Major Diseases Research

MAJOR DISEASES RESEARCH

Catalogue of Research Projects (2005-2006) in the Sixth Framework Programme Volume II project synopses project Interested in European research?

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European Commisssion Directorate-General for Research Directorate F – Health Unit F.2 – Medical and Public Health Research Contact: Manuel Hallen European Commission Ofﬁ ce CDMA 2/22 B-1049 Brussels MAJOR DISEASES RESEARCH

Catalogue of Research Projects (2005-2006) in the Sixth Framework Programme Volume II

Edited by Manuel Hallen

2008 Medical and Public Health Research EUR 23346 EN Europe Direct is a service to help you ﬁ nd answers to your questions about the European Union

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Luxembourg: Ofﬁ ce for Ofﬁ cial Publications of the European Communities, 2008

ISBN 978-92-79-08526-0

Printed in Belgium PRINTED ON WHITE CHLORINE-FREE PAPER TABLE OF CONTENTS

INTRODUCTION 7

CARDIOVASCULAR 9 ➞ 44 Cardiogenics 10 CONTICA 13 CVDIMMUNE 15 EUGeneHeart 17 GENECURE 20 HDLomics 22 HeartRepair 24 IMMUNATH 27 InGenious HyperCare 29 NORMACOR 31 PolyALA 33 PROCARDIS 34 PULMOTENSION 36 SOUTH 39 VASOPLUS 42

DIABETES & OBESITY 45 ➞ 57 EuroDia 46 HEPADIP 48 InterAct 51 PREDICTIONS 54 SAVEBETA 56

RARE DISEASES 59 ➞ 78 EURAMY 60 EuroCareCF 62 EuroGrow 65 EURO-Laminopathies 67 HUE-MAN 70 MYASTAID 73 TREAT-NMD 76

3 ANTI-MICROBIAL DRUG RESISTANCE 79 ➞ 110 ACE 80 CRAB 82 DRESP2 84 EACCAD 86 EAR 88 EURESFUN 91 GRACE 94 MagRSA 98 MANASP 100 MOSAR 102 NPARI 105 REBAVAC 107 StaphDynamics 109

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES 111 ➞ 160 AGLAEA 112 cNEUPRO 114 DB workshop 116 ENINET 118 Enough sleep 120 EPICURE 122 EUSynapse 125 IMAGEN 127 INCF 129 INDABIP 131 INTELLIMAZE 133 MEMORIES 136 NEOBRAIN 138 NEURODYS 141 NeuroproMiSe 143 NovelTune 146 PHECOMP 148 PHOTOLYSIS 150 PNS-EURONET2 152

4 RATstream™ 154 SGENE 157 TAMAHUD 159

HUMAN DEVELOPMENT AND AGEING 161 ➞ 181 ARIG 162 CRESCENDO 164 ECONAG 167 ELAST-AGE 169 EWA 171 LifeSpan 174 PROTEOMAGE 176 RespViruses 179 SENECA 180

CANCER 183 ➞ 265 Anti-tumor targeting 184 Apotherapy 186 BAMOD 188 CancerGrid 190 CANCERIMMUNOTHERAPY 193 CAPPELLA 195 CHEMORES 197 CHILDHOPE 199 CONTICANET 202 DASIM 205 DEPPICT 206 DISMAL 208 E.E.T.-Pipeline 210 EPCRC 212 EPITRON 214 EuroBoNet 216 EuroCSC 218 GenoMEL 220 GROWTHSTOP 222

5 HERMIONE 224 HI-CAM 226 HighReX 228 Immuno-PDT 230 INCA 232 KidsCancerKinome 234 LIGHTS 236 MAMMI 238 MCSCs 240 MMR-related cancer 242 MSCNET 243 NORMOLIFE 245 ONCASYM 247 ONCODEATH 249 OVCAD 251 POLYGENE 253 PROMET 255 TCAC in Cancer 257 TRIDENT 259 TRKCancer 261 Tumor-Host Genomics 262 VITAL 264

ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES 267 ➞ 272 ECRIN-TWG 268 EUROIRON1 271

INDEX BY ACRONYM 274 ➞ 275

FP6 INSTRUMENTS 276 ➞ 277

6 INTRODUCTION

The “Sixth Framework Programme for RTD (FP6 – 2002-2006) of the Five different funding schemes, each with a different objective, were European Union” has dedicated some € 800 million to supporting research available in FP6 for providing support to research projects. Integrated in the fi eld of “Major Diseases: Application-orientated Genomic Approaches Projects (IP) and Specifi c Targeted Research Projects (STREP) were aimed to Medical Knowledge and Technologies”, with the aim of improving the at generating, demonstrating and validating new knowledge through quality of life and the health of patients in Europe (1) and around the world. research and development. Networks of Excellence (NoE) supported strategic research coordination through extensive networking. Coordination The greatest disease burden in Europe is represented by non-communicable Actions (CA) and Specifi c Support Actions (SSA) promoted collaboration diseases (NCD): a group of conditions that includes cardiovascular disease and coordination of smaller scale projects, and other activities such as (CVD), cancer, mental health problems, diabetes mellitus, chronic respiratory conferences and studies. In addition, collaboration between industry and diseases and musculoskeletal conditions. This broad group of disorders; academia was strongly encouraged, in particular the involvement of small which are linked by common risk factors, underlying determinants and and medium-sized enterprises (SMEs). opportunities for intervention, cause 86 % of all deaths and 77 % of the disease burden in Europe. In 2005 the fi rst Volume on project synopses was published, covering 87 projects selected from the fi rst and second call for proposals in The ultimate objective of any health research funding should, therefore, 2002-2004. This publication is available at ftp://ftp.cordis.europa.eu/ be to reduce instances of, and eventually eliminate these diseases. The pub/lifescihealth/docs/major_catalogue_complet.pdf. task is huge since conditions such as CVD, neurological disorders and cancer are highly complex and thus can often be understood and ad- The present Volume 2 of project synopses on “Major Diseases Research” dressed only by multidisciplinary teams. For more than 25 years, European is a complementary publication featuring an additional 113 projects Union Framework Programmes have provided fi nancial support to medi- selected under the third and fourth calls for proposals in the afore- cal and life sciences research, including the training of new scientists. mentioned research areas. The budget of some € 800 million was divided Europe is fortunate in having an excellent research base and scientists unevenly across funding schemes. Approximately half of the budget went able to collaborate across national borders, producing top quality results to large integrated projects (IP), which represented less than a quarter of and a mutual respect of their respective neighbours’ skills and qualities. the number of projects funded. On average, an IP involved 20 partners with a mean EC contribution of around € 9 million per project. On the Due to the availability of the full sequence of the human genome and other hand, around one third of the major diseases budget was devoted many other model organisms at the beginning of the Millennium, the to smaller, specifi c targeted research projects (STREP), which accounted Sixth Framework Programme placed an emphasis on exploiting the for more than half of the total number of projects supported. These con- unprecedented opportunities for generating new knowledge. This know- sortia gathered an average number of 10 partners with a mean project ledge could then be used for the prevention and management of human budget in the region of € 2.5 million. disease and for advice on living and ageing healthily. Although the programme focused primarily on basic research, some areas, such as cancer, The structure of this “Volume 2” follows the structure of the fi rst were more directed towards translational research. This enabled the basic Volume, and brief reference is made to all 87 projects described in the research already completed to be transferred to medical and therapeutic fi rst Volume, including complete and coherent “Tables of Contents” and applications. “Indices” arranged by project acronym, contract number and name of project coordinator. This allows us to provide the full picture of all Close to 850 research proposals were evaluated by peer review and some research funded under the Sixth Framework Programme in this area. 200 projects have been funded across the seven priority areas of: Each research area has a chapter devoted to it and is introduced by the • Cardiovascular disease current Scientifi c Offi cer(s) in charge. • Diabetes • Rare diseases We would like to acknowledge the Scientifi c Offi cers in charge, praising • Anti-microbial drug resistance their competence, professionalism and commitment as well as the • Brain, neurological and psychiatric diseases invaluable input provided by the previous Scientifi c Offi cers in charge • Human development and ageing, and (Philippe Cupers, Mary Fitzgerald, Elmar Nimmesgern, Jürgen Sautter and • Cancer Christian Wimmer). We would also like to acknowledge Anna Lönnroth, Deputy Head of Unit “Infectious Diseases” and the preceding Head of An average success rate of around 25 % was achieved for proposals across Unit “Major Diseases”, Alain Vanvossel. all four calls. This rate varied depending on the type of funding scheme and area of research, with a lower success rate for cancer topics overall (between 18 and 22 %), but a higher rate for small projects (coordination actions and specifi c support actions) and Networks of Excellence (above 35 % of successful proposals). Maria Vidal-Ragout Manuel Hallen (1) WHO Europe fact sheet EURO/03/06, www.euro.who.int/document/mediacentre/fs0306e.pdf Deputy Head of Unit Head of Unit

9 ➞ 44

CARDIOVASCULAR Cardiogenics

Identifi cation of genetic roots of coronary artery disease by combining stepwise genome wide association studies with transcriptomic and functional genomic investigation of relevant genetic variants

Keywords Myocardial infarction, coronary artery disease, been fraught with issues of replication and limited ability to interrogate genetic, association, genome wide, gene expression, the genome systematically. Nonetheless, the fi ndings to date provide functional genomics fi rm grounds for believing that further analysis of the genetic basis of CAD with more powerful approaches will yield important novel insights into the etiology of CAD which may translate into clinical benefi ts in terms of better assessment of patients, improved strategies for prevention, novel treatments and better tailoring of treatments to those likely to benefi t the most. Summary Aim We hypothesise that genome-wide, large-scale, stringent and stepwise linkage disequilibrium (LD) mapping of cases with familial coronary The recent identifi cation and cataloguing of a large number of single artery disease (CAD) will result in unbiased, systematic and reliable nucleotide polymorphisms (SNPs) (http://snp.cshl.org) and establish- discovery of CAD-related gene variants as well as detection of regulatory ment of the degree of linkage disequilibrium (LD) between SNPs non-coding regions. Extended exploration in large pan-European cohorts (www.hapmap.org) offer unprecedented opportunities for high-resolu- will document the diagnostic yield of these markers in the aetiology of tion genome-wide studies to be successful in identifi cation of genomic CAD. Finally, characterisation of the crucial genes with a range of markers associated with CAD. Nonetheless, there is a need to analyze functional genomic approaches will defi ne novel targets and pathways, large numbers of samples with strong genetic signals to maximize the which can be used in improving the assessment, prevention and treatment chances. In addition, coordinated replication studies in several addition- of CAD and its sequelae. al cohorts are essential to ensure that the fi ndings are robust. Coupled to a strong programme in functional genomics, Cardiogenics has the highest chance of meeting the ultimate objective of LSH-2005-2.1.1-1 call: “to develop new treatments for this disease that is of major importance for European citizens“.

The unique potential of Cardiogenics builds on extensive historic achievements, powerful pan-European bioresources and currently ongoing genome-wide SNP genotyping. The consortium consists of existing Problem European academic and clinical partnerships to establish a long-term co-operative framework dedicated to the improvement of cardiovascu- In the European Community, CAD is the leading cause of disability and lar health. The initiative proposed by Cardiogenics is novel but has death (n = 744,000 annually). The direct (51 %) healthcare costs, indirect clearly defi ned and achievable objectives. The decision to integrate this productivity losses (34 %) and informal care costs (15 %) of CAD amount translational programme with the leading European centre for genom- to € 45 billion yearly in the EU. Despite signifi cant advances in the ics research, the WTSI, provides access to facilities, knowledge and management of CAD and its major complication, myocardial infarction resources unequalled in Europe. As a result of the high level of integra- (MI), the burden of disease continues to increase, particularly in the tion and progress already achieved over the last 12 months and the Eastern European countries. Worldwide CAD is predicted to be the large-scale ongoing SNP genotyping we will be globally competitive and leading cause of morbidity and mortality by 2020. Novel approaches to in a position to make signifi cant contributions to the understanding of prevention, diagnosis, and therapy are critical if the burden of CAD- the genetic aetiology of CAD. The clinical usefulness of the data will related disability is to be reduced. These measures need to be targeted result in the development and commercial exploitation of Intellectual to those who are most likely to benefi t in order to maximize the benefi t Property Rights (IPR) by partnering SMEs and companies. for individual citizens and the society at large.

Numerous studies have established a consistent and strong role for Expected results genetic factors in the etiology of CAD, particularly when it is of early onset. First-degree relatives of MI patients carry a risk that is 2 to 8 times The main scientiﬁ c and technological objectives of this highly focused that of their peers, independently of other risk factors. Recently, many RTD project are: attempts have been made to identify genetic variants that affect this • to carry out well-integrated genome-wide and functional genomics susceptibility of CAD. With a few notable exceptions these studies have research in order:

10 | CARDIOVASCULAR – to identify genetic risk markers for CAD; – to link these with transcriptome data and cellular function in Genome wide association Functional Genomics atherosclerosis; Phase 1: GWA (500+175 k) Build RNA Bioresource – to discover new candidates and systems for drug development; – to exploit Intellectual Property Rights (IPR) in partnership with SMEs and the pharmaceutical industry; – to develop integrated novel information systems; Phase 2: Repilation (24 k) Genome wide expression • to establish a world-leading European consortium on the basis of Monocytes/macrophages Atherosclerotic plaque genuine and equal partnership between leading clinical centres, academic groups, cell biologists, bioinformaticians and biostatisti- Phase 3: Optimisation (0.5 k) Various Bioresources cians to provide further knowledge into the genetic basis of CAD Phase 3a: 10-20 priority genes for the beneﬁ t of members of the European Community. Phase 3b: Cardiogenics SNP chip

Effect of Strong Candidate Potential applications Genes (SCGs) on: Phase 4: Globalisation Monocyte transcriptome SCG-encoded proteins CAD is the Number One killer in the EU with a cost of billions of € to CAD risk factors and related phenotypes Society. Targeted prevention of premature CAD is the most effective Specific SCG-related phenotypes method to reduce such enormous burden of disease. In addition, one of Aetiology the most important social objectives of the Community is to improve the Mechanistic insight quality of life and healthy ageing. One of the key elements in reaching Candidates for drugability testing Diagnostic kits this goal is prevention of a chronic and often disabling disease. Conven- tional risk factors like smoking, diabetes, hypertension, and high cholesterol explain only about half of the risk to develop CAD and inter- individual relevance of these risk factors is low. Additional markers for the risk of CAD need to be discovered so that prevention can be targeted to those at highest risk. This is particularly true for those with early onset of CAD and familial predisposition. The resources for preven- proteins in biological samples (plasma, serum, etc.). We foresee a sce- tion are limited, and reliable risk identifi cation is required to make nario in which the prescription of novel antibody-based monocyte/ preventive programmes achievable from an economic and public accept- macrophage compounds will be based on the level of a selective number ance point of view. of SCG markers and the monocyte transcriptome. EUROIMMUN AG will generate murine or rat monoclonal antibodies against a selected number The functional genomics programme of Cardiogenics has a strong focus (~10-20) of SCGs. Propriety technologies and platforms will be used to on monocytes/ macrophages. By this approach we will identify novel develop bedside based assays for the rapid and reliable measurement of target molecules on monocytes. Until the completion of phase 1 and of SCG levels. the functional genomics studies, there will be no list of confi rmed targets. However, we foresee in month 18, a convergence between the TRIUM AG has developed over the last two years a clinical information genome wide analysis and functional genomics studies and will have system for CAD (as part of the EU-FP6 funded BLOODOMICS project) identifi ed pathways and novel key molecules in the monocyte lineages which can support the complexity of post-genome research projects. amenable for therapeutic antibody development. We have a public/pri- The system has been developed with scalability in mind and will be the vate partnership funded ‘in-house’ and European Clinical Trial directive main tool to integrate epidemiological, clinical and biomarker data from compliant pipeline to take human phage antibodies from the bench to the over 80,000 CAD patients and controls. During the lifetime of bedside. Cardiogenics will exploit this unique resource and lead human Cardiogenics, the patient informatics management system (PIMS) will antibodies will be transferred to European pharmaceutical companies after be provided with additional functionality to estimate the risk of CAD completion of phase I human studies. associated morbidity and mortality on basis of clinical history, classic risk factors (including family history), genetic markers and existing and novel The future prescription of drugs will be increasingly tailored to sub- biomarkers (including the classical ones like CPK, troponins, etc.). Also groups of patients. The selective use of statins in a subset of CAD functionalities will be developed to support future stratifi cation of patients with hyperlipidaemia is an explicit example. Easy-to-use assays prescription of drugs for CAD. The decision to house the PIMS data at will be developed to quantitate strong candidate genes (SCG) -encoded the WTSI data-centre provides ample scope for expandability.

CARDIOVASCULAR | 11 Coordinator Francois Cambien C.E. van der Schoot Alun Evans INSERM U525 Sanquin Blood Queen’s University Belfast Heribert Schunkert Faculté de Médecine Supply Foundation Belfast, United Kingdom Universität zu Lübeck, Pierre & Marie Curie Amsterdam, The Netherlands Medizinische Klinik II, UK S-H, Paris, France Alistair Hall Campus Lübeck, Thomas Meitinger BHF Heart Research Centre Ratzeburger Allee 160, Willem Ouwehand GSF-Forschungszentrum Leeds General Infi rmary 23538 Lübeck, Germany University of Cambridge & National fuer Umwelt und Leeds, United Kingdom Phone: +49 451 5002501 Blood Service Cambridge Gesundheit GmbH Fax: +49 451 5006437 Cambridge, United Kingdom Muenchen-Neuherberg, Germany Ulf Steller heribert.schunkert@innere2. EUROIMMUN Medizinische uni-luebeck.de Christian Hengstenberg Stefan Blankenberg Labordiagnostica AG Klinik und Poliklinik für Klinikum der Johannes Gutenberg- Lübeck, Germany Innere Medizin II Universität, II. Medizinische Klinik Partners Universitätsklinikum Regensburg Verfügungsgebäude für Forschung Michael Scholz Regensburg, Germany und Entwicklung TRIUM Analysis Online GmbH Nilesh Samani Mainz, Germany Munich, Germany University of Leicester Panos Deloukas Clinical Sciences Wing Wellcome Trust Sanger Institute Ann-Christine Syvännen Dietlind Zohlnhöfer Glenfi eld General Hospital Wellcome Trust Genome Campus Uppsala University Deutsches Herzzentrum München Leicester, United Kingdom Hinxton, Cambs, United Kingdom Uppsala, Sweden Technische Universität München Munich, Germany

Project number LSHM-CT-2006-037593 EC contribution € 9 999 902 Project Duration 48 months Starting date 01/10/2006 Instrument IP Project website www.cardiogenics.eu/web/

12 | CARDIOVASCULAR CONTICA

Control of Intracellular Calcium and Arrhythmias

Keywords Ion channels, mutations, arrhythmias, calcium Problem handling, sarcoplasmic reticulum calcium release channel, ryanodine receptor Ventricular arrhythmias are among the leading causes of cardiovascular morbidity and mortality in patients affected by cardiovascular disease in Europe. A major cause of arrhythmias may be inherited or acquired dysfunction of the cardiac ryanodine receptor. However, the molecular and cellular mechanisms underlying these arrhythmias are only poorly understood and neither effective diagnosis nor pharmacological treatments are available yet. Summary

Ventricular arrhythmias are a major cause of mortality. Currently there is Aim no effective treatment available, largely because our understanding of their molecular basis is poor. Altered intracellular Ca2+ handling may be The major scientiﬁ c objective of this project is to determine the subcel- a fi nal common pathway predisposing to ventricular tachycardias and lular mechanisms underlying fatal inherited arrhythmias related to sudden death in acquired or congenital heart diseases. In particular, mutations in the cardiac SR Ca2+ release channel (ryanodine receptor, dysfunction of the SR Ca2+ release channel (ryanodine receptor, RyR2) RyR2). We will apply state-of-the-art technology, including genetics, has been implicated in arrhythmogenesis and sudden death in heart genotype-phenotype correlations, generation of RyR2 mutants and failure and in catecholaminergic polymorphic ventricular tachycardia expression in cell systems, transgenic animals and gene chip technology (CPVT), a fatal inherited arrhythmogenic disease characterised by for proﬁ ling mRNA, in order to achieve in vitro and in vivo phenotyping. mutations in RyR2. Understanding the complex function of the RyR2 Ca2+ Based on initial evidence that arrhythmias observed in patients with channel and its regulatory mechanisms, therefore, holds the promise heart failure (acquired arrhythmias) are also caused by defective intracel- to develop new diagnostic and therapeutic strategies for effective lular Ca2+ handling and RyR2 dysfunction, we will compare the basic treatment of these lethal arrhythmias. The CONTICA investigators are mechanisms of inherited arrhythmias in transgenic animals harbouring a multidisciplinary team composed of cardiologists, molecular biologists, RyR2 mutations to those of acquired arrhythmias in animal models of biophysicists and physiologists with established and complementary hypertrophy and heart failure, as well as in native human myocardium. interests and expertise in the study of intracellular Ca2+ handling and In addition, a new and unique mathematical model for the assessment arrhythmias. The goal pursued by the CONTICA group is the elucidation of of the excitation-contraction coupling process in the heart has been the molecular mechanisms linking defective RyR2 function to the developed. We anticipate that this model is able to predict the sus- development of arrhythmias. Employing a wide range of state-of-the-art ceptibility to triggered arrhythmias. The employed algorithms will be techniques, the group will perform in depth studies on RyR2 dysfunction advanced in CONTICA using data from animal models and patients with in animal and human arrhythmias on the molecular, cellular and whole inherited and acquired arrhythmias. The model will help to gain a better organism level. Of particular importance, data gathered from the world’s largest collective of individuals affected by mutations in RyR2 will reveal novel insights into the genetics of CPVT and enable genotype-phenotype Catecholaminergic polymorphic ventricular tachycardia correlations and expression studies of mutated RyR2 in cell lines and For men animals. The combined knowledge gained from these studies will be used to develop and test novel diagnostic approaches and anti-arrhythmic drugs to help alleviate one of the largest health burdens to the European society, i.e. malignant ventricular arrhythmias. Cardiac Myocyte to cell

Ca2+ sparks and waves

Single RyR2 channel to single molecules O

CARDIOVASCULAR | 13 understanding of the arrhythmogenic mechanisms studied and to Coordinator Prof. Sylvain Richard predict the anti-arrhythmic potential of novel compounds designed Institut National de la Santé to modulate cardiac ion channel function. Ultimately, the CONTICA Prof. Burkert Pieske et de la Recherche Médicale investigators will develop novel diagnostic and therapeutic concepts Dept of Cardiology and Pneumology Montpellier, France Bereich Humanmedizin and test new pharmacological compounds that may be effective in pre- der Georg-August-Universitaet Prof. F. Anthony Lai venting inherited and acquired arrhythmias related to abnormalities in Goettingen – Stiftung Cardiff University 2+ intracellular Ca handling. Oeffentlichen Rechts Cardiff, United Kingdom Robert-Koch-Str. 40 37075 Goettingen, Germany Prof. Alexandra Zahradníková Expected results Phone: +49 551 39 8925 Ústav molekulárnej fyziológie Fax: +49 551 39 19127 a genetiky Slovenskej The CONTICA consortium will develop novel mechanistic insights, [email protected] Akadémie Vied diagnostic strategies, and therapeutic targets in patients with inherited Bratislava, Slovakia or acquired Ca2+-related arrhythmias. Therefore, CONTICA will contribute Partners Prof. Gottfried Brem to the development of new diagnostic and therapeutic guidelines of Agrobiogen GmbH national and international clinical or scientifi c societies. The better Prof. Marc A. Vos Hilgertshausen, Germany understanding of the molecular cause of the disease will also help to University Medical Center Utrecht improve standards for identifi cation, risk stratifi cation and therapy of Utrecht, The Netherlands Dr. Marion Hirt affected patients. Dr. Ralph Oehlmann Prof. Silvia G. Priori IMGM Laboratories GmbH Not only will we strengthen our basic knowledge concerning inherited IRCCS Fondazione Salvatore Maugeri Planegg-Martinsried, Germany and acquired forms of ventricular arrhythmias due to improper intracel- Pavia, Italy lular calcium handling, but we aim at developing novel diagnostic tests and identify new targets for drug therapy. Such an initiative will eventually reduce the number of ventricular arrhythmias and thus alleviate the risk of sudden cardiac death in these patient populations.

Major results

• Determination of the functional consequences of cardiac ryanodine receptor (RyR2) mutations. • Generation of transgenic animals featuring different genetic abnormalities and combinations of defects. • Development of a novel risk stratiﬁ cation method that will account for intracellular Ca2+ abnormalities. • Identiﬁ cation of new pharmaceutical targets in inherited and acquired cardiac disease. • Development of drugs that we expect to impact on the occurrence of sudden cardiac death. Project number LSHM-CT-2005-018802 Potential applications EC contribution € 2 755 000 • New animal models for the study of inherited and acquired Project Duration arrhythmias. 36 months • New methods for diagnosis of patients at risk to develop fatal Starting date arrhythmias. 01/02/2006 • New drugs for treatment of fatal arrhythmias. Instrument STREP Project website www.contica.eu

14 | CARDIOVASCULAR CVDIMMUNE

Immonmodulation and autoimmunity in cardiovascular disease and atherosclerosis

Keywords Atherosclerosis, atherothrombosis, natural Problem antibodies, antiphospholipid antibodies, SLE, immunomodulation Cardiovascular disease, mainly caused by atherosclerosis, is the main cause of death in the Western world and also increasingly in developing countries. Even though progress has been made in prevention of the disease, the infl ammatory and immunological nature of atherosclerosis is not refl ected by available diagnostic measures. Furthermore, treatment has been improved by lipid lowering drugs, e.g. statins, but novel Summary treatment modalities aiming at the pathological infl ammatory and immune reactions characterizing atherosclerosis are yet to be developed. Atherosclerosis, the major cause of cardiovascular disease (CVD) is an Likewise, the understanding of the mechanisms leading to atherosclerosis infl ammatory disease, where the immune system plays an important role. and causing the immune reactions are poorly characterized. This applies Autoantibodies as protection or risk markers and therapy through to the general population but also to SLE, an autoimmune disease immunomodulation could be a major advance in prevention and treatment where the risk of CVD is very high. of CVD and is a focus of this project. One type of antibodies is natural antibodies against phosphorylcholine (aPC), an antigen exposed in some bacteria and in phospholipids with platelet activating factor (PAF)-activity, Aim e.g. in oxidized LDL. Our clinical and experimental studies indicate that aPC, mainly of IgM type are protection factors against human This project aims at developing: atherosclerosis and CVD. Our plan is therefore to develop aPC as • a novel protection marker for atherosclerosis, natural IgM antibodies a diagnostic tool, which will be tested in several unique cohorts, including against phosphorylcholine (PC) and novel risk markers, IgG autoan- healthy individuals and high risk individuals. We also think aPC could tibodies against phospholipids (aPL): platelet activating factor (PAF) represent a novel therapeutic modality, where both polyclonal and and PAF-like lipids (aPAF). monoclonal aPC are possibilities. Both kinds of aPC antibodies will be • these markers will be investigated in unique patient cohorts. Docu- developed and tested in mouse animal models for atherosclerosis. mentation for registration of diagnostic ELISA kits in EU and the In contrast to natural antibodies (which are germ line encoded), U.S. will be developed. antiphospholipid antibodies (aPL) are risk markers for both arterial and • novel immunomodulation therapy against atherosclerosis and CVD in venous thrombosis, and our main focus is novel types of aPL against direct association with these factors will be developed: administration platelet activating factor (PAF) or PAF-like lipids (aPAF). These novel risk of atheroprotective aPC and increasing of Annexin A5 binding to markers (aPAF) will be tested in the same cohorts as aPC and in both cases, robust kits will be developed. The genetic background of aPC and aPAF will be studied, e.g. in the Swedish Twin Registry. In a prototypic Resting levels of EC intracellular calcium before leukocytes autoimmune disease, SLE where the risk of CVD is very high, such aPL are were allowed to interact with ECs is shown in individual cells through continuous registration of fl uorescence raised. Annexin A5 is a plasma protein, interfering with phospholipid intensity with a widefi eld microscope. Endothelial cell surfaces and acting as an anticoagulant. We have demonstrated that borders are shown by labelling ECs with Alexa 488- aPL inhibit Annexin A5 binding to endothelium, an effect neutralized by conjugated anti VE Cadherins antibodies. ECs (GREEN) intravenous immunoglobulins (IVIG) and that Annexin A5 is present in were loaded with Calcium sensitive probe. atherosclerotic lesions especially at sites prone to rupture. Based on our fi ndings, we have hypothesized that raising Annexin A5 binding, either by Leukocytes, freshly isolated from venous blood of healthy administration of neutralizing antibodies from IVIG in individuals with donors on Ficoll/Hypaque gradients were allowed to settle on the monolayers at 37 °C were allowed to transmigrate high aPL levels, or by administration of Annexin A5 per se, could prevent through IL-1 ß treated EC at 37 ºC. Leukocytes,labeled plaque rupture and atherothrombosis. (Red) and are at different stages of diapedesis process. Taken together, our project combines diagnostic and therapeutic projects and is focused on the role of the immune system, especially autoantibodies, in atherosclerosis and atherothrombosis. Changes in EC intracellular calcium were measured during leukocytes extravasation on individual cells through continuous registration of fl uorescence intensity with a widefi eld microscope. Leukocytes were allowed to transmigrate through IL-1 ß treated EC monolayers at 37 °C. A transient increase in Enthothelial cells- [Ca2+]i is shown. ECs (GREEN) were loaded with Calcium sensitive probe. Localized increase in EC Calcium in individual cells is shown (GLOWING GREEN).

CARDIOVASCULAR | 15 endothelium through neutralizing of aPAF and other aPL or admin- Potential applications istration of Annexin A5 per se, a plasma protein that may prevent rupture of atherosclerotic plaques and CVD. We expect our novel protection and risk markers to reﬂ ect inﬂ ammatory • proof of concept for at least one of these pharmaceutical product and immunological factors in plaques and to be useful as tools for risk candidates. assessment for CVD, as a complement to classical risk factors as dyslipidemia, hypertension, diabetes, and smoking. We also propose that treatment relating to these factors could be a major step forward in Expected results addition to established ones as lipid lowering drugs.

• Development of robust and quantitative test kits in ELISA format for analysis of IgM antibodies to PC and IgG antibodies to PAF or PAF-like lipids in serum. • Epidemiological data on aPC and aPAF from several well character- Coordinator Jörg Blaeser ized and in several cases unique cohorts of healthy and high-risk Sweden Diagnostics GmbH individuals. Johan Frostegård Freiburg, Germany • Assessment of genetic variability of aPC and aPAF and identiﬁ cation Karolinska Institutet Stockholm, Sweden Paul Quax of candidate genes. Netherlands Organization • Establishment of in vitro and in vivo models for studies of aPC and for Applied Scientifi c Research Annexin A5 relating to atherosclerosis and atherothrombosis. Partners Leiden, The Netherlands • In vivo data for drug candidates for atherosclerosis and atherothrombosis and data compiled to submit an IND application from Hans Grönlund Marta E. Alarcón-Riquelme one of the drug candidates investigated in the project. Athera Biotechnologies AB Uppsala Universitet Stockholm, Sweden Uppsala, Sweden

Peter Sever Thomas Norberg Imperial College London IsoSep AB London, United Kingdom Uppsala, Sweden

Stefan Blankenberg Wouter Jukema Johannes Gutenberg-University Mainz Leiden University Medical Center Mainz, Germany Leiden, The Netherlands

Ewa Ninio Institut National de la Santé et de la Recherche Médicale Paris, France

Project number LSHM-CT-2006-037227 EC contribution € 2 700 000 Project Duration 36 months Starting date 01/08/2006 Instrument STREP – SME Project website www.contica.eu

16 | CARDIOVASCULAR EUGeneHeart

Genomics of Cardiomyocyte Signalling to Treat and Prevent Heart Failure

Keywords Heart failure, benefi cial and maladaptive Problem hypertrophy, myocardial infarction, signal transduction, proteomics, microarrays, drug development Congestive heart failure is a leading cause of cardiovascular morbidity and mortality and the main cause of hospitalization among patients older than 65 years in Europe. Due to the aging of industrialized populations, the number of hospitalizations on account of heart failure is predicted to increase by 17 % and pose an increasing burden in the 21st century. However, even young patients may suffer form heart failure. Heart failure is one of the most frequent disabling diseases in Europe and therefore carries a tremendous socio-economical relevance. Summary

Heart failure is one of the most frequent diseases and in Europe about Aim 4 million of humans are affected. The patients are suffering from limited ability to cope with physical stress and shortage of breath and have The EUGeneHeart project aims to develop new treatment strategies, by a reduced expectancy of life. Heart attack, high blood pressure and which maladaptive signalling can be prevented and adaptive signalling valvular defects are the main causes for heart failure. The consequences can be promoted to inhibit heart failure. are abnormal changes (‘remodelling’) in the heart, which cause hypertrophy and weakening of the myocardial muscle. Hypertrophy (thickening of the The 5 specifi c aims of the EUGeneHeart project are: myocardial wall) occurs also due to exercise but without transition to • to break down and identify benefi cial and harmful aspects of heart failure. hypertrophic signalling. This includes analysing how hypertrophic EUGeneHeart will develop new approaches to prevent and treat heart stimulation is perceived and connected into intracellular signals and failure through analysis of the genomics of signalling. The strategy is how these signals change the transcriptional and translational based on the hypothesis that benefi cial and maladaptive forms of hyper- program of the cells. Dissection of signalling will be carried out in trophy exist and that heart failure is frequently preceded by maladaptive mouse models of hypertrophy and heart failure including models hypertrophy. The dissection of both, adaptive and maladaptive signalling with genetic manipulations leading to an adaptive type of hypertrophy. in hypertrophy will allow the identifi cation of benefi cial and maladaptive The models will be subjected to different stressors provoking either components of signal transduction in hypertrophy and heart failure. physiological or pathological hypertrophy and phenotyping of The analysis of murine models subjected to pressure overload, myocardial signalling events in a standardised and comparable manner will be infarction, and chronic exercise will allow identifying the models with performed. Analysis will also be extended to tissue from human a benefi cial response. Their differences in signal transduction will then be hearts. Gender aspects will be especially considered. High levels of analyzed. By means of screening techniques including proteomics and synergy will be achieved by development of a phenotyping matrix; microarrays, forward and reverse genetics in drosophila and zebrafi sh • screening techniques will identify new players in genomics of and validation in murine models and patient cohorts, new players in signalling. Different screening techniques will be applied to uncover different forms of hypertrophy and failure will be identifi ed. The results of new candidate targets which will be exploited to enhance both the the signal transduction and screening experiments will be exploited with understanding of signalling and to develop new treatment strategies the aim to generate new candidate drugs. In addition, specifi c questions in a joint translational research activity. In particular, these screening regarding sex differences relating to signalling in hypertrophy will be approaches will include forward genetics in drosophila and zebrafi sh addressed. Last but no least, a training initiative to provide education for and genome wide screens in cardiomyocytes. Also, small molecules young scientists in the specifi c fi eld is also integrated in the project. The will be screened for their potential to alter the activation of signalling EUGeneHeart project has brought together a team of 20 research centres pathways in cardiomyocytes and in engineered heart tissue. across Europe (academia and industry) to elucidate the questions Screening will also include a transcriptome and proteome based regarding the genomics of cardiomyocyte signalling in the treatment and analysis to identify differentially expressed genes and proteins. In prevention of heart failure. a bench-to-bedside-to-bench approach, we will identify candidate genes in forward genetics screens in zebrafi sh and drosophila and verify their signifi cance via analysis of genetic polymorphisms in these (and additional) genes in patient cohorts. This will be followed by development of gene targeted treatments (using conventional drug development and RNAi technology); • to exploit the knowledge on key signalling events. We will develop new strategies to inhibit detrimental and to promote benefi cial signals of the hypertrophic programme. This strategy includes

CARDIOVASCULAR | 17 Task 6 Management existing pharmacological interventions

Task 2 Screening for new genes maladptive hypertrophy and failure

in drosophila, Prevention and treatment of zebrafish, mouse, human Task 3 Translation drug development proof of concept. Phase I and IIa trials Task 1 Outlicensing Dissection Adaptative and Adaptative Hypothesis based geno and phenotyping in mouse and human maladaptive signaling and phenotyping maladaptive

Task 4 Gender Aspects

Task 5 Training and Demonstration career development, conference, PR The organisation of the EUGeneHeart project as different Tasks.

translational research on existing pharmacological interventions, Expected results proof of principle studies with molecules, already developed by the partners of the consortium and the development of new candidate The ultimate accomplishment will include improvement of health, molecules identifi ed during the course of the integrated project. reduction of health expenses, as well as creation of new jobs and Candidate targets (genes or proteins) will be tested for their effects economic benefi t. on hypertrophy and signalling in a series of model platforms ranging from an immortalised cardiomyocyte cell line via engineered heart tissue and transgenic animals to larger animal models. Small Potential applications molecules will be screened in the same systems together with the SME Greenpharma; The major goal of the IP is represented by Task 3 which accomplishes • since signifi cant gender differences exist in the physiology and exploitation of knowledge into new treatments of heart failure. This pathophysiology of adaptive and maladaptive myocardial hypertro- includes intellectual property and licensing management as well as plan- phy, these will investigated. Specifi c questions regarding sex differ- ning to spin-off companies. Task 3 is based on three different strategies: ences related to signalling in hypertrophy will help to unravel, • use of existing pharmacological interventions for further development whether future therapy needs to be sex-specifi c so as to yield the of drugs; best therapeutic results. Our approach takes account of national • use of previous fi ndings and developments from members of the research activities and recruits knowledge, patients and tissues at consortium to modulate signal transduction pathways associated a European level; with heart failure development; • this task includes a training initiative for scientists and also deals • new targets identifi ed by Task 1 and Task 2. with the dissemination of knowledge achieved during the course of the EUGeneHeart project. Amongst the organization of various The established matrix structure is a major achievement, which will scientifi c meetings, the consortium will create a European Scientifi c allow to characterise transgenic animal models within 14 weeks or more Conference in the thematic area of its expertise that would be the extensively including pressure overload, infarction and exercise hyper- equivalent of the US-based Gordon Conferences. Such a high level trophy in 12 months. This matrix may be of high interest to pharmaceu- scientifi c conference currently does not exist in Europe. The main tical companies and other research groups and by itself could make goals of this new initiative are: a spin-off enterprise. – to expose European scientists to top level science in the thematic area of the consortium – to provide young European scientists with a platform to communicate their own scientifi c work.

18 | CARDIOVASCULAR Coordinator Prof. Gianluigi Condorelli Prof. Vera Regitz-Zagrosek Dr. Alexandra Zahradníková Fondazione Parco Biomedico Charité Universitätsmedizin Ústav molekulárnej fyziológie Prof. Gerd Hasenfuss San Raffaele Berlin, Germany a genetiky Slvenskej Dept of Cardiology and Pneumology Rome, Italy Akadémie Vied Bereich Humanmedizin der Prof. Ajay Shah Bratislava, Slovakia Georg-August-Universitaet Goettingen Dr. Leon De Windt King’s College Stiftung oeffentlichen Rechts Dr. Jeroen Bakkers London, United Kingdom Dr. Quoc-Tuan Do Robert-Koch-Str. 40 Koninklijke Nederlandse Akademie Greenpharma S.A. 37075 Goettingen, Germany van Wetenschappen Prof. Karin Sipido Orléans, France Phone: +49 551 396351 Utrecht, The Netherlands Katholieke Universiteit Leuven Fax: +49 551 396389 Leuven, Belgium Prof. Josef Penninger [email protected] Prof. Pieter Doevendans Dr. Hans Loibner University Medical Center Utrecht Prof. Guido Tarone Apeiron Biologics Forschungs und Utrecht, The Netherlands Prof. Emilio Hirsch Entwicklungs GmbH Partners Università degli studi di Torino Vienna, Austria Prof. Thomas Eschenhagen Torino, Italy Prof. Wolfgang Wuttke University Medical Center Prof. Josef Penninger Prof. Hubertus Jarry Hamburg-Eppendorff, Germany Dr. Marc Van Bilsen Institute of Molecular Prof. Ralph Knöll Universiteit Maastricht Biotechnology of the Austrian Faculty Human Medicine Prof. Jean-Jacques Mercadier Maastricht, The Netherlands Academy of Sciences (IMBA) Georg-August-Universitaet Prof. Rodolphe Fischmeister Vienna, Austria Goettingen, Germany Prof. Frederic Jaisser Prof. Juleen Zierath Institut National de la Santé Karolinska Institutet Dr. Kader Thiam Prof. Jean-Luc Balligand et de la Recherche Médicale Stockholm, Sweden genOway S.A. Université catholique de Louvain Paris, France Lyon, France Louvain, Belgium Prof. Hugh Watkins Prof. Julius Papp University of Oxford Dr. Lars Kattner University of Szeged Oxford, United Kingdom Endotherm Pharmaceuticals GmbH Szeged, Hungary Saarbrücken, Germany

Project number LSHM-CT-2005-018833 EC contribution € 11 400 000 Project Duration 60 months Starting date 01/01/2006 Instrument IP Project websites www.eugeneheart.com www.eugeneheart.eu

CARDIOVASCULAR | 19 GENECURE

Applied genomic strategies for treatment and prevention of cardiovascular death in uraemia and end stage renal disease

Keywords Cardiovascular, renal, genetics Problem

CV risk is greatly increased in renal failure. The increase in CV risk is already detectable in mild renal failure and amounts to a 10-100-fold elevated CV mortality in ESRD. The number of patients with ESRD is increasing progressively, and a further rise is anticipated due to the effects of ageing and the epidemic of patients with type II diabetes. Better elucidation of the pathophysiology of CV damage in renal patients is Summary needed. It is presumably multifactorial with involvement of common CV risk factors, such as hypertension and dyslipidemia (that cluster in renal In renal failure cardiovascular risk (CV) is greatly enhanced, posing a large patients), as well as factors specifi c for renal failure such as cardiovascu- burden on both patient and society. Elucidation of its genetic basis is lar calcifi cation and the malnutrition-infl ammation syndrome. The mode important to allow innovative prevention strategies in these patients by: of renal replacement therapy (RRT) is also relevant to CV risk, as shown • early identifi cation of subjects at risk; by differential risk patterns in pre-dialysis patients, dialysis and trans- • better allocation of available prevention and treatment modalities and; plantation. Thus, the impact of treatment modality should be explicitly • identifi cation of novel pathways of organ damage as new targets for considered. Genetic factors are involved in the high CV risk in renal intervention. patients, but knowledge on their impact is scattered and far from sys- GENECURE will pursue these aims by a coherent set of studies and tematic. Moreover, infrastructure for collaborative studies is still lacking, initiatives that will: hampering rapid independent confi rmation of results from association • link existing initiatives into a joint infrastructure for collaborative studies, and precluding studies of suffi cient sample size to cover the genetic studies in renal populations, consisting of a phenotype bank, range of relevant gene-environmental interactions. The GENECURE a DNA bank, and a Metabase application; project consortium is hosted by the larger ReGeNet consortium, that • identify genes relevant to accelerated CV damage in end stage renal caters for over 25,000 renal patients all over Europe. disease, in terms of natural course and response to intervention, with emphasis on identifi cation of targets for primary and secondary prevention. Candidate genes from established pathophysiological Aim pathways will be studied, and novel candidates identified from hypothesis free studies in rodent models and human tissue; The general aim is to develop an integrated strategy for better primary • provide a statistical risk model on impact of genetic and environmental and secondary prevention of the CV complications of renal failure by factors over time, for clinical use in risk stratifi cation and decision translating genomic approaches to patient care. To this purpose the spe- making (to be tested in future prospective studies) and for scientifi c cifi c aims are twofold. First, to identify genes relevant to the patho- use for identifi cation of research targets likely to have the largest physiology of the enhanced CV risk in renal failure as to natural course clinical impact. This will provide a systematic overall assessment on and as to impact of interventions. Second, to establish an infrastructure the role of genetic factors in CV risk in renal failure, to be made for collaborative genetic studies in renal populations that optimizes use available for guideline development and health economic evaluation. of existing and future resources. Moreover, a scientifi c and infrastructural basis for future collaborative genetic studies will be established. Expected results

• A common data-bank for renal populations, containing a DNA-bank, standardized data on phenotype and ICT infrastructure, i.e a Metabase application for web-based access and analyses. • A network infrastucture for analysis of outcome of interventions in relation to genetic background. • New candidate genes for the accelerated cardiovascular damage in the renal patients. • A systematic analysis of the impact of known and novel candidate genes for cardiovascular outcome in renal populations representing the whole spectrium of renal impairment, both for natural course and outcome of interventions. • Statistical risk models for the impact of genetic factors on cardiovascular outcome in renal populations.

20 | CARDIOVASCULAR Potential applications Coordinator Hans Neumayer Klemens Budde The systematic overall assessment on the role of genetic factors in CV risk Gerjan Navis Charité in renal failure will serve: Dept Medicine, Division of Nephrology Berlin, Germany • improvement of clinical risk stratifi cation in renal patients; University Medical Centre Groningen Hanzeplein 1 Olivier Devuyst • identifi cation of research targets likely to have the largest clinical NL-9700 RB Groningen Université Catholique de Louvain impact; The Netherlands Louvain, Belgium • guideline development; Phone: +31 503614441/2621 • health economic analysis. Fax: +31 503619310 Koos Zwinderman [email protected] Academic Medical Center The established infrastructure for genetic studies will enable the renal Amsterdam, The Netherlands community to make optimal use of the rapid developments in the fi eld of molecular genetics for clinical studies Partners Marina Noris Mario Negri Institute Bergamo Bengt Lindholm Bergamo, Italy Martin Schalling Karolinska Institute Jurgen Floege, Markus Ketteler Stockholm, Sweden University Hospital Aachen Aachen, Germany Paul Brenchley University of Manchester Ivar Roots Manchester, United Kingdom CENIMED Berlin, Germany Els Boeschoten Hans Mak Instituut Naarden, The Netherlands

Project number LSHM-CT-2006-037697 EC contribution € 2 246 000 Project Duration 36 months Starting date 01/01/2007 Instrument STREP Project website www.genecure.eu

CARDIOVASCULAR | 21 HDLomics

Functional genomics of inborn errors and therapeutic interventions in high density lipoprotein (HDL) metabolism

Keywords Animal models, atherosclerosis, cardiovascular Problem disease, genetics, high density inborn errors of metabolism, lipoproteins, lipids, lipidomics, prevention, proteomics Fifty per cent of the population of Europe dies of coronary heart disease (CHD). Of these, current preventive and therapeutic measures, notably lowering of low density lipoprotein (LDL) cholesterol and blood pressure can probably save 30 %. For the remaining seventy percent there will be only hope if new targets for therapeutic intervention are identifi ed. One of the most interesting targets is high density lipoprotein (HDL) cholesterol. The great clinical interest in HDL has been generated by numerous Summary epidemiological studies that have found an increased risk of fatal and non-fatal CHD events, such as myocardial infarction, being associated Despite considerable progress in prevention and therapy, coronary artery with low HDL cholesterol. In addition, HDL particles as well as HDL-asso- disease remains the most frequent cause of death. High density lipoproteins ciated proteins and lipids exert a broad scope of anti-atherogenic effects. (HDL) are promising targets to further reduce morbidity and mortality In agreement with these protective effects, the development of athero- because low HDL cholesterol plasma concentrations increase cardiovascular sclerotic lesions could be inhibited or even reverted in animal models by risk and because HDL exert many cardio- and vasoprotective functions. elevation of HDL cholesterol levels. With the exception of nicotinic acid, There is also great need in improving the diagnostic and prognostic value which increases HDL cholesterol by about 20 % (but with signifi cant of the biomarker HDL cholesterol as HDL function is determined by the side effects), the currently available lipid modifying drugs have little quality rather than by the quantity of HDL. Therefore, this HDLomics project effect on HDL cholesterol levels. Hence, there exists a great interest in will apply functional genomics approaches including genomics, proteomics fi nding treatment modalities that increase HDL cholesterol concentra- and lipidomics as well as new clinical diagnostic tools to study the effects tion in plasma. Moreover, there is strong evidence that the atheropro- of differential regulation of HDL metabolism on pathophysiological events tective effect of HDL is determined by the quality rather than by the relevant for atherosclerosis: quantity indicating that there is also great need in improving the diag- • using genome wide linkage analysis in families with Mendelian nostic and prognostic value of the biomarker HDL cholesterol. Therefore inheritance of low HDL cholesterol we will search new major genes and in agreement with the call LSH-2005-2.1.1.4, this HDLomics project regulating HDL cholesterol; will apply functional genomics approaches including genomics, pro- • using high throughput sequencing and allele specifi c genotyping teomic and lipidomics as well as new clinical diagnostic tools to improve technology we will elucidate the impact of mutations in candidate insight in HDL metabolism in disease states and pathophysiological genes for the variation of HDL cholesterol as well as other lipid traits events relevant to atherosclerosis. The ultimate goal of HDLomics is the and the occurrence of cardiovascular events; identifi cation of new targets to reduce the burden of CVD. • using knock-out and transgenic mouse models we will study the metabolic and vascular impact of mutated candidate genes on HDL metabolism; Aim • we will employ proteomic and lipidomic tools for the characterisation of HDL and fi nding specifi c effects of mutations or interventions on • Using genome wide linkage analysis approaches in families with the structure and function of HDL as well as on lipid metabolism and Mendelian inheritance of low HDL cholesterol, we will try to iden- atherosclerosis; tify new major genes regulating HDL cholesterol. • we will develop and evaluate new therapies including gene therapy • Using high throughput sequencing and allele specifi c genotyping protocols for the correction of inborn errors of HDL metabolism in technology, we will elucidate the impact of rare and frequent poly- humans. The ultimate goals of HDLomics are hence the identifi cation morphisms in candidate genes for the regulation of HDL choles- and validation of new targets, which can be used in differential terol as well as other lipid and lipoprotein traits and the occurrence diagnosis, prognosis, therapy, prevention, and therapy monitoring of of cardiovascular events in families and the population. low HDL cholesterol and atherosclerosis. • Using a large well-characterized prospective cohort study with incident CHD events and non-invasive diagnostic imaging tools, we will investigate the impact of mutations in pivotal genes of HDL metabolism on atherogenesis as well as the effect of experimental HDL modulating therapies on the course of subclinical atherosclerosis. • Using gene therapy in mouse models of HDL metabolism, we will study the impact of mutated candidate genes on HDL metabolism, lipid homeostasis and atherosclerosis.

22 | CARDIOVASCULAR • We will develop and evaluate new therapies including gene therapy ↔ HDL Proteomics / Lipidomics HDL↔ Physiology / Function protocols for the correction of inborn errors of HDL metabolism in ↔ humans. • We will develop and use new proteomics and lipidomics tools for Population / Family Genetics the characterization of HDL and ﬁ nding associations between mutations, HDL function, and atherosclerosis in both humans and mouse models. Improving Insight in HDL Metabolism • The ultimate goal of HDLomics is the identiﬁ cation and validation Develop Diagnostic Tools of new targets, which can be used in differential diagnosis, Develop Therapy prognosis, therapy, prevention, and therapy monitoring of low HDL cholesterol and atherosclerosis.

ReduceReduce Cardiovascular DeathDeath Expected results

• High-throughput methods and tools for a better characterization Coordinator Angelika Chroni of HDL metabolism and function are established. National Centre of Scientifi c • New proteomic, lipidomic and functional markers of HDL function Arnold von Eckardstein Research ‘Demokritos’ and metabolism are identiﬁ ed. University of Zurich, Switzerland Athens, Greece • Novel genes and mutations affecting HDL metbolism are discovered; Vassilis I. Zannis • Novel clinical and animal models for a better characterization of Partners Foundation for Research HDL metabolism and function are established. and Technology • The effects of HDL-related genes and mutations on atherosclerosis John Kastelein Hellas, Iraklion, Greece and coronary artery disease are determined. Universiteit van Amsterdam • Low HDL cholesterol and atherosclerosis in mice can be corrected Academisch Ziekenhuis Jaap Twisk by therapeutic interventions which are useful for further clinical Amsterdam, The Netherlands Amsterdam Molecular development. Therapeutics Inc. • The results of the consortium are disseminated and exploited Anne Tybjaerg-Hansen Amsterdam, The Netherlands with regard to development of diagnostic tests or therapies which Rigshospitalet ultimately help to reduce morbidity and mortality associated with University of Copenhagen Copenhagen, Denmark cardiovascular diseases. Mats Lindahl Linköping University Potential applications Linköping, Sweden

We hope that the results of the consortium applied with regard to the development of both diagnostics and therapies which ultimately help to reduce morbidity and mortality associated with cardiovascular diseases. In more detail we expect the development of laboratory tests which can be used for the identiﬁ cation of patients with increased cardiovascular risk and for the monitoring of treatment effects in these Project number patients. In addition newly identiﬁ ed genes important in HDL metabo- LSHM-CT-2005-037631 lism may turn out as important targets for the development of drugs EC contribution which help to prevent, stop or even cure atherosclerosis. Finally, we € 2 748 674 expect to develop gene therapy protocols to treat inborn errors of HDL Project Duration metabolism. 36 months Starting date 01/01/2007 Instrument STREP Project website www.hdlomics.org HeartRepair

Heart Failure and Cardiac Repair

Keywords Myocardial infarction, heart failure, stem cells, Problem cardiomyocyte differentiation Cardiovascular diseases (CVD) cause 4 million deaths each year in Europe. Moreover CVD causes, in comparison with other life-threatening diseases such as cancer, the most deaths in Europe and in the European Union (cancer: 26 %, CVD: 43 %). In some countries in Central and Eastern Europe, CVD are already responsible for more than 50 % of deaths in men. Summary By far the leading cause of congestive heart failure (HF) is myocardial infarction. In certain extreme, though not too uncommon, cases of HF up HeartRepair is an integrated cross-border European project set-up to to 30 % (about 100 g) of working cardiac muscle is lost which usually fatally scientifi cally tackle one of the leading causes of congestive heart failure, perturbs functioning of the heart leading to death. Fundamentally then, the namely myocardial infarction. Pulling together and centralizing the chief basis for an effective therapeutic approach is just as simple: replacement knowledge and expertise of twenty two laboratories from eight European of damaged/dead myocardium with new viable cardiomyocytes. Community countries, it sets out to implement the lessons the embryo Although the past and recent advances in the fi eld of stem cell replace- teaches us during cardiac development to facilitate muscular reconstruction ment and rejuvenation therapies, specifi cally cardiomyocyte regeneration, of the infarcted heart. are rapidly gaining grace in the published scientifi c literature as a realistic Although the underlying causes of myocardial infarction may be varied, the future alternative for congestive heart repair strategies, the promises of subsequent heart failure which usually follows is straight forward, massive this therapy cannot yet be met. The major obstacle is lack of suffi cient cardiomyocyte necrosis which fatally perturbs heart function. Fundamentally knowledge on the pathophysiology of heart disease and the scientifi c then, HeartRepair sees the chief basis for an effective therapeutic approach expertise underlying the development of cellular replacement and rejuve- as clear-cut: replacement of damaged myocardium with viable nation therapies. Therefore, in order to realize and implement effective cardiomyocytes. Recent advances in the fi eld of stem cell replacement stem cell therapies for heart repair and in particular myocardial infarction therapies, although still in their infancy, offer a realistic future alternative we need to gather the expertise of leading research groups in Europe. for congestive heart failure repair. Scientifi c publications drawing most attention revolve around transplantation trials carried out using either embryonic stem cells or bone marrow-derived stem cells. Useful as such Aim trials may seem, this approach is often a black box and naive harbouring many dangerous pitfalls, which at the very least may result in a loss of The raison d’être of HeartRepair is to gather suffi cient scientifi c critical public faith if clear positive results are not forthcoming. Realization of this mass in Europe to address the fundamental issues on the genomics of aim will only ever be achieved by understanding the underlying principles cardiac muscle cell formation and repair. By pooling and sharing of cardiac muscle cell formation. knowledge R&D questions that are crucial for the scientifi c foundation In a major effort to deal this issue, our HeartRepair’s central goal is to of effective future therapies can be addressed that are presently being scientifi cally address the underlying biological questions surrounding put aside. For the fi rst time in the fi eld of European cardiac research, cardiomyocyte differentiation with the ultimate aim of correctly harnessing leading research groups will come together, sharing their knowledge on stem cells for cardiac repair. cardiac muscle cell formation, in an attempt to solve the ongoing For a clear coordinated approach HeartRepair is formulated into four work themes: • Genes for heart repair and plasticity, integrating clinical knowledge based genome sequencing to identify genes involved in heart development. • Diversifi cation of cardiac progenitor cells, examining the genetics of cardiac muscle cell formation by exploring the process of cardiomyogenesis and subsequent differentiation. • Cell interaction and cardiac reprogramming, exploring the details and signals necessary to facilitate and redirect the cardiac fi broblast cell lineage for repair recruitment. • Cardiac rejuvenation, to develop techniques to facilitate and speed repair of damaged though not yet necrotic myocardium.

A predictive protein model of the heart regulatory factors TBX5 NKX2-5.

24 | CARDIOVASCULAR Preserved adult human heart.

questions surrounding the applicability of cell replacement therapies in Potential applications cardiac repair. HeartRepair’s mission statement will be fulfi lled by addressing the following scientifi c objectives: Myocardial infarction is the leading cause of congestive heart failure in • fi nding the key genes involved in cardiac muscle cell formation by Europe. Furthermore, cardiac diseases are one of the major causes of genome mapping and sequencing; mortality in the western society; it causes huge social and economic • understanding the mechanisms underlying muscle cell origin and costs. The generation of functional contracting myocardial tissue with diversifi cation; new muscle cells is a promising prospect that may provide opportunities • development of tools to permit the reprogramming of cardiac for novel therapies for the protection and rescue of the myocardium fi broblasts already present in infarcted tissue; from ischemia and failure. Fundamental and translational research to • development of tools to enhance the capacity of the infarcted unravel the differentiation from stem cell to a well functioning heart heart to respond to cellular repair techniques. muscle cell is essential for the clinical application of stem cells in cell transplantation therapies. Moreover, therapies against cardiac disorders are the biggest market for the pharmaceutical and biotechnology sector. Expected results HeartRepair will allow the European industry to make a major advance in the development of effective cellular replacement and rejuvenation Four interwoven but independent research and development lines have therapies for myocardial infarction. been initiated to achieve a maximal knowledge acquisition: Line 1 uses genome and transcriptome discovery techniques from both clinical and non-clinical approaches which will lead to the identifi cation of genes and their protein products involved in heart development. Line 2 focuses not only on the genes which are steering development, but also on the DNA binding elements and the down/upstream effector genes which are being activated or deactivated as the cell travels its developmental journey along the cardiac muscle lineage. This will identify the controlling elements necessary to drive a progenitor cell to the desired cardiac muscle lineage. Line 3 attempts to unravel the signals involved in cellular fate decisions. This knowledge will be necessary if one is to attempt, for example, large scale culturing of cells for cardiac repair, or recruiting stem cells and/or differentiated cells, such as cardiac fi broblasts, to assist in the repair process. Line 4 will focus its attention on discovering the body’s own natural factors which assist in cellular repair and rejuvenation. The addition of such factors during cellular replacement and rejuvenation may prove quintessential in avoiding repair complications such as cellular rejection and apoptosis.

Project number LSHM-CT-2005-018630 EC contribution € 11 400 000 Project Duration 48 months Starting date 01/01/2006 Instrument 3 day chicken embryo. Linear Cardio-myocyte (in vitro). IP heart tube (green) with at the Project website inﬂ ow the pro-epicardium (red) protruding into pericardial cavity. www.heartrepair.eu

CARDIOVASCULAR | 25 Coordinators Dr. V. M. Christoffels Prof. B. J. M. Mulder Academic Medical Centre Cardiology, Academic Medical Centre Prof. Antoon F.M. Moorman Amsterdam, The Netherlands Amsterdam, The Netherlands Dr. Ruth van der Gaag Dept Anatomy & Embryology Dr. G. Cossu Prof. C. Mummery Academic Medical Centre Stem Cell Research Institute Hubrecht Laboratory L2-106 Meibergdreef 9 Milan, Italy Utrecht, The Netherlands 1105 AZ Amsterdam The Netherlands Dr. J. L. De la Pompa Prof. R. Munoz Chapuli Phone: +31 20 5664647 Centro Nac. de Biotecnologia CSIC Animal Biology Fax: +31 20 6976177 Univ. Autonoma Madrid Faculty of Science [email protected] Madrid, Spain University of Malaga [email protected] Malaga, Spain Prof. S. Dimmeler University of Frankfurt Dr. J. M. Perez Pomares Partners Frankfurt, Germany Animal Biology Faculty of Science Dr. U. Bauer Prof. D. Franco University of Malaga Competence Network Congenital University of Jaen Malaga, Spain Heart Defects, National Register Jaén, Spain Congenital Heart Defects Prof. N. Rosenthal Berlin, Germany Prof. J. A. Goodship Mouse Biology Programme Institute of Human Genetics- EMBL-Monterotondo-Scalo Prof. N. A. Brown International Centre for Life Heidelberg, Germany St George’s, University of London University of Newcastle London, United Kingdom Newcastle, United Kingdom Dr. P. Sartipy Cellartis AB Prof. M. E. Buckingham Dr. P. A. C. ‘t Hoen Gothenburg, Sweden Institut Pasteur Centre for Human and Clinical Genetics Paris, France Leiden University Medical Centre Prof. S. Schiaffi no Leiden, The Netherlands Scienze Biomediche Sperimentali Prof. J. Burn University of Padova Institute of Human Dr. W. Huber Padova, Italy Genetics-International European Bioinformatics Institute Centre for Life Cambridge, United Kingdom Dr. S. Sperling University of Newcastle Cardiovascular Genetics United Kingdom Dr. I. M. C. Kamerling Max-Planck-Institute Netherlands Heart Foundation for Molecular Genetics Dr. M. Campione The Hague, The Netherlands Berlin, Germany Inst of Neurosciences Consiglio Nazionale delle Ricerche Dr. R. G. Kelly Dr. M. J. B. van den Hoff Padova, Italy Institute of Developmental Academic Medical Centre Biology of Marseille Amsterdam, The Netherlands Marseille, France Prof. R. Zimmermann Prof. A. Kispert University Hospital Zurich Institut fur Molekularbiologie Zurich, Switzerland Medizinische Hochschule Hannover, Germany Dr. A.H. Zisch University Hospital Zurich Zurich, Switzerland

26 | CARDIOVASCULAR IMMUNATH

Translating innate immune receptor function into diagnostic and therapeutic applications for atherosclerosis

Keywords Atherosclerosis, immune system, Problem toll like receptor, vaccination Atherosclerotic cardiovascular disease remains as number one killer of the aging population in Western Society and numbers of cardiovascular events are strongly increasing in developing countries. Worldwide about 17 million deaths are caused by this infl ammatory disease and the costs for health care and loss or productivity exceeds 169 billion Euro a year in Europe. Fortunately, increasing knowledge on the mechanisms of Summary atherosclerotic disease resulted in preventive strategies and a subsequent decrease in rate of mortality in industrialized countries since 1950. The immune system has a major role in atherosclerosis and its innate and However, the increasing prevalence of type II Diabetes in developed and adaptive arms jointly and separately co-determine atherosclerotic developing countries deserves careful consideration since this will surely disease initiation and progression. The search for approaches to modulate infl uence morbidity and mortality rates due to cardiovascular disease. the infl ammatory response in atherosclerotic disease is still in its infancy. Improvement of our understanding of the mechanisms that lead to Infl ammatory diseases like rheumatoid arthritis (RA) have much longer atherosclerotic disease has resulted in innovative modalities that may been recognized as immune disorders and hence are much ahead in help diagnose, prevent and treat this life threatening disease. development of therapeutics targeting infl ammation. A multidisciplinary We now know that atherosclerosis is an infl ammatory disease and that approach will stimulate the discovery of immune modulating compounds the body’s immune system plays a central role in the initiation and pro- to treat atherosclerotic disease. Thereto, the current project joins forces gression of atherosclerotic lesion development. The recent insights in of three SMEs owning unique proprietary complementary technology how the immune system recognizes endogenous and exogenous lig- together with four academic groups, which are actively involved in the ands and how ligation of innate immune receptors results in a local fi eld of cardiovascular disease and immune modulation. Work of the infl ammatory response, has opened exciting new therapeutic avenues research groups is dedicated to SMEs to develop new and test pre- which we would like to implement. Atherosclerosis bears many simi- existing lead compounds that modulate the innate or adaptive immune larities to other infl ammatory diseases like rheumatoid arthritis or response and subsequent atherosclerotic disease. In addition, diagnostic Crohn’s disease. Experimental studies revealed that such diseases may markers will be exploited that refl ect the severity of atherosclerotic be treated by different approaches in humans: blockade of innate disease based on innate receptor ligands and responsiveness, focused immunity (anti-TNFalpha) and also by immunisation in mouse models. on TLR and NOD receptors. In atherosclerotic mice it has been shown that the vaccination approach In work packages (WP) 1 and 2 targets for intervention, that have been may also be applied to atherosclerotic disease. A successful vaccination discovered using an integrated genomics or proteomics approach, will be strategy to prevent adverse outcomes due to atherosclerotic disease validated and antagonists constructed using both small molecule and will have enormous impact on healthcare. antibody technology. WP 1 will focus on innate receptor signalling, WP 2 will address the therapeutic and diagnostic value of TLR ligands and responsiveness. In WP 3 and 4, the properties of therapeutic lead Aim compounds and diagnostic modalities that are based on modifi cation of the innate and adaptive immune response will be assessed using state of General objective: to develop and validate therapeutic approaches that the art in vitro and animal model systems. This STREP will signifi cantly modulate the innate and adaptive immunological responses in athero- advance the position of three SMEs based on a multidisciplinary sclerotic disease. approach, and will promote therapy and diagnosis for a disease with high This general objective can be specifi ed according to 4 Work packages: socio-economic impact. WP 1 Receptors. To identify targets for intervention and test antagonizing compounds in the signalling cascade of the innate receptors, TLR and NOD, to inhibit atherosclerotic lesion development. WP 2 Ligands. To identify endogenous ligands for TLR and NOD that are involved in atherogenesis and assess diagnostic value of ligand expression and receptor responsiveness following ligation. WP 3 Innate Immunity. To inhibit atherosclerotic lesion progression by lead compounds, targeting TLR ligation or signalling and TNFalpha, that have been developed and licensed by participating SMEs. WP 4 Adaptive immunity. To inhibit atherosclerotic lesion progression by immunisation targeting lipoproteins that have been shown to induce a vascular infl ammatory response and plaque formation.

CARDIOVASCULAR | 27 Expected results Medium TNFα 10 ng/ml TNFα + Remicade Sups + Remicade

We expect to detect: • molecules/proteins that serve as a therapeutic target in atherosclerotic disease; • targets that may serve as surrogate endpoints for adverse cardiovascular events in clinical trials or as a prognostic marker for Sups IL-1 10 ng/ml IL-1 + IL-1 Ra Sups + IL -1 Ra clinical events due to atherosclerotic disease; • therapeutic interventions to prevent initiation or progression of atherosclerotic disease by: – antagonising receptors, – antagonising ligands, Monaco C. et al, 2007 (unpublished data). – altering receptor response, – immunisation.

Potential applications Coordinator M. Feldmann C. Monaco • Therapeutic interventions based on immuno modulation that may Prof. Dr. G. Pasterkamp Kennedy Reserach Institute Interuniversity Cardiology Institute Imperial College prevent initiation of and complications by pre-existent atherosclerotic of the Netherlands London, United Kingdom disease. Postbox 19258 • Prognostic measures that may help diagnosing the patients prone 3501 DG Utrecht R. Carlsson to develop acute myocardial infarction. The Netherlands Bioinvent www.icin.knaw.nl Lund, Sweden [email protected] www.vascularbiology.nl J. Nilsson University of Lund Lund, Sweden List of participants G. Hansson G. Pasterkamp Y. Zhou D. de Kleijn Karolinska Institute A. Heijkamp Stockholm, Sweden Interuniversity Institute of The Netherlands J. Laman Utrecht, The Netherlands Erasmus University Rotterdam, The Netherlands M. Hefferman L. O’Neill R. Brands OPSONA Therapeutics Alloksys Dublin, Ireland Utrecht, The Netherlands

G. Pasterkamp D. de Kleijn Project number Utrecht University Medical Centre LSHM-CT-2006-037400 Utrecht, The Netherlands EC contribution € 2 500 000 Project Duration 36 months Starting date 01/12/2006 Instrument STREP – SME

28 | CARDIOVASCULAR InGenious HyperCare

Integrating Genomics, Clinical Research and Care in Hypertension

Keywords Hypertension, autonomic nervous system, organ damage and in exploring genetics, genomics and proteomics of infl ammation, oxidative stress, sodium handling, stroke, proneness to hypertension and hypertension-related cardiovascular dis- renal dysfunction, heart failure. ease. A powerful instrument of integration will be the Programme of Joint Research Activities. A fi rst group of three research packages will be addressed to Mechanomics of Hypertension, i.e. identifying genetic, genomic and proteomic markers of disturbances in the major mechanisms controlling blood pressure (A1: autonomic control; A2: infl ammation and oxidative stress, A3: renal sodium handling), as indicators of the risk of becoming hypertensive. The second group of three research Summary packages will be addressed to Mechanomics of Hypertension-Related Diseases, i.e. identifying genetic, genomic and proteomic markers of A better prevention of hypertension and its cardiovascular consequences the risk of developing a hypertension-related event (B1: stroke; B2: renal is an essential public health goal in Europe, where cardiovascular dysfunction; B3: heart failure). diseases are the major cause of mortality and morbidity. The Network plans to integrate the research efforts of 31 research teams (including a SME) and 1SME experienced in EC project management. A powerful Expected results instrument of integration will be the Programme of Joint Research Activities. A fi rst group of research packages will be addressed to • Identifi cation of polymorphisms in the most important genes Mechanomics of Hypertension, i.e. identifying genetic, genomic and belonging to defi nite pathways (autonomic genes, genes within proteomic markers of disturbances in the major mechanisms control- the infl ammatory and oxidative stress pathways, genes interfering ling blood pressure, as indicators of the risk of becoming hypertensive. with renal sodium handling, renal susceptibility genes, cardiac The second group of research packages will be addressed to Mecha- genes, etc.) involved in the development of hypertension and nomics of Hypertension Related Diseases, i.e. identifying the genetic, hypertension-related damage. genomic and proteomic markers of the risk of developing a hypertension- • Identifi cation of protein biomarkers of early cardiac and renal related event. Spreading of excellence beyond the Network will be done damage. with the support of the European Society of Hypertension. • Creation of very large databases of hypertensive phenotypes and genotypes. • Creation of a lasting network, where genotyping and phenotyping are pursued in close associations by groups using common genetic platforms and common methods of phenotyping. • Spreading of excellence beyond the Network to the European and international community of investigators in the area of hypertension and cardiovascular diseases. Problem Potential applications A better prevention of hypertension and hypertension-related cardiovascular events is an essential public health goal in Europe. Despite the • A better identifi cation of normotensive subjects genetically prone widely shared opinion that hypertension has an important heriditary com- to developing hypertension will allow concentration of primary ponent, genetic and genomic research has not yet been fully exploited by prevention efforts on subjects at a greater potential risk of becom- medical research on hypertension, which has limited progresses of pri- ing hypertensive. mary prevention of hypertension, and the prevention of the cardiac, • A better identifi cation of hypertensive patients at a greater genetic vascular and renal consequences of hypertension. risk of developing a cardiovascular complication (and, potentially, a specifi c type of complication) will make treatment of hypertension, a widespread condition, more cost-effective, and may lead to Aim the development of new therapeutic approaches. • Identifi cation of protein biomarkers of early cardiac and renal damage The problem will be approached by integrating complementary but still is expected to provide new diagnostic and therapeutic tools. fragmented experience in the mechanisms of blood pressure control and hypertension development, in phenotyping initiation and progression of

CARDIOVASCULAR | 29 Coordinator Università Vita-Salute Medizinische Hochschule Medical University of Gdansk San Raffaele Hannover Gdansk, Poland Prof. Alberto Zanchetti Milano, Italy Hannover, Germany Istituto Auxologico Italiano (AU) Jagiellonian University Milan, Italy Institut National de la Santé Westfälische Wilhelms Medical College Fax: +39 02619112480 et de la Recherche Médicale Universität Krakow, Poland [email protected] Paris, France Muenster, Germany Institute for Clinical and Centre Hospitalier Fundacion de Investigacion Experimental Medicine Partners Universitaire de Nancy del Hospital Clinico Prague, Czech Republic Nancy, France Universitario de Valencia Università degli Studi Valencia, Spain Charles University in Prague di Milano-Bicocca University of Glasgow Pilsen, Czech Republic Milano, Italy Glasgow, United Kingdom Fundación para la Investigación Médica Aplicada Mosaiques diagnostics GmbH Università degli Studi University of Manchester Pamplona, Spain Hannover, Germany di Milano Manchester, United Kingdom Milano, Italy Hospital 12 de Octubre Hospices Cantonaux University of Leicester Unidad HTA (HC/CHUV) Università degli Studi di Brescia Leicester, United Kingdom Madrid, Spain Lausanne, Switzerland Brescia, Italy Katholieke Universiteit Leuven Hospital Clinic y Provincial Institute of Internal Medicine Università degli Studi di Roma Leuven, Belgium de Barcelona Siberian Branch of Russia Academy of ‘La Sapienza’ (RM2) Barcelona, Spain Medical Sciences Roma, Italy Universiteit Maastricht Novosibirsk, Russian Federation Maastricht, The Netherlands Lunds Universitet Università degli Studi Lund, Sweden Shanghai Institute of Hypertension di Padova Charité-Universitätsmedizin Shanghai, China Padova, Italy Berlin, Germany Helsingin yliopisto (UH.DPH) Helsinki, Finland CF consulting Finanziamenti Unione Europea s.r.l. Milano, Italy

Project number LSHM-CT-2006-037093 EC contribution € 10 000 000 Project Duration 48 months Starting date 01/11/2006 Instrument NoE Project website www.hypercare.eu

30 | CARDIOVASCULAR NORMACOR

Normal Cardiac Excitation: Generation, Propagation and Coupling to Contraction

Keywords Cardiac arrhythmias, hyperpolarization-activated Scientifi c and technological objectives channels, channelopathies, atrial fi brillation, pacemaking, cardiac remodelling, transgenic mice, myofi brils, modelling tools • To develop suitable experimental tools, including HCN transgenic mice, novel bradycardic and antiarrhythmic agents, control of mechano-electrical interactions, and the symbiosis of the above. • To acquire new knowledge on molecular mechanisms and pathways controlling generation and spread of excitation in normal and diseased cardiac tissue. • To combine advanced engineering techniques for ‘top-down’ Summary development of novel experimental tools with mathematical ‘bottom-up’ integration of data from the molecular level to fundamen- Cardiac arrhythmia is a major cause of morbidity and mortality in Europe tal and clinical relevance. and of growing socio-economic concern. Recent data suggest that novel mechanisms such as HCN channels, mechanical effects on Ca handling Based on the combination of skills and techniques not currently availa- and ion fl ux balances, and cellular heterogeneity may trigger and/or ble in any single lab worldwide, we will provide novel insight into novel permit the sustenance of arrhythmias. The novelty of our approach is to arrhythmogenic mechanisms, quantify their interrelation, target preven- focus on these potentially crucial, but ill-investigated, mechanisms by tive and therapeutic interventions to reduce the societal and economical uniting 8 leading labs from 5 EU countries who focus on individual impact of cardiac arrhythmia. arrhythmogenic aspects, in a bid to combine their expertise and shed light on how physiological or compensatory mechanisms may turn arrhythmogenic, and how this may be controlled or corrected. Problem Prime targets are structured along three levels of functional integration: • membrane level: Role of HCN channels in the generation of normal This project faces several problems dealing with relevant EC societal and and abnormal rhythm; policy objectives: • cellular level: Role of electro-mechanical interaction in modulating • the discovery of new antiarrhythmic strategies and the identiﬁ cation contractile activity and electrophysiology; of vulnerable parameters (proteins and pathways) tricking the • multicellular level: Role of heterogeneity in normal and remodelled propensity to abnormal cardiac excitation, as it occurs in a variety tissue on electrical excitation and conduction. of cardiac diseases associated to high arrhythmic propensity (heart failure, diabetes, hypertension); • development of novel drugs able to prevent cardiac arrhythmias or reduce the propensity to recurrence/chronicization of them, thus improving the quality of life and health.

Aim

This STREP is a research and technological development project which brings together 8 expert European research laboratories in 5 different EU countries and integrates a range of multidisciplinary investigations into cardiac arrhythmia mechanisms and ion channel exploration. Meas- urable and veriﬁ able scientiﬁ c and technological objectives can be sum- marized as follows: • to develop suitable experimental tools, including HCN transgenic mice, novel bradycardic and antiarrhythmic agents, control of mechano-electrical interactions, and the symbiosis of the above; • to acquire new knowledge on molecular mechanisms and pathways controlling generation and spread of excitation in normal and diseased cardiac tissue; • to combine advanced engineering techniques for ‘top-down’ development of novel experimental tools with mathematical ‘bottom-up’ integration of data from the molecular level to clinical relevance.

CARDIOVASCULAR | 31 Expected Results Coordinator Prof. David Eisner University of Manchester The expected result consists in the development of new experimental Prof. Elisabetta Cerbai Manchester, United Kingdom tools for studying normal/abnormal rhythm generation, such as: Biol. Sc., PhD Centro Interuniversitario di Medicina Dr. Peter Kohl • HCN Transgenic animals; Molecolare e Biofi sica Royal Society Research Fellow • HCN-selective bradycardic drugs; Applicata (CIMMBA) Oxford, United Kingdom • generation of a biological pacemaker; Università degli Studi di Firenze • application of the single-myofi bril technique to perform mechanical Viale G. Pieraccini 6 Prof. Ursula Ravens experiments in samples from human and animal hearts; 50139 Firenze, Italy Medical Faculty Carl Gustav Carus • adaptation of the multi-microelectrode array technique to sections Dresden, Germany of animal and human atrial tissue. Partners Dr. Ulrich Schotten University of Maastricht Potential applications Prof. Martin Biel Maastricht, The Netherlands Ludwig Maximilians University München, Germany Prof. Andras Varró Scientifi c advancements will consist in the elucidation of molecular University of Szeged mechanisms and pathways controlling rhythm generation and electrical Prof. Dario DiFrancesco Szeged, Hungary propagation in healthy and diseased cardiac tissue, such as: Università degli Studi di Milano • role of HCN channels in the generation of normal and abnormal Milano, Italy rhythm. Three strategies will be used: – assessment of the molecular composition and sub-cellular localization of native HCN channels; – effect of mislocalization, over-expression, or abolition of cardiac HCN channels in terms of altered pacemaking or cardiac conduction, and propensity to arrhythmias in the normal and diseased heart; – identifi cation of cardiac channelopathies linked to mutations of human HCN channels. • integrated knowledge of electrical and structural atrial remodelling. The project will address the relationship among electrical, contractile and conduction abnormalities in atrial cells/tissues challenged with different stressing conditions (in vivo atrial pacing, in vitro mechanical stimulation and axial stretch, exposure to neu- rohumoral factors such as endothelin-1) and in human atrial specimens.

Societal advancements and applications are the discovery of new antiarrhythmic strategies and the identiﬁ cation of vulnerable parameters (proteins and pathways) tricking the propensity to abnormal cardiac excitation, as it occurs in a variety of cardiac diseases associated to high arrhythmic propensity (heart failure, diabetes, hypertension). The ﬁ nal objective is to propose newly developed drugs and/or therapeutic strategies aimed to prevent cardiac arrhythmias or reduce the propensity to Project number recurrence/chronicization of them, thus contributing to quality of life LSHM-CT-2006-037093 and health and to reduce the socio-economic impact of cardiac diseas- EC contribution es. Finally, the project will contribute to educational training and mobil- € 10 000 000 ity in Europe, as a number of young researchers, both postgraduate and Project Duration postdoctoral, will be employed to carry out the research. 1 November 2006 Starting date 01/12/2006 Instrument NoE

32 | CARDIOVASCULAR PolyALA

Insights into novel therapeutic strategies for a nuclear inclusion disease caused by polyalanine expansion

Keywords Molecular genetics, gene therapy, OPMD Potential applications

To provide the Pharma Industry with novel intervention drug targets and structuring a clinical trial for gene therapy for OPMD which may ultimately lead to improved quality of life of OPMD patients. OPMD, although a rather rare disease, has several parallels with more common degenerative disorders such as Huntington’s disease. OPMD has the Summary advantage of being the only trinucleotide expansion protein aggregation disorder with easy access to symptomatic and presymptomatic A consortium of world-renowned scientists and their teams will carry out tissue (muscle biopsies). studies to unravel the molecular mechanisms involved in oculopharyngeal muscular dystrophy (OPMD) pathogenesis and to develop innovative gene therapy strategies for this disease in order to fi nd a clear way forward to Coordinator Dr. Silvere van der Maarel providing the pharmaceutical industry with possible intervention drugs Leiden University Medical Center which may improve the quality of life of OPMD patients. Prof. Egas Moniz Leiden, The Netherlands Institute of molecular medicine Faculty of medicine Dr. Martine Simonelig 1649-028 Lisboa, Portugal Centre national de Scientist: Prof. Maria Carmo Fonseca la Recherche scientifi que Phone: +351 21 7999411 Institut de Génétique humaine Fax: +351 21 7999412 Montpellier, France [email protected] Prof. Elmar Wahle Martin-Luther Universitat Partners Halle-Wittenberg Institut für Biochemie Problem Dr. Michael Antoniou Halle, Germany King’s College London A broad range of degenerative diseases is associated with intracellular London, United Kingdom inclusions formed by toxic, aggregation-prone mutant proteins. Intra- Prof. J. G. Dickson nuclear inclusions constitute a pathological hallmark of OPMD, a rare Royal Holloway inherited disease caused by (GCG) repeat expansions in the gene that and Bedford New College encodes for nuclear poly(A) binding protein (PABPN1). The mutation School of Biological Science results in an extended polyalanine stretch that has been proposed to University of London, Egham include protein aggregation and formation of intranuclear inclusions. Surrey, United Kingdom

Aim

To study the molecular mechanisms of oculopharyngeal muscular dystrophy (OPMD) and to develop new strategies for its treatment with a main emphasis on gene therapy.

Project number Expected results LSHM-CT-2005-018675 EC contribution We expect to obtain novel structural and functional knowledge of normal € 1 805 000 00 and expanded PABPN1; to identity cellular pathways affected by PABPN1 Project Duration expansion and formation of nuclear inclusions; to identify suppressors of 36 months OPMD phenotype; and to develop gene therapy strategies for OPMD. Starting date 01/01/2006 Instrument STREP

CARDIOVASCULAR | 33 PROCARDIS

A functional genomic approach to elucidating Precocious Coronary Artery Disease

Keywords Procardis, coronary artery disease, myocardial genomics programme. Each of the principal areas of genetic exploration infarction, angina, genetics, genome wide association study, Gwas in complex traits is represented by leading expertise; thus the programme is made powerful by the involvement of world-class epidemiology, statistical genetics and bioinformatics. The programme will characterise the functions and physiological roles of susceptibility variants. Finally, the involvement of SME and pharmaceutical company partners provides unrivalled capability for the translation of basic knowledge through to clinical application. Summary Although the emphasis is on application-oriented genomics approaches, PROCARDIS will also generate new tools that will be exploited and thus Procardis will exploit advances in complex trait genetics and functional made widely available. These will include, for example, derivation of new genomics to discover novel susceptibility genes for coronary artery disease information technology platforms for genomic research, bioinformatic (CAD). PROCARDIS will incorporate a defi nitive genome-wide association tools, and statistical genetics approaches. analysis and measurement of novel intermediate phenotypes that will yield biomarkers for CAD risk and quantitative traits for genetic analysis. Expected results

Identifi cation of novel genes and genetic variants that confer susceptibility to CAD, using genome-wide linkage and association mapping; this will be successful if at least 10 susceptibility genes and genetic variants that confer susceptibility to CAD are identifi ed. The studies in population-based and clinical cohorts will be seen as successful if the quantitative impact on CAD susceptibility has been determined for a minimum of 10 genetic variants. The outcome of the intermediate Problem phenotyping experiments will be seen as successful if novel metabolite or peptide biomarker profi les can be confi rmed. The critical importance of the burden of cardiovascular mortality and morbidity in Europe hardly needs to be underlined. The need for targeted studies with the aim of exploring the specifi c interaction between Potential applications genetic and environmental risk factors is recognised as a priority. Two well recognised general features characterise the overall cardiovascular The programme is expected to generate new knowledge eminently mortality fi gures (which represent 1/3 to 1/2 of overall mortality rates) as suitable for subsequent exploitation, as witnessed by the active well as the mortality specifi cally from CAD (which accounts for 2/3 to participation of the SME and pharmaceutical company partners. This is 1/3 of CV mortality): a great inter-country variability, with decreasing expected to include: new software algorithms for bioinformatics, new East-West and North-South gradients, and a falling prevalence in the biomarkers for predicting CAD risk, validated SNPs for predicting CAD Western and Southern countries contrasting the stable/increasing rates risk, and new targets for drug discovery. PROCARDIS will signifi cantly in many Eastern countries. contribute to improvements in diagnostic tools, prevention and treatment methodologies in CAD. This will impact favourably on all major stakeholders: Aim • the European Community, through overall innovation aspects; • reduction in patients suffering from CAD, thus helping to solve The aim is to identify novel proteins and pathways implicated in societal problems; atherosclerosis and arterial thrombosis in order to defi ne new targets for • the scientifi c community, through exploitation/dissemination of prevention and treatment and to devise diagnostic tools that identify new knowledge and tools; disease mechanisms in individuals. The PROCARDIS programme seeks • health authorities and policy makers in Europe, thus improving to “improve prevention and management of important causes of welfare. mortality and ill health in Europe” through an application-oriented

34 | CARDIOVASCULAR Coordinator Prof. Gianni Tognoni Istituto di Ricerche Farmacologiche Prof. Hugh Watkins ‘Mario Negri’ Chancellor, Masters & Scholars Milan, Italy of the University of Oxford Dept of Cardiovascular Medicine and Prof. Mark Lathrop Wellcome Trust Centre for Human Centre National de Genotypage Genetics, University of Oxford Evry, France Oxford, United Kingdom hugh.watkins@cardiovascular-medicine. Dr. Jose Manuel Soria oxford.ac.uk Institut de Recerca del Hospital de la Santa Creu i Sant Pau Hospital de la Santa Creu i Sant Pau Partners Barcelona, Spain

Prof. Anders Hamsten Professor Elena Tremoli Karolinska Institutet Università degli Studi di Milano King Gustaf V Research Milan, Italy Institute (KI.GV) Centre of Molecular Medicine Dr. Johan Björkegren (KI.CMM) Clinical Gene Networks AB Centre of Gender-Related Medicine Karolinska Science Park (KI.CGRM) Stockholm, Sweden Stockholm, Sweden Prof. Gunnar Olsson AstraZeneca Prof. Gerd Assmann Mölndal, Sweden Universität Münster Leibniz Institute for Atherosclerosis Dr. Petra Budde Research Digilab BioVisioN GmbH Münster, Germany Hannover, Germany

Dr. Frank Bonner Metabometrix Limited London, United Kingdom

Dr. Carla Finocchiaro CF consulting s.r.l. Milan, Italy

Project number LSHM-CT-2007-037273 EC contribution € 10 000 000 Project Duration 48 months Starting date 01/04/2007 Instrument IP Project website www.procardis.org

CARDIOVASCULAR | 35 PULMOTENSION

Pulmonary Hypertension: Functional Genomics and Therapy of Lung Vascular Remodelling

Keywords Pulmonary hypertension, cor pulmonale, lung Problem vascular remodelling, functional genomics Pulmonary hypertension is a disease developing from many different clinical backgrounds ranging from primarily genetic causes to intake of drugs and toxins. For all their differences in background, the various forms of PH share similarities in pathogenetic mechanisms, clinical presentation and therapeutic options, allowing clinicians to conduct trials using homogeneous Summary groups of patients. • Pulmonary arterial hypertension (PAH). This includes fi rst subgroups Pulmonary hypertension (PH) describes a group of devastating chronic without identifi able cause, so-called idiopathic PAH (IPAH; spo- diseases, comprising idiopathic and secondary forms, which cause radic appearance) and familial PAH (FPAH). For these subgroups, breathlessness and premature death, representing a major burden on mutations in the bone morphogenetic protein (BMP)/Transforming healthcare systems. Extensive lung vascular remodelling with loss of Growth Factor beta (TGF-b) superfamily are now known to play vessel patency is the underlying pathomechanism in PH. PULMOTENSION a major role. However, additional genetic/environmental factors integrates the top European Centres in PH and lung vascular biology in are likely to be the ‘second hit’, favouring the start of the disease. a multidisciplinary approach, with the aim to combat and possibly fi nd The other subgroups include a number of conditions or diseases of a cure for PH. The expertise of all members extends from the initial known causes associated with the appearance of PAH (APAH). All discovery of gene mutations causative of PH to having established new PAH forms have in common the localization of remodelling lesions therapeutic regimen of PH. We will uncover molecular pathways underlying to the small pulmonary muscular arterioles. In addition, they may disease development, identify distinct targets for anti-remodelling be linked with signifi cant venous or capillary involvement. therapy, foster drug development based on these targets, and test these • Pulmonary hypertension with left heart disease. This category new treatment options in preclinical and clinical trials. consists predominantly of left-sided valvular or myocardial diseases, The presented project includes: exerting mechanical stress on the pulmonary vasculature due to • genetics of PH; congestion and/or hypercirculation (cardiac shunt fl ow). • functional genomics and proteomics to unravel the molecular • Pulmonary hypertension associated with lung diseases and/or pathways underlying vascular remodelling; hypoxia. Within this category, the predominant cause is inadequate • characterisation of the cross-talk between different cells and oxygenation of arterial blood as a result of either lung disease, signalling pathways involved; impaired control of breathing, or residence at high altitude. Hypoxia • preclinical trials in PH models including transgenics; driven molecular mechanisms are in the focus of the pathogenetic • set up of a European PH Tissue Bank and Registry; sequelae in this group. In addition, infl ammatory mechanisms • conversion of target discovery into drug design and exploitation; linked to the lung disease may come into play. • development of non-invasive clinical tools for monitoring lung • Pulmonary hypertension due to chronic thrombotic and/or embolic vascular remodelling in patients; disease (CTEPH). This category includes either chronic thromboembolic • set up of a Clinical Trial Network to conduct phase I/II(III) studies PH due to proximal organized clots in major pulmonary arteries, focussing on prevention and/or reversal of remodelling in PH which can benefi t from pulmonary endarterectomy, or more patients; peripheral emboli or thrombi similar to those appearing in the small • promotion of a world-leading long-lasting infrastructure for scientifi c vessels in IPAH. Abnormalities of coagulation and fi brinolysis control, and technical competence in the fi eld of PH, including training and either systemic or endothelium-based, are assumed to represent the mobility programs, technology transfer in alliance with industry and decisive trigger events. exploitation facilities. • Miscellaneous. This category involves PH linked with various, mostly Gender issues will be particularly addressed, as many PH forms have infl ammatory disorders. a female preponderance. Hence, this ambitious translational research programme will cover the entire spectrum ‘from molecules to patients’ Epidemiological data are not available for the entire fi eld of PH. Estimates eliciting new insights into the development of PH and thus allowing of the incidence of its ‘prototype’ entity, IPAH/FPAH, range from 1-2 targeted development of new treatments of PH, a prototype disease of cases per million people per year, thus fulfi lling the criteria of a rare vascular remodelling. disease, with markedly higher appearance in women as compared to men. Gender differences are also true for other categories of PAH, such as collagen vascular disease-associated PAH, being more frequent in women as compared to men. On the other hand, ~ 15 % of all patients suffering from chronic obstructive lung disease, the world’s third most common cause of death, develop serious PH, and ~ 4 % of all patients

36 | CARDIOVASCULAR Pulmotension smooth muscle cells: Isolated mouse pulmonary artery smooth muscle cells, 20x, anti-a-smooth muscle actin staining.

undergoing acute pulmonary embolisms. Overall, ~ 5 % of all patients Aim dying from heart failure are assumed to have predominantly right-sided heart failure, with pulmonary hypertension representing the underlying Deciphering of the functional genomics of lung vascular remodelling mechanism. Moreover, pulmonary hypertension in congestive heart and identifi cation of new molecular targets to alleviate or even cure failure is a cause of exercise intolerance and poor prognosis. These pulmonary hypertension is the main objective of this project. fi gures demonstrate the substantial, hitherto largely underestimated We will characterise the molecular mechanisms underlying the dramatic burden which PH exerts on mankind. remodelling processes in the lung vasculature, which result in an increase in vascular resistance by more than one order of magnitude. To this end, Life-expectancy of non-treated IPAH/FPAH is extremely poor, ranging genetics, functional genomics, and proteomics will be combined with between 2 and 3 years. This is similarly true for many other categories cell co-culture studies, experimental pulmonary hypertension animal of PH, such as collagen vascular disease-associated PAH (citation). Over models, and analysis of clinical tissue bank material. A European PH the past years, various therapeutic strategies were developed, which Registry will be set up and diagnostic tools for non-invasive diagnosis of include different type prostanoids, endothelin receptor antagonists and lung vascular and right heart remodelling be developed. Finally, based phosphodiesterase inhibitors. These agents were shown to relieve on the identifi cation of new genes and signalling pathways as novel dyspnoea, improve exercise capacity and – though not proven for all therapeutic targets, preclinical and clinical studies with focus on anti- approaches – survival in these patients. Notably, several of the clinical remodelling treatment represent a leading goal of the Consortium. In all scientists contributing to this Consortium inspired and led the aspects, gender differences will be particularly addressed. Hence, the development of these treatment concepts However, we are still far away members of the Consortium will jointly carry out an ambitious from curing PH patients, but further progress in drug development is translational research programme, with the intent to perform an analysis urgently needed to achieve long-term survival with good quality of life. ‘from molecules to patients’.

PH has a multifactorial pathobiology. Apart from vasoconstriction and According to the scientifi c nature of our project, generation of new in-situ thrombosis, remodelling of all layers of the pulmonary vessel wall knowledge and improvement of PH patient therapy are the primary represents the hallmark of this disease, with proliferating resident cell goals of the Consortium. Publications in international peer-reviewed types (endothelial [EC], smooth muscle [SMC], and adventitial fi broblast Journals, patents, evaluation of new diagnostic and therapeutic [FB]) and infl ammatory cells, platelets as well as circulating progenitor approaches, standardization of these techniques throughout Europe, cells coming into close interplay. Endothelial dysfunction characterized and information to society are important measurable objectives in this by a lack of vasodilatory and antiproliferative mediators such as nitric respect. Due to the strong translational research infrastructure of the oxide (NO) and prostacyclin and their downstream cyclic nucleotides is Consortium, we expect innovative therapies to become available for a key event in PH, providing a starting point for therapies based on patients suffering from PH, as a result of the work of this Consortium. these agents. Activation of these signalling pathways is closely linked to The objectives also include the promotion of a long-lasting European the regulation of – partially oxygen sensitive K+ – and Ca2+ channels in infrastructure for scientifi c and technical competence in the fi eld of the smooth muscle cells. In contrast, potent vasoconstrictors such as vascular remodelling and PH. Training and mobility programs, especially endothelin (ET)-1 and the serotonin pathway are known to be for young researchers, mechanisms for technology transfer and upregulated, again serving as a basis for new therapeutic approaches. educational programs, and establishment of exploitation facilities Most importantly, going far beyond their immediate effect on vasomotor represent essential parts of this infrastructure. control, all these pathways have major impact on the long term balance of proliferation and apoptosis of the lung vascular cells, i.e. affect remodelling events. On this level, there is a close but not yet fully Expected results characterized interplay between the BMP/TGF-b and the angio¬poietin/ vascular endothelial growth factor (VEGF) signalling pathways, the Based on the competence, complementary knowledge and critical mass decisive role of which was recently demonstrated in genetic and assembled within the Consortium, major progress in the fi eld of PH is to functional genomic studies. Moreover, coagulation and infectious be anticipated, going far beyond the present state of the art. This project processes, possibly linked with each other, have an impact on the represents the fi rst attempt to unify currently fragmented European process of lung vascular remodelling. As soon as hypoxia comes into expertise in this fi eld. We expect that: play, hypoxia-inducible factor (HIF)-dependent oxygen sensing and • additional signalling pathways, contributing to the development of signalling is of central importance, further contributing to the above PH, will be unravelled; mentioned pathways. Notably, next to the resident vascular cell types • advanced knowledge in functional genomics will shed light on the involved in the remodelling (EC, SMC, FB), recruitment of circulating complex interplay between different cell types and molecular pathways progenitor cells and different leukocyte types is controlled by these in this devastating disease; pathways and by a hitherto only partially characterized lung vascular • new molecular targets for therapeutic interventions will be identifi ed; chemokine system.

CARDIOVASCULAR | 37 The pulmonary vascular pathology observed in pulmonary hypertension is characterized by a remodeling process, affecting all vessel layers (intima, media and adventitia). The image shows a rat pulmonary artery in monocrotaline-induced pulmonary hypertension (diameter 50μ). Red indicates proliferating cells (PCNA staining), blue indicates cell nuclei (DAPI staining).

• due to the outstanding expertise in translational research of this Coordinator Consortium, new therapies will evolve through preclinical and clinical studies to the market, providing new treatment options for Prof. Dr. Werner Seeger patients with pulmonary hypertension. Justus-Liebig University Giessen University of Giessen Lung Centre (UGLC) Giessen, Germany Potential applications [email protected]

Pulmonary hypertension is a chronic, prolonged crippling and fatal disease for which there is no cure. Current therapies aim at the alleviation Partners of symptoms and may prolong survival. PULMOTENSION with its vast scope of approaches to understanding the disease process will contribute Alphalyse – Denmark to and initiate the development of new therapeutic strategies that will Altana Pharma – Germany greatly improve quality of life for patients and their families. Apart from Dr. Riethmueller MRS GmbH – Germany Dualsystems – Switzerland this humanitarian impact, PULMOTENSION will prove a valuable asset to Genolyze Ltd – Finland the European Research Area by assembling a great amount of scientifi c Hôpital Antoine Béclère, Université Paris Sud – France and clinical expertise in the fi eld of pulmonary hypertension in one co- Hospital Clinic I Provincial de Barcelona – Spain operative effort that is expected to outlast the duration of the project, Imperial College London – United Kingdom thereby reinforcing Europe’s ability to compete internationally. INSERM Institute – France Due to the progress in treatment of pulmonary hypertension that has Institute of Physiology, Charité – Germany been made in recent years – and can be expected to be furthered from LEA Medizintechnik – Germany the PULMOTENSION project – and the socioeconomic burden of the Luxcel Bioscience – Ireland different variants of PH, the pharmaceutical industry is becoming most Medical School Hannover – Germany interested in this area. New drugs that may be developed as a result of Medical University Graz – Austria Medical University of Vienna – Austria the project may well be of commercial interest, not only in the fi eld of BioSpring GmbH – Germany PH, but also of vascular therapeutics in general. Transfer into commercial Scottish Pulmonary Vascular Unit – United Kingdom exploitation by European pharmaceutical companies will be fostered by University of Oxford – United Kingdom the PULMOTENSION project. TransMIT GmbH – Germany Universitätsklinik Heidelberg – Germany Université Libre de Bruxelles – Belgium University College London – United Kingdom University of Bologna – Italy University of Cambridge – United Kingdom University of Duisburg-Essen – Germany University of Glasgow – United Kingdom University of Ioannina – Greece King’s College London – United Kingdom University of Sevilla – Spain University of Zurich – Switzerland

Project number LSHM- CT-2005-018725 EC contribution € 11 399 999 Project Duration 48 months Starting date 01/01/2006 Instrument IP Project website www.pulmotension.de

38 | CARDIOVASCULAR SOUTH

Application-oriented studies on regulatory networks involved in lipid homeostasis and atherosclerosis

Keywords Gene expression and regulation, cholesterol and Problem lipid metabolism, atherosclerosis Because cardiovascular diseases are the leading cause of death and severe disabilities in European and other industrialised countries and also an emerging problem in developing countries, it is crucial to have a suitable arsenal of clinical diagnostic tools and intervention strategies to prevent and minimize their consequences on the affected population. A number of medications such as statins, ezetimibe, an inhibitor of Summary intestinal cholesterol absorption, and ligands of the nuclear receptors PPAR a and g (i.e., fi brates and thiazolidinediones) have proven effective Cholesterol and lipid homeostasis is achieved through the action of a complex in the treatment and prevention of atherosclerosis via multifactorial regulatory network (regulome) that controls the expression of genes involved mechanisms including improvements in circulating lipids, systemic and in these metabolic pathways. Our recent studies suggest that: local anti-infl ammatory effects, and, potentially, inhibition of vascular • high in the hierarchy of the regulatory network is the nuclear receptor cell proliferation. However, a more pronounced decrease of plasma cho- HNF-4, whose activity on cholesterol metabolism genes is selectively lesterol levels would be desirable particularly for patients at high risk of affected by histone deacetylase 7 (HDAC7) recruitment; cardiovascular diseases. Therefore, the discovery of novel pharmaco- • targeting HDAC7 lowers serum cholesterol levels. Given this premise, logical targets and of molecules regulating their activity represents the general objective of this proposal is to bridge these new basic a new frontier to widen the choice of treatments that could be used science concepts to clinical applications by analysing the transcriptome alone, or in combination with established medications, to ameliorate and the regulome controlling cholesterol and lipid homeostasis and the lipid profi le and consequently the cardiovascular risk in those by pharmacologically targeting the HNF-4/HDAC7 regulatory axis. patients not responding to drugs affecting lipid metabolism. We will develop improved strategies for the prevention and treatment of atherosclerosis by pursuing the following specifi c aims: On the other hand, a global reduction of the cardiovascular risk in the • rational design of high affi nity ligands for HNF-4a and inhibitors of population must also rely on appropriate diagnostic tools that can aid HDAC7 based on the analysis of their 3D-structures; both the early detection of atherosclerotic disease and monitoring the • analysis of the global pattern of gene expression (Transcriptome) effectiveness of the therapeutical interventions in patients undergoing and transcription factor network (Regulome) involved in lipid pharmacological treatments. Angiography has endured for more than homeostasis in control mice and in mice treated with HDAC inhibitors 40 years as the predominant method used to defi ne arterial anatomy. and HNF-4 ligands and defi nition of their pharmacological profi le; However, many studies have challenged the accuracy and reproducibility • development of clinical diagnostic tools. of this technique. Several imaging modalities have been developed to The multidisciplinary nature of the proposal will exploit and integrate the evaluate the atherosclerotic plaque, its distribution and its morphology. expertise in molecular/cell biology, pharmacology, toxicology, medicine, Among these, Intravascular Ultrasound (IVUS), by imaging arterial physics/crystallography, chemistry, biochemistry and bioinformatics to atheroma directly, allows measurement of atheroma size and, to some identify new molecules affecting lipid metabolism and study their effects extent, composition. Since the IVUS technology still requires an invasive on pathophysiological events underlying atherosclerosis. procedure, the Magnetic Resonance Imaging (MRI) technique, fully non- Upon completion of the project we expect to: invasive, certainly offers an attractive alternative. MRI allows serial • identify HNF-4 ligands and HDAC7 selective inhibitors affecting lipid repetitions without the risk of ionizing radiation, provides high-resolution metabolism; images of the arterial wall and lumen. Application of MRI opens new • defi ne the effects of these molecules on the transcription network areas for diagnosis, prevention, and treatment of atherosclerosis in all regulating cholesterol and lipid metabolism and their pharmacological arterial locations. profi le in animal models of atherosclerosis; • develop non-invasive tools for evaluating the atherosclerotic burden. Aim

In the previous FP5 NORTh project we deﬁ ned the central role of the HNF-4a/HDAC7 axis in the regulation of cholesterol catabolism to bile acids, which is a very promising target to develop new hypolipidemic agents. As a result of this ﬁ nding, we were able to demonstrate that the administration of the generic HDAC inhibitors valproic acid and trichostatin A to Ldl-r KO mice reduces dramatically plasma total and LDL-cholesterol, as a consequence of the increased hepatic conversion of cholesterol to bile acids. Therefore, based on the very promising

CARDIOVASCULAR | 39 results obtained by several members of the consortium in the FP5 NORTh project, the general goal of this proposal is to determine the effects of known HDAC inhibitors on the transcriptome and on the transcription factor network (regulome) controlling cholesterol and lipid metabolism and on atherogenesis. We will also make effort to design and identify synthetic ligands for HNF-4a and selective inhibitors of HDAC7 on the basis of the 3D-structure of these target proteins. The new molecules will be then tested in animal models of atherosclerosis and in other animal models available within the consortium, which are characterised by alterations of lipid metabolism. Finally, clinical diagnostic tools will be developed by using non-invasive imaging techniques (MRI) to monitor plaque progression during the treatment with the molecules.

Expected results

Upon completion of the proposed objectives we expect to: • identify ligands of HNF-4a and HDAC7 selective inhibitors affecting lipid metabolism and to solve the crystal structure of these molecules in the absence and presence of the identifi ed ligands and inhibitors; • defi ne the effects of HDAC inhibitors and HNF-4a ligands on the transcription factor network regulating cholesterol and triglyceride metabolism; • defi ne the effects of these molecules at the global genomic level and their pharmacological and in vitro toxicological profi le; • develop a non-invasive imaging method and a biomolecular tool for the evaluation of the effect of pharmacological treatments on atherogenesis.

As a whole, the successful implementation of this project will provide an important contribution in the prevention and management of cardiovascular diseases and, consequently, will have a major socio-economical impact on lives of the affected population as well as on the healthcare systems of European countries.

Potential applications Model of feedback regulation of the gene cholesterol 7a-hydroxylase (CYP7A1) by bile acids (BA). Bile acids cause the translocation of HDAC7 By using functional genomics approaches integrated with new diagnostic into the nucleus where it is recruited, along with the other coregulators SMRT and HDAC3, on the nuclear receptor HNF-4 bound to the CYP7A1 tools, the SOUTH consortium will further characterise novel targets for promoter. This event results in the dephosphorylation of phosphoserine 2 new hypolipidemic drugs and will identify small molecules from libraries at the C-terminal domain repeat of RNA polymerase II and ultimately in the designed on the basis of the 3D-structures of these promising targets. transcriptional repression of CYP7A1 gene. HDAC inhibitors, by targeting The molecules identiﬁ ed in this study may be the precursors of future the HDACs recruited on CYP7A1 promoter, can relieve the physiological repression of bile acids on CYP7A1 gene transcription and increase bile acid medications aimed at decreasing plasma lipid levels and lower the risk synthesis, thus leading to the reduction of blood cholesterol. of developing atherosclerosis and cardiovascular disease.

40 | CARDIOVASCULAR Coordinator Peter Baráth Cancer Research Institute Maurizio Crestani, Ph.D. Slovak Academy of Sciences Professor of Biochemistry Bratislava, Slovakia Dept of Pharmacological Sciences University of Milan Sean McSweeney Milan, Italy Macromolecular Crystallography Group European Synchrotron Radiation Facility Partners Grenoble, France

Iannis Talianidis Béatrice Desvergne BSRC ‘Al. Fleming’ Center for Integrative Genomics Vari, Greece University of Lausanne Lausanne, Switzerland Gilles Salbert University of Rennes I Darren Hart Rennes, France High Throughput Protein Expression Lab Marco Bertolotti EMBL Outstation The bile acid chenodeoxycholic acid (CDCA) causes the nuclear translocation University of Modena Grenoble, France of HDAC7 in liver cell culture. The translocation seems to be FXR- and Reggio Emilia independent, as the synthetic ligand (GW4064) for the bile acid receptor Modena, Italy Paola Tarroni FXR does not cause the nuclear translocation of HDAC7. The phosphatase Axxam s.r.l. inhibitor Calyculin A blocks the dephosphorylation of HDAC7 and consequently prevents the CDCA-induced translocation into the nucleus. Target Biology Milano, Italy

Project number LSHM-CT-2006-037498 EC contribution € 2 935 582 Project Duration 36 months Starting date 01/10/2006 Instrument STREP

CARDIOVASCULAR | 41 VASOPLUS

Placental Growth Factor (PlGF): new diagnostic and therapeutic applications in cardiovascular disease

Keywords Placental growth factor, PlGF, ischemia, Problem cardiovascular disease Ischemic heart disease (IHD) and peripheral arterial occlusive disease (PAOD) are the major cause of severe morbidity and mortality in West- ern societies. Patients who presently survive acute coronary events as a result of coronary intervention (angioplasty, stenting or coronary bypass surgery) or pharmacological coronary artery reperfusion often develop congestive heart failure that is resistant to intervention or phar- Summary macological treatment. Thus, the progress in the treatment of cardiovascular disease has converted acute lethal syndromes into a chronic The proposed Strep evaluates the potential of PlGF as a pharmaceutical for debilitating disease. Therapeutic angiogenesis is a novel treatment para- major chronic progressive ischemic cardiovascular disorders (ischemic heart digm but ongoing clinical trials with the angiogenic proteins VEGF and disease, heart failure) and arterial insuffi ciency (intermittent claudication bFGF have not achieved convincing primary endpoints of improved tis- and critical limb ischemia) and as a biomarker for cardiovascular disease. sue perfusion and functional recovery. Therefore, current strategies The proposed Strep is based on basic research on the VEGF-signaling must be reconsidered and alternative angiogenic targets validated. pathway (via PlGF) performed over the last fi ve years (CTG, VIB-3), prototype research performed with recombinant PlGF for pro-angiogenesis (GT and TG/ The pro-angiogenic drug candidate PlGF, unlike VEGF, has in proof of TX), basic research on the association of plasma PlGF and VEGF levels and concept studies been demonstrated to target pathological angiogenesis human diseases (IGB) and the development of a diagnostic test to evaluate and not physiological angiogenesis and to be devoid of most of the PlGF as a biomarker for cardiovascular disease. systemic side effects associated with administration of VEGF. In initial The comprehensive strategy encompasses the validation of the ‘proof of proof of concept studies, PlGF levels in blood have been shown to be concept’ observations made by the academic partners of the consortium into a distinct biomarker for cardiovascular disease. therapeutic concepts in new relevant small animal models, development of industrial scale GLP/GMP quality materials and confi rmation of their safety and effi cacy in new, relevant large animal models. The strategy will be applied Aim to the development of pro-angiogenic recombinant PlGF in ischemic heart disease models and in peripheral arterial insuffi ciency models. In parallel the The aim is threefold and summarised below. value of PlGF as a predictive biomarker and as a diagnostic test for cardiovascular disease will be explored in a collaborative effort with a large 1. Determine the potential of PLGF as therapeutic for major chronic diagnostic company. progressive ischemic cardiovascular disorders. The principal aim of the project is to develop a new, safe and effi cacious • Determine the pro-angiogenic effects of PlGF on: medicine to enhance the formation of blood vessels and in doing so, – revascularization of ischemic limbs. This will be studied in to develop new treatment paradigms for heart failure and critical mice, rats and in rabbits; limb ischemia. – Revascularization of ischemic cardiac tissue. This will be investigated in mice. These models will be of critical importance as these diseases are prevalent in patients eligible for therapeutic angiogenesis, and the limited success of clinical trials with VEGF and basic ﬁ broblast growth factor (bFGF) has indeed been in part attributed to refractoriness of such disease groups to angiogenic growth factors. These models and strategies will not only be of value for the present project but also for the development of pro-/anti-angiogenic medicines in general. • Once the pro-angiogenic effect of PlGF will be established in rodent models, these effects need to be conﬁ rmed and validated in large animal models that are physiologically more closely related to the situation in patients. Therefore newly established models of cardiac ischemia in pigs and in baboons will be used. Demonstration that PlGF stimulates vessel growth in diseases conditions closely related to the situation in patients will markedly increase the likelihood of success in humans.

42 | CARDIOVASCULAR 2. Further development of the production process. • Perfusion studies to quantitate the degree of myocardial perfusion • The production process has been transferred from its original site both in the endo- and epicardial myocardium. The effect of of development to an industrial manufacturer for further develop- intervention on LV function will be expected within 1.5 year. ment so as to fi t with fi nal production scale and GMP constraints. • State of the art application of ‘in process’ and ‘fi nal’ quality and 2. Further development of the production process. stability control will require additional validation of new assays. • Material qualifi ed for toxicology studies and GMP qualifi ed rhPlGF-1. • Discovery research will be performed on compound improvements, • High yield production of the mutein. involving Cys-to-Gly substitution for improved stability and alterna- • Stability of the mutein in solution and gel analyzed. tive isoforms (glycosylated PlGF-1 and PlGF-2 produced in Chinese • Comparative biological activity of the mutein and different isoforms hamster ovary (CHO) cells). of PlGF verifi ed by in vitro assays. • Small scale preparations available of mammallian-cell-produced 3. Determine the potential of PlGF as biomarker for cardiovascular rhPlGF-1, rhPlGF-2 and rhPlGF-1CG. disease. • Establish and validation assay assay for quality control, stability • Population study: A database, recently developed by IGB, holds the studies and batch release. description of the health status, the genealogy and the genome- • GLP compliant certifi cation/recognition of the Belgian Monitoring wide microsatellite marker scan of approximately 4,000 individuals authorities based on a fi ctive GLP stability study with the aim to from genetically isolated villages in Italy. This database will be determine the shelf life and stability on rhPlGF-1. studied to determine the contribution of PlGF and VEGF to specifi c human cardiovascular diseases and cancer. • Development of a reliable diagnostic test for measurement of PlGF 3. Determine the potential of PlGF as biomarker for cardiovascular concentrations in human blood samples based on compounds disease. from Geymonat and ThromboGencics/Thromb-X. It is intended • Correlation of the plasma levels of VEGF and PlGF (-1 and -2) and to evaluate the degree of angiogenesis based on PlGF blood health status of 2 800 samples by analysis of our database. levels together with other biomarkers of cardiovascular disorders in • Defi nition of the VEGF and PlGF haplotypes in 2 800 individuals. a well-characterized Italian cohort with the intend to stratify • Association analysis of VEGF and PlGF variants and haplotypes with patients who might benefi t from proangiogenic treatment. a trait or disease. • Status of PlGF assay development including pre-analytical conditions. • Completion of sample measurements using PlGF prototype assay, Expected results sCD40L and NT-pro-BNP diagnostic assays. • Correlation of plasma levels of PlGF, sCD40L, NT-pro-BNP and Expected results for the different objectives are as follows. health status of the study individuals. • Multi-variate analysis of the study completed. 1. Determine the potential of PLGF as therapeutic for major chronic progressive ischemic cardiovascular disorders. • Effi cacy analysis of rhPlGF-1 for revascularization of ischemic Potential applications myocardium and limb muscle. • Comparative analysis of rhPlGF-1 effi cacy for revascularization of Two possible applications are being pursued with this program: a new, ischemic tissue in mouse and rabbit ischemia models. safe and effective medicine to enhance the formation of new blood • Comparative analysis of rhPlGF-1 effi cacy for revascularization of vessels in ischemic tissues and a new diagnostic tool for cardiovascular ischemic tissue in a mouse model of ischemic cardiac failure. disease. • Data on the percentage of animals that develop a dysfunctional and ischemic myocardium versus those that develop signifi cant transmural necrosis caused by acute occlusion of the stented segment. This will allow to determine the number of animals required to enter the study. • An evaluation of the size of dysfunctional but viable myocardium by magnetic resonance imaging with delayed enhancement as well as dobutamine stress echo and quantitation of global LV function allowing power calculations for the NNT in order to observe a signifi cant increase in global LV function (increase in Ejection Fraction by 4-6 %).

CARDIOVASCULAR | 43 Coordinators

Dr. Jean-Maris Stassen ThromboGenics Ltd 14 Bridgecourt Offi ce Park Walkinstown Avenue Dublin 12 Ireland [email protected]

ThromboGenics N.V. previously Thromb-X NV Placebo PIGF treatment Herestraat 49 3000 Leuven More visible collateral vessels after 7 days of PIGF treatment in rabbit hindlimb ischemia. Belgium

Partners

Dr. Domenico Maglione Geymonat SpA Anagni, Italy

Prof. Florence Xhonneux Eurogentec SA Seraing, Belgium

Prof. Stefan Janssens KULeuven Leuven, Belgium

Dr. Maria Persico IGB-ABT Naples, Italy

Prof. Philip Badenhorst University of the Free State Bloemfontein, South Africa

Dr. Georg Hess Roche Diagnostics GmbH Mannheim, Germany

Project number LSHM-CT-2006-037254 EC contribution € 1 955 856 Project Duration 24 months Starting date 01/01/2007 Instrument STREP Project website www.vasoplus.eu

44 | CARDIOVASCULAR 45 ➞ 57

DIABETES & OBESITY EuroDia

Functional genomics of pancreatic beta cells and of tissues involved in control of the endocrine pancreas for prevention and treatment of type 2 diabetes

Keywords Type 2 diabetes, pancreatic beta cell, apoptosis, Problem animal models, human Type 2 diabetes mellitus (T2DM) has a high prevalence and is a major cause of mortality in Europe. The diagnostic feature of diabetes is a raised blood glucose level that results when a patient’s insulin-producing pancreatic beta cells secrete amounts of insulin inadequate for the tissue requirements. Understanding in greater molecular detail the normal regulation of the number and function of beta cells and how they fail in T2DM is crucial. This increased knowledge will lead to the development of new strategies for the treatment and prevention of T2DM. Summary

Failure of pancreatic beta cells to maintain adequate functional capacity Aim leads to increasing blood glucose levels and subsequent type 2 diabetes (T2DM). This disease imposes a huge and growing socio-economic The aim of this Integrated Project is to clarify key pathophysiological burden on European and global societies. However, the pathophysiological mechanisms and sites that are responsible for the dysfunctional insulin mechanisms underlying beta cell dysfunction remain poorly understood, secretion in type 2 diabetes (T2DM) and to identify and assess potential limiting the availability of novel approaches to treat or prevent T2DM. targets for prevention and treatment. This Integrated Project will strengthen the understanding of the factors infl uencing the maintenance and loss of normal beta cell functional This will be achieved through development of research that is divided capacity. This will be realised through the application of functional into three global sequences: genomics technologies in an integrated and systematic approach which • dissection of the molecular pathways and identiﬁ cation of key employs studies in cellular and animal models as well as genetic analysis regulatory events that control the physiological ability of the pancreatic of human monogenic and polygenic T2DM patients. The project integrates beta cell to preserve its secretory function over a lifetime; European experts in islet diabetes research, human geneticists, • identiﬁ cation of the key pathophysiological events in the above bioinformaticians and computational scientists as well as SMEs. pathways that become dysfunctional in T2DM, and determination of Specifi c aims are to: the pathogenic roles of lipotoxicity, glucotoxicity, oxidative stress and • dissect the molecular pathways and identify key regulatory events the impact of genetic variations in the induced beta cell dysfunction; – including those contributed by tissues located outside the • selection of genes and proteins which could be likely candidates in endocrine pancreas – that control the ability of the beta cell to the pathogenesis of beta cell diabetic dysfunction, and assessment maintain its secretory function over a lifetime; of their physiological role and potential value as novel drug targets • identify key pathophysiological events in the above pathways that for prevention and treatment of T2DM. become dysfunctional in T2DM and determine the contributions of lipotoxicity, glucotoxicity, oxidative stress and the impact of genetic variations in the induced beta cell dysfunction; • select genes and proteins that are candidates in the pathogenesis of beta cell dysfunction and assess their physiological role and potential value as novel drug targets for prevention and treatment of T2D. This project will strengthen the understanding of beta cell molecular physiology and pathophysiology to improve health in Europe.

46 | DIABETES & OBESITY Expected results Coordinator Cecilia Holm Cell and Molecular Biology Pathophysiology of beta cells: Sigurd Lenzen Lund University • the discovery of novel mechanisms regulating beta cell mass and Institute of Clinical Biochemistry Lund, Sweden Hannover Medical School function and sites that are dysregulated in T2DM; Hannover, Germany C. Victor Jongeneel • specifi c gene variants that predispose to T2DM in the human Phone: +49 511 532 6525 Institut Suisse de Bioinformatique population. Fax: +49 511 532 3584 Lausanne, Switzerland Target identifi cation for prevention or treatment: [email protected] • the identifi cation of genes that play a critical role in pathways Piero Marchetti regulating beta cell mass and function and which, when activated University of Pisa or inhibited, prevent beta cell diabetic dysfunctions; Partners Pisa, Italy • identifi cation of gene variants that can be used to diagnose the susceptibility to T2DM and/or which can provide critical pharma- Frances Mary Ashcroft Mark McCarthy cogenomic information. University of Oxford Endocrinology and Metabolism Oxford, United Kingdom University of Oxford Oxford, United Kingdom Naama Barkai Potential applications Weizmann Institute of Science Patrik Rorsman Rehovot, Israel Endocrinology and Metabolism Due to the increasing incidence of T2DM in Europe new preventive and University of Oxford curative therapies of this disease must be developed. The elucidation of Per-Olof Berggren Oxford, United Kingdom the mechanisms underlying beta cell dysfunction and loss will allow the Karolinska Institutet identifi cation of new targets for prevention, diagnosis and treatment of Stockholm, Sweden Jorgina Satrústegui T2DM. This will permit the development of new therapies against this Universidad Autónoma de Madrid metabolic disease. Helena Edlund Madrid, Spain Umea University Umeå, Sweden Frans C. Schuit Katholieke Universiteit Leuven Decio L. Eizirik Leuven, Belgium Université Libre de Bruxelles Brussels, Belgium Bernard Thorens University of Lausanne Jorge Ferrer Lausanne, Switzerland Endocrinology Institut d’Investigacions Biomèdiques Claes B. Wollheim August Pi i Sunyer University of Geneva Barcelona, Spain Geneva, Switzerland

Philippe Froguel Alexander Kel Genetic of Multifactorial Diseases Biobase GmbH Institut de Biologie de Lille Wolfenbüttel, Germany Institut Pasteur de Lille Lille, France Kader Thiam Project number Genoway SA LSHM-CT-2006-518153 Andrew Hattersley Lyon, France EC contribution Molecular Genetics € 9 150 000 Peninsula Medical School Sophie Chappuis The University of Exeter Transat SA Project Duration Exeter, United Kingdom Lyon, France LSHM-CT-2006-518153 Starting date 01/03/2006 Instrument IP Project website www.eurodia.info

DIABETES & OBESITY | 47 HEPADIP

Hepatic and Adipose Tissue and Functions in the Metabolic Syndrome

Keywords Metabolic syndrome, hepatic tissue, adipose tissue, Problem lipid metabolism, insulin signaling, glucose homeostasis The Metabolic Syndrome is a major risk marker for type 2 diabetes and for cardiovascular disease. Recent evidence suggests a crucial role for fat accumulation in the liver in the development of the Metabolic Syndrome. The hypothesis of HEPADIP is that this reﬂ ects an alteration in the metabolic relationships or the signaling between adipose tissue and liver.

Summary Aim

The Metabolic Syndrome is a major risk marker for type 2 diabetes and for The aims of the HEPADIP are to address the role of the adipose tissue cardiovascular disease, and recent evidence suggests a crucial role for fat and the liver and their integrated functions in the development of the accumulation in the liver in the development of the Metabolic Syndrome. disturbances of the lipid metabolism, insulin signaling, and glucose The LSH-2004-2.1.1-7 topic “Adipose tissue and liver dysfunction in the homeostasis in the Metabolic Syndrome. On this basis, novel targets for Metabolic Syndrome” therefore sets the goal “to clarify the relationships diagnosis, characterization, prevention and treatment of the Metabolic between dysfunction of lipid metabolism, insulin signaling and regulation Syndrome will be identifi ed, validated and developed. of glucose level and to provide advances to combat the Metabolic Syndrome, with a particular emphasis on adipose tissue and liver”. On this basis, novel targets for diagnosis, characterization, prevention, and treatment of the Expected results Metabolic Syndrome will be identifi ed, validated and developed. Five lines of research (RLs) are defi ned in HEPADIP (fi gure 1) for the study of the adipose tissue (RL 1), the liver (RL 2), the integrated functions of both organs in animal models (RL 3) and in humans (RL 4), and the clinical and genetic epidemiological studies of human populations (RL 5). There are several direct interactions between the RLs, and they are also linked by horizontal activities dealing with the applied and novel ‘omics’ technology (RL 6), target validation and development (RL 7), development of new standardized diagnostic criteria and gender comparisons. Each of the RLs has a series of specifi c objectives, and the expected results of HEPADIP are therefore described in relation to each RL.

• RL1: Biology of the adipose tissue. Inﬂ uence fatty acid metabolism in various human adipose tissue (HAT) depots. Find novel secreted signal peptides in HAT inﬂ uencing insulin action in fat cells and liver. Study alterations in insulin signaling using cellular, animal and human models.

• RL 2: Biology of liver tissue. Use a Systems Biology approach to study nonalcoholic fatty liver disease (NAFLD) in humans. To use a Candidate Gene approach to study NAFLD in humans. To develop blood tests markers of early stages of hepatic steatosis and steatohepatitis and predictors of evolution towards Metabolic Syndrome.

• RL 3: Integrative studies of the adipose tissue – liver axis in rodents. Use a Systems Biology approach to identify early molecular mechanisms linking lipotoxicity and genetic factors with insulin resistance. Use a Candidate Gene approach to unravel the roles of PPARα and PPARγ in the control of carbohydrate, lipid metabolism and inﬂ ammation in liver and adipose tissue.

48 | DIABETES & OBESITY Lipidomic analysis of ob/ob mouse tissue.

• RL 4: Integrative studies of the adipose tissue – liver axis in humans. • RL7: Target validation and development. Select targets from candidate Establish methods for studying the adipose tissue-hepatic axis in proteins identifi ed in the HEPADIP applying scientifi c, medical and vivo in humans. Defi ne how adipose tissue and liver traffi cking and technical criteria. Validate potential targets in critical experiments in signaling are altered in individuals with the Metabolic Syndrome vitro and in vivo in collaboration with the different partners in HEPADIP. and in weight-matched individuals without the Metabolic Prepare a ‘drug target package’ for a selected target including an Syndrome. Identify in humans target pathways for intervention, activity assay and recombinant, purifi ed protein and to prepare for the to validate these targets and to develop strategies for treatment. decision whether the particular target should be further developed by a third partner or proceeded within HEPADIP. • RL 5: Clinical and genetic epidemiological studies. Quantify the mutual relationship and dynamic development over time of components of the Metabolic Syndrome and factors related to the Potential applications function of adipose tissue and the liver. Identify variants in candidate genes expressed in liver and fat tissue and to elucidate the role of The objectives of the HEPADIP project contribute to the scientifi c, these variants in the pathogenesis of common phenotypes of the technical, wider societal, and policy objectives of the LifeSciHealth Metabolic Syndrome. Validate novel biomarkers and therapeutic/ program by exploiting the united effort of the European academic preventive targets related to function of the liver and adipose tissue research community in concert with several capable Small and Medium- associated with the disturbances of the lipid and glucose Sized Enterprises, to focus on a specifi c priority area for policy-relevant metabolism in the Metabolic Syndrome. research. To bring this strategic line to maturity, the HEPADIP Consortium will later, if appropriate, engage a larger company that has the • RL 6: Integration and development of omics and bioinformatics. competence and capacity to bring novel targets discovered by the Establish a discussion forum at the network level to harmonize HEPADIP research further towards target and drug development. The methods and tools in omics and bioinformatic techniques, and for Consortium represents a comprehensive European integrated approach training scientists. Establish tools for trans-experimental transcriptomic able to tackle the Metabolic Syndrome problem from all angles relevant data analysis and integration of transcriptomic analysis with other to the topic of the call. omic techniques, proteomic and lipidomics.

DIABETES & OBESITY | 49 A patient undergoing metabolic studies to elucidate adipose tissue and hepatic function.

Coordinator Prof. José María Mato Prof. Marja-Riitta Taskinen Scientifi c Director Dean Hum Centro de Investigacion Cooperativa en Helsinki University Genfi t SA Prof. Thorkild I. A. Sørensen Biociencias, Metabolomics Central Hospital Loos, France Institute of Preventive Medicine Derio (Bizkaia), Spain Helsinki, Finland H:S Bispebjerg Hospital Dr. Azucena Castro Øster Søgade 18, 1357 Copenhagen K Prof. Albert Emmanuel Geerts Prof. Pirjo Nuutila One Way Liver Genomics Denmark Faculty: Medicine and Farmacy Turku PET Centre Derio, Spain Phone: +45 33 38 38 60 Laboratory: Cell Biology University of Turku Fax: +45 33 32 42 40 Vrije Universiteit Brussel Turku, Finland Dr. Gorka Ochoa [email protected] Brussels, Belgium Dr. Daniel Nagore Prof., Dr. Luc van Gaal Proteomika S.L. Senior Lecturer Universiteit Antwerpen Derio, Spain Partners Dr. Antonio Vidal-Puig University Hospital Antwerp University of Cambridge Edegem, Belgium Dr. Philip Just Larsen Prof. Peter Arner Cambridge, United Kingdom Rheoscience A/S Karolinska University Prof. Oluf Pedersen Rødovre, Denmark Hospital Huddinge Prof. Bernard Thorens Steno Diabetes Center Stockholm, Sweden Université de Lausanne Gentofte, Denmark Prof. Kim Overvad Lausanne, Switzerland Aalborg Hospital Prof. Dominique Langin Dr. Petra Budde Aarhus University Hospital Unité de recherches sur les obésités Prof. Bart Staels Digilab BioVisioN GmbH Aalborg, Denmark Inserm-UPS U586 U 545 INSERM Hannover, Germany Institut de Médecine Moléculaire de Institut Pasteur Rangueil (I2MR), Institut Louis Bugnard Lille, France CHU Rangueil Toulouse, France Prof. Folkert Kuipers Institute for Drug Exploration (GUIDE) Prof. Karine Clément Centre for Liver and Metabolomics INSERM Nutriomique U755 Groningen University Paris, France Groningen, The Netherlands

Dr. Yannick Le Marchand-Brustel Dr. Matej Oresic Signalisation moléculaire et obésité VTT: Technical Research INSERM-UPS U568 Centre of Finland Faculté de Médicine VTT Espoo, Finland Nice, France Prof. Keith N. Frayn Prof. Hans J. Hauner University of Oxford Else Kröner-Fresenius-Zentrum Oxford, Great Britain für Ernäherungsmedizin Technical University of Munich Prof. Hannele Yki-Järvinen Freising-Weihenstephan, Germany Helsinki University Central Hospital Chief physician, prof. Ulf Smith Helsinki, Finland Göteborg University Project number Göteborg, Sweden LSHM-CT-2005-018734 EC contribution Prof. Vladimir Stich € 12 000 000 Charles University Project Duration Praha, Czech Republic 60 months Starting date 01/11/2005 Instrument IP Project website www.hepadip.org

50 | DIABETES & OBESITY InterAct

An examination of the interaction of genetic and lifestyle factors on the incidence of type 2 diabetes

Keywords Diabetes, genetic factors, lifestyle factors, high, not only because ﬁ ndings have been replicated in many studies in prevention, intervention, interaction different populations, but also because interventions aimed at changing these factors have led to reductions in the incidence of diabetes in randomised controlled trials. The importance of genetic factors in leading to type 2 diabetes has been demonstrated by studies of familial aggregation, high concordance in twin studies, and the impact of different degrees of high-risk heritage on diabetes risk in high-prevalence populations. Progress in discovering the genetic basis of diabetes is accelerating as more powerful genotyping technologies are applied to Summary larger study samples.

The pattern of variation of the incidence of type 2 diabetes suggests that The global variation in the prevalence of type 2 diabetes is marked and the disorder originates from an interaction between genetic and lifestyle suggests that an interaction between genes and lifestyles is central to behaviour factors. The overall goal of the InterAct integrated project is to determining diabetes risk. The geographical pattern of variation suggests investigate how biologically plausible genetic and lifestyle behavioural that prevalence is lowest in rural areas of developing countries, is factors, particularly dietary intake and physical activity, interact to lead to generally intermediate in developed countries, but is highest in certain the development of type 2 diabetes and to study the relevance of these ethnic groups who have adopted western lifestyle patterns. One discoveries for preventive action. This overall goal will be achieved by explanation for this variation is the ‘thrifty genotype’ hypothesis which analysis of gene-lifestyle interaction in an existing large-scale multi- postulates that genetic variation that is advantageous in times of food cohort observational study (EPIC) and by the construction of a Consortium scarcity will become disadvantageous in times of food abundance. The of existing trials in which differential response to lifestyle intervention can human genome, tailored to function in a hostile environment, now be tested. Finally InterAct will investigate the implications of gene-lifestyle faces novel stressors and, depending on the genetic subgroup, copes interaction for preventive strategies. variably with this challenge. The InterAct project is aimed at investigating the basis of these interactions.

Aim

The InterAct Consortium will construct a case-control study nested within an existing large cohort – the EPIC study. The cohort for this project is large, comprising 350 000 participants from 8 European countries. Standardised dietary and physical activity information is Problem available at baseline, and there is an available DNA source and plasma for nutritional biomarkers. Follow-up has been completed for an average Diabetes mellitus is a chronic disorder of glucose metabolism and is the of eight years. The collaborators will amass a case set of 10 000 single biggest cause of preventable blindness, the leading cause of non- individuals and a similarly sized control cohort free of diabetes at traumatic lower extremity amputation, and a major cause of end-stage baseline and during follow-up. DNA extraction and genotyping will be renal disease and coronary heart disease in European populations. The undertaken using centralised high throughput methods. lifetime cumulative incidence of diabetes in European populations is 35-40 %; it is a major cause of premature mortality and accounts for The choice of polymorphisms in candidate genes will be undertaken by 5-10 % of health care expenditure. The prevalence of diabetes is rising a group leading efforts to discover diabetes genes through a variety of in European populations, thus efforts to understand the aetiology of approaches including whole genome association studies. The causal the condition and develop preventive strategies are key public health inference from the discovery of gene-lifestyle interactions will be targets. strengthened by testing the same genes in existing intervention trials, which have examined the effectiveness of lifestyle changes on diabetes The overall importance of environmental factors in the aetiology of incidence. These include the Finnish Diabetes Prevention Study, the type 2 diabetes is evidenced by observations of increased diabetes risk Danish Inter99 project and the Indian Diabetes Prevention Study. The among individuals from at-risk populations who migrate to countries demonstration of differential response to lifestyle change by genotype where lifestyles are more westernised, as well as from cohort studies will not only provide greater aetiologic understanding, but will also where the importance of major non-genetic risk factors such as obesity, provide the opportunity to investigate possibilities to use genotypic data physical inactivity, and dietary intake of fat have been demonstrated. in risk stratifi cation and identifi cation of individuals who have the The strength of causal inference concerning these aetiological factors is potential to benefi t most from targeted lifestyle prevention.

DIABETES & OBESITY | 51 This shows a series of liquid nitrogen tanks that contain specimens from volunteers, stored at -196°C. The samples will be used for biochemical and genetic analysis. The tanks are sited in a specialist facility. All the specimens are handled according to strict procedures.

The objectives of the InterAct Project will be met by four research lines. Potential applications • Analysis of gene-lifestyle interaction in observational studies of diabetes incidence. The identifi cation of genetic variants that interact with key lifestyle Aim: to construct a nested case-control study for type 2 diabetes exposures to modify the risk of type 2 diabetes enhances the possibility within the EPIC observational cohort and to use this study as the of fi nding alternative therapeutic options or preventive strategies. The basis for studying gene-lifestyle interaction. realisation of these prospects following the InterAct project will require • Analysis of nutritional factors and physical activity in observational the translation of research knowledge into clinical practice. As a prelude studies of diabetes incidence. to these activities, InterAct has a work package exploring how genetic Aim: to study the association of nutritional, dietary and physical information on risk can be used. This part of the project aims, fi rst of all, activity behaviours with incident diabetes in the nested case-control to increase our understanding of the effect of the provision of study and to contribute to the analysis of gene-lifestyle interaction. information about risk of type 2 diabetes on emotion, cognition, health • Analysis of genetic factors in observational studies of diabetes behaviour and other outcomes such as use of health services. It will incidence. increase our understanding of the effect of the provision of genetic risk Aim: to study the association of genetic variation with incident information and information concerning gene-environment interactions diabetes in the nested case-control study and to contribute to the on emotion, cognition, health behaviour and other outcomes such as analysis of main genetic effects and gene-lifestyle interaction. the use of health services. It will assess the extent to which the impact • Analysis of gene-lifestyle interaction in intervention studies. of the provision of risk information concerning type 2 diabetes is Aim: to construct a Consortium of lifestyle intervention diabetes mediated by the method and content of the risk communication, for prevention trials in order to study the association of the genetic example genetic versus non-genetic risk information, and moderated by variation with differential response to the lifestyle intervention and the socio-cultural context. Finally it will explore the policy implications to consider the implications of these fi ndings for preventive action. for key stakeholders (government, health services, the food industry, the scientifi c community, and ethicists) of the new knowledge concerning associations between genes and lifestyle factors and the risk of type 2 Expected results diabetes, in particular the potential responses to fi ndings concerning the strength of associations and the population frequencies of genetic • InterAct will produce evidence of the association of genetic variants and behavioural risk factors. with risk of type 2 diabetes and will investigate interaction between these genes. • InterAct will provide evidence of the association between dietary patterns and specifi c foods and nutrients with risk of diabetes. • The project will describe the association of physical activity with incident diabetes and will investigate the combined effects of activity and nutritional and dietary factors. • InterAct will demonstrate how the effect of genetic variants on diabetes risk is modifi ed by lifestyle behavioural factors. • InterAct will show how these genetic variants modify response to lifestyle interventions.

Project number LSHM-CT-2006-037197 EC contribution € 10 000 000 Project Duration 48 months Starting date 01/10/2006 Instrument IP Project website www.inter-act.eu

52 | DIABETES & OBESITY Plastic straws are used for the storage of each blood specimen. Each straw contains half a millilitre of sample. Red straws are used for plasma, orange straws for serum, blue straws for white blood cells (from which DNA will be isolated) and green straws for red blood cells. Each straw is stored and recorded by an identiﬁ able number.

Coordinator Dr. Yvonne T. van der Schouw Prof. Kim Overvad Dr. Miren Dorronsoro Julius Center for Health Sciences Aalborg Hospital Public Health Department Prof. Nick Wareham and Primary Care Aalborg, Denmark of Gipuzkoa Medical Research Council University Medical Center Utrecht Basque Government Epidemiology Unit Utrecht, The Netherlands Prof. Göran Berglund Donostia-San Sebastian, Spain Institute of Metabolic Science University Hospital MAS Box 285, Addenbrooke’s Hospital Dr. H. Bas. Bueno-de-Mesquita Malmö, Sweden Dr. Aurelio Barricarte Hills Road National Institute for Health Institute of Public Health Navarra GB-Cambridge CB2 0QQ, and Environment Prof. Leif Groop Pamplona, Navarra, Spain United Kingdom Centre for Prevention and Health University Hospital Malmö Phone: +44 1223 330315 Bilthoven, The Netherlands Malmö, Sweden Dr. Carmen Martínez-García Fax: +44 1223 330316 Granada Cancer Registry [email protected] Dr. Caroline A. Baan Prof. Göran Hallmans Andalusian School of Public Health National Institute for Health Umeå University Hospital Granada, Spain and Environment Umeå, Sweden Partners Centre for Prevention and Health Dr. J.Ramón Quirós Bilthoven, The Netherlands Dr. Jakob Linseisen J.S. Información Sanitaria Prof. Mark McCarthy German Cancer Research Center Consejeria de Salud y Servicios Oxford Centre for Diabetes Dr. Salvatore Panico Heidelberg, Germany Sanitarios del Principado de Oviedo Endocrinology and Metabolism Federico II University of Naples Oviedo, Spain University of Oxford Napoli, Italy Prof. Heiner Boeing Oxford, United Kingdom German Institute Dr. Inês Barroso Dr. Domenico Palli of Human Nutrition The Wellcome Trust Sanger Institute Prof. Tim Key Scientifi c Institute of Tuscany Bergholz-Rehbruecke, Germany The Wellcome Trust Genome University of Oxford Firenze, Italy Cambridge, United Kingdom Oxford, United Kingdom Dr. Joachim Spranger Dr. Rosario Tumino German Institute Dr. Panos Deloukas Dr. Edith Feskens Ragusa Cancer Registry of Human Nutrition Wellcome Trust Sanger Institute Wageningen University Azienda Ospedaliera Nuthetal, Germany The Wellcome Trust Genome Wageningen, The Netherlands ‘Civile M.P. Arezzo’ Cambridge, United Kingdom Ragusa, Italy Dr. Beverley Balkau Prof. Jaakko Tuomilehto INSERM U780-IFR169 Prof. Oluf Pedersen University of Helsinki Dr. Vittorio Krogh Epidemiological Steno Diabetes Center Helsingin Yliopisto, Finland National Cancer Institute and Biostatistical Research Gentofte, Denmark Milano, Italy Villejuif, France Prof. Elio Riboli Prof. Ambady Ramachandran Imperial College London Dr. Carlotta Sacerdote Dr. Carlos A. González Indian Diabetes Research Foundation London, United Kingdom Fondazione I.SI. – Institute for Scientifi c Catalan Institute of Oncology WHO Collaborating Centre Interchange Foundation Barcelona, Spain for Research, Education and Prof. Philippe Froguel Torino, Italy Training in Diabetes Imperial College Dr. María-José Tormo Chennai, India London, United Kingdom Dr. Anne Tjønneland Consejeria de Sanidad CARM Institute of Cancer Epidemiology Murcia, Spain Prof. Markku Laakso Dr. Nadia Slimani Danish Cancer Society University of Kuopio International Agency Copenhagen, Denmark Kuopio, Finland for Research on Cancer Lyon, France

DIABETES & OBESITY | 53 PREDICTIONS

PREvention of DIabetic ComplicaTIONS: the Identifi cation of Risk Factors for the Development of Diabetic Nephropathy

Keywords Diabetes, nephropathy, genetic association, Problem protein glycation, oxidative stress, therapeutic agent A worldwide epidemic of renal failure from DN is in progress. DN has become the leading cause of end stage renal failure in the western world. Approximately 19 million people live with diabetes in the EU countries. All these patients are at risk of developing DN and approximately 30 % of them will get the complication. Hence, DN patients will be a burden to the health care system, demanding expensive haemodialysis and an enormous number of kidney transplantations. Considering the size of the Summary impending epidemic it seems unlikely that we can get through by treating with antihypertensive drugs and creating more dialysis facilities alone. Approximately 30 % of all patients with type 2 diabetes will develop diabetic Earlier intervention is required. Therefore, we need to identify the nephropathy (DN). Still there are no effective methods to determine the individuals at high risk and to provide individualised treatments. individual risk of developing DN. Duration of diabetes as well as glycaemic and blood pressure control do not suffi ciently explain the risk of developing Although many (merely minor) genetic and biochemical markers have DN. About 31 genes and many other factors have been suspected to been suggested to be associated with DN, this knowledge is not usable contribute to the development of DN. A major gene (CNDP1) has been in clinical practice. The reason is that it is very difﬁ cult to verify and mapped to 18q and was identifi ed to cause a 3-fold elevated risk of DN. quantify the inﬂ uence of isolated minor factors. Here, we intend to The aim of this project is to identify pathophysiologically relevant genes and simultaneously investigate all potentially relevant biomarkers (genetic biomarkers correlated to the development, progression and therapy of DN. and non-genetic). Markers previously predicted to be major factors (such as CNDP1 and SLC12A3) will be included.

Aim

The major overall objective is to develop novel diagnostic and prognostic tools for identifying patients with a genetic predisposition to develop DN early after diabetes onset. Furthermore, genomic and proteomic methods will be applied to identify factors associated with the response to current intervention therapies. Concepts for new intervention strategies will be developed. The new diagnostic tools and risk estimation procedures will be made available for practicing physicians within the EU and beyond.

Expected results

• Previous studies have suggested that the CNDP1 gene encoding serum-carnosinase is a major risk factor for DN. PREDICTIONS aims to provide independent conﬁ rmation of these data. Genetic association studies on CNDP1 will be performed and carnosine levels and serum carnosinase activity will be determined in blood samples of all study patients.

54 | DIABETES & OBESITY • Re-evaluation of other previously proposed genetic variants. Coordinator Prof. Dr. GJ Navis Because CNDP1 is not expected to be the only DN-causing factor, Groningen University some additional factors may play an important role in ‘CNDP1- PD Dr. Bart Janssen Medical Center independent’ DN. Institute of Human Groningen, The Netherlands Genetics Heidelberg • Expression profi ling for the identifi cation of genes associated with Im Neuenheimer Feld 366 Prof. Paul J. Thornalley the pathogenesis of DN. 69120 Heidelberg, Germany Protein Damage and Systems Biology • Analysis of the urinary proteome for the identifi cation of a secretion Phone: +49 6221 5639568 Research Group – Clinical pattern associated both with a specifi c genetic trait and a high-risk Fax: +49 6221 565091 Sciences Research Institute vascular profi le of diabetic patients. [email protected] University of Warwick • Assessment of biomarkers of protein glycation and oxidative stress as Coventry, United Kingdom prognostic factors. Various AGEs and molecules involved in AGE formation and oxidative stress play in important role in the pathogenesis Partners Prof. Dr. Ivan Rychlík of DN. To this end, AGEs and several markers of oxidative stress will be Charles University at Prague assessed in the study patients. It will be evaluated, which of the Prof. Dr. F.J. van der Woude Prague, Czech Republic Fakultät für Klinische selected biomarkers may serve as prognostic factor. Medizin Mannheim Dr. Lise Tarnow • Exploration of the association between biomarkers and response to Mannheim, Germany Novo Nordisk (Steno Diabetes Center) treatment. In a therapeutic trial on Benfotiamine, patients will be Gentofte, Denmark classifi ed in two groups: ‘responders’ and ‘escapers’. We will identify Prof. Brigitte M. Winklhofer-Roob genomic and proteomic biomarkers associated with the response to M.D. – The University of Graz Prof. Harald Mischak treatment. As the Consortium has access to materials and data of Graz, Austria Mosaiques Diagnostics GmbH trials on ACE-inhibitors and ATR-1 blockers (Irbesartan) conducted Hannover, Germany previously, similar data will be obtained for these substances. Dr. Emile De Heer Leiden University Medical Center Dr. Marcus Woerwag Leiden, The Netherlands Woerwag Pharma GmbH & Co. KG Potential applications Boeblingen, Germany

In summary, the main question underlying the work is: “Which biomarkers are associated with the risk to develop DN”. The challenge is to identify these markers and to fi nd out how they contribute to the pathogenesis of the disease. In the future these markers may facilitate early recognition of persons at risk and early diagnosis of DN. Of the variety of possibly relevant biomarkers, those with a signifi cant predictive value will be identifi ed and used to develop a risk model to be used in clinical practice. Moreover, the acquired knowledge on the pathways involved in the pathogenesis of the disease will enable the development of new therapeutical strategies. Both the early identifi cation of individuals at risk and the availability of novel options for therapeutical intervention will signifi cantly change the lives of people at risk.

Project number LSHM-CT-2005-018733 EC contribution € 1 757 500 Project Duration 36 months Starting date 01/12/2005 Instrument STREP Project website www.predictions-project.eu SAVEBETA

Molecular pathways underlying decreased beta cell mass in diabetes mellitus

Keywords Diabetes mellitus, pancreatic beta cells, Problem apoptosis, regeneration, cytokines, ER stress The two main forms of diabetes mellitus are T1D and T2D. They are a major cause of morbidity and mortality, decreasing both life quality and life expectancy of nearly 30 million affected individuals in Europe. T1D is characterized by a near complete lack of insulin production due to specifi c destruction of the pancreatic beta cells that typically develops Summary over several years. Although some immune-related biomarkers can identify individuals at risk to develop T1D, the process by which the beta A reduction in pancreatic beta cell mass, caused by increased apoptosis cells are destroyed is not well understood. As a consequence, there are and defective regeneration, is a key component of both type 1 (T1D) and no adequate strategies for preservation of beta cell mass and prevention type 2 (T2D) diabetes mellitus. The molecular mechanisms underlying of the disease. Accumulating evidence suggests that beta cell loss in decreased beta cell mass remain to be clarifi ed. Molecular signalling in the T1D is the result of an autoimmune mediated process, where a chronic beta cells is decisive for their survival or death in diabetes. We hypothesize infl ammation called insulitis causes beta cell destruction. This is mediated that crosstalk between key gene networks and insufficient protective by cytokines and other mediators released by the activated immune cells responses trigger the apoptosis program and prevent regeneration. The aim invading the islets, which activate secondary pathways of cell death in of this proposal is to utilize functional genomics to identify pathways the target beta cells. responsible for the reduction of beta cell mass in diabetes, and use this knowledge to defi ne targets for intervention to preserve beta cell mass. T2D results from a reduced ability of the pancreatic beta cells to secrete enough insulin to stimulate glucose utilization by peripheral tissues. Initially, this causes impaired glucose tolerance, i.e. a reduced capacity to clear glucose from the blood following a glucose load. As beta cell mass decreases and beta cell secretory capacity further deteriorates, there is a progressive increase in the fasting glucose concentration, eventually culminating in overt hyperglycaemia. Defects in both insulin secretion and action contribute to the pathogenesis of T2D, but it is now acknowledged that T2D is an insulin defi ciency syndrome associated with a progressive reduction in beta cell mass. The loss of beta cell mass in T2D is probably secondary to chronic exposure to high glucose and free fatty acid (FFA) levels (glucolipotoxicity).

In conclusion, a reduction in beta cell mass is a key component of diabetes mellitus and the molecular mechanisms underlying beta cell loss remain to be clariﬁ ed.

Aim

The aim of this proposal is to utilize functional genomics to identify pathways responsible for the reduction of beta cell mass in diabetes, and use this knowledge to defi ne targets for intervention to preserve beta cell mass. The specifi c aims are: • identifi cation of the regulatory molecular pathways that control physiological beta cell mass through regeneration, differentiation and apoptosis; • use of functional genomics to identify key pathophysiological events in the above pathways that are responsible for reduction of beta cell mass in diabetes, with focus on the mechanisms regulating cytokine- and glucolipotoxicity-induced beta cell apoptosis; • intervention in the defective signal transduction pathways identifi ed above using genetically modifi ed mice, long-acting viral vectors and small interfering RNAs. This step should identify and validate targets to preserve beta cell mass in diabetes.

56 | DIABETES & OBESITY Expected results Potential applications

The expected results from this project include: Overall costs of diabetes mellitus, comprising both direct and indirect • the elucidation of key molecular pathways regulating beta cell costs, have been calculated in various countries and are enormous. For mass through regeneration and differentiation and identifi cation instance, the cost of treating one patient over a 25-year period is in the of sites that are dysregulated in T1D and/or T2D; range of 100 000-200 000 EU. These problems are recognised in all • the identifi cation of gene networks regulating cytokine-induced European communities. They pose an increasing challenge to the health beta cell apoptosis through use of functional genomics and care systems that needs to be addressed urgently. Facing this challenge clarifi cation of their role for beta cell loss in diabetes mellitus; will require fundamentally innovative approaches. Against this back- • the identifi cation of molecular pathways and the metabolic defects ground, we propose a strategy to understand the key molecular path- contributing for beta cell damage due to glucolipotoxicity and their ways underlying decreased beta cell mass in both type 1 and type 2 role for beta cell loss in diabetes mellitus; diabetes mellitus. We will employ a multidisciplinary approach, includ- • the identifi cation of genes and proteins that play a critical role in ing functional genomics on both in vivo and in vitro models, with the pathways regulating beta cell mass and which, when activated or aim to identify possible targets for intervention to preserve beta cell inhibited, preserve beta cell mass in diabetes mellitus. mass in diabetes mellitus. Hopefully this will lead to the identifi cation and assessment of novel targets for intervention to preserve beta cell mass in diabetes mellitus, to halt or slow the development of the disease. The subsequent exploitation of these targets for drug development may have a signifi cant impact upon the general health status of the population and a very positive effect on health costs in Europe.

Coordinator Prof. Sigurd Lenzen Institute of Clinical Biochemistry Prof. Decio L. Eizirik Hannover Medical School Laboratory of Hannover, Germany Experimental Medicine Faculty of Medicine Prof. Chantal Mathieu Université Libre de Bruxelles (ULB) Katholieke Universiteit Leuven Brussels, Belgium Leuven, Belgium Phone: +32 2 555 62 42 Fax: +32 2 555 62 39 Dr. Danielle Melloul [email protected] Hadassah University Hospital Jerusalem, Israel

Partners Prof. Timo Otonkoski University of Helsinki Dr. Jolanta Jura Helsinki, Finland Jagiellonian University Krakow, Poland Prof. Guy Rutter Imperial College of Science, Project number Dr. Gavin Kelsey Technology and Medicine LSHM-CT-2006-036903 Laboratory of Developmental Genetics London, United Kingdom EC contribution and Imprinting € 2 750 000 The Babraham Institute Prof. Torben Orntoft Project Duration Cambridge, United Kingdom AROS Applied Technology 36 months ApS Science Park Skejby Starting date Aarhus N, Denmark 01/11/2006 Instrument STREP Project website www.savebeta.eu

DIABETES & OBESITY | 57

59 ➞ 78

RARE DISEASES EURAMY

Systemic Amyloidoses in Europe

Keywords Amyloid, amyloidosis, protein fi bril, aggregation, alternatives for systemic amyloidoses are directed against the production toxicity, degradation of the substrate for the amyloid fi bril protein. There is presently no treatment to the amyloid deposits themselves. The way by which amyloid deposits causes cellular and tissue injury is still not clear but it is evident that the amyloid masses may interfere severely with normal function by mechanical effects on tissues. Recent research indicates that smaller, abnormal protein aggregates, often referred to as oligomers, are toxic Summary to cells in their vicinity. Exactly how the oligomers exert their toxic effects is not well understood. Whether or not toxic effects of oligomeric In systemic amyloidoses deposition of misfolded and aggregated plasma aggregates are important in the genesis of cellular lesions is of great proteins occurs in most organs and tissues and cause serious effects on practical importance for the development of therapeutical strategies of organ function and ultimately death. With the aid of in vitro models, systemic amyloidosis. cellular systems and animal models, the program aims to elucidate how proteins misfold and aggregate to amyloid, how this substance interacts with other molecules and how it causes tissue damage. This knowledge Aim will be used for the development of new diagnostic procedures and updated therapeutic strategies. The aim of this project is to make an effort to elucidate mechanisms as to how and why certain proteins misfold and assemble into amyloid fi brils in systemic forms of the disease and to understand the mechanisms by which fi brils or preamyloid aggregates are deleterious to tissues and organs. Based on clinical biopsies and on cellular and animal models of disease the programme also aims to elucidate which other genes and molecules are interacting with the main fi bril protein in amyloidogenesis and how these interactions lead to the very varying Problem distribution of deposits. The aim is also to use this knowledge to refi ne diagnostic methods and to develop synthetic compound or defi ne The systemic amyloidoses are a heterogeneous group of diseases, all naturally occurring substances that interfere with the assembly proc- characterized by deposition in many tissues and organs of a principally ess, thereby for the fi rst time fi nding direct methods to treat the extracellular, pathological substance, called amyloid. The amyloid is an life-threatening systemic amyloidoses. insoluble, fi brillar form of one out of more than 25 normally soluble autologous proteins. The fi bril protein in the amyloid is more or less misfolded and contains an unusually high degree of beta-sheet, allow- Expected results ing molecules to assemble into long, rigid and resistant fi brils. In systemic amyloidoses, the mother protein is a plasma protein, trans- • Biophysical characterisation of misfolding intermediates and ported as a soluble product that forms fi brils at the deposition sites by oligomers by assessment of amyloidogenic state and mechanism yet not well understood mechanisms. In addition to the major protein of aggregation. fi brils there are in the amyloid deposits always additional components, • Full biochemical characterisation of proteins and peptides, gly- bound to the fi brils. These include amyloid P-component (SAP) and cosaminoglycans and lipids in amyloid fi brils. heparan sulfate proteoglycan. In the systemic amyloidoses, the distribu- • Identifi cation of genes important for hereditary and AA forms of tion of deposits in tissues varies strongly between the different systemic amyloidoses and establishment of a web-based register biochemical forms. The reasons for the variations are unknown as are for amyloidogenic mutations. the mechanisms that determine where amyloid deposits occur. Inter- • Development of transgenic mouse and/or Drosophila models of actions between local tissue factors and the amyloid fi bril proteins have AL, ATTR, AApoAI and ALys amyloidoses and a mouse model for been suggested but no such factors have been identifi ed so far. the study of heparan sulfate in amyloidogenesis. • Development of cell systems for studies of amyloidogenesis in vitro The symptoms and clinical consequences of the systemic amyloidoses and identifi cation of proteases and autoantibodies on formation depend on which tissues are affected. The very pronounced variation in and degradation of amyloid fi brils. amyloid distribution between the amyloid types and also within each form • Further development of diagnostic procedures for systemic amy- in combination with the relative rareness of systemic amyloidosis leads loidoses and of methods to determine amyloid distribution and very frequently to a late diagnosis. This is unfortunate since early treat- effects of treatment. ment is often very important for the outcome. Lethal complications often • Identifi cation and development of drugs and antibodies for treatment depend on renal or cardiac deposits. Presently available therapeutical of systemic amyloidoses in animal models and fi nally in humans.

60 | RARE DISEASES Potential applications Coordinator Prof. Philip Hawkins Royal Free and University College The systemic amyloidoses are rare diseases which often are diagnosed Professor Per Westermark Medical School late, thereby causing unnecessary suffering for the patients and costs Uppsala University National Amyloidosis Centre Dept of Genetics and Pathology London, United Kingdom for the society. One important outcome of the project will be an Rudbeck Laboratory increased awareness of these lethal disorders, allowing earlier diagno- SE 751 85 Uppsala, Sweden Dr. Bouke Hazenberg sis and treatment. Diagnostic procedures are presently insuffi cient, Phone: +46 18 6113849 University of Groningen particularly regarding determination of amyloid distribution in the Fax: +46 18 552739 Groningen, The Netherlands body. Therefore, the methods that will be developed are fi lling a gap. [email protected] Present treatment of systemic amyloidoses is generally directed against Associate Prof. Jin-ping Li the production of the amyloidogenic protein. Understanding of the Uppsala University misfolding process, the assembly of the proteins into fi brils and the Partners Uppsala, Sweden interaction of aggregated proteins with cells will give us new opportunities to develop new therapy, directed against the aggregated protein Prof. Michel Cogné Prof. Erik Lundgren Hôpital Universitaire Umeå University itself. This is presently a fi eld in which the pharmaceutical industry in Lab Immunologie Umeå, Sweden Europe and elsewhere is very interested and in addition to have a great UMR CNRS 6101 potential impact for the suffering group of patients with systemic Limoges, France Prof. Giampaolo Merlini amyloidosis, the program also facilitates interaction with the industry I.R.C.C.S. Policlinico S. Matteo in the European Community. Prof. Ana Margarida Damas Pavia, Italy University of Porto Instituto de Ciencias Biomedicas Prof. Christoph Röcken de Abel Salazar Charité University Hospital Porto, Portugal Berlin, Germany

Prof. Chris Dobson Prof. Maria João Saraiva University of Cambridge University of Porto Cambridge, United Kingdom Porto, Portugal

Prof. Gennaro Esposito Prof. Ole Suhr Università di Udine Umeå University Udine, Italy Umeå, Sweden

Prof. Gilles Grateau Associate Prof. Gunilla Westermark INSERM Linköping University Paris, France Linköping, Sweden

Project number LSHM-CT-2006-037525 EC contribution € 3 054 000 Project Duration 36 months Starting date 01/11/2006 Instrument STREP

RARE DISEASES | 61 EuroCareCF

European Coordination Action for Research in Cystic Fibrosis

Keywords CFTR, cystic fi brosis, CFTR-opathies, lung disease, genetic form of chronic bronchitis, the treatment of respiratory diseases bacterial infections, clinical diagnosis, patient care, clinical affl icting EU citizens (e.g. chronic obstructive pulmonary disease) will also trials, novel therapies, animal models ultimately benefi t from advances in CF research and patient care. In recent years, there has been a surge of advances in both fundamental and clinical CF research. Disappointingly, these advances have not been translated effectively to the clinic nor disseminated widely across Europe. Furthermore, European CF research efforts are fragmented. In the USA, the role of coordinating and fi nancing CF research is principally provided by the private Cystic Fibrosis Foundation (www.cff.org). In Europe there Summary is no equivalent coordinating organisation.

Cystic Fibrosis (CF) is a life-shortening, genetic disease affl icting about 30 000 individuals in the European Union (EU). Mutations in a single gene Aim encoding the cystic fi brosis transmembrane conductance regulator (CFTR) cause CF and a spectrum of related disorders. Despite impressive advances EuroCareCF aims to create a European forum for CF and CF-related in understanding the disease, patient life expectancy and quality of diseases in which all stakeholders will have the opportunity to interact, life remain limited because resources are scattered in Europe. Building exchange and collaborate to achieve common goals. These goals include: on the success of the FP5-funded Cystic Fibrosis Thematic Network • to promote: (www.cfnetwork.be), the three-year Coordination Action, EuroCareCF – optimal standards of care for CF patients in all EU countries, (European Coordination Action for Research in Cystic Fibrosis) will unite CF – the distribution of resources for CF research; research in Europe. EuroCareCF will translate research results into • to produce: optimised clinical management and therapy development to promote good – a uniform registry of European CF patients, standards of care for European CF patients. – consensus guidelines, – patient information; • to evaluate: – the impact of scientifi c research on patient care, – the translation of scientifi c progress to the clinic, – the quality of clinical diagnosis, – the diversity of patient care, – the possible role of industry in CF management/therapy; • to participate in designing applications for scientifi c progress; Problem • to improve: – the survival and quality of life of CF patients, CF is a life-shortening inherited disorder that predominantly affects chil- – the quality of clinical diagnostic procedures, dren and young adults. In the European Union about 30 000 individuals – the quality of care provided to CF patients, live with CF today and there is one new case in approximately every – clinical management of the disease, 2,500-3 000 newborn infants. Life expectancy and quality of life are – therapy development and assessment. limited in CF with progressive lung disease leading to premature death (median survival in Europe is ~27 years). Thus, CF is a serious medical Through its activities, EuroCareCF aims to provide ‘proof of concept’ for issue for Europe. the coordination of European CF research and patient care, setting the stage for future coordination of public and private activities in this area. Although mutations in a single gene encoding CFTR cause CF, the disease is clinically heterogeneous. Moreover, a number of CFTR-associated diseases have been identifi ed (e.g. bilateral absence of the vas defer- Expected results ens). These diseases (termed ‘CFTR-opathies’) are distinct from CF. How- ever, they share clinical features with CF and are usually caused by CFTR To achieve its objectives, EuroCareCF is organised into a series of eight mutations. Identifying CFTR-opathies and distinguishing them from workpackages that will perform tasks leading to a series of specifi c atypical cases of CF is a signifi cant challenge for clinicians. Thus, CF, deliverables and products: a single gene disorder, exemplifi es the complex challenges of genetic • Optimising Patient Care & CF Team Work: diseases in the post-genomic era. Of special note, CF is a paradigm for – recommendations on good standards of care for CF patients, studies of disease pathogenesis, clinical management and therapy – specialist training in CF Team Work, development in other genetic diseases. Because CF is the most common – the establishment of clinical networks for each speciality;

62 | RARE DISEASES • European CF Patient Registry: the creation of a uniform and ethically compliant European registry of CF patients; • Coordination of Clinical Research: – recommendations on clinical diagnostic procedures, clinical trials, the identifi cation of microbiological pathogens, neonatal screening and the classifi cation of CF-related diseases, – specialist training in the diagnosis of CF, – the establishment of a network of reference laboratories to identify microbial pathogens; • Small and Middle-Sized Enterprises (SMEs) Group: – identifi cation of the needs of SMEs and the obstacles that they face to place ‘orphan drugs’ into clinical trials, – the organisation of partnering meetings to promote co-operation between SMEs and academic centres; • Novel Therapies: – recommendations for the transfer of innovative CF General Scheme of EuroCareCF: This Coordination Action consists of a series therapies from research laboratory to clinical trials, of Workpackages designed to integrate fundamental and clinical research with – specialist training in translation research; the goals of (1) improving the survival and quality of life of CF patients and (2) optimising clinical management and therapy development. • Animal Models: – guidelines for standard laboratory techniques using CF mouse models, – specialist training in the use of animal models for CF research, – the distribution of CF mouse models for fundamental and translational research; • Integration of Fundamental Research: – guidelines for good laboratory techniques to evaluate Potential applications novel diagnostics/therapies, – specialist training in laboratory techniques, The potential impact of the EuroCareCF project is wide-ranging: – the distribution of resources (e.g. cell lines, cDNAs and • CF Patients and their Families: the implementation of recommendations antibodies) for fundamental and translational research; for good standards of patient care will have a very positive impact on • Ethical/Legal/Social Issues: CF patients and their families across Europe; – recommendations for the introduction of informed consent • Partner and Collaborator Centres: a key theme of EuroCareCF is to place personal/clinical data in the European registry of the interaction of participating groups. This will automatically lead CF patients, to the wide dissemination of information which will impact upon – review of the ethics and legislation on clinical trials involving the quality of services and/or research undertaken by partners and CF minors. collaborators participating in the EuroCareCF project; • Fundamental and Clinical Research in the CF Field: by fostering inter- The above eight ‘core’ workpackages will be supported by two further actions between fundamental researchers, clinical scientists and workpackages with their own specifi c deliverables and products: SMEs, EuroCareCF will initiate and promote collaborations that will • Telenetworking: facilitate the effective translation of research results from the labora- – the establishment of tele/video-conferencing facilities tory to the clinic. Moreover, resources generated or validated by for regular communication amongst the project partners, EuroCareCF will be made available to partners and collaborators for – the development of a telenetworking environment to their use in CF diagnosis and/or research; interconnect partners and permit the secure exchange • Other Major Respiratory Diseases: by identifying drugs to treat of information in real-time; lung disease in CF, the activities of EuroCareCF will have an impact • General Coordination: on the clinical management of other major respiratory diseases – a EuroCareCF website, (e.g. chronic obstructive pulmonary disease); – quarterly EuroCareCF newsletters, • Economics: through the development of a registry of European – a generic material transfer agreement, CF patients, the overall impact of CF in Europe in terms of preva- – intellectual property rights guidelines for lence, mortality, morbidity and economic costs will be known on novel CF therapies and clinical trials of these agents. a comparative basis among CF centres across Europe;

RARE DISEASES | 63 • National Authorities and the EU: the consensus guidelines produced Coordinator Dr. Dorota Sands by EuroCareCF will be made available to national authorities and to Instytut Matki i Dziecka the EU to advise their decision making. Endorsement of the guidelines Dr. David N. Sheppard Warszawa, Polska by European CF patients associations and by the European CF Society University of Bristol Dept of Physiology Prof. Eitan Kerem will strengthen their impact; and Pharmacology Hadassah University Hospital • The International Community: international quality standards and School of Medical Sciences Jerusalem, Israel procedures are required for CF research and patient care. Data University Walk, GB-BS8 1TD generated by EuroCareCF will be disseminated to the CF community Bristol, United Kingdom Prof. Milan Macek Jr. worldwide to contribute to the development of international Phone: +44 117 331 2290 Charles University Prague standards and procedures; Fax: +44 117 331 2288 Institute of Biology • Science and Society: through the activities of EuroCareCF, the [email protected] and Medical Genetics general public will become better informed about CF. University Hospital Motol and Second School of Medicine Partners Prague, Czech Republic

Prof. Margarida D. Amaral Prof. Dr. Peter A. R. Vandamme University of Lisboa Universiteit Gent Lisboa, Portugal Gent, Belgium

Prof. Carla Colombo Prof. Pier Franco Pignatti University of Milan Università di Verona Milan, Italy Verona, Italy

Dr. Anil Mehta Prof. Frédéric Becq University of Dundee Université de Poitiers Dundee, United Kingdom Poitiers, France

Prof. Dr. Kris De Boeck PD Dr. Robert Bals University Hospital of Leuven Philipps-Universitaet Marburg Leuven, Belgium Marburg, Germany

Dr. Kris Dierickx Dr. Bill Colledge Catholic University of Leuven University of Cambridge Leuven, Belgium Cambridge, United Kingdom

Dr. Andreas Reimann Dr. Marcus Mall Mukoviszidose e.V. University of Heidelberg Bonn, Germany University Hospital Heidelberg, Germany Dr. Massimo Conese San Raffaele Scientifi c Institute Dr. Martin J. Hug Milano, Italy Universitätsklinikums Freiburg Freiburg, Germany Project number Dr. Bob J. Scholte LSHM-CT-2005-018932 Erasmus University Medical Dr. Eva Hladká EC contribution Centre Rotterdam Masarykovy Univerzity € 1 757 500 Rotterdam, The Netherlands Brno, Czech Republic Project Duration Prof. Burkhard Tuemmler 36 months Medizinische Hochschule Hannover Starting date Hannover, Germany 01/01/2006 Instrument CA Project website www.eurocarecf.eu

64 | RARE DISEASES EuroGrow

Pathophysiology of the Cartilage Growth Plate

Keywords Cartilage, chondrodysplasia, gene mutation, mouse Problem model, growth plate, bone All long bones in the skeleton grow by a process known as endochondral ossifi cation, which is a complex procedure in which chondrocytes form a cartilage template known as a growth plate. Growth plate chondrocytes then undergo a coordinated and highly regulated process of cell proliferation, hypertrophy and apoptosis, at which stage vascular Summary invasion brings osteoblasts that begin to replace the cartilage matrix with trabecular bone. Disruptions to this intricate and highly coordinat- The chondrodysplasias are an extremely diverse and complex group of rare ed process results in a group of genetic bone diseases known as the genetic disorders, which affect the development of the skeleton. There are chondrodysplasias. The chondrodysplasias are an extremely diverse and over 200 unique and well-characterised phenotypes, which range in severity complex group of rare disorders, which primarily affect the develop- from relatively mild to severe and lethal forms. They have an overall ment of the osseous skeleton and their aetiology is both diverse and prevalence of at least 4 per 10 000 and this extrapolates to a minimum of complex. Although individually rare, as a group of conditions they have 178 000 people in the 25 EU member states that suffer from an overall birth prevalence of at least 3.9 per 10 000 and to put this in chondrodysplasia. Many of the individual skeletal dysplasia phenotypes context the prevalence of cystic fi brosis is around 4 per 10 000. Based have been grouped into ‘bone dysplasia families’ on the basis of similar on a European population of 4 489 million, there are at least 191 000 clinical and radiographic features and it has been proposed that members individuals in the 27 EU Member States who have a bone dysplasia, and of the same family will share a common disease pathophysiology. Therefore, the clinical complications associated with chondrodysplasia represent as a group of heterogeneous diseases, the chondrodysplasias have a very signifi cant social and economic burden in Europe. a complex aetiology but are likely to share similar basic mechanisms of disease initiation, progression and end-stage pathology. In this context the As a group of heterogeneous diseases the chondrodysplasias have principle objective of this project is to determine the molecular, cell and a complex aetiology, but are likely to share similar basic mechanisms of extracellular matrix pathology of a number of distinct chondrodysplasia disease initiation, progression and end-stage pathology. Over the last phenotypes, which result from mutations in a variety of different gene decade, remarkable progress has been made in identifying the under- products that are important for normal bone development. To achieve this lying genetic defect in many chondrodysplasias, and causative objective we have developed a series of mouse models of chondrodysplasia mutations have now been identifi ed in over 50 different genes. These that closely mimic the relevant human phenotype. We will use genes encode transcription factors, growth factor receptors, structural a multidisciplinary systems biology approach to determine the molecular macro-molecules, enzymes and transporter proteins and molecules mechanisms that underpin the pathophysiology of these distinct with an as yet unknown function. All of these gene products are vital chondrodysplasias. From this approach we can expect to identify common for either the morphogenesis or development of the cartilage anlagen, disease mechanisms and learn general principles about genotype- in coordinating endochondral ossifi cation, the development and main- phenotype correlations in chondrodysplasia phenotypes. These data will tenance of the extracellular matrix or for bone mineralisation. Studying ultimately pave the way for developing common therapeutic strategies that the disease processes in skeletal dysplasias has led to major advances might be targeted to a range of individual phenotypes. in understanding fundamental pathways of bone morphogenesis, development and homeostasis. However, most of these studies have been performed in vitro, and whilst these have proved useful in studying defi ned pathways and in identifying specifi c pathomolecular mechanisms for some chondrodysplasias, for many phenotypes they have failed to establish the link between an abnormal gene product and the resulting growth plate dysplasia. In order to enhance the state-of-the-art and increase our current knowledge of the pathobiology of endochondral ossifi cation we will focus on determining in vivo the pathophysiology of selected chondrodysplasia phenotypes. As a consortium we have recently generated, or obtained via collaboration, over 10 different relevant mouse models of chondrodysplasia, in addition to other specifi c in vivo models that will allow us to also study defi ned aspects of growth plate biology, such as the role of hypoxia and its associated pathology. Preliminary phenotypic and biochemical Transmission electron microscopy and serial section reconstruction of a 4-cell analysis has confi rmed that these models closely mimic the relevant chondron. A) TEM image of a 4-cell chondron from the proliferative zone of human chondrodysplasia and therefore provide a very valuable and a wt growth plate. B) Fully rendered image showing the plasma membrane (red). C) Partial rendering showing nucleus (blue), rER (yellow) and cilia (cyan). novel resource for these studies. In addition, the diverse nature of the

RARE DISEASES | 65 Imunohistochemistry (IHC) of the growth plate of a murine model of pesudoachondroplasia 21 days of age. The growth plate in the mutant mouse (m/m) is disorganised with an enlarged proliferative zone when compared with a wild type littermate (wt/wt). IHC with an anti-COMP antibody showing little or no retention of mutant protein, but with distinct areas of hypocellularity.

mutated or deleted gene products, which include structural proteins, Coordinator Prof. Dr. med. Bernhard Zabel a transporter molecular, growth receptor, molecules implicated in cell sig- Universitaetsklinikum Freiburg nalling and those currently with an unknown function, provide a unique Dr. Michael Briggs Freiburg, Germany opportunity to compare the different molecular mechanisms that can Wellcome Trust Centre for Cell-Matrix Research Prof. Stefan Mundlos result in growth plate pathophysiology. Faculty of Life Sciences Institut für Medizinische Genetik University of Manchester Charité – Universitäts-Medizin Michael Smith Building Manchester Berlin, Germany Aim M13 9PT, United Kingdom Phone: +44 161 275 5642 Prof. John F. Bateman The principle aim of EuroGrow is to determine the molecular mecha- Fax: +44 161 275 5082 Murdoch Children’s nisms that underpin the pathophysiology of a number of distinct [email protected] Research Institute chondrodysplasia phenotypes that result from mutations in different University of Melbourne gene products all of which are important for normal bone development. Melbourne, Australia To achieve this objective we have developed a series of mouse models Partners Prof. Geert Mortier of chondrodysplasia that closely mimic the relevant human phenotype. Dr. Laurence Legeai-Mallet Ghent University Hospital We will now study in-depth the growth plate of these mice to deter- INSERM U393 Gent, Belgium mine the molecular, cell and extracellular matrix pathology of these Paris, France distinct phenotypes. By the end of the project this multidisciplinary Dr. Brian Lings approach will allow us to generate a virtual representation of a cartilage Dr. Antonio Rossi Certus Technology Associates growth plate in health and disease and pave the way for developing University of Pavia Exeter, United Kingdom therapeutic strategies that might be targeted to a range of individual Pavia, Italy phenotypes. Prof. Dr. Erwin R. Schmidt Prof. Judith Goodship Institut für Molekulargenetik Institute of Human Genetics Johannes Gutenberg-Universität Expected results Newcastle upon Tyne, United Kingdom Mainz, Germany Dr. Victor Luis Ruiz Perez • To comprehensively deﬁ ne the clinical and radiographic phenotype Centro de Investigaciones Biologicas of the mouse models during skeletal development. Madrid, Spain • To generate a visual interpretation of the different changes in growth plate morphology resulting from a range of diverse disease- causing mutations. • To determine the changes in the spatio-temporal location of proteins within the extracellular matrix and the effect on cell phenotype and function of disease-causing mutations. • To determine how speciﬁ c disease-causing mutations affect chondro- cyte gene expression and the resulting protein composition (proteome) of the growth plate. • To compare molecular mechanisms between the different mouse models to identify common basic mechanisms and learn general principles about genotype-phenotype correlations. Project number LSHM-CT-2005-018932 Potential applications EC contribution € 1 757 500 This multidisciplinary research project will allow us generate a virtual Project Duration representation of a cartilage growth plate in health and disease. More 36 months importantly it will allow us to identify the disease mechanisms which are Starting date a cause of reduced bone growth in skeletal dysplasias and to determine 01/01/2006 if there are any common diseases mechanisms. Ultimately this knowledge Instrument will pave the way for the development of targeted therapies that may CA be applicable to a range of different phenotypes. Project website www.eurocarecf.eu

66 | RARE DISEASES EURO-Laminopathies

Nuclear Envelope-linked Rare Human Diseases: from Molecular Pathophysiology towards Clinical Applications

Keywords Adult stem cells, animal disease models, Problem cell cycle, cell differentiation, chromatin organization, drug screening, drug targets, mutations, nuclear architecture, Lamins are major architectural proteins in the nuclei of eukaryotic cells. nuclear lamina, lamins, structural biology, tissue regeneration Mutations in one type of lamins that are preferentially expressed in differentiated cells cause a variety of disease phenotypes, collectively called laminopathies. These diseases can affect muscle, adipose, nerve, bone, and skin tissues or cause premature ageing. Based on known and proposed functions of lamins, various disease hypotheses have been proposed to explain the molecular basis of laminopathies, but it remains unclear how much a particular disease mechanism can contribute to Summary a given clinical phenotype.

Laminopathies are rare human diseases linked to mutations in genes The mechanical hypothesis predicts that mutations in lamins and lamin- encoding nuclear envelope proteins, such as A-type lamins (LMNA) and binding proteins weaken lamin structure and stability, either by inter- lamin-binding proteins (EMD, LBR, LAP2). Laminopathies are clinically fering with proper folding of the proteins or by affecting assembly of manifested after birth, can afffect different tissues and progressively lamin complexes, all predisposing cells and tissues to physical damage. develop during childhood or adolescence, often leading to early death. This model seems reasonable, as lamins provid mechanical stability to Effi cient therapies have been hampered by the lack of understanding the the nucleus, and patient cells often reveal structural abnormalities of molecular disease mechanisms. the nucleus. Interestingly, fi broblasts from Lmna-/- mice, which repre- Using a variety of clinical and basic research approaches the EURO- sent an animal model for some laminopathies, possess less resistance Laminopathies team analyses how disease-causing mutations in A-type to deformation and compression forces and an attenuated, mechanical lamins or in one of their prominent binding partners LAP2alpha affect stress-induced NF-kB signalling. However, the structural disease model atomic structure, interactions, and assembly properties of lamins. cannot explain all clinical pathologies, as unaffected tissues also show By means of patient cells and animal models the consortium tests how deformed nuclei. mutations affect chromatin organization, gene expression, and differentiation of adult muscle- and adipose stem cells. Data obtained in these systems The gene expression hypothesis proposes that mutations in lamins dis- as well as chemical compound screening using the zebrafi sh model system rupt interactions of lamins with transcriptional regulators, such as the will identify novel drug targets and drugs for potential therapeutic adipocyte-specifi c transcription factor SREBP1, and affect tissue-specifi c intervention. Furthermore, the consortium extends and evaluates clinical patterns of gene expression. Furthermore, lamins are also involved in trials on the treatment of lipodystrophy-type laminopathy patients and epigenetic mechanisms regulating heterochromatin formation through develops theranostic tests for the validation of therapies. numerous interactions of lamin complexes with DNA and chromatin proteins. Mutations in A-type lamins may cause gross changes in higher order chromatin structure and gene expression. In line with this hypothesis nuclei from patient cells often lack the peripheral heterochromatin. The cell proliferation/differentiation hypothesis is mainly based on the in vivo interaction between lamins A/C and LAP2a and the tumour suppressor retinoblastoma protein (pRb), which is required for muscle and adipocyte differentiation. Expression of a disease lamin A mutant inhibited myoblast differentiation in vitro, probably by destabilizing pRb. It has been suggested that stem cells in laminopathy patients have a defective differentiation potential and cannot effectively regenerate tissues.

Aim

The EURO-Laminopathies team focuses on three principal aims: • understanding the basic functions of lamins at the molecular level and their contributions to the different types of laminopathies; • increasing patients’ quality of life by opening new avenues of therapeutic approaches; • attracting health professionals’ attention to these rare diseases in order to improve diagnosis and patient treatment.

RARE DISEASES | 67 Immunofl uorescence micrograph of an in vitro differentiated muscle fi ber, depicting the lamin A binding protein LAP2a in red and the muscle specifi c protein Myosin Heavy Chain in green.

Expected results

• Identifi cation of new mutations in LMNA, FACE-1, LAP2 and in novel laminopathy-linked genes, and new laminopathy-like dis- Electron microscopic images of fi broblasts isolated from orders and novel biochemical pathways potentially involved in control persons (panel A) and from Hutchinson Gilford laminopathies. Progeria patients (panels B and C). Patient cells in panel C were treated with farnesyl transferase inhibitors, • Establishment of a structural model for the ‘head-to-tail’ lamin a potential drug for therapeutic treatment of patients. assembly based on atomic structures of domains of lamin, the Note that the nucleus in patient cells has an abnormal structural characterization of the interaction of lamins A/C with distorted shape and heterochromatin inside the nucleus (dark regions at periphery) is lost. These phenotypes are LAP2a and the identifi cation of the structural/functional conse- partially rescued upon drug treatment. quences of disease-related mutations in lamins A/C and LAP2a. • Description of the effects of lamins A/C-binding proteins on lamin assembly, characterization of effects of disease-relevant mutated residues on lamin assembly and characterization of the effects of disease-relevant mutated residues on lamins A/C:LAP2a:pRb To reach these aims the interdisciplinary team of human geneticists, interactions. clinical researchers, structural biologists, molecular cell biologists, and • Identifi cation of the phenotype of knock-in, knock-out, and trans- industrial partners will: genic disease mouse models and mutant C. elegans strains, and • identify and/or analyse molecular functions of lamina proteins in evaluation how phenotypes correlate with the pathophysiology of nuclear architecture, chromatin organization, gene expression, cell patients. cycle regulation, differentiation, and aging; • Evaluation of the effi ciency of muscle regeneration in animal dis- • test how laminopathy-causing mutations in lamins affect these ease models, and identifi cation of molecular defects in the control functions; of the cell cycle, differentiation and aging in laminopathic cells and • unravel, how mutation-linked defects cause laminopathy pheno- mutant C. elegans strains. types and pathologies in cells and tissues of animal models and • Description of lamin complex stability and analyses of defects in patients; chromatin organization and dynamics in laminopathic human and • identify molecular targets and pharmaceutical tools for therapeutic mouse cells and in mutated disease relevant C.elegans strains. intervention in laminopathy patients; • Evaluation of the therapeutic effects of PPARg activators and adi- • develop clinical protocols for the treatment of patients with pocytokines in lipodystrophies, and identifi cation of novel drug laminopathies and in particular in lipodystrophy syndromes and targets and drugs in a zebrafi sh-based screening, to be tested in identify novel drugs to be tested in animal models and patients. animal models and patients.

68 | RARE DISEASES 100 nm

Electron microscopic image of in vitro reconstituted lamin ﬁ laments.

Potential applications adipocyte-specifi c transcription factor PPAR-g. Clinical researchers within EURO-Laminopathies will extend these studies and develop theranostic The EURO-Laminopathies network project gathers key scientists in the tools with an industrial partner to evaluate the effi ciency of these thera- relevant research fi elds. Many of the expected results may be considered pies. This will allow the optimization of existing therapies and the as signifi cant conceptual advances in understanding lamin function and reduction of side effects and risks for patients. Furthermore the screening atomic structure. Furthermore, numerous tools and animal models will of a compound library in the zebrafi sh system and the identifi cation of be generated that will be valuable assets for future research in these novel pathways, in which lamins are involved, will lead to the identifi ca- areas by scientists world-wide. tion of novel potential drug targets and drugs, which will set the basis for the development of effi cient therapies also for other laminopathies. The human geneticists collect genetic and clinical data of patients and extend the Universal Mutation Database (see http://www.umd.be:2000/ The concept of EURO-Laminopathies spans the bridge from basic _top ), which is unique in Europe. This will strengthen and further devel- science-oriented analyses of molecular disease mechanisms towards op the role of the French partners as a European reference centre for applied research identifying novel drug targets and drugs, and testing genetic diagnosis of laminopathies. The clinical researchers of the con- therapeutic interventions. Therefore, the project is also a great momen- sortium are in contact with patients and their families as well as tum for research into the rapidly growing group of laminopathy disorders physicians throughout Europe. The developed clinical protocols for diag- on the European level, forming a critical mass of knowledge and creating nosis and treatment of specifi c patient groups, and cost-effective interdisciplinarity. This will strengthen the competitiveness of European methodologies for identifi cation of inherited genetic diseases and evalu- science in this fi eld. Newly generated knowledge on laminopathies may ation of treatments will be offered to physicians on the project website. also move into mainstream health care areas, such as cardiovascular, This will help diagnosing laminopathies at early disease stages and thus neurological and metabolic diseases. increase the quality of patients’ life.

Speciﬁ c targeting of molecular defects in laminopathies in therapeutic approaches has not been described so far. Treatment was mostly restricted to implantation of a pacemaker to prevent sudden cardiac death in lami- Coordinator Dr. Harald Herrmann nopathy patients with muscle pathologies. Only recently, trials have been Deutsches Krebsforschungszentrum started to treat fat loss in lipodystrophy patients with drugs affecting the Prof. Roland Foisner Heidelberg, Germany Medizinische Universität Wien Dept of Medical Biochemistry Prof. Christopher John Hutchison Dr. Bohrgasse 9/3 University of Durham 1030 Vienna, Austria Durham, United Kingdom Phone: +43 1 4277 61680 Fax: +43 1 4277 9616 Prof. Nadir Mario Maraldi [email protected] Istituti Ortopedici Rizzoli Bologna, Italy

Partners Prof. Giuseppe Novelli Universita’ degli Studi Prof. Ueli Aebi die Roma Tor Vergata University of Basel Rome, Italy Basel, Switzerland Project number Prof. Brigitte Rohner LSHM-CT-2005-018690 Dr. Gisèle Bonne Brigitte Rohner punkt EC contribution Institut National de la Santé Vienna, Austria € 2 565 000 et de la Recherche Médicale Project Duration U 582, Institut de Myologie Dr. Beatriz Romero 36 months G.H. Pitié-Salpêtrière ZF Biolabs S.L. Starting date Paris, France Tres Cantos, Spain 01/02/2006 Instrument Prof. Yosef Gruenbaum Dr. Elisa Gargiullo The Hebrew University of Jerusalem DIATHEVA s.r.l. STREP Jerusalem, Israel Fano, Italy Project website www.project.mfpl.ac.at/ euro-laminopathies

RARE DISEASES | 69 HUE-MAN

Towards the development of an effective Enzyme Replacement Therapy (ERT) for human α-Mannosidosis

Keywords lysosomal storage disease, α-Mannosidosis, ERT, Problem Gaucher, Fabry Human α-Mannosidosis is a genetic disorder, caused by the lack of the lysosomal enzyme α-Mannosidase. The lack of α-Mannosidase causes a disorder of glycoprotein catabolism associated with abnormal levels in different tissues and excretion of small mannose-rich oligosaccharides. These biochemical alterations are secondary to a defi ciency of the lysosomal enzyme, α-Mannosidase. Lysosomal α-Mannosidosis (LAMAN) Summary is an enzyme that cleaves α-mannosidic linkages during the ordered degradation of oligosaccharides. Only after degradation, the sugars can Lysosomal storage disorders (LSDs) represent a large group of rare genetic leave the lysosomes. The defi ciency in Mannosidase activity causes a block metabolic diseases characterized by progressive course, manifestation of in the degradation of glycoproteins. This results in lysosomal accumulation several organ systems and variable phenotypic expression. The defect of mannose-rich oligosaccharide chains in all tissues. Consequently, these involves the lysosomal system of the cell which is responsible for the sugars are accumulated in the lysosomes. Finally, the lysosomes increase degradation of macromolecules and exogenous compounds by activity of in size, producing huge vacuoles and an impaired cell function is induced. lysosomal acid hydrolases. A failure in the system caused by a defi ciency The progressive accumulation of oligosaccharides is somehow toxic to the of one or more lysosomal enzymes leads to a gradual accumulation cells, some types of cells are more sensitive than others. of undegraded metabolites, such as lipids, oligosaccharides, and mucopolysaccharides in all cells and tissues of the body. The overall Children are often born apparently normal, and their conditions develop prevalence of lysosomal storage diseases is estimated at 11-15 per progressively, without any possibility to prevent this evolution. The clinical 100 000 in the general population. In the last ten years enzyme replacement fi ndings in α-Mannosidosis include a broad range of symptoms. From an therapy (ERT) has been evolved as the therapy of choice for several LSDs. early lethal form to less symptomatic forms initially diagnosed in children The enzyme lacking in the patient is introduced into the blood stream, from α-Mannosidosis always seems to have its onset before the age of two where it is internalised by the cells and reaches the lysosomes, acting as years, probably at birth. It is frequently associated with corneal opacities, the endogeneous enzyme. hearing loss, aseptic destructive arthritis, metabolic myopathy and immune α-Mannosidosis is a rare inborn disorder caused by the lack of the lysosomal defi ciency. α-Mannosidosis is classifi ed into two forms. A more severe enzyme α-Mannosidase (LAMAM) resulting in mental retardation, skeletal infantile (type 1) phenotype which includes rapid, progressive mental retar- changes, hearing loss and recurrent infections. The children are often born dation; hepatosplenomegaly; severe dysostosis multiplex; and often death apparently normal, and their conditions worsen progressively, without any between 3 and 12 years of age. The juvenile-adult phenotype (type 2), possibility to prevent this evolution. In children, who are born healthy, which is characterized by a milder and more slowly progressive course with a therapy initiated at an early age could contribute to a normal development. survival into adulthood. The distinctions are not absolute, and symptoms In pilot studies which were also supported by the FP5 (EURAMAN project) and lethality may vary. In the children that are born healthy, an enzyme the proof of principle of an enzyme replacement therapy for a mouse replacement therapy initiated at an early age might contribute to normal model of α-Mannosidase was demonstrated. A correction of storage in development. many tissues including brain was found after administration of lysosomal acid α-Mannosidosis from bovine kidney, and human and mouse To obtain a more accessible model for the study of therapeutic modalities recombinant LAMAN. The main objective of the HUE-MAN project is to in α-Mannosidosis a mouse model for α-Mannosidosis was generated by transfer and expand the knowledge obtained from the EURAMAN project disrupting the gene for LAMAN. The morphological and biochemical alter- studies to investigate and establish clinical parameters in the mouse ations in mouse α-Mannosidosis closely resemble those reported in human model and a natural history study of the human disease in order to defi ne α-Mannosidosis and the phenotype corresponds to a mild form of the clinical endpoints for the future clinical trials in α-Mannosidase. human disease. Using an enzyme replacement therapy a correction of stor- Furthermore, in parallel, HUE-MAN will establish conditions for the age in a mouse model of α-Mannosidosis after intravenous administration production of recombinant human (rh) LAMAN that can pave the way for of lysosomal acid α-Mannosidosis from bovine kidney, and human and a fi rst clinical trial in man. mouse recombinant LAMAN was observed.

70 | RARE DISEASES Aim Assessment of genotype/phenotype relationships and collection of patient data. The project has been divided into fi ve main objectives: • Construction and analysis of a mutation database that will aid • to explore the effi cacy of enzyme replacement therapy in the to understand the natural history of α-Mannosidosis (mutation mouse model of alpha-mannosidosis; database). • to systematically determine the natural history of patients suffering from human alpha mannosidosis; Production of human recombinant LAMAN to provide the enzyme • to fi nd new diagnostic parameters which are suitable for analysis needed for the studies using the mouse model. of a possible benefi t of LAMAN ERT-treatment; • Production and improvement of enzyme production by development • to systematically defi ne genotype/phenotype relationships and to of effi cient fermentation and purifi cation protocols. centrally collect patient data; • to establish the production of human recombinant LAMAN to provide the enzyme needed for the studies using the mouse model.

Expected results A Increased knowledge about the effi cacy of enzyme replacement therapy in the mouse model of α-Mannosidosis. • Development of an effective long term ERT protocol by using different doses of rhLAMAM and administration of the drug at different intervals. • Biochemical,immunological and histological analysis of ERT-treated mice by the determination of activity, storage and immunological response after ERT of the mouse model. • Behavioral analysis of α-Mannosidase defi cient mice with and without ERT. • Transcriptome profi le of the mouse model with and without ERT. B Complete genome-wide profi ling, determination of epigenetic effects of ERT in the mouse model and biochemical and cell biological evaluation.

Systematic determination of the natural history of patients suffering from human α-Mannosidosis. • A natural history study in patients with α-Mannosidosis will be performed with the objective to determine the natural cause of α-Mannosidosis in order to defi ne the baseline for effi cacy assessment of potential therapeutics (collection of medical data Disappearance of storage vacuoles in brain and statistical report). after intravenous administration of recombinant human (rh) LAMAN in an a-Mannosidosis mouse model. (A) Microscopical brain section Determination of new diagnostic parameters which are suitable for of an a-Mannosidosis mouse not treated shows analysis of a possible benefi t of LAMAN ERT. numberous neurons with lysosomal storage; (B) Enzyme Replacement Therapy of the mouse • Development of mass spectrometry analysis for the quantifi cation model leads to a considerable reduction of and fi ne structure determination of oligomannosides in patients’ storage lysosomes within the brain; arrowheads serum and urine (establishment of methology). point to neurons with lysosomal storage.

RARE DISEASES | 71 Typical lysosomal storage in testis of an a-Mannosidosis mouse model. The abnormal swollen lysosomes contains typical storage vacuoles. The pathological storage, which is also observed in many other tissues leads to defects in the immune system, bone and mental development.

Potential applications Coordinator Torben Lübke Georg-August-University The ultimate impact of the proposal is in providing the basis for Prof. Dr. Paul Saftig Göttingen, Germany therapeutic strategy to treat the rare disorder alpha-mannosidosis. The Institute of Biochemistry Christian –Albrechts-University Kiel Ed Wraith principle feasibility of ERT in the mouse model of alpha-mannosidosis Eduard Buchner Haus, Central Manchester Manchester has been shown. In the treated mice corrections of lysosomal storage in Otto Hahn Platz 9 Children’s University Hospitals NHS viceral organs but also -most importantly- brain has been demonstrated 24118 Kiel, Germany Trust, United Kingdom making the introduction of ERT for human alpha mannosidosis into Phone: +49 431 8802216 clinical trials a very promising project. HUE-MAN has been and will be of Fax: +49 431 8802238 Michael Beck substantial importance to establish preclinical and clinical therapy [email protected] Johannes-Gutenberg-University protocols, to set out the conditions for a large scale and toxicity Mainz, Germany monitored drug production, to better understand and deﬁ ne the typical hallmarks of this disease and to evaluate epigenetic factors inﬂ uencing Partners Jiri Zeman the disease progression. All these goals of HUE-MAN will add new Charles University Prag Jens Fogh Prag, Czech Republic important informations which will pave the way to realise a causative Christian Friis cure of this still untreatable disease. Zymenex Oivind Nilssen Copenhagen, Denmark Dag Malm The major technological prospect of this research would be the achieve- University Tromsoe ment of a medicament for curing alpha-mannosidosis. This research is Rudi D’Hooge Tromsoe, Norway intended to decrease the economical burden, and to improve the pros- Katholieke Universiteit Leuven pects of a new medicament, to the point where there is an incentive Leuven, Belgium Jean-Claude Michalski among biotechnological companies to produce this drug. Centre National de la Recherche Scientifi que Lille, France

Project number LSHM-CT-2006-018692 EC contribution € 2 374 854 Project Duration 36 months Starting date 01/04/2006 Instrument STREP Project website www.biochem.uni-kiel.de/hue-man/

72 | RARE DISEASES MYASTAID

Development of models to improve management of Myasthenia Gravis: from basic knowledge to clinical application

Keywords Myasthenia gravis, rare disease, chemokines, Problem hormones, transcriptome, thymus, muscle, diagnosis, anti-TCR antibodies, therapy Acquired Myasthenia Gravis is a rare autoimmune disease with a prevalence around 0.1/2000. This disease is due to autoantibodies directed against components of the neuromuscular junction and leading to disabling fatigability of muscles. MG is mainly (85 % of the patients) caused by anti-acetylcholine receptor (AChR) autoantibodies and in some patients (about 5 %) by autoantibodies against a muscle-specifi c receptor tyrosine kinase (MuSK). The remaining population for which the Summary autoimmune target is not yet identifi ed is defi ned as double negative MG patients. Autoimmune Myasthenia Gravis (MG) is a rare, chronic and heterogeneous neuromuscular disease characterised by severe muscle weakness. In If the muscle is the target organ in this disease, the thymus is clearly most patients, it is due to auto-antibodies to the acetylcholine receptor involved in the pathogenesis of MG with anti-AChR antibodies. Indeed, (AChR). The detrimental effects of the autoimmune attack on the muscle 50-60 % of the patients present a thymic hyperplasia with germinal cen- are not fully understood. The current treatments of MG induce severe tres and around 20 % of the patients have a thymoma. The titre of side-effects with no permanent clinical remission. The early-onset anti-AChR is not correlated with the severity of the disease but is associ- patients are mostly females with thymic hyperplasia, while the existing ated with thymic abnormalities. The hyperplastic thymus includes all the experimental model for MG (EAMG) does not present thymic pathology. components of the anti-AChR response: the autoantigen (AChR), B cells This project intends to develop models to progress in knowledge, producing anti-AChR antibodies and anti-AChR autoreactive T cells. The monitoring, diagnosis and therapy of MG: thymus is thus clearly involved in the development of MG and thymec- • videomicroscopic models to explore the motility of lymphocytes tomy is often advised for MG patients improving slowly but effi ciently involved in thymic remodelling in young females; over a few years their symptoms. The other pharmacological treatments • new transgenic models overexpressing the CXCL13 chemokine in proposed are: the thymus, to better mimic the human disease which involves • symptomatic drugs such as cholinesterase inhibitors preventing the thymic abnormalities and increased thymic CXCL13 expression; degradation of acetylcholine; • mouse model of estrogen defi ciency, to determine the infl uence of • glucocorticoids and immunosuppressor drugs which are used for estrogens in predisposition and progression of MG; many autoimmune diseases and act in a global way on the immune • humanized NOD/SCID mouse model transferred with human MG response; thymocytes to test therapy by human regulatory T cells; • plasmapheresis or intravenous human immunoglobulins in crisis • T cell receptor-based signature, for better classification and period. monitoring of MG patients; • rat EAMG models immunized with Torpedo AChR, to test an array of Although progress has been made in developing therapies for MG, this therapies (IVIg subfractions, chemokine inhibitors, regulatory cells) disease is still incapacitating and treatments are not satisfactory. Many • rat models immunized with recombinant human AChR subunits, to questions remain unanswered: why is there no correlation between the determine the contribution of each subunit to the pathogenicity and anti-AChR antibody titer and the disease severity? Which are the other develop immunotherapies; autoantigens? Why is the thymus pathologic in most cases? Why are • monkey models to test protective anti-AChR antibodies. females predominant in the early onset disease? What occurs in the Since the research teams involved in this program also participate in the muscle after the autoimmune attack? and fi nally – how to improve the large ‘European Myasthenia Gravis Network’ supported though the EU therapy of MG? Public Health Programme 2003-2008, interactions between the two networks will promote effi cient dissemination of the obtained knowledge.

RARE DISEASES | 73 Aim Expected results

Myasthenia gravis is a well-defi ned antibody-mediated disease, but the • Biophysical characterisation of misfolding intermediates and oli- pathogenic mechanisms at the effector organ (the thymus) and at the gomers by assessment of amyloidogenic state and mechanism of target organ (the muscle) are still puzzling. The aims are to improve aggregation. knowledge, diagnosis, monitoring and management of MG patients. The • Full biochemical characterisation of proteins and peptides, gly- main scientifi c and technological objectives of the project are as follows. cosaminoglycans and lipids in amyloid fi brils. • Identifi cation of genes important for hereditary and AA forms of To improve knowledge on the mechanisms of pathogenicity occurring in systemic amyloidoses and establishment of a web-based register myasthenia gravis. for amyloidogenic mutations. • Mechanisms of thymus remodelling in young patients. This topic • Development of transgenic mouse and/or Drosophila models of will be addressed by a genomic approach using cDNA microarrays AL, ATTR, AApoAI and ALys amyloidoses and a mouse model for and a post-genomic approach exploring the role of chemokines the study of heparan sulfate in amyloidogenesis. previously identifi ed as key molecules in the development of the • Development of cell systems for studies of amyloidogenesis in vitro disease. New transgenic models overexpressing CXCL13 in the and identifi cation of proteases and autoantibodies on formation thymus will be established to investigate whether germinal centres and degradation of amyloid fi brils. will be generated after inducing the disease in this animal model. • Further development of diagnostic procedures for systemic amy- • Determination of the consequences of the autoimmune attack on loidoses and of methods to determine amyloid distribution and the muscle organization, gene expression and function. effects of treatment. – The transcriptome analysis of muscle biopsies should identify • Identifi cation and development of drugs and antibodies for treatment a specifi c signature for the different subgroups of patients: of systemic amyloidoses in animal models and fi nally in humans. patients with anti-AChR antibodies or with anti-Musk antibodies, and double negative patients. – Identifi cation of new genes and proteins deregulated as Potential applications a result of the autoimmune attack by analysis of muscle transcriptome in MG patients, rat EAMG, and in vitro model. This project aims to improve diagnosis and monitoring. The collaboration – Investigating the specifi city and pathogenicity of each subunit with 6 industrial partners included in this project will help developing domain. New in vivo models will be created by immunizing the following clinical applications: the rodents with each subunit ectodomain as well as their • a new, easier diagnostic assay for anti-AChR antibody titers is combination to evaluate the pathogenicity of each subunit. expected to be developed; • Understanding the female predominance in the early onset disease • a biological diagnosis is still expected for double seronegative MG by analysing the transcriptome of the human thymus in young patients. The proposed research fi rst aims at identifying the normal males and females and by exploring the infl uence of sexual autoantigen. The use of the technology developed by one of our hormones on the susceptibility of MG disease in animal models. industrial partner to analyse the T cell repertoire in MG patients will provide a pioneering work as an example for studies in other diseases, To improve diagnosis and monitoring of Myasthenias therefore increasing the existing market for immune signatures. This • By developing a new, easier diagnostic assay and a monitoring technology is based on a genomic approach, exploiting sequences of assay of the autoimmune and regulatory status. the entire TCR locus; • By searching for the autoantigen in the anti-MuSK and anti-AChR • the development of a new tool named antiTracKeR, to analyse the negative patients by using proteomics approaches. anti-TCR repertoire; • By analysing the complete repertoire of the T cells and the diversity • the arrays of therapies to be used in animal models and particularly of the anti-T cell receptor (TCR) antibodies. in the humanized mice model should result in novel therapeutic approaches. To improve therapy for MG using several approaches, all of which are based on modulation of the adverse autoimmune response and/or activation of the muscle response to the autoimmune attack in animal models.

74 | RARE DISEASES Thymic Hyperplasia in early onset myasthenia gravis patient.The staining in red (anti-keratin) represents the epithelial network all over the thymus, and the staining in green (anti-CD21) labels B cells and follicular dendritic cells visualizing the germinal centers. The picture was obtained by an original technique using a scanner for microarray.

Coordinator

Sonia Berrih-Aknin Université Paris Sud (UPS) Le Plessis Robinson, France [email protected]

Partners

Socrates Tzartos Hellenic Pasteur Institute Athens, Greece

Marc De Baets University of Maastricht Maastricht, The Netherlands

Sara Fuchs Miry Souroujon Tsvee Lapidot Weizmann Institute of Sciences Rehovot, Israel

Amnon Peled Biokine Therapeutics Ltd Jerusalem, Israel

Eugene Bosmans Epsilon Biotech Antwerp, Belgium

Paul Parren Genmab B.V. Utrecht, The Netherlands

Nicolas Pasqual ImmunId Technologies Grenoble, France

Orgad Laub OMRIX biopharmaceuticals Nes-Ziona, Israel

Project number Michael Roberts LSHM-CT-2006-037833 Regulon EC contribution Athens, Greece € 2 700 000 Project Duration 36 months Starting date 01/10/2006 Instrument STREP-SME Project website www.euromyasthenia.eu

RARE DISEASES | 75 TREAT-NMD

Translational Research in Europe Assessment and Treatment of Neuromuscular Diseases

Keywords Neuromuscular diseases, molecular genetics, Problem translational research, network of excellence, Duchenne muscular dystrophy, spinal muscular atrophy Inherited neuromuscular diseases (NMD) form a large group of diseases, each of which is individually rare (prevalence < 5/10 000). They are present in all populations and affect both sexes and children and adults. Most NMD result in chronic long-term disability posing a signifi cant health care burden for society. Death may result from cardiac and respiratory muscle involvement. The goal of existing management is to minimise the impact of complications such as joint or spinal Summary deformity and improve cardiac and respiratory function as there are currently no curative treatments for any NMD. Consequently this is an TREAT-NMD is a European neuromuscular network addressing the area where there has been a relative dearth of clinical trials and little fragmentation currently hindering translational research for cutting-edge concerted activity for the development of standards of care or vali- therapies in rare neuromuscular diseases (NMD). TREAT-NMD will address dated outcome measures for assessment of effi cacy. At the same time, this fragmentation by establishing a common road map for the progression knowledge of disease-causing genes has begun to allow the elucida- of cutting-edge therapies from laboratory to clinic, from the assessment of tion of the molecular pathological mechanisms underlying NMD, cellular and animal models, via issues of delivery, production and toxicology, leading to plans for specifi c gene based therapies or targeted pharma- to clinical outcome measures. The TREAT-NMD coordination centre (TNCC) ceutical approaches. Some of these treatment options are beginning will develop and integrate organisations and networks comprising the top to move to limited human studies. Examples include antisense oligo- researchers, clinicians and industries working in Europe (representing nucleotide treatment for Duchenne Muscular Dystrophy (DMD), 11 countries) in partnership with patient organisations in order to deliver myostatin inhibition in a range of muscular dystrophies, gene therapy the dream of treatments for these devastating disorders. approaches to DMD and pharmacological approaches to survival motor neuron gene (SMN) upregulation in Spinal Muscular Atrophy (SMA). These developments, while universally welcomed amongst scientists, clinicians and patient organisations, have exposed the lack of harmonisation of approaches to possibly benefi cial therapeutics in NMD which is hindering a smooth move into clinical trials. TREAT-NMD is a European neuromuscular network initiative addressing the fragmentation currently hindering translational research for cutting-edge therapies in rare NMD.

Aim

TREAT-NMD’s ultimate goal is to accelerate the progress of the development of curative treatments for patients with neuromuscular diseases. To reach this goal the network will address the fragmentation currently hindering translational research for cutting-edge therapies in rare NMD. It will aim to strengthen European excellence in the assessment and treatment of rare NMD. TREAT-NMD will be committed to delivering innovative treatments for rare neuromuscular diseases, starting with muscular dystrophies and spinal muscular atrophy, from laboratory development to clinical practice via the generation of a durable network coordination centre (TREAT-NMD Co-ordination Centre, TNCC). The deliverables of TREAT-NMD will include the development of common protocols for animal assessment, the delivery of compounds to muscle and production and toxicology of novel agents. Preparing the patient population for trials will involve the development of integrated databases and biobanks, standardised protocols for diagnosis and standards of care and assessment methodologies. The network will be supported by a programme of education and outreach.

76 | RARE DISEASES Towards integrating research and creating a TREAT-NMD Coordination Centre.

Expected results • The development of a training, exchange and mobility programme to educate clinicians, researchers and students in all these previously • The establishment of a network Co-ordination Centre (TREAT-NMD listed processes, thereby ensuring a long-term leading position for Co-ordination Centre, TNCC) and communication platform, within European research in this fi eld. which International Centres of Excellence for research and • The extension of the network to Eastern European countries. management of NMD will operate. The TNCC will be a permanent and self-sustaining structure, which will ensure the long-term utility of the Network’s research and clinical outcomes and continue to Potential applications apply the tools and methods developed into future cutting-edge therapeutics for NMD as a whole. The objective of TREAT-NMD was set to accelerate the pathway of promis- • The development of Biobanks and patients databases as an unifi ed ing treatments to the clinic. To achieve this goal, the Network brings resource for the defi nition of signifi cant and well-defi ned patients together the best basic researchers, clinicians, involved industries, regula- cohorts and biological materials. This will represent a durable tory agents and patient organisations in the NMD fi eld. TREAT-NMD part- resource for future clinical trials. ners and participants will tackle all fragmented areas preventing the • The implementation of a Clinical Trial Co-ordination Centre (CTCC) patients from rapidly accessing to relevant therapies (pharmacological, which will provide advice on issues of protocol design, regulatory cell and gene therapies) through strong integration of materials, tech- and statistical support, liaison with orphan drugs authorities, and nologies and resources. It includes work on the standardisation of assess- EU clinical trials authority. ment of animal and cell models, the harmonisation of production, • The development of standardised assessment methodologies and toxicology and systemic delivery methods, the assessment of patients in screening techniques in different selected animal models and cell clinical trials, databases, databases and biobanks and standards of care. systems relevant to neuromuscular disorders. The initial emphasis of the NoE will be on the muscular dystrophies and • The development of a large scale support for production of clinical SMA, but the common tools developed will enable extension of the net- grade cutting-edge therapeutic materials for clinical trials in NMD work to other conditions as the network proceeds. TREAT-NMD will pave including toxicology, safety assessment and the development and the way to a new era of international neuromuscular basic and clinical evaluation of methods for their systemic delivery. research collaboration to accelerate the pathway of the NMD cutting- • The defi nition of outcome measures for the assessment of muscle edge therapies to the clinic. Finally, TREAT-NMD will develop training pro- morphology (imaging), strength, function, quality of life measures, grammes based on the outcomes of all activity areas (diagnosis, care, and other biomarkers of disease progression. outcome measures, production, toxicology) for experienced and young • The defi nition of standards of care and diagnosis, including all researchers and clinicians of the fi eld so to ensure the excellence of aspects of multidisciplinary management. present and tomorrow’s workforce and place Europe permanently at the forefront of the NMD fi eld.

RARE DISEASES | 77 Coordinators Prof. Hanns Lochmüller Prof. Markus Ruegg Prof. Kay Davies MD-NET Biozentrum, University of Basel Medical Research Council Prof. Volker Straub Friedrich-Baur-Institute Basel, Switzerland Oxford, United Kingdom Prof. Kate Bushby Dept of Neurology Institute of Human Genetics Ludwig-Maximilians-University Dr. Fabrizia Bignami Dr. Anna Ambrosini University of Newcastle upon Tyne of Munich European Organisation Fondazione Telethon International Centre for Life Munich, Germany for Rare Diseases Milan, Italy Central Parkway Paris, France Newcastle upon Tyne – NE1 3BZ Dr. Veronika Karcagi Prof. Jean-Marie Gillis United Kingdom Fodor Jozsef National Dr. Thomas Sejersen Université Catholique de Louvain Phone: +44 (0)191 241 8762/8655 Center for Public Health Karolinska Institute Brussels, Belgium Fax: +44 (0)191 241 8757/8737 Budapest, Hungary Astrid Lindgren Paediatric Hospital [email protected] Stockholm, Sweden Dr. Miguel Chillon Prof. Francesco Muntoni Universitat Autònoma Faculty of Medicine, Imperial College Prof. Richard Hughes de Barcelona Partners Hammersmith Campus King’s College London Center of Animal Biotechnology London, United Kingdom Guy’s Hospital and Gene Therapy (CBATEG) Dr. Christoph Beroud London, United Kingdom Belleterra (Barcelona), Spain Laboratoire de Genetique Moleculaire Annette Boersen Montpellier, France European Neuromuscular Centre Dr. Thomas Meier Dr. Muriel Audit Baarn, The Netherlands Santhera Pharmaceuticals GenoSafe SAS Dr. Annemieke Aartsma-Rus LTD Liab.Co Evry, France Center for Human and Clinical Genetics Dr. Jon Tinsley Liestal, Switzerland Leiden University Medical Center VASTOX Dr. David Koubi Leiden, The Netherlands Oxfordshire, United Kingdom Prof. Carina Wallgren-Pettersson ACIES The Folkhalsan Institute of Research Promotion Dr. Serge Braun Genetics and the Department and Management Consulting Association Française of Medical Genetics Lyon, France contre les Myopathies University of Helsinki Evry, France Helsinki, Finland

Project number LSHM-CT-2006-036825 EC contribution € 10 000 000 Project Duration 60 months Starting date 01/01/2007 Instrument NoE Project website www.treat-nmd.eu

78 | RARE DISEASES 79 ➞ 110

ANTI-MICROBIAL DRUG RESISTANCE ACE

Approaches to control multi-resistant enterococci: studies on molecular ecology, horizontal gene transfer, fi tness and prevention

Keywords Enterococcus faecium, enterococcus faecalis, sewage, farm animals (pigs, calves and poultry), healthy humans and VRE, antibiotic drug resistance, horizontal gene transfer, hospitalized patients. Interestingly, E. faecium strains colonizing differ- population structure, genetic evolution, niche adaptation, ent reservoirs have markedly different genetic characteristics (host- colonisation modulation, immunotherapy specifi city) and a specifi c clonal complex (CC17), apparently well adapted to the hospital environment is associated with global spread within and between healthcare settings. In such settings, enterococci disseminate through high-effi cient patient-to-patient transfer; via temporarily colonized staff or through contaminated medical instruments and surfaces. The abundance of antibiotic-resistant enterococci among farm animals in Europe has been linked to the massive use of antibiotics for therapy, prophylaxis and growth promotion. Importantly, Summary E. faecium belonging to CC17 have never been isolated from animals. From the animal reservoir, enterococci spread to humans through the In European hospitals up to 60 % of nosocomial infections are nowadays food chain and, once colonizing the human gut, animal-derived resist- caused by antibiotic-resistant pathogens, including vancomycin-resistant ance determinants (such as vancomycin resistance genes) can be trans- Enterococcus faecium and Enterococcus faecalis (VRE). VRE are among ferred to human-specifi c enterococci. Driven by the selective pressure in the most resistant opportunistic pathogens and have become the paradigm hospitals, acquisition of resistance determinants and other adaptive for the post-anti-microbial era. Genetic population analyses of Enterococcus mechanisms like virulence genes, have favoured the emergence and faecium revealed the presence of a High-Risk Enterococcal Clonal Complex spread of High-Risk Enterococcal Clonal Complexes (HiRECC) in which (HiRECC) resistant to multiple antibiotics and responsible for the vast enterococci have progressively changed from innocent commensals to majority of nosocomial VRE infections and hospital outbreaks worldwide. In feared multi-resistant hospital-adapted pathogens. this proposal, the evolutionary development of HiRECC in E. faecium and E. faecalis will be further unraveled and combined with new knowledge on intra- and inter-species gene transfer (including antibiotic resistance Aim genes), biological fi tness costs of hospital adaptation and novel approaches to prevent further spread of multi-resistant enterococci. The main project objectives are to determine the population structure of enterococci and the evolutionary development of HiRECC, to identify yet unknown HiRECC in E. faecalis and improve our understanding of E. faecium, to improve our understanding of transfer of resistance within Enterococcus species and between Enterococci and other bacterial species, and to improve our understanding of the biological fi t- ness costs of hospital adaptation of Enterococci. Furthermore, we aim to modulate human enteric colonisation with HiRECC by applying protective commensals and select viable candidates for immunotherapy. Problem Elucidation of the population structure of both enterococcal species is Vancomycin-reistant enterococci (VRE) have become the paradigm of a prerequisite for effective control of multi-resistant enterococci. Such the post-antibiotic era. Nosocomial VRE-infections are rising in all Euro- knowledge also enables reconstruction of the evolutionary past of pean countries, with proportions >10 % among enterococcal blood- a species and prediction of its success in the future. Furthermore, stream infections in 9 countries in 2005. From a microbial perspective, essential insights in species adaptability and ecology (distribution, emergence of VRE represents a dual-edged sword: they are amongst survival, and persistence (fi tness) of bacteria and their genes in different the most resistant opportunistic nosocomial pathogens with an increas- environments) will be derived. For all these reasons identifi cation of ing impact on patients’ health care. Moreover, and maybe even more HiRECC is a fundamental aspect of our approach. Understanding important, their unprecedented capacity of genetic exchange make processes that have lead to adaptation would provide fundamental them perfect hubs for resistance genes facilitating horizontal gene insights in bacterial pathofysiology and will contribute to intervention transfer amongst bacterial species, most notably to MRSA. The emer- strategies to reduce fi tness c.q. adaptability of strains and identifi cation gence of VRE in European hospitals, where MRSA has been fi rmly estab- of novel targets for antibiotics and immunotherapy. Knowledge on lished, creates a dooming perspective of large-scale horizontal transfer horizontal transfer of resistance is crucial to assess the risk of emergence of resistance genes. In Europe different reservoirs of antibiotic-resistant of multiple antibiotic resistance among HiRECC and other pathogens. enterococci (Enterococcus faecium and Enterococcus faecalis) exist:

80 | ANTI-MICROBIAL DRUG RESISTANCE Population snapshot of Enterococcus faecium.

Expected results Potential applications

• Improved MLST scheme for E. faecium and E. faecalis with an Antibiotic resistance clearly constitutes a major threat to public health. improved resolution based on polymorphism in housekeeping Drug resistance among bacteria is increasing, both in Europe and in genes and surface and extra cellular protein genes leading to the North-America. Ironically, those persons admitted to hospitals are amongst identifi cation of HiRECC, community and animal associated CCs. the most vulnerable populations exposed to increasingly intractable • A typing scheme for resistant plasmids to construct a catalogue of Enterococcus infections, where VRE are causing deaths across the resistance determinants, transposons, and plasmids present in developed world each year. More and more European countries report different host groups (hospital and non-hospital, animals, and the hospital outbreaks of VRE, with nine European countries showing environment). prevalence rates of VRE among nosocomial bloodstream infections above • In vitro and in vivo models to determine fi tness costs related to 10 %. Identifi cation of HiRECC as distinct genetic subpopulations, different hospital environmental subspaces. improved knowledge on molecular ecology of mobile genetic elements • Insight in ecological and bacterial determinants that promote hospital and improved understanding of biological fi tness costs of hospital adaptation of enterococci in hospital environmental subspaces. adaptation of HiRECC will enable new strategies to reduce spread of • A list of HiRECC associated genes, a collection of HiRECC mutants, resistance and infections among hospitalised patients. Furthermore, the and a redesigned E. faecium microarray, containing more genes identifi cation of HiRECC-specifi c candidates for immunotherapy represents from other E. faecium strains including plasmid encoding genes a unique opportunity to develop vaccination (passive and active and the E. faecium PAI associated genes. immunization) as a new concept to prevent infection and colonization, • A list of candidate molecules for immunotherapy and knowledge respectively, with multi-resistant HiRECC. on the relations between virulence factor expression and growth conditions.

Coordinators Dr. Lars Jensen Prof. Fernando Baquero Danish Technical University Fundacion para la Investigacion Prof. Marc J. M. Bonten Lyngby, Denmark Biomedica Hospital Universitario Dr. Rob J. L. Willems Ramon y Cajal Eijkman-Winkler laboratory for Prof. Arnfi nn Sundsfjord Madrid, Spain Microbiology, Infectious Diseases University of Tromso and Infl ammation Tromso, Norway Prof. Waleria Hryniewicz The Netherlands National Institute of Public Health [email protected] Prof. Ingolf Nes Warsaw, Poland [email protected] Norwegian University of Life Sciences Ås, Norway Ir. Marco van Es Winclove Bio Industries BV Partners Dr. Johannes Huebner Amsterdam, The Netherlands Universitätsklinikum Freiburg Dr. Anette Marie Hammerum für die Medizinische Fakultät Herman Groen Statens Serum Institut der Albert-Ludwigs-Universität IQ Corporation National Center for Antimicrobials Freiburg, Germany Groningen, The Netherlands and Infection Control Copenhagen, Denmark Dr. Guido Werner Dr. Tjibbe Bosma Robert Koch Institute Stichting Biomade Technology Project number Wernigerode Branch Biomade Technology Research LSHM-CT-2006-037410 Wernigerode, Germany Groningen, The Netherlands EC contribution € 3 148 000 Project Duration 36 months Starting date 01/02/2007 Instrument STREP Project website www.aceproject.eu

ANTI-MICROBIAL DRUG RESISTANCE | 81 CRAB

COMBATING RESISTANCE TO ANTIBIOTICS

Keywords Antibiotics, horizontal gene transfer, transposition, A coherent picture, which could explain the road to multiple AR, is conjugation, integron, toxin beginning to emerge (see Figure). Superintegrons could be considered as the ‘cradles’ of AR since many of the integron cassettes are often silent, and can be subjected to random mutational drift, cassette amplifi cation and, if transferred to the expression region, periodic selection. These proto AR genes therefore contribute to diversity at low cost to the bacterial host. Subsequently, integron cassettes are recruited into integron platforms carried by dispersion modules (transposons, Summary plasmids, genomic islands…) where they can be transferred horizontally to other bacterial species. Insertion sequences (IS), small (<2kb) Combating bacteria with antibiotics is an endless race because bacteria independently mobile DNA segments capture AR genes or integron acquire antibiotic resistance (AR) genes easily from unknown environ- platforms in various ways to form transposons. These are subsequently mental sources. We think that an appropriate long-term public health translocated to transmissible genetic elements such as conjugative objective would be to elucidate the molecular mechanisms behind the plasmids. In doing so transposons shuffl e genes, modulate their observed AR spread in a concerted strategy targeting the dissemination expression and in general contribute to the adaptation of the acquired modules, from AR recruitment to their ultimate acquisition by bacterial gene to the pathogenic host. Conjugative plasmids transfer AR genes pathogens. The focus of our project is to explore a mechanistic approach and establish themselves with variable effi ciency in pathogenic bacteria. to combat AR by tackling each of the dissemination modules in this chain Finally, plasmids need to be stably inherited once they have established – integrons, transposons, conjugative plasmids and stability modules – themselves in a recipient cell. in a concerted approach. We concentrate on the as yet insuffi ciently understood aspects of these mechanisms. Aim

The principal aim of this proposal is to explore the mechanisms and process dynamics at work in each of the dissemination modules of the chain of AR genes dissemination – integrons, transposons, conjugative plasmids and stability modules – in a concerted approach. We concentrate on the as yet improperly studied aspects of these mechanisms. Problem Our work will be divided in 4 work package (WP): WP1: mechanism of integron cassette recombination, signals triggering Antibiotic resistance has represented a serious impediment to antibiotic integrase expression and molecular evolution of gene cassettes. therapy for as long as antibiotics have been used. Although mutations are WP2: propensities of three major classes of IS to acquire, stabilize and ve- responsible for some specifi c cases of AR, the driving force behind the hicle AR genes and identifi cation of host factors that affect transposition. problem of multiresistance to antimicrobials is gene acquisition by human WP3: identifi cation of host factors that affect conjugation, development pathogens. In the past, understanding of AR spread and its control was of in vitro models for conjugation on cultured mammalian cells. based largely on a unique approach: the precise description of AR genes WP4: identifi cation of novel stabilization systems and their intracellular presently found in hospitals, and inference from this of the working targets and identifi cation of host factors that trigger these systems. mechanisms of dispersion. This approach resulted in the accumulation of an impressive knowledge base of individual elements, modules and clones of Although the main avenues for AR dissemination can be accounted for by bacteria that underpin AR dissemination. Nevertheless, and in spite of the the description above, we still lack both a deep knowledge of many of the vast number of publications on the subject, after over 40 years of study, we detailed mechanisms involved as well as of the infl uence that different are as yet unable to circumvent or even simply restrain AR dissemination. factors (genetic and environmental) have on the modes of AR dissemination and fi nal acquisition by pathogens. In a few cases where a more intimate AR genes are usually carried by transposons, plasmids and other mobile knowledge of these mechanisms is available, for instance, the mechanisms genetic elements, which provide mechanisms assuring their sequestration of plasmid stability, this knowledge is already offering new tools for and dissemination (dispersion modules). Interestingly, in gram-negative biotechnology. In summary, we propose to investigate on the mechanisms species, individual AR genes are often in the form of integron cassettes. of AR transmission in the hope of identifying and characterizing targets Resistance integrons (RI) are frequently found in clinical isolates of resistant for novel biotechnological intervention. Enterobacteriaceae and pseudomonads It is thought that RIs and their cassettes derive from superintegrons (SIs), such as those of Vibrio species. SIs contain several hundred tandem integron cassettes and are carried on bacterial chromosomes.

82 | ANTI-MICROBIAL DRUG RESISTANCE Vibrio cholerae expressing a fusion between the integrase gene and the gfp.

Expected results Coordinator Dr. Ferenc Olasz Agricultural Biotechnology Center • The establishment of the precise mechanisms of cleavage and Dr. Didier Mazel Institute of Environmental Biosafety strand transfer reactions involved in integron cassette insertion Unité Plasticité du Genome Bactérien Gödöllö, Hungary Dept Structure et and excision. dynamique des génomes Prof. Dr. Ellen L. Zechner • The identifi cation of the conditions that trigger cassette Institut Pasteur Institut fuer Molekulare recombination. Paris, France Biowissenschaften • The determination of the model driving integron cassette [email protected] Universitaet Graz evolution. www.pasteur.fr/recherche/unites/pgb Graz, Austria • A description of the different ways by which the three major classes of IS acquire, stabilise and vehicle AR genes. Dr. Laurence Van Melderen • A quantitative evaluation of the impact of IS elements on Partners Laboratoire de Génétique horizontal transfer within and between chosen bacterial genera. des Procaryotes • The precise description of the mechanism of conjugative DNA Dr. Michael Chandler Universite Libre de Bruxelles Centre National de IBMM-DBM processing. la Recherche Scientifi que Gosselies, Belgium • The establishment of the host mutations that affect transposition Laboratoire de Microbiologie et and conjugation. Génétique Moléculaire Dr. Finbarr Hayes • A list of environmental factors affecting plasmid transfer and Toulouse, France The University of Manchester biofi lm formation on cultured human cells. Manchester, United Kingdom • The identifi cation of novel stabilisation modules (toxin / antitoxin) Prof. Fernando De La Cruz and of their functional characterization. Universidad de Cantabria Dr. Philippe Gabant • The development of new molecular biological tools. Santander, Spain Delphi Genetics SA Charleroi, Belgium

Potential applications

The application of state-of-the-art functional genomics will ultimately facilitate the translation of genomic data into novel products, such as new stabilization operons, which would be developed by Delphi Genetics.

In addition, several of the novel approaches such as the system of bacterial attachment to biotic surfaces, in situ monitoring of bacterial conjugation and induction of expression of bacterial and plasmid genes, will likely lead to further developments with commercial potential.

Project number LSHM-CT-2003-019023 EC contribution € 1 795 500 Project Duration 36 months Starting date 01/12/2005 Instrument STREP

ANTI-MICROBIAL DRUG RESISTANCE | 83 DRESP2

Role of Mobile Genetic Elements in the Spread of Antimicrobial Drug Resistance

Keywords Drug resistance, epidemiology, molecular diagnostics, Problem in vitro diagnostic medical device, mobile genetic element Antibiotic resistance remains a major clinical and public health problem despite the availability of very potent antibiotics. While it is clear that antibiotics are pivotal in the selection of bacterial resistance, the mechanisms of spread of both resistance genes and of resistant bacteria can be very diverse. Mobile DNA provides a major contribution to the spread of antimicrobial resistance, by recruiting new resistance genes in Summary bacterial pathogens and facilitating their horizontal spread among microbial populations. Although the acquisition of new resistance genes Mobile DNA provides a major contribution to the spread of antimicrobial is an important factor in the increasing incidence of resistant strains, the resistance, by recruiting new resistance genes in bacterial pathogens and nature of the elements carrying the resistance genes and their transfer facilitating their horizontal spread. While much is known about individual mechanisms have been clarifi ed only in a few cases, and very little is resistance genes and mechanisms, the nature of the mobile elements and known about their molecular epidemiology. Moreover, a critical, but their transfer mechanisms have been clarifi ed only in few cases, and very often under-appreciated feature of elements carrying resistance genes is little is known about their molecular epidemiology. A comprehensive their stability: these elements are often able to be integrated into and understanding of these aspects could provide a sinifi cant breakthrough maintained by new hosts without antibiotic selection, demonstrating that toward combating dissemination of resistance determinants. resistance can be very hard to reverse. This feature explains the spread of The DRESP project aims at investigating these aspects, focussing on few resistance among community-acquired pathogens not commonly under major families of genetic elements that carry a number of important and antibiotic pressure. emerging resistance determinants: • the double resolvase elements (DR-elements) of gram-positive cocci, A comprehensive understanding of these aspects, beyond the mere epi- involved in spread of macrolide resistance genes in streptococci; demiology of resistance genes, could provide a signifi cant breakthrough • the related DR-elements in the anaeronic species Acidaminococcus, in combating drug-resistant pathogens by clarifying the dynamics and an entirely novel type of mobile element; patterns of spreading of the various mobile elements. This could possibly • the conjugative transposons encoding vancomycin resistance; lead to the identifi cation of new unconventional targets for antimicrobial • the integrons; chemotherapy. • plasmids which are the principal carrier for mobile and non mobile elements. The GENERAL OBJECTIVES of DRESP are: Aim • to investigate the molecular epidemiology of the selected families of mobile elements in clinical and environmental situations; DRESP2 is a multidisciplinary project in which the scientifi c synergism • to carry out structural and functional analysis of these genetic and technical complementarities of the participants is coordinated for elements to elucidate transfer mechanisms and their regulation, and a innovative proposal on the characterisation of the molecular to identify possible common molecular themes underlying these mechanism(s) underlying mobility of genetic elements carrying antimi- functions; crobial resistance genes. This characterisation will be performed apply- • to promote Information-Sharing and dialogue to stakeholders as to ing up to day post-genomic technologies (Comparative genomics, increase the social value and impact on pubic health of the knowledge DNA and RNA micro-arrays, gene chip technology, PCR rep typing, gained. quantitative and high throughput real-time PCR), in addition to more established biomedical (antimicrobial resistance surveillance) and biotechnological approaches (mutant construction and recombinant protein production), to the analysis of selected classes of genetic elements. The mobile genetic elements chosen are the prototype elements responsible for the spread of antimicrobial resistance in the last decade. The genetic elements selected are: • the double resolvase element responsible for the recent spread of the macrolide effl ux determinant (mef(A)) in Gram-positive Streptococci; • the still un-described double resolvase element responsible for beta-lactam resistance in anaerobic Acidaminococcus; • the integrons responsible for spread of a broad repertoire of resistance genes in Gram-negative bacilli;

84 | ANTI-MICROBIAL DRUG RESISTANCE Microarray analysis is one upon the tools used both for molecular epidemiology of mobile genetic elements and for their characterisation.

• the conjugative transposons responsible for spread of vancomycin Potential applications resistance in Enterococci; • plasmids. • In vitro diagnostic medical device. • Epidemiology of drug resistances. While there are a plethora of resistance determinants, the number of • Antimicrobial treatment target discovery. genetic elements responsible for their sensational evolutionary success • Evaluation of the impact of treatment schemes. is limited. Unfortunately, most industrially fi nanced research focuses • Prediction of drug resistance emergence to novel compounds. on determinants of resistance, mechanisms of resistance, and novel compounds, neglecting the possibly most important feature for public health which is the effi ciency of spread which is intimately and exclusively Coordinators Fernando Baquero linked to the genetic elements. The DRESP proposal through: Juan Carlos Galan • fi ne characterisation of molecular mechanisms behind mobilisation; Marco R. Oggioni Ramón y Cajal Hospital • cross linking of epidemiological and evolutionary data; Gianni Pozzi Madrid, Spain Gian Maria Rossolini • broad spectrum gene expression profi ling, in elements of evolution- LA.M.M.B. (Laboratorio di Microbiologia Lars Sundström ary very distant organisms, is expected to produce innovative results. Molecolare e Biotecnologia) Uppsala University The value of the results of the DRESP proposal, including licensable Dipartimento di Bilogia Molecolare Uppsala, Sweden tools for diagnosis and public health monitoring, will not be restrict- Università degli Studi di Siena ed to the elements studied, but are expected to add fundamental Policlinico Le Scotte (lotto 5, piano 1) Alex van Belkum knowledge to the fi eld of study encompassing all mobile genetic 53100 Siena, Italy Erasmus University Rotterdam elements and on antimicrobial resistance spread in general. Phone: +39 0577 233 101 Rotterdam, The Netherlands [email protected] Phone: +39 0577 233 430 Alessandra Carattoli Expected results [email protected] Annalisa Pantosti Phone: +39 0577 233 326 Istituto Superiore di Sanità [email protected] Roma, Italy • New information on the spread of mobile elements carrying Fax: +39 0577 233 334 resistance genes, and mechanisms thereof. Patrice Nordmann • New techniques for molecular epidemiology studies. Hopital de Bicetre • Theoretical schemes for prediction of the potential spread of Partners University Paris XI resistance to specifi c drugs. Le-Kremlin-Bicetre, France David J Farrell Medical and Molecular Jose-Luis Martínez Microbiology Teresa M. Coque GR Micro Limited Centro Nacional de Biotecnologia London, United Kingdom Madrid, Spain

Stefania Stefani Balázs Libisch Maria Santagati Miklos Fuzi University of Catania National Center for Epidemiology Catania, Italia Budapest, Hungary

Timothy R Walsh Project number Cardiff University LSHM-CT-2005-018705 Cardiff, United Kingdom EC contribution € 1 852 000 Project Duration 36 months Starting date 01/01/2006 Instrument STREP Project website dresp2.net

ANTI-MICROBIAL DRUG RESISTANCE | 85 EACCAD

European approach to combat outbreaks of clostridium diffi cile associated diarrhoea by development of new diagnostic tests

Keywords Clostridium diffi cile, hypervirulent type 027, CDAD, type BI and PCR-ribotype 027. The strain produces toxins A and B and diagnostics, outbreaks contains a 18 bp deletion in the toxin regulator gene TcdC (fi gure). It is imperative that an overall strategy to combat this and similar novel variant(s) is devised. Central to control of epidemics by such new C. diffi cile strains with increased virulence is a strategy that will include not only a rapid detection but also a specifi c recognition of virulent strains. Diagnostic assays for CDAD rely either on the detection of C. diffi cile products (toxins), C. diffi cile genes, or the culture and toxin testing of the Summary isolates. Toxins can be detected either by their biological properties (cell cytotoxicity assay) or by immunological methods (latex agglutination, Clostridium diffi cile-associated disease (CDAD) has become the most immunoassay). The cell cytotoxicity test is the ‘gold standard’. However, frequent nosocomial infection in many European hospitals. In 2004, the there are signifi cant drawbacks. Laboratories must have access to cultured situation was exacerbated by the arrival in Europe of a new hypervirulent monolayers, and results vary according to the cell line, dilution factors, strain (PCR ribotype 027) previously confi ned to N. America, where it has reagents used and storage conditions. Additionally, the turnaround time been responsible for a massive increase in CDAD incidence and associated is very slow. Enzyme immunoassays consist of conventional enzyme deaths. Central to the control of epidemics are the deployment of assays immunoassays and a membrane immunochromatographic tests. The able to rapidly diagnose and monitor the presence and spread of the sensitivities and specifi cities of enzyme immunoassays are within 85-95 % organism. No such tests currently exist for these new hypervirulent of the cell cytotoxicity test, and numerous commercial kits are available. C. diffi cile strains. It is the overall objective of this proposal to develop the However, their performance relative to each other has not been subjected urgently required rapid, diagnostic assays. to a rigorous meta-analysis. Immuno-chromatography tests are extremely simple, can be performed at the bedside and give a result within 30 minutes. This may be of crucial importance in an epidemic situation to recognise infected patients. DNA-based tests have focussed on the detection of the C. diffi cile genes encoding 16S RNA, GDH or the toxin genes (tcdA & tcdB).

Problem Aim

C. diffi cile is resistant to various antibiotics and capitalizes on the ensuing The recognition of suitable targets and development of a commercial rapid disruption of the normal intestinal fl ora to colonization and cause test that will distinguish variant hypervirulent and antibiotic resistant strains disease. The spectrum of disease ranges from asymptomatic carriage to from ordinary C. diffi cile strains is the main aim of this project. Our objective a fulminant, relapsing, and increasingly fatal colitis. The effects of CDAD will be achieved through the complementary skills of a consortium are devastating, both in terms of morbidity/ mortality and the high costs composed 3 SMEs and 4 public sector institutes, each experts in their of disease management. Presently, C. diffi cile may only be treated with fi elds. There will be a close collaboration in the form of subcontractors either metronidazole or vancomycin. A number of factors have contributed with the European Study Group of C. diffi cile (ESGCD). Suitable diagnostic to the worrying escalation in the incidence of CDAD. The elderly and markers (DNA- and antigen-based) will be identifi ed using a combination immuno-compromised are particularly at risk (80 % of cases occur in the of targeted approaches (focusing on toxin and antibiotic resistance over 65s). The proportion of the population in these high-risks groups is determinants), and empirical approaches (DNA arrays and HT-AFLP) to rapidly rising. Strains exhibiting greater virulence are also beginning to identify unique strain differences. Assay development will be led by the emerge, which in some instance has been attributed to the production of SMEs, using proprietary technologies. Clinical validation will be performed additional virulence factors, eg., binary toxin. The situation has now been in a certifi cated reference laboratory. The programme outputs will allow exacerbated by the arrival in Europe during 2004 of a new hypervirulent the formulation of a European strategy to combat the considerable threat strain and antibiotic resistant strain (ribotype 027, toxinotype III) previously now being posed to European quality of life by C. diffi cile. confi ned to Canada and the USA. The occurrence of this strain is associated with an excessive use of quinolone antibiotics, and has been responsible for a massive increase in CDAD incidence in N. America and Expected results associated deaths, eg., the proportion of patients with CDAD who died within 30 days after diagnosis rose from 4.7 % in 1991-92 to 13.8 % in • Recognition of targets for new diagnostic tests by characterisation of 2003. This epidemic strain isolated in Canada, USA, United Kingdom, hypervirulent and drug resistant C. diffi cile strains. The targets are Belgium, France and The Netherlands was characterized as toxinotype III, based on toxins, toxin coding regions, or unique other genes of North American PFGE type 1, restriction-endonuclease analysis group C. diffi cile.

86 | ANTI-MICROBIAL DRUG RESISTANCE • The availability of molecular tests and rapid membrane immunoassays for detection of the target in patient material and in bacterial isolates. • Validation of new developed tests for clinical diagnostics and strain characterization. The new molecular tests and rapid membrane Toxin A (TcdA) and toxin B (TcdB) are encoded on large chromosomal region assays will be investigated in faeces samples of patients with various PaLoc, which encompasses two toxin genes (tcdA and tcdB) and three additional genes coding for regulatory and putative transport function (tcdR,E,C). forms of C. diffi cile associated diarrhoeae. The new developed tests In nontoxigenic strains, PaLoc is replaced by 115 bp sequence. will also be used to recognize hypervirulent isolates and antibiotic resistance of clinical isolates obtained by prospective surveillance studies in Europe. Coordinator Prof. Nigel Minton Institute of Infection, Immunity Dr. Ed J. Kuijper and Infl ammation Potential applications Dept of Medical Microbiology University of Nottingham Leiden University Medical Center, PO Centre for Biomolecular Science Box 9600, 2300 RC Nottingham, United Kingdom The new diagnostic tests will overcome the lack of sensitivity and Leiden, The Netherlands specifi city of current diagnostic tests. The new immunoassay offers an Phone: +31 71 5263574 Dr. Paola Mastrantonio alternative for those laboratories which lack capability in molecular Fax: +31 71 5248148 Istituto Superiore di Sanità, ISS biology. Recognition of variant 027 is not possible with the currently [email protected] Rome, Italy available diagnostic tests. Rapid recognition of CDAD and its variant 027 will result in appropriate patient treatment and specifi c measures to Prof. Cristoph von Eichel-Streiber prevent nosocomial spread and the development of outbreaks. The rapid Partners Mainz, Germany molecular test will provide the fi rst indication of the presence of type 027, allowing adequate infection control measures to be undertaken Dr. Maja Rupnik Dr. Thierry Leclipteux and prevent an outbreak. The genomic approaches (DNA Array and HT- University of Maribor Coris BioConcept Maribor, Slovenia Gembloux, Belgium AFLP) will provide fundamental information on the genetic make-up of hypervirulent C. diffi cile strains and will lead to greater insight into Dr. Guus Simons pathogenesis which will in turn allow the development of more effective Pathofi nder B.V therapeutic countermeasures and IPR spin-offs. Additionally, European Maastricht, The Netherlands guidelines will be formulated to diagnose CDAD and to combat outbreaks. The introduction of these tests and European guidelines increase the awareness of CDAD as an important nosocomial infection and will be of help to prevent the development of large outbreaks by new hypervirulent variants, as currently occur in USA and Canada. Application of the new tests and updated guidelines will contribute to insights in the epidemiology of CDAD and will be of general support to prevent over-prescription of antibiotics in hospitals. Application of the new developed tests will provide a platform for automation of diagnosis. A further stimulant for effective DNA-based diagnostic tests is to extend testing on the same faecal sample for other non-bacterial causes of infectious diarrhoea, such as viral diarrhoea and parasitic diarrhoea. It is possible to automate nucleic acid extraction and use a common extraction Project number for all subsequent analyses. LSHM-CT-2006-037870 EC contribution € 1 771 000 Project website Project Duration 36 months The clostridium website at www.clostridia.net) will act as the focal point for Starting date the dissemination of general information. A specifi cs website (www.cdiff.nl) 01/12/2006 will be used for information and progress of the EACCAD study. The website Instrument will contain a hierarchical organisation with three main levels of accessibility: STREP one for the general public, one for all of the clostridial scientifi c community Project website and one for the consortium. www.clostridia.net

ANTI-MICROBIAL DRUG RESISTANCE | 87 EAR

Effects of antibiotic resistance on bacterial fi tness, virulence and transmission

Keywords Antibiotic resistance, fi tness, genetic Problem compensation, transmission, virulence Ever since antibiotics were ﬁ rst introduced 60 years ago they have been a remarkable success story giving us the opportunity to treat and cure most infectious diseases. However, the intensive use and inappropriate use of antibiotics has also resulted in the many important human pathogens developing resistance. There is a concern that in time that the loss of therapeutic options will present us with a post-antibiotic era Summary where present and future medical advances are threatened. The combination of a worldwide rapid increase in resistant bacteria and the A major factor that affects the emergence and survival of resistant strains downward trend in the development of new antibiotics has serious is the biological cost of resistance. Thus, to reduce the rate of spread of implications. Resistant bacteria dramatically reduce the possibilities of resistant bacteria we need to identify antibiotic targets and antibiotics for treating infections effectively and increase the risk of complications and which the resistance mechanisms have the most negative effects on fatal outcome for patients with severe infections. Those most vulnerable bacterial fi tness. Thus, the overall aims of this proposal are to experimentally patients are children, the elderly and the economically disadvantaged. examine and defi ne in several medically important species how fi tness, Individuals with compromised immune defences, such as cancer patients virulence and transmission are affected by different types of antibiotic and those living with HIV require antibiotic therapy to prevent and treat resistance. Such knowledge is a prerequisite for: severe infections and these drugs are necessary for their survival. In • predicting the rate and stability of resistance development; addition, antibiotic resistance jeopardises advanced medical procedures • developing novel diagnostic test systems for resistant bacterial clones such as organ transplantation and implants of prostheses, where with a high risk of resistance development; infective complications are common and antibiotic therapy is necessary • forecasting the value of intervention strategies; to prevent or treat complications. Thus, antibiotic resistance represents • rational design of antibiotics and choice of antibiotic targets where a major public health concern and economic problem. the potential for resistance development is minimized. Aim

• The fi rst objective is to experimentally determine how different types of antibiotic resistances affect fi tness (growth and survival within and outside hosts) of several pathogenic bacterial species. In particular, we will determine if antibiotic resistance can truly be cost free for a bacterium. Putative cost-free mutations are expected to be especially problematic and stable in the population. • The second objective is to determine the physiological reasons for why fi tness is reduced in antibiotic resistant mutant bacteria. Using global approaches (microarrays and proteomics) we intend to determine how gene expression and metabolism is changed as a result of the acquisition of a resistance determinant. In particular, we will examine why the same resistance mechanism/mutation might have very different physiological effects in different species. An understanding of the specifi c nature of the fi tness defects could reveal weaknesses in the resistant bacteria exploitable either by alternative or supplementary therapies or by novel antibiotics. • The third objective is to determine if the fi tness costs of resistance can be reduced by mutation and/or environmental conditions. Thus, we will examine the rate at which compensatory mutations ameliorate fi tness defi cit both in vitro and in vivo. Thus, the objective will be to understand the link between the fi tness cost of resistance and cost compensation making use of appropriate animal model systems.

88 | ANTI-MICROBIAL DRUG RESISTANCE • The fourth objective is to develop animal experimental models to Potential applications study the impact of resistance on transmission rates. • The fi fth objective is to use volunteer studies to examine the impact Antibiotic resistance is a serious problem that creates a substantial of antibiotic resistance on transmission rates. mortality, morbidity and health care cost. In intensive care units recent • The sixth objective is to use epidemiological data to examine in studies have shown that inadequate antibiotic therapy related to a real clinical situation if and how the basic reproductive number antibiotic resistance causes up to four times higher mortality as well as (the number of secondary infections generated from an infected considerable added costs. From this perspective, the completion of the individual) is altered due to the acquisition of antibiotic resistance. objectives of this project should certainly infl uence several social • The seventh objective is to perform a unique clinical intervention objectives of the Community. Antibiotic resistant organisms should study where the use of an antibiotic will be signifi cantly curtailed in thus be considered as dangerous spreading organisms that may affect a community in an attempt to reduce the frequency of resistant the health of the community. In particular, an important part of the bacteria. currently available medical technology, including advanced surgery, • Finally, in the eighth objective we want to investigate two new transplantation, therapies for cancer and immuno-defi ciencies, and concepts experimentally in the development and selection of drugs intensive care units, depends on the successful control of microorganisms and drug targets where the likelihood of resistance development is by antimicrobial agents, and the safety of all these procedures depends reduced. First, the ‘multiple targets concept’ where the drugs have on the maintenance of effective antibiotic treatments. The deliverables multiple targets in the bacterial cell, making the development of of this proposal will aid in the development of guidelines for the clinical resistance due to target alterations very diffi cult. Second, the ‘high use and regulation of antibiotics which may help free resources for pleiotropic biological cost’ concept. That is, novel drugs and drug other important health issues of EU citizens. targets are designed and chosen such that the resistance mechanisms severely reduce pathogen fi tness by interfering with During the antibiotic era the molecular mechanisms for resistance to bacterial physiology at many different levels. We will explore as a large variety of antibacterial agents have been elucidated. However, a model system how resistance to different variants of fusidic acid it is only recently that population dynamics and evolutionary affects fi tness. engineering approaches have been taken to describe the development and reversibility of bacterial resistance. Recent advances from the HIV fi eld have taught us that population sizes, rates of growth, and rates Expected results of genetic variation, within different cellular compartments in a host, determines the likelihood of resistance development and hence the • The results obtained from this network of researchers will provide prognosis of the patient. By adopting population dynamic and the experimental knowledge required to model and perform risk evolutionary approaches also towards bacterial antibiotic resistance assessment for the development, and spread of resistance to any we have the possibility to develop tools as a base for any future given antibiotic. predictions of the spread of antibiotic resistance. For that a purpose • The achievements made here will also form the knowledge base we need to consider the size of any given bacterial population, required to formulate and to interpret intervention strategies mutation rates, rate of genetic lateral fl ow between bacteria, biological which attempt to reduce the rate of resistance development and costs of resistance, transmission rates and virulence in order to achieve a reversal of the rising tide of resistance in the society. The formulate mathematical models describing resistance development approaches suggested in this proposal will be useful for pharma- under various assumptions. Without an understanding of the above ceutical companies and drug-licensing agencies when they assess parameters it will not be possible to properly develop local, national or the potential risk for resistance development towards both new European strategies to counteract the rapidly increasing problem of and established antibiotics. antibiotic resistance. • Finally, the methodology and approaches proposed in this application will make it possible to identify particular attributes in high-risk resistant bacteria (e.g. clones that have no fi tness cost for being resistant) allowing for their rapid dissemination in the community, and hence provide the basis for early warning diagnostic systems forecasting rapidly spreading resistant clones. By identifying attributes in susceptible bacteria predisposing for rapid and stable resistance development this proposal promises to provide the knowledge base on which to develop diagnostic systems forecasting resistance development within susceptible bacterial communities.

ANTI-MICROBIAL DRUG RESISTANCE | 89 Coordinator Prof. Stephen H. Gillespie University College Prof. Dan I Andersson London, United Kingdom Uppsala University Dept of Medical Biochemistry Dr. Jose Luis Martinez and Microbiology Centro Nacional de Biotecnología Box 582, BMC Madrid, Spain S-751 23 Uppsala, Sweden Phone: +46 18 471 4175 Prof. Erik C. Böttger Fax: +46 18 509876 Institute of Medical Microbiology, [email protected] University of Zurich Zurich, Switzerland

Partners Prof. Patrice Courvalin Institut Pasteur Dr. Niels Frimodt-Møller Paris, France The National Centre for Surveillance and Control of Infectious Diseases Dr. Monique Twynholm Copenhagen, Denmark European Clinical Development, Anti-infective/Antiviral Therapeutic Prof. Diarmaid Hughes Group GlaxoSmithKline Uppsala University London, United Kingdom Uppsala, Sweden Dr. Tore Duvold Dr. Fernando Baquero LEO Pharma Ramón y Cajal University Hospital Ballerup, Denmark Madrid, Spain

Project number LSHM-CT-2005-518152 EC contribution € 2 755 000 Project Duration 36 months Starting date 01/12/2005 Instrument STREP

90 | ANTI-MICROBIAL DRUG RESISTANCE EURESFUN

Integrated post-genomic approaches for the understanding, detection and prevention of antifungal drug resistance in fungal pathogens

Keywords Fungal pathogens, antifungal drug, antifungal The fi ght against fungal infections necessitates the use of antifungal resistance, diagnostic agents. Current available antifungal drugs belong to 4 major different classes: the polyenes, the azoles, pyrimidine analogues and the echinocandins. Each of these classes contains different compounds with specifi c range of activities against the major fungal pathogens. The exposure of fungal pathogens to antifungal agents has different outcomes, one of them being the development of resistance. Studies investigating Summary mechanisms of resistance to specifi c antifungal agents could reveal the cellular and genetic basis of resistance. However, some of these studies The EURESFUN (EUropean RESistance FUNgal) network will utilize were initiated with hypothesis-based approaches and thus only give genomics-based integrated approaches to study antifungal resistance in a limited view on the diversity of possible resistance mechanisms to relevant fungal pathogens (Candida, Aspergillus). This network will study specifi c agents. The availability of collection of mutants with systematic distinct resistance mechanisms (target mutations, drug effl ux, signal gene deletions has opened the way to procedures for screening genes transduction, transcriptional activation, cell wall alterations, biofi lm involved in susceptibility of resistance to a given agent. This genome- formation) all of which can contribute individually or in combination to based procedure was implemented in the S. cerevisiae collection of antifungal resistance in clinical isolates. Using microarray strategies, mutants deleted in non-essential genes with compounds such as systematic deletion/overexpression approaches, the network will also fl uconazole and caspofungin. Many of the genes identifi ed in these elucidate new resistance mechanisms. This approach will unravel studies are not only candidates genes that might be altered in drug- potential novel targets for antifungal drug discovery, but also yield resistant strains of other fungi, but they also provide valuable information diagnostic tools and mutations suitable for the use in resistance on potential primary and secondary target genes for new agents. monitoring and surveillance. Aim

Microarray strategies and gene deletion approaches performed mainly with C. albicans will help the design of new therapeutic strategies to improve effi cacy of existing antifungal therapy, including combination drug therapies to generate synergism. Novel intervention strategies to Problem minimise or bypass resistance will be validated using animal models. Based on this knowledge, cell-based assays for drug target genes will be According to the WHO data, infectious diseases represent the most established and used for drug discovery by a SME. common cause of death in the world (WHO). Among infectious diseases, fungal infections also take a prominent position, ranking for instance To monitor progression of systemic infections and monitor resistance, the number 4 in infections of hospitalized patients acquiring microbial consortium will establish a collection of clinical strains representing a wide infections. The frequency of fungal infections has been steadily increasing range of antifungal susceptibilities. SMEs will pursue the development in the human population worldwide over the past decades. Several and validation of reliable diagnostic tools with commercial potential for fungal pathogens cause severe fungal infections in hospitals. Among rapid clinical strain identifi cation and resistance testing, employing them, the most important are Candida albicans, C. glabrata and microarray technologies in combination with PCR technologies. Aspergillus fumigatus. C. albicans accounts for more than 50 % of all fungal infections, causing both superfi cial and disseminated infections, Finally this network will establish data on resistance incidence and while C. glabrata infections account for 10-20 % of the cases. Although prevalence and will link clinical data on susceptibility to known A. fumigatus infections are less frequent, the clinical outcome is fatal antifungals with therapy outcome. Major European reference centers in 80-90 % of the cases. C. albicans and C. glabrata infections are will jointly set up and recommend European standards for resistance responsible for systemic infections in a signifi cant proportion of cancer defi nition, resistance testing/prediction/defi nition, as well as improved and transplant patients and over half of these infections are still fatal. breakpoint identifi cation.

ANTI-MICROBIAL DRUG RESISTANCE | 91 Candida albicans cells showing different morphologies (blastospores and hyphae). Hyphae shown as elongated tubes have a size of 30-40 μm.

Expected results exploited in order to generate novel diagnostic tools using post- genomic approaches (eg oligonucleotide arrays). These tools will Considering the present status of knowledge on antifungal resistance enable genotyping, species identifi cation and antifungal resistance mechanisms, the incidence of antifungal resistance and problems monitoring. associated with the prediction, detection, and surveillance of resistance, the EURESFUN consortium was assembled to undertake in order to undertake the several actions with the following expected results: Potential applications • a better understanding of the molecular basis of antifungal drug resistance is required in order to facilitate the development of diag- Fungal infections are a major source of nosocomial infections associ- nostic tools and effective intervention strategies to prevent resist- ated with a high mortality. They have steadily increased over the last ance. Therefore specifi c mutations linked to antifungal resistance two decades and this will continue because of the increasing number will be identifi ed; of immuno-suppressive treatments and the aging of the population. • current approaches to antifungal therapy are limited by a restricted This together with the increasing treatment costs makes fungal infec- number of available agents and by their mono-therapeutic use. tions a major concern to the European health system. In this context, New approaches using drug combinations could improve resistance of human pathogenic fungi towards the currently available therapeutic outcome and overcome resistance. Cellular components antifungal agents, whether intrinsic or acquired, has a strong impact involved in the response of fungal cells to antifungal drugs could on the length of hospitalization as well as the number of invasive pro- provide alternative targets for compounds that are able to cedures that are associated with patient treatment. Therefore, potentiate the action of known antifungal drugs. Therefore, these antifungal resistance contributes to an increase in the social and fi nan- components will be identifi ed and their inhibitors isolated in order cial cost of fungal infections. Approaches developed by this Network to test their effects in susceptibility to known antifungal drugs are aimed at providing diagnostic tools to rapidly detect mechanisms in vitro and in animal models; of resistance. These tools will undoubtedly help tailor the treatments • only a few European data are available on the epidemiology of in such a way that resistance does not emerge and that their duration antifungal resistance. Moreover antifungal susceptibility tests are is at a minimum. Hence, they will have a strong impact on the costs not fully standardized among European countries. Therefore, the associated with the treatment of fungal infections and reduce the network will build up a collection of strains displaying a wide range social burden of fungal infections. Furthermore, development of these of susceptibility to known antifungals and associated with molecular innovative tools will reinforce the competitiveness of the European epidemiology data. This strain collection will be used: industry in the fi eld of diagnostics. This Network will develop original – to establish a distribution of susceptibility data in order to research programs on the molecular mechanisms involved in antifun- propose microbiological breakpoints of resistance and gal tolerance or antifungal resistance. The Network is organized to – to survey mutations in known resistance genes in the strain translate the knowledge gained on these molecular mechanisms into collection in order to develop optimal diagnostic tools for novel approaches to potentiate existing antifungals. Establishment of resistance surveillance; screening assays and the identifi cation of lead compounds will pave • current methodologies for strain identifi cation and susceptibility the way to innovative combined antifungal therapies. testing are time consuming and not fully reliable. Molecular diagnostic tools especially those able to detect resistance are needed. The knowledge obtained from previous objectives will be

92 | ANTI-MICROBIAL DRUG RESISTANCE Aspergillus fumigatus conidiophores. Conidia stained in blue have a diameter size of 2-3 μm.

Coordinator Prof. Karl Kuchler Prof. Steven Kelly Medizinische Universität Wien University of Wales Swansea Prof. Dominique Sanglard Dept Medical Biochemistry Wolfson Lab. of P450 Biodiversity Centre Hospitalier Universitaire Vaudois Division of Molecular Genetics Swansea Clinical School Institut de Microbiologie Max F. Perutz Laboratories Swansea, Wales, United Kingdom Rue du Bugnon 48 Campus Vienna Biocenter 1011 Lausanne, Switzerland Vienna, Austria Dr. Emilia Mellado Phone: +41 21 314 40 83 Servicio de Micologia Fax +41 21 314 40 60 Prof. Uwe Gross Centro Nacional de Microbiologia [email protected] Bereich Humanmedizin Istituto de Salud Carlos III Georg-August Universitaet Goettingen Madrid, Spain German National Reference Center for Partners Systemic Mycoses Prof. Frank Odds Goettingen, Germany The University Court Dr. Christophe D’Enfert of the University of Aberdeen Institut Pasteur Dr. Steffen Rupp School of Medical Sciences Département Structure Fraunhofer Gesellschaft Institute of Medical Sciences et Dynamique des Génomes Institut für Grenzfl ächen Aberdeen, United Kingdom Unité Postulante Biologie et und Bioverfahrenstechnik Pathogénicité Fongiques Stuttgart, Germany Dr. Johannes Regenbogen Paris, France GATC Biotech AG, R&D Konstanz, Germany

Dr. Derek Law F2G Limited Microbiology Department Manchester, United Kingdom

Project number LSHM-CT-2005-518199 EC contribution € 2 755 000 Project Duration 36 months Starting date 01/11/2005 Instrument STREP Project website www.chuv.ch/imul/euresfun

ANTI-MICROBIAL DRUG RESISTANCE | 93 GRACE

Genomics to combat Resistance against Antibiotics in Community-acquired LRTI in Europe

Keywords Lung, infection, resistance, genomics, Problem therapeutic agent Community-acquired lower respiratory tract infections (LRTI) are the leading reason for seeking medical care. Yet, there are few conditions in medicine that are so controversial. These uncertainties have resulted in prescriptive promiscuity, which largely explains the escalating antibiotic resistance of common bacterial respiratory pathogens in the community. Invasive S. pneumoniae represent a major threat of serious morbidity Summary and mortality. The continued development of antibiotic resistance to an increasing number of different antibiotic classes by this organism has GRACE is a Network of Excellence focusing on the complex and controversial made the treatment of community-acquired LRTI more diffi cult. There fi eld of community-acquired lower respiratory tract infections (LRTI), which is are no good studies of suffi cient size on detecting bacterial aetiology of one of the leading reasons for seeking medical care. The promiscuous use of LRTI and on diagnosis of CAP in primary care. The epidemiological antibiotics to treatment of LRTI accounts for a major part of the community situation of infections, especially in the outpatient sector is almost burden of antibiotic use and contributes dramatically to the rising prevalence completely unknown, because microbiological testing with an of resistance among major human pathogens. The overall objective of GRACE appropriate quality is almost never performed. Indeed, the knowledge is to combat antimicrobial resistance through integrating centres of research concerning the ‘so-called’ atypicals is largely incomplete in terms of excellence and exploiting genomics in the investigation of community- incidence of infections, sub clinical infections, and the existence of acquired LRTI. Microbial and human genomics will be integrated with health carriers. However, nucleic acid amplifi cation based molecular diagnostic sciences research consisting of clinical observational and intervention methods could be used to identify the causative organisms or to study studies, health economics and health education to specifi cally change the prevalence of these pathogens, such as the ‘atypical’ bacterial and practice in managing community-acquired LRTI. GRACE is exceptional as it viral organisms. These methods could also be used to detect resistance brings together investigators from basic laboratory sciences, clinical medicine, genes. There has been no comprehensive, in-depth qualitative study and health economics. In the jointly executed research programme, identifying the perspective of patients and clinicians on management of 17 academic groups, spread widely across 9 EU Member States and 5 SMEs community-acquired LRTI. Specifi cally, no study has sought to achieve will participate. GRACE will organise professional education, including web- a deep understanding of the variation in presentation, investigation and based teaching and practical courses, through two leading European scientifi c management across a range of European cultures and clinical setting. societies (European Society of Clinical Microbiology and Infectious Diseases Very few, if any, interventions aimed to enhance clinical care have and European Respiratory Society) to disseminate the excellence of the been based on thorough development work that incorporates a deep programme. A high level of co-ordination will be obtained through understanding of clinicians’ and patients’ perspectives. Most patients a professionally IT-supported and rigorous management structure, so as to with acute bronchitis receive antibiotics, because there is still uncertainty achieve optimal synergy of the components of GRACE. We aim to develop about the indications for antibiotics in important subgroups of patients a genomic laboratory network in 8 European countries and a primary care (e.g. the elderly with systemic symptoms; chest signs) and often the research network in 11 European countries during the fi rst 18 months of the distinction between viral and bacterial infections and between project, and then to build on the infrastructure to create the jointly executed pneumonia and bronchitis is diffi cult in daily practice. There is a tendency research programme. The consortium will become a virtual ‘European LRTI to ‘over-treat’ and unnecessarily admit relatively mild cases to hospitals Research Centre’ potentially leading to a forum promoting research and good but also to inappropriately treat severely compromised and other high- practice in the fi eld of community-acquired LRTI. risk patients. Some patients develop a severe CAP, despite appropriate antimicrobial treatment and this could be related to host genetic susceptibility, as shown for other fatal infectious diseases. Prediction rules for poor outcome in patients with community-acquired LRTI in primary care, that could help to restrict antibiotic treatment to high-risk patients, are lacking. Costs will become an important issue for the reimbursement of diagnostic tests and for new antimicrobials, which will depend upon their benefi t and effectiveness. Technologies and solutions are available to address these issues, and the individual areas of expertise do exist in Europe, but the problem is in integrating these. It is obvious that the expected achievements of applicable microbial and genomic deliverables for improved patient care require the joint efforts of a multidisciplinary consortium.

94 | ANTI-MICROBIAL DRUG RESISTANCE Aim • Information on pathogenicity islands and horizontally acquired DNA among pneumococcal isolates causing community-acquired This proposal aims to strengthen European human and microbial LRTI by performing comparative genomics with micro-array genomic research excellence, focussing on an infection, which is technology. a leading reason for seeking medical care and consuming antibiotics. • Identifi cation of potential target pathways for new immunomodu- This antibiotic consumption leads to a potent selective force for latory approaches. antimicrobial resistance among common bacterial respiratory pathogens • Potential genetic risk profi le for community-acquired LRTI and in the community. There is currently considerable fragmentation in assessment of whether these polymorphisms identify individuals at European research efforts into community-acquired LRTI and this risk of various presentations and outcomes of community-acquired application represents a unique opportunity to address this head-on. LRTI in several European populations. GRACE will develop a durable network established over an initial fi ve- • Interaction of the human genetic risk factors identifi ed with each year time frame. We aim to integrate research groups and organisations other or with key microbial genetic or other environmental risk on a large pan-European with an ambitious scale. The close collaboration factor for community-acquired LRTI. that will fl ow from GRACE aims to generate comprehensive, creative • Description of current community-acquired LRTI management and and more practical thinking through novel integration of cutting edge analysis of the determinants of antibiotic use in 10 to 15 primary basic genomic science with research practitioners working in the care networks across the EU, using qualitative and quantitative community at the bedside and training such that the evidence informs approaches. future generations of practitioners. The hallmark of the network of • Internationally agreed evidence-based defi nitions of the major excellence created by GRACE will be the integration of research community-acquired LRTI, which are the leading reasons for platforms creating a European-wide infrastructure to investigate and antibiotic prescribing. improve the management of community-acquired LRTI. • Appropriate clinical outcome measures for evaluating interventions. • Clinical models to differentiate viral from bacterial infections and identify pneumonia. Expected results • Clinical models to identify patients at risk for adverse outcomes including severe and prolonged illness. • Molecular based techniques for the detection of pathogens • Assessment of the effectiveness of antibiotics among patients with implicated in community-acquired LRTI. community-acquired LRTI in order to fi ll the current gap in evidence • Elucidate the role of ‘atypical’ bacteria and viruses, including novel about which subgroups selectively benefi t from antibiotic treatment, pathogens, in patients with community-acquired LRTI. and which subgroups do not benefi t. • A European repository of specimens and strains linked to a database • Assessment of a practice based intervention using the novel data including microbial (e.g. antibiotic resistance, typing) and patient generated from the network in reducing inappropriate antibiotic information for development of novel rapid genome based diagnostic use in patients with community-acquired LRTI and effect on tests. antibiotic resistance. • Assessment of risk factors for infection with resistant S. pneumoniae • A model for the cost-effectiveness of the management strategies and H. infl uenzae in patients with community-acquired LRTI. developed in the observational studies. • The relationship between the integrating and conjugating antibiotic • Economic evaluations in parallel with the intervention studies. resistance elements (ICEs) and non-conjugating resistance elements, • A model for the macroeconomic impact of antibiotic resistance and and the exposure to antibiotics at the individual and population policies to contain resistance. level among, in nasopharyngeal commensal Haemophilus spp. • Economic evaluations of molecular diagnostics. • Correlation between the prevalence of resistance genes present • Information aimed at increasing awareness among the public and among commensal Haemophilus spp. (the resistance gene pool) policy makers of the importance, economic impact and threat of and the prevalence of ICE and chromosomal housekeeping genes antimicrobial resistance. encoded resistance in clinical isolates of H. infl uenzae. • Education and training support to disseminate awareness and • Steering antibiotic treatment policies in Europe based on analysis knowledge relevant to antibiotic resistance and its control. of the resistance gene pool found in commensal haemophili. • Educational packages including web-based resources and work- • Correlation between antibiotic resistance, virulence characteristics and shops to inform postgraduate lifelong learning needs of prescribing pneumococcal genotype to severity of community-acquired LRTI. professionals. • Pneumococcal serotype distribution across Europe in community- acquired LRTI.

ANTI-MICROBIAL DRUG RESISTANCE | 95 Potential applications The integration of medical laboratories and molecular biology laboratories provides an ideal basis that brings together a broad array of expertise, By restructuring and integrating the existing research capacities to and will also serve as a basis for long-term collaborative projects. The combat antimicrobial resistance in community-acquired LRTI through information that GRACE will generate on the aetiology and genetic a Network of Excellence, GRACE will strategically achieve a European background of community-acquired LRTI will enable future development leadership on this globally important topic, with a huge and increasing on new diagnostic tools and management strategies in daily practice in burden on public health. The proposed NoE includes an effective plan for Europe. A successfully outcome of this programme would provide spreading excellence, exploiting results and disseminating knowledge to compelling evidence for the wisdom of further investment in Europe at those outside the network, and is likely to have a durable structuring this interface of genomics and health care. Existing isolated research impact on European research. The GRACE concept could serve as networks have limited capacity to perform the large trials that are a model of how different disciplines can be integrated, for translating necessary to provide the evidence base for action. By co-ordinating genomics into better patient care, or for translating basic science into networks in different countries and paying close attention to the clinical care. The current project combines epidemiological with molecular diffi culties of recruiting in everyday practice, which are a major diffi culty biological expertise for the study of the aetiology of LRTI and antibiotic of randomised trials internationally, GRACE will provide an unparalleled resistance profi les in key pathogens. High-throughput technology will be opportunity to generate the data that is needed to improve practice. used to provide, for the fi rst time, a rapid genome-wide test platform for There is no doubt that GRACE is unique in deploying a structure, which monitoring causative agents and antibiotic resistance on a large scale. links elegantly laboratory science with improved European health care.

Project number LSHM-CT-2005-518226 EC contribution € 11 500 000 Project Duration 60 months Starting date 01/03/2006 Instrument NoE

96 | ANTI-MICROBIAL DRUG RESISTANCE Coordinator Birgitta Henriques Normark Roger Finch Guido Krupp Dept of Bacteriology School of Clinical AmpTec GmbH Herman Goossens Solna, Sweden Laboratory Sciences Hamburg, Germany Medical Microbiology University of Nottingham Universiteit Antwerpen Christopher C. Butler Clinical Sciences Building Peter van den Broek Campus Drie Eiken University of Wales College of Medicine Nottingham, United Kingdom Primagen Holding Universiteitsplein 1 S3 Llanedeyrn Health Centre Amsterdam, The Netherlands B-2610 Wilrijk, Belgium Cardiff, United Kingdom Fransesco Blasi Phone: +32 3 821 37 89 Instituto di Tisiologia e Malattie Jan Schouten Fax: +32 3 825 42 81 Theo Verheij dell’Apparato Respiratorio MRC-Holland [email protected] Julius Center for Health Universita degli Studi di Milano Amsterdam, The Netherlands Sciences and Primary Care Milano, Italy Universitair Medisch Guus Simons Partners Centrum Utrecht Peter Garred Pathofi nder Utrecht, The Netherlands Tissue Typing Laboratory-Section 7631 Ede, The Netherlands Greet Ieven Copenhagen, Denmark Medical Microbiology Robert van Veen Thierry Leclipteux Universiteit Antwerpen Julius Center for Health Staffan Normark Coris BioConcept Campus Drie Eiken Sciences and Primary Care Karolinska Institutet, MTC Gembloux, Belgium Wilrijk, Belgium Universitair Medisch Centrum Utrecht Solna, Sweden Utrecht, The Netherlands Javier Garau Willy Spaan Regine Hakenbeck ESCMID Leids Universitair Medisch Centrum Anton van Loon Universität Kaiserslautern University of Barcelona Leiden, The Netherlands Universitair Medisch Kaiserslautern, Germany Hospital Mutua de Terrassa Centrum Utrecht Terrassa Barcelona, Spain Adrian V.S. Hill Utrecht, The Netherlands Hermínia de Lencastre Wellcome Trust Centre Instituto de Tecnologia Eiselé Jean-Luc for Human Genetics Paul Little Química e Biológica ERS Oxford, United Kingdom Faculty of Medicine Laboratory of Molecular Genetics Lausanne, Switzerland Health and Biological Oeiras, Portugal Derrick Crook SciencesPrimary Medical Care John Radcliffe Hospital University of Southampton José Campos University of Oxford Aldermoor Health Centre Bacteriology Oxford, United Kingdom Southampton, United Kingdom Instituto de Salud Carlos III Majadahonda, Spain David Holden Richard Smith Imperial College London Health Economics Group Joanna Coast London, United Kingdom School of Medicine Health Economics Facility Health Policy and Practice University of Birmingham University of East Anglia Birmingham, United Kingdom Norwich, United Kingdom

ANTI-MICROBIAL DRUG RESISTANCE | 97 MagRSA

Fully automated and integrated Microfl uidic Platform for Real-time Molecular Diagnosis of Methicillin-resistant Staphylococcus Aureus

Keywords Magnetic nanoparticles, Microfl uidics, Problem Quantitative PCR, Antimicrobial Resistance, Methicillin-Resistant Staphylococcus Aureus (MRSA), Methicillin-resistant Staphylococcus Aureus (MRSA), an organism Molecular Diagnostics, quantitative PCR, DNA-array, resistant to many drugs, is seen with increasing frequency in hospitals Microfl uidics, magnetic particles, Molecular Diagnostics, and long-term care facilities. It can cause life-threatening disease, and automated platform treatment options are limited. MRSA infections are indeed associated with a 40 % mortality when found in the blood of patients suffering from severe staphylococcal infection. According to the World Health Organization (WHO), resistance of Staphylococcus Aureus (Staph A) to methicillin, its usual antibiotic, increased from 2 % in 1975 to 60 % today and no new antibiotic is expected on the market before at many years. Whereas MRSA is considered as a nosocomial pathogen, recent Summary reports showed an increasing number of outbreaks in the community, despite the absence of known risk factors (prior hospitalization, Methicillin-resistant Staphylococcus aureus (MRSA), a virulent organism antibiotic use or household contacts from the healthcare system). resistant to many drugs, is responsible for most nosocomial and community- Indeed, a relatively large spread of MRSA strains within the gay acquired infections. It can cause life-threatening disease, and treatment community was recently reported several European countries and the options are limited. Effective diagnostics is a strategic key element in the United States. Such atypical MRSA is known to produce a potent toxin campaign against the spread of MRSA, allowing better infection surveillance causing severe skin infections and necrotising pneumonia in both and control measures as well as more effi cient patient treatment and/or immunocompromised and immunocompetent individuals. isolation options. The MagRSA project aims at the development of a new diagnostics platform that will provide a fast, simple and accurate With such critical health issues, an early detection of MRSA carriers is identifi cation of MRSA from clinical samples. crucial for infection control strategies but also to take appropriate therapeutic decisions, avoiding non-appropriate utilization of last barrier antimicrobial agents. Strategies to fi ght MRSA transmission are indeed well-described, and can effi ciently reduce subsequent colonization and infection. However, for cost issues and better patient management, these strategies need to be focused on patients with confi rmed MRSA and defi ned resistance phenotype.

Aim

The MagRSA project aims at the development of a new diagnostics platform that will provide a fast, simple, automated and accurate identiﬁ cation of MRSA from clinical samples.

The diagnostic protocol that we propose to develop relies on a new and clinically validated procedure that consists of a direct one-step enrichment of MRSA present in either nasal or inguinal swabs, followed by DNA extraction of immunocaptured bacteria and their identifi cation by multiplex sequence amplifi cation using real-time quantitative PCR. This protocol will be implemented with a simple ‘hands-off’ system based on: • novel strategies for the integration of full operations required for the entire nucleic acid analysis chain in a microfl uidic platform; • advanced microfl uidic magnetic nanoparticles manipulation technology allowing effi cient capture and extraction of target bacteria and nucleic acids. The separate steps of sample preparation, signal amplifi cation by multiplex PCR, and simultaneous detection of multiple genes, will be performed as one single step using a ready- to-use disposable fl uidic chip.

98 | ANTI-MICROBIAL DRUG RESISTANCE Staphylococci growing on agar plates (yellow colonies are Staphylococcus aureus, and white ones are Staphylococcus epidermidis).

In light of the above, this project aims at providing hospitals and care Coordinator Partners units with a fast, easy and automated test for the rapid diagnostic of MRSA. Moreover, the simplicity of the proposed technology concept Prof. Jacques Shrenzel Dr. Amar Rida integrating cost effective and widely available components allows to Geneva University Hospitals Spinomix SA Division of Infectious Diseases Scientifi c Park EPFL, PSE-C provide low cost systems, a prerequisite condition for the large adoption Genomic Research Lab Lausanne, Switzerland of molecular tests by hospitals. Rue Micheli-du-Crest 24, CH-1211 Geneva, Switzerland Dr. Neven Zoric Phone: +41 22 372 7301 TATAA Biocenter AB Expected results Fax: +41 22 372 7304 Göteborg, Sweden [email protected] The MagRSA project measurable and quantifi able objectives can be Dr. von Stetten Felix classifi ed in three categories: Faculty of Applied Sciences • new molecular diagnostics protocol allowing effi cient and reliable Laboratory for MEMS Applications MRSA diagnostics and genotyping; Freiburg, Germany • new assay reagents including magnetic nanoparticles for sample Dr. Emanuel Gaboyard preparation and quantitative PCR (Q-PCR) related reagents; ADEMTECH SA • fully automated systems, mainly based on advanced microfl uidics Pessac, France and nanoparticles handling technologies, for MRSA diagnostics and genotyping.

Potential applications

The MagRSA project will address the unmet need for new diagnostics tools for management and control of antimicrobial resistance in general and Methicillin-resistant Staphylococcus Aureus (MRSA) in particular. Moreover, MagRSA project will provide a diagnostics platform with potential applications in molecular diagnostics as the most growing segment within the global in-vitro-diagnostics market.

Project number LSHM-CT-2006-037957 EC contribution € 2 095 800 Project Duration 36 months Starting date 01/10/2006 Instrument STREP Project website www.MagRSA.org

ANTI-MICROBIAL DRUG RESISTANCE | 99 MANASP

Development of novel management strategies for invasive aspergillosis

Keywords Immunotherapy, vaccine, antigens, T cells, dendritic vivo-generated, donor-derived, Aspergillus-specifi c T-cells might be cells, A. fumigatus, PCR diagnostics, monoclonal antibodies, benefi cial for recipients of allogeneic HSCT. GMP-conditions, aspergillosis, transplantation There are only raw data regarding immunotherapy of patients with invasive fungal infection, and minimal data relating to IA. This might be due, at least in part, to the complex antigenic properties of A. fumigatus, which have been less well characterized compared to viruses such as CMV or EBV. Only a minority of the hundreds of (glyco)proteins of Summary A. fumigatus reported in the literature have been characterized at either a molecular and/or biochemical level. • Our goal is to develop new treatment strategies for Invasive Aspergillosis (IA) which has become the major infectious complication The diagnosis of IA is a particular challenge. Conventional microbiological of treating haematological malignancies with intensive chemotherapy methods fail to detect most cases so that current diagnostic categories or haematopoietic stem cell transplantation (HSCT). are based on a combination of clinical, imaging, and histopathological • Ineffective host immune responses facilitate fungal invasion of the (often post-mortem) criteria. pulmonary system and other vital organs leading to death. In order to redress this immunological imbalance we propose to develop new There have been some advances with non-culture techniques based on immunotherapeutic strategies which will augment current antifungal PCR based amplifi cation of fungal DNA or detection of Aspergillus treatments and reduce morbidity and mortality. antigens in blood samples. However these have not been validated as • Facilitated by recent publication of the A. fumigatus (the principal tools for identifying patients at risk or for monitoring responses to pathogen of the genus) genome sequence we will exploit new treatment by determination of fungal loads. knowledge and techniques in genomics and post-genomics. We will identify the immunologically important fungal molecules to design immunotherapies based on vaccines using fungus-primed innate Aim immune cells, monoclonal antibodies or adoptive transfer of specifi c T lymphocyte clones. The overall goal of our STREP is to develop more effective management • We will also generate new nucleic acid based diagnostic tests to strategies for IA with the following aims: inform when and how immunotherapy can be optimally applied. • Development of immunotherapeutic strategies for Invasive The outcomes will have strong commercial applications which will be Aspergillosis. This will be achieved by identifying the T-cell clones delivered by three leading European SMEs within the consortium. that react with specifi c Aspergillus targets and evaluating their effi cacy for prevention and treatment of IA. Success will be measured by characterization of the generated T-cells, and demonstration of their functional properties against Aspergillus. • Development of improved diagnostic tests for IA with commercial potential. This will be achieved by identifying appropriate nucleic acid targets, extraction and amplifi cation protocols and demonstrating their sensitivity and specifi city in clinical samples. Problem • Validation of a dendritic cell based vaccine immunotherapy strategy in animal model to generate protective immunity against Aspergillus. Over the past decade, invasive aspergillosis (IA) has emerged as the most • Use of genomic and proteomic techniques to identify new serious life-threatening infectious complication of intensive remission- Aspergillus targets that interact with the host’s immune system. induction chemotherapy and allogeneic HSCT in patients with a variety of haematological malignancies. Aspergillus fumigatus is the most commonly isolated species from cases of IA and is the focus of our research although we believe the intended outcomes will act as a paradigm for management of IA due to less common species such as A fl avus and the emerging A terreus. Despite the limited improvements that have been made with preventative strategies and the development of new antifungal drugs, IA has an incidence of 10-30 % and is still associated with high mortality rates as high as 90 % in some surveys. T lymphocytes provide a critical secondary defense against this and other fungal pathogens. Therefore, Germination of a conidium in a macrophage Conidiophores of A. fumigatus. Aspergillus-specifi c T-cell immunity, transferred through the infusion of ex of an immunocompromised host.

100 | ANTI-MICROBIAL DRUG RESISTANCE Macrophage – conidia interaction.

Coordinator Prof. Luigina Romani Expected results University of Perugia Prof. Hermann Einsele Perugia, Italy • Identifi cation of different pattern recognition receptors in response University of Wuerzburg Medizinische Klinik II Dr. Jean Kadouche to A. fumigatus and their role in activating DCs. Klinikstrasse 6-8 Monoclonal Antibody Therapeutics • Identifi cation of PAMPs of A. fumigatus useful for immunotherapy 97070 Wuerzburg, Germany Evry, France strategies. Phone: +49 931 20170010 • Identifi cation of murine DC subsets capable of inducing antifungal [email protected] Prof. Axel Brakhage Th responses. Leibniz Institute for Natural Product • Adoptive transfer of manipulated DC into mice suffering from IA. Research and Infection Biology • Characterization of Aspergillus-specifi c T-cell response in healthy Partners Jena, Germany individuals. • Identifi cation of a protective T-cell response in patients surviving IA. Dr. Birger Jansson Dr. Georg Rauser • Development of a clinical protocol under current cGMP-conditions Cepheid AB Miltenyi Biotec Bromma, Sweden Bergisch Gladbach, Germany for treatment of patients with IA.

• Modulation of Aspergillus-specifi c immune response by transduced Prof. Tom Rogers Prof. Jean Marie Francois T-cells. Trinity College Institut National des Sciences • Preparation of a bank of monoclonal antibodies against A. Dublin, Ireland Appliquées, Département de Génie fumigatus. Biochimique et Alimentaire • Selection of the most effi cient antibodies by in vitro and in vivo Prof. Jean Paul Latgé Toulouse, France analysis. Institute Pasteur • Development of an assay to detect Aspergillus DNA with high Unité Aspergillus specifi city. Paris, France • Development of an assay to detect Aspergillus RNA with high sensitivity. • Clinical evaluation to confi rm sensitivity in detecting Aspergillus infection using standardised defi nitions produced by International consensus and comparison to different well established assays. • Commercialization of the assay into an affordable and rapid diagnostic test.

Potential applications

• Surveillance of patients being treated for haematological malignancies by chemotherapy or HSCT using DNA/RNA based molecular diagnostics. This will generate greater ascertainment of possible and probable cases of IA and earlier therapeutic intervention leading to improved survival rates. • Incorporation of these diagnostic tests into internationally accepted EORTC/MSG criteria for clinical application in categorising cases of Project number IA to facilitate research trials of new antifungal agents or other LSHM-CT-2006-037899 novel therapies. EC contribution • Development of immunotherapeutic strategies based on demonstrated € 2 914 000 efﬁ cacy against Aspergillus in vitro and in animal models. This will Project Duration either be through adoptive transfer of donor T-cells, or administration 36 months of monoclonal antibodies targeted against immunologically relevant Starting date Aspergillus antigens/epitopes. 01/01/2007 • Wider application of this technology to the treatment of other Instrument groups of patients (outside the Haematological Malignancy ﬁ eld). STREP Project website www.manasp.org

ANTI-MICROBIAL DRUG RESISTANCE | 101 MOSAR

Mastering Hospital Antimicrobial Resistance and its Spread into the Community

Keywords Antimicrobial resistance, cross-transmission, spp have been described recently in many European hospitals. There is clearly diagnostic tests, infection control, prevention, typing, a need for recommendations to combat the emergence of these organisms statistical models in Europe with respect to recognition, detection, and prevention of transmission.

Although most authorities agree that control of AMRB such as MRSA, VRE, extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, and Acinetobacter spp. is desirable, the most appropriate strategies for Summary control remain elusive. While interventions to reduce patient-to-patient transmission are considered the cornerstone of infection prevention, their Infections caused by antimicrobial-resistant bacteria (AMRB) account for feasibility and cost-effectiveness have not been determined rigorously. an increasing proportion of healthcare-associated infections in European Evidence from prospective, multi-facility studies regarding the relative hospitals, particularly in high-risk units, e.g., intensive care units and effi cacy and cost-effectiveness of different control strategies is lacking. The surgery. Patients discharged to rehabilitation care units often remain long- lack of strong scientifi c support to recommendations for control of AMRB term carriers of AMRB, contributing to their dissemination into other care substantially weakens their impact and contributes to the marked areas and within the community. Increased prevalence of resistance to heterogeneity of preventive practices across institutions. antibiotics of major hospital pathogens is associated with adverse outcomes of patients affected, which include delayed appropriate therapy or even failure of therapy, prolonged length of hospital stay, and increased Aim mortality. While the optimal strategies for control of AMRB remain debated, understanding the dynamics of resistance and the relative contribution of The overall objective of MOSAR is to provide advanced knowledge in the most important determinants of dissemination of AMRB (cross- the dynamics of transmission of AMRB, and address the controversies transmission via contacts, epidemicity of strains and antimicrobial selective surrounding control measures by testing different strategies to combat pressure), is needed to better defi ne these strategies. The integration in the emergence and spread of antimicrobial resistance, focusing on these approaches of rapid molecular diagnostic testing for AMRB carriage endemic or emerging AMRB in hospitals, now spreading into the may improve the timeliness and effi cacy of control measures. community. Microbial genomics and human response to carriage of AMRB will be integrated in MOSAR with health sciences research, including interventional controlled clinical studies of strategies for control of AMRB in ICUs, surgical wards and rehabilitation centers, incorporating the use of new molecular diagnostic tools for rapid detection of carriage; medico-economic evaluation of the strategies tested will be conducted in parallel to inform policy makers. The data Problem gathered during the clinical trials will be used to advance our understanding of the dynamics of resistance through mathematical The prevalence of AMRB has been rising worldwide during the past decades. modelling, integrating several factors contributing to the spread of AMR Data gathered within the European surveillance system (EARSS project) show strains to inform on: that resistance rates for major nosocomial pathogens have increased up to • the real-time analysis of the relative contribution of exposure to alarming levels in many countries, despite persistently low rates among a few antibiotics and resulting selective pressure; others, mostly from northern Europe. Each of the AMRB listed below can • the intrinsic characteristics of epidemic clones that contribute to colonise hospitalised patients in its specifi c niche, who then become occult inter-individual transmission (‘epidemicity’ of strains); carriers and may contribute to the further dissemination of AMRB within • the identifi cation of factors contributing to the transmission of facilities or upon transfer to other facilities and into the community. strains between individuals in the hospital population and com- Rehabilitation centres could serve as a large reservoir of AMRB, with the munity (e.g., ‘transmission hubs’ linked to caregivers and direct potential for amplifi cation and dissemination into the community. Indeed, contacts between patients). there is growing concern with e.g., community-acquired methicillin-resistant Staphylococcus aureus (MRSA), causing small outbreaks in specifi c populations. To achieve these objectives, MOSAR brings together experts in basic Exchange of genetic material and resistance traits can also occur between laboratory sciences, hospital epidemiology, clinical medicine, behavioural related or unrelated species, further worsening the problem. For example, co- sciences, quantitative analysis and modelling, and health economics. colonization with MRSA and vancomycin-resistant enterococci (VRE), which MOSAR involves 11 institutions recognized for their leadership in these frequently occurs in patients with prolonged stay, facilitates horizontal transfer fi elds from 10 EU Member or Associated States, as well as 7 SMEs to of vancomycin-resistance genes, transforming MRSA into vancomycin-resistant develop and validate high-throughput automated molecular tools for S. aureus (VRSA). Several outbreaks with VRE and multi-resistant Acinetobacter detection of AMRB.

102 | ANTI-MICROBIAL DRUG RESISTANCE Expected results The results of the observational and interventional studies contained within the MOSAR integrated project will provide evidence to base • Development of standards for conventional methods for detection additional support for, or the modifi cation of, the standards on which to of AMRB in screening samples. base interventions to combat dissemination of antibiotic resistance • Development and validation in the clinical setting of high- within healthcare facilities, against which local practices can be audited. throughput molecular-based methods for detection of resistant Mathematical modelling will facilitate the generalizability and imple- bacteria in screening for carriage of AMRB in clinical samples, and mentation of these interventions to make these standards relevant to all assessment of their cost-utility. healthcare systems across Europe. These interventions will enhance • Establishment of a bank of well-characterised clinical isolates of MRSA, infection control practices and are likely to subsequently impact on VRE, and ESBL-producing Enterobacteriaceae and Acinetobacter spp. antibiotic use and resistance. Initiatives for change in practice can be • Characterisation of subtypes of the MRSA and VRE associated with directed towards these standards. Knowledge gained through MOSAR hospital outbreaks, and analysis the molecular basis of the will also help formatting education and training of healthcare personnel epidemicity of corresponding MRSA and VRE strains. and beyond. • Assessment from prospective, multi-centre studies in areas with high prevalence of antimicrobial resistance (ICUs, surgical units, Through development of models forecasting levels of antimicrobial rehabilitation centers), of the relative effi cacy and cost-effectiveness resistance, accounting for the major players (control measures, antibiotic of different control strategies including enhanced standard pressure and strains involved), MOSAR will provide healthcare staff and precautions, screening for carriage using conventional or high- infection control personnel with user-friendly instruments designed to throughput molecular methods, and decolonisation in surgical provide short-term and real-time forecasting of antimicrobial resistance patients. within a given setting. These instruments will be developed to interpret • Defi nition of the cost-effectiveness and applicability of strategies outcomes from intervention studies and help design future strategies for control of AMRB in high-risk areas in the hospital setting. for control. • Development of mathematical models to assess the potential value of the tested control measures in various environments and their long-term effects, allowing prediction of the effi cacy of interventions in different populations, settings, timescales, and variants of interventions. • Development of mathematical models integrating the contribution of the intrinsic epidemicity of MRSA, of cross-transmission and of antibiotic use in the dynamics of resistance, to be translated into user-friendly interfaces for use by infection control personnel.

Potential applications

Results from MOSAR will inform healthcare workers and decision- makers on strategies for forecasting and mastering antimicrobial resistance. The results from MOSAR should increase awareness of nosocomial pathogens such as VRE as an emerging cause of hospital acquired infections. Project number MOSAR will contribute to develop the next generation state-of-the art LSHM-CT-2006-037941 technologies of diagnostic tests. The currently available molecular tests EC contribution and the newly developed technologies will be adapted to the different € 10 000 000 needs of the laboratories and countries participating in MOSAR. Project Duration 60 months In addition, the standardisation of conventional tests and high Starting date throughput assays for the detection of AMR will facilitate a common 1/02/2007 approach to microbiological practice, and allow the meaningful Instrument comparison of results from different settings. IP Project website www.mosar-ip.org

ANTI-MICROBIAL DRUG RESISTANCE | 103 Coordinator Prof. Jordi Vila Prof. Isabelle Durand-Zaleski Institut d’Investigacions Université Paris Val de Marne Prof. Christian Brun-Buisson Biomèdiques August Pi I Sunyer Créteil, France Institut National de la Santé University of Barcelona et de la Recherche Médicale (INSERM) Barcelona, Spain Prof. Eric Fleury INSERM U657 & Université Paris Institut National de la Recherche en Val de Marne (UPVM) Prof. Marc Bonten Informatique et Automatique Hôpital Henri Mondor University Medical Center Utrecht CITI/ARES – INSA Lyon Assistance Publique-Hopitaux de Paris Julius Center for Health Sciences Villeurbanne, France 51, Ave Mal De Lattre de Tassigny and Primary Care Créteil, France Utrecht, The Netherlands Dr. Patrick Trieu-Cuot Phone: +33 1 4981 2391 Institut Pasteur Fax: +33 1 4207 9943 Dr. Stephan Harbarth Paris, France [email protected] University of Geneva Hospitals Infection Control Program Dr. Jérôme Weinbach Geneva, Switzerland INSERM Transfert SA Partners Paris, France Dr. Yehuda Carmeli Dr. Didier Guillemot Medical research Fund of the Tel Aviv Dr. Camille Cyncynatus Institut National de la Santé Sourasky Medical Center AbAg SA et de la Recherche Médicale Division of Epidemiology Chilly-Mazarin, France INSERM U657 & CeRBEP Tel Aviv, Israel Institut Pasteur Dr. Paul Wallace Paris, France Dr. Robert Veen British Biocell International Ltd University Medical Center Utrecht Cardiff, United Kingdom Prof. Herman Goossens Julius Center for Health Sciences University of Antwerp and Primary Care Dr. Guido Krupp Antwerp, Belgium Utrecht, The Netherlands AmpTec GmbH Hamburg, Germany Prof. Waleria Hryniewicz Dr. Ben Cooper National Medicines Institute Health Protection Agency Dr. Dirk Fischer Warsaw, Poland Statistics, modelling and Array-on GmbH economics unit Gatersleben, Germany London, United Kingdom

104 | ANTI-MICROBIAL DRUG RESISTANCE NPARI

Tailoring of Novel Peptide coatings and therapeutics derived from a newly identifi ed component of the human innate immunity Against Resistant Infections

Keywords Peptide, resistance, infection antibacterial, rate of up to 40 %. These organisms are the same organisms where antifungal, therapeutic agent, coating agent resistance is a major issue. The incidence of resistant bacterial and fungal nosocomial infections is high. The European Study Group on Nosocomial Infections (ESGNI) reports on blood stream infections indicated 72.8 % of infections were nosocomial and mortality due to bacteremia was 7.1 %. The most frequently isolated microorganisms from BSI were S. aureus (15.1 %), E. coli (14.5 %), S. epidemidis and coagulase negative staphs (CNS) (17.8 %), P. aeruginosa/K. pneumo- Summary niae (both 5.3 %) and Candida spp and enterococci (both 4.6 %) (ESGNI-001, ESGNI-002). The apoE and apoB human proteins have recently been linked to the innate immune system. Peptide sequences derived from these proteins have been shown to have varied anti-infective properties which can be Aim modifi ed by small changes to the core peptide sequence. Thus the apoE and apoB peptides exhibit antibacterial, antifungal and antiviral The aims of the NPARI consortium are to fully exploit the exciting properties and present and excellent opportunity to develop novel properties of this novel peptide class. Speciﬁ cally the consortium aims to therapeutics and medical device coatings. Specifi cally the exploitation of target peptide sequences into two areas; coating agents for medical these novel peptides allows for the potential development of a new array devices and therapeutics agents. of agents targeting against the growing problems of antibiotic resistant microorganisms. A major application for the apoE peptides is as coatings for medical devices such as catheters. ApoE derivative peptides are already in development as coatings for contact lenses. We aim to extend this application to use active peptides as coating agents for catheters. Catheter related infection causes signiﬁ cant morbidity and mortality across the EU and resistant bacteria and fungi are often responsible.

Another focus for the application of apoE peptides is as therapeutic Problem agents. We will focus on targeting specifi c resistant bacteria and fungi in order to rapidly establish in vivo effi cacy in a variety of animal models. Infectious diseases represent the commonest cause of morbidity in the We will specifi cally target respiratory infections caused by Pseudomonas world (WHO). Over the last 40 years major advances have been made in and Apsergillus. This will have direct clinical relevance to the 30 000 the development of numerous classes of antimicrobial agents to treat European CF patients where resistant Pseudomonas aeruginosa infec- serious life threatening infections. This is particularly true for antibacterial tions are a major cause of mortality. Colonisation with Aspergillus is also agents. However microorganisms are slowly turning the tide and a major problem is this patient group. becoming increasingly resistant to the agents developed by man. Long term and indiscriminate use of antibacterials has led to resistance developing for all the major classes of therapeutic agents. Increasingly Expected results clinicians are fi ghting a rearguard action with a dwindling armoury of drugs to combat serious life threatening infections. Nosocomial or • The design of a small peptide library which will be tailored to the hospital acquired infections (HAIs) represent an increasingly serious proposed exploitable application of the project. problem across Europe and the rest of the world. • Determination of the activity spectrum of active peptides and the ranking of peptide variants. Data from the SENTRY Antimicrobial Surveillance program of 25 Euro- • Optimisation and toxicity profi ling of active peptides. pean university hospitals highlighted the fi ve most common bacterial • Effi cacy profi les against a panel of resistant organisms growing as blood isolates. The most prevalent organisms (E. coli, S. aureus, biofi lms. P. aeruginosa and K. pneumoniae) are also the most prevalent CVC • Pharmacological and effi cacy evaluation of peptides in a range of associated infections (Clin Inf Dis 30; 3). Candida species were also models. common organisms isolated from blood and carried a crude mortality • Effi cacy data for candidate coatings in dynamic biofi lm models.

ANTI-MICROBIAL DRUG RESISTANCE | 105 Potential applications Coordinator Dr. Jean-Marc Ghigo Groupe de Génétique des Biofi lms The exploitation of this new class of antimicrobial peptides offers the Dr. Mike Birch CNRS URA 2172, Institut Pasteur potential to develop new therapeutic against a range of the most F2G Ltd Paris, France Po Box 1, Lankro Way resistant and problematic organisms facing European Infectious Disease Eccles Dr. Chiara Bigogno Clinicians. Manchester NiKem Research s.r.l. M30 OBH, United Kingdom Giulio Dondio, Analytical Department The rate infection by resistant bacteria is increasing but new chemical [email protected] Baranzate, Italy agents are some way from the clinic. This project aims to develop candidate peptide therapeutics which target the most problematic Prof. Niels Høiby organisms and develop them to the point were they show sufﬁ cient Partners University Hospital of Copenhagen potential for further development. Copenhagen, Denmark Dr. Christophe d’Enfert In addition the project focuses on the prevention of infection by the Biologie et Pathogénicité Fongiques Dr. Curtis Dobson INRA USC 201 Ai2 Ltd. same resistant organisms through the coating of medical devices with Institut Pasteur Manchester Incubator active peptides. Paris, France Manchester, United Kingdom

By preventing the colonisation of catheters by resistant microorganisms it is expected that serious life threatening infections can be avoided.

Project number LSHM-CT-2006-037692 EC contribution € 2 800 000 Project Duration 36 months Starting date 01/12/2006 Instrument STREP Project website www.npari.org

106 | ANTI-MICROBIAL DRUG RESISTANCE REBAVAC

Novel opportunities to develop vaccines to control antibiotic resistant bacteria: from the trials back to the laboratory

Keywords Antibiotic resistance, vaccine, infection Another important pathogens for which antibiotic resistance is increasing and spreading is Streptococcus pneumoniae (Spn). Vaccines currently in use to fi ght encapsulated bacteria like Spn are based on polysaccharides constituting the capsule conjugated with carrier proteins. The 7-valent conjugated vaccine against Spn appears not only to protect against infection but also decreasing the rate of antibiotic-resistant invasive Summary pneumococcal infections in young children and older persons. However after vaccination campaigns there was an increase in infections caused Vaccination appears to be the best way to defeat antibiotic resistant by serotypes not included in the vaccine. In addition the Spn conjugated bacteria. Conjugated polysaccharide vaccines are quite effi cacious and vaccine is very expensive and consequently un-affordable for the poorest represent a good solution to elicit protective immune responses to population, where the higher number of Spn infections occur. encapsulated bacteria. However, the analysis of the effects of using for several years conjugated polysaccharide vaccines to encapsulated The analysis of the effects of using conjugated vaccines against Spn, bacteria is raising questions on how immunity to polysaccharide is Haemophilus infl uenzae b (Hib) and Neisseria meningitidis (Men) elicited following vaccination. showed some paradoxes and some interesting aspects that lead to a With this project we aim to organise a Workshop involving the most re-thinking about how immunity to polysaccharide is elicited following important vaccine experts worldwide to address several questions raised vaccination, and how memory is acquired. from the analysis of the vaccination trial results in order to develop new ideas to improve vaccination to antibiotic resistant bacteria. For all these reasons the Workshop proposed by REBAVAC that will see The main objectives of the Workshop are: the presence of important experts in vaccination, in immunology and in • to evaluate the current status of conjugated vaccines against bacterial resistance, both from industry and academy, will represent encapsulated bacteria; a very important occasion in Europe to discuss the implication of the • to verify the parameters used to define immune memory after above mentioned results and of the results of ongoing research on vaccination; vaccine development and on vaccination campaigns against antibiotic • to indicate ways to improve protection against antibiotic resistant resistant bacteria. The current status of protein-based vaccines will be bacteria. In addition, the role of carrier proteins and the current analysed and the importance of innovative vaccination strategies status of protein-based vaccines will be analysed. evaluated to give directions to the European research for the development In a training session young scientists will be given the opportunity to of more effi cacious, universal and affordable vaccines to defeat antibiotic present their research, whereas a round table with industries resistant bacteria. representatives, policy makers and scientists will be organised to discuss the effi cacy of vaccination and the economical and political aspects related with the development, use and distribution of vaccines to Aim encapsulated bacteria. A fi nal Consensus Document on “Future trends in vaccine development to defeat antibiotic resistant bacteria” will be The overall aim of REBAVAC is to organise a European Workshop in prepared and made available to the public. which the worldwide most important experts in vaccinology and immunology will meet with healthcare providers, industry representatives, and public health experts to: • discuss if the currently available conjugate polysaccharide vaccines can still be considered the best tool to fi ght antibiotic resistant bacteria; • discuss the results of clinical trials on development and persistence of immunity to polysaccharides in infants, children, adults and eld- Problem erly people after vaccination with conjugate polysaccharide vaccines and the impact on polysaccharide switching; Recent years have witnessed a rapidly growing crisis in antimicrobial • better understand the contribution of innate and adaptive compo- resistance, especially among microorganisms that cause nosocomial nents of the immune system in eliciting and boosting immune infection: Methicillin-resistant Staphylococci, vancomycin resistant responses to conjugate vaccines; Enterococci and extended-spectrum beta-lactamase (ESBL) producing • discuss the role and the function of the carrier protein(s) in the Gram negative bacilli. Vaccines against these pathogens are either not elicitation and persistence of protective immune responses and to available yet or demonstrate just a limited duration of effectiveness of evaluate if there a risk of carrier proteins- overload in fi rst-second the antibodies produced. Development and testing of more effective year of life; vaccines is under way.

ANTI-MICROBIAL DRUG RESISTANCE | 107 Scientists at work to improve human health. By Ivo Saglietti.

• evaluate if the currently used parameters to defi ne memory are In addition the discussion on the defi nition of the parameters to measure appropriate to defi ne the protection elicited by vaccination; protective immunity after vaccination will be considered a bench-mark • check if the vaccination schedules in use are appropriate; for any study aiming to the development any future vaccine for antibiotic • discuss the indirect effects of infant and children vaccination on resistant bacteria, while the observations on the vaccination schedules the epidemiology of the diseases (herd immunity) and how that might infl uence the vaccination campaigns of European and non- can infl uence the vaccination policy; European countries. • assess the current status of protein-based vaccines; • evaluate novel vaccination strategies for antibiotic resistant bacteria. Project website

Expected results A dedicate page will be created in the www.altaweb.it area in which the summary of the talk of invited speakers and the results of the Conference The outcomes of the Workshop will surely give an input to European should be available for young investigators, interested organisations, research and industry to move forward towards more effi cient/effi cacious public audience. The Consensus Document mentioned above will be vaccines and vaccination strategies, to fi nd novel immunization ways to inserted into it. Contracts with companies will be encouraged, in particular optimise the use and formulation of currently available vaccines. with small biotechnology companies. Consensus Document on “Future trends in vaccine development to defeat antibiotic resistant bacteria” will The Workshop aims indeed to provide European researchers with the be made available to the extended public on the project website. newest trends and directions of research in the area of vaccines to antibiotic resistant bacteria and at envisaging possible exploitation for the design of new vaccination strategies. Coordinator The expected global achievement at the end of the Workshop is a Consensus Document on “Future trends in vaccine development to Dr. Aldo Tagliabue defeat antibiotic resistant bacteria” that will be available in the project CEO ALTA Srl website to allow for measuring the success of the project and it will Via Fiorentina 1 include critical indications of the future trends and directions to be 53100 Siena, Italy followed by European academic and industrial players and public health Phone: +39 0577 719064 organizations in the fi eld of vaccines to defeat antibiotic resistant Fax: +39 0577 278515 bacteria. [email protected]

Potential applications

The impact of the Scientifi c Workshop and of the correlated activities planned by REBAVAC project will be very strong in the fi eld of developing new strategies against antibacterial resistant infections. The REBAVAC project will have an important impact on European industry and research because: • it will capitalise on an existing base of business and skills in Europe in the vaccine fi eld; Project number • it will design vaccination strategies that will increase the market LSHM-CT-2006-037163 and therefore the output and work for European workforce; EC contribution • it will contribute to the control of infectious diseases, giving a new € 121 200 input to the fi ght against antibacterial resistance, therefore Project Duration contributing to the well being of the European Union and the 12 months whole world. Starting date 01/12/2006 Instrument SSA Project website www.altaweb.eu/rebavac

108 | ANTI-MICROBIAL DRUG RESISTANCE StaphDynamics

Functional genomic characterisation of molecular determinants for staphylococcal fi tness, virulence and drug resistance

Keywords Staphylococcus, epidemiology, antibiotic Problem resistance, fi tness, virulence, target identifi cation, vaccine The quality of life of patients infected with antibiotic-resistant microorganisms is dramatically decreased. Hospital stays of such patients are prolonged and infections often show very dramatic effects on the individual health situation. Moreover, infections with antibiotic resistant micro-organisms even lead to a higher morbidity in specifi c risk groups, such as elderly, immune-suppressed patients and children. Accordingly, Summary the StaphDynamics programme addresses the general need for generating specifi c knowledge and tools to signifi cantly enhance the Staphylococcus aureus is a dangerous community- and hospital-acquired quality of life of infected patients, and it contributes to meeting the pathogen. Therefore, the spread, survival and prevalence of antibiotic societal challenge of an ageing European population. This is particularly resistant clones of S. aureus represent an important problem for human relevant in view of the cost burden for the national health systems health. The selective pressure for antibiotic resistance is a driving force incurred by these untreatable bacteria. Since medical knowledge and due to the massive use of such drugs throughout the world. However, the technical standards are continuously increasing and the European overall fitness and virulence of the bacteria is determined by their population is ageing at the same time, the demand for high quality underlying genetic makeup. The traits, which determine the ability of medical care is growing. Therefore, the costs for national health systems a clone to become widespread, are largely unknown. These are likely to will increase even more over the next years. Supporting research in the be multifactorial, as S. aureus is widespread in the environment due to its area of antibiotic-resistance is therefore not only a response to ability to survive outside the host and to be carried as a commensal expectations of society, but will also contribute to ensuring sustainability bacterium in the human nose. It is therefore crucial to determine the key of the European public health systems. parameters required for virulence, nasal colonisation and survival in the environment in order to elucidate how these combine to produce epidemic strains. This requires a detailed knowledge of the bacterial components Aim necessary for the above processes. The StaphDynamics partnership aims to defi ne these bacterial components, which in themselves may form The primary aim of the StaphDynamics project is to identify important novel targets for prevention and control. Specifi cally, the partnership will molecular determinants for fi tness, virulence and drug resistance of investigate the fi tness and epidemiology of resistant clones, characterise S. aureus that may serve as future targets for drug and vaccine the most important clones phenotypically, and identify molecular development and to fi ght staphylococcal infections. The specifi c determinants for fi tness, virulence and resistance. To reach these goals, technical and scientifi c objectives include: the full potential of bioinformatics, genomics, and proteomics approaches • Application and further development of new methods to identify will be exploited. The StaphDynamics partnership has a proven track novel determinants for staphylococcal fi tness, virulence and drug record in the application of these cutting-edge technologies. resistance using applied genomics, proteomics, and transcriptomics. StaphDynamics will establish strong interactive links with the European These can be used in novel tools for strain typing and profi ling. Antimicrobial Resistance Surveillance System (EARSS) which, together • Characterization of the function of novel gene functions in with the full participation of three SME companies, will guarantee a rapid S. aureus, which are essential for growth or critical for bacterial translation of fundamental research data into practical clinical fi tness, virulence and drug resistance. applications. • Validation of the role of potential bacterial virulence and fi tness factors in animal models of S. aureus infections and in assays for bacterial persistence in or inactivation by human phagocytes. • Development of novel vaccines to preclude staphylococcal infections. This will involve a SME from the Netherlands (Stichting Biomade Technology). • Development of assay systems for a few promising novel drug targets that allow high throughput screening for inhibitory compounds. Such assays will be employed in high-throughput screenings using different compound libraries with the ultimate goal to identify a fi rst set of new lead molecules that should give raise to new antibiotics. This will involve two SMEs from Germany (EMC Microcollections GmbH and Dr. Petry Genmedics GmbH).

ANTI-MICROBIAL DRUG RESISTANCE | 109 Scanning electron microscopic image of Staphylococcus aureus.

• Establishment of strong interactive links with the European com- Coordinator Prof. Dr. Michael Hecker munity behind the European Antimicrobial Resistance Surveillance Dr. Susanne Engelmann System (EARSS) in order to guarantee a rapid translation of Prof. Dr. Jan Maarten van Dijl Ernst-Moritz-Arndt Universität fundamental research data into practical clinical applications. Dept of Medical Microbiology Institut für Mikrobiologie University Medical Center Groningen Greifswald, Germany (UMCG) Hanzeplein 1 Dr. Iñigo Lasa Expected results P.O. Box 30 001 Instituto de agrobiotecnologia 9700 RB Groningen Universidad Publica de Navarra • Novel analytical and diagnostic tools for the characterisation of The Netherlands Consejo Superior de Investigaciones strains in order to predict their virulence and epidemic behaviour. Phone: +31 50 3633079 Científi cas This will include the identifi cation of novel molecular signatures of Fax: +31 50 3633528 Pamplona, Spain resistant clones. [email protected] • Improved understanding of fi tness and epidemiology of resistant Prof. Dr. Tarek Msadek clones. This will serve to fi ll major gaps in our current knowledge Biology of Gram-positive Pathogens Partners Institut Pasteur about the dynamic epidemiological behaviour of drug resistant Paris, France S. aureus. Prof. Dr. Friedrich Götz • Novel targets for drug and vaccine development. University Tübingen Prof. Dr. Hajo Grundmann • Informed strategies for combating resistant clones at the European Microbial Genetics European Antimicrobial Resistance level. Tübingen, Germany Surveillance System

Prof. Dr. Jiri Doskar Dr. Karl-Heinz Wiesmüller Potential applications Dept of Genetics Dr. Renate Spohn and Molecular Biology EMC microcollections GmbH The StaphDynamics programme focuses on the need to translate Brno, Czech republic Tübingen, Germany genome data into practical applications, both in the ﬁ elds of medicine Prof. Dr. Simon J. Foster Dr. Kees Leenhouts (controlling infectious disease) and biotechnology. The project has been University of Sheffi eld Biomade Technology formulated to provide innovative solutions to the three industrial Sheffi eld, United Kingdom Groningen, The Netherlands StaphDynamics partners (EMC, Biomade, Genmedics). Furthermore, the partnership will create an important knowledge base needed to foster Prof. Dr. h.c. Jörg Hacker Dr. B. Krismer European competitiveness in the area of antibiotics research. Dr. Knut Ohlsen Genmedics GmbH Institut für Molekulare Tübingen – Germany Infektionsbiologie Würzburg, Germany

Project number LSHM-CT-2006-019064 EC contribution € 2 375 000 Project Duration 48 months Starting date 01/04/2006 Instrument STREP Project website www.staphdynamics.net

110 | ANTI-MICROBIAL DRUG RESISTANCE 111 ➞ 160

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES AGLAEA

Development of novel animal models of glutamatergic central nervous system disorders using in vivo siRNA and transgenic approaches

Keywords Animal models, glutamate, siRNA, transgenic mice Problem

Modulation of glutamate signalling levels is associated with many psychiatric disorders. Creation of reliable and practical models of altered brain glutamate function has been diffi cult. Glutamate is the most abundant excitatory transmitter in the brain and interacts with numerous receptors and transporters responsible for both fast synaptic transmission and modulatory functions. However, development of knockout (KO) Summary rodents is expensive and time-consuming, and gene KO is often associated with developmental alterations. Inducible and conditional knockout Glutamate is the most abundant excitatory transmitter in the brain. However, technologies have helped to alleviate this problem; however, the gene is very few models exist to explore the potential of drugs that modulate nearly completely suppressed when the induction occurs. Because gluta- glutamate transmission. Because altered glutamate transmission is involved mate is a ubiquitous excitatory transmitter, full knockout of a specifi c in numerous psychiatric diseases, there is a strong need for such models glutamate receptor or transporter often yields a phenotype incompatible to characterise the effects of hypo- and hyper-glutamatergic states on the with behavioural testing. onset and development of such diseases. AGLAEA will develop and characterise models of selective, partial knockdown of specifi c components of the brain glutamatergic system in mice. This will provide better comprehension of the implication of glutamate signalling in Aim diseases such as schizophrenia, anxiety and cognitive disorders. AGLAEA will lead to breakthrough research on the neurobiological and neurochemical AGLAEA aims to develop models of selective, partial knockdown of spe- bases of psychiatric disorders and will enable the further testing of new cifi c components of the brain glutamatergic system that will result in drugs for treatment. In order to selectively turn off specifi c components of hypo- and hyper-glutamatergic states, yielding animals amenable to the glutamatergic pathways, an siRNA approach will be used. The effect of behavioural investigation. The results will then drive the creation of spe- modulation of glutamate signalling will be characterised using functional cifi c transgenic lines with targeted knockdown of components of the MRI (fMRI) and microdialysis/microsensor analysis. Specifi c behavioural brain glutamate system, yielding mice that can be studied over long tests will be carried out on live animals for the assessment of glutamate- periods of time. related psychiatric disorders. Furthermore, the data collected from siRNA experiments will be applied to the generation of transgenic mice, in which modulation of glutamate signalling will be induced at different stages of development. Therefore, Expected results AGLAEA will provide both the pharmaceutical and the academic world with potent models. These models will be used to test novel compounds and Access to improved animal models of specifi c psychiatric disorders will assess their therapeutic value and will benefi t researchers investigating the enhance the development of improved treatments, in particular for dis- neurobiological and neurochemical bases of those diseases. orders such as schizophrenia, where existing models are often based on The consortium set up to reach the objectives of AGLAEA brings together limited pharmacological and neurobiological information about the dis- three high-tech SMEs, 2 academic groups and one large group for the order. Targeting the glutamatergic system should lead to models that management of the project (ALMA), representing altogether four European more closely resemble the human situation with regard to the symp- countries (FR, HU, UK, NL). toms that are at present not treated by existing drugs (e.g., cognition in schizophrenia) or when existing drugs have side-effects and/or dependence issues (anxiety). The creation of these models will also increase fundamental research into the basic mechanisms of glutamate function. In addition, the techniques used will expand the use of siRNA in vivo for the creation of new models in CNS disease, providing new solutions for the exploration of such pathologies.

112 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Mouse embryonic stem cell colonies, with C57Bl/6 genetic background.

Potential applications

A certain number of CNS disorders, including schizophrenia, anxiety and cognitive disorders, can be linked to glutamatergic dysfunction. The existing treatments for patients suffering from these diseases have serious side-effects and are not always efﬁ cient. For example, existing drugs are unable to treat the social, emotional and cognitive symptoms of schizophrenia and there are few marketed treatments for the cognitive deﬁ cits of Alzheimer’s disease. By developing and characterising animal models with hypo- and hyper-glutamatergic states, AGLAEA will provide valuable data on the impact of glutamatergic signalling on such disorders, thereby opening the way for better treatment and care of patients.

Coordinator Partners

Mark Epping-Jordan Prof. Andras Janos Dinnyes ADDEX Pharmaceuticals France SAS BioTalentum Ltd [email protected] Godollo, Hungary Archamps, France Charles Alexander Marsden University of Nottingham Nottingham, United Kingdom

Prof. Ben Westerink Groningen Research A 2.3 Tesla magnetic resonance imaginging (MRI) machine used to investigate Institute of Pharmacy the effects of drugs on neuronal activity in regions of the mouse brain. The method will be used in the present project to identify the effects of Groningen, The Netherlands altering glutamate activity on brain region function. Dr. Thomas Cremers Brains-On-Line Groningen, The Netherlands

Mr. Benjamin Questier ALMA Consulting Group Lyon, France

Project number LSHM-CT-2007-037554 EC contribution € 1 198 900 Duration 36 months Starting date 01/01/2007 Instrument STREP – SME

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 113 cNEUPRO

Clinical Neuroproteomics of Neurodegenerative Diseases

Keywords Alzheimer’s Dementia, biomarker, neurochemical Problem dementia diagnostics, cerebrospinal fl uid, medical proteomics, blood-based bioassays, multiplex assays, predictive diagnostics Europe has to face a threatening increase in the prevalence of the most for preventive therapy, quality control common dementia, i.e. Alzheimer’s Dementia (AD). There is an urgent need for novel disease- modifying treatments, which will be most effective if offered at a very early dementia stage, preferentially as preventive therapy during the prodromal stage of mild cognitive impairment (MCI). However, the reliable clinical differential diagnosis of very early AD is not satisfactory and conventional diagnostic tools do not offer predictive diagnostics for AD. Here, recent research has clearly demonstrated that Summary multiparametric neurochemical dementia diagnostics (NDD) in cerebrospinal fl uid (CSF) clearly improves the early and differential diagnosis of Alzheimer’s Dementia (AD) is one of the most common brain diseases AD. Moreover, recent studies have also indicated that incipient AD can in the elderly. Dementias pose a major health problem in European be predicted several years prior to dementia onset. However, these countries, with currently 5.7 million affected dementia patients in the promising results of CSF-based NDD need to be validated by additional EU25. No curative therapies are currently available for these dementias; studies. Futhermore, additional neurochemical biomarkers have to be however, fi rst disease-modifying treatment strategies such as Ab- identifi ed for the improved early differential diagnosis of dementias and, immunisation have entered clinical trials. These novel treatments will be most importantly, fi rst assays for blood-based NDD need to be devel- most effective if they are offered at a very early dementia stage. Currently, oped. Moreover, only blood-based NDD will allow a widely applicable however, the reliable clinical differential diagnosis of very early dementia monitoring of therapy effi cacy. stages is not satisfactory. Here, recent research has clearly demonstrated that multiparametric neurochemical dementia diagnostics (NDD) in Meanwhile, CSF-based NDD has entered neurochemical routine diag- cerebrospinal fl uid (CSF) clearly improves the early and differential nostics of dementias in European expert centres. However, there is diagnosis of dementias. a strong need to develop fi rst standard operation procedures (SOPs) for cNEUPRO will apply advanced proteomic tools to discover novel CSF-based NDD, which signifi cantly depends on the quality of preana- neurochemical dementia markers (BIOMARKERS) in blood and CSF for lytical sample handling, storage conditions and sample storage. It is the improved early, as well as predictive, diagnosis of AD. A predictive noteworthy that the success of this novel approach for the improved dementia diagnosis is essential in order to optimise the effectivity diagnosis of very early dementias has also stimulated strong research of forthcoming preventive therapeutic strategies. Our initiative will activities in the US. establish European standard operating procedures (SOPs) for current neurochemical dementia diagnostics (NDD) and we will establish the fi rst NDD reference centres in Portugal and Hungary. Aim A strong methodological impact is placed on the quality of pre- analytical sample handling and clinical phenotyping, which has been cNEUPRO is designed to discover innovative protein biomarkers and to neglected in industry-driven discovery studies. cNEUPRO integrates develop novel CSF and blood multiplex assays for the accurate early, innovative biotech and bioinformatic companies with leading clinical differential and possibly predictive diagnosis of AD. Moreover, cNEURPO and proteomic dementia research centres. cNEUPRO will also support will develop fi rst European SOPs for CSF-based NDD. the discovery of new diagnostic targets and, furthermore, will provide a promising approach to identify novel scaffolds for advanced molecular neuroimaging. Accordingly, cNEUPRO will contribute considerably to the Project design and expected results welfare, health and quality of life in Europe, and concomitantly it will reinforce the competitiveness of the European biotech industry. cNEUPRO consists of two R&D modules: Module I is dedicated to the discovery of novel protein biomarkers, the generation of high-affi nity ligands and the implementation of biomarkers into novel ELISA or Multiplex Assays. The discovery of biomarkers is differentiated into the development activities: defi nition of high-quality samples, identifi cation of candidate biomarkers in blood and CSF by multi-dimensional state-of-the-art proteomics, and validation of the candidate biomarkers. The validation of a biomarker comprises its protein chemical (structural) validation by advanced mass spectrometry and its clinical validation within a re-test study.

114 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Core facility mass spectrometry unit of one of the laboratories of cNEUPRO partners for identiﬁ cation of biomarkers. © 2007 Dr. Oliver Ratajczak – strukturwelt.de

For validated biomarkers we will devise novel poly- and monoclonal Coordinator Prof. Edgar F. da Cruz e Silva antibodies as well as high-affi nity molecules. The latter highly stable Proteomics small binding proteins are designed using a proprietary technology and Prof. Dr. Jens Wiltfang Prof. Odete A.B. da Cruz e Silva may possibly also be used as therapeutic tools or scaffolds for molecular University of Erlangen-Nuremberg NDD reference center Portugal Germany University of Aveiro neuroimaging. Finally, the biomarkers will be integrated into novel ELISA [email protected] Aveiro, Portugal and, most importantly, Multiplex-Assays. Applying advanced bioinfor- Phone: +49 9131 85 34262 matics tools (proprietary technology), cNEUPRO will design a prototype Fax: +49 9131 85 34862 Prof. Laszlo Vescei predictor system for AD. University of Szeged Szeged, Hungary Module II is dedicated to improving the performance of current CSF-based Partners NDD, by defi ning European SOPs for pre-analytical sample handling, sam- Dr. Johannes Schuchhardt ple storage and assay conditions. As part of module II, CSF quality surveys Prof. Sylvain Lehmann MicroDiscovery GmbH will be conducted, ‘gold standard’ CSF samples devised, and we will estab- Centre Hospitalier Universitaire Berlin, Germany lish two new NDD reference centres in Hungary and Portugal. de Montpellier Montpellier, France Prof. Lucilla Parnetti University of Perugia Prof. Kaj Blennow Perugia, Italy Potential applications Sahlgrenska Academy at Göteborg University Prof. Helmut E. Meyer • Improved early and predictive diagnosis of Alzheimer’s Dementia. Mölndahl, Sweden Prof. Katrin Marcus • Development of fi rst blood-based NDD for dementia diagnostics Ruhr-Universitaet Bochum and therapy monitoring. Dr. Charlotte Teunissen Bochum, Germany • Identifi cation of potential therapy responders by indicative biomarker VU University medical centre phenotypes. Amsterdam, The Netherlands Dr. Michel Bergh • Discovery of novel scaffolds, which may possibly also be used as Farallone Therapeutics BV Dr. Connie Jimenez Amsterdam, The Netherlands therapeutic tools or scaffolds for molecular neuroimaging. Vrije Universiteit Amsterdam • Elucidation of the molecular mechanisms involved in AD. Amsterdam, The Netherlands Prof. Carsten Korth • Implementation of fi rst European SOPs for CSF-based NDD. Heinrich Heine University Prof. Markus Otto of Düsseldorf Universitätsklinik Ulm Düsseldorf, Germany Ulm, Germany Prof. Tuula Pirttila Dr. David Burn (clinics) University of Kuopio Dr. Chris Morris (proteomics) Kuopio, Finland University of Newcastle upon Tyne Newcastle, United Kingdom Prof. Luc Buee Institut de la Santé et Dr. Martin Wiesenfeldt de la Recherche Médicale Matrix Advanced Solutions Lille,France Germany GmbH London, United Kingdom Dr. Stefan Müllner Protagen Dortmund, Germany

Project number LSHM-CT-2006-037950 EC contribution € 3 000 000 Duration 36 months Starting date 01/01/2007 Instrument STREP – SME

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 115 DB workshop

Databasing the brain – Workshop

Keywords Neuroinformatics, databases, data sharing, Problem tools sharing, standards, journals Two main web portals provide an overview of neuroscience-related databases, data archives and analytical software. One is the neuroinformatics site (www.neuroinf.org), originally funded by the EU Thematic network on Computational Neuroscience and Neuroinformatics (QLG3- CT 1999-00763). The other is the Neuroscience Database Gateway (www.sfn.org/ndg). Judging from these portals, there has been a large Summary increase in the number of database resources, as well as in the amount of data made available via these resources. An increasing number of New technological opportunities for data sharing have gradually emerged ‘smart’ databases (database applications providing direct access to ana- with the introduction of the Internet with gradually faster networks, lytical tools) are being established. But only a small proportion of improved database technologies, and affordable massive data storage neuroscience databases available today are based in Europe. Further, capabilities. The fi eld of bioinformatics has exploited these opportunities only few neuroscientists in general are involved in the establishment of for essential aspects of data sharing. But far from all the relevant gene database resources. Hence, there is a potential for more data sharing, and molecular level data, from the brain or other systems, are today re-use of data, and novel analysis based on new combinations of data shared in databases. Moreover, databases are used only sparingly in many that can be performed via database systems. subfi elds of the neurosciences. Hence, there is a potential for more data sharing, re-use of data, and novel analysis based on new combinations of data that can be performed via database systems. Aim The overall objective of the proposed workshop ‘Databasing the brain’ was to accelerate existing research and development efforts in the fi eld of Broadly deﬁ ned, the objectives of the workshop ‘Databasing the brain’ neuroscience databasing. Two specifi c areas were selected for a detailed were to accelerate existing research and development efforts that will study. First, the workshop focused on standardised data formats and facilitate data sharing, tools sharing and integration of multiple catego- annotations of data at the level of the laboratory workbench; it explored ries of data from the brain. The meeting was used as a vehicle to inform how the industry (companies producing software and equipment for data European researchers about the ﬁ eld of database applications to neuro- acquisition, storage and data management) could help establish data science in general, and to increase the interest of researchers in contrib- and metadata formats via interactions with the scientifi c communities. uting to and using relevant databases. Topics selected for in-depth Second, the workshop focused on the role of journals in providing analysis were: information about databasing initiatives, in encouraging scientists to share • standardised data formats and annotation of data at the workbench; persistent data via databases, and in contributing to novel publishing • the role of journals in providing information about databasing formats. The workshop brought together established scientists in the fi eld initiatives and in encouraging scientists to share persistent data via of brain databasing, expert neuroscientists from many sub-areas of the databases. neurosciences, and representatives of SMEs, as well as journal editors and other representatives of journals.

116 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Expected results and potential applications Coordinator

The workshop explored how the industry (companies producing software Prof. Jan G. Bjaalie and equipment for data acquisition, storage and data management) Centre for Molecular Biology and Neuroscience could help establish data and metadata formats via interactions with the University of Oslo scientifi c communities. A natural format for this, already taking place in Oslo, Norway other fi elds, is the establishment of expert panels that include represen- [email protected] tatives from the industry. Companies are often willing to contribute to developments of standards and thereby facilitate data analyses. Partner There is a fundamental need, in the laboratories producing experimental data, for tools and methods for organising primary data to be uploaded Dr. Erik De Schutter into database applications. Rich metadata of high accuracy are needed Theoretical Neurobiology to make use of the experimental data and facilitate re-analysis. University of Antwerp Antwerp, Belgium While databases represent novel approaches for data sharing and analysis, journals provide the only established mechanism for peer review and proper referencing of data and interpretations. Mechanisms for publication in journals and simultaneous posting of data in data repositories have been established. Success stories exist, particularly in the imaging fi eld. Journals are in a position to communicate and emphasise new developments and encourage the establishment of new routes for the dissemination and sharing of data. Efforts to couple existing database developments to journal publications now appear to be starting up in more fi elds.

Project number LSSM-CT-2005-019083 EC contribution € 77 400 Duration 18 months Starting date 01/10/2005 Instrument SSA Project website www.nesys.uio.no/Workshop2006

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 117 ENINET

Network of European Neuroscience Institutes

Keywords Neuroscience, network, Young Investigators, professorship in most European Member States. The neurosciences, student exchange, workshop, symposia, European which are characterised by rapid development of both concepts Research Area and techniques and a high degree of interdisciplinary, cannot do without the input of these highly fl exible and motivated people. Unfortunately in Europe, unlike the United States, opportunities for independent work that allows creativity to manifest itself have been rare for these young investigators. More recently, fellowship programmes by some of the European Member States and also by the European Union have opened up funding sources for the Summary research of independent Young Investigator teams, usually limited to a three- to fi ve-year funding period. The members of this European Research, particularly in the neurosciences, depends critically Consortium are aware of their role (and chance!) of hosting such on the creative contributions of young investigators. In recognition of this teams and optimising their working conditions. The networking important need, fi fteen major European Neuroscience Institutes (ENIs) have proposed here is an essential element of this endeavour. formed a network dedicated to the promotion of the independent work of • Young Investigators are the ideal ‘carriers‘ for networking. It is young investigators. The institutes supply laboratory space, infrastructure, a matter of experience that encounters among young investigators a nurturing environment and other support, enabling young investigators are the most effi cient means of initiating collaborations. Graduate to build small research teams and to perform independent work. Through students meeting each other may be even more interactive than the Coordination Action the network provides funds, such that the young the age group we are addressing. However, they are bound to their investigator groups can organise regular meetings, exchange students and thesis projects and usually are not free to redirect their research share expertise. From the interactions among groups we expect a major goals as a consequence of an insight gained at a meeting. More impact on the research of the individual teams, a stronger integration of senior researchers may well be in a position to redirect research in research between participating institutions, mobilisation of major resources their departments, but this usually happens on a long timescale for neuroscience, and a signifi cant structuring effect within the neurosciences and not on the basis of some networking activity. The fl exibility and of the European Research Area. mobility of young investigators, combined with their personal presence in the laboratory, constitutes what is needed for a fruitful collaboration in a research area moving as fast as the neurosciences.

Structure of the Network

16 European institutions, each conducting a strong neuroscience program, have joined forces to set up ENI-net, a network of European Aim Neuroscience Institutes dedicated to the promotion of the independent work of young researchers. The partners have agreed to provide Brain diseases and neurological disorders are among the most debilitat- infrastructure and laboratory space for at least two independent Young ing health conditions presently known. Most of these, such as Parkinson‘s Investigator Groups. disease, Alzheimer‘s and schizophrenia, many forms of epilepsy, chronic pain and migraine, have been considered incurable for decades and Each ENI has a local coordinator, who functions as a mentor for the ENI- centuries. Tackling the enormous task of understanding the function net Young Independent Group Leaders. The ENI-net group leaders have and dysfunction of the whole or even minute subassemblies of neurons been guaranteed a high level of independence by their hosting institu- requires the best brains and will gain enormously from a close collaborations: this independence provides the necessary training in administration within Europe. With this in mind, the major neuroscience research tive and organisational matters that each group leader needs for his or centres in Europe have agreed to collaborate at an organisational lev- her future career. ENI-net Young Investigators are selected on the basis el beyond that of specifi c research projects. The primary means of the of their scientifi c track record and excellence. They are approved by the collaboration is the interaction among Young Investigator Groups. steering committee and elected for a period of fi ve years. At the time of • The creativity and intellect of young researchers is highly needed in their appointment, ENI-net group leaders should not have spent more Europe – particularly in the neurosciences. The young investigators than 8-12 years in science after receiving their PhD. ENI-net Young supported by the network are typically researchers in the age group Investigators have access to all ENI-net resources and receive funds 30-40 years, at the peak of their productivity. They usually have for participation in ENI-net meetings, workshops and conferences. three to fi ve years experience of postdoctoral training – very often Furthermore, mobility funds are provided to conduct collaborations and abroad – but are too young to be considered eligible for a tenured joint activities between the different ENI-net labs.

118 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Expected results Coordinator Prof. David Attwell University College London The dedicated goal of the network as a whole is to encourage Prof. Erwin Neher London, United Kingdom participants to improve the conditions for young researchers within their Max Planck Institute for Biophysical Chemistry Prof. Carlos Belmonte institution. Furthermore, the network aims to facilitate and enrich the Am Fassberg 11 Universidad Miguel Hernandez work of the individual groups (45 in number by August 2006) by D-37077 Göttingen Alicante, Spain organising workshops, annual general meetings, student exchange and Germany mutual visits. Last not least, an aim of the joint meetings is to stimulate Phone: +49 551 2011630 Prof. José Lopez-Barneo future collaboration. Fax: +49 551 2011688 University of Seville [email protected] Seville, Spain We expect the number of laboratories available for Young Investigators to increase and the quality of the research being performed to improve. Prof. Sten Grillner Furthermore, we hope that exciting collaborative research will evolve Partners Karolinska Institutet through the regular meetings of 45 Young Investigator Groups. From the Stockholm, Sweden Prof. Diethelm W. Richter interactions among groups, we expect a major impact on the research Bereich Humanmedizin Prof. Eva Sykova of the individual teams, a stronger integration of research between par- der Georg-August-Universität Institute of Experimental Medicine ticipating institutions, mobilisation of major resources for neuroscience, Göttingen, Germany Academy of Sciences and a signiﬁ cant structuring effect on the research ﬁ eld in the future of the Czech Republic European Research Area. Prof. Christophe Mulle Prague, Czech Republic Institut National de la Santé et de la Recherche Médicale Prof. Leszek Kaczmarek Bordeaux, France Nencki Institute of Experimental Biology of the Polish Academy Prof. Pierre-Marie Lledo of Sciences Institut Pasteur Warsaw, Poland Paris, France Prof. Henry Markram Bernard Poulain Ecole Polytechnique Fédérale Centre National de de Lausanne la Recherche Scientifi que Lausanne, Switzerland Strasbourg, France Prof. Antonio Cattaneo Prof. Alain Marty European Brain Research Institute Université René Descartes Rome, Italy Paris, France Prof. Rodrigo A. Cunha Prof. Jeremy Henley University of Coimbra University of Bristol Portugal Bristol, United Kingdom

Project number LSH-CT-2005-019063 EC contribution € 1 282 500 Duration 36 months Starting date 01/11/2005 Instrument CA Project website www.eni-net.eu

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 119 Enough sleep

Disorders of sleep regulation: basic mechanisms and therapeutic perspectives

Keywords Sleep, sleep restriction, insomnia, depression Aim

General objectives • To spearhead the fi rst integrated effort at understanding the different mechanisms of sleep regulation using a combined genetic, molecular, electrophysiological and systems approach. • To develop novel diagnostic tools (genetic, molecular and electrophysiological) for identifying disorders of sleep regulation and for Summary characterising their pathophysiology. • To apply such tools in the diagnosis and cure of disordered sleep, The regulation of sleep is essential for our wellbeing; unfortunately, the with special emphasis on insomnia and depression. relevant homeostatic control mechanisms often fail. The affected individuals suffer from fatigue, decreased motivation and performance, and an Specifi c objectives increased risk of accidents. Insomnia may lead to depression and other • Identify cortical mechanisms of sleep regulation. medical problems, and can even reduce the expected life span. Improved • Identify sub-cortical mechanisms of sleep regulation. understanding and more accurate diagnostics of cortical dysfunction in • Identify humoral and genetic mechanisms of sleep regulation. sleep disorders and more effective and better-targeted treatment would give • Explore the role of glia in sleep regulation. enormous benefi ts to both patients and to society. • Develop new diagnostic tools specifi cally to study sleep regulation This project presents the fi rst integrated effort to study the different aspects in humans. of sleep regulation at the genetic, molecular, cellular and network levels. • Understand the mechanisms responsible for the alterations of sleep The Consortium consists of highly qualifi ed scientifi c groups covering all regulation in insomniac/depressed patients in order to envisage areas of basic sleep research and complemented by specialists in human new treatments. genomics, clinical and adenosine research. One SME company is included in the Consortium. The research makes extensive use of the existing, representative DNA data- Expected results bank recently collected in Finland and studies the correlations between polymorphisms in genes associated with sleep regulation and the behav- We expect to identify genes, anatomical sites and physiological mole- ioural phenotype assessed by extensive questionnaires and by a health cules that are involved in the regulation of sleep duration and homeo- examination. static control. We also expect to be able to identify genes that are in To study cortical functionality and state in humans at the network level, we common with sleep regulation and susceptibility for depression. use and will further develop novel tools, including high-density EEG and MRI- assisted navigated TMS/EEG. The latter technique allows one to study local The software developed in this project by the SME Nexstim, will be used cortical excitability and time-resolved area-to-area functional connectivity. for the evaluation of excitability changes in various brain disorders such The project aims at simplifying the TMS/EEG technology, especially its as insomnia and depression. software, in order to make the technique suitable for routine diagnostic use. Clinical and commercial exploitation will be seriously addressed to enable a wide impact of the work. Potential applications

Nexstim will develop clinical applications for the brain navigation system, which currently is mainly used for research purposes. NBS/EEG provides detailed information about the functional state and connectivity of the cortex; the present project represents a signiﬁ cant step in learning to use these measures in the evaluation of cortical state and functionality. Clinical applications will be based on such evaluations.

If the present project indicates that, using NBS/EEG, one can differentiate between different disorders in regulatory mechanisms in insomnia or depression (and if the effects of medication can be individually assessed), the market for such evaluations may turn out to be very large, both within Europe and worldwide.

120 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Moisture absorption capsules for chemicals and tissues.

Coordinator Dr. Irene Tobler University of Zurich Dr. Tarja Porkka-Heiskanen (Stenberg) Zurich, Switzerland University of Helsinki Helsinki, Finland Dr. Tiina Paunio [email protected] National Public Health Institute Helsinki, Finland

Partners Dr. Marcello Massimini University of Milan Dr. Bertil Fredholm Milan, Italy Karolinska Institutet Stockholm, Sweden Dr. Risto Ilmoniemi Nexstim Oy Dr. Marina Bentivoglio Helsinki, Finland University of Verona Verona, Italy

Dr. Tom de Boer Leiden University Medical Center Identiﬁ cation of sleep and wakefulness periods (states) in animal EEG. Leiden, The Netherlands

Project number LSHM-CT-2005-518189 EC contribution € 1 425 000 Duration 36 months Starting date 01/12/2005 Instrument STREP Project website www.enoughsleep.fi

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 121 EPICURE

Functional genomics and neurobiology of epilepsy: a basis for new therapeutic strategies

Keywords Neurobiology, neurology, genetics, epilepsy, epileptogenesis, channelopathies, ion channels, antiepileptic drugs, development Summary Problem Epilepsies are chronic disorders with a prevalence of 1 % worldwide, including Europe. They severely affect the quality of life of patients, especially Epilepsy is a serious and common neurological disorder that has major of those (one-third) who are drug-resistant. Epilepsies are multifactorial implications not only for health but also for independent living, education disorders sharing common pathophysiological mechanisms, in which dys- and employment, mobility, personal relationships, and prospects for insur- functions in voltage-gated and ligand-gated (receptors) ion channels and ance. It is estimated to affect currently 6 million European citizens, with alterations in the structure and function of neuronal networks play a major a resultant economic burden of € 18 billion per year, if we do not cure role. A better knowledge of these epileptogenic mechanisms is the essen- the underlying causative disorder. The main remaining problems are: tial precondition to developing more effective antiepileptic treatments. • the signifi cant proportion of patients (60-70 %) whose seizures are With this aim, the EPICURE collaborative project has been developed, refractory to the presently available antiepileptic drugs; involving several groups from 13 European countries. The project will • the lack of effective strategies capable of preventing the develop- identify disease-causing genes and their functional role in the patho- ment of the epileptogenic process set in motion by genetic or physiology of neuronal excitability and network synchronisation, using environmental risk factors; complementary advanced methods. The work will be focused on volt- • the tendency of several very active epilepsies of developmental age age-gated Na+ channels and GABAergic synaptic inhibition. Their crucial to progress towards a condition of drug-refractory encephalopathy epileptogenic role is recognised for both inherited acquired epilepsies (infantile epileptogenic encephalopathies); and will be investigated in animal models and human tissue, with a focus • the stage in which a surgical treatment (when indicated) is capable on drug-resistant temporal lobe epilepsy and epilepsies due to develop- of stopping a drug-resistant epileptogenic process and of preventing mental cortical abnormalities. its negative cognitive and psychosocial consequences. A main effort is devoted to investigating the developmental aspects of epileptogenesis that account for the high frequency of epilepsies in children, and for their often severe prognosis. Strictly related to the above Aim research lines are the pharmacological approaches, by which EPICURE aims to develop more effective antiepileptic strategies, to understand the To design novel strategies to cure epilepsy and prevent its progressive biological bases of pharmacoresistance, and to evaluate the potential course by acting on the underlying biological mechanisms. A key role in of innovative therapeutic approaches based on synaptic modulation by epilepsy and epileptic seizures is attributed to changes in neuronal neurotrophic factors and cell cycle kinase inhibitors. excitability due to altered voltage- or ligand-gated ion channels. This The research activity is organised in fi ve sub-projects: contention is strongly supported by human genetic and molecular studies • genetics of human epilepsies; of genes coding for channel proteins, showing that defects in individual • functional consequences of mutations in ion channel genes associ- ion channel genes can give rise to chronic hyperexcitability and epilepsy. ated with idiopathic epilepsy and genetically determined pharma- The phenotype resulting from an altered ion channel depends on the coresistance; neuronal systems and the maturational stage, during which such changes • acquired channelopathies and neuronal network reorganisation exert their effects. In the common forms of familial epilepsies, however, underlying temporal lobe epilepsy; the underlying genetic mechanism is still unknown; moreover whether • epilepsy and development; and how genetic risk factors interact with the environmental ones in • pharmacogenetics of refractory epilepsy, mechanisms of drug determining acquired epilepsies and their response to the antiepileptic resistance and new therapeutic strategies. drugs is largely unexplored. By a multidisciplinary approach based on an Despite the internationally recognised role of epilepsy research in Europe, integration of the clinical and basic science domains, EPICURE will seek to the leading research groups lack a central coordination to take advan- answer these open questions. tage of cross-fertilisation and the synergistic effects. EPICURE intends to bring together expertise from different research areas, both clinical and Specifi c aims are: laboratory-based, with the common goal of advancing European epilepsy • identifi cation of epilepsy genes and characterisation of the research to ensure novel therapeutic benefi ts for patients. functional alteration affecting the encoded proteins, in relation to their epileptogenic function. These will be applied both to monogenic, as well as common epilepsies with complex inheritance. The EPICURE Consortium fulfi ls key prerequisites, without which such an approach would not be possible:

122 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES – the ability to collect a statistically signifi cant number of • Abnormalities of cortical development (namely focal cortical informative families, whose phenotypes are well characterised dysplasia) are the major cause of intractable epilepsies in children. according to common criteria, Etiologies of the precise mechanisms linking cortical malformations – a documented experience of novel, powerful methods for genetic to epileptogenesis remain elusive. The coordinated work of the analysis (linkage, association and sequencing techniques), EPICURE partners is aimed at resolving the pathogenetic role of – considerable expertise in the functional analysis of altered mutations associated with cortical malformations and at defi ning gene products in cells and animal models; their epileptogenic potential. This can be achieved thanks to the • identifi cation of acquired channelopathies and network reorgani- statistical power of the observations and to the combination of sation in human temporal lobe epilepsy and clinically relevant animal physiological, morphological and biomolecular methods applied models, with the aim of identifying protective strategies and new to human tissue and animal models. therapeutic targets; availability of a signifi cant amount of human tis- • Groups in the EPICURE Consortium have already identifi ed sue from patients with well-defi ned epilepsies for morphological, a number of changes in ion channels and drug transporters: the electrophysiological and biomolecular analyses, which can be com- relevance of these to decreasing AED sensitivity and intraparenchimal pared with data obtained from suitable animal models; concentration will be investigated. By this integrated effort, genetic • identifi cation of mechanisms controlling age-related epileptogenesis factors (including polymorphisms in a number of candidate genes) in children suffering from brain maldevelopment. Because a number that may play an important role in determining the function of of epilepsies either occur during specifi c developmental stages or are both drug transporters and some drug targets will potentially related to developmental abnormalities, a considerable effort in the be identifi ed. Integrated collaboration between clinical and EPICURE project will be devoted to analysis of the role of genes that experimental centres, as well as the collection of large numbers regulate cortical development and the pathophysiological mechanisms of well characterised patients according to common classifi cation accounting for dysplasia-associated epileptogenesis, both in human criteria and study protocols, will enable EPICURE to overcome the tissue and experimental models; shortcomings of previous studies. • development and testing of new strategies for prevention and treat- • A European Epilepsy Brain Bank initiative (EEBB) will be developed to ment of epilepsies and identifi cation of mechanisms that account for examine and collect systematically histopathologically identifi ed pharmacoresistance. Virtually all the EPICURE research lines are rele- specimens from patients operated on for chronic, intractable drug- vant to the therapeutic approach and will contribute to a better resistant epilepsies. This will lead to harmonisation and standardisation understanding of cellular mechanisms responsible for drug-resistant of a number of procedures, including: epilepsies and the underlying genetic profi les. In addition to predict- – tissue sampling and preservation, ing and overcoming drug resistance by novel pharmacotherapeutic – neuropathological diagnosis, strategies, the potential of innovative therapeutic approaches tar- – acquisition and distribution of tissue specimens. geted to infl ammatory mechanisms, or based on synaptic modulation by the neurotrophic factor, will be investigated. Potential applications

Expected results • The most direct application of the results of this research project is represented by its impact on the prevention of epileptogenesis and • A combined molecular genetic and neurophysiological approach will on the therapy of epilepsies as specifi ed above. provide a solid basis for the potential of the EPICURE Consortium to • EPICURE will help to establish an extnsive European epilepsy research disentangle the complex genetic basis of the common IGE syn- network based on a synergistic integration of multidisciplinary com- dromes. Moreover the functional information drawn from genetically petences that cannot be achieved at any national level. determined channel dysfunctions will give new insights into cellular • EPICURE will contribute to the creation of a new generation of signalling mechanisms that underlie neuronal excitability, and is European researchers that will look at epilepsy with a novel therefore also relevant to acquired epilepsy. It will also potentially perspective and a solid understanding of the potentials and limits lead to new avenues for anticonvulsive therapies. of technological and clinical progress, thus reinforcing the European • The multidisciplinary approach to acquired channelopathies, studied tradition in epilepsy research. both in human tissue resected from patients with drug-resistant • EPICURE will signifi cantly contribute to raising public participation epilepsies and in experimental animal models, will be particularly and awareness through a wide dissemination plan which involves focused on plastic phenomena that lead to the development of mesial all relevant stakeholders. This plan will include: periodical e-news- temporal lobe epilepsy. Identifi cation of the underlying mechanisms letters in lay languages, distributed through lay organisations and will potentially lead to strategies to prevent epileptogenesis in patients patient association channels, web-based information campaigns who are at risk. In addition, new therapeutic targets for the treatment and participation at European events also involving the media of these otherwise pharmacoresistant patients may be identifi ed. (TV and newspapers).

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 123 • Finally the expected results will lead to potential industrial developments such as: – encapsulation of cell biodelivery technologies for suppression of epileptic seizures; – methods for genetic tests; – new gene therapy approaches.

Coordinator Merab Kokaia Michele Simonato Tomris Cesuroglu Lunds Universitet Università degli Studi Advance Medical Technologies Giuliano Avanzini Lund, Sweden di Ferrara Ankara, Turkey Fondazione IRCCS Istituto Neurologico Ferrara, Italy ‘Carlo Besta’ INNCB Bobby Koelemann Lars U. Wahlbeg Milan, Italy Universitair Medisch Centrum Gunther Sperk NsGene A/S [email protected] Utrecht, The Netherlands Medizinische Universität Innsbruck Ballerup, Denmark Fax: +39 02 7060077 Innsbruck, Austria Anna-Elina Lehesjoki Benedicte Menn Helsingin Yliopisto Matthew Walker Neurokin SARL Partners Helsinki, Finland University College London Marseille, France London, United Kingdom Michel Baulac Holger Lerche Carla Finocchiaro Institut national de la Santé et Universitätsklinikum Ulm Annamaria Vezzani CF consulting Finanziamenti Unione de la Recherche médicale Ulm, Germany Istituto di Ricerche Europea srl Paris, France Farmacologiche ‘Mario Negri’ Milano, Italy Heiko Luhmann Milano, Italy Marina Bentivoglio Johannes Gutenberg Università degli Studi di Verona Universitat Mainz Federico Zara Verona, Italy Mainz, Germany Istituto Giannina Gaslini Genova, Italy Ingmar Blumcke Ugur Ozbek Friedrich Alexander University of Istanbul Universitesi Olivier Zelphati Erlangen-Nurnberg Istanbul, Turkey OZ Biosciences Erlangen, Germany Marseille, France Emilio Perucca Tamas Freund Fondazione Istituto Neurologico Magyar Tudomanyos Akademia Casimiro Mondino Kiserleti Orvostudomanyi Pavia, Italy Kutatointezet Budapest, Hungary Asla Pitkanen Kuopion Yliopisto Renzo Guerrini Kuopo, Finland Institute of Developmental Project number Neurosciences Stella Maris Foundation Felix Rosenow LSHM-CT-2006-037315 and University of Pisa Calambrone Philipps-Universitaet Marburg EC contribution Pisa, Italy Marburg, Germany € 9 883 259 Duration Armin Heils José Serratosa 48 months Universitätsklinikum Bonn Fundación Jiménez Díaz Starting date Bonn, Germany UTE Madrid, Spain 01/01/2007 Uwe Heinemann Instrument Charité Universitätsmedizin IP Berlin, Germany Project website www.epicureproject.eu

124 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES EUSynapse

From molecules to networks: understanding synaptic physiology and pathology in the brain through mouse models

Keyword Synapse An international consortium of renowned scientists and small and medium-size enterprises (SMEs) has joined forces to assist the European Commission in addressing and solving the problems out- lined above. Signiﬁ cant progress is anticipated during the duration of four years, thus getting closer to the overall goal of combating and treating neurological and psychiatric disorders that arise from synaptic Summary dysfunction.

The involvement of synapses in many neurological diseases (synaptopathies) is becoming increasingly apparent in recent years. Yet, despite considerable Aim advances in our understanding of synaptic signal transmission processes, much remains to be delineated with respect to the molecular details. The overall aim of the project is to build on the treasure trove of infor- Our aim is to further our understanding of synaptic function using multiple mation provided by the complete genome sequences of humans and systems. Starting from cell free systems (in vitro) we also want to take full other species. One of the major goals is to stimulate and sustain multi- advantage of genetically modifi ed mice (in vivo), particularly those serving as disciplinary and basic research, in order to underpin applications to models for synaptic dysfunction in neurological diseases. human health. Both forward and reverse genetic approaches have been Innovative and state-of-the-art technologies will be applied and optimised, immensely facilitated by the availability of genomic databases, affecting including biochemical, molecular, electrophysiological, and optical tools. We virtually all fi elds of the life sciences. will derive detailed knowledge of molecular machineries that drive synaptic transmission and of mechanisms responsible for changes in the synaptic The EUSynapse consortium will signifi cantly further the understanding properties (synaptic plasticity). We expect that these studies will provide of synaptic function by using the mouse as the prime model organism. invaluable insights into synaptic function and dysfunction, and into their Based on the ability to manipulate the expression levels of synaptic contribution to complex brain functions in health and disease. proteins, their role in synaptic transmission and in more complex brain functions will be better understood, providing links to human diseases and identifying candidate drug targets for therapeutic intervention in synaptopathies. Major milestones include: • development of novel optical methods, generation of reporter mouse models, and the adaptation of pre-existing high-resolution techniques (capacitance patch clamping, multiple electrode recordings) to mouse preparations; Problem • establishment of genetic procedures to manipulate protein expression levels in mouse model synapses, complemented with pro- Neurological and psychiatric disorders are among the most prevalent teomic and DNA-array analyses to monitor changes induced by and debilitating diseases of modern societies. Most of these, promi- altered expression; nently including Alzheimer’s and Parkinson’s disease, epilepsy, schizo- • determination of the mechanisms by which active zones and postsyn- phrenia, stroke, chronic pain, and dementia are long-lasting diseases, aptic densities (PSD) are assembled, taking advantage of mouse incapacitating affl icted people for years and even decades, and resulting models containing key proteins of active zones and PSDs tagged to in human suffering and immense socio-economic costs. It is becoming fl uorescent proteins; apparent that seemingly complex diseases can often be traced to the • identifi cation of genes involved in autism, bipolar disease, schizo- dysfunction of single or a very few molecules. A growing list of neuro- phrenia and language disorders, using material derived from human logical disorders is caused by mutations in proteins, either directly causa- patients. Initial characterisation of the role of the candidate genes in tive for the disease process or increasing susceptibility to developing synaptic function by introducing mutations/changing expression clinical symptoms. levels in the mouse; • determination of the calcium dynamics and calcium-dependent The European Commission has recently underlined the urgency of molecular dynamics in the pre-synaptic terminal. Development of research in one of he most central areas of neurosciences where con- standardised screening assays for identifi cation and quantitation of nection to diseases is evident but not well understood at the mechanistic pre-synaptic protein complexes; level. While understanding the cause of a disease is a long way from • refi nement of techniques to measure short-term plasticity, and devising an effective treatment, it is clear that therapeutic strategies can application of these techniques to mouse models with synaptic be developed only by research aimed at an in-depth analysis of the aber- phenotypes; rant proteins and their impact on the hierarchy of simple and increasingly • determination of the rules of assembly, polarised traffi cking and complex neuronal functions. signal transduction during physiologically relevant network activities;

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 125 Unravelling the molecular details of synaptic processes is the mission of the EUSynapse project: a new antibody labels the vesicular GABA Transporter vGaT in cultured neurons.

• development of new approaches to study the function of small Coordinator Angus Silver University College London neural networks in mice, using one sensory and two cortical regions Reinhard Jahn London, United Kingdom as models. Transgenic reporter lines will be used to identify neuronal Max Planck Institute subpopulations. for Biophysical Chemistry Henry Markram Am Fassberg 11 Ecole Polytechnique Fédérale D-37077 Göttingen de Lausanne Expected results Germany Lausanne, Switzerland Phone: +49 551 2011634 The work of the Consortium will make signiﬁ cant contributions: Fax: +49 551 2011639 Zoltan Nusser • by identifying synaptic genes that are likely to cause neurological [email protected] Institute of Experimental Medicine disease caused by pathology of synaptic proteins; Budapest, Hungary • by generating mouse mutants that copy genetic aberrations associ- Partners Michela Matteoli ated with human disease (e.g. deletion mutants lacking neuroligins Università degli Studi di Milano or complexins); Reinhard Jahn Milano, Italy • by generating and characterising mouse mutants affecting pro- Erwin Neher teins that result either in a phenocopy of a disease or at least mimic Max-Planck-Gesellschaft zur Yael Stern-Bach certain aspects of a disease. Förderung der Wissenschaften e.V. The Hebrew University of Jerusalem Furthermore, standardisation of techniques for studying synaptic function Max Planck Institute for Jerusalem, Israel will be directly applicable both for diagnostics and the screening/evalua- Biophysical Chemistry tion of potential therapeutics: Göttingen, Germany Guillermo Alvarez de Toledo • by providing standardised assays for expression proﬁ ling that are Universidad de Sevilla Nils Brose Seville, Spain applicable for biopsy material and that may be further developed Max Planck Institute for into standardised diagnostic kits; Experimental Medicine Rostislav Turecek • by providing standardised synaptic preparations for the measure- Göttingen, Germany Institute of Experimental Medicine ment of synaptic parameters that can be utilised for testing drug Prague, Czech Republic effects on synaptic transmission; Peter Seeburg • by developing criteria, used in cell-based or in vitro assays, to Thomas Kuhner Yukiko Goda screen for the neurological side-effects of candidate drugs. Max Planck Institute Medical Research Council for Medical Research London, United Kingdom Heidelberg, Germany Thomas Bourgeron Matthijs Verhage Institut Pasteur Vrije Universiteit Amsterdam Paris, France Amsterdam, The Netherlands Alfred Bach Hannah Monyer Axaron Bioscience AG University Hospital Heidelberg Heidelberg, Germany Heidelberg, Germany Henrik Martens Christophe Mulle Synaptic Systems GmbH Centre National de la Göttingen, Germany Project number Recherche Scientifi que LSHM-CT-2005-019055 Bordeaux, France Pascal Neuville Faust Pharmaceuticals EC contribution Gerard Borst Illkirch, France € 8 000 000 Erasmus University Duration Medical Center Rotterdam 48 months Rotterdam, The Netherlands Starting date 01/12/2005 Instrument IP Project website www.eusynapse.mpg.de

126 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES IMAGEN

Reinforcement-related behaviour in normal brain function and psychopathology

Keywords Functional neuroimaging, genes, reinforcement by the shortcomings in their classifi cation, which is often merely descrip- behaviour, adolescence, epidemiology tive and groups heterogeneous disorders under a single diagnostic category. The use of brain imaging methods makes it possible to understand brain mechanisms that underlie the various disorders and link them to genetic variation and behavioural changes that are characteristic of disease processes. Recent advances in genomics (e.g. complete catalogues of candidate genes and candidate genetic variants, high throughput genotyping Summary methodologies, etc.) provide new opportunities to identify the genes that infl uence these processes, and to examine their interaction with A fundamental function of the brain is to evaluate the motivational and environmental factors. One of the major challenges addressed in this emotional signifi cance of stimuli and to adapt behaviour accordingly. We programme is to assemble key biological resources and the brain imag- hypothesise that genetically infl uenced individual differences in brain ing data, from human volunteers and in animal models, that is required responses to reward, punishment and emotional cues in adolescents to take advantage of these opportunities. We will use the biological mediate the risk of mental disorders, with a major public health impact. resources and phenotype data collected here to identify major genetic Neuroimaging permits the measurement of specifi c brain functions determinants of core processes of motivational and emotional behav- implicated in the aetiology of mental disorders, and links them to genetic iours, in order to understand abnormal changes in these processes that variations and behavioural characteristics relevant to disease processes. lead to many mental disorders. The goals of assembling and investigat- The goal of the present study is to identify the neurobiological and ing these materials can be achieved only through a large, pan-discipli- genetic basis of these traits and to assess their relevance to mental nary collaboration that integrates groups specialised in neuroimaging, disorder. To this end, we will perform the fi rst multicentre functional and human genetics and behaviour. structural genetic-neuroimaging study of a cohort of 2,000 + 14 year- old adolescents. Intermediate phenotypes of risk for adolescent mental illness will be explored based on cognitive, behavioural, clinical and Aim neuroimaging data. To determine the predictive value of intermediate phenotypes and genetics for development of mental disorders, our cohort Identifi cation and characterisation of specifi c genetically infl uenced alter- will be psychometrically assessed during recruitment and longitudinally ations in reinforcer sensitivity and executive control which are manifest in at year 4 (age 16-18) of this proposal. adolescence and carry the risk of overt psychopathology later in life. DNA samples and a phenotype database for the cohort will create a powerful resource for present and future genetic investigations. In the present study, we will assess association with markers of genes chosen from existing Expected results genomic information and from our studies of animals selected for extreme phenotypes of impulsivity and other relevant behavioural traits. Results Using post-genomic methodological approaches while establishing and obtained will be validated in 1,000 siblings from the Canadian Saguenay applying new neuroimaging technologies for multicentre functional and youth neuroimaging study. The IMAGEN study will help elucidate the neural structural neuroimaging studies, we will generate the knowledge to basis of mental disorders and will lay the groundwork for development bridge genetic and neural functions and develop novel prevention and of treatments that target specifi c pathological processes across mental treatment strategies of disorders such as addiction, depression or anxiety disorders, rather than heterogeneous categories of mental illness. that are related to defi cient reinforcer sensitivity and executive control. This proposal addresses two major societal issues: • neurobiological characterisation of mental disorders to identify novel prevention and treatment strategies; • mental and behavioural disorders in adolescence. Considering the overall prevalence of >20 % of mental and behavioural disorder among children and adolescents in European countries, inadequate attention is paid to mental and behavioural disorders during child- Problem hood and adolescence. In addition to drug use disorders and affective/anxiety disorders, which frequently begin during adolescence but have their peak Mental disorders are a major health problem with a lifetime prevalence incidence in adulthood, behavioural and emotional disorders in adoles- of more than 25 % (WHO report 2001). In Europe, the prevalence of cents include hyperkinetic disorders, conduct disorders and emotional dis- addiction, affective and anxiety disorders alone amounted to 34 million orders. All these diagnostic categories involve dysfunctional reinforcer people in 2004, with an annual cost to society of at least € 204 billion. sensitivity and executive control. Our contributions to the identifi cation of The analysis of genetic factors in mental disorders has been hampered the neural basis of these disorders will add to the new risk intervention

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 127 General Electric 1.5 Tesla MRI Scanner at the Centre for Neuroimaging Sciences, a joint venture of the King’s College London Institute of Psychiatry (IoP) and the South London and Maudsley Trust.

research demanded in the WHO report 2002 – reducing risks, promoting Coordinator Patrick Constant healthy lives, and strengthen the scientifi c evidence base for new preven- Pertimm Pertinent tion and treatment strategies of disorders related to reinforcer sensitivity Prof. Gunter Schumann, MD et Immediat and executive control. Chair of Addiction Biology Asnières sur Seine, France Institute of Psychiatry This project will reinforce European competitiveness in the emerging fi eld King’s College David N. Stephens of gene-neuroimaging analyses and will help to understand the neuro- London, United kingdom University of Sussex biological basis of clinically relevant behavioural traits. The multicentre [email protected] Brighton, United Kingdom character of this research project will enable a unique mobilisation of resources and expertise across Europe, including leading academic cen- Christian Büchel tres in neuroimaging, clinical and preclinical neurobiology, psychiatry, Partners Universitätsklinikum genetics, ethics as well as multiple SMEs. Due to its size and sophisticat- Hamburg-Eppendorf ed neuroimaging, neuropsychological and psychometric analysis, this Gunter Schumann Hamburg, Germany project will provide a reference databank, both for neuroimaging-related Institute of Psychiatry and genetic research. King’s College London Birgit Fuchs London, United Kingdom GABO Geselllschaft While in the context of this proposal, we are focusing on genotype für Ablauforganisation x phenotype analyses related to drug use disorders, the general psycho- Herta Flor miliarium mbH & Co. KG pathological relevance of traits related to the processing of reward and Central Institute of München, Germany punishment and behavioural control, as well as the comprehensive psy- Mental Health chometric assessment of personality traits and mental disorders, will Mannheim, Germany John Rogers result in the generation of a major resource for European and interna- Delosis Ltd. tional neurobiological research. The project will, thus, have a strong Trevor W. Robbins London, United Kingdom structuring effect on European neurosciences. The Chancellor, Masters and Scholars of the University of Cambridge Tormod Thomsen UCAM-DEXP NordicNeuroLab AS Potential applications Cambrigde, United Kingdom Bergen, Norway Romain Valabrègue Tomas Paus Identifi cation and neurobiological characterisation of risk groups for SCITO S.A. University of Nottingham neuropsychiatric disorders, allowing for development of early primary Paris, France Nottingham, United Kingdom intervention or secondary pharmacological and psycho-therapeutical interventions. Jean Baptiste Poline Dirk Lanzerath Commissariat à l’Énergie Atomique German Reference Centre Paris, France for Ethics in Life Sciences Bonn, Germany Mark Lathrop Consortium National Hugh Garavan de Recherche en Génomique Trinity College Dublin Evry, France Dublin, Ireland

Andreas Heinz Jean-Luc Martinot Charité – Universitätsmedizin Berlin, Unit INSERM – CEA ERM Campus Charité Mitte 0205 Orsay Unit Project number Berlin, Germany Orsay, France LSHB-CT-2007-037286 EC contribution € 10 000 000 Duration 60 months Starting date 01/02/2007 Instrument IP Project website www.imagen-europe.com

128 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES INCF

Neuroinformatics

Keyword Neuroinformatics Aim

The large area of neuroscience that extends from genes and molecules to systems neuroscience, psychology, psychiatry, neurology and neuro- inspired technology will promote Neuroinformatics. The diseases of the nervous system are responsible for about 35 % of the total cost of disease and health problems. Databases extending over this vast area from Summary basic science to disease would be markedly facilitated by the development of databases which are linked to each other. Similarly a development Neuroinformatics (NI) is a new research fi eld devoted to the development of towards a routine of repository of investigator data, available to other neuroscience data and knowledge bases, together with computational models researchers, will be very important to save resources and time, at least in and analytical tools, for the sharing, integration and analysis of experimental certain areas. Modelling at all levels from the single molecule and recep- data and the advancement of theories of neural function. To develop this tor structure, to the cell, network and cognitive levels is increasingly area a new ‘International Neuroinformatics Coordinating Facility’ (INCF) has becoming an indispensable method in the quantitative analyses of neu- been instigated within OECD through Global Science Forum. It was formed ral systems. Infrastructure for computational neuroscience is a primary on 1 August 2005 – currently with seven EU countries, Japan, Switzerland goal for INCF as is the development of IT base tools. and the US as member states – but more countries are likely to join. The secretariat is located at the Karolinska Institute campus in Stockholm, and The purpose of INCF is to coordinate the different global efforts, to avoid Professor Jan Bjaalie is the newly appointed executive director. duplication between researchers in different parts of the world and to INCF will have a secretariat consisting of an executive director, two associate catalyse a rapid development of these areas of neuroinformatics. directors and a staff of seven, including programmers. The secretariat salaries and operation costs will be covered by the contributions of the member states as specifi ed in the understanding signed with each state. Within this SSA, Expected results the purpose is to grant support for an initiative to coordinate NI in a global perspective, which is the explicit goal of INCF. This support will help INCF implement its plans and goals, and will facili- The objective of this proposal is to promote the INCF objectives by: tate the necessary interaction between the INCF central offi ce and the • organising small workshops (5-15 experts) in different NI areas national nodes in the different member states. This involves a global to be implemented (the latter can be databases concerned with neuroinformatics database structure and infrastructure for computa- a particular disease or neural function, and the workshops will be tional neuroscience. The promotion of NI will speed up the development held at the INCF secretariat); of neuroscience and ensure more effective research. One goal of neuro- • making an inventory of all NI resources available in Europe (scientifi c science is to uncover the bases of many diseases of the nervous system environments, training activities at PhD and postdoctoral level, and to fi nd ways either to prevent them or remedy their symptoms once different national or international funding schemes); they have occurred, and ultimately to fi nd a cure. Reducing the signifi - • analysing the most effective strategy to implement INCF objectives cant costs of diseases will obviously be benefi cial for the economy and promote an effective interaction with the national nodes (each worldwide. More importantly, it will reduce the suffering of patients and member state has agreed to make a national node to further the their relatives. objectives of INCF); • analysing the most effi cient way to develop NI training programmes, both for students with a basic training in the IT area, and for those with Potential applications a neuroscience training that need to develop NI skills to model e.g. a neural function at any level from the molecular to the behavioural. Basic and clinical neuroscience.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 129 Coordinator Global Neuroinformatics Coordination Prof. Sten Grillner INCF and Karolinska Institutet INCF tasks • Coordination of tasks Partner • Guidelines for: - Data sharing Prof. David Willshaw - Software sharing University of Edinburgh • Development of: Edinburgh, United Kingdom - Ontologies - NI-data bases - Standards • Maintenace of: INCF tasks - Portal Secr. host - Data repository - Funding scheme

Professional Organisations National Scientists Research Bodies Scientific Journals

Node tasks • Dissemination Node tasks • Integration • National infrastructure • Mirror sites • Software distribution • National training • Implementation Project number • INCF tasks LSSM-CT-2006-037452 EC contribution € 500 000 Duration 36 months Starting date 01/01/2007 Instrument SSA Project website www.incf.org

130 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES INDABIP

Innovative diagnostic approaches for biomarkers in Parkinson’s disease

Keywords Parkinson’s Disease, diagnostics, Aim drug target identifi cation While biomarkers can take many forms, the INDABIP project aims to identify relevant proteins, mRNA, miRNAs, differentially matured RNAs and methylated DNA, whose analysis can be transformed in diagnostic tests. The brain damage in Parkinson’s Disease and other neurodegenerative diseases is progressive and, by opening a window for therapeutic treatment and prevention, the early detection of biomarkers may help to identify individuals that have started to develop the disease. Summary While most biomarkers are mere reporters of the disease state, others The INDABIP project aims to identify biomarkers for the early diagnostics of may actually be key factors in the processes that lead to the onset and Parkinson’s disease. Biomarkers are any type of biological molecules that development of the disease. The second aim of the INDABIP project is are present in people suffering a specifi c disease, even when the symptoms therefore to identify these key factors and to evaluate their potential as are mild, but that are not present in people free from the disease or drug targets. suffering from other diseases. The identifi cation of the early indications of the development of a neurodegenerative disease is the basis for preventive To study the development and evaluate potential targets and therapies treatment strategies, which aim to control the disease at early stages rather for Parkinson’s Disease, biological models are needed, in which the than when irreparable neurological damage has been caused. events that occur in the patient are mimetised. A target is any molecular entity (transcript, protein, etc.) that can be interfered with to interrupt the chain of events leading to the onset or development of the disease. We will develop novel in vitro models for Parkinson’s Disease based on cell cultures for the development of a high throughput screening platform, and use these for in vitro target validation experiments.

Finally, we will initiate a screen for potential drugs interacting with selected molecular targets.

Expected results

Problem The INDABIP project aims to deliver an early diagnostic assay for Parkinson’s Disease validated in three independent laboratories. We will Parkinson’s disease (PD), a specifi c type of a group of neurodegenerative work closely together with Parkinson’s Disease Patients Organizations to disorders called synucleinopathies, is the second most common neuro- discuss and promote the possible future implantation of this diagnostic degenerative disorder worldwide. Many neurodegenerative disorders assay in the national health systems. are preceded by a pre-symptomatic phase, probably lasting years, during which degeneration and death of neurons occurs before any clinical symptoms appear. One major challenge of clinical research is to improve Potential applications early detection of these diseases by developing tools to move diagnosis backward in the neurodegeneration temporal course. Thus, the central Early diagnostics becomes really helpful when appropriate treatments objective of this INDABIP proposal is the identifi cation of molecular become available that can prevent the further development of the incipi- markers that hallmark the onset of a cellular dysfunction in the brain ent disease. The INDABIP project will therefore develop the tools to areas involved in PD, and that permit the identifi cation of at-risk groups, screen for drugs that can prevent the further development of Parkinson’s both for disease onset and progression during the pre-clinical period. Disease and initiate the process of drug development.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 131 PD DLBD Coordinator

Tamara Maes C.S.O. Oryzon genomics [email protected] Barcelona, Spain

Partners

Oryzon genomics SA Barcelona, Spain

Genfi t SA Loos, France

Prof. Isidro Ferrer Fundació Privada Institut d’Investigació Biomèdica de Bellvitge Lewy bodies: optical and e.m. Hospitalet de Llobregat, Spain LEWY BODIES Spherical intracytoplasmic inclusions with concentric eosinophilic core Prof. Hans Kretschmar and peripheral unique or multilayered narrow, Ludwig-Maximilians- pale-stained, hallo. Diameter ranging from 8 to University of Munich 30 μm. May bwe unique or multiple.The core is composed of densely packed ﬁ laments Zentrum für Neuropathologie and granular material. The hallo is formed und Prionforschung by radially-arranged 7-20 nm intermediate Münich, Germany ﬁ laments associated with granular electron- dense coating material and vesicular structures. Prof. Irina Alafuzoff Kuopio University Kuopio, Finland

Prof. Regis Bordet University Hospital Lille, France

Project number LSHM-CT-2006-037050 EC contribution € 1 600 000 Duration 36 months Starting date 01/12/2006 Instrument STREP

132 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES INTELLIMAZE

High-throughput, fully automated and cost-effective behavioural phenotyping of normal, clinical and genetic mouse models

Keywords Mouse phenotyping, testing apparatus, Problem high-throughput testing, transponder technology, automation, Alzheimer’s disease, depression, anxiety, brain lesions, Too many mice. During the past 15 years, molecular biology and ENU neurology, transgenic mice mutagenesis programmes have led to an explosion of mouse models, many of them needing behavioural characterisation. Behavioural characterisation (‘phenotyping’) of laboratory animals is an established tool in neurobiology and neuropharmacology, but may fi nd much wider application in drug development, ecotoxicology and other fi elds of biomedicine dealing with changes in body physiology. However, there is Summary no appropriate technology to deal with the large number of mice. High- throughput behavioural phenotyping (HTBP) would be needed, either Behavioural change is the most sensitive biological end-point signalling for mass screening of mice, or, equally important, for the application of any alteration in the organism of a mouse. However, large-scale bio-assays numerous tests to selected samples. of behaviour face limitations: outdated technology, need for space and specialised manpower, lack of standardisation, and increasing legal demands Simplistic technology. Traditionally, animals are subjected manually to on animal husbandry. This has limited the use of behavioural methods to a battery of tests designed for demonstrating changes in spatial or fear- specialised laboratories, limiting the market as well. related memory, motivation, exploration or learning abilities thought to In order to advance scientifi c progress and expand the market, two SMEs and refl ect the operation of specifi c brain system. In addition, tests have an academic partner will combine their existing expertise and products in been designed to model processes observed in human diseases of the the fi eld of behavioural phenotyping to generate a compact modular system, CNS. Thus, every laboratory specialising in behavioural analysis must INTELLIMAZE, based on a recently developed technology of in-cage testing of maintain an expensive collection of test set-ups, all of them being microchipped mice, INTELLICAGE. This system should fi t into a single small conducted with single mice. mouse room, where it will: • assess home cage activity and learning of transponder-tagged mice Economic limits. A standardized preliminary behavioural screen of living in social groups; a knockout mouse requires testing of about 100 mice in 4-6 different • automatically analyse social behaviour; tests, equalling three person-months; a more detailed characterisation • guide individual mice to a battery of traditionally used tests; requires up to 6 person-months, even when using highly standardized • show ongoing learning on-line to a supervisor working in his offi ce; procedures and automated data analysis. Thus, the main obstacles for • analyse data according to expert knowledge-based rules; high-throughput behavioural phenotyping are costs in terms of space, • provide a web-based analysis of results for user groups. salaries and expertise. The bottleneck of such technological development is functional validation and comparison with traditional tests. Three reputed academic partners will Hidden problems in mouse testing. Traditional behavioural characterisation use the novel technology for: of mice faces a number of problems well known to experts but rarely • generating new mouse models of depression; discussed publicly: • profi ling malfunctions of specifi c brain systems; • mice are far less cooperative than rats and other laboratory animals, • monitoring the effects of age-dependent neurodegeneration in existing thus requiring lengthy testing; and new mouse models. • there is limited knowledge about the behavioural biology of mice, Finally, an SME partner in need of effi cient behavioural phenotyping will at least with respect to the needs of behavioural assessment in validate the novel systems for drug discovery and development. biomedicine; We expect that the availability of simplifi ed, rapid and thorough behavioural • species-specifi c functions of structures in the mouse brain are testing of mice without need for specialised personnel will open new and poorly known; much larger markets for the SMEs. Moreover, we predict signifi cant scientifi c • the development of clinical mouse models for psychopathology discoveries in the fi elds of drug development, psychopharmacology, faces problems in recognising how a behavioural impairment rele- neurodegeneration, neural plasticity and repair, and genetic engineering. vant to the human disorder would manifest itself in a mouse; • all these problems are compounded by the lack of standardisation and comparability of test results.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 133 Example of traditional test to be included in INTELLIMAZE food burrowing test after Deacon. This test recognises brain malfunction by the unwillingness of mice to remove food morsels from a tube.

Animal welfare. For behavioural testing, mice are often kept isolated Expected results over prolonged periods. Yet, mice are social animals and isolation in an impoverished environment can cause many physiological and neuro- What is proposed here is almost a dream for mouse behavioural biolo- chemical changes producing confounded results. Moreover, European gists: a fully automated behavioural test system for mice inside a home legislation is likely to ban animal housing under isolated conditions. This cage and outside in traditional test arenas, working without supervision calls for social in-cage alternatives in behavioural testing. and outputting data in familiar format and ready for statistics. Scientiﬁ cally, it will permit the implementation of almost all those studies that should have been done but were never realised. Moreover, Aim cross-laboratory standardisation is achieved with minimal effort and animal welfare guaranteed. Commercially, the system will certainly pene- This SME project allies three SMEs and four academic partners in devel- trate the market, and has the potential of opening new markets in the oping and validating a compact, economic and fully automated modular biotechnology and pharmaceutical industries, which have been hesitant platform, INTELLIMAZE, that will permit both high-throughput and about using costly behavioural assessment. Finally, as the products com- detailed behavioural characterisation of current and future mouse mod- bine state-of-the-art technology with expert behavioural knowledge, els in biomedicine. In accordance with legislative trends in Europe, the the project will offer a sustainable perspective for the SMEs, providing system will operate with mice living in social groups in a home cage. a technological edge on the US, and keeping production competence in Europe. The technical development will be done by two SMEs (NewBehavior and Frank Buschmann International) and one academic partner (Laboratory of Systemogenesis at the Anokhin Institute of Normal Physiology of the Potential applications Russian Medical Academy of Science). We expect these systems will be employed by the pharmaceutical indus- The commercial objective is to obtain a signiﬁ cant market share of the try, by small biotech companies producing and testing their own developing market of automated test systems, to expand the present compounds, by companies offering phenotyping services for mice, and market to include new categories of customers, and to tailor such sys- by academic behavioural research laboratories. tems for small biotech SMEs in need of behavioural characterisation.

The joint scientifi c objectives of the academic partners are validation of this novel technology and inter-laboratory standardisation. In line with their expertise, they will also implement the novel technology specifi cally for: • in-depth behavioural profi ling of brain lesions in mice, focusing on hippocampus, striatum and prefrontal (University of Zurich); • development of novel behavioural paradigms for mouse models of anxiety and depression (Istituto Superiore di Sanita in Rome); • monitoring the progress of brain malfunction in mouse models of Alzheimer’s disease and premature ageing (EVOTEC Hamburg and NEUROTEC at the Karolinska Institute in Stockholm).

134 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Coordinator

Prof. Hans-Peter Lipp NewBehavior AG Zürich, Switzerland [email protected]

Partners

Frank Buschmann International GmbH Bochum, Germany

Enrico Alleva Istituto Superiore di Sanita Roma, Italy

Assessing learning abilities of individual transponder-tagged mice in an Abdul Mohammed automated home-cage (INTELLICAGE). Each cage house up to 16 mice Neurotec Karolinksa Institute and contains 4 operant conditioning units allowing for assessment of spatial, temporal and operant learning abilities. Stockholm, Sweden

David Wolfer University of Zürich Zürich, Switzerland

Kostya Anokhin Anokhin Institute Russian Academy of Medical Science Moscow, Russian Federation

Antje Willuweit Evotec Neurosciences AG Hamburg, Germany

Project number LSHM-CT-2007-037965 EC contribution € 1 299 697 Duration 36 months Starting date 01/11/2006 Instrument STREP – SME

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 135 MEMORIES

Development, characterisation and validation of new and original models for Alzheimer’s Disease

Keywords Alzheimer’s disease, transgenic mouse model, Recently, memantine, an uncompetitive antagonist of the N-methyl-D- neurotrophins aspartate receptor, was approved for the treatment of moderate-to-severe AD. These agents can benefi t some AD patients for a limited period of time. However, due to the extensive and multifocal nature of AD degeneration, the effects of these neurotransmitter modulators are modest and the need of a truly disease-modifying drug persists. Although there have been signifi cant advances in understanding the biology and subse- Summary quent diagnosis of AD, such research has not been translated into a disease-modifying treatment. Alzheimer’s disease (AD) is a progressive brain disorder that gradually destroys a person’s memory and ability to learn, reason, make judgments, One limit in translating basic research fi ndings into therapeutical agents communicate and carry out daily activities. The disease is the most is the lack of suitable animal models fully reproducing the AD neuro- important type of dementia affecting elderly people, and the percentage degeneration. Having animal models reduces the risk, and thus the cost, of patients is going to increase exponentially over the next few years. of developing drugs. Currently, no aetiology and cure has been found. One major hurdle in drug screening and target discovery in AD is the lack of a suitable animal In the past decade, AD research has been fundamentally infl uenced by model, as these fail to fully reproduce the characteristics of the disease. the development of genetically modifi ed animal models of amyloid-driv- The MEMORIES project aims to develop new mouse models based on en or tau-driven neurodegeneration. These in vivo models – exploited a defi cit of neurotrophic signalling, potentially useful for developing new on the basis of early-onset disease determined by rare, inherited muta- therapeutic tools. tions – are important in understanding the involvement of amyloid or tau in the onset of the disease, but have failed to reproduce the hallmarks of the AD pathology fully.

One other potential pitfall in these transgenic mice is that these models might not be applicable to sporadic AD. Indeed sporadic forms of AD, which account for more that 95 % of the population affl icted by AD, are Problem multifactorial diseases, the progression of which is infl uenced by gender and epigenetic factors (such as neuroinfl ammation, growth factor defi cits, Alzheimer’s disease (AD) is a neurological disorder and is the most autoimmunity/autotoxicity). The use of animal models derived from the common form of dementia in later life. It is estimated that, by 2050, the genetic forms of AD to screen drugs, instead of more appropriate animal number of people aged 80 years or older will approach 370 million models, have revealed the limitations of their use for sporadic AD. Indeed, worldwide and that 50 % of those aged 85 years and older will be the vaccine approach seemed to work well in mice, but brain infl ammation affected by AD. in a few patients triggered an abrupt halt to the clinical trial.

AD is clinically characterised by short-term memory loss and cognitive dementia, associated with language and behavioural impairments. The Aim pathological hallmarks of AD include the presence of extracellular amyloid plaques, intracellular neurofi brillary tangles (NFT), neurodegeneration The MEMORIES hypothesis-driven project brings together eight partners and cell loss. One severely affected region of the AD brain is the basal from fi ve countries with the aim of developing, characterising and forebrain (which includes the nucleus basalis of Meynert, the medial validating new animal models that have a real potential for becoming septum and the diagonal band of Broca), a group of cholinergic neu- a ‘gold standard’ in the AD fi eld. rons that are connected to areas of the neocortex and hippocampus and that are important for learning, memory and attention. Expected results The complex degeneration in AD has been fertile ground for the formulation of hypotheses on the pathogenesis of the disorder. Methodological Using a multidisciplinary approach, a panel of mouse models will be pro- advances have shed light on alterations of the various neurotransmitter duced and analysed for the presence of neurodegeneration. These mice systems in the AD brain. The discovery of the degeneration in the basal will express specifi c antibodies neutralising TrkA receptors or mutated forebrain, in the context of experimental evidence for the role of ace- form of pro-NGF. AD11 anti-NGF, which already represent a good model tylcholine in memory, have led to the development of a symptomatic for sporadic AD, will be crossed to mice in which the human APP or Tau therapy for AD, based on enhanced acetylcholine availability determined are over-expressed or to mice in which pro-convertases or the TrkB or by cholinesterase inhibitors. SorLA receptors are knocked out. Mice will be analysed using standardised

136 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES methodology for neuroanatomy and behavioural analysis. We anticipate Coordinator Liliana Minichiello that blocking different signalling pathways will help in ameliorating the European Molecular currently available experimental mouse models, and will also be useful for Simona Capsoni Biology Laboratory developing new therapeutic tools for this disease and strengthening Lay Line Genomics SpA Monterotondo, Italy Rome, Italy European competitiveness in the war against AD. [email protected] Antonino Cattaneo European Brain Research Institute Foundation Potential applications Partners Rome, Italy

The creation of a mouse model – which, by fully reproducing the hall- Manuel Gaviria Eero Castren marks of the disease, will not only provide insights into the neurobiology Neuréva Inc. University of Helsinki of the disease, but will also permit the evaluation of the response to Montpellier, France Helsinki, Finland drugs, especially in relation to the effects on pathology – is one of the most challenging aims of AD research. Thomas E. Willnow Daniel Constam Max Delbrück Centrum Swiss Federal institute

Für Molekulare Medizin of Technology – ‘Daniel Constam’ The potential medical benefi ts that will derive from the production of Berlin, Germany Ecole Polytechnique this mouse model are immense. The identifi cation of protein linked to Fédérale de Lausanne specifi c cellular pathways will provide the possibility of developing new Lausanne, Switzerland diagnostic assays and new drug targets for the treatment of AD. Mouse models will also make it possible to visualise neural circuits in their nor- David Koubi mal and abnormal states, which is likely to have a large impact on the ACIES diagnosis of disease and the evaluation of the effectiveness of therapy. Lyon, France

Project number LSHM-CT-2007-037831 EC contribution € 2 374 689 Duration 36 months Starting date 01/01/2007 Instrument STREP

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 137 NEOBRAIN

Neonatal Estimation Of Brain Damage Risk and Identifi cation of Neuroprotectants

Keywords Newborns, hypoxia/ischemia, infl ammation, Problem perinatal brain damage neuroprotectants Prevention of perinatal brain damage is of major importance for public health and obviously for individual wellbeing. Both white and grey brain matter are affected in perinatal brain damage observed in pre-term infants. Long-term consequences of extreme prematurity are devastating, and perinatal brain damage clearly plays a role in this scenario. The current pathogenetic paradigm of perinatal brain damage in pre-term infants has multiple interrelated aspects and includes infection/infl am- Summary mation, hypoxia-ischemia, excitotoxicity, and free radicals. It is likely that these mechanisms do not act alone, but in concert. NEOBRAIN brings together small and medium-size enterprises (SMEs), industry and academic groups devoted to the diagnosis, management The absolute number of neurological handicaps of perinatal origin is and neuroprotection in newborns with perinatal brain damage. The focus increasing in western countries due to the increasing survival of pre-term of NEOBRAIN is the prevention of brain damage mainly observed in pre- infants. The major brain lesions associated with cerebral palsy (CP) and term newborns. cognitive impairment are white matter damage (WMD), mostly occurring The objectives of NEOBRAIN are: in very pre-term infants (born in less than 32 weeks of gestation), and • to generate marker profi les of damage in multiple animal models and cortico-subcortical lesions mostly observed in term infants. For fi nancial, in human pre-term infants using genomic/genetic, proteomic and technical and ethical reasons, the pharmaceutical industry has diffi culties metabolomic approaches, as well as imaging and electrophysiologic in making substantial investments in this area, and this has left perinatolo- modalities; gists with a limited therapeutic arsenal. At the present time, despite major • to develop neuroprotective strategies by identifying candidate improvements in neonatal care, there are no established therapeutic regi- molecules for intervention in animals; mens for the prevention or treatment of perinatal brain lesions that are • to implement a platform for an observational clinical epidemiologic successful. Nevertheless, epidemiological and experimental data have study in human infants designed to contribute to objective 1 above, allowed the identifi cation of potential targets for neuroprotection. New and to transfer from the animal to the human level insights gained animal models, such as those employed in NEOBRAIN, clearly show the in objectives 1 and 2; pathophysiology involved in neurodegeneration and will help identify • to prepare for drug testing by using the project structure as neuroprotective strategies in the newborn. a platform for initial steps in clinical testing of potential interventions discovered in NEOBRAIN. We further envisage developing the clinical platform in such a fashion Aim that it can serve as the basis for subsequent large-scale pan-European perinatal neuroprotective research initiatives (Euro-Neo-Net, EURAIBI). Objective 1 – Generate marker profi le(s). To help reduce the enormous individual, familial and societal burden that perinatal brain damage represents, it is our fi rst objective to identify early damage markers and novel pathways for interventional neuroprotection. We will study, in various established animal models, the mechanisms that lead to perinatal brain damage in order to identify genomic, proteomic and metabolomic biomarkers to generate biomarker profi les. We will also establish biomarker profi les in human newborns (see objective 3, below) in an observational clinical study involving extremely pre-term infants born before completion of 28 weeks gestation (normal pregnancy duration: 40 weeks).

Objective 2 – Develop neuroprotective strategies. Once we have identiﬁ ed biomarkers of damage and potential avenues for neuroprotection, we can begin to develop an intervention. Again, we will initially use multiple animal models to pursue this goal. Only the most promising strategies will be considered worthy of being translated from bench to bedside.

Objective 3 – Implement clinical platform. We see the need to implement a clinical platform for two purposes. First, we want to design a biomarker proﬁ le of perinatal brain damage in experimental animals and in human

138 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES NEOBRAIN researchers are committed to protect the preterm brain.

newborns (see above). Thus, we will need to establish a functional Economic impact – Economic burden of prematurity-associated brain network of institutions caring for newborns that can serve as the basis damage: the potential savings to the EU and worldwide are worth men- for such a clinical study, designed to identify human biomarker profi les tion. Almost half of all cerebral palsy cases are due to prematurity (41 %). based on genetic and biochemical markers, electroencephalographic The estimated total lifetime cost for one case of cerebral palsy in the (EEG) patterns and magnetic resonance imaging (MRI). Secondly, we United States in 1992 US dollars is $ 500 000. About 80 % of extremely will use and expand this platform for clinical drug testing both within low gestational age newborns will be disabled in such a fashion that the 36 months of NEOBRAIN and thereafter. they will lead to more than some € 6-7 billion of newly incurring prospective healthcare costs annually in the EU. These costs could be saved Objective 4 – Prepare for drug testing. After biomarker profi les of early if neurodisability among extremely preterm infants alone could be brain damage are established in animals and newborns, and neuropro- prevented. tective strategies have been identifi ed, NEOBRAIN will pave the way for clinical drug development. In essence, our fourth objective is to design the clinical platform in a way that allows for quick expansion (i.e. recruit- Potential market ment of further centres), so that bench-to-bedside translational steps (i.e. a clinical trial) can be taken quickly after NEOBRAIN is fi nished. The close cooperation and integration of enterprises and academic Indeed, we will prepare for the possibility that this might be the case centres offers the unique chance to exploit new business areas and even within the three years of NEOBRAIN. position the participating companies as market leaders not only in Europe, but also in the USA, Australia and Asia.

Potential applications New multi-parametric biomarker measurement tools will be developed based on metabolomic and proteomic techniques. These tools, in turn, Societal impact: the most obvious potential impact of NEOBRAIN is its will help improve clinical diagnostics with regard to perinatal brain inju- potential to help reduce mortality and stamp out developmental ry, and this will contribute to a greater potential for neonatologists to disabilities associated with perinatal brain damage. The most important consult with parents and within their therapeutic teams about the indi- source of societal suffering from perinatal brain damage is at the vidual child’s prognosis. As such, we consider the growing medical sub- individual and family level. Four out of fi ve pre-term infants are limited discipline of ‘neonatology’ a potential market for tools to be developed in their everyday activities. Moreover, brain-damage-associated cognitive in NEOBRAIN. and learning diffi culties represent a potentially preventable source of suffering. It is NEOBRAIN’s goal to contribute to an improvement of this Another potential market among sick and pre-term newborns is called situation for future generations. ‘theranostics’, the individualised surveillance of therapy effi cacy and results. NEOBRAIN will contribute to this fi eld by offering improved Economic impact – economic burden of prematurity: this is immense, as strategies for biomarker measurements, including imaging and electro- it is estimated that, in 2005, a cumulative cost of hospital inpatient encephalographic markers of infant wellbeing. admissions during the fi rst 10 years of life of about £ 20 000 was incurred for children born at <28 weeks gestation. This estimate does not include non-hospital inpatient health care costs, such as costs incurred through family practitioners, educational and social services. An average total two-year health care cost of € 104,635 was estimated, based on surviving infants with a birth weight <1000 g, from a regional cohort at Helsinki University’s catchment area in Finland.

Approximately 60 000 infants who sustain some sort of brain injury are born each year in the EU. Improved capabilities to protect or even intervene in this scenario are much desired. The high-risk group of pre-term infants ranges from 4-6 % to up to 10 % of all newborns in developed western countries. Annually, approximately four million live births occur in the EU. Thus, approximately 200 000 infants will be pre-term, and about 15-20 000 will be extremely pre-term (<28 weeks gestation). In the USA, approximately four million live births occur annually. Among these are 499 000 (12.3 %) pre-term infants, 30 000 being <28weeks gestation. Thus, the potential market for any diagnostic modality and neuroprotective intervention in pre-term infants is very large.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 139 Coordinator University Medical Center Utrecht Prof. Olaf Dammann Utrecht, The Netherlands Medizinische Hochschule Hannover Hannover, Germany Göteborgs universitet [email protected] Göteborg, Sweden

Institut National de la Santé Partners et de la Recherche Médicale Paris, France Medizinische Hochschul Hannover Hannover, Germany Université de Genève-Hôpitaux Universitaires de Genève BIOCRATES life sciences GmbH Genève, Switzerland Innsbruck, Austria Charité Universitätsmedizin BrainZ Instruments Limited Berlin, Germany Auckland, New Zealand Università degli Bioanalyt GmbH Studi di Siena Potsdam, Germany Siena, Italy

THERAPTOSIS S.A. Lunds Universitet Paris, France Lund, Sweden

NEUROPHARMA Tres Cantos – Madrid, Spain

Project number LSHM-CT-2006-036534 EC contribution € 3 299 809 Duration 36 months Starting date 15/10/2006 Instrument STREP Project website www.neobrain.eu

140 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES NEURODYS

Dyslexia genes and neurobiological pathways

Keywords Dyslexia, functional imaging, event-related school dropouts, higher incidence of long-term unemployment. Other potential, association, linkage disequilibrium, mapping, consequences of dyslexia are that it increases the risk for psychiatric gene cloning, DNA databank, gene-environment, DTI, disorders like emotional and affective disorder, and conduct disorders. early risk factors, longitudinal Although some individuals show considerable amelioration of symp- tomatology with development and treatment, the core symptoms persist into adult life and the disorders are associated with signifi cant morbidity across Europe. The development of novel specifi c treatments or strategies to prevent or ameliorate reading and spelling ability requires a proper understanding of the underlying molecular and neu- Summary rological mechanisms. These advances are likely to be of general interest to society. In part that is because there is evidence that suscep- The NEURODYS project has been initiated as a major new European effort tibility loci predispose to dyslexia and to dyslexia- related traits, such as to clarify the biological bases of developmental dyslexia, or specifi c reading phonological and orthographic processing. It will be necessary for disability. Dyslexia is a common, severe learning disorder that affects at a proper appreciation of the complexity of the intervening mechanisms least 5 % of schoolchildren and it has a strong biological basis. Confi rming to understand the different dyslexia-related quantitative traits. relations between dyslexia, candidate genes and brain regions requires large samples from diverse cultures and languages – a characteristic So far the aetiology of dyslexia is poorly understood. Therefore, it is European feature. mandatory to tackle the problem from several directions. NEURODYS links 15 top research groups and clinics from nine different countries. It also covers the most common European languages of the 2.5 million dyslexic schoolchildren. The project combines innovative Aim analyses of how the reading problems relate to genes, environment, brain structure, and brain function. Nearly 4 000 children will be assessed in In a multicentre, multidisciplinary project we will investigate the bio- this large coordinated effort, which is supplemented by several major logical basis of dyslexia by collecting powerful samples of subjects national projects. consistently characterised across EU populations on three different lev- Building the largest biological database on dyslexia worldwide will put els: genetics, environment, and neuroscience. Our aim is to under- Europe at the forefront of dyslexia research in less than three years. The stand the aetiology of the disorder by integrating the results of the integration of new fi ndings from the molecular, brain and behavioural three levels. studies will facilitate a thorough understanding of universal and language- specifi c aspects in dyslexia. This provides a scientifi c basis for more On the genetic level, we will use a systematic two-stage approach to effective targeted diagnosis and treatment. map and clone dyslexia susceptibility genes in samples of 800 families and 1 900 dyslexic cases and 1 900 controls. The identifi ed risk-conferring genes will also be used to understand gene-gene and gene-environment interactions, as well as gene-specifi c contributions to a variety of neurobiological correlates of dyslexia. Identifying susceptibility alleles will be a key for developing an understanding of the molecular and cellular nature of the brain dysfunction, for discovering the factors infl u- encing phenotypic expression, and for developing rational preventative Problem and treatment strategies to improve quality of life.

Dyslexia is one of the very common learning disorders around the world Environmental risk factors will be investigated in two samples, a longi- with prevalence in about at least 5-10 % of school-age children. At tudinal sample of 4 000 twin pairs and a longitudinal sample of least 22 750 000 EU citizens are affected and perhaps another million 100 at-risk and 100 non-at-risk children. Speciﬁ cally, we will test for in those countries seeking to join. Recent estimates of the Dyslexia the presence of gene-gene and gene-phenotype relationships (interac- Institute in the UK estimate that poor literacy and basic skills, as the tions, epistasis/co-actions). The environmental factors include selected result of unrecognised dyslexia, cost the UK economy £1 billion per preschool home environmental factors such as articulation, vocabulary, year, which is a staggering £2.75 million daily. This cost for 2003/2004 and parent-child interaction, which are candidate environmental risks for an individual taxpayer equates to approximately £34 per annum. factors in dyslexia. We will use a prospective longitudinal sample to investigate the potential early predictors of dyslexia, including neuro- The societal impact of dyslexia is as follows: higher incidence of dyslex- physiological and home environment measures, taking account of the ia within the prison and probation populations, higher incidence of new susceptibility genes identiﬁ ed.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 141 Molecular genetic studies.

On the neuroscience level we will investigate the prerequisites of read- Coordinator Juha Kere ing and spelling development, and the central stages of becoming Karolinska Institutet a fl uent reader. The proposal combines structural and functional brain Gerd Schulte-Körne Stockholm, Sweden information at different levels, in order to understand better the func- Professor of Child and Adolescent Psychiatry Karin Landerl tioning and dysfunctioning of the brain, and in order to gain new insight Dept of Child and Universität Salzburg into mental processes. It is necessary to highlight the brain correlates of Adolescent Psychiatry Salzburg, Austria speech perception, grapheme-phoneme associations and visual word University of Munich recognition in order to identify genetically driven brain dysfunctions Munich, Germany Heikki Lyytinen related to important reading acquisition milestones that may lead to gerd.shulte-koerne@med. University of Jyväskylä dyslexia. The ERP studies focus on the main prerequisite necessary for uni-muenchen.de Jyväskylä, Finland starting reading acquisition, i.e. effi cient speech perception and phoneme representations. The fMRI studies complement this approach by Anthony Monaco directly focusing on the processes that build on these spoken language Partners Lon Cardon milestones to acquire fl uent written language skills like reading. University of Oxford Gerd Schulte-Körne Oxford, United Kingdom Ludwig-Maximilians-Universität München Markus Nöthen Expected results Munich, Germany Universität Bonn Bonn, Germany The knowledge gained in this project will lead to the discovery of the Leo Blomert underlying causes of dyslexia and to the development of early diagnostic Rainer Goebel Robert Plomin instruments as well as specifi c treatments. The exploitation of new University of Maastricht King’s College London treatment approaches will improve health and quality of life in Europe. Maastricht, The Netherlands London, United Kingdom Additionally, this offers new possibilities for wealth creation, as new knowledge is exploited by industries developing rehabilitation, therapy, Daniel Brandeis Franck Ramus University of Zürich CNRS and diagnostic tools, and as the fi nancial burden on our health care Zürich, Switzerland Paris, France systems is diminished. Valéria Csépe Julie Williams We will provide information that will enable society to understand this Institute for Psychology of the Cardiff University disease, increasing public and governmental awareness of the scale of Hungarian Academy of Sciences Cardiff, United Kingdom the problem, the underlying cause, the ongoing research, and the hope Budapest, Hungary for new therapy. Our collective research efforts will raise awareness at the national and European levels, as part of our mission to help prevent Jean-Francois Démonet Europe from experiencing a high incidence of dyslexic children, similar Institut National de la Santé to that already seen in the US. et de la Recherche Médicale Paris, France

Potential applications

NEURODYS will translate the discoveries into effective therapy and prevention of developmental disorders. It is of great value that the partners in NEURODYS have experience in the validation of diagnostic instruments Project number as well as treatments. In later stages, it is anticipated that newly discovered LSHM-CT-2005-018696 predictors of dyslexia will be the bases for the development of an early EC contribution diagnostic instrument. In addition, the functional brain studies will lead to € 3 040 000 the development of new training modules that will be validated in Duration controlled treatment evaluation studies. 36 months Starting date 01/07/2006 Instrument STREP Project website www.neurodys.com

142 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES NeuroproMiSe

Neuroprotective strategies for multiple sclerosis

Keywords Neurodegeneration, infl ammation, neuroprotection, Aim genomics, proteomics, transgenesis, disease models The overall aim of the NeuroproMiSe project is to elucidate the molecular mechanisms underlying infl ammation-driven injury of the CNS and to defi ne and validate novel targets for the development of therapies for debilitating CNS infl ammatory diseases. NeuroproMiSe will achieve this goal through a disease-oriented approach by identifying the essential Summary genes and pathways leading to MS and by direct protection of axons/ neurons through targeting of critical signalling pathways. The NeuroproMiSe project represents a highly focused and integrated effort to investigate in-depth the genetic and mechanistic pathways involved in Within the NeuroproMiSe project, several European laboratories with pro- infl ammation-induced neurodegeneration, and exploit this knowledge to found expertise in genetics, basic neuroscience, neuropathology, neuroim- develop novel candidate drugs for effective neuroprotective therapy. munology, protein chemistry and experimental models of neuroinfl ammation Specifi c NeuroproMiSe objectives are: and neurodegeneration are collaborating with industrial partners with • to identify the major genes and critical pathways associated with a strong interest in developing therapeutics for infl ammatory and neuro- Multiple Sclerosis (MS) and infl ammatory neurodegeneration. This logical diseases. This Consortium will integrate their research potential to goal will be achieved through a comparative genetic approach, identify genes and pathways associated with disease susceptibility and using an established animal platform suitable for identifi cation of course into experimental models of MS and neurodegeneration, and will experimental autoimmune encephalomyelitis susceptibility genes, and use this information to defi ne synonymous genes in homogeneous and well-characterised, ethnically homogeneous cohorts of MS patients. well characterised cohorts of MS patients and population-based controls. Through genomic and proteomic screens carried out in animal disease By transcriptome and proteome analyses, new molecules and pathways models, further knowledge will be obtained on genes and molecular involved in infl ammatory and non-infl ammatory neurodegeneration will be pathways involved in infl ammatory neurodegeneration; identifi ed in selected disease models. • to elucidate essential immunopathological mechanisms of neurodegeneration, focusing on innate and adaptive immune responses The Consortium will validate the role of candidate genes in a range of and on the use of humanised animal models to defi ne new rational disease and in vitro models and, based on this knowledge, will develop ways of developing disease modifying drugs; new therapeutic compounds. New, genetically engineered mice will be • to develop novel neuroprotective drugs based on targets validated generated that represent invaluable tools for both basic research and in animal models. In a fi rst step, new therapeutics targeting critical disease modelling. By detailed analysis of human material and relevant pathways (Ncf-1, TREM-2, TNFR subtypes, ion channels, glutamate disease models, and by exploring the involvement of the innate and receptors, cell apoptosis) will be tested and used in combination adaptive arms of the immune system in CNS tissue destruction, the therapies. NeuroproMiSe project also aims to provide new insights into infl amma- The project is conducted in alliance with four biotechnology companies tory mechanisms responsible for neuronal and glial cell damage and to and has potential for direct transfer of basic research results into clinical defi ne biomarkers for evaluation of pathogenic processes and respon- studies and economic and society benefi ts. siveness to therapeutic treatments. Moreover, the project will use anti-infl ammatory and neuroprotective compounds, recently generated in the applicants’ laboratories, to develop novel and combined therapeutic strategies to prevent infl ammation-driven neurodegeneration. Although a large part of the work will be performed in MS patients and experimental models of MS, the knowledge and tools generated will also enhance understanding of pathogenetic mechanisms and allow development of new therapeutic approaches in other acute and chronic Problem neuroinfl ammatory disorders.

Despite intensive research on the causes and pathogenesis of MS, the To accelerate the achievement of the NeuroproMiSe objectives, horizon- genetic basis of this common cause of neurological disability is still tal activities will be developed through the establishment of neuropa- unknown and the complex immunopathological mechanisms underlying thology and genomics/proteomics facilities and training programmes. brain injury are only beginning to be understood. To date, no curative This will ensure the uniform availability and distribution of technological therapy is available and none of the existing drugs can stop the know-how in cutting-edge technologies of lesion analysis/imaging and neurodegenerative process responsible for disease progression. functional genomics.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 143 Expected results Potential applications

A unique platform of mouse and rat congenic strains will be used for The NeuroproMiSe Project as a whole focuses on translational research identifi cation of disease-susceptibility genes and associated molecular and uses basic and functional genomic and proteomic knowledge for pathways in experimental models of MS and neurodegeneration, and translation into therapeutic concepts and further into preclinical appli- for the study of gene-gene and gene-environment interactions. To char- cation. It is expected that, with the knowledge acquired about specifi c acterise the allelic diversity of known MS risk genes and of selected genes and pathways involved in infl ammatory neurodegeneration and genes identifi ed in the experimental genetic platforms, high-throughput neuroprotection, and with the compounds developed on the basis of single nucleotide polymorphism (SNP) genotyping and haplotype analy- this knowledge, within the fi ve years of the project candidate therapeu- sis will be carried out in selected cohorts of Finnish and Swedish patients tic molecules will be made available and proposed for subsequent with MS. High-throughput gene expression profi ling using cDNA micro- clinical evaluation. Knowledge and products generated will speed up arrays and proteomic analysis of CNS tissue will be performed in relevant effective treatment of neuroimmune diseases, contributing to improve- experimental models, in order to search specifi cally for molecules/path- ments in the quality of life and health and promoting the biotechnology ways involved in neurodegeneration and identify new targets for and pharmaceutical industries. neuroprotective strategies as well as novel biomarkers to monitor MS progression and response to therapy.

Validation of candidate genes and mechanistic pathways will be performed by gene and protein expression analysis and bioinformatics, and by using a wide array of already existing and to-be-developed genetically modiﬁ ed mice in relevant disease models and functional assays. Targets identiﬁ ed and validated in the above studies will provide seeds for the development of new therapeutic compounds.

Improved transgenic models will be generated to investigate the role of innate and adaptive immunity in mediating CNS tissue damage. Based on the information obtained, new disease-modifying strategies and compounds will be developed and tested in laboratory models of MS. A number of critical pathways of neurodegeneration and neuroprotection will be investigated further and different approaches will be undertaken to achieve neuroprotection, including: • development and testing of compounds targeting molecules that are involved in the regulation of myeloid cell function; • application of genetic engineering and nanotechnology to develop compounds targeting tumor necrosis factor receptor subtypes and intracellular signalling; • evaluation of the effi cacy of new sodium and calcium channel blockers and of glutamate receptor antagonists in axonal protection; • manipulation of intracellular pathways involved in neuronal apoptosis with neurotrophic factors, pharmacological approaches or new protein transfer tools; Project number • evaluation of the therapeutic effi cacy of combined neuroprotective LSHM-CT-2005-018637 and anti-infl ammatory compounds in disease models. The genera- EC contribution tion of new compounds, which will be tested for their anti-infl am- € 11 400 000 matory and neuroprotective effi cacy, and of bi-functional molecules Duration for targeting of neuroprotective activities specifi cally to neurons 60 months and glial cells, will permit the application of innovative approaches Starting date for the treatment of neuroinfl ammatory diseases. 1 November 2005 Instrument IP Project website www.neuropromise.eu

144 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Coordinator Klaus A. Nave Max-Planck Institute of Francesca Aloisi Experimental Medicine Istituto Superiore di Sanità Münich, Germany Rome, Italy [email protected] Harald Neumann University of Bonn Bonn, Germany Partners Peter Olofsson Mathias Bähr Biovitrum AB University of Göttingen Stockholm, Sweden Göttingen, Germany Tomas Olsson Wim Buurman Karolinska Institute HyCult biotechnology bv Stockholm, Sweden Uden, The Netherlands Leena Peltonen Wolfgang Brück National Public Health Institute University of Göttingen Helsinki, Finland Göttingen, Germany Toshal Patel Uli Eisel Eisai Groningen University London, United Kingdom Groningen, The Netherlands Victor H. Perry Lars Fugger University of Southampton Medical Research Council Southampton, United Kingdom London, United Kingdom Klaus Pfi zenmaier Rikard Holmdahl University of Stuttgart Lund University Stuttgart, Germany Lund, Sweden Lesley Probert Ulrich Kettling The Hellenic Pasteur Institute Direvo Biotech AG Athens, Greece Köln, Germany Kenneth Smith Hans Lassmann King’s College London Medical University of Vienna London, United Kingdom Vienna, Austria

Roland Liblau Institut National de la Santé et de la Recherche Médicale France

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 145 NovelTune

Novelty Tuning: behavioural, electrophysiological and molecular mechanisms of novelty detection

Keywords Novelty detection, behaviour, electrophysiology, Problem genetic engineering, auditory cortex, transcriptome The main scientifi c question that underlies this project is to understand the mechanisms of brain plasticity and how the brain discriminates and adapts to a stimulus that is novel compared to a non-novel stimulus. This question is at the forefront of neuroscience research today. Brain plasticity enables humans and other animals to adapt to the changing environment. Electrical activity is the way by which the environment infl uences genetic mechanisms, but Summary the relationships between electrical activity and the resulting molecular changes are unclear. The project integrates in a unique way the force of the This proposal addresses one of the most profound determinants of brain new post-genomic approaches in order to study the information processing function and cognition: how is the brain affected by a stimulus that is and output of the sensory nervous system with state-of-the-art classical novel and is therefore potentially signifi cant? electrophysiology, genetics and behaviour. The proposal, centred around the auditory system, will decipher the pathways that start with the detection of a novel stimulus and lead through a cascade of inter-related signals to the induction of the synaptic Aim plasticity. We hypothesise that a novel sensory stimulus is detected by the conjunction of the sensory activity it evokes in the cortex with The aim of this project is to study the interactions between the behavioural, inputs from the neurotransmission systems. This conjunction activates molecular and electrophysiological aspects of the processing of novel electrophysiological novelty responses and the associated expression stimuli. More precisely, our objective is to understand the implication of of immediate-early genes (IEGs). The IEGs can be used to identify electrophysiological responses, the induction of IEGs, and c-Fos in particular, network components within auditory and motor cortices that subserve the modulation by neurotransmitters and the resulting long-term synaptic sound frequency discrimination in the behavioural training cortex. They changes which, together, combine to adapt the sensory responses. Within orchestrate changes in the profi les of gene expression, alternative this framework, our proposal focuses on auditory cortex and relevant motor splicing and microRNAs leading to synaptic modifi cations that ultimately cortical areas by studying the pathways that lead from novelty detection to change the animal behaviour. cellular and molecular mechanisms underlying synaptic plasticity. Finally, it To test this model we will study novelty detection using three different will give a complete circle of predictions for the processing of novel vs. non- readouts: behavioural, electrophysiological and molecular. Genetic novel stimuli, including the electrophysiological, behavioural and molecular engineering manipulations and molecular techniques will be used effects that follow. to assess the relationships between these readouts. Brain disorders such as schizophrenia are associated with abnormal sensory gating of novel vs. non-novel stimuli. The expression of such pathologies requires Expected results interactions between environmental, electrophysiological and molecular mechanisms. The NovelTune teams aim to link these various processes • Establish the behavioural tuning curve for novelty detection. and elucidate the underlying mechanisms through an interdisciplinary • Identify the electrophysiological tuning curve for novelty detection, strategy. The network integrates strong expertise including state-of-the- as modifi ed by behavioural training. This tuning curve will be art and high-throughput gene expression technologies and the most compared with the behavioural and the molecular tuning curves advanced bioinformatics competence. The combination of these powerful for novelty detection. The most promising animal model will be methods is expected to yield profound insights into the cascade of events then used, in order to study the single-neuron correlates of changes underlying the processing of novelty. in the novelty-processing pathway in the auditory cortex. • Establish the novelty tuning curve of c-Fos activation, an IEG that is hypothesised to refl ect novelty detection in order to: – analyse, at the transcriptomic level, the neurons in the primary auditory cortex in which immediate early genes (IEGs) are activated in response to novel auditory stimulus; – understand neuronal plasticity and adaptative processes triggered by alteration of dopaminergic, serotonergic and cholinergic neurotransmissions; – investigate the role of dopaminergic, serotonergic and cholinergic neurotransmissions in the brain’s control of novelty response; – determine the contribution of the glucocorticoid receptor (GR) in pre- and post-synaptic neurons to these phenomena.

146 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES • Obtain animal models in which neurotransmitter systems, stress Coordinator Israel Nelken response and IEGs expression have been modiﬁ ed, and to assess the The Hebrew Univeritsity consequences on novelty detection and biology of the auditory cortex. Jacques Mallet Jerusalem, Israel Centre National de la Recherche Scientifi que Hermona Soreq Paris, France The Hebrew Univeritsity Potential applications [email protected] Jerusalem, Israel

The present proposal addresses essential and clinically important Jan Schnupp determinants of brain function: the processing of cortical information Partners Oxford University required to discriminate the novelty from background stimuli and to adapt Oxford, United Kingdom to environmental changes. Our approach will help in generating high- François Tronche resolution cortical maps and in defi ning auditory-motor cortex areas that Centre National Hans-Peter Lipp are jointly involved in the control of a specifi c behaviour. Thus, the present de la Recherche Scientifi que NewBehavior project represents multidisciplinary basic research, which aims at leading Paris, France Zurich, Switzerland to applications to human health. Leszek Kazmarek Nencki Institute The ultimate objective of the proposed research should provide novel Warsaw, Poland targets that could favour the development of new therapies for those clinical conditions in which specifi c defi cits in novelty detection and information gating play a role. Among these conditions are chronic neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, and psychiatric disorders, such as schizophrenia. All these conditions are prevalent in Europe and will become an even more serious health problem as the population ages.

Project number LSHM-CT-2006-037378 EC contribution € 2 400 000 Project Duration 36 months Starting date 01/11/2006 Instrument STREP

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 147 PHECOMP

Phenotypical characterisation of animal models for neuropsychiatric disorders related to compulsive behaviour

Keywords Compulsive disorders, animal models, behavioural Problem paradigms, Positron Emission Tomography (PET), drug abuse, eating disorders, glucocorticoid receptors Both drug addiction and food intake disorders constitute increasingly serious health care and social problems in the EU and are also responsible of the loss of millions of working hours every year. However, there is still a lack of suitable speciﬁ c animal models to further elucidate the neurobiological substrate of such disorders. In this context, PHECOMP offers an impact, in new animal models, for probing these and other related psychiatric disorders and compulsive behaviours.

Summary The present proposal is a multidisciplinary project bringing together new and sophisticated behavioural methodologies as well as cutting-edge Compulsive disorders, including drug abuse and compulsive overeating, molecular and imaging techniques which will be applied to the represent prevalent neuropsychiatric diseases that have a large health and phenotypical characterisation of targeted and refi ned animal models. socio-economic impact in the European population. These disorders are The characterisation of new animal models and their transfer to mice produced by an alteration of the capability to control seeking for reward, when needed will allow the use of these rodent behavioural models for and seem linked by common neurobiological substrates. However, there the study of the compulsive components in addiction and related is an important gap in the availability of reliable behavioural models in psychiatric disorders. animals that permit investigation of compulsion towards reward in the perspective of human pathologies. The present proposal will use new sophisticated behavioural and Aim neuroimaging techniques for the characterisation of four new and complementary animal models of compulsive disorders, allowing us to There are three main aims of the specifi c-targeted research project: analyse precisely the main components of those behavioural alterations. • deliver four phenotypically well-characterised animal models, namely The study will be performed in mice and rats, including the transfer of the modifi ed confl ict, the deprivation and the reinstatement models rat models to mice when necessary. The behavioural and molecular of compulsive drug intake, and the compulsive food-seeking/-taking characterisation of the models, along with parallel neuroimaging (PET), will model, addressing different components of compulsion and using provide a complete anatomical and functional illustration of the reward rats and mice; pathways imbalance in the above-mentioned pathological situations. Novel • elucidate the role of selected gene activities and protein receptors in behavioural paradigms will be proposed, tested and validated within the the neurobiological mechanisms involved in compulsive disorders; project, taking advantage of cutting-edge imaging technologies. Molecular • provide complete new structural and functional illustration of the studies will characterise changes induced in several key elements of the reward pathways imbalance found in compulsion, using cutting- reward circuits during these behavioural disorders. edge imaging techniques (PET). After the full characterisation of the models, they will be used on genetically modifi ed mice for glucocorticoid receptors to ascertain correlations between The ultimate objective of this STREP Project is to provide new knowledge behavioural and genetic components of compulsion in drug addiction and that can be used by the participants, by other research groups or by eating disorders. Hence, reliable and predictive animal models will be fully European pharmaceutical companies, in order to develop new drugs characterised and employed, in order to understand better the mechanisms for the treatment of both drug craving and relapse, core features of the involved in these alterations, and to design new therapeutic strategies in compulsive components of addictive disorders, or for providing new neuropsychiatric disorders related to compulsive behaviour. drugs for treating compulsive eating leading to obesity and metabolic imbalance.

Expected results

The neurobiological bases of compulsive behaviours share common mechanisms, as drug-seeking or compulsive food intake promote activation and neuro-adaptations within the common neuronal networks. The general strategy of the project is, ﬁ rst, to carry out

148 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Cocaine sensitization and reward are inﬂ uenced by circadian genes and rhythm (Abarca et al., Proc Natl Acad Sci USA 99, 9026-30, 2002).

a complete phenotypical characterisation of four animal models, Coordinator Pier Vincenzo Piazza addressing different components of compulsive disorders that are Institut National de la Santé already being studied in partner laboratories, namely: Prof. Rafael Maldonado et de la Recherche Médicale u588 • the modiﬁ ed conﬂ ict model (Dr. Piazza’s group at Bordeaux); Universitat Pompeu Fabra Paris, France Barcelona, Spain • the deprivation model (Dr. Spanagel’s group at Mannheim); [email protected] Andrea Heyne • the reinstatement model (Dr. Maldonado’s group at Barcelona); Medimod Pharmacology • the compulsive food-seeking/-taking model (Dr. Heyne’s group Services GmbH at Reutlingen). Partners Reutlingen, Germany

These models will be transferred from rat to mouse when needed Mara Dierssen Olga Millán (involving the former groups as well as that of Dr. Dierssen at Barcelona). Centre de Regulació Genómica Institut d’Alta Tecnologia The proposal will investigate selected genes and proteins (Dr. Przewlocka’s Barcelona, Spain Barcelona, Spain group at Krakow) which could play a role in compulsive-derived brain plasticity and its pathophysiological response. Moreover, the availability Barbara Przewlocka François Tronche Institute of Pharmacology Centre National de la of specifi c feeding pattern evaluation tools (Dr. Célérier’s group at Polish Academy of Sciences Recherche Scientifi que Cornellà) and the set-up of potent neuroimaging technologies adapted Krakow, Poland Paris, France to small experimental animals (Dr. Millán’s group at Barcelona) will allow correlating anatomic-functional events with behavioural and genetic Rainer Spanagel Evelyne Célérier paradigms. Finally, a mechanistically targeted-oriented array of genet- Central Institute of Mental Health Panlab ically modifi ed mice (Dr. Tronche’s group in Paris) will be used to Mannheim, Germany Cornellà, Spain specifi cally address the elucidation at molecular level of the neurobiological basis of compulsive disorders, i.e. the role of glucocorticoid receptors.

Potential applications

The optimisation of targeted animal models will render them suitable for ﬁ nding new treatment approaches for these compulsive disorders and ready-to-use in pre-clinical research to test putative anti-obesity and anti-addictive compounds. It is recognised that both opioid, dopamine and cannabinoid systems play key roles in regulating not only addiction but also other aspects of limbic and motor function. The potential for the use of these animals to investigate other CNS diseases as well as to develop and test new pharmacotherapies is accordingly very high.

Moreover, PHECOMP offers three SMEs, leaders in their fi elds, the opportunity of participating in a project at the European level, thus enabling the possible outputs of the project to fi nally benefi t the same (and other) SMEs, thus increasing their know-how and commercial competitiveness in and outside Europe. In particular, at the end of the project, Medimod (DE) will have a fully-operative food-seeking/food- Project number taking model in rat ready for the preclinical search for new pharmaco- LSHM-CT-2007-037669 therapeutic solutions in food intake disorders. Also, IAT (ES) will develop EC contribution and validate new PET built-in methodologies for neuroimaging analysis € 2 170 000 of mice, correlated with behavioural and molecular data. Finally, Panlab Project Duration (ES) will set-up and validate specifi c devices that will improve the quality 36 months and effi ciency of animal feeding patterns evaluation tools. Starting date 01/11/2006 Instrument STREP – SME

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 149 PHOTOLYSIS

Development of fl ash photolysis for deep uncaging in vivo and high-throughput characterisation of neurotransmitter gated ion channels in drug discovery

Keywords Photolysis, caged neurotransmitters, synaptic Problem transmission, synaptic plasticity, neurovascular regulation, wave front engineering, optical imaging, high throughput Experimentally it is diffi cult to separate the contributions of presynaptic screening, channelopathies and postsynaptic mechanisms to the effi ciency and strength of a synaptic connection. The method of fl ash photolysis can provide a way of determining the properties of synapses between neurones in situ that are currently studied only in recombinant receptors in vitro, and would allow the testing of putative mediators of local vasoregulation in the brain. These applications of photolysis in neuroscience are impeded by the poor photochemical properties and stability of caged neuro- Summary transmitters, by the unavailability of many mediators in photolabile caged form, and by the unsuitability of present optical methods. Flash photolysis is widely applied in cell physiology to initiate neurotransmitter and other ligand-receptor interactions in conditions Currently, high throughput screening methods to fi nd ligands acting at that are subject to poor diffusional access and receptor desensitisation, brain synapses use conventional perfusion methods to apply the and for ligands that are particularly labile. It has the potential to initiate activating neurotransmitter or analogue. These methods are too slow to reactions on physiological time and spatial scales (sub-msec and sub- activate receptors on a physiological timescale because of diffusional micron) in complex tissues such as brain slices and in vivo, and is often access to the cell surface and, for this reason, potential medicines that combined with electrophysiology and optical imaging. affect the degree of activation of synapses and act in a use-dependent However, this potential is unrealised in neuroscience and medicine in way will not be optimally detected. The application of this method in several areas: high-throughput screening to test for new medicines has the potential • photolabile ‘cages’ optimised to make use of the localisation achievable to detect use-dependent ligands acting at the rapidly desensitising with excitation by the two-photon effect; amino acid receptors that mediate neuronal signalling. • wavefront modulation of photolysis light to make z axis location and spot size readily changeable; • application in high-throughput screening for drug discovery of ligands Aim acting at rapidly desensitising neurotransmitter receptors in the brain. The PHOTOLYSIS consortium comprises neurophysiologists, photochemists, • To develop new photochemical probes and optical methods in optical physicists, specialists in high-throughput patch clamp screening localising and extending the range of photolysis for use in and ion channel targeted drug discovery, to address these areas. New neuroscience. photochemistry of cages combined with new pulsed lasers and new • To adapt photolysis to current parallel patch clamp methods in the adaptive optics will optimise the effi ciency, depth and location of photolysis development of new medicines. in whole brain in vivo and in vitro. These developments will be combined with deep imaging to: • identify mediators and cell types in neurovascular coupling of blood Expected results perfusion to neuronal activity; • when applied to synaptic transmission to study postsynaptic channels • Improved photochemical properties of probes, particularly making in situ, identify their role in synaptic plasticities; better use of the two-photon effect for localisation and extending • and investigate the interactions between metabotropic receptors and the range of synaptic and local messenger systems to which the fast transmitter channels. method can be applied. • fi nally, adapt near-UV fl ash photolysis to patch clamp HTS technology, • Improved methods in drug discovery of ligands active at brain in order to characterise drugs acting at fast activating and desensitising synapses. neurotransmitter receptors, and to study the functional pharmacology • Specifi c therapeutic targeting of genetically linked channelopathies of genetically-linked channelopathies and in developing somatic cell and applications in developing somatic cell replacement therapies. replacement therapies. Potential applications

• To investigate neurotransmitter receptors in synaptic transmission in situ. • To investigate mediators in neurovascular regulation. • To improve methods of deep imaging in vivo. • To develop better drugs acting at synaptic receptors in the brain.

150 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Coordinator Michael Fejt Flyion GMBH David Ogden Tübingen, Germany CNRS UMR 8118 Physiologie Cerebrale Duncan Crawford Universite Rene Descartes Paris 5 Tocris-Cookson 45 Rue des Saints Peres Bristol, United Kingdom F-75006 PARIS France Ladislav Vyklycky [email protected] Dept of Cellular Neurophysiology Institute of Physiology Prague, Czech Republic Partners Yair Feld Serge Charpak GeneGraft Institut National de la Santé Haifa, Israel et de la Recherche Médical Paris, France Isabelle Geahel Inserm-Transfert Gail McConnell Paris, France University of Strathclyde Glasgow, United Kingdom

Justin Molloy Medical Research Council/NIMR London, United Kingdom

Project number LSHM-CT-2007-037765 EC contribution € 2 700 000 Duration 36 months Starting date 01/01/2007 Instrument STREP

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 151 PNS-EURONET2

Paraneoplastic Neurological Syndromes (PNS): strengthening the European Network

Keywords paraneoplastic neurological disorders, database, Problem sample bank, neuro-oncology Paraneoplastic neurological disorders (PNS) represent a number of characteristic syndromes that are associated with systemic cancer, without invasion of tumour cells into, or opportunistic infections of, the nervous system. However, making a certain diagnosis of PNS is diffi cult, as at the time of presentation a tumour cannot be found in more than two-thirds of patients. A search for the underlying cancer can be Summary frustrating as the tumour mass in most patients is small and hard to detect. Moreover, neuro-imaging or other ancillary techniques for Paraneoplastic Neurological Syndromes (PNS) are severe disorders of the establishing the diagnosis are often normal or non-specifi c. nervous system that arise as remote effects of neoplasia. Diagnosis of PNS is currently aided by detection, in patients’ serum and cerebrospinal fl uid, This combination of factors has made it a diffi cult task to establish of specifi c autoantibodies that recognise antigens shared by the tumour a reliable diagnosis of PNS. This is all the more signifi cant considering and nervous system cells. In most cases, correct diagnosis of PNS permits that, in the majority of cases, these syndromes run a subacute course early detection of the tumour, before it can metastasise. The rarity of PNS and may leave the patient incapacitated within weeks, without much prompt the need for multicentric studies to collect suffi cient numbers of hope of neurological improvement later on. Treatment attempts are patients on which to base specifi c studies. often initiated once irreversible neurological loss has occured and this With the support of an EC grant (QLG1-CT-2002-01756), a Network of inevitably results in failure of therapy. reference centres located in over ten European nations was established for these pathologies (see www.pnseuronet.org). During the fi rst two years of the This untoward sequence of events has changed since the detection of scheduled three years’ activity, a database was developed and validated for anti-neuronal antibodies associated with PNS. These antibodies can be the standardised collection of information on PNS patients dating from 2000, identifi ed in serum and provide the opportunity for an early diagnosis of and a central bank was set up to store biological material. This Network PNS. Since the detection of these auto-antibodies, the fi eld of has defi ned and published guidelines for the fi rst time on PNS diagnosis, paraneoplastic neurological syndromes has evolved rapidly and a number which will facilitate clinical diagnosis of the diseases and the collection of of specifi c auto-antibodies has been discovered (anti-Hu, -Yo, -Ri, -VGCC, uniform groups of patients for specifi c studies. Over 500 cases have now -Ma1 – Ma2 -CV2/CRMP5, -ANNA3), together with identifi cation of the been collected in the database which, considering the rarity of the diseases, genes that express the corresponding neuronal and tumour proteins. is a very signifi cant number, unmatched in the international literature. These developments have resulted in the production of recombinant The aim of the project is to: proteins that can be applied for auto-antibody recognition in the serum of • continue, for three more years, to input patients in the database, patients with paraneoplastic neurological syndromes. making the necessary quality controls on incoming data, and to maintain centralised storage of biological material; • diffuse information yielded by the database, and identify and design Aim specifi c studies on uniform groups of these diseases; • clinically characterise new PNS pathologies emerging from analysis of • To consolidate use of the European database developed by the database information. participants in a previous European Commission project, in order We stress that no other international group is able to gather such an that suffi cient, comparable data can be collected in order to: extensive case series. By continuing this cooperation, the EU will become – control the quality of the data inserted in the database and world leader in PNS research. data insertion procedure; – make useful correlations between paraneoplastic neurological syndromes and tumour types and specifi c antineuronal antibodies; – identify previously unrecognised paraneoplastic neurological syndromes; – identify relevant clinical issues that could be answered in prospective studies. • To consolidate use of the sample bank (serum, cerebrospinal fl uid, lymphocytes) of patients with PNS in order to: – test the relevance of new antibodies initially detected in one or a few patients with paraneoplastic neurological syndromes;

152 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES – identify and characterise new antineuronal autoantibodies Coordinator Division of Neuropathology that may be relevant in the pathogenesis or diagnosis of Institute of Neurology paraneoplastic neurological syndromes; Dr. Bruno Giometto UCL – collect high-quality samples in the sample bank for U.O. Neurologia London, United Kingdom Azienda ULSS 9 Treviso future studies. Treviso, Italy Ludwig Boltzmann Insitute • To consolidate and update the research inventory and exploit the [email protected] for Neuro-Oncology database for scientifi c research in order to: Wien, Austria – set up multicentric prospective clinical trials; – obtain epidemiological data; Partners Dept of Clinical Medicine – update the guidelines; Section for Neurology – identify possible other projects. Institute of Neurology Neurology Research Lab. Università Cattolica del Sacro Cuore Bergen, Norway Expected results Roma, Italy Institute of Clinical Neurophysiology Università di Torino Clinical Centre The presence of a consolidated group of experts on these diseases, an Torino, Italy Ljubljana, Slovenia established database, a serum bank, and publication of the very fi rst set of guidelines drawn up by the same panel of specialists will have a major Neuropathology Laboratory University Dept impact on scientifi c knowledge on PNS, not only at European but also at INSERM U. 433 of Neurological Science world level. Hôpital Neurologique Walton Centre for Neurology Lyon, France and Neurosurgery Liverpool, United Kingdom Potential applications Dept Biologie des Interactions Neurone-glie, Inserm U 495 Azienda Ospedaliera ‘M. Mellini’ The products of the project will permit the diffusion of clinical data that Hôpital de la Salpêtrière Brescia, Italy Paris, France have been collected in a standardised, centralised, controlled manner. Istituto Mario Negri These will enable specifi c studies to be designed on related topics, given University Hospital Villa Camozzi the suffi ciently large numbers of patients. of the University of Cologne Lab. Clinical Epidemiology All the collected information can be made available to the scientifi c Koeln, Germany Bergamo, Italy community through the website. European neurologists will achieve leadership in this fi eld of diseases. Istituto d’Investigacions Biomediques Dipartimento di Neuroscienze August Pi I Sunyer Azienda Ospedaliera Universitaria Barcelona, Spain Policlinico di Messina Messina, Italy Facultni nemocnice Brno Brno, Czech Republic Justus-Liebig-University Giessen Giessen, Germany Fondazione Centro San Raffaele del Monte Tabor Universitatsklinikum Freiburg fur Milano, Italy die Medizinische Fakultat der Albert-Ludwigs-Universitat Erasmus University Medical Centre Freiburg, Germany Project number Rotterdam, The Netherlands LSSM-CT-2005-518174 Weatherall Institute of EC contribution Leiden University Medical Centre Molecular Medicine € 475 000 Leiden, The Netherlands University of Oxford Project Duration Oxford, United Kingdom Université Jean Monnet 36 months St. Étienne, France Starting date 01/01/2006 Instrument SSA Project website www.pnseuronet.org

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 153 RATstream™

European project on the characterisation of transgenic rat models for neurodegenerative and psychiatric diseases: Automated home cage analyses, live imaging and treatment

Keywords Brain research, neurodegeneration, Problem/Aim transgenic animal models, comprehensive phenotyping, automated behavioral phenotyping, in vivo imaging, RATstream™ is an ambitious European project that aims to characterise pre-clinical treatment studies and use three transgenic rat models of neurological diseases which – in humans – present with a wide range of neurological and psychiatric phenotypes: • transgenic rat model of HD (von Hörsten et al. 2003, Hum Mol Genet 12:617-624); • transgenic rat model of PD overexpressing alpha-Synuclein with the A30P mutation; Summary • transgenic rat model of SCA17 with 64 expanded CAG repeats in the TATA binding protein. Such transgenic rat models are unique The RATstream Consortium will concentrate on the comprehensive phe- worldwide. notypical characterisation of rat models of neurodegenerative diseases such as Huntington’s disease (HD), Parkinson’s disease (PD) and spinoc- The project aims to pursue a completely novel gene-to-function erebellar ataxia type 17 (SCA17). Ultimately, the project will deliver approach resulting in a comprehensive phenotyping process which will a procedure for low-cost automated drug screening along with a set of comprise four components: data describing the phenotype for each of the models. • classical phenotyping; To achieve this goal, automated home cage systems for behavioural • monitoring of behavioural and physiological performance in fully and physiological phenotyping will be developed by two SMEs and vali- automated physiological and behavioural home cage test systems; dated independently by two academic partners, and individual data will • non-invasive imaging technologies which have been adapted to be incorporated into an integrated database developed by a third SME. small animals; In a joint effort, the groups will develop a comprehensive set of behav- • neuropathology; ioural and physiological phenotyping procedures, including PET and DTI • microarray analysis. technologies, in order to detect systematically neuropsychiatric corre- The project will apply this approach to all rat models in order to achieve lates of neuronal dysfunction and disease progression in rat models of comprehensive high-quality characterisation of models. HD, PD, and SCA17. The resulting set of biomarkers will lead to a valid set of minimised experiments and markers best suited to provide read-out Two members of the consortium (TSE, NewBehavior) aim to develop parameters in pre-clinical studies applying novel substances delaying or automated home cage test systems for rat models which do not exist preventing neurodegeneration. yet, but which are imperative in view of the upcoming large number of transgenic rat models in both industry and academia. Collaboration with the academic partners FAU and Uni Tübingen will provide an optimal environment for development and reﬁ nement of home cages which are validated via correlation with data from classical read-outs and by cross comparison between two experienced academic partners. Cage systems will be suitable for continuous monitoring of spontaneous, social, cognitive, emotional and physiological measures (drinking, feeding, metabolic performance/calorimetry, telemetry for temperature and bio-potentials) in home-cage-like environments for rats.

This phenotyping set up (1-5) will be used to develop a minimised and essential set of biomarkers, in order to monitor disease progression reliably in the transgenic rat models of PD, HD, and SCA17. Phenotype data are also correlated to neuropathological features such as protein aggregates, neuronal cell loss, and neurotransmitter alteration at different disease stages. The phenotyping approach will be used to characterise for each disease model a minimised set of markers best suited as read-outs in pre-clinical studies, applying novel compounds delaying or preventing neurodegeneration.

The objective of this project will be to provide the proof-of-principle that it is possible in the rat:

154 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES Workﬂ ow of RATstream™ integrating all components of development of automated home cage systems, transgenic rat model characterisation, preclinical treatment trials, and data mining. The contribution of the partner in the RATstream™ consortium is indicated. The HD model has already been studied extensively by classical phenotyping and by PET analysis. Legend: BS_ Basic screen (to exclude integration effects of the transgene, done before ofﬁ cial initiation of RATstream™, P1-4_products of RATstream™, CP_classical phenotyping, AP_Automated home cage phenotyping, DrS Disease-related screen.

• to develop, validate, and use standardised automated home cage • Standardised progression features of tg rats. systems, and to adapt in vivo imaging techniques for phenotyping • Minimised parameter sets for pre-clinical trials in transgenic rat neurological and cognitive function (1st year); models. • to harvest large data sets on gene functions and corresponding • Power calculation for pre-clinical trials. phenotypes thereof (2nd year); • Pharmocokinetic and toxicity data for compounds to be applied in • to determine and validate a minimised set of predictive parameters pre-clinical trials. (2nd year) and experiments as well as of appropriate time slots for • Study reports on preclinical trials. each rat model (low-cost approach); and • Data base for phenotyping and treatment studies of transgenic rats. • to develop and apply specifi c quality standards applicable for phenotyping tools and models (over the entire project duration). Potential applications Furthermore, these rat models will be used: • to scrutinise novel experimental, pre-clinical treatments (year 1-3), The commercial impact of the project can be estimated explicitly for the chiefl y with regard to effectiveness, side-effects, applicability, and two SMEs engaged in development of automated home cage test transferability. systems. NewBehavior forecasts a growing share (10-25 %) of the market for rat behavioural equipment, estimated to be about € 5 million per annum in Europe and € 15 million worldwide. TSE forecasts, for Expected results 2008, a share of the European market valued at € 1.5 million and, for the rest of the world, € 0.15 million. In 2009, this is expected to increase • Validated novel automated behavioural and physiological test to € 3.75 million in Europe and € 1.8 million in the rest of the world systems. respectively. While the market itself is not large in comparison with • Behavioural markers derived from correlation analysis of data from other fi elds of biotechnology, one can expect sustainability over a long automated and classical screening for transgenic rats. period, since automated home-cage testing systems will gradually • Data on MRI (DTI), PET and Microarray expression profi ling as replace existing rat testing equipment in every university and larger regards disease progression in transgenic rats. pharmaceutical company. • Full scale phenotyping data for tg rats.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 155 Behavioral analysis of rat models by means of Rotarod.

Big pharmaceutical companies such as Boehringer Ingelheim and Coordinator Dr. Silvia Brenda Novartis have already expressed interest in the generated transgenic rat TSE Systems GmbH models of PD and HD. With widespread distribution of these models Prof. Olaf Riess Bad Homburg, Germany and their proven validity for therapeutical studies, this interest and thus Eberhard-Karls-Universität Tübingen Tübingen, Germany Prof. Bertrand Tavitian interest in the behavioural test systems will increase. For instance, based [email protected] Commissariat à on the publication of the rat HD model (von Hörsten et al. 2003, Hum l’Énergie Atomique Mol Genet 12:617-624), more than 40 academic and pharmaceutical Paris, France partners were interested in investigating treatment tools (stem cells, Partners drugs, viruses) with the help of this model. With the publication of the Dr. Rebecca Pruss PD and SCA17 models, an even greater interest is foreseeable (for PD Prof. Stephan von Hoersten Trophos SA because of the limitations of the mouse models and because of the Friedrich-Alexander-Universität Marseille, France impact in drug development for pharmaceutical companies). Erlangen, Germany Dr. Ronald Naninga For Trophos, the commercial gain consists of a potential reduction in the Prof. Hans-Peter Lipp CrossLinks BV NewBehavior AG Rotterdam, The Netherlands risk to further clinical development of its drug candidate for the Zürich, Switzerland indication targeted in this STREP, in the event that the models are Prof. Annemie van der Linden validated and found predictive of the human disease. This can lead to University of Antwerp signiﬁ cant advantage to other commercial competitors, once outcomes Antwerp, Belgium can be approved in clinical trials in humans, and can point to signiﬁ cant potential earnings.

CrossLinks’ product, namely an integrated database and software tool for complex phenotyping of transgenic animals, will bridge a gap in the market. To our knowledge, such a tool does not exist.

Project number LSHM-CT-2006-037846 EC contribution € 3 400 000 Project Duration 36 months Starting date 01/02/2007 Instrument STREP – SME Project website www.ratstream.eu

156 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES SGENE

A large-scale genome-wide association study of schizophrenia addressing variation in expressivity and contribution from environmental factors

Keywords Schizophrenia, association analysis, eye tracking, Problem cannabis, eye movements, neuropsychological tests More susceptibility genes for schizophrenia have to be identiﬁ ed. Phenotypic markers as well as environmental factors have to be studied in order to understand how the phenotype and the environmental factors correlate with the genetic variants identiﬁ ed.

Summary Aim

Schizophrenia is a complex and disabling disorder that continues to exact We aim at fi nding schizophrenia genes by typing a large sample of a heavy toll, not only upon those affl icted but also on relatives and on schizophrenia patients for 317 000 SNPs. The isolation of schizophrenia our health and welfare systems. Family history signifi cantly increases the susceptibility variants allows for the study of whether they contribute to risk of developing the disease and evidence from epidemiological and twin other psychiatric disorders or risk of certain endophenotypes. studies continues to support the notion that the genetic risk is the most substantial contributor to its aetiology. SGENE consists of several leading groups in the genetics of schizophre- The SGENE project aims to identify genetic variants associating with nia, groups that previously have successfully identifi ed susceptibility schizophrenia, study their impact on phenotype and their interactions with genes, have a track record in studying endophenotypes and have environmental factors contributing to the pathogenesis of the disease. The the expertise necessary to examine the interaction between prevalent availability of ultra-high throughput genotyping platforms at manageable environmental factors, including cannabis use, and schizophrenia. The cost now allows for a direct genome-wide search for susceptibility variants. Consortium aims to: We propose a two-stage design for cost effi ciency where, in Phase I, we will • identify susceptibility genes and variants for schizophrenia; genotype 1 600 schizophrenia patients and 1 600 controls using TagSNP • identify new genetic mechanisms that are involved in schizophrenia; CHIPs with 317,000 markers, and search genome-wide for association • identify new molecular targets for the treatment of schizophrenia; to schizophrenia. In this phase we will aim to identify a large number of • generate and study potentially informative phenotypic markers in markers with relative risk greater than 1.3 that have frequencies ranging the context of genetics and neurobiology of schizophrenia; from 10-50 %. • study the interaction between cannabis use and genetic variants The most signifi cant markers from Phase I will be carried over to Phase II infl uencing the risk of schizophrenia and related psychotic disorders. and typed on a larger, independent sample. We expect most associations in Phase I and Phase II to be detected indirectly, by linkage disequilibrium (LD), rather than directly as causal variants. Therefore, in Phase III we will Expected results search for causal variants in LD with the markers reaching genome-wide signifi cance after Phase I and II. The isolation of schizophrenia susceptibility While the evidence for a genetic contribution to schizophrenia is strong, variants allows the study of their contribution to other psychiatric disorders the genetics is complex, with environmental factors contributing as or risk of certain endophenotypes. Gene-environment interaction is at the well. Molecular genetic studies have identifi ed several regions of heart of pathogenesis of schizophrenia, and this calls for the inclusion of potential linkage: chromosomal abnormalities confer risk in some environmental factors in the study. Genes identifi ed in this project may pedigrees, and evidence is accumulating in favour of a few positional uncover dysfunction of biochemical pathways in schizophrenia and provide candidate genes, including the neuregulin 1 gene identifi ed by the new targets for the development of novel therapies. SGENE Consortium partners. Identifi cation of susceptibility genes opens up new avenues for research aimed at understanding the pathogenesis of schizophrenia because schizophrenia is not associated with clear anatomical lesions or known specifi c causative agents.

The isolation of susceptibility genes may even re-order the nosological system for classiﬁ cation of psychiatric disorders; this is already evident from evidence suggesting that genes for schizophrenia are also associated with bipolar disorder. Thus the isolation of schizophrenia susceptibility variants allows for the study of whether they confer risk of other psychiatric disorders (such as bipolar illness) or risk of certain endophenotypes (such as abnormal eye tracking or impaired cognitive performance).

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 157 Coordinator Leena Peltonen National Public Health Institute Hreinn Stefansson, PhD Helsinki, Finland deCODE genetics Reykjavik, Iceland Hannes Petursson [email protected] Landspitali University Hospital Reykjavík, Iceland A B C Partners Dan Rujescu Analysis with Fluorescence in Situ Hybridization (FISH): Ludwig-Maximilians University two BAC clones 2.0 Mb apart, one labeled red and the other labeled green, David St Clair Munich, Germany were used as probes for FISH analysis. A) A cell from a normal control in University of Aberdeen interphase. Both chromosomes are labeled with one red probe and one green Aberdeen, United Kingdom Hans-Jürgen Möller probe. B) Deletion associating with schizophrenia, one of the chromosomes has only the red probe, the green is missing (1 Mb deletion) C) The same region Genetic Research Centre duplicated, two green labels on one of the chromosomes. David Collier Munich, Germany King’s College London London, United Kingdom

Gene-environment interaction is at the heart of schizophrenia etiology, and this calls for the inclusion of relevant and growing environmental factors into the study, i.e. cannabis use. This interplay is best studied by analysing genotype-environment interactions once new genetic associations with the schizophrenia phenotype have been established. The main expected results are 1-10 susceptibility genes for schizophrenia and their effect on expressivity and their interaction with environmental factors.

Potential applications

The public health care cost of schizophrenia in Europe runs into thousands of millions of euro per year. Advances in our understanding of the genetics of schizophrenia may have dramatic implications for diagnosis and prevention as well as for treatment. It is not far- fetched to envisage the day when a patient suffering from psychosis undergoes a battery of tests, including genetic tests, to deﬁ ne a disease and to choose the most effective course of treatment. It might also be possible to identify and educate individuals who are particularly vulnerable to an environmental factor, such as a speciﬁ c drug, in scenarios where even brief exposure to a drug poses a substantial risk of developing schizophrenia.

As genomic efforts succeed in identifying genes and proteins involved in normal behaviour and in psychiatric diseases such as schizophrenia, several tangible benefi ts will result. The most obvious is the identifi cation Project number of novel targets for drug discovery, and the challenge is to fi nd these LSHM-CT-2007-037761 treatment targets. Genomic approaches can identify genes conferring EC contribution risk of complex disorders. Interestingly, most of the newly identifi ed € 2 499 958 schizophrenia susceptibility genes point to the role of glutamatergic Project Duration pathways, whereas current drugs mainly have an impact on dopaminergic 36 months and serotoninergic systems. This highlights the need for the identifi cation Starting date of multiple genes, as this solidifi es the identifi cation of the key biochemical 01/11/2006 pathways, providing the pharmaceutical industry with several potential Instrument drug targets in addition to the risk-conferring genes identifi ed. STREP Project website www.sgene.eu

158 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES TAMAHUD

Identifi cation of early disease markers, novel pharmacologically tractable targets and small molecule phenotypic modulators in Huntington’s Disease

Keywords Huntington’s Disease, HT-RNAi, functional genomics, Problem validated target, chemical hit, metabonomics, biomarker, text-mining, data-mining, data visualisation No curative therapy is currently available for patients with HD, and the pathophysiology of HD is still not well understood. Generally, the current treatment of HD combines non-pharmacological therapy with management of the symptoms of the disease. Pharmacological treatment of the disease must be tailored to the speciﬁ c needs of the patient. Physicians may prescribe a number of medications to help control emotional and movement problems associated with HD, although no drug is yet available Summary to stop or reverse the progression of the disease.

Huntington’s Disease (HD) is a devastating neurodegenerative disease The signiﬁ cant unmet medical need for HD includes the following: with many unmet patient needs. There are no known ways of slowing or • better understanding of pathophysiology to yield more relevant preventing the neurodegeneration associated with the disease, and clinical targets; trials in man are hampered by the slow disease progression and the • improved symptomatic treatments; absence of suitable biomarkers of short-term progression. The genetics of • drugs that slow, halt or reverse the disease progression; HD is characterised, and involves the expansion of a polyglutamine tract at • diagnostics of disease onset/progression. the amino-terminus of the Huntington gene (HTT). However, the translation of this knowledge into therapeutic and diagnostic approaches is hampered by the scarce knowledge of HTT biology, the paucity of information on Aim how cellular signalling pathways interact with the HD mutation, and the lack of systematic and modern approaches aimed at identifying useful This project addresses the challenge of identifying novel treatable biopredictors of disease progression in individuals diagnosed with HD. targets causally associated with the pathology, in order to support the The proposal addresses key areas of HD patient need, namely the discovery and development of disease-modifying therapeutics for the cure of HD, and development of therapeutically meaningful novel targets and biomarkers. To of discovering novel early biomarkers leading to the development of achieve these aims, a Consortium of specialist partners has been assembled, new diagnostic tools. representing complementary and specifi c know-how and expertise which will be integrated and further developed in the course of the project. High throughput-RNAi, focusing on genes encoding pharmacologically tractable proteins rather than on a whole-genome approach, will be employed on a novel and robust HD cellular disease model to identify genes whose inhibition of expression is protective against the HD mutation. Following a stringent target validation approach, selected validated targets will be progressed to assay development and primary screening activities, to identify druggable compounds active on the target and effi cacious against the HD mutation in cellular disease models. The complete process will be accompanied by an extensive data and text mining workfl ow providing background information for the HD knowledge network. This network will guide the experts in the generation of hypotheses about possible relationships (proteins, genes, regulation, and pathology). Therefore, TAMAHUD aims at delivering novel targets causally associated with aspects of the HD pathology in model systems, where biological target validation is accompanied by demonstration of target relevance through small-molecule pharmacological modulation. In parallel, the biomaterial repository made available to the Consortium from a disease-specialist academic partner will be investigated through state-of-the-art metabolomics approaches in order to identify biomarkers predictive of disease onset.

BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES | 159 Expected results Coordinator Partners

The expected results can be summarised as: Dr. Andrea Caricasole Prof. David Rubinsztein • the identiﬁ cation of novel, treatable targets causally associated Siena Biotech SpA University of Cambridge [email protected] Cambridge, United Kingdom with the pathology, along with small molecules capable of Siena, Italy modifying aspects of the pathology in cellular disease models via Dr. David Grainger modulation of target activity, and TCP Innovations Ltd • biomarkers of disease onset/progression. Cambridge, United Kingdom

Dr. Christian Blaschke Potential applications Alma Bioinformatics S.L. Madrid, Spain If met with success, the scope of the project goes beyond the 3.5 year granting period and the research outcomes are relevant to: Dr. Reinhard Schneider EMBL • further development of therapeutic compounds through preclinical Heidelberg, Germany and clinical studies; • reduction of costs in clinical development: the lack of reliable and predictive biomarkers of disease onset requires clinical trials of higher complexity in order to reach statistical signiﬁ cance; • creation and development of a network of competencies aimed at progressing drug discovery in such a specialist ﬁ eld as the neuroscience area.

TAMAHUD aims to terminate its activities by the constitution of a solid base for the later completion of pre-clinical development of potential drugs, comprising the demonstration of in vivo effi cacy of TAMAHUD- derived chemical series, also through the use of TAMAHUD-derived biomarkers. At even later times, clinical studies on candidate drugs originating from TAMAHUD activities will again benefi t from the inclusion of TAMAHUD biomarkers to determine clinical effi cacy.

Project number LSHM-CT-2007-037472 EC contribution € 3 000 000 Project Duration 42 months Starting date 01/02/2007 Instrument STREP

160 | BRAIN, NEUROLOGICAL AND PSYCHIATRIC DISEASES 161 ➞ 181

HUMAN DEVELOPMENT AND AGEING ARIG

Conference on European research on ageing: “Ageing research in immunology: The impact of genomics”

Keywords Ageing, immunology, genomics/proteomics Problem

Worldwide, the segment of the population aged 60 or over is increasing rapidly. Increased life expectancy, combined with three decades of falling birth rates, will raise the proportion of the EU’s population aged 60 and over from 21 % today to 34 % in 2050. The average age is rising fast in all Member States, and even faster in the candidate countries of Central Summary and Eastern Europe. The ratio of retired people to those of working age is increasing. Ageing therefore represents a huge challenge to health care, Recent technological advances in genomics, proteomics and bioinformatics social service and pension scheme, as well as to the medical profession. have yielded new insights into the molecular mechanisms that underlie Both, policy-makers and practitioners, rely heavily upon research – to immune cell signalling and function, and opened up new avenues for extend understanding of ageing’s genetic, biological and psychological biological investigation. Although interesting results have emerged by the processes, to develop new medicines and technologies for the old, and to application of genomics/proteomics in basic studies performed in various substantially improve the life quality of the aged segment of population models of ageing, these new technologies have only recently been introduced and thus to relieve the burden of the institutions on which they depend. in studies on the ageing immune system. The goal of the two day conference The goal of biomedical research on ageing is to help people grow old with for 150 participants, which was held from September 4-5, 2006 in Paris right dignity and in good health. The ARIG conference intended at improving before the 16th European Congress of Immunology, was therefore to assess interactions among a large fraction of all European researchers working in the present standing of European research in this field, by providing the fi eld of the ageing immune system and represented the continuation a forum for scientifi c exchange among the most outstanding European of a series of four very successful conferences which initialised the scientists working in immunogerontology, representatives of key stakeholders exchange of knowledge, the start of new collaborations, the exchange of of industry, including SMEs, and fi eld leaders from the United States (funded programmes and the design of future strategies for joint interactions. The by the NIH), where a large number of projects on genomics and the ageing specifi c aim of ARIG was to further discuss a new and very important immune system have recently been funded and are in progress. The conference dimension, namely the use of technologies such as genomics and provided an excellent basis for constructive discussions on recent proteomics in the specifi c fi eld of immunogerontology. Due to the interest achievements in research on the ageing immune system, advantages and of leading U.S. scientists in this area of research, who obtained funding limits of the usage of genomics/proteomics, as well as methodological from the NIH to allow their participation at the conference and formed an developments and clinical application in this field. It will contribute advisory committee for the planning of the programme, it was possible substantially to a more integrated and better coordinated European research to make comparisons between the state of the art in Europe and the structure in immunogerontology which will at the same time greatly profi t United States in this particular discipline. Exchange of knowledge between from new technical input by industry as well as from intensifi ed cooperation European and U.S. scientists in a fi eld as specifi c as the application of programmes with leading U.S. researchers in the fi eld. a new technology in research on the ageing immune system will greatly help European research to become even more competitive and to establish itself as an internationally leading force. Strengthening research on the ageing immune system will also contribute to ensuring the health and welfare of the ageing European population. The activity thus also has a strong economic as well as social perspective.

Aim

• The ﬁ rst objective of the conference was to summarise recent progress in research on the immunology of ageing, addressing the most important research areas such as adaptive, innate and clinically applied immunology. Special emphasis was given to presentations on work so far performed with the help of genomics/ proteomics. These lectures were given by specialists in immunology of ageing as well as by experts from mainstream immunology with no previous experience in ageing research (WP 1-3).

162 | HUMAN DEVELOPMENT AND AGEING T-Cells.

• The second objective was to trigger a critical discussion on the Coordinator Partners advantages, limits and potential problems of the application of genomics/proteomics in the research on the ageing immune system. Prof. Beatrix Grubeck-Loebenstein Prof. Graham Pawelec The discussion focused on: Institute for Biomedical Aging Research University of Tuebingen Austrian Academy of Sciences Tübingen, Germany – methodological questions; Rennweg 10 – potential problems concerning the acquisition, administration A-6020 Innsbruck, Austria Prof. Arne Akbar and usage of data; Phone: +43 512 58 39 19 0 University College – possibilities of using joint data banks in this specifi c fi eld [email protected] London, United Kingdom in the future. Present and future possibilities of commercial exploitation were also discussed (WP 3). Dr. Rosalyn Forsey • The third, and also very important objective of the project was to Bourn Hall Ltd design future strategies on: Cambridge, United Kingdom – specifi c research fi eld in Europe; – how to organise continuous interchahnge between European and US scientists; – how to use and to take advantage of new techniques such as genomics/proteomics for a quick transfer of basic research data into clinical application; – how to disseminate scientifi c and technical know-how in this specifi c fi eld (WP 3 and 4).

Results

• Further interest in the ﬁ eld. • Dissemination of knowledge. • Further contacts to researchers in the US. • Stimulation of young scientists to specialize in the ﬁ eld. • Special issue in Experimental Gerontology containing short summaries of the presentations at the meeting (Exp Gerontol 2007; 42: 379-456). • Meeting report in EMBO Reports (EMBO Rep. 2007; 8:220-223). • Reports for societies, industry and politicians.

Potential applications

Project number LSSM-CT-2005-018924 EC contribution € 80 365 Project Duration 18 months Starting date 01/10/2005 Instrument SSA

HUMAN DEVELOPMENT AND AGEING | 163 CRESCENDO

Consortium for Research into Nuclear Receptors in Development and Aging

Keywords Nuclear receptor, development aging Problem

The overarching objective of CRESCENDO is to exploit new opportunities provided by advances in genomics and bioinformatics, and associated technological developments, to enhance understanding of the basic mechanisms underlying NR actions and their translation into the physiological regulation of developmental and aging processes. Summary Increased understanding of the basic mechanisms of nuclear research signalling and the integration of these signalling pathways into the Nuclear receptors function throughout development and aging as molecular networks underlying developmental, aging and (patho)-physiological integrators of complex physiological regulations, such as growth, reproduction regulations will help resolve two problems: and metabolism. To translate genetic knowledge from the Human Genome • the molecular basis of NR – related diseases; into therapy requires intimate understanding of fundamental regulatory • the need to reduce the economic burden of these diseases by mechanisms. CRESCENDO will build on existing knowledge and infrastructures better prevention and innovative therapy. by drawing together European excellence to focus on signalling dynamics, integration of target gene responses during development and aging, and human genetics and pathophysiology, emphasising key nuclear receptors. Aim State-of-the-art genomic and post-genomic techniques will lever advances on mechanisms and kinetics of nuclear receptor signalling in processes CRESCENDO has 4 major aims: underlying development and aging. Information on spatial and temporal • to enhance understanding of NR signalling in the context of regula- aspects of signalling dynamics will feed into a broader vision of how nuclear tory networks; receptors integrate multiple inputs in whole organisms across the life span. • to study the roles of NRs in the continuum of development to aging, Focus will be on developmental targets, on brain development and aging, and with emphasis on complex links between NR signalling, metabolic the complex interplay between metabolism, particularly glucose and lipid disease, as well as brain development and aging; signalling, and brain function. The brain is emphasised, as its disorders often • to increase knowledge of how NR-related information in the genome cause most distress in unhealthy aging. A strong investment in bioinformatics impacts on phenotype; will facilitate data extraction from multi-level analyses (genes to cells to • to transfer this knowledge into therapeutic leads. organisms) and accelerate determination of targets, regulatory networks and integration of physiological responses within individuals and populations. The The project will produce greatly increased physiological and genomic consortium includes molecular and developmental biologists, physiologists level knowledge and innovative tools to address NR regulation of and human geneticists, and has access to major national and transnational developmental and aging processes. It will generate novel concepts and data banks and resources. Nuclear receptor research is being revolutionised therapeutic leads stimulating integrated European research on this by the combination of technological advances with novel insights into critical topic with far-reaching socio-economical consequences. signalling processes. CRESCENDO will ensure that European research in the fi eld accelerates its momentum, fulfi lling its potential to catalyse therapeutic innovation and to create substantial socio-economic benefi t. Potential applications

The main category of potential technological transfers and applications that can be envisaged is through reduction of the economic burden of NR related disease, both in understanding mechanisms of disease and by taking ideas from fundamental research in NR signalling through to Drug discovery.

Nuclear receptors (NR) signalling dysfunction leads to proliferative, reproductive and metabolic disease. Indeed, combinations of NRs are implicated in every major chronic disease associated with aging, from cardiovascular disorders (30 % of global deaths and correlated morbidity) to obesity (300 million obese adults world wide) and its associated disorder type-2

164 | HUMAN DEVELOPMENT AND AGEING diabetes, to cancer and brain disorders (neurodegenerative disease, A B depression, anxiety etc.). Pharmaceutical NR agonists or antaognists such as tamoxifen for ERs (targeted in breast cancer) thiazolidinediones for PPARg (targeted in type 2 diabetes) or dexamethasone for GR (targeted in inﬂ ammatory disease) and thyroxine (a TR ligand) are among the most commonly prescribed drugs.

Three main arguments point strongly towards a vision wherein current research into NR signalling is situated at a turning point for innovative drug discovery. These arguments are: • the dramatic new insights into the complex dynamics of NR signalling and the protein-protein and protein-DNA interactions that govern NR-related transcriptional control. Increased understanding of these

kinetic parameters will open up new concepts and windows of C possibilities for therapeutic intervention; • the fact that chemistry is providing us with not only with receptor selective and various types of full, partial or inverse agonists, but also with compounds that activate only a subset of the functions induced by the cognate (usual) ligand or even with ligands that act in a cell speciﬁ c manner, reducing side effects. These innovations and increased understanding of the chemistry of signalling are open up new possibilities for design, screening and validation of ligands and targets; D E • the immense potential of bioinformatic approaches with gene ontologies, which provide a standardised vocabulary for data exploration and new software programmes that make it possible to handle enormous amounts of data and build realistic models of complex systems.

NRs constitute the second largest class of drug targets; it has been estimated that the current market of nuclear receptor targeted drugs is 10-15 % of the global pharmaceutical market of about 366 € billions in 2003. It is conceivable, however, that given the involvement of NRs in so Amphibian metamorphosis provides a parallel to the mammalian perinatal period and implicates signalling through key NR particularly the many different age-related physiological regulation the share of the thyroid hormone receptor: using transgenic Xenopus with either general market will increase in the coming years. It is essential that Europe acquires expression (A) or speciﬁ c expression (B), the balance between apoptosis a leading position in this highly dynamic and developing market. (D) and proliferation (E) in maturation of the nervous system (B, C) during the critical period of metamorphosis can be analysed.

CRESCENDO will constitute a unique platform to generate knowledge in NR research through the integration of European laboratories and to translate this new information into EU-owned patents and innovation which will then constitute the basis for the identiﬁ cation of new therapeutic compounds by large EU pharmaceutical industries.

CRESCENDO will thus contribute to closing the gap between US and Europe providing EU-based pharmaceutical industries new concepts and ideas originated through R&D developed in large parts of the public sector using common EU funding.

HUMAN DEVELOPMENT AND AGEING | 165 Coordinators Jan-åke Gustafsson Martin Göttlicher Ingemar Pongratz Forschungszentrum Für Barbara Demeneix Juha Kere Umwelt und Gesundheit, Gmbh Giovanni Levi Karolinska Institutet Neuherberg, Germany Centre national de la Huddinge, Sweden Recherche scientifi que Roberto Di Lauro Paris, France Olli A. Janne Stazione Zoologica Phone: +33 1 4079 3607 Helsingin Yliopisto Anton Dorhn Fax: +33 1 4079 3618 Helsinki, Finland Napoli, Italy [email protected] Frank Gannon Günther Schutz Stefanie Denger Deutsches Krebsforschungszentrum Partners European Molecular Biology Laboratory Heidelberg, Germany Heidelberg, Germany Barbara Demeneix Hinrich Gronemeyer Giovanni Levi Osborne Almeida Centre européen de Recherche en Laurent Sach Dietmar Spengler Biologie et Médecine Franck Delaunay Max Planck Institute of Psychiatry Illkirch, France Centre national de la Münich, Germany Recherche scientifi que Philippe Amouyel Paris, France Adriana Maggi Aline Meirhaeghe-hurez University of Milan Institut National de la Santé Vincent Laudet Milan, Italy et de la Recherche Médicale Jacques Samarut Paris, France Frédéric Flamant Denis Duboule Michela Plateroti University of Geneva Catherine Dacquet Centre national de la Geneva, Switzerland Servier Research Institute Recherche scientifi que Suresnes, France Paris, France Juan Bernal Ana Aranda Michael R. Witt Consejo Superior de Karo Bio Investigaciones Cientifi cas Huddinge, Sweden Madrid, Spain Alessandro Weisz Petr Bartunek Seconda Università Institute of Molecular Degli Studi di Napoli Genetics Napoli, Italy Prague, Czech Republic Edison T. Liu Hilde Nebb Genome Institute of Singapore University of Oslo Singapore Oslo, Norway Marc Robinson-Rechavi Université de Lausanne Project number Lausanne, Switzerland LSHM-CT-2005-018652 EC contribution € 9 999 343 Project Duration 60 months Starting date 01/03/2006 Instrument IP Project website www.crescendoip.org ECONAG

European Conference on Aging

Keywords Ageing research, translational research, Problem dissemination of knowledge The current demographic development predicts a rapid increase in the proportion of elderly people in the European societies and this has considerable impact on the society as a whole. In particular, age- associated frailty and health problems, along with an increasing number of patients suffering from age-related diseases, causes a huge economical burden for the health care and social systems. A way to counteract Summary these developments is to promote healthy ageing and delay age- associated dysfunctions. The current project aims to summarize current We will prepare a conference program that refl ects the most advanced research into mechanisms of ageing and their implications to the health parts of European Ageing Research and provide a forum for discussions status of the European population. concerning future directions of this research. From the technological point of view, a particular emphasis will be put on the aspect of translational research. The planned scientifi c conference is to be held in Aim close conjunction with the “Genetics, Longevity and Demography Scientific Workshop” of the ERA AGE Network that will be held in The overall aim of this project is to provide a forum for scientiﬁ c Innsbruck at the same time during the year 2006, probably by the end of exchange and, in addition, for attempts to inﬂ uence the European September 2006. The latter one is coordinated by Prof. Beatrix Grubeck- Research on Ageing. Particular emphasis of the conference will be on Loebenstein, who serves as a director of the applicant’s institution. We a synopsis of current achievements in ageing research with particular anticipate a signifi cant impact for the conference from this constellation reference to genomic and post-genomic technologies. and expect that this activity will contribute substantially towards an integrated structure for European Ageing Research, taking into account both stakeholders’ interests and commercial applications. Expected results

• One objective is to provide a forum for scientifi c exchange and to provide a synopsis of current achievements in ageing research, with particular emphasis on genomic and post-genomic technologies. • A second objective is to evaluate potential applications of ageing research that has undergone tremendous progress at the scientifi c and technological level in the last couple of years; however, in comparison to the United States of America (USA), the attempts to generate products and services from this research are underdevel- oped at the present time in Europe. To facilitate this transition, in addition to the scientifi c sessions, the symposium will feature a session where qualifi ed people from the European biotechnology industry and of small and middle enterprises (SMEs) working in that area will have the chance to discuss potential practical outcomes of current ageing research. • A further objective of the project is to provide a forum for discussions concerning future directions of this research. We anticipate that the combination with the “Genetics, Longevity and Demography Scientifi c Workshop” of the ERA AGE Network will lead to mutual benefi t for the participants of both events and as a whole will provide an ideal forum where researchers, funding organisations, policy makers and representatives of the biotechnology community can exchange ideas.

Potential applications

n.a.

HUMAN DEVELOPMENT AND AGEING | 167 Coordinator Partners

Univ. Doz. Dr. Pidder Jansen-Dürr Barbara Fitzky Austrian Academy of Sciences Amynon Biotech GmbH p.jansen-dü[email protected] Innsbruck, Austria Phone: +43 512 583919 44 Fax: +43 512 583919 8 Gordon Koell Kompetenzzentrum Medizin Tirol GmbH Innsbruck, Austria

Efstathios Gonos National Hellenic Research Foundation Athens, Greece

Thomas Nyström Gothenburg University Gothenburg, Sweden

Project number LSSM-CT-2005-518161 EC contribution € 75 600 Project Duration 18 months Starting date 01/11/2005 Instrument SSA Project website www.econag2006.com

168 | HUMAN DEVELOPMENT AND AGEING ELAST-AGE

Targeting the elastic tissues ageing to improve the quality of ageing

Keywords Ageing, elastin, connective tissues, amyloid, Problem tissues, cardio-vascular, dermatology, tissue engineering, toxico-genomics Our understanding of the cellular and molecular mechanisms underlying ageing is increasing tremendously. However, the complex features of in vivo ageing at the tissue and organ levels are far from being understood. Moreover, ageing is accompanied by several ‘age-associated’ diseases, most of them involving a tissue disorganization associated with manifestation of connective tissue ageing. One of the most remarkable manifestations of ageing is the loss of tissue elasticity, with its consequences for breathing, regulation of pulse pressure and, consequently, cardiac Summary function. Therefore, ageing of elastic tissues might be related to general ageing, and reciprocally. Ageing is accompanied by several ‘age-associated’ diseases, most of them involving tissue disorganization associated with a loss of tissue elasticity, with multiple physiological, mechanical and social Aim consequences. The goal of this project is to target the ageing of elastic tissue to cope with ageing, or at least some manifestations of ageing. As The main objectives of the project are centred on: the mechanisms that govern correct elastogenesis during development, • the understanding of ageing of elastic tissues in animal as which are lost in adulthood and ageing, are still unknown, and as it is a piece of global ageing; also unknown how to reactivate these processes, the project introduces • the search of ‘positive modulators’ of elastic tissue ageing a coordinated action to fi nd ‘positive modulators’ (protection against (protection against degradation and post-modifi cation, degradation and post-translational modifi cation, re-induction of optimal re-induction of optimal elastogenesis). elastogenesis) of elastic tissue ageing. The originality of the project lies in the Coordination of strategies that will Therefore, the technical contributions of this project will be to defi ne defi ne: rodent models and tissue-equivalents to study the potential impact of • rodent and tissue-equivalent models to study the impact of therapeutic approach on elastic tissue ageing and global ageing, the therapeutic approaches on elastic tissue ageing; development of strategies to assess the neosynthesis of elastic tissues • the development of diverse elastic fibre-derived peptides and to defi ne biomarkers at the cross-roads between global ageing and – proteoglycan conjugates based on the elastic fi bre components elastic tissue ageing. structure and its alterations with age, in order to protect or delay elastic fi bre degradation or post-translational modifi cation; • the development of strategies to assess neosynthesis of elastic Expected results tissues and to defi ne biomarkers at the cross-roads between ageing and elastic tissue ageing. Assays on cells from humans displaying The expected results of the project will be the following: genetically-based ‘exaggerated’ ageing will be used as indicators of • the consortium want to develop mimetics of elastic fi bres components the effectiveness of the selected ‘modulators’. that could be protectors of elastic fi bres against degradation; The expected impacts should be a breakthrough on skin and vessel • the project will also search global protectors of elastic fi bres post- ageing treatments with a special interest with the improvement of translation modifi cations, using proteoglycan – peptide based mimics chronic vein insuffi ciency and, longer term, other disorders of joints, or anti-oxidants; arteries and macula and the improvement of healing. The project brings • as Elastic fi bres do not spontaneously assemble in adult (in contrast to together 12 participants with complementary expertise from 6 different young), an important and coordinated effort will be made to fi nd countries, including basic scientists, clinicians and three SMEs. activators of elastogenesis in cells from adult and aged persons, through a screening of active extracts library; • scaling up of a skin equivalent model and development of a dedicated DNA arrays will be expected to improve assays aiming to fi nd extracts active on cell and tissue elastogenesis; • the expression analysis will be tested on 3 disorders characteirisc of accelarzated ageing of elastic tissues: cutis laxa, pseudoxanthum elasticum and varicosis; • the selected compounds will be checked in tissue-equivalents (skin and cardiac valves) and in rodents. The consortium will use Senescence Accelerated Mice and defi cient heterozygous elastin +/- mice.

HUMAN DEVELOPMENT AND AGEING | 169 1. Young skin: staining of elastin by Immuno-ﬂ uorescence. 2. Young skin: Electronic microscopy observations. 3. Old skin: staining of elastin by Immuno-ﬂ uorescence. 4. Old skin: Electronic microscopy observations. 1 1 2 3 3 4

Potential applications Coordinators Ulrich Stock Charité Universitätsmedizin The expected positive modulators (against degradation and post- Pascal Sommer Berlin, Germany translational modifi cation, and to induce neoelastogenesis) might have Agnès Borel Centre National Valérie André potential applications for treating several complications of aged associated de la Recherche scientifi que COLETICA disorders, as elastic fi bre synthesis occurs during development and early Délégation Régionale Rhône-Auvergne Lyon, France adult growth, and is scarce in adults. While age-related dysfunction of 2, avenue Albert Einstein – BP1335 elastic tissues can be observed at different levels of the human body. 69609 Villeurbanne Cedex, France Bruna Parma [email protected], [email protected] OPOCRIN One of the most spectacular manifestations of human ageing is the Corlo di Formigine, Italy apparition of skin wrinkles. Intrinsically aged skin (chrono-ageing) shows a decrease of elastin and disintegration of elastic fi bres. In contrast, Partners Marie-Paule Jacob photo-ageing is characterized by wrinkles, an increase of fragility of the Gilles Faury skin and an increase of elastin staining intensity, with accumulation in Daniela Quaglino Institut National de la University of Modena Recherche Médicale facial skin of morphologically abnormal (amyloidal) elastin structures. Modena, Italy Paris, France It should be noted the risk of post-operative skin hernia with age, due to structurally altered elastic fi bres. Julia Bujan Armando Di Donato University of Alcala de Henarès Giannina Gaslini Hospital The cardio-vascular apparatus is highly sensitive to ageing of elastic Henarès, Spain Genova, Italy tissues, with atherosclerosis, aneurysms, hypertension, varicosis. Elastin degradation within the alveolar wall is an important event in the Antonio Tamburro Gorka Ochoa development of pulmonary emphysema. University della Basilicata PROGENIKA Biopharma Potenza, Italy Derio, Spain Late age-related maculopathy (ARM), a leading cause of untreatable Cay Kielty Stephan Baydanoff new vision loss in elderly individuals, is a consequence of the thickening Manchester University University Hospital of Pleven of the Bruch’s membrane (eye’s thin connective tissue) mainly due to the Manchester, United Kingdom Pleven, Bulgaria deposition of fi brillar and amorphous elastin material and an increased proportion of heparan sulphate and lipids.

It is observed an increase of elastin expression in diabetic persons’ (type 1) kidneys.

Many other tissues are also concerned by elastin decrease: ligaments, tendons and intervertebral discs.

It was also described that presence of elastic peptides produced by elastase activity, in elastin rich tissues (lung, skin) encourages tumours progression

The acquisition of knowledge brought by the consortium should help to defi ne new targets, in response to a growing interest in pharmacology, Project number cosmetology and bioengineering domains. The potential innovation aspects LSHM-CT-2005-018960 will be on the development of new peptide-proteoglycan complexes, on EC contribution the improvement of tissue-equivalents, on the identifi cation of new € 2 374 787 compounds or molecules favouring elastic fi bre tissue homeostasis and on Project Duration the development of new ‘post-genomic read-out grid’ to evaluate the 36 months effects of such new compounds. The potential domains of interest are the Starting date cardio-vascular disorders, the cosmeto-dermatology, the toxico-genomic 01/01/2006 testing, and the bio-engineering domain. Beside, other domains might be Instrument concerned in a more distant future, for instance on the age-related macular STREP degeneration. Project website www.elastage.org

170 | HUMAN DEVELOPMENT AND AGEING EWA Estrogens and Women Aging A project of the European Union coordinated by Adriana Maggi

Keywords Menopause, estrogen action, women aging, Problem diabetes, neural diseases, skin diseases, molecular genetics A common feature of female physiology is the cessation of ovarian functions (menopause) occurring when women are in their late forties- early fi fties. Therefore menopause is not a disease, but rather a normal physiological event in a woman’s life. It can be associated, however, with a signifi cant decrease in quality of life, and an increase in the risk of developing illnesses such as osteoporosis, coronary heart diseases, asthma, Summary stroke, eye dysfunctions and neurological disorders. Today most women live long enough to reach menopause, with life expectancy throughout In women estrogens and their cognate receptors regulate reproductive Europe in the range 80-82 years so that more than one-third of a woman’s activities and exert protective functions in skeletal, cardiovascular and life will be lived in menopause. The mean age of the population will increase nervous systems. With the continuing increase in life expectancy, women even faster in the next half century and it is projected that there will be now spend more than one third of their life in menopause, a physiological almost 2 billion (1,970 million) older persons in 2050 compared with state in which ovaries stop producing sexual hormones. To date, the risks 580 million in 1998: the majority of these elderly will be women. The and benefi ts of post-menopause hormone replacement therapy (HRT) are quality of life of older women in the aging population will depend in large still unclear. Indeed, clinical findings are in strong conflict with measure on the abilities of societies to cope with the economics and experimental results, the latter showing a clear protective effect of medical challenges of the postmenopausal years. estrogens against bone, vessel, skin and brain malfunctions. EWA will build on existing knowledge, reagents and infrastructure landmarks of The main consequence of menopause is an abrupt drop in levels of the cir- European excellence in estrogen research to focus on: culating sex hormones: estradiol and progesterone. This phenomenon is • generation of innovative cell and animal models to identify the only observed in women; in men, gonadal activity does not suddenly stop, safest compounds to be used in HRT; but declines slowly with age; thus in aged men the levels of circulating sex • evaluation of the therapeutic effi cacy and risks of known and novel hormones (including estrogens) are much higher than in post-menopausal molecules to be used for HRT and testing diverse modalities of HRT women. The augmented susceptibility to disorders of the brain, immune to mimic at best the physiological cycle in fertile women; system, bone, urogenital tract, cardiovascular tissues, eye and gastroin- • investigation on the anti-infl ammatory activity of estrogens which testinal tract (colon in particular) are not explained with a diminished are of therapeutic benefi t in diseases associated with aging. production of progesterone because this hormone preferentially targets In EWA, pharmacologists, molecular biologists, endocrinologists, reproductive organs, where its cognate receptors are expressed. Conversely, neuroscientists and clinicians from the main European groups in the fi eld the decline of estradiol is believed to be of functional relevance for repro- will co-operate to assure a multidisciplinary approach indispensable for ductive as well as non reproductive organs such as heart and blood vessels, a successful research programme and for the full access to the state-of- bone, brain, immune cells: indeed it has been demonstrated that all of the-art technologies available in the fi eld, in a collaboration that is based these tissues and organs express estrogen receptors (ERalpha or ERbeta) on a successful history of joint research. Moreover, the involvement of and therefore are targeted by this hormone. In addition, a large number of a SME will assure the prompt exploitation of the results of the project, animal studies have shown that estradiol, but not progesterone, exerts while disseminating the knowledge gained will hopefully strengthen links protective functions in neural, immune, muscle and endothelial cells. Still with other Companies. an object of heated debate is the usefulness of HRT for women in menopause. The most recent clinical studies failed to show clear benefi cial effects of hormone replacement therapy for cardiovascular and neurological disorders claimed in several of the preceding analyses. These more recent results raise one important question: can HRT be modifi ed to recapitulate the benefi cial effects of ovarian hormone production in pre-menopausal women?

To design novel, more effi cacious treatments we should increase signifi - cantly our knowledge of estrogen action in the context of woman physiology. Indeed, at present time, we know very little about the activity of estrogens and their cognate receptors in non-reproductive organs. This is mainly due to the fact that the concept of estrogenic action expounded outside the boundaries of reproductive functions is novel, thus we should focus our efforts towards an understanding of the effects of this sex hormone in organs where the benefi cial effects of estradiol are lacking in the post-menopausal years.

HUMAN DEVELOPMENT AND AGEING | 171 Aim

The main purpose of EWA research project is to understand why current HRTs cannot reproduce estrogen’s protective activities found in original epidemiological and experimental studies and to generate novel knowledge that will provide a basis upon which to build novel, safer therapies.

Thus the research carried out by the EWA consortium will focus on: • generation of new tools which will be indispensable for the study of the activity of estrogens and their receptors in living organisms; • evaluating the effects of estrogens and synthetic estrogenic compounds in a range of model systems to gain the knowledge necessary to develop novel replacement therapies; • study of the relevance of the anti-inﬂ ammatory activity of estrogens in the onset of the major pathologies associated with menopause.

EWA’s aim will be pursued through a multidisciplinary approach combin- Reporter mice to shed light into the complexity of estrogen action. ing the expertise of molecular biologists, geneticists, endocrinologists, neuroscientists, pharmacologists, and clinicians.

• the pharmacologists located in Milan and at the collaborating SME Expected results in Turku will verify the effi cacy of selected protocols of hormone replacement therapy providing suggestions for further trials to be EWA will induce several of the European groups leader in the fi eld of carried out in humans; estrogen action to share expertise and reagents and focus their research • the collaboration among the groups in Manchester, Stockholm, on topics more related to woman aging. We expect that this group will Milan and Madrid will provide very relevant novel information on act as a catalyst to attract new researchers to the fi eld. the activity of estrogenic compounds in brain and skin likely providing the basis for the design of novel therapies; Our research will also provide signifi cant advancements: • the involvement of the Lorenzini Foundation will be instrumental for • the research lead by the group based in Heidelberg will identify nov- the dissemination of the results of the groups, for increasing the public el, cell specifi c, genes targeted by natural and synthetic estrogens, awareness of the necessity of research in the fi eld and for increasing thus providing knowledge potentially applicable to the analysis the collaborations with other researchers located in Europe. of the effi cacy of estrogenic treatments in humans; the collaboration of this group with clinicians located in Milan and Manchester will The participation of the Milan group to research sponsored by the NIH enable a very preliminary testing on the applicability of the results Institute of Aging (MADRI RFA R011AG027713-01 P.I. Adriana Maggi) obtained; will facilitate exchanges with overseas researchers providing the EWA • collaborative studies carried out between the groups located in program with an even greater breadth of research. Strasbourg and Milan will result in the generation of very innovative reporter animals in which we will be able to study in real time the effects of natural and synthetic estrogens on each ER subtype and Potential applications on mutants of the two receptors in which selected functions will be ablated the tools generated will provide novel reagents that will be Generation of kits for the study of the effi cacy of replacement therapies indispensable in gaining a full understanding of estrogenic action; in women; generation of novel tools for drug screening.

172 | HUMAN DEVELOPMENT AND AGEING Coordinator Prof. Pierre Chambon Centre Européen de la Recherche en Prof. Adriana Maggi Biologie et Medecine University of Milan Groupment d’Intérêt Economique Center of Excellence on Illkirch, France Neurodegenerative Diseases Milan, Italy Prof. Livio Luzi [email protected] San Raffaele Scientifi c Institute Consortium for Research Prevention and Treatment of Metabolic Partners and Endocrine Diseases Milan, Italy Prof. Gillian Ashcroft University of Manchester Prof. Jan-Åke Gustafsson Manchester, United Kingdom Karolinska Institutet Stockholm, Sweden Dr. Frank Gannon Dr. Mikko Unkila, HORMOS Medical European Molecular Corporation, Turku, Finland Biology Laboratory Heidelberg, Germany Giovanni Lorenzini Medical Science Foundation Prof. Luis Miguel García-Segura Milan, Italy Consejo Superior de Investigaciones Houston, USA Centífi cas – Instituto Cajal Madrid, Spain

Expression of hERa isoforms in human osteoblasts.

Project number LSHM-CT-2005-518245 EC contribution € 2 375 000 Project Duration 36 months Starting date 01/01/2006 Instrument STREP Project website www.ewa.unimi.it

HUMAN DEVELOPMENT AND AGEING | 173 LifeSpan

Integrating research into Development and Ageing

Keywords Development, ageing, model systems, genetics, Problem epigenetics, plasticity, environment, stochasticity, human health Why linking research into development and ageing? The answer lies in the notion that ageing is complex and requires an integrated approach that builds together medical science, genetics, and a framework of evolutionary theory. To date, the focus of ageing research has primarily been on which genes, physiological and metabolic processes exert their effects in adult life. However, there is a growing need for research in the fi eld of ageing to include the study of developmental processes within Summary the context of reproduction, longevity, and ageing. The participants in this Network of Excellence (NoE) take this stance not only because there Although human ageing has many dimensions it is a biological process that is ample evidence that developmental processes affect adult life but also we share with nearly all living organisms. Therefore, we must comprehend because evolutionary theory predicts that natural selection has worked the broad principles of the ageing process beyond current preconceptions to integrate the whole of an organism’s life history, which includes about the ageing process that it is an ineluctable part of our biology. In line development. with the continuous increase of our lifespan, ageing may be intrinsically far more malleable than previously thought. The challenging focus of our To date only little is known about the mechanisms and their functions endeavour is to reveal how conditions in early development infl uence that underlie such links. Evolutionary conservation of the genetic regula- ageing across a diverse spectrum of species, including humans. tion of ageing among model-organisms opens unique opportunities for Ageing can only be understood by considering the entire human life history. understanding human ageing and age-related health problems. LifeSpan Our life span is determined by genetic, environmental and stochastic will use the power of key (in)vertebrate model-organisms to uncover the factors, and evidence suggests that early-life events determine the mechanisms that are relevant in mankind. individual status at the end of life. Therefore, we aim to bridge research on development with research on ageing, and to integrate these two disciplines that do not have a tradition of close interaction. We will move to and fro Aim between experiments in (in)vertebrate models and observations in humans to test effects detected in one species as candidate longevity mechanism • Integration: LifeSpan will serve as a real and virtual working in the other. This approach has only been marginally explored and has environment that enables expertise building in the emerging research never been attempted in one collaborative effort. Some of us have begun fi eld “Integrating research into Development and Ageing”. LifeSpan to probe this unexplored fi eld of research, but fragmentation in Europe should result in an European research community where all participants must be avoided. LifeSpan ensures the future framework of ageing research have mutual and essential cross links. This community as a whole is that allows the appropriate questions to be asked, using the most suitable much more than the sum of its parts. organisms, and taking the appropriate experimental and observational • Scientifi c excellence: LifeSpan aims to identify the mechanisms that approach. This can only be done effectively when the research groups in connect the developmental and the adult life phase and how they Europe have the proper expertise and carry out their research in an infl uence ageing and health at old age. These mechanisms will be integrative manner, in terms of science, organisation, and of a common described in terms of genetics, epigenetics, development, and physi- education programme. All participants excel in science including the ology. The discovery of these mechanisms and how they interact will expertise and resources necessary for this Network. Crucially then, LifeSpan be propelled mostly by using experimental manipulations in model will provide the essential framework to reach European scientifi c excellence organisms. Crucially, these observations will be combined with by “Integrating research into Development and Ageing”. unique observations in human cohorts to establish the contribution of these mechanisms to variation in human ageing and longevity. • Spread results: Knowledge of the LifeSpan initiative, its results and impact will be made available to professionals and the general public. The professionals include scientists from outside the network and outside Europe, experts working in the healthcare sector, and experts from industry. This is essential for implementing the scientifi c results in healthcare programmes, and to help develop sustainable solutions.

174 | HUMAN DEVELOPMENT AND AGEING Translational research: from experiments in model organisms to solutions for humans.

Expected results Coordinators M. Hertweck R. Baumeister • Identify genetic pathways and mechanisms that link development Prof. Dr. R.G.J. Westendorp Albert-Ludwigs University to ageing and longevity. The effects will be determined in different Dr. B.J. Zwaan of Freiburg, Bio 3, Bioinformatics and environments to include descriptions of developmental plasticity and Leiden University Medical Molecular Genetics, ALU.FR Center (LUMC) Freiburg, Germany gene by environment interactions. Dept of Gerontology & Geriatrics • Establish for the identiﬁ ed pathways and mechanisms whether The Netherlands T. Kirkwood they contribute to variation in life span and life histories in human [email protected] or University of Newcastle populations. [email protected] Institute for Ageing and Health Newcastle, United Kingdom

Potential applications Partners K. Christensen University of Southern Denmark LifeSpan is aimed at understanding a fundamental process of metazoan B. Zwaan Odense, Denmark life, ageing and its relation with development. The identifi cation of P. Brakefi eld Leiden University S. Cnattingius genetic, bio-molecular and physiological pathways that co-ordinate Leiden, The Netherlands Karolinska Institutet developmental processes and adult longevity and ageing, and the envi- Stockholm, Sweden ronmental factors that infl uence their expression, opens the possibility R. de Kloet for non-invasive intervention in these pathways by means of a changed R. de Rijk R. Aamodt life-style, dietary intake, medication, or other. A full understanding of Leiden University, Leiden/ S. Omholt the genetic and environmental factors and of the mechanisms by which Amsterdam Center Norwegian University they exert their effects will facilitate an individual-tailored healthcare for Drug Research of Life Sciences scheme, including both medicinal and non-medicinal treatments. Leiden, The Netherlands Aas, Norway The exploitation of the results is twofold: • development of healthcare schemes by the industrial and scientifi c B. Grubeck-Loebenstein D. Gems Austrian Academy of Sciences L. Partridge participants, as well as ‘outsourcing’ such developments to industry; Vienna, Austria University College London • the results can also be exploited by healthcare professionals who can London, United Kingdom change their advisory and prevention plans by informing their patients A. Metspalu and potential future patients how they can change their way of living University of Tartu M. Remm and child-care to increase the likelihood of healthy ageing. Tartu, Estonia OSAÜHING BioData Tartu, Estonia M. Holzenberger Hopital Saint-Antoine T. Flad Paris, France PANATecs GmbH Tübingen, Germany L. Keller University of Lausanne J. Hoeijmakers Lausanne, Switzerland Erasmus University Rotterdam G. Pawelec Rotterdam, The Netherlands Eberhard-Karls-Universität Tübingen Project number Tübingen, Germany R. Strijker LSHG-CT-2007-036894 Dnage EC contribution Leiden, The Netherlands € 10 000 000 Project Duration 60 months Starting date 01/01/2007 Instrument NoE Project website www.lifespannetwork.nl

HUMAN DEVELOPMENT AND AGEING | 175 PROTEOMAGE

Functional Analysis of Evolutionarily Conserved Mechanisms of Ageing Based on Advanced Proteome Analysis

Keywords Ageing, proteomics, proteoasome, 2d-page, Aim mass-spectrometry, phage display, bioinformatics, cell culture, mice, yeast, podospora anserina, c. elegans The aim of PROTEOMAGE is to gain novel insight into molecular mechanisms of healthy ageing. Based on a proteomic analysis of ageing processes in a variety of ageing models including model organisms and model systems (e.g. human cell cultures), we will address the question how: • changes in protein concentration and protein modifi cation; • protein-protein interactions and protein networks; Summary • signaling mediated by extracellular proteins; • protein turnover and degradation via the proteasomal system play Ageing can be defi ned as the progressive loss of function accompanied by a functional role in the ageing process. an increase in frailty, morbidity and mortality with advancing age. In humans, improvements in medicine and health care have resulted in PROTEOMAGE is based on complementary advanced technological drastical increase of the mean life span of the individual over the centuries, platforms with centralized management, in order to address biological and lifespan maybe still increasing. According to a statistics from the year questions that cannot be addressed at the level of a single partner. 2000 mean life span of a boy born in 2001 in one of 15 selected European PROTEOMAGE will integrate state-of-the-art proteomic facilities and countries will be 75.3 years and a girl will reach 81.4 years. As a phage display platform, supported with bioinformatics analysis. In a consequence of a higher death than birth rate, the population of Europe a supplementary platform, we will test in vitro models of ageing based is ‘greying’. Whereas, as an example, today approximately 29 % of German on human cell cultures in normoxic conditions. The project focuses on individuals are over 60, it is estimated that in 2050 the percentage fi gure age-associated changes in protein abundance, post-translational modi- will rise to 44. This situation already generates severe social tensions fi cations and maintenance. The work will also address the question to because the younger population does not seem to be able to support the what extent modifi cations are just diagnostic of ageing or are actual growing number of elderly which need extensive help due to disabilities or causes. PROTEOMAGE will also focus on protein-protein interactions severe diseases. Solving this immense social problem clearly requires and protein networks in relation to the ageing process. This work also drastic changes in our social system. However, such changes alone will not aims at unraveling changes of protein-protein interactions as a function be suffi cient unless effi cient interventions in the ageing process result in of age to describe the interactions of structurally and phylogenetically a higher proportion of ‘healthy’ old people. The prerequisite towards this conserved mechanism in ageing and to explore the functional protein goal is clear: a detailed understanding of the basis of biological ageing. repertoire in different experimental systems. We will explore the role Key questions in the ageing fi eld remain unanswered. The PROTEOMAGE of core protein regulatory network components in ageing and ana- project will elucidate molecular pathways underlying the ageing process in lyze them as putative biomarkers and lifespan modulating targets. human cell lines and to some degree in model organism. PROTEOMAGE will also investigate age-associated changes in extracel- Today proteome analysis is viewed as the analysis of an entire protein set lular signalling complexes, which modulate the activity of key regulators expressed by a biological system in a given condition. In the current project, of ageing processes in model organisms such as the insulin/IGF pathway. advanced proteomics technology will be applied to establish age-associated PROTEOMAGE will also determine the extent to which extracellular changes in proteomes and this will be used to reveal functional circuits that signalosomes have been conserved in evolution and contribute to the control or trigger the ageing process. The unavailability of human beings for regulation of lifespan in lower eukaryotes. Additionally, PROTEOMAGE biological experimentation will be dealt with by the strategy to compare aims to study alterations of proteasome activity and effects on protein ageing processes in cultured human cells and model organisms from yeast degradation and stress sensitivity during ageing. We will also answer to mice; Following this strategy, we will defi ne proteins in human systems the question whether increased proteasome activity is a longevity that are likely to play a key regulatory role in ageing. assurance mechanism and will try to develop experimental strategies to reactivate proteasome function in aged cells.

176 | HUMAN DEVELOPMENT AND AGEING Expected results Potential applications

By using a centralized cutting-edge proteomic technological platform Ageing as the progressive loss of function and consequently an increase and a new platform for cell culture at physiological low oxygen pres- in morbidity is a serious social problem. Already today, social security sure, a high resolution proteomic analysis of unmatched precision will and health insurance systems are collapsing. With the increasing be applied to well-established in vitro models of replicative senescence proportion of the elderly in the near future, this problem will continue and cells and tissues from people of different age including centenari- to grow and a generation war is forecast. A solution is only possible by ans. Novel technology will also allow identifi cation of age-associated an integrated approach: political decisions (e.g. restructuring social and post-translational modifi cations and cleavage events that are known to health insurance systems) and substantial scientifi c advances to infl uence greatly the activity of proteins. Trans-species comparisons will understand the basis of biological ageing and to use this understanding reveal candidates for proteins that play a functional role in driving age- to intervene in the development of age-associated dysfunctions. ing processes, allowing for the fi rst time a delineation of cause-effect Increasing life span is not the major goal as this is rightly criticized when relationships (instead of mere correlations) governing proteome chang- discussing the importance of ageing research. The clear goal is to es that lead to age-associated phenotypes. The work described above increase healthy ageing: in other words to “add life to years and NOT will also lead to new protocols for early diagnosis and prevention of years to life” (B. Strehler). age-associated dysfunctions, based for example on strategies to reactivate proteasome activity in senescent cells. The economic impact of PROTEOMAGE is two fold: since a large part of the research carried out by PROTEOMAGE is basic research, the Proteome analysis of ageing will be used to shed new light on molecular advancement of knowledge about the ageing process will give rise mechanisms underlying the ageing process in various model organisms. concomitantly to commercial exploitation in a sense that we envisage This will enable us to: that in the midterm new diagnostic kits and preventive measures can be • establish age-associated changes in the proteomes of several cell developed and turned into products that increase the health and wealth types, such as human fi broblasts, keratinocytes, endothelial cells and of the European population. lymphocytes, during replicative senescence, in comparison with lymphocytes, fi broblasts and postmitotic cells and tissues (brain) In addition, the same facts will have an impact on the competitiveness from subjects of different ages, including centenarians; of European industry, in particular the involved SMEs and research • establish targets for age-associated posttranslational modifi cations institutes in the sense that a successful project will help them to mark of proteins along with a molecular characterization of the protein their position on the international scene, as a prerequisite for future modifi cation. For selected cases, it is anticipated to clarify the role business development and exploitation plans. of the modifi cation for age-associated functional changes; • defi ne the role of extracellular components in controlling signalling We also expect some direct commercial exploitation of work described in through the IGF and TGFβ signalling pathways and their changes PROTEOMAGE. On the one hand, the SMEs Amynon Phyto Research and during ageing; Senetek will try to identify compounds from plant extracts that stimulate • study intracellular and extracellular chaperones, as well as other proteasome activity and will be helpful for preventing or delaying age- protein maintenance (i.e. repair and degradation) systems, as mod- associated dysfunctions of the skin and vascular system, respectively. The ulators of cellular ageing processes; innovation that is pursued by the company WOW will result in a prototype • better understand age-associated decrease in proteasome activity in laboratory for continous normal oxygen cell culture conditions, which will evolution. New procedures to activate the proteasome in vitro and in have a large market potential for all institutes working on ageing research vivo will give information about its potential to delay cellular ageing; worldwide and can be extended since cell culture facilities with defi ned • better understand protein networks that are involved in lifespan oxygen will be useful for many other disciplines of biomedical research and control in various model organisms and model systems. technology, for example organ transplantation.

HUMAN DEVELOPMENT AND AGEING | 177 TGFβ LAP Coordinator David Gems HSP LTBP University College London IGF/IGFBP/ALS Brian F. C. Clark London, United Kingdom University of Aarhus MMP2 Aarhus, Denmark Peter Csermely signalosome [email protected] Semmelweis Egyetem HSP Budapest, Hungary

Partners Thomas Nyström Goteborgs Universitet Efstathios S. Gonos Goteborgs, Sweden National Hellenic Research Foundation Athens, Greece Claudio Franceschi

20S proteasome CIG – University of Bologna Pidder Jansen-Duerr Bologna, Italy clusterin, extracellular chaperones Oesterreichische Akademie der Wissenschaften Manuel Serrano Vienna, Austria Fundación Centro Nacional de Investigaciones oncológic cas Carlos III Olivier Toussaint Mardid, Spain Facultés Universitaires Notre-Dame de la paix de Namur Alan Fersht Namur, Belgium University of Cambridge Cambridge, United Kingdom Bertrand Friguet University of Paris 7/Denis Diderot Rong Zeng Paris, France Shanghai Institutes for Biological Sciences Karin Scharffetter-Kochanek Shanghai, China Universitaet Ulm Medizinische Fakultaet Ulm, Germany Joel Demarteau WOW Company s.a. Jennifer Rivett Naninne, Belgium University of Bristol Bristol, United Kingdom Suresh Rattan SENETEK Aps Heinz D. Osiewacz Aarhus, Denmark Johann Wolfgang Goethe University Frankfurt am Main, Germany Sylvia Dürr Amynon Phyto Research GmbH Peter Roepstorff Innsbruck, Austria University of Southern Denmark Odense M, Denmark

Project number LSHM-CT-2005-518230 EC contribution € 10 700 000 Project Duration 60 months Starting date 01/03/2006 Instrument IP Project website www.proteomage.dk

178 | HUMAN DEVELOPMENT AND AGEING RespViruses

Immune response to viral respiratory infections and vaccination in the elderly

Keywords New respiratory viruses, immune response of the Potential applications elderly, mouse models, diagnosis, siRNA, EGS New diagnostic assays; new antiviral therapies; optimized treatment and vaccination strategies; new software tool for surveillance and clinical data mining.

Summary Coordinator Catherine Manoha University Hospital Dijon During the last years some new respiratory viruses (HMPV/hCoV-NL63/ Oliver Schildgen CHU Dijon SARS/Bocavirus/fl u H5N1) have emerged. In addition to other viruses, Institute for Medical Microbiology, Dijon, France such as RSV, EBV, and Paramyxoviruses, they are able to induce severe Immunology, and Parasitology respiratory diseases in high-risk patients, in particular young children Department of Virology Matthias Hamann and elderly. University of Bonn Softwareentwicklung Sigmund-Freud-Strasse 25 Nidderau, Germany D-53105 Bonn Germany Brian Sproat RNA-Tec Partners Leuven, Belgium

Maria Grazia Beatriz Lazaro Cusi University of Siena INGENASA UNISI MC Madrid, Spain Problem Siena, Italy Michael Kleines We will study the innate and acquired immune response of the elderly Lia van der Hoek University Hospital Aachen against the new and known respiratory viruses. Clinical and basic Academic Medical Aachen, Germany research aspects will be addressed equally, and new diagnostic assays to Center Amsterdam Amsterdam, The Netherlands evaluate the immune status of the elderly will be developed. Within the project a mouse model for the investigation of the elderly’s immune response will be established. In this model antiviral agents will be tested for their ability to support the patient’s immune response. Up to 40 000 elderly sera collected during the last 9 years will be tested for antibodies to emerging respiratory viruses (HCoV-NL63/HMPV/RSV/EBV/fl uH5N1), providing a wide view of the epidemiology of these viruses. For a precise view on the elderly’s immune response to the viruses, a defi ned and uniform panel of relevant clinical data related to respiratory infections will be collected. Internationally standardized prospective data mining and a new multi-lingual software tool will be developed within the project and used by the partners. Project number LSHM-CT-2006-037276 EC contribution Expected results € 1 770 361 Project Duration We expect to gain detailed knowledge on the innate and acquired 36 months immune response to emerging respiratory viruses in the elderly. A thor- Starting date ough basic investigation of emerging respiratory viruses will provide 01/01/2007 further handles to develop antiviral strategies based on siRNA and exter- Instrument nal guide sequences (EGSs). Furthermore, known antivirals will be STREP – SME evaluated with the fi nal goal to support the elderly’s immune response Project website whilst reducing mortality in the elderly caused by respiratory infections. www.lifecompetence.eu/ index.php/kb_370/io_622/io.html

HUMAN DEVELOPMENT AND AGEING | 179 SENECA

From Cellular Senescence and Cell Death to Cancer and Ageing

Keywords Cancer, Ageing, Cellular Senescence, Cell Death, Problem DNA Damage, Telomerase, Immunosurveillance Age is the most important demographic risk factor for many life- threatening human cancers. Over two-thirds of all diagnosed cancers occur in people over the age of 65. According to IARC, worldwide cancer rates increase sharply with age: for the age group 65 years and older incidents of cancer are 2-3 times higher than in the 45-64 age group and 12-36 times higher than in the 25-44 age group. The increased incidence of cancer in elderly people has been related to age- Summary associated changes occurring with time in the whole system. The elderly are more vulnerable than younger individuals to environmental The main objective of the SENECA project is to improve the awareness of carcinogens not only because of potentially greater exposure time, but ageing research among cancer researchers, stimulating cooperation for other reasons, including impaired macromolecular repair and defense between the two disciplines. To achieve this objective a conference will against reactive oxygen species, age-dependent changes in tissue be organized to provide a forum for scientifi c exchange among leading environment facilitating increased pro-inﬂ ammatory status, possibly scientists working in the fi elds of ageing and cancer, as well as to diminished immuno-surveillance of malignant transformation and pro- stimulate cooperation aimed at redefining molecular targets and cancerogenic activity of senescent cells. improving cancer prevention and therapeutics in the ageing population. The discussion will focus on such issues as DNA damage, telomeres and Since tumours include cancer cells with an extensive proliferative history, telomerase in cancer and ageing, effects of tissue environment in tumour subject to senescence and senescence-avoidance mechanisms, cancer formation, impact of the ageing immune system on cancer researchers commonly study various aspects of biological ageing. immunosurveillance and immunotherapy, links between stem cells and However, many cancer specialists, clinicians, and industry representatives cancer and ageing, links between tumour suppression and cellular remain unaware of what ageing research can offer for cancer prevention senescence, and cellular senescence as a new target in anticancer and therapy. Presently the research ﬁ elds of biological ageing and cancer therapy. Attendance of approx. 300 scientists and representatives of in Europe remain largely fragmented, without structured links or other key stakeholder groups from around the world is anticipated. widespread interdisciplinary approaches.

Aim

The main goal of the project is to improve the awareness of ageing research among cancer researchers, stimulating cooperative research between the two disciplines. The enhanced cooperation should aim at redeﬁ ning molecular targets and improving cancer prevention and therapeutics in the ageing population. This goal will be achieved by organ- izing an international conference for approximately 150 participants October 4-6, 2007.

180 | HUMAN DEVELOPMENT AND AGEING Expected results Coordinator Partners

The conference will provide a forum for scientiﬁ c exchange among Prof. Ewa Sikora Prof. Graham Pawelec outstanding European scientists working in the ﬁ elds of ageing and Nencki Institute of University of Tübingen Experimental Biology Tübingen, Germany cancer. The discussion will focus around such issues as: DNA damage, Polish Academy of Sciences telomeres and telomerase in cancer and ageing, effects of tissue Warsaw, Poland Prof. Vladimir Anisimov environment in tumour formation, impact of the ageing immune system [email protected] N.N. Petrov Research on cancer immunosurveillance and immunotherapy, links between stem Institute of Oncology cells and cancer and ageing, links between tumour suppression and St. Petersburg cellular senescence, and cellular senescence as a new target in anticancer Russian Federation therapy. The conference will also bring together other key stakeholder groups such as policy makers, clinicians and industry.

The proposed event will contribute to attracting scientists from cancer research and other disciplines to ageing research. It will help to establish sustainable organizational links between these two closely related scientiﬁ c ﬁ elds, structuring European research in oncogerontology.

Potential applications

The planned conference is expected to: • stimulate cancer specialists to use the insights, results and methodologies stemming from ageing research in studying aetiology, prevention and therapy of cancer, especially in the elderly. • design strategies as to how basic research results in cancer and ageing can be best transferred into industrial exploitation and clinical practice. This may be an important starting point for the development of new preventive and therapeutic measures. It may give a whole new dimension to geriatric medicine, as it will shift the emphasis from care – which is extremely expensive – to maintenance of function and prevention of disease as long as possible.

Project number LSSM-CT-2006-037312 EC contribution € 142 800 Project Duration 24 months Starting date 01/10/2006 Instrument STREP – SME Project website www.SENECA2007.eu

183 ➞ 265

CANCER Anti-tumour targeting

Modulation of the Recruitment of the Vessels and Immune Cells by Malignant Tumours: Targeting of Tumour Vessels and Triggering of Anti-Tumour Defence Mechanisms

Keywords Tumour-host interactions, tumour targeting Problem

Despite significant improvements in diagnosis, surgical techniques, general patient care, and local and systemic adjuvant therapies, many solid tumours remain a major cause of death. Among the most prevalent and fatal forms are carcinomas of the breast, colon and prostate. Most Summary deaths from cancer are due to metastases, seeded from the primary tumour via the blood and lymph vasculature, which are resistant to All malignant tumours acquire the capacity for efficient recruitment of blood conventional therapies. The main barrier to the non-surgical treatment vessels, which are absolutely necessary for tumour growth beyond a certain of the primary neoplasm and its metastases is the genetic instability and size. They also frequently stimulate lymphangiogenesis supporting biological heterogeneity of cancer cells leading to the rapid development dissemination of tumour cells, not only via the blood vasculature but also via and growth of resistant cells. Since the expansion of solid tumours and the lymphatic system, leading to metastasis. Vessel growth is promoted by their metastases beyond a minimal size is absolutely dependent on the sprouting angiogenesis and the homing of bone marrow progenitor cells into formation of new blood vessels, anti-angiogenesis therapies constitute the tumours and tumour vessels. The extensive vascularisation facilitates the a promising alternative. In this case the genetically normal endothelial invasion of cells of the innate and adaptive immune system, which stay largely cells of the tumour vessels are targeted, avoiding the problem of functionally suppressed by the tumour environment, and even contribute to resistance development. Furthermore, immune therapies are based on angiogenesis and tumour growth by cytokine and growth factor secretion. the ability of the immune system, evolved over evolutionary times, to We propose in this application to: cope with an almost unlimited number of antigens, thus opening the • further investigate key regulatory pathways by which tumour-secreted possibility to find a mechanism to target any tumour variant as long as molecules promote vascularisation and inhibit immune cell function; the inhibitory milieu of the tumour environment can be overcome. • develop methods to inhibit tumour growth and metastasis by blocking Therefore angiogenesis and immune therapies remain among the most vessel and tumour cell growth; promising fields of cancer therapy. • achieve tumour clearance by additionally promoting activation and homing of functional immune cells to the tumours. The project will comprise the collaboration of laboratories with complementary Aim expertise. It will includeexperts inbloodvesselandlymph vessel angiogenesis, metastasis formation, progenitor cell incorporation into tumours and tumour The general aim of the project is to design and evaluate strategies of vessels, anti-tumour defence mechanisms of the immune system and viral anti-tumour angiogenesis and anti-tumour immune therapies and their transduction techniques. The final goal will be the preclinical evaluation of combination in murine models of some of the most prevalent forms of strategies in murine models of three of the most prevalent forms of human human solid tumours. It will include the identification, modification and cancer, i.e. carcinomas of the breast, colon and prostate. use of key regulatory molecules of vessel growth and immune defence The strategies to target the tumour will be based on gene, cell and immune and the development of methods to specifically and efficiently target therapy methods. They will include the use of : tumours and their metastasis. • adenoviruses for the expression of angiogenesis inhibitors following targeted delivery of the viruses to the tumour vasculature; • the genetic modification of murine embryonic and human umbilical Expected results cord/bone marrow progenitor cells and their directed homing into the tumour; We will undertake a concentrated effort to identify targets and develop • the use of genetically-engineered immune cell products or the methods for interference with vessel growth. Furthermore, we will transduction of immune cells to activate targeting of the tumours by develop techniques to target tumour endothelium by viruses and pro- innate and adaptive anti-tumour defence mechanisms. We expect that genitor cells,methodsthat could be usedto also reachdistant metastases this project will contribute to innovation on three levels. Firstly, we will via the blood stream and not only the primary tumour. In addition, we gain basic additional novel knowledge on important pathways and will explore the use of innate receptors of NK cells to detect malignant regulatory molecules for the recruitment of host cells to the tumours cells and to boost the T-cell response of the immune system with the and their functional interaction with the tumour. Secondly, we will use help of dendritic cells. Weanticipate thatasingle method,depending on this knowledge to test novel ways of targeting viruses and (transduced) the individual tumour, may not be sufficient, but the development of cells to the tumours. Finally, we will evaluate whether, by a combination several techniques based on different principles and their tumour- of anti-angiogenesis therapy with directed anti-tumour immunotherapy, specific or combined application may be successful. it would be possible not only to inhibit tumour growth, but also to eradicate residual disease.

184 | CANCER For this purpose we combine laboratories with differential expertise, each Coordinator Hidde Haisma having either identified a specific target molecule or developed a specific University of Groningen technique for targeting tumours.We will combine the expertise, molecules Erhard Hofer Groningen, The Netherlands and techniques and comparatively evaluate different strategies in Dept of Vascular Biology and Thrombosis Research Antonis Hatzopoulos corresponding models of human carcinomas. Centre for Biomolecular Medicine and Institute for Clinical Molecular Biology Pharmacology & Tumour Genetics GSF Medical University Vienna Munich, Germany Potential applications Vienna, Austria [email protected] Ofer Mandelboim The results of this project will be disseminated and exploited on three Hebrew University-Hadassah levels: Medical School Jerusalem • basic molecular medicine research level: all expected findings with Partners Jerusalem, Israel the angiogenesis inhibitors and immune stimulators will be important to improve understanding of the role of vessel formation and Seyedhossein Aharinejad Alexander H. Enk University of Vienna Karsten Mahnke of anti-tumour immune responses for cancer. These basic findings Vienna, Austria University of Heidelberg will be published in quality scientific journals; Heidelberg, Germany • clinical level: it is anticipated that our findings and developed Michael Detmar preclinical methods will have impact on the design and further Institute of Pharmaceutical Sciences Melvyn Little development of clinical protocols for the treatment of cancer; Swiss Federal Institute of Technology Affimed Therapeutics AG Heidelberg • company level: key novel molecules, findings and techniques will Zurich, Switzerland Heidelberg, Germany be patented and, together with patents available, used to develop reagents and protocol for gene-therapy of solid tumours. Karl-Heinz Preisegger EccoCell Biotechnology and Stem Cell Therapy GmbH Graz, Austria

Project number LSHC-CT-2005-518178 EC contribution € 3 005 000 Project Duration 36 months Starting date 01/11/2005 Instrument STREP

CANCER | 185 Apotherapy

CD40 ligand-based modalities for the treatment of cancer

Keywords Cancer, gene therapy, signalling, apoptosis, CD40, Problem phosphoinositide 3-kinase, metastasis Cancer has a major health, social and fi nancial impact on Europe and its people. Current treatments include primary tumour resection and/or aggressive chemotherapy and radiotherapy in order to achieve both local control and effective therapy for distant metastases. Despite improved therapeutic regimens, mortality rates are still high. Moreover, the frequency of cancer incidence is predicted to increase and, as Summary a result, its social and economic toll may reach even higher levels in the next decades. The growing cancer burden in Europe underscores the Cancer is a major disease according to the WHO Mortality Data base need to develop more effi cient anti-tumour agents with a view to (2004) and is responsible for approximately 25 % of all deaths in the increasing survival and improving the quality of life of cancer sufferers. European Union. Epithelial tumours, such as those of the ovary, lung and oesophagus, have particularly poor prognosis, with only a minority of patients achieving a fi ve-year survival. Conventional treatments, including Aim chemotherapy and radiotherapy, have limited effi cacy and frequently cause severe side-effects in patients. Apotherapy aims to combat cancer through an innovative combination The Apotherapy project brings together expertise from academic and strategy which targets cancer cells at multiple levels. One arm of this biotechnology sectors across seven European countries with the aim of strategy is to encourage carcinoma cell apoptosis and immune developing and validating novel anti-cancer agents with a wide therapeutic recognition through the optimal activation of the CD40 pathway and index and minimal side-effects. The focal point of our research is the the other is to suppress tumour cell survival mediated by the PI3 kinase utilisation of combined approaches which attack the tumour cell at multiple signaling pathway. This strategy has been designed to achieve maximal levels, achieving maximal apoptosis while limiting the risk of drug inhibition of tumour growth while limiting the risk of side-effects. resistance. These approaches involve the efficient delivery of a pro- apoptotic molecule to cancer cells in combination with inhibitors of anti- apoptotic signal transduction pathways. Expected results Specifi cally, the project will formulate therapeutic strategies which exploit the ability of CD40 ligand (CD40L), a TNF family member, to reduce proliferation, • The Apotherapy project will provide new information about promote apoptosis and activate anti-tumour immune responses selectively in targeting the CD40 and PI3 kinase pathways in solid tumours. cancer cells. Apotherapy will develop state-of-the-art vehicles for the effi cient • The project will develop and evaluate viral and non-viral vectors for the delivery of CD40L to cancer cells, such as CD40L-encapsulated liposome effi cient and tumour-specifi c delivery of anti-cancer agents in vivo. formulations and recombinant adenoviruses expressing CD40L, and examine • Apotherapy will characterise novel antagonists and inhibitors of their in vitro and in vivo effects on tumour cell growth and metastasis. the anti-apoptotic PI3 kinase signalling pathway. Apoptosis induced by CD40 engagement is dramatically augmented in the • An extensive pre-clinical evaluation of CD40L delivery systems in presence of inhibitors of the phosphoinositide 3-kinase (PI3 kinase) pathway, combination with PI3 kinase pathway antagonists will be performed, which is frequently found activated in human tumours. The Apotherapy aided by in vivo imaging technology. project will expand on the development of novel PI3 kinase antagonists and evaluate their in vitro and in vivo capacity to kill tumour cells and to amplify the CD40L-mediated effects on carcinoma cell growth, angiogenesis and Potential applications metastasis. The Apotherapy project will lead to the development of novel anti- cancer strategies which will be directly applicable to the clinic for the benefi t of cancer sufferers. The strong focus on translational research will translate R&D results into tangible benefi ts for health, science and economy in Europe.

186 | CANCER Coordinator Akseli Hemminki University of Helsinki Aristides Eliopoulos Helsinki, Finland University of Crete Medical School Heraklion,Greece Jiri Ehrmann [email protected] Univerzita Palackeho v Olomouci Olomouc, Czech Republic

Partners Angelica Loskog Uppsala University Marco Falasca Uppsala, Sweden University College London London, United Kingdom Steffen Panzner Novosom AG Massimo Broggini Halle, Germany Istituto Mario Negri Milano, Italy

Green-ﬂ uorescence protein- expressing lung tumours in mice are visualised by in vivo imaging. This technology is used to monitor tumour growth following treatment with recombinant adenoviruses.

Project number LSHC-CT 2006-037344 EC contribution € 1 895 900 Project Duration 36 months Starting date 01/10/2006 Instrument STREP Project website apotherapy.med.uoc.gr

CANCER | 187 BAMOD

Breath-Gas Analysis for Molecular- Oriented Detection of Minimal Diseases

Keywords Exhaled breath analysis, lung cancer, oesophageal Problem cancer, medical diagnosis, therapeutic monitoring, gas chromatography, mass spectrometry, PTR-MS, SIFT-MS, Cancer is one of the leading causes of death in the western world. More laser spectrometry, ion mobility spectrometry than 940 000 people died of lung cancer in Europe in the year 2000. These diseases have a tremendous impact on healthcare systems and economics. At present, the diagnosis of cancer often happens late in the course of the disease; early diagnosis would improve prognosis and treatment and could save thousands of lives.

Summary Aim

Cancer is one of the leading causes of death in Europe and the western The objectives of the project are: world. At present, diagnosis of cancer occurs late in the course of the • the identification of sensitive and specific molecular marker sets in disease since available diagnostic methods are not sufficiently sensitive human breath for the detection of early cancer stages; and specific. An early diagnosis of cancer would improve prognosis and • the development of reliable analytical methods to detect these treatment, and could save thousands of lives a year. markers in the clinical environment; There is strong evidence to suggest that particulate cancers can be detected • the production of easy-to-use and non-expensive equipment by a molecular analysis of exhaled air. Breath analysis represents a new to facilitate breath analysis as a novel cancer screening tool. diagnostic technique that is without risk for the patient, even if repeated frequently, and can provide information beyond conventional analysis of The proposal is centred on five studies: in vivo lung cancer and oesopha- blood and urine. Recent results suggest that detection of different kinds of geal cancer studies, a study of cancer cell lines in vitro, a study of cancer is possible by means of breath analysis in the very early stages of immune-system related cells and a study of bacterial cell lines. the disease. This project is focused on the diagnosis of minimal disease and early The SMEs in our consortium will develop analytical methodology for stages of lung and oesophageal cancer. The analytical techniques will be subsequent use in clinical applications. gas chromatography with mass spectrometric detection (GC-MS), proton transfer reaction mass spectrometry (PTR-MS), selected ion flow tube mass spectrometry (SIFT-MS), laser spectrometry and ion mobility Expected results spectrometry (IMS). In order to establish a reliable clinical method for the diagnosis of minimal The major objective is the establishment of a non-invasive, breathbased residual cancer diseases, clinical expertise, basic research and technical test for early detection of lung and oesophageal cancer. The development development is necessary. The European consortium set up to pursue this of sensitive detection technology, a database of molecular markers research work involves specialists with skills in the fields of basic and related to cancer, and the development of specific sensors for such clinical research, and analytical instrument development. Thus, the markers is anticipated. consortium has the expertise to investigate and screen exhaled breath for many molecular species, to identify specific cancer markers and the statistical tools to treat the data. Potential applications

The developed breath-based tests for the detection of lung and oesophageal cancer should be introduced to assist clinical diagnosis and therapeutic monitoring.

188 | CANCER Detection of lung and oesophageal cancer based on exhaled breath.

Coordinator Manfred Mürtz Universtitätsklinikum Düsseldorf Anton Amann Düsseldorf, Germany Innsbruck Medical University Dept of Anaesthesia and Boguslaw Buszewski General Intensive Care Nicolaus Copernicus University Innsbruck, Austria Torun, Poland [email protected] Ireneusz Sliwka Polish Academy of Sciences Partners Krakow, Poland

Jochen Schubert Viktor Witkovsky Universität Rostock Slovak Academy of Sciences Rostock, Germany Bratislava, Slovakia

Rosa Margesin Kerstin Wex Leopold-Franzens- IT Dr. Gambert GmbH Universtität Innsbruck Wismar, Germany Innsbruck, Austria Armin Hansel Karl Unterkofl er IONIMED Analytik GmbH Vorarlberg University Innsbruck, Austria of Applied Sciences Dorbirn, Austria David Smith Trans Spectra Limited Nandor Marczin Keele, United Kingdom Imperial College London London, United Kingdom Jörg Ingo Baumbach Institute for Analytical Sciences Dortmund, Germany

Project number LSHC-CT-2005-019031 EC contribution € 2 998 228 Project Duration 36 months Starting date 01/02/2006 Instrument STREP Project website www.eu-proposal.voc-research.at

CANCER | 189 CancerGrid

Grid-aided computer system for rapid anti-cancer drug design

Keywords Bioinformatics, pharmacology, grid technology, seems most of the targets can be classiﬁ ed into large target families library design, in-silico prediction of drug-like properties, such as kinases and GPCRs: thus, development of target focused libraries prediction of ADME parameters, predictive toxicology, could dramatically increase the hit rate as well as open the way to creation of virtual libraries identifying selective inhibitors/antagonists within the target families.

The idea of ‘focused libraries’ or ‘targeted libraries’ of molecules emerged in recent years as a ‘compromise’, or as an attempt to bridge between two seemingly confl icting approaches to drug discovery: • High Throughput Screening (HTS), by which hundreds of thousands Summary of compounds, mainly in big pharma, were tested against a (hopefully validated) biological target such as a protein or a cellular system. In the three years of this multidisciplinary research project, the 10-member The basic assumption of HTS is that large numbers and diversity SME-led Consortium plans to develop and refi ne methods for the enrichment should cover chemical space well enough to fi nd, at least, ‘hits’ (that of molecular libraries to facilitate discovery of potential anti-cancer agents. are active in micromolar concentrations) which may subsequently be Using grid-aided computer technology, the likelihood of fi nding anti-cancer transformed to ‘leads’ (with affi nities in the nanomolar range and novel leads will substantially increase the translation of basic knowledge to with reasonable drug-like properties) and fi nally to drug candidates. application stage. Combinatorial chemistry has also been on the side of HTS, present- In particular, through the interaction with novel technologies and biology, ing the ability to synthesise huge amounts of derivatives based on the R&D consortium aims at: specifi c ‘scaffolds’. • developing focused libraries with a high content of anti-cancer leads; • Rational drug design approaches such as structure-based design • building models for prediction of disease-related cytotoxicity and of and ligand-based design. The fi rst takes into consideration the kinase/HDAC/MMP and other enzyme (i.e. HSP90) inhibition or detailed atomic structure of the target and the possibilities for receptor antagonism using HTS results; forming physical interactions (i.e., hydrogen bonds, Van der Waals • developing a computer system based on grid technology, which helps interactions, electrostatic complementarity, hydrophobicity, etc.) to accelerate and automate the in silico design of libraries for drug between small molecules and specifi c sites on the targets, while discovery processes, and which is also suitable for future design of the second depends more on properties of known active molecules libraries for drug discovery processes that have different biological and uses similarity ideas (including ‘pharmacophore’ searches) to targets (the result is a new marketable technology). discover new actives. The substantial reduction in discovering new chemical entities by big pharma in recent years has been in part attributed to the failures due to very low hit discovery in both the HTS and Combichem, on the one hand, and on the inability to properly account for pharmacokinetic (ADME/TOX) effects as well as for entropy, solvation and target fl exibility in structure- and ligand-based designs. Problem A landmark in introducing pharmacokinetic considerations to drug After the completion of the sequencing stage of the human genome design and development has been the ‘Rule of 5’ of Lipinski. This idea, project, the major focus of discovery efforts turned to the identifi cation which is now less than a decade old, also provided an immediate tool to of the druggable portion of the genome that is linked to pathological reduce the size of combinatorial libraries and of HTS candidates by states and is able to interact with the drug-like chemical space, restoring ‘fi ltering’, i.e., requiring that all molecules must pass the Lipinski rule normal functions. (three out of four conditions for the limiting of molecular weight, calculated lipophilicity, and the numbers of H-bond donors and Apparently, the druggable genome is a subset of the 30 000 genes in the acceptors) in order to be in the proper bioavailability range. human genome that express proteins and represent, in many ways, an unprecedented gift and exceptional opportunity for drug discovery scientists The molecules that passed the Lipinski fi lter were thus targeted on and for patients who are hoping for therapies of diseases currently uncured. bioavailability, and their numbers were much smaller than those for the That subset (estimated as ca. 3 000 proteins) is able to bind drug-like initially planned experiments. The idea of ‘fi lters’ thus gained momentum, molecules as characterised by the Lipinki’s rule-of-5 criteria. and additional fi lters such as those of Veber (limiting the number of rotatable bonds and the size of polar surface area), also for bioavilability, In order to fi nd more rapidly small molecule modulators to the newly were suggested. Both Lipinski and Veber rules did not consider directly emerging validated targets, the high-throughput screening provides any conformational aspects (3-dimensional descriptors of the molecules a reasonable solution to screen large compound libraries. However, it to be tested, but pharmacophore searches (ligand-based design) and

190 | CANCER virtual docking and scoring (structure based design) serve as subsequent fi ltering processes in 3D that cover the ‘affi nity’ part of drug action, while the other fi lters mostly deal with ‘drug transport’ issues.

These two properties are, to a large extend, orthogonal. Thus, one may regard the ﬁ ltering process as beginning with huge numbers of molecules, which a few descriptors serve to reduce to a smaller set which may then be studied with more detailed conformations at the pharmacophore level, reducing it further to a group of molecules which may be docked virtually to the assumed target, ﬁ nally leaving a small set of substantially ‘focused’ or ‘targeted’ lead candidates. The main ‘focusing’ activity has been however concentrating on molecular scaffolds that are useful for probing families of targets such as GPCRs, tyrosine kinases, MMPs etc.

But, even that approach suffers from many drawbacks. Lipinski and Veber rules can not distinguish well between drugs and non-drugs, and are clearly not appropriate indicators of ‘drug-likeness’. Neural networks have been applied speciﬁ cally to this problem and managed to distinguish properly between drugs and non-drugs, but have the disadvantage of ‘hidden layers’ which do not enable to plan and design novel molecules.

A drug-like index has been suggested but is based on fragment identifi cation and therefore limited in its ability to discover novel structures. Structure-based approaches can consider small molecule fl exibility, but are still inappropriate for dealing with the fl exibility of the protein targets, especially with the fl exibility of backbone and of larger loops. The scorings in docking methods have recently been exposed to much criticism. Using single conformations in pharmacophore searches is clearly inappropriate, because it has been shown that small molecules bind to proteins in conformations that are higher in energy than their global minima. Toxicity predictions have not yet reached enough reliability to prevent major toxicity threats by drugs. The need for selectivity has not yet been properly addressed in the preparation of focused libraries.

Therefore, although many companies nowadays are offering focused libraries for kinases, GPCRs and other families of molecules, there is a great need to improve the production of such libraries in order to short- en the time for discovery and to save enormous expense. A main stumbling block on the way to solving such issues is the complex combinatorial nature of the problem of library construction and drug design.

In this proposal, we include methods that deal directly with the combinatorial nature of the problems, that have been shown to solve combinatorial problems in a highly satisfactory manner, that discover the global minimum in most cases and retain a large set of best results, many of them excellent alternatives to the global minimum.

CANCER | 191 Aim Coordinator Balazs Borsi GKI Economic Research Co • Developing focused libraries with a high content of anti-cancer leads. István Bágyi, PhD Budapest, Hungary • Building models for prediction of disease-related cytotoxicity and IPR and Grant Coordinator AMRI Hungary Peter Kacsuk of kinase/HDAC/MMP and other enzyme inhibition or receptor Budapest, Hungary Computer and Automation antagonism, using the results of screenings in cell-based and Phone: +36 1 6666 100 Research Institute speciﬁ c target based experiments. Direct: +36 1 6666 142 Hungarian Academy of Sciences • Developing a computer system based on grid technology: Fax: +36 1 6666 110 Budapest, Hungary – which helps to accelerate and automate the in silico [email protected] design of libraries for drug discovery processes; Amiram Goldblum – which is also suitable for future design of libraries for University of Jerusalem drug-discovery processes that have different biological Partners Jerusalem, Israel targets (the result is a new marketable technology). Thierry Langer Saverio Minucci Inte:Ligand DAC Expected results Maria Enzersdorf, Austria Milano, Italy Mati Karelson Angelo Carotti Novelties and added values of the IT part of the project: Tallinn University of Technology University of Bari • virtual focused libraries of anti-cancer agents; Tallin, Estonia Bari, Italy • potential anti-cancer agents; • HTS technology; Mart Saarma Ferran Sanz • data for model building purposes; University of Helsinki University Pompeu Fabra • models able to predict anti-cancer properties; Helsinki, Finland Barcelona, Spain • CancerGrid: a grid-based system able to provide anti-cancer candidates faster and in a more efﬁ cient way, also suitable to develop candidates for other targets.

Potential applications

The models developed within the framework of this project can be used for fi ltering large discovery libraries to fi nd anti-cancer drug candidates, and to design anti-cancer focused libraries. The CancerGrid™ computer system will be able to support the design of lead compounds in general, not only in the anti-cancer fi eld, but in any other activity area. Thanks to its grid-based architecture, the system will be able to predict molecular descriptors for compound libraries, containing a large number of molecular structures, in a short time. When calculating 3D molecular descriptors, the system will take all major conformers into account. This enables the calculation of information-rich molecular descriptors, and the development of reliable linear and non-linear models. The system Project number will also be able to apply these models to predict the biological activity LSHC-CT-2006-037559 or chemical/physical property of the compounds. EC contribution € 2 800 000 Project Duration 36 months Starting date 01/01/2007 Instrument STREP – SME Project website www.cancergrid.eu

192 | CANCER CANCERIMMUNOTHERAPY

Cancer immunology and immunotherapy

Keywords Therapeutic vaccine, immunomonitoring, DC, CTL, Aim CD4,Treg, melanoma, tumour escape, Ag processing The ultimate objective of this Integrated Project is to develop a therapeutic cancer vaccine with defined tumour antigens that would provide a clinical benefit in at least 40 % of patients. This threshold of 40 % of vaccinated patients showing an objective tumour response, in the absence of unacceptable toxicity, would definitely qualify immunothera- Summary py as a standard cancer treatment. Further improvements could come from refining the vaccinations, and from combining tumour vaccines The first part of the project consists of clinical trials of vaccination, to compare with other modalities of cancer treatment. various vaccines, such as peptides and RNA, with different types of immunological adjuvants and dendritic cells. Safety and clinical efficacy will be the primary endpoints of these trials. A large effort will be devoted to Expected results monitoring the anti-vaccine T-cell responses, as examining the correlation between immunological and clinical responses to the vaccines will be crucial We believe that the principal objective of our project is reachable, for to understanding which factor(s) limit tumour regression. A second part of the following reasons: the project,tightly connected to the clinical trials as it uses biological material • the preliminary observation that vaccination with tumour antigens from the vaccinated patients, consists of optimising tumour vaccines and can be associated with tumour regressions, and in a few cases with combating immune evasion. Foreseeable mechanisms of tumour escape will sustained remissions, is encouraging, as it indicates that the vaccines be analysed and correlated with the clinical results. Improved modalities of tested so far have an anti-tumoural activity. Considering that vac- vaccination will be tested and new target antigens will be identified. All these cine-induced immune responses and tumour regressions seem to be results will help to design improved vaccines. Finally, considering the correlated, and that the immune responses that have been detected complexity of mechanisms that may lead to or prevent tumour regression in so far appear to be quantitatively weak, it is reasonable to hypothe- vaccinated patients, we propose to explore more fundamental aspects of the sise that vaccines with a greater immunogenicity, such as those we anti-tumour immune response. This includes the cross-presentation of tumour plan to investigate, will also have a greater clinical efficacy; antigens by dendritic cells, recruitment of cells of the innate immune system, • our project will build a close interaction between the research involvement of suppressor T-cells, and development of murine models of laboratory and the clinic, which allows new ideas emerging from inducible tumours. If new concepts emerge from this work, they will also help observations made in either of these two fields to be integrated in the design of better vaccines. rapidly into new projects; • our consortium comprises groups with an excellent record in clinical trials, T-cell immunology, dendritic cell biology, and mechanisms of tumour resistance. Many of these groups have a longstanding experience of collaborative programmes with each other, both in the laboratory and the clinical trial fields.

Problem Potential applications

Cancer is a major life-threatening disease and the second greatest cause Development and validation of surrogate end-points is a high priority in of mortality in Europe after cardio-vascular diseases. Classical cancer cancer vaccine research. The project will contribute to this goal by pro- treatment still relies on surgery, chemotherapy and radiotherapy. Despite moting standardised assays and methods for the immunomonitoring of clear progress in some cancer types, cancer therapy in general often fails clinical trials. In practice, validation of these surrogate markers may prove to prevent disease progression to metastatic disease. In addition, these extremely useful for meaningful comparisons of various immunisation approaches are by themselves very toxic, imposing a heavy burden of modalities and as markers of consistency for a given product. side effects on the patient. There is clearly a need for new therapeutic approaches that would be more efﬁcient and less toxic.

CANCER | 193 Coordinator Cornelis Melief Leiden University Medical Center Thierry Boon Leiden, The Netherlands Christian de Duve Institute of Cellular Pathology Giorgio Parmiani Brussels, Belgium Istituto Nazionale per lo Studio [email protected] e la Cura dei Tumori Milan, Italy

Partners Gerold Schuler Friedrich Alexander Universität Hans-Georg Rammensee Erlangen-Nürnberg Eberhard-Karls-Universität Tübingen Erlangen, Germany Tübingen, Germany Thomas Wölfel Sebastian Amigorena Johannes Gutenberg-Universität Mainz Institut Curie Mainz, Germany Paris, France Eric Tartour Marc Bonneville Université René Descartes Cancer immunotherapy with defi ned tumor antigens: Institut Nationale de la Santé Paris, France antigens that have been identifi ed as specifi cally expressed by the tumor cells are used in therapeutic vaccination trials to stimulate anti-tumor et de la Recherche Médicale lymphocytes. Patients are monitored for signs of tumor regression and Paris, France Muriel Moser for the induction of anti-tumor immune responses. Université Libre de Bruxelles Jean-Charles Cerottini Brussels, Belgium Ludwig Institute for Cancer Research Epalinges, Switzerland Kris Thielemans Vrije Universiteit Brussel Vincenzo Cerundolo Brussels, Belgium University of Oxford Oxford, United Kingdom Hélène Sicard Innate Pharma SA Alexander Eggermont Marseille, France Erasmus MC Rotterdam Rotterdam, The Netherlands Catia Traversari Molecular Medicine Carl Figdor Milan, Italy Stichting Katholieke Universiteit Nijmegen, The Netherlands Brigitte Dreno Centre Hôspitalier Federico Garrido Universitaire de Nantes Fundacion Virgen de las Nieves Nantes, France Granada, Spain Steve Pascolo Project number Günter Hämmerling CureVac the RNA people LSHC-CT-2006-518234 Deutsches Krebsforschungszentrum Tübingen, Germany EC contribution Heidelberg, Germany € 12 185 102 Vincenzo Russo Project Duration Ulrich Keilholz Fondazione San Raffaele Charité Universitatsmedizin Berlin del Monte Tabor 48 months Berlin, Germany Milan, Italy Starting date 01/03/2006 Instrument IP Project website www.cancerimmunotherapy.eu

194 | CANCER CAPPELLA

Combating cancer through novel approaches to protein-protein interaction inhibitor libraries

Keywords Identifi cation of novel compounds, natural Problem products, inhibition of protein-protein interactions, high throughput screening Most protein-protein interactions occur within the cell and thus can only be targeted by small molecules. Furthermore, PPI differ structurally from more classic drug targets such as enzymes and receptors, and consequently existing compounds have generally delivered disappointing results. Therefore, new approaches are needed to develop novel small molecules which inhibit PPI in cancer. Summary Aim The inhibition of protein-protein interactions (PPI) is one of the most promising approaches to the development of novel cancer therapies. This The objective of this project is to develop a series of innovative small- project brings together some of Europe’s leading biotech SMEs and several ligand tools and libraries that allow new approaches to the inhibition of highly recognised academic institutes. By combining fi ve distinct chemical protein-protein interactions in cancer. A key theme is the utilisation of approaches and testing them on three different targets (all from different structural motifs found in natural PPI-inhibitor compounds. This is cou- partners) a series of innovative small-ligand tools and libraries that allow pled with high content testing of the resultant structures on three distinct new approaches to the inhibition of PPI in cancer will be developed. The PPI targets relevant to different types of cancer, to allow compound rule- project is a unique opportunity to integrate novel in silico, chemical, genetic sets to be developed and improved. We want to develop small-ligand and ADME-based approaches to the design, synthesis and optimisation of libraries focused on PPI inhibitors of relevance to cancer. Furthermore, we libraries and compounds. will develop innovative tools that allow improved library design in this area by integrating in silico approaches, bio-informatics, new approaches to compound synthesis and pharmacology. The project will also cover the scientiﬁ c areas such as in silico prediction of drug-like properties, prediction of ADME parameters, predictive toxicology and creation of virtual libraries.

Expected results

Innovative tools for designing PPI inhibitors • Five different PPI-inhibitor library creation tools, based on ﬁ ve complementary approaches: – in silico, – genetic chemistry, – advanced natural product technologies, – retro-synthesis of natural scaffolds, – ADME improvement. • Cross-fertilisation of approaches so that each of the ﬁ ve approaches learns lessons from the others and incorporates relevant leanings into its approach. • Three high-content assay systems for three important PPI cancer targets (p53-Mdm2, Beta catenin-TCF4, BRCA2-RAD51). • Design rules for PPI inhibitor compound libraries (mass, diversity composition, lipophilicity, compound class etc.) generated from 15 complementary data sets.

Novel small-ligand libraries and pre-clinical candidates • Several ‘PPI inhibitor’ compound libraries. • Different candidate compound families from within these libraries that can subsequently be taken forward into pre-clinical testing by the SME partners.

CANCER | 195 Libraries containing extracts of biological material offer great chemical diversity.

Coordinator Prof. Dr. Birger Lindberg Møller Royal Veterinary Dr. Melyha Hughes & Agricultural University Evolva SA Copenhagen, Denmark Basel, Switzerland [email protected] Dr. Carmen Cuevas Pharmamar Sociedad Anonima Sociedad Unipersonal Partners Madrid, Spain

Dr. Hajo Schiewe Prof. Dr. Ashok Venkitaraman AnalytiCon Discovery GmbH University of Cambridge Potsdam, Germany Cambridge, United Kingdom

Dr. Lise Madsen Prof. Dr. Ariel Ruiz i Altaba BioLigands ApS University of Geneva Odense, Denmark Geneva, Switzerland

Prof. Thierry Langer Prof. Dr. Juhan Sedman Inte:Ligand University of Tartu Software-Entwicklungs- Tartu, Estonia und Consulting GmbH Vienna, Austria

Project number LSHC-CT-2006-037251 EC contribution € 3 361 300 Project Duration 36 months Starting date 01/01/2007 Instrument STREP – SME Project website www.cappellabio.eu

196 | CANCER CHEMORES

Molecular mechanisms underlying chemotherapy resistance, therapeutic escape, effi cacy and toxicity

Keywords Cancer chemotherapy, drug resistance, toxicity, Problem lung cancer, melanoma Resistance to systemic chemotherapy still remains one of the greatest problems in clinical oncology, and contributes to the death of a large number of cancer patients. Despite extensive efforts, no signifi cant progress has been made in solving this fundamental problem during the last decades. Not only have effi cient means to overcome chemotherapy resistance not been identifi ed, but also the development of clinically useful tools Summary to predict response to chemotherapy has been largely unsuccessful.

The CHEMORES project aims to improve the outcome of cancer chemotherapy The different aspects of the chemoresistance problem are well illustrated by developing novel tools to predict tumour response to treatment as well as in the two model tumours that will be studied. The two main subtypes of individual toxicity to chemotherapy. The project will thus seek to identify and lung cancer are small cell lung cancer (SCLC) which is highly chemosensitive validate mechanisms of intrinsic and acquired chemotherapy resistance, as with response rates of 80 % to chemotherapy; and non-small cell lung well as predictors of effi cacy and of individual toxicity. cancer (NSCLC) that is moderately chemosensitive with response rates of This is achieved by integrating the work of groups conducting large clinical 30-60 %. For all stages and types of lung cancer (NSCLC/SCLC), relapse trials with preclinical research groups, as well as with state-of-the-art after primary therapy is common, at which stage most patients have platforms for genomic and proteomic analyses and bioinformatics. The developed an acquired resistance to chemotherapy and seldom respond participants have chosen to focus on melanoma and lung cancer as model to second line treatment. In lung cancer, a therapeutic plateau has thus tumours of separate histogenetic types exhibiting intrinsic resistance and/ been reached with existing cytotoxic drugs and further improvements in or a high degree of acquired resistance to chemotherapy. survival are dependent on our understanding of the molecular mechanisms Candidate mechanisms of drug resistance and therapeutic effi cacy will be of chemoresistance. identified using genomic and proteomic analyses of sequential tumour samples and paired sera obtained before and after chemotherapy, as well as There is no effective systemic therapy for metastatic melanoma and only experimental systems, including in vitro studies of tumuor cell lines, a small minority of patients with melanoma respond to chemotherapy, transplanted human tumours and novel animal tumour models. These putative which also has no discernible impact upon median overall survival. In mechanisms will then be validated in further analyses of larger sets of tumour the adjuvant setting interferon-alfa (IFN) is the only agent that has biopsies from patients. Functional studies of novel mechanisms and pathways demonstrated a consistent effect on relapse-free survival, but without will be also performed using in vitro systems and animal models. The result a signiﬁ cant impact on overall survival. We have as yet no tools to of these activities will thus be a set of clinically and functionally validated identify the patient population that beneﬁ ts from treatment with IFN. mechanisms of chemotherapy resistance and therapeutic effi cacy. Thus, improved knowledge regarding mechanisms of resistance is Likewise, large-scale genomic analyses of patients receiving chemotherapy needed in order both to develop predictive tests and to modulate as part of clinical trials will be performed, in order to validate novel markers resistance and thus improve the therapeutic outcome in melanoma. of individual toxicity following chemotherapy. Cancer chemotherapy is frequently associated with severe toxic side- effects. Novel tools to identify individuals with an increased risk of developing severe side-effects are required in order to avoid such adverse events.

Aim

The overall aim of CHEMORES is to improve the outcome of cancer chemotherapy by developing novel tools to predict tumour response to treatment as well as individual toxicity to chemotherapy.

CANCER | 197 Expected results Coordinator Curt Peterson Linköping University The novel knowledge obtained through the project will lead to new Johan Hansson Linköping, Sweden tools for prediction of treatment outcome as well as toxicity of Karolinska Institutet Stockholm, Sweden Joakim Lundeberg chemotherapy. This knowledge may also be used to identify and prepare [email protected] Kungliga Tekniska Högskolan for pre-clinical development of potential novel modulators of drug Stockholm, Sweden resistance based on validated mechanisms and pathways. The new information obtained in CHEMORES will be disseminated to the medical Partners Joost van den Oord profession and other key stakeholders, such as health care providers, Katholieke Universiteit Leuven patient organisations and policy-makers. Alan Spatz Leuven, Belgium Institut Gustave Roussy Villejuif, France Stefano Fais Potential applications Istituto Superiore di Sanità Guido Kroemer Roma, Italy Institut National de la Santé Predictive tests of response to chemotherapy may be used in medical et de la Recherche Médicale Manfred Schmitt oncology to select patients with an increased likelihood to beneﬁ t from Paris, France Technical University of Munich therapy. The novel information on molecular mechanisms responsible München, Germany for resistance to chemotherapy may lead to development of modulators Mariano Barbacid of resistance. Both these advances may in turn lead to improved results Fundación Centro Nacional de Mark Middleton of cancer chemotherapy. The development of tools to predict severe Investigaciones Oncológicas Carlos III University of Oxford drug toxicity will lead to safer cancer chemotherapy. Madrid, Spain Oxford, United Kingdom

Alexander Eggermont Geoff Margison Erasmus Medical Centre Rotterdam University of Manchester Rotterdam, The Netherlands Manchester, United Kingdom

Marco Pierotti Anna Eidefors Istituto Nazionale per lo Studio Magnetic Biosolutions AB e la Cura dei Tumori Stockholm, Sweden Milano, Italy Marie-Claire Beckers Piet Borst Eurogentec SA Netherlands Cancer Institute Seraing, Belgium Amsterdam, The Netherlands Alexandre Passioukov European Organization for Research and Treatment of Cancer Brussels, Belgium

Project number LSHC-CT-2006-037665 EC contribution € 8 710 300 Project Duration 60 months Starting date 01/02/2007 Instrument IP Project website www.chemores.org

198 | CANCER CHILDHOPE

Chimaeric T cells for the treatment of paediatric cancers

Keywords Children, cancer, leukaemia, T lymphocytes, Problem chimaeric T-cell receptor Intensiﬁ cation of post remission therapy and addition of autologous or allogeneic haematopoietic stem cell transplantation (HSCT) have marginally improved outcome in children with relapsed leukaemia or lymphoma. Failure to induce prolonged remissions in a large portion of relapsed children, and the toxicity of current regimens in this age category with unique physiological parameters, has led to a resurgence Summary of interest in immunotherapies.

Leukaemias are the most common cancers affecting children while malignant Administration of tumour-specifi c T cells (adoptive immunotherapy) has lymphomas, including non-Hodgkin lymphomas (NHL), come in third position proven to be an effective cancer treatment: allogeneic HSCT and donor- after brain tumours. A significant number of children with leukaemia/ leukocyte infusion (DLI) can induce lasting remissions in patients, including lymphomas still fail current therapies. children, with a range of malignancies; and Epstein-Barr virus (EBV)-specifi c The aim of the CHILDHOPE project is to develop a safe and effi cient adoptive T cells can cure patients with post-transplant lymphoproliferative disorder immunotherapy for children with advanced or refractory malignancies. (PTLD). However, considerable barriers hinder wider application of this CHILDHOPE particularly focuses on three paediatric tumours: acute B-lineage promising form of immunotherapy. It is diffi cult and often impossible to lymphoblastic leukaemia, non-Hodgkin B-lineage lymphoma and acute select and expand tumour-specifi c T cells or, in an allogeneic setting, to myeloid leukaemia. separate T cells causing graft-versus-host disease (GVHD) from tumour- The CHILDHOPE project is a new approach in paediatric cancer treatment reactive ones. Further, tumour cells often lack infl ammatory cues and the since it brings from bench to bedside (and back) an innovative technology as appropriate environment to maintain a persistent immunological rejection. yet never applied in children with advanced or refractory haematopoietic Absence of a specifi c means of destroying adoptively transferred T cells in malignancies. the face of unacceptable toxicity has also stalled clinical studies. The CHILDHOPE translational research project will focus on: • improving and testing the effi cacy and the safety of anti-leukaemia/ Many of these barriers can be now overcome by ex vivo genetic engineering lymphoma chimaeric T cells in relevant preclinical models in vitro and in of T cells using gene-transfer technology, thanks to pioneering work vivo in mice; performed by us and others in the last decade. Initially cumbersome and • scaling-up this technology to numbers suitable for a clinical application impractical, this technology has matured to the point of effective clinical in children with haematopoietic malignancies; application. However little is known about the homing and function of the • based on biological material obtained from our preclinical models and different subsets of tumour-specifi c T cells once re-injected in patients. from children treated with these genetically engineered T cells, dissecting Dissecting the killing mechanisms of these tumour-specifi c T cells – especially the interface between the host’s tumour and immune cells and using this once they have been administered in vivo in children – and elucidating some knowledge to understand the mechanisms of anti-tumour action, of the reasons for their failure in certain patients will ultimately improve our validate novel targets and diagnostic tools specifi c to children affected knowledge of the host-tumour interface and help design enhanced with leukaemia or lymphomas. strategies using engineered T cells against paediatric and adult cancers. The CHILDHOPE project is built on the excellence of a network of EU-based partners with a broad experience in the fi eld of paediatric haematology and oncology, immunology and cell and gene therapies, and integrates the Aim International Confederation of parents of children with cancer and an SME specialised in project management. The CHILDHOPE project builds on the excellence of a network of EU- based partners with broad experience in the fi eld of paediatric haematology and oncology, immunology and cell and gene therapies. The CHILDHOPE project is unique since it brings from bench to bedside (and back) an innovative technology as yet never applied in children with advanced or refractory haematopoietic malignancies. The CHILDHOPE translational research project will focus on: • improving and testing effi cacy and the safety of anti-leukaemia/ lymphoma chimaeric T cells in relevant preclinical models in vitro and in vivo in mice; • scaling-up this technology to numbers suitable for a clinical application in children with haematopoietic malignancies;

CANCER | 199 • based on biological material obtained from our preclinical models In parallel, the CHILDHOPE project will provide preclinical data comparing and from children treated with these genetically engineered T cells, the cytotoxic potency of different T-cell subsets (EBV-CTLs, Cytokine dissecting the interface between the host’s tumour and immune Induced Killer T cells and chimaeric g9d2 T cells) in an effort to identify cells and using this knowledge to understand the mechanisms of the best immune effector for future clinical application. anti-tumour action, validate novel targets and diagnostic tools specifi c to children affected with leukaemias or lymphomas. Potential applications The fi nal aim is to develop a safe and effi cient adoptive immunotherapy for children with advanced or refractory malignancies. CHILDHOPE No animal model can fully dissect the tremendous complexity of the particularly focuses on three paediatric tumours: acute B-lineage interactions between a human tumour and its host. The CHILDHOPE lymphoblastic leukaemia, non-Hodgkin B-lineage lymphoma and acute project is the fi rst comprehensive attempt at administrating anti-tumour myeloid leukaemias. chimaeric T cells in children with haematopoietic malignancies. It represents a unique opportunity to address in vivo in a complete human environment some of the most fundamental hypothesis in the fi eld of Expected results anti-tumour immunology. Our hypothesis is that targeting CD19 or CD33 will not only kill target cells but will also contribute to the release The originality of the CHILDHOPE project is to exploit the immunostimu- of yet unknown tumour-associated antigens (TAAs), which in turn may latory properties of EBV-CTLs and retarget them to leukaemia/lymphoma generate further immune activation. This mechanism known as antigen cells, which themselves lack many of the costimulatory molecules needed spreading may be of particular interest in tumours known for their pro- to activate CTLs. Thus, our underlying general hypothesis is that pensity to induce antigen-loss variants. As these new TAAs may be expression of chimaeric antileukaemia/lymphoma receptors on EBV- either leukaemia/lymphoma-associated or have a broader scope of specifi c CTLs will allow these cells to retain their known safety and paediatric tumours, they may provide new targets for future immuno- functionality in vivo (ability to expand and regress in response to antigen therapeutic approaches. load, to persist as memory cells, and to retain antiviral activity) in children with haematopoietic malignancies while adding safety, specifi city While the CHILDHOPE project focuses at this stage on three of the most and effector function directed to the residual leukaemia cells. frequent paediatric tumours (acute B-lineage lymphoblastic leukaemias, non-Hodgkin B-lineage lymphoma and acute myeloid leukaemias), our This strategy seems highly feasible since EBV-specifi c CTL lines have been innovative technology should pave the way for the generation of generated and reinfused in numerous patients using robust and clinically T lymphocytes with an antibody-dictated specifi city toward other validated methodologies developed by Brenner, one of our key scientifi c tumour-associated antigen for which a monoclonal antibody exists. This advisors in the CHILDHOPE project. Unmodifi ed CD19 molecules have in turn should allow us to redirect immune effectors towards other been manufactured and incorporated into EBV-CTLs, and tested in vitro. malignancies, including solid tumours, for which current therapeutic Our pioneering project now proposes to extend this approach against strategies are limited or have failed. This includes – but is not limited to CD33+ malignancies, which represent a signifi cant part of hard-to-treat – metastatic disease where T-cell therapies have had some success due paediatric leukaemias. Another major innovation is that we will investigate to their capacity to migrate and infi ltrate distant tumour sites. modifi cations to the chimaeric receptor molecule itself, in efforts to augment its capacity to enhance the transduction of signals that increase In fact, it is likely that only a combination of therapies that act at key cytotoxic and memory effector function, while maintaining a high-level of points of tumour escape pathways may help to eradicate tumour cells safety. The T-cell proliferation should be strictly antigen-dependent and and also to lessen the dose-intensity of current chemotherapy regimen, dwindle after elimination of the antigen. a critical element of any therapeutic approach in developing individuals or in elderly patients who have reached maximum tolerable doses of Finally, the CHILDHOPE project moves the fi eld forward as it brings to anticancer drugs when their disease reoccur. Hence, our innovative clinic this therapeutic modality in a phase I setting. We are confi dent therapeutic approach may provide hope for a cure for young as well as that the chosen methods of gene transfer (i.e. retroviral vs. electropora- older patients with advanced or refractory tumours. tion), in conjunction with our suicide gene system, will provide a safe albeit suffi ciently prolonged antileukaemia effect in vivo in children with relapsed/refractory leukaemia/lymphoma. We anticipate that the antileukaemia/lymphoma effects – and consequently the survival of the treated patients – will increase with the chimaeric T-cell doses.

200 | CANCER Coordinator Gustav Gaudernack The Norwegian Radium Hospital Raphaël F. Rousseau Oslo, Norway MD, PhD Oncologie Pédiatrique Joël Plumas INSERM U590 Etablissement Français Centre Léon Bérard du Sang Lyon, France Grenoble, France Assistant: +33 478 782 642 Phone: +33 478 785 933 Martin Pule Mobile: +33 682 662 979 University College of London Fax: +33 478 782 703 London, United Kingdom [email protected] Claudia Rossig Universitätsklinik Münster Partners Münster, Germany Chimaeric TCR shown in context of native TCR signalling complex. Single-chain variable region (scFv) is composed of heavy and light chain variable domains (VH and VL) which are connected by a short peptide linker. A ﬂ exible spacer connects Christophe Caux Marianne C. Naafs-wilstra VL allowing the antigen binding region to orient in different directions, improving Centre Léon Bérard International Confederation antigen recognition. The spacer is then connected to the transmembrane and Lyon, France of Childhood Cancer intracellular portion of CD3-z. The native TCR a and b chains are also shown Parent Organisation alongside CD4/CD8. An unmodiﬁ ed CD3-z chain and CD3-e and CD3-g chains are Persis Amrolia Nieuwegein, The Netherlands also represented. Antigen binding to the native TCR leads to phosphorylation of CD3 immunoreceptor tyrosine-based activation motif (ITAMS, red boxes) which activate Great Ormond Street ZAP70, which in turn propagates further downstream signalling. Co-stimulation Children’s Hospital Costa Starvelakis (in this case via CD28) is required for full T-cell activation. London, United Kingdom ACIES Lyon, France Ettore Biagi Università Milano Bicocca Milano, Italy

Project number LSHC-CT-2006-037381 EC contribution € 3 208 760 Project Duration 48 months Starting date 01/11/2006 Instrument STREP Project website www.childhope.eu

CANCER | 201 CONTICANET

Connective Tissue Cancers’ Network to integrate the European Experience in Adults and Children

Keywords Oncology, anti-cancer therapy, clinical pharmacology, Problem uncommon cancer, sarcomas, GIST, children, adults Scientifi c challenges • Connective tissue tumours: a collection of rare tumours. • Molecular characterisation is routine for few tumours, in progress for other tumours but unknown for most tumours. • There is a need for harmonisation of research activities in the following ﬁ elds: Summary – molecular characterisation of these tumours, – functional genomics. Connective tissue cancers, and more specifically sarcomas, GIST, aggressive • Pharmacology: target characterisation and validation. fibromatosis and hamartomas, are uncommon cancers with an incidence • Promotion of clinical and translational research. below 2/100 000 per year in the EU affecting children, young adults and • Improvement of medical practices. adults. At a national level,the limited number of cases (about 7/10 000 in • Education of the physicians for CPGs. Europe each year) does not allow Europe to have a critical mass of researchers • Epidemiology and medical economics. and the supporting environment to progress in the disease understanding and management and to have access to new drugs and treatments. Europe, Medical challenges however, has proven with research made on GIST, through the EORTC-STBSG • Demonstrated ‘proof of concept’ for several connective group, that gathering workforces can achieve significant progress. CONTICANET tissue subtypes. will create the critical mass of key stakeholders to overcome the current • Targeted oncogene treatments are active treatments. difficulties in terms of lack of data and data fragmentation, mobility of • Harmonisation of research activities, medical practices and education researchers, heterogeneity of methodologies and legislations. in a context where: CONTICANET will: – patients are not always treated in specialised centres, • start by providing the required environment for developing joint research – most patients are not treated according to CPGs, activities through the development of standard operating procedures – structured clinical research and TR in large centres (SOP), distributed databases, tissue banks and harmonisation of ongoing and network. and previous research projects, to promote complementarity and perform • Medical practices do not follow clinical practice guidelines for more parallel research programmes according to similar SOPs; than 60 % of patients in most western countries so there is a need • provide the infrastructure to test several drug candidates and perform for education of physician and patients. European wide exploratory clinical trials while implementing a federated research policy; Economical challenges • further lead to an integrated sustainable structure – a European research Health: foundation – able to support integrated research actions and make new Inappropriate management results in increased costs. treatments available. Survival and functional disabilities in adults and children. Involving 20 partners – major comprehensive cancer centres, academia and Identiﬁ cation of novel targets and treatment: private companies – over a period of five years, CONTICANET will gather the Generate intellectual propriety. critical mass of resources and knowledge in the understanding, diagnosis and Commercialisation of new agents. clinical management of connective tissue cancers, at the same time opening Transfer towards industrial partners: new therapeutic options. Perform rapid proof of concept clinical trials. CONTICANET will spread excellence in several directions: enlarging the Generate synergy between European academia and industry. network with other academic and private organisations; developing working sessions with EMEA, health authorities and insurances in order to optimise Political challenges the availability of new therapeutic options; supporting and collaborating with Strengthen and harmonise European research in these rare tumours: patients through advocacy groups and cancer leagues. Establish the network as the worldwide leading force for research in these rare tumours. Promote academic and industrial collaborations: Generate a network with integrated joint research programmes. Include new partners from academia, industry and patients advocacy groups. Ensure the long term viability of the network.

202 | CANCER Aim and expected results • inform, get feedback from, collaborate with and support patients advocacy groups (SOS-Desmoides, Jeunes Solidarité Cancer, Life Raft) CONTICANET aims at setting up a Network of Excellence as a vehicle to • interact with EMEA, health authorities, social security and insurances create the critical mass of researchers, clinicians and industrialists able to: • involve other pharmaceutical, biotech and academic centres in the • improve the understanding of carcinogenesis and tumour progression EU to enlarge the capacity of the network of connective tissue cancers in adults and children and specifi cally of • reinforce collaborations outside Europe, in particular with Canada sarcomas, GIST, aggressive fi bromatosis and hamartomas; (McGill University), Russia (NCI Moscow), Israel (Weizman Inst.), • develop new diagnostic tools, prevention strategies and treatments USA (MSKCC) and Australia (Peter MacCallum Cancer Institute) for these connective tissue cancers in adults and children. Finally CONTICANET will generate three different types of results: • An organisation gathering key stakeholders in connective tissue The main goals of CONTICANET are to: cancer research and able to perform signifi cant progress in the • create the critical mass of key stakeholders from translational, disease understanding and management through jointly performed pharmacological and clinical research to make a real breakthrough studies in the fi elds of epidemiology, molecular biology research, in connective tissue cancer diagnosis and management, both for clinical research, drug development. This organisation will take the adults and children; form of an international non-profi t association gathering all • capitalise on pre-existing collaborations to set up, implement and network participants. This association will then in a second step update a joint research programme and then a European-wide evolves towards a research foundation open to new members and organisation covering pre-clinical and clinical research on connective to sponsors. By involving pharmaceutical companies and with the tissue cancers with a patient to patient approach; objective of extending the approach to other uncommon cancers, • make available to all participants a strong network of facilities such a foundation will be self-sustained through the contributions accelerating research output. of its members and the support to be obtained form the industry through the development of orphan disease models. From this integration, CONTICANET will provide a basis for the development • The delineation of methods, tools and activities which will be of new treatments and, from an industrial point of view, to: developed within the network and supporting the integration of • establish synergisms between data and models acquired by aca- the research activities of the participants and which will be demics and drug development from the pharmaceutical industry to shared among participants and ultimately beyond the network to improve therapeutic options for both adults and children; strengthen research in this fi eld: • validate some of those options with competent authorities as – distributed standardised repository of tissues, data, a model for orphan disease. information supporting research activities; – standardised Operating Procedures (SOP) at European level; CONTICANET will spread the excellence achieved in the network to: – common pool of facilities: instruments, platforms, drugs, • improve training of health professionals in charge of diagnosis diagnostic tools, etc.; and multidisciplinary primary care of the European patients – collaborative platform for data transfer and validation and through the production of guidelines and reference for medical collaborative work; practice. In particular, strong relationships already exist – and will – network for pharmacodynamics based clinical trials; be reinforced – between the consortium and: – standardised validated screening tools (ex vivo & in vivo); – the European Society of Medical Oncology – ESMO (all) – pharmacovigilance network. – the EORTC (ERASM, UCBL, IGR, INT, ICR, MAUNIHEI) • Public health research coupled with education aiming to understand – the European Society for Surgical Oncology – ESSO the determinants of medical practice in order to propose innovative (INT, UCBL, IGR, ICR, MAUNIHEI) education strategies to improve patient management and follow – the European Society for Therapeutic Radiation Oncology – up. Knowledge gained in this fi eld will be disseminated within the ESTRO (UCBL, SLS, IGR, UNIPD, ICR) network and beyond by the key opinion leaders present in the – the European Musculo Skeletal Oncology Society – EMSOS network in their respective countries. (INT, IGR, SLS) – the European paediatric Soft tissue Sarcoma Group – EpSSG Once these goals are achieved for sarcomas, GIST, aggressive fi bromatosis (UNIPD, ICR, IGR, CURIE, BERGONIE) and hamartomas, the consortium will choose other types of uncommon – but also with international societies: the world wide connective tissue tumours (primary CNS tumours, mesotheliomas) and Connective Tissue Oncology Society – CTOS (UCBL, IGR, ultimately aim at contributing to integrated research in the fi eld of uncom- ICR, ERASM, INT, MAUNIHEI) mon epithelial cancers (neuroendocrine cancers, adenoid cystic carcinomas). – and European organisations such as orphanet Europe, research Thus the CONTICANET network is expected to develop on the long term directorate by contributing to the European effort on rare a stable structured European research organisation on uncommon cancers diseases (www.cordis.lu/lifescihealth/major/rare-diseases.htm) and could possibly serve as a model for other rare diseases.

CANCER | 203 Coordinator Javier Olaiz Jean-Pierre Bizzari EZUS Sanofi -Aventis General Affairs Jean-Yves Blay Villeurbanne, France Valorisation & Innovation Université Claude Bernard Lyon 1 Bagneux, France Villeurbanne, France Ian Judson [email protected] The Institute of Cancer Research Michel Marty [email protected] London, United Kingdom Centre Hospitalier Universitaire Saint-Louis Axel Lecesne Paris, France Partners Institut Gustave Roussy Villejuif, France Jean-Yves Blay Eric Papon Université Claude Bernard Lyon Arttic Nadia Zaffaroni Lyon, France Paris, France Istituto Nazionale per lo Studio e la Cura dei Tumouri Carlo Ricardo Rossi Gerald O’Sullivan Milano, Italy Università degli Studi di Padova National University of Ireland Padova, Italy University College Cork Fabienne Hermitte Cork, Ireland Ipsogen SAS Patrick Schöffski Marseille, France Katholieke Universiteit Leuven Jaap Verweij Leuven, Belgium Erasmus University Medical Centre Christian Auclair Rotterdam, The Netherlands Laboratory for Biotechnology J-M. Coindre & Applied Pharmacology Institut Bergonié Cachan, France Bordeaux, France

Zvonimir Rudolph Olivier Delattre Institute of Oncology Ljubljana Institut Curie Ljubljana, Slovenia Paris, France

Arcadio Garcia de Castro Peter Hohenberger PharmaMar SA Universitätsklinikum Mannheim Madrid, Spain Mannheim, Germany

Project number LSHC CT-2005-018806 EC contribution € 9 432 600 Project Duration 60 months Starting date 01/02/2006 Instrument NoE

204 | CANCER Keywords xxxxxxxxxx DASIM

Diagnostic Applications of Synchrotron Infrared Microspectroscopy

Keywords Clinical diagnosis, cancer, infrared spectroscopy, Expected results infrared microscopy, infrared imaging, synchrotron radiation • Signiﬁ cant acceleration of the rate of progress in the ﬁ eld by coordinating the research effort. • Dissemination of knowledge through meetings, courses, website and publications, informing clinicians in particular. • Reliable assessment of the potential of this technique as a guide for future EU science funding policy. Summary

DASIM is a Specifi c Support Action to coordinate research effort of all Potential applications Europe’s synchrotron light sources in the fi eld of infrared microspectroscopy of pathological samples as an aid to clinical diagnosis. Clinical diagnosis, particularly typing, staging and grading of tumours.

Coordinator Michel Manfait Université de Reims David Moss Reims, France Forschungszentrum Karlsruhe Problem Karlsruhe, Germany Marco Colombatti [email protected] Università di Verona Verona, Italy Diagnosis of disease is the basis for all clinical medicine. The primary requirement is reliability of diagnosis in order to ensure that therapies are Partners Sheila Fisher appropriate and successful. However, the modern requirements of clinicians University of Leeds from diagnostic services go beyond a simple ‘yes’ or ‘no’ to the presence of Augusto Marcelli Leeds, United Kingdom a particular indication. Successful therapy requires information on disease INFN – LNF subtype classiﬁ cation, assessment of the disease stage and extent such as Frascati, Italy the grading of tumours, as well as the monitoring of disease progress and therapeutic success. The speed of pathological analysis can also be amongst the requirements arising as a result of a time-limited therapeutic window beyond which therapy may be less or no longer effective. Such constraints are particularly apparent in the treatment of cancer and infectious diseases. Post-mortem diagnosis is also an aspect to be included, since there remain some diseases that can only be unequivocally diagnosed post mortem, and in these cases the retrospective diagnosis plays a central role in improving therapies through the accumulation of clinical experience.

Aim Project number In the last ten years there has been considerable progress in the application LSHC-CT-2005-005326 of infrared microspectroscopy to the analysis of human tissues in the context EC contribution of disease diagnosis, and it has been convincingly demonstrated in many € 280 000 studies that infrared spectroscopy has the potential to contribute signiﬁ cantly Project Duration to this ﬁ eld. The aim of DASIM is to coordinate this research effort by 36 months networking the existing centres of excellence across Europe, by providing Starting date a forum for the necessary multidisciplinary exchange of expertise between 01/07/2005 clinicians, spectroscopists, biologists and physicists on the European scale, Instrument and by facilitating access to synchrotron facilities for scientists and clinicians SSA in countries that do not have their own synchrotron facility. Project website www.dasim.eu

CANCER | 205 DEPPICT

Designing Therapeutic Protein-Protein Inhibitors for Brain Cancer Treatments

Keywords Protein-Protein interactions, small molecule Problem inhibitors, knowledge driven drug design, blood/brain, tumour penetrant Amongst the range of cancer types, brain and perhaps pancreatic cancers are especially lacking in effective treatments. In particular brain tumours are: • the leading cause of death from childhood cancers among persons under 19; • the second leading cause of cancer-related deaths in males aged 20-39; Summary • the fi fth leading cause of cancer-related deaths in women aged 20-39. Protein-Protein interactions (PPI) are central elements in cellular Although onset of disease varies with tumour type, it can occur at processes and important targets for selective therapeutic agents. They a relatively young age causing additional and signifi cant social and constitute a rich area for discovery of novel small ligand-based therapies. economic problems for both patients and their families. This proposal seeks to utilise such interactions, in particular those featuring a a-helix binding groove such as p53-MDM2, or more novel Current standard treatments include surgery, radiation therapy and targets, e.g. nm23-prune, to develop targeted small-molecule libraries chemotherapy. These may be used either individually or typically in with physico-chemical properties appropriate for therapeutic effect combination. Brain cancers however present unique problems due to against various tumour types such as the brain cancers of glioblastoma the location of the tumours: surgery and radiotherapy carry considerable and medulloblastoma. risk to the patient and resection is not always possible. Chemotherapy is Combination of the concepts below should provide an opportunity to faced with the problem of penetration of drugs across the blood-brain unlock the potential of protein interactions as key components in barrier (BBB) and of lack of specifi city. The focus for these patients is signalling pathways via design of selective small-molecule modulators therefore on more effective therapies to prevent relapse, and on more targeting the kinase-effector interaction instead of the ATP active site. effi cient screening and diagnosis to halt the disease at an early stage. • Develop an understanding of the elements controlling selectivity in Protein-Protein signalling networks by developing approaches for design of small molecules that target a-helix binding groove Aim interactions through use of structure-based and fragment-based approaches. The main objective of the proposed project is to provide more effective • Data-mining of ADME and drug-drug interactions to build a predictive anti-tumour therapies by developing targeted small ligand libraries with database for library design. appropriate physico-chemical properties for therapeutic effect targeted • Develop quantitative structure/property relationships, with an against Protein-Protein interactions implicated in various tumour types. emphasis on CYP-mediated metabolism, ABC transporters at the The research activities will concentrate on knowledge-based approaches blood/brain and brain/tumour interfaces, mutagenicity, solubility, supporting translational research aimed at bringing basic knowledge pKa, and passive permeability, and predictive tools for mutagenicity through to applications in clinical practice and public health. The project and other genetic toxicology end-points. will thus focus on providing small molecule ligands with minimal side • Develop predictive PK and PBPK models to improve understanding effects as treatments for the brain tumours glioblastoma (GBM) and of BBB and tumour penetration. medulloblastoma. • In vitro and in vivo PK/PD and TK/TD characterisation of compounds, to increase understanding of their mechanism of action and reduce the use of laboratory animals. Expected results Such knowledge-based approaches will also be applicable to design of small molecules for other Protein-Protein interactions utilising a a-helix The successful integration of the various aspects of this proposal will binding groove both for peripheral tumours and other therapeutic areas. provide a robust knowledge-based strategy for exploiting Protein- Protein interactions as drug targets in the treatment of brain tumours. The strategy should however be suffi ciently generic to be transferable to other disease areas, especially within the CNS, where Protein-Protein interactions provide an entry point into disease-modifying therapies.

206 | CANCER Iterative Research Cycles where data will be used to construct robust testable hypotheses in the context of the pathophysiology of brain tumours. To achieve this it is necessary to look for trends – Structure Activity Relationships. The discovery and exploitation of site / mechanism of action and developing knowledge at multiple levels – Target, Cell, and Pathway will be key to achieving this.

Potential applications Coordinator Dr. Juan Aymami Crystax The design of selective small molecule modulators of Protein-Protein Dr. Graeme Robertson Barcelona, Spain interactions should provide the basis for developing new therapeutic Siena Biotech SpA Siena, Italy Prof. Roberto Pellicciari strategies against brain cancers. They will allow for improving current [email protected] University of Perugia libraries and for building predictive databases for library design of new Perugia, Italy speciﬁ c drugs and therapeutic agents with high central nervous system penetration, necessary to reach a higher efﬁ cacy, selectivity, responsiveness Partners Dr. Sophie Ollivier and lower toxicity. In addition these approaches will represent a powerful Aureus Pharma system for preclinical data collection, leading to an optimisation of Prof. Gabriele Cruciani Paris, France clinical trials setting and development. Molecular Discovery Pinner Middlesex, United Kingdom Prof. Herbie Newell University of Newcastle Prof. Massimo Valoti Newcastle, United Kingdom University of Siena Siena, Italy

Structure Driven Drug Design Predictive ADME and Drug-Drug Interactions Quantitative Structure/Property Relationships

Predictive PK & PBPK Models

In vitro and in vivo PK/PD Protein-Protein Interaction and TK/TD Characterisation Inhibitors

The individual components needed to achieve successful knowledge driven drug discovery will be combined to harness the opportunity for Protein-Protein inhibitors as potential small molecule therapies for brain tumours.

Project number LSHC-CT-2007-037834 EC contribution € 3 640 293 Project Duration 36 months Starting date 01/03/2007 Instrument STREP – SME Project website www.deppict.eu

CANCER | 207 DISMAL

Molecular Signatures as Diagnostic and Therapeutic Targets for Disseminated Epithelial Malignancies

Keywords Tumour cell dissemination, micrometastases, Problem minimal disease, biobanking, genomic profi ling, microarray analysis, circulating nucleic acids, bioinformatical synopsis Investigations during the last decades have shown that the metastatic cascade is a complex pathobiological process and highly dependent on the type of tumour. Some tumours, such as breast cancers, have a proclivity for parallel dissemination to the haematogenous and lymphatic compartment, starting in the earliest phases of tumour development. In other tumour types, such as in head and neck cancer, this pattern is very different and haematogeneous dissemination appears Summary to result from metastases in the lymphatic compartment. For these reasons we selected three tumour types: one as an extreme example of Disseminated tumour cells (DTC) occur at very low numbers in the blood parallel, independent dissemination (breast cancer), one as an extreme and particularly in the bone marrow, and can be detected using sensitive example of sequential dissemination (head and neck cancer), and one immunostaining and PCR methods. However, the sensitivity and specifi city intermediate type (colorectal cancer). of this approach need to be improved. Combining the expertise of 11 academic partners with long-term expertise in micrometastasis research, the DISMAL project will establish an improved platform for DTC Aim detection with an increased sensitivity and, in particular, with a greatly improved specifi city. Besides DTC detection, the combination with analysis The main objective of the DISMAL-project is to improve the specifi city and of circulating tumour DNA and RNA as well as the expression profi ling of sensitivity of current platforms for DTC detection in patients with epithelial tumours are being explored to improve the assessment of minimal disease. tumours, the predominant form of cancer in Europe, whilst also identifying We will focus on epithelial tumours, as they are the most common types of novel markers at the DNA, RNA or protein level that allows a more precise solid tumours in the EU, investigating the carcinoma types that display detection of DTC with a high risk for metastatic progression. different modes of metastatic spread. Dissemination via the blood circulation will be analysed using bone marrow as this is the most important The programme will focus on markers associated with haematogenous indicator organ epithelial tumour cells home in on. Using genomic-based dissemination (using bone marrow as the most well defi ned indicator approaches, novel diagnostic target molecules will be identifi ed on these organ) because these markers have the potential to become novel cells and validated in functional models. We will complement targets, not only for diagnostic purposes but also for therapeutic immunocytochemical DTC detection by additional genotypic and phenotypic interventions. The markers functionally associated with metastatic markers relevant for metastatic progression. To further increase diagnostic progression of DTC are likely to be promising targets for therapy. These precision, we will analyse whether this improved platform can be combined specifi c markers could be further developed as therapeutic targets in with the analysis of tumour characteristics that were revealed by microarray collaboration with SMEs and larger pharmaceutical industries. profi ling, and with evaluation of circulating tumour-associated DNA or RNA. Besides the primary focus on improvement of DTC-based diagnostic With regard to cancer therapy, two key issues will be addressed. First, it platforms, it is important to realise that these cells are the target cells for will be evaluated whether it is possible to eradicate DTC by an any kind of adjuvant systemic therapy, including chemotherapy and targeted immunotherapeutic approach, optimised for success in a mouse model. biological therapies. In innovative DTC models, the effi cacy of DTC treatment Secondly, we will investigate whether DTC are susceptible to current will therefore be analysed and potential improvements studied. The systemic therapies (for example chemotherapy) and develop novel translation of scientifi c knowledge into commercial products will be ensured approaches for their specifi c eradication. Current adjuvant chemo- or by three SMEs with unique technological capabilities. radiation therapy aims at hitting the most overt property of tumour cells their unrestricted potential to proliferate. According to our current knowledge, DTC in the bone marrow are non- or slowly proliferating.

Expected results

Primarily, the result of DISMAL will lead to improved disease staging and when implemented in clinical practice to personalised medicine. As a long-term goal, reaching beyond the duration of this proposal, the newly discovered diagnostic markers will be further evaluated for their suitability as targets for therapeutic intervention, speciﬁ cally aimed to

208 | CANCER eradicate minimal residual disease (MRD) in patients with solid tumours. Potential applications DISMAL will provide: • knowledge about the biology of the metastatic process, particularly Apart from the ultimate goal of DISMAL (the delivery of an improved genes that determine early dissemination, homing and survival of diagnostic platform for minimal disease detection), the sensitive detec- epithelial tumour cells at different sites; tion tools might also be applied to a broad range of other biotechnology • identifi cation of relevant genes and signalling pathways associated applications that involve the detection of rare cells. One fi eld might be, with tumour cell dissemination via the blood vessels to distant organs for example, the screening for cancer cells in body fl uids as a tool for (bone marrow); primary cancer diagnosis, or more sensitive and specifi c detection of • improved estimation of prognosis and need for therapy of cancer tumour cells in the lymph compartment. patients with minimal disease through the development of a novel diagnostic platform for DTC detection which includes the information on the biology of micrometastasis and metastatic progression; • basic information about new therapeutic strategies for preventing Coordinators Monique Slijper metastasis formation through the identifi cation of functionally Utrecht University relevant therapeutic targets that are frequently expressed on DTC. Klaus Pantel Utrecht, The Netherlands These targets might therefore be explored for the development of Institute for Tumour Biology University Medical Centre Roland Eils new forms of adjuvant cancer therapy aimed at specifi cally Hamburg-Eppendorf German Cancer Research Centre eradicating minimal disease with less severe side effects than current Hamburg, Germany Heidelberg, Germany chemotherapy; [email protected] • a unique, large-scale biobank of freshly frozen and paraffi n- Wolfgang Deppert embedded epithelial primary tumours and autologous samples Ruud Brakenhoff Heinrich Pette Institut of common sites of micrometastatic spread, including bone VU University Medical Center Hamburg, Germany marrow (cells) and blood (cells and serum). The biobank will be Dept Otolaryngology complemented by computerised storage of data on patients and Head-Neck Surgery Raoul Charles Coombes tumour characteristics, including histopathological analyses and Amsterdam, The Netherlands Imperial College London clinical follow up information. [email protected] London, United Kingdom

Jean-Pierre Vendrell Partners Lapeyronie Hospital Montpellier, France Björn Naume Radium Hospital Oslo Paddy O’Kelly Oslo, Norway Applied Imaging Newcastle, United Kingdom Laura van ’t Veer Netherlands Cancer Institute Rainer Uhl Amsterdam, The Netherlands TILL Photonics Grätelfi ng, Germany Hans Tanke Leiden University Medical Center Bernhard Sixt Leiden, The Netherlands Agendia Amsterdam, The Netherlands Project number LSHC-CT-2005-018911 Michael Speicher EC contribution Medical University of Graz € 4 200 000 Graz, Austria Project Duration 36 months Starting date 01/11/2005 Instrument STREP Project website www.dkfz.de/dismal

CANCER | 209 E.E.T.-Pipeline

European Embryonal Tumour Pipeline

Keywords Neuroblastoma, medulloblastoma, retinoblastoma, Problem nephroblastoma, rhabdoid, Ewing, translational research Second to accidents, cancer is still the leading cause of death for children in Europe. Approximately 30 % of childhood malignancies are embryonal tumours (ET), often demonstrating resistance to conventional treatment approaches and being associated with lower survival rates compared to other childhood cancers. Thus, novel diagnostic and therapeutic options are urgently needed in particular for this group of tumours in order to Summary improve survival rates and quality of life of pediatric cancer patients. The limitations of current treatment approaches include, in particular, a lack Treatment of embryonal tumours (ET) is a challenge for the pediatric of validated post-genomic technology available for routine diagnostics oncologist. Innovative translational research is required to exploit available and a large gap between target identifi cation in basic research efforts genomic data and implement state-of-the-art technologies to overcome and resulting pre-clinical development of novel drugs. the defi cits of current diagnostic and treatment strategies. We will set up a Consortium of leading European institutions and SMEs with extensive Beyond defi ning characteristic gene expression signatures, only a limited clinical and technological expertise in order to establish a unique pipeline number of studies have addressed the functional analysis of identifi ed for the comprehensive development and validation of novel diagnostic target genes. The genetic low complexity of ET tumours provides tools in addition to effi cient preclinical drug development for ET. a suitable system to identify druggable targets. Conclusive diagnosis Our holistic approach includes: using histology alone is diffi cult due to the uniform morphologies of the • validation of a chip-based diagnostic platform tailored specifi cally different ET entities, identifying ET as an important area to complement for ET, including analysis of genes previously shown by the current strategies with modern post-genomic approaches. Consortium to be affected in ET; • validation of a chip-based diagnostic platform tailored specifi cally Our approach is of particular importance for ET, as the ET entities are for ET, including analysis of genes previously shown by the orphan diseases, meaning each entity does not have a large enough Consortium to be affected in ET; market to justify the costs of drug development by a private company. • extension of an existing database designed to warehouse complete The identifi cation of potential targets for all ET entities should be more clinical and experimental data for neuroblastoma to include all ET economically attractive for private companies. Common molecular entities; pathways such as myc- and RB-signalling and chromosomal deletions • implementation of a virtual ‘ET-Biobank’ to improve sharing of including 1p36 and 11q loss have been previously identifi ed in different patient samples; ET entities by the Consortium members and others, supporting • functional characterisation of the most promising molecular targets a rationale integrating all ET types. previously identifi ed by the partners as a foundation for entry into a drug development pipeline; • integration of existing disease-specifi c mouse models to evaluate Aim new treatment modalities in vivo; • initial evaluation of a screening method and antibody affecting ET The multimodal genomic and proteomic approaches proposed here cell invasion; provide a promising alternative for more successful tumour diagnosis, • application of novel bioinformatic solutions for meta-analysis; subclassifi cation and drug development for ET. This STREP will provide • dissemination of the novel tools to researchers and clinical study a coordinated strategy for the translation of basic research results into centres in Europe. the pre-clinical arena, focusing on the implementation of state-of-the- Our coordinated effort can achieve the critical mass to facilitate the art diagnostic tools, improvement of access to clinical material, and necessary integration of research capacities for translating ET genome the effi cient combination of post-genomic research approaches with data into signifi cant medical progress. Involvement of clinical study pre-clinical drug development. centres will ensure a direct link to the bedside, aimed at improving child health and quality of life.

210 | CANCER Expected results Coordinator Prof. Heinrich Kovar Children’s Cancer The E.E.T.-Pipeline provides a comprehensive, multi-team approach for Prof. Angelika Eggert Research Institute improving ET diagnostics and treatment by the integration, assessment University Children’s Vienna, Austria Hospital Essen and validation of information generated by basic research utilising Essen, Germany Prof. Olivier Delattre high-throughput technologies. In this integrated post-genomic research [email protected] Institut Curie effort, we conceive dual pipelines concentrating on: Paris, France • state-of-the-art diagnostics; • innovative drug development and pre-clinical testing, in order to Partners Dr. Massimo Zollo channel these efforts. We aim to close the gap between basic and Ceinge-Biotecnologie clinical research by focusing on the initial evaluation and charac- Prof. Manfred Schwab Avanzate S.C. a.r.l. terisation of a selected subset of identiﬁ ed genes, proteins and German Cancer Research Center Napoli, Italy biological pathways contributing to malignant transformation or Heidelberg, Germany progression of ET. Frank Speleman Prof. Roland Eils Centre for Medical Genetics German Cancer Research Center Ghent, Belgium Heidelberg, Germany Potential applications Dr. Björn Cochlovius Prof. Saso Dzeroski Affi tech AS This integration of research capacities is expected to enable the Jozef Stefan Institute Oslo, Norway participating SMEs to develop novel, biology-based therapies for Ljubljana, Slovenia disseminated disease, which will complement current protocols with Dr. Alessandro Bulfone more speciﬁ c and less toxic alternatives. We aim to provide a platform Prof. Michael Grotzer Biofl ag Srl to support the initial phase of development of new, well tolerated University Children’s Pula, Italy disease- and patient-adapted anti-tumour strategies. Hospital of Zurich Zurich, Switzerland

A B

C D

Project number LSHC-CT-2006-037260 EC contribution € 4 000 000 Project Duration Custom-made tissue microarray for validation of identiﬁ ed novel target proteins 36 months in embryonal tumor tissue. Starting date 01/01/2007 Instrument STREP Project website www.eet-pipeline.eu

CANCER | 211 EPCRC

The European Palliative Care Research Collaborative: improved treatment of pain, depression and fatigue through translation research

Keywords Cancer, palliative care, pain, depression, fatigue, Problem cachexia, opioid response, genetic marker, classifi cation, assessment, guidelines Even though pain, fatigue and depressive symptoms are the most common symptoms in advanced cancer, there is still inadequate understanding of the inter-individual variability of these problems. Cachexia is a major reason for fatigue in palliative care patients, but there is no internationally agreed assessment tool or classifi cation system for either cachexia or depression in this patient group. Although there are several publications and agreements on the classifi cation of pain, Summary there is still no consensus on how to assess or measure it. In addition, there is a lack of internationally developed and updated clinical Pain, depression and fatigue are subjective clinical manifestations of guidelines for the treatment of these conditions and symptoms. advanced cancer. Control of these symptoms is pivotal for the quality of life of millions of palliative care patients. Pain is the most feared symptom for many patients. The prevalence of depression varies from 6-58 % in Aim palliative care patients, reflecting lack of a standardised validated methodology for its assessment in these patients. The overall objectives are to develop novel genetic methods for predic- Cachexia is likely to be the most important contributor to fatigue in tion of opioid responses and individual variation of fatigue (cachexia), palliative care. The plethora of other symptoms, co-morbidities, and and methods for assessment and classifi cation of pain, fatigue (cachexia), the advanced age of most palliative patients support the need for and depression. evidence-based management strategies. To improve management of pain, depression and cachexia in cancer patients demands new knowledge • To identify genes and genetic variation relevant for inter-individual through research in several areas. variation in opioid responses and genetic variation that may identify The research plan of the EPCRC will be based upon questions raised in patients at particular risk for developing cachexia. a clinical setting, with focus on palliative care cancer patients, thus • To improve classifi cation and assessment of pain, depression and a ‘true’ translational approach is chosen. The research will focus both on cachexia by computer-assisted approaches. diagnosis and classifi cation of these symptoms and on an understanding • To combine the new knowledge of symptoms, genomics and of the underlying mechanisms. To reach the aims of EPCRC requires assessment in an Internet-based system for implementation of a multidisciplinary approach, including basic scientists and clinicians European evidence-based guidelines, which will include standard- who will translate human genome data into practical applications for ised assessment and individualised treatment plans for pain, these patients. depression and cachexia. Assessment and classification of pain, depression and cachexia • To develop a long lasting European Collaborative in palliative care (fatigue) are the basis for diagnosis and subsequent treatment. By use cancer research. of modern molecular biology methods in this project, we will increase the understanding of the role of genetic variability for pain and cachexia. European evidence-based Internet guidelines will be developed by members of the EPCRC, supported by an international advisory board. By recruiting a pan-European panel, cultural, social and language barriers will be taken into consideration in the early phase of guideline development.

212 | CANCER Identiﬁ cation of proﬁ les of genetic markers that best predict pain treatment responses.

Expected results Coordinators Partners • Identifi cation of profi les of genetic markers that best predict pain treatment responses, with specifi c emphasis on opioids. Stein Kaasa Prof. Geoffrey Hanks • Increased understanding of the molecular basis for cachexia and Frank Skorpen Bristol Haematology Marianne Jensen Hjermstad & Oncology Centre identifi cation of genetic factors that may predict patients at Jon Håvard Loge Bristol, United Kingdom particular risk. Norwegian University • Increased understanding of the value of serum parameters as an of Science and Technology Dr. Franco De Conno indicator of pain response. Trondheim, Norway National Cancer Institute of Milan • Better methods for classifi cation and assessment of pain, depression [email protected] Milano, Italy and cachexia in palliative care. [email protected] • International European-adopted and agreed clinical guidelines for [email protected] Prof. Irene Higginson classifi cation, assessment and treatment of pain, depression and [email protected] King’s College London cachexia in palliative care. London, United Kingdom • Guidelines available and regularly updated on the EPCRC website, Dr. Florian Strasser the EAPC (European Association for Palliative Care) website, and Spitalregion St. Gallen Rorschach other relevant websites. Kantonsspital • Guidelines translated and disseminated in relevant European St. Gallen, Switzerland languages. Prof. Lukas Radbruch Rheinisch-Westfälische Technische Potential applications Hochshule Aachen Aachen, Germany The aim of the EPCRC is to have the guidelines disseminated and universally accepted within the palliative care community in Europe, Prof. Kenneth Fearon The University of Edinburgh both in clinical work and research. Edinburgh, United Kingdom

Prof. Hellmut Samonigg Medical University of Graz Graz, Austria

Mr. Ketil Bø Trollhetta AS Trondheim, Norway

Mr. Michael Schaude The Bender Med System Vienna, Austria

Project number LSHC-CT-2006-037777 EC contribution € 2 799 910 Project Duration 36 months Starting date 01/11/2006 Instrument STREP Project website www.EPCRC.org

CANCER | 213 EPITRON

Epigenetic treatment of neoplastic disease

Keywords Epigenetic, apoptosis, differentiation Aim

The overall goal of EPITRON is to validate and extend the concept of ‘epigenetic therapy’ of cancer. For this a pipeline will be established, which extends from the analysis of epigenetic (de)regulation in cancer to the study and generation of epi-drugs in a multiplicity of in vitro, ex vivo and in vivo mouse models. We will develop and use mouse models that accurately reproduce the human disease. The particular goals of EPITRON are: Summary • to study the epigenetics of cancer cells (with a focus on leukaemia, breast and colon cancer), and defi ne the mechanisms Chromatin is epigenetically modified to regulate gene expression. of (cancer selective) action of epi-drugs; Upstream signals induce complex patterns of enzyme-catalysed • to establish the basis of the cancer-selectivity of TRAIL/TRAIL modifi cations of DNA and histones, key protein components of chromatin. receptor action; These epigenetic modifi cations create docking sites and form a code • to identify novel epi-drug targets; that specifi es transient or permanent (and heritable) patterns of genome • to synthesise novel epi-drugs with increased effi cacy/tumour function. In addition, epigenetic enzymes modify the activity of major selectivity; transcription factors. Emerging evidence causally links altered epigenetic • to validate epi-drug target therapy of cancer in vitro (primary functions to oncogenesis, and suggests that chromatin regulators and normal and tumour cells), ex vivo (leukaemic blasts vs. normal upstream pathways are critical targets for developing novel anti-cancer progenitors) and in vivo (mouse models which accurately drugs (epi-drugs). EPITRON will defi ne and validate the concept of reproduce human cancer; the focus will be on APL/AML but ‘epigenetic cancer treatment’ from the molecular mechanism(s) to also solid cancer models will be used or established). animal models reproducing human cancers. EPITRON will establish a programme from drug target exploration and drug development to pre- Taken together, EPITRON will not only provide information about clinical validation in vitro, ex vivo and in vivo. Epi-drugs are amongst the epigenetic modifi cation imposed upon cancer cells, validate existing and most important novel drugs that have been generated to treat cancer, as generate novel epi-drugs, but most importantly engage upon a major can be concluded already from the existing results obtained with HDACi’s, challenge of cancer therapy by devising treatments that kill cancer, but some of which performed very well in phase I and phase II clinical trials. not normal cells. EPITRON is unique in its efforts to strengthen European biomedical and pharmaceutical competitiveness. It fosters an exchange of biomedical knowledge, technology and materials among European laboratories, Expected results provides opportunities for education and training – and creates jobs. To validate and extend the concept of ‘epigenetic cancer therapy’, EPITRON will follow six axes of research, focused on preclinical models. • Mechanisms of anti-leukaemic action of epigenetic drugs. We will defi ne the anti-leukaemogenic potential and the corresponding mechanistic basis of existing epigenetic drugs used alone or in combination, and in combination with other signalling drugs, such as nuclear receptor ligands. The impact of chromatin modifi - cation (DNA, histones) that correlates with tumourigenesis and underlying recognition principles will be studied. Problem • Oncofusion genetic and epigenetic programmes. We will use cell lines, patients’ blasts and mouse models to understand the altered Elucidating the ‘signatures’ of cancer cells is one of the four so-called gene programming due to the oncogenic fusion protein(s). ‘extraordinary opportunities for immediate investment’ defi ned by the • Decryption of the leukaemia cell-selective apoptogenic action of National Cancer Institute of the United States of America. These four TRAIL. Based on the original fi nding of members of this consortium priorities (defi ning the signatures of cancer cells, cancer genetics, that several anti-leukaemogenic treatments activates the TRAIL death preclinical models of cancer and imaging technologies) were selected as pathway, and the observation that TRAIL signalling induces apoptosis top priorities to receive privileged attention and funding. Thus the in tumour, but not normal, cells, the molecular mechanism(s) under- EPITRON project aims to contribute at multiple levels to the defi nition of lying this fascinating potential will be defi ned in suitable cellular epigenetic signatures of cancer. models using a plethora of genomic technologies.

214 | CANCER MS275 • Therapeutic potential and toxicities of TRAIL in animal models. Based on regulable TRAIL expression systems, EPITRON will establish mouse models to assess the spectrum of anti-cancer activities and possible toxicities of TRAIL in vivo using both ubiquitous and tissueselective expression paradigms. At the same time ‘reporter mice’ will be created, which will allow monitoring activation of the TRAIL signallin pathway by (epi-)drugs. • Generation and validation of novel epigenetic drugs. Crystal structures and innovative chemistry will be used to generate compounds that (selectively) modulate the activity of epigenetic enzymes/machineries.

• Models for epigenetic therapy of solid cancers. Among the several AML patient 1 AML patient 2 tumour mouse models used by EPITRON, studies will be performed to assess efﬁ cacy and mechanisms of HDACi and novel EPITRON- generated epi-drug actions in breast cancer models (e.g. MMTV-neu, MMTV-wnt, etc.). In these studies, primary cell cultures derived from the above mouse models (and their normal counterparts) will also be used. Moreover, EPITRON will establish as a novel tool matched pairs of primary normal and cancer cells from the same patients to assess (epi-)drug action, especially tumourselective activities. As an example The HDAC inhibitor MS275 induces differentiation and apoptosis in blasts of a gender cancer, primary patient-matched cultures of normal and of acute myeloid leukemia (AML) patients. breast cancer epithelial cells will be studied. The photos show ex vivo cultures of blasts of two AML patients (left and right panels) treated with MS275 (top panel), resulting in blast differentiation and death, or with vehicle (bottom panels).

Potential applications Coordinator Hugues de The Centre Nationale pour la In their entirety, the studies performed in AML, breast, skin and colon Hinrich Gronemeyer Recherche Scientifi que cancer preclinical models will provide a framework for a detailed CERBM-GIE Centre Européen Paris, France molecular deﬁ nition of ‘epigenetic therapy’, which will pave the way to de Recherche en Biologie et Médecine – Groupement Adriana Maggi more focused and appropriate protocols for future clinical trials. d’Intérêt Economique Università degli Studi di Milano Illkirch, Strasbourg, France Milano, Italy [email protected] Tony Kouzarides University of Cambridge Partners Cambridge, United Kingdom

Saverio Minucci Olli Kallioniemi Pier Giuseppe Pelicci University of Turku Istituto Europeo di Oncologia Turku, Finland Milano, Italy Kurt Berlin Henk Stunnenberg Epigenomics Project number Stichting Katholieke Universiteit Berlin, Germany LSHC-CT-2004-518417 Nijmegen, The Netherlands EC contribution Tiziana Cataudella € 10 904 474 Angel De Lera Congenia Project Duration Universidad de Vigo Milano, Italy 60 months Vigo, Spain Starting date Holger Hess-Stumpp 01/11/2005 Lucia Altucci Schering AG Seconda Università Berlin, Germany Instrument degli Studi di Napoli IP Napoli, Italy Abbie Harris Project website Abcam www.epitron.eu Cambridge, United Kingdom

CANCER | 215 EuroBoNeT Keywords xxxxxxxxxx

European Network to Promote Research into Uncommon Cancers in Adults and Children: Pathology, Biology and Genetics of Bone Tumours

Keywords Bone tumour, chondrosarcoma, osteosarcoma, The 2002 WHO classifi cation recognises 32 different entities of bone giant cell tumour of bone, Ewing sarcoma, osteochondroma, tumours. Achieving signifi cant numbers to study the different types of Paget disease of bone, chondrogenesis, osteogenesis bone tumours, which are already rare as a group, is diffi cult. The research into these tumours is often performed in relatively small research groups, which are inherently hampered by the lack of availability of substantial numbers of cases, as well as lacking a critical technical and/or multidisciplinary mass. Interestingly, despite their rareness, these tumours provide excellent examples for unravelling oncogenic mechanisms. The main problems are: Summary • collecting enough tumours to obtain reliable signifi cant statistical results, also when comparing subtypes based on locations, grade, etc.; Primary bone tumours are rare, accounting for ~0.2 % of the cancer burden. • since osteogenic and Ewing’s family of tumours are considered Children and young adolescents are frequently affected. The aggressiveness orphan diseases, pharmaceutical companies will not invest in devel- of these tumours has a major impact on morbidity and mortality. Though oping new drugs; progress has been made in pathological and genetic typing, the aetiology • chemotherapeutic treatment can be toxic for the patient or not is largely unknown. Advances in therapeutic approaches have increased effective, and there are no tools that predict which patients are survival rates, but a signifi cant numbers of patients (~40 %) still die. hypersensitive or which tumours are refractory; Within the EuroBoNeT, staff exchange, share of material and technologies, • cartilaginous tumours are resistant to treatment other than surgery. as well as the organisation of training courses, will be used to increase and This can be mutilating and it is not always feasible to remove the disseminate knowledge of primary bone tumours. Exchange of material, whole tumour (for instance in the pelvic area); standard operating protocols, and the use of technology platforms will • a small percentage of benign cartilaginous tumours develop into enable us to obtain statistically signifi cant datasets. A joint programme will malignant chondrosarcomas, but there are no clues to recognising contribute in obtaining molecular portraits of tumours, separated into four the ones that will deteriorate; research lines: RL1: cartilaginous tumours; RL2: osteogenic tumours and • the biological behaviour of giant cell tumours is variable and cannot related sarcomas; RL3: osteoclastogenesis and giant cell tumours of bone; be predicted so far. In a small proportion of tumours, synchronous or and RL4: the Ewing family of tumours. metachronous metastases develop; The tumours will be examined by genome-wide expression, genomic • the biology of normal chondrogenesis and osteogenesis is complex. aberration studies, and specifi c hypothesis-driven approaches (RNA/ A better under-standing of the pathways involved could provide protein expression and mutation analysis). In vitro studies will be used to clues to the biology of bone tumours. obtain knowledge of normal growth and differentiation, and this may help to identify markers for malignant transformation and/or progression, as well as identifi cation of therapeutic targets. Aim Dissemination of knowledge will be achieved by training courses on bone and soft tissue pathology. This is essential since patients do not normally The above-mentioned major problems will be handled by EuroBoNeT in present themselves at centres, and it is important to share such knowledge. the following ways: • to overcome the spread of samples and lack of critical technical and/or multidisciplinary mass in some of the institutes, the main objective will be to reach integration through sharing of samples, sharing of technologies and incorporating all information gathered by the different partners. This will be done through staff exchange, core facilities, a virtual BioBank and combining each others’ experimental results; • the EuroBoNeT will characterise the tumours in the different Problem research lines by proteomics, genome-wide expression analysis, and genomic array. This will provide a large data set, which can be Bone sarcomas are rare and represent a group of cancers that occur compared within and between the different tumour (sub)types. predominantly in children and young adults. Intrinsic to their aggressive These data will be used for: behaviour, these tumours are lethal in about 40 % of patients despite – identifi cation of loci involved in conferring hypersensitivity or modern multimodality therapy. Although substantial progress has been chemotherapy resistance; made over the last 10 years in understanding these tumours at the – identifi cation of molecular targets for therapy; biological, pathologic and genetic level, this has not been translated – identifi cation of markers involved in malignant transformation; into more effective therapies so far. – identifi cation of genes/pathways involved in disease progression.

216 | CANCER A

Images of the main tumours investigated in the EuroBoNeT NoE. In Research line 1, cartilage tumours are the subject, of which, in A en B, B C an example is given of an osteochondroma, Research line 2 focuses on osteogenic tumours such as osteosarcomas (C), Research line 3 on giant cell tumors (D) and osteoclastogenesis, while Reseach Line 4 investigates the Ewing family of tumours such as the Ewing Sarcomas (E).

D E

Coordinator Wim Wuyts Søren Daugaard Wiltrud Richter University of Antwerp Rigshospitalet Stiftung Orthopadische Pancras CW Hogendoorn Antwerp, Belgium Copenhagen, Denmark Universitatsklinik Heidelberg Leiden University Medical Center Heidelberg, Germany Dept of Pathology Antonio Llombart-Bosch Fredrik Mertens Leiden, The Netherlands University de Valencia Lund University Jos Joore [email protected] Valencia, Spain Lund, Sweden Pepscan Systems BV Lelystad Chris Poremba Thomas Aigner The Netherlands Partners Heinrich-Heine-University Düsseldorf Friedrich-Alexander- Düsseldorf, Germany University Erlangen Jan Schouten Piero Picci Erlangen, Germany MRC-Holland Istituti Ortopedici Rizzoli Pierre Mainil Amsterdam, The Netherlands Bologna, Italy University of Bern Raf Sciot Bern, Switzerland Catholic University of Leuven Zsolt Hollo Sakari Knuutila University Hospital Solvo Biotechnology, Inc. University of Helsinki Nick Athanasou Leuven, Belgium Budapest, Hungary Helsinki, Finland University of Oxford Oxford, United Kingdom Horst Bürger Peter Riegman Lars-Gunnar Kindblom Westfalische Wilhelmuniversity Münster Erasmus Medical Center Royal Orthopaedic Enrique de Alava Münster, Germany Rotterdam, The Netherlands Hospital NHS Trust University de Salamanca-CSIC Birmingham, United Kingdom Centro de Investigacion del Cancer Angelo Paolo Dei Tos Salamanca, Spain Hospital of Treviso Ramses Forsyth Treviso, Italy N Goormaghtigh Institute Ola Myklebost of Pathology University of Oslo Miklös Szendröi Gent, Belgium Norwegian Radium Hospital Semmelweis University Budapest Oslo, Norway Budapest, Hungary

• by studying the molecular mechanisms involved in osteoclastogenesis and chondrogenesis (using in vitro models), EuroBoNeT aims to identify new mechanisms that could also play a role in bone tumourigenesis, and provide clues for the identiﬁ cation of new diagnostic and/or prognostic markers. Project number LSHC-CT-2006-018814 Expected results EC contribution € 13 218 960 The establishment of a lasting network in which samples will be Project Duration exchanged and results combined and discussed, and that will be able to: 60 months • provide uniform guidelines for bone tumour pathology; Starting date • play a role in the development of: 01/02/2006 – new diagnostic markers, Instrument – new prognostic markers, NoE – markers to predict the effect of chemotherapeutics, Project website – new drug treatment. www.eurobonet.eu

CANCER | 217 EuroCSC

Targeting Cancer Stem Cells for Therapy

Keywords Cancer, stem cells, leukaemia, breast cancer Problem

Human tumours are currently treated primarily with drugs with cytotoxic effects against proliferating cells. This therapy is effi cient against cells with the properties of rapidly proliferating progenitors. However, the realisation that many tumour types contain malignant cells with stem cell properties, in that they are able to initiate and sustain a tumour but Summary proliferate infrequently, provides a potential explanation for the ability of many tumours to recur even after the eradication of the bulk tumour Cancer remains one of the leading causes of death in the western world mass. The ability to identify and pharmacologically target these cancer and, while chemotherapy has provided a major improvement in survival stem cells would signifi cantly enhance the effi cacy and reduce the side- for a wide array of malignant diseases, lethality remains high in most effects of cancer therapy. cancers and side-effects are severe, including developmental impairment when used in childhood malignancies, infertility as well as damage to non-malignant tissues with resulting diminished quality of life for a large Aim proportion of survivors. Recently, the realisation that several tumour types contain rare populations We will use functional analysis and gene profi ling of purifi ed human of cancer stem cells (CSCs) which are capable of reforming the tumour cancer stem cells and genetic modelling in the mouse to identify upon transplantation while their progeny are not, have opened the molecular targets that may be used to selectively eradicate or inactivate possibility of using CSCs as targets for directed molecular therapies that the malignant stem cells that sustain tumours. These targets will be could lead to improved tumour eradication, as well as reduced side- validated by knockdown and genetic ablation in mouse model systems. effects of treatment. Finally, we will initiate the identifi cation of lead compounds with activity The goal of the present project is to perform a thorough characterisation against these targets. of AML, cALL and breast cancer CSCs, as well as a systematic comparison of these with their normal stem cell and progenitor counterparts, using gene profi ling to identify putative molecular targets in CSCs. In parallel, Expected results we will use mouse genetic modelling to obtain information about genes regulated by oncogenic changes in stem and progenitor cell populations. We expect to identify and validate target molecules with activity against Directly oncoprotein-regulated CSC targets will be validated in vitro and, cancer stem cells in AML, call and breast carcinoma. where relevant, in vivo. The fi nal outcome will be identifi cation of the nature and hierarchical position of CSCs in three major cancers, and a set of identifi ed and validated CSC molecular targets with activity Potential applications against the effects of leukemogenic oncoproteins on hematopoietic stem cell/progenitor populations. These results are directly applicable to the development of drugs targeting human cancer stem cells.

218 | CANCER Coordinator Dr. Tariq Enver A MRC Molecular Hematology Unit Dr. Claus Nerlov Oxford, United Kingdom European Molecular Biology Laboratory LT-HSC Monterotondo, Italy Dr. Roger Patient MEP + [email protected] MRC Molecular Hematology Unit Oxford, United Kingdom LT-HSC CMP GMP Partners Dr. Ole W. Petersen MPP University of Copenhagen Dr. Sten Eirik W. Jacobsen Copenhagen, Denmark CLP University of Lund Lund, Sweden Jon Tinsley VASTOX B Dr. Dominique Bonnet Oxford, United Kingdom Cancer Research UK London, United Kingdom CSC MEP + Partially CSC CMP differentiated myeloid cells GMP MPP*

CLP

“L-CMP” + “L-CMP” MEP Partially “CMP” differentiated myeloid cells GMP

The Cancer Stem Cell hypothesis: Human tumors contain cells that are able to re-initiate tumor formation at distant sites (when metastases form) or when tumors relapse after chemotherapy. As illustrated in the Figure using acute myeloid leukemia as an example, these Cancer Stem Cells are self-renewing Project number (CSC; panel B), and may phenotypically resemble the normal self-renewing stem LSHC-CT-2006-037632 cells (Long-term hematopoietic stem cells; LT-HSC; panel A) of the tissue from which they arise. However, Cancer Stem Cells may also have phenotypes of EC contribution more committed progenitors (as exempliﬁ ed by the leukemic Common Myeloid € 1 900 000 Progenitor, or L-CMP; panel C), which have acquired ectopic self-renewal Project Duration capacity, and thus able to perpetuate the malignant clone. Both normal and 36 months cancer stem cells reside at the top of a differentiation hierachy; however, while normal stem cells maintain the tissue by providing the cells which sustain its Starting date function Cancer Stem Cells give rise to defective progeny that when allowed to 01/01/2007 accumulate can lead to tissue failure. Instrument LT-HSC: Long-term hematopoietic stem cell; MPP: multipotent progenitor; STREP CLP: common lymphoid progenitor; CMP: common myeloid progenitor; GMP: Project website granulocyte-macrophage progenitor; MEP: megakaryocyte-erythroid progenitor. www.embl-monterotondo.it/ research/projects/nerlov/ eurocsc.html

CANCER | 219 GenoMEL

Genetic and Environmental Risk Factors for Melanoma: Translation into Behavioural Change

Keywords Melanoma, genetics, naevi, sun exposure Problem

Melanoma is an important health issue within Europe because it continues to increase in incidence in many Member States and because it has a relatively fl at-age incidence curve, so that the tumour is disproportionately frequent in young persons. Furthermore, in the new European Member States, there are concerns that as affl uence increases Summary incidence levels will rise precipitously if action is not taken to discourage risky behaviours in the sun. There are already, unfortunately, data GenoMEL, formerly known as the Melanoma Genetics Consortium, has suggesting that survival from melanoma is poorer in these European focused on the identifi cation of familial high-penetrance melanoma countries than in western European countries. genes. To date, the consortium has been very successful in identifying susceptibility genes and developing joint data collection for gene/ environment interaction studies. In order to continue its proactive role, Aim GenoMEL is developing a multidisciplinary European platform with relevant input from international participants. This platform will also The objective of GenoMEL is to understand the genetic causes of enable the investigation of attitudes to risk of melanoma in Europe, and melanoma and how the identifi ed susceptibility genes interact with the translate that risk perception into behavioural change. environment, predominantly with sun exposure. The intent is then to use the information obtained to improve on the ranking of risk factors for melanoma and to understand the phenotypic markers of those susceptibility genes. These fi ndings will then be converted into a webbased tool, called a content management system (CMS), for use by the general public, in order to calculate individual risks of melanoma. The CMS will be assessed and correlated with reported behaviour in the sun. We will then ‘close the loop’ by designing materials for use primarily on the web but also as written materials to educate European people about the primary and secondary prevention of melanoma.

The CDKN2A locus – the ﬁ rst high penetrance melanoma susceptibility A malignant melanoma. gene to be identiﬁ ed.

220 | CANCER A GenoMEL researcher at work.

Coordinator Florence Demenais David Hogg INSERM, Institut National University of Toronto Julia A. Newton Bishop de la Santé et de Toronto, Canada Genetic Epidemiology Division la Recherche Medicale Cancer Research UK Paris, France Esther Azizi Leeds, United Kingdom Tel Aviv University Giovanna Libera Bianchi-Scarrà Tel Aviv, Israel Universita degli Studi di Genova Partners Genova, Italy Martyn Rainford Naked Design Consultancy Nelkele Gruis Johan Hansson Leeds, United Kingdom Leiden University Medical Centre Karolinska Institutet Leiden, The Netherlands Stockholm, Sweden Olita Heisele Biomedical Research and Nicholas Kim Hayward Andre Wijfjes Study Centre Queensland Institute ServiceXS B.V. University of Latvia of Medical Research Leiden, The Netherlands Riga, Latvia Brisbane, Australia Antony Young Brigitte Bressac-de Paillerets Graham Mann King’s College London Institut Gustave Roussy University of Sydney London, United Kingdom Villejuif, France Westmead Institute of Cancer Research Håkan Olsson Evert van Leeuwen Sydney, Australia University Hospital VU Medical Center Lund, Sweden Amsterdam, The Netherlands Susana Puig Sarda Hospital Clinic i Provincial David Elder Marko Hocevar de Barcelona University of Pennsylvania Institute of Oncology Barcelona, Spain Philadelphia, USA Ljubljana, Slovenia

Sancy Leachman Jan Lubinski University of Utah Pomeranian Medical University Salt Lake City, USA Szczecin, Poland

Project number LSHC-CT-2005-018702 EC contribution € 10 452 723 Project Duration 60 months Starting date 01/12/2005 Instrument NoE Project website www.genomel.org

CANCER | 221 GROWTHSTOP

Identifi cation, development and validation of novel therapeutics targeting programmed cell death in tumours

Keywords Apoptosis, cell death, cell imaging, tumour models, kinase inhibitors, pharmacophore inhibitors, scaffold inhibitors

Summary

Mounting evidence indicates that the acquired ability to resist apoptosis is a hallmark of most, and perhaps all types of cancer. As scientists learn more about how apoptosis is thwarted by cancer cells, they are also gaining a greater understanding of why many tumours are resistant to the apoptosis-inducing effects of radiation and chemotherapy. These insights will guide efforts to overcome treatment resistance and offer important clues about new drugs that target genes and protein products in the apoptosis pathways to encourage selective cell death. GROWTHSTOP is exploring how apoptosis is regulated and how it can be selectively triggered to induce suicide in cancer cells while sparing normal cells. The GROWTHSTOP Consortium applies a combination of high resolution bio-imaging techniques, proteomics, cellular models, and in vivo tumour models towards: • the understanding of the pathways that signal apoptosis in solid tumours; • their validation as viable targets for tumour suppression or regression in animal models in vivo; • the discovery and validation of a novel, alternative class of inhibitors that specifically targets protein interactions rather than, or in addition to, enzyme activity. The goal of the GROWTHSTOP project is Compartmentalized signal transduction along the MAP kinase through scaffold to exploit apoptotic pathways as a viable therapeutic strategy. proteins and subcellular localization. Importantly, more than 30 % of the applied EU budget will be reserved for SMEs that deliver expertise and chemical screening in order to ensure a rapid translation of novel screens and assays into an effi cient search economic impacts, both for the individual and for the health care provider. for specifi c drugs manipulating pro-apoptotic pathways. As a result, improvements in cancer therapy remain of prime importance for the wellbeing of Europeans and for the future development of the Union.

Cancer is caused by mutations in a relatively small and identiﬁ able number of genes, which fall into two categories: proto-oncogenes, which provide critical proliferative or survival signals to cells and which are inappropriately activated by mutation during tumourigenesis; and tumour-suppressor genes, which restrain cell growth and proliferation, and which are lost or inactivated by mutations during the development of a tumour. Problem Importantly, mutations in individual genes do not cause tumours, since the Cancer is a major challenge to European health care. Each year nearly two human genome harbours failsafe mechanisms that protect normal cells million people are diagnosed with cancer in the EU, and over one million from the consequences of deregulated proliferative stimuli. Two such deaths result from this disease. Each case can have a tremendous impact on failsafe mechanisms are known. The ﬁ rst is an irreversible growth arrest, the health and wellbeing of the affected person, his or her family and termed cellular senescence, which is activated by deregulated oncogenic personal environment. In addition, a high percentage of cases have major signals through the Ras pathway: one key example is the often lifelong

222 | CANCER X-ray diffraction of target drug complexes at high resolution.

lack of proliferation of melanocytic naevi despite the presence of mutations Coordinator Piero Crespo Baraja in B-Raf, a downstream effector of Ras proteins. The second is apoptosis, Consejo Superior de or programmed cell death, which is activated by many forms of de-regulated Lukas Huber Investigaciones Cientifi cas proliferative signals. Tumours can only develop when secondary mutations Innsbruck Medical University Santander, Spain Innsbruck, Austria that disable these failsafe programmes arise; as a consequence, many [email protected] Walter Kolch mutations that are found in human tumours are involved in pathways that Beatson Institute control either senescence or apoptosis. for Cancer Research Partners Glasgow, United Kingdom

Aim Manuela Baccarini György Kéri University of Vienna Vichem Chemie Research Ltd. Strategies that aim at restoring these failsafe programmes, in particular Vienna, Austria Budapest, Hungary apoptosis, in established solid tumours have emerged as an important approach to cancer therapy. The promise of this approach is that such Martin Eilers Martin Ried Philipps University of Marburg CRELUX GmbH strategies create a therapeutic window, killing tumour cells while sparing Marburg, Germany Munich, Germany normal cells. The key aim of this project is therefore to devise, test and implement strategies that restore apoptosis as a failsafe programme to Maria Sibilia Dr. Armin Peter Czernilofsky solid human tumours. Medical University of Vienna Baden, Austria Vienna, Austria More specifi cally, the following aims will be addressed: Fionnuala McKelvey • advanced cancer mouse models, hepatocellular carcinoma and Rony Seger Kompetenzzentrum Medizin GmbH squamous cell carcinoma (SCC) will be used to dissect the contribu- Weizmann Institute of Science Innsbruck, Austria tion of individual kinase pathways to the survival of tumour cells; Rehovot, Israel • understanding of the signalling mechanisms used by human Tony Ng tumours to counteract apoptosis will be improved; King’s College London • a lucid and structured pathway to exploit these fi ndings for thera- London, United Kingdom peutic intervention will be provided, by addressing the problems that hinder the effi cient translation of knowledge about kinase pathways into therapeutic approaches.

Expected results

Through the integrated approach of the project, the following results are expected: • an understanding of the pathways that signal apoptosis in solid tumours; • their validation as viable targets for tumour suppression or regression in animal models in vivo; • and the discovery and validation of a novel, alternative class of inhibitors that speciﬁ cally targets protein interactions rather than, Project number or in addition to, enzyme activity, with the ﬁ nal goal of establishing LSHC-CT-2006-037731 the manipulation of apoptotic pathways as a viable strategy for EC contribution cancer therapy. € 3 531 507 Project Duration 48 months Potential applications Starting date 01/10/2006 Proliferative diseases. Instrument STREP Project website www.growthstop.kmt.at

CANCER | 223 HERMIONE

Novel Anticancer Therapeutics based on Modulation of Apoptosis through Dependence Receptors

Keywords Dependence receptors, apoptosis, and advanced stage cancer patients treated with cytotoxic therapeutics tumour suppressor are often subject to debilitating side- effects. Therefore, there is a great need to develop more precise therapeutics, to reduce side-effects and improve the overall wellbeing of treated patients.

Aim

Summary The aim of HERMIONE is to understand better how dysfunctions in DR- signalling can lead to tumour formation and/or progression. The Receptors are usually seen as inactive unless bound by their ligand. generated knowledge will be applied to the development of targeted However, a new concept has emerged since 1998, by which some receptors drugs, to provide safer and more effective treatments for patients. can in fact mediate two different signalling pathways, depending on the presence or absence of their ligand. Studies of such receptors have indeed The following models, by which DRs regulate tumour initiation, revealed that, in the absence of ligand, signalling initiates an active process progression and metastasis, can be surmised from current knowledge leading to cell death via apoptosis, whereas programmed cell death is on DR-signalling and illustrate the project’s hypothesis that these inhibited in the presence of the ligand. Therefore, expression of these receptors act as tumour suppressors via their pro-apoptotic activity: receptors leads to a state of cellular dependence on their respective in a normal context: the receptor is bound by its ligand and induces ligands, which is why they have been named ‘Dependence Receptors’ a positive signal (survival, differentiation, etc.). In the case of genetic (DRs). The different DRs trigger apoptosis in the absence of ligand, alteration leading to cell transformation and thus cell proliferation, the suggesting that they may all act as regulators of tumourigenesis. concentration of ligand in the extracellular environment becomes In addition, expression of DRs is lost or decreased in many tumours, insufﬁ cient to bind all receptors. In some cases, cells acquire the property suggesting that they act as tumour suppressors and that their loss to migrate into the blood circulation and/or invade other tissues where represents a selective advantage for tumour cells. the ligand is absent. Unbound dependence receptors consequently HERMIONE proposes that unravelling the link between different DRs (DCC, trigger apoptosis. UNC5H, KAI1 and RET), downstream molecules and apoptosis will lead to the identifi cation of new potential targets for anti-cancer drugs. The project will provide a better understanding of the signalling pathways acting downstream of DRs, observe the association of mutations with the onset and progression of tumours (grade, prognosis), and generate murine models in which the apoptotic signalling of the DR is turned off to study the implication of DRs in tumourigenesis in vivo. Through this, HERMIONE will generate knowledge on DR-signalling pathways involved in the apoptosis of tumoural cells (colorectal, breast, thyroid and prostate cancers) and use the general concept of Dependence Receptors to select and perform pre-clinical testing of novel anti-cancer drugs.

Problem

Colorectal, breast and prostate cancer are among the commonest forms of cancer in Europe. Breast cancer is the main cause of death by cancer, killing over 400 000 women a year. There is no efﬁ cient treatment against metastatic breast tumours and available anti-cancer treatments are imperfect, therefore the probability of cure is not high. Existing treatments are highly toxic to normal tissue and cause substantial loss of life quality. Surgery, chemotherapy and radiotherapy are very invasive, Screen for apoptosis genes by highly specialised custom-made robots.

224 | CANCER Keywords xxxxxxxxxx

Immunohistochemistry on mouse small intestine using anti-netrin-1 antibody showing localisation of the netrin-1 in the crypts.

According to these models, a selective advantage for a tumour cell Coordinator Dr. Olivier Donze would be to lose the death activity of DRs by mutation, to curb their Apotech Corporation expression or to acquire autocrine expression of the ligand. Therefore, Dr. Patrick Mehlen Epalinges, Switzerland Dependence Receptors appear to be original targets for combating Centre Léon Bérard cancer and potential candidates to be studied in the aim of developing Lyon, France Prof. Stefan Grimm novel cancer therapeutics. In order to achieve this, proper understanding [email protected] Imperial College of Science, Technology and Medicine of the way these receptors induce apoptosis, i.e. deciphering the London, United Kingdom signalling pathways operating downstream, is of great importance. Partners Dr. John Hickman Prof. Jurg Tschopp Institut de Recherche Servier Expected results Université de Lausanne Suresnes, France Lausanne, Switzerland • Understanding the fundamental phenomena: Dr. Raffaella Catena – to yield better understanding of the signalling pathways that act Prof. Giovanni Romeo Alma Consulting Group downstream of dependence receptors. Understanding the way Alma Mater Studiorum Lyon, France Dependence Receptors are linked to the trigger of apoptosis will Universita di Bologna Bologna, Italy provide targets for drug development; – to analyse the status of DR/ligand pairs in human tumourigenesis: is there a selective advantage to losing expression of the receptor, gaining autocrine expression of the ligand or losing death function by mutation? From these studies, clear links between DRs and tumour progression will be established, thereby generating new markers for prognosis.

• Applying scientiﬁ c knowledge to the identiﬁ cation and pre-clinical validation of novel anti-cancer drugs: – generation of new markers for tumours, valuable tools for prognosis; – development of peptides and monoclonal antibodies: by studying different DRs and their respective ligands, the Consortium will validate targets in mouse models through the development of recombinant proteins and monoclonal antibodies; – development of anti-cancer agents: the data collected through this project will provide the basis for the development of small molecules as anti-cancer agents.

Potential applications

HERMIONE will enable better understanding of how dysfunctions in dependence receptors signalling can lead to tumour formation and/or progression. The knowledge generated will be applied to the generation of targeted drugs, providing safer and more effective treatments for Project number patients. The work performed within this project will include the LSHC-CT-2006-037530 development of new biomarkers that will provide means for both EC contribution diagnosis and prognosis. The amount of fundamental knowledge € 2 364 909 generated within the project will allow better comprehension of the Project Duration events linking dependence receptors to tumour progression and provide 36 months targets to modulate apoptosis of tumour cells. This will provide valuable Starting date data to identify Dependence Receptors that could be involved in other 01/11/2006 types of cancer. Instrument STREP

CANCER | 225 HI-CAM

Development of a high-resolution Anger camera for diagnosis and staging of cancer diseases based on state of the art detector technology

Keywords Technological sciences, health sciences, Problem physical sciences, medical imaging The state-of-the-art in the fi eld is represented by a range of commercial systems, usually having large fi eld detectors (~40x50 cm2). These systems are best exploited while performing whole-body SPECT studies since their large, heavy PMT-based detector heads and bulky gantries present diffi culties in operating close to the patient’s skin for dedicated studies of specifi c, small tissues such as in parathyroid imaging, brain Summary scanning and investigation of kidney cancer in infants. In a realistic clinical setting, at an imaging distance usually rather greater than 20 cm, The purpose of the project is the development of a compact and high- the overall effective spatial resolution is typically 10-16 mm (7-10 mm resolution Anger camera to be used in clinical and research environments for brain studies). When a single detector head is used for dedicated and which allows earlier and more reliable diagnosis and therapy planning scintigraphic studies of small organs, permitting a closer imaging of cancer diseases in specifi c applications where high overall spatial reso- distance, the overall effective spatial resolution is limited by both the lution (less than 3 mm) and system compactness (less than 10 x 10 cm2 intrinsic spatial resolution of the system and the collimator. fi eld of view) are required. The gamma camera is based on the well-established Anger architecture, where a collimator acts as a mechanical sieve for incoming gamma pho- Aim tons, a continuous scintillator uses the energy of each selectively passed gamma photon to generate visible photons, and an array of photodetectors The aim of the project is therefore the development of a new compact emits electric signals in response to the absorption of the visible photons. and high position resolution (<1mm) gamma camera based on the new The improvement of performances is based on the use of a particular type SDD photodetector technology. The fi rst technological objective is the of photodetector, the Silicon Drift Detector (SDD), which has recently dem- development of an extended array of SDDs with large cell size (1 cm2), onstrated its ability to provide better performance, by comparison with characterised by high detection effi ciency to the scintillation light and commonly used photomultiplier tubes. low electronic noise. The low noise level is a result of specialised The camera is intended for use both single-handedly for planar scintigraphic advanced semiconductor processing, as well as of the integration of an studies and inserted in an annular holder (gantry) of small diameter for on-chip JFET in the detector chip which allows us to fully exploit the SPECT imaging. Thanks to its compactness and high spatial resolution, it intrinsic low capacitance of the SDD, by the minimisation of parasitic offers potential applications in early diagnosis of cancer diseases affecting capacitances of the connection between detector and electronics. areas of the human body which can only be imaged with diffi culty using the The other key technological objectives addressed by the project are: large and heavy imaging heads and gantries of commercial Anger cameras. • the realisation of a high-resolution collimator. The aim is to obtain The camera to be developed in the present project also offers promising a parallel hole collimator whose spatial resolution equals ~2 mm at perspectives of integration at the system level with MRI instrumentation, an imaging distance of 5 cm with a sensitivity higher than 20cpm/ thanks to the relative insensitivity of the SDD photodetectors to large uCi. Pinhole collimators will be also realised; magnetic fi elds. • a very compact geometry of the detection module, based on a thin The research activity will be organised as follows: the fi rst two years of substrate where the SDD array and VLSI readout circuits will be the three-year project will be dedicated to the development of the SDD- assembled, and a single CsI(Tl) crystal (potentially substituted by based Anger camera, while the third year will be dedicated to the experi- the more recently introduced LaBr3:Ce) will be coupled to the mentation of the camera in selected imaging applications related to photodetector array; cancer diagnosis and research. • the introduction of a thermoelectric system to attain moderate cooling (~ -20°C) during operation of the gamma camera; • the development of dedicated VLSI electronics for amplifi cation and fi ltering of the detector signals, followed by processing electronics based on FPGA for the event reconstruction; • the design of a compact assembly of the complete Anger Camera. The compactness of the assembly will allow ease of positioning of the instrument close to the patient’s body surface; • the realisation of image-reconstruction algorithms and user interface software running on a common personal computer.

226 | CANCER Expected results Coordinator Nuclear Fields International B.V. Vortum Mullem, The Netherlands • Development of large-areas low-noise Silicon Drift Detectors. Carlo Fiorini Politecnico di Milano Max-Planck-Gesellschaft zur • Development of high-resolution collimators. Milano, Italy Foerderung der Wissenschaften eV • Development of a high-resolution and compact Anger Camera Phone: +39 03 23993733 Munich, Germany based on state-of-the-art technologies. carlo.fi [email protected] • Improved diagnostic capabilities thanks to the use of the camera. University College London London, United Kingdom Partners Ospedali Riuniti di Bergamo CF consulting Finanziamenti Bergamo, Italy Unione europea srl Milano, Italy Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau L’ACN L’accessorio Nucleare S.R.L. Barcelona, Spain Cerro Maggiore (MI), Italy Università degli Studi di Milano PNSensor GmbH Milano, Italy Muenchen, Germany

Principle scheme of the Anger camera based on Silicon Drift Detectors technology developed in HICAM.

Potential applications

• Improved possibility to implement an effective therapy with higher capability to detect as small as possible concentrations of tumour cells. • Effective imaging on reduced volume of the biological tissues: less than 10 x 10 cm2 area of the planar view (in scintigraphic investigations with one single imaging head); less than 10 cm total extent on the coronal plane of the patient’s body being imaged by the acquired scans (after appropriate rotation of the camera/s on the annular holder in the tomographic arrangement). • Measurements on anatomical sites of the target which usually lead to severe space constraints during acquisition of the scan. • Improved use of imaging instrumentation in those conditions where the patient’s age, condition or physical disabilities (e.g. patient on wheelchair) prevent an effective use of large detector heads and gantries without compromising patient comfort. Project number • Brain tumours. LSHC-CT-2006-037737 • Thyroid cancer, particularly differentiated carcinomas, with 99mTc- EC contribution perthecnetate. € 1 715 000 • Parathyroid cancer with 99mTc-sestamibi for preoperative localisation Project Duration of parathyroid adenomas with the aim of surgical planning in MIP 36 months interventions (minimally invasive parathyroidectomy). Starting date • Breast cancer with 99mTc-labelled lipophilic cations (SestaMIBI or 01/03/2007 tetrofosmin). The most promising application of the proposed Instrument system concern its use in preoperative or postoperative sites. STREP – SME Project website In addition, the project offers innovative approaches to the diagnosis of lifecompetence.eu/index.php/kb_1/ tumours in infants and children. io_740/io.html

CANCER | 227 HighReX

High Resolution X-Ray Imaging for Improved Detection and Diagnosis of Breast Cancer

Keywords Mammography, breast imaging, photon counting, Problem tomosynthesis, contrast mammography, breast cancer, dual-energy The incidence of breast cancer currently increases in all European countries: according to the European Breast Cancer Network (EBCN), every year 50,000 women are diagnosed with breast cancer. Around 40 % of these women will die from the disease, making it the second most common cause of death for women between the ages of 20 and 70. The most effi cient weapon against breast cancer is currently early Summary detection through mammography screening. An early detection makes the subsequent therapy more successful and also mitigates bi-effects Breast cancer is currently the most common cause of death from cancer and facilitates breast conserving surgery in contrary to mastectomy. for women below 70 years of age, and currently over 100 million European There is currently scientifi c evidence for a decrease of mortality of women are screened every year for early detection through mammogra- between 30 % and 40 % in the screened population. phy. The objective of the proposal is to increase the effi ciency in detection and diagnosis of breast cancer and thus to decrease the mortality in Currently fi lm is the most common image receptor in mammography, breast cancer. but this is now being replaced by digital mammography. In mammog- To achieve this we will develop novel imaging methods based on recent raphy screening, 70-90 % of the cancers are detected. The undetected results in the research fi elds of nano-technology, x-ray optics, detector technology and integrated electronics. The new modality, which will be designed by leading European industries and SMEs in these areas, will develop the only European detector platform for digital mammography commercially available today into a leading technology platform for tomorrow. The novel method will provide signifi cantly increased contrast and spatial resolution, compared to current state-of-the-art breast imaging, through elimination of noise from electronics as well as from overlapping tissue and by way of utilising the signal more effi ciently through fast single photon counting integrated circuits. To make sure that the project targets the right issues in breast imaging, experienced mammography doctors from several European breast imaging centres are involved in the project and they will also test and evaluate the new imaging system and compare it to current state-of-the- art mammography, as well as ultrasound and MR imaging of the breast. The clinical trials will involve an enriched population of symptomatic women and the potential impact on European screening for and diagnosis of breast cancer will be estimated from the results.

228 | CANCER Vision of clinical application for breast cancer detection with 3D photon counting tomosynthesis based on research in the Highrex project.

cancers are mainly in women with dense breasts where the contrast Coordinator Walter Heindel resolution of state-of-the-art equipment is limited by overlapping tissue. Münster Universität Recent results from the so called ACRIN trial show that this challenge Mats Danielsson Münster, Germany can to some extent be met with the advent of digital mammography. Sectra Mamea Stockholm, Sweden Ulrich Bick The improvements are however modest and the problem remains. What [email protected] Charité Universitätsmedizin Berlin makes the problem worse is that the risk of breast cancer is almost Humboldt University a factor of three higher for women with dense breasts. Berlin, Germany Partners One way of solving the problem would be to increase the radiation dose Brigitte Séradour to increase contrast resolution. However, in dense breasts, this would also Mikko Juntunen Association pour la Recherche et le increase the noise caused by overlying tissue. Moreover, the radiation Detection Technology Dépistage des Cancers du Sein dose in mammography is a growing concern, and increasing the radiation Li, Finland Marseille, France dose would mean an increased risk of radiation-induced cancers. This is particularly true for women below the age of 50 who, on average, have Edvard Kälvesten Nico Karssemeijer Silex Microsystems AB Radboud University much denser breasts and who, because of their age, are signiﬁ cantly Järfälla, Sweden Nijmegen, The Netherlands more radiation-sensitive compared to older women. Due to the limitations of the current technology, in many EU countries breast cancer screening Roeland van der Burght Ruben van Engen is presently only offered to women older than 50. Artinis Stichting Landelijk Referentie Centrum Zetten, The Netherlands voor Bevolkingsonderzoek Nijmegen, The Netherlands Aim Matthew Wallis Addenbrooke’s Hospital Karin Leifl and We propose to solve the current dilemma in mammography by increasing University of Cambridge Capio Diagnostics the image quality in terms of contrast and spatial resolution while Cambridge, United Kingdom Radiology Sweden Stockholm, Sweden lowering the radiation dose. This will be achieved by using results from fundamental research in nano-technology, x-ray optics and detector Kenneth Young Ulf Strand technology obtained over the last few years. The only European detector Royal Surrey County Avalon Product Development platform currently available commercially for digital mammography will Hospital NHS Trust Helsingborg, Sweden be drastically improved and developed into a detector system for the Guildford, United Kingdom next generation of breast imaging equipment.

Expected results

An increase in breast cancer detection rate in screening of just 1 % in Europe would mean that in the order of 500 otherwise undetected cases would be diagnosed annually, with a potential of 100-300 lives saved. It may however be expected that the increase in detection rate is signiﬁ cantly higher than 1 %, maybe even exceeding 10 %. Project number LSHC-CT-2007-037642 Potential applications EC contribution € 3 635 200 We believe that the competitiveness of the European technology Project Duration platform will manifest itself more strongly in the second generation of 36 months mammography and that the proposed project will be able to deliver Starting date a standard for 3D mammography that will be unsurpassed for quite 01/02/2007 some time to come. There is also no reason why the photon-counting Instrument advantages in mammography should not be beneﬁ cial as well in other STREP – SME imaging applications, such as CT and chest x-ray imaging. This project Project website may provide the example needed for the technology to spread also to www.sectra.se/medical/ those areas. mammography/highrex

CANCER | 229 Immuno-PDT

Immunophotodynamic therapy of cancer: concepts and applications

Keywords Photodynamic therapy, antibody-photosensitiser photosensitisers to the tumour neo-vasculature mediates a rapid conjugates, tumour neovasculature occlusion of blood vessels, thus depriving tumour cells of oxygen and nutrients and triggering an avalanche of tumour cell deaths. As an additional beneﬁ t, lower doses of photosensitiser can be administered, thus reducing problems of skin photosensitivity.

Aim Summary The present project has as objectives the synthesis and conjugation of Photodynamic therapy of cancer, i.e. the generation of reactive oxygen novel infrared photosensitisers to the most promising antibodies against species in the tumour environment which follows the irradiation of suitable vascular tumour antigens obtained by human antibody technology, the photosensitising molecules, is an attractive modality for the selective immunohistochemical characterisation, the biodistribution and imaging ablation of inoperable superfi cial neoplastic lesions. In this project, we targeting in vivo, in order to select the best antibody-photosensitiser have put together a network of academic research groups and companies conjugates to be taken forward into clinical trials as a fi nal objective. for the development of antibody-based targeted photodynamic therapy modalities. In details, the objectives are: The planned research activity starts with the synthesis of novel photo- • the synthesis of novel infrared photosensitisers with suffi cient water sensitising molecules suitable for conjugation to antibodies, and with the solubility (i.e., not sticky to unwanted cells and tissues), which absorb identifi cation of novel human monoclonal antibodies capable of a selective in the near-infrared light spectrum and which effi ciently generate targeting of the tumour neovasculature for immuno-PDT applications. singlet oxygen and/or other reactive oxygen species; Following an extensive in vitro characterisation of the most promising • screening of phage display libraries to fi nd the most suitable antibody-photosensitiser conjugates, the therapeutic potential of the best antibodies for the project; conjugates will be tested in rodent models of cancer, paving the way for • investigation of a novel method for the conjugation of the antibody future clinical applications. and photosensitiser molecules. We propose to investigate the coupling of photosensitisers to antibodies, based on the non- covalent but stable interaction of photosensitising molecules with specifi c antibody fragments or suitable single domain binders; • evaluation of the conjugates in vitro e in vivo. Our novel PDT agents will be extensively tested in vitro, in order to ascertain whether non-covalently bound photosensitisers can retain singlet oxygen production activity upon irradiation.

Problem The agents will be tested in rodent models of cancer and, if successful, will open novel therapeutic opportunities for the selective treatment of Cancer chemotherapy is generally accompanied by severe side effects, superfi cial tumors in accessible body cavities. mainly due to unspecifi c cytotoxicity of classic antineoplastic treatments. Photodynamic Therapy contributes to a signifi cant effi cacy in the treatment of neoplastic and abnormal tissues, using a combination of Expected results photosensitiser, such as porphyrin, chlorin, bacteriochlorin or phthalocy- anine derivatives, and tissue-penetrating visible laser light. Laser light Most importantly, there is a reasonable expectation of a medical benefi t promotes the photosensitiser to its excited state. The photosensitiser, in for cancer patients stemming both directly and indirectly from this turn, interacts with molecular oxygen and returns to its ground state, project: resulting in the generation of the highly localised cytotoxic agent, singlet • directly, since immuno-PDT procedures promise to be invaluable oxygen, which ultimately affords tumour destruction. for the selective ablation of inoperable superfi cial neoplastic lesions, such as certain head&neck, gastrointestinal, urogenital and PDT is a modality of cancer treatment that causes cytotoxic action only gynecological tumours; locally in the region of exposure to laser light with a specifi c wavelength • indirectly, since the knowledge generated by the validation of matching the absorption profi le of the photosensitiser, thus leading to novel antibodies for vascular targeting applications is likely to have very site specifi c toxicity. The targeted delivery of photosensitisers to an impact in other forms of immunotherapy, including the use of suitable neoplastic sites is likely to increase the scope and the effi cacy of full IgGs and antibody-cytokine fusions for cancer therapy. PDT therapy still further. The antibody-mediated targeted delivery of

230 | CANCER Keywords xxxxxxxxxx

Potential applications

When considering immuno-PDT applications in oncology in a strict sense, the potential market is directly determined by the incidence of inoperable superﬁ cial neoplastic lesions, such as certain head&neck, gastrointestinal, urogenital and gynecological tumors, which would beneﬁ t from a PDT-based ablation.

In a broader sense, discoveries in terms of new tumour targets, new antibodies, new coupling methodologies and new photosensitisers will beneﬁ t several areas of biomedical development, including non- oncological indications such as potentially-blinding angiogenesis-related ocular disorders (age-related macular degeneration, diabetic retinopathy).

Coordinators Dr. Gokhan Yahioglu Photobiotics Chiara Falciani London, United Kingdom Reinerio Gonzalez Philogen Luciano Zardi Siena, Italy Centro Biotecnologie Avanzate [email protected] Genova, Italy [email protected] Dario Neri Swiss Federal Institute of Technology Partners Zurich, Switzerland

Ross Boyle Peter Vajkoczy University of Hull University of Heidelberg Hull, United Kingdom Heidelberg, Germany

Mahendra Deonarain Imperial College London London, United Kingdom

Christina Kousparou Trojantec Nicosia, Cyprus

Project number LSHC-CT-2006-37489 EC contribution € 3 000 000 Project Duration 36 months Starting date 01/10/2006 Instrument STREP – SME Project website www.immunopdt.net

CANCER | 231 INCA

The role of chronic infections in the development of cancer

Keywords Virus, bacteria, HPV, HTLV, HCV, EBV, KSHV, Aim HHV8, Helicobacter pylori The INCA project will investigate the role of six of these infectious agents – EBV, KSHV/HHV8, HPV, HTLV-I, HCV, and HP – in the pathogenesis of infection-associated cancer. In addition, the co-factor role of enterohepatic HP will also be investigated.

The INCA Integrated Project aims towards a better understanding of the Summary molecular and cellular circuits involved in the development of cancers caused by these infectious agents, of the mechanisms of long-term Approximately 17 % of the human cancer cases occurring worldwide are persistence of these infectious agents in apparently healthy hosts, and of caused by infectious agents, in particular by viruses, bacteria and some genetic factors contributing to the development of these types of cancer. parasites. Using a multidisciplinary approach, the INCA project will investigate the role of six of these infectious agents in the pathogenesis of infection-associated cancers. Expected results

Based on this knowledge, INCA will develop and validate animal models to study chronic infl ammation and cancer progression, and new diagnostic procedures for the identifi cation of infected individuals likely to develop infection-associated malignancies. This will ultimately lead to the identifi cation of new drugs and procedures to interfere with processes that are central to the development of infection-associated cancer. The results of this joint effort will contribute to the understanding Problem of malignant transformation and provide new tools to address an urgent socio-economic and human need. To date, nine infectious agents have been recognised as human carcinogens by the International Agency for the Research on Cancer: Epstein-Barr virus (EBV), Kaposi sarcoma herpes virus (KSHV), Human papillomavirus (HPV), Potential applications Human T-cell lymphotropic virus (HTLV-I), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Helicobacter pylori (HP), Schistosoma haemotobium and liver Diagnostics and therapy. fl ukes (Opisthorchis viverrini, Clonorchis sinensis). These nine infectious agents are responsible for about 17 % of cancer cases worldwide, i.e. approximately 1.6 million newly diagnosed cases of cancer annually. In addition, more recent epidemiological evidence suggests that Chlamydiae could play a co-factor role in the development of cervical and lung cancer, and an involvement of enterohepatic Helicobacter in hepatobiliary tumours has been suggested. Moreover, infection-associated cancer is also of increasing importance in immunosuppressed individuals, i.e. transplant recipients and AIDS patients.

Project number LSHC-CT-2005-018704 EC contribution € 12 400 000 Project Duration 48 months Starting date 01/01/2006 Instrument IP Project website www.inca-project.org

232 | CANCER Coordinator Agnès Labigne Werner Zwerschke Mario Luppi Institut Pasteur Austrian Academy of Sciences Università degli Studi Thomas F. Schulz Paris, France Institute for Biomedical di Modena e Reggio Emilia Hanover Medical School Ageing Research Experimental Haematology Laboratory Dept of Virology Francis Megraud Innsbruck, Austria Modena, Italy Hanover, Germany Université Victor Segalen [email protected] Bordeaux 2 Luc Willems Olle Nilsson C.H.U. Bordeaux National Fund for CanAg Diagnostics AB Bordeaux, France Scientifi c Research Majnabbe Terminal Partners Faculté Universitaire des Gothenburg, Sweden Jean Crabtree Sciences Agronomiques Sebastian Suerbaum University of Leeds Gembloux, Belgium Bruno Cucinelli Hanover Medical School St James’ University Hospital Annette Ringwald Institute of Medical Microbiology Leeds, United Kingdom Vincenzo Ciminale Carole Amroune Hanover, Germany Università degli Studi di Padova ARTTIC SA Jose Machado Padua, Italy Paris, France Charles Bangham Institute of Molecular Paul Farrell Pathology and Immunology Ralph Grassmann Harald Mischak Imperial College of Science University of Porto Friedrich-Alexander Universität Mosaiques Diagnostics Technology and Medicine Porto, Portugal Erlangen-Nürnberg & Therapeutics AG Wright Fleming Wing Institute of Clinical and Hanover, Germany London, United Kingdom Susanne Kruger-Kjaer Molecular Virology Danish Cancer Society Erlangen, Germany Michael Pawlita Maria Masucci Institute of Cancer Deutsches Krebsforschungszentrum Karolinska Institutet Epidemiology Lawrence Young Heidelberg, Germany Stockholm, Sweden Copenhagen, Denmark University of Birmingham Medical School György Kéri Thomas F. Meyer Massimo Tommasino Institute of Cancer Studies Vichem Chemie Research Ltd Max Planck Society for Silvia Franceschi Birmingham, United Kingdom Budapest, Hungary the Advancement of Science International Agency for MPI für Infektionsbiologie Research on Cancer George Mosialos Jochen Koenig Berlin, Germany World Health Organisation Aristotle University of Thessaloniki Translating 135 Lyon, France Thessaloniki, Greece Genedata AG Thomas Iftner Basel, Switzerland Frank Stubenrauch Pidder Jansen-Dürr Paivi Ojala Eberhard-Karls-Universität Tübingen Amynon Biotech GmbH University of Helsinki Staffan Normark Institut für Med. Virologie Innsbruck, Austria Biomedicum Helsinki Birgitta Henriques-Normark Tübingen, Germany & Haartman Institute Swedish Institute for Infectious Helsinki, Finland Disease Control Solna, Sweden

CANCER | 233 KidsCancerKinome

Selecting and validating drug targets from the Human kinome for high risk paediatric cancers

Keywords Kinases, Pediatric Cancers, New Drugs Aim

The overall aim of the KidsCancerKinome project is to systematically explore the human kinase family for targeted therapy development for children with cancer can be broken down in the following objectives.

• In silico analysis of the ITCC microarray database (in which we Summary established the expression profi les of 600 childhood tumours), for expression profi les of all protein kinase family members. The Selecting and validating drug targets from the human kinome for high risk expression patterns will be used to prioritize the analyses of the pediatric cancers. KidsCancerKinome will make a comprehensive analysis protein kinases. of the human protein kinase family. Protein kinases are already excellent • Functional high-throughput screening of the full >500 member targets for many small inhibitory molecules and antibodies designed for kinase gene family for essential protein kinases in 24 childhood adult tumors. cancer cell lines, using a kinase-specifi c viral siRNA library and Six aggressive childhood tumors (neuroblastoma, medulloblastoma, bar-coding-based read out system. rhabdomyosarcoma, osteosarcoma, Ewing tumor, acute lymphocytic • Screening of extended cell line panels of the six selected tumour leukemia) will be addressed, which are responsible for 50 % of childhood types for dependency on known cancer-related protein kinases and cancer deaths. Viral shRNA libraries will be applied to test the entire human the protein kinases identifi ed in step 2, by single-kinase siRNA. kinase gene family for tumor-driving kinases in cell lines. They will • Mutation analysis of ‘tumour-driving’ protein kinases (identifi ed in subsequently be analyzed for mutations and functional parameters in large steps 1-3) in series of paediatric tumour samples. cohorts of tumor samples. siRNA mediated inactivation in larger cell line • Analysis of large clinical series of tumour samples for presence panels will critically validate suitable kinases as drug targets. and activation status of protein kinases identifi ed in step 1-3 by Novel kinase inhibitors being developed for adult oncology will be tested immunohistochemistry. for in vitro activity against the tumor-driving kinases. When no inhibitor is • In vitro testing of available small molecules for inhibition of the available, a novel generation of siRNA based nucleic acid drugs (LNAs) will protein kinases identifi ed in step 1-3. be applied. Successful compounds will be taken further to in vivo validation • In vitro testing of LNA kinase inhibitors for protein kinases identifi ed in established xenograft models of the six childhood tumor types. in step 1-3 for which no small molecule drugs are available. • Validation of selected small molecule and LNA kinase inhibitors in nude mouse transplants of childhood tumours. • Pharmacokinetic and pharmacodynamic studies of LNA’s for protein kinases selected in step 1-7 to identify LNA antagonist kinase inhibitor for the drug development pipeline of Santaris.

Problem Expected results

Each year 15 000 European children are diagnosed with cancer and KidsCancerKinome will contribute to a better understanding of the 25 % die of this disease. Survivors frequently suffer from late side-effects unique pediatric tumor biology and to the development of new drugs. of current treatments regimes.

Translational research of childhood tumors to identify molecular targets Potential applications for novel generations drugs is therefore urgently needed. In addition, novel targeted drugs currently developed for adult tumors have to Identiﬁ cation of new drugable targets and development of new speciﬁ c become available for children. Indeed, the EU will in 2007 launch treatments for childhood malignancies. a Pediatric Medicines Regulation to stimulate drug evaluation in children. Nine European research centers devoted to molecular-biologic and pharmacologic studies of childhood cancers and two SMEs therefore engaged in the KidsCancerKinome project.

234 | CANCER Coordinator Dr. Janet Shipley The Institute Of Cancer Research Gilles Vassal Royal Cancer Hospital Institut Gustave Roussy London, United Kingdom Villejuif, France [email protected] Prof. Olivier Delattre Institut Curie Paris, France Partners Dr. Claudia Lanvers-kaminsky Prof. Huib Caron Westfälische Wilhelms- Academisch Medisch Centrum Universität Münster Emma Children’s Hospital Amc Münster, Germany Amsterdam, The Netherlands Prof. Torsten Pietsch Expression of human kinases in 6 pediatric malignancies (neuroblastoma, Dr. Steve Clifford Universitätsklinikum Bonn medulloblastoma, rhabdomyosarcoma, osteosarcoma, Ewing tumours University Of Newcastle Upon Tyne Bonn, Germany and acute lymphoblastic leukemias). Newcastle Upon Tyne, United Kingdom Dr. Massimo Serra Dr. Monique Den Boer Istituti Ortopedici Rizzoli Erasmus Mc Bologna, Italia Sophia Children’s Hospital Rotterdam, The Netherlands Olivier Degrand France Europe Innovation Le Vesinet, France

Troels Koch Santaris Pharma A/s Horsholm, Denmark

Project number LSHC-CT-2006--037390 EC contribution € 3 415 414 Project Duration 36 months Starting date 01/11/2006 Instrument STREP Project website www.KidsCancerKinome.org

CANCER | 235 LIGHTS

Small ligands to interfere with Thymidylate Synthase dimer formation as new tools for development of anti-cancer agents against ovarian carcinoma

Keywords Ovarian cancer, thymidylate synthase inhibitor, Problem small molecule inhibitor design Ovarian cancer is the fi fth most common cause of death from cancer and the most common cause of death from gynecologic cancer in women of all ages in the Western world. Single-agent carboplatin (cDDP) has been considered a reasonable option for fi rst-line chemotherapy for ovarian cancer. However the occurrence of resistant cell populations in the tumour, limiting the usefulness of the platinum drug, represents Summary a growing problem. Resistant cells often become refractory to the initially used drugs and are extremely diffi cult to eradicate, therefore the Ovarian cancer is the fi fth most common cause of death from cancer in use of drug combinations is necessary. women. The standard fi rst-line treatment is a combination of paclitaxel and carboplatin (DDP) or carboplatin alone. In the case of progressive The combination of cDDP and antifolates such as azidothymidine (AZT), disease or drug resistance treatment with platinum, either alone or in a deoxythymidine analogue or, more recently, pemetrexed (Alimta) that combination, especially investigational compounds should be used. The inhibits three enzymes in the de novo purine and pyrimidine pathways, mechanisms behind acquired resistance to cDDP and its derivatives are has been shown to synergistically affect the growth of human ovarian not clear yet, although it is evident that the process is multifactorial, carcinoma cells resistant to cDDP. Due to the role played by the enzymes including enhanced DNA repair. In the human ovarian carcinoma cell of DNA synthesis and repair in the occurrence of cDDP-resistance, it line A2780, a three-fold-DDP- resistance was associated with cross- seems of great priority to develop clinical reagents designed to limit the resistance to the thymidylate synthase (TS) inhibitor 5-fl uorouracil and intracellular level of TS protein which is associated with clinical resistance, to methotrexate, a 2.5-fold increase in TS, and an increase in the thus sensitising even resistant cells to the effects of anti-cancer drugs. intracellular pools of the TS cofactor 5, 10-methylentetrahydrofolate The ultimate aim of LIGHTS is to directly halt tumour progression and and of tetrahydrofolate. interfere with the development of drug resistance upon treatment with The ultimate goal of LIGHTS is to directly halt tumour progression and the platinum-derived drugs by inhibiting the protein regulatory function of development of drug resistance upon treatment with platinum-derived TS through small molecule cellular perturbation. drugs, by inhibiting the protein regulatory function of monomeric TS through small molecule cellular perturbation. The scientific and technological objectives will be to design small-ligand libraries to bind to Aim the TS monomer (dimer interface) and thereby disrupt TS. The strategy will include, systems pathway analysis, protein SH-labelling to identify The project is clearly oriented to halt directly the progression of ovarian low-affi nity ligands, peptide mimic design and synthesis, and fi ltering for cancer and to interfere with the development of drug resistance, upon ADME properties. treatment with platinum-derived drugs, by inhibiting the protein The multidisciplinary approach will be carried out by a Consortium regulatory function of monomeric TS. The intermediate objectives are integrating molecular modelling, chemistry, chemoinformatics, structural based on employing novel medicinal chemistry strategies to identify Biology and pharmacology, and will apply the knowledge being created potential drug candidates with new mechanisms of action. LIGHTS by genomics and other fi elds of basic research to the problem of discovery specifi cally addresses early-phase medicinal chemistry issues that can of anti-cancer agents. The Consortium consists of six groups from fi ve critically infl uence the time schedule for obtaining an investigational different countries, including three SMEs. drug candidate. Nevertheless it is also expected as products of our project that potential drug candidate(s) with a high-quality in vitro activity profi le can be obtained ready for in vivo pharmacology profi ling.

In particular, LIGHTS objectives are: • derivation of small-ligand libraries with ligands designed to bind to the thymidylate synthase monomer/monomer interface affecting dimer formation and TS- mRNA interactions; • validation of the integrated, multidisciplinary drug design strategy necessary to achieve objective 1, which poses a highly challenging design problem. The strategy includes systems pathway studies, protein cysteine SH-labelling to identify low-afﬁ nity ligands, peptide mimetic design, and ﬁ ltering for ADME properties;

236 | CANCER Cisplatin-resistant human ovarian carcinoma cells stained with Toluidine blue and observed with a Zeiss axiophot light microscope in a phase contrast mode.

• identifi cation of small-ligands identifi ed in a chemical-biology Coordinator Paolo Lombardi approach as effective perturbing agents to investigate the mecha- Naxospharma srl nism of resistance against a panel of cis-platinum-resistant ovarian Maria Paola Costi Cesate (MI), Italy carcinoma cell-lines; University of Modena and Reggio Emilia Hannu Myllykallio • development of potential drug candidate(s) with new mechanisms Modena, Italy Institut de Génétique of action for further development as safer therapeutic agent(s) for [email protected] et Microbiologie the treatment of ovarian carcinoma. Université Paris-Sud Orsay, France Partners Expected results Massimo Baroni Rebecca Wade Molecular Discovery The project is clearly oriented to halt directly the progression of ovarian EML Research GmbH Ponte San Giovanni (PG), Italy cancer and to interfere with the development of drug resistance, upon Heidelberg, Germany treatment with platinum-derived drugs, by inhibiting the protein regula- Robert Stroud UCSF-Genentech Hall, UCSF tory function of monomeric TS. The intermediate objectives are based San Francisco, USA on employing novel medicinal chemistry strategies to identify potential drug candidates with new mechanisms of action. LIGHTS specifi cally addresses early-phase medicinal chemistry, critically infl uencing the time schedule for obtaining an investigational drug candidate. Nevertheless, it is also expected that potential drug candidate(s) with high quality in vitro activity profi le can be obtained for in vivo pharmacology profi ling.

Our Consortium is clearly committed to this ﬁ nal aim, and the involvement of the SMEs Naxospharma, Molecular Discovery and EML provides expertise in discovery and synthetic chemistry support, lead development, intellectual property issues, searching for out-licensing and/or co-operative opportunities for the inventive aspects of the project.

Potential applications

The proposed research can lead to technical developments and innovation, which could have a large impact on the biotech industry. The selection of the participating SME partners guarantees that the new knowledge – methods and potential drug(s) candidate(s) – will be transformed into new technology and new products. The Consortium, in addition to its contribution to the project’s basic research by providing a suitable discovery chemistry programme, will be responsible for further development of the selected promising new chemical entities, comprising intellectual property protection, and chemical and pharmaceutical development. Project number LSHC-CT-2006-037852 EC contribution € 1 902 150 Project Duration 36 months Starting date 01/10/2006 Instrument STREP – SME Project website www.lights-EU.org

CANCER | 237 MAMMI

Mammography with molecular imaging

Keywords PEMT, PET mammography, molecular imaging, Problem breast cancer diagnosis, chemotherapy Breast cancer is the most common non-skin cancer and the leading cause of cancer death in women. The best condition for successful breast cancer treatment is early detection. Some studies indicate that early breast cancer detection has reduced the disease mortality by about 29 %. The ability to deﬁ ne the extent of disease, to monitor response, and to predict tumour behaviour in patients with breast cancer are Summary therefore important public health problems.

The proposed project focuses on the development of a PET prototype Conventional methods for breast cancer imaging like X-ray mammography, dedicated to the examination of breast cancer, using a gamma ray sensor ultra-sound and Magnetic Resonance Imaging (MRI) produce morphologic based on an innovative design and the new generation of photo-detectors and structural images, show lesions (like micro-calciﬁ cations), but not and scintillating crystals. The innovative features of the PEMT (Positron cancers. On the contrary, imaging methods based on Molecular Imaging Emission MammoTomography) system we propose will imply a high show functional images, metabolism. This implies that they are much resolution (pushed to the physical limit), higher sensitivity and lower cost. more sensitive and hence can be used to detect and locate malign It includes integrated analogue and digital electronics through the design tumours at an early stage. For instance, PET (Positron Emission Tomography) of an ASIC chip. The main application will be early breast cancer diagnosis is a powerful tool for non-invasive molecular imaging diagnostic based on and evaluation of chemotherapy response. New radio-tracer molecules will gamma ray (emitted by an isotope compound, previously administered to be searched for the detection and visualisation of the pharmacokinetics of the patient) detection. breast tumours, more specifi c than glucose (FDG) for breast cancer, and based on human amino and fatty acids. Phase I Clinical trials will be performed with the new radio-tracers. A clinical multi-centric validation will Aim be performed for the PEMT prototypes. • Design and development of a dedicated low cost PET camera prototype for breast examination with an intrinsic resolution of less than 1 mm, high sensitivity, and tomographic 3D reconstruction. • Study of new and more speciﬁ c radio-pharmaceuticals for breast cancer detection and therapy monitoring (FLT, FAS, etc.). Perform phase I clinical trials of the radio-tracers. • Clinical multi-centric validation of the new PEMT prototype.

Expected results

MAMMI proposes a new PET device speciﬁ cally designed for breast cancer diagnosis and evaluation of therapy response. The dedicated breast cancer PET camera will improve the position resolution of current whole-body PET cameras (about 5 mm) and will push it to the physical limit (slightly below 1 mm).

The new detector design will also have the ability to detect the depth of interaction of the gamma ray interactions within the crystal with a resolution better than 3 mm. This is an essential feature since it allows for an improvement of the ﬁ nal image resolution by almost eliminating the parallax error present in current PET detectors. This is essential in the case of breast examination since the detector cameras are placed close to the body, to increase the sensitivity.

238 | CANCER The advantages of the new generation of photo-detectors (silicon Coordinator Johann Hauer photo-multipliers), such as their compactness, will be explored. The Fraunhofer-Institut für design of the electronics, including an ASIC, will allow an acquisition Jose Mª Benlloch Integrierte Schaltungen rate capability of the order of 1 MHz, with minimum dead time, to cope Consejo Superior Erlangen, Germany de Investigaciones científi cas with the higher sensitivity. IFIC- Instituto de Física Corpuscular Pedro Branco

Valencia, Spain DIGI-UTOPIKA LTD Moreover MAMMI will develop and study more speciﬁ c than FDG radio- benlloch@ifi c.uv.es Lourinhã, Portugal tracers for breast cancer diagnostic and therapy monitoring, based on human amino acids (such as FLT) and fatty acids (such as FAS). Norberg Gunnar Partners C-Rad Imaging AB Frösön, Sweden Potential applications Sibylle Ziegler Klinikum Rechts der Isar der Anders Brahme The main application will be early breast cancer diagnosis and evaluation Technischen Universität München Karolinska Institutet München, Germany Stockholm, Sweden of chemotherapy response. New radio-tracer molecules will be searched for the detection and visualisation of the pharmacokinetics of breast Angels Bernabeu Renato A. Valdes Olmos tumours, more speciﬁ c than glucose (FDG) for breast cancer, and based General Equipment for Medical Netherlands Cancer Institute on human amino and fatty acids. Imaging, S. L. Amsterdam, The Netherlands Paterna, Spain

Project number LSHC-CT-2006-037555 EC contribution € 2 500 000 Project Duration 48 months Starting date 01/01/2007 Instrument STREP – SME Project website ifi c.uv.es/mammi

CANCER | 239 MCSCs

Migrating cancer stem cells (MCSCs) in breast and colon cancer

Keywords Colon cancer, breast cancer, migrating cancer Problem stem cells, mouse models, beta catenin, miRNA, kinome, phosphatome, single-cell analysis, epigenetics • Which signal transduction pathways underlie the onset of CSCs? • Which additional genetic and epigenetic factors modulate their invasive behaviour? • How does the tumour micro-environment, and therefore the cancer patient’s genetic background, affect the capacity of MCSCs to successfully invade and metastatise distant sites thus determining good vs. poor prognosis? Summary

Carcinomas of the colon-rectum and breast represent among the most Aim prevalent malignancies in the western world. A stepwise accumulation of genetic alterations in oncogenes and tumour suppressor genes is considered • To prospectively isolate and characterise intestinal and mammary as the driving force behind tumour initiation, progression and metastasis. cancer stem cells. However, although formally correct, this model does not take into account • To elucidate the mechanisms underlying the aberrant behaviour other essential characteristics of human cancers, i.e. tumour heterogeneity of cancer stem cells during tumour initiation and progression to and the role played by a subpopulation of tumour cells, the cancer stem cells malignancy. (CSCs), in determining local invasion into surrounding tissues and distant metastasis. Tumours are not autonomously acting proliferation machines, but are very heterogeneous, both in their morphological and functional aspects. Expected results In fact, an individual tumour shows distinct sub-areas of proliferation, cell cycle arrest, epithelial differentiation, cell adhesion and dissemination. The isolation of intestinal and breast CSCs from both experimental According to this more dynamic model (see Figure), the majority of tumour mouse models and cancer patients. types, and in particular breast and colon cancer, arise within stem cell niches characterised by a tightly coordinated balance between self-renewal, The establishment of expression, genomic and epigenetic signatures for migration, proliferation, differentiation and apoptosis. The initial and rate- CSCs and their micro-environment. limiting mutation affects this balance and leads to a relative increase in stem cells without drastically compromising their differentiation capacity. This The generation of new animal models for breast and colon cancer that imbalance eventually leads to the formation of a partially differentiated and closely reproduce the natural history of cancer stem cells and their heterogeneous tumour mass that, in response to additional somatic mutations progression towards malignancy and metastasis. and micro-environmental factors, progresses towards malignancy. Tumour cells are shed from this heterogeneous mass into the micro-environment. The development of diagnostic and prognostic tests based on the early However, they will refl ect the heterogeneity of the primary tumour, and only detection of MCSCs and the prediction of metachronous metastases in few will retain the necessary plasticity to undergo trans-differentiation and breast and colon cancer patients by speciﬁ c antibodies. enable homing and metastasis in distal organs. This ‘Migrating Cancer Stem Cells’ (MCSCs) model is central to our proposal and experimental plans. Our MCSCs consortium has been designed and assembled to address these issues by exploiting the unique expertise, experimental models and collections of human cancer samples of the different participants, in order to develop tailor-made diagnostic and therapeutic strategies for breast and colorectal cancer patients.

240 | CANCER Coordinator Partners

Riccardo Fodde Alberto Bardelli Erasmus MC/JNI University of Torino Dept of Pathology Torino, Italy Rotterdam, The Netherlands [email protected] Thomas Brabletz University of Erlangen Erlangen, Germany

Manuel Esteller Badosa Fundación Centro Nacional de Investigaciones Oncológicas Carlos III (CNIO) Madrid, Spain

Christoph Klein University of Regensburg Regensburg, Germany

Tumor initiation, progression to malignancy, and metastasis: the ‘cancer stem cell’ view.

Project number LSHC-CT-2006-037297 EC contribution € 2 169 569 Project Duration 36 months Starting date 01/11/2006 Instrument STREP

CANCER | 241 MMR-related cancer

Prevention, Detection and Molecular Characterisation of Mismatch Repair-Related Hereditary Cancers of the Digestive System

Keywords HNPCC, familial gastric cancer, mismatch repair Expected results

Our proposal will thus improve genetic testing, improve early detection of polyps/tumours in individuals at risk for these cancer syndromes and improve clinical management of HNPCC and FGC patients. Summary Potential applications This project focuses on hereditary cancers of the digestive system associated with microsatellite instability, hereditary non-polyposis The assays and the data generated will be set up and used in diagnostic colorectal cancer (HNPCC) and familial gastric cancer (FGC). laboratories and by clinical geneticists all over Europe and the world. Microsatellite instability is the result of a defective mismatch repair (MMR) system. Germline mutations in MMR genes are found in families with HNPCC characterised by development of colorectal cancer and Coordinator Lene Juel Rasmussen extracolonic malignancies, particularly cancer of the endometrium. Roskilde University Evidence is accumulating that also a subset of FGC is MMR-related as Robert M.W. Hofstra Roskilde, Denmark University Medical families have been identifi ed with tumours showing microsatellite instability. Center Groningen Niels de Wind MMR gene mutations have not yet been identifi ed in these families. Dept of Medical Genetics Leiden University Medical Center Groningen, The Netherlands Leiden, The Netherlands [email protected] Raquel Seruca University of Porto Partners Institute of Molecular Pathology and Immunology Lauri A. Aaltonen Cancer Genetics Group Haartman Institute Porto, Portugal Problem Helsinki, Finland

In HNPCC, identiﬁ cation of MMR gene mutations has helped in identifying individuals at risk when a clear pathogenic mutation was found.

Many families, however, in particular those with less penetrant HNPCC, remain genetically unresolved. Furthermore, in a large proportion of families, mutations are identiﬁ ed whose pathogenic nature is uncertain (unclassiﬁ ed variants). Although we have made great progress in the genetic delineation of this cancer syndrome, it has scarcely improved early diagnosis or treatment of cancer.

Project number Aim LSHC-CT-2005-018754 EC contribution The objectives are to improve genetic testing by: € 2 620 200 • determining the role known MMR genes play in both cancer Project Duration syndromes and identifying new HNPCC or FGC-related genes; 36 months • setting up comprehensive functional assays to determine the role Starting date of unclassifi ed variants in MMR related genes; 01/02/2006 • identifying tumour cells at very early stages in faeces by enhancing Instrument the sensitivity of MSI determination; STREP • profi ling mutations accumulating in tumours as a consequence Project website of MMR defi ciency, in order to get a better insight in tumour devel- www.rug.nl/umcg/onderzoek/ opment, which can be instrumental in clinical management/tumour internationaleprojecten/ treatment. europeseprojecten/mmr/index

242 | CANCER MSCNET

Myeloma Stem Cell Network: a translational programme identifying and targeting the myeloma stem cell

Keywords Multiple myeloma, cancer stem cell, Problem clonal disease progression MM is at present an incurable disease, for which effective new therapies are being actively sought. It is by no means clear, however, what the nature of clonal propagation is in MM. To progress work in this area, the MSCNET has set out to identify the nature of the cell underlying disease survival and persistence.

Summary One of the most striking concepts emerging in cancer biology is a role for cancer stem cells (CSCs) in feeding malignant cell growth and tumour Multiple myeloma (MM) is a disease where malignant plasma cells maintenance. By defi nition, these CSCs have an indefi nite self-renewal accumulate in the bone marrow. Normal plasma cell development at this potential, and are able to populate both their own pool and the growth site is thought to refl ect a synchronous terminal differentiation of B cells of the tumour. Although the fi rst indication that such a cell might exist that have followed sequential stages of maturation. In MM, however, came from studies in leukaemia, evidence for CSCs in solid tumours lends disease characterisation has revealed a number of phenotypic and further support for the concept of a myeloma stem cell. The question for molecular features that suggest the existence of a clonally related ‘less the MSCNET is whether such a stem cell exists in MM. mature’ cell, and the question arises whether this may be a stem cell critical to propagating disease. To address this, the MSCNET has formulated a strategy which includes Aim genomic and proteomic approaches, in order to examine the nature of the cell underlying MM disease origins and progression. This will utilise Based on the above hypothesis, the aims will be: both in vitro and in vivo models of the disease, and examine MM at • to study whether the putative myeloma stem cell (MSC) exists as presentation and during its advance, in order to track factors governing a less differentiated clonally related memory B-cell or as a more disease behaviour in this regard. mature plasma blast/plasma clonogenic cell; • to identify genes of potential impact on stem cell function; • to propose new therapeutic strategies.

Expected results

The results of the scientifi c programme are expected to redefi ne stem cell characteristics and especially to characterize the myeloma stem cell compartment and its progeny, as well as its relationship to the tumour micro-environment. This insight will allow us to examine whether there are any MSC-related features that can be targeted by future specifi c therapies to ablate malignant disease.

Potential applications

Identifying the nature of the MSC will have a profound impact on our understanding of pathogenesis, not only for MM but for all malignant B-cell diseases. Importantly, the MSCNET is well placed to identify potential drug-based approaches to attack MSC and disease progression.

CANCER | 243 Coordinator Alberto Orfao University of Salamanca Hans E. Johnsen Salamanca, Spain Aalborg Hospital, Aarhus University Hospital Surinder S. Sahota Dept of Haematology School of Medicine Aalborg, Denmark Southampton, United Kingdom [email protected] Nico A. Bos University Medical Center Partners Groningen, Netherland

Karin Vanderkerken Bernhard Klein Vrije Universiteit Brussel University of Montpellier Brussels, Belgium Montpellier, France

Pieter Sonneveld Dirk Hose Erasmus University Medical Center University of Heidelberg Rotterdam, The Netherlands Heidelberg, Germany

Zojer Niklas Wilheminenhospital Wien, Austria

Project number LSHC-Ct-2006-037602 EC contribution € 2 734 000 Project Duration 36 months Starting date 01/11/2006 Instrument STREP Project website www.myeloma-europe.org

244 | CANCER NORMOLIFE

Development of new therapeutic substances and strategies for treatment of pain in patients with advanced stages of cancer

Keywords Cancer pain, analgesic medicines, Problem pain pharmacology The approach in which compounds are designed to interact with a wide spectrum of targets involved in pain signal formation and transmission is a new and original therapeutic strategy, opposite to current strategies that use drugs which are as receptor-selective as possible. The project’s major goal is chemical design of new multitarget molecules and analysis of their pharmacological properties that will result in the selection of Summary several new compounds for further clinical evaluation as a new generation of potent analgesics for multicomponent cancer pain treatment. Prolonging the life expectation of patients with advanced cancer could be done by modern medicine. However, progressive pain that is associated with progression of the disease is the major factor that destructs the last Aim moments of life. Severe progressive and uncontrolled pain is a major reason of requesting euthanasia. The applications of oral (morphine) pills The project will involve three general complementary scientifi c objectives: or transdermal patches with lipophilic analgesic drugs are the most chemistry, in vitro biopharmacology and in vivo pharmacology that will common treatments of cancer pain. These compounds penetrating into be accomplished by multidisciplinary teams integrated in the project. central nervous system produce side effects (respiratory depression, constipation, tolerance, sedation, etc.) to such extent that pain treatment is Synthesis of new compounds, designed by theoretical (SAR) analysis, reduced by doctors or refused by the patients. The discoveries of the last will be synthesized in chemical laboratories. Hundred new compounds years indicated the changes in expressions of pro- and antinociceptive will be designed and synthesized on initial stages of the project. These receptors in pathologically changed peripheral tissues as well as in the compounds will be preselected in in vitro tests. The in vitro tests comprise central nervous system. The concerted modulation of these receptors in receptor affi nity evaluation and functional cell-based assays. The combination with designed receptor ligands may block nociceptive signal selected (expected 6) compounds will be fi nally characterized in vivo in formation and transmission more effectively than traditional monotherapies. an animal model of cancer pain. The objective of this project is to focus on the development of new multitarget compounds and methods which will interact with opioid receptors expressed in infl amed and/or pathologically modifi ed tissues. Partial penetration into the central nervous system will result in synergistic pain suppression via interaction between the peripheral and central nervous system. Alternatively, newly developed compounds could be applied directly into central nervous system to interact with the specifi c, pathological set of receptors. The developed compounds will be screened in vitro in a cell silicon hybrid biosensor and selected compounds, in vivo in rodent’s cancer pain models. The project will yield new basic data on structural requirements of analgesics for treatment of persistent cancer pain in advanced stages and will develop new compounds characterized to the stage that will allow to promote them for further clinical phase testing.

Nerve cells cultured on a micro-electrode chip for testing analgetics. (Foto C. and M. Kage)

CANCER | 245 At new cell laboratory (Medical Research Centre, Warsaw).

Expected results Coordinator Dr. Zafi roula-Iro Georgoussi Institute of Biology Prof. Andrzej W. Lipkowski National Centre of Scientifi c The application of a new generation of medicines designed under the Medical Research Centre Research ‘Demokritos’ NORMOLIFE project will reduce side effects generated by traditional Polish Academy of Sciences Athens, Greece opioids in central nervous system, including tolerance, dependence, Warsaw, Poland constipation, euphoria, etc. The major prospective application of the [email protected] Prof. Zofi a Lipkowska results will be a more effective treatment of acute as well as chronic, Institute of Organic Chemistry neuropathic and inﬂ ammatory cancer pain of patients with advanced Polish Academy of Sciences stages of the disease. We predict that the project will be able also to Partners Warsaw, Poland deﬁ ne the differences between the spectrum of pain symptoms and pain progression in progressive cancer typical for female (ovarian) and Prof. Giuseppe Ronsisvalle Dr. Wieslawa Faculty of Pharmacy Walisiewicz-Niedbalska male (prostate) patients. The establishment of such differences will help University of Catania Industrial Chemistry to propose different pain treatment for women and men. Catania, Italy Research Institute Warsaw, Poland Prof. Dirk Tourwe Potential applications Free University of Brussels Prof. Dieter G. Weiss Brussels, Belgium University of Rostock The experimental preclinical data of the newly-developed compounds Rostock, Germany will allow for selection and proposition of new medicines for further Prof. Aleksandra Misicka-Kesik clinical evaluation. Warsaw University Olaf Schroeder Warsaw, Poland Pattern Expert Borsdorf, Germany Prof. Geza Toth Biological Research Centre Frank Graage Hungarian Academy of Sciences Steinbeis-Forschungs- und Szeged, Hungary Entwicklungs-zentrum GmbH Rostock, Germany

Project number LSHC-CT-2006-037733 EC contribution € 2 182 325 Project Duration 36 months Starting date 01/12/2006 Instrument STREP

246 | CANCER ONCASYM

Cancer stem cells and asymmetric cell division

Keywords Drosophila, breast cancer, colorectal cancer, Problem mammospheres, crypt, tissue array, larval neuroblast, sensory organ precursor Cell types within a tumour vary in their ability to form the whole tumour mass. In fact, only very few cells can give rise to all cells present in the tumour. These so-called tumour stem cells have been characterised in a variety of tumours, including leukaemia, breast cancer and brain tumours. Their existence challenges conventional tumour therapy, which is targeted at destroying rapidly proliferating cells. Stem cells often Summary proliferate slowly and might not be eliminated by such therapies, which might explain the high relapse rate observed for some cancers. An intense line of current investigation into cancer is based on the Alternative therapies that target stem cells are not available. This is connection between tumourigenesis and stem cell biology. Some tumours partly due to our limited understanding of proliferation control in stem may originate from the transformation of normal stem cells, at least in the cells and the lack of appropriate cancer models which mimic the case of blood, breast, skin, brain, spino-cerebellar and colon cancers. In development of tumours from deﬁ ned stem cell populations. addition, tumours may contain ‘cancer stem cells’, rare cells with indefi nite potential for self-renewal that drive tumourigenesis. Interestingly, the same signalling pathways (TGF-beta/BMP, Wnt and Notch pathways) appear to Aim regulate self-renewal in stem cells and cancer cells. Self-renewal occurs through the asymmetric cell division of stem cells, The goal of this project is to develop new therapeutic strategies that tar- which thereby generate a daughter stem cell and another daughter cell that get tumour stem cells. Stem cells are characterised by their ability to divide contributes to populate the developing organ or the growing tumour. In one asymmetrically and thereby form self-renewing and differentiating daugh- of the best understood asymmetric cell division models, the Drosophila ter cells at the same time. Even a slight change in the balance between nervous system, asymmetry is mediated by a biased Notch-dependent these two cell types could dramatically increase the number of daughter signalling event between the two daughter cells. cells created by a stem cell and thereby contribute to tumour formation. ONCASYM partners have recently shown: • that the process of biased signalling during asymmetric cell division is controlled by endocytosis; • that tumours can be induced in mutants with altered stem-cell asymmetric division. In human normal and cancer stem cells, asymmetric cell division is supposed to take place, but it has not directly been proven yet. Furthermore, the role of biased signalling by endocytosis in these stem cells has not been addressed to date. The aim of this project is threefold: • to screen for genes involved in asymmetric cell division of human cancer stem cells; • to characterise the asymmetric cell division of these stem cells by using these candidate genes as markers; • to study functionally the role of the identifi ed candidate genes during asymmetric cell division of cancer stem cells. Our ultimate goal is to untangle the molecular machinery of cancer stem cell asymmetric division, thereby providing druggable targets for cancer therapy.

Asymmetric cell division of a Drosophila sensory organ precursor cell. In red: the determinant Pon is asymmetrically distributed. In green: early endosomes. The sensory organ precursor is used in our consortium as a test tube to identiﬁ y factors involved in asymmetric cell division and stem cell division. ONCASYM pays special attention to the role of endosomal dynamics during asymmetric cell division in stem cells and its relevance during tumourigenesis.

CANCER | 247 Mammospheres. These spheroid 3-D structures are clonally derived from a mammary stem cell ex vivo and recapitulate the differentiation programme of mammary tumours. In ONCASYM, mammospheres are used as a suitable ex vivo model system for studying mammary stem cell biology.

Expected results

In our project we plan: • to discover the genes involved in asymmetric cell division and tumour suppression in normal and malignant stem cells, using Drosophila as a model system, and to fi nd their human and mouse homologues; • to use this candidate gene list to fi nd markers for the identifi cation of normal and cancer stem cells in the mammary gland and the intestinal crypts and use these markers to image the asymmetric cell division event.

Potential applications

We will directly translate the acquired knowledge into the clinical practice. • We plan to validate the newly identifi ed ‘stemness’ signature as a clinical tool for genomic grading and prognostic evaluation. This task will be initially accomplished by retrospectively performing meta-analysis, using the currently available public databases as well as the wide collection of cases from our tumour registry. • Tissue microarrays will be used to further validate the relevance of candidate ‘stemness’ genes to the diagnostic routine, by establishing Drosophila as a model system for tumourigenesis. Transplanted tissue into their predictive strength in relation to the common clinical-prognostic Drosophila abdomen (black scars) do not cause tumours (labelled in green) parameters. unless the transplanted tissue is mutant for factors involved in asymmetric cell • Prospectively, the identifi ed ‘stemness’ genes will be introduced as division (right fl y). ONCASYM uses this assay as a gene discovery tool for the ‘biomarkers’ for the clinical and prognostic evaluation of cancer genes involved in tumourigenesis. patients enrolled in ad hoc clinical trials at the European Institute of Oncology, even including their use in the procedure of the sentinel Coordinator Pier Paolo de Fiore node in order to identify the presence of stem cells in metastasis. Istituto FIRC Marcos Gonzalez-Gaitan di Oncologia Molecolare Dept of Biochemistry Milan, Italy University of Geneva Geneva, Switzerland Umberto Veronesi European Institute of Oncology Milan, Italy Partners Jürgen Knoblich Hans Clevers Institute of Molecular The Netherlands Institute Biotechnology of the Austrian for Developmental Biology Academy of Sciences Project number Hubrecht Laboratory Vienna, Austria LSHC-CT-2006-037398 Royal Netherlands EC contribution Academy of Sciences Cayetano Gonzalez € 2 823 800 Utrecht, The Netherlands Institut de Reçerca Project Duration Biomédica 36 months Barcelona, Spain Starting date Galapagos NV 01/10/2006 SME Instrument Mechelen, Belgium STREP Project website www.Oncasym.unige.ch

248 | CANCER ONCODEATH

Sensitisation of solid tumour cells to death receptor-related therapies

Keywords TRAIL, Ras, CRC-colorectal cancer, PI3K, DISC, • Assessment of a role of mitochondrial ﬁ ssion and fusion in TRAIL- choline kinase, aurora inhibitors, caspase-2, mitochondria, mediated apoptosis. mouse models • Selection of PI3 kinase and Aurora inhibitors that cooperate with TRAIL in inducing apoptosis of colon cancer cells. • Assessment of sensitivity of tumours induced by activated oncogenes in transgenic mice and in mouse xenografts.

Summary Expected results

TNF-related apoptosis-inducing ligand (TRAIL) is currently a promising We believe that the principal objective of our project is reachable, for anti-tumour agent with proven activity on several cell and animal cancer the following reasons. The novelty and innovative potential of the models. A unique feature of TRAIL is its specifi city towards malignant ONCODEATH project is based on the strength of its partners, a highly cells while sparing normal cells. At present, preclinical studies with effi cient management and organisation plan, and a novel technology recombinant TRAIL are in progress as well as with an anti-TRAIL-R2 development strategy: all of these address R&D possibilities for cancer monoclonal antibody with no hepatotoxicity. In order to identify specifi c therapy, which derive from very recent fi ndings for the role of additional cell death determinants induced by KRAS, BRAF and PIK3CA oncogenes, mutations in colorectal cancer formation, as well as that the fact that examination of specifi c pathways like B-RAF, MEK, PI3K, Rho, and BCL-2 tumours have intrinsic defective apoptotic pathways, like ‘Achilles’ will be performed. Studies will be further extended to animal models of heal’, that can be exploited per case. tissue-specifi c K-ras-induced carcinogenesis. Combined action of TRAIL with other therapeutic molecules will be ONCODEATH can offer possibilities to test novel signalling drugs alone utilised, in order to succeed optimum effects with minimum concentrations and in combination with a very potent apoptotic ligand TRAIL, in order and toxicity. The potential fi ndings of our study will be determined by to enhance potency and overcome resistance/non-responsiveness of the present research project and will provide mechanistic basis for tumours and fi nally induce tumour-selective death. a pharmacogenomic approach which could be further therapeutically exploited, in order to provide cancer patients with novel personalised therapies in the near future.

Problem

In the last decade, an encouraging decline of the death rate from cancer has been observed, as a result of recent advances in prevention, early diagnosis and therapy. Efforts are made towards the generation of ‘smart’ anti-cancer drugs that will target speciﬁ c molecules, depending on the molecular phenotyping of the patient’s tumour.

Aim

• Panel on new cell lines with up- and down-regulated colon cancer- related oncogenes. • Map of TRAIL-induced proximal signalling pathways per system. TRAIL apoptotic signalling overview in type I and type II cells. • Determinants of caspase-2 activation and Bax in TRAIL-induced Targeted agents in clinical trials are indicated. apoptosis of tumour cells.

CANCER | 249 Keywords xxxxxxxxxx

Incorporation of various inhibitors to help overcome possible kinase resistance to TRAIL induced apoptosis.

Normal cell growth and epithelial like morphology are severely altered once cells enter apoptosis.

Potential applications

We will investigate resistance of pathways to apoptosis present in tumour Coordinator Paul Workman cells and we will aim to sensitise these to cell death by treatment with Cancer Research UK Centre a combination of agents. These pathways will be studied by genomic Alexander Pintzas for Cancer Therapeutics National Hellenic The Institute of Cancer Research technologies, imaging technologies, biochemistry, tumour biology and Research Foundation Sutton, United Kingdom novel therapeutic agents which are prepared on specifi c target and Institute of Biological Research pathway inhibition. The analysis will be performed on representative cell and Biotechnology Spiros Linardopoulos as well as mouse models for colorectal carcinogenesis. Specifi c oncogenic Laboratory of Signal Cancer Research UK, Institute of mutations will be analysed according to the research plan described. Mediated Gene Expression Cancer Research If successful, the efforts should provide landmark discoveries in the fi eld, Athens, Greece Breakthrough Breast which would greatly advance our current knowledge and exploitation of [email protected] Cancer Research Centre defective apoptotic pathways in tumour cells. London, United Kingdom

Partners Jean-Claude Martinou University of Geneva Ladislav Andera Geneva, Switzerland Czech Academy of Sciences Institute of Molecular Genetics Juan Carlos Lacal Laboratory of Cell Signaling Instituto de Investigaciones and Apoptosis Biomédicas Prague, Czech Republic Madrid, Spain

Boris Zhivotovsky Sylvie Robine Karolinska Institutet Institut Curie Project number Institute of Environmental Medicine UMR 144 CNRS/Institut Curie LSHG-CT-2006-037278 Stockholm, Sweden Paris, France EC contribution Georgios Nasioulas € 2 344 900 DTCA HYGEIA Hospital Project Duration Maroussi 36 months Athens, Greece Starting date 01/11/2006 Instrument STREP Project website www.eie.gr/nhrf/institutes/ibrb/ eu-projects/oncodeath/index-en.html

250 | CANCER OVCAD

Ovarian Cancer – Diagnosis of a Silent Killer

Keywords Ovarian cancer, molecular diagnosis, minimal Problem disease, chemotherapy, gene expression, signature, proteomics, CGH, mutation, methylation, targeted therapy Diagnosis at advanced stages and a high mortality rate is the tragedy of ovarian cancer. After initial surgical therapy, 25 % of patients relapse within six months and 75 % die within fi ve years, mainly due to resistance to chemotherapy. Available methods for the detection of recurrent disease lack both sensitivity and specifi city and usually miss minimal disease as a fi rst sign of therapy resistance. Summary Aim In Europe each year, 63 000 ovarian cancer cases are diagnosed and 41 000 ovarian cancer patients die. Seventy-fi ve per cent of patients are The aim of this project is to defi ne clinically useful molecular-orientated diagnosed at advanced stages due to an asymptomatic course and 75 % of early detection of minimal residual disease (MRD) in ovarian cancer that can these patients die within fi ve years. Treatment involves surgery followed by identify patients not responding to the standard (state-of-theart) therapy at chemotherapy. However, 25 % of patients relapse within six months after the time of surgery. This will disburden the patients from the very toxic and initial treatment and there is doubt as to whether these patients benefi t ineffi cient standard chemotherapy and eventually lead to alternative from this therapy at all. Recurrent disease is diagnosed by clinical evidences therapy modalities, which can really bring benefi ts to this group of patients. or by CA125 dynamics. But detection is limited due to a lack of sensitivity ‘Signatures’ that signal the presence of MRD will be investigated at various and specifi city, as is the case with primary diagnosis. molecular levels (DNA, RNA and protein) and in a broad spectrum of Currently, there is no method to detect minimal disease, the fi rst indicator biological materials (tumour tissue, disseminated tumour cells, sera, white of therapy failure and a precursor of recurrence, which inevitably leaves blood cells, ascites) from ovarian cancer patients. specifi c traces throughout the body. There is a strong need for molecular- Specifi cally, the project is aiming at: oriented research to detect minimal disease in order to disburden patients • development and/or validation of several molecular diagnostic from an ineffi cient and toxic therapy. methods to identify MRD in ovarian cancer patients; • defi nition of a new ‘diagnostic state-of-the-art’ by correlating the diagnostic results with the clinically-defi ned response of the patients to standard therapy, consisting of primary surgery, followed by standard platinum/Taxol-based chemotherapy; • early discovery and characterisation of MRD by molecular diagnostics leading to additional therapeutic interventions, ultimately improving patients’ prognosis and quality of life; • better understanding of the mechanisms that cause MRD and therapy failure; • identifi cation and evaluation of new potential therapy targets.

Expected results

Deﬁ nition of a diagnostic method consisting of one or several molecular tests for early detection of minimal disease as an early indicator of therapy failure.

Potential applications

Diagnostic molecular tests and immunotherapy. Ovarian carcinoma – immunohistochemical staining of L1on ovarian carcinoma and its metastases in adjacent organs (Source: J. Sehouli, Berlin; M. Fogel, Rehovot and P. Altevogt, Heidelberg).

CANCER | 251 Coordinator Mina Fogel Kaplan Medical Centre Robert Zeillinger Institute of Pathology Medizinische Universität Wien Rehovot, Israel Dept of Obstetrics and Gynaecology Vienna, Austria Lothar Prix, Andreas Schuetz [email protected] Biofocus GmbH Recklinghausen, Germany

Partners Charles Theillet Centre Régional de Lutte contre Jalid Sehouli le Cancer de Montpellier Dominique Koensgen Montpellier, France Immunohistochemical staining of disseminated tumour cells isolated from blood Alexander Mustea – Comparative genomic hybridisation to detect genomic loss and gain. Charité Viktoria Weber – GeneStiX Imager for analysis of gene methylation (Sources: G. Hager and R. Zeillinger, Vienna; C. Theillet, Montpellier; Biofocus GmbH, Recklinghausen). Universitätsmedizin Berlin Centre for Biomedical Technology Berlin, Germany Danube University Krems Krems, Austria Ignace B. Vergote Toon van Gorp Bernd Mayer Katholieke Universiteit Leuven Emergentec biodevelopment GmbH Leuven, Belgium Vienna, Austria

Jean-Paul Borg Fritz Kury Anthony Gonçalves Labordiagnostika GmbH Institut Paoli-Calmettes Vienna, Austria Institut de Recherches sur le Cancer de Marseille Els Berns Marseille, France Erasmus Medical Center Rotterdam, The Netherlands Peter Altevogt German Cancer Research Centre Hans Dieplinger Heidelberg, Germany Vitateq Biotechnology GmbH Innsbruck, Austria

Burkhard Brandt Universitätsklinikum Hamburg-Eppendorf Institute of Tumour Biology Hamburg, Germany

Project number LSHC-CT-2005-018698 EC contribution € 4 259 625 Project Duration 36 months Starting date 15/12/2005 Instrument STREP Project website www.ovcad.org

252 | CANCER POLYGENE

Inherited risk of breast and prostate cancer

Keywords Breast, prostate, gene, association studies Aim

This project has two major aims: to determine the contribution of polymorphic variants in a large number of candidate genes to the risk of breast and prostate cancer, and to develop efﬁ cient statistical and computational methods for the analysis of genetic and association data. Summary Expected results Studies of cancer families have identifi ed high-penetrance cancer genes such as BRCA1 and BRCA2. However, although these genes have resulted We expect to conﬁ rm or exclude the association of multiple candidate in novel insights into cancer genes and pathways, it is clear that a large cancer genes with breast and prostate cancer in the Icelandic and Dutch component of inherited cancer risk remains unaccounted for. It has been populations. Also, the study may identify novel candidate cancer genes, proposed that common low penetrance cancer susceptibility genes which can translate into novel diagnostic markers for breast or prostate contribute signifi cantly to the genetic predisposition of cancer in a polygenic cancer and possible targets for therapy. In addition, we expect to model of inheritance. Association studies have been suggested as the develop novel statistical algorithms and software for analysis of genetic method of choice for fi nding susceptibility alleles of high frequency but low association data. penetrance. Here we propose to take advantage of accumulating genomic data and two European populations of different history and structure to determine the contribution of candidate cancer susceptibility genes Potential applications to different clinical forms of breast and prostate cancer. We will use a population-based association study in Iceland and Holland to map the Potential applications include a commercial software package for risk profi les associated with common polymorphic variants in and near the analysis of complex genetic data and novel cancer genes to be candidate cancer susceptibility genes in breast and prostate cancer used as predictive markers for breast or prostate cancer risk, or for patients. We will also develop methods for statistical analysis of the developing drugs. resulting data. The proposed study has the potential to cast light on how genetic variants affect the risk of cancer initiation, and how it affects progression and response to treatment. Finally, the results may serve as a starting point for building models of genetic risk of these cancers.

Problem

Although several important genes have been shown to contribute to cancer susceptibility, multiple studies suggest that a major portion of such genes remain to be found. This is particularly true for prostate cancer. The major obstacles to ﬁ nding those genes are the limited size of most studies and the inadequacy of the statistical methods available. Here we Participant in the Icelandic Cancer Project donating a blood sample. will address these problems by studying samples from two large Samples and data are collected by registered nurses. populations of breast and prostate cancer patients and developing novel methods for the analysis of the resulting data.

CANCER | 253 DNA extraction in progress.

Coordinator Partners

Eirikur Steingrimsson Leif Schauser Iceland Genomics Corporation/Urður Bioinformatics ApS Verðandi Skuld Århus C, Denmark Reykjavík, Iceland [email protected] Jotun Hein University of Oxford Oxford, United Kingdom

L.A.L.M. Kiemeney Radboud University Nijmegen Medical Center Epidemiology and Biostatistics Nijmegen, The Netherlands An IGC researcher examines cells in tissue culture under the microscope.

Project number LSHC-CT-2005-018827 EC contribution € 2 962 908 Project Duration 36 months Starting date 01/11/2005 Instrument STREP Project website www.polygene.eu

254 | CANCER PROMET

Prostate cancer molecular-oriented detection and treatment of minimal residual disease

Keywords Prostate cancer, micrometastasis, minimal Problem residual disease, bioluminescence, multiphoton microscopy, nanotechnology, optoacoustic technology, detection, treatment, Prostate cancer is one of the most common malignancies in men: in Europe MegaFasL, human glandular kallikrein 2 approximately 40 000 men die of it each year. Due to the aging population, this number will increase signifi cantly to around 60 000 men by the year 2020. Therefore, prostate cancer is a major medical problem with which the European Community will be increasingly confronted in the forthcoming decades. There are a number of initiatives ongoing to reduce the mortality by detecting the disease earlier, the so-called screening programmes.The clinical evaluation of the usefulness of prostate cancer screening is being Summary examined in the European randomised study of screening for prostate cancer and is expected to answer this question sometime in 2006. Even In the European Union, about 200 000 men are diagnosed with prostate though there is a signifi cant stage migration in the patient population cancer every year and that number is likely to increase due to a growing identifi ed with this disease, mortality has still not dropped in Europe. population at risk due to ageing. Because of the progress made in the This is primarily because when the tumour has locally spread, no curative treatment of the primary tumour, mortality in cancer patients is intervention is available. If the patients are given the time to live, they will increasingly linked to metastatic disease, often hidden (micrometastasis ultimately develop bone metastatic disease that is unresponsive to the or ‘minimal residual disease’) at the time of diagnosis/therapy of the currently available androgen ablation-based therapies. Bone metastases primary tumour. Understanding the complex mechanisms of metastasis cause considerable morbidity characterised by severe bone pain and high (circulating tumour cells – micrometastasis – metastasis) at the molecular incidence of skeletal, neurological and haematopoietic complications and physiological level is crucial for the successful detection of minimal (hypercalcaemia, fracture, spinal cord compression and bone marrow residual disease and for evolving possible strategies for the prevention of aplasia). These, together with the chronic character of terminal CaP disease, their development into overt metastasis. have a severe impact on the socio-economical costs for healthcare. In this project, we intend to elucidate the mechanisms and signature of The objective of this project therefore meets directly with one of the minimal residual disease in prostate cancer and develop novel therapeutic priorities of the life science health programme, namely to combat a major approaches to prevent the development of minimal residual disease to disease, in this case prostate cancer. Prostate cancer is rather unique in its overt metastasis. In close collaboration of basic scientists with clinical clinical behaviour and its molecular genetic background. Relatively few researchers, the pathways of minimal residual disease will be explored consistent mutations have been found, which do not occur in other cancers, using functional genomics and expression profi ling as technology plat- so many cases of indolent tumours are described. forms, advanced experimental models of minimal residual disease using bioluminescence, multiphoton microscopy, nanotechnology and optoacoustic technology for detection and treatment. Innovative imaging and Aim therapeutic strategies developed by the industry and selected for their potential to enhance detection and eradicate minimal residual disease will Because of the progress made in the treatment of the primary tumour by be tested in preclinical models for subsequent clinical evaluation. surgery or radiotherapy, mortality in cancer patients is increasingly linked to The goal is to identify at least two signal transduction targets, to develop metastatic disease. Malignant tumours are known to be heterogeneous, a diagnostic test for the detection of the presence of minimal residual and subpopulations with different invasive and metastatic potential may disease and to defi ne a novel therapeutic strategy for the treatment of this alter their biological properties over time and under treatment due to disease in prostate cancer. Thus, earlier detection and diseasespecifi c genetic instability and epigenetic infl uences. treatment may decrease morbidity and mortality, and ultimately have an The primary tumour releases a large number of cells into the blood stream. impact on socio-economical costs. However, only a small minority (approx. 0.01 %) of the tumour cells entering the blood are thought to be capable of developing into metastatic deposits. The future ability to detect minimal residual disease early, to understand the natural history of micrometastasis and, consequently, to predict outcome, and ultimately to treat adequately will rely on investigational efforts in a context as close as possible to the clinical situation. For this a close interaction between clinical experience and basic research, together with the availability of human tumour tissue specimens from established tumour tissue banks and adequate experimental models are crucial to improve current treatment modalities or even develop innovative therapeutic strategies. In this targeted approach to combat minimal residual disease in prostate cancer, we will pursue various levels at which we attack the

CANCER | 255 malignant process and validate these at a phenotypic and functional level. • the development of a more sensitive bioluminescence-based imaging We will be developing novel means of detecting and treating minimal system for the preclinical investigation of the biology of minimal residual disease. By integrating a variety of state of the art approaches, we residual disease and the in vivo evaluation of novel diagnostic and aim to: therapeutic methods. • identify and validate at least two target genes for detection of minimal residual disease in prostate cancer; • develop an integral in vivo model of minimal residual disease allowing the study of the mechanisms and signatures; Coordinator Dieter Schweizer • evaluate the in vivo detection of minimal residual disease by means of Kontron Medical S.A.S. nanoparticles and optoacoustics; George N. Thalmann Basel, Switzerland • develop a therapeutic strategy for the treatment of minimal residual Dept of Urology University of Bern Manfred Hennecke disease in prostate cancer. Bern, Switzerland Berthold GmbH & Co. KG [email protected] Bad Wildbad, Germany

Expected results Partners Marc Dupuis Apoxis S.A. We expect to identify genes up- or down-regulated in minimal residual Gabri van der Pluijm Lausanne, Switzerland disease with a potential for use in diagnostics and therapeutic strategies. Leiden University Medical Center Furthermore, the expression pattern might increase our understanding of Leiden, The Netherlands Petra Zalud the mechanisms and reveal potential novel therapeutic targets. With this tp21 GmbH work we expect to provide a detection assay with the potential for use in Freddi Hamdy Saarbrücken, Germany University of Sheffi eld clinical practice based on blood, urine or bone marrow aspirate and Sheffi eld, United Kingdom David Deperthes evidence that optoacoustics can be applied in the clinical context. Med Discovery S.A. Novel treatment strategies will be developed and we expect to validate at Marc Colombel Crans-près-Céligny, Switzerland least one treatment strategy in the treatment of minimal residual disease Philippe Clézardin (MRD) that can be applied in the clinical setting. Finally we expect to INSERM Walter Pyerin establish a confocal and deconvolution-based dorsal chamber metatarsal University of Lyon Deutsches Krebsforschungszentrum model for the study of homing and growth support of minimal residual Lyon, France Heidelberg, Germany disease. Further we intend to establish a dual wavelength bioluminescent imaging system for the simultaneous study of two indicators, enabling the evaluation of the interrelation between these.

Potential applications

The innovative potential and impact on industry, the health system and the market lies in: • the development of novel diagnostic methods for the detection of minimal residual disease; • the implementation of optoacoustics with the help of nanoparticles for diagnosis and therapy; Project number • novel targeted therapeutic strategies for micrometastases that take LSHC-CT-2006-018858 into account the particular knowledge about speciﬁ c biology of the EC contribution disease gained from animal models that more closely mimic MRD € 4 034 200 (translational research); Project Duration • the optimisation of experimental imaging of living cells by coupling 48 months multi-photon microscopy with quantum dot nanoparticle cell tracking Starting date to study early pathophysiological pathways involved in MRD. This will 01/04/2006 complement other methods used by the group, such as whole body Instrument animal bioluminescent imaging; STREP Project website www.fp6-promet.net

256 | CANCER TCAC in Cancer

Defects in the Tricarboxylic Acid (Krebs) Cycle

Keywords Hereditary, tricarboxylic acid cycle, fumarase, Aim succinate dehydrogenase, renal, paraganglioma, leiomyoma The two key tasks to achieve this project’s objective are: • characterising the natural history and prevalence of TCAC-defi cient cancers; • unravelling the molecular mechanisms driving TCAC-associated tumourigenesis. Summary Expected results For this project, top European cancer research groups working on the association between defects in the tricarboxylic acid cycle (TCAC) and The impact of the proposed research is two-fold. First, it provides a basis cancer form a consortium to profoundly characterise the human for cancer detection, prevention and treatment in high-risk individuals phenotypes to enable identifi cation and effi cient cancer prevention, and with TCAC defects. The discoveries by us and others linking TCAC defects to unravel the underlying molecular mechanisms. Recent breakthrough to a high risk of tumours are so recent that even the very basic data on the fi ndings by the consortium participants and others have shown that natural history of the respective syndromes is missing and, without this defects in at least four TCAC genes – fumarate hydratase (FH, fumarase), knowledge, evidence-based measures to fi ght this novel tumour type and succinate dehydrogenase (SDH) B, C, and D – can confer susceptibility cannot be developed. Second, the proposed research will create data on to cancer. We shall take these studies forward by characterising the the frequency of TCAC-defi cient hereditary and sporadic cancers, and will natural history of the syndromes in large European materials. characterise the molecular mechanisms underlying their genesis. This will Simultaneously, we shall perform functional studies to elucidate the cellular be a major advancement for cancer research in general. events induced by these defects by systematic biology approaches including functional studies in cell lines, model organisms, and transcription profi ling of TCAC defi cient and profi cient tumours and models. Potential applications TCAC defect-associated expression patterns will be utilised to examine other cancer types for such defects. We have evidence that modifying This project will create data for management of TCAC-defi cient tumours. genes play a key role in TCAC defect-associated tumourigenesis and candidate regions have been identifi ed. Following gene identifi cation, the Diagnosis possible role of these modifi ers in low penetrance cancer predisposition • Improved molecular diagnosis of hereditary susceptibility. in the general population will be examined. The rationale to form this • Classifi cation of tumours. consortium is simple and strong. The consortium brings together the key Also, it will create an expression profi le-based classifi er for TCAC defi cient European cancer researchers studying TCAC-associated tumourigenesis. cancers. Studying the tumourigenic effects of FH and the different units of SDH separately would be ineffective, and formation of the consortium will Management ensure that Europe will maintain the initiative in this new and exciting • Hereditary susceptibility: it goes without saying that an exact fi eld of research. The deliverables arising from the work packages will diagnosis of the syndromes involved is a very signifi cant factor in contribute to the common goals; prevention of TCAC-associated cancers improving the management of the patient, and the relatives. and learning the lessons these lesions can teach to cancer research. Appropriate follow-up strategies and much more accurate genetic counselling on cancer risk will become available. • TCAC-defi cient tumours: whether hereditary or sporadic, TCAC- defi cient tumours are likely to display special biological properties which are relevant for clinical management, including response to drug treatment. We anticipate that the more detailed molecular classifi cation of tumours provided by efforts of this proposal will considerably improve the standard care of such lesions.

CANCER | 257 Immunoﬂ uorescence staining of FH in HeLa cells shows strong mitochondrial (and weak cytoplasmic) protein expression.

Coordinator Hartmut P.H. Neumann Medizinische Universitätsklinik Freiburg Lauri A. Aaltonen Freiburg, Germany University of Helsinki Dept of Medical Genetics Ian Tomlinson Biomedicum Helsinki Molecular and Population Helsinki, Finland Genetics Laboratory [email protected] London Research Institute Cancer Research UK London, United Kingdom Partners Andrzej Januszewicz Peter Devilee National Institute of Cardiology Leiden University Medical Center Warsaw, Poland Human Genetics, Pathology Leiden, The Netherlands Richard Houlston Institute of Cancer Research University of London London, United Kingdom Down left corner is seen papillary renal tumor tissue, which remains negative in FH immunohistochemistry staining. Normal kidney (up right corner) displays FH protein expression (brown).

Project number LSHC-CT-2005-518200 EC contribution € 2 751 000 Project Duration 48 months Starting date 01/01/2006 Instrument STREP

258 | CANCER TRIDENT

Therapeutic molecules for treatment of solid tumours by modulating death receptor-mediated apoptosis

Keywords Solid tumours, apoptosis (programmed cell death), Problem tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), receptor-selective TRAIL mutants, TNF ligand-mimicking With almost 3 million new cases each year and 1.7 million deaths, peptides, intracellular peptide inhibitors, computational cancer is an important public health problem in Europe. The ageing of rational protein design, directed evolution, tailor-made therapy, the European population will cause these numbers to continue to primary tumour culture, cell-based delivery systems of anti- increase. The most common cancers are lung, colorectal and breast cancer genes, GLP/GMP procedures cancers. The available treatments for these solid cancers and the outcome of the treatments are not satisfactory. New therapeutic strategies and novel tumour-selective anti-cancer agents are necessary in order to improve the treatment of these and other solid tumours.

Aim Summary The ultimate aim of the TRIDENT project is to develop novel molecules The effi cacy of current treatments for some types of solid tumours is that target critical apoptotic signalling pathways important in the disappointingly poor. New therapies using novel tumour-selective anti- formation of various solid tumours. cancer agents are necessary. A major aim of anti-tumour therapies is to inhibit proliferation and induce death of tumour cells without affecting The speciﬁ c objectives are: normal cells. In this regard, members of TNF ligand/receptor family are • to develop of novel apoptosis-inducing agonists and proliferation of interest since they regulate both apoptosis (programmed cell death) antagonists using state-of-the art, computer based rational design and cell proliferation. One TNF family member, TRAIL, is of particular and directed evolution techniques; interest since it selectively induces death of tumour cells without affecting normal cells. Currently, TRAIL and TRAIL-specifi c antibodies are being investigated as anti-cancer agents. However, a major drawback to TRAIL’s effi cacy is that it binds to multiple receptors, not all of which transduce an apoptotic signal. Previously, we developed receptor-selective TRAIL variants which are potent inducers of apoptosis in various tumour cells, are more effi cacious than native TRAIL, and display synergistic effect in combination with other chemotherapy treatments or radiotherapy. This proposal will investigate and characterise TRAIL variants pre-clinically in solid tumour models and defi ne new treatment protocols in combination with already proven treatments. Furthermore, we will elucidate the role of other TNF receptor family members in signalling in tumour cell survival/ death. Biochemical and structural characterisation will identify new targets for molecular cancer therapy while computational design and molecular evolution techniques will be used to develop novel receptor- selective apoptosis-inducing agonists. We will also develop intracellular acting agents (intracellular-acting peptides inhibiting pro-survival signals; intracellular peptide inhibitors), which will block the unwanted proliferative/anti-apoptotic signals activated by some TNF members. The tumoricidal activity of these lead molecules will be tested using conventional, viral and cell based delivery strategies which will utilise peptide sequences to specifi cally target them to tumour cells. The objective of TRIDENT is to channel death receptor activation towards tumour cell apoptosis. Activation of cell surface death receptors do not only induce apoptosis of the receptor carrying cell but can simultaneously activate compensatory, pro-survival pathways, which limits the use of this pathway to kill cancerous cells. The TRIDENT project aims to generate TNF ligand variants that only bind to the death-inducing members of the TNF receptor family, not to decoy TNF receptors (selective ligand) and design cell permeable peptide inhibitors (intracellular inhibitors; IC inh) against anti-apoptotic molecules. These novel molecules used in combination can maximize tumour cell killing.

CANCER | 259 • to gain in-depth knowledge on the role of the TNF ligand-receptor level by individual partners. The participants involved are from both family interactions in the carcinogenesis of solid tumours, both at industrial and academic backgrounds and are experienced with working a molecular and structural level; in research networks at a European level. Our application of state-of- • identify high efﬁ ciency combinations of the novel lead molecules the-art technologies will facilitate and accelerate the development of with chemotherapy and/or radiation and conduct pre-clinical new tools and processes in general, for development and production of studies; novel protein/peptide therapeutics. • develop a prediction model and high-throughput primary tumour characterisation to identify patient-speciﬁ c (tailor-made) targeted TRIDENT will assist in the advancement of genetic and protein engineering, treatments; in silico protein design, molecular evolution, protein microarray • devise new tumour targeting techniques by exploiting gene and technology and lab-on-a-chip formats in the development and production cell therapy-based approaches; of lead therapeutic molecules. A greater understanding of molecular • development of new computational and experimental methods mechanisms involved in the control of apoptotic signal transduction in in order to advance potential protein therapeutics faster from tumour cells will be also gained. The integration of this derived knowledge pre-clinical to clinical stage; will ultimately be useful in the context of post-genomic research for • development of an exploitation strategy involving a start-up SME. human health activities. Our focus will be on the market need for alternative, more effective therapies in the treatment of solid tumours, and more particularly for therapeutics with minimal toxic-side effects. Expected results

The anticipated deliverables include the development of novel diagnosis Potential applications technologies and novel therapeutics for intervention in cancer progression through activation of apoptosis as well as technologies to advance Treatment of solid tumours and possibly leukemias, identifi cation of a more rational approach to the design of ‘tailor-made’ therapeutic most effi cient treatments for individual patients, high effi ciency design drugs. This proposal takes a multi-disciplinary approach to develop new of protein mutants and specialised-variants (e.g. receptor selective methodologies and will specifi cally generate unique scientifi c knowledge ligands, agonistic, antagonistic ligands). and industrial technologies that could not be created on a national

Coordinator Wim Quax University of Groningen Afshin Samali Groningen, The Netherlands National University of Ireland Galway, Ireland Steven de Jong [email protected] University Medical Center Groningen Groningen, The Netherlands

Partners Guillermo Montoya Fundación Centro Nacional Ralf Zwacka de Investigaciones Project number National University of Ireland, Galway Oncológicas Carlos III LSHC-CT-2006-037686 Galway, Ireland Madrid, Spain EC contribution € 2 069 000 Luis Serrano Ian Hayes Project Duration Centre de Regulació Genòmica Triskel Therapeutics Ltd. Barcelona, Spain Galway, Ireland 36 months Starting date 01/10/2006 Instrument STREP Project website www.trident-biotech.com

260 | CANCER TRKCancer

The anoikis suppressor TrkB as a target for novel anti-cancer agents

Keywords Metastase, xenografts, tyrosine kinases, biomarkers, Expected results tumour profi ling, siRNA, animal models, drug design • Novel animal models. • Novel TrkB inhibitors as potential anti-cancer drugs. • Novel biomarkers of metastasis.

Potential applications Summary • Novel anti-cancer agents. The failure of anti-cancer therapies generally results from locally intractable • New diagnostic tools and indices of therapeutic efﬁ cacy. invasive growth or from the presence of metastases refractory to treatment with curative intent. A novel therapeutic strategy is to develop new anti- cancer drugs specifi cally targeting the invasive or metastatic phenotype of tumour cells. We propose to validate at the pre-clinical level a strategy that targets the Coordinator Partners critical mechanism allowing apoptosis evasion and survival of invasive or metastatic tumour cells. Anoikis is a process by which a cell detached Dr. Pierre Sokoloff Dr. Daniel Peeper from its resident tissue undergoes apoptosis as a result of loss of normal Institut De Recherche Pierre Fabre Nederlands Kanker Instituut 17, Avenue Jean Moulin Amsterdam, The Netherlands cell-matrix interactions. Loss of anoikis allows survival of cancer cells in Castres Cedex, France abnormal micro-environments, such as tissue compartments invaded by [email protected] Dr. Niko Bausch the primary tumour, and the intravascular compartment, during the meta- Institute For Experimental static process. The BDNF receptor TrkB is a potent suppressor of anoikis Oncology (Oncotest) and is responsible for apoptosis evasion that occurs in aggressive human Freiburg, Germany tumours overexpressing TrkB. The aim of the present proposal is to validate TrkB as a target for new anti- Dr. Wolter Mooï cancer drugs, aiming to restore anoikis and thereby destroy the invasive Vrije Universiteit Medical Center and metastatic cancer cells. Amsterdam, The Netherlands

Problem

• Metastasis is a cause of cancer relapse. • TrkB, as an anoikis suppressor, may favour metastasis, but the relevance of this target has not been assessed in relevant cancer models. • There are no TrkB inhibitors ready for clinical trials. • Biomarkers of metastasis are lacking. Project number LSHC-CT-2006-037758 EC contribution Aim € 2 368 715 Project Duration • To validate TrkB as a target for anti-metastasis agents by using 36 months human tissue-derived animal models. Starting date • To identify novel TrkB inhibitors. 01/01/2007 • To explore mechanisms of action of TrkB inhibitors and to identify Instrument biomarkers of metastasis. STREP

CANCER | 261 Tumour-Host Genomics

Genome-wide Analysis of Signalling Pathways in Regulation of the Interactions between Tumour and Host Cells: Applications of Cancer Therapy

Keywords Tumour, host, signalling, stem cell, Problem angiogenesis, lentivirus Tumour cell growth depends on interactions with its microenvironment. The development of cancer therapies targeting these interactions is hampered by the fact that the molecular mechanisms behind tumourhost interactions are often poorly understood.

Summary Aim

In addition to oncogenic mutations that act cellautonomously, tumour cell • To identify endothelial/BM cell-specifi c cis-regulatory elements for growth depends on interactions with its microenvironment. The tumour use in lentiviral in vivo targeting vectors. microenvironment consists of cells of haematopoietic and mesenchymal • To develop a targeted lentiviral library for the inhibition of selected origin, including infl ammatory cells, stem and progenitor cells, fi broblasts, major cell signalling pathways. endothelial cells and vascular mural cells. Tumour cell growth is known to • To identify tumour-derived factors that lead to increased angiogen- depend on the interaction of tumour cells with such stromal cells. For esis and recruitment of stromal cells contributing to a microenviron- example, a growing tumour needs to recruit normal endothelial and ment permissible for tumour growth. vascular mural cells to form its blood vessels. In addition, tumour cells • To identify host-derived factors that induce tumour cell growth and induce stromal cells to secrete factors that contribute to tumour cell tumour stem cell self-renewal. growth and invasion. Stromal cell-dependent interactions represent an • To test in vivo the effect of targeted lentiviruses in inhibition of attractive target for cancer therapy, because normal cells are genetically tumour growth and metastasis. stable, and would not be expected to develop resistance to therapeutic agents. The development of such therapies is hampered by the fact that the molecular mechanisms behind tumour-stroma interactions are often Expected results poorly understood. In summary, the work plan entails development of novel advanced functional The project aims to develop novel tools and methods to study tumourhos genomic instruments, technologies and methods to study tumour-host interactions in cancer, and to apply these techniques to the identifi cation interactions in cancer, and to apply these techniques to the identifi cation of molecules and processes in normal cells, which could be targeted by of molecules and processes in normal cells, which could be targeted by novel anti-cancer therapeutic agents. In addition, we also propose to novel anti-cancer therapeutic agents. In addition, we will develop targeted develop targeted lentiviruses that specifi cally express genes in bone lentiviruses which would allow in vivo delivery of therapeutic agents into marrow-derived cells and/or in endothelial cells, which would allow tumours. Functional validation of the discovered targets and developed in vivo delivery of therapeutic agents into tumours. delivery systems will be performed in in vivo models of murine tumour growth and dissemination. For purely technical reasons, melanoma and prostate cancer models are planned to be utilised fi rst. However, tumourhost interactions are universally. essential for the growth and dissemination of any malignant disease, and the results of the experiments will be applicable for any kind of human cancer. The work has signifi cant exploitation potential and relevance for health in the understanding of the molecular mechanisms of tumourhost interactions, and in the treatment of cancer.

A model of in vivo bone marrow stem cell differentiation and tumour angiogenesis in mice. The studied bone marrow-derived stem cells originate from GFP-tagged transgenic mice, and are labelled with green ﬂ uorescence. The stem cells are transplanted in syngeneic recipient mice. Syngeneic melanoma tumours are used to induce angiogenesis and cancer growth in the mice, and to study the mobilisation and functions of the bone marrow-derived cells during tumour growth. Endothelial cells (ECs) are detected by using antibodies against EC-speciﬁ c surface markers (red).

262 | CANCER Potential applications Coordinator

Potential target genes for the treatment of cancer will allow the search, Petri Salven and preclinical and clinical validation of respective lead compounds. University of Helsinki Helsinki, Finland [email protected] Tumour-host interactions are universally essential for the growth and dissemination of any malignant disease, and the results of this project could in principle ultimately be applicable for any kind of human cancer. Partners

Peter ten Dijke WP5: Angionic factors, Factors recruiting stromal Leiden University Medical Center and BM derived cells Leiden, The Netherlands

Lentiviral targering (WP7,8) Luigi Naldini San Raffaele Telethon Selected (WP2) and RNAi screen Institute for Gene Therapy identifi ed (WP3,6) signaling pathway Tumor Host Milan, Italy inhibitors Kari Alitalo Validited host-cell Selected (WP2) specifi c regulatory and RNAi screen Jussi Taipale elements (WP4) identifi ed (WP3) signalising pathway University of Helsinki inhibitors WP6: Tumor growth factors Helsinki, Finland

Summary of the science and technology objectives of the project, and the work package structure. The project aims to develop novel tools and methods to study tumour-host interactions in cancer, and to apply these techniques to the identiﬁ cation of molecules and processes in normal cells, which could be targeted by novel anti- cancer therapeutic agents. In addition, we will also develop targeted lentiviruses that speciﬁ cally express genes in bone marrow-derived cells and/or in endothelial cells, which would allow in vivo delivery of therapeutic agents into tumours. Abbreviations: BM, bone marrow; RNAi, RNA interference; WP, work package.

Project number LSHC-CT-2005-518198 EC contribution € 2 700 000 Project Duration 36 months Starting date 01/11/2005 Instrument STREP Project website http://research.med.helsinki.fi / tumorhostgenomics/default.htm

CANCER | 263 VITAL

Development of optimised recombinant idiotypic vaccines for subset-specifi c immunotherapy of B cell lymphomas

Keywords Lymphoma, leukaemia, vaccine, idiotype, Although in the vast majority of patients complete or partial remissions can immunotherapy be obtained with either single agents or combination chemotherapy, the clinical course is characterised by a high relapse rate. After relapse, both the response rate and relapse-free survival after subsequent salvage treatment regimens steadily decrease, resulting in a median survival of only 4-5 years after the fi rst relapse. These clinical fi ndings, coupled with the substantial toxicities of standard treatments, have stimulated the search for novel Summary and more tumour-selective therapies. Therapeutic vaccines targeting B cell lymphoma idiotype (Id) represent a promising immunotherapeutic approach Therapeutic vaccines targeting B cell non-Hodgkin lymphoma (NHL) idiotype for a better clinical control of these malignancies. This strategy is based (Id) represent a promising approach against these malignancies. A broad on the observation that immunoglobulins (Ig) expressed by neoplastic use of Id-based vaccination, however, is hampered by the complexity and B lymphocytes carry unique determinants in their variable regions (idiotypes), costs due to the individualised production of these vaccines. Recent which can be recognised as tumour-specifi c antigens. evidence indicates that these limitations may be overcome. In fact, distinct sets of stereotyped immunoglobulins have been identified in various Indeed, both protein- and dendritic cell-based vaccines that use the B-NHL, suggesting that patients share Id with a higher frequency than patient-specifi c Id have resulted in clinically signifi cant tumour-specifi c appreciated previously. cellular responses with very little toxicity. A broad use of Id-based vacci- Through the complementary and synergistic work of academic partners and nation for B cell lymphomas, however, is hampered by the fact that these three SMEs, we plan to exploit the molecular features of Id proteins of approaches are patient-specifi c, so that the vaccine must be individually distinct B cell lymphomas/leukemias, particularly those pathogenically produced for each patient. On these grounds, new strategies obviating associated with antigen stimulation and/or selection, to develop pre-made, the need to produce customised vaccines would further simplify clinical recombinant Id proteins to vaccinate subgroups of lympho-proliferative applications of idiotypic vaccines. disorders expressing molecularly correlated idiotypes. A database of Id sequences expressed by different B-NHL will be constructed to identify subgroups of tumours expressing molecularly correlated Id Aim proteins. Selected Id proteins will be characterised for their immunogenicity and, particularly, for the ability to induce cross-reactive immune responses The objective of VITAL is the development and production of optimised against related Id proteins. B and T cell epitopes will be identifi ed using recombinant idiotypic vaccines for the treatment of subgroups of lym- innovative approaches, and dedicated assays for immunomonitoring will be phoproliferative disorders expressing molecularly correlated idiotypes. developed. Optimised versions of selected Id vaccines will be produced using These vaccines will be included in new formulations for innovative trials new strategies and validated in animal models. New adjuvants and delivery of immunotherapy potentially targeting a large fraction of lymphoma/ systems for improved Id vaccine formulations and administration will be also leukemia patients. evaluated and validated. The most promising Id proteins will be produced and purifi ed according to GMP standards and included in new vaccine formulations for innovative trials of ‘cross-reactive’ immunotherapy. Expected results

• Establishment of a large database including sequences of idiotypic VH and VL genes expressed by a variety of lympho-proliferative disorders, including low grade B-NHL, autoimmunity-associated lympho-proliferations, and chronic lymphocytic leukemia. This will allow the identifi cation of candidate Id proteins for ‘cross-reactive’ Problem immunotherapy. • Pre-clinical characterisation of the immunogenicity of selected Non-Hodgkin’s lymphomas (NHL) constitute a heterogeneous group of natural Id proteins, with particular regard to their ability to induce malignancies whose incidence has signifi cantly increased in recent decades. immune responses against lymphoma cells expressing molecularly In the year 2000, more than 145 000 cases of NHL were diagnosed in correlated Id proteins. The characterisation will include the developed countries, representing the sixth most common cancer occurring identifi cation of B cell epitopes and HLA Class I-restricted cytotoxic among men and the eighth among women. Low-grade B-cell NHLs, in T cell epitopes using innovative approaches and will allow the particular, are incurable diseases characterised by relatively slow growth and development of dedicated assays for immunomonitoring. excellent initial responsiveness to chemotherapy but also by continuous • Design and validation of optimised Id vaccines. relapses. In particular, for patients with follicular lymphoma, median overall • Evaluation and validation of new adjuvants and innovative delivery survival (7-10 years) has not improved over the past 30 years. systems for improved Id vaccine formulations and administration.

264 | CANCER • ‘Clinical-grade’ production and puriﬁ cation of optimised Id proteins for patient vaccination.

The SMEs are an integral part in the project in making the new diagnostic and therapeutic tools available, not only for Europe but also for the world market. The close integration between clinical and research activities at several university hospitals and cancer centres with the SMEs will form new centres of excellence where European SMEs will beneﬁ t from close collaboration, at the same time as new diagnostic and therapeutic products will be developed to the beneﬁ t of patients with lymphoid malignancies.

Potential applications

The results obtained in the present project will allow the design and activation of phase I/II clinical trials aimed at validating the use of optimised, pre-made vaccines for the treatment of a relatively broad spectrum of lymphoid malignancies. The proposed Id vaccination may be beneﬁ cial also for patients with pre-neoplastic B-cell lymphoproliferations, such as mixed cryoglobulinaemia. These vaccines, in fact, may be used with the purpose of alleviating symptoms and, ultimately, preventing a possible evolution towards an overt B cell malignancy. Once validated as drugs, the vaccines will have the advantage of being easily distributed to all hematology and oncology departments, including those of peripheral hospitals/universities. Thus, results obtained in the present project will have an important strategic impact in solving, at least in part, the dramatic social and health problem represented by NHL. Flow chart, outlining the main expected results, leading from identiﬁ cation of shared idiotypes to the development of optimized vaccines for the treatment of B-cell lymphoproliferations.

Coordinator Maria Masucci Karolinska Institutet Riccardo Dolcetti Stockholm, Sweden Centro di Riferimento Oncologico – IRCCS Antonio Rosato National Cancer Institute University of Padova Aviano, Italy Padova, Italy Project number [email protected] LSHC-CT-2006-037874 Hans Petrus Maria Langedijk EC contribution Pepscan Systems BV € 2 050 000 Partners Lelystad, The Netherlands Project Duration 36 months Bjarne Bogen Nikolai Schwabe Starting date University of Oslo ProImmune Limited and Rikshospitalet Oxford, United Kingdom 01/01/2007 University Hospital Instrument Oslo, Norway Maria Luisa Nolli STREP – SME Areta International Srl Project website Gerenzano, Italy www.cro.sanita.fvg.it/ progetti/vital/index.htm

CANCER | 265

267 ➞ 272

ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES ECRIN-TWG

European Clinical Research Infrastructures Network – Transnational Working Groups

Keywords Clinical research, infrastructure, ethics, regulation, Problem pharmacovigilance, monitoring, data management, education, quality assurance Development of diagnostic and therapeutic innovation, and delivering improved health care to EU citizens requires clinical research during the whole process extending from understanding the mechanism of disease, genetic studies or identifi cation of biomarkers, clinical development and evaluation, and to post-marketing strategy trials. The recent development of therapeutic innovation is mainly based on biopharma- Summary ceuticals and on personalized treatments, on pharmacogenetics and toxicogenetics, on the use of biomarkers, and requires access to large This project is the second step of the ECRIN programme, whose objective populations of patients, enabling clinical trials adapted to these new consists of integrating national clinical research facilities into a EU-wide therapeutic strategies with a need to focus on specifi c patient subpopu- network, able to provide support to clinical research in any medical fi eld, lations. Further, a very large number of rare diseases are without effec- and for any type of clinical research. Through transnational working tive interventions. In addition, the quality of clinical trials and other groups in charge of defi ning procedures and guidelines for multinational clinical investigations, the quality of clinical and biological data, and the studies in the EU, the current project is designed to build an infrastructure rate of enrolment of patients into clinical trials are all requiring urgent for EU-wide clinical trials, which represents the third step of the improvements. Hence, the quality of the clinical research infrastructure programme. This distributed infrastructure, based on the integration of will be one of the main factors determining the competitiveness of competence centres, will provide access to clinical research projects European clinical research. European academic research, as well as the through a set of fl exible services based on its know-how. These services pharmaceutical and biotechnology research and development need are particularly relevant for academic clinical research, or for clinical an effi cient, integrated, and professionalized organization of clinical trials steered by biotechnology SMEs who often lack the capacity to act research, based on competence centers able to provide effi cient support as a sponsor in EU-wide studies. In addition, ECRIN will enable effi cient through a consistent set of services for clinical trials. Infrastructures sup- implementation of the Innovative Medicines Initiative (IMI) strategic porting clinical trials, data management, quality assurance, monitoring, research agenda, providing support to academic sponsors in public- ethics, and regulatory affairs are required for improving the quality and private partnership biomarker or post-marketing, EU-wide studies. raising the credibility of data. An integrated, EU-wide infrastructure will allow the conduct of multinational studies in Europe, taking advantage of the EU population and competencies, unlocking latent expertise and patients currently scattered across the EU member states.

EU-wide disease-oriented networks EU-wide infrastructures cancer brain diabetes infl ammation infection networks Clinical Infrastructures for multinational clinical research: (ECRIN) clinical research centres, cohorts, biobanks, databanks, imaging

Infrastructures for premedical R&D: toxicology models and databases, Preclinical biopharmaceutical manufacturing facilities

Infrastructures for drug discovery: high throuput ‘omic’ facilities, Drug discovery in silico and animal models

Research networks Investigators networks Patients’ registries

Implementation of the Innovative Medicines Initiative strategic research agenda requires Europe-wide infrastructures and disease-oriented networks.

268 | ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES Aim Staring 2008, ECRIN will provide a set of ‘one-stop shop’ services to investigators and sponsors in multinational studies: The European Clinical Research Infrastructures Network (ECRIN) is • support in the interaction with ethics committees; designed to improve the capacity of the European Union to perform • support in the interaction with competent authorities and in high-quality clinical research, and to promote innovative pharmaceutical regulatory affairs; and biotechnology development as well as development of other inter- • support in adverse event reporting; ventions. This integrated clinical research infrastructure bridges the • support in drug dispensing; fragmentation of clinical research in Europe through the interconnec- • support in the circulation of biological samples; tion of national networks of clinical research centers and clinical trial • support in study monitoring; units. ECRIN participants are currently Austria, Denmark, France, • data management; Germany, Hungary, Ireland, Italy, Spain, Sweden, Switzerland, and the • GMP manufacturing of biotherapy products; United Kingdom, with the European Organisation for Research on • patients recruitment and investigation. Treatment of Cancer (EORTC), and the European Forum for Good Clinical Practice (EFGCP). ECRIN plans extension to other existing national networks in other member states, and stimulates the set-up of new (1) Demotes-Mainard J, Ohmann C. European Clinical Research Infrastructures Network: promoting harmonisation and quality in European clinical research. Lancet 2005; 365, national networks for further connections to ECRIN. 107-108. (2) Demotes-Mainard J, Ohmann C, Gluud C, Chene G, Fabris N, Garattini S, Carné X, Lafolie P, Collet JP, Crawley F. European Clinical Research Infrastructures Network Meeting report: ‘Towards an integration of clinical research infrastructures in Europe’, Expected results Brussels, Feb 14-15th, 2005. Int J Pharm Med, 19:43-45, 2005.

The ECRIN project consists of three steps: Potential applications 1 – The fi rst step (ECRIN-RKP 1) was supported by a FP6 grant (health priority, 2004-2005) and resulted in a survey on clinical research Users of this integrated and distributed EU clinical research infrastructure in the participating countries, leading to compare the regulation, will be disease-oriented scientifi c networks, clinical research projects tools and practice, hence to identify the bottlenecks to multina- sponsored by public institutions, SMEs or pharmaceutical companies, tional clinical research 2 (see reports and comparative analyses on and public-private partnership programs. Such an integrated infrastructure www.ecrin.org). will benefi t the scientifi c community, enabling it to further understand 2 – Based on the outcome of the fi rst programme, the ongoing second the mechanism of diseases, to identify new targets of innovative step (ECRIN-TWG), also funded by the FP6 (health priority, 2006- treatments and new biomarkers, to identify responder populations, to 2008), consists of building an infrastructure able to provide support develop innovative diagnostic and therapeutic tools. This integrated to EU-wide clinical studies. In this phase seven transnational work- infrastructure will be of particular relevance in rare diseases, however, ing groups are in charge of defi ning guidelines and procedures to the personalized approach regarding effi cacy and safety of treatments support multinational studies, in any medical fi eld, in any patient requires extended networking as well. In addition, ECRIN will develop population, and for any type of study including medicinal trials, synergy with other EU-wide biomedical infrastructures for translational medical device or surgery trials, diagnostic or prognostic studies, research (EATRIS) and biobanks (BBMRI). genetic studies and studies on the mechanism of disease. These working groups cover: interaction with ethics committees, interac- The Innovative Medicines Initiative (IMI) project requires integrated tion with competent authorities and regulatory affairs, adverse infrastructures for drug development at the drug discovery, preclinical event reporting, data management, study monitoring, quality and clinical steps. As IMI will support public-private partnership research assurance, and education. These tasks are supported by European at the precompetitive step, clinical research will mainly focus on the correspondents embedded in the national network of each mem- identifi cation and validation of biomarkers predictive for effi cacy of for ber state, and coordinated by a multinational Coordination Team. safety, and on post-marketing studies. Typically this precompetitive 3 – Competence and know-how accumulated by the Coordination research will be sponsored by academic institutions, which are currently Team and European correspondents in each participating country lack the capacity to act as a sponsor for clinical studies at the EU level. will allow ECRIN, during the next step of its development, to pro- This project is therefore critical for the implementation of the IMI vide integrated support to multinational studies during the third strategic research agenda. In addition, tools provided by ECRIN will help phase of ECRIN. This next step is funded by the FP7 research infra- connect the national disease-oriented networks across the borders, structures programme (contract 211738), as the ESFRI roadmap particularly in the fi elds covered by IMI (cancer, brain, metabolism- European infrastructure for clinical trials and biotherapy. This diabetes, infl ammation, and infectious diseases). project plans development of datacenters and of GMP facilities for biotherapy coupled to clinical research centres and networks.

ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES | 269 Europe-wide infrastructures and disease-oriented Coordinator Pierre Meulien networks in drug development Irish Clinical Research Jacques Demotes-Mainard Infrastructures Network INSERM-DRCT Dublin, Ireland ECRIN participates in the discussion on the revision of the EU Directive 101 rue de Tolbiac on clinical trials to promote an efﬁ cient and harmonized framework for F-75654 PARIS cedex 13, France Nicola Fabris investigator-driven clinical science in the EU. ECRIN also promotes the [email protected] Consorzio Italiano per active participation of patients and citizens, and transparency in clinical Phone: +33 1 4423 6285 la Ricerca in Medicina research. For this purpose ECRIN has launched a yearly communication Milano, Italy event (May 20, the International Clinical Trials Day) on the challenges raised by clinical research. Partners Silvio Garattini Istituto di Ricerche Christian Gluud Farmacologiche Mario Negri Danish Clinical Research Milano, Italy Infrastructures Network Copenhagen, Denmark Xavier Carné Spanish Clinical Research Network Jacques Demotes Madrid, Spain Réseau des Centres d’Investigation Clinique INSERM-Hôpitaux Pierre Lafolie Paris, France Swedish Clinical Research Infrastructures Network Geneviève Chêne Stockholm, Sweden Réseau Français des Unités d’Essais Cliniques Peter Selby Bordeaux, France Maxine Stead UK Clinical Research Network Christian Ohmann Leeds, United Kingdom Netzwerk der Koordinierungszentren für Klinische Studien Jean-Paul Collet Köln, Germany University of British Columbia Vancouver, Canada Csaba Farsang Hungarian ECRIN Committee Jean-Pierre Tassignon Budapest, Hungary European Forum for Good Clinical Practice Brussels, Belgium

Project number LSHM-CT-2006-037199 EC contribution € 700 000 Project Duration 24 months Starting date 01/10/2006 Instrument SSA Project website www.ecrin.org

270 | ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES EUROIRON1

Genetic Control of the Pathogenesis of Diseases Based on Iron Accumulation

Keywords Iron, iron overload, haemochromatosis, anaemia of Expected results chronic disease, infl ammation, gene, hepcidin To answer the following speciﬁ c questions concerning iron mismanagement of genetic iron overload and/or anaemia of chronic disease: • role of the heme, versus non heme, iron pathway in intestinal iron absorption; • roles of HFE and Iron Regulatory Protein/Iron Responsive Element systems; Summary • role of hepcidin as a key regulator in iron homeostasis; • identiﬁ cation of the genes accounting for phenotypic variability Diseases involving iron accumulation in humans are frequent and potentially in experimental models and in humans. severe. The present project will focus on the genetic understanding of both genetic iron overload diseases and the anaemia of chronic disease which are, from the pathophysiological viewpoint, ‘mirror’ situations. This project Potential applications joins together 12 scientifi c groups, from 6 countries, including 11 academic teams and 1 Small Medium Enterprise. • to determine novel diagnostic and prognostic markers of these iron accumulation diseases; • to identify new therapeutic targets in order to counteract these abnormal iron distribution processes, especially through inhibition of intestinal iron absorption and/or promotion of macrophagic iron egress.

Problem

In humans, disorders of iron distribution are of two main types: Modifier genes (WP5) • chronic iron overload diseases of genetic nature, especially HFE haemochromatosis which is one the most frequent hereditary recessive diseases affecting both quality of life and vital prognosis; • anaemia of chronic disease which is also a frequent condition worldwide, mainly related to inﬂ ammatory diseases whatever their cause and source of considerable disability. These two types of diseases of Iron cellular iron accumulation, systemic for haemochromatosis and localised Iron ingress trafficking Iron ingress within macrophages for anaemia of chronic disease, present with (WP1) & regulation (WP2) (WP4) marked phenotypic variability which is, at least in part, related to genetic abnormalities affecting the expression of numerous proteins involved in iron metabolism.

Systemic regulation Aim (WP3)

The scientiﬁ c aims of the project are: • to elucidate the genetic factors involved, directly or indirectly, in the regulation of the expression of key genes of iron metabolism associated with pathological iron accumulation; Schematic representation • to assess the functional consequences of their abnormal expression of the 5 workpackages of EuroIron1. in cellular and animal models.

ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES | 271 Iron accumulation within liver cells (seen as blue deposits within hepatocytes – black arrow- and kupffer cells- yellow arrow –).

Coordinator Clara Camaschella Universita Vita-salute Pierre Brissot San Raffaele, Italy University of Rennes1 Rennes, France Martina Muckenthaler Universität Klinikum Heidelberg Heidelberg, Germany Partners Matthias Hentze Pierre Brissot European Molecular Olivier Loreal Biology Laboratory University of Rennes 1 Heidelberg, Germany Rennes, France Guenter Weiss Andrew Mckie Medical University King’s College London of Innsbruck London, United Kingdom Innsbruck, Austria

Ioav Cabantchik Marie-paule Roth The Hebrew University Sophie Vaulont of Jerusalem Institut National de la Santé Jerusalem, Israel et de la Recherche Médicale Paris, France Antonello Pietrangelo Universita degli Studi Pascal Soularue di Modena e Reggio Emilia Partnerchip Modena, Italy Evry, France

Paolo Arosio University of Brescia Brescia, Italy

Project number LSHM-CT-2006-037296 EC contribution € 2 796 000 Project Duration 36 months Starting date 01/01/2007 Instrument STREP Project website www.euroiron1.univ-rennes1.fr

272 | ADDITIONAL PROJECTS RELATED TO MAJOR DISEASES INDEX BY ACRONYM 274 ➞ 275

FP6 INSTRUMENTS 276 ➞ 277 INDEX BY ACRONYM

ACE 80 ENINET 118 AGLAEA 112 Enough sleep 120 Anti-tumor targeting 184 EPCRC 212 Apotherapy 186 EPICURE 122 ARIG 162 EPITRON 214 BAMOD 188 EUGeneHeart 17 CancerGrid 190 EURAMY 60 CANCERIMMUNOTHERAPY 193 EURESFUN 91 CAPPELLA 195 EURO-Laminopathies 67 Cardiogenics 10 EuroBoNet 216 CHEMORES 197 EuroCSC 218 CHILDHOPE 199 EuroCareCF 62 cNEUPRO 114 EuroDia 46 CONTICA 13 EuroGrow 65 CONTICANET 202 EUROIRON1 271 CRAB 82 EUSynapse 125 CRESCENDO 164 EWA 171 CVDIMMUNE 15 GENECURE 20 DASIM 205 GenoMEL 220 DB workshop 116 GRACE 94 DEPPICT 206 GROWTHSTOP 222 DISMAL 208 HDLomics 22 DRESP2 84 HeartRepair 24 E.E.T.-Pipeline 210 HEPADIP 48 EACCAD 86 HERMIONE 224 EAR 88 HI-CAM 226 ECONAG 167 HighReX 228 ECRIN-TWG 268 HUE-MAN 70 ELAST-AGE 169 IMAGEN 127

274 IMMUNATH 27 ONCODEATH 249 Immuno-PDT 230 OVCAD 251 INCA 232 PHECOMP 148 INCF 129 PHOTOLYSIS 150 INDABIP 131 PNS-EURONET2 152 InGenious HyperCare 29 POLYGENE 253 INTELLIMAZE 133 PREDICTIONS 54 InterAct 51 PROCARDIS 34 KidsCancerKinome 234 PROMET 255 LifeSpan 174 PROTEOMAGE 176 LIGHTS 236 PULMOTENSION 36 MagRSA 98 PolyALA 33 MAMMI 238 RATstream™ 154 MANASP 100 REBAVAC 107 MCSCs 240 RespViruses 179 MEMORIES 136 SAVEBETA 56 MMR-related cancer 242 SENECA 180 MOSAR 102 SGENE 157 MSCNET 243 SOUTH 39 MYASTAID 73 StaphDynamics 109 NEOBRAIN 138 TAMAHUD 159 NEURODYS 141 TCAC in Cancer 257 NeuroproMiSe 143 TREAT-NMD 76 NORMACOR 31 TRIDENT 259 NORMOLIFE 245 TRKCancer 261 NovelTune 146 Tumor-Host Genomics 262 NPARI 105 VASOPLUS 42 ONCASYM 247 VITAL 264

275 FP6 INSTRUMENTS

Instruments are the means of Commission intervention – the structures Integrated Project (IP) and parameters that have been agreed at the political level within which projects can be funded. FP6 introduces three new instruments as well as IPs are multipartner projects to support objective-driven research, where adaptations of existing instruments. You are not free to choose among all the primary deliverable is generating the knowledge required to available instruments of FP6 for a given topic. The work programmes and implement the thematic priorities. IPs should bring together a critical calls will defi ne which instruments are available for which topics. mass of resources to reach ambitious goals aimed either at increasing Europe’s competitiveness or at addressing major societal needs. They To make a conscious decision if one of the available instruments in your must contain a research component and may contain technological topic is suitable for your ideas you have to understand their basic features development and demonstration components, as appropriate, as well as and underlying concepts. perhaps a training component. A project may be at any point in the research spectrum. A single project may indeed span large parts of the Network of Excellence (NoE) spectrum, i.e. from basic to applied research.

NoEs are multipartner projects aimed at strengthening scientifi c and Integration within an integrated project may take several forms: technological excellence on a particular research topic by integrating at • Vertical integration of the full “value-chain” of stakeholders from European level the critical mass of resources and expertise needed to those involved in knowledge production through to technology provide European leadership and to be a world force in a given domain. development and transfer. This expertise will be networked around a joint programme of activities • Horizontal integration of a range of multidisciplinary activities. aimed primarily at creating a progressive and durable integration of • Activity integration: integrating various research activities from fun- research capacities of network partners while at the same time advancing damental to applied research and with other types of activity, includ- knowledge on the topic. ing take-up activities, protection and dissemination of knowledge, training, etc., as appropriate. NoEs are more than just schemes for the co-ordination of research and • Sectoral integration of actors from private and public sector research information exchange; and the research itself is not their main focus organisations, and in particular between academia and industry, either. Participating institutions have to invest seriously in structural including SMEs. change aiming at a durable integration of their research capacities. This • Financial integration of public and private funding, with overall requires the commitment of all levels of decision-making in an institution, fi nancing plans that may involve the European Investment Bank including top management, supervising and fi nancing bodies. and co-operation with Eureka.

The main result should be a durable restructuring and reshaping of the Funding will take the form of a grant to the budget, as a contribution to way research is carried out in Europe in a given area. costs incurred, with specifi ed maximum rates of support for the different types of activity within the project. The fi nancial regime for NoEs has been built on the following principles: IPs will be applied in the 7 priority thematic areas and in the specifi c • a grant for integration, as a fi xed amount to support the joint programme for nuclear research. In duly justifi ed cases they may also be programme of activities; used in the research areas supporting policies and anticipating scientifi c • to be calculated taking into account: and technological needs. – the degree of integration proposed by the consortium, – the number of researchers that all participants intend to Specifi c Targeted Research Projects (STREP) integrate, and Specifi c Targeted Innovation Projects (STIP) – the characteristics of the fi eld of research concerned and – the joint programme of activities; STREPs and STIPs are multipartner research, demonstration or innovation • to be disbursed in installment, with payment depending primarily projects. They are an evolved form of the shared-cost RTD projects and on the network’s progress towards achieving a durable integration demonstration projects used in FP5. Their purpose is to support research, and on condition that the costs incurred in implementing the joint technological development and demonstration or innovation activities programme of activities are greater than the grant itself. of a more limited scope and ambition than IPs. The Community contributionmay range from hundreds of thousands of Euros to a few Details on calculation of the grants will be provided in the model millions of Euros and is paid as a grant to the budget (percentage of contracts. NoEs will be applied in the 7 priority thematic areas and in the total costs of the project). There must be a minimum of three participants specifi c programme for nuclear research. They may also be used, in duly from three different Member States or Associated States of which at justifi ed cases, in research areas supporting policies and anticipating least two are from Member States or Associated Candidate States. scientifi c and technological needs. Different minimum numbers may be specifi ed in the calls for proposals.

276 Special conditions for minimum numbers of participants apply for the Commission support. The latter activities will not be welcome if they do “Specifi c international co-operation activities (INCO)” part of the not serve the programme’s strategic objectives, (in the sense of the programme (to be specifi ed in the work programme). European Research Area, improved co-ordination, public awareness, preparation of future Community initiatives, etc.). SSAs can be proposed STREPs are used in implementing the priority thematic areas, in other by a single participant or by a consortium of several participants. The areas supporting Community policies and anticipating scientifi c and activities of a specifi c support action will be supported through a grant to technological needs, in specifi c international co-operation research the budget of up to 100 % of the budget or, if necessary, as a lump sum. activities, and in research activities developing harmonious relations between science and society. STIPs are used in activities exploring, Specifi c research projects for SMEs validating and disseminating new innovation concepts and methods at European level. The Commission wishes to encourage the participation of SMEs in all Framework programme activities. While SMEs can participate in any Coordination Action (CA) project consortium, it is recognised that SMEs that do not have research facilities need special routes to participate in FP6. The following CAs are multi-partner actions intended to promote and support the instruments are foreseen to meet this need. networking and co-ordination of research and innovation activities. They are a reinforced form of the concerted actions/thematic networks Co-operative research projects (CRAFT) used in FP5. They will cover the defi nition, organisation and management They are projects whereby a number of SMEs (minimum three SMEs of joint or common initiatives as well as activities such as the organisation from two different countries) having specifi c problems or needs assign of conferences, meetings, the carrying out of studies, exchanges of a signifi cant part of the required scientifi c and technological research personnel, the exchange and dissemination of good practice, setting up activities to RTD performers. These activities may also be carried out by common information systems and expert groups. EU funding is given innovative and high-tech SMEs in co-operation with research centres for the costs of co-ordination (not for the research) in the form of and universities. The SMEs retain ownership of the results. a grant to the budget of up to 100 % of the budget. There must be a minimum of three participants from three different Member States Collective research projects or Associated States of which at least two are from Member States They are carried out by RTD performers on behalf of industrial or Associated Candidate States. Different minimum numbers may associations or groupings in sectors where SMEs are prominent. The aim be specifi ed in the calls for proposals. Special conditions for minimum is to expand the knowledge base of large communities of SMEs and numbers of participants apply for the “Specifi c international co- thus their general standard of competitiveness. The ownership of the operation activities (INCO)” part of the programme (to be specifi ed results lies with the industrial associations. in the work programme). Specifi c Support Action (SSA)

Support activities are more limited in scope than the accompanying measures of the previous Framework Programmes. These projects aim to contribute actively to the implementation of activities of the work programme, the analysis and dissemination of results or the preparation of future activities, with a view to enabling the Community to achieve or deﬁ ne its RTD strategic objectives. Therefore, a signiﬁ cant emphasis has been placed on Support Actions: • to promote and facilitate the dissemination, transfer, exploitation, assessment and/or broad take-up of past and present programme results (over and above the standard diffusion and exploitation activities of individual projects); • to contribute to strategic objectives, notably regarding the European research area (e.g. pilot initiatives on benchmarking, mapping, networking, etc.); • to prepare future community RTD activities, (e.g. via prospective studies, exploratory measures. pilot actions etc.); as opposed to awareness and information exchange activities, e.g. annual Workshops and Conferences, that would take place anyway without

277

European Commission

Major Diseases Research – Catalogue of Research Projects (2005-2006) in the Sixth Framework Programme – Volume II

Luxembourg: Ofﬁ ce for Ofﬁ cial Publications of the European Communities

2008 – 277 pp. – 21.0 x 29.7 cm

ISBN 978-92-79-08526-0

HOW TO OBTAIN EU PUBLICATIONS

Our priced publications are available from EU Bookshop (http://bookshop.europa.eu/), where you can place an order with the sales agent of your choice.

The Publications Ofﬁ ce has a worldwide network of sales agents. You can obtain their contact details by sending a fax to (352) 29 29-42758. KI-NA-23346-EN-C eld of “Major Diseases: Application- eld health of patients in Europe and around the world.health of patients in Europe and around The “Sixth Framework of the European Union” has Programme for RTD (FP6 – 2002-2006) 800 million to supporting research in the fi dedicated some € with the aim of Knowledge and Technologies”, orientated Genomic Approaches to Medical and the the quality of life improving is caused by non-communicable diseases (NCD): a The greatest disease burden in Europe group mental health problems, cancer, disease (CVD), of conditions that includes cardiovascular chronic respiratorydiabetes mellitus, group of diseases and musculoskeletal conditions. This underlyingdisorders; determinants are linked by common risk factors, which and opportunities % of the disease burden in Europe. % of all deaths and 77 for intervention, cause 86 be to reduce instances of, therefore, funding should, The ultimate objective of any health research and eventually these diseases. eliminate nancial fi from the ted of projects which have benefi This catalogue presents the second batch support Union Framework of the “Sixth European Programme for RTD” in this area.