Scientific Report 2013 PDF file (10 Mb)

Scientific Report 2013 Scientific Report 2013 Cover image by Massimo Degano (see Biocrystallography Unit, page 151, figure 22): Active site of the IAGNH enzyme from T. brucei bound to a nanomolar inhibitor. Knowledge of the active site cavity features (green transparent surface) allows the modification of the compound (yellow sticks) to enhance its activity. Some of the images in this book has been published in scientific papers: Figure 2, p. 16: Modified from: Leukemia: 2013; 27(11): 21962199 doi: 10.1038/leu.2013.98 Figure 6, p. 50: Modified from: J. Clin. Invest. [published online ahead of print June 17, 2014] doi:10.1172/JCI74664 Figure 8, p. 55: Modified from: Neurobiol. Aging: 2013; 34(11): e13-e18 doi: 10.1016/j.neurobiolaging.2013.05.020 Figure 9, p. 57: Neuropsychopharmacology: 2013; 38(2): 313327. doi: 10.1038/npp.2012.17 Figure 15, p. 88: J. Transl. Med. 2013 Dec 13;11:310 doi: 10.1186/1479-5876-11-310 Figure 20, p. 125: J. Allergy Clin. Immunol.: 2014; 133(3): 799-806.e10 doi: 10.1016/j.jaci.2013.12.1043 Figure 34, p. 199: Cover image in: Tissue Engineering - Part A: 2014; 20(5-6) Figure 36, p. 216: FEBS J.: 2014; 281(1): 216-231 doi: 10.1111/febs.12588 Figure 37, p. 217: Curr. Biol.: 2014; 24(4): 393-403 doi: 10.1016/j.cub.2014.01.007 Figure 40A, p. 230: Modified from: Mol. Cell. Biol.: 2013; 33(18): 3644-3658 doi: 10.1128/MCB.00302-13 Figure 43, p. 251: Neurology: 2013; 81(2): 134-143 doi: 10.1212/WNL.0b013e31829a33f8 Figure 46, p. 263: Proteomics: 2013; 94: 401-412 doi: 10.1016/j.jprot.2013.10.007 Edited by the San Raffaele Library Layout project by Rolando Cassinari Cover design by the San Raffaele Marketing, Fundraising & Communication Office Printed by Grafiche Parole Nuove, Brugherio INDEX III INDEX INTRODUCTION IX Scientific Directors' Introduction XII A short visual history of the Institute XX San Raffaele Scientific Retreat 2013 XXI 2013 Seminars and lectures XXIV DIVISION OF MOLECULAR ONCOLOGY 5 Introduction by the Directors Research Units 11 Lymphoid malignancies Unit Biology of multiple myeloma Cell activation and signalling Tumour microenvironment Immuno-biotherapy of melanoma and solid tumors Unit Cancer gene therapy B-cell neoplasia Unit Functional genomics of cancer Unit Lymphoid organ development Unit Molecular histology and cell growth Unit Preclinical models of cancer Unit Tumor biology and vascular targeting Unit 12 12 13 13 14 15 15 16 17 18 18 19 Clinical Research Units Digestive and pancreatico-biliary endoscopy Unit 20 Endosonography: diagnostic and therapeutic endoscopic ultrasound 20 Gastrointestinal surgical oncology Unit 21 Head and neck oncology Unit 21 Multidisciplinary group for thoracic surgical oncology 22 Oncogenesis in liver neoplasms Unit 23 Pancreatic cancer Unit: biology and new therapeutic approaches 23 Pathology Unit 24 Clinical lymphoid malignancies 24 Gynecologic oncology 25 Medical oncology Unit - Clinical trials 26 Medical oncology Unit - Phase I and lung cancer clinical trials 26 Onco-hematology 27 Urological Research Institute (URI) Introduction by the Director Research Units 28 28 Urological pre-clinical research Unit Functional urology 29 29 Clinical Research Units Prostate cancer Bladder cancer Unit 30 30 Renal cancer Unit Sexual medicine Unit 31 31 Selected publications Selected publications, URI 32 33 DIVISION OF NEUROSCIENCE 37 Introduction by the Directors Research Units 44 Neuropsychopharmacology Unit Rett syndrome research group Cell adhesion Unit Cellular neurophysiology Unit Developmental neurogenetics Unit Neurobiology of learning Unit Proteomics of iron metabolism Unit Stem cells and neurogenesis Unit Molecular genetics of mental retardation Unit 45 46 46 47 48 49 49 50 51 Clinical Research Units Acute brain protection, Acute post-operative pain, Drugs and central nervous system Unit Cognitive neuroscience Unit Experimental neurosurgery Unit Eye repair Unit Functional neuroradiology Unit In vivo Human molecular and structural neuroimaging Unit Neuroothology Unit Psychiatry and clinical psychobiology Sleep medicine Clinical psychology Motor function rehabilitation 55 56 56 57 58 59 Institute of Experimental Neurology (INSPE) Introduction by the Director Research Units 60 60 61 Experimental neuropathology Experimental neurophysiology Molecular genetics of behaviour Neuromuscular repair Neuroimmunology Unit Clinical neuroimmunology Immunobiology of neurological disorders Neuroimaging research Unit Neuroimaging of CNS white matter Human inherited neuropathies Unit Axo-glia interactions Unit 61 61 62 62 63 64 65 66 66 67 68 52 52 52 53 54 Clinical Research Units Inflammatory CNS disorders Unit 69 V INDEX Cerebrovascular disorders Memory disorders Movement disorders Neuromuscular disorders Paroxysmal events 69 70 70 71 71 Selected publications Selected publications, INSPE 72 73 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES 81 Introduction by the Directors Research Units 85 Coagulation and platelet biology Unit Cardiodiabetes & core Lab Complications of diabetes Metabolism, nutrigenomics and cell differentiation Bone metabolism Unit Coagulation service & thrombosis research Unit Pediatric endocrinology research 86 87 87 88 89 89 90 Clinical Research Units Diabetes and endocrinology Unit 91 Cardio-metabolism and clinical trials 91 Fetal-maternal medicine 92 Clinical pediatric endocrinology 92 Diabetes and metabolic diseases in children and adolescents 93 Neonatology 93 Structural heart disease Unit 94 Cardiopulmonary clinical physiology Unit 95 Cardiovascular interventions Unit 95 Center for arrhythmia research 96 Echocardiography Unit 96 Ischaemic heart disease, heart failure and echocardiography Unit 97 Organ protection in critically ill patients, Advanced cardiac failure and mechanical supports Unit 98 Strategic research on heart failure Unit 99 Study and treatment of aortic disease Unit 99 Vision first Unit 100 Selected publications 101 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY 107 IIntroduction by the Directors Research Units 112 Angiogenesis and tumor targeting Unit 113 Functional genetics of muscle regeneration Neural stem cell biology Autoimmunity & vascular inflammation Unit Innate immunity and tissue remodelling Experimental hematology Unit Gene expression and muscular dystrophy Unit Leukemia immunotherapy Unit Molecular and functional immunogenetics Unit 113 114 114 115 116 117 118 118 Clinical Research Units Hematology and hematopoietic stem cell transplantation Unit Immunohematology and transfusion medicine Unit The San Raffaele-Telethon Institute for Gene Therapy (HSR-TIGET) Introduction by the Director Research Units Gene transfer technologies and new gene therapy strategies Unit Gene/Neural stem cell therapy for lysosomal storage diseases Gene therapy for WASP/Omenn Unit Gene transfer into stem cells Unit Hematopoietic stem cell based gene therapy for the treatment of lysosomal storage disorders Unit Immunological tolerance Unit Genetic autoimmune diseases Pathogenesis and therapy of ADA-SCID Unit Safety of gene therapy and insertional mutagenesis Unit Tolerogenic dendritic cells Unit 119 119 120 120 121 121 122 122 123 123 124 125 126 126 Clinical Research Units PCRU - Gene therapy for Wiskott-Aldrich Syndrome 127 PCRU - ADA gene transfer into hematopoietic stem cells for the treatment of ADA-SCID 127 PCRU - Clinical trial of gene therapy in metachromatic leukodystrophy 128 Selected publications Selected publications, HSR-TIGET 129 130 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES 139 Introduction by the Directors Research Units 146 Immunopathology Unit Cellular and molecular allergology Human virology 147 147 148 INDEX Protein engineering and therapeutics γδ T cells in innate and adaptive immunity Immunobiology of HIV AIDS immunopathogenesis Unit Biocrystallography Unit Cellular immunology Unit Dynamics of immune responses Unit Emerging bacterial pathogens Unit Experimental immunology Unit Infection and cystic fibrosis Unit Leukocyte biology Unit Lymphocyte activation Unit Tumor immunology Unit Viral evolution and transmission Unit Viral pathogens and biosafety Unit 148 149 150 150 151 152 152 153 154 154 155 156 156 157 158 Protein transport and secretion Unit Age related diseases Molecular immunology Chromatin dynamics Unit In vivo Chromatin and transcription Molecular dynamics of the nucleus Biology of myelin Unit Biomolecular mass spectrometry Unit European Institute for Research in Cystic Fibrosis (IERFC) Genetics of common disorders Unit Intracellular signaling pathways Unit Molecular basis of polycystic kidney disease Unit NeuroGlia Unit Regulation of iron metabolism Unit Molecular genetics of renal disorders Unit 159 159 Clinical Research Units Clinical Research Units Management and antiretroviral treatment of HIV infection Neurovirology Study and treatment of hepatotropic viruses related diseases Vaccine and immunotherapy Clinical immunopathology and advanced medical therapeutics Unit Clinical transplant Unit Pancreatic tumors Unit: immunotherapy and β cell function substitution Gynecological cancers immunology Immunology in liver neoplasms Obesity and bariatric surgery Clinical hepato-gastroenterology Digestive pathophysiology 187 188 188 189 190 191 191 192 192 193 193 194 194 196 196 160 161 Dento-facial histopathology Unit Genomics of renal diseases and hypertension Unit Reproductive sciences Lab Tissue engineering and biomaterials 197 198 199 161 162 Selected publications 201 162 163 163 164 164 165 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS 211 Diabetes Research Institute (DRI) Introduction by the Directors Research Units 166 166 Experimental diabetes β cell biology Unit Immune-mediated diseases Unit: from pathogenesis to treatment 167 167 168 Clinical Research Units 197 Introduction by the Directors Research Units 213 Neurogenomics Unit Genome function Unit Biomolecular NMR Laboratory Genomic Unit for the diagnosis of human pathologies Organelle biogenesis and motility Unit Proteome biochemistry Unit 214 215 215 Selected publications 219 216 217 218 Prevention in Type 1 diabetes Unit Epidemiology & data management Islet transplantation Childhood diabetes 169 169 169 170 EXPERIMENTAL IMAGING CENTER 225 Selected publications Selected publications, DRI 171 172 Introduction by the Directors Research Units DIVISION OF GENETICS AND CELL BIOLOGY Introduction by the Directors Research Units 183 186 Advanced fluorescence microscopy and nanoscopy research Unit Dynamic fluorescence spectroscopy in biomedicine Mouse functional genetics Unit 228 229 230 231 Clinical Research Units VII INDEX Clinical and experimental radiology Unit High technology in radiation therapy Unit Medical physics Unit Molecular imaging Unit Neuroradiology research group 232 232 233 233 234 Service Units 235 236 237 238 Selected publications 239 243 Brain Regeneration usIng medical Devices, Gene vectors and stEm cells (BRIDGE) Program in Immunology and Bio-immunotherapy of Cancer (PIBIC) Islet Trasplantation Program (ITP) Human Brain Invivo Mapping with neuroimaging (BRAINMAP) Bone Physiopathology Program (BoNetwork) Correlates of HIV-Associated Immune Response Modulation program (CHARM) Microenvironment and Genes in Cancers of the Blood (MAGIC) FACILITIES Selected publications 276 279 280 282 284 Selected publications 286 Maternal and child health Department 287 Selected publications 289 Department of internal and specialistic medicine 291 Selected publications 292 Department of clinical neuroscience 294 Selected publications 297 Department of neurology Selected publications 243 Department of onco-haematology 245 247 Department of medical oncology 249 Department of radiology 252 Department of urology Selected publications Selected publications Selected publications Selected publications 299 300 301 302 304 305 307 309 310 312 254 256 259 CFCM, San Raffaele-Telethon Core Facility for Conditional Mutagenesis 260 FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory 261 CERMAC, Centre of Excellence of High Field Magnetic Resonance 262 ProMiFa, Protein Microsequencing Facility 262 Mouse histopathology 263 THE ETHICS COMMITTEE 266 THE CLINICAL DEPARTMENTS Selected publications Head and neck Department Department of infectious diseases ALEMBIC, Advanced Light and Electron Microscopy BioImaging Center Intravital microscopy Preclinical MRI and US facility Pre-clinical PET facility RESEARCH PROGRAMMES Department of general and specialistic surgery CLINICAL SERVICES 313 Pathology Nuclear medicine Radiotherapy Laboratory medicine Immunohematology and transfusion medicine service Emergency medicine General intensive care General anaesthesia and neurointensive care Unit 315 316 316 317 Selected publications 320 317 318 318 319 WHO Collaborating Centre for Integrated Laboratory Strengthening on Tuberculosis, WHO Supranational Reference Laboratory and ESCMID collaborative Centre 324 269 Cardio-thoracic-vascular Department 271 PUBLICATIONS 325 Selected publications 274 List of 2013 publications 327 INTRODUCTION IX SAN RAFFAELE SCIENTIFIC INSTITUTE Scientific Director: Francesco Montorsi*, since September 2013 Deputy Scientific Director: Luca G. Guidotti, since September 2013 Scientific Director: Maria Grazia Roncarolo*, until September 2013 * Professor at: Università Vita-Salute San Raffaele XI INTRODUCTION Scientific Directors’ Introduction The San Raffaele Scientific Institute (SRSI) gathers basic, translational and clinical research activities conducted within Ospedale San Raffaele (OSR) and Fondazione Centro San Raffaele (FCSR). OSR is a forprofit organization that is governed by Prof. Gabriele Pelissero (President) and Dr. Nicola Bedin (CEO); FCSR is a non-profit entity headed by Prof. Giuseppe Banfi (General Director). 2013 was a year of high scientific productivity for SRSI. Indeed, SRSI scientific productivity continued its upward trend as per i) quantity and quality of publications, ii) total impact factor and iii) number of clinical trials. All of this reflects the dedication and the excellence of our scientists and clinical investigators, who continue to be highly recognized at the national and international level. 2013 was also a year in which the SRSI governance was re-organized: on September 15th, Prof. Francesco Montorsi was appointed as the new Scientific Director (in place of Prof. Maria Grazia Roncarolo) and Prof. Luca G. Guidotti was appointed as Deputy Scientific Director. Research Divisions and Centers During 2013, SRSI has maintained its previous research organization. Six Research Divisions and two Research Centers provide critical mass and synergy in existing areas of excellence. RESEARCH DIVISIONS Director Associate Director Molecular Oncology Federico Caligaris-Cappio Giorgio Parmiani Neuroscience Gianvito Martino Flavia Valtorta Regenerative Medicine, Stem Cells Luigi Naldini Fabio Ciceri Immunology, Transplantation Ruggero Pardi/ Adriano Lazzarin and Infectious Diseases Luca G. Guidotti Genetics and Cell Biology Roberto Sitia Metabolic and Cardiovascular Sciences Ottavio Alfieri and Emanuele Bosi (Co-directors); and Gene Therapy Marco E. Bianchi Zaverio Ruggeri (Coordinator for Basic Research) RESEARCH CENTERS Director Co-Director Experimental Imaging Carlo Tacchetti Alessandro Del Maschio Translational Genomics and Bioinformatics Giorgio Casari Elia Stupka Research Institutes Four internal Research Institutes continue to focus on specific and coherent research themes RESEARCH INSTITUTE Director San Raffaele Telethon Institute for Gene Therapy (TIGET) Luigi Naldini Institute for Experimental Neurology (INSPE) Giancarlo Comi Diabetes Research Institute (DRI) Emanuele Bosi Urological Research Institute (URI) Francesco Montorsi (ad interim) INTRODUCTION • The “San Raffaele Telethon Institute for Gene Therapy” (TIGET), a joint venture with the Telethon Foundation, is an internationally renowned center pioneering cell and gene therapy in genetic diseases. • The Institute for Experimental Neurology (INSPE), based on an agreement with Merck-Serono, constitutes one of the major European Institutes primarily dedicated to translational research in neuroscience. • The Diabetes Research Institute (DRI) is part of the International DRI Federation (an organization that includes 12 other DRIs around the world) and mainly promotes basic and clinical research on Type 1 Diabetes (T1D). • The Urological Research Institute (URI) is an internationally recognized center carrying out basic and clinical research in oncological and non-oncological diseases of the genito-urinary tract. Research Programs Research Programs represent strategic projects focused on specific scientific goals. During 2013, seven Research Programs have remained active at SRSI, and they relate to dedicated areas in the fields of Neuroscience, Oncology, Immunology, Metabolic or Infectious Diseases. Research Programs Head Brain Regeneration usIng medical Devises, Gene vectors Head: and stEm cells (BRIDGE) Gianvito Martino (ad interim) Deputy Head: Luigi Naldini (ad interim) Program in Immunology and Bio-immunotherapy of Cancer (PIBIC) Co-Heads: Paolo Dellabona Giorgio Parmiani Pancreatic Islet Trasplantation Co-Heads: Lorenzo Piemonti Paola Maffi Human Brain In vivo Mapping with neuroimaging (BRAINMAP) Head: Massimo Filippi Deputy Head: Andrea Falini Bone Physiopathology Program (BoNetwork) Co-Heads: Roberto Sitia Enrico Gherlone Correlates of HIV-Associated Immune Response Modulation (CHARM) Co-Heads: Paola Cinque Guido Poli Microenvironment and Genes in Cancers of the Blood (MAGIC) Co-Heads: Paolo Ghia Giovanni Tonon XIII INTRODUCTION Scientific Directors’ Introduction Institutional Facilities Traditionally, strategic investments in facilities and technological platforms have been a top priority at SRSI. As such, SRSI continue to offer state-of-the art services covering different research areas. Eight Institutional Facilities are operational at SRSI, including proteomics, flow cytometry, conditional mutagenesis, mouse histopathology, high field magnetic resonance, light and electron microscopy and statistics. Facility Scientific Manager Supervisor Promifa Angela Bachi/ Annapaola Andolfo Facility Manager Marco E. Bianchi Fractal Alessio Palini Chiara Villa Head of Facility Experimental models Luca G. Guidotti Enzo Oriani Facility Manager Cfcm Marco E. Bianchi Lorenza Ronfani Facility Manager Mhp Claudio Doglioni Francesca Sanvito Facility Manager Alembic Fabio Grohovaz Fabio Grohovaz Head of Facility Cermac Enrico Smeraldi Andrea Falini Head of Facility Cussb Clelia Di Serio Clelia Di Serio Head of Facility of diseases Human resources at SRSI As of December 2013 over 1,640 individuals - distributed throughout the 6 Research Divisions, the 2 Research Centers and the 8 Institutional Facilities - have been working at SRSI: • 964 people, including scientists, technicians, postdoctoral fellows, PhD students and undergraduate students, have been working on preclinical and translational research; • 665 people, including physicians, research nurses, residents and clinical fellows have been working on clinical research projects; • 25 people, including heads of facility, lab managers, research associates and technicians have been working in Institutional Facilities. Highlights of scientific activities in 2013 News from Basic Research • A study published in Science reports encouraging results using lentivirus gene therapy to treat the fatal genetic disorder Metachromatic Leukodystrophy (MLD) that lacks any effective treatments. Hematopoietic stem cells were collected from children with the most severe variant of MLD, exposed to a lentiviral vector expressing the corrective gene and re-injected in the patients after a short course of chemotherapy. Almost two years after treatment, sustained engraftment of gene corrected stem cells and prevention of neurological disease manifestation or progression were observed (Biffi et al., Science 2013; 341, 1233158). • A parallel study published in Science reports promising results of gene therapy in Wiskott-Aldrich syndrome (WAS), a genetic disorder characterized by thrombocytopenia and immune deficiency. Three patients received infusion of autologous hematopoietic stem/progenitor cell, transduced with a lentiviral vector encoding WAS, following reduced chemotherapy conditioning. Molecular studies showed high level engraftment of gene corrected stem cells (25-50%) with multilineage hematopoiesis and a safe profile of vector integration. Gene therapy resulted in improved platelet counts with decreased bleeding, INTRODUCTION improved immune functions and reduced infections (Aiuti et al., Science 2013; 34, 1233151). • A study published in Nature Immunology disclosed an essential function of autophagy in the immune system. Autophagy ensures sustainable antibody production through selective digestion of the plasma cell secretory compartment. Moreover, autophagy is required for the maintenance of memory plasma cells in bone marrow. This study furthers our understanding of immunity and offers a framework for identifying new targets against multiple myeloma, the cancer of plasma cells (Pengo et al., Nat Immunol. 2013; 14:298-305). • A study published in Nature Medicine demonstrated that a common variant in the promoter of the UMOD gene encoding uromodulin, the most abundant protein in urine, triggers a cascade of events that end up in hypertension and signs of chronic kidney disease. This study expands our knowledge on the complex mechanisms that determine hypertension and chronic kidney disease and it shows how genetic information could be exploited for personalized medicine (Trudu et al., Nat Med. 2013; 19:1655-1660). • A study published in Nature Medicine described a new set of surface markers (CD49b and lymphocyte activation gene 3) enabling the identification and isolation of a particularly important subset of human and mouse CD4+ T regulatory cells (Tr1 cells). The discovery makes it feasible to track Tr1 cells in vivo and to purify Tr1 cells for cell therapy approaches aimed at inducing or restoring tolerance in subjects with immune-mediated diseases (Gagliani et al., Nat Med. 2013; 19:739-746). • A study published in Nature Medicine shed new light in the pathogenesis of Autosomal Dominant Polycystic Kidney Disease (ADPKD), a common genetic disorder characterized by bilateral renal cyst formation. This work showed that the epithelia lining the cysts have an aberrant glucose metabolism, characterized by anaerobic glycolysis. Glucose deprivation impairs their growth and survival. A glucose analogue, 2-Deoxy-D-Glucose, administered to murine models of PKD effectively retarded disease progression, providing a proof-of-concept for the use of this molecule as a safe therapeutical approach (Rowe et al., Nat Med. 2013; 19:488-493). • A study published in Nature Methods utilized a novel and highly mutagenic lentiviral vector to uncover genes that are involved in hepatocellular carcinoma (HCC) development. Four cancer genes were identified and validated as “altered genes” in HCC samples isolated from patients. This innovative approach should help identify new prognostic markers and new therapeutic targets for this and perhaps other relevant human cancers (Ranzani et al., Nat Methods 2013; 10:155-161). • A study published in Immunity identified specific multipotent mesodermal precursors that give rise to different stromal cell subsets of the spleen and support injury-induced regeneration. These results begin to define the cellular mechanisms responsible for the generation of stromal diversity in secondary lymphoid organs and for the remodeling of lymphoid tissue (Castagnaro et al. Immunity: 2013; 38: 782791). News from Translational and Clinical Research The vibrant interaction between basic research and clinical areas represents one of the greatest values of SRSI and, undoubtedly, this is the peculiarity that makes the Institute so unique. In the various chapters included in this scientific report one can follow in detail the lines of research developed in all the laboratories and clinical units of the hospital and understand the deepest sense of their value. All major areas in medicine and biology, which are actively developed at SRSI, are covered by translational and clinical research projects. As mentioned above, top-level research was conducted in the pediatric arena with gene therapy-based approaches for MLD and WAS. A new paradigm of treatment with excellent results in terms of efficacy, safety and tolerability was developed. XV INTRODUCTION Scientific Directors’ Introduction The area of oncology brought in a large number of state-of- the-art areas of research including oncohematology, cancers of the genito-urinary tract, breast cancer and cancers of the digestive system. Namely, genomics of chronic lymphocytic leukemia, pancreatic islet auto-transplantation for pancreatic cancer, innovative medical treatment for breast cancer, prognostic factors and multimodal therapy for prostate cancer, the relationship between inflammation-immunity and cancer are only a few of the many topics that were protagonists of our research. Radiology and Nuclear Medicine contributed significantly in the areas of central nervous system disease, oncology in almost all systems, cardiovascular medicine. Laboratory medicine was able to produce top-level research in many areas including pharmacotherapy of venous thromboembolism and management of infections. Neuroscience confirmed its leadership with a large number of different research projects in MR imaging, management of sleep disorders, multiple sclerosis and genomics, management of degenerative disease of the central nervous system, use of stem cells for different conditions including eye disease. Cardiovascular research was significant in many areas including percutaneous vs. open surgical management of coronary artery disease and valve disease, pathophysiology of ischemic heart disease, hypertension and heart failure, and management of arrhythmias. Diabetes has remained a historically proven area of major scientific research, and contributions in the field of pancreatic islet transplantation and pharmacotherapy for type 2 Diabetes have been substantial. Last but not least in the area of dentistry research of substantial interest was led in many areas. It is expected that both translational and clinical research will continue to flourish in the coming years. INTRODUCTION Scientific productivity in 2013 In 2013 the number of SRSI scientific publications and the total impact factor continued to rise, as compared to data from previous years. The total number of scientific publications was 1,142, with a total Impact Factor of 6,451.729 (Figure 1). In 2013 more than to 10% of all SRSI publications were in top-level scientific journals (Impact Factor over 10, Figure 2). Based on these results, SRSI has been recognized - once more - as the most highly ranked Italian IRCCS (among the 47 Italian IRCCS) in terms of scientific quality and scientific productivity. Figure 1. Publications and total Impact Factor of SRSI in the past 4 years 6,451.729 6,118.243 5,467.350 4,624.629 2010 (average IF: 5.558) Total IF 2010-2013 2011 (average IF: 5.798) 1,142 1,107 943 832 2012 (average IF: 5.527) 2013 * (average IF: 5.650) Publications 2010-2013 * based upon IF 2012 Figure 2. % of SRSI publications sorted according to Impact Factor ranges Percentage of publications 100% 80% 65.3% 65.2% 65.1% 60% 61.0% 40% 27.3% 28.6% 26.9% 24.2% 20% 7.6% 10.4% 7.8% 10.6% 0% < 5.000 > _10.000 5.000-9.999 Impact Factor ranges 2010 2011 2012 2013 Highlighting the notion that clinical research is a major strength at SRSI, the 2013 output in clinical trial activity was simply excellent. The Ethics Committee examined a total of 243 clinical protocols: 110 (45.3 %) sponsored by Pharmaceutical Companies, 72 (29.6%) SRSI investigator initiated trials and 61 (25.1%) no-profit groups sponsored trials (Figure 3). XVII INTRODUCTION Scientific Directors’ Introduction Figure 3. Clinical trials evaluated by the OSR Ethics Committee in 2013 Clinical trials evaluated by EC, 2013 72 (29.6%) 110 (45.3%) 61 (25.1%) Biotechnology Transfer Activity in 2013 The “Office of Biotechnology Transfer” (OBT) acts as the interface between SRSI and the business community. Its mission is to generate value from know-how, intellectual property, technologies and research facilities. In the last 10 years of activity OBT obtained the following results: 368 sponsored research or industrial collaboration contracts with about 130 biotech/pharmaceutical companies were successfully finalized; 68 licenses/options and evaluation agreements with industrial partners were executed; 135 patent families (patented technologies) were filed; 47 patent families (corresponding to a total of 221 patents and patent applications) are alive as of December 2013. Funding for research activities As in the past, the Ministry of Health structural funds (Ricerca Corrente) contributed significantly to the 2013 total research revenues of SRSI. Important research revenues were also obtained from external grants, which were assigned through internationally recognized peer-reviewed processes. Below is a list of the most representative funding agencies and of their contribution to SRSI (both OSR and FCSR) in 2013. INTRODUCTION Contributions from Main Funding Agencies YEAR 2013 data in € Ricerca Finalizzata (Ministry of Health) 6,095,030 AIRC (Italian Association for Cancer Research) 4,513,590 EU (European Union) 3.541.731 Other Private Funding Agencies 1,206,305 Telethon Foundation 1,205,492 ISS (Italian National Institute of Health) 977,431 Armenise Harvard Foundation 603,521 Lombardy Region 546,556 MIUR (Ministry of Education, University and Research) 382,215 JDRF (Juvenile Diabetes Research Foundation) 290,962 FISM (Italian Federation for Multiple Sclerosis) 265,220 AICR (Association for International Cancer Research) 185,365 WHO (World Health Organization) 157,526 INAIL (National institute for insurance against industrial injuries) 124,186 Conclusions and future perspectives The 2013 Scientific Report of SRSI clearly shows that research in all its forms is lively and vibrant. We would like to take this opportunity to thank Prof. Maria Grazia Roncarolo who acted as Scientific Director until September 2013 and who was instrumental in the organization of the various Research Divisions, Institutes and Centers. This structure is currently being revamped in order to improve the workflow of research but the overall skeleton, being robust and efficient, will be maintained. At present the team of the Scientific Directorate has expanded to include Prof. Giuseppe Banfi, General Director of Fondazione Centro San Raffaele (FCSR) and Dr. Anna Flavia d‘Amelio Einaudi as representative of the CEO of OSR. The first objective of this new team is to serve the scientific community in order to facilitate the every day work of all scientists and clinicians involved in research activities. Creating liaisons among different groups to foster the development of new research projects, helping the process of grant writing, facilitating the entrance of students as active participants in prestigious research groups, supporting educational programs for all those involved in research, also represent targets that we want to hit. We are convinced that the quality of health care provided to patients is better in hospitals where research is actively practiced; curing our patients remain our first goal. Francesco Montorsi Scientific Director Luca G. Guidotti Deputy Scientific Director XIX INTRODUCTION A short visual history of the Institute... The hospital, 1970 Construction site of Dibit1, 1991 The hospital in the 1990s Dibit1 in 1992 The hospital today The Dibit2 project Work in progress: Dibit2 and the new hospital quarter Dibit2 today INTRODUCTION San Raffaele Scientific Retreat 2013 XXI INTRODUCTION San Raffaele Scientific Retreat 2013 INTRODUCTION Best poster award Rocco Baccaro Efficacy of different anti-hypertensive drugs to reverse coronary microvascular remodeling in the spontaneously hypertensive rat Best paper award Davide Gabellini Cabianca, DS; Casà, V; Bodega, B; Xynos, A; Ginelli, E; Tanaka, Y; Gabellini, D. A long ncRNA links copy number variation to a polycomb/trithorax epigenetic switch in fshd muscular dystrophy. Cell: 2012; 149(4): 819-831 XXIII INTRODUCTION 2013 Seminars and Lectures ■ Division of molecular oncology ■ Division of neuroscience ■ Division of metabolic and cardiovascular sciences ■ Division of regenerative medicine, stem cells and gene therapy ■ Division of immunology, transplantation, and infectious diseases ■ Division of genetics and cell biology ■ Center for translational genomics and bioinformatics ■ Experimental imaging Center ■ February 4, 2013 Mechanisms and consequences of NFAT signaling pathway activation downstream of PRRs in innate immune cells Granucci Francesca, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan Guest: Paolo Dellabona ■ March 4, 2013 Stochastic gene expression in single mammalian cells Nacho Molina, The Institute of Bioengineering (IBI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland Guest: Samuel Zambrano ■ March 6, 2013 Redox processes in the mammalian endoplasmic reticulum: sensors, mechanisms, and consequences Christian Appenzeller-Herzog, Division of Molecular & Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Switzerland Guest: Eelco Van Anken ■ March 11, 2013 Bone and mineral metabolism: from gene to treatment to gene Maria Luisa Brandi, Bone and Mineral Metabolism Unit, Department of Surgery and Translational Medicine, University of Florence Guest: Simone Cenci March 18, 2013 Chromatin, gene regulation and Cancer Luciano Di Croce, Center for Genomic Regulation, ICREA, Spain Guest: Vania Broccoli & Giovanni Tonon ■ March 18, 2013 Gene expression changes induced by the HIV-1 TAT protein. Role of the second exon José Alcami, Centro National de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Guest: Elisa Vicenzi ■ March 18, 2013 Functions of histone H4-K20 methyltransferases in genome replication and stability, a link with cancer? Eric Julien, Montpellier Institute of Molecular Genetics (IGMM) and Montpellier University, France Guest: Giovanni Tonon ■ March 21, 2013 The innate function of human IgM memory B cells: production of secretory IgA in the gut Carsetti Rita, B-cell development Unit, Immunological Diagnosis Unit, Bambino Gesù Children Hospital, Rome Guest: Paolo Dellabona ■ March 25, 2013 Che-1 inhibiting mTor pathway in response to cellular stress sustains autophagy and multiple myeloma cells growth Maurizio Fanciulli, Epigenetic Laboratory, Regina Elena Cancer Institute, Rome Guest: Giovanni Tonon INTRODUCTION ■ March 28, 2013 The liver: a site of primary activation leading to tolerance? Patrick Bertolino, Liver Immunology group, Centenary Institute in Sydney, Australia Guest: Luca G. Guidotti ■ July 16, 2013 Tuberous sclerosis complex, sometimes it takes a zoo Kevin Ess, Vanderbilt University, Nashville, Tennessee, USA Guest: Rossella Galli ■ April 08, 2013 Mechanisms of myelination in the central nervous system Michael Simons, Experimental Medicine, Max Planck Institute, Göttingen, Germany Guest: Alessandra Bolino ■ July 22, 2013 The ion channel CFTR as a platform for protein kinase cross-talk: the ins and outs of CFTR signalling Anil Mehta, CVS Diabetes Lung, Ninewells Hospital and Medical School, University of Dundee, UK Guest: Luigi Maiuri ■ April 15, 2013 How integrins control cell fate decisions and the orientation of polarity in epithelia Charles Streuli, Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, UK Guest: Ivan De Curtis ■ April 15, 2013 Revealing how WASp stings in immunodeficiency Lisa Westerberg, Department of Medicine, Translational Immunology Unit, Karolinska Institutet, Stockholm, Sweden Guest: Anna Villa ■ April 17, 2013 Scurvy is the cost to pay for a defective oxidative folding Ester Zito, Metabolic Research laboratory, University of Cambridge, UK Guest: Roberto Sitia ■ April 29, 2013 Regulation of normal and leukemic human stem cells Tsvee Lapidot, Department of Immunology, Edith Arnoff Stein Professorial Chair in Stem Cell Research, Weizmann Institute of Science, Israel Guest: Luigi Naldini ■ May 27, 2013 Neuronal endosomes: powerful sorting stations in health and disease Bettina Winckler, Department of Neuroscience, University of Virginia, USA Guest: Alessandra Bolino ■ July 22, 2013 Prevention of HIV transmission: what can we learn from non human primate studies Roger Le Grand, CEA - Division of Immunovirology, IDMIT and University Paris Sud, France Guest: Gabriella Scarlatti ■ September 19, 2013 Transcriptional control of glutamatergic differentiation during adult neurogenesis Rebecca Hodge, Center for Integrative Brain Research, Seattle Children’s Research Institute, USA Guest: Francesco Bedogni ■ September 23, 2013 Genetic and non genetic determinants drive tumor heterogeneity John Dick, Department of Molecular Genetics, University of Toronto, Canada Guest: Luigi Naldini ■ October 03, 2013 DNA methylation shapes the Polycomb landscape Richard Meehan, MRC Human Genetics Unit (HGU), University of Edinburgh, UK Guest: Davide Gabellini ■ October 07, 2013 The synapsins: alterations of release dynamics and synaptic plasticity in neuro-psychiatric disorders Fabio Benfenanti, Department of Neuroscience and Brain Technologies, University of Genova Medical School Guest: Flavia Valtorta XXV INTRODUCTION 2013 Seminars and Lectures ■ October 07, 2013 Advanced optical imaging for biology and medicine Gianluca Valentini, Politecnico di Milano Guest: Davide Mazza ■ October 28, 2013 Regulation of cancer cell motility and adhesion by integrins and intracellular adapters Bernhard Wehrle-Haller, Department of Cell Physiology and Metabolism, University of Geneva, Switzerland Guest: Ivan de Curtis ■ November 04, 2013 Quantitative imaging of hematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment César Nombela-Arrieta, Experimental Hematology Department, University Hospital Zurich, Switzerland Guest: Matteo Iannacone ■ November 18, 2013 Aquaporins: a never ending story of new isoforms and surprising regulations and functions Gerd Patrick Bienert, Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany Guest: Roberto Sitia ■ November 22, 2013 KRAB’n’KAP: from the control of mobile genetic elements to the regulation of mammalian homeostasis Didier Trono, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne and “Frontiers in Genetics” National Center of Competence in Research, Switzerland Guest: Luigi Naldini ■ November 25, 2013 Minimising transplant immunosuppression by the harnessing of tolerance mechanism Herman Waldmann, Sir William Dunn School of Pathology, Oxford University Guest: Anna Mondino ■ December 02, 2013 Ciliary trafficking and epithelial cell polarity defects as pathophysiological mechanisms in Nephronophthisis and associated ciliopathies Sophie Saunier, Université Paris DescartesHôpital Necker-Enfants Malades, France Guest : Alessandra Boletta ■ December 05, 2013 Blocking IL-1 in a broad spectrum of inflammatory diseases Charles Dinarello, University of Colorado, Denver, USA Guest: Roberto Sitia ■ December 09, 2013 The new mesenchymal stem cell Arnold Caplan, Skeletal Research Center, Case Western Reserve University, Cleveland, USA Guest: Massimo Candiani ■ December 10, 2013 Life without H3K9 methylation: loss of heterochromatin and genome stability Susan Gasser, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Guest: Davide Gabellini XXVI SCIENTIFIC REPORTS La “Squadra” The Scientific Director, the Deputy Scientific Director, and the past Scientific Director, the Directors and Associated Directors of the Research Divisions and Centers, and the Directors of the Institutes 1 Divisions, Centers, Institutes and Research programmes DIVISION OF MOLECULAR ONCOLOGY DIVISION OF NEUROSCIENCE URI UROLOGICAL RESEARCH INSTITUTE INSPE INSTITUTE OF EXPERIMENTAL NEUROLOGY Microenvironment and Genes in Cancers of the Blood (MAGIC) Human Brain Invivo Mapping with neuroimaging (BRAINMAP) DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Brain Regeneration usIng medical Devices, Gene vectors and stEm cells (BRIDGE) DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY HSR-TIGET THE SAN RAFFAELE-TELETHON INSTITUTE FOR GENE THERAPY DIVISION OF IMMUNOLOGY, TRANSPLANTATION AND INFECTIOUS DISEASES DIVISION OF Program in Immunology and Bio-immunotherapy of Cancer (PIBIC) DRI DIABETES RESEARCH INSTITUTE Islet Trasplantation Program (ITP) GENETICS AND CELL BIOLOGY CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS IMAGING EXPERIMENTAL CENTER Correlates of HIV-Associated Immune Response Modulation Program (CHARM) Bone Physiopathology Program (BoNetwork) 3 DIVISION OF MOLECULAR ONCOLOGY Director: Federico Caligaris-Cappio* Associate Director: Giorgio Parmiani Research Units Lymphoid malignancies Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 12 HEAD OF UNIT: Federico Caligaris-Cappio* POST-DOCTORAL FELLOWS: Benedetta Apollonio (since November 2013), Maria Teresa Sabrina Bertilaccio, Cristina Scielzo PHD STUDENT: Benedetta Apollonio**(until October 2013) FELLOW: Elisa ten Hacken TECHNICIANS: Federica Barbaglio, Pamela Ranghetti Biology of multiple myeloma ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 12 GROUP LEADER: Marina Ferrarini POST-DOCTORAL FELLOW: Daniela Belloni Cell activation and signalling –––––––––––––––––––––––––––––––––––––––––––––––––––––––– 13 GROUP LEADER: Marta Muzio FELLOWS: Eleonora Fonte, Maria Giovanna Vilia VISITING FELLOW: Stavroula Ntoufa Tumor microenvironment ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 13 GROUP LEADER: Elisabetta Ferrero POST-DOCTORAL FELLOW: Daniela Belloni Immuno-biotherapy of melanoma and solid tumors Unit ––––––––––––––––––––––––––––– 14 HEAD OF UNIT: Giorgio Parmiani RESEARCHER: Cristina Maccalli (until October 2013) PHYSICIAN: Carolina Cimminiello POST-DOCTORAL FELLOW: Elisabetta Zambon TECHNICIANS: Filippo Capocefalo, Nathascia Chiodo 5 DIVISION OF MOLECULAR ONCOLOGY Research Units Cancer gene therapy –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 15 GROUP LEADER: Vincenzo Russo POST-DOCTORAL FELLOWS: Raffaella Fontana, Laura Raccosta, Matias Soncini PHD STUDENTS: Marta Moresco**, Aida Paniccia** TECHNICIAN: Daniela Maggioni B-cell neoplasia Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 15 HEAD OF UNIT: Paolo Ghia* POST-DOCTORAL FELLOWS: Andreas Agathangelidis, Claudia Fazi PHD STUDENTS: Maria Gounari, Giorgia Simonetti FELLOW: Lydia Scarfò** TECHNICIAN: Tania Veliz Rodriguez Functional genomics of cancer Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 16 HEAD OF UNIT: Giovanni Tonon POST-DOCTORAL FELLOWS: Michela Frenquelli, Manuela Occhionorelli, Beatrice Rondinelli, Simona Segalla PHD STUDENTS: Marco Gaviraghi**, Alessio Lupi**, Benedetta Santoliquido** TECHNICIAN: Elena Antonini INFORMATICIAN: Stefano Ventura Lymphoid organ development Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––– 17 GROUP LEADER: Andrea Brendolan POST-DOCTORAL FELLOWS: Elisa Lenti, Monika Wozinska FELLOWS: Diego Farinello, Maria Teresa Palano** Molecular histology and cell growth Unit –––––––––––––––––––––––––––––––––––––––––––––––– 18 HEAD OF UNIT: Stefano Biffo POST-DOCTORAL FELLOWS: Daniela Brina, Piera Calamita, Elia Ranzato, Sara Ricciardi PHD STUDENTS: Simone Gallo**, Elisa Pesce FELLOWS: Marilena Mancino, Stefania Oliveto, Jianglin Tan TECHNICIAN: Annarita Miluzio Preclinical models of cancer Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 18 GROUP LEADER: Rosa Bernardi, Armenise-Harvard Career Development Award POST-DOCTORAL FELLOW: Nadia Coltella PHD STUDENTS: Manfredi Ponente**, Roberta Valsecchi Tumor biology and vascular targeting Unit –––––––––––––––––––––––––––––––––––––––––––––– 19 HEAD OF UNIT: Angelo Corti RESEARCHER: Flavio Curnis FELLOW: Mimma Bianco TECHNICIANS: Barbara Colombo, Anna Gasparri, Angelina Sacchi DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units Clinical oncology Unit HEAD OF UNIT: Eugenio Villa Digestive and pancreatico-biliary endoscopy Unit ––––––––––––––––––––––––––––––––––––– 20 HEAD OF UNIT: Pier Alberto Testoni* PHYSICIANS: Giulia Martina Cavestro*, Lorella Fanti, Alberto Mariani, Edi Viale RESIDENTS: Matteo Alessandri , Milena Di Leo, Giorgia Mazzoleni, Chiara Notaristefano, Gemma Rossi, Raffaella A. Zuppardo FELLOW: Marco Le Grazie Endosonography: diagnostic and therapeutic endoscopic ultrasound ––––– 20 CLINICAL GROUP LEADER: Paolo Giorgio Arcidiacono PHYSICIANS: Emanuele Dabizzi, Maria Chiara Petrone RESIDENT: Sabrina G. Testoni FELLOW: Maria Emilia Traini Gastrointestinal surgical oncology Unit –––––––––––––––––––––––––––––––––––––––––––––––––– 21 HEAD OF UNIT: Gianfranco Ferla* ad interim, since November 2013; Carlo Staudacher* until October 2013 PHYSICIANS: Paola De Nardi, Saverio Di Palo, Elena Orsenigo, Andrea Marco Tamburini, Andrea Vignali RESIDENTS: Massimiliano Bissolati**, Damiano Chiari**, Guido Fiorentini**, Paolo Giovanni Gazzetta**, Francesca Muffatti** Head and neck oncology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 21 HEAD OF UNIT: Mario Bussi* PHYSICIANS: Stefano Bondi, Leone Giordano, Francesca Lira Luce, Matteo Trimarchi CONSULTANTS: Giulia Danè, Francesca Palonta RESIDENTS: Matteo Biafora, Davide Di Santo, Francesco Pilolli, Daniela Sarandria, Salvatore Toma TECHNICIANS: Daniela Gherner, Barbara Ramella Multidisciplinary group for thoracic surgical oncology ––––––––––––––––––––––––––––––– 22 HEAD OF UNIT: Piero Zannini* PHYSICIANS: Alessandro Bandiera, Angelo Carretta, Paola Ciriaco, Giulio Melloni, Giampiero Negri*, Armando Puglisi RESIDENTS: Piergiorgio Muriana, Silvia Raimondi Cominesi, Stefano Viscardi Oncogenesis in liver neoplasms Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––– 23 HEAD OF UNIT: Gianfranco Ferla* PHYSICIAN: Marco Catena CONSULTANT: Mvunde Mukenge RESIDENTS: Federica Cipriani**, Francesca Ratti** Pancreatic cancer Unit: biology and new therapeutic approaches ––––––––––––––––– 23 HEAD OF UNIT: Gianpaolo Balzano RESIDENTS: Giovanni Luigi Capretti**, Cristina Gilardini** FELLOW: Jacopo Nifosi 7 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units Pathology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 24 HEAD OF UNIT: Claudio Doglioni* PHYSICIANS: Giacomo Dell’Antonio, Massimo Freschi, Roberta Lucianò, Federica Pedica, Maurilio Ponzoni, Nathalie Rizzo, Francesca Sanvito, Isabella Sassi BIOLOGISTS: Maria Giulia Cangi, Lorenza Pecciarini PHD STUDENTS: Daniela Clavenna, Valeria De Pascale, Greta Grassini, Gilda Magliacane FELLOWS: Francesca Invernizzi, Lara Mainetti TECHNICIANS: Elena Dal Cin, Stefano Grassi, Anna Innocenzi, Martina Rocchi, Graziella Santambrogio, Anna Talarico Clinical lymphoid malignancies –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 24 HEAD OF UNIT: Federico Caligaris-Cappio* CLINICAL GROUP LEADER: Andrés José Marìa Ferreri PHYSICIANS: Giovanni Donadoni, Giovanni Citterio, Marco Foppoli, Marianna Sassone, Lydia Scarfò RESIDENTS: Antonella Capasso, Chiara Giudice, Gabriele Todisco, Alessandro Vignati DATA MANAGERS: Eleonora Piraino, Costanzo Sarracino, Eloise Scarano Gynecologic oncology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 25 HEAD OF UNIT: Massimo Candiani* CLINICAL GROUP LEADER: Giorgia Mangili PHYSICIANS: Patrizia De Marzi, Davide Ferrari, Emanuela Rabaiotti, Micaela Petrone RESIDENTS: Alice Bergamini, Veronica Giorgione, Jessica Ottolina, Francesca Pella, Cristina Sigismondi Medical oncology Unit HEAD OF UNIT: Luca Gianni Clinical trials –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 26 CLINICAL GROUP LEADER: Michele Reni PHYSICIANS: Stefano Cereda, Elena Mazza CONSULTANT: Carmen Belli RESEARCH NURSE: Domenica Ceraulo Phase I and lung cancer clinical trials ––––––––––––––––––––––––––––––––––––––––––––– 26 CLINICAL GROUP LEADER: Vanesa Gregorc PHYSICIANS: Alessandra Bulotta, Maria Grazia Viganò Onco-hematology ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 27 HEAD OF UNIT: Fabio Ciceri CLINICAL GROUP LEADER: Massimo Bernardi PHYSICIANS: Matteo Carrabba, Fabio Giglio, Elena Guggiari, Francesca Lunghi, Magda Marcatti RESIDENTS: Carlo Messina**, Elisa Sala** STUDY COORDINATOR: Stefania Trinca RESEARCH NURSE: Margherita Brambilla DATA MANAGER: Ambra Malerba TECHNICIAN: Fernanda Tripiciano DIVISION OF MOLECULAR ONCOLOGY URI Urological Research Institute Director (ad interim): Francesco Montorsi* Research Units Urological pre-clinical research Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 29 HEAD OF UNIT: Petter Hedlund PHYSICIANS: Umberto Capitanio, Federico Dehò, Massimo Freschi, Andrea Gallina, Roberta Lucianò, Nazareno Suardi RESIDENTS: Paolo Capogrosso**, Fabio Castiglione**, Paolo Dell’Oglio**, Ettore Di Trapani**, Nicola Fossati**, Giorgio Gandaglia**, Giovanni La Croce**, Alessandro Nini**, Lorenzo Rocchini**, Andrea Russo**, Manuela Tutolo**, Luca Villa** POST-DOCTORAL FELLOW: Ilaria Cavarretta TECHNICIANS: Arianna Bettiga, Giorgia Colciago, Anna Maria Ferrara Functional urology ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 29 GROUP LEADER: Fabio Benigni PHYSICIANS: Federico Dehò, Andrea Gallina, Alessandro Mistretta, Nazareno Suardi RESIDENTS: Fabio Castiglione**, Ettore di Trapani**, Nicola Fossati**, Giorgio Gandaglia**, Giovanni La Croce**, Alessandro Nini**, Andrea Russo**, Luca Villa** POST-DOCTORAL FELLOW: Roberta Buono TECHNICIANS: Arianna Bettiga, Giorgia Colciago, Anna Maria Ferrara Clinical Research Units Prostate cancer ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 30 HEAD OF UNIT: Francesco Montorsi CLINICAL GROUP LEADER: Alberto Briganti PHYSICIANS: Firas Abdollah, Umberto Capitanio, Andrea Gallina, Giovanni Lughezzani, Vincenzo Scattoni, Nazareno Suardi RESIDENTS: Marco Bianchi**, Fabio Castiglione**, Paolo Dell’Oglio**(since August 2013), Ettore Di Trapani**, Nicola Fossati**, Giorgio Gandaglia**, Alessandro Larcher**, Giuliana Lista**, Alessandro Nini** (since August 2013), Manuela Tutolo**, Luca Villa** DATA MANAGERS: Elena Farina, Nadia Finocchio, Marta Picozzi 9 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units, URI Bladder cancer Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 30 HEAD OF UNIT: Renzo Colombo PHYSICIANS: Nazareno Suardi, Giuseppe Zanni RESIDENTS: Paolo Capogrosso**, Giovanni La Croce**, Lorenzo Rocchini** POST-DOCTORAL FELLOW: Marco Moschini DATA MANAGER: Giusy Burgio Renal cancer Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 31 HEAD OF UNIT: Roberto Bertini PHYSICIANS: Umberto Capitanio, Ryan Matloob RESIDENTS: Fabio Castiglione**, Ettore Di Trapani**, Andrea Russo** DATA MANAGER: Cristina Carenzi Sexual medicine Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 31 HEAD OF UNIT: Andrea Salonia RESIDENTS: Paolo Capogrosso**, Giulia Maria Castagna (since August 2013), Fabio Castiglione**, Giovanni La Croce** DATA MANAGER: Donatella Moretti * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele DIVISION OF MOLECULAR ONCOLOGY Introduction by the Directors Mission and vision - to overcome the biological barriers that prevent clinical advances in the treatment of cancer innovative strategies are needed in the areas of diagnosis, patient risk stratification and treatment. Along this line we are building onto two fundamental concepts: 1) cancer is a collection of many diseases most requiring specialized research, diagnostic and treatment; 2) the development of new treatment strategies depends on the seamless and multi-disciplinary interaction between researchers and specialized clinicians. Accordingly we are fostering extensive collaborations between basic scientists and clinicians and improving international connections to develop translational and clinical research programs. Organization - The Division is organized into Basic Research Units (URB) and Groups of Clinical Research (GRC) trying to funnel their activities into Research Programs. Overall the Personnel is close to 190 people. The Lab space allocated to the Division is about m2 1800. To favor a more mature organization we are developing, together with Clinical Departments and other Research Divisions, infrastructure facilities that include an annotated data base of patients with blood tumors linked to a bio-bank (in collaboration with the Dept of Pathology). We have also established mouse and cell line registers. Goals - The scientific goals of the Division are based on the approach “build on strength”. Two major areas of excellence have been identified: Cancer Microenvironment and Cancer Immunology and Immunotherapy. The scientific aims within these areas have been directed into two specific Research Programs, Microenvironment and Genes in Cancers of the Blood (MAGIC) and Immunology and Bio-Immunotherapy of Cancer (PIBIC), which span from basic science to clinical application and focus on specific malignancies for which we are internationally renowned. Moreover Dr Luca Gianni and his group have opened a new perspective in the development of Phase I clinical trials and the investigation of Breast Cancer, Prof F. Montorsi and his group are focusing onto the investigation of Prostate Cancer. Achievements - MAGIC bridges the Division of Molecular Oncology with the Clinical Department of OncoHematology, entails the participation of numerous other Research Divisions and is essentially centered upon Chronic Lymphoid Malignancies. The program combines and compares the genetic and microenvironment analysis into a fully integrated genotype-phenotype and tumor-microenvironment scientific approach utilizing a whole range of mouse models, many established by different members of the Division. We expect to translate the findings into a comprehensive blueprint for the risk stratification of individual patients and to develop novel therapeutic strategies based upon new molecular targets. PIBIC is co-organized with the Division of Immunology, its scientific theme being an Institutional historical strength. PIBIC’s focus is the role of the immune system and of its manipulation in specific cancers, especially melanoma and epithelial cancers. The basic/pre-clinical results start to be translated into novel, proof of principle clinical trials. Training Opportunities - The Division hosts the Section of Biology and Biotherapy of Cancer which is part of the Institutional PhD Program in Molecular Medicine, offers numerous post-doc opportunities at national and international level and is well prepared to host, train and mentor Physician Scientists. 11 DIVISION OF MOLECULAR ONCOLOGY Research Units Lymphoid malignancies Unit Monocyte/macrophage lineage cells significantly influence the survival and proliferation of CLL cells in mouse models The development and progression of CLL are dependent upon a complex microenvironmental network of cellular and molecular signals. As an example, the in vitro survival of CLL cells is supported by nurse-like cells, which have been identified as a CLL-specific tumor-associated macrophage (TAM) population. However little is known regarding the role of TAMs in CLL development and progression. We characterized the macrophage composition in two CLL mouse models: a) a xenograft model based on the engraftment of the MEC1 human CLL cell line into Rag2-/-gc-/- mice; and b) the TCL1 transgenic mouse model of CLL. We analyzed the whole-genome transcriptome of TAMs and leukemic B cells isolated from the bone marrow of MEC1-bearing mice and found an enrichment of genes involved in inflammation and interaction between TAMs and B-cells. We then employed an in vivo macrophage killing approach based on liposome-mediated internalization of clodronate (clodrolip) by macrophages. We transplanted Rag2-/-gc-/-mice intravenously (i.v.) with MEC1 cells and depleted macrophages by i.v. clodrolip delivery. Macrophage depletion resulted in a drastically reduced accumulation of CLL cells in all tissues. Moreover, when C57BL/6 mice were transplanted with leukemic cells from TCL1 transgenic mice and macrophage-depleted, significantly fewer leukemic B cells were detected in the lymphoid tissues analyzed. Finally, to explore the possibility that manipulating leukemic cell/macrophage interactions might be exploited as a potential novel therapeutic strategy, Rag2-/-gc-/- animals transplanted subcutaneously with MEC1 cells and carrying visible leukemic lymph nodes (LN) were injected with clodrolip in the proximity of the LNs. This treatment drastically reduced LN size and favourably impacted on the overall mouse survival. In summary, our data open up novel therapeutic avenues for CLL based on interfering with leukemicmacrophage interactions. Federico Caligaris-Cappio Biology of multiple myeloma Role of angiopoietins in multiple myeloma-associated angiogenesis Our Unit is investigating the role of Bone Marrow (BM) microenvironment, and particularly of BMassociated vasculature, in Multiple Myeloma (MM) biology and progression, to identify biomarkers and possibly new therapeutic targets. The Tie-2/Angiopoietins (Ang) receptor-ligand axis has been recognized as a key regulator of angiogenesis in both solid tumors and haematological malignancies. In MM, serum Ang-2 correlates with disease progression and response to therapy. Ang-1 and Ang-2 are the major members of the Angiopoietin family and exert antagonistic roles in angiogenesis, via the competitive binding to the common tyrosine kinase receptor Tie-2 on endothelial cells (EC). In particular, Ang-1 ensures vascular stability, while Ang-2 destabilizes neoforming vessels. In collaboration with Elisabetta Ferrero (Tumor Microenvironment Unit), we are assessing expression and pro-angiogenic functions of Angs in MM. Ang-2 levels in the BM allowed to discriminate patients with active disease, suggesting a pathogenetic role for the molecule. Accordingly, MM BM sera with high Ang-2 concentration specifically contributed to EC activation in vitro, while Ang-1 containing sera maintained EC stabilization/proliferation. The functional dichotomy of Ang-1 and Ang-2 was confirmed by the triggering of distinctive pathways of Tie-2 phosphorylation and downstream signaling through the Akt or the ERKphosphorylation pathway. Notably, Ang-2 but not VEGF serum levels correlated with BM micro-vessel density, further underscoring the key role of Ang-2 in dictating angiogenesis. Western Blot, RT-PCR analysis and immunocytochemistry identified MMEC as the major source of Ang-2 inside the BM, at variance with MM cells, BM stromal cells and CD14+ BM monocytes. These data indicate that Ang-2 produced in the BM milieu contributes to MM angiogenesis in vitro and in vivo and suggest that the molecule can be further exploited both as angiogenesis biomarker and as a potential therapeutic target. We next plan to determine the prognostic value of Angs, in association with other angiogenic molecules, in MM. This might help to define an “angiogenic profile” of MM patients and in turn allow the identification of patients who could benefit from targeted anti-angiogenic approaches. Marina Ferrarini DIVISION OF MOLECULAR ONCOLOGY Cell activation and signalling Toll-like receptors in chronic lymphocytic leukemia Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by the accumulation of small clonal B-lymphocytes in the peripheral blood and lymphoid tissues. Several lines of evidence support the hypothesis that, in addition to specific genetic alterations within leukemic cells, microenvironmental interactions may also play a role in the biology of the disease. Toll like receptors (TLR) are expressed by normal and leukemic B cells; that notwithstanding, different patients with CLL show different patterns of expression of TLR and signaling molecules in leukemic lymphocytes. Previous data suggested that the stimulation of CLL cells with TLR ligands can trigger specific signaling pathways and can regulate the expression of surface molecules and spontaneous apoptosis. During 2013 we focused our attention onto chemoresistance; we hypothesized that TLR stimulation could modify the response of CLL cells to subsequent drug treatment in vitro. We isolated leukemic cells from the peripheral blood of CLL patients, and cultured them with or without the addition of specific TLR ligands; following, we treated the cells with fludarabine, and we analyzed cell viability, apoptosis and molecular pathways involved. Heterogeneity was observed among samples, and TLR stimulation caused a significant protection to fludarabine treatment in leukemic cells isolated from patients bearing adverse prognostic factors. To identify which molecular mechanisms accounted for this heterogeneous response, we performed an RT-PCR apoptosis gene expression array on leukemic cells either unstimulated or stimulated with TLR9 ligand. Strikingly, TLR9 stimulation upregulated the expression of lymphotoxin-α and miR155-3p specifically in “chemoresistant” cells which resulted protected from in vitro fludarabine treatment. These results help to better understand the specific contribution of distinct TLR within distinct groups of CLL samples, and suggest that among microenvironmental interactions TLR ligands may play a role in CLL pathobiology and chemoresistance. Marta Muzio Tumour microenvironment Ex-vivo dynamic culture in bioreactor of human MM samples and MM cellsseeded scaffolds allows the study of MM biology and response to drugs Multiple Myeloma (MM) cells depend on the interactions with the Bone Marrow (BM) microenvironment for their survival and progression. Accordingly, a better understanding of MM biology and response to drugs require the existence of human models able to properly reflect the dynamic complexity and spatial organization of the native milieu. In collaboration with Marina Ferrarini (Unit of MM Biology), we have recently developed and validated a human, dynamic model of culture in RCCS TM, suitable to keep in culture MM samples for up to two weeks; samples maintained their viability and intact overall architecture, including vasculature and bone lamellae. This technology allowed also to monitor over time specialized functions of MM cellular components, in particular vessels, and, notably, to assess response to drugs. As an alternative to the use of MM samples, we are now exploiting the Bioreactor technology as a tool to support the reconstruction of a MM-associated microenvironment. The reconstruction is based on the use of selected scaffolds pre-seeded with cellular components of MM microenvironment, namely endothelial cells and BM stromal cells, to be repopulated with MM cells. The integrated use of scaffolds and Bioreactor sustains an efficient cellular pre-seeding and MM viability, proliferation and specialized functions for weeks. Our final achievement is to recreate an autologous, patient-derived system, for the identification of personalized therapies. Elisabetta Ferrero 13 DIVISION OF MOLECULAR ONCOLOGY Research Units Figure 1. Scanning Electron Microscopy (SEM) analysis of scaffolds populated with endothelial cells and myeloma cells and cultured in RCCS TM bioreactor (in collaboration with Consorzio MIA). Immuno-biotherapy of melanoma and solid tumors Unit During 2013, the research activity of our Unit has been focused on: 1. Pre-clinical work • Mutated antigens of colorectal cancer (CRC). During last year we have continued and concluded the first part of our project aimed at testing the immunogenicity of somatically mutated new CRC antigens, previously identified by sequence analysis of the mutations of CRC cells and of their cancer stem cells (CSC). This work involves the collaboration with the Unit of Exp. Immunology (P. Dellabona) within the framework of the PIBIC. • Cancer Stem Cells. CSC have been isolated in vitro from glioblastoma and CRC and their immune profile defined. We have previously shown that CSC of human glioblastoma may be immune-suppressive. In the 2013 we have identified potential mechanisms of such a phenomenon, in the CSC-mediated activation and release of the indolamine 2,3-dioxigenase by cancer cells, and by expression of IL-4 by CSCs. • Immune monitoring of melanoma patients treated with kynase inhibitors or ipilimumab. In collaboration with other groups (M. Maio, Siena; L. Rivoltini, Milan) we have analyzed the phenotype and function of patients’ T cells before, during and after therapy in order to identify immune biomarkers associated with the clinical response. It was found that a large fraction of melanoma pts who have received ipilimumab show an increased T cell response against defined melanoma antigens. 2. Clinical studies • Adoptive immunotherapy of melanoma. New studies allowed to identify the best procedure of in vitro expansion for patients’ anti-tumor T cells. In 2013 we have validated the procedure of T cell preparation under GMP in 3 cases as requested by Italian regulatory authorities. The dossier to be submitted to AIFA is an advanced stage of preparation. A new PI (Dr. Jacopo Peccatori) was recruited since Dr. Parmiani retired from OSR at the end of 2013; a re-evaluation of the different aspects of the project is ongoing to allow to initiate the clinical trial. • A new protocol based on the combination of DIVISION OF MOLECULAR ONCOLOGY NGR- hTNF and peptide vaccine has been closed; 8 pts have concluded the treatment program. Immune response to the vaccine and to other melanoma antigens (antigenic spread) has been found partly associated with the clinical outcome. • Another spontaneous trial combining Ipilimumab and Fotemustine was approved for metastatic melanoma; the patients accrual was closed in March 2012. Results were collected during the first part of the 2013 and then published in Lancet Oncology. Giorgio Parmiani Cancer gene therapy It is recognized that the formation of human cancers requires the progressive acquisition of oncogene activating mutations and oncosuppressor alterations of normal cells, together with the establishment of immunosuppressive networks within the tumor microenvironment. In recent years, several and distinct immunosuppressive mechanisms dampening antitumor immune responses have been characterized. These mechanisms play a key role in affecting antitumor immune responses, as demonstrated by recent clinical experiences with immune checkpoint blocking antibodies (i.e., anti-CTLA4 and anti-PD1 monoclonal antibodies), which have prolonged the overall survival of metastatic melanoma patients. Our group has recently identified a mechanism of immune escape based on the release by tumor cells of metabolites of cholesterol, namely oxysterols (Traversari and Russo. Curr. Op. Pharmacol, 2012). These molecules and their receptors (i.e., Liver X Receptors), primarily identified as regulators of cholesterol homeostasis, are currently recognized as pleiotropic and multifaceted molecules controlling several functions of immune cells. In this context, we have shown that tumorderived oxysterols inhibit the antitumor immune response by recruiting pro-tumor neutrophils within the tumor microenvironment (Raccosta et al. J. Exp. Med, 2013). Recruited neutrophils promote tumor growth by inducing neo-angiogenesis or by suppressing antigen-specific T cells. Notably, the use of drugs interfering with oxysterol generation restores the spontaneous antitumor immune response and the control of tumor growth in mouse tumor models. In addition, we are investigating new strategies of active immunotherapy to improve antitumor immune responses (Russo et al. Trends Mol. Med, 2012). We have developed a novel strategy of active immunotherapy based on the in vivo loading of antigen presenting cells (i.e., dendritic cells) with tumor antigens (Russo et al. J Clin Invest, 2007). The clinical translation of this approach led to the development of a clinical protocol showing a favorable correlation between the increase of antitumor effectors and overall survival in melanoma patients (Fontana et al. Blood, 2009; Russo et al. Int. J. Cancer, 2013). Vincenzo Russo B-cell neoplasia Unit Non-random association of novel gene mutations with stereotyped CLL subsets Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. During 2013, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were 15 DIVISION OF MOLECULAR ONCOLOGY Research Units found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy. Paolo Ghia Cumulative events Genetic defects in Subsets are not random Subset 1 Subset 2 Subset 8 Subsets Ghi t l Bl d 2005 Figure 2. Different stereotyped subsets show distinct genetic abnormalities (frequency of somatic mutations on NOTCH1 and SF3B1 genes). Modified from: Strefford, JC et al., Leukemia: 2013; 27(11): 2196-2199 doi: 10.1038/leu.2013.98 Functional genomics of cancer Unit Our laboratory is currently pursuing the following lines of research: 1. Role of histone methylation and demethylation in cancer development In the nuclei of eukaryotic cells, DNA is packed into chromatin. The basic constituent of chromatin, the nucleosome, is comprised of 147bp of DNA wound around an octamer of core histone proteins. Post-translational modifications of the N-terminal tails of these core histone proteins, modulate chromatin configuration, resulting in changes in transcriptional regulation. These epigenetic modifications are controlled by complex enzymatic machinery that, when deregulated, disrupt normal transcriptional programs. Indeed, genomic alterations of MLL, NSD1, CREBBP, WHSC1L1 and WHSC1, all encoding proteins involved in histone modification, have been implicated in several cancer types. Through genomic analysis and large-scale highthroughput sequencing, we have identified genetic lesions affecting histone methylases and demethylases and we are exploring the role of the genes implicated in these genomic rearrangements in the development of multiple myeloma, lung cancer and renal carcinoma. DIVISION OF MOLECULAR ONCOLOGY 2. RNA metabolism in cancer The pathways leading to RNA catabolism are poorly characterized, and even less their potential role in cancer. We have identified genetic lesions affecting genes at the crossroad of various RNA metabolism pathways. Ongoing experiments are evaluating their role in dysregulating oncosuppressive and oncogenic mechanisms in cancer cells. 3. Receptor tyrosine kinase receptors in the development of Multiple Myeloma The protein kinase is the most commonly represented Pfam domain encoded by cancer genes. Many carcinogenic tyrosine kinases are receptors, which are often overexpressed and/or mutated in a variety of tumors. Therapeutic agents targeting this class of receptors have proven to be highly effective in the clinical setting. We have conducted a genomic and proteomic survey of primary multiple myeloma samples, a deadly hematological cancer and have identified two overexpressed RTKs. The goal of this project is to elucidate the nature of the signaling mediated by these RTKs in MM, both in vitro and in vivo, with the final goal of identifying more effective drugs against this deadly disease. Giovanni Tonon Lymphoid organ development Unit Development and function of the lymphoid stromal microenvironment Research in our laboratory focuses on understanding the mechanisms underlying the development of lymphoid organs with emphasis on stromal cells. Current research projects aim: 1. To identify signaling pathways underlying specification of stromal progenitors. The signaling pathways required for specification and differentiation of stromal progenitors remain largely unknown. We use mouse genetics coupled to microarray and ChIP-seq analyses to identify pathways controlled by spleen and lymph node-specific transcription factors during early spleen and lymph node development. We are currently assessing the contribution of different signaling pathways in organogenesis of lymphoid tissues. 2. To uncover the origin of mature lymphoid stromal cells. The origin of the different stromal cell subsets remains elusive. By performing lineage-tracing analysis, we demonstrated that spleen, but not lymph node stromal cells, originate from embryonic Nkx2-5+Isl1+ mesodermal progenitors. This lineage includes lymphoid tissue organizer cells capable to promote the formation of artificial lymphoid-like structures with features of native tissues. We discovered that resident stromal cells of this lineage proliferate and regenerate the stromal microenvironment following resolution of a viral infection. These findings provided novel mechanistic insights into how stromal diversity originates and stromal repair occurs, and the basis for developing artificial lymphoid tissues for pre-clinical applications (Castagnaro et al., Immunity 2013). We are currently assessing the origin of lymph node stromal cells. 3. To dissect the role of the stromal microenvironment in leukemia. It is increasingly clear that stromal cells play a crucial role in B-cell malignancies including chronic lymphocytic leukemia (CLL) survival and expansion. However, the precise nature of stromal cells and the mechanism by which these cells contribute to leukemia progression remain unclear. We performed a comprehensive in vivo analysis on murine and human CLL biopsies and identified changes occurring within the stromal compartment during disease progression. We are currently identifying the signaling pathways contributing to the remodeling of the stromal microenvironment during leukemia progression. Andrea Brendolan 17 DIVISION OF MOLECULAR ONCOLOGY Research Units Molecular histology and cell growth Unit Translational control in cancer Gene expression is controlled at the level of translation by the combination of Initiation Factors and RNA binding proteins. It has now emerged that translational control is altered in tumors and it can be targeted by specific drugs. Our laboratory has shown that eIF6 is rate-limiting in tumor formation by a combination of cellular, biochemical and genetic techniques. In summary, some tumors express high levels of hyperphosphorylated eIF6, and its targeting results in reduced tumor growth. Through a combination of High Throughput studies and specific investigator-driven hypothesis, we have been able to identify the molecular signature that accompanies tumorigenesis mediated by eIF6 activation. In parallel, our laboratory has contributed to the identification of a) translational pathways altered in multiple myeloma, b) new therapeutic strategies in malignant mesothelioma, and c) new cellular models for the validation of drugs acting in a patient specific fashion. In summary, we have contributed to the new notion that gene expression is shaped by translational mechanisms that can be exploited for targeting cancer cells. Stefano Biffo Preclinical models of cancer Unit Role of HIF factors in hematological malignancies We aim to understand the role of hypoxia-inducible transcription factors (HIF) in hematological malignancies, and to test the efficacy of their targeting as a novel therapeutic approach in combination with standard therapeutic agents. HIF factors are often upregulated in solid tumors because of hypoxia or oxygen-independent mechanisms, and promote tumor progression by regulating metabolic adaptive pathways, neo-angiogenesis, cell migration and invasion, and maintenance of cancer stem cells. In the past year we have terminated earlier studies where we had identified HIF-1α as a critical proleukemogenic factor in acute myeloid leukemia (AML), particularly in the M3 subtype acute promyelocytic leukemia (APL), and in chronic lymphocytic leukemia (CLL). We had found that in APL cells HIF-1α is functionally activated because of direct cooperation with the oncogenic fusion protein PML-RARα while in CLL it is highly expressed at the mRNA level. Inhibition of HIF-1α by shRNA led to reduced leukemia progression in both pathologies. However, interestingly HIF-1α plays different roles in distinct hematopoietic neoplasms: in APL HIF-1α regulates intrinsic and chemokine-directed cell migration, neoangiogenesis in the bone marrow and leukemia stem cells self-renewal, while in CLL HIF-1α especially regulates the interaction of leukemic cells with bone marrow and splenic microenvironments. As a consequence, inhibition of HIF-1α by compounds with HIF-inhibitory activity blunted leukemia progression in both pathologies, but in APL it also affected maintenance of leukemia initiating cells while in CLL it induced the mobilization of CLL cells from bone marrow and spleen and cooperated with chemotherapeutic compounds in inducing CLL cells apoptosis. In this past year we have found that in APL expression of HIF-1α is further upregulated by treatment with all-trans retinoic acid (ATRA), the treatment of choice for APL patients. ATRA treatment does not fully eradicate APL, and in vitro increases clonogenicity of leukemic blasts. We found that concomitant treatment of APL cells with ATRA and inhibitors of HIF-1α exquisitely cooperates in reducing leukemia-initiating cells in serial replating and serial transplantation experiments. Rosa Bernardi DIVISION OF MOLECULAR ONCOLOGY Tumor biology and vascular targeting Unit Regulation and manipulation of tumor vessels and microenvironment Our research activities focus on studies of tumor vascular biology and of the development of new strategies for cancer therapy based on the manipulation of the tumor vasculature and microenvironment. We have found that peptides containing the NGR or isoDGR sequences can be exploited for delivering TNF-α and other cytokines to tumor vessels and, consequently, for altering the endothelial barrier function and increasing the penetration of chemotherapeutic drugs and infiltration of lymphocytes in tumors. Because of these properties, one of these compounds, called NGR-TNF, is currently tested in various Phase II and III clinical studies, alone and in combination with chemotherapy or immunotherapy. Other lines of research are focused on the role of proteins containing the NGR, and isoDGR sequences in tumor vascular biology and angiogenesis. Regarding NGR (asparagine-glycine-arginine) this sequence is present in many molecules of the extracellular matrix. We have demonstrated that NGR can undergo rapid deamidation reactions generating the isoDGR (isoAsp-Gly-Arg) sequence in fibronectin and in other proteins of the extracellular matrix. This post-translational modification can work as a “molecular switch” for the time-dependent generation of new binding sites for integrins. In particular, we have found that peptides and fibronectin fragments containing the isoDGR motif can bind αvβ3, an integrin expressed in the angiogenic vasculature, can affect endothelial cell functions and inhibit tumor growth. We have also developed head-to-tail cyclic isoDGR peptides that can be easily coupled to proteins and gold nanoparticles and demonstrated that they can be exploited as ligands for delivering cytokines and nanodrugs to the tumor vasculature. Finally, we have observed that circulating CgA, a protein secreted by many neuroendocrine cells and granulocytes, is elevated in patients with cancer and other inflammatory diseases, can regulate the endothelial barrier function and the TNFinduced vascular leakage, can regulate angiogenesis (by working as an angiogenic switch activated by thrombin), and can reduce tumor cell trafficking and metastasis development in animal models. Angelo Corti 19 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units Digestive and pancreatico-biliary endoscopy Unit The unit is involved in the development and clinical application of advanced endoscopic imaging and endoluminal therapy for recognition and treatment of high grade dysplasia and early cancer of gastrointestinal tract (G.I.) and pancreatico-biliary system. Advanced endoscopic imaging In these years the research has been focused mainly on the use of advanced imaging techniques for detecting and characterizing mucosal and submucosal lesions of gastrointestinal tract and differential diagnosis of strictures of unknown aetiology of the main pancreatic and common bile duct. Surface and contrast enhancement endoscopy has been shown effective in the recognition and treatment of mucosal pre-neoplastic and neoplastic lesions; the technique appears particularly useful to investigate more accurately Barrett’s esophagus, gastric dysplasia, colonic flat lesions. Confocal endomicroscopy is currently used in clinical practice since the beginning of 2010. It is a new advanced imaging technique which enables in vivo microscopy with subcellular resolution during ongoing endoscopy. The technique is very effective in detecting neoplastic lesions at a very early stage, identifies pre-neoplastic lesions, and gives information about living cells in human beings. Confocal endoscopy can be carried out also by probes that can be inserted into the pancreatico-biliary system, during ERCP procedures, and plays a pivotal role in the endoluminal diagnosis and staging of cholangiocarcinoma. Endoluminal therapy Endoscopy is increasingly used for treatment of neoplastic or pre-neoplastic lesions of the GI tract confined in mucosa and submucosa layer, since it allows to remove tissue by mucosal resection, submucosal dissection, and radiofrequency ablation. Radiofrequency can also be used into pancreatico-biliary ductal system to treat neoplastic strictures. The unit is involved in the development of new devices for tissue removal and radiofrequency delivering for both GI tract and pancreatico-biliary system. These devices consent the complete removal of pre-neoplastic or early neoplastic lesions, and treatment of inflammatory and neoplastic ductal strictures. Circulating biomarkers Biomarkers should be useful in clinical surveillance in digestive diseases, in prevention of malignancy, and recognition of cancers in early stage. Circulating levels dosage of apoptosis inhibitory proteins and mRNA will be analyzed. In particular, QSOX-1, survivin, XAF-1 and BCL2 will be tested in Barrett’s esophagus and gastrointestinal cancer, IPMN, and pancreatic adenocarcinoma. Pier Alberto Testoni Endosonography: diagnostic and therapeutic endoscopic ultrasound During 2013, our Unit focused on the following Research Projects: • EUS-guided cryothermal ablation in patients with stage III (Locally advanced and borderline resectable) pancreatic adenocarcinoma. Phase II/III trial. Grant ERBE gmbh Germany. Local treatment of pancreatic cancer with vessel infiltration to increase the resection rate was the end points of a trial on the use of a new prototype of ablation device. The preliminary results have shown the safety of this device and a trend in favor of tumor reduction after treatment. Now a Randomized Controlled Trial was designed to show the efficacy of the probe to increase resection rate of this tumor. • IPMN branch duct follow up. Multicenter Trial (Mayo Clinic Charlottsville Principal Investigator) Adherence of Sendai criteria to the EUS follow up data in branch duct IPMN. Presentation Award Best Poster Presentation DDW 2014 Chicago. • ASPRO Study. Randomized Controlled Trial on comparison of 20 G pro-core needles vs 25 needles in FNA (Erasmus Rotterdam Principal Investigator) (Funded by Cook Medical) • EUS FNA is considered the best modality to target lesions in different organs In some cases the difficulty in specimen interpretation by pathologist hampers the diagnosis. Histology could provide easier interpretation. The EUS Unit was involved in a European Group (Pro Core) to study new histology needles. In 2013 the European Group has expanded to an International Group and the ASPRO Study is the first DIVISION OF MOLECULAR ONCOLOGY step of this International cooperation on this field. • CAPS International. International Program on Pancreatic Cancer Screening (John Hopkins Baltimore Principal Investigator). There is nowadays a great interest in the definition of the characters that could lead to an early diagnosis of Pancreatic Adenocarcinoma and the definition of screening modalities on high risk populations is very important. In 2011 our Group has participated to the 1st International Meeting on Pancreatic Cancer Screening, a consensus paper published in 2013. After that the CAPS Group established an international screening of high risk patients. Paolo Giorgio Arcidiacono Gastrointestinal surgical oncology Unit Consistently at the forefront of surgical innovation, the Gastrointestinal and Peritoneal surface Surgery Unit is transforming the multidimensional field of surgery through delivering compassionate clinical care, making groundbreaking discoveries and training future surgeons. From basic science to clinical investigations, the Unit is working in a wide range of research that is transforming the field of surgery. The Section of Gastrointestinal and Peritoneal Surface Surgery has maintained its prominence as a national and international leader in the surgical management of patients with complex diseases by building on its rich heritage while continuously evolving to define today’s and tomorrow’s standards of care. In 2013, the Unit expanded its faculty in critical areas of colorectal and upper GI surgical oncology, and peritoneal surface malignancy with wide use of minimally invasive techniques exploring new surgical approaches to a variety of difficult clinical problems. The Unit maintains a national and regional presence in clinical trials that aim at improving treatment for patients with esophageal, gastric, colorectal and peritoneal surface malignancy. Over the past years the Unit has pursued the following projects: 1. Role of induction of innate immunity in nonmetastatic rectal cancer after neo-adjuvant radio-chemotherapy and its relationship with patient survival and pathological response. 2. Tumor regression grade after neo-adjuvant chemotherapy plus surgery in Upper GI malignancy. 3. HER-2 expression after neo-adjuvant chemotherapy for Upper GI malignancy. 4. Single- site surgery application in staging laparoscopy in order to reduce the rate of port metastasis. 5. Application of a novel surgical technique (APPEAR) in the treatment of rectal cancer. 6. Sentinel node mapping in Upper GI Cancer. 7. Clinical application of cyto-reductive surgery and HIPEC (intra peritoneal chemotherapy) in the treatment of peritoneal carcinomatosis in Upper GI and colorectal cancer. 8. Role of radio chemotherapy in the esophagealgastric junction (EGJ) tumors. Gianfranco Ferla Head and neck oncology Unit During 2013 our research activity focused on the following areas: • Patient recovery after surgery, with the implementation of an Enhanced recovery after surgery (ERAS) program in order to reduce morbidity, improve recovery, and shorten hospital stays of surgical patients. In our Unit this program has been applied in patients undergoing major oncologic surgical procedures. • New technologies for early diagnosis of head and neck cancer. In 2013, we used Narrow Band Imaging (NBI) technology in the evaluation of precancerous laryngeal and oropharyngeal lesions. We gathered preliminary data on the reliability of this technology that will undergo further investigation during patient follow up over the following years. • HPV detection in oral and oropharyngeal cancer. We aimed at replicating the findings of previous studies on detection of HPV DNA in lymph node FNABs in head and neck cancer of unknown origin (CUP-syndrome) patients. • Biomarkers associated with relapse of Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). In collaboration with the Immunology and Allergology department, we investigated the role of Long Pentraxin 3 (PTX3) as a possible relapse biomarker in CRSwNP after surgical treatment. Our results show that PTX3 is a promising biomarker that could be implemented for use in 21 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units clinical settings to guide therapeutic decision. • New technologies for diagnosis of Granulomatosis with Polyangiitis (GPA, Wegener’s). We conducted a pilot investigation on the possible role of NBI technology in the recognition of submucosal vascular patterns possibly associated with GPA. We identified and classified some mucosal vascular pattern visible with NBI light in GPA patients and we evaluated their diagnostic potential. • Multicenter study concerning the head and neck manifestations of GPA. • Prediction of outcomes in aesthetic surgery with the development of a mathematical model to assess adherence of pre-rhinoplasty projects with surgical results. • Mini invasive new surgical approaches to parapharyngeal space. Mario Bussi Multidisciplinary group for thoracic surgical oncology New Positron Emission Tomography-derived parameters as predictive factors for recurrence in resected Stage I non-small cell lung cancer (Giulio Melloni) The recurrence rate for stage I non-small cell lung cancer is high with 20-40% of patients that relapse after surgery. We evaluated new F-18 fluorodeoxyglucose positron emission tomography derived parameters, such as standardized uptake value index, metabolic tumor volume and total lesion glycolysis as predictive factors for recurrence in resected stage I non-small cell lung cancer. Solitary fibrous tumors of the pleura: himmunohistochemical analysis and evaluation of prognostic factors after surgical treatment (Alessandro Bandiera) Solitary fibrous tumors of the pleura (SFTP) are rare neoplasms with unusual histological and clinical features. Although surgery is the treatment of choice tumor recurrence may occur after complete resection. The aim of the study was to identify the clinical and pathological features of SFTP associated with a higher risk of recurrence after surgical treatment. Results of surgical treatment of large cell and large cell neuroendocrine carcinomas of the lung: a clinico-pathological study (Angelo Carretta) Large cell neuroendocrine carcinomas (LCNEC) of the lung are a group of tumors with peculiar histological and clinical features. Aim of the study is to analyze the results of surgical treatment of LCNEC and to assess the prognostic role of clinical and histological factors. Pediatric parapneumonic empyema: a correlation between preoperative chest ultrasonography and surgical evidence (Paola Ciriaco) The purpose of this study was to evaluate the role of chest ultrasonography in staging pediatric parapneumonic empyema in terms of a correlation between ultrasound patterns and surgical evidence. Dumbbell mediastinal neurogenic tumors: diagnostic and surgical strategy (Giampiero Negri) Neurogenic mediastinal tumors presents an intraspinal extension in 10% of the cases and rarely an intramediastinal development, becoming the so called Dumbbell neurogenic tumor (DNT). Aim of the study was to analyze diagnostic and surgical strategy of the mediastinal DNT in terms of surgical approach end long term results. Piero Zannini DIVISION OF MOLECULAR ONCOLOGY Oncogenesis in liver neoplasms Unit The Hepatobiliary Unit is a multidisciplinary clinical unit where the traditional gap between physicians and surgeons has been overcome to reach the optimal standards of patient care. Indeed, the medical staff includes hepatologists and hepatobiliary surgeons who share their specific competences to treat in an unique environment both “medical” and “surgical” patients. Clinical activities are mainly focused on hepatobiliary tumors and chronic liver diseases, including primary (hepatocellular carcinoma and cholangiocarcinoma) and metastastic liver tumors (mainly liver metastases from colorectal cancer), liver cirrhosis and related complications (portal hypertension, ascites, esophageal varices), benign and malignant diseases of the biliary tract, acute and chronic hepatitis. As far as liver tumors are concerned, the research activities are mainly focused on the outcome (in term of overall survival and disease-free survival) of patients affected by liver metastases from colorectal cancer and treated by surgery and adjuvant/neo adjuvant chemotherapy. Studies regarding the prognostic factors of survival after surgery and the impact of locoregional chemotherapy on the outcome after surgery have been published (World J Surg, Ann Surg Oncol, J Gastroint Surg, J HBS). Among the speculative studies about the liver metastases from colorectal cancer, the hepatobiliary unit have been involved in cooperative protocols of study with the Unit of Immunobiotherapy of Melanoma and Solid Tumors. Surgical specimens have been collected after liver resection for colorectal liver metastases to characterize the expression of OX40 and 4-1BB on peripheral blood lymphocytes and in tumor and lymphoid tissue of oncological patients. The study has ended the phase of case collection and it is still ongoing for the final analysis of the results. During 2013, further attention has been focused on hilar cholangiocarcinoma: indeed, patients affected by this kind of neoplasm still have a severe long term outcome despite surgical treatment, which presently represents the only potentially curative treatment. In this setting, studies regarding oncogenesis may contribute to amelioration of overall and disease free survival of these patients. Gianfranco Ferla Pancreatic cancer Unit: biology and new therapeutic approaches Pancreatic cancer is the fourth cause of cancer mortality in the Western world and it has the worst prognosis among malignancies, with a 5-years survival around 5%. The San Raffaele Hospital is one of the leading Italian Institutes for the treatment of pancreatic cancer and our Unit is devoted to the identification of new therapeutic approaches against this devastating disease. One of the mainstay to improve the results of surgery is chemotherapy (CT). Jointly with the Medical Oncology Unit, we are currently evaluating the efficacy of different adjuvant CT schemes in resectable pancreatic cancer, including preoperative (neoadjuvant) CT. The San Raffaele Hospital is the Italian leading centre in patients enrollment in an ongoing multicentre, randomized, phase II-III study about perioperative CT. In this setting, we recently demonstrated, through a case-match study, the safety of neoadjuvant approach in the short-term postoperative outcome of pancreatic resections. In patients with locally advanced pancreatic cancer we are evaluating further approaches such as radiation therapy and cryothermal ablation. A phase I study has been completed to determine the maximum tolerated radiation dose of an integrated boost to the tumour subvolume infiltrating vessels, delivered simultaneously with radical dose to the whole tumour. Cryothermal ablation of the tumour can be applied under Endoscopic Ultrasound guidance to reduce the tumour mass, potentially increasing the resection rate of locally advanced cancer and patient survival. We are collaborating with the Endosonography Unit to develop a prospective randomized study on this matter. Further, in collaboration with the Tumor Immunology Unit, we are investigating the role of tumor immune infiltrates in pancreatic cancer regression or promotion. Finally, we have evaluated the oncologic safety of islet autotransplantation (IAT) in the liver of patients with pancreatic and periampullary malignancy. We applied IAT in 17 patients with malignancy, and none of them developed metastases in the transplantation site. Gianpaolo Balzano 23 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units Pathology Unit Our research activity is mainly focused on three major topics: hematopathology, pancreatic neoplasms and urologic malignancies. In particular, during 2013, our hematopathology group has been actively involved in two major fields, the link between infectious agents, mainly bacteria, and lymphoproliferative disorders and the relation between neoplastic cells and microenvironment in lymphomas. In the latter area, our investigation involves the relationship between neoplastic cells and microenvironment in hematologic neoplasms, including indolent forms (such as marginal zone lymphomas, chronic lymphocytic leukemia, follicular lymphomas, plasma cell dyscrasias, chronic myeloproliferative disordes and myelodysplastic syndromes) and aggressive hemopathies. In this context several studies are currently active and in particular the role of accessory cells - T lymphocytes subclasses, monocyte-macrophages cells, dendritic and stromal cells - in these heterogeneous malignancies. Regarding T lymphocytes, we are currently characterizing their immunomodulatory role in follicular lymphomas and a cooperative study with Prof. C. Vinuesa (Immunological Institute, Australian National University, Canberra, Australia) for the characterization of the neuroendocrine features of T follicular helper cells, is in progress. In addition, we recently investigated the morphology and the immunophenotypic and molecular features of Erdheim-Chester disease. This is a rare, progressive and fatal disease of monocytes/macrophages cells, involving bone marrow and several other tissues and organs. In this disease monocytes/macrophages are mutated in Braf and display unique phenotypic and functional properties. We have also identified the presence of mutated monocytes not only in involved organs, but also in the peripheral blood, paving the way for a new diagnostic approach in this disease. We have performed, in cooperation with the COMP of the Dana Farber Cancer Institute in Boston, a massive sequence analysis of a series of neuroendocrine pancreatic neoplasms with the identification of a number of putative relevant gene alterations. Furthermore, the scientific activity of the Pathology unit relies also on the collaboration with Research Groups at our institution, but also with National, European and Intercontinental leading Institutions, where some of our staff members act as chairmen and coordinators of international studies. The scientific production from Our Unit is sound and mostly devoted on the translational implications of basic discoveries, as witnessed by the several high-quality publications present within top scientific journals with high Impact Factor. Beside the research carried on patients, Our Unit incorporates the Mouse Histopathology Unit, with the aim of supporting research groups in the advancement of scientific projects, by providing morphological expertise and immunophenotypic analysis. In particular, part of these skills contributed to the development of the HSR-TIGET Test Facility, thus enabling to obtain the certification of compliance with the Principles of Good Laboratory Practice (GLP) for preclinical toxicity/tumorigenicity studies of gene therapy medicinal products. Claudio Doglioni Clinical lymphoid malignancies The approach of the Clinical Unit of lymphoid malignancies against the primary CNS lymphomas Ten years ago, we established a multidisciplinary scientific group focused on primary CNS lymphomas (PCNSL), the International PCNSL Collaborative Group (IPCG). Since then, over 100 researchers and clinicians working on PCNSL from 19 countries have been actively involved in this group established under the sponsorship of the International Extranodal Lymphoma Study Group with conference grant support from the NCI, USA. Since 2003, this multidisciplinary group has met annually or biannually, in Europe or the USA and meetings have been focused on multiple topics relevant for PCNSL including neuropsychological assessment, new targets and drugs, neuroimaging, the role of HDC/ASCT and many other important topics. The continuous updates, exchange of ideas and debates among IPCG members has resulted in the design and execution of key studies in this field. Debates on new concepts and clinical trials have been the fertile background for relevant biological and clinical studies on a challenging malignancy that straddles the border between hematology and neuro-oncology. In fact, the IPCG has become the primary scientific group DIVISION OF MOLECULAR ONCOLOGY focused on improving molecular, diagnostic and therapeutic knowledge and outcomes in PCNSL. IPCG members have written consensus diagnostic and therapeutic guidelines and have standardized baseline evaluation and response criteria as well as neurocognitive assessment recommendations. They have chaired several international studies that contributed to improve the knowledge on PCNSL T-cell, intraocular lymphoma, PCNSL in children and adolescents, and many others. Although it is quite challenging to obtain research funding for investigator-driven clinical research on orphan malignancies like PCNSL, cooperation within the IPCG has ushered in the era of randomized trials in this subtype of lymphoma. In fact, IPCG members have chaired the first two most successfully executed randomized trials in PCNSL, and are coordinating five ongoing randomized trials aimed to improve therapeutic efficacy and minimize neurotoxicity in the PCNSL population. In the next decade, the primary objectives of IPCG collaboration are to increase the proportion of PCNSL patients cured of their disease and to preserve long-term neurocognitive function in PCNSL survivors. Andrés José Marìa Ferreri Gynecologic oncology A retrospective multi-institutional review of patients with granulosa cell tumors (GCTs) of the ovary treated or referred to MITO centres was conducted. Surgical outcome, intraoperative and pathological findings and follow-up data were analyzed. Kaplan-Meier and Cox proportional hazards analyses were used to determine the predictors for survival and recurrence.A total of 97 patients with primary GCT of the ovary were identified. The median follow-up period was 88 months (range 6-498). Of these, 33 patients had at least one episode of disease recurrence, with a median time to recurrence of 53 months (range 9332). Also, 47% of recurrences occurred after 5 years from initial diagnosis. At multivariate analysis, age and stage were independent poor prognostic indicators for survival; surgical treatment outside MITO centres and incomplete surgical staging retained significant predictive value for recurrence in both univariate and multivariate analyses.This study confirms the favorable prognosis of GCTs of the ovary, with 5-year overall survival approaching 97%. Nevertheless, prognosis after 20 years was significantly poorer, with 20-year survival rate of 66.8% and a global mortality of 30- 35. These findings support the need for lifelong follow-up even in early-stage GCT. Treatment with anticancer agents induces dysregulation of specific Wnt signaling pathways in human ovarian luteinized granulosa cells in vitro. Chemotherapy has been associated with premature ovarian failure and infertility in women with cancer. It is well known that anticancer drugs reduce the primordial follicle pool and harm the ovarian blood vascularization leading to ovarian atrophy. However, their mechanism of injury still remains unclear. Treatment with doxorubicin (DXR), paclitaxel (PC), and cisplatin (CP) affected LGCs viability by inducing apoptosis and downregulating both estrogen receptor β and follicle-stimulating hormone receptor in a dose-dependent manner. Several members of the WNT signaling pathway are expressed in granulosa cells where they regulate follicle development, ovulation, and luteinization. Here we show that treatment with DXR, PC, and CP induced upregulation of WNT4 expression, whereas WNT3 expression was downregulated by DXR and PC and upregulated by CP. Giorgia Mangili 25 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units Medical oncology Unit - Clinical trials The Unit is committed to design and conduct investigator-driven clinical trials, is involved in new drugs development and cooperate with teams of laboratory researchers to identify molecular prognostic or predictive biomarkers in the therapeutic management of gastro-intestinal and central nervous system (CNS) cancer. Ongoing projects can be listed under four main topics: 1. Pancreatic cancer: a) definition of molecular prognostic/predictive tools on markers generated by assays of gene expression and single nucleotide polymorphisms involved; b) analysis of the role of inflammation as prognostic/predictive variable; c) development of new therapies and therapeutic strategies, including the role of adjuvant and neoadjuvant polichemotherapy in stage I-II disease; of taxanes and metformin in stage III and IV disease; of maintenance therapy in stage IV disease as well as of salvage therapies in patients with progressive disease. 2. Biliary tract cancer: assessment of new drug combinations in upfront and second-line therapeutic management of patients with metastatic disease and of the role of K-Ras mutations in predicting the activity of anti-EGFR agents. 3. Colo-rectal cancer: evaluation of the feasibility of an adaptive strategy in preoperative radiochemotherapy for rectal cancer and development of new combination therapies in metastatic colon cancer. 4. CNS tumors: assessment of a) the role of target therapies in meningiomas; b) the combination treatment for elderly patients with glioblastoma multiforme; c) the concomitant and sequential chemoradiation in grade III gliomas. Michele Reni Medical oncology Unit - Phase I and lung cancer clinical trials The research objective is to improve patients outcome by applying personalized therapies. The activities of the group are divided into two main areas: clinical trials and translational studies (molecular targets, systemic response against tumor and tumor mico-environment). There are 20 ongoing clinical trials (n) focusing on following targets: EGFR pathway and resistant mechanism (3), K-ras (1), ALK (4), FGFR (1), B-raf (2), immunotherapy (anti PD-L1 target, 4), genotype guided therapies (ITACA and EPIC study), NGR-hTNFα in mesothelioma (2) and palliative radiotherapy + pan-histonedeacetylase (1). We developed a serum multivariate protein-based test, VeriStrat, that classifies NSCLC patients into 2 VeriStrat categories Good and Poor, according to the overall survival with EGFR-TKIs. The predictive value of the test was prospectively evaluated in the independent multi-center trial: PROSE. This is the first prospective biomarker-stratified study in thoracic oncology to test treatment/biomarker interaction. NSCLC patients previously treated platinum based chemotherapy were randomized between erlotinib or chemotherapy (pemetrexed or docetaxel). The trial reached its primary objective of showing significant interaction between VeriStrat classification and therapy. The results showed (ASCO oral presentation) that VeriStrat poor classified patients had shorter survival on erlotinib compared with chemotherapy (3 vs 6.4 months, respectively); HR 1.72 (95% CI 1.08-2.74), p 0.021, while no difference was observed in VeriStrat good classified patients (11 vs 11 months, respectively); HR 1.06 (95% CI 0.77-1.46), p 0.714. The study also confirmed a strong prognostic value of the test. EGFR and KRAS mutation analyses were also performed that confirmed independent predictive value of the proteomic test. A longitudinal analysis shoved that 34% baseline VeriStrat good patients treated with erlotinib who changed to poor had worse PFS compared with those whose remained good. In conclusion, our studies indicate that VeriStrat test can guide treatment decision making in NSCLC patients with unknown or wild type EGFR status in 2nd line setting. The possible clinical impact of changes in VeriStrat classification during therapy requires further investigation. Vanesa Gregorc DIVISION OF MOLECULAR ONCOLOGY Onco-hematology The San Raffaele Onco-Hematology Group participates to several original, national and international clinical and traslational studies, at our Institute and within disease networks (NILG, GIMEMA, EORTC, EBMT). During 2013 we continued to enroll patients with AML, ALL, MM, MDS and MPN in local and multicentric phase II/III trials aimed at evaluating the efficacy of new drugs and/or new treatment strategies. We also partecipated to several cooperative studies within the regional hematologic network (REL), for the diagnosis and treatment of acute leukemias, MDS, CML and MM. Within the REL, we contributed to implement the MDS web-based registry which is collecting multiple epidemiologic, clinical, biological and genetic data from MDS patients followed at the regional haematologic centers. This registry already includes hundreds of patients and keeps on enrolling new patients every year, being periodically updated on their clinical and therapeutic outcomes. In parallel, the network of local biological banks, which has been set up in 2012, has provided cells/nucleic acids from MDS patients for the first biological studies approved and started up within the REL. Data from the registry and the biological samples are always available to participating centers for ongoing and future biological studies. The OSR AML bio-bank was further developed in 2013 providing samples for molecular characterization with new generation assays. This bio-bank also provides samples to several laboratories in San Raffaele-DIBIT thus supporting several important pre-clinical studies in the field of AML and gene therapy. Our group went on in 2013 with studies on metabolomic and angiopoiesis of MGUS and multiple myeloma and in studies on WT1 monitoring to predict MRD persistence or recurrence after allogeneic transplantation and evaluating WT1 levels, as a quantitative marker of leukemic contamination, in autologous PBSC used for autotransplant after myeloablative chemotherapy. A paper reporting the results of this last study is in preparation and will be submitted in early 2014. Our Group has also particularly focused and distinguished in the curative treatment of elderly patients (older than 65-70 yrs) with AML and MDS, utilizing adapted strategies, including autologous or allogeneic transplantation. Massimo Bernardi 27 DIVISION OF MOLECULAR ONCOLOGY Introduction by the Director URI Urological Research Institute URI’s mission is to develop translational projects in clinically relevant topics of uro-oncology and functional urology. In order to effectively develop a translational research program, i.e. to create a close relationship between preclinical researchers and the physicians and surgeons who are faced with patients’ needs and expectations on a daily basis, URI has been structured into Disease Units. Five different disease units currently make up URI. Among these, the Prostate Cancer Unit is devoted to translationally investigating prostate cancer, from bench to bedside, having the unique opportunity to use clinical data from one of largest and most comprehensive patient datasets in Europe. Likewise, the Bladder Cancer Unit is mainly devoted to investigating transitional cell carcinomas of the bladder, along with cancer of the upper urinary tract system. In this specific context, URI provides the unique opportunity to study these issues from multiple approaches, from in-vitro models of tumour cells to large patients datasets, including both data from non-muscle invasive and muscle-invasive bladder cancers. Third, the Kidney Cancer Unit provides excellent research opportunities in terms of projects on renal cell carcinoma, with one of the largest single-institutional datasets in the world. These 3 datasets have been rigorously organized in a web-based system which also provides a user-friendly and private way for patients to update their prospective followup themselves. Moreover, URI has a robust experience in the field of Functional Urology, which includes the translational study of the pathology of the lower urinary tract, the kidney and the ureter. In this specific context, URI has been involved in many multinational preclinical research projects, which have also been translated into close relationships with pharma-companies, thus enabling the development of potentially novel molecules and the corroboration of existing drugs. Lastly, URI has a solid Sexual Medicine Unit which is mainly devoted to the translational study of male sexual and reproductive health, with a clear vision of the real-life setting and the transfer of preclinical know-how into daily management of issues relating to the desire for parenthood, sexual health as a proxy of general male health and overall quality of life. As a whole, these five Disease Units have achieved an impressive number of research goals, with collaborations across the world, and numerous publications in peer-reviewed indexed journals. This is made possible thanks to the structural organization of the units themselves. Indeed, under the guidance of the various heads of units, each group has provided research and educational opportunities for Medical students, residents in training and research and clinical fellows. DIVISION OF MOLECULAR ONCOLOGY Research Units, URI Urological pre-clinical research Unit The URI preclinical research unit encompasses translational research modalities in clinically relevant topics of functional urology and uro-oncology. During 2013, we have implemented several research projects addressing for instance the issue of erectile dysfunction after radical prostatectomy, innovative therapeutic approaches to treat lower urinary tract symptoms and surgery-induced neuropraxia, novel potential targets for the treatment of bladder cancer as well as studies on markers of prognostic and therapeutic relevance in prostate cancer. To facilitate the translational interaction between clinicians and basic scientists we are investing resources in the collection of biological materials obtained from informed patients, which will be preserved in a dedicated biobank, and implemented several Disease Units that will comprehensively address research projects on prostate, bladder, and renal cancer as well as on functional urology and sexual medicine. A particularly relevant project that succeeded in 2013 is the investigational study on a novel treatment in Peyronie’s disease (PD). PD is a connective tissue disorder of the tunica albuginea (TA). Currently, no gold standard has been developed for the treatment of the disease in its active phase. We have assessed the effects of a local injection of adipose tissue-derived stem cells (ADSCs) in the active phase of a rat model of PD on the subsequent development of fibrosis and elastosis of the TA and underlying erectile tissue. This study is the first to test stem cell therapy in an animal model of PD. Injection of ADSCs into the TA during the active phase of PD prevents the formation of fibrosis and elastosis in the TA and corpus cavernosum. Petter Hedlund Functional urology Urological dysfunctions and lower urinary tract symptoms still represent a major problem in the management of genitourinary organs pathologies. Goal of the Unit is to study the patho-physiology as well as to identify novel therapeutic opportunities for the overactive bladder (OAB), prostatic and ureter disorders and improving functional recovery after urological cancer surgery by means of experimental modelling. In 2013, we have demonstrated that the α1-adrenoceptor antagonist silodosin has excellent functional selectivity both in vivo to relieve pressure-load of the rat obstructed ureter and exvivo to inhibit contractions of human isolated ureters, suggesting that this medical expulsive therapy may also facilitate ureter stone passage in humans. Furthermore, we have confirmed the role of the endocannabinoid system in controlling the urinary functions; indeed chronic treatment with the cannabinoid degrading enzyme fatty acid amide hydrolase (FAAH) inhibitor oleoyl ethyl amide (OEtA) alters sensory urodynamic parameters and reduces experimental bladder overactivity. The presence of FAAH and cannabinoid receptors (CB) in the bladder and activation of CB-associated intracellular signals after intravesical OEtA in turn suggest a local role for FAAH in micturition control, even if OEtA might also act on central sensory pathways to contribute to these effects. Along this, we have assessed whether spinal inhibition of FAAH would have urodynamic effects both in normal rat and rats with experimental OAB induced by partial urethral obstruction or prostaglandin E2. After quantification of FAAH and CB1 and CB2 receptors levels in the sacral spinal cord, we have shown that FAAH blockade in the sacral spinal cord resulted in urodynamic effects in normal rats and diseased rats. Thus, the spinal endocannabinoid system may be involved in normal micturition control and it appears altered in OAB. Finally, we have started several corporate collaborations to investigate the role of TRPM8 receptor in modulating urinary functions upon the presence of LUTS, to evaluate the effects of s.repens extracts in the experimental BPH, and to assess the effects of the bladder GAG layer replenishment in experimental cystitis occurring after chemical and surgical mucosal damages. Fabio Benigni 29 DIVISION OF MOLECULAR ONCOLOGY Clinical Research Units, URI Prostate cancer Pre-clinical research: We have started a novel research project addressing the use of human decellularized tissue specimens to identify molecular markers associated with metastatic events in PCa patients. Unmasking which primary tumors harbor cells with an aggressive phenotype may improve prediction of metastasis. We have purified human prostate-derived ECM preserving the stromal-derived (glyco)-protein scaffold and observed that pathological ECMs are differently infiltrated by PCa cells in a way strictly dependent on the inherent cancer cell invasive potential. We are exploiting this methodology to unveil the metastatic phenotype in organ-confined, low grade tumors. Clinical research: We have investigated the impact of the stage migration phenomenon on the risk of lymph node invasion in patients over the last 20 years. Our analyses shows that patients with intermediate- and high-risk PCa still harbor significant risk of lymph node invasion at final pathology. Thus, extended lymph node dissection should not be omitted in these individuals. Additionally, we tested the accuracy of currently available risk stratification tools to predict the risk of lymph node invasion. We also studied the impact of adjuvant radiotherapy on cancer-specific mortality according to PCa features and developed a novel risk score based on pathological Gleason, pathological stage, and lymph node invasion to determine the ideal candidates for adjuvant radiotherapy following radical prostatectomy. This novel prognostic tool will help clinicians better counsel their patients, plan followup protocols, and select candidates for multimodal treatments. We also identified the predictors of cancer-specific survival in patients who experience biochemical recurrence after adjuvant radiotherapy. Our analyses showed that not all patients who recur after adjuvant radiotherapy die from PCa. However, patients with more aggressive disease are at higher risk of experiencing cancer-specific mortality and, thus, may benefit from additional secondary therapies. Finally, we focused on quality of life and functional outcomes after radical prostatectomy and evaluated the impact of robotic approaches on the probability of recovering urinary continence and erectile dysfunction after surgery. Alberto Briganti Bladder cancer Unit The Uro-Oncology Unit for urothelial neoplasms handles the diagnosis, treatment and follow-up of bladder cancer (BC) and upper urinary tract tumors. Performing nearly 350 endoscopic resections and more than 120 radical cystectomies per year, it is one of the highest volume surgical oncology units world-wide. • Diagnostics: We use exclusively flexible, highdefinition tools (video endoscopy and cystoscopy in HD) and routinely use the latest diagnostic methods including early biological diagnosis by F.I.S.H., and diagnostic techniques using PDD (Photodynamic Diagnosis) or NBI (Narrow Band Imaging). The diagnostic techniques for muscle-invasive BC include the latest imaging tools (MRI, CT/PET) that allow for reliable clinical staging. • Therapeutics: For non-muscle invasive BC Synergo® technology is now available for local chemo-hyperthermia (RITE) and ablative treatment with a Holmium laser for small neoplasms. For local muscle-invasive BC, many new techniques of post-cystectomy bladder reconstruction have recently been developed to spare the nerves responsible for sexual potency, particularly complex surgical procedures suitable primarily for young subjects with organ-confined disease. Our unit was among the first to perform robot-assisted laparoscopic radical cystectomies, gaining considerable experience also in the reconstructive phase. • Scientific Activities: No information is currently available on the molecular events resulting from cannabinoid receptor (CB) activation in urothelial cancer. We investigated the expression of CBs in primary human bladder carcinoma, quantified the anti-proliferative, ceramide-mediated activity of CB2 agonists in BC cell lines, and provided insight to the mechanisms of action of glycoshingolipids-mediated effects induced by CB2 agonists. The data support the use of CB2 agonists to treat BC and possibly limit disease progression. Our intense clinical research is evidenced by the numerous publications and communications at International Conferences. The Unit has also established close collaborations with prestigious European and American Urological centers, providing a fruitful exchange of expertise and enabling many young residents to acquire valuable experience in renowned centers of Urological excellence. Renzo Colombo DIVISION OF MOLECULAR ONCOLOGY Renal cancer Unit When to perform lymph node dissection in patients with renal cell carcinoma: a novel approach to the preoperative assessment of risk of lymph node invasion at surgery and of lymph node progression during follow-up The question of whether to perform lymph node dissection (LND) in RCC is still under debate. Indeed, although level one evidence showed no benefit in performing LND at the time of nephrectomy, several retrospective reports seem to suggest that those results should be applied to lowrisk cases only. A population-based analysis recently showed that there has been a persistent decline in the use of LND at radical nephrectomy over the last decade. In this context, it appears crucial to identify which patients may benefit from LND at the time of nephrectomy, but to date only two predictive models have been proposed to accurately identify patients at risk of lymph node invasion (LNI). Unfortunately, those reports were not able to address the major limitation of a nonquantifiable rate of false-negative LNI cases owing to their inclusion of patients treated with limited LND and the exclusion of patients who did not undergo LND. By relying on a unique approach, combining the risk of harbouring LNI and/or LN progression during the follow-up period, we provided the first clinical presurgery statistical model predicting the need for LND in RCC setting. The model showed excellent accuracy and calibration with a significant benefit in decision curve analyses. Those performance characteristics are remarkable given the fact that no pathological variables, which traditionally show a higher accuracy, were used. For all those reasons, our model can be considered the first attempt to identify before surgery, based exclusively on clinical variables, those cases in which the tumor shows a LN trophism during their natural history and that might benefit from a LND at the time of surgery. Roberto Bertini Sexual medicine Unit • Pre-clinical research: The exact role of the endocannabinoid-regulatory enzyme fatty acid amide hydrolase (FAAH), expressed in the human and rat ejaculatory system, is yet unknown. We investigated the effect of an acute treatment with a peripherally-acting FAAH inhibitor (URB937) by measuring the ejaculatory latencies and intraspongiosus suprasystolic pressures after apomorphine-induced ejaculation reflex in vivo. The data show that pharmacological inhibition of FAAH enzyme significantly and dose-dependently prolongs both the latency and inter-ejaculatory times after apomorphine, without changing the single reflex strength. Thus, FAAH and endocannabinoids may represent interesting drug targets for premature ejaculation. • Clinical research: In a comprehensive investigation of the link between men’s sexual dysfunction and general health status, we found that sexual health declines and concomitant morbidities increase with age. We also found that the severity of ED accounts for a higher association of health significant comordities as scored with the Charlson comorbidity index (CCI), a reliable proxy of lower general health status, regardless of the etiology of ED. • Seeking to evaluate prevalence and predictors of ED in young men, we found that one out of four suffering from ED is younger than 40, with almost half of these suffering from severe ED. As a proxy of general male health status, these results highlight the potential role of ED severity as a harbinger of medical disorders in men under 40. • In the field of reproductive medicine, we conducted a survey of opinions concerning preoperative sperm banking among Prostate Cancer (PCa) patients prior to surgery and found that this is relevant for one fifth of patients. Considering the incidence of PCa and prevailing attitudes toward delayed parenthood, providing information on the risk of infertility and possible fertility preservation interventions is critical at the time of diagnosis. • Finally, a deranged metabolism has been shown to actively affect male reproductive function. Thus, we assessed prevalence of Metabolic Syndrome (MetS), correlations of MetS with clinical characteristics, and its impact on semen and hormonal parameters in men presenting for primary couple’s infertility. Andrea Salonia 31 DIVISION OF MOLECULAR ONCOLOGY Selected publications Abdollah, F and Boorjian, S; Cozzarini, C; Suardi, N; Sun, M; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Montorsi, F; Karnes, RJ; Briganti, A. Survival following biochemical recurrence after radical prostatectomy and adjuvant radiotherapy in patients with prostate cancer: The impact of competing causes of mortality and patient stratification. Eur. Urol.: 2013; 64(4): 557-564 - Article IF 2012: 10,476 Abdollah, F; Suardi, N; Cozzarini, C; Gallina, A; Capitanio, U; Bianchi, M; Sun, M; Fossati, N; Passoni, NM; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Selecting the Optimal Candidate for Adjuvant Radiotherapy After Radical Prostatectomy for Prostate Cancer: A Long-term Survival Analysis. Eur. Urol.: 2013; 63(6): 998-1008 - Article IF 2012: 10,476 Abdollah, F; Suardi, N; Gallina, A; Bianchi, M; Tutolo, M; Passoni, N; Fossati, N; Sun, M; dell’Oglio, P; Salonia, A; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Extended pelvic lymph node dissection in prostate cancer: A 20-year audit in a single center. Ann. Oncol.: 2013; 24(6): 1459-1466 - Article IF 2012: 7,384 Apollonio, B; Scielzo, C; Bertilaccio, MT; Ten Hacken, E; Scarfò, L; Ranghetti, P; Stevenson, F; Packham, G; Ghia, P; Muzio, M and Caligaris-Cappio, F. Targeting B-cell anergy in chronic lymphocytic leukemia. Blood: 2013; 121(19): 3879-3888 - Article IF 2012: 9,060 Castagnaro, L; Lenti, E; Maruzzelli, S; Spinardi, L; Migliori, E; Farinello, D; Sitia, G; Harrelson, Z; Evans, SM; Guidotti, LG; Harvey, RP; Brendolan, A. Nkx2-5+islet1+ mesenchymal precursors generate distinct spleen stromal cell subsets and participate in restoring stromal network integrity. Immunity: 2013; 38(4): 782-791 - Article IF 2012: 19,795 Crippa, L; Bianco, M; Colombo, B; Gasparri, AM; Ferrero, E; Loh, YP; Curnis, F; Corti, A. A new chromogranin A-dependent angiogenic switch activated by thrombin. Blood: 2013; 121(2): 392-402 - Article IF 2012: 9,060 Fonte, E; Apollonio, B; Scarfò, L; Ranghetti, P; Fazi, C; Ghia, P; Caligaris-Cappio, F; Muzio, M. In vitro sensitivity of CLL cells to fludarabine may be modulated by the stimulation of toll-like receptors. Clin. Cancer Res.: 2013; 19(2): 367-379 - Article IF 2012: 7,837 Gianni, L; Romieu, GH; Lichinitser, M; Serrano, SV; Mansutti, M; Pivot, X; Mariani, P; Andre, F; Chan, A; Lipatov, O; Chan, S; Wardley, A; Greil, R; Moore, N; Prot, S; Pallaud, C; Semiglazov, V. AVEREL: a randomized phase III Trial evaluating bevacizumab in combination with docetaxel and trastuzumab as first-line therapy for HER2-positive locally recurrent/metastatic breast cancer. J. Clin. Oncol.: 2013; 31(14): 1719-1725 - Article IF 2012: 18,038 Hu, S; Xu-Monette, ZY; Balasubramanyam, A; Manyam, GC; Visco, C; Tzankov, A; Liu, WM; Miranda, RN; Zhang, L; Montes-Moreno, S; Dybkær, K; Chiu, A; Orazi, A; Zu, Y; Bhagat, G; Richards, KL; Hsi, ED; Choi, WW; Han van Krieken, J; Huang, Q; Huh, J; Ai, W; Ponzoni, M; Ferreri, AJ; Zhao, X; Winter, JN; Zhang, M; Li, L; Møller, MB; Piris, MA; Li, Y; Go, RS; Wu, L; Medeiros, LJ; Young, KH. CD30 expression defines a novel subgroup of diffuse large B-cell lymphoma with favorable prognosis and distinct gene expression signature: a report from the International DLBCL Rituximab-CHOP Consortium Program Study. Blood: 2013; 121(14): 2715-2724 Article IF 2012: 9,060 Raccosta, L and Fontana, R; Maggioni, D; Lanterna, C; Villablanca, EJ;Paniccia, A; Musumeci, A; Chiricozzi, E; Trincavelli, ML; Daniele, S; Martini, C; Gustafsson, J; Doglioni, C; Feo, SG; Leiva, A; Ciampa, MG; Mauri, L; Sensi, C; Prinetti, A; Eberini, I; Mora, M; Bordignon, C; Steffensen, KR; Sonnino, S; Sozzani, S; Traversari, C and Russo, V. The oxysterol-cxcr2 axis plays a key role in the recruitment of tumor-promoting neutrophils. J. Exp. Med.: 2013; 210(9): 1711-1728 - Article IF 2012: 13,214 DIVISION OF MOLECULAR ONCOLOGY Ranzani, M; Cesana, D and Bartholomae, CC; Sanvito, F; Pala, M; Benedicenti, F; Gallina, P; Sergi Sergi, L; Merella, S; Bulfone, A; Doglioni, C; Von Kalle, C; Kim, YJ; Schmidt, M; Tonon, G; Naldini, L; Montini, E. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat. Methods: 2013; 10(2): 155-161 - Article IF 2012: 23,565 Russo, V and Pilla, L; Lunghi, F; Crocchiolo, R; Greco, R; Ciceri, F; Maggioni, D; Fontana, R; Mukenge, S; Rivoltini, L; Rigamonti, G; Mercuri, SR; Nicoletti, R; Del Maschio, A; Gianolli, L; Fazio, F; Marchianò, A; Di Florio, A; Maio, M; Salomoni, M; Gallo-Stampino, C; Del Fiacco, M; Lambiase, A; Coulie, PG; Patuzzo, R; Parmiani, G; Traversari, C; Bordignon, C; Santinami, M and Bregni, M. Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3genetically modified lymphocytes. Int. J. Cancer: 2013; 132(11): 2557-2566 - Article IF 2012: 6,198 ten Hacken, E and Scielzo, C; Bertilaccio, MT; Scarfò, L; Apollonio, B; Barbaglio, F; Stamatopoulos, K; Ponzoni, M; Ghia, P; Caligaris-Cappio, F. Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia. Blood: 2013; 121(12): 2264-2273 - Article IF 2012: 9,060 Vardi, A; Dagklis, A; Scarfò, L; Jelinek, D; Newton, D; Bennett, F; Almeida, J; Rodriguez-Caballero, A; Allgood, S; Lanasa, M; Cortelezzi, A; Orlandi, E; Veronese, S; Montillo, M; Rawstron, A; Shanafelt, T; Orfao, A; Stamatopoulos, K; Ghia, P. Immunogenetics shows that not all MBL are equal: the larger the clone, the more similar to CLL. Blood: 2013; 121(22): 4521-4528 - Article IF 2012: 9,060 Von Hoff, DD; Ervin, T; Arena, FP; Chiorean, EG; Infante, J; Moore, M; Seay, T; Tjulandin, SA; Ma, WW; Saleh, MN; Harris, M; Reni, M; Dowden, S; Laheru, D; Bahary, N; Ramanathan, RK; Tabernero, J; Hidalgo, M; Goldstein, D; Van Cutsem, E; Wei, X; Iglesias, J; Renschler, MF. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. New Engl. J. Med.: 2013; 369(18): 1691-1703 - Article IF 2012: 51,658 Selected publications, URI Abdollah, F; Suardi, N; Cozzarini, C; Gallina, A; Capitanio, U; Bianchi, M; Sun, M; Fossati, N; Passoni, NM; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Selecting the Optimal Candidate for Adjuvant Radiotherapy After Radical Prostatectomy for Prostate Cancer: A Long-term Survival Analysis. Eur. Urol.: 2013; 63(6): 998-1008 - Article IF 2012: 10,476 Capitanio, U; Abdollah, F; Matloob, R; Suardi, N; Castiglione, F; Di Trapani, E; Capogrosso, P; Gallina, A; Dell’Oglio, P; Briganti, A; Salonia, A; Montorsi, F; Bertini, R. When to perform lymph node dissection in patients with renal cell carcinoma: a novel approach to the preoperative assessment of risk of lymph node invasion at surgery and of lymph node progression during followup. BJU Int: 2013; 112(2): E59-E66 - Article IF 2012: 3,046 Castiglione, F; Hedlund, P; Van der Aa, F; Bivalacqua, TJ; Rigatti, P; Van Poppel, H; Montorsi, F; De Ridder, D; Albersen, M. Intratunical Injection of Human Adipose Tissue-derived Stem Cells Prevents Fibrosis and Is Associated with Improved Erectile Function in a Rat Model of Peyronie’s Disease. Eur. Urol.: 2013; 63(3): 551-660 - Article IF 2012: 10,476 33 DIVISION OF MOLECULAR ONCOLOGY Selected publications, URI Salonia, A; Capogrosso, P; Castiglione, F; Russo, A; Gallina, A; Ferrari, M; Clementi, MC; Castagna, G; Briganti, A; Cantiello, F; Damiano, R; Montorsi, F. Sperm banking is of key importance in patients with prostate cancer. Fertil. Steril.: 2013; 100(2): 367-372.e1 - Article IF 2012: 4,174 Villa, L; Buono, R; Fossati, N; Rigatti, P; Montorsi, F; Benigni, F; Hedlund, P. Effects by silodosin on the partially obstructed rat ureter in vivo and on human and rat isolated ureters. Br. J. Pharmacol.: 2013; 169(1): 230-238 - Article IF 2012: 5,067 DIVISION OF MOLECULAR ONCOLOGY Tumor biology and vascular targeting Unit Functional genomics of cancer Unit 35 DIVISION OF NEUROSCIENCE Director: Gianvito Martino Associate Director: Flavia Valtorta* Research Units Neuropsychopharmacology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 45 HEAD OF UNIT: Flavia Valtorta* RESEARCHER: Giovanni Piccoli* POST-DOCTORAL FELLOWS: Serena Bellani**, Maria Maddalena Valente** PHD STUDENTS: Maria Daniela Cirnaru, Fabrizia Guarnieri **, Francesca Pischedda FELLOW: Marco Tramarin TECHNICIAN: Elena Monzani Rett syndrome research group –––––––––––––––––––––––––––––––––––––––––––––––––––––– 46 GROUP LEADER: Nicoletta Landsberger POST-DOCTORAL FELLOWS: Francesco Bedogni, Maria Maddalena Valente PHD STUDENTS: Clementina Cobolli Gigli, Anna Gandaglia FELLOW: Elisa Bellini Cell adhesion Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 46 HEAD OF UNIT: Ivan de Curtis* POST-DOCTORAL FELLOWS: Elena Chiricozzi**, Romina Macco**, Roberta Pennucci** PHD STUDENTS: Veronica Astro**, Sara Chiaretti, Kristyna Hanusova** FELLOW: Anna Falco TECHNICIAN: Diletta Tonoli Cellular neurophysiology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 47 HEAD OF UNIT: Fabio Grohovaz* RESEARCHERS: Franca Codazzi*, Daniele Zacchetti POST-DOCTORAL FELLOWS: Barbara Bettegazzi, Ilaria Pelizzoni FELLOWS: Irene Bertolini, Elisabeth Mangiameli 37 DIVISION OF NEUROSCIENCE Research Units / Clinical Research Units Developmental Neurogenetics Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––– 48 HEAD OF UNIT: Gian Giacomo Consalez POST-DOCTORAL FELLOW: Grazia Iaffaldano FELLOWS: Renato Arnese, Camilla Bosone TECHNICIANS: Aurora Badaloni, Laura Croci Neurobiology of learning Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 49 HEAD OF UNIT: Antonio Malgaroli* RESEARCHERS: Carley R. Benton*, Adrian Scultoreanu*, Vincenzo Zimarino POST-DOCTORAL FELLOWS: Mattia Ferro **, Jacopo Lamanna **, Maddalena Ripamonti** PHD STUDENTS: Alessandro Arena, Sara Spadini ** Proteomics of iron metabolism Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 49 HEAD OF UNIT: Sonia Levi* RESEARCHERS: Anna Cozzi, Paolo Santambrogio FELLOWS: Francesca Cignarella, Michela Guaraldo**, Ida Luisa Rotundo Stem cells and neurogenesis Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 50 HEAD OF UNIT: Vania Broccoli PHD STUDENTS: Luca Massimino**, Federica Ungaro** FELLOWS: Maxim Bespalov, Massimiliano Caiazzo, Ernesto Ciabatti, Gaia Colasante, Maria Teresa Dell’Anno, Serena Giannelli, Matteo Perino, Ida Luisa Rotundo, Alessandro Sessa Molecular genetics of mental retardation Unit –––––––––––––––––––––––––––––––––––––––– 51 (Dulbecco Telethon Institute) GROUP LEADER: Patrizia D’Adamo* POST-DOCTORAL FELLOWS: Maila Giannandrea, Lorenzo Morè PHD STUDENTS: Veronica Bianchi**, Maria Lidia Mignogna** FELLOW: Michela Masetti Clinical Research Units Acute brain protection, Acute postoperative pain, ––––––––––––––––––––––––––––––––––– 52 Drugs and central nervous system Unit HEAD OF UNIT: Luigi Beretta* PHYSICIANS: Massimo Agostoni, Maria Rosa Calvi, Assunta De Vitis, Marco Gemma, Francesco Ruggieri FELLOW: Massimiliano Greco** Cognitive neuroscience Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 52 HEAD OF UNIT: Stefano F. Cappa* RESEARCHERS: Jubin Abutalebi**, Nicola Canessa** PHYSICIANS: Maria Cristina Giusti, Sandro Iannaccone, Alessandra Marcone, Michele Zamboni PHD STUDENTS: Chiara Crespi**, Alessandra Dodich** DIVISION OF NEUROSCIENCE Clinical Research Units Experimental neurosurgery Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 52 HEAD OF UNIT: Pietro Mortini* PHYSICIANS: Stefania Acerno, Lina Raffaella Barzaghi, Nicola Boari, Alberto Franzin, Marco Losa, Carlo Mandelli PHD STUDENTS: Filippo Gagliardi**, Silvia Snider ** RESIDENTS: Michele Bailo, Jody Capitanio, Andrea Cavalli, Elena Colombo, Lucio Aniello Mazzeo, Pietro Panni, Giorgio Spatola, Alfio Spina Eye repair Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 53 HEAD OF UNIT: Paolo Rama POST-DOCTORAL FELLOW: Fabio Bignami PHD STUDENT: Louise Louro FELLOW: Chiara Giacomini CONSULTANTS : Oriella Ambrosio, Giulio Ferrari, Federica Ferrario, Chiara Insacco, Stanislav Matuska, Giorgio Paganoni, Elena Scandola, Maurizia Viganò TECHNICIAN: Anna Lorusso Functional neuroradiology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 54 HEAD OF UNIT: Andrea Falini* PHYSICIANS: Nicoletta Anzalone, Cristina Baldoli, Valeria Blasi, Silvia Pontesilli, Roberta Scotti, Paolo Vezzulli PHD STUDENT: Antonella Castellano** RESIDENT: Claudia Godi** TECHNICIAN: Antonella Iadanza In vivo Human molecular and structural neuroimaging Unit ––––––––––––––––––––––––– 55 HEAD OF UNIT: Daniela Perani* RESEARCHER: Marco Tettamanti POST-DOCTORAL FELLOWS: Chiara Cerami**, Angela Coliva**, Pasquale Della Rosa** PHD STUDENT: Leonardo Iaccarino FELLOWS: Silvia Caminiti**, Danilo Spada Neuroothology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 56 HEAD OF UNIT: Mario Bussi* PHYSICIANS: Lucia Oriella Piccioni, Roberto Teggi CONSULTANTS: Fabrizio Ferrario, Omar Gatti Psychiatry and clinical neurosciences Unit ––––––––––––––––––––––––––––––––––––––––––––– 56 HEAD OF UNIT: Francesco Benedetti Psychiatry and clinical psychobiology ––––––––––––––––––––––––––––––––––––––––––––– 56 HEAD OF UNIT: Francesco Benedetti PHYSICIANS: Sara Angelone, Cinzia Arancio, Barbara Barbini, Laura Bellodi*, Alessandro Bernasconi, Laura Bianchi, Marta Bosia, Stefania Cammino, Marco Catalano, Roberto Cavallaro, Maria Cristina Cavallini, Federica Cocchi, Silvia Cocchi, Cristina Colombo*, Sara Dallaspezia, Danilo Dotoli, Stefano Erzegovesi, Linda Franchini, Carmelo Guglielmino, Marta Henin, Marco Locatelli, Adelio Lucca, Ernestina Politi, Laura Sforzini, Francesca Siliprandi, Enrico Smeraldi*, Raffaella Zanardi POST-DOCTORAL FELLOWS: Elisa Galimberti**, Sara Poletti**, Daniele Radaelli** PHD STUDENTS: Irene Bollettini, Paola Canali, Emma Fadda, Riccardo Martoni, Roberta Riccaboni, Cecilia Smeraldi, Benedetta Vai 39 DIVISION OF NEUROSCIENCE Clinical Research Units FELLOWS: Margherita Bechi, Vittoria Bottelli, Chiara Brambilla, Maria Chiara Buonocore, Ursula Catenazzi, Daniele Cavadini, Mara Ciracì, Francesco Fresi, Chiara Gavinelli, Stefania Ozino, Giulia Paredi, Marco Piantanida, Liana Ricci, Andrea Zanoletti RESIDENTS: Giampiero Bottero, Silvia Brioschi, Dario Delmonte, Charlotte De Santis, Valentina Ferrari, Clara Locatelli, Antonella Rita Mastromatteo, Angelo Notaristefano, Marco Spangaro, Irene Vanelli LAB MANAGERS: Cristina Lorenzi, Adele Pirovano** Sleep medicine ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 57 CLINICAL GROUP LEADER: Luigi Ferini-Strambi* RESEARCHERS: Vincenza Elena Castronovo, Enrico Giora *, Sara Marelli, Alessandro Oldani, Marco Zucconi PHD STUDENT: Andrea Galbiati ** TECHNICIANS: Massimo Antonio, Daniele Bizzozero, Elisa Dallabà, Cristina Martinelli Clinical psychology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 58 CLINICAL GROUP LEADER: Cesare Maffei* RESEARCHERS: Andrea Fossati*, Mariagrazia Movalli, Laura Vanzulli, Raffaele Visintini EXTERNAL RESIDENTS: Serena Artesani, Antonella Di Biase, Michela Adele Pozzi, Emanuela Roder, Manuela Sanvito, Edoardo Vassallo Motor function rehabilitation ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 59 CLINICAL GROUP LEADER: Roberto Gatti PHYSICAL THERAPIST: Andrea Tettamanti DIVISION OF NEUROSCIENCE INSPE Institute of Experimental Neurology Director: Giancarlo Comi* Research Units Experimental neurology Unit HEAD OF UNIT: Giancarlo Comi* Experimental neuropathology ––––––––––––––––––––––––––––––––––––––––––––––––––––––– 61 GROUP LEADER: Angelo Quattrini POST-DOCTORAL FELLOWS: Federica Cerri, Nilo Riva PHD STUDENT: Ignazio Diego Lopez ** FELLOW: Teuta Domi TECHNICIAN: Giorgia Dina Experimental neurophysiology –––––––––––––––––––––––––––––––––––––––––––––––––––––– 61 GROUP LEADER: Letizia Leocani PHD STUDENTS: Ninfa Amato**, Valerio Castoldi**, Raffaella Chieffo **, Francesca Spagnolo ** FELLOWS: Loris Camaleonti**, Elise Houdayer, Laura Straffi RESIDENTS: Giovanni Di Maggio**, Mario Fichera**, Arturo Nuara**, Roberto Santangelo** Molecular genetics of behaviour ––––––––––––––––––––––––––––––––––––––––––––––––––– 62 GROUP LEADER: Riccardo Brambilla PHD STUDENTS: Raffaele d’Isa**, Nicola Solari** FELLOWS: Stefania Fasano, Ilaria Morella, Alessandro Papale TECHNICIAN: Marzia Indrigo Neuromuscular repair ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 62 GROUP LEADER: Stefano Carlo Previtali PHD STUDENTS: Emanuela Porrello**, Cristina Rivellini**, Daniela Triolo** RESIDENT: Daniele Velardo** TECHNICIANS: Isabella Lorenzetti, Rossana Tonlorenzi 41 DIVISION OF NEUROSCIENCE Research Units, INSPE Neuroimmunology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 63 HEAD OF UNIT: Gianvito Martino RESEARCHERS: Linda Chaabane, Luca Muzio POST-DOCTORAL FELLOWS: Marco Bacigaluppi**, Erica Butti, Emanuela Colombo, Melania Cusimano PHD STUDENTS: Benedetta Arnò**, Roberta De Ceglia, Donatella De Feo**, Francesca Grassivaro**, Cecilia Laterza**, Amir Malvandi**, Ramesh Menon, Chiara Rossi** RESIDENTS: Marco Di Dario, Arianna Merlini TECHNICIANS: Andrea Bergamaschi, Elena Brambilla, Eleonora Capitolo, Francesca Ruffini Clinical neuroimmunology ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 64 GROUP LEADER: Roberto Furlan PHD STUDENTS: Federico Colombo**, Livia Garzetti RESIDENT: Dacia Dalla Libera** FELLOW: Giacomo Casella TECHNICIAN: Annamaria Finardi Immunobiology of neurological disorders –––––––––––––––––––––––––––––––––––––––– 65 GROUP LEADER: Cinthia Farina POST-DOCTORAL FELLOWS: Emanuela Colombo, Ramesh Menon FELLOW: Marco Di Dario Neuroimaging research Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 66 HEAD OF UNIT: Massimo Filippi* BIOENGINEERS: Elisabetta Pagani, Paola Valsasina POST-DOCTORAL FELLOWS: Federica Agosta, Elisa Canu PHD STUDENTS: Martina Absinta**, Francesca Caso**, Laura Parisi**, Evelina Prudente RESIDENTS: Giulia Longoni**, Roberta Messina**, Paolo Preziosa**, Lidia Sarro**, Edoardo Gioele Spinelli** FELLOWS: Pilar Maria Ferraro, Sebastiano Galantucci, Maria Elisa Morelli TECHNICIANS: Luca Dall’Occhio, Alessandro Meani, Melissa Petrolini, Stefania Sala, Roberto Vuotto Neuroimaging of CNS white matter –––––––––––––––––––––––––––––––––––––––––––––––– 66 GROUP LEADER: Maria Assunta Rocca CONSULTANT: Gianna Riccitelli PHD STUDENT: Sara Cirillo** TECHNICIANS: Paolo Misci, Mauro Sibilia Human inherited neuropathies Unit (Dulbecco Telethon Institute) ––––––––––––––––– 67 HEAD OF UNIT: Alessandra Bolino POST-DOCTORAL FELLOWS: Marta Guerrero Valero, Françoise Piguet**, Ilaria Vaccari PHD STUDENT: Roberta Noseda** FELLOW: Antonietta Carbone TECHNICIANS: Valeria Alberizzi, Fiammetta Viganò Axo-glia interactions Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 68 GROUP LEADER: Carla Taveggia, FISM POST-DOCTORAL FELLOWS: Marta Pellegatta, Amelia Trimarco PHD STUDENTS: Maria Grazia Forese**, Evelien Fredrickx** TECHNICIANS: Rosa La Marca, Alessandra Lucente DIVISION OF NEUROSCIENCE Clinical Research Units, INSPE Inflammatory CNS disorders Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 69 HEAD OF UNIT: Vittorio Martinelli RESEARCHERS: Filippo Martinelli-Boneschi, Federica Esposito, Lucia Moiola, Mariaemma Rodegher POST-DOCTORAL FELLOWS: Ferdinando Clarelli, Elisabetta Mascia, Ana Maria Osiceanu, Marta Radaelli, Melissa Sorosina, Andrea Zauli PHD STUDENTS: Simona Maida**, Silvia Santoro** RESIDENTS: Valeria Barcella**, Gloria Dalla Costa**, Laura Ferrè** , Giacomo Giacalone**, Clara Guaschino**, Giuseppe Liberatore**, Maria Josè Messina**, Marzia Romeo**, Francesca Sangalli** Cerebrovascular disorders ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 69 CLINICAL GROUP LEADER: Maria Sessa PHYSICIANS: Silvia Mammi, Antonella Poggi, Luisa Roveri PHD STUDENTS: Marco Bacigaluppi**, Raffaella Chieffo**, Grazia Maria Nuzzaco** (until June 2013) RESIDENTS: Giovanni Di Maggio, Giacomo Giacalone, Sara La Gioia, Luca Peruzzotti Jametti, Davide Strambo Memory disorders –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 70 CLINICAL GROUP LEADER: Giuseppe Magnani PSYCHOLOGISTS: Claudia Arcari, Alessandra Barbieri, Maria Paola Bernasconi, Rosalinda Cardamone, Chiara Dallatomasina, Monica Falautano, Agnese Fiorino, Francesca Possa, Elisa Riboni RESIDENTS: Elisabetta Coppi, Laura Ferrari, Roberto Santangelo Movements disorders ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 70 CLINICAL GROUP LEADER: Ubaldo Del Carro PHYSICIANS: Stefano Amadio, Calogera Butera, Roberta Guerriero, Maria Antonietta Volonté RESIDENT: Francesca Bianchi Neuromuscular disorders –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 71 CLINICAL GROUP LEADER: Raffaella Fazio PHYSICIAN: Marina Scarlato POST-DOCTORAL FELLOW: Nilo Riva RESIDENTS: Francesca Bianchi , Daniele Velardo** Paroxysmal events ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 71 CLINICAL GROUP LEADER: Fabio Minicucci PHYSICIANS: Bruno Colombo, Giovanna Franca Fanelli, Paolo Marchettini RESIDENT: Giulia Pavan ENGINEER: Marco Cursi TECHNICIAN: Claudia Paleari * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele 43 DIVISION OF NEUROSCIENCE Introduction by the Directors Mission and vision - The mission of the Division of Neuroscience is to understand the inner workings of the human brain and to find the causes of, and treatments for, neurological and psychiatric diseases, i.e. some of humanity’s most disabling disorders. To accomplish this aim, we engage in studies on the molecular and cellular mechanisms – either in vitro or in vivo – of neural cells (both neuronal and glial cells) dysfunction, as well as in translational and clinical research projects aimed at understanding the pathogenic mechanisms of the nervous system disorders. Our aim is to discover and/or validate novel therapeutic approaches for neurological and psychiatric disorders. Since the beginning, the fundamental approach of the Division has been the integration of basic research with clinical applications. Organization - The Division of Neuroscience is made up of more than 300 scientists primary or secondary affiliated to 21 basic research units and 17 clinical research groups. Given the diverse backgrounds of the scientists affiliated to the Division, considerable effort has been dedicated to the creation of a neuroscientific community, by implementing divisional seminars and journal clubs. Goals - Fostering translational research by implementing both basic and clinical research is the main focus of the research activity of the Division of Neuroscience. This aim has been pursued at both the scientific and organizational levels and the followings are the achievements accomplished. Achievements - Organizational achievements: To foster cross-fertilization between clinical and wet labs we have continued to organize weekly meetings – named ‘Neuroclub’ – for senior and junior scientists as well as under- and post-graduate students referring to the Division. In 2013 the Neuroclub activity has been intense, going from progress reports to focus on diseases, PhD student’s reports, and meetings dedicated to research programs. Wet labs have been set up allowing physician scientists from clinical wards (Neurology, Psychiatry, Neurosurgery, Ophthalmology, Neurophysiology) affiliated to the Division to have laboratory space for preclinical research and collaborations with basic research scientists. Scientific achievements: As outlined in the reports of the single Units, important achievements have been obtained in areas of basic research as diverse as myelin biology, developmental and regenerative neurobiology, biomaterials for nerve regeneration, stem cells and gene delivery, in vitro and in vivo molecular mechanisms of neurodegeneration. Scientific achievements obtained in clinical research areas have been noteworthy as those of the basic research; the most productive areas have been human brain imaging, genetics of psychiatric disorders, inflammatory and degenerative neurological diseases, clinical neuropsychology, eye disorders and cognitive neuroscience. Funding: In addition to numerous grants and industrial contracts, previously set strategic alliances have been continuing (Telethon’s program project; INSpe-MerckSerono Strategic Alliance; POMS-bioengineering) and phase I/II/III clinical trials (both spontaneous and sponsored), mainly focusing on neurological and psychiatric disorders, have been initiated and/or continued. Training Opportunities - The Division is also actively involved in the training of graduate students, organizing Ph.D. Courses in Neuroscience, Experimental Neurology, Neurobiotechnology, Developmental Psychopathology, and taking part in the Ph.D. Course in Cellular and Molecular Biology. In addition, numerous undergraduate students, enrolled in the Schools of Medicine, Biotechnology, Psychology or Physiotherapy, have worked in the Research Units of the Division for the preparation of their thesis. No formal training for post-doctoral fellows have been organized, but post-docs have been encouraged to participate in the numerous institutional seminars and to national or international meetings. DIVISION OF NEUROSCIENCE Research Units Neuropsychopharmacology Unit Excitation/inhibition imbalance in epilepsy and autism spectrum disorders The correct equilibrium between excitatory and inhibitory neuronal inputs is necessary for the normal functioning of the entire nervous system, and a variety of neurological and psychiatric disorders are currently thought to involve disruption of this delicate equilibrium. Among these pathologies are epilepsy, autism spectrum disorders, intellectual disability. A growing number of mutations in genes coding for proteins involved in neurotransmission have been found in familiar cases of these pathologies. Although the number of patients suffering from these rare conditions is limited, monogenic disorders are invaluable models for clarifying the molecular mechanisms that produce these diseases, allowing also the development of animal models that help in elucidating the pathogenetic role of mutations affecting neuron-specific genes. We have studied the synapsins, neuronal phosphoproteins involved in neurotransmitter release and neuronal development. We and others have recently identified an array of mutations in human synapsin genes associated with epilepsy, autism spectrum disorder and/or mental retardation. We expressed synapsin mutations identified in human in mouse hippocampal synapsin knock-out (KO) neurons. The Q555X mutation induces higher network excitability and firing activity, recapitulating the KO phenotype. The presence of a premature termination codon in W356X synapsin renders the transcript susceptible to nonsense-mediated mRNA decay. These data support the value of synapsin KO mice as an experimental model mimicking the human pathology. Neuronal networks formed in vitro from synapsin KO mice show diffuse hyperexcitability, associated with decreased strength of inhibitory transmission and increased strength of excitatory transmission, suggesting that excitation/inhibition imbalance might be at the basis of the synapsin KO phenotype and possibly of the corresponding human pathology. Electroencephalographic recordings of synapsin triple knocked-out (TKO) mice show spontaneous and evoked epileptic discharges as well as changes in background cortical activity. In addition, deletion of synapsins impairs social behavior, resulting in autism-related phenotypes which become manifest before the onset of seizures. Flavia Valtorta Figure 3. Hippocampal neurons grown in culture, expressing GFP and stained for the presynaptic marker Bassoon and the vesicular glutamate transporter VGLUT1. Color-inverted image. 45 DIVISION OF NEUROSCIENCE Research Units Rett syndrome research group Rett syndrome (RTT) is a devastating genetic disease mainly caused by mutations in the epigenetic transcriptional regulator MeCP2, represents one of the most common causes of severe mental retardation in girls worldwide. In 2007 the reversibility of the RTT condition in mice was proved, however, there is still no effective therapy, demonstrating the urge of a thorough comprehension of MeCP2 neuronal role. MeCP2 binds methylated DNA thereby affecting chromatin structure and transcription; in mature neurons, due to its high abundance, MeCP2 acts as a wide transcriptional modulator controlling the expression of virtually all genes. Possibly due to this reason and to technical limitations, so far only a few direct targets of MeCP2 action have been discovered. Further, although several clinical evidences suggest a role played by MeCP2 in early development, almost no studies have so far investigated its functions during brain maturation. We have produced an exhaustive analysis of the transcriptional defects displayed by the murine embryonic Mecp2-null neocortex. These studies have permitted to demonstrate the existence of an embryonic maturation delay that persists in the perinatal age and that we hypothesize might be responsible, at least some extent, of the neuronal defects that typically arise during postnatal life in RTT patients. Further, we have demonstrated that, through bioanalytic approaches never used in RTT before, high throughput screenings can produce precious insights over molecular pathways affected by MeCP2 (Bedogni, 2014; Bedogni, submitted). In a more translational approach, in collaboration with Dr. James Eubanks (University of Toronto, Canada) we have started a project of gene therapy aimed at producing a novel lentivirus capable of reproducing physiological levels of MeCP2. We prepared (and are still preparing) several constructs to produce viruses that will be soon injected in a specific Mecp2-null mouse brain area selected for its potential rescue benefits. The expression of MeCP2, the tropism of the virus and the potential benefits will be then tested. Promising vectors will be also tested in an AAV background. Eventually, we are ending a project describing the role of MeCP2 in skeletal muscle development and homeostasis (to be submitted). Nicoletta Landsberger Cell adhesion Unit Molecular networks regulating migration, invasion and neuronal development Rac GTPases interact with networks of regulators and effectors to regulate cell motility. Analysis of single and double knockout (KO) mice for Rac1 (Rac1N, conditional KO of Rac1) and Rac3 (Rac3KO) has revealed a role of these proteins in the regulation of the migration and differentiation of GABAergic interneurons. Rac1N or Rac3KO adult animals show differential defects in the maturation of cortical and hippocampal parvalbuminpositive (PV+) GABAergic cells. A robust phenotype is observed in Rac1N mice that develop epilepsy and stronger cognitive defects compared to Rac3KO. The inhibitory input by PV+ basket cells on hippocampal CA1 pyramidal cells is decreased in both single KOs, although only Rac1N mice show a parallel strong decrease of the GABA-producing enzyme GAD67. Moreover, Rac1N, but not Rac3KO mice, show more perisomatic terminals by other types of interneurons on the CA1 pyramidal cells. The morphological alterations observed in the Rac1N mice correlate with their altered hippocampal circuitry, and may explain their specific behavioral and cognitive deficits. We have set up primary cultures of interneuron precursors to identify protein regulators and effectors that mediate the effects of Rac on the maturation of interneurons. Candidate molecules analyzed include genes mutated in developmental disorders leading to impaired cognition. Our analysis of the molecular networks functionally linked to Rac GTPases, has led to the identification of liprin-α1, a scaffold protein regulating the protrusive activity of migrating cells. Liprin-α1 is highly expressed in some human tumors, and our studies in vitro have shown that this protein positively regulates tumor cell migration and invasion, by affecting the turnover of focal adhesions and invadopodia. By using distinct assays for invasion in vivo we are also addressing the requirement of liprin-α1 for the formation of metastases in mice, and the mechanisms used by liprin to regulate protrusion. In this direction we have identified a complex of adaptor proteins that is needed for liprin-promoted migration and invasion. Ivan De Curtis DIVISION OF NEUROSCIENCE Figure 4. Primary culture of developing interneurons isolated from mouse embryonic MGE. Staining for GABA (green) and for nuclei (blue, DAPI) to show the increased expression of the GABAergic phenotype in vitro (DIV, days in vitro). Cellular neurophysiology Unit Physiopathological mechanisms in neuroprotection, neuroinflammation and neurodegeneration The work of our unit is aimed to understand the molecular and cellular mechanisms underlying neuroprotection, neuroinflammation and neurodegeneration. In 2013 we focused our research on the role of astrocytes by investigating two main aspects. We studied primary astrocytes activated by pro-inflammatory stimuli showing that they have the potential to buffer an excess of extracellular iron, thereby protecting neurons from iron overload in pathological conditions (Pelizzoni et al., 2013). We also showed that activated astrocytes acquire a phenotype characterized by a specific gene expression pattern and a higher resistance to stressing stimuli with particular regards to oxidative stress and iron overload (Macco et al., 2013). Therefore, we continued the gene expression profiling of primary glial cultures activated by various pathological stimuli, in order to identify putative biomarkers, related to specific pathological cellular processes and aimed to become a diagnostic and prognostic tool for neuroinflammatory and neurodegenerative diseases. This activity will be pursued within the framework of the Ivascomar project of the “Cluster Tecnologico Nazionale Scienze della Vita” (Ministry of Re- search). Regarding our investigations on neurons, iPSCsderived neurons derived from patients affected by Friedreich’s ataxia were employed to characterize intracellular iron homeostasis and susceptibility to different oxidative stress conditions, in order to gain new insights in this neurodegenerative disorder. The control of intracellular iron homeostasis was also studied in an animal model for hereditary human neuroferritinopathy (the pathogenic L-ferritin variant 498-499InsTC). A comprehensive study of this transgenic animal model, performed at the biochemical, morphological and physiological level, in collaboration with S. Levi and O. Cremona, demonstrated a higher neuronal susceptibility to oxidative stress. Finally, we continued the characterization of the phosphorylation of the translation initiation factor eIF4B upon neuronal activation and evaluated its possible role in the translational control of BACE1, the neuronal βsecretase responsible for the production of the neurotoxic amyloid-β peptides that accumulate in the brain of Alzheimer’s disease patients. Fabio Grohovaz 47 DIVISION OF NEUROSCIENCE Research Units Developmental neurogenetics Unit A “translational” approach to the study of amyotrophic lateral sclerosis and other neurodegenerative disorders In 2013, our lab has started a new project in the field of axonal biology. We study axonal protein synthesis and its regulation, in wild type mice and mouse models of motor neuron diseases (MNDs). While the genetic basis of familial and sporadic amyotrophic lateral sclerosis (ALS) is being unraveled at a fast pace, our understanding of its molecular pathogenesis remains largely incomplete. One distinctive feature of ALS is the fact that it primarily affects long range fiber tracts, i.e. the cortico-spinal tract and the peripheral nerve. In these pathways, the axon accounts for >99% of the corresponding neuron’s total volume, a feature that is somewhat overlooked by many neurobiologists. Profiling studies have revealed that both growing and mature axons possess complex and dynamic transcriptomes. Axonal mRNA translation is tightly regulated and plays a role in neuronal homeostasis affecting important biological processes, including energy production, cytoskeletal remodeling and retrograde transport. Recent evidence indicates that axonal regeneration in the peripheral nervous system is impaired in the absence of local mRNA translation. Accordingly, many patients affected by familial and sporadic forms of ALS carry mutations of genes involved in RNA metabolism. Taken together, this evidence points to axonal protein synthesis as a potentially crucial factor in the pathogenesis of MNDs. In this project, we have generated molecular tools and in vivo/in vitro models to study compartmentalized protein synthesis and its regulation in the axon. Thanks to fluorescent ribosomal proteins (RPs) expressed in genetically defined neurons and glial cells, we will analyze cell-autonomous and cell-nonautonomous contributions to the pathogenesis of MNDs. This will be achieved through immunoprecipitation of tagged polysomes in gray and white matter lysates, and by RNAseq analysis of RNAs loaded onto polysomes (the neuronal and axonal “translatome”). Gian Giacomo Consalez Figure 5. Confocal images of hippocampal neurons transduced with a lentivirus encoding a green fluorescent ribosomal protein (green); red, ankyrin G, a marker of the axon initial segment; magenta, 3-tubulin (TuJ1), a marker of the neuronal cytoskeleton; blue, DNA (DAPI staining). The fusion protein localizes to dendrites and the axon (inset) in a discrete pattern compatible with incorporation into ribosomes and polyribosomes. DIVISION OF NEUROSCIENCE Neurobiology of learning Unit LTP modulates fractal behaviour of quantal releases at hippocampal synapses the generation of voltage and Ca2+ oscillations known to be important for GCPs differentiation. Spontaneous quantal release series were analysed before and after the induction of LTP. We found that the long-term enhancement of quantal release seen with LTP is accompanied by the occurrence and/or modulation of a 1/f behaviour, with a significant increase in the fractal exponent. Similarly we found that α-latrotoxin, a powerful promoter of exocytosis, modulates the 1/f behaviour. Our results suggest that some pre-synaptic mechanism strictly related to the dynamics of vesicular pools is actively involved in the generation of 1/f behaviour and LTP expression. Analysis of the effects of general anesthetics on the activity of rat visual cortex Even if anaesthetics are widely used in the medical practice, there is still an unresolved issue which relates to their mechanism of action. Rats were anesthetised with either sevofluorane or propofol and visual evoked potentials, both ON and OFF responses and EEG activity recorded by superficial electrodes. Results show that while basal cortical activity is reduced with both drugs, the effects on VEPs exhibit clear differences, suggesting two very distinct circuital mechanisms of action. HCN Channels and cerebellar Maturation We found that the hyperpolarization-activated cyclic nucleotide-gated channels HCN 1, 3, 4 are expressed on Bergmann glia and GCs starting from early postnatal days. We investigated the role of these channels by applying specific blockers (Procolaran and ZD7288) both in vivo and in vitro. We found that blockade of HCN channels unbalances this process favouring GCs proliferation. This suggests that the activation of HCN channels by c-AMP and membrane potential participate in Novel indicators for synaptic activity in vivo The establishment of a causal relationships between the activity of subgroups of synapses is essential for understanding the basis of animal behaviour. Our laboratory is currently developing a series of innovative genetically encoded indicators to measure synaptic trasmission both in vitro and in vivo. We are using these tools to define the pattern of synaptic activation in the visual cortex by light. Antonio Malgaroli Proteomics of iron metabolism Unit L-ferritin deficiency is associated to idiopathic generalized seizures and atypical restless leg syndrome Our main research interest is to understand the relationship between brain iron dysregulation and the process of neurodegeneration. Alterations of brain iron homeostasis is a hallmark of various neurodegenerative diseases. Localized iron overload is associated with Alzheimer and Parkinson Disease and with a group of genetic disorders, termed Neurodegeneration with Brain Iron Accumulation. Moreover, brain iron deficiency is related to neurodegenerative processes like Restless Leg Syndrome and Epilepsy. In particular, last year, we investigated the effects of L-ferritin deficiency in a human, demonstrating an association between alteration of functionality of the iron storage proteins ferritin and Restless Leg Syndrome and Epilepsy. Cytosolic ferritins are composed of Heavy (H) and Light (L) subunits that co-assemble into a hollow spherical shell with an internal cavity where iron is stored. The ferroxidase activity of the ferritin H-chain is critical to store iron in its Fe3+ oxidation state, while the L-chain shows iron nucle- ation properties. We described a patient with a complete loss of L-ferritin function due to a homozygous mutation (310G>T), located in exon 3 of the L-ferritin gene. We showed that L-chain ferritin was undetectable in primary fibroblasts from the patient and thus ferritin consisted only of Hchains. Increased iron incorporation into the H-ferritin homopolymer leaded to reduced cellular iron availability, diminished levels of cytosolic catalase, SOD1 protein levels, enhanced ROS production and higher levels of oxidized proteins. Importantly, key phenotypic features observed in fibroblasts were also mirrored in reprogrammed neurons from the patient’s fibroblasts. Our results demonstrated for the first time the pathophysiological consequences of L-ferritin deficiency in a human and help to define the concept for a new disease entity hallmarked by idiopathic generalized seizure and atypical restless leg syndrome. Sonia Levi 49 DIVISION OF NEUROSCIENCE Research Units Stem cells and neurogenesis Unit Genetic conversion of mouse and human fibroblasts into induced neuronal subtypes with therapeutic prospective for neurological disorders Direct lineage reprogramming through geneticbased strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal sub-types. We previously identified a set of three transcription factors (Asl1, Nurr1 and Lmx1a) that are sufficient to generate induced dopamine (iDA) neurons by direct conversion of skin fibroblasts (Caiazzo et al. Nature, 2011). These findings lay the ground for a straightforward and efficient generation of human neurons in vitro with elaborated functional properties instrumental for disease modeling and cell-based approaches of brain repair. In particular, transplantation of iDA neurons can potentially improve the clinical outcome of Parkinson’s disease by direct replacement of the degenerated mesencephalic DA neurons. For instance, transplantation of embryonic-stemcell-derived DA neurons is efficient in restoring motor symptoms in conditions of DA deficiency (Kirkeby et al. Cell Reports, 2012). However, current protocols for in vitro differentiation of human induced pluripotent stem cells (hiPSCs) to generate DA neurons are laborious and time-expensive. In order to accelerate the overall process, we have established a fast protocol by the expression of our three reprogramming factors. With this method, we were able to generate mature and functional DA neurons in as few as 21 days, skipping all the intermediate steps of embryoid bodies and rosette neural precursors (Theka et al., Stem Cells Translational Medicine, 2013). Strikingly, the resulting neuronal conversion process was very proficient up to 93% of overall efficiency. hiPSC-derived DA neurons expressed all the critical molecular markers of the DA molecular machinery and exhibited sophisticated functional features including spontaneous electrical activity and dopamine release. This one-step protocol holds important implications for in vitro disease modeling and is particularly amenable for exploitation in high-throughput screening protocols. Expired by these results, we are currently working in developing new reprogramming sets of factors able to induce skin fibroblasts into inhibitory GABAergic interneurons aiming for a cell therapy of pharmacological-resistant forms of intractable human epilepsy. Vania Broccoli Figure 6. Wheat germ agglutinin (WGA) synaptic tracing of induced DA (iDA) neurons. (A) iDA neurons were infected with WGA-IRES-mCherry lentivirus 1 day after sorting and then replated on mouse MGE primary cell culture for in vitro analysis. (B) iDA neurons show a dense arborization after 21 days in vitro (DIV) and are able to establish functional synaptic contacts with mouse primary cells. (C-F) Staining for WGA clearly revealed WGA+/GFP- cells demonstrating that WGA from infected iDA neurons was uptaken from surrounding GAD+ neural cells through functional synapses. Modified from: Dell'Anno MT, et al. Remote control of induced dopaminergic neurone in parkinsonian rats [published online ahead of print June 17, 2014]. J Clin Invest. doi:10.1172/JCI74664 DIVISION OF NEUROSCIENCE Molecular genetics of mental retardation Unit Intellectual Disability (ID) is a common and highly heterogeneous paediatric disorder characterized by an IQ lower than 70, with a frequency of 3% in the population and a very severe social impact. Family studies have demonstrated a relatively large number of X-linked forms of ID (XLID) with an incidence of about 0.9-1.4 over 1000 males. The work of many laboratories has identified 117 different genes encoding proteins with a large variety of functions that affect the normal cellular processing leading to pre- and/or post-synaptic neuronal terminals dysfunction. The identification of all the genes responsible for human ID is a though and ambitious mission but the accomplishment of that task will shed light about the molecular basis of ID since genetic defects can be linked through common molecular pathways. We proposed to focus genomic studies on specific intracellular pathways linked to XLID in both ID patients and murine pre-clinical models. We identified as XLID genes, GDI1 and RAB39B that encode for two functionally related proteins involved in vesicular trafficking: GDI1 encodes for αGDI that, regulating RAB GTPases, is involved in the spatial and temporal control of intracellular trafficking. The molecular and behavioural characterisation of Gdi1 null mouse revealed a complex phenotype comprehending a marked reduction in the total number of glutamatergic synaptic vesicles in adult hippocampus and alterations in different endosomal RAB GTPases leading to deficits in hippocampus-dependent forms of short-term memory. Additionally we are demonstrating that the absence of αGDI allows a misregulation of the glucose metabolism effecting the correct neuronal functioning, giving us a possible new explanation on the onset of cognitive disorders. Furthermore, using genetic, biochemical and behavioural approaches we are unravelling the specific role of RAB39B protein in neuronal intracellular trafficking mediating cognitive function. The availability of Rab39b null mouse, together with Gdi1 null mouse, will give us a useful tool to greatly improve our understanding of the causes and progression of neurodevelopmental disorders and to discovery new therapeutic drugs. Patrizia D’Adamo 51 DIVISION OF NEUROSCIENCE Clinical Research Units Acute brain protection, Acute post-operative pain, Drugs and central nervous system Unit Clinical research areas: • Prognosis of diffuse axonal injury after trauma by MRI (prospective observational study). • Troponine and BNP after major elective neurosurgery (prospective observational study). • Cerebral perfusion after major elective neurosurgery in patients submitted to normo- or hyperventilation during surgery (randomized controlled study) • Participation to the multicenter prospectic database “Neurolink” of patients admitted to neurosurgical Intensive Care Units after severe head injury. • Participation to the multicenter prospectic study “Neuromorfeo” (randomized comparison between total intravenous and inhalation anesthesia during major elective neurosurgery). • Effects of propofol on “functional MRI” in 4-8 ys old children submitted to sedation to accomplish neuroradiological examination (prospective observational study). • Effects of propofol and thiopental on “functional MRI” in 1-4 ys old children submitted to sedation to accomplish neuroradiological examination (randomized controlled study). • Physiopathology of mechanical ventilation during ENT surgery with laser-proof endotracheal tubes (prospective observational study). • Systemic complications during catharact surgery under local anesthesia (prospective observational study). • Assessment of airway protection during sedation (prospective interventional study) • Study of complications in children sedated for neuroradiologic procedures (prospective observational study). • Sedation in gastroenterology procedures. Analysis of a large database. • Acute postoperative pain: ERAS (Enhanced recovery After Surgery). Luigi Beretta Cognitive neuroscience Unit This is a multidisciplinary Unit which combines theoretical knowledge and research skills from different backgrounds (neurology, neuropsychology, linguistics, cognitive psychology), with the general aim to investigate the neural mechanisms of language, memory and high-order cognitive functioning, particularly decision-making and social cognition processes. To this purpose, we use and integrate different experimental approaches, including behavioural studies in normal subjects and in neurological patients, brain imaging (magnetic resonance techniques and positron emission tomography), and neurophysiological investigations based on event-related potentials (ERPs), transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS). Stefano F. Cappa Experimental neurosurgery Unit Clinical and basic research in neurosurgery: • Results of Gamma Knife Radiosurgery in the treatment of pituitary adenomas • Multisession Gamma Knife treatment for perioptic tumors • Gamma Knife treatment for vestibular schwannomas: analysis of vestibular and acoustic function • Experimental studies on neuroregeneration with collagen scaffold and autologous olfactory cell implant • Collaboration with Prof. Comi, Dott. Martino, Dept. of Neurology, INSPE, Dott. Ciceri Dept. of Hematology OSR, in the design of clinical trial regarding cell therapy with blood monocytes in traumatic spinal cord injury • Role of cancer stem cells in brain tumours biology (in collaboration with Dott. Galli, SCRI OSR) • Study of pathogenetic mechanisms involved in human pituitary adenomas (in collaboration with Prof. G.K. Stalla, Department of Endocrinology, Max Planck Institute of Psychiatry, Munich, Germany) • Molecular biology and genetics of chordomas (in collaboration with Prof. P. Riva, Department of Biology and Genetics, Medical Faculty, University of Milan) • Molecular biology and genetics of craniopharyn- DIVISION OF NEUROSCIENCE giomas (in collaboration with Prof. P. Riva, Department of Biology and Genetics, Medical Faculty, University of Milan) • Microanatomical studies for new skull base approaches and relative reconstructive techniques (in collaboration with Prof. A.J. Caputy, Prof. F. Roberti, George Washington University Neuro- logical Institute, Washington DC, USA) • Study of microcerebral blood flow (in collaboration with Prof. Agabiti Rosei, Prof. D. Rizzoni, Dott. G. Boari, Clinica Medica, Department of Medical and Surgical Sciences, University of Brescia) Pietro Mortini Eye repair Unit CLINICAL RESEARCH 1. In vivo corneal confocal microscopy of corneal nerves: normal anatomy and pathological changes in corneal infections and neurologic diseases. 2. Epidemiology of corneal neovascularization. 3. Retrospective evaluation of the safety and efficacy of autologous cultivated limbal stem cells transplantation for restoration of corneal epithelium in patients with limbal stem cell deficiency due to ocular burns. 4. A 10-year retrospective study of patients treated with polyhexamethylene biguanide (PHMB) for Acanthamoeba keratitis to develop PHMB as the first approved drug medicinal product (orphan drug). Project funded by the EU under the Seventh Framework Programme (GA N 305661) in collaboration with the Moorfields Eye Hospital, London (UK). 5. Release of neuromediators in the lacrimal fluid after corneal surgery in patients affected by keratoconus. TRANSLATIONAL RESEARCH 1. Role of various macrophage populations in animal models of corneal neovascularization, in collaboration with Dr. R. Mazzieri and Prof. L. Naldini. 2. Safety and efficacy of NK1Ra as a treatment of corneal neovascularization. 3. The role of corneal nerve morphology as a biomarker for diabetes nephropathy and retinopathy, in collaboration with Dr. G. Zerbini. 4. Proteomic and cellular fingerprint of corneal neovascularization. 5. Local anti–TNF therapy in ocular surface inflammation with implications for conjunctival scarring diseases: the project preludes clinical application. 6. The effect of corneal inflammation on central nervous system (trigeminal ganglion) in a mouse animal model with in vivo MRI and ex vivo techniques, in collaboration with Dr. L. Chaabane. 7. Retrospective study on the incidence of corneal neovascularization in a population presenting at the Cornea and Ocular Surface Unit. 8. Modulation of nerve growth factor (NGF) levels and related pathways in the retina following optic nerve crush for developing new therapies. TRIALS 1. Multicentric European Clinical trial (REPARO STUDY), phases I-II, on the safety and efficacy of recombinant NGF for the treatment of neurotrophic keratitis 2. Monocentric observational study of corneal nerves, electrophysiology of peripheral nerves and clinical evaluation in peripheral neuropathies (CONFONEUR) Paolo Rama 53 DIVISION OF NEUROSCIENCE Clinical Research Units Functional neuroradiology Unit The Unit is composed by different groups applying advanced MR techniques to study brain structure and function during physiological and pathological development, during normal and pathological aging, in neuro-oncology and neurovascular diseases. The neuropediatric group studied myelination in normal and preterm neonates; diffusion techniques have been employed to follow the modification of white matter over time. Functional MRI has been used to investigate auditory and language areas and their maturation over the time (in collaboration with In vivo Human molecular and structural neuroimaging Unit). fMRI has been used to investigate lateralization of the mirror neuron system in normal adult subjects and to verify the influence of experience on some specific motor functions, like digit skill, in musicians (in collaboration with Experimental Neurophysiology Unit). Volumetric techniques (VBM analysis) and diffusion based techniques have been employed to study subjects affected by neurodegenerative diseases like MCI, AD and FTD, trying to identify early marker of disease associated with the conversion of MCI to dementia and to characterize selective involvement of brain areas in FTD (with Cognitive Neuroscience and Neuroimaging research Unit). The neurodegenerative changes related to motor neuron disease have been studied in Patients with ALS and PLS. The vascular group applied new high resolution techniques to identify vulnerability characteristics in patients with severe carotid stenosis and their correlation with cerebral ischemia and to test new contrast media. In collaboration with Sleep Medicine Unit, combined neuropsychologic testing and MR VBM analysis was used to assess cognitive deficits and the corresponding brain morphology changes before and after treatment in patients with OSA) The neuro-oncology group focused on the clinical validation and utility of tractography, and on the development of new algorithm based on tensorial diffusion, potentially useful to better characterize cellular and extracellular components of brain tumors. Other topics like the study of inflammatory diseases or optic nerve degeneration have been performed in collaboration with Neuroimaging Research Unit, Neuroimaging of CNS White Matter Unit, Eye Repair Unit. Andrea Falini Figure 7. Whole brain language network tractography using naming fMRI activations as seed region of interest, based on high-angular-resolution diffusion imaging (HARDI) in a Patient harboring a brain tumor. DIVISION OF NEUROSCIENCE In vivo Human molecular and structural neuroimaging Unit Researches addressed brain functional and molecular parameters in neurodegenerative conditions by using PET techniques. We assessed the amyloid load using PET and fluorinated tracers in subject with MCI/prodromal AD and compared with FDG PET functional derangements. These research results support divergent pathways for these biomarkers that might guide clinical applications. The clinical expression of Alzheimer’s disease (AD) is thought to occur as neuropathology exceeds the brain “reserve capacity”. [11C]-MP4 and PET measures of the cholinergic system allowed the estimation of AchE activity in AD and MCI subjects and the testing of reserve proxies, such as education and occupation, as modulator factors on the cholinergic system. We found significant positive correlations in the left hippocampus, lateral temporal cortex and posterior cingulate gyrus, suggesting that reserve proxies modulate specific cholinergic projections involved in learning and memory. We validated an optimized voxel-based SPM analysis based on a new atlas for normalization to be applied at single subject level for 18FDG PET studies in neurodegenerative disease associated with cognitive decline and dementia. We showed high sensitivity and specificity in the detection of brain functional alterations at the single subject level. This optimized SPM methods for FDG PET analysis, was applied in series of patients with neurodegenerative disorders and brain functional, clinical and neuropsychological correlations were obtained, useful for research purposes and clinical applications. MRI structural measurements were applied to study morphometric changes in developmental disorders (subjects with sporadic and genetic association dyslexia, subjects with DCDC2 mutation causing altered neuronal migration and associated cognitive disturbances). The results provided evidence for strucutural alterations of neural systems in these individuals supporting reading and specific cognitive processes. Daniela Perani Figure 8. Correlation between acetylcholinesterase (AChE) activity and education (LEFT) and occupation (RIGHT) proxies. The images show regions of interest of significant positive correlation, superimposed on a standard anatomical template, and the corresponding scatterplot of AChE activity against individual scores. Modified from: Garibotto, V et al. Neurobiol. Aging: 2013; 34(11): e13-e18 doi: 10.1016/j.neurobiolaging.2013.05.020. 55 DIVISION OF NEUROSCIENCE Clinical Research Units Neuroothology Unit Four papers were published during 2013 on topics related to inner ear disorders. One more paper on Vestibular Schwannomas has been accepted and will be soon published. Bruno Colombo and Roberto Teggi participated in 2013 as guest editors of the book Vestibular Migraine and related disorders that will be published by Springer Edition during 2014. Audiovestibular research has at present the following lines of research: • Study of the phenotypes of episodic vertigo, in a consortium of the Barany Society (Principal Investigator Alexandre Bisdorff) • Genetics of Menière’s Disease: the study is carried on both in collaboration with Prof Lopez Escamez (Centre Genyo – Almeria) and with Prof Manunta for genetics of ionic transporters. • Phenotypes and genetics of Vestibular Migraine: a collaboration has been carried on both with Dr Bruno Colombo and Prof Paolo Manunta. A grant of the Italian Headache Society has been obtained for the purpose • Functional MRI in patients with Vestibular Migraine, in collaboration with Bruno Colombo and Massimo Filippi • S-Loreta EEG mapping in patients with chronic tinnitus in collaboration with MAGICS (Chief Dr Leocani). Cocaine Induced Midline Destructive Lesions (CIMDL) research on with the following projects: • Position paper concerning CIMDL scheduled to be published in 2014 (accepted on Rhinology Journal) • Multicenter study on links between CIMDL and autoimmunity. • Analysis of genetic polymorphisms possibly associated with CIMDL through exome sequencing in collaboration with the San Raffaele Center for Translational Genomics and Bioinformatics. A government grant from the Italian Dipartimento Politiche Antidroga della Presidenza del Consiglio has been obtained for the purpose. Mario Bussi Psychiatry and clinical psychobiology We continued our research activities at the interface between neuroscience and behavioral disorders, to increase knowledge and develop effective diagnosis and treatment options in the broad field of Psychiatry and Clinical Psychobiology. We provided the first reports on the effects of the simple cation lithium, the mainstay of the treatment of Bipolar Disorder, on white matter microstructure. We showed that the less active GSK-3β rs334558*C gene promoter variant, and the long-term administration of the GSK-3β inhibitor lithium, were associated with increases of DTI measures of axial diffusivity in several WM fiber tracts contributing to the functional integrity of the brain, thus counteracting the detrimental influences of BD on WM structure of the brain. We also showed that widespread changes of white matter microstructure are common to ObsessiveCompulsive Disorder, and a target for drug treatment. Using translational approaches in the study of gene-environment interactions, we showed that COMT genotype biases neural correlates of empathy and perceived personal distress in schizophrenia; and contributed to large international studies of genome-wide association of OCD, and of the heritability of OCD and of Tourette syndrome. We also continued the reasoning about the role of intracellular pathways which are part of the biological clock machinery in mood disorders. We continued the profiling of psychopatological conditions by studying social interactions, executive functioning, and startle reflex in eating disorders; reality monitoring deficits in schizophrenic and affective psychosis; and neural responses to moral valence decision in depression. Refining diagnostic and treatment options for our patients, we studied the cortical effects of electroconvulsive therapy by means of transcranial magnetic stimulation and electroencephalography in severe depression; Theory of Mind interventions for outpatients with schizophrenia; and current drug options to achieve remission in mood disorders and psychotic psychiatric conditions. Finally, we continued our commitments within the frame of the EU funded MOODINFLAME research project, investigating the clinical and brain imaging correlates of neuroinflammation in mood disorders. Francesco Benedetti DIVISION OF NEUROSCIENCE Figure 9. White matter areas where carriers of a low activity variant of the glycogen synthase kinase 3-b promoter gene showed significantly higher values of axial diffusivity than homozygotes for the wild variant. Voxels of significant group differences are mapped on the mean fractional anisotropy template of the studied sample. Cover image courtesy of Dr Francesco Benedetti & Dr Irene Bollettini, Ospedale San Raffaele, Division of Neuroscience, Milano, Italy. Published in: Neuropsychopharmacology: 2013; 38(2): 313-327. doi: 10.1038/npp.2012.17 Sleep medicine The main objective of our research program was the evaluation of pathogenetic mechanisms and treatment strategies of abnormal motor activity during sleep. In particular, we studied patients with Restless legs syndrome (RLS), periodic leg movements during sleep (PLMS), and REM sleep behavior disorder (RBD). We compared night-to-night variability of PLMS and Periodicity indices in patients with RLS or periodic limb movement disorder (PLMD). In both patient groups, the Periodicity index showed a significantly lower degree of variability than that of PLMS index, being >6.5 times lower in RLS patients and 2 times lower in PLMD patients. These data support the use of the Periodicity index in the evaluation of PLMS in RLS and PLMD and indicate that this parameter seems to be more stable than the widely used PLMS index which has higher night-to-night variability. Dopamine agonists (DAs) represent the first-line 57 DIVISION OF NEUROSCIENCE Clinical Research Units treatment in RLS; however, in the long term, a substantial proportion of patients will develop augmentation, which is a severe drug-related exacerbation of symptoms and the main reason for late DA withdrawal. We evaluated consecutive outpatients affected by RLS with clinically significant augmentation during treatment with immediate-release DA. A switchover from immediate-release DAs to the long-acting, extended-release formula of pramipexole was performed and in all patients resolution of augmentation was observed within two to four weeks. This observation supports the hypothesis that the duration of action of the drug plays a key role in the mechanism of augmentation. Idiopathic RBD is a parasomnia characterized by dream enactment and is commonly a pre-diagnostic sign of parkinsonism and dementia. We participated to a multicenter case-control study to assess the family history in RBD. A total of 632 participants (316 patients, 316 controls) were recruited. We found increased odds of proxy-reported family history of presumed RBD among individuals with confirmed idiopathic RBD. This suggests the possibility of a genetic contribution to this parasomnia. We analyzed sleep characteristics in drug-free RBD patients compared to controls, and we evaluate the changes following the long-term use of clonazepam in RBD patients. Slow transient electroencephalography (EEG) events were increased in RBD, while fast transient events decreased in these patients. After clonazepam treatment, sleep stages 1 and 2 NREM instability significantly decreased and the duration of EEG transients significantly decreased. A decreased sleep instability after clonazepam might indicate a factor contributing to the restorative properties of sleep in RBD. Luigi Ferini-Strambi Clinical psychology The Unit of Clinical Psychology developed research programs according to the characteristics of each Sub-Unit: 1. Personality and Personality disorders: different aspects of emotional and behavioral dysregulation remained the main topic of interest and were studied both in non-clinical and clinical subjects. According to the plasticity of personality over time, in different periods of life, studies were conducted in adolescent and adult subjects. In particular, predictive characteristics of DSM-5 Section III traits and psychopathy measures were assessed in nonclinical adolescents and adults, as well as in adult clinical participants (Prof. A. Fossati). According to the increasing interest in the role of mindfulness both in pathogenesis and treatment of borderline personality disorder, the hypothesis of a failure in mindfulness was investigated. Results showed that mindfulness capacities were reduced in personality disorders in general, but the most important difficulties were found in borderline subjects. Interest in mindfulness was a natural expansion of the area of mentalisation present in previous research projects and still going on. The project on elicitation of emotions in borderline subjects using standardized videoclips was extended with the insertion of psychophysiological characteristics (HRV- Heart Rate Variability) and saccadic eye movements. The collaboration with the Unit of Sleep Medicine made that possible. 2. Alcohol addiction (Dr. M. Movalli): a pilot study on the effect of an innovative treatment based on the Skills Training of the Dialectical Behavior Therapy (DBT) was started. Preliminary results were promising and showed how mindfulness capacities, together with personality characteristics, play a relevant role in relapse prevention. 3. HIV infection (Dr. R. Visintini): the multicenter national study coordinated by the Health Ministry (National AIDS Center -Istituto Superiore di Sanità) on the vaccine for HIV infection (TAT) was going on under the direction of our Unit as to the psychological area. Neuropsychological tests were administered and preliminary findings showed that in a sub-population of subjects signs of precocious brain atrophy were detectable. Cesare Maffei DIVISION OF NEUROSCIENCE Motor function rehabilitation The 2013 research activities focused on 1) the effectiveness of Action Observation (AO) treatment in rehabilitation of subjects with neurological diseases, 2) the effect of AO on postural control and balance, 3) the connection between motor training and language and 4) the study of muscle innervation zone (IZ) and muscular painful spot. The second activity was about the possibility to use cognitive facilitation (such as AO) in the rehabilitation of postural and balance disorders was also studied in non-pathologic subjects. These studies addressed the feasibility of using cognitive facilitation to improve some implicit aspects of human movement (as balance and postural control). The first activity was developed in collaboration with the Functional Unit of Quantitative Neuroimaging of San Raffaele Hospital (OSR) and the Department of Neurology OSR. Two RCT have been started. The first one was about the effectiveness of AO for improving the gait of subjects with Parkinson disease and freezing. This study involved both clinical measures and the study of brain cortical activation by fMRI. These were collected prior to and after a period of physiotherapy treatment based on AO of videos inherent to the situation that normally provokes the freezing phenomenon or sham videos. The second study addressed the rehabilitation of hand dexterity in subjects with multiple sclerosis. In this case the subjects in the experimental group look at videos related to functional hand tasks. Even in this case the outcomes were both clinical measures and measures of fMRI. The third activity was inherent the connection between motor performance and cognitive processes, such as language. The study was about the effect of linguistic training on hand motor abilities congruent at the trained task. The study was realized in collaboration with the experts of language of the Psychology Faculty of the Vita-Salute San Raffaele University and using the OSR Lab of movement analysis for the acquisition and analysis of the hand kinematics. The fourth activity was developed in collaboration with the SUPSI University of Lugano (CH). It was inherent the study of the relationship between muscle IZ and muscular painful spots, using matrix surface electromyography. Roberto Gatti 59 DIVISION OF NEUROSCIENCE Introduction by the Director INSPE Institute of Experimental Neurology The Institute of Experimental Neurology (INSPE) constitutes one of the major European institutes primarily dedicated to translational research in the neuroscience field. The overall ambition is to conjugate high level of basic research with the specific competences in pre-clinical and clinical research. This is achievable considering that INSPE includes both research laboratories and the Neurological Department of the San Raffaele Scientific Institute. Inflammatory and neurodegenerative disorders of the central and peripheral nervous system, stroke, spinal cord nerve and traumatic injures represents the primary targets of the INSPE research. The primary goal is to better understand the molecular mechanisms that sustain inflammatory and degenerative pathogenic mechanisms underlying neurological disorders so to identify therapeutic targets, validate in co-operation with pharma companies new treatments, develop new disease biomarkers for both clinical trials and patients monitoring. The neurological department is deeply involved in the definition of new treatment algorithms for stroke, inflammatory and neurodegenerative diseases. The molecular and functional bases for central and peripheral nervous system recovery processes and the potential to modulate them by cellular and gene therapy and by specific rehabilitation interventions are another area of interest of INSPE. The INSPE laboratories spans on four different levels in DIBIT2 buildings. One floor is dedicated to neuropathology, experimental neurophysiology, neurogenetics of monogenic and complex diseases. Another floor is dedicated to basic and clinical neuroimmunology, neurobiology, neural stem cell research and neuroembriology. The third floor is focused on neuroimaging of neuroinflammatory and neurodegenerative disorders. The fourth floor includes labs on experimental imaging and molecular biology. Other laboratories involved in behavioral research and in signals that regulate myelination and in clinical neurophysiology are also part of the Institute. Finally the MAGICS Center which studies effects on magnetic stimulation of the brain complete the research area. DIVISION OF NEUROSCIENCE Research Units, INSPE Experimental neuropathology Defining peripheral nerve environment in amyotrophic lateral sclerosis Growing evidence indicate that alterations within the peripheral nervous system (PNS) are involved at an early stage in the amyotrophic lateral sclerosis (ALS) pathogenetic cascade. Moreover, the hypothesis that some of the peripheral nerve environment (PNE)-resident cells, such as endothelial or Schwann cells, could be involved in or could “sense” an early axonal damage is to be considered. Our aim is to characterize in details the natural history of the PNS damage in the hSOD-1G93A ALS rat model using both in vivo and ex vivo readouts. We will obtain an exhaustive functional and pathological description of the PNS involvement in hSOD-1G93A ALS rat model that could be useful for a reliable and quantitative analysis in preclinical research in ALS. Moreover, human motor nerve diagnostic biopsies previously obtained for diagnostic purposes from ALS patients will also be characterized and studies using different readouts. Our studies will establish the PNS signature of the disease, shedding new lights into ALS pathogenesis and providing a rationale basis for translating future systematic functional and pathological studies aimed at unrevealing the PNS involvement in human ALS. We believe that our combined, innovative rat-human approach will provide rationale for the development of new therapeutic approaches and to identify new diagnostic biomarkers. Angelo Quattrini Experimental neurophysiology Our unit is involved in the development and validation of non invasive neurophysiological techniques, such as electroencephalography and evoked potentials in assessing and monitoring neurological diseases. We also investigate the usefulness of neurostimulation techniques (deep brain stimulation, transcranial magnetic-TMS and direct current stimulation-tDCS) with the aim of promoting therapeutic neuromodulation and brain plasticity. These objectives are searched through a translational approach, with application in people with neurological disorders and animal models. During 2013, we explored functional interhemispheric motor connections using TMS in Parkinson’s disease, demonstrating different degrees of involvement according to disease severity (Spagnolo et al. 2013). We applied topographic source analysis of cognitive event related potentials to the Stroop test, demonstrating a more severe involvement of executive function in amyotrophic lateral sclerosis with respect to primary lateral sclerosis (Amato et al 2013). Our results suggest that the covert version of the Stroop task used in the present study, may be useful to assess cognitive state in the very advanced stage of the disease, when other cognitive tasks are not applicable. The cognitive event-related potentials to the Stroop test have been integrated on anatomical brain reconstructions from magnetic resonance imaging in order to provide a better individual source localization to be further applied in the investigation of cognitive involvement in neurological disorders (Gonzalez-Rosa et al 2013). In pre-clinical research, we used EEG to demonstrate dysfunction of brain circuits involved in the genesis of resting EEG in models of epilepsy, such as those associated with synapsyn I/II/III knock-out (Cambiaghi et al Neuropharmacology 2013) and to investigate mechanisms of rapamycin treatment in a mouse model of tuberous sclerosis complex (Cambiaghi et al Neuropharmacology 2013). On the side of brain stimulation as a treatment or neurological disorders, we tested safety and potential efficacy of a newly developed method of non invasive transcranial brain magnetic stimulation in an open label study on motor symptoms in Parkinson’s disease (Spagnolo et al. 2013) and we applied this technique in a case of progressive supranuclear palsy with progressive aphasia (Spagnolo et al. 2013). We also implemented the application of non invasive neuro-stimulation techniques in stroke. In particular, in an explorative pilot study we tested the efficacy of different protocols of deep repetitive transcranial magnetic stimulation to improve naming in chronic post-stroke aphasia (Chieffo et al 2013). Moreover, we evaluated whether different stimulation protocols of transcranial direct current stimulation could have a neuroprotective effect in the acute phase of experimental brain ischemia (Peruzzotti Jametti et al 2013). Letizia Leocani 61 DIVISION OF NEUROSCIENCE Research Units, INSPE Molecular genetics of behaviour Our laboratory has focussed for a number of years on the role of the Ras-ERK intracellular cascade in neuronal cell signalling and behavioural plasticity. Once neurotransmitter receptors are activated, specific guanine exchange factors (GEFs) are able to catalyse the exchange of GDP for GTP on Ras proteins, thus activating this class of small GTPases. Activated Ras proteins then stimulate the core element of the signalling pathway, the Ras-MEK-ERK protein kinase cascade which is crucially involved in transducing signals to the nucleus, hence controlling chromatin remodelling and gene expression, key steps involved in various forms of behavioural plasticity. In recent years, it has become clear that deregulation of this signalling pathway in the brain may lead to brain dysfunction. The work of our laboratory in 2013 has focussed on the cellular mechanisms of L-DOPA induced Dyskinesia (LID), a severe disorder associated to the current pharmacotherapy of Parkinson’s Disease (PD). The key brain structure implicated in LID is the dorsal portion of the striatum, the input nucleus of the basal ganglia system. During this year we have extended previous work of the laboratory on Ras-GRF1, a neuronal specific activator of Ras proteins, by showing that a specific ablation of this factor in the striatum is sufficient to significantly attenuate LID symptoms in mice. In fact, similarly to the Ras-GRF1 KO animals, Ras-GRF1 knockdown mice in the striatum appear to be protected toward abnormal involuntary movements (AIM), the behavioural correlate of dyskinesia in rodents, in a neurotoxin model of LID. In addition, we have shown that Ras-GRF1 and ERK signalling modulate synaptic plasticity processes in the striatum, including long-term potentiation (LTP), in a complex manner and that in the PD neurotoxin model, chronic L-DOPA administration causes a substantial synaptic reorganisation, likely representing the cellular correlate of LID. Finally, we have patented cell permeable peptides (CPPs) able to modulate ERK signalling in vivo. Such CPPs are able to alter several ERK mediated behavioural processes, including responses to psychostimulants and to L-DOPA in dyskinetic animals, providing a new interesting approach to therapies of neuropsychiatric disorders. Riccardo Brambilla Neuromuscular repair Our unit is interested in the role of adhesion in neuromuscular regeneration, focusing on integration of cellular and extracellular signals in order to regulate development, function and regeneration of muscles and nerves. We recently reported the role of vimentin, a cytoskeleton component of PNS neurons, in the regulation of peripheral nerve myelination. Vimentin modulates the levels of Neuregulin 1 type III through the shedding activity of the TACE enzyme. We also reported evidence that proper peripheral nerve development and Schwann cell-axon interaction is regulated by the nuclear molecule Jab1. Loss of Jab1 in Schwann cells compromises cell proliferation, differentiation and survival, causing a dysmyelinating peripheral neuropathy. Jab1 integrates and modulates laminin2-derived signals in Schwann cells, thus suggesting to be involved in the pathogenesis of Congenital Muscular Dystrophy Finally, we provided evidence that stem cell mesoangioblasts, engineered to release miniagrin, can ameliorate muscular dystrophy in mouse model of Congenital Muscular Dystrophy type 1A (CMD1A). Mini-agrin links laminin2 receptors expressed by skeletal muscle to laminin isoforms overexpressed in the absence of laminin2. Stefano Carlo Previtali DIVISION OF NEUROSCIENCE Figure 10. abnormal axon sorting in nerves of Jab1 null mice Neuroimmunology Unit The central nervous system (CNS) has the intrinsic capability to repair itself when injured. Over the years a series of molecular and cellular innate repair mechanisms have been discovered and their ability to prevent irreversible tissue damage and irreversible neurological deficits has been characterized. Among cellular mechanisms, those sustained by neural stem/precursor cells (NPCs) - the self-renewing and multipotent cells of the CNS capable of driving neurogenesis and gliogenesis during development and adult life - play a crucial role. As a matter of fact, both endogenous and transplanted NPCs are capable of migrating into damaged CNS areas to promote functional and structural tissue repair via different mechanisms of action spanning from cell replacement to the release of soluble neuroprotective molecules (i.e. bystander effect). The Neuroimmunology Unit is particularly interested in understanding the role of endogenous NPCs in both the developing and the adult healthy CNS in order to develop alternative therapies based on NPC transplantation for acute and chronic CNS inflammatory disorders characterized by irreversible neurodegeneration (i.e. multiple sclerosis, stroke and spinal cord injury). Endogenous NPCs in the adult brain. We are studying the role of microglia - the immune relevant cells of the CNS - during brain development. So far, we obtained results indicating that microglia patrol and protect - possibly via their phagocytizing activity - neuronal precursor positioning within the cortex. Transplantation of adult NPCs. We have found that both adult and embryonic-like NPCs are able to protect from inflammation-induced neurodegeneration - as that occurring is ischemic stroke and multiple sclerosis - via a series of different therapeutic strategies encompassing both immunomodulation (by restraining DC function) and neuroprotective mechanisms. Among neuroprotective mechanisms, we found that NPCs protect neurons from excitotoxicity by up regulating glutamate transporters on endogenous astrocytes and oligodenrdocytes by secreting neurotrophic factors such as LIF. Gianvito Martino 63 DIVISION OF NEUROSCIENCE Research Units, INSPE Figure 11. GFP positive miPSC colonies expressing SSEA1 marker. Shown are GFP positive miPSC colonies (green) stained with SSEA1 (red). Nuclei are counterstained with dapi (blue). Clinical neuroimmunology The Group of Clinical Neuroimmunology is active in both experimental and clinical neuroimmunology with the aim to have a bi-directional transfer of scientific questions and experimental data between the two. Concerning our work on mice, during 2013 we have continued our gene therapy approach to deliver cytokines in the inflamed brain by completing the studies on IL-35 and investigating also the effects of IL-27. Both of them are able to modulate microglia and modify the clinical and neuropathological features of neuroinflammation, but with different efficiency and slightly different mechanisms. Further, by using IL-4 as comparison, we highlighted a new, suppressive role for IL- 6. Concerning microvesicles, in humans we have characterized their release from a microglia cellline, learning their dependence from the stimulation of the P2X7 purinergic receptor. From a clinical point of view, we studied the number and relation to clinical and para-clinical data of microvesicles in the CSF in demented patients. Finally, we immunologically profiled patients affected by major psychoses such as schizophrenia, major depression, and bipolar disorder. We found that depressed patients display an M1 activation of monocytes in the blood. Further studies on this issue are ongoing. Roberto Furlan DIVISION OF NEUROSCIENCE Figure 12. A human microglia cell line stimulated with ATP modifies its shape and starts to release shed vesicles in a polarized manner. Immunobiology of neurological disorders Our lab studies the role of the immune system in the physiology and pathology of central and peripheral nervous system. To tackle this issue we make use of complementary disciplines, including immunology, neurobiology, histology, animal modelling, cellular and molecular biology, genomics, bioinformatics and systems biology. Thus, taking the moves from clinical observations and from human samples (blood or cerebral tissue), our research explores the activity of immune/glial/neuronal cells in vitro, sums up the high-throughput generated data with systems biology approaches, goes to the in vivo mouse models, until it is ready to go back to the clinic via the identification of novel diagnostic and prognostic biomarkers and the proposition of new therapeutic targets and approaches. Recently, we developed a statistics and bioinformatics platform for the analysis of high-throughput transcriptomics data in multiple sclerosis. The gender-based functional genomics analysis allowed the definition of biomarkers for multiple sclerosis in peripheral blood and emphasized the role of the transcription factor SP1 in neuroinflammation (Menon et al J. Autoimmunity 2012). During the year 2013 we isolated transcriptional signatures in peripheral blood which stratified distinct multiple sclerosis stages and identified a series of novel transcriptional regulators as potential therapeutic targets. Regarding the inflammatory processes in peripheral nervous system, during the last years we acquired novel information about the physiology of skeletal muscle and extended this knowledge to the study of idiopathic inflammatory myopathies. Starting from evidences published by our group (Colombo et al J. Neuropath. Exp. Neurol. 2011, J. Neuroimmunology 2012, Neuropath Appl Neurobiol 2012), in 2013 we demonstrated that neurotrophins regulate myogenesis and tissue regeneration in skeletal muscle (Colombo et al. J. Pathology 2013). Moreover, we found that in in65 DIVISION OF NEUROSCIENCE Research Units, INSPE flammatory myopathies infiltrating T lymphocytes and macrophages produce the neurotrophic factor BDNF and get localized near regenerating myofibers which express the neurotrophin receptor p75NTR(Colombo et al. J. Pathology 2013), supporting the hypothesis of a tissue-protective immune response in inflamed skeletal muscle. Cinthia Farina Neuroimaging research Unit Main achievements • Normal brain. The organization of functional brain networks differ between genders, likely contributing to the abnormalities detected in diseases with a gender prevalence. • Multiple sclerosis. In MS, imaging features of cortical lesions differ from those of white matter (WM) lesions and may reflect neuronal damage and microglial activation. • Both trans-synaptic degeneration secondary to optic nerve damage and Wallerian degeneration due to T2 lesions contribute to optic radiation damage in MS. • Cervical cord voxel-based assessment contributes to a better characterization of the clinical heterogeneity of MS. • In MS, cognitive reserve independently protects against disease-related cognitive decline over and above brain reserve. Lifestyle choices protect against cognitive impairment independently of genetic factors. • Migraine. Cortical abnormalities occur in migraine representing the results of a balance between an intrinsic predisposition and diseaserelated processes. • Dementia. Early onset Alzheimer’s disease patients had a more severe and distributed WM damage relative to late onset AD. • The nonfluent and semantic variants of primary progressive aphasia share an overlapping pattern of dorsal and ventral WM pathway abnormalities. Variant-specific WM changes were also found, contributing to the specific language deficits. • Resting state fMRI abnormalities in the behavioural variant of frontotemporal dementia (bvFTD) involve the salience, default mode and frontoparietal networks. Functional changes distinguish bvFTD from AD. • Graph analysis showed that global and local functional networks are altered in bvFTD, suggesting a loss of efficiency in information exchange between both distant and close areas. • Amyotrophic lateral sclerosis. In ALS, an increased parietal connectivity may have a role in maintaining cognitive efficiency despite structural frontotemporal injury. • WM damage to interhemispheric, limbic and associative tracts contribute to cognitive deficits in primary lateral sclerosis. • Parkinson’s disease. In PD, WM abnormalities beyond the nigrostriatal system accumulate with increasing motor and nonmotor severity. • WM damage in PD patients with GBA gene mutations may have an impact on the clinical manifestations, including cognitive impairment. Massimo Filippi Neuroimaging of CNS white matter Research activity Multiple Sclerosis • We evaluated the role of brain and cognitive reserve in limiting onset of cognitive deficits in cross-sectional and longitudinal investigations. • We assessed the contribution of involvement of strategic regions of the gray matter (GM) and white matter (WM) to the pathogenesis of fatigue and depression. • In patients with pediatric MS, we found a peculiar pattern of structural involvement of the GM and WM in posterior regions of the brain. • Differently from adult MS patients, pediatric MS patients experience only limited abnormalities of resting state (RS) functional connectivity (FC) and internetwork connectivity. • Using voxel-wise analysis, we defined the contribution of structural damage to the cerebellar peduncles to cerebellar deficits in a large cohort of patients. • Deficits in memory and visuospatial learning correlate with regional hippocampal atrophy. • In a 13-year longitudinal study, we found that baseline measures of GM damage predicted worsening of clinical disability and cognitive deficits in patients with relapse-onset MS. • Modifications of RS FC of cognitive-related re- DIVISION OF NEUROSCIENCE gions predicted the persistence of cognitive rehabilitation effects in patients with relapsing-remitting MS. • Both trans-synaptic degeneration secondary to optic nerve damage and Wallerian degeneration due to local T2 lesions contribute to optic radiation damage. • We developed two different methods for the assessment of regional cervical cord atrophy. Migraine • Similarly to adult patients, pediatric patients with migraine have GM structural abnormalities involving regions of the pain matrix. • Patients with Fahr’s disease and migraine have structural and functional abnormalities similar to those observed in migraineours. • We explored the patterns of cortical thickness and cortical surface area abnormalities in patients with migraine. Pediatric Brain Injury • Using RS MRI and diffusion tensor imaging (DTI), we identified the predictors of clinical improvement following constraint-induced movement therapy (CIMT) in pediatric patients with chronic hemiplegia. Neuropathies • In patients with peripheral neuropathy, RS FC modifications extend beyond the sensorimotor network and involve other sensory and cognitive networks. Maria Assunta Rocca Human inherited neuropathies Unit Research in our group is focused on CharcotMarie-Tooth (CMT) neuropathies, which constitute the most common form of human inherited neuromuscular disorders. At present, no known therapy is available for any CMT neuropathy. CMTs are commonly characterized by distal wasting, weakness and sensory loss, and are the consequence of mutations in at least 70 different genes. Among them, several CMT genes encode proteins regulating the endo-lysosomal trafficking, thus providing the opportunity to study basic mechanisms of the cell biology of myelination in Schwann cells, which are largely unknown. Our laboratory contributed to identify some of these CMT genes, whose function has been then investigated using several animal models that we generated in the last 10 years. These models have been exploited to a) study the natural history and progression of the disease; b) to unravel pathogenetic mechanisms at the basis of these neuropathies and c) to prove the efficacy of therapeutic strategies aimed at restoring proper myelination and favor nerve repair and regeneration. In particular, during this year: a) we investigated the cell autonomy of FIG4 function in Schwann cells and motor neurons. In human, recessive mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and CharcotMarie-Tooth type 4J neuropathy (CMT4J). Loss of FIG4 phospholipid phosphatase causes decreased PtdIns(3,5)P2 levels and defects in multiple pathways in the endomembrane system. b) we assessed the role in the regulation of PNS and CNS myelination of KIF13B motor protein and of its interactor the DLG1 scaffold, which we identified in Schwann cells in complex with MTMR2, a phospholipid phosphatase mutated in autosomal recessive demyelinating CMT4B1. c) we tested the hypothesis that modulation of the NRG1 (Neuregulin) type III signaling, through the control of its primary inhibitor TACE secretase, could constitute an efficient treatment for hypermyelinating CMTs. This hypothesis relies on the instructive role of NRG1 to initiate and regulate the amount of myelination, its role in the pathogenesis of some CMT neuropathies, and its potential role in nerve repair. Alessandra Bolino 67 DIVISION OF NEUROSCIENCE Research Units, INSPE Figure 13. Schwann cell/DRG neuron primary co-cultures from FIG4 knock out mouse embryos display abnormally enlarged and vacuolated late endosomal-lysosomal compartments marked with LAMP1green (red is phalloidin) Axo-glia interactions Unit Myelin is a highly specialized membrane, which wraps around axons in peripheral (PNS) and central nervous system (CNS) and is required to facilitate efficient and rapid propagation of nerve impulses. Myelin formation is tightly regulated and while the molecular events controlling the development of Schwann cells and oligodendrocytes have been characterized, the axonal mechanisms directing its production have been only recently identified. Indeed, gliogenesis and the development of nervous system strictly depend upon interaction between neurons and glial cells. Glial cells promote neuronal survival, regulate the organization of myelinated axons and promote axonal differentiation and myelin sheath production. Axons promote glial cells survival and development. Alteration in myelination can have dramatic consequences ranging from reduction of the conduction of nerve impulses to neuronal cell death, with a significant impairment of normal functionality in patients affected by demyelinating disorders. The main interest in our laboratory is to investigate the axonal molecules and the downstream signaling pathways intermediates they activate in myelinating glia. In the CNS several factors contribute to myelin sheet formation and maintenance. Unlike the PNS, where NRG1 type III controls all the aspects linked to myelination, in the CNS no specific factor(s) has been identified, suggesting that axonal control of myelination is achieved via cooperation of several molecules. The majority of the growth factors implicated in CNS myelination are subjected to regulated processing by secretases. We recently showed that the α-secretase ADAM17 is a key protease controlling PNS myelination. We have now evidences that ADAM17 promotes oligodendrocytes development and CNS myelination. In vitro and in vivo experiments indicate that axonal ADAM17 promotes oligodendrocytes differentiation and myelination. Further, conditional ablation of ADAM17 in transgenic models indicates that ADAM17 is important also in neuronal survival. Our observations suggest the existence of posttranscriptional mechanism(s) regulating myelin formation and axonal survival and identify secretases as a potential therapeutic target to modulate myelination and possibly neuronal survival. Carla Taveggia DIVISION OF NEUROSCIENCE Clinical Research Units, INSPE Inflammatory CNS disorders Unit Clinical Trials (PI: M. Rodegher). We participated in many International RCTs (phase II, III) to evaluate the safety and efficacy of new immunoactive drugs in pts with MS with different disease courses. We analyzed clinical and laboratory data on more than 1500 MS pts, treated with first-line disease modifying drugs (DMD),to identify the early predictors of long-term disability progression. We have carried on a prospective study to characterize clinical, MRI and Vitamin D as prognostic factors in pts at first clinical attack. Finally, we concluded a study on the prevalence of CCSVI in a large cohort of MS pts. Genetics of Complex Neurological Diseases (PI: F. Martinelli-Boneschi). The laboratory was involved in the identification of genetic risk factors and molecular mechanisms implicated in the susceptibility to complex common neurological diseases such as MS, Alzheimer’s disease and neuropathy. An additional activity was the identification of predictive and diagnostic, disease activity and treatment-response biomarkers involved in inflammatory and degenerative diseases. The laboratory was also involved in the processing and bioinformatic analyses of Illumina array and sequencing data. Clinical Research (PI: L. Moiola). We participated in studies evaluating the efficacy and safety of natalizumab and of different DMDs in paediatric MS. We were involved in a project on implementation of a web-information aid for newly diagnosed MS pts and in a multicentre study evaluating MS patients’ risk propensity to accept risks associated to different DMDs. We analyzed clinical, immunological and MRI data of “atypical” forms of MS, CNS Vasculitis and of NMO. Finally, we carried out a prospective study to evaluate safety and efficacy of long-term rituximab treatment. Neuro-rehabilitation. We evaluated the efficacy of different aspects of intensive multidisciplinary rehabilitation in MS pts and we concluded a study on the identification of the predictors of disability improvement. We were involved in studies to assess the effect of fampridine on QoL and sativex on spasticity in progressive MS. Finally, we concluded a research aiming at evaluating the usefulness of an objective assessment of the ambulatory skills in MS outpatients using GPS technology. Vittorio Martinelli Cerebrovascular disorders The cerebrovascular patient: from bedside to bench and vice versa Epidemiological, clinical and instrumental data of patients admitted to SU are collected in a database, and blood samples are stored. This represents an invaluable tool for clinical research and for collaboration within national and international networks. Spontaneous projects: • Study of clinical/radiological factors predictive of haemorrhagic transformation in acute phase of ischemic stroke. • Identification of specific neurological deficits at stroke onset predictive of persistent disability, regardless the low total NIHSS. • Study of the role of circulating monocytes in the neurobiological processes of acute cerebral ischemia, through analysis of candidate genes expression such as aging gene p66Shc and plasma proteins like cytokines/chemokines. National and international collaborations: • IPSYS: Italian study aiming at the definition of risk factors and genetic deteminants of young stroke; • MUCH-Italy: Italian study aiming at the definition of risk factors and genetic deteminants of hemorrhagic stroke; • CADISP: international study on whole-genome genetic association with clinical characteristics of pts affected by cervical arterial dissection. Interventional trials • OSR SU is the coordinator center of the WakeUp Stroke trial, designed to evaluate safety/efficacy of thrombolysis in pts with arousal stroke. • IRIS: randomized vs placebo, multicentric, international, double-blind trial evaluating efficacy of DU-176b vs warfarin in reducing the risk of stroke in stroke pts with atrial fibrillation 69 DIVISION OF NEUROSCIENCE Clinical Research Units, INSPE • ENGAGE: randomized vs placebo, multicentric, international, double-blind trial evaluating efficacy of pioglitazone in reducing the risk of stroke. Morevoer, we have continued the optimization of the diagnostic-therapeutic process, implementing guidelines that involve all the staff from Emer- gency Department to Stroke Unit and Rehabilitation. In this respect, our unit is involved in a project with the 118-Milano to better recognize and dispatch pts for thrombolysis. Maria Sessa Memory disorders In last few years the increasing of population ageing is associated with an increase of incidence of secondary and primary forms of dementia and our Unit is involved in some interesting studies about these diseases. The research activity of Memory Disorders Unit in 2013 continued the work started during the previous years, which was designed to improve the diagnosis at an early stage in the various forms of dementia. Particularly in the patient with the onset of dementia in early stage (age less than 65 years old) we decided to make routine a series of advanced diagnostic tests such as the dosage of cerebrospinal fluid (CSF) biomarkers (β-amyloid, protein τ and phospho-τ), 3 Tesla brain MRI, [18F] FDG-PET brain, that, associated with the complete neuropsychological evaluation, have allowed us to improve our diagnostic capabilities in vivo by limiting the risk of diagnostic errors since, as is well known, the diagnosis of certainty in different forms of dementia is only confirmed with a post-mortem examination. The close collaboration with other research units of our hospital (Neuroimaging Research Unit, Functional Neuroradiology Unit, Experimental Neurophysiology Unit, In vivo Human Molecular and Structural Neuroimaging Unit, Laboratory of Genetics of Complex Neurological Diseases, Proteome Biochemistry Unit) and other external centres (Negri Institute, Policlinico Hospital of Milan, Memory and Aging Centre in San Francisco) has enabled us to published several scientific papers aimed at improving knowledge of the ultrastructural alterations of white and grey matter in the behavioural variant of fronto-temporal dementia (FTD), in rare variant of FTD called Primary Progressive Aphasia and in AD. Much emphasis has been given to projects with the clinical neurogenetic unit to improve the pharmacogenomics knowledge: our collaboration with the Laboratory of Genetics of our hospital also continued, in order to search for new diagnostic and pharmacogenetics markers in AD, with the collection of DNA from AD subjects treated with Achei divided into responders and non-responders to therapy for the identification of genetic characteristics indicative of a better response to anticholinesterase inhibitors therapy. Giuseppe Magnani Movement disorders The Movement Disorders Unit is focused on understanding the physiopathological mechanisms responsible for different movement disorders, arising from both central and peripheral nervous system dysfunctions. We are involved in the clinical and neurophysiological validation studies on safety and efficacy of innovative therapeutic strategies, both in clinical practice and in experimental settings. In particular we are studying several animal models of human acquired and inherited diseases in order to characterize underlying physiopathological mechanism and to provide a sensitive model to verify the response to experimental therapies. Further, we are committed in the translation process of these new therapeutic approach in human pathology, like the deep brain stimulation in several basal ganglia disorders or lentiviral hematopoietic stem cell gene therapy in metachromatic leukodystrophy. Notably we are studying the functional recovery after botulinum toxin type A (BT-A) therapy in patients with focal spasticity in multiple sclerosis (MS), the tuning of the treatment of neurogenic bladder by BT-A injection into the detrusor muscle, as well as the cortical excitability modifications in dystonic diseases. In details, we are studing the transcranial magnetic stimulation (TMS) effect on the exteroceptive suppression (“geste antagoniste”) in cervical dystonia (CD). A peculiar finding of focal dystonia, particularly of CD is the inhibitory effect of sensory tricks, such as touching the face or chin, holding the nape with the hand, which were reported to decrease or even abolish dystonia in up to 70% of the patients. Whether the inhibitory ef- DIVISION OF NEUROSCIENCE fect of sensory tricks is related to exteroceptive rather than proprioceptive afferent commands is still a matter of debate. TMS is a well-established method for the non-invasive study of the motor cortex. Stimulating the motor cortex with a double magnetic stimulation over the scalp (paired-pulse TMS, ppTMS) or with an electrical-magnetic paradigm (sensory afferent inhibition, SAI), allows to modulate the motor output from the cortex through intracortical as well as cortico-subcortical network. Ubaldo Del Carro Neuromuscular disorders Clinical studies in Neuromuscular Diseases are addressed to give the best medical supports to patients affected by neuromuscular disorders. We are involved in several clinical studies either spontaneous or sponsored, mono-centric and multicentric, national and international. We completed: 1. Epidemiological Study about the possible association between anti-flu vaccine and onset of Guillain-Barrè syndrome. We are working on: 1. A Randomized, Double-Blind, Placebo-Controlled, Multicentre Study to Assess the Efficacy on Spasticity Symptoms of a Cannabis Sativa Extract in Motor Neuron Disease Patients. 2. Evaluation of corneal confocal microscopy, neurophysiological studies and skin biopsy in sensory peripheral neuropathy. 3. Double blind controlled and randomized study to evaluate the efficacy of high dose immunoglobulins in treatment of painful diabetic polyneuropathy resistant to conventional therapy. 4. IGOS : International GBS outcome study. 5. CFTY720I2201: A double-blind, randomized, multicenter, placebo controlled, parallel-group study to evaluate the efficacy and safety of fingolimod 0.5 mg administered orally once daily versus placebo in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). 6. Functioning and quality of life in patients with peripheral neuropathy associated with antiMAG antibodies. 7. SCIG in disimmune chronic peripheral neuropathies: a retrospective national study. Raffaella Fazio Paroxysmal events Our unit is principally involved in the evaluation of epileptic patterns and single status epilepticus seizures. Starting from the data contained in the database of the Center for Clinical Epilepsy, we currently have in progress a study to identify the clinical and neurophysiological markers indicating the possibility of maintaining the antiepileptic therapy only for a limited number of years. Data in literature are currently generic and of little help for the individual patient in clinical practice. We have completed the evaluation of the neurophysiological characteristics of the experimental model of Tuberous Sclerosis Complex in mice. Our future program will be to assess the impact of inflammation in the genesis and maintenance of epilepsy. In particular, we are interested in the evaluation of inflammatory diseases that develop on the basis of autoimmune responses as a possible mechanism at the basis of at least a part of the drug-resistant epilepsy. The other side of activities involves neurophysiological studies related to vascular disease in the acute phase. We acquired simultaneous EEG recordings and Doppler TC in the course of thrombolytic therapy of acute ischemic stroke. The work program includes an evaluation of the actual feasibility of these systems of monitoring during both their real clinical utility in the description of the procedure performance and the ability to provide data of short-and long-term prognosis already in the acute phase of the treatment. Fabio Minicucci 71 DIVISION OF NEUROSCIENCE Selected publications Cozzi, A; Santambrogio, P; Privitera, D; Broccoli, V; Rotundo, LI; Garavaglia, B; Benz, R; Altamura, S; Goede, JS; Muckenthaler, MU and Levi, S. Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome. J. Exp. Med.: 2013; 210(9): 1779-1791 - Article IF 2012: 13,214 Laterza, C; Merlini, A; De Feo, D; Ruffini, F; Menon, R; Onorati, M; Fredrickx, E; Muzio, L; Lombardo, A; Comi, G; Quattrini, A; Taveggia, C; Farina, C; Cattaneo, E and Martino, G. iPSCderived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF. Nat. Commun.: 2013; 4: 2597 - Article IF 2012: 10,015 Benedetti, F; Bollettini, I; Barberi, I; Radaelli, D; Poletti, S; Locatelli, C; Pirovano, A; Lorenzi, C; Falini, A; Colombo, C; Smeraldi, E. Lithium and GSK3-β promoter gene variants influence white matter microstructure in bipolar disorder. Neuropsychopharmacology: 2013; 38(2): 313-327 - Article IF 2012: 8,678 Agosta, F; Sala, S; Valsasina, P; Meani, A; Canu, E; Magnani, G; Cappa, SF; Scola, E; Quatto, P; Horsfield, MA; Falini, A; Comi, G; Filippi, M. Brain network connectivity assessed using graph theory in frontotemporal dementia. Neurology: 2013; 81(2): 134-143 - Article IF 2012: 8,249 Filippi, M; Preziosa, P; Copetti, M; Riccitelli, G; Horsfield, MA; Martinelli, V; Comi, G; Rocca, MA. Gray matter damage predicts the accumulation of disability 13 years later in MS. Neurology: 2013; 81(20): 1759-1767 - Article IF 2012: 8,249 Colombo, E; Bedogni, F; Lorenzetti, I; Landsberger, N; Previtali, SC; Farina, C. Autocrine and immune cell-derived BDNF in human skeletal muscle: Implications for myogenesis and tissue regeneration. J. Pathol.: 2013; 231(2): 190-198 - Article IF 2012: 7,585 Canessa, N; Crespi, C; Motterlini, M; Baud-Bovy, G; Chierchia, G; Pantaleo, G; Tettamanti, M; Cappa, SF. The functional and structural neural basis of individual differences in loss aversion. J. Neurosci.: 2013; 33(36): 14307-14317 - Article IF 2012: 6,908 Noseda, R; Belin, S; Piguet, F; Vaccari, I; Scarlino, S; Brambilla, P; Martinelli-Boneschi, F; Feltri, ML; Wrabetz, L; Quattrini, A; Feinstein, E; Huganir, RL; Bolino, A. DDIT4/REDD1/RTP801 is a novel negative regulator of schwann cell myelination. J. Neurosci.: 2013; 33(38): 15295-15305 Article IF 2012: 6,908 Martinelli-Boneschi, F; Giacalone, G; Magnani, G; Biella, G; Coppi, E; Santangelo, R; Brambilla, P; Esposito, F; Lupoli, S; Clerici, F; Benussi, L; Ghidoni, R; Galimberti, D; Squitti, R; Confaloni, A; Bruno, G; Pichler, S; Mayhaus, M; Riemenschneider, M; Mariani, C; Comi, G; Scarpini, E; Binetti, G; Forloni, G; Franceschi, M; Albani, D. Pharmacogenomics in Alzheimer’s disease: A genomewide association study of response to cholinesterase inhibitors. Neurobiol. Aging: 2013; 34(6): 1711.e7-1711.e13 - Article IF 2012: 6,166 Consonni, M; Cafiero, R; Marin, D; Tettamanti, M; Iadanza, A; Fabbro, F; Perani, D. Neural convergence for language comprehension and grammatical class production in highly proficient bilinguals is independent of age of acquisition. Cortex: 2013; 49(5): 1252-1258 - Article IF 2012: 6,161 Filippi, M; Agosta, F; Scola, E; Canu, E; Magnani, G; Marcone, A; Valsasina, P; Caso, F; Copetti, M; Comi, G; Cappa, SF; Falini, A. Functional network connectivity in the behavioral variant of frontotemporal dementia. Cortex: 2013; 49(9): 2389-2401 - Article IF 2012: 6,161 Amato, N; Riva, N; Cursi, M; Martins-Silva, A; Martinelli, V; Comola, M; Fazio, R; Comi, G; Leocani, L. Different Frontal Involvement in ALS and PLS Revealed by Stroop Event-Related Potentials and Reaction Times. Front. Aging Neurosci.: 2013; 5: 82 - Article DIVISION OF NEUROSCIENCE IF 2012: 5,224 Pelizzoni, I; Zacchetti, D; Campanella, A; Grohovaz, F; Codazzi, F. Iron uptake in quiescent and inflammation-activated astrocytes: a potentially neuroprotective control of iron burden. Biochim. Biophys. Acta Mol. Basis Dis.: 2013; 1832(8): 1326-1333 - Article IF 2012: 4,910 Tagliavacca, L; Colombo, F; Racchetti, G; Meldolesi, J. L1CAM and its cell-surface mutants: New mechanisms and effects relevant to the physiology and pathology of neural cells. J. Neurochem.: 2013; 124(3): 397-409 - Article IF 2012: 3,973 Losa, M; Donofrio, CA; Barzaghi, R; Mortini, P. Presentation and surgical results of incidentally discovered nonfunctioning pituitary adenomas: Evidence for a better outcome independently of other patients’ characteristics. Eur. J. Endocrinol.: 2013; 169(6): 735-742 - Article IF 2012: 3,136 Selected publications, INSPE Filippi, M; Preziosa, P; Copetti, M; Riccitelli, G; Horsfield, MA; Martinelli, V; Comi, G; Rocca, MA. Gray matter damage predicts the accumulation of disability 13 years later in MS. Neurology: 2013; 81(20): 1759-1767 - Article IF 2012: 8,249 Colombo, E; Bedogni, F; Lorenzetti, I; Landsberger, N; Previtali, SC; Farina, C. Autocrine and immune cell-derived BDNF in human skeletal muscle: Implications for myogenesis and tissue regeneration. J. Pathol.: 2013; 231(2): 190-198 - Article IF 2012: 7,585 Noseda, R; Belin, S; Piguet, F; Vaccari, I; Scarlino, S; Brambilla, P; Martinelli-Boneschi, F; Feltri, ML; Wrabetz, L; Quattrini, A; Feinstein, E; Huganir, RL; Bolino, A. DDIT4/REDD1/RTP801 is a novel negative regulator of schwann cell myelination. J. Neurosci.: 2013; 33(38): 15295-15305 Article IF 2012: 6,908 Martinelli-Boneschi, F; Giacalone, G; Magnani, G; Biella, G; Coppi, E; Santangelo, R; Brambilla, P; Esposito, F; Lupoli, S; Clerici, F; Benussi, L; Ghidoni, R; Galimberti, D; Squitti, R; Confaloni, A; Bruno, G; Pichler, S; Mayhaus, M; Riemenschneider, M; Mariani, C; Comi, G; Scarpini, E; Binetti, G; Forloni, G; Franceschi, M; Albani, D. Pharmacogenomics in Alzheimer’s disease: A genomewide association study of response to cholinesterase inhibitors. Neurobiol. Aging: 2013; 34(6): 1711.e7-1711.e13 - Article IF 2012: 6,166 Peruzzotti-Jametti, L and Cambiaghi, M; Bacigaluppi, M; Gallizioli, M; Gaude, E; Mari, S; Sandrone, S; Cursi, M; Teneud, L; Comi, G; Musco, G; Martino, G and Leocani, L. Safety and efficacy of transcranial direct current stimulation in acute experimental ischemic stroke. Stroke: 2013; 44(11): 3166-3174 - Article IF 2012: 6,158 73 DIVISION OF NEUROSCIENCE Research Units Cellular neurophysiology Unit Developmental neurogenetics Unit DIVISION OF NEUROSCIENCE Neuropsychopharmacology Unit Molecular genetics of mental retardation Unit 75 DIVISION OF NEUROSCIENCE Proteomics of iron metabolism Unit Clinical neuroimmunology DIVISION OF NEUROSCIENCE Immunobiology of neurological disorders Memory disorders 77 DIVISION OF NEUROSCIENCE Neuromuscular repair Axo-glia interactions Unit DIVISION OF NEUROSCIENCE Human inherited neuropathies Unit Neuroimmunology Unit 79 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Scientific Coordinator: Zaverio M. Ruggeri Associate Directors: Ottavio Alfieri*, Emanuele Bosi* Research Units Coagulation and platelet biology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––– 86 HEAD OF UNIT: Zaverio M. Ruggeri RESEARCHER: Chiara Foglieni FELLOWS: Maria Lombardi, Maria Elena Mantione Type 2 diabetes and diabetic complications –––––––––––––––––––––––––––––––––––––––––––– 87 HEAD OF UNIT: Emanuele Bosi* Cardiodiabetes & core Lab GROUP LEADER: Lucilla D. Monti POST-DOCTORAL FELLOW: Elena Galluccio FELLOW: Serena Spadoni TECHNICIANS: Sabrina Costa, Barbara Fontana Complications of diabetes ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 87 GROUP LEADER: Gianpaolo Zerbini POST-DOCTORAL FELLOW: Silvia Maestroni FELLOWS: Valentina Martina, Alice Spinello CONSULTANT: Mara Lorenzi TECHNICIAN: Daniela Gabellini Metabolism, nutrigenomics and cell differentiation ––––––––––––––––––––––––––– 88 GROUP LEADER: Ileana Terruzzi POST-DOCTORAL FELLOW: Nausicaa Mazzocchi 81 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Research Units / Clinical Research Units Bone metabolism Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 89 HEAD OF UNIT: Alessandro Rubinacci RESEARCHER: Isabella Villa POST-DOCTORAL FELLOW: Simona Bolamperti FELLOW: Alice Spinello CONSULTANTS: Giovanna Mignogna, Gianluigi Moro, Marcella Sirtori TECHNICIAN: Rita Masullo Coagulation service & thrombosis research Unit –––––––––––––––––––––––––––––––––––––– 89 HEAD OF UNIT: Armando D’Angelo* RESEARCHERS: Patrizia Della Valle, Annalisa Fattorini PHYSICIANS: Luciano Crippa, Silvana Viganò TECHNICIAN: Francesca Sampietro Pediatric endocrinology research –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 90 HEAD OF UNIT: Giuseppe Chiumello* GROUP LEADER: Stefano Mora FELLOWS: Silvia Capelli, Cecilia Diceglie, Katia Maruka, Ilaria Zamproni TECHNICIAN: Maria Puzzovio Clinical Research Units Diabetes and endocrinology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 91 HEAD OF UNIT: Emanuele Bosi* PHYSICIANS: Alberto Davalli, Gabriella Galimberti, Roberto Lanzi, Marco Federico Manzoni, Matteo Rocco Pastore, Alessandro Saibene, Maurizio Storti RESIDENTS: Andrea Bolla**, Amelia Caretto**, Anna Dolcetta Capuzzo, Ilaria Formenti**, Laura Frosio**, Alessandra Gandolfi**, Tania Garito**, Chiara Molinari**, Valentina Villa** FELLOWS: Raffaele Di Fenza, Andrea Laurenzi, Alessandro Rossini NUTRITIONIST: Monica Marchi Cardio-metabolism and clinical trials –––––––––––––––––––––––––––––––––––––––––––––– 91 CLINICAL GROUP LEADER: Piermarco Piatti FELLOWS: Valentina Giulia Crippa, Francesca Perticone, Emanuela Setola RESEARCH NURSE: Michela Stuccillo Fetal-maternal medicine ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 92 HEAD OF UNIT: Massimo Candiani* CLINICAL GROUP LEADER: Maria Teresa Castiglioni RESEARCHERS: Paolo Cavoretto, Susanna Rosa, Maddalena Smid, Luca Valsecchi FELLOWS: Lara Di Piazza, Annalisa Inversetti, Federica Pasi Pediatrics Unit HEAD OF UNIT: Giuseppe Chiumello* Clinical pediatric endocrinology –––––––––––––––––––––––––––––––––––––––––––––––––––– 92 CLINICAL GROUP LEADER: Giovanna Weber* RESEARCHERS: Gisella Garbetta, Sara Osimani, Gabriella Pozzobon, Gianni Russo, Maria Cristina Vigone DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units RESIDENTS: Silvana Caiulo**, Chiara Damia**, Marianna Di Frenna**, Alessandra Di Lascio**, Valentina Donghi**, Maria Piera Ferrarello**, Moira Gianninoto**, Silvia Meroni**, Elena Peroni**, Sarah Rabbiosi**, Giulia Maria Tronconi** Diabetes and metabolic diseases in children and adolescents –––––––––––––– 93 CLINICAL GROUP LEADER: Franco Meschi RESEARCHERS: Stefania Di Candia, Andrea Rigamonti, Paola Sgaramella Neonatology ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 93 CLINICAL GROUP LEADER: Graziano Barera RESEARCHERS: Gisella Garbetta, Antonella Poloniato, Rosanna Rovelli Structural heart disease Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 94 HEAD OF UNIT: Ottavio Alfieri* PHYSICIANS: Stefano Benussi, Michele De Bonis, Francesco Maisano FELLOWS: Andrea Guidotti, Davide Schiavi Cardiopulmonary clinical physiology Unit –––––––––––––––––––––––––––––––––––––––––––––––– 95 HEAD OF UNIT: George Cremona RESIDENTS: Barbara Calcaterra, Sara Dal Farra, Anna Chiara Ogliari CONSULTANT: Gabriella Gambaro TECHNICIAN: Antonella Fumagalli Cardiovascular interventions Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 95 HEAD OF UNIT: Antonio Colombo PHYSICIANS: Mauro Carlino, Alfredo Castelli, Alaide Chieffo, Filippo Figini, Azeem Mohamed Latib, Matteo Montorfano FELLOWS: Chiara Bernelli, Jaclyn Chi Lin Chan, Georgina Fuertes, Gennaro Giustino, Caroline J. Magri, Tadashi Miyazaki, Toru Naganuma, Charbel A. Naim, Vasilis Panoulas, Katsumasa Sato, Paola Spatuzza TRIAL COORDINATOR: Angela Ferrari Center for arrhythmia research –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 96 HEAD OF UNIT: Paolo Della Bella PHYSICIANS: Francesca Baratto, Caterina Bisceglia, Simone Gulletta, Giuseppe Maccabelli, Patrizio Mazzone, Gabriele Paglino, Andrea Radinovic, Simone Sala, Nicola Trevisi, Pasquale Vergara Echocardiography Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 96 HEAD OF UNIT: Giovanni La Canna PHYSICIANS: Emanuela Alati, Giovanna Di Giannuario FELLOW: Chiara Sordelli TECHNICIAN: Marta Martino Ischaemic heart disease, heart failure and echocardiography Unit –––––––––––––––– 97 HEAD OF UNIT: Alberto Margonato* PHYSICIANS: Eustachio Agricola, Alberto Capelletti, Gabriele Fragasso, Michele Oppizzi, Cristina Pedrigi RESIDENT: Claudia Montanaro ** 83 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units Organ protection in critically ill patients, Advanced cardiac –––––––––––––––––––––––– 98 failure and mechanical supports Unit HEAD OF UNIT: Alberto Zangrillo* PHYSICIANS: Pier Carlo Bergonzi, Tiziana Bove, Luca Cabrini, Maria Grazia Calabrò, Remo Daniel Covello, Antonella Crescenti, Martina Crivellari, Annalisa Franco, Chiara Gerli, Giovanni Landoni*, Carlo Leggieri, Giulia Maj, Daniela Mamo, Giulio Melisurgo, Fabrizio Monaco, Giacomo Monti, Federico Pappalardo, Anna Mara Scandroglio, Massimo Zambon POST-DOCTORAL FELLOWS: Francesca Isella, Diana Taddeo RESIDENTS: Giovanni Borghi, Andrea Montisci, Marina Pieri, Laura Ruggeri, Valentina Testa FELLOWS: Alessandro Belletti**, Alessandro Putzu**, Omar Saleh**, Dario Winterton** QUALITY ASSURANCE AND REGULATORY AFFAIRS: Rosalba Lembo, Simona Massani, Lara Sussani, Paola Zuppelli Strategic research on heart failure Unit –––––––––––––––––––––––––––––––––––––––––––––––––– 99 HEAD OF UNIT: Paolo Camici* PHYSICIANS: Enrico Ammirati, Marco Magnoni, Roberto Spoladore** POST-DOCTORAL FELLOWS: Massimiliano Mancini**, Paolo Musarò, Angela Scavone** RESIDENTS: Alessandro Durante**, Alessia Faccini**, Alessandra Laricchia, Francesco Maranta**, Damiano Regazzoli** FELLOWS: Rocco Baccaro, Veronica Buia, Ilaria My, Isabella Scotti, Valentina Turco Study and treatment of aortic disease Unit –––––––––––––––––––––––––––––––––––––––––––––– 99 HEAD OF UNIT: Roberto Chiesa* PHYSICIANS: Domenico Astore, Renata Clotilde Castellano, Efrem Civilini, Laura Dordoni, Gloria Esposito, Enrico Maria Marone, Massimiliano Marrocco-Trischitta, Germano Melissano*, Yamume Tshomba RESIDENTS: Luca Apruzzi **, Serena Frezza**, Jessica Lanza, Davide Logaldo, Daniele Mascia **, Girolomina Mazzeo, Enrico Rinaldi **, Sara Spelta, Renato Vitale** FELLOWS: Domenico Baccellieri, Luca Bertoglio, Chiara Brioschi, Andrea Kahlberg Vision first Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 100 HEAD OF UNIT: Francesco Bandello* PHYSICIANS: Nicola Baccelli, Piero Barboni, Maurizio Battaglia Parodi, Paolo Bettin, Loredana Bonisolli, Elena Bruschi, Raffaele Di Fenza**, Maria Lucia Cascavilla, Carlo Ciampi, Marco Codenotti, Umberto De Benedetto, Federico Di Matteo, Marina Fiori, Maddalena Forti, Marco Gagliardi, Matteo Ghidoni (until March 2013), Silvia Giatsidis, Antonio Giordano Resti, Lauretta Guarisco, Ugo Introini, Rosangela Lattanzio, Francesca Legorini, Francesco Loperfido, Gisella Maestranzi, Angela Malegori, Maria Pia Manitto, Elisabetta Martina, Paolo Mauceri, Elisabetta Miserocchi, Giulio Modorati, Veronica Odazio, Luisa Pierro, Matteo Prati, Giuseppe Querques, Andrea Ramoni, Carmen Rojo, Alessandra Spinelli, Monica Stoppani, Alessandra Tavola, Ilaria Zucchiatti RESIDENTS: Luigi Berchicci**, Ingrid Bianchi**, Giuseppe Casalino**, Giulia Corradetti**, Claudia Del Turco**, Giovanni Fogliato**, Lorenzo Iuliano**, Karl Anders Knutsson**, Carlo La Spina**, Jacopo Milesi**, Davide Panico**, Lea Querques**, Giacinto Triolo** TECHNICIANS: Giorgio Alto, Adriana Angiolini, Alessio Buzzotta, Silvia Giganti (until March 2013), Silvia Marcaggi (from June 2013), Antonella Ribecca * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Introduction by the Directors Mission and Vision - It is unquestionable that some of the most relevant diseases with high social, economic and clinical impact in developed societies link disorders of metabolism to chronic and acute alterations of cardiovascular function. Metabolic and cardiovascular diseases represent a major area of clinical care and research at San Raffaele Research Institute. Thus, the Division of Metabolic and Cardiovascular Sciences is devoted to planning an integrated approach to treat patients and understand mechanisms based on a structured view of the pathophysiological connections between metabolic and cardiovascular alterations. Such a vision could set a strong example of how to tackle complex problems with beneficial outcomes. Aim of the Division is therefore not only to exploit the obvious possibility of synergy at the level of basic research, but also the creation of clinical programs in which experts in the study of altered metabolism are integrated in the team of cardiologists, cardiac surgeons, vascular surgeons and cardiac emergency physicians treating acute or chronic cardiovascular diseases. Conversely, cardiovascular specialists should be part of the clinical team evaluating and treating patients with metabolic disorders. Goals - The biology of endothelium may be considered as a theme of unifying interest and particular relevance to research bridging the metabolic and cardiovascular disease areas. This could easily be expanded to include the biology of other cellular components of the vessel wall – smooth muscle cells, fibroblasts, inflammatory cells and adipocytes – which play a concerted and fundamental role in controlling all responses to acute and chronic vascular injuries. Along with this unifying theme, areas of interest span from the mechanisms of myocardial damage; thrombosis; hemostatic imbalance (post-surgery bleeding in emergency patients and patients with ventricular assisting devices); pathobiology of the atherosclerotic plaque; metabolic alterations leading to cardiovascular diseases, in particular insulin resistance and type 2 diabetes; material sciences and bioengineering with respect to developing new devices; and cell therapies. The outcomes of this integrated approach will lay new grounds for innovative treatments of established metabolic and cardiovascular diseases, personalized preventive medicine programs and new generations of clinical and nutritional studies. Achievements - Work performed in the Division is internationally recognized in several areas of excellence including arrhythmology, cardiac valve and aortic surgery, acute and chronic myocardial damage, coronary revascularization, vascular inflammation, insulin resistance, diabetes and diabetic macro- and microangiopathy, intermediary and energy metabolism, bone pathophysiology. Training Opportunities - The Division, through the intertwined connections with several Clinical Care Departments, gives ample support to the Postgraduate Schools in Anesthesia and Intensive Care, Cardiovascular Diseases, Cardiac Surgery, Vascular Surgery, Endocrinology and Metabolic Diseases, Obstetrics and Gynecology, Pediatrics, and Ophthalmology. 85 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Research Units Coagulation and platelet biology Unit Activity of the Coagulation and Platelet Biology Unit has been limited to the group coordinated by Dr. Chiara Foglieni owing to the lack of dedicated facilities. According to a reorganization implemented by the Scientific Direction, only this group will remain operative after 2013. The Foglieni laboratory focuses on the molecular and cellular vascular components responsible for atherosclerotic plaque destabilization and the onset of arterial thrombosis. These studies involve the Vascular Surgery Unit (prof. R. Chiesa) and the Structural Heart Disease Unit (prof. O. Alfieri). Platelet aggregates and fibrin resulting from coagulation activation are key components of the thrombi that occlude arteries. Thus, cellular and molecular components of the atherosclerotic vessel might participate in the development and progression of atherothrombosis by directly or indirectly interacting with platelets and/or mediators of coagulation. We have optimized human vessels tissue and primary human smooth muscle cells culture models, and developed methods for the analysis of P2X7 and MMP9 suitable for functional studies of the pathogenesis of atherosclerosis and atherothrombosis. Our findings are highlighting distinct thrombogenic roles of the vascular layers, depending on the degree and type of alteration, as well as new synergistic functions of tissue factor and coagulation contact phase pathways in thrombus formation. We are also collaborating with Dr. F. Canducci (Insubria University), Prof. R. Burioni and Prof. M. Clementi (Vita-Salute San Raffaele University), and Dr. A. Rubinacci (Bone metabolism Unit) to study the molecular immune response in experimental atherosclerosis. Collaborative relationships have been established also with Dr. G. Fragasso and Prof. A. Margonato (Heart Failure Clinic-Clinical Cardiology) to investigate heart failure mechanisms. We anticipate obtaining results that will help improve diagnosis and treatment of atherothrombotic disorders. Zaverio M. Ruggeri Figure 14. Structural and functional analysis of atherosclerotic carotid artery tissue cultured after explant. a) Atherosclerotic macro-sections for tissue culture dissected from a carotid artery specimen obtained by endarterectomy. b) Histological examination of the carotid artery sections after 30 min (t 0), 3, 7, 10 and 15 days of culture displaying morphology preservation over time. c) Visualization and quantification by real-time confocal videomicroscopy of platelet aggregates and fibrin strands formed over carotid artery cryosections following blood perfusion; note the comparable response of specimens after 30 min (t 0) and 3 days (t 3) of culture, but the different response of the diverse vascular wall layers. d) Confocal microscopy characterization of smooth muscle cells explanted from atherosclerotic carotid artery fragments and analysis of their response to P2X7 specific antagonists (A740003, KN62) and to the physiologic agonist, ATP. DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Cardiodiabetes & core Lab Translational studies on gene polymorphysms and type 2 diabetes and cardiovascular disease Our group demonstrated a phenotype characterized by increased fasting nitric oxide levels but reduced response after an insulin stimulus. Further, we found a strong association between eNOS gene variants and T2DM and CAD. The polymorphism in intron 19 of eNOS affects mRNA maturation, hence yielding a consistent skipping of exons 20 and 21. This generates a stable truncated form of eNOS that exerts a dominant negative effect. These stable eNOS isoforms with altered properties were present in platelets and endothelial cells with a characteristic pro-atherogenic phenotype and alteration of vascular function in healthy subjects mutated for eNOS gene variants. The proposed mechanism provides a suggestive explanation for the increase of early restenosis in mutated CAD patients. The negative contribution of these mutation both in term of mortality or in term of early restenosis in subjects with eNOS variants were associated with increased risk of restenosis/amputation after PTA in patients with limb ischemia. Moreover, these patients showed significantly lower circulating EPCs. The pharmacogenetics analysis of the present study showed that the best effect in stimulating EPCs and in turn reducing restenosis was found in patients not presenting eNOS variants and treated by Intensified Insulin treatment. Recent data seems to demonstrate, in healthy subjects, that eNOS gene variants correlate with a reduced number of EPCs. While these data open to new intriguing perspectives in viewing different diseases involving vasculature response to NO, further studies are needed to clarify the molecular mechanisms of action and regulation of truncated eNOS protein both in CAD patients and in mutated healthy subjects, a new potential class at risk for early atherosclerosis and glucose intolerance (GI). In fact, in a large cohort of FDR healthy subjects, we found that eNOS gene mutation associates with GI, suggesting an increased susceptibility for these patients to become diabetic. An international patent was posted (PCT/EP2011/068471, number WO2012052555) on the beneficial effects of a bar with a low content in sugars and proteins but added with L-arginine. Lucilla D. Monti Complications of diabetes Prevention of the microvascular complications of diabetes: new approaches The only feasible way to prevent the microvascular complications of diabetes is to start the specific preventive treatment as soon as possible after the onset of diabetes, when irreversible, glucose-induced abnormalities, are still not present. Diabetic retinopathy. To find a way to prevent this complication during the last year we used a double strategy: a) early identification of the patients at risk to develop the complication and b) setting up of new preventive pharmacologic approaches. a) In this field we have demonstrated that an increased activity of circulating endothelial progenitor cells (cells originated from the bone marrow with the aim to maintain the turnover of mature endothelial cells) characterizes the patients at risk to develop the complication and that a similar dysfunction predicts also the development of a disease closely related to diabetic retinopathy, i.e. age-related macular degeneration, thus further validating the role of the biomarker. b) Based on recent evidences suggesting that the first component of the retina affected by glucotoxicity is the neuroretina, we have set up a study (presently ongoing), aimed to verify the effect of early neuroprotection on the pathogenesis of diabetic retinopathy in a mouse model of type 1 diabetes. Diabetic nephropathy. The progression of this complication is characterized by the gradual decrease of the number of intraglomerular podocytes (the cells in charge of the size-selectivity of the glomerular filtration), paralleled by an increased urinary excretion of these same cells, suggesting a direct role of podocyturia in the progression of the disease. During the last year we have demonstrated that podocyturia is characterized by the presence of a large number of immature podocytes suggesting that glucose might directly induce the dedifferentiation of resident podocytes with consequent detachment from the basement membrane. We are now involved in a new study aimed to verify whether, by inhibiting the process of in vivo dedifferentiation it is also possible to reduce the number of immature podocytes excreted with urine. Gianpaolo Zerbini 87 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Research Units Metabolism, nutrigenomics and cell differentiation Nutrigenomics is the aspect of our research interested in understanding the molecular-level interaction between nutrients and genome. This area is organized in translational projects following our identification of specific natural substances with insulin-mimetic effects. Among these, conglutin γ study has led to a patent deposition recognizing its insulin-mimetic properties, providing a possible nutraceutical/ pharmaceutical development of a food compound to prevent and treat insulin resistance and related diseases. Because insulin anabolic effect, we are studying the cellular action of natural molecules as resveratrol and betaine on skeletal muscle metabolism, differentiation and hypertrophy. Our published results show that these molecules might regulate cell cycle and promote muscle differentiation and myotubes size: Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation; RSV regulates cell cycle exit, induces C2C12 muscle differentiation and might control MRFs and muscle-specific proteins synthesis. In addition, RSV stimulates IGF-1 signaling pathway, in particular AKT and ERK 1/2 protein activation, AMPK protein level and induces hypertrophic morphological changes in neo-formed myotubes modulating cytoskeletal proteins expression. indicating a possible new drug/integrator strategy in sport performance and clinical conditions characterized by muscle impairment. Currently, an aim of our resaearch project is to assess the ability of RSV to preserve muscle, fat and liver function in relation to glucose metabolism in mice subjected to a high fat diet (HFD). Another aspect of our research is the study the effects of these substances on primary skeletal muscle cultures/skeletal muscle biopsies from patients with muscle diseases. Both patients with type 2 diabetes (T2DM) and myotonic dystrophy (MD) show insulin resistance and skeletal muscle damage. This project aims to investigate the possible relationship between defects in insulin signalling and muscle atrophy and the effects of insulin mimetic natural compounds Ileana Terruzzi Figure 15. Resveratrol (0,1 and 25 μM) action on MyHC and p21 expression, myotubes morphology, nuclei arrangement in hypertrophy at the end of differentiation process. Immunofluorescence analysis shows hypertrophic morphological changes in MyHC-positive neo-formed myotubes after RSV treatment. Scale bar 50 μm. p21 Immunofluorescence images and DAPI also confirms the nuclei arrangement in neo-formed myotubes after RSV treatments in respect to DM condition. Scale bar 50 μm. In: Montesano A, Luzi L, Senesi P, Mazzocchi N, Terruzzi I Resveratrol promotes myogenesis and hypertrophy in murine myoblasts. J Transl Med. 2013 Dec 13;11:310 doi: 10.1186/1479-5876-11-310. DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Bone metabolism Unit The Bone Metabolism Unit is actually engaged in two major study lines: One line is focused on the biomolecular signalings and the endocrine milieu that characterize femural fracture: a major public health treat representing the most devastating outcome of osteoporosis. The study attempts to characterize genes expression in bone tissue as well as the miRNAs presence in both bone and serum of osteoarthritic and fractured patients admitted to our Orthopaedic Unit with the ultimate aim to define the potential value of serum miRNAs as biomarkers of bone diseases state, in particular of the osteoporosis severity state (i.e femural fracture vs primary osteoarthritis). Preliminary results have identified an OPG/RANKL ratio promoting osteoclastogenesis and a disregulation of the Wnt signaling. The other study line is focused on the physiological role of the osteocyte- lining cells syncitium with the aim to understand its contribution to the mechanisms underlying plasma calcium homeostasis, mechano-transduction and targetted remodelling which are all potentially affected by aging, microgravity and renal insufficiency. This study is done through collaborative studies with the Endocrine Unit, Harvard, Boston, and it has been recently inserted in the Joint Universities Summer Teaching Laboratory (JUSTL) program at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, USA. Animals lacking PTH/PTHrp receptor (PTH1R) expression in osteocytes (OcyPTHR1-KO), developed at Harvard, and littermate controls are used to investigate calcium efflux and influx from bone using a scanning ion-selective electrode technique (SIET). Preliminary results have outlined the role of PTH. The Bone Metabolism Unit is also engaged in collaborative studies with IRCCS Istituto Ortopedico Galeazzi, Milano, Italy for the evaluation of calcium metabolism in fish scales and with the Department of Molecular Medicine, Section of Biochemistry, University of Pavia, Italy for the evaluation of the impaired osteoblastogenesis in murine models of osteogenesis imperfecta. Alessandro Rubinacci Coagulation service & thrombosis research Unit Our Unit is currently involved in the extensive analysis of the thrombin generation test (TGT), in the attempt to mirror the clotting potential within the microcirculation. As opposed to the routine PT and aPTT tests, the TGT makes use of a low tissue factor concentration (TF, 1 to 5 pmol), which permits analysis of the amplification/propagation phase of the clotting process. This renders the test sensitive to defects of the intrinsic coagulation pathway while triggering the physiological hemostatic response via the FVIIa/TF complex. To insure comparability of the results we have shown that reference to the normal pooled plasma’s thrombogram is mandatory, and we have also identified numerical parameters which may independently better describe the clotting status of the test plasma (lag-time ratio, peak velocity ratio, acceleration ratio, true-tail ratio, endogenous thrombin potential ratio, with ratios >1.0 indicating hypercoagulability versus normal pooled plasma). To mimic the setting of microcirculation, we have identified optimal conditions to detect defects in natural anticoagulant systems (protein C, antithrombin) by the addition of selected concentration of thrombomodulin (protein C system) and fondaparinux (which selectively activates the FXa neutralizing activity of antithrombin). Finally, we have also developed a software which permits the graphical representation of the test plasma’s thrombogram as percentage values – positive or negative - of the normal pooled plasma’s thrombogram. By analysis of apparently healthy volunteers and patients with established thrombophilia markers (FV Leiden, prothrombin mutation, protein C, protein S and antithrombin deficiencies, or combinations) we show the potential of the modified TGT to identify patients with hypercoagulability inspite of the absence of established thrombophilia markers. Patients on anti-vitamin K drugs may experience major bleeding or thrombosis episodes while on therapeutic INR values. We show that our modification of the TGT has also the potential to identifiy patients who are either hypercoagulable or excessively anticoagulated inspite of therapeutic INR values. The modified TGT may alsoidentify patients with severe sepsis/septic shock requiring antithrombin and/or protein C supplementation. Armando D’Angelo 89 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Research Units Pediatric endocrinology research Our group, in collaboration with the Universities of Milan, Insubria, and Catania, is participating in a study aimed to the identification of genetic markers of Congenital Hyperinsulinism of Infancy (HI,) a rare disorder of glucose metabolism. We established a National Registry for this disease, and we enrolled and studied over 40 families with HI. Molecular studies lead to the identification of several novel mutations responsible for the disease. We are also actively involved in the study of bone health in several pediatric disorders. Osteoporosis is regarded as a condition of the elderly, but it is now clear that it has its antecedents during childhood and adolescence. A major role in the development of osteoporosis is the bone mass gain occurring during growth. The concerted action of bone-forming cells (osteoblasts), and bone resorbing cells (osteoclasts) is crucial for the development of a healthy and competent skeleton. Although genetic factors play a crucial role in the determination of bone mass gain, bone metabolism impairment during childhood and adolescence leads to a deficient skeletal development We are investigating the role of antiretroviral treatment on bone mass in HIV-infected youths, in collaboration with Vania Giacomet and Gian Vincenzo Zuccotti at L. Sacco Hospital in Milan. We are also studying bone mass and bone metabolism in young patients with congenital adrenal hyperplasia, a disease leading to disturbances of sexual differentiation. Hypophosphatemic rickets is a rare condition leading to impaired bone mineralization, skeletal deformities, and metabolic disturbances. The cause of the disease have been described recently, but they remain unknown in some patients. Our group is involved in a collaborative study with the group of Maria Luisa Brandi, University of Florence, to identify the genetic causes of this type of rickets. Stefano Mora DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units Diabetes and endocrinology Unit Prevention and new therapies of type 2 diabetes. This line of investigation is pursued within the Cardio-Diabetes and Clinical Trials Unit. A recent accomplishment is the demonstration of improvement of glucose metabolism, insulin sensitivity, insulin secretion and endothelial function in subjects with impaired glucose tolerance and metabolic syndrome by a nutritional approach based on L-Arginine-enriched biscuits with low sugar and protein content. This study opens innovative avenues for the prevention of type 2 diabetes by non pharmacological approaches. Moreover, a large number of phase III and some phase II clinical trials are being conducted, with more than 400 patients on follow up during the year 2013, having as a major focus new drugs for type 2 diabetes, including GLP-1 agonists, DPP-4 inhibitors, SGLT2 inhibitors and insulin analogues. Self-monitoring of blood glucose (SMBG). The largest clinical trial ever performed on SMBG in type 2 diabetes (PRISMA Study) has been conducted under the coordination of the San Raffaele group. The results indicated that the use of SMBG, structured in timing and frequency, improves glycemic control and provides guidance in prescribing diabetes medications in noninsulin-treated patients with type 2 diabetes. This study provides relevant information into the controversial issue of clinical benefits of SMBG in this patient population. New treatments of diabetic complications. Following a preliminary pilot trial performed in the recent past, we conducted a multicenter European clinical trial that validated the Frequency Modulated Electro-Magnetic neural Stimulation (FREMS) as a novel and original non pharmacological treatment for symptomatic diabetic neuropathy. During the year 2013, this innovative technology has also been successfully applied in Scleroderma Diabeticorum, a rare and otherwise untreatable complication of diabetes. Emanuele Bosi Cardio-metabolism and clinical trials New and innnovative treatments of type 2 diabetes and cardiovascular disease In the light to evaluate innovative treatments to revert IR and endothelial dysfunction (ED), as part of a nutritional planning, we demonstrated that among persons with Impaired Glucose Tolerance (IGT) and Metabolic Syndrome (MS), the supplementation of L-arginine for 18 months significantly increased regression to Normal Glucose Tolerance and the results were maintained during an extended follow-up of 30 months. This interventional study demonstrated that dietary L-arginine supplementation in conjunction with lifestyle intervention could significantly increase the conversion of impaired glucose tolerance and metabolic syndrome to normal glucose tolerance. This study provides a potential population-directed approach to prevention of type 2 diabetes. In the line of a nutritional approach to prevent type 2 diabetes, a functional food was invented by the group, i.e. a biscuit with a low content in sugars and proteins and added with L-arginine that enhances endothelial function and improves glucose metabolism, insulin sensitivity and secretion in subjects with IGT and MS. An international patent was posted (PCT/EP2011/068471, number WO2012052555) in collaboration with Cardio-Diabetes and Core Lab Unit. We are also evaluating the role of environmental factors, i.e. smoking, in the development of insulin resistance. The group is also involved in the largest and major worldwide sponsored Clinical Trials for the prevention and cure of DM2 and CAD and we have implemented our performance in these Trials and in October 2012 our Centre was defined one of the “Premier Site” in the world in collaboration with Sanofi-Aventis. The Unit collaborate with the Cardio-Thoraco-Vascular and the Neurology Departments. Piermarco Piatti 91 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units Fetal-maternal medicine Study of new predictors of pregnancy outcomes in women with high risk pregnancies 1. Identification of novel markers of local and systemic inflammation able to anticipate pregnancy outcome in patients with recurrent pregnancy losses In collaboration with the Innate Immunity and Tissue Remodelling and the Inflammation and vascular remodelling Units (Division of Regenerative Medicine, Stem Cells and Gene Therapy) we are verifying the ability of novel markers of vascular activation, systemic inflammation and coagulation to predict maternal and foetal outcome in women with the Anti-Phospholipid Syndrome and/or with unexplained recurrent miscarriages, second trimester abortions or intrauterine foetal deaths. In the same cohort of patients we are also verifying the presence of novel auto-antibodies specificities (i.e. the acute phase protein PTX3, protein S, protein C) and of polymorphisms in genes that control the development of immune tolerance versus inflammation (e.i. HLAG). In addition we are testing how the treatment with low molecular weight heparins interfere with the markers analysed. transcripts of fetal and placental genes potentially involved in the pathogenesis of preeclampsia and intrauterine growth restriction. The identification of early markers of these complications of pregnancy is important for prevention and early diagnosis. For this study we are collecting blood samples at different gestational ages and clinical data from patients at increased risk to develop preeclampsia and intrauterine growth restriction. 3. Screening for gestational diabetes in Lombardy: a population-based study 2. Study of feto-placentar nucleic acids in maternal plasma in preeclampsia and intrauterine growth restriction In Italy screening for gestational diabetes is recommended for women at increased risk, i.e., women with previous gestational diabetes or diabetes in first-degree relatives, obese or older than 35 years of age or from geographical areas at increased risk of type 2 diabetes. Current practice and compliance to screening guidelines has never been documented. In collaboration with the Department of Biostatistics of the University of Milano Bicocca and the Diabetes Research Institute we are analyzing regional Health Care System archives to document the screening practice for gestational diabetes in over 360.000 pregnancies in the Lombardy Region in the period 2007-2010. We are collaborating with the Genomic Unit for Diagnosis of Human Pathologies to test a panel of Maria Teresa Castiglioni Clinical pediatric endocrinology The main fields of research of the pediatric endocrinology unit are thyroid diseases, short stature, Congenital Adrenal Hyperplasia (CAH), Hypogonadotrophic Hypogonadism, Disorders of sex differentiation (DSD). As regards Congenital Hypothyroidism (CH) we continued a collaborative research project with the University of Milan to expand our knowledge on the genetic, epigenetic and environmental factors contributing to CH. We also focused on the thyroid function in twins and preterm babies positive at screening programme for CH. Concerning this topic, our attention has been focused on the aetiological and peculiar clinical aspects of CH in twins, and we have studied the outcome of thyroid function in the group. As co-investigator of AIFA Research we continued the study that evaluates the influence of initial Levothyroxine dose at diagnosis on neurodevelopmental, growth, cardiovascular and skeletal outcomes in children with CH. We participated in some multicentric studies on hyperthyroidism, evaluating the possible prognostic factors for recovery after medical treatment. We conducted a study on relevant clinical and auxologic aspects in Noonan patients: we evaluated the response to Growth Hormone in short syndromic children, in terms of growth velocity and short term height gain (3 years of therapy) and its correlation to genotype (PTPN11 + and PTPN11 - patients). Congenital Adrenal Hyperplasia (CAH) is a field of application of less-invasive and more accurate methods. We are continuing to determine serum, salivary and urinary steroid profile through LC-MS for diagnosis and follow-up of various forms of CAH. Hypogonadotrophic Hypogonadism is a family of diseases that could have a neonatal, pu- DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES bertal or adult onset. We continued to study these patients from a clinical and a genetic perspective. In some patients, mutations in candidate genes have been found. Disorders of sex differentiation (DSD) comprehend congenital conditions in which chromosomal, gonadic and anatomic development is atypical. We aim to standardize pharmacologic tests execution and results interpretation in order to obtain a better diagnostic definition, together with genetic analysis. A diagnosis is obtained in 66% of patients with DSD and 46XY karyotype. Giovanna Weber Diabetes and metabolic diseases in children and adolescents The aim of the diabetes unit is clinical research in children and adolescents with type 1 diabetes in a large outpatients clinic attended by 750 subjects with diabetes. We have 3 main lines of study: 1) prevention of diabetes; 2) technology in diabetes; 3) Genetic and metabolic study in infants with neonatal insulin resistant diabetes. More than 250 children treated with pump for continuous subcutaneous insulin infusion (CSII) are in follow-up for many years and we have compared CSII and multiple daily injections (MDI) in children < 6 yrs and investigated technical and clinical failures with a review of a large number of patients. In cooperation with Prof Barbetti ( Bambino Gesù Hospital - Rome ) we are studying correlation phenotype-genotype in insulin resistant diabetes due to insulin receptor gene mutations and incidence of neonatal/infancy onset diabetes in Italy. We are an active part of SIEDP’s group in pediatric diabetology and we participate in different multicenter studies. We collaborate in Trialnet (directed by Prof. Bosi), primary and secondary prevention study, and with Dr. Battaglia (of Diabetes Research Institute ) in evaluating innate cells role in children affected by type 1 diabetes. The obesity unit is performing clinical research in children and adolescents with Prader-Willi Syndrome (PWS), the most common genetic cause of obesity, characterized by elevated morbidity and mortality in all ages. In collaboration with others Italian Research centre we investigated different aspects: • the effects of long-term GH therapy on sleepdisordered breathing and adenotonsillar hypertrophy in children with PWS • the prevalence of central adrenal insufficiency in Prader-Willi syndrome using the metyrapone test • the occurrence of metabolic syndrome and its components in a large group of PWS, according to obesity status. Another field of interest is congenital hyperinsulinism of infancy (CHI) a rare disease that needs prompt treatment to avoid brain damage. We studied the clinical and molecular features of 33 patients with CHI and their parents. This is the first report on hyperinsulinism of infancy in Italy. Moreover, in order to search for novel loci contributing to the pathogenesis of CHI, we combined a family-based association study, using the transmission disequilibrium test on 17 CHI patients lacking mutations in ABCC8/KCNJ11, with a whole-exome sequencing analysis performed on 10 probands. This strategy allowed the identification of the potential causative mutations in genes implicated in the regulation of insulin secretion. Franco Meschi Neonatology The main clinical activity of our Unit is the assistance to healthy and premature newborn (about 2.100/ year) with support of the local intensive care unit. Offspring of mothers with gestational diabetes and neonatal endocrine disease are clinical excellence area of our unit. We pay specially attention to quality of care as preterm infant developmental care in ELBW infants,through strict control of all the environmental challenges. Research Fields The main research interest in Neonatology deals with three clinical fields. Maturative study of the Central Nervous System. Study of CNS is a very fascinating field of research and using innovative approaches, such as Diffusion Tensor Imaging and Functional MRI, it is possible to quantify the anatomic and functional evolution of the brain of the preterm babies. Our 93 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units group co-operates with the department of Pediatric Neuroradiology for the application of the latest imaging techniques and to valuate the clinical feasibility. Metabolic and clinical outcome of infants born pre-term or from mothers with diabetes. Infants born from pregnancies complicated by pre-gestational or gestational diabetes, whose clinical conditions are often similar to those of uncomplicated pregnancies, need more characterization in order to better define their long-term clinical and metabolic outcome. Body composition and bone mineralization are innovative fields of research, that we are exploring in such babies along with growth and neuro-psychologic development. The aim is to evaluate the impact of pre-gestational conditions on extra-uterine growth. Results are than compared with normal babies an with infants born pre-term or small for gestational age. New molecular methods for detecting severe infections in newborn infants. Early diagnosis of sepsis is an important challenge in newborns: validation of molecular assay detecting bacteria DNA in blood could be an useful tool for early diagnosis and therapy. Preliminary datas, obtained by collaborating with Microbiology Department suggest that molecular microbiological techniques are both instruments for possible microbiological diagnosis and potential markers potentially used in the clinical management of non-infection conditions as gut perforation in necrotising enterocolitis Graziano Barera Structural heart disease Unit Heart failure Ischemic heart disease Surgical strategies to treat patients with heart failure are investigated. Original procedures to correct secondary mitral and tricuspid regurgitation have been developed. Mid- and long-term results of the procedures on tricuspid valve have been evaluated and a randomized study is ongoing to establish indications for the correction of tricuspid pathology. Surgical approaches alternative to transplant have been investigated as therapies for the late stage heart-failure including ventricular assist devices. Surgical repair of the mitral valve has been deeply investigated in the context of dilatative cardiomyopathy, even associated to atrial fibrillation ablation and resynchronization therapy. Active contribution has been given to the SYNTAX study, the FREEDOM study and the EXCEL study, all comparing PCI and CABG in multivessel and main stem disease. Heart valve disease Innovative techniques to repair mitral and aortic valve have been systematically evaluated. New imaging modalities have been applied and correlated to the operative findings. Experimental adjustable devices for mitral valve repair have been designed and evaluated. Very long term results of the edge-to-edge repair (an original technique of MR correction introduced by our group) have been assessed and reported at international meetings. Atrial fibrillation New surgical methods to increase the effectiveness of the atrial fibrillation ablation procedures, during open heart surgery, have been developed. A program for the treatment of lone atrial fibrillation via a minimally invasive approach has been implemented, and new technologies have been introduced and meticulously tested. The left atrial remodeling has been studied not only structurally but also at molecular and biochemical level. Morphologic and structural changes of the cardiomyocytes in atrial fibrillation have been investigated. Devices to exclude the left atrial appendage are tested and evaluated. Transcatheter valve therapy A multidisciplinary program for transcatheter aortic, mitral and tricuspid valve therapy has been developed using a wide spectrum of techniques and technologies. Guidelines have been prepared and extensive investigation in this area are ongoing. Ottavio Alfieri DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Cardiopulmonary clinical physiology Unit Research has focused on early diagnosis of small airway disease using impulse oscillometry and multiple breath nitrogen washout in subjects with asthma and in recipients of bone marrow transplantation with bronchiolitis obliterans The gold standard for the diagnosis of bronchial asthma in patients with a normal spirometry is currently the methacholine challenge test (MCT) test. IOS is a promising technique to assess airway function able to quantify changes in peripheral airway resistance undetected by traditional spirometry. Non smoking patients were studied by spirometry and IOS prior to MCT. Mean baseline FEV1 was normal. Ten subjects had a positive MCT. IOS showed higher baseline R5-20Hz values in MCT+ subjects (p2O/l/s. Rp may be a useful marker in predicting MCT response and provide a screening tool for detecting bronchial asthma. Bronchiolitis Obliterans (BO) initially affects terminal and respiratory bronchioles, a region of the lung largely unexplored by spirometry. Multiple nitrogen wash out (N2-MBW ) is shows a high sensitivity to peripheral airway changes potentially more suited to early detection of small airways disease. Healthy controls (n=41), bone marrow transplant candidates (n=47), haematopoietic stem cell transplantation (HSCT) recipients (n=65) and patients with COPD (n=8), were assessed by N2MBW and spirometry. HSCT enhanced ventilation inhomogeneity both in conductive (Scond*VT) and acinar (Sacin*VT) airways. Patients with BO (n=8) were characterized by a further 3-fold increase in Sacin*VT more than twice that in COPD patients. At 321% of predicted, Sacin*VT could distinguish the subjects with BO from recipients, with 87% accuracy, 88% sensibility and 90% specificity. N2-MBW is able to detect changes following HSCT as well as those specific to BO. Data presented at the annual meeting of the European Respiratory Society Barcelona September 2013 and the 55th ASH Annual Meeting and Exposition (December 7-10, 2013) in New Orleans, LA. George Cremona Cardiovascular interventions Unit Coronary artery disease: PARTICIPATE is a prospective observational study evaluating polymer Cre8 stent ”de-novo”coronary artery. Acute Coronary Syndromes, ACS: MATRIX is a randomized clinical trial evaluating in patients with ACS trans-radial intervention as compared to femoral access site and bivalirudin vs. unfractionated heparin±GP IIb/IIIa inhibitor. High risk patients, LEADERS FREE is a prospective randomized clinical trial evaluating bare metal stent versus Biofreedom stent in patients at high risk for bleeding. Complex Coronary Lesions:The EXCEL trial is a randomized trial comparing Everolimus eluting coronary stent system vs. coronary artery bypass graft surgery in subjects with unprotected left main coronary artery disease and low to intermediate SYNTAX scores. Treatment of bifurcation coronary lesions: The OPEN II is a prospective observational study evaluating the long-term safety and efficacy of the STENTYS stent. The Sideguard Coronary Sidebranch Registry(SGR1) is a prospective observational registry evaluating Bare Metal Sidebranch Stent. Bioabsorbable vessel scaffolds (BVS):PABLOS Prospective observational registry evaluating the use of Absorb BVS specifically in patients with bifurcation lesions. Adjunctive therapy trials: the duration of dual antiplatelet therapy (6 vs.12 months) is currently being investigated in the SECURITY study. STATIPLAT is a randomized trial evaluating the effect on platelet reactivity of the high dosage of Atorvastatin vs Rosuvastatin administrated before PCI. Structural Heart Disease/Transcatheter valve therapy: the DIRECT FLOW IP 010 is a prospective, multicenter, non-randomized clinical trial to determine the safety and performance of the Direct Flow Medical Percutaneous Aortic Valve 18F System for the treatment of severe aortic stenosis. The primary endpoint is freedom from all-cause mortality from procedure to 30 days. WIN TAVI is a prospective observational multinational study evaluating TAVI procedures specifically in women. Others: OneShot is a prospective observational study evaluating One Shot Ablation System for renal denervation in patient with refractory hypertension. EKOSONIC is a prospective observational registry evaluating safety and efficacy of loco regional trombolysis using Ekosonic catheter in patients with sub and massive pulmonary embolism. Antonio Colombo 95 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units Center for arrhythmia research The main research topic of our group is the improvement in the management of patients with complex arrhythmias. The following projects about VT management are currently in progress at our Institution: storm (Group B). The main objective of this second B Phase is to compare the 2-year rate of worsening Heart Failure hospitalizations, deaths, cardiac death, VT recurrences and Electrical Storm recurrences between the two groups. 1) Role of substrate modification aimed to the electroanatomic definition of areas with abnormal potential and their complete elimination 3) Radio-frequency ablation as primary management of well-tolerated sustained monomorphic ventricular tachycardia in patients with structural heart disease and left ventricular ejection fraction over 30% The inconsistent inducibility of the index VT at baseline programmed stimulation study may limit the value of programmed electrical stimulation. We investigated the ventricular tachycardia substrate in patients affected by myocarditis, Ischemic heart disease, Idiopathic Dilated Cardiomyopathy, Arrhythmogenic Right Ventricular Cardiomyopathy. 2) Identification of the appropriate timing for VT ablation in patients with structural heart disease. The PARTITA trial We planned a multicentric, controlled, singleblinded trial to evaluate if the timing of VT ablation significantly affects the patient’s prognosis. The PARTITA study will consist of a prospective data collection (Phase A) and a prospective randomized study period (Phase B). After the first appropriate ICD shock for VT, patient will enter the Phase B and they will be randomized to immediate VT ablation (Group A) or to ablation after the first arrhythmic Patients with well-tolerated sustained monomorphic ventricular tachycardia (SMVT) and left ventricular ejection fraction (LVEF) over 30% may benefit from a primary strategy of VT ablation without immediate need for a ‘back-up’ implantable cardioverter-defibrillator (ICD). This approach was evaluated in a multicenter study. 4) Bipolar radiofrequency ablation Unipolar radiofrequency ablation is known to produce superficial lesions; this approach may be ineffective in the treatment of patients with tachicardias originating from deep myocardial tissue. Bipolar radiofrequency ablation is supposed to produce more deep lesion and increase the percentage of transmural ablations. We will assess if bipolar ablation allows better clinical results compared to unipolar technique. Paolo Della Bella Echocardiography Unit The Echo Unit focuses on clinical imaging research: • accuracy of 3-Dimensional Echo for functional mitral valve anatomy in patients with mitral regurgitation, using standard anatomical surgical findings; • outcome research on 3D echo-guided surgical mitral valve repair; • Left ventricle fluid dynamics analysis; • clinical outcome research on surgical myectomy or percutaneous septal alcoholization for the treatment of Obstructive Hypertrophic Car- diomyopathy; • 3-Dimensional Echo in the setting of percutaneous valve therapy, including MitraClip Therapy and Transcatheter Aortic Valve Implantation; • Clinical Outcome research on MitraClip Therapy for functional mitral regurgitation in patients with congestive heart failure; • Tricuspid Valve Assessment using 3D Echo to predict the mechanism and late valve regurgitation after correction of left-sided valve disease; • Imaging support for technical innovation in the surgical department. Giovanni La Canna DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Figure 16. Left Ventricle Fluid dynamics analysis. Ischaemic heart disease, heart failure and echocardiography Unit Study of cellular and metabolic adaptation and evaulation of potential prognostic and therapeutic implications in heart failure 1. Investigations on the potential presence and the functional role of specific mitochondrial proteins in patients with chronic heart failure. More specifically, we intend to continue the performance of endomyocardial biopsies in patients with various degrees of cardiac dysfunction. The samples will be analyzed with a specific antibody against intramitochondrial ferritin. In presence of reduced contractile function, the heart could evidence increased expression of mitochondrial ferritin either as a protective effect or as a marker of intracellular iron storage. In both conditions, monitoring intramitochondrial ferritin and its correlation with cardiac function could be very useful in order to evaluate the role of cellular expression of this protein in both ischemic and non ischemic dilatative cardiomyopathy. 2. Additional studies we intend to continue in the years 2014-2015 are aimed at evaluating the effects of metabolic drugs on cardiac function and metabolism in patients with chronic heart failure. In facts, the observed gross metabolic derangements in patients with heart failure, are often increased by a state of insulin resistance and increased levels of xanthine-oxidase. These conditions worsen the metabolic and functional adaptation of the failing heart. Furthermore, we intend to evaluate the effects of ivabradine versus beta blockers in patients with heart failure and functional mitral regurgitation. The aim is to evaluate the efficacy in terms of reduction of the regurgitant fraction associated to improved left and right ventricular function, and reduced pulmonary artery pressure. 97 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units Aims: • Role of pulmonary ultrasound and non invasive determination of central venous pressure in the optimization of medical therapy in the clinical follow-up of patients with chronic heart failure. The primary end-point is the reduction of hospital admissions for worsening heart failure, while the secondary end-point is the reduction of mortality. More specifically, subgroups analysis will evaluate the adjunctive value in patients without clinical signs of congestion and negative heart failure biomarkers. • We will continue the research in the field of characterization and outcome of functional mitral regurgitation in patients with left ventricular dysfunction. • Diagnostic characterization and outcome of functional tricuspid regurgitation in patients with left ventricular dysfunction. • Finally, echocardiographic evaluation of atrial function and its significance in terms of clinical symptoms in patients with organic mitral valve regurgitation will be evaluated by strain analysis. Alberto Margonato Organ protection in critically ill patients, Advanced cardiac failure and mechanical supports Unit Organ failure in critically ill patients is associated to high morbidity and mortality. Our research focuses on the implementation of advanced mechanical supports to the cardiovascular and respiratory system and on the reduction of perioperative mortality. • ECMO (Extracorporeal Membrane Oxygenation), VAD (Ventricular Assist Device), artificial heart and other advanced mechanical and pharmacological supports of the circulation for the treatment of acute heart failure or for refractory hypoxia. We coordinate national networks performing mRCTs and we collaborate with international centers. • Non-invasive ventilation in critically ill patients even outside the intensive care unit including the development of new devices. • Protective intra-operative ventilation: participation in multicenter studies to clarify the role of mechanical ventilation settings during general anesthesia on post-operative pulmonary complications. • Alternative procoagulant and anticoagulants in the perioperative period with participation to mRCTs. • The role of volatile anesthetics in perioperative cardioprotection coordinating mRCTs. • Reduction of perioperative mortality and perioperative acute myocardial infarction: role of betablockers, inotropic agents, antiplatelet agents, and clonidine including the participation in international studies as Italian national coordinator center. • Prevention and treatment of acute renal failure and perioperative organ damage in critically ill patients, coordinating large mRCTs at the Italian level. • The role of metabolomics in the identification of different metabolic phenotypes associated with the development of specific outcomes. • Role of ultrasound in the study of diaphragmatic function. • Cardiac biomarkers (proBNP, cardiac troponin) and Renal biomarkers (ouabaine). • Sepsis in intensive care. • Intra and extra-hospital emergencies. • Anesthesiological management of the patient with a very high periprocedural risk. • Antibiotic therapy in intensive care. • Control of perioperative pain. • Coordination of an international network through innovative web-based consensus conferences; • Indexing on Pubmed of the international journal “Heart, Lung and Vessels”, freely available at www.heartlungandvessels.org With over 50 manuscripts published on the above described topics in indexed journals yearly we are the most prolific Italian group publishing in anesthesia and intensive care journals. Alberto Zangrillo DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Strategic research on heart failure Unit CORONARY MICROVASCULAR DYSFUNCTION Hypertrophic Cardiomyopathy (HCM) Pilot multicenter, double blind, randomized study to assess the effect of Ranolazine on functional capacity and diastolic function in patients (n=10) with HCM which has been completed. Systemic Sclerosis (SS) To identify prevalence of In patients with SS coronary microvascular dysfunction (CMD) is more prevalent in patients than in controls (10/19 vs 0/20; p Hypertensive Heart Disease 1. Clinical. Multicenter randomized study to evaluate the effect of the direct renin inhibitor aliskiren on CMD in hypertension as compared with ACEinhibitor enalapril. The primary endpoint of the study is the change in myocardial blood flow and flow reserve measured with PET (positron emission tomography). 2. Spontaneously hypertensive rat (SHR). To study effect of several drugs used in hypertension such as ACE inhibitor, beta blockers and calcium antagonist on microcirculation in SHR in addition to molecular characterization of several genes known to be involved in remodelling processes. Prospective validation of PET/computerized (CT) with 11C-PK11195 and contrast-enhanced ultrasound imaging of intraplaque neovascularization in asymptomatic patients with carotid atherosclerosis as a predictor of plaque vulnerability assessed by burden of cerebral ischemic lesions detected by repeated magnetic resonance (RM) studies. Number of patients enrolled: 52/60 Asymptomatic patients with carotid plaque; 9/20 symptomatic patients with carotid plaque. MYOCARDIAL ISCHEMIA AND LEFT VENTRICULAR DYSFUNCTION 1. Repetitive stunning and left ventricular dysfunction: effect of Ivabradine To evaluate the effect of ivabradine on post-ischemic stunning during exercise echocardiography in patients with known CAD. Number of patients enrolled: 13/15. 2. Ischemia-Reperfusion Injury: No Reflow Phenomenon Prospective study to compare no reflow diagnostic accuracy and prognostic stratification value of cardiac magnetic resonance versus coronary angiography in patients with ST-segment elevation acute myocardial infarction. Number of patients enrolled: 62. NON INVASIVE STRATEGY FOR DETECTION OF VULNERABLE ATHEROSCLEROTIC PLAQUES Paolo G. Camici Study and treatment of aortic disease Unit In 2013 the Vascular Surgery Unit of the IRCCS Ospedale San Raffaele has been involved in several clinical research studies regarding vascular pathology, in particular on the treatment of aortic disease, including: • RESTORE TRAUMA Sub-Study, focused on the endovascular treatment of traumatic injuries of the thoracic aorta (results published in Zipfel B, Chiesa R, Kahlberg A, et al. Ann Thorac Surg. 2014 Mar;97(3):774-80). • INNOVATION Trial: it is a multicenter, open label, prospective, non-randomized trial of INCRAFTT stent-graft in subjects with abdominal aortic aneurysms (results published in Coppi G, Njila M, … Chiesa R, et al. J Cardiovasc Surg (Torino). 2014 Feb;55(1):51-9.) • The Ovation Abdominal Stent Graft System Post-Market Study, an international study to evaluate the safety and performance of the TriVascular AAA Stent Graft System. • STABLE Trial: endovascular treatment of complicated type B aortic dissection with a new composite device design (results published in Lombardi JV, Cambria RP, …Chiesa R, et al. J Vasc Surg. 2014 Feb 19, in press.) • SAFROS: Patient Safety in Robotic Surgery. Aim of the study is to define patient safety metrics, to develop methods that abide by safety requirements and to demonstrate that a properly controlled robotic surgery can improve the level of patient’s safety. • Evaluation of the effect of renal perfusion with histidine-tryptophan-ketoglutarate hypothermic solution during thoracoabdominal aortic repair (results published in Tshomba Y, Kahlberg A, Melissano G,… Chiesa R. J Vasc Surg. 2014 99 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Clinical Research Units Mar;59(3):623-33) • Use of “GORE Hybrid Vascular Graft” for renal artery revascularization during thoracoabdominal aortic repair in order to reduce the renal ischemia time during surgery and the postoperative renal complications (results currently in press). • European Registry of Endovascular Aortic Repair Complications (EuREC 4), on aortoesophageal fistulae developing after thoracic aortic endovascular repair (results published in Czerny M, Eggebrecht H, … Chiesa R, et al. Eur J Cardiothorac Surg. 2014 Mar;45(3):4527. During 2013 the Vascular Surgery Unit has also published 14 papers on peer reviewed journals, regarding the treatment of aortic disease and other aspects of vascular pathology. Roberto Chiesa Vision first Unit Surgical retina Orbital Surgery CR: Characterization of myopic macular hole with posterior pole detachment after surgical closure with inverted flap technique. Vitreoretinal interface prognostic characterization in vitreomacular traction. BR: Dynamic Vessel Analyzer (DVA) of retinal vessels after macular vitreoretinal surgery. CR: New therapeutic strategies of ocular MALT lymphomas. Medical retina CR: DVA retinal vessel changes after Ozurdex/anti-VEGF treatment for Retinal Vein Occlusion and diabetic macular edema. DVA retinal vessel and EDI-OCT choroid characterization in reticular pseudodrusen. Influence of refractive status, caffein and wine intake on DVA. New therapeutic options and algorithms with intravitreal compounds for macular disorders. Applications of Drug Delivery Systems. Diagnosis and imaging of macular dystrophies BR: Retinal and choroidal structure in-vivo longlasting diabetes mouse model. Citicoline topical treatment. Chromogranin A-derived Vasostatin-1 on laser-induced choroidal neovascularization in the mouse. Ocular immunology and uveitis CR: New biologic agents for intraocular inflammation. Ocular Oncology CR: Gamma Knife Radiosurgery (GKR) treatment in uveal melanoma and intralesional injection of Rituximab in conjunctival lymphoma. Imaging CR: GCC and choroidal thickness in patients with diabetes and connective tissue disease with Reynaud phenomenon. DVA of retinal vessels in patients with Reynaud phenomenon. GCC thickness in AMD after anti-VEGF intravitreal injection. Choroidal thickness in treated posterior uveitis. Anterior segment surgery CR: Quality of life in low vision subjects implanted with a +8D multifocal lens. European population large scale database of human ocular biometry values. Intracameral anaesthetic and mydriatic solution for cataract surgery. Glaucoma CR: New medical and surgical therapeutic options in glaucoma. Neuro-ophthalmology CR: Surgical decompression and GKR on pituitary tumors compressing visual pathways. Brain damage and visual network plasticity in neurodegenerative diseases. Citicoline treatment and neurodegenerative disorders. Orbital RM alterations in patients with Graves disease. Ophthalmic artery, central retinal artery and vein, short posterior ciliary arteries Ecocolordoppler in LHON and DOA patients. Francesco Bandello DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Selected publications Bosi, E; Scavini, M; Ceriello, A; Cucinotta, D; Tiengo, A; Marino, R; Bonizzoni, E; Giorgino, F; on behalf of the PRISMA STUDY GROUP. Intensive structured self-monitoring of blood glucose and glycemic control in noninsulin-treated type 2 diabetes: the PRISMA randomized trial. Diabetes Care: 2013; 36(10): 2887-2894 - Article IF 2012: 7,735 Bosi, E; Bax, G; Scionti, L; Spallone, V; Tesfaye, S; Valensi, P; Ziegler, D; on behalf of the FREMS European Trial Study Group. Frequency-modulated electromagnetic neural stimulation (FREMS) as a treatment for symptomatic diabetic neuropathy: results from a double-blind, randomised, multicentre, long-term, placebo-controlled clinical trial. Diabetologia: 2013; 56(3): 467-475 - Article IF 2012: 6,487 Taramasso, M; Latib, A; Denti, P; Candreva, A; Buzzatti, N; Giannini, F; La Canna, G; Colombo, A; Alfieri, O; Maisano, F. Acute kidney injury following MitraClip implantation in high risk patients: Incidence, predictive factors and prognostic value. Int. J. Cardiol.: 2013; 169(2): e24-e25 Letter IF 2012: 5,509 Franzoni, I and Latib, A; Maisano, F; Costopoulos, C; Testa, L; Figini, F; Giannini, F; Basavarajaiah, S; Mussardo, M; Slavich, M; Taramasso, M; Cioni, M; Longoni, M; Ferrarello, S; Radinovic, A; Sala, S; Ajello, S; Sticchi, A; Giglio, M; Agricola, E; Chieffo, A; Montorfano, M; Alfieri, O; Colombo, A. Comparison of incidence and predictors of left bundle branch block after transcatheter aortic valve implantation using the corevalve versus the edwards valve. Am. J. Cardiol.: 2013; 112(4): 554-559 - Article IF 2012: 3,209 Piscopiello, M; Sessa, M; Anzalone, N; Castellano, R; Maisano, F; Ferrero, E; Chiesa, R; Alfieri, O; Comi, G; Ferrero, ME; Foglieni, C. P2X7 receptor is expressed in human vessels and might play a role in atherosclerosis. Int. J. Cardiol.: 2013; 168(3): 2863-2866 - Letter IF 2012: 5,509 Della Bella, P; Baratto, F; Tsiachris, D; Trevisi, N; Vergara, P; Bisceglia, C; Petracca, F; Carbucicchio, C; Benussi, S; Maisano, F; Alfieri, O; Pappalardo, F; Zangrillo, A; Maccabelli, G. Management of ventricular tachycardia in the setting of a dedicated unit for the treatment of complex ventricular arrhythmias: Long-term outcome after ablation. Circulation: 2013; 127(13): 1359-1368 Article IF 2012: 15,202 Godino, C; Lauretta, L; Pavon, AG; Mangieri, A; Viani, G; Chieffo, A; Galaverna, S; Latib, A; Montorfano, M; Cappelletti, A; Maisano, F; Alfieri, O; Margonato, A; Colombo, A. Heyde’s syndrome incidence and outcome in patients undergoing transcatheter aortic valve implantation. J. Am. Coll. Cardiol.: 2013; 61(6): 687-689 - Letter IF 2012: 14,086 Grimaldi, A; Figini, F; Maisano, F; Montorfano, M; Chieffo, A; Latib, A; Pappalardo, F; Spagnolo, P; Cioni, M; Vermi, AC; Ferrarello, S; Piraino, D; Cammalleri, V; Ammirati, E; Sacco, FM; Arendar, I; Collu, E; La Canna, G; Alfieri, O; Colombo, A. Clinical outcome and quality of life in octogenarians following transcatheter aortic valve implantation (TAVI) for symptomatic aortic stenosis. Int. J. Cardiol.: 2013; 168(1): 281-286 - Article IF 2012: 5,509 Chieffo, A and Buchanan, GL; Van Mieghem, NM; Tchetche, D; Dumonteil, N; Latib, A; Van Der Boon, RMA; Vahdat, O; Marcheix, B; Farah, B; Serruys, PW; Fajadet, J; Carrie, D; De Jaegere, PPT; Colombo, A. Transcatheter aortic valve implantation with the Edwards SAPIEN versus the medtronic corevalve revalving system devices: A multicenter collaborative study: The PRAGMATIC plus initiative (Pooled-RotterdAm-Milano-Toulouse in Collaboration). J. Am. Coll. Cardiol.: 2013; 61(8): 830836 - Article IF 2012: 14,086 Barile, L; Landoni, G; Pieri, M; Ruggeri, L; Maj, G; Nigro Neto, C; Pasin, L; Cabrini, L; Zangrillo, A. Cardiac index assessment by the pressure recording analytic method in critically ill unstable patients after cardiac surgery. J. Cardiothorac. Vasc. Anesth.: 2013; 27(6): 1108-1113 - Article IF 2012: 1,448 101 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Selected publications Godino, C; Bassanelli, G; Economou, FI; Takagi, K; Ancona, M; Galaverna, S; Mangieri, A; Magni, V; Latib, A; Chieffo, A; Carlino, M; Montorfano, M; Cappelletti, A; Margonato, A; Colombo, A. Predictors of cardiac death in patients with coronary chronic total occlusion not revascularized by PCI. Int. J. Cardiol.: 2013; 168(2): 1402-1409 - Article IF 2012: 5,509 Agricola, E and Slavich, M; Tufaro, V; Fisicaro, A; Oppizzi, M; Melissano, G; Bertoglio, L; Marone, E; Civilini, E; Margonato, A; Chiesa, R. Prevalence of thoracic ascending aortic aneurysm in adult patients with known abdominal aortic aneurysm: An echocardiographic study. Int. J. Cardiol.: 2013; 168(3): 3147-3148 - Letter IF 2012: 5,509 Fragasso, G; Rosano, G; Baek, SH; Sisakian, H; Di Napoli, P; Alberti, L; Calori, G; Kang, SM; Sahakyan, L; Sanosyan, A; Vitale, C; Marazzi, G; Margonato, A; Belardinelli, R. Effect of partial fatty acid oxidation inhibition with trimetazidine on mortality and morbidity in heart failure: Results from an international multicentre retrospective cohort study. Int. J. Cardiol.: 2013; 163(3): 320-325 - Article IF 2012: 5,509 Marrocco-Trischitta, MM; Cremona, G; Lucini, D; Natali-Sora, MG; Cursi, M; Cianflone, D; Pagani, M; Chiesa, R. Peripheral baroreflex and chemoreflex function after eversion carotid endarterectomy. J. Vasc. Surg.: 2013; 58(1): 136-144.e1 - Article IF 2012: 2,879 Bertoglio, L; Melissano, G; Civilini, E; Chiesa, R. Stent misalignment of the Zenith Dissection Endovascular System. J. Vasc. Surg.: 2013; 57(2): 515-517 - Article IF 2012: 2,879 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Coagulation and platelet biology Unit Complications of diabetes 103 DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Pediatric endocrinology research Diabetes and metabolic diseases in children and adolescents DIVISION OF METABOLIC AND CARDIOVASCULAR SCIENCES Neonatology Echocardiography Unit 105 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Director: Luigi Naldini* Associate Director: Fabio Ciceri Research Units Angiogenesis and tumor targeting Unit ––––––––––––––––––––––––––––––––––––––––––––––––– 113 HEAD OF UNIT: Luigi Naldini* POST-DOCTORAL FELLOW: Erika Zonari PHD STUDENT: Giulia Escobar** TECHNICIAN: Anna Ranghetti Functional genetics of muscle regeneration ––––––––––––––––––––––––––––––––––– 113 GROUP LEADER: Silvia Brunelli POST-DOCTORAL FELLOWS: Valentina Conti, Thierry Touvier, Paola Zordan PHD STUDENT: Mario Tirone Neural stem cell biology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 114 GROUP LEADER: Rossella Galli PHD STUDENTS: Katayoun Hemmesi**, Ashwin Narayanan Autoimmunity & vascular inflammation Unit ––––––––––––––––––––––––––––––––––––––––––– 114 HEAD OF UNIT: Angelo A. Manfredi* POST-DOCTORAL FELLOWS: Chiara Gualteroni, Norma Maugeri PHD STUDENT: Lucia Cottone** TECHNICIAN: Annalisa Capobianco Innate immunity and tissue remodelling –––––––––––––––––––––––––––––––––––––––– 115 GROUP LEADER: Patrizia Rovere-Querini* POST-DOCTORAL FELLOWS: Gianfranca Corna, Elena Rigamonti PHD STUDENT: Imma Caserta** TECHNICIANS: Antonella Monno, Clara Sciorati 107 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units/Clinical Research Units Experimental hematology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 116 HEAD OF UNIT: Chiara Bonini POST-DOCTORAL FELLOWS: Nicoletta Cieri, Sara Mastaglio, Maddalena Noviello PHD STUDENT: Giacomo Oliveira** TECHNICIANS: Barbara Camisa, Zulma Magnani Gene expression and muscular dystrophy Unit (Dulbecco Telethon Institute) –– 117 GROUP LEADER: Davide Gabellini, ERC Starting Grant POST-DOCTORAL FELLOWS: Mathivanan Jothi, Marie Victoire Neguembor PHD STUDENTS: Valentina Casà**, Claudia Huichalaf**, Valeria Runfola** FELLOW: Giulia Ferri TECHNICIANS: Roberta Caccia, Stefano Micheloni Leukemia immunotherapy Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 118 GROUP LEADER: Attilio Bondanza* POST-DOCTORAL FELLOW: Monica Casucci PHD STUDENTS: Benedetta Nicolis di Robilant**, Margherita Norelli FELLOWS: Laura Falcone, Fabiana Gullotta Molecular and functional immunogenetics Unit –––––––––––––––––––––––––––––––––––––– 118 HEAD OF UNIT: Fabio Ciceri (ad interim) POST-DOCTORAL FELLOWS: Gabriele Bucci, Cristina Toffalori, Luca Vago PHD STUDENT: Giacomo Oliveira FELLOW: Lara Crucitti TECHNICIAN: Laura Zito Clinical Research Units Hematology and hematopoietic stem cell transplantation Unit ––––––––––––––––––– 119 HEAD OF UNIT: Fabio Ciceri PHYSICIANS: Andrea Assanelli, Fabio Giglio, Maria Teresa Lupo Stanghellini, Sarah Marktel, Mara Morelli, Jacopo Peccatori RESIDENTS: Raffaella Greco**, Luca Vago** Immunohematology and transfusion medicine Unit –––––––––––––––––––––––––––––––––– 119 HEAD OF UNIT: Fabio Ciceri RESEARCHERS: Laura Bellio, Simona Malato, Oriana Perini, Michela Tassara FELLOW: Annika Calloni DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY HSR-TIGET The San Raffaele Telethon Institute for Gene Therapy Director: Luigi Naldini* Research Units Gene transfer technologies and new gene therapy strategies Unit –––––––––––––– 121 HEAD OF UNIT: Luigi Naldini*, ERC Advanced Grant PROJECT LEADERS: Bernhard Gentner, Anna Kajaste-Rudnitski, Angelo Lombardo POST-DOCTORAL FELLOWS: Alessio Cantore, Amy Patel** PHD STUDENTS: Angelo Amabile, Francesco Boccalatte**, Claudia Firrito, Pietro Genovese**, Alice Giustacchini**(until September 2013) FELLOW: Carolina Petrillo LAB MANAGERS: Mauro Biffi, Tiziano Di Tomaso TECHNICIANS: Sara Bartolaccini, Tiziana Plati, Lucia Sergi Sergi Gene/Neural stem cell therapy for lysosomal storage diseases –––––––––––– 121 GROUP LEADER: Angela Gritti POST-DOCTORAL FELLOWS: Chiara Cavazzin (until October 2013), Daniela Corno (until November 2013), Vasco Meneghini PHD STUDENTS: Giacomo Frati, Annalisa Lattanzi (until March 2013), Alessandra Ricca TECHNICIANS: Luigi Tiradani (since November 2013) Gene therapy for WASP/Omenn Unit –––––––––––––––––––––––––––––––––––––––––––––––––––– 122 HEAD OF UNIT: Anna Villa POST-DOCTORAL FELLOWS: Ileana Bortolomai, Marita Bosticardo, Maria Carmina Castiello PHD STUDENT: Lucia Sereni TECHNICIAN: Elena Draghici Gene transfer into stem cells Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––– 122 HEAD OF UNIT: Giuliana Ferrari* RESEARCHERS: Maria Rosa Lidonnici**, Francesca Salvatori PHD STUDENTS: Annamaria Aprile, Ylenia Paleari** TECHNICIANS: Giacomo Mandelli, Claudia Rossi, Francesca Tiboni 109 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units, HSR-TIGET Hematopoietic stem cell based gene therapy for lysosomal storage –––––––––––– 123 disorders Unit GROUP LEADER: Alessandra Biffi POST-DOCTORAL FELLOWS: Alessia Capotondo, Francesca Cecere, Martina Cesani, Ilaria Visigalli PHD STUDENTS: Stefania Delai, Rita Milazzo**, Silvia Ungari** FELLOWS: Eleonora Cavalca, Francesca Ferro, Daniela Redaelli TECHNICIAN: Serena Acquati Immunological tolerance Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 123 HEAD OF UNIT: Maria Grazia Roncarolo* POST-DOCTORAL FELLOW: Andrea Annoni PHD STUDENT: Mahzad Akbarpour TECHNICIANS: Grazia Andolfi, Eleonora Tresoldi (until September 2013) Genetic autoimmune diseases ––––––––––––––––––––––––––––––––––––––––––––––––––––– 124 GROUP LEADER: Rosa Bacchetta POST DOCTORAL FELLOWS: Laura Passerini, Francesca Santoni De Sio** PHD STUDENT: Eva Rossi Mel** RESIDENT: Federica Barzaghi** TECHNICIANS: Silvia Restelli , Claudia Sartirana Pathogenesis and therapy of ADA-SCID Unit –––––––––––––––––––––––––––––––––––––––––– 125 HEAD OF UNIT: Alessandro Aiuti POST-DOCTORAL FELLOWS: Luca Biasco, Immacolata Brigida, Aisha Vanessa Sauer, Samantha Scaramuzza PHD STUDENTS: Nicola Carriglio, Maddalena Migliavacca, Serena Scala** TECHNICIANS: Cristina Baricordi (until April 2013), Luca Basso Ricci, Francesca Dionisio, Stefania Giannelli, Raisa Jofra Hernandez, Michela Vezzoli (since October 2013) Safety of gene therapy and insertional mutagenesis Unit –––––––––––––––––––––––––– 126 GROUP LEADER: Eugenio Montini POST-DOCTORAL FELLOWS: Andrea Calabria, Daniela Cesana, Marco Ranzani PHD STUDENT: Monica Volpin** FELLOWS: Stefano Annunziato, Laura Rudilosso TECHNICIANS: Fabrizio Benedicenti, Pierangela Gallina, Erika Tenderini BIOINFORMATICIANS: Stefano Brasca, Giulio Spinozzi Tolerogenic dendritic cells Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 126 GROUP LEADER: Silvia Gregori PHD STUDENTS: Giada Amodio**, Michela Comi, Monica Gianolini, Grazia Locafaro TECHNICIANS: Fabio Russo, Daniela Tomasoni DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Clinical Research Units PCRU - Pediatric Clinical Research Unit –––––––––––––––––––––––––––––––––––––––––––––––– 127 HEAD OF UNIT: Alessandro Aiuti PROJECT LEADERS: Maria Grazia Roncarolo* and Alessandro Aiuti (ADA-SCID; WAS), Maria Sessa and Alessandra Biffi (MLD) PHYSICIAN: Maria Pia Cicalese RESIDENTS: Francesca Ferrua**, Marta Frittoli**, Mila Kalapurackal (since July 2013)**, Laura Lorioli**, Sara Napolitano**, Roberta Pajno** TCTO COORDINATOR: Luciano Callegaro REGULATORY AFFAIRS: Marco Bonopane, Antonio Martelli RESEARCH NURSES: Gigliola Antonioli, Miriam Casiraghi DATA MANAGERS: Laura Castagnaro (since November 2013), Sara Di Nunzio (until September 2013), Marcella Facchini (since November 2013), Emanuela Mrak (since October 2013) * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele 111 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Introduction by the Directors Mission and Vision - The mission of the Division of Regenerative Medicine, Stem Cells and Gene Therapy is to develop novel cell and gene therapy strategies for immuno-hematologic, metabolic, muscle and neuro-degenerative diseases and cancer. By bringing together researchers with complementary expertise, it enables effective synergy between projects that share common methodological approaches and face similar scientific/technological and regulatory hurdles. Organization - The Division comprises three major pillars. The first pillar is the San Raffaele-Telethon Institute for Gene Therapy (HSR-TIGET). This is a multi-disciplinary research Institute with an established leadership in the development of gene and cell therapy strategies and their application to the treatment of inherited immuno-hematologic and metabolic diseases. The second pillar embraces basic and translational research on genetic and cellular mechanisms of muscle development, repair and regeneration in response to inherited dystrophies, inflammatory and vascular disease. The third pillar is the clinical Hematopoietic Stem Cell (HSC) Transplantation Unit, which has one of the largest case records in EU, and the Experimental Hematology and Functional Immunogenetics Units, which have long been engaged in innovative immunotherapy approaches for hematologic malignancies. The governance is implemented by Luigi Naldini, Director, and Fabio Ciceri, co-Director. Goals - The Division hosts internationally recognized basic and translational research with the following goals: isolate, characterize and devise improved transplantation strategies for stem and progenitor cells from the hematopoietic, muscle and nervous system; unravel the genetic bases and molecular pathogenesis of immunodeficiencies and autoimmune diseases, lysosomal storage diseases and muscular dystrophies; develop new gene transfer and gene editing strategies for treating genetic diseases as well as for the immunotherapy of cancer based on genetic engineering of T-cell specificity against tumor-associated antigens or targeted progenitor based therapy; investigate the role of angiogenesis, inflammation and immune responses in modulating regeneration of diseased tissues, engraftment of transplanted cells, survival of gene-corrected cells and response of tumors to immunotherapy. The clinical units translate preclinical studies into innovative clinical trials and develop novel readouts to monitor disease progression and response to therapy. Main Achievements - Three new first-in-human phase I/II trials are active: two are among the first ever to exploit lentiviral vectors for HSC gene transfer and aim to the treatment of Metachromatic Leukodystrophy and Wiskott-Aldrich Syndrome; the third is the first cell therapy using mesangioblasts for Duchenne Muscular Dystrophy. All trials have treated several patients and report no serious adverse event related to the therapy. Major clinical benefits have been observed in all MLD and WAS patients together with substantial levels of stable long term genetic engineering of reconstituted hematopoiesis. Mesangioblast therapy has proven safe and able to reconstitute some dystrophin expression in vivo. A major alliance with GlaxoSmithKline is helping TIGET to translate its pioneering HSC-based gene therapies into novel medicines to be made available worldwide. In translational research, a surface signature was identified for CD4 type 1 T regulatory (Tr1) cells. It was also shown that FOXP3 gene transfer into CD4+ T cells convert them into T(reg) cells displaying stable phenotype and suppressive function. The use of the new surface markers and gene transfer strategies makes it feasible to purify regulatory T cells for cell therapy to induce or restore tolerance in subjects with immune-mediated diseases. New chimeric antigen receptors have been tested for genetically redirecting T-cell specificity against tumors and used in the context of innovative gene transfer platforms to broaden the reach of adoptive immunotherapy of cancer. A new insertional mutagenesis platform based on lentiviral vectors was developed to discover oncogenes and allowed identifying new genes associated with hepatocellular carcinoma in human cancer. Training opportunies - Graduate and Ph.D. programs, post-doctoral fellowships and clinical residency. DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units Angiogenesis and tumor targeting Unit Exploiting tumor-infiltrating monocyte/macrophages to fight tumors Tumor-infiltrating hematopoietic cells play important and sometimes contrasting biological roles within the tumor. We previously characterized, both in mice and humans, a subset of tumor associated monocyte/macrophages (TAMs) that express the Angiopoietin receptor TIE2 and are specifically recruited to tumors (Tie2-expressing monocytes, TEMs). Selective elimination of TEMs inhibits angiogenesis and tumor growth in several mouse tumor models. We thus hypothesized that genetic engineering of hematopoietic stem cells (HSC) with anti-tumor transgenes transcriptionally targeted to TEMs could provide a novel gene therapy strategy to treat tumors. Using a combination of transcriptional and microRNA-mediated control, we achieved selective expression of an interferon-α (IFN-α) transgene in the monocyte progeny of engineered HSC transplanted into mice. The regulated IFN-α transgene did not impair engraftment and long-term multilineage repopulation. By applying our strategy to mouse breast cancer models, we achieved inhibition of tumor progression and experimental metastases through enhanced generation of effector T cells and their recruitment to the neoplastic tissues. By forcing IFN-α expression in tumor-infiltrating macrophages, we blunted their innate pro-tumoral activity and reprogrammed the tumor microenvironment towards more effective dendritic cell activation and immune effector cell cytotoxicity. In a parallel study, we investigated the role of miR-155 in TAMs, as miR-155 upregulation in hematopoietic cells has been implicated in the deployment of effective innate and adaptive immunity. We stably knocked down (KD) miR-155 in the myeloid compartment of a mouse model of spontaneous breast carcinogenesis. Notably, miR-155/KD significantly accelerated tumor growth by impairing classical activation of TAMs. This created an imbalance towards a pro-tumoral microenvironment as evidenced by a lower proportion of CD11c+ TAMs, reduced expression of activation markers and skewing of immune cells within the tumor towards an M2/Th2 response. This study highlights the importance of tumor infiltrating hematopoietic cells in constraining carcinogenesis and establishes an anti-tumoral function of a prototypical oncomir (Zonari et al., Blood 2013). Overall, our studies illustrate the biological complexity of tumor infiltrating myeloid cells, some of which constrain while other promote carcinogenesis, and highlight new therapeutic strategies to manipulate these cells to treat tumors. Luigi Naldini Functional genetics of muscle regeneration Vascular progenitors fate and role during muscle development and regeneration Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is sustained by generation of new myofibers, Progenitors in the muscle comprise quiescent myogenic cells, the satellite cells, that are primary stem cells responsible for skeletal muscle regeneration after damage and vessel-associated progenitors, such as the mesoangioblasts (MAB). Using a genetic lineage tracing approach, a transgenic mouse expressing an inducible Cre under the control of an endothelial specific promoter we have provided new insights on the distinctive characteristics of the extraembryonic and embryonic hemogenic endothelium and we have identified for the first time the in vivo counterpart of embryonic MAB (Azzoni et al., Development, in press). We have also been studying the relationship between the innate immune system and muscle and vascular progenitors during acute and chronic muscle regeneration progenitors. We have shown that the Nitric Oxide donor molsidomine can modulate the characteristics of the macrophages that infiltrate the dystrophic muscle, enhancing their healing function and reducing fibrosis (Zordan et al, European Journal of Pharmacology 2013; Rovere-Querini et al, 2013, European Journal of Pharmacology 2013). We have also shown that macrophages are necessary for efficient vascular remodeling in the injured muscle. When phagocyte infiltration is compromised endothelial-derived progenitors undergo a significant endothelial to mesenchymal transition (EndoMT). This together with an inefficient tissue remodeling contributes to the accumulation of collagen, fat and significant fibrosis. Our findings provide new insights in En113 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units doMT in the adult skeletal muscle, and suggest that endothelial cells in the skeletal muscle may represent a new target for therapeutic intervention in fibrotic diseases (Zordan et al, Cell Death and Disease in press). Silvia Brunelli Neural stem cell biology Glioblastoma multiforme (GBM) is the most common and malignant brain tumor of adults. GBM comprise subpopulations of cancer stem cells (CSCs) that can generate experimental gliomas, characterized by an infiltrative growth pattern, typical of the human disease. By comparing the transcriptome of invasive GBM CSCs vs. non-invasive serum-grown glioma cell lines, we identified two candidate genes that actively promote GBM invasion and angiogenesis, in vivo. We are currently defining their role by modulating their expression through gain-of-function and loss-offunction strategies and interfering with the activation of molecular pathways involved in their function. Medulloblastoma (MB) arises from mutations in stem/progenitor cells located in restricted hindbrain territories. To identify novel molecular mediators involved in medulloblastomagenesis, we compared distinct types of postnatal hindbrainderived neural stem cells (NSCs) with compound Ptch/p53 mutant MB CSCs, which faithfully phenocopy the different variants of human MB in vivo. Transcriptome analysis of hindbrain NSCs and MB CSCs resulted in the generation of well-defined gene signatures, each reminiscent of a specific human MB molecular subclass (Corno et al., Cancer Discovery, 2012). From these signatures, we selected top-ranking genes and looked for miRNAs regulating all of them at the same time. We have now clearly defined the expression and function of one specific candidate miRNA, which acts as tumor suppressor miRNA in vivo by inhibiting the expression of an oncogene that was never implicated previously in medulloblastomagenesis. Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by hamartomatous neurological lesions that exhibit abnormal cell proliferation and differentiation. mTOR pathway hyperactivation by mutations in either Tsc1 or Tsc2 genes underlies TSC pathogenesis. We deleted Tsc1 in both embryonic neuroepithelial progenitors (NEPs) as well as radial glial cells (RGCs) and found that mutant mice faithfully recapitulated TSC neuropathological lesions (Magri et al., Cell Stem Cell, 2011; Magri et al., Disease Models & Mechanisms, 2013). We are currently generating additional mouse models of TSC by neural stem cells (NSC)-restricted inducible conditional mice. Rossella Galli Autoimmunity & vascular inflammation Unit Inflammation encompasses diverse events that are required to eradicate invading pathogens and to repair injured tissues. Despite its homeostatic function, unyielding inflammation shapes the clinical features and determines the persistence of most diseases of higher vertebrates. Unrelenting inflammation often depends on defects in the initiation and in the execution of cell death programs. In particular the clearance of cell debris and the activation of autophagic pathways appear as crucial players in the pathogenesis of systemic autoimmune and rheumatic diseases, in which they behave as initiators and amplifying circuits. Stem and precursor cells represent a preferential target of the autoimmune response, which in turn jeopardize their ability to regenerate injured tissues. Vascular inflammation fulfills homeostatic roles and humoral innate immunity finely tunes the cross-talk among circulating leukocytes, platelets and endothelial cells. We are investigating the molecular mechanisms underlying the vicious circle that maintains and amplifies vessel and tissue injury, with specific attention to the role of injuryassociated signals (Damage-Associated Molecular Patterns, DAMPS) and of acute phase proteins as environmental cues that transform regenerating cells in a trigger for self-sustaining autoimmune responses. Our recent work has allowed identifying a self-sustaining circuit by which the cross-talk between platelets and leukocytes in Systemic Sclerosis regulate the oxidation state and the inflammatory action of the prototypical DAMP, HMGB1, contributing to the generation of Neutrophil Extracellular Traps (NETs) and to the DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY persistence of vascular inflammation. Conversely, the ability of endogenous HMGB1 to recruit leukocytes and to modify the function of endothelial cells and pericytes contributes to various features of dermatomyositis and to sustain the growth and the spreading of neoplastic lesions within the peritoneal cavity. Angelo A. Manfredi Figure 17. HMGB1 as well as RAGE agonist induce the formation of neutrophil extracellular traps. Innate immunity and tissue remodelling The response of tissues to injury: macrophages are in control Macrophages play various roles in damaged tissues: they amplify local injury, either directly or indirectly, and they favor remodelling and repair. To break the molecular code by which macrophages shape the tissue response to microbial or sterile stressful events, which can be partially mimicked or tampered with, will provide novel strategies for the treatment of a variety of human diseases. We focus on the events that determine the action of infiltrating polarized macrophages in experimental models of acute and chronic injury of skeletal muscle (inflammatory myopathies and muscular dystrophies). Moreover, we are interested in the role of the innate immune responses in shaping the interaction between the mother and the developing embryo, with specific attention to pregnancy complications. During the last year we have in particular focused on the dissection of molecular events that are involved in the physiopathologic actions of macrophages in the remodeling tissue, specifically the regulation of the nitric oxide synthase expression and of the ability to provide iron to nascent myofibers. Moreover we have been involved in the dissection of the mechanisms by which low molecular weight heparin influences specific features of the innate immune responses in patients with high-risk pregnancies. Patrizia Rovere-Querini 115 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units MHC -IIA MHC -IIB MHC -I Figure 18. Extensorium Digitorum Longus (EDL) mouse muscle staining for the different Myosin Heavy Chain (MHC) isotypes. Type I (blue) are oxidative fibers, Type IIA (green) have both oxidative and glycolitic metabolism, while Type IIB (red) are glycolitic fibers. Experimental hematology Unit Cancer immunotherapy with genetically engineered T lymphocytes Cancer immunotherapy aims at harnessing the power and specificity of the immune system, well documented by its ability to combat infectious pathogens, to treat cancer. This approach has been limited, for several years, by a combination of biological and technological factors, including immune escape mechanisms adopted by tumors, the lower immunogenicity of cancer cells compared to infectious pathogens and pre-existing tolerance toward tumor-associated (TAA), but often not tumor-specific, antigens. We exploit gene transfer techniques to overcome these limitations and to generate long lasting tumor-reactive T lymphocytes to treat patients affected by hematological malignancies (Cieri Immunol. Rev. 2013). 1. Memory T cells with stem cell-like properties in health and disease. The ability to remember and respond more robustly in a second encounter with a pathogen is a critical property of the adaptive immune system. This process has been proposed to involve a stem cell-like memory T-cell subset (TSCM), able to rapidly differentiate in effectors and self-renew upon antigen re-encounter. We identified conditions to genetically modify and expand long-living TSCM with the ability to self renew and the plasticity to differentiate into potent effectors. (Cieri et al., Blood 2013). 2. Suicide gene therapy in allogeneic stem cell transplantation. The transfer of a suicide gene into donor lymphocytes promotes rapid and effective immune reconstitution and control of GvHD. Building on clinical results of suicide gene therapy trials, we are currently designing innovative approaches of T cell manipulation designed to potentiate the graftversus-leukemia effect of donor lymphocytes (Casucci et al., Mol. Ther. 2013). 3. TCR gene editing To completely and permanently substitute T cell specificity, we developed the TCR gene editing approach based on: i. Somatic knockout of the endogenous TCR genes (by transient exposure to α and/or β chain specific Zinc Finger NucleasesZFN), and ii. Introduction of a tumor-specific TCR by lentiviral vectors. This approach, was validated in vitro and in vivo with different TCRs specific for antigens expressed by acute leukemias and multiple myelomas (Provasi, Genovese, et al., Nat. Med 2012). Chiara Bonini DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Gene expression and muscular dystrophy Unit The Gene Expression and Muscular Dystrophy Unit is interested in understanding the regulation of muscle-specific gene expression. We are studying this in normal physiology and in disease using FSHD muscular dystrophy as a paradigm. FSHD is the second most common muscular dystrophy in adults. It is an autosomal dominant disorder that is not due to a mutation within a protein-coding gene. Instead, FSHD patients carry deletions of 3.3 kilobase macrosatellite repeats, termed D4Z4, located at chromosome 4q35. During 2013, we investigated the biology and mechanism of action of the FSHD candidate gene FRG1. We showed that FRG1 is selectively overexpressed in FSHD patients. Accordingly, FRG1 transgenic mice develop a muscular dystrophy with physiological, histological, ultra structural and molecular features analogous to FSHD patients. We have also shown that the transcriptome of FRG1 mice is remarkably similar to that of FSHD patients, further supporting a role for FRG1 in the disease and validating our animal model. We showed that FRG1 primarily inhibits the activation, clonogenic and differentiation of myogenic stem cells. In classical muscular dystrophies, myogenic stem cells are affected late in the disease as a secondary effect of muscle wasting. On the contrary, our data suggest that defects in myogenic stem cells could be at the basis of FSHD. We have shown that FRG1 operates through two molecular mechanisms: reduced expression of the splicing factor Rbfox1 and interference with the activity of the epigenetic repressor Suv420h1. Among extra-muscular manifestations of FSHD are mental retardation, epilepsy and autism. Interestingly, RBFOX1 mutation or downregulation is also associated to the same symptoms identifying a potential mechanism for the neurological involvement in FSHD. Collectively, our results provided a long awaited molecular explanation for the muscle differentiation defects that have been frequently reported in FSHD and identified molecular pathways and FRG1 targets altered in FSHD that can be exploited for therapeutic purposes. Davide Gabellini Figure 19. Transplantation of muscle stem cells during muscle regeneration in mice. Laminin (red), Hoechst (blue) and transplanted muscle stem cells (green) staining of tibialis anterior cryosections, 10 days after muscle injury. 117 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units Leukemia immunotherapy Unit The Leukemia Immunotherapy Group led by Dr. A. Bondanza is actively engaged in the development of novel gene therapy strategies that can be readily translated to the clinic for the treatment of hematological malignancies and, possibly, solid tumors. The main focus is on chimeric antigen receptor (CAR)-modified T cells, i.e. T cells modified with a synthetic receptor that combines the antigenbinding moiety of a monoclonal antibody with the signal transduction machinery of a T cell. In this endeavor, the Group takes advantage of cuttingedge gene transfer technologies available within the Division and innovative humanized mouse models originally developed over the last few years (Bondanza et al, Blood 2006 & 2011, Kaneko et al, Blood 2009). During 2013, a CAR specific for the tumor-initiation antigen CD44v6 has been developed and preclinically validated for the treatment of acute myeloid leukemia and multiple myeloma (Casucci et al, Blood 2013). Moreover, different novel CAR targets have been identified. In parallel, the Group researches for elucidating the anti-inflammatory and regenerative properties of mesenchymal stromal cells from different sources (bone marrow, adipose tissue, cord blood) with the final aim of identifying critical factors and over-expressing them by genetic means for the treatment of human diseases (graftversus-host disease, autoimmunity). Finally, the Group is engaged in a wide European project (TRANSCAN Haploimmune) on the validation of immune biomarkers predictive of clinical outcome after allogeneic hematopoietic stem cell transplantation for leukemia. Attilio Bondanza Molecular and functional immunogenetics Unit The scientific activity of this Unit is aimed at characterizing the biological bases of the interplay between immune system and leukemia in the context of allogeneic hematopoietic stem cell transplantation (HSCT). During the year 2013, the following main achievements were made: • we retrospectively evaluated 233 transplants to determine the incidence, risk factors and clinical outcome of leukemia relapses with genomic loss of the mismatched HLA. HLA loss variants accounted for 33% of the relapses after family donor HSCT and were significantly associated with disease aggressiveness and clinical manifestations of graft-versus-host alloreactivity, further suggesting their relevance as a mechanism of immune evasion and warranting their routine screening after transplant (Crucitti et al., submitted); • we developed an innovative diagnostic tool based on quantitative PCR (qPCR) for the early detection of HLA loss relapses, and validated it in our translpant series (Vago et al., in preparation); • by using an innovative system, we dissected the molecular bases of T cell recognition of HLA-DPB1 alleles, demonstrating the relative impact of individual amino acids on alloreactivity patterns, and by this defining the “functional distance” amongst HLA-DPB1 alleles, a promising tool to evaluate the permissiveness of HSCT mismatches (Crivello et al., submitted); • by comparing the mRNA expression profile of patients’ leukemic cells harvested at diagnosis and at relapse after allogeneic HSCT we demonstrated in the latter the selective deregulation of immune-related processes, comprising antigen processing and presentation and T cell costimulation, and functionally validated that these mechanisms concur in prompting leukemia immune evasion from donor-derived immunity (Toffalori et al. in preparation); • by combining immunogenetic and next-generation sequencing analyses, we deciphered the complex disease history of a patient with leukemia experiencing subsequent relapses upon serial treatments. We documented radical changes in the disease genetic features at each presentation, selectively evolving to escape from therapies, and throughout these changes identified leukemia founder mutations, which were tracked back to pre-leukemic precursors (Vago et al., in preparation). Fabio Ciceri DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Clinical Research Units Hematology and hematopoietic stem cell transplantation Unit The Unit is developing transplantation from partially HLA-incompatible (haploidentical) family members to offer a cure to all patients affected by high-risk hematologic malignancies who are candidate to allogeneic Hematopoietic Stem Cell Transplantation. Over the last years, we developed both T-cell depleted and T-cell replete platforms for haploSCT. Extensive application of T-cell depletion is limited by high rate of late transplant related mortality (TRM) and relapse associated with the inadequate immune reconstitution (IR) due to ex vivo T cell depletion or in vivo post-transplant cyclophosphamide administration for severe graft-vs-host disease (GvHD) prevention. We extensively developed HSV-TK+ suicide gene donor lymphocytes after haploidentical transplantation (haplo-HSCT). A phase III trial (TK008, NCT00914628) is ongoing in 8 centres in Europe and US and preliminary results confirm safety and potential benefit in improving survival of the T-cell gene transfer technology integrated with T-cell depleted haplo-HSCT We developed unmanipulated peripheral blood stem cells haploSCT by exploring a calcineurin inhibitor-free rapamycin-based GvHD prophylaxis regimen (TrRaMM study, Eudract 2007-5477-54). We showed that rapamycin allowed the preferential expansion of donor-derived natural T regulatory cell (Tregs), ultimately protecting from the severe GvHD that would have been expected in this context. Building on this promising experience, we are currently testing several new rapamycin-based regimens to improve the efficacy and feasibility of haploidentical HSCT. In more than fifty patients with high-risk malignancies we combined a treosulfan- and fludarabinebased chemotherapeutic regimen to low-dose Total Body Irradiation (4Gy TBI) modulating in vivo T cell depletion with ATG-Fresenius on the basis of donor-recipient matching and underlying disease risk (TrRaMM4Gy study, Eudract 2011-001534-42). Specific effort is dedicated to the biological characterization of immune reconstitution, comprising the detailed analysis of the early and late immune events. Cell therapy of DMD by intra-arterial delivery of HLA-identical allogeneic mesoangioblasts (MABs) Five pediatric patients were treated (age: 8-12 years) at our Institution. Fabio Ciceri Immunohematology and transfusion medicine Unit The Hospital San Raffaele (OSR) Immunohematology and Transfusion Medicine Service (ITMS) provides clinical services to support OSR patients in need of blood component therapy, cellular therapy, therapeutic apheresis, and specialized laboratory diagnostics. This ISO 9002 Certified unit collects and prepares the blood components and cellular therapy products used in patient care at the OSR, maintain an accredited Immunohematology Reference Lab and provides education in the field of Transfusion Medicine. ITMS is conducting a project, funded by Regione Lombardia, on the appropriateness of blood transfusion. The ITMS is subdivided into two distinct subunits, each responsible for a particular process: Blood Donation Center The blood donation center subunit is responsible for the collection and testing of blood to be transfused into patients at the San Raffaele Hospital. It is responsible for handling all aspects of donor recruitment for whole blood products, apheresis products, the auto-transfusion program and therapeutic phlebotomy. During 2013 were presented to make a donation 8424 candidates of these only 6820 have donated. 5535 were donations of whole blood and the rest were donations by apheresis, plateletpheresis or plasmapheresis. Therapeutic Apheresis and Cellular Therapy The Therapeutic Apheresis and Cellular Therapy subunit is responsible for collecting and processing hematopoietic stem cells and performs the necessary diagnostic testing required for bone marrow transplantation procedures. The lab supplies hematopoietic stem cells and relevant cells in support of clinical trials for allogenic bone marrow and peripheral blood transplantation and offers a Stem Cell Cryo Banking service for autologous and allogeneic bone marrow products. Bone Marrow from qualified donors is collected in accordance and recognition of the Italian Bone Marrow Donor Registry (IBMDR). Therapeutic apheresis procedures to treat patients with neurologic and blood diseases, including photopheresis, red cell and plasma exchange procedures are also performed routinely. The unit of therapeutic apheresis is part of the bone marrow transplants program and underwent Jacie Accreditation Inspection in October 2013. During 2013, were performed 212 apheresis for the collection HSCT. Fabio Ciceri 119 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Introduction by the Director HSR-TIGET The San Raffaele Telethon Institute for Gene Therapy The San Raffaele-Telethon Institute for Gene Therapy (HSR-TIGET) is a joint-venture between the San Raffaele Scientific Institute and the Telethon Foundation established to perform research on gene transfer and cell transplantation and translate its results into clinical application of gene and cell therapy for genetic diseases. The research projects aim to: a) identify the genetic bases and pathophysiology of primary immunodeficiencies and autoimmune disorders, leukodystrophies and other lysosomal storage disorders and thalassemia; b) develop new gene transfer and editing technologies for efficient and safe genetic correction of disease ex vivo and in vivo; c) characterize the biological properties of stem cells and establish procedures for their isolation, genetic modification and transplantation; d) investigate cell types mediating innate and adaptive immunity and modulate the immune response to gene and cell products to improve efficacy and stability of the therapy. The gene and cell therapy strategies being developed are then tested in pre-clinical disease models under GLP conditions within the TIGET “Centro di Saggio” in preparation for clinical trials. The clinical studies are conducted within the TIGET Clinical Research Unit, which is devoted to the diagnosis, treatment and follow-up of patients with primary immunodeficiencies, hematologic and metabolic disorders, in close collaboration with the Pediatric Immunohematology Unit, the Bone Marrow Transplantation Unit, and the GMP Facility of MolMed SpA. TIGET pioneering gene therapy of a severe form of primary immunodeficiency (ADA-SCID) has provided the most successful demonstration today that gene transfer into human hematopoietic stem cells (HSC) can safely result in long-term disease correction. This therapy is now undergoing development for drug registration by GlaxoSmithKline (GSK) and is expected to become the first ex vivo gene therapy product on the market. We also made essential contributions to the development of lentiviral vectors, which have become one of the most widely used tool in biomedical research today. Since 2010 TIGET has been conducting two first-in-human trials of lentiviral vector mediated HSC gene therapy for Metachromatic Leukodystrophy and Wiskott-Aldrich syndrome. Several patients have already been safely treated in each trial and continue to show stable and remarkably high levels of hematopoietic reconstitution with gene corrected cells and evidence of major therapeutic benefit. The molecular and clinical results observed in the first 3 patients treated in both trials were reported in 2 back to back full research articles in the Science magazine on August 2013 and were deeply covered by the scientific and lay press and news agencies worldwide. At the end of 2013, GSK exercised its rights of option on these two other TIGET gene therapies and has since embarked in their development towards drug registration. In parallel, we are investigating the pathophysiological mechanisms leading to immune dysfunction and autoimmunity in primary immunodeficiencies, or underlying disease correction in leukodystrophies, such as microglia turnover, enzyme overexpression and biodistribution to the CNS. In these neurodegenerative diseases, we are also investigating the potential of intra-CNS gene delivery and neural stem cell transplantation. We continue to improve vector design by limiting the impact on cellular transcription and exploiting microRNA regulation to stringently control transgene expression. Improvements are validated by sensitive in vivo models to score genotoxicity and high-throughput vector integration site analyses to monitor clonal behaviour of vector-transduced cells in experimental models and in the clinical trial patients. HSC biology is investigated to identify novel regulators of cell growth and quiescence, such as microRNAs, that help designing better strategies for ex vivo expansion, genetic modification and transplantation. Meanwhile, new gene targeting and editing approaches are developed based on artificial endonucleases, such as Zinc Finger Nucleases, TALENs and Crispr/Cas9 RNA-guided nucleases, which brings the possibility of targeted integration, gene editing and correction of mutations within the reach of gene therapy. The potential of in vivo gene delivery is investigated to treat hemophilia, exploiting a lentiviral platform stringently targeted to hepatocytes by transcriptional and microRNA-regulated control. By using this strategy, we could overcome the immunological barrier to in vivo gene transfer and establish long-term correction of hemophilia in small and large animal models and induce active tolerance to the transgene products. In parallel, significant advances were made in the field of immunological tolerance mediated by regulatory T cells and the potential application of these cells as cell therapy of immune-mediated diseases. DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units, HSR-TIGET Gene transfer technologies and new gene therapy strategies Unit Our research has long been investigating new strategies to improve the therapeutic reach, efficiency and safety of gene transfer. Over the years we have developed a versatile gene transfer platform based on lentiviral vectors (LV), which has become one of the most widely used tool in biomedical research and has recently entered clinical testing in T-cell and Hematopoietic Stem Cells (HSC)-based gene therapy. In 2013, we reported the results of two first-in-human clinical trials of LV-mediated HSC gene therapy undergoing in our Institute for the treatment of two rare inherited diseases, metachromatic Leukodystrophy (MLD) and Wiskott-Aldrich syndrome (WAS). All treated patients showed stable and high levels of polyclonal gene marking and therapeutic gene expression in the reconstituted hematopoiesis with remarkable therapeutic benefit. Data from both trials support the feasibility and safety of extensive genetic engineering of human hematopoiesis, essentially producing a stable “transgenic” hematopoietic system in humans (Biffi et al., Science, 2013; Aiuti et al., Science 2013). These encouraging results offer the perspective of a treatment for MLD and WAS and open the avenue to develop new therapeutic strategies for other diseases. LV, however, integrate semi-randomly in the genome, which leads to variegated transgene expression and is associated with insertional mutagenesis, the risk of which has been substantially alleviated by improved vector design but cannot be fully abrogated. We have shown that artificial nucleases, such as engineered Zinc Finger Nucleases (ZFN) can be used to edit specific gene sequences. We are exploiting this strategy for targeted integration of transgenes at preselected and safe genomic sites and for in situ correction of inherited mutations in human cells, which allows restoring both gene function and physiological expression. Concerning transgene expression control, we have shown that endogenous microRNA can be exploited to stringently regulate transgene expression and target it to desired cell types (Amendola et al, Mol Ther 2013). We are exploiting miR-126 to improve the safety and efficacy of HSC gene therapy. By making LV responsive to miR-126 we can suppress transgene expression in HSC, sparing them from potential toxicity, and target it to selected lineages of differentiated progeny. MiR-regulated LV are also being exploited for liver gene transfer. Using miR142-regulated hepatocyte-targeted LV we have established long-term Factor IX expression in mouse and dog models of hemophilia B. We now showed that this strategy can eradicate preexisting anti-FIX antibodies and inhibitors in hemophilia B mice and induce active tolerance in an already primed immune system (Annoni*, Cantore* et al., EMBO Mol Med 2013). These data position gene therapy as an attractive treatment option also for inhibitors-positive hemophilic patients. Luigi Naldini Gene/Neural stem cell therapy for lysosomal storage diseases Leukodystrophies are genetic neurodegenerative pediatric disorders that affect the CNS, PNS and visceral organs. Gene/cell therapy strategies are promising approaches for the treatment of the CNS pathology in leukodystrophies, as they have the potential to provide a permanent source of the deficient enzyme. Our previous studies demonstrated that neonatal intracerebral delivery of a therapeutic vector, intracerebral transplantation of neural stem cells (NSCT) and hematopoietic cell transplantation (HCT) are safe and effective as independent treatments in preventing/delaying pathology in relevant mouse models of leukodystrophies. Interestingly, we have substantial evidence that a combinatorial strategy based on NSC gene therapy (GT) or intracerebral GT coupled to HSC transplant, is safe and effective in providing relevant and timely enzymatic levels in all the affected organs, providing benefits when compared to single treatments in terms of amelioration of pathology and enhancement of survival. In order to scale up these approaches to larger brains, we have tested the feasibility and tolerability of the intracerebral GT platform in juvenile non-human primates (NHP). Our preliminary data show robust biodistribution of vector and transgene in animals injected with a LV.ARSA. We are now addressing potential safety concerns. Finally, a better knowledge of the pathogenesis of LSD is instrumental to develop 121 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units, HSR-TIGET effective therapies. We have established a collection of induced pluripotent stem cells (iPSCs) derived from the somatic reprogramming of normal donor and patients affected by Metachromatic and Globod Leukodystrophy (MLD, GLD) that we have characterized for their bona fide pluripotency. We have optimized a protocol to obtain iPSCderived neural stem/progenitor cells to be used as a renewable source of patient-derived neural cells that will be further differentiated in cultures enriched in oligodendrocytes and mature neurons. These cultures will offer an unprecedented opportunity to recapitulate the LSD CNS pathology in vitro and to test gene/cell replacement strategies. Angela Gritti Gene therapy for WASP/Omenn Unit Wiskott-Aldrich Syndrome protein deficiency perturbs the homeostasis of B-cell compartment in humans Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21−/low B cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients. Anna Villa Gene transfer into stem cells Unit Preclinical evaluation of safety and efficacy of gene therapy for β-thalassemia: the first GLP toxicology study for an advanced therapy -thalassemia is a severe congenital anemia caused by deficiency of β-globin production. At present, the only curative approach is represented by allogeneic hematopoietic stem cell transplantation (HSCT), which, however, is limited by HLA compatibility and toxicity. Gene therapy for β-thalassemia is based on the autologous transplantation of hematopoietic stem cells (HSPCs) engineered to express the human β-globin gene and lentiviral vectors are the vectors of choice. Starting from the demonstration of proof of efficacy in thalassemic mice and in hematopoietic cells harvested by thalassemic patients, we moved towards the clinical development by assessing the risk/benefit, in comprehensive in vivo pre-clinical studies, testing toxicology and tumorigenicity of genetically modified murine HSPCs and biodistribution of transduced human CD34+ progenitors following transplantation in immunodeficient mice. Evaluating the biosafety of gene therapy medicinal products under regulatory compliance, utilizing GLPs (good laboratory practices), provides results of scientific significance within regulatory standards, and paves the way towards future market registration. We developed a GLP Test Facility according to OECD Principles on GLP, combining the different expertise of personnel trained in research, R&D, pathology and safety assessment, and quality assurance. Several laboratories dedicated to GLP activities were appropriately equipped, documentation required for quality DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY management, GLP procedures and analytical methods were assembled and all personnel involved were specifically trained. Instruments were qualified and most of the analytical procedures were developed and validated according to EMA, CBER/FDA and ICH guidelines. In this context, we treated by gene therapy thalassemic mice, using cell dose and vector copy number/cell superior to that expected in human cells. Mice have been monitored for 12 months and at termination full necropsy and histopathology examination were performed along with clinical chemistry, hematological evaluation and integration site analysis. Overall, the final results show absence of increased tumorigenicity and genotoxicity associated to gene therapy and represent the proof of safety and efficacy for the clinical trial. Giuliana Ferrari Hematopoietic stem cell based gene therapy for the treatment of lysosomal storage disorders Unit In most Lysosomal Storage Disorders (LSD) hematopoietic stem cell (HSC) transplantation is not or poorly effective. HSC gene therapy could ameliorate the outcome of allogeneic transplant and provide an expectation of efficacious treatment for these LSD. HSC can be genetically modified to express supra-normal levels of the therapeutic enzyme, and become a quantitatively more effective source of functional enzyme than normal donor’s cells. Moreover, autologous HSC are immediately available, thus saving precious time in rapidly progressing forms, and can significantly reduce transplant-related morbidity and mortality. We are thus implementing an innovative approach based on the transplantation of autologous, gene corrected HSC for the treatment of severe LSD lacking efficacious and safe therapeutic opportunities. To this goal, we exploit the features of lentiviral vectors (LV). By using LV for HSC gene correction, we proved the therapeutic potential of HSC gene therapy in the murine model of three different LSDs. In the case of metachromatic leukodystrophy (MLD), a severe dysmyelinating LSD, preclinical research led to Phase I/II clinical testing. Indeed, a clinical trial of HSC gene therapy for MLD is currently open to patient’s recruitment and thus far 9 patients have been enrolled and treated. Evidence of tolerability and safety of the proposed approach, as well as of therapeutic efficacy in the treated patients have been obtained. The same approach has been applied with success to the murine models of type I Mucopolysaccharidosis (MPS I), a LSD characterized by visceral organ, skeleton and nervous system involvement, and of globoid leukodystrophy (GLD), a demyelinating LSD similar to MLD. A clinical development plan for these two diseases has been started with a Phase I/II trial of HSC gene therapy for MPSI patients planned for late 2014-early 2015. We are also experimentally addressing the critical need of enhancing brain microglia turnover with donor cells following HCT in order to anticipate the time of clinical benefit and improve the efficacy of the transplant procedure. This work thus far provided hints for designing novel and less invasive approaches for treating LSDs having a prevalent or exclusive CNS involvement. Alessandra Biffi Immunological tolerance Unit The goal of our Unit is to investigate the mechanisms that promote tolerance, and to identify novel therapies to prevent/cure immune-mediated diseases. We focus on the role of T regulatory cells (Tregs); specifically on Tr1 and FOXP3+ Tregs. The specific aims of our research are: 1) To define Tr1 cell-gene signature; 2) To develop a platform to generate Ag-specific Tr1 cells using lentiviral vectors (LVs) encoding for human IL-10 (LV-hIL-10); 3) To test the efficacy of IL-10engineered T cells in humanized mouse models; 4) To investigate the mechanisms underlying tolerance induced by in vivo delivery of miRNA-regulated LVs; 5) To evaluate the efficacy of the miRNA-regulated LV platform for prevention or treatment of autoimmune diseases. Achievements: 1) Defined the Tr1-specific gene signature; 2) Demonstrated that enforced expression of hIL-10 by LVs confers a Tr1 phenotype and function to human CD4+ (CD4IL-10) T cells; 3) Showed that CD4IL-10 cells suppress T-cell responses in vitro and in vivo; 4) Induced Ag-spe123 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units, HSR-TIGET cific FOXP3+ Tregs in vivo by injection of LVs that target Ag expression to hepatocytes by combining transcriptional (ET promoter), and post-transcriptional (miR142-target sequences (142T)) gene regulation; 5) Determined that Ag-specific FOXP3+ Treg-induction is TGF-β-mediated; 6) Demonstrated that a single injection of LV.ET.142T encoding for Insulin-B9-23 prevents diabetes development in NOD mice. Conclusions and future plans: 1) The identification of Tr1-specific biomarkers is a major step forward to understand the biology and to monitor these cells in vivo. The Tr1-specific gene signature will be investigated using different Tr1 cell sources and functional studies will be performed to delineate the role of the identified genes; 2 and 3) The ability to generate large numbers of human CD4IL-10 cells with regulatory properties open the possibility to use these cells for cellular therapy. The potential of LV-hIL-10 to convert Ag-specific effector T cells into Tr1 cells will be investigated; 4 and 5) The LV.ET.142T platform can be used to prevent immune responses and induce tolerance to transgenes in gene therapy or to selfAgs in autoimmune diseases. We will investigate the efficacy of this platform in blocking ongoing autoimmune diseases. Maria Grazia Roncarolo Genetic autoimmune diseases Novel insights into the pathogenesis and diagnosis of immune deficiencies with autoimmunity: towards innovative therapeutic strategies In the past years, our studies on Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome have significantly advanced the understanding of the physiological role of FOXP3 in human regulatory T cells (Treg) and effector T cells, and provided insights in the diagnosis and pathogenesis of this devastating genetic autoimmune disease. More recently, our research focused on defining new therapeutic approaches for IPEX patients, as alternative to hematopoietic stem cell (HSC) transplantation, which is not always feasible. We showed that, upon lentivirus-mediated FOXP3 gene transfer CD4+ T cells are converted into Treg. The resulting CD4FOXP3 T cell population displays stable phenotype and suppressive function, especially when naïve T cells are converted. CD4FOXP3 T cells are stable in inflammatory conditions not only in vitro, but also in vivo in a model of xenogeneic graft-versus-host-disease. IPEX-derived CD4FOXP3 T cells mirrored Treg cells from healthy donors in terms of cellular markers, anergic phenotype, cytokine production, and suppressive function. These findings suggest the feasibility of adoptive cell therapy with genetically engineered Treg cells for the treatment of IPEX patients. With the ultimate goal of replacing the mutated gene into autologous HSC, we are currently studying the effect of FOXP3 overexpression or knock-down in HSC in vitro and in vivo in humanized-mice. This will allow us to track FOXP3 in shaping human (effector and regulatory) T cell development and better define the best gene transfer strategy to be adopted to restore immune tolerance and cure the disease. To get a complete picture, we are also assessing the contribution of epigenetic regulators in controlling human T cell development. In parallel, we are extending our studies to a wider cohort of patients with early onset genetic autoimmunity, with or without enteropathy, and several of them also affected by B cell dysfunction. We are applying a system biology approach, combining genomic analysis with immuno-functional studies to identify the causative genes and altered pathways in these diseases. Altogether, results from these research projects will be instrumental for the definition of new targeted therapeutic intervention to re-establish immune tolerance. Rosa Bacchetta DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Pathogenesis and therapy of ADA-SCID Unit Genetic defects in adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID). Our studies investigate the immune alterations and onset of autoimmunity in ADA-SCID. We also study vector integrations (IS) in patients treated with hematopoietic stem cell (HSC) gene therapy (GT) to understand the dynamics of genetically modified cells. In addition, we develop new therapeutic strategies using lentiviral vectors for ADA-SCID and other immunodeficiencies. Since B-cell tolerance defects contribute to autoimmunity in this disease, we compared the efficacy of various treatments on B-cell development and function. HSC-GT with a retroviral vector carrying the ADA cDNA reverted the block in bone marrow development and progressively increased B-cell numbers. Gene-corrected B cells displayed a strong selective advantage after egress into the periphery (Figure 20A-B), leading to restored B-cell proliferative responses and antibody secretion. Levels of correction correlated with the percentage of vector-positive cells (Figure 20C), indicating an advantage for endogenous ADA expression in B cells to recover B-cell development and function. Increased gene transfer efficiency through a lentivirus-based platform might further accelerate B-cell reconstitution and reduce the risk of autoimmunity. To evaluate the long-term activity of transplanted HSC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients. Identical IS were consistently detected at multiple lineages level for up to 6 years after GT. By semiquantitative PCR on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSC without phases of clonal quiescence. Since a gammaretroviral vector was used in this trial, which is able to transduce only actively replicating cells, we provided the first evidence that in vitro activated HSC, “awaken” from dormancy may retain in vivo long-term activity in humans. We are exploiting IS similarities among the lineages from ADA-SCID and other datasets to reconstruct the hematopoietic hierarchy. Preliminary data unveiled a link between myeloid progenitors and mature lymphoid cells that supports the recently suggested model of delayed branching of these lineages during hematopoiesis. Alessandro Aiuti Figure 20. Selective advantage and functionality of gene-corrected B cells in patients with ADA-SCID. Adapted from: Brigida, I et al., J. Allergy Clin. Immunol.: 2014; 133(3): 799806.e10 doi: 10.1016/j.jaci.201 3.12.1043 125 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units, HSR-TIGET Safety of gene therapy and insertional mutagenesis Unit We developed and validated novel highly sensitive tumor prone mouse models able to test the genotoxicity of gene therapy vector integrations in hematopoietic and liver tissues and to identifiy novel genes involved in cancer (Ranzani M. et al., 2013 Nature Methods, Nowrouzi A. et al., 2013 Molecular Therapy and Ranzani M. et al., 2013 Molecular Cancer Research). In this context we also generated double knockout mice harboring the null mutations for the Cdkn2a and WiskottAldrich syndrome (WAS) genes. The study of these mice were instrumental also to address WAS protein deficiency in natural killer and dendritic cells affects antitumor immunity (Catucci M et al., 2013 European Journal of Immunology). Moreover, we generated novel protocols and algorithms for integration site analysis for preclinical testing and clinical trials. These advancements in technologies and know-how have been instrumental to investigate the crosstalk between integrated proviral and cellular host genome, study basic mechanisms of insertional mutagenesis and to test and validate, at the molecular level and in vivo, novel LV designs with a superior safety profile and that could be potentially adopted future clinical trials. Finally, we successfully setup a centralized core for standardized integration site retrieval and analysis and performed the molecular monitoring of the hematopoiesis in gene therapy patients from our two recent clinical trials for the therapy of metachromatic leukodystrophy and WAS. Our data provided evidence that efficient ex vivo gene transfer occurred and was followed by substantial engraftment and sustained clonogenic activity of the transduced hematopoietic stem cells in the patients, allowing polyclonal reconstitution of hematopoiesis with gene corrected cells to near homogeneity (Biffi A.*, Montini E.* et al., 2013 Science and Aiuti A. Et al., 2013 Science). Eugenio Montini Tolerogenic dendritic cells Unit The final goal of our group is to better characterize the role of HLA-G-expressing DC-10, tolerogenic DC that promote T regulatory type 1 (Tr1) cells, via the IL-10-dependent HLA-G/ILT4 pathway, in dictating immune tolerance induction, and to identify their clinical therapeutic application. Aims: 1) To dissect the role of HLA-G in modulating tolerogenic properties of DC-10; 2) To define the correlation between HLA-G genetic and its expression on DC-10 and to investigate the impact of miRNA in post-transcriptional regulation of HLA-G in DC10; 3) To identify biomarkers for DC-10 selection in vivo and the specific DC-10 gene signature; 4) To outline new mechanisms of DC-10-mediated immune-modulation; 5) To elucidate the localization and mode of action of DC-10 in vivo. Achievements: 1) The expression of HLA-G on DC-10 is donor-dependent, and high expression of membrane-bound HLA-G is required for efficient IL-10-mediated induction of Tr1 cells via DC-10. 2) Specific 3’UTR HLA-G genotypes are associated with the expression of membranebound HLA-G on DC-10 and, consequently with their tolerogenic properties. 3) The genomic and miRNA profiles of DC-10 have been delineated. 4) DC-10 modulate not only CD4+ but also CD8+ T cell responses in vitro. 5) DC-10 are present at high frequency in human decidua during the first trimester of pregnancy and in peripheral blood of AML patients. Conclusions: The expression of membranebound HLA-G on DC-10 is genetically defined, and is needed for reinforce DC-10-mediated generation of Tr1 cells. DC-10 are characterized by a specific gene signature that put them as central determinants of a tolerogenic path; future studies will better delineate the multiple roles of DC-10 in promoting tolerance. High frequency of DC-10 is associated with tolerance in pregnancy. These findings open new perspectives for DC-10 clinical application: DC-10 are currently used to generate allo-specific Tr1 cells for adoptive Treg-based cell therapy, but they represent an interesting therapeutic tool to induce or re-establish immunological tolerance in different clinical settings including allogeneic transplantation or autoimmune diseases. Silvia Gregori DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Clinical Research Units, HSR-TIGET PCRU - Gene therapy for Wiskott-Aldrich Syndrome Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, and an increase risk to develop autoimmunity and lymphomas. Despite progress in allogeneic transplantation of hematopoietic stem/progenitor cells (HSC), the use of mismatched donors is still associated with substantial complications. Gene therapy with ex vivo transduced HSC could represent a valid therapeutic option for patients lacking an HLA-identical donor. We developed an approach based on a lentiviral vector (LV) encoding for WAS under the control of the homologous 1.6 kb WAS promoter (Aiuti et al., Science 2013). A phase I/II clinical trial based on infusion of autologous transduced HSC cells and reduced intensity conditioning started in June 2010. Six patients were treated with autologous bone marrow (BM) or mobilized peripheral blood (PB) derived CD34+ cells transduced with highly purified LV. Results relating to the first four patients followed for up to 3.5 years, showed that transduction of clonogenic progenitors in vitro was highly efficient (>90%). This led to robust and stable engraftment observed in BM (including clonogenic progenitors), PB myeloid cells and lymphocytes. Accordingly, WASP expression, measured by flow-cytometry, was observed in monocytes and, as expected due to the selective advantage, in a higher percentage of B and T cells cells. TCR driven proliferation was improved also at low doses of anti-CD3 monoclonal antibody, which are typically defective in WAS. TCR repertoire and immunological functions, including NK cytotoxic ability, and Treg function, were also improved after GT. WASP expression was also restored in the majority of platelets, leading to an increase in their number. All patients are currently clinically well, independent from platelet transfusions. Insertions analyses confirm that hematopoiesis remains highly polyclonal, in the absence of aberrant clonal expansion. In conclusion, the high level of gene transfer obtained with LV-WAS results in stable engraftment of transduced HSC and restored WAS expression even when combined to reduced intensity conditioning. Although a longer observation is required to establish the long-term safety, lentiviral gene therapy represents a promising treatment for WAS. Maria Grazia Roncarolo and Alessandro Aiuti PCRU - ADA gene transfer into hematopoietic stem cells for the treatment of ADA-SCID SCID due to adenosine deaminase deficiency (ADA-SCID) is characterized by impaired lymphoid development and function, and systemic manifestation of metabolic toxicity. Since year 2000, we have treated 18 children with autologous CD34+ cells transduced with a gammaretroviral encoding ADA combined to reduced intensity conditioning with busulfan. Median age at treatment was 1.7 years (range: 0.5 to 6.1); 83% of patients had received enzyme replacement therapy (ERT) before gene therapy. As of December 31st, 2013, all patients are alive and well, with a median follow up of 7.2 years. Three subjects required reintroduction of ERT or allogeneic bone marrow (BM) transplantation while 15 patients remain off ERT. ADA transduced cells continue to be stably detected by PCR in multiple lineages of the BM and peripheral blood, leading to an effective reduction of systemic dAXP. T cell counts, including CD4+ naïve T cells, and in vitro T-cell proliferative responses have improved significantly after gene therapy. IVIg infusions have been discontinued in 12 patients, with positive response to vaccinations. Severe infections have progressively decreased after gene therapy. No event of insertional oncogenesis has been detected during the follow up, in agreement with the polyclonal pattern of vector integration by insertion site analyses. Study on patients receiving infusions of transduced lymphocytes or CD34+ cells allowed tracking of single T-cell clones and investigating the survival potential and hierarchical relationships of naive and memory subpopulations directly in vivo. In summary, these data confirm the favourable risk-benefit profile of gene therapy for ADA-SCID and support further development of this therapy. Alessandro Aiuti and Maria Grazia Roncarolo 127 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Clinical Research Units, HSR-TIGET PCRU - Clinical trial of gene therapy in metachromatic leukodystrophy Phase I/II clinical trial of hematopoietic stem cell gene therapy for the treatment of Metachromatic Leukodystrophy Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder caused by Arylsulfatase A (ARSA) deficiency and leading to severe demyelination, neurodegeneration and premature death of the affected patients. Currently, no treatment can halt the progression of this devastating disease. According to relevant preclinical data, and based on the experience we acquired on the natural clinical course of the disease, on March 2010 a clinical trial based on transplantation of autologous hematopoietic stem cells transduced with LVs encoding ARSA was approved by the Italian Regulatory Authorities. The clinical protocol foresees the enrollment of 8 late infantile (LI) and 2 early juvenile (EJ) patients, in pre- and, in the case of EJ patients, early-symptomatic stage, in order to provide them a reasonable expectation of clinical benefit. The study objectives are the evaluation of the safety of the treatment, related to the myeloablative conditioning regimen employed and to the use of LVs, and of its efficacy by measuring patients’ motor abilities and demyelination occurring in the nervous system through the use of validated instrumental readouts. By end of 2013 nine patients have been enrolled and treated. Thus far, we can report a favorable outcome of the transplant procedure with a good bone marrow recovery and the short/medium-term safety of both the conditioning regimen and stem cell transduction with LVs. Moreover, we report stable sustained ARSA gene replacement in the reconstituted hematopoiesis of the patients, and ARSA activity reconstitution in the cerebrospinal fluid, the latter thus far documented in the first three treated patients. These findings are associated with evidence of therapeutic benefit. These data are extremely encouraging, even if only the long-term follow-up of all the treated patients will confirm this favorable indication. Maria Sessa and Alessandra Biffi DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Selected publications Aiuti, A; Biasco, L and Scaramuzza, S; Ferrua, F; Cicalese, MP; Baricordi, C; Dionisio, F; Calabria, A; Giannelli, S; Castiello, MC; Bosticardo, M; Evangelio, C; Assanelli, A; Casiraghi, M; Di Nunzio, S; Callegaro, L; Benati, C; Rizzardi, P; Pellin, D, Di Serio, C; Schmidt, M; Von Kalle, C; Gardner, J; Mehta, N; Neduva, V; Dow, DJ; Galy, A; Miniero, R; Finocchi, A; Metin, A; Banerjee, P; Orange, J; Galimberti, S; Valsecchi, MG; Biffi, A; Montini, E; Villa, A; Ciceri, F; Roncarolo, MG and Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome. Science: 2013; 341(6148): 123351 - Article IF 2012: 31,027 Biffi, A and Montini, E; Lorioli, L; Cesani, M; Fumagalli, F; Plati, T; Baldoli, C; Martino, S; Calabria, A; Canale, S; Benedicenti, F; Vallanti, G; Biasco, L; Leo, S; Kabbara, N; Zanetti, G; Rizzo, WB; Mehta, NA; Cicalese, MP; Casiraghi, M; Boelens, JJ; Del Carro, U; Dow, DJ; Schmidt, M; Assanelli, A; Neduva, V; Di Serio, C; Stupka, E; Gardner, J; von Kalle, C; Bordignon, C; Ciceri, F; Rovelli, A; Roncarolo, MG; Aiuti, A; Sessa, M; Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy. Science: 2013; 341(6148): 1233158 - Article IF 2012: 31,027 Casucci, M; Perna, SK; Falcone, L; Camisa, B; Magnani, Z; Bernardi, M; Crotta, A; Tresoldi, C; Fleischhauer, K; Ponzoni, M; Gregori, S; Caligaris-Cappio, F; Ciceri, F; Bordignon, C; Cignetti, A; Bondanza, A and Bonini, C. Graft-versus-leukemia effect of HLA-haploidentical central-memory t-cells expanded with leukemic APCs and modified with a suicide gene. Mol. Ther.: 2013; 21(2): 466-475 - Article IF 2012: 7,041 Casucci, M; Nicolis di Robilant, B; Falcone, L; Camisa, B; Norelli, M; Genovese, P; Gentner, B; Gullotta, F; Ponzoni, M; Bernardi, M; Marcatti, M; Saudemont, A; Bordignon, C; Savoldo, B; Ciceri, F; Naldini, L; Dotti, G; Bonini, C; Bondanza, A. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood: 2013; 122(20): 3461-3472 - Article IF 2012: 9,060 Patel, AS; Smith, A; Nucera, S; Biziato, D; Saha, P; Attia, RQ; Humphries, J; Mattock, K; Grover, SP; Lyons, OT; Guidotti, LG; Siow, R; Ivetic, A; Egginton, S; Waltham, M; Naldini, L; De Palma, M and Modarai, B. TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb. EMBO Mol. Med.: 2013; 5(6): 858-869 - Article IF 2012: 7,795 Gagliani, N and Magnani, CF and Huber, S; Gianolini, ME; Pala, M; Licona-Limon, P; Guo, B; Herbert, DR; Bulfone, A; Trentini, F; Di Serio, C; Bacchetta, R; Andreani, M; Brockmann, L; Gregori, S and Flavell, RA and Roncarolo, MG. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat. Med.: 2013; 19(6): 739-746 - Article IF 2012: 24,302 Passerini, L; Rossi Mel, E; Sartirana, C; Fousteri, G; Bondanza, A; Naldini, L; Roncarolo, MG; Bacchetta, R. CD4+ T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer. Sci. Transl. Med.: 2013; 5(215): 215ra174 - Article IF 2012: 10,757 Ranzani, M; Cesana, D and Bartholomae, CC; Sanvito, F; Pala, M; Benedicenti, F; Gallina, P; Sergi Sergi, L; Merella, S; Bulfone, A; Doglioni, C; Von Kalle, C; Kim, YJ; Schmidt, M; Tonon, G; Naldini, L; Montini, E. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat. Methods: 2013; 10(2): 155-161 - Article IF 2012: 23,565 Zonari, E; Pucci, F; Saini, M; Mazzieri, R; Politi, LS; Gentner, B; Naldini, L. A role for miR-155 in enabling tumor-infiltrating innate immune cells to mount effective antitumor responses in mice. Blood: 2013; 122(2): 243-252 - Article IF 2012: 9,060 Cieri, N; Camisa, B; Cocchiarella, F; Forcato, M; Oliveira, G; Provasi, E; Bondanza, A; Bordignon, C; Peccatori, J; Ciceri, F; Lupo-Stanghellini, MT; Mavilio, F; Mondino, A; Bicciato, S; Recchia, A; Bonini, C. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood: 2013; 121(4): 573-584 - Article 129 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Selected publications, HSR-TIGET IF 2012: 9,060 Sundarasetty, BS; Singh, VK; Salguero, G; Geffers, R; Rickmann, M; Macke, L; Borchers, S; Figueiredo, C; Schambach, A; Gullberg, U; Provasi, E; Bonini, C; Ganser, A; Woelfel, T; Stripecke, R. Lentivirus-induced dendritic cells for immunization against high-risk WT1+ acute myeloid leukemia. Hum. Gene Ther.: 2013; 24(2): 220-237 - Article IF 2012: 4,019 Rigamonti, E; Touvier, T; Clementi, E; Manfredi, AA; Brunelli, S and Rovere-Querini, P. Requirement of inducible nitric oxide synthase for skeletal muscle regeneration after acute damage. J. Immunol.: 2013; 190(4): 1767-1777 - Article IF 2012: 5,520 Lo-Coco, F; Avvisati, G; Vignetti, M; Thiede, C; Orlando, SM; Iacobelli, S; Ferrara, F; Fazi, P; Cicconi, L; Di Bona, E; Specchia, G; Sica, S; Divona, M; Levis, A; Fiedler, W; Cerqui, E; Breccia, M; Fioritoni, G; Salih, HR; Cazzola, M; Melillo, L; Carella, AM; Brandts, CH; Morra, E; Von Lilienfeld-Toal, M; Hertenstein, B; Wattad, M; Lubbert, M; Hanel, M; Schmitz, N; Link, H; Kropp, MG; Rambaldi, A; La Nasa, G; Luppi, M; Ciceri, F; Finizio, O; Venditti, A; Fabbiano, F; Dohner, K; Sauer, M; Ganser, A; Amadori, S; Mandelli, F; Dohner, H; Ehninger, G; Schlenk, RF; Platzbecker, U; for Gruppo Italiano Malattie Ematologiche dell’ Adulto; the German-Austrian Acute Myeloid Leukemia Study Group; and Study Alliance Leukemia. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. New Engl. J. Med.: 2013; 369(2): 111-121 - Article IF 2012: 51,658 Todisco, E; Ciceri, F; Oldani, E; Boschini, C; Mico, C; Vanlint, MT; Donnini, I; Patriarca, F; Alessandrino, PE; Bonifazi, F; Arcese, W; Barberi, W; Marenco, P; Terruzzi, E; Cortelazzo, S; Santarone, S; Proia, A; Corradini, P; Tagliaferri, E; Falcioni, S; Irrera, G; Dallanegra, L; Castagna, L; Santoro, A; Camboni, A; Sacchi, N; Bosi, A; Bacigalupo, A; Rambaldi, A. The CIBMTR score predicts survival of AML patients undergoing allogeneic transplantation with active disease after a myeloablative or reduced intensity conditioning: A retrospective analysis of the Gruppo Italiano Trapianto di Midollo Osseo. Leukemia: 2013; 27(10): 2086-2091 - Letter IF 2012: 10,164 Neguembor, MV; Xynos, A; Onorati, MC; Caccia, R; Bortolanza, S; Godio, C; Pistoni, M; Corona, DF; Schotta, G; Gabellini, D. FSHD muscular dystrophy region gene 1 binds Suv4-20h1 histone methyltransferase and impairs myogenesis. J.Mol. Cell Bio.: 2013; 5(5): 294-307 - Article IF 2012: 7,308 Selected publications, HSR-TIGET (see also the list under Division of Regenerative Medicine, Stem Cell and Gene Therapy) Amendola, M; Giustacchini, A; Gentner, B; Naldini, L. A double-switch vector system positively regulates transgene expression by endogenous microRNA expression (miR-ON vector). Mol. Ther.: 2013; 21(5): 934-946 - Article IF 2012: 7,041 Annoni, A and Cantore, A; Della Valle, P; Goudy, K; Akbarpour, M; Russo, F; Bartolaccini, S; D’Angelo, A; Roncarolo, MG and Naldini, L. Liver gene therapy by lentiviral vectors reverses antifactor IX pre-existing immunity in haemophilic mice. EMBO Mol. Med.: 2013; 5(11): 1684-1697 - Article IF 2012: 7,795 Lattanzi, A and Gentner, B; Corno, D; Di Tomaso, T; Mestdagh, P; Speleman, F; Naldini, L; Gritti, A. Dynamic Activity of miR-125b and miR-93 during Murine Neural Stem Cell Differentiation In Vitro and in the Subventricular Zone Neurogenic Niche. PloS One: 2013; 8(6): e67411 - Article IF 2012: 3,730 McMurchy, AN; Gillies, J; Gizzi, MC; Riba, M; Garcia-Manteiga, JM; Cittaro, D; Lazarevic, D; Di DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Nunzio, S; Piras, IS; Bulfone, A; Roncarolo, MG; Stupka, E; Bacchetta, R and Levings, MK. A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells. Blood: 2013; 121(8): 1265-1275 - Article IF 2012: 9,060 Scaramuzza, S and Biasco, L; Ripamonti, A; Castiello, MC; Loperfido, M; Draghici, E; Hernandez, RJ; Benedicenti, F; Radrizzani, M; Salomoni, M; Ranzani, M; Bartholomae, CC; Vicenzi, E; Finocchi, A; Bredius, R; Bosticardo, M; Schmidt, M; von Kalle, C; Montini, E; Biffi, A; Roncarolo, MG; Naldini, L; Villa, A; Aiuti, A. Preclinical Safety and Efficacy of Human CD34(+) Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome. Mol. Ther.: 2013; 21(1): 175-184 - Article IF 2012: 7,041 131 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Functional genetics of muscle regeneration Neural stem cell biology DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Autoimmunity & vascular inflammation Unit Gene expression and muscular dystrophy Unit 133 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units Molecular and functional immunogenetics Unit Hematology and hematopoietic stem cell transplantation Unit DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Gene therapy for WASP/Omenn Unit Hematopoietic stem cell based gene therapy for the treatment of lysosomal storage disorders Unit 135 DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Research Units Pathogenesis and therapy of ADA-SCID Unit Tolerogenic dendritic cells Unit DIVISION OF REGENERATIVE MEDICINE, STEM CELLS AND GENE THERAPY Safety of gene therapy and insertional mutagenesis Unit 137 DIVISION OF Immunology, Transplantation, and Infectious Diseases Director: Luca G. Guidotti, since June 2013 Director: Ruggero Pardi*, until May 2013 Associate Director: Adriano Lazzarin* Research Units Immunopathology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 147 HEAD OF UNIT: Luca G. Guidotti, ERC Advanced Grant RESEARCHER: Giovanni Sitia POST-DOCTORAL FELLOW: Mario Catarinella PHD STUDENT: Roberto Aiolfi** FELLOW: Andrea Monestiroli TECHNICIANS: Tiziana Cataudella, Diego Covarello, Pietro Di Lucia, Amleto Fiocchi, Bruno Fiore, Marta Mainetti, Michele Raso Cellular and molecular allergology ––––––––––––––––––––––––––––––––––––––––––––––– 147 GROUP LEADER: Samuele E. Burastero TECHNICIANS: Daniela Breda, Marco Rossi Human virology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 148 GROUP LEADER: Mauro S. Malnati FELLOW: Davide De Battista TECHNICIAN: Francesca Sironi Protein engineering and therapeutics –––––––––––––––––––––––––––––––––––––––––––– 148 GROUP LEADER: Luca Vangelista POST-DOCTORAL FELLOW: Massimiliano Secchi γδ T cells in innate and adaptive immunity ––––––––––––––––––––––––––––––––––––– 149 RESEARCHER: Maria Raffaella Zocchi FELLOWS: Caterina Camodeca, Paolo Canevali, Silvia Catellani 139 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Infectious diseases Unit HEAD OF UNIT: Adriano Lazzarin* Immunobiology of HIV ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 150 GROUP LEADER: Lucia Lopalco POST-DOCTORAL FELLOW: Assunta Venuti FELLOW: Riccardo Miglietta TECHNICIAN: Claudia Pastori AIDS immunopathogenesis Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 150 HEAD OF UNIT: Guido Poli* RESEARCHER: Massimo Alfano POST-DOCTORAL FELLOW: Elisa Saba PHD STUDENTS: Marylinda Famiglietti**, Luca Genovese**, Francesca Graziano** Biocrystallography Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 150 HEAD OF UNIT: Massimo Degano POST-DOCTORAL FELLOWS: Giovanna Avella, Claudia Minici, Marco Patrone TECHNICIAN: Paola Tornaghi Cellular immunology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 152 HEAD OF UNIT: Matteo Bellone POST-DOCTORAL FELLOW: Elena Jachetti PHD STUDENTS: Arianna Calcinotto**, Alessia Ricupito** TECHNICIAN: Matteo Grioni Dynamics of immune responses Unit ––––––––––––––––––––––––––––––––––––––––––––––––––– 152 GROUP LEADER: Matteo Iannacone, Armenise-Harvard Career Development Award and ERC starting grant POST-DOCTORAL FELLOWS: Jessica Fioravanti, Nereida Jiménez de Oya, Mirela Kuka, Stefano Sammicheli, Laura Sironi PHD STUDENTS: Marco De Giovanni**, Donato Inverso** FELLOWS: Silvia Burati**, Lorenzo Fossati**, Lucia Ganzer Emerging bacterial pathogens Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 153 HEAD OF UNIT: Daniela Maria Cirillo POST-DOCTORAL FELLOWS: Emanuele Borroni, Lucinda Furci, Paolo Miotto, Elisa Tagliani, Enrico Tortoli PHD STUDENTS: Andrea Cabibbe, Elisa Schena FELLOWS: Riccardo Alagna, Alberto Trovato, IIaria Valente TECHNICIAN: Rossella Baldan, Paola Mantegani Experimental immunology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 154 HEAD OF UNIT: Paolo Dellabona RESEARCHERS: Giulia Casorati, Claudia De Lalla PHD STUDENTS: Michela Consonni**, Filippo Cortesi**, Francesca Gorini** FELLOWS: Maya Fedeli, Daniele Mennonna TECHNICIAN: Claudio Garavaglia DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Infection and cystic fibrosis Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 154 GROUP LEADER: Alessandra Bragonzi POST-DOCTORAL FELLOWS: Irene Bianconi, Cristina Cigana**, Ida De Fino, Marcella Facchini (until April 2013), Nicola Ivan Lorè PHD STUDENTS: Beatriz Alcalà-Franco, Maura De Simone, Lorenza Spagnuolo FELLOWS: Camilla Riva, Alice Rossi Leukocyte biology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 155 HEAD OF UNIT: Ruggero Pardi* RESEARCHER: Monica Fabbri POST-DOCTORAL FELLOWS: Carolina Lage Crespo, Raffaella Molteni, Michela Palmisano, Martina Panattoni FELLOW: Marcello Delfini TECHNICIAN: Barbara Clissi Lymphocyte activation Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 156 HEAD OF UNIT: Anna Mondino POST-DOCTORAL FELLOW: Karolina Pilipow PHD STUDENT: Tabea Sturmheit** FELLOW: Valentina Guida TECHNICIAN: Veronica Basso Tumor immunology Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 156 HEAD OF UNIT: Maria Pia Protti RESEARCHER: Lucia De Monte POST-DOCTORAL FELLOW: Giulia Di Lullo PHD STUDENT: Emanuela Brunetto TECHNICIAN: Silvia Heltai Viral evolution and transmission Unit ––––––––––––––––––––––––––––––––––––––––––––––––––– 157 HEAD OF UNIT: Gabriella Scarlatti POST-DOCTORAL FELLOWS: Mariangela Cavarelli, Stefania Dispinseri TECHNICIAN: Monica Tolazzi Viral pathogens and biosafety Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 158 HEAD OF UNIT: Elisa Vicenzi PHD STUDENTS: Andrea Di Pietro**, Filippo Turrini** TECHNICIANS: Silvia Ghezzi, Lucia Nicora 141 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units Infectious diseases Unit HEAD OF UNIT: Adriano Lazzarin* Management and antiretroviral treatment of HIV infection –––––––––––––––––– 159 CLINICAL GROUP LEADER: Antonella Castagna PHYSICIAN: Nicola Gianotti FELLOW: Vincenzo Spagnuolo** RESEARCH NURSES: Alba Bigoloni, Concetta Vinci STUDY COORDINATOR: Elisabetta Carini DATA MANAGER: Stefania Salpietro STATISTICIAN: Laura Galli TECHNICIAN: Andrea Galli BIOMEDICAL ENGINEER: Andrea Poli** Neurovirology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 159 CLINICAL GROUP LEADER: Paola Cinque FELLOWS: Francesca Ferretti**, Valeria Longo STUDY COORDINATORS: Maria Rita Parisi, Ester Tuveri Study and treatment of hepatotropic viruses related diseases ––––––––––––– 160 CLINICAL GROUP LEADER: Caterina Uberti-Foppa RESEARCHER: Giulia Morsica POST-DOCTORAL FELLOWS: Sabrina Bagaglio, Hamid Ibrahim Hasson FELLOWS: Marco Merli, Emanuela Messina Vaccine and immunotherapy ––––––––––––––––––––––––––––––––––––––––––––––––––––––– 161 CLINICAL GROUP LEADER: Giuseppe Tambussi PHYSICIAN: Silvia Nozza FELLOWS: Stefania Chiappetta**, Manuela Pogliaghi**, Marco Ripa** RESEARCH NURSE: Liviana Della Torre TRIAL COORDINATOR: Maria Rita Parisi TECHNICIAN: Andrea Galli Clinical immunopathology and advanced medical therapeutics Unit ––––––––––––– 161 HEAD OF UNIT: Maria Grazia Sabbadini* PHYSICIANS: Enrica P. Bozzolo, Giselda Colombo, Lorenzo Dagna*, Massimo Memoli, Luisa Praderio, Moreno Tresoldi POST-DOCTORAL FELLOWS: Patrizia Aiello, Emmanuel Della Torre**, Stefano Franchini, Mirta Tiraboschi, Mona Rita Yacoub FELLOWS: Alvise Berti, Corrado Campochiaro, Valentina Canti, Andrea Duca, Luca Ferrante, Andrea Segalini, Cristina Sorlini, Enrico Tombetti CONSULTANT: Elena Baldissera Clinical transplant Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 162 HEAD OF UNIT: Antonio Secchi* PHYSICIANS: Rossana Caldara, Chiara Gremizzi, Ennio La Rocca, Vera Paloschi POST-DOCTORAL FELLOWS: Francesca D’Addio, Silvia Foti CONSULTANT: Paolo Fiorina RESEARCH NURSE: Barbara Pontiroli DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units Pancreatic tumors Unit: immunotherapy and β cell function substitution –––––– 162 HEAD OF UNIT: Marco Braga* PHYSICIAN: Renato Castoldi RESIDENT: Nicolò Pecorelli Gynecological cancers immunology ––––––––––––––––––––––––––––––––––––––––––––––––––––– 163 HEAD OF UNIT: Massimo Candiani* CLINICAL GROUP LEADER: Massimo Origoni* PHYSICIAN: Luigi Caputo RESIDENTS: Guia Carminati**, Chiara Stefani** Immunology in liver neoplasms ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 163 HEAD OF UNIT: Gianfranco Ferla* CLINICAL GROUP LEADER: Luca Aldrighetti PHYSICIANS: Marco Catena, Renato Finazzi RESIDENTS: Federica Cipriani **, Francesca Ratti** Obesity and bariatric surgery –––––––––––––––––––––––––––––––––––––––––––––––––––––– 164 RESEARCHER: Michele Paganelli Gastroenterology Unit HEAD OF UNIT: Pier Alberto Testoni* Clinical hepato-gastroenterology ––––––––––––––––––––––––––––––––––––––––––––––––– 164 RESEARCHER: Mario Guslandi Digestive pathophysiology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 165 RESEARCHER: Sandro Passaretti 143 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES DRI Diabetes Research Institute Director: Emanuele Bosi* Associate Directors: Manuela Battaglia, Lorenzo Piemonti Research Units Diabetes research Unit HEAD OF UNIT: Emanuele Bosi* Experimental diabetes ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 167 GROUP LEADER: Marika Falcone PHD STUDENTS: Caterina Di Pietro**, Chiara Sorini** FELLOWS: Jayashree Dolpady, Alessandra Mandelli TECHNICIAN: Alessandra Caputo β cell biology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 167 HEAD OF UNIT: Lorenzo Piemonti RESEARCHER: Rita Nano POST-DOCTORAL FELLOWS: Paolo Monti, Valeria Sordi PHD STUDENTS: Elisa Cantarelli**, Antonio Citro, Erica Dugnani, Simona Marzorati, Silvia Pellegrini FELLOWS: Daniela Liberati, Alessia Mercalli, Valentina Pasquale, Debora Vignali TECHNICIAN: Raffaella Melzi DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units/Clinical Research Units, DRI Immune-mediated diseases Unit: from pathogenesis to treatment ––––––––––––––– 168 HEAD OF UNIT: Manuela Battaglia POST-DOCTORAL FELLOW: Georgia Fousteri PHD STUDENTS: Gian Maria Giamporcaro, Bechara Mfarrej, Celia Peral de Castro**, Andrea Valle** FELLOWS: Roberta Di Fonte, Antonio Martelli, Cristina Morsiani TECHNICIANS: Tatiana Jofra, Angela Stabilini Clinical Research Units Prevention in Type 1 diabetes Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 169 HEAD OF UNIT: Emanuele Bosi* PHYSICIANS: Sabina Martinenghi, Matteo Rocco Pastore RESIDENT: Andrea Laurenzi TRIAL COORDINATOR: Pauline Grogan RESEARCH NURSE: Eleonora Bianconi Epidemiology & data management ––––––––––––––––––––––––––––––––––––––––––––––– 169 RESEARCHER: Marina Scavini Islet transplantation –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 169 HEAD OF UNIT: Antonio Secchi* CLINICAL GROUP LEADER: Paola Maffi DATA MANAGER: Paola Magistretti RESEARCH NURSE: Luisa Zamberletti Childhood diabetes –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 170 HEAD OF UNIT: Giuseppe Chiumello* CLINICAL GROUP LEADER: Riccardo Bonfanti RESIDENTS: Roseila Battaglino, Clara Bonura, Valeria Favalli, Giulio Frontino * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele 145 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Introduction by the Directors The Division of Immunology, Transplantation and Infectious Diseases (DITID) stems from a deeply integrated area of Institutional research at the San Raffaele Scientific Institute, dating back to the inception of DIBIT and making up the scientific core of areas of intense clinical investigation, which include, but are not limited to, cancer, type 1 diabetes, allergy, cell and solid organ transplantation and infectious diseases such as those caused by HIV, HBV and HCV. Based on these premises, the unifying mission of the DITID is to harness the immune response for the benefit of patients. To this aim, DITID fosters research efforts and development of new technologies aimed at dissecting the mechanisms underlying the immune response to infectious agents, cancer- and transplantation-associated antigens, as well as the deregulation of processes controlling immunological tolerance. Basic discoveries are validated through a large variety of dedicated animal models and human immune cells/tissues, with the goal of translating them into proof-of-concept clinical trials. Over the last few years, DITID has also structured selected research activities into a limited set of Research Programs, aimed at fostering interdisciplinary research and strong integration of pre-clinical and clinical investigations. The Islet Transplantation Program (ITP) aims at achieving long-lasting insulin independence in patients with type 1 diabetes (T1D) undergoing islet transplantation. The Program of Immunology and Bio-immunotherapy of Cancer (PIBIC) seeks to provide a deeper understanding of the mechanisms underlying the tumor/immune system interaction and designs new immunotherapy strategies against different solid tumors. A third Program termed Correlates of HIV-Associated Immune Response Modulation (CHARM) focuses on the role of the chemokine receptor CCR5 and its ligands in HIV infection and related clinical entities. DITID personnel includes Faculty, Tenured Research Associates, Post-doctoral fellows, PhD Students, Technicians, and Clinicians. Graduate training is currently provided within the frame of the Institutional PhD Programs. PhD students are given the opportunity to participate to “ad hoc” courses mainly organized in the format of frontal Lectures by Invited Speakers followed by interactive Journal Club sessions. Students are also offered the possibility to meet visiting scientists and to participate to national and international meetings. Post-graduate education is mainly provided by the possibility to attend and present data to weekly-held DITID Meetings or Journal Clubs. Participation to the institutional San Raffaele Scientific Retreat (held once a year) is also strongly encouraged and particularly useful. DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Immunopathology Unit Pathogenesis of viral hepatitis and liver cancer Hepatitis B virus (HBV) and hepatitis C virus (HCV) are unique in their capacity to cause persistent infection, fibrosis/cirrhosis and hepatocellular carcinoma (HCC) in humans. The main objective of our research is to define the cellular and molecular mechanisms responsible for viral clearance and disease pathogenesis during HBV or HCV infection. We expect that that results emerging for this work will help the design of novel therapeutic approaches to prevent and cure these diseases. The program takes advantage of infected patients, proprietary mouse models of viral hepatitis and liver cancer, and new technological advances in the field of live imaging (e.g. 7Tesla MRI and single- or two-photon-based intravital microscopy). Using these resources, we have recently demonstrated that platelets play a central role in the pathogenesis of viral hepatitis (i.e. they facilitate the accumulation of pathogenic virusspecific T cells into the organ) and that daily lowdoses of anti-platelet drugs reduce the severity of liver immunopathology and the development of fibrosis/cirrhosis and HCC in a mouse model of immune-mediated chronic hepatitis. Additional activities of our Unit include the implementation of cell-based approaches attempting to cure established primary or secondary liver cancers. One such approach involves the use of specialized monocytes - termed Tie2 monocytes -, which accumulate preferentially in the proximity of tumors. In collaboration with the Naldini’s lab, we reasoned to utilize Tie2 monocytes at the host advantage, i.e. to genetically manipulate them (so that they produce anti-tumoral drugs such as IFNα) and use them as Trojan Horses delivering high doses IFN-α only at tumor sites. Thus far, our preclinical efforts mainly involved mouse models of colorectal cancer (CRC) liver metastases and showed that mice reconstituted with bone marrow cells engineered to express IFN-α only in Tie2 monocytes display reduced tumor burden, increased signs of anti-tumoral immune activation and overall improved survival. As these remarkable results are not associated to systemic signs of toxicity, this work suggests that Tie2 monocytes expressing IFN-α might represent a new and valuable cell therapy platform for the treatment of CRC liver metastases in humans. Luca G. Guidotti Cellular and molecular allergology In the last decade, single allergen components have been progressively substituting allergen extracts in allergy diagnosis and treatment. We previously characterized structural aspects of “allergoids”, i.e., allergens mutated in order to loose allergenicity while fully maintaining T-cell stimulatory potential, to be used as candidates for immunotherapy in allergic diseases. Presently, we are exploring (in collaboration with the Protein Biochemistry Unit) the presence of IgE to allergen components which, although infrequently found in the allergic population based on presently available allergen micro-array based diagnosis, represent sensitizations with a clinical impact in single patients. In parallel, we are investigating the role of diamine oxidase (DAO), the main enzyme for the metabolism of histamine ingested with foods and/or released following IgE dependent reactions, in histamine intolerance. Histamine intolerance is a disputed clinical syndrome potentially capable to jus- tify up to 34% of the requests of the patients referred to the Allergy Clinic. The altered balance between histamine production and catabolism can follow ingestion of histamine-rich food, drugs releasing histamine and drugs blocking diaminoxidase. Several pathogenic steps follow, including smooth muscle contraction, vasodilatation, extravasation of plasma from capillaries, and stimulation of gastric acid secretion. Together, these mechanisms are responsible for such heterogeneous symptoms as diarrhea, epigastralgia, meteorism, headache, hypotension, arrhythmias, urticaria, pruritus, flushing, rhinitis and asthma. Association of functional polymorphisms in the genes coding for DAO and histamine intolerance will be also investigated. Evaluation of these clinical parameters in association with serum diamine oxidase levels and expression of DAO polymorphisms will help to shed light on the pathogenesis of histamine intolerance. Samuele E. Burastero 147 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Human virology The failure of anti HIV-1 vaccine strategy based on the exclusive induction of a broad T-cell response against structural antigens suggests that the most effective defence against HIV-1 transmission might require the combination of HIV-1-specific Tcell responses and anti-HIV-specific antibody responses. Our unit is involved in characterizing the anti HIV-1 T-cell mediated immune responses as well as in the individuation of the genetic polymorphisms that favour the long term survival of particular HIV-1 infected individuals without sign of immune deterioration (LTNP) and/or that naturally control viral replication below the level of detection of the clinical lab tests (Elite controllers= EC) Main results obtained in 2013 In collaboration with the Unit of Molecular Medicine led by Dr Roberto Biassoni at the G. Gaslini Institute we characterized a cohort of 80 HIV-1 infected Natural controllers (41 EC and 39 LTNPs), 175 HIV1 progressors and 111 HIV-1 negative blood donors for a set of genetic markers involved in shaping differential immune-responses in autoimmune diseases and chronic infections (snp IL7R/rs987106 A/T, rs987107 C/T, IL28/rs8099917 T/G, IL28/rs2979860 T/C HLA B and C supertyping, HLAC/rs9264942 T/C HLAC/rs67384697 G/miRNA 148 a/b in combination with a complete KIR genotype). All the genetic tests have been performed and a database containing clinical and routine laboratory analysis as well as the results obtained by our genetic screening have been realized to perform the necessary statistical evaluations. A screening system of statistical evaluation based on Chi square and fisher exact test have been set up for fishing out the most significant variations in markers distribution. Regarding both IL7 R and IL-28 snp the data show a trend for a significant increased only in Primary HIV-1 infected individuals that deserve further investigation. On the contrary both HLA-C associates markers and Bw4 I80 supratype are significantly increased in NC. More interestingly a few KIR genes are significantly overrepresented only in EC individuals. Given the existing linkage disequilibrium (LD) from HLA genes as well as from KIR genes a more complex analysis that take in consideration LD and epistatic models is under evaluation. Mauro S. Malnati Protein engineering and therapeutics We focus most of our efforts on the production and development of HIV-1 entry inhibitors based on derivatives of CCL5/RANTES (a natural ligand of CCR5, the major HIV-1 co-receptor) acting as CCR5 antagonists. In the context of competitive grants from the EU, the NIH and the Italian Ministry of Health, potent anti-HIV-1 full-length CCL5 mutants and short peptides have been developed for their prospective implementation as mucosal microbicides. We are involved also in the “live microbicides” field, an approach based on the engineering of human commensal bacteria (e.g., lactobacilli) to produce anti-HIV-1 proteins. Lactobacilli were successfully implemented also as a platform to screen conceptually novel CCL5 mutants. By fusing our best CCL5 full-length derivative with an HIV-1 fusion inhibitor, we recently obtained a crossclade CCR5 antagonist with low/sub-picomolar IC50% activity. Furthermore, the combination of many different HIV-1 inhibitors with full-length or short peptide CCL5 derivatives presented full additivity or synergism in vitro. Our CCL5 derivatives will be tested also on other pathologies where CCL5 is of major relevance (e.g., inflammation and cancer). For our work on CCL5 derivatives, Massimiliano Secchi received the “G.B. Rossi prize 2013” by the Italian Association to fight AIDS (ANLAIDS). In a different research area (co-directed with Antonio Siccardi), we investigate the role of IgE as anti-tumor agent, studies empowered by our unique collection of IgE-related transgenic mice. We found that IgE is a potent adjuvant in anti-tumor vaccination and that Fc ε RI (the high affinity receptor for IgE) is the key mediator of the anti-tumor effect. This system was evolved by recruiting a truncated version of membrane IgE (tmIgE), capable to bind and activate Fc ε RI, using a safe recombinant MVA (modified vaccinia virus Ankara) engineered to express tmIgE. We also investigated the role of IgE as natural anti-tumor surveillant, a long-lasting research project producing exciting results being assembled for publication. Finally, we plan to implement commensal bacteria engineering to combat various worldwide pathological threats (such as HIV-1, allergy and avian influenza). Luca Vangelista DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Figure 21. Rational design of a sub-picomolar HIV-1 inhibitor based on the fusion between a CCL5 derivative (targeting CCR5 as antagonist) and C37 (targeting gp41). γδ T cells in innate and adaptive immunity Targeting ADAM10 and ERp5 in Hodgkin and non Hodgkin lymphomas CD8+T and γδ T cells, mediators of stress-related immunity, respond through the NKG2D receptor to NKG2D-ligands (NKG2D-L), as the MHC class-I related molecules MICA/B and the UL16-binding proteins 1-4 (ULBPs). We reported that circulating γδT lymphocytes in chronic lymphocytic leukemia and non Hodgkin lymphomas (NHL), react to autologous cancer cells expressing MICA or ULBPs and this correlates with disease stage and progression (Blood 2007, 109:2078). Due to NF-κB activation via NKG2D, leading to transcription of Bcl-xL antiapoptotic protein, γδ T cells are quite resistant to apoptosis (Int J Cancer 2011,129:387). NKG2D-L can be upregulated in vivo by all-transretinoic acid or sodium valproate (VPA) (Leukemia 2009, 23:642); however, NKG2D-L can be shed, interact with NKG2D on effector lymphocytes and hinder the recognition of tumor cells. Shedding of soluble (s) MIC-A depends on the thiol isomerase ERp5and the disintegrins ADAM10/17, which also cleave ULPBs. In Hodgkin’s lymphoma (HL), the LN stroma displayed in situ high levels of ERp5 and ADAM10, mainly in Reed-Sternberg (RS) and in LN-mesenchymal stromal cells (MSC), and sMICA and sULBP3 were detectable in patients’sera. Upon co-culture with LNMSC, CD8+T and γδ T cells reduced their cytolytic activity against NKG2DL+ targets, due to TGFβ produced by LNMSC and able to down-regulate the expression of NKG2D. In addition, CD8+T and γδ T cells from the lymph nodes of cHL patients co-cultured with LNMSC, underwent TGF-mediated down regulation of NKG2D (Blood 2012, 1196:1479). Of note, exposure of LNMSC to aminobisphosphonates prevented the effects of LNMSC on γδ T cell function (Hematologica 2014, 99:131). We are currently preparing specific ADAM10/ADAM17 inhibitors in order to prevent sNKG2D-L; preliminary results of experiments with siRNA point to a predominant role of ADAM10 vs ADAM17 in RS cells and at least one specific ADAM10 inhibitor has been synthesized able to interfere with the sheddase activity. We are also preparing specific peptides to inhibit ERp5 enzymatic activity Maria Raffaella Zocchi 149 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Immunobiology of HIV CCR5 modulation as target to block HIV transmission Our Unit focuses on the structure-immunogenicity applied to the host-virus relationship. The last few years have provided documentary evidence of the existence of subjects who, despite multiple exposures to HIV-1, remain seronegative (referred to as ESN). The possibility that some of these subjects may be “spontaneously vaccinated/cured” cannot be excluded, and so the study of their antiviral response may reveal as yet unknown resistance mechanisms that may be reproducible in others. The analyses the immune factors involved in immune controls could be extremely relevant to generate strategies able to prevent HIV transmission. CCR5 cellular protein is one the main coreceptors for HIV and CCR5 downregulating immunoglobulins were found in sera and mucosal samples in ESN suggesting a role for such antibodies in controlling viral replication in vivo. Natural antibodies (Abs) to CCR5 showed HIV-blocking properties, both in CD4+ T lymphocytes and in CCR5 transfected cells lines. It is very relevant to underline that the mechanism of action of these Abs is different from that reported for other CCR5 specific molecules. Natural Abs to CCR5 specifically inhibit HIV replication by inducing a full and long lasting CCR5 downregulation with a very slow kinetic. Indeed, in an experimental BalB/c mouse model, full in vivo CCR5 downregulation, as well as receptor backregulation, follow a very slow kinetics (similar to that induced by Abs in humans) that was gradually recovered in 4 weeks after immunization. We found that anti-CCR5-ECL1 Abs induce receptor internalization and recycling mediated by G-protein dependent ERK activity. These events require β-arrestins, which may couple ligand-activated receptors to clathrin, and lead to a cytoplasmatic CCR5 accumulation with consequent activation of ERK1/2. Our findings suggest that natural anti CCR5 antibodies, bind receptor and reach CCR5 intracellularly, thus inducing a degradation of the receptor, which prevent interaction with HIV. Thus, our research is aimed at creating such immune barrier by targeting the host rather than the virus establishing a durable elimination of CCR5. Lucia Lopalco AIDS immunopathogenesis Unit Central theme of our Unit is the regulation of HIV-1 replication by host factors, primarily of immunological nature. In this scenario, in this year we have been focusing our research primarily on the topics listed below. Macrophage polarization and HIV-1 infection. After our original observation that that both M1 and M2 polarized monocyte-derived macrophages (MDM) support less efficiently virus propagation, we have observed that M2-MDM upregulate selectively the surface molecule DC-SIGN, a capture receptor for HIV involved in trans-infection of CD4+ T cells, which is, conversely, downregulated in M1-MDM. On the other hand, M1-MDM showed the upregulation of APOBEC-3A, an intracellular factor that might contribute to the retroviral restriction in these cells. Identification of host factors influencing HIV-1 replication in the female genital tract. We have established a model of infection of human cervical tissue explants (CTE) established form women undergoing hysterectomy for benign tumors. Tissues are subdivided in small regular 2-mm3 blocks and infected in the absence of exogenous stimulation by mitogens or cytokines. By a post-hoc analysis we have observed that only CTE established form women in their secretory phase of the menstrual cycle were productively infected, implying a direct or indirect contribution of progesterone to HIV replication in this anatomical tract. Our findings support epidemiological evidence of a higher susceptibility of women in this phase of the menstrual cycle to become infected by HIV-1 as well as of transmitting the virus to their partners. Genetic studies on LTNP. As a follow-up of the concerted effort of a European consortium (“GISHEAL”) investigating the genetic background of individuals with a “long-term nonprogressing” (LTNP) phenotype (defined as maintenance of >500 CD4+ T cells/µl for >8 years of infection and good healthy conditions without ever receiving antiretroviral therapy) we have participated to a metanalysis study involving 6,300 infected and 7,200 uninfected control individuals. The results of the analysis indicated that, except for the Δ-32 deletion mutation in CCR5, there is no contribution of genetic factors to HIV-1 acquisition. Guido Poli DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Biocrystallography Unit The Biocrystallography Unit is devoted to the biochemical, structural and functional characterization of macromolecules of biomedical interest. The main areas of activity include: a) Infectious diseases We have a longstanding interest in the study of nucleotide-metabolizing enzymes for the rational development of drugs. We showed that enzymes with nucleosidase (NH) activity are essential for the life cycle of trypanosomes, parasites that cause life-threatening pathologies such as sleeping sickness or Chagas’ disease. We recently determined the crystal structure of the purine-specific NH from Trypanosoma brucei bound to several inhibitors, including a compound that eradicates parasites in infection models. Using a similar approach, we are also identifying targets for novel antibiotics against multidrug-resistant Staphylococcus aureus and Mycobacterium tuberculosis. b) Cancer We are characterizing the role of a novel cyclindependent kinase in the invasiveness of glioblastoma multiforme, the deadliest brain tumor. The goal is to determine the crystal structure of the enzyme, and to use a combination of in silico and in vitro methodologies to identify selective, tightbinding inhibitors. These compounds may reduce the aggressiveness of the neoplasy, and enhance the success of surgical removal. We are also characterizing the structures of B-cell receptors involved in the pathogenesis of chronic lymphocytic leukemia, in order to develop novel, effective therapeutic strategies. c) Autoimmune diseases We are studying the molecular mechanisms underlying the immunomodulatory activity of fingolimod, a prodrug that upon phosphorylation by sphingosine kinases prevents the egress of T lymphocytes from the lymph nodes. This compound can be administered orally, and is effective in reducing the progress of disability in patients affected by multiple sclerosis. We are currently determining the crystal structures of both the activatory enzyme SPK2, and the five members of the sphingosine 1-phosphate receptor family. These G-protein coupled receptors are expressed differentially in tissues, and the structures will allow the design of selective agonists to enhance the immune suppression, and reduce systemic adverse effects in treated patients. Massimo Degano Figure 22. Active site of the IAGNH enzyme from T. brucei bound to a nanomolar inhibitor. Knowledge of the active site cavity features (green transparent surface) allows the modification of the compound (yellow sticks) to enhance its activity. 151 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Cellular immunology Unit Harnessing the immune system against malignancies Our research is focused on defining the dynamic interactions between a growing tumor (i.e., transformed cells and surrounding stroma) and the immune system. This knowledge is then implemented to identify means whereby induce a therapeutic tumor-specific immune response. We have recently focused our research on cancer stem cells (CSCs) as drivers of prostate cancer development and progression. From transgenic adenocarcinoma of the mouse prostate (TRAMP) mice harboring oncogene-driven prostate cancer, we have established stage-specific prostate CSC lines that are endowed with the critical features expected from malignant bona fide CSCs, namely, self-renewal, multipotency, and tumorigenicity. Long-term population analyses ruled out the possibility that our CSCs were transient amplifying progenitors. Transcriptome analysis showed that genes upregulated in each stage-specific CSC were significantly associated with distinct clinical subgroups of prostate cancer patients, thus indicating that mouse CSCs define human prostate cancer progression signatures. Although CSCs were killed in vitro by NK cells and cytotoxic T lymphocytes (CTLs), they generated tumors when injected in immunocompetent mice, thus suggesting they possess mechanisms of immune evasion. We are investigating this issue. In the same model as well as in the transplantable B16 melanoma, we have also assessed the ability of dendritic cell-based vaccines to induce a longlasting tumor-specific CTL response in either prophylactic or therapeutic settings. Our results indicate that booster vaccinations are indispensable in prophylactic settings to sustain the pool of vaccine-induced central memory T cells, which associate with potent protection against tumor development. Conversely, booster vaccinations are detrimental in tumor-bearing subjects because negatively impact on central memory T cells. Thus, our results suggest that the need for boosting should be evaluated in any given cancer patient depending on the state of the disease. Matteo Bellone Dynamics of immune responses Unit Our research program seeks to dissect the complex dynamics of host-virus interactions with a particular focus on the development and function of adaptive immune responses. Since it is still beyond the reach of even the most sophisticated in vitro methodology to simulate the complex interplay of physical, cellular, biochemical, and other factors that influence cell behavior in microvessels and interstitial tissues, we make use of intravital microscopy. This technique is complemented by more traditional molecular, cellular and histological approaches, thus characterizing host-virus interactions at the molecular-, single cell- and whole animal-level. Ongoing projects in the lab include: 1) To understand how virus-specific effector CD8 T cells migrate to the liver and recognize intrahepatic antigens in the context of viral hepatitis and hepatocellular carcinoma. 2) To provide the first complete in vivo imaging survey of virus-specific B cell activation, from the first minutes of viral entry into lymph nodes to the generation of high affinity neutralizing antibody-secreting cells and to identify virus-induced mechanisms interfering with neutralizing antibody responses. 3) To characterize the newly discovered adjuvant activity of bisphosphonates 4) To assess the role of antigenic stimulation in chronic lymphocytic leukemia pathogenesis. Matteo Iannacone DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Figure 23. Confocal micrograph of a mouse popliteal lymph node cross-section. CD169+ macrophages are highlighted in red, B220+ B cells in white and LCMV-infected cells in green. Scale bar represents 200 μm. Emerging bacterial pathogens Unit Fighting drug resistant Tuberculosis and other multidrug resistant nosocomial pathogens Drug resistant (DR) bacteria are an emerging plague worldwide. Our research program seeks to understand the mechanisms leading to DR and increased bacterial virulence in two main areas: Tuberculosis (TB) and Health Care Associated DR Pathogens (HCAP). TB: The future universal regimens for TB and DRTB, will be based on three main drugs(rifampicin, moxifloxacyn and pyrazinamide). By Next Generation Sequencing of a large collection of TB strains we have identified panels of genes and mutations associated to resistance to specific classes of antibiotics. For Pyrazinamide we have mapped the mutations on pncA gene and shown that resequencing of this gene is the most accurate method to predict resistance to this drug. We showed that not all mutations confer the same DR level. The progress in biomarkers research has lagged behind that of tuberculosis diagnostics: we have identified and validated a specific signature based on circulating miRNAs able to discriminate people with active TB from healthy controls. Small non-coding RNAs (sRNAs) play an important role in the stress response and the pathogenicity of many bacteria. We identified and published sRNAs in M. tuberculosis. To better understand their role in the response to stress conditions induced by antibiotics, RNAseq approach has been used to provide novel insights in mycobacterial trascriptomic networks (sRNA + mRNA) and showed that selective sRNAs are expressed during exposure to ofloxacin. HCAP: We have previously shown that the MRSA clone ST22-IV is the predominant MRSA clone in OSR. We have identified as factor contributing to his success several virulence properties including biofilm production, growth rate and invasiveness. 153 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units KPC producers Gram negative bacteria have been a major cause of nosocomial infections in 2013. We identified 2 main epidemic KPC-Klebsiella pneumoniae clones, ST512 and ST258. We showed that both clones are able to transfer the blaKPC gene horizontally to E. coli. The spread of such resistance to other species is worrisome and must be closely monitored. C. difficile is a major cause of nosocomial diarrhea. We demonstrated that defensins (HNP-1 HD5) may play an important role during the infection disrupting the bacterial cell membrane. Daniela Maria Cirillo Experimental immunology Unit T cells restricted for CD1 and MHC molecules in the immunosurveillance of tumors Our research proceeds along two main complementary axis addressing the role of T-cell immune responses in the tumor immunesurveillance. 1. Development and function of CD1-restricted T cells specific for lipid antigens We investigate the development and function of CD1d-restricted invariant (i)NKT cells, a unique subset of T lymphocytes with innate effector functions that plays and important role in tumor immune surveillance. Specific miRNAs that control the development and effector functions of iNKT cells were identified and the effects of the modulation of their targets are being investigated. We have also gained further mechanistic insights into the iNKT-cell control of pro-tumor macrophages infiltrating the prostate cancer developing in the TRAMP transgenic mouse model. Furthermore, we have established a clear prognostic and functional role for the iNKT/CD1d axis in CLL, both in patients and in the TCL1 mouse transgenic model. A second type of CD1 restricted T cells recognizes endogenous lipid antigens presented by CD1a, b, c, which are not expressed in mice. We have characterized a new lipid antigen that is enriched in acute leukemia and is recognized by CD1c-restricted T cells with anti-leukemia activity. T cells with comparable reactivity develop in transgenic mice expressing human CD1c, providing a valuable pre-clinical model to study this newly discovered T cell response. 2. Definition of the somatically mutated T cell-defined antigen landscape of colorectal cancer We have further implemented an integrated platform based on next generation sequencing and reverse immunology, to identify somatically mutated genes expressed by colorectal cancer (CRC) and CRC cancer stem cells (CSC), and to define the immunogenicity of the corresponding unique epitopes recognized by both CD4+ and CD8+ T cells. Paolo Dellabona & Giulia Casorati Infection and cystic fibrosis Unit Host-pathogen interactions in cystic fibrosis: implications for pathogenesis and therapy The goal of our program is to dissect hostpathogen interactions during persistent infection in cystic fibrosis (CF) with the aim of devising new therapeutic approaches to treat respiratory infections. 1. Cellular and molecular mechanisms involved in host-pathogen interactions. Polymicrobial infections by Pseudomonas aeruginosa, Staphylococcus aureus and Burkholderia cenocepacia are difficult to treat and responsible for the decline of lung function in CF. Taking advantage of wholegenome analysis, human specimens and unique mouse model of infection, developed within the lab, we have recently demonstrated that these pathogens adapt to the CF niche and often coexist influencing the CF pathogenesis. A repertoire of adaptive changes in the bacterial genome accumulated with the length of persistence in the CF airways and affected the persistent lifestyle of the pathogens in chronic infection. CF bacterial patho-adaptive variants revisit their interaction with host by dampening the inflammatory response and activating pathways relevant for damage and remodelling process. Pathological analysis of murine lungs infected with adaptive strains showed advanced chronic pulmonary disease, inflammation and mucus secretory cells hyperplasia, mimicking many key features of pulmonary DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES exacerbation observed in CF patients. This new knowledge will favor much-needed insight into aspects of bacteria/CF interaction and lead to novel biomarkers and therapeutic approaches. 2. Evaluation of novel molecules for treating respiratory infection and inflammation. Murine model for acute and chronic infection, along with mice genetically modified for the Cftr gene, are a key asset in CF research. The CF Animal Core Facility (CFaCore) provides murine models and supports the CF community testing novel therapeutic molecules. The aim is to favor the translation of basic research projects into pre-clinical applications. Pre-clinical murine models of P. aeruginosa chronic lung infections have been used to demonstrate that the flucytosine, an anti-virulence drug discovered by a drug-repurposing approach, and myriocin, an inhibitor of the sphingolipids, are potential therapeutic tools in controlling infection and inflammation supporting further development in translational applications. Alessandra Bragonzi Figure 24. Non-CF and CF human lungs stained by AB-PAS for globet cells. Leukocyte biology Unit Harnessing oncogene-induced responses in liver tumorigenesis to identify novel therapeutic strategies Aberrant DNA replication induced by deregulated or excessive proliferative stimuli evokes a “replicative stress response” leading to cell cycle restriction and/or apoptosis. This robust fail-safe mechanism is eventually bypassed by transformed cells, due to ill-defined epistatic interactions. The COP9 Signalosome (CSN) is an evolutionarily conserved regulator of Cullin Ring Ligases (CRLs), the largest family of ubiquitin ligases in metazoans. Conditional inactivation of the CSN in several tissues leads to activation of S- or G2phase checkpoints resulting in irreversible cell cycle arrest and cell death. Herein we ablated COPS5, the CSN’s catalyitc subunit, in the liver, to investigate its role in cell cycle re-entry by differentiated hepatocytes. Lack of COPS5 in regenerating livers causes substantial replicative stress, which triggers a CDKN2A-dependent genetic program leading to cell cycle arrest, polyploidy and apoptosis. These outcomes are phenocopied by acute overexpression of c-Myc in COPS5 null hepatocytes of adult mice. We propose that combined control of proto-oncogene product levels and proteins involved in DNA replication origin licensing may explain the deleterious consequences of CSN inactivation in regenerating livers and give insight into the pathogenic role of the frequently observed overexpression of the CSN in hepatocellular carcinoma. Ruggero Pardi 155 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Figure 25. DNA re-replication in CSN-deficient hepatocytes in vivo. Shown is a DNA fiber undergoing aberrant rereplication, as determined by the merged fluorochromes. Arrows denote stalled replication forks. Lymphocyte activation Unit Understanding the cellular and molecular mechanisms at the basis of adaptive T cell immunity Our group is interested in the cellular and molecular events controlling CD4 and CD8 T cell homeostasis, activation, proliferation, survival and differentiation. We take advantage of primary T cell cultures, and predictive murine models allowing the tracking of antigen-specific T cells. Ongoing research activities focus on the TCR-induced intracellular signaling events impacting on T cell proliferation and differentiation, and on strategies to ameliorate T-cell-mediated responses to solid tumors. Over the past year we have analyzed the impact of TSC1 deletion in mouse T cell in vitro and in vivo and also in human T cells, and found that this unbalances the control of the mammalian target Of Rapamycin Complex 1 (mTORC1) and mTORC2, and together with that proper T cell homeostasis. We also characterized immune responsiveness of patients with heterozygous germline mutations of TSC1 to start understanding functional consequences of TSC1 loss in humans.In addition, to ameliorate protective T cell immunity we have exploited the engineering of T cells with TCR directed to tumor-associated antigen and to minor histocompatibility antigen, in combinatorial adoptive and active immunotherapy in the Transgenic Adenocarcinoma of the Mouse Prostate cancer (TRAMP) model. We are currently testing additional combination, including vascular targeting strategies to envisage clinical translation of the defined approach for the immuno-therapy of refractory solid tumors. Anna Mondino Tumor immunology Unit Role of the different CD4+ T cell subsets in tumor immunity CD4+ T helper (Th) cells exist in several subsets, such as Th1, Th2, Th17 and Th22, which are defined based on the profile of cytokines produced (IFN-γ for Th1; IL-4, IL-5 and IL-13 for Th2; IL-17 and IL-22 for Th17; and IL-22, IL-13 and TNF-α for Th22, respectively). CD4+ T cells are frequently found within tumor immune infiltrates and it is now well established that infiltration by Th1 cells DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES associates with favorable clinical outcome while predominant Th2 infiltration is mostly associated with tumor progression. Conflicting results are reported for the Th17 subset, depending on the tumor studied, and very little is known about Th22 cells, which have been recently identified as a independent Th subset. Polarization towards one or another CD4+ Th cell subset depends on the cytokine(s) milieu present in the tumor and in draining lymph nodes, where CD4+ T cell priming occurs. Thus, the network of cytokine/chemokine in the tumor microenvironment dictates polarization of CD4+ T cells towards the different subsets that in turn, through cytokine release, may further condition the tumor microenvironment with reciprocal influence. Our studies are focused on pancreatic cancer and multiple myeloma (MM). Specific aims are: a) to evaluate tumor infiltrating CD4+ T cell subsets and to define possible correlation between the features of the Th subsets identified and the clinical outcome (i.e., predictive markers) and to study b) cells and mechanisms dictating Th polarization and c) how the different CD4+ T cell subsets exert pro- versus anti-tumor activity in these pathologies. Our studies are conducted in collaboration with surgeons, clinical oncologists and pathologists of the Institute. In the last year we focused on the study of the relevance of the different Th subsets in both peripheral blood and bone marrow aspirate of MM patients with asymptomatic disease, MM patients at diagnosis and MM patients with relapsed/refractory disease. We also continued experiments to deepen our understanding of the cells and factors involved in driving Th2-type inflammation in pancreatic cancer. Maria Pia Protti Viral evolution and transmission Unit Antigenic and phenotypic characterisation of HIV-1 variants in transmission and disease progression as target for vaccine development New immunogens capable of inducing broadly neutralizing antibodies (bNAbs) are a priority in HIV-1 vaccine research. We hypothesized that viral variants from patients, who display bNAb responses, may harbor specific Env structures that could elicit bNAbs if formulated into an appropriate vaccine immunogen. Thus, we studied the kinetics of Nabs and antibodies reactive to HIV-1 Env constant and variable regions as well as childhood vaccine antigens from birth on for five years of follow up in infected children with rapid or slow disease progression. We show that newborns display antibodies directed towards HIV-1 immunodominant, constant epitopes, however, antibodies do not neutralize the transmitted virus. The Nab response directed to the autologous transmitted/founder virus tends to increase during follow up, induces continuous emergence of escape variants and appears in parallel with antibody responses to peptides representing the Env variable region of transmitted/ founder virus, suggesting that these might contribute to the autologous Nabs response. Interestingly, Nabs to heterologous viruses may be developed within 2 years of age, but their subsequent increase in potency and breadth is a trait of slow progressors. However, kinetics of antibody responses to the viral immunodominant constant and childhood vaccination antigens is preserved and independent of disease progression. Thus, immunocompetence appears essential for the ability to mature the Nab response. Furthermore, transmitted/founder viruses of slow progressors may represent an attractive target for vaccine design, which aims to induce cross-Nabs, and therefore we selected the Env of two transmitted/founder viruses to produce Env gp140 trimers for vaccination in rabbits. Comparison of their immunogenicity side-by-side with other five gp140 trimers selected from adult infected individuals showed that all groups of immunized rabbits develop antibody reactivity to Env, which is followed by Nabs against heterologous HIV-1. Thus, we have selected new Env from transmitted/founder viruses of children suitable for immunization in animals to study protection from transmission. Gabriella Scarlatti 157 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Viral pathogens and biosafety Unit Restriction factors of Immunodeficiency virus (HIV) and Influenza A virus replication: role of tripartite motif containing protein 22 (TRIM22) Replication of the human immunodeficiency virus (HIV) is heavily dependent on components of the host cell machinery. These may encompass active resistance governed by innate immune circuits that aim at eliminating pathogen burden and collectively defined as “restriction factors (RF)”. In addition, a group of constitutively active cellular components can curtail the levels of pathogen replication (“set point factors, SPF”). We hypothesize that these latter enable the host to tolerate infection by attenuating the cytopathic effects of unrestricted replication. While viruses almost invariably have developed countermeasures to cellular RF, some, but not all, SPF appear to be targeted for viral inactivation. The activities of both host cell viral resistance factors contribute to determining the capacity of a virus to establish a pathogenic infection in the host. We have previously shown that the interferon-inducible Tripartite Motif-22 (TRIM22) acts as SPF for HIV-1 by inhibiting HIV-1 replication by acting at the transcriptionl level (A. Kajaste-Rudnitski et al., J. Virology 2011). We are currently determining the molecular mechanisms by which TRIM22 exerts these functions and have identified two single nucleotide polymorphisms (SNPs) in the coding region of TRIM22 and located in the coiledcoil domain of the protein that enhance its capacity to inhibit HIV transcription. Furthermore, we have reported that these TRIM22 isoforms are associated with the severity of HIV disease (S. Ghezzi et al., AIDS 2013). In addition to HIV-1, we have demonstrated that TRIM22 can also restrict influenza A virus (IAV) infection (A. Di Pietro et al., J. Virology 2013). In this case TRIM22 mechanism of restriction relies on its E3-ubiquitin ligase activity upon interaction with the viral nucleoprotein that promotes its degradation in a proteasome-dependent fashion. We are currently studying the potential role of TRIM22 in the acquisition of nucleoprotein adaptive mutations during human-to-human viral spreading also in the context of understanding the nature of pandemic IAV infection. Elisa Vicenzi DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units Management and antiretroviral treatment of HIV infection Clinical Trial Unit (CTU) is a clinical research unit at the Department of Infectious Diseases, focused on addressing clinically relevant questions that may improve HIV patient care. Cohort Studies: CTU is collaborating with national and international cohorts studies through the Infectious Diseases Database (IDD-OSR) with the aim to conduct epidemiological research on the prognosis and outcome of HIV-infected people from across Italy and Europe including adults, children and pregnant women. The research is focus on scientific questions requiring a large sample size of patients which the contributing cohorts cannot answer individually. The IDD-OSR is an observational, longitudinal, clinical database on adult patients receiving primary HIV care at the Clinic Infectious Diseases. This database was developed in 1999. IDD-OSR manage a wide set of data of 8821 recorded patients. Clinical Trials: CTU is involved in conducting and coordinating national and international industry sponsored phase II-IV clinical trials. Main activities contribute to the registration of new compounds, new indications and to the organization of early access program for patients with limited therapeutic options. Randomised clinical trials are focused on two research areas: 1) Development of new antiretroviral strategies in order to overcome HIV resistance and to favour patient quality of life. In the next tree years we will study new drugs for the treatment of naïve and experienced patients; we will evaluate efficacy and safety of simplification strategies in patients with suppressed viral replication, salvage strategies in patients resistant to all the standard therapies holding regimens based on lamivudine monotherapy in failing patients harbouring the M184V mutation 2) Long-esposure to antiretroviral drugs and HIV resistance are frequently associated with longterm non HIV complications such as cardiovascular risk, cirrosis, cancer, diabetes. In the next three years CTU will contribute to evaluate pathogenesis, diagnosis, therapy and prevention of type 2 diabetes during HIV infection. Research activity of CTU led to 45 scientific publications during 2013. Antonella Castagna Neurovirology Central nervous system (CNS) HIV infection. HIV brain infection may be associated with neurocognitive impairment (NCI); in addition CNS cells are a potential viral reservoir. We are characterizing mild NCI by radiological and cerebrospinal fluid (CSF) and blood markers. In neuro-asymptomatic untreated patients, MR-DTI showed white matter microstructural alterations that did not correlate with NCI, suggesting that NCI may be associated to HIV-unrelated conditions. Alterations did not improve after years of therapy, providing a marker of subclinical tissue injury in chronic infection. By the study of cases of “CSF viral escape”, i.e., CSF HIV replication despite no or low systemic replication, we identified different HIV populations between the two compartments, indicating that HIV may evolve autonomously in CNS and supporting the hypothesis of CNS as virus reservoir. Progressive multifocal leukoencephalopathy (PML). PML is a progressive demyelinating disease caused by JC virus (JCV) reactivation and infection of oligodendrocytes that affects patients with impaired immunity or taking immunomodulant drugs. We are investigating a pathogenesis model by which JCV escapes the host immune system by selection of mutations in its major surface protein VP1. We identified several immunogenic VP1 peptides binding with high affinity with most HLA-DRB1 molecules and showed ex-vivo IFN-γ Elispot responses against wildtype, but not mutated peptides. Similarly, analysis of serum from PML patients revealed the presence of neutralizing antibodies against wild-type, but not mutated viruses. These findings may be relevant for developing immune-based treatments and vaccines. Chronic inflammation in HIV infection. Chronic diseases are a major cause of mortality and disability in treated chronic HIV infection and are associated with persistent low-grade inflammation. Following a moderate intensity physical exercise program we observed significant improvements of plasma IL-6, hsCRP, D-dimer, IL-18 and myostatin, and of CD8+/CD38+/HLA-DR+ cell frequencies, in parallel with improvement of morphometric measures, and blood lipids. These findings are prompting us to develop protocols for exercise as treatment of chronic inflammation in HIV infection. Paola Cinque 159 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units Study and treatment of hepatotropic viruses related diseases Dynamics of mixed HCV infection in different compartments, plasma and peripheral blood mononuclear cells, during treatment with Peg-IFN plus ribavirin in HIV/HCV coinfected patients: possible implication for treatment in the era of direct antivirals According to the World Health Organization, 130 to 170 million people are persistently infected with hepatitis C virus (HCV) and are at risk of developing severe liver disease and hepatocellular carcinoma. HCV is an enveloped, positive strand RNA virus belonging to the Hepacivirus genus in the Flaviviridae family. Seven confirmed genotypes are generally distinguished by phylogenetic methods and pair-wise distance calculations. It is well known that antiviral treatment with pegylated interferon (P) plus ribavirin (R) at initiation of HCV infection achieves sustained virological response (SVR) rates of 30-50% in HIV/HCV coinfected individuals, depending by the infecting genotype. In clinical trials a number of direct antivirals (DA) in association or not with P-R (figure1)showing a dramatic increase of SVR in HCV mono infection as in HIV/HCV coinfection, will revolutionize the HCV treatment.Virological studies suggested that drug resistance variants to DA may pre-exist as minor quasispecies and can serve as a rapid source of drug resistance. We have studied this issue and showed that natural resistant strain may revert to wild type, and that resistant strain may be present in liver compartment irrespective of wild type in plasma compartment. These ourfindings hampered the importance to perform the genotyping resistanceassay beforeanti-HCV treatment. The majority of patients attending our Department,need new drugs treatment becausefailed PR treatment. In this context, it is unknown the effect of P-R treatment on the virus heterogeneity in HIV/HCV coinfection. We investigated the viral population by ultra deep pyrosequencing before and during HCV treatment with P-R and surprisingly we found a high frequency of mixed HCV genotype with alternance of different genotypes inplasma and peripheral blood mononuclear cells. Failure to P-R treatment was associated with mixed infection in plasma compartment. These findings may have important clinical implications in light of the new therapeutic approaches in HIV/HCV coinfected individuals that may harbour unrecognised mixed infection, suggesting that triple therapy including DA could be ineffective in individuals with mixed genotypes. Caterina Uberti-Foppa Figure 26. HCV genome organization and target regions of direct antivirals compounds. DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Vaccine and immunotherapy Treatment of primary HIV infection The potential benefit of an early intervention could pave the way for future therapies aimed at HIV eradication, are currently widely studied In patient with primary HIV infection (PHI). An early treatment could potentially preserve the HIV-specific immune response, thus increasing the chances of a better control of viral replication. Examples of HIV-1 positive individuals treated in PHI who maintain viral suppression when ART is stopped have received much attention as the mechanisms behind ‘post-treatment control’ (PTC) might inform strategies for achieving drugfree remission. The timing and duration of therapy required to induce PTC is unclear, as are host and viral factors that may contribute. The SPARTAC (Short Pulse ART at Seroconversion) trial is the largest international randomised study of short-course therapy in PHI (N Engl J Med. 2013 Jan 17;368(3):207-17) The impact of therapy at PHI on subsequent viraemia and clinical progression remains a clinically and mechanistically important question. SPARTAC demonstrated clinical benefit and suppression of viraemia with 48 (but not 12) weeks of short-course ART (PLoS One. 2013 Oct 25;8(10):e78287) CCR5 is pivotal both as co-receptor for the majority of HIV quasispecies and as part of the inflammatory response cascade, and several authors suggested a possible role of maraviroc (MVC) as immune modulator. B-cells compartment is also impaired since the earliest phases of infection and these alterations are not restored by ART initiated during chronic infection (7th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Kuala Lumpur, Malaysia, 30 June-3 July 2013; abs# WEPE449). We conducted a randomized proof of concept clinical trial aimed at assessing whether ART intensified with MVC initiated during PHI would be harnessed to improve immunological and virological parameters (Keystone Symposia: Immune Activation in HIV Infection: Basic Mechanisms and Clinical Implications. April 3, 2013 - April 8, 2013 - Beaver Run Resort - Breckenridge, Colorado, USA; abs# 3032). In addition, we investigated the influence of ART on the immune system perturbation established in this early phase of HIV pathogenesis (paper submitted). Giuseppe Tambussi Clinical immunopathology and advanced medical therapeutics Unit Systemic inflammatory diseases represent a major unmet medical need: despite the growing insight on the molecular basis of the immunological response, our understanding of the events associated to autoimmunity in each given patient remains scant and the treatment are mainly selected based on empirical knowledge. A more lucid insight on the molecular events underlying the pathogenesis of these diseases is required to identify diagnostic and predictive markers of activity, organ involvement and outcome. In the last period we have specifically focused on the characterization of clinical features associated with the prototypic systemic autoimmune disease, Systemic Lupus Erythematosus (SLE) with attention to the kidney involvement and to the associated pregnancy complications; the identification and validation of novel markers to assess the activity of systemic large vessel vasculitides, Takayasu’s arteritis in particular, and the validation of novel imaging approaches and possibly of novel criteria to assess disease activity; the immunopathogenesis of fibrosis and vascular damage in systemic sclerosis and associated conditions, with attention to the molecular determinants involved in the leukocytes/endothelial cells/ platelets interaction; among fibroinflammatory conditions we are also particularly interested in immunoglobulin G4 (IgG4)-related disease, a recently described immune-mediated disease that can cause progressive fibrosis and damage of virtually every organ. Maria Grazia Sabbadini 161 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units Clinical transplant Unit The main fields of research activities of the Unit are focused on: • Islet transplantation (see description in the DRI) • Chronic allograft nephropathy (CAN) after kidney transplantation • Cell therapy in living-donors kidney transplantation (The One Study) CAN CAN remains the leading cause of late graft loss after kidney transplantation. Characterized by progressive tubular atrophy and interstitial fibrosis (TA/IF) as well as microvascular and glomerular damage in the setting of declining graft function months to years after transplantation. • Group 1: patients with normal renal function • Group 2: patients who underwent kidney transplant and developed a chronic allograft nephropathy (CAN). We are now monitoring the following parameters: 1) renal function (eGFR, serum creatinine, urine exam) 2) kidney biopsies. 3) Immunosuppression level. 4) Cytokine profile and inflammation. The ONE Study (www.onestudy.org) This is a large-scale, collaborative project funded by the Seventh Framework Programme (FP7) of the European Commission. The consortium is composed of academic institutions and industrial partners. It is aimed to test several distinct purified haematopoietic immunoregulatory cells as clinical therapies in solid organ transplantation by initiating a series of independent clinical trials. The ultimate goal is the reduction of long-term immunosuppressive therapy. Reference group trial: It is aimed to investigate the progression of the immunological response in living-donor kidney transplant recipients treated with a standard immunosuppressive regimen. Cellular therapy group At San Raffaele it will be based on TR1 cell therapy (MariaGrazia Roncarolo and Manuela Battaglia). The clinical protocol is under development. Clinical trials already ongoing: 1. RAD001 multicenter, randomized, open label study to evaluate the impact of early vs delayed introduction of everolimus on wound healing in kidney transplant (NEVERWOUND) 2. A phase 2 multicenter pilot study to assess the efficacy and safety of reparixin following islet transplantation. 3. Planned Transition to Sirolimus-based Therapy Versus Continued Tacrolimus-based Therapy in Renal Allograft Recipients 4. Pilot study to assess feasibility and safety of bone-marrow as alternative site in islet transplant. Antonio Secchi Pancreatic tumors Unit: immunotherapy and β cell function substitution Autologous Islet Transplantation (AIT) is performed to improve glycaemic control after extended pancreatectomy. In our center, in addition to chronic pancreatitis, indications for IAT are: grade C pancreatic fistula (treated with completion or left pancreatectomy); total pancreatectomy as an alternative to high-risk anastomosis during pancreaticoduodenectomy; distal pancreatectomy for benign/borderline neoplasm of pancreatic bodyneck. Malignancy is not an exclusion criterion. In 34 transplanted patients, insulin independence was reached in 44% of cases and 47% had partial graft function. Seventeen IAT recipients had malignancy (14 had pancreatic or periampullary adenocarcinoma) and two of them had already liver metastases at surgery. After a median follow- up of 750 days, thirteen patients were diseasefree and only one developed metastases in the transplantation site. Another important result achieved in 2013 was the first unequivocal successful engraftment of endocrine tissue in the Bone Marrow (BM) in humans. Four patients who developed diabetes after total pancreatectomy were candidates for autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable post-transplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Gianpaolo Balzano Gynecological cancers immunology Human papillomavirus (HPV) infection and cervical cancer Main field of investigation of the Clinical Research Group is the correlation between Human Papillomavirus (HPV) and cervical cancer. In cooperation with the Tumor Immunology Unit we focused on HPV-18 and found that low frequencies of CD4+ T cells specific for the E6 and E7 proteins are present in the majority of HPV-18+ CIN patients. We also found HPV-18-specific CD4+ T cells in 50% of patients tested, irrespective of the presence of HPV-18 in their lesions, suggesting that HPV-18-responsive patients might have cleared the infection. Consistent with this hypothesis, we found that a robust Th1/Th2 immune response against E7 but not E6 was associated with the HPV-18- status. On the other hand, a robust Th1/Th2 immune response against E6 but not against E7 correlated with lack of relapses. Our data demonstrate a positive role for anti-HPV-18 E6 and E7 CD4+ T immunity in CIN patients. Moreover, we found a significantly higher relapse rate of preneoplastic lesions (CIN2 - CIN3) after surgical treatment in cases with reduced CD4,CD8,CD11C,T-bet and GATA-3 in the cervix. On the other side, high levels of CD4+ and GATA-3+ in cervical tissues correlated with disease regression. These data strenghten the tight correlation between immune response and HPVrelated carcinogenesis.Recent experiences of the group have lead to the publication of interesting results concerning the correlation between HPV infection and HPV-induced cervical lesions in graft recipients; long-term follow up in multi-drug immunosuppressed transplanted women did not correlate with an increased prevalence of HPV genital diseases. A recent cooperation with the AIDS Immunopathogenesis Unit started and preliminary results, already published in outstanding journals, indicate that productive HIV infection in female cervical tissue samples is more likely to occur in the secretive phase of the menstrual cycle. In 2014 a new project of vaginal microbiome analysis in cases of HPV-correlated cervical intraepithelial neplasia will start. Ongoing research projects deal with: • Cervical mucosal immunity; • HPV DNA identification; • Analysis of cervical immune subpopulations. Massimo Origoni Immunology in liver neoplasms Chemotherapy associated liver injury and major hepatic resection for colorectal liver metastases: role of APRI score and endothelin-1 levels. A prospective study Chemotherapy associated liver injury (CALI) is recognized as a risk factor for development of liver failure (LF) following hepatic resection. APRI score, based on preoperative AST level and platelet count, has been proposed as a marker of oxaliplatin induced parenchymal damage in patients affected by liver metastases. Endothelin-1 is a vasocostrictor peptide which might play a significant in PLF pathogenesis, as a mediator of acute phase shear stress. Aim of this study was to evaluate correlation between APRI score and Endothelin-1 level in pre- and postoperative period. Patients who required major liver resection for CLM between 1st January 2011 and 31st June 2013 and who had undergone a period of preoperative oxaliplatin based CT entered this prospective study. In all of them preoperative APRI score was calculated. In all the patients liver function tests including AST, ALT, bilirubin and PT were dosed preoperatively and in POD1, 2 and 5, as well as ET-1 serum levels. 26 patients entered the study. They received meanly 6 ± 2 cycles of CT with a mean interval of 31 ±5 days from surgery. 13 (50%) were treated with bevacizumab. 14 of the whole series (53.8%) underwent right hepatectomy, 4 (15.4%) right trisectionectomy, 6 (23.1%) left hepatectomy and 2 163 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units (7.7%) left trisectionectomy. 12 (46.2%) patients underwent portal vein occlusion before surgery. Mortality was nil. Morbidity was 38.5%: 4 patients (15,4%) showed signs of LF. Mean APRI score of the entire series was 0.59 ± 0.13. Patients developing PLF had a significantly higher APRI score (0.66 ± 0.11 vs 0.49 ± 0.12, p<0.05), number of CT cycles (9 ± 2 vs 5 ± 2, p<0.0) and baseline ET-1 levels (18 ± 4.4 vs 9.7 ± 3.2, p<0.05) compared to others. Pearson analysis showed a significant correlation of APRI score with preoperative and POD2 ET-1 levels and with POD5 ET-1. Correlation of APRI with pre and postoperative ET1 may reflect parenchymal damage induced by CT, resulting in a higher baseline risk of LF. Patients with elevated APRI and ET-1 levels should therefore be evaluated with caution when a major resection is planned. Luca Aldrighetti Obesity and bariatric surgery Laparoscopic gastric banding for morbid obesity Laparoscopic adjustable gastric banding (LAGB) is performed in case of primary obesity with Body Mass Index (BMI) >40 or BMI >35 with concomitant serious medical obesity-related conditions, unresponsive to dietary treatment. Preoperative evaluation included screening for endocrine disease, endoscopic and radiological study of the upper digestive tract and ultrasound study of the upper abdomen. Between June 1996 and December 2013, 450 patients (78 males/372 females, ranging 18-65 years) received LAGB. Mean body weight was 116.0 ± 18.8 Kg (range 90-195 Kg), with a BMI ranging between 35 and 65.7 (mean 42.8 ± 5.9). The laparoscopic procedure was completed in 441 patients (98 %). Eight cases (2.4%) had to be converted to the laparotomic procedure either because of hepatomegaly (1 case) or because of gastric lesion during the laparoscopic approach (7 cases). In one patient the procedure was performed with open technique together with ventral hernia repair. Michele Paganelli Clinical hepato-gastroenterology Manipulation of the gut microbiota in the treatment of chronic intestinal disorders The role of the gut microbiota in promoting and maintaining intestinal inflammation is by now well recognized and the object of a great deal of speculation and research, both in the basic science and in the clinical area. Antibiotics such as ciprofloxacin and metronidazole can be effectively employed in the treatment of inflammatory bowel disease, but side effects are common. Both non-adsorbable antibiotics such as rifaximin and probiotic agents can represent a safer alternative. Probiotics have been tested in the treatment of ulcerative colitis and Crohn’s disease (both in the acute phase and in the maintenance of remission) with variable but promising results. The choice of the specific probiotic agent is a critical point because the mechanisms of action of the various Lactobacillus strains, Bifidobacteria or yeasts such as Saccharomyces boulardii are quite different. To further expand our previous published experience in this area, we are currently evaluating the precise role of single probiotic agents, probiotic mixtures and rifaximin as well as symbiotics (combinations of prebiotics and probiotics) in the short and longterm treatment of chronic intestinal disorders both of overt inflammatory nature (ulcerative colitis and Crohn’s disease) and where microscopic inflammation may have a pathogenetic role (microscopic colitis, irritable bowel syndrome). In addition we are endeavouring to identify and develop proper study protocols in order to evaluate the efficacy of probiotic agents in clinical trials carried out according to a correct methodology able to generate sound evidence-based data. Mario Guslandi DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Digestive pathophysiology The gastro-esophageal-laryngopharyngeal reflux: from diagnosis to therapy Gastroesophageal reflux disease (GERD) is defined as a condition that develops when reflux of stomach contents causes troublesome symptoms and/or complications. The manifestations of GERD are classically described as heartburn and reflux, which are often referred to as “typical GERD”. However, GERD may also present atypically and is referred to as extraesophageal syndrome. Common extraesophageal manifestations are hoarseness, globus, throat clearing, and cough; many evidences show that the gastroesophageal reflux (GERD) may play a pivotal role in the pathogenesis of the symptoms the so called laryngopharyngeal Reflux (LPR). Diagnostic approach to patients with extraesophageal reflux disease involved the use of insensitive tools, which have hampered the ability to correctly identify patients at risk. In fact, empiric trial using proton pump inhibitors (PPI) is still the recommended initial approach to the patients suspected of having reflux as the cause for extraesophageal symptoms. However, research shows conflicting evidence of utility of empiric acid suppression for these symptoms and an objective test is necessary. The ambulatory impedance/pH monitoring performed on or off therapy is the best test for studying the gastro-esophageal reflux but can be misleading when the pH is measured in the pharynx. In addition, up to now is unexplained why in some patients the reflux reach the proximal esophagus. Our aim is to study in this patients the esophageal motility with a new manometric system with 36 pressure sensors that allows to perform an high resolution manometry. In addition we study the proximal reflux with a new pH catheter for the detection of oropharyngeal acid reflux. The Restech pH catheter uses a nasopharyngeal catheter to measure the pH in either liquid or aerosolized droplets. The goal of the study is to understand the physiopathology of the gastroesophageal-laryngopharyngeal reflux. Sandro Passaretti 165 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Introduction by the Directors DRI Diabetes Research Institute Type 1 diabetes is a disease resulting from the inability to produce insulin, a hormone essential to regulate glucose and the entire metabolism. The cause of insulin deficiency is the destruction of insulin producing β cells located within the islets of pancreas by the immune system. This process is known as autoimmune disease and the cause is still unknown. Type 1 diabetes, if left untreated, represents a potentially lethal disease. Type 1 diabetes is predominantly diagnosed in children and young adults and requires life long multiple daily injections of insulin and blood glucose monitoring by way of finger pricks for survival and control. This constant burden greatly affects the quality of life of both patients and family members. Moreover, despite major and constant advancements of insulin therapy and glucose monitoring, the normalization of blood glucose over the 24 hour period is not an easy goal to achieve and maintain. In Europe, the number of new cases is currently two to four times more than in the 1950s. In mainland Italy, the incidence of type 1 diabetes is 6 to 10 cases per 100.000 inhabitants per year in the 0-14 age group, whilst in Sardinia the incidence is four times higher. This means that in Italy 4 children or adolescent develop diabetes every day. At present, there is neither a cure nor prevention for type 1 diabetes. At San Raffaele research in diabetes and related topics has been consistent and innovative over the years. With the objective of reinforcing and expanding the commitment on diabetes research, in 2007 a specialized Research Institute entirely devoted to type 1 diabetes, denominated Diabetes Research Institute (DRI), was established. Formally incorporated within the Division of Immunology, the DRI is an independent Research Institute The overall objective of DRI is to prevent and cure T1D. To achieve this objective, two specific programs are pursued, both of which take advantage of patients and animal models: 1) Prediction and prevention of T1D: this program aims to define mechanisms of induction and perpetuation of autoimmunity and to develop strategies for halting/reverting the progression to T1D. 2) β cell replacement and cell therapy in T1D: this program pursues novel strategies promoting immune tolerance and islet-regeneration to prolong the survival of native and/or transplanted β cells. DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units, DRI Experimental diabetes Role of innate immune cells in the pathogenesis of autoimmune type 1diabetes The pathogenesis of autoimmune diseases such as Type 1 Diabetes (T1D) is regulated by genetic and environmental factors. Those factors act through modulation of innate immune cell subsets that are crucial for the immune system’s decision to initiate autoimmunity or tolerate self-antigens. Our research is focused on studying the mechanisms through which environmental factors affect the pathogenesis of autoimmune T1D. Specifically, we aim at investigating how environmental factors modulate autoimmunity by acting on three innate immune cell subsets that play important immune regulatory function such as dendritic cells (DCs), natural killer T cells (iNKT cells) and mast cells (MCs). We recently demonstrated that intestinal DCs of T1D patients are defective in their tolerogenic function and fail to induce peripheral differentiation of regulatory FoxP3+ T cells. We are now investigating in human T1D patients as well as in pre-clinical models of T1D, how alteration of the gut microbiota induced by diet modifications and gluten-induced inflammation, affect Teff/Treg cell ratio by modulating the tolerogenic potential of intestinal DCs. iNKT cells represent an important innate immune cell subsets that play modulatory function in T1D. We recently demonstrated that regulatory iNKT cells contribute to maintenance of immune tolerance through the induction of tolerogenic DCs and in the past year we performed experiments with reporter mice (iNKT cells purified from Ds-Red mice and injected into CD11cCreXRosa26:EYFP) to analyze by confocal microscopy and 2P intravital microscopy the in vivo dynamics of the iNKT-DC interaction. MCs represent the third innate immune cell subset that we are currently studying to assess its role in T1D pathogenesis. By using a conditional MC deficient murine model (mcpt5-Cre-iDTR mice) backcrossed into the NOD background we have demonstrated that MC are actively recruited at the site of autoimmune inflammation, i.e., the pancreatic islets of diabetic NOD mice, fail to acquire a tolerogenic IL-10-secreting phenotype upon Treg cell- modulation and contribute to T1D pathogenesis by secreting IL-6 and favoring Th17 cell differentiation (Betto et al. manuscript under revision). Marika Falcone β cell biology Unit Our project aims to create favorable conditions for the survival and expansion of β cells under native conditions and after transplant. Specifically we will: 1) Improve islet transplantation. We started a phase 3, international, multicenter, randomized, double-blind trial to evaluate the efficacy and tolerability of an inhibitor of CXCR1/2 receptors after islet transplantation in patients with T1D (NCT01817959). We have completed the retrospective observational study aimed at understanding the role of allograft rejection and recurrence of autoimmunity in long-term clinical outcome of islet transplantation, showing that the increase in DSA or autoantibodies after islet transplantation is an important prognostic marker and its identification has the potential to improve the outcome of islet cell transplantation. We started a randomized clinical trial (NCT01722682 ) to evaluate the safety and effectiveness of bone marrow as a site of islet transplantation. We have completed a pilot study to evaluate the possibility of using the autologous islet transplantation (IAT) in a population of patients undergoing pancreatectomy (NCT01702051). 2) Create insulin-producing cells starting from stem cells. This part of the project investigates the possibility of isolating stem cells from adult/fetal tissue and assesses their differentiation potential toward the insulin-secreting cell phenotype. The main expected result is a renewable source of cells that can be used to augment or replace the transplantable β cell mass. We are actively studying as β cells alternative source: pancreatic CD133+ cells and reprogrammed fibroblasts. 3) Identify the mechanisms of immunization against β cell autoantigens. The objective is to determine the mechanisms of immunization resulting in the production of pancreatic islet autoimmuniy. Among the various hypotheses under consideration, we are currently evaluating the role of the human influenza virus in the processes of development of diabetes mellitus type 1. Lorenzo Piemonti 167 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units, DRI Figure 27. Human pancreatic islet (brown:insulin staining). Immune-mediated diseases Unit: from pathogenesis to treatment Aims: Ultimate goal of our group is to better understand immune-mediated diseases (i.e., autoimmune type 1 diabetes (T1D) and rejection after transplantation) and to define new approaches for their prevention and/or cure. Specifically we aim at: a) characterize the immune responses occurring in the target organ of T1D patients; b) dissect the immunological players in charge of T1D development; c) understand the immunological functions of autoimmunity-predisposing genes; d) explore the mechanisms of transplant tolerance and e) perform clinical trials with regulatory lymphocytes to re/establish immune tolerance. Achievements: a) Pancreatic lymph nodes were collected from patients with T1D. The genomic and miRNA profiles of T regulatory (Treg) and effector cells have been delineated. miRNA-specificaly expressed only in Tregs isolated from PLN of patients with T1D and not in their peripheral blood or in healthy controls have been found and represent an interesting therapeutic target. b) Neutrophils are reduced in individuals at risk of developing T1D and at onset and are localized in their pancreas. c) PTPN22 is a protein tyrosine phosphatase expressed by the majority of the immune cells and polymorphisms in PTPN22 are associated with autoimmunity. A novel role for PTPN22 in mediating transplant tolerance is under active investigation. d) We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3+ Treg and Tr1 cells. We have now showed that these Treg cells exert their regulatory function at different time points after transplantation and from different sites. e) A clinical trial with donor specific T regulatory type 1 (Tr1) cells is under development for preventing allograft rejection after kidney transplantation (THE ONE study). Conclusions: a) A specific genomic and miRNA signature is present in Treg cells; b) the innate immunity is involved in the pathogenesis of T1D; c) a previously unreported role for PTPN22 in transplant tolerance is becoming evident; d) a novel relationships between Treg-cell subsets after transplantation is now clear; and e) Tr1 cells are going to be used in vivo after transplantation in patients. Manuela Battaglia DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units, DRI Prevention in Type 1 diabetes Unit This line of investigation is pursued within TrialNet, an international consortium aiming at the prevention or delay of type 1 diabetes. TrialNet is a NIHsupported network of clinical centers, investigators and core support facilities in the US, Canada, United Kingdom, Italy, Finland and Australia. In 2002, with the support of the Juvenile Diabetes Research Foundation (JDRF) a TrialNet Clinical Center was established at San Raffaele. TrialNet researchers are working to better understand the natural history of the disease, identify persons at risk, and evaluate novel therapies. The fundamental goal of TrialNet is to interdict the T1D disease process by immune modulation and/or enhancement of β cell proliferation and regeneration. Studies currently ongoing at the San Raffaele TrialNet Center include: the “Pathway to Prevention Study”, investigating the natural history of development of type 1 diabetes and the identification of individuals at risk; and the “oral insulin trial” for the prevention of diabetes in relatives at risk. Based on the same JDRF-funded infrastructure, other clinical trials, in addition to those performed within TrialNet, are excepted to be performed in the next future in new onset type 1 diabetes. Emanuele Bosi Epidemiology & data management The mission of the Epidemiology and Data Management Core is to provide methodological support to investigator-initiated clinical research and translational research projects within the Department of Immunology, Transplantation and Infectious Diseases (including the Diabetes Research Institute and the Islet Transplant Program). In the past year the Epidemiology and Data Management Core has been providing support to the following major projects: • Islet autotransplantation: a randomized multicenter clinical trial to assess whether total pancreatectomy with islet autotransplantation is a superior alternative to pancreaticoduodenectomy in patients at very high-risk of complications of the pancreatic anastomosis (soft pancreas and pancreatic duct diameter • Self-monitoring of blood glucose in Type 2 diabetes: after publishing the main results of the PRISMA Study, we are working on additional analysis on the effect of self-monitoring of capillary glucose on the prescription of diabetes medication, post-prandial glucose control, and impact on quality of life and locus of control. • Diabetes & pregnancy: our clinical research projects in diabetes and pregnancy include observational studies on the fetal and maternal outcomes of pregnacies complicated by gestational diabetes in first generation immigrants, incidence of diabetes or impaired glucose tolerance post-partum in women with a history of gestational diabetes, transition from the twostep to the single step strategy for the screening of gestational diabetes. The Epidemiology and Data Management Core is also conducting a study on the screening practice for the screening of gestational diabetes in the population of the Lombardy region. Current screening practice and compliance to screening guidelines has never been documented at a population-based level. In collaboration with the Department of Biostatistics of the University of Milano Bicocca and the Unit of Maternal and Fetal Health we are analyzing the regional Health Care System archives, including the Certificate of Delivery Care (CEDAP) archive to document the screening practice for gestational diabetes in over 360.000 pregnancies in the Lombardy Region in the period 2007-2010. Marina Scavini Islet transplantation The Clinical Islet Transplantation Program activity has continued the project of developing translational clinical trials. The close interaction between Clinical Unit and β Cell Biology Unit is the dominant trait of this program. On going experimental protocols: • Phase 2 single center, randomized open study: bone marrow versus liver as site for islet transplantation in patients with type 1 diabetes: pilot study to evaluate efficacy. 3 patients received islet into bone marrow; 2 patients received islet into the liver as control. • A phase 3, multicenter, randomized, doubleblind, parallel assignment study to assess the 169 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical Research Units, DRI efficacy and safety of reparixin in pancreatic islet transplantation. 8 patients were enrolled; 6 patients received completed transplant . Previous experimental protocols: The patients recruitment of 2 pilot studies that had been started up in previous years was completed; the follow-up was ongoing: • Intra bone marrow islet infusion in patients not eligible for islet infusion in the liver • A phase 2 multicenter, randomized, open label, parallel assignment, pilot study to assess the efficacy and safety of reparixin following a singleinfusion of islet in patients with type 1 diabetes mellitus: 9 patients were included at our center, they received islet transplant and the infusion of reparixin. The follow-up of patients who received islet allotransplant in previous clinical trials and islet autotransplant protocols has been continued. Followup of clinical trials: Islet Allotransplant • Edmonton protocol • Pre transplant rapamycin treatment associated to the Edmonton protocol • Calcineurin inhibitor free immunosuppression protocol • Effect of islet transplant alone on long-term diabetes complications (early retinopathy,neuropathy, carotid artery disease) • Prospective study of kidney function and incidence of cancer in islet transplant alone • Islet and kidney transplant. Islet Autotransplant • Total pancreatectomy with islet autotransplant as a superior alternative to pancreatoduodenectomy in patients at very high-risk of complications of the pancreatic anastomosis: a single-center prospected randomised clinical trial; • Islet autotransplant in patients who underwent total or partial pancreatectomy. Paola Maffi Childhood diabetes The Childhood Diabetes Unit is mainly focused on clinical research aimed to prevent and cure type 1 diabetes in children and adolescents. The Unit presently represents one of the biggest centers for the study of type 1 diabetes in Italy as it follows more than 900 diabetic children, including 300 patients treated with intensive insulin therapy. More than 70 new cases of diabetes are diagnosed every year. The Unit actively collaborates with other research groups both inside and outside the San Raffaele Scientific Institute, in particular: 1. collaboration with the Units of Dr. Battaglia and Dr. Piemonti (Diabetes Research Institute, San Raffaele Scientific Institute) aimed to study newly diagnosed children in order to better understand the immunological and virological bases of type 1 diabetes. Along with the same research groups we are also involved in a study aimed to clarify whether patients with different β cell residual secretion also differ for immunological markers. On this regard we submitted a grant application to the Juvenile Diabetes Research Foundation aimed to study long term residual β cell secretion in type 1 diabetic patients. 2. we have a collaboration with Prof. Lorenzi (Schepen’s Eye Research Institute, Harvard Medical School, Boston, MA, USA) and Dr. Zerbini (Diabetes Research Institute, San Raf- faele Scientific Institute) to understand if biomarkers for microvascular complications are detectable in children with diabetes. We submitted a grant application to the National Institute of Health (NIH) to study the progression of diabetic complications taking advantage of a novel, non-invasive approach. 3. The Unit, as a member of an active research network (SIEDP), has been previously involved in multicenter trials recently published (VIPKIDS, SHIPD), and it is now part of a large study aimed to investigate diabetic chetoacidosis at diagnosis of type 1 diabetes and to verify insulin pump malfunctioning in children treated with intensive insulin therapy. 4. The Unit is an active member of the Italian Group of Diabetes Technology and last year we performed a study on intensive insulin therapy in diabetic patients in Italy (IMITA study), that will be published soon. 5. We have an ongoing collaboration with Prof. Barbetti (Rome) and Prof. Iafusco (Naples) to study children with rare genetic cause of diabetes. 6. We are part of the writing team of the Società italiana di endocrinologia pediatrica (SIEDP), our aim is to define the guidelines for insulin therapy and chetoacidosis treatment in children with type 1 diabetes. Riccardo Bonfanti DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Selected publications Canducci, F; Ceresola, ER; Saita, D; Castagna, A; Gianotti, N; Underwood, M; Burioni, R; Lazzarin, A; Clementi, M. In vitro phenotypes to elvitegravir and dolutegravir in primary macrophages and lymphocytes of clonal recombinant viral variants selected in patients failing raltegravir. J. Antimicrob. Chemother.: 2013; 68(11): 2525-2532 - Article IF 2012: 5,338 Capua, I; Mercalli, A; Pizzuto, MS; Romero-Tejeda, A; Kasloff, S; De Battisti, C; Bonfante, F; Patrono, LV; Vicenzi, E; Zappulli, V; Lampasona, V; Stefani, A; Doglioni, C; Terregino, C; Cattoli, G; Piemonti, L. Influenza A viruses grow in human pancreatic cells and cause pancreatitis and diabetes in an animal model. J. Virol.: 2013; 87(1): 597-610 - Article IF 2012: 5,076 Cassetta, L; Kajaste-Rudnitski, A; Coradin, T; Saba, E; Della Chiara, G; Barbagallo, M; Graziano, F; Alfano, M; Cassol, E; Vicenzi, E; Poli, G. M1 polarization of human monocyte-derived macrophages restricts pre and postintegration steps of HIV-1 replication. AIDS: 2013; 27(12): 18471856 - Article IF 2012: 6,407 Cassol, E; Cassetta, L; Rizzi, C; Gabuzda, D; Alfano, M and Poli, G. Dendritic Cell-Specific ICAM3 Grabbing Nonintegrin mediates HIV-1 Infection of and Transmission by M2a-Polarized Macrophages In Vitro. AIDS: 2013; 27(5): 707-716 - Article IF 2012: 6,407 Castagnaro, L; Lenti, E; Maruzzelli, S; Spinardi, L; Migliori, E; Farinello, D; Sitia, G; Harrelson, Z; Evans, SM; Guidotti, LG; Harvey, RP; Brendolan, A. Nkx2-5+islet1+ mesenchymal precursors generate distinct spleen stromal cell subsets and participate in restoring stromal network integrity. Immunity: 2013; 38(4): 782-791 - Article IF 2012: 19,795 Cavarelli, M; Foglieni, C; Rescigno, M; Scarlatti, G. R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5. EMBO Mol. Med.: 2013; 5(5): 776-794 - Article IF 2012: 7,795 Cieri, N; Camisa, B; Cocchiarella, F; Forcato, M; Oliveira, G; Provasi, E; Bondanza, A; Bordignon, C; Peccatori, J; Ciceri, F; Lupo-Stanghellini, MT; Mavilio, F; Mondino, A; Bicciato, S; Recchia, A; Bonini, C. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood: 2013; 121(4): 573-584 - Article IF 2012: 9,060 Ghezzi, S; Galli, L; Kajaste-Rudnitski, A; Turrini, F; Marelli, S; Toniolo, D; Casoli, C; Riva, A; Poli, G; Castagna, A; Vicenzi, E. Identification of TRIM22 single nucleotide polymorphisms associated with loss of inhibition of HIV-1 transcription and advanced HIV-1 disease. AIDS: 2013; 27(15): 2335-2344 - Article IF 2012: 6,407 Giannese, F; Berg, M; Van Der Veken, P; Castagna, V; Tornaghi, P; Augustyns, K; Degano, M. Structures of purine nucleosidase from Trypanosoma brucei bound to isozyme-specific trypanocidals and a novel metalorganic inhibitor. Acta Crystallogr. Sect. D Biol. Crystallogr.: 2013; 69(8): 1553-1566 - Article IF 2012: 14,103 Hess Michelini, R and Manzo, T; Sturmheit, T; Basso, V; Rocchi, M; Freschi, M; Listopad, J; Blankenstein, T; Bellone, M and Mondino, A. Vaccine-instructed intratumoral IFN-g enables regression of autochthonous mouse prostate cancer in allogeneic T-cell transplantation. Cancer Res.: 2013; 73(15): 4641-4652 - Article IF 2012: 8,650 Maffi, P and Balzano, G; Ponzoni, M; Nano, R; Sordi, V; Melzi, R; Mercalli, A; Scavini, M; Esposito, A; Peccatori, J; Cantarelli, E; Messina, C; Bernardi, M; Del Maschio, A; Staudacher, C; Doglioni, C; Ciceri, F; Secchi, A; Piemonti, L. Autologous Pancreatic Islet Transplantation in Human Bone Marrow. Diabetes: 2013; 62(10): 3523-3531 - Article IF 2012: 7,895 Napolitano, A; Pittoni, P; Beaudoin, L; Lehuen, A; Voehringer, D; MacDonald, HR; Dellabona, P; Casorati, G. Functional education of invariant NKT cells by dendritic cell tuning of SHP-1. J. Immunol.: 171 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Selected publications, DRI 2013; 190(7): 3299-3308 - Article IF 2012: 5,520 Pensieroso, S; Galli, L; Nozza, S; Ruffin, N; Castagna, A; Tambussi, G; Hejdeman, B; Misciagna, D; Riva, A; Malnati, M; Chiodi, F and Scarlatti, G. B-cell subset alterations and correlated factors in HIV-1 infection. AIDS: 2013; 27(8): 1209-1217 - Article IF 2012: 6,407 Ricupito, A; Grioni, M; Calcinotto, A; Hess Michelini, R; Longhi, R; Mondino, A; Bellone, M. Booster vaccinations against cancer are critical in prophylactic but detrimental in therapeutic settings. Cancer Res.: 2013; 73(12): 3545-3554 - Article IF 2012: 8,650 Tonti, E; Jimenez de Oya, N; Galliverti, G; Moseman, E; Di Lucia, P; Amabile, A; Sammicheli, S; De Giovanni, M; Sironi, L; Chevrier, N; Sitia, G; Gennari, L; Guidotti, LG; Von Andrian, U; Iannacone, M. Bisphosphonates Target B Cells to Enhance Humoral Immune Responses. Cell Rep.: 2013; 5(2): 323-330 - Article IF 2012: Indexed by JCR 2012 Selected publications, DRI Balzano, G; Maffi, P; Nano, R; Zerbi, A; Venturini, M; Melzi, R; Mercalli, A; Magistretti, P; Scavini, M; Castoldi, R; Carvello, M; Braga, M; Del Maschio, A; Secchi, A; Staudacher, C; Piemonti, L. Extending indications for islet autotransplantation in pancreatic surgery. Ann. Surg.: 2013; 258(2): 210-218 - Article IF 2012: 6,329 Gagliani, N; Jofra, T; Valle, A; Stabilini, A; Morsiani, C; Gregori, S; Deng, S; Rothstein, DM; Atkinson, M; Kamanaka, M; Flavell, RA; Roncarolo, MG; Battaglia, M. Transplant tolerance to pancreatic islets is initiated in the graft and sustained in the spleen. Am. J. Transplant.: 2013; 13(8): 19631975 - Article IF 2012: 6,192 Maffi, P and Balzano, G; Ponzoni, M; Nano, R; Sordi, V; Melzi, R; Mercalli, A; Scavini, M; Esposito, A; Peccatori, J; Cantarelli, E; Messina, C; Bernardi, M; Del Maschio, A; Staudacher, C; Doglioni, C; Ciceri, F; Secchi, A; Piemonti, L. Autologous Pancreatic Islet Transplantation in Human Bone Marrow. Diabetes: 2013; 62(10): 3523-3531 - Article IF 2012: 7,895 Piemonti, L; Everly, MJ; Maffi, P; Scavini, M; Poli, F; Nano, R; Cardillo, M; Melzi, R; Mercalli, A; Sordi, V; Lampasona, V; De Arias, AE; Scalamogna, M; Bosi, E; Bonifacio, E and Secchi; A and Terasaki; PI. Alloantibody and autoantibody monitoring predicts islet transplantation outcome in human type 1 diabetes. Diabetes: 2013; 62(5): 1656-1664 - Article IF 2012: 7,895 Valle, A and Giamporcaro, GM and Scavini, M; Stabilini, A; Grogan, P; Bianconi, E; Sebastiani, G; Masini, M; Maugeri, N; Porretti, L; Bonfanti, R; Meschi, F; De Pellegrin, M; Lesma, A; Rossini, S; Piemonti, L; Marchetti, P; Dotta, F; Bosi, E; Battaglia, M. Reduction of circulating neutrophils precedes and accompanies type 1 diabetes. Diabetes: 2013; 62(6): 2072-2077 - Article IF 2012: 7,895 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Protein engineering and therapeutics AIDS immunopathogenesis Unit 173 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Cellular immunology Unit Emerging bacterial pathogens Unit DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Experimental immunology Unit Lymphocyte activation Unit 175 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Viral evolution and transmission Unit Viral pathogens and biosafety Unit DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Management and antiretroviral treatment of HIV infection Neurovirology 177 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Research Units Study and treatment of hepatotropic viruses related diseases Clinical immunopathology and advanced medical therapeutics Unit DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Clinical transplant Unit Experimental diabetes 179 DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES β cell biology Unit Immune-mediated diseases Unit: from pathogenesis to treatment DIVISION OF IMMUNOLOGY, TRANSPLANTATION, AND INFECTIOUS DISEASES Infection and cystic fibrosis 181 DIVISION OF GENETICS AND CELL BIOLOGY Director: Roberto Sitia* Associate Director: Marco E. Bianchi* Research Units Protein transport and secretion Unit –––––––––––––––––––––––––––––––––––––––––––––––––––– 187 HEAD OF UNIT: Roberto Sitia* RESEARCHER: Tiziana Anelli POST-DOCTORAL FELLOWS: Iria Medraño Fernandez**, Maria Francesca Mossuto, Edgar Yoboue PHD STUDENTS: Milena Bertolotti**, Sara Sannino FELLOW: Stefano Bestetti TECHNICIAN: Claudio Fagioli Age related diseases ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 188 GROUP LEADER: Simone Cenci POST-DOCTORAL FELLOWS: Nicola Napoli, Laura Oliva PHD STUDENT: Enrico Milan** FELLOW: Ugo Orfanelli TECHNICIANS: Elisabetta Mariani, Massimo Resnati Molecular immunology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 188 GROUP LEADER: Antonio Siccardi RESEARCHER: Maddalena Panigada POST-DOCTORAL FELLOWS: Francesca Bosè, Francesco Gubinelli PHD STUDENTS: Andrea Barbieri, Marta Recagni TECHNICIAN: Elisa Soprana Chromatin dynamics Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 189 HEAD OF UNIT: Marco E. Bianchi* POST-DOCTORAL FELLOWS: Elena Gatti, Michelle Monasky**, Emilie Venereau FELLOW: Giovanni Pietrogrande TECHNICIAN: Francesco De Marchis 183 DIVISION OF GENETICS AND CELL BIOLOGY Research Units In vivo Chromatin and transcription –––––––––––––––––––––––––––––––––––––––––––––– 190 GROUP LEADER: Alessandra Agresti POST-DOCTORAL FELLOW: Samuel Zambrano PHD STUDENT: Ilario De Toma ** Molecular dynamics of the nucleus ––––––––––––––––––––––––––––––––––––––––––––––– 191 RESEARCHER: Massimo Crippa Biology of myelin Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 191 SCIENTIFIC ADVISOR: Lawrence Wrabetz HEAD OF UNIT: Roberto Sitia* POST-DOCTORAL FELLOWS: Maurizio D’Antonio, Nicolò Musner, Cristina Scapin PHD STUDENTS: Domenica Vizzuso**, Vera Giulia Volpi** TECHNICIANS: Cinzia Ferri, Emanuela Pettinato Biomolecular mass spectrometry Unit –––––––––––––––––––––––––––––––––––––––––––––––––– 192 HEAD OF UNIT: Marco E. Bianchi* (ad interim) POST-DOCTORAL FELLOW: Umberto Restuccia PHD STUDENT: Vittoria Matafora FELLOW: Simone Tamburri TECHNICIAN: Angela Cattaneo European Institute for Research in Cystic Fibrosis (IERFC) –––––––––––––––––––––––– 192 HEAD OF UNIT: Luigi Maiuri POST-DOCTORAL FELLOWS: Emanuela Bruscia (until March 2013), Daniela De Stefano, Speranza Esposito, Konstantina Kyritsi, Valeria Villella FELLOWS: Eleonora Ferrari, Romina Monzani Genetics of common disorders Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––– 193 HEAD OF UNIT: Daniela Toniolo PHD STUDENT: Michela Traglia FELLOWS: Caterina Maria Barbieri, Corrado Masciullo TECHNICIAN: Cinzia Sala Intracellular signaling pathways Unit ––––––––––––––––––––––––––––––––––––––––––––––––––– 193 GROUP LEADER: Eelco Van Anken, Armenise-Harvard Career Development Award POST-DOCTORAL FELLOWS: Anush Bakunts, Andrea Orsi PHD STUDENT: Milena Vitale Molecular basis of polycystic kidney disease Unit ––––––––––––––––––––––––––––––––––– 194 HEAD OF UNIT: Alessandra Boletta* POST-DOCTORAL FELLOWS: Maddalena Castelli, Isaline Rowe PHD STUDENT: Monika Pema** FELLOWS: Chiara De Pascalis, Luca Drusian TECHNICIANS: Marco Chiaravalli, Gianfranco Distefano NeuroGlia Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 194 SCIENTIFIC ADVISOR: Maria Laura Feltri HEAD OF UNIT: Roberto Sitia* RESEARCHER: Daniela Talarico POST-DOCTORAL FELLOW: Ernesto Pavoni PHD STUDENTS: Cristina Colombelli, Monica Ghidinelli**, Marilena Palmisano** FELLOW: Alberto Merli TECHNICIAN: Desirée Zambroni DIVISION OF GENETICS AND CELL BIOLOGY Research Units/Clinical Research Units Regulation of iron metabolism Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––– 196 HEAD OF UNIT: Clara Camaschella* POST-DOCTORAL FELLOWS: Alessandro Campanella, Antonella Nai**, Alessia Pagani, Laura Silvestri PHD STUDENT: Marco Rausa Molecular genetics of renal disorders Unit (Dulbecco Telethon Institute) –––––– 196 GROUP LEADER: Luca Rampoldi POST-DOCTORAL FELLOWS: Céline Schaeffer, Matteo Trudu PHD STUDENT: Martina Brunati** FELLOW: Anna Creatore TECHNICIAN: Elena Pasqualetto Clinical Research Units Dento-facial histopathology Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 197 HEAD OF UNIT: Enrico Gherlone* PHYSICIANS: Enrico Agliardi, Paolo Capparé, Giuseppe Cardaropoli, Roberto Crespi, Maria Gabriella Grusovin, Alessandra Lucchese*, Simona Tecco, Raffaele Vinci Genomics of renal diseases and hypertension Unit –––––––––––––––––––––––––––––––––– 197 HEAD OF UNIT: Paolo Manunta* CLINICAL GROUP LEADER: Donatella Spotti RESEARCHER: Laura Zagato PHYSICIANS: Teresa Arcidiacono, Chiara Lanzani, Maria Teresa Sciarrone Alibrandi, Marco Simonini, Giuseppe Vezzoli CONSULTANTS: Giuseppe Bianchi*, Mara Ferrandi, Isabella Molinari RESIDENTS: Guido Gatti**, Lino Merlino**, Marina Nuzzo**, Simona Pozzoli**, Stefano Tentori**, Francesco Trevisani** FELLOWS: Lorena Citterio, Simona Delli Carpini, Elisabetta Messaggio TECHNICIAN: Nunzia Casamassima RESEARCH NURSES: Elena Brioni, Marie Jankaricova Reproductive sciences Lab –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 198 HEAD OF UNIT: Massimo Candiani* CLINICAL GROUP LEADER: Paola Panina RESEARCHERS: Enrico Papaleo, Paola Viganò POST-DOCTORAL FELLOWS: Alessandra Mugione**, Ana Maria Sanchez** FELLOWS: Laura Corti, Luca Pagliardini Tissue engineering and biomaterials –––––––––––––––––––––––––––––––––––––––––––––––––––– 199 HEAD OF UNIT: Gianfranco Fraschini CLINICAL GROUP LEADER: Giuseppe M. Peretti PHYSICIAN: Marco Domenicucci POST-DOCTORAL FELLOWS: Elena Arrigoni, Elisa Benasciutti, Daniela Deponti, Celeste Scotti RESIDENTS: Marco Agnoletto, Marco Melato * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele 185 DIVISION OF GENETICS AND CELL BIOLOGY Introduction by the Directors Mission and vision - The Division of Genetics and Cell Biology (DGCB) aims at the mechanistic comprehension of biological and pathological phenomena, to acquire basic knowledge and provide fellow clinicians with novel concepts, tools and protocols. Understanding physiology and pathology in cellular and molecular terms is fundamental to cure disease and create novel biotechnologies. The creation of new tools impacts directly on diagnostics. Scientific training and outreach are also DGCB priorities. Organization - DGCB consists of 15 basic, 4 clinical research groups and 2 facilities, totaling about 150 staff. Scientists are encouraged to foster synergies and engage in competitive projects. Areas of particular strength include the pathophysiology of stress and inflammatory responses, kidney and iron metabolism, the genetics of complex disorders, chromatin dynamics and epigenetics, cell polarity. Our staff are active in numerous Research Programs and Facilities. Goals - DGCB fosters curiosity-driven research projects that aim at understanding the molecular and cellular bases of biological phenomena and human diseases for the benefit of knowledge, technology and patients. The iterative process of translation is a key asset for DGCB, which aims providing clinical sciences with novel concepts and protocols and robust cell or animal models for their testing. As important is reverse translation, where detailed patient analyses can unravel physiological mechanisms. Research on genetic diseases is an example: identifying mutational hotspots in patients, for instance, sheds light on basic functions and allows for identification of novel bio-markers and therapeutic targets. Another important aim is to develop strong, reliable and quantitative and high-throughput assays. To this end we work in close collaboration with institutional Centers and Facilities to develop state-of-the-art technological platforms. The Proteomic service and the Center for conditional mutagenesis represent successful examples of our strategy: they are at the technological forefront and are serving a vast number of intramural and external scientists and clinicians. Achievements - 2013 has been a particularly successful year for DGCB scientists. A number of projects have come to fruition as high-impact papers appearing in top journals. The reader is referred to reports of the single laboratories for details on these accomplishments. Suffice here to recall the demonstration of the essential role of autophagy in plasma cell differentiation and long term antibody production; the unexpected finding that uromodulin levels impact blood pressure, besides causing kidney diseases when mutated; the further mechanistic characterization of the iron metabolism circuitry in human cells; the characterization of a novel, pH-dependent mechanism of protein quality control; the definition of new roles for redox control in inflammation and tyrosine kinase signaling; and, the observation that defective glucose metabolism is intimately involved in the pathobiology of polycystic kidney disease, paving the way for novel therapeutic strategies with existing drugs. The Core Facility for Conditional Mutagenesis generated 7 gene-targeted and 4 transgenic new lines, and cryopreserved/rederived over 40 lines for intra- and extra-mural scientists. The Protein Microsequencing Facility increased the number of internal (>15) and external users (≥20). Having acquired new instruments, ProMiFa can now further extend its portfolio. DGCB staff contributed significantly to developing metabolomics and imaging protocols. Training Opportunities - DGCB hosts many undergraduate students for their experimental theses and attractive PhD and PostDoctoral training programs, offering positions on competitive bases. Many scientists visited DGCB laboratories for sabbatical or training periods. DGCB organised numerous well-attended and lively seminars, delivered internal and external invitees. DIVISION OF GENETICS AND CELL BIOLOGY Research Units Protein transport and secretion Unit Proteostasis, redoxtasis and signal integration in the early secretory compartment A fundamental question in biology is how cells coordinate the size and activity of the different compartments. Our lab investigates the integration of signalling, protein quality control and sorting at the endoplasmic reticulum (ER)-Golgi interface. We discovered that ERp44 is a master regulator in this hub, regulating Ca2+ signalling, redox homeostasis (redoxtasis) and proteostasis. ERp44 acts as a pH-dependent chaperone that cycles within the early secretory compartment (ESC) and interacts with other key regulatory molecules (Ero1α, peroxiredoxin 4, IP3R1, KDEL receptors) to guarantee membrane homeostasis, transport efficiency and fidelity of the secretory proteome (Anelli et al., 2012; Vavassori et al. 2013; Kakihana et al., 2013; Benham et al., 2013; Wang et al., 2014; MedrañoFernandez et al., 2014). We developed powerful models of ER storage disorders and novel technologies to investigate what controls protein aggregation in ESC (Anelli and Sitia, 2010), and design suitable treatments. We confirmed that the degradative load vs. capacity ratio dictates sensitivity to bortezomib, a drug currently used against myeloma and other plasma cell dyscrasias and showed how autophagy and iron metabolism impact ESC proteostasis (Cenci, van Anken and Sitia 2011; Campanella et al., 2013; Pengo et al. 2013). We developed metabolomic platforms (Garcia-Manteiga et al., 2011) to identify markers of myeloma progression (Fontana et al., submitted). We demonstrated that aquaporin 8 mediates H2O2 transport across the endoplasmic reyiculum and plasma-membranes, allowing entry of H2O2 generated by NOXes to amplify tyrosine kinase signal transduction (Bertolotti et al., 2013). Modulating redox signaling may open novel possibilities of intervention to control inflammation, cancer cell migration and apoptosis. Our findings open the way to develop combinatorial treatments – i.e. targeting autophagy or redoxtasis - for overcoming drug resistance in the treatment of multiple myeloma. Deciphering the connections between ESC proteostasis, redoxtasis and signaling should identify novel targets for manipulating (mal)adaptive stress responses (Masciarelli et al., 2010; Rubartelli and Sitia, 2009a, b), and treating ER storage and conformational diseases. Roberto Sitia Figure 28. IgM subunits and other client proteins (in dark blue) bind to ERGIC53 at the neutral pH of the endoplasmic reticulum (>7). Oligomerization takes place as clients proceed to the cisGolgi. Also ERp44 binds to ERGIC53 (Anelli et al., 2007, EMBO J; Cortini and Sitia, 2010, Traffic). As the pH drops below 6.6 in the cisGolgi, ERGIC53 releases cargo glycoproteins while ERp44 and KDEL receptors are activated (red to orange shift). Movements of the C-tail of ERp44 expose the substrate-binding site (thick white arrow) and RDEL motif (yellow anchor), allowing capture of the clients and their retrieval to the ER by KDEL-R (Vavassori et al., 2013, Mol. Cell; Sannino et al., submitted). 187 DIVISION OF GENETICS AND CELL BIOLOGY Research Units Age related diseases New insights in plasma cell ontogenesis The CenciLab studies the biology of normal plasma cells (PC), terminally differentiated effectors of Ab responses, and their malignant degeneration, multiple myeloma (MM), responsible for 2% of all cancer deaths. In particular, we are interested in the role of autophagy, a crucial asset ensuring proteome plasticity, in PC ontogenesis and myeloma. Our goal is to identify new prognostic markers and molecular targets against PC malignancies. Synergic studies on the skeletal microenvironment provide the framework to explore the vicious relationship of myeloma with the bone marrow. Cancer cells rely on protein homeostasis, providing therapeutic targets: proteasome inhibitors (PI), the prototypical negative proteostasis regulators, emerged as potent anti-cancer drugs, especially against MM (Cenci, Semin Hematol 2012; Cenci, Mol Immunol 2014). We recently contributed to clarify the bases underlying the exquisite sensitivity of MM to PI, establishing the concept that high proteasme workload and limited proteasome capacity are key determinants of PI sensitivity (Bianchi et al, Blood, 2009; Cenci et al, J Leuk Biol 2012). In 2013, we disclosed a novel, specific and essential function of autophagy in PC ontogenesis. In differentiating PCs, autophagy contains the size of the endoplasmic reticulum, PC differentiation signals (XBP-1, Blimp-1), and Ab production, to optimize energy metabolism and sustain Ab responses. Moreover, autophagy is required for the maintenance of bone marrow memory PCs, the normal counterpart of MM (Pengo et al, Nat Immunol 2013). This discovery provides a framework for identifying new prognostic markers and molecular targets against PC malignancies (Pengo & Cenci, Autophagy 2013; Oliva & Cenci, Front Immunol 2014). Simone Cenci Molecular immunology Adjuvant IgE, headless hemagglutinins, survivin vaccines 1) Endogenous IgE against cancer (with L. Vangelista & A. Brini, Farmacologia, Unimi). Balb/c mutant KN1, a “high IgE responder” carrying a γ-1/ε gene substitution, shows minimal growth of subcutaneous tumors and of lung dissemination of i.v. administered tumors. The effect was shown to be due to immunization by cancer antigens and anti-tumor IgE-priming of effector cytotoxic cell, as demonstrated by CD8 depletion studies. Normal tumor growth is restored in the double mutant KN1/ Fc-ε RI-KO, indicating that anti-tumor immunity depends on IgE-driven antigen processing and presentation, in analogy to the adjuvanticity mechanism previously described for exogenous IgE. 2) Recombinant headless hemagglutinins as “universal” infuenza vaccines (with E. Vicenzi). Wide-spectrum neutralizing antibodies directed to stalk hemagglutinin conformational epitopes are normal, but very rare, because of the immunodominance of strain-specific epitopes in the globular head. Headless mutants could overcome the dominance, but loose the conformation necessary to be exported to the cell surface and fail to elicit protective responses. Compensatory mutations which allow the surface expression of epitopes reactive with anti-stalk monoclonal antibodies would allow the construction of wide-spectrum vaccines. Their selection from recombinant mutagenized poxvirus has been so far elusive, but new strategies employing artificial random mutant repertoires and stable lentiviral transfectants have been developed (with E. Montini). 3) Survivin as a “universal” tumor marker (with L. Piemonti). Recombinant poxviruses expressing human or murine survivin have been produced and are currently tested as preventive or curative vaccines in a number of mouse tumor models, in collaboration with several groups. Significant protection from tumor growth in immunized animals has been obtained in a mouse orthotopic model of pancreatic carcinoma. A study on therapy of murine mesothelioma by FPV-survivin vaccination has been published (with P. Bertino, Hawai’i University). Survivin vaccines have been included among candidate tumor vaccines for the innovative Self-Amplifying RNA vaccination platform (collaboration with D. Maione, Novartis Research, Siena). Antonio Siccardi DIVISION OF GENETICS AND CELL BIOLOGY Figure 29. Lung tumor dissemination Chromatin dynamics Unit HMGB1 as a DAMP (Damage Associated Molecular Pattern) The state of chromatin determines the identity of different (but genetically identical) cells in the same organism. Notably, we discovered in 2002 that a chromatin protein (HMGB1) is also a Damage Associated Molecular Pattern (DAMP) and a key player in immunity. HMGB1 is released by dead cells to signal tissue damage, and is also actively secreted by cells under severe stress, for example by hypoxic cells,or by activated monocytes/macrophages. HMGB1 contains 3 cysteines and has multiple redox states, each of which behaves in distinctly different ways: completely reduced HMGB1 forms a complex with the chemokine SDF-1/CXCL12, which engages the CXCR4 receptor and recruits monocytes to the site of tissue damage; HMGB1 containing one disulfide bond between C23 and C45 interacts with Toll-like receptor 4 (TLR4) and promotes the synthesis of various pro-inflammatory cytokines and chemokines; sulfone HMGB1 (in which at least one cysteine is completely oxidized to sulfonate) has no activity either as chemoattractant or proinflammatory signal. We are currently investigating how the different redox forms of HMGB1 alert the immune system to tissue damage, trigger inflammation and then direct tissue reconstruction. We are also investigating a novel concept: that cancer cells secrete HMGB1 to enlist the help of the immune system, as if they were a damaged tissue in need of repair. We have already demonstrated that an anti-HMGB1 monoclonal antibody developed by us slows mesothelioma growth in a mouse model. Our findings provide mechanistic links between HMGB1 release, acute and chronic inflammation, and carcinogenesis. We are now developing different agonists or inhibitors of HMGB1 for therapy. Marco E. Bianchi 189 DIVISION OF GENETICS AND CELL BIOLOGY Research Units In vivo Chromatin and transcription Nucleosome modulation in the cellular response to inflammation and stress HMGB1 (High Mobility Group Box 1) is a very abundant and conserved protein that localizes mainly in the cell nucleus, where it facilitates nuclear transactions (Bianchi & Agresti, 2005). Remarkably, cells lacking HMGB1 from their nuclei show a 20% decrease in nucleosome content, possibly because they lack HMGB1-driven DNA bending activity for nucleosome deposition (Celona et al,2011). The reduction in nucleosome number results in a global 30% increase in transcription and increased susceptibility of DNA to damage. HMGB1 also has an extracellular signalling function as a Damage Associated Molecular Pattern (DAMP) molecule (Bianchi, 2007). Necrotic cells release HMGB1 passively while cells undergoing severe stress and immune cells stimulated with LPS or IFN-γ secrete it actively (Bonaldi et al, 2003). Upon stimulation, HMGB1 relocates from the nucleus to the cytoplasm leaving nuclei partially or totally devoid of HMGB1. The immune functions of HMGB1 have traditionally been attributed to its actions as a secreted protein. We asked, however, whether the depletion of HMGB1 from the nuclei of secreting cells might also play a role in the overall response of inflammatory cells to trauma and stress. We found that Hmgb1–/– MEFs, inflammation non-proficient cells, show an inflammatory transcription phenotype and can be considered a model for cells that have lost HMGB1 from their nuclei (DeToma et al, 2014, in press). We then asked whether cells secreting HMGB1 also rearrange their chromatin. We found that unstimulated macrophages lacking HMGB1 show a molecular phenotype associated to the response to stress and inflammation. Remarkably, wild type macrophages that secrete HMGB1 following exposure to LPS and IFN-γ, reduce their histone content almost as much as macrophages where HMGB1 has been genetically deleted. We envisage chromatin rearrangement due to nucleosome loss as an important event in the cellular response to inflammation and stress (Figure 30). We expect to find similar outcomes upon a variety of insults where HMGB1 is secreted: infection, ischemia/reperfusion and neuronal hyperexcitation are likely candidates. Overall, modulation of nucleosome number may emerge as a critical event in many physiological and pathological conditions of clinical interest. Alessandra Agresti Figure 30. HMGB1-driven nucleosome depletion facilitates the chemotactic response in M1 macrophages Macrophages patrolling tissues for enemies express PAMPs receptors (Pathogen Associated Molecular Pattern, e.g. LPS) and chemokines (Cxcl12) (A, Left). Upon infection, LPS activates the TLR4 receptor on macrophages membranes., HMGB1 is then secreted eliciting a concomitant loss of histones from the nucleus. When activated, macrophages unpackage their chromatin in order to access and transcribe the necessary genes. Among the hundreds of LPS activated genes, those involved in chemotaxis and extravasation are also expressed (A, Right). Macrophages genetically lacking HMGB1 (B) contain 20% fewer nucleosomes similarly to LPS-activated macrophages. Remarkably, the transcriptional phenotype overlaps with a subset of the LPS-response containing chemokines, cytokines and CAMs molecules. DIVISION OF GENETICS AND CELL BIOLOGY Molecular dynamics of the nucleus A syngeneic approach to malignant mesothelioma Malignant mesotehlioma (MM) is a very aggressive cancer with poor prognosis, because of late stage diagnosis and resistance to current conventional therapies. Asbestos-exposed, primary human mesothelial cells release High Mobility Group Box1 (HMGB1) protein, a Damage-Associated Molecular Pattern (DAMP) polypeptide, that establishes an autocrine circuit making MM cells “addicted” to HMGB1 for their viability, proliferation and survival. Accordingly, anti-HMGB1 antibodies reduce the growth of MM xenografts in Severe-Combined ImmunoDeficient (SCID) mice and extend host survival, suggesting that ablation of HMGB1 may be sufficient to elicit therapeutic activity. As DAMP, extracellular HMGB1 plays a role in sterile inflammation, associated with several types of cancer. HMGB1 acts both as a chemoattractant for leukocyes and as a proinflammatory mediator inducing the release of cytokines and chemokines by leukocytes and resident immune cells. Thus a comprehensive approach to the role of HMGB1 in MM entails a whole-animal system in which the immune response is fully functional. We have set up such a system using murine MM cells (AB1), obtained from tumors induced by asbestos in BALB/c mice. Intraperitoneal injection of 3 x 104 – 106 AB1 cells in syngeneic BALB/c mice induces the formation of tumors in approximately one week. We monitor the tumors by ultrasound and PET scans, providing information on their growth rate, vascularization, expansion in the peritoneal cavity, association with residing organs and metabolism. The cellular composition of explanted tumors is analysed by immunohistochemistry and related to the above observations. We plan to add optical imaging (IVIS) by using AB1 cells expressing either the luciferase or the infraRed Fluorescent Protein gene. Further molecular work will be aimed at understanding which redox form of HMGB1 is responsible for the observed phenotype and through which mechanism (chemoattraction of leukocytes or proinflammatory cytochine production). Furthermore, this system will allow testing a number of molecules as potential therapeutics for MM. Massimo Crippa Biology of myelin Unit Genesis and maintenance of myelin We have a long-standing interest in myelin, which permits rapid conduction of impulses and guarantees axonal health in the nervous system. We have explored the role of axonal signals, adhesion and transcriptional regulation in myelination. In particular, we study inherited neuropathies, which reveal important determinants of myelin formation. Recently, we have produced/studied seven mouse models of Myelin Protein Zero (MPZ) neuropathies that cause widely varying neuropathy phenotypes in patients. Mice overexpressing wildtype P0 develop congenital hypomyelination; mice expressing P0 with a myc-epitope tag at the amino terminus unexpectedly model two more severe subtypes of CMT1B neuropathy; and mice expressing P0 with any of five known human MPZ mutations model CMT1B or Congenital Hypomyelination Neuropathy. We validated these models, confirmed that the mutations operate through gain of function, and showed that the mutant proteins have their “toxic” effect from various locations in the Schwann cell, many times away from myelin itself. For example, in MpzS63del mice, mutant P0 is retained in the endoplasmic reticulum (Figure 31) and activates protein quality control pathways in myelinating Schwann cells of peripheral nerve. Our recent data suggest that protein quality control alters trafficking of the P0 wildtype counterpart, the translation of myelin proteins, or impairs proteasome degradation of myelin proteins, thereby impairing myelin stability in CMT nerves. More recently, we have strong evidence that endoplasmic reticulum stress alters translational homeostasis of myelinating Schwann cells, and this could explain the developmental defect seen in S63del nerves. In addition, in another model of CMT1B, MpzR98C, we found that endoplasmic reticulum stress alters the balance of Krox20 and cJUN transcription factors that regulate differentiation of myelinating Schwann cells. Another mutant P0 protein, P0Q215X, may be mistrafficked to the surface of Schwann cells where it may interfere with appropriate signals from its extracellular matrix. Finally, P0S63C may form oxidized dimers in trans in the myelin sheath, altering the fluidity of the myelin sheath. These data reveal both pathogenesis of neuropathy, and biological clues about the normal myelination. Lawrence Wrabetz 191 DIVISION OF GENETICS AND CELL BIOLOGY Research Units Figure 31. P0S63del is retained in the endoplasmic reticulum of myelinating Schwann cells in S63del sciatic nerve. Biomolecular mass spectrometry Unit The Unit was closed down in 2013 following the relocation of Angela Bachi to IFOM in Milan. The unit has applied technological innovations in proteomics to achieve a comprehensive qualitative and quantitative description of complex molecular mechanisms. In particular, the unit focused on novel proteomic approaches that can be applied to cells under physiological and pathological states. Marco E. Bianchi European Institute for Research in Cystic Fibrosis (IERFC) Cystic fibrosis (CF) patients harboring the most common F508del-CFTR mutant are poor responders to potentiators of CFTR channel activity. Misfolded F508del-CFTR can be rescued at the cell surface by pharmacological agents (correctors) preventing its intracellular retention and degradation, but it is rapidly dismissed from the plasma membrane (PM) and redirected from endosomal recycling towards lysosomal degradation. A number of CFTR-corrector molecules have been identified by highthroughput screening, but their efficacy in ameliorating the CF lung phenotype in pre-clinical models or in CF patients, is controversial. Restoration of a functional proteostasis network by proteostasis regulators (PRs) is a novel approach to correct protein misfolding in conformational diseases, among which CF constitutes the quintessential example. We previously reported that defective CFTR function induces reactive oxygen species-dependent and transglutaminase-2-mediated sequestration of BECN1, leading to defective autophagy and increased lung inflammation. Our results provide evidence that functional perturbation of CFTR affects endosomal trafficking of cell-surface proteins, in turn sequestrating BECN1. This leads to local generation of PtdIns3P, which is pivotal in regulating endosomal fusion/maturation and trafficking, and to disabled autophagy. We demonstrated that PRs can rescue F508delCFTR trafficking and stabilize a functional CFTR mutant at the PM in primary bronchial epithelial cells, in brushed nasal epithelial cells from CF patients and in F508del-CFTR homozygous mice. The effects of cystamine extend well beyond drug washout thus enabling the beneficial action of CFTR potentiators in controlling lung inflammation. These results outline a novel approach for the treatment of CF indicating that prior re-establishment of autophagy prolongs the PM residence of a sufficient amount of F508del-CFTR to facilitate the action of CFTR potentiators. Our data suggest that a functional CFTR might orchestrate peripheral proteostasis and regulate its own PM residence and function. Moreover, manipulating the cellular mechanisms that ultimately link protein misfolding to protein malfunction, might constitute a therapeutic strategy for the treatment of conformational diseases. Luigi Maiuri DIVISION OF GENETICS AND CELL BIOLOGY Genetics of common disorders Unit Genetics of complex disorders: toward a personalized medicine The projects of the Unit aim at the elucidation of genetic loci underlying the risk for complex disorders by analysis of the genome of genetically isolated populations and case control cohorts from the general population. We have collected a large set of clinical and family data of 1800 descendants from the ancient founders of a genetically isolated population living in a valley in the Apennine, Val Borbera (VB) and we have established a network of Italian Genetic Isolates (INGI) largely representative of the general Italian population. With the information deriving from the dense genotypes of >5000 people we were able to participate in international consortia and contribute to the identification of common variants and genetic loci responsible for complex phenotypes and disease risks, including fertility and reproduction, thyroid pathology, kidney disorders, blood pressure, tumors of the elderly, iron parameters and blood cells parameters, food preferences and others. Among the phenotypes studied we were directly involved in the genetics of reproductive traits, with special interest in menopause age and its extremes, Early Menopause (EM) and Premature Ovarian Insufficiency (POI), traits that have become relevant due to the postponement of first child bearing in all developed countries and the consequent infertility problems. We have identified several genes associated to age of menopause and to the risk of EM and POI. We were also directly involved on the study of genetics of Anti Mullerian Hormone (AMH) and of thyroid disorders and we have recently completed the first meta analysis of common variants associated to thyroid stimulating hormone (TSH) and anti-thyroid peroxidase (antiTPO) antibodies. Larger meta analysis are ongoing and they include less common and rare variants identified from imputation of sequenced DNAs of different origin, also from the INGI populations. This kind of studies are expected to better explain the risk of early menopause and to provide genetic markers that can be used to establish protocols for personalized medicine. Daniela Toniolo Intracellular signaling pathways Unit Live cell imaging of endoplasmic reticulum stress sensing Homeostasis of the protein folding machinery in the endoplasmic reticulum (ER) is maintained via unfolded protein response (UPR) pathways that in large part are conserved in all eukaryotes. Like all stress responses, the UPR allows cells to adapt to novel environmental conditions or physiological needs. When excessive or prolonged, however, stress responses become maladaptive. The UPR thus plays a role in several degenerative disorders and other diseases related to disturbances in ER function. We established earlier that ER stress signaling involves clustering of the UPR sensor/transducer Ire1 in yeast and that this phenomenon is conserved in man. Through insertion of fluorescent tags GFP and mCherry in the transmembrane UPR transducers, IRE1 and its distant family member PERK, we have been able to create transgenes that report on ER stress signaling. By use of lentiviruses we deliver the tagged transgenes. Crucial is that expression levels of the transgenes can be kept lower or on a par with the endogenous counterparts as not to alter the circuitry of the UPR. We therefore express fluorescent transgenes under the doxicyclin inducible Tet promoter. By expressing the tagged transgenes of UPR transducers in corresponding knockout cell lines, we have verified that they can restore downstream signaling, in particular splicing of xbp-1 in the case of IRE1 and expression of CHOP in case of PERK, both upon ER stress induction. Using UPR eliciting drugs such as tunicamycin, tagged IRE1 and PERK transgenes faithfully report on how the endogenous UPR transducers cluster into foci. Moreover, the two UPR sensors do not co-cluster, but form distinct foci. We now employ a second lentiviral inducible expression system, which is under control of mifepristone (Mif). As such, we express secretory IgM heavy chain (µs). In the absence of the light chain, µs will never be assembled into antibodies, accumulates in the ER lumen and causes mild ER stress, as evidenced by xbp-1 splicing. More importantly, µs expression provoked IRE1-GFP clustering–the first proof that UPR sensor clustering occurs under more physiological (i.e. non-drug induced) ER stress conditions. Our findings will be useful to study (patho-)physiological conditions of UPR activation. Eelco Van Anken 193 DIVISION OF GENETICS AND CELL BIOLOGY Research Units Molecular basis of polycystic kidney disease Unit Unraveling the molecular basis of polycystic kidney diseases and identification of potential targets for therapy Polycystic Kidney Diseases (PKD) is a class of pathologies characterized by abnormally enlarged tubules eventually causing renal failure. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common form of PKD and a very frequent genetic disease affecting 1:1000. ADPKD is caused by mutations in either the PKD1 (in 85% of cases) or the PKD2 genes (in 15%) encoding for Polycystin-1 (PC-1), a large plasma membrane receptor, and Polycystin-2 (PC-2), a calcium channel, respectively. ADPKD is caused by loss of function of either of the two genes, but the normal function of the two genes or why their loss-of-function causes cystogenesis remains elusive. Recent identification of signaling cascades de-regulated in ADPKD has led to the initiation of several clinical trials, but an approved therapy is still lacking. Likewise, the precise molecular mechanism of how a cyst forms is still lacking. In 2013 we have shown that Polycystin-1 is involved in the establishment of proper tubular diameter during renal development. Renal tubules are generated through a very complex and diverse type of events. During embryonic development the final diameter of the tubules is established through a mechanism similar to convergent extension involving cellular intercalation. We have shown that mice mutants of the Pkd1 gene, encoding for a truncated form of PC-1, show a defect in tubular diameter and in the capability of cells to properly achieve mediolateral orientation. We have further shown that PC-1 regulates the capability of cells to polarize during cell migration. Finally, we have described that all these activities of PC-1 are due to its capability to interact with the Par3/aPKC complex (Castelli et al, Nat Comm, 2013). During 2013 we have also described a novel alteration in ADPKD which might be targeted for therapy. We have shown that defective glucose metabolism is intimately involved in the pathobiology of ADPKD and we have provided evidence that a glucose analogue, 2-deoxyglucose (2DG) has a beneficial effect on murine models of the disease providing a strong rationale for a novel therapeutic approach (Rowe et al, Nat Med, 2013). Alessandra Boletta NeuroGlia Unit Adhesion molecules and signaling in nervous system development and myelin diseases Schwann cells and oligodendocytes myelinate axons, and contribute to neuronal development, differentiation, integrity and regeneration. All these functions are modulated by interactions of the glial cells with the extracellular matrix (ECM) and with axons. Our laboratory studies these extracellular interactions and how they can be exploited to facilitate remyelination in neurological diseases. In 2013 the following goals were achieved: 1) In the ECM, laminins regulates myelination and permit axonal sorting, the process by which large axons are segregated by a single Schwann cell to be myelinated. We identified α6β1, α7β1 integrins as the receptors important for radial sorting. 2) By studying the signalling molecules activated by these receptors, we found that a specific isoform of P38 MAPK represents a potential target for remyelination, because it inhibits oligodendrocyte myelination and it is overexpressed in brains from Multiple Sclerosis patients. 3) Another aspect controlled by the ECM in peripheral nerves is the formation of Nodes of Ranvier. In peripheral neuropathies due to laminin deficiency patients have abnormal clustering of voltage-gated-sodium channels at Nodes. These effects are mediated by the receptor dystroglycan. We showed that dystroglycan recruits perlecan at nodes, which in turn binds gliomedin, a molecule required for clustering of sodium channels 4) Sodium channels are also clustered at the axon initial segment (AIS), the region where action potentials are generated. We are investigating the role of a guanosine exchange factor for the small GTPase ARF6, in the clustering of sodium channels at the AIS and in the establishment of the AIS compartment. 5) By performing epistasis experiments in mice we have shown that laminins prevents myelination driven by the growth factor neuregulin in fibers that should not be myelinated, either because DIVISION OF GENETICS AND CELL BIOLOGY they are not at the correct developmental time or because they are too small. 6) Only few molecules that mediate interactions between glial cells and axons are known. Using a subfractionation in vitro system and proteomic we have identified novel molecules that prevents myelination when they are deleted in mice. Maria Laura Feltri Figure 32. Perlecan and gliomedin induce the formation of sodium channel clusters (red) on axons from dorsal root ganglia sensory neurons (cyan blue). 195 DIVISION OF GENETICS AND CELL BIOLOGY Research Units Regulation of iron metabolism Unit Our Unit is involved in the study of systemic iron homeostasis and especially in the relationship between iron and erythropoiesis. In the last years we focused on transferrin receptor 2 (TfR2), a transmembrane protein, mainly expressed in the liver and in the erythroid precursors, that is mutated in hemochromatosis type 3, a rare recessive disorder leading to iron overload. Liver TfR2 activates hepcidin (HAMP) transcription in response to increased plasma iron, although the molecular mechanism remains uncertain. In erythroid cells TfR2 associates with the erythropoietin receptor (EpoR) and is required for its surface transport. TfR2 knockdown in vitro delays the erythroid terminal differentiation (Foretnikova et al, Blood 2010;116:5357-67). To discriminate between liver and erythroid function of Tfr2 we first crossed iron deficient mice lacking the hepcidin inhibitor transmembrane serine protease 6 (Tmprss6-/-) with Tfr2 total (Tfr2-/-) or liver-specific (Tfr2LCKO) knockout animals. All double KO animals have iron deficiency as Tmprss6-/-, demonstrating that liver TfR2 acts upstream TMPRSS6, but only mice with complete Tfr2-/- develop erythrocytosis, in the presence of normal Epo levels suggesting that this was consequent to the loss of erythroid Tfr2 (Nai et al Haematologica DOI: 10.3324/haematol.2013.103143). In order to better clarify this finding, in collaboration with G. Ferrari at Tiget, we have generated a mouse specifically lacking Tfr2 in the erythropoietic compartment by transplanting (T) wild-type animals with the bone marrow (BM) of Tfr2-/- donors. Four months after BMT mice transplanted with Tfr2-/- BM have normal iron homeostasis, normal Epo levels but higher hemoglobin and red cell count than animals transplanted with wild type BM. BM conditional Tfr2-/- have higher number of nucleated erythroid precursors, which show decreased apoptosis. Our results suggest that erythroid cells have increased Epo sensitivity in the absence of TfR2, in keeping with its function of EpoR partner. We propose that TfR2 is a limiting factor for erythropoiesis especially in conditions of iron-restriction through mechanisms that are under investigation. Clara Camaschella Molecular genetics of renal disorders Unit Our main reserch interest is to understand the role of uromodulin in renal function and in chronic diseases of the kidney. Uromodulin, the most abundant protein found in urine, is exclusively expressed in the kidney by the thick ascending limb (TAL) of Henle’s loop and plays a protective role against urinary tract infections and calcium oxalate crystals-induced damage (Rampoldi et al, Kidney Int 2011). Mutations in the uromodulin gene UMOD lead to uromodulin-associated kidney disease (UAKD), an autosomal dominant disease characterised by, tubulo-interstitial nephritis and renal failure. We demonstrated that uromodulin mutations have a gain-of-function effect as they lead to retention of mutant protein in the ER (Bernascone et al, Hum Mol Genet 2010). This is a key pathogenetic event, eventually leading to TAL damage, inflammation and fibrosis. Interestingly, common variants in the UMOD gene have been shown to give independent susceptibility for chronic kidney disease (CKD) and hypertension in several genome-wide association studies (GWAS). Such variants map in the UMOD gene promoter and are in full linkage disequilibrium. We recently demonstrated that UMOD risk alleles are associated with increased levels of urinary uromodulin and directly increase gene expression in vitro and in vivo (Trudu et al, Nat Med 2013). We modeled this effect in transgenic mice and showed that uromodulin overexpression leads to salt-sensitive hypertension and to agedependent renal lesions that are similarly observed in elderly subjects homozygous for UMOD risk variants. We demonstrated that the link between uromodulin and hypertension is caused by activation of the renal sodium co-transporter NKCC2. This mechanism is relevant in humans, as pharmacological inhibition of NKCC2 is more effective in lowering BP in hypertensive patients homozygous for UMOD risk variants. Our findings establish a link between the genetic susceptibility to hypertension and CKD, the control of uromodulin expression and its role in salt-reabsorption in the TAL segment. These data point to uromodulin as a novel therapeutic target to lower BP and preserve renal function. Luca Rampoldi DIVISION OF GENETICS AND CELL BIOLOGY Clinical Research Units Dento-facial histopathology Unit Biomaterials and implant-prosthetic rehabilitations in dentistry Our activity focused on biological features and clinical applications of bone substitues and dental implants for oral rehabilitations. Moreover, we studied histological features of bone healing in oral surgery procedures. To evaluate the different behavior of 3dimensional biomaterial scaffolds-Bovine Bone (BB; Bio-Oss) and Hydroxyapatite (HA;ENGIpore)-during initial bone healing and development. Human dental papilla stem cells (hDPaSCs) were selected with FACsorter cytofluorimetric analysis, cultured with osteogenic medium, and analyzed with Alizarin red stained after differentiation. The obtained osteoblast-like cells (OCs) were cultured with BB and HA. alkaline phosphatase (ALP), OC, MEPE, and runt-related transcription factor 2 (RUNX2) expression markers were investigated performing Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis. After 40 days, samples were analyzed by light and electron microscopy. All the samples showed high in vitro biocompatibility and qualitative differences of OCs adhesion. RTPCR and Western blot data exhibited similar marker rate, but ALP, OC, MEPE, and RUNX2expression, during initial healing and bone regeneration phase, was higher and faster in human dental papilla onto BB than in HA scaffolds. In biomaterials growth, RUNX2 seems to play an important role as a key regulator in human OCs from dental papilla bone development. Different surface BB scaffold characteristics seem to play a critical role in OCs differentiation showing different time of bone regeneration morphological characteristics as well as higher and faster levels of all observed markers. Moreover, we studied innovative and microinvasive implant-prosthodontics procedures from a clinical point of view. In clinical studies we also investigated the use of a magnetic mallet in bone condensing procedures, lowering postoperative complications, and testing new dental implant materials, as sandblasted and acid etched titanium surfaces. In conclusion, we focused our clinical activity on new implant surgical protocols, to shorten treatment time (e.g. immediate loading procedures) and improve the outcome, applying esthetic and functional prostheses. Enrico Gherlone Genomics of renal diseases and hypertension Unit 1. Pharmacogenomics (PGX) of Hypertension In hypertensive population only 1/3 of patients reaches blood pressure (BP) targets with the main classes of anti-hypertensive drugs. This variability is, in part, under genetic control. PGX aims to identify genetic markers predicting the individual drug response. We evaluated two current anti-hypertensive drugs: Hydrochlorothiazide (HCTZ) and Perindopril (ACEi). Polymorphisms of genes affecting renal sodium transport and signaling (NKAIN2, NKAIN3, NEDD4L, WNK1, SLC12A3; SLC12A1, SIK1, EGFR), drug metabolism (CYP7A1), vasodilation state (CPS1, PRKG1, ITPR2, MYO16), dopaminergic and RAAS systems (DRD1; REN) associated to BP decrease after 4 weeks of therapy. We developed two pathway-based algorithms to create genetic profiles of response to HCTZ and ACEi in naïve hypertensive patients (see figure). This innovative approach allows an a priori choice of the treatment in hypertensive patients. 2. Acute Kidney Injury (AKI) predicting model AKI is a frequent complication of cardiac surgery. We focused on milder AKI not requiring dialysis (AKI-ND), which is very common and contributes to several in-hospital outcomes. High pre-operative Endogenous Ouabain (EO) levels, an adrenal stress hormone with hemodynamic and renal effects, are associated with a worse renal outcome after cardiac surgery. We developed two risk models of AKI-ND using both clinical aspects, based on 8 pre-operative variables, (CLIN-AKI) and the EO plasma levels (CLIN-EO-AKI). Both these models are straightforward, useful and readily applicable at the bedside to provide more precisely the relative risk of development of AKI after cardiac surgery. 3. Salt-inducible kinase 1 (SIK1) and Nephrin Nephrin is a crucial component of the slit diaphragm. We linked Adducin, a cytoskeletal protein, and EO levels to hypertension and, through nephrin down-regulation, to kidney injury. We discovered that SIK1 colocalizes with nephrin in podocytes. SIK1 may act as a component of the slit diaphragm membrane required to maintain the filtration barrier functions of glomerular podocytes, facilitating nephrin signaling activities. rs3746951 SIK1 polymorphism has been associated with hypertension that may also affect nephrinuria, a potential new marker for kidney injury development. Paolo Manunta 197 DIVISION OF GENETICS AND CELL BIOLOGY Clinical Research Units Figure 33. Effect on Systolic Blood Pressure (SBP) response after 4 weeks of HCTZ or ACEi treatment: all patients in light blue box plot, according to the absence (white, Profile NO) or presence (green, Profile YES) of the respective genetic profiles. A genetic profile is the combination of genotypes of pathwaybased genes associated to phenotype. The box plot graph summarizes the statistical measures (median, the 75th and 25th percentiles, and minimum and maximum data values) of the distribution of the SBP decreases (mmHg). OR= Odds ratio. Reproductive sciences Lab Molecular and genetic aspects of female infertility Endometriosis, an estrogen-dependent chronic inflammatory disease characterized by the ectopic growth of endometrial tissue, is the most frequent occurrence in infertile women. In affected women, granulosa cells, the sex steroid producing cells in the ovarian follicle, undergo apoptosis in vitro. A dysregulation of the WNT/β-catenin signaling pathway and an aberrant gene expression of the downstream targets survivin and BMP4 suggest a role of this pathway in granulosa cell atresia (Sanchez et al, Fertil Steril 2014). A most common form of endometriosis is characterized by the presence of one or more ovarian cysts containing high concentrations of cellular stress factors, including iron (Sanchez et al, Hum Reprod Update 2013; Sanchez et al, Reprod Sci 2014). We have shown that the lower numbers of oocytes retrieved from follicles adjacent to the cyst are associated with increased iron levels, expression of its major storage protein H/L ferritin in the follicular fluid and increased expression of H ferritin and transferrin receptor-1 in the granulosa cells (Sanchez et al., Hum Reprod 2014). Our results demonstrate that the ovary has the ability to store excess iron, however prevention of ovarian iron overload is needed to allow the maturation of a functional oocyte. We are also interested in the identification of novel genetic risk factors for endometriosis (Pagliardini et al, J Med Genet 2013). In a replication study (305 endometriosis and 2710 controls) we have shown that the SNP located near the VEZT gene (coding for vezatin, adherens junctions protein) is strongly associated with endometriosis (p=2x105). Ongoing analysis of VEZT expression and function will provide new insights into the pathogenesis of the disease. Premature ovarian failure can also be induced by DIVISION OF GENETICS AND CELL BIOLOGY chemotherapy. We demonstrated that human granulosa cells treated in vitro with doxorubicin or paclitaxel undergo rapid apoptosis, downregulate ERβ and FSHR expression, and switch off the WNT3/β-catenin signaling pathway. Restoration of the β-catenin signaling, by inhibiting its proteo- somal degradation, protects granulosa cells from chemotoxicity (Sanchez et al, Toxicol Sci 2013). This approach will help to identify novel molecular targets to limit chemotherapy-induced gonadotoxicity. Paola Panina Tissue engineering and biomaterials Tissue engineering studies for osteo-cartilaginous, meniscal and tendon tissue Our research activity focuses on the development of tissue engineering strategies for the repair and regeneration of damaged structural tissues. In this regard, articular cartilage, meniscus tissue and tendon tissue do not possess innate capacity of self-repair. Additionally, some clinical situation involves the lack of bone tissue. We have developed of an engineered osteochondral composite for the repair of the cartilage and osteo-chondral lesions and tested in pre-clinical model demonstrating the great potential of such a material in the repair of these complex lesions. Having as ultimate goal the developing an engineered meniscal substitute, we are also conduct- Figure 34. Collagen I and collagen II double immunofluorescent staining of engineered cartilage samples (collagen II in red, collagen I in green). Samples at 1 week of culture; many chondrocytes are immunopositive to collagen II in the cytoplasm (red) and are immersed in an intensely collagen II immunopositive ECM (red), while a very scarce immunoreactivity to collagen I (green) is evident in the fibrin glue, as evidenced by the co-expression of both collagen types (yellow stain in the bottom of the image). This image has been published as cover image in the March 2014 issue of the journal Tissue Engineering - Part A. 199 DIVISION OF GENETICS AND CELL BIOLOGY Clinical Research Units ing a series of experiments with the attempt of characterizing the native juvenile and adult meniscus. We have demonstrated the presence of at least three cell lines within the meniscus tissue and a different biochemical composition in the young and adult meniscus. These data may represent the base for meniscus tissue engineering. On this regard, adipose derived stem cells (ASC) have been tested as possible source for engineering this important structure. We are also conducting a series of experiments with the goal of create a tissue engineered tendon. We believe that a tendon engineered in vitro with a biological scaffold seeded with autologous fibroblasts could represent an important solution. As possible cell source, we have tested tendon, ligament, peritenon and dermal fibroblasts. Once the ideal cell source and culture condition will be identified, pre-clinical studies will be conducted in order to test the potential of this model. In collaboration with the University Hospital Basel, Basel, Switzerland, we have developed a strategy for bone tissue engineering based on the recapitulation of the embryonic paths during the bone formation through the endochondral ossification process. Several studies are on their way for the characterization of the newly bone formed in a heterotopic environment and test the potential for clinical applications. Giuseppe M. Peretti DIVISION OF GENETICS AND CELL BIOLOGY Selected publications Berndt, SI; Gustafsson, S; Magi, R; Ganna, A; Wheeler, E; Feitosa, MF; Justice, AE; Monda, KL; Croteau-Chonka, DC; Day, FR; Esko, T; Fall, T; Ferreira, T; Gentilini, D; Jackson, AU; Luan, J; Randall, JC; Vedantam, S; Willer, CJ; Winkler, TW; Wood, AR; Workalemahu, T; Hu, YJ; Lee, SH; Li,ang, L; Li,n, DY; Min, JL; Neale, BM; Thorleifsson, G; Yang, J; Albrecht, E; Amin, N; Bragg-Gresham, JL; Cadby, G; Heijer, MD; Eklund, N; Fischer, K; Goel, A; Hottenga, JJ; Hu,ffman; JE; Jarick, I; Johansson, A; Johnson, T; Kanoni, S; Kleber, ME; Konig, IR; Kristiansson, K; Kutalik, Z; Lamina, C; Lecoeur, C; Li, G; Mangino, M; McArdle, WL; Medina-Gomez, C; Muller-Nurasyid, M; Ngwa, JS; Nolte, IM; Paternoster, L; Pechlivanis, S; Perola, M; Peters, MJ; Preuss, M; Rose, LM; Shi, J; Shungin, D; Smith, AV; Strawbridge, RJ; Surakka, I; Teumer, A; Trip, MD; Tyrer, J; Van Vliet-Ostaptchouk, JV; Vandenput, L; Waite, LL; Zhao, JH; Absher, D; Asselbergs, FW; Atalay, M; Attwood, AP; Balmforth, AJ; Basart, H; Beilby, J; Bonnycastle, LL; Brambilla, P; Bruinenberg, M; Campbell, H; Chasman, DI; Chines, PS; Collins, FS; Connell, JM; O Cookson, W; De Faire, U; De Vegt, F; Dei, M; Dimitriou, M; Edkins, S; Estrada, K; Evans, DM; Farrall, M; Ferrario, MM; Ferrieres, J; Franke, L; Frau, F; Gejman, PV; Grallert, H; Gronberg, H; Gudnason, V; Hall, AS; Hall, P; Hartikainen, AL; Hayward, C; Heard-Costa, NL; Heath, AC; Hebebrand, J; Homuth, G; Hu, FB; Hu,nt; SE; Hypponen, E; Iribarren, C; Jacobs, KB; Jansson, JO; Jula, A; Kahonen, M; Kathiresan, S; Kee, F; Khaw, KT; Kivimaki, M; Koenig, W; Kraja, AT; Kumari, M; Kuulasmaa, K; Kuusisto, J; Laitinen, JH; Lakka, TA; Langenberg, C; Launer, LJ; Lind, L; Lindstrom, J; Liu, J; Liuzzi, A; Lokki, ML; Lorentzon, M; Madden, PA; Magnusson, PK; Manunta, P; Marek, D; Marz, W; Leach, IM; McKnight, B; Medland, SE; Mihailov, E; Milani, L; Montgomery, GW; Mooser, V; Muhleisen, TW; Munroe, PB; Musk, AW; Narisu, N; Navis, G; Nicholson, G; Nohr, EA; Ong, KK; Oostra, BA; Palmer, CN; Palotie, A; Peden, JF; Pedersen, N; Peters, A; Polasek, O; Pouta, A; Pramstaller, PP; Prokopenko, I; Putter, C; Radhakrishnan, A; Raitakari, O; Rendon, A; Rivadeneira, F; Rudan, I; Saaristo, TE; Sambrook, JG; Sanders, AR; Sanna, S; Saramies, J; Schipf, S; Schreiber, S; Schunkert, H; Shi,n; SY; Signorini, S; Sinisalo, J; Skrobek, B; Soranzo, N; Stancakova, A; Stark, K; Stephens, JC; Stirrups, K; Stolk, RP; Stumvoll, M; Swift, AJ; Theodoraki, EV; Thorand, B; Tregouet, DA; Tremoli, E; Van Der Klauw, MM; Van Meurs, JB; Vermeulen, SH; Viikari, J; Virtamo, J; Vitart, V; Waeber, G; Wang, Z; Widen, E; Wild, SH; Willemsen, G; Winkelmann, BR; Witteman, JC; Wolffenbuttel, BH; Wong, A; Wright, AF; Zillikens, MC; Amouyel, P; Boehm, BO; Boerwinkle, E; Boomsma, DI; Caulfield, MJ; Chanock, SJ; Cupples, LA; Cusi, D; Dedoussis, GV; Erdmann, J; Eriksson, JG; Franks, PW; Froguel, P; Gieger, C; Gyllensten, U; Hamsten, A; Harris, TB; Hengstenberg, C; Hicks, AA; Hingorani, A; Hinney, A; Hofman, A; Hovingh, KG; Hveem, K; Illig, T; Jarvelin, MR; Jockel, KH; Keinanen-Kiukaanniemi, SM; Kiemeney, LA; Kuh, D; Laakso, M; Lehtimaki, T; Levinson, DF; Martin, NG; Metspalu, A; Morris, AD; Nieminen, MS; Njolstad, I; Ohlsson, C; Oldehinkel, AJ; Ouwehand, WH; Palmer, LJ; Penninx, B; Power, C; Province, MA; Psaty, BM; Qi, L; Rauramaa, R; Ridker, PM; Ripatti, S; Salomaa, V; Samani, NJ; Snieder, H; Sorensen, TI; Spector, TD; Stefansson, K; Tonjes, A; Tuomilehto, J; Uitterlinden, AG; Uusitupa, M; Van Der Harst, P; Vollenweider, P; Wallaschofski, H; Wareham, NJ; Watkins, H; Wichmann, HE; Wilson, JF; Abecasis, GR; Assimes, TL; Barroso, I; Boehnke, M; Borecki, IB; Deloukas, P; Fox, CS; Frayling, T; Groop, LC; Haritunian, T; Heid, IM; Hu,nter; D; Kaplan, RC; Karpe, F; Moffatt, MF; Mohlke, KL; O’Connell, JR; Pawitan, Y; Schadt, EE; Schlessinger, D; Steinthorsdottir, V; Strachan, DP; Thorsteinsdottir, U; Van Duijn, CM; Visscher, PM; Di Blasio, AM; Hirschhorn, JN; Li,n,dgren; CM; Morris, AP; Meyre, D; Scherag, A; McCarthy, MI; Speliotes, EK; North, KE; Loos, RJ; Ingelsson, E. Genome-wide meta-analysis identifies 11 new loci for anthropometric traits and provides insights into genetic architecture. Nature Genet.: 2013; 45(5): 501-512 - Article IF 2012: 35,209 Bertolotti, M and Bestetti, S and García-Manteiga, JM and Medraño-Fernandez, I; Dal Mas, A; Malosio, ML; Sitia, R. Tyrosine kinase signal modulation: a matter of H2O2 membrane permeability? Antioxid. Redox Signal.: 2013; 19(13): 1447-1451 - Review IF 2012: 7,189 Camaschella, C. Treating iron overload. New Engl. J. Med.: 2013; 368(24): 2325-2327 - Article IF 2012: 51,658 Castelli, M; Boca, M; Chiaravalli, M; Ramalingam, H; Rowe, I; Distefano, G; Carroll, T; Boletta, A. Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis. Nat. Commun.: 2013; 4: 2658 - Article IF 2012: 10,015 201 DIVISION OF GENETICS AND CELL BIOLOGY Selected publications Citterio, L and Ferrandi, M; Delli Carpini, S; Simonini, M; Kuznetsova, T; Molinari, I; Dell’Antonio, G; Lanzani, C; Merlino, L; Brioni, E; Staessen, JA; Bianchi, G; Manunta, P. cGMP-dependent protein kinase 1 polymorphisms underlie renal sodium handling impairment. Hypertension: 2013; 62(6): 1027-1033 - Article IF 2012: 6,873 D’Antonio, M; Musner, N; Scapin, C; Ungaro, D; Del Carro, U; Ron, D; Feltri, ML; Wrabetz, L. Resetting translational homeostasis restores myelination in charcot-marie-tooth disease type 1B mice. J. Exp. Med.: 2013; 210(4): 821-838 - Article IF 2012: 13,214 Dormoy-Raclet, V and Cammas, A; Celona, B; Lian, XJ; Van Der Giessen, K; Zivojnovic, M; Brunelli, S; Riuzzi, F; Sorci, G; Wilhelm, BT; Marco, SD; Donato, R; Bianchi, ME; Gallouzi, IE. HuR and miR-1192 regulate myogenesis by modulating the translation of HMGB1 mRNA. Nat. Commun.: 2013; 4: 2388 - Article IF 2012: 10,015 Kottgen; A; Albrecht, E; Teumer, A; Vitart, V; Krumsiek, J; Hundertmark, C; Pistis, G; Ruggiero, D; O’Seaghdha, CM; Haller, T; Yang, Q; Tanaka, T; Johnson, AD; Kutalik, Z; Smit,h, AV; Shi, J; Struchalin, M; Middelberg, RPS; Brown, MJ; Gaffo, AL; Piras,tu, N; Li, G; Hayward, C; Zemunik, T; Huffman, J; Yengo, L; Zhao, JH; Demirkan, A; Feitosa, MF; Li,u; X; Malerba, G; Lopez, LM; Van Der Harst, P; Li, X; Kleber, ME; Hicks, AA; Nolte, IM; Johansson, A; Murgia, F; Wild, SH; Bakker, SJL; Peden, JF; Dehghan, A; Steri, M; Tenesa, A; Lagou, V; Salo, P; Mangino, M; Rose, LM; Lehtimaki, T; Woodward, OM; Okada, Y; Tin, A; Muller, C; Oldmeadow, C; Putku, M; Czamara, D; Kraft, P; Frogheri, L; Thun, GA; Grotevendt, A; Gislason, GK; Harris, TB; Launer, LJ; McArdle, P; Shuldiner, AR; Boerwinkle, E; Coresh, J; Schmidt, H; Schallert, M; Martin, NG; Montgomery, GW; Kubo, M; Nakamura, Y; Tanaka, T; Munroe, PB; Samani, NJ; Jacobs, DR; Li,u; K; D’Adamo, P; Ulivi, S; Rotter, JI; Psaty, BM; Vollenweider, P; Waeber, G; Campbell, S; Devuyst, O; Navarro, P; Kolcic, I; Hastie, N; Balkau, B; Froguel, P; Esko, T; Salumets, A; Khaw, KT; Langenberg, C; Wareham, NJ; Isaacs, A; Kraja, A; Zhang, Q; Wild, PS; Scott, RJ; Holliday, EG; Org, E; Viigimaa, M; Bandinelli, S; Metter, JE; Lupo, A; Trabetti, E; Sorice, R; Doring, A; Lattka, E; Strauch, K; Theis, F; Waldenberger, M; Wichmann, HE; Davies, G; Gow, AJ; Bruinenberg, M; Stolk, RP; Kooner, JS; Zhang, W; Winkelmann, BR; Boehm, BO; Lucae, S; Penninx, BW; Smit, JH; Curhan, G; Mudgal, P; Plenge, RM; Portas, L; Persico, I; Kirin, M; Wilson, JF; Leach, IM; Van Gilst, WH; Goel, A; Ongen, H; Hofman, A; Rivadeneira, F; Uitterlinden, AG; Imboden, M; Von Eckardstein, A; Cucca, F; Nagaraja, R; Piras, MG; Nauck, M; Schurmann, C; Budde, K; Ernst, F; Farrington, SM; Theodoratou, E; Prokopenko, I; Stumvoll, M; Jula, A; Perola, M; Salomaa, V; Shin, SY; Spector, TD; Sala, C; Ridker, PM; Kahonen, M; Viikari, J; Hengstenberg, C; Nelson, CP; Meschia, JF; Nalls, MA; Sharma, P; Singleton, AB; Kamatani, N; Zeller, T; Burnier, M; Attia, J; Laan, M; Klopp, N; Hillege, HL; Kloiber, S; Choi, H; Pirastu, M; Tore, S; Probst-Hensch, NM; Volzke, H; Gudnason, V; Parsa, A; Schmidt, R; Whitfield, JB; Fornage, M; Gasparini, P; Siscovick, DS; Polasek, O; Campbell, H; Rudan, I; Bouatia-Naji, N; Metspalu, A; Loos, RJF; Van Duijn, CM; Borecki, IB; Ferrucci, L; Gambaro, G; Deary, IJ; Wolffenbuttel, BHR; Chambers, JC; Marz, W; Pramstaller, PP; Snieder, H; Gyllensten, U; Wright, AF; Navis, G; Watkins, H; Witteman, JCM; Sanna, S; Schipf, S; Dunlop, MG; Tonjes, A; Ripatti, S; Soranzo, N; Toniolo, D; Chasman, DI; Raitakari, O; Kao, WHL; Ciullo, M; Fox, CS; Caulfield, M; Bochud, M; Gieger, C. Genome-wide association analyses identify 18 new loci associated with serum urate concentrations. Nat. Genet.: 2013; 45(2): 145-154 - Article IF 2012: 35,209 Pellegatta, M; De Arcangelis, A; D’Urso, A; Nodari, A; Zambroni, D; Ghidinelli, M; Matafora, V; Williamson, C; Georges-Labouesse, E; Kreidberg, J; Mayer, U; McKee, KK; Yurchenco, PD; Quattrini, A; Wrabetz, L; Feltri, ML. α6β1 and α7β1 Integrins Are Required in Schwann Cells to Sort Axons. J. Neurosci.: 2013; 33(46): 17995-18007 - Article IF 2012: 6,908 Pengo, N; Scolari, M; Oliva, L; Milan, E; Mainoldi, F; Raimondi, A; Fagioli, C; Merlini, A; Mariani, E; Pasqualetto, E; Orfanelli, U; Ponzoni, M; Sitia, R; Casola, S; Cenci, S. Plasma cells require autophagy for sustainable immunoglobulin production. Nat. Immunol.: 2013; 14(3): 298-305 Article IF 2012: 26,199 DIVISION OF GENETICS AND CELL BIOLOGY Rowe, I; Chiaravalli, M; Mannella, V; Ulisse, V; Quilici, G; Pema, M; Song, XW; Xu, H; Mari, S; Qian, F; Pei, Y; Musco, G; Boletta, A. Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat. Med. : 2013; 19(4): 488-493 - Letter IF 2012: 24,302 Sanchez, AM; Giorgione, V; Viganò, P; Papaleo, E; Candiani, M; Mangili, G; Panina-Bordignon, P. Treatment with anticancer agents induces dysregulation of specific Wnt signaling pathways in human ovarian luteinized granulosa cells in vitro. Toxicol. Sci.: 2013; 136(1): 183-192 - Article IF 2012: 4,328 Trudu, M; Janas, S; Lanzani, C; Debaix, H; Schaeffer, C; Ikehata, M; Citterio, L; Demaretz, S; Trevisani, F; Ristagno, G; Glaudemans, B; Laghmani, K; Dell’Antonio, G; Swiss Kidney Project on Genes in Hypertension (SKIPOGH) Team; Bochud, M; Burnier, M; Devuyst, O; Martin, PY; Mohaupt, M; Paccaud, F; Pechère-Bertschi, A; Vogt, B; Ackermann, D; Ehret, G; Guessous, I; Ponte, B; Pruijm, M; Loffing, J; Rastaldi, MP; Manunta, P; Devuyst, O and Rampoldi, L. Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression. Nat. Med.: 2013; 19(12): 1655-1660 - Article IF 2012: 24,302 Vavassori, S and Cortini, M and Masui, S and Sannino, S; Anelli, T; Caserta, IR; Fagioli, C; Mossuto, MF; Fornili, A; van Anken, E; Degano, M; Inaba, K; Sitia, R. A pH-Regulated Quality Control Cycle for Surveillance of Secretory Protein Assembly. Mol. Cell: 2013; 50(6): 783-792 - Article IF 2012: 15,280 Villella, VR; Esposito, S; Bruscia, EM; Vicinanza, M; Cenci, S; Guido, S; Pettoello-Mantovani, M; Carnuccio, R; De Matteis, MA; Luini, A; Maiuri, MC; Raia, V; Kroemer, G; Maiuri, L. Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator. Cell Death Differ.: 2013; 20(8): 1101-1115 - Article IF 2012: 8,371 203 DIVISION OF GENETICS AND CELL BIOLOGY Age related diseases Protein transport and secretion Unit DIVISION OF GENETICS AND CELL BIOLOGY Molecular immunology European Institute for Research in Cystic Fibrosis (IERFC) 205 DIVISION OF GENETICS AND CELL BIOLOGY Intracellular signaling pathways Unit Molecular basis of polycystic kidney disease Unit DIVISION OF GENETICS AND CELL BIOLOGY Regulation of iron metabolism Unit Molecular genetics of renal disorders Unit 207 DIVISION OF GENETICS AND CELL BIOLOGY Research Units Genomics of renal diseases and hypertension Unit Tissue engineering and biomaterials DIVISION OF GENETICS AND CELL BIOLOGY Reproductive sciences Lab 209 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Director: Giorgio Casari* Co-Director: Elia Stupka Research Units Neurogenomics Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 214 HEAD OF UNIT: Giorgio Casari* RESEARCHERS: Giovanni Lavorgna, Francesca Maltecca* POST-DOCTORAL FELLOWS: Laura Cassina, Mimma Vizzuso PHD STUDENT: Francesco Consolato FELLOW: Elisa Baseggio TECHNICIAN: Maurizio De Fusco Genome function Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 215 HEAD OF UNIT: Elia Stupka RESEARCHERS: Davide Cittaro, Arek Kasprzyck, Dejan Lazarevic, Paolo Provero POST-DOCTORAL FELLOWS: Silvia Bonfiglio, Gabriele Bucci**, Jose Manuel Garcia-Manteiga, Francesca Giannese, Michele Loi, Vincenza Maselli, Davide Rambaldi, Michela Riba FELLOWS: Giulia Barbiera, Iwan Buetti, Stefania Merella, Luca Pandini TECHNICIANS: Donatella Biancolini, Eleonora Capitolo**, Celia Pardini, Arianna Rezzonico**, Valeria Rossella, Serenella Sartori Biomolecular NMR Laboratory (Dulbecco Telethon Institute) –––––––––––––––––––––– 215 HEAD OF UNIT: Giovanna Musco POST-DOCTORAL FELLOWS: Davide Gaudesi, Michela Ghitti, Valeria Mannella, Andrea Spitaleri, Chiara Zucchelli PHD STUDENTS: Andrea Berardi, Dimitrios Spiliotoupulos** FELLOWS: Giacomo Quilici, Cristina Paissoni 211 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Research Units Genomic Unit for the diagnosis of human pathologies –––––––––––––––––––––––––––––– 216 HEAD OF UNIT: Maurizio Ferrari* RESEARCHERS: Sara Benedetti, Paola Carrera, Vito Lampasona FELLOWS: Angela Brisci, Chiara Di Resta, Silvia Galbiati, Stefania Stenirri TECHNICIAN: Nadia Soriani Organelle biogenesis and motility Unit ––––––––––––––––––––––––––––––––––––––––––––––––– 217 HEAD OF UNIT: Maria Vittoria Schiaffino POST-DOCTORAL FELLOWS: Massimo Lazzaro, Ilaria Palmisano Proteome biochemistry Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 218 HEAD OF UNIT: Massimo Alessio POST-DOCTORAL FELLOW: Massimo Lazzaro PHD STUDENTS: Sheila Maria Alvarez-Fernandez, Marco Barbariga, Alan Zanardi** TECHNICIAN: Antonio Conti * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Introduction by the Directors Mission and vision - The Center promotes and contributes genomics and bioinformatics approaches to biomedicine research. By taking advantage of the excellent clinical environment coupled to outstanding basic science locally present, the Center works with the San Raffaele Scientific Institute as a whole to advance towards integrative translational research. Biomedicine is quickly transforming the diagnostic and cure-delivering processes, which are being further enhanced by both –omics and bioinformatics methodologies and the complex network analyses. The Center supports as well as initiates novel, interdisciplinary research that endorses omics-driven translational research approaches. The Center acts as a catalyst for innovative research projects by complementing existing research lines across the Institute with high throughput technologies, quantitative methods, and powerful data mining approaches. Organization - The Center for Translational Genomics and Bioinformatics (CTGB) is located in the DIBIT2 building, and includes 80 scientists. The Center houses six research units, with different expertise ranging from cell biology, protein chemistry, structural biology and genetics, as well as a genome function unit which is involved in both research and provision of genomics and bioinformatics services. Goals - The goal of the Center is to produce original research in the fields of translational -omics and bioinformatics, to enrich and enhance research occurring within other divisions and departments with these disciplines, and to develop novel clinical protocols which bring –omics from bench to bedside. Achievements See the Research Unit Reports of: • Neurogenomics Unit • Biomolecular NMR Spectroscopy Unit • Proteome Biochemistry Unit • Genomic Unit for the Diagnosis of Human Pathologies • Organelle Biogenesis and Motility Unit • Genome Function Unit 213 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Research Units Neurogenomics Unit Three main achievements with translational potential are reported. Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disorder characterized by unbalanced standing, gait incoordination, nystagmus, ophthalmoparesis and pyramidal signs. Several disease-causing mutations have been identified in the AFG3L2 gene. The haploinsufficient Afg3l2+/- mouse recapitulates the features of SCA28 patients, displaying motor incoordination due to dark degeneration of Purkinje cells (PC-DCD). We estabilished in cultured PCs that Afg3l2-depleted mitochondria ineffectively buffer the evoked calcium peaks, thus enhancing cytoplasmic calcium levels and finally triggering PC-DCD. This defect is caused by the negative synergism between mitochondrial depolarization and altered trafficking of the organelles to PC dendrites. These data, besides disclosing the pathogenetic mechanism of SCA28, demonstrate for the first time the impact of defective mitochondrial calcium uptake on local calcium signaling in neurons. Autosomal dominant cortical myoclonus and epilepsy (ADCME) is characterized by distal, fairly rhythmic myoclonus and epilepsy. We have recently demonstrated that a causative mutation of the α2-adrenergic receptor subtype B (α2B-AR) associates to ADCME by identifying a novel inframe insertion/deletion in two Italian families. The mutation alters several conserved residues of the third intracellular (3i) loop, thus altering the binding with the scaffolding protein spinophilin upon neurotransmitter activation. This work implicates for the first time the α-adrenergic system in human epilepsy and opens new ways for understanding the molecular pathway of epileptogenesis, widening the spectrum of possible therapeutic targets. A polymorphisms of the endothelial NO synthase (eNOS) gene associates with a new variant of eNOS originating by the skipping of exons 20-21. This variant is insensitive to calcium stimulation and displays increased basal NO production. In fact, carriers of the minor allele express the truncated form of eNOS with altered enzymatic activity that influences NO production and endothelial function. These findings open to new intriguing perspectives to several diseases involving vascular response to NO. Giorgio Casari Figure 35. Mitochondria in Purkinje’s cell dendrites (green, caldindin; red, COX1-mitochondria) CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Genome function Unit The mission of the Genome Function Unit is to improve our knowledge of genome function and dysfunction in health and disease. The unit operates on two levels: as a research group undertaking its own original and collaborative research as well as a unit providing services in both genomics and bioinformatics within the Institute as well as to customers and collaborators internationally. During the 2013, the GFU focused mostly on developing research projects as well as clinical protocols. It consists now of approximately 20 members, with highly interdisciplinary educational background. The unit operates 2 NGS sequencers, the Illumina HiSeq 2500 and MiSeq, and utilizes a computing cluster consisting of ~600 CPUs. We processed and analyzed more than 2,800 samples. In terms of research the unit has a strong interest in epigenomics, in particular DNA Methylation. In this area we have several important collaborations exploring the heritability of environmentally induced epigenetic traits, some of them involved in psychiatrics disorder, other in nutrition and allergy, as for example the FP7 project ATOPICA. In 2013 the Unit also published 13 articles (IF 148,75) and was involved in 8 awarded grants revolving around clinical and translational genomics from the Ministry of Health, in collaboration with other OSR units as well as external collaborators. The Unit applied coordinates a COST Action named CHIP ME, focused on ethical, legal and social issues in genomics. The unit is also actively involved in 2 clinical protocols, one operating on rare diseases and the other studying the familiar multiple sclerosis. The unit has worked on the implementation of a web tool for data mining and visualization of large NGS datasets. Last, we collaborated with the unit of Dr. Cirillo to implement a method to evaluate the feasibility of using sequencing technologies for surveillance of drug resistance in TB and with Dr. Montini at TIGET on the use of NGS for vector integration analysis. In terms of service activity the ISO90001 certified unit provides routine NGS protocols such as sequencing of whole human genomes and exomes, RNA-Seq, Medip-Seq and aptamer sequencing. Elia Stupka Biomolecular NMR Laboratory We study the structure and dynamics of biomolecular complexes in solution by NMR spectroscopy in combination with a wide range of biochemical, biophysical and computational techniques. Main projects are: 1) Structural characterization of ligand-receptor interactions; 2) Structural and dynamic characterization of chromatin-interacting modules; 3) metabolomics. 1. Integrin αVβ3 is involved in angiogenesis, inflammation, and cancer. It exerts its role interacting with proteins containing an RGD motif. Recent studies have shown that isoDGR motif can compete with RGD in the binding to αVβ3, paving the way for isoDGR based drugs. We are currently working on computational methods (molecular dynamics, metadynamics, MMPBSA methods) to predict the conformational effects of the flanking residues of RGD and isoDGR based cyclopeptides. The effects of the flanking residue on integrin selectivity is also under investigation. 2. Methylation of lysine residues on histone H3 tails regulates transcription. The PHD finger is a histone binding module able to decode the histone H3 methylated status. We solved the structure of the PHD fingers of Autoimmune Regulator, a protein expressed in mTEC and responsible for autoimmune polyendocrinopathy-candidiasisectodermal dystrophy and characterized their bindingto histone tails by means of biochemical, biophysical and computational methods. We have also solved the PHD finger structure of Sp140 an other transcriptional activator, involved in CLL and demonstrated that it binds to Prolylisomerase Pin1. We are currently characterizing the PHD fingers of NSD1 an other transcription factor involved in Sotos Syndrome and CLL. 3. Metabolomics is an emerging ‘omics’ field that provides an analytical description of the metabolites in complex biological samples under disparate biological or environmental conditions. Ongoing projects focus on metabolomics of osteolytic lesions (S. Cenci, R4R), metabolic profiling of PKD1 -/- MEF cells and mice upon pharmacological treatment (Boletta) software development for multivariate analysis of metabolomics data. Giovanna Musco 215 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Research Units Figure 36. Solution structure of Sp140-PHD domain. Published in: Structure of human Sp140 PHD finger: An atypical fold interacting with Pin1. Zucchelli, C; Tamburri, S; Quilici, G; Palagano, E; Berardi, A; Saare, M; Peterson, P; Bachi, A; Musco, G. FEBS J.: 2014; 281(1): 216-231 © 2013 FEBS doi: 10.1111/febs.12588 Genomic Unit for the diagnosis of human pathologies Our investigation is focused to the identification of new disease genes and to a variety of predisposing sequence variations in order to improve genotype-phenotype correlation in both monogenic and multifactorial traits. We developed high throughput genotyping to perform case-control association studies as well as targeted or exome re-sequencing by Next Generation Sequencing (NGS). Here we summarize our main results published in 2013: i) in order to classify new variants with respect to their pathogenicity, in-vitro functional studies were carried out: a variant from migraine patients was identified in the synprint site of the CaV2.1 channel which increased function. This gain-of-function occurs via alterations in inactivation and SNARE protein regulation in the CaV2.1 channel; ii) in order to identify genetic markers for risk stratification of Brugada Syndrome, we genotyped 73 candidate SNPs in a cohort of 92 patients and correlated the presence of 5 SNPs with the occurrence of major arrhythmic events by allelic association and survival analysis. This allowed us to elaborate a pilot risk stratification algorithm and a weighted genetic risk score. Moreover, our research is involved in the analysis of circulated minority mutated alleles in the plasma of pregnant women to perform noninvasive prenatal diagnosis through protocols based on CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) combined with Sanger sequencing and highly sensitive microarray substrates which could allow for the detection of fetal minority sequences without any enrichment strategy. Both these methodologies were also applied for the detection of KRAS minority mutations in colon cancer. A further line of research is focused on the development of tools for the detection of circulating biomarkers of disease. This include the development of advanced immunoassays for antibody measurement. These assays were applied to: 1) the characterization of autoantibody responses against enterocyte’s autoantigens in the IPEX syndrome, a monogenic disease associated with the development of severe intestinal autoimmunity; 2) antibodies to classical diabetes autoantigens; 3) antibodies to enteroviruses in type 1 diabetes, including in the clinical setting of islet transplantation in patients. Maurizio Ferrari CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Organelle biogenesis and motility Unit Our group is focused into the study of secretory organelle biogenesis and motility in mammalian cells, as alteration of these processes represents an important cause of human disease. In particular, we are focused into the study of melanosomes, i.e. lysosome-related organelles of pigment cells, devoted to the synthesis, storage and transport of melanins, and into their aberrant function in albinism. Indeed, in this genetic disease, melanosomes can lose their ability to synthesize melanin, or can undergo abnormal biogenesis and transport processes. However, in addition to organelles belonging to the secretory pathway, mammalian cells also contain other subcellular organelles, such as mitochondria, which are essential for multiple cellular functions, including ATP production and calcium signaling. Despite the reciprocal crosstalk between different subcellular organelles and their spatial organization relative to each other is relevant for their proper function, secretory organelles and mitochondria have been traditionally considered distinct. Indeed, physical contacts between mitochondria and the secretory pathway have been demonstrated so far only with the endoplasmic reticulum (ER), through structural and functional interorganellar connections. We recently uncovered that mitochondria physically contact melanosomes as well, through fibrillar bridges resembling the protein tethers linking mitochondria and the ER (Figure 1). Mitofusin (Mfn) 2, which bridges ER to mitochondria, specifically localises also to melanosome-mitochondrion contacts, and its knockdown significantly reduces the interorganellar connections. These contacts are associated to the melanogenesis process, since they are enhanced both where melanosome biogenesis takes place in the perinuclear area, and when it is actively stimulated by OA1, the G protein-coupled receptor implicated in ocular albinism and organellogenesis. Conversely, the interorganellar connections are reduced in conditions of abnormal melanosome biogenesis, either due to a primary melanosomal defect, or due to a primary mitochondrial dysfunction. Overall, melanosome-mitochondrion contacts appear relevant for melanogenesis and might play a role in physiological pigmentation and pigmentary diseases. Maria Vittoria Schiaffino Figure 37. Three-dimensional tomographic model of ERmitochondria and melanosome-mitochondria interorganellar contacts, as obtained by manually contouring the limiting membranes and internal structures (melanin fibers and mitochondrial inner membranes) of a melanosome (red), a mitochondrion (yellow) and a ER tubule (green). Fibrillar bridges between the mitochondrion and the ER (light blue) and between the mitochondrion and the melanosome (dark blue) are shown (modified from: Daniele et al. Curr. Biol.: 2014; 24(4): 393-403 doi: 10.1016/j.cub.2014.01.007). 217 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Research Units Proteome biochemistry Unit Onco-proteomics Serological Proteome Analysis of colorectal carcinoma: Immunologic tolerance is broken in pathologic conditions such as cancer. To identify tumoral antigens, we performed a screening of the colorectal tumor proteoma by exploiting auto-antibodies contained in the sera of patients. AutoAbs directed against a surface metallo-protease have been found with high frequency in patients. Similar reactivity was observed in patients affected by others cancers of epithelial origin, but not in cancers of hematopoietic origin. The presence of auto-Abs anti-metalloprotease give an advantages in patients follow up, prolonging the disease-free condition. Auto-Abs affect the metalloprotease function reducing its sheddase activity. Antibody-based proteomics to study cellular signalling networks: Reverse phase protein microarrays is a technique that allow to analyze the abundance of proteins and their phosphorylation status in patient tumor exploiting specific sets of Abs able to dissect different signalling pathways. We are using this approach in B-CLL investigating the BCR signalling in patients having stable or progressive disease. By recombinant protein microarray screening we are also trying to define the reactivity of stereotyped-BCRs cloned from B-CLL patients. Neuro-proteomics Protein pattern changes in cerebrospinal fluid (CSF) of neurodegenerative diseases: Being in contact with the brain, the CSF analysis is important for pathological processes understanding and diagnosis. We found that in the CSF of Parkinson’s (PD) and Alzheimer’s (AD) diseases the protein ceruloplasmin (Cp) is modified in comparison to healthy subject and others neurological diseases. The Cp-modifications are due to protein oxidation that causes a decrease in Cp ferroxidase activity, promoting intracellular iron retention in neurons, that contributes to pathological mechanisms. Now we are studying by mass spectrometry approach the post-translation modifications induced by the pathological oxidative environment on Cp (e.g. carbonylation, deamidation, thiol oxidation, etc.), and the effects on Cp functions. In particular we found that deamidation of the NGR-motifs present in the Cp sequence promotes loss of ferroxidase function and the gain of integrin-binding properties. Massimo Alessio Figure 38. Molecular dynamic modelling of deamidated ceruloplasmin (in red) docking onto αV/β6 integrin (in blue). In licorice are shown the most important interactions between the two proteins. CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Selected publications Webb, EA and Almutair, A; Kelberman, D; Bacchelli, C; Chanudet, E; Lescai, F; Andoniadou, CL; Banyan, A; Alsawaid, A; Alrifai, MT; Alahmesh, MA; Balwi, M; Mousavy-Gharavy, SN; Lukovic, B; Burke, D; McCabe, MJ; Kasia, T; Kleta, R; Stupka, E; Beales, PL; Thompson, DA; Chong, WK; Alkuraya, FS; Martinez-Barbera, JP; Sowden, JC; Dattani, MT. ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies. Brain: 2013; 136(10): 3096-3105 - Article IF 2012: 9,915 Burgoyne, T; Jolly, R; Martin-Martin, B; Seabra, MC; Piccirillo, R; Schiaffino, MV; Futter, CE. Expression of OA1 limits the fusion of a subset of MVBs with lysosomes - a mechanism potentially involved in the initial biogenesis of melanosomes. J. Cell Sci.: 2013; 126(Pt 22): 5143-5152 - Article IF 2012: 5,877 Condliffe, SB; Fratangeli, A; Munasinghe, NR; Saba, E; Passafaro, M; Montrasio, C; Ferrari, M; Rosa, P; Carrera, P. The E1015K variant in the synprint region of the CaV2.1 channel alters channel function and is associated with different migraine phenotypes. J. Biol. Chem.: 2013; 288(47): 33873-33883 - Article IF 2012: 4,651 Drago, D; Cossetti, C; Iraci, N; Gaude, E; Musco, G; Bachi, A; Pluchino, S. The stem cell secretome and its role in brain repair. Biochimie: 2013; 95(12): 2271-2285 - Review IF 2012: 3,142 Elvers, KT; Geoghegan, I; Shoemark, DK; Lampasona, V; Bingley, PJ; Williams, AJK. The core cysteines, (C909) of islet antigen-2 and (C945) of islet antigen-2β, are crucial to autoantibody binding in type 1 diabetes. DIABETES: 2013; 62(1): 214-222 - Article IF 2012: 7,895 Magnoni, R; Palmfeldt, J; Christensen, JH; Sand, M; Maltecca, F; Corydon, TJ; West, M; Casari, G; Bross, P. Late onset motoneuron disorder caused by mitochondrial Hsp60 chaperone deficiency in mice. Neurobiol. Dis.: 2013; 54: 12-23 - Article IF 2012: 5,624 Mancini, C; Roncaglia, P; Brussino, A; Stevanin, G; Lo Buono, N; Krmac, H; Maltecca, F; Gazzano, E; Bartoletti Stella, A; Calvaruso, MA; Iommarini, L; Cagnoli, C; Forlani, S; Le Ber, I; Durr, A; Brice, A; Ghigo, D; Casari, G; Porcelli, AM; Funaro, A; Gasparre, G; Gustincich, S; Brusco, A. Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways. BMC Med. Genomics: 2013; 6: 22 - Article IF 2012: 3,466 Massa, O and Alessio, M and Russo, L; Nardo, G; Bonetto, V; Bertuzzi, F; Paladini, A; Iafusco, D; Patera, P; Federici, G; Not, T; Tiberti, C; Bonfanti, R; Barbetti, F. Serological Proteome Analysis (SERPA) as a tool for the identification of new candidate autoantigens in type 1 diabetes. J. Proteomics: 2013; 82: 263-273 - Article IF 2012: 4,088 Minelli, C and De Grandi, A; Weichenberger, CX; Gogele, M; Modenese, M; Attia, J; Barrett, JH; Boehnke, M; Borsani, G; Casari, G; Fox, CS; Freina, T; Hicks, AA; Marroni, F; Parmigiani, G; Pastore, A; Pattaro, C; Pfeufer, A; Ruggeri, F; Schwienbacher, C; Taliun, D; Pramstaller, PP; Domingues, FS; Thompson, JR. Importance of Different Types of Prior Knowledge in Selecting Genome-Wide Findings for Follow-Up. Genet. Epidemiol.: 2013; 37(2): 205-213 - Article IF 2012: 4,015 Parker, J and Tsagkogeorga, G; Cotton, JA; Liu, Y; Provero, P; Stupka, E; Rossiter, SJ. Genomewide signatures of convergent evolution in echolocating mammals. Nature: 2013; 502(7470): 228231 - Letter IF 2012: 38,597 Privitera, D; Corti, V; Alessio, M; Volonté, A; Lampasona, V; Comi, G; Martino, G; Franciotta, D; Furlan, R and Fazio, R. Proteomic identification of aldolase A as an autoantibody target in patients with atypical movement disorders. Neurol. Sci.: 2013; 34(3): 313-320 - Article IF 2012: 1,412 Rowe, I; Chiaravalli, M; Mannella, V; Ulisse, V; Quilici, G; Pema, M; Song, XW; Xu, H; Mari, S; 219 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Selected publications Qian, F; Pei, Y; Musco, G; Boletta, A. Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat. Med. : 2013; 19(4): 488-493 - Letter IF 2012: 24,302 Sanges, R; Hadzhiev, Y; Gueroult-Bellone, M; Roure, A; Ferg, M; Meola, N; Amore, G; Basu, S; Brown, ER; De Simone, M; Petrera, F; Licastro, D; Strahle, U; Banfi, S; Lemaire, P; Birney, E; Muller, F; Stupka, E. Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development. Nucleic Acids Res.: 2013; 41(6): 3600-3618 - Article IF 2012: 8,278 Sommariva, E; Pappone, C; Martinelli-Boneschi, F; Di Resta, C; Carbone, MR; Salvi, E; Vergara, P; Sala, S; Cusi, D; Ferrari, M; Benedetti, S. Genetics can contribute to the prognosis of Brugada syndrome: A pilot model for risk stratification. Eur. J. Hum. Genet.: 2013; 21(9): 911-917 Article IF 2012: 4,319 Vita, F; Lucarotti, V; Alpi, E; Balestrini, R; Mello, A; Bachi, A; Alessio, M; Alpi, A. Proteins from Tuber magnatum Pico fruiting bodies naturally grown in different areas of Italy. Proteome Sci.: 2013; 11: 7 - Article IF 2012: 2,420 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Neurogenomics Unit Genome function Unit 221 CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Biomolecular NMR Laboratory Proteome biochemistry Unit CENTER FOR TRANSLATIONAL GENOMICS AND BIOINFORMATICS Genomic Unit for the diagnosis of human pathologies 223 EXPERIMENTAL IMAGING CENTER Director: Carlo Tacchetti Associate Director: Alessandro Del Maschio* Research Units Advanced fluorescence microscopy and nanoscopy research Unit ––––––––––––––– 229 HEAD OF UNIT: Carlo Tacchetti POST-DOCTORAL FELLOW: Davide Mazza** Dynamic fluorescence spectroscopy in biomedicine ––––––––––––––––––––––––– 230 GROUP LEADER: Valeria R. Caiolfa POST-DOCTORAL FELLOWS: Valeria Corti, Giulia Ossato, Antonio Trullo FELLOW: Moreno Zamai Mouse functional genetics Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 231 HEAD OF UNIT: Ottavio Cremona* RESEARCHER: Giuseppina Di Giacomo* POST-DOCTORAL FELLOWS: Marina Cardano**, Luca Ruggiero**, Elisa Sala** Clinical Research Units Clinical and experimental radiology Unit ––––––––––––––––––––––––––––––––––––––––––––––– 232 HEAD OF UNIT: Francesco De Cobelli* PHYSICIANS: Antonio Esposito*, Claudio Losio, Roberto Nicoletti, Massimo Venturini 225 EXPERIMENTAL IMAGING CENTER Clinical Research Units/Service Units High technology in radiation therapy Unit –––––––––––––––––––––––––––––––––––––––––––––– 232 HEAD OF UNIT: Nadia Di Muzio PHYSICIANS: Genoveffa Berardi, Anna Chiara, Cesare Cozzarini, Italo Dell’Oca, Andrei Fodor, Paolo Passoni, Najla Slim RESIDENTS: Barbara Noris Chiorda, Flavia Zerbetto TECHNICIANS: Alessandro Capelli, Giovannella Salvadori, Andrea Sbalchiero, Simone Selli Medical physics Unit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 233 HEAD OF UNIT: Riccardo Calandrino PHYSICISTS: Sara Broggi, Giovanni Mauro Cattaneo, Antonella Del Vecchio, Claudio Fiorino, Barbara Longobardi, Paola Mangili, Lucia Perna, Patrizia Signorotto RESEARCHERS: Giancarmelo Agnello, Maria Luisa Belli, Marco Bianchini, Carmen Gigliotti, Federica Palorini, Roberta Raso, Carla Sini, Antonello Spinelli Molecular imaging Unit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 233 HEAD OF UNIT: Luigi Gianolli RESEARCHERS: Sara Belloli, Valentino Bettinardi, Assunta Carpinelli, Isabella Castiglioni, Giuseppe Di Grigoli, Francesca Gallivanone, Maria Carla Gilardi, Elena Incerti, Rosa Maria Moresco, Maria Picchio, Ornella Rimoldi, Paola Scifo, Silvia Valtorta PHD STUDENTS: Elena Maria Andreolli, Luca Presotto PHYSICIANS: Elena Busnardo, Carla Canevari, Federico Fallanca, Claudio Landoni, Patrizia Magnani, Andrea Panzacchi, Ana Maria Samanes Gajate, Pietro Spagnolo, Paola Todeschini, Giovanna Vanoli RADIOCHEMISTS: Valeria Masiello, Maria Grazia Minotti, Cristina Monterisi TECHNICIANS: Antonia Compierchio, Pasquale Simonelli, Francesco Sudati Neuroradiology research group ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 234 HEAD OF UNIT: Andrea Falini* CLINICAL GROUP LEADER: Letterio Salvatore Politi PHYSICIANS: Simonetta Gerevini, Claudio Righi, Francesco Scomazzoni, Franco Simionato FELLOWS: Chiara Cianciaruso, Antonella Pagani Service Units ALEMBIC, Advanced Light and Electron Microscopy BioImaging Center –––––––– 235 HEAD OF FACILITY: Fabio Grohovaz* COORDINATORS: Fabio Grohovaz* (Light microscopy); Carlo Tacchetti (Electron microscopy) RESEARCHER: Maria Carla Panzeri LAB MANAGER: Andrea Raimondi TECHNICIANS: Cesare Covino, Andrea Menegon, Desirée Zambroni Intravital microscopy ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 236 HEAD OF FACILITY: Luca G. Guidotti RESEARCHERS: Matteo Iannacone, Giovanni Sitia TECHNICIANS: Tiziana Cataudella, Pietro Di Lucia EXPERIMENTAL IMAGING CENTER Service Units Preclinical MRI and US facility –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 237 HEAD OF UNIT: Antonio Esposito* PHYSICIANS: Letterio Salvatore Politi, Massimo Venturini PHYSICIST: Antonello Spinelli BIOLOGIST: Laura Perani RESIDENTS: Giulia Agostini**, Giulia Cristel, Paolo Marra**, Anna Palmisano** TECHNICIANS: Tamara Canu Pre-clinical PET facility –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 238 HEAD OF FACILITY: Rosa Maria Moresco RESEARCHERS: Sara Belloli, Paola Scifo POST-DOCTORAL FELLOWS: Giuseppe Di Grigoli, Silvia Valtorta FELLOW: Isabella Raccagni TECHNICIAN: Pasquale Simonelli, Francesco Sudati * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele 227 EXPERIMENTAL IMAGING CENTER Introduction by the Directors Mission and vision - The mission is threefold: 1. Facility - Organizing and providing imaging platforms able to support independent basic, pre-clinical and clinical research, and to foster translational research in OSR. 2. Technology development - Continuously developing and providing up-to-date technology through R&D activities involving biologists, chemists, physicists, computer scientists and clinician scientists. 3. Research - Hosting independent basic and clinical research groups. The vision is to create an environment in which the expertise provided by the R&D and research groups helps the facilities to provide the most up-to-date service possible. Organization - The Center is composed of 4 Facility Units (Small animal MR and Ultrasound; Small animal PET; Intravital Microscopy; and Light and Electron Microscopy), 3 Basic Research Unit, 5 Clinical Research Units, for a total of about 70 people. Several members of the CIS are Faculty members of the Università Vita - Salute San Raffaele. The Center is also partner of the European Consortium for Nanomedicine, having access to high resolution cryo-EM, EM-tomography and other state-of-the-art EM technologies. The small animal imaging facilities are integrated within the “Mouse Clinic” project in the campus. The Service Units operate on a fee for service basis for internal and external users. Finally, due to CIS capabilities and organization, the Light and Electron Microscopy, the pre-clinical MR and US, and the pre-clinical PET facilities are part of the Italian national nodes of the European Consortium EurobioImaging, a network of facility nodes scattered in Europe. Goals - (a) foster R&D in Bioimaging; (b) develop research in cardiovascular diseases, oncology, genetic diseases, metabolic diseases, urology, neurological diseases by mean of Bioimaging; (c) offer technical and scientific expertise in Bioimaging. The involvement of interdisciplinary competences, including biology, chemistry, physics, informatics and clinical research, will be pivotal to these aims. Achievements - Significant achievements in all fields of biology, medicine and R&D studied at CIS have been obtained: • Clinical and Experimental Radiology: Innovative breast cancer screening program tailored on personal risk factors. Cardiac MR prospective study of acute myocardial infarction. Development of Diffusionweighted MR technique to characterize lesions and to monitor tumour response to therapy. • Neuroradiology: Development, optimization and validation of MRI techniques for diagnosis and followup of genetic disease and tumor lesions in pre-clinical models and in clinical applications. • Molecular Imaging: Clinical research activity using molecular Imaging (SPECT and PET/CT), preclinical characterization of radiopharmaceuticals, and validation of animal models for different diseases. • High Technology in Radiation Therapy: Application of Adaptive approach in rectal, head and neck, and lung cancer. In particular pilot studies are ongoing to: obtain an escalation of tumor dose, for better tumor regression and low grade side effects in the second half of treatment of rectal cancer; to reduce the dose to normal tissue in lung and head and neck cancers. • Medical physics: Development of a preclinical molecular optical imaging Cerenkov method, to image β+, β-, α and γ emitters. Multimodal imaging acquisition during Radiotherapy treatment to allow the definition of adaptive strategies to optimize treatment. • Mouse functional genetics: study of the role of endophilin in mouse genetic. • Advanced fluorescence microscopy and nanoscopy: Assembly and validation of a new 3D nanoscopy microscope to study TF/chromatin interaction dynamics. • Dynamic fluorescence spectroscopy in biomedicine: Set up detection of protein stoichiometry in complexes and optimization of new approaches to analyze time-stack fluorescence multiphoton raster scanning and total internal reflection images. EXPERIMENTAL IMAGING CENTER Research Units Advanced fluorescence microscopy and nanoscopy research Unit The behavior of a protein can be determined by measuring how the protein moves within the intercellular milieu and how it interacts with its partners, or in other words by quantifying its dynamics. Especially in the field of transcription, it is now demonstrated that different dynamic behaviors of transcription factors (TFs) are crucial in producing specific cellular responses to physiological or pathological stimuli. Unfortunately, the conventional biochemical and microscopical methods are unable to capture the wide heterogeneity of dynamical behaviors typically observed for proteins in living cells, underscoring the need for methods capable of detecting and following single molecules. The microscope can be used to quantify molecule by molecule kinetic properties such as the diffusion rates of proteins in living cells, their binding to other proteins or to immobile scaffolds (such as the cytoskeleton or chromatin), and their oligomerization state. Further the capability of the microscope to visualize individual molecules, can be used to overcome the resolution limit, by applying approaches such as PALM or dSTORM to obtain fluorescence images with a spatial resolution below 40 nm (5 times better then conventional fluorescence microscopy). We have applied the developed prototype to study how TFs search for their target sequences on DNA in living cells, a long-standing question in the transcription dynamics field. By focusing on the tumor suppressor p53 we were able to measure that only a small fraction of the TF is bound specifically to DNA, and that its binding is very transient (on the timescale of seconds). Further our data supports the facilitated diffusion mechanism: when searching for its binding sites p53 first binds non specifically to DNA by means of its Cterminal tail, and then scans the genome for its specific targets by means of 1D diffusion. We have recently extended the microscope to twocolor single-molecule analysis, which has allowed to identify that p53 binds more strongly to transcriptionally active DNA regions. Carlo Tacchetti Figure 39. Two-color super-resolution (dSTORM) image obtained on H1299 lung carcinoma cells, labelled with phalloidin-ATTO488 and anti-TubulinAlexa568. Details of 40nm and below can be resolved in the dSTORM image. 229 EXPERIMENTAL IMAGING CENTER Research Units Dynamic fluorescence spectroscopy in biomedicine Quantitative imaging of protein clustering We are involved in developing quantitative imaging approaches for elucidating the spatio-temporal dynamics of protein clustering and protein-protein interaction at the cell membrane and in intracellular compartments. Despite the importance and ubiquity of protein clustering mechanisms in controlling a variety of cell signaling cascades, very few methods can be employed for real-time analysis in live cells. Our approach includes the combination of phasor-FLIM-FRET and Single Molecule imaging by the Number of Molecules and Brightness (N&B) analyses. By these means, we have uncovered the regulatory role of tetraspanins at the immune synapse (IS) in live T lymphocytes, by showing that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling (Figure 40A). In a second project, we have addressed the issue of the fast activation/deactivation of membranebound proteases (MMPs) that are involved in matrix proteins degradation or adhesion receptors shedding. MMPs display high basal expression but negligible activity that can be rapidly activated by unknown mechanisms beyond classical transcriptional or internalization regulation. Different MMPs associate with tetraspanins, and alterations in tetraspanin expression levels demonstrate that tetraspanins are negative regulators of protease activity. To elucidate the unknown molecular mechanism by which this regulation occurs we are studying the rapid association/dissociation of MMPs in tetraspanin-enriched microdomains (TEM). The combination of phasor-FLIM-FRET and N&B analysis is unrevealing the stoichiometry of MMPs homo complexes and their interaction with TEMs (Figure 40B) Valeria R. Caiolfa Figure 40. (A) CD81-mCherry interacts with ICAM-1-mEGFP during conjugation between T cells and antigen-presenting cells (APCs), and the interaction increases over the time course of IS formation, as the molecules redistribute throughout the contact area. J77 cells were co-transfected with mEGFPmCherry pairs, conjugated with Raji/SEE cells, and analyzed by phasorFLIM-FRET at the IS, revealing the area of tight interaction (red mask) between the two proteins. Early IS after 5 min from conjugation; Late IS after 30 min from conjugation. (Molecular and Cellular Biology, 2013) (B) In migrating cells, the association of MMPs with TEMs at the basal cell membrane (high FRET eff %) disrupts the MMPs oligomers (low brightness) (unpublished results). EXPERIMENTAL IMAGING CENTER Mouse functional genetics Unit Mice mutants for endocytosis and neuroferritinopathy Our research activities have been focused on two topics: 1. Role of Epsin family members in the function and homeostasis of human and mouse skin. In recent years, endocytosis has been characterized as a major platform for cell signaling activation and regulation. Our laboratory is characterizing the function of a newly family of endocytic adaptors – the Epsin family – in cell signaling and, possibly, in cancerogenesis. We previously showed that the ubiquitously expressed Epsin1 and Epsin2 are essential factors for the activation of the Notch signaling pathway during mammalian development. Currently, we focused on Epsin function in epithelial tissue homeostasis. We found that acute depletion of Epsins by gene knockdown techniques led to a significant decrease in Notch and Wnt signaling both in vitro and in vivo. These alterations tightly correlate with subversion of skin differentiation and proliferation and with early endocytic defects in these pathways. Moreover, mice with gene dosage defects of Epn1 and 2, spontaneously develop pre/paraneoplastic lesions and frankly carcinomatous lesions during aging. These data strongly suggest an essential role of the Epsin family members in skin homeostasis. 2. Animal models of neuroferritinopathies for the study of iron in neurodegeneration. Mutations of ferritin L−chain gene (FTL-1) lead to brain iron accumulation and neurodegeneration in humans. In collaboration with the S. Levi and F. Grohovaz Units, we generated and characterized mouse transgenic mice for a human FTL-1 pathogenic gene. Transgene expression correlates with increased accumulation of iron and extensive oxidative damage in neurons both in vitro and in vico. The mutated FTL-1 protein showed reduced capacity to incorporate iron and increased ability in generating reactive oxygen species. Brain tissues from transgenic mice accumulate lipofuscin containing iron in GABAergic neurons, as in early stages of the human disease. Behavioral analyses of mutant mice show motor deficits with a developmental profile of a progressive pathology, as observed in the human disease. In sum, these data show that our FTL-1 mice mutants are excellent models to study neuroferritinopathy and may serve to develop specific therapeutic agents to target iron dysregulation in brain. Ottavio Cremona 231 EXPERIMENTAL IMAGING CENTER Clinical Research Units Clinical and experimental radiology Unit 1. Cardiac Imaging 3. Breast Imaging a. Myocarditis: in order to find prognostic imaging markers for patients at risk of ventricular tachyarrhythmias and sudden death. b. Cardiac CT and MR assessment of structural substrate of ventricular tachycardia; 3D imaging based modeling of the heart/coronary anatomy and scars localization as a tool to be merged with electro-anatomic maps for a guide of ventricular tachycardia ablation. c. A cardiac MR-study to evaluate the effect of GH-deficiency in patients’ heart. d. Characterization of cardiac masses. e. Optimization of Cardiac-CT protocols for the assessment of coronary arteries in re-vascularized patients. f. Patients with AMI submitted to PCI in order to evaluate the microvascular obstruction and to compare our results with coronary angiography and to find new prognostic factors. a. Two large multicenter studies are ongoing investigating the role of Breast-MRI in two different settings: • as an alternative tool to mammography and ultrasound in the screening of women at intermediate risk of breast cancer (MRIB study) • to demonstrate the performance of MRI over traditional imaging in the pre-surgical planning of patients diagnosed with breast cancer (MIPA study) b. Assessment of tumor response in women with locally-advanced breast cancer undergoing neoadjuvant chemotherapy. c. Characterization of breast architectural distortions by means of MRI. 2. Diffusion-weighted MR 5. Interventional Radiology We aimed to evaluate the accuracy of DWI as a marker of tumour aggressiveness and tumour response to chemotherapy. We demonstrated the correlation between ADC and tumour grading factors in prostate, gastroesophageal and rectal cancer and that changes in ADC before and after neoadjuvant-treatment can be used as reliable marker of tumor response. Analysis of innovative therapeutic interventional approaches for liver metastases, such as a new TACE using drug eluting beads (DEBIRI technique) and portal embolization for the two-stage hepatectomy and a study on the extending indications for portal islet autotransplantation after pancreatic surgery. Francesco De Cobelli 4. Imaging of islet transplantation MR functional assessment of micro-vascular events occurring after pancreatic islets transplantation and relationship with graft outcome. High technology in radiation therapy Unit Among its various research project the Radiation Therapy Dept is collaborating with Nuclear Medicine to define treatment volume on the basis of biological target characteristics, while the last Linac generation, e.g. Tomotherapy and Rapid Arc permits extreme precision in delivering radiation treatment. The most important result of this combination is improved tumor control with reduced side effects. In radical treatment of prostate cancer we obtain 5 year biochemical disease free survival of 100% in Low Risk patients and 97.5% in Intermediate and High Risk patients. In prostate cancer patients with lymphnodal relapse diagnosed with Pet choline our radical radiation treatment protocol using high dose to treat the entire lymphnodal chain with a supplementary boost on Pet positive nodes has resulted in controlled psa and disease in 50% of treated patients after two years from treatment end. In some cases psa has been controlled without pharmacological therapy such as hormonal(HT)or chemotherapy(CT). This result is important in terms of delaying the use of drugs(HT or CT)in selected patients for many years. In rectal cancer treatment using combined radiochemotherapy with hypofractionated radiotherapy(RT),75% of patients achieved residual tumor cell vitality lower than 5%, as shown in histopathological examination. It was attempted to increase the total dose delivered in these patients by means of the reduction of the treated volume in the last six sessions of RT administered in adaptive modality. All patients underwent Diffusion MR at the start and after ten treatment sessions. A boost dose to the residual tumor is delivered, reducing the target volume and the involvement of the surrounding tissue. This modality allows us to decrease side effects even with increased total dose in combination with chemotherapy. Some of EXPERIMENTAL IMAGING CENTER these patients refused surgery and are disease free after 30 months from treatment end. In the next months the patients will undergo not only Diffusion MR but also FdG PET/TC in order to evaluate the metabolic volume and compare MR and Pet volume. The ultimate aim of our research in these rectal cancer patients is to identify a subgroup of patients that can avoid surgery considering that such interventions are frequently demolitive in nature. Nadia Di Muzio Medical physics Unit The main areas of interest of the Medical Physics Department (MPD) are: 1. Radiotherapy Physics The main working area of the MPD is the knowledge of advanced methodologies for the optimization of the radiotherapy treatments. Sophisticated treatment units such as tomotherapy and rapid-arc are continuously monitored and tuned by the senior staff of the Department. The outcomes were monitored during patients follow up over several years. Multi-modal images were acquired before and during the treatment in order to obtain: 1) a more accurate definition of the real extension of neoplastic disease 2) a better visualization of healthy tissues 3) the evaluation of breathing related tumor/organ mobility (4D technology) 4) The definition of adaptive strategies to modify treatment plans. A number of studies have been conducted evaluating the impact of multi-modal approach in treatment planning optimization. Several collaborations with national and international groups have been consolidated over the last years; two large projects have been funded by AIRC during the last two years and are currently in progress. 2. Physics for Imaging Cerenkov luminescence imaging (CLI) has been developed by the MPD in collaboration with the University of Verona and has been applied to small animals and humans. The MPD also provides support to optical imaging research carried out at the pre-clinical imaging facility of the centre for experimental imaging. A second research topic is the CT dose monitoring and reduction using state of the art iterative reconstruction algorithms. 3. Physics applied to Radioprotection, Laser and NMR Safety At OSR is present one of the largest Italian Nuclear Medicine Department (NMD) with two Cyclotrons. The radiation protection of the NMD is carried out by the MPD with the use of the most advanced methodologies for radiation monitoring. Since the beginning of the 80’s the MPD staff is also enrolled in the safety and quality assurance of Laser and NMR equipments installed at OSR. Riccardo Calandrino Molecular imaging Unit The main research activities are focused on: 1) Validation of PET/CT with 18F-FAZA for the definition of hypoxia in tumour tissue in patients with NSCLC. The laboratories are now able to ensure the quality and safety of the production of the radiopharmaceutical complying with the regulations in the field of production of radiopharmaceuticals for diagnostic use. The activities for the implementation of PET laboratories according to Good Manufacturing Practice (GMP) are also continuing. 2) Preclinical imaging activities for: a) preclinical evaluation of radioligand for the in vivo imaging of microglia activation in brain neuroinflammatory and neurodegenerative disorders; b) combined use of 18F-FAZA with 18F-FDG or 18F-FLT for the in vivo characterization of preclinical model of breast, brain and ovarian cancer; c) evaluation of the potential use of PET radioligand as biomarkers of treatment efficacy. 3) In clinical oncological PET studies, the diagnostic accuracy of 18F-FDG and 11C-Choline PET tracers have been evaluated in several neoplastic diseases. In particular, in gestational trophoblastic disease, 18F-FDG PET/CT have been proved to identify the sites of primary and/or metastatic disease in patients with persistent high levels of 233 EXPERIMENTAL IMAGING CENTER Clinical Research Units βHCG after first-line chemotherapy being of additional value in patient management for guiding alternative treatment. The role of 11C-Choline PET/CT for the detection of prostate recurrent disease have been demonstrated also at low PSA serum value, particularly when PSA doubling time is less than 6 months. This modality may thus be indicated in the early phases of recurrent disease to guide personalized treatments. 4) In clinical cardiological studies, 13N-NH3 myocardial blood flow quantification with PET/CT produced comparable results over different image reconstruction techniques as well as by using different analysis software programs. The variability between commercial software programs for the analysis of cardiac volume and ejection fraction in 13N-NH3 PET/CT were found to be limited. The prognostic role of SPECT in patients with cardiac syndrome X and the utility of SPECT for the evaluation of the effect of stem cell transplantation on cardiac function and perfusion have been demonstrated. Luigi Gianolli Neuroradiology research group The Neuroradiology Research Group is committed to applying innovative imaging technologies toward more comprehensive understanding of pathologies of the central and peripheral nervous systems at both preclinical and clinical levels, and to developing new neuro-interventional procedures and protocols for minimally-invasive treatment of neurovascular diseases. During 2013 the research activity of the Group was focused on 3 main areas: preclinical studies, clinical diagnostic research activity and neuro-interventional trials. In the first area, the Group has a well-established expertise in conventional and advanced MR-based imaging techniques of brain tumors and of inflammatory, demyelinating and dysmyelinating murine models of central nervous system diseases. Using the small-animal 7T MRI we evaluated and quantified structural changes in the brains of murine models of several neurological pathologies, such as inherited demyelinating disorders, stroke and brain tumors. Further, innovative cellular and molecular imaging techniques were developed for monitoring stem cell transplantation in neurological diseases and for tracking the recruitment of hematopoietic and stromal cells inside tumors. We explored several cell labeling strategies based on superparamagnetic iron oxides particles and on different MR-reporter genes, allowing iron accumulation within cells. Transgenic mice expressing a mutated form of the human L-ferritin chain were also analyzed. In the field of clinical research, the Group worked on the assessment of structural and functional aspects of brain and orbital inflammatory, infectious and tumoral disorders. Specific fields of interest were HIV-related infections and lymphomas. A diffusion tensor imaging study of the cervical spinal cord was performed on adrenomyeloneuropathy patients. Additionally, MRI was also applied for studying hereditary neuromuscular diseases such as Duchenne muscular dystrophy within a trial based on mesoangioblast intra-arterial transplantation. The research in the neuro-interventional area was focused at the optimization of new endovascular techniques and at the validation of new angiographic devices for treatment of intracranial aneurysms, vascular stenosis/occlusions and artero-venous malformations or shunts. Letterio Salvatore Politi EXPERIMENTAL IMAGING CENTER Service Units ALEMBIC, Advanced Light and Electron Microscopy BioImaging Center The Advanced Light and Electron Microscopy BioImaging Center, ALEMBIC, has been operating for more than 10 years at San Raffaele Scientific Institute as an environment in which biological experimentation requiring optical or electronic imaging is expedited through the ready access to state-of-the-art instrumentation. Within the facility, the staff: instructs researchers in the most effective and independent use of microscope setups; provides continuous support for resolution of scientific problems through the use of the best available technology and correct planning of experimental protocols; promotes technical updates to keep the facility on the forefront of available technology; performs, when required, full service for fees. Alembic aims to stay at the forefront of microscopy innovation to maintain its leadership position in Italy (member of ItaBio) but also in Europe (member of the EuroBioImaging Italian node). Thanks to consistent investment by OSR, ALEMBIC is currently undergoing a relevant increase of its microscopy fleet with the acquisition of new and important technologies: Figure 41. Electron microscopy image of the endomebrane system of a primary human plasma cell. (Nu= Nucleus; GA= Golgi Apparatus; RER= Rough Endoplasmic Reticulum; Cen= Centrosomes. Scale bar 500nm). Credits: Cenci Lab - Age related diseases Unit 235 EXPERIMENTAL IMAGING CENTER Service Units • TIRF microscopy, to investigate plasma membrane events • GSD (Ground State Depletion), a super-resolution approach • Multiphoton microscopy, for deep imaging living tissues • White Light Laser Confocal microscopy with FLIM-FRET to study protein-protein interactions These instruments will be available in 2014. There is also an area in which the staff develops new methodologies and conducts research on techniques with significant potential to enhance bioimaging research. This R&D activity has mainly converged around the use of voltage sensitive dyes along two main directions: • integration of optical recordings with a multi electrode system to monitor membrane potential changes in neuronal networks (with Politecnico di Milano and San Raffaele Units) • use in drug discovery screening (patent application n. EP09165872 “Method for optical measuring variations of cell membrane conductance”) Our numbers for 2013: 821 registered users (331 active), 125 persons attending theoretical/practical courses; ~6000 hours of independent microscopes use; 15 papers spontaneously acknowledging that results were in part enabled by Alembic infrastructures. Web resources: www.hsr.it/research/alembic Fabio Grohovaz Intravital microscopy Up until few years ago the visualization of cellular or subcellular processes that control physiological and/or pathological responses in animal models has been limited to the static imaging of explanted tissues. The advent of wide field epifluorescence single photon intravital microscopy (1P-IVM) and two photon IVM (2P-IVM) has given us the opportunity to follow those processes within live hosts. The inability of 1P-IVM to collect images in a “confocal-like fashion” represents both a limitation (narrow image quality as photons are collected also from out-of-focus planes) and an advantage (fast speed of acquisition of up to 200 frames/sec) when compared to 2P-IVM, which has high-resolution and 3-D sectioning capabilities but it usually acquires a frame every 15-30 sec. Because of limited surgical access and/or intrinsic anatomical features, the much more limited depth penetration of 1P-IVM - as compared to 2P-IVM - restrains IVM to certain districts (e.g. lymph nodes, spleen, bone marrow, pancreas, brain, etc) but not others (e.g. liver, cremaster muscle and various vascular beds). As such, 1P-IVM represents the best option for studying events that occur rapidly (e.g. cell adhesion to blood and lymph vessels), while 2PIVM is best suited to follow slow-occurring phenomena (e.g. cell extravasation and cell-cell interaction). Surgical access also imposes the visualization of districts such as the popliteal lymph nodes, the bone marrow, the cremaster muscle or the brain with up-right microscopes, while the visualization of the liver is better performed by the use of inverted machines. The IVM Facility at OSR is intended to be a resource for the scientific community and, at present, it counts on an upright microscope dedicated to 1P-IVM and two microscopes (one up-right and one inverted) dedicated to 2P-IVM. The facility provides consulting activity ruling out necessity/feasibility of a given IVM approach and it is primarily devoted to highly selected projects that effectively benefit from the use of this time-consuming and complex technology. Luca G. Guidotti EXPERIMENTAL IMAGING CENTER Preclinical MRI and US facility Small animal imaging is an integral part of a modern translational research Several modalities allow to visualize organs, tissues, or cells, in living animals. Some imaging modalities are exclusive for preclinical imaging, as the optical imaging, instead most of them are the same applied in the clinical setting. The Preclinical MRI and US Facility is equipped with a 7T Magnetic Resonance scanner (Bruker; BioSpec 70/30), a dedicated Ultrasound scanner (Vevo 2100; Visualsonic) and a combined Optical Computed Tomography scanner (IVIS SpectrumCT® Perkin Elmer), therefore as the capability to offer tailored assistance to researchers needing to characterize their small animal models or to perform longitudinal imaging studies. All the modalities are non-invasive and are managed with the scientific collaboration of imaging experts, which may propose the best imaging protocol for each specific task: Antonio Esposito (Head of the Facility and Coordinator of body-MRI); Letterio Salvatore Politi (Coordinator of neuroMRI); Massimo Venturini (Coordinator of US); Antonello Spinelli (Coordinator of Optical Imaging). In 2013 the Facility collaborated with both internal and external researchers on several issues: • MRI monitoring of acute and chronic muscle injury and regeneration • MRI monitoring of liver neoplastic burden (both HCC and metastasis) • MRI staging of disease in mice genetically predisposed to develop pancreatic cancer • MRI and US monitoring of disease burden in mouse models of polycystic kidney disease • MRI and US monitoring transgenic models of prostate cancer • MRI and US monitoring of lymphedema after lymph-node dissection and transplantation • MRI and CT characterization of a mouse model of osteoarthritis • In-vivo and ex-vivo MRI assessment of atherosclerotic burden and plaques instability • MRI of cerebral ischemic stroke models • MRI characterization of mouse models of neuroferritinopathies • MRI study on specificity of SPIO signal for cellular imaging purposes • In vivo MRI monitoring of SPIO labeled leukocytes migration into nodules of peritoneal carcinogenesis • Brain and spinal MRI in mouse models of multiple sclerosis • US monitoring of models of peritoneal carcinogenesis • US assessment of splenomegaly in Wiscott Aldrich syndrome models • US assessment of leukemic mice • Preliminary experience of US guided punctures in mice. Antonio Esposito Figure 42. Collection of images obtained with the 7T MRI scanner: On the left coronal color coded map of a Diffusion Tensor Imaging study performed on mouse brain; each color represent the preferential direction white matter fibers. In the center the diastolic frame of a long axis cine view of the mouse heart; are clearly depicted the left and right ventricles and the aortic valve with ascending aorta and aortic arch. On the right a 3D reconstruction of the liver of a mouse model of chronic hepatitis B; in transparent lightgreen is represented the normal liver parenchyma and in dark-green are segmented the HCC nodules. 237 EXPERIMENTAL IMAGING CENTER Service Units Pre-clinical PET facility The Laboratory of Preclinical PET Imaging, localized within the Nuclear Medicine Department of the San Raffaele Hospital, operates and has access through formal collaborations with personnel and instrumentation installed inside the laboratory belonging to the Institute of Bioimaging and Molecular Physiology (IBFM) of the National Research Council and the Tecnomed Foundation of University of Milan Bicocca. Imaging studies are performed taken advantage of the single photon or positron emitters labeled radiopharmaceuticals developed by the laboratory Radiochemistry of the Nuclear Medicine Department. Main activities of the laboratory are focused on the use of emission tomography techniques for: a) characterization of kinetics and biological properties of novel radiopharmaceuticals; b) characterization of preclinical model of diseases using emission tomography techniques in comparison of the different clinical presentation of patients evaluated with PET or SPECT in clinical practice as well as during research protocols; c) design and set up of preclinical imaging studies for drug the characterization of novel therapeutic strategies. Studies are performed on animal or cellular model prepared by other research groups or directly by the personnel of the laboratory. Radiopharmaceutical development includes the characterization and measurement of the circulating and tissue fraction of radiolabeled metabolites and the evaluation of the potential diagnostic sensitivity of the probes using adequate animal model of diseases. The laboratory is equipped with: • Small animal dedicated PET/SPECT tomograph • Equipment for administration of volatile anesthetic • Phosphor-imager for authoradiography • HPLC system for the analysis of radiolabeled metabolites • Gamma counter • Cryostat for tissue dissection • Stereotaxic surgery apparatus for mice and rats • Work stations e software for image analysis and quantification The laboratory is also equipped with ventilated cabinet for animal housing authorized by local and national institutions and is connected with the general facility for animal housing and care. Rosa Maria Moresco EXPERIMENTAL IMAGING CENTER Selected publications Cattaneo, GM; Passoni, P; Longobardi, B; Slim, N; Reni, M; Cereda, S; Di Muzio, N; Calandrino, R. Dosimetric and clinical predictors of toxicity following combined chemotherapy and moderately hypofractionated rotational radiotherapy of locally advanced pancreatic adenocarcinoma. Radiother. Oncol.: 2013; 108(1): 66-71 - Article IF 2012: 4,520 Fellin, F; Azzeroni, R; Maggio, A; Lorentini, S; Cozzarini, C; Di Muzio, N; Fiorino, C; Calandrino, R; Schwarz, M. Helical tomotherapy and intensity modulated proton therapy in the treatment of dominant intraprostatic lesion: A treament planning comparison. Radiother. Oncol.: 2013; 107(2): 207-212 - Article IF 2012: 4,520 Sitia, G; Iannacone, M; Guidotti, LG. Anti-platelet therapy in the prevention of hepatitis B virus-associated hepatocellular carcinoma. J. Hepatol.: 2013; 59(5): 1135-1138 - Article IF 2012: 9,858 Trullo, A; Corti, V; Arza, E; Caiolfa, VR; Zamai, M. Application limits and data correction in number of molecules and brightness analysis. Microsc. Res. Tech.: 2013; 76(11): 1135-1146 - Article IF 2012: 1,593 Mazza, D; Mueller, F; Stasevich, TJ; McNally, JG. Convergence of chromatin binding estimates in live cells. Nat. Methods: 2013; 10(8): 691-692 - Letter IF 2012: 23,565 Cortese, K; Howes, MT; Lundmark, R; Tagliatti, E; Bagnato, P; Petrelli, A; Bono, M; McMahon, HT; Parton, RG; Tacchetti, C. The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments. Mol. Biol. Cell.: 2013; 24(2): 129-144 - Article IF 2012: 4,803 Spinelli, AE; Ferdeghini, M; Cavedon, C; Zivelonghi, E; Calandrino, R; Fenzi, A; Sbarbati, A; Boschi, F. First human Cerenkography. J. Biomed. Opt.: 2013; 18(2): 20502 - Letter IF 2012: 2,881 Zonari, E; Pucci, F; Saini, M; Mazzieri, R; Politi, LS; Gentner, B; Naldini, L. A role for miR-155 in enabling tumor-infiltrating innate immune cells to mount effective antitumor responses in mice. Blood: 2013; 122(2): 243-252 - Article IF 2012: 9,060 Venturini, E; Losio, C; Panizza, P; Rodighiero, MG; Fedele, I; Tacchini, S; Schiani, E; Ravelli, S; Cristel, G; Panzeri, MM; De Cobelli, F; Del Maschio, A. Tailored breast cancer screening program with microdose mammography, us, and mr imaging : Short-term results of a pilot study in 4049-year-old wome. Radiology: 2013; 268(2): 347-355 - Article IF 2012: 6,339 Esposito, A and Campana, L; Palmisano, A; De Cobelli, F; Canu, T; Santarella, F; Colantoni, C; Monno, A; Vezzoli, M; Pezzetti, G; Manfredi, AA; Rovere-Querini, P; Del Maschio, A. Magnetic Resonance Imaging at 7T Reveals Common Events in Age-Related Sarcopenia and in the Homeostatic Response to Muscle Sterile Injury. PLoS ONE: 2013; 8(3): e59308 - Article IF 2012: 3,730 De Cobelli, F; Giganti, F; Orsenigo, E; Cellina, M; Esposito, A; Agostini, G; Albarello, L; Mazza, E; Ambrosi, A; Socci, C; Staudacher, C; Del Maschio, A. Apparent diffusion coefficient modifications in assessing gastro-oesophageal cancer response to neoadjuvant treatment: comparison with tumour regression grade at histology. Eur. Radiol.: 2013; 23(8): 2165-2174 - Article IF 2012: 3,548 Passoni, P; Reni, M; Cattaneo, GM; Slim, N; Cereda, S; Balzano, G; Castoldi, R; Longobardi, B; Bettinardi, V; Gianolli, L; Gusmini, S; Staudacher, C; Calandrino, R; Di Muzio, N. Hypofractionated image-guided IMRT in advanced pancreatic cancer with simultaneous integrated boost to infiltrated vessels concomitant with capecitabine: A phase i study. Int. J. Radiat. Oncol. Biol. Phys.: 2013; 87(5): 1000-1006 - Article IF 2012: 4,524 Picchio, M; Piert, M. Prostate cancer imaging. Eur. J. Nucl. Med. Mol. Imaging: 2013; 40(Suppl 1): S1-S4 - Editorial IF 2012: 5,114 239 EXPERIMENTAL IMAGING CENTER Selected publications Garibotto, V; Tettamanti, M; Marcone, A; Florea, I; Panzacchi, A; Moresco, R; Virta, JR; Rinne, J; Cappa, SF; Perani, D. Cholinergic activity correlates with reserve proxies in Alzheimer’s disease. Neurobiol. Aging: 2013; 34(11): e13-e18 - Article IF 2012: 6,166 Esposito, A; Colantoni, C; De Cobelli, F; Del Vecchio, A; Palmisano, A; Calandrino, R; Del Maschio, A. Multidetector computed tomography for coronary stents imaging: High-voltage (140KVP) prospective ecg-triggered versus standard-voltage (120-kvp) retrospective ecg-gated helical scanning. J. Comput. Assisted Tomogr.: 2013; 37(3): 395-401 - Article IF 2012: 1,582 EXPERIMENTAL IMAGING CENTER ALEMBIC Dynamic fluorescence spectroscopy in biomedicine 241 RESEARCH PROGRAMMES Brain Regeneration usIng medical Devices, Gene vectors and stEm cells (BRIDGE) Head of Research Program: Gianvito Martino (ad interim) Deputy Head of Research Program: Luigi Naldini* Participating investigators: Alessandra Biffi, HSR-TIGET - The San Raffaele Telethon Institute for Gene Therapy Alessandra Bolino, INSPE - Institute of Experimental Neurology Vania Broccoli, Division of Neuroscience Gian Giacomo Consalez, Division of Neuroscience Roberto Furlan, INSPE - Institute of Experimental Neurology Rossella Galli, Division of Regenerative medicine, Stem cells, and Gene therapy Angela Gritti, HSR-TIGET - The San Raffaele Telethon Institute for Gene Therapy Letizia Leocani, INSPE - Institute of Experimental Neurology Gianvito Martino, INSPE - Institute of Experimental Neurology Pietro Mortini, Division of Neuroscience Luigi Naldini, HSR-TIGET - The San Raffaele Telethon Institute for Gene Therapy Stefano Pluchino, INSPE - Institute of Experimental Neurology Letterio Salvatore Politi, Experimental Imaging Center Stefano Carlo Previtali, INSPE - Institute of Experimental Neurology Angelo Quattrini, INSPE - Institute of Experimental Neurology Carla Taveggia, INSPE - Institute of Experimental Neurology Flavia Valtorta, Division of Neuroscience 243 RESEARCH PROGRAMMES Vision - The characterization, dissection and experimental manipulation of the molecular and cellular events sustaining reactive brain repair might provide an attractive conceptual framework to foresee more efficacious therapies for neurological diseases. Indeed, fostering and/or resetting ‘spontaneous’ neural tissue regenerative processes may lead to ‘natural’ therapies characterized by higher efficacy and less toxicity. As a matter of fact, sspontaneous neural tissue repair occurs in patients affected by inflammatory and degenerative disorders of the nervous system. However, this process is not robust enough to promote a functional and stable recovery of the 3D neural tissue architecture. The development of stem celland gene therapy-based approaches capable of promoting the restoration and/or regeneration of the diseased or injured nervous system is thus foreseeable. Goals - Experimental strategies aiming at either replacing damaged vs. injured neural cells or delivering therapeutic genes have been developed in the last 30 years. However, most of these experimental approaches have failed to consistently foster repair in multifocal neurological diseases where the anatomical and functional damage is widespread. Novel stem cell- and gene therapybased protocols might partially overcome some of these limitations, including (a) the poor capability to grow and differentiate in vitro large number of neural cells from progenitors; (b) the anatomical, immunological and ethical barriers to cell transplantation into the CNS; (c) the limited bio-distribution of gene transfer products to the neural tissues, as well as (d) the genotoxic and immunological risks associated with conventional cell and gene delivery approaches. Novel therapeutic approaches that overcome these important hurdles might therefore represent valid alternatives for the treatment of multifocal nervous system diseases. These novel approaches may be complemented by the development of biocompatible medical devices providing the appropriate microenvironment to foster functional integration of the transplanted or endogenous gene-modified cells into the damaged tissues. Main achievements - The program has capitalized the scientific expertise and clinical excellence available at OSR in the areas of interest. Premiere knowledge derived from state-of-the-art stem cell and gene therapy experimental and human trials conducted in our Institute for a number of inflammatory and neurodegenerative conditions has been further expanded. New phase I/II human trials have been initiated in different myelin disorders, including multiple sclerosis and leukodystrophies, and new therapeutic pre-clinical strategies have been further developed in relevant animal model of neurodegenerative diseases. All of this work has been conducted with the main aim of combining powerful new stem cell isolation and gene transfer approaches to state-of-the-art neuroimaging, immunological and neurophysiological readouts. Finally, biological models has been developed to allow tracking gene expression and neural activity in the transplanted cells and the treated tissues. RESEARCH PROGRAMMES Program in Immunology and Bio-immunotherapy of Cancer (PIBIC) Co-Heads of Research Program: Paolo Dellabona and Giorgio Parmiani Participating investigators: Matteo Bellone, Division of Immunology, Transplantation, and Infectious Diseases Chiara Bonini, Division of Regenerative medicine, Stem cells, and Gene therapy Giulia Casorati, Division of Immunology, Transplantation, and Infectious Diseases Angelo Corti, Division of Molecular oncology Paolo Dellabona, Division of Immunology, Transplantation, and Infectious Diseases Angelo A. Manfredi, Division of Regenerative medicine, Stem cells, and Gene therapy Anna Mondino, Division of Immunology, Transplantation, and Infectious Diseases Giorgio Parmiani, Division of Molecular oncology Maria Pia Protti, Division of Immunology, Transplantation, and Infectious Diseases Patrizia Rovere-Querini, Division of Regenerative medicine, Stem cells, and Gene therapy Vincenzo Russo, Division of Molecular oncology Vision - The Program in Immunology and Bio-Immunotherapy of Cancer (PIBIC) combines expertise in immunology, cancer biology and medical onco-hematology to investigate fundamental aspects of tumor immunology and translate them into innovative immunotherapy strategies for phase I/II clinical trials. Goals - PIBIC aims at: a) gaining a deeper understanding of the mechanisms underlying the tumor/immune system interactions; b) providing rationally designed immunotherapy strategies that may significantly increase the clinical response. Main achievements - The immunogenicity of somatically mutated antigens in colon cancer was demonstrated by massive sequencing of the cancer transcriptome and reverse immunology (P. 245 RESEARCH PROGRAMMES Dellabona, G. Parmiani). We also obtained evidence that T cells specific for CD1c-restricted methylated lyso-phospholipid antigens can control leukemia progression (G. Casorati). New mechanisms of immunosuppression in the tumor microenvironment were defined: a) inhibition of antitumor immune responses by tumor-derived oxysterols via recruiting pro-tumor neutrophils in the tumor microenvironment (V. Russo); b) frequency of bone marrow pro-tumor Th22 cells greater in multiple myeloma patients with poor prognosis than low-risk patients and healthy controls (M.P. Protti). Studies focusing on targeting angiogenesis and tumor-microenvironment demonstrated the efficacy of delivering TNF and other cytokines to tumors via peptide-tagged nanoparticles capable of homing to tumor vessels. Chromogranin A was also found to act as angiogenic switch activated by proteolytic cleavage in damaged tissues and in tumors (A. Corti). Improvement of immunotherapy strategies was also obtained: booster vaccinations with a tumorspecific vaccine were detrimental in tumor-bearing subjects because of the negative impact on central memory T cells (M. Bellone). Remarkable control of a realistic mouse prostate cancer model was achieved by adoptive T cell therapy with T cells redirected against tumor and stroma by TCR gene transfer, or by combining tumor-redirected T cells with tumor-targeted NGR-TNF (A. Mondino). Significant advancements in adoptive T cell therapy were obtained: a) with CAR-engineered human T cells by developing a new chimeric antigen receptor specific for the CD44v6 isoform to target acute myeloid leukemia and multiple myeloma (A. Bondanza); b) by defining conditions to generate and expand long-lived human memory stem T cells for adoptive cell therapy by IL-7 and IL-15 (C. Bonini). The characterization of cancer stem cells (CSC) as targets for immunotherapy defined gene expression signatures of mouse stage-specific prostate CSC lines that associated with subgroups of progressing prostate cancer patients (M. Bellone). The suppressive mechanisms of human colorectal CSC were found to depend on indolamine 2,3-dioxigenase and/or on increased expression of cell surface IL-4 (G. Parmiani). New clinical trials that incorporate the conceptual advancements conceived within PIBIC were conducted: • A new strategy of vaccination by loading dendritic cells with tumor antigens in vivo was applied to 23 MAGE-A3+ stage IIIC or IV melanoma patients by transferring autologous lymphocytes engineered with MAGE-A3 and HSV-TK. Disease control rate of 26.3% (1 CR, 4 SD) and positive correlation between the increase of MAGE-A3-specific effectors and overall survival was observed in 19 patients with measurable disease. • A protocol based on the combination of NGRhTNF and peptide vaccine was conducted on 8 metastatic melanoma patients. Immune response to the vaccine and to other melanoma antigens, partly associated with the clinical outcome, and OS close to 30 months in 4 subjects were found (G. Parmiani). • -A phase I study combining Ipilimumab and Fotemustine in metastatic melanoma was performed in 19 patients. The treatment induced PRs of brain metastases in 50% of patients. A phase III protocol is now ongoing (G. Parmiani). RESEARCH PROGRAMMES Islet Trasplantation Program (ITP) Co-Heads of Research Program: Lorenzo Piemonti and Paola Maffi Participating investigators: Manuela Battaglia, DRI - Diabetes Research Institute Luca G. Guidotti, Division of Immunology, Transplantation, and Infectious Diseases Emanuele Bosi, DRI - Diabetes Research Institute; Division of Metabolic and Cardiovascular sciences Paola Maffi, DRI - Diabetes Research Institute Rita Nano, DRI - Diabetes Research Institute Marina Scavini, DRI - Diabetes Research Institute Vision - The mission of this program is to achieve long-lasting insulin independence in patients with type 1 diabetes (T1D) undergoing portal vein islet transplantation. Recognized experts in islet transplantation, immunological tolerance, liver immunopathology and non-invasive imaging have been brought together to address and modify innate and adaptive immune responses to islet transplants that together prevent lifelong persistence of functional islets within the liver. Goals - Aims of this program are: 1) to improve donor management, donor selection criteria, organ recovery techniques and islet cell processing techniques. Expected results: identification of more efficient islet cell processing to maximize islet recovery; 2) to identify and standardize methods for the evaluation of islet preparations intended for transplantation in humans (i.e. cell composition, cell viability, insulin secretion). Expected results: identification and validation of predictor for islet engraftment and post transplant function; 3) to identify strategies and drugs able to improve 247 RESEARCH PROGRAMMES islet “engraftment”. Expected results: achieve successful islet transplantation from one donor to one recipient; 4) to develop single or multi centre clinical trial to test new immunosuppression and tolerogenic strategies. Expected results: achieve successful islet transplantation without immunosuppression or with less toxic immunosuppressive regimens; 5) to provide islets for researchers. Expected results: support research activity related to cell biology in Europe. Main achievements - During the last year we: • have implemented a calcineurin inhibitor-free, anti-IL-2R-free protocol to clinical islet transplantation (NCT01346085). This “tolerance-accommodating” protocol resulted feasible, safe and efficient in term of maintaining patients insulin-free at 3 years; • demonstrate that immune monitoring with frequent posttransplant assessment of allo- and autoantibodies could be helpful in clinical islet transplantation. This approach to active immune monitoring should allow for the use of more-tailored (and potentially milder) immunosuppression combinations and prompt intervention for acute immunological events. In addition, such monitoring may provide a better understanding and characterization of the various mechanisms of destruction involved in the loss of islet grafts; • performed the first clinical study aiming to investigate in noninvasive way liver perfusion modifications occurring during islet engraftment and attempting to correlate them with clinical parameters in T1DM patients. Dynamic contrast-enhanced magnetic resonance imaging (DCEMRI) identifies various adaptive responses of liver microvasculature in patients submitted to islet transplantation; these responses could have an impact on islet engraftment; • conducted a phase 3, multicenter, randomized, double-blind, parallel assignment study to assess the efficacy and safety of the CXCR1/2 inhibitor reparixin in pancreatic islet transplantation (NCT01817959); • conducted a phase 2 study to compare bone marrow vs. liver as site for islet transplantation in patients (NCT01722682); • extended the clinical indication of islet autotransplantation to a broader population of patients undergoing pancreatic surgery including subjects with technically unfeasible or high risk pancreatic anastomosis during partial pancreatectomy and subjects undergoing completion pancreatectomy because of anastomosis leakage after pancreatoduodenectomy for nonmalignant or malignant diseases (NCT01702051). RESEARCH PROGRAMMES Human Brain Invivo Mapping with neuroimaging (BRAINMAP) Head of Research Program: Massimo Filippi* Deputy Head of Research Program: Andrea Falini* Participating investigators: Francesco Benedetti, Division of Neuroscience Luigi Beretta, Division of Neuroscience Stefano F. Cappa, Division of Neuroscience Paola Cinque, Division of Immunology, Transplantation, and Infectious Diseases Andrea Falini, Division of Neuroscience Massimo Filippi, INSPE - Institute of Experimental Neurology Roberto Gatti, Division of Neuroscience Letizia Leocani, INSPE - Institute of Experimental Neurology Giuseppe Magnani, INSPE - Institute of Experimental Neurology Vittorio Martinelli, INSPE - Institute of Experimental Neurology Daniela Perani, Division of Neuroscience Maria Assunta Rocca, INSPE - Institute of Experimental Neurology Maria Sessa, INSPE - Institute of Experimental Neurology 249 RESEARCH PROGRAMMES Vision - Due to its exquisite sensitivity, relative non-invasiveness, and major technical advances, neuroimaging has become in the past couple of decades an irreplaceable way for the in vivo assessment of the CNS in healthy and diseased humans. Goals - The “Human Brain In-vivo Mapping with Neuroimaging” (BRAINMAP) Research Program is aimed at joining and strengthening the decennial neuroimaging expertise developed at our Institute; and developing new research lines through a multidisciplinary and inter-departmental approach. Main achievements • MS and other WM disorders: imaging features of cortical lesions and cervical cord atrophy were assessed in patients with different MS phenotypes; advanced MRI techniques identified predictors of cognitive and clinical deterioration in MS; the role of brain and cognitive reserves in limiting onset of cognitive deficits in MS was demonstrated; in pediatric MS, structural and functional MRI improved the understanding of the correlates of cognitive impairment; GM regional abnormalities were explored in adult and pediatric migraine. • Neurodegenerative diseases: DT MRI revealed specific patterns of WM damage in patients with early onset AD, primary progressive aphasia, PD, and MND; resting state fMRI abnormalities were assessed in FTD and MND; graph analysis showed that global and local functional networks are altered in FTD; social cognition impairment were assessed in the FTD spectrum as well as their relationship with GM and WM damage. • Psychiatric diseases: the GSK-3 inhibitor lithium, and the less active GSK-3 gene promoter variants, add on to counteract the detrimental effects of bipolar disorder on WM; widespread WM changes are common to obsessive-compulsive disorder, and a target for drug treatment; fMRI reveals gene-environment interactions between COMT genotype and the neural correlates of empathy and perceived personal distress in schizophrenia. • Infectious diseases: DT MRI revealed WM abnormalities in patients with asymptomatic HIV infection, including treated chronic infection representing a potential marker to study cognitive deficits in this population; in PML, MRI enhancement patterns and evolution is being studied in the context of the relationship between JC virus replication and host response providing a useful means for clinical disease monitoring. • Developmental disorders: Voxel-basedmorphometry and DT MRI were applied in subjects with sporadic dyslexia and genetic association, subjects with DCDC2 mutation associated with altered neuronal migration and cognitive disturbances, providing evidence for structural alterations of neural systems supporting reading and specific cognitive processes. • Clinical neuroscience: psycholinguistic, neuropsychological and neuroimaging studies of abstract word processing in normal subjects; assessment of the functional and structural correlates of loss of aversion in normal subjects, including the investigation of individual differences; functional correlates of innovative decision-making in managers and entrepreneurs. • Neurophysiological techniques: using advanced source localization of cognitive event-related potentials to the Stroop test, the electrophysiological and psychophysiological correlates (reaction times) of frontal executive involvement were explored in MND. • Nuclear medicine techniques: the amyloid load was assessed using PET and fluorinated tracers in MCI/prodromal AD and compared with FDG PET functional derangement, supporting divergent pathways for these biomarkers that might guide clinical applications; the mechanisms of neuroinflammation were explored using 11CPK-PET in PD and parkinsonisms, AD and prion diseases; FDG PET scans in patients with neurodegenerative disorders were evaluated providing brain functional, clinical and neuropsychological correlations useful for research purposes and clinical applications. RESEARCH PROGRAMMES Figure 43. Graph analysis of resting state functional MRI in patients with the behavioural variant of frontotemporal dementia (bvFTD). (A) Cortical hubs of the functional networks of healthy controls (i, ii) and patients with bvFTD (iii, iv). (B) Regions showing decreased integrated nodal degree (i, ii) in patients with bvFTD compared to healthy controls. Node size is proportional to the difference in the value of the integrated nodal parameters between the 2 groups. Abbreviations: ACC = anterior cingulate cortex; Cal = calcarine cortex; Caud = caudate nucleus; Cun = cuneus; Fus = fusiform gyrus; Hes = Heschl gyrus; Ins = insula; IOG = inferior occipital gyrus; ITG = inferior temporal gyrus; Lin = lingual gyrus; MCC = middle cingulate cortex; MFG = middle frontal gyrus; MOG = middle occipital gyrus; MTG = middle temporal gyrus; OFC = orbitofrontal cortex; Prec = precuneus; PoCG = postcentral gyrus; PreCG = precentral gyrus; Rec = gyrus rectus; Rol = rolandic operculum; SFG = superior frontal gyrus; SOG = superior occipital gyrus; SPL = superior parietal lobule; STG = superior temporal gyrus; TPO = temporal pole. Reproduced with permission from Agosta F, Sala S, Valsasina P, Meani A, Canu E, Magnani G, Cappa SF, Scola E, Quatto P, Horsfield MA, Falini A, Comi G, Filippi M. Brain network connectivity assessed using graph theory in frontotemporal dementia. Neurology 2013;81:134-43. 251 RESEARCH PROGRAMMES Bone Physiopathology Program (BoNetwork) Co-Heads of Research Program: Roberto Sitia* and Enrico Gherlone* Participating investigators: Alessandra Biffi, HSR-TIGET - The San Raffaele Telethon Institute for Gene Therapy Clara Camaschella, Division of Genetics and Cell biology Francesco Camnasio, Department of general and specialistic surgery Simone Cenci, Division of Genetics and Cell biology Roberto Crespi, Division of Genetics and Cell biology Marina Ferrarini, Division of Molecular oncology Elisabetta Ferrero, Division of Molecular oncology Gianfranco Fraschini, Division of Genetics and Cell biology Federico Furlan, Emergency medicine Roberto Gatti, Division of Neuroscience Enrico Gherlone, Division of Genetics and Cell biology Stefano Mora, Division of metabolic and cardiovascular sciences Giuseppe M. Peretti, Division of Genetics and Cell biology Patrizia Rovere-Querini, Division of Regenerative medicine, Stem cells, and Gene therapy Alessandro Rubinacci, Division of metabolic and cardiovascular sciences Roberto Sitia, Division of Genetics and Cell biology Giovanni Tonon, Division of Molecular oncology Giuseppe Vezzoli, Division of Genetics and Cell biology Anna Villa, HSR-TIGET - The San Raffaele Telethon Institute for Gene Therapy Raffaele Vinci, Division of Genetics and Cell biology RESEARCH PROGRAMMES Vision - The skeleton evolved to empower locomotion, shield the neuraxis and the hematopoietic marrow, and amass minerals. Efficient mechanosensing ensures skeletal homeostasis by co-ordinating the remodeling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. Bone metabolic diseases are frequent and disabling: involutional osteoporosis affects older people (35% postmenopausal white women, 19% white men), leading to pathologic fractures (>1.3 million/year), high costs (14 billion USD direct expenditures per year in the US), severe disability, and reduced lifespan. These figures rise as life expectancy grows, warranting new strategies to promote healthy aging. Understanding how osteoblasts and osteoclasts differentiate and operate, their derangement in bone wasting diseases, and how cancers divert the bone microenvironment will provide targets for osteoporosis and osteolytic cancers. The integrated study of bone biology, stemness, and immunity will expand our understanding of skeletal homeostasis, and lead to identify new specific therapeutic targets for regenerative medicine. Goals - The San Raffaele Bone Pathophysiology Program (BoNetwork) aims to integrate basic, translational and clinical research on bone pathophysiology to generate strong scientific synergies. Specific objectives include: identifying the molecular and cellular bases of bone homeostasis, including determinants of bone mass gain; promoting joint efforts in cartilage and bone engineering in orthopaedics and odontoiatrics; identifying potential molecular markers and targets of diseases; establishing robust cellular and animal disease models, and solid core technologies; providing an attractive interface for pharmaceutical and nutriceutical companies. Main recent achievements • Development and preclinical validation of strategies for cartilage and osteochondral repair, including: an engineered osteochondral composite; human cartilage fragments as a cell source; fibrin glue and collagen scaffold as a carrier; • Development of animal models of skeletal aging and to develop tissue engineering strategies for articular repair; • Evaluation of the effects of chronic diseases and associated treatments (HIV and antiretrovirals) on skeletal modeling in children/adolescents; • Identification of genetic variants associated with osteoporosis; • Identification of mechanisms conferring therapeutic resistance to multiple myeloma, the prime bone cancer; • Discovery of the role of autophagy in plasma cell ontogenesis and in the maintenance of the resident pool of bone marrow plasma cells, the normal myeloma counterpart; • Identification and characterization of gene variants of the calcium-sensing receptor associated with human disease; • Studies of biological features and clinical applications of bone substitutes and dental implants for oral rehabilitation. 253 RESEARCH PROGRAMMES Correlates of HIV-Associated Immune Response Modulation program (CHARM) Co-Heads of Research Program: Paola Cinque and Guido Poli* Participating investigators: Massimo Alfano, Division of Immunology, Transplantation, and Infectious Diseases Antonella Castagna, Division of Immunology, Transplantation, and Infectious Diseases Paola Cinque, Division of Immunology, Transplantation, and Infectious Diseases Nicola Gianotti, Division of Immunology, Transplantation, and Infectious Diseases Luca G. Guidotti, Division of Immunology, Transplantation, and Infectious Diseases Adriano Lazzarin, Division of Immunology, Transplantation, and Infectious Diseases Mauro S. Malnati, Division of Immunology, Transplantation, and Infectious Diseases Giulia Morsica, Division of Immunology, Transplantation, and Infectious Diseases Ruggero Pardi, Division of Immunology, Transplantation, and Infectious Diseases Guido Poli, Division of Immunology, Transplantation, and Infectious Diseases Gabriella Scarlatti, Division of Immunology, Transplantation, and Infectious Diseases Giuseppe Tambussi, Division of Immunology, Transplantation, and Infectious Diseases Caterina Uberti-Foppa, Division of Immunology, Transplantation, and Infectious Diseases Luca Vangelista, Division of Immunology, Transplantation, and Infectious Diseases Elisa Vicenzi, Division of Immunology, Transplantation, and Infectious Diseases Vision - After the discovery of HIV-1 as the cause of AIDS (1983-1984), CD4 was soon identified as the primary receptor utilized by the virus to infect T lymphocytes and mononuclear phagocytes. However, 10 years passed before the identification of the second co-receptor required for viral entry in the molecule known as CCR5. Genetic variants of CCR5 include a deletion of 32 base pairs leading to misfolding of the protein and lack of surface expression. Individuals who are homozygotes for such a mutation are naturally resistant to HIV-1 infection, whereas heterozygotes RESEARCH PROGRAMMES are frequently “long-term nonprogressors”, thus underscoring the importance of this receptor in the natural history of HIV-1 infection. CCR5 binds to 3 natural CC chemokines and these observations have led to the rapid discovery of small molecules antagonizing HIV infection at the entry step. CCR5 antagonists are indeed the first drugs approved for their use in regimens of combination anti-retroviral therapy (cART) of HIV-1 infection that are targeting a host rather than a viral protein. The specific clinical use of CCR5 antagonists in the context of potent cART might be significantly enhanced by their potential “immunomodulatory/anti-inflammatory” effect resulting from the interference with the chemokine receptor. If proven, this activity could be exploited in other clinical conditions where reduction of the inflammatory cell state by interference with CCR5 could be desirable. Goals - General goals of CHARM are the investigation and exploitation of the role of CCR5 and its natural or chemical ligands in HIV-1 infection and in infection-related inflammation. The program stems from the recognition of a critical mass of basic and applied investigators already ongoing in this Division and in the Department of Infectious and Tropical Diseases on the role of CCR5 and its ligands in HIV-1 infection and related clinical entities. The projects and their individual leaders have already proven their validity and vitality in terms of dedicated scientific publications, grants and patents. However, they have never been coordinated as an internal network or program up to date. Therefore, CHARM has the general goal to create and foster such a network of knowledge and mutual exchange of information, and collaborations by promoting their synergy, avoid duplicating efforts, and seeking for additional funding and support both to individual projects and, hopefully, to the Program as a whole. 2013 Achievements - In addition to several publications related to CHARM objectives by the program participants, we have mostly focused on the use of Maraviroc (MVC), a CCR5 inhibitor, in the treatment of primary HIV infection (PI: Dr. Giuseppe Tambussi). In this regard, the potential benefit of an early intervention could pave the way for future therapies aimed at HIV eradication, such as strategies to mobilize virus from latently infected CD4 cells or vaccine- and immune-based therapies, which would have a greater probability of success in patients whose reservoir has been reduced by starting ART during PHI. An early treatment could also potentially preserve the HIV- specific immune response, thus increasing the chances of a better control of viral replication. Although the impact of viral production on the replenishment of HIV reservoir remains controversial, adopting an entry inhibitor molecule in the course of chronic infection could potentially reduce the pool of latently infected cells to a lesser extent if compared to standard ART regimens, thus enhancing the infection prognosis and increasing future possibilities of viral eradication. In a prospective trial (MAIN study, EUDRACT n° 2008-007004-29) conducted in our department we enrolled PHI patients and assigned them to receive either MVC + standard cART or standard cART alone for 48 weeks. At week 48, all the patients achieved plasma viremia levels <50 RNA copies/ml. Both intention-to-treat and on-treatment analyses showed no differences between groups in terms of mean CD4 T cell numbers and plasma viremia at week 48. However, a better performance in terms of CD4 T cell number gain from baseline was observed in the MVC + cART vs. cART alone groups. cART, whether or not associated with MVC, determined a significant decrease in the number of activated CD4 and CD8 T lymphocytes. Furthermore, we demonstrated a significant increase in terminally differentiated CD8 T cells, usually reduced in chronic HIV infection. Terminally differentiated CD4 T lymphocytes expressing CD127 (IL7-R ) were reduced in both treatment groups, with patients who had received MVC + ART showing a greater decrease (manuscript submitted). Overall, these findings suggest that inhibition of CCR5 function by MVC may exert additional immunological benefit during cART early immune reconstitution. The longer-term potential effects of these therapy regimens on virus reservoirs are the object of ongoing follow-up studies. The preliminary results of this study have been presented at the following meetings: • 20th Conference on Retroviruses and Opportunistic Infections, March 3-6 2013 Atlanta, Georgia, USA (abs# H-124, M. Ripa winner of young investigator award). • Keystone Symposia: Immune Activation in HIV Infection: Basic Mechanisms and Clinical Implications (D2) April 3, 2013 - April 8, 2013 Beaver Run Resort - Breckenridge, Colorado, USA (abs# 3032). • 5th Italian Conference on AIDS and retrovirusICAR 2013 - May 12-14 2013, Turin, Italy (abs# CO13). • 7th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Kuala Lumpur, Malaysia, 30 June-3 July 2013 (abs# WEPE449). 255 RESEARCH PROGRAMMES Microenvironment and Genes in Cancers of the Blood (MAGIC) Co-Heads of Research Program: Paolo Ghia* and Giovanni Tonon Participating investigators: Massimo Alessio, Center for Translational Genomics and BioInformatics Angela Bachi, Division of Genetics and Cell biology Matteo Bellone, Division of Immunology, Transplantation, and Infectious Diseases Rosa Bernardi, Division of Molecular oncology Stefano Biffo, Division of Molecular oncology Attilio Bondanza, Division of Regenerative medicine, Stem cells, and Gene therapy Chiara Bonini, Division of Regenerative medicine, Stem cells, and Gene therapy Andrea Brendolan, Division of Molecular oncology Valeria R. Caiolfa, Experimental Imaging Center Federico Caligaris-Cappio, Division of Molecular oncology Giulia Casorati, Division of Immunology, Transplantation, and Infectious Diseases Simone Cenci, Division of Genetics and Cell biology Fabio Ciceri, Division of Molecular oncology; Division of Regenerative medicine, Stem cells, and Gene therapy Angelo Corti, Division of Molecular oncology Paolo Dellabona, Division of Immunology, Transplantation, and Infectious Diseases Claudio Doglioni, Division of Molecular oncology Marina Ferrarini, Division of Molecular oncology Andrés José Marìa Ferreri, Division of Molecular oncology Elisabetta Ferrero, Division of Molecular oncology Katharina Fleischhauer, Division of Regenerative medicine, Stem cells, and Gene therapy Paolo Ghia, Division of Molecular oncology Luca G. Guidotti, Division of Immunology, Transplantation, and Infectious Diseases Matteo Iannacone, Division of Immunology, Transplantation, and Infectious Diseases RESEARCH PROGRAMMES Cristina Maccalli, Division of Molecular oncology Eugenio Montini, HSR-TIGET - The San Raffaele Telethon Institute for Gene Therapy Marta Muzio, Division of Molecular oncology Ruggero Pardi, Division of Immunology, Transplantation, and Infectious Diseases Giorgio Parmiani, Division of Molecular oncology Maurilio Ponzoni, Division of Molecular oncology Maria Pia Protti, Division of Immunology, Transplantation, and Infectious Diseases Vincenzo Russo, Division of Molecular oncology Roberto Sitia, Division of Genetics and Cell biology Giovanni Tonon, Division of Molecular oncology Vision - Cancer development is due to a complex interplay between genetic abnormalities arising within the neoplastic clone and favourable stimuli originating in the surrounding microenvironment. Progress toward the cure of cancer can only come from a deep understanding of all these factors and of the mechanisms through which they concur toward tumor pathogenesis. In this program, we focus on blood cancer involving B lymphocytes at different stages of differentiation namely acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), NonHodgkin Lymphomas (NHL) and multiple myeloma (MM). Despite major therapeutic advances, all these tumors are presently incurable. Interestingly, in all these malignancies, the interaction between genetically abnormal malignant cells and a dysregulated microenvironment is playing a crucial role in their pathogenesis. In details, all these malignancies carry a number of genetic abnormalities in multiple genes that control cell division, differentiation, survival or programmed cell death, leading to unrestrained proliferation and accumulation. In addition, in each case the tumor microenvironment provides critical stimuli conferring to malignant cells a growth advantage and an extended survival. The rationale of this program relies on the idea that effective and ultimately curative therapies for these diseases could only emerge from targeting simultaneously the tumour cell and its microenvironment interactions. Goals - The aim of the Program is the identification of novel therapeutic targets at the forefront between tumor cells and the microenvironment that will allow defining new personalized treatment strategies based on molecularly-based prognostic and predictive cards. We have devised 4 different interconnected Programs. Programs 1 (Novel Therapies) and 2 (Optimizing existing therapies and improving quality of life) deal with preclinical proof-of-principle studies of targeted approaches and the definition of novel prognostic and predictive factors. Programs 3 (Novel targets) and 4 (New cellular protagonists) are exploratory in nature and aim at dissecting the role of novel microenvironment molecular and cellular components whose significance has been strongly implied by preliminary data. 2013 achievements - During 2013, we have achieved three main conclusions: Conclusion 1. Chronic hematological neoplams are characterized by active and dynamic microenvironments co-evolving with the leukemic clone thereby supporting the evolution of more aggressive subclones and ultimately promoting disease progression. The key role of active microenvironments acquires a clinical relevance especially in the present era of non-genotoxic drugs. Conclusion 2. The stratification of cancer patients based upon gene/microenvironment interactions may lead to more adequate treatment strategies for specific categories of patients. Conclusion 3. Our overall strategy aiming at unravelling the interaction between cancer cells and their environment has led to the identification of new promising therapeutic targets as well as the potential therapeutic significance of some accessory cell populations that warrant further exploitation and pre-clinically validation. During 2013, investigators participating in the program have produced data toward the development of a number of options that are ready to be proposed to the scientific community. These include drugs targeting by-stander cells in CLL and MM (conclusion 1), novel stratification strategies for CLL patients incorporating stereotypy of the Immunoglobulin receptors (conclusion 2), small molecular drugs targeting intracellular pathways in CLL and MM (conclusion 3) and chimeric antigen receptors (CARs) redirecting T-cell specificity against CLL and MM cells (conclusion 3). All these have the potential to be moved rapidly toward clinical translation as new treatment and prognostic approaches. 257 FACILITIES CFCM, San Raffaele Core Facility for Conditional Mutagenesis ––––––––––––––––––– 260 SCIENTIFIC SUPERVISOR: Marco E. Bianchi* FACILITY MANAGER: Lorenza Ronfani FELLOWS: Lorenzo Benini, Ivana Benzoni TECHNICIANS: Maria Luisa Pintonello, Rosanna Rinaldi FRACTAL, Flow cytometry Resource, Advanced Cytometry –––––––––––––––––––––––– 261 Technical Applications Laboratory SCIENTIFIC SUPERVISOR: Alessio Palini FACILITY MANAGER: Chiara Villa BIOLOGISTS: Simona Di Terlizzi, Federico Sizzano TECHNICIANS: Emanuele Canonico, Ivan Muradore (until July 2013) CERMAC, Centre of Excellence of High Field Magnetic Resonance –––––––––––––– 262 SCIENTIFIC SUPERVISOR: Enrico Smeraldi* HEAD OF FACILITY: Andrea Falini* PHYSICIAN: Valeria Blasi RESIDENT: Claudia Godi** POST-DOCTORAL FELLOWS: Sofia Crespi**, Roberta Longaretti PHD STUDENTS: Antonella Castellano**, Sara Cirillo** FELLOW: Paola Scifo TECHNICIAN: Antonella Iadanza PROMIFA, PROtein MIcrosequencing FAcility ––––––––––––––––––––––––––––––––––––––––– 262 SCIENTIFIC SUPERVISOR: Marco E. Bianchi* (ad interim) FACILITY MANAGER: Annapaola Andolfo TECHNICIAN: Cinzia Magagnotti Mouse histopathology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 263 SCIENTIFIC SUPERVISOR: Claudio Doglioni* FACILITY MANAGER: Francesca Sanvito PHYSICIAN: Maurilio Ponzoni TECHNICIANS: Anna Innocenzi, Martina Rocchi * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele 259 FACILITIES CFCM, San Raffaele-Telethon Core Facility for Conditional Mutagenesis Transgenic and knock-out mice are widely used in the research because of their high impact in the understanding of the proteins functional role and because they constitute models to study genetic diseases. The San Raffaele Core Facility for Conditional Mutagenesis (CFCM) has been operative since 1999. During this time CFCM has provided to the Scientific Community more than 400 murine models. CFCM performs pronuclear injections and lentiviral infections to generate transgenic mice, offers the electroporation of Embryonic Stem cell (ES cells) and morulae and blastocysts injections of recombinant clones to generate gene targeted models. Moreover, CFCM carries out rederivations of mice via embryo transfer. In addition, CFCM helps Researchers to plan experiments, to manipulate and genotype mice. Other services provided by CFCM are the screening of resistant clones by Southern Blotting. Figure 44. Microinjection of ES cells into a morula CFCM performs the De novo Isolation of ES cells from wild type and mutant bastocysts. ES cells derived from transgenic mice represent important tools for the analysis of the mutation of interest. Moreover, Researchers have the possibility to differentiate ES cells in all cell lineages. Very important, CFCM performs Embryo Cryopreservation. This is a basic service because Researchers spent time and money to maintain murine lines, which are not necessary for their current studies. Moreover, these murine lines occupy precious space in the Animal House. During 2013, CFCM generated 7 new gene-targeted murine models and 4 new transgenic lines. 11 and 30 murine lines were, respectively, cryopreserved and rederived. More information can be found at the site http://www.hsr.it/research/organization/servicesopen-labs/cfcm/. Lorenza Ronfani FACILITIES FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory The Flow cytometry Resource and Advanced Cytometry Technical Applications Laboratory (FRACTAL) is a core facility that offers state-of-the-art instrumentation and analysi stechniques to the scientific community. Flow cytometry is an evolving field and scientists approach this technology to answer a multitude of questions in their discovery work. FRACTAL supports researchers in obtaining verifiable results for such applications as Immunophenotyping, Cell division and Apoptosis, Cell activation, Intracellular pH shifts, Phagocytosis, Oxidative burst and many more. So wide ranging is the applicability of this technology thatpractitioners may include, in addition to biologists, physicians, microbiologists, marinebiologists, veterinarians and research chemists to name a few. In line with this inter-disciplinary environment, FRACTAL is now implementing a “Translational Cytometry” program aimed to develop its own projects that bridge research and clinical protocols, for which cytometry analysis is essential. In addition to the analytical capabilities of this technique, the core facility offers assisted cell Sorting services for characterizing, separating and purifying populations of particles as diverse as beads, bacteria, micro-particles, cells and chromosomes. In 2010 the facility achieved ISO 9001:2008 Certification for the processes of Cell Sorting, Assisted Analysis and Training. Additionally, the facility supported over 400 Researchers, performed more than 1361 cell sorts and logged over 8,000 hours of analytical instrument time. Chiara Villa Figure 45. Interrogation point on Moflo XDP 261 FACILITIES CERMAC, Centre of Excellence of High Field Magnetic Resonance The Centre of Excellence High Field MRI or CERMAC start operations in 2003 with testing of the 3 Tesla magnet that constitutes the instrumental core of the Center. Around the high field magnet develops a two-story structure housing offices equipped with all the workstations and computers necessary to post-process images and their analysis. The building is located within the Unit of Neuroradiology at the Hospital San Raffaele in Milan. The creation of a Centre of Excellence based on the use of a high-field MRI has become possible thank to the initial presence at the Faculty of Medicine and Psychology, University Vita-Salute San Raffaele and at the homonymous Scientific Institute of personnel with high expertise in neuroradiology, functional neuroimaging, neurology, cognitive neuroscience, psychiatry, neurosurgery and oncology, and integration of these disciplines with physical and engineering skills. The integration of the different afferents having great scientific and cultural significance have made vital and highly productive in terms of research, teaching and training a multidisciplinary center based on neuroimaging. Scientists of different disciplines work together carrying on projects concerning development and validation of functional MR techniques and their application in the field of neuroscience (neuroradiology, neurology, neurosurgery, psichiatry, psichology and cognitive neurosciences). Advanced MR techniques are employed to investigate normal brain development and function with special focus on myelination process, music comprehension, white matter connections, language areas and mirror neuron system.The same techniques are used to characterize morphological, structural and functional modifications related to degenerative, inflammatory, neoplastic and vascular diseases. A novel approach to psychiatric diseases has been enabled by the noninvasive MR capabilities. More than hundred papers have been published in major international journals during the last five years. The Center is site for postgraduate and PhD training in neuropsychology, cognitive neuroscience, neurology and psychiatry. Medicine, Psichology, Biotechnology and Physiotherapy students complete their thesis at CERMAC. Andrea Falini ProMiFa, Protein Microsequencing Facility During the last year, the facility could further extend its portfolio by increasing the number of internal (up to 15) and external users (4 companies and a total of 16 between academic and no profit institutions). The activities of the facility can be described as below: 1. Protein characterization • MW determination of peptides or proteins by MALDI-MS or ESI-MS and sequencing by tandem mass spectrometry (MS/MS). 2. Protein identification • Protein identification in gel bands or spots by MALDI-MS or nanoLC followed by ESI- MS/MS. • Post-translational modifications (PTMs)-characterization by MS/MS, including type and site. • Phosphorylation characterization: phosphorylated peptides are enriched using affinity columns (TiO2 or IMAC) and then analyzed by MS. 3. Biomarker profiling in biological samples by 2DE, image analysis and statistical evaluation • Analysis of the entire proteome in complex samples by 2D-electrophoresis, followed by acquisition of high definition images to evaluate the differential protein expression pattern in tissues and biological fluids. 4. Protein quantification in biological samples by MS/MS analyses • Protein quantification in complex samples by label-free or labelling approaches, including metabolic labelling of living cells using Stable isotope labelling by amino acids in cell culture (SILAC) and dimethyl-labelling of digested proteins. 5. Instrumentation • LTQ Orbitrap XL mass spectrometer equipped with an Easy nLC • Voyager DE-STR MALDI-TOF mass spectrometer Moreover, in order to develop a metabolomics/ lipidomics platform, the acquisition of two new instrumentations was indicated. More information can be found at the site: www.promifa-protein-microsequencing-facility/ Annapaola Andolfo FACILITIES Figure 46. Typical workflow for the analysis of the entire proteome in complex samples by 2D-electrophoresis, followed by acquisition of high definition images to evaluate the differential protein expression pattern in biological fluids. Mass spectrometry identification of proteins and their validation by an independent technique help to specifically define the treatment strategy for patients. Reprinted from: Calcium signaling-related proteins are associated with broncho-pulmonary dysplasia progression. Magagnotti, C and Matassa, PG; Bachi, A; Vendettuoli, V; Fermo, I; Colnaghi, MR; Carletti, RM; Mercadante, D; Fattore, E; Mosca, F; Andolfo, A. J. Proteomics: 2013; 94: 401-412 doi: 10.1016/j.jprot.2013.10.007 © 2013, with permission from Elsevier. Mouse histopathology The principal aim of the Mouse Histopathology Unit is to support, complement and favour the advancement of scientific projects, by providing conventional morphological analysis and immunophenotyping, in order to evaluate the presence of morphological alterations in mouse models of human disease. The facility is located in the Department of Pathology and offers the technical and the interpretative experience for the analysis of tissues from animal models, evaluating the best histological or immunocytochemical procedures, based on the expected results. The services provided include: • frozen tissues and cryostatic section preparation • immunohistochemistry (commercial antibody and new ones to be set up) • cytospin and paraffin cytoblock • final report 1. macroscopic examination including perfusion and necropsy To date multiple collaborations within our Institute and with research groups from other Institutes have been settled in order to analyze the morphological and immunophenotypical patterns of murine models of diseases, treated with different therapeutic approaches and to analyze the efficacy of gene therapy and safety of the use of viral vectors. Francesca Sanvito 2. microscopic analysis • paraffin embedding and inclusion • microtome sectioning and standard HE staining • special histochemical staining (Perls, PAS, Masson, Gomori stainings, etc.) 3. image analysis The role of pathology in the field of experimental studies on laboratory animal is of interest for: • indentification and evaluation of experimentally induced lesions • setting of animal models of human diseases • efficacy and safety studies • phenotyping of transgenic mice 263 FACILITIES FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory CFCM, San Raffaele-Telethon Core Facility for Conditional Mutagenesis FACILITIES PROMIFA, PROtein MIcrosequencing FAcility 265 THE ETHICS COMMITTEE The Ethics Committee of the Scientific Institute Hospital San Raffaele - Milano Established Pursuant to M.D. 08022013 Until October 2013 CHAIRMAN: Gianna Zoppei, San Raffaele Scientific Institute VICE CHAIRMAN: Guido Pozza, Physician, Professor Emeritus of Internal Medicine, Università VitaSalute San Raffaele SECRETARY: Alfredo Anzani, Expert in Bioethics, San Raffaele Scientific Institute MEMBERS: Giliola Calori, Biostatistician, San Raffaele Scientific Institute Ilaria Carretta, Psychologist and Psychoterapist, San Raffaele Scientific Institute Francesco Caviezel, Physician, Professor Emeritus of Endocrinology, University of Milan Maurizio Chiodi, Theologian, Professor at the Theological Faculty of Northern Italy, Milan Francesco Clementi, Pharmacologist, Professor Emeritus of Pharmacology, University of Milan Maria Grazia Fusi, Regional Area GPs, Milan Andrea Gentilomo, Medical Examiner, Professor of Legal Medicine, University of Milan Roberts Mazzuconi, Hospital Director, San Raffaele Scientific Institute, ex officio Massimo Reichlin, Expert in Bioethics, Professor of Phylosophy, Università Vita-Salute San Raffaele Maria Grazia Roncarolo, Scientific Director, San Raffaele Scientific Institute, ex officio Claudio Rugarli, Physician, Professor Emeritus of Internal Medicine, Università Vita-Salute San Raffaele Patrizia Tadini, Pharmacist, San Raffaele Scientific Institute, ex officio Cesare Triberti, Expert in Legal Procedures Liliana Villa, Representative of the Fondazione Centro San Raffaele del Monte Tabor Ewa Wysocka, Representative of Nursing Area Maria Elisabetta Zanardelli, Volunteer association member, Associazione per l’Aiuto ai Giovani Diabetici (Onlus) CONSULTANTS: Cesarina Curti, Expert in Medical Devices, San Raffaele Scientific Institute Gianvincenzo Zuccotti, Head of Pediatrics Unit, Luigi Sacco Hospital, Milan Since November 2013 MEMBERS: Anzani Alfredo, Physician, San Raffaele Hospital, President of the Ethic’s Committee Pozza Guido, Physician, San Raffaele Hospital, Vice-President of the Ethic’s Committee Arrigoni Cristina, Representative of healthcare professions area, University of Pavia Basile Giuseppe, Legal Doctor, Galeazzi Orthopaedic Institute Cabitza Paolo, Expert in Medical Devices, San Donato Polyclinic Calori Giliola, Biostatistician, San Raffaele Hospital Carretta Ilaria, Expert in Psychology, San Raffaele Hospital Chiodi Maurizio, Expert in Moral Theology, Faculty of Theology, Milan University Chiumello Giuseppe, Head Physician in Paediatrics, San Raffaele Hospital – University Vita-Salute San Raffaele Clementi Francesco, Pharmacologist, Professor Emeritus of Pharmacology, University of Milan Colloca Stefano, Bioethicist, University of Pavia Ferrari Maurizio, Geneticist, San Raffaele Hospital – University Vita-Salute San Raffaele Martinelli Vittorio, Physician, San Raffaele Hospital Meola Giovanni, Physician, San Donato Polyclinic – University of Milan Micieli Giuseppe, Physician, Mondino Neurological Institute Orlando M. Elisabetta, Member of Volunteer association Procopio Giuseppe, Physician, National Cancer Institute, Milan Reichlin Massimo, Bioethicist, University Vita-Salute San Raffaele Richelmi Plinio, Pharmacologist, University of Pavia Sorghi Massimo, General Practitioner, Milan 2 Area Tadini Patrizia, Pharmacist, San Raffaele Hospital Zoppei Gianna, Expert in Humanization of Care, San Raffaele Hospital MEMBERS IN RELATION TO QUALIFICATION Luciani Diego, Clinical Engineer, San Raffaele Hospital Pontiroli Antonio, Expert in Diabetes and Nutrition, University of Milan Triberti Cesare, Expert in Health Law MEMBERS OF THE SCIENTIFIC INSTITUTE HOSPITAL SAN RAFFAELE Mazzuconi Roberts, Health Care Director, San Raffaele Hospital Mandelli Paolo, Substitute of the Health Care Director, San Raffaele Hospital Tadini Patrizia, Pharmacist, San Raffaele Hospital Franzin Michela, Substitute of the Pharmacist, San Raffaele Hospital Montorsi Francesco, Scientific Director, San Raffaele Hospital Guidotti Luca, Substitute of the Scientific Director, San Raffaele Hospital MEMBERS OF THE SCIENTIFIC INSTITUTE SAN DONATO Cuppone M. Teresa, Health Care Director, San Donato Polyclinic Carpinelli Luca, Substitute of the Health Care Director, San Donato Polyclinic Cavallazzi Mario, Pharmacist, San Donato Polyclinic Felisatti Monica, Substitute of the Pharmacist, San Donato Polyclinic Tettamanti Guido, Scientific Director, San Donato Polyclinic Menicanti Lorenzo, Substitute of the Scientific Director, San Donato Polyclinic MEMBERS OF THE ORTHOPAEDIC SCIENTIFIC INSTITUTE GALEAZZI Pregliasco Fabrizio, Health Care Director, Galeazzi Orthopaedic Institute Vinci Anna, Substitute of the Health Care Director, Galeazzi Orthopaedic Institute 267 THE ETHICS COMMITTEE Cavallazzi Mario, Pharmacist, Galeazzi Orthopaedic Institute Felisatti Monica, Substitute of the Pharmacist, Galeazzi Orthopaedic Institute Banfi Giuseppe, Scientific Director, Galeazzi Orthopaedic Institute Cittera Elena, Substitute of the Scientific Director, Galeazzi Orthopaedic Institute MEMBERS OF THE NEUROLOGICAL SCIENTIFIC INSTITUTE MONDINO Moneta Angela, Health Care Director, Mondino Institute Gervasio Luisa, Pharmacist, Mondino Institute Nappi Giuseppe, Scientific Director, Mondino Institute Blandini Fabio, Substitute of the Scientific Director, Mondino Institute MEMBERS OF SAN RAFFAELE TURRO INSTITUTE Mazzitelli Salvatore, Health Care Director, San Raffaele Turro Institute Benedetti Francesco, Substitute of the Health Care Director, San Raffaele Turro Institute Tadini Patrizia, Pharmacist, San Raffaele Turro Institute Franzin Michela, Substitute of the Pharmacist, San Raffaele Turro Institute MEMBERS OF SANT’AMBROGIO E SAN SIRO INSTITUTES Bissolati Morena, Health Care Director, Sant’Ambrogio e San Siro Institutes Cirri Silvia, Substitute of the Health Care Director, Sant’Ambrogio e San Siro Institutes Felisatti Monica, Pharmacist, Sant’Ambrogio e San Siro Institutes Cavallazzi Mario, Substitute of the Pharmacist, Sant’Ambrogio e San Siro Institutes THE CLINICAL DEPARTMENTS 269 CARDIO-THORACIC-VASCULAR DEPARTMENT Head of Department: Ottavio Alfieri* DEPARTMENT AREA COORDINATORS: Stefano Benussi, Renata Clotilde Castellano, Domenico Cianflone*, Guglielmo Cornero, Stefano Gerosa, Giovanni La Canna, Silvio Magrin, Enrico Maria Marone, Germano Melissano*, Stefano Moriggia Cardiac surgery HEAD OF UNIT: Ottavio Alfieri* CLINICAL UNIT LEADERS: Stefano Benussi, Andrea Blasio, Michele De Bonis, Francesco Maisano, Stefano Moriggia, Alessandra Rossodivita CLINICAL UNIT COORDINATOR: Alessandro Castiglioni PHYSICIANS: Andrea Blasio, Maria Chiara Calabrese, Micaela Cioni, Paolo Denti, David Ferrara, Andrea Fumero, Giuseppe Iaci, Elisabetta Lapenna, Simona Nascimbene, Maria Grazia Pala, Alessandro Verzini RESIDENTS: Nicola Buzzati, Marta Casamassima, Andrea Giacomini, Mikel Kamami, Teodora Nisi, Elena Portinaro, Alberto Pozzoli, Maurizio Taramasso, Cinzia Trumello FELLOWS: Andrea Guidotti, Davide Schiavi Echocardiography Unit HEAD OF UNIT: Giovanni La Canna PHYSICIANS: Emanuela Alati, Giovanna Di Giannuario FELLOW: Chiara Sordelli TECHNICIAN: Marta Martino Cardiovascular rehabilitation and prevention HEAD OF UNIT: Domenico Cianflone* CLINICAL UNIT LEADER: Carlo Meloni PHYSICIANS: Maria Avitabile, Nicole Cristell, Marco Magnoni RESIDENTS: Francesco Ancona, Letizia Fausta Bertoldi, Alessandro Durante, Alessandra Laricchia, Giovanni Peretto, Marco Spartera FELLOW: Luca Rosario Limite Cardiovascular interventions Unit HEAD OF UNIT: Antonio Colombo CLINICAL UNIT LEADERS: Mauro Carlino, Alaide Chieffo, Matteo Montorfano PHYSICIANS: Alfredo Castelli, Filippo Figini, Azeem Mohamed Latib FELLOWS: Chiara Bernelli, Jaclyn Chi Lin Chan, Georgina Fuertes, Caroline J. Magri, Charbel A. Naim, Vasilis Panoulas, Paola Spatuzza TRIAL COORDINATOR: Angela Ferrari 271 THE CLINICAL DEPARTMENTS TECHNICIANS: Gianfranco Accarino, Raimondo Bellanca, Salvatore Cannavale, Andrea Di Marco, Fanny Lavazza, Matteo Longoni, Davide Maccagni, Massimo Angelo Messa, Marcello Murino, Vittorio Romano, Mario Squilla Clinic for primary and secondary cardiomyopathy HEAD OF UNIT: Paolo Camici* PHYSICIANS: Sara Benedetti, Roberto Spoladore** RESIDENTS: Alessandro Durante**, Alessia Faccini**, Francesco Maranta**, Damiano Regazzoli** FELLOWS: Alberto Castella, Umberto Gianni Arrhythmia Unit and electrophysiology laboratories HEAD OF UNIT: Paolo Della Bella CLINICAL UNIT LEADERS: Giuseppe Maccabelli, Patrizio Mazzone PHYSICIANS: Caterina Bisceglia, Manuela Cireddu, Simone Gulletta, Gabriele Paglino, Simone Sala, Nicola Trevisi CONSULTANTS: Francesca Baratto, Alessandra Marzi, Andrea Radinovic, Nicoleta Sora, Pasquale Vergara POST-DOCTORAL FELLOWS: Amr Nawar, Miki Yamase, Luck Yonguanijchit Clinical cardiology and outpatient clinic HEAD OF UNIT: Alberto Margonato* CLINICAL UNIT LEADERS: Fabrizio Bonetti, Alberto Cappelletti, Andrea Conversano, Gabriele Fragasso, Stefano Gerosa, Cosmo Godino, Michele Oppizzi PHYSICIANS: Eustachio Agricola, Chiara Camesasca, Cristina Canciani, Barbara Demarchi, Valeria Magni, Alessandra Mailhac, Cinzia Nitti, Alessandra Pancaldi, Marco Papa, Cristina Pedrigi, Patrizia Puccetti, Carmen Silipigni RESIDENTS: Monica Mazzavillani, Claudia Montanaro, Anna Salerno ** Coronary Care Unit (CCU) HEAD OF UNIT: Alberto Margonato* CLINICAL UNIT LEADERS: Carlo Ballarotto, Giuseppe Pizzetti PHYSICIANS: Michela Cera, Massimo Slavich, Roberto Spoladore Functional rehabilitation HEAD OF UNIT: Alessandra Raschi Intensive Care Unit (ICU) and Post Operatory Care Unit (POCU) HEAD OF UNIT: Alberto Zangrillo* CLINICAL UNIT COORDINATORS: Tiziana Bove, Maria Grazia Calabrò, Giovanni Landoni*, Federico Pappalardo, Anna Mara Scandroglio PHYSICIANS: Elena Bignami, Francesco Cama, Andrea Carozzo, Francesca Cavenago, Guglielmo Cornero, Remo Daniel Covello, Antonella Crescenti, Martina Crivellari, Monica De Luca, Greta Fano, Rossana Fiori, Annalisa Franco, Giovanna Frau, Chiara Gerli, Silvio Magrin, Giovanni Marino, Giulio Melisurgo, Roberta Mennella, Melissa Messina, Fabrizio Monaco, Jaques N’Zepa Batonga, Nicola Pasculli, Ornella Sottocorna Thoracic surgery HEAD OF UNIT: Piero Zannini* CLINICAL UNIT LEADERS: Giulio Melloni, Giampiero Negri*, CLINICAL UNIT COORDINATOR: Angelo Carretta PHYSICIANS: Alessandro Bandiera, Paola Ciriaco, Armando Puglisi RESIDENTS: Piergiorgio Muriana, Silvia Raimondi Cominesi, Stefano Viscardi THE CLINICAL DEPARTMENTS Vascular surgery HEAD OF UNIT: Roberto Chiesa* CLINICAL UNIT LEADERS: Efrem Civilini, Enrico Maria Marone, Germano Melissano*, Yamume Tshomba CLINICAL UNIT COORDINATOR: Renata Clotilde Castellano PHYSICIANS: Domenico Astore, Laura Dordoni, Gloria Esposito, Massimiliano Marrocco-Trischitta RESIDENTS: Luca Apruzzi**, Serena Frezza**, Jessica Lanza, Davide Logaldo, Daniele Mascia**, Girolomina Mazzeo, Enrico Rinaldi**, Sara Spelta, Renato Vitale** FELLOWS: Domenico Baccellieri, Luca Bertoglio, Chiara Brioschi, Andrea Kahlberg * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The clinical activity of the Department is devoted to the diagnosis and treatment of cardiac, thoracic and vascular disease. New techniques and technologies have been introduced to offer to the patients the entire spectrum of the therapy options in a multidiciplinary environment. Patients-centered care is the philosophy behind the daily clinical practice. The interaction among different specialists (anesthesiologists, cardiovascular and thoracic surgeons, interventional and clinical cardiologists) is favored by the contiguity of the areas devoted to surgery, cath lab procedures and intensive care. Patients with acute disease coming on emergency basis as well as patients with chronic diseases admitted electively are submitted to diagnostic investigation, treatment and then rehabilitation if needed. A well structured rehabilitation program is available providing complete recovery even for the sickest patients. An active outpatient clinic is functioning for new referrals and follow-up. Clinical activity and areas of excellence In regard to cardiac diseases, the main pathologies are ischemic and valvular heart diseases, which are treated either with catheter based interventions or minimally invasive procedures or conventional surgery. Grown-up patients with congenital heart disease have been increased recently. A large number of patients with major aortic and vascular pathologies are also increasingly referred as well as patients with complex oncological problems of the thoracic organs. Transcatheter aortic valve implantation has been widely carried out to treat inoperable or high risk patients with severe aortic stenosis and today the largest experience in Italy has been accumulated in this field by our multidisciplinary team. The percutaneous treatment of mitral insufficiency is performed in patients with heart failure and our series represents one of the very first in Europe. Innovative modalities of medical treatment for patients with congestive heart failure have been recently introduced, particularly in the field of improvement of cardiac metabolism, achieving a significant increaseof survival/quality of life as compared to figures reported in the literature. A structurized program for the treatment of patients with end-stage heart failure has been developed. Aim of the program is to provide a comprehensive treatment for the failing heart, including mechanical circulatory support (short and long term) in different clinical settings: post cardiotomy cardiogenic shock, acute myocardial infarction, bridge to transplant, destination therapy. Also treatment of acute hypoxia and ARDS by ECMO is now offered as a life-saving procedure. A wide spectrum of aortic pathologies, including aneurysms, dissection, traumatic injuries, coarctation, etc., are treated using both conventional and endovascular approaches. For high risk patients with complex aortic arch and thoracoabdominal pathology, hybrid open and endovascular branch-technology is currently under evaluation. Both open and endovascular procedures are also used for carotid and lower limb revascularization. Great experience has been achieved in the treatment of patients with ventricular tachycardia, who are re273 THE CLINICAL DEPARTMENTS ferred from all over the country. Other major arrhythmias are treated by electrophysiologists and surgeons in the cath lab and in the operating room. A great recent achievement in thoracic surgery has been the treatment of lung cancer in patients with emphysema. The respiratory impairment due to this disease used to be a surgical contraindication in the past. Today different strategies have been introduced in our Department to extend surgical indication in lung cancer including parenchyma-sparing bronchoplasty techniques, lung volume reduction surgery and bronchoscopic lung volume reduction. Finally, 3-D echocardiography has been introduced as a routine imaging modality to assess patients with structural heart disease, with considerable enhancement of diagnostic capabilities. Ottavio Alfieri Selected publications Ammirati, E; Cristell, N; Cianflone, D; Vermi, AC; Marenzi, G; De Metrio, M; Uren, NG; Hu, D; Ravasi, T; Maseri, A; Cannistraci, CV. Questing for circadian dependence in ST-segment-elevation acute myocardial infarction: a multicentric and multiethnic study. Circ. Res.: 2013; 112(10): e110e114 - Article Buzzatti, N; Maisano, F; Latib, A; Cioni, M; Taramasso, M; Mussardo, M; Colombo, A; Alfieri, O. Computed tomography-based evaluation of aortic annulus, prosthesis size and impact on early residual aortic regurgitation after transcatheter aortic valve implantation. Eur. J. Cardiothorac. Surg.: 2013; 43(1): 43-51 - Article Chieffo, A and Buchanan, GL; Van Mieghem, NM; Tchetche, D; Dumonteil, N; Latib, A; Van Der Boon, RMA; Vahdat, O; Marcheix, B; Farah, B; Serruys, PW; Fajadet, J; Carrie, D; De Jaegere, PPT; Colombo, A. Transcatheter aortic valve implantation with the Edwards SAPIEN versus the medtronic corevalve revalving system devices: A multicenter collaborative study: The PRAGMATIC plus initiative (Pooled-RotterdAm-Milano-Toulouse in Collaboration). J. Am. Coll. Cardiol.: 2013; 61(8): 830836 - Article Chiesa, R; Tshomba, Y; Mascia, D; Rinaldi, E; Logaldo, D; Civilini, E. Open repair for juxtarenal aortic aneurysms. J. Cardiovasc. Surg.: 2013; 54(1 SUPPL.1): 35-45 - Review Citro, R; Mirra, M; Baldi, C; Prota, C; Palumbo, B; Piscione, F; La Canna, G. Concomitant dynamic obstruction and endocarditis after “valve in valve” TAVI implantation. Int. J. Cardiol.: 2013; 167(2): e27-e29 - Letter De Bonis, M; Lapenna, E; Buzzatti, N; Taramasso, M; Calabrese, MC; Nisi, T; Pappalardo, F; Alfieri, O. Can the edge-to-edge technique provide durable results when used to rescue patients with suboptimal conventional mitral repair?. Eur. J. Cardio-thorac. Surg.: 2013; 43(6): e173-e179 Article Della Bella, P; Baratto, F; Tsiachris, D; Trevisi, N; Vergara, P; Bisceglia, C; Petracca, F; Carbucicchio, C; Benussi, S; Maisano, F; Alfieri, O; Pappalardo, F; Zangrillo, A; Maccabelli, G. Management of ventricular tachycardia in the setting of a dedicated unit for the treatment of complex ventricular arrhythmias: Long-term outcome after ablation. Circulation: 2013; 127(13): 1359-1368 Article Farooq, V and Van Klaveren, D; Steyerberg, EW; Meliga, E; Vergouwe, Y; Chieffo, A; Kappetein, AP; Colombo, A; Holmes ,Jr DR; MacK, M; Feldman, T; Morice, MC; Stahle, E; Onuma, Y; Morel, MA; Garcia-Garcia, HM; Van Es, GA; Dawkins, KD; Mohr, FW; Serruys, PW. Anatomical and clinical characteristics to guide decision making between coronary artery bypass surgery and percutaneous coronary intervention for individual patients: Development and validation of SYNTAX score II. Lancet: 2013; 381(9867): 639-650 - Article Fragasso, G; Rosano, G; Baek, SH; Sisakian, H; Di Napoli, P; Alberti, L; Calori, G; Kang, SM; Sahakyan, L; Sanosyan, A; Vitale, C; Marazzi, G; Margonato, A; Belardinelli, R. Effect of partial fatty acid oxidation inhibition with trimetazidine on mortality and morbidity in heart failure: Results from an international multicentre retrospective cohort study. Int. J. Cardiol.: 2013; 163(3): 320-325 - Article THE CLINICAL DEPARTMENTS Godino, C; Lauretta, L; Pavon, AG; Mangieri, A; Viani, G; Chieffo, A; Galaverna, S; Latib, A; Montorfano, M; Cappelletti, A; Maisano, F; Alfieri, O; Margonato, A; Colombo, A. Heyde’s syndrome incidence and outcome in patients undergoing transcatheter aortic valve implantation. J. Am. Coll. Cardiol.: 2013; 61(6): 687-689 - Letter Gould, KL; Johnson, NP; Bateman, TM; Beanlands, RS; Bengel, FM; Bober, R; Camici, PG; Cerqueira, MD; Chow, BJ; Di Carli, MF; Dorbala, S; Gewirtz, H; Gropler, RJ; Kaufmann, PA; Knaapen, P; Knuuti, J; Merhige, ME; Rentrop, KP; Ruddy, TD; Schelbert, HR; Schindler, TH; Schwaiger, M; Sdringola, S; Vitarello, J; Williams Sr, KA; Gordon, D; Dilsizian, V; Narula, J. Anatomic versus physiologic assessment of coronary artery disease. Role of coronary flow reserve, fractional flow reserve, and positron emission tomography imaging in revascularization decision-making. J. Am. Coll. Cardiol.: 2013; 62(18): 1639-1653 - Review Lancellotti, P; Rosenhek, R; Pibarot, P; Iung, B; Otto, CM; Tornos, P; Donal, E; Prendergast, B; Magne, J; La Canna, G; Pierard, LA; Maurer, G. ESC Working Group on Valvular Heart Disease Position Paper-heart valve clinics: Organization, structure, and experiences. Eur. Heart J.: 2013; 34(21): 1597-1606 - Article Magnoni, M; Figini, F; Piraino, D; Cianflone, D. Coexistence of multiple and widespread cardiovascular complications in a patient with Marfan syndrome. J. Clin. Ultrasound: 2013; 41(3): 195-198 - Article Mancini, M; Petretto, E; Kleinert, C; Scavone, A; De, T; Cook, S; Silhavy, J; Zidek, V; Pravenec, M; d’Amati, G; Camici, PG. Mapping genetic determinants of coronary microvascular remodeling in the spontaneously hypertensive rat. Basic Res. Cardiol.: 2013; 108(1): article 316 - Article Melloni, G; Samanes Gajate, AM; Sestini, S; Gallivanone, F; Bandiera, A; Landoni, C; Muriana, P; Gianolli, L; Zannini, P. New positron emission tomography derived parameters as predictive factors for recurrence in resected stage i non-small cell lung cancer. Eur. J. Surg. Oncol.: 2013; 39(11): 1254-1261 - Article Mizuno, H; Vergara, P; Maccabelli, G; Trevisi, N; Colombo; S; Brombin, C; Mazzone, P; Della Bella, P. Contact force monitoring for cardiac mapping in patients with ventricular tachycardia. J. Cardiovasc. Electrophysiol.: 2013; 24(5): 519-524 - Article Negri, G; Bandiera, A; Carretta, A; Puglisi, A; Mandelli, C; Ciriaco, P; Zannini, P. Unusual presentation of mediastinal neurogenic tumours. Case Rep. Surg.: 2013; 2013: 414260 - Case report Pappalardo, F; Pieri, M; Greco, T; Patroniti, N; Pesenti, A; Arcadipane, A; Ranieri, VM; Gattinoni, L; Landoni, G; Holzgraefe, B; Beutel, G; Zangrillo, A; on behalf of the Italian ECMOnet. Predicting mortality risk in patients undergoing venovenous ECMO for ARDS due to influenza A (H1N1) pneumonia: the ECMOnet score. Intensive Care Med.: 2013; 39(2): 275-281 - Article Pasin, L; Landoni, G; Zangrillo, A. Glutamine and antioxidants in critically ill patients. New Engl. J. Med.: 2013; 369(5): 482-484 - Letter Scheinert, D; Pratesi, C; Chiesa, R; Coppi, G; Brunkwall, JS; Klarenbeek, G; Cebrian, A; Torsello, G. First-in-human study of the INCRAFT endograft in patients with infrarenal abdominal aortic aneurysms in the INNOVATION trial. J. Vasc. Surg.: 2013; 57(4): 906-914 - Article 275 DEPARTMENT OF GENERAL AND SPECIALISTIC SURGERY Department coordinator: Gianfranco Ferla*, since November 2013 Head of Department: Carlo Staudacher* until October 2013 DEPARTMENT AREA COORDINATORS: Marco Braga*, Francesco Deni, Renato Finazzi, Emiliano Giorgi, Gilberto Mari, Michele Paganelli, Danilo Parolini, Sandro Passaretti, Carlo Socci Gastroenterologic surgery HEAD OF UNIT: Gianfranco Ferla* (ad interim) since November 2013; Carlo Staudacher*, until October 2013 CLINICAL UNIT LEADERS: Saverio Di Palo, Elena Orsenigo, Danilo Parolini, Carlo Socci CLINICAL UNIT COORDINATOR: Paola De Nardi PHYSICIANS: Andrea Marco Tamburini, Andrea Vignali RESIDENTS: Massimiliano Bissolati**, Damiano Chiari**, Guido Fiorentini**, Paolo Giovanni Gazzetta**, Luca Ghirardelli**, Francesca Muffatti** General and pancreatic surgery HEAD OF UNIT: Gianfranco Ferla* (ad interim) since November 2013; Carlo Staudacher*, until October 2013 CLINICAL UNIT LEADERS: Gianpaolo Balzano, Marco Braga* RESEARCHER: Lorenzo Piemonti PHYSICIAN: Renato Castoldi RESIDENTS: Riccardo Ariotti**, Giovanni Luigi Capretti**, Cristina Gilardini** Hepatobiliary and week surgery HEAD OF UNIT: Gianfranco Ferla* CLINICAL UNIT LEADERS: Luca Aldrighetti, Edoardo Beretta, Alberto Marassi, Gilberto Mari PHYSICIANS: Marco Catena, Enrico Fiacco, Renato Finazzi, Michele Paganelli CONSULTANTS: Mvunde Mukenge, Veronica Zuber RESIDENTS: Federica Cipriani**, Annalisa Gagliano**, Francesca Ratti** FELLOW: Fabio Ferla THE CLINICAL DEPARTMENTS Orthopaedics HEAD OF UNIT: Gianfranco Fraschini* CLINICAL UNIT LEADERS: Francesco Camnasio, Maurizio De Pellegrin, Giuseppe Gioia, Crispino Grispigni, Eliseo Mainetti RESEARCHER: Alessandro Rubinacci PHYSICIANS: Arianna Banfi, Erica Bulgheroni, Carlo Maria Castoldi, Alessandro De Ponti, Dario Fracassetti, Valerj Maltsev, Davide Mandelli, Umberto Mezzadri, Gianluigi Moro, Marco Ometti, Paola Rivoltini, Matteo Vitali RESIDENTS: Laura Mangiavini, Alessandro Pozzi POST-DOCTORAL FELLOW: Daniela Deponti Gastroenterology-Endoscopy HEAD OF UNIT: Pier Alberto Testoni* CLINICAL UNIT LEADERS: Paolo Giorgio Arcidiacono, Mario Guslandi, Sandro Passaretti, Maria Chiara Petrone PHYSICIANS: Giulia Martina Cavestro*, Emanuele Dabizzi, Lorella Fanti, Alberto Mariani, Edi Viale CONSULTANT: Elena Radice RESIDENTS: Matteo Alessandri, Milena Di Leo, Giorgia Mazzoleni, Chiara Notaristefano, Gemma Rossi, Sabrina G. Testoni, Raffaella A. Zuppardo FELLOWS: Marco Le Grazie, Maria Emilia Traini Anaesthesiology HEAD OF UNIT: Luigi Beretta* CLINICAL UNIT COORDINATORS: Massimo Agostoni, Massimo Caldi, Eleonora Colnaghi, Laura Comotti, Francesco Deni, Emiliano Giorgi, Daniela Giudici, Valeria Perotti PHYSICIANS: Barbara Airaghi, Felicia Adalgisa Antonino, Tania Capocasa, Domenica Carrieri, Mariela Decembrino, Cinzia Elisabetta De Grandis, Alessandra Garassino, Renato Meani, Alessandra Mello, Silvia Morero, Laura Pasin, Francesca Presti, Lorenzo Quario Rondo, Raffaella Reineke, Stefano Turi, Federico Vinciguerra General intensive care HEAD OF UNIT: Alberto Zangrillo* CLINICAL UNIT LEADER: Sergio Colombo PHYSICIANS: Paolo Federico Beccaria, Pier Carlo Bergonzi, Luca Cabrini, Carlo Leggieri, Daniela Mamo, Elena Moizo, Giacomo Monti, Milena Mucci, Davide Salaris, Massimo Zambon * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The Department of general and specialistic surgery comprehends the following branches: Upper Gastrointestinal Surgery, Colorectal Surgery, Pancreas Surgery, Hepatobiliary Surgery, Bariatric Surgery, Lymphatic Surgery, Breast Surgery, Endocrine Surgery, General Surgery, Pancreas and Kidney Transplantation Surgery, Gastroenterology & Endoscopy Procedures, Orthopaedic Surgery, Anaesthesiology and Intensive Care Unit. The Department has three core missions: excellence clinical care, outstanding research productivity and high level educational program imparting. Clinical activity and Areas of excellence The Upper Gastrointestinal Surgery Unit has a dedicated program in treating cancers and benign diseases of the upper gastrointestinal tract. Patients are treated by a multidisciplinary team of experts. The research 277 THE CLINICAL DEPARTMENTS program aims to improve the pre-operative workup of esophagus and stomach tumors including magnetic resonance imaging, sentinel node identification in early cancer stage and neoadjuvant chemotherapy and hyperthermic intraperitoneal chemotherapy (HIPEC) in the advanced ones. The Colorectal Surgery Unit offers the most advanced surgical and minimally invasive options not only to eradicate the disease, but also to preserve patients’ ability to normal function. Among the programs there are the sentinel node mapping in colorectal and anal canal cancer as well as the perioperative evaluation of anorectal function and pelvic floor biofeedback rehabilitation method. The Pancreatic Surgery Unit presents one of the highest surgery volume in Europe with more than 160 pancreatic resections performed per year. In order to treat the post-surgical diabetes, secondary to total pancreatectomy, there is an active program of islet autotransplantation. In the Hepatobiliary Surgery Unit the activities are mainly focused on hepato-biliary tumors and chronic liver diseases, including primary and metastatic liver tumors, benign and malignant diseases of the biliary tract, acute and chronic hepatitis. Concerning the Hepatic Surgery the unit is a leading reference in our country for liver resections, performing about 170 liver resection per year. A clinical program of laparoscopic liver surgery has been started on January 2007, being at the moment the most active program in Italy. Data regarding the Bariatric Surgery reveals a successful activity in the past years. The current program foresees an activity increase, with special interest to the obese diabetic patient and the metabolic surgery. Lymphatic Surgery: the primary and secondary lymphoedema surgical therapy finds in the lympho-venous anastomosis as well as in the lymphatic grafting a reliable procedure. In the Breast Surgery Unit the International Standard of Excellence stated by EUSOMA (European Society of Mastology) is applied. Following this criteria, 300 new malignant cases per year are enrolled and more than 2000 patients per year are treated in the unit. The Endocrine Surgery Unit treats endocrine tumors in children and adults. Surgeons working in this section employ cutting edge technology, includ ing minimally invasive parathyroid surgery with intraoperative parathormone (PTH) assay determination, and laparoscopic adrenalectomy. The clinical activity of Transplant Surgery Unit includes the following programs: Simultaneous Pancreas and Kidney Transplantation and Pancreas Alone Transplantation in insulin dependent diabetes mellitus (IDDM) patients; double kidney transplantation from marginal donors; laparoscopic living donor nephrectomy; islet transplantation. The clinical activity of the Gastroenterology and Gastrointestinal Endoscopy Unit includes four main sections: clinical gastroenterology and hepatology, gastrointestinal and pancreatic biliary endoscopy, ultrasound endoscopy, and digestive motility. The unit is a tertiary referral center for therapeutic pancreatic biliary and gastrointestinal tract endoscopy, as well as for endoscopic ultrasound, diagnosis and treatment of motility disorders (mainly esophagus and rectum), and pancreatic diseases. Advanced endoscopic procedures, as the endoscopic retrograde cholangiopancreatography (ERCP), endoscopic ultrasound, and therapeutic gastrointestinal tract procedures are carried out under deep sedation and anesthesiologist assistance; two anesthesiologists are involved daily in two operatory rooms. The Orthopaedic Surgery Unit cares for all injuries and diseases of the musculoskeletal system. The unit provides subspecialty clinics in the specific areas of major joint reconstruction, sport medicine, paediatric orthopaedics, foot, hand, spine, trauma oncology, reconstructive microsurgery, and rehabilitation. The clinical activity of the Orthopaedics and Traumatology Unit aims to cover all the needs related to traumatic, degenerative, oncologic, infectious and metabolic aspects of the musculoskeletal system. The spine surgery, the shoulder and elbow surgery, the treatment of the degenerative disease of the joints, the pelvis surgery, the diagnosis and treatment of the pathologies of the bone metabolism represent areas of distinction of the Orthopaedics and Traumatology Unit. The Orthopaedics and Traumatology Unit has also an intense basic research activity with the Laboratory for Tissue Engineering and Biomaterials. The Anesthesiology and Intensive Care Unit reflects the comprehensive effort of a team of anesthesia physicians, nurses, and staff engaged in the advanced patient care. Preadmission diagnosis and staging program focus on decrease in the hospital stay. The main fields of research are: medical emergency team; non invasive ventilation in innovative fields; percutaneous tracheostomy in difficult cases; activated or Zymogen Protein C in sepsis; use of simulation in medical training; extracorporeal support in acute respiratory distress syndrome (ARDS) patients (in particular in influenza A H1N1 related ARDS). The team publishes an international journal “Heart, Lung and Vessels”, freely available on www.itacta.org. THE CLINICAL DEPARTMENTS Fields of research The Department of Surgery has a long and distinguished history of surgical research, nationally and internationally recognized. Unique clinical trials are offered for a variety of surgical diseases, giving life expectancy to patients with critical illness who were once considered untreatable. The research mission is to enhance new scientific knowledge to surgically related disease and to provide outstanding scientific training for future surgeons and surgical scientists. The key focus of the Department of Surgery is the development and application of molecular/genetic biomarkers for the diagnosis, prognosis and prediction of treatment response of the digestive system. The primary objectives are: 1) to be a reference to surgical research; 2) to help develop new applications to clinical care; and 3) to provide outstanding research training for surgical residents and surgical scientists. Gianfranco Ferla Selected publications Balzano, G; Maffi, P; Nano, R; Zerbi, A; Venturini, M; Melzi, R; Mercalli, A; Magistretti, P; Scavini, M; Castoldi, R; Carvello, M; Braga, M; Del Maschio, A; Secchi, A; Staudacher, C; Piemonti, L. Extending indications for islet autotransplantation in pancreatic surgery. Ann. Surg.: 2013; 258(2): 210-218 - Article Bolamperti, S; Mrak, E; Moro, G; Sirtori, P; Fraschini, G; Guidobono, F; Rubinacci, A; Villa, I. 17β-Estradiol positively modulates growth hormone signaling through the reduction of SOCS2 negative feedback in human osteoblasts. Bone: 2013; 55(1): 84-92 - Article Masci, E; Viale, E; Notaristefano, C; Mangiavillano, B; Fiori, G; Crosta, C; Dinelli, M; Maino, M; Viaggi, P; Della Giustina, F; Teruzzi, V; Grasso, G; Manes, G; Zambelli, S; Testoni, PA. Endoscopic mucosal resection in high- and low-volume centers: a prospective multicentric study. Surg. Endosc.: 2013; 27(10): 3799-3805 - Article Mukenge, S; Negrini, D; Catena, M; Ratti, F; Dosio, F; Paesano, P; Rigatti, P; Ferla, G. Development of functionally patent lymphatic meshes in postsurgical long-term resolution of peripheral secondary lymphedema. Journal of Vascular Surgery: Venous and Lymphatic Disorders: 2013; 1(3): 280-288 - Article Pecorelli, N; Braga, M; Doglioni, C; Balzano, G; Reni, M; Cereda, S; Albarello, L; Castoldi, R; Capretti, G; Di Carlo, V. Preoperative Chemotherapy Does Not Adversely Affect Pancreatic Structure and Short-Term Outcome after Pancreatectomy. J. Gastrointest. Surg.: 2013; 17(3): 488-493 Article Ratti, F; Cipriani, F; Ferla, F; Catena, M; Paganelli, M; Aldrighetti, LAM. Hilar cholangiocarcinoma: Preoperative liver optimization with multidisciplinary approach. Toward a better outcome. World J. Surg.: 2013; 37(6): 1388-1396 - Article Testoni, PA; Notaristefano, C; Di Leo, M; Vailati, C; Mazzoleni, G; Viale, E. High-definition with iScan gives comparable accuracy for detecting colonic lesions by non-expert and expert endoscopists. Dig. Liver Dis.: 2013; 45(6): 481-486 - Article Vignali, A; De Nardi, P; Ghirardelli, L; Di Palo, S; Staudacher, C. Short and long-term outcomes of laparoscopic colectomy in obese patients. World J. Gastroenterol.: 2013; 19(42): 7405-7411 Article Vignali, A; Ghirardelli, L; Di Palo, S; Orsenigo, E; Staudacher, C. Laparoscopic treatment of advanced colonic cancer: A case-matched control with open surgery. Colorectal Dis.: 2013; 15(8): 944-948 - Article 279 HEAD AND NECK DEPARTMENT Head of Department: Luigi Beretta* DEPARTMENT AREA COORDINATORS: Antonio Dell’Acqua, Marco Gemma, Susanna Piccoli, Sandra Pieralli, Claudio Righi, Francesco Scomazzoni Head and neck anaesthesia and neurointensive care HEAD OF UNIT: Luigi Beretta* CLINICAL UNIT LEADERS: Silvano Cozzi, Cristina Mattioli CLINICAL UNIT COORDINATORS: Antonio Dell’Acqua, Marco Gemma, Susanna Piccoli PHYSICIANS: Fabio Bernasconi, Maria Rosa Calvi, Marco Cerri, Antonella Cipriani, Assunta De Vitis, Cristina Frascoli, Luigi Gioia, Elisabetta Grandi, Karin Iemi, Maurizio Mungo, Alfredo Ravizza, Francesco Ruggieri, Luisa Sacchi RESIDENT: Massimiliano Greco Neuroradiology HEAD OF UNIT: Andrea Falini* CLINICAL UNIT LEADERS: Nicoletta Anzalone, Cristina Baldoli, Sandra Pieralli, Claudio Righi, Letterio Salvatore Politi, Franco Simionato PHYSICIANS: Simonetta Gerevini, Silvia Pontesilli, Francesco Scomazzoni, Roberta Scotti, Paolo Vezzulli Ophthalmology HEAD OF UNIT: Francesco Bandello* CLINICAL UNIT LEADER: Francesco Fasce CLINICAL UNIT COORDINATORS: Gianluigi Bolognesi, Luisa Pierro PHYSICIANS: Maurizio Battaglia Parodi, Paolo Bettin, Marco Codenotti, Antonio Giordano Resti, Ugo Introini, Rosangela Lattanzio, Francesco Loperfido, Gisella Maestranzi, Giulio Modorati CONSULTANTS: Nicola Baccelli, Piero Barboni, Elena Bruschi, Maria Lucia Cascavilla, Carlo Ciampi, Umberto De Benedetto, Federico Di Matteo, Marina Fiori, Maddalena Forti, Marco Gagliardi, Matteo Ghidoni (until March 2013), Silvia Giatsidis, Lauretta Guarisco, Francesca Legorini, Angela Malegori, Maria Pia Manitto, Elisabetta Martina, Paolo Mauceri, Elisabetta Miserocchi, Veronica Odazio, Matteo Prati, Giuseppe Querques, Andrea Ramoni, Carmen Rojo, Alessandra Spinelli, Monica Stoppani, Ilaria Zucchiatti RESIDENTS: Luigi Berchicci**, Ingrid Bianchi**, Giuseppe Casalino**, Giulia Corradetti**, Claudia Del Turco**, Giovanni Fogliato**, Lorenzo Iuliano**, Karl Anders Knutsson**, Carlo La Spina**, Jacopo Milesi**, Davide Panico**, Lea Querques**, Giacinto Triolo ** TECHNICIANS: Giorgio Alto, Adriana Angiolini, Alessio Buzzotta, Silvia Giganti (until March 2013), Silvia Marcaggi (since June 2013), Antonella Ribecca THE CLINICAL DEPARTMENTS Cornea and ocular surface Unit HEAD OF UNIT: Paolo Rama CONSULTANTS: Oriella Ambrosio, Giulio Ferrari, Federica Ferrario, Chiara Insacco, Stanislav Matuska, Giorgio Paganoni, Elena Scandola, Maurizia Viganò TECHNICIANS: Gloria Badin, Enrico Delfino Otorhinolaryngology Unit HEAD OF UNIT: Mario Bussi* PHYSICIANS: Stefano Bondi, Leone Giordano, Francesca Lira Luce, Lucia Oriella Piccioni, Roberto Teggi, Matteo Trimarchi CONSULTANTS: Giulia Danè, Fabrizio Ferrario, Omar Gatti, Francesca Palonta, Rosaria Taverna RESIDENTS: Matteo Biafora, Davide Di Santo, Francesco Pilolli, Daniela Sarandria, Salvatore Toma SPEECH THERAPISTS: Daniela Gherner, Barbara Ramella TECHNICIAN: Federica Beltrando Neurosurgery HEAD OF UNIT: Pietro Mortini* CLINICAL UNIT LEADERS: Stefania Acerno, Camillo Ferrari Da Passano, Alberto Franzin, Marco Losa, Carlo Mandelli, Aleandro Rocca PHYSICIANS: Lina Raffaella Barzaghi, Nicola Boari, Lorenzo Gioia, Marzia Medone, Micol Valle PHD STUDENT: Filippo Gagliardi**, Silvia Snider ** RESIDENTS: Michele Bailo, Jody Capitanio, Andrea Cavalli , Elena Colombo, Jaime Mandelli, Lucio Aniello Mazzeo, Pietro Panni, Luca Ruggeri, Giorgio Spatola, Alfio Spina * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The Head and Neck Department integrates the clinical and research activities of six clinical “operative units” dedicated to pathology of: head and neck, auditory system, Central Nervous System (brain and spinal cord), visual system. Clinical activity and areas of excellence From diagnosis to treatment, the areas of clinical excellence of the Department are: Advanced neuroradiological techniques Four high field magnetic resonance units (three 1.5T and one 3.0T MR) perform more than 13.000 MRI per year in children and adults. Presurgical functional studies (activation, tractography) are performed on a regular basis for surgery of brain gliomas. Advanced MR techiniques are routinely applied in cerebrovascular diseases, in the main neurodegenerative conditions such as AD, FTD and ALS and in inflammatory diseases. Pediatric MRI studies are performed in fetuses at risk, premature newborns, infants and children, both on an outpatient basis in sedated or awake children and in emergency and inpatient basis. Considerable advances have been performed in the field of Peripheral Nervous System imaging. High resolution and advanced MR techniques are used in the evaluation of orbital and optic nerve diseases. Interventional Neuroradiology addresses endovascular treatment of aneurysms, arteriovenous malforma tions of the brain and spinal cord, fistulae, carotid stenosis. A wide variety of coils, stents and different types of glue are in regular routine use. All procedures are performed in strict consultation and cooperat ion with neurosurgery and neurointensive care units. 281 THE CLINICAL DEPARTMENTS Neurosurgery The clinical hallmarks of neurosurgery are: • pituitary and base of the skull tumors. The treatment of pituitary lesion is coordinated with the clinicoendocrinological support available within the department unit. Brain gliomas are operated with the support of functional MRI studies and tractography when needed and are followed in cooperation with neurooncology for chemiotherapy and radiotherapy units. Aggressive and extensive skull base tumors are operated in cooperation with otolaryngology department. • Radiosurgery is performed on a wide range of lesions from brain AVM’s to skull base tumors and brain metastasis with a very active gamma knife up to date equipment operated on a multidisciplinary basis. Spine surgery and peripheral nervous system surgery are also performed. Otolaryngology The unit is characterized by a very intense surgical activity mainly addressed to neck malignancies (tumors of the larynx, pharynx, thyroid), paranasal sinuses, oropharyngeal cavity, base of the skull. Rehabilitation of patients submitted to laryngectomy and evaluation and treatment of patient with dis turbed vestibular system and vertigo are among the non surgical clinical activities. Ophtalmology Organised in two vision units, one of which dedicated to the cornea and ocular surface and the other one to all the other fields of ophthalmology: pediatric ophthalmology, retinal surgery, ocular oncology with a particular interest in orbital lymphomas and uveal melanoma treatment with Gamma Knife radiosurgery, ocular immunology, advanced diagnostic imaging with evaluation of nerve fiber layer thickness and retinal ganglion cells in the development of epiretinal membrane. Neurophthalmology is particularly active in studying and treating inflammatory and hereditary optic neuropathies. Neurointensive Care The neurointensive care unit provides support to the surgical activity of all the units of the department, neurosurgery, ophthalmology, otolaryngology and of the interventional procedures in Neuroradiology as well as sedation of children and adults for diagnostic neuroradiological procedures. Treatment of comatose patients and of severe brain trauma cases is one of the fields of excellence of the unit. Luigi Beretta Selected publications Battaglia Parodi, M; Iacono, P; Papayannis, A; Kontadakis, SD; Cascavilla, M; Pierro, L; Gagliardi, M; Bandello, F. Intravitreal ranibizumab for pigment epithelium detachment with subfoveal occult choroidal neovascularization: A prospective 24-month case series. Am. J. Ophthalmol.: 2013; 155(1): 103-108 - Article Bondi, S; Limardo, P; Toma, S; Bussi, M. Non-vestibular head and neck schwannomas: a 10year experience. Eur. Arch. Oto-Rhino-Laryngol.: 2013; 270(8): 2365-2369 - Article Codenotti, M; Fogliato, G; Iuliano, L; Querques, G; Maestranzi, G; Prati, M; Ramoni, A; De Benedetto, U; Bandello, F. Influence of intraocular tamponade on unintentional retinal displacement after vitrectomy for rhegmatogenous retinal detachment. Retina: 2013; 33(2): 349-355 - Article Fanti, L; Agostoni, M; Gemma, M; Rossi, G; Azzolini, ML; Viale, E; Guslandi, M; Beretta, L; Testoni, PA. Two dosages of remifentanil for patient-controlled analgesia vs. meperidine during colonoscopy: A prospective randomized controlled trial. Dig. Liver Dis.: 2013; 45(4): 310-315 - Article Filippi, M; Agosta, F; Scola, E; Canu, E; Magnani, G; Marcone, A; Valsasina, P; Caso, F; Copetti, M; Comi, G; Cappa, SF; Falini, A. Functional network connectivity in the behavioral variant of frontotemporal dementia. Cortex: 2013; 49(9): 2389-2401 - Article THE CLINICAL DEPARTMENTS Filippi, M; Rocca, MA; Bastianello, S; Comi, G; Gallo, P; Gallucci, M; Ghezzi, A; Marrosu, MG; Minonzio, G; Pantano, P; Pozzilli, C; Tedeschi, G; Trojano, M; Falini, A; De Stefano, N. Guidelines from The Italian Neurological and Neuroradiological Societies for the use of magnetic resonance imaging in daily life clinical practice of multiple sclerosis patients. Neurol. Sci.: 2013; 34(12): 20852093 - Review Mortini, P; Gagliardi, F; Boari, N; Losa, M. Surgical strategies and modern therapeutic options in the treatment of craniopharyngiomas. Crit. Rev. Oncol. Hematol.: 2013; 88(3): 514-529 - Review Mortini, P; Gagliardi, F; Boari, N; Roberti, F; Caputy, AJ. The Combined Interhemispheric Subcommissural Translaminaterminalis Approach for Large Craniopharyngiomas. World Neurosurg.: 2013; 80(1-2): 160-166 - Review Pellegrini, G; Rama, P; Matuska, S; Lambiase, A; Bonini, S; Pocobelli, A; Colabelli, RG; Spadea, L; Fasciani, R; Balestrazzi, E; Vinciguerra, P; Rosetta, P; Tortori, A; Nardi, M; Gabbriellini, G; Traverso, CE; Macaluso, C; Losi, L; Percesepe, A; Venturi, B; Corradini, F; Panaras, A; Di Rocco, A; Guatelli, P; De Luca, M. Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells. Regen. Med.: 2013; 8(5): 553-567 - Article Rama, P; Knutsson, KA; Razzoli, G; Matuska, S; Viganò, M; Paganoni, G. Deep anterior lamellar keratoplasty using an original manual technique. Br. J. Ophthalmol.: 2013; 97(1): 23-27 - Article Sigurtà, A; Zanaboni, C; Canavesi, K; Citerio, G; Beretta, L; Stocchetti, N. Intensive care for pediatric traumatic brain injury. Intensive Care Med.: 2013; 39(1): 129-136 - Article Trimarchi, M; Bussi, M; Sinico, RA; Meroni, P; Specks, U. Cocaine-induced midline destructive lesions - an autoimmune disease?. Autoimmun. Rev.: 2013; 12(4): 496-500 - Review 283 DEPARTMENT INFECTIOUS DISEASESOF Head of Department: Adriano Lazzarin* DEPARTMENT AREA COORDINATORS: Nicola Gianotti, Paolo Scarpellini Infectious diseases HEAD OF UNIT: Adriano Lazzarin* CLINICAL UNIT LEADERS: Antonella Castagna, Massimo Cernuschi, Paolo Scarpellini, Giuseppe Tambussi, Caterina Uberti-Foppa RESEARCHERS: Laura Galli, Lucia Lopalco, Giulia Morsica, Claudia Pastori PHYSICIANS: Paola Cinque, Anna Danise, Luca Fumagalli, Nicola Gianotti, Monica Guffanti, Myriam Maillard, Silvia Nozza CONSULTANTS: Laura Alagna, Simona Bossolasco, Barbara Castiglioni, Giovanni Gaiera, Andrea Galli, Hamid Ibrahim Hasson, Flavia Salmaso, Stefania Salpietro, Vincenzo Spagnuolo, Chiara Tassan Din RESIDENTS: Sabrina Bagaglio, Alessia Carbone**, Stefania Chiappetta**, Francesca Ferretti**, Valeria Longo, Marco Merli**, Emanuela Messina**, Chiara Oltolini**, Manuela Pogliaghi**, Marco Ripa** TECHNICIAN: Manuela Testa RESEARCH NURSES: Alba Bigoloni, Liviana Della Torre, Concetta Vinci STUDY COORDINATORS: Elisabetta Carini, Maria Rita Parisi * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The activity of the Department of Infectious Diseases is organized in five Functional Units (Ordinary Admission, Day-Hospital, HIV-Infected Outpatient Service, Infectious Diseases Consultancy Service for the whole Hospital, Experimental Therapies) and two Coordination Areas (Quality and Information Technology). The Department includes the Admission Unit with 34 beds (26 of which currently active), the DayHospital Unit with 2 beds, 13 rooms for outpatient evaluation also including gastroenterology and fibroscan examination. Clinical activity and Areas of excellence These include HIV, HBV/HCV and central nervous system (CNS) infections. Management of HIV infection includes both in-hospital and outpatient care, with 4700 patients currently being followed (approximately 90% on treatment with antiretroviral drugs). The Department also follows 860 HIV-infected patients with HBC or HCV infection, many of them treated with standard or experimental therapies and it is an active reference centre for CNS infections, particularly JCV-related progressive multifocal leukoen- THE CLINICAL DEPARTMENTS cephalopathy (PML). Outpatient services have also been set for TB, tropical diseases and travel medicine, and following patient hospital discharge. The Department collaborates with the Hospital Infections Committee, to survey incidence and implement diagnostic and treatment guidelines for management of infectious diseases, and with the Hospital Pharmacy and the Microbiology Laboratory within the Antimicrobial Management Program, on control of quality and appropriateness of antimicrobials prescriptions in hospital. Fields of research The Management and ARV therapy of HIV infection Clinical Research Group is involved in the conduction and coordination of national and international industry sponsored phase III and IV clinical trials contributing to the registration of new antiretroviral compounds. In addition, they have coordinated investigator-driven, no-profit clinical studies, including on salvage strategies and simplification treatments, such as monotherapy with protease inhibitors. The Unit also carries on observational and cohort studies through the HIV database, which contains information from 8800 patients and collaborates with national and international networks of HIV cohorts (ICONA, ARCA, NEAT, EuroSida, D:A:D, COHERE, EURO CHAVI). These focus on clinically relevant areas, including long-term comorbidities associated with HIV infection and antiretroviral treatment, such as diabetes, cancer and osteoporosis. The Vaccine and Immunotherapy Clinical Research Group (CSQ/Certiquality/ISO 9001:2000 accreditation), with a staff well trained in the immunology and infectious diseases fields and experienced in the conduction of clinical trials, carries out preventive, therapeutic, and vaccine trials: amongst vaccine trials, VIU is conducting studies in both HIV (Canaripox plus Remune vaccination in acute HIV infection, anti-TAT phase I and II studies, Bionor CT-BI vacc-4X2007/01) and non- HIV field (Novartis seasonal flu, and H5N1 vaccination in immunocompromised host). Clinical trials are also the main activity of the Hepatotropic Viruses Clinical Research Group. Trials have included phase III studies in patients with HCV monoinfection or HIV/HCV coinfection using new directacting antiviral agents in combination with standard treatments, but also interferon-free regimens. Clinical laboratory research has moved in parallel with treatment advances by setting up methodologies for detection and interpretation of resistance to new anti- HCV drugs. Studies on CNS infections (Neurovirology Clinical Research Group) have involved both laboratory and clinical investigations aiming to optimize disease management by identification and validation of diseases biomarkers. Main research areas have been HIV CNS infection and PML. Studies on HIV encephalopathy have assessed the frequency of neurocognitive impairment in treated patients, and correlates of selective HIV replication in CNS. PML studies have focused on viral and immune determinants of PML, including sequencing of key JCV regions and studies on B-cell and T-cell responses against specific JCV epitopes. The Department is also involved in programs in concert with HIV patients’ associations, aiming to improve early diagnosis and management of HIV infection and its complications. These have included the free and anonymous administration of salivary test for HIV and HCV, an internal counseling service and structured programs of physical activity. The Department Director is the National Coordinator of the Healthy Ministry Italian Guidelines on the use of antiretroviral drugs and diagnostic-clinical management of persons with HIV-1 infection. Members of the Department are involved as committee members in the Italian guidelines on HIV infection, the European AIDS Clinical Society Guidelines for treatment of HIV infected adults and the NIH-CDC HIVMA/IDSA Guidelines for the Prevention and Treatment of Opportunistic Infections in HIV-Infected Adults and Adolescents. The Department organizes residential stages in this field for doctors coming from all Italy. In 2013 more than 40 papers have been published in international scientific journals. Adriano Lazzarin 285 THE CLINICAL DEPARTMENTS Selected publications Bagaglio, S; Messina, E; Uberti-Foppa, C; Merli, M; Della Torre, L; Lazzarin, A; Hasson, H; Morsica, G. Reversion of naturally occurring high-level resistance mutations to NS3 protease inhibitors in two treatment-naive individuals infected with hepatitis C virus. J. Antimicrob. Chemother.: 2013; 68(6): 1448-1450 - Letter Canducci, F; Ceresola, ER; Saita, D; Castagna, A; Gianotti, N; Underwood, M; Burioni, R; Lazzarin, A; Clementi, M. In vitro phenotypes to elvitegravir and dolutegravir in primary macrophages and lymphocytes of clonal recombinant viral variants selected in patients failing raltegravir. J. Antimicrob. Chemother.: 2013; 68(11): 2525-2532 - Article Clifford, DB; Nath, A; Cinque, P; Brew, BJ; Zivadinov, R; Gorelik, L; Zhao, Z; Duda, P. A study of mefloquine treatment for progressive multifocal leukoencephalopathy: Results and exploration of predictors of PML outcomes. J. Neurovirol.: 2013; 19(4): 351-358 - Article Eron, JJ; Clotet, B; Durant, J; Katlama, C; Kumar, P; Lazzarin, A; Poizot-Martin, I; Richmond, G; Soriano, V; Ait-Khaled, M; Fujiwara, T; Huang, J; Min, S; Vavro, C; Yeo, J; for the VIKING Study Group. Safety and efficacy of dolutegravir in treatment-experienced subjects with raltegravir-resistant HIV type 1 infection: 24-Week results of the VIKING study. J. Infect. Dis.: 2013; 207(5): 740-748 Article Eron, JJ; Cooper, DA; Steigbigel, RT; Clotet, B; Gatell, JM; Kumar, PN; Rockstroh, JK; Schechter, M; Markowitz, M; Yeni, P; Loutfy, MR; Lazzarin, A; Lennox, JL; Strohmaier, KM; Wan, H; Barnard, RJ; Nguyen, BY; Teppler, H; for the BENCHMRK Study Teams. Efficacy and safety of raltegravir for treatment of HIV for 5 years in the BENCHMRK studies: final results of two randomised, placebocontrolled trials. Lancet Infect. Dis.: 2013; 13(7): 587-596 - Article Ganor, Y; Drillet-Dangeard, AS; Lopalco, L; Tudor, D; Tambussi, G; Delongchamps, NB; Zerbib, M; Bomsel, M. Calcitonin gene-related peptide inhibits langerhans cell-mediated HIV-1 transmission. J. Exp. Med.: 2013; 210(11): 2161-2170 - Article Pensieroso, S; Galli, L; Nozza, S; Ruffin, N; Castagna, A; Tambussi, G; Hejdeman, B; Misciagna, D; Riva, A; Malnati, M; Chiodi, F and Scarlatti, G. B-cell subset alterations and correlated factors in HIV-1 infection. AIDS: 2013; 27(8): 1209-1217 - Article Rubenstein, JL and Wong, VS and Kadoch, C; Gao, HX; Barajas, R; Chen, L; Josephson, SA; Scott, B; Douglas, V; Maiti, M; Kaplan, LD; Treseler, PA; Cha, S; Hwang, JH; Cinque, P; Cyster, JG; Lowell, C. CXCL13 plus interleukin 10 is highly specific for the diagnosis of CNS lymphoma. Blood: 2013; 121(23): 4740-4748 - Article Spagnuolo, V and Travi, G; Galli, L; Cossarini, F; Guffanti, M; Gianotti, N; Salpietro, S; Lazzarin, A; Castagna, A. Clinical, virologic, and immunologic outcomes in lymphoma survivors and in cancer-free, HIV-1-infected patients: A matched cohort study. Cancer: 2013; 119(15): 2710-2719 Article The SPARTAC Trial Investigators. Short-course antiretroviral therapy in primary HIV infection. N. Engl. J. Med.: 2013; 368(3): 207-217 - Article MATERNAL AND CHILD HEALTH DEPARTMENT Head of Department: Giuseppe Chiumello* DEPARTMENT AREA COORDINATORS: Alessandro Aiuti, Riccardo Bonfanti, Maria Pia Guarneri Pediatrics and neonatology HEAD OF UNIT: Giuseppe Chiumello CLINICAL UNIT LEADERS: Graziano Barera, Franco Meschi, Gianni Russo, Giovanna Weber* PHYSICIANS: Valentina Biffi, Riccardo Bonfanti, Maddalena Bove, Stefania Di Candia, Gisella Garbetta, Sara Osimani, Antonella Poloniato, Gabriella Pozzobon, Andrea Rigamonti, Rosanna Rovelli, Paola Sgaramella, Maria Cristina Vigone RESEARCHER: Stefano Mora RESIDENTS: Roseila Battaglino**, Clara Bonura**, Silvana Caiulo**, Bruna Cammarata**, Patrizia Corsin**, Chiara Damia**, Marianna Di Frenna**, Alessandra Di Lascio**, Valentina Donghi**, Valeria Favalli**, Maria Piera Ferrarello**, Giulio Frontino**, Moira Gianninoto**, Elena Grechi**, Silvia Meroni**, Elena Peroni**, Sarah Rabbiosi**, Giulia Maria Tronconi** Gynaecology and obstetrics HEAD OF UNIT: Massimo Candiani* CLINICAL UNIT LEADERS: Ferdinando Bombelli, Maria Teresa Castiglioni, Enrico Conti, Giorgia Mangili, Enrico Papaleo, Daniele Spagnolo, Luca Valsecchi, Riccardo Viganò CLINICAL UNIT COORDINATORS: Guido Candotti, Gabriella Colombo, Moreno Dindelli, Stefano Ferrari, Stefania Luchini RESEARCHERS: Massimo Origoni*, Stefano Salvatore* PHYSICIANS: Giada Almirante, Luigi Caputo, Anna Cardani, Paolo Cavoretto, Patrizia De Marzi, Davide Ferrari, Luca Gandini, Elisabetta Garavaglia, Paolo Giardina, Elena Marsiglio, Fabio Mauro, Michela Molgora, Federica Pasi, Paola Persico, Micaela Petrone, Maria Teresa Potenza, Laura Privitera, Emanuela Rabaiotti, Susanna Rosa, Maddalena Smid, Simona Vailati BIOLOGISTS: Laura Corti, Lucia De Santis, Giulia Intra, Paola Panina, Elisa Rabelotti, Paola Viganò CONSULTANTS: Rossana Cairone, Raffaella Chionna, Rossana Favia, Susanna Filippis, Fiorenza Lagona, Silvia Maddalena, Guido Marelli, Valentina Mariotti, Mara Zanirato RESIDENTS: Alice Bergamini**, Guia Carminati**, Diana Del Prato**, Lara Di Piazza**, Veronica Giorgione**, Eleonora Iachini**, Annalisa Inversetti**, Jessica Ottolina**, Marta Parma**, Francesca Pella**, Lavinia Quaranta**, Audrey Serafini**, Cristina Sigismondi**, Chiara Stefani**, Iacopo Tandoi** FELLOWS: Luca Pagliardini, Umberto Leone Roberti Maggiore, Ana Maria Sanchez 287 THE CLINICAL DEPARTMENTS Pediatric immuno-hematology Unit HEAD OF UNIT: Maria Grazia Roncarolo* PHYSICIANS: Alessandro Aiuti, Rosa Bacchetta, Maria Pia Cicalese PHYSICIAN SCIENTIST SENIOR: Alessandra Biffi PHYSICIAN: Maddalena Migliavacca (since July 2013) RESIDENTS: Federica Barzaghi**, Francesca Ferrua**, Marta Frittoli**, Mila Kalapurackal** (since July 2013), Laura Lorioli**, Sara Napolitano**, Roberta Pajno** CHARGE NURSE: Clara Soliman RESEARCH NURSES: Gigliola Antonioli, Miriam Casiraghi DATA MANAGER: Laura Castagnaro (since November 2013), Sara Di Nunzio (until September 2013), Marcella Facchini (since November 2013), Emanuela Mrak (since October 2013) REGULATORY AFFAIRS OFFICE: Marco Bonopane TCTO COORDINATOR: Luciano Callegaro * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele Obstetrics and gynecology Unit The scientific and clinical activities involve: 1. Gynecological Surgery. The Unit is pilot centre for the diagnosis and treatment of endometriosis with a multidisciplinary approach and the most advanced minimally invasive surgical techniques. 2. Reproductive Sciences. The Infertility Unit completed two multicentre randomised control clinical trials on the efficacy of subcutaneous progesterone, and rFSH/rLH treatment on the outcome of ovulation induction protocols. One major research focus is the identification of new genomic biomarkers involved in controlling embryo implantation after IVF, including the HLAG antigen and its functional polymorphisms in relationship with tolerogenic cells. 3. Cancer Prevention. Two new projects have been developed in order to better deal with problems regarding fertility in female cancer patients. This collaboration between the fertility and gynecology oncology unit aims to translate these new options for preservation of fertility in oncological patients into clinical practice. Pediatrics and neonatology Neonatology The research activities involve: 1. Metabolic-clinical outcome of infants born preterm or from mothers with diabetes. We are studying the effect of specific intrauterine environments on extra-uterine growth (body composition and bone mineralization). 2. Development study of the Central Nervous System. Our group co-operates with the Department of Pediatric Neuroradiology for clinical application of Diffusion Tensor Imaging and Functional MRI and their feasibility in newborns. Diabetes, obesity and metabolic diseases in children and adolescents The areas research are: prevention of diabetes, technology in diabetes, genetic-metabolic studies in neonatal insulin resistant diabetes. In cooperation with Bambino Gesù Hospital (Rome) we are studying phenotype-genotype correlations in insulin-resistant diabetes due to insulin receptor gene mutations and incidence of neonatal/infancy onset diabetes in Italy. We collaborate in Trialnet primary and secondary prevention study, and with Diabetes Research Institute in evaluating the role of innate immunity in type 1 diabetes . Clinical research regarding Prader-Willi Syndrome (PWS): • effects of long-term GH therapy on sleep-disordered breathing and adenotonsillar hypertrophy • prevalence of central adrenal insufficiency THE CLINICAL DEPARTMENTS We also studied the clinical/molecular features of patients with congenital hyperinsulinism and their parents, allowing the identification of the potential causative gene mutations. Pediatric endocrinology The fields of research are thyroid diseases, short stature, Congenital Adrenal Hyperplasia (CAH), Hypogonadotrophic Hypogonadism (HH) and Disorders of sex differentiation (DSD). Regarding Congenital Hypothyroidism (CH) we collaborate with the University of Milan to assess the genetic, epigenetic and environmental factors contributing to CH. As co-investigator of AIFA Research we investigate the influence of initial Levothyroxine dose at diagnosis on neurodevelopmental outcomes in children with CH. We also studied the response to Growth Hormone in terms of growth velocity and short term height and its correlation to genotype in Noonan patients. We assess serum, salivary and urinary steroid profiles through LC-MS for diagnosis and follow-up of various forms of CAH. We study patients with HH from a clinical/genetic perspective. We aim to standardize pharmacologic tests execution and results interpretation in order to obtain a better diagnostic definition, together with genetic analysis. Pediatric immuno-hematology Unit Clinical research involves immunological, hematological, autoimmune, lysosomal storage disorders and other genetic diseases. Experimental therapeutic options are also offered. We performed more than 70 pediatric allogeneic stem cell transplantations for thalassemia, sickle cell anemia, primary immunodeficiencies and malignancies, in cooperation with the San Raffaele Stem Cell Programme. The Unit collaborates with HSR-TIGET on advanced diagnosis, pathogenetic studies and development of novel therapeutic approaches for genetic diseases, with particular regards to primary immunodeficiencies, autoimmune diseases with known or unknown genetic defect, lysosomal storage disorders and hemoglobinopathies. Results from long-term follow up in ADA-SCID patients treated with retroviral-mediated gene therapy confirm its efficacy and safety. Initial results of clinical trials for Metachromatic Leukodystrophy and WiskottAldrich Syndrome have shown that the infusion of transduced hematopoietic stem cells is safe and efficacious. Patients affected by Duchenne muscular dystrophy have been treated with cell therapy and their follow up is currently ongoing. Clinical trials of gene therapy for β-thalassemia and Mucopolisaccaridosis I are in preparation. Giuseppe Chiumello Selected publications Aiuti, A; Biasco, L and Scaramuzza, S; Ferrua, F; Cicalese, MP; Baricordi, C; Dionisio, F; Calabria, A; Giannelli, S; Castiello, MC; Bosticardo, M; Evangelio, C; Assanelli, A; Casiraghi, M; Di Nunzio, S; Callegaro, L; Benati, C; Rizzardi, P; Pellin, D; Di Serio, C; Schmidt, M; Von Kalle, C; Gardner, J; Mehta, N; Neduva, V; Dow, DJ; Galy, A; Miniero, R; Finocchi, A; Metin, A; Banerjee, P; Orange, J; Galimberti, S; Valsecchi, MG; Biffi, A; Montini, E; Villa, A; Ciceri, F; Roncarolo, MG and Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome. Science: 2013; 341(6148): 123351 - Article Berini, J; Spica Russotto, V; Castelnuovo, P; Di Candia, S; Gargantini, L; Grugni, G; Iughetti, L; Nespoli, L; Nosetti, L; Padoan, G; Pilotta, A; Trifiro, G; Chiumello, G; Salvatoni, A; on behalf of the Genetic Obesity Study Group of the Italian Society of Pediatric Endocrinology and Diabetology (ISPED). Growth hormone therapy and respiratory disorders: Long-term follow-up in PWS children. J. Clin. Endocrinol. Metab.: 2013; 98(9): E1516-E1523 - Article Biffi, A and Montini, E; Lorioli, L; Cesani, M; Fumagalli, F; Plati, T; Baldoli, C; Martino, S; Calabria, A; Canale, S; Benedicenti, F; Vallanti, G; Biasco, L; Leo, S; Kabbara, N; Zanetti, G; Rizzo, WB; Mehta, NA; Cicalese, MP; Casiraghi, M; Boelens, JJ; Del Carro, U; Dow, DJ; Schmidt, M; Assanelli, A; Neduva, V; Di Serio, C; Stupka, E; Gardner, J; von Kalle, C; Bordignon, C; Ciceri, F; Rovelli, A; Roncarolo, MG; Aiuti, A; Sessa, M; Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy. Science: 2013; 341(6148): 1233158 - Article 289 THE CLINICAL DEPARTMENTS Goudy, K and Aydin, D; Barzaghi, F; Gambineri, E; Vignoli, M; Mannurita, SC; Doglioni, C; Ponzoni, M; Cicalese, MP; Assanelli, A; Tommasini, A; Brigida, I; Dellepiane, RM; Martino, S; Olek, S; Aiuti, A; Ciceri, F; Roncarolo, MG; Bacchetta, R. Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity. Clin. Immunol.: 2013; 146(3): 248-261 Article Mangili, G; Ottolina, J; Gadducci, A; Giorda, G; Breda, E; Savarese, A; Candiani, M; Frigerio, L; Scarfone, G; Pignata, S; Rossi, R; Marinaccio, M; Lorusso, D. Long-term follow-up is crucial after treatment for granulosa cell tumours of the ovary. Br. J. Cancer: 2013; 109(1): 29-34 - Article Pagliardini, L; Gentilini, D; Viganò, P; Panina-Bordignon, P; Busacca, M; Candiani, M; Di Blasio, AM. An Italian association study and meta-analysis with previous GWAS confirm WNT4, CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis. J. Med. Genet.: 2013; 50(1): 43-46 - Article Rabbiosi, S; Vigone, MC; Cortinovis, F; Zamproni, I; Fugazzola, L; Persani, L; Corbetta, C; Chiumello, G; Weber, G. Congenital hypothyroidism with eutopic thyroid gland: Analysis of clinical and biochemical features at diagnosis and after re-evaluation. J. Clin. Endocrinol. Metab.: 2013; 98(4): 1395-1402 - Article Sanchez, AM; Giorgione, V; Viganò, P; Papaleo, E; Candiani, M; Mangili, G; Panina-Bordignon, P. Treatment with anticancer agents induces dysregulation of specific Wnt signaling pathways in human ovarian luteinized granulosa cells in vitro. Toxicol. Sci.: 2013; 136(1): 183-192 - Article Valle, A and Giamporcaro, GM and Scavini, M; Stabilini, A; Grogan, P; Bianconi, E; Sebastiani, G; Masini, M; Maugeri, N; Porretti, L; Bonfanti, R; Meschi, F; De Pellegrin, M; Lesma, A; Rossini, S; Piemonti, L; Marchetti, P; Dotta, F; Bosi, E; Battaglia, M. Reduction of circulating neutrophils precedes and accompanies type 1 diabetes. Diabetes: 2013; 62(6): 2072-2077 - Article DEPARTMENT OF INTERNAL AND SPECIALISTIC MEDICINE Head of Department: Emanuele Bosi* DEPARTMENT AREA COORDINATORS: Giselda Colombo, Matteo Rocco Pastore, Moreno Tresoldi General medicine, diabetes, endocrinology and metabolic diseases HEAD OF UNIT: Emanuele Bosi* CLINICAL UNIT LEADERS: Gabriella Galimberti, Roberto Lanzi, Marco Federico Manzoni, Matteo Rocco Pastore, Piermarco Piatti PHYSICIANS: Alberto Davalli, Sabina Martinenghi, Alessandro Saibene, Maurizio Storti RESIDENTS: Andrea Bolla**, Amelia Caretto**, Anna Dolcetta Capuzzo**, Ilaria Formenti**, Laura Frosio**, Alessandra Gandolfi**, Tania Garito**, Chiara Molinari**, Valentina Villa** FELLOWS: Valentina Giulia Crippa, Raffaele Di Fenza, Andrea Laurenzi, Francesca Perticone, Alessandro Rossini, Emanuela Setola NUTRITIONIST: Monica Marchi TRIAL COORDINATOR: Pauline Grogan RESEARCH NURSE: Eleonora Bianconi Clinical transplantation HEAD OF UNIT: Antonio Secchi* CLINICAL UNIT LEADERS: Rossana Caldara, Ennio La Rocca, Paola Maffi CONSULTANTS: Chiara Gremizzi, Vera Paloschi RESIDENTS: Francesca D’Addio, Silvia Foti RESEARCH NURSE: Barbara Pontiroli General medicine, clinical immunology and rheumatology HEAD OF UNIT: Maria Grazia Sabbadini* CLINICAL UNIT LEADERS: Giselda Colombo, Luisa Praderio, Moreno Tresoldi PHYSICIANS: Enrica P. Bozzolo, Lorenzo Dagna*, Angelo A. Manfredi*, Massimo Memoli, Patrizia Rovere-Querini*, Chiara Salmaggi, Nicoletta Saporiti, Raffaella Scotti, Magda Vecellio CONSULTANTS: Patrizia Aiello, Elena Baldissera, Teresa D’Aliberti, Mirta Tiraboschi RESIDENTS: Nicola Bettera, Margherita Bevilacqua, Andrea Duca, Luca Ferrante, Andrea Segalini, Cristina Sorlini, Enrico Tombetti 291 THE CLINICAL DEPARTMENTS Nephrology and dialysis HEAD OF UNIT: Donatella Spotti CLINICAL UNIT LEADERS: Paolo Manunta*, Giorgio Slaviero, Giuseppe Vezzoli PHYSICIANS: Teresa Arcidiacono, Chiara Lanzani, Marco Melandri, Rita Quartagno, Maria Teresa Sciarrone Alibrandi, Marco Simonini RESIDENTS: Guido Gatti**, Lino Merlino**, Marina Nuzzo**, Simona Pozzoli**, Stefano Tentori**, Francesco Trevisani** * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The Department of Internal Medicine incorporates all the areas and clinical activities of General and Specialized Medicine of the San Raffaele Hospital and Scientific Institute. Within the Department, physicians, nurses, technicians, students and volunteers are dedicated to patients clinical care, with a continuum of treatments encompassing preventive medicine, acute care, palliative therapies and rehabilitation. The objective of the Department is to integrate clinical care, research and education with the aim of assisting patients at the best of current medical knowledge and technological expertise. Clinical activity and Areas of excellence The Department of Internal Medicine is composed of four inpatient Clinical Units, a Day Hospital Service and many Outpatient Clinics covering General Medicine and the medical specialties of Allergology, Clinical Immunology, Clinical Transplantation, Diabetes, Dialysis, Endocrinology, Hypertension, Metabolic Diseases, Nephrology, Nutrition and Rheumatology. Moreover, the Department of Internal Medicine works in close interaction with the Emergency Department, representing the main structure for hospitalization of patients presenting at the Emergency Medicine. The Department of Internal Medicine offers a complete range of teaching for students at the Medical School and hosts the Post Graduate Medical Schools of Allergology and Clinical Immunology, Endocrinology, Nephrology and Emergency Medicine, being also recognized as a centre of excellence in these specific fields. Fields of research Integration between clinical care and research is a general theme across the Department. Relevant activities include: phase II/III clinical trials on novel pharmacological treatments and diagnostic tools in the fields of diabetes, rheumatoid arthritis, metabolism, hypertension, cardiovascular and immune-mediated diseases; and some important projects of translational medicine in the fields of islet transplantation and immunotherapies of type 1 diabetes and other autoimmune diseases. The Department is embedded in a remarkable clinical and scientific environment at San Raffaele, which offers unique opportunities for interdisciplinary collaboration and translational research. The scientific production by physicians and clinical investigators from the Department is remarkable, with internationally recognized areas of excellence in diabetes and metabolism, clinical immunology, hypertension, islet and pancreas transplantation. Emanuele Bosi Selected publications Bosi, E; Bax, G; Scionti, L; Spallone, V; Tesfaye, S; Valensi, P; Ziegler, D; on behalf of the FREMS European Trial Study Group. Frequency-modulated electromagnetic neural stimulation (FREMS) as a treatment for symptomatic diabetic neuropathy: results from a double-blind, randomised, multicentre, long-term, placebo-controlled clinical trial. Diabetologia: 2013; 56(3): 467-475 - Article Bosi, E; Scavini, M; Ceriello, A; Cucinotta, D; Tiengo, A; Marino, R; Bonizzoni, E; Giorgino, F; on behalf of the PRISMA STUDY GROUP. Intensive structured self-monitoring of blood glucose and THE CLINICAL DEPARTMENTS glycemic control in noninsulin-treated type 2 diabetes: the PRISMA randomized trial. Diabetes Care: 2013; 36(10): 2887-2894 - Article Bozzolo, EP; Ramirez, GA; Bonavida, G; Lanzani, C; Scotti, R; Dell’Antonio, G; Baldissera, E; Canti, V; Manfredi, AA; Rovere-Querini, P; Sabbadini, MG. Efficacy and toxicity of treatments for nephritis in a series of consecutive lupus patients. Autoimmunity: 2013; 46(8): 537-546 - Article Della-Torre, E; Passerini, G; Furlan, R; Roveri, L; Chieffo, R; Anzalone, N; Doglioni, C; Zardini, E; Sabbadini, MG; Franciotta, D. Cerebrospinal fluid analysis in immunoglobulin G4-related hypertrophic pachymeningitis. J. Rheumatol.: 2013; 40(11): 1927-1929 - Letter Dogliotti, E; Vezzoli, G; Nouvenne, A; Meschi, T; Terranegra, A; Mingione, A; Brasacchio, C; Raspini, B; Cusi, D; Soldati, L. Nutrition in calcium nephrolithiasis. J. Transl. Med.: 2013; 11: 109 - Review Maffi, P and Balzano, G; Ponzoni, M; Nano, R; Sordi, V; Melzi, R; Mercalli, A; Scavini, M; Esposito, A; Peccatori, J; Cantarelli, E; Messina, C; Bernardi, M; Del Maschio, A; Staudacher, C; Doglioni, C; Ciceri, F; Secchi, A; Piemonti, L. Autologous Pancreatic Islet Transplantation in Human Bone Marrow. Diabetes: 2013; 62(10): 3523-3531 - Article Pierobon, ES; Sandrini, S; De Fazio, N; Rossini, G; Fontana, I; Boschiero, L; Gropuzzo, M; Gotti, E; Donati, D; Minetti, E; Gandolfo, MT; Brunello, A; Libetta, C; Secchi, A; Chiaramonte, S; Rigotti, P. Optimizing utilization of kidneys from deceased donors over 60 years: Five-year outcomes after implementation of a combined clinical and histological allocation algorithm. Transplant Int.: 2013; 26(8): 833-841 - Article Vezzoli, G and Terranegra, A and Aloia, A; Arcidiacono, T; Milanesi, L; Mosca, E; Mingione, A; Spotti, D; Cusi, D; Hou, J; Hendy, GN; Soldati, L. Decreased transcriptional activity of calciumsensing receptor gene promoter 1 is associated with calcium nephrolithiasis. J. Clin. Endocrinol. Metab.: 2013; 98(9): 3839-3847 - Article 293 DEPARTMENT OF CLINICAL NEUROSCIENCE Head of Department: Enrico Smeraldi* DEPARTMENT AREA COORDINATORS: Barbara Barbini, Francesco Benedetti, Maria Cristina Cavallini, Ernestina Politi, Paolo Ronchi General psychiatry HEAD OF UNIT: Enrico Smeraldi* CLINICAL UNIT LEADERS: Ernestina Politi, Paolo Ronchi PHYSICIANS: Sara Dallaspezia, Marta Henin, Francesca Siliprandi, Laura Sforzini PSYCHOLOGISTS: Vittoria Bottelli, Daniele Cavadini, Tomaso Siccardi, Cecilia Smeraldi RESIDENTS: Giampiero Bottero**, Valentina Ferrari**, Antonella Rita Mastromatteo** Clinical health psychology HEAD OF UNIT: Lucio Sarno* PSYCHOLOGISTS: Letizia Carnelli, Valentina Di Mattei*, Claudia Yvonne Finocchiaro, Camilla Giulia Andrea Ghidini, Serena Giuliani, Luca Leardini, Maria Rita Milesi, Chiara Motta, Liliana Novella, Sara Peluso, Maria Monica Ratti RESIDENT: Stefano Clerici Clinical psychology and psychotherapy HEAD OF UNIT: Cesare Maffei* CLINICAL UNIT LEADERS: Mariagrazia Movalli, Laura Vanzulli CLINICAL UNIT COORDINATORS: Andrea Fossati*, Raffaele Visintini CONSULTANTS: Roberta Alesiani, Silvia Boccalon, Serena Borroni, Paola Broggi, Elena Campanini, Ilaria Carretta, Paolo Casati, Rosaria Devoti, Michela Donini, Cinzia Facchi, Marina Fiore, Nicolò Gaj, Laura Giarolli, Salvatore La Viola, Alessandro Pieri, Sergio Premoli, Martina Testa, Roberto Vanni, Daniele Villa EXTERNAL RESIDENTS: Serena Artesani, Antonella Di Biase, Michela Adele Pozzi, Emanuela Roder, Manuela Sanvito, Edoardo Vassallo Neurology HEAD OF UNIT: Stefano F. Cappa* (until October 2013), Sandro Iannaccone (since November 2013) SCIENTIFIC CONSULTANT: Stefano F. Cappa*(since November 2013) CLINICAL UNIT LEADER: Alessandra Marcone RESEARCHERS: Jubin Abutalebi*, Nicola Canessa*, Chiara Cerami PHYSICIANS: Maria Cristina Giusti, Valeria Golzi, Barbara Sferrazza, Michele Zamboni PSYCHOLOGISTS: Federica Alemanno, Giada Caramatti, Valentina Esposito THE CLINICAL DEPARTMENTS Eating disorders HEAD OF UNIT: Laura Bellodi* CLINICAL UNIT LEADER: Daniela Di Molfetta, Stefano Erzegovesi CLINICAL UNIT COORDINATOR: Maria Cristina Cavallini PHYSICIAN: Alessandro Bernasconi Mood disorders HEAD OF UNIT: Cristina Colombo* CLINICAL UNIT LEADERS: Linda Franchini, Marco Locatelli, Raffaella Zanardi PHYSICIANS: Barbara Barbini, Francesco Benedetti, Euridice Campori, Danilo Dotoli, David Rossini RESIDENTS: Silvia Brioschi**, Dario Delmonte**, Charlotte Desantis**, Clara Locatelli** Psychotic disorders disease Unit HEAD OF UNIT: Roberto Cavallaro PHYSICIANS: Laura Bianchi, Marta Bosia** RESIDENT: Marco Spangaro** PSYCHOLOGIST: Margherita Bechi Sleep disorders HEAD OF UNIT: Luigi Ferini-Strambi* PHYSICIANS: Alessandro Oldani, Marco Zucconi PSYCHOLOGISTS: Vincenza Elena Castronovo, Laura Giarolli, Sara Marelli TECHNICIANS: Massimo Antonio, Daniele Bizzozero, Elisa Dallabà, Cristina Martinelli Out patients services (Ambulatory and day care) HEAD OF UNIT: Enrico Smeraldi* Alcoholism HEADS: Enrico Smeraldi*, Cesare Maffei* CLINICAL UNIT LEADERS: Sara Angelone, Mariagrazia Movalli RESIDENT: Angelo Notaristefano** Eating disorders HEAD: Laura Bellodi* PHYSICIAN: Cinzia Arancio Anxiety disorders HEAD: Laura Bellodi* PHYSICIANS: Marco Catalano, Silvia Cocchi, Angela Gabriele FELLOWS: Emma Fadda, Elisa Galimberti, Riccardo Martoni Psychotic disorders HEAD: Roberto Cavallaro CLINICAL UNIT LEADER: Federica Cocchi PHYSICIAN: Carmelo Guglielmino PSYCHOLOGIST: Maria Chiara Buonocore Personality disorders HEAD: Cesare Maffei CLINICAL UNIT LEADER: Raffaele Visintini PHYSICIAN: Laura Sforzini 295 THE CLINICAL DEPARTMENTS Mood disorders HEAD: Cristina Colombo* CLINICAL UNIT LEADERS: Linda Franchini, Marco Locatelli, Raffaella Zanardi PHYSICIANS: Barbara Barbini, Francesco Benedetti, Euridice Campori, Danilo Dotoli, David Rossini Obsessive compulsive disorders HEAD: Enrico Smeraldi* PHYSICIANS: Sara Dallaspezia, Marta Henin PSYCHOLOGISTS: Giulia Mazza, Cecilia Smeraldi RESIDENT: Irene Vanelli** * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The first and main committment of the Department is to define and to develop common guideline for the clinical approach to the different aspects of behavioural disorders, according to the funding principles of our Institution, in order to overcome the strict meaning of each symptom and to adequately consider the nature and the whole of the individual suffering. The public psychiatric structure is based on the model of Mental Health Department (DSM). Our department, in its psychiatric and psychological component, is based on long term therapeutical assistance, hence resembling DSM and their mode of function, but it differs from DSM in its objectives. In DSM, the main aim is to take care of the mental health of a community (differently defined) and it must be addressed in terms of prevention too. Treating patients in wards dedicated to acute treatment for strictly necessary periods and relying on ambulatory and residential/semiresidential servicesfor th remaing part of the therapeutical process is a choice which can be considered also as a therapy in social environment (= territory, in the language of social psychiatry), that will be useful and produce mental health. The activity of our Department is different in that it is mainly focused on the individual suffering and is addressed to the patient: the possible interest for the environment in which he lives or for his relationships is only aimed at improving assistance and therapeutical approach to the patient, who freely chooses our structure instead of the public one, even if located in his territory. The relationship between these two types of Psychiatric Departments has not yet been established and we are trying to find a different model of assistance according to the psychopathological “quality” of each disease. Translational research is a proved reality in our department with results of basic and clinical research rapidly transferred to everyday treatment protocols. In the opposite direction clinical activity is a constant inspiration for new research with the aim to continuously ‘raise the bar’ of possible treatment efficacy, in a virtuous circle of which the primary beneficiary is the patient. Another innovative feature is to put together in the same Department cognitive neurology and psychiatry, since behavioural disorders are frequent and common and the same therapeutical principles can be applied. Clinical activity and Areas of excellence Following the principle that expertise is based on the widest experience possible in a selected area of treatment/research, the clinical structure relies on disease-dedicated units: • Mood disorders • Psychotic disorders • Anxiety disorders • Eating disorders • Personality disorders • Alcohol abuse We have 120 beds for acute treatment and rehabilitation treatment where the therapeutical environment in which all mental health workers operate is studied and maintained to optimize pharmacological and non THE CLINICAL DEPARTMENTS pharmacological treatments. The way in which treatments are delivered is in our principles the key feature for a successful therapy and is, then, a part of them. Ambulatory and day-care activities are similarly structured as activities of the disease-dedicated units. Fields of research The department produces research in all the neurosciences areas encompassed by the activity of the disease-dedicated units, with the inspiring ‘leit motiv’ of getting results for personalized medicine rather than for ‘average medicine’. This is the consequence of the strong psychiatric genetic vocation on which the department was founded and that is easily readable in any research program and its results publication. Areas of recognized excellence are: • Molecular Biology and Genetics • Brain imaging • Clinical Psychopharmacology • Cronobiology • Neuropsychology • Neurocognitive remediation and rehabilitation Enrico Smeraldi Selected publications Bechi, M; Spangaro, M; Bosia, M; Zanoletti, A; Fresi, F; Buonocore, M; Cocchi, F; Guglielmino, C; Smeraldi, E; Cavallaro, R. Theory of Mind intervention for outpatients with schizophrenia. Neuropsychol. Rehabil.: 2013; 23(3): 383-400 - Article Benedetti, F; Bollettini, I; Barberi, I; Radaelli, D; Poletti, S; Locatelli, C; Pirovano, A; Lorenzi, C; Falini, A; Colombo, C; Smeraldi, E. Lithium and GSK3-β promoter gene variants influence white matter microstructure in bipolar disorder. Neuropsychopharmacology: 2013; 38(2): 313-327 - Article Benedetti, F; Giacosa, C; Radaelli, D; Poletti, S; Pozzi, E; Dallaspezia, S; Falini, A; Smeraldi, E. Widespread changes of white matter microstructure in obsessive-compulsive disorder: Effect of drug status. Eur. Neuropsychopharmacol.: 2013; 23(7): 581-593 - Article Canessa, N; Crespi, C; Motterlini, M; Baud-Bovy, G; Chierchia, G; Pantaleo, G; Tettamanti, M; Cappa, SF. The functional and structural neural basis of individual differences in loss aversion. J. Neurosci.: 2013; 33(36): 14307-14317 - Article Carlotta, D; Borroni, S; Maffei, C; Fossati, A. On the relationship between retrospective childhood ADHD symptoms and adult BPD features: The mediating role of action-oriented personality traits. Compr. Psychiatry: 2013; 54(7): 943-952 - Article Dauvilliers, Y; Postuma, RB; Ferini-Strambi, L; Arnulf, I; Hogl, B; Manni, R; Miyamoto, T; Oertel, W; Fantini, ML; Puligheddu, M; Jennum, P; Sonka, K; Zucconi, M; Leu-Semenescu, S; Frauscher, B; Terzaghi, M; Miyamoto, M; Unger, M; Desautels, A; Wolfson, C; Pelletier, A; Montplaisir, J. Family history of idiopathic REM behavior disorder a multicenter case-control study. Neurology: 2013; 80(24): 2233-2235 - Article Davis, LK; Yu, D; Keenan, CL; Gamazon, ER; Konkashbaev, AI; Derks, EM; Neale, BM; Yang, J; Lee, SH; Evans, P; Barr, CL; Bellodi, L; Benarroch, F; Berrio, GB; Bienvenu, OJ; Bloch, MH; Blom, RM; Bruun, RD; Budman, CL; Camarena, B; Campbell, D; Cappi, C; Cardona Silgado, JC; Cath, DC; Cavallini, MC; Chavira, DA; Chouinard, S; Conti, DV; Cook, EH; Coric, V; Cullen, BA; Deforce, D; Delorme, R; Dion, Y; Edlund, CK; Egberts, K; Falkai, P; Fernandez, TV; Gallagher, PJ; Garrido, H; Geller, D; Girard, SL; Grabe, HJ; Grados, MA; Greenberg, BD; Gross-Tsur, V; Haddad, S; Heiman, GA; Hemmings, SMJ; Hounie, AG; Illmann, C; Jankovic, J; Jenike, MA; Kennedy, JL; King, RA; Kremeyer, B; Kurlan, R; Lanzagorta, N; Leboyer, M; Leckman, JF; Lennertz, L; Liu, C; Lochner, C; Lowe, TL; Macciardi, F; McCracken, JT; McGrath, LM; Mesa Restrepo, SC; Moessner, R; Morgan, J; Muller, H; Murphy, DL; Naarden, AL; Ochoa, WC; Ophoff, RA; Osiecki, L; Pakstis, AJ; Pato, MT; Pato, CN; Piacentini, J; Pittenger, C; Pollak, Y; Rauch, SL; Renner, TJ; Reus, VI; Richter, MA; Riddle, 297 THE CLINICAL DEPARTMENTS MA; Robertson, MM; Romero, R; Rosario, MC; Rosenberg, D; Rouleau, GA; Ruhrmann, S; Ruiz-Linares, A; Sampaio, AS; Samuels, J; Sandor, P; Sheppard, B; Singer, HS; Smit, JH; Stein, DJ; Strengman, E; Tischfield, JA; Valencia Duarte, AV; Vallada, H; Van Nieuwerburgh, F; Veenstra-VanderWeele, J; Walitza, S; Wang, Y; Wendland, JR; Westenberg, HGM; Shugart, YY; Miguel, EC; McMahon, W; Wagner, M; Nicolini, H; Posthuma, D; Hanna, GL; Heutink, P; Denys, D; Arnold, PD; Oostra, BA; Nestadt, G; Freimer, NB; Pauls, DL; Wray, NR; Stewart, SE and Mathews, CA and Knowles, JA and Cox, NJ and Scharf, JM. Partitioning the Heritability of Tourette Syndrome and Obsessive Compulsive Disorder Reveals Differences in Genetic Architecture. PLoS Genet.: 2013; 9(10): e1003864 - Article Fadda, E; Galimberti, E; Cammino, S; Bellodi, L. Smoking, physical activity and respiratory irregularities in patients with panic disorder. Riv. Psichiatr.: 2013; 48(4): 293-300 - Article Ferini-Strambi, L. A need for new treatments in narcolepsy. Lancet Neurol: 2013; 12(11): 10391040 - Comment Fossati, A; Krueger, RF; Markon, KE; Borroni, S; Maffei, C. Reliability and Validity of the Personality Inventory for DSM-5 (PID-5): Predicting DSM-IV Personality Disorders and Psychopathy in Community-Dwelling Italian Adults. Assessment: 2013; 20(6): 689-708 - Article Innocenti, I; Cappa, SF; Feurra, M; Giovannelli, F; Santarnecchi, E; Bianco, G; Cincotta, M; Rossi, S. TMS interference with primacy and recency mechanisms reveals bimodal episodic encoding in the human brain. J. Cogn. Neurosci.: 2013; 25(1): 109-116 - Article Poletti, S; Radaelli, D; Cavallaro, R; Bosia, M; Lorenzi, C; Pirovano, A; Smeraldi, E; Benedetti, F. Catechol-O-methyltransferase (COMT) genotype biases neural correlates of empathy and perceived personal distress in schizophrenia. Compr. Psychiatry: 2013; 54(2): 181-186 - Article Sarno, L; Di Mattei, V; Ratti, MM; Franchini, E; Ferrara, S; Rossi, A; Finocchiaro, CY; Grimoldi, M. The rule of health psychology Services and their diffusion in Lombardia’s hospitals: main activities and intervention setting. 27th Conference of the European Health Psychology Society, Bordeaux, France, 16-20 July 2013, Proceedings Sarno, L; Ratti, MM; Delli Zotti, GB; Rossi, A; Franchini, E; Ferrara, S; Spotti, D. Quality of life and psychological issues of hemodialyzed patients. 27th Conference of the European Health Psychology Society, Bordeaux, France, 16-20 July 2013, Proceedings Vita, A; De Peri, L; Siracusano, A; Sacchetti, E; for the ATOM group. Efficacy and tolerability of asenapine for acute mania in bipolar I disorder: meta-analyses of randomized-controlled trials. Int Clin Psychopharmacol: 2013; 28(5): 219-227 - Review Zanardi, R; Colombo, L; Marcheggiani, E; Rossini, D; Delmonte, D; Cigala Fulgosi, M; Gavinelli, C; Colombo, C. Paroxetine drops versus paroxetine tablets: Evaluation of compliance in a sixmonth study. Riv. Psichiatr.: 2013; 48(3): 261-267 - Article DEPARTMENT OF NEUROLOGY Head of Department: Giancarlo Comi* CLINICAL UNIT LEADERS: Mauro Comola, Ubaldo Del Carro, Vittorio Martinelli, Fabio Minicucci DISEASE UNIT COORDINATORS: Bruno Colombo, Raffaella Fazio, Letizia Leocani, Giuseppe Magnani, Paolo Marchettini, Vittorio Martinelli, Lucia Moiola, Mariaemma Rodegher, Maria Sessa, Giulio Truci, Maria Antonietta Volonté PHYSICIANS: Stefano Amadio, Marco Bacigaluppi, Federica Cerri**, Raffaella Chieffo**, Maria Grazia Deriu**, Federica Esposito**, Giovanna Franca Fanelli, Francesca Fumagalli**, Roberta Guerriero**, Silvia Mammi, Filippo Martinelli-Boneschi, Stefania Medaglini, Maria Grazia Natali Sora, Grazia Maria Nuzzaco**, Antonella Poggi, Marta Radaelli, Nilo Riva, Luisa Roveri, Marina Scarlato, Francesca Spagnolo** RESIDENTS: Martina Absinta**, Valeria Barcella**, Damiano Baroncini**, Francesca Bianchi**, Francesca Caso**, Elisabetta Coppi**, Gloria Dalla Costa**, Dacia Dalla Libera**, Donatella De Feo**, Giovanni Di Maggio**, Laura Ferrari**, Laura Ferrè**, Mario Fichera**, Sebastiano Galantucci**, Giacomo Giacalone**, Clara Guaschino**, Matteo Impellizzeri**, Sara La Gioia**, Emanuela Leopizzi**, Giuseppe Liberatore**, Giulia Longoni**, Ignazio Diego Lopez**, Arianna Merlini**, Maria Josè Messina**, Roberta Messina**, Arturo Nuara**, Giulia Pavan**, Luca Peruzzotti Jametti**, Paolo Preziosa**, Marzia Romeo**, Francesca Sangalli**, Roberto Santangelo**, Lidia Sarro**, Edoardo Gioele Spinelli**, Laura Straffi**, Davide Strambo**, Daniele Velardo** * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The Department of Neurology consists of the Neurology Unit (45 beds), the Neurorehabilitation Unit (42 beds), the laboratory of Clinical Neurophysiology and the Neuropsychology Service. The out patient area, day hospital service and the Centres for Epilepsy, Multiple Sclerosis, Headache, Pain, ALS and Multiple Sclerosis are the other activity characterising the Department. Clinical activities are organized in disease units in order to provide patients an integrated assistance going from the diagnostic aspects to the advanced therapeutic interventions, including rehabilitation. The Department of Neurology is mostly qualified for advanced treatment strategies in inflammatory diseases of the Central and Peripheral Nervous System, stroke, neurodegenerative disorders and is the clinical arm of the Institute of Experimental Neurology. A large number of phase I-IV clinical trials performed in cooperation with pharmaceutical industries and generated by the Department itself make it possible to provide patients with more advanced therapeutic strategies. Points of excellence of the clinical research are etiologic and symptomatic treatment of multiple sclerosis, epidemiological studies on multiple sclerosis, and the evaluation of the disease pathogenesis and pathophysiology. Ischemic stroke researches are focused on the risk factors for the occurrence in young adults and on the organisational aspect of the early intervention. Studies on de mentia are conducted in co-operation with Imaging and clinical Neurophysiology laboratories and aims 299 THE CLINICAL DEPARTMENTS to define biomarkers specific for dementia subtypes and to assess the risk for evolution to Alzheimer Disease in patients with minimal cognitive impairment. In Amyotrophic Lateral Sclerosis the contribution of instrumental test to the early diagnosis, the characterisation of cognitive dysfunction, the assessment of the value of new treatments are the key research activities. The recovery medicine is one of the more recent area of research activated in the Department, because of the possibility to follow neurological dysfunctions due to acute central nervous system lesions, from the onset to the recovery phase. The impact of various therapeutic strategies to enhance recovery, the functional and molecular bases underlying brain plasticity after acute damage with imaging and neurophysiological examinations are deeply investigated. Giancarlo Comi Selected publications Agosta, F; Sala, S; Valsasina, P; Meani, A; Canu, E; Magnani, G; Cappa, SF; Scola, E; Quatto, P; Horsfield, MA; Falini, A; Comi, G; Filippi, M. Brain network connectivity assessed using graph theory in frontotemporal dementia. Neurology: 2013; 81(2): 134-143 - Article Cambiaghi, M and Cursi, M and Magri, L; Castoldi, V; Comi, G; Minicucci, F; Galli, R; Leocani, L. Behavioural and EEG effects of chronic rapamycin treatment in a mouse model of Tuberous Sclerosis Complex. Neuropharmacology: 2013; 67: 1-7 - Article Canu, E and Agosta, F; Spinelli, EG; Magnani, G; Marcone, A; Scola, E; Falautano, M; Comi, G; Falini, A; Filippi, M. White matter microstructural damage in Alzheimer’s disease at different ages of onset. Neurobiol. Aging: 2013; 34(10): 2331-2340 - Article Caso, F; Onofrio, F; Falautano, M; Todeschini, P; Migliaccio, R; Comi, G; Perani, D; Magnani, G. From primary progressive aphasia to corticobasal syndrome: two clinical and rCBF functional reports. Neurocase: 2013; 19(2): 201-207 - Article Comi, G. Disease-modifying treatments for progressive multiple sclerosis. Mult. Scler.: 2013; 19(11): 1428-1436 - Review Comi, G; Martinelli, V; Rodegher, M; Moiola, L; Leocani, L; Bajenaru, O; Carra, A; Elovaara, I; Fazekas, F; Hartung, HP; Hillert, J; King, J; Komoly, S; Lubetzki, C; Montalban, X; Myhr, KM; Preziosa, P; Ravnborg, M; Rieckmann, P; Rocca, MA; Wynn, D; Young, C; Filippi, M. Effects of early treatment with glatiramer acetate in patients with clinically isolated syndrome. Mult. Scler.: 2013; 19(8): 1074-1078 - Article Dalla Costa, G; Colombo, B; Dalla Libera, D; Martinelli, V; Comi, G. Parry Romberg Syndrome associated with chronic facial pain. J. Clin. Neurosci.: 2013; 20(9): 1320-1322 - Article Filippi, M; Agosta, F; Scola, E; Canu, E; Magnani, G; Marcone, A; Valsasina, P; Caso, F; Copetti, M; Comi, G; Cappa, SF; Falini, A. Functional network connectivity in the behavioral variant of frontotemporal dementia. Cortex: 2013; 49(9): 2389-2401 - Article Martinelli-Boneschi, F; Giacalone, G; Magnani, G; Biella, G; Coppi, E; Santangelo, R; Brambilla, P; Esposito, F; Lupoli, S; Clerici, F; Benussi, L; Ghidoni, R; Galimberti, D; Squitti, R; Confaloni, A; Bruno, G; Pichler, S; Mayhaus, M; Riemenschneider, M; Mariani, C; Comi, G; Scarpini, E; Binetti, G; Forloni, G; Franceschi, M; Albani, D. Pharmacogenomics in Alzheimer’s disease: A genome-wide association study of response to cholinesterase inhibitors. Neurobiol. Aging: 2013; 34(6): 1711.e71711.e13 - Article Spagnolo, F; Coppi, E; Chieffo, R; Straffi, L; Fichera, M; Nuara, A; Gonzalez-Rosa, J; Martinelli, V; Comi, G; Volonté, MA; Leocani, L. Interhemispheric balance in Parkinson’s disease: A transcranial magnetic stimulation study. Brain Stimul.: 2013; 6(6): 892-897 - Article DEPARTMENT ONCO-HAEMATOLOGYOF Head of Department: Federico Caligaris-Cappio* DEPARTMENT AREA COORDINATORS: Gianfranco Ciboddo, Consuelo Corti Internal medicine HEAD OF UNIT: Federico Caligaris-Cappio* CLINICAL UNIT LEADER: Marco Foppoli CLINICAL UNIT COORDINATORS: Andrés José Marìa Ferreri, Paolo Ghia* PHYSICIANS: Marta Bruno Ventre (until June 2013), Gianfranco Ciboddo, Giovanni Citterio, Giovanni Donadoni, Silvia Govi (until September 2013), Marianna Sassone (since September 2013) RESIDENTS: Serena Antoniolli, Elena Brioschi, Gaetano Di Terlizzi, Chiara Giudice, Chiara Miggiano, Sara M. Rosa Previtali, Carlo Rossi, Lydia Scarfò, Paolo Strati, Gabriele Todisco, Alessandro Vignati Haematology and bone marrow transplantation HEAD OF UNIT: Fabio Ciceri CLINICAL GROUP LEADER: Massimo Bernardi PHYSICIANS: Andrea Assanelli, Matteo Carrabba, Consuelo Corti, Fabio Giglio, Elena Guggiari, Francesca Lunghi, Maria Teresa Lupo Stanghellini, Magda Marcatti, Sarah Marktel, Sara Mastaglio, Mara Morelli, Jacopo Peccatori RESIDENTS: Simone Claudiani**, Alessandra Forcina**, Barbara Forno**, Bernhard Gentner**, Stefania Girlanda**, Raffaella Greco**, Francesca Lorentino**, Carlo Messina**, Francesca Pavesi**, Simona Piemontese**, Elisa Sala**, Luca Vago** STUDY COORDINATOR: Stefania Trinca RESEARCH NURSE: Margherita Brambilla DATA MANAGER: Ambra Malerba TECHNICIAN: Fernanda Tripiciano * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The aim of the Department is to achieve optimization of care and acceleration of cure of blood cancers by implementing the transformation of everyday practice into protocol-based and clinical-trial-based clinical activity. To this end we are strengthening the clinical application of Translational Research by establishing interactions with other Departments involved in the diagnosis and treatment of cancer (especially the Departments of Oncology and Pathology) and joining efforts with several Research Divisions 301 THE CLINICAL DEPARTMENTS especially the Division of Molecular Oncology. Specific goals are: 1) to maintain/reach “state of the art” clinical care for all types of blood cancers; 2) to improve logistic and organization for ameliorating patient care; 3) to become second to none in specific cancers such as lymphoid malignancies. Clinical activity and Areas of excellence The Department includes the quality-certified Divisions of Medicine 1QB and of Hematology and Bone Marrow Transplantation (BMT) with ample out-patient (and Day Hospital) activity, research nurses and data managers. Fruitful interactions with other Departments and with Research Divisions allow to bridge the clinical experience in applying new therapies in close contact with the development of new potential therapeutic tools systematically analysed in preclinical settings. Furthermore a) the Department is full member of the ROL (Oncology Network of Lombardia Region) and the REL (Hematology Network of Lombardia Region), b) the Departmental Area of Lymphoid Tumors has been blooming with more than 200 new patients/year and more than 40 ongoing clinical trials, c) the BMT Unit has ranked among the firsts in Italy as for the number of allogeneic transplantations performed and d) new clinical trials, including Phase I, Phase II and Phase III trials with innovative molecules, usually in the context of international collaborations, are constantly approved by the Ethical Committee. The Department is member of the European Organization of Cancer Centers (OECI), the Southern Europe for New Drugs Organization (SENDO), the Cancer Research UK (CRUK), the Swiss Group for Clinical Cancer Research (SAKK), the EBMT (European Bone Marrow Transplantation), the International Extranodal Lymphoma Study Group (IELSG), the International PCNSL Collaborative Group (IPCG), the European PCNSL Collaborative Group (EPCG), the Lymphoma Study Association (LYSA), the Nordic Lymphoma Group (NLG), the European Splenic Lymphoma Group (ESLG), the European Gastrointestinal Lymphomas Study Group (EGILS), the ERIC (European Research Initiative on CLL) and the CLL US/Europe Alliance Organization (driven by the MD Anderson Cancer Center). Fields of research We keep on building teams of laboratory-based and clinical investigators to define molecular endpoints in clinical material, develop studies on the pato-biology of specific tumours and organize pilot studies and investigator-driven clinical trials. The currently ongoing projects can be summarized under two main headings: 1) Molecular mechanisms of disease and their clinical translation in terms of prognostic/predictive factors with the definition of new biologically-based tools; 2) Novel treatment strategies developed by a) increasing the number and quality of clinical studies and b) clinically translating the results of preclinical investigations, examples being immunotherapy and BMT, anti-angiogenesis-based treatments and the use of several new investigational drugs. Federico Caligaris-Cappio Selected publications Apollonio, B; Scielzo, C; Bertilaccio, MT; Ten Hacken, E; Scarfò, L; Ranghetti, P; Stevenson, F; Packham, G; Ghia, P; Muzio, M and Caligaris-Cappio, F. Targeting B-cell anergy in chronic lymphocytic leukemia. Blood: 2013; 121(19): 3879-3888 - Article Bertilaccio, MTS; Scielzo, C; Simonetti, G; Ten Hacken, E; Apollonio, B; Ghia, P; CaligarisCappio, F. Xenograft models of chronic lymphocytic leukemia: Problems, pitfalls and future directions. Leukemia: 2013; 27(3): 534-540 - Review Ciceri, F; Lupo-Stanghellini, MT; Korthof, ET; on behalf of the SAA-WP EBMT. Haploidentical transplantation in patients with acquired aplastic anemia. Bone Marrow Transplant.: 2013; 48(2): 183-185 - Review Cieri, N; Camisa, B; Cocchiarella, F; Forcato, M; Oliveira, G; Provasi, E; Bondanza, A; Bordignon, C; Peccatori, J; Ciceri, F; Lupo-Stanghellini, MT; Mavilio, F; Mondino, A; Bicciato, S; Recchia, A; Bonini, C. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood: 2013; 121(4): 573-584 - Article Fonte, E; Apollonio, B; Scarfò, L; Ranghetti, P; Fazi, C; Ghia, P; Caligaris-Cappio, F; Muzio, THE CLINICAL DEPARTMENTS M. In vitro sensitivity of CLL cells to fludarabine may be modulated by the stimulation of toll-like receptors. Clin. Cancer Res.: 2013; 19(2): 367-379 - Article Ghielmini, M; Vitolo, U; Kimby, E; Montoto, S; Walewski, J; Pfreundschuh, M; Federico, M; Hoskin, P; McNamara, C; Caligaris-Cappio, F; Stilgenbauer, S; Marcus, R; Trneny, M; Dreger, P; Montserrat, E; Dreyling, M; on behalf of the Panel Members of the 1st ESMO Consensus Conference on Malignant Lymphoma. ESMO guidelines consensus conference on malignant lymphoma 2011 part 1: Diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL) and Chronic Lymphocytic Leukemia (CLL). Ann. Oncol.: 2013; 24(3): 561-576 - Article Lo-Coco, F; Avvisati, G; Vignetti, M; Thiede, C; Orlando, SM; Iacobelli, S; Ferrara, F; Fazi, P; Cicconi, L; Di Bona, E; Specchia, G; Sica, S; Divona, M; Levis, A; Fiedler, W; Cerqui, E; Breccia, M; Fioritoni, G; Salih, HR; Cazzola, M; Melillo, L; Carella, AM; Brandts, CH; Morra, E; Von Lilienfeld-Toal, M; Hertenstein, B; Wattad, M; Lubbert, M; Hanel, M; Schmitz, N; Link, H; Kropp, MG; Rambaldi, A; La Nasa, G; Luppi, M; Ciceri, F; Finizio, O; Venditti, A; Fabbiano, F; Dohner, K; Sauer, M; Ganser, A; Amadori, S; Mandelli, F; Dohner, H; Ehninger, G; Schlenk, RF; Platzbecker, U; for Gruppo Italiano Malattie Ematologiche dell’Adulto; the German-Austrian Acute Myeloid Leukemia Study Group; and Study Alliance Leukemia. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. New Engl. J. Med.: 2013; 369(2): 111-121 - Article Maffi, P and Balzano, G; Ponzoni, M; Nano, R; Sordi, V; Melzi, R; Mercalli, A; Scavini, M; Esposito, A; Peccatori, J; Cantarelli, E; Messina, C; Bernardi, M; Del Maschio, A; Staudacher, C; Doglioni, C; Ciceri, F; Secchi, A; Piemonti, L. Autologous Pancreatic Islet Transplantation in Human Bone Marrow. Diabetes: 2013; 62(10): 3523-3531 - Article ten Hacken, E and Scielzo, C; Bertilaccio, MT; Scarfò, L; Apollonio, B; Barbaglio, F; Stamatopoulos, K; Ponzoni, M; Ghia, P; Caligaris-Cappio, F. Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia. Blood: 2013; 121(12): 2264-2273 - Article Todisco, E; Ciceri, F; Oldani, E; Boschini, C; Mico, C; Vanlint, MT; Donnini, I; Patriarca, F; Alessandrino, PE; Bonifazi, F; Arcese, W; Barberi, W; Marenco, P; Terruzzi, E; Cortelazzo, S; Santarone, S; Proia, A; Corradini, P; Tagliaferri, E; Falcioni, S; Irrera, G; Dallanegra, L; Castagna, L; Santoro, A; Camboni, A; Sacchi, N; Bosi, A; Bacigalupo, A; Rambaldi, A. The CIBMTR score predicts survival of AML patients undergoing allogeneic transplantation with active disease after a myeloablative or reduced intensity conditioning: A retrospective analysis of the Gruppo Italiano Trapianto di Midollo Osseo. Leukemia: 2013; 27(10): 2086-2091 - Letter 303 DEPARTMENT MEDICAL ONCOLOGY OF Head of Department: Luca Gianni DEPARTMENT AREA COORDINATORS: Gianni Bordogna, Vanesa Gregorc, Michele Reni, Cristiana Sessa, Milvia Zambetti Medical oncology HEAD OF UNIT: Luca Gianni CLINICAL UNIT LEADERS: Daniela Aldrighetti, Vanesa Gregorc, Giovanna Petrella, Monica Ronzoni, Cristiana Sessa, Aurelio Vicari, Milvia Zambetti PHYSICIANS: Gianni Bordogna, Stefano Cereda, Gianluca Del Conte, Angelica Fasolo, Elena Mazza, Manuela Pacchioni, Antonella Perotti, Michele Reni, Giordano Pietro Vitali, Patrizia Zucchinelli PHYSICIAN SCIENTIST: Giampaolo Bianchini CONSULTANTS: Carmen Belli, Alessandra Bulotta, Francesca Colonese, Vincenzo Ricci, Alessia Rognone, Gilda Rossoni, Maria Grazia Viganò, Stefania Zambelli The Department of Medical Oncology has the goals of 1) providing clinical care based on “state of the art” treatment for all types of solid tumors through the activity of interdisciplinary Disease Units dedicated to the coordinated diagnosis and therapy of specific neoplasms; 2) fostering high level basic, translational and clinical research; 3) educating teaching and tutoring students, nurses, fellows, and residents with active participation to the activity of Università Vita-salute San Raffaele. The Department has continued and increased all its activities in 2013 within the Ospedale San Raffaele and the Scientific Institute. Clinical activity and Areas of excellence The Department has two in-patient units including one for emergencies in oncologic patients for total 27 beds; outpatient activities in an Ambulatory Service of 14 visit rooms; and a Day Hospital for delivery of anticancer therapies. The personnel counts 50 people including clinical trials assistants and research nurses. Clinical activity is conducted through interdisciplinary Disease Units. Currently Disease Units are operational for patients with tumors of the breast, lung, pancreas, biliary tract, gastrointestinal, liver, central nervous system, genito-urinary, head and neck, and melanoma. In each Unit a group of Physicians is dedicated to the treatment and follow up of patients according to guidelines or to approved experimental protocols. There also is a Phase I Unit. The collaboration with the Division of Molecular Oncology is strengthened by the clinical experience in applying new therapies to patients and is favoring the development of new potential therapeutic tools that are systematically analysed in preclinical settings. In 2013 the Department has taken care of more than 3000 cases in inpatient and outpatient services. The Department is full member of the ROL (Oncology Network of Lombardia Region); has had 11 new clinical trials approved by the Ethical Committee, and has been conducting overall 53 trials. Four newly approved protocols are investigators initiative trials. Overall 21investigators initiative trials are ongoing in the THE CLINICAL DEPARTMENTS Department. Over 60 lesson hours have been taught to students of the faculty of Medicine and of Dental Hygiene, to nurses and fellows. Overall 35 scientific manuscripts were published. The Department is member of the Michelangelo Foundation for cooperative studies in breast cancer, the European Organization Research and Treatment of Cancer Brain Tumor Group (EORTC), the pancreas Aide et Recherche en CAncérologie Digestive (ARCAD) group, the Associazione Italiana Studi Pancreas (AISP), the Gruppo Italiano Cooperativo di Neuro-Oncologia (GICNO), and the Italian Melanoma Intergroup. Fields of research The Department is committed to conduct clinical trials in collaboration with the pharmaceutical industry, and to design and conduct investigator-driven clinical trials. Special emphasis is given to new drugs development and collaborations with laboratory researchers to identify molecular prognostic or predictive biomarkers. Ongoing projects can be listed under three headings: 1) Molecular mechanisms of disease driven phase I trials; 2) Definition of molecular prognostic/predictive tools on markers generated by assays of gene expression, proteomics and single nucleotide polymorphisms involved; 3) New treatment strategies developed by phase I/II studies and by translation of the results of preclinical investigations. In all these fields the Department has reached relevant results in 2013. Phase I trials were opened with PI3K inhibitors in different combinations according to the type of neoplastic disease to be treated (platinating agents, aromatase inhibitors, MEK inhibitors). Based on analyses of gene expression profiles conducted within the Department, it has been shown that the immune system plays a key role in women with breast cancer in determining the probability of tumor eradication with drug treatment. In particular this analysis has led to the design of a study that will use the immune check point directed monoclonal antibody antiPDL1 in comination with HER2-directed monoclonals in HER2 positive breast cancer in an attempt to improve efficacy without use of chemotherapy. Another similar study has been designed and approved for women with triple negative breast cancer. Also notable is the continuous contribution of the Department to the development of new therapies in patients with pancreatic carcinoma and lung cancers. The role of combination chemotherapy in stage IV disease; of maintenance therapy in stage IV disease; and of predictive role of single nucleotide polymorphisms in advanced disease were explored in pancreatic cancer. In lung cancer the use of proteomic analysis to select patients who will or will not benefit of specific therapies has seen the completion of an investigator-initiative study with promising results on the utility of approach. Luca Gianni Selected publications Bianchini, G; Pusztai, L; Karn, T; Iwamoto, T; Rody, A; Kelly, CM; Müller, V; Schmidt, M; Qi, Y; Holtrich, U; Becker, S; Santarpia, L; Fasolo, A; Del Conte, G; Zambetti, M; Sotiriou, C; Haibe-Kains, B; Symmans, WF; Gianni, L. Proliferation and estrogen signaling can distinguish patients at risk for early versus late relapse among estrogen receptor positive breast cancers. Breast Cancer Res.: 2013; 15: R86 - Article Belli, C; Cereda, S; Anand, S; Reni, M. Neoadjuvant therapy in resectable pancreatic cancer: A critical review. Cancer Treat. Rev.: 2013; 39(5): 518-524 - Review Corti, A; Curnis, F; Rossoni, G; Marcucci, F; Gregorc, V. Peptide-mediated targeting of cytokines to tumor vasculature: the NGR-hTNF example. BioDrugs: 2013; 27(6): 591-603 - Review Fasolo, A; Sessa, C; Gianni, L; Broggini, M. Seminars in clinical pharmacology: An introduction to met inhibitors for the medical oncologist. Ann. Oncol.: 2013; 24(1): 14-20 - Review Gianni, L; Romieu, GH; Lichinitser, M; Serrano, SV; Mansutti, M; Pivot, X; Mariani, P; Andre, F; Chan, A; Lipatov, O; Chan, S; Wardley, A; Greil, R; Moore, N; Prot, S; Pallaud, C; Semiglazov, V. AVEREL: a randomized phase III Trial evaluating bevacizumab in combination with docetaxel and trastuzumab as first-line therapy for HER2-positive locally recurrent/metastatic breast cancer. J. Clin. Oncol.: 2013; 31(14): 1719-1725 - Article Goldhirsch, A; Gelber, RD; Piccart-Gebhart, MJ; De Azambuja, E; Procter, M; Suter, TM; Jackisch, 305 THE CLINICAL DEPARTMENTS C; Cameron, D; Weber, HA; Heinzmann, D; Lago, LD; McFadden, E; Dowsett, M; Untch, M; Gianni, L; Bell, R; Kohne, CH; Vindevoghel, A; Andersson, M; Brunt, AM; Otero-Reyes, D; Song, S; Smith, I; Leyland-Jones, B; Baselga, J. 2 years versus 1 year of adjuvant trastuzumab for HER2positive breast cancer (HERA): An open-label, randomised controlled trial. Lancet: 2013; 382(9897): 1021-1028 - Article Passoni, P; Fiorino, C; Slim, N; Ronzoni, M; Ricci, V; Di Palo, S; De Nardi, P; Orsenigo, E; Tamburini, A; De Cobelli, F; Losio, C; Iacovelli, NA; Broggi, S; Staudacher, C; Calandrino, R; Di Muzio, N. Feasibility of an adaptive strategy in preoperative radiochemotherapy for rectal cancer with image-guided tomotherapy: Boosting the dose to the shrinking tumor. Int. J. Radiat. Oncol. Biol. Phys.: 2013; 87(1): 67-72 - Article Pecorelli, N; Braga, M; Doglioni, C; Balzano, G; Reni, M; Cereda, S; Albarello, L; Castoldi, R; Capretti, G; Di Carlo, V. Preoperative Chemotherapy Does Not Adversely Affect Pancreatic Structure and Short-Term Outcome after Pancreatectomy. J. Gastrointest. Surg.: 2013; 17(3): 488-493 Article Sessa, C; Del Conte, G; Christinat, A; Cresta, S; Perotti, A; Gallerani, E; Lardelli, P; Kahatt, C; Alfaro, V; Iglesias, JL; Fernandez-Teruel, C; Gianni, L. Phase I clinical and pharmacokinetic study of trabectedin and cisplatin given every three weeks in patients with advanced solid tumors. Invest. New Drugs: 2013; 31(5): 1236-1243 - Article Tartarone, A; Lazzari, C; Lerose, R; Conteduca, V; Improta, G; Zupa, A; Bulotta, A; Aieta, M; Gregorc, V. Mechanisms of resistance to EGFR tyrosine kinase inhibitors gefitinib/erlotinib and to ALK inhibitor crizotinib. Lung Cancer: 2013; 81(3): 328-336 - Review DEPARTMENT OF RADIOLOGY Head of Department: Alessandro Del Maschio* DEPARTMENT AREA COORDINATORS: Francesco De Cobelli*, Maurizia Del Maschio, Roberto Nicoletti, Pierluigi Paesano Radiology OSR HEAD OF UNIT: Alessandro Del Maschio* CLINICAL UNIT LEADERS: Francesco De Cobelli*, Maurizia Del Maschio, Roberto Nicoletti, Pierluigi Paesano, Maria Grazia Rodighiero, Massimo Venturini PHYSICIANS: Laura Brasca, Stefano Cappio, Giulia Maria Crespi, Angela De Gaspari, Antonio Esposito*, Elda Garuti, Domenico Ghio, Simone Gusmini, Claudio Losio, Carlo Martinenghi, Renata Mellone, Marco Salvioni, Simona Irma Tacchini, Roberto Varagona CONSULTANTS: Elena Contrino, Silvia Ravelli, Elena Schiani Radiology OSRT HEAD OF UNIT: Giuseppe Balconi CLINICAL UNIT LEADER: Gianpiero Cardone PHYSICIANS: Elena Capitelli, Roberto Lanzi, Massimo Mandelli, Maurizio Papa CONSULTANT: Rossana Favia RESIDENT: Audrey Serafini TECHNICIANS: Cecilia Bertocchi, Enza Galvano, Caterina Parolo, Alessandra Poggi, Maria Pia Rapallo, Stefano Rovani * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The activity of the Department of Radiology is based upon both inpatient and outpatients Services covering all the main fields of general Radiology. The Department of Radiology works in tight interaction with the most important research groups of the Hospital allowing to reach very important results in terms of number and quality of research lines. The objective of the Department is to integrate clinical activity, research and education with the aim of helping patients at the best of current medical knowledge and technological expertise. Clinical activity and Areas of excellence The Department includes six sections of clinical activity and areas of excellence: 1. Conventional and Digital Radiology 2. Breast Imaging 3. Ultrasound 307 THE CLINICAL DEPARTMENTS 4. Computed Tomography 5. Magnetic Resonance Imaging 6. Diagnostic and Interventional Radiology/Angiography Fields of research 1. Interventional Radiology. • Transarterial chemoembolization (TACE) with Drug-Eluting-Beads preloaded with Irinotecan (DEBIRI)in the treatment of unresectable hepatic metastases confined to the liver from uveal melanoma (first line treatment), cholangiocarcinoma (second line treatment), colorectal cancer (third line treatment). • First phase-1 study in the treatment of Duchenne Muscular Dystrophy (DMD) by multiple intra-arterial transplantations of mesoangioblasts in dystrophic children. • Percutaneous intraportal islet auto-transplantation to prevent diabetes after extensive pancreatic surgery. • Endovascular treatment of visceral artery aneurysms by trans arterial embolization, covered stents and combined technique (embolization associated with covered/uncovered stent). • Drug-eluting-stent in peripheral occlusive disease due to Takayasu arteritis. 2. Ultrasound • Contrast-enhanced ultrasound (CEUS) in the quantitative assessment of uveal melanoma (UM) response to gamma knife radiosurgery (GKR). • Orbital Color Doppler Ultrasound application in dural fistulas, ischemic optic neuropathy, type 1 diabetes, and as monitoring during cardiac surgery. 3. Imaging of islet transplantation MRI based functional assessment of micro-vascular events occurring after pancreatic islets transplantation and relationship with graft outcome. 4. Cardiac Imaging The main cardiac study projects were focused on: • Myocarditis: in order to find prognostic imaging markers for patients at risk of ventricular tachyarrhythmias and sudden death. • Cardiac CT and Cardiac MRI assessment of structural substrate of ventricular tachycardia; 3D imaging based modeling of the heart/coronary anatomy and scars localization as a tool to be merged with electro-anatomic maps for a real time anatomical/structural/electrical guide of ventricular tachycardia ablation. • A cardiac MR-study to evaluate the effect of GH-deficiency in patients’ heart. • Characterization of cardiac masses. • Optimization of Cardiac-CT protocols for the assessment coronary arteries in re-vascularized patients. • Patients with AMI submitted to PCI in order to evaluate the microvascular obstruction and to compare our results with coronary angiographyand to find new prognostic factors. 5. Oncologic MR Imaging We aimed to evaluate the accuracy of Diffusion-weighted Imaging (DWI) as a marker of tumour aggressiveness and tumour response to chemotherapy. We demonstrated the correlation between ADC and tumour grading factors in prostate, gastroesophageal and rectal cancer and that changes in ADC before and after neoadjuvant-treatment can be used as reliable marker of tumor response. Moreover, we implemented the use of imaging in radiotherapy planning, in particular MR and DWI-MR to improve target volume definition in pancreas and rectal tumors. 6. Breast Imaging • Two large multicenter studies are ongoing investigating the role of Breast-MRI in two different settings: • a. as an alternative tool to mammography and ultrasound in the screening of women at intermediate risk of breast cancer (MRIB study) • b. to demonstrate the outperformance of MRI over traditional imaging in the pre-surgical planning of patients diagnosed with breast cancer (MIPA study) THE CLINICAL DEPARTMENTS • Assessment of tumor response in women with locally-advanced breast cancer undergoing neoadjuvant chemotherapy: • a. by multimodality imaging: trying to identify the best technique to monitor tumor shrinkage and residual disease according to morphologic features of different tumor types • b. by means of MRI: investigating all parameters that could predict subsequent response or resistance to chemotherapy, including Diffusion-Weighted Imaging • c. Characterization of breast architectural distortions by means of MRI. Alessandro Del Maschio Selected publications Balzano, G; Maffi, P; Nano, R; Zerbi, A; Venturini, M; Melzi, R; Mercalli, A; Magistretti, P; Scavini, M; Castoldi, R; Carvello, M; Braga, M; Del Maschio, A; Secchi, A; Staudacher, C; Piemonti, L. Extending indications for islet autotransplantation in pancreatic surgery. Ann. Surg.: 2013; 258(2): 210-218 - Article De Cobelli, F; Giganti, F; Orsenigo, E; Cellina, M; Esposito, A; Agostini, G; Albarello, L; Mazza, E; Ambrosi, A; Socci, C; Staudacher, C; Del Maschio, A. Apparent diffusion coefficient modifications in assessing gastro-oesophageal cancer response to neoadjuvant treatment: comparison with tumour regression grade at histology. Eur. Radiol.: 2013; 23(8): 2165-2174 - Article Esposito, A and Campana, L; Palmisano, A; De Cobelli, F; Canu, T; Santarella, F; Colantoni, C; Monno, A; Vezzoli, M; Pezzetti, G; Manfredi, AA; Rovere-Querini, P; Del Maschio, A. Magnetic Resonance Imaging at 7T Reveals Common Events in Age-Related Sarcopenia and in the Homeostatic Response to Muscle Sterile Injury. PLoS ONE: 2013; 8(3): e59308 - Article Esposito, A; Colantoni, C; De Cobelli, F; Del Vecchio, A; Palmisano, A; Calandrino, R; Del Maschio, A. Multidetector computed tomography for coronary stents imaging: High-voltage (140KVP) prospective ecg-triggered versus standard-voltage (120-kvp) retrospective ecg-gated helical scanning. J. Comput. Assisted Tomogr.: 2013; 37(3): 395-401 - Article Losa, A; Gadda, GM; Lazzeri, M; Lughezzani, G; Cardone, G; Freschi, M; Lista, G; Larcher, A; Nava, LD; Guazzoni, G. Complications and quality of life after template-assisted transperineal prostate biopsy in patients eligible for focal therapy. Urology: 2013; 81(6): 1291-1296 - Article Maffi, P and Balzano, G; Ponzoni, M; Nano, R; Sordi, V; Melzi, R; Mercalli, A; Scavini, M; Esposito, A; Peccatori, J; Cantarelli, E; Messina, C; Bernardi, M; Del Maschio, A; Staudacher, C; Doglioni, C; Ciceri, F; Secchi, A; Piemonti, L. Autologous Pancreatic Islet Transplantation in Human Bone Marrow. Diabetes: 2013; 62(10): 3523-3531 - Article Passoni, P; Fiorino, C; Slim, N; Ronzoni, M; Ricci, V; Di Palo, S; De Nardi, P; Orsenigo, E; Tamburini, A; De Cobelli, F; Losio, C; Iacovelli, NA; Broggi, S; Staudacher, C; Calandrino, R; Di Muzio, N. Feasibility of an adaptive strategy in preoperative radiochemotherapy for rectal cancer with image-guided tomotherapy: Boosting the dose to the shrinking tumor. Int. J. Radiat. Oncol. Biol. Phys.: 2013; 87(1): 67-72 - Article Piatti, PM; Marone, E; Mantero, M; Setola, E; Galluccio, E; Lucotti, P; Shehaj, E; Villa, V; Perticone, F; Venturini, M; Palini, A; Airoldi, F; Faglia, E; Del Maschio, A; Colombo, A; Chiesa, R; Bosi, E; Monti, LD. Effect of normalization of fasting glucose by intensified insulin therapy and influence of eNOS polymorphisms on the incidence of restenosis after peripheral angioplasty in patients with type 2 diabetes: a randomized, open-label clinical trial. Acta Diabetol.: 2013; 50(3): 373-382 - Article Russo, V and Pilla, L; Lunghi, F; Crocchiolo, R; Greco, R; Ciceri, F; Maggioni, D; Fontana, R; Mukenge, S; Rivoltini, L; Rigamonti, G; Mercuri, SR; Nicoletti, R; Del Maschio, A; Gianolli, L; Fazio, F; Marchianò, A; Di Florio, A; Maio, M; Salomoni, M; Gallo-Stampino, C; Del Fiacco, M; Lambiase, A; Coulie, PG; Patuzzo, R; Parmiani, G; Traversari, C; Bordignon, C; Santinami, M and Bregni, M. Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3genetically modified lymphocytes. Int. J. Cancer: 2013; 132(11): 2557-2566 - Article Venturini, E; Losio, C; Panizza, P; Rodighiero, MG; Fedele, I; Tacchini, S; Schiani, E; Ravelli, S; Cristel, G; Panzeri, MM; De Cobelli, F; Del Maschio, A. Tailored breast cancer screening program with microdose mammography, us, and mr imaging : Short-term results of a pilot study in 4049-year-old wome. Radiology: 2013; 268(2): 347-355 - Article 309 DEPARTMENT OF UROLOGY Francesco Montorsi* Clinical Unit of Urology - San Raffaele Hospital Giorgio Guazzoni* Clinical Unit of Urology - San Raffaele Turro Urology OSR HEAD OF UNIT: Francesco Montorsi* CLINICAL UNIT COORDINATORS: Roberto Bertini, Alberto Briganti, Renzo Colombo, Federico Dehò (since October 2013), Andrea Salonia, Vincenzo Scattoni PHYSICIANS: Lina Bua, Umberto Capitanio, Andrea Gallina, Massimo Ghezzi (until June 2013), Caterina Lania, Arianna Lesma, Carmen Maccagnano (until April 2013), Ryan Matloob (since August 2013), Nazareno Suardi (until April 2013), Giuseppe Zanni RESIDENTS: Marco Bianchi**, Paolo Capogrosso**, Giulia Maria Castagna** (since August 2013), Fabio Castiglione**, Stefano Corti**, Dario Di Trapani**, Ettore Di Trapani**, Matteo Ferrari** (until March 2013), Giorgio Gandaglia**, Giovanni La Croce**, Ryan Matloob (until July 2013), Alessandro Nini** (since August 2013), Lorenzo Rocchini**, Andrea Russo**, Manuela Tutolo**, Luca Villa** Urological endoscopy service and day surgery HEAD OF UNIT: Valerio Di Girolamo Strategic Program for uro-andrological research HEAD OF UNIT: Francesco Montorsi* THE CLINICAL DEPARTMENTS Urology OSRT HEAD OF UNIT: Giorgio Guazzoni* CLINICAL UNIT LEADERS: Piera Bellinzoni, Luigi Broglia, Andrea Cestari (until June 2013) PHYSICIANS: Nicolò Maria Buffi , Antonia Centemero, Andrea Losa, Tommaso Maga, Lorenzo Rigatti, Giovanni Lughezzani CONSULTANTS: Vincenzo Dell’Acqua, Girolamo Fiorini RESIDENTS: Paolo Dell’Oglio**, Nicola Fossati **, Giulio Maria Gadda**, Ella Kinzikeeva **, Alessandro Larcher**, Giuliana Lista** * Professor at: Università Vita-Salute San Raffaele ** Università Vita-Salute San Raffaele The Department of Urology at Vita-Salute San Raffaele University, Milan, is composed of two clinical and research units (Ospedale San Raffele Sede Centrale and San Raffaele Turro) chaired by Professor Francesco Montorsi and Professor Giorgio Guazzoni, respectively. The Department was founded in 1985 and it has been directed by Professor Patrizio Rigatti until November 2012. Clinical activity The surgical activity is based on 25 operating theatre sessions weekly and it is serving 80 beds for ordinary recovery. Every year, about 1000, 500 and 250 surgical procedures are performed for prostate, bladder and kidney cancer, respectively. There is a continue effort to set the highest standards of care for patients with urological conditions, ground-breaking surgical techniques, professional education, teaching and training. Specific interest is directed to robotic surgery which is currently used to perform radical prostatectomy, partial nephrectomy for cancer, radical cystectomy and reconstructive. Likewise, the two units have been involved in highly challenging surgical procedures (extra-corporeal circulation and deep hypothermic circulatory arrest for cavo-atrial neoplastic thrombosis of kidney or adrenal cancer). In 2012, our department surpassed its objectives in terms of ward organization, clinical information organization and management, quality of clinical outcomes, and the level of patients’ satisfaction. Fields of research The clinical investigations performed in the most recent years leaded to the publication of 283 scientific contributions throughout the last three years (2010-2012) for a overall citation index of 7752; h-index: 58 (citation index 2012: 439; h-index: 11) [Source SCOPUS June 2013]. During the last 4 years the Department of Urology ranked first in terms of abstracts presented at the official annual meetings of the European Association of Urology and American Urological Association. The main clinical research areas consist of prostate cancer, kidney cancer, bladder cancer, infertility and andrology, female sexual medicine, benign prostatic hyperplasia. Ongoing and new translational researches have been starting after the foundation of the Urological Research Institute (URI), headed by Professors Francesco Montorsi and Petter Hedlund. The main basic research areas consist of prostate and bladder cancer, functional urology, reproductive and erectile dysfunction. In 2010, a biological bank was founded to permit collecting and analyzing tissue, blood, and urine samples from virtually all oncological patients for scientific research purpose. Francesco Montorsi and Giorgio Guazzoni 311 THE CLINICAL DEPARTMENTS Selected publications Abdollah, F; Suardi, N; Cozzarini, C; Gallina, A; Capitanio, U; Bianchi, M; Sun, M; Fossati, N; Passoni, NM; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Selecting the Optimal Candidate for Adjuvant Radiotherapy After Radical Prostatectomy for Prostate Cancer: A Long-term Survival Analysis. Eur. Urol.: 2013; 63(6): 998-1008 - Article Briganti, A; Bianchi, M; Sun, M; Suardi, N; Gallina, A; Abdollah, F; Bertini, R; Colombo, R; Di Girolamo, V; Salonia, A; Scattoni, V; Karakiewicz, PI; Guazzoni, G; Rigatti, P; Montorsi, F. Impact of the introduction of a robotic training programme on prostate cancer stage migration at a single tertiary referral centre. BJU Int.: 2013; 111(8): 1222-1230 - Article Buffi, N; Lista, G; Larcher, A; Lughezzani, G; Cestari, A; Lazzeri, M; Guazzoni, G; Ficarra, V. Re: “Trifecta” in Partial Nephrectomy: A. J. Hung, J. Cai, M. N. Simmons and I. S. Gill J Urol 2013; 189: 36–42. J. Urol.: 2013; 190(2): 810-811 - Reply Capitanio, U; Abdollah, F; Matloob, R; Suardi, N; Castiglione, F; Di Trapani, E; Capogrosso, P; Gallina, A; Dell’Oglio, P; Briganti, A; Salonia, A; Montorsi, F; Bertini, R. When to perform lymph node dissection in patients with renal cell carcinoma: a novel approach to the preoperative assessment of risk of lymph node invasion at surgery and of lymph node progression during followup. BJU Int: 2013; 112(2): E59-E66 - Article Capogrosso, P; Colicchia, M; Ventimiglia, E; Castagna, G; Clementi, MC; Suardi, N; Castiglione, F; Briganti, A; Cantiello, F; Damiano, R; Montorsi, F; Salonia, A. One patient out of four with newly diagnosed erectile dysfunction is a young man—worrisome picture from the everyday clinical practice. J. Sex. Med.: 2013; 10(7): 1833-1841 - Article Castiglione, F; Hedlund, P; Van der Aa, F; Bivalacqua, TJ; Rigatti, P; Van Poppel, H; Montorsi, F; De Ridder, D; Albersen, M. Intratunical Injection of Human Adipose Tissue-derived Stem Cells Prevents Fibrosis and Is Associated with Improved Erectile Function in a Rat Model of Peyronie’s Disease. Eur. Urol.: 2013; 63(3): 551-660 - Article Lazzeri, M; Haese, A; Abrate, A; De La Taille, A; Redorta, JP; McNicholas, T; Lughezzani, G; Lista, G; Larcher, A; Bini, V; Cestari, A; Buffi, N; Graefen, M; Bosset, O; Corvoisier, PL; Breda, A; De La Torre, P; Fowler, L; Roux, J; Guazzoni, G. Clinical performance of serum prostate-specific antigen isoform [-2]proPSA (p2PSA) and its derivatives, %p2PSA and the prostate health index (PHI), in men with a family history of prostate cancer: Results from a multicentre European study, the PROMEtheuS project. BJU Int.: 2013; 112(3): 313-321 - Article Lazzeri, M; Haese, A; De La Taille, A; Palou Redorta, J; McNicholas, T; Lughezzani, G; Scattoni, V; Bini, V; Freschi, M; Sussman, A; Ghaleh, B; Le Corvoisier, P; Alberola Bou, J; Esquena Fernandez, S; Graefen, M; Guazzoni, G. Serum isoform [-2]proPSA derivatives significantly improve prediction of prostate cancer at initial biopsy in a total PSA range of 2-10 ng/ml: A multicentric european study. Eur. Urol.: 2013; 63(6): 986-994 - Article Losa, A; Gadda, GM; Lazzeri, M; Lughezzani, G; Cardone, G; Freschi, M; Lista, G; Larcher, A; Nava, LD; Guazzoni, G. Complications and quality of life after template-assisted transperineal prostate biopsy in patients eligible for focal therapy. Urology: 2013; 81(6): 1291-1296 - Article Scattoni, V; Lazzeri, M; Lughezzani, G; De Luca, S; Passera, R; Bollito, E; Randone, D; Abdollah, F; Capitanio, U; Larcher, A; Lista, G; Gadda, GM; Bini, V; Montorsi, F; Guazzoni, G. Headto-head comparison of prostate health index and urinary PCA3 for predicting cancer at initial or repeat biopsy. J. Urol.: 2013; 190(2): 496-501 - Article Villa, L; Buono, R; Fossati, N; Rigatti, P; Montorsi, F; Benigni, F; Hedlund, P. Effects by silodosin on the partially obstructed rat ureter in vivo and on human and rat isolated ureters. Br. J. Pharmacol.: 2013; 169(1): 230-238 - Article CLINICAL SERVICES Pathology –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 315 HEAD OF UNIT: Claudio Doglioni* CLINICAL UNIT COORDINATORS: Gianluigi Arrigoni, Giacomo Dell’Antonio, Massimo Freschi, Maurilio Ponzoni, Isabella Sassi, Gianluca Taccagni, Maria Rosa Terreni RESEARCHER: Francesca Sanvito PHYSICIANS: Luca Albarello, Nathalie Rizzo BIOLOGISTS: Maria Giulia Cangi, Lorenza Pecciarini POST-DOCTORAL FELLOWS: Daniela Clavenna, Tiziana De Marino, Valeria De Pascale, Ilaria Francaviglia, Greta Grassini, Gilda Magliacane CONSULTANTS: Graziana Famoso, Gabriella Leone, Roberta Lucianò, Federica Pedica TECHNICIANS: Mara Bertolazzi, Marina Brambilla, Roberto Cairella, Diana Ciscato, Elena Dal Cin, Stefania Demasi, Barbara Di Ruvo, Patrizia Ferrari, Marilena Flore, Franco Galli, Stefano Grassi, Laura Labbate, Camilla Lambiente, Anna Mauri, Michela Pensato, Martina Rocchi, Graziella Santambrogio, Carlo Silva, Francesca Simonetti, Anna Talarico, Melissa Tonani CYTOTECHNOLOGISTS: Teresa Carbone, Miriam Cosciotti, Grazia Lamonaca, Giulio Legnani, Franca Toffolo CONSULTANT CYTOTECHNOLOGIST: Loredana Alasio Nuclear medicine –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 316 HEAD OF UNIT: Luigi Gianolli CLINICAL UNIT COORDINATOR: Daniela Perani* PHYSICIANS: Elena Busnardo, Carla Canevari, Flaviano Dosio, Federico Fallanca, Claudio Landoni, Patrizia Magnani, Ursola Pajoro, Andrea Panzacchi, Gino Pepe, Maria Picchio, Ana Maria Samanes Gajate, Paola Todeschini, Giovanna Vanoli PHYSICISTS: Valentino Bettinardi, Maria Carla Gilardi, Luca Presotto, Annarita Savi RADIOCHEMISTS: Valeria Masiello, Maria Grazia Minotti, Cristina Monterisi PHD STUDENTS: Elena Maria Andreolli, Ada Apollaro, Flavio Mortarino, Isabella Raccagni TECHNICIANS: Luca Brioschi, Patrizia Cerè, Antonia Compierchio, Paola Cordisco, Valeria Crippa, Silvia Debbia, Andrea Fabro, Davide Gatti, Paola Lanzoni, Lorenzo Lecchi, Stefania Longari, Claudia Maddè, Claudio Mannu, Raffaele Menichini, Giacomo Orlandi, Jacopo Perego, Alice Piana, Riccardo Rigamonti, Lucia Rozza, Francesco Sudati, Mauro Vaghi, Raffaele Vannulli Radiotherapy –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 316 HEAD OF UNIT: Nadia Di Muzio CLINICAL UNIT COORDINATOR: Angelo Bolognesi PHYSICIANS: Saverio Beatrice, Genoveffa Berardi, Anna Chiara, Cesare Cozzarini, Aniko Maria Deli, Italo Dell’Oca, Andrei Fodor, Marcella Pasetti, Paolo Passoni, Najla Slim FELLOWS: Barbara Noris Chiorda, Flavia Zerbetto TECHNICIANS: Fabio Baratto, Nietta Barricella, Roberta Bin, Alessandro Capelli, Alberta De Leonardis, Caterina Fiordelisi, Laura Longoni, Marilena Martulano, Lidio Palumbo, Diana Parutto, Vincenzo Sacco, Giovannella Salvadori, Andrea Sbalchiero, Simone Selli, Antonella Soccio, Alessandro Tavilla, Andrea Viale Laboratory medicine –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 317 HEAD OF DIVISION: Fernanda Dorigatti HEADS OF UNIT: Ferruccio Ceriotti, Massimo Clementi*, Maurizio Ferrari*(*), Massimo Locatelli, Laura Soldini CLINICAL UNIT LEADERS: Paola Cichero, Stefania Del Rosso, Fulvio Ferrara, Anna Maria Girardi, Cristina Ossi, Marina Pontillo, Sara Racca, Armando Soldarini, Silvana Viganò 313 CLINICAL SERVICES PHYSICIANS: Enzo Boeri, Luciano Crippa, Rita Daverio, Annalisa Fattorini, Nicasio Mancini*, Andrea Motta, Maria Grazia Patricelli, Serena Rolla, Loredana Tomassini, Mladen Trbos BIOLOGISTS: Elena Bazzigaluppi, Sara Benedetti, Silvia Carletti, Anna Carobene, Arianna Crepaldi, Nadia Ghidoli, Rossella Ieri, Rosanna Latino, Marcello Marinelli, Gabriella Passerini, Elisabetta Pattarini, Flavia Piccini, Barbara Maria Pirola, Laura Seghezzi, Barbara Sioli, Ivana Spiga, Annunziata Spina, Monica Zanussi BIOENGINEER: Davide Alessio CONSULTANTS: Emanuele Bosi *(#), Roberto Burioni *, Armando D’Angelo (#) FELLOWS: Angela Brisci, Filippo Canducci, Nicola Clementi, Roberta Diotti, Chiara Di Resta, Silvia Galbiati, Gisella Moreno, Diego Saita, Giuseppe Andrea Sautto, Laura Solforosi, Stefania Stenirri TECHNICIANS: Michela Sampaolo, Francesca Sampietro,(#), Nadia Soriani (*) (*) reporting to the Center for Translational Genomics and BioInformatics (#) reporting to the Division of Metabolic and Cardiovascular Sciences Immunohematology and transfusion medicine –––––––––––––––––––––––––––––––––––––––– 317 HEAD OF UNIT: Fabio Ciceri CLINICAL UNIT LEADERS: Lorena Barzizza, Laura Bellio PHYSICIANS: Cinzia Bargiggia, Milena Coppola, Salvatore Gattillo, Lucia Malabarba, Simona Malato, Raffaella Milani, Paola Ronchi, Michela Tassara BIOLOGISTS: Oriana Perini, Cristina Tresoldi, Elisabetta Zino CHEMIST: Benedetta Mazzi TECHNICIANS: Cristina Parisi, Gabriele Torriani, Matilde Zambelli Emergency medicine –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 318 HEAD OF UNIT: Michele Carlucci DIRECTOR OF EMERGENCY MEDICINE PROGRAM: Marzia Spessot CLINICAL UNIT COORDINATORS: Pietro Bisagni, Anna Borri, Roberto Faccincani, Maria Vittoria Lavorato, Federica Mariani PHYSICIANS: Aldo Beneduce, Giuseppe Capasso, Laura Ferrario, Manuela Fortunato, Federico Furlan, Stefano Franchini, Simona Mauri, Enrico Ortolano, Cecilia Piani, Simona Rocchetti, Maria Vittoria Taglietti, Valentina Tomajer RESIDENTS: Nicola Bettera, Massimiliano Bissolati, Barbara Calcaterra, Giovanni Luigi Capretti, Gabriella Cicenia, Sara Dal Farra, Andrea Duca, Luca Ferrante, Annalisa Gagliano, Paolo Giovanni Gazzetta, Luca Ghirardelli, Cristina Gilardini, Barbara Guglielmi, Maria Lemma, Alessandro Marinosci, Carlo Messina, Francesca Motta, Jacopo Nifosi, Francesca Pavesi, Nicolò Pecorelli, Francesca Ratti, Maria Chiara Salandini, Eleonora Setti, Mirta Tiraboschi, Anna Chiara Uccellatore General intensive care –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 318 HEAD OF UNIT: Alberto Zangrillo* CLINICAL UNIT COORDINATOR: Paolo Silvani General anesthesia and neurointensive care Unit –––––––––––––––––––––––––––––––––––– 319 HEAD OF UNIT: Luigi Beretta* CLINICAL UNIT COORDINATORS: Massimo Caldi, Daniela Giudici * Professor at: Università Vita-Salute San Raffaele CLINICAL SERVICES Pathology The OSR Pathology Unit provides complete diagnostic services in anatomic/surgical pathology and autopsy service. Our Unit receives internal samples from Ospedale San Raffaele Scientific Institute and from several other hospital members of the San Donato Group, with approximately 48.000 surgical pathology cases per year; cytological samples are 31.000 per year. We perform also a same day diagnostic service for prostatic biopsies, in which sampling, biopsy processing and reporting are completed in about three hours. Our activity encompasses all the wide variety of pathology scenario. Moreover, approximately 1250 cases are reviewed either by our Unit when patients, whose pathology specimens were originally examined elsewhere, are referred to OSR for treatment or when other pathologists refer difficult cases for second opinions. The number of specimens examined in surgical pathology has increased by almost 30% per year in the last 2 years. The specialized medical and surgical practices at San Raffaele Hospital are reflected in the diversity of the specimen materials in our activity. Pathologists with particular interest and expertise in different specialty areas (e.g., Breast Pathology, Dermatopathology, Gastrointestinal&Hepatic Pathology, Gynecologic Pathology, Hematopathology, Neuropathology, Pulmonary Pathology, Soft Tissue Pathology, and Urologic Pathology) interpret and signout these cases. This approach ensures excellent diagnostic interpretations, enhancing collaboration with the specialized clinical services present in San Raffaele Hospital. Diagnosis and complementary services are provided by experienced and board-certified faculty and technical staff, represented by 15 pathologists, 2 biologists, 26 technicians. Many of our staff pathologists exert their specific activity also when they are active members of Disease Units present at Our Istitutions, where the individual patients are specifically managed by a team including different specialists. Available tools include frozen sections, formalinfixed paraffin-embedded tissues, light microscopy, immunohistochemistry and immunofluorescent staining, tissue micro- and macro-arrays, in-situ hybridization, and laser-capture assisted microdissection, with a continuously expanding and updated wide range of reagents. The Oncologic Molecular Diagnostics Laboratory performs testing at the forefront of molecular medicine using both molecular biology and molecular cytogenetics techinques. This rapidly growing lab provides a wide range of diagnostic tests for solid tumors and hematolymphoid malignancies for both OSR and external patients. Many years of close interaction with our internal pathologists and our referring physicians have provided experience in understanding the diagnostic complexities (histotype evaluation, sample selections, neoplastic component estimation) and practical implications associated with these kind of tests. The molecular biology tests at present performed include: • KRAS, NRAS, EGFR, BRAF, PIK3CA, IDH1/2 genes mutation analysis • MGMT methylation analysis Methods and technologies utilized: standard PCR, real-time PCR, Sanger sequencing, Pyrosequencing, Sequenom MassArray Genotyping. We perform around 1,500 tests a year on genomic DNA extracted from a variety of specimen types, including formalin-fixed paraffin-embedded tissues, fresh and frozen tissues, cytological samples and body fluids. The molecular cytogenetics tests include: • HER2, FGFR1, MET,gene amplification • ALK and ROS1 gene rearrangement • EGFR gene copy number • 1p and 19q chromosomal region deletions • FISH analysis in hematological malignancies (MYC, BCL1, BCL2, BCL6, BCL10, p53, ATM, etc) We perform around 1,000 tests a year on a variety of specimen types, including formalin-fixed paraffin-embedded tissues, frozen tissues, cytological samples and blood. Claudio Doglioni 315 CLINICAL SERVICES Nuclear medicine The Nuclear Medicine Unit clinical activity may be generally divided into two main fields: the “conventional” nuclear medicine activity, using single photon emitting isotopes, such as 99m-Tc, 111In, and the positron emission tomography (PET) nuclear medicine activity, using positron emitting isotopes, such as 18F and 11C. As for “conventional” nuclear medicine, the current main clinical indications are the cardiovascular evaluation of ischemic patients, the study of skeletal system for both oncological and non-oncological disorders, the functional evaluation of urinary, respiratory and endocrinal systems, and the assessment of inflammatory and infectious processes. For these examinations, the Centre is provided with advanced equipment and techniques, including 3 SPECT and 1 SPECT/CT. The principal clinical activity in Nuclear Medicine Unit is PET imaging. In particular, in our Department, the integrated modality PET/computed tomography (PET/CT) scan is commonly used. The Nuclear Medicine Unit / PET Centre provided with the most advanced technology. Currently, there are two cyclotrons (18 MeV and 11 MeV), 4 PET/CT tomographs, five radiochemical laboratories equipped with hot cells and automatic systems for the synthesis of PET radiopharmaceuticals, according to “Norme di Buona Preparazione dei Radiofarmaci in Medicina Nucleare (NBPMN)”. The main clinical indication of PET imaging is the evaluation of oncological patients. PET/CT is particularly used in different phases of diagnostic processes, including the staging and the re-staging of the disease, the planning of radiotherapy treatment, the monitoring and the evaluation of therapies. In addition to oncology, by using PET/CT, cardiac and neurological studies are also performed, and particularly for the evaluation of coronary diseases and degenerative disorders, respectively. Luigi Gianolli Radiotherapy Radiotherapy (RT) currently represents one of the main treatments in oncologic patients, 60-80% of whom are candidate to RT as exclusive treatment ( radical or palliative) ,or as adjuvant treatment after surgery . In particular today Radiotherapy could be used as a valid alternative to surgery or chemotherapy in selected groups of patients. In our Department we treat up to 2000 new patients/year and all cancer types except for paediatric disease . The installation in 2004 of the new generation of Linac allowed us to improve the quality of our treatments by means of Intensity Modulated Radiation Therapy ( IMRT) and Image Guided Radiation Therapy (IGRT). Thanks to greater precision in delivering the curative dose in our clinical activity we are able to use hypofractionated schemes for the principal tumor pathologies, reducing the total length of the treatment. The use of metabolic imaging by means of PET/CT, permits an improved identification of disease sites with elevated cellular activity. These images are useful, both for better definition of treatment volume and in particular the identification of sub-volumes to which higher radiation doses can be delivered thanks to the treatment technique known as SIB (Simultaneous Integrated Boost). MR imaging has been shown to be particularly effective in the identification of organs at risk during the planning process, and of specific characteristics of the target to be irradiated (especially in the head/neck, prostate and brain districts). These treatment modalities have permitted the improvement of the therapeutic index as well as the decrease of iatrogenic sequels with a concurrent reduction in the number of treatment sessions. PET/TC and MR repeated during the radiation treatment, in combination with daily IGRT, can address tumor volume and positional changes, as well as other pathologic changes and deformations occurring during the RT treatment course, introducing the potential for individualized plan adaptation based on imaging feedback, including bulky residual disease, tumor progression, and physiological changes that occur during the treatment course, thereby enabling better normal tissue sparing while allowing radical target doses with the possibility of maximising local control. Finally for lesions subject to organ motion (e.g. lung, liver , pancreatic lesions) four-Dimensional PET/TC allows more accurate target definition, limiting dose to nearby organs at risk. In addition, advances in dose calculation algorithms have allowed for more accurate dosimetry in heterogeneous media, and intensity modulated and arc/ helical delivery techniques can help spare organs at risk. Nadia Di Muzio CLINICAL SERVICES Laboratory medicine Diagnostica e Ricerca San Raffaele (Laboraf) is the Laboratory Medicine Unit that provides clinical laboratory services to the San Raffaele Hospital. Laboraf is organized as a department that includes: Clinical Chemistry - Toxicology - Immunochemistry, Haematology (in conjunction with the Immunohematology and Transfusion Medicine Service), Coagulation, Microbiology - Virology and Serology, Clinical Molecular Biology and Cytogenetic, Autoimmune diseases. All these sectors are highly specialized and continuously evolving. In 2013 Laboraf produced about 7.2 millions of clinical analyses, for a total of 990,000 patients. In 2013 21 new types of tests were introduced in various fields (microbiology and virology, molecular biology, new biomarkers and toxicology tests). Besides clinical services Laboraf produces also research work in the field of Standardization in Clinical Chemistry, in Microbiology and Virology. 1. Standardization in Clinical Chemistry. Development of a reference measurement procedure for the determination of Pancreatic Lipase catalytic activity. Definition of reference intervals and biological variation. 2. Microbiology. Molecular diagnosis of sepsis; molecular diagnosis of systemic fungal infections; analysis of gut and oral microbiome in different clinical conditions. 3. Virology. Molecular diagnosis of infections by viral agents of acute respiratory diseases (myxoviruses, coronaviruses, respiratory syncytial virus and viruses identified recently in infants with acute respiratory diseases); molecular monitoring of antiviral therapies, including detection of drug-resistant variants; in vitro characterization of neutralizing anti-HCV and anti-Influenza A human monoclonal antibodies; cross-reacting and neutralizing human monoclonal antibody directed against the HCV\E2 protein; Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia; antiviral Response Elicited by Anti-Idiotype Monoclonal Antibodies. Laboraf collaborates also in research projects with the research Center for Translational Genomics and BioInformatics, with the research Division of metabolic and cardiovascular sciences and with the Department of Internal and specialistic medicine. Fernanda Dorigatti Immunohematology and transfusion medicine service The Hospital San Raffaele (OSR) Immunohematology and Transfusion Medicine Service (ITMS) provides clinical services to support OSR patients in need of blood component therapy, cellular therapy, therapeutic apheresis, and specialized laboratory diagnostics. This ISO 9002 Certified unit collects and prepares the blood components and cellular therapy products used in patient care at the OSR, maintain an accredited Immunohematology Reference Lab and provides education in the field of Transfusion Medicine. ITMS is conducting a project, funded by Regione Lombardia, on the appropriateness of blood transfusion. The ITMS is subdivided into three distinct subunits, each responsible for a particular process: Blood Donation Center The blood donation center subunit is responsible for the collection and testing of blood to be transfused into patients at the San Raffaele Hospital. It is responsible for handling all aspects of donor recruitment for whole blood products, apheresis products, the auto-transfusion program and therapeutic phlebotomy. During 2013 were presented to make a donation 8376 candidates of these only 7021 have donated. 5550 were donations of whole blood and 1471 were donations by apheresis, plateletpheresis or plasmapheresis. Therapeutic Apheresis and Cellular Therapy The Therapeutic Apheresis and Cellular Therapy subunit is responsible for collecting and processing hematopoietic stem cells and performs the necessary diagnostic testing required for bone marrow transplantation procedures. The lab supplies hematopoietic stem cells and relevant cells in support of clinical trials for allogenic bone marrow and peripheral blood transplantation and offers a Stem Cell Cryo Banking service for autologous and allogeneic bone marrow products. Bone Marrow from qualified donors is collected in accordance and recognition of the Italian Bone Marrow Donor Registry (IBMDR). Therapeutic apheresis procedures to treat patients with neurologic and blood diseases, including photopheresis, red cell and plasma exchange procedures are also performed routinely. The unit of therapeutic apheresis is part of the 317 CLINICAL SERVICES bone marrow transplants program and underwent in October 2013, accreditation Jacie inspection. During 2013, were performed 2010 apheresis for the collection of autologous or allogeneic hematopoietic stem cells. The procedures of photopheresis were 150 while the plasma exchange procedures were 70. Clinical Laboratory Diagnostics The Clinical Laboratory Diagnostics subunit includes the following specialized laboratories: Immunohematology, Hematology, Flow Cytometry, Hematological Molecular Biology and an EFI Certified and accredited HLA tissue typing laboratory. New technologies such as genetic red blood cell typing are being investigated. A special emphasis is given to onco-hematologic malignancies and to the development of minimal-residual-disease tools. Fabio Ciceri Emergency medicine In 2013 the Emergency Department of San Raffaele Hospital provided care for 62,157 patients. 1,346 of the triaged patients were given a red code (that is, to be seen immediately in the resuscitation area). 11,610 of them were given a yellow code (that is, to be seen within 30 minutes of arrival). The largest amount of them (46,988) were given lower priority (green code, that means to be seen within 1 hour of arrival). Only 2,213 of them were given a white code (that is, patients whose conditions are not true emergencies). In 2013 we have seen 20,498 patients with medical problems, 15,800 with surgical problems and 10,628 with minor trauma. 7,450 children have been treated in the pediatric area. In the dedicated area for obstetrics 5,186 women received treatment. 10,449 patients, after initial evaluation, were admitted to different wards for further investigations and treatments. In 2013 608 patients received surgery in the Emergency room. Major trauma (patient with multiple injuries) is treated by a trauma team who has been trained using the principles taught in the internationally recognized Advanced Trauma Life Support course. Medical emergencies are treated as taught in the Advanced Life Support and Trauma Advanced Life Support courses. Some members of the Emergency Room staff are ALS and ATLS instructors and such courses are regularly held in our Hospital every year. Staff members receive Emergency Medicine upto-date meetings every two weeks. Medical students from Università Vita-Salute are trained on application of classical emergency medicine principals in a human and supportive patient environment. In 2013 physicians attended the following meetings and courses: American Heart Association Dallas, USA, November) Il medico d’urgenza nella gestione della syndrome coronarica acuta (Torino, April) 14th European Congress of Trauma 6 Emergency Surgery (Lyon, May), Trauma Update (Milano, December), Medical Response to Major Incidents (Stockholm, August), Mass Fatality Incidents (Rome, May), FIMEUC National Meeting (Rome, November). International Conference on Health Care System Engeneering (Milan, May), 24th SMART (May); Le urgenze neurologiche: dalla diagnosi alla terapia (Milan, October), Campus in medicina d’Urgenza (Torino, April). Michele Carlucci General intensive care Our General Intensive Care Unit (ICU) admitted 436 patients and the occupational rate was 86% with a mean ICU lenght of stay 6.2+9.8) days. Overall, patients were 61 y, 56% had acute kidney injury and mean SAP II was 35+20.8. Nearly 60% of these patients required an advanced intensive treatment, while the others were subjects who needed a postoperative monitoring after major elective surgery. In order to guarantee the care for in-hospital or territorial emergencies and to improve availability towards patients from lower level hospitals, we have recently developed a strategy to optimize ICU admissions, improving an early postoperative care in recovery room for the postoperative patients. The principal critical illnesses managed in our unit (sum is >100% because more than one diagnosis was allowed) included: • Trauma (10% of intensive treatments). As front line of a 2nd level hospital in Milan county, general ICU accept a significant number of subject involved in traffic and work accident; CLINICAL SERVICES • Respiratory failure (77% of intensive treatments). Primary and secondary ARDS (acute respiratory distress syndrome) are frequent and undesirable evolutions of pneumonia or systemic sepsis, especially in immunocompromised patients like in autoimmune pathology or after transplantation; • Cardiovascular failure or multiple organ dysfunctions (43.8 % of intensive treatments). Patients rescued from cardiac arrest or with cardiac congestive failure; • Cardiocirculatory arrests (8.4%) • Septic shock (47% of intensive treatments). Severe evolution of sepsis, complication in patients submitted to major surgery or transplantation; • Neurologic, neuromuscolar and neurophysiological disorders; • Intensive treatment in potential donor patients. In our general ICU we can provide a wide range of therapeutic options and protocols for these specific pathologies, updated to the last international guidelines: the newest strategies in mechanical ventilation including extracorporeal life support, updated antibiotic therapy, hypothermic therapy post cardiac arrest and developments in continuous renal replacement therapy. Improving ultrasound use in ICU through an intensive staff training leaded to the organization of a centralized system for ultrasound central line catheter positioning for more than 600 in-hospital patients/year. The general ICU physicians also provide a medical emergency team (MET) 24 hours a day, which is involved in hospital emergencies and consulting. This kind of organization allows MET to safely perform non invasive ventilation treatment for mild or chronic respiratory failure in non intensive areas. The same ICU staff provides all the non-cardiosurgery emergency surgical procedures. Our ICU promotes and supports a significant number of teaching courses (BLSD, ALS, ATLS, maxiemergency, sepsis campaign diffusion, CVC ultrasound positioning, non invasive ventilation). Alberto Zangrillo General anaesthesia and neurointensive care Unit Neurointensive Care is a 6 beds unit. 300 patients are admitted every year, 50% from the Casualty Department (severe head injury, stroke and subarachnoid haemorrhage) and 50% from the Neurosurgery Unit (tumor, vascular). 20 patients with brain death are treated and 12 of them become organ donors. The Head and Neck Anaesthesia Staff provides for general anaesthesia in neurosurgical, interventional neuroradiology, ENT and ophthalmic surgery for approximately 3,000 cases per year and for conscious sedations in children and adults (1,000 cases per year) submitted to diagnostic or therapeutic procedures in Neuroradiology. The General Anaesthesia Staff provides for anaesthesia in the Surgical Department (Gastroenterology, Haepatology, Endocrine, Pancreatic), Orthopaedics, Obstetrics and Gynaecology, Plastic Surgery (approximately 20,000 cases/per year). Outside the O.R. sedation and anaesthesia are performed for diagnostic and therapeutic interventions in Gastroenterology. A staff is dedicated to the treatment of the postoperative acute pain and chronical neoplastic pain (“Hospital without Pain” Committee). Luigi Beretta 319 CLINICAL SERVICES Selected publications Abdollah, F; Suardi, N; Cozzarini, C; Gallina, A; Capitanio, U; Bianchi, M; Sun, M; Fossati, N; Passoni, NM; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Selecting the Optimal Candidate for Adjuvant Radiotherapy After Radical Prostatectomy for Prostate Cancer: A Long-term Survival Analysis. Eur. Urol.: 2013; 63(6): 998-1008 - Article Aiuti, A; Biasco, L and Scaramuzza, S; Ferrua, F; Cicalese, MP; Baricordi, C; Dionisio, F; Calabria, A; Giannelli, S; Castiello, MC; Bosticardo, M; Evangelio, C; Assanelli, A; Casiraghi, M; Di Nunzio, S; Callegaro, L; Benati, C; Rizzardi, P; Pellin, D; Di Serio, C; Schmidt, M; Von Kalle, C; Gardner, J; Mehta, N; Neduva, V; Dow, DJ; Galy, A; Miniero, R; Finocchi, A; Metin, A; Banerjee, P; Orange, J; Galimberti, S; Valsecchi, MG; Biffi, A; Montini, E; Villa, A; Ciceri, F; Roncarolo, MG and Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome. Science: 2013; 341(6148): 123351 - Article Argentieri, MC; Pilla, D; Vanzati, A; Lonardi, S; Facchetti, F; Doglioni, C; Parravicini, C; Cattoretti, G. Antibodies are forever: A study using 12-26-year-old expired antibodies. Histopathology: 2013; 63(6): 869-876 - Article Azzeroni, R; Maggio, A; Fiorino, C; Mangili, P; Cozzarini, C; De Cobelli, F; Di Muzio, NG; Calandrino, R. Biological optimization of simultaneous boost on intra-prostatic lesions (DILs): Sensitivity to TCP parameters. Phys. Med.: 2013; 29(6): 592-598 - Article Hartmann, S; Döring, C; Jakobus, C; Rengstl, B; Newrzela, S; Tousseyn, T; Sagaert, X; Ponzoni, M; Facchetti, F; de Wolf-Peeters, C; Steidl, C; Gascoyne, R; Küppers, R; Hansmann, ML. Nodular lymphocyte predominant hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma - endpoints of a spectrum of one disease?. PLoS One: 2013; 8(11): e78812 - Article Biffi, A and Montini, E; Lorioli, L; Cesani, M; Fumagalli, F; Plati, T; Baldoli, C; Martino, S; Calabria, A; Canale, S; Benedicenti, F; Vallanti, G; Biasco, L; Leo, S; Kabbara, N; Zanetti, G; Rizzo, WB; Mehta, NA; Cicalese, MP; Casiraghi, M; Boelens, JJ; Del Carro, U; Dow, DJ; Schmidt, M; Assanelli, A; Neduva, V; Di Serio, C; Stupka, E; Gardner, J; von Kalle, C; Bordignon, C; Ciceri, F; Rovelli, A; Roncarolo, MG; Aiuti, A; Sessa, M; Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy. Science: 2013; 341(6148): 1233158 - Article Briganti, A; Joniau, S; Gandaglia, G; Cozzarini, C; Sun, M; Tombal, B; Haustermans, K; Hinkelbein, W; Shariat, SF; Karakiewicz, PI; Montorsi, F; Van Poppel, H; Wiegel, T. Patterns and predictors of early biochemical recurrence after radical prostatectomy and adjuvant radiation therapy in men with pT3N0 prostate cancer: implications for multimodal therapies. Int. J. Radiat. Oncol. Biol. Phys.: 2013; 87(5): 960-967 - Article Broggi, S; Cantone, MC; Chiara, A; Di Muzio, N; Longobardi, B; Mangili, P; Veronese, I. Application of failure mode and effects analysis (FMEA) to pretreatment phases in tomotherapy. J. Appl. Clin. Med. Phys: 2013; 14(5): 265-277 - Article Brunetto, E and Ferrara, AM; Rampoldi, F; Talarico, A; Dal Cin, E; Grassini, G; Spagnuolo, L; Sassi, I; Ferro, A; Veronica Cuorvo, L; Barbareschi, M; Piccinin, S; Maestro, R; Pecciarini, L; Doglioni, C; Cangi, MG. CDC25A protein stability represents a previously unrecognized target of HER2 signaling in human breast cancer: Implication for a potential clinical relevance in trastuzumab treatment. Neoplasia: 2013; 15(6): 579-590 - Article Caganova, M; Carrisi, C and Varano, G; Mainoldi, F; Zanardi, F; Germain, PL; George, L; Alberghini, F; Ferrarini, L; Talukder, AK; Ponzoni, M; Testa, G; Nojima, T; Doglioni, C; Kitamura, D; Toellner, KM; Su, IH; Casola, S. Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis. J Clin Invest: 2013; 123(12): 5009-5022 - Article Calandrino, R; Ardu, V; Corletto, D; Del Vecchio, A; Origgi, D; Signorotto, P; Spinelli, A; Tosi, G; Bolognesi, A; Cariati, M; Kluzer, A; Muscarella, S. Evaluation of second cancer induction risk by CT follow-up in oncological long-surviving patients. Health Phys.: 2013; 104(1): 1-8 - Article Canducci, F; Ceresola, ER; Saita, D; Castagna, A; Gianotti, N; Underwood, M; Burioni, R; Lazzarin, A; Clementi, M. In vitro phenotypes to elvitegravir and dolutegravir in primary macrophages and lymphocytes of clonal recombinant viral variants selected in patients failing raltegravir. J. Antimicrob. Chemother.: 2013; 68(11): 2525-2532 - Article CLINICAL SERVICES Carbone, A; Spina, M; Gloghini, A; Ponzoni, M; Doglioni, C; Tirelli, U. Nodular lymphocyte predominant Hodgkin lymphoma with non-invasive or early invasive growth pattern suggests an early step of the disease with a highly favorable outcome. Am. J. Hematol.: 2013; 88(2): 161-162 - Letter Carobene, A; Braga, F; Roraas, T; Sandberg, S; Bartlett, WA. A systematic review of data on biological variation for alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase. Clin. Chem. Lab. Med.: 2013; 51(10): 1997-2007 - Article Carobene, A; Guerra, E; Ceriotti, F. A mechanism-based way to evaluate commutability of control materials for enzymatic measurements. The example of gamma-glutamyltransferase. Clin. Chim. Acta: 2013; 424: 153-158 - Article Castellucci, P; Picchio, M. 11C-Choline PET/CT and PSA kinetics. Eur. J. Nucl. Med. Mol. Imaging: 2013; 40(1 Suppl): 36-40 - Article Casucci, M; Nicolis di Robilant, B; Falcone, L; Camisa, B; Norelli, M; Genovese, P; Gentner, B; Gullotta, F; Ponzoni, M; Bernardi, M; Marcatti, M; Saudemont, A; Bordignon, C; Savoldo, B; Ciceri, F; Naldini, L; Dotti, G; Bonini, C; Bondanza, A. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood: 2013; 122(20): 3461-3472 - Article Casucci, M; Perna, SK; Falcone, L; Camisa, B; Magnani, Z; Bernardi, M; Crotta, A; Tresoldi, C; Fleischhauer, K; Ponzoni, M; Gregori, S; Caligaris-Cappio, F; Ciceri, F; Bordignon, C; Cignetti, A; Bondanza, A and Bonini, C. Graft-versus-leukemia effect of HLA-haploidentical centralmemory t-cells expanded with leukemic APCs and modified with a suicide gene. Mol. Ther.: 2013; 21(2): 466-475 - Article Cattaneo, GM; Passoni, P; Longobardi, B; Slim, N; Reni, M; Cereda, S; Di Muzio, N; Calandrino, R. Dosimetric and clinical predictors of toxicity following combined chemotherapy and moderately hypofractionated rotational radiotherapy of locally advanced pancreatic adenocarcinoma. Radiother. Oncol.: 2013; 108(1): 66-71 - Article Clementi, N and Mancini, N; Castelli, M; Clementi, M; Burioni, R. Characterization of epitopes recognized by monoclonal antibodies: Experimental approaches supported by freely accessible bioinformatic tools. Drug Discov. Today: 2013; 18(41556): 464-471 - Review Dvir, R and Mancini, N; Assanelli, A; Racca, S; Rolla, S; Clementi, N; Piemontese, S; Ciceri, F; Burioni, R; Clementi, M. Acute respiratory distress in a neutropenic febrile patient after hematopoietic cell transplantation. J. Clin. Virol.: 2013; 57(1): 1-4 - Note Fayad, HJ; Lamare, F; Le Rest, CC; Bettinardi, V; Visvikis, D. Generation of 4-dimensional CT images based on 4-dimensional PET-derived motion fields. J. Nucl. Med.: 2013; 54(4): 631-638 - Article Fellin, F; Azzeroni, R; Maggio, A; Lorentini, S; Cozzarini, C; Di Muzio, N; Fiorino, C; Calandrino, R; Schwarz, M. Helical tomotherapy and intensity modulated proton therapy in the treatment of dominant intraprostatic lesion: A treament planning comparison. Radiother. Oncol.: 2013; 107(2): 207-212 - Article Folli, C; Consonni, D; Spessot, M; Salvini, L; Velati, M; Ranzani, G; Maiavacca, R; Monzani, V. Diagnostic role of copeptin in patients presenting with chest pain in the emergency room. Eur. J. Intern. Med.: 2013; 24(2): 189-193 - Article Garibotto, V; Tettamanti, M; Marcone, A; Florea, I; Panzacchi, A; Moresco, R; Virta, JR; Rinne, J; Cappa, SF; Perani, D. Cholinergic activity correlates with reserve proxies in Alzheimer’s disease. Neurobiol. Aging: 2013; 34(11): e13-e18 - Article Gianotti, N; Galli, L; Salpietro, S; Cernuschi, M; Bossolasco, S; Maillard, M; Spagnuolo, V; Canducci, F; Clementi, M; Lazzarin, A; Castagna, A. Virological rebound in human immunodeficiency virus-infected patients with or without residual viraemia: Results from an extended follow-up. Clin. Microbiol. Infect.: 2013; 19(12): E542-E544 - Article Giovacchini, G; Picchio, M; Garcia-Parra, R; Mapelli, P; Briganti, A; Montorsi, F; Gianolli, L; Messa, C. [(11)C]Choline Positron Emission Tomography/Computerized Tomography for Early Detection of Prostate Cancer Recurrence in Patients with Low Increasing Prostate Specific Antigen. J. Urology: 2013; 189(1): 105-110 - Article Goudy, K and Aydin, D; Barzaghi, F; Gambineri, E; Vignoli, M; Mannurita, SC; Doglioni, C; Ponzoni, M; Cicalese, MP; Assanelli, A; Tommasini, A; Brigida, I; Dellepiane, RM; Martino, S; Olek, 321 CLINICAL SERVICES S; Aiuti, A; Ciceri, F; Roncarolo, MG; Bacchetta, R. Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity. Clin. Immunol.: 2013; 146(3): 248-261 Article Hess Michelini, R and Manzo, T; Sturmheit, T; Basso, V; Rocchi, M; Freschi, M; Listopad, J; Blankenstein, T; Bellone, M and Mondino, A. Vaccine-instructed intratumoral IFN-g enables regression of autochthonous mouse prostate cancer in allogeneic T-cell transplantation. Cancer Res.: 2013; 73(15): 4641-4652 - Article Ichihara, K; Ceriotti, F; Tam, TH; Sueyoshi, S; Poon, PMK; Thong, ML; Higashiuesato, Y; Wang, X; Kataoka, H; Matsubara, A; Shiesh, SC; Muliaty, D; Kim, JH; Watanabe, M; Lam, CWK; Siekmann, L; Lopez, JB; Panteghini, M. The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes. Clin. Chem. Lab. Med.: 2013; 51(7): 14291442 - Article Ichihara, K; Ceriotti, F; Kazuo, M; Huang, YY; Shimizu, Y; Suzuki, H; Kitagawa, M; Yamauchi, K; Hayashi, S; Tsou, CC; Yamamoto, Y; Ishida, S; Leong, L; Sano, M; Lim, HS; Suwabe, A; Woo, HY; Kojima, K; Okubo, Y. The Asian project for collaborative derivation of reference intervals: (2) results of non-standardized analytes and transference of reference intervals to the participating laboratories on the basis of cross-comparison of test results. Clin. Chem. Lab. Med.: 2013; 51(7): 1443-1457 - Article Klunk, WE; Perani, D. Amyloid and neurodegeneration: Converging and diverging paths. Neurology: 2013; 81(20): 1728-1729 - Comment Maffi, P and Balzano, G; Ponzoni, M; Nano, R; Sordi, V; Melzi, R; Mercalli, A; Scavini, M; Esposito, A; Peccatori, J; Cantarelli, E; Messina, C; Bernardi, M; Del Maschio, A; Staudacher, C; Doglioni, C; Ciceri, F; Secchi, A; Piemonti, L. Autologous Pancreatic Islet Transplantation in Human Bone Marrow. Diabetes: 2013; 62(10): 3523-3531 - Article Maggio, A; Carillo, V; Cozzarini, C; Perna, L; Rancati, T; Valdagni, R; Gabriele, P; Fiorino, C. Impact of the radiotherapy technique on the correlation between dose-volume histograms of the bladder wall defined on MRI imaging and dose-volume/surface histograms in prostate cancer patients. Phys. Med. Biol.: 2013; 58(7): N115-N123 - Article Mancini, N; Burioni, R; Sanguinetti, M; Clementi, M. Risks of “blind” automated identification systems in medical microbiology. J. Clin. Microbiol.: 2013; 51(11): 3911 - Letter Mapelli, P; Mangili, G; Picchio, M; Gentile, C; Rabaiotti, E; Giorgione, V; Spinapolice, EG; Gianolli, L; Messa, C; Candiani, M. Role of 18F-FDG PET in the management of gestational trophoblastic neoplasia. Eur. J. Nucl. Med. Mol. Imaging: 2013; 40(4): 505-513 - Article Marinova, M; Altinier, S; Caldini, A; Passerini, G; Pizzagalli, G; Brogi, M; Zaninotto, M; Ceriotti, F; Plebani, M. Multicenter evaluation of hemoglobin A1c assay on capillary electrophoresis. Clin. Chim. Acta: 2013; 424: 207-211 - Article Melloni, G; Samanes Gajate, AM; Sestini, S; Gallivanone, F; Bandiera, A; Landoni, C; Muriana, P; Gianolli, L; Zannini, P. New positron emission tomography derived parameters as predictive factors for recurrence in resected stage i non-small cell lung cancer. Eur. J. Surg. Oncol.: 2013; 39(11): 1254-1261 - Article Passoni, P; Fiorino, C; Slim, N; Ronzoni, M; Ricci, V; Di Palo, S; De Nardi, P; Orsenigo, E; Tamburini, A; De Cobelli, F; Losio, C; Iacovelli, NA; Broggi, S; Staudacher, C; Calandrino, R; Di Muzio, N. Feasibility of an adaptive strategy in preoperative radiochemotherapy for rectal cancer with image-guided tomotherapy: Boosting the dose to the shrinking tumor. Int. J. Radiat. Oncol. Biol. Phys.: 2013; 87(1): 67-72 - Article Passoni, P; Reni, M; Cattaneo, GM; Slim, N; Cereda, S; Balzano, G; Castoldi, R; Longobardi, B; Bettinardi, V; Gianolli, L; Gusmini, S; Staudacher, C; Calandrino, R; Di Muzio, N. Hypofractionated image-guided IMRT in advanced pancreatic cancer with simultaneous integrated boost to infiltrated vessels concomitant with capecitabine: A phase i study. Int. J. Radiat. Oncol. Biol. Phys.: 2013; 87(5): 1000-1006 - Article Picchio, M; Piert, M. Prostate cancer imaging. Eur. J. Nucl. Med. Mol. Imaging: 2013; 40(Suppl 1): S1-S4 - Editorial Ponzoni, M; Govi, S; Licata, G; Mappa, S; Giordano Resti, A; Politi, LS; Spagnuolo, L; Di Cairano, E; Doglioni, C; Ferreri, AJM. A reappraisal of the diagnostic and therapeutic management of CLINICAL SERVICES uncommon histologies of primary ocular adnexal lymphoma. Oncologist: 2013; 18(7): 876-884 Review Raccosta, L and Fontana, R; Maggioni, D; Lanterna, C; Villablanca, EJ;Paniccia, A; Musumeci, A; Chiricozzi, E; Trincavelli, ML; Daniele, S; Martini, C; Gustafsson, J; Doglioni, C; Feo, SG; Leiva, A; Ciampa, MG; Mauri, L; Sensi, C; Prinetti, A; Eberini, I; Mora, M; Bordignon, C; Steffensen, KR; Sonnino, S; Sozzani, S; Traversari, C and Russo, V. The oxysterol-cxcr2 axis plays a key role in the recruitment of tumor-promoting neutrophils. J. Exp. Med.: 2013; 210(9): 1711-1728 - Article Ranzani, M; Cesana, D and Bartholomae, CC; Sanvito, F; Pala, M; Benedicenti, F; Gallina, P; Sergi Sergi, L; Merella, S; Bulfone, A; Doglioni, C; Von Kalle, C; Kim, YJ; Schmidt, M; Tonon, G; Naldini, L; Montini, E. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat. Methods: 2013; 10(2): 155-161 - Article Russo, V and Pilla, L; Lunghi, F; Crocchiolo, R; Greco, R; Ciceri, F; Maggioni, D; Fontana, R; Mukenge, S; Rivoltini, L; Rigamonti, G; Mercuri, SR; Nicoletti, R; Del Maschio, A; Gianolli, L; Fazio, F; Marchianò, A; Di Florio, A; Maio, M; Salomoni, M; Gallo-Stampino, C; Del Fiacco, M; Lambiase, A; Coulie, PG; Patuzzo, R; Parmiani, G; Traversari, C; Bordignon, C; Santinami, M and Bregni, M. Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3genetically modified lymphocytes. Int. J. Cancer: 2013; 132(11): 2557-2566 - Article Soliman, C; Casiraghi, M; Antonioli, G; Ciceri, F; Roncarolo, MG; Callegaro, L; Aiuti, A. Percorso assistenziale nel bambino ADA SCID sottoposto a terapia genica con cellule staminali ematopoietiche: Il ruolo dell’infermiere di ricerca. Pediatric Reports: 2013; 5(SUPPL.1): 127-128 Article Todisco, E; Ciceri, F; Oldani, E; Boschini, C; Mico, C; Vanlint, MT; Donnini, I; Patriarca, F; Alessandrino, PE; Bonifazi, F; Arcese, W; Barberi, W; Marenco, P; Terruzzi, E; Cortelazzo, S; Santarone, S; Proia, A; Corradini, P; Tagliaferri, E; Falcioni, S; Irrera, G; Dallanegra, L; Castagna, L; Santoro, A; Camboni, A; Sacchi, N; Bosi, A; Bacigalupo, A; Rambaldi, A. The CIBMTR score predicts survival of AML patients undergoing allogeneic transplantation with active disease after a myeloablative or reduced intensity conditioning: A retrospective analysis of the Gruppo Italiano Trapianto di Midollo Osseo. Leukemia: 2013; 27(10): 2086-2091 - Letter 323 CLINICAL SERVICES WHO Collaborating Centre for Integrated Laboratory Strengthening on Tuberculosis, WHO Supranational Reference Laboratory and ESCMID collaborative Centre Tuberculosis (TB) causes nearly 2 million deaths each year. Current TB treatment is long, and drug resistance (DR) continues to increase globally with multiple and extensively drug-resistant (MDR and XDR) TB being major threats to TB Control worldwide. In this context, the Global TB Department of the World Health Organization (WHO) gives high priority to the prevention of the emergence of additional drug resistance, for which the laboratory surveillance of drug resistance represents an essential tool in evaluating and monitoring quality and effectiveness of TB control programmes. In May 2013, on the basis of the successful collaborative activities with the WHO Stop TB department, and of the acknowledged experience in providing technical assistance in the field of mycobacteriology the Fondazione Centro San Raffaele was reconfirmed by the WHO and the Italian Ministry of Health “WHO Collaborating Centre for Integrated Laboratory Strengthening on Tuberculosis” (WHOCC, ITA98). This recognition is in addition to the status of member of Supranational Reference Laboratory Network. The activities performed by the Center during 2013 cover three main areas: Laboratory training courses on laboratory techniques and management for laboratory staff from high incidence Countries and for laboratory consultants, with the aim to increase the knowledge and skills of laboratory staff in assisted Countries. Five training courses were offered no different topics including a national training in advanced mycobacteriology. We have organized an advanced national training course on mycobacteriology in Milan and supported the molecular diagnostics implementation in Ethiopia, Ivory Coast and Burkina Faso. Planning and supporting high burden Countries in conducting Operational Research on new diagnostic tools for drug resistant TB detection. This study gathers answers to scientific research questions and will serve as a basis for improving fast detection and treatment of drug resistant tuberculosis cases. Two studies are ongoing: the first aiming at the implementation of new regimens for shorter treatment of drug resistant tuberculosis (in collaboration with the University of Brescia WHOcc and other international partners), the second on the rational use of new diagnostics in several high burden Countries. In support to the Global Program for TB drug resistance surveillance we have optimized a protocol based on Next Generation Sequencing for fast detection of mutation. The protocol was used to detect the pharmacoresistance to floroquinolones and pyrazinamide on 1400 TB strains collected in Pakistan and Bangladesh. Laboratory technical assistance to Countries: in order to contribute to meet the local national TB programme’s objectives in the area, we provide long term support in the implementation of laboratory activities at countries level according to the WHO global plan guidelines. Through co-funding by WHO and other international partners, the Centre has performed 14 monitoring and assessment visits providing technical assistance and supervision to the TB programs and the TB National Reference laboratories in 10 Countries (Mozambique, Ethiopia, Nigeria, Albania, Kosovo, Turkey, Iraq, Burkina Faso, Ivory Coast). We have participated to national review missions of the National TB Control program in Pakistan and in Nigeria. In addition, in the role of ESCMID center and supported by the ESCMID short visit exchange program, 4 scientists were hosted by EBPU for completion of their research projects. Main focus of the four ESCMID sponsored observerships was the typing of multidrug resistant pathogens emerging worldwide. Daniela Maria Cirillo Head, WHO Collaborating Centre ITA 98 TB Supranational Reference laboratory PUBLICATIONS 325 PUBLICATIONS P.1. Abbas, AK and Benoist, C and Bluestone, JA and Campbell, DJ and Ghosh, S and Hori, S and Jiang, S and Kuchroo, VK and Mathis, D and Roncarolo, MG and Rudensky, A and Sakaguchi, S and Shevach, EM and Vignali, DAA and Ziegler, SF. Regulatory T cells: Recommendations to simplify the nomenclature. Nat. Immunol.: 2013; 14(4): 307-308 - Letter P.2. Abdollah, F; Abdo, A; Sun, M; Schmitges, J; Tian, Z; Briganti, A; Shariat, SF; Perrotte, P; Montorsi, F; Karakiewicz, PI. Pelvic lymph node dissection for prostate cancer: Adherence and accuracy of the recent guidelines. Int. J. Urol.: 2013; 20(4): 405-410 - Article IF 2012: 1,734 P.3. Abdollah, F and Boorjian, S; Cozzarini, C; Suardi, N; Sun, M; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Montorsi, F; Karnes, RJ; Briganti, A. Survival following biochemical recurrence after radical prostatectomy and adjuvant radiotherapy in patients with prostate cancer: The impact of competing causes of mortality and patient stratification. Eur. Urol.: 2013; 64(4): 557-564 - Article IF 2012: 10,476 P.4. Abdollah, F; Cozzarini, C; Sun, M; Suardi, N; Gallina, A; Passoni, NM; Bianchi, M; Tutolo, M; Fossati, N; Nini, A; Dell’Oglio, P; Salonia, A; Karakiewicz, P; Montorsi, F; Briganti, A. Assessing the most accurate formula to predict the risk of lymph node metastases from prostate cancer in contemporary patients treated with radical prostatectomy and extended pelvic lymph node dissection. Radiother. Oncol.: 2013; 109(2): 211-216 - Article IF 2012: 4,520 P.5. Abdollah, F and Gandaglia, G; Thuret, R; Schmitges, J; Tian, Z; Jeldres, C; Passoni, NM; Briganti, A; Shariat, SF; Perrotte, P; Montorsi, F; Karakiewicz, PI; Sun, M. Incidence, survival and mortality rates of stage-specific bladder cancer in United States: A trend analysis. Cancer Epidemiol.: 2013; 37(3): 219-225 - Article IF 2012: 2,232 P.6. Abdollah, F; Suardi, N; Capitanio, U; Gallina, A; Sun, M; Villa, L; Scattoni, V; Bianchi, M; Tutolo, M; Fossati, N; Karakiewicz, P; Rigatti, P; Montorsi, F; Briganti, A. Spatial distribution of positive cores improves the selection of patients with low-risk prostate cancer as candidates for active surveillance. BJU Int.: 2013; 112(4): E234-E242 Article IF 2012: 3,046 P.7. Abdollah, F; Suardi, N; Cozzarini, C; Gallina, A; Capitanio, U; Bianchi, M; Sun, M; Fossati, N; Passoni, NM; Fiorino, C; Di Muzio, N; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Selecting the Optimal Candidate for Adjuvant Radiotherapy After Radical Prostatectomy for Prostate Cancer: A Long-term Survival Analysis. Eur. Urol.: 2013; 63(6): 998-1008 - Article IF 2012: 10,476 P.8. Abdollah, F; Suardi, N; Gallina, A; Bianchi, M; Tutolo, M; Passoni, N; Fossati, N; Sun, M; dell’Oglio, P; Salonia, A; Karakiewicz, PI; Rigatti, P; Montorsi, F; Briganti, A. Extended pelvic lymph node dissection in prostate cancer: A 20-year audit in a single center. Ann. Oncol.: 2013; 24(6): 1459-1466 - Article IF 2012: 7,384 P.9. Abdollah, F; Sun, M; Suardi, N; Gallina, A; Capitanio, U; Bianchi, M; Tutolo, M; Fossati, N; Castiglione, F; Freschi, M; Karakiewicz, P; Rigatti, P; Montorsi, F; Briganti, A. Presence of positive surgical margin in patients with organ-confined prostate cancer equals to extracapsular extension negative surgical margin. A plea for TNM staging system reclassification. Urol. Oncol.: 2013; 31(8): 1497-1503 - Article IF 2012: 3,647 P.10. Abdollah, F; Sun, M; Suardi, N; Gallina, A; Tutolo, M; Passoni, N; Bianchi, M; Salonia, A; Colombo, R; Rigatti, P; Karakiewicz, PI; Montorsi, F; Briganti, A. A novel tool to assess the risk of urinary incontinence after nervesparing radical prostatectomy. BJU Int.: 2013; 111(6): 905-913 - Article IF 2012: 3,046 P.11. Abraham, AB; Bronstein, R; Chen, EI; Koller, A; Ronfani, L; Maletic-Savatic, M; Tsirka, SE. Members of the high mobility group B protein family are dynamically expressed in embryonic neural stem cells. Proteome Sci.: 2013; 11(1): 18 - Article IF 2012: 2,420 P.12. Abutalebi, J. Bilingualism beyond languages: The impact of bilingualism upon the brain. Comment on “The bilingual brain: Flexibility and control in the human cortex” by Buchweitz and Prat.. Phys. Life Rev.: 2013; 10(4): 444445 - Comment P.13. Abutalebi, J; Della Rosa, PA; Castro Gonzaga, AK; Keim, R; Costa, A; Perani, D. The role of the left putamen in multilingual language production. Brain Lang.: 2013; 125(3): 307-315 - Article IF 2012: 3,386 P.14. Abutalebi, J; Della Rosa, PA; Ding, G; Weekes, B; Costa, A; Green, DW. Language proficiency modulates the engagement of cognitive control areas in multilinguals. Cortex: 2013; 49(3): 905-911 - Article IF 2012: 6,161 P.15. Acquati, F; Lualdi, M; Bertilaccio, S; Monti, L; Turconi, G; Fabbri, M; Grimaldi, A; Anselmo, A; Inforzato, A; Collotta, A; Cimetti, L; Riva, C; Gribaldo, L; Ghia, P; Taramelli, R. Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis. Proc. Natl. Acad. Sci. U. S. A.: 2013; 110(20): 8140-8145 - Article IF 2012: 9,737 P.16. Adorno, M; Sikandar, S; Mitra, SS; Kuo, A; Nicolis Di Robilant, B; Haro-Acosta, V; Ouadah, Y; Quarta, M; Rodriguez, J; Qian, D; Reddy, VM; Cheshier, S; Garner, CC; Clarke, MF. Usp16 contributes to somatic stem-cell defects in Down’s syndrome. Nature: 2013; 501(7467): 380384 - Article+F12 IF 2012: 38,597 P.17. Agathangelidis, A; Ntoufa, S; Stamatopoulos, K. B Cell receptor and antigens in CLL. Adv. Exp. Med. Biol.: 2013; 792: 1-24 - Article IF 2012: 1,825 P.18. Agnelli, G; Buller, HR; Cohen, A; Curto, M; Gallus, AS; Johnson, M; Masiukiewicz, U; Pak, R; Thompson, J; Raskob, GE; Weitz, JI; for the AMPLIFY Investigators. Oral apixaban for the treatment of acute venous thromboembolism. N. Engl. J. Med.: 2013; 369(9): 799-808 Article IF 2012: 51,658 P.19. Agnelli, G; Buller, HR; Cohen, A; Curto, M; Gallus, AS; Johnson, M; Porcari, A; Raskob, GE; Weitz, JI; for the AMPLIFY-EXT Investigators. Apixaban for Extended Treatment of Venous Thromboembolism. N. Engl. J. Med.: 2013; 368(8): 699-708 - Article IF 2012: 51,658 P.20. Agosta, F; Canu, E; Stojkovic, T; Pievani, M; Tomic, A; Sarro, L; Dragasevic, N; Copetti, M; Comi, G; Kostic, VS; Filippi, M. The topography of brain damage at different stages of parkinson’s disease. Hum. Brain Mapp.: 2013; 34(11): 2798-2807 - Article IF 2012: 6,878 P.21. Agosta, F; Canu, E; Valsasina, P; Riva, N; Prelle, A; Comi, G; Filippi, M. Divergent brain network connectivity in amyotrophic lateral sclerosis. Neurobiol. Aging: 2013; 34(2): 419-427 - Article IF 2012: 6,166 P.22. Agosta, F; Caso, F; Filippi, M. Dementia and neuroimaging. J. Neurol.: 2013; 260(2): 685-691 - Article IF 2012: 3,578 327 PUBLICATIONS P.23. Agosta, F; Galantucci, S; Canu, E; Cappa, SF; Magnani, G; Franceschi, M; Falini, A; Comi, G; Filippi, M. Disruption of structural connectivity along the dorsal and ventral language pathways in patients with nonfluent and semantic variant primary progressive aphasia: A DT MRI study and a literature review. Brain Lang.: 2013; 127(2): 157-166 - Article IF 2012: 3,386 P.24. Agosta, F; Kostic, VS; Davidovic, K; Kresojevic, ´ N; Sarro, L; Svetel, M; Stankovi´c, I; Comi, G; Klein, C; Filippi, M. White matter abnormalities in Parkinson’s disease patients with glucocerebrosidase gene mutations. Mov. Disord.: 2013; 28(6): 772-778 - Article IF 2012: 4,558 P.25. Agosta, F; Sala, S; Valsasina, P; Meani, A; Canu, E; Magnani, G; Cappa, SF; Scola, E; Quatto, P; Horsfield, MA; Falini, A; Comi, G; Filippi, M. Brain network connectivity assessed using graph theory in frontotemporal dementia. Neurology: 2013; 81(2): 134-143 - Article IF 2012: 8,249 P.26. Agretti, P and Segni, M; De Marco, G; Ferrarini, E; Di Cosmo, C; Corrias, A; Weber, G; Larizza, D; Calcaterra, V; Pelizzo, MR; Cesaretti, G; Vitti, P; Tonacchera, M. Prevalence of activating thyrotropin receptor and Gsa gene mutations in paediatric thyroid toxic adenomas: A multicentric Italian study. Clin. Endocrinol.: 2013; 79(5): 747-749 - Letter IF 2012: 3,396 P.27. Agricola, E and Slavich, M; Tufaro, V; Fisicaro, A; Oppizzi, M; Melissano, G; Bertoglio, L; Marone, E; Civilini, E; Margonato, A; Chiesa, R. Prevalence of thoracic ascending aortic aneurysm in adult patients with known abdominal aortic aneurysm: An echocardiographic study. Int. J. Cardiol.: 2013; 168(3): 3147-3148 - Letter IF 2012: 5,509 P.28. Aiuti, A; Biasco, L and Scaramuzza, S; Ferrua, F; Cicalese, MP; Baricordi, C; Dionisio, F; Calabria, A; Giannelli, S; Castiello, MC; Bosticardo, M; Evangelio, C; Assanelli, A; Casiraghi, M; Di Nunzio, S; Callegaro, L; Benati, C; Rizzardi, P; Pellin, D; Di Serio, C; Schmidt, M; Von Kalle, C; Gardner, J; Mehta, N; Neduva, V; Dow, DJ; Galy, A; Miniero, R; Finocchi, A; Metin, A; Banerjee, P; Orange, J; Galimberti, S; Valsecchi, MG; Biffi, A; Montini, E; Villa, A; Ciceri, F; Roncarolo, MG and Naldini, L. Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome. Science: 2013; 341(6148): 123351 - Article IF 2012: 31,027 P.29. Aiuti, A; Cossu, G; de Felipe, P; Galli, MC; Narayanan, G; Renner, M; Stahlbom, A; Schneider, CK; Voltz-Girolt, C. The committee for advanced therapies’ of the European Medicines Agency reflection paper on management of clinical risks deriving from insertional mutagenesis. Hum. Gene Ther. Clin. Dev.: 2013; 24(2): 47-54 - Article IF 2012: No Impact Factor P.30. Alasker, A and Meskawi, M; Sun, M; Ismail, S; Hanna, N; Hansen, J; Tian, Z; Bianchi, M; Perrotte, P; Karakiewicz, PI. A contemporary update on rates and management of toxicities of targeted therapies for metastatic renal cell carcinoma. Cancer Treat. Rev.: 2013; 39(4): 388-401 - Review IF 2012: 6,024 P.31. Alberici, L; Roth, L; Sugahara, KN; Agemy, L; Kotamraju, VR; Teesalu, T; Bordignon, C; Traversari, C; Rizzardi, GP; Ruoslahti, E. De Novo design of a tumor-penetrating peptide. Cancer Res.: 2013; 73(2): 804-812 - Article IF 2012: 8,650 P.32. Alessandrino, EP and Porta, MGD and Malcovati, L and Jackson, CH and Pascutto, C; Bacigalupo, A; Teresa van Lint, M; Falda, M; Bernardi, M; Onida, F; Guidi, S; Iori, AP; Cerretti, R; Marenco, P; Pioltelli, P; Angelucci, E; Oneto, R; Ripamonti, F; Rambaldi, A; Bosi, A and Cazzola, M; and on behalf of Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Optimal timing of allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome. Am. J. Hematol.: 2013; 88(7): 581-588 - Article IF 2012: 4,138 P.33. Alfano, M; Graziano, F; Genovese, L; Poli, G. Macrophage polarization at the crossroad between hiv-1 infection and cancer development. Arterioscler. Thromb. Vasc. Biol.: 2013; 33(6): 1145-1152 - Article IF 2012: 6,338 P.34. Alfieri, O; De Bonis, M. Tricuspid valve surgery for severe tricuspid regurgitation. Heart: 2013; 99(3): 149-150 Comment P.35. Alfieri, O; Pozzoli, A. Mitral valve surgery in octogenarians: Insights and perspectives. J. Cardiothorac. Vasc. Anesth.: 2013; 27(2): 201-202 - Comment P.36. Amato, MP; Langdon, D; Montalban, X; Benedict, RHB; Deluca, J; Krupp, LB; Thompson, AJ; Comi, G. Treatment of cognitive impairment in multiple sclerosis: Position paper. J. Neurol.: 2013; 260(6): 1452-1468 - Review IF 2012: 3,578 P.37. Amato, N; Riva, N; Cursi, M; Martins-Silva, A; Martinelli, V; Comola, M; Fazio, R; Comi, G; Leocani, L. Different Frontal Involvement in ALS and PLS Revealed by Stroop Event-Related Potentials and Reaction Times. Front. Aging Neurosci.: 2013; 5: 82 - Article IF 2012: 5,224 P.38. Amendola, M; Giustacchini, A; Gentner, B; Naldini, L. A double-switch vector system positively regulates transgene expression by endogenous microRNA expression (miR-ON vector). Mol. Ther.: 2013; 21(5): 934-946 - Article IF 2012: 7,041 P.39. Ammirati, E; Cristell, N; Cianflone, D; Vermi, AC; Marenzi, G; De Metrio, M; Uren, NG; Hu, D; Ravasi, T; Maseri, A; Cannistraci, CV. Questing for circadian dependence in ST-segment-elevation acute myocardial infarction: a multicentric and multiethnic study. Circ. Res.: 2013; 112(10): e110-e114 - Article IF 2012: 11,861 P.40. Ammirati, E; Musca, F; Oliva, F; Garascia, A; Pacher, V; Verde, A; Cipriani, M; Moreo, A; Martinelli, L; Frigerio, M. Levosimendan reverted severe pulmonary hypertension in one patient on waiting list for heart transplantation. Int. J. Cardiol.: 2013; 168(4): 4518-4519 - Letter IF 2012: 5,509 P.41. Amodio, G and Mugione, A; Sanchez, AM; Viganò, P; Candiani, M; Somigliana, E; Roncarolo, MG; PaninaBordignon, P and Gregori, S. HLA-G expressing DC-10 and CD4(+) T cells accumulate in human decidua during pregnancy. Hum. Immunol.: 2013; 74(4): 406-411 - Article IF 2012: 2,298 P.42. Amzulescu, MS; Slavich, M; Florian, A; Goetschalckx, K; Voigt, JU. 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Autores/miembros del Grupo de Trabajo: Camm, AJ; Lip, GY; Caterina, RD; Savelieva, I; Atar, D; Hohnloser, SH; Hindricks, G; Kirchhof, P; Comité de la ESC de Guías para la Práctica Clínica (CGPC): Bax, JJ; Baumgartner, H; Ceconi, C; Dean, V; Deaton, C; Fagard, R; Funck-Brentano, C; Hasdai, D; Hoes, A; Kirchhof, P; Knuuti, J; Kolh, P; McDonagh, T; Moulin, C; Popescu, BA; Reiner, Z; Sechtem, U; Anton Sirnes, P; Tendera, M; Torbicki, A; Vahanian, A; Windecker, S; Revisores del documento: Vardas, P; Al-Attar, N; Alfieri, O; Angelini, A; Blömstrom-Lundqvist, C; Colonna, P; De Sutter, J; Ernst, S; Goette, A; Gorenek, B; Hatala, R; Heidbüchel, H; Heldal, M; Dalby Kristensen, S; Kolh, P; Le Heuzey, JY; Mavrakis, H; Mont, L; Perrone Filardi, P; Ponikowski, P; Prendergast, B; Rutten, FH; Schotten, U; Van Gelder, IC; Verheugt, FW. 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