron metabolism (ferroportin, TFR1, DMT1, ferritin and hephaestin) were decreased in SCD mice kidney. In contrast, we found increased protein levels of transferrin receptor (iron importer), ferritin (iron storage protein) and slightly increased level of ferroportin (iron exporter). We also observed significant renal macrophages infiltration in SCD mice. Conclusions: Increased levels of renal hepcidin expression in SCD mice may be associated with renal inflammation. Higher levels of locally expressed hepcidin may lead to the partial degradation of ferroportin. Increased levels of iron importers (TFR1 and DMT1) and no significant change in ferroportin expression can cumulatively saturate iron storage in ferritin and lead to the accumulation of intracellular iron. Acknowledgments: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.
Seventh Congress of the International BioIron Society Page 254 Poster Abstracts IBIS
Poster #140
ENHANCED MEDULLARY IRON TRAFFICKING MAY EXPLAIN RESISTANCE OF KIDNEY TO IRON OVERLOAD INJURY Avital Weiss, PhD, Lior Spector, MSc, Inbar Gold, MSc, Lena Lifshitz, PhD, Abraham Nyska, PhD, Sefi Addadi, PhD, Diana Goldberg, Yael Haimovich, MSc, Tracey A. Rouault, MD and Esther G. Meyron-Holtz Technion - Israel Institute of Technology Presented By: Esther Meyron-Holtz, PhD
Background: In the kidney, iron bound to transferrin (Tf) passes the glomerulus and is reabsorbed through epithelial cells. The kidney is extremely sensitive to hemolysis and the oxidative damage elicited by heme-iron, filtered through the glomerulus. In contrast, it is protected from damage of iron overload that harms many other tissues, including liver, spleen, heart and pancreas, by an unknown mechanism. Methods: We studied the effect of iron overload on iron distribution and on the regulation of iron transport proteins in the kidney. To do this, we used a novel correlative light- and electron-microscopy approach, coupled with atomic detection and standard biochemical assays. Results: Transferrin Receptor 1 (TfR1) was down-regulated as expected during iron overload, but the multi-ligand heterodimeric receptor-complex megalin/cubilin, that is known to internalize Tf as well, was highly up-regulated and surprisingly was also present in the renal medulla of iron overloaded mice. Cubilin up-regulation was dependent on transcriptional regulation of megalin by iron. Moreover, intracellular ferritin distribution shifted from an apical to a punctate location throughout the epithelial cells and the ferrous iron exporter ferroportin was not reduced by iron overload. Iron accumulated mainly in the interstitial macrophages, and more prominently in the medulla than in the cortex. Conclusions: This suggests that during iron overload, the megalin/cubilin complex, the iron storage protein ferritin and ferroportin participate in efficient iron trafficking through kidney epithelial cells, and spare these cells from iron overload. Much iron trafficking takes place in the relatively hypoxic renal medulla. This may explain why the kidney is not suffering from oxidative damage during iron overload as much as other tissues.
Seventh Congress of the International BioIron Society Page 255 Poster Abstracts IBIS
Poster #141
ELEVATIONS IN FGF23 PRECEDE DISRUPTIONS IN EITHER PHOSPHATE OR IRON HOMEOSTASIS IN THE EBF1- KO MODEL OF RENAL INSUFFICIENCY Sarah Jenkins¹, Xuiqi Li, BS¹, Tracy Nelson², Karin Finberg, MD, PhD¹ and Jackie Fretz, PhD¹ ¹Yale School of Medicine; ²Yale School of Medicne Presented By: Jackie Fretz, PhD
Development of anemia during chronic kidney disease (CKD) is a multifactorial process, but the exact mechanisms regulating the onset of anemia are not completely understood. Phosphate and iron metabolism are linked intimately through the phosphate-regulating hormone FGF23. It has been observed that Iron status is inversely correlated to the level of circulating FGF23, and improvement in iron bioavailability within individual patients correlates with a decrease in FGF23. Also, elevated FGF23 has been shown to negatively regulate erythropoiesis. Independently, FGF23 has arisen as an early biomarker of renal dysfunction, and its regulation in early CKD may be independent of the parathyroid hormone (PTH), 1,25(OH)2 Vitamin D3 signaling axis. To better understand the events regulating early dysfunctions of iron bioavailability, phosphate balance, and FGF23 expression in CKD we profiled the changes in iron homeostasis that accompany the onset of a mouse model of congenital CKD. Our model employed targeted deletion of the transcription factor Early B cell Factor 1 (Ebf1) from the kidney stromal progenitors (using Foxd1-cre). This results in a developmental abrogation of outer cortex beginning at postnatal day 14 (P14), and animals present with glomerulonephritis, phosphate wasting, moderate elevations in FGF23, and anemia. We profiled the sequential presentation of indicators of renal dysfunction (NGAL, albuminuria, hematuria, inflammatory markers), phosphate imbalance (PTH, 1,25(OH)2 Vitamin D3, serum phosphate, phosphaturia), and regulators of iron bioavailability and transport (hepcidin, transferrin, erythrocyte counts, Epo, and splenic erythropoiesis) to better understand the order of events that initiate and drive abrogation of the phosphate-iron-FGF23 regulatory axis during the onset of CKD. We report here that elevations in circulating intact-FGF23 coincide with the earliest indicators of renal dysfunction, and precede changes in urinary phosphate wasting or changes in iron homeostasis. This was apparent as soon at kidney development was visibly abrogated, and coincided with locally produced markers of damage and inflammation within the kidney. Histological abnormalities of kidney development are apparent at P14, yet phosphaturia is only present at P21, anemia (assessed by erythrocyte counts and hemoglobin content) trended down at P21 but was not significantly altered until P24. Splenomegaly arose at P28. Both circulating intact-FGF23 and FGF23 mRNA in osteocytes was elevated at P14. Serum Epo was normal until disease was well established, at 1 month, but was preceded by transferrin loss in the urine (P20), and elevations in Epo-transcript levels within the kidney (P14). Neither 1,25(OH)2 Vitamin D3 nor serum PTH were changed within the first month. We conclude that early CKD resembles a situation of primary FGF23 excess. Furthermore, elevations in FGF23 occur weeks prior to imbalances in iron or phosphate homeostasis, and are secondary to elevated FGF23. Although it remains to be determined if altered mineral availability can later modulate FGF23 levels in early CKD, they are not the driving stimulus for the initial upregulation of FGF23.
Seventh Congress of the International BioIron Society Page 256 Poster Abstracts IBIS
Poster #142
IRIDA DUE TO HETEROZYGOUS MUTATIONS OF TMPRSS6 AND ACVR1A ENCODING THE BMP RECEPTOR ALK2 Alessia Pagani, Silvia Colucci, Renata Bocciardi, Roberto Ravazzolo, Laura Silvestri and Clara Camaschella Vita Salute University and Division of Genetics and Cell Biology, San Raffaele Scientific Institute Milano, Italy Presented By: Alessia Pagani, PhD
Introduction: Iron Refractory Iron Deficiency Anemia (IRIDA) is an autosomal recessive disorder due to loss of function mutations of TMPRSS6 encoding the transmembrane serine protease matriptase 2, which inhibits hepcidin expression by cleaving from the hepatocyte surface the BMP co-receptor hemojuvelin (HJV). The disease is characterized by inappropriately high levels hepcidin for body iron content, microcytic hypochromic red cells and anemia refractory to oral iron treatment. Case report: Here we reevaluate a child patient with IRIDA we previously reported with an incomplete TMPRSS6 genotype (De Falco et al, Hum Mut 2010). The proband was compound heterozygote for two TMPRSS6 mutations leading to I212T (a causal mutation) and R271Q (a silent one) substitutions. In the follow up the patient was diagnosed with the rare dominant disorder Fibrodysplasia Ossificans Progressiva (FOP), with R258S mutation in ACVR1A encoding the BMP type I receptor ALK2. Methods: Cell surface localization of ACVR1Awt, R206H and R258S was evaluated in HeLa cells by streptavidin-biotin pull down according to standard procedures. Hepcidin activation by ACVR1Awt, R206H and R258S pathogenic variants was analyzed in hepatoma cells using the hepcidin promoter luciferase assay in the presence of BMP6 or HJV. TMPRSS6 and ACVR1A expressing vectors, carrying proband mutations, were transfected in hepatoma cells and hepcidin activation evaluated using the hepcidin promoter luciferase assay. Immunoprecipitation was performed in HeLa cells transfected with ACVR1AFLAG, wt and mutants, HJV and TMPRSS6HA: briefly, ACVR1A was pull down by using the anti-FLAG affinity gel and HJV and TMPRSS6 detected using the anti-HJV and the anti-HA antibodies, respectively. Results: ACVR1A wt and variants are immunoprecipitated with TMPRSS6 and HJV, suggesting a functional interaction. FOP-associated ACVR1A mutations R258S, present in the proband, and R206H, common in other FOP patients, activate hepcidin in hepatoma cells, while wild type ACVR1A receptor does not. In vitro reconstruction of the proband genotype expressing mutants in the presence of the BMP coreceptor hemojuvelin show that in cells expressing the inactive TMPRSS6I212T and the ACVR1AR258S variants hepcidin is significantly higher than in the presence of TMPRSS6wt. Conclusions: Our results demonstrated the role of ACVR1AR258S mutant on a constitutive hepcidin activation and IRIDA development in the presence of a partially inactive protease TMPRSS6I212T. We speculate that patients with ACVR1AR206H do not develop IRIDA because of normal protease activity, considering how rare TMPRSS6 mutations are. Our results indicate a role for ACVR1A mutations in the liver, emphasize the importance of TMPRSS6 in the control of the hepatic BMP/SMAD signaling pathway, provide a novel tool to diagnose rare cases of IRIDA and novel insights into hepcidin regulation. Acknowledgements: Supported by Telethon Grant GGP15064 to LS
Seventh Congress of the International BioIron Society Page 257 Poster Abstracts IBIS
Poster #143
COMPARISON OF MATERNAL AND FETAL IRON REGULATORS IN IRON DEFICIENCY ANEMIA OF PREGNANCY Sreenithi Santhakumar, MTech¹, Rekha Athiyarath, PhD¹, Anne George Cherian, MD², Vinod Joseph Abraham, MD² and Eunice Sindhuvi Edison, PhD¹ ¹Department of Haematology, Christian Medical College, Vellore, India; ²Department of Community Health, Christian Medical College, Vellore, India Presented By: Eunice Sindhuvi Edison, PhD
Iron Deficiency Anaemia in Pregnancy (IDAP) occurs when optimal iron requirement is not met for the developing fetal- placental unit and increased maternal erythrocyte mass expansion. IDAP remains a constant public health problem in our country, where all pregnant women receive routine iron supplementation. Anaemia associated with iron depletion leads to premature delivery, low birth weight, maternal and infant mortality. Trans-placental iron is regulated by hepcidin whose levels are decreased in IDAP. The critical role of maternal iron status and its regulation of foetal iron metabolism and transport across placenta need to be elucidated. To analyse this further, we compared the maternal and foetal hepcidin, GDF15 and ferritin levels in IDAP and normal subjects. Subjects who presented to the Department of Community health for child birth were screened and consenting subjects (age 35 years and a gestational age≥36weeks) were included in the study. Maternal blood sample and cord blood was collected. Complete blood counts (CBC) were done using an automated cell counter (Sysmex KX21). Serum ferritin was measured using chemiluminescent immunoassay. Maternal and cord blood serum hepcidin and GDF15 levels were measured using ELISA (DRG Diagnostics and Ray Biotech, respectively). Statistical analysis was performed using SPSS software. Of the one hundred and twenty four subjects (n=124) who were included in the study, twenty nine were anaemic (Hb<11g/dL) and ninety five were non-anaemic controls. The haematological and biochemical parameters of these groups are tabulated (Table.1). Of the anaemic subjects, 13 were primi gravida (G1), 14 in 2nd gravida (G2), and 2 in multigravida (>G2) whereas in non-anaemic subjects 46, 39, 10 were in G1, G2,>G2 respectively. Notably, the median log hepcidin levels were significantly reduced in G2 (0.785; -0.1-1.97) when compared with G1 (1.011; -0.05-1.88). In addition maternal hepcidin levels were low in subjects with low iron stores (ferritin<20ng/ml) in comparison to subjects with high ferritin (p=0.038). Hepcidin and ferritin correlated positively in both maternal (r= 0.184, p=0.042) and foetal (r=0.288, p=0.001) samples in subjects with anaemia; this was not observed in controls. GDF15 levels were higher in anaemic subjects as compared to control group. Interestingly, a negative correlation was observed between maternal GDF15 and foetal hepcidin-ferritin ratio (H:F) (r=- 0.439, p=0.002) and also fetal GDF15 with maternal H:F (r=-0.228, p=0.015); this relationship and its significance has to be further validated. Of the twenty nine anaemic subjects, only one foetus (1/29, 3.4%) had IDA. These findings suggest that foetal iron regulation is independent of maternal iron status. Even when the maternal iron stores are low, the foetus gets the priority according to the hierarchy of iron usage. The role of iron regulatory molecules which facilitate this hierarchy needs to be elucidated. Table.1 Maternal and Foetal parameters in anaemic and non-anaemic subjects (Values of Age, Gestational age, Hb, MCV are represented as Mean±S.D, Ferritin, Hepcidin and GDF15 are represented as Median, Range)
Seventh Congress of the International BioIron Society Page 258 Poster Abstracts IBIS
Poster #144
A NOVEL IN VITRO MODEL OF SIMULATED TWICE A DAY IRON SUPPLEMENTATION FOR ASSESSMENT OF IRON UPTAKE AND CELLULAR DAMAGE Petia Apostolova¹, Voni Blesia, MSc², Christelle Tossou, MPharm², Derek Renshaw, MSc, PhD³, Sebastien Farnaud, MSc, PhD³, Robert Evans, BA, PhD⁴ and M.Gulrez ari ala, BPharm, MSc, PhD² ¹University of Westminster; ²Faculty of Science & Technology / University of Westminster; ³Centre for Applied Biological and E ercise Sciences Coventry University; ⁴Centre For Electronic Systems Research Brunel University Presented By: Petia Apostolova
Iron deficiency is the most common and widespread preventable nutritional deficiency in the world. Programs using ferrous sulphate (FeSO4) are routinely carried out to reduce prevalence of iron deficiency. Current guidelines recommend treatment with doses varying from 60 - 120 mg/day elemental iron (Fe) prescribed once or twice a day. Average daily intake of dietary iron with a regular diet is ~ 10 - 20 mg/adult. This equates to a duodenal concentration of approximately 10 µM Fe. In contrast, with therapeutic oral iron supplementation, the duodenal iron concentration is likely to be 200 µM (60 mg Fe dose). Free unbound iron promotes formation of reactive oxygen species (ROS) that produces oxidative stress which manifests as gastrointestinal side effects associated with poor patient compliance. The aim of this study was to assess iron uptake with varying doses and evaluate cellular effects of iron supplemented once and twice a day in vitro. The study was conducted in two stages. First stage compared iron uptake in caco-2 cells (n=6) with varying doses (20 µM, 100 µM and 200 µM) of FeSO4, equating to 6mg, 30mg and 60mg of Fe. Highest iron uptake (intracellular ferritin as marker) was observed in caco-2 cells treated with 20 µM followed by 100 µM (275 ± 13 ng ferritin/mg protein vs 223 ± 9 ng ferritin per mg protein). Iron uptake with concentration of 200 µM was significantly lower than at 20 µM treatment (17 ± 4 ng ferritin/mg protein; p<0.04). Doses with highest absorbency (20 µM, 100 µM) were used for the second stage where cells were treated at 0 and 12 or 0 and 24 hours (n=6), mimicking once or twice a day iron administration. Total iron concentration, following two-fold exposure was measured by FerroZine® based assay. There was no significant difference in iron uptake between the two concentrations (p=0.26) consistent with first stage results. Cellular damage was recorded by measuring necrosis and lipid peroxidation throughout. Lactate dehydrogenase release analysis suggests higher concentration (100 µM) of Fe may increase lipid peroxidation (p=0.09). These effects were time bound; peak cytotoxicity was observed at 12 h, prior to second supplementation (p=0.03). Corresponding results were recorded with PrestoBlueTM cell viability assay. At both concentrations, cell viability was reduced post second exposure at 12 h (42% ± 1) than at 24 hours (71% ± 4)(100 µM Fe treated cells). Accordingly, 20 µM iron treatment resulted in 42% ± 1 and 57% ± 2 at 12 and 24 hours. Caco-2 monolayers treated with Fe 20 µM twice a day displayed peak in lipid peroxidation (malondialdehyde as marker) pre-second exposure. However, using the same concentration but with once a day treatment the peak occurred following the second supplementation. Our results demonstrated that lower dose less often may be more favourable for enterocytes. These results are also supported by our protein oxidation analysis (OxyBlotTM). Our findings are consistent with findings by Khalid et al., (2012) and Viteri et al., (2012) (randomised control clinical trials) which suggest less frequent supplementation with lower Fe dose may improve patient compliance due to reduction of side effects. In summary, we consider this preliminary study an optimisation of a novel in vitro model that demonstrates that FeSO4 supplementation may be more effective and less cytotoxic at smaller doses and at lower frequency of administration.
Seventh Congress of the International BioIron Society Page 259 Poster Abstracts IBIS
Poster #145
IRONWOMAN TRIAL: THE IMPACT OF INTRAVENOUS IRON ON EXERCISE PERFORMANCE IN IRON DEFICIENT, EXERCISING WOMEN Georgie Bruinvels⁵, Charles Pedlar, PhD¹, Richard Burden, PhD², Nicola Bro n, PhD³, Anna Butcher, MD⁴, Marisa Chau, BSc⁴ and oby Richards, MD⁴ ¹Massachusetts General Hospital, St Mary's University, Orreco Ltd; ²St Mary's University, Orreco Ltd, English Institute of Sport; ³St Mary s University; ⁴University College London; ⁵UCL Presented By: Georgie Bruinvels
Introduction: The impacts of iron deficiency anaemia (IDA) on exercise performance are well known, however the effects of iron deficiency without anaemia (IDNA) are less established, and research is historically conflicting. Haemoglobin levels are not compromised in IDNA, therefore oxygen carrying capacity should not be affected. However, iron is also essential for other physiological pathways including mitochondrial respiration and enzyme activity. Part of the ambiguity surrounding IDNA is likely to be caused by a lack of clarity in the criteria used for IDNA diagnosis, with ferritin cut off values ranging from 15-35µg/L. As a toxic element, unnecessary supplementation or iron is not advisable. This study aimed to determine the impact of iron deficiency in a group of iron deficient exercising women using a ferritin <30 µg/L for the classification of iron deficiency. Methods: In this observational study, thirty-two iron deficient (ferritin <30 µg/L) exercising women were recruited (mean age: 35± 8.9 years, height: 165.2± 5.5 cm and weight: 61.7± 7.6 kg and received an intravenous injection of iron isomaltoside (20mg/kg bodyweight). They completed a maximal exercise test, had their haemoglobin mass measured and had blood samples collected both pre- injection and two weeks post-injection. Study approval was received from both the National Health Service (15/LO/1570) and St Mary’s University Ethics Committee. Results: Mean baseline haemoglobin concentration was 12.4± 0.65g/dL and serum ferritin was 15.7± 7.0µg/L. When comparing pre- and post- injection tests serum ferritin and haemoglobin concentration significantly increased (p<0.05). Exercise performance also increased; VO2max changed from 49.1± 6.3 kg/ml/min to 50.7± 7.0 kg/ml/min (t=2.43, p<0.05) and time to exhaustion from 10 min: 8s to 10min: 56s (t=5.67, p<0.05). Total haemoglobin mass increased from 602.7± 61.0g to 617.6± 60.1g (t=2.38, p<0.05). Conclusions: In summary, a single dose of intravenous iron increased iron status, aerobic power and total haemoglobin mass in this group of iron deficient exercising females suggesting that a criterion of ferritin <30 µg/L for the diagnosis of IDNA may be appropriate. However, more work is required to establish whether, within this populous response to intravenous iron can be predicted from baseline characteristics.
Seventh Congress of the International BioIron Society Page 260 Poster Abstracts IBIS
Poster #146
THE IMPACT OF HEAVY MENSTRUAL BLEEDING AND IRON STATUS ON FATIGUE IN MENSTRUATING WOMEN Georgie Bruinvels¹, Charles Pedlar, PhD², Richard Burden, PhD³, Anna Butcher, MD⁴, Marisa Chau, BSc⁴, imothy Cush ay⁵ and oby Richards, MD⁴ ¹UCL; ²Massachusetts General Hospital, St Mary's University, Orreco Ltd.; ³St Mary's University, Orreco Ltd., English Institute of Sport; ⁴University College London; ⁵ ifor Pharma Presented By: Georgie Bruinvels
Introduction: Fatigue is the most common reason to seek medical advice in primary care. It is more commonly reported in women than their male counterparts. Onset of fatigue can be slow and insidious often causing it to go unnoticed. It is often associated with iron deficiency and anaemia. Due to menstrual blood loss, anaemia is particularly prevalent in women of a reproductive age. It would therefore seem likely that those with heavy menstrual bleeding (HMB) would be more likely to have iron deficiency and/or anaemia and therefore are more susceptible to fatigue. This study aimed to identify the association between HMB and iron deficiency with or without anaemia and fatigue. Methods: Four hundred and eighty-six menstruating women who attended routine medical screening appointments completed a ‘Female Health Questionnaire’ which assessed menstrual cycle patterns alongside awareness of iron status amongst other factors, the Multidimensional Fatigue Inventory (MFI-20), and had venous blood samples for measurement of iron status. Results: Nearly a quarter of the populous had HMB (23.0%), while 15.2% had iron deficiency anaemia (IDA; [Hb] <12.0g/dL and serum ferritin <30µg/L), 18.1% had anaemia [Hb] <12.0g/dL), and 28.8% were iron deficient (serum ferritin <16µg/L). HMB was associated with an increased risk of IDA, anaemia and iron deficiency (p<0.05). Those with HMB, IDA and anaemia reported higher levels of fatigue (p<0.05). However, there was no associated between iron deficiency without anaemia and fatigue (p>0.05). Amongst those with HMB, iron status had no impact on fatigue (p>0.05). Awareness of both HMB and iron status appeared poor. Conclusions: HMB, IDA and anaemia all independently increase fatigue in menstruating women. However, in this study, the symptoms secondary to HMB appeared to dominate the symptoms secondary to IDA and anaemia as fatigue was not related to iron status in those with HMB. However further research is required as to the impact of iron deficiency without anaemia on fatigue. Awareness of both iron status and HMB was found to be poor amongst both individuals and medical professionals and needs to be improved.
Seventh Congress of the International BioIron Society Page 261 Poster Abstracts IBIS
Poster #147
EFFECTS OF THE IRON CHELATOR CURCUMIN IN THE DSS-INDUCED COLITIS MOUSE MODEL Macha Samba Mondonga, MSc, Marco Constante, PhD, Gabriela Fragoso, PhD and Manuela M. Santos, PhD CRCHUM, Universite de Montreal Presented By: Macha Samba Mondonga, MSc
Introduction: Patients with inflammatory bowel disease (IBD) have a major increased risk for colorectal cancer (CRC). Curcumin, an active ingredient in the dietary spice tumeric, has been shown to reduce inflammation and colonic tumor burden in mouse models of IBD. However, curcumin has also been shown to chelate iron and is sufficient to induce iron deficiency in vivo. The iron chelating properties of curcumin may therefore contribute to the development and severity of iron deficiency in IBD settings. Methods: To evaluate the effects of curcumin on systemic iron balance, colitis was induced by administration of dextran sodium sulfate (DSS) (1% W/V) in drinking water to C57Bl/6 or Balb/c mice fed an iron sufficient diet (50 ppm iron) with or without supplemented curcumin (2% w/w). Results: We found that curcumin supplementation in the DSS mouse model of colitis caused mild anemia and aggravated diarrhea, depleted splenic iron storage, and decreased survival in C57Bl/6 mice. Furthermore, we have confirmed that the impact of curcumin on anemia and survival is reproducible in Balb/c mice. Discussion and Conclusions: The anemia observed in mice fed the diet supplemented with curcumin and subjected to DSS may be due to the chelation of iron by curcumin concomitantly with the use of the an iron sufficient diet (50 ppm of iron). Our finding that curcumin decreases survival in DSS-treated mice contrasts with previous data in which curcumin was shown to improve the survival of DSS-challenged Balb/c mice. A contributing factor to this discrepancy may be the high iron content of diets used in other studies (>250 ppm of iron) compared with the iron sufficient diet (50 ppm of iron) used here. These findings suggest that curcumin use as a supplement may exacerbate the iron deficiency anemia in IBD patients.
Seventh Congress of the International BioIron Society Page 262 Poster Abstracts IBIS
Poster #148
OSTEOMALACIA AFTER TREATMENT WITH INTRAVENOUS IRON Benedikt Schaefer, MD¹, André Viveiros, MD¹, Irina Nardin², Bernhard Glodny, MD² and Heinz Zoller, MD¹ ¹Medical University of Innsbruck, Department of Medicine II, Gastroenterology and Hepatology; ²Medical University of Innsbruck, Department of Radiology Presented By: Benedikt Schaefer, MD
Background: Ferric carboxymaltose (FCM) and iron isomaltoside 1000 (IIM) allow complete correction of iron deficiency in a single infusion. A transient decrease in plasma phosphate concentrations, which is mediated by the phosphaturic hormone FGF-23, has been reported as a side effect of FCM. As highlighted in a recent cohort study severe hypophosphatemia (<0.6 mmol/L) occurs in 32.7% of patients treated with FCM. Case reports have shown that devastating musculoskeletal complications like osteomalacia can occur after severe and prolonged hypophosphatemia. Methods: Electronic medical records of patients attending the University Hospital of Innsbruck were searched for the keywords ferric carboxymaltose or iron isomaltoside. Eighty-one patients with documented administration of FCM or IIM with plasma phosphate concentrations before and after treatment were included in the study. All available radiological studies since 2000 were analysed for changes of the bone associated with prolonged and severe hypophosphatemia after treatment with i.v. iron. Results: Of 26 patients with iron-induced hypophosphatemia four patients had documented atraumatic pathological fractures after FCM. In the most severe case the patient suffered from chronic gastrointestinal blood loss and iron deficiency anemia was treated with a total of 19 g of FCM over two years. This caused hyperphosphaturic hypophosphatemia with elevated concentrations of intact FGF-23. Magnetic resonance imaging showed symmetric bilateral linear “Looser zones” that extended perpendicularly to the weight-bearing axis. In total, review of all imaging studies in this patient revealed 23 fractures. The diagnosis of osteomalacia was confirmed by a bone biopsy, showing broad seams of uncalcified osteoid upon Goldner staining. Prolonged hyperphosphaturic hypophosphatemia during repeated i.v. iron with FCM due to chronic intestinal blood loss in another patient was associated with chronic back pain and pain in his lower extremities. Magnetic resonance imaging revealed symmetrical looser zones in both calcanei and sacral wings. In the remaining two patients, multiple atypical bone fractures were found but not considered to be exclusively caused by iron-induced hypophosphatemia. In one patient vitamin D deficiency was identified and in the other patient the fractures were considered to be primarily caused by high-dose steroid treatment. Conclusions: Prolonged and severe hypophosphatemia caused by certain intravenous iron preparations can be associated with severe osteomalacia and atraumatic fractures. Patients with repeated i.v. iron dosing appear to be at particularly high risk of developing bone disease. In such patients, proactive management of risk factors such as vitamin D deficiency and monitoring of plasma phosphate concentrations is therefore advocated.
Seventh Congress of the International BioIron Society Page 263 Poster Abstracts IBIS
Poster #149
PREDICTORS OF IRON ABSORPTION FROM A NOVEL NANO IRON SUPPLEMENT (IHAT) IN GAMBIAN WOMEN IN THE ABSENCE OF INFLAMMATION Dora Pereira, PhD, MEng¹, Rita Wegmuller, PhD², Carla Cerami, PhD, MD² and Andrew Prentice, PhD² ¹University of Cambridge; ²MRC Unit, The Gambia Presented By: Dora Pereira, PhD MEng
The novel nano iron supplement IHAT was developed to simulate dietary iron absorption and slowly release its iron into the circulation so as not to cause an abnormal rise in transferrin saturation (TSAT). This feature of IHAT has a number of advantages over ferrous sulphate which induces an un-physiological iron bolus release into the circulation: (i) the lower rise in serum Fe and TSAT after each daily dose of IHAT should not cause a bolus rise in hepcidin which has been recently shown to inhibit absorption of the next-day dose with ferrous sulphate, (ii) less likelihood of formation of ‘true’ redox-active non-transferrin bound iron (NTBI) when TSAT is maintained below 40%, (iii) less ‘free’ iron highly available to sustain growth of systemic extracellular pathogens. The study presented here was conducted at the MRC Unit, The Gambia in accordance with the principles set forth in the ICH Harmonised Tripartite Guideline for Good Clinical Practice. The design was a cross-over, double-blind, single-dose comparison against ferrous sulphate in mildly anaemic and non-anaemic pre-menopausal non-pregnant women. The iron single dosage for IHAT and ferrous sulphate was 60 mg elemental iron, each compound was labelled with a stable iron 58 57 isotope (i.e. 2 mg Fe for IHAT and 10 mg Fe for FeSO4), and their absorption was determined from the red blood cell incorporation of the stable isotope 14 days after the single dose. Thirty women were randomly assigned to receive one of the 2 iron compounds (IHAT or FeSO4) in their first study visit (day 1) and the other in the second study visit (day 14). Participants were blind to which compound they received on day 1 and day 14. The route of administration was oral and the compounds were encapsulated into identical gelatin capsules. Investigators and analysist were also blinded to the treatments. In healthy women without iron deficiency anaemia, IHAT’s iron absorption was reduced by around 50% when compared to the iron absorption in mildly anaemic women (9 Seventh Congress of the International BioIron Society Page 264 Poster Abstracts IBIS Poster #150 ENTERAL IRON SUPPLEMENTATION INCREASES ERYTHROPOIESIS IN PREWEANLING WILD TYPE AND TMPRSS6 KNOCKOUT MICE Ramya Bharathi, Nermi Parrow, Princy Prasad and Robert Fleming Saint Louis University School of Medicine Presented By: Nermi Parrow, PhD Introduction: Inactivating mutations in the Tmprss6 gene cause iron-resistant iron deficiency anemia (IRIDA) due to excess hepcidin expression and decreased enteral iron absorption in humans and in mice. Lactoferrin (Lf) is an iron binding protein which has been reported to undergo enteral absorption by transcytosis and distribute to the liver and other tissues. These observations suggest that absorption of iron-bound lactoferrin (FeLf) might not be subject to hepcidin- mediated downregulation. We thus compared the ability of ferrous sulfate (FeSO4) and iron-loaded lactoferrin (FeLf) to correct iron-deficiency anemia in Tmprss6 knockout mice. Because reported evidence suggests that in suckling mice enterocyte ferroportin is resistant to hepcidin-mediated downregulation, mice were analyzed both prior to and after weaning. Methods: Tmprss6-/- mice were bred as heterozygotes and maintained on standard chow. Mice were supplemented with 4 mg/kg/d of iron as either FeSO4 or FeLf (or carrier) by gavage in two doses for 3 days. Supplementation of pre-weanling mice began on day 12, and post-weanling mice on day 23. Twelve hours after the last dose, mice were sacrificed and samples collected. Hematocrits, splenic indices (spleen weight/body weight), tissue iron concentrations, and expression of selected genes were measured. Because values from heterozygous and wild type mice across all parameters were similar, they were combined (control). Results: At day 15, carrier-treated Tmprss6 knockout mice demonstrated expected elevated liver Hamp1 mRNA compared with control; splenic index was also greater in the knockout mice (0.0076 +/- 0.0002 vs control 0.0051+/- 0.0002). However there was no statistical difference in tissue (liver or spleen) non-heme iron concentrations, or in hematocrits (29.4 +/- 0.96 vs 30.7 +/- 0.39). In response to either FeSO4 or FeLf, hematocrits increased in knockout (FeSO4 38.4 +/- 0.7, FeLf 36.1 +/- 1.2) and wild type mice (FeSO4 41.7 +/- 1.2, FeLf 38.4 +/- 0.8). Splenic indices increased with either form of iron in control mice (FeSO4 0.008 +/- 0004, FeLf 0.010 +/- 0.0005). Splenic indices were higher in the iron treated knockout mice (FeSO4 0.0083 +/- 0.0003, FeLf 0.012 +/- 0.0008). Splenic size was greater in mice treated with FeLf than with FeSO4. In post-weanling mice (26 days) control mice had higher hematocrits compared with knockout mice (47.4 +/- 0.8 vs 31.2 +/- 2.3). There was no statistical difference in the hematocrits of control mice treated with FeSO4 (45.8 +/- 1.7) or FeLf (47.9 +/- 0.7) compared with carrier. Likewise the hematocrits of the knockout mice treated with either FeSO4 (32.21 +/- 1.25) or FeLf (30.5 +/- 2.1) were not different from control. The splenic index was greater in knockout (0.0074 +/- 0.0003) compared with control mice (0.0051 +/- 0.0001); however, splenic index did not statistically differ in either wild type or knockout mice treated with FeSO4 or FeLf. Conclusions: These data demonstrate that both wild type and Tmprss6 knockout preweanling mice increased hematocrits in response to enteral iron supplementation. This suggests that erythropoiesis at this age may be iron-limited. Iron delivered as lactoferrin was equally or more effective than ferrous sulfate in increasing erythropoiesis in control and knockout mice at this age. However, enteral iron supplementation by similar regimen in post-weanling mice resulted in no significant change in measured erythropoietic parameters with either form of iron. Seventh Congress of the International BioIron Society Page 265 Poster Abstracts IBIS Poster #151 PANCREATIC IRON IN PATIENTS WITH HEMOCHROMATOSIS: NOT AS RARE AS CARDIAC IRON Jin Yamamura, MD², Roland Fischer, PhD³, B rn Sch nnagel, MD², Regine Grosse, MD⁴ and Peter Nielsen, MD, PhD¹ ¹University Medical Centre Hamburg, Dept. of Biochemistry and Molecular Cell Biology; ²University Medical Centre Hamburg, Dept. of Diagnostic and Interventional Radiology; ³UCSF Benioff Children's Hospital and Research Centre akland, Dept. of Hematology ncology; ⁴University Medical Centre Hamburg, Pediatric Hematology ncology Presented By: Peter Nielsen, MD, PhD HFE-associated hereditary Hemochromatosis (HFE-HH) is the most frequent monogenic genetic disorder in the Caucasian population. Due to a disturbed hepcidin-ferroportin axis, the food iron absorption is permanently increased leading to an excessive iron storage in organs. The liver is mostly affected (> 90 %) resulting in a potentially severe liver damage (fibrosis, cirrhosis). The exact distribution of iron into other organs is not well established and may also be variable among affected patients. In the last years, noninvasive techniques for iron quantification have been established. In this study we used biomagnetic liver susceptometry (BLS) for measuring liver iron (LIC) and quantitative MRI using multiple gradient-echo sequences (echo time 1.3–25.7 ms) for the determination of transverse relaxation rates R2* (=1/T2*). In the heart, septal R2* was determined from signal intensities by a mono-exponential model, while pancreatic iron (R2*) and fat was assessed by fat-water chemical shift relaxometry. So far, these measurements were performed in 21 patients with HFE-HH in a wide range of iron loading. As shown in Fig.1, the presence of heart iron is rare in patients with HFE-HH even in severely iron loaded patients. Among about 500 patients, we have seen only one patient with very severe iron overload presenting clinically impressive heart failure which recovered very quickly under DFO and phlebotomy treatment. As seen from the R2*-rates in the pancreas, the pancreas, especially the tail, is more frequently affected in severely loaded subjects but also in some milder cases. A larger prospective study must be awaited to document a pancreatic risk also in HFE-HH patients under routine diagnostic and treatment procedure. Fig.1: R2* rates in heart and pancreas in 21 patients with HFE-HH. Control values with this technique: cardiac R2*: 25-38 s-1; pancreatic R2*: 22-40 s-1 (Pfeiffer et. al. J Magn Reson Imaging. 2015 Jul;42(1):196-203). Seventh Congress of the International BioIron Society Page 266 Poster Abstracts IBIS Poster #152 WITHDRAWN Poster #153 HEME AND IRON SHAPE THE PHENOTYPE OF MACROPHAGES IN THE TUMOR MICROENVIRONMENT OF NON- SMALL CELL LUNG CANCER Milene Costa da Silva¹,⁵, Carl Ma imilian hielmann¹,⁵, Margareta P. Correia², Michael . Breck oldt³, Michael Meister⁴, homas Muley⁴, Adelheid Cer enka² and Martina U. Muckenthaler¹,⁵ ¹Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Germany; ²Innate Immunity Group, German Cancer Research Center , Heidelberg, Germany; ³Department of Neuroradiology, University Hospital Heidelberg, Heidelberg, Germany; ⁴ ranslational Research Unit, Thoraxklinik at University Hospital Heidelberg, Heidelberg, Germany; ⁵Molecular Medicine Partnership Unit (MMPU), Heidelberg University EMBL, Heidelberg, Germany Presented By: Milene Costa Da Silva, PhD The tumor microenvironment, which impacts significantly on cancer progression and therapy response, is characterized by high cellular complexity, including fibroblasts, stroma, blood vessels and infiltrates of immune cells. Tumor-associated macrophages (TAMs) are a critical component of the tumor microenvironment. They represent up to 50% of the mass of infiltrated cells and were shown to be of prognostic value. TAMs generally acquire an anti-inflammatory M2 phenotype, contributing to immune suppression, angiogenesis, tumor growth and remodeling of the tumor microenvironment. Here we show that tumor samples from patients with non-small cell lung cancer and from experimental murine lung tumors accumulate iron in TAMs, whereas cancer cells are relatively iron spared. Iron loaded macrophages are located close to sites of red blood cell extravasation in hemorrhagic areas of the tumor microenvironment. Hemorrhagic areas not only show increased numbers of iron-loaded TAMs, but also enhanced infiltration of neutrophils and increased expression of cytokines and chemokines responsible for macrophage and neutrophil recruitment. Consistent with in vivo observations, treatment of macrophages with conditioned media from tumor cells polarizes macrophages towards an M2-like phenotype. Interestingly, incubation of these M2-like macrophages with hemolytic red blood cells triggers polarization towards an M1-like inflammatory phenotype and increases the expression of cytokines responsible for neutrophil recruitment. Functionally, macrophages treated with hemolytic red blood cells and co-cultured with T cells, show enhanced killing activity. In conclusion, our data suggest that iron and heme derived from leaking red blood cells of fragile vessels shape the phenotype of TAMs and trigger cytokine production. We propose that delivering iron to TAMs may be of value therapeutically to increase anti-cancer immune responses. Seventh Congress of the International BioIron Society Page 267 Poster Abstracts IBIS Poster #154 THE ROLE OF IRON IN THE PROGRESSION OF SOFT TISSUE SARCOMAS Michela Asperti, PhD, Silvia Codenotti, PhD student, Paolo Crotti, student, Paolo Arosio, PhD, Alessandro Fanzani, PhD and Maura Poli, PhD University of Brescia Presented By: Maura Poli, PhD Iron is an essential micronutrient for life, but if present in excess it is toxic for the formation of free radicals (ROS) with irreversible cell damages. Several epidemiological studies have indicated that excess iron can contribute to the development of cancer cells that require much iron to proliferate. Many types of cancer present increased levels of TfR1, high hepcidin expression and a reduction of ferroportin (FPN). Also the tumor microenvironment contributes to provide iron to nearby cancer cells with tumor associated macrophages (TAM) expressing high FPN and low ferritin to improve the release of iron, made it available for tumor. Consequently iron increases in tumor cells (known as "iron addition") and it is used for their growth and for stimulating the formation of new endothelial vessels (angiogenesis), that help the tumor proliferation and aggressiveness. Although this process is widely studied in the most common cancers caused by ecto- and endodermal precursor cells, it is surprising how little attention has been paid to the iron metabolism in sarcomas, derived from mesenchymal precursors. The aim of this study is to establish the iron contribution to the growth and differentiation of sarcomas and how it can influence the evolution of the tumor. Sarcomas are rare tumors (1% incidence of all cancers) that affect the soft tissues, such as adipose tissue, muscle or cartilage, and predominantly affect adults although there are types restricted to the childhood. In this study we focus our attention on RD, MLS1765 and SKLMS-1 cell lines as a models of the most common sarcomas, specifically Rhabdomiosarcoma (RMS), Liposarcoma (LPS) and Leiomiosarcoma (LMS). The preliminary data showed that these cell lines express all the mRNA and proteins related to iron homeostasis and they were regulated by iron supplementation. These tumor cells are characterized by a higher expression of TfR1 in the proliferative state than in the differentiated cells which supports the hypothesis of "iron addiction" of proliferating tumor cells. Furthermore iron supplementation (FAC or Olotransferrin) caused a slower differentiation rate and the cells remained proliferating longer and more aggressive. In fact the marker of muscle differentiation, MyHC (Myosin Heavy Chain) is activated earlier in non treated (day 2) than in iron-treated cells (day 4), during the differentiation procedure and it does not reach the levels of untreated cells. This is a first clear evidence of the role of iron in tumor progression of sarcoma cells. The characterization of the proteins related to iron homeostasis in sarcomas at different stages of proliferation and aggressiveness could help to identify new diagnostic and therapeutic targets, aimed to increase the survival rate and the quality of life for patients with sarcomas. Seventh Congress of the International BioIron Society Page 268 Poster Abstracts IBIS Poster #156 INDUCTION OF IRON EXCESS IN PROSTATE CANCER: A NOVEL POTENTIAL THERAPEUTIC APPROACH Federica Morisi, Jessica Bordini, Gianluca Venturi, Alessia Fulli, Matteo Bellone, Francesco Montorsi, Alberto Briganti, Clara Camaschella and Alessandro Campanella IRCCS Ospedale San Raffaele Presented By: Alessandro Campanella, PhD Introduction and Objectives: Treatment of metastatic prostate cancer (PCa) is still a major challenge. Despite the introduction of novel therapeutic approaches, androgen deprivation therapy remains the mainstay treatment of PCa. However, virtually all patients become resistant to this treatment and develop castration resistant disease over time. Disruption of iron homeostasis has been recently demonstrated in PCa cells. These cells rearrange their iron homeostasis, limiting iron export by ferroportin and increasing intracellular iron by transferrin receptor 1 (TfR1) to favor malignant cell growth. However, excess iron may be toxic since it catalyses ROS formation. In this study we explored the effect of high dose iron administration to cellular and murine models of PCa. Material and Methods: We analyzed human androgen-dependent LNCaP and androgen-independent PC3 cell lines and murine TRAMP-C1 and TRAMP-C2 cells. Cells were grown in 10% or 2% serum media in combination with iron (200 μM Ferric ammonium citrate) or bicalutamide (2,5-5 μM), a specific inhibitor of cellular androgen pathway. Additionally, we generated PCa models by subcutaneously injecting TRAMP-C2 or PC3 cells in wild-type and immunodeficient mice, respectively. Mice were treated with combinations of iron dextran (250 mg/Kg, IP) and bicalutamide (10 mg/Kg, PO). Results: Among the PCa cells analyzed LNCaP cells and both murine cell lines reduced their proliferation when exposed to iron excess in vitro (p<0.01). On the contrary, PC3 cells maintained a cell growth rate similar to untreated cells, however they acquired iron sensitivity when grown in low serum medium (p<0.01) a condition that decreases androgen availability. To target androgen pathway more specifically, experiments were replicated in the presence of bicalutamide. As expected, LNCaP proliferation was strongly reduced in this condition (p<0.001). The combination of bicalutamide and iron excess produced the strongest impairment of cell proliferation in all the cell lines analyzed (p<0.01), suggesting that limitation of androgen availability exacerbates iron toxicity. Indeed, also PC3 cells were affected by iron excess upon bicalutamide exposure and reduced proliferation (p<0.001). In vivo experiments confirmed the in vitro results: high dose iron administered to mice injected with TRAMP-C2 reduced tumor growth (p<0.01). In mice injected with PC3 cells both iron and bicalutamide, used as single agents, did not affect tumor growth, while the combination successfully controlled the disease expansion (p<0.05). Preliminary experiments showed that both iron sensitive LNCaP cells and iron resistant PC3 cells grown in vitro are characterized by high TfR1. LNCaP cells showed higher levels of ferroportin, ferritin, catalase and SOD1 than PC3 cells, suggesting high basal iron content and the need to favor iron export and safe storage in ferritin to limit ROS increase. Bicalutamide did not prevent TfR1 downregulation and ferritin increase after iron addition, however it increased catalase in PC3 suggesting increased oxidation. In this condition, further oxidative stress induced by iron is the likely cause of cell viability reduction. Conclusions: Iron excess negatively affects androgen-dependent PCa proliferation. Androgen limitation promotes intracellular oxidative status, increased iron toxicity and allows iron excess to affect also PC3 cell proliferation. Our data suggest that interfering with PCa cell growth is possible combining androgens deprivation and modulation of iron status. Seventh Congress of the International BioIron Society Page 269 Poster Abstracts IBIS Poster #157 IRON ADMINISTRATION INCREASES BORTEZOMIB EFFICACY IN MULTIPLE MYELOMA MURINE MODELS Jessica Bordini, Federica Morisi, Alessia Fulli, Gianluca Venturi, Clara Camaschella and Alessandro Campanella Vita-Salute San Raffaele University Presented By: Jessica Bordini Background: Multiple myeloma (MM) is a malignancy of bone marrow (BM) plasma cells (PC) characterized by bone disease, anemia, renal insufficiency and the release of monoclonal component (M-spike) in serum. In the transgenic Vk*MYC MM mouse model, we showed that BM macrophages sequester iron restricting local iron availability (Bordini et al, 2015). Since redox homeostasis is fundamental for PC biology, we hypothesized that PCs balance the need to limit iron with the increased demand for proliferation and may benefit from iron deficiency. In in vitro experiments, we demonstrated that addition of high dose iron reduced MM cell lines proliferation inducing ROS mediated cell death. In vivo, we showed that iron administration overcomes macrophages sequestering capacity leading to iron accumulation in PC with consequent delay of disease development in xenograft and Vk*MYC MM models (Bordini et al, 2016). We aimed at investigating whether iron toxicity may favor the efficacy of pharmacological therapies based on the proteasome inhibitor bortezomib, a drug that has the side effect of interfering with cellular iron homeostasis maximizing iron toxicity (Campanella et al, 2013). Methods: Xenograft mice were generated by intravenous injection of MM.1S cells (5 x106) and disease expansion evaluated by flow cytometry of BM tissues. Transgenic Vk*MYC were treated when M-spike was at least 7% of total serum proteins. Transplanted Vk*MYC (tVk*MYC) were generated by syngeneic transplantation of BM derived from Vk*MYC bortezomib-resistant donors into wild-type recipients. Treatment efficacy was determined by measuring M-spike variation. Mice were treated with various combinations of bortezomib (0,25 or 0,43 mg/Kg, IP), melphalan (3 mg/kg, IP), prednisone (20 mg/kg, PO) and iron dextran (250 or 100 mg/Kg, IP). Results: In a first set of experiments we evaluated whether high dose iron increases low dose bortezomib efficacy. Xenograft and Vk*MYC mice were treated with 0,25 mg/Kg bortezomib on days 1,4,8,11 plus or minus 250 mg/Kg iron dextran on day 5. In xenograft mice iron administration upon bortezomib reduced PC expansion compared with bortezomib alone or control mice. In Vk*MYC mice bortezomib treatment induced response in 33% of the cases, however progressive disease was still observed in 55% of the mice. On the contrary, mice treated with bortezomib plus iron showed 80% of response and no case of progressive disease. The low dose bortezomib used in our experiments has a clinical value especially for elderly MM patients affected by comorbidities. In a second set of experiments we are evaluating whether a lower dose of iron administration may increase the efficacy of the same dose of bortezomib when in combination with the other drugs commonly present in the pharmacological cocktail. Vk*MYC mice will be treated with 0,25 mg/Kg bortezomib on days 1,4,8,11 plus melphalan and prednisone on days 1-4 and plus or minus 100 mg/Kg iron dextran on day 5. To test whether high dose iron administration is able to overcome resistance to high dose bortezomib we are planning to treat tVk*MYC mice characterized by homogeneous bortezomib resistant disease with 0,43 mg/Kg bortezomib, melphalan and prednisone plus the high iron dose (250 mg/Kg). Conclusions: High dose iron administration increases bortezomib efficacy in MM murine models. Experiments are ongoing to evaluate whether different iron/bortezomib doses might be of clinical value. This work was supported by AIRC Fondazione Cariplo TRIDEO 2014 no. 15465 (to AC) and by AIRC Program Grant in Molecular Oncology-Extension no. 9965 (to AC) Seventh Congress of the International BioIron Society Page 270 Poster Abstracts IBIS Poster #158 ELEVATED SYSTEMIC HEME AND IRON LEVELS AS RISK FACTORS FOR VASCULOPATHY AND ATHEROSCLEROSIS: EVIDENCE FROM A B-THALASSEMIA COHORT STUDY Francesca Vinchi¹, Graca Porto, MD², Gregory M Vercellotti, MD³, John Belcher, PhD³, Richard Sparla, BTA¹, Eitan ibach, PhD , ala reid, MD , ushdy asras, MD , ussam hoti, MD , Martina . Muckenthaler, PhD and Elie er A. Rachmilewit , MD ¹Molecular Medicine Partnership Unit , University of Heidelberg & EMBL, Heidelberg, Germany; ²University of Porto, Porto, Portugal; niversity of Minnesota, Minneapolis, SA; The ebrew niversity adassah Medical Center, erusalem, srael; AlShifa ospital, a a, Palestine; European ospital of a a, a a, Palestine; Assuta Medical Center, Tel Aviv, srael; The Edith olfson Medical Center, olon, srael Presented By: Francesca Vinchi, PhD ß-thalassemia is an inherited hemoglobinopathy due to reduced synthesis of ß globin chains. The clinical manifestations are mainly the result of chronic anemia and iron overload. The latter is due to increased iron absorption, induced by accelerated but ineffective erythropoiesis, and recurrent red blood cell transfusions. Alpha chains and iron excess promote oxidative damage of red blood cell membrane, resulting in macrophage sequestration and extravascular hemolysis, and to a lower extent, in intravascular hemolysis, with consequent release of hemoglobin (Hb), heme and iron. Increasing evidence suggests that free heme exerts vasculotoxic, pro-inflammatory and procoagulant effects due to its ability to trigger endothelial and immune cells activation. In mouse models of ß-thalassemia and sickle cell disease, circulating heme levels are elevated and correlate with the exhaustion of systemic scavengers for hemoglobin and heme, haptoglobin and hemopexin, respectively, as well as with severe endothelial dysfunction and inflammation. Hemopexin- based therapies significantly ameliorate endothelial damage, vascular oxidative stress and inflammation in these mice. Whereas more data are reported on sickle patients in this regard, few data are available in patients with ß-thalassemia. In the present study, we examined serum samples from a cohort of 60 patients with ß-thalassemia major and 7 patients with ß-thalassemia intermedia. 10% of the patients received inconsistent iron chelation therapy. Serum from 17 healthy subjects served as control. Both groups of patients show high systemic heme levels, which associate with a severe drop in serum haptoglobin and hemopexin. Moreover, serum iron levels as well as transferrin saturation, non-transferrin bound iron (NTBI) and serum ferritin are strongly elevated. As a consequence of severe anemia, these patients show high levels of Erythropoietin and increased but still inappropriately low levels of Hepcidin, when compared to the iron burden. Interestingly, these patients present with high systemic levels of the soluble adhesion molecules sVCAM-1, sICAM-1, sE- selectin and sP-selectin, which suggest a state of enhanced endothelial activation. In addition, they show increased levels of serum malondialdehyde, a well-known marker of lipid peroxidation and oxidative stress, and high levels of circulating oxidized low density lipoproteins (oxLDL). All parameters significantly correlate with increased systemic heme and iron indices as well as decreased haptoglobin and hemopexin and elevated transferrin saturation. In conclusion, ß-thalassemia patients show a strong correlation between systemic heme and iron overload, scavenger depletion, and markers of oxidative stress, inflammation and endothelial dysfunction, thus confirming studies in animal models. These results emphasize the involvement of serum hemoglobin, heme and iron in the pathophysiology of ß-thalassemia, including vascular dysfunction and inflammation. Importantly, our data also suggest a pro-atherosclerotic role for heme and iron, as recently addressed in an iron-overload mouse model of hemochromatosis. Therefore these observations are relevant for disorders hallmarked by vasculopathy, such as sickle cell disease and ß-thalassemia, as well as for cardiovascular diseases, such as heart attack and stroke, when systemic heme and iron are elevated. Our findings highlight the key protective role of hemoglobin and heme scavengers and support the potential therapeutic benefit of scavenger administration along with efficient iron chelation therapy to counteract heme- and iron-driven toxicity. Seventh Congress of the International BioIron Society Page 271 Poster Abstracts IBIS Poster #159 IN FIFTY YEARS: A UNIQUE WINDOW ON HEME AND IRON METABOLISM FROM THE HEMOPEXIN SYSTEM Ann Smith, BSc, PhD SBS-UMKC Presented By: Ann Smith, BSc, PhD The heme-binding and transporting protein, hemopexin is being developed as a therapeutic in a novel plasma protein replenishment therapy for conditions including sepsis as well as other hemolytic conditions and genetic diseases. Each of the previous five decades can be considered to have one or more milestones in heme and iron biology that was revealed by and linked to research on hemopexin. In fact, the concept of hemopexin deficiency states was noted in the 1960’s and proposed to assess the extent of severe hemolysis during cardiac surgery in the late 1970’s. Receptor- mediated heme transport by hemopexin, shown in the 1970’s, was linked to the concept of heme as a regulatory molecule for gene expression, and specifically heme oxygenase-1, in the 1980’s. Subsequently, the 3-D crystal stricture of hemopexin revealed a unique heme binding site for attenuating the high chemical reactivity of heme. Hemopexin-null mice, among other experimental systems and models of human diseases, revealed the protection against heme toxicity that is now know to drive much of the pathology in hemolytic and inflammatory states for example, after ischemic stroke, in sickle cell disease, sepsis and even after extracorporeal circulation of blood and multiple blood transfusions. Recent research is confirming the historical human studies. Hemopexin levels respond to plasma heme and hemopexin is not an acute phase reactant, unlike haptoglobin, thus hemopexin is now recognized as the best heme toxicity biomarker for patients. Toll like- receptor 4 is activated by heme and additional mechanisms of heme toxicity and hemopexin deficiency states that are under investigation will likely be fully discerned early in the next 50 years of heme and iron biology. These data were collected and collated by Dr. Ann Smith from publications from the groups of Dr.s Ursula Muller-Eberhard, Ronald J. Elin & Margaret Foidart, Zbynek Hrkal, David A. Sears, David L. Drabkin, József and Gyorgy Balla, William T. Morgan, Ted N. Baker, Soren Moestrup, Sylvain Dore, Emmanuela Tolosano, Joris Delanghe, Miguel Soares & Rasmus Larsen, Gregory ercelotti ohn Belcher, and . Shaw arren as well as from her own group’s research ( Peter ahl, awed Alam, im Rish, Roberto Vanacore, Jeff Eskew and LokMan Sun). This is a reprise and update of a poster presented at the 50th anniversary of the GRC on “The Chemistry & Biology of Tetrapyrroles,” July 2016, and at the International Society of Blood Transfusions, Dubai, September 2016. Seventh Congress of the International BioIron Society Page 272 Poster Abstracts IBIS Poster #160 HEPCIDIN AS A PREDICTOR TO TREAT PREOPERATIVE ANEMIA EFFECTIVELY WITH INTRAVENOUS IRON Christina Wittkamp², Lisa Traeger², Ines Ellermann³, Maria Eveslage, Dipl-Stat⁴ and Andrea Steinbicker, MD MPH¹ ¹University Hospital Muenster, Department of Anesthesiology, Intensive Care and Pain Medicine; ²Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Muenster, University of Muenster, Muenster, Germany; ³Department of Pharmacy, University Hospital Muenster, University of Muenster, Muenster, Germany; ⁴Institute of Biostatistics and Clinical Research, University of Muenster, Germany Presented By: Andrea Steinbicker, MD, MPH Preoperative anemia occurs in about one third of patients who undergo elective surgery. Anemia is associated with a poor outcome after surgery such as postoperative infections and higher mortality. Therefore a screening of preoperative anemia was established in the context of a multidisciplinary Patient Blood Management (PBM) programme in four university hospitals in Germany, whereof one is in the university hospital of Muenster. If there were no contraindications, anemic patients were treated with intravenous (iv) iron (ferric carboxymaltose). The aim was to increase the hemoglobin level prior to surgery, reduce the need for red blood cell transfusions and hence improve the patient’s outcome after surgery. Different responses in the sense of responders and non-responders to the iv iron administration were observed. We therefore investigated, if hepcidin, the key regulator of iron homeostasis and also a marker of inflammation, can serve as a predictor for the response of anemic patients to iv iron treatment. The study was approved by the ethical committee. Patients gave their informed consent that their preoperatively withdrawn blood sample could be analyzed for hepcidin. The measurement of samples from non-anemic patients, anemic patients treated with iv iron and anemic patients that were not treated with iv iron due to contraindications was performed with the hepcidin ELISA from DRG Hepcidin 25 (bioactive) ELISA EIA-5782 (DRG Instruments GmbH, Marburg, Germany) diagnostics. Patients, who received iv iron and responded well to iv iron presented a delta b ≥0,6g/dl between the day of iv iron treatment and the day of elective surgery. A poor-responder was defined with deltaHb <0,6g/dl. The data indicates that serum hepcidin levels were significantly higher in anemic patients without iv iron treatment compared to non-anemic patients or patients treated with iv iron. In addition, a significant difference in serum hepcidin levels of well-responders and poor-responders was observed. The mean value of serum hepcidin levels was higher among the poor responders. Our findings implicate that hepcidin is a possible biomarker for effective preoperative anemia treatment with iv iron. Seventh Congress of the International BioIron Society Page 273 Poster Abstracts IBIS Poster #161 INVESTIGATION OF MITOCHONDRIAL FUNCTION WITH IRON AND LIPID LOADING IN AML12 CELLS Abhishek Singh¹, Cyril Mamotte, BSc, PhD², Leon Adams, MB BS PhD WAust, FRACP³ and Ross Graham, PhD, CBiol² ¹Curtin University/ Biomedical Science; ²Curtin University/ School of Biomedical Science and Curtin Health Innovation Research Institute; ³University of Western Australia/ School of Medicine and Pharmacology Presented By: Abhishek Singh Introduction: Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver disorders associated with abnormal fat deposition in the liver. It is the most common cause of chronic liver disease in the developed world. The liver is the metabolic centre of the body and is the most important site for iron and lipid metabolism and their interaction. Even though iron is necessary for biological processes, any dysregulation of iron may lead to cellular damage and serious clinical consequences. It is known that mitochondria play an important role in hepatocyte metabolism, being the primary site for fatty acid oxidation and oxidative phosphorylation. Accumulating evidence indicates that mitochondrial dysfunction is central to the pathogenesis of NAFLD. Data from our laboratory indicates that liver iron loading leads to a decrease in expression of genes associated with mitochondrial fatty acid oxidation. The present study is focussed on understanding how liver iron and lipid loading leads to changes in mitochondrial fatty acid oxidation in NAFLD. Methods: The mouse hepatocyte cell line, AML12, was iron and lipid loaded for 24 h using ferric ammonium citrate (FAC; 30µg/mL) and oleic acid conjugated to albumin (0-300µM). Mitochondrial bioenergetics was measured using a Seahorse XFe Extracellular Flux Analyser. Results: There was an increase in ATP production (71.5pmol/min) in cells treated with 300µM oleic acid and FAC as compared to control (47.5pmol/min). Reserve respiratory capacity is a measure of the ability of substrate supply and electron transport to respond to increase in energy demand. Reserve respiratory capacity (325pmol/min) was also increased compared to control (270.6pmol/min) under the same conditions. Discussion and Conclusions: These findings are consistent with the hypothesis that cells loaded with oleic acid have an increased capacity to respond to stress compared to unloaded cells. The study also gives insight into the role of mitochondria in NAFLD. Seventh Congress of the International BioIron Society Page 274 Poster Abstracts IBIS Poster #162 UNCOUPLED IRON HOMEOSTASIS IN TYPE 2 DIABETES MELLITUS Sandro Altamura, PhD¹,³, Stefan Kopf², Julia Schmidt¹,³, Ana Rita da Silva¹,³, Peter Nawroth² and Martina Muckenthaler¹,³ ¹Department of Pediatric Hematology, Oncology and Immunology - University of Heidelberg, Heidelberg, Germany; ²Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany; ³Molecular Medicine Partnership Unit, Heidelberg, Germany Presented By: Sandro Altamura, PhD Diabetes mellitus is a comorbidity associated with iron overload conditions, such as primary and secondary hemochromatosis. Vice versa, patients affected by type 2 diabetes mellitus (T2DM) show elevated ferritin levels, a biomarker for increased body iron stores. Despite these documented associations between dysregulated iron metabolism and T2DM, the underlying mechanisms are poorly understood. Here we show that patients affected by T2DM without an inflammatory burden have reduced serum levels of hepcidin, the iron-regulated hormone that maintains systemic iron homeostasis. Consistently, circulating iron and ferritin levels are increased. The analysis of db/db mice recapitulates the systemic findings in patients. Interestingly, db/db mice show an overall hepatic iron deficiency despite having unaltered expression of the iron storage protein ferritin and of the iron importer TfR1. In addition, the liver correctly senses increased circulating iron levels by activating the BMP/SMAD signaling pathway. However, hepcidin expression is decreased. We show that increased AKT phosphorylation may override active BMP/SMAD signaling and decrease hepcidin expression in 10-week old db/db mice. We conclude that metabolic alterations in T2DM, impact on the regulation of iron homeostasis on multiple levels and induce an “iron resistance” phenotype in that signals that translate increased circulating iron levels to hepcidin production are dysregulated. Seventh Congress of the International BioIron Society Page 275 Poster Abstracts IBIS Poster #163 CROSSTALK BETWEEN ACIDOSIS AND IRON METABOLISM: DATA ON HEPCIDIN AND H+/K+ ATPASE Raed Daher¹, hibaud Lefebvre²,⁵, Nicolas Ducrot², Philippe Lett ron³, Anne Couvelard⁴, Herv Puy²,⁵ and oubida Karim² ¹INSERM U1149, Université Paris Diderot, Laboratory of Excellence, GR-Ex; ²INSERM U1149, Université Paris Diderot, Laboratory of Excellence, GR-Ex, Paris, France; ³INSERM U1149, Université Paris Diderot, Paris, France; ⁴INSERM U1149, Université Paris Diderot, AP-HP, D partement d anatomo-pathologie, H pital Bich t, Paris, France; ⁵AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France Presented By: Raed Daher Background: Gastric acidic secretion is an important intraluminal factor for inorganic iron solubility and absorption by the intestinal enterocytes. Indeed, iron transport activity of the duodenal DMT1 (Divalent Metal Transporter 1) that operates as a symporter of metals and proton, is optimal only at high acidic environment. Gastric acidity is largely achieved by the gastric proton pump H/K ATPase and mutation of its alpha subunit (ATP4A) causes iron-deficiency anemia in mice1. Iron metabolism is controlled by hepcidin, a small peptide mainly synthesized in the liver that negatively acts on the duodenal DMT1 and on the iron exporter FPN (Ferroportin), thus reducing iron absorption. Previous studies of Kulaksiz group (in parietal cells)2 and ours (in renal cells)3 have shown that expression of ATP4A is strongly repressed in mice lacking hepcidin gene (Hepcidin KO) resulting in a major alkalization of gastric juice and urine. However, whether ATP4A is regulated by an iron regimen and conversely whether hepcidin is regulated by acid-base variations is not yet known. Our aim was to investigate these issues using mouse model of metabolic acidosis (MA) and iron rich diet (IRD). Methods: MA was induced in mice by giving ammonium chloride (280 mM NH4Cl) in distilled drinking water during 2 weeks. IRD was induced in mice by feeding with 2g iron/kg diet for 3 weeks. Serum iron parameters were analyzed and serum hepcidin was measured by LC-MS/MS. Expressions of hepcidin, iron transporters and proton pump in the different organs, were studied by quantitative RT-PCR, western blot or immunohistochemistry. Results: Following MA, as was expected, the expression of gastric ATP4A was significantly increased at mRNA and protein levels. Serum hepcidin was elevated and that was through increase of its mRNA level in the liver without change in the parietal cells. Both duodenal DMT1 and FPN were significantly downregulated under this condition. These responses may oppose the increased availability of intraluminal iron and of DMT1 activity in MA condition. Indeed, serum iron level and transferrin saturation remained unchanged and serum ferritin was significantly increased sign of tissue iron retention. Following IRD, the mRNA level of hepcidin was increased both in liver and parietal cells, the protein expression of ATP4A was significantly increased with no changes in the mRNA abundance. In the kidney, hepcidin expression remained unchanged in IRD while renal ATP4A protein level was significantly enhanced with no modification of its mRNA level, suggesting a post translational regulation of ATP4A by systemic iron disorder. Further in vitro studies are in progress to investigate ATP4A regulatory mechanisms by iron and/or hepcidin. Since iron promotes pathogen growth, increased acid secretion both in gut and kidney may be relevant for protection against bacterial infection eventually favored by IRD. Conclusions: Our results provide evidences that ATP4A takes place in iron metabolism and that hepatic hepcidin is induced during acidosis in vivo. Taken together, these results reinforce the hypothesis of a crosstalk between acidosis and iron metabolism. References: 1. Krieg L et al, Blood. Blood. 2011 Dec 8;118(24):6418-25. 2.. Schwarz P et al, Gut. 2012 Feb;61(2):193- 201. 3. Houamel D et al, J Am Soc Nephrol. 2016 Mar;27(3):835-46 Seventh Congress of the International BioIron Society Page 276 Poster Abstracts IBIS Poster #164 TRANSFERRIN: A PREDICTOR OF SURVIVAL IN EARLY STAGE LIVER CIRRHOSIS André Viveiros, MD, Armin Finkenstedt, MD, Benedikt Schaefer, MD, Konrad Lehner and Heinz Zoller, MD Medical University Innsbruck Presented By: Andre Viveiros, MD Background: The best available prognostic model to predict survival in patients with liver cirrhosis is the Model for End- stage Liver Disease (MELD). The MELD score is based on serum bilirubin, creatinine, and the international normalized ratio for prothrombin time (INR). Additional parameters have been proposed to improve the prognostic accuracy of the model and transferrin was recently identified as a predictor of survival in patients with advanced and decompensated cirrhosis. The aim of this study was to understand if altered iron metabolism in liver cirrhosis is a consequence of inflammation and impaired liver function or an independent predictor of survival. Methods: Clinical, demographic and biochemical data was retrospectively analyzed from a cohort of 995 patients with liver cirrhosis (age ≥ 18 years) who presented from 01/08/2004 to 31/12/2014 at the niversity ospital of nnsbruck. Patients with hepatocellular carcinoma, extrahepatic malignancy at diagnosis or a follow-up time < 6 months (unless death occurred within 6 months) were excluded. Results: During a median follow-up of 3.13 years, 193 deaths occurred and 234 patients underwent liver transplantation. Median transplant-free follow-up was 2.17 years. Multivariate Cox regression analysis was carried out in search for predictors of survival in this cohort. In a model including C-reactive protein, ferritin, transferrin, transferrin saturation and MELD, only transferrin and MELD remained as independent predictors of transplant-free survival. ROC curve analysis showed that a transferrin concentration of 180 mg/dl is at the highest prognostic accuracy to predict 3-months and 1-year survival. hen patient subgroups with MELD ≥ 15 and 15 were analy ed, transferrin remained significantly associated with transplant-free survival in the subgroup with a MELD score < 15. Conclusions: In conclusion, our study shows that the association of transferrin with survival is independent of MELD score even at early stages of liver cirrhosis. This finding can improve the prognostic evaluation in patients with liver cirrhosis and suggests that interventions targeted at iron handling might improve outcome. Seventh Congress of the International BioIron Society Page 277 Poster Abstracts IBIS Poster #165 ERYTHROFERRONE DOES NOT CONTRIBUTE TO GLUCOSE HOMEOSTASIS AND DOES NOT AFFECT INSULIN SENSITIVITY IN RESPONSE TO CHRONIC ERYTHROPOIETIN TREATMENT R. Coffey, V. Gabayan, E. Nemeth and T. Ganz University of California, Los Angeles Presented By: Richard Coffey Erythropoietin (EPO), a hormone produced in response to hypoxia, acts on erythroid progenitor cells to promote red blood cell development and is required for erythropoiesis. Along with the canonical role of EPO in promoting erythropoiesis, elevated levels of EPO are also reported to modulate aspects of energy homeostasis resulting in improved glucose tolerance and insulin sensitivity. EPO also stimulates the production of the hormone erythroferrone (ERFE) which acts to suppress hepcidin production, thus increasing dietary iron absorption and mobilizing stored iron for use in erythropoiesis. ERFE was originally identified as myonectin, a myokine that is suggested to promote the clearance of free fatty acids from circulation. As elevated levels of serum free fatty acids are associated with insulin resistance and impairments in glucose homeostasis, elevated levels of ERFE during EPO administration may have a beneficial effect on glucose metabolism. The aim of the current study was to determine if the metabolic effect of EPO treatment on glucose homeostasis could be attributed to increased ERFE production. Glucose and insulin tolerance were measured in wild type (WT) and ERFE knockout (ERFE-/-) mice chronically treated with either EPO or saline. 6 wk old mice were injected intraperitoneally with 200 U recombinant mEPO on alternate days 3 times a wk for 2 wk prior to, and 16 h immediately before, testing. EPO treatment reduced fasting glucose levels and improved glucose tolerance to a similar degree in both WT and ERFE -/- mice. No difference in glucose tolerance was detected in ERFE-/- mice relative to WT controls during either EPO- stimulated or saline-treated conditions. Additionally, no differences in insulin sensitivity were detected between WT and ERFE-/- mice under either EPO-stimulated or saline-treated conditions. Upon completion of testing, hemoglobin was elevated in both WT and ERFE-/- mice treated with EPO relative to saline-treated controls. Erfe mRNA expression was also increased in EPO treated WT mice relative to saline-treated controls, indicating that the stimulatory effect of EPO on ERFE production was preserved during chronic EPO administration. These findings suggest that the changes observed in glucose metabolism during EPO treatment are not attributable to an accompanying increase in ERFE production, and that ERFE does not exert a detectable effect on glucose homeostasis. Seventh Congress of the International BioIron Society Page 278 Poster Abstracts IBIS Poster #166 GENOME WIDE ASSOCIATION STUDY (GWAS) OF STEATOSIS DUE TO DIETARY IRON OVERLOAD IN INBRED STRAINS OF MICE Dongyi Lu, Brie Fuqua, Simon Hui, Frode Norheim, Calvin Pan, Sarada Charugundla, Nam Che, Brian Parks, Eleazar Eskin, Chris Vulpe and Aldons Lusis University of California, Los Angeles Presented By: Dongyi Lu Non-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases. Iron is not only deposited in the liver with NAFLD in about 1/3 of the cases, but is also suggested to contribute to the progression of NAFLD. Specifically, previous studies have shown that iron overload increases hepatic triglyceride and/or cholesterol levels by enhancing lipid synthesis in the liver; however, the evidence for such upregulation is inconsistent and the mechanism of such upregulation is far from being clear. Our lab is implementing GWAS in 114 strains of hybrid mouse diversity panel (HMDP) male mice to identify novel genetic loci associated with iron metabolism. Mice on a high iron diet develop iron overload in the liver. Although the mice eat less and have lower body fat, steatosis of various severity in different strains has been observed histologically, with fairly consistent severity within strains, co-localizing with regions with iron loading, suggesting a genetic contribution to these pathophysiologic phenotypes. This study aims to integrate GWAS, transcriptomic (RNAseq), and metallomic (ICP-MS) data to identify candidate genes and pathways, to shed light on the mechanism of how iron overload influences lipid metabolism in the liver. Over 200,000 high quality SNPs are used for the GWAS. Lipids are extracted from livers of over 700 mice from 114 strains, and triglycerides, total cholesterol, unesterified cholesterol, esterified cholesterol (calculated by subtracting unesterified cholesterol from total cholesterol), and phosphatidylcholine are quantified and mapped. At present, with data from 394 mice (88 strains), some loci have been identified for some of the phenotypes quantified. Esterified and total cholesterol are mapped to the same significant locus on chromosome 15, and phosphatidylcholine to a locus on chromosome 16. Compared to the mice on a high fat, high sucrose diet in a previous HMDP GWAS, mice on the high iron diet tend to have less unesterified cholesterol, less phosphatidylcholine, and more esterified cholesterol in the liver, suggesting that while both diets lead to steatosis, they affect cholesterol metabolism and perhaps the plasma membrane differently. While as expected, iron loaded mice generally have less triglyceride in the liver than mice from the previous study, the mean triglyceride level for each strain has a weak positive correlation among the mice in the two studies, suggesting that the mechanisms by which the two diets lead to steatosis may share pathways in part. The previous study mapped liver triglyceride level to a locus on chromosome 7, but this locus was not significant in our mapping, suggesting differences in the mechanism of how the two diets lead to steatosis. Seventh Congress of the International BioIron Society Page 279 Poster Abstracts IBIS Poster #167 GENETIC LOSS OF TMPRSS6 MODIFIES THE PHYSIOLOGICAL RESPONSE TO A HIGH FAT DIET Abitha Sukumaran, PhD, Larisa Lozovatsky, MS, Xiuqi Li, BS, Luis Gonzalez, BS and Karin Finberg, MD, PhD Yale School of Medicine Presented By: Karin Finberg, MD, PhD Systemic iron parameters, including levels of the iron regulatory hormone hepcidin, have been associated with obesity in children and adults, but the mechanisms underlying these associations are not yet well understood. The hepatic transmembrane serine protease TMPRSS6 plays a key role in systemic iron homeostasis by down-regulating hepcidin production by the liver. Accordingly, both humans and mice with TMPRSS6 mutations show inappropriately elevated hepcidin levels that lead to impaired dietary iron absorption, systemic iron deficiency, and anemia. In this study, we examined (1) if the administration of a high fat diet alters systemic iron parameters in mice with pre-existing abnormalities in iron homeostasis due to homozygous loss of Tmprss6, and (2) if the physiological response to a high fat diet is altered in mice with homozygous loss of Tmprss6. By intercrossing Tmprss6+/- mice, we generated Tmprss6+/+, Tmprss6+/-, and Tmprss6-/- littermates, which were raised on a standard diet. At 8 weeks of age, mice of all three genotypes were transferred to either a high fat (60% kcal as fat) or control (10% kcal as fat) diet. Mice were weighed weekly, and at 12 weeks of age, blood and tissues from sex-matched mice of each genotype were collected for analysis. Liver non-heme iron concentration of Tmprss6-/- mice fed either the high fat or control diet remained significantly lower than liver non-heme iron concentrations of diet-matched Tmprss6+/+ and Tmprss6+/- littermates. Splenic non-heme iron concentration was lower in Tmprss6-/- mice fed the high fat diet compared to the control diet. Compared to littermate controls, Tmprss6-/- mice fed either the high fat or control diet showed microcytic anemia. While the severity of anemia in Tmprss6-/- mice was not significantly altered by dietary fat content, the high fat diet nevertheless induced a marked increase in splenomegaly in Tmprss6-/- mice, raising the possibility that expanded extramedullary hematopoiesis could be required in this dietary setting to prevent a further exacerbation of anemia. Intriguingly, although the high fat diet induced weight gain in all three genotypes, Tmprss6-/- mice showed significantly smaller gains in percent total body weight and in the weights of several body fat depots compared to Tmprss6+/+ and Tmprss6+/- littermates. In summary, four weeks administration of a high fat diet was sufficient to induce alterations in a subset of iron-related parameters in Tmprss6-/- mice. Furthermore, administration of a high fat diet produced smaller weight gains in Tmprss6-/- mice compared to Tmprss6+/+ and Tmprss6+/- littermates, suggesting that Tmprss6 genotype may impact the physiological response to dietary fat content. We suggest that the attenuated fat-induced weight gain observed in Tmprss6-/- mice may indicate (1) that mechanisms of dietary fat- induced weight gain are altered in the setting of systemic iron deficiency and/or hepcidin elevation, or (2) that the TMPRSS6 protein possesses an iron-dependent function that impacts metabolism. As single nucleotide polymorphisms in the TMPRSS6 gene have been reported to be associated with the risk of type II diabetes in humans, our findings in the Tmprss6 mouse model may have relevance to understanding obesity and diabetes risk in patient populations. Seventh Congress of the International BioIron Society Page 280 Poster Abstracts IBIS Poster #168 STUDY OF FERRITIN SELF-ASSEMBLY AND HETEROPOLYMER FORMATION BY FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) Fernando Carmona, PhD, Maura Poli, PhD, Magdalena Gryzik, PhD student, Andrea Denardo, Student and Paolo Arosio, PhD Department of Molecular and Translational Medicine. University of Brescia. Italy Presented By: Andrea Denardo The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. To study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from <0.3 of the disassembled to >0.7 in the assembled allowed to study the self-assembly kinetics of human ferritin homo- and heteropolymers. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. These data reveal the preferred formation of H/L heterodimers over homodimers during ferritin assembly, and thus contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until a number above eight, when they start to co-localize, and that this distribution correlates with the iron uptake activity of the protein. This observation suggests that participation of a single H-chain at 3-fold channel is sufficient for the highest ferritin activity, at least under the conditions of high iron increments. Seventh Congress of the International BioIron Society Page 281 Poster Abstracts IBIS Poster #169 HEPARIN BINDING DOMAINS IN BMP6: STUDY ON THE SYNTHETIC PEPTIDES AND THE RECOMBINANT PROTEIN Paola Ruzzenenti, PhD student¹, Andrea Denardo, Student, Fernando Carmona, PhD, Stefania Federici, PhD², Michela Asperti, PhD, Paolo Bergese, PhD, Maura Poli, PhD and Paolo Arosio, PhD ¹Department of Molecular and Translational Medicine, University of Brescia, Italy; ²INSTM and Department of Mechanical and Industrial Engineering, University of Brescia, via Branze 38 25123 Brescia, Italy Presented By: Paola Ruzzenenti Hepcidin is the master regulator of iron homeostasis and its expression is mainly attributed to BMP6 protein which triggers the phosphorylation of SMAD1/5/8. Our previous results have shown that heparins strongly repress the BMP6/SMAD signaling resulting in a decrease of hepcidin expression. This finding suggested that not only heparin, but also endogenous heparan sulfates (HSs), ubiquitous heparin-like polysaccharides exposed on cells surface, may participate in the BMP6/SMAD pathway by facilitating the binding of BMP6 to its receptors. How BMP6 binds heparin is not clear, but different Heparin Binding Domains (HBDs) have been already characterized in BMP2 and BMP4 isoforms as lysine and arginine-rich regions.The aim of this work is to define the heparin/HS binding domains of BMP6 to clarify the mechanism of action of heparin in hepcidin inhibition and to verify if HSs are involved in hepcidin expression.BMP6 presents three basic regions rich in Arg and Lys, that are putative HBDs. The first is located at the unstructured N-terminus (HBD1), a second in central portion (HBD2) and the third at C-Terminus (HBD3). Synthetic 15-residue peptides representing the three domains were produced in fusion with biotin. They were used to track the energetics of binding of each HBD peptide to heparin adsorbed onto microcantilever biosensors. We found that N-terminus domain (HBD1) showed higher heparin affinity than the others and that it was dose dependent. Then we assayed their binding to heparan sulfates (HS) using monolayers of cells that express or not membrane HS. The results showed that also with this approach the N-terminus HBD peptide bound with higher affinity the surface of cells expressing HS, and that this binding was reduced in the presence of heparin. These findings prompted us to produce the recombinant mature form of human BMP6 in a prokaryotic system in order to study its heparin and HS binding activity in a completely folded protein. Human mature BMP6 with his-tag was cloned into the pASK-IBA43plus vector and expressed in E. Coli Rosetta™ (DE3) host strain, as insoluble inclusion bodies. BMP6 was solubilized and then purified obtaining a highly pure denatured BMP6 on its monomeric form. Finally, we used an in vitro oxidative renaturation protocol to obtain the dimeric functional form of BMP6.We found that the produced dimeric BMP6 was able to bind heparin and that it binds more effectively to cells expressing HS. The biological activity of BMP6 seems to require the glycosylation, thus we cloned the mature human and mouse BMP6 in eucariotic vectors. We transfected the Hek293 with these plasmids and treated HepG2 cells with their supernatant. The transcription of hepcidin was induced by the BMP6 transfected Hek supernatant supporting the expression and secretion of BMP6. Mutagenesis of arginine in these prokaryotic and eukaryotic clones are in progress to identify the major HBD in the BMP6; the results will be present. Seventh Congress of the International BioIron Society Page 282 Poster Abstracts IBIS Poster #170 COMPARATIVE CHARACTERISATION OF THE FERROUS IRON TRANSPORT SYSTEMS EFEUOB AND FEOABC IN E. COLI SALEM AL-AIDY and Simon Andrews, Professor University of Reading Presented By: Salem Al-Aidy Iron is an essential element for bacterial growth. However, it is a dangerous metal because it has the ability to catalyse reactive oxygen species (ROS) through the Fenton reaction [1,2]. The oxidation status of iron in the environment is largely 3+ determined by the pH and oxygen levels, with the poorly soluble ferric (Fe ) form persisting with high pH and O2, and the 2+ more soluble ferrous form (Fe ) favoured by low pH and O2 [3]. This study explores the differences in the activities of the two ferrous-iron transporters (Feo and Efe) of E. coli in their responses to hydrogen peroxide. E. coli mutants devoid of iron-transport systems were employed along with low-copy number plasmids, or inducible plasmids (pBAD), carrying 55 either efeUOB or feoABC. Results showed that H2O2 enhances Fe uptake for efeUOB transformants whereas provision of exogenous catalase caused strong inhibition. In contrast, FeoABC dependent iron-uptake was enhanced by catalase but inhibited by H2O2. The iron uptake results were found to correlate with the growth phenotypes of EfeUOB and FeoABC-dependent E. coli strains, where FeoABC dependent low-iron growth was inhibited by peroxide whereas EfeUOB-dependent low-iron growth was enhanced by peroxide. The above findings indicate that H2O2 is required by EfeUOB for iron transport whereas the absence of H2O2 is required for the FeoABC system to function effectively in iron uptake. Previous work has shown that FeoABC is regulated by oxygen (Fnr induced anaerobically) and is actively degraded under oxic conditions through a FeoC-controlled mechanism [4]. The results presented above indicate that Feo-dependent iron uptake is sensitive to H2O2, rather than O2. These findings also match the predicted role of EfeB as a periplasmic haem-peroxidase mediating ferrous iron oxidation, using peroxide as oxidant, during the EfeUOB iron-uptake process. In conclusion, EfeUOB and FeoABC are ferrous-iron transporters that appear to be required under distinct conditions relating to peroxide abundance; this requirement would thus explain the need for two alternate ferrous-iron uptake systems in E. coli. References: [1] Cornelis, P. et al (2011) Iron uptake and homeostasis in Microorganisms, Metallomics 3, 540–549 [2] Andrews, S. C. et al. (2003). Bacterial iron homeostasis. FEMS Microbiology Reviews 27, 215-237. [3] Cao, J. et al. (2007). EfeUOB (YcdNOB) is a tripartite, acid induced and CpxAR regulated, low pH Fe2+ transporter that is cryptic in Escherichia coli K 12 but functional in E. coli O157: H7. Molecular Microbiology 65, 857-875. [4] Kim, H. et al. (2015) Lon- mediated proteolysis of the FeoC protein Prevents‐ Salmonella enterica‐ from accumulating‐ the Fe(II) transporter FeoB under high-oxygen‐ conditions. J. Bacteriol. 197, 92-98. Seventh Congress of the International BioIron Society Page 283 Poster Abstracts IBIS Poster #171 HEME OVERLOAD IN MICE PROMOTES IRON EXPORT FROM MACROPHAGES VIA ROS-MEDIATED FERROPORTIN ACTIVATION Maja Vujic Spasic, PhD¹, Naveen Kumar Tangudu, MSc², Betuel Alan, MSc², Dilay Lai², Francesca Vinchi, PhD³, atharina Woehrle, MSc⁴, Sabine ettorazzi, PhD² and erstin Leopold, PhD⁴ ¹Institute of Comparative Molecular Endocrinology; ²Institute of Comparative Molecular Endocrinology, Ulm, Germany; ³Molecular Medicine Partnership Unit, Heidelberg, Germany; ⁴Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany Presented By: Maja Vujic Spasic, PhD Heme, a prosthetic group consisting of a protoporphyrin ring coordinated to ferrous iron, is an essential molecule with double-sward actions. Under homeostatic conditions, the reactivity of heme is controlled by its insertion into the hemoproteins such as hemoglobin, myoglobin and cytochromes, which are involved in numerous processes essential for life. During oxidative stress conditions however, and in certain pathological conditions such as haemolytic diseases, large amounts of free heme are released into the circulation promoting the pro-oxidant, cytotoxic and pro-inflammatory responses. Reactive oxidant species (ROS) are considered critical for the ability of heme to promote oxidative stress, which poses a serious threat to cellular and systemic redox homeostasis. We here demonstrate that overproduction of ROS by iron-protoporphyrin complex, heme, critically impairs cellular iron balance. This effect is ascribed to the activation of the ferroportin that communicates iron export from macrophages. Importantly, using total-reflection x-ray fluorescence spectrometry we measured a significant decrease in the intracellular iron pool under these conditions. This effect could not be counter-acted by blocking the Tlr4-signaling. Furthermore, we show that ROS production and ferroportin induction are abolished when macrophages are stimulated with iron-free heme precursor. We next addressed the pathophysiological relevance of ROS-mediated ferroportin regulation in vivo. We infused mice with heme (35umol/kg;1h) to establish an acute model of heme overload and as a model of chronic heme overload we used sickle cell anemia mice HbS. Ferroportin expression was analysed by real-time PCR and Western blot analysis and measured the levels of heme and non-heme iron in the spleen. We show that despite hemolysis and heme-driven inflammation, both mouse models presented high ferroportin expression levels, an effective iron export into the circulation and developed iron-deficiency in the spleen. Interestingly, the expression of hepatic iron-hormone hepcidin was not changed in the model of acute heme overload in contrast to decreased hepcidin expression measured in HbS mice. Finally, we show the observed effects of heme, in vitro and in acute heme overloaded mice, could be blocked by the treatment with pharmacological anti-oxidant such as N-acetylcysteine (NAC). We propose that the use of anti-oxidants may contribute to re-establish cellular and systemic iron disturbances in conditions where heme and ROS levels are high. Seventh Congress of the International BioIron Society Page 284 Poster Abstracts IBIS Poster #172 INTRACELLULAR SYSTEMS OF IRON COFACTOR DELIVERY: ANALYSIS OF MURINE MODELS OF IRON CHAPERONE DEFICIENCY Caroline Philpott, MD, Moon-Suhn Ryu, PhD, Fengmin Li, PhD, Avery Frey, PhD, Olga Protchenko, PhD and Minoo Shakoury-Elizeh, MS NIDDK, NIH Presented By: Caroline Philpott, MD Iron is essential because it is incorporated into iron cofactors— heme, iron-sulfur clusters, and mono- and dinuclear iron centers— that are essential for the normal functioning of cells. Although hundreds of cellular proteins require iron- containing cofactors for activity, the machinery responsible for distributing these cofactors remains incompletely defined. Studies from our laboratory indicate that proteins of the poly rC-binding protein family (Pcbps) can function as iron chaperones, binding iron and delivering it to ferritin and other iron-dependent enzyme. Pcbps are multifunctional adaptor proteins that can bind cellular RNA, DNA, proteins, and iron, altering the fate of their binding partnersThe two most abundant Pcbps, Pcbp1 and Pcbp2, can bind up to three iron atoms and deliver them to client proteins via direct ligand exchange, facilitated by protein-protein interactions. Although the role of Pcbps in iron delivery is apparent in general cell culture models, especially when cells are iron deficient, whether the iron chaperone activity is important in specific mammalian tissues or under physiological or pathophysiological conditions remains unknown. Mouse cells and tissues have proven to be excellent models of human iron metabolism. We have developed conditional models of murine Pcbp1 deficiency by engineering mice carrying PCBP1 alleles flanked by loxP sites. Because the erythron is the major consumer of iron in mammals, we examined whether Pcbp1 and Pcbp2 function iron chaperones in the erythroid system in mice. Using an inducible form of the Cre recombinase, we have confirmed partial deletion at the PCBP1 locus in hematopoietic tissues. Data from these animals indicate that iron homeostatic systems and erythropoiesis are perturbed, as they exhibit impaired iron delivery to ferritin and impaired heme and hemoblobin synthesis in erythropoietic cells. These defects are manifest as microcytic anemia. The liver is also an important organ for iron storage and utilization in mammals. It is the site of production for many proteins, lipids and metabolites, for the degradation and excretion of xenobiotics and metabolic products, and for the maintenance of body energy stores in mammals. We have developed a murine model of liver- specific PCBP1 deletion. Preliminary data from these animals indicate disturbances of iron regulatory systems and lipid homeostasis. These studies will allow us to more precisely define the functions of iron chaperones and understand intracellular systems of iron distribution in mammals. Seventh Congress of the International BioIron Society Page 285 Poster Abstracts IBIS Poster #173 THE MAIN PHYSIOLOGIC SUBSTRATE FOR ZIP14 IS NEITHER IRON NOR ZINC Supak Jenkitkasemwong, PhD and Mitchell Knutson, PhD University of Florida Presented By: Mitchell Knutson, PhD ZIP14 (SLC39A14) is a transmembrane metal-ion transporter that, when overexpressed in cells, can mediate the uptake of various divalent metals including zinc, iron, manganese, and cadmium (Pinilla-Tenas et al., AJP Cell Physiol, 2011). The physiologic functions of ZIP14, however, are only starting to be revealed, primarily by studies of ZIP14 knockout (Slc39a14-/-) mice. ZIP14 was first proposed to play a physiologic role in zinc metabolism based on the observation that Slc39a14-/- mice displayed diminished hepatic zinc concentrations (30% less than normal) and mild growth retardation (Hojyo et al., PLoS One, 2011). Recently we demonstrated that ZIP14 is required for the uptake of non-transferrin-bound iron by hepatocytes and pancreatic acinar cells and for iron loading of the liver and pancreas during iron overload (Jenkitkasemwong et al., Cell Metab., 2015). In the course of our studies with Slc39a14-/- mice, we made the unexpected observation that these mice have dramatic perturbations in manganese homeostasis. Specifically, we found that livers of Slc39a14-/- mice are manganese deficient whereas most other tissues and blood show elevated manganese concentrations. The brain and bone are particularly affected, with manganese concentrations being approximately 10 and 65 times normal in Slc39a14-/- mice at 4 weeks of age. The alterations in tissue manganese concentrations persist throughout life up to at least 1 year of age. The defect is specific for manganese because concentrations of iron and zinc in the brain and bone are unaffected. Moreover, the concentrations of iron and zinc in other tissues (e.g., kidney, pancreas, heart) are also unaffected by loss of ZIP14. Studies using intravenously or orally 54 -/- administered [ Mn]MnCl2 indicate that the reduced concentrations of manganese in the liver of Slc39a14 mice reflect an impairment in manganese uptake from the plasma. Based on these and other data, we hypothesize that when ZIP14 is absent, manganese in the plasma cannot be efficiently taken up by the liver and subsequently secreted into the bile, the normal route of manganese excretion. As a consequence, manganese levels increase in the blood, eventually leading to manganese accumulation in peripheral tissues including the brain and bone. In humans, excess manganese in the brain deposits in regions of the basal ganglia and can lead to a Parkinsonian-like disorder known as manganism. Our findings in Slc39a14-/- mice are consistent with a recent report showing that human patients with loss-of-function mutations in SLC39A14 accumulate manganese in the brain but not the liver (Tuschl et al., Nat Comm. 2016). We therefore conclude that Mn2+—rather than Fe2+ or Zn2+—is the main physiologic substrate for hepatic ZIP14. However, in a pathological condition such as iron overload, Fe2+ from non-transferrin-bound iron is also transported by hepatic ZIP14. Seventh Congress of the International BioIron Society Page 286 Poster Abstracts IBIS Poster #174 INTESTINAL HEPHAESTIN IS NECESSARY FOR OPTIMAL IRON ABSORPTION IN PREGNANT MICE, BUT IS DISPENSABLE DURING LACTATION Caglar Doguer, PhD¹, Jung-Heun Ha, PhD¹, Shireen RL. Flores, MS¹, Tao Wang, MS¹,² and James F. Collins, PhD¹ ¹University of Florida; ²Sichuan University Presented By: Caglar Doguer, PhD Dietary nonheme (Fe3+) iron absorption into duodenal enterocytes requires reduction and subsequent import by divalent metal-ion transporter 1 (Dmt1). At the basal surface, release of ferrous iron (Fe2+) into the portal circulation is mediated by the iron export protein, ferroportin 1 (Fpn1), which is functionally coupled to iron oxidation by the multi-copper ferroxidase (FOX) hephaestin (Heph), allowing ferric iron to bind to transferrin in the interstitial fluids. Another circulating FOX, ceruloplasmin (Cp), may also participate in this oxidation process. Our previous studies showed that rapidly growing male and female neonatal mice lacking intestinal Heph (Hephint) had impaired iron absorption compared to WT littermates. Interestingly, although enterocyte FOX activity was not different between genotypes, serum Cp activity was lower in the KOs (p<0.05), perhaps providing a mechanistic explanation for diminished iron flux. In the current study, we tested the hypothesis that intestinal Heph is required for iron absorption during pregnancy and lactation. This is a logical postulate since these physiologic states are associated with increased iron absorption due to greater demands for iron to supply the growing embryo and placenta, and to deliver iron to developing offspring through milk. Iron absorption and tissue distribution were thus determined in pregnant and lactating mice 24 hours after oral administration of 59Fe. As we predicted, iron absorption was markedly impaired in pregnant mice lacking intestinal Heph compared to pregnant WT littermates. Duodenal enterocyte FOX activity and serum Cp activity were also diminished in the pregnant KOs as compared to pregnant WT mice. Moreover, serum Cp activity was elevated in pregnant mice as compared to non- pregnant littermates, irrespective of genotype. Further, the amount of 59Fe transferred from mother to the developing fetuses across the placenta was also diminished in pregnant mice lacking intestinal Heph. Unlike during pregnancy, however, 59Fe absorption, tissue iron distribution and iron transferred to the pups through milk were not altered in Hephint mice during lactation. Overall, this investigation has provided novel evidence that intestinal Heph is necessary for optimal iron absorption during pregnancy, but is dispensable during lactation. We postulate that non-Heph FOXs compensate for the lack of Heph in enterocytes and that serum FOX activity may complement intestinal FOX activity, thus allowing enhancement of iron absorption during lactation (this study) and in adult mice deprived of dietary iron or during acute hemolytic stress (our previous studies). These compensatory mechanisms are likely less effective, however, in mice during the rapid neonatal growth period and during pregnancy, when intestinal Heph is clearly more important from a physiological perspective. Supported by NIH grant 1R01 DK074867 (to J.F.C.). Seventh Congress of the International BioIron Society Page 287 Poster Abstracts IBIS Poster #175 TRANSCRIPTIONAL REGULATION OF TRANSFERRIN RECEPTOR BY HEME IN ERYTHROID CELLS Daniel Garcia dos Santos, PhD¹, Amel Hamdi, PhD², Matthias Schranzhofer, PhD¹, Nam Chun Lok, PhD³ and Prem Ponka, MD, PhD² ¹Lady Davis Institute, Jewish General Hospital; ²Lady Davis Institute, Jewish General Hospital/ Physiology Department, McGill University; ³Department of Chemistry, The University of Hong Kong Presented By: Daniel Garcia-Santos, PhD The function of cell surface transferrin receptor (TfR) is to mediate cellular iron uptake from plasma glycoprotein, transferrin (Tf). In general, TfR expression is negatively post-transcriptionally regulated by intracellular “free” iron (also referred to as “labile iron pool”, LIP) through iron-responsive elements (IREs) in the 3' untranslated region (UTR) of TfR mRNA. IREs are recognized by iron regulatory proteins (IRP-1 and IRP-2), which are specific cytoplasmic RNA-binding proteins that respond to cellular iron levels. At low iron levels IRPs bind to TfR mRNA IREs preventing degradation of the mRNA. On the other hand, high levels of cellular iron in the LIP inactivate the binding of IRPs to IREs resulting in rapid degradation of TfR mRNA. Erythroid cells are the largest consumers of iron which is delivered to them exclusively by Tf via TfR. Developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription1. Here we provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. ncubation of erythroid cells with 5 aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone blocks ALA dehydratase or methylprotoporphyrin blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Additionally, TfR promoter was activated in vitro by addition of ALA and Hemin in Murine Erythroleukemia Cells (MEL) differentiated with DMSO. Finally, direct site specific mutation in erythroid active element1 in TfR promoter abolished the heme mediated effects of ALA and Hemin. These results suggest that heme might directly or indirectly interact with TfR promoter activating gene expression. Hence, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. 1Nam-Lok C., Ponka P. (2000) Identification of an Erythroid Active Element in the Transferrin Receptor Gene. J. Biol. Chem. 275: 24185-24190. Seventh Congress of the International BioIron Society Page 288 Poster Abstracts IBIS Poster #176 FURTHER EVIDENCE THAT INTESTINAL COPPER ABSORPTION DOES NOT OCCUR THROUGH DMT1 BUT THROUGH CTR1 AND A CHLORIDE DEPENDENT UPTAKE TRANSPORTER, USING THE CACO2 CELL MODEL Adrian Ricarte, BS, Aleksander Wertz, undergraduate, Yuseinis Gutierrez, undergraduate, Angelica Valadez, undergraduate, Theodros Kidane, MS and Maria Linder, PhD California State University Fullerton Presented By: Adrian Ricarte, BS It is well established that divalent metal transporter 1 (DMT1) is the main transporter involved in uptake of dietary non- heme iron by enterocytes, and that it is also capable of taking up some other divalent metals. The potential role of DMT1 in dietary copper uptake is still in dispute. In the human Caco2 intestinal cell model, it was shown that knockdown of DMT1 mRNA was accompanied by a decreased rate of Cu(I) as well as Fe(II) uptake1, although the effect on iron uptake was greater. Over-expression of mouse DMT1 in HEK293 cells increased Cu (II) uptake2, and the same was true for cells transfected with rat DMT1 mRNA3. However, in the former studies, rates of uptake fell rather than increased with increasing proton concentrations (decreasing pH from 6.5), which is not characteristic of DMT1, and in the latter studies, the increase in copper uptake only occurred in iron deficiency, whereas rates of iron uptake failed to increase with iron deficiency. We reported that although copper inhibited iron uptake by Caco2 cell monolayers, the kinetics were not competitive4; Mackenzie’s group reported that less current was obtained during copper uptake by Xenopus oocytes expressing human DMT1 mRNA than with Fe(II), Cd(II), Mn(II) and Ni(II)5,6, and rates of Cu(I) or Cu(II) uptake were not stimulated by DMT1 overexpression6. To further test the involvement of DMT1 in intestinal copper uptake, we tested the effects of excess Mn(II) and Ni(II), using 5 uM 67Cu(I)-NTA applied to the apical medium of Caco2 cell monolayers with tight junctions. Additions of 50 or 200 uM concentrations did not inhibit rates of copper uptake over 60 min. The contributions of CTR1 and a potential chloride-dependent transporter7 were also examined in monolayers pretreated (or not) for 24 h with extra copper or iron (5 uM), or depleted of these by addition of TETA or DFO, respectively. Addition of Ag(I) (50 uM) was used to identify the contribution of CTR1 to rates of Cu(I) uptake, and substitution of 150 mM Na sulfate for NaCl in the medium was used to assess chloride-dependent uptake. Ag(I) caused a small but statistically significant inhibition of copper uptake (averaging 20%). Substitution of 150 mM sulfate for chloride markedly reduced uptake (averaging about 70%). In the presence of sulfate plus silver, rates of uptake were still about 15% of the total rate without these inhibitors suggesting an additional transport mechanism was present. Pretreatment with extra Cu or Fe failed to change uptake rates; however, copper deficiency decreased rates about 18%, while Fe deficiency had no effect. We conclude that at least in this intestinal model, chloride-dependent transport is the major means by which copper ions are taken up across the brush border, that CTR1 is only a minor contributor, and that DMT1 is unlikely to be involved. 1Arredondo et al (2003) Am J Physiol 284:C1525 2Arredondo et al (2014) Biometals 27:115 3Jiang et al (2013) J Nutr 143:1927 4Linder et al (2006) Biol Res 39:143 5Gunshin et al (1997) Nature 388:482 6Illing et al (2012) J Biol Chem 287:30485. 7Zimnicka et al (2011) Am J Physiol 300:C588 Seventh Congress of the International BioIron Society Page 289 Poster Abstracts IBIS Poster #177 STEPS IN THE MOBILIZATION AND RELEASE OF FERRITIN IRON DURING STIMULATION OF HEMATOPOIESIS Maria Linder, PhD¹, Alice La, BS Biochem¹, Trinity Nguyen, BS Biochem¹, Ketson Tran, BS Biochem¹, Eric Sauble, MS¹, David Tu, BS Biochem¹, Angelica Gonzalez, MS¹, Theodros Kidane, MS¹, Cesar Soriano, BS Biochem¹, Jessica Morgan, BS Biochem¹, Michael Doan, BS Biochem¹, Diane Jun, Undergraduate¹, Cia-Yu Wang, PhD² and Mitchell Knutson, PhD² ¹California State University Fullerton; ²University of Florida Presented By: Maria C. Linder, PhD Despite some controversy, numerous studies have now confirmed that for mobilization of the iron stored in ferritin to meet cell needs and hematopoiesis, ferritin moves from the cytosol into lysosomes, where degradation of its protein “coat” releases the iron inside. Now we have demonstrated that treating HepG2 cells containing 59Fe-labeled ferritin with mM concentrations of reducing/chelating agents, ranging from GSH and ascorbate to thioglycolate, lipoic acid and FMN, for hours or days failed to release iron from ferritin, as determined by rocket immunoelectrophoresis and autoradiography. Confocal microscopy confirmed movement of ferritin into lysosomes in response to iron deprivation with desferrioxamine (DFO), with ferritin accumulating in lysosomes when lysosomal proteases were inhibited. Quantitation of the particles representing the merge of ferritin and the lysosomal marker LAMP2 demonstrated a 2-3-fold increase in the number of merged particles and their total area in cells exposed to leupeptin or chloroquine. We then investigated how the iron in the ferrihydrite crystallites (“iron cores”) released by ferritin degradation might be solubilized by substances likely to be present in lysosomes. The iron in purified suspensions of ferritin iron cores was readily dissolved by 1-10 mM concentrations of GSH, ascorbate and citrate, at both pH 5 and 7. Preparations of lysosomal extracts purified from murine macrophages (J774a.1) and mouse liver and spleen, also dissolved the ferritin iron. Evidence for the next step, namely that DMT1 is involved in the return of ferritin iron to the cytosol was obtained by following its co-localization with LAMP2 in HepG2 cells by confocal microscopy during iron deprivation, which indicated a rapid movement of DMT1 to lysosomes (a 10-fold increase within 2.5h). Although attempts to knock down expression of DMT1 in these and rat hepatoma cells by RNAi almost obliterated DMT1 mRNA (determined by qPCR), it failed to have much effect on DMT1 protein expression (determined by immunoblotting) even after multiple treatments. However, a specific inhibitor of DMT1 (Nramp2) (Xen 485; Xenon Pharmaceuticals) consistently reduced transfer of lysosomal iron to the cytosol in J774a.1 macrophages. In these cells, further addition of specific siRNA for another potential iron transporter (Nramp1) reduced release even further, implying that both transporters were involved. However, no expression of Nramp1 mRNA was detected in hepatic cells. To verify involvement of DMT1 in iron mobilization from ferritin in whole animals, we used 129S6 mice (WT) and those in which DMT1 had been knocked out just in hepatocytes (kindly obtained from Mitch Knutson and Chia Yu Wang). Bleeding about 10% of the blood from the submandibular vein resulted in a statistically significant 25% drop in liver total iron and ferritin in the WT, but no significant change in the case of the conditional DMT1 knockout mice. Concentrations of ferritin in spleen decreased 30-40% in both kinds of mice. Very similar results were obtained for these mice when erythropoiesis was stimulated by daily injections of EPO over 5 days. We confirm that mobilization of ferritin iron in response to iron need and enhanced erythropoiesis drives ferritin into lysosomes for proteolytic degradation, and conclude that once ferritin protein is removed, the ferrihydrite crystals inside are dissolved by GSH and other constituents of the lysosomes, allowing return of ferritin iron to the cytosol with the help of DMT1 (in hepatocytes) and both DMT1 and Nramp1 in macrophages. Seventh Congress of the International BioIron Society Page 290 Poster Abstracts IBIS Poster #178 A MATHEMATICAL MODEL OF IRON DYNAMICS IN A MOUSE Jignesh Parmar, PhD and Pedro Mendes, PhD Center for Quantitative Medicine, UCONN Health Presented By: Jigneshkumar Parmar, PhD We developed a computational model of mouse iron physiology to gain insights into its complex hormonal regulations. Model parameters were estimated from experimental data previously published on radioactive iron distribution. Model calibration revealed an essential role of NTBI uptake by the liver under iron-rich conditions, without this the model would not reproduce iron accumulation in the liver and would instead have excess iron accumulate in red blood cells . The model was validated by its ability to simulate the pathophysiology of several iron disorders such as hemochromatosis, β- thalassemia and anemia of inflammation. We also tested various other experimental observations under normal and pathological states which not only further validated our model but also provided better understanding of underlying mechanisms. This physiological model paves the way for a more comprehensive multiscale model across organs, cells, and molecules. The present model contributes to a deeper understanding of iron physiology and can be used for predictive exploration of therapeutic interventions in iron disorders. Seventh Congress of the International BioIron Society Page 291 Poster Abstracts IBIS Poster #179 A GLUTAREDOXIN-BOLA COMPLEX SERVES AS AN IRON-SULFUR CLUSTER CHAPERONE FOR THE CYTOSOLIC CLUSTER ASSEMBLY MACHINERY Avery Frey, PhD¹, Dan Palenchar¹, Kym Zumbrennen¹, Toshiki Yabe¹, Ajay Vashisht², Justin Wildemann¹, James Wohlschlegel² and Caroline Philpott¹ ¹National Institutes of Health-NIDDK; ²Department of Biological Chemistry, UCLA Presented By: Avery Frey, PhD Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apo-proteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]- coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live-cell, proximity-dependent biotinylation, to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3-BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, while Glrx3 homodimer formation did not. Cellular Glrx3-BolA2 complexes increased 6-8-fold in response to increasing iron, forming a rapidly-expandable pool of Fe-S clusters. Fe-S coordination by Glrx3-BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3-BolA2 is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apo-proteins in human cells. Future studies will detail the molecular mechanism by which the Glrx3-BolA2 complex acquires its [2Fe-2S] clusters Seventh Congress of the International BioIron Society Page 292 Poster Abstracts IBIS Poster #180 NOVEL SENSORS FOR SIMULTANEOUS DETECTION OF LABILE Fe2+ AND Fe3+ BASED ON IN VITRO- SELECTED DNAZYMES Ryan Lake, BA¹, Seyed-Fakhreddi Torabi, PhD², Peiwen Wu, PhD² and Yi Lu, PhD¹ ¹Department of Chemistry, University of Illinois Urbana-Champaign; ²Department of Biochemistry, University of Illinois Urbana-Champaign Presented By: Ryan Lake, BA Iron plays an important role in many aspects of biology, ranging from oxygen transport to DNA synthesis to electron transfer to catalysis. Although the role of iron within these systems is well understood, much less is known about how the labile iron pool makes iron available to these systems, while also limiting its ability to produce reactive oxygen species (ROS). Monitoring labile iron within biological systems, especially both Fe2+ and Fe3+ simultaneously, can thus provide a better understanding of its regulated homeostasis, including during the nutritional immune response, as well as its role in various diseases such as anemia, cancer, and ROS-mediated cell death. However, current iron sensors lack high selectivity, have difficulty overcoming the non-specific fluorescence quenching effect of iron, and lack the ability to measure both Fe2+ and Fe3+ simultaneously. To overcome such limitations, the Lu lab has developed a novel class of sensors for the detection of metal ions using metal-dependent catalytic DNA, called DNAzymes. DNAzymes that are highly selective for either Fe2+ or Fe3+ have been selected from a D A library of 1015 random DNA sequences to undergo catalytic cleavage of a ribonucleotide phosphodiester bond in the presence of either Fe2+ or Fe3+, such that the DNAzymes only catalyze the reaction in the presence of this specific cofactor, with high selectivity and sensitivity. Following the catalytic beacon method developed by the Lu lab, I have transformed these iron-dependent DNAzymes into fluorescent sensors through the conjugation of fluorophores and quenchers, such that cleavage of a substrate strand produces a turn-on fluorescent signal (Fig a.). This modular design allows for simultaneous detection of Fe2+ and Fe3+ by incorporating different fluorophores on different sensors. The sensors were first demonstrated in buffer by annealing the DNAzyme enzyme strand with the cleavable substrate strand. The fluorescence activity rate was measured after adding iron in various concentrations, and these activity curves were then fit to a bimolecular activity curve. The initial rates (kobs) derived from these curves are proportional to the iron concentration present during the reaction (Fig b.). After demonstrating in vitro iron-dependent fluorescence activity of the sensors, they were then tested intracellularly. The DNAzymes were transfected into HepG2 cells using the G6 cationic polypeptide, developed by JianJun Cheng’s lab, which electrostatically associates with the negative DNA. Successful transfection to lysosomes was confirmed by confocal laser-scanning microscopy (CLSM) on a Zeiss 880 Airyscan confocal microscope (Fig d). Furthermore, iron-dependent intracellular fluorescence turn-on of the sensors was confirmed by flow cytometry, as compared with non-cleavable substrate or inactive enzyme negative controls (Fig c.). Following demonstration of these fluorescent sensors in the HepG2 model, they can further be applied to cellular models for ferroptosis, cancer, anemia, and nutritional immunity to better understand the role of abnormal iron homeostasis during these processes. Additionally, because of the modular design of these catalytic beacon iron sensors, they can be applied to different sensing modalities that have already been demonstrated by the Lu lab, such as a colorimetric point-of-care device which would be useful for monitoring anemia, or for in vivo 3D imaging using MRI to monitor abnormal labile iron levels in anemia and various cancers or neurological diseases. Seventh Congress of the International BioIron Society Page 293 Poster Abstracts IBIS Poster #181 ERYTHROFERRONE EXPRESSION LEVELS IN MYELODYSPLATIC SYNDROMES SHOW CLINICAL RELEVANCE Augusta Di Savino, Arianna Savi, Student, Daniela Gallo, MD, Valentina Gaidano, MD, Patrizia Scaravaglio, BSc, Mauro Mezzabotta, MD, Giuseppe Saglio, MD, PhD, Daniela Cilloni, MD, PhD and De Gobbi Marco, MD, PhD Department of Clinical and Biological Sciences Presented By: Augusta Di Savino, PhD Introduction: Dysregulation of hepcidin, a key iron regulating hormone, is important in the pathogenesis of iron overload in patients with myelodysplatic syndromes (MDS), heterogeneous clonal myeloid disorders characterized by ineffective hematopoiesis, especially erythropoiesis, cytopenia and deregulated iron homeostasis. Recently, Erythroferrone (ERFE) was discovered as a new erythroid-regulator of hepcidin in the context of erythropoietic stress. ERFE, encoded in humans by the FAM132B gene, induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of FAM132B in MDS patients and controls and correlated the differential expression data with clinical parameters and expression level of others iron genes. Methods: Bone marrow samples from 65 newly diagnosed MDS and 8 non-malignant hematological patients were collected. The diagnosis of MDS was based on the 2008 World Health Organization (WHO) classification. Total RNA was extracted with Trizol reagent. Reverse transcription and PCR amplification were performed according to manufacturer’s instruction. The expression level of each gene was normalized to the housekeeping gene ABL1 and analyzed by 2- CT relative quantitative method. Statistical analyses were performed using GraphPad 4.0 software. P-values of <0.05 were considered statistically significant. Unpaired t-test or Mann Whitney test was used to study difference between two groups. Correlation analysis was performed using the Spearman’s rank correlation coefficient rs. Results: We first evaluated the expression of FAM132B in total bone marrow (BM) cells of 65 newly diagnosed MDS patients. FAM132B expression had a more variable expression pattern in MDS than controls and was highly expressed in 31 out of 65 patients. In BM cells, expression levels of FAM132B correlated inversely with those of HAMP and positively with those of FPN1 and EPOR. We next evaluated FAM132B expression in the different subgroups of WHO classification. ERFE expression was significantly higher in RA (p=0.0013) and RAEB-1 (p=0.001) subtypes when compared with controls, but not in RAEB-2 or in RCMD. To investigate the potential role of ERFE in a clinical setting, we next conducted Spearman’s correlation analysis on the mRNA expression of FAM132B and laboratory parameters. Our results showed that FAM132B was positively correlated with serum iron, ferritin, bone marrow erythroblasts and MCV and inversely correlated with peripheral RBC. Discussion and Conclusion: This study analyzes the FAM132B expression in MDS, clonal conditions of the bone marrow with impaired hematopoiesis, especially erythropoiesis. The observed high overexpression of FAM132B in MDS patients, its positive correlation with BM erythroblasts but its negative correlation with peripheral RBC might be related to an abnormal and ineffective erythropoiesis. Moreover, the inverse correlation between FAM132B and BM HAMP confirms that ERFE is able to modulate, through hepcidin suppression, the iron homeostasis, not only at systemic level as shown by the correlation with the laboratory markers of iron metabolism, but also at the BM level. These finding suggest that ERFE may play an important role in the physiopathology of the dysfunctional erythropoiesis and it could be assessed as new biomarker in MDS. Seventh Congress of the International BioIron Society Page 294 Poster Abstracts IBIS Poster #182 EFFECT OF ERYTHROPOIETIN ON IRON HOMEOSTASIS IN TMPRSS6-MUTATED MASK MICE Jana Frydlova², Zuzana Rychtarcikova³, Iuliia Gurieva², Martin Vokurka², Jaroslav Truksa¹ and Jan Krijt² ¹Czech Academy of Sciences, Institute of Biotechnology; ²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic; ³Institute of Biotechnology, BIOCEV Research Center, Czech Academy of Sciences, Vestec, Czech Republic Presented By: Jaroslav Truksa, PhD The ENU-mutagenesis generated mutant Tmprss6msk/msk mouse, also known as mask mouse shows significant alterations in the iron metabolism and severe microcytic anemia. The underlying cause is inapropriately high expression of hepcidin, resulting in the inability to normally respond to iron deficiency, leading to insufficient erythropoiesis. We have analyzed the response of the wild type and mask mice to erythropoietin (EPO) and we have studied the levels of splenic erythroferrone and ferroportin as well as liver hemojuvelin. In agreement with previous studies, mask mice are not responding to EPO by hepcidin downregulating, showing critical role of TMPRSS6 in this process. Our data show that mask mice are responding to EPO stimulus by erythroferrone induction similarly to the wild type mice, yet they show a different pattern of hemojuvelin cleavage compared to wild type animals, with additional cleavage product detected in mask mice. Interestingly, mask mice treated with EPO show lower level of splenic ferroportin compared to the wild type animals The results provide support for the interaction between TMPRSS6 (also known as matriptase-2) and hemojuvelin in vivo; it also shows that hemojuvelin could be cleaved by another as yet unknown protease in the absence of functional TMPRSS6. Intriguingly, the observed HJV fragments do not support cleavage at arginines R121 or R288. The lack of effect of erythropoietin on hepcidin expression in mask mice cannot be explained by changes in erythroferrone synthesis, yet it demonstrates that lack of matriptase-2 is blocking the response to EPO despite normal erythroferrone synthesis in vivo. The exact crosstalk between TMPRSS6 and erythroferrone remains to be clarified as our data together with other in vivo data (Nai et al., Blood, 2016) suggest a critical role of TMPRSS6 in response to EPO while data observed on isolated hepatocytes suggest that erythroferrone inhibits the BMP/SMAD pathway independently of TMPRSS6 (Kautz et al. ASH2016, poster 3616). Grant Support: This work has been supported by Czech Science Foundation (GA CR), grant 15-16803S to JK and grant 13-28830S to JT; by the Ministry of Education, Youth and Sports of CR within the LQ1604 National Sustainability Program II (Project BIOCEV-FAR) and by the project „BIOCEV“(CZ.1.05/1.1.00/02.0109). by the Grant Agency of the Charles University in Prague, grant GAUK IG 186315 to IG and by institutional grants PRVOUK P24/LF1/3 and SVV 260157. Seventh Congress of the International BioIron Society Page 295 Poster Abstracts IBIS Poster #183 THE ROLE OF GDF11 IN THE TREATMENT OF ß-THALASSEMIA Rea Oikonomidou, MD¹, Amaliris Gonzalez, PhD, Ping La, PhD, Vania Lo Presti, Ritama Gupta, Carla Casu, PhD, Laura Breda, PhD, Rajasekhar NVS Suragani, PhD², Ravi Kumar, PhD and Stefano Rivella, PhD¹ ¹Children's Hospital of Philadelphia; ²Acceleron Presented By: Stefano Rivella, PhD The investigational drug Luspatercept (ACE-536) is a ligand trap in phase-3 clinical trial. It contains the extracellular domain of activin receptor IIB (ACVR2B) and induces red blood cell production in an erythropoietin independent pathway. t binds with high affinity to members of the transforming growth factor (T ) β superfamily and alters activin signaling through the intracellular SMAD complex. Recent studies in a mouse model of beta thalassemia intermedia identified growth differentiation factor-11 (GDF11) as a possible target of the drug. It has been proposed that GDF11 is overexpressed in thalassemic erythroblasts and inhibits terminal erythroid maturation via SMAD complex phosphorylation. In order to recapitulate the phenotype of thalassemic mice treated with RAP-536, the murine counterpart of ACE-536, we generated and analyzed animals with GDF11 deletion in erythroid cells (Hbbth3/+Gdf11fl/flEpoR-Cre) and in all hematopoietic tissues (Hbbth3/+Gdf11fl/flVav-Cre). We did not detect any differences in RBC number, reticulocyte count, hemoglobin or hematocrit levels compared to thalassemic mice. Administration of RAP-536 in these mouse models significantly improved hematopoietic parameters in the peripheral blood, decreased spleen size and ameliorated ineffective erythropoiesis indicated by an increased ratio of mature to immature erythroblasts in the spleen analyzed by FACS. Therefore, the lack of GDF11 did not prevent a response to the drug. To assess the effect of complete lack of D 11, we are generating β-thalassemic mice with total GDF11 deletion and if this modifies activation of the Smad pathway in presence and absence of RAP-536. In addition, we are further investigating the role of this drug on erythropoietin synthesis, iron metabolism, globin synthesis and generation of BFU-e/CFU-e colonies. To investigate the possible effects of Luspatercept on erythroid cells, we treated cells derived from normal or thalassemic patients with the drug. We did not observe any alteration on erythroid cell viability, number, differentiation or cell cycle. Interestingly, we did not detect significant expression of GDF11/Gdf11 in human and mouse erythroid cells. To investigate the possibility of an alternate cell source of GDF11 production that would affect erythroid cells, we treated in vitro murine erythroleukemia (MEL) cells with exogenous GDF11. After treatment we observed phosphorylation of the SMAD2/3 complex by western blot. This effect was hindered after co-administration of GDF11 with Luspatercept. Additional analyses are being performed to investigate pathways that are modulated by administration of RAP536. Thus, our findings suggest that GDF11 is not the sole ligand of RAP-536 in thalassemic mice or that GDF11 is required but not sufficient to promote improvement of erythropoiesis. Furthermore, erythroid cells do not produce but can respond to exogenous GDF11, synthesized by non-erythroid cells, under conditions of ineffective erythropoiesis. Even though we detected in vitro effects, these may not be physiologically relevant as experimental conditions may not correlate with GDF11 concentrations in vivo. These results reveal a potential alternative target of action for ACE-536 and may lead to the discovery of new therapeutic molecules. Seventh Congress of the International BioIron Society Page 296 Poster Abstracts IBIS Poster #184 THE HIGH-RESOLUTION CRYSTAL STRUCTURE OF THE GLOBULAR DOMAIN OF MOUSE ERFE Kirsty McHugh, DPhil Clinical Medicine, Jing Jin, Joao Arezes, Reema Jasuja, Alexander Drakesmith, Simon Draper and Matt Higgins Oxford University Presented By: Kirsty McHugh, PhD Summary: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. To date, atomic-level structures are available for the globular C1q domains of several family members (adiponectin, CTRP10, CTRP13 and CTRP14), which show that their C1q domains adopt a classic ‘jelly-roll’ topology and form a trimeric quaternary structure stabilised by a central hydrophobic interface. Here we present the structure of the globular C1q domain of mouse ERFE to 1.32A and discuss some similarities and differences between the structure of ERFE C1q and those of other CTRP members. Methods: mouse ERFE C1q DNA, with an added mouse IgG signal sequence and c-tag, was cloned into an in house mammalian expression vector, which then underwent site-directed mutagenesis to substitute cysteine for alanine at position 194. The protein was expressed in HEK293E cells in the presence of 5uM Kifunensine, and then purified via a CaptureSelect c-tag affinity column followed by size exclusion chromatography. Purified protein was concentrated to 10mg/ml and crystallised in a reservoir solution of 20% glycerol ethoxylate, 10% tetrahydrofuran and 0.1M Tris pH 8.0. Crystals were transferred into a drop containing 25% glycerol and 75% well solution and were cryo-cooled in liquid nitrogen for storage and data collection. Data were collected at beamline I02 (Diamond Light Source, UK) using radiation with a wavelength of 0.98Å and a Pilatus 6M-F detector. These data were indexed, refined and scaled using XDS in xia2 to a resolution of 1.32Å. Fugue was used to potential search models and molecular replacement using Phaser-MR (CCPN4, 1994) found one copy in the asymmetric unit. The model was built into the remaining electron density using an iterative cycle of refinement using autobuster (Bricogne et al., 2011) and manual model building using Coot (Emsley et al., 2010). Seventh Congress of the International BioIron Society Page 297 Poster Abstracts IBIS Poster #185 IDENTIFICATION OF RNAi TECHNOLOGY TO TARGET TRANSFERRIN RECEPTOR 1 FOR REDUCTION OF HEMICHROME FORMATION IN ß-THALASSEMIA Amaliris Gonzalez, PhD¹, alentina Ghiaccio², Laura Breda, PhD, elena Ginzburg, MD³, Robert Fleming, MD⁴ and Stefano Rivella, PhD ¹Children's Hospital of Philadelphia; ²2Università degli studi di Cagliari; ³Mount Sinai Medical Center, N ; ⁴Saint Louis University Presented By: Amaliris Gonzalez, PhD Iron overload is the principal cause of morbidity and mortality in individuals with -thalassemia. Disruptions of the stoichiometry between - and -globin chains result in excessive free -chains, which form hemichromes (HMC) that precipitate in erythroid precursors1 and leads to both altered red blood cell (RBC) viability and ineffective erythropoiesis. Studies on the effect of administration of transferrin show a reversal of this phenotype in the experimental murine model Hbbth1/th1 of -thalassemia2. Increasing the concentration of circulating transferrin results in the overall net decrease of iron uptake by RBC precursor cells via transferrin receptor 1 (TFRC) and subsequent reduction of the formation of HMCs. As HMCs are reduced, the lifespan of RBCs increases and, therefore, the total amount of hemoglobin (Hb) in circulation also increases2. In this study, we aim to decrease iron uptake of erythroid progenitors by lowering TFRC expression using RNAi technology. We have identified several low-affinity, lentivirally-expressed RNAi constructs targeting both human TFRC and mouse Tfrc receptors. We have tested the RNAi constructs on immortalized Human Erythroid Progenitor (HUDEP) and Murine Erythroleukemia (MEL) cell lines, and obtained successful targeting of TFRC/Tfrc without perturbing erythroid maturation or cell viability, as determined by FACS analysis. Also, the RNAi constructs were tested in vitro (using patient -thalassemic CD34+cells) and in vivo (using a -thalassemia mouse model) to evaluate whether moderate reduction of TFRC/Tfrc by our lentiviral vectors expressing our shRNAs and microRNAs can reduce HMCs production and improve ineffective erythropoiesis. Next, we will incorporate our RNAi constructs into a lentiviral vector containing the - globin gene. We aim to moderately reduce iron uptake by erythroid progenitors to prevent HMC accumulation, while also decreasing -globin aggregation by restoring the - and -globin stoichiometry in both mice and patient cells. References: 1. Rachmilewitz EA, Thorell B. Hemichromes in single inclusion bodies in red cells of ß thalassemia. Blood 1972;39:794- 800. 2. Li H, Rybicki AC, Suzuka SM et al. Transferrin therapy ameliorates disease in ß-thalassemic mice. Nat.Med. 2010;16:177-182. Seventh Congress of the International BioIron Society Page 298 Poster Abstracts IBIS Poster #186 EX VIVO GENE THERAPY APPROACH BY TARGT TECHNOLOGY FOR THE TREATMENT OF ß –THALASSEMIA INTERMEDIA Carla Casu, Vania Lo Presti, Emir O'Hara, Garry Neil, Reem Miari, Nir Shapir and Stefano Rivella The Children’s Hospital of Philadelphia Presented By: Carla Casu, PhD ß-thalassemia intermedia or non-transfusion dependent thalassemia (NTDT) is a genetic blood disorder characterized by ineffective erythropoiesis, anemia, splenomegaly and systemic iron overload. In theory, agents that can stimulate erythropoiesis (such as erythropoietin or EPO) could improve anemia by increasing the production of RBC. and, under conditions of reduced iron absorption, correct the iron overload by consuming the excess of stored iron stored. We used an ex vivo technology (TARGT-Transduced Autologous Restorative Gene Therapy) for prolonged production and secretion of therapeutic agents. Primary dermal fibroblasts transduced with helper dependent adenovirus expressing mouse Epo were implanted Hbbth3/+ and wild-type (WT) C57BL/6 mice. We generated three groups that received 10e5, 5x10e5 or 1x10e6 of genetically modified fibroblasts, while one group received untransduced fibroblasts as control. In WT mice, one week after implantation, hemoglobin (Hb) levels rose 2/3 g/dL, showing erythrocytosis. At the end of the treatment (six weeks) animals showed significantly increased Hb (18.5±1.7 g/dL, 19.4±0.5 g/dL and 19.8±0.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 13.5±0.8 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases up to 39%. In Hbbth3/+ mice Hb levels rose about 3 g/dl after the first week and remained elevated until the end (8.7±0.3 g/dL, 10.7±1.0 g/dL and 10.6±1.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 7.7±0.7 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases up to 27%. At the end of the treatment, serum Epo levels were markedly increased at all doses both in Hbbth3/+ and WT animals (more than 50% and up to 90%) when compared with their respective controls. As expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin, preventing the beneficial effect of erythroid-mediated consumption of stored iron in Hbbth3/+ animals. We postulated that, in presence of agents that increase erythropoiesis (Epo), some level of iron restriction is required to improve the anemia, prevent exacerbation of the splenomegaly and decrease iron overload. TARGTEPO was then combined with low iron diet or iron chelation. In WT animals, after three weeks, the combination of TARGTEPO with low iron diet significantly reduced Hb levels (-40%), HTC (-42%), RBC number (-38%) and reticulocytes (-80%), when compared to animals overexpressing Epo and receiving normal iron diet or iron chelation. This indicates that in these animals the stored iron is insufficient to support the increased erythropoiesis. In Hbbth3/+ animals this effect was not seen, suggesting a delay due to the highest level of stored iron. Furthermore, in several Hbbth3/+ animals overexpressing Epo and under iron restriction, the improvement in the anemia was associated with reduced splenomegaly. These effects were not seen in Hbbth3/+ animals treated with iron chelation. We are now finalizing our study by analyzing RBC lifespan, hemichrome formation, serum Erythroferrone (Erfe) levels and tissue iron concentration and distribution. In conclusion, these preliminary results suggest that the TARGT platform could be utilized, in combination with drugs that limit iron intake, to improve anemia and decrease iron overload in NTDT. Seventh Congress of the International BioIron Society Page 299 Poster Abstracts IBIS Poster #187 A NOVEL ROLE FOR ZIP8 IN ERYTHROPOIESIS Supak Jenkitkasemwong, PhD, Wei Zhang, PhD and Mitchell Knutson, PhD University of Florida Presented By: Supak Jenkitkasemwong, PhD ZIP8 (SLC39A8), similar to its closest homologue ZIP14, is capable of transporting a number of metals including zinc, iron, manganese, and cadmium. Unlike ZIP14, however, global deletion of ZIP8 is embryonic lethal, indicating that ZIP8 serves an essential, non-redundant function. Mice with ~90% reduction in ZIP8 levels (so-called Zip8 hypomorphic mice) display severe anemia in utero, suggesting that ZIP8 plays a role in maternofetal transfer of iron or in erythropoiesis (Gálvez-Peralta et al., PLOS One, 2012). A role for ZIP8 in erythropoiesis is additionally suggested by our observation that ZIP8 mRNA levels are consistently elevated in the spleen of anemic mice (4 different models of anemia) and that ZIP8 mRNA levels increase in parallel to those of transferrin receptor 1 (TfR1) during differentiation of murine erythroleukemia (MEL) cells. To test the hypothesis that ZIP8 plays a role in erythropoiesis, we used CRISPR-Cas9 to disrupt the Slc39a8 gene (which encodes ZIP8) in MEL cells. We found that ZIP8-deficient MEL cells displayed significantly lower hemoglobin and total iron levels 5 days after differentiation. Cellular concentrations of zinc, manganese, and copper, however, were unaffected. ZIP8-deficient cells also exhibited lower mRNA levels of transferrin receptor 1 (TfR1), delta-aminolevulinate synthase 2 (ALAS2), and hemoglobin alpha chain (Hb-a). To investigate the role of ZIP8 in erythropoiesis in vivo, we disrupted the Slc39a8 gene in adult Zip8flox/flox;Rosa26-cre mice by tamoxifen administration. Sixty-days after tamoxifen administration, ZIP8-deficient mice exhibited significantly lower Hb levels (12.1 ± 0.1 g/dL for ZIP8-null (n=4) vs 13.5 ± 0.2 g/dL for WT (n=6); P = 0.003) and higher reticulocytes (4.5 ± 0.3 % for ZIP8-null (n=4) vs 1.4 ± 0.5 % for WT (n=5); P < 0.001) when compared with control mice. Together, our data suggest that ZIP8 plays a role in normal erythropoiesis. Future study will be needed to define the exact role(s) of ZIP8 and at which stage it may function in erythropoiesis. Seventh Congress of the International BioIron Society Page 300 Poster Abstracts IBIS Poster #188 GLYCINE TRANSPORTER 1 PLAYS A CRUCIAL ROLE IN HEMOGLOBINIZATION Daniel Garcia dos Santos, PhD¹, Matthias Schranzhofer, PhD¹, Richard Bergeron, MD², Alex D Sheftel, PhD³ and Prem Ponka, MD, PhD⁴ ¹Lady Davis Institute, Jewish General Hospital; ²Ottawa Hospital Research Institute, University of Ottawa; ³Spartan Bioscience Inc High Impact Editing; ⁴Lady Davis Institute for Medical Research, Je ish General Hospital Physiology Department, McGill University Presented By: Daniel Garcia-Santos, PhD Vertebrate heme synthesis requires three substrates: succinyl-CoA, which regenerates in the tricarboxylic acid cycle, iron and glycine. For each heme molecule synthesized, one atom of iron and eight molecules of glycine are needed. It is well known that inadequate delivery of iron to immature erythroid cells leads to a decreased production of heme, but virtually nothing is known about the consequence of an insufficient supply of glycine on the process of hemoglobinization. To address this issue, we exploited mice in which the gene encoding glycine transporter 1 (GlyT1) was disrupted. Primary erythroid cells isolated from fetal livers of GlyT1 knockout (GlyT1-/-) and GlyT1-haplodeficient (GlyT1+/-) embryos exhibited a substantial decrease in the cellular uptake of [2-l4C]glycine and heme synthesis as revealed by a considerable decrease in [2-l4C]glycine and 59Fe incorporation into heme. Since GlyT1-/- mice die during the first postnatal day, we analyzed blood parameters of newborn pups and found that GlyT1-/- animals develop hypochromic microcytic anemia. These results provide the first evidence that GlyT1 is essential for appropriate hemoglobinization of developing erythroid cells both in vitro and in vivo. Our finding that Glyt1-deficiency causes decreased heme synthesis in erythroblasts is unexpected, since glycine is a non-essential amino acid. It also suggests that GlyT1 represents a limiting step in heme and, consequently, hemoglobin production. GlyT1 inhibitors are currently tested in clinical trials as an alternative therapy for the treatment of schizophrenia. Importantly, our study provides strong evidence that such an approach may also target erythroid precursors and cause hypochromic microcytic anemia. Seventh Congress of the International BioIron Society Page 301 Poster Abstracts IBIS Poster #189 HEME OXYGENASE 1 DIRECTS STRESS-INDUCED REMODELING OF IRON-RECYCLING MACROPHAGES Carine Fillebeen, PD¹, Daniel Garcia-Santos, PhD¹, Konstantinos Gkouvatsos, MD¹, Saljoughian Noushin, PhD², Louis- Philippe Leroux, PhD², Maritza Jaramillo, PhD², Tina Haliotis, MD¹, Marzel Buffler, PhD³, Christiane Becker, PhD³, Klaus Schümann, MD³, Prem Ponka, MD¹ and Kostas Pantopoulos, PhD¹ ¹Lady Davis Institute for Medical Research/McGill University; ²INRS Institut Armand Frappier Research Centre; ³Science Center Weihenstephan, Technical University Munich Presented By: Kostas Pantopoulos, PhD Macrophages regulate systemic iron balance by recycling heme iron from senescent erythrocytes via heme oxygenase 1 (HO-1). We generated Hmox1LysM-Cre mice to explore implications of myeloid-specific HO-1 disruption in iron homeostasis. Bone marrow-derived or thioglycollate-elicited peritoneal Hmox1LysM-Cre macrophages lack any considerable HO-1 expression, which correlates with a prolonged pro-inflammatory phenotype and decreased viability following in vitro erythrophagocytosis. Hmox1LysM-Cre mice do not develop anemia, but manifest progressive splenomegaly, disruption of splenic architecture and proliferation of cells from the white pulp B cell areas. Despite an early delay in clearance of injected 59Fe-labeled erythrocytes from the blood, Hmox1LysM-Cre mice maintain physiological iron recycling by partially shifting heme catabolism from the liver to the spleen. Importantly, 59Fe-heme degradation occurs predominantly in the liver of control Hmox1fl/fl animals, and the contribution of the spleen is low. Consistently with these findings, Hmox1LysM-Cre mice exhibit profound HO-1 ablation in Kupffer cells but retain considerable HO-1 expression in splenic and bone marrow macrophages. Hmox1LysM-Cre mice respond to phenylhydrazine-induced hemolysis by recruiting HO-1-expressing macrophages from the bone marrow to the liver, and by inducing residual HO-1 expression in the spleen. Similar responses were documented in Hmox1fl/fl controls. Nevertheless, Hmox1LysM-Cre mice show increased sensitivity to sub- lethal phenylhydrazine doses compared to Hmox1fl/fl controls. Furthermore, they upregulate HO-1 in renal proximal tubule cells following chronic administration of phenylhydrazine, indicating a failure to adapt to this challenge. In conclusion, myeloid-specific HO-1 deficiency triggers remodeling of erythrophagocytic macrophages. The plasticity of these cells depends on HO-1 expression, as a small population of monocytes that is resistant to LysM-Cre-mediated excision of HO- 1 readily expands in response to hemolytic stress and populates the liver, to preserve iron homeostasis. Our data also highlight the role of Kupffer cells as the primary site of erythrophagocytosis in vivo, and demonstrate the importance of splenic macrophages and bone marrow-recruited liver macrophages as a critical alternative reserve. Seventh Congress of the International BioIron Society Page 302 Poster Abstracts IBIS Poster #190 IDENTIFICATION OF NOVEL LOCI ASSOCIATED WITH HEMATOLOGICAL TRAITS IN MICE Brie Fuqua, Stela McLachlan, Richard C. Davis, Simon T. Hui, Nam Che, Zhiqiang Zhou, Carmen Ng, Claire Yukhtman, Raffi Gharakhanian, Hannah Qi, Milagros C. Romay, Calvin Pan, Yehudit Hasin-Brumshtein, Dongyi Lu, Brian W. Parks, Eleazar Eskin, Aldons J. Lusis and Chris D. Vulpe University of Florida and University of California, Los Angeles Presented By: Brie Fuqua, BS, PhD Association and linkage studies in humans and mice have identified loci associated with iron and red cell traits, but in many cases the causal variants are not well delineated. In order to refine and discover new loci involved in iron metabolism, we are performing two genome wide association studies in mice using the Hybrid Mouse Diversity Panel (HMDP), a renewable resource of inbred and densely genotyped mouse strains. In the first study, female mice at 3-4 weeks of age were put on an iron sufficient (50 ppm) defined diet. In the second study, male mice at 4 weeks of age were put on an iron overloaded (20,000 ppm iron, as 2% iron carbonyl) defined diet. For both studies, tissues were collected at 10 weeks of age, from 112 and 114 mouse strains at an average of 7.8 and 6.6 mice per strain, for the female and male studies, respectively. Hematological parameters were measured in lithium heparin anticoagulated blood using a Heska Hematrue Hematology Analyzer for mice. Association mapping was performed using the FaST-LMM algorithm with a kinship matrix that accounts for the HMDP population structure. Mapping results were compared with those from previous HMDP studies of mice on chow and high fat/high sucrose diets. Female mice on the iron sufficient diet appeared healthy overall while strains on the iron loaded diet were generally stunted in growth, showed visible evidence of tissue iron overload, and developed various degrees of normocytic anemia associated with pallor. Significant inter-strain variation was observed in both studies for the blood traits, and most traits mapped significantly to a least one locus. Of note, in the female and male iron studies, hematocrit (HCT), red cell distribution width (RDW), mean cell hemoglobin concentration (MCHC), and mean cell hemoglobin (MCV) all mapped strongly to a locus on chromosome 7 near a hemoglobin beta cluster of genes, as previously reported. Uniquely in the iron sufficient female study, hemoglobin (HGB) mapped to a locus on Chromosome 5, with the peak SNP rs32279386 located in an intron of Fbxo24 within 5kB of Tfr2. The homologous locus in humans has been reported to associate with erythrocyte phenotypes. Uniquely in the iron loaded male HMDP study, red blood cell count (RBC), HCT, and HGB traits mapped to a region on Chromosome 9 (see figure: Manhattan plot of results for RBC; significance cutoff shown in red), with the peak SNP rs29689367 falling within 1 Mb of several genes including known iron metabolism gene Fdx1. Further studies will focus on the refinement and functional validation of these and other loci. Using a systems genetics approach, we will integrate future RNA-Seq gene expression data, tissue metal results, and other data with these hematological findings to gain better insight into the underlying biology linking these loci to traits. Seventh Congress of the International BioIron Society Page 303 Poster Abstracts IBIS Poster #191 IMATINIB AND SPIRONOLACTONE SUPPRESS HEPCIDIN EXPRESSION Katarzyna Mleczko-Sanecka¹, Debora Call², Ana Rita da Silva², Nikolai Schmeer, Melanie Kiessig, Georg Damm and Martina Muckenthaler ¹International Institute of Molecular and Cell Biology, Warsaw, Poland; ²Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Germany Presented By: Katarzyna Mleczko-Sanecka, PhD Disorders of iron metabolism are largely attributed to an excessive or insufficient expression of hepcidin, the master regulator of systemic iron homeostasis. Here, we investigated whether drugs targeting genetic regulators of hepcidin can affect iron homeostasis. We focused our efforts on drugs approved for clinical use to enable repositioning strategies and/or to reveal iron-related side effects of widely prescribed therapeutics. To identify hepcidin-modulating therapeutics, we reevaluated data generated by a genome-wide RNAi screen for hepcidin regulators. By applying RNAi assays of potential drug targets in combination with small molecule testing we identified spironolactone, diclofenac, imatinib and SAHA as hepcidin modulators in Huh7 cells and primary hepatocytes. We further show that administration of imatinib and spironolactone suppresses liver hepcidin expression in mice. Our results demonstrate that a commonly used anti- hypertensive drug, spironolactone, which is prescribed for the treatment of heart failure, acne and women hirsutism, as well as imatinib, a first-line, lifelong therapeutic option for some frequent cancer types, act as inhibitors of hepcidin expression. We expect these results to be of relevance for patient management, which needs to be addressed in prospective clinical studies. Seventh Congress of the International BioIron Society Page 304 Poster Abstracts IBIS Poster #192 FASTING INCREASES HEPATIC HEPCIDIN EXPRESSION AND CAN OVERCOME THE EFFECT OF HFE DELETION IN MICE Cornel Mirciov, BSc, Sarah Wilkins, BSc, Greg Anderson, PhD and David Frazer, PhD Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: David Frazer, PhD Introduction: Several prominent disorders, such as hereditary hemochromatosis and beta-thalassemia, are associated with the inappropriately low expression of the iron regulatory hormone hepcidin. Pharmaceutical agents that increase hepcidin production would, therefore, be of benefit in these conditions. A recent study has demonstrated that fasting can increase hepcidin production in mice, although the potential effects of circadian variations in hepcidin expression were not taken into account. We have examined the effect of fasting in more detail to determine whether the pathway(s) responsible might provide novel targets for pharmaceutical intervention in disorders of iron homeostasis Methods: C57BL/6 mice were fasted for 5, 10, 16 or 24 hours prior to euthanasia, then blood and tissue samples were collected for analysis. Tissue collection was performed at the same time of day for all time points to avoid the potential effects of circadian variations in hepcidin production. Other models examined included mice maintained on an iron deficient diet for six weeks, mice injected with erythropoietin and Hfe knockout mice. Hepatic Hamp1 expression was determined by real-time quantitative PCR, as was the expression of bone morphogenetic protein 6 (Bmp6) and cAMP responsive element binding protein 3-like 3 (Creb3l3), and the expression of erythroferrone in bone marrow. Serum iron levels and liver iron concentration was determined using colorimetric assays. Results: We have confirmed that fasting increases hepatic Hamp1 expression in mice, with maximal expression seen after 16 hours (2.3-fold). This was despite a significant decrease in serum iron levels and an increase in bone marrow erythroferrone expression, although a significant increase in Bmp6 expression (1.4-fold) was observed in mice fasted for 16 hours. Hamp1 expression increased in both iron deficient fasting mice and in fasted mice treated with erythropoietin, however, these results did not reach statistical significance when compared with unfasted animals. Surprisingly, fasting of Hfe knockout mice resulted in a 7.7-fold increase in Hamp1 expression when compared to unfasted knockout mice and a 3.7-fold increase when compared to unfasted wild-type mice. In this case, there was no significant change in Bmp6 expression. In all fasted mice, a significant increase in hepatic Creb3l3 was observed. Discussion and Conclusions: These studies have confirmed that fasting increases the expression of hepatic Hamp1 in mice. Although the effect of fasting was not able to overcome the stimuli to decrease Hamp1 expression in chronically iron deficient mice or following erythropoietin injection, fasted Hfe knockout animals expressed higher levels of Hamp1 than both unfasted Hfe knockout and wild-type mice. This indicates that the induction of Hamp1 expression in response to fasting is independent of Hfe. Our data also suggest that the effect is independent of changes to serum iron levels, erythroferrone expression or Bmp6 production. Creb3l3 encodes a transcription factor known to bind the Hamp1 promotor and its induction in all fasting conditions studied supports previous suggestions that hepcidin production is regulated by gluconeogenic signals. In conclusion, our results suggest that targeting the pathway(s) involved in the regulation of hepcidin production by fasting could be of clinical benefit in conditions such as hemochromatosis and beta-thalassemia. Seventh Congress of the International BioIron Society Page 305 Poster Abstracts IBIS Poster #193 EFFECT OF DIETARY VITAMIN D SUPPLEMENTATION ON SERUM HEPCIDIN LEVELS IN MICE WITH ACUTE INFLAMMATION Abitha Sukumaran, Larisa Lozovatsky, MS and Karin E Finberg, MD, PhD Yale University School of Medicine Presented By: Abitha Sukumaran, PhD Introduction: Disorders of iron balance lead to various diseases in humans. Deficiency of iron causes anemia, while iron overload results in damage to tissues including liver, heart and endocrine glands. Hepcidin, a peptide hormone synthesized by the liver and secreted into the circulation, plays a central role in maintaining systemic iron homeostasis, by limiting iron absorption from the intestine and efflux of iron from the reticuloendothelial system. Aim of the Study: In anemia of inflammation, hepcidin is induced by inflammatory stress, resulting in decreased iron availability in the circulation, thereby contributing to the development of anemia. Recent studies have shown an inverse correlation between serum levels of hepcidin and vitamin D in patients with chronic kidney disease. Oral vitamin D supplementation decreased serum hepcidin levels in healthy volunteers, suggesting a role for vitamin D in modulating systemic iron status. In addition, vitamin D deficiency has been observed in elderly patients associated with anemia of inflammation. However, the precise molecular mechanism involved in the regulation of hepcidin by vitamin D is not known. Given that murine models have been useful for deciphering mechanisms of systemic iron regulation, the aim of the present study was to determine if oral vitamin D supplementation could acutely suppress serum hepcidin levels in mice. Methods: Female C57BL/6N mice were raised on a standard rodent diet (2U vitamin D3/g). At eight weeks of age, mice were divided into 2 groups, which were placed on either a vitamin D3 enriched diet (20U/g) or control diet (2U vitamin D3/g). Forty-eight hours later, each group of mice was then further subdivided into two groups, receiving either turpentine oil (to induce acute inflammation) or saline (control) injection. Mice were sacrificed 16 hours after injection. Blood was collected and serum was isolated for hepcidin measurement by ELISA. Liver and spleen were harvested to measure non- heme iron levels in the tissues. Results: Serum hepcidin levels were significantly higher (p < 0.00001) in mice with acute inflammation compared to saline-injected controls. However, high vitamin D supplementation did not significantly alter the levels of hepcidin, when compared to mice that received control diet. In addition, spleen weights were found to be significantly lower (p < 0.01) in mice injected with turpentine, as compared to saline-injected controls, irrespective of the diets used. There were no significant differences in the levels of iron in the liver between different groups studied. Compared to saline-injected controls, levels of splenic iron were increased (p< 0.08) in mice injected with turpentine, compatible with iron restriction within the macrophages of the spleen. This effect was observed in mice receiving either high vitamin D or control diet. Discussion: Acute inflammation induced by turpentine oil injection increased serum hepcidin levels after 16 hrs in mice fed either a vitamin D enriched or control diet. However, we did not observe any significant change in the levels of hepcidin in response to the high vitamin D diet within this timeframe. We hypothesize that the reduction in spleen weight observed in the inflamed mice might be due to the mobilization of white blood cells from the spleen. Seventh Congress of the International BioIron Society Page 306 Poster Abstracts IBIS Poster #194 THE PROTECTIVE ROLE OF HAPTOGLOBIN/HEMOPEXIN AXIS IN ANKYLOSING SPONDYLITIS-DRIVEN SPINAL CORD INJURY Ana Catarina Vaz Carreto Martins, MSc², Illyane Sofia Martins Lima, MSc² and Raffaella Gozzelino, PhD¹ ¹Chronic Diseases Research Center /NOVA Medical School; ²CEDOC/NOVA Medical School Presented By: Raffaella Gozzelino, PhD Ankylosing spondylitis (AS) is a chronic inflammatory disease, which compromises spine motility and leads patients to a complete disability. Despite the advances in the treatment of AS, no therapy is capable so far to restore spine motility. Therefore, the identification of biomarkers for AS prognosis is of utmost importance to prevent AS-driven spinal cord injury (SPI). By using a loss of function approach, our preliminary experiments demonstrate the existence of a strict correlation between the expression of the circulating Hemoglobin and Heme scavenger, Haptoglobin (Hp) and Hemopexin (Hx) respectively, and AS severity. Impaired heme/iron homeostasis is shown, in our findings, to be crucially involved in AS progression and observed to contribute to the infiltration of activated immune cells in the entheses of affected mice and the accumulation of iron in the spine of AS-induced animals. Exacerbated AS symptoms have been found in Hp- and/or Hx-deficient mice, which is consistent with the observation that enhanced bone formation and inflammatory lesions to spine were detected in response to heme administration. Similar data were also obtained in vitro, where the absence of Hp and/or Hx significantly modulates the proliferation of primary isolated osteoblasts in response to heme/iron. Thus, our findings indicate that the expression of Hp and Hx might dictate the outcome of AS progression and, as such, could be used as prognostic markers for disease severity. Seventh Congress of the International BioIron Society Page 307 Poster Abstracts IBIS Poster #195 INCREASING DIETARY IRON INTAKE ANTAGONIZES COPPER METABOLISM IN A DOSE-DEPENDENT FASHION IN WEANLING RATS Jung-Heun Ha, PhD¹, Caglar Doguer, PhD¹, Shireen RL. Flores, MS¹, Tao Wang, MS¹,², Sun Young Jeong, MS¹ and James F. Collins, PhD¹ ¹University of Florida; ²Sichuan University Presented By: Jung-Heun Ha, PhD High-iron feeding (1-2% Fe) is a widely used approach to induce iron overload in rats and mice, modeling human genetic iron-loading disorders. We recently utilized this approach to create a rat model of iron overload. Unexpectedly, however, consumption of a high iron diet (0.8%; 8800 ppm) impaired growth and caused copper-deficiency anemia and cardiac hypertrophy in weanling rats (Ha et. al. PLoS One, 11(8): e0161033, 2016). Importantly, adding extra copper to this high- iron diet prevented these copper-related physiologic perturbations. Since the iron content in this diet was very high, the current study was designed to test the hypothesis that lower iron levels would similarly perturb copper metabolism in growing rats. The experimental design was to feed three-week-old, male, weanling Sprague-Dawley rats 6 different AIG- 93G-based diets for 7-8 weeks. The diets varied only in iron content (88 [adequate Fe], 320, 756, 1475, 3021, and 8834 ppm) with copper being held constant (i.e. ~8 ppm; adequate levels). Thereafter, iron and copper-related biochemical and physiologic parameters were quantified in experimental animals. The data reported in this study were derived from 6 rats/group and all reached statistical significance (p<0.05; 1-way ANOVA with Tukey’s post hoc analysis), unless otherwise stated. Consumption of the diet containing 8834 ppm Fe impaired growth and caused severe anemia, cardiac hypertrophy, and hematomegaly and nephromegaly, consistent with our previous study. A noticeable trend towards heart and liver enlargement was also noted in rats fed the diets with 1475 and 3021 ppm Fe. Further, tissue copper levels gradually decreased as iron content of the diets increased. For example, hepatic, splenic and cardiac copper depletion was noted when rats consumed the diets with the 2 highest iron levels. Representing a more subtle biomarker of body copper levels, serum ceruloplasmin (Cp) (i.e. amine oxidase) activity also trended lower as the iron content of the diets increased, with the decrease in the rats consuming the diets with the 3 highest Fe levels reaching statistical significance. Complementary molecular analyses demonstrated that expression of copper transporter 1 mRNA decreased as the iron content of the diets increased, possibly providing an explanation for hepatic copper depletion and depressed Cp activity with high-iron feeding. Overall then, this investigation supports the postulate that higher iron intake dose-dependently perturbs copper metabolism. The suggestion that copper can antagonize iron absorption has been proposed previously (Klevay, Nutr. Res. Rev., 28:1-8, 2016), and our studies support this possibility. Although longer-term feeding studies are required to assess the impact of more moderate iron levels on copper metabolism, it seems likely that iron supplementation in humans could also cause similar physiologic perturbations. Supported by NIH grant 1R01 DK074867 (to J.F.C.). Seventh Congress of the International BioIron Society Page 308 Poster Abstracts IBIS Poster #196 A SINGLE ADAPTABLE COCHAPERONE-SCAFFOLD COMPLEX DELIVERS NASCENT IRON-SULFUR CUSTERS TO MAMMALIAN RESPIRATORY CHAIN COMPLEXES I-III Nunziata Maio, PhD¹, Ki Soon Kim, PhD², Anamika Singh² and Tracey Rouault, MD² ¹National Institutes of Health; ²NIH/NICHD Presented By: Nunziata Maio, PhD The iron-sulfur (Fe-S) cluster of the Rieske protein, UQCRFS1, is essential for acquisition of mitochondrial Complex III (CIII) activity, though the mechanism for Fe-S cluster transfer has not previously been elucidated. Recent studies have shown that the co-chaperone HSC20, essential for Fe-S cluster biogenesis of SDHB in mammalian cells, directly binds LYRM7, formerly described as a chaperone that stabilizes UQCRFS1 prior to its insertion into CIII. Here we report that a novel transient subcomplex involved in CIII assembly, composed of LYRM7 bound to UQCRFS1, interacts with components of an Fe-S transfer complex, consisting of HSC20, its cognate chaperone HSPA9, and the holo-scaffold ISCU. Direct binding of HSC20 to the LYR motif of LYRM7 in a pre-assembled UQCRFS1-LYRM7 intermediate in the mitochondrial matrix facilitates transfer of a Fe-S cluster from holo-ISCU to UQCRFS1. Numerous Fe-S cluster subunits of mitochondrial Complex I also interact with HSC20 to acquire their clusters, highlighting the crucial role of the HSC20 co- chaperone in the assembly and function of the mitochondrial electron transport chain. Seventh Congress of the International BioIron Society Page 309 Poster Abstracts IBIS Poster #197 ENTRY OF IRON AND OTHER METAL IONS INTO MITOCHONDRIA MichaeL Garrick, PhD¹, Natascha Wolff, PhD², Lin Zhao, MD¹, Laura Garrick, PhD¹ and Frank Thévenod, MD, PhD² ¹UB; ²Witten/Herdecke University Presented By: Michael Garrick, PhD A major portion of metal ion metabolism occurs in mitochondria, yet there is little agreement on how metal ions enter mitochondria. We have previously reported convincing evidence that Divalent Metal Transporter (DMT1) is on the outer mitochondrial membrane (OMM) [1,2]. Its presence there is not likely to be an accident, so we have tested hypotheses about its function and tissue distribution. The kidney is one tissue where expression of DMT1 is high so we tested metal ion import into isolated renal mitochondria from rats, comparing homozygous Belgrade (b/b) preparations to +/b and +/+ ones. Mn uptake increased in a graded fashion across the 3 genotypes, indicating that Mn2+ import into mitochondria depended on having functional DMT1 on the OMM. Erythroid cells differentiate to synthesize vast quantities of heme with the initial and final steps occurring in the mitochondria; we predict that the last step creates demand for Fe2+. We therefore cultured human CD34 cells under conditions where they renew or commit to erythroid differentiation. Commitment to erythroid differentiation leads to increased levels of OMM DMT1. We have also prepared mitochondria from HEK293 cells engineered to overexpress DMT1 in an inducible fashion. Uninduced cells have little OMM DMT1; while induced cells have much more OMM DMT1 [1,2]. Preloading the mitochondria with Phen-Green-SK (PGSK) allows detection of iron import via PGSK quenching. We see a rapid initial phase of import that does not depend on OMM DMT1 levels then a slower phase that does. Addition of 0.1mM of the specific DMT1 inhibitor 2 (3 carbamimidoylsulfanylmethylbenzyl) isothiourea (CISMBI) blocks the DMT1-dependent slower phase leading even to a reversal of PGSK quenching but does not affect the rapid initial phase for mitochondria from induced cells. CISMBI has‐ ‐a much smaller effect on the slower‐ phase for mitochondria from uninduced cells, consistent with lower levels of DMT1 being present and/or a CISMBI inhibited pathway that does not involve DMT1. The slow phase is also responsive to a pH gradient imposed on the mitochondrial preparation. Results to date are consistent with Fe2+ and Mn2+ ions entering mitochondria via DMT1 and probably other transporters or channels, changing the paradigm for this organelle. acknowledgments Funding from ZBAF and the Laura and Michael Garrick fund. References: [1] Wolff, N. A. et al. (2014) FASEB J. 28, 2134-2145. [2] Wolff, N. A. et al. (2014) Channels 8, 458-466. Seventh Congress of the International BioIron Society Page 310 Poster Abstracts IBIS Poster #198 A NOVEL PLAYER IN THE REGULATION OF HEPCIDIN REVEALED BY THE MTOR INHIBITOR RAPAMYCIN Laura Silvestri, PhD, Silvia Colucci, BSc, Alessia Pagani, PhD, Irene Artuso, BSc, Mariateresa Pettinato, BSc, Antonella Nai, PhD and Clara Camaschella, MD IRCCS San Raffaele Scientific Institute & San Raffaele Vita-Salute University Presented By: Laura Silvestri, PhD Introduction: The main signaling regulating hepcidin and iron homeostasis is the BMP/SMAD pathway. In the liver, BMP- type I receptors (BMPRI) ALK2 and ALK3, essential for ligand responsiveness and hepcidin basal levels maintenance (Steinbiecker et al., 2012), are phosphorylated in the glycine/serine rich domain by BMP-type II receptors (BMPRII), BMPR2 and ACVR2A), thus activating the kinase activity of BMPRI and SMAD1/5/8 phosphorylation. Among other stimuli, as erythropoiesis expansion, inflammation and hypoxia, hepcidin expression is influenced by drugs, as the mTOR inhibitor rapamycin (RAPA)(Mleczko-Sanecka et al., Blood 2014). Indeed hepcidin increases in primary hepatocytes treated with RAPA. However, the mechanisms involved in hepcidin and mTOR crosstalk are unknown. Methods: Hepcidin, BMP-SMAD and mTOR were analyzed in hepatoma cells and murine primary hepatocytes (HCs), treated with RAPA, Torin 1, tacrolimus (TAC), or GPI1046 in the presence or absence of the BMP-pathway inhibitor DMH1 or ligands BMP6, BMP2, and Activin A. ALK2 mutants were generated by mutagenesis. Phospho-SMAD1/5/8 was analyzed by WB. C57BL/6 (8 weeks-old) wild-type male mice were treated with TAC and sacrificed at different time points. Hepcidin, LIC, SIC, serum iron and hematological parameters were analyzed. Results: We have analyzed hepcidin in Hep3B cells and primary HCs treated with RAPA, which inhibits mTORC1, and Torin1, an ATP-competitive inhibitor of mTORC1 and 2. Hepcidin is increased by RAPA but not Torin1, suggesting that mTOR does not play a direct role in hepcidin regulation. To exert its function, RAPA binds FKBP12, an immunophilin that interacts with several proteins, including BMPRI, to avoid uncontrolled meglio leakage activation of the pathway in the absence of ligands. Since RAPA-dependent hepcidin activation is SMAD1/5/8 dependent, we hypothesized that FKBP12 displacement from BMPRI, and not mTOR inhibition, regulates hepcidin. To explore this possibility, we mutagenized ALK2 to impair its binding to FKBP12 (ALK2mut). Overexpression of ALK2mut in Hep3B cells and primary hepatocytes (HCs) increases hepcidin in a SMAD1/5/8 dependent way and DMH1 abrogates this effect. Then we displaced FKBP12 by TAC, a calcineurin inhibitor that binds FKBP12, and GPI1046, a molecule without immunosuppressive effect. TAC and GPI1046 treatment of hepatoma cells and primary HCs increases hepcidin in a SMAD1/5/8 manner, suggesting a common mechanism with RAPA. FKBP12-dependent hepcidin regulation is independent from the BMP-coreceptor HJV since TAC upregulates hepcidin also in primary HCs from HjvKO mice. The FKBP12 effect is conserved in vivo since hepcidin is increased at 6 hrs post-injection in TAC-treated wt mice. FKBP12 displacement regulates BMP receptors selectivity to ligands. Despite ALK2wt binds preferentially BMP6, ALK2mut become responsive to Activin A, a TGF-β ligand increased in inflammation that usually signals through SMAD2/3. Activin A activates hepcidin only in ALK2mut-transfected cells through SMAD1/5/8 phosphorylation. This effect is due to loss of FKBP12 binding to ALK2mut since Activin A increases hepcidin also in ALK2wt transfected cells treated with TAC to displace FKBP12. Conclusions: In conclusion, FKBP12 contributes to hepcidin regulation through modulation of ALK2 activity, both in vitro and in vivo, thus adding a new player to the BMP-dependent hepcidin activation and a potential pharmacologic target for hemochromatosis and beta-thalassemia, characterized by low hepcidin and iron overload. Furthermore the ability of ALK2 to respond to Activin A, released in inflammation, links the BMP pathway-hepcidin activation to the liver inflammatory response. Acknowledgements: Supported by Telethon Grant GGP15064 to LS Seventh Congress of the International BioIron Society Page 311 Poster Abstracts IBIS Poster #199 WORLDWIDE STANDARDIZATION OF SERUM HEPCIDIN ASSAYS Dorine Swinkels, MD, PhD Radboudumc, Nijmegen, The Netherlands Presented By: Dorine W. Swinkels, MD, PhD Absolute serum hepcidin concentrations measured by various methods differ considerably, complicating interpretation of results and rendering reference intervals method dependent. A previous hepcidin harmonization study identified a commutable secondary reference material. Here we aim to i) validate the commutability of this secondary reference material and its functionality to increase the degree of equivalence between methods (i.e. harmonize methods), ii) to produce a large batch of this material for international use and iii) to establish worldwide standardization (i.e. harmonization with results traceability to the SI units) through value assignment of hepcidin concentrations of the matrix material with a candidate reference method that is itself calibrated with a primary reference material. We applied technical procedures to achieve harmonization, developed by the International Consortium for Harmonization of Clinical Laboratory Results. Nine hepcidin assays (4 mass spectrometry- and 5 immunochemical-based) quantified native individual serum samples (n=16) and native serum pools (n=8) to assess analytical performance, current- and achievable equivalence, as well as the reference material (2 levels, n=2), consisting of native lyophilized serum with cryolyoprotectant. The latter was assessed to validate its suitability, most notably in terms of commutability, to serve as secondary reference material. Homogeneity of the reference material was tested according to ISO 13528:2005 by means of duplicate measurements of hepcidin of 12 randomly selected low and middle calibrator samples.Value assignment of the secondary reference material was performed by measurement of primary reference material by a validated Weak-Cation-eXchange MALDI Time-of-Flight – mass spectrometry assay. Commercially obtained synthetic hepcidin was used as candidate primary reference material. Comprehensive impurity analysis of the candidate primary reference material was performed by liquid chromatography high-resolution tandem mass spectrometry, and used to correct value assignment by amino-acid analysis. Analytical characteristics of the participating methods allowed harmonization in terms of reproducibility, mutual correlations and linearity. Current equivalence between methods (inter-assay CV 40.4%) was mainly attributable to differences in calibration and could therefore be improved by harmonization. We validated the commutability of the reference material and simulated harmonization with this calibrator set, which resulted in a maximum achievable equivalence of 11.0%. Vials of both the low and middle calibrator passed the homogeneity criteria as defined by ISO 13528:2005. Periodical assessment of hepcidin concentrations in stored vials of the secondary reference material showed no significant decrease for at least a year at temperatures ranging from -80°C to 4°C. Using the candidate primary reference material we assigned a mean value of 0.91 and 4.04 nM to the low and middle secondary calibrators, respectively. In conclusion, we identified, produced and – using a candidate primary reference material - assigned a value to secondary hepcidin reference material. This material will be made available in the course of 2017 and has the potential to substantially improve equivalence between hepcidin assays worldwide by its ability to provisionally standardize hepcidin assays, i.e. assign “true” values to hepcidin concentrations obtained by various validated hepcidin measurement procedures. Ongoing testing of the candidate secondary and primary reference materials and the candidate primary reference method will ultimately lead to certification of reference materials and reference method and full standardization. Seventh Congress of the International BioIron Society Page 312 Poster Abstracts IBIS Poster #200 MARINE SPONGE EXTRACTS AS A PROMISING SOURCE OF IRON CHELATORS Hector Aguilar Vitorino, PhD¹ and Breno Pannia Espósito, PhD² ¹University of São Paulo - USP; ²University of São Paulo Presented By: Hector Aguilar Vitorino, PhD Different forms of labile iron such as non-transferrin bound iron (NTBI), labile plasma iron (LPI) or labile iron pool (LIP) can cause toxicity to different organs through the production of reactive oxygen species (ROS). Iron overload therapy relies on high affinity chelating agents, including the so-called siderophores, peptides produced by microorganisms often associated to other organisms, such as sponges. The marine sponge Hymeniacidon heliophila was collected in São Sebastião, Brazil, in a search for compounds with iron affinity. H. heliophila presented 67 μg/g of iron (total) but no metal in its organic extracts. The crude ethanol extract presented iron affinity in the calcein competition test, as well as antioxidant activity in the iron-ascorbate setup. Ultrafiltrates (<1 kDa) and chromatography separation (Sep – Pak C18) showed no loss of chelating activity. The final fraction separated through the Amberlite® IR 400 resin maintained the chelating activity and its infrared analysis showed carboxylate groups. Our data showed that in these sea sponge extracts, we found compound with iron-chelating activity and antioxidant proprieties. This marine sponge, H. heliophila, can be a natural source of promising of iron chelator and possible used for a chelation therapy. Seventh Congress of the International BioIron Society Page 313 Poster Abstracts IBIS Poster #201 NUCLEAR HORMONE RECEPTOR-14 INTEGRATES IRON UPTAKE AND INNATE IMMUNITY IN CAENORHABDITIS ELEGANS THROUGH PQM-1 SIGNALING Elizabeth Leibold, PhD, Cole P. Anderson, PhD, Steven J. Romney, BS and Paul Rindler, PhD University of Utah Presented By: Elizabeth A Leibold, PhD Iron uptake and storage in the nematode C. elegans is maintained by hypoxia-inducible transcription factor-1 (HIF-1). Our previous study showed that hif-1 null mutants are developmentally delayed when grown under iron limitation. Here, we identify nhr-14 encoding nuclear hormone receptor-14 (NHR-14), a homolog of vertebrate 4 , in a genetic screen to identify genes that rescue the developmental delay of hif-1 mutants. Loss of NHR-14 increased iron content in hif-1 null mutants by up-regulation of the intestinal iron transporter SMF-3. nhr-14 null mutants also show increased expression of innate immune genes and DAF-16/FOXO down-regulated Class 2 genes, and resistance to Pseudomonas aeruginosa and Salmonella enterica infections. NHR-14 regulation of iron uptake and innate immunity is mediated by the insulin/IGF- like signaling (IIS) pathway transcription factor PQM-1. Loss of NHR-14 function enhances the translocation of PQM-1 to the nucleus where it activates smf-3 and innate immune genes via DAF-16-associated elements (DAE) in the promoters of these genes. Our data support a model in which C. elegans respond to bacterial infection by reducing NHR-14, thus allowing PQM-1 to translocate to the nucleus and activate genes critical for the innate immune response and iron uptake. We also propose that the induction of smf-3 by PQM-1 in nhr-14 mutants is a mechanism to limit iron bioavailability in the intestinal lumen in response to pathogenic infection. The regulation of iron uptake by NHR-14 is conserved as the SMF-3 ortholog, vertebrate divalent metal transporter 1 (DMT1), is repressed by 4 in human T29 colorectal cells. Our data provide insight into how C. elegans, and likely vertebrates, utilize nuclear receptors to coordinate iron availability and innate immunity. Seventh Congress of the International BioIron Society Page 314 Poster Abstracts IBIS Poster #202 IMPROVED PATIENT-REPORTED OUTCOMES WITH A FILM-COATED VERSUS DISPERSIBLE TABLET FORMULATION OF DEFERASIROX: RESULTS FROM THE RANDOMIZED, PHASE II ECLIPSE STUDY Ali T. Taher¹, Raffaella riga², Silverio Perrotta³, Ale andra ouraklis⁴, Giovan Battista Ruffo⁵, Antonis attamis⁶, Ai-Sim Goh⁷, Annelore Cortoos⁸, icky Huang⁸, Marine Weill , Ra uel Merino Herranz and John B. Porter¹ ¹American University of Beirut Medical Center; ²University of Cagliari; ³Second University of Naples; ⁴University of Patras Medical School; ⁵U. .C. Ematolog. Con alassemia; ⁶University of Athens; ⁷Hospital Pulau Pinang; ⁸Novartis Pharmaceuticals Corporation; Novartis Pharma AG; ¹ University College London Presented By: John B. Porter, MA, MD, FRCP DRCPath Background: The iron chelator deferasirox (DFX) has been available as once-daily dispersible tablet (DT) formulation since 2005, offering an alternative to parenteral deferoxamine. However, limitations in patient adherence, including the need to take the drug in a fasting state, palatability and gastrointestinal (GI) tolerability led to development of DFX film- coated tablet (FCT). DFX FCT can be taken orally, once-daily with or after a light meal, and is potentially less burdensome to patients than the DT formulation. Here we present results from patient-reported outcomes, during the 24-week ECLIPSE study. Methods: Iron chelation therapy (ICT)-naïve or pre-treated patients aged ≥10 years, re uiring CT and with serum ferritin (SF) >1000 ng/mL, were eligible for enrollment. ICT-naïve patients were requested to start on DFX DT 20 mg/kg/day or DFX FCT 14 mg/kg/day while pre-treated patients were requested to start on a dose equivalent to their pre-washout dose. Outcomes were assessed with 3 different PRO instruments: 1) The modified satisfaction with ICT (modified SICT; adherence, satisfaction/preference and concern domain scores), 2) the palatability questionnaire (taste, aftertaste, ability to consume medicine, perception of medicine); both were completed at weeks 2, 3, 13, and end of treatment. 3) The GI symptom diary (belly pain, nausea, vomiting, constipation, diarrhea) was completed daily. Results: Of the 173 transfusion-dependent patients (thalassemia, n=140; myelodysplastic syndromes, n=32; missing, n=1) enrolled, 87 received FCT and 86 received DT. Completion rates for questionnaires (~80% at the start reducing to ~70% by week 24) and GI daily diary (~70% at the start reducing to ~35% by week 24) were similar for each formulation. Throughout the 24-week study period, FCT patients consistently reported better adherence, greater satisfaction/preference, and fewer concerns compared to DT patients (Figure). At end of treatment, 53/60 (88.3%) evaluable patients receiving FCT and 41/63 (65.1%) evaluable patients receiving DT indicated they would prefer FCT rather than DT. FCT patients reported higher satisfaction on palatability scores than DT patients (Figure). The difference in score between DT and FCT was >1 point (minimal important difference) for all modified SICT domains and for palatability at most visits, indicating a clinically meaningful difference between formulations. In this study, overall GI summary scores were low for both formulations indicating patients experienced very little trouble/concern associated with GI symptoms; DT patients reported more trouble/concern than those receiving FCT. Conclusions: These results show a clear preference in favor of DFX FCT in all domains for the modified SICT as well as for palatability. More patients were satisfied with DFX FCT compared with DT at all visits. DFX FCT offers patients an improved formulation that does not require administration in a fasting state, has better palatability, and very little concern associated with GI tolerability. Enhanced patient satisfaction with the new DFX FCT formulation may improve adherence, thereby, reducing iron overload-related complications. Seventh Congress of the International BioIron Society Page 315 Poster Abstracts IBIS Poster #203 LONG-TERM IRON CHELATION THERAPY WITH DEFERASIROX IN PEDIATRIC PATIENTS WITH TRANSFUSIONAL HEMOSIDEROSIS (ENTRUST) Elliott Vichinsky¹, Amal El-Beshla y², Azzam Alzoebie³, Annie amdem⁴, Suzanne oussa⁵, hirachit Chotsampancharoen⁶, Andreas Bruederle⁷, Jackie Han⁸ and Mohsen Elalfy ¹Children s Hospital Research Center; ²Cairo University; ³Sheikh halifa Medical City; ⁴Centre Hospitalier Intercommunal; ⁵Chronic Care Center; ⁶Songklanagarind Hospital; ⁷Novartis Pharma AG; ⁸Novartis Pharmaceuticals; Ain Shams University Presented By: Elliott Vichinsky, MD Background: Pediatric patients with β thalassemia major (βTM), sickle-cell disease (SCD) and Diamond–Blackfan anemia (DBA) often require regular transfusion therapy from early age and consequently accumulate iron. Iron overload (IOL) can result in organ damage and delayed growth/development, therefore optimal management of iron loading is required. Methods: ENTRUST is a 5-year multinational, observational study in patients (enrolment age, 2–<6 years) with transfusional IOL. Patients were prescribed deferasirox according to local labels and dose was adjusted based on serum ferritin (SF) levels, therapeutic goals, tolerability and weight gain. Primary objective reported safety, specifically renal and hepatic function. Secondary objectives included analysis of SF, and growth/development during long-term deferasirox treatment. Results: 267 patients (βTM, 65.9%; SCD, 19.5%; DBA, 4.5%; other anemias, 10.1%) with mean age, 3.2 years were enrolled and received ≥1 deferasirox dose (median exposure, 59.9 (range 1.2–65.6) months; mean±SD dose, 25.8±6.5 mg/kg/day). 122 (45.7%) patients discontinued, 55 (20.6%) due to ‘Other’ reasons, 19 (7.1%) due to lost to follow-up and 18 (6.7%) due to adverse events (AEs; increased ALT [n=7]; increased AST [n=7]). 172 (64.4%) patients received an average blood intake ≥7 mL/kg/month. Median SF level (ng/mL) decreased from 1702 (range 334–9577) at baseline (n=243) to 1127 (range 38–6428) after 5 years (n=84). Patients initially received suboptimal deferasirox doses to manage iron intake and were infrequently dose adjusted for weight gain. Eight (3.1%) patients had two consecutive serum creatinine (SCr) increases >age-adjusted ULN and >33% from baseline; two of them received deferasirox dose adjustments and none discontinued. 11 (4.2%) patients had two consecutive ALT increases >5 or >10xULN, three of whom received deferasirox dose interruptions. There were no unexpected AEs or laboratory abnormalities. Increased ALT (21.1%), increased AST (11.9%), vomiting (5.4%) and rash (5.0%) were the most common AEs (≥5.0%) suspected to be deferasirox-related. Although liver lab parameters met Hy's law criteria in few patients, none of them was consistent with a Hy's law case. Overall, number of patients experiencing drug-related AEs decreased with each year of deferasirox exposure. Mean growth velocity was 4.86–5.95 cm/yr in male and 5.44–6.08 cm/yr in female patients (Table), in line with public growth curves for children aged 2–≤11 years. Mean weight increased steadily over time in line with mean growth velocity. Median SCr (μmol/L) increased steadily over time (28.0 at baseline to 39.4) in line with median height, and by association with increasing muscle mass. One βTM patient experienced an AE of below normal height, not suspected to be drug-related. Conclusions: This long-term, ‘real-life’ study indicates favorable safety and efficacy of deferasirox in pediatric patients with transfusional IOL. Over half the patients completed the five-year study, with limited discontinuations due to AEs. In accordance with previous deferasirox trials, pre-pubertal population showed expected growth velocities and weight gain. Thus, careful monitoring and appropriate deferasirox dose adjustments based on iron burden, safety, and weight gain ensure effective management of long-term ICT in majority of pediatric patients in clinical practice. Seventh Congress of the International BioIron Society Page 316 Poster Abstracts IBIS Poster #204 LABILE PLASMA IRON PREDICTS FOR SURVIVAL IN PATIENTS UNDERGOING ALLOGENIC-STEM CELL TRANSPLANTATION – RESULTS FROM THE PROSPECTIVE MULTICENTER GERMAN-AUSTRIAN ALLIVE TRIAL Martin Wermke, Julia Eckoldt, Katharina Goetze, Stefan Klein, Gesine Bug, Friedrich Stoelzel, Malte von Bonin, Johannes Schetelig, Michael Laniado, Verena Plodeck, Wolf-Karsten Hofmann, Gerhard Ehninger, Martin Bornhaeuser, Dominik Wolf, Igor Theurl and Uwe Platzbecker TU Dresden, Medical Faculty, Department of Medicine I Presented By: Martin Wermke Allogeneic stem cell transplantation (allo-SCT) offers the potential for cure to acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) patients. It can, however, be associated with high non-relapse mortality (NRM). The impact of systemic, mainly transfusional iron overload (SIO) on NRM and post-transplant outcome is matter of constant debate, which may be explained by the paucity of prospective trials and the use of interference-prone surrogate marker such as serum ferritin to quantify SIO in many studies performed so far. We therefore set out to study the impact of SIO on post allo-SCT outcome using MRI derived liver iron content (LIC) as an unbiased marker of SIO and enhanced labile plasma iron (eLPI) as the putative mediator of iron toxicity. ALLIVE was a prospective multicenter observational trial (NCT01746147). LIC was determined prior to and on day +100 and day +360 after allo-SCT. Enhanced labile plasma iron (eLPI) was measured using the Ferros eLPI Kit (Afferix) prior to, during and after conditioning and an eLPI above 0.4 was defined as positive. A total of 112 patients were evaluable for the final analysis (last patient last visit). Median LIC prior to conditioning was 120 µmol/g. There was a significant correlation between pre-transplant eLPI levels and LIC (p < 0.001). Mean eLPI levels increased continuously during the course of the conditioning regimen and then gradually decreased starting on day +7, while most patients had negative eLPI levels by day +100 and +360 after allo-SCT (Figure A). Patients with a positive eLPI prior to conditioning had a higher incidence of bacterial infections and liver toxicity. There was, however, no difference between eLPI positive and negative patients with respect to neutrophil engraftment or the occurrence of relapse. A LIC above the pre-specified threshold of >=125 µmol/g (7 mg/g) was associated with a significantly increased cumulative incidence (CI) of NRM at day 360 (29.1 % vs. 12.5 %, p = 0.025). This association was even more pronounced in patients tested positive for eLPI prior to the initiation of conditioning, who had a CI of NRM at day +360 of 32.6 % compared to 9.7 % in those being eLPI negative (p = 0.003). Moreover the patients with positive eLPI levels prior to conditioning had a significantly lower probability of overall survival (OS, day +360: median 171 days vs. not- reached; p < 0.001, Figure B). These associations of eLPI positivity and adverse outcome were preserved in multivariate models controlling for other factors with known impact on NRM and OS, respectively. The results of the prospective ALLIVE trial provide evidence that SIO in AML and MDS patients prior to allo-SCT is associated with an increased mortality after transplant patients. Given our results we believe that there is a reasonable probability that LPI is the mediator of toxicity in this setting and peri-transplant LPI-scavenging strategies should be tested in future clinical trials. Seventh Congress of the International BioIron Society Page 317 Poster Abstracts IBIS Poster #205 EXPANDING THE CLINICAL AND MOLECULAR SPECTRUM OF FERROPORTIN DISEASE André Viveiros, MD, Benedikt Schaefer, MD, Armin Finkenstedt, MD, Benjamin Henninger, MD, Karin Nachbaur, MD, Herbert Tilg, MD and Heinz Zoller, MD Medical University Innsbruck Presented By: Andre Viveiros, MD Background: Ferroportin disease is an autosomal dominant hepatic iron overload syndrome where two distinct clinical and biochemical phenotypes have been reported. Classical ferroportin disease is due to a loss of iron export function and characterized by low to normal transferrin saturation with iron overload in Kupffer cells and splenic macrophages. Non- classical ferroportin disease is due to a resistance of ferroportin to hepcidin mediated internalization and degradation of the protein. Histologically and biochemically, non-classical ferroportin disease resembles HFE associated hemochromatosis. The aim of this study was to characterize the functional consequences of the ferroportin gene (SLC40A1) mutation p.Arg178Gln on iron export and hepcidin binding activity of the protein. To characterize the natural history of ferroportin disease, long-term follow up and comprehensive assessment of liver function as well as liver stiffness are required. Methods: To assess the functional consequences, the p.Arg178Gln mutant protein was recombinantly expressed as fusion protein to GFP (green fluorescent protein) in HEK (human embryonic kidney) 293T cells, which were characterized by confocal microscopy, 59Fe export, ferritin ELISA, 125I hepcidin binding assays and flow cytometry. Results: Although normal and p.Arg178Gln mutant ferroportin localized to the plasma membrane, iron export was reduced. This finding was confirmed by ferritin ELISA. Hepcidin binding assays showed that hepcidin binds the mutant ferroportin with normal affinity but altered binding kinetics. Flow cytometry studies showed that hepcidin induces the internalization of normal and mutant ferroportin. The affected patient is a 72-year old female patient with chronic HCV infection. At presentation, the patient also had isolated hyperferritinemia and normal transferrin saturation. Magnetic resonance imaging showed markedly increased R2* frequencies in the spleen but only mild iron overload in the liver. Transient elastography of the liver by fibroscan and MRE showed no evidence of liver fibrosis. Conclusions: In conclusion, p.Arg178Gln is a loss of function ferroportin mutation, where reduced iron export causes subtle changes in hepcidin binding kinetics. This molecular disease mechanism is in accord with the clinical presentation as classical ferroportin disease, which can present as predominantly splenic iron overload, but appears to confer no additional risk for liver fibrosis. Seventh Congress of the International BioIron Society Page 318 Poster Abstracts IBIS Poster #206 HEPCIDIN IN HEALTHY AND ANEMIC PREGNANT WOMEN Valerii Demikhov, MD, Manel Ayari, Igor Mirov, MD, Prof, Elena Zinovieva, Elena Demikhova and Olga Zhurina, PhD Ryazan State Medical University Presented By: Valerii Demikhov, MD Now it is known that pathogenesis of anemia in pregnancy (AP) is multifactorial. One of them or combination of two or three iron deficiency syndromes (absolute iron deficiency, functional iron deficiency, iron supplementation) can observe in AP. That is why differential diagnosis of AP is difficult and oral iron therapy is ineffective often. Hepcidin is key regulator of iron metabolism and main factor of anemia of inflammation pathogenesis. There are data that hepcidin can predict nonresponsiveness to oral iron in non-pregnant patients with iron deficiency anemia (IDA). But hepcidin function during normal pregnancy and in AP pathogenesis little-studied. The aim of our investigation was to evaluate hepcidin values during normal pregnancy and in anemic pregnants. Total sera of 78 normal Hb level pregnant women were examined including 9 pregnants in first trimester of pregnancy, 25 – in second trimester and 44 – in third trimester. In addition sera of 10 nonpregnant women with normal Hb level were tested. In the present study, we investigated 116 anemic pregnant women too. Anemia was defined as hemoglobin less than: 10.5 g/dl – second trimester, 11.0 g/dl – first and third trimester. All anemic pregnants were divided into 2 groups on the basis of iron status: group 1 - iron deficiency anemia (IDA) – n=93 (80.2%), group 2 - anemia with normal iron status – n=23 (19.8%). Anemia verified as IDA under serum ferritin (SF) <20 ng/ml and transferrin saturation (Tsat) <17%. Hepcidin values were measured immunoenzymometrically by using commercial kits Hepcidin-25 (Human, Peninsula Laboratories, LLS, USA). SF concentrations were determined with immunochemiluminescent analysis on automated immunchemiluminescent analyzer Access using commercial kits Ferritin (Beckman Coulter, USA). Multiple linear regression was used to examine the strongest determinants of hepcidin, adjusted for gestational age of pregnancy. Mean serum hepcidin concentration in 78 normal Hb level pregnant women was significant less than in group of 10 nonpregnant women with normal Hb level: 9.2±1.89 ng/ml and 27.4±2.69 ng/ml respectively (p= .0000003). Maternal serum hepcidin concentrations were: 1st trimester – 26.4±8.78 ng/ml; 2nd trimester – 15.2±4.04 ng/ml; 3rd trimester – 2.3±0.47 ng/ml. Maternal hepcidin concentrations were significantly correlated with indicator of maternal iron status: ferritin (r = - 0.44, p = 0.00006). Mean serum hepcidin value in anemic pregnants with normal iron status (group 2) was significant higher than in IDA pregnants (group 1): 32.3±6.64 ng/ml and 3.5±1.12 ng/ml respectively (p= .00003). Hepcidin in pregnant women is lower than in nonpregnant healthy women and hepcidin levels decrease as pregnancy progresses, with the lowest hepcidin levels observed in the third trimester. Hepcidin level in nonIDA pregnants above IDA pregnant women one. That is why hepcidin test could be beneficial for differential diagnostic of iron deficiency syndromes during pregnancy. Moreover hepcidin is a regulator of iron homeostasis and may be a useful biomarker to determine iron bioavailability in pregnancy. Elevated hepcidin values may predict a resistance to oral iron therapy in anemic pregnant women and help clinicians prescribe alternative therapy appropriately (intravenously iron or erythropoiesis stimulating agents). Seventh Congress of the International BioIron Society Page 319 Poster Abstracts IBIS Poster #207 HOMOMERIC AND HETEROMERIC RECEPTOR COMPLEX FORMATION OF BONE MORPHOGENETIC TYPE I RECEPTORS ALK2 AND ALK3 Lisa Traeger¹, Inka Gallitz, PhD¹, Rohit Sekhri¹, Nicole Bäumer, PhD², Tanja Kuhlmann, MD³, Claudia Kemming³, Thomas B. Bartnikas, MD, PhD⁴ and Andrea U. Steinbicker, MD, MPH¹ ¹Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Muenster, Muenster, Germany; ²Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany; ³Institute for Neuropathology, University Hospital Muenster, Muenster, Germany; ⁴Department of Pathology and Laboratory Medicine, Brown University, Providence, USA Presented By: Lisa Traeger The bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 are predominantly expressed in the liver and required for hepcidin induction by BMP signaling. We demonstrated previously that hepatocyte-specific Alk2 deficiency leads to moderate iron overload with basal hepcidin expression and periportal iron accumulation while hepatocyte-specific deficiency of Alk3 leads to loss of basal hepcidin expression and severe iron overload with centrilobular liver iron accumulation. Whether the two receptors have redundant, synergistic or additive functions in hepcidin induction, is still under investigation. In zebrafish, orthologues of ALK2 and ALK3 were shown to assemble into a ligand dependent heteromeric receptor complex that patterns the embryonic dorsoventral axis. To elucidate their precise function in hepcidin signaling, in vitro experiments with co-immunoprecipitations of ALK2 and ALK3 in HUH7 cells and in vivo experiments in KO mice were performed. Co-immunoprecipitation analyses provided evidence for ALK3 homomeric complexes while ALK2-ALK3 complexes assembled only in the presence of BMP6. To distinguish the role of ALK2 and ALK3 in vivo, hematological and iron parameters as well as hepatic mRNA and protein levels of mice with hepatocyte- specific double deficiency for Alk2/3 compared to mice with hepatocyte-specific Alk3 deficiency and the appropriate controls were characterized. Male mice with hepatocyte-specific Alk2/3 double deficiency developed an iron overload phenotype similar to mice with hepatocyte-specific Alk3 deficiency in terms of serum iron levels, transferrin saturation and organ iron loading. In hepatocyte-specific Alk2/3 double deficient mice, iron accumulation in the liver occurred predominantly in the centrilobular area with expansion into the periportal zone. Ferroportin was expressed in both areas in the liver and determined the histological iron overload phenotype. The in vivo and in vitro results support the role of hepatic ALK3 as the dominant BMP type I receptor in hepcidin expression. ALK2 only assembles into heteromeric receptors complexes with ALK3 in the presence of BMP6 in order to induce hepcidin by iron. ALK3 also forms ligand- independent preformed homomeric receptor complexes that are essential for basal as well as IL-6 mediated hepcidin induction. Seventh Congress of the International BioIron Society Page 320 Poster Abstracts IBIS Poster #208 THE STRUCTURAL AND FUNCTIONAL RELATIONSHIPS OF TRANSFERRIN RECEPTOR 2-MEDIATED HEPCIDIN SIGNALING Mark Kleven and Caroline Enns, PhD Oregon Health & Science University Presented By: Mark Kleven, PhD Transferrin (Tf) the major serum iron-binding protein that serves as the main source of iron for most tissues. There are two plasma membrane receptors for transferrin, TfR1 and TfR2. The Tf-TfR1 complex is endocytosed and the iron is released from Tf and taken up by the cell. The main function of TfR2, however, is not in iron uptake, but in the regulation of the systemic iron through its interaction with transferrin. Through a mechanism that remains ill-defined, Tf-TfR2 induces an increase in hepcidin expression in hepatocytes. Secreted hepcidin binds to and inhibits the only cellular iron exporter, ferroportin, leading to its degradation and effectively reducing circulating iron levels. Disruption of TfR2 expression results in a rare form of hereditary hemochromatosis, a disorder characterized by severe iron overload from insufficient hepcidin expression. To better understand how TfR2 is able participate in hepcidin signaling rather than iron uptake, a structure- function examination of TfR2 was performed. Mutants of TfR2 were generated and characterized at both in vitro and in vivo levels for their ability to interact with Tf and stimulate hepcidin expression. Purified full-length TfR2 interacted with holo-Tf near physiological concentrations (KD of 4 µM). In contrast to the nanomolar affinity of TfR1 for Tf, this result indicates that Tf preferentially interacts with TfR1 and the Tf-TfR2 interaction only occurs when Tf saturation levels are high. This further complements previous studies, providing a basis for the iron-sensing role for TfR2. Additionally, in knockout mice expressing transgenic TfR2, the cytoplasmic domain, including the AP-2 internalization motif, was critical to hepcidin signaling. This complements previous studies that showed that the internalization motif is critical for Tf- responsiveness. Together, these findings suggest that differences between TfR1 and TfR2 in both their affinity for Tf and their cytoplasmic domains are root causes for their separate functions. This work contributes to our understanding of how the dynamic interactions at the hepatocyte plasma membrane provide responsiveness to systemic iron levels. Seventh Congress of the International BioIron Society Page 321 Poster Abstracts IBIS Poster #209 HAMP1 mRNA AND PLASMA HEPCIDIN LEVELS ARE INFLUENCED BY SEX AND STRAIN BUT DON’T PREDICT TISSUE IRON LEVELS IN INBRED MICE Stela McLachlan, PhD¹, Kathryn Page, PhD², Seung-Min Lee, PhD³, Erika alore, Ms⁴, Simon Hui, PhD⁴, Brie Fu ua, PhD⁵, Aldons Lusis, PhD⁴, omas Ganz, PhD⁴, Elizabeta Nemeth, PhD⁴ and Chris ulpe, MD PhD⁶ ¹University of Edinburgh; ²University of California, Berkeley; ³ onsei University, Seoul, orea; ⁴University of California at Los Angeles; ⁵University of California at Los Angeles and University of Florida; ⁶University of Florida Presented By: Brie Fuqua, BS, PhD Iron homeostasis is tightly regulated and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex and strain dependent differences in mRNA and serum levels of hepcidin. Similarly, conflicting data on the relationship between hepcidin (Hamp1) mRNA levels and iron status as assessed by transferrin saturation and tissue iron concentrations were reported. Our aim was to clarify the relationships between strain, sex and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron and plasma diferric transferrin levels were measured in fourteen inbred strains on a control iron diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes, as well as iron levels in the heart, kidneys, liver, pancreas and spleen in males, were measured in nine inbred/recombinant inbred strains raised on an iron sufficient or high iron diet. We examined the effect of strain and sex on each trait separately for a given diet. We applied the aligned rank transform to a factorial model in order to conduct nonparametric analyses of variance and followed up any significant interaction effects with a test of simple effects. Bonferroni correction was used to correct for multiple comparisons. Hierarchical multiple linear regression was employed to examine if hepcidin mRNA levels were predictive of plasma hepcidin levels or if either of them were predictive of any of the four tissue iron levels in male mice only. Vice versa, we looked at whether liver iron levels predict hepcidin mRNA and plasma hepcidin levels in both sexes. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily strain effect). Hepatic hepcidin mRNA level is a highly significant predictor of plasma hepcidin levels. However, liver iron or diferric transferrin levels are not a predictor of Hamp1 mRNA levels in iron sufficient or high iron diet, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. Figure 1: Shown are Hamp1 mRNA, liver iron (µg/g; A) and plasma diferric transferrin (%; B) levels in 14 inbred strains of mice on control diet in Study 1 and Hamp1 mRNA levels and plasma hepcidin levels in eight strains of mice (six inbred and two recombinant inbred) on control diet (C) and nine strains on high iron diet (high Fe; six inbred and three recombinant inbred; D) in Study 2. Mean values (and SD for Hamp1 mRNA levels) were calculated for a given strain and sex. Strains are ordered my increasing values of mean Hamp1 levels in females. Seventh Congress of the International BioIron Society Page 322 Poster Abstracts IBIS Poster #210 DEVELOPMENT OF A NOVEL ASSAY FOR STUDYING THE HEPCIDIN-FERROPORTIN AXIS AND ITS APPLICATION TO CHARACTERIZATION OF A NEW FERROPORTIN DISEASE-CAUSING MUTATION Nathan Subramaniam, PhD, Cameron McDonald, PhD, Lesa Ostini, BSc, Diploma, Gautam Rishi, PhD, David Iser, MD, Annabel Iser, MD and Daniel Wallace, PhD Institute of Health and Biomedical Innovation, Queensland University of Technology and QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: Daniel Wallace, PhD Objective: The hepcidin-ferroportin axis underlies the pathophysiology of many iron-associated disorders. The clinical presentation and treatment of ferroportin disease is highly dependent on the functional consequences of the causative mutation: transport-deficient or hepcidin-resistant. In this study we developed a novel antibody directed against an extracellular loop of the multi-transmembrane protein and iron exporter ferroportin. Using this antibody we developed an assay to characterize ferroportin variants and examine their sensitivity to hepcidin. This assay was applied to characterize the molecular basis of disease in a patient with iron overload. Design and Methods: Targeted next-generation sequencing utilising an iron metabolism gene panel developed in our group was used to identify variants in a patient with iron overload. Antibodies against the largest extracellular loop of ferroportin were generated and characterized by western blotting, immunofluorescence, confocal microscopy and flow cytometry. Wild-type and mutant ferroportin constructs were transfected into HEK293 cells. The effects on hepcidin- mediated internalisation and iron transport were studied using a novel flow cytometry-based assay. Results: We identified a novel ferroportin variant (p.D84E) associated with iron overload. An antibody specific for cell surface ferroportin was characterized and a novel flow cytometry-based assay developed which can measure the dynamics of hepcidin-mediated ferroportin internalization and accurately and efficiently characterize the functional consequences of ferroportin mutations. Using this assay we showed that this ferroportin variant is unable to transport iron, and is also insensitive to hepcidin-mediated internalization. Conclusions: The p.D84E mutation results in the classical form of ferroportin disease and is predicted to affect a key residue involved in ferroportin function, resulting in an inability to transport iron. Our novel ferroportin antibody and flow cytometry assay will be a valuable tool for investigating the dynamics of ferroportin trafficking, response to hepcidin or other agonists/antagonists and structure-function relationships. Seventh Congress of the International BioIron Society Page 323 Poster Abstracts IBIS Poster #211 INAPPROPRIATE EXPRESSION OF HEPCIDIN IN THE LIVER OF PROFILIN-2 KNOCK OUT MICE Sara Lucieti², Bruno Galy, PhD³, Lucia Gutierrez, PhD⁴, Michael Reinke⁵, Jorge Couso², Maya Shvartsman, PhD⁶, Antonio Di Pascale, PhD⁷, Walter Witke, PhD⁵, Matthias W. Hentze, PhD, MD⁸, Pietro Pilo Boyl, PhD⁵ and Mayka Sanchez, Dr¹ ¹Josep Carreras Leukaemia Research Institute ; ²Josep Carreras Leukaemia Research Institute , Iron Metabolism: Regulation and Disease group; ³Division of Virus-associated Carcinogenesis, German Cancer Research Centre (DKFZ), Heidelberg, Germany; ⁴Department of Biomaterials and Bioinspired Materials, Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Madrid, Spain; ⁵Institute of Genetics, University of Bonn, Bonn, Germany; ⁶European Molecular Biology Laboratory (EMBL), Monterotondo, Italy; ⁷Department of Pharmacy, University of Naples Federico II, Naples, Italy; ⁸European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Presented By: Mayka Sanchez Fernandez, PhD Introduction: The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. We are studying Profilin2 (Pfn2) as a new IRP-binding mRNA. Previously, we have discovered a functional and conserved 3’ IRE in Pfn2 mRNA and in vivo data using mice with IRP1 and IRP2 intestinal specific ablation shows that Pfn2 mRNA levels are controlled by IRPs. We show that overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces the labile iron pool and ROS. Interestingly, Pfn2-KO mice present a striking phenotype with iron accumulation in discrete areas of the brain and liver iron store depletion without anaemia. Urine iron losses were discarded. Spleen non-heme iron content was found not to be changed. We are now studying more in deep the liver and spleen of Pfn2 KO mice. Methods: Iron quantification measured by colorimetric assay and Perls Prussian staining. mRNA levels by qPCR and by expression profile array analysis using the Affymetrix GeneChip Mouse Gene 2.0 St platform in the case of liver samples. Protein levels of iron-related genes by immunoblotting. Upstream regulator analysis was done using IPA software. Results and Discussion: Liver microarray data from Pfn2 KO mice revealed very few changes in the expression profile, with 20 genes up-regulated and 28 genes down-regulated by more than 2 folds compared with WT controls, representing a 0.06% and a 0.08%, respectively, of the tested transcriptome. Among the top up-regulated genes we found Lipocalin2 (2.16 fold up-regulated; data was confirmed by qPCR with ~10 fold induction). In line with liver iron deficiency present in Pfn2 KO mice, we detected by qPCR a ~30% reduction of Bmp6 mRNA levels. L-ferritin protein levels were decreased by ~35% in Pfn2 KO mice, mirroring the reduction of iron content in this organ. By qPCR we detected that liver Slc11a2-IRE isoform mRNA (but not non-IRE isoform) was specifically up-regulated. Surprisingly, hepatic Hamp1 mRNA levels were found to be inappropriately unchanged in Pfn2 KO mice, as liver iron deficiency (and reduced Bmp6 levels) should reduce hepcidin mRNA levels in order to increase duodenal iron absorption and macrophage iron release to restore liver iron deposits. Liver expression data and upstream regulator analysis indicates that IL6 pathway is mildly activated with increase of some liver IL6 targets (Saa1, Saa3, Lcn2). Ferroportin mRNA and protein levels in spleen of Pfn2 KO mice were found to be increased. Other iron-related genes/proteins were not significantly changed. Conclusions: We hypothesize that the up-regulation of Dmt1 and Lcn2 is a liver compensatory feedback signal to re- establish the iron stores by increasing the expression of the iron and siderophore transporters, respectively. Ferroportin splenic up-regulation would indicate higher iron delivery by spleen to compensate for liver lost of iron storage capacity and to sustain hematopoiesis (mice are not anaemic). Hepatic hepcidin inappropriately normal levels could be due to a mild chronic inflammatory state since it has been shown that inflammatory signals (possibly via IL6) can up-regulate hepcidin expression, thus overtaking the down-regulatory signal excerted by low hepatic iron content and Bmp6 signalling. Funding: Work supported by grant SAF2015-70412-R (MINECO), DJCLS R14/04 from Deutsche José Carreras leukämie Stiftung, 2014 SGR225 (GRE) Generalitat de Catalunya and from Fundació Internacional Josep Carreras and Obra Social “la Caixa” Spain to M.S. All work on the Pfn2 knockout mouse model was supported by the Deutsche Forschungsgemeinschaft (DFG) SFB1089 grant. Seventh Congress of the International BioIron Society Page 324 Late-Breaking Abstracts IBIS Poster #LB1 MORE HARM THAN HELP: DOES EARLY-LIFE IRON EXPOSURE INCREASE THE RISK OF AGE-RELATED NEURODEGENERATION? Dominic Hare, PhD The Florey Institute of Neuroscience and Mental Health Presented By: Dominic Hare, PhD Iron is an essential element required at every stage of life, particularly during development. Iron deficiency during the post-natal period can have severe implications for neurodevelopment. During the early 1900s, iron deficiency and iron deficiency anemia were endemic, and in response peak medical advisory bodies advocated for high levels of iron supplementation. Though effective in reducing prevalence of deficiency, recent studies have suggested that long-term negative health outcomes may arise from early-life iron overload. A major prospective public health concern is that arising from the contribution of early-life iron exposure to increased retention of iron in the ageing brain. Iron accumulation is a common feature to several neurodegenerative diseases, most notably Parkinson’s disease. Combined with deficits to iron metabolic processes, an exacerbation of normal iron accumulation with age may initiate biochemical reactions that precede neuron death. Central to testing this hypothesis is the identification of novel biomarkers of early-life iron exposure, combined with contemporary technologies used to assess brain iron levels in vivo. Identifying individuals at risk from elevated brain iron will provide new avenues to intervene and prevent aberrant reactions of iron, and potentially mitigate neurodegeneration before clinical symptoms appear. Figure 1: Age-adjusted mortality rates for Parkinson’s disease (PD) in males and females from selected high-income countries. All data were obtained from the WHO mortality database (http://www.who.int/healthinfo/mortality_data/en/) and were age-adjusted to the year 2000 population for each country. Increasing PD mortality is most marked in the UK and USA approximately 50-65 years following initiation of mandatory iron fortification of grains and milled flour (1980-1995). Japan, with no policy on iron fortification, shows relatively stable mortality rates. PD as the cause of death was defined per the ICD10 code G20 or ICD9 code 332 (both PD). Figures from the UK for 1984-1992 were corrected for dual listing of PD as a chronic condition and a cause of death reported by the Office of Population Censuses and Surveys to the WHO, as suggested by Clarke1. Although WHO mortality data extend only to 1979, Clarke showed that age-adjusted PD mortality in the UK was relatively static in the decades preceding 1980 (†; dashed blue line)1. Pre-1979 mortality data for the US (‡) was obtained from Hinz et al.2 and Lilienfield et al.3 Note that these figures are estimates only and are not age-adjusted to the population at 2000. Mortality was used in place of prevalence or incidence rates to remove confounding effects of improving diagnosis or increasing disease duration though medical intervention, such as the introduction of L-DOPA treatment in the early 1980s 1. C. E. Clarke, J. Neurol. Neurosurg. Psychiatry, 1993, 56, 690-693. 2. M. Hinz, A. Stein and T. Cole, Clin. Pharmacol, 2014, 6, 161-169. 3. D. E. Lilienfeld, E. Chan, J. Ehland, J. Godbold, P. J. Landrigan, G. Marsh and D. P. Perl, Arch. Neurol., 1990, 47, 731-734. Seventh Congress of the International BioIron Society Page 325 Late-Breaking Abstracts IBIS Poster #LB2 NANOSTRUCTURED IRON COMPOUNDS FOR NUTRITION – IN VITRO AND IN VIVO ASSESSMENT OF SAFETY AND ABSORPTIVE PATHWAYS Hans Christian Winkler, DVM, PhD, Lea M. von Moos, PhD, Julian Kornprobst, MSc, Mirjam Schneider, Florentine M. Hilty, PhD, Monika Hilbe, DVM, Myrtha Arnold, Nathalie Ziegler, Diogo Sales Mato, Elisabeth M. Schraner, Peter Wick, PhD, Barbara Bathke, Shana J. Sturla, PhD, Ioannis A. Trantakis, PhD, Hubertus Hochrein, PhD, Wolfgang Langhans, DVM, Mark Suter, DVM, Hanspeter Naegeli, DVM and Michael B. Zimmermann, MD, MSc Swiss Federal Institute of Technology ETH, Laboratory of Human Nutrition Presented By: Hans Winkler Iron-deficiency anemia (IDA) is a major contributor to the global burden of disease and food fortification can be an effective strategy to prevent IDA. Effective iron fortification of foods is difficult, because water-soluble compounds that are well absorbed, such as ferrous sulfate (FeSO4), often cause unacceptable changes in the color or taste of foods. Poorly water-soluble compounds, on the other hand, cause fewer sensory changes, but are not well absorbed. Poorly water soluble nanostructured Fe compounds (specific surface area 190 m2/g) made by flame aerosol technology have in vivo iron bioavailability in rats comparable to FeSO4 and cause less color change in reactive food matrices than conventional iron fortificants. However, the safety of these promising compounds∼ is uncertain: their high surface area could potentially also increase their immunogenicity and alter their absorption pathways in the gastrointestinal tract. We studied the effects of nanoscale iron compounds on human epithelial cell lines, dendritic cells generated from mouse bone marrow cultures and in a 90-day feeding study in rats. The endpoints analyzed include cellular uptake demonstrated by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX), cell viability, oxidative stress, secretion of pro-inflammatory cytokines (interleukin (IL)-1α, I -1 and tumor necrosis factor α determined by immunoassays, dendritic cell maturation assessed by changes in the surface markers cluster or differentiation (CD)40, CD62L, CD69, CD86 analyzed by flow cytometry, organ damage, excess iron accumulation in organs, or histological changes. To corroborate these data, iron bioavailability for hemoglobin synthesis is assessed in intestinal divalent metal transporter 1 knockout (DMT1int/int) mice fed nanostructured Fe compounds. Progeny from floxed DMT1 mice carrying a Villin-Cre transgene selectively lacks duodenal DMT1. Since DMT1 is necessary for intestinal Fe2+ absorption, its loss is utilized to determine iron uptake by DMT1-independent absorptive pathways. Similar to nanostructured silica (E 551), a commonly employed food-additive used as a comparator, nanostructured FePO4 treatment resulted in cellular uptake in vitro without changes in cell viability or oxidative stress. In contrast, cytokine secretion and dendritic cell maturation were not affected by nanostructured FePO4. Iron bioavailability for hemoglobin synthesis data is currently being completed and a full description will be reported at the meeting. Feeding rats with diets containing nanostructured FePO4 at doses at which FeSO4 has been shown to induce adverse effects, did not result in organ damage, excess iron accumulation in organs, or histological changes. Our findings suggest that nanostructured FePO4 appears to be as safe for human consumption as FeSO4. Seventh Congress of the International BioIron Society Page 326 Late-Breaking Abstracts IBIS Poster #LB3 WITHDRAWN Poster #LB4 EFFECT OF A NOVEL ORALLY ACTIVE IRON CHELATOR: 1-(N-ACETYL-6-AMINOHEXYL)-3-HYDROXY-2- METHYLPYRIN-4-ONE (CM1) IN IRON-OVERLOADED AND NON-OVERLOADED MICE Somdet Srichairatanakool, PhD¹, Nittaya Chansiw, PhD², Kanjana Pangjit, PhD³, Chada Phisalaphong, PhD⁴, John Porter, FRCP, FRCPath, MD⁵, Patricia vans, PhD⁵, Robert Hider, PhD⁶ and Suthat Fucharoen, MD⁷ ¹Chiang Mai University; ²Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Thailand; ³College of Medicine and Public Health, Ubon Ratchathani University, Thailand; ⁴Government Pharmaceutical rgani ation, Thailand; ⁵Department of Haematology, University College London, United Kingdom; ⁶Institute of Pharmaceutical Science, King s College London, United Kingdom; ⁷Institute of Molecular Bioscience, Mahidol University, Thailand Presented By: Somdet Srichairatanakool, PhD Iron is a trace element essential for many biochemical functions. Iron chelators are required for b-thalassemia patients with iron overload to get negative iron balance; nonetheless, over-dose administration of the drugs may deplete functional iron and some trace elements in the body, possibly resulting in organ malfunctions and symptoms. Our orally active bidentate iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) have shown nontoxic and effective in treatment of b-thalassemic mice with iron overload. This study aimed to evaluate efficacy and toxicity of the CM1 in mice under normal and iron overload conditions. Wild type C57BL/6 mice were fed with normal (N) and 0.2% (w/w) ferrocene-supplemented (Fe) diets, respectively for 240 days and orally given the CM1 for 180 days. CM1 treatment lowered plasma levels of labile plasma iron and non-transferrin bound iron, but not ferritin in the Fe-fed mice. However, the treatment did not impact blood hemoglobin level, white blood cell and platelet numbers in both N diet and Fe diet-fed mice. Interestingly, CM1 treatment did not markedly elevate plasma aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities in the N diet-fed mice but it tended to increase the levels of the liver enzymes slightly in the Fe-fed mice. Hematoxylin and eosin staining result showed no abnormal pathological changes in heart, liver and spleen tissues. It is clear that CM1 would not be toxic to bone marrow and liver cells under normal and iron-overload conditions. Key words: Iron chelation, Iron overload, Thalassemia, Toxicity, Liver enzyme Seventh Congress of the International BioIron Society Page 327 Late-Breaking Abstracts IBIS Poster #LB5 ROLE OF EFEUOB IN IRON ACQUISITION AND PATHOGENICITY OF ESCHERICHIA COLI O157:H7 Arvindkumar Salunkhe and Simon Andrews University of Reading, UK Presented By: Arvindkumar Salunkhe Iron acquisition is important for pathogens since iron withdrawal is key for innate immune defense. Escherichia coli O157:H7 is a potentially lethal intestinal pathogen. It possesses a ferrous transporter, EfeUOB, that functions at low pH aerobically; this system is cryptic in some E. coli strains (e.g. K-12). EfeUOB of E. coli O157:H7 represent the ideal target for further investigation of the role of this type of iron transport system in bacterial pathogenicity and survival. efeUOB and feoABC (anaerobic ferrous-iron uptake) single and double mutants of E. coli O157:H7 were constructed. Growth studies showed that iron restriction reduces growth of the feo mutant under anaerobic and aerobic conditions. At low pH, with competing divalent metals and reducing agents, Mn2+ markedly inhibited growth of the efe mutant, although other metals failed to do so. However, although the feo mutant was unaffected by Mn2+, the efe feo double mutant exhibited an enhanced iron-limited growth defect with respect to the efe mutant. In addition, Ni2+, Zn2+, Co2+and Mo inhibited growth of the feo mutant under iron restriction but not the efe mutant. The poor growth of the efe mutant under low iron, reducing and acidic conditions with Mn2+ was found to be dependent on the presence of hydrogen peroxide; various catalases caused reversal of the low iron growth advantage provided by the EfeUOB system. The effect on the FeoABC system was opposite, with catalase enhancing the ability of the Feo system to improve growth under aerobic iron restriction conditions. The results provided indicate that EfeUOB plays a major role in Fe acquisition for E. coli O157:H7 under aerobic, low-pH, low iron conditions in the presence of Mn2+. FeoABC has no apparent role under these conditions but partially compensates for lack of EfeUOB. The above experiments thus suggest that EfeUOB is the major Fe transporter employed at low pH when Mn2+ is a competing metal; and suggest a clear rationale for the presence of two dedicated ferrous iron uptake systems in E. coli O157:H7, with EfeUOB operational when peroxide is available (i.e. aerobically) at low pH (favouring ferrous iron stability) and FeoABC operational under low oxygen conditions when peroxide is not likely to be generated (again favouring ferrous iron stability). We used the Galleria mellonella as an infection model and killing assays were performed to test efe and feo mutations impact on E. coli O157:H7 pathogenicity. Kaplan–Meier survival curves showed significantly reduced pathogenicity for the feo mutant while the efe and efe feo mutation did not have any clear effect. This result indicates the role for feo in E. coli O157:H7 pathogenicity. With survival in macrophage studies, the feo and efe feo double mutant showed statistically significant reductions in survival; survival was reduced for the efe mutant but this was not significant. Thus, so far, no role for EfeUOB in pathogenicity of E. coli O157 has been established. Seventh Congress of the International BioIron Society Page 328 Late-Breaking Abstracts IBIS Poster #LB6 IRON INDUCES INSULIN RESISTANCE IN CARDIOMYOCYTES VIA REGULATION OF OXIDATIVE STRESS Hyekyoung Sung and Gary Sweeney, PhD York University Presented By: Hyekyoung Sung Inadequate supply of iron can elicit health defects yet there is also strong evidence that too much iron induces detrimental cellular effects. Accordingly, disturbed iron homeostasis is associated with various pathological conditions, and here we focus on diabetes and heart failure. We contend that disturbed iron homeostasis is an important and underappreciated contributor to development of heart failure. In primary adult and neonatal cardiomyocytes as well as H9c2 cells, derived from rat heart ventricle, iron Fe 4, 1 /ml induced insulin resistance as determined by estern blotting and immunofluorescent detection of Akt phosphorylation. Using CellROX Deep Red assay we also observed that iron increased generation of reactive oxygen species (ROS), and that using anti-oxidants to attenuate ROS production reduced iron-induced insulin resistance. Various complementary assays indicated that iron also suppressed autophagy. To study the functional significance of changes in autophagy we created an autophagy-deficient cell model by overexpressing a dominant-negative Atg5 mutant. Iron or autophagy deficiency both induced insulin resistance. We translated these studies to an animal model of iron overload and preliminary data has indicated development of insulin resistance in iron overload mice. In conclusion, our study indicated that iron induced insulin resistance in cardiomyocytes and this involved regulation of the crosstalk between autophagy and oxidative stress. Further studies will investigate mechanisms via which iron regulates cardiac remodeling and their physiological significance. We anticipate that our findings will provide new knowledge relevant to current diagnostics and therapeutics related to altered iron status in clinical settings. Seventh Congress of the International BioIron Society Page 329 Late-Breaking Abstracts IBIS Poster #LB7 MITOCHONDRIAL FERRITIN DELETION EXACERBATES ß-AMYLOID-INDUCED NEUROTOXICITY IN MICE Peina Wang, Qiong Wu, Wenyue Wu, Haiyan Li, Yuetong Guo, Peng Yu, Guofen Gao, Zhenhua Shi, Baolu Zhao and Yan-Zhong Chang Hebei Normal University Presented By: Peina Wang Mitochondrial ferritin (FtMt) is a mitochondrial iron-storage protein which protects mitochondria from iron-induced oxidative damage. ur previous studies indicate that Ft t attenuates -amyloid- and 6-hydroxydopamine-induced neurotoxicity in SH- cells. o explore the protective effects of Ft t on -amyloid-induced memory impairment and neuronal apoptosis, and the mechanisms involved, 10-month-old wild-type and Ftmt knockout mice were infused intracerebroventricularly IC with -35 to establish an Alzheimer’s disease model. Knockout of Ftmt significantly exacerbated -35-induced learning and memory impairment. The Bcl-2/Bax ratio in mouse hippocampi was decreased and the levels of cleaved caspase3 and PARP were increased. The number of neuronal cells undergoing apoptosis in the hippocampus was also increased in Ftmt knockout mice. In addition, the levels of L-ferritin and FPN1 in the hippocampus were raised, and the expression of TfR1 was decreased. Increased MDA levels were also detected in Ftmt noc out mice treated with -35. In conclusion, this study demonstrated that the neurological impairment induced by -35 was exacerbated in Ftmt knockout mice, and that this may relate to increased levels of oxidative stress. Acknowledgments: This work was supported by the National Natural Science Foundation of China (grant numbers 31520103908, 31471035) Seventh Congress of the International BioIron Society Page 330 Late-Breaking Abstracts IBIS Poster #LB8 THE PROTECTIVE ROLE OF MITOCHONDRIAL FERRITIN ON ERASTIN-INDUCED FERROPTOSIS Yueqi Wang, Shiyang Chang, Qiong Wu, Yujing Gou, Linpei Jia, Yanmei Cui, Peng Yu, Zhenhua Shi, Wenshuang Wu, Guofen Gao and Yan-Zhong Chang Hebei Normal University Presented By: Shiyang Chang Ferroptosis, a newly identified form of regulated cell death, is characterized by overwhelming iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). Preventing cellular iron overload by reducing iron uptake and increasing iron storage may contribute to inhibit ferroptosis. Mitochondrial ferritin (FtMt) is an iron-storage protein that is located in the mitochondria, which has a significant role in modulating cellular iron metabolism. Recent studies showed that FtMt played inhibitory effects on oxidative stress-dependent neuronal cell damage. However, the potential role of FtMt in the progress of ferroptosis in neuronal cells has not been studied. To explore this, we established ferroptosis models of cell and drosophila by erastin treatment. We found that overexpression of FtMt in neuroblastoma SH-SY5Y cells significantly inhibited erastin-induced ferroptosis, which very likely was achieved by regulation of iron homeostasis. Upon erastin treatment, significant increases of cellular labile iron pool (LIP) and cytosolic ROS were observed in wild-type SH- SY5Y cells, but not in the FtMt-overexpressed cells. Consistent with that, the alterations of iron-related proteins in FtMt- overexpressed cells were different from that of the control cells. We further investigated the role of FtMt in erastin-induced ferroptosis in transgenic drosophila. We found that the wild-type drosophilas fed an erastin-containing diet didn't survive more than 3 weeks. In contrast, the FtMt overexpressing drosophilas fed the same diet were survival very well. These results indicated that FtMt played a protective role in erastin-induced ferroptosis. Acknowledgments: This work was supported by the National Natural Science Foundation of China (grant numbers 31520103908, 31471035) Seventh Congress of the International BioIron Society Page 331 Late-Breaking Abstracts IBIS Poster #LB9 CHARACTERIZATION OF MITOCHONDRIAL FERRITIN-OVEREXPRESSION MICE Peina Wang, Xin Li, Shiyang Chang, Weina Kong and Yan-Zhong Chang Hebei Normal University Presented By: Peina Wang Mitochondrial ferritin (FtMt) is a H-ferritin-like protein which localizes in the mitochondrial. More and more studies have shown that this protein can protect mitochondrial from iron-induced oxidative damage and it’s overexpression in cultured cells protected against oxidative damage and reduced cytosolic iron availability. To investigate the in vivo role of FtMt , we established Ftmt overexpression mouse by using pronucleus microinjection and examined the characterization of this mouse. We confirmed Ftmt overexpression using PCR and primers specific to the transgene regions. To measure Ftmt expression, we used reverse transcriptasepolymerase chain reaction (RT-PCR) and control reactions without reverse transcription were performed to ensure that PCR products did not represent amplification of genomic DNA. Our data show that there were no significant differences between wild type and transgene mice on body weight and organ coefficient. To determine the effect of Ftmt overexpression on baseline murine iron metabolism and hematology, we measured serum, spleen, heart, kidney, testis, liver and brain iron concentrations, liver hepcidin expression and red blood cell parameters. There were no significant differences between wild type and transgene mice. In conclusion, these results suggest that Ftmt overexpression mice didn’t have over phenotype and we predict the effect of Ftmt overexpression can be represented under stress conditions. This work was supported by the National Natural Science Foundation of China (grant numbers 31520103908, 31471035) and the National Natural Science Foundation of Hebei Province (grant number C2016418006) Seventh Congress of the International BioIron Society Page 332 Late-Breaking Abstracts IBIS Poster #LB10 MITOCHONDRIAL FERRITIN PROTECTS HYDROGEN PEROXIDE-INDUCED NEURONAL CELL DAMAGE Guofen Gao, Nan Zhang, Yueqi Wang, Zhen Li, Peng Yu, Zhenhua Shi, Baolu Zhao, Wenshuang Wu, Yan-Zhong Chang and Shiyang Chang Hebei Normal University Presented By: Shiyang Chang Oxidative stress and iron accumulation are tightly associated with neurodegenerative diseases. Mitochondrial ferritin (FtMt) is identified as an iron-storage protein that is located in the mitochondria, and its role in regulation of iron hemostasis in neurodegenerative diseases has been reported. However, the role of FtMt in hydrogen peroxide (H2O2) induced oxidative stress and iron accumulation in neuronal cells has not been studied. Here, we overexpressed FtMt in neuroblastoma SH-SY5Y cells, and induced oxidative stress by treating with extracellular H2O2. We found that overexpression of FtMt significantly prevented cell death induced by H2O2, particularly the apoptosis-dependent cell death. The protection effects involved inhibiting the generation of cellular reactive oxygen species, sustaining mitochondrial membrane potential, and maintaining the level of anti-apoptotic protein Bcl-2 and inhibiting the activation of pro-apoptotic protein caspase3. We further explored the mechanism of these protection effects, and found that FtMt expression markedly alters iron homeostasis of the H2O2 treated cells as compared to that of controls. The FtMt overexpression significantly reduces cellular labile iron pool (LIP) and protects H2O2-induced elevation on LIP. While in H2O2 treated SH-SY5Y cells, the increased iron uptake and reduced iron release, in correlation with higher DMT1(-IRE) and lower ferroportin 1, resulted in heavy iron accumulation, the FtMt protected cells didn’t show any significant changes in levels of iron transport proteins, and in the level of LIP. These results implicate a neuroprotective role of FtMt on H2O2- induced oxidative stress, and may provide insights into the treatment of iron accumulation associated neurodegenerative diseases. This work was supported by the National Natural Science Foundation of China (grant numbers 31520103908, 31371073) and the National Natural Science Foundation of Hebei Province (grant number C2016418006) Seventh Congress of the International BioIron Society Page 333 Late-Breaking Abstracts IBIS Poster #LB11 WITHDRAWN Poster #LB12 FERRIC CARBOXYMALTOSE VERSUS IRON SUCROSE COMPLEX IN WOMEN WITH IRON DEFICIENCY ANEMIA – A RANDOMISED CONTROLLED TRIAL Garima Chaudhry Obstetrics & Gynaecology Presented By: Garima Chaudhry Introduction: To compare safety and efficacy of Ferric Carboxymaltose (FCM) with Iron Sucrose complex (ISC) regarding improvement in haematological parameters and adverse effects in women with iron deficiency anemia (IDA). Methods: Prospective randomized controlled study conducted in department of Obstetrics & Gynecology, in a tertiary care hospital in Delhi. 60 women having Iron deficiency Anaemia with Hb 6-8 g% were randomized 1:1 into two groups and were given 1000mg parenteral iron. One group received intravenous 500mg FCM on day 0 and 8. 200mg Iron Sucrose complex was given in second group on alternate days for 5 doses. Haematological parameters - Hb. Reticulocyte count, RBC indices, S. ferritin; clinical parameters - fatigue, dyspnoea on exertion and adverse effects were studied on day 0, 7, 14 & 28. Results: wo FC infusions vs five I C infusions were re uired. n day b increment g seen in , and MCV>80FL seen in 100% of FCM group vs. 0% and 43.33% in ISC group. FCM group had 3.17 g/dl increment in Hb vs. 1.9 g/dl in ISC group. S. Ferritin increased to 147ng/ml in FCM group vs. 98 ng/ml in ISC group. Significant improvement in RBC indices & rectic count was seen in FCM group. Earlier and significant improvement in fatigability & dyspnoea on exertion was observed in FCM group. Both groups had similar safety profile except for thrombophlebitis was observed in 6.67% FCM group vs. 50.00% ISC group. Discussion: The serum ferritin showed a pattern of peak rise followed by gradual fall thus reflecting iron utilization in terms of increase in haemoglobin, normalization of red cell indices and increase in reticulocyte count. At the end of the study an increment in haemoglobin of >3g/dl was achieved by 64% of women and normalization of MCV (>80fl) was attained by 100% women. At the end of the study an increment in haemoglobin of >3g/dl was achieved by 64% of women and normalization of MCV (>80fl) was attained by 100% women in FCM group. Conclusions: Intravenous Ferric Carboxymaltose is more effective and safer than Iron Sucrose complex in treatment of Iron deficiency anaemia. Seventh Congress of the International BioIron Society Page 334 Late-Breaking Abstracts IBIS Poster #LB13 INTRA- AND INTER-MOLECULAR SIGNAL TRANSDUCTION MECHANISMS OF HEME SENSING SYSTEMS Hitomi Sawai, PhD¹, Gareth S. A. Wright, PhD², Akane Saeki, MSc¹, Takaaki Hikima, PhD³, Masaki Yamamoto, PhD³, Svetlana Antonyuk, PhD², S. Samar Hasnain, PhD² and Yoshitsugu Shiro, PhD¹,³ ¹Graduate School of Life Science, University of Hyogo; ²Institute of Integrative Biology, University of Liverpool; ³RIKEN SPring-8 Center Presented By: Hitomi Sawai, PhD Heme-regulated transporter HrtAB is required for survival in toxic heme concentrations in pathogenic bacteria and heme auxotrophs. HrtA is an ATPase that drives heme efflux by HrtB permease. Expression of the heme efflux pump HrtAB is regulated by two types of heme sensing systems: one is a heme-sensing transcriptional regulator HrtR, and another is a two component regulatory system (TCS) proteins, a histidine kinase HssS and a response regulator HssR, especially in Gram-positive pathogens. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates hrtAB gene expression for the heme efflux system, we previously determined crystal structures of heme-bound, heme- unbound, and DNA-bound HrtR at the resolutions of 1.9, 3.1, and 2.0 Å, respectively [H. Sawai, et al. (2012) J. Biol. Chem. 287, 30755-30768.]. These structures and biochemical studies revealed that HrtR adopts a unique mechanism for heme sensing by a coil-to-helix transition. To further understanding of the other heme-responsive TCS, we recently established the Asian first size-exclusion chromatography integrated small-angle X-ray scattering (SEC-SAXS) in SPring- 8. To investigate the molecular dynamics of the heme-responsive TCS in solution states, we have studied on the TCS structures by a combinational technique of SEC-SAXS, crystallography, in silico modelling. Key questions of TCS signaling are how sensor histidine kinases are activated by stimuli (heme, O2, nutrient, etc.) and how they interact with their specific response regulators for the transcriptional regulation. Answers to these question have been hampered by a lack of structural information on full-length TCS proteins and their complexes at molecular and atomic levels. To overcome this issue, we used rhizobial heme-based O2 sensing system FixL/FixJ as a complete model of TCS proteins. The full- length FixL structures in the active and inactive forms suggest that intra-molecular signal transduction upon O2 sensing is driven by a series of localized structural rearrangement. O2 dissociation from the heme iron induces twisting of the helical structures to activate the signal transduction. To detect a transient structure of FixL-FixJ complex in solution state, SEC- SAXS data collection was performed with FixJ-saturated buffer. The data indicated homodimeric FixL binds two FixJ molecules in solution to form a heterotetramer that facilitates signal transfer between FixL and FixJ (Fig. 1). These structures provide a mechanistic understanding of TCS signal relay, and our technique can be applied to reveal the molecular mechanisms of heme-responsive TCS proteins with/without heme in solution. Seventh Congress of the International BioIron Society Page 335 Late-Breaking Abstracts IBIS Poster #LB14 STRUCTURAL INSIGHTS INTO ASCORBATE-DEPENDENT FERRIREDUCTASE, DCYTB, IN HUMAN Menega Ganasen¹, Hitomi Sawai, PhD², Hiromi Togashi, PhD³, Hanae Takeda, MSc³, Yoshitsugu Shiro, PhD²,³ and Hiroshi Sugimoto, PhD³ ¹Graduate School of Life Science, University of Hyogo; ²Grad. Sch. of Life Sci., Univ. Hyogo; ³RIKEN SPring-8 Center Presented By: Menega Ganasen, MS Iron overload and deficiency are the most pervasive micronutritional disorder worldwide. A tight control over systemic and cellular iron balance is required to overcome this issue. Systemic iron levels are controlled by dietary iron absorption in duodenal enterocyte. Dietary iron comes in two main forms, heme iron (iron-protoporphyrin IX) from meat and non-heme iron from vegetables. Heme iron has high bioavailability, while non-heme iron primarily exists in water insoluble and poorly absorbed ferric (Fe3+) form. Hence, reduction of Fe3+ to soluble divalent ferrous iron (Fe2+) is prerequisite for succesful iron uptake by divalent metal transporter-1 (DMT-1). A membrane-intergrated ascorbate-dependent ferrireductase, known as duodenal cytochrome b (Dcytb), is highly expressed in the intestinal brush border membrane, and reduces Fe3+ at duodenal surface prior to uptake by DMT-1 (Fig. 1). Fe3+ is the only iron source in vegetarian meal. Therefore, Fe3+ reduction is critical for sufficient iron absorption in vegetarian diets. We have interest in understanding how Fe3+ reduction is efficiently regulated by Dcytb. We used x-ray crystallography and biochemical approaches to study the Fe3+ reduction mechanism of human Dcytb. We determined the crystal structures of human Dcytb in its apo form and Asc bound form, at 2.6 and 2.8 Å resolution, respectively. As predicted, Asc is bound on the cytoplasmic surface of Dcytb, contacting with the heme methyl group and three positively charged residues, K79, K83 and R152. This binding coincides with Asc’s role as the major electron donor for the reduction reaction. The importance of these three residues in Asc binding was further investigated by stopped flow measurement using several single mutants. All of these single mutations showed some effects on the reaction rate and affinity of Asc, but the effect was not critical. This suggest that electron transfer from Asc involves a tunnelling to heme edge rather than along then side chain of one of these amino acid residues. Another electron density was observed on the luminal side of Dcytb. This site corresponds to the second Asc-binding position. Dcytb belong to cytochrome b561 (Cytb561) family. Cytb561 is an oxidoreductase, that transfer electrons to ferric-chelate for ferric reduction or to monodehydroascorbate (MDA) for Asc recycling. Asc binding on the luminal side is consistent with the multiple role of Cytb561 family members. It also highlights the possible role of Asc as Fe3+ chelator in duodenal lumen. The present anaysis provides an insight into the long-standing nutrition question of how ascorbate in vegetable meal enhances the iron absorption in our body. Seventh Congress of the International BioIron Society Page 336 Late-Breaking Abstracts IBIS Poster #LB15 NASAL DELIVERY OF NANOLIPOSOME-ENCAPSULATED FERRIC AMMONIUM CITRATE CAN INCREASE THE IRON CONTENT OF RAT BRAIN Xueling Guo, MS², Hong Zheng, MS³, Yuetong Guo, MS², Yan Wang, MS², Gregory J. Anderson, PhD⁴, Yun he Ci, MS², Peng Yu, PhD¹, Lina Geng, PhD³ and Yanzhong Chang, PhD² ¹Laboratory of Molecular Iron Metabolism, Key laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University; ²College of Life Sciences, Hebei Normal University; ³College of Chemistry and Material Science, Hebei Normal University; ⁴Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute Presented By: Peng Yu, PhD Background: Iron deficiency in children can have significant neurological consequences, and iron supplementation is an effective treatment of choice. However, traditional routes of iron supplementation do not allow efficient iron delivery to the brain due to the presence of the blood-brain barrier. So an easily delivered iron formulation with high absorption efficiency potentially could find widespread application in iron deficient infants. Results: In this study, we have developed and characterized a nanovesicular formulation of ferric ammonium citrate (ferric ammonium citrate nanoliposomes, FAC-LIP) and have shown that it can increase brain iron levels in rats following nasal administration. FAC was incorporated into liposomes with high efficiency (97%) and the liposomes were small (40nm) and stable. Following intranasal delivery in rats, FAC-LIP significantly increased the iron content in the olfactory bulb, cerebral cortex, striatum, cerebellum and hippocampus, and was more efficient at doing so than FAC alone. No signs of apoptosis or abnormal cell morphology were observed in the brain following FAC-LIP administration, and there were no significant changes in the levels of SOD and MDA, except in the cerebellum and hippocampus. No obvious morphological changes were observed in lung epithelial cells or tracheal mucosa after nasal delivery, suggesting that the formulation was not overtly toxic. Conclusions: In this study, nanoscale FAC-LIP proved an effective system delivering iron to the brain, with high encapsulation efficiency and low toxicity in rats. Our studies provide the foundation for more detailed investigations into the applications of niosomal nasal delivery of liposomal formulations of iron as a simple and safe therapy for iron deficiency anemia. Key words: nasal delivery – FAC – nanoliposomes – ICP-MS – micro-X-ray fluorescence Seventh Congress of the International BioIron Society Page 337 Late-Breaking Abstracts IBIS Poster #LB16 FERRITIN IN THE CEREBROSPINAL FLUID PREDICTS RATE OF AMYLOID BETA ACCUMULATION IN BIOMARKER-CONFIRMED ALZHEIMER’S DISEASE Scott Ayton, PhD¹, Ibrahima Diouf, PhD² and Ashley Bush, MB BS, PhD³,⁴,⁵ ¹Florey Institute of Neuroscience and Mental Health; ²CSIRO Health and Biosecurity/Australian E-Health Research Centre, Brisbane, Australia; ³The Florey Institute for Neuroscience and Mental Health; ⁴The University of Melbourne, Australia; ⁵Cooperative Research Center for Mental Health, Victoria, Australia Presented By: Scott Ayton, PhD Introduction: In the natural history of l heimer s disease, -amyloid accumulation in cortical pla ue, detected either by imaging or measuring falling C F 1-42 levels in CSF, progresses in a decades-long prodromal period. Once amyloid pathology has begun, there is considerable variability on its rate of progression between individuals, and the factors that impact on this rate are uncertain. a or genetic factors, such as familial l heimer s mutations, or the 4 isoform of the ma or genetic ris for sporadic l heimer s disease , cause amyloid to commence accumulation at an earlier age, but they have little impact on the rate of accumulation. Brain iron has been implicated in plaque pathogenesis iron is enriched in pla ue pathology iron increases the aggregation of in vitro, and promotes deposition of in mouse models. here has not yet been a prospective clinical study of the impact of iron on deposition. We recently showed that elevated CSF ferritin, a reporter of brain iron load, was associated with worse cognitive performance over a 7-year period (controlling for inflammation and blood leakage), and with earlier onset of dementia (Nat Comms, 2015, JAMA Neurology 2017). We investigated whether CSF ferritin, was associated with changes in the C F biomar ers, -amyloid and tau, over 5-years in participants along the l heimer s disease spectrum in the l heimer s Disease Neuroimaging Infinitive DNI cohort. Results: In subjects with biomarker-confirmed l heimer s pathology C F <192 ng/ml), high CSF ferritin was associated with accelerated depreciation of C F 1-4 levels reporting increased -plaque) over 5-years (P=0.007; Fig 1A). CSF ferritin was neither associated with changes in CSF tau in the same subjects, nor changes in CSF tau or 1-42 levels in subjects with low baseline pathology. Using the calculated rates of change in our modelling, we estimated that pla ue accumulates over a -year period, consistent with prior estimates. High CSF ferritin levels once the amyloid pathology is manifest was associated with accelerated C F 1-42 decline, shortening the time taken to reach mean levels of C F 1-4 in l heimer s disease from 1 . years to 1 .1 years Fig 1 . Discussion: levated brain iron levels in l heimer s disease might facilitate the deposition of and accelerate the disease process. Iron chelators, such as permeable deferiprone, may therefore slow l heimer s disease progression – which we will investigate in a Phase IIb clinical trial beginning in 2017. Seventh Congress of the International BioIron Society Page 338 Late-Breaking Abstracts IBIS Poster #LB17 HAEMOCHROMATOSIS AUSTRALIA - A POTENTIAL RESEARCH PARTNER? Dianne Prince Haemochromatosis, Australia Presented By: Dianne Prince Haemochromatosis Australia (HA) is the peak body in Australia supporting people with hereditary haemochromatosis This poster presents a number of case vignettes outlining different pre and post diagnosis experiences of Australian individuals with the condition, with the intent of providing insights for researchers on possible future research directions. It also summarises the work Haemochromatosis Australia undertakes to ensure no Australian suffers harm from hereditary haemochromatosis, highlighting key activities over recent years and providing a brief snapshot of factors impacting its performance. For more than 25 years HA has worked with researchers to increase the body of knowledge about haemochromatosis and its impact on the health and well-being of people with Haemochromatosis, and with healthcare professionals to ensure early diagnosis and access to appropriate treatment. HA has built, and continues to build, connections across the research community, the medical community, allied health practitioners, pathology providers and other agencies including similar groups in the nonprofit sector. HA also works to raise awareness of Haemochromatosis in the general community and with the Australian Department of Health to ensure access to testing and treatment, and a number of resources have been developed to support this work. These include videos and booklets for use with patients and the general public, accredited training modules for nurses and general practitioners, and HealthPathways to assist in the diagnosis of Haemochromatosis and referrals for patients to appropriate care, HA is a completely volunteer run organization. Its managing committee is elected and endorsed by members at the Annual General meeting each year. HA’s patron, Margaret Rankin AM, RN, GradDip Health Sciences, founded the group in 1990. Margaret's contribution to haemochromatosis support was recognised with a Member of the Order of Australia (AM) medal in 2005. The group of eminent medical and scientific advisors providing guidance and advice to HA, includes: Dr Katie Goot, MBBS BSc FACRRM Professor Lawrie Powell AC, Emeritus Professor, UQ Centre for Clinical Research, University of Queensland, Brisbane, QLD Professor John Olynyk, Director of Gastroenterology, Fremantle Hospital, Fremantle WA Professor Martin Delatycki, Director of Clinical Genetics, Austin Health, Director, Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute.Melbourne VIC Professor Katie Allen, Director, Population Health, Genes and Environment Research Theme, Murdoch Children’s Research Institute, Melbourne VIC Professor Darrell Crawford, Head, Discipline of Medicine, University of Queensland, Brisbane QLD Professor Greg Anderson, Co-ordinator, Chronic Disorders Program, Head, Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane Qld. Seventh Congress of the International BioIron Society Page 339 Late-Breaking Abstracts IBIS Poster #LB18 FERRIC CITRATE AND FERRIC EDTA DRIVE PROLIFERATION OF GUT EPITHELIAL CANCER CELLS VIA AMPHIREGULIN MEDIATED ACTIVATION OF THE MAP KINASE PATHWAY Nathalie Scheers¹,², Dora I.A. Pereira²,³, Nuno Faria²,⁴ and Jonathan J.J. Powell²,⁴ ¹Chalmers University of Technology; ²Elsie Widdowson Laboratory, Medical Research Council, Cambridge, UK; ³Department of Pathology, University of Cambridge; ⁴Department of Veterinary Medicine, University of Cambridge Presented By: Natalie Scheers, PhD Ferric chelates are used as well tolerated oral iron supplements but some have been shown to promote tumor burden in murine models of colon cancer. In contrast, we have previously observed a growth inhibitory effect of a specific iron compound in gut epithelial adeno-carcinoma cell lines. Collectively these data indicate that the form of iron may be a determinant for influencing cancerous growth. Therefore, the aim of this study was to investigate the mechanisms for specific iron compounds on cell growth at typical iron levels encountered in the gastrointestinal tract. Caco-2 and Hutu-80 cells were exposed to different forms of iron and outcomes were assessed using protein arrays, western blot, and ELISA. Ferric EDTA and ferric citrate supplementation increased the cellular levels of the onco-protein amphiregulin and its receptor (EGFr) which in turn stimulated the activation of the MAP kinase pathway. Simultaneously, the expression of the negative Wnt regulator DKK-1 increased, suggesting that ferric EDTA and ferric citrate do not drive proliferation through the Wnt pathway. In contrast, another form of supplemental iron, ferrous sulfate, did not increase cellular amphiregulin or EGFr, nor the levels of Wnt inhibitor DKK-1, which supports previous data in which ferrous sulfate was shown to increase the transcription factor c-MYC, associated with the Wnt signaling pathway. We conclude that specific iron compounds affect cell proliferation differently and some may increase the risk of colon cancer advancement in an amphiregulin- dependent fashion. Further scrutiny is required for safe oral iron supplementation. Seventh Congress of the International BioIron Society Page 340 Late-Breaking Abstracts IBIS Poster #LB19 SHORT FORM HEALTH SURVEY (SF-36) DIFFERS BETWEEN GENOTYPIC GROUPS OF PATIENTS WITH SUSPECTED HEREDITARY HEMOCHROMATOSIS Paula Fernanda, MSc², Rodolfo Can ado, MD, PhD³, Fl vio Naoum, MD, PhD⁴, Carla Dinardo, MD, PhD⁵,⁹, Guilherme Fonseca, MD, MSc⁶, Sandra Gualandro, MD, PhD⁷, Jose Krieger, MD, PhD², Alexandre Pereira, MD, PhD², Pierre Brissot, MD, PhD⁸ and Paulo Santos, PhD¹ ¹Federal University of Sao Paulo; ²Laboratory of Genetics and Molecular Cardiology, Heart Institute , University of São Paulo Medical School, São Paulo, Brazil; ³Hematology and Hemotherapy Section, Santa Casa Medical School, São Paulo, Bra il; ⁴Academia de Ci ncia e Tecnologia, S o Jos do Rio Preto, Bra il; ⁵Funda o Pr -Sangue, Hemocentro de S o Paulo, S o Paulo, SP, Bra il; ⁶Hematology Service, Hospital das Clinicas, Medical School, University of S o Paulo, S o Paulo, Bra il; ⁷Hematology and Hemotherapy Discipline, Hospital das Clinicas, Medical School, University of S o Paulo, S o Paulo, Bra il; ⁸Liver Disease Unit, Pontchaillou University Hospital, University of Rennes, and National Reference Centre for Rare Iron verload Diseases of Genetic rigin, Rennes, France; ⁹Universidade de S o Paulo (USP), São Paulo, SP, Brazil Presented By: Paulo Caleb J L Santos, PhD Background: Hereditary hemochromatosis (HH) encompasses a group of autosomal recessive disorders mainly characterized by enhanced intestinal absorption of iron and its accumulation in parenchymal organs. HH diagnosis is based on iron biochemical and magnetic resonance imaging (MRI) assessment, and genetic testing. Questionnaires, such as SF-36 (short form health survey), have been increasingly used to assess the impact of diseases on the patient's quality of life (QL). In addition, different genotypes are identified as results of genetic tests in patients with suspected primary iron overload. In the present study, our aim was to evaluate whether domains of QL are different according to genotypic groups in patients suspected of HH. Methods: Seventy-nine patients with primary iron overload were included and two genotypic groups were formed (group 1: homozygous genotype for the HFE p.Cys282Tyr mutation; group 2: other genotypes). Results: Group 1 had higher means of plasma transferrin saturation (86±19%) and serum ferritin (1669±1209 ng/mL) compared to group 2 (71±12%, 1252±750 ng/mL, respectively; p=0.001). Four domains were significantly different among groups 1 a nd 2: physical functioning (p=0.03), bodily pain (p=0.03), vitality (p=0.02) and social functioning (p=0.01). Conclusions: our main finding was that patients with p.Cys282Tyr homozygosity had a worse QL scenario assessed by SF-36, compared with patients with iron overload without the same genotype. Being aware of this relationship between genotypes and QL might be helpful in the overall management of patients suspected of hereditary hemochromatosis. Seventh Congress of the International BioIron Society Page 341 Late-Breaking Abstracts IBIS Poster #LB20 HDAC INHIBITION AND GAIN-OF-FUNCTION P53 MUTATION DICTATES ONCOGENIC PHENOTYPE AND INFLUENCES IRON METABOLISM IN THE SW13 CELL LINE McKale Montgomery¹ and Elizabeth Hull, PhD² ¹Midwestern University; ²Midwestern Universty Presented By: McKale Montgomery Histone deacetylase inhibitors (HDACi) are powerful epigenetic regulators that have vast therapeutic potential. Unfortunately, their pleiotropic in vivo effects greatly complicate efforts to harness this potential. Our lab has previously shown that this may be because HDAC inhibitors can induce changes in oncogenic properties. For example, in SW13 cells, HDAC inhibition induces a phenotype switch from a rapidly growing highly tumorigenic subtype (SW13-) to a slow growing, but highly invasive subtype (SW13+). Importantly, this does not appear to be limited to the SW13 cell line. In a screen of more than 30 cancer cell types, we have identified a significant association between gain-of-function p53 mutations and a negative response to HDAC inhibitor treatment. This is of interest because wild-type p53 was recently shown to be involved in the maintenance of iron homeostasis in cancer cells, and iron plays a significant role in both tumor growth and metastasis. To begin to understand the influence of HDAC inhibition and gain-of-function p53 mutation on cellular iron metabolism, SW13 cells were treated with an HDAC inhibitor (FK228) for 24 hr. Western blot and DQ gelatin assays revealed FK228 treatment resulted in ~ 50% reduction in p53 protein expression, and a 5-fold increase in MMP activity, respectively. Microarray analysis of changes in gene expression of iron-related genes following 24 hours of FK228 treatment revealed that the expression of genes involved in iron uptake (STEAP1B and ferroportin) and utilization (EPAS1 and HO1) were significantly increased, while the expression of genes important for iron-sulfur cluster biogenesis (IBA57, NFU1, and HSCB) was significantly decreased. Importantly, these changes in gene expression corresponded with ~35% increase in total intracellular iron levels. To further interrogate influence of mutant p53 expression alone on cellular iron levels, shRNA containing plasmids were then used to generate stable knockdowns of mutant p53 in the SW13 cell line. Interestingly, knockdown of mutant p53 expression alone was sufficient to significantly increase total intracellular iron levels. The ability of cancer cells to respond, adapt, migrate, and invade is made possible by the cell’s ability to undergo rapid epigenetic remodeling in response to environmental cues, and can contribute to lack of responsiveness to treatment. Findings from this study suggest that gain-of-function p53 mutation may contribute to cancer cell responsiveness epigenetic therapy, and that it may do so by manipulating pathways involved in iron acquisition and utilization. These findings are clinically relevant as p53 is mutated in some 50% of all cancers and many of the hotspot mutations display the gain-of-function phenotype. As several HDAC inhibitors are already FDA approved for the treatment of certain types of cancer, understanding how to manipulate iron availability in these cells could ultimately allow for the development of more effective combinatorial approaches to cancer treatment. Seventh Congress of the International BioIron Society Page 342 Late-Breaking Abstracts IBIS Poster #LB21 FERROPORTIN INHIBITORS PREVENT IRON LOADING IN A MOUSE MODEL OF HEREDITARY HEMOCHROMATOSIS Naja Nyffenegger, PhD, Anna Flace, Camillo Canclini, Franz Duerrenberger, PhD and Vania Manolova, PhD Vifor Pharma Ltd. Presented By: Naja Nyffenegger, PhD In hereditary hemochromatosis mutations in genes encoding components that regulate iron homeostasis, such as hepcidin, the hemochromatosis protein HFE, hemojuvelin and transferrin receptor 2 cause excessive intestinal absorption of dietary iron resulting in pathological iron overload in mice and men. HFE is a MHC class I-like membrane protein that associates with beta 2-microglobulin and it is believed to participate in hepcidin transcriptional regulation through the bone morphogenetic protein receptor pathway. Mice deficient in beta 2-microglobulin (beta 2m-/-) develop hyperferremia and hemochromatosis with similar iron metabolism-related parameters as HFE deficient animals (HFE-/-), due to the fact that beta 2-microglobulin is necessary for the cell-surface expression and function of HFE1. Iron restriction by activation of endogenous hepcidin2 has been shown to reduce serum iron and liver iron overload in a mouse model of genetic hemochromatosis (HFE-/-). We have discovered orally bioavailable small molecular weight Ferroportin Inhibitors with a similar mode of action as the physiological ferroportin ligand hepcidin. Oral Ferroportin Inhibitors caused transient hypoferremia in wild-type mice with similar kinetics as intraperitoneally injected hepcidin. Administration of a single dose of the Ferroportin Inhibitor to beta 2m-/- mice corrected serum iron to wild-type levels. Repeated dosing of Ferroportin Inhibitor in young beta 2m-/- mice prevented liver iron loading and induced retention of iron in spleen and duodenal enterocytes. Direct evidence that Ferroportin Inhibitors block dietary iron absorption was demonstrated by their ability to prevent an increase of serum iron following oral application of ferrous sulfate to anemic rats. Therefore, reducing excessive iron absorption by pharmacologically blocking ferroportin with oral Ferroportin Inhibitors provides a novel therapeutic opportunity in hemochromatosis. 1 Zhou, 1998, PNAS; Rothenberg and Voland, 1996, PNAS. 2 Schmidt, 2013, Blood; Guo, 2013, JClinInvest. Seventh Congress of the International BioIron Society Page 343 Late-Breaking Abstracts IBIS Poster #LB22 GINGER NANOPARTICLE-DERIVED LIPID VECTOR SIRNA DELIVERY SYSTEM TARGETING INTESTINAL DIVALENT METAL-ION TRANSPORTER 1 (DMT1) AS A NOVEL TREATMENT FOR HEREDITARY HEMOCHROMATOSIS Xiaoyu Wang¹, Mingzhen Zhang, PhD², Didier Merlin, PhD² and James Collins, PhD¹ ¹University of Florida; ²Georgia State University Presented By: Xiaoyu Wang Introduction: Nanotechnology is being applied to the drug delivery area because of its ability to deliver hydrophobic drugs and biologics. Nanoparticles produced from edible plants did not cause detectable toxicity and have proven to be effective siRNA delivery vehicles. Intestinal DMT1 is the primary entry point for iron into the body. Intestinal DMT1 is thus a logical target for therapeutic intervention in hereditary hemochromatosis (HH). This study was thus designed to test the hypothesis that DMT1 expression can be attenuated by oral administration of siRNA carrying ginger nanoparticle-derived lipid vectors (GNLVs), which will ameliorate iron loading in a mouse model of HH (hepcidin KO mice). Methods: Nanoparticles from ginger juice were isolated from a sucrose density gradient (30-45%) followed by ultracentrifugation. Then, total lipids were extracted from ginger-derived nanoparticles (GDNP) to get GNLVs. GNLVs were characterized and tested for toxicity in vitro. GNLVs were then loaded with FITC-tagged (green) or DMT1 specific (or scrambled) siRNA and transfected into Colon-26 cells. Colon-26 cells and HEK 293 (overexpressing mouse DMT1) cell lines were used to select a functional DMT1-specific siRNA. C57BL/6 adult mice were orally gavaged with folic acid- conjugated GNLVs loaded with siRNAs. Results: TEM images of GNLVs showed a generally spherical shape with an average size of ~179 nm, with lower polydispersity. These data were also confirmed by Atomic Force Microscopy, which showed that GNLVs were highly dispersed with a uniform size distribution. When exposed to mouse intestinal epithelial (Colon-26) cells after 24 or 48 h, GNLVs showed much less toxicity compared to a cationic liposome, DC-Chol/DOPE, which has been used extensively for the in vivo delivery of therapeutic reagents. Also, no changes in intestinal barrier function were noted in fully differentiated Caco-2 cell monolayers exposed to GNLVs. A functional DMT1 siRNA was selected from three commercial siRNAs (from two companies). Colon-26 and HEK 293 (overexpressing mouse DMT1) cells were used for selection, and the functional DMT1 siRNA decreased DMT1 mRNA expression by 70-80 % in both cell lines. GNLV-mediated siRNA delivery to the cytoplasm was confirmed by incubation of FITC-tagged siRNA and visualization of green fluorescence. To target the duodenum and proximal jejunum where intestinal iron absorption occurs, the GNLVs were conjugated to folic acid (FA- GNLVs) to target folic acid receptors in the proximal small bowel. FA-GNLVs showed enhanced retention in the duodenum. Furthermore, mice that were orally gavaged with FA-GNLVs loaded with DMT1 siRNAs showed reduction of DMT1 mRNA expression by ~40%. Conclusions: We developed a GNLV siRNA delivery system targeting intestinal DMT1. GNLVs showed less toxicity and effectively delivered siRNA into cells. GNLVs delivered functional DMT1 siRNA and significantly decreased DMT1 mRNA expression in vitro and in vivo. Further optimization may allow a greater suppression. Future proof-of-principal studies will be carried out in mouse and rat models of human HH. Since iron loading in HH involves only incremental increases in iron absorption over time, this level of DMT1 inhibition will likely be effective to reduce tissue iron burden and decrease comorbidities associated with iron toxicity. Seventh Congress of the International BioIron Society Page 344 Late-Breaking Abstracts IBIS Poster #LB23 LOSS OF LIVER SPECIFIC IRON CHAPERONE PCBP1 LEADS TO STEATOSIS AND HYPERLIPIDEMIA Shyamalagauri Jadhav, Olga Protchenko, Fengmin Li, Oksana Gavrilova, James Cox, Howard Smith, Minoo Shakoury- Elizeh and Caroline Philpott National Institute of Diabetes and Digestive and Kidney Diseases Presented By: Shyamalagauri Jadhav Iron is an essential cofactor required for the activity of hundreds of proteins regulating cellular processes. Despite its crucial role, iron at higher concentrations is toxic. Therefore, it is very important to tightly regulate the intracellular iron levels. Poly (rC)-binding protein 1 (PCBP1) functions as an iron chaperone. Initially, PCBP1 was isolated as a heterogeneous nuclear ribonucleoprotein that binds single stranded RNA and regulates RNA processing, stability and translation. Further studies showed that PCBP1 also binds cytosolic iron and delivers it to ferritin and other iron enzymes. Most of the PCBP1 studies are done in cultured cells; therefore the role of PCBP1 in tissue is not very well understood. Liver is an iron storage organ and plays an important role in regulating iron balance, which makes liver an ideal model to study iron homeostasis. To study the role of PCBP1, a liver specific knockout of PCBP1 was generated. Preliminary studies show that hepatocyte-specific knockout of PCBP1 leads to increased lipid accumulation in the liver, and specifically increased triglyceride levels in the liver and increased cholesterol in the serum. In order to further understand the dis-regulated lipid metabolism in the PCBP1-KO,we carried out a lipidomic and transcriptomic study. Lipidomics analysis reveled that deletion of PCBP1 increased diacylglycerides, free fatty acids and membrane phospholipids (phosphatidylcholine and phosphatidylglycerol), carnitine and ceramide and decreased phosphatidylethanolamine and coenzyme Q levels. Consistent with the preliminary and lipidomics data, transcriptomics data revealed that PCBP1 deletion up-regulated genes involved in cholesterol, unsaturated fatty acid and triglyceride synthesis as well as retinol and omega-6 fatty acid metabolism. Taken together these data suggest that PCBP1 deletion increases lipid synthesis and lipoprotein secretion. Previous studies reveal that mitochondrial dysfunction causes altered lipid metabolism. Therefore, we plan to investigate whether deletion of PCBP1 affects mitochondrial function. Seventh Congress of the International BioIron Society Page 345 Late-Breaking Abstracts IBIS Poster #LB24 THE HEPCIDIN KNOCKOUT RAT: A NOVEL MODEL OF JUVENILE HEREDITARY HEMOCHROMATOSIS IN HUMANS Shireen Flores, MS, Xiaoyu Wang, MS, Tao Wang, Jungheun Ha, PhD, Caglar Doguer, PhD and James Collins, PhD University of Florida Presented By: Shireen Flores, MS Genetically-modified mouse models of altered iron homeostasis have made invaluable contributions to our understanding of the regulation of iron metabolism in humans. Mice may not, however, be the best model for all aspects of human physiology/pathophysiology. For example, mice are resistant to developing liver cirrhosis and diabetes, which is important in relation to investigation of iron-overload disorders, since dysfunction of the endocrine pancreas and liver damage typify excess iron deposition. Fortunately, recent advances in technology have now made it more feasible to produce genetically-modified rats, which may better recapitulate some (but not all) aspects of human diseases, including genetic iron-loading disorders. We have thus developed a hepcidin knockout (Hamp-/-) Sprague-Dawley (SD) rat. The rationale for this experimental approach was based upon the numerous pathophysiologic differences noted between mouse models of hereditary hemochromatosis (HH) and humans with HH. We hypothesized that the Hamp-/- rats would exhibit iron loading, in the liver, heart and pancreas, similar to humans with juvenile HH. Moreover, since rats live longer, we postulated that they would be more likely to develop tissue damage associated with iron loading (which does not occur in mice). We confirmed deletion of the hepcidin gene, and showed that liver hepcidin mRNA expression is very low and ferroportin protein levels are high (consistent with low hepcidin expression). Hamp-/- rats develop early and severe multivisceral iron overload, with splenic sparing. Importantly, Hamp-/- rats displayed a periportal pattern of hepatic iron deposition, similar to what is seen in humans, but distinct from the iron-loading pattern seen in Hamp-/- mice (which is centrolobular). In addition, when Hamp-/- rats were fed a high-iron diet, they incurred severe cardiac injury as well as unexplained, altered morphology of the spleen. These data demonstrate that rats lacking hepcidin do indeed load iron, and further that key differences may exist between Hamp-/- mice and rats in terms of iron-loading patterns and dysregulation of iron metabolism. It is thus possible that SD rats may better model disturbances of iron metabolism in humans. The Hamp-/- rat may thus be an invaluable model that will ultimately provide novel mechanistic insight into the dysregulation of iron metabolism in HH. Supported by NIH grant 1R01 DK074867 (to J.F.C.). Seventh Congress of the International BioIron Society Page 346 Late-Breaking Abstracts IBIS Poster #LB25 PLUTONIUM-TRANSFERRIN BINDING MONITORED BY CE-ICP-MS Robert Evans, BA, PhD¹, Sandrine Sauge-Merle²,³,⁴, David Lemaire²,³,⁴, Catherine Berthomieu²,³,⁴ and Jean Aupiais²,⁵,⁶ ¹Brunel University; ²C A; ³CNRS; ⁴Aix-Marseille University; ⁵DAM; ⁶DIF Presented By: Robert William Evans, BA, PhD A variety of techniques, including fluorescence spectroscopy, isothermal titration calorimetry, time-resolved laser spectroscopy and capillary electrophoresis linked to an inductively coupled plasma mass spectrometer (CE-ICP-MS), are available to monitor the binding of a metal to a protein and to determine the binding affinity of the protein for the metal. In view of the sensitivity of CE-ICP-MS it is ideally suited to studies of metal-protein interactions which require the handling of toxic metals or radionuclides. We have therefore explored, for the first time, the use of CE-ICP-MS to monitor the binding of the radionuclide tetravalent plutonium (Pu) to human transferrin (Tf). As a result of the continued development of the civil and military industries there is the possibility of release of radionuclides into the environment, with Pu being one of the major radionuclide hazards to human health and ecosystems. Pu is known to accumulate in bone and liver but is only slowly excreted in urine and faeces. In the healthy adult only about 30% of the metal-binding sites on Tf are occupied with iron. Tf therefore has the ability to bind and transport a wide range of other metal ions (1), including Pu, and possibly deliver them to mammalian cells via the receptor-mediated endocytic pathway. With an accurate knowledge of the binding affinity for Pu it would be possible to design chelating agents which can compete effectively in plasma with Tf for Pu. Human Tf was purified from outdated plasma (2) and made iron-free by dialysis against 0.1 M citric acid, pH 4.7. CE-ICP-MS was carried out using a Beckman Coulter P/ACE 800 Plus commercial Capillary Electrophoresis system equipped with a diode array detector coupled to a Thermo X Series ICP-MS. The experiments were made possible by using the nitrilotriacetate (NTA) anion as an effective competitor with Tf for Pu and to prevent the hydrolysis of Pu. In preliminary studies, CE-ICP-MS was shown to be able to resolve Pu(NTA)2 from Pu-Tf. The binding constant of Tf for Pu was then determined by investigating its competition with Pu-NTA using CE-ICP-MS to resolve and quantify the Pu-Tf and Pu-NTA complexes at fixed concentrations of Pu and NTA but with Tf concentrations over the range 0.627 mM to 12.5 nm. On the basis of the data from two series of independent competition experiments, using two different types of coated capillaries, we determined a value of log K1* = 22.50 +/- 0.19 for the first stepwise conditional bicarbonate-free binding constant of Pu to Tf (3). Our results are in agreement with those obtained using UV-Visible spectroscopy and ultrafiltration (4). The results of our studies confirm that Tf has a high affinity for Pu but also demonstrate that CE-ICP-MS is ideally suited for the determination of binding constants of proteins for metals when the toxicity or radioactive nature of the metal necessitates the handling of metals at extremely low concentrations. (1) Sargent PJ, Farnaud S, Evans RW. Curr Med Chem (2005) 12, 2683-2693. (2) Devanur LD, Evans RW, Evans PJ, Hider RC. Biochem J (2008) 409, 43-447. (3) Sauge-Merle S, Lemaire D, Evans RW, Berthomieu C, Aupiais J. Dalton Trans (2017) 46, 1389-1396. (4) Yule L. (1991) PhD Thesis, University of Wales, Cardiff. Seventh Congress of the International BioIron Society Page 347 Late-Breaking Abstracts IBIS Poster #LB26 IMMUNOASSAY FOR HUMAN SERUM ERYTHROFERRONE Tomas Ganz, PhD, MD, Grace Jung, MS, Arash Naeim, MD, Yelena Ginzburg, MD, Zahra Pakbaz, MD, Patrick Walter, PhD, Leon Kautz, PhD and Elizabeta Nemeth, PhD UCLA, Los Angeles Presented By: Tomas Ganz, PhD, MD Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to stimulation by erythropoietin. We previously demonstrated that murine ERFE mRNA expression and serum protein concentration increase in mice subjected to hemorrhage or erythropoietin therapy, that ERFE acts on hepatocytes to suppress hepcidin, and that the resulting decrease in hepcidin augments iron delivery for intensified erythropoiesis. We also showed that ERFE contributes to pathological hepcidin suppression and iron overload in a mouse model of -thalassemia intermedia. We now report the development and technical validation of a rabbit monoclonal antibody-based sandwich immunoassay for human erythroferrone. Using this assay we showed that blood loss or erythropoietin administration increased serum ERFE concentrations in humans, and that patients with both non-transfused and transfused -thalassemia had very high serum ERFE levels, which decreased after blood transfusion (Figure). Serum hepcidin concentrations were inversely proportional to serum erythroferrone concentrations. The assay should be useful for human studies of normal and disordered erythropoiesis and its effect on iron homeostasis. Figure erum erythroferrone in patients with -thalassemia, non-transfused (Non), before transfusion (Pre) or after transfusion (Post). Box plots show median, box 25% to 75%, whiskers 10% to 90%, and outliers. Serum ERFE levels were massively increased in thalassemic patients, non-transfused and pre-transfusion, and returned to normal after transfusion, *p<0.05, One Way ANOVA on Ranks, comparing to normal reference group. B: Serum hepcidin in the same samples, vs erythroferrone, scatterplot is fitted by an inverse third order equation, R2 = 0.7. Seventh Congress of the International BioIron Society Page 348 Late-Breaking Abstracts IBIS Poster #LB27 ANTI-INFLAMMATORY EFFECT OF FGF-23 INHIBITION PREVENTS LIVER HEPCIDIN UPREGULATION IN A CHRONIC KIDNEY DISEASE MOUSE MODEL Rafiou Agoro, PhD¹, Anna Montagna, PhD¹, Onyedikachi Aligbe, BA¹, Regina Goetz, PhD¹, Moosa Mohammadi, PhD¹, Stefano Rivella, PhD² and Despina Sitara, PhD¹ ¹New York University; ²Children's Hospital of Philadelphia Presented By: Rafiou Agoro, MSc Introduction: Chronic kidney disease (CKD) is a global public health problem characterized by gradual loss of kidney function over time. We have reported that Fibroblast Growth Factor-23 (FGF-23), the primary regulator of phosphate homeostasis and vitamin D metabolism, also regulates erythropoiesis. High FGF-23 levels are associated with CKD and correlate with declining renal function and disease progression. We have previously reported that administration of FGF- 23 rapidly decreases erythropoiesis, whereas genetic ablation of Fgf-23 results in increased red blood cell production. CKD patients develop anemia of inflammation associated with abnormal activation of immune cells and overexpression of inflammatory cytokines, which correlate with increased cardiovascular mortality. Furthermore, expression of hepcidin, the main hormone responsible for maintaining systemic iron homeostasis, is induced by inflammatory cytokines resulting in iron deficiency and anemia. The aim of this work is to better understand the role of FGF-23 in iron homeostasis by assessing whether high FGF-23 levels induce expression of inflammatory markers and blocking FGF-23/FGFR signaling rescues iron deficiency in nephrectomy-induced renal insufficiency. Material and Methods: In this study, we used the 5/6 nephrectomy (5/6 Nx) mouse model in which C57BL/6 male mice underwent a 2-step surgery to reduce the original functional renal mass by five-sixths to induce renal insufficiency. Control mice underwent sham nephrectomy (Sham) at the same time. Nine weeks after the second surgery, mice were treated with a single intraperitoneal (ip) injection of an FGF-23 antagonist which blocks FGF-23 signaling by competing with endogenous FGF-23, or PBS as vehicle, and sacrificed 12 hours post-injection. Results and Discussion: Our data show that hepatic hepcidin expression is significantly upregulated in 5/6 Nx mice in comparison to sham mice, and it is negatively correlated with serum iron levels and transferrin saturation in 5/6 Nx mice. Our data also confirm the presence of inflammation in response to 5/6 nephrectomy in mice, as demonstrated by induction of hepatic IL-6, TNF-α, and IFN-γ expression, and increased number of bone marrow macrophages (CD11b+/F4-80+). Importantly, we show that a single ip injection of the FGF-23 antagonist significantly increased serum iron and transferrin saturation and abrogated hepatic upregulation of hepcidin in 5/6 Nx mice within 12 hours post- injection. Furthermore, we observed that treatment of Sham mice with the FGF-23 antagonist also increased serum iron and transferrin saturation independently of liver hepcidin expression. In addition, blocking FGF-23 signaling by the use of the FGF-23 antagonist abolished nephrectomy-induced inflammation in 5/6 Nx mice by strongly attenuating hepatic expression of IL-6, TNF-α, and IFN-γ expression, and significantly reducing the number of bone marrow CD11b+/F4-80+ cells. Conclusions: In conclusion, our data highlight that renal insufficiency, induced by nephrectomy in a mouse model, promotes inflammation, particularly with macrophage recruitment in the bone marrow and upregulation of cytokines such as IL-6, TNF-α and IFN-γ in the liver. Inflammation results in upregulation of hepatic hepcidin which binds to ferroportin, thereby regulating hepatocyte and macrophage iron efflux. Importantly, in this study we demonstrate that inhibiting FGF- 23/FGFR signaling has an anti-inflammatory effect in a CKD mouse model by preventing hepatic hepcidin overexpression and restoring physiological serum iron levels and transferrin saturation. Seventh Congress of the International BioIron Society Page 349 Late-Breaking Abstracts IBIS Poster #LB28 ROLE OF C-JUN NH(2)-TERMINAL KINASE AND IRON IN CELLULAR MODEL OF ACUTE PANCREATITIS Zbigniew Sled i ski, MD PhD¹, Michal Wozniak, MD PhD², Maciej Sledzinski, MD² and Jedrzej Antosiewicz, PhD³ ¹Medical University of Gda sk; ²Medical University of Gdansk, Poland; ³Gdansk University of Physical ducation and Sport, Gdansk, Poland Presented By: Zbigniew Sledzinski, MD, PhD Involvement of reactive oxygen species (ROS) in pathogenesis of acute pancreatitis (AP) has been recognize long time ago. However, the source of ROS is still not well defined. NADPH-oxidase has been proposed to be important source of ROS during AP development. Recently it has been shown that activation of c-jun terminal kinase (JNK) plays important role in pathogenesis of AP. Moreover we demonstrated that JNK controls iron metabolism by regulating ferritin degradation in cancer cells. Thus we hypothesized that JNK activation will lead to increase iron-dependent ROS formation. The study was performed on AR42J cell line treated with cerulean, this is a cellular model of AP. Changes in labile iron pool has been measured by fluorescence microscope after calcein staining. Intracellular ROS generation was evaluated by flow cytometry monitoring of oxidation of H2DCFDA. Cells treated with cerulein demonstrated higher level of ROS and desferrioxamine completely abrogated this effect. Cerluein treatment lead to decreased in ferritin protein level and this was accompanied by increase in LIP. Next we designed experiments to determine if JNK kinase is responsible for cerulean-induced ferritin degradation. AR42J cells were transfected with the plasmid encoding inactive variants of JNK1 or JNK2 or empty pcDNA3.1. No ferritin degradation was observed in cells expressing inactive JNK1. In conclusion, our data suggest that the cerulein stimulates degradation of ferritin in acinar AR42J cells. c-Jun NH(2)-terminal kinase 1 dependent ferritin degradation is accompanied by an increase in LIP and ROS formation. These data strongly suggest that iron may be important source of ROS during AP. Seventh Congress of the International BioIron Society Page 350 Late-Breaking Abstracts IBIS Poster #LB29 EXERCISE INDUCED-INCREASE IN BLOOD HEPCIDIN IS THE SAME IN WT AND HETEROZYGOTES FOR HFE H63D Jedrzej Antosiewicz, PhD¹, Jakub Kortas, PhD², Maja Tomczyk, MS², Krzysztof Prusik,, PhD², Katarzyna Prusik, PhD², Joanna Jaworska, MS², Barbara Kaczorowska, MD² and Ewa Ziemann, PhD² ¹Gdansk University of Physical Education and Sport, Ka imier a Go rskiego ,; ²Gdansk University of Physical Education and Sport, Gdansk, Poland Presented By: Jedrzej Antosiewicz, PhD Excess body iron accumulation has been associated with ageing. Although, there are several studies suggesting that regular exercise may induce changes in iron metabolism, we are still far from complete understanding the mechanism and effects of exercise on iron metabolism. The main objective of this study was to determine combine effect of aerobic training and HFE mutation on blood hepcidin among different groups of ages. First group involved 35 elderly women participated in 12 weeks of Nordic walking (NW) training. Among them 11 were carrier’s heterozygotes for HFE H63D. Second group involved 41 young men, performed marathon (11 carrier’s heterozygotes for HFE H63D and 30 wild type (WT). We demonstrated that the training in elderly women caused the significant reduction in blood ferritin 99.4 ± 62.7 vs. 81.4 ± 61.7 ng/ml) indicating for drop body iron stores. These changes did not correlate with changes in blood hepcidin. Applied training program induced the rise in hepcidin concentration in 17 women, whereas in 18 a decrease was noted. Among subjects with blood hepcidin HFE H63D and WT group, effects of the training was the same. Similar results were observed in group of running marathon. In 20 of them an increase of blood hepcidin was recorded and in 21 opposite tendency was observed. Heterozygotes for HFE H63D were represented in both groups. In conclusion, NW training reduced body iron stores but these changes were not associated with changes blood hepcidin. The effects of regular or single exercise on blood hepcidin seems to be the same in carrier’s heterozygotes for HFE H63D and WT. Seventh Congress of the International BioIron Society Page 351 Late-Breaking Abstracts IBIS Poster #LB30 BACTERIAL DYSBIOSIS IN COLON AND BREAKDOWN OF MUCOSAL PERMEABILITY BARRIER IN A MOUSE MODEL OF HEMOCHROMATOSIS Sathish Sivaprakasam¹, Bojana Ristic, BSs¹, Abdul N Hamood2, PhD¹, Nithya S Mudaliar, MS¹, Jane Colmer Hamood, PhD¹, Kameswara Rao Kottapalli, PhD² And Vadivel Ganapathy, PhD¹ ¹Texas Tech University Health Sciences Center; ²Texas Tech University Presented By: Sathish Sivaprakasam, PhD Inflammatory bowel disease (IBD) is a chronic disorder characterized by recurrent intestinal inflammation. Pathogenesis of IBD involves excessive and pathological immune response to colonic bacteria. Even though the co-evolution of colonic bacteria and the host is mutually beneficial under normal physiological conditions, genetic and environmental factors can potentially disrupt this symbiotic relationship and cause IBD. Bacterial dysbiosis, defined as microbial imbalance with significant alterations in bacterial species and abundance, as well as breakdown of mucosal permeability barrier are closely associated with IBD. Recent studies have shown that iron is a key regulator of immunity and that excess iron/heme impacts on colonic bacteria. Hemochromatosis is a genetic disease with iron overload, primarily caused by mutations in the iron-regulatory protein HFE. Hfe-null mice, a model for hemochromatosis, exhibit iron overload in colonic epithelial cells and show evidence of a pro-inflammatory environment in colon. We have shown that these mice are also at increased risk of colitis and colon carcinogenesis in experimental model systems. Based on these findings, we hypothesized that hemochromatosis impacts on colonic bacteria and mucosal permeability barrier. To test this hypothesis, we analyzed fecal and adherent bacteria in colons of wild type mice and Hfe-null mice. 16S metagenomic analysis of fecal bacteria showed increased incidence of Proteobacteria in Hfe-null colon than in age- and gender-matched wild type mouse colon. Further analysis revealed that Desulfovibrio, Prevetolla, Sutterella, Enterococcous, Streptococcus, and unclassified TM7 bacterial species were enriched in feces from Hfe-null mice. Furthermore, there were more bacteria adherent to colonic mucosal surface in Hfe-null mice than in wild type mice. This was demonstrable by culture of the adherent bacteria and by scanning electron microscopy. There was also evidence of bacterial biofilm formation on the colonic mucosal surface in Hfe-null mice, which was not seen in wild type mice. Further characterization of adherent bacteria revealed increased incidence of coliform, indole-positive bacterial species, Enterococcus faecium, and Lactobacillus spp. in Hfe-null mice. Analysis of expression of antibacterial peptides in colon showed decreased generation of Cryptidin-2, Reg3b, and Reg3d in Hfe-null mice compared to their expression in wild type mice; these peptides are effective blockers of gram-positive and gram-negative bacteria. But, there was no difference in the production of the mucosal secretory antibody IgA between Hfe-null mice and wild type mice. We also investigated the mucosal permeability barrier by measuring the appearance of orally administered FITC-dextran in serum. We found strong evidence for breakdown of the intestinal barrier in Hfe-null mice. These data provide convincing evidence for significant alterations in colonic bacteria and for disruption of intestinal mucosal barrier in the iron-overload disease hemochromatosis, which correlates with accelerated progression of experimentally induced colitis and colon carcinogenesis. We conclude that hemochromatosis is likely to be associated with bacterial dysbiosis and breakdown of intestinal barrier in humans, thereby increasing the risk for colitis and colon cancer. Seventh Congress of the International BioIron Society Page 352 Late-Breaking Abstracts IBIS Poster #LB31 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL NA+-COUPLED, CITRATE-DEPENDENT TRANSPORT SYSTEM FOR NON-TRANSFERRIN-BOUND IRON Jiro Ogura, PhD¹, Ellappan Babu, PhD¹, Seiji Miyauchi, PhD² and Vadivel Ganapathy, PhD¹ ¹Texas Tech University Health Sciences Center; ²Toho University Presented By: Jiro Ogura, PhD There is very little free iron in circulation as most of it is transferrin-bound. This provides protection against oxidative stress inducible by free iron. However, in certain pathological conditions associated with iron overload (e.g., hemochromatosis, sickle cell disease, frequent blood transfusion), the circulating levels of free, non-transferrin-bound iron (NTBI) increase. Mammalian cells take up iron from transferrin via transferrin receptor. NTBI is also taken by mammalian cells, but very little is known on the transport systems responsible for this uptake process. ZIP8 (SLC39A8) and ZIP14 (SLC39A14) mediate cellular uptake of NTBI, but whether there are additional mechanisms for NTBI uptake in mammalian cells remains unexplored. During the course of our work on citrate uptake in liver and mammary epithelial cells, we identified a novel Na+-coupled, citrate-dependent transport system for NTBI that is distinct from ZIP8 and ZIP14. When human liver cell lines (HepG2 and THLE3) and mammary cell lines (MCF10A, MCF7, and MB231) were pretreated with ferric ammonium citrate (FAC; 250 µg/mL) for two or more passages, uptake of citrate increased >10-fold compared to untreated cells. This phenomenon was unrelated to NaCT (SLC13A5), a Na+-coupled citrate transporter present in HepG2 cells but not in MCF7 cells. Pretreatment with equivalent concentrations of citrate or NH4Cl did not increase citrate uptake, showing that the effect was selective for ferric ion. When tested for the effect of Fe3+ during uptake without involving pretreatment, Fe3+ stimulated citrate uptake markedly in a dose-dependent manner. The stimulation was significant even at 5 µM Fe3+, which kept increasing when [Fe3+] increased to 100 µM, at which the stimulation of citrate uptake was >20-fold. Furthermore, citrate stimulated the uptake of Fe3+ to a significant extent in HepG2 cells; no other intermediates of the citric acid cycle had this ability. To determine why citrate uptake was higher in FAC-treated cells though Fe3+ was not included during uptake, the effect of the iron-chelator deferiprone in the uptake medium was examined in FAC-treated HepG2 cells. Deferiprone abrogated the stimulation of citrate uptake, indicating that Fe3+ was released from FAC-treated cells to the extracellular medium that stimulated citrate uptake. The phenomenon was not due to Fe3+ adherent to the cell surface in FAC-treated cells because pretreatment of the cells with deferiprone failed to abolish the stimulation of citrate uptake. There was no role for the Fe3+ exporter ferroportin as treatment of FAC-treated HepG2 cells with hepcidin decreased ferroportin levels but the FAC-dependent stimulation of citrate uptake remained unchanged. In untreated cells, only Fe3+ and Fe2+ stimulated citrate uptake; Zn2+, Mn2+, and Mg2+ had no effect. The Fe3+- stimulated citrate uptake was obligatorily dependent on Na+, suggesting co-transport of Fe3+-citrate with Na+. Zn2+ had no effect on this uptake process, indicating non-involvement of ZIPs. Bioinformatic analysis has identified a new member of the citrate transporter gene family (SLC13) in mammals, but this transporter (SLC13A6) failed to transport citrate in the presence or absence of Fe3+. These studies identify a novel, hitherto unknown, transport system for the uptake of not only NTBI but also citrate in mammalian cells, but the molecular identity of the transporter remains to be established. Seventh Congress of the International BioIron Society Page 353 Late-Breaking Abstracts IBIS Poster #LB32 UNCOMMON IRON CHELATORS AURINTRICARBOXYLIC ACID (ATA) AND LIPOIC ACID (LA) AS ANTICANCER AGENTS Michal Wozniak¹, Alicja Kuban-Jankowska, PhD², Magdalena Gorska-Ponikowska, PhD², Kamlesh Sahu, PhD³ and Jack Tus ynski, PhD⁴ ¹Medical Univeristy of Gdansk, Poland; ²Medical Chemistry Department, Medical University of Gdansk, Gdansk, Poland; ³Department of Medical Microbiology and Immunology, University of Alberta, dmonton, Canada; ⁴Department of Oncology, University of Alberta, Edmonton, Canada Presented By: Matt Wozniak Iron is an essential element for cell physiology being critical for DNA synthesis. Rapidly dividing cancer cells have higher requirement for Fe comparing to nontransformed counterparts, making them particularly sensitive to Fe depletion. Our previous studies (Biometals, 2015, vol. 28, nr 6, s.975-986) revealed the selective sensitivity of PTP1B to inhibitory effect of Fe2+. Protein tyrosine phosphatase PTP1B is over-expressed in breast cancer cells, thus the inhibition of its activity can be potentially useful in breast cancer therapy. Here we investigated the effect of aurintricarboxylic acid (ATA) and lipoic acid (LA) on the viability of MCF-7 breast cancer cells and enzymatic activity of PTP1B phosphatase. ATA and LA has been found to decrease the viability and proliferation of breast cancer cells. Both iron chelators has been found to inhibit the activity of PTP1B. Iron chelator modality of ATA and LA enables iron to enter catalytic site located cysteine essential for enzyme activity. The studies was supported from the project IP2015 038774 from Polish Ministry of Science and Higher Education Seventh Congress of the International BioIron Society Page 354 Late-Breaking Abstracts IBIS Poster #LB33 SMART IRON DELOCALIZATORS IN COMBINATION WITH NEW GPRX4 PROTECTOR PREVENT DAMAGE OF TRANSPLANTED KIDNEY FROM FERROPTOTIC INSULT Narcyz Knap¹, Michal Wozniak, MD PhD², Magdalena Gorska-Ponikowska, PhD², Alicja Kuban-Jankowska, PhD², Rys ard Smolenski, MD PhD³, Michal Zmijewski, PhD⁴, Jedr ej Antosiewic , PhD⁵ and Zbigniew Sled inski, MD PhD⁶ ¹Medical Univeristy of Gdansk, Poland; ²Department of Medical Chemistry, Medical University of Gdansk; ³Department of Biochemistry, Medical University of Gdansk; ⁴Department of Histology, Medical University of Gdansk; ⁵Department of Bioenergetics and Physiology of Exercise Medical University of Gdansk; ⁶Department of General, ndocrine Surgery and Transplantation, Medical University of Gdansk Presented By: Narcyz Knap Endothelial and epithelial cell death by regulated necrosis induce dangerous tissue damage in ischemia-reperfusion injury upon organ transplantation, being iron dependent as ferroptosis. Herein, we demonstrate the involvement of smart iron delocalizators namely inosine and ATA together with alpha-ketobutyrate acid (hydrogen peroxide scavenger and GPRX-4 protector) as essential compounds of new innovative transplant fluid in order to protect transplanted kidney from ferroptosis (PNAS, November 25, 2014, 111, 16836-41). A solution for protecting, storing and perfusing organs during transplantation and other surgical procedures containing osmolytes and oncolytes, electrolytes, substances with antioxidant activity, cryoprotectants, cytoskeleton stabilizers, precursors for the synthesis of high-energy adenylates, ferroptosis inhibitors, and a stimulator of endothelial cell regeneration. A solution according to the present invention contains: trehalose at a concentration from 1 mM to 50 mM, inosine at a concentration from 0.1 mM to 5 mM, a- ketobutyric acid or its salts at a concentration from 0.1 mM to 25 mM, aurintricarboxylic acid or its salts at concentrations from 0.01 mM to 1 mM and N-methylnicotinamide at a concentration from 0.1 mM to 5 mM. The scope of uses of said solution encompasses perfusion and storage at low temperature of organs collected from a donor or one subjected to a surgical procedure. Our formulation – Transmedium Transplant Fluid, demonstrates high efficiency to protect from ferroptosis at physiological as well protective 4 degree preservative temperature. 2 kidney origin cell lines namely HRG and NRK were exposed to ischemia reperfusion challenge. Since ferroptosis can be categorized as a molecular mechanism of disease with localized iron accumulation, utilizing iron redistributors seems to be a novel therapeutic strategy to prevent ischemia-reperfusion injury. Seventh Congress of the International BioIron Society Page 355 Late-Breaking Abstracts IBIS Poster #LB34 INDEPENDENT RISK FACTORS FOR BIOPSY-PROVEN CIRRHOSIS IN HEMOCHROMATOSIS: A STUDY OF 110 HFE p.C282Y HOMOZYGOTES WITH GNPAT p.D519G GENOPTYING James Barton, MD²,¹³, Christine McLaren, PhD³, Wen-pin Chen, MS⁴, Grant Ramm, PhD⁵, Gregory Anderson, PhD⁶, Lawrie Powell, MD, PhD⁶, V. Nathan Subramaniam, PhD⁷, Paul Adams, MD⁸, Pradyumna Phatak, MD⁹, Lyle Gurrin, PhD¹ , John D. Phillips, MD¹¹, Charles J. Parker, PhD¹¹, Mary J. dmond, PhD¹² and Gordon McLaren, MD¹,³ ¹Tibor Rubin VA Medical Center; ²Southern Iron Disorders Center; ³University of California, Irvine; ⁴Chao Family Comprehensive Cancer Center; ⁵QIMR Berghofer Medical Research Institute; ⁶The University of Queensland; ⁷Queensland University of Technology; ⁸London Health Sciences Centre; ⁹Rochester General Hospital; ¹ The University of Melbourne; ¹¹University of Utah School of Medicine; ¹²University of Washington; ¹³University of Alabama at Birmingham Presented By: Gordon McLaren, MD Introduction: We sought to identify risk factors associated with cirrhosis in HFE p.C282Y homozygotes. Methods: Each participant underwent GNPAT p.D519G genotyping. We compared participants with and without biopsy- proven cirrhosis. We performed model selection using multivariable logistic regression with backward selection on biopsy- proven cirrhosis with: age at diagnosis; sex; iron supplement use; whole blood units donated; erythrocyte units received; mean daily alcohol intake, g; diabetes; body mass index; swollen/tender 2nd/3rd metacarpophalangeal joints; elevated alanine aminotransferase activity; elevated aspartate aminotransferase activity; steatosis/fatty liver; GNPAT p.D519G positivity; and iron removed by induction phlebotomy, g. Results: The mean age of 110 participants (70.0% men) was 50 ± 12 (SD) y. Forty-six participants (41.8%) had GNPAT p.D519G. Seventy-nine participants (71.8%) underwent liver biopsy. Nineteen participants (17.3%) had biopsy-proven cirrhosis. Participants with cirrhosis had significantly greater mean daily alcohol intake, prevalence of swollen/tender metacarpophalangeal joints, serum ferritin, and elevated aminotransferase activity, and mean iron removed. In a multivariable model for cirrhosis, there were significant associations with: age (odds ratio 1.096 [95% confidence interval 1.009, 1.190]); mean daily alcohol intake (1.047 [1.006, 1.089]); elevated aspartate aminotransferase activity (10.142 [1.742, 59.028]); and iron removed (1.244 [1.092, 1.416]). There was no significant association of cirrhosis with GNPAT p.D519G. There was no statistical evidence of any two-way interactions between these four variables determining the risk of cirrhosis. As shown in Figure 1, the risk of cirrhosis increases by 6.2 times with daily alcohol intake of 40 g, compared to that of no alcohol consumption [1.29, 29.98], adjusted for age at diagnosis, elevated AST activity, and amount iron removed to achieve iron depletion. For an estimated daily alcohol intake of 50 g, the risk of cirrhosis increases 9.8-fold [1.38, 70.15], adjusted for the additional predictors. In participants whose iron removed to achieve iron depletion was 10 g, the risk of cirrhosis increases by 8.4 times [2.41, 32.43]. With 15 g of iron removed to achieve iron depletion, the risk of cirrhosis increases by 26.3 times [3.74, 184.68], adjusted for age at diagnosis, mean daily alcohol intake, and elevated AST. Conclusions: Cirrhosis in HFE p.C282Y homozygotes is significantly associated with age, daily alcohol intake, elevated aspartate aminotransferase activity, and iron removed by induction phlebotomy, taking into account the effect of other variables. Seventh Congress of the International BioIron Society Page 356 Late-Breaking Abstracts IBIS Poster #LB35 IDENTIFICATION OF THE NUCLEATION SITE FOR IRON BIOMINERALIZATION IN L-FERRITIN BY X-RAY CRYSTALLOGRAPHY Silvia Ciambellotti¹,³, Cecilia Pozzi², Caterina Bernacchioni¹,³, Flavio Di Pisa², Stefano Mangani² and Paola Turano¹,³ ¹Center for Magnetic Resonance , University of Florence; ²Department of Biotechnology, Chemistry, and Pharmacy, University of Siena; ³Department of Chemistry, University of Florence Presented By: Silvia Ciambellotti Ferritins are 24-mer iron-storage nanocage proteins that concentrate iron in their inner compartment as a bioavailable iron oxide biomineral. They are generally heteropolymers composed by two different types of subunits, the heavy H and the light L (183 and 175 amino acids, respectively, in the human chains). The relative ratios of the two types of subunits vary in different tissues. H-subunit contains a di-iron ferroxidase center, capable of rapidly oxidizing Fe2+ to Fe3+ and H-richer ferritins are abundant in organs with fast iron metabolism like heart and brain. The L-subunit lacks catalytic activity but is proposed to facilitate the growth of the ferric biomineral via iron oxidation at specific, yet unobserved nucleation sites. This subunit is prevalently found in ferritins from organs involved in long-term iron storage, such as liver and spleen. The multistep biomineralization process in ferritin is governed by a number of specific, but weak, interactions between the protein shell and the iron species moving across the cage. This situation constituted the major problem for crystallography. Time-lapse anomalous X-ray diffraction technique let us to solve crystal structures of the iron-loaded homopolymeric L-ferritin (1.98 Å resolution) obtained by freezing protein crystals at increasing exposure times to a ferrous solution. We detected for the first time iron bound at the inner cage sites of L-subunits in the form of a tri iron(III) triperoxo cluster. Structures captured during iron uptake show that the formation of the iron biomineral proceeds via the assembly of a tri-nuclear iron cluster, anchored to the protein via glutamic acid side chains of Glu60, Glu61 and Glu64 and involving oxo and peroxo ligands that are produced during the iron(II) oxidation by dioxygen. To corroborate X-ray data and establish a functional significance for the observed patch of carboxylate side chains and the resulting cluster, site-directed mutagenesis was applied to produce the E60AE61AE64A mutant in order to prevent iron coordination at these sites. Both homopolymeric wild type and mutant L-ferritin samples were produced by heterologous expression in E.coli competent cells. The comparison of the iron oxidation kinetics in the wild type and E60AE61AE64A variant of human L-ferritin nanocage showed a significantly lower reaction rate for the mutant. All together, these results provide a direct evidence of the glutamate-bound triiron cluster as the mineral nucleation site in L-ferritins. A detailed understanding of the biomineralization mechanism in L-cages might help elucidating abnormal iron metabolism in neurodegenerative disorders, such as neuroferritinopathy, caused by mutations in the ferritin L chain. Seventh Congress of the International BioIron Society Page 357 Late-Breaking Abstracts IBIS Poster #LB36 IDENTIFICATION OF STAPHYLOCOCCUS AUREUS IRON ACQUISITION LOCI REQUIRED FOR REPLICATION WITHIN MACROPHAGES David Heinrichs and Ron Flannagan, PhD University of Western Ontario Presented By: David Heinrichs, PhD Staphylococcus aureus is an important bacterial pathogen capable of infecting virtually every tissue of the body. Although phagocytes, like macrophages, play an important role in immunity, they also can contribute to the dissemination of S. aureus disease. We have demonstrated that S. aureus is capable of intracellular replication within macrophages and that replication proceeds inside mature phagolysosomes. Interestingly the onset of bacterial proliferation is delayed, occurring several hours (6 to 12h) post-infection, suggesting the bacteria must first overcome growth restriction mechanisms within this niche. We hypothesize this delay is due to the need for S. aureus to overcome nutrient restriction and acquire iron intracellularly. Using in vitro models of macrophage infection and fluorescence microscopy, we demonstrate that S. aureus is indeed confined to CD63 and LAMP1-positive phagosomes where at 12h post-infection bacterial proliferation can be detected. Previously it has been suggested that phagolysosome acidification is required for S. aureus growth in macrophages. Conceivably acidification could also impact on the bioavailability of iron within this niche. To test this notion infected macrophages were treated with inhibitors of the vacuolar ATPase which effectively cause alkalinization of the S. aureus-containing phagosome as revealed by lysotracker staining. Importantly, using a fluorescent indicator dye to report on bacterial proliferation, we find that inhibitors of phagolysosome acidification neither enhance nor inhibit S. aureus growth within macrophages. However, since modulation of acidification events will impact on the genetic loci required by S. aureus to acquire iron and proliferate, it is not surprising that S. aureus is highly evolved to acquire iron in different forms, as it possesses acquisition mechanisms for heme, and both ferric and ferrous iron. We tested several mutants, containing knockouts in established iron acquisition loci, for their ability to replicate within the intraphagosomal compartment. Using fluorescence microscopy to follow bacterial replication within the mature phagolysosome, we found that mutants impaired for uptake of either ferrous or ferric iron alone were unimpaired for replication within macrophages, however, combining mutations in both ferrous and ferric iron acquisition genes resulted in a strain unable to replicate within mature phagolysosomes. Understanding these mechanisms in detail, and the timing of their involvement in bacterial proliferation, will yield information useful for the development of novel deliverables that could aid in combating S. aureus infections. Seventh Congress of the International BioIron Society Page 358 Late-Breaking Abstracts IBIS Poster #LB37 THE CORE COMPONENT OF THE MITOCHONDRIAL CALCIUM UNIPORTER (MCU) PROMOTES ACETAMINOPHEN HEPATOXICITY AND IS REQUIRED FOR MITOCHONDRIAL Fe2+ UPTAKE Jiangting Hu, MD, PhD², James Weemhoff, PhD³, Andaleb Kholmukhamedov, MD², Hartmut Jaeschke, PhD³, Anna-Liisa Nieminen, PhD² and John Lemasters, MD, PhD¹ ¹Medical University of South Carolina; ²Medical Univeristy of South Carolina; ³University of Kansas Medical Center Presented By: John Lemasters, MD, PhD Background: Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and the mitochondrial permeability transition (MPT). Previous studies show that iron translocation from lysosomes into mitochondria by the electrogenic mitochondrial calcium uniporter promotes the MPT after APAP. Here, our Aim was to investigate the role of MCU, the core protein of the mitochondrial calcium uniporter complex, in mitochondrial iron uptake during APAP-induced hepatotoxicity both in vitro and in vivo. Methods: Mouse hepatocytes were isolated from MCU knockout mice and wild type (CD-1) mice. Mitochondrial membrane potential and cell death were visualized by confocal microscopy of rhodamine 123 and propidium iodide, respectively. Mitochondrial chelatable Fe2+ was monitored by fluorescence quenching of mitoferrofluor (MFF), a mitochondrially targeted reporter. For in vivo studies, MCU knockout mice and wild type mice were treated with an overdose (300 mg/kg, i.p.) of APAP or vehicle. Liver injury was assessed by ALT and histology at 24 h after APAP. Results: In cultured hepatocytes, progressive quenching of MFF began at ~4 h after APAP (10 mM), becoming maximal within 11 h, signifying increased mitochondrial chelatable Fe2+. Dipyridyl, a membrane-permeable iron chelator, partially restored quenched MFF fluorescence. Mitochondria then depolarized after ~11 h. Cells subsequently lost viability, as indicated by nuclear PI staining. By contrast, mitochondrial MFF quenching, mitochondrial depolarization and cell death were suppressed in MCU knockout hepatocytes. In vivo at 24 h after APAP overdose to wild-type mice, serum ALT increased to 5838 ± 1658 units/L, indicating severe liver injury. In MCU knockout mice, ALT rose to only 559 ± 216 units/L (p<0.01). Histology at 24 h after APAP administration revealed centrilobular necrosis with hemorrhage in 35.7% of cross sectional areas in wild type mice but only 2.5% in MCU knockout mice. CYP2E1 expression and APAP-protein adduct formation were not different between two groups, indicating equivalent APAP metabolism in the two mouse strains. MCU knockout mice also had similar JNK activation at 2 h after APAP as wild type mice. 2+ Conclusions: MCU is required for uptake of chelatable Fe into mitochondria during APAP hepatotoxicity, which promotes the MPT and cell killing both in vitro and in vivo. Seventh Congress of the International BioIron Society Page 359 Late-Breaking Abstracts IBIS Poster #LB38 PRETREATMENT OF RICE SEEDS WITH IRON AND ZINC DECREASES CADMIUM TRANSPORT IN RICE DURING GROWTH Di Guan1, Xionghui Ji1, and Xi Huang2 1Hunan Academy of Agricultural Sciences, Changsha, China and 2Innovative FLM, LLC, New Jersey, USA Presented By: Di Guan Rice is the one of the main food crops for human and China's rice production ranks first in the world. Among them, Hunan production is 23.2×106 tones, accounting for 12.7% of the national’s total yield. Hunan province is abundant in mineral resources. However, 72.6% of the cultivated land is acidified and this leads to increased levels of metal ions in soils. For example, the total cultivated land contaminated by heavy metals, such as Cd, Pb, Hg, As, and Cr, are approaching to 9.4×105 hectares and especially, cadmium levels could be as high as 354 times of the government permissible levels in certain industrial and mining areas. When cadmium enters into the human body, it triggers adverse health effects. In the present study, we have tested a hypothesis that cadmium shares the same absorption and transport pathways with iron and zinc and, pretreatment of rice seeds with iron and zinc downregulates these genes and therefore decrease cadmium absorption and transport during growth. After an initial screening, rice species Yuzhenxiang with highest capacity of absorbing Cd was selected for this study. Various concentrations of iron (40, 100, 200 mM), zinc (0.32, 0.75, and 1.5 mM), and iron and zinc (100 + 0.75 and 200 + 1.5 mM) mixtures were added to the micronutrients growth media. Rice seeds were placed in the media for 30 days and parts of seedlings were collected for Fe and Zn analyses, and the remaining were transferred into a media containing 50 mM Cd for 5 days. We have found that iron and zinc levels significantly increase in a Fe- and Zn-dose dependent manner in the roots and the iron level can reach as high as 4,000 mg/kg and zinc level can reach 160 mg/kg in the highest dose treatments, respectively. These trends remain in Zn treatment but are less evident in iron and iron/zinc mixture treatments in the stems. After transferring rice seedling into the Cd-containing media for 5 days, Cd levels reach 1,600 mg/kg in the roots and 600 mg/kg in the stems in Cd treatment alone. Cd level in the stems of highest iron treatment is the lowest. By dividing the levels of Cd in the stem to the root, the translocation factor is approximately 18.5% in both highest iron treatment and iron/zinc (100 + 0.75 mM) treatments as compared to 36% at the Cd alone treatment. In conclusion, our study suggests that pretreatment of rice seeds with iron/zinc appears no effect on Cd absorption, but could inhibit Cd transport from the root to the stem. Because of the cold weather, rice grains cannot be collected for Cd measurement in this preliminary study. Therefore, pretreatment of the seeds with Fe and/or Zn on Cd level in the rice grains remains unknown and this awaits further investigation. Seventh Congress of the International BioIron Society Page 360 Late-Breaking Abstracts IBIS Poster #LB39 ERFE-INDEPENDENT REGULATION OF HEPCIDIN IN A MOUSE MODEL OF INFLAMMATORY BOWEL DISEASE Ugo Sardo, Benjamin Billoré, BSc, Perrine Rousset, MSc and Leon Kautz, PhD Inserm U1220/IRSD Presented By: Ugo Sardo Ulcerative colitis and Crohn’s disease, collectively termed inflammatory bowel disease (IBD), are immunologic disorders that represent the prototypes of chronic intestinal inflammation. Their combined prevalence is more than 300/100.000 individuals in Western populations. Notably, iron-deficiency anemia is the most common complication of inflammatory bowel disease, both at diagnosis and during flare-ups, exceeding by far the frequency of extraintestinal manifestations (e.g., rheumatic, dermatologic, and ophthalmologic). It is only partly explained by iron loss from bleeding due to gastrointestinal inflammation and results essentially from an inflammation-driven blockade of intestinal iron acquisition and macrophage iron reutilization, a hallmark of anemia of chronic disease. The liver-produced hormone hepcidin regulates the body iron stores. Its expression is induced by inflammatory cytokines but repressed by the erythroid regulator erythroferrone (ERFE) when erythropoietic activity intensifies during anemia. However, the exact contribution of hepcidin and its regulation during IBD are not known and inappropriately high or low hepcidin levels have been observed in different IBD murine models. Dextran sulfate sodium (DSS)–induced colitis in WT mice is accompanied by a severe anemia with elevated EPO levels and decreased hepcidin expression. Moreover, inhibition of erythropoiesis with carboplatin prevented the suppression of hepcidin in DSS-treated mice (Shanmugam, 2014). We therefore investigated the regulation of hepcidin and erythroferrone during anemia caused by DSS administration in mice. C57BL/6 mice were given 3% (w/v) DSS in drinking water for 3, 5 or 7 days. The mice exhibited an IBD-like phenotype similar to the symptoms observed in human patients and a severe anemia 7 days after treatment with DSS. Although hepcidin levels were unchanged at days 3 and 5, hepatic hepcidin mRNA expression was significantly reduced 10-fold 7 days after treatment with DSS without any significant change in liver, spleen or serum iron concentration. Similarly, our results indicate that the hepcidin-regulatory pathways IL6/STAT3 and BMP/SMAD are not impaired by the DSS treatment. However, Erfe mRNA expression was significantly increased in the bone marrow and the spleen 7 days after treatment with DSS. To determine whether ERFE is responsible for the DSS-induced repression of hepcidin, we compared WT and Erfe-deficient after DSS-induced colitis. Interestingly, liver hepcidin mRNA expression was similarly suppressed in WT and Erfe-deficient mice 7 days after treatment with DSS compared to control mice. Furthermore, Erfe-deficient exhibited iron and hematological parameters comparable to those of WT mice. Finally, assessment of colon damage by macroscopic scoring did not reveal any difference between WT and Erfe-deficient mice. Our results suggest that ERFE is not responsible for hepcidin suppression during DSS-induced colitis. Further work will be necessary to determine the mechanism involved in the regulation of hepcidin production in this mouse model of inflammatory bowel disease Seventh Congress of the International BioIron Society Page 361 Late-Breaking Abstracts IBIS Poster #LB40 RESTORED IRON TRANSPORT BY A SMALL MOLECULE PROMOTES GUT IRON ABSORPTION AND HEMOGLOBINIZATION IN MICE, RATS, AND ZEBRAFISH Alexander Cioffi, Anthony Grillo, Anna SantaMaria and Martin Burke, MD/PhD University of Illinois at Urbana-Champaign Presented By: Alexander Cioffi There are more than 25 currently known hereditary diseases caused by deficiencies in iron regulation and homeostasis, making disorders of iron metabolism some of the most prevalent human diseases worldwide. Given all of the advantageous features that make small molecules effective therapeutics, we questioned whether small molecules might also restore physiology by autonomously replicating the functions of deficient proteins. Because endogenous iron- dependent networks of transporters and regulators remain active, we hypothesized a build-up of labile iron would occur upstream of the missing iron transporters. We thus probed whether a small molecule could harness these gradients to move iron into, within, and out of cells, thereby restoring physiology in models of iron metabolic disorders (Fig. 1A). To find such a molecule, we designed a modified functional complementation experiment, in which compounds known or predicted to bind iron were tested for their capacity to restore growth in a strain of S. cerevisiae missing the iron transporting complex FetFtr1. We discovered the small molecule natural product, hinokitiol, can autonomously transport iron and thereby restore physiology to this iron-deficient yeast. To test the hypothesis that deficiencies of iron transporters lead to a build-up of labile iron upstream of the deficiency, we chose to study DMT1-deficient mouse erythroleukemia (MEL) cells and FPN1-deficient J774 macrophages. Using spatiotemporal imaging with iron-sensitive fluorescent dyes, we observed increased endosomal iron and reduced cytosolic and mitochondrial iron in DMT1-deficient MEL cells. We discovered hinokitiol could leverage these gradients to release iron from endosomes and increase cytosolic and mitochondrial iron levels (Fig. 1C). Further fluorescent studies also revealed a build-up of labile iron in FPN1-deficient J774 macrophages relative to wild type cells. Hinokitiol direction-selectively promotes both iron influx (Fig. 1B) and efflux from J774 macrophages depending on the presence of high extracellular or intracellular iron, respectively. Finally, we tested if hinokitiol treatment could restore hemoglobinization and reverse anemia in Mfrn1-deficient frascati (frs/frs) embryos (Fig. 1D). Addition of hinokitiol to the water of Mfrn1-deficient zebrafish embryos 24 hours post fertilization and incubation for an additional 48 hours reversed the severe anemia observed in this protein-deficient zebrafish. Thus, a small molecule restores site- and direction-selective iron transport in cells deficient in distinct iron-transport proteins. Mechanistic studies support the role of transmembrane ion gradients that build up in the setting of missing iron transporters, enabling hinokitiol to utilize these gradients to restore physiology. These findings suggest small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases. Seventh Congress of the International BioIron Society Page 362 Late-Breaking Abstracts IBIS Poster #LB41 RESTORED IRON TRANSPORT BY A SMALL MOLECULE PROMOTES GUT IRON ABSORPTION AND HEMOGLOBINIZATION IN MICE, RATS, AND ZEBRAFISH Anna SantaMaria, Anthony Grillo, Alex Cioffi and Martin Burke, MD/PhD University of Illinois at Urbana-Champaign Presented By: Anna SantaMaria More than twenty-five human diseases are associated with aberrant iron transport, homeostasis, or metabolism. In many cases, a deficiency of a critical iron transporting protein leads to the inability to move iron into, within, or out of cells. As the endogenous iron-dependent networks of transporters and regulators remain active, we hypothesized a build-up of labile iron would occur upstream of the membrane that normally hosts these missing proteins. In this vein, we hypothesized a small molecule mimic of these missing iron transporters may be able to restore the movement of iron in a site- and direction-selective manner, thereby restoring physiology (Fig. 1A). To test this, we chose three disease-relevant iron transporter deficiencies that disrupt iron movement in different directions, cellular locations, and tissues. Deficiencies of DMT1 reduce apical iron uptake into duodenal enterocytes and prevents endosomal iron release in erythroid precursors. Mfrn1 deficiencies in the inner mitochondrial membrane impair iron import into the mitochondrial matrix. FPN1 deficiencies reduce iron efflux from gut epithelium and macrophages. We questioned whether a lipophilic small molecule that performs transmembrane iron transport (Fig. 1B) could promote physiology in cells and animals missing each of the aforementioned proteins. We discovered the natural product hinokitiol can restore growth to yeast deficient in an iron transporting complex (Fet3Ftr1). We further developed DMT1-deficient Caco-2 gut epithelia and DMT1- and Mfrn1- deficient mouse erythroleukemia (MEL) cells, which exhibit reduced transepithelial transport and hemoglobinization, respectively. Hinokitiol restored iron uptake into and transport across DMT1-deficient monolayers. Further, hinokitiol treatment restored hemoglobinization in DMT1- and Mfrn1-deficient MEL cells. Finally, transient knockdown of FPN1 in Caco-2 gut epithelia and J774 macrophages with quercetin and hepcidin, respectively, led to decreased transepithelial transport in FPN1-deficient Caco-2 monolayers and reduced iron release in FPN1-deficient J774 macrophages. Treatment of hinokitiol restored transport and iron release in these cells. These results collectively suggest hinokitiol can restore the transport of iron into, within, and/or out of cells missing three distinct iron transporting proteins. We finally asked if hinokitiol treatment can restore gut iron absorption or hemoglobinization in leading animal models of these protein deficiencies. Treatment of 1.5 mg/kg hinokitiol via oral gavage promoted the gut absorption of 59Fe into the blood of DMT1-deficient Belgrade (b/b) rats and FPN1-deficient Flatiron (ffe/+) mice (Fig. 1C, D). Furthermore, addition of hinokitiol to the water of DMT1- and Mfrn1-deficient zebrafish embryos 24 hours post fertilization and incubation for an additional 48 hours reversed the severe anemia observed in these protein-deficient zebrafish (Fig. 1E). Collectively, these results support that a small molecule iron transporter may have untapped potential in the treatment of diseases resulting from a deficiency of iron transporting proteins. Seventh Congress of the International BioIron Society Page 363 Late-Breaking Abstracts IBIS Poster #LB42 TREATING DARK CIRCLE UNDER THE EYE BY ADDRESSING IRON IN FERRITIN AND HEMOGLOBIN Xi Huang, PhD Marivan Skincare Inc. Presented By: Xi Huang, PhD We have previously shown that ferritin increases during menopausal transition and iron released from ferritin contributes to skin aging and photo-aging. By using an iron removal technology (IRT), skin wrinkles can be significantly reduced after four weeks use of the topical cream. In the present study, the dark circles under the eye and their cause and treatment have been investigated. While dark circles may not be pathologic, women are highly concerned about this cosmetic impairment and dark circles clearly are a concern for quality of life for women. Despite its prevalence and cosmetic importance, there are few published studies in identifying the etiological factors and providing treatment solutions. In the present study, we tested a hypothesis that dermal melanin deposition in association with ferritin and hemoglobin accumulation as a result of inflammation-mediated blood leakage contribute to the formation of the dark circle under the eye. Therefore, treatment of skin with IRT and natural heme oxygenase inducer should reduce the darkness of skin under the eye. We have previously shown in our research that normal human epidermal keratinocytes (NHEK) contain higher levels of hydrogen peroxide (H2O2) than melanocytes. Nevertheless, despite lower concentrations of H2O2, higher levels of oxidative DNA in melanocytes as indicated by increased levels of 8-oxo-2’-deoxyguanosine is observed when compared to those in NHEK. Cellular bioavailable iron was also measured as a function of ferritin concentration and found to be nearly four-fold higher in melanocytes than in NHEK. Further, ferritin levels in melanocytes were also higher than in HepG2 cells, an iron-rich hepatocarcinoma cell line, and indicate that higher relative iron levels may be characteristic of melanocytes. By formulating a cream containing both IRT and heme oxygenase-1 inducer, ferritin and hemoglobin are targeted. We have shown that dark circle under the eye can be significantly reduced by 50% and the effect can be seen within six days of treatments. Taken together, our results indicate that high level of ferritin in melanocytes and hemoglobin accumulations in the epidermis are important etiological factors in the dark circle under the eye. In consideration of that iron is the most abundant transition metals in the human body and skin is a major route to excrete iron, skin as an important target of iron and the role of iron in aging, photo-aging, excessive pigmentation, and even melanoma should draw greater attention in the Bio Iron community. By identifying iron as the cause with further investigation, we may be able to provide the appropriate treatments according to the cause. Seventh Congress of the International BioIron Society Page 364 Late-Breaking Abstracts IBIS Poster #LB43 RETINAL IRON OVERLOAD ACCELERATES THE PROGRESSION OF DIABETIC RETINOPATHY Kapil Chaudhary, MD, PhD², Wanwisa Promsote, PhD³, Sudha Ananth, MSc⁴, Rajalakshmi Veeranan-Karmegam, MSc⁴, Amany Tawfik, PhD⁴, Pamela Martin, PhD⁴, Sylvia Smith, PhD⁴, Vadivel Ganapathy, PhD⁵ and Jaya P. Gnana-Prakasam, PhD¹ ¹Saint Louis University, St Louis, MO; ²Washington University, St Louis, MO; ³National Institute of Health, Bethesda, MD; ⁴Augusta University, Augusta, GA; ⁵Texas Tech Health Sciences University, Lubbock, TX Presented By: Jaya P. Gnana-Prakasam, PhD Diabetic retinopathy (DR) is a leading cause of blindness among adults in U.S. and worldwide. During DR, retina may accumulate iron secondary to inflammation and hemorrhages. Iron accumulation due to dysregulation of local iron homeostasis has been associated with several degenerative diseases. Surprisingly, the potential role of impaired iron metabolism in the pathogenesis of DR has not been elucidated. Although there are clinical reports on positive link between iron levels and proliferative retinopathies, the mechanism by which iron exacerbates the progression of DR is unknown. Iron is required for the normal function of many proteins in retina but excess iron is toxic. Hence stringent mechanisms maintain iron levels by regulating proteins involved in iron homeostasis. In this study, we found increased iron accumulation in the retinas of type 1 (streptozotocin, STZ-induced) and type 2 (C57BLKS-Leprdb, db/db) diabetic mice. We found a marked increase in ferritin levels by ELISA, western blot and immunofluorescence in the diabetic mice retina compared to the non-diabetic control retina. The expression levels of iron regulatory proteins were also drastically altered in the retinas of type I and type II mice models of diabetes. Similarly, the expression profile of iron regulatory proteins in postmortem retina samples obtained from human diabetic patients were altered. HFE, an important iron regulatory protein, senses cellular iron status and regulates iron uptake by competitively inhibiting transferrin receptor and regulating hepcidin expression. We have previously reported that HFE is expressed predominantly in the basolateral membrane of retinal pigment epithelium and HFE knockout (KO) mice have age related accumulation of iron in retina with significant retinal degeneration by 18 months of age. To elucidate the mechanism by which iron accumulation affects the progression of DR, we induced diabetes in wild type (WT) mice and HFE KO mice model of iron overload using STZ. Diabetic HFE KO mice show increased neuronal cell death, vascular alterations and loss of outer retinal barrier integrity compared to diabetic WT mice. Accelerated progression of DR in HFE KO diabetic mice was accompanied by increase in retinal prorenin/renin expression mediated by G-protein coupled succinate receptor GPR91. Our results reveal a novel and important relationship between diabetes, iron and renin-angiotensin system unraveling new therapeutic targets for the treatment of diabetic retinopathy and likely other vascular complications of diabetes. Seventh Congress of the International BioIron Society Page 365 Late-Breaking Abstracts IBIS Poster #LB44 MITOFERRIN 2 ASSOCIATES WITH MCU, THE CORE PROTEIN OF THE MITOCHONDRIAL CALCIUM UNIPORTER Anna-Liisa Nieminen, Monika Gooz, MD, PhD and John Lemasters, MD, PhD Medical University of South Carolina Presented By: Anna-Liisa Nieminen, PhD Excess mitochondrial accumulation of calcium and iron promotes mitochondrial dysfunction in many pathological conditions. Iron-dependent formation of reactive oxygen species (ROS) and/or calcium uptake induce the mitochondrial permeability transition (MPT), a pathological state of mitochondria that leads to both necrosis and apoptosis. Mitoferrin (Mfrn) 1 and 2 mediate mitochondrial iron uptake in hematopoietic and non-hematopoietic tissues, respectively (Mol Cell Biol 2009;29:1007), whereas the CCDC109A gene product MCU is the core protein of the electrogenic calcium uniporter complex that catalyzes mitochondrial Ca2+ uptake driven by the negative-inside mitochondrial membrane potential (Nature 2011;476:336&341). Ru360, a specific inhibitor of the mitochondrial calcium uniporter, also inhibits Fe2+ translocation into mitochondria after release from lysosomes (Hepatology 2008;48:1644, FRBM 2013;63:243, FRBM 2016;97:418, Tox Sci 2010;117:101). Here, our aim was to determine whether the apparently shared pathway of mitochondrial Fe2+ and Ca2+ uptake occurs via a physical association of Mfrn 2 and MCU. In HeLa cells co-transfected with MCU-V5 and Mfrn 2-GFP, anti-GFP beads pulled down MCU-V5, whereas anti-V5 beads pulled down Mfrn 2-GFP. Subunit 4 of cytochrome oxidase (COX-IV), a mitochondrial inner membrane protein, was not pulled down, indicating that the interaction between MCU and Mfrn 2 was specific. Duolink proximity ligation assays (PLA) also showed a molecular association of MCU and Mfrn 2 in HeLa cells. By contrast, PLA showed no molecular proximity of Mfrn 2 and COX IV. In conclusion, these findings indicate a direct physical interaction between Mfrn 2 and MCU, consistent with the conclusion that Mfrn 2 is a component/regulator of the electrogenic mitochondria calcium uniporter complex. Therefore, the mitochondrial calcium uniporter might more appropriately be called the mitochondrial Ca,Fe uniporter (MCFU). Seventh Congress of the International BioIron Society Page 366 Late-Breaking Abstracts IBIS Poster #LB45 DISTAL TUBULAR IRON LOADING IN EXPERIMENTAL FOCAL SEGMENTAL GLOMERULOSCLEROSIS Rachel van Swelm, Henry Dijkman, Jack Wetzels, Erwin Wiegerinck, Rian Roelofs, Johan van der Vlag, Bart Smeets and Dorine Swinkels Radboud University Medical Center Presented By: Rachel Van Swelm, PhD Chronic kidney disease (CKD) affects 5-7% of the worldwide population. The most important risk factor for disease progression is proteinuria, which is strongly associated with tubulointerstitial injury. Current treatment consists primarily of proteinuria reduction, which is, however, not adequate to prevent disease progression in many patients. There is an unmet need for effective treatment modalities for patients with persistent proteinuria, which could be addressed by more targeted interventions focused at preventing the tubulointerstitial injury that is associated with proteinuria. The mechanisms of proteinuria-induced tubulointerstitial injury have not been fully elucidated yet, but may include cellular injury catalyzed by reactive iron. In an experimental model for proteinuric CKD, the Thy-1.1 mice, we recently observed increasing distal tubular iron accumulation in line with progressive tubulointerstitial injury. Here, we studied the molecular mechanisms of distal tubular iron loading in progressive CKD in Thy-1.1 mice as potential targets for intervention. Moreover, we assessed the effect of iron deficiency and iron loading on disease progression. Transgenic Thy-1.1 mice were injected with a monoclonal anti-Thy-1.1 antibody (mAb) to induce CKD or saline as control. Mice were placed in a metabolic cage to collect 24h urine and sacrificed subsequently at the following time points: immediately after injection (D1), day 5 (D5) day 8 (D8) and day 22 (D22). Localization and expression of proteins involved in iron handling were assessed using immunohistochemistry, Western blot and QPCR. Some mice were given an iron-deficient or iron-rich diet to assess the role of iron in renal injury. Injection mAb resulted in immediate and persistent proteinuria indicated by increased albuminuria (p<0.05 D5; p<0.001 D8 and D22) compared to control, accompanied by polyuria (p<0.01 D5; p<0.001 D8 and D22). Renal injury was confirmed by standard histology and elevated renal mRNA expression of kidney injury markers IL-6 (p<0.01 D1; p<0.001 D5, D8 and D22) and HO-1 (p<0.05 D1, D4, D8) in mAb-injected mice, compared to control. Enhanced distal tubular iron deposition was observed with increasing renal injury in mAb-treated mice as assessed by Perl’s staining. MAb-injected mice demonstrated increased mRNA expression of lipocalin-2 receptor (p<0.001; D7), which is localized in the distal tubules and can facilitate transferrin-bound iron uptake. Although mRNA expression of ferroportin was increased (p<0.001 D1; p<0.01 D5, D8; p<0.05 D22), immunohistochemistry revealed ferroportin localization exclusively in the proximal tubules. The absence of ferroportin in the distal tubules in combination with the increased expression of lipocalin-2 receptor may account for the observed distal iron loading during proteinuric CKD in Thy-1.1 mice. Mice subjected to either an iron-deficient or iron-rich diet demonstrated significantly reduced polyuria compared to normal diet on D8 (p<0.001 and p<0.05, respectively) and D22 (p<0.001 for both), although albuminuria was not significantly changed. Nevertheless, preliminary results showed that mice on iron deficient-diet presented with reduced histological distal iron loading and renal injury (D8) compared to mice on normal diet, whereas the opposite was observed for mice on iron-rich diet (D8). Ongoing analysis will further demonstrate the effect of iron status on disease progression. Moreover, we will use in vivo silencing to functionally evaluate the mechanisms of distal tubular iron loading in mAb-injected Thy-1.1 mice. Seventh Congress of the International BioIron Society Page 367 Late-Breaking Abstracts IBIS Poster #LB46 IRON REGULATORY PROTEINS REGULATE FRATAXIN AND ISCU TO SECURE MITOCHONDRIAL FUNCTION Huihui Li, BS and Kuanyu Li Nanjing University Presented By: Kuanyu Li Iron is essential for growth and proliferation of mammalian cells. The maintenance of cellular iron homeostasis is regulated by iron regulatory proteins (IRPs), which can bind the iron-responsive elements (IREs) of target mRNAs and regulate the expression of these genes. Here, we report that Irp1 or Irp2-null mutation not only reduces the level of cellular iron by decreasing TfR1 and increasing ferritin, but also causes the downregulation of Fxn and IscU, two of the core components of iron sulfur cluster (Fe-S) biogenesis. Interestingly, the activities of some of Fe-S-containing enzymes including mitochondrial aconitase and cytosolic xanthine oxidase are not affected, but the activities of respiratory complexes are specifically drastically diminished to make mitochondria dysfunction. Overexpression of human ISCU and FXN in Irp1 or Irp2-null cells rescues the deficit of Fe-S cluster biogenesis and mitochondrial quality, whereas addition of iron is not able to accomplish it though IscU and Fxn were upregulated by iron to a certain level. Our results indicate that IRPs modulate mitochondrial iron metabolism, at least partially, through regulating the expression of the core components for Fe-S cluster biogenesis and for Fe-S directed delivery to electron transport chain complexes. Key words: Iron regulatory proteins (IRPs), iron homeostasis, iron-sulfur cluster biogenesis, mitochondrial function Seventh Congress of the International BioIron Society Page 368 Late-Breaking Abstracts IBIS Poster #LB47 CHARACTERIZATION OF IRON HANDLING IN HEALTHY AND DISEASED HUMAN RENAL BIOPSIES Sanne van Raaij¹, Rachel van Swelm, PhD¹, Karlijn Bouman¹, Maaike Bouman¹, Paul Bass, MD², Robert Unwin, PhD², Jeanne Pertijs¹, Dorine Swinkels, PhD¹ and Kaila Srai, PhD³ ¹Radboud University Medical Center, Nijmegen, the Netherlands; ²Royal Free Hospital, London, United Kingdom; ³University College London, London, United Kingdom Presented By: Sanne van Raaij Increasing preclinical evidence suggests a detrimental effect of iron in the onset and/or progression of chronic kidney disease (CKD). Increased exposure of renal tubular epithelial cells to iron may result in oxidative stress-mediated cellular damage. However, human-based evidence for detrimental effects of iron in CKD, as well as knowledge on physiological renal iron handling, is scarce. Specifically, localization and expression of proteins involved in cellular iron handling in the normal kidney remains unclear. Here, we characterized renal iron handling in health and in CKD by using available normal renal tissue from kidneys unsuitable for transplantation and from consented patient renal biopsy material. After histological assessment by a trained renal pathologist, biopsies were classified as healthy control (n=6), established diabetic nephropathy (DNE; n=8), advanced diabetic nephropathy (DNA; n=5), focal segmental glomerulosclerosis (FSGS; n=5), lupus nephritis (LN; n=5) and IgA nephropathy (IgAN; n=5). Perl’s staining assessed iron deposition, whereas localization and intensity of various proteins involved in cellular iron handling were determined by immunohistochemistry and quantified using ImageJ. We observed that iron deposition was absent in biopsies of healthy controls, but moderately present in 1-3 biopsies for each of the various nephropathies. In healthy control biopsies we found the putative cellular iron importers DMT-1, ZIP8 and ZIP14 in proximal and distal tubules with apical and intracellular localization. ZIP8 appeared more intense in the distal tubules compared with the proximal tubules. Interestingly, iron storage proteins L- and H-ferritin, as well as the cellular iron exporter ferroportin, were found in proximal tubules only; L- and H-ferritin localized intracellularly and ferroportin was present at the basolateral membrane. In proximal tubules, iron deposition in DNA and DNE was associated with increased H-ferritin intensity (p<0.001 in DNA and p<0.05 in DNE, relative to control) and decreased ferroportin intensity (p<0.01 in DNA and p<0.05 in DNE), in contrast to increased ZIP14 intensity in IgAN and increased ZIP14 and L-ferritin intensity in FSGS (all p<0.001). Furthermore, iron deposition in distal tubules was associated with increased ZIP8 intensity in DNE (p<0.01), as well as increased ZIP14 expression in IgAN and FSGS (both p<0.001). In conclusion, we are the first to characterize in detail renal iron handling proteins in the human healthy kidney. Furthermore, our data show renal iron accumulation in human nephropathies, which could be the result of increased iron uptake and decreased iron export. These initial findings provide a basis for beginning to unravel iron’s handling and role in the kidney and the potential detrimental effect of iron deposition and retention in kidney disease. Seventh Congress of the International BioIron Society Page 369 Late-Breaking Abstracts IBIS Poster #LB48 ZIP14 DEFICIENCY PREVENTS IRON LOADING OF THE ANTERIOR PITUITARY AND ADRENAL GLAND Joseph Olivera, Master of Science in Food Science and Human Nutrition, Supak Jenkitkasemwong, PhD in Nutritional Sciences and Mitchell Knutson, PhD in Nutrition University of Florida Presented By: Joseph Olivera The blood disorder thalassemia major is usually diagnosed in the first two years of life and affected patients require regular blood transfusions to survive. Repeated blood transfusions eventually lead to iron overload, which predominantly affects the liver, pancreas, heart, and other endocrine tissues. These tissues load iron mainly by taking up non-transferrin- bound iron (NTBI), which appears in the plasma in iron overload. We have previously shown that NTBI uptake and iron loading by the liver and pancreas requires ZIP14 (SLC39A14), a cell-surface metal-ion transport protein. The aim of the present study was to investigate the role of ZIP14 in iron loading of the pituitary gland and adrenal gland. Iron deposition in the anterior pituitary, and subsequent damage to pituitary gonadotrophs, is thought to be a main contributor to hypogonadotrophic hypogonadism, the most common endocrinopathy in thalassemia major. Iron loading in the adrenal glands is thought to contribute to adrenal insufficiency, which is also commonly observed in thalassemia major patients. To study iron loading of the pituitary and adrenal gland, we used hemojuvelin knockout (Hfe2-/-) mice, which rapidly develop iron overload. To study the role of ZIP14 in iron loading, we used hemojuvelin and ZIP14 double-knockout mice (Hfe2-/-;Slc39a14-/-). Perls’ Prussian blue staining of paraffin-embedded pituitary gland sections revealed that Hfe2-/- mice, but not wild-type controls, accumulated iron in cells of the anterior pituitary, with little or no staining observed in the posterior pituitary. By contrast, Hfe2-/-;Slc39a14-/- mice showed no iron staining in the anterior pituitary, suggesting that ZIP14 is required for iron loading of the anterior region. Interestingly, significant iron staining was observed in the posterior pituitary of Hfe2-/-;Slc39a14-/- mice—a pattern opposite to that of Hfe2-/- mice. Iron staining in anterior pituitary of Hfe2-/- mice was found to partially co-localize with luteinizing hormone (LH) and follicle stimulating hormone (FSH), indicating that some iron deposited in gonadotrophs, as has been reported for iron-loaded human pituitary glands. As for adrenal glands, we found that Hfe2-/- mice load iron primarily in the adrenal cortex, whereas iron loading is nearly absent in adrenals of Hfe2-/-;Slc39a14-/- mice. Collectively, our data suggest that ZIP14 is required for iron loading of the anterior pituitary and adrenal gland of mice. Therefore, it is possible that therapeutic inhibition of ZIP14 may help to mitigate iron-related endocrinopathies observed in iron overload disorders such as thalassemia major. Seventh Congress of the International BioIron Society Page 370 Late-Breaking Abstracts IBIS Poster #LB49 DELETION OF BMP6 WORSENS THE PHENOTYPE OF HJV-ASSOCIATED HEMOCHROMATOSIS Chloé Latour, Céline Besson-Fournier, Ophélie Gourbeyre, Delphine Meynard, PhD, Marie-Paule Roth, MD and Hélène Coppin, PhD IRSD Inserm Presented By: Marie-Paule Roth, MD, PhD Liver sinusoidal endothelial cells (LSEC) produce bone morphogenetic proteins BMP2 and BMP6 which both act on their hepatocyte co-receptor hemojuvelin (HJV) to regulate hepcidin production in a paracrine fashion. All these molecules seem required to ensure optimal maintenance of iron homeostasis. Bmp6 and Hjv knockout mice have a severe iron loading phenotype with extrahepatic iron accumulation in males. Mice lacking LSEC Bmp2 appear to have a slightly milder phenotype than Bmp6 knockout mice, possibly due to differences in genetic background. However, males also have iron loading in the heart and the pancreas, a feature characteristic of Bmp6 deficiency but absent in Hfe, Tfr2, or Alk2 knockout males. This suggests that Bmp2 could be a hepcidin regulator as important as Bmp6. Notably, interaction of Bmp2/6 heterodimers with Hjv seems necessary for optimal production of hepcidin. Indeed, in the absence of Bmp6 (or Bmp2), activation of Smad1/5/8 is ensured in a less effective way by the interaction of Bmp2 (or Bmp6) homodimers with Hjv. Whether Bmp6 or Bmp2 may also signal to hepcidin independently of Hjv is still a topic of discussion. To move forward on this issue, we intercrossed Hjv and Bmp6 knockout mice and looked whether deletion of both Bmp6 and Hjv in mice of the F2 progeny was aggravating the phenotype of single knockout animals. We observed that loss of Bmp6 further repressed hepcidin mRNA expression in the liver of Hjv knockout mice. Hepcidin quantified by ELISA in the serum of Hjv knockout mice was also significantly reduced by concomitant deletion of Bmp6. Gene expression of Id1 and Smad7, two targets of Smad1/5/8 signaling, was further repressed in double knockout mice. Finally, loss of Bmp6 considerably worsened the iron overload phenotype of Hjv knockout females which now accumulate iron in all extrahepatic tissues examined as do single Bmp6 or Hjv knockout males. It is known that the N-terminal domain of HJV mimics the structure of BMP type I receptors and that HJV competes with BMP type I receptor for BMP binding. Therefore, BMPs such as BMP2 or BMP6 can be recruited to the membrane through their interaction with HJV. The BMP2/BMP6/HJV complexes then associate with BMP type II receptors and undergo endocytosis into endosomal compartments. Owing to the acidification of endosomes and the pH dependence of the HJV-BMP interaction, HJV dissociates from the complex and is replaced by BMP type I receptors, leading to activation and signaling from these compartments. In the absence of co-receptors, activation of the SMAD pathway is also possible via preformed BMP receptor complexes. Our data suggest that this is the case for Hjv knockout mice. In the absence of Hjv, Bmp2/6 homo or heterodimers, by binding preformed BMP type I/type II receptor complexes, retain some capacity to trigger the Smad1/5/8 pathway and hepcidin production, although in a less efficient way than when they interact with hemojuvelin. This alternative possibility to stimulate Smad1/5/8 signaling is suppressed when both Hjv and Bmp6 are lacking. Altogether, our data suggest that, when one actor of the major hepcidin signaling pathway is lacking (Bmp6, Bmp2, or Hjv), there are alternative pathways (initiated by the interaction of Hjv with homodimers instead of heterodimers, or by the binding of hetero or homodimers to preformed receptor complexes) that, although less efficient to activate Smad1/5/8, succeed in maintaining hepcidin to a mininum level, at least in females. Their suppression, as in Bmp6/Hjv double knockout mice, leads to a greater repression of hepcidin and an aggravation of the iron overload phenotype. Seventh Congress of the International BioIron Society Page 371 Late-Breaking Abstracts IBIS Poster #LB50 HUMAN HEPATIC HEPARAN SULFATE REGULATES HEPCIDIN EXPRESSION Ferdous Anower-E-Khuda, PhD¹, Maura Poli, PhD², Philip Gordts, PhD³, Paolo Arosio, PhD² and Jeffrey sko, PhD⁴ ¹University of California, San Diego; ²Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; ³Department of Medicine, University of California, San Diego, La Jolla, CA; ⁴Department of Cellular and Molecular Medicine, University of California, San Diego, CA Presented By: Ferdous Anower-E-Khuda, PhD Anemia, an iron metabolism disorder, is a common complication in patients with malignancies and inflammatory disease. Iron homeostasis is a complex process that involves regulation of iron uptake from the diet, recycling of iron from red blood cell turnover, and mobilization from stores in the liver and macrophages. Hepcidin, a hepatic peptide hormone, regulates systemic iron availability by altering iron efflux. Hepcidin expression is regulated by BMP6/SMAD pathway, which in turn is modulated by the sulfation of heparan sulfate. Previous studies showed that exogenous addition of heparin, a highly sulfated form of heparan sulfate, strongly inhibits hepcidin expression; however, the ability of hepatic heparan sulfate to modulate hepcidin expression and iron metabolism is yet to be known. We used the human hepatoma line Hep3B and CRISP/Cas9 gene editing to genetically ablate the heparan sulfate biosynthetic enzymes EXT1 (a subunit of the heparan sulfate copolymerase complex), NDST1 (a member of the N-deacetylase/N-sulfotransferase family of enzymes), HS6ST1 and HS6ST2 (two isozymes that transfer sulfate residue to the C6 position of glucosamine residues in heparan sulfate ). Biallelic inactivation of EXT1 completely abolished heparan sulfate expression and hepcidin expression. Inactivation of NDST-1 resulted in undersulfation of the chains and partial reduction of hepcidin expression. Deletion of HS6ST1 and HS6ST2 results in partial reduction of 6-O-sulfation, which also translated into a modest reduction in hepcidin expression. The mechanism by which alterations in heparan sulfate modulates hepcidin expression will be discussed. These observations are the first genetic evidence that human hepatic heparan sulfate regulates hepcidin expression and iron homeostasis. Seventh Congress of the International BioIron Society Page 372 Late-Breaking Abstracts IBIS Poster #LB51 URATE OFFSETS MANGANESE-INDUCED IRON DYSHOMEOSTASIS AND NEURONAL DEGENERATION Rachit Bakshi, PhD², Vivek Venkataramani, MD³,⁹, Sanghamitra Bandyopadhyay, PhD⁴, Yadong Huang, PhD¹, Yanyan Liu, PhD¹, Xudong Huang, PhD¹, Ashley Bush, MD¹, Catherine Cahill, PhD¹, Debomoy Lahiri, PhD⁵, Hong Jiang, MD⁶, Fudi Wang, MD⁷, Ann Smith, PhD⁸ and Jack Rogers, PhD¹ ¹MGH; ²Neurochemistry lab, Depts. Neurology and Psychiatry MGH; ³Department of Hematology and Medical Oncology, University Medicine Göttingen; ⁴Developmental Toxicology Division, CSIR-IITR Campus, India; ⁵Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, USA; ⁶Dept. Physiology, Qingdao University, China; ⁷Department of Nutrition Zhejiang University, China; ⁸School of Biological Sciences, University of Missouri-K.C., Kansas City, MO, USA,; ⁹Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, USA Presented By: Jack Rogers, PhD Both iron and manganese (Mn) are essential nutrients but elevated levels can be toxic. Mn poisoning has life-long consequences and ‘Manganism’ is an occupational disease affecting those who breath in Mn-containing welding fumes, causing psychiatric and movement problems related to, though distinct from, Parkinson’s disease (PD). Excessively high levels of ferrous iron and Mn can be toxic due to the catalytic generation of destructive intracellular hydroxyl radicals by the Fenton reaction to enhance the production of reactive oxygen species. We report that Mn disrupts the translational regulation of proteins that help control iron homeostasis, for example the Alzheimer’s amyloid precursor protein (APP) that stabilizes the iron exporter ferroportin thus facilitating neuroprotective iron and Mn efflux. Translation of both APP and the iron storage ferritin light and heavy chains are controlled by the modulated interaction of iron-regulatory proteins (IRP1, IRP2) binding to Iron-responsive Elements (IREs) that are RNA stem loops in their cognate mRNAs. APP mRNA has an active uniquely folded 5’UTR that exhibits homology with the IREs encoded by the light (L) and heavy (H) transcripts of ferritin. We have found that that Mn inhibits APP and H-ferritin translation prior to toxicity to human neurons and also in Mn exposed rat brains. Manganese decreased the expression of APP and ferritin protein in human neuroblastoma SH- SY5Y cells likely resulting from Mn-induced increased IRP1 binding to the cognate APP and H-ferritin IREs, that share 75% homology. As we showed to be the case for Lead (Pb) neurotoxicity (Rogers et al., 2016 JNC 138(3), 479-494), increased levels of intracellular unstored iron may also to be toxic to neurons after Mn exposure. Supplementation therapy with uric acid, as an antioxidant, has recently been shown to be protective to alleviate midbrain neurodegeneration in PD with restored gait in rodent models. We now can report recent findings that urate (a.k.a uric acid) treatment also shields neurons from Mn-induced/ catalyzed toxicity. We discuss urate’s therapeutic potential to relieve Mn-dependent interference with IRP/IRE-mediated repression of APP and ferritin to thus enhance neuroprotection in these circumstances. We will define urate’s mechanism of action, with that of novel like-acting small molecules, to therapeutically offset Mn’s capacity to interfere with IRE dependent translation pathways when embargoing the protective export of iron and Mn by an APP-ferroportin complex. Urate, and other IRE targeting translational activators, may reverse the inhibitory effects of Mn on ferritin translation to relieve toxic iron overload in neuronal cells. These agents would provide a treatment for manganism with implications for Parkinson’s disease therapies. Seventh Congress of the International BioIron Society Page 373 Late-Breaking Abstracts IBIS Poster #LB52 HEPCIDIN DETERMINATION IN BETA THALASSAEMIA MAJOR CHILDREN BY ELISA AND QPCR Yvonne Elliott, PhD¹, Shradda Bhange, BSc¹, Rob Evans, PhD² and Sebastien Farnaud, PhD¹ ¹Coventry University; ²Brunel University Presented By: Sebastien Farnaud, PhD Beta thalassaemia major is an inherited blood disorder that arises due to mutations that reduce or prevent the synthesis of beta globin chains. Whereas in wildtype tetrameric haemoglobin A (HbA), the two beta globin chains are associated with two alpha globin chains, in new born beta thalassaemic patients, normal HbA is absent and fetal haemoglobin HbF is the major haemoglobin form. Unfortunately, since HbF waned off at 6 months after birth, severe anaemia can be present as early as the age of 6 month old. Although the marked reduction in oxygenation to the tissues results in production of the hormone erythropoietin EPO, ineffective erythropoiesis leads to erythroid marrow expansion, and to the expansion of subsequent organs, such as liver and spleen in extramedullary hematopoiesis. As a result, these patients become transfusion-dependent from a young age, with major consequences to their health; generally, their organs suffer from iron overload due to transfusion but also due to excess duodenal iron-uptake, leading to heart failure being the major cause of morbidity in these patients. Patients often undergo splenectomies to alleviate worsening anaemia as red blood cells can be sequestered for destruction. In healthy individuals, hepcidin produced by liver hepatocytes, regulates iron-uptake by acting on ferroportin, the baso-lateral iron transporter in the enterocyte therefore controlling dietary iron absorption, plasma iron concentrations, and tissue iron distribution. In beta thalassemia major patient, due to high erythropoietic activity, hepcidin expression has been shown to be reduced. This deficiency in the iron hormone is proposed to be one of the main contributing factors to iron overload in beta-thalassemia, in particular in non-transfused beta thalassemia major patients, as shown by liver biopsies indicative of hyperabsorbed dietary iron. Transfusions can cause hepcidin level to increase due to the suppression of EPO. Similarly, higher levels of hepcidin have also been shown in patients with sickle cell disease. This pilot study explores levels of hepcidin expression in children and young adults between the ages of 10 to 20 years of age, suffering from beta thalassemia. HAMP expression was found to be decreased in beta thalaessemia major patients compared to normal blood samples, with positive fold change of qPCR of 0.184 (p=0.013). Serum hepcidin levels were also determined in these patients to define correlation between HAMP expression and peptide serum levels. Seventh Congress of the International BioIron Society Page 374 Late-Breaking Abstracts IBIS Poster #LB53 HFE mRNA EXPRESSION IS NOT DIRECTLY RELATED TO IRON SENSING BUT IS RELATED TO TFRC EXPRESSION Kosha Mehta, PhD², Robert Evans, PhD³, Vinood Patel, PhD² and Sebastien Farnaud, PhD¹ ¹Coventry University; ²University of Westminster; ³Brunel University Presented By: Sebastien Farnaud, PhD Objective: The HFE protein takes part in the regulation of cellular and systemic iron homeostasis by modulating cellular iron-uptake, and is proposed to play a role in modulating the iron-hormone hepcidin expression in response to systemic iron elevation. However, the full mechanism of iron sensing in hepatocytes remains controversial. In this study, the effect of distinct extracellular and intracellular iron-loading on HFE and hepcidin (HAMP) expressions are being investigated. Materials and Methods: Wild-type HepG2 cells and previously characterised recombinant TFRC-expressing HepG2 cells, which carry constitutively expressed TFRC gene, were treated with holotransferrin at 0, 1, 2 and 5g/L for 6 h, where 2g/L represents the physiological range. Intracellular iron content was determined by ferrozine assay and gene expression analysis was determined using qPCR. Results: Expression of TfR1 gene, TFRC, in wildtype HepG2 varies as expected with a decrease in expression following increased exposure to holo-Tf. In the TFRC-expressing HepG2, expression of TfR1 gene increases constitutively despite increased iron exposure, as expected. In wild-type HepG2 cells, following increased exposure to extracellular iron, intra- cellular iron remains unchanged in all treatments due to the decrease in endemic TFRC. Since HFE expression pattern does not follow proportionally any of both trends, it can be concluded that HFE expression is not directly regulated by iron level either externally or internally. Similarly variation of HAMP expression does not follow either of the trends, therefore indicating that HAMP expression is neither regulated directly by external or internal iron level. In TFRC-expressing HepG2 cells, whereas the variation pattern of HAMP expression is simply an amplification of the variation observed in wildtype cells HepG2, for HFE the variation pattern is the opposite of the pattern observed with the wild-type HepG2. Conclusions: the disproportionate increased expression of HFE mRNA observed in HepG2 wild-type following increased iron exposure suggests a role in iron sensing but not directly related to iron levels. The opposite decreased expression in HFE observed in TFRC-expressing HepG2 with increased iron content, suggests an additional mechanism for iron sensing. This decrease in HFE expression, which is observed even at the highest level of iron exposure, is paradoxically characteristic of low iron levels. Therefore, the HFE gene in TFRC-expressing HepG2 is not sensing increased iron levels, but to the contrary, is reacting as if iron levels were low. This confirm that HFE expression is not directly sensing iron levels. In this instance it is suggested that HFE expression is following TFRC expression, hence is increasing because of the increased constitutive expression of TFRC. The different expression pattern between HFE and HAMP confirms that they both respond to different iron sensing mechanisms. Seventh Congress of the International BioIron Society Page 375 Late-Breaking Abstracts IBIS Poster #LB54 PHARMACOKINETICS AND PHARMACODYNAMICS MODEL OF SYNTHETIC HUMAN HEPCIDIN LJPC-401 FOLLOWING SINGLE DOSE ADMINISTRATION IN DOGS Hong Jin, PhD¹, Wojciech Krzyzanski, PhD², Sima Patel, MS¹, Robin Marsden, BS¹, James Rolke, BS¹ and Andrew Seacat, PhD¹ ¹La Jolla Pharmaceutical Company; ²University at Buffalo Presented By: Hong Jin, PhD Objectives: LJPC-401 is a synthetic human hepcidin drug product intended for the treatment of iron overload. A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe serum concentrations of LJPC-401 and serum iron following a single escalating subcutaneous (SC) and intravenous (IV) dose administration in dogs. Methods: The data were obtained from studies of LJPC-401 in dogs consisting of 6 dose groups (n=20 dogs total). Single SC injections of placebo, 0.2, 1, and 5 mg/kg (n=4 per group), and 30 min IV infusions of 1.1 and 3.1 mg/kg (n=2 per group) were administered at time 0. Blood samples for LJPC-401 serum concentration (PK) and serum iron (PD) measurements were collected at pre-dose, 1, 2, 4, 8, 24, 48, and 72 h from the SC groups, and pre-dose, 15 and 30 min (during infusion) and at 35, 40, 50 min, 1, 1.5, 2.5, 4.5, 8, 12, 24, 36, 48, and 72 h (post infusion) from the IV groups. A two-compartment model with first-order absorption and zero-order endogenous hepcidin production rate was applied to describe the PK data. An indirect response model with a precursor was selected to characterize the PD data. The precursor pool represents iron stores in macrophages and liver, and the response is the serum iron concentration. LJPC- 401 inhibits iron release from the precursor pool into the serum at the maximal inhibition Imax and potency IC50. The PK and PD data were fitted simultaneously resulting in estimates of model parameters and their variances in the population. Results: LJPC-401 exhibited linear kinetics with typical clearance Cl=256 mL/h/kg and volume of distribution V=178 mL/kg, with a half-life of 45 min following IV administration. The absorption rate of ka = 0.15 h-1 for SC administration was slower than the elimination rate of kel = 1.4 h-1 resulting in the flip-flop kinetics and the terminal half-life of 4.6 h. Bioavailability was F=60%. Estimated endogenous hepcidin concentration was 3.1 ng/mL. Baseline serum iron was 162 µg/dL. Serum iron decreased in a dose-dependent manner with maximal decrease at ~ 5 h (IV group) and ~8 h (SC groups), with a return to the baseline within 24 h (IV groups) and 48 h (SC groups). The maximal decrease in serum iron was ~88% with an IC50 = 8.0 ng/mL, implying a 25% inhibition of the iron release from the precursor pool at the baseline conditions. Conclusions: Following SC dosing, LJPC-401 exhibited linear kinetics with a flip-flop absorption, and displayed a 6-fold longer half-life than IV infusion. A single SC dose of 5 mg/kg resulted in the maximal 88% decrease of serum iron within 5- 8 h followed by a return to baseline within 24-48 h. This PKPD model developed from acute dose PKPD data in dogs can be used to design LJPC-401 repeat dose studies as well as the data analysis. Seventh Congress of the International BioIron Society Page 376 Late-Breaking Abstracts IBIS Poster #LB55 INHIBITION OF INTESTINAL NON-HAEM IRON ABSORPTION BY QUERCETIN IS MEDIATED BY BOTH LUMINAL AND SYSTEMIC MECHANISMS M. Lesjak1,2, S. Balesariaa, V. Skinnera, E. S. Debnamc, S. K. S. Sraia 1Research Department of Structural and Molecular Biology, Division of Biosciences, University College London, Darwin Building, London, United Kingdom; 2Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia; 3Research Department of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, Royal Free Campus, London, United Kingdom Presented By: M. Lesjak Inhibitors of iron absorption are a great threat to human health as the rate of non-haem iron absorption is generally low. Dietary polyphenols block iron uptake, but the mechanism by which they exert this action is not fully understood. Since the polyphenol quercetin is ingested daily in significant amounts it was considered worthwhile to investigate the chelation effect of quercetin on duodenal non-haem iron absorption in vivo, as well as its effect on systemic iron metabolism. Rats were subject to gavage and systemic quercetin treatments. Treatments were followed with uptake studies using radiolabeled iron, serum iron and transferrin saturation measurements, LC–MS/MS analysis of quercetin metabolites in serum, determination of tissue non-haem iron content and analysis gene expression of iron-related proteins. Both oral and intraperitoneal (IP) quercetin caused iron deficiency in vivo by two mechanisms, firstly by increasing mucosal iron uptake and inhibiting iron efflux from duodenal mucosa and secondly by decreasing levels of duodenal DMT1, Dcytb and FPN. Additionally, IP quercetin induced highly significant increased expression of hepcidin (175- and 1031-fold after single and double quercetin administration, respectively). Only after IP quercetin administration, did some quercetin metabolites were detected with LC-MS/MS in serum. Thus, the possibility that quercetin metabolites could also be involved in the observed effects on iron uptake and metabolism should not be overlooked. Oral quercetin had a great effect on iron absorption and a minor effect on systemic iron regulation, while IP quercetin mainly affected iron-related genes. These results could lead to development of new effective ways of preventing and treating iron deficiency anaemia, the most frequent and widespread nutritional disorder in the world. Seventh Congress of the International BioIron Society Page 377 Alphabetical Index of Presenters IBIS Alphabetical Index of Presenters Author/Presenter, Date, Time, and Abstract Placement Due to time limitations, authors who do not have a time and date listed will not be presenting their abstracts at this meeting. Agoro, Rafiou Boero, Martina 5/ 8/17 16:45 Poster #LB27 5/10/17 16:45 Poster #135 Aguilar Vitorino, Hector Bordini, Jessica 5/10/17 16:45 Poster #200 5/10/17 16:45 Poster #157 Al-Aidy, Salem Borges, Marina Dorigatti 5/10/17 16:45 Poster #170 5/ 8/17 16:45 Poster #28 Altamura, Sandro Bourne, Gregory Thomas 5/10/17 16:45 Poster #115 5/11/17 11:45 Podium #36 5/10/17 16:45 Poster #162 Bruinvels, Georgie Angmo, Stanzin 5/10/17 16:45 Poster #145 5/11/17 12:15 Podium #38 5/10/17 16:45 Poster #146 Anower-E-Khuda, Ferdous Bullock, Grant 5/ 8/17 16:45 Poster #LB50 5/ 8/17 16:30 Podium #13 Antosiewicz, Jedrzej Butcher, Anna 5/ 8/17 16:45 Poster #LB29 5/ 8/17 16:45 Poster #10 Aparicio, Ana Cairo, Gaetano 5/ 8/17 16:45 Poster #17 5/10/17 14:00 Podium #25 Apostolova, Petia Campanella, Alessandro 5/10/17 16:45 Poster #144 5/10/17 16:45 Poster #156 Artuso, Irene Canali, Susanna 5/ 8/17 16:45 Poster #91 5/ 8/17 14:15 Podium #4 Aryal, Ritambhara Carrilho, Patricia 5/ 8/17 14:00 Podium #9 5/ 8/17 16:45 Poster #37 Aschemeyer, Sharraya Castagna, Annalisa 5/10/17 15:15 Poster #3 5/ 8/17 16:45 Poster #19 5/10/17 16:45 Poster #3 Casu, Carla Asperti, Michela 5/10/17 16:45 Poster #186 5/10/17 15:00 Podium #27 5/11/17 11:15 Podium #34 Ayton, Scott Chang, Shiyang 5/ 8/17 16:45 Poster #LB16 5/ 8/17 16:45 Poster #LB8 5/ 8/17 16:45 Poster #LB10 Balusikova, Kamila 5/ 8/17 16:45 Poster #65 Chang, Yan-Zhong 5/11/17 16:25 Podium #53 Bartnikas, Thomas 5/ 8/17 16:45 Poster #77 Chaudhry, Garima 5/ 8/17 16:45 Poster #LB12 Baumann, Bailey 5/ 8/17 14:45 Podium #12 Chen, Chih-Lung 5/ 8/17 16:45 Poster #40 Seventh Congress of the International BioIron Society Page 378 Alphabetical Index of Presenters IBIS Chessa, Roberta Diez Ricote, Laura 5/ 8/17 16:45 Poster #56 5/11/17 16:35 Podium #49 Chiabrando, Deborah Doguer, Caglar 5/ 8/17 16:45 Poster #90 5/10/17 16:45 Poster # 74 Chiou, Brian Doty, Raymond 5/ 8/17 16:45 Poster #32 5/ 8/17 16:45 Poster #85 Chitambar, Christopher Dunaief, Joshua L. 5/10/17 14:15 Podium #26 5/ 8/17 16:45 Poster #76 Ciambellotti, Silvia Dziuba, Nathaniel 5/ 8/17 16:45 Poster #LB35 5/ 8/17 16:45 Poster #75 Cioffi, Alexander Edison, Eunice Sindhuvi 5/ 8/17 16:45 Poster #LB40 5/ 8/17 16:45 Poster #46 5/10/17 16:45 Poster #143 Coffey, Richard 5/10/17 16:45 Poster #165 Eisenstein, Richard 5/ 8/17 16:45 Poster #114 Collingwood, Joanna 5/ 8/17 16:45 Poster #35 Esposito, Breno P. 5/ 8/17 16:45 Poster # 69 Connor, James 5/ 8/17 16:45 Poster # 96 5/ 8/17 16:45 Poster #30 5/ 8/17 16:45 Poster #31 Evans, Robert William 5/ 8/17 16:45 Poster # LB25 Constante, Marco 5/10/17 16:45 Poster #126 Farnaud, Sebastien 5/ 8/17 16:45 Poster #LB53 Costa Da Silva, Milene 5/ 8/17 16:45 Poster #LB52 5/10/17 16:45 Poster #153 Finberg, Karin Cruz, Tania 5/10/17 16:45 Poster #167 5/ 8/17 16:45 Poster #23 Flores, Shireen Daher, Raed 5/ 8/17 16:45 Poster #LB24 5/10/17 16:45 Poster #163 Francis, Connor Das, Nupurkanti 5/11/17 16:40 Podium #54 5/11/17 16:55 Podium #55 Frazer, David Del Castillo-Rueda, Alejandro 5/10/17 16:45 Poster #192 5/ 8/17 16:45 Poster #47 Fretz, Jackie Delatycki, Martin 5/10/17 16:45 Poster #141 5/11/17 14:40 Podium #46 Frey, Avery Demikhov, Valerii 5/10/17 16:45 Poster #179 5/10/17 16:45 Poster #206 Fujiwara, Tohru Denardo, Andrea 5/ 8/17 16:45 Poster #84 5/10/17 16:45 Poster #168 Fuqua, Brie Di Savino, Augusta 5/10/17 16:45 Poster #209 5/10/17 16:45 Poster #181 5/10/17 16:45 Poster #190 Seventh Congress of the International BioIron Society Page 379 Alphabetical Index of Presenters IBIS Gahagan, Sheila Gupta, Ritama 5/11/17 15:15 Podium #44 5/ 8/17 16:45 Poster #25 Ganasen, Menega Ha, Jung-Heun 5/ 8/17 16:45 Poster #LB14 5/10/17 16:45 Poster #195 Ganz, Tomas Hamdi, Amel 5/ 8/17 16:45 Poster #LB26 5/ 8/17 16:45 Poster #74 Garcia, Alejandra Handley, Simon 5/ 8/17 16:45 Poster #109 5/ 8/17 16:45 Poster #107 5/ 8/17 16:45 Poster #106 Garcia-Santos, Daniel 5/11/17 11:30 Podium #35 Hanudel, Mark 5/10/17 16:45 Poster #175 5/ 8/17 16:45 Poster #87 5/10/17 16:45 Poster #188 Hara, Yuichi Garrick, Michael 5/11/17 11:40 Podium #30 5/10/17 16:45 Poster #197 Hare, Dominic Gassmann, Max 5/ 8/17 16:45 Poster #LB1 5/10/17 16:45 Poster #121 Haschka, David Gerhard, Glenn 5/ 8/17 16:45 Poster #26 5/11/17 11:55 Podium #31 Haselwandter, Kurt Ghareeb, Abdulameer 5/ 8/17 16:45 Poster #12 5/ 8/17 16:45 Poster #101 Heidari, Moones Ghosh, Manik 5/ 8/17 16:45 Poster #133 5/ 8/17 16:45 Poster #82 Heinrichs, David Gnana-Prakasam, Jaya P. 5/ 8/17 16:45 Poster #LB36 5/ 8/17 16:45 Poster #LB43 Hider, Robert Gonzalez, Amaliris 5/ 8/17 16:45 Poster #86 5/10/17 16:45 Poster #185 Huang, Xi Gozzelino, Raffaella 5/ 8/17 16:45 Poster #LB42 5/10/17 16:45 Poster #130 5/10/17 16:45 Poster #194 Ito, Fumiya 5/10/17 16:45 Poster #122 Grillo, Anthony 5/11/17 16:10 Podium #52 Jacob, Molly 5/ 8/17 16:45 Poster #61 Gryzik, Magdalena 5/ 8/17 16:45 Poster #68 Jadhav, Shyamalagauri 5/ 8/17 16:45 Poster #LB23 Guan, Di 5/ 8/17 16:45 Poster #LB38 Jain, Anshika 5/ 8/17 16:45 Poster #99 Guo, Shanshan 5/11/17 15:10 Podium #48 James, Jithu 5/ 8/17 16:45 Poster #64 Guo, Shuling 5/10/17 15:15 Poster #7 James, Simon 5/10/17 16:45 Poster #7 5/10/17 16:45 Poster #5 5/10/17 15:15 Poster #5 Seventh Congress of the International BioIron Society Page 380 Alphabetical Index of Presenters IBIS Jenkitkasemwong, Supak Lesjak, M. 5/10/17 16:45 Poster #187 5/10/17 17:15 Poster #LB55 Jeong, Suh Young Levi, Sonia 5/ 8/17 13:45 Podium #8 5/ 8/17 13:30 Podium #7 5/10/17 16:45 Poster #132 Jin, Hong 5/ 8/17 16:45 Poster #LB54 Li, Kuanyu 5/ 8/17 16:45 Poster #LB46 Kallo, Veronika 5/ 8/17 16:45 Poster #33 Li, Xiuqi 5/ 8/17 16:15 Podium #18 Khoja, Kholoud 5/ 8/17 16:45 Poster #55 Lichota, Jacek 5/10/17 16:45 Poster #129 Kim, Ki Soon 5/10/17 15:15 Poster #6 Lim, Pei Jin 5/10/17 16:45 Poster #6 5/ 8/17 13:45 Podium #2 Kleven, Mark Lindahl, Paul 5/10/17 16:45 Poster #208 5/ 8/17 16:45 Poster #73 Knap, Narcyz Linder, Maria C. 5/ 8/17 16:45 Poster #LB33 5/10/17 16:45 Poster #177 Knutson, Mitchell Lipinski, Pawel 5/10/17 16:45 Poster #136 5/ 8/17 16:45 Poster #45 5/10/17 16:45 Poster #173 5/ 8/17 16:45 Poster #44 Krijt, Jan Litvinkova, Liubov 5/10/17 15:15 Poster #2 5/ 8/17 16:45 Poster #43 5/10/17 16:45 Poster #2 Liu, Jing Kumari, Namita 5/11/17 14:55 Podium #47 5/10/17 16:45 Poster #127 5/ 8/17 16:45 Poster # 98 Lake, Ryan Liu, Yuqian 5/10/17 16:45 Poster #180 5/ 8/17 16:45 Poster #60 Lal, Ashutosh Lo Presti, Vania 5/ 8/17 16:45 Poster #57 5/10/17 16:45 Poster #9 5/10/17 15:15 Poster #9 Lasocki, Sigismond 5/ 8/17 16:45 Poster #42 Loreal, Olivier 5/ 8/17 16:45 Poster #93 Latunde-Dada, Gladys 5/ 8/17 16:45 Poster #63 5/ 8/17 16:45 Poster #18 Lu, Dongyi Lee, Sang 5/10/17 16:45 Poster #166 5/ 8/17 16:45 Poster #52 Ly, Seak Lin Leibold, Elizabeth A. 5/ 8/17 14:15 Podium #10 5/10/17 16:45 Poster #201 5/ 8/17 15:15 Podium #14 Ma, Juan 5/ 8/17 16:45 Poster #83 Lemasters, John 5/ 8/17 16:45 Poster #LB37 Macdonald, Ramsay 5/10/17 16:45 Poster #125 Seventh Congress of the International BioIron Society Page 381 Alphabetical Index of Presenters IBIS Mackenzie, Bryan Mura, Catherine 5/10/17 15:15 Poster #4 5/ 8/17 16:45 Poster #21 5/10/17 16:45 Poster #4 5/ 8/17 16:45 Poster #22 Maio, Nunziata Nai, Antonella 5/10/17 16:45 Poster #196 5/11/17 11:00 Podium #33 Nakanishi, Takeshi Malerba, Mariangela 5/ 8/17 16:45 Poster #88 5/10/17 14:35 Podium #21 Nechushtai, Rachel Manolova, Vania 5/10/17 13:30 Podium #23 5/11/17 14:00 Podium #37 Nekhai, Sergei Marques, Oriana 5/ 8/17 16:45 Poster #111 5/10/17 16:45 Poster #123 5/10/17 16:45 Poster #139 Martin, Kristy Neves, Joana 5/ 8/17 16:45 Poster #15 5/ 8/17 16:45 Poster #13 McHugh, Kirsty Nielsen, Peter 5/10/17 16:45 Poster #184 5/10/17 16:45 Poster #151 5/ 8/17 16:45 Poster #78 McLaren, Gordon 5/10/17 16:45 Poster #120 Nieminen, Anna-Liisa 5/ 8/17 16:45 Poster #LB34 5/ 8/17 16:45 Poster #LB44 Meadowcroft, Mark Nixon, Anne 5/10/17 16:45 Poster #131 5/ 8/17 16:45 Poster #24 Meynard, Delphine Noble, Charles 5/ 8/17 15:45 Podium #16 5/10/17 16:45 Poster #116 Meyron-Holtz, Esther Nyffenegger, Naja 5/10/17 14:50 Podium #22 5/ 8/17 16:45 Poster #LB21 5/10/17 16:45 Poster #140 Ogura, Jiro Milward, Elizabeth 5/ 8/17 16:45 Poster #LB31 5/10/17 16:45 Poster #134 Olivera, Joseph Miyanishi, Koji 5/ 8/17 16:45 Poster #LB48 5/ 8/17 16:45 Poster #102 Pagani, Alessia Mleczko-Sanecka, Katarzyna 5/ 8/17 16:45 Poster #142 5/10/17 16:45 Poster #191 Pai, Amy Montgomery, McKale 5/ 8/17 16:45 Poster #58 5/ 8/17 16:45 Poster #LB20 5/ 8/17 16:45 Poster #59 Moos, Torben Pantopoulos, Kostas 5/ 8/17 14:30 Podium #11 5/10/17 16:45 Poster #189 5/ 8/17 13:30 Podium #1 Moretti, Diego 5/10/17 16:45 Poster #124 5/ 8/17 16:45 Poster #48 Parmar, Jigneshkumar Mrowczynski, Oliver 5/10/17 16:45 Poster #178 5/ 8/17 16:45 Poster # 51 Parrow, Nermi 5/10/17 16:45 Poster #150 5/ 8/17 14:00 Podium #3 Seventh Congress of the International BioIron Society Page 382 Alphabetical Index of Presenters IBIS Pasricha, Sant-Rayn Ramm, Grant A. 5/ 8/17 16:45 Poster #41 5/11/17 14:25 Podium #45 5/11/17 14:45 Podium #42 5/ 8/17 16:45 Poster #108 Ricarte, Adrian 5/10/17 16:45 Poster #176 Paw, Barry H. 5/ 8/17 16:45 Poster #79 Ristic, Bojana 5/ 8/17 16:45 Poster #80 5/10/17 14:45 Podium #28 5/11/17 11:25 Podium #29 Rivella, Stefano Pek, Rini 5/10/17 16:45 Poster #183 5/ 8/17 16:45 Poster #89 Roetto, Antonella Pellegrino, Rosa Maria 5/ 8/17 16:45 Poster #11 5/ 8/17 16:45 Poster #20 Rogers, Jack Pereira, Dora 5/ 8/17 16:45 Poster #LB51 5/10/17 16:45 Poster #149 Roth, Marie-Paule Petrillo, Sara 5/ 8/17 16:45 Poster #LB49 5/10/17 15:15 Poster #8 5/10/17 16:45 Poster #8 Routhe, Lisa Juul 5/ 8/17 16:45 Poster #36 Philpott, Caroline 5/10/17 16:45 Poster #172 Ruzzenenti, Paola 5/10/17 16:45 Poster #169 Pietrangelo, Antonello 5/10/17 16:45 Poster #117 Salman, Ahmad 5/ 8/17 16:45 Poster #67 Piga, Antonio 5/ 8/17 16:45 Poster #105 Salunkhe, Arvindkumar 5/ 8/17 16:45 Poster #LB5 Piubelli, Chiara 5/10/17 16:45 Poster #118 Samba Mondonga, Macha 5/10/17 16:45 Poster #147 Poli, Maura 5/ 8/17 14:45 Podium #6 Sanchez Fernandez, Mayka 5/10/17 16:45 Poster #154 5/10/17 16:45 Poster #211 Popp, Andreas Sangkhae, Veena 5/ 8/17 16:45 Poster #92 5/ 8/17 16:45 Poster # 113 5/ 8/17 16:45 Poster #29 5/11/17 14:00 Podium #39 Porter, John B. SantaMaria, Anna 5/10/17 16:45 Poster #202 5/ 8/17 16:45 Poster #LB41 5/ 8/17 16:45 Poster #104 Santos, Paulo Caleb J L Pratt, Raymond D. 5/ 8/17 16:45 Poster #LB19 5/ 8/17 16:45 Poster #70 5/11/17 15:00 Podium #43 Sara Helgudottir, Steinunn 5/10/17 16:45 Poster #128 Prince, Dianne 5/ 8/17 16:45 Poster #LB17 Sardo, Ugo 5/ 8/17 16:45 Poster #LB39 Protchenko, Olga 5/ 8/17 15:30 Podium #15 Sawai, Hitomi 5/ 8/17 16:45 Poster #LB13 Pulfer, Maria Dolores 5/ 8/17 16:45 Poster #110 Seventh Congress of the International BioIron Society Page 383 Alphabetical Index of Presenters IBIS Schaefer, Benedikt Tarantino, Germano 5/10/17 16:45 Poster #148 5/ 8/17 16:45 Poster #49 Scheers, Natalie Tjendana Tjhin, Vindy 5/ 8/17 16:45 Poster #LB18 5/ 8/17 16:45 Poster #34 Schwartz, Andrew Tong, Wing-Hang 5/10/17 13:45 Podium #24 5/ 8/17 16:45 Poster #27 Sharp, Paul A Torti, Frank 5/ 8/17 16:45 Poster #72 5/ 8/17 16:45 Poster #54 Shinoda, Shoko Torti, Suzy 5/ 8/17 16:45 Poster #100 5/ 8/17 16:45 Poster #53 Silvestri, Laura Traeger, Lisa 5/10/17 16:45 Poster #198 5/10/17 16:45 Poster #207 Singh, Abhishek Truksa, Jaroslav 5/10/17 16:45 Poster #161 5/10/17 16:45 Poster #182 Sivaprakasam, Sathish van Raaij, Sanne 5/ 8/17 16:45 Poster #LB30 5/ 8/17 16:45 Poster #LB47 5/10/17 16:45 Poster #138 Sledzinski, Zbigniew 5/ 8/17 16:45 Poster #LB28 Van Swelm, Rachel 5/ 8/17 16:45 Poster #LB45 Smith, Ann 5/ 8/17 16:45 Poster #39 5/ 8/17 16:45 Poster #95 5/10/17 16:45 Poster #159 Varghese, Joe 5/ 8/17 16:45 Poster #62 Sobh, Amin 5/ 8/17 16:45 Poster #71 Vera-Aviles, Mayra 5/ 8/17 16:45 Poster #38 Srichairatanakool, Somdet 5/ 8/17 16:45 Poster #LB4 Vichinsky, Elliott 5/10/17 16:45 Poster #203 Stefanova, Debora 5/10/17 14:20 Podium #20 Vinchi, Francesca 5/10/17 16:45 Poster #158 Steinbicker, Andrea 5/ 8/17 16:00 Podium #17 5/10/17 16:45 Poster #160 Viveiros, Andre Stoffel, Nicole 5/10/17 16:45 Poster # 205 5/11/17 14:15 Podium #40 5/10/17 16:45 Poster # 164 Strong, Roland Vlachodimitropoulou, Evangelia 5/ 8/17 16:45 Poster #66 5/ 8/17 16:45 Poster #103 Sukumaran, Abitha Vujic Spasic, Maja 5/10/17 16:45 Poster #193 5/10/17 16:45 Poster #171 5/11/17 17:05 Podium #51 Sung, Hyekyoung 5/ 8/17 16:45 Poster #LB6 Wallace, Daniel Frederick 5/10/17 16:45 Poster #119 Swinkels, Dorine W. 5/10/17 16:45 Poster #210 5/10/17 16:45 Poster #199 Walter, Patrick 5/10/17 16:45 Poster #137 Seventh Congress of the International BioIron Society Page 384 Alphabetical Index of Presenters IBIS Wang, Chia-Yu 5/10/17 15:15 Poster #1 5/10/17 16:45 Poster #1 Wang, Peina 5/ 8/17 16:45 Poster #LB9 5/ 8/17 16:45 Poster #LB7 Wang, Xiaoyu 5/ 8/17 16:45 Poster #LB22 Wang, Yongwei 5/ 8/17 16:45 Poster #81 Ward, Diane 5/11/17 12:10 Podium #32 Wermke, Martin 5/10/17 16:45 Poster #204 Weyhmiller, Marcela G. 5/ 8/17 16:45 Poster #50 Whatcott, Clifford 5/ 8/17 16:45 Poster #112 White, Desley 5/ 8/17 16:45 Poster #16 Winkler, Hans 5/ 8/17 16:45 Poster #LB2 Wozniak, Matt 5/ 8/17 16:45 Poster #LB32 Yamamoto, Masayo 5/ 8/17 16:45 Poster #94 Yu, Peng 5/ 8/17 16:45 Poster #LB15 Zacharski, Leo 5/ 8/17 16:30 Podium #19 Zhang, An-Sheng 5/ 8/17 14:30 Podium #5 Zhang, Deliang 5/11/17 16:50 Podium #50 Zhu, Hao 5/ 8/17 16:45 Poster #14 Zimmermann, Michael 5/11/17 14:30 Podium #41 Seventh Congress of the International BioIron Society Page 385 INTERNATIONAL BIOIRON SOCIETY International BioIron Society (IBIS) Two Wood eld Lake 1100 E. 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