Annual Report 2007 - Istituto di Ricerche Farmacologiche Mario Negri

IRFMNEdited by Isabella Bordognaprinted May 20082ANNUAL REPORT 2007

IRFMNPREFACEThis booklet provides a brief description of the research and training work done at the MarioNegri Institutes in Milan and Bergamo. It is divided by departments, and in some cases bysingle laboratories. Details of the results are provided in the text itself, so here we shall justdraw a few general remarks.Compared to previous years, our research programs are increasingly spreading out to involvethe collaboration among several laboratories within each department, and some areinterdepartmental. We have slimmed down the number of research topics in order to focusmore closely, to reach a “critical mass” of scientists and resources employed on each theme.The tendency nowadays is towards widespread use of molecular biology techniques, especiallyfor studying the mechanism of action of drugs.In vitro studies are an essential basis for thorough investigations although a significant numberof in vivo experiments are still needed as this is the only mean we have of validating in vitrofindings and establishing models that resemble human diseases as closely as possible. Thishas led to a substantial increase in the use of transgenic animals.The Institute is still concentrating on its traditional research lines: oncology, neurosciences,cardiovascular and renal diseases, organ transplants – with strong cell biology and molecularbiochemistry connotations. Significant work has been carried out on the environment andhealth as a whole. Experimental, clinical and epidemiological research on rare diseases andorphan drugs is growing all the time.The Mario Negri Institute strives to develop a multifaceted approach to all these researchthemes, ranging from basic research, to pharmacokinetics, pharmacology, controlled clinicaltrials, epidemiological analysis and – whenever possible – the epidemiology of services.So far we have published more than 11.000 articles on peer reviewed scientific journals.If research is to continue young scientists must be continually trained. Working in the laboratorynot only gives them an outlet for their ideas, but enables them to obtain worthwhilequalifications by taking part in the Institute’s training schemes, which are recognised by theLombardy Region in Italy, or by working for a Ph.D. awarded by the Open University, UK .Training courses are also available on biomedical statistics, for general practitioners, familypediatricians, and clinical trial nurses.A vital part of the Institute’s work involves providing information, at all levels. This is done, inparticular, through the Rare Diseases Information Center, the Center for Information onMedicinal Drugs and the Website (www.marionegri.it) .Scientific activities have been affected by the recent move to the new headquarters in via G.Masa 19 in Milan, where there is more space and new and modern technologies.These are difficult years for research in Italy, and the Institute is most grateful to all those publicand private bodies, foundations and private citizens who generously support the work outlinedhere, with their steady flow of contributions. Our sincere thanks to all of them.Silvio GarattiniDirector3ANNUAL REPORT 2007

IRFMN4ANNUAL REPORT 2007

IRFMNMario Negri INSTITUTE FORPHARMACOLOGICAL RESEARCH MilanANNUALREPORT 2007departments and laboratories5ANNUAL REPORT 2007

IRFMNDEPARTMENT OF ONCOLOGYSTAFFChiefMaurizio D’INCALCI, M.D.Oncological Studies Office and DocumentationScientific DocumentalistStefania FILIPPESCHI, ChemistLaboratory of Cancer PharmacologyHeadBiophysics UnitHeadFlow Citometry UnitHeadCancer Clinical Pharmacology UnitHeadMaurizio D’INCALCI, M.D.Paolo UBEZIO, Phys.D.Eugenio ERBA, Biochem.DMassimo ZUCCHETTI, Chem.Pharm.D.Laboratory of Molecular PharmacologyHeadDNA Repair UnitHeadMassimo BROGGINI, Ph.D.Giovanna DAMIA, M.D.Laboratory of Biology and Therapy of MetastasisHeadTumor Angiogenesis UnitHeadMolecular Cancer Therapeutics UnitHeadRaffaella GIAVAZZI, Biol.Sci.D., Ph.D.Giulia TARABOLETTI, Biol.Sci.D.Maria Rosa BANI, Biol.Sci.D., Ph.D.6ANNUAL REPORT 2007

IRFMNLaboratory for the development of new pharmacological strategiesHeadValter TORRI, M.D.Laboratory of Clinical TrialsHeadIrene FLORIANI, Dr.Sci.Biol., Dr.Stat., Ph.D.Laboratory of Translational and Outcome Research in OncologyHeadGynecology Oncology UnitHeadGiovanni APOLONE, M.D.Roldano FOSSATI, Dr.Med.Chir.CERP:Center for the Evaluation and Research on PainHeadGiovanni APOLONE, M.D.Laboratory of Medical Research and Consumer InvolvementHeadPaola MOSCONI, Biol.Sci.D.7ANNUAL REPORT 2007

IRFMNSelected publications• Fossati R, Apolone G, Negri E., Compagnoni A, La Vecchia C, Mangano S, Clivio L, Garattini S, A double-blind,placebo controlled, randomized trial of bupropion for smoking cessation in primary care, Arch Intern Med 2007; 67:1791-179• Bertele' V, Banzi R, Capasso F, Tafuri G, Trotta F, Apolone G, Garattini S Haematological anticancer drugs in Europe:any added value at the time of approval?, Eur J Clin Pharmacol 2007; 63: 713-719• Apolone G, Mangano S, Compagnoni A, Negri Emanuele, Mosconi P, Mannino S, Villa M, Zuccaro P, Amultidisciplinary project to improve the quality of cancer pain management in Italy. Background, methods andpreliminary results J Ambul Care Manage 2006; 29: 332-341• Apolone G, La Vecchia C, Garattini S. Targeted kinase inhibitors in lung cancer: from EGFR to patients Eur J ofCancer, 2006; 42: 124-125• Apolone G, Joppi R, Bertelè V & Garattini S. Ten years of marketing approvals of anti-cancer drugs in Europe.Regulatory policy and guidance documents need to find a balance between different pressures BJC 2005; 93: 504-509• Gallus S, Colombo P, Apolone G, Zuccaro P, La Vecchia C. A tax to prevent the epidemic of lung cancer. Lancet 2005;366: 288Massimo Broggini followed the faculty of Science of the University of Milan, got the specialization inBiochemistry at Mario Negri Institute, and the PhD degree at the Open University, London,UK.He worked for a period in the laboratory of Molecular Pharmacology of the National Cancer Institute ofBethesda, Md, in 1986. From 1991 is the head of the Molecular Pharmacology Unit of the Mario NegriInstitute and from 1999 of the Laboratory of Molecular Pharmacology of the same Institute.His main fields of interest are the study of the mechanism of action of new anticancer agents, the searchof proteins and genes altered in human cancer and the study of oncosuppressor genes. He is member ofthe "Pharmacology and Molecular Mechanisms Group" of the European Organisation for the Researchand Treatment of Cancer (EORTC) and of the American Association for Cancer Research. He is in theEditorial board of the European Journal of Cancer.He is author of more than 100 articles published in international journals.Principali pubblicazioni• Zangrossi S, Marabese M, Broggini M, Giordano R, D'Erasmo M, Montelatici E, Intini D, Neri A, Pesce M, Rebulla P,Lazzari L. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker. Stem Cells.2007;25(7):1675-80.• Marrazzo E, Marchini S, Previdi S, Broggini M. Questioning the oncogenic role of DeltaNp73alpha in different cell linesexpressing p53 or not. Cancer Biol Ther. 2006;5(7):794-803.• Polato F, Codegoni A, Fruscio R, Perego P, Mangioni C, Saha S, Bardelli A, Broggini M. PRL-3 phosphatase isimplicated in ovarian cancer growth. Clin Cancer Res. 2005 11:6835-9.• Maffucci T, Piccolo E, Cumashi A, Iezzi M, Riley AM, Saiardi A, Godage HY,Rossi C, Broggini M, Iacobelli S, PotterBV, Innocenti P, Falasca M. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway by inositol pentakisphosphateresults in antiangiogenic and antitumor effects. Cancer Res. 2005;65:8339-49.• Marabese M, Marchini S, Sabatino MA, Polato F, Vikhanskaya F, Marrazzo E, Riccardi E, Scanziani E, Broggini M.Effects of inducible overexpression of DNp73alpha on cancer cell growth and response to treatment in vitro and in vivo.Cell Death Differ. 2005;12:805-14.• Sabatino MA, Colombo T, Geroni C, Marchini S, Broggini M. Enhancement of in vivo antitumor activity of classicalanticancer agents by combination with the new, glutathione-interacting DNA minor groove-binder, brostallicin. ClinCancer Res. 2003;9:5402-8.Irene Floriani got her degree in Biological Sciences at the University of Milan in 1988, her degree inBiostatistics and Experimental Statistics at the University of Milan in 2003 and her phD in Life Sciencesat Open University of London (UK) in 2005. After ten-year experience in pharmaceutical industry, in2002 she became Head of the Biometry and Data Management Unit of the Laboratory of ClinicalResearch in Oncology and since 2006 she is Head of Laboratory of Clinical Trials. She is also member asbio-statistician of three Italian Ethics Committees. Her main fields of interest are: statistical aspects ofmethodology of clinical research with focus on Controlled Clinical Trials in Oncology; SystematicOverview of the medical literature and Methodological aspects of diagnostic test evaluation.Selected publications• Cascinu S, Labianca R, Barone C, Santoro A, Carnaghi C, Cassano A, Beretta GD, Catalano V, Bertetto O, Barni S,Frontini L, Aitini E, Rota S, Torri V, Floriani I. Adjuvant treatment of high-risk, radically resected gastric cancerpatients with 5-fluorouracil, leucovorin, cisplatin, and epidoxorubicin in a randomized controlled trial.J Natl Cancer Institute (2007); 99: 601-7• Ruzzo A, Graziano F, Loupakis F, Rulli E, Canestrari E, Santini D, Catalano V, Ficarelli R, Maltese P, Bisonni R, MasiG, Schiavon G, Giordano P, Giustini L, Falcone A, Tonini G, Silva R, Mattioli R, Floriani I, and Magnani M.9ANNUAL REPORT 2007

IRFMNPhamacogeneic profiling in patients with advanced colorectal cancer treated first-line FOLFOX-4 chemotherapyAnnals of Oncology (2007); 25: 1247-1254• Cazzaniga M, Mustacchi G, Pronzato P, De Matteis A, Di Costanzo F, Floriani I on behalf of the NORA Study GroupAdjuvant treatment of early breast cancer: do the St Gallen recommendations influence clinical practice? Results fromthe NORA study. Annals of Oncology (2007); 18: 1976-80• Mandalà M, Reni M, Cascinu S, Barni S, Floriani I, Cereda S, Berardi R, Mosconi S, Torri V, Labianca R.Venous thromboembolism predicts poor prognosis in irresectable pancreatic cancer patients.Annals of Oncology (2007); 10: 1660-5• Mustacchi G, Cazzaniga ME, Pronzato P, De Matteis A, Di Costanzo F, Floriani I on Behalf of the NORA StudyGroup. Breast cancer in elderly women: a different reality? Results from the NORA study. Annals of Oncology (2007);18: 991-6• Gregorc V, Spreafico A, Floriani I, Colombo B, Ludovini V, Pistola L, Bellezza G, Vigano MG, Villa E, Corti A.Prognostic value of circulating chromogranin A and soluble tumor necrosis factor receptors in advanced nonsmall celllung cancer. Cancer (2007); 110; 845-53Raffaella Giavazzi got her Biological Sciences degree (1979) at the University of Milan, and her Ph.D.in Pharmacology at the Mario Negri Institute of Milan (1984), followed by a specialization inpharmacology (1994) at the University of Milan. From 1981 to 1983 she was a Fellow in the CancerMetastasis and Treatment Laboratory, NCI-FCRDC, Frederick, MD., and from 1983 to 1985 AssistantProfessor at the Department of Cell Biology of M.D. Anderson Hospital and Tumour Institute,University of Texas System Cancer Centre in Houston (TX).Raffaella Giavazzi’s research interests are in the field of tumour biology and pharmacology. Specifically,she is studying aspects related to the metastatic process and angiogenesis. She is involved in the preclinicalevaluation of new therapeutic strategies against cancer focusing on the angiogenesis inhibitorsand combination therapies.From 1986 to 1993 she was Head of the Cancer Metastasis Treatment Unit and since 1993 she has beenthe Head of the Laboratory of Biology and Treatment of Metastasis at Mario Negri Institute forPharmacological Research.She is also adjutant Professor in Oncology, Medical School-University of Brescia, member of theTeaching Committee for the PhD course in Physiology-Pharmacology-Molecular and CellularTossicology-University of Siena, member of the Executive Committee at SENDO (South Europe NewDrug Development Organization) and member of the Pezcoller Foundation Scientific Committee.She was consulting scientist for the NCI-Drug Therapeutics Program, USA (1996-2006);She is a member of the American Association for Cancer Research (AACR), International MetastasesResearch Society, EORTC-Screening and Pharmacology Group, European Association for CancerResearch (EACR), Italian Cancer Society (SIC) of which she was President (2006-2007).She is on the Editorial Board of the European Journal of Cancer, Journal of Clinical ExperimentalMetastasis, Journal Exp. Therapeutic & Oncology, The International Journal of Biological Markers,Current Cancer Therapy Reviews and Journal of Chemotherapy. She has published approximately 150articles on “peer reviewed” journals and 34 book chapters.Selected publications• Martinelli M., Bonezzi K., Riccardi E., Kuhn E., Frapolli R., Zucchetti M., Ryan A.J., Taraboletti G.,Giavazzi R.: Sequence dependent antitumour efficacy of the vascular disrupting agent ZD6126 in combination withpaclitaxel. British Journal of Cancer 97:888-94, 2007.• Giavazzi R., Bani M.R.,Taraboletti G.: Tumor–host interaction in the optimization of paclitaxel-based combinationtherapies with vascular targeting compounds. Cancer Metastasis Rev. 26:481–88, 2007• Naumova E., Ubezio P., Garofalo A., Borsotti P., Cassis L., Riccardi E., Scanziani E., Eccles S.A., Bani M.R., GiavazziR.: The vascular targeting property of paclitaxel is enhanced by SU6668, a receptor tyrosine kinase inhibitor, causingapoptosis of endothelial cells and inhibition of angiogenesis. Clin. Cancer Research 12(6):1839-49, 2006.• Bani M.R., Nicoletti M.I., Alkharouf N.W., Ghilardi C., Petersen D., Erba E., Sausville E.A., Liu E.T., Giavazzi R.: Geneexpression correlating with response to paclitaxel in ovarian carcinoma xenografts. Molecular Cancer Therapeutics3(2):111-121, 2004.• Belotti D., Paganoni P., Manenti L., Garofalo A., Marchini S., Taraboletti G., Giavazzi R.: Matrix Metalloproteinases(MMP9 and MMP2) Induce the Release of Vascular Endothelial Growth Factor (VEGF) by Ovarian Carcinoma Cells:Implications for Acites Formation. Cancer Res. 63:5224-5229, 2003.• Micheletti G., Poli M., Borsotti P., Martinelli M., Imberti B., Taraboletti G., Giavazzi R.: Vascular-targeting Activity ofZD6126, a Novel Tubulin-binding Agent. Cancer Res. 63:1534-37, 2003.Paola Mosconi got his Biological Science degree (Milan 1982) and the specialisation in PharmacologicalResearch (Milan 1984). Her main areas of interest are: development of strategies to involve patients-10ANNUAL REPORT 2007

IRFMNconsumer associations in health debate, and research projects; assessment of quality of life, translationand cultural adaptation of questionnaires for quality of life; studies to evaluate the type of information ondiseases and treatments received by patients, mainly in cancer patients; set-up of websites targeted onconsumers/patients www.partecipasalute.it, www.paincare.it; studies to evaluate the consumers’ level ofsatisfaction with the health services and cure.Paola Mosconi has participated as a teacher, or coordinator, to the realization of training course on“Methodological aspects of clinical research” or “Evaluation of quality of life” for health careprofessionals and representatives of voluntary associations.Selected publications• O’Connel D, Mosconi P. An active role for patients in clinical research? Drug Development Research 67 (3): 188-192, 2006.• Mosconi P, Colombo Cinzia, La Bianca R, Apolone G. Oncologists' opinions about research ethics committees in Italy: anupdate, 2004. Eur J Cancer Prev 2006; 15: 91-94• Leone M A, Beghi E, Righini C, Apolone G, Mosconi P. Epilepsy and quality of life in adults: a review of instruments.Epilepsy Research 66: 23-44, 2005.• Mosconi P, Poli P, Giolo A, Apolone G. How health consumers feel about clinical research: a questionnaire survey. EuropeanJournal of Public Health 15: 372-379, 2005.• Mosconi P, Buchanan M, Kyriakides S, Fernandez-Marcos A, Horvatin J, O'Connell D, Zernik N, on behalf of EUROPADONNA. EUROPA DONNA: has strength in its heterogeneity. European J Cancer 40: 1145-1149, 2004.• Mosconi P, Apolone G. Fixed and dynamic health related quality of life measurements. J Headache Pain S31-S34, 2003.• Domenighetti G, D’Avanzo B, Egger M, Berrino F, Perneger T, Saracci R, Mosconi P. and M. Zwahlen.Women’s perceptionof the benefits of mammography screening: population-based survey in four countries. Int J Epidem 32: 816-821, 2003.Valter Torri got his Medical degree in 1985 and the specialization in medical Oncology in 1989 at theUniversity of Milano.Education: 1985: MD Degree with full honors cum Laude, University of Milano; 1988 Post-DoctoralDegree in Pharmacological Research, Mario Negri Institute, Milano; 1989 Post-Doctoral Degree inMedical Oncology, University of Milano; 1989-1991 Research Fellow at the Biometric Research Branchof Cancer Treatment Evaluation Program, NCI, Bethesda, MD (USA)Areas of Interest: Statistical aspects of clinical research methodology with focus on Controlled ClinicalTrials in Oncology; Systematic Overview of the medical literature; Methodological aspects of diagnostictest evaluation.Present Position: Head of Laboratory of Clinical Research In Oncology, Oncology Department, MarioNegri Institute, Milano.Chronology of Professional Appointments: 1983-1985: Clinical research Fellow in Internal Medicine atthe University Hospital, University of Milan; 1985-1989: Research assistant at the Clinical Trial Unit ofthe Laboratory of Clinical Epidemiology, Mario Negri Institute for Pharmacological Research, Milano;1989-1991: Research fellow at the Biometric Research Branch of Cancer Treatment Evaluation Program,NCI, Bethesda, MD (USA); 1994: Head of Biometric Unit of the Laboratory of Cancer ClinicalEpidemiology, Oncology Department, Mario Negri Institute for Pharmacological Research, Milano, Italy;1995 Vice Director of the Italian “Cochrane” Center; 2001: Head of Laboratory of Clinical Research InOncology, Oncology Department, Mario Negri Institute, Milano. 2006: Head of Laboratory for thedevelopment of new pharmacological strategies , Oncology Department, Mario Negri Institute, Milano.Selected publications• Mandalà M, Reni M, Cascinu S, Barni S, Floriani I, Cereda S, Berardi R, Mosconi S, Torri V, Labianca R. Venousthromboembolism predicts poor prognosis in irresectable pancreatic cancer patients. Ann Oncol. 2007;18(10):1660-5.• Malesci A, Laghi L, Bianchi P, Delconte G, Randolph A, Torri V, Carnaghi C, Doci R, Rosati R, Montorsi MRoncalli M,Gennari L, Santoro A. Reduced likelihood of metastases in patients with microsatellite-unstable colorectal cancer. Clin CancerRes. 2007;13(13):3831-9.• Garassino MC, Hollander L, Torri V. Bevacizumab for non-small-cell lung cancer. N Engl J Med. 2007;356(13):1373• Cascinu S, Labianca R, Barone C, Santoro A, Carnaghi C, Cassano A, Beretta GD, Catalano V, Bertetto O, Barni S, FrontiniL, Aitini E, Rota S, Torri V, FlorianiI; Italian Group for the Study of Digestive Tract Cancer, Adjuvant treatment of high-risk,radically resected gastric cancer patients with 5-fluorouracil, leucovorin, cisplatin, and epidoxorubicin in a randomisedcontrolled trial. J Natl Cancer Inst. 2007;99(8):601-7.• Graziano F, Kawakami K, Ruzzo A, Watanabe G, Santini D, Pizzagalli F, Bisonni R, Mari D, Floriani I, Catalano V, Silva R,Tonini G, Torri V, Giustini L, Magnani M Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancersusceptibility and genomic DNA hypomethylation in an at-risk Italian population. Int J Cancer 2006 118 : 628-632• Torri V: Clinical trials and data management In: Oxford textbook of oncology, 2nd. ed. Vol. 1. Oxford Univ. Press, Oxford;2002 : 1123-113411ANNUAL REPORT 2007

IRFMN• Carrassa L, Broggini M, Vikhanskaya F, Damia G. Characterization of the 5' flanking region of the human chk1 gene.Identification of E2F1 functional sites. Cell Cycle 2003; 2: 604-609• Damia G, Sanchez Y, Erba E, Broggini M. DNA damage induces p53-dependent down-regulation of hCHK1. J BiolChem 2001; 276: 10641-10645Eugenio Erba has obtained his Biological and Biochemmistry Analysis Degree at the University ofUrbino. He worked as a research fellow in the Laboratory of Cancer Chemotherapy at the Mario NegriInstitute and since 1984 he is head of the Flow Cytometry Unit in the Department of Oncology at theMario Negri Institute of Milan. He has worked as a visiting fellow in the Department of Istochemistryand Cytochemistry of the University of Leiden, The Netherlands in 1983. Since 1997 he is Teacher ofPost-Graduate Studies in Cytometry at the University of Milan and Co-ordinator and Teacher of Post-Graduate Studies in Cytometry for the Italian Cytometry Group. He has been President of the ItalianCytometry Group from 1999 to 2001. Since 2001 he is member of the Executive Board of the ItalianCytometry Group.Scientific areas of interest: studies on the mechanism of action of different compounds with providedantitumoral activity evaluating the mechanism of cell death and cell cycle phase perturbations inducedon different human cancer cell lines by using flow cytometry. Co-ordinator of working-group in aquality control study on flow cytometric DNA content analysis in human tumors.Selected publications• Paulis M., Bensi M., Orioli D., Mondello C., Mazzini G., D’Incalci M., Falcioni C., Radaelli E., Erba E., RaimondiE., De Carli L. Transfer of a Human Chromosomal Vector from a Hamster Cell Line to a Mouse Embryonic StemCell Line. Stem Cell , 25:2543-2550 (2007)• Tavecchio M., Natoli C., Ubezio P., Erba E., D’Incalci M. Dynamics of cell cycle perturbations by trabectedin (ET-743) in nucleotide excision repair (NER) –deficient and NER-proficient cells, unravelled by a novel mathematicalsimulation approach. Cell Prolif., 40:885-904 (2007)• Dolfini E., Roncoroni L., Dogliotti E., Sala G., Erba E., Sacchi N., Ghidoni R. Resveratrol impairs the formation of MDA-MB-231 multicellular tumor spheroids concomitant with ceramide accumulation. Cancer Lett 2007; 249: 143-147.• Tognon G., Bernasconi S., Celli N., Faircloth G.T. Cuevas C., Jimeno J., Erba E., D’Incalci M. Induction of resistanceto Aplidin® in a human ovarian cancer cell line related to MDR expression. Cancer Biology and Therapy, 4(12):1325-1330 (2005).• Minuzzo M., Ceribelli M., Pitarque-Martì M., Borrelli S., Erba E., di Silvio A., D’Incalci M., Mantovani R. Selectiveeffects of the anti-cancer drug Yondelis (ET-743) on cell-cycle promoters. Mol. Pharmacol., 68: 1496-1503 (2005).• Simone M., Erba E., Damia G., Vikhanskaya F., Di Francesco A.M., Riccardi R., Bailly C., Cuevas C., FernandezSousa-Faro J.M., D’Incalci M. Variolin B and its derivate deoxy-Variolin B: new marine natural compounds with acyclin-dependent-kinase inhibitor activity. Eur. J. Cancer, 41: 2366-2377 (2005).• Allavena P., Signorelli M., Chieppa M., Erba E., Bianchi G., Marchesi F., Garbi A., Lissoni A., de Braud F., JimenoJ. and D’Incalci M. Anti-inflammatory properties of the novel antitumor agent Yondelis (Trabectedin): inhibition ofmacrophage differentiation and cytokine production. Cancer REs., 65(7):2964-2971 (2005).• Tognon G., Frapolli G., Zaffaroni M., Erba E., Zucchetti M., Faircloth G.T., M. D’Incalci, Fetal bovine serum, but not humanserum inhibits the in vitro cytotoxicity of ET-743 (Yondelis TM; trabectedin). An example of potential problems forextrapolation of active drug concentrations from in vitro studies. Cancer Chemother Pharmacol., 53(1): 89-90 (2004).• Erba E., Cavallaro E., Damia G., Mantovani R., Di Silvio A, Di Francesco A.M., Riccardi R., Cuevas C., FairclothG.T., D’Incalci M. The unique biological features of the marine product YondelisTM (ET-743, Trabectedin) areshared by its analog ET-637, which lacks the C ring. Oncology Research, 14: 579-587 (2004).Roldano Fossati got his Medical Degree cum Laude from the University of Milan in 1980, his Post-Doctoral Degree in Endocrinolgy cum Laude from the University of Verona in 1983 and his Post-Doctoral Degree in Medical Statistics from the University of Milan in 1992. He has been consultant atthe Mario Negri Institute since 1983 and, at present, he is head of the Gynecology and Oncology Unit ofthe Laboratory of Translational and Outcome Research.Areas of Interest: Statistical and methodologic aspects of clinical research with focus on ControlledClinical Trials in Oncology; Systematic Overview of the medical literature.Selected publications• F.E. Johnson, K.S. Virgo, R. Fossati. Follow-up for patients with colorectal cancer after curative-intent primarytreatment. J Clin Oncol 2004; 22:1363-65• Roberto Labianca, Roldano Fossati , Alberto Zaniboni, Valter Torri , Silvia Marsoni, Donato Nitti, Lamberto Boffi,Marco Scatizzi, Berardino Tardio, Nicola Mastrodonato, Stefano Banducci, Giampiero Consani, Gianfranco Pancera onbehalf of ACOI/GIVIO/GISCAD investigators. Randomized Trial of Intraportal and/or Systemic AdjuvantChemotherapy in Patients with Colon Carcinoma. J Natl Cancer Inst 2004; 96:750-8• P. Benedetti Panici, A. Maggioni, N. Hacker, F. Landoni, S. Ackermann, E. Campagnutta, K. Tamussino, R. Winter, A.Pellegrino, S. Greggi, R. Angioli, N. Manci, G. Scambia, T. Dell'Anna, R. Fossati, I. Floriani, R.S. Rossi, R. Grassi, G.13ANNUAL REPORT 2007

IRFMNFavalli, F. Raspagliesi, D. Giannarelli, L. Martella, C. Mangioni. Systematic Aortic and Pelvic Lymphadenectomy versusResection of Bulky Nodes Only in Optimally Debulked Advanced Ovarian Cancer: A Randomized Clinical Trial J NatlCancer Inst 2005; 97:1-6• Buda A, Fossati R, Colombo N, Fei F, Floriani I, Gueli Alletti D, Katsaros D, Landoni F, Lissoni A, Calzoni C, SartoriE, Scollo P, Torri V, Zola P, Mangioni C. Randomized trial of neoadjuvant chemotherapy comparing paclitaxel,ifosfamide, and cisplatin with ifosfamide and cisplatin followed by radical surgery in patients with locally advancedsquamous cell cervical carcinoma: The SNAP01 (Studio Neo-Adjuvante Por. J Clin Oncol 2005; 23: 4137-4145.• Maggioni A, Benedetti Panici P, Dell'anna T, Landoni F, Lissoni A, Pellegrino A, Rossi RS, Chiari S, Campagnutta E,Greggi S, Angioli R, Manci N, Calcagno M, Scambia G, Fossati R, Floriani I, Torri V, Grassi R, MangioniC.Randomised study of systematic lymphadenectomy in patients with epithelial ovarian cancer macroscopically confinedto the pelvis. Br J Cancer. 2006; 18;95(6):699-704.• Maggi R, Lissoni A, Spina F, Melpignano M, Zola P, Favalli G, Colombo A, Fossati R. Adjuvant chemotherapy vsradiotherapy in high-risk endometrial carcinoma: results of a randomised trial. Br J Cancer. 2006; 7;95(3):266-71• Fruscio R, Colombo N, Lissoni AA, Garbi A, Fossati R, Ieda' N, Torri V, Mangioni C.A phase II randomised clinicaltrial comparing cisplatin, paclitaxel and ifosfamide with cisplatin, paclitaxel and epirubicin in newly diagnosed advancedepithelial ovarian cancer: long-term survival analysis. Br J Cancer. 2008; [Epub ahead of print]• Marabese M, Marchini S, Marrazzo E, Mariani P, Cattaneo D, Fossati R, Compagnoni A, Signorelli M, Moll UM,Codegoni AM, Broggini M. Expression levels of p53 and p73 isoforms in stage I and stage III ovarian cancer. Eur JCancer. 2008 Jan;44(1):131-141.• Marchini S, Marabese M, Marrazzo E, Mariani P, Cattaneo D, Fossati R, Compagnoni A, Fruscio R, Lissoni AA,Broggini M. {Delta}Np63 expression is associated with poor survival in ovarian cancer. Ann Oncol. 2007 Nov 12;[Epub ahead of print]• Fossati R, Apolone G, Negri E, Compagnoni A, La Vecchia C, Mangano S, Clivio L, Garattini S; for the GeneralPractice Tobacco Cessation Investigators Group. A double-blind, placebo-controlled, randomized trial of bupropion forsmoking cessation in primary care. Arch Intern Med. 2007; 10;167(16):1791-7.Giulia Taraboletti got her degree cum laude in Biological Sciences at the University of Pavia (Pavia,Italy) in 1983, and the specialization in Pharmacological Research at the Mario Negri Institute, Milano,Italy in 1986. From 1986 to 1988 she was a post-doctoral fellow at the Laboratory of Pathology, NCI,NIH, Bethesda, MD, and from 1988-1995 research scientist at Mario Negri Institute in Bergamo, Italy.Since 1995 she is Head of the Unit of Tumor Angiogenesis, at Mario Negri Institute, in Bergamo.Research interests include tumor angiogenesis, endogenous inhibitors of angiogenesis (thrombospondin-1) and preclinical studies of antiangiogenic and vascular disrupting compounds, including tubulintargetingagents. She is member of Metatasis Research Society (MRS, Board of Directors), AmericanAssociation for Cancer Research (AACR), European Association for Cancer Research (EACR), and theItalian Society of Oncology (SIC). She is on the editorial board of European Journal of Cancer.Selected publications• Giavazzi R., Bani M.R.,Taraboletti G. Tumor–host interaction in the optimization of paclitaxel-basedcombination therapies with vascular targeting compounds. Cancer Metastasis Rev, 26:481–88, 2007.• Martinelli M., Bonezzi K., Riccardi E., Kuhn E., Frapolli R., Zucchetti M., Ryan A.J., Taraboletti G., Giavazzi R.Sequence dependent antitumour efficacy of the vascular disrupting agent ZD6126 in combination with paclitaxel. Br JCancer 97:888-94, 2007.• Margosio B., Marchetti D., Vergani V., Giavazzi R., Rusnati M., Presta M., and Taraboletti G. Thrombospondin-1 as ascavenger for matrix-associated fibroblast growth factor-2. Blood 102: 4399-4406, 2003.• Taraboletti G, D’Ascenzo S, Borsotti P, Giavazzi R, Pavan R, and Dolo V Shedding of MMP-2, MMP-9 and MT1-MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol,160: 673-680, 2002• Taraboletti G. Micheletti G, Rieppi M, Poli M, Turatto M, Rossi C, Borsotti P, Roccabianca P, Scanziani E, Nicoletti MI,Bombardelli E, Morazzoni P, Riva A, and Giavazzi R. Antiangiogenic and antitumor activity of IDN 5390, a new taxanederivative. Clin Cancer Res. 8: 1182-1188, 2002• Taraboletti G., Morbidelli L., Donnini S., Parenti A., Granger H.J., Giavazzi R., and Ziche M.The heparin binding 25kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production inendothelial cells. FASEB J., 14: 1674-1676, 2000.Paolo Ubezio got his B.Sc. degree in Physics at the University of Milan, in 1982, and the specialisationin Pharmacological Research Specialist" at the Mario Negri Institute for Pharmacological Research in1986.Main activities are: i) Study of cell-cycle mathematical models; ii) Development of flow cytometricmethods; iii) Optimization of anticancer drug scheduling.Since 1991 is Head of the Unit of Biophysics at the Mario Negri InstituteSelected publications• Basse, B., Ubezio, P. (2007) A generalised age and phase structured model of human tumour cell populations bothumperturbed and exposed to a range of cancer therapies. Bull. Math. Biol. 69:1673-90.14ANNUAL REPORT 2007

IRFMN• Lupi, M., Matera, G., Natoli, C., Colombo, V., Ubezio, P. (2007) The Contribution of p53 in the Dynamics of Cell CycleResponse to DNA Damage Interpreted by a Mathematical Model. Cell Cycle 6:943-950.• Spinelli, L., Torricelli, A., Ubezio, P., Basse, B. (2006) Modeling the balance between quiescence and cell death innormal and tumor cell populations Math Biosciences 202: 349-370.• Lupi, M., Matera, G., Branduardi, D., D'Incalci M. and Ubezio, P. (2004) Cytostatic and cytotoxic effects of topotecandecoded by a novel mathematical simulation approach. Cancer Res. 64: 2825-2832.• Matera, G., Lupi, M.,and Ubezio, P. (2004) Heterogeneous cell response to topotecan in a CFSE-based proliferation test.Cytometry 62A:118-128.• Ubezio, P. (2004) Unraveling the complexity of cell cycle effects of anticancer drugs in cell populations.Discrete andContinuous Dynamical Systems-Series B 4:323-335.• Tomasoni, D., Lupi, M., Bekkal Brikci, F. and Ubezio, P. (2003) Timing the changes of cyclin E cell content in G1 inexponentially growing cells. Exp Cell Res.; 288: 158-167.• Montalenti, F., Sena, G., Cappella, P., and Ubezio, P. (1998) Simulating cancer-cell kinetics after drug treatment:Application to cisplatin on ovarian carcinoma. Phys. Rev. E, 57:5877-5887.Massimo Zucchetti obtained his Chem. Pharm. Degree from the University of Milan in 1982. Afterspecializing in Pharmacology at the Mario Negri Institute of Milan (1988), he worked in the Laboratoryof Clinical Pharmacology of Department of Oncology at San Giovanni Hospital, Bellinzona, Switzerland(1988-1990). Since 1996 he has been chief of the Cancer Clinical Pharmacology Unit at the Mario NegriInstitute. He is member of the Pharmacology and Molecular Mechanisms Group of the EuropeanOrganization for Research and Treatment of Cancer (EORTC) from 1988 up to date. His main field ofinterest are:- Clinical pharmacology, phase I and Phase II studies- Analysis of drugs, pharmacokinetic and pharmacodynamic studies in humans in GCP and GLPconditions- Pharmacokinetic and metabolic studies in animals- Pharmacokinetic drug interactionDr Zucchetti is author of more than 80 papers on pre-clinical and clinical cancer chemotherapy publishedin peer reviewed international journals.Selected publications• Frapolli R., Marangon E., Zaffaroni M., Colombo T., Falcioni C., Bagnati R., Simone M., D’Incalci M., Manzotti C.,Fontana G., Morazzoni P., Zucchetti M. Pharmacokinetics and metabolism in mice of IDN 5390 (13-(N-Boc-3-ibutylisoserinoyl)-C-7,8-seco-10-deacetylbaccatinIII), a new oral C-seco-taxane derivative with antiangiogenic propertyeffective on paclitaxel-resistant tumors. Drug Metabolism and Disposition, 34(12):2028-2035 (2006).• Rizzari C., Citterio M., Zucchetti M., Conter V., Chiesa R., Colombini A., Malguzzi S., D’Incalci M. Pharmacologicalstudy on pegylated asparaginase used in front-line treatment of children with acute lymphoblastic leukemia.Hematologica, 91: 24-31 (2006).• Fruscio R., Lissoni A.A., Frapolli R., Corso S., Mangioni C., D’Incalci M., Zucchetti M. Clindamycin-Paclitaxelpharmacokinetic interaction in ovarian cancer patients. Cancer Chemother. Pharmacol., 58(3): 319-325 (2006).• Gambacorti Passerini C, Zucchetti M, Russo D, Frapolli R, Verga M, Bungaro S, Tornaghi L, Rossi F, Pioltelli P,Pogliani E, Alberti D, Corneo G, D'Incalci M Alpha 1 acid glycoprotein binds to imatinib (STI571) and substantiallyalters its pharmacokinetics in chronic myeloid leukemia patients Clin Cancer Res 2003; 9: 625-632• Pratesi G, Laccabue D, Lanzi C, Cassinelli G, Supino R, Zucchetti M, Frapolli R, D'Incalci M, Bombardelli E,Morazzoni P, Riva A, Zunino F IDN 5390: An oral taxane candidate for protracted treatment schedules Br J Cancer2003; 88: 965-972• Zaffaroni M, Frapolli R, Colombo T, Fruscio R, Bombardelli E, Morazzoni P, Riva A, D'Incalci M, Zucchetti MHigh-performance liquid chromatographic assay for the determination of the novel C-Seco-taxane derivative (IDN 5390)in mouse plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 780: 93-9815ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe Oncology Department comprises three preclinical experimental laboratories (Laboratory ofCancer Pharmacology, Laboratory of Molecular Pharmacology and Laboratory of Biology andTherapy of Metastasis) and four laboratories dealing with clinical research and clinical trials(Laboratory for the Development of New Pharmacological Strategies, Laboratory of ClinicalTrials, Laboratory of Translational and Outcome Research in Oncology and Laboratory forMedical Research and Consumer Involvement).The Oncology department hosts the coordination center of two networks of hospitals that carryon clinical research in gynecologic cancer (MaNGO: Mario Negri Gynecologic Oncology) andin cancer pain (CPOR-SG: Cancer Pain Outcome Research Study Group) and a center forcancer pain assessment and research (CERP:Center for the Evaluation and Research on Pain).In some cases research projects are carried out by single laboratories or research units, in othercases by collaborations between different laboratories of the Oncology Department or otherdepartments, or other groups outside the Institute (see National and InternationalCollaborations).Preclinical laboratories focus on the discovery and development of new antitumor andantimetastatic drugs and their new combinations; on tumor biology, not only to acquire newscientific knowledge, but particularly as a base for more selective therapeutic approaches and toidentify and evaluate experimental models for discovering and studying new drugs ortreatments.Clinical new drug development involves close participation in the activity of SENDO (SouthEurope New Drug Development Organization) and studies driven by the Laboratory of CancerPharmacology, the Laboratory of Molecular Pharmacology and the Laboratory of Biology andTherapy of Metastasis. The Laboratory for the Development of New PharmacologicalStrategies, the Laboratory of Clinical Trials, the Laboratory of Translational and OutcomeResearch in Oncology and the Laboratory for Medical Research and Consumer Involvement areinvolved in the evaluation of the effects of new therapeutic modalities in phase I/II and in phaseIII comparative and effectiveness outcome studies.Outcome Research implies organizing trials to clarify the results of certain health care practicesand interventions in clinical practice. Observational (surveys) and outcome research(effectiveness) studies are carried out, in collaboration with regional and national healthauthorities and other scientific associations.At the preclinical and clinical level there are studies of various human tumors, with particularemphasis on ovarian tumors and more recently on soft tissue sarcomas.FINDINGS/MAIN RESULTSAt nanomolar concentrations, ET-743 (Yondelis, Trabectedin) affects the regulatorymechanisms of the transcription. Nucleotide excision repair deficient cells that arehypersensitive to UV rays and to other DNA damaging drugs are resistant to Trabectedin.Use of mathematical models of tumor growth and anticancer treatment to interpret experimentaldata and to manage the complexity of underlying biological phenomena.The theoretical relationship between proliferation, quiescence and cell loss leading to growthcontrol of tumor cell populations was found.16ANNUAL REPORT 2007

IRFMNVariolin and some of its derivatives induce rapid apoptosis in some tumor cell lines that have alow sensitivity to other anticancer drugs.Gene profiling analysis shows specific molecular signatures according to the histotype andprognosis of stage I ovarian carcinoma.The expression of a truncated form of p63 (DNp63) increases with the increased malignancy ofovarian cancer. Patients expressing high levels of DNp63 have a worst prognosis. DNp63represents therefore a new potential target for selective therapies in this malignancy.A new and efficient system to selectively downregulate CHK1 in vivo has been developed. Thesystem is suitable to test combinations of drugs in vivo and to study new checkpoints inhibitors.An anthracycline derivative, Nemorubicin, has a peculiar mechanism of action and is activeagainst tumors resistant to drugs such as cisplatin. The combination of the two drugs is highlysynergic.Embryo fibroblasts isolated from DRAGO KO mice show a reduced response to treatment withdifferent anticancer agents.DRAGO gene is particularly responsive to p73.Inositol pentaphosphate analogues interfere with the PI3-kinase-induced phosphorylation of aktand possess antitumor activity in vitro and in vivo.Human umbilical cord-derived stem cells express checkpoints proteins only in specificdifferentiation stages. It is likely that this is related to the different susceptibility of the cells.Inhibition of PLC gamma, through siRNA technology, reduces the in vivo growth of tumors andreduces the formation of metastasis.Identification of genes preferentially expressed by tumor associated endothelial cells.VEGF released by cancer cells modulates the gene expression in the tumor microenvironment(stroma).The production of VEGF influence the response to paclitaxel of an ovarian carcinoma xenograftmodel; the anti-VEGF antibody bevacizumab (Avastin®) is of benefit in improving therapeuticefficacy.VEGF produced by ovarian tumor cells stimulates host MMP9 expression; the anti-VEGFantibody bevacizumab (Avastin®) inhibited MMP9 expression and abolished ovarian tumorinvasion.A new antiangiogenic domain of thrombospondin-1 (an endogenous inhibitor of angiogenesis)that binds the angiogenic factor FGF-2 has been identified and characterized.New antineoplastic compounds directed against the tumor vasculature (vascular disruptingagents) have been selected.The sequence of drug administration determines the efficacy of combination treatments withtubulin-binding vascular disrupting agents and cytotoxic drugs.17ANNUAL REPORT 2007

IRFMNThe histone deacetylase inhibitor SAHA potentiates the cytotoxic effect of paclitaxel in humanovarian carcinoma cells resistant to paclitaxel. The effect is mediated by the acetylation oftubulin.The expression of protease-activated receptor-1 (PAR-1) correlates with the malignantphenotype of human melanomas and is accountable for their motility and invasise featuresICON4, a, randomised trial of second-line chemotherapy in advanced ovarian cancer,coordinated by the Mario Negri Institute and by MRC, showed for the first time a reduction inmortality in favour of platinum and paclitaxel chemotherapy.The response to chemotherapy was a good surrogate endpoint of survival in patients withlocally advanced cervix carcinoma.Adjuvant chemotherapy with the regimen vindesin, mitomycin C and cisplatin (MVP) did notimprove survival of non small cell lung cancer (NSCLC) patients compared with surgery alone.The website of the project PartecipaSalute (www.partecipasalute.it) has a very innovativecharacter in comparison with the other health Italian sites because introduces and develops withad-hoc instruments the information transfer in an active way.The LYMPHADENECTOMY trial in advanced ovarian cancer: “two decades of uncertaintyresolved” (editorial). This trial showed that systematic lymphadenctomy does not improveoverall survival and these results will spare many patients the unduly toxicity of this surgicalprocedure.Bupropion more than doubled the odds of continuous abstinence from smoking from week 4 to7 and from week 4 to 12 months in a way similar to that observed in academic studies. Theadherence of GPs and participants to the protocol was excellent, making our findings robust andeasy to generalize to the context of primary care.A randomised phase II trial showed that chemotherapic multidrug regimens containing eitherifosfamide or epirubicin are effective in advanced ovarian cancer, though ifosfamide is moretoxic. This trial had an exceedingly long follow up and showed a median overall survivalnoticeably higher than usually reported in similar clinical settings. Authors suggest that anaggressive therapeutic approach to this disease, with several lines of chemotherapies, could havebenefitted these patients.A survey carried out in Italy that collected data in 110 clinical centers about 1801 patientsshowed that as many as 40% of patients with cancer enter the oncologic/palliative careprograms while clearly undertreated mostly because of sub optimal use of morphine or otheropiate drugs. Data collected at three-month follow up (1461 patients) allowed to describe thetrend over time of many analgesic and palliative endpoints and to find the proportion of nonresponderto analgesic therapy (25-30%).Most of the drugs recently approved by the European Agency (EMEA) for hematologicmalignancies had some information debt about their true risk-benefit profiles and,notwithstanding they are prescribed to thousands of patients. A survey of all the assessmentscarried out by EMEA over the last decade gave evidence that in as many as 2/3 of new drugswere approved by EMEA without a real therapeutic benefit over the old ones18ANNUAL REPORT 2007

IRFMNA randomized trial of patients with high risk (stage IcG3, IIG3 with myometrial invasion >50%,and III) endometrial carcinoma showed the substantial equivalence between radiotherapy orchemotherapy as an adjuvant therapy after surgery. Although both radiotherapic andchemotherapic approaches are still unsatisfactory, since the risk of progression or death remainshigh, this encouraging evidence of clinical activity suggest a possible use of their concurrent orsequential use in an adjuvant setting.The estimates of the prevalence and impact of cancer pain in a large and representative sampleof cancer patients (1800) recruited by several Italian centers (more than 120), with theevaluation of the actual proportion of cases that received a substantial analgesic under-treatment(about 25%), mainly attributable to a sub-optimal utilization of opiods.An evaluation of the activity of the EMEA over the last 10 years, has documented that most ofthe new anti-cancers drugs has actually received an approval on the basis of very preliminaryfindings: in 48% of case the approval was based on studies using surrogate endpoints, and in40% of cases the design of the study was non-comparative and non-randomized.The activity of training and information organized with the associations of citizens & patients inthe framework of the PartecipaSalute project has been finalized to the organization of the Paritatask “Participate to the research project with the associations”. Parita is organised to discusswith the scientific community the grey areas of the medical assistance and clinical researchidentified from the patients and their associations, and to develop ad hoc protocols for futureresearch programs.Development and validation of a new short questionnaire, the PGWBI-short version available tobe used in large sample of citizens or patients.NATIONAL COLLABORATIONSASR, Agenzia Sanitaria Regionale, BolognaAIFA, Agenzia Italiana del Farmaco (Roma)Assessorato Sanità, Regione Emilia RomagnaAzienda Sanitaria Locale (Rimini)Azienda Sanitaria Unica Regionale, (Marche)Casa Sollievo della Sofferenza, San Giovanni Rotondo (IRCCS)CNPDS, Centro Nazionale per la prevenzione e Difesa Sociale, MilanoCNR IGBE, PaviaCNPDS, Centro Nazionale per la prevenzione e Difesa Sociale, MilanoCNR, Istituto di Chimica del Riconoscimento Molecolare, MilanoCochrane CollaborationEUROPA DONNAFederazione Italiana Società ScientificheFondazione LUVI, MilanoFondazione Nerina e Mario Mattioli Onlus, MilanoFondazione Salvatore Maugeri, PaviaFondazione SmithKline (FSK), MilanoFondo Edo Tempia, Laboratorio di Farmacogenomica, BiellaI.A.S.I., RomaIstituto Clinico Humanitas, Rozzano MI19ANNUAL REPORT 2007

IRFMNIstituto Dermopatico dell'Immacolata, RomaIstituto Ortopedico Galeazzi, MilanoIstituti Ortopedici Rizzoli, BolognaIstituto dei Tumori di MilanoIstituto Europeo di Oncologia (IEO), MilanoIstituto di Fisica, Politecnico di MilanoIstituto di Genetica Molecolare CNR, Sezione di Istochimica e Citometria, PaviaIstituto Nazionale per la Ricerca sul Cancro (IST), GenovaIstituto Regina Elena, RomaLaboratorio Cell factory, Policlinico di MilanoOspedale San Gerardo, Monza, MilanoOspedale San Matteo, PaviaUnità di Tossicologia e Scienze Biomediche, ENEA Centro Ricerche, RomaUniversità Cattolica del Sacro Cuore, RomaUniversità di BariUniversità di BresciaUniversità di ChietiUniversità di L’AquilaUniversità di MilanoUniversità di Modena e Reggio EmiliaUniversità di MonzaUniversità di CataniaUniversità di PadovaUniversità di PisaUniversità di SienaUniversità “La Sapienza”, RomaZadig, Agenzia di Giornalismo ScientificoINTERNATIONAL COLLABORATIONSARCAGY (Association de Recherche sur les Cancers Gynécologiques), FranceBreakthrough Breast Cancer Center, Instutite of Cancer Reasearch, London, UKCancer Biomarkers and Prevention Group, University of Leicester, UKCancer Research UK, London, UKCancerdegradome Consortium, IP 6th FP, ECEORTC, Bruxelles, BelgiumEUROPA DONNAEuropean Agency for the Evaluation of Medicinal Products (EMEA), London, UKEuropean Regulatory Issues on Quality of life Assessment (ERIQA), Paris, FranceExecutive Board of GCIG (Gynecologic Cancer Intergroup)Genome Institute of Singapore (GIS), SingaporeGerman Cancer Research Center, Division of Toxicology and Cancer Risk Factors, Heidelberg,GermanyGoteborg University, Lundberg Laboratory for Cancer Research, Goteborg, SwedenHelios Klinikum Erfurt GmbH, Institute of Pathology, GermanyIstituto Oncologico della Svizzera ItalianaJohns Hopkins University, USALudwig Institute for Cancer Research, London, UKNational Cancer Center, Singapore20ANNUAL REPORT 2007

IRFMNStony Brook University, NY USAMassachusetts General Hospital and Harvard Medical School, USAMD Anderson Cancer Center, Houston, Texas, USAMRC, London, UKNational Cancer Institute (NCI), Bethesda and Frederick, MD, USAOspedale San Giovanni, Bellinzona, SvizzeraPaterson Institute for Cancer Research, Manchester, UKSouthern Europe New Drug Organization (SENDO), Milan, ItalyStroma Consortium, IP 6th FP, ECSwiss Federal Institute of Technology, Zurigo, SwizelandThe Sackler Institute, University College London, UKTumor Biology and Metastasis Institute of Cancer Research, Sutton, UKUniversity College, London Medical School, London, UKUniversity of Birmingham, UKUniversity of Cincinnati, USAUniversity of Crete Medical School, GreeceUniversity of Newcastle, UKUniversity of Pau, FranciaUniversity of Wisconsin, Madison, WI, USAKyoto University, JapanWeizmann Institute of Science, IsraelEDITORIAL BOARD MEMBERSHIPAttualità in Senologia (Paola Mosconi)British Journal of Cancer (Maurizio D’Incalci)Chemotherapy (Maurizio D’Incalci)Clinical Experimental Metastasis (Raffaella Giavazzi)Current Opinion in Oncologic, Endocrine and Metabolic Drugs (Maurizio D’Incalci)Current Cancer Therapy Reviews (Raffaella Giavazzi)European Journal of Cancer (Maurizio D’Incalci, Giovanna Damia, Raffaella Giavazzi,MassimoBroggini and Giulia Taraboletti)Health and Quality of Life Outcomes (Giovanni Apolone, Paola Mosconi)Health and Quality of Life Outcomes (Giovanni Apolone, Paola Mosconi)International Journal of Biological Markers (Raffaella Giavazzi)International Journal for Quality in Health Care (Giovanni Apolone)Journal of Ambulatory Care and Management (Giovanni Apolone)Journal of B.U.ON. (Maurizio D’Incalci)Journal of Chemotherapy (Raffaella Giavazzi)Journal of Experimental Therapeutics and Oncology (Raffaella Giavazzi)Journal of Medicine and the Person (Giovanni Apolone)Journal of Preventive Medicine and Hygiene (Giovanni Apolone)Molecular Cancer Therapeutics (Maurizio D’Incalci)Oncology Research (Maurizio D’Incalci)Tumori (Maurizio D’Incalci, Raffaella Giavazzi)www.senology.it (Paola Mosconi)www.PartecipaSalute.it (Paola Mosconi)21ANNUAL REPORT 2007

IRFMNPEER REVIEW ACTIVITIESAmerican Journal of Pathology, Annals of Oncology, Anti-cancer Drugs, BiochemicalPharmacology, British Journal of Cancer, British Journal of Pharmacology, British MedicalJournal, Cancer Chemotherapy and Pharmacology, Cancer Detection and Prevention, CancerLetters, Cancer Research, Carcinogenesis, Chemico-Biological Interactions, Clinical &Experimental Metastasis, Clinical Cancer Research, Cytometry, European Journal of Cancer,European Journal of Immunology, Faseb Journal, Gynecologic Oncology, Health and Quality ofLife Outcomes, Intensive Care Medicine, International Journal of Biological Markers,International Journal of Cancer, International Journal for Quality in Health Care, Journal ofAmbulatory Care and Management, Journal of Biological Chemistry, Journal of BiologicalMarkers, Journal of Cell Biochemistry, Journal of Clinical Oncology, Journal of ExperimentalTherapeutics and Oncology, Journal of Medicine and the Person, Journal of the National CancerInstitute, Journal of Neurology, Journal of Preventive Medicine and Hygiene, Journal of theNational Cancer Institute, Leukaemia, Molecular Cancer Therapeutics, Nature Biotechnology,Nature Reviews, Oncology Research, PharmacoEconomics, Quality of Life Research, Science,TumoriNATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPEthical Committee, Centro di Riferimento Oncologico, Aviano PN, ItalyEthical Committee, Ente Ospedaliero San Paolo, Milan, ItalyEthical Committee, Istituto Europeo di Oncologia, Milan, ItalyEthical Committee, Istituto Neurologico Carlo Besta, Milan, ItalyEthical Committee, Istituto Scientifico Eugenio Medea, Bosisio Parini, Lecco, ItalyEthical Committee, Ospedale San Gerardo, Monza, Milan, ItalyEthical Committee, Ospedale Sant’Anna, Como, ItalyEthical Committee, Ospedale della Valtellina e Valchiavenna, Sondrio, ItalyEthical Committee, IRCCS MultiMedica, Sesto San Giovanni, Milan, ItalyExecutive Board of GCIG (Gynecologic Cancer Intergroup)Scientific Committee, Associazione Italiana Ematologia e Oncologia Pediatrica, Monza, Milan,ItalyScientific Committee, Pezcoller Foundation, Trento, ItalyTechnical-Scientific Commitee, Associazione Italiana per la Ricerca sul Cancro, Milan, ItalyBoard of Directors, Metastasis Research Society (MRS)Board of Directors, Società Italiana di Cancerologia (SIC)Board of Directors, Società Italiana di Citometria (GIC)Directional Council Areas-CCINational Advisory Board 8th World Congress of Psycho-OncologyDevelopmental Therapeutics Program, National Cancer Institute (NCI)Decision Network and Executive Committee, South Europe New Drug Organization (SENDO)Executive Board, Europa DonnaExternal Scientific Committee, Angiotargeting Consortium – EU Sixth Framework Programme,University of Bergen, NorvegiaNHS R&D National Coordinating Centre for Health Technology Assessment, UKScientific Committee, Swiss Cancer LeagueUniversity Medical School of Siena, Italy22ANNUAL REPORT 2007

IRFMNEVENT ORGANIZATIONConference: “International Conference on Vascular Targeted Therapies in Oncology”.Mandelieu (France), October 4-6, 2007.Investigator Meeting “Cancer Pain” Istituto di Ricerche Farmacologiche Mario Negri,december 17, 2007 - MilanPARTICIPATION IN EVENTS IN WHICH THE DEPARTMENT WASINVOLVEDMeeting: 28th Winter PAMM Meeting “New vicinal diaryl-substituted imidazole derivativeswith vascular disrupting activity”. Berlin (Germany), 31 January - 3 February 2007.Conference: 4th International Conference on Tumor Microenvironment: Progression, Therapy& Prevention. “Identification of novel markers of tumor vasculature”. Florence (Italy) , 6 March- 10 March 2007.Conference: XVI Convegno Nazionale M. Gioia - Le nuove frontiere del diritto e dellamedicina legale: quale errore nella medicina estrema? “ Identificazione delle responsabilità nelpercorso tra ricerca preclinica e l’applicazione clinica in ambito oncologico”. Pisa (Italy), 25May -26 May 2007.Conference: European Conferences on Biomedical Optics (ECBO). “Study of antiangiogenicdrugs by fluorescence imaging and spectroscopy of a contrast agent in mice”. Munich(Germany), 17 June - 21 June 2007.Symposium: International Symposium on New Directions in Cancer Management “Targetingthe Tumor Vasculature. strategies for Combination Therapy”. Buenos aires (Argentina), 6 June- 8 June 2007.Meeting: V° Meeting NIBIT- Network Italiano per la Bioterapia del Tumori. AngiogenicTargets and Combination Therapies”. Siena (Italy), 21 September 2007 .Conference: XXV National Conference of Citometria “ La Citometria nell’era delleBiotecnologie”, Pontificia Università Lateranense, Rome, Città del Vaticano, 3-6 October 2007.Conference: International Conference on Vascular Targeted Therapies in Oncology.“Pharmacological approaches with vascular Targeting Agents in Combinations withChemiotherapy”. Mandelieu (France), 4 October - 6 October 2007.Conference: International Conference on Vascular Targeted Therapies in Oncology. “VEGFreleased by ovarian cancer cells stimulates organ specific MMP9 expression and invasion”.Mandelieu (France), 4 October - 6 October 2007.23ANNUAL REPORT 2007

IRFMNConference: International Conference on Vascular Targeted Therapies in Oncology. “Molecularcharacterization of tumor endothelium and identification of novel markes”. Mandelieu (France),4 October -6 October 2007.Conference: International Conference on Vascular Targeted Therapies in Oncology. “Binding ofthrombospondin-1 type III repeats to fibroblast grown factor-2 as an exploitable mechanism ofangiogenesis inhibition”. Mandelieu (France), 4 October - 6 October 2007.Conference: International Conference on Vascular Targeted Therapies in Oncology. “Newtubulin-binding imidazole derivatives as vascular disrupting agents”. Mandelieu (France), 4October - 6 October 2007.Conference: International Conference on Vascular Targeted Therapies in Oncology. “Molecularprofile of stroma derived from human ovarian carcinoma xenograft tumors equipped of differentangiogenic phenotypes”. Mandelieu (France), 4 October - 6 October 2007.Meeting: IX Congresso Nazionale di Oncologia Medica (AIOM). “Endpoints Farmacologicinello sviluppo e nell’applicazione clinica degli agenti antiangiogenici”. Palermo (Italy),12October - 15 October 2007.Conference: AACR/NCI/EORTC International Conference Molecular Targets and CancerTherapeutics: Discovery, Biology and Clinical Applications. “Influence of VEGF production onpaclitaxel response in a model of ovarian carcinoma xenograft”. S. Francisco (USA), 22October - 26 October 2007.Conference: 4th International Conference on Thrombosis and Hemostasis Issues in Cancer.“Protease-activated receptor-1 (PAR-1) expression correlates with a malignant phenotype inhuman melanoma”. Bergamo (Italy), 26 October - 28 October 2007.Gynecologic Cancer Intergroup (GCIG), General AssemblyOctober 28, 2007, Charité Ruine, Campus Charité Mitte, BerlinVISEAR II, Vienna Initiative to Save European Academic Research IIVienna, november 12, 2007Congress: 24° Congresso della Società Italiana di Chemioterapia. “Pharmacological endpointsin the development of angiogenesis inhibitors”. Verona (Italy), 25 November - 28 November2007.Meeting: 49th Annual Meeting of the Italian Cancer Society (SIC) “Modulation of endothelialcell gene expression by angiogenic factors”. Pordenone (Italy), 26 November - 29 November2007.Meeting: 49th Annual Meeting of the Italian Cancer Society (SIC) “Modulation of tubulinacetylation affects the sensitive of endothelial cells to the antimotility effects of paclitaxel”.Pordenone (Italy), 26 November - 29 November 2007.Congress: Swiss Proteomic Society “Large scale comparative proteomic study of accessiblevascular proteins in mouse liver metastases and normal liver”. Losanna (Switzerland), 3December -5 December 2007.24ANNUAL REPORT 2007

IRFMNGRANTS AND CONTRACTSAgenzia Italiana del FarmacoAIL Associazione Italiana contro le Leucemie, PadovaAmgem SpA, MilanAIRC Associazione Italiana per la Ricerca sul CancroASL PadovaASL Provincia di LodiAstra Zeneca SpAAstra Zeneca UKAzienda Sanitaria Locale - RiminiAzienda Sanitaria Unica Regionale - MarcheBracco Imaging SpA, MilanCentro Cochrane ItalianoChiesi Farmaceutici SpACNPDS, Centro Nazionale per la prevenzione e Difesa Sociale, MilanoCNR Consiglio Nazionale delle RicercheCNR-MIUR Ministero Istruzione Università e RicercaCompagnia di San Paolo, TorinoCTI Cell Therapeutics, Inc.Cyclacel Ltd.DompéEli Lilly Italia SpAElsevier Science Ltd.EORTC-European Organization for Research and Treatment of CancerEOS SpAEuropean Commission - 6th Framework Programme (Cancerdegradome, STROMA)FIRB-MIUR Fondo per gli Investimenti della Ricerca di Base-Ministero Istruzione Università eRicercaFIRC Fondazione Italiana per la Ricerca sul CancroFondazione Cassa di Risparmio delle Province LombardeFondazione Lu.V.I.Fondazione Nerina e Mario Mattioli OnlusFondo Edo TempiaGrunenthal, MilanoGlaxoSmithKline, VeronaIndena SpAInstitut de Recherche Pierre FabreIstituto Superiore di SanitàItalfarmacoKomen Italia OnlusLottomaticaMadaus SrlMedacMerck Sharp & DomeMinistero della SanitàNCI –SAIC FrederickNerviano Medical Science S.r.l.25ANNUAL REPORT 2007

IRFMNNovartis Farma SpAOptigenex Inc.Pfizer Global Research and DevelopmentPharma Mar, SAPharminox Ltd, UKPoliclinico di Padova / C.O.R.PTC Pharma AGRegione Emilia RomagnaRegione VenetoSara Bet, RomaSENDO-Tech SrlSigma-Tau SpAUniversità degli Studi di PadovaSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Mandalà M, Reni M, Cascinu S, Barni S, Floriani I, Cereda S, Berardi R, Mosconi S, Torri V, Labianca R. Venousthromboembolism predicts poor prognosis in irresectable pancreatic cancer patients. Ann Oncol 2007; 18: 1660-5.Cascinu S, Labianca R, Barone C, Santoro A, Carnaghi C, Cassano A, Beretta GD, Catalano V, Bertetto O, Barni S,Frontini L, Aitini E, Rota S, Torri V, Floriani I; Italian Group for the Study of Digestive Tract Cancer, Pozzo C,Rimassa L, Mosconi S, Giordani P, Ardizzoia A, Foa P, Rabbi C, Chiara S, Gasparini G, Nardi M, Mansutti M,Arnoldi E, Piazza E, Cortesi E, Pucci F, Silva RR, Sobrero A, Ravaioli A. Adjuvant treatment of high-risk, radicallyresected gastric cancer patients with 5-fluorouracil, leucovorin, cisplatin, and epidoxorubicin in a randomizedcontrolled trial. J Natl Cancer Inst 2007; 99: 601-7.Gregorc V, Spreafico A, Floriani I, Colombo B, Ludovini V, Pistola L, Bellezza G, Vigano MG, Villa E, Corti A.Prognostic value of circulating chromogranin A and soluble tumor necrosis factor receptors in advanced nonsmallcell lung cancer. Cancer 2007; 110: 845-53.Mustacchi G, Cazzaniga ME, Pronzato P, De Matteis A, Di Costanzo F, Floriani I on Behalf of the NORA Study Group. Breast cancer inelderly women: a different reality? Results from the NORA study. Ann Oncol 2007; 18: 991-6.Mosconi P., Colombo C., Satolli R., Liberati A. PartecipaSalute, an Italian project to involve lay people, patients’associations and scientific-medical representatives on the health debate. Health Expect 2007; 10: 194-204.Gallus S., Zuccaro P., Colombo P., Apolone G., Pacifici R., Garattini S., Bosetti C., La Vecchia C. Smoking in Italy2005-2006: Effects of a comprehensive National Tobacco Regulation. Prev Med 2007; 45: 198-201.Fossati R., Apolone G., Negri E., Compagnoni A., La Vecchia C., Mangano S., Clivio L., Garatini S., GeneralPractice Tobacco Cessation Investigators Group. Arch Intern Med 2007; 167: 1791-1797.Bianchi R., Gilardini A., Rodriguez-Menendez V., Oggioni V., Canta A., Colombo T., De Michele G., Martone S.,Sfacteria A., Piedemonte G., Grasso G., Beccaglia P., Ghezzi P., D’Incalci M., Lauria G., Cavaletti G. Cisplatininducedperipheral neuropathy: neuroprotection by erythropoietin without affecting tumour growth. Eur J Cancer2007; 43: 710-717.Salvati E., Leonetti C., Rizzo A., Scarsella M., Mottolese M., Galati R., Sperduti I., Stevens M.F.G., D’Incalci M.,Blasco M., Chiorino G., Bauwens S., Horard B., Gilson E., Stopacciaro A., Zupi G., Biroccio A. Telomere damageinduced by the G-quadruplex ligand RHPS4 has an antitumor effect. J Clin Invest 2007; 117: 3236-3247.Tavecchio M., Natoli C., Ubezio P., Erba E., D'Incalci M. Dynamics of cell cycle phase perturbations by trabectedin(ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematicalsimulation approach. Cell Prolif 2007; 40: 885-904.Lupi M., Matera G., Natoli C., Colombo V., Ubezio P. The contribution of p53 in the dynamics of cell cycle responseto DNA damage interpreted by a mathematical model. Cell Cycle 2007; 6: 943-950.26ANNUAL REPORT 2007

IRFMNSessa C., Cresta S., Cerny T., Baselga J., Rota Caremoli E., Malossi A., Hess D., Trigo J., Zucchetti M., D'Incalci M.,Zaniboni A., Capri G., Gatti B., Carminati P., Zanna C., Marsoni S., Gianni L. Concerted escalation of dose anddosing duration in a phase I study of the oral camptothecin gimatecan (ST1481) in patients with advanced solidtumors. Ann Oncol 2007; 18: 561-568.D'Incalci M., Steward W.P., Gescher A.J. Modulation of response to cancer chemotherapeutic agents by dietconstituents. Is the available evidence sufficiently robust for rational advice for patients? Cancer Treat Rev 2007; 33:223-229.Bagnati R., Bianchi G., Marangon E., Zuccato E., Fanelli R., Davoli E. Direct analysis of isopropylthioxanthone(ITX) in milk by high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun MassSpectrom 2007; 21: 1998-2002.Cavaletti G., Gilardini A., Canta A., Rigamonti L., Rodriguez-Menendez V., Ceresa C., Marmiroli P., Bossi M.,Oggioni N., D'Incalci M., De Coster R. Bortezomib-induced peripheral neurotoxicity: A neurophysiological andpathological study in the rat. Exp Neurol 2007; 204: 317-325.Gordon M.O., Torri V., Miglior S., Floriani I., Miller J.P., Gao F., Adamsons F., Poli D., D'Agostino R.B., KassM.A. Validated prediction model for the development for the development of primary open-angle glaucoma inindividuals with ocular hypertension. Ophthalmology 2007; 114: 10-19.Basse B., Ubezio P. A generalised age- and phase-structured model of human tumour cell populations bothunperturbed and exposed to a range of cancer terapies. Bull Math Biol 2007; 69: 1673-1690.Vikhanskaya F., Lee M.K., Mazzoletti M., Broggini M., Sabapathy K. Cancer-derived p53 mutants suppress p53-target gene expression-potential mechanism for gain of function of mutant p53. Nucleic Acids Res 2007; 35: 2093-2104.D'Incalci M., Brunelli D., Marangon E., Simone M., Tavecchio M., Gescher A., Mantovani A. Modulation of genetranscription by natural products. A viable anticancer strategy? Current Pharmaceutical Design 2007; 13: 2744-2750.Di Francesco A.M., Meco D., Torella A.R., Barone G., D'Incalci M., Pisano C., Carminati P., Riccardi R. The novelatypical retinoid ST1926 is active in ATRA resistant neuroblastoma cells acting by a different mechanism. BiochemPharmacol 2007; 73: 643-655.Bertele' V., Banzi R., Capasso F., Tafuri G., Trotta F., Apolone G., Garattini S. Haematological anticancer drugs inEurope: any added value at the time of approval? Eur J Clin Pharmacol 2007; 63: 713-719.Grassi R., Lombardi G., Reginelli A., Capasso F., Romano F., Floriani I., Colacurci N. Coccygeal movement:Assessment with dynamic MRI. Eur J Radiol 2007; 61: 473-479.Torri V., Floriani I., Poli D., European Glaucoma Prevention Study Group (EGPS). Central corneal thickness in theEuropean Glaucoma Prevention Study. Ophthalmology 2007; 114: 454-459.Gambacorti Passerini C., Tornaghi L, Marangon E., Franceschino A., Pogliani E., D'Incalci M., Zucchetti M.Imatinib concentrantrations in human milk. Blood 2007; 109: 1790Ruzzo A., Graziano F., Loupakis F., Rulli E., Canestrari E., Santini D., Catalano V., Ficarelli R., Maltese P., BisonniR., Masi G., Schiavon G., Giordani P., Giustini L., Falcone A., Tonini G., Silva R., Mattioli R., Floriani I., MagnaniM. Pharmacogenetic profiling in patients with advanced colorectal cancer treated with first-line FOLFOX-4chemotherapy. J Clin Oncol 2007; 25: 1247-1254.European Glaucoma Prevention Study Group (EGPS), Torri V., Floriani I., Rulli E., Poli D. Predictive factors foropen-angle glaucoma among patients with ocular hypertension in the European Glaucoma Prevention Study.Ophthalmology 2007; 114: 3-9.Grosso F., Jones R.L., Demetri G.D., Judson I.R., Blay J-Y., Le Cesne A., Sanfilippo R., Casieri P., Collini P., DileoP., Spreafico C., Stacchiotti S., Tamborini E., Tercero J.C., Jimeno J., D'Incalci M., Gronchi A., Fletcher J.A., PilottiS., Casali P.G. Efficacy of trabectedin (ecteinascidin-743) in advanced pretreated myxoid liposarcomas: aretrospective study. Lancet Oncol 2007; 8: 595-602.27ANNUAL REPORT 2007

IRFMNZangrossi S., Marabese M., Broggini M., Giordano R., D'Erasmo M., Montelatici E., Intini D., Neri A., Pesce M.,Rebulla P., Lazzari L. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cellmarker. Stem Cells 2007; 25: 1675-1680.Martinelli M., Bonezzi K., Riccardi E., Kuhn E., Frapolli R., Zucchetti M., Ryan A.J., Taraboletti G., Giavazzi R.Sequence dependent antitumor efficacy of the vascular disrupting agent ZD6126 in combination with paclitaxel Br JCancer 2007; 97: 888-894.Giavazzi R., Bani M.R., Taraboletti G. Tumor-host interaction in the optimization of paclitaxel-based combinationtherapies with vascular targeting compounds. Cancer Metastasis Rev 2007; 26: 481-488.Marabese M., Vikhanskaya F., Broggini M. p73: A chiaroscuro gene in cancer. Eur J Cancer 2007; 43: 1361-1372.Damia G., D'Incalci M. Targeting DNA repair as a promising approach in cancer therapy. Eur J Cancer 2007; 43:1791-1801.Dolfini E., Roncoroni L., Dogliotti E., Sala G., Erba E., Sacchi N., Ghidoni R. Resveratrol impairs the formation ofMDA-MB-231 multicellular tumor spheroids concomitant with ceramide accumulation. Cancer Lett 2007; 249: 143-147.Apolone G., Mosconi P. Techniques for assessing the quality of life with a particular emphasis on physical exercise.Springer-Verlag Italia, Milano, 2007; 183-190.Mosconi P., Satolli R., Colombo C., Liberati A., Donati S., Mele A. Hormone replacement therapy and information:in Italy a Consensus Conference to help woman decision. BMJ 2007.Malesci A., Laghi L., Bianchi P., Delconte G., Randolph A., Torri V., Carnaghi C., Doci R., Rosati R., Montorsi M.,Roncalli M., Gennari L., Santoro A. Reduced likelihood of metastases in patients with microsatellite-unstablecolorectal cancer. Clin Cancer Res 2007; 13: 3831-3839.Miglior S., Torri V., Zeyen T., Pfeiffer N., Vaz J.C., Adamsons I., European Glaucoma Prevention Study Group(EGPS) Intercurrent factors associated with the development of open-angle glaucoma in the European GlaucomaPrevention Study. Am J Ophthalmol 2007; 144: 266-275.Ubezio P., Lupi M., Matera G. Antiproliferative activity of cisplatin detected by CFSE in p53-proficient and p53-deficient cells. Immunol Invest 2007; 36: 847-859.Paulis M., Bensi M., Orioli D., Mondello C., Mazzini G., D’Incalci M., Falcioni C., Radaelli E., Erba E.,Raimondi E., De Carli L. Transfer of a Human Chromosomal Vector from a Hamster Cell Line to a MouseEmbryonic Stem Cell Line. Stem Cell , 2007; 25:2543-2550.28ANNUAL REPORT 2007

IRFMNLAY PRESS SELECTION PUBLISHED IN 2007Apolone G, Colombo CinziaIl dolore non necessarioPartecipa Salute 2007Apolone G, Mosconi PLe agenzie regolatorie: FDA, EMEA e AIFAIn : La dispensa di PartecipaSalute, 2007; 101-107Apolone GIl dolore nel paziente con cancro: attività in corso e primi risultatiCancer Aging 2007; 5: s10-s12Mosconi P, Colombo CinziaQuale informazione per la donna in menopausa sulla terapia ormonale sostitutiva? Rispondere attraverso unaConferenza di ConsensoNewsletter Ospedali Riuniti di Bergamo, Number 11 - September 2007; 7Colombo Cinzia, Mosconi PaolaL’associazionismo sta cambiando: dall’assistenza alla nascita di un progettoIn: La dispensa di PartecipaSalute, Mario Negri Institute 2007; 109115Mosconi P, Colombo CinziaDalla parte dei cittadini: al via il progetto “farmaci equivalenti: facciamo chiarezza”Newsletter Ospedali Riuniti di Bergamo, Number 8 - June 2007; 7Mosconi P, Colombo Cinzia, Satolli R, Liberati A.La ricerca clinica risponde ai bisogni dei pazienti? Risultati di un’indagine di PartecipaSaluteRicerca & Pratica 2007;23:192-203Mosconi P.Quando la medicina arriva prima della malattia.Consumers’ Magazine Ottobre 2007 pag. 5Mosconi P.Esperienze internazionali di coinvolgimento di associazioni di pazienti nella ricercaLe Donne per le Donne, Centro Congressi Villa Olmo (CO), 27-29 September 2007; 12-14Mosconi P.Discussione: il coinvolgimento del paziente nei sistemi sanitariIn: Rischio clinico e sicurezza del paziente. Modelli e soluzioni nel contesto internazionale. Società editrice IlMulino, Bologna, March 2007Mosconi P.La Commissione oncologica nazionale e la rappresentanza dei pazienti: occasione persa?http://www.partecipasalute.it/cms/?q=node/645; 2007; 24 AgoustMosconi P, Colombo Cinzia, Satolli R, Liberati A.Il corso di PartecipaSalute 2007 allo specchio: i commenti di chi ha organizzatohttp://www.partecipasalute.it/cms/?q=node/619 ; 2007; 12 JuneMosconi P, Colombo Cinzia, Satolli R.Le associazioni di pazienti e la ricerca clinica: un’indagine di PartecipaSalutehttp://www.partecipasalute.it/cms/?q=node/605 ; 2007; 28 May29ANNUAL REPORT 2007

IRFMNMosconi P, Colombo CinziaI tuoi diritti quando partecipi alla ricerca clinicahttp://www.partecipasalute.it/cms/?q=dirittiricerca; 2007; 2 FebruaryMosconi P, Colombo CinziaDalla parte dei cittadini: la ricerca clinica risponde ai bisogni dei pazienti? Dati presentati durante la secondagiornata internazionale della ricerca clinicaNewsletter Ospedali Riuniti di Bergamo, Numero 7 - May 2007; 6Mosconi P, Colombo CinziaClinical Trial Day 2007: seconda giornata internazionale della ricerca clinicahttp://www.partecipasalute.it/cms/?q=node/568 2007; 2007; 31 MayMosconi P, Apolone GSei domande sui comitati eticihttp://www.partecipasalute.it/partecipa/comitati001.php 2007; 15 January30ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Cancer PharmacologyMode of action of EcteinascidinsA project ongoing since several years is about the characterization of marine natural productspossessing antitumor activity. In particular we carried on the studies on the effects of ET-743 incells defective for some DNA repair mechanisms. Cells deficient for HomologousRecombination (HR) are very sensitive to the drug, while cells deficient for Non HomologousEnd-Joining (NHEJ) are only slightly more sensitive, but surprisingly cell lines defective forNucleotide Excision Repair (NER) are less sensitive to ET-743. Flow cytometric analysiscoupled to a software of computer simulation, developed in our laboratory, has demonstratedthat NER defective cells showed, after ET-743 treatment, cell cycle perturbations different thanthose occurring in NER proficient cells, probably for the activation of different and moreefficient repair mechanisms.We study also a functional evaluation of the DNA repair mechanisms by the cell capacity torecognize and repair double helix breaks with a recently introduced test that is very sensitive todetect the phosphorylation of histone H2AX. An in vitro study is ongoing with flow cytometryand immunofluorescence techniques to evaluate in different tumor cell lines the phosphorylationlevel of histone H2AX in relation to the distribution of the cells in the different phases of thecell cycle and the cytotoxic effect induced after treatment with ET-743.Studies are in progress on the mechanism of action of new ET-743 derivates compounds thathave shown antitumoral activity on cell lines with different DNA repair mechanisms.A new project is the study of the selective action of ET-743 on mixoid lyposarcoma, apathology representing 10% of all soft tissue sarcomas, trying to understand if the significativeantitumor effect is due to a selective action of the compound on pathogenetic alterationscharacteristic of this pathology. In particular we are trying to evaluate how ET-743 interact withthe transcriptional modifications of specific genes due to the translocation FUS-CHOP thatcharacterizes mixoid sarcomas or those caused by the interaction host-tumor, modifyinginflammatory and angiogenetic processes. Studies are in progress to obtain cell lines andxenografts of mixoid lyposarcomas exhibiting the same molecular features of the patients’tumors.Combinations of natural products of marine origin with other anticancerdrugsWe have observed additive or synergistic activity of ET-743 combined with other anticancerdrugs such as cisplatin, doxorubicin, campthotecin and inhibitors of telomerase.Flow Cytometric analysis of the DNA content in human ovarian cancer:clinical correlationsConflicting results have been published on the prognostic significance of DNA aneuploidy onadvanced ovary carcinoma (stage III or IV). The Citometry unit has reported one of the largeststudies of the scientific literature indicating that the aneuploidy in the advanced ovarycarcinoma is not an independent prognostic factor. In a large number of cases of stage I and IIovary tumors DNA content and the percentage of cells in S phase of the cell cycle, has beenmeasured with flow cytometry, demonstrating that in the early stages of the disease DNAcontent is a prognostic factor important for ovary tumor.31ANNUAL REPORT 2007

IRFMNAnalysis of cell cycle data and interactions of different drugsThe Biophysics Unit is engaged in theoretical and methodological studies aimed at a criticalevaluation of current techniques of investigation of drug effects on heterogeneous cellpopulations. Several computing tools have been produced to simulate the cell proliferation atdifferent levels (from molecular interactions to in vivo growth of solid tumours) and the processof measure.Collaborations are ongoing with other research groups for design and data analysis of drugcombination studies in vitro. In this field, a number of computer programs have been developed,allowing comparative data analysis with the most common models of drug interaction.Evaluation of the complexity of the response of cell populations totreatment with anticancer drugsThis project of the Biophysics Unit addresses the issue of establishing a connection between theintracellular drug interactions and the resulting cell cycle perturbations. It starts from the singlecelllevel of investigation to reach the cell-population level where the relevant end points oftreatment efficacy are evaluated by flow cytometry and growth inhibition/cytotoxicity assays.The model adopted for data analysis and interpretation is the result of the merging of twomathematical models. One model describes the cell cycle, exploiting the results of the theory ofage-structured cell population dynamics. The second model describes the response to the drug'schallenge, using distinct parameters ("effect descriptors") measuring either the strength of cellcycle arrest, damage repair or cell death in every phase (G1, S and G2M). In this way, it ispossible to reach a quantitative interpretation of the experimental results, overcoming thecurrent qualitative and partial approaches to this problem, which are unable to resolve theoverlapping of cytostatic and cytotoxic effects, and to establish a connection with phase-relatedevents.Applying this procedure we demonstrated complex but biologically consistent patterns of timeand dose-dependence for each cell cycle effect descriptor, following a short treatment withmelphalan on a reference cell line of ovarian carcinoma. These results add to the previouslyreported studies on topotecan, cisplatin and taxol. Eventually, this project will produce adatabase containing the values associated to the new effect descriptors, related to fewcompounds but rich of information about them, especially in the dose and time dependence ofthe effects. This database will be used to compare the treatment response of the most commondrugs adopted in the ovarian carcinoma.Cell cycle dysregulation in erlotinib-based treatments decoded by flowcytometry and mathematical modellingEpidermal growth factor receptor (EGFR) inhibitors represent one of the most promising classof anticancer compounds, some of them, like erlotinib, are already used for clinical therapy.Nevertheless, so far, the research has focused on molecular interaction of these compounds,somewhat neglecting the study of the dynamics of cell cycle perturbations and underscoring theimportance of this issue for the optimization of both single and multidrug therapies.In order to fill this gap, the Biophysics Unit will apply the multidisciplinary approach,exploiting cell cycle simulation tools, to the study of cell cycle perturbations induced byerlotinib as a representative EGFR inhibitor. Studies of the time- and dose-dependence ofperturbations induced by treatments are ongoing for single erlotinib treatment or for erlotinibcombined with gemcitabine, irinotecan and oxaliplatin. The appreciation and the quantificationof these effects will provide an important contribution to the comprehension of the mode ofaction of erlotinib alone or in combination. Such perspectives are vital if the events are to bedecoded and used in predictive models for the exploration of pharmacodynamic actions and inunderstanding the origins of treatment failure.32ANNUAL REPORT 2007

IRFMNAnticancer Drug Effects Decoded by Time-Lapse Imaging, Flow Cytometryand ModellingWe aim to use flow cytometric (cell-population based analysis) and time-lapse imaging (singlecell lineage based analysis) techniques to generate data that will be used to predict drugresponses in term of the two major determinant of cytostatic/cytotoxic actions of anticancerdrugs: specific cell cycle perturbations (detecting accumulation or depletion of cells in G1, Sand G2M phases) and the commitment to cell death (apoptosis).The response to a treatment with the anticancer drugs will be investigated in a humanosteosarcoma cell line U2OS. engineered in order to express fluorescent reporter (a fusioncyclin B1-GFP protein), so that it is now possible to follow the cells through G1, S and G2Musing time-lapse microscopy. The working hypothesis is that quantitative analysis of time-lapsemicroscopy data could be integrated with the information provided by flow cytometry -allowing for the first time a joint interpretation of both kind of experiments through a commonmathematical model that simulate the underlying phenomena. The final goal is to provide newlevels of understanding and simulation tools to the cancer research community.Timing the changes of the cellular content of specific proteins inside G1,in exponentially growing cellsWe developed a method for measuring the content of immunocytochemically detected proteinsin individual cells progressing through G1 phase. The feasibility was demonstrated in theanalysis of cyclin E levels. The sequence of G1 events is tracked in unaltered cyclingconditions, in a cell line in the phase of balanced growth in vitro, to avoid the pitfalls ofsynchronization.The method is based on i) a bromodeoxyuridine (BrdUrd) pulse-and-chase experimental plan;ii) triparametric flow cytometric detection of DNA, BrdUrd and cyclin E; iii) data analysissupported by the basic mathematical theory of asynchronous growing populations with variablecell cycle phase durations.Establishing a CFSE-based method for a quantitative measure ofcytostatic effects of drugs on tumor cell populationsCarboxyfluorescein diacetate succinimidyl ester (CFSE) is currently used to investigatemigration and proliferation of hematopoietic cells. Several technical problems had precludeduntil now its use in the studies of the antiproliferative activity of anticancer drugs. We analysedseveral critical steps of the procedure, providing the way to overcome potential pitfalls. Theproject was eventually successful and the previous limitations were overcome. The outcomewas a new standardised procedure of cytometry and data-analysis allowing a measure of thedynamics of cell cycle blockades after drug treatment. The new method was applied in the studyof the drug topotecan, measuring the variability of response to treatment in terms percentage ofcells immediately blocked, divided once or more times and then blocked or unaffected by thetreatment.Pharmacokinetic of new taxane derivatesPreclinical studies have been done on new taxane derivatives, chemically and biologicallydifferent from the conventional ones. For three of these compounds we evaluated thebioavailability after oral administration and the kinetic and metabolic profile has been entirelycharacterized by applying analytical methods based on HPLC/MS/MS technique developed inthe Cancer Clinical Pharmacology Unit. They showed biological activity even in tumors withlow susceptibility to other taxanes (cerebral tumor), suggesting a potential clinical interest.According to the hypothesis that these drugs act through an antiangiogenic mechanism, putforward by the Laboratory of the Biology and Therapy of Metastasis, it would be important to33ANNUAL REPORT 2007

IRFMNinvestigate prolonged chronic treatments and therefore we are investigating the pharmacokineticproperties after oral administration and after prolonged daily treatment.An interesting pk profile was found for the 14-β-hydroxy-10-deacetylbaccatin III derivativeIDN 6140. He showed good bioavailability and high distribution in brain achieving highconcentration in comparison to that of plasma, demonstrating high ability to cross the bloodbrain-barrier.These data make this compound of great potential interest for the therapy ofCentral Nervous System tumors and metastasis.Clinical pharmacokinetics of gimatecanA study in collaboration with SENDO has shown the clinical pharmacokinetics of a newderivative of camptothecin (Gimatecan) in patients with sarcoma under phase II investigation.Studies still in progress show that this compound is rapidly absorbed after oral administrationand has a long half life, with consequent long exposure to the drug.Pharmacokinetic parameters will be correlated to the clinical data, i.e. toxicity and antitumoractivity, to characterize the properties of this new drug.Other relevant clinical studiesST 1926. The clinical pharmacokinetic of ST1926 a new oral retinoid derivative is underinvestigation in women with ovarian cancer .In particular we studied the pharmacokinetics, thebioavailability of the drug and its metabolism during a Phase I study, providing for the firsttime data on the main plasma metabolite of ST 1926 (the glucuronide conjugate). Our data showvariable absorption of the drug and high conversion to the glucuronide.Antitumoral activity and pharmacokinetic properties of new drugs andcombinationsThe antitumor activity, pharmacokinetic properties and toxicity of novel anticancer drugs withspecific targets (e.g. different kinase inhibitors), conventional anticancer drugs (camptothecin)and combinations is being investigated using rodent tumors and human tumor xenografts.Laboratory of Molecular PharmacologyG2 checkpoint and cell cycleA new system able to specifically inhibit CHK1 expression in vivo in nude mice transplantedwith human tumors has been developed. The system has been proved to be able to reduce theexpression only in cancer cells. This plasmid allows the expression of the siRNA only afterinduction with tetracycline and is therefore a unique tool to determine the effect of CHK1inhibition in human tumors growing in nude mice following treatment with anticancer agents.Characterization of new potential oncosuppressor genesDRAGO gene, identified and cloned in our laboratory is one of the most interesting projects ofthe group. The characterization of the response of KO mice for DRAGO to ionising radiation issimilar to normal mice. The characterization of the transcriptional regulation of DRAGOindicated that the gene is not only a p53-responsive gene, but, inside the p53 family, p73 has astrong ability to induce the transcription. Drago therefore, represents a new p73 responsivegene.Molecular characterization of ovarian carcinomaThe molecular characterization of stage I ovarian carcinomas has been further studied.The gene expression profile analysis has showed interesting results.It is possible to identify genes able to discriminate in ovarian cancer the different histotypes,suggesting that specific biological and molecular characteristics are responsible for the34ANNUAL REPORT 2007

IRFMNdifferences in morphology and clinical behaviour. These studies can help in identifying newspecific molecular targets for the different subclasses of ovarian cancer that have a differentclinical outcome.It is possible to identify patients with higher probability of relapse because there are genes ableto differentiate these two classes of patients.We have demonstrated that stage I borderline patients have a gene expression profile similar tothat of grade 1 patients, while they are well distinguishable from grade 2 and grade 3 patients.This can have important clinical implications for the treatment of this particular subgroup ofpatients.Expression of p63 in ovarian tumorsP63 belongs to the family of p53 and its role is particularly relevant in embryo development. Itsrole as tumor suppressor has not been clarified yet. The gene encoding for p63 has a complexorganisation which generates, among the others, a truncated isoform (DNp63), lacking thetransactivation domain, which could act as a dominant negative for either p63 or p53. We haveanalysed the expression of the entire form (TAp63) and of DNp63 in approximately 90 ovariancancer patients at stage I (early) and 90 at stage III (advanced tumor). The levels of TAp63 aresimilar in the two groups of patients, while the levels of DNp63 were higher in stage III tumorsthan in stage I tumors. The ratios between DNp63 and TAp63 increased consequently with theincrease in malignancy. As a result, patients with higher ratio between DNp63 and TAp63 havea worst probability to survive. This has been observed not only in the whole population, but thetendency has been observed also analysing separately the two groups of patients. Altogetherthese results suggest that DNp63 can be considered as a new target to inhibit in this malignancy.Inhibition of the signal mediated by PI3K/aktIn ovarian carcinoma cells PI3K gene is often overexpressed or mutated and this kinase is thenconstitutively activated. The consequence is the presence of proteins involved in cell survival,like akt, constitutively phosphorylated and then active. This project evaluated the capacity oftetra and pentaphosphate inositols and of some analogues to inhibit akt recruitment to themembrane and its further phosphorylation. IP5 and some new synthetic molecules, were shownto induce a reduction of the phosphorylation of akt and therefore to reduce cell growth. Theactivity of these molecules is not restricted to ovarian cancer, but has been shown also for othertumors such as breast and prostate. The possibility to combine PI3K inhibitors and mTOR ( akinase downstream to PI3K and akt) inhibitors has been evaluated. It has in fact been shownthat some mTOR inhibitors induce an aberrant phosphorylation, and hence activation of akt, andthe combination of these inhibitors can block these undesired effects. The in vitro results showthat the combination has at least an additive effect and open the way to find out new treatmentschedules to verify whether the sequence of combination can be important for theantiproliferative effects.Oncosuppressors p53 and p73The p53 analogue, p73 is present in different isoforms derived from alternative splicing of theC-terminal. Among these isoforms there is one called DNp73 in which the transactivationdomain in the N-terminal of the protein is absent. This DN form of p73 is an antagonist of p53.The DNp73 isoforms can be further processed through alternative splicing, to generate a numberof isoforms with a not yet clarified biological activity. We have previously shown that the alphaform of DNp73 does not modify either the growth in vitro and in vivo of cancer cells or theresponse to treatment with anticancer agents.We have therefore generated clones derived from the human lung cancer derived cell lineH1299 which express DNp73 beta following induction. These clones show, upon induction,interesting effects on cell growth suggesting that the presence of high levels of this isoform canhave unpredicted effects. Since the data generated on the expression of p73 isoforms in cancer35ANNUAL REPORT 2007

IRFMNpatients indicate that the beta isoform of DNp73 is indeed present in human cancer, the resultsobtained have a particular relevance and will be further analysed to verify which are the effectslinked to the overexpression of this isoform.Identification of a new proteolytic mechanism of activation of p73Another member of the p53 family, p73, has been characterised in the laboratory for its abilityto modify the growth and response to therapy of cancer cells. We have further studied thispotential tumor suppressor by studying new possible mechanisms of activation. We have foundan additional regulation mechanism for the p73-alpha form that occurs through proteolyticcleavage connected to the activity of the serine protease HtrA2. Following apoptotic stimuli,HtrA2 accumulates in the nucleus and cleaves p73alpha in the C-terminal portion, enabling theprotein to increase its transactivation activity on the apoptotic gene bax but not on the cell-cycleregulator gene p21. In the presence of HtrA2, p73 is more prone to cause caspase activation andnuclei fragmentation: p73 needs HtrA2 to activate and enhance its apoptotic functions. This newrelation between p73 and HtrA2 may help to understand the different behavior of the p73protein in cell physiology and in the responses of cancer cells to chemotherapy.Mechanisms of action of new antitumor drugsThe mechanism of action of a new anthracycline derivative, nemorubicin (methoxy morpholinodoxorubicin) has been characterised. Nemorubicin presents a pattern of antitumor activity invitro and in vivo different from that of doxorubicin.We have found that cell lines with defects in DNA repair mechanisms, and particularly in thenucleotide excision repair (NER), are resistant to the treatment with this molecule. It isinteresting to note that the majority of the drugs interacting with DNA, like cisplatin, showexactly the opposite: defects in NER are associated with high sensitivity. Moreover, a possibleresistance mechanism for these drugs, is associated with an increased activity of NER in sometumors.Nemorubicin resistant cells have instead a reduced NER activity and show a collateralsensitivity to cisplatin. These result represent the molecular rational for the combined treatmentbetween cisplatin and nemorubicin and /or for the treatment of cisplatin resistant tumors withnemorubicin.Generation of new cellular systems for in vivo imagingWe have generated new cell clones derived from human cancer cells growing in vitro, whichstably express fluorescent or luminescent probes which can allow us to follow in vivo thegrowth of primary tumors and metastasis in mice. These systems generated in human ovarian ,breast and prostate cancer cell lines, can be implanted in nude mice and the growth and responseto therapy followed by either optical and luminescent imaging or microTAC analysis.These systems will be particularly useful to study the antimetastatic potential of new drugs.Characterization of response of stem cells to damageThe therapeutic use of stem cells is continuously increasing. This project aims to investigate theability of stem cells isolated from umbilical cord to respond to stress induction with particularemphasis on their ability to activate checkpoint proteins. Stem cells isolated and maintained invitro with specific cytokine cocktails able to induce partial differentiation, have shown apeculiar expression of proteins controlling the cell cycle. In particular, there is a specific timepoint in the differentiation process in which cell cycle checkpoints proteins are present at highlevels. This could imply that this represents the time point in which these cells are moresusceptible and must be “protected” from external damages. The characterization of theseimportant molecular aspects will be further studied.36ANNUAL REPORT 2007

IRFMNIdentification of cancer stem cells from ovarian cancerThis project is aimed at isolating and characterizing a possible cancer stem cell from ovariancancers. There are increasing evidences supporting the idea that few important multipotentcancer cells, termed cancer stem cells, are among the most relevant cells to be killed in a tumor.Normally present as quiescent cells inside the tumors, they are able to rapidly generate dividingand growing cancer cells. The current hypothesis is that normally dividing cancer cells can bepreferentially killed by chemotherapy while the cancer stem cells would be more difficult to killand would be responsible for the relapse following treatment. The possibility to identify andcharacterize the cancer stem cell would theoretically open the way to the selection of newgeneration molecules able to preferentially kill these cells. Cancer stem cells have been alreadyidentified in different human tumors including breast and hematopoietic tumors. We will focusour attention on human ovarian cancer by the use of antibodies directed against surface markerproteins to identify such potentially relevant cancer sub populations.Determination of the impact of EGFR mutations in the activity of tyrosinekinase inhibitors in patients with NSCLCWe have started a multicenter, three year project aimed at identifying those patients who canhave the chance to respond to tyrosine kinase inhibitors.The study will define whether patients without mutations in the EGFR have more chance torespond to conventional therapy rather than to treatment with TKI.Our laboratory is involved in the detection of mutations of EGFR in patients with NSCLC eitherin the tumor tissue or in the blood of patients with a secondary aim of defining whether ispossible to detect mutations in the blood circulating DNA which are representative for thetumor. DNA sequencing and scorpion arms-based PCR methods are used for these studies.Laboratory of Biology and Therapy of MetastasisPhysiologic regulation of angiogenesisAngiogenesis, the formation of blood vessels from existing ones is a fundamental process intumor progression. A delicate balance between pro- and antiangiogenic factors finely tunes thisprocess. In the last years we have been interested in endogenous angiogenesis-regulatoryfactors. During 2007 we have continued the study of trombospondin-1 (TSP-1), an endogenousinhibitor of angiogenesis. TSP-1 directly binds to angiogenic factors, in particular FGF-2(Fibroblast Growth Factor-2), inhibiting their bioavailability and activity. In particular, we arestudying the structure/function relationship of the different active domains of TSP-1, with themain aim to identify the FGF-2 binding site and design antiangiogenic compounds based on theactive sequence of TSP-1.We studied the involvement of matrix metalloproteinases (MMPs) in angiogenesis andtumor progression. In particular in 2007, we studied the cross-talk between MMPs and VascularEndothelial Growth Factor (VEGF), a factor that stimulates angiogenesis and vesselpermeability, during ovarian carcinoma progression. In addition we evaluated the possibilityof using VEGF inhibitors to affect MMP-dependent ovarian tumor invasion.We are analyzing modifications in metastasis/invasion-related gene expression profile in stromacells induced by tumor VEGF.Lymphangiogenesis in ovarian carcinomaLymphatic spread in early ovarian cancer is a predictor of outcome with potential clinical value.With the aim to clarify the molecular mechanisms involved in the process of lymphangiogenesisin ovarian cancer, the expression of VEGFC, a factor that stimulates lymphangiogenesis, has37ANNUAL REPORT 2007

IRFMNbeen analyzed in serum and ascites of mice bearing human ovarian carcinoma xenografts andcorrelated with lymphonodes infiltration by neoplastic cells .How the microenvironment affects endothelial cell gene expressionIt is fundamental to understand qualitative and functional differences between tumor and normaltissue endothelial cells (EC) and the molecular mechanisms that drive the angiogenic process.This could lead to the identification of selective markers of the vascular endothelium associatedto pathologies and/or of target molecules for the development of new drugs. To this purpose weanalysed the gene expression profile of endothelial cells isolated from ovarian carcinoma andadrenal glands exposed or not to an “angiogenic/tumor” environment reconstituted in vitro. Wehave found that:i) the “angiogenic/tumor” environment is indeed able to modulate EC gene expression;ii) few genes are preferentially expressed by tumor associated endothelial cells.Preliminary results suggest that few of the genes might be of interest as markers of tumorendothelium. Their expression is higher in endothelial cells from tumor specimens than fromnormal tissues and they are not expressed by tumor cells. The putative new tumor endothelialmarkers have been further validated as anti-cancer / vascular targets by in situ hybridizationanalysis of normal and tumor tissues. The putative targets (identified as mRNA transcript) arebeing produced as recombinant proteins, with the aim to isolate antibodies, directed against therecombinant proteins, suitable for immunohistochemical analyses.Preclinical models: role of Vascular Endothelial Growth Factor (VEGF) ontumor growth, vascularization and response to therapyTo study the role of VEGF induced angiogenesis in the resistance of cancer tochemotherapy we have generated a variant of the human ovarian carcinoma A2780/1A9 bystable transfection with the VEGF121 isoform (1A9-VS1) or the antisense (1A9-VAS3). 1A9-VS1cells express VEGF mRNA and the protein is secreted in the culture supernatant. 1A9-VS1xenografts i) show dilated blood vessels when implanted sc ii) produce ascites when implantedorthotopically in the peritoneal cavity, iii) release human VEGF in the plasma, iv) the level ofcirculating VEGF correlates with tumor burden.We have found that the response to chemotherapy (e.g. paclitaxel) might differ in xenograftsproducing high levels of VEGF. The blockade of VEGF, by the administration of Avastin®,greatly improved the antitumor activity by paclitaxel treatment of 1A9-VS1 (T/C=11% vsT/C=40%).Studies are ongoing to elucidate the mechanism responsible of these differences. The hypotesisbeing that by producing/releasing VEGF, the cancer cells modify the microenvironmentof the growing tumor, and in doing so, influence the response to therapy.This model is therefore being used to study gene expression. Based on circulating VEGF levelsand morphological analysis of the tumor vasculature, samples were chosen and tissue slices ofthe 1A9-VS1 and 1A9-VAS3 were xenografts microdissected (PALM Microlaser system, incollaboration with the Institute of Pathology at Helios Klinikum, Germany) in order to isolatethe stroma compartment to be evaluated by mean of Affymetrix’s GeneChip® Arraystechnology. The microarray hybridisation data were analyzed with the aid of specializedsoftwares (i.e. GeneSpring and Rosetta Resolver) and differentially expressed genes wereidentified using a one-way, modified and error-weighted Analysis of Variance (ANOVA) witha P-value cut-off of 0.01. Specifically, we discovered that in the stroma of tumors 227 were upregulatedand 167 were down-regulated in consequence of the VEGF produced by the cancercells. Gene Ontology (GO) enquiry (using Expression Analysis Systematic Explorer EASE),identified over-represented categories of potentially biologically relevant genes in the stroma of1A9VS1 (high VEGF) tumors. Structural molecule activity, cell organization and biogenesis,38ANNUAL REPORT 2007

IRFMNand basement membrane were among the up-regulated categories, with EASE score lower than0.07.Vascular targeting agentsAntineoplastic therapies directed against the tumor vascular system may be designed with twodifferent strategies. Antiangiogenic therapy prevents the formation of new vessels, whilevascular disrupting treatment aims to selectively destroy the already formed tumor vessels. In2007 we have focused on the identification of molecules with vascular targeting properties.Among the anti-vascular compounds, we have studied the properties of tubulin binding agents(analogues of colchicines and combretastatins), which cause microtubules depolymerization,selective damage to tumor blood vessels and tumor necrosis in experimental models in vivo. Incollaboration with Prof. Bellina at the Department of Chemistry, University of Pisa (Prof.Bellina and Prof. Rossi), we have screened classes of compounds with such properties. The leadcompound/s will be further characterized for pharmacological and anti-vascular/antineoplasticproperties.Antineoplastic combination TherapiesThe optimization of biological therapies against selective targets in combinations withchemotherapy is one of the interest of this laboratory. The main class of compounds under studyare the angiogenesis inhibitors and vascular disrupting agents that are thought to achieveoptimal therapeutic efficacy when combined with conventional therapies.We have shown that in combination treatments, the vascular disupring activity of the tubulinbinding ZD6126 is blocked by pretreatment with paclitaxel, a tubulin binding drug withopposite mechanism of action. An optimized schedule of administration of the two agents notonly avoids the interference among drugs, but greatly improves the antineoplastic efficacy of thecombination in experimental models. Studies are undergoing to further otpimize schedules oftreatment on the basis of the mechanism of action of the drugs given in combination.There is clear evidence that histone deacetylase inhibitors are a new potential class of anticanceragents; recent reports indicate a greater antitumor effect of these compounds combined withradiation and chemotherapy. We investigated the effect of suberoylanilide hydroxamic acid(SAHA) on the paclitaxel-sensitive 1A9 and -resistant 1A9PTX22 cells.SAHA induced a comparable growth inhibition at micromolar concentrations and in a dosedependentmanner in both 1A9 and 1A9PTX22 cells. Inhibition of cell proliferation, evaluatedby combining doses of SAHA and paclitaxel ranging from IC30 to IC0, produced a synergisticeffect. These findings show that SAHA has an antitumor activity in ovarian cancer cells withdifferent sensitivity to paclitaxel. Furthermore, SAHA seems to be capable to partially restoresensitivity to paclitaxel in the 1A9PTX22 resistant cells. The effect is not due to a cell cycleblokade or augmented apoptosis, but is instead associated to enhanced tubulin acetylation.High PAR-1 expression is associated to a malignant phenotypeProtease-activated receptor-1 (PAR-1) over-expression has been associated to a variety ofhuman cancers, and increasing evidence implicates PAR-1 as a contributor to human melanomamalignancy. We investigated human melanoma cells, isolated from lesions representing variousstages of disease progression, for the expression of PAR-1 (in collaboration with Prof. Naldiniat the University of Siena) and evaluated their migratory and metastatic capabilities. Cells fromadvanced stage melanomas expressed higher levels of PAR-1 than those from early stages. Themetastatic capability showed by the melanoma cells which overexpressed PAR-1 allowed thesecells to colonize the lungs in 70-100% of the mice. Accordingly, melanoma cells overexpressingPAR-1 had higher migrated (chemotaxis assay) and invading (invasion assaymatrigel)cell counts than those expressing low PAR-1. Migration and invasion were decreasedby both, PAR-1 siRNA and SCH9797 (PAR-1 specific inhibitor).39ANNUAL REPORT 2007

IRFMNLaboratory for the development of new pharmacologicalstrategiesThe laboratory was born out of the consideration that the advent of oncological drugs endowedwith mechanisms of action different from those of traditional chemiotherapics, introduces newtreatment opportunities. At the same time, new problems arise concerning the choice of themost appropriate and effective design for research into the clinical activity profile of these newtreatments.The traditional paradigm where the choice of dose is based on the maximal tolerated toxicity,and the screening of therapeutic activity focus on tumor mass reduction, may not necessarily besuitable for the evaluation of new agents whose targets may include the extracellularcompartment or specific molecular targets.The clinical development of ‘non toxic’ anti tumor molecules requires a critical review of theexisting models as well as of all the aspects relative to the conduction of clinical trialsincluding: dose selection criteria, methods for determination and confirmation ofpharmacological activity, and the validation of new technologies and laboratory methods.This is where the need for a profound integration of the ‘clinical screening’ and the preclinicalresearch lies. It is a prerequisite for the construction of the pharmacological rationale for theidentification of the most interesting molecules, the choice of dose, the hypotheses ofcombination with other drugs, and of the most appropriate indicators of clinical activity.The acquisition of know how and the development and application of new designs for clinicalactivity studies, including the use of randomization, the introduction of groups of patientstreated with placebo, and new discontinuation designs, proceed in parallel to the above.Another fundamental issue in laboratory research is the recognition that the genomiccharacterization of any single tumor may now play a more relevant role in drug developmentand treatment identification.This notwithstanding, numerous uncertainties remain regarding the role of biomarkers in drugdevelopment and in the implementation of genomic technologies in clinical trials. It is thereforenecessary to improve the methodology and more biomarkers evaluation already in the earlystages of research, thus shifting translational research from a simple process of correlationsearch to one producing knowledge regarding the predictive role of the clinical activity of theinvestigational treatments.Therefore, the primary focus of the laboratory is the optimization of the methods for evaluatingthe activity of cytotoxic drugs, but mostly for those therapies aimed at specific moleculartargets, as well as the identification of factors predictive of therapeutic response.Laboratory of Clinical TrialsThe Laboratory of Clinical Trials is involved in the planning, coordination and analysis ofrandomized clinical trials in oncology, conducted in cooperation with a network of medicaloncologists. Main covered research areas are gastric, colo-rectal, breast and lung cancer.Gastric cancerITACAS ”Intergruppo Nazionale Adiuvante Gastrico” study is a randomised, open-label,multicenter, trial aimed at assessing the role of adjuvant chemotherapy in the treatment ofgastric cancer. It compares the efficacy and safety of a sequential treatment (campto plusflurouracil/leucovorin, followed by taxotere and cisplatin) versus flurouracil/leucovorinregimen, used as standard reference in patients with radically resected adenocarcinoma of thestomach or gastroesophageal junction. The study, sponsored by Mario Negri Institute, involves40ANNUAL REPORT 2007

IRFMN11 oncological collaborative groups and is being conducted in more than 110 Italianexperimental centers. From February 2005, more than 750 patients out of the planned 1100 havebeen enrolled and it is expected to conclude the recruitment in the first half of 2009.Lung cancerThe epidermal growth factor (EGFR) is overexpressed in many solid tumors including nonsmall cell lung cancer (NSCLC) and is associated with disease progression and poor prognosis.EGFR is therefore a promising target for anticancer therapy. The development of agents thattarget the EGFR signal transduction pathways has provided a novel class of therapeutic agents.Erlotinib, an orally active, selective EGFR tyrosine kinase inhibitor (TKI) recently approved inNSCLC pre-treated patients is with gefitinib the most studied agent of this class of compounds.Recently, several authors reported results of analyses on retrospective data suggesting arelationship between response to TKIs and specific mutations in the EGFR tyrosine kinasedomain, increased EGFR and Her-2 gene copy number and to the expression of p-AKT andpoorer clinical outcomes in patients with K-ras mutant when treated with erlotinib andchemotherapy.All these data are suggesting that a "tailored therapy" based on individual molecular featuresmay result in better responses and optimization of sources and costs. Evidences so far availableare based on retrospective or "post hoc" analyses of trials and are drawn on samples, nonproviding adequately powered tests. Furthermore, ongoing studies assess possibleprognostic/predictive factors only as explorative analyses. As a consequence, in those trials noformal hypothesis primarily focused on a differential effect of treatment based on selectedspecific patient subgroups has been addressed with an adequate power.TAILOR AIFA trial is aimed at comparing the efficacy in terms of overall survival of erlotinib vs.docetaxel given as second line therapy in pts with advanced NSCLC. In particular, thepredictive value of K-ras mutation, EGFR protein expression and EGFR gene amplification indetermining the effect of erlotinib as compared to chemotherapy will be assessed. The study,sponsored by Azienda Ospedaliera Fatebenefratelli e Oftalmico of Milan and supported by theAgenzia Italiana del Farmaco (AIFA), started in late 2007 and will enroll approximately 1500patients in three years.Colon cancerA randomised, phase III clinical trial aimed at identifying the best therapeutic adjuvant strategyin radically resected colon cancer patients is starting. The study, sponsored by FondazioneGiscad per la Cura dei Tumori and supported by the Agenzia Italiana del Farmaco (AIFA), willassess the following two questions:1) Optimal duration of FOLFOX-4 regiomen (3 vs 6 months)2) Efficacy of the addition of Bevacizumab to FOLFOX-4 regimen (only in high risk stageIII patients)For both questions, primary efficacy endpoint will be recurrence free survival.Breast cancerTOP (Trastuzumab Optimisation trial) study is aimed at increasing the knowledge on theefficacy of herceptin in the treatment of locally advanced or metastatic breast cancer patients. Itincludes two randomised, open label, phase III clinical trials: the first addresses the impact of amaintenace therapy with herceptin in patients previously treated with chemotherapy plusherceptin, the second is focused on the efficacy in term of overall survival of a second-line ofchemotherapy plus herceptin versus chemotherapy alone in patients progressed to a first-linecontaining herceptin. The results of TOP study will allow to evaluate the cost/benefit ratio ofherceptin treatment and to optimise the therapeutic effectiveness of such a drug, already41ANNUAL REPORT 2007

IRFMNapproved in Italy, but used without clear data supporting evidence of benefit in the consideredsetting.Head and neekThis is a randomized multicenter open label phase 3 factorial trial evaluating the overallsurvival in patients with locally advanced squamous cell carcinoma of head and neck treatedwith locoregional treatment (radiotherapy plus concomitant chemotherapy or cetuximab) with orwithout neoadjuvant chemotherapy. Patients will be randomized to receive 3 cycles ofneoadjuvant chemotherapy (TPF) followed by radiotherapy plus concomitant chemo orcetuximab, or radiotherapy plus concomitant chemo or cetuximab alone. The primaryobjectives of the study are to compare the overall survival between neoadjuvant and noneoadjuvant arm and to compare the toxicity between concomitant chemoradiotherapy andradiotherapy plus Cetuximab. The study will be conducted by a multidisciplinary team,composed by oncologists, radiotherapists and othorinolaryngologists and will enrollapproximately 350 patients in Italian centers.Laboratory of Translational and Outcome Research in OncologyThe Laboratory is mainly aimed at documenting, by using either Randomized or OutcomeResearch studies, the value of new diagnostic and therapeutic interventions in oncology, payingparticular attention to two critical steps: the passage from early to late clinical research (fromthe activity to efficacy evaluation) and from phase III to clinical practice (from efficacy toeffectiveness). In order to facilitate the research activities and optimize the outputs, theLaboratory hosts the Coordination Centers of two multi-disciplinary Groups (MANGO: MarioNegri Gynecologic Oncology and the CP-OR: Cancer Pain Outcome Research Study Groups).As from 2007 on, all the activities of research and training in the field of chronic pain has beencoordinated by a dedicated center (CERP:Center for the Evaluation and Research on Pain).CERPThe objective of this newly set up center is in line with the Mario Negri Institute’s purpose:CERP is aimed at advancing the scientific knowledge of chronic pain and particularly the cancerpain and at improving the quality of palliative care. Activities will be concerning three fields:research, formation and information. It’ll be used a multidisciplinary and multi-institutionalapproach with special care to pharmacologic theraphy. Multidiscipinary teams will be created tovalue any contribution from either physicians or patients and with the involvement of scientificsocieties and patients’ associations. Several clinical activities focusing on patients resistant tostandard analgesic treatments are currently on going and are being conducted with both privateand public funds.The collaborative group in clinical gynecologic oncology named MaNGOThe Mario Negri Gynecologic Oncology group (MaNGO) is a new name for a collaborativegroup that has been active in clinical gynecologic oncology for several years. Infact, this groupconsolidated its network and logistics while running the ICONs studies which were conductedin very close partnership with researchers at the Medical Research Council, Clinical Trial Unit,UK. MaNGO was formally set up in May 2006 is mainly representative of the northern part ofItaly, although there are important sites in the central and southern part of the country too.Participating centers are either general public and private hospitals or university clinics. One ofMaNGO’s main statutory objectives was to foster an active collaboration with the GynecologicCancer Intergroup (GCIG), a true International Forum that circulates the scientific proposalsfrom fifteen collaborative groups through ten countries. Currently, two randomized clinicaltrials in ovarian cancer have already started recruitment, one sponsored by a French group42ANNUAL REPORT 2007

IRFMN(CALYPSO study) and the other sponsored by EORTC. Other collaboration with Dutchcollaborative groups has been activated to conduct randomized clinical trials in endometrialcancer (PORTEC3 study).In 2007, several meetings of the Technical-Scientific Committe were held while MaNGOaffiliates were conveyed in two General Assemblies.Colorectal cancerThe assessment of efficacy of screening for relapses of colorectal carcinoma has been debatedfor a long time, with controversial results. GILDA is an open label, international, randomisedstudy comparing two different strategies of post surgical surveillance in colorectal cancer(Dukes B2-C stage): minimalist versus intensive. Primary endpoints of this trial are disease freesurvival (which is used to assess diagnostic anticipation of metastases), overall survival, healthrelated quality of life, direct and indirect costs evaluation. At present, GILDA trial is the largestrandomised study evaluating the efficacy of two follow-ups in colorectal carcinoma. The trialwas closed to patient entry in September 2006 when a total of 1200 patients had been enrolled.Follow up of patients is ongoing and preliminary results in terms of diagnostic anticipation andpattern of relapses are expected in 2008.Smoking cessationBupropion is an antidepressant with dopaminergic and noradrenergic activity and has shownclinical efficacy for smoking cessation in specialized clinics when combined with high levels ofpsycosocial support. Aims of this trial is to confirm the efficacy of bupropion in the setting offamily medical practice, within a simplified strategy of counselling not provided by trainedcounsellors. The research design is the double blind, placebo controlled, randomised clinicaltrial. Eligible for inclusion are subjects who have smoked an average of 10 cigarettes or moreper day for the past year. The length of treatment is 7 weeks with three clinic visits scheduledover this period. Clinic visits for follow up assessments will occur at 26 and 52 weeks. Primaryoutcome measures are continuous abstinence and point prevalence rate of abstinence (self reportof abstinence during the seven days preceding assessment); changes in weight, blood pressure,heart rate and the incidence of adverse events will be recorded and analysed. The study hasreached the required number of patients and has been closed. A network of 71 Italian generalpractitioners (GPs) enrolled 593 participants between April 2004 and May 2005. Forty-onepercent of the group given bupropion were continuously abstinent from week 4 to 7 ascompared with 22% of the placebo group (OR, 2.37; 95% CI, 1.60 to 3.53). The continuousabstinence rates from week 4 to 12 months were 25% in the bupropion group and 14% in theplacebo group (OR, 2.13; 95% CI, 1.33 to 3.41). The adherence of GPs and participants to theprotocol was excellent, making our findings robust and easy to generalize to the context ofprimary care. This study, moreover, highlighted the great research potential of Primary Caresetting which, in Italy, has been overlooked for a long time because of legislative vacuum.The study was published in Archives Internal Medicine in September 2007.Lymphoangiogenesis in epithelial ovarian cancer: clinical aspects andexperimental studiesEpithelial ovarian carcinoma is the leading cause of death from gynecologic malignancies in thedeveloped world. Lymphatic spread in early ovarian cancer is a predictor of outcome withpotential clinical value. In fact, the presence of node metastases upstages the patients withovarian cancer apparently limited to the pelvis to FIGO stage IIIc disease and these patients areappropriate candidates for postoperative chemotherapic treatments. Even in advanced diseasethe metastatic involvement of aortic and pelvic nodes is still a predictor of survival. This projectis aimed at providing further insight into the lymphatic spread of ovarian cancer. Twoapproaches will be followed to study this pathogenetic aspect of ovarian cancer: a clinical43ANNUAL REPORT 2007

IRFMNapproach where the lymphoangiogenetic activity of primary ovarian cancer will be quantifiedand correlated with the nodal status of women who underwent systematic lymphadenectomy(task 1) and an experimental approach where a model of human ovarian cancer xenograftoverexpressing VEGF C will be set up and used to study the mechanisms of lymphatic spreadand possible therapeutic approaches (task 2).Ovarian cancer: clinical and translational studiesDuring 2007, the Unit of Gynecology-Oncology has coordinated the participation of a selectednetwork of Italian hospitals to two international randomized clinical trials. The CALYPSO trialwas sponsored by ARCAGY, France and compared two chemotherapy regimens (carboplatintaxolvs carboplatin- pegylated liposomal doxorubicin ) in patients with epithelial ovariancancer in late relapse. The TARCEVA trial was sponsored by EORTC and evaluated the impactof adding erlotinib for two years in patients with no evidence of progression after first line,platinum-base chemotherapy for ovarian cancer. Both studies reached their target sample sizein 2007 well in advance to the anticipated date of recruitment closure, thanks to really succesfullinternational collaboration. Results will be available in about three years.The Gynecology-Oncology Unit has started collaboration with the University of Newcastle tocollect histologic specimens of patients enrolled in ICON3 randomized trial. This research aimsat analyzing the p53 gene to test whether we can predict how much a patient with ovariancancer will benefit from chemotherapy by looking at the genetic make-up of the tumour. Inparticular, we wish to see if it would be possible to predict whether certain patients' cancercould be treated equally well with either one drug (carboplatin alone) or with the addition of asecond chemotherapy drug (carboplatin and paclitaxel). Patients experience more side effects ifthey are treated with the combination of two drugs. If we can tell which patients will do just aswell without the second drug, then we can make their treatment safer and more tolerable whilststill being effective.During 2007, the Gynecology-Oncology Unit has finalized the protocol of an observationalstudy aimed to describe the therapeutic approaches used to manage the ovarian cancerrecurrence that develop between 6 and 12 months from the end of a first line platinum-basedchemotherapy. This study should give clues to optimize the choise of drug combination in thisspecific clinical setting.Endometrial cancer: clinical studiesIn 2007, the Gynecology Oncology Unit registered in the Italian Clinical Trial Registry thePORTEC 3 protocol. This is an international randomized phase III sponsored by the DutchCooperative Gynecologic Oncology Group and aimed at comparing concurrent chemoradiationand adjuvant chemotherapy with pelvic radiation alone in high risk and advanced stageEndometrial Carcinoma. In late 2007 this protocol has been approved by the first Local EthicsCommittee and it will be submitted to the other Ethics Committees of the interested sites inearly 2008. During 2007 an observational study was also designed and it is ready to launchwithin spring 2008. Objective of this study is to assess the value of trans-vaginal ultrasoundimaging in predicting the myometrial infiltration of endometrial carcinoma. Should thisdiagnostic procedure show satisfactory concordance rate with final histopathologicalexamination clinicians could use it to improve surgical planning. During the whole 2007 twoimportant collaborations were considered and finally drawn up with the Sapienza University,Rome and the Nordic gynaecologic oncology group. Such partnerships aimed at sharing andmerging the database of very similar randomised clinical trials that singularly had been unableto reach the appropriate sample size. Such prospective poolings of the data should allow to haveenough power to detect clinically important differences between treatments (i.e.lymphadenectomy versus no-lymphadenectomy and chemoradiotherapy versus radiotherapyalone, in stage I and high risk endometrial cancer, respectively).44ANNUAL REPORT 2007

IRFMNOutcome research project in cancer painIn the context of a wide multidisciplinary project (J. Ambulatory Care Manage 29:332-341,2006), a nationwide multicenter, prospective outcome research study was launched in Italyin 2006 to investigate the epidemiology of cancer pain, the pattern and quality of analgesic-drugtherapy, and the evolution of health outcomes over time.In a large, prospective, cohort of advanced cancer patients reporting pain, investigators collectedpredictive and prognostic variables, information about type of care, as well as several patientreported-outcomes,such as pain, quality of life, satisfaction with analgesic care usingstandardized questionnaires and data collections forms.110 centers recruited 1801 patients from February 2006 to March 2007. Subjects weremonitored for 28 days and then with a simplified scheme for further 8 weeks.At inclusion, 50% had bone metastasis, 73% a level of pain classified as moderate-severe, 48%reported episodes of breakthrough pain, 49% were still on active anti-cancer treatments and60% were already on treatment with strong opioids. When the Index of Pain Management wascomputed to provide a rough estimate of how pain was treated (Cleeland et al, NEJM 330:592-596, 1994), up to 45% had negative values suggesting a possible analgesic under-treatment,with large variations according to a selected list of clinical variables. In the sub-sample ofpatients with a complete follow-up at 28 days (no.=1461), all pain and palliative outcomes didsignificantly improve on average, with variations according to case mix, type of treatments andtype of recruiting centers. Outcomes based on worst and average pain intensity showed thehigher effect size estimates (0.84 and 0.69) when compared to patients' satisfaction and qualityof life (0.44, 0.35). Up to 26% of patients were classified as non-responders.This outcome research study carried out at national level produced data to help implementfuture educative and research activities in Italy.Other research activitiesDuring 2007, other activities on patient-oriented clinical translational research in oncology werecarried out. The focus was on the improvement of transferring information from the pre-clinicalto clinical and research setting, and from clinical research to clinical practice. Most of this workinvolves data-entry, storage and other computerized applications for the management of largeand complex databases of biological and clinical data. In addition to the methodological andbio-informatics issues that are relevant in this area, particular attention was also given to issuesrelated to the ethical and legal issues pertaining the collection, storage and utilization ofbiological samples from patients and citizens.Finally, we would like to mention a research project about fairness in Health Care, sponsored byRegione Lombardia and run in collaboration with the “Centro Nazionale di Prevenzione eDifesa Sociale (CNPDS)” and the “Centro Studi e Ricerche Medicina Generale (CseRMEG)”.This project aims to identify “social”, i.e. not-clinical, determinants of a unfair access to theNational Health Care system, to evaluate the measurability in real conditions of suchdeterminants, gauge their impact on care profiles and propose interventions for possibleimprovements.Specific objective of this Project is thus the creation of a classification system of the “socialvulnerability”. That would be a pragmatic tool that will allow social researchers to preciselylocate a single subject inside the “social continuum” and relate such vectorial space to a specificrisk of missed of inappropriate access to Health services. A pilot phase with about 100 subjectsis currently on going and it will be followed in 2008 by a survey made by about 100 GeneralPractitioners.45ANNUAL REPORT 2007

IRFMNLaboratory of Medical Research and Consumer InvolvementThis Laboratory promotes activities of research in the field of involvement of citizens andpatients and their associations in decision making in health and medical issues. Moreover theactivities of this laboratory include: researches on information conveyed to patients on illnessand treatment, implementation of web site on the topics of the health and the information(www.partecipasalute.it; www.paincare.it; www.fondazionemattioli.it); strategy of involvementof groups of patients for the publication of educational material; research on the evaluation ofthe quality of the life and satisfaction with care through studies on ad hoc selected groups, andimplementation on validated ad hoc questionnaires.Strategic alliance between consumers and the scientific communityPartecipaSalutePartecipaSalute ("Participate in Health Care") – developed with the support of Compagnia diSan Paolo foundation - is a project initiated at September 2003 to foster a strategic alliancebetween patients’ groups and professional societies with the common goal of promoting betterhealth and shared decision-making. The project’s main aims are:- to increase patients’ associations’ involvement in the healthcare debate and in decisionmaking processes, also in the research field;- to promote a partnership between patients’ associations and medical societies solicitingmedical societies’ attention to patients’ need and perspectives as far as concern both theproduction of medical information (clinical studies), and the dissemination of information.In 2007 the second edition of the Partecipasalute training course was organized: participantswere 31 patients’ associations representatives. In this occasion a manual was given to allparticipants, dealing with the main arguments developed during the course.Regarding the website (http://www.partecipasalute.it/), a new form was designed, with newtools useful for readers; at the end of 2007 the website’s visits reached 40.000 a month.New exploratory projects were defined together with patients’ associations in the PARITAworking group/area. One example is the pilot project discussed and developed with volunteers’associations interested in severe brain injury.Study on the appropriateness and appraisal of effectiveness inoncological setting, follow-upA timely oncological diagnosis and the appropriate surgical therapies remain the bench mark ofthe primary therapy of many oncological diseases. Effective adjuvant therapies have allowed toimprove the survival of the patients, and have moreover supplied the base in order to set upprograms of surveillance (follow-up). In such programs, also in absence of good results on theefficacy and effectiveness, the follow-up it is practiced with the assumption that the anticipateddiscovery of one relapse allows the activation of effective therapies and therefore improve theprognosis, with benefits in terms of health, consumption of resources and costs. In some cases,as an example in the breast and colon cancer, the follow-up it is pursued in the clinical practice,also in presence of randomised clinical studies, meta-analyses, consensus conference, editorialsand statements of scientific associations and societies that suggest that obvious and greatbenefits in leading many diagnostic examinations after the primary therapies do not exist. Inother cases (like in the case of the melanoma and some gynaecological tumours), because thelack of valid information on follow-up, exist a wide variability of behaviours between centres,doctors and patients.The research project is proposed to build a network of researchers, cancer centers and regionalassociations of patients who, after reviewing the literature, share an analysis on practical followupand on the results obtained, to set lines for shared research optimize assets and the practice,even through the production of documents on how to address follow-up more appropriate. The46ANNUAL REPORT 2007

IRFMNactivities cover patients with carcinoma of the breast, colon-rectum, endometrium. During 2007were organised a series of working meetings that led to the development of a protocol forworking gynaecology cancer and two protocols for breast cancer, one of which to be conductedin collaboration with general practioners. The project has been endorsed by the Ministry ofHealth.Conference of Consent on the Hormonal Replacement TherapyAt the end of 2006 a Conference of Consent on Hormonal Replacement Therapy has beensponsorized by a bank foundation. The project will be articulated with multidiscliplinaryworking groups and it will carry out to a consent document on a topic still extremelycontroversial for public health, in particular regarding the information that is supplied to citizensand patients. During the 2007 the process leading to the celebration of the consensus conferencewas fully activated: definition of the scientific technical committee and three working groupsmultidisciplinary (clinical, media, citizens) who worked through review of the literature andcollection of ad hoc data for the development of material to be presented to the jury of theconsensus conference (May 2008). Numerous meetings have been working for the developmentof procedures to give room for discussion and organization of the preparatory work.SNAP project - Smoke, Nutrition, Alcohol and Physical ActivitySNAP is a campaign for the health addressed to all the population of the province of Como,with particular attention to the young people between 11 and 20 years. This project, for want ofFSE - Frontier Science & Technology Research Foundation, Southern Europe, a foundation forthe support to the independent search - in collaboration with Istituto Mario Negri, has beenlaunched with the attempt to increase knowledge and to modify the opinions, the attitudes andthe behaviors of the young people on the four topics examined, through the circulation of papermaterial, a website ad hoc and a public event. In order to estimate the effectiveness of theformation-information plan, a form on knowledge, opinions and behaviors of the young peoplebetween the 11 and 20 years will be proposed, before and after the event. Throughout the lastyear it has been prearranged the informative material and the evaluation form to submit to thepopulation and has been set up the website of the project.Project generic drugs, USL BergamoAs part of the work of the collaborative project between ASL Bergamo, AO ospedali Riuniti ofBergamo, Istituto di Ricerche Farmacologiche Mario Negri and Regione Lombardia, as well asbetween the goals, was activated a research project on generic drugs.The project has been the development and organization of an initiative - given to citizens in theprovince of Bergamo and their representatives (groups, associations of patients and citizens), thePrimary Care Doctors and Pharmacists - on the subject of generic drugs whose consumption inthe province of Bergamo to 31 December 2006 was 28%. The project, proposed by Mario Negriin activities related to the project PartecipaSalute, had intended to increase awareness amongcitizens about drugs equivalent and encourage good use of medicines. An evaluation after thefirst-consumption of medicines equivalent assess the effectiveness of this intervention-traininginformation. The project started in May with a survey on a representative sample of thepopulation in the province of Bergamo to assess knowledge, opinions and attitudes towardsdrugs equivalent. In autumn was organised the "Equivalent Drug Week" with brochures andposters distributed at pharmacies, studies of General Parctitioners, clinics and facilitiesaccredited districts. The "Week of Drug Equivalent" had its high point in the one-day open tothe public entitled "Who's afraid of generic drug?".In collaboration with Dr Alessandro Nobili, Laboratory of assessement of quality of care andservice for Elder.47ANNUAL REPORT 2007

IRFMNA population based evaluation of an intervention to improve cancer painmanagementThe purpose of the project, designed in 2005 on the basis of preliminary results of the activitiescarried out in the context of the Outcome Research project above summarized (see the part b:evaluation of the volume and quality – appropriateness - of prescription of analgesic drugs in alarge administrative data base ) is to evaluate the effect of a community-oriented multimodalintervention to change the quantity and quality of analgesic drugs for the treatment of cancerpain. The project, after the pilot phase carried out in a smaller geographic area (Cremona:350,000 citizens) will be carried out in an Italian Region (Le Marche:1.500.000 citizens). Theproject , sponsored by he Italian Agency for Drug evaluation (AIFA), was started in 2006 withthe creation of the integrated system (data-bases) that will be used to monitor the efefct of theintervention using pre-dfined indicators of opiods prescriptions, and the -assembling of relevantpanels and working groups of experts (methodologists, clinicians, general practitioners, healthcare managers, representative of citizens and patients, etc). During the year we organised theoperational phases of the project. Several organizational meetings aimed to define themethodology of the project and its feasibility in the Marche region, have been organised.In collaboration with Dr Giovanni Apolone, Laboratory of Research Translational and Outcomein Oncology.Quality of life projectsNo specific research projects have been carried out on quality of the life evaluation. Howeverare on going the activities of support and coordination of other groups using the instruments ofquality of life translated and validated by our research group, SF-36, SF-12, PGWBI. During theyear it has been periodically up-to-date the specific website http://crc.marionegri.it/qod.48ANNUAL REPORT 2007

IRFMN49ANNUAL REPORT 2007

IRFMNDEPARTMENT OF ENVIRONMENTALHEALTH SCIENCESSTAFFHeadRoberto FANELLI, Biol.Sci.D.Laboratory of Analytical BiochemistryHeadChiara CHIABRANDO, Biol.Sci.D.Laboratory of Environmental Chemistry and ToxicologyHeadEmilio BENFENATI, Chem.D.Industrial and Environmental Health UnitHeadLaboratory of Food ToxicologyMarco LODI, ChemistHeadEttore ZUCCATO, M.D.Laboratory of Mass SpectrometryHeadEnrico DAVOLI, Anim.Sci.D.Laboratory of Molecular ToxicologyHeadProtein and Gene Biomarkers UnitHeadLuisa AIROLDI, Pharm.D.Roberta PASTORELLI, Biol.Sci.DDepartment’s UnitsEnvironmental Pollutants Risk Assessment UnitHeadElena FATTORE, Biol.Sci.DAnalytical Instrumentation UnitHeadRenzo BAGNATI, Chem.D.50ANNUAL REPORT 2007

IRFMNCURRICULA VITAERoberto Fanelli, Head of the Environmental Health Sciences Department since 1997, Laboratory Head1978-97, Researcher 1975-78, Research fellow 1969-74 at the Mario Negri Institute.Doctoral Degree in Biological Sciences (University of Milan, 1973), Assistant Professor in Biochemistryat Baylor College of Medicine (Houston, Texas). Member of the Commissione Consultiva ProdottiFitosanitari (Ministero Salute), Member of the Scientific Panel on Contaminants in the Food Chain(European Food Safety Authority, 2003-2006), Certified Italian Toxicologist. Member of the ComitatoScientifico Ente Risi.Research areas: Sources, diffusion, toxicology, human exposure and risk assessment of persistentenvironmental pollutants. Environmental risk of plant protection products. Development of analyticalmethods for identification and measurement of biomarkers in toxicology. Mechanisms of toxic action byproteomic techniques.Selected publications:1. Pastorelli R, Carpi D, Campagna R, Airoldi L, Pohjanvirta R, Viluksela M, Hakansson H, Boutros P C, Moffat I D, Okey A B,Fanelli R. Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation withgenomic and transcriptomic analyses. Mol Cell Proteomics 2006; 5: 882-8942. Zuccato E, Chiabrando C, Castiglioni S, Calamari D, Bagnati R, Schiarea S, Fanelli R. Cocaine in surface waters: a newevidence-based tool to monitor community drug abuse. Environ Health 2005; 4: 14(http://www.ehjournal.net/content/4/1/14 2005)3. Pastorelli R, Carpi D, Airoldi L, Chiabrando C, Bagnati R, Fanelli R, Moverare S, Ohlsson C. Proteome analysis for theidentification of in vivo estrogen-regulated proteins in bone. Proteomics 2005; 5: 4936-49454. Zuccato E, Castiglioni S, Fanelli R. Identification of the pharmaceuticals for human use contaminating the Italian aquaticenvironment. J Hazard Mater 2005; 122: 205-2095. Airoldi L, Magagnotti C, Pastorelli R, Fanelli R. Enzyme polymorphisms influencing the metabolism of heterocyclic aromaticamines. J Chromatogr B Analyt Technol Biomed. Life Sci 2004; 802: 175-1816. Fattore E, Di Guardo A, Mariani G, Guzzi A, Benfenati E, Fanelli R. Polychlorinated dibenzo-p-dioxin and dibenzofurans inthe air of Seveso, Italy, 26 years after the explosion. Environmental Science Technology 2003; 37: 1503-1508Luisa Airoldi, Head of the Molecular Toxicology Laboratory since 1994, Unit Head 1987-94, Researcher1978-87, Technician 1967-75 at the Mario Negri Institute.Doctoral Degree in Pharmacy (University of Milan, 1975), Postdoctoral fellow at the MassachusettsInstitute of Technology (Cambridge, MA, 1976) and at the Northwestern University Medical School(Chicago, Il, 1977), Researcher at the Yale University Medical School (New Haven, CT, 1980-81).Research areas: Proteomics in toxicology with particular interest on the study of proteome changes intissues and biological fluids from animals and humans after exposure to toxic compounds; molecularepidemiology focused on the identification and measurement of biomarkers of exposure to environmentalcarcinogens and disease susceptibility; chemical carcinogenesis centered on the study of carcinogens’mechanism of action.Selected publications:1. Vineis P, Hoek G, Krzyzanowski M, Vigna-Taglianti F, Veglia F, Airoldi L, Overvad K, Raaschou-Nielsen O, Clavel-Chapelon F, Linseisen J, Boeing H, Trichopoulou A, Palli D, Krogh V, Tumino R, Panico S, Bueno-De-Mesquita HB, PeetersPH, Lund E E, Agudo A, Martinez C, Dorronsoro M, Barricarte A, Cirera L, Quiros JR, Berglund G, Manjer J, Forsberg B,Day NE, Key TJ, Kaaks R, Saracci R, Riboli E. Lung cancers attributable to environmental tobacco smoke and air pollution innon-smokers in different European countries: a prospective study. Environ Health. 2007 15; 6:7.2. Pastorelli R, Saletta F, Campagna R, Carpi D, Dell'Osta C, Schiarea S, Vineis P, Airoldi L, Matullo G Proteomecharacterization of a human urothelial cell line resistant to the bladder carcinogen 4-aminobiphenyl Proteome Sci 2007 5: 63. Pastorelli R, Carpi D, Campagna R, Airoldi L, Pohjanvirta R, Viluksela M, Hakansson H, Boutros P C, Moffat I D, Okey A B,Fanelli R. Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation withgenomic and transcriptomic analyses. Mol Cell Proteomics 2006; 5: 882-8944. Airoldi L, Vineis P, Colombi A, Olgiati L, Dell'Osta C, Fanelli R, et al. 4-Aminobiphenyl-hemoglobin adducts and risk ofsmoking-related disease in never smokers and former smokers in the European Prospective Investigation into Cancer andNutrition Prospective study. Cancer Epidemiol Biomarkers Prev 2005; 14: 2118-21245. Pastorelli R, Carpi D, Airoldi L, Chiabrando C, Bagnati R, Fanelli R, Moverare S, Ohlsson C. Proteome analysis for theidentification of in vivo estrogen-regulated proteins in bone. Proteomics 2005; 5: 4936-49456. Magagnotti C, Pastorelli R, Pozzi S, Andreoni B, Fanelli R, Airoldi L. Genetic polymorphisms and modulation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in human lymphocytes. Int J Cancer 2003; 107: 878-88451ANNUAL REPORT 2007

IRFMNEmilio Benfenati, Head of the Laboratory of Environmental Chemistry and Toxicology since 1997, UnitHead 1987-97, Researcher 1986-87, Research fellow 1981-86 at the Mario Negri Institute. Researcher atIstituto Biochimico Italiano 1979-1981.Doctoral Degree in Chemistry (University of Milan, 1979).Member of Commissione Consultiva Prodotti Fitosanitari (Ministero Salute 1997-99), Certified ItalianChemist.Resarch areas: Computer-based models for chemistry and toxicology; Molecular descriptors; QSAR;Toxicity prediction; Metabolism studies; Characterization and assessment of wastes, industrial effluents,emissions from landfill and incinerator; Integration of chemical analysis and eco-toxicological data;Chemical analysis of organic compounds by mass spectrometry.Principali pubblicazioni:1. Lo Piparo E, Koehler K, Chana A, Benfenati E, Virtual screening for aryl hydrocarbon receptor binding prediction, J MedChem 2006; 49: 5702-57092. Casalegno M, Sello G, Benfenati E, Top-priority fragment QSAR approach in predicting pesticide aquatic toxicity, Chem ResToxicol 2006; 19: 1533-15393. Lo Piparo E, Fratev F, Lemke F, Mazzatorta P, Smiesko M, Fritz J I, Benfenati E, QSAR models for Daphnia magna toxicityprediction of benzoxazinone allelochemicals and their transformationproducts, J Agric Food Chem 2006; 54: 1111-1115.Benfenati E. (Ed.), Quantitative Structure-Activity Relationships for Pesticide Regulatory Purposes, Elsevier, pp. 1-510(2007).4. Benigni R, Netzeva T, Benfenati E, Bossa C, Franke R, Helma C, Hulzebos E, Marchant C, Richard A, Woo Y-T, Yang C.The expanding role of predictive toxicology: An update on the (Q)SAR models for mutagens and carcinogens. J Environ SciHealth C 25: 53-97 (2007).5. Roncaglioni A, Benfenati E. In silico-aided prediction of biological properties of chemicals: oestrogen receptor-mediatedeffects. Chem Soc Rev 37: 441-450 (2008).6. Porcelli C, Boriani E, Roncaglioni, A, Chana A, Benfenati. Regulatory perspectives in the use and validation of QSAR. A casestudy: DEMETRA model for daphnia toxicity. Environ Sci Technol 42: 491-496 (2008).Chiara Chiabrando, Head of the Analytical Biochemistry Laboratory since 1997, Unit Head 1987-97,Researcher 1978-87, Research fellow 1975-78 at the Mario Negri Institute.Doctoral degree in Biological Sciences (University of Milan, 1974), Postdoctoral fellow at the BaylorCollege of Medicine (Houston, Texas, 1974-75). Postgraduate degree in Pharmacological Research,Mario Negri Institute (1977).Research areas: Development and application of bio-analytical methods based on mass spectrometry inthe fields of biochemistry, metabolism, clinical chemistry and pharmacology. Identification andcharacterization of proteins and peptides of biomedical interest by proteomic approaches and massspectrometry. Proteomics in toxicology.Selected publications1. Castiglioni S, Zuccato E, Crisci E, Chiabrando C, Fanelli R, Bagnati R. Identification and measurement of illicit drugs andtheir metabolites in urban wastewaters by liquid chromatography tandem mass spectrometry (HPLC-MS-MS). Anal Chem2006, 78: 8421-8429.2. Pastorelli R, Carpi D, Airoldi L, Chiabrando C, Bagnati R, Fanelli R, Moverare S, Ohlsson C. Proteome analysis for theidentification of in vivo estrogen-regulated proteins in bone. Proteomics. 2005;5:4936.3. Zuccato E, Chiabrando C, Castiglioni S, Calamari D, Bagnati R, Schiarea S, Fanelli R. Cocaine in surface waters: a newevidence-based tool to monitor community drug abuse. Environ Health. 2005;4:14.4. Chiabrando C, Avanzini F, Rivalta C, Colombo F, Fanelli R, Palumbo G, Roncaglioni MC; PPP Collaborative Group on theantioxidant effect of vitamin E. Long-term vitamin E supplementation fails to reduce lipid peroxidation in people atcardiovascular risk: analysis of underlying factors. Curr Control Trials Cardiovasc Med. 2002;3:5.5. Chiabrando C, Rivalta C, Bagnati R, Valagussa A, Durand T, Guy A, Villa P, Rossi JC, Fanelli R. Identification ofmetabolites from type III F2-isoprostane diastereoisomers by mass spectrometry. J Lipid Res. 2002;43:495.6. Chiabrando C, Valagussa A, Rivalta C, Durand T, Guy A, Zuccato E, Villa P, Rossi JC, Fanelli R. Identification andmeasurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha.J Biol Chem. 1999 Jan 15;274:1313.52ANNUAL REPORT 2007

IRFMNEnrico Davoli, Head of the Mass Spectrometry Laboratory since 1997, Unit Head 1994-97, Researcher1989-94, Research Fellow 1985-87 at the Mario Negri Institute. Fellow at USDA, Beltville, MD 1977-78.Doctoral Degree in Animal Sciences (University of Milan, 1983), Postdoctoral fellow at the University ofNebraska (Lincoln, NE, 1987) and at the University of Colorado Health Sciences Center (Denver, CO,1988). Postgraduate degree in Pharmacological Research, Mario Negri Institute (1988). Member of theAmerican Association for Mass Spectrometry of the Environment and Safety Commission of IGQ and ofthe ETS (Emission Trading System) commission. Member of the National Biomass Research CenterScientific Committee.Research areas: Development of methodology, instrumentation and software for environmental research.Studies of urban air pollution and characterization of environmental odor annoyance.Selected Publications1. Riservato M, Rolla A, Davoli E . An isotopic dilution approach for 1,3-butadiene tailpipe emissions and ambient airmonitoring. Rapid Commun Mass Spectrom 2004; 18: 399-4042. Davoli, L. Cappellini, M. Moggi and R. Fanelli. Automated, high speed analysis of selected organic compounds in urban airby on line isotopic dilution cryofocussing GC/MS. J. Am. Soc. Mass Spectrometry. 5: 1001-1007.19943. E. Davoli, L. Cappellini, M. Moggi S. Ferrari and R. Fanelli. On-Line Monitoring of Benzene Air Concentrations WhileDriving in Traffic by Isotopic Dilution GC/MS. Int. Arch. Occup. Environ. Health 1996; 68: 262-2674. E. Davoli, L. Cappellini, R. Fanelli, M. Bonsignore, M. Gavinelli. On-Site Analysis of World War II Cylinders and Barrelswith Unknown Contents. Field Anal. Chem. Technol. 2001; 5: 313-3195. E. Davoli, L. Gangai, P. Morselli, D. Tonelli, Characterisation of Odorant emissions from Landfills by SPME and GC/MS.Chemosphere 2003; 51: 357-3686. E. Zuccato, P. Grassi, E. Davoli, L. Valdicelli, D. Wood, G. Reitano, R. Fanelli. PCB concentrations in some foods from fourEuropean countries. Food Chem Toxicol 2008 ; 46 : 1062-10677. R. Bagnati, G. Bianchi, E. Marangon, E. Zuccato, R. Fanelli, E. Davoli. Direct analysis of isopropylthioxanthone (ITX) inmilk by high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2007 ; 21 :1998-2002Ettore Zuccato, Head of the Food Toxicology Laboratory since 2005, Unit Head 1997-2005, Researcher1986-97, Technician 1975-86 at the Mario Negri Institute.Doctoral degree in Medicine (University of Milan, 1986), Postdoctoral degree in Human Nutrition(1999), Postdoctoral fellow at the King’s College School of Medicine (London, UK, 1988-89).Member of the ANSISA, EMEA expert, member of the Commissione Consultiva per i ProdottiFitosanitari, and expert for the evaluation of plant protection products for registration within the EU.Research areas: Food safety, including the study of dietary chemical contaminants, safety assessment ofGMO in human nutrition, food allergens and toxicants, emerging issues in food toxicology, riskperception and risk communication to the consumers, and evaluation of plant protection products forregistration within the European Union. Environmental pollution by pharmaceuticals, and monitoring ofillicit drugs in surface waters to estimate community drug abuse.Selected publications1. Castiglioni S, Zuccato E, Crisci E, Chiabrando C, Fanelli R, Bagnati R. Identification and measurement of illicit drugs andtheir metabolites in urban wastewaters by liquid chromatography tandem mass spectrometry (HPLC-MS-MS). Anal Chem2006, 78: 8421-8429.2. Pomati F, Castiglioni S, Zuccato E, Fanelli R, Rossetti C and Calamari D. Effects of Environmental Contamination byTherapeutic Drugs on Human Embryonic Cells. Environ. Sci. Technol. 2006, 40, 2442-2447.3. Zuccato E, Calamari D, Castiglioni S, Chiabrando C, Bagnati R, Fanelli R. Cocaine in surface water: a new evidence-basedtool to monitor community drug abuse. Environmental Health: A Global Access Science Source 2005, 4:144. Calamari D, Zuccato E, Castiglioni S, Bagnati R, Fanelli R. Strategic survey of therapeutic drugs in the rivers Po and Lambroin northern Italy. Environ Sci Technol 2003; 37: 1241-12485. Zuccato E, Calamari D, Natangelo M, Fanelli R. Presence of therapeutic drugs in the environment. Lancet 2000; 355: 1789-17906. Zuccato E, Calvarese S, Mariani G, Mangiapan S, Grasso P, Guzzi A, Fanelli R. Level, sources and toxicity of polychlorinatedbiphenyls in the Italian diet. Chemosphere 1999; 38 (12): 2753-2765.53ANNUAL REPORT 2007

IRFMNRenzo Bagnati, Head of the Analytical Instrumentation Unit since 2005, Researcher 1992-2005,Research fellow 1986-92 at the Mario Negri Institute.Doctoral degree in Chemistry (University of Turin, 1985), Postgraduate degree in PharmacologicalResearch, Mario Negri Institute (1989).Research areas: Mass spectrometry applied to the analysis of biological and environmental relevantsubstances (proteins, peptides, hormones, pharmaceuticals, drugs of abuse, pesticides).Selected Publications1. Castiglioni S, Zuccato E, Crisci E, Chiabrando C, Fanelli R, Bagnati R. Identification and measurement of illicit drugs andtheir metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry. Anal Chem 2006; 78: 8421-8429.2. Castiglioni S, Bagnati R, Fanelli R, Pomati F, Calamari D, Zuccato E. Removal of pharmaceuticals in sewage treatment plantsin Italy. Environmental Science Technology 2006; 40: 357-363.3. Zuccato E, Chiabrando C, Castiglioni S, Calamari D, Bagnati R, Schiarea S, Fanelli R. Cocaine in surface waters: a newevidence-based tool to monitor community drug abuse. Environ Health. 2005;4:14.4. Pastorelli R, Carpi D, Airoldi L, Chiabrando C, Bagnati R, Fanelli R, Moverare S, Ohlsson C. Proteome analysis for theidentification of in vivo estrogen-regulated proteins in bone. Proteomics 2005; 5: 4936-4945.5. Bagnati R, Ramazza V, Zucchi M, Simonella A, Leone F, Bellini A, Fanelli R. Analysis of dexamethasone and betamethasonein bovine urine by purification with an 'on-line' immunoaffinity chromatography-HPLC system and determination by GC-MS.Anal Biochem 1996; 235: 119-126.6. Davoli E, Fanelli R, Bagnati R. Purification and analysis of drug residues in urine samples by on-line immunoaffinitychromatography/high-performance liquid chromatography/continuous-flow fast atom bombardment mass spectrometry. AnalChem 1993; 65: 2679-2685.Elena Fattore, Head of the Environmental Pollutants Risk Assessment Unit since 2005, Researcher2001-2004, Research fellow 1991-1997 at the Mario Negri Institute.Doctoral Degree in Biological Sciences (University of Milan, 1991), Postgraduate degree inPharmacological Research, Mario Negri Institute (1994), Postdoctoral fellow at the National Institute ofEnvironmental Medicine, Karolinska Institutet, Stockholm (1998-2000).Member of the Group of Experts of the Italian Presidency of the Council of Ministers for the Surveillanceof Exposure to Endocrine Disrupters.Research areas: Environmental chemistry, toxicology, assessment of human exposure and risk fromenvironmental pollutants with emphasis on dioxins and dioxin-like compounds.Selected publications1. Carubelli G, Fanelli R, Mariani G, Nichetti S, Crosa G, Calamari D, Fattore E. PCB contamination in farmed and wild sea bass(Dicentrarchus labrax L.) from a coastal wetland area in central Italy. Chemosphere 2007 ; 68 : 1630-1635.2. Fattore E, Fanelli R, Turrini A, Di Domenico A. Current dietary exposure to polychlorodibenzo-p-dioxins,polychlorodibenzofurans, and dioxin-like polychlorobiphenyls in Italy. Mol Nutr Food Res 2006; 50: 915-9213. Fattore E, Guardo A, Mariani G, Guzzi A, Benfenati E, Fanelli R. Polychlorinated Dibenzo-p-Dioxins and Dibenzofurans inAir of Seveso, Italy, 26 years after the accident. Environ Sci Technol 2003; 37: 1503-10584. Fattore E, Fanelli R, La Vecchia C. Persistent organic pollutants in food: Public health and implications. J EpidemiolCommunity Health 2002; 56: 831-8325. Fattore E, Trossvik C, Håkansson H. Relative potency values derived from hepatic vitamin A reduction in male and femaleSprague-Dawley rats following subchronic dietary exposure to individual polychlorinated dibenzo-p-dioxin and dibenzofurancongeners, and a mixture thereof. Toxicol Appl Pharmacl 2000; 165: 184-1946. Fattore E, Benfenati E, Mariani G, Fanelli R, Evers E H. Pattern and Sources of Polychlorinated dibenzo-p-dioxins andDibenzofurans in Sediment from Venice Lagoon. Environ Sci Technol 1997; 31: 1777-178454ANNUAL REPORT 2007

IRFMNMarco Lodi, Head of the Industrial and Environmental Unit since 2002, Consultant 1997-2002 at theMario Negri Institute.General Certificate of Education in Industrial Chemistry (Milan, 1974).Member of AIDII (Italian Industrial Hygiene Association), certified by ACGIH (American Conference ofGovernmental Industrial Hygienist).Research areas: Emission sources, environmental diffusion, toxicology, human exposure and riskassessment of persistent environmental pollutants. Environmental risk of chemical pollution products.Development of sampling methods for environmental toxic compounds.Selected publications1. Benfenati E, Azimonti G, Auteri D, Lodi M Environmental and ecological toxicology: computational risk assessment.Computational toxicology. Risk assessment for pharmaceutical and environmental chemicals John Wiley, Hoboken, NJ, 2007;625-6502. Grosso M, Cernuschi S, Palini E, Lodi M, Mariani G. PCDD/Fs release during normal and transient operation of a full scaleMSWI plant. Organohalogen Compounds 2004; 66: 1243-12493. Benfenati E, Mariani G, Lodi M, Reitano G, Fanelli R. Is bioexsiccation releasing dioxins? Organohalogen Compounds2004; 66: 955-9614. Fattore E, Mariani G, Guzzi A, Di Guardo A, Benfenati E, Lodi M, Fanelli R. Air dioxin concentrations in Seveso area. In:Halogenated Environmental Organic Pollutants, 18th. Symp., Stockholm, Sweden, august 17-21, 1998. 1998 : 237-2405. Benfenati E, Mariani G, Schiavon G, Lodi M, Fanelli R. Diurnal, weekly and seasonal air concentrations of PCDD and PCDFin an industrial area. Fresenius Journal Analytical Chemistry 1994; 348: 141-1436. Benfenati E, Pastorelli R, Castelli M G, Fanelli R, Carminati A, Farneti A, Lodi M. Studies on the tetrachlorodibenzo-pdioxins(TCDD) and tetrachlorodibenzofurans (TCDF) emitted from an urban incinerator. Chemosphere 1986; 15: 557-561Roberta Pastorelli, Head of Protein and Gene Biomarkers Unit since 2004, Researcher 1992-2003,Research fellow 1983-92 at the Mario Negri Institute.Doctoral Degree in Biological Sciences (University of Milan, 1982), Postgraduate degree inPharmacological Research, Mario Negri Institute (1986), Postdoctoral fellow at the MassachusettsInstitute of Technology, Cambridge, MA (1987-89 and 1991).Research areas: Toxicoproteomic investigation of global protein expression profiles and their modulationevoked by environmental compounds in different biological compartments. Critical targets and pathwaysin toxicology. Pharmacogenetics: effects of genetic polymorphisms in the human population on theindividual susceptibility to environmental xenobiotic and carcinogen effects.Selected publications:1. Pastorelli R, Saletta F, Campagna R, Carpi D, Dell?osta C, Schiarea S, Vineis P, Airoldi L, Matullo G. Proteomecharacterization of a human urothelial cell line resistant to the bladder carcinogen 4-aminobiphenyl. Protome Science 2007.2. Moretti M, Dell'omo M, Villarini M, Pastorelli R, Muzi G, Airoldi L, Pasquini R. Primary DNA damage and geneticpolymorphisms for CYP1A1, EPHX and GSTM1 in workers at a graphite electrode manufacturing plant. BMC Public Health.2007 7: 2703. Pastorelli R, Carpi D, Campagna R, Airoldi L, Pohjanvirta R, Viluksela M, Hakansson H, Boutros P C, Moffat I D, Okey A B,Fanelli R. Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation withgenomic and transcriptomic analyses. Mol Cell Proteomics 2006; 5: 882-8944. Pastorelli R, Carpi D, Airoldi L, Chiabrando C, Bagnati R, Fanelli R, Moverare S, Ohlsson C.Proteome analysis for theidentification of in vivo estrogen-regulated proteins in bone. Proteomics 2005; 5: 4936-49455. Airoldi L, Magagnotti C, Pastorelli R, Fanelli R. Enzyme polymorphisms influencing the metabolism of heterocyclic aromaticamines. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 802: 175-1816. Vineis P,V eglia F, Anttila S, Benhamou S, Clapper M L, Dolzan V, Ryberg D, Hirvonen A, Kremers P, Le Marchand L,Pastorelli R, Rannug A, Romkes M, Schoket B, Strange R C, Garte S, Taioli E. CYP1A1, GSTM1 and GSTT1 polymorphismsand lung cancer: a pooled analysis of gene-gene interactions. Biomarkers 2004; 9: 298-30555ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe Department works to investigate environmental factors and their effects on human health. The mainresearch lines focus on the survey of environmental contaminants, the assessment of human exposurewith related health risks, and toxicity mechanisms of pollutants.The assessment of environmental contamination is carried out not only for well-known and widespreadcompounds, like dioxins and PCBs, but also for new classes of "unconventional" pollutants, e.g.,endocrine disruptors, potentially toxic "natural" compounds, and drugs entering the environment afterhuman or veterinary use. The identification –for the first time– of illicit drugs in urban waste and riverwaters, led to a new original tool for the evidence-based monitoring of community drug abuse. For allthese survey activities sophisticated analytical methods based on advanced mass spectrometric techniquesare developed.The Department is active in the assessment of human exposure to toxic compounds in the atmosphere andthe diet, which is the main source of priority pollutants (PCBs, dioxins and other endocrine disruptors).Assessment of the risk associated to contamination in real-life scenarios has recently gained muchimportance. In order to respond to the growing demand for information, the Department is more and moreinvolved in toxicological and ecotoxicological risk analysis, based on studies in field and predictivemodels of toxicity.Molecular epidemiology studies are used to identify genetic and/or environmental factors posing risks tohuman health. By this approach, we search for new useful “biological markers" to identify susceptiblesubjects, in view of finding appropriate preventive strategies.The Department has implemented an advanced technological proteomic platform, in order to identifyproteins differentially expressed in biological compartments in various experimental and clinicalconditions. This approach is particularly relevant in toxicology, since it can contribute to find newbiomarkers of toxicity or pathology, and to identify molecular targets and toxic effect mechanisms ofpollutants and drugs.FINDINGS/MAIN RESULTSThe lack of retinoic acid (knock-out mice for the retinal dehydrogenase 1 gene, RALDH1) modifies theproteome profile of the bone, through changes in the expression of proteins involved in chondrogenesisand osteoclastogenesis.Resistance to the bladder carcinogen 4-aminobiphenyl in human urothelial cells is mediated byderegulation of apoptosis and membrane trafficking proteins.Bone protein profile in a murine model of osteoporosis.Identification of novel protein targets responsive to the effects of estrogens in bone.TCDD's effect on the liver proteome profile of exposed rats. Determination of a subset of rat hepaticproteins indicative of differences in dioxin susceptibility.The presence of 4-aminobiphenyl-hemoglobin adducts may help identify nonsmokers at high risk ofcancers related to environmental tobacco smoke exposure.Reference values of allele and genotype frequency of several metabolic genes in 15,000 control subjects.CYP1A1 polymorphism affects lung tumor risk.Identification of CYP2C9 genetic polymorphism as a determinant of severe adverse reactions tophenytoin.On-line in silico models to predict ecotoxicity of pesticides for regulatory purposes.A new model for identification of endocrine disruptors using molecular docking.A method aimed at characterizing environmental odors to identify odor sources in complex environments.Moderate-to-high fish consumption can result in exceeding the daily intake safety levels of PCBs anddioxin-like compounds established by the European Commission.The same food type may contain significantly different concentrations of PCBs and dioxin-likecompounds, depending on the geographic origin (this may help lower the risk for the consumers byunderstanding and controlling the causes of the differences).The environmental levels of several drugs exceed the “safety limits” established for them.Environmental pollution from pharmaceuticals is a general phenomenon that can be described by56ANNUAL REPORT 2007

IRFMNcontrollable variables (environmental load and mass balance).Illicit drug residues and their metabolites were found in urban waste and river waters. Environmentallevels can be used as a new tool to estimate illicit drugs consumption in the population.The distribution of dietary intake values of dioxins, dioxin-like PCBs and non dioxin-like PCBs wascharacterized for the general Italian population.The higher intake of PCBs due to consumption of farmed fish vs. wild fish is mainly due to the higher fatcontent in farmed fish.Development of novel mass spectrometric methods for the selective measurement of therapeutic andillicit drugs in environmental samples.NATIONAL COLLABORATIONSARPA VenetoASL di BresciaCESTEC , Regione Lombardia, MilanoCNR – IRSACSPO-FirenzeCSRA-AstiFondazione 'S. Maugeri'Fondazione ISI, TorinoINRAN-Istituto Nazionale di Ricerca sugli Alimenti e la NutrizioneIstituto Clinico Humanitas, MilanoIstituto Superiore di SanitàI.Z.S.L.T - Istituto Zooprofilattico Sperimentale del Lazio e ToscanaMinistero dell'AmbientePolitecnico di MilanoPolitecnico di TorinoProvincia di VercelliProvincia PordenoneUniversità degli Studi di CagliariUniversità degli Studi di GenovaUniversità degli Studi di Napoli "Federico II"Università degli Studi di PalermoUniversità degli Studi di PaviaUniversità degli Studi di PerugiaUniversità degli Studi di Roma "La Sapienza"Università degli Studi di TorinoUniversità dell’Insubria, Varese57ANNUAL REPORT 2007

IRFMNINTERNATIONAL COLLABORATIONSBASF Agricultural Centre, Limburgerhorf, GermanyCentral Science Laboratory, York, UKDanish Institute of Agricultural Sciences, Research Centre Foulum, Tjele, DenmarkDepartment of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy,DenmarkDepartment of Anatomy and Cell Biology, University of Oulu, Oulu, FinlandDepartment of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy,DenmarkDepartment of Computer Science and Engineering, University of Galati, RomaniaDepartment of Electrical and Computer Engineering, University of Patras, GreeceDepartment of Environmental Health, National Public Health Institute, Kuopio, FinlandDepartment of Epidemiology & Public Health, Imperial College, London, United KingdomDepartment of Inland Fisheries, Institute of Freshwater Ecology and Inland Fisheries, Berlin, GermanyDepartment of Molecular Biology, University of Bergen, Bergen, NorwayDepartment of Organic Chemistry, Universidad de Cadiz, SpainDivision of Endocrinology, Department of Internal Medicine, Sahlgrenska University Hospital, Goteborg,SwedenEnvironmental Chemistry, IIQAB-CSIC, Barcelona, SpainEnvironmental Hygiene and Chemistry Department, Institute of Environmental Medicine and HospitalEpidemiology, University of Freiburg, GermanyEnvironmental Hygiene and Chemistry Department, Institute of Environmental Medicine and HospitalEpidemiology, University of Freiburg, GermanyFaculté de Médicine et de Pharmacie, Université de Mons-Hainaut, Mons, BelgiumFaculty of Veterinary Medicine, Utrecht University, Utrecht, The NetherlandsForschungzentrum Jülich Gmbh, Jülich, GermanyGruppo Collaborativo sulla Suscettibilità Genetica ai Cancerogeni Ambientali (GSEC), Milano, ItalyInstitute of Environmental Medicine. Karolinska Institute, Stockholm, SwedenInstitute of Pharmaceutical Chemistry, University of Pécs, Pecs, HungaryInstitute of Phytomedicine, Biological Control, Horticulture and Nematology, Wien, AustriaInstitute of Soil Science and Plant Cultivation, Pulawy, PolandInteruniversitaeres Forchunginstitut fuer Agrarbiotechnologie, Tulln, AustriaIstituto di Chimica di São Carlos, Università di São Paulo, BrazilKnowledgeMiner Software, Berlin, GermanyIn Vitro Testing Industrial Platform, Tres Cantos (Madrid), SpainLaboratory of Chemometrics & Bioinformatics, University of Orléans, Orléans, FranceLithuanian Institute of Agricultrure, Vilnius, LithuaniaLiverpool John Moores University, Liverpool, United KingdomNational Institute of Chemistry, Kemijski Institut Ljubljana, Ljubljana, SloveniaNatural Resources Research Institute, University of Minnesota, Duluth, MNNational Institute for Public Health and the Environment (RIVM), Bilthoven, The NetherlandsPesticide Safety Directorate, York, UKPlant Protection Institute, Hungarian Academy of Sciences, Budapest, HungarySchool of Biomedical Sciences, University of Ulster, Coleraine, Northern IrelandSyngenta Crop Protection AG, Basel, SwitzerlandUFZ Leipzig, GermanyUniversity of Tartu, Tartu, Estonia58ANNUAL REPORT 2007

IRFMNEDITORIAL BOARD MEMBERSHIPJournal of Environmental Science and Health, Part B (Emilio Benfenati), Journal of EnvironmentalScience and Health, Part C (Emilio Benfenati), International Journal of Computational Intelligence(Emilio Benfenati), International Journal of Information Technology (Emilio Benfenati), InternationalJournal of Signal Processing (Emilio Benfenati), Chemistry Central Journal (Emilio Benfenati), CurrentComputer Aided Drug Design (Emilio Benfenati), Advances in Chemoinformatics and ComputationalMethods (Emilio Benfenati)..PEER REVIEW ACTIVITIESAddiction, Analytical and Bioanalytical Chemistry, Chemometrics and Intelligent Laboratory Systems,CHEMOLAB, Chemosphere, Chemical Biology & Drug Design, Environmental Modelling &Software, Environmental Science & Technology, Journal of Chemical Information andModeling, International Journal of Molecular Science, Journal of Hazardous Materials,Molecular Diversity, Proteomics, Waste Management.NATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPCCPF – Commissione Consultiva Prodotti Fitosanitari (Ministero della Salute, Ministero dell'Ambiente)IGQ - Commissione Ambiente e SicurezzaIGQ - ETS CommissionEVENT ORGANIZATIONNOSE2008. INTERNATIONAL CONFERENCE ON ENVIRONMENTAL ODOUR MONITORINGand CONTROL. Rome, Italy 6-8 July 200859ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE DEPARTMENT WASINVOLVEDSETAC Europe, 17th annual meeting, 20-24 May 2007, Porto, Portugal55th American Society for Mass Spectrometry Conference, June 3-7, 2007, Indianapolis, IN, USAThe International Society of Pharmacovigilance. 7 th Annual Meeting. 21-24 October 2007,Bournemouth, UK.ASMS sponsored meeting. The Art of Open Air Ionization on Surfaces. November 8 - 9. 2007.ChemicalHeritage Foundation. Philadelphia, PA, USAProteomix, XI Meeting, November 29-30, 2007, Torino, Italy.Workshop of the ATHON EC project, Valencia, Spain, November 29- December 30, 2007..GRANTS AND CONTRACTSACEGAS S.p.A, TriesteASL MantovaCSRAAssociazione Italiana Ricerca sul CancroComune di LomelloComune di Mazzano e RezzatoConsorzio Quadrifoglio S.p.A.ECODECO, PaviaEuropean Commission (MEBFOOD; SAFEFOODNET; DEMETRA; EUFRAM; HERBICBIOREM;BONETOX, ATHON, CASCADE, HAIR, CAESAR, RAINBOW, CHEMOMENTUM)Ferrero, Alba (Cuneo)FIAT Auto S.p.A.Fondazione CARIPLO, MilanoFondazione Italo Monzino, MilanoI.Z.S.L.T - Istituto Zooprofilattico Sperimentale del Lazio e ToscanaLucart, Cartiera Lucchese S.p.A. Porcari, LuccaMinistero dell'Istruzione, dell'Università e della Ricerca, ItaliaMinistero della Salute, ItaliaMinistero dell'Ambiente, ItaliaProvincia di PordenoneProvincia di VercelliSO.GE.NU.S. S.p.ATelethonTM.E. S.p.A60ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Pastorelli R, Saletta F, Campagna R, Carpi D, Dell?osta C, Schiarea S, Vineis P, Airoldi L, Matullo G. Proteomecharacterization of a human urothelial cell line resistant to the bladder carcinogen 4-aminobiphenyl. ProteomeScience 2007Moretti M, Dell'omo M, Villarini M, Pastorelli R, Muzi G, Airoldi L, Pasquini R.Primary DNA damage and genetic polymorphisms for CYP1A1, EPHX and GSTM1 in workers at a graphiteelectrode manufacturing plant. BMC Public Health. 2007 7: 270Vineis P, Veglia F, Garte S, Malaveille C, Matullo G, Dunning A, Peluso M, Airoldi L, Overvad K, Raaschou-Nielsen O, Clavel-Chapelon F, Linseisen JP, Kaaks R, Boeing H, Trichopoulou A, Palli D, Crosignani P, Tumino R,Panico S, Bueno-De-Mesquita HB, Peeters PH, Lund E, Gonzalez CA, Martinez C, Dorronsoro M, Barricarte A,Navarro C, Quiros JR, Berglund G, Jarvholm B, Day NE, Key TJ, Saracci R, Riboli E, Autrup H. Geneticsusceptibility according to three metabolic pathways in cancers of the lung and bladder and in myeloid leukemias innonsmokers. Ann Oncol. 2007 Jul;18:1230-42.Vineis P, Hoek G, Krzyzanowski M, Vigna-Taglianti F, Veglia F, Airoldi L, Overvad K, Raaschou-Nielsen O,Clavel-Chapelon F, Linseisen J, Boeing H, Trichopoulou A, Palli D, Krogh V, Tumino R, Panico S, Bueno-De-Mesquita HB, Peeters PH, Lund E E, Agudo A, Martinez C, Dorronsoro M, Barricarte A, Cirera L, Quiros JR,Berglund G, Manjer J, Forsberg B, Day NE, Key TJ, Kaaks R, Saracci R, Riboli E. Lung cancers attributable toenvironmental tobacco smoke and air pollution in non-smokers in different European countries: a prospective study.Environ Health. 2007 15; 6:7.Manuguerra M, Matullo G, Veglia F, Autrup H, Dunning AM, Garte S, Gormally E, Malaveille C, Guarrera S,Polidoro S, Saletta F, Peluso M, Airoldi L, Overvad K, Raaschou-Nielsen O, Clavel-Chapelon F, Linseisen J, BoeingH, Trichopoulos D, Kalandidi A, Palli D, Krogh V, Tumino R, Panico S, Bueno-De-Mesquita HB, Peeters PH, LundE, Pera G, Martinez C, Amiano P, Barricarte A, Tormo MJ, Quiros JR, Berglund G, Janzon L, Jarvholm B, Day NE,Allen NE, Saracci R, Kaaks R, Ferrari P, Riboli E, Vineis P. Multi-factor dimensionality reduction applied to a largeprospective investigation on gene-gene and gene-environment interactions. Carcinogenesis. 2007 28: 414-22.Toropov A A, Benfenati ESMILES in QSPR/QSAR modeling: Results and perspectivesCurrent Drug Discovery Technologies 2007 4 : 77-116Benigni R, Netzeva T, Benfenati E, Bossa C, Franke R, Helma C, Hulzebos E, Marchant C, Richard A, Woo Y-T,Yang CThe expanding role of predictive toxicology: An update on the (Q)SAR models for mutagens and carcinogensJ Environ Sci Heal C 2007 25 : 53-97Mandich A, Bottero S, Benfenati E, Cevasco A, Erratico C, Maggioni S, Massari A, Pedemonte F, Viganò LIn vivo exposure of carp to graded concentrations of bisphenol AGen Comp Endocr 2007 153 : 15-24Benfenati EThe specificity of the QSAR models for regulatory purposes: the example of the DEMETRA projectSAR QSAR Environ Res 2007 18 : 209-220Maran U, Sild S, Mazzatorta P, Casalegno M, Benfenati E, Romberg MGrid computing for the estimation of toxicity: Acute toxicity on fathead minnow (Pimephales promelas)Lect Notes Comput Sci 2007 4360 : 60-74Urbatzka R, van Cauwenberge A, Maggioni S, Viganò L, Mandich A, Benfenati E, Lutz I, Kloas WAndrogenic and antiandrogenic activities in water and sediment samples from the river Lambro, Italy, detected byyeast androgen screen and chemical analysesChemosphere 2007 67 : 1080-1087Fratev F, Lo Piparo E, Benfenati E, Mihaylova EToxicity study of allelochemical-like pesticides by combination of 3DQSAR, docking, local binding energy (LBE)and GRID approachesSAR QSAR Environ Res 2007 18 : 675-69261ANNUAL REPORT 2007

IRFMNToropov A A, Benfenati EOptimisation of correlation weights of SMILES invariants for modelling oral quail toxicityEur J Med Chem 2007 42 : 606-613Kahn I, Maran U, Benfenati E, Netzeva T I, Schultz T W, Cronin M T DComparative quantitative structure-activity-activity relationships for toxicity to Tetrahymena pyriformis andPimephales promelasAltern Lab Anim 2007 35 : 15-24Toropov A A, Benfenati ESMILES as an alternative to the graph in QSAR modelling of bee toxicityComput Biol Chem 2007 31 : 57-60Benfenati E, Azimonti G, Auteri D, Lodi MEnvironmental and ecological toxicology: Computational risk assessmentin: Ekins S (Ed.), Computational Toxicology: Risk Assessment for Pharmaceutical and Environmental Chemicals,John Wiley & Sons Inc., Hoboken NJ, USA (2007), 625-650Benfenati E, Craciun M, Neagu C DThe use of the DEMETRA modelsin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 303-313Benfenati EThe quality criteria of the DEMETRA models for regulatory purposes: Specificity, general lessons and futureperspectivesin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 283-301Amaury N, Benfenati E, Boriani E, Casalegno M, Chana A, Chaudhry Q, Chretien J R, Cotterill J, Lemke F, PiclinN, Pintore M, Porcelli C, Price N, Roncaglioni A, Toropov A AResults of DEMETRA modelsin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 201-281Benfenati E, Chretien J R, Gini G, Piclin N, Pintore M, Roncaglioni AValidation of the modelsin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 185-199Amaury N, Benfenati E, Bumbaru S, Chana A, Craciun M, Chretien J R, Gini G, Guo G, Lemke F, Minzu V,Mueller J A, Neagu C D, Pintore M, Stroia S A, Trundle PHybrid systemsin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 149-183Benfenati E, Casalegno M, Cotterill J, Price N, Spreafico M, Toropov A ACharacterization of chemical structuresin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 83-109Benfenati E, Boriani E, Craciun M, Malazizi L, Neagu C D, Roncaglioni ADatabases for pesticide ecotoxicityin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 59-81Benfenati E, Clook M, Friday S, Hart AQSARs for regulatory purposes: the case for pesticide authorizationin: Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes,Elsevier Science Ltd, Amsterdam, The Netherlands (2007), 1-5762ANNUAL REPORT 2007

IRFMNLo Piparo E, Mazzatorta P, Benfenati E Computational methods to study properties of allelochemicals and modellingof molecular interactions in allelopathy in: Roshchina V V, Narwal S S (Eds.), Plant Cell Diagnostics - Images,Biophysical and Biochemical Processes in Allelopathy, Science Publishers, Enfield NH, USA (2007), 169-183Pomati F, Cotsapas C, Castiglioni S, Zuccato E, Calamari D. Gene expression profiles in zebrafish (danio rerio) livercells exposed to a mixture of pharmaceuticals at environmentally relevant concentrations. Chemosphere 2007, 70: 65-73.Castiglioni S, Zuccato E, Chiabrando C, Fanelli R, Bagnati R. Detecting illicit drugs and metabolites in wastewaterusing high performance liquid chromatography-tandem mass spectrometry. Spctroscopy Europe 2007, 19 (4): 11-13.Bagnati R, Bianchi G, Marangon E, Zuccato E, Fanelli R, Davoli E. Direct analysis of isopropylthioxanthone (ITX)in food by HPLC/MS/MS. Rapid Commun Mass Spectrom 2007, 21: 1998-2002.Berrie C P, Iurisci C, Piccolo E, Bagnati R, Corda D. Analysis of phosphoinositides and their aqueous metabolites.Methods Enzymol 2007; 434 : 187-231.Carubelli G, Fanelli R, Mariani G, Nichetti S, Crosa G, Calamari D, Fattore E. PCB contamination in farmed andwild sea bass (Dicentrarchus labrax L.) from a coastal wetland area in central Italy. Chemosphere 2007 ; 68 : 1630-1635.LAY PRESS SELECTION PUBLISHED IN 2007Zuccato E, Castiglioni S, Bagnati R, Fanelli R. I farmaci: inquinanti ambientali ubiquitari. Quaderni acp 2007. 14(5):203-206.Zuccato E, Castiglioni S, Fanelli R, Bagnati R. Inquinamento da farmaci: le evidenze (parte I). Ricerca & Pratica2007; 23 (134): 67-73.Zuccato E, Castiglioni S, Fanelli R, Bagnati R. Inquinamento da farmaci: la regolamentazione e gli interventi (parteII). Ricerca & Pratica 2007; 23 (135): 113-118.Fattore E, Davoli E. La qualità dell'ambiente e le problematiche tossicologiche relative alle emissioni dimicroinquinanti organ ici In: Recupero di energia e materia da rifiuti solidi: i processi, le tecnologie, le esperienze, lenorme. AMRA, Napoli, 2007; 299-311E. Davoli, G. Bianchi, A. Colombo, M. Lodi, R. Marras, G. Rotella, V. Senese, E. Benfenati. Studi ambientali suilivelli di microinquinanti per e post insediamento di impianti di termovalorizzazione. Nuova Gea- QuaderniAmbiente 2007 ; 2 : 106-120OTHER PRODUCTS PUBLISHED IN 2007Benfenati E (Ed.), Quantitative Structure-Activity Relationships (QSAR) for Pesticide Regulatory Purposes, ElsevierScience Ltd, Amsterdam, The Netherlands (2007), 1-510.63ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Molecular ToxicologyToxicoproteomicsStudies are ongoing on the characterization of changes in the proteome profile induced byenvironmental toxic compounds, with the aim of obtaining protein biomarkers with the abilityto differentiate two or more biological states. Proteome changes in tissues and target organs ofanimals, and cells treated with endocrine disruptors, estrogens, or environmental carcinogens,are related to functional changes during toxicological processes. Qualitative and quantitativeproteome changes resulting from the exposure to environmental toxic compounds or inpathological conditions are monitored also in humans and focused on plasma and urine.Proteome analysis includes protein separation by two-dimensional gel electrophoresis andprotein identification by mass spectrometry (MALDI-TOF-MS, LC-ESI-MS/MS) coupled to theuse of existing databases. Alternatively, peptides resulting from the digestion of proteinmixtures with specific proteases are separated by two-dimensional liquid chromatography.Molecular EpidemiologyThe laboratory works mainly on the measurement of biological markers used to assess humanexposure to environmental toxic compounds. Our studies include DNA- and blood proteinadductformation by several environmental carcinogens. In addition, we study whether thepolymorphism of genes coding for enzymes involved in the activation and detoxification ofcarcinogens are determinants of adduct formation. Genotypes are detected by restrictionfragment length polymorphism analysis, after the amplification by polymerase chain reaction ofspecific nucleotide sequences of the genes under study.The laboratory participates in an international cooperation study aimed at the collection ofreference values on allele and genotype frequency of the most common metabolic enzymepolymorphisms in control populations.PharmacogeneticsThe study of the human genome has established that part of the observed individual variabilityin the reaction to pharmacological therapies is due to genetic traits. The analysis of the geneticpolymorphism of drug metabolism and disposition allows optimizing the therapy according tothe individual genetic makeup. This provides useful information not only for a correct drugdosage, but also for the prevention of adverse drug reactions.Laboratory of Analytical BiochemistryIdentification and characterization of proteins by mass spectrometryOur laboratory is developing different analytical and instrumental techniques for theidentification and characterization of proteins and peptides in biological samples. This activityis mainly aimed at 1) characterizing different protein isoforms that are found significantlyaltered in “differential proteomics” studies with two dimensional gel electrophoresis, 2)profiling proteins in biological fluids for discovery and identification of biomarkers ofphysiopathological and toxicological relevance, 3) identifying and characterizing endogenousdegradation products of proteins, 4) identifying proteins produced by cells in vitro in responseto given stimuli, 5) selectively isolating biologically relevant proteins by immunoaffinity-basedmicro-techniques for subsequent identification by mass spectrometry, and characterization ofpost-translational modifications by LC-MS/MS.64ANNUAL REPORT 2007

IRFMNOngoing projects include the study of exogenous protein degradation in renal tubular cells inrelation to antigen presentation mechanisms, the search for a protease responsible of theabnormal cleavage of von Willebrand Factor in patients with thrombotic thrombocytopenicpurpura, and the characterization of the secretome of cancer cell lines in vitro to identify factorsaffecting immune cells.Method development in proteomicsThe laboratory works on the optimization of various analytical methodologies for proteomics,i.e. various complementary techniques of protein identification by mass spectrometry (MALDI-TOF-MS, LC-ESI-MS/MS), isolation and purification of proteins, and characterization of theirpost-translational modifications.Laboratory of Environmental Chemistry and ToxicologyDevelopment and use of analytical methods to evaluate contamination inwater bodies, soil, biota, human samples in exposed populationAnalytical methods are developed to study environmental pollutants in water ecosystems,landfills, contaminated sites. Qualitative and quantitative analyses of organic pollutants are doneby mass spectrometry (GC-MS, LC-MS, LC-MS/MS). Typical analyses include PCDD/F, PCB,PAH, polybrominated diphenylethers, pesticides, endocrine disruptor chemicals, and industrialpollutants.Studies on environmental, toxicological and ecotoxicological propertiesof chemicalsResearch is carried out on pollutant properties, searching literature data, comparing andevaluating different sources, and mainly developing predictive models to cope with the lack ofexperimental data. Thus, we develop models starting merely from the chemical structure. Theresearch addresses the different kinds of chemical descriptors and chemical fragments, obtainedwith different software. Then, we develop models using algorithms such as neural network,fuzzy logic, genetic algorithms, classifiers, multivariate analysis, etc. Different methods arecompared and integrated within a structured ensemble. Standardized methods for pesticideswere developed and validated according to OECD guidelines.Risk assessment of pollutantsStudies are aimed at assessing the risk of pollutants for human population and environment. Forthis we model transport and diffusion of pollutants, to obtain a predicted concentration on givenspace and time scales. Such an activity is integrated with those above described on chemicalanalyses and toxicity prediction, to achieve a continuous transfer of data and research.Research on pollutants emitted in the atmosphere (Unit of Industrial andEnvironmental Hygiene)Studies address different aspects of atmospheric pollution. Research deals with: sampling areasaround the pollution source, chemical analyses, transport modeling depending onmeteorological conditions and orography, risk assessment for population and environment.Qualitative and quantitative analyses are done by gas chromatography-mass spectrometry usinghigh resolution for PCDDs/PCDFs, and negative ion-chemical ionization for PCBs.65ANNUAL REPORT 2007

IRFMNLaboratory of Mass SpectrometryParticulate air pollutionEpidemiological studies consistently show an association between an increasing number ofpathologies, both acute and chronic, and particulate air pollution. This has been shown not onlyin respiratory, but in cardiovascular diseases as well. Airborne particulate sampling and analysisstrategies are developed to characterize both adsorbed compounds and exposition in differentactivities.Method development in environmental sciencesMethods, analytical methodologies, instrumentation and software for data acquisition andreduction, are developed for environmental studies. High-sensitivity instrumentation, mainlybased on mass spectrometry, is developed for trace and ultra-trace analysis. Also, transportableinstrumentation is developed for field studies or continuous monitoring.Characterization of environmental odor annoyanceCharacterization of odors poses several analytical problems because they result from a complexmixture of compounds (odorants) stimulating receptors in the nasal cavity. Most odorants arevolatile organic compounds (VOC) generated by bacterial degradation of organic matter. Theyare often present at trace levels, while numerous sources can contribute to the total odor. Usingsampling techniques specifically developed for olfactometry, solid phase microextraction andGC/MS analysis, we can detect traces (low ppb to high ppt) of a wide polarity/volatility range ofairborne VOC odorant compounds. With a chemometric approach, we can characterize thesources of emissions, assess odor control methods, and identify emissions that contribute toodors in ambient air.Laboratory of Food ToxicologyChemical contaminants in foodWe are studying human exposure to dietary PCBs and dioxins. PCBs were measured in fooditems in different European countries, showing differences in PCBs exposure of Europeanconsumers. Further studies were aimed at measuring PCBs and dioxins in food from an Italianarea at high risk of contamination. Ongoing studies are focused on other emerging foodcontaminants.Therapeutic and illicit drugs in the environmentPharmaceuticals are a class of emerging environmental pollutants. We have organized acampaign to detect the presence of pharmaceuticals and their metabolites in Italian rivers andsewage treatment plants, with the aim of better characterizing the contamination and assessingrelated risks.Human and environmental risks are evaluated by studying the toxic effects of pharmaceuticalsat environmental levels, on cultures of human and zebra fish cells. Further ongoing studies areaimed at investigating a possible relationship between antibiotic occurrence and resistance inenvironmental bacteria.The possible presence of illicit drugs in water samples from sewage treatment plants and riverswas investigated, starting with cocaine and its metabolites. Their levels, used to estimate drugabuse in the local population, revealed that cocaine consumption greatly exceeds officialestimates.66ANNUAL REPORT 2007

IRFMNFarming methods and emerging pollutantsA project has been outlined to set up new methods for the assessment of GMO safety in humannutrition. This project is based on the use of new techniques to detect possible changes inducedin living organisms by GMO-derived foods.Regulatory activitiesOn behalf of the Ministry of Health, we carried on the evaluation of the dossiers required forpesticide registration within the European Union.Unit of Environmental Pollutants Risk AssessmentExposure to environmental pollutantsResearch activities include both quantitative measurement of contaminants in environmentalsamples, and assessment of exposure. Specific projects are the following:Risk assessment on health effects due to exposure to pollutants from emissions of incineratorsand landfillsMeasurments of persistent organic pollutants such as dioxins and furans polybrominated, andperfuorooctanoic acid and perfluorooctanoic acid (PFOA) and perfluorooctane sulphonate(PFOS) in farmed and wild fish coming from Mediterranean Sea. These data will be used toassess dietary exposure of the general Italian population through fish consumption.Exposure assessment methodology developmentNew methods for exposure assessment are developed, employing probabilistic approaches andmore refined statistical models, starting from real cases of contamination. A current study dealswith the exposure of the Seveso population to dioxin from contaminated soil.Evaluation of toxicological dataToxicological data resulting from in vivo sub-chronic studies in rats exposed to individualdioxin-like and non dioxin-like PCBs are evaluated in detail, in order to investigate the doseresponserelationship and the applicability of the “benchmark dose” approach.Unit of Analytical InstrumentationDevelopment and application of analytical methods for compounds ofbiological and environmental interestMethods are developed mainly using solid phase extraction (SPE) followed by liquidchromatography - mass spectrometry (LC-ESI-MS/MS) or gas chromatography - massspectrometry (GC-MS). Substances of interest include proteins, peptides, hormones,pharmaceuticals, drugs of abuse, pesticides, and other environmental contaminants (PCBs,hydroxy-PCBs, perfluorinated compounds).67ANNUAL REPORT 2007

IRFMNDEPARTMENT OF NEUROSCIENCESTAFFHeadGianluigi FORLONI, Biol.Sci.D.Laboratory of Biology of Neurodegenerative DisordersHeadCell Death and Neuroprotection UnitHeadGianluigi FORLONI, Biol.Sci.D.Tiziana Borsello, Biol.Sci.D.Laboratory of Drug MetabolismHeadSilvio CACCIA, Farm.D.Laboratory of Experimental NeurologyHeadAnnamaria VEZZANI, Biol.Sci.D.Laboratory of Experimental PsychopharmacologyHeadLuigi CERVO, Ph.D.Laboratory of Geriatric NeuropsychiatryHeadUgo LUCCA, MScEpidemiology and Social Psychiatry UnitHeadBarbara D’AVANZO, Philos.D.Geriatric Epidemiology UnitHeadGeriatric Pharmacology UnitHeadMauro TETTAMANTI, Biol.Sci.D.Emma RIVA, M.D.Laboratory of Inflammation and Nervous System DiseasesHeadMaria Grazia DE SIMONI, Biol.Sci.D.Laboratory of Molecular NeurobiologyHeadCaterina BENDOTTI, Farm.D.68ANNUAL REPORT 2007

IRFMNLaboratory of Neurochemistry abd BehaviorHeadRoberto William INVERNIZZI, Biol. Sci DPharmacology of Cognitive Behavior UnitHeadMirjana CARLI, Ph.D.Laboratory of Neurological DisordersHeadEttore BEGHI, M.D.Laboratory of Quality Assessment of Geriatric ServicesHeadAlessandro NOBILI, M.D.69ANNUAL REPORT 2007

IRFMNCURRICULA VITAEGianluigi Forloni, obtained the Degree of Biological Science at the University of Milan in 1985. Aftertwo years of post doc at the Department of Neuroscience and Psychiatry at Johns Hopkins University inBaltimore, USA, he came back to the Mario Negri Institute and between 1992 and 1996 he was the headof the Neurobiology of Alzheimer's disease Unit; since 1996 he is the Head of the Biology ofNeurodegenerative Diseases Lab and since 2002 the Head of the Neuroscience Department. His scientificinterest is focused on the biological and genetic bases of aging-related disorders in particular Alzheimer’sdisease, Prion-related encephalopathies and Parkinson’s disease. He has been member of severalEuropean committees for the examination of projects in the neuroscience field. He is now member of thecoordination group and thematic leader on drugs development of European Network of Excellence“Neuroprion”. He is President of the Italian Association on Brain Aging Research (AIRIC) and memberof the European Academy of Sciences. He is the author of more than 150 peer-reviewed scientific articlesand about 30 reviews or book chapters.Selected publications• Forloni G. Angeretti N., Chiesa R., Monzani E., Salmona M., Bugiani O.,Tagliavini F. Neurotoxicity of a prion proteinfragment. Nature 362: 543-546 (1993)• Forloni, G., Tagliavini, F.,Bugiani, O. and Salmona, M. Amyloid in Alzheimer’s disease and prion-relatedencephalopathies: Studies with synthetic peptides. Progr. Neurobiol. 49: 287- 315 (1996)• Forloni, G., Bertani, I. Calella, AM., Thaler, F.Invernizzi. R. Alpha-synuclein and Parkinson's disease selectiveneurodegeneration effect of alpha synuclein fragment on dopaminergic neurons in vitro. Ann. Neurol. 47: 632-640(2000)• Forloni G. Iussich, S. Awan T. Colombo L. Angeretti, N. Girola, L. Bertani, I. Poli, G. Caramelli, M. Bruzzone,MG.Farina, L. Limido, L. Rossi, G. Giaccone G. Ironside, JW. Bugiani, O.Salmona M. and Tagliavini, F. Tetracyclinesaffect prion infectivity Proc. Natl. Acad. Sci . New York 99: 10849-10854 (2002)• Pesaresi M, Lovati C, Bertora P, Mailland E, Galimberti D, Scarpini E, Quadri P, Forloni G, Mariani C. Plasma levels ofbeta-amyloid (1-42) in Alzheimer's disease and mild cognitive impairment. Neurobiol Aging., 27:904-5 (2006)• Fioriti, L. Angeretti, N.. Colombo, L., De Luigi A., Manzoni, C., Colombo A., Morbin, M., Tagliavini, F., Salmona, M.Chiesa, R. Forloni, G. Neurotoxic and gliotrophic activity of a synthetic peptide homologous to Gerstmann-Sträussler-Scheinker disease amyloid protein. J. Neurosci. 27: 576-83 (2007)Ettore Beghi (EB) graduated in Medicine in 1972 and received his specialty in neurology in 1976 at theUniversity of Milan. He trained in epidemiology with a fellowship at the Department of statistics andEpidemiology of the Mayo Clinic in Rochester, MN (USA). He is Head of the Laboratory ofNeurological Disorders at the Mario Negri Institute, Director of the Neurophysiology/Epilepsy Unit andProfessor of Neuroepidemiology at the University of Milano-Bicocca, Monza. He is member of theeditorial board of the journals Epilepsia, Neuroepidemiology, Inpharma, Drugs in R & D, Clinical DrugInvestigation, Neurological Sciences and is a referee of several national and international medicaljournals. The main areas of interest and research include studies on the descriptive, analytic, andexperimental epidemiology in the field of epilepsy, peripheral neuropathies, headache, and amyotrophiclateral sclerosis.Selected publications• Leone, MA. Solari, A.,Beghi, E. for the FIRST Group. Treatment of the first tonic-clonic seizure does not affect longtermremission of epilepsy. Neurology 2006; 67: 2227-2229• Millul, A., E. Beghi, G. Logroscino, A. Micheli, E. Vitelli, A. Zardi, for the “Registro Lombardo SLA”(SLALOM).Survival of patients with amyotrophic lateral sclerosis in a population-based registry. Neuroepidemiology 2005; 25: 114-119.• Tonini, C., E. Beghi, A.T. Berg, G. Bogliun, L. Giordano, R.W. Newton, A. Tetto, E. Vitelli, D. Vitezic, S. Wiebe.Predictors of epilepsy surgery outcome: a meta-analysis. Epilepsy Res 2004; 62: 75-87.• Van den Broek, M., and Beghi E., for the RESt-1 Group. Morbidity in patients with epilepsy: type and complications. AEuropean Cohort Study. Epilepsia 2004; 45: 71-76.• Van den Broek, M. and Beghi E. for the RESt-1 Group. Accidents in patients with epilepsy: type and complications. AEuropean Cohort Study. Epilepsia 2004; 45: 667-672.• Musico, M., E. Beghi, A. Solari, F. Viani for the First Seizure Trial Group. Treatment of the first tonic-clonic seizuredoes not improve the prognosis of epilepsy. Neurology 1997; 49: 991-998.70ANNUAL REPORT 2007

IRFMNCaterina Bendotti, got her degree in Pharmacy at the University of Milano in 1984; In 1986 -1988 she waspost doc at the Genetic developmental Lab, Dept. of Physiology of the Johns Hopkins University, Baltimore,USA. In 1988 -1992 she was research fellow in the laboratory of Neuropharmacology and in the 1992, shebecame head of the Molecular Neurobiology Unit in Institute, since 1998 she is head of laboratory. Themajor research interest is the study of pathogenetic mechanisms of familial Amyotrophic Lateral Sclerosis..Since 2002 she is a member of the editorial board of Journal of Neurochemistry. In 2002-2003 has beenMember of Scientific Committees of the International Symposia on ALS held in Milano, 17-19Novembre,2003. In 2003-2007 has been member of the Italian Ministry of Health Committees for thediagnosis, cure, care and assistance of patients with ALS. Since 2005 is member of the Board of Directors ofthe Italian Society of Neuroscience. Since 2006 is member of the Research Advisory Panel of the MNDAssociation, UK. Scientific reviewer of 11 international scientific journals. In 2007 she has co-organised thefirst international meeting on” Mutant SOD1 and familial ALS:from the molecule to man” held inMilano(13-16 September). She is author and co-author of 110 articles 100 of which with peer-review.Rapporteur of many communications in national and international meetings.Selected publications• Sau D, De Biasi S, Vitellaro-Zuccarello L, Riso P, Guarnieri S, Porrini M, Simeoni S, Crippa V, Onesto E, Palazzolo I,Rusmini P, Bolzoni E, Bendotti C, Poletti A. Mutation of SOD1 in ALS: a gain of a loss of function. Hum Mol Genet.16(13):1604-18, 2007.• Massignan T, Casoni F, Basso M, Stefanazzi P, Biasini E, Tortarolo M, Salmona M, Gianazza E, Bendotti C, Bonetto V.Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse.Biochem BiophysRes Commun. 353(3):719-25, 2007• Peviani M, Cheroni C, Troglio F, Quarto M, Pelicci G, Bendotti C. Lack of changes in the PI3K/AKT survival pathwayin the spinal cord motor neurons of a mouse model of familial amyotrophic lateral sclerosis.Mol Cell Neurosci. 34:592-602, 2007• Carri MT, Grignaschi G, Bendotti C. Targets in ALS: designing multidrug therapies. Trends Pharmacol Sci. 27(5):267-73, 2006• Cheroni C., Peviani M., Cascio P., Debiasi S., Monti C. and Bendotti C. Accumulation of human SOD1 andubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with adecrease of proteasome. Neurobiol. Disease. 18(3): 509-522, 2005• Bendotti C and MT Carri. Lessons from models of SOD1-linked familial ALS. Trends Mol Med. 10(8):393-400, 2004• Bendotti C., Tortarolo M., Suchak S.K., Calvaresi N., Carvelli L., Bastone A., Rizzi M., Rattray M. and Mennini T.Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamatelevels. J. Neurochem.,79, 737-746, 2001• Migheli A., Atzori C., Piva R., Tortarolo M., Girelli M., Schiffer D. and Bendotti C. Lack of apoptosis in mice withALS. Nature Medicine: 5, 966-967, 1999.Silvio Caccia. Laurea in Pharmacy at the University of Milan. Diploma in Industrial Chemistry at the L.Cobianchi Technical Institute (Verbania, NO) and Diploma in Biochemical Research at the Istituto diRicerche Farmacologiche “Mario Negri” (Milan).Research fellow, Laboratory of General Pharmacology at the Mario Negri Institute, 1970-1973;Permanent Researcher, Laboratory of Neuropharmacology, 1975; Head of Pharmacokinetic Unit, 1983;Head of Drug Metabolism Laboratory, 1988 to date, doing research in the field of pharmacology andtoxicology with particular focus on pharmacokinetic aspects, both at the pre-clinical and clinical level.He has been member of the scientific assessment teams (acting as expert) for the evaluation of marketingauthorisation applications submitted to the European and Italian Agencies.He is author and co-author ofmore than 200 articles, including reviews, monographs and book chapters.Selected publications• Caccia S. N-dealkylation of arylpiperazine derivatives: disposition and metabolism of the 1-aryl-piperazines formed.Curr Drug Metab 2007 ; 8 : 612-622.• Caccia S. Main active components of St. John's Wort (Hypericum Perforatum) extracts: current analytical procedures forpharmacokinetics and concentration-response studies. Curr Pharm Anal 2006; 2: 59-68.• Caccia S. Antidepressant-like components of Hypericum perforatum extracts: An overview of their pharmacokineticsand metabolism. Curr Drug Metab 2005; 6: 531-543• Caccia S. Metabolism of the newest antidepressants: Comparisons with related predecessors IDrugs 2004; 7: 143-150.• Caccia S. Biotransformation of post-clozapine antipsychotics. Pharmacological implications. Clin Pharmacokinet2000; 38: 39.• Caccia S. Metabolism of the newer antidepressants. An overview of the pharmacological and pharmacokineticimplications. Clin Pharmacokinet 1998; 34: 281-302.71ANNUAL REPORT 2007

IRFMNLuigi Cervo got the PhD degree from the Open University, Milton Keynes, United Kingdom in 2005 inrecognition of a program of research undertaken at the “Mario Negri” Institute for PharmacologicalResearch. From 2006 he is the head of the Laboratory of Experimental Psychopharmacolgy. Since 1997he has been the chief of the Behavioural Pharmacology Unit. From 1978 to 2001 he has been a researchfellow in the laboratory of Neuropharmacology and in 1981 he was awarded the degree in BiochemicalResearch from the “Mario Negri” Institute. Between 1981 and 1983 he spent two years as a researchfellow in the Department of Psychiatry at the Chicago University, Illinois, U.S.A. His main researchinterest concerns Neuropsychopharmacology and the mechanism of action of psychotropic drugs. Inparticular the role of receptors subtypes for serotonin, dopamine, noradrenaline and glutamate in drugdependence and drug craving, depression and anxiety. Mechanisms underlying acute as well as chronicpain are also of interest. He is author of numerous peer-reviewed articles in international scientificjournals, author of communications in international meetings, and ad-hoc reviewer for severalinternational scientific journals.Selected publications• Cervo L, Carnovali, F, Stark JA, Mennini T. Cocaine-seeking behavior in response to drug-associated stimuli in rats:involvement of D 3 and D 2 dopamine receptors. Neuropsychopharmacology 2003; 28: 1150-1159• Cervo L, Cocco A, Carnovali F. Effects on cocaine and food self-administration of (+)-HA-966, a partial agonist at theglycine/NMDA modulatory. Psychopharmacology (Berl) 2004; 173: 124-131• Grignaschi G, Burbassi S, Zennaro E, Bendotti C, Cervo L.A single high dose of cocaine induces behaviouralsensitization and modifies mRNA encoding GluR1 and GAP-43 in rats. Eur J Neurosci. 2004, 20:2833-7.• Cervo L, Mennini T, Rozio M, Ekalle-Soppo CB, Canetta A, Burbassi S, Guiso G, Pirona L, Riva A, Morazzoni P,Caccia S, Gobbi M. Potential antidepressant properties of IDN 5491 (hyperforin-trimethoxybenzoate), a semisyntheticester of hyperforin. Eur Neuropsychopharmacol. 2005, 15:211-8.• Cervo L, Canetta A, Calcagno E, Burbassi S, Sacchetti G, Caccia S, Fracasso C, Albani D, Forloni G, Invernizzi RW.Genotype-dependent activity of tryptophan hydroxylase-2 determines the response to citalopram in a mouse model ofdepression. J Neurosci. 2005 Sep 7;25(36):8165-72.• Cervo L, Burbassi S, Colovic M, Caccia S. Selective antagonist at D3 receptors, but not non-selective partial agonists,influences the expression of cocaine-induced conditioned place preference in free-feeding rats. Pharmacol BiochemBehav. 2005, 82:727-34.• Cervo L, Cocco A, Petrella C, Heidbreder CA. Selective antagonism at dopamine D3 receptors attenuates cocaineseekingbehaviour in the rat. Int J Neuropsychopharmacol. 2007, 10:167-81..• Calcagno E, Canetta A, Guzzetti S, Cervo L, Invernizzi RW. Strain differences in basal and post-citalopram extracellular5-HT in the mouse medial prefrontal cortex and dorsal hippocampus: relation with tryptophan hydroxylase-2 activity. JNeurochem. 2007, 103:1111-20..Maria Grazia De Simoni got the Doctoral Degree in Biological Sciences in 1977 at the University ofMilano, Italy. 1981: Research Specialist in Pharmacology (PhD), Mario Negri Institute, Milan, Italy.1981-1982: European Community fellowship for "Advanced Professional Training", INSERM U 171,Universitè Claude Bernard, Lyon, France; 1984 Department of Histology, Karolinska Institute,Stockholm. Working experience:1987-1997: Chief of the Neurochemistry Unit, Mario Negri Institute,Milano; 1998-present: Chief of the Laboratory of Inflammation and Nervous System Diseases, MarioNegri Institute. Scientific interests: pathogenesis of cerebral ischemia/reperfusion and traumatic braininjury; inflammatory response and apoptotic mechanisms as targets of therapeutic strategies; animalmodels and clinical studies. She is member of the board of “Master in Tecnologie Avanzate Applicatealle Patologie Neurodegenerative", University of Milan and member of the board of “AssociazioneItaliana per la Ricerca sull’Invecchiamento Cerebrale” (AIRIC).Selected pubblications• De Simoni MG, Storini C, Barba M, Catapano L, Arabia AM, Rossi E, Bergamaschini L. Neuroprotection bycomplement (C1)-inhibitor in mouse transient brain ischemia. J Cereb Blood Flow Metab, 23: 232-239, 2003.• De Simoni M G, Rossi E, Storini C, Pizzimenti S, Echart C, Bergamaschini L. The powerful neuroprotective action ofC1-inhibitor on brain ischemia-reperfusion injury does not require C1q. Am J Pathol., 164: 1857-1863, 2004.• Bergamaschini L, Rossi E, Storini C, Pizzimenti S, Distaso M, Perego C, De Luigi A, Vergani C and De Simoni MG.Peripheral treatment with enoxaparin, a low-molecular weight heparin, reduces plaques and β-amyloid accumulation in amouse model of Alzheimer’s disease. J. Neurosci. 24: 4181-4186, 2004• Troglio F, Echart C, Gobbi A, Pawson T, Pelicci PG, De Simoni MG & Pelicci G. The neuron-specific Rai (Shc C)adaptor regulates the PI3K-Akt pathway in vivo and protects against cerebral ischemia. Proc Natl Acad Sci U S A101(43): 15476-15481, 2004.• Storini C, Bergamaschini L, Gesuete R, Rossi E, Maiocchi D, De Simoni MG. Selective inhibition of plasma kallikreinprotects brain from reperfusion injury. JPET 318: 849-854, 200672ANNUAL REPORT 2007

IRFMN• Capone C, Fabrizi C, Piovesan P, Principato MC, Marzorati C, Ghirardi O, Fumagalli L, Carminati P and De SimoniMG. 2-Aminotetraline derivative protects from ischemia/reperfusion brain injury with a broad therapeutic window,Neuropsychopharmacology, 32: 1302-1311, 2007• Capone C, Frigerio S, Fumagalli S, Gelati M, Principato M C, Storini C, Montinaro M, Kraftsik R, De Curtis M, ParatiE, De Simoni MG. Neurosphere - derived cells exert a neuroprotective action by changing the ischemicmicroenvironment. PLoS ONE 2 e373, 2007.Roberto W. Invernizzi started his career in the laboratory of Neuropharmacology of the “Istituto diRicerche Farmacologiche “Mario Negri” in 1976, where, at present, he heads the Laboratory ofNeurochemistry and Behavior. In 1986 he got his degree in Biological Sciences at the Università Statale diMilano and in 1996 he was nominated head of the Intracerebral Microdialysis Unit. Of particular interestto Invernizzi’s research team is the study of the neurochemical mechanisms and neuronal circuitriesinvolved in the pathology of the main psychiatric diseases, such as depression and schizophrenia and inthe mechanism of action of psychotropic drugs. Since 1987 he applied the intracerebral microdialysistechnique to study the in vivo release of monoamines. Using this technique, Invernizzi’s team firstcontributed to clarifying the role of serotonergic and adrenergic autoreceptors in the effect ofantidepressant drugs suggesting new hypotheses on their mechanism of action. Currently, Invernizzi’steam is involved in two main collaborative projects aimed at clarifying the neurochemical mechanismsinvolved in the “resistance” to antidepressant drugs and the role of glutamatergic and serotonergicmechanisms in attentional processes. Reviewer for various international journals in the field ofpharmacology and neurochemistry. Author and co-author of more than 60 peer-reviewed articles. Memberof the Italian Society of Neuroscience and the Italian Society of Pharmacology.Selected publications• Baviera M, Invernizzi RW, Carli M. Haloperidol and clozapine have dissociable effects in a model of attentionalperformance deficits induced by blockade of NMDA receptors in the mPFC. Psychopharmacology (Berl) 2007 publishedonline.• Calcagno E, Canetta A, Guzzetti S, Cervo L, Invernizzi RW. Strain differences in basal and post-citalopram extracellular5-HT in the mouse medial prefrontal cortex and dorsal hippocampus: relation with tryptophan with tryptophanhydroxylase-2 activity. J Neurochem 2007; 103 : 1111-1120• Invernizzi RW, Pierucci M, Calcagno E, Di Giovanni G, Di Matteo V, Benigno A, Esposito E. Selective activation of5HT2C receptors stimulates GABA-ergic function in the rat substantia nigra pars reticulata: a combined in vivoelectrophysiological and neurochemical study. Neuroscience 2007 144 : 1523-1535• Carli M, Baviera M, Invernizzi RW, Balducci C Dissociable contribution of 5-HT1A and 5-HT2A receptors in themedial prefrontal cortex to different aspects of executive control such as impulsivity and compulsive perseveration inrats Neuropsychopharmacology 2006; 31: 757-767• Calcagno E, Carli M, Invernizzi RW The 5-HT1A receptor agonist 8-OH-DPAT prevents prefrontocortical glutamateand serotonin release in response to blockade of cortical NMDA receptors J Neurochem 2006; 96: 853-860• Cervo L, Canetta A, Calcagno E, Burbassi S, Sacchetti G, Caccia S, Fracasso C, Albani D, Forloni G, Invernizzi RWGenotype-dependent activity of tryptophan hydroxylase-2 determines the response to citalopram in a mouse model ofdepression J Neurosci 2005; 25: 8165-8172• Greco B, Invernizzi RW, Carli M Phencyclidine-induced impairment in attention and response control depends on thebackground genotype of mice: reversal by the mGLU2/3 receptor agonist, LY379268 Psychopharmacology (Berl) 2005;179: 68-76Ugo Lucca got his Master of Science, University of Aberdeen - UK, 1999. At the Mario Negri Institutehe was investigator from 1986- 1995, head of the "Clinical Evaluation of Antidementia Drugs Unit"(1995-1996) and, since 1996, head of the "Laboratory of Geriatric Neuropsychiatry".The main areas of interests include epidemiology and clinic features of dementia; natural history ofdementia; neuropsychiatric disorders of the elderly; instruments for the screening diagnosis and clinicalcourse assessment of dementia; clinical evaluation of anti dementia treatments and CNS active drugs(phase I, II, III, IV and observational studies).Selected publications• Spagnoli A, Lucca U, Menasce G, Bandera L, Cizza G, Forloni G, Tettamanti M, et al. Long-term acetyl-L-carnitinetreatment in Alzheimer's disease. Neurology 1991; 41:1726-1732• Lucca U, Comelli M, Tettamanti M, Tiraboschi P, Spagnoli A. Rate of progression and prognostic factors in Alzheimer’sdisease: a prospective study. J Am Geriats Society 1993; 41: 45-49.• Lucca U, Tettamanti M, Forloni G, Spagnoli A. Nonsteroidal anti-inflammatory drug use in Alzheimer’s disease.Biological Psychiatry 1994; 36: 854-856.73ANNUAL REPORT 2007

IRFMN• Imbimbo BP, Martelli P, Troetel WM, Lucchelli F, Lucca U, Thal LJ, and the Eptastigmine Study Group. Efficacy andsafety of eptastigmine for the treatment of patients with Alzheimer’s disease. Neurology 1999; 52: 700-708.• Quadri P, Fragiacomo C, Pezzati R, Zanda E, Forloni G, Tettamanti M, Lucca U. Homocysteine, folate, and vitamin B-12 in mild cognitive impairment, Alzheimer’s disease and Vascular Dementia. Am J Clinical Nutr 2004; 80: 114-122.• Lucca U, Tettamanti M, Quadri P. Homocysteine lowering and cognitive performance. New England Journal ofMedicine 2006; 355: 1390.Alessandro Nobili got his degree in Medicine (Milan, 1990). Master in Biotechonological Research,Regione Lombardia, Milan 1988. International School of Pharmacology, 31° Course on: DrugEpidemiology and Post-marketing Surveillance, Erice, September 1990. Course on: Methods inEpidemiological Research, Milan, October 1990. Course: Long Term Clinical Trials, Cogne January1991.Main areas of interest Methodology of Randomized Clinical Trials; Pharmacoepidemiology and postmarketingsurveillance research; Drug utilization studies; Quality assessment of geriatric services;Qualitative studies on caregiver role in the care of patients with dementia; Methodological evaluation ofthe Special Care Unit for Alzheimer Disease patients; Methodology of drug information. Employmentand research experience Chief of the Unit of Quality Assessment of Geriatric Services Chief of the DrugInformation Services for the Elderly, Laboratory of Geriatric Neuropsychiatry, Istituto di RicercheFarmacologiche “Mario Negri”, Milan. Editorial Board of the MICROMEDEX Inc., Englewood,Colorado 80111-4740 USA. National Expert accredited by Italian Ministry of Health for The Italian(AIFA) and European Agency for the Evaluation of Medicinal Products (EMEA). Head of theLaboratory of the Quality Assessment of Geriatric Services at the MarioNegri Institute since 2007.Selected publications• Nobili A, Tettamanti M, Frattura L, et al. Drug use in the elderly in Italy. Ann Pharmacother 1997; 31:416-422.• Nobili A, Gebru F, Rossetti A, et al. Doctorline a private toll-free telephone medical information service. AnnPharmacother 1998; 32:120-5.• Nobili A, Riva E, Tettamanti M, et al. The effect of a structured intervention on caregivers of patients with dementia andproblem behavior: a randomized controlled pilot study. Alzheimer Dis Assoc Disord 2004; 18: 75-82.• Lucca U, Nobili A, Riva E, Tettamanti M. Low level of B vitamins and the risk of cognitive and functional decline in thevery-old: results from the Monzino 80-Plus Study. Neurobiol Aging 2004; 25: 31.• Lucca U, Nobili A, Riva E, Tettamanti M Cholinesterase inhibitor use and age in the general population Arch Neurol2006; 63: 154-155.• Tettamanti M, Garri' M T, Nobili A, Riva E, Lucca U Low folate and the risk of cognitive and functional deficits in thevery old: The Monzino 80-plus study J Am Coll Nutr 2006; 25: 502-50874ANNUAL REPORT 2007

IRFMNAnnamaria Vezzani got her Degree in Biological Science at the University of Milan in 1978 and shespecialized in Neuropharmacology at the Mario Negri Institute in 1982. She spent her post-doctoralperiod in Baltimore at the University of Maryland in 1983-1984 working on the mechanisms ofepileptogenesis in experimental models of epilepsy. She spent additional post-doctoral periods at theUniversity of Stockholm and at the Karolinska Institute between 1985 and 1999. She was on sabbatical atthe Albert Einstein College of Medicine in 2002 in the laboratory of Developmental Epilepsy. She isinvolved in studies on the biochemical and molecular mechanisms involved in the etiopathogenesis ofseizures disorders using experimental models of epilepsy. The present research is focused on thefunctional role of neuroactive peptides and inflammatory mediators in the modulation of neuronalexcitability and seizure-related neurodegeneration. Focus of the research is also on the mechanisms ofpharmacoresistance. Since 1997 she is the Head of the Laboratory of Experimental Neurology at theMario Negri Institute. She is member of the Editorial Board of Epilepsy Currents and Neuroscience andAssociate Editor for Exp Models of Epilepsia. She is appointed of the Chair of the Commission onNeurobiology of International League Against Epilepsy which is promoting initiatives for improvingtranslational research in epilepsy.Selected publications• Balosso S, Ravizza T, Perego C, Peschon J, Campbell I, De Simoni MG, Vezzani A. TNF-alpha inhibits kainic acidinducedseizures in mice via p75 receptors (2005) Ann Neurol, 57, 804• Dube’ C., Vezzani A., Behrens M., Bartfai T., Baram TZ. (2005) Interleukin-1beta contributes to the generation ofexperimental febrile seizures. Ann Neurol, 57,152.• Richichi C, E-J. D. Lin, D. Stefanin, D. Colella, T. Ravizza,G. Grignaschi, G. Sperk, M. J. During and A. Vezzani “Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression inthe rat hippocampus” (2004) J Neurosci, 24,3051• Rizzi M, Caccia S, Guiso G, Richichi C, Gorter JA, Aronica E, Aliprandi M, Bagnati R, Fanelli R, D'Incalci M,Samanin R, Vezzani A.“Limbic seizures induce P-glycoprotein in rodent brain: functional implications forpharmacoresistance” (2002) J Neurosci, 22, 5833• Vezzani A., Moneta D., Conti M., Richichi C., Ravizza T., De Luigi A., De Simoni M.G., Sperk, Andell-Jonsson S.,Lundkvist J., Iverfeldt K. and Bartfai T. (2000) "Powerful anticonvulsant action of IL-1 receptor antagonist uponintracerebral injection and astrocytic overexpression in mice" Proc. Natl. Acad. Sci. USA, 97, 11534.• Vezzani A., Moneta D., Conti M., Richichi C., Ravizza T., De Luigi A., De Simoni M.G., Sperk, Andell-Jonsson S.,Lundkvist J., Iverfeldt K. and Bartfai T. (2000) "Powerful anticonvulsant action of IL-1 receptor antagonist uponintracerebral injection and astrocytic overexpression in mice" Proc. Natl. Acad. Sci. USA, 97, 11534.Tiziana Borsello got her Degree in Biological Science at the University of Torino in 1990 and she thenobtained a PhD in Neuroscience at the University of Turin Medical School. She won 1 year fellow of theEuropean Science Foundation scholarship for work at the Netherlands Research Institute of Amsterdam. From1997 to 1999 she was a Researcher at the Institute of Neurobiology, CNR, Rome Italy. In the period 1999-2003she was Premier Assistant, Département de Biologie Cellulaire et de Morphologie, Université de Lausanne,Switzerland, and then became Maitre Assistant and group leader in the same institute. In 2004 joined the Biol.Neurodeg. Disorders Lab at the "Mario Negri” Institute. In 2005 won the Prize of the Pfizer Foundation,Neuroscience and Diseases Nervous System. Since 2006 she is the Head of the Unit: Neuronal Death andNeuroprotection. Her main scientific interest is understanding the role of signalling pathways in neuronaldeath after different stress-stimuli and the neuroprotection. In particular, the present research is focused on thestudy of the mechanisms of excitotocic stress, ischemia, Traumatic Brain Injury and the cell death pathways inneurodegenerative diseases as Alzheimer, with the challenge to define the neuronal death pathways to designmore specific methods of neuroprotection.Selected pubblications• Repici M, Centeno C, Tomasi S, Forloni G, Bonny C, Vercelli A, Borsello T.Activation After Cerebral Ischemia AndEffect Of D-Jnki1 On C-Jun And Caspase-3 Activation. Neuroscience. 2007, 150: 40-9• Borsello T., Centeno C., Riederer IM, Haeflinger JA and Riedere BM. Phosphorylation-dependent dimerization andsubcellular localization of islet-brain 1/c-Jun N-terminal kinase-interacting protein 1. J Neurosci Res. 2007, 85:3632-41.• Borsello T Ed “Neuroprotection: Methods In Molecular Biology” Published By Humana Press, Usa Humana Press,USA, Methods in Molecular Biology, June 2007• Colombo A, Repici M, Pesaresi M, Santambrogio S, Forloni G, Borsello T. The Tat-Jnk Inhibitor Peptide Interferes WithBeta Amyloid Protein Stability Cell Death Differ. 2007, 14:1845-8.• Borsello T and Forloni G. JNK signalling: a possible target to prevent neurodegeneration. Current PharmaceuticalDesign 2007, 13, 1875-1886• Centeno C., Repici M., Chatton J. Y., Riederer B. M., Bonny C., Nicod P., Price M., Clarke P. G., Papa S., Franzoso G.and Borsello T. Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons. Cell Death Differ ,2007, 14: 240-253.75ANNUAL REPORT 2007

IRFMN• Borsello T. and Bonny C.Use of cell-permeable peptides to prevent neuronal degeneration. Trend in Mol. Med. 2004, 10:239-44• Borsello T, Clarke PG, Hirt L, Vercelli A, Repici M, Schorderet DF, Bogousslavsky J, Bonny C. A peptide inhibitor ofc-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nature Med. 2003, 9: 1180-6Mirjana Carli started his scientific career in the laboratory of Neuropharmacology of the “Istituto diRicerche Farmacologiche Mario Negri” Milan in 1977, where, at present, she is head of thePharmacology of Cognitive Behaviour Unit. She spent a few years in the laboratory of CognitiveNeuroscience, Dept. of Experimental Psychology, University of Cambridge (UK) directed by Prof.Trevor W. Robbins. Here she took interest in the role of brain monoamines in attention, and for thispurpose developed several behavioral tests for rats. In 1986 she returned to the laboratory ofNeuropharmacology of the “Istituto di Ricerche Farmacologiche Mario Negri”. Here she devoted herefforts to the study of the role played by neuronal mechanisms in cognitive processes such as memory,attention and executive functions. Her work has improved the knowledge of the role played by someserotonin receptors in cognitive processes.Selected publications• Carli M, Baviera M, Invernizzi R, Balducci C Dissociable contribution of 5-HT1A and 5-HT2A receptors in the medialprefrontal cortex to different aspects of executive control such as impulsivity and compulsive perseveration in ratNeuropsychopharmacology 2006; 31: 757-767• Greco B, Carli M Reduced attention and increased impulsivity in mice lacking NPY Y2 receptors: Relation to anxiolyticlikephenotype Behav Brain Res 2006; 169: 325-334• Carli M, Baviera M, Invernizzi R, Balducci C The serotonin 5-HT2A receptors antagonist MI00907 prevents impairmentin attentional performance by NMDA receptor blockade in the rat prefrontal cortex Neuropsychopharmacology 2004;29: 1637-1647• Balducci C, Nurra M, Pietropoli A, Samanin R, Carli M Reversal of visual attention dysfunction after AMPA lesions ofthe nucleus basalis magnocellularis (NBM) by the cholinesterase inhibitor donepezil and by a 5-HT(1A) receptorantagonist WAY 100635 Psychopharmacology (Berl) 2003; 167: 28-36• Carli M, Balducci C, Samanin R. Stimulation of 5-HT1A receptors in the dorsal raphe ameliorates the impairment ofspatial learning caused by intrahippocampal 7-chloro-kynurenic acid in naive and pretrained rats Psychopharmacology(Berl) 2000; 158: 39-47• Carli M, Samanin R The 5-HT1A receptor agonist 8-OH-DPAT reduces rats' accuracy of attentional performance andenhances impulsive responding in a five-choice serial reaction time task: Role of presynaptic 5-HT1A receptorsPsychopharmacology (Berl) 2000; 149: 259-268Barbara D’Avanzo obtained her master in Philosophy in 1989 at the University of Milan. Her mainfield of interest is epidemiologic research in mental health. She was involved in the analysis of theimplementation of the psychiatric reform in Italy and quality evaluation of services and their recentmodifications with specific attention to the role of psychiatric residential facilities in the communityservice networks; evaluation of effectiveness of the most common psychosocial interventions; suicidetrend monitoring and study of suicide prevention programs and initiatives. More recently, she is workingon issues like recovery-oriented services, consumers’ empowerment, and methods of participation ofconsumers to evaluation of services, and acknowledgment of the value of the consumers’ point of viewabout psychiatric treatments and services.She worked as researcher in the Laboratory of General Epidemiology between 1991 and 1996, and she isChief of the Unit of Epidemiology and Social Psychiatry since 2002. Member of the Scientific NationalBoard of WAPR Italy and of the World Head Office of the WAPR.Selected publications• Barbato A, D'Avanzo B. Marital therapy for depression. Cochrane Database Systematic Reviews 2006; Issue 2.• Parabiaghi A, Barbato A, D'Avanzo B, Erlicher A, Lora A. Assessing reliable and clinically significant change on Healthof the Nation Outcome Scales: method for displaying longitudinal data. Aust N Z J Psychiatry 2005; 39: 719-725.• Barbato A, D'Avanzo B. Involuntary placement in Italy. Br J Psychiatry 2005; 186: 542-543.• Guaiana G, Andretta M, Corbari L, Mirandola M, Sorio A, D'Avanzo B, Barbui C. Antidepressant drug consumption andpublic health indicators in Italy, 1955-2000. J Clinical Psychiatry 2005; 66: 750-755.• D'Avanzo B, Battino R N, Gallus S, Barbato A. Factors predicting discharge of patients from community residentialfacilities: A longitudinal study from Italy. Aust N Z J Psychiatry 2004; 38: 619-628.• D'Avanzo B, Barbato A, Barbui C, Battino N, Civenti G, Frattura L. Discharges of patients from public psychiatrichospitals in Italy between 1994 and 2000. Int J Social Psychiatry 2003; 49: 27-376ANNUAL REPORT 2007

IRFMNEmma Riva, Medical Doctor degree in 1984 University of Milan, PhD in 1990 in CardiovascularPathophysiology at the University of London (UK) Training: Research Assistant, Department ofPharmacology, Medical School, University of Ottawa, Canada; Internship in Internal Medicine, OspedaleLuigi Sacco, Milan; Cardiac Fellow, St Thomas' Hospital, London, UK. Field of interest: Prevalence andeffects of anemia on cognitive, functional and clinical variables in the elderly; Problem behaviors indementia; Burden for care-givers of Alzheimer Disease patients; End of life care. Present and past rolesin Institute Head of the Geriatric Pharmacology Unit, Istituto "Mario Negri", Milan; Scientific Directorof the hospice “Via di Natale Franco Gallinin”, Aviano, Italy; Consultant Istituto Geriatrico “Pio AlbergoTrivulzio”, Milan: Project member of PREDICT (Policy Review and Evaluation of Dementia andInstitutional Care Trends): a Transnational Comparison.Selected publications• Tettamanti M, Garrì MT, Nobili A, Riva E, Lucca U. Low folate and risk of cognitive and functional deficits in the veryold: The Monzino 80-plus study. J Am Coll Nutr 2006;25:502-508• Lucca U, Nobili A, Riva E, Tettamanti M. Cholinesterase inhibitor use and age in the general population. Arch Neurol2006;63:154-155• Nobili A, Riva E, Tettamanti M, Lucca U, Liscio MR, Petrucci B, Salvini Porro G. The effect of a structured interventionon caregivers of patients with dementia. Results of a Randomized Controlled Study. Alzheimer Dis and AssociatedDisorders 2004;18:75-82• Il malato terminale oncologico. Esperienze dall’hospice. Ed. Emma Riva. Il Pensiero Scientifico, 2001• Riva E, Tettamanti M, Gallini C. Il ruolo del medico di medicina generale nella gestione dei malati terminali oncologici.Indagine svolta tra i medici di medicina generale in Friuli Venezia Giulia. Ricerca & Pratica 2001• Riva E, Nobili A, Trecate F. Per un impiego "ragionato" dei neurolettici, per la gestione dei disturbi del comportamentoin corso di Malattia di Alzheimer. Rec Prog Med 1998;89:598-603Mauro Tettamanti got his Biology Degree at the Università degli Studi di Milano in 1986, and thespecialisation in Epidemiology and Medical Statistics in 1993, at the Università degli Studi di Pavia.Teaching experience Introduction course to statistics, Master in Ergonomy, Politecnico di Milano, years2001-2004 Areas of interest: Planning, conduction and analysis of clinical trials and epidemiologicresearches in the geriatric field: Phase I, II, III and observational studies on the efficacy of drugs onneurologic disorders, with special emphasis on dementia; Effects of multi-disciplinary interventions ongeriatric/dementia patients; Epidemiology and risk factors of dementia; Care of patients with terminalillness; Association of anemia with prevalence of diseases and cognitive problems Scholarship between1989 and 1998, Senior Researcher since 1999 and Head of the Unit of Geriatric Epidemiology at theMario Negri Institute since 2001.Selected publications• Spagnoli A, Lucca U, Menasce G, Bandera L, Cizza G, Forloni G, Tettamanti M, et al. Long-term acetyl-L-carnitinetreatment in Alzheimer's disease. Neurology 1991; 41:1726-1732.• Lucca U, Comelli M, Tettamanti M, Tiraboschi P, Spagnoli A. Rate of progression and prognostic factors in Alzheimer'sdisease: A prospective study. J Am Geriatr Soc 1993; 41:45-49• Quadri P, Fragiacomo C, Pezzati R, Zanda E, Forloni G, Tettamanti M, Lucca U. Homocysteine, folate, and vitamin B-12 in mild cognitive impairment, Alzheimer disease, and vascular dementia. Am J Clin Nutr 2004; 80: 114-122• Lucca U, Nobili A, Riva E, Tettamanti M. Cholinesterase inhibitor use and age in the general population. Arch Neurol2006; 63:154-155• Lucca U, Tettamanti M, Quadri P. Homocysteine lowering and cognitive performance. N Engl J Med 2006; 355:1390• Tettamanti M, Garri' M T, Nobili A, Riva E, Lucca U. Low folate and the risk of cognitive and functional deficits in thevery old: The Monzino 80-plus study. J Am Coll Nutr 2006; 25: 502-50877ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe Department of Neuroscience is formed by ten Laboratories; the activities of research are devoted tothe study of neurological and psychiatric diseases, evaluated by the biological point of view, clinical andepidemiological aspects and the quality of care. Together with these activities, in the Department othermore general expertise are present. Pharmacokinetics studies, drug information service and preparation ofprotocols for clinical trial and epidemiological studies are activities in charge of the NeuroscienceDepartment. Traditionally part of the Department was devoted to the creation of experimental models forthe pharmacological, neurochemical and pathogenetic studies in Alzheimer or prion's diseases, epilepsy,depression and cognitive impairment. More recently, consolidated expertise were created in thepathogenesis of amyotrophic lateral sclerosis (ALS), cerebral stroke and drug abuse. Some of thesedisorders, like epilepsy, ALS and Alzheimer's disease are investigated from the clinical andepidemiological points of view for the evaluation of drug and care efficacy. The activities of theDepartment are aimed to an integration of the different expertise to develop multidisciplinary approaches.The purpose is to address at different levels, knowledge, therapy and clinical practice to the numerousquestions, largely unresolved, proposed by the disorders of nervous system.FINDINGS/MAIN RESULTS 2007In a cellular model it has been shown that the peptide of D-JNK-1-TAT inhibiting the JNKphosphorylation activity mediated by the enzyme JNK, exerts a control on β amyloidproduction, these data indicate possible therapeutic perspectives in Alzheimer’s diseaseThe transgenic mice overexpressing the mutated form of human protein precursor of amyloid(APP) have a cognitive deficit associated to glutametergic dysfunction independent from the βamyloid depositsIn cellular model the presence of α synuclein mutations associated to Parkinson’s disease, doesnot influence the neuroprotective activity of the protein, this indicates that these mutationsaffected the aggregation state more then physiological activity of α synuclein.In the transgenic mice overexpressing a mutated form of human prion protein associated to CJDgenerated in the Institute exhibit some behavioral features reminiscent of the clinical conditionin humans carrying the same mutation.In an epidemiological study we found that the values of some plasmatic parameters (IGF, IL-6)showed an age-dependent trend until 90 years old, for the older population the correlation withage disappeared.In a prospective population-based study in the very old (Monzino 80-plus Study), depressivesymptoms were common prior to dementia. However, the risk of developing dementia associatedwith depression, if present, seemed modest.In the same prospective population-based study in the very old (Monzino 80-plus Study),walking one hour or more a day was associated with an almost 40% decreased risk of developingdementia.78ANNUAL REPORT 2007

IRFMNIn a rural population of elderly aged 75 years and older, dividing the subjects in three groups onthe basis of disability (no disability at all, instrumental disability and basic activity disability) gavegroups with similar size. People with major disability were older, had a higher number ofdiagnoses, received more specialist visits (but a similar number of primary care physician visitsand hospitalizations) and needed more paid assistance (double than those with minor disability andten times more than those without disability).In a prospective ambulatory population of cognitively normal or mildly cognitively impairedelderly, individuals with baseline elevated homocysteine concentrations have an almost 3-foldincreased risk of developing dementia in the following 3-4 years.More than 1 out of 10 elderly persons (65-84 years) were anemic and most of the cases had a mildgrade anemia. Hemoglobin concentrations decreased and prevalence of (mild) anemia increasedwith increasing age. After controlling for many potential confounders, mild anemia was found tobe associated with poorer health conditions and with increased risk of clinically relevant outcomesuch as hospitalization and mortality.Patients with dementia resident in Alzheimer’s special care units (ASCU) had a lower rate ofhospitalisation and use of physical restraints than those in traditional nursing homes.In ASCU 60% of patients with dementia were taking at least one antipsychotic, 49% typical and51% atypical. More than 50% of patients exposed to antipsychotics at baseline, were still takingthe drug after 18 months of follow-up. The use of antipsychotic agents was strongly related tothe presence of agitation, irritability, delusions, anxiety, night-time behaviour and aberrantmotor behaviour.From January to December 2003, in the Lecco Local Health Authority, 15.5% of elderlypatients were exposed to potential severe drug-drug interactions (60.8% were women). The riskof drug interaction increased with age and the number of prescription and chronic therapies.Marked differences in the quality of acute care offered across various acute wards in Italy. Seriuosinadequancies were reported in physical and process indicators in the private wards. Whereasefficacy on crisis was evaluated as rather good, satisfaction of patients with the care and assistancereceived during the admission was modest.The Natural Networks patients, followed according to a naturalistic design, showed significantimprovements in quality of life, needs satisfaction, and social functioning.In amyotrophic lateral sclorosis loss of ambulation, percutaneous gastrostomy and non-invasivemechanical ventilation are outcome measures to be used in epidemiological surveys andtherapeutic trials.The long-term prognosis of epilepsy is the same in patients treated at the first seizure and thosetreated at the recurrence. These findings suggest that treatment should be started at the first seizureonly on a case-by-case basis. The use of a third drug in children refractory to two anticonvulsantsdoes not affect the chance of seizure remission, suggesting that drug resistance in epilepsy can beidentified at the time of failure of two drugs.Focal dystonia in the adult is a rare age-related clinical condition. Blepharospasm is thecommonest disease variety.79ANNUAL REPORT 2007

IRFMNThe proteomic analysis of the blood cells of ALS patients has evidenced variations of someproteins that are also found alterated in the spinal cord of SOD1G93A mice at thepresymptomatic stage. Therefore, these proteins can be considered potential candidates for theidentification of the pathogenetic mechanisms of ALS and as useful biomarkers for the earlydiagnosis of the diseaseWe evidenced a specific proteasomal dysfunction in the motor neurons of SOD1G93A mice atthe symptomatic stages which may contribute to the accumulation of intracellular proteinaggregates. In addition the proteomic characterization of the insoluble proteins from the spinalcord of SOD1G93A mice has identified a series of oxidated proteins suggesting a possible linkbetween oxidative stress and aggregation pathways in ALS pathogenesis.We have demonstrated that specific neuropeptide Y and somatostatin receptor subtypes mediatethe anticonvulsant activity of these endogenous peptides. Thus, this knowledge may beexploited to develop new anticonvulsant drugs with a novel mechanism of actionWe have demonstrated the modulatory role of some pro- and anti-inflammatory cytokines inseizures using experimental models of epilepsy in rodents, thus describing a newetiopathological mechanism which may be relevant for human epilepsyWe have demonstrated that membrane-bound drug transport proteins are functionally activatedby seizures and have a significant role in decreasing the brain concentrations of antiepilepticdrugs in experimental models. Pharmacological intervention to block the activity of theseproteins may contribute to reverse multidrug resistance in epilepsyComplement inhibitor (C1-INH) has powerful neuroprotective actions in brainischemia/reperfusion injuryMicroglia can explain protective actions in the ischemic environmentNeural stem cell reduce ischemia/reperfusion injury by changing the ischemicenvironment.Agonists at serotonin 2C receptors as well non-selective opioid receptor antagonists selectivelymodulate, in laboratory rodents, the drug seeking behaviour induced by the environmentalstimuli predictive of cocaine availability.DBA/2J and BALB/c mice are not responder to serotonin selective uptake blockers in an animalmodel predictive of the antidepressant activity and may represent an animal model of resistanceto SSRIAgonists at serotonin 2C receptors as well non-selective opioid receptor antagonists selectivelymodulate, in laboratory rodents, the drug seeking behaviour induced by the environmentalstimuli predictive of cocaine availabilityGenetic differences in serotonin synthesis contribute to the efficacy of SSRIs in miceCo-treatment with tryptophan restores the antidepressant-like effect in mice non-responder tothe SSRI alone80ANNUAL REPORT 2007

IRFMNBasal and SSRI-induced serotonin release is strongly suppressed in mice carrying the mutationof TPH-2, the limiting step enzyme in brain serotonin synthesisThe blockade of NMDA receptors of the rat prefrontal cortex induces an increase of glutamaterelease and is deleterious for prefrontal cortex-dependent cognitive functionsNATIONAL COLLABORATIONSAssociazione Familiari Insonnia Familiare Fatale malattie da prioni, TrevisoAssocizione Italiana GIST A.I.G.Associazione per la Ricerca Neurogenetica, Lamezia Terme (CS) e ASL 6, Regione CalabriaAgenzia di Sanità Pubblica del Lazio, Regione LazioAssessorato alla Salute, Comune di MilanoAzienda Ospedaliera Ospedali Riuniti di BergamoAzienda Sanitaria Locale di BergamoCEND, Centro Eccellenza per le Malattie Neurodegenerative, Università di MilanoCentro Fatebenefratelli San Giovanni di Dio, Cernusco sul NaviglioCentro di Neurofarmacologia, Dipartimento di Scienze Farmacologiche, Università di MilanoCentro Studi in Psichiatra, ASL 2, TorinoCentro Parkinson-Istituti Clinici di PerfezionamentoClinica IRCSS S. Maria Nascente, MilanoClinica Neurologica III Università di Milano, Azienda Ospedaliera S. Paolo, MilanoClinica Psichiatrica, Università Milano BicoccaConsorzio Ricerche Luigi Amaducci, CRIC, Arcugnano (Vc)Consorzio MIA, MilanoDIBIT, San Raffaele Scientific Insitute, Milano.Dipartimento di Chimica Biologica, Università di PadovaDipartimento di Chimica, Università egli Studi di FirenzeDipartimento Endicronologia, Università di MilanoDipartimento Farmaco Chimico Tecnologico, Università di SienaDipartimento di Farmacologia Medica, Università di MilanoDipartimento di Fisiologia Umana, Facoltà di Medicina, Università di MilanoDipartimento di Medicina e Sanità Pubblica, Sezione di Psichiatria e Psicologia Clinica,Università di VeronaDip. di Morfofisiologia, Scuola di medicina Veterinaria, Università di Torino, Grugliasco (TO).Dip. Neurologia, IRCCS Fondazione Maugeri, PaviaDipartimento Neurologia, Ospedale Molinette, TorinoDipartimento di Neurologia Università di Milano, Ospedale Luigi Sacco.Dipartimento di Neuroscienze, Università di Parma, ParmaDipartimento di Salute Mentale di Niguarda, MilanoDipartimento di Salute Mentale ASL 3 ”Genovese”, GenovaDipartimento di Salute Mentale ASL 4, TorinoDipartimento di Salute Mentale, Azienda Ospedaliera Carlo Poma di Mantova, MantovaDipartimento di Salute Mentale San Carlo, MilanoDip. di Scienze Biomolecolari e Biotecnologie, Università di MilanoDipartimento Scienze Neurologiche, Università di Genova, GenovaDipartimento Scienze Neurologiche, Ospedale Maggiore Policlinico di MilanoDirezione Generale Famiglia e Solidarietà Sociale, Regione Lombardia, MilanoDirezione Generale Sanità, Regione Lombardia, Milano81ANNUAL REPORT 2007

IRFMNDirezione Regionale Sanità e Servizi Sociali, Regione UmbriaDivisione Neurologica, Università di BolognaEvidentia Medica, Grottaferrata, RomaFederazione Alzheimer Italia, MilanoFranco Calori Cell Factory, Centro Trasfusionale e di Immunologia dei Trapianti, IRCCSOspedale Maggiore, MilanoFondazione Clelio AngelinoFondo Edo TempiaHospice “Via di Natale Franco Gallini”, Aviano (PN)IRCSS "Casa Sollievo della Sofferenza", San Giovanni RotondoIRCCS Istituto Auxologico Italiano, MilanoIstituto di Ricovero e Cura a Carattere Scientifico IRCCS (I.N.R.C.A.), AnconaIRCSS "San Raffaele", MilanoIstituto Europeo di Oncologia, IRCCS, MilanoIstituto di Farmacologia e Farmacognosia, Università di UrbinoIstituto di Farmacologia, Università di MilanoIstituto “G. Ronzoni”, MilanoIstituto Nazionale Neurologico “Carlo Besta”, MilanoIstituto Scientifico HumanitasIstituto di Scienze e Tecnologie della Cognizione, CNR, RomaIstituto "Stella Maris", IRCCS, Calambrone (PI)Istituto Superiore di Sanità, RomaIstituto Zooprofilattico Piemonte Liguria Val D'Aosta,TorinoLaboratorio di Immunopatologia Renale, Ospedale San Carlo, MilanoLaboratorio di Neuroscienze, Centro Dino Ferrari, Università di MilanoLega Italiana per la Lotta contro i TumoriOspedale del Bambin Gesu’, RomaOspedale Regionale Ca Fondello, TrevisoOspedale "Molinette", TorinoPolo Oncologico, ASL 12, BiellaProvincia Lombardo-Veneta Ordine Ospedaliero San Giovanni di Dio, Fatebenefratelli diCernusco sul NaviglioScuola di Specializzazione in Psicoterapia IRIS-Insegnamento e Ricerca Individuo e Sistemi,MilanoScuola di Terapia Cognitiva “Studi Cognitivi”, MilanoSocietà Italiana Medicina Interna, RomaUnione Nazionale delle Associazioni per la Salute Mentale (UNASAM), MilanoUnità di Geriatria, Ospedale Maggiore IRCCS, Università di MilanoUnità Operativa di Psichiatria, Azienda Ospedaliera Luigi Sacco di Milano, MilanoUnità Operativa di Psichiatria, Azienda Ospedaliera San Gerardo di Monza, MonzaUnità Operativa di Psichiatria di Garbagnate, Azienda Ospedaliere Salvini di Garbagnate,Garbagnate MilaneseUnità Operativa di Psichiatria, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagallie Regina Elena di Milano, MilanoUniversità degli Studi di FoggiaUniversità Cattolica del Sacro Cuore di RomaUniversità del Piemonte Orientale, NovaraUniversità di Milano, IRCCS Ospedale Maggiore, MilanoUniversità Milano-Bicocca, MonzaUniversità La Sapienza, RomaU.O. Neurologia, Clinica S. Maria, IRCCS, Castellanza (VA).82ANNUAL REPORT 2007

IRFMNINTERNATIONAL COLLABORATIONSAlbert Eistein College of Medicine, Bronx, NY, USAAtomic Energy Commission, Service de Neurovirologie, Fontenay-aux-Roses, FranceBeaumont Hospital, Dublin, IrelandCambridge Centre for Brain Repair, University of Cambridge, UKCentre for Neuroscience Research and Division of Biomolecular Sciences, GKT School, King’sCollege, London, UKChorley & South Ribble General Hospital, Chorley,Columbia Univ, Haverstraw, NY, USADepartment of Anatomy and Physiology, Laval University, QuebecDepartment of Cell Biology, Washington University, St Louis, USADepartment of Chemistry,The Australian National University, Canberra City, AustraliaDepartment of Experimental Psychology, University of Cambridge, UKDepartment of Pathology and Infectious Diseases Royal Veterinary College, Herts, UKDepartment of Psychiatry, Medical Center University of Mississippi, Jackson, USADirectorate General for the Health and Consumer Protection, European Commission,LuxembourgDivision of Medical Genetics, CHUV Lausanne, SwitzerlandEuropean Union of Family Associations of People with Mental Illness (EUFAMI)Geriatric Division and Department of Metabolic Diseases, Ospedali Regionali of Lugano andMendrisio, SwitzerlandHSPH Harvard University, Boston, USAIBCM, University of Lausanne, Lausanne, SwitzerlandInstitut de Génétique Humaine du CNRS, Montpellier, FranceJefferson Med Coll, Philadelphia, USAKarolinska Institutet, Stockholm, SwedenKing’s College Hospital, London, UKMax-Delbrück-Center for Molecular Medicine, Berlin, GermanyNational Insitute on Aging, NIH, Baltimore, USANeuroprion, Network of Excellence, WP VI, ECNeurological Department of the University of Tirana, AlbaniaNinewells Hospital and Medical School, Dundee, Scotland UKNorthern Illinois University, DeKalb, IL, USANovartis Pharma, Basel, SwitzerlandRobarts Research Institute, London, Ontario, CanadaRoyal Manchester Children's Hospital, Manchester, UKRoyal Preston Hospital, Preston, UKSergievsky Center, Columbia University, New York, NY, USAServizio di Geriatria, Ospedale della Beata Vergine, Mendrisio, SwitzerlandThe Scripps Research Institute, Jupiter, Florida, USAUniversity of Alberta, CanadaUniv of California at Irvine, Irvine, CA, USAUniversity of Cardiff, United KingdomUniv of Colorado, Denver, USAUniversity Hospital, London, ON, CanadaUniv of Innsbruck, Innsbruck, AustriaUniv of Maryland, Baltimore, USA83ANNUAL REPORT 2007

IRFMNUniversity of Maastricht, the NetherlandsUniversity of Rijeka Medical School, Rijeka, CroatiaUniversity of Szeged, UngaryUniversité Victor Segalen, Bordeaux, FranceVirtanen Institute for Molecular Sciences, University of Kuopio, FinlandVrije Universiteit Medical Center, Amsterdam, The NetherlandsWalton Hospital, Liverpool, UKWAPR (World Association for Psychosocial Rehabilitation)Weill Cornell Medical College, New York, USAWorld Mental Health, Department of Mental Health and Substance Abuse, Geneva,SwitzerlandEDITORIAL BOARD MEMBERSHIPBiochemical Journal (Chiesa, Forloni)Brain Aging (Forloni)Clinical Drug Investigation (Beghi)Clinical Neurology and Neurosurgery (Beghi)Cochrane Collaboration, Epilessia (Beghi)Dialogo sui Farmaci (Nobili)Drugs in the R&D (Beghi)Epidemiologia e Prevenzione (Lucca)Epilepsia (Beghi, Vezzani Assistant editor)Epilepsy Current (Vezzani)Epilepsy Research (Vezzani)Inpharma (Beghi)International Journal of Mental Health (Barbato)Journal of Neurochemistry (Bendotti)Neurological Sciences (Beghi)Neuroepidemiology (Beghi)Neuroscience (Vezzani)Open Aging Journal (Forloni)Open Geriatric Medicine Journal (Forloni)Psichiatria di Comunità (Barbato)Quality of Life Research (Barbato)Ricerca & Pratica (Nobili)Stroke (De Simoni, Associate editor)84ANNUAL REPORT 2007

IRFMNPEER REVIEW ACTIVITIESActa Neurologica ScandinavicaActa Psychiatrica ScandinavaAlzheimer Disease and Associated DisordersAmerican Journal of Clinical NutritionAmerican Journal of Human GeneticsAmerican Journal of PathologyAmerican Journal of PhysiologyAnnals of NeurologyAnnals of PharmacotherapyBehavioural Brain ResearchBiochimica et Biophysica ActaBiochemical JournalBiochemistryBioMed Central NeurologyBiological PsychiatryBrain ResearchBrain Research BulletinBrain Research ReviewClinical Drug InvestigationClinical Neurology and NeurosurgeryClinical PharmacokineticsClin Pharm TherapyCNS DrugsDialogo sui farmaciDrugsEpidemiologia e Psichiatria SocialeEpilepsiaEpilepsy & BehaviorEuropean Journal of ImmunologyEuropean Journal of NeuroscienceEuropean Journal of PharmacologyEuropean Journal of Public HealthExperimental NeurologyEuropean NeuropsychopharmacologyExpert Opinion on PharmacotherapyFASEB JournalFEBS lettersFundamental Clinical PsychopharmacologyFuture DrugsGiornale di Neuropsichiatria dell’Età EvolutivaGliaInternational Journal of NeuropsychopharmacologyJournal of the American Board of Family PracticeJournal of Biological ChemistryJournal of Cell. BiologyJournal of Cerebral Blood Flow and MetabolismJournal of Chemical NeuroanatomyJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Science85ANNUAL REPORT 2007

IRFMNJournal of Headache and PainJournal of Histochemistry and CytochemistryJournal of ImmunologyJournal of NeurochemistryJournal of NeuroimmunologyJournal of Neurology, Neurosurgery and PsychiatryJournal of NeuroscienceJournal of Pharmacy and PharmacologyJournal of PsychopharmacologyJournal of Psychosomatic ResearchJournal of Structural BiologyLife SciencesLancetLancet NeurologyMolecular Brain ResearchMolecular and Cellular NeuroscienceMolecular TherapyNeuroepidemiologyNeurologyNeurological SciencesNerobiology of AgingNeurobiology of DiseasesNeuropharmacologyNeuropsychopharmacologyNeuroscienceNeuroscience LettersN.S. Archives PharmacologyParkinsonism & Related DisordersPharmacological ResearchPharmacoepidemiology and Drug SafetyPharmacology Biochemistry & BehaviorPlosOneProc Natl Acad Sci, USAPsychopharmacologySynapseTrends Molecular Medicine86ANNUAL REPORT 2007

IRFMNNATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPAdvisory Board of the Italian League Against EpilepsyBoard of "Master in Advanced Technologies for the Study of Neurodegenerative Diseases",University of MilanCommission on Health Care Policy of the International League Against Epilepsy (ILAE)Commission of Italian Minister of Health for the study of the problems associated to diagnosis,therapy and assistance ALS patientsCoordination Group of NoE Neuroprion ECCoordination Group of the European project ”Quelles professionnalités en santé mentale.Perspectives croisées, usagers, élus professionnels”.Council of Italian Association on Brain Aging Research (AIRIC)Expert for European Agency for the Evaluation of Medicines (EMEA)Expert reviewer for the Medical Research Council (MRC), UKExpert of the Minister of Health to EMEAItalian Association of Neuroepidemiology (Past President)Italian Association on Brain Aging Research (AIRIC, President)Italian Society of Neuroscience (Council)International Committee on “Epilepsy and the Law”International Organizing Committee e coordinator of the Secretariat al Global Forum forCommunity Mental Health, Department of Mental Health of WHOInternational Subcommittee of the American Academy of NeurologyMedical Research Council Strategic Grant ApplicationMental Health Working Party of DG-SANCO, Directorate General – Public Health andConsumer Protection – of the European Union, Brussels, BelgiumNational expert accredited by AIFA (Italian Medicines Agency) for the European Agency forthe Evaluation of Medicinal Products (EMEA)Neurobiology Commission of the International League against EpilepsyNeuroepidemiology Section of the American Academy of Neurology (past Chair)Research Advisory Panel, MND Association, UK Scientific Advisory Board of SheffieldInstitute Foundation for MND87ANNUAL REPORT 2007

IRFMNEVENT ORGANIZATIONEpidemiology and research methods course.“Medicina basata sull’evidenza inneuropsichiatria dello sviluppo: la diagnosi come strumento di codifica per una banca-dati.”Istituto Scientifico Stella Maris, Calabrone (Pisa) June 7-9, 2007.59th Annual Meeting of American Academy of Neurology – Breakfast Seminar – How tomanage a patient with a first epileptic seizure: An evidence-based approach – April 28 – May 5,2007, Boston, MA, USA.International meeting on “Mutant SOD1 and familial ALS: from the molecule to man”.Milan, September 13-16, 2007Course for physicians: The traps of literature: looking for, selecting and reading an article.May 23 rd , 2007 - Villa Camozzi, Ranica (BG)Course for physicians: What one knows when a new drug is approved.September, 27 th 2007 - ASL Bergamo.Information week: Who is afraid of generic (bioequivalent) drugs?November 17 th -23 rd 2007- ASL Bergamo.5 a Giornata di studio sulla malattia di AlzheimerI disturbi del comportamento nelle demenze. La gestione dei disturbi del comportamento nelledemenze, March 24, 2007, Ateneo Veneto, Venezia3 ° Corso di formazione e aggiornamento per operatori socio-sanitari:La malattia di Alzheimer e le altre demenz e (10 days)October 1 st – November 5 th , 2007, IRE Venezia, VeneziaRehabilitation, empowerment, recoveryNovember 30 – December 1 st , 2007, Istituto di Ricerche Farmacologiche Mario Negri, Milano4th Conference on Epileptogenesis, Pisa May 23-26 200727th International Epilepsy Congress, Singapore, July 8-12, 200788ANNUAL REPORT 2007

IRFMNGRANTS AND CONTRACTSAbbott GmbH & Co. KGAmgen, MilanoASL 2 Piemonte.Assessorato alla Salute, Comune di MilanoAssociation pour la recherché sur la SLA, FranceBristol-Myers SquibbBoehringer IngelheimCURE EpilepsyDipartimento di Salute Mentale, Azienda Ospedaliera Niguarda Ca’ Granda, MilanoDana FoundationEvidentia Medica, Grottaferrata (Roma)Fondazione Cariplo, MilanoFondazione Mariani, MilanoFondazione Italo Monzino, MilanoFP6, European UnionGlaxo-SmithKline, ItalyHospice "Via di Natale Franco Gallini", Aviano (PN)Human Frontiers Scientific ProgrammeIMPHA II, DG-SANCO, Public Health and Consumers' Protection (Directorate GeneralIstituto Comprensivo Statale "G.D. Romagnosi", Carate Brianza (MI)I.R.I.SIstituto Superiore di SanitàJanssen-CilagH. Lundbeck A/S, DenmarkMinistero della Ricerca ScientificaMinistero della SaluteMND Association, UKNewronOspedale “Casa Sollievo” di San Giovanni RotondoPharmingRegione Lombardia, Assessorato alla Famiglia e Solidarietà Sociale e Assessorato alla Sanità,MilanoRimoldi e BergaminiSanofi-AventisSELECTA MEDICA, PaviaServier Laboratories, ParigiSigma-TauTelethonUnione Nazionale Associazioni per la Salute Mentale – UNASAMVertex89ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Albani D., Batelli, S., Pesaresi, M. Prato, F., Polito L. Forloni G. Pantieri R. A novel PSENEN mutation in a MCIpatient with positive family history for Alzheimer’s Disease. Alzheimer & Dementia 3: 235-238 (2007)Albani, D., Artuso V. Roiter, I. Batelli, S., Prato, F. Pesaresi, M. Galimberti, D. Scarpini, E., Bruni, A., Franceschi,M. Piras MR., Confaloni, A. and Forloni G. Presenilin-1 mutation E318G represents a risk factor for familialAlzheimer’s disease in the Italian population and affects A(1-40) production. Neurobiol Aging, 28:1682-8 (2007)Barbato A, Agnetti G, D'Avanzo B, Frova M, Guerrini A, Tettamanti M. Outcome of a community-basedrehabilitation program for people with mental illness who are considered difficult to treat. J Rehabil Res Devel 44:775-784 (2007)Bartfai T, Sanchez-Alavez M, Andell-Jonsson S, Schultzberg M, Vezzani A, Danielsson E, Conti B. Interleukin-1system in CNS stress: seizures, fever, and neurotrauma. Ann N Y Acad Sci. 1113:173-7 (2007)Baviera M, Invernizzi RW, Carli M. Haloperidol and clozapine have dissociable effects in a model of attentionalperformance deficits induced by blockade of NMDA receptors in the mPFC. Psychopharmacology (Berl) publishedonline. (2007)Beghi. E. Epilepsy. Current Opinion in Neurology 20: 169-174 (2007)Beghi, E., A. Millul, A. Micheli, E Vitelli, G. Logroscino, and SLALOM Group. Incidence of ALS in Lombardy,Italy. Neurology 68: 141-145 (2007)Beghi, E., T. Mennini, C.Bendotti, P. Bigini, G. Logroscino, A.Chiò, O. Hardiman, D. Mitchell, Swingler, B.J.Traynor, A. Al Chalaby. The heterogeneity of amyotrophic lateral sclerosis: a possible explanation of treatmentfailure. Curr Med Chem 14: 3185-3200 (2007)Begley, CE., G.A. Baker, E. Beghi, J. Butler, D. Chisholm, J.T. Langfitt, P. Levy, C. Pachlatko, S. Wiebe, K.L.Donaldson on behalf of the ILAE Commission on Healthcare Policy. Cross-country measures for monitoringepilepsy care. Epilepsia 48: 990-1001 (2007)Biasini E, Medrano AZ, Thellung S, Chiesa R, Harris DA.Multiple biochemical similarities between infectious and non-infectious aggregates of a prion protein carrying anoctapeptide insertion. J Neurochem. 2007 [Epub ahead of print]Bigini P, Repici M, Cantarella G, Fumagalli E, Barbera S, Cagnotto A, De Luigi A, Tonelli R, Bernardini R,Borsello T, Mennini T. Recombinant human TNF-binding protein-1 (rhTBP-1) treatment delays both symptomsprogression and motor neuron loss in the wobbler mouse. Neurobiol Dis. 2007 Nov 12; [Epub ahead of print]Borsello, T. Forloni, G. JNK signaling: a target to prevent neurodegeneration. Current Pharmaceutical Design. 13:875-886 (2007)Borsello T, Centeno C, Riederer IM, Haefliger JA, Riederer BM.Phosphorylation-dependent dimerization and subcellular localization of islet-brain 1/c-Jun N-terminal kinaseinteractingprotein 1. J Neurosci Res. 85: 3632-41 (2007)Burattini C, Burbassi S, Aicardi G, Cervo L.Effects of naltrexone on cocaine- and sucrose-seeking behaviour inresponse to associated stimuli in rats.Int J Neuropsychopharmacol. Epub 2007 Mar 5Burbassi S, Cervo L. Stimulation of serotonin(2C) receptors influences cocaine-seeking behavior in response todrug-associated stimuli in rats.Psychopharmacology (Berl). Epub 2007 Sep 27Caccia, S. N-dealkylation of 1-arylpiperazine derivatives: disposition and metabolism of the formed 1-arylpiperazines.Curr. Drug Met., 8: 612-622 (2007)Calcagno E, Canetta A, Guzzetti S, Cervo L, Invernizzi RW. Strain differences in basal and post-citalopram90ANNUAL REPORT 2007

IRFMNextracellular 5-HT in the mouse medial prefrontal cortex and dorsal hippocampus: relation with tryptophanhydroxylase-2 activity.J Neurochem. 103:1111-20 (2007)Capone C, Fabrizi C, Piovesan P, Principato M C, Marzorati P, Ghirardi O, Fumagalli L, Carminati P, De Simoni MG. 2-Aminotetraline derivative protects from ischemia/reperfusion brain injury with a broad therapeutic window.Neuropsychopharmacology 2007 ; 32 : 1302-1311Capone C, Frigerio S, Fumagalli S, Gelati Maurizio, Principato M C, Storini C, Montinaro M, Kraftsik R, De CurtisM, Parati E, De Simoni M G. Neurosphere-derived cells exert a neuroprotective action by changing the ischemicmicroenvironment. PLoS One 2007; 2(4):e373Cavestro, C., A. Rosatello, G.M. Micca, M. Ravotto, M.P. Marino, G. Asteggiano. E. Beghi. Insulin metabolism saltered in migraineurs: a new pathogenic mechanism for migraine? Headache 47: 1436-1442 (2007)Colombo, A., Repici M., Pesaresi M. Santambrogio S. Forloni G. and Borsello T. The Amyloid Precursor Proteinprocessing is regulated by JNK phosporylation in the cytoplasmic domain: D-JNKI1 inhibits its proteolytic cleavage,Cell Death & Diff. 14: 1845-48 (2007)Cornaggia, CM., Beghi, M. Beghi E. for the REST-1 Group. Psychiatric events in epilepsy. Seizure 16: 586-592(2007)De Girolamo G, Barbato A, Bracco R, Gaddini A, Miglio R, Morosini P, Norcio B, Picardi A, Rossi E, Rucci P,Santone G, Dell’Acqua G. The Characteristics and Activities of Acute Psychiatric Inpatient Facilities: NationalSurvey in Italy. British J. Psych 191:170-177 (2007)Del Bo, R., Ghezzi, S., Scarlato, M., Albani, D. Galimberti, D., Lucca, U., Tettamanti, M., Scarpini, E., Forloni, G.,Bresolin, N, Comi, GP. Role of VEGF gene variability in longevity: A lesson from the Italian population NeurobiolAging, 2007 June 15 [Epub ahead of print]Fenoglio C, Galimberti D, Piccio L, Scalabrini D, Panina P, Buonsanti C, Venturelli E, Lovati C, Forloni G, MarianiC, Bresolin N, Scarpini E. Absence of TREM2 polymorphisms in patients with Alzheimer's disease andFrontotemporal Lobar Degeneration. Neurosci Lett. 411:133-7 (2007)Fioriti, L. Angeretti, N.. Colombo, L., De Luigi A., Manzoni, C., Colombo A., Morbin, M., Tagliavini, F., Salmona,M. Chiesa, R. Forloni, G. Neurotoxic and gliotrophic activity of a synthetic peptide homologous to Gerstmann-Sträussler-Scheinker disease amyloid protein. J. Neurosci. 27: 576-83 (2007)Fossati, R., G. Apolone, E. Negri, A. Compagnoni, C. La Vecchia, S. Mangano, L. Clivio, S. Garattini, for theGeneral Practice Tabacco Cessation Investigators Group. A double-blind, placebo-controlled, randomized triail ofBupropion for smoking cessation in primary care. Arch Intern Medicine 167: 1791-1797 (2007).Furlan R, Bergami A, Brambilla E, Butti E, De Simoni M G, Campagnoli M, Marconi P, Comi G, Martino G. HSV-1-mediated IL-1 receptor antagonist gene therapy ameliorates MOG35-55 induced experimental autoimmuneencephalomyelitis in C57BL/6 mice. Gene Ther 14 : 93-98 (2007)Gallucci M., Amici, GP. Ongaro F. Gajo G.B., De Angeli, S. Forloni G., Albani, D. Prato F. Polito , L. Zanardo Aand Regini C. Associations of the plasma interleukin 6 (IL-6) levels with disability and mortality in the elderly in thetreviso longeva (trelong) study. Arch Geriatr. Geront. 44 Suppl: 193-8. (2007)Gallucci M., Ongaro F. Bresolin F. Bernardi, U. Salvato, C. Minello A., Amici, GP., Barasciutti, E., Mazzucco, S.,Gajo G.B, De Angeli, S. Forloni G., Albani, L. Zanardo A and Regini C. The treviso longeva (trelong) study: abiomedical, demographic, economic and social investigation on people 70 years and over in a typical town of northeastof Italy. Arch Geriat Geront 44 Suppl: 173-92. (2007)Grignaschi G, Zennaro E, Tortarolo M, Calvaresi N, Bendotti C. Erythropoietin does not preserve motor neurons in amouse model of familial ALS. Amyotroph Lateral Scler. 8:31-5, (2007).Invernizzi R Role of TPH-2 in brain function: news from behavioral and pharmacologic studies J Neurosci Res 200785: 3030-303591ANNUAL REPORT 2007

IRFMNInvernizzi R, Pierucci M, Calcagno E, Di Giovanni G, Di Matteo V, Benigno A, Esposito E Selective activation of5HT2C receptors stimulates GABA-ergic function in the rat substantia nigra pars reticulata: a combined in vivoelectrophysiological and neurochemical study Neuroscience 144: 1523-1535 (2007)Li A, Christensen HM, Stewart LR, Roth KA, Chiesa R, Harris DA. Neonatal lethality in transgenic mice expressingprion protein with a deletion of residues 105-125. EMBO J. 26:548-58. (2007)Lo Coco D, Veglianese P, Allievi E, Bendotti C. Distribution and cellular localization of high mobility group boxprotein 1 (HMGB1) in the spinal cord of a transgenic mouse model of ALS. Neurosci Lett. 412(1):73-7 (2007)Logroscino, G.,E. Beghi, O. Hardiman, A. Chiò, J.D. Mitchell, R.J. Swingler, B. Traynor, for EURALS. Effects ofreferral bias on assessing survival in ALS. Neurology 69: 939 (2007)Ludolph AC, Bendotti C, Blaugrund E, Hengerer B, Loffler JP, Martin J, Meininger V, Meyer T, Moussaoui S,Robberecht W, Scott S, Silani V, Van Den Berg LH; ENMC Group for the Establishment of Guidelines for theConduct of Preclinical and Proof of Concept Studies in ALS/MND Models. Guidelines for the preclinical in vivoevaluation of pharmacological active drugs for ALS/MND: report on the 142nd ENMC international workshop.Amyotroph Lateral Scler. 8:217-23 (2007)Massignan T, Casoni F, Basso M, Stefanazzi P, Biasini E, Tortarolo M, Salmona M, Gianazza E, Bendotti C,Bonetto V. Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse.Biochem Biophys Res Commun. 353:719-25 (2007)Noe' F, Nissinen J, Pitkänen A, Gobbi M, Sperk G, During M, Vezzani A. Gene therapy in epilepsy: the focus onNPY. Peptides. 28:377-83 (2007)Parabiaghi A, Lasalvia A, Bonetto C, Cristofalo D, Salvi G, Ruggeri M. Predictors of caregiving burden in relativesof people with schizophrenia. A 3-year follow-up in a community mental health service. Acta Psych Scand Suppl437: 66-76 (2007)Pesaresi M, Batelli S, Prato F, Polito L, Lovati C, Scarpini E, Quadri P, Mariani C, Albani D, Forloni G. Theurokinase-type plasminogen activator polymorphism PLAU_1 is a risk factor for APOE-epsilon4 non-carriers in theItalian Alzheimer's disease population and does not affect the plasma Abeta(1-42) level. Neurobiol Dis. 25: 609-613(2007)Peviani M, Cheroni C, Troglio F, Quarto M, Pelicci G, Bendotti C. Lack of changes in the PI3K/AKT survivalpathway in the spinal cord motor neurons of a mouse model of familial amyotrophic lateral sclerosis. Mol CellNeurosci. 34:592-602 (2007)Renoldi G, Calcagno E, Borsini F, Invernizzi R Stimulation of group I mGlu receptors in the ventrotegmental areaenhances extracellular dopamine in the rat medial prefrontal cortex J Neurochem 100 : 1658-1666 (2007)Repici M, Centeno C, Tomasi S, Forloni G, Bonny C, Vercelli A, Borsello T.Time-course of c-Jun N-terminal kinase activation after cerebral ischemia and effect of D-JNKI1 on c-Jun andcaspase-3 activation. Neuroscience.150: 40-9 (2007)Ruggeri M, Salvi G, Bonetto C, Lasalvia A, Allevi L, Parabiaghi A, Bertani M, Tansella M. Outcome of patientsdropping out from community-based mental health care: a 6-year multiwave follow-up study. Acta Psych ScandSuppl 437:42-52 (2007).Savica, R., E. Beghi, G. Mmazzaglia, F. Innocenti, O. Brignoli, C. Cricelli, A.P. Caputi, R. Musolino, E. Spina, G.Trifirò. Prescribing patterns of antiepileptic drugs in Italy: a natiowide population-based study in the years 2000-2005. Eur J Neurology 14: 1317-1321 (2007)Sau D, De Biasi S, Vitellaro-Zuccarello L, Riso P, Guarnieri S, Porrini M, Simeoni S, Crippa V, Onesto E, PalazzoloI, Rusmini P, Bolzoni E, Bendotti C, Poletti A. Mutation of SOD1 in ALS: a gain of a loss of function. Hum MolGenet. 16: 1604-18 (2007)Vezzani A.On Demand Up-regulation of Therapeutic Genes in the Brain: Fiction or Reality?Epilepsy Curr. ;7:88-90. (2007)Vezzani A, Baram TZ.New roles for interleukin-1 Beta in the mechanisms of epilepsy.92ANNUAL REPORT 2007

IRFMNEpilepsy Curr.7:45-50 (2007)Zoccolella, S., E. Beghi, G. Palagano, A. Fraddosio, V. Guerra, V. Samarelli, V. Lepore, I.L. Simone, P. Lamberti,L.Serlenga and G. Logroscino for the SLAP registry. Riluzole and amyotrophic lateral sclerosis survival: apopulation-based study in southern Italy. Eur J Neurol. 14: 262-268 (2007)Zoccolella, S., E. Beghi, G. Palagano, A. Fraddosio, V. Guerra, V. Lepore, I.L. Simone, P. Lamberti, L. Serlenga, G.Logroscino. ALS multidisciplinary clinic and survival: results from a population-based study in Southern Italy. JNeurol 254: 1107-1112 (2007)..LAY PRESS SELECTION PUBLISHED IN 2007Albani D, Polito L, Vittori A, Forloni GFattori di rischio genetici per le demenze e screening per la loro indentificazione pre-clinicaRicerca & Pratica 2007 ; n.137 : 204-211Barbato A, D’Avanzo B, Ferrannini L, Parabiaghi A, Vaggi M. Un’occasione per la ricerca clinica in Italia. Lo studioGISAS su aripipr, olanz e aloper nel trattamento dei disturbi schizofrenici, Psichiatria di Comunità, in press.Cerzani M, Pasina L, Clavenna A, Nobili A, Garattini LRevisione critica degli studi italiani di farmacoeconomia sull'uso dei farmaci antinfiammatori non steroidei inmedicina generaleQuaderni Farmacoeconomia 2007 ; 3 : 17-28D’Avanzo B. L’empowerment come orizzonte di lavoro con il disagio psichico. Animazione Sociale 2007; 10:55-61.Dell’Acqua G, Norcio B, de Girolamo G, Barbato A, Bracco R, Gaddini A, Miglio R, Morosini P, Picardi A, Rossi E,Rucci P, Santone G. Caratteristiche e attività delle strutture di ricovero per pazienti psichiatrici acuti: i risultatidell’indagine nazionale ‘Progres-Acuti’, Giornale Italiano di Psicopatologia 2007; 13:26-39.Clavenna A, Bonati M, Campi R, Labate L, Nobili A, Pasina L, Tettamanti M, Monesi L, Marzona I, Roncaglioni MC, Bortolotti A, Fortino I, Locateli G W, Giuliani GLa prescrizione di farmaci per i bambini e gli anziani nella ASL di LeccoRicerca & Pratica 2007 ; n.135 : 100-112La Vecchia C, Beghi E Epidemiologia delle demenzeLa demenza in Italia. Aggiornamento e casi clinici interattivi UTET, Torino, 2007; 20-24Nobili A, Garattini S La CaffeinaAggiornamento Medico 2007 ; 31 : 30-33Nobili A, Pasina L, Garattini S Il paziente allergicoAggiornamento Medico 2007 ; 31 : 98-102Nobili A, Pasina L, Garattini S. Il paziente con emicrania.Aggiornamento Medico 2007; 31: 168-172.Nobili A, Pasina L, Garattini S Il paziente con dolore cronico: gli analgesici oppioidiAggiornamento Medico 2007 ; 31 : 238-244Nobili A, Pasina L, Garattini S. I fluorochinoloni. Aggiornamento Medico 2007; 31: 312-315.Nobili A, Piana I, Balossi L, Tettamanti M, Trevisan S, Lucca U, Tarantola MIl malato di demenza in RSA: risultati di una valutazione comparativa tra i pazienti degenti nei Nuclei Alzheimer ein RSA senza Nuclei AlzheimerIn-formazione Alzheimer. Alla ricerca di nuove connessioni nella rete lombarda dei servizi alle demenzeFranco Angeli, Milano, 2007; 109-126Sterzi R, Toso V, Guidetti D, Provinciali L, Consoli D, Leone M A, Beghi EIndagine epidemiologica italiana su "La neurologia nell'emergenza-urgenza": il Progetto NEUNeurol Sci 2007 ; 28 : S1-S3693ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Biology of Neurodegenerative DisordersAlzheimer's disease: genetic studies and clinical investigationsIn collaboration with different neurological centers and the laboratory of GeriatricNeuropsychiatry it has been created a bank of blood samples for DNA of patients withAlzheimer’s disease (AD), in familial (FAD) or sporadic form (SAD), and patients withvascular dementia (VD). In all subjects the diagnosis of dementia is performed according to theinternational guidelines. Since 2005 we started also the collection of blood samples fromsubjects with fronto-temporal dementia. The genetic studies are aimed to the identification ofcausal factors in FAD and risk factors in SAD. Mutations on genes encoding proteins involvedin the physiopathology of AD were investigated. The pathogenic role of these mutations isunder investigation using fibroblasts obtained from skin biopsy. Furthermore, we continued thescreening of FAD samples for the genes encoding for presenilin 1 and 2 (PS-1 and PS-2) andAPP, missense mutations in these three genes were associated with AD.Based on the evidence obtained in experimental studies, in collaboration with the GeriatricNeuropsychiatry lab and the Inflammation and CNS diseases lab, we elaborated a protocol for aclinical trial to investigate the effects low molecular weight heparin on the CSF and plasma βamyloid levels in Alzheimer patientsAlzheimer's disease: preclinical studiesThe formation of β amyloid (Aβ) deposits in brain parenchyma and on the wall of cerebralblood vessels is an early event in AD and there are now numerous genetic, biochemical andneuropathological studies pointing to a causal role of Aβ in the pathogenesis of AD. Thus,prevention the formation of Aβ aggregates or their elimination once formed is a potentialtherapeutic approach to the disease. This aim is strongly persecuted with different strategiesincluding the regulation of enzymes responsible of the synthesis and degradation of Aβ and theenzymes influencing the metabolism of amyloid precursor protein (APP). In the lab, wedeveloped the idea to interfere directly with the Aβ deposits formation using anti-amyloidogenicdrugs. The experimental studies have shown the potential therapeutic activity of these drugs inAD, and now they will be tested in a clinical setting. In collaboration with the Department ofBiochemistry and Molecular Pharmacology, we tested new molecules that can bind the amyloidaggregates to identify either anti-dementia drugs or potential diagnostic markers. Furthermore,using in vitro and in vivo models, we are evaluating new approaches to reduce the beta amyloidproduction either by affecting directly on beta secretase or through the modulation of JNKpathway, Furthermore, the modulation of this pathway has been considered for the potentialneuroprotective activity in AD and other neurodegenerative disorders.Genetics of agingIn collaboration with Geriatric Neuropsychiatry Lab for the Monzino 80-plus study and with dr.Maurizio Gallucci from the ARGel Association in Treviso for Trelong study we collected alarge number of blood samples from subjects over seventy. In these samples we are performinga genetic analysis to identify genetic profiles associate to the longevity and /or to the agingassociatedpathologies with specific attention to the dementias. The aim is to cross thegenotype/phenotype profile with pathologies and environmental aspects including style of life,diet and economical conditions to identify risks and protective factors. Initially the subjectswere genotypized for ApoE, whom allele E4 is a well-known risk factor for Alzheimer’s diseaseand several other disorders and sirt-1 a gene codified for protein member of a enzymatic family94ANNUAL REPORT 2007

IRFMNof sirtuins associated to the longevity in several experimental models. The results are interestingbut before drawing any conclusion we need to consider the numerous other parameters collectedin our database.Prion's disease: in vitro studiesPrion’s diseases (TSE) are neurodegenerative disorders of sporadic inherited origin but alsotransmissible, they have different clinic and neuropathological features but all are characterizedby the cerebral accumulation of an altered form of prion protein (PrPsc). TSE are rare diseasesbut the transmission from bovine (BSE) to humans induced a public health alarm in UK andsuccessively in all Europe. PrPsc is involved in the pathogenesis of the disease and it is also anessential component of the infective agent. In the lab numerous projects were developed tounderstand the association between the presence of PrPsc and the neurodegenerative process.The biological effects of peptides homologous to large fragment of PrP were investigated. Inparticular PrP 82-146, synthesized in the Protein Chemistry and Biochemistry lab, homologousto the fragment found in the cerebral deposits in TSE patients, is neurotoxic and spontaneouslystructured in beta sheet conformation. The pathogenetic role of PrP was investigated not onlythrough the external application of peptides but also by the evaluation of intracellularmechanisms potentially involved in the formation of PrP aggregates.Prion’s diseases: studies in vivoThe lab has the facilities to study the experimental scrapie, mice and hamsters are inoculatedwith infected brain homogenate. The hamsters after intracerebral inoculation develop thedisease in 60-70 days and died within a month. The histopathological analysis of the brain showthe presence of PrPsc deposits, neuronal damage, diffuse astrogliosis and the typical spongiosisat cortical and thalamic level. The anti-amyloidogenic activity of tetracyclines has beeninvestigated also in this experimental contest. After the ex-vivo approach, where thehomogenate was treated before the inoculation, the curative effect of tetracyclines was tested incollaboration with the lab of Chemistry and Biochemistry of Proteins by treatment theexperimental scrapie in hamsters with intramuscular doxycycline, the treatment prolonged thesurvival of the animals. Other drugs are now under investigation to verify the curative effects inTSE.Other animal models of TSE are available in the lab, transgenic mouse developed by dr. Chiesa.These mice express the mutated forms of PrP associated to the familial TSE. The homozygotesexhibit at different level of severity the symptoms reminiscent of the pathologies associatedwith the mutations. From the neuropathological point of view some lines exhibit anaccumulation of PrP and clear neurodegeneration of the cerebellar granular cells, in the otherbrain regions no evident alterations were found. In the brain tissue of the mice was found a PrPwith some of the characteristic of PrPsc, however the brain material is not infected. Thepathogenesis of TSE is investigated in these models through different approaches including theproteomic approach and the electronic microscopy.Parkinson’s Disease: genetic studiesParkinson’s disease (PD) is the second more diffuse neurodegenerative disorder with anunknown pathogenesis, however for PD several therapies are available and, although at thesymptomatic level, their efficacies is well-established. In the etiological studies on PD thegenetic component has been traditionally considered with scarce interest whereas theenvironmental causes were carefully evaluated. This orientation was based on the evidence thatthe exposure to several toxins can mimic the PD pathology. However the genetic studies in thelast few years have completely changed the perspective with the identification of mutations ontwo genes, encoding for alpha-synuclein and parkin, associated to the juvenile forms of thedisease. A mutation on alpha synuclein gene is an event extremely rare, only three mutations95ANNUAL REPORT 2007

IRFMNidentified until now, the parkin mutations are numerous ether in puntiform or in deletion form.The mutations on alpha-synuclein gene are dominant while the parkin mutations are associatedwith PD in recessive form. We collected, in collaboration with several neurological centers,blood samples from PD subjects and the screening of the samples involved genes like alphasynuclein,parkin, DJ-1 and other factors potentially involved in PD.Parkinson’s disease: studies in vitroThe identification of the mutations associated to Parkinson’s disease (PD) gave a substantialcontribute to understand the disease and allowed the develop of cellular models to investigatethe pathogenesis of the disease. In past we showed the potential neurotoxic activity of alphasinucleinusing the synthetic peptide homologous to the fibrillogenic fragment 61-95 (NAC) ofthe protein. Successively with help of dr. Negro at the Department of Biochemistry at theUniversity of Padova we prepared cDNA vectors including the sequence of wild type andmutated alpha-synuclein Their transfection to the PC12 cells induced in specific conditions acellular damage. More recently alpha-synuclein was associated to a TAT sequence capable totransport inside the cells the protein. With this method the intracellular concentration of alphasinucleinwas better controlled. In a micromolar range alpha-synuclein was toxic, but innanomolar range, it exerted neuroprotective effect against oxidative stress induced by hydrogenperoxide. This double effect dose-dependent was confirmed in an “inducible” model. Morerecently again in collaboration with Dr. Negro, we obtained the recombinant form of DJ-1associated with TAT (TAT-DJ-1). This protein is similar to alpha-synuclein, mutations of itssequence has been associated to PD. TAT-DJ-1 silencing by small interference RNA (siRNAi)were used to study the interaction between DJ-1 and alpha synuclein..Laboratory of Neurological DisordersEpidemiological studies on amyotrophic lateral sclerosis (ALS)Included are studies on the incidence, risk factors and mortality of ALS. The data are obtainedfrom a regional registry of the disease activated in 1998 and including all patients with newlydiagnosed ALS identified in eight provinces of Lombardy. Using similar study protocols, thesame data are collected in two additional regional registries (from Piemonte and Puglia)included in a network with the Lombard registry. Information obtained from patients enrolled inthe Lombard registry and from cases examined by members of the Italian ALS Study Group hasbeen used to assess the validity and reliability of diagnostic criteria for ALS and selecteddisability scales. Based on the data recorded, the annual incidence of ALS is comparable to thatobtained in other Western countries where ALS registries have been activated, and is among thehighest ever published (1.9 per 100,000). Mortality of ALS has been found to be comparable tothat of studies from similar populations studied with the same protocol. The study on thevalidation of the current diagnostic criteria for ALS (the El Escorial criteria) showed that to beconsidered valid and reliable, the criteria should be used after proper training of theinvestigators.In October 2004, the Laboratory of Neurological Disorders has started a European collaborativegroup for the ALS registries (EURALS) with the intent to create a common database(completed in the year 2005) with the participation of the existing regional and national diseaseregistries. Three major scientific activities are in course: 1. A comparative study of the clinicalcharacteristics of patients with ALS enrolled in the registries as compared to those seen insecondary and tertiary health care facilities; 2. A meta-analysis of the incidence of ALS,performed by pooling data from the 1998-99 cohorts of patients enrolled in the populationbasedregistries; 3. A case-control study on trauma and risk of ALS; 4. A case-control study onALS, physical exercise and sport. Study # 3 is ongoing only in Italy. In 2007 a scientific report96ANNUAL REPORT 2007

IRFMNwas completed (now in press) on the progression of ALS (marked by loss of ambulation,percutaneous gastrostomy and non-invasive assisted ventilation).Innovative therapeutic strategies in patients with epilepsyA cohort of patients with a first unprovoked seizure, randomised since 1988 by several Italiancenters to immediate treatment or to treatment only at the time of a seizure relapse, wasfollowed to verify the impact of the two therapeutic strategies on the long-term prognosis ofepilepsy, measured by the chance of achieving 5-year remission.To provide a pragmatic definition of drug resistance in childhood epilepsy, children refractoryto two antiepileptic drugs (in sequence or in combination) were randomised to the use of a thirddrug or to the optimization of the existing treatment and followed for up to three years.Therapeutic response was measured by the achievement of a six-month period of remission. Thestudy has been conducted in collaboration with the IRCCS “Stella Maris” of Calambrone (PI).Epidemiology of neurological disorders in AlbaniaWith the collaboration of the Fondazione Mariani and the Neurological Department of theUniversity of Tirana, an epidemiological survey has been started to assess the prevalence andincidence of several neurological conditions (stroke, epilepsy, headache, dementia, peripheralneuropathy, multiple sclerosis) comparing an urban and a rural community (Tirana andSaranda). A study on the validation of the diagnostic criteria is in course.Quality of life in children with neuromuscular disordersWith a financial support of the Telethon organization, a study has been completed on thevalidation of a questionnaire on the quality of life in children and adolescents with differentneuromuscular disorders. This is an Italian multicenter study coordinated to the ChildNeurology Clinic of Pavia.Cerebrovascular disorders and risk of epilepsyEpilepsy is a frequent complication of stroke. Acute symptomatic seizures (i.e. seizuresoccurring in the seven days after stroke) can occur in up to two-thirds of cases and epilepsy (i.e.repeated unprovoked seizures) in 2-4%. There are no consistent findings on the risk factors foracute symptomatic seizures, unprovoked seizures and epilepsy in patients with stroke. For thesereasons, in 2007 a multicenter national prospective survey has been started to assess the risk ofseizures and epilepsy (and the main risk factors) in a cohort of patients with a first ischemic orhemorrhagic stroke followed for a maximum period of 24 months. The study was alsoimplemented to assess the feasibility of a pragmatic therapeutic trial on the prophylaxis ofseizures and epilepsy in stroke.Diagnosis and prevalence of dystoniaAs part of a collaboration with the institute “San Raffaele” of Milano, a study has beencompleted on the prevalence of adult focal dystonia in the province of Foggia. Cases areascertained through different sources (hospital admissions, neurology, ENT, ophthalmology,and ortopedics outpatient visits) in the two major local hospitals. A total of 41 women (59%)and 28 men (41%) with a mean age of 58 years (range 23-87) were identified as having aprimary focal or segmental dystonia. The crude prevalence rate was 12.6 per 100,000 (95% CI9.8-16.0) The age-adjusted prevalence rate was 13.5 (95% CI 10.3-16.7). Blepharospasm wasthe commonest variety. The prevalence of the disease increases up to 27.8 at age 55-74 yearsand declines thereafter.97ANNUAL REPORT 2007

IRFMNTherapeutic trials in neurological disordersDuring the year 2007 three therapeutic trials sponsored by the Italian Drug Agency (AIFA) werestarted or continued. Included are: 1. A randomized double-blind parallel-group placebocontrolledtrial on the efficacy and tolerability of L-acetylcarnitine in ALS; 2. A randomizedopen-label parallel-group trial comparing Erythropoietine to Metyl-prednisolone in patients withacute spinal cord injury; 3. A randomized double-blind parallel-group placebo-controlled trialon the efficacy and safety of valproate in medication-overuse headache. The first trial aims atfinding a potentially effective drug in a clinical condition for which there is only one product(Riluzole) with at best modest efficacy on survival. L-acetylcarnitine has been found to improvesurvival in experimental models of motor neuron disease. The second trial intends to verify theefficacy of erythropoietin, a drug shown to mitigate the effects of traumatic spinal shock andaccelerate recovery in experimental animals. The drug chosen for comparison(Methylprednisolone at high doses) has been selected for being the present gold standard inclinical practice. The third trial aims at verifying whether valproate (a drug commonly used forthe prophylaxis of migraine) abates symptoms occurring in drug-overuse headache, a commonand frequently invalidating variety of chronic idiopathic headache.All three trials are multicenter and nationwide. The laboratory of neurological disorders is thecoordinator of the first trial and a partner in the other two trials, where the main tasks includeprotocol and CRF preparation, statistical analysis, and preparation of the final scientific report..Laboratory of Drug Metabolism1-Aryl-piperazine as active metabolites of centrally acting drugDichloro(substituted) phenylpiperazine has been incorporated in the structure of drugs andpharmacologically active compounds which, in analogy with structurally related compounds, mayundergo CYP3A4-mediated N-dealkylation (oxidative N-C cleavage) resulting in the formation ofphenylpiperazine derivatives. However, there was no information on how these metabolites contributeto the pharmacological activity of their parent compound(s), which include psychotropic drugs andexperimental compounds currently under pre-clinical evaluation. We therefore developed a reliablehigh-performance liquid chromatography-electrospray ionization mass spectrometric method for thedetection of dichlorophenylpiperazine isomers and their parent compound(s) in body fluids and braintissue. This enabled us to confirm that the metabolism of these phenylpiperazine derivatives includesN-dealkylation of the piperazinyl nitrogen to 2,3-, 2,4- or 2,5-dichlorophenyl-piperazine, although withdifferences in the rate and extent of the process. As phenylpiperazines concentrate in the brain, causinga variety of serotonin receptor-related pharmacological effects, we also examined their concentrationsin brain of rodents given the various phenylpiperazine derivatives, and the metabolite-to-parent drugratios at steady state. The metabolites had higher brain uptake than the parent compound(s), so themetabolite-to-parent drug ratio was higher in brain than in blood. For the recently introducedantipsychotic aripiprazole the concentrations of the metabolite 2,3-dichlorophenylpiperazine averagedonly 0.08 in serum but about 0.4 in whole brain (on a molar basis) at approximately the time of thearipiprazole maximum concentration in the rat.Obviously, these measurements cannot be made in humans but assuming that the compoundsconcentrate in human brain to the same extent as in the rat, at the site of action 2,3-dichlorophenylpiperazine should account for only a small percentage of aripiprazole. It is thereforeconceivable that this metabolite contributes to the central effects of aripiprazole in rats, but it should beof less clinical significance for human patients, although its pharmacological profile is still largelyunknown and there may be physiological and pathological factors that raise the metabolite-to-parentdrug ratio at the site of action.98ANNUAL REPORT 2007

IRFMNPharmacological role of the constituents of Hypericum perforatumextractsThe chemical composition of extracts of hypericum perforatum L. (St. John’s wort) is essentiallyknown but is still not clear which constituent(s) account, wholly or in part, for the antidepressantactivity of the extracts, and through what neurochemical mechanism(s). The phloroglucinol hyperforinshares most of the in vitro and in vivo pharmacological properties of the extracts, and is possibly amain “antidepressant” component, but there is also evidence for other pharmacologically activecomponents. However, identifying the roles of the various derivatives and the mechanism(s) of theiractivity is complicated by the scarcity of information about their ability to cross the blood-brainbarrier and the concentrations reached in brain after administration of the extracts. This is also true forthe biflavone biapigenin and particularly its I3’,II8 analog amentoflavone which, although present insmaller amounts in extracts, shows a multitude of pharmacological actions in vitro and in vivo inanimal models. The lack of pharmacokinetic data in man and animals and questions about the brainuptake of amentoflavone and biapigenin prompted us to examine their brain uptake and concentrationsand the relationships with plasma concentrations after pharmacologically effective doses in mice.After doses of Hypericum perforatum extracts the brain concentrations of biapigenin andamentoflavone were below the limit of quantification. The same was true for amentoflavone after abiflavone-enriched extract of Ginkgo biloba. Levels were consistently detected only afterintraperitoneal biapigenin or amentoflavone but were low and mostly related to the residual biflavonein the circulation. Poor brain-to-blood permeability is common to other polar components ofHypericum perforatum, resulting in brain concentrations generally too low for any direct interactionwith neurotransmitter transporters and receptors which are obviously important for the action ofconventional antidepressants. Likewise, the in vitro interactions of biapigenin and amentoflavone withknown central mechanisms are apparently not relevant for the in vivo effects of the extracts becausethey occur at biflavone concentrations far exceeding those found in the brain after pharmacologicallyeffective doses. However, this does not exclude that tissues other than brain may concentratebiapigenin or amentoflavone sufficiently to exert beneficial effects after daily intake of the extracts.Resistence to antidepressant drugs: studies in animal modelsThe selective serotonin reuptake inhibitors are the drugs of choice in the treatment of depression.However, they are not or only partially effective in a fraction of depressed patients. The reasons aresubstantially unknown, though pharmacogenetic studies have linked the response to serotonin reuptakeinhibitors to polymorphisms in various genes coding for serotonin mechanisms, particularly thepromoter of the serotonin transporter molecule. These studies are therefore aimed to investigate theneurobiological mechanism(s) of resistance to antidepressant drugs in strains of mice carryingdifferent isoforms of tryptophan hydroxylase-2, the enzyme responsible for the synthesis of brainserotonin.These studies are conducted in collaboration with the laboratory of Experimental Psycopharmacology(L. Cervo) and the laboratory of Neurochemistry and Behaviour (R.W. Invernizzi), who will provide abrief description of recent results with the potent serotonin reuptake inhibitors citalopram andparoxetine.99ANNUAL REPORT 2007

IRFMNLaboratory of Experimental NeurologyRole of inflammatory molecules in ictogenesis and epileptogenesisWe are studying the role of IL-1beta and TNF-alpha systems in the genesis and propagation ofseizures and in the associated neurodegenerative phenomena. We have demonstrated thatepileptic activity induces the synthesis of these cytokines and related molecules involved ininflammatory processes. IL-1beta has proconvulsant actions while its naturally occurringantagonist (IL-1Ra) and TNF-alpha have anticonvulsant activities. We are now evaluating therole of these molecules in seizure-associated neuronal damage in mature brain and duringpostnatal development. We are studying pharmacological approaches to block IL-1betasignalinginvolved in the proconvulsant effects of this cytokine. Research activity 2:Mechanisms of drug resistance in epilepsy: This study is aimed at investigating whethermultidrug resistance in epilepsy is dependent on the activity of membrane bound glycoproteinsable the extrude antiepiletic drugs from the brain tissue back into the blood stream. Theseproteins (in particular those produced by the MDR or MPR genes) appear to have a significantrole in determining the resistance to some anticancer agents. Our studies indicate that the MDRgene product, P-glycoprotein or P-gp, is increased in specific brain regions during epilepticactivity. The increase in protein determines a decreased brain concentration of phenytoin andtransgenic mice lacking this protein accumulate higher concentration of this antiepileptic drug intheir brain. Since an increased production of this protein occurs in the brain of human epileptics,it is possible that it contributes to drug refractoriness by impairing the attainment of clinicalrelevant concentrations of these drugs in the epileptic tissue.We recently found that the inhibition of the activity of P-gp in experimental models of epilepsyusing specific blockers of its function, enhances the efficacy of anticonvulsant treatments. Wehave also demonstrated a role P-gp in brain drug disposition in experimental models of focalcortical dysplasia, a clinical condition associated with drug-refractory seizures.Mechanisms of drug resistance in epilepsyThis study is aimed at investigating whether multidrug resistance in epilepsy is dependent on theactivity of membrane bound glycoproteins able the extrude antiepiletic drugs from the braintissue back into the blood stream. These proteins (in particular those produced by the MDR orMPR genes) appear to have a significant role in determining the resistance to some anticanceragents. Our studies indicate that the MDR gene product, P-glycoprotein or P-gp, is increased inspecific brain regions during epileptic activity. The increase in protein determines a decreasedbrain concentration of phenytoin and transgenic mice lacking this protein accumulate higherconcentration of this antiepileptic drug in their brain. Since an increased production of thisprotein occurs in the brain of human epileptics, it is possible that it contributes to drugrefractoriness by impairing the attainment of clinical relevant concentrations of these drugs inthe epileptic tissue.We recently found that the inhibition of the activity of P-gp in experimental models of epilepsyusing specific blockers of its function, enhances the efficacy of anticonvulsant treatments. Wehave also demonstrated a role P-gp in brain drug disposition in experimental models of focalcortical dysplasia, a clinical condition associated with drug-refractory seizures.New therapeutic approaches of In vivo gene transferThis study concerns the use of adeno-associated viral vectors to introduce genes with therapeuticpotential in the brain, thus increasing the synthesis of specific proteins to produce long-lastinganticonvulsant effects. We have demonstrated that adeno-associated viral vectors carrying thehuman neuropeptide Y gene under the control of a neuronal promoter, significantly increase thebrain concentration of this peptide after intrahippocampal injection for a prolonged time (up to 3months after a single intracerebral injection). The rats overexpressing this peptide are less100ANNUAL REPORT 2007

IRFMNsusceptible to limbic seizures and to epileptogenesis. Future development of this study concernsthe optimization of the transgene transfer technology to inhibit spontaneously recurring seizuresand envisaging a possible clinical application.Laboratory of Geriatric NeuropsychiatrySurvey on the health status of old people living in the rural community ofMoltrasioData on the recruited subjects (95% of the target population) were cleaned and analyzedtogether with the primary care physicians. A full report and a short one dedicated to alarger public were written. The availability of the information to the local administratorsenabled them to identify which interventions were most requested/needed and to begin toimplement them. The results were also the subject of a conference specifically held for thepopulation which was the target of survey.Population study on the prevalence of dementias in the older-oldParallel to the progressive increase of individuals aged 80 years or older within the elderlypopulation (65+), the number of demented patients of 80 years or older makes up an everincreasing fraction of the total population affected by dementia. As very often happens,the exclusion from studies of subjects in the oldest age classes tends to inevitablyunderestimate the total number of individuals affected by dementia present in thepopulation. To fill this gap, a door-to-door population study on the prevalence, incidence,risk factors and evolution of dementias and age-associated cognitive deficits has been setup in an elderly population aged 80 years or older living in eight small towns of VareseProvince. The study is funded by a grant from the Fondazione Italo Monzino, Milano.Effects of anemia in the elderlyA previous large survey in old resident of Biella (65-84 years old) has been conducted incollaboration with the Local Health Authority of Biella (ASL 12) to determine theprevalence of anemia. We have now extended the investigation to the oldest old residents(85 years or older; n= 1.775) in order to estimate the prevalence and impact of mildanemia also in this segment of the elderly population.Evaluating risk profiles in hospitalised elderly subjectsIn collaboration with the Geriatric Division of the Beata Vergine Hospital, Mendrisio,Switzerland, hospitalized and ambulatory patients are evaluated from aneuropsychological, functional and mobility point of view in order to estimate the impactof these factors on heath-related outcomes and disease progression.Longitudinal follow-up of individuals with mild cognitive impairment (MCI)In collaboration with the Geriatric Unit of the Beata Vergine Hospital, Mendrisio,Switzerland, follow-up study to estimate the rate of conversion to dementia of all MildCognitive Impairment or Questionable Dementia (CDR 0.5) patients seen at the MemoryClinic of the Hospital.Quality of care of terminally ill oncological subjectsIn 1999 we started a collaborative programme with the hospice “via di Natale FrancoGallini” in Aviano (PN). The aim of the research project was to assess the quality of caregiven in hospice to terminally ill oncological patients at the end of life. Two studies havebeen planned, one retrospective and one prospective. Present aim of the study is the101ANNUAL REPORT 2007

IRFMNassessment of the hospice activities after its opening and to provide continuous training tonurses on use of databank.Randomised controlled trial of the Italian Group for the Study of the SecondGeneration Antipsychotics (GISAS)The study compares three antipsychotics, aloperidole, olanzapine, aripiprazole, accordingto efficacy and tolerability through a pragmatic experimental design on 800 patients,identified and randomized in 80 centres (10 patients per centre with about 120000inhabitants catchment areas). The study is the first one conducted on such large numbersin Italy on this topic. Tools and procedures were defined and procedures for the EthicalCommittees approval started. The study was presented in its scientific background inseveral occasions, and recruitment of centres was conducted, mainly in Northern Italy. Atend of 2007, 38 centres were recruited, some of which are already active, some waitingfor the ethical committee’s approval, some organizing the setting for the recruitment andrandomization phases.SuicidesWe are working on two sides: one concerns temporal trends analyses in the Italianpopulation, currently with particular attention to suicides in adolescents and genderdifferences; the second one concerns the definition of a multicenter study on attemptedsuicides aiming at improving identification, first contact, registration, quantification at theemergency services and referral to adequate services. A network of participating servicesis in course of definition. At present, the study consists of the administration of astructured questionnaire to the professionals responsible of the emergency services(currently tested in three services), extraction and analysis of information from the clinicalrecords of cases of attempted suicide identified in the emergency services (one service),comparison of data from clinical records and those from the informative system whereavailable, adoption of specific codes to be added into the informative system whereavailable, and a follow-up of cases seen at the emergency services.The ProgresAcuti Project of care in psychiatric acute servicesAnalysis of data from the second phase of the descriptive study of acute psychiatricservices from all Italy. In the second phase, a cluster randomization selected a subsampleof 64 services. An analysis was conducted on outcomes of patients with short admissionsin these services.Innovative Regional Program. The Empowerment ProjectThe Department of Mental Health of the Niguarda Hospital in Milan continues theactivity to spread and introduce empowerment values and practices in all the psychiatriccommunity services of Milan in the perspective to encourage real opportunities for users’autonomy and group identity. Tools to investigate expectations and motivations of theprofessionals involved in the project were designed and applied, and other tools weredesign for self-evaluation of the professionals in their activity of sensibilisation and toevaluate quality usefulness of this activity according to the targeted service professionals.We are conducting an observational prospective evaluation of the project Natural SocialNetworks (patients are accompanied by friends, acquaintances, neighbors, or colleaguesfor emotional support or support in various activities); we have started the observationalprospective evaluation of patients admitted to housing facilities in Milan.102ANNUAL REPORT 2007

IRFMNThe revolving door effect in psychiatric residential facilities: monitoring theproblem and identifying strategiesThe second and third phases of the project are going on: we evaluate follow-up pathwaysof care and clinical and functional outcomes of patients of the psychiatric residentialfacilities of the community service networks of several areas (for a total catchment area of500,000 inhabitants). A total of 97 patients were recruited in the cohort of the dischargepatients (discharged patients are followed up since their discharge from a communityresidential facility, with concern to their pathway of care in the community), and 85 in thecohort of the admitted patients (patients are followed since their admission to psychiatricresidential facilities with concern to their care pathways in the facility, and, if dischargedin the two years of prospective observation, to their pathway of care in the community),and evaluation tools are administered every six months. The aim is to investigateoutcomes of patients during and after residential treatment, follow the care projects andpathways, identify barriers to discharge, and, for those discharged, assess quality ofcommunity tenure and subsequent admissions to residential facilities or acute services.Self-accreditation of quality of the housing facilities in the Health Trusts 1and 2 of TurinGroups composed by users of the housing facilities, family members, professionalsworking in the same housing facilities and professional working in the communityservices discuss and the key features of the housing facilities according to their ownpoints of view. From this material, a group of professionals develops a scheme forattribution of scores to the housing facilities. The scores will be periodically attributed tothe housing facilities by the multidisciplinary groups. We assist and train the groups in thewhole process.Satisfaction of users of mental health services of Milan who receive supportfrom the Social Fund provided by the City of MilanThe investigation was commissioned by the City of Milan, which provides the communitypsychiatric services with a fund to be used for patients considered in need, and aimedeither at describing the main features of the patients assisted through the fund, and toestimate their satisfaction with it. Of the 18 community service networks of Milan, 8provided data for more than 70% of the subjects supported by the social fund, and 4 forless than 50%. We evaluated a total of 633 patients with reference to satisfaction, and 627of them with reference to their general and social features.UNASAM Project – Quality improvement and evaluation in mental healthwith the active participation of the associationsDuring the first semester a questionnaire investigating family members’ satisfaction withpsychiatric services, professionals and interventions was designed and discussed withfamily members from four Italian regions in various focus groups. Groups of familymembers were also trained about basic concepts and procedures in service evaluation andconduction of the investigation. A total of 100 family members administered thequestionnaire in 41 community mental health services of four regions, and 1450questionnaires at end of 2007 had been collected.103ANNUAL REPORT 2007

IRFMNLaboratory of Inflammation and Nervous System DiseasesRole of selected aspects of the inflammatory responsein ischemia/reperfusion injuryPrevious studies of ours have indicated that complement and related inflammatory systems suchas contact/kinin and fibrinolytic systems may represent novel targets for reducingischemia/reperfusion injury. We have shown that C1-INH, an endogenous serine-proteaseinhibitor that acts as a major regulator of both complement and kinin systems, markedlyimproves neurological deficits and reduces infarct volume in mice with focal transient as well aspermanent ischemia induced by middle cerebral artery occlusion. We have further extended thisfinding defining its effectivness on different strains of mice (displaying different levels ofcomplement expression), the time-window and the dose-response curves. Since C1-INH mayact on different substrates, we have evaluated the specific involvement of the differentcomplement pathways and of the other inflammatory systems, ie kinin and coagulation systems.To explore the mechanisms of C1-INH neuroprotection, we have also investigated theexpression (protein and mRNA) of inflammatory cytokines, adhesion molecules, NO synthaseisoforms, apoptosis markers.The results obtained show that: i) C1-INH effectively and markedly reduces brainischemia/reperfusion injury, inducing a decrease of the 90% of the ischemic lesion; ii) C1-INHactions lead to inhibition of cell recruitment, inflammation and apoptosis; iii) C1-INHneuroprotection is at least partially independent from the complement classical pathway andinhibition of other complement pathways, of other inflammatory systems such as thecontact/kinin system, or of thrombotic events associated with ischemia-reperfusion injury maybe involved in its powerful protective action (De Simoni et al. 2003; De Simoni et al. 2004;Storini et al. 2005; Storini et al. 2006).Thus C1-INH, which is presently used as replacement therapy in patients with C1-INHdeficiency, represent a novel promising strategy for stroke therapy. Studies are presently goingon to further define the mechanism of neuroprotection by C1-INHStem cells as a therapeutic approach in strokeThe aim of the project is to verify the conditions for the effectivenss of microsphere-derivedstem cells (MSC) in reducing the ischemic injury and to investigate the mechanisms triggeredby their infusion in the ischemic brain. MSC, isolated from newborn mice are infused tosyngenic mice in which transient ischemia is induced by middle cerebral artery occlusion. Atdifferent time points, up to 14 days, several parameters are evaluated: distribution andphenotype of injected cells, neurodegeneration, behavioral deficits, cytokine and trophic factorgene expression, microglia activation. The results obtained show that: i) MSC effectivelycounteract ischemia / reperfusion injury: they can decrease neuronal loss and reverte functionalimpairments related to exploratory behaviour and sensory/motor activity; ii) they elicite an earlyresponse: 24 h after infusion, chemokines, angiogenic and neurotrophic factor transcripts areactivated; these factors are no more activated at longer times or when cells are infused after 7days; iii) the protective mechanism of MSC in this model of mild ischemia seems to be mainlydue to the induction of beneficial factors; iv) activation of microglial cells is one of the aspectsof changes in ischemic environment induced by MSC; v) their presence in the brain tissue isenhanced by the ischemic injury. Thus the reciprocal interaction between MSC and ischemicenvironment is crucial for stem cells protective actions (Capone et al, 2007). Ongoing studiesinclude: 1) definition of the mechanisms of homing of stem cells in the injuried brain; 2)analysis of the protective/toxic role of microglia; 3) investigation of the early events triggeredby stem cells in the ischemic brain104ANNUAL REPORT 2007

IRFMNIdentification of ischemic tolerance mediators in cerebral ischemiaRecent studies indicate that cell death resulting from ischemic injury can be reduced when asublethal ischemic episode occurs hours or days before a severe ischemic insult. Thisphenomenon is known as Ischemic PreConditioning (IPC) and the induced neuroprotection iscalled Ischemic Tolerance (IT). Several models of induction and maintenance of tolerance havebeen described, but the molecular mechanisms of the IPC-induced neuroprotection are notidentified yet and a clear view of the mechanisms responsible for ischemic preconditioning isstill lacking. Specific aims of the project are: i) to define the characteristics of IT induced bysmall transient ischemic attack (TIA) in experimental models of cerebral ischemia; ii) toelucidate the pathways and/or the mediators involved in ischemic tolerance that may representpotential therapeutic targets for stroke; iii) to study of direct effect of relevant protectivemolecules in “in vitro” and “in vivo” ischemia models. Thus ischemic preconditioning providesan opportunity to identify the putative candidate that can confer neuroprotection against stroke.A major goal is to identify the underlying endogenous protective cellular receptor/signalingcascades, with the long-term goal to allow therapeutic augmentation of the endogenousprotective mechanisms in cerebral ischemia.Blood-brain barrier and preconditioningAlthough most attention has focused on the neuronal effects of ischemic preconditiong (IPC,see above) recent studies have shown that IPC reduces also ischemia-induced damage of thecerebrovascular unit. Together with astrocytes, pericytes and microglia, the endothelial cells inthe cerebral microvasculature are the main component of the Blood-Brain Barrier (BBB). Theintegrity of this complex structure is essential for the maintenance of the nervous systemmicroenvironment and its dysfunction is involved the pathophysiology of cerebral ischemia.The major goal of the research is to identify possible endogenous protective cellularreceptor/signalling cascades, activated by IPC on BBB. To this purpose we have established abidimensional BBB model by means of co-cultures of mouse brain endothelial cells and glialcells. In this model we can show the typical BBB features: presence of tight junctions, hightransendothelial electrical resistance, typical paracellular and transcellular permeability values.We are specifically addressing the following aspects: 1) changes in BBB characteristics andprotein expression following IPC ex vivo; 2) identification of mediators and/or pathwaysinvolved in activation and maintenance of ischemic tolerance in the cerebrovascular unit.Traumatic brain injury in mice: pathophysiological mechanismsTraumatic brain damage is the result of a primary injury (irreversible and due to thebiomechanical effects of the impact) and of secondary molecular and cellular events that areinitiated minutes after the injury and that interact in a complex network leading to cell death orrecovery. In patients who survive the initial injury, this secondary injury cascade is nowbelieved to account for the majority of brain damage observed following trauma. In addition theinjured brain is vulnerable to systemic and intracranial secondary insults that may occur andexacerbate traumatic brain damage. Since the major part of the damage evolves over daysfollowing the impact, there is an opportunity for pharmacological therapeutic intervention.Animal models of TBI have been developed to recapitulate many clinical and pathologic aspectsof head injury and have provided the basis for the dramatic increase in the understanding of thepathophysiology of brain damage after trauma. We have recently set up a mouse model ofcontrolled cortical impact (CCI) brain injury, which yields a consistent and predictable degree ofinjury and recapitulates many aspects of human cerebral contusion, including increasedintracranial pressure, changes in cerebrovascular reactivity (regional hypoperfusion), cerebraledema and neuronal damage in the cortex and hippocampus resulting in neurological motordeficits and cognitive dysfunction. Even if to date no specific neuroprotective therapies havebeen proven to be effective in attenuating the neurological sequelae of TBI patients, a betterunderstanding of the molecular and cellular mechanisms leading to post-traumatic cell death and105ANNUAL REPORT 2007

IRFMNrecovery and their relation to functional impairment remains an important goal of experimentalTBI research.Current areas of our research include: 1) neuroinflammatory response following TBI by usinggenetically engineered mice with the aim of investigating the role of selected genes; 2) cellularand molecular mechanisms of post-traumatic brain vulnerability to secondary insults; 3)identification of possible endogenous protective response activated by trauma similar to theischemic preconditioning; 4) translational research in which we investigate the molecularmechanisms of damage and recovery in our model and in human brain samples obtained frompatients during surgical removal of cerebral contusions.Laboratory of Molecular NeurobiologyStudy on pathogenic mechanisms of Amyotrophic Lateral SclerosisRole of protein aggregationA pathological feature of ALS is the accumulation of protein aggregates in the perykaria andaxons of motor neurons. Our hypothesis is that this may be due to an impairment of theubiquitin/proteasome system (UPS). To investigate whether motor neurons of SOD1 mutantmice may specifically display an impairment of the proteasome activity we have developeddouble transgenic mice expressing the SOD1G93A and carrying in all cells a fusion proteinfunctioning as marker for the activity of the proteasome, the UbG76V-GFP. We have recentlyconcluded the project with the following results: in some motor neurons of symptomatic doubletransgenic mice, showing neuropathological features such as the vacuolisation and accumulationof phosphorylated neurofilaments, there is an increase in the immunoreactivity for GFPindicating a dysfunction of the proteasome. However, the accumulation of GFP in the motorneurons does not occur in the presymptomatic mice indicating that this phenomenon is likelyinvolved in the rapid progression rather than in the pathogenesis of the disease. This iscompatible with the massive accumulation of insoluble proteins found in the spinal cord ofSOD1G93A at the advanced stage of the disease. The results are reported in the PhD thesis ofCristina Cheroni and in a manuscript submitted for publication.In addition, in collaboration with the Proteomic Unit of the Molecular Biochemistry andPharmacology department we have completed the proteomic characterization of the insolubleproteins components of the aggregates isolated from the spinal cord of SOD1G93A mice. Theyare cytoskeletal proteins, mainly intermediate filaments, several mitochondrial proteins,chaperones, proteins of the endoplasmic reticulum, proteins involved in metabolic pathways andsignaling. Post-translational modification such as nitration and carbonylation have also beenfound in some proteins suggesting a possible link between oxidative stress and aggregation.Role of glutamate AMPA receptors in the pathogenesis of ALSTo further study the role of glutamate AMPA receptors in the susceptibility of motor neuronswe have examined the expression and distribution of other two proteins regulating thetrafficking of the AMPA receptor and in particular that of the subunit GluR2, which plays themajor role in the homeostasis of intracellular calcium. The proteins are GRIP-1 and alsin whoseinteraction determine a stabilisation of the GluR2 subunit to the membrane. We observed anincrease of both proteins in the motor neurons of presymptomatic SOD1G93A mice. Theseresults, together with the previous one showing an increase of NSF, which is mainly involvedin the transport of the GluR2 subunit from the cytosol to the membrane, suggest that thisresponse may be an attempt of the motor neurons to protect them from the excitototoxity byincreasing the calcium impermeable subunit of AMPA receptor at the membrane.106ANNUAL REPORT 2007

IRFMNIn vitro studies on neuron-glia interactionThe interaction neurons-glia plays an important role in the inflammatory and excitotoxicmechanisms leading to motor neuron degeneration. To assess these mechanisms we set up an invitro paradigm made by co-colture of astrocytes and spinal neurons from SOD1G93A miceembryos and non transgenic mice. We have found that motor neurons degenerate selectivelywhen the mutant SOD1 is expressed only in the motor neurons or only in the astrocytes or inboth cell type, suggesting an active role of the astrocytes in this process. Selective inhibitors ofp38MAPK exhibited a neuroprotective effect on motor neuron when applied to the differentcoltures confirming the hypothesis of an important involvement of this signalling protein in thepathogenesis of ALS. However, we have not observed significant alteration in the expressionand release of TNF alpha , as found in the in vivo studies, suggesting that high levels of thiscytokine found in the spinal cord of SOD1G93A mice might derive from microglia and/or otherimmuno cells. Further studies are in progress to assess the involvement of other cytokines andtheir interaction with the mechanisms of excitotoxicity.Studies on the axo-nuclear communication in the motor neurons of FALSmodels.The aim of this unit is to investigate whether an impairment of axonuclear communication maycontribute to the degeneration of motor neurons or if it is possible to use this mechanism tovehicle a potential neuroprotective gene therapy from the nerves to the nucleus by a retrogradetransport. To this purpose we plan to analyse the expression of some proteins contributing tothis process in the spinal cord and in the nerves(sciatic and frenic) of SOD1G93A mice.Initially, we mainly focused our attention to the analysis of importin beta which play a crucialrole in assembly dinein and other signaling proteins that must be rapidly transported from theaxon to the nucleus after an axon injury. In the motor neurons which show clear signs ofdegeneration as the accumulation of phosphorylated neurofilaments , the immunostaining forimporting beta appeared remarkably increased in the nucleus in respect to the cytoplasmsuggesting that this may be a response signal to the axonal sufferance of these motor neurons.Also in the sciatic nerves of presymptomatic SOD1G93A mice, analysed byimmunohistochemistry we have found an increase of this protein. Whether this may representan accumulation or a slower transport of the protein in the axons triggered by the presencemutant SOD1 remains needs to be elucidated. We are now confirming these results in theaxoplasm extract from sciatic and frenic nerves of SOD1G93A transgenic rats that allow to havemore material to be analized. A colony of these rats that develop a phenotype similar to that ofSOD1G93A mice have been recently introduced in our laboratory.Therapeutic interventions in mouse model of ALSDevelopment of gene therapies for motor neuron protectionThe results obtained so far in our laboratory and in other groups indicate that one of the earlyresponse occurring in the motor neurons of SOD1G93A mice is the activation of the MAPkinases cascade while the mechanisms of defence such is that mediated by PI3K/Akt is notactivated. Based on these observations we plan to develop a neuroprotective intervention formotor neurons using two strategies: one aimed to inhibit the intracellular activation of MAPkinases and the other aimed to activate the intracellular mechanisms of survival. In the first casewe have developed viral vector carrying the shRNA against p38MAPK. Preliminary tests onneuron and astrocytes coltures have allowed to select sequences of shRNA able to silencingalmost completely the expression of p38 and to inhibit its activation by cytokines.For the second strategy we developed viral vectors expressing the activated form of Akt,importing for the inhibition of cell death process. At the present we are developing new vectorswith specific promoters in attempt to vehicle the Akt selectively to the motor neurons.107ANNUAL REPORT 2007

IRFMNStudies aimed to identify biomarkers for the diagnosis and progression ofthe disease in ALS patients.The diagnosis of ALS is based mainly on neurophysiological parameters associated to theprogression of the disease and requires about one year to be formulated with certainty. Suchinterval is quite long considering that the prognosis of the disease is two-five years since theonset of symptoms. This has a negative impact on the possibility to apply a prompt therapeuticintervention. In collaboration with the department of Neurology of the Fondazione SalvatoreMaugeri, IRCCS, of Pavia, we have started a series of studies aimed to investigate through theproteomic analysis, the protein profile in the PBMC and CSF of SLA patients at different stagesof the disease in comparison to health controls. So far we have observed a series of interestingproteins differentially expressed potentially involved in the disease progression and useful aspotential biomarkers. We are now validating the changes in some of these proteins in a widernumber of ALS patients by western blot.In parallel, we are completing the characterization of nitrated proteins in the PBMC and in theCSFof ALS patients compared to healthy controls. The protein nitration on tyrosine is anoxidative mechanism that alters the function of proteins inducing their inactivation or a gain oftoxic functions.Laboratory of Experimental PsychopharmacologyDrug Abuse. Neural basis of drug self-administration, “drug craving” and“relapse” in the drug abuse assumptionDrug craving, defined as “the desire to experience the effect(s) of a previously experiencedpsychoactive substance” is a cardinal feature of drug addiction and is clinically significantbecause of its potential link to relapse. To provide useful indications to the development ofnovel therapeutic approaches to prevent the use and abuse and the relapse of drug assumptionfollowing the outcome of “craving”, we elaborated experimental models of self-administrationand “relapse” induced by cocaine-associated cues, after a period of abstinence. It was found thatagonists at serotonin 2C receptors and non selective antagonists at opioids receptors selectivelymodulate rats’ seeking behaviour induced by cocaine-associated cues after a long period ofabstinence and in the absence of any further cocaine. Ongoing studies are evaluating whetherthis modulation is peculiar for cocaine or could be generalized to other abused drugs. The roleof several other neurochemical mechanisms potentially involved in the drug-seeking behaviourare also in progress.Resistance to antidepressant drugs: experimental and clinical studiesThis project arises from a collaboration between the laboratories of Neurochemistry andBehavior (R.W. Invernizzi), Drug Metabolism (Silvio Caccia), Biology of NeurodegenerativeDisorders (Gianluigi Forloni) and focus on behavioral and biochemical characterization of anexperimental model of resistance to the antidepressant drugs.Using an animal model predictive of the antidepressant activity, the effects of selectiveserotonin reuptake blockers (SSRI) was evaluated in several mice strains. It was found thatDBA/2J and BALB/c do not respond to the antidepressant-like activity of the SSRI. The lack ofeffect was attributed to genotype-dependent impairment of 5-HT synthesis since DBA/2J andBALB/c carrying a single nucleotide polymorphism (C1473G mice) in the gene for the brainspecificisoform of tryptophan hydroxylase-2, the rate-limiting enzyme in the synthesis ofserotonin are characterized by a decreased serotonin synthesis. This hypothesis seems to besupported by the observation that DBA/2J and BALB/c mice had less dialysate 5-HT in themedial prefrontal cortex and dorsal hippocampus than C57BL/6J amice. Moreover, in DBA/2J108ANNUAL REPORT 2007

IRFMNand BALB/c the SSRI raised significantly less extracellular 5-HT when compared to C57BL/6Jmice. Studies are ongoing to clarify whether 1) whether DBA/2J and BALB/c are responder toSSRI effects other than the anti-depressant-like 3) whether DBA/2J and BAB/c are responder toother class of antidepressant drugs.Laboratory of Neurochemistry and Behavior“Resistance” to antidepressant drugsDespite the wide range of antidepressant drugs available for the treatment of mood disorders thedelayed onset of the antidepressant effect and the partial or no response in a considerableportion of patients still limits their efficacy. Studies in mice show that the response toantidepressant drugs is strain-dependent, suggesting that genetic differences influence theantidepressant response.Ongoing studies in collaboration with the Laboratories of “Experimental Psychopharmacology”and “Drug Metabolism” are aimed at assessing the neurobiological mechanisms involved in theresistance to antidepressant drugs in mice. The gene for the brain-specific isoform of tryptophanhydroxylase-2 (TPH-2), the rate-limiting enzyme in the synthesis of serotonin, is mutated inDBA/2J and BALB/c mice. These mice synthesize less serotonin than C57BL/6 mice (that carrythe “wild type” form of TPH-2) and do not respond to antidepressants inhibiting selectively thereuptake of serotonin (SSRI) in the forced swimming test, a procedure used to screencompounds for antidepressant effects. In addition, mutation of TPH-2 attenuated the effect ofSSRI on the extracellular concentrations of the neurotransmitter These results suggest thatgenetic differences in serotonin synthesis could contribute to the efficacy of SSRI. Ongoingstudies are aimed at assessing the molecular mechanisms involved in the antidepressantresponse and identify pharmacological strategies to restore the response in non-responder to theSSRI alone.Animal model of cognitive deficit of schizophrenia; typical and atypicalantipsychoticsThe cognitive deficit is a core symptom of schizophrenia, which has been linked to functionaloutcome and is relatively independent of psychotic symptoms. The antipsychotics, either typicalor atypical, are able to control positive symptoms such as delirium, hallucinations and paranoia.However, the currently available atypical antipsychotics when compared to conventionalantipsychotics show somewhat superior efficacy for the management of cognitive deficits inpatients with schizophrenia.The cognitive deficit of schizophrenia was modelled in rats, by using a test of attention such asthe 5-choice serial reaction time task (5-CSRTT) and injections of glutamate NMDA receptorantagonists into the medial prefrontal cortex (mPFC). This model makes clear links withpsychopathology as dysfunctional glutamate neurotransmission in the mPFC has beenimplicated in cognitive deficits of schizophrenia and the 5-CSRTT is the rat analogue of thecontinuous performance test used to assess attention and vigilance in schizophrenic patients.Antipsychotics possess a complex pharmacology across the biogenic amine receptor families asshown by affinity constants derived from radioligand-binding techniques. The ability toantagonise the DA D 2 receptor function is shared by the conventional and by the atypicalantipsychotics. However, atypical antipsychotics show a high affinity also for serotonin 5-HT 2A , 5-HT 2C and 5-HT 1A receptors. Our studies compared the effects of conventional andatypical antipsychotics in this model of cognitive deficit of schizophrenia. The results show thatantipsychotics may be differentiated by a selective effect of typical antipsychotics oncompulsive perseveration, and atypical antipsychotics on impulsivity. Intracerebralmicrodialysis studies show that attentional deficits induced by NMDA receptor antagonists is109ANNUAL REPORT 2007

IRFMNassociated with excessive glutamate in the medial prefrontal cortex of the rat and this effect wasprevented by atypical antipsychotics.110ANNUAL REPORT 2007

IRFMN111ANNUAL REPORT 2007

IRFMNDEPARTMENT OF CARDIOVASCULARRESEARCHSTAFFHeadMaria Grazia FRANZOSI, Biol.Sci.D.Laboratory of Cardiovascular Clinical PharmacologyHeadRoberto LATINI, M.D.Bio-imagin UnitHeadCardiovascular Endocrine UnitHeadTissue Culture UnitHeadFabio FIORDALISO, Biol.Sci.D.Serge MASSON, Ph.D.Giovanna BALCONI, Univ.Dipl.Laboratory of Clinical Drug EvaluationHeadMaria Grazia FRANZOSI, Biol.Sci.D.Bioinformatics UnitHeadEnrico NICOLIS, Comp.Sci.Stud.Laboratory of General Practice ResearchHeadM.Carla RONCAGLIONI, Biol.Sci.D.Laboratory of Medical StatisticsHeadSimona BARLERA, Dr.Sci.Pol., MSc.Laboratory of Clinical PharmacologyHeadNursing Research UnitHeadGianni TOGNONI, M.D.Paola DI GIULIO, R.N.112ANNUAL REPORT 2007

IRFMNCURRICULA VITAEMaria Grazia Franzosi got her Biological Science degree in 1972 at the University of Milan.Education1972 Doctoral degree in Biological Sciences, University of Milan, Italy1978 Postdoctoral degree in Pharmacological Research, Istituto di Ricerche Farmacologiche "MarioNegri” di Milano, ItalyMain fields of activityCoordination of multicentric randomised clinical trials. Relationship between genetic and environmental riskfactors in coronary events. Pharmacogenetics. Pharmacoeconomics. Drug Epidemiology and Post-MarketingSurveillance.Positionfrom 2002 Director of the Department of Cardiovascular Research, Istituto di Ricerche Farmacologiche"Mario Negri", Milano, Italyfrom 2004 Member of Steering Committee, Studio GISSI-AF Study, Milano, Italyfrom 2001from 1998Member of Steering Committee, Studio GISSI-HF Study, Milano, ItalyMember of Steering Committee of the PROCARDIS Research Programme - A genome-widestrategy to identify susceptibility loci in precocious coronary artery disease - University ofOxford, UKfrom 1997 Member of “Antithrombotic Trialists’ Collaboration”, Oxford, UKfrom 1996 Member of Steering Committee and National Coordinator for Italy of the Organization to AssessStrategies for Ischemic Syndromes (OASIS-2, OASIS-5, OASIS 6, OASIS-7, CURE, INTER-HEART studies), Canadian Cardiovascular Collaboration, McMaster University, Hamilton,Canada and of ACTIVE and RELY studies, Canadian Cardiovascular Collaboration, McMasterUniversity, Hamilton, Canada1994-1996 Director of European Coordinating Centre and Member of Steering Committee, Collaborativefrom 1993from 2002Organization for RheothRx Evaluation (CORE), McMaster University, Hamilton, CanadaMember of Steering Committee, Studio GISSI-Prevenzione, Milano, ItalyMember of “Fibrinolytic Therapy Trialists’s Collaboration”, Oxford, UK e del “CollaborativeGroup on Angiotensin Converting Enzyme Inhibitors Trials”, National Institutes of Health,Bethesda, Washington, USA1989-2001 Head of the Laboratory of Clinical Drug Evaluation, Istituto di Ricerche Farmacologiche "MarioNegri"1985-1988 Head of the Clinic Drug Evaluation Unit of the Laboratory of Clinical Pharmacology, Istituto diRicerche Farmacologiche "Mario Negri"from 1984Member of the Scientific and Organising Secretariat, Gruppo Italiano per lo Studio dellaSopravvivenza nell'Infarto Miocardico (GISSI-1, GISSI-2, GISSI-3 studies) Milano, Italy1975-1984 Researcher at the Laboratory of Clinical Pharmacology, Istituto di Ricerche Farmacologiche"Mario Negri" and at the Regional Center for Drug Information of the Lombardy RegionSelected publications• Anand SS, Xie CC, Mehta S, Franzosi MG, Joyner C, Chrolavicius S, Fox KA, Yusuf S, CURE Investigators.Differences in the management and prognosis of women and men who suffer from acute coronary syndromes. J Am CollCardiol 2005, 46: 1845-1851• Yusuf S, Howken S, Ounpuu S, Bautista L, Franzosi MG, Commerford P, Lang CC, Rumboldt Z, Onen CL, Lisheng L,Tanomsup S, Wangai Jr P, Razak F, Shama AM, Anand SS, on behalf of the INTERHEART Study Investigators.Obesity and the risk of myocardial infarction in 27000 participants from 52 countries: a case-control study. Lancet 2005,336: 1640-1649• Levantesi G, Macchia A, Marfisi R M, Franzosi M G, Maggioni A P, Nicolosi G L, Schweiger C, Tavazzi L, TognoniG, Valagussa F, Marchioli R, on behalf of the GISSI-Prevenzione Investigators. Metabolic syndrome and risk ofcardiovascular events after myocardial infarction. J Am Coll Cardiol 2005; 46: 277-283• Franzosi MG. Should we continue to use BMI as a cardiovascular risk factor? Lancet 2006; 368: 624-625• Farrall M, Green FR, Peden JF, Olsson PG, Clarke R, Hellenius ML, Rust S, Lagercrantz J, Franzosi MG, Schulte H,Carey A, Olsson G, Assman G, Tognoni G, Collins R, Hamsten A, Watkins H, on behalf of the PROCARDISConsortium. Genome-wide mapping of susceptibility to coronary artery disease identifies a novel replicated locus onchromosome 17. PLoS Genet 2006 - http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020072• Chiodini B, Franzosi M G, Barlera S, Signorini S, Lewis C M, D'Orazio A, Mocarelli P, Nicolis E, Marchioli R, TognoniG, GISSI, SIBioC-GISSI Prevenzione Group. Apolipoprotein E polymorphisms influence effect of pravastatin onsurvival after myocardial infarction in a Mediterranean population: the GISSI-Prevenzione study. Eur Heart J 2007 ; 28:1977-1983113ANNUAL REPORT 2007

IRFMNSimona Barlera got her degree in Political Science, area Statistics at the “Università degli Studi diMilano” in Milano in 1992, followed by a master in Medical Statistics at the London School of Hygieneand Tropical Medicine, “University of London” in 1998.Education1987-1992 Degree in Political Science, area Statistics at the “Università degli Studi di Milano” inMilano.1997-1998 Master post-lauream in Medical Statistics at the London School of Hygiene and TropicalMedicine, University of London, London.1998-1999 Visiting Scientist in the Department of Statistical Genetics, Wellcome Trust Centre forHuman Genetics, University of Oxford (UK).Main fields of activityMethodology of Clinical Trials in the cardiovascular field. Preparation and viewing of research protocols,planning and conduct of statistical analyses and the reporting of findings on scientific journals.Genetic epidemiology: genome-wide strategies (linkage analysis) to identify susceptibility genes incoronary artery disease; case-control studies in order to identify candidate genes involved in thecardiovascular pathology.Positionfrom Oct 2006Head of the Laboratory of Medical Statistics, Department of Cardiovascular Research,Istituto di Ricerche Farmacologiche "Mario Negri", Milano, ItalyMay 99-Sept 06 Head of the Medical Statistics Unit, Department of Cardiovascular Research, Istituto diRicerche Farmacologiche "Mario Negri", Milano, Italy1998-1999 Visiting Scientist in the Department of Statistical Genetics, Wellcome Trust Centre forHuman Genetics, University of Oxford (UK).1992-1997 Researcher in the Unit of Applied Statistics and Information Technology, Istituto diRicerche Farmacologiche "Mario Negri", Milano, ItalySelected publications• Chiodini B, Barlera S, Franzosi M G, Labarta V, Introna M, Tognoni G.APO B gene polymorphisms with coronaryartery disease: A meta-analysis. Atherosclerosis 2003; 167: 355-366• Latini R, Masson S, Anand I, Salio M, Hester A, Judd D, Barlera S, Maggioni AP, Tognoni G, Cohn J N, Val-HeFTInvestigatore. The comparative prognostic value of plasma neurohormones at baseline in patients with heart failureenrolled in Val-HeFT. Eur Heart J 2004; 25: 292-299• Roncaglioni M C, Avanzini F, Roccatagliata D, Monesi L, Tamayo-Benitez D, Tombesi M, Caimi V, Longoni P, LauriD, Barlera S, Tognoni G, Collaborative Group Risk Prevention Study. How general practitioners perceive and grade thecardiovascular risk of their patients. Eur J Cardiovasc Prev Rehabil 2004; 11: 233-238• Maggioni AP, Latini R, Carson PE, Singh SN, Barlera S, Glazer R, Masson S, Cere` E, Rognoni G, Cohn JN. Valsartanreduces the incidence of atrial fibrillation in patients with heart failure: Results from the Valsartan Heart Failure Trial(Val-HeFT). Am Heart J 2005; 149: 1-10• Masson S, Latini R, Anand IS, Vago T, Angelici L, Barlera S, Missov ED, Clerico A, Tognoni G, Cohn JN, Val-HeFTInvestigators. Direct comparison of B-type natriuretic peptide (BNP) and amino-terminal proBNP in a large populationof patients with chronic and symptomatic heart failure. The Valsartan Heart Failure (Val-HeFT) data. Clin Chem 2006;52: 1528-1538• Barlera S, Specchia C, Farrall M, Chiodini BD, Franzosi MG, Rust S, Green F, Nicolis E, Peden J, Assmann G, CollinsR, Hamsten A, Tognoni G, PROCARDIS Consortium. Multiple QTL influence the serum Lp(a) concentration: agenome-wide linkage screen in the PROCARDIS study. Eur J Hum Genet 2007; 15: 221-227114ANNUAL REPORT 2007

IRFMNRoberto Latini got his Medical Doctor degree in 1978 at the University of Milan.Education1970-1978 University of Milan School of Medicine, degree in Medicine1981-1983 Merck Sharp & Dohme International Fellow in Clinical PharmacologyMain fields of activityMechanisms of cardiac damage following ischemia, with focus on eurohumoral activation. Use of stemcells for cardiac repair. Biohumoral investigations within large scale clinical trials in heart failure andatrial fibrillation.Positionsfrom 1990 Head of the Cardiovascular Clinical Pharmacology Laboratory (Cardiovascular ResearchDepartment) Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italyfrom 2001 Member of the GISSI-HF Steering Committeefrom 2004 Member of the GISSI-AF Steering Committeefrom 2005 Member of the CandHeart Steering Committeefrom 1999 Visiting Professor Dept of Medicine, New York Medical College, Valhalla, NY, USA1981-1983 Cardiology Fellow (Dr. R. E. Kates, Laboratory) Stanford University Medical Center,CA, USA1976-1981 Member of the Sub-Group RMs for Drugs (Community Bureau of Reference,Commission of the European Communities)1973-1990 Fellow at the Laboratory of Clinical Pharmacology of the Istituto di RicercheFarmacologiche "Mario Negri", Milano, ItalySelected publications• Latini R, Maggioni AP, Peri G, Gonzini L, Lucci D, Mocarelli P, Vago L, Pasqualini F, Signorini S, Soldateschi D, TarliL, Schweiger C, Fresco C, Cecere R, Tognoni G, Mantovani A, LATIN Investigators. Prognostic significance of the longpentraxin PTX3 in acute myocardial infarction. Circulation 2004; 110: 2349-2354• Corada M, Chimenti S, Cera M R, Vinci M, Salio M, Fiordaliso F, De Angelis N, Villa A, Bossi M, Staszewsky L,Vecchi A, Parazzoli D, Motoike T, Latini R, Dejana E. Junctional adhesion molecule-A-deficient polymorphonuclearcells show reduced diapedesis in peritonitis and heart ischemia-reperfusion injury. Proc Natl Acad Sci USA 2005; 102:10634-10639• Galli D, Innocenzi A, Staszewsky L, Zanetta L, Sampaolesi M, Bai A, Martinoli E, Carlo E, Balconi G, Fiordaliso F,Chimenti S, Cusella G, Dejana E, Cossu G, Latini R. Mesoangioblasts, vessel-associated multipotent stem cells, repairthe infarcted heart by multiple cellular mechanisms. A comparison with bone marrow progenitors, fibroblasts, andendothelial cells. Arterioscler Thromb Vasc Biol 2005; 25: 692-697• Latini R, Masson S, Anand I S, Missov E, Carlson M, Vago T, Angelici L, Barlera S, Parrinello G, Maggioni A P,Tognoni G, Cohn J N, Val-HeFT Investigators. Prognostic value of very low plasma concentrations of troponin T inpatients with stable chronic heart failure. Circulation 2007; 116: 1242-1249• Sarto P, Balducci E, Balconi G, Fiordaliso F, Merlo L, Tuzzato G, Pappagallo G L, Frigato N, Zanocco A, Forestieri C,Azzarello G, Mazzucco A, Valenti M T, Alborino F, Noventa D, Vinante O, Pascotto P, Sartore S, Dejana E, Latini R.Effects of exercise training on endothelial progenitor cells in patients with chronic heart failure. J Card Fail 2007; 13:701-708115ANNUAL REPORT 2007

IRFMNMaria Carla Roncaglioni got her Biological Science degree in 1987 at the University of Milan.Education1987 Doctoral degree in Biological Sciences, University of Milan, Italy1982-1983 “Research Fellow” at the Dept. of Biochemistry, Faculty of Medicine, Rijksuniversiteit ofLimburg, Maastricht , The Netherland (Prof. C.Hemker);1998-1999 “Visiting Scientist” at the Cardiovascular Research Unit, Hammersmith Hospital, London,UK (Prof. A. Maseri)Main fields of activityCoordination of multicenter clinical trials and observational studies in different cardiovascular areas(neurological, angiological, cardiological). Coordination of a network of more than 1000 GPs activelyinvolved in epidemiological and experimental studies in the prevention of cardiovascular diseases.Positionfrom 2001 Head of the Laboratory for General Practice Research, Istituto di Ricerche Farmacologiche"Mario Negri", Milano, Italyfrom 1989 Senior Researcher in the Clinical Pharmacology Laboratory, Istituto di RicercheFarmacologiche "Mario Negri", Milano, Italyfrom 1974 Researcher in the Laboratory for the Study of Haemostasis and Thrombosis, Istituto diRicerche Farmacologiche "Mario Negri", Milano, ItalySelected publications• Avanzini F, Palumbo G, Alli C, Roncaglioni MC, Ronchi E, Cristofari M, Capra A, Rossi S, Nosotti L, Costantini C,Pietrofeso R, PPP Primary Prevention Project. Effects of low-dose aspirin on clinic and ambulatory blood pressure intreated hypertensive patients. Am J Hypertens 2000; 13: 611-616• Tognoni G, Avanzini F, Pangrazzi J, Roncaglioni M C, Bertele V, de Gaetano G, Caimi V, Tombesi M, Colombo Fabio,Barlera S, PPP - Primary Prevention Project. Low-dose aspirin and vitamin E in people at cardiovascular risk: Arandomized trial in general practice. Lancet 2001; 357: 89-95• Sacco M, Pellegrini F, Roncaglioni MC, Avanzini F, Tognoni G, Nicolucci A, PPP - Primary Prevention Project.Primary prevention of cardiovascular events with low-dose aspirin and vitamin E in type 2 diabetic patients. Results ofthe Primary Prevention Project (PPP) trial. Diabetes Care 2003; 26: 3264-3272• Roncaglioni MC, Avanzini F, Roccatagliata D, Monesi L, Tamayo-Benitez D, Tombesi M, Caimi V, Longoni P, LauriD, Barlera S, Tognoni G, Collaborative Group Risk Prevention Study. How general practitioners perceive and grade thecardiovascular risk of their patients Eur J Cardiovasc Prev Rehabil 2004; 11: 233-238• Monesi L, Avanzini F, Barlera S, Caimi V, Lauri D, Longoni P, Roccatagliata D, Tombesi M, Tognoni G, RoncaglioniMC. Appropriate use of antiplatelets: is prescription in daily practice influenced by the global cardiovascular risk? Eur JClin Pharmacol 2005; 61: 595-601• Berger JS, Roncaglioni MC, Avanzini F, Pangrazzi J, Tognoni G, Brown DL. Aspirin for the primary prevention ofcardiovascular events in women and men: A sex-specific meta-analysis of randomized controlled trials. JAMA 2006;295: 306-313116ANNUAL REPORT 2007

IRFMNGianni Tognoni got his Medical Doctor degree in 1970, University of Milan.Education1964 Philosophy Doctor, University of Rome1970 Medical Doctor degree, University of MilanMain fields of activityRandomised Clinical Trials. Pharmacoepidemiology and pharmacoeconomics. Outcomes researchGenetic epidemiology. Technology transfer and drug policy in developing countries. Cardiocerebrovasculardisorders; primary and secondary prevention; heart-failure; diabetes; aging; psychiatricepidemiology. Health and human rights.Positionfrom 2004 Member, Commission of Human Experimentation of the Italian Drug Agency (AIFA)2001-2003 Member, Commissione Unica del Farmaco (CUF), Ministry of Healthfrom 2002 Director, Consorzio Mario Negri Sud, S. Maria Imbaro, Chieti.1992-2002 Coordinator, Department of Clinical Pharmacology and Epidemiology, Consorzio MarioNegri Sud, S. Maria Imbaro, Chieti.1996-2002 Coordinator, Department of Cardiovascular Research, Istituto di Ricerche Farmacologiche"Mario Negri", Milanofrom 1990 Co-Director, Scuola Superiore di Ricerca in Medicina Generale (CSeRMEG)from 1988 Coordinator of training and research programs in community medicine, clinicalepidemiology, pharmaco-epidemiology in Bolivia, Argentina, Chile, Brazil, Ecuador, Perùfrom 1986 Founding member of the International Society of Drug Bulletins (ISDB)Coordinator, Commission of Human Experimentation, Regione Lombardiafrom 1983 Member of the Editorial Board of the nursing research Journal Rivistadell'Infermiere/Assistenza Infermieristica e Ricercafrom 1977 Consultant to WHO and other UN agencies for drug selection and policy; training inmethods of clinical and epidemiological research in developing countries mainly in LatinAmerica (Costa Rica, Nicaragua, Bolivia, Perù) and Africa (Angola, Ethiopia, BurkinaFaso)from 1976 Head, Laboratory of Clinical Pharmacology of the Istituto di Ricerche Farmacologiche"Mario Negri", Milanofrom 1975 Head, Regional Centre for Drug Information (CRIF), Regione Lombardia, Istituto diRicerche Farmacologiche "Mario Negri", Milano1969-1974 Research Assistant, Laboratory of Clinical Pharmacology, Istituto di RicercheFarmacologiche "Mario Negri", MilanoSelected publications• Anand I S, Fisher L D, Chiang Y T, Latini R, Masson S, Maggioni A P, Glazer R D, Tognoni G, Cohn J N, Val-HeFTInvestigators. Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in theValsartan Heart Failure (Val-HeFT). Circulation 2003; 107: 1278-1283• Franciosi M, Pellegrini F, De Berardis G, Belfiglio M, Di Nardo B, Greenfield S, Kaplan S H, Rossi M C E, SaccoM, Tognoni G, Valentini M, Nicolucci A, QuED Study Group. Impact of physicians' beliefs and practices on cholesterollevels in patients with type 2 diabetes: A longitudinal assessment. Am Heart J 2005; 149: 104-111• Maggioni A P, Latini R, Carson P, Singh S N, Barlera S, Glazer R, Masson S, Cere' E, Tognoni G, Cohn J N.Valsartanreduces the incidence of atrial fibrillation in patients with heart failure: Results form the Valsartan Heart Failure Trial(Val-HeFT). Am Heart J 2005; 149: 548-557• Monesi L, Avanzini F, Barlera S, Caimi V, Lauri D, Longoni P, Roccatagliata D, Tombesi M, Tognoni G, RoncaglioniM C. Appropriate use of antiplatelets: is prescription in daily practice influenced by the global cardiovascular risk? Eur JClin Pharmacol 2005; 61: 595-601• Evangelista V, De Berardis G, Totani L, Avanzini F, Giorda CB, Brero L, Levantesi G, Marelli G, Pupillo M, Iacuitti G,Pozzoli G, Di Summa P, Nada E, De Simone G, Dell'Elba G, Amore C, Manarini S, Pecce R, Maione A, Tognoni G,Nicolucci A. Persistent platelet activation in patients with type 2 diabetes treated with low doses of aspirin. J ThrombHaemost 2007; 5: 2197-2203• Silletta MG, Marfisi RM, Levantesi G, Boccanelli A, Chieffo C, Franzosi MG, Geraci E, Maggioni AP, Nicolosi GL,Schweiger C, Tavazzi L, Tognoni G, Marchioli R, GISSI-Prevenzione. Coffee consumption and risk of cardiovascularevents after acute myocardial infarction. Results from the GISSI (Gruppo Italiano per lo Studio della Sopravvivenzanell'Infarto miocardico)-Prevenzione trial. Circulation 2007; 116: 2944-2951117ANNUAL REPORT 2007

IRFMNGiovanna Balconi got her degree at the School for Technicians of Biomedical Institutes of theUniversity of Milan, with a specialisation in Histology in the Pathological Anatomy Laboratory of thesame University (1968).Main fields of interestIsolation, culture and characterization of peripheral blood circulating progenitor cells of patients withheart failure.“In vitro” culture and characterization of stem cells for repair of myocardial infarction in experimentalanimal models.Positionsfrom July 2005 Head of Tissue Culture Unit, Cardiovascular Clinical Pharmacology Laboratory,Istituto di Ricerche Farmacologiche "Mario Negri", Milano, ItalyOct 1995 - June 2005 Head of Tissue Culture Unit, Vascular Biology Laboratory, Istituto di RicercheFarmacologiche "Mario Negri", Milano, ItalyDec 1983 - Oct 1995 Head of Tissue Culture Unit, Anticancer Chemotherapy Laboratory, Istituto diRicerche Farmacologiche "Mario Negri", Milano, ItalyOct 1968 - Nov 1983 Researcher, Anticancer Chemotherapy Laboratory, Istituto di RicercheFarmacologiche "Mario Negri", Milano, ItalySelected publications• Balconi G, Spagnuolo R, Dejana E Development of endothelial cell lines from embryonic stem cells. A tool for studyinggenetically manipulated endothelial cells in vitro Arterioscler Thromb Vasc Biol 2000; 20: 1443-1451• Condorelli G, Borello U, De Angelis L, Latronico M, Sirabella D, Coletta M, Galli R, Balconi G, Follenzi A, Frati G,Cusella De Angelis M G, Gioglio L, Amuchastegui C S, Adorini L, Naldini L, Vescovi A, Dejana E, Cossu GCardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: Implications for myocardiumregeneration Proc Natl Acad Sci USA 2001; 98: 10733-10738• Cattelino A, Liebner S, Gallini R, Zanetti A, Balconi G, Corsi A, Bianco P, Wolburg H, Moore R, Oreda B, Kemler R,Dejana E The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern andincreased vascular fragility J Cell Biol 2003; 162: 1111-1122• Cusella De Angelis M G, Balconi G, Bernasconi S, Zanetta L, Boratto R, Galli D, Dejana E, Cossu G Skeletal myogenicprogenitors in the endothelium of lung and yolk sac Exp Cell Res 2003; 290: 207-216• Galli D, Innocenzi A, Staszewsky L, Zanetta L, Sampaolesi M, Bai A, Martinoli E, Carlo E, Balconi G, Fiordaliso F,Chimenti S, Cusella G, Dejana E, Cossu G, Latini R Mesoangioblasts, vessel-associated multipotent stem cells, repairthe infarcted heart by multiple cellular mechanisms. A comparison with bone marrow progenitors, fibroblasts, andendothelial cells Arterioscler Thromb Vasc Biol 2005; 25: 692-697• Sarto P, Balducci E, Balconi G, Fiordaliso F, Merlo L, Tuzzato G, Pappagallo G L, Frigato N, Zanocco A, Forestieri C,Azzarello G, Mazzucco A, Valenti M T, Alborino F, Noventa D, Vinante O, Pascotto P, Sartore S, Dejana E, Latini REffects of exercise training on endothelial progenitor cells in patients with chronic heart failure J Card Fail 2007; 13:701-708Paola Di Giulio got her Nursing Diploma in the Nursing School of Istituto Nazionale dei Tumori inMilano and her Master in Oncology Nursing at Guildford University (UK) in 1995.Main fields of activityCoordination of multicentre and observational studies studies in cardiology and palliative care.Coordination of nursing networks.Positionfrom March 2001 Associated professor at the Turin University.from 1997 Responsible of the Nursing Research Unitfrom 1995 Senior researcher of the Cardiovascular Research Departmentfrom 1989 Consultant of the Clinical Phrmacology Laboratorysince 1998 Coordinator of the Editorial Board of Assistenza Infermieristica e RicercaSelected publications• Laquintana D, Di Giulio P: Per un ruolo infermieristico nella farmacovigilanza. Assistenza Infermieristica e ricerca2002; 21: 198-210• Di Giulio P, Saiani L, Laquintana D, Palese A, Gruppo PARI-ETLD. Studio clinico randomizzato controllato in doppiocieco sull’efficacia dei trattamenti delle lesioni da decubito. Assistenza Infermieristica e Ricerca 2004; 23: 201-218• Toscani F, Brunelli C, Miccinesi G, Costantini M, Gallucci M, Tamburini M, Paci E, Di Giulio P, Peruselli C and theItalian Co-operative Research Group on Palliative Medicine Predicting survival in terminal cancer patients: clinicalobservation or quality of life evaluations? Palliative Medicine 2005; 19: 220-227• Toscani F, Di Giulio P, Brunelli C, Miccinesi G. Laquintana D. How How people die in hospital general wards: adescriptive study. J Pain Symptom Manage 2005; 30: 33-40118ANNUAL REPORT 2007

IRFMN• Saiani L, Di Giulio P, Gruppo PARI-FV (Percorsi Assistenziali e Ricerca Infermieristica-Farmaco Vigilanza)Epidemiologia dei problemi assistenziali legati a farmaci e presidi in RSA e distretto. Assistenza Infermieristica eRicerca 2007; 26: 123-164• Lepore V, Cecchetto G, Di Giulio P, Saiani L, Samarelli V, Saugo M, Romero M, Scurti V, Tognoni G, Valerio M. Etàanziana-molto-anziana, e “aspettativa di vita”? Assistenza Infermieristica e Ricerca 2007; 26: 234-242Fabio Fiordaliso got his Biological Science degree in 1995 at the University of Milan.Education1998 Postdoctoral degree in Pharmacological Research, Istituto di Ricerche Farmacologiche“Mario Negri”, Milan, Italy1995 Doctoral degree in Biological Sciences, University of Milan, ItalyMain fields of activityTherapeutical potential of stem cell and antioxidant treatments in experimental model of diabeticcardiomyopathy and in primary myocyte cultures exposed to hyperglycemia.Morphological and structrural analysis of cells and tissue by optical, confocal and electron microscopy.Positionsfrom 2007from 2006from 2005from 2005from 2001Head of Bio-imaging Unit, Department of Cardiovascular Research, Istituto di RicercheFarmacologiche “Mario Negri”, MilanMember of the Heart Failure Association (HFA) of the European Society of CardiologyMember of the Working group on myocardial function (WG 4) of the European Society ofCardiologyMember of the steering committee of the Consorzio of Microscopy and Image Analysis(MIA)Senior Research Scientist, Laboratory of Cardiovascular Clinical Pharmacology(Department of Cardiovascular Research), Istituto di Ricerche Farmacologiche “MarioNegri”, Milan1997-2001 Post-Doctoral Research Fellow at Cardiovascular Research Institute (Department ofMedicine), New York Medical College, Valhalla, New York1994-1997 Research Fellow, Laboratory of Cardiovascular Clinical Pharmacology (Department ofCardiovascular Research), Istituto di Ricerche Farmacologiche “Mario Negri”, Milan1992-1994 Research training, Institute of General Pathology, University of Milan (Italy)Selected publications• Fiordaliso F, Li B, Latini R, Sonnenblick EH, Anversa P, Leri A, Kajstura J. Myocyte death in streptozotocin-induceddiabetes in rats is angiotensin II dependent. Laboratory Investigation 2000; 80: 513-527• Fiordaliso F, Leri A, Cesselli D, Limana F, Safai B, Nadal-Ginar B, Anversa P, Kajstura J. Hyperglycemia activates p53and p53-regulated genes leading to myocyte cell death. Diabetes 2001; 50: 2355-2362• Fiordaliso F, Bianchi R, Staszewsky .,Cuccovillo I, Doni M, Laragione T, Salio M, Savino C, Melucci S, Santangelo F,Scanziani E, Masson S, Ghezzi P, Latini R. Antioxidant N-Acetyl-L-Cysteine attenuates hyperglycemia-inducedcardiomyocyte death in rats. Journal of Molecular and Cellular Cardiology 2004; 37: 959-968• Fiordaliso F, Chimenti S, Staszewsky L, Bai A, Carlo E, Cuccovillo I, Doni M, Mengozzi M, Tonelli R, Ghezzi P,Coleman T, Brines M, Cerami A, Latini R. A non-erythropoietic derivative of EPO effectively ameliorates experimentalcardiac ischemia with reperfusion. Proceedings National Academy Sciences (PNAS) 2005; 102: 2046-2051• Fiordaliso F, Cuccovillo I, Bianchi R, Bai A, Doni M, Salio M, De Angelis N, Ghezzi P, Latini R, Masson S.Cardiovascular oxidative stress is reduced by an ACE inhibitor in a rat model of streptozotocin-induced diabetes . LifeSciences 2006; 79: 121-129• Fiordaliso F, De Angelis N, Cuccovillo I, Bai A, Salio M, Serra DM, Bianchi R, Razzetti R, Latini R, Masson S. Effectof β-adrenergic and renin-angiotensin system blockade on myocyte apoptosis and oxidative stress in diabetichypertensive rats. Life Sciences 2007; 81: 951-959Serge Masson obtained his doctorate (PhD) in Biochemistry and Cellular Biology in 1990 at theUniversity of Marseilles (France), followed by a postdoctoral stay at the Panum Institute in Copenhagen(Denmark)Education1988-1990 Doctorate fellow, Faculty of Medicine, University of Aix-Marseilles, France119ANNUAL REPORT 2007

IRFMN1990-1993 Post-doctoral Researcher, Panum Institute and Assistant Lecturer, University ofCopenhagen, Denmark1993 Research Scientist, NMR Laboratory, Hospital “San Raffaele”, Milan, Italyfrom 1994Research Scientist, Department of Cardiovascular Research, Istituto di RicercheFarmacologiche "Mario Negri", Milano, ItalyMain fields of activityPhysiopathology, diagnostic and prognostic role of the activation of neuroendocrine systems incardiovascular diseasePositionfrom 2002from 2002from 2002Head of the Cardiovascular Endocrine Unit, responsible for Quality Assurance for theDepartment of Cardiovascular Research, Istituto di Ricerche Farmacologiche "MarioNegri", Milano, ItalyTutor of fellows of the School of Specialists in Pharmacological Research, Istituto diRicerche Farmacologiche "Mario Negri", Milano, ItalyFellows of the American Heart Association (Basic Council) and the Working Group onMyocardial Function of the European Society of CardiologySelected publications• Latini R, Masson S, Anand IS, Salio M, Hester A, Judd D, Barlera S, Maggioni AP, Tognoni G, Cohn NJ, for the Val-HeFT Investigators. The comparative prognostic value of plasma neurohormones at baseline in patients with heart failureenrolled in Val-HeFT. Eur Heart J 2004; 25: 292-299• Anand IS, Latini R, Florea VG, Kuskowski MA, Masson S, Signorini S, Mocarelli P, Hester A, Glazer R, Cohn JN, forthe Val-HeFT Investigators. C-reactive protein in heart failure: prognostic value and the effect of valsartan. Circulation2005; 112: 1428-1434• Masson S, Latini R, Anand I S, Barlera S, Judd D, Salio M, Perticone F, Perini G, Tognoni G, Cohn JN, on behalf of theVal-HeFT Investigators. The prognostic value of Big endothelin-1 in more than 2,300 patients with heart failure enrolledthe Valsartan in Heart Failure Trial (Val-HeFT). J Card Fail 2006; 12: 375-380• Masson S, Latini R, Anand IS, Vago T, Angelici L, Barlera S, Missov ED, Clerico A, Tognoni G, Cohn JN, on behalf ofthe Val-HeFT Investigators. Direct comparison of B-type natriuretic peptide (BNP) and amino-terminal proBNP in alarge population of patients with chronic and symptomatic heart failure. The Valsartan Heart Failure (Val-HeFT) data.Clin Chem 2006; 52: 1528-1538• Latini R, Masson S, Anand I S, Missov E, Carlson M, Vago T, Angelici L, Barlera S, Parrinello G, Maggioni AP,Tognoni G, Cohn J N, Val-HeFT Investigators. Prognostic value of very low plasma concentrations of troponin T inpatients with stable chronic heart failure. Circulation 2007; 116: 1242-1249• Staszewsky L, Wong M, Masson S, Barlera S, Carretta E, Maggioni AP, Anand IS, Cohn JN, Tognoni G, Latini R,Valsartan Heart Failure Trial Investigators. Clinical, neurohormonal, and inflammatory markers and overall prognosticrole of chronic obstructive pulmonary disease in patients with heart failure: data from the Val-HeFT Heart Failure Trial.J Card Fail 2007; 13: 797-804Enrico Bjørn Nicolis has attempted some courses in Computer Science at the University of Milan.Education1991-1999 “Research fellow”, Istituto di Ricerche Farmacologiche "Mario Negri", Milano, ItalyMain fields of activityData management and analysis of randomized clinical trials. Developing of database and tools for studiesof population genetics, particularly for linkage analysis.Positionfrom 2001 Head of the Bioinformatics Unit, Istituto di Ricerche Farmacologiche "Mario Negri",Milano, Italyfrom 1999 Research fellow of the Laboratory of Clinical Drugs Evaluationfrom 1997 System administrator at the EDP center, Istituto di Ricerche Farmacologiche "Mario Negri",Milano, Italyfrom 1991 Research fellow at the Medical Informatics and Applied Statistics Unit, Istituto di RicercheFarmacologiche "Mario Negri", Milano, ItalySelected publications• GISSI-Avoidable Delay Study Group. Epidemiology of avoidable delay in the care of patients with acute myocardialinfarction in Italy. A GISSI-generated study. Arch Intern Med 1995; 155: 1481-1488• Nobili A, Gebru F, Rossetti A, Schettino F, Zahn R W, Nicolis E, Macario G, Celani L, Acik V O, Farina M L, Naldi L.Doctorline: A private toll-free telephone medical information service. Five years of activity: Old problems and newperspectives. Ann Pharmacother 1998; 32: 120-125120ANNUAL REPORT 2007

IRFMN• Santoro E, Nicolis E, Franzosi MG.Telecommunication technology for the management of large scale clinical trials: TheGISSI experience. Comput Methods Programs Biomed 1999; 60: 215-223• Santoro E, Nicolis E, Franzosi M G, Tognoni G. Internet for clinical trials: Past, present, and future. Control ClinTrials 1999; 20: 194-201• Tognoni G, Franzosi MG, Nicolis E, Barlera S, Specchia C, Chiodini B, Crociati L, Ferrario L, PROCARDISConsortium. A trio family study showing association of the lymphotoxin-alfa N26 (804A) allele with coronary arterydisease. Eur J Hum Genet 2004; 12: 770-774• Barlera S, Specchia C, Farrall M, Chiodini BD, Franzosi MG, Rust S, Green F, Nicolis E, Peden J, Assmann G, CollinsR, Hamsten A, Tognoni G, PROCARDIS Consortium. Multiple QTL influence the serum Lp(a) concentration: agenome-wide linkage screen in the PROCARDIS study. Eur J Hum Genet 2007; 15: 221-227121ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe areas of interest of the Department of Cardiovascular Research include the experimental,clinical, genetic, epidemiological aspects of acute myocardial infarction, cardiac failure, cardiacarrhythmias, as well as the clinical and epidemiological investigation of cardiovascularprevention, hypertension and stroke. Following the successful experience of the GISSI-trials(Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto), the activation of largecollaborative networks in the setting of the National Health Service hospitals and in generalpractice has become a key characteristics of the Department, which can now rely on thepermanent collaboration of over 300 clinical groups and of several hundred generalpractitioners. Over the years, firm links have also been established with international leadingresearch groups.The experimental research activity concerns the physiopathology, the pharmacologicalmodulation and the prognostic role of the activation of the renin-angiotensin-aldosteronesystem, as well as other neurohormonal systems, in myocardial infarction and heart failure, thephysiopathology, the pharmacological modulation and prognostic role of the activation of theinflammatory processes in myocardial infarction and heart failure; a more recent research topicis the cell therapy of myocardial infarction.The activity in clinical research includes the clinical assessment of therapeutic strategies withlarge scale clinical trials in the field of acute coronary syndromes, congestive heart failure andatrial fibrillation. A recently developing area is the genetic epidemiology of myocardialinfarction and heart failure. Several studies have been conducted in the area of clinicalepidemiology and risk factors assessment of myocardial infarction.The collaboration with a large network of General Practitioners in the area of cardiovascularprevention allowed to test new hypotheses through large scale clinical trials and to evaluate theactual transferability of evidence based interventions in the every day practice throughepidemiological or outcome research studies. Pharmacoepidemiological studies through theanalysis of a large sample of Local Health Units drug prescriptions were also performed. Aresearch network of nurses has been developed with the main focus on the assessment of healthrelatedquality of life of patients and on the epidemiology of nursing interventions and theirimplications for patients' well being and outcomes.FINDINGS/MAIN RESULTSThe short pentraxin C-reactive protein (CRP) and the long pentraxin (PTX3) are markers ofcardiovascular risk and of severity of heart disease. However, the pthophysiologic role ofpentraxins in these diseases is largely unknown. Using knockout mice for pTX3 we have shownthat PTX3, a highly conserved protein from mouse to man, reduces inflammatory response inacute myocardial infarction and consequently ischemia/reperfusion injury. Thus PTX3 is morethan a simple marker of cardiovascular risk. The study has been done in collaboration withIstituto Clinico Humanitas, Rozzano, Milano (Prof Alberto Mantovani).We conducted a genetic study in the patients of the GISSI-Prevenzione trial to assess the effectof the APOE gene on mortality and on the response to statin treatment in secondary preventionafter myocardial infarction (MI). We analysed 3304 Italian patients with MI randomized topravastatin or no treatment, with a median follow-up time of 24.3 months. We found thatepsilon-4 allele is a determinant of pravastatin response in terms of survival. Thoughin the entire population investigated we found a beneficial effect of pravastatin in terms ofsurvival, only the epsilon-4 carriers seemed to have gained a significant benefit from thistreatment. We suggest that the effect of statins is of particular interest in this fraction of the122ANNUAL REPORT 2007

IRFMNpopulation and that genetic markers can help in identifying patients that benefit more fromstatin treatment.PROCARDIS data were analyzed with the goal to identify chromosomal regions associated tothe variability of quantitative phenotypes.The serum concentration of lipoprotein Lp(a), known to be highly heritable and associated withcardiovascular risk, was analyzed using a genome-wide linkage analysis. A highly significantlinkage was confirmed at LPA locus on chromosome 6q27, as well as another region ofsignificant linkage was detected on chromosome 13q22-31. These results have been publishedon the European Journal Human Genetics 2007: 15, 221-227 and provide a basis for furtherstudies of candidate genes in these regions.NATIONAL COLLABORATIONSAngiogenesis and Tumor Targeting Research Unit & Telethon Institute for Gene Therapy,Ospedale San Raffaele, MilanoANMCO (Associazione Nazionale Medici Cardiologi Ospedalieri)CINECA (Consorzio Interuniversitario per il Calcolo Automatico dell'Italia Nord-Orientale)CSeRMEG (Centro Studi e Ricerche in Medicina Generale)Gruppi organizzati di MMG (FIMMG, CoS, Ass.Cu.M.I., AMISI)IEO (Istituto Europeo di Oncologia), MilanoIFOM-FIRC, MilanoIstituto di Ricerca in Cure palliative Lino MaestroniLaboratorio di Endocrinologia, Ospedale Luigi Sacco, MilanoEmatologia, Ospedale Sant’Anna, TorinoRegione LombardiaSIBioC (Società Italiana di Biochimica Clinica e Biologia Molecolare)SIFO (Società Italiana di Farmacia Ospedaliera)Stem Cell Research Institute, Ospedale San Raffaele, MilanoUniversità degli Studi di Milano, Dipartimento di Medicina InternaUniversità degli Studi di Torino, Dipartimento di Anatomia, Farmacologia e Medicina ForenseUniversità degli Studi di Torino, Dipartimento di Sanità Pubblica e MicrobiologiaUniversità degli Studi di Verona, Dipartimento di Sanità PubblicaUniversità degli Studi di Verona, Istituto di Anatomia UmanaINTERNATIONAL COLLABORATIONSCecomet (Centro de Epidemiologia comunitaria y Medicina tropical, Esmeraldas) EcuadorCochrane Collaboration, Oxford, UKClinical Trial Research Unit, Auckland University, New ZealandCTSU (Clinical Trial Service Unit) /ISIS (International Studies on Infarct Survival), Oxford,UKDivision of Genetics and Development, Guy's, King's and St Thomas' School of Medicine,King's College, London, UKECLA (Estudios Cardiologicos de Latino-America)EVGN (European Vascular Genomics Network) VI Framework Program, Unione EuropeaJW Goethe University, Department of Cardiology, Frankfurt, GermanyKarolinska Institutet, Stockholm, SwedenPHRI (Population Health Research Institute), McMaster University, Hamilton, Ontario, Canada123ANNUAL REPORT 2007

IRFMNSIOP (International Society of Paediatric Oncology) EuropeUniversity of Minnesota, Minneapolis, USAUniversity of Oslo, NorwayWellcome Trust Centre for Human Genetics, University of Oxford, UKWONCA (World Organization of Family Doctors), EuropeEDITORIAL BOARD MEMBERSHIPCirculation, International Journal of Health Services, Journal of Clinical Epidemiology (GianniTognoni)Journal of Cardiac Failure, Journal of Cardiovascular Medicine (Roberto Latini)Assistenza Infermieristica e Ricerca, European Journal of Cancer Care, European Journal ofOncology Nursing, International Nursing Perspectives (Paola Di Giulio)PEER REVIEW ACTIVITIESAmerican Heart Journal, Atherosclerosis Thrombosis and Vascular Biology, CardiovascularResearch, Circulation, Circulation Research, Clinical Pharmacology and Therapeutics,European Heart Journal, European Journal of Cardiovascular Nursing, International Journal ofCardiology, International Journal of Obesity, Italian Journal of Cardiology, Journal of CardiacFailure, Journal of Internal Medicine, Journal of Cardiovascular Medicine, Life Sciences, TheLancet, PharmacoEconomics.NATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPEthical Committee of Lombardy RegionEthical Committee of the Provincia of VeronaAgenzia Italiana del Farmaco, Direzione Generale Farmaci e Dispositivi MediciEVENT ORGANIZATIONInvestigator's Meeting - Stato di avanzamento dello studio RE-LY02/03/07, Boheringer Ingelheim, Reggello (Firenze)Master di I° Livello in Ricerca Clinica dell’Università degli Studi di Milano, Facoltà diMedicina e Chirurgia, Dipartimento di Medicina Interna.Lezioni di Metodologia e Statistica:16/04/07 La ricerca clinica oggi: profit e no-profit. Il protocollo dello studio17/04/07 Il disegno degli studi clinici18/04/07 Legislazione sulla sperimentazione clinica e ruolo dei Comitati Etici26/04/07 Statistica descrittiva08/05/07 Statistica inferenziale (1)124ANNUAL REPORT 2007

IRFMN10/05/07 Statistica inferenziale (2)16/05/07 Monitoraggio degli studi clinici22/05/07 Analisi della sopravvivenzaIstituto di Ricerche Farmacologiche “Mario Negri”, MilanoInvestigator's Meeting - Stato di avanzamento dello studio Prono/Supino II01/06/07, Sala Gialla, Milano Convention Centre-MIC, SMART Fiera, MilanoInvestigator's Meeting – Valutazione di differenti strategie anestesiologiche per interventineurochirurgici sopratentoriali "NeuroMorfeo"14/11/07, Aula E, Istituto di Ricerche Farmacologiche “Mario Negri”, MilanoPARTICIPATION IN EVENTS IN WHICH THE DEPARTMENTWAS INVOLVEDAmerican College of Cardiology. 56th Annual Scientific Session, 24-27/03/07, New Orleans,USA- Microalbuminuria in chronic heart failure: prevalence and determinants in more that 2000patients enrolled in the GISSI-HF trial- Brain natriuretic peptide and severity of diastolic dysfunction: interaction with LV geometryand function in mild-moderate heart failure. The AREA-IN-CHF StudyIUSS Ferrara Università degli studi di Ferrara – Master di II° Livello, Scuola di RicercaClinica ed Epidemiologica, 28/03/07, Università di Ferrara, Italy- Introduzione alla farmacocinetica- Farmacocinetica di fattori endogeni e biomarcatoriAnnual Meeting of the Norwegian Society of Cardiology. Kardiologisk Varmote 2007,Radisson SAS Plaza Hotel, 3-5/05/07 Oslo, Norway- Changes over time vs single measurements of proBNP in risk stratification and monitoring ofpatients with chronic heart failure. Recent data with use of Roche TnT in relation to HFEuropean Society of Cardiology. Annual Meeting of the Working Group: Myocardial FunctionAdverse Cardiac Remodelling: mechanism and repair, 4-6/05/07, Kracow, Polonia- Cardiac mesoangioblasts are committed, self renewable progenitors, associated with smallvessels of adult mouse ventricle- Modulation of homing and survival of cardiac mesoangioblasts transplanted in the infarctedmouse heartRoche Diagnostics GmbH. proCardio Symposium. 5th International Symposium on NTproBNP.NT-proBNP and other biomarkers for the management of cardiac patients, 11-12/05/07, Seville, Spain- The prognosti value of high sensitive troponin T in patients with stable chronic heart failure.Data from the Val-HeFT trialConvegno Regionale ANCE Lombardia. Il cuore nella donna. “Un appuntamento annuale”.26/05/07, Hotel Michelangelo, Milano, Italy- Scarsa presenza femminile nella popolazione dei trial rispetto al mondo reale. Rischio di unindagine deformata e possibili correzioni125ANNUAL REPORT 2007

IRFMNAssociazione Nazionale Medici Cardiologi Ospedalieri. XXXVIII° Congresso Nazionale diCardiologia, 3-5/06/07, Firenze, Italy- Le evidenze sperimentali- Marcatori biologici circolanti in pazienti con bronco-pneumopatia cronica ostruttiva edinsufficienza cardiaca sintomatica. Dati dello studio GISSI-HF- La microalbuminuria nell’insufficienza cardiaca: prevalenza e correlati clinici in più di 2000pazienti dello studio GISSI-HF- Implementazione di un protocollo di infusione di insulina a gestione infermieristica per ilcontrollo intensivo della glicemia nel diabete con sindrome coronarica acuta- Validazione di un protocollo standardizzato per la transizione della terapia insulinica infusivaa quella sottocutanea nel diabetico con sindrome coronarica acutaHeart Failure Association of the European Society of Cardiology. Heart Failure Congress 2007,9-12/06/07, Hamburg, Germany- Prevalence and significance of microabuminuria in a large population of patients withchronich heart failure. Data from the Italian GISSI-HF trial- Metabolic syndrome in chronic heart failure: prevalence and relation to clinical features andaetiology- Cardioprotection by erythropoietin and its derivativeseMeeting. Cardiac Stem Cells: what we need for clinical translation, 27-28/06/07, Roma, Italy- Cardiac mesoangioblasts are committed, self renewable progenitors, associated with smalvessels of adult mouse ventricle- Modulation of homing and survival of cardiac mesoangioblasts transplanted in the infarctedmouse heartEuropean Society of Cardiology. ESC Congress 2007, 1-5/09/07, Vienna, Austria- Circulating biomarkers in patients with chronic obstructive pulmonary disease andsymptomatic heart failure. Data from the GISSI-HF trialEuropean Vascular Biology Organization. 4th European Meeting on Vascular Biology andMedicine, 17-20//09/07, Bristol, United Kingdom- Workshop V, Vascular Progenitors 2WONCA, CNGE. 13th WONCA 2007 Europe Conference – 1er Congrès de la MédicineGénérale en France. Re-thinking primary care in the European context. A new challenge forGeneral Pratice, 17-20/10/07, Paris, France.- Feasibility of a large scale randomised trial in General Practice: The Risk & Prevention StudySocietà’ Italiana Attività Regolatorie, Società Italiana di Farmacia Ospedaliera, Società diScienze Farmacologiche Applicate. La sperimentazione clinica dei farmaci e dispoistivi medici.I° corso di aggiornamento, 18-19/10/07, Roma, Italy- Gli studi no-profitAzienda Ospedaliera S. Giovanni Addolorata. I primi 30 giorni dopo l’infarto, 19-20/10/07,Roma, Italy- Ruolo della inibizione neurormonale (betabloccanti – inibizione del sistema renina –angiotensina – aldosterone)Casa di Cura Polispecialistica Presidio Ospedaliero ASL 22, Regione Veneto. Strategie diprevenzione cardiovascolare, 20/10/07, Peschiera del Garda (VR), Italy- Terapia cellulare cardiaca. A che punto siamo?126ANNUAL REPORT 2007

IRFMNAmerican Heart Association. AHA Scientific Session 2007, 4-7/11/07, Orlando, Florida- Levels of circulating progenitor cells in patients with chronic heart failure predict outcomesClinical Forum Srl, Next Stop Knowledge, 9-10/11/07, Istituto di Ricerche Farmacologiche“Mario Negri”, Milano, Italy- Statine in studi di popolazione e nel cardiopatico non ischemico con insufficienza cardiaca:dati disponibili- Sottoprogetti del GISSI-HF: obiettivi e dati basaliRegione Toscana–Consiglio Regionale “Pianeta Galileo” - La sperimentazione dei farmacisull’uomo, 17/11/07, Lucca, Italy- La sperimentazione dei farmaci sull’uomoEuropean Section of the Aldosterone Council ESAC Meeting 2007 – 1st International Meetingof the French Society of Hypertension, 13-14/12/07, Paris, France- Effects of canrenone on BNP, PIIINP and LV remodeling in patients with mild chronic heartfailure (AREA-IN-CHF study): final results.GRANTS AND CONTRACTSAIFA (Agenzia Italiana del Farmaco), AstraZeneca, Azienda Ospedaliera San Gerardo Monza,Chiesi Farmaceutici, Boehringer Ingelheim Italia SpA, BRAHAMS AG, CollegioInterprovinciale Milano-Lodi, Fondazione Cariplo, Fondazione Don Gnocchi Milano,Fondazione San Raffaele Milano, Heart Care Foundation, Istituto Auxologico di Milano,Istituto Nazionale Neurologico C. Besta, Kinetic Concepts Inc., Ministero della Salute, NovartisPharma, Oslo University, Oxford University, Population Health Research Institute-Mc MasterUniversity, Pfizer Italia, Roche Diagnostics, Sigma Tau, SPA Società Prodotti AntibioticiS.p.A., Takeda Italia S.p.A.127ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Abdulla J, Barlera S, Latini R, Kjoller-Hansen L, Sogaard P, Christensen E, Kober L, Torp-Pedersen CA systematic review: Effect of angiotensin converting enzyme inhibition on left ventricular volumes and ejectionfraction in patients with a myocardial infarction and in patients with left ventricular dysfunctionEur J Heart Fail 2007; 9: 129-135Barlera S, Specchia C, Farrall M, Chiodini BD, Franzosi MG, Rust S, Green F, Nicolis E, Peden J, Assmann G,Collins R, Hamsten A, Tognoni G, PROCARDIS ConsortiumMultiple QTL influence the serum Lp(a) concentration: a genome-wide linkage screen in the PROCARDIS studyEur J Hum Genet 2007; 15: 221-227Boccanelli A, Cacciatore G, Mureddu GF, De Simone G, Clemenza F, De Maria R, Di Lenarda A, Gavazzi A, LatiniR, Masson S, Porcu M, Vanasia M, Gonzini L, Maggioni AP, AREA IN-CHFBaseline characteristics of patients recruited in the AREA IN-CHF study (Antiremodelling Effect of AldosteroneReceptors Blockade with Canrenone in Mild Chronic Heart Failure)J Cardiovasc Med 2007; 8: 683-691Chiodini B, Franzosi MG, Barlera S, Signorini S, Lewis CM, D'Orazio A, Mocarelli P, Nicolis E, Marchioli R,Tognoni G, GISSI, SIBioC-GISSI Prevenzione GroupApolipoprotein E polymorphisms influence effect of pravastatin on survival after myocardial infarction in aMediterranean population: the GISSI-Prevenzione studyEur Heart J 2007; 28: 1977-1983Cicoira M, Maggioni AP, Latini R, Barlera S, Carretta E, Janosi A, Soler Soler J, Anand I, Cohn J, Val-HeFTInvestigatorsBody mass index, prognosis and mode of death in chronic heart failure: Results from the Valsartan Heart FailureTrialEur J Heart Fail 2007; 9: 397-402Evangelista V, De Berardis G, Totani L, Avanzini F, Giorda CB, Brero L, Levantesi G, Marelli G, Pupillo M, IacuittiG, Pozzoli G, Di Summa P, Nada E, De Simone G, Dell'Elba G, Amore C, Manarini S, Pecce R, Maione A, TognoniG, Nicolucci APersistent platelet activation in patients with type 2 diabetes treated with low doses of aspirinJ Thromb Haemost 2007; 5: 2197-2203Fiordaliso F, De Angelis N, Bai A, Cuccovillo I, Salio M, Serra DM, Bianchi R, Razzetti R, Latini R, Masson SEffect of Beta-adrenergic and renin-angiotensin system blockade on myocyte apoptosis and oxidative stress indiabetic hypertensive ratsLife Sci 2007; 81: 951-959Grasselli G, Gattinoni L, Kavanagh B, Latini R, Laupacis A, Lemaire F, Pesenti A, Suter P, Slutsky A, Tognoni GFeasibility, limits and problems of clinical studies in Intensive Care UnitMinerva Anestesiol 2007; 73: 595-601Krum H, Latini R, Maggioni AP, Anand I, Masson S, Carretta E, Ingrilli' F, Pettinati G, Glazer R, Tognoni G, Cohn JStatins and symptomatic chronic systolic heart failure. A post-hoc analysis of 5010 patients enrolled in Val-HeFTInt J Cardiol 2007; 119: 48-53Latini R, Masson S, Anand I S, Missov E, Carlson M, Vago T, Angelici L, Barlera S, Parrinello G, Maggioni AP,Tognoni G, Cohn J N, Val-HeFT InvestigatorsPrognostic value of very low plasma concentrations of troponin T in patients with stable chronic heart failureCirculation 2007; 116: 1242-1249Sarto P, Balducci E, Balconi G, Fiordaliso F, Merlo L, Tuzzato G, Pappagallo GL, Frigato N, Zanocco A, ForestieriC, Azzarello G, Mazzucco A, Valenti M T, Alborino F, Noventa D, Vinante O, Pascotto P, Sartore S, Dejana E,Latini REffects of exercise training on endothelial progenitor cells in patients with chronic heart failureJ Card Fail 2007; 13: 701-708Saunders CL, Chiodini B, Sham P, Lewis CM, Abkevich V, Adeyemo AA, de Andrade M, Arya R, Berenson GS,Blangero J, Boehnke M, Borecki IB, Chagnon YC, Chen W, Comuzzie AG, Deng HW, Duggirala R, Feitosa MF,128ANNUAL REPORT 2007

IRFMNFroguel P, Hanson RL, Hebebrand J, Huezo-Dias P, Kissebah AH, Li W, Luke A, Martin LJ, Nash M, Ohman M,Palmer LJ, Peltonen L, Perola M, Price RA, Redline S, Srinivasan SR, Stern MP, Stone S, Stringham H, Turner S,Wijmenga C, Collier DAMeta-analysis of genome-wide linkage studies in BMI and obesityObesity 2007; 15: 2263-2275Silletta MG, Marfisi RM, Levantesi G, Boccanelli A, Chieffo C, Franzosi MG, Geraci E, Maggioni AP, Nicolosi GL,Schweiger C, Tavazzi L, Tognoni G, Marchioli R, GISSI-PrevenzioneCoffee consumption and risk of cardiovascular events after acute myocardial infarction. Results from the GISSI(Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico)-Prevenzione trialCirculation 2007; 116: 2944-2951Staszewsky L, Wong M, Masson S, Barlera S, Carretta E, Maggioni AP, Anand IS, Cohn JN, Tognoni G, Latini R,Valsartan Heart Failure Trial InvestigatorsClinical, neurohormonal, and inflammatory markers and overall prognostic role of chronic obstructive pulmonarydisease in patients with heart failure: data from the Val-HeFT Heart Failure TrialJ Card Fail 2007; 13: 797-804LAY PRESS SELECTION PUBLISHED IN 2007Avanzini F, Marelli G, Donzelli W, Sorbara L, Palazzo E, Bellato L, Colombo E, De Martini M, per il Gruppo diStudio DDDProtocollo di infusione di insulina a gestione infermieristica per il controllo intensivo della glicemia. G It DiabetolMetab 2007; 27: 202-211.Clavenna A, Bonati M, Campi R, Labate L, Nobili A, Pasina L, Tettamanti M, Monesi L, Marzona I, Roncaglioni MC, Bortolotti A, Fortino I, Locateli G W, Giuliani GLa prescrizione di farmaci per i bambini e gli anziani nella ASL di LeccoRicerca & Pratica 2007; n.135: 100-112Marchioli R, Marfisi R M, Borrelli G, Chieffo C, Franzosi M G, Levantesi G, Maggioni A P, Nicolosi G L, ScaranoM, Silletta M G, Schweiger C, Tavazzi L, Tognoni GEfficacy of n-3 polyunsaturated fatty acids according to clinical characteristics of patients with recent myocardialinfarction: insights from the GISSI-Prevenzione trialJ Cardiovasc Med 2007; 8: s34-s37Masson S, Latini RAn update on N-terminal pro-brain-type natriuretic peptide for risk stratification in chronic heart failureEur Cardiov Dis 2007; 1: 35-38Masson S, Latini R, Tacconi M T, Bernasconi RIncorporation and washout of n-3 polyunsaturated fatty acids after diet supplementation in clinical studiesJ Cardiovasc Med 2007; 8 suppl 1: s4-s10129ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Cardiovascular Clinical PharmacologyThe effects of mesoangioblasts and of different progenitor cells on injuryafter experimental myocardial infarction in the mouseMany studies have demonstrated that autologous and homologous cells of various origins canrepair myocardium damaged due to an acute ischemic insult. Mesangioblasts are potentiallyinteresting when compared with bone marrow precursors because (a) they are easily expandedand (b) obtainable by a biopsy of skeletal muscle in man. Mesoangioblasts isolated from humanheart biopsies migrate and home in the heart of immunodeficient mice after coronary ligation,and they survive for at least 4 weeks. Studies are ongoing to establish their protective andregenerative potential, as a basis for possible clinical studies. The same model of coronaryligationin immunodeficient mice is being used for testing in vivo the angiogenic potential ofother progenitor cells such as mononuclear cells Tie-2+ (collaboration with Michele De Palma,HSR, Milano), CD34+ (collaboration with Franca Fagioli,Osp S. Anna, Torino), CD133+(collaboration with Giulio Pompilio, Centro Cardiologico Monzino, Milano). This research ispartly done within the sixth EU program, European Vascular Genomics Network (EVGN,www.evgn.org).Characterization of the cardiac phenotype of PTX3 in knock-out micePTX3 has been shown to have a non redundant role in reproduction and in the resistance tofungal infection (Aspergillus fumigatus). However, its role in the cardiovascular apparatus hasnot been well defined, though it is an independent prognostic marker after acute myocardialinfarction in man. The role of PTX3 in myocardial infarction appears to be protective, in factPTX3-null mice had larger infarct size than mice wild type. The observation can be explainedby the decrease in inflammatory response induced by PTX3 through complement cascade.Therapeutic potential of stem cell therapy in animal models of diabeticcardiomyopathyDiabetes is a major risk factor for the development of various cardiovascular complications.Morbidity and mortality associated with diabetes are essentially related to vascular lesions. Theaccumulation of extracellular matrix in the diabetic aorta is involved in increases in wallstiffness and eventually in the development of cardiac dysfunction. Neural Stem Cells (NSC)are multipotent cells which have the ability to differentiate also to endothelial progenitors cells(EPCs). Decreased levels of EPCs have been reported in both type 1 and type 2 diabeticpatients. The large decrease in elastin/collagen ratio, index of aortic elasticity, and alteredangiogenesis found in diabetic rats in our study could reflect the progressive deterioration ofvascular structure and function that may lead to turbulent blood flow and eventually hamperingthe hemodynamic function. We observed, for the first time, that treatment with mouse NSCattenuated the remodeling of the arterial extracellular matrix and restored the correct angiogenicresponse, and eventually attenuated the mild hypertensive state of diabetic rats suggesting apotential role of these cells in reducing the vascular dysfunction in diabetes.A new study has been already planned to evaluate the mobilization and homing of endogenousprogenitor cells (EPCs) and exogenous (mesoangioblast) progenitor cells injected into the leftventricle chamber in a genetic model of obesity and type II diabetes, and by this way theircontribution to the regeneration of myocardium and blood vessels after an experimentalmyocardial infarction.130ANNUAL REPORT 2007

IRFMNPulmonary injury by hydrochloric acid in the mouse: a model ofAspiration pneumonitis to test protective interventionsAspiration pneumonitis (AP) occurs when the acid content of the stomach makes his waythrough the larynx in the lower respiratory tract. Patients with consciousness disturbance are atrisk for this event. Specifically, it has been shown that Pulmonary Aspiration can complicatebetween 0.47-1.41% general anesthesia procedures.The main injurious mechanism of AP is the chemical insult due to the low pH of gastricsecretions, which causes a chemical burn of the airway tree and of the alveolar structures. Thecourse of AP can be extremely variable, ranging from the “silent aspiration” characterized by amodest desaturation to the dramatic sequelae of Acute Lung Injury (ALI) and Acute RespiratoryDistress Syndrome (ARDS), requiring prolonged mechanical ventilation and potentially leadingto death. The purpose of this research is to establish and characterize under different aspects amurine model of regional AP, allowing a two week survival of the animals and the evaluation ofthe long term evolution of the injury by multiple tests, computed tomography, biochemicalassays, histological evaluation, lung function and gas-exchange.Conditional transgenic model of cardiac HGF expression to promoteneovascularization and to recruit stem cells in the myocardial infarctionGrowth factors such as hepatocyte growth factor (HGF) through angiogenic and anti-apoptoticeffects may promote cardiac repair after myocardial infarction and in heart failure. The cardiacspecificα-Myosin Heavy Chain (MHC-α) transactivator was used to direct expression of HGFto cardiomyocytes: by this way the effects of HGF can be tested under cardiac ischemia andreperfusion, without the need for administration of exogenous HGF. The Heart-HGF transgenicmodel is being used to verify the ability of HGF in vivo to promote neovascularisation, toprotect cardiomyocytes from apoptosis, to recruit and activate endogenous or transplanted stemcells and to sustain their cellular replication and differentiation into cardiomyocytes afterischemic damage and reperfusion.Roles of macrophages in cardiac ischemia/reperfusion injury and incardiac repairMacrophages either resident or from blood-borne monocytes play several key roles in theresponse of the heart to ischemic injury. They may be useful in particular during cardiac repair,when collagen deposition occurs and neonagiogenesis, stimulated by growth factors producedby macrophages. The aim of the project is to assess the relevance of macrophages in myocardialrepair and scar formation after myocardial infarction, in the attempt to dissect the role ofinflammatory cells in myocardial injury vs repair.The effects of coronary ligation (with/without reperfusion) will be tested in mice in whichmachrophages can be ablated by administration of Difteria toxin: transgenic mice expressinghuman difteria toxin receptor (CD11b-DTR) will respond to the toxin. Since machrophagespeak in mouse heart on day 1 after MI and return to baseline by day 7, the ablation should lastno more than 7 days coronary ligation and reperfusion. The heart will be evaluatedhistologically for infarct size, myocyte loss, immunostain for machropages and PMN,capillarydensity (angiogenic response to ischemia). Cardiac function will be evaluated byechocardiography at week 6-8. Other experiments might be envisaged to assess the role ofmachrophages in modulating homing and/or retention of stem cells administered to mice withinfarction.GISSI-HF: biohumoral sub-study and microalbuminuriaThe clinical trial GISSI-HF (follow up has been completed by 2007) is designed to assesswhether two treatments (a statin and n-3 polyunsaturated fatty acids or PUFA) can improve theprognosis of patients with heart failure of any etiology, with preserved or compromised left131ANNUAL REPORT 2007

IRFMNventricular ejection fraction. A biohumoral substudy aims at exploring the pathophysiology ofheart failure and mechanisms of action of the treatments in study. In 1200 patients enrolled inthe substudy, blood samples are collected at enrolment and after three months to measureplasma PUFA, and markers of ventricular myocyte stress (brain natriuretic peptides, BNP andN-terminal proBNP), myocardial damage (cardiac troponin T, measured with the standard assayor with a newly developed high sensitive method) and inflammation (C-reactive protein, CRP,pentraxin-3, PTX3). Baseline fraction of n-3 PUFA averages 3.4 and increases by about 30%over 3 months. Baseline BNP concentration in 1223 patients is 141 pg/mL (median) andcorrelates with the severity of heart failure. The number of circulating endothelial progenitor hasbeen measured in a smaller group of 68 patients, in collaboration with the laboratory of E.Dejana (IFOM). Number of endothelial progenitor cells in patients with heart failure is 30-50%lower than in healthy volunteers was inversely correlated to morbidity/mortality in a populationof patients from 5 Italian centers and a Cardiology center in Frankfurt (collaboration withAndreas Zeiher). Microalbuminuria (defined as the ratio between urinary concentrations ofalbumine and creatinine) is being measured in more than 2000 patients enrolled in the GISSI-HF trial as an indicator of renal endothelial dysfunction. Microalbuminuria (albumine/creatinine= 30-299 mg/g) is present in 19% of the patients and is associated to inflammation, endothelialdysfunction and neurohormonal activation. Novel and more stable circulating biomarkers, havebeen successively assayed. They belong to the family of natriuretic peptides (mid-regional proatrialnatriuretici peptide, MR-proANP), endothelin (C-terminal pro-endothelin-1, CT-proET-1),vasopressin (C-terminal pro-vasopressin, CT-proAVP) and adrenomedullin (mid-regional proadrenomedullin,MR-proADM). Other markers related to infection and innate immunity arecurrently measured.Anti-remodeling effect of aldosterone receptors blockade with canrenonein mild chronic heart failure: the AREA IN-CHFThe RALES study has shown that spironolactone, an aldosterone receptor antagonist, reducesthe risk of morbidity and mortality both from progressive heart failure (HF) and sudden death inpatients with moderate to severe HF. Aldosterone antagonists may be effective because theyoppose the effects of aldosterone on sodium retention, loss of magnesium and potassium,sympathetic activation, vascular compliance and cardiac fibrosis. In AREA IN-CHF, amulticentre randomized, placebo-controlled clinical trial, the effect of canrenone, a metaboliteof spironolactone, is assessed in patients with mild chronic HF (n= 500) in terms ofechocardiographic left ventricular remodeling and a combined end-point of mortality andmorbidity. The design of the study includes blood collection in all patients at randomization andafter 6 months to determine the plasma concentration of aldosterone, brain natriuretic peptideand a circulating marker of collagen turnover (propeptide N-terminal of collagen III). Thesebiohumoral factors are assayed centrally at the Department of Cardiovascular Research andshould give insight on the effect of canrenone on these markers and on their relation with studyoutcome. There is no significant difference between canrenone and placebo for the primaryendpoint, changes in end-diastolic left ventricular volume. On the other hand, canrenoneimproves systolic function and reduces left ventricular mass compared to placebo. BNPconcentration, but not PIIINP, is drastically reduced in patients randomized to canrenone.PTX-3, a novel long pentraxin is an marker of severity of disease and ofoutcome in cardiovascular diseases, independent of C-reactive proteinPTX-3 is a novel long pentraxin whose expression is induced by cytokines in endothelial andmononuclear cells, mostly in striated muscle and heart, while C-reactive protein (CRP) ismainly synthesized in the liver. PTX3 was shown to peak in plasma around 7 h after onset ofsymptoms of MI and to be an independent predictor of 3-month mortality. PTX3 has beenassayed with a more accurate method in 1200 patients with symptomatic heart failure (GISSI-132ANNUAL REPORT 2007

IRFMNHF), and will be assayed in 350 patients with atrial fibrillation (GISSI-AF) and in 1400 patientswith heart failure (CandHeart) to explore its role in other cardiovascular diseases. First resultsindicate that PTX3 is associated with different clinical characteristics in patients with heartfailure, including advanced age, ventricular dysfunction, functional class (NYHA class), andcomorbidities such as atrial fibrillation and diabetes.Echocardiographic and biohumoral substudy – GISSI-AF trialThe GISSI Atrial Fibrillation trial (GISSI-AF) tests the efficacy of Valsartan, an angiotensin IIAT1-receptor blocker, in the prevention of atrial fibrillation recurrence in 1400 patients. Asubstudy of the GISSI-AF is currently ongoing to evaluate the potential role of biohumoralfactors and cardiac structural remodeling in the reoccurrence and severity of atrial fibrillation. Inapprox. 400 patients three serial echocardiographic exams (at randomization, 6 months and 1year) and contemporaneous blood collection are performed. Left ventricular and atrialdimensions will be determined by echocardiography, whereas plasma levels of BNP, NTproBNP,troponin T (high sensitive method), endothelin-1 and inflammatory markers (CRP,PTX3) will be measured. Besides giving clues on the pathophysiology of atrial fibrillation, themost common arrhythmia in elderly, this substudy aims at providing mechanical insights of thepotential benefits of the study drug. The study was completed on January 31, 2008. Centralreadings of echocardiograms and assay of circulating biomarkers are ongoing.CandHeart: effects of candesartan on BNP and left ventricular function inpatients with symptomatic heart failureCandesartan, an antagonist of angiotensin II type 1 receptors, significantly reduces mortalityand morbidity in heart failure, as shown by the CHARM trials. The principal objective of thetrial is to assess the effects Candesartan on circulating levels of brain natriuretic peptide (BNP)in patients suffering from CHF with depressed or preserved left ventricular (LV) systolicfunction. The patients (approx. 1400) will be followed for 1 year. Serial biohumoral andechocardiographic determinations will be performed at randomization, and after 3 and 12months (end of study). Besides BNP, other biohumoral markers such as aldosterone, C-reactiveprotein, PTX3 and the microalbuminuria will be measured. The first patient was randomized inDec. 2005. About 450 patients have been randomized by the end of 2007.Trial Prone/Supine 2: effects of prolonged prone position on survival insevere acute respiratory distress syndrome (ARDS)This trials extends the findings of the previous Prone/Supine trial that showed in 304 patientswith ARDS that 6-hour pronation in the first 10 days improved blood gas parameters, but didnot reduce mortality (around 50% in the first 6 months). This was one of the rare example of amulticenter trial in Intensive Care performed entirely in Italy. The same group of Centers(headed by the Istituto di Anestesia e Rianimazione from the Policlinico di Milano, L Gattinoniwith data management by Mario Negri) has launched a new trial that aims at evaluating theeffect of a protocol of prolonged pronation (20 hours a day) in patients with severe ARDS, asubgroup that showed a trend towards improved survival on pronation in the previous study.Three hundred and twenty five patients out of the target of 340 have been enrolled in 20 centers;enrollment should be concluded by May 2008 and data analyzed by the statisticians at theDepartment of Cardiovascular Research.DyDa: left ventricular dysfunction in diabetes. Prevalence and incidenceof left ventricular dysfunction in diabetics patients without clinical cardiacdiseaseThis is a prospective, multicentric, national and epidemiological trial aimed at evaluating theprevalence of left ventricular dysfunction (systolic or diastolic) in 1000 patients with type 2133ANNUAL REPORT 2007

IRFMNdiabetes mellitus but no clinical cardiovascular disease at enrolment. The incidence of leftventricular dysfunction will be monitored during a 2-year follow-up using ECG andechocardiography. The Biomarker Core Laboratory will evaluate the biohumoral profile of thesepatients at study entry, measuring the circulating levels of brain natriuretic peptide, C-reactiveprotein, microalbuminuria and glycated hemoglobin. Enrolment started in July 2006 will beconcluded in March 2008. In a subgroup of patients from the study, levels of circulatingprogenitor cells and their angiogenic potential in vitro will be measured to assess whether theyare associated to risk of developing cardiovascular disorders in diabetics.Albumin Italian Outcome Sepsis Study. The ALBIOS Study (AIFA)ALBIOS is a multicenter, controlled, randomized clinical trial that compares the efficacy ofhuman albumin and a crystalloid solution for volume replacement in patients with severe sepsisor septic shock. The primary endpoint is survival at 28 and 90 days after enrolment. Secondaryendpoints include the number of organ dysfunctions, severity of organ dysfunction (SOFAscale), and lengths of stay in (intensive care unit) ICU and in hospital. More than 150 ICU inItaly are expected to participate to this large study, coordinated by the Ospedale MaggiorePoliclinico in Milan and the Consorzio Mario Negri Sud. In a substudy coordinated by theLaboratory of Cardiovascular Clinical Pharmacology from the Mario Negri Institute in Milan, acentralized blood bank will be collected to provide insights on the potential effects of albuminon markers of inflammation, infection, cardiac function and coagulation in about 700 patients.The first patient should be enrolled in the first semester of 2008.Clinical, biochemical and instrumental predictors of outcome inrehabilitation after cardiac surgery (MIUR)Due to the short length of hospital recovery in patients with cardiac disease, the period ofrehabilitation becomes crucial and is indicated for almost all the cardiomyopathies, includingmyocardial revascularization and surgery of cardiac valves. The objective of this study is toevaluate the prognostic role of circulating biomarkers in 250 patients enrolled in 5 Centers forrehabilitation after cardiac surgery. A plasma bank will be collected under the responsibility ofthe Laboratory of Cardiovascular Clinical Pharmacology from the Mario Negri Institute to assaypotential markers of interest. The project is coordinated by the Fondazione Don Gnocchi inMilan. The first patient should be enrolled in the first semester of 2008.PUFAxStatin: a clinical study on the interaction between statins and n-3polyunsaturated acids (PUFA) on the lipid and inflammatory profile ofdiabetic patients at cardiovascular riskThere are potential beneficial pharmacological interactions between statins and n-3 PUFA onlipid and inflammatory metabolism. The present study assesses the effects of these two drugs,alone or in combination on circulating biomarkers of inflammation and on the lipid profile ofdiabetic patients at high cardiovascular risk (hypertension, ischemic heart disease, smoking). Atotal of 123 patients have been enrolled. In collaboration with the Laboratory ofNeuroimmunology, the effect of statin and n-3 PUFA on the ex-vivo production of proinflammatorycytokines by stimulated whole blood has been evaluated. First results indicatethat the serum concentrations of LDL-cholesterol and triglycerides are significantly reduced inpatients receiving the statin, alone or in association with n-3 PUFA. Markers of ventricularfunction (N-terminal probrain natriuretic peptide, NT-proBNP) and inflammation (C-reactiveprotein) are significantly improved in patients enrolled in the group treated with statin. Thereare no overt differences among the experimental groups for the other markers of inflammationand endothelial dysfunction. The study is conducted in collaboration with the Laboratory ofClinical Drug Evaluation.134ANNUAL REPORT 2007

IRFMNEvaluation of different anesthesiological strategies for supratentorialneurosurgery. The NeuroMorfeo Study (AIFA)The aim of the study is to evaluate whether an anesthesia with volatile anesthetics is equivalentto endovenous anesthetics for elective supratentorial surgery. This is a multicenter, randomized,controlled and opened study, based on a design of equivalence for comparison of differentanesthesiological strategies. Patients to be included (n= 393) will be selected in 14neurosurgical centers in Italy for elective intracranial surgery with supratentorial lesions withoutsigns of endocranial hypertension (range of age 18-75 years). The biohumoral response tosurgical stress will be measured as an indicator of homeostasis and neurovegetative status. Theurinary excretion of catecholamines and cortisol and the plasma concentration of cortisol will bemeasured in a central laboratory. The study is coordinated by the San Gerardo Hospital inMonza and by the Department of Cardiovascular Research from the Mario Negri Institute. Thefirst patient has been enrolled in December 2007.Laboratory of Clinical Drug EvaluationPROCARDIS: A genome-wide strategy to identify susceptibility loci inprecocious coronary artery diseaseThe PROCARDIS research programme, a genome-wide strategy to identify susceptibility lociin precocious coronary artery disease (CAD) supported by the 5th Framework Programme ofthe EC, was initiated as a collaboration between the universities of Oxford and Münster, theKarolinska Institute, the Mario Negri Institute with the support of the GISSI group, Oxagen, abiotechnology company, and AstraZeneca. The objectives of the first stage of this programmewere to collect a minimum of 2000 affected sibling pairs (ASPs) and families with precociousCAD and to apply genome-wide linkage mapping techniques, by typing anonymous highlyinformative markers spaced throughout the genome, to identify chromosomal regions which arelinked to the susceptibility to early-onset CAD. The study design is based on familyascertainment, to allow non-parametric linkage analyses (in ASPs). The PROCARDIS collected2,036 CAD families from four European countries, in order to maximise the power of detectinggenes that confer modest risks. A genome-wide linkage scan identified three promising regionsfor intensive study; one of the linked regions (Chromosome 17) was confined to families withmultiple cases of myocardial infarction and was replicated in a second independent series offamilies. In addition the linkage scan confirmed a previously identified locus on Chromosome 2.These results demonstrate that novel CAD susceptibility genes are tractable to positionalcloning which promises to lead to the identification of new molecular insights into thiscondition, and hopefully, new treatments.Additionally, the program has collected nuclear families (e.g. parent-offspring trios) fortransmission-based tests of association for fine mapping by linkage disequilibrium analysis andtesting of positional candidate genes.Extensive clinical and biochemical intermediate phenotype data have also been collected bybringing study subjects in to dedicated clinics; these data will allow mapping of known andnovel quantitative risk factors which will be important in prioritising positional candidate genesat mapped chromosomal loci. The serum concentration of lipoprotein Lp(a), known to be highlyheritable and associated with cardiovascular risk, was analyzed using a genome-wide linkageanalysis. A highly significant linkage was confirmed at LPA locus on chromosome 6q27, aswell as another region of significant linkage was detected on chromosome 13q22-31. Theseresults provide a basis for further studies of candidate genes in these regions. Further quantitavephenotypes have been already analyzed (Body Mass Index) or are in progress (Homocisteine).The second stage of PROCARDIS, supported by the EC 6th Framework Programme, isconducting a large “genome wide association” study, where the patients with myocardialinfarction enrolled in the first stage will be compared with control subjects to identify novel135ANNUAL REPORT 2007

IRFMNcandidate genes. The results will be replicated in different populations. The collaboration hasbeen extended to include complementary expertise, as proteomics, bioinformatics, functionalanalysis.GISSI-HF: A large scale clinical trial testing the effects of n-3 PUFA andRosuvastatin on mortality/morbidity of patients with symptomaticCongestive Heart FailureThe GISSI (Gruppo Italiano per lo Studio della Sopravvivenza nell'Insufficienza cardiaca) is acollaborative group endorsed by ANMCO (Associazione Nazionale Medici CardiologiOspedalieri) and by the Istituto Mario Negri, active from 20 years in the cardiovascular researchfield. The GISSI-HF is the fifth large scale clinical trial conducted by the Group.While pharmacological treatments specifically targeted to the cardio-circulatory system havebeen largely investigated, scanty controlled data are available concerning the role of dietary andmetabolic approaches in the management/outcome of patients with heart failure. The GISSI-HFis a prospective, multicenter, randomized, double blind, placebo controlled study, withrandomized allocation of patients with a clinical diagnosis of heart failure to:Randomization 1 (R1): n-3 PUFA vs corresponding placebo;Randomization 2 (R2): rosuvastatin vs corresponding placebo.The primary objective of the GISI-HF is to demonstrate that, in patients with heart failuretreated at the best of recommended treatment, long term administration of n-3 PUFA and/orrosuvastatin is more effective than the corresponding placebo in the reduction of all-causemortality and all-cause mortality or cardiovascular hospitalizations.The study follow-up will last at least 3 years. The study started in September 2002 with theparticipation of 364 departments of cardiology. The recruitment phase has been completed withmore than 7000 patients randomized.Several substudies focus on possible mechanistic effects of the study treatments:ventricular remodeling (echo); biohumoral; genetic; arrhythmic and autonomic pattern (Holtermonitoring); exercise capacity; cognitive function; burden of care.The follow-up was completed and final results are expected to be presented in 2008. The projectis conducted in collaboration with the Laboratory of Cardiovascular Clinical Pharmacology.GISSI-HF Genetic SubstudyThe role of genetic factors in causes, evolution, prognosis and treatment of heart failure islargely unexplored, with the exception of heart failure originated by specific cardiomyopathies(such as dilated, hypertrophic, arrhythmogenic right ventricular cardiomyopathies), for whichthe role of heritable gene mutations is increasingly well understood. Heart failure (HF) is asyndrome with different etiologies, and more than one half is caused by coronary heart disease(CHD). A genetic substudy to GISSI-HF (see "ad hoc" section) offers the opportunity toimprove knowledge on the role of genetic factors involved in heart failure, through a collectionof blood samples of a large population of patients, involving cases of heart failure of differentetiologies, i.e. non-ischaemic and ischaemic heart disease.The objective of the genetic substudy is 1) to assess the relationships between thepolymorphysms of various candidate genes and the clinical outcome in patients enrolled inGISSI-HF study; 2) to assess whether these relationships are modified by the experimentaltreatments.The genetic markers to be studied will initially focus on polymorphisms of genes involved inlipid metabolism and inflammation. However, as new genetic information is being accumulatedcontinually, at present it is not known if other polymorphisms/genes will be important at thetime of study completion. The possibility to assay other allelic variants is then foreseen. TheGISSI-HF genetic substudy is conducted by nearly 100 Centres that have included 2200patients.136ANNUAL REPORT 2007

IRFMNGISSI-Prevenzione-Genetic StudyMyocardial infarction is a multifactorial disease. While the role of known risk factors oncoronary heart disease susceptibility is well defined, the impact of the genetic components andits interaction with environmental factors need investigation. The GISSI-Prevenzione trialinvestigated the effects of pharmacological treatments with n-3 PUFA and pravastatin onmorbidity and mortality after myocardial infarction. During the study more than 8000 samplesof a large population of patients affected by this disease has been collected and stored with thecollaboration of SIBioC (Società Italiana di Biochimica Clinica e Biologia Molecolare). TheGISSI-Prevenzione-Genetic Study investigates the role of genetic factors in ischaemic heartdisease. The objectives of the project are 1) to assess the relationships between thepolymorphysms of various candidate genes and the clinical outcome in patients enrolled in thelarge clinical trial GISSI-Prevenzione study; 2) to assess whether these relationships aremodified by the pharmacological treatments. According to these objectives, we investigated therelationship between APOE, mortality and the response to treatment in 3304 myocardialinfarction survivors randomized to pravastatin or no treatment. We found that epsilon 4 allele isa determinant of pravastatin response in terms of survival. Though in the entire populationinvestigated we found a beneficial effect of pravastatin in terms of survival, only the epsilon 4carriers seemed to have gained a significant benefit from this treatment. We suggest that theeffect of statins is of particular interest in this fraction of the population and that genetic markerscan help in identifying patients that benefit more from statin treatment.Association studies in the same population on the adiponectin gene and C-reactive protein genevariants are in progress.GISSI-AF. Clinical trial testing the efficacy of an angiotensin II AT1-receptor blocker in Atrial FibrillationThe GISSI-AF is a randomized, prospective, parallel group, placebo-controlled, multicenterstudy on the use of valsartan, an angiotensin II AT1-receptor blocker in the prevention of AtrialFibrillation (AF) recurrence.Atrial fibrillation is the most frequent form of arrhythmia in clinical practice, affecting 6% ofpeople over 65 years old. The traditional therapies are often able to restore the sinus rhythm butare subject to a very high percentage of relapses, above all when the AF has been present for along time and when there are structural changes at both atrial and ventricular level. The reninangiotensin-aldosteronesystem (RAAS) plays a key role in the “remodeling” phenomenon,which makes increasingly irreversible the electrical, mechanical and structural properties of theatrial tissue. Existing experimental and clinical data do not allow any definite conclusionregarding the efficacy of an angiotensin II AT1-receptor blocker in the prevention of AF. TheGISSI group decided to conduct a specific trial of adequate size versus placebo aimed to assessif the addition of valsartan on top of established therapies can reduce the recurrence of atrialfibrillation in patients with a history of recent AF associated with cardiovasculardiseases/comorbidities. The study has recruited and treated for 12 months 1400 patients . Abiohumoral and echo substudy including 400 patients will be integral part of the main studyfocusing on how possible prognostic factors can be affected by the study treatment.According to the GISSI philosophy, GISSI-AF is a pragmatic trial, with broad selection criteriato mimic real clinical practice as much as possible. The final results are expected to bepresented in 2008. The project is conducted in collaboration with the Laboratory ofCardiovascular Clinical Pharmacology.The Population Health Research Institute (PHRI), McMaster University, Hamilton, Ontario, isthe coordinating center of a multinational network of cardiology clinics that collaborate to the137ANNUAL REPORT 2007

IRFMNconduction of multicenter large scale clinical trials (nearly 40 Countries and more than 600cardiology clinics). The Laboratory of Clinical Drug Evaluation is responsible for the scientificcoordination in Italy of some of these trials (ACTIVE, RE-LY, CURRENT).ACTIVE study (Atrial Fibrillation Clopidogrel Trial with Irbesartan forprevention of Vascular Events)The aims of the study were to evaluate whether clopidogrel plus acetylsalicylic acid (ASA) issuperior to ASA alone (A study) and non-inferior to standard oral anticoagulant therapy (Wstudy) in preventing vascular events in patients with atrial fibrillation and to evaluate whetherblood pressure lowering with irbesartan is superior to placebo in preventing vascular events inpatients with atrial fibrillation (I study). The primary efficacy outcome is the first occurrence ofstroke, myocardial infarction, vascular death over the duration of follow-up, (a minimum of 2and maximum of 4 years approximately). A sample size of 14000 patients was planned, 6500 inthe W study (testing the efficacy of warfarin vs ASA + clopidogrel) and 7500 in the A study(testing the efficacy of ASA + clopidogrel vs ASA alone). The W study was stopped early bythe Data and Safety Monitoring Board in September 2005 after an interim analysis showing asignificant difference in favor of warfarin over the combination of ASA + clopidogrel. Thedetails of the ACTIVE W have been published (Lancet 2006; 367:1903-1912). The A and the Istudies are continuing.RE-LY study (Randomized Evaluation of Long term anticoagulant therapY)Non-valvular atrial fibrillation is implicated in nearly 15% of strokes. Dose-adjusted warfarindecreases the risk of stroke by 62%. However, in practice, the risk of bleeding, the variability ofanticoagulation intensity and the need of frequent monitoring and dose adjustments limitstreatment with warfarin, leaving patients outside the therapeutic range almost half the time.Underuse of warfarin in patients with atrial fibrillation at high risk of bleeding calls for safer,more reliable alternatives. The direct thrombin inhibitor dabigatran could offer fixed oral dosingwithout need for coagulation monitoring, rapid onset and offset of action, stablepharmacokinetics with little potential for drug interactions, and no known food interactions. Forthese reasons an international multicentre, randomized, active controlled, parallel group, noninferiority,clinical trial (RE-LY study) was designed to evaluate the efficacy and safety ofdabigatran etexilate compared with open label adjusted warfarin for the prevention of stroke andsystemic embolism in patients with non-valvular atrial fibrillation. The recruitment of patientswas completed and the long-term follow-up is ongoing.CURRENT – OASIS-7 studyOASIS 7 is a randomized, multinational, 2X2 factorial design, parallel-group, double-blindstudy, comparing a high loading dose regimen of clopidogrel versus standard dose and highdose regimen of aspirin versus standard dose, in patients with acute coronary syndromemanaged with an early invasive strategy. The study involves approximately 14,000 patients in800 clinical centers worldwide. The primary objective of the study is to determine whether ahigh dose regimen of clopidogrel is superior to a standard dose of clopidogrel in preventing CVdeath, myocardial infarction or stroke and to determine if high dose of aspirin is as safe as lowdose in terms of TIMI major bleeding rate. Secondary objective is to evaluate the safety of theclopidogrel high dose regimen compared to the standard dose regimen in terms of TIMI majorbleeding.INTER-HEART StudyINTER-HEART is an epidemiological study sponsored by the World Health Organization andthe World Heart Federation aimed to determine the association between risk factors and acutemyocardial infarction within populations defined by ethnicity and/or geographic region, and to138ANNUAL REPORT 2007

IRFMNassess the relative importance of risk factors across these populations. The project is coordinatedby the PHRI, McMaster University, Hamilton, Canada. The Clinical Drug EvaluationLaboratory serves as National Coordination Office for Italy.INTER-HEART was conducted in 52 countries in Asia, Europe, Middle East, Africa, Australia,and North and South America, utilizing a standardized protocol. The study evaluated theimportance of conventional and emerging risk factors within each geographic region, andwhether their impact varies by region in a population of nearly 28000 individuals (cases withacute myocardial infarction and matched controls). The study collected data on demographicfactors (country of origin, first language), socioeconomic status (education, occupation,income), lifestyle (tobacco use, physical activity, dietary patterns), and personal and familyhistory of cardiovascular disease and risk factors.The INTER-HEART has documented that nine simple modifiable risk factors can account forover 90% of the population attributable risk for heart disease globally. More importantly, theserisk factors appear to have similar predictability in all regions of the world, as well as in allethnic groups. A further analysis published in the 2005 redefined obesity: traditional definitionshave been based on Body Mass Index (BMI), but this paper very clearly showed that,irrespective of BMI, the waist-to-hip ratio (WTHR) is a far better predictor of myocardialinfarction risk across diverse populations. The observations carry important implications forpeople and populations hitherto considered to be low risk on the basis of their BMIs.In a further analysis, the INTERHEART study established that all forms of tobacco exposure,such as smoking, chewing tobacco or inhaling second hand smoke, increase the risk of heartattack. Compared to people who had never smoked, smokers had a three-fold increased risk of aheart attack. An ad hoc assessment of the gender influence, comparing risk factors for acutemyocardial infarction (MI) between women and men globally, the INTER-HEART showed thatwomen experience their first acute MI on average 9 years later than men. Nine modifiable riskfactors are significantly associated with acute MI in both men and women andexplain greater than 90% of the PAR. The difference in age of first MI is largely explained bythe higher risk factor levels at younger ages in men compared to women. The approach toprevention of MI in men and women can be based on similar principles in all regions of theworld.Laboratory of General Practice ResearchRisk and Prevention Study (R&P)R&P is a study on the optimization of cardiovascular prevention of subjects at high riskperformed at national level by General Practitioners.Study objective and design- Controlled clinical trial, double-blind and randomised, of the efficacy of a n-3 PUFA treatmentin reducing the incidence of cardiovascular events, both fatal and non-fatal, in a populationdefined as at high risk by participating GPs.- Practicability and overall yield of the preventive interventions adopted (outcome study) Theepidemiological and care history of this population shall form the object of a specific evaluationaccording to a plan of formal predefined analyses.Study populationInclusion criteriaAmong the subjects deemed by GPs to be at high cardiovascular risk, patients are selected ifpresenting:- multiple risk factors (e.g. hypertension, hypercholesterolemia, diabetes, smoking, familyhistory of myocardial infarction, obesity, sex and old age)- previous cardio-cerebrovascular events or clinical manifestations of atherosclerotic disease(stroke, TIA, peripheral arteriopathy, previous arterial revascularisation procedures, angina139ANNUAL REPORT 2007

IRFMNpectoris).Exclusion criteria- serious co morbidity with an unfavorable prognosis over the short term (e.g. cancer);- expected non-compliance over a long period of time; contraindications (known allergies to n-3PUFA)- indications (previous MI) for treatment with n-3 PUFA.Efficacy measuresThe primary objective is to evaluate if a long-term administration of n-3 PUFA is more effectivethan placebo in reducing: overall mortality and major cardiovascular events (non fatalmyocardial infarction and stroke) overall major cardiovascular events (cardiovascular mortality,non-fatal strokes and myocardial infarction); coronary-related mortality and sudden death.Randomisation is central, stratified by GP.The experimental treatment consists of one capsule containing 1g of n-3 PUFA, or thecorresponding placebo, to be taken daily.The duration of follow-up is 5 years.In order to document with sufficient statistical reliability that the experimental treatment with n-3 PUFA reduces the incidence of events [-15% total death, non fatal myocardial infarction andstroke; -20% cardiovascular death and non fatal myocardial infarction and stroke; -30%cardiovascular death; -40% sudden death] a total of 12,000 patients is required.Up-date of the study: From February 2004 to March 2007 12,521 patients have been enrolledby a network of 860 GPs. The Local Health Authorities involved are 57 and in each oneinvestigator’s meeting has been organized.The characteristics of the population so far enrolled are the following: mean age 65 years, males62%, hypertension 79%, hypercholesterolaemia 62%, diabetes 56%, smokers 16%, obesity35%, family history of premature myocardial infarction 20%. Twenty five% of patients have aclinical manifestation of atherosclerotic diseases, 50% have diabetes in association with anotherrisk factor and 23% have multiple risk factors.The “attributability”of cardiovascular events in the GP’s perceptionA cardiovascular event is the sudden manifestation - more or less expected - of clinical history,life-style habits, social environment, patient and medical attitudes and health strategies adopted.To carefully examine the reasons why a cardiovascular event occurred is part of a good care inthe context of general practice. To render explicit this process for all the representative casesthat occurred during a defined period of care could transform a usual attitude of good clinicalpractice into an “epidemiology of the attributability”.The aim of this study is to evaluate the “epidemiology of the attributability” of cardiovascularevents through GPs’ perception. Clinical, socio-economic, psychological, risk factors and lifestylevariables will be investigated. The study started in October 2007 and a network 167 GPsare actively involved.Pharmaco-epidemiology studies on administrative databases: the “Drugdruginteractions" ProjectThis is a study aimed to develop and implement a database on drug-drug interaction to be putinto a prescription computerised system of the Regione Lombardia (Italy) as a support for thegeneral practitioners (GPs) to assess the risk of potential drug interactions. The study was alsoset up to assess the prevalence of drug-drug interaction in a sample of prescriptions of a cohort ofGPs. A model for the epidemiological analyses of the drug prescriptions’ database of LeccoLocal Health Authority has been developed. The analyses have been performed on the cohort ofsubjects > 65 years (58.800 subjects, mean age of 75.2 years) on a total of 964.858 prescriptionrecords. Eighty-six % of the subjects received al least one prescription per year, overall menwere more treated than woman and the class of drugs most frequently prescribed was the140ANNUAL REPORT 2007

IRFMNcardiovascular one (63.8% of the population). ). The prevalence of potentially severe drug-druginteractions was 16 %, and increased at rising of the patient’s age and of the number or chronicdrug prescribed.Epidemiological and clinical profile of diabetic patients in LombardyRegion using administrative databases.The study is part of an ongoing pharmacoepidemiological project in collaboration with theHealth Department of the Lombardy Region. Its main objective is the definition of a model toassess and control the use of health resources of diabetic patients by means of integratedadministrative database.Specific aims of the study are:• To identify the diabetic population by specific drug consumption (ATC group = A10)• To assess the co morbidities by co-administered cardiovascular and non-cardiovascular drugs• To describe the diagnostic procedures by the assessment of laboratory, instrumental andspecialist examinations• To estimate the rate and causes of hospitalization by the analyses of hospital admission dataThe stratification of global cardiovascular risk in hypertensive patients ofthe district of Borbon – EcuadorThe Laboratory is involved in a collaborative project with the Cecomet (Centro deEpidemiologia comunitaria y Medicina tropical) in Esmeralda, Ecuador, on the prevalence andtreatment of hypertension in the district of Borbon, a rural zone of Ecuador in the northern partof the country.In this area, 36% of the adult population is affected by hypertension and more than half ofhypertensive patients present blood pressure levels > 160/110 mmHg.From 2001, in the District is ongoing an intensive follow-up of the hypertensive population withthe following aims: to evaluate the global cardiovascular risk of the population, to better controlblood pressure levels increasing the number of subjects treated with hypertensive therapy (inparticular those at high cardiovascular risk) and monitoring of the clinical complications.Preliminary data show that:• Patients treated with hypertensive therapy are increased from 39% to 59%• Antihypertensive drugs are mainly prescribed to subjects with high blood pressure levels(80% of those with systolic blood pressure >180mmHg are actually under treatment) or athigh cardiovascular risk (82%)• Blood pressure control is improved (patients with systolic blood pressure levels> 180mmHg decreased from 33% to 24% and those with levels

IRFMNExamination, Kansas City Cardiomiopathy Questionnaire) with the clinical perception of thenurses; describe if assessed or perceived patients' problems (low quality of life, high depressionor compromised cognitive function) lead to any caring intervention.The baseline clinical characteristics of the 1564 patients included in the QDF study are closelycomparable with those of the GISSI-HF population. The study instruments could be validlyadministered to the greatest majority of patients (KCQQ 97.2%, GDS 94.9%, MMSE 80.6% ofpatients >70 years).The nurses network nested in a major clinical trial, has produced one of the largest prospectivecohort of HF patients who are comprehensively assessed and prospectively monitored, to allowan integrated evaluation of the relevance and implications of QDF measurements also on theclinical outcomes of this population.Epidemiology of nursing problems and drug-surveillance in nursinghomes and home careThe aim of the study is to describe problems nurses that require nursing decisions, and nursesencounter in the care of patients admitted to nursing home and home care, with specificattention to drug-related problems. In fact an adverse drug reaction is likely to be the cause ofmost unexpected problems that arise in elderly patients.The project has been completed and more than 350 nurses have been involved in 95 NursingHomes and Districts. In the 6 observation days overall 1500 patients were observed. Overall2224 problems were identified and 25% attributed to drugs and devices.Knowledge that patients have on their drugs at discharge from hospitalThe project is in its initial phase and involves several hospitals in the northen regions of Italy.Data on a first group of 163 patients from 6 hospitals were collected interviewing patients atdischarge on their old and new therapies: 78% of patients is not informed on drugs/behavioursto avoid and 70% don’t know side effects. No major differences were observed between old andnew therapies. The data collection is still ongoing in other centres.142ANNUAL REPORT 2007

IRFMN143ANNUAL REPORT 2007

IRFMNDEPARTMENT OF MOLECULARBIOCHEMISTRY ANDPHARMACOLOGYSTAFFHeadMario SALMONA, Sci.Prep.Alim.D., Ph.D.Laboratory of Biochemistry and Protein ChemistryHeadMario SALMONA, Sci.Prep.Alim.D.,Ph.D.Medical Biochemistry UnitHeadValentina BONETTO, Chem.Pharm.D.Synaptic Transmission UnitHeadMarco GOBBI, Pharm.D.Laboratory of Molecular BiologyHeadEnrico GARATTINI, M.D.Pharmacogenomics UnitHeadMaddalena FRATELLI, Biol.Sci.D.Gene Structure and Regulation UnitHeadMineko TERAO, Bioch.D., Ph.D.Laboratory of Receptor PharmacologyHeadTiziana MENNINI, Pharm.D.Laboratory of NeuroimmunologyHeadPietro GHEZZI, Ph.D.Pharmacology of Septic Shock UnitHeadPia VILLA, Pharm.D.Metabolic Neuropathies UnitHeadRoberto BIANCHI, Biol.Sci.D.Laboratory of Molecular PathologyHeadLavinia CANTONI, Biol.Sci.D.Laboratory of Systems BiologyHeadGianfranco BAZZONI, M.D.144ANNUAL REPORT 2007

IRFMNCURRICULA VITAEMario Salmona obtained his doctorate degree in Biochemistry and Food Technology at the University ofMilan in 1971. His background is in biochemistry, biophysics and pharmacology. His scientific interestsrelate to problems of human and animal diseases originating from the aberrant folding of proteins. In thiscontext, a major portion of his studies was devoted to the etiopathogenesis and therapy of prion diseases.He has published over 200 articles on peer reviewed scientific journals.1971-1975 Ph.D in Pharmacology, Mario Negri Institute1975 Visiting Fellow in the Department of Biology of the Weizmann Institute of Science, Rehovot, Israel1976-1997 Head, Laboratory of Enzyme Research, Mario Negri Institute1995 to date Dean of the School of Advanced Pharmacology, Mario Negri Institute1997 to date Head, Department of Biochemistry and Molecular Pharmacology, Mario Negri Institute2003 to date Member of the American Society of Biochemistry and Molecular BiologyReferee for international scientific journalsSelected publications• Gerstmann-Straussler-Scheinker disease amyloid protein polymerizes according to the "dock-and-lock" model. Gobbi M,Colombo L, Morbin M, Mazzoleni G, Accardo E, Vanoni M, Del Favero E, Cantù L, Kirschner D A, Ceci P, Ubezio P,Manzoni C, Forloni G, Tagliavini F, Salmona M J Biol Chem. 2006; 281: 843-9• Tetracycline and its analogues as inhibitors of amyloid fibrils: searching for a geometrical pharmacophore by theoreticalinvestigation of their conformational behaviour in aqueous solution U. Cosentino, M.R.Varì, A.A. G. Saracino, D. Pitea,G. Moro, M. Salmona Journal of Molecular Modelling, 2005, 11: 17-25• Role of Plasminogen in Propagation of Scrapie Salmona M., Capobianco R., Colombo L., De Luigi A., Rossi C.,Mangieri M., Giaccone G., Quaglio E., Chiesa R., Donati M.B., Tagliavini F. and Forloni G. Journal of Virology 2005,79: 11225-11230• The role of platelet activating factor in prion and amyloid-beta neurotoxicity Bate C, Salmona M, Williams ANeuroreport 2004; 15: 509-513• Squalestatin cures prion-infected neurons and protects against prion neurotoxicity. Bate C, Salmona M, Diomede L,Williams A. J Biol Chem. 2004; 279:14983-90• Channels formed with a synthetic mutant prion protein PrP(82-146) homologous to a 7 kDa fragment in diseased brain ofGSS patients Bahadi R., Farrelly P.V., Kenna B.L., Kourie J.I., Tagliavini F., Forloni G., Salmona M. Am.J.Physiol.(Cell Physiology) 2003; 285: C862-C872Gianfranco Bazzoni got his Medicine and Surgery degree in 1988 (at the University of Milan) and thespecialisation in Pharmacological Research in 1992 (at the Mario Negri Institute, Milan). His area ofexpertise is cell biology, with focus on the processes of cell adhesion and migration.1988-2000 Research Fellow, Mario Negri Institute1993-1997 Post-doctoral Fellow, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA2000-2002 Research Scientist, Mario Negri Institute2003 Head, Unit of Cell Adhesion, Mario Negri Institute2004 to date Head, Laboratory of Systems Biology, Mario Negri Institute2004 Regular Member of The American Physiological Society, Bethesda, MDReferee for international scientific journalsSelected publications• Paris L, Tonutti L, Vannini C, Buzzoni G. Structural organization of the tight junction. Biochim Biophys Acta 2007;(Epub ahead of print)• Huang H, Cruz F, Bazzoni G. Junctional Adhesion Molecule-A Regulates Cell Migration and Resistance to Shear Stress.J Cell Physiol 2006; 209: 122-130.• Bazzoni G. Endothelial tight junctions: permeable barriers of the vessel wall. Thromb Haemost 2006; 95(1): 36-42.• Martinez-Estrada OM, Manzi L, Tonetti P, Dejana E, Bazzoni G. Opposite effects of Tumor Necrosis Factor and solublefibronectin on Junctional Adhesion Molecule-A in endothelial cells. Am J Physiol (Lung Cell Mol Physiol) 2005; 288:L1081-L1088.• Bazzoni G, Tonetti P, Manzi L, Cera MR, Balconi G, Dejana E. Expression of Junction Adhesion Molecule-A preventsspontaneous and random motility. J Cell Sci 2005; 118: 623-632.• Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. PhysiolRev 2004; 84(3): 869-901.145ANNUAL REPORT 2007

IRFMNLavinia Cantoni obtained her degree in Biological Sciences in 1973 at the University of Milan. Thenshe specialized in pharmacological research at the Mario Negri Institute (1974-1977).Research areas 1) biochemical-molecular mechanisms activated by oxidative stress 2) drug metabolism3) porphyrias.1977-1978 Post-doctoral Fellow, Medical Research Council, Toxicology Unit, Carshalton, UK (Winnerof a Welcome Trust Research Fellowship)1979-1982 Research Scientist, Mario Negri Institute1980-1990 for short periods Visiting Scientist, Toxicology Unit, Carshalton, UK, and Cornell MedicalCenter, New York, USA1983-1998 Head, Unit of Heme and Hemoprotein Metabolism, Mario Negri Institute.1998 to date, Head, Laboratory of Molecular Pathology at the Mario Negri Institute.Member of the National Roll of Biologists and of the Italian Toxicology Society.Referee for international scientific journals, tutor for university degree theses and teacher in thePharmacological Research Specialisation Course for graduates held at the Mario Negri Institute.Selected publications• Rizzardini M., Chiesa R., Angeretti N., Lucca E., Salmona M., Forloni G., Cantoni L. Prion protein fragment 106-126differentially induces heme oxygenase-1 mRNA in cultured neurons and astroglial cells. J.Neurochem. 68: 715-720,1997• Rizzardini M., Zappone M., Villa P, Gnocchi P., Sironi M., Diomede L., Meazza C., Monshouwer M., Cantoni L.Kupffer cell depletion partially prevents hepatic heme oxygenase 1 messenger RNA accumulation in systemicinflammation in mice: role of interleukin 1 beta. Hepatology 27: 703-710, 1998• Cantoni L.,Valaperta R.,.Ponsoda X., Castell, J.V., Barelli D., Rizzardini M., Mangolini A., Hauri L., Villa P. Inductionof hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity. J.Hepatology, 38: 776-783, 2003• Babetto E., Mangolini A., Rizzardini M., Lupi M., Conforti L., Poletti A., Rusmini P., Cantoni L. Tetracycline-regulatedgene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases. Mol. Brain Res. 140: 63-72,2005• Rizzardini M., Lupi M., Mangolini A., Babetto E., Ubezio P., Cantoni L. Neurodegeneration induced by complex Iinhibition in a cellular model of familial amyothrophic lateral sclerosis. Brain Res. Bull., 69: 465-474, 2006• Raimondi A., Mangolini A., Rizzardini M., Tartari S., Massari S., Bendotti C., Francolini M., Borghese N., Cantoni L.,Pietrini G. Cell culture models to investigate the selective vulnerability of motoneuronal mitochondria to familial ALSlinkedG93ASOD1. Eur. J. Neurosci. 24: 387-399, 2006Enrico Garattini obtained his degree in Medicine and Surgery with full marks (110/110) in 1982 at theUniversity of Milan. His scientific interests relate to problems of Cellular Biology and MolecularBiology.1982-1990 Research Fellow of the National Research Council, Mario Negri Institute1983-1987 Postdoctoral Researcher at the Roche Institute of Molecular Biology, Department ofNeurosciences Nutley, New Jersey, US1991-1997 Senior Researcher Regione Lombardia and Head of the Molecular Biology Unit, Mario NegriInstitute1997 to date Head, Laboratory of Molecular Biology, Mario Negri InstituteFrom 2005 Dean, Advanced School of Pharmacology (Philosophy Doctor), Mario Negri InstituteMember of the Editorial Board of the European Journal of Cancer and of Current Cancer TherapyReviewsMember of the American Society of Biochemistry and Molecular Biology (ASBMB)Selected publications• Gianni M, Parrella E, Raska I Jr, Gaillard E, Nigro EA, Gaudon C, Garattini E, Rochette-Egly C. P38MAPK-dependentphosphorylation and degradation of SRC-3/AIB1 and RARalpha-mediated transcription. EMBO J. 2006 Feb22;25(4):739-51• Parrella E, Gianni M, Fratelli M, Barzago MM, Raska I Jr, Diomede L, Kurosaki M, Pisano C, Carminati P, Merlini L,Dallavalle S, Tavecchio M, Rochette-Egly C, Terao M, Garattini E.Antitumor activity of the retinoid-related molecules (E)-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid (ST1926)and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) in F9 teratocarcinoma: Role ofretinoic acid receptor gamma and retinoid-independent pathways.Mol Pharmacol. 2006 Sep;70(3):909-24• Garattini E, Parrella E, Diomede L, Gianni M, Kalac Y, Merlini L, Simoni D, Zanier R, Ferrara F F, Chiarucci I,Carminati P, Terao M, Pisano C. ST1926, a novel and orally active retinoid-related molecule inducing apoptosis inmyeloid leukemia cells: Modulation of intracellular calcium homeostasis. Blood 2004; 103: 194-207• Vila R, Kurosaki M, Barzago M M, Kolek M, Bastone A, Colombo L, Salmona M, Terao M, Garattini E. Regulation andbiochemistry of mouse molybdo-flavoenzymes. The DBA/2 mouse is selectively deficient in the expression of aldehyde146ANNUAL REPORT 2007

IRFMNoxidase homologues 1 and 2 and represents a unique source for the purification and characterization of aldehyde oxidase.J Biol Chem 2004; 279: 8668-8683• Kurosaki M, Terao M, Barzago M M, Bastone A, Bernardinello D, Salmona M, Garattini E. The aldehyde oxidase genecluster in mice and rats: Aldehyde oxidase homologue 3, a novel member of the molybdo-flavoenzyme family withselective expression in the olfactory mucosa. J Biol Chem 2004; 279: 50482-50498• Pisano C, Kollar P, Gianni M, Kalac Y, Giordano V, Ferrara F F, Tancredi R, Devoto A, Rinaldi A, Rambaldi A, PencoS, Marzi M, Moretti G, Vesci L, Tinti O, Carminati P, Terao M, Garattini E. Bis-indols a novel class of moleculesenhancing the cytodifferentianting properties of retinoids in myeloid leukemia cells. Blood 2002; 100: 3719-3730Pietro GhezziResearch Areas: Cytokines and inflammation; redox regulation1979-1990: Researcher, Mario Negri Institute1991 to date: Head, Laboratory of Neuroimmunology, Mario Negri Institute1998-2000: Research Associate at Stanford University School of Medicine, Department of Genetics2000 to date: Member, Kenneth Warren Laboratory, Ossining, NY (USA)Referee for international scientific journalsSelected publications• Leist M, Ghezzi P, et al.. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science. 2004 Jul9;305(5681):239-42.• Fratelli M, Goodwin LO, Orom UA, Lombardi S, Tonelli R, Mengozzi M, Ghezzi Gene expression profiling reveals asignaling role of glutathione in redox regulation.Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13998-4003.• Villa P, Bigini P, Mennini T, Agnello D, Laragione T, Cagnotto A, Viviani B, Marinovich M, Cerami A, Coleman TR,Brines M, Ghezzi P. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemiaby targeting neuronal apoptosis. J Exp Med. 2003 Sep 15;198(6):971-5.• Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, Keenan S, Gleiter C, Pasquali C, Capobianco A,Mennini T, Heumann R, Cerami A, Ehrenreich H, Ghezzi P. Erythropoietin prevents neuronal apoptosis after cerebralischemia and metabolic stress. Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):4044-9.• Laragione T, Bonetto V, Casoni F, Massignan T, Bianchi G, Gianazza E, Ghezzi P. Redox regulation of surface proteinthiols: identification of integrin alpha-4 as a molecular target by using redox proteomics. Proc Natl Acad Sci U S A.2003 Dec 9;100(25):14737-41.Tiziana Mennini got her degree in Pharmacy at the University of Milano (1975). In the same year sheobtained a fellowship from the European Molecular Biology Organization, to learn sub-cellularfractionation techniques and synaptosomes utilization in neurochemistry, at the laboratory of Prof. VPWhittaker ( Stockholm, Sweden). In 1882 she spent a further period in Prof. Whittaker’s laboratories(Max-Plank-Institut fur Biophysikalische Chemie, Abteilung Neurochemie Am Fassberg, Gottingen,Germany). She spent all her scientific career at the Mario Negri Institute:1967- 1975 Research Assistant in the Laboratory of Drug Metabolism1975-1987 Chief of the Unit of Neurochemical Transmission,1988 to date Chief of the Laboratory of Receptor PharmacologySpeaker, chairman and organizer at many congresses and courses, author of more than 200articles published in international journals in the area of receptor pharmacology andneuropharmacology.Selected publications• Beghi E, Bendotti C, Mennini T. 2005. Merits of a new drug trial for ALS? Science 308:632-633• Gobbi M, Mennini T. 2001. Is St John's wort a 'Prozac-like' herbal antidepressant? Trends Pharmacol Sci 22:557-559.• The Italian ALSSG. 1996. Ceftriaxone in amyotrophic lateral sclerosis. Eur J Neurol 3:295-298.• Mennini T, Mocaer E, Garattini S. 1987. Tianeptine, a selective enhancer of serotonin uptake in rat brain. Naunyn-Schmiedebergs Arch Pharmacol 336:478-482.• Mennini T, Garattini S. 1983. Benzodiazepines receptor binding in vivo: pharmacokinetic and pharmacologicalsignificance. Advances Biochemical Psychopharmacol 38:189-199.• Mennini T, Bernasconi S, Manara L, Samanin R, Serra G. 1977. The effect of intracerebral 6-hydroxy dopamine on 3Hreserpinebinding to different brain regions of the rat. Pharmacol Res Commun 9:857-862.147ANNUAL REPORT 2007

IRFMNRoberto Bianchi got his degree in Biological Sciences at University of Milan, Italy in 1992. Since 1975he served as student teacher and supervisor at Mario Negri Institute and from 1989 to 1997 at HoustonUniversity. His main interests are diabetic complications (peripheral and autonomic neuropathies) andneurodegenerative disorders (Multiple Sclerosis, drugs induced neuropathies).1971 Technician in the Laboratory of Biochemical Pharmacology, Mario Negri Institute1980-1981 Visiting Scientist in the Center for Neurosciences Behavioral Research, The WeizmannInstitute of Science, Rehovot, Israel1981-1995 Research Assistant in the Laboratory of Biochemical Pharmacology, Mario Negri Institute1988-1997 Research Fellow in the Dept. Biochemical Biophysical Sciences, University of Houston, US1993-1995 Research Fellow in the Dept. Medicine, Case Western Reserve University, Cleveland, USSince 1996 Head, Unit of Metabolic Neuropathies, Mario Negri InstituteSelected publications• Bianchi R., Berti-Mattera L.N., Fiori M.G., Eichberg J.:Correction of altered metabolic activities in sciatic nerves ofstreptozotocin-induced diabetic rats. Effects of ganglioside treatment. Diabetes 39: 782-788 (1990).• Scarpini E., Bianchi R., Moggio M., Sciacco M., Fiori M.G., Scarlato G.: Decrease of nerve Na+,K+-ATPase activity inthe pathogenesis of diabetic neuropathy. J. Neurol. Sci. 120: 159-167 (1993).• Conti G., Scarpini E., Baron P.L., Livraghi S., Tiriticco M., Bianchi R, Vedeler C., Scarlato G.: Macrophage infiltrationand death in the nerve during the early phases of experimental diabetic neuropathy: a process concomitant withendoneurial induction of IL-1 and p75NTR. J. Nuerol. Sci. 195: 35-40 (2002)• Bianchi R., Buyukakilli B., Brines M., Savino C., Cavaletti G., Oggioni N., Lauria G., Borgna M., Lombardi R., CimenB., Comelekoglu U., Kanik A., Tataroglu C., Cerami A., Ghezzi P. Erythropoietin both protects from and reversesexperimental diabetic neuropathy. Proc Natl Acad Sci USA 101: 823-828 (2004)• Leist M., Ghezzi P., Grasso G, Bianchi R., Villa P., Fratelli M., Savino C., Bianchi M., Nielsen J., Gerwien J., KallunkiP., Larsen A.K., Helboe L., Christensen S., Pedersen L.O., Nielsen M., Troup L., Sager T., Sfacteria A., Erbayktar S,Erbayktar Z., Gokmen N., Yilmaz O., Cerami-Hand C., Xie, Q-W., Coleman T., Cerami A., Brines M. Erythropoietinderivedtissue-protective cytokines that do not bind to the classical erythropoietin receptor. Science, 305(5681) 239-242,2004.• Savino C., Pedotti R., Baggi F., Furlan R., Ubiali F., Gallo B, Nava S., Bigini P., Barbera S., Fumagalli E., Mennini T.,Vezzani A., Rizzi M., Coleman T., Cerami A.,Brines M., Ghezzi P., Bianchi R.Delayed administration of erythropoietinand its non-erythropoietic derivatives ameliorates chronic murine autoimmune encephalomyelitis. Journal ofNeuroimmunology, 2005, E-pub December 6, 2005Valentina Bonetto has got the degree in Pharmaceutical Chemistry and Technology at the University ofPadua, Italy in 1993. She has got the Ph.D. in Medical Biochemistry and Biophysics at KarolinskaInstitutet, Stockholm, Sweden.Her principal lines of research are: 1) Study of the pathogenetic mechanisms at the basis of amyotrophiclateral sclerosis (ALS); 2) Identification of biomarkers of ALS; 3) Role of the oxidative modification inneurological disorders. These issues are investigated by different experimental approaches, includingproteomics and mass spectrometry.Since 2000 she is at the Mario Negri Institute in the Laboratory of Biochemistry and Protein Chemistry,from 2002 is also Assistant Telethon Scientist at Dulbecco Telethon Institute. Since October 2007 she isthe Head of the Unit of Medical Biochemistry inside the Laboratory of Biochemistry and ProteinChemistry. She is author of 26 publications from 1994 to 2007, in peer-reviewed journals. Among themshe is first author in 11 and last author in 4. She is also author of 2 reviews. She is reviewer for scientificjournals in the field of Proteomics and Neuroscience.Selected publications• Massignan, T., Casoni, F., Basso, M., Stefanazzi, P., Biasini, E., Tortarolo, M., Salmona, M., Gianazza, E., Bendotti, C.,Bonetto V. (2007) Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1mouse. Biochem. Biophys. Res. Commun., 353:719-25.• Basso, M., Massignan, T., Samengo, G., Cheroni, C., De Biasi, S., Salmona, M., Bendotti, C., Bonetto, V. (2006)Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice. J. Biol. Chem., 281:33325-33335.• Ghezzi, P., Casagrande, S., Massignan, T., Basso, M., Bellacchio, E., Mollica, L., Biasini, E., Tonelli, R., Eberini, I.,Gianazza, E., Dai, W.W., Fratelli, M., Salmona, M., Sherry, B., Bonetto. V. (2006) Redox regulation of cyclophilin A byglutathionylation. Proteomics, 6: 817-825.• Casoni, F., Basso, M., Massignan, T., Gianazza, E., Cheroni, C., Salmona, M., Bendotti, C., Bonetto, V. (2005) Proteinnitration in a mouse model of familial amyotrophic lateral sclerosis: Possible multifunctional role in the pathogenesis. J.Biol. Chem., 280: 16295-16304.• Laragione, T., Bonetto, V., Casoni, F., Massignan, T., Bianchi, G., Gianazza, E., Ghezzi, P. (2003) Redox regulation ofsurface protein thiols: identification of integrin alpha-4 as a molecular target by using redox proteomics. Proc. Natl.Acad. Sci. U S A 100: 14737-14741.148ANNUAL REPORT 2007

IRFMN• Casagrande, S.* , Bonetto, V.*, Fratelli, M., Gianazza, E., Eberini, I., Massignan, T., Salmona, M., Chang, G., Holmgren,A., Ghezzi, P. (2002) Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione andthioredoxin systems. Proc. Natl. Acad. Sci. U S A 99, 9745-9749. *These authors contributed equally to the study.Maddalena Fratelli got her degree in Biological Sciences at the University of Pisa and at the ScuolaNormale Superiore di Pisa in 1983. Then the specialization in Pharmacological Research at the MarioNegri Institute in 1986.Her main fields of interest are: 1. High throughput genomic systems for the study of drug action andpharmacoresistance. 2. Redox regulation of protein function and gene expression: glutathionylation andgene expression profiling of glutathione dependent responses to oxidant challenge.1988-1989 Postdoctoral Research Fellow in the Medical Research Council, Neurobiology Unit,Cambridge, UK.Since 1995, Head, Unit of Mediators of inflammation, Laboratory of Neuroimmunology, Mario NegriInstituteSince 2005, Head, Unit of Pharmacogenomics, Laboratory of Molecular Biology, Mario Negri InstituteSelected publications• Fratelli M, Goodwin LO, Orom UA, Lombardi S, Tonelli R, Mengozzi M, Ghezzi P. Gene expression profiling revealsa signaling role of glutathione in redox regulation. Proc Natl Acad Sci U S A. 2005;102:13998-4003.• Brines M, Grasso G, Fiordaliso F, Sfacteria A, Ghezzi P, Fratelli M, Latini R, Xie QW, Smart J, Su-Rick CJ, Pobre E,Diaz D, Gomez D, Hand C, Coleman T, Cerami A. Erythropoietin mediates tissue protection through an erythropoietinand common beta-subunit heteroreceptor. Proc Natl Acad Sci U S A. 2004; 101:14907-12.• Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, Savino C, Bianchi M, Nielsen J, Gerwien J, Kallunki P,Larsen AK, Helboe L, Christensen S, Pedersen LO, Nielsen M, Torup L, Sager T, Sfacteria A, Erbayraktar S,Erbayraktar Z, Gokmen N, Yilmaz O, Cerami-Hand C, Xie QW, Coleman T, Cerami A, Brines M. Derivatives oferythropoietin that are tissue protective but not erythropoietic. Science. 2004; 305:239-42• Fratelli M, Minto M, Crespi A, Erba E, Vandenabeele P, Del Soldato P, Ghezzi P. Inhibition of nuclear factor-kappaBby a nitro-derivative of flurbiprofen: a possible mechanism for antiinflammatory and antiproliferative effect. AntioxidRedox Signal. 2003; 5:229-35• Fratelli M, Demol H, Puype M, Casagrande S, Eberini I, Salmona M, Bonetto V, Mengozzi M, Duffieux F, Miclet E,Bachi A, Vandekerckhove J, Gianazza E, Ghezzi P. Identification by redox proteomics of glutathionylated proteins inoxidatively stressed human T lymphocytes. Proc Natl Acad Sci U S A. 2002; 99:3505-10• Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, Keenan S, Gleiter C, Pasquali C, Capobianco A,Mennini T, Heumann R, Cerami A, Ehrenreich H, Ghezzi P. Erythropoietin prevents neuronal apoptosis after cerebralischemia and metabolic stress. Proc Natl Acad Sci U S A. 2001; 98:4044-9Marco Gobbi got his degree in Pharmacy at the University of Milan, Italy, in 1989.Currently, his main fields of interest are: 1) the study of presynaptic mechanisms, such asneurotransmitter release/reuptake with a particular focus on plasma membrane transporters; and 2) thestudy of protein misfolding and aggregation and in particular the characterization of the prion proteinamyloid formation, investigated by different approaches including surface plasmone resonance.Since 1981, Researcher in the Laboratory of Neuropharmacology and, from 1988, in the Laboratory ofReceptor Pharmacology, Mario Negri InstituteStarting from 1989 Chief, Unit of Synaptic Transmission, Mario Negri InstituteIn Jan 2006, his group joined the Laboratory of Biochemistry and Protein Chemistry, Mario Negri InstituteCo-author in more than 70 scientific publications on peer-reviewed international journals. First or lastauthor in more than 40 of them. Reviewer for international scientific journals operating in theNeuroscience/Neuropharmacology fields.Selected publications• Gerstmann-Straussler-Scheinker disease amyloid protein polymerizes according to the "dock-and-lock" model. Gobbi M,Colombo L, Morbin M, Mazzoleni G, Accardo E, Vanoni M, Del Favero E, Cantù L, Kirschner D A, Ceci P, Ubezio P,Manzoni C, Forloni G, Tagliavini F, Salmona M J Biol Chem. 2006; 281: 843-9• Funicello M, Conti P, De Amici M, De Micheli C, Mennini T, Gobbi M. 2004. Dissociation of [3H]L-glutamate uptakefrom L-glutamate-induced [3H]D-aspartate release by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid, twoconformationally constrained aspartate and glutamate analogs. Mol Pharmacol 66:522-529.• Gobbi M, Moia M, Pirona L, Ceglia I, Reyes-Parada M, Scorza C, Mennini T. 2002. p-Methylthioamphetamine and 1-(m-chlorophenyl)piperazine, two non-neurotoxic 5-HT releasers in vivo, differ from neurotoxic amphetaminederivatives in their mode of action at 5-HT nerve endings in vitro. J Neurochem 82:1435-1443.• Gobbi M, DallaValle F, Ciapparelli C, Diomede L, Morazzoni P, Verotta L, Caccia S, Cervo L, Mennini T. 1999.Hypericum perforatum L. extract does not inhibit 5-HT transporter in rat brain cortex. Naunyn Schmiedebergs ArchPharmacol 360:262-269.149ANNUAL REPORT 2007

IRFMN• Gobbi M, Gariboldi M, Piwko C, Hoyer D, Sperk G, Vezzani A. 1998. Distinct changes in peptide YY binding to, andmRNA levels of, Y1 and Y2 receptors in the rat hippocampus associated with kindling epileptogenesis. J Neurochem70:1615-1622.• Crespi D, Mennini T, Gobbi M. 1997. Carrier-dependent and Ca(2+)-dependent 5-HT and dopamine release induced by(+)-amphetamine, 3,4-methylendioxymethamphetamine, p-chloroamphetamine and (+)-fenfluramine. Br J Pharmacol121:1735-1743.Mineko Terao obtained her doctorate degree in Pharmaceutical Science from the Kobe Women’sCollege of Pharmacy, Japan in 1978. Her scientific interests relate to problems of Cellular Biology andMolecular Biology.1983 Ph.D in Molecular Biology, Kyoto University, Japan1982-1983 Research Fellow, Department of Medical Chemistry, Kyoto University Faculty of Medicine,Japan1983-1987 Postdoctoral Associate of the Institute for Cancer Research, Philadelphia, USFrom 1987 Visiting Scientist of Mario Negri InstituteFrom 1998 Head of the Unit of Gene Structure and Regulation, Mario Negri InstituteSelected publications• Terao M, Kurosaki M, Barzago MM, Varasano E, Boldetti A, Bastone A, Fratelli M, Garattini E.Avian and canine aldehyde oxidases. Novel insights into the biology and evolution of molybdo-flavoenzymes.J Biol Chem. 2006 Jul 14;281(28):19748-61• Garattini E, Parrella E, Diomede L, Gianni M, Kalac Y, Merlini L, Simoni D, Zanier R, Ferrara F F, Chiarucci I,Carminati P, Terao M, Pisano C. ST1926, a novel and orally active retinoid-related molecule inducing apoptosis inmyeloid leukemia cells: Modulation of intracellular calcium homeostasis. Blood 2004; 103: 194-207• Vila R, Kurosaki M, Barzago M M, Kolek M, Bastone A, Colombo L, Salmona M, Terao M, Garattini E. Regulation andbiochemistry of mouse molybdo-flavoenzymes. The DBA/2 mouse is selectively deficient in the expression of aldehydeoxidase homologues 1 and 2 and represents a unique source for the purification and characterization of aldehyde oxidase.J Biol Chem 2004; 279: 8668-8683• Kurosaki M, Terao M, Barzago M M, Bastone A, Bernardinello D, Salmona M, Garattini E. The aldehyde oxidase genecluster in mice and rats: Aldehyde oxidase homologue 3, a novel member of the molybdo-flavoenzyme family withselective expression in the olfactory mucosa. J Biol Chem 2004; 279: 50482-50498• Parrella E, Gianni’ M, Cecconi V, Nigro E, Barzago MM, Rambaldi A, Rochette-Egly C, Terao M and Garattini E.Phosphodiesterase 4 inhibition by piclamilast potentiates the cyto-differentiating action of retinoids in myeloid leukemiacells. J Biol Chem 2004; 279: 42026-42040• Garattini E, Gianni’ M and Terao M. Retinoid related molecules an emerging class of apoptotic agents with promisingclinical potential in oncology: pharmacological activity and mechanisms of action. Curr Pharm Design 2004, 10: 433-448Pia Emilia Villa got her degree in Pharmaceutical Chemistry and Technology at the University of Paviain 1975 and the specialisation in Pharmacological Research at the Mario Negri Institute, Milan in 1978.Her scientific interests are the physiopathologic factors of sepsis and their pharmacological modulation,the cellular and molecular mechanisms of neurodegeneration and neuroprotection in experimentalcerebral ischemia.1976-1992: Research fellow, laboratory of Perfusion of Isolated Organs and Toxicology, Mario NegriInstitute1979-1980: Visiting fellow, laboratory of Cultured Hepatocytes, Toxicology Unit, MRC, Carshalton,England1983: Visiting fellow, Unité de Recherche Hépatologique, Rennes, France1993-1995: Research Scientist, laboratory of Neuroimmunology, Mario Negri Institute1995 to date: Head, Unit of Pharmacology of Septic Shock, Mario Negri Institute1982 Regular member of the Italian Society of Toxicology1992 Regular member of Celltox1996 Regular member of the International Cytokine Society.Selected publications• Villa P, Shaklee CL, Meazza C, Agnello D, Ghezzi P, Senaldi G. Granulocyte colony-stimulating factor and antibioticsin the prophylaxis of a murine model of polymicrobial peritonitis and sepsis. J Infect Dis. 1998; 178: 471-7.• Villa P, Saccani A, Sica A, Ghezzi P. Glutathione protects mice from lethal sepsis by limiting inflammationand potentiating host defense. J Infect Dis. 2002; 185: 1115-20.• Erbayraktar S, Grasso G, Sfacteria A, Xie QW, Coleman T, Kreilgaard M, Torup L, Sager T, Erbayraktar Z,Gokmen N, Yilmaz O, Ghezzi P, Villa P, Fratelli M, Casagrande S, Leist M, Helboe L, Gerwein J,Christensen S, Geist MA, Pedersen LO, Cerami-Hand C, Wuerth JP, Cerami A, Brines M.Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc NatlAcad Sci U S A. 2003;100 (11):6741-6.150ANNUAL REPORT 2007

IRFMN• Villa P, Bigini P, Mennini T, Agnello D, Laragione T, Cagnotto A, Viviani B, Marinovich M, Cerami A,Coleman TR, Brines M, Ghezzi P. Erythropoietin selectively attenuates cytokine production andinflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med. 2003;198 (6):971-5.• Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, Savino C, Bianchi M, Nielsen J, Gerwien J, Kallunki P,Larsen AK, Helboe L, Christensen S, Pedersen LO, Nielsen M, Torup L, Sager T, Sfacteria A, Erbayraktar S,Erbayraktar Z, Gokmen N, Yilmaz O, Cerami-Hand C, Xie QW, Coleman T, Cerami A, Brines M. Derivatives oferythropoietin that are tissue protective but not erythropoietic. Science. 2004;305: 239-42.• Garau A, Bertini R, Colotta F, Casilli F, Bigini P, Cagnotto A, Mennini T, Ghezzi P, Villa P. Neuroprotection with theCXCL8 inhibitor repertaxin in transient brain ischemia. Cytokine. 2005;30:125-31.151ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe Department comprises six laboratories. Research is heterogeneous in terms of scientificinterests and aims, but it is unified by the structural and functional study of specific,pharmacologically important gene products, using a common body of techniques. Classicalbiochemistry and molecular biology methods are used to define proteins that might be targetsfor the pharmacological activity of drugs. Potential direct interactions between drugs andproteins are studied at the molecular level by a variety of approaches ranging from animalstudies to computer simulation.FINDINGS/MAIN RESULTSIdentification of tetracylines as potential anti-prion drugs.Synthesis and physicochemical and biological characterization of peptides deduced from theprimary sequence of prion protein.Identification of a relationship between cholesterol synthesis and production of prion protein.Protein identifications by mass spectrometry and data base searching using a combination oftechniques.Characterization of the Pentraxin 3 role in the organization of the cumulus oophorusextracellular matrix and in female fertility.Characterization of the role of Junctional Adhesion Molecule-A (JAM-A) in the control of cellmotility.Characterization of the effect of inflammatory cytokines on JAM-A function.Development of a constitutive and of an inducible motor neuron cellular model to unravel thetoxicity of mutant G93A superoxide dismutase 1 responsible for some forms of familialamyotrophic lateral sclerosis.Conditions of oxidative stress or the presence of compounds impairing the electron transportchain are a risk factor to motor neurons of individuals carrying mutant forms of superoxidedismutase 1.Mitochondrial damage due to mutant G93A superoxide dismutase 1 occurs selectively in motorneurons.Mitochondrial damage by mutant G93A superoxide dismutase 1 in motor neurons is modulatedby the level of expression of the mutant protein.Identification and characterization of a novel class of retinoids endowed with strong andselective apoptogenic acivity on the neoplastic cell. Pre-clinical development of these agents forthe treatment of acute leukemia.Identification and characterization of novel retinoid-based pharmacological combinations forthe treatment of acute myelogenous leukemia.Molecular cloning and characterization of the cDNAs and genes of four novel members of themammalian molybdo-flavoprotein family. Definition of a novel gene cluster on humanchromosome 2 and mouse chromosome 1.Development of knok-out animals for molybdo-flavoproteins: AOX1, AOH1, AOH2, AOH3.Identification of erythropoietin as a neuroprotective agent and of new molecules withneuroprotective activity.Identification of the pharmacological action of erythropoietin against peripheral neuropathy ofdiabetes.Identification of erythropoietin derivatives that retain its neuroprotective actions but have lostits hemopoietic ones.Discovery of proteins that are regulated by the redox state through the formation of reversible152ANNUAL REPORT 2007

IRFMNdisulfide bonds with glutathione (protein glutathionylation).Identification of exofacial proteins undergoing thiol redox regulation.The use of antioxidant molecules in models of sepsis and inflammation diminishes theinflammatory response while potentiates the innate immune response.Identification of the gene expression profile regulated by thiols.The treatment with a non haematopoietic derivate of Erythropoietin (CEPO) reduces motorneuron loss and clinical progression in a mouse model of ALS related to alterations in vesicletrafficking, the wobbler mouse.Treatment with a soluble TNF receptor in the wobbler mouse, reduces motor neurondegeneration and the phosphorylation of the two main stress kinases (p38 e JNK) activated byTNF receptors.Riluzole treatment reduces motor neuron loss and clinical progression of wobbler mouse byincreasing the endogenous BDNF expression .Oxidative stress, glial activation and inflammation occur in the retinopathy as well as incerebral and spinal cord dysfunction in the mnd mouse, a model of progressive epilepsy withmental retardation related to mutation in the CLN8 gene. These findings provide furtherevidence for the implication of TNF death receptor signaling in the pathology of NeuronalCeroid LipofuscinosisThe affinity of pergolide for human cloned 5-HT 2A and 5-HT 2B receptors is similar and higherthat that for the human cloned D 2L receptor. These findings, together with the fact that it acts asagonist at both h5-HT 2A and h5-HT 2B receptors, could explain its potential toxicity mediated byactivation of cardio-pulmonary 5-HT 2B receptor.New conformationally constrained aspartate and glutamate analogues dissociate glutamateuptake inhibition and reverse transport-mediated release.Dimethyl sulfoxide, a solvent commonly utilized to dissolve hydrophobic compound for invitro experiments, interferes with the 5-HT6 agonists activity when the scintillation proximityassay is used for evaluating 35S-GTP-γ-S binding; but does not interfere with the europiumlabelled GTP binding determined by “time-resolved fluorescence” .NATIONAL COLLABORATIONSAdvanced Biology Center, GenoaDip. Anatomia, Farmacologia, Medicina Legale, University of TurinDip. Biotecnologie, Università degli Studi, MilanDip. Chimica Biochimica e Biotecnologie per la Medicina, Università degli Studi, MilanDip. Chimica Farmaceutica e Tossicologica, Università degli Studi, MilanDip. Farmaco-Chimico, Università degli Studi, MessinaDip. Farmaco-Chimico-Tecnologico, University of SienaDip. Farmacologia Medica, Università degli Studi, MilanDip. Scienze Biochimiche, University of FlorenceDip. Scienze Farmaceutiche, University of CataniaDip. Scienze Farmaceutiche, University of GenoaDip. Scienze Farmacologiche, Università degli Studi, MilanDip. Scienze Fisiologiche e Farmacologiche,University of PaviaDip. Scienze Molecolari, University of MilanDip. Studi pre-clinici, University of MilanFacoltà di Biologia, Università degli Studi, MilanFacoltà di Chimica, Università degli Studi, MilanFacoltà di Chimica, University of Ferrara153ANNUAL REPORT 2007

IRFMNGlaxoSmithkline, VeronaHarlan, MilanIRCCS Fondazione "Istituto C. Mondino", Laboratorio di Neurobiologia Sperimentale, PaviaIstituto di Biologia Molecolare Buzzati Traverso, NaplesIstituto di Biomedicina e Immunologia Molecolare CNR, PalermoIstituto di Endocrinologia, Centro di Eccellenza per le Malattie Neurodegenerative, Universitàdegli Studi, MilanIstituto di Clinica Neurologica, Ospedale Maggiore Policlinico, MilanIstituto Clinico Humanitas, MilanIstituto di Neuroscienze C.N.R., PisaIstituto Nazionale dei Tumori, MilanIstituto Nazionale dei Tumori, NaplesIstituto Nazionale Neurologico "C. Besta", MilanIstituto Oncologico Europeo, MilanIstituto Regina Elena, RomeNewron Pharmaceuticals, MilanOspedale Maggiore Policlinico, MilanOspedale Pediatrico Bambino Gesu', RomeOspedale Pediatrico "Gaslini", GenoaOspedale S. Gerardo, Monza, MilanSigma-Tau, Pomezia, RomeZambon, MilanINTERNATIONAL COLLABORATIONSBabraham Institute, Cambridge, UKBoston College, Boston, MA, USACase Western Research University, Cleveland, OH, USADept. of Neurology, Keio University, Tokyo, JapanDept. de Quimica-Fisica de Macromoleculas Biologicas, CSIC, Madrid, SpainFaculdad de Ciencias Medicas, Universidad de Santiago de Chile, ChileDept. of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, TheHebrew University of Jerusalem, Jerusalem, IsraelFlanders Interuniversity Institute for Biotechnology (VIB) University of Gent, BelgiumFMP, Berlin, GermanyGiessen Polyclinic University, Giessen, GermanyHouston University, TX, USAIBSN CNRS, Marseille, FranceIndiana University, Indianapolis, IN, USAInstitut de Genetique et Biologie Moleculaire et Cellulaire, Strasbourg, FranceInstitut Pasteur, Paris, FranceJohn Innes Centre, Norwich, UKKenneth S. Warren Institute, Ossining, NY, USAMax-Planck-Institut für experimentelle Medizin, Göttingen, GermanyNational Institute of Health, Bethesda, MD, USANippon University, Tokyo, JapanNorth Shore University Hospital, Manhasset, NY, USAPepscan System BV, Lelystad, HollandPolichem S.A., Lugano, SwitzerlandStanford University School of Medicine, Stanford, CA, USA154ANNUAL REPORT 2007

IRFMNTechnical University Braunschweig, GermanyTrinity College, Dublin, IrelandUniversidad de La Laguna, Tenerife, SpainUniversidad Nova, Lisbon, PortugalUniversitat des Saarlandes, Hamburg, GermanyUniversitat Freiburg, GermanyUniversité Paris, FranceUniversité Victor Segalen Bordeaux 2, Bordeaux, FranceUniversity of Birmingham, UKUniversity of Cardiff, UKUniversity of Colorado, School of Medicine, Denver, CO, USAUniversity of Glasgow, UKUniversity of Gottingen, GermanyUniversity of Muenster, GermanyUniversity of Southampton, UKUniversity of Sussex, UKUniversity of Vienna, AustriaWaring-Webb Institute, University of Colorado, Denver CO, USAWeizmann Institut, Rehovot, IsraelWestfaelische Wilhelms-Universitaet Muenster, GermanyEDITORIAL BOARD MEMBERSHIPNeurobiology of Lipids (L. Diomede)Neuroimmunomodulation (P. Ghezzi)Newsletters of the International Cytokine Society (P. Ghezzi)European Journal of Cancer (E. Garattini)PEER REVIEW ACTIVITIESAmerican Journal Physiology, Biochemical Journal, Biochemical Pharmacology, BiochimicaBiophysica Acta, Brain Research, Cancer Research, Cell Death and Differentiation, CellResearch, Circulation, Drug Investigation, European Journal of Cancer, European Journal ofImmunology, European Journal of Neuroscience, International Journal of Cancer, Journal ofCell Biology, Journal of Hepatology, Journal of Immunology, Journal of InvestigativeDermatology, Journal of Lipid Mediators, Journal of Neurochemistry, Journal of TranslationalMedicine, Neuroscience Letters, Pharmacological Research, Physiological Genomics,Proceedings of the National Academy of Sciences, Life Sciences.155ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE DEPARTMENT WASINVOLVEDSymposium: “Keystone Symposia on Molecular and Cellular Biology - Molecular Mechanismsof Neurodegeneration”, “Synaptic dysfunction in the cerebellum of transgenic mice expressing aprion protein with an insertional mutation”, 16-21 January, Taos, New MexicoMeeting : “III Meeting on the Molecular Mechanisms of Neurodegeneration”, ”Use ofsteroidogenic acute regulatory protein (STAR) in study of mitochondrial ATP-dependentproteases potentially involved in familial amyotrophic lateral sclerosis”; “Increased formation ofglutathione mixed disulfides in a motor neuron-like cell model for familial amyotrophic lateralsclerosis”; “Adaptive response to stable expression of human mutant G93A superoxidedismutase 1 in motor neuron-like cells involves alterations of the glutathione pool”, “In vitroMechanisms of Neuroinflammation in a Co-culture system of Astrocytes and Microglia fromNeonatal Hamsters” , PrP82-146 amyloid: the role of oligomers in triggering neuronaldegeneration”, “Early alterations of glutamate exocytosis in the cerebellum of transgenic miceexpressing a PrP insertional mutation, 19-21 May, Milan, ItalyMeeting: “Joint Annual Meeting ISMRM – ESNR”, “Neural stem cells tracking by MRI inwobbler mouse, a model of motoneuron disease”, 19-25 May, Berlin, GermanyCongress: “5° Congresso della Società Italiana di Immunologia, Immunologia Clinica eAllergologia”, “The long pentraxin PTX3 recognizes Neisseria meningitidis, binds selectedouter membrane proteins and amplify the induced inflammatory response”, 6-9 June, Trieste,ItalyMeeting: “5th Joint Meeting Medical Chemistry”, “Arylpiperazinylalkylthio benzothiazole orbenzoxazole derivatives as selective 5-HT1A serotonin receptor ligands”, “(-)-HIP-A and (+)-HIP-B: investigation of the pharmacological profile and potential therapeutic applications”, 17-21 June, Portoroz, SloveniaMeeting: “Mutant SOD1 and familial ALS: from the molecole to man”, “Altered redoxenvironment in a motor neuron-like cell model for SOD1-related forms of amyotrophic lateralsclerosis”, Glutathione and protein-glutathione mixed disulfides compartmentalization in intactmotor neuron-like cells”, 13-16 September, Milan, ItalyCongress: “XVIII Convegno Nazionale della Divisione di Chimica Farmaceutica della SocietàChimica Italiana”, “Nuovi Arilpiperazinilalchilpirrolopirimidindioni come ligandi potenti eselettivi per i recettori alfa1-adrenergici”, 16-20 September, Chieti, ItalyConference: “Prion 2007”, “Evaluation of neuroinflammation mechanisms in Co-cultures ofneurons, astrocytes and microglia from newborn hamsters”, “Use of heparin derivatives fortherapeutic intervention of transmissible spongiform encephalopathies”, “Role of oligomers inthe neurotoxicity of Gerstmann-Straussler-Scheinker disease amyloid protein”, 26-28September, Edinburgh, ScotlandCongress: “Congresso Nazionale della Società italiana di Neuroscienze”,”Mitochondrialtoxicity in cell models for familial amyotrophic lateral sclerosis”, 27-30 September, Verona,Italy156ANNUAL REPORT 2007

IRFMNSymposium: “18th International Symposium on ALS/MND”, “Proteomic analysis of nitratedproteins from peripheral blood mononuclear cells of patients with sporadic ALS”,“Characterization of protein aggregates in the G93A SOD 1 mouse reveals a possible linkbetween oxidative stress and aggregation pathogenetic pathways”, 1-3 December, Toronto,CanadaGRANTS AND CONTRACTSAgenzia Italiana del Farmaco, Rome, ItalyBiotecnologies BT - Perugia, ItalyDompè, L' Aquila, ItalyEuropean Union, Bruxelles, BelgiumIstituto Auxologico Italiano, Milan, ItalyIstituto Nazionale Neurologico "C. Besta", Milan, ItalyItalian Association for Cancer Research (AIRC), Milan, ItalyItalian Ministry of University and Research (MIUR), Rome, ItalyKenneth S. Warren Institute, NY, USALundbeck A/S, Copenhagen, DenmarkCariplo Foundation, Milan, ItalyDon Gnocchi Foundation, Milan, ItalyMariani Foundation, Milan, ItalyMonzino Foundation, Milan, ItalyMinistry of Health, Rome, ItalyNational Research Council (CNR), Palermo, ItalyNorth Shore University Hospital, NY, USAPerfetti-Van Melle, Lainate (Mi), ItalySigma Tau, Pomezia (Rome), ItalyTelethon, Milan, ItalyUniversity of Florence, ItalyUniversity of Milan-Bicocca, ItalyUniversity of Siena, ItalyWeizmann-Pasteur-Negri Foundation, Paris, FranceZambon Group, Bresso (Mi), Italy157ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Beghi E, Mennini T, Bendotti C, Bigini P, Logroscino G, Chiò A, Hardiman O, Mitchell D, Swingler R, Traynor BJ,Al-Chalabi A.The heterogeneity of amyotrophic lateral sclerosis: a possible explanation of treatment failure.Curr Med Chem. 2007 14: 3185-200Bernhagen J, Krohn R, Lue H, Gregory JL, Zernecke A, Koenen RR, Dewor M, Georgiev I, Schober A, Leng L,Kooistra T, Fingerle-Rowson G, Ghezzi P, Kleemann R, McColl SR, Bucala R, Hickey MJ, Weber C.MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.Nat Med. 2007 13: 587-96Bianchi R, Gilardini A, Rodriguez-Menendez V, Oggioni N, Canta A, Colombo T, De Michele G, Martone S,Sfacteria A, Piedemonte G, Grasso G, Beccaglia P, Ghezzi P, D'Incalci M, Lauria G, Cavaletti GCisplatin-induced peripheral neuropathy: neuroprotection by erythropoietin without affecting tumour growthEur J Cancer 2007 43 : 710-717Bigini P, Atzori C, Fumagalli E, Cagnotto A, Barbera S, Migheli A, Mennini TLack of caspase-dependent apoptosis in spinal motor neurons of the wobbler mouseNeurosci Lett 2007 426 : 106-110Bolchi C, Pallavicini M, Rusconi C, Diomede L, Ferri N, Corsini A, Fumagalli L, Pedretti A, Vistoli G, Valoti EPeptidomimetic inhibitors of farnesyltransferase with high in vitro activity and significant cellular potencyBioorg Med Chem Lett 2007 17 : 6192-6196Fiordaliso F, De Angelis N, Bai A, Cuccovillo I, Salio M, Serra D M, Bianchi R, Razzetti R, Latini R, Masson SEffect of Beta-adrenergic and renin-angiotensin system blockade on myocyte apoptosis and oxidative stress indiabetic hypertensive ratsLife Sci 2007 81 : 951-959Fioriti L, Angeretti N, Colombo L, De Luigi A, Colombo Alessio, Manzoni Claudia, Morbin M, Tagliavini F,Salmona M, Chiesa R, Forloni GNeurotoxic and gliotrophic activity of a synthetic peptide homologous to Gerstmann-Straussler-Scheinker diseaseamyloid proteinJ Neurosci 2007 27 : 1576-1583Garattini E, Gianni M, Terao MRetinoids as differentiating agents in oncology: A network of interactions with intracellular pathways as the basis forrational therapeutic combinationsCurrent Pharmaceutical Design 2007 13 : 1375-1400Garattini E., Giannì M., Terao M.Cytodifferentiation by retinoids, a novel therapeutic option in oncology: rational combinations with other therapeuticagents.Vitam Horm. 2007 75: 301-54Ghezzi P, Di Simplicio P.Glutathionylation pathways in drug response.Curr Opin Pharmacol. 2007 7: 398-403. ReviewGhezzi P, Mengozzi MActivities of erythropoietin on tumors: An immunological perspectiveEur J Immunol 2007 37 : 1427-1430Leonelli E, Bianchi R, Cavaletti G, Caruso D, Crippa D, Garcia-Segura L M, Lauria G, Magnaghi V, Roglio I,Melcangi R CProgesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: a multimodalanalysisNeuroscience 2007 144 : 1293-1304Luo X Y, Sharma D, Inouye H, Lee D, Avila R L, Salmona M, Kirschner D A158ANNUAL REPORT 2007

IRFMNCytoplasmic domain of human myelin protein zero likely folded as Beta structure in compact myelinBiophys J 2007 92 : 1585-1597Luo X, Inouye H, Gross AA, Hidalgo MM, Sharma D, Lee D, Avila RL, Salmona M, Kirschner DA.Cytoplasmic domain of zebrafish myelin protein zero: adhesive role depends on beta-conformation.Biophys J. 2007, 93: 3515-28Massignan T, Casoni F, Basso M, Stefanazzi P, Biasini E, Tortarolo M, Salmona M, Gianazza E, Bendotti C, BonettoVProteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouseBiochem Biophys Res Commun 2007 353 : 719-725Mereghetti I, Cagnotto A, Mennini TDimethyl sulfoxide: An antagonist in scintillation proximity assay [35S]-GTPgammaS binding to rat 5-HT6 receptorcloned in HEK-293 cells?J Neurosci Methods 2007 160 : 251-255Modica M, Salerno L, Pittala' V, Romeo G, Siracusa M, Mereghetti I, Cagnotto A, Mennini TSynthesis and binding properties of new endothelin receptor ligandsLett Drug Des Discov 2007 4 : 232-238Noe F, Nissinen J, Pitkanen A, Gobbi M, Sperk G, During M, Vezzani AGene therapy in epilepsy: the focus on NPYPeptides 2007 28 : 377-383Prezzavento O, Campisi A, Ronsisvalle S, Li Volti G, Marrazzo A, Bramanti V, Cannavo' G, Vanella L, Cagnotto A,Mennini T, Ientile R, Ronsisvalle GNovel sigma receptor ligands: Synthesis and biological profileJ Med Chem 2007 50 : 951-961Roglio I, Bianchi R, Giatti S, Cavaletti G, Caruso D, Scurati S, Crippa D, Garcia-Segura L M, Camozzi F, Lauria G,Melcangi R CTestosterone derivatives are neuroprotective agents in experimental diabetic neuropathyCell Mol Life Sci 2007 64 : 1158-1168Salerno L, Guerrera F, Modica M, Romeo G, Pittalà V, Siracusa MA, Mereghetti I, Cagnotto A, Mennini T.Synthesis of 1,2,4-triazole derivatives: binding properties on endothelin receptors.Med Chem. 2007 3: 551-60Scarchilli L, Camaioni A, Bottazzi B, Negri V, Doni A, Deban L, Bastone A, Salvatori G, Mantovani A, Siracusa G,Salustri APTX3 Interacts with inter-alpha- trypsin inhibitor. Implications for hyaluronan organization and cumulus oophorusexpansionJ Biol Chem 2007 282 : 30161-30170Setola V, Terao M, Locatelli D, Bassanini S, Garattini E, Battaglia GAxonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesisProc Natl Acad Sci USA 2007 104 : 1959-1964Villa P, Triulzi S, Cavalieri B, Di Bitondo R, Bertini R, Barbera S, Bigini P, Mennini T, Gelosa P, Tremoli E, SironiL, Ghezzi PThe interleukin-8 (IL-8/CXCL8) receptor inhibitor reaparixin improves neurological deficits and reduces long-terminflammation in permanent and transient cerebral ischemia in ratsMol Med 2007 13 : 125-133159ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Receptor PharmacologyStudies on the role of dysfunction of the endoplasmic reticulum (ER) orthe Golgi apparatus (GA) in neurodegenerative diseaseThe secretory pathway starts at the endoplasmic reticulum (ER) where proteins are synthesizedand folded and chaperone–mediated quality control prevents misfolded proteins to reach theirdestination and interfere with normal metabolism. Protein transport by mean of vesiclescontinues through the Golgi Apparatus (GA), that is most abundant in neurons, and finishes inthe plasma membrane, secretory vesicles or lysosomes. The endocytic pathway enablesinternalized macromolecules to be delivered via endosomes to lysosomes where they areenzymatically digested.In mammalian cells, the Golgi-associated retrograde protein (GARP) complex is involved inretrograde transport of endosomes to the trans GA network. Defective intracellular membranetrafficking is common to several neurodegenerative diseases. Among the neuronal population,motor neurons, due the their high energy requirement and long axons are, together with retinalcells, the most sensitive ones.The Laboratory of Receptor Pharmacology utilizes two mouse models of neurodegenerationrelated to cellular transport disruption, carrying mutation in proteins resident in the ER (themnd mouse) or in the GARP complex (the wobbler mouse).The neuronal ceroid lipofuscinosis (NCLs) are a group of autosomal recessiveneurodegenerative diseases and a significant cause of childhood progressive intellectual andneurological deterioration, for which no curative or preventive treatment are available. Amongthem, the progressive epilepsy with mental retardation is the newest form with mutation in theCLN8 gene encoding a novel ER transmembrane protein with undefined function. Anorthologue of CNL8 is mutated in the motor neuron degeneration mouse (mnd) which showsearly retinopathy and delayed motor neuron dysfunction without degeneration. How CLN8mutation leads to NCL defect is unknown. Our laboratory is studying mnd mice since manyyears: we have already reported decreased spinal cord GLT-1 glial glutamate transporter andincreased plasma glutamate concentration already at presymptomatic stage in mnd mice, withincreased GluR2 and lowered GluR3 AMPA receptor subunits in the lumbar spinal cord. TheAMPA receptor antagonists ZK 187638 (non-competitive), like NBQX (competitive),ameliorate motor behavior in mnd mice. We also found that TNF and TNFR1 is increased inthe spinal cord of mnd mice already at presymptomatic stage, when intensive astrocytes andmicroglial proliferation occurs. The rate of oxygen consumption (QO2), and mitochondrialfunctions were decreased in mnd spinal cord. The level of lipid peroxide derivatives reactingwith thiobarbituric acid (TBARS) were increased in mnd spinal cord and retina. L-carnitinetreatment delayed the onset of motor behaviour impairment, increased the mitochondrialenzyme activities and was effective in enhancing QO 2 and decreasing TBARS levels.At present we are testing the susceptibility to convulsions in mnd mice at pre-symptomaticstage , in order to find a further link to the human pathology. In these studies new non-invasiveapproaches, like Optical Imaging , MRI, MicroCT and Confocal Angiography are applied, inorder to allow a better translation of the results to the human disease.Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder involvingprimarily motor neurons in the spinal cord, brainstem and cerebral motor cortex and leading todenervation, muscular atrophy and paralysis. The disease is sporadic in approximately 90% of160ANNUAL REPORT 2007

IRFMNcases, and the mechanism(s) responsible for the selective motor neuron degeneration are farfrom being elucidated.A missense mutation in Vps54 has been described in the wobbler mouse, which share manypathological features with ALS patients. In mammalian cells Vps54 forms heterotrimericcomplexes with Vps52 and Vps53 to form the GARP complex, involved in retrograde transportof endosomes to the trans GA network. Thus we use the wobbler mouse as a reliable tool tounderstand the interplay between endosomal dynamics and the selective loss of motor neurons.A series of experiments are in progress in cultured neural cells obtained from wobbler andhealthy homozygous mice to investigate the possible effect of Vps54 mutation on intracellulartrafficking, mitochondrial activity, and lysosome accumulation.We have already reported that wobbler mice are sensitive to riluzole treatment, withoutmarked changes in AMPA/NMDA receptor subunits expression in motor neurons of earlysymptomatic mice. In addition we found increased levels of TNF and TNFR1 in the cervicalspinal cord and a significant effect of chronic treatment with a soluble h-TNF binding protein,resulting in slower clinical symptoms progression, reduced motor neuron loss and selectiveinhibition of the two main stress-kinases (p38 and JNK) associated to TNF receptors activation.Finally we investigated two different approaches of cell therapy. Undifferentiated adult neuralstem cells (in collaboration with Dr. Parati, Istituto Besta) produced a weak and transientprotective effect in clinical progression but significantly reduced motor neuron loss occurring inthe wobbler mouse. Transplantation of mononucleate cells from human cord blood (incollaboration with Dott. Lazzari, Policlinico), although did not replace degenerating motorneurons, produced a marked neuroprotective effect by slowing the clinical progression andreducing motor neuron loss, biceps atrophy and neuroinflammation (reactive gliosis).Neuropharmacology of the glutammatergic and serotonergic systemsGlutamate is the major excitatory amino acid in the central nervous system; the extracellularglutamate concentration has to be maintained at physiological level by active uptake mediatedby specific transporters (Excitatory Amino Acid Transporters, EAATs) located on the plasmamembranes of neurons and glia. Alterations in this process might lead to a relevant increase inextracellular glutamate concentrations, that is highly toxic for neurons in the central nervoussystem. A research project is in progress in order to better characterize the involvement and therole of the neuronal compartment in the general process of glutamate homeostasis. Thefunctional properties are evaluated using biochemical assays and the quantitative characteristicsare evaluated by western blot for specific neuronal and glial proteins and flow cytometryanalysis (in collaboration with Dr. Bernasconi, Department of Oncology, IRFMN). Theseevaluations are done on purified preparations from mouse spinal cord. The same characteristicsare evaluated on two different animal models of motor neuron degeneration, the wobbler mouseand the transgenic SOD1G93A mouse (in collaboration with Dott. Bendotti, Department ofNeuroscience, IRFMN), in order to understand the possible involvement in neurodegenerativediseases.Results obtained to date indicate that there is an active neuronal component in the glutamateuptake. The comparison between the two animal models of neurodegeneration suggest that thiscomponent has a different importance in the motor neuron death processes. In the wobblermouse the excitotoxicity related to alterations of glutamate uptake is likely to be not involved,while in the transgenic SOD1G93A mouse we found a significant reduction of glutamateuptake in the neuronal fraction. This alteration probably contribute to the exacerbation ofprocesses leading to motor neuronal death in this animal model.A research project is in progress in collaboration with professor Bonanno (University ofGenova) to characterize mechanisms of glutamate release from synaptosomes obtained fromspinal cord of wobbler mouse, in order to evaluate if the motor neuronal death might depend onaltered processes of neurotransmitter release in the nerve endings.161ANNUAL REPORT 2007

IRFMNWe recently concluded a project focused on the study of the interaction between glutamatetransporters and riluzole. Riluzole is a drug with a complex mechanism of action, and is theonly drug approved for the treatment of amyotrophic lateral sclerosis (ALS). We used asexperimental model cell cultures that stably express the main three glutamate transporters(GLT1, GLAST and EAAC1) and we demonstrated for the first time that riluzole acts on theglutamate uptake mediated by the three EAAT subtypes, significantly increasing the efficiencyof the process. This mechanism might be relevant in pathological conditions characterized byglutamate concentrations over the physiological threshold, as occurs in ALS.AMPA receptor-mediated excitotoxicity is one of the main events involved in motor neurondegeneration in ALS pathogenesis. We established a new model of primary cocultures ofpurified motor neurons over a glial layer. This in vitro model allows to obtain healthier motorneurons maintained in more physiological conditions and was used to demonstrate that AMPAreceptor agonists can induce both apoptotic or non-apoptotic death pathways depending on theirconcentrations. We studied the interaction between excitotoxicity and other potentiallyneurotoxic factors, such as the inflammatory mediators, and we demonstrated that the proinflammatorychemokine IL-8 induces motor neuron death through the CXCR2 receptor.Preliminary studies on the effect of TNF-α, whose levels were found increased in the spinalcords of animal models of motor neuron degeneration, indicated the pivotal role of glial cells inmediating neurotoxicity of this cytokine. At present, we are studying the main intracellularbiochemical pathways involved in neurodegenerative mechanisms (such as calcium influx) andtesting new potential treatments to selectively interfere with each of them. We have also starteda study on the neurotoxic effect of serum from professional soccer players aimed at identifyingpotential risk factors present in the blood which could support the high incidence of ALS in thisgroup of athletes.Studies on primary spinal cord or hippocampal neuronal cultures from wild type or mnd miceare in progress in order to investigate their sensitivity to AMPA receptor agonists andantagonists, and to evaluate the role of astrocytes obtained from mnd mice in affecting motorneuron viability. Studies on astrocytes derived from neural stem cells of wobbler or controlmice are in progress in order to evaluate whether typical biochemical alterations of wobblermice are already evident in precursor-derived cells : we study the role of glutamate transportersand possible effects of this kind of cells on healthy motor neurons. Finally, studies on the roleof Vsp54 mutation on intracellular trafficking are in progress in primary cultures of astrocytesobtained from the spinal cord of adult wobbler or control mice.The Laboratory of Receptor Pharmacology is maintaining, since many years, collaborationswith medicinal chemistry laboratories to characterize the affinity and selectivity of newlysynthesized compounds on neurotransmitter receptors using in vitro binding methods. Theresults are utilized for molecular modelling (QSAR) studies and/or for furtherpharmacological evaluations. Particularly, a collaboration is ongoing with Prof. S. Grasso(University of Messina) and Prof. C. De Micheli (University of Milan), for the development ofnew non-competitive AMPA receptor antagonists.An useful application of this technique is also the evaluation of the agonist/antagonist activityof compounds acting on G-protein coupled receptors (GPCR), measuring their effects on 35S-GTP-γ-S binding. New non-radioactive methods based on “time-resolved fluorescence” areunder characterization as functional assays to monitor GPCR activity on cell membranes.Recently we have verified the effect of dimethyl sulfoxide, a solvent commonly utilized todissolve hydrophobic compound for in vitro experiments, on different in vitro assays utilizingHEK 293 cells expressing the 5-HT6 serotonin receptors. Our results indicate that dimethylsulfoxide interferes with the agonist activity on 5-HT6 receptor when the scintillationproximity assay 35S-GTP-γ-S binding is used. On the contrary, it does not interferes witheuropium labelled GTP binding determined by “time-resolved fluorescence”.162ANNUAL REPORT 2007

IRFMNIn addition, the laboratory perform autoradiography binding studies in order to evaluate ex vivodrug-receptor occupancy.Finally, we have ongoing collaborative studies with Dr. Gobbi (Lab. biochemistry and proteinchemistry, IRFMN), to characterize the role of the allosteric site at the serotonin transporter onthe mechanism of action of SSRI (selective serotonin reuptake inhibitors) antidepressant, likeescitalopram.Laboratory of NeuroimmunologyRedox regulationThe study of the so-called oxidative stress has led to the identification of biochemical eventsthat are modified by antioxidant molecules even in the absence of oxidative stress intended asoverproduction of toxic oxygen intermediates (free radicals). We use the term redox regulationto define the pattern of cell functions (gene expression, activity of enzymes or transcriptionfactors) that are in some way modified by the redox state of the cell, defined as the ratiobetween oxidizing and reducing species (usually: the oxidized glutathione / reduced glutathioneratio). Our work focuses on the molecular mechanisms by which small changes in the redoxstate can affect proteins, with particular attention to the reversible modification of cysteines toform disulfide bonds (with proteins or with small molecular weight thiols such as glutathione).We recently focused our attention on the identification of the redox state of proteins present onthe outside of the plama membrane since these often have key functions (e.g.: transporters,receptors) and are the closest target of extracellular oxidants. We also apply proteomicstechniques and gene expression profiling using DNA microarrays to identify the pathwayssusceptible of redox regulation.Neuroprotection and erythropoietinThe pathologies of the central or peripheral nervous systems studied in the lab are: cerebralischemia, experimental autoimmune encephalomyelitis, and diabetic neuropathy). Using animalmodels and cell culture, we try to clarify the relationships between neuronal death andinflammation, and to intervene with protective agents. Among the latter, we are studyingendogenous molecules that have shown an unexpected anti-apoptotic action on neuronal cells,particularly erythropoietin and anti-inflammatory drugs.Laboratory of Molecular BiologyNovel retinoids for the treatment of acute myeloid leukemiaThe synthetic and natural derivatives of retinoic acid (retinoids) have shown promising activityin the treatment of leukemia and solid cancer. Retinoids exert their therapeutic activity throughthree distinct types of effects: cyto-differentiation, growth inhibition and apoptosis. The threeeffects can be dissociated, albeit partially, as retinoids endowed primarily with cytodifferentiatingor anti-proliferative activities are known. Recently, we identified andcharacterized a novel class of retinoids with strong and selective apoptotic activity towards theneoplastic cell. These compounds (RRMs, retinoid related molecules), which were originallydeveloped as selective agonists of the gamma-types of the nuclear receptors of retinoic acid(RARy), induce apoptosis in different types of leukemia and solid cancer cells through a largelyunknown mechanism. The process of apoptosis set in motion by RRMs is different from that ofother known chemotherapeutic agents and does not require the activation of the nuclear retinoicacid receptors. RRMs are active not only in vitro but also in vivo on a number of pre-clinicalmodels of acute myeloid leukemia. Currently, some of these innovative molecules are in an163ANNUAL REPORT 2007

IRFMNadvanced phase of pre-clinical development.Novel retinoic-acid-based pharmacological combinations for the treatmentof acute leukemiaThe clinical use of retinoic acid for the treatment of acute promyelocytic leukemia (APL) isbased on the ability of this compound to induce the maturation of the leukemic blast along thenormal myelocytic/granulocytic pathway. At present, the clinical use of retinoic acid for thetreatment of patients suffering from APL is the sole example of differentiation therapy.Differentiation therapy is worth pursuing as it is theoretically associated with a lower level oftoxicity relative to what observed following treatment with the classical cyto-toxic agents.However, the clinical use of retinoic acid is still burdened by a number of problems includingnatural and induced resistance as well as systemic and local toxicity. One of the possible waysto increase the therapeutic index of retinoic acid is based on the identification of compounds ordrugs that potentiate the pharmacological activity of the retinoid. We have recentlydemonstrated that a series of agents, such as G-CSF, interferons, cAMP analogs,phosphodiesterase IV inhibitors and a number of other novel compounds sensitize the leukemicblast to the pharmacological activity of retinoic acid. In the long run, it is our objective todevelop novel combinations and bring some of the above mentioned retinoic-acid-basedcombinations to the clinic. In addition we intend to use some of the combinations aspharmacological tools to dissect the intricacies of the cyto-differentiation process set in motionby retinoic acid in the leukemic blast.The family of molybdo-flavoproteinsMolybdo-flavoenzymes are proteins of medical and industrial interest. They are the soleenzymes that require molybdenum, in the form of the molybdenum cofactor, for their catalyticactivity. The laboratory has a long-standing and specific interest in the biochemistry andbiology of mammalian molybdo-flavoproteins. In the past, the laboratory contributed to theelucidation and characterization of the primary structure of the two mammalian molybdoflavoproteins,aldehyde oxidase (AOX1) and xanthine oxidoreductase (XOR). In the last fewyears, the group identified and cloned the cDNAs and the genes coding for three novel mousemolybdo-enzymes (AOH1, AOH2 and AOH3) belonging to the sub-family of molybdoflavoproteins.The long-term goal of our studies is to define the structure, the substratespecificity, the mechanisms of catalysis as well as the physo-pathological function of the threenew proteins. We will also continue the biochemical and functional studies on mammalianAOX1 and XOR. To achieve our aims, we have recently developed cell lines over-expressingXOR in a tetracycline inducible fashion. In addition, we have generated knock-out mice for thegenes encoding AOH2 and AOH3.Laboratory of Biochemistry and Protein ChemistryChimico-physical and molecular-biochemical studies on the prion proteinand on the peptides deduced from its aminoacid sequencePrion protein fibril formation is associated with neuronal cytotoxicity and astrogliosis observedin prion diseases. The formation of fibrils is the consequence of a conformational switchbetween the structure of the native and the pathological proteins and it is considered of pivotalimportance for the appearance and progression of prion protein diseases.Identifying the molecular determinants responsible for the conformational transition is likely togive insight into the pathogenetic process leading to prion diseases. A reductionist appraoch tothe problem calls for the generation of simple experimental models in which the dynamics ofthe conformational transition can be studied in detail. In our laboratory we developed syntheticpeptides that mimic the fibrillogenic properties of pathological prion protein. With the use of164ANNUAL REPORT 2007

IRFMNvarious types of biochemical and biophysical techniques we studied the conformation of thesepeptides through the evaluation of their secondary structure, the resistance to protease digestionas well as the aggregating and amyloidogenic properties. Our approach gave detailedinformations on the conformational plasticity of various types of prion protein fragments.To understand the correlation between the chemico-physical properties of prion protein-derivedpeptides and their biological effects we used appropriate cellular models. These experiments,performed in collaboration with the Laboratory of the Biology of Neurodegenerative Disorders,gave informations on the sub-cellular distribution of the peptides to identify the intracellularbiological targets.Development of a therapeutical strategy against prion-related diseasesCurrently, no therapeutic options for the cure of prion-related diseases are available and theidentification of new molecules for the prevention and treatment of the infettivity is of greatinterest. Althought some compounds gave positive results in prion cellular models, in humanthey exhert low efficacy for toxicity and for their inability to pass the blood brain barrier. A firstapproach for the development of anti-prion candidates involved molecules capable of interferingwith the fibrils formation, directly interacting with the β-sheet conformation of PrPSc causingits disaggregation. In collaboration with the Laboratory of the Biology of the NeurodegenerativeDisorders we aimed to identify compounds endowed with anti-fibrillogenic activity and testtheir efficacy in different in vitro and in vivo pre-clinical models of prion diseases.Our studied indicated that tetracyclines are new good candidates as anti-prion drugs since theyact as anti-fibrillogenic compunds increasing the prion sensitivity to protease K digestion.Moreover, they inhibited neuronal cell death and astrogliosis caused by prion peptides andprolonged the survival of prion-infected animals.Another therapeutic approach is based on the development of molecules that inhibited the PrPformation by interacting with the lipid metabolism and destabilizing specific cellular membranedomains. In collaboration with the Department of Infective Pathology and Diseases of theRoyal Veterinary School (Hawkshead, UK) we reported that statins, inhibiting cholesterolsynthesis, reduced the in vitro prion production. On the other hand, compounds such aspolyunsaturated fatty acids, known for their reducing effects on cholesterol levels, increased thePrP production. Our future studies are aimed at understanding the relationship betweencholesterol cellular membrane distribution, lipid domain stability and the conversion of cellularprion protein in its pathologic form.Oxidative stress and protein aggregation in amyotrophic lateral sclerosis:a proteomic approachThe molecular mechanisms at the basis of neurodegenerative diseases including the geneticallylinkedones, such as amyotrophic lateral sclerosis (ALS), are still unknown. However, there isevidence that oxidative stress and protein aggregation play central roles in the pathogenesis ofsuch diseases. The Unit of Medical Biochemistry, conducted proteome analysis of an animalmodel of familial ALS. In collaboration with the laboratory of Molecular Neurobiology, wefocused the attention on the analysis of protein expression changes and protein modifications,such as tyrosine nitration and ubiquitination, in a transgenic mouse, which over-express humanmutated (G93A) superoxide dismutase (SOD1). We analyzed, by proteomic tools, spinal cord ofpresymptomatic G93A SOD1 mice, we identified nitrated proteins and quantified the level ofnitration for each protein in comparison with healthy controls. We have revealed that there is asubstantial increase of the nitration level in at least five proteins: actin, alpha and gammaenolase, ATP synthase and a chaperone protein, HSC71. The alteration of the function of theseproteins may have important consequences on the cellular metabolism and catabolism, andtherefore may be at the basis of the molecular mechanisms leading to neurodegeneration. Inaddition, by mass spectrometry, we have identified the specific nitrated tyrosines for a number165ANNUAL REPORT 2007

IRFMNof proteins. We have observed that al least enolase and glyceraldehyde 3-phosphatedehydrogenase are nitrated at the same tyrosine site known to be phosphorylated. This is animportant finding, which is worthwhile further studying. In fact, it may indicate a possibleinvolvement of nitration in signaling pathways and phosphorylation cascades. Regarding theaggregation studies, we have isolated detergent insoluble-protein fractions from spinal cord ofG93A SOD1 mice and we have completed the comprehensive characterization of all theproteins contained. With this study we have identified the protein constituents of aggregates,still unknown, and therefore have contributed to the comprehension of the role of proteininclusions in ALS pathogenesis.Laboratory of Molecular PathologyNovel in vitro models for investigating motor neuron pathologiesPresence of mutant forms of specific proteins plays a key role in many neurodegenerativediseases. Experimental models in vivo and in vitro are sorely needed to study the effects ofthese toxic proteins. Recently it was developed a new methodology (pTet-On/pTet-Off) tocontrol gene expression through the level of tetracyclines. We have applied the pTet-Off systemto a motor neuron-like cell line (NSC-34) establishing NSC-34 tTA cell lines for tetracyclineregulatedgene expression. These lines are suitable to study the pathogenetic mechanisms ofmotor neuron diseases after transient/stable transfection with genes of interest for thesepathologies.We further used this approach to establish NSC-34 tTA cell lines that express in a doxycyclineinducible fashion the human G93A mutant Cu/Zn superoxide dismutase. Mutant forms ofsuperoxide dismutase 1 are responsible for some of the familial forms of amyotrophic lateralsclerosis. This model allows to develop novel approaches to study pathogenic mechanisms ofamyotrophic lateral sclerosis.Novel intracellular targets in the selective degeneration of motor neuronsin amyotrophic lateral sclerosisAmyotrophic lateral sclerosis is a rapidly fatal neurodegenerative disease characterized by lossof motor neurons. The management remains essentially supportive and symptomatic.Understanding the mechanisms underlying the disease is a way to favor more efficienttherapeutic strategies. Mitochondrial morphological alterations were observed in the earlystages of the disease in the motor nerve terminals of ALS patients and in murine experimentalmodels. For these reasons we addressed our studies to determine biochemical-molecularalterations involved in the mitochondrial damage utilizing different cellular models. We havestudied the toxicity of mutant forms of superoxide dismutase 1, responsible for some familialforms of amyotrophic lateral sclerosis. We showed that mutant superoxide dismutase 1(G93ASOD1) altered the mitochondrial morphology selectively in motor neuronal cells. Thisdamage was modulated by the extent of expression of G93ASOD1. Furthermore presence ofG93ASOD1 increased the susceptibility of motor neurons to inhibitors of the electron transportchain (ETC) and to oxidants. Exposure to drugs or exogenous compounds impairing the ETCcould thus be a risk to motor neurons of individuals carrying mutant superoxide dismutase 1.Cytochrome P-450 superfamilyCytochrome(s) P-450 have evolved into a large superfamily that varies enormously in substrateaffinity and product formation. This system plays a major role in the metabolism of drugs andother chemicals. The majority of existing drugs depends on the P-450 system for terminatingtheir biological effects or for side effects or adverse reaction. The laboratory of MolecularPathology has a long-standing interest in the induction/degradation mechanisms of specificcytochrome P-450 families due to drug administration or to disease states. Our recent research166ANNUAL REPORT 2007

IRFMNfocused on cytochrome P-450 induction by herbal remedies such as Hypericum perforatumextracts (St. John’s Wort), which have an alleged activity in mild to moderate depression butinterfere with the effect of several drugs.Activation of enzymes of the heme metabolic pathway (heme oxygenasesystem, biliverdin reductase) as a protective response to stressThe enzymatic system of heme oxygenase (HO) is devoted to cellular degradation of hemecontaining molecules, like cytochromes and hemoglobin, and to recycling of iron. Productsformed by the catalytic activity of HO - carbon monoxide and bile pigments - are importantregulating factors in the cell. An increase of HO activity (which is usually sustained byactivation of the inducible form HO-1) is now considered a protective mechanism againstuntoward stimuli particularly when oxidative stress is involved. In the past, the laboratory ofMolecular Pathology identified cytokines as inducers of HO activity and as transcriptionalactivators of the HO-1 gene. We are currently investigating the functional significance of HO-1activation in neurodegeneration.Laboratory for the Study of Biological SystemsNovel regulators of cell motilityCell motility plays a central role in several biological processes, under both normal (e.g.embryonic development) and pathological conditions (e.g. tumor cell dissemination). Thus, it isimportant to identify the molecular mechanisms that regulate cell motility. In recent years, wehave characterized Junctional Adhesion Molecule-A (JAM-A), a membrane molecule thatlocalizes to the intercellular tight junctions and binds PDZ-type intracellular proteins. In thecourse of these studies, we have discovered that JAM-A expression reduces cell motility. Inaddition, we have found that JAM-A enhances microtubule stability and focal adhesionformation, which are the adhesive points of contact between cells and extracellular matrix. Allthese functional changes require amino acid residues that mediate binding to PDZ-typeintracellular proteins. These findings have highlighted a novel mechanism of motility inhibitionthat requires the interaction between a membrane protein and PDZ-type intracellular proteins.Effect of inflammatory cytokines on Junctional Adhesion Molecule-A(JAM-A)In the course of inflammatory responses, JAM-A contributes to the leakage of plasma proteinsand the transmigration of circulating leukocytes. Although it has been reported that theinflammatory cytokine Tumor Necrosis Factor (TNF) causes the disassembly of JAM-A fromthe intercellular junctions, the mechanism has not been elucidated fully. Recently, we found thatTNF enhances the solubility of JAM-A in non-ionic detergents and increases the amount ofdetergent-soluble JAM-A at the cell surface. In addition, we found that, upon cell treatment withTNF, higher levels of JAM-A become detectable at the cell surface (by FACS analysis). Asthese higher levels of JAM-A derive from the intercellular junctions (and not from intracellularstores), we propose that TNF causes not only the disassembly of JAM-A from the junctions andits subsequent redistribution to the cell surface, but also its dispersal in such a way that JAM-Abecomes more easily accessible to the antibodies used for FACS analysis. These findings areimportant to highlight potential mechanisms of permeability regulation during inflammationthat might be modulated by inflammatory interventions.167ANNUAL REPORT 2007

IRFMNDEPARTMENT OF EPIDEMIOLOGYSTAFFHeadCarlo LA VECCHIA, M.D.LABORATORY OF GENERAL EPIDEMIOLOGYHeadCarlo LA VECCHIA, M.D.Cancer Epidemiology UnitHeadCristina BOSETTI, Mat.Sci.D.Lifestyle Habits and Prevention UnitHeadLiliane CHATENOUD, Biol.Sci.D.Epidemiology for Clinical Research UnitHeadSilvano GALLUS, Comp.Sci.D.LABORATORY OF EPIDEMIOLOGICAL METHODSHeadEva NEGRI, Mat.Sci.D.LABORATORY OF EPIDEMIOLOGY AND CHRONIC DISEASESHeadAlessandra TAVANI, Biol.Sci.D.LABORATORY OF MEDICAL INFORMATICSHeadEugenio SANTORO, Comp.Sci.D.168ANNUAL REPORT 2007

IRFMNCURRICULA VITAECarlo La Vecchia holds a Doctor of Medicine from the University of Milan, a Master of Science inClinical Medicine (epidemiology) from Oxford University and a Diploma from the Post-Graduate Schoolof Pharmacological Research at the “Mario Negri” Institute for Pharmacological Research in Milan.Work experiences: he is Head of the Department of Epidemiology at the Mario Negri Institute forPharmacological Research in Milan, Italy. He is also Associate Professor of Epidemiology at theUniversity of Milan, Adjunct Professor of Epidemiology, University of Lausanne, Switzerland andAdjunct Professor of Medicine, School of Medicine, Vanderbilt University, Nashville, TN.Dr. La Vecchia is an Honorary Senior Lecturer in Oral Medicine at the Eastman Dental Institute at theUniversity College of London, a temporary advisor at the International Agency for Research on CancerIARC/WHO in Lyon and at the WHO in Geneva, and a registered journalist in Milan. He was AdjunctAssociate Professor of Epidemiology at the Harvard School of Public Health between 1996 and 2001. Heis Associate Editor to: European J. of Cancer Prevention and presently serves on the editorial boards ofthe: American J. of Epidemiology, Asian Pacific J. of Cancer Prevention, Cancer Causes and Control,Current Cancer Therapy Reviews, Digestive and Liver Disease, Economia Politica del Farmaco,European J. of Cancer, European J. of Cancer Prevention, European J. of Clinical Nutrition, European J.of Nutrition, Scope Oncology & Hematology, Int. J. Cancer, J. of Nephrology, Nutrition and Cancer,Oncology, Oral Oncology, Revisiones en Ginecología y Obstetricia, Revista Española de NutriciónComunitaria, Revue d’Epidémiologie et de Santé Publique, Sozial und Praeventivmedizin, The Lancet(Italian edition), Tumori, Alimentazione e Prevenzione. In 1993, he received the Glaxo Prize for medicalpublication.He has authored or co-authored over 1,360 publications in peer reviewed journals, with over 35,000quotations.Eva Negri got a degree in Mathematics in 1985 at the University of Milan, School of Mathematics.Awards: EEC scholarship for postgraduate training in Epidemiology (1988).Areas of interest: Design, conduction and analysis of epidemiologic studies on chronic diseases (e.g.cancer and myocardial infarction) and injuries, analysis of mortality of cohorts of workers, analysis oftemporal trends and geographic distribution of mortality from cancer, cardiovascular disease, injuries andother selected conditions, analysis of national health surveys, application of linear modeling techniques tothe analysis of epidemiological data, collaborative re-analyses and meta-analyses of epidemiologicalstudies.Work experiences: Since 2007: Laboratory Chief, Unit of Epidemiologic Methods, Department ofEpidemiology; 1992-2006: Unit Chief, Unit of Epidemiologic Methods, Laboratory Epidemiology; since1990-1992: Researcher at the Laboratory of Epidemiology; 1984-1990: Collaborator of the Laboratory ofEpidemiology.Selected publications• Negri E, La Vecchia C, Pelucchi C, Tavani A The risk of acute myocardial infarction after stopping drinking Prev Med2005; 40: 725-728• Negri E, Pelucchi C, Talamini R, Montella M, Gallus S, Bosetti C, Franceschi S, La Vecchia C Family history of cancerand the risk of prostate cancer and benign prostatic hyperplasia Int J Cancer 2005; 114: 648-652• Negri E, Little D, Boiocchi M, La Vecchia C, Franceschi S. B-cell non-Hodgkin’s lymphoma and hepatitis C virusinfection: A systematic review Int J Cancer 2004; 111: 1-8• Negri E, Ron E, Franceschi S, La Vecchia C, Preston-Martin S, Kolonel L, et al. Risk factors for medullary thyroidcarcinoma: A pooled analysis Cancer Causes Control 2002; 13: 365-372• Levi F, La Vecchia C, Boyle P, Lucchini F, Negri E Western and eastern European trends in testicular cancer mortalityLancet 2001; 357: 1853-1854169ANNUAL REPORT 2007

IRFMNAlessandra Tavani degree in Biological Sciences, University of Milan, Italy (July 1977);Pharmacological Research Specialist, “Mario Negri” Institute for Pharmacological Research, Milan,Italy (July 1979).Work experiences: 1979-81: Researcher at the laboratory of Drug Metabolism, “Mario Negri” Institutefor Pharmacological Research. 1981: Researcher at the Unit for Research on Addictive Drugs (directorprof. H.W. Kosterlitz), University of Aberdeen, Scotland, U.K. 1982-1990: Head of the Unit of OpioidNeuropharmacology, “Mario Negri” Institute for Pharmacological Research. 1990: Researcher at theUnit of Clinical Perinatal Pharmacology, “Mario Negri” Institute for Pharmacological Research. From1991-2006: Head of the Unit of Epidemiology of Chronic Diseases of the Laboratory of Epidemiology,“Mario Negri” Institute for Pharmacological Research. 2007-: Head of the Laboratory of Epidemiologyof Chronic Diseases of the Department of Epidemiology, “Mario Negri” Institute for PharmacologicalResearch.Awards: "Rafaelsen Scholar Award" from the Collegium Internationale Neuro-Psychopharmacologicum(CINP), 16th Meeting, Munich (F.R.G.), 1988.Areas of interest: Epidemiology of cancer and coronary heart disease.Selected publications• Tavani A, Zucchetto A, Dal Maso L, Montella M, Ramazzotti V, Talamini R, Franceschi S, La Vecchia C. Lifetimephysical activity and the risk of renal cell cancer. Int J Cancer 2007 ; 120 : 1977-1980• Dal Maso L, Zucchetto A, Tavani A, Montella M, Ramazzotti V, Talamini R, Canzonieri V, Garbeglio A, Negri E,Tonini A, La Vecchia C, Franceschi S . Renal cell cancer and body size at different ages: An Italian multicentre casecontrolstudy. Am J Epidemiol 2007 ; 166 : 582-591• Tavani A, Pelucchi C, Parpinel M T, Negri E, Franceschi S, Levi F, La Vecchia C. n-3 Polyunsaturated fatty acid intakeand cancer risk in Italy and Switzerland. Int J Cancer 2003; 105: 113-116• Herrero R, Castellsague X, Pawlita M, Lissowska J, Kee F, Balaram P, Rajkumar T, Sridhar H, Rose B, PintosJ, Fernandez L, Idris A, Sanchez M J, Nieto A, Talamini R, Tavani A, et al. Human papillomavirus and oral cancer: TheInternational Agency for Research on Cancer Multicenter Study. J Natl Cancer Inst 2003; 95: 1772-1783• Tavani A, Pelucchi C, Negri E, Bertuzzi M, La Vecchia C. n-3 polyunsaturated fatty acids, fish, and nonfatal acutemyocardial infarction. Circulation 2001; 104: 2269-2272Eugenio Santoro got his degree in Computer Science in 1990 at the Milan University. He started towork at the “Mario Negri” Institute in 1985 as a research fellow. He was Head of the Applied Statisticsand Informatics Unit and of the Applied Statistics and Informatics laboratory, which was part of theDepartment of Cardiovascular Research. Since 2001 he is Head of the Laboratory of MedicalInformatics that is currently part of the Department of Epidemiology. His main areas of interest havebeen biostatistics and clinical informatics with the development of software for data management anddata analyses of large scale clinical trials in cardiology, such as the GISSI studies (Gruppo Italiano perlo Studio della Sopravvivenza nell’Infarto miocardico). His main current area of interest is the Internet,and more recently the web 2.0, and their application in the medical field, in clinical research, and inmedical education through the development of health related websites. He is author or co-author of morethan 140 scientific papers published in peer reviewed journals, and of more than 70 scientific abstractssubmitted to the main international meetings in the cardiology and in the computer science fields. He isalso author of two books (available in Italian) about the use of the Internet in medicine (“Guida allamedicina in rete” and “Internet in medicina. Guida all’uso e applicazioni pratiche”, both published bythe Pensiero Scientifico Editore, Rome) and of one section about Internet and medicine, included in oneof the most important italian medical encyclopedia (“Enciclopedia Medica Italiana”, UTET 2007). Healso collaborates to the publication of the Italian National Bioethics Committee’s guidelines aboutethics, health, and the new information technologies. He is a member of the Society for Clinical Trialsand of the Society for Internet in Medicine.Selected publications• Santoro E. Podcast, wiki e blog: il web 2.0 al servizio della formazione e dell’aggiornamento del medico. Recenti ProgMed 2007;98:484-494.• Santoro E, Rossi Valentina, Pandolfini C, Bonati M. DEC-NET: The development of the European register of clinicaltrials on medicines for children. Clin Trials 2006; 3: 366-375• Clivio L, Tinazzi A, Mangano S, Santoro E. The contribution of information technology: Towards a better clinical datamanagement. Drug Dev Res 2006; 67: 245-250• Santoro E. Internet and information on breast cancer: an overview. Breast 2003; 12: 424-431170ANNUAL REPORT 2007

IRFMN• Santoro E, Nicolis E, Franzosi M G, Tognoni G. Internet for clinical trials: Past, present, and future. Control Clin Trials1999; 20: 194-201• Franzosi M G, Santoro E, Zuanetti G, Latini R, Maggioni A P, Tognoni G, GISSI. Indications for ACE inhibitors in theearly treatment of acute myocardial infarction. Systematic overview of individual data from 100.000 patients inrandomized trial. Circulation 1998; 97: 2202-2212• Franzosi M G, Santoro E, De Vita C, Geraci E, Lotto A, Maggioni A P, Mauri F, Rovelli F, Santoro L, TavazziL, Tognoni G, GISSI.Ten-year follow-up of the first megatrial testing thrombolytic therapy in patients with acute myocardial infarction.Results of Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto-1 study. Circulation 1998; 98: 2659-2665• Franzosi M G, Latini R, Maggioni A P, Barlera S, Negri E, Nicolis E, Santoro E, Santoro L, Tognoni G, GarattiniS, GISSI-3.GISSI-3: Effects of lisinopril and transdermal glyceryl trinitrate singly and together on six-week mortality andventricular function after acute myocardial infarction. Lancet 1994; 343: 1115-1122• Maggioni A P, Maseri A, Fresco C, Franzosi M G, Mauri F, Santoro E, Tognoni G, GISSI-2. Age-related increase inmortality among patients with first myocardial infarctions treated with thrombolysis. N Engl J Med 1993; 11: 1442-1448Cristina Bosetti got her degree in Mathematics in 1994 at the University of Milan, School ofMathematics, and the Post-Graduate Diploma in Pharmacological Research in 1999 at the “Mario Negri”Institute for Pharmacological Research in Milan.Areas of interest: Epidemiology of cancer, cardiovascular diseases and other chronic conditions. Inparticular case-control studies on cancers of the upper respiratory and digestive sites, thyroid, breast,hormone-related cancers, and on ischemic heart disease. Analysis of risk related to diet, alcohol, tobacco,reproductive and hormonal factors, occupational and environmental exposure to toxic substances, throughthe application of generalized linear models.She is author/coauthor of more than 130 publications on these issues on peer-reviewed scientific journals.Work experiences: Since Sept. 2005: Unit Head, Unit of Cancer Epidemiology, Department ofEpidemiology; 1998-2005: Researcher at the Laboratory of Epidemiology; 1996-1997: Researcher at theLaboratory of Mother and Child Health.Selected publications:• Bosetti C, Gallus S, Peto R, Negri E, Talamini R, Tavani A, Franceschi S, La Vecchia C.Tobacco Smoking, SmokingCessation, and Cumulative Risk of Upper Aerodigestive Tract Cancers.Am J Epidemiol. 2007 Dec 4; [Epub ahead ofprint]• Bosetti C, Malvezzi M, Chatenoud L, Negri E, Levi F, La Vecchia C. Trends in cancer mortality in the Americas, 1970-2000. Ann Oncol 2005; 16: 489-511.• Bosetti C, Spertini L, Parpinel M T, Gnagnarella P, Lagiou P, Negri E, et al. Flavonoids and breast cancer risk in Italy.Cancer Epidemiol Biomarkers Prev 2005; 14: 805-808.• Bosetti C, Micelotta S, Dal Maso L, Talamini R, Montella M, Negri E, et al. Food groups and risk of prostate cancer inItaly. Int J Cancer 2004; 110: 424-428.• Bosetti C, Negri E, Kolonel L, Ron E, Franceschi S, Preston-Martin S, et al. A pooled analysis of case-control studies ofthyroid cancer. VII. Cruciferous and other vegetables (International). Cancer Causes Control 2002; 13: 765-775.• Smith J S, Herrero R, Bosetti C, Munoz N, Bosch F X, Eluf-Neto J, et al. IARC Multicentric Cervical Cancer StudyGroup Herpes simplex virus-2 as a human papillomavirus cofactor in the etiology of invasive cervical cancer. J NatlCancer Inst 2002; 94: 1604-1613.Liliane Chatenoud, Doctor in Science Biology, University of Milan (1987); Postgraduate Doctor inHealth Statistics University of Milan (1995).Areas of interest: Epidemiological studies on obstetric diseases. Dermato-epidemiology. Cancerepidemiology (case-control studies on cancers of the breast, female genital tract). Analysis of temporaltrends and geographical distribution of perinatal, infant mortality, cancer and other selected conditions(over 100 publications on these topics, 1993-2005).Work experiences: Since Sept. 2005: Unit Head, “Lifestyle and Prevention”, Department ofEpidemiology. 1993-2005: Researcher at the Laboratory of Epidemiology; 1988-1990: Staff StatisticianBracco S.p.A., Milan.Selected publications• Chatenoud L, Mosconi P, Malvezzi M, Colombo P, La Vecchia C, Apolone G. Impact of a major thermoelectric plant onself-perceived health status. Prev Med. 2005;41:328-33.• Naldi L, Chatenoud L, Linder D, Belloni Fortina A, Peserico A, Virgili AR, et al. Cigarette smoking, body mass index,and stressful life events as risk factors for psoriasis: results from an Italian case-control study. J Invest Dermatol.2005;125:61-7.171ANNUAL REPORT 2007

IRFMN• Bosetti C, Malvezzi M, Chatenoud L, Negri E, Levi F, La Vecchia C. Trends in cancer mortality in the Americas, 1970-2000. Ann Oncol 2005; 16: 489-511.• Chatenoud L, Tavani A, La Vecchia C, Jacobs D R, Negri E, Levi F, Franceschi S. Whole grain food intake and cancerrisk. Int J Cancer 1998; 77: 24-28.• Chatenoud L, Parazzini F, Di Cintio E, Zanconato G, Benzi G, Bortolus R, La Vecchia C. Paternal and maternal smokinghabits before conception and during the first trimester: Relation to spontaneous abortion. Ann Epidemiol 1998; 8: 520-526.Silvano Gallus was born in Milan on the 20th of November 1970, and got his degree in ComputerScience in 1999 at the Milan University.Areas of interest: Design, data managing, and statistical analyses of case-control studies on theassociations between several risk factors (including in particular tobacco smoking, alcohol drinking andMediterranean diet) and risk of cancer, coronary heart disease and several other conditions. Analyses ofoccupational cohort studies. Monitoring of prevalence and trends of smoking habit and obesity in Italy.Author/coauthor of over 130 publication in peer-reviewed journals since 1998.Work experiences: Chief of the Unit of Epidemiology for Clinical Research of the Department ofEpidemiology (since 2006); computer analyst, graphic designer, and statistical and epidemiologicalconsultant, Milan and Bergamo (since 2002); researcher at the Laboratory of Epidemiology (since 1997);creator, designer and webmaster of the website of one of the major Italian public hospital, Milano (1999-2002).Selected publications• Gallus S, Foschi R, Negri E, Talamini R, Franceschi S, Montella M, Ramazzotti V, Tavani A, Dal Maso L, La VecchiaC. Dietary zinc and prostate cancer risk: a case-control study from Italy. Eur Urol. 2007;52:1052-6.• Gallus S, Naldi L, Carli P, La Vecchia C; Italian Group for Epidemiologic Research in Dermatology (GISED). Nevuscount on specific anatomic sites as a predictor of total body count: a survey of 3,406 children from Italy. Am JEpidemiol. 2007;166:472-8.• Gallus S, Scotti L, Negri E, Talamini R, Franceschi S, Montella M, Giacosa A, Dal Maso L, La Vecchia C. Artificialsweeteners and cancer risk in a network of case-control studies. Ann Oncol. 2007;18:40-4.• Gallus S, Schiaffino A, La Vecchia C, Townsend J, Fernandez E. Price and cigarette consumption in Europe. TobControl. 2006;15:114-9.• Gallus S, Zuccaro P, Colombo P, Apolone G, Pacifici R, Garattini S, La Vecchia C. Effects of new smoking regulationsin Italy. Ann Oncol. 2006;17:346-7.• Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJ, Vaccarella S, Anh PT, Ferreccio C, Hieu NT, Matos E,Molano M, Rajkumar R, Ronco G, de Sanjosé S, Shin HR, Sukvirach S, Thomas JO, Tunsakul S, Meijer CJ, FranceschiS; IARC HPV Prevalence Surveys Study Group. Worldwide distribution of human papillomavirus types in cytologicallynormal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis.Lancet.2005;366:991-8.172ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT’S ACTIVITIESThe Department of Epidemiology is involved in the epidemiology of several common cancers(including cancers of the breast, female genital tract, respiratory and digestive sites, prostate andurinary organs, lymphoid malignancies, melanoma, etc.) and of cardiovascular diseases, boththrough a descriptive and an analytical approach. Among the activities of descriptiveepidemiology are the analysis of temporal trends and geographical distribution of mortalityfrom cancer, cardiovascular diseases, and other selected conditions, in Italy and Europe; theanalysis of trends in tobacco consumption in the Italian population, and the correspondingeffects on incidence and mortality from lung and other tobacco-related neoplasms. The analyticepidemiology activities include the conduction and analysis of case-control studies, aimed atidentifying and better quantifying the association between genetic factors (family history),selected lifestyle habits (diet, tobacco, alcohol, etc.), use of exogenous hormones and exposureto various substances and the development of various forms of cancers and cardiovasculardiseases. In particular, the Department works on the analysis of dietary correlates of cancer andcardiovascular disease risk; quantification of health effects of tobacco smoking, alcoholconsumption and implications for prevention; epidemiological studies on the risk related to oralcontraceptive and hormone replacement therapy use; evaluation of the impact of screening inthe early diagnosis and prevention of cancer. Other activities include: the conduction ofquantitative reviews and meta-analysis of published data; the re-analysis of original data fromepidemiological studies of cancers of the thyroid, breast, ovary, and cervix; epidemiological andclinical studies in dermatology in collaboration with the “Gruppo Italiano per gli StudiEpidemiologici in Dermatologia” (GISED); the analysis of historical cohort studies ofoccupational exposures to aromatic amines, asbestos, herbicides and other known carcinogens;monitoring and prevention of injuries. Other Department’s activities are related to thedevelopment of medical websites, the study of the quality of medical information available onthe Internet, and the training and research on issues related to medical informatics and thoseconcerning the use in the medical field of the Internet and web 2.0 applications.FINDINGS/MAIN RESULTSIn the Italian population, men who stopped smoking before age 50 years avoided morethan half of the excess risk of upper aerodigestive tract cancer as men who did not, andmen who stopped smoking before age 30 years avoided more than 90% of the risk.Flavonoids, particularly flavanones, are inversely related to the risk of cancers of the upperaero-digestive tract, including oral cavity and pharynx, oesophagus and larynx.A family history of oral, pharyngeal and laryngeal cancer is a strong determinant of oral andpharyngeal cancer risk, independent from tobacco and alcohol use.Among never drinkers, cigarette smoking is associated with an increased risk of head and neckcancer, and among never users of tobacco, alcohol consumption is associated with an increasedrisk of head and neck cancer only when alcohol is consumed at high frequency.Compared to never smokers, exclusive pipe smokers have a 9-fold increased risk of all upperdigestive tract cancers. Pipe smoking and heavy alcohol drinking appear to interact at least on amultiplicative model.173ANNUAL REPORT 2007

IRFMNHistory of thyroid diseases and goiter does not appear to be a relevant cause of gastric cancer inItaly.There is no relevant role of fried foods consumption on colorectal cancer risk, while there is apossible favorable effect of (fried) olive oil on colon but not on rectal cancer risk.Coffee has a favourable effect on hepatocellular carcinoma.There is an inverse relation between a diet rich in linoleic acid and beta-carotene intake andhepatocellular carcinoma risk.The lifetime number of ovulatory cycles is directly associated with ovarian cancer risk, whilepregnancy and oral contraceptive use have a stronger protective effect on ovarian cancer thanother anovulatory factors.A starch-rich dietary pattern is an unfavorable indicator of risk for both breast and ovariancancers, while the animal products and the vitamins and fiber patterns might be associated witha reduced risk of breast and ovarian cancers, respectively.Wine and other types of alcoholic beverages do not influence ovarian cancer risk in the Italianpopulation.High energy and cholesterol intake might increase endometrial cancer risk in Italy.Prostate cancer risk is increased for subjects at the highest quintile of zinc intake, as comparedto the lowest one. The direct association is stronger for advanced than for early stage cancers.The major risk factors for prostate cancer observed in middle-aged men (i.e.,

IRFMNThere are no significant associations between intakes of folate nor other micronutrients involvedin the one-carbon metabolism and non-Hodgkin lymphoma risk. However, a significant inverseassociation is observed for all the nutrients examined among abstainers and former drinkers,supporting the possibility of an antagonist effect of alcohol on the one-carbon metabolism innon-Hodgkin lymphoma etiology.Non-Hodgkin lymphoma risk is elevated for individuals who reported first-degree relatives withlymphomas and leukemia. The pattern of non-Hodgkin lymphoma heritability appear to beuniform across non-Hodgkin lymphoma subtypes, but risk patterns differ by specifichematopoietic malignancies and the sex of the relative, revealing critical clues to diseaseetiology.There is no evidence to support the hypothesis that obesity is a determinant of all types of non-Hodgkin lymphoma combined, while the association observed between severe obesity anddiffuse large B-cell lymphoma may warrant further investigation.No direct association emerged between use of saccharin, aspartame and other sweeteners andthe risk of various common neoplasms.High dietary levels of glycemic index and load increase the risk of thyroid cancer.Subjects with at least a first-degree relative with cancer of the bladder, kidney or prostate have a6-fold increased risk of bladder cancer, a 2-fold increased risk of renal cell cancer and a 2-foldincreased risks of prostate cancer, respectively.The reliability of the information on family history of cancer in first-degree relatives in ourhospital-based case-control study of digestive tract cancers is satisfactory.No substantial reduction in acute myocardial infarction risk resulted from the decrease ofcigarette tar yield.Parity and irregular menstrual cycle increased acute myocardial infarction risk, mainly insmokers.Lung cancer mortality in European women is levelling off in Germany, Italy and Poland over the lastyears, while it is still steadily increasing in France and Spain.Mortality from cirrhosis shows favorable trends in most countries of the world, but is stillsteadily upward in the UK and central and eastern European countries, due to the persistentincrease in the prevalence of alcohol consumption.Mortality from testicular cancer in (young) men remains exceedingly high in most LatinAmerican countries.Body mass index affects early clinical response to systemic treatment for psoriasis irrespectively ofthe administered drug.Examining the upper limbs only, particularly the lateral arms, is a practical and suitable tool forpredicting total nevus count in children.The BPCO.CARE website (www.bpcocare.it, a web based medical index that collect, classify,evaluate, and describe the most useful medical information on the web related to the chronic175ANNUAL REPORT 2007

IRFMNobstructive pulmonary disease) has been developed, including a collection of the web 2.0 toolsand applications available in the medical field.NATIONAL COLLABORATIONSAssociazione Nazionale dei Medici Cardiologi Ospedalieri (ANMCO)Centro di Riferimento Oncologico, Servizio di Epidemiologia, Aviano (PN)Fondazione LuVIFondazione SmithKline, MilanoGruppo Italiano per lo Studio della Sopravivenza nell’Infarto miocardico (GISSI)Gruppo Italiano Studi Epidemiologici in Dermatologia GISED, BergamoInternational Centre for Pesticides and Health Risk Prevention, MilanoIstituto Auxologico Italiano, Divisione Malattie Metaboliche III, IRCCS, Piancavallo (VB)Istituto Auxologico Italiano, Laboratorio Sperimentale di Ricerche Endocrinologiche (LSRE),IRCCS, MilanoIstituto di Medicina del Lavoro, CTO, TorinoIstituto Europeo di Oncologia, Divisione di Epidemiologia e Biostatistica, MilanoIstituto Europeo di Oncologia, Divisione di Chirurgia Cervico Facciale, MilanoIstituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), RomaIstituto Nazionale Neurologico "Carlo Besta", MilanoIstituto Nazionale per lo Studio e la Cura dei Tumori, Oncologia Sperimentale, Unità di EreditàPoligenica, MilanoIstituto Tumori “Fondazione Pascale”, Servizio di Epidemiologia, NapoliNovartis Vaccines SpA, SienaOspedale Niguarda Ca’ Granda, Dipartimento Trapianti di Fegato, MilanoOspedale Niguarda Ca’ Granda, Istituto di Fisiologia Clinica CNR, Sezione di Milano, MilanoPoliclinico di Monza, Unità Operativa di Endoscopia I, Monza (MI)Prima Clinica Ostetrico Ginecologica, Mangiagalli, MilanoSocietà Italiana Attività RegolatorieUniversità degli Studi di Milano - Bicocca, Dipartimento di Statistica, MilanoUniversità degli Studi di Milano, MilanoUniversità degli Studi di Milano-Bicocca, I Clinica Otorinolaringoiatria, DNTB, MonzaUniversità di Milano, Clinica Pediatrica De Marchi, MilanoUniversità di Milano, Istituto di Statistica Medica e Biometria “G.A. Maccacaro”, MilanoUniversità di Milano, Prima Clinica Ostetrico Ginecologica, MilanoUniversità di Verona, Clinica Ostetrico Ginecologica, VeronaINTERNATIONAL COLLABORATIONSCatalan Institute of Oncology, Institut d’Investigaciò Biomédica de Bellvitge (IDIBELL),Cancer Prevention and Control Unit, L’Hospitalet de Llobregat, SpainCenter of Oncology, Dept. of Epidemiology and Cancer Prevention, Varsavia, PolandCentre for Research in Environmental Epidemiology (CREAL) and Municipal Institute ofMedical Research (IMIM), Barcellona, SpainHarvard School of Public Health, Department of Epidemiology, Boston, USAHôpital Necker - Enfants Malades, Centre of the Association Claude Bernard on Auto-immunesdiseases, Parigi, France176ANNUAL REPORT 2007

IRFMNInternational Agency for Research on Cancer, Lione, FranceInternational Epidemiology Institute (IEI), Rockville, USAInternational Life Science Institute (ILSI), Bruxelles, BelgiumNational Cancer Institute, Environmental Studies Section, Bethesda, USANational Cancer Institute, Radiation Epidemiology Branch, Bethesda, USANational School of Public Health, WHO, Atene, GreeceRegistre Vaudois des Tumeurs, Institut Universitaire de Médecine Sociale et Préventive,Losanna, SwitzerlandSenologic International SocietySociety for Internet in MedicineUniversitat Pompeu Fabra, Department of Experimental and Health Sciences, Barcellona, SpainUniversity of Athens Medical School, Department of Hygiene and Epidemiology, Atene,GreeceUniversity of Las Palmas de Gran Canaria, Department of Clinical Sciences, Las Palmas deGran Canaria, SpainVanderbilt University, Department of Medicine, School of Medicine, Nashville, USAEDITORIAL BOARD MEMBERSHIPAdvances in Therapy (Eva Negri)Alimentazione e Prevenzione (Carlo La Vecchia)Asian Pacific Journal of Cancer Prevention (Carlo La Vecchia)Digestive and Liver Disease (Carlo La Vecchia)European Journal of Cancer Prevention (Carlo La Vecchia, Associate Editor)European Journal of Nutrition (Carlo La Vecchia)Evidence Based Dermatology (Carlo La Vecchia, Liliane Chatenoud)International Journal of Cancer (Carlo La Vecchia)Journal of Nephrology (Carlo La Vecchia)Nutrition and Cancer (Carlo La Vecchia)Portale Partecipasalute.it – http://www.partecipasalute.it (Eugenio Santoro)Revisiones en Ginecologìa y Obstetricia (Carlo La Vecchia)Revista Española de Nutriciò Comunitaria (Carlo La Vecchia)Revue d’Epidémiologie et de Santé Publique (Carlo La Vecchia)Società Italiana Attività Regolatorie News, SIARNews (Eugenio Santoro)The Lancet, edizione italiana (Carlo La Vecchia)Tumori (Carlo La Vecchia)PEER REVIEW ACTIVITIESActa Psychiatrica Scandinavica, American Journal of Clinical Nutrition, American Journal ofEpidemiology, Annals of Epidemiology, Annals of Oncology, Archives of Internal Medicine,BMC-Public Health, British Journal of Cancer, British Journal of Nutrition, British MedicalJournal, Canadian Journal of Physiology and Pharmacology, Cancer, Cancer Causes andControl, Cancer Epidemiology Biomarkers and Prevention, Computer Methods and Programs inBiomedicine, Diabetes/Metabolism Research and Reviews, Digestive Liver Disease,Epidemiologia & Prevenzione, Epidemiology, Epidemiology & Biostatistic, European HeartJournal, European Journal of Cancer, European Journal of Cancer Prevention, European Journalof Clinical Nutrition, European Journal of Epidemiology, European Journal of Public Health,Evidence-Based Healthcare & Public Health, Gynecological Endocrinology, Gut, Hepatology,177ANNUAL REPORT 2007

IRFMNHuman Reproduction, International Journal of Cancer, International Journal of Epidemiology,International Journal of Obesity, JAMA, Journal of American College of Nutrition, Journal ofClinical Endocrinology and Metabolism, Journal of Clinical Epidemiology, Journal ofEpidemiology and Community Health, Journal of the National Cancer Institute, Maturitas,Nicotine & Tobacco Research, Nutrition and Cancer, Obstetric and Gynecology, Oncology,Preventive Medicine, Radiation Research, Revue d’Epidèmiologie et de Santé Publique, TheBreast, The Cancer Journal, The Lancet, Tumori.NATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPAdvisory Committee of the Oxford Collaborative group on Aetiological Factors in Cancers ofthe Female Genital TractComitato Scientifico del Gruppo Italiano Studi Epidemiologici in DermatologiaComitato Scientifico della Società Italiana di Colposcopia e Patologia Cervico VaginaleConsiglio Superiore di Sanità, Gruppo di lavoro sul Metodo Di BellaData and Safety Monitoring Board of the “Phase II therapeutic trial with a humanizednonmitogenic CD3 (ChAgly CD3) monoclonal antibody in recently diagnosed type I diabeticpatients”IARC/WHO, Lyon, and WHO, GenevaIstituti Clinici di Perfezionamento, MilanoProgetto Menopausa Italia, Associazione Ostetrici Ginecologi ItalianiScientific Review Committee of UND/WHO/World Bank Human Reproduction ProgramSocietà Italiana della RiproduzioneMinistero della Salute, Sottocomitato fumo.Ministero della Salute, Commissione Oncologica Nazionale.178ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE LABORATORY WASINVOLVEDWorkshop on Environment & Health: Air quality research needs and opportunities in the EUseventh framework programme of research (FP7). CONCAWE. “European studies on ambientparticulate matter and health, with a focus on long-term exposure”. Brussels, Belgium. 15January 20074th Annual Inhance Meeting. IARC. “Family history of oral cancer”. Lyon, France. 18-19January 2007Press Lunch AIRC. “Le sostanze che fanno bene: ricerca sui flavonoidi e vitamina C perdifendersi dal cancro”. Milan, Italy. 25 January 2007IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 96: Alcoholicbeverage consumption, acetaldehyde and urethane. Lyon, France. 6-13 February 2007Hi-wate Kick-Off Meeting. Barcelona, Spain.13-15 February 2007HEM – Closing the Gap in central Europe. Meeting of country coordinators. Warsaw, Poland.5-6 March 2007Incontro Conferenza stampa. Presentazione dell’analisi dei risultati a 5 anni di tre studi, condottida Mayo Clinic, Lee Moffit Center e INT/IEO, eseguita dagli epidemiologi dello SloanKettering, sull’utilità della diagnosi precoce con TAC spirale come strumento di individuazioneprecoce del tumore al polmone, pubblicata su The Journal of American Medical Association ecommentata su Nature. Milan, Italy. 7 March 2007IARC Fellowships Committee Meeting 2007. Lyon, France. 26-27 March 2007IV Convegno Nazionale. La tutela della salute nelle attività sportive e la lotta contro il doping.“Un’indagine sulla conoscenza, attitudine e prevalenza del doping nell’opinione degli atletiitaliani. Rome. 3 April 2007ESMO International Symposium. ESMO. “The smoking epidemic and lung cancer”. Geneva,Switzerland. 30 Marzo-1 April 2007.Porto Cancer Meeting. XVI Edition. Cancer etiology: Brindging worlds. “Cancer mortalitytrends in the European Union: priorities for cancer control”. Porto, Portugal. 20-21 April 2007.Giornata mondiale senza tabacco. IX Convegno nazionale tabagismo e servizio sanitarionazionale. “Politiche di controllo del fumo: quali azioni da intraprendere”. Rome. 31 May 2007The future of medical sciences. “Infectious diseases: new frontiers and migration pathologies”.Milan. 8 June 2007.6° National Congress SIB – Italian Biometric Society. “New perspective on parametricconfidence intervals for the cost-effectiveness ratio”. Pisa, Italy. 20-22 June 2007Forum dei centri per la disassuefazione dal fumo. “Epidemiologia della disassuefazione estratificazione delle popolazioni che accedono all’ambulatorio specialistico”. Rome. 5 July2007.Insediamento della Commissione Oncologica Nazionale. Rome. 12 July 2007.1st Meeting of Collaborators. Collaborative Group on epidemiological studies of endometrialcancer. St Hilda’s College, Oxford, UK. 13-14 July 2007179ANNUAL REPORT 2007

IRFMN56° International congress ISI - International Statistical Institute. “New perspective onconfidence intervals for the ratio of means of Normal Variables, jointly distributed as aBivariate Normal”. Lisbon, Portugal. 22-29 August 2007.ERA-net-2007-rtd evaluation. “Evaluation”. Brussels, Belgium. 3-4 September 200726 Journèes Internationales Huiles Essentielles & Extraits. “L’Huile d’olive: Santè, bien-être etprevention du cancer”. Digne Les Bains, France. 13-14 September 2007.IV National Congress SISMEC – Italian Society of Medical Statistics and ClinicalEpidemiology. “Bioequivalence assessment from crossover data: a new approach for theconstruction of confidence intervals for the ratio of two formulation means”. “Epidemiologia”.Monreale. 19-22 September 2007.I determinanti della salute: una valutazione quantitativa. Sassari, Italy. 5-6 October 2007.Conferenza. Osteonecrosi della mandibola (ONJ) in corso di terapia con bisfosfonati. Milan. 8October 2007.62° Congresso Nazionale FIMMG-METIS. “Caffè e cancro, con riferimento ad altre patologiedell’apparato digerente “. Villasimius. 8-14 October 2007.Conferenza Lega Italiana per la Lotta contro i Tumori. “L’alimentazione nella prevenzione deitumori”. Castelfranco Veneto. 11 October 2007.Le giornate nazionali di nutrizione pratica 2007. “Dieta mediterranea e cancro. Elementi diprevenzione”. Milan.18 October 2007.Closing the Gap final conference. The evolution of health following enlargement HEM.“Cardiovascular disease”. Brussels, Belgium. 23 October 2007.PSONET European registry of psoriasis. “International safety review board”. Rome. 25 October2007.XXXI Congresso Annuale AIE. “Fattori di rischio per le cadute nell’anziano: scopo per unarevisione sistematica”, “Interventi di prevenzione delle cadute negli anziani con particolareattenzione agli aspetti relativi alla partecipazione dei soggetti”. Ostuni. 17-19 October 2007.Giornata Nazionale per la Ricerca sul Cancro organizzata Associazione Italiana Ricerca sulCancro. “I danni al nostro DNA provocati dall’ambiente”. 10 November 2007.7 th Scietific Meeting of the International Epidemiology Association. EMR: epidemiologytranslating science to health care. “Diet and cancer risk in mediterranean countries” Riyadh,Saudi Arabia. 27-29 November 2007.Vino e salute. Convegno scientifico divulgativo. “L’aumento della longevità associato alcorretto consumo di vino” “Perché occorre evitare l’abuso di bevande alcoliche”. GrinzaneCavour. 24 November 2007XXXXIX Congresso nazionale AIPO. Malattie respiratorie: emergenza sociale. “Epidemiologyof lung cancer”. Florence. 4-7 December 2007.Convegno regionale AIOM Lombardia. “Riduzione della mortalità per cancro in Europa:illusione o realtà?”. Milan. 14 December 2007.XII World Congress on Internet in Medicine. “GCPBASE: a web-based tool for remote datacapture in a clinical trial”. Lipsia. 7-10 October 2007.180ANNUAL REPORT 2007

IRFMNXXII Workshop GIDIF, RBM (Gruppo Italiano Documentalisti dell’Industria Farmaceutica edegli Istituti di Ricerca Biomedica), Palazzo delle Stelline. “Tecnologia RSS e aggregatori dinotizie: un nuovo modo di distribuire l’informazione”. Milan. 4 December 2007Master Universitario di II livello in “Informatica medica”, Università di Udine, annoaccademico 2006-2007. Ruolo di docenza nel modulo “Internet e sanità; strumenti eapplicazioni al servizio del medico e del cittadino”. Udine. 24 May 2007.Corso, “Internet e l’aggiornamento professionale in ambito biomedico” promosso da ULSS n.6Vicenza. “Siti e applicazioni Internet in ambito medico”. Vicenza. 6 June 2007Corso, “Medicina basata sulle prove di efficacia” promosso dall’Azienda Unità SanitariaLocale n.1 di Sassari. “Siti e applicazioni Internet in ambito medico: tipologia diinformazione e modalità di reperimento”. Sassari. 7 May 2007.Corso, “Medicina basata sulle prove di efficacia” promosso dall’Azienda Unità SanitariaLocale n.1 di Sassari. “Siti e applicazioni Internet in ambito medico: tipologia diinformazione e modalità di reperimento”. Sassari. 21 September 2007.AIFAAssociazione Italiana per la Ricerca sul CancroAstraZeneca ItaliaComune di MilanoLega Italiana Lotta contro i TumoriEuropean Commission (FP6)GISEDMinistero della SaluteRegione LombardiaGRANTS AND CONTRACTS181ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007La Vecchia C.Hormone replacement therapy in menopause and breast, colorectal and lung cancer: an update.In: Treatment of the Postmenopausal Woman: Basic and Clinical Aspects, ed 3, Lobo R.A. ed.Academic Press, 2007, pp. 599-608 (2007).Gallus S, La Vecchia C.Is there a link between diet and esophageal cancer?Nature Clin. Practice Gastroenterol. & Hepatol., 4: 2-3 (2007).Dal Maso L, La Vecchia C, Augustin LSA, Mantzoros CS, Kendall CWC, Franceschi S.Relationship between a wide range of alcohol consumptions, components of the insulin-like growth factor system andadiponectin.Eur. J. Clin. Nutr., 61: 221-225 (2007).Galeone C, Talamini R, Levi F, Pelucchi C, Negri E, Giacosa A, Montella M, Franceschi S, La Vecchia C.Fried foods, olive oil and colorectal cancer.Ann. Oncol., 18: 38-39 (2007).Randi R, Pelucchi C, Gallus S, Parpinel M, Dal Maso L, Talamini R, Augustin LSA, Giacosa A, Montella M,Franceschi S, La Vecchia C.Lipid, protein and carbohydrate intake in relation to body mass index: an Italian study.Publ. Health Nutr., 10: 306-310 (2007).La Vecchia C., Bosetti C.Diet and cancer risk in Mediterranean countries.Hungarian Med. J., 1: 13-23 (2007)Colosio C, Fustinoni S, Corsini E, Bosetti C, Birindelli S, Boers D, Campo L, La Vecchia C, Liesivuori J, PennanenS, Pennanen S, Vergieva T, van Amelsvoort L.G.P.M., Steerenberg P, Swaen GMH, Zaikov C, van Loveren H.Changes in serum markers indicative of health effects in vineyard workers following exposure to the fungicidemancozeb: an Italian study.Biomarkers, 12: 574-588 (2007).Bravi F, Bosetti C, Negri E, Lagiou P, La Vecchia C.Family history of cancer provided by hospital controls is satisfactorily reliable.J. Clin. Epidemiol., 60: 171-175 (2007).Chiaffarino F, Parazzini F, Bosetti C, Franceschi S., Talamini R, Canzonieri V, Montella M, Ramazzotti C, LaVecchia C.Risk factors for ovarian cancer histotypes.Eur. J. Cancer., 43: 1208-1213 (2007).Scotti L, Tavani A, Bosetti C, Dal Maso L, Talamini R, Montella M, Franceschi S, La Vecchia C.Diabetes and risk of non-Hodgkin lymphoma: a case-control study.Tumori, 93: 1-3 (2007).Galeone C, Negri E, Pelucchi C, La Vecchia C, Bosetti C, Hu J.Dietary intake of fruit and vegetable and lung cancer risk: a case-control study in Harbin, northeast China.Ann. Oncol., 18: 388-392 (2007).Bosetti C, Boffetta P, La Vecchia C.Occupational exposures to polycyclic aromatic hydrocarbons, and respiratory and urinary tract cancers: a quantitativereview to 2005.Ann. Oncol., 18: 431-446 (2007).Pelucchi C, Galeone C, Talamini R, Bosetti C, Montella M, Negri E, Franceschi S, La Vecchia C.Lifetime ovulatory cycles and ovarian cancer risk in 2 Italian case-control studies.Am. .J Obstet. Gynecol., 196: 83.e1-83.e7 (2007).La Vecchia C, Tavani A.Coffee and cancer risk: an update.182ANNUAL REPORT 2007

IRFMNEur. J. Cancer Prev., 16: 385-389 (2007).La Vecchia C., Negri E., International Collaboration of Epidemiological Studies of Cervical Cancer.Cervical cancer and hormonal contraceptives: collaborative reanalysis of individual data on 16573 women withcervical cancer and 35509 women without cervical cancer from 24 epidemiological studies.Lancet, 370: 1609-1621 (2007).Montella M, Polesel J, La Vecchia C, Dal Maso L, Crispo A, Crovatto M, Casarin P, Izzo G, Tommasi LG, TalaminiR, Franceschi S.Coffee and tea consumption and risk of hepatocellular carcinoma in Italy.Int. J. Cancer, 120: 1555-1559 (2007).Gallus S, Scotti L, Negri E, Talamini R, Franceschi S, Montella M, Giacosa A, Dal Maso L, La Vecchia C.Artificial sweeteners and cancer risk in a network of case-control studies.Ann. Oncol., 18: 40-44 (2007).Bertuccio P, Tavani A, Gallus S, Negri E, La Vecchia C.Menstrual and reproductive factors and risk of non-fatal acute myocardial infarction in Italy.Eur. J. Obst. Gynecol. Repr. Biol., 134: 67-72 (2007).Pira E, Pelucchi C, Piolatto PG, Negri E, Discalzi G, La Vecchia C.Role of first and subsequent asbestos exposures on mesothelioma and lung cancer.Br. J. Cancer, 97: 1300-1304 (2007).Polesel J, Dal Maso L, La Vecchia C, Montella M, Spina M, Crispo A, Talamini R, Franceschi S.Dietary folate, alcohol consumption, and risk of non-Hodgkin Lymphoma.Nutr. Cancer, 57: 146-150 (2007).Gallus S., Foschi R, Negri E, Talamini R, Franceschi S, Montella M, Ramazzotti V, Tavani A, Dal Maso L, LaVecchia C.Dietary zinc and prostate cancer risk: a case-control study from Italy.Eur. Urol., 52: 1052-1057 (2007).Tavani A, Zucchetto A, Dal Maso L, Montella M, Ramazzotti V, Talamini R, Franceschi S, La Vecchia CLifetime physical activity and the risk of renal cell cancer.Int. J. Cancer, 120: 1977-1980 (2007)Pelucchi C, Galeone C, Dal Maso L, Talamini R, Montella M, Ramazzotti V, Negri E, Franceschi S, La Vecchia C.Dietary acrylamide and renal cell cancer.Int. J. Cancer, 120: 1376-1377 (2007).Gallus S, Zuccaro P, Colombo P, Apolone G, Pacifici R, Garattini S, Bosetti C, La Vecchia C.Smoking in Italy 2005-2006: Effects of a comprehensive national tobacco regulation.Prev. Med., 45: 198-2001 (2007).Gallus S, Naldi L, Carli P, La Vecchia C, and the Italian Group for Epidemiologic Research in Dermatology(GISED).Nevus count on specific anatomic sites as a predictor of total body count: a survey of 3,406 children from Italy.Am. J. Epidemiol., 166: 472-478 (2007).Levi F, Randimbison L, Maspoli M, Te VC, La Vecchia C.Second neoplasms after oesophageal cancer.Int. J. Cancer, 121, 694-697 (2007).Bosetti C. Levi F. Lucchini F, Zatonski WA, Negri E, La Vecchia C.Worldwide mortality from cirrhosis: An update to 2002.J. Hepatol., 46: 827-839 (2007).Fossati R, Apolone G, Negri E, Compagnoni A, La Vecchia C, Mangano S, Clivio L, Garattini S for the GeneralPractice Tobacco Cessation Investigators Group.A double-blind, placebo controlled, randomized trial of bupropion for smoking cessation in primary care.Arch. Intern. Med., 167: 1791-1797 (2007).183ANNUAL REPORT 2007

IRFMNRossi M, Garavello W, Talamini R, La Vecchia C, Franceschi S, Lagiou P, Zambon P, Dal Maso L, Bosetti C, NegriE.Flavonoids and risk of squamous cell esophageal cancer.Int. J. Cancer, 120: 1560-1564, (2007).Bertuccio P, Malvezzi M, Chatenoud L, Bosetti C, Negri E, Levi F, La Vecchia C.Testicular cancer mortality in the Americas (1980-2003).Cancer, 109: 776-779 (2007).Galeone C, Pelucchi C, Talamini R, Negri E, Dal Maso L, Montella M, Ramazzotti V,Franceschi S, La Vecchia C. Onion and garlic intake and the odds of benign prostatic hyperplasia.Urology, 70: 672-676 (2007).Zucchetto A, Dal Maso L, Tavani A, Montella M, Ramazzotti V, Talamini R, Canzonieri V, Garbeglio A, Negri E,Franceschi S, La Vecchia C.History of treated hypertension and diabetes mellitus and risk of renal cell cancer.Ann. Oncol., 18: 596-600 (2007).Wang SS, Slager SL, Brennan P, Holly EA, De Sanjose S, Bernstein L, Boffetta P, Cerhan JR, Maynadie M, SpinelliJJ, Chiu BCH, Cocco PL, Mensah F, Zhang Y, Nieters A, Dal Maso L, Bracci PM, Seniori Costantini A, Vineis P,Severson RK, Roman E, Cozen W, Weisenburger D, Davis S, Franceschi S, La Vecchia C, Foretova L, Becker N,Staines A, Vornanen M, Zheng T, Hartge P.Family history of hematopoietic malignancies an risk of non-Hodgkin lymphoma (NHL): a pooled analysis of 10 211cases and 11 956 controls from the International Lymphoma Epidemiology Consortium. (InterLymph).Blood, 109: 3479-3488 (2007).Bosetti C, Rossi M, McLaughlin JK, Negri E, Talamini R, Lagiou P, Montella M, Ramazzotti V, Franceschi S, LaVecchia C.Flavonoids and the risk of renal cell carcinoma.Cancer Epidemiol. Biomarkers Prev., 16: 98-11 (2007).Garavello W, Rossi M, McLaughlin JK, Bosetti C, Negri E, Lagiou P, Talamini R, Franceschi S, Parpinel M, DalMaso L, La Vecchia C.Flavonoids and laryngeal cancer risk in Italy.Ann. Oncol., 18: 1104-1109 (2007).Bravi F., Bosetti C., Tavani A., Bagnardi V., Gallus S., Negri E., Franceschi S., La Vecchia C.Coffee drinking and hepatocellular carcinoma risk: a meta-analysis.Hepatology, 46: 430-435 (2007)La Vecchia C.Alcohol and liver cancer.Eur. J. Cancer Prev., 16: 495-497 (2007).Levi F, Lucchini F, Negri E, La Vecchia C.Continuing declines in cancer mortality in the European Union.Ann. Oncol., 18: 593-595 (2007).Dal Maso L, Zucchetto A, Tavani A, Montella M, Ramazzotti V, Talamini R, Canzonieri V, Garbeglio A, Negri E,Tonini A, La Vecchia C, Franceschi S.Renal cell cancer and body size at different ages: an Italian multicenter case-control study.Am. J. Epidemiol., 166: 582-591 (2007).Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C, Curado MP, Dal Maso L, Daudt AW, Fabianova E,Wünsch-Filho V, Franceschi S, Hayes RB, Herrero R, Koifman S, La Vecchia C, Lazarus P, Levi F, Mates D, MatosE, Menezes A, Muscat J, Eluf-Neto J, Olshan AF, Rudnai P, Schwartz SM, Smith E, Sturgis EM, Szeszenia-Dabrowska N, Talamini R, Wei Q, Winn DM, Zaridze D, Witold Zatonski W, Zhang ZF, Berthiller J, Boffetta P.Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer:pooled analysis in the International Head and Neck Cancer Epidemiology Consortium (Inhance).JNCI, 99: 777-789 (2007)Gallus S., Foschi R., Talamini R., Altieri A., Negri E., Franceschi S., Montella M., Dal Maso L., Ramazzotti V., LaVecchia C.184ANNUAL REPORT 2007

IRFMNRisk factors for prostate cancer in men aged less than 60 years. A case-control study from Italy.Urology, 70: 1121-1126 (2007).Levi F, Bosetti C, Fernandez E, Hill C, Lucchini F, Negri E, La Vecchia .Trends in lung cancer in young European women: the rising epidemic in France and Spain.Int. J. Cancer, 121: 462-465 (2007).Naldi L, Chatenoud L, Bertuccio P, Zinetti C, Di Landro A, Scotti L, La Vecchia C, and the Oncology CooperativeGroup of the Italian Group for Epidemiologic Research in Dermatology (GISED).Improving sun protection behavior among children: results of a cluster-randomized trial in Italian elementary schools.The "SoleSi SoleNo-GISED" project.J. Invest. Dermatol., 127: 1871-1877 (2007).Randi G, Scotti L, Bosetti C, Talamini R, Negri E, Franceschi S, Levi F, La Vecchia C.Pipe smoking and cancers of the upper digestive tract.Int. J., Cancer, 121: 2049-2051 (2007).Rossi M., Garavello W., Talamini R., Negri E., Bosetti C., Dal Maso L., Lagiou P., Tavani A., Polesel J., Barzan L.,Ramazzotti V., Franceschi S., La Vecchia C.Flavonoids and the risk of oral and pharyngeal cancer: a case-control study from Italy.Cancer Epidemiol. Biomarkers Prev., 16: 1621-1625 (2007).Polesel J., Talamini R., Montella M., Dal Maso L., Crovatto M., Parpinel M., Izzo F., Tommasi LG, Serraino D, LaVecchia C, Franceschi S.Nutrients intake and the risk of hepatocellular carcinoma in Italy.Int. J. Cancer, 43: 2381-2387 (2007).La Vecchia C., Fabbri L.M.RE: Prevention of death in COPD.N Engl J Med. 2007 May 24;356(21):2211-2.Galeone C, Pelucchi C, Talamini R, Negri E, Montella M, Ramazzotti V, Zucchetto A, Dal Maso L, Franceschi S, LaVecchia C.Fibre intake and renal cell carcinoma: a case-control study from Italy.Int. J. Cancer, 121: 1869-1862 (2007).Boffetta P, McLaughlin JK, La Vecchia C, Autier P, Boyle P.“Environment” in cancer causation and aetiological fraction: limitations and ambiguities.Carcinogenesis, 28:913-5 (2007).Gallus S, Scottti L, Talamini R, Franceschi S, Dal Maso L, Negri E, La Vecchia C.Re: Alcohol consumption and ovarian cancer risk in a population-based case-control study by Peterson et al.Int. J. Cancer, 121: 2578-2579 (2007).Gallus S., La Vecchia C.Reply to L.C. Costello, R.B. Franklin’s Letter to the Editor re: Gallus S., Foschi R., Negri E., et al. Dietary zinc andprostate cancer risk: a case-control study from Italy.Eur. Urol., 52: 1263-1264 (2007)Deandrea S, Bertuccio P, Chatenoud L, Franceschi S, Serraino D, La Vecchia C.Reply to “Alcohol consumption and risk of Hodgkin’s lymphoma and multiple myeloma: a multicentre case-controlstudy” by Gorini et al,Ann. Oncol., 18: 1119-1121 (2007).Ferlay J, Randi G, Bosetti C, Levi F, Negri E, Boyle P, La Vecchia C.Declining mortality from bladder cancer in Europe.BJU Int., 101: 11-19 (2007).Levi F, Te VC, Maspoli M, Randimbison L, Bulliard JL, La Vecchia CTrends in breast cancer incidence among women under the age of forty.Br. J. Cancer, 97: 1013-1014 (2007).Corrao G, Zambon A, Nicotra F, Fornari C, La Vecchia C, Mezzanzanica M, Nappi RE, Merlino L, Cesana G.185ANNUAL REPORT 2007

IRFMNPersistence with oral and transdermal hormone replacement therapy and hospitalisation for cardiovascular outcomes.Maturitas, 57:315-324 (2007).Lucenteforte E, Bosetti C, Talamini R, Montella M, Zucchetto A, Pelucchi C, Franceschi S, Negri E, Levi F, LaVecchia C.Diabetes and endometrial cancer: effect modification by body weight, physical activity and hypertension.Br. J. Cancer, 97: 995-998 (2007).Bravi F, Bosetti C, La Vecchia C.Re: Coffee and hepatocellular carcinoma: cause or confounding?Hepatology, 28: 46:2047 (2007).Randi G, Pelucchi C, Negri E, Talamini R,Galeone C, Franceschi S, La Vecchia C.Family history of urogenital cancers in patients with bladder, renal cell and prostate cancers.Int. J. Cancer, 121: 2748-2752 (2007).Levi F, La Vecchia C.Epidemiologie et tendances du cancer en Suisse.Bull. Cancer, 94: 775-780 (2007).Boccardi D, Menni S, Ferraroni M, Stival G, Bernardo L, La Vecchia C, Decarli A.Birthmarks and transient skin lesions in newborns and their relationship to maternal factors: a preliminary report fromNorthern Italy.Dermatology, 215: 53-58 (2007).Santoro E, Clivio L, Mangano S.GCPBASE: a web-based tool for remote data capture in a clinical trial.Technology and Health Care, 15:355 (2007).SELEZIONE PUBBLICAZIONI DIVULGATIVE APPARSENELL’ANNO 2007La Vecchia C. Tumori. L’Eco-difesa. Salviamoci dall’autoinquinamento da fumo e alcol. Repubblica, no. 555: 9-10(2007).La Vecchia C, Beghi E. Epidemiologia delle demenze. In: La demenza in Italia. Aggiornamento e casi cliniciinterattivi. UTET, Torino, 2007; 20-24.La Vecchia C. Mediterranean diet and cancer. Element of prevention. Rivista Nutrizione Pratica, no. 1: 26-31 (2007).La Vecchia C. European studies on ambient particulate matter and health, with a focus on long-term exposure.In: Workshop on environment & health: air quality research needs and opportunities in the EU seventh framework.Programme of research (FP7). Proc. Symp. Brussels, Jan. 15-17, 2007. Concawe, Brussels; 2007: 13-15.Bertuccio P, Foschi RLa salute del bambino nel primo anno di vita in Italia: che cosa è cambiato?NEWS & OPINIONS in Ginecologia 2007 2 : 4-9.Santoro E. Podcast, wiki e blog: il web 2.0 al servizio della formazione e dell’aggiornamento del medico. RecentiProg Med , 98:484-494 (2007).Santoro E. Il sito del British Medical Journal. Ricerca&Pratica, n. 136: 169-170 (2007).Santoro E. Strumenti di rating al servizio del pubblico: è vero empowerment? Ricerca&Pratica, n. 137: 189-91(2007).Santoro E. Il web 2.0: dalla partecipazione alla in-formazione. Ricerca&Pratica, n. 138:266-269 (2007).Santoro E. Web 2.0 e medicina. CARE, 6: 33-37 (2007).186ANNUAL REPORT 2007

IRFMNSantoro E. Open access e ricerca biomedica: un nuovo modo di diffondere i risultati della ricerca. SIAR News, 51:29-31 (2007).Santoro E. My NCBI: uno strumento per l’aggiornamento periodico dei risultati di una ricerca eseguita sul databaseMedline. SIAR News, 50:47-49 (2007).Santoro E. Informazione sanitaria: ecco le linee guida. Medici Oggi, n. 6: 8-9 (2007).Santoro E. Revolution Health e il coinvolgimento dei cittadini: uno strumento di empowerment? PartecipaSalute,2007; http://www.partecipasalute.it/cms/node/621.Santoro E. I blog come strumento di condivisione di esperienze tra pazienti. PartecipaSalute 2007;http://www.partecipasalute.it/cms/node/652.Santoro E. Open source e web 2.0: una guida per gli utenti di Partecipasalute. PartecipaSalute 2007;http://www.partecipasalute.it/cms/node/704.Santoro E. Meglio Google o PubMed? II puntata. Va’ Pensiero 2007;http://www.pensiero.it/attualita/articolo.asp?ID_sezione=31&ID_articolo=553Santoro E. “Internet e medicina”. In Enciclopedia Medica Italiana, aggiornamento III, Tomo 1, pp. 1691-1714. UTET2007 (L. Vella, ed.).Pistotti V, Santoro E. “Navigare sulla rete alla ricerca di informazioni di salute”. In La dispensa di PartecipaSalute,pp. 85-89. Istituto di Ricerche Farmacologiche “Mario Negri”, Milano 2007.RESEARCH ACTIVITIESData analysis and managementCollection of epidemiological data has continued as planned, and also data checking, entry andinput have been pursued. The overall dataset now includes 1,250 cases of oral and pharyngealcancers, 700 of esophageal, 1,100 of stomach, 6,500 of colorectal, 600 of primary liver, 120 ofgallbladder and bile duct cancers, 600 of pancreatic, 850 of laryngeal, 500 of cutaneousmalignant melanoma, 7,000 of breast, 1,000 of cervical and 1,000 of endometrial cancers, 200of gestational trophoblastic disease, 200 of vulvar, 2,000 of ovarian, 1,300 of prostatic, 700 ofbladder, 800 of kidney and renal pelvis, 600 of thyroid cancer, 200 of Hodgkin’s disease and500 of other lymphomas, 150 sarcomas, 300 myelomas and about 18,000 controls. Biologicalsamples have been collected for cancers of the upper digestive and respiratory tract, bladder andcolorectal cancer to investigate genetic polymorphisms.Flavonoid intake and upper aerodigestive tract cancersWe investigated the effect of selected flavonoids in relation to the risk of upper aerodigestivetract neoplasms, and found inverse associations with each cancer site considered. The study oforal and pharyngeal cancer included 805 cases and 2081 hospital controls. The ORs for thehighest versus the lowest quintile of intake were 0.51 for flavanones, 0.62 for flavonols, and0.56 for total flavonoids. The study of esophageal cancer included 304 cases and 743 hospitalcontrols. An inverse association emerged between flavanone intake and esophageal cancer risk(OR=0.38). This study suggested that flavanone intake may account, with vitamin C, for theprotective effect of fruit on esophageal cancer. The study of laryngeal cancer included 460 casesand 1088 hospital controls. Significant inverse relations were found with intake of flavan-3-ols(OR=0.64, for the highest versus the lowest quintile), flavanones (OR=0.60), flavonols187ANNUAL REPORT 2007

IRFMN(OR=0.32) and total flavonoids (OR=0.60), providing support for a beneficial effect of selectedflavonoids on laryngeal cancer, too.Family history of cancers and oral and pharyngeal cancersWith reference to history of neoplasms in first-degree relatives, the OR of oral and pharyngealcancer was 3.1 for a family history of oral, pharyngeal and laryngeal cancers combined.Significant increases in risk of oral and pharyngeal cancer were also observed for a familyhistory of melanoma (OR=5.8) and lung cancer (OR=1.4). Our study found that a family historyof oral, pharyngeal and laryngeal cancer is a strong determinant of oral and pharyngeal cancerrisk, independent from tobacco and alcohol use.Alcohol drinking in never-smokers, cigarette smoking in never-drinkers,and upper aerodigestive tract cancersOur data on head and neck cancers were integrated in the International Head and Neck CancerEpidemiology Consortium, a pooled analysis of 15 case-control studies that included a total of10,244 cancer case subjects and 15,227 control subjects. One of the aims of the pooled analysiswas to analyze the extent to which head and neck cancers are associated with cigarette smokingamong never drinkers and with alcohol drinking among never users of tobacco. We found that,among never drinkers, cigarette smoking is associated with an increased risk of head and neckcancer (OR=2.13), and among never users of tobacco, alcohol consumption is associated withan increased risk of head and neck cancer only when alcohol is consumed at high frequency(OR=2.04 for three or more drinks per day versus never drinking).Pipe smoking and upper aerodigestive tract cancerWe analysed the association between exclusive pipe smoking and cancers of the upper digestivetract, using data from a series of case-control studies conducted in Italy and Switzerlandbetween 1984 and 1999. After excluding cigarette and cigar smokers, 41 male oral andpharyngeal cancer cases, 52 male oesophageal cancer cases and 1,032 male controls wereincluded in the present analysis. Compared to never smokers, exclusive pipe smokers had anOR of 8.7 of all upper digestive tract cancers. The OR was 12.6 for oral and pharyngeal and 7.2for oesophageal cancer. Pipe smokers who were also heavy alcohol drinkers had an OR of 38.8as compared to never smokers and light drinkers. Thus, pipe smoking and heavy alcoholdrinking appears to interact at least on a multiplicative model.Cumulative risk of upper aerodigestive tract cancer in relation to smokingcessationWe estimated the effect of smoking cessation on the cumulative incidence of thesecancers by age 75 years (in the absence of competing causes of death), combining oddsratios for males from a network of Italian hospital-based case-control studies (1984-2000) with 1993-1997 incidence data for Italian men. The studies included 961 caseswith oral/pharyngeal cancer, 618 cases with esophageal cancer, and 613 cases withlaryngeal cancer, plus 3,781 controls. For all upper aerodigestive tract cancers, thecumulative risks by 75 years of age were 6.3% for men who continued to smoke anytype of tobacco, 3.1% and 1.2% for men who stopped smoking at around 50 and 30years of age, respectively, and 0.8% among lifelong nonsmokers. Corresponding figureswere 3.3%, 1.4%, 0.5%, and 0.2% for oral/pharyngeal cancer; 1.0%, 0.5%, 0.4%, and0.2% for esophageal cancer; and 2.1%, 1.1%, 0.2%, and 0.2% for laryngeal cancer. Inthis Italian population, men who stopped smoking before age 50 years avoided morethan half of the excess risk of upper aerodigestive tract cancer as men who did not, andmen who stopped smoking before age 30 years avoided more than 90% of the risk.188ANNUAL REPORT 2007

IRFMNGoiter and autoimmune thyroid disease and gastric cancerWe analyzed data from a hospital-based case-control study conducted between 1985 and 1997in Milan, including 769 incident cases of gastric cancer and 2081 control subjects. The OR ofgastric cancer was 0.70 for history of goiter, 0.61 for thyroid nodules/adenomas, 1.10 forhyperthyroidism, and 0.71 for other benign thyroid diseases. None of the results was statisticallysignificant. Therefore, goiter and other thyroid diseases do not appear to be a relevant cause ofgastric cancer in Italy.Fried foods and colorectal cancerThis investigation was based on 1394 cases of colon cancer, 886 cases of rectal cancer and 4765controls, enrolled in Italy and Switzerland between 1992 and 2000. Our results did not indicatea relevant role of fried foods on colorectal cancer risk, while we found a possible favorableeffect of (fried) olive oil on colon but not on rectal cancer risk.Selected dietary factors and hepatocellular carcinomaA case-control study of hepatocellular carcinoma was conducted in Italy in 1999-2002,including 185 histologically confirmed cases and 412 control subjects admitted to the samenetwork of hospitals. The present study supported the hypothesis of a favorable effect of coffee(OR=0.4 for high consumption), though not decaffeinated coffee and tea, on the risk ofhepatocellular carcinoma. Using the same dataset, we considered the effect of various nutrientson the risk of hepatocellular carcinoma, and found an inverse relation with a diet rich in linoleicacid (OR=0.35, for highest versus lowest tertile) and beta-carotene intake (OR=0.48).Lifetime ovulatory cycles and ovarian cancerWe considered data from 2 multicentric Italian studies of ovarian cancer, including a total of2002 cases and 4914 controls, in relation to the number of lifetime ovulatory cycles and todifferent anovulatory factors. As compared with the lowest quartile, the ORs of ovarian cancerwere 1.60, 1.65, and 1.81 for increasing quartiles of lifetime ovulatory cycles. For 1 year ofovulation avoided, the continuous ORs were 0.91 for parity-related anovulations, 0.90 forabortions, 0.92 for oral contraceptive use, 0.99 for age at menarche, and 0.97 for age atmenopause. This study found that pregnancy and oral contraceptive use had a strongerprotective effect on ovarian cancer than other anovulatory factors.Dietary patterns and breast and ovarian cancersDietary patterns were identified on a selected set of nutrients through principal componentfactor analysis. The starch-rich pattern emerged as an unfavorable indicator of risk for bothbreast and ovarian cancers, while the animal products and the vitamins and fiber patterns mightbe associated with a reduced risk of breast and ovarian cancers, respectively.Alcohol consumption and ovarian cancerIn a separate analysis, we examined the relation between alcohol consumption and ovariancancer risk. In a population characterized by regular and frequent wine consumption in women,wine was not materially related to ovarian cancer risk, the continuous estimate for wine being1.02. Our investigation also confirmed results of earlier studies that none of the other types ofalcoholic beverages was associated to ovarian cancer risk.Macronutrients and cholesterol intake and endometrial cancerPrevious studies found some evidence that dietary habits may influence the risk of endometrialcancer independently of body mass. In our investigation, significant direct associations wereobserved with intake of energy (OR=1.7 for the highest versus the lowest quintile), and189ANNUAL REPORT 2007

IRFMNcholesterol (OR=2.1), while a direct borderline association emerged with saturated fatty acids(OR=1.3). On the other hand, there was no association with proteins, sugars, starch, total fat andother selected fatty acids. Energy and cholesterol intake increased endometrial cancer risk.Dietary zinc and prostate cancerIn a multicentric case-control study of prostate cancer, including 1294 cases and 1451 malecontrols, we investigated the relation with dietary zinc intake. This was computed from avalidated and reproducible food frequency questionnaire, through the use of an Italian foodcomposition database. Compared with the lowest quintile of intake, the OR of prostate cancerfor subjects at the highest quintile of zinc intake was 1.56, with a significant trend in risk. Thedirect association was stronger for advanced than for early stage cancers.Prostate cancer in men aged less than 60 yearsWe considered the same dataset of prostate cancer to investigate the major risk factors in menyounger than 60 years. This age group comprised 219 cases and 431 controls. The major riskfactors observed in middle-aged men were family history of prostate cancer (OR=5.5), highlevel of education (OR=3.3), and low physical activity (OR=0.5, for active versus inactivesubjects).Dietary factors and renal cell carcinomaWe evaluated the relation between selected risk factors and renal cell cancer (RCC) in an Italiancase-control study including 767 patients younger than 79 years with RCC and 1,534 controls.The results of this study suggested that a diet rich in refined cereals and poor in vegetables mayhave an unfavorable role on RCC. No relation was found for coffee and tea, eggs, red meat, fish,cheese, fruits, desserts and sugars. After allowing for energy and other major covariates, asignificant inverse association was found for vitamin E (OR=0.56, for the highest quintile ofintake versus the lowest one), and vitamin C (OR = 0.72), although the trend in risk for vitaminC was of borderline significance. No significant trend of decreasing risk was found for othermicronutrients analyzed. We also investigated in depth the topic of dietary fibers and RCC, andfound an inverse association with vegetable fibers that may reflect a real favorable effect, or bean indicator of a beneficial role of a diet rich in vegetable on RCC risk.Body size at different ages and renal cell carcinomaAn increased risk of RCC has been reported in overweight persons in previous studies. In ourinvestigation, we found an OR of 1.3 among obese persons (i.e., those with a body mass index≥30) as compared to normal-weight persons (body mass index

IRFMNHypertension, diabetes mellitus and the risk of renal cell cancerIn the same RCC dataset, patients with a history of treated hypertension reported an excess riskof RCC (OR=1.7), and the pattern was confirmed in different strata of sex, education, smokinghabits, body mass, tumor histological type, stage, or grade. Therefore, we estimate that theattributable risk of RCC for treated hypertension in this population is 16%. A slight, nonsignificantincreased risk was found also for history of diabetes mellitus (OR=1.3).Alcohol consumption and renal cell carcinomaThere is some evidence that alcohol consumption is inversely associated with RCC. Since theissue is still unclear, we investigated the relation by pooling data from two Italian case-controlstudies, including a total of 1115 incident, histologically confirmed cases and 2582 controls.Compared with non-drinkers, the ORs of RCC were 0.87 for ≤4 drinks per day, 0.76 for >4 to≤8 drinks per day and 0.70 for >8 drinks per day of alcoholic beverages. Thus, this pooledanalysis found an inverse association between alcohol drinking and RCC. Risks continued todecrease even above 100 g/day of alcohol intake, with no apparent leveling in risk.Diabetes and non-Hodgkin lymphomaDuring year 2007, we examined the role of selected risk factors for non-Hodgkin lymphoma(NHL) using data from two Italian case-control studies conducted in 1985-1997 and 1999-2002,including 671 cases with incident, histologicallyconfirmed NHL and 1799 controls. Weanalysed the combined datasets in relation to diabetes. Epidemiological results on the issue arecontroversial, and diabetes has been related to the risk of several neoplasms. However, theresults of our study allow to exclude a strong association between diabetes and NHL. The smallnumber of cases with diabetes in this investigation left open the possibility of a moderate directrelation.Folate, alcohol and non-Hodgkin lymphomaWith reference to this issue, we examined our recent case-control study, i.e., the only one withdetailed information on dietary habits, including 190 cases of NHL and 484 controls. Nosignificant association emerged between NHL risk and intakes of folate (OR=0.9) and othermicronutrients involved in the one-carbon metabolism, i.e., vitamin B2 (OR=0.9), vitamin B6(OR=0.8), and methionine (OR=0.7). However, a significant inverse association was observedfor all the nutrients examined among abstainers and former drinkers. Our findings support thepossibility of an antagonist effect of alcohol on the one-carbon metabolism in NHL etiology.However, the lack of an overall effect for one-carbon nutrients and the small sample sizesuggested caution in interpreting our results.InterLymph Pooled Analysis on non-Hodgkin lymphomaOur data on hematopoietic malignancies were included in a pooled analysis of 10,211 NHLcases and 11,905 controls from the International Lymphoma Epidemiology Consortium(InterLymph). During year 2007, this pooled analysis examined family history of hematopoieticmalignancies and obesity as potential risk factors for NHL. The pattern of NHL heritabilityremains poorly understood, though there is accumulating evidence of common geneticvariations altering NHL risk. In the InterLymph pooled analysis, NHL risk was elevated forindividuals who reported first-degree relatives with NHL (OR=1.5), Hodgkin lymphoma(OR=1.6), and leukemia (OR=1.4). The pattern of NHL heritability appeared to be uniformacross NHL subtypes, but risk patterns differed by specific hematopoietic malignancies and thesex of the relative, revealing critical clues to disease etiology. Another investigation from theInterLymph Consortium considered the role of obesity on NHL risk. This concluded that thereis no evidence to support the hypothesis that obesity is a determinant of all types of NHL191ANNUAL REPORT 2007

IRFMNcombined, while the association observed between severe obesity and diffuse large B-celllymphoma may warrant further investigation.Artificial sweeteners and cancer riskThe role of sweeteners on cancer risk has been widely debated over the last few decades. Toprovide additional information on saccharin and other sweeteners (mainly aspartame), weconsidered data from a large network of case-control studies, including upper aero-digestivetract, colon, rectum, breast, ovary, prostate and kidney. The ORs for consumption of saccharinwere 0.83 for cancers of the oral cavity and pharynx, 1.58 for oesophageal, 0.95 for colon, 0.93for rectal, 1.55 for laryngeal, 1.01 for breast, 0.46 for ovarian, 0.91 for prostate and 0.79 forkidney cancer. This work indicated a lack of association between saccharin, aspartame and othersweeteners and the risk of these neoplasms.Glycaemic index and load and thyroid cancerRisk of thyroid cancer has been related to refined cereals and starch foods, but the associationhas never been studied in terms of glycemic index and load. We analyzed the issue using datafrom an Italian study of 399 cases of thyroid cancer and 616 control subjects. Our study showedthat high dietary levels of glycemic index (OR=1.73) and glycemic load (OR=2.17) areassociated with thyroid cancer risk.Family history of cancer and urogenital cancersHistory of urogenital cancers in first-degree relatives was investigated among a total of 1356subjects with cancer of the bladder, kidney and prostate, and 1067 controls. ORs of urogenitalcancers for subjects with at least a first-degree relative with cancer at the same site were 6.1 forbladder, 2.0 for renal cell and 2.0 for prostate cancer.Reliability of information on family history of cancerThe reliability of the information on family history of cancer in first-degree relatives in ourhospital-based case-control study of digestive tract cancers was tested by re-interviewing athome 294 controls. A satisfactory agreement between the two interviews was reported forfamily history of any cancer and of cancers of the digestive tract. We found a systematictendency to report a history of cancer more frequently in the hospital setting than in the homesetting. Thus, data on family history of cancer provided by hospital controls show a goodreliability.Tar yield in cigarettes and risk of acute myocardial infarctionControversial information is available with reference to the role of type or tar yield of cigaretteson the risk of cardiovascular disease. We considered the issue in a combined dataset of threecase-control studies of acute myocardial infarction (AMI) conducted in Italy between 1983 and2003, including 1990 subjects with a first episode of non-fatal AMI, and 2521 controls. Ascompared to never smokers, the multivariate OR was 2.70 for smokers of low tar cigarettes (

IRFMN(OR=1.45), but without a linear trend in risk with number of children. The association wasmuch stronger in smokers, with an OR of 5.98 for smokers with an irregular menstrual cyclecompared with non smokers with a regular one and an OR of 4.77 in smokers who had childrencompared with non smokers nulliparae. There was no relation with menopausal status, age atmenarche and menopause, age at first and last birth and number of abortions.Record-linkage for cohort analysesWith reference to our project to create an Italian cohort study based on record-linkage, during2007 we prepared the archives that will allow to link data between our network of case-controlstudies (conducted since 1982) and those from the historical archives of the Local Health Unit(ASL) of Milan, that include several health information such as vital status and date of death ofthe subjects. Several stages of the project have been undertaken and completed during the year.In particular, we finished the identification of subjects recruited in the case-control studiesamong the historical archives of the ASL, and a database with univocal information on subjectsfrom the two sources is now available, thus allowing a linkage of the archives; at the same time,we completed data collection from all case-control studies in a unique database (using originaldata and through the preparation of a codebook that allowed to re-codify studies conducted invarious periods and on several diseases) that includes information from interviews toapproximately 27,000 subjects. During 2007, we searched in various other historical databasesindividuals that were not linked in historical archives of the ASL. On 18,257 residents inLombardy, 15,860 (86.9%) subjects, including 7,680 cases (82.2%) and 8,911 controls (91.8%)were linked.Lung cancer in young European womenWe analyzed age-standardized trends in lung cancer mortality in young women (20-44) for the 6major European countries, using joinpoint regression. In the early 1970s the highest lung cancermortality in young women was in the UK (2.1/100,000). UK rates, however, steadily declinedand in 2000-2004 they were the lowest of all 6 major EU countries (1.2/100,000). The secondlowest rate in 2000-2002 was in Italy, whose rates remained around 1.1/100,000 between 1970and 1994, and increased to 1.4 thereafter. In Germany and Poland, lung cancer rates in youngwomen rose from 0.8-1.0/100,000 in the early 1970s to 1.7-1.9 in the mid 1990s and levelled offduring the last decade. Major rises over recent years were observed in France (from 0.8/100,000in 1985-1989 to 2.2 in 2000-2003) and in Spain (from 0.8 in the 1985-1989 to 1.7 in 2000-2004). Thus, France showed both the highest rate observed over the last 3 decades and thelargest rise over the last 2 decades.Worldwide mortality from cirrhosisCirrhosis mortality was analyzed for 41 countries worldwide over the period 1980-2002 usingdata from the WHO. In the early 1980s, the highest rates were in Mexico, Chile (around55/100,000 men and over 14/100,000 women), France, Italy, Portugal, Austria, Hungary andRomania (around 30-35/100,000 men and 10-15/100,000 women). Mortality from cirrhosis hasbeen steadily declining in most countries worldwide since the mid or late 1970s. In southernEurope, rates in the early 2000s were less than halved compared to earlier decades. In contrast,rates have been rising in eastern European countries to reach extremely high values in the mid1990s, and declined only thereafter. In the UK rates were still steadily rising. Mortality fromcirrhosis shows favourable trends in most countries of the world, following the reduction inalcohol consumption and hepatitis B and C virus infection. The steady upward trends observedover more recent calendar periods in the UK and central and eastern European countries areattributed to the persistent increase in the prevalence of alcohol consumption.193ANNUAL REPORT 2007

IRFMNTesticular cancer mortality in the AmericasDeath rates and trends from testicular cancer were compared over the 1980-2003 period in allthe American countries that provide data. In the early 1980s the highest testicular cancermortality rates were observed in Chile (1.7/100,000 at all ages, 3.6/100,000 at 20-44 years) andArgentina (0.9/100,000 at all ages, 1.7/100,000 at 20-44 years), as compared with 0.4/100,000for all ages and 0.6/100,000 at 20 to 44 years in Canada, and 0.3/100,000 for all ages and0.7/100,000 at 20 to 44 years in the US. In 2001-2003, testicular cancer mortality had fallen to0.2/100,000 in men aged 20 to 44 years in Canada, and to 0.4/100,000 in the US. Conversely,rates were still 1.6/100,000 in Argentina, 2.2/100,000 in Chile and 1.2/100,000 in Mexico, andwere around 0.5-0.6/100,000 in most other Latin American countries that provide data.Mortality from testicular cancer in (young) men remains exceedingly high in most LatinAmerican countries. Urgent intervention is required to provide treatment (essentially modernintegrated platinum-based chemotherapy) for this largely curable neoplasm in young men.Smoking prevalence in ItalyOn 10 January 2005, Italy became the first large European country adopting a comprehensivesmoke-free legislation. To provide information on smoking prevalence in Italy and evaluate theeffects of the 2005 regulation, we considered data from three companion surveys on smokingconducted in 2004, 2005 and 2006 in Italy. Each survey included more than 3,039 subjects(1,461 men and 1,578 women) aged 15 or over, representative of the general Italian adultpopulation. Current smokers declined from 26.2% (30.0% of men, 22.5% of women) in 2004, to25.6% (29.3% of men, 22.2% of women) in 2005, and to 24.3% (28.6% of men, 20.3% ofwomen) in 2006. While no significant difference was found comparing smoking prevalence in2003-2004 vs 2001-2002, the drop in smoking prevalence in 2005-2006 vs 2003-2004 wassignificant (p

IRFMNthe association with various DBPs, including in particular trihalomethanes (THMs). The studywill be conducted in the greater Milan area and in the Provinces of Pordenone and Udine, andwill include approximately 500 incident, histologically confirmed cases enrolled in the majorgeneral and teaching hospital of the study area, and 500 frequency-matched controls, admittedto the same hospital as cases for acute, nonneoplastic conditions. Cases and controls will beinterviewed using an extensive questionnaire. Detailed information on water use and waterrelatedhabits for etiologically relevant time periods will be collected, including not only waterconsumption, but also water related activities, such as showering, bathing and swimming, thatmay influence exposure assessment. Moreover, in order to assess the subjects’ exposure toDBPs and THMs in water, current and historical THM levels, water source and year of startingchlorination in the study area will be collected from local companies, authorities andmunicipalities. The relative and attributable risks of colorectal cancer for exposure to THMs andother DBPs will be estimated. Giving the large number of people exposed to chlorinateddrinking-water and its DBPs, the study has potentially important implications in terms of publichealth. Even modest excess risk in relation to DBP exposure may in fact have a relevant impacton a population level, and may be responsible of a considerable number of colorectal cancercases.Medical History, Drug Exposure and the Risk of PsoriasisThe association of psoriasis with selected medical conditions and a number of drugs used beforediagnosis was evaluated in a multicenter case-control study involving outpatient services of 20general and teaching hospitals, conducted in collaboration with the “Gruppo Italiano per gliStudi Epidemiologici in Dermatologia” (GISED). Entry criteria for cases were a first diagnosisof psoriasis made by a dermatologist and a history of skin manifestations of no more than 2years after the reported onset of the disease. Controls were the first eligible dermatologicalpatients observed on randomly selected days in the same centers as cases. A total of 560 casesand 690 controls were recruited. The OR of psoriasis was 0.8 in hypertensive subjects, 1.1 indiabetics and 1.1 in hyperlipidemic subjects. Histamine 2 receptor antagonist exposure wasnegatively associated with psoriasis, with an OR of 0.3. Our study rules out a strong associationof psoriasis with common chronic conditions. The reported associations of psoriasiswith relatively common conditions such as diabetes mellitus, hypertension and hyperlipidemiamay represent a late effect of well-known risk factors for psoriasis, such as smoking andoverweight, or reflect factors related to the long course of psoriasis itself.Impact of body mass index and obesity on clinical response to systemictreatment for Psoriasis. Evidence from the PSOCARE projectTo assess the role of body mass index (BMI) on early clinical response to systemic treatmentfor psoriasis, we analyzed data from a nationwide registry and cohort study involvingcompelling registration of patients receiving for the first time in their life a new systemictreatment for psoriasis at reference centres in Italy. Information was gathered by treatingphysicians with the aid of a web-based electronic form. Patients with a clinical diagnosis of chronicplaque psoriasis and with 8 and 16 weeks of completed follow-up by March 31 th , 2007 were eligible.A relative reduction of Psoriasis Area Severity Index (PASI) of at least 75% at follow-upcompared to baseline was evaluated as clinical endpoint (PASI-75). A total of 2,368 patients wereanalysed at 8 weeks and 2,042 at 16 weeks. PASI-75 was achieved by 819 (34.5%) patients at 8weeks and 1,034 (50.6%) patients at 16 weeks. The proportion steadily decreased with increasedvalues of BMI, from 41.7% in patients with BMI < 20 to 29.1% in patients with BMI ≥30 at 8weeks, and from 59% in patients with BMI

IRFMNto the drug prescribed at entry. Thus, BMI affects early clinical response to systemic treatment forpsoriasis irrespectively of the administered drug. This finding has implications for individual patientcare and the design of future randomised clinical trialsNevus count on specific anatomic sites as a predictor of total body countScanty information is available on the relation between nevus count on specific anatomic areasand the total body surface, particularly in children. We analyzed this issue by using data from auniquely large study conducted in 1997 on 3,406 schoolchildren (1,746 boys and 1,660 girls)aged 13-14 years in 13 cities from northern, central, and southern Italy. Children were examinedby trained dermatologists who counted melanocytic nevi (≥2 mm in diameter) on 19 differentanatomic sites. Overall, the mean number of nevi was 17.3 (18.6 in boys and 15.8 in girls). Theadjusted correlation coefficients (r) with number of nevi on the whole body were 0.74 for headand neck, 0.83 for anterior and 0.84 for posterior trunk, and 0.88 for upper and 0.80 for lowerlimbs. With reference to single anatomic sites, the best predictor of total nevus count was thelateral arms (r=0.80), overall and in strata of sex and pigmentary characteristics. This largestudy provides definite evidence that examining the upper limbs only, particularly the lateralarms, is a practical and suitable tool for predicting total nevus count in children.Histological types and anatomic sites of cutaneous basal-cell carcinomaSince different clinico-pathologic subtypes and anatomic sites of basal-cell carcinoma (BCC)may display distinct characteristics and mechanisms of development, we analyzed data from anItalian case–control study, including 528 subjects with BCC and 512 controls. The risk ofnodular (OR=1.53) but not superficial (OR=0.71) BCC was increased for occupational exposureto sunlight. Considering the anatomic site of BCC, the corresponding values were 1.46 forhead/neck and 0.74 for truncal location. Direct associations were observed with recreationalsunlight exposure, eye color, red hair, and number and early age of severe sunburn episodes,along with some differences in risk between histotypes and anatomic sites. Therefore, this studyconfirmed the role of (intermittent) sun exposure and phenotypic characteristics as risk factorsfor BCC, and suggested etiological differences between nodular and superficial histotypes andbetween head/neck and truncal locations.Anthropometric factors and cutaneous malignant melanomaSeveral studies have investigated the effect of various anthropometric factors on the risk ofcutaneous malignant melanoma (CMM). As the results are controversial, we analyzed the issuein a case-control study conducted in Italy between 1992 and 1994, including 542 patients withCMM and 538 controls. The ORs for the highest versus the lowest quartile were 2.06 forweight, 1.16 for height, 1.90 for BMI and 1.87 for body surface area (BSA). When allowing forBMI and BSA in the same model, the ORs were 1.55 for BMI and 1.41 for BSA. The presentfindings confirm that obesity increases the risk of CMM. BSA is also related to the risk ofCMM. In terms of population attributable risks, overweight and obesity would account for 31%of the cases of CMM in this Italian population, indicating the scope of prevention.Number of naevi and cutaneous malignant melanomaIn the same case-control study, we analyzed the association between CMM at a specificanatomical site and number of nevi at the same site. Cases and controls were examined bytrained dermatologists who counted the number of melanocytic nevi. The ORs of CMM for thehighest versus the lowest tertile of number of nevi at the corresponding site was 1.4 at face andneck, 2.3 at anterior trunk, 4.9 at posterior trunk, 2.9 at upper limbs and 5.0 at lower limbs. In acase-case analysis, comparing CMM cases at a specific site and CMM cases at all other sites,the only excess risk was found for the posterior trunk, the OR being 2.1 for the highest versus196ANNUAL REPORT 2007

IRFMNthe lowest tertile of number of nevi. Our data do not support the hypothesis of a specific effectof nevi at each single anatomical site.197ANNUAL REPORT 2007

IRFMNapplied criteria and principles for the evaluation of health websites and the evidence basedmedicine tools, an interdisciplinary team of reviewers is involved in the evaluation of thequality of information offered by the websites included in the PAIN.CARE medical index. Thesame study will also allow to compare the level of the quality of health related informationamong different classes of websites. The study, in collaboration with the Laboratory of MedicalResearch and Consumer Involvement, is ongoing and the results will be available in 2008.Training activitiesIn 2007, the Laboratory of Medical Informatics continued its training activity on issues relatedto the use of the Internet in medicine, and extended it to the use of the recent web 2.0technologies and tools in the medicine area. The members of the laboratory staff activated (orattended as invited teachers) a number of training courses, workshops, and master courses.Public health prevention and informationThe major products of our activity, mainly related to diet, tobacco and alcohol have also beenpublished in the lay press, in order to increase the project impact on prevention and publichealth.199ANNUAL REPORT 2007

IRFMN200ANNUAL REPORT 2007

IRFMNLABORATORY OF REGULATORYPOLICIESSTAFFHeadVittorio BERTELE’, M.D.201ANNUAL REPORT 2007

IRFMNCURRICULUM VITAEVittorio Bertele’ is a clinical pharmacologist. He got his MD degree in 1977 and the specialization inInternal Medicine in 1982, both at the Milan University Medical School. He was research fellow at theHarvard Medical School and then worked at the Milan University and the “Mario Negri” Institute.His main areas of interest have been clinical pharmacology of drugs active on the hemostatic andvascular system 1,2 , epidemiology of interventions in the cardiovascular area, and clinical trials and drugutilization studies in the cardiovascular area 3,4 . He was CPMP expert at the EMEA, and member of theCommittee for Drug Price Negotiation at the Italian Ministry of Health 5,6 .At present he is Head of the Regulatory Policies Laboratory at the "Mario Negri" Institute, and memberof the Technical-Scientific Committee at the Italian Drug Agency.Selected publications• Bertele' V., Falanga A., Tomasiak M., Dejana E., Cerletti C., De Gaetano G. Platelet thromboxane synthetase inhibitorswith low doses of aspirin: Possible resolution of the "aspirin dilemma". Science 1983; 220: 517-519• Bertele' V., Falanga A., Tomasiak M., Chiabrando C., Cerletti C., De Gaetano G. Pharmacological inhibition ofthromboxane synthetase and platelet aggregation: Modulatory role of cyclooxygenase products. Blood 1984; 63: 1460-1466 (1984).• Bertele' V, Mussoni L, Pintucci G, Del Rosso G, Romano G, de Gaetano G, Libretti A. The inhibitory effect of aspirin onfibrinolysis is reversed by iloprost, a prostacyclin analogue. Thromb Haemost 1989; 61: 286-288• The i.c.a.i. Group (Gruppo di studio dell'Ischemia cronica Critica degli Arti Inferiori). Prostanoids for chronic critical legischemia: A randomized, controlled, open-label trial with prostaglandin E 1 . Ann Int Med 1999; 130: 412-421• Collaborative Group of the Primary Prevention Project (PPP). Low-dose aspirin and vitamin E in people atcardiovascular risk: a randomised trial in general practice. Lancet 2001; 357: 89-95• Garattini S, Bertele’ V. Adjusting regulatory rules to public health needs. Lancet 2001; 358: 64-67• Garattini S, Bertele' V. Efficacy, safety, and cost of new anticancer drugs. BMJ 2002; 325: 269-271• Garattini S, Bertele’ V, Li Bassi L. How can research ethics committees protect patients better? BMJ 2003; 326:1199–201• Joppi R, Bertele' V, Garattini S. Disappointing biotech. BMJ 2005; 331: 895-897• Garattini S, Bertele' V. Non-inferiority trials are unethical because they disregard patients' interests. Lancet 2007; 370 :1875-1877202ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE LABORATORY'S ACTIVITIESCritical appraisal of clinical methodologyOptimisation of drug use and healthcare fund stewardshipCritical appraisal and recommendations for European Pricing and Reimbursement systemsEvaluation of marketing authorization applications submitted to the European regulatory agency(EMEA) and of subsequent variationsEvaluation of the appropriateness of drug legislation, institutions, and regulatory procedureswith respect to public health needs.Cooperation to the development and to the solution of regulatory issues in developing countries.FINDINGS/MAIN RESULTSCritical appraisal of clinical research methodological aspects as the adoption ofequivalence/non-inferiority design in clinical trialsDevelopment of Pan-European strategies for rational use of drugsRecommendations for Pan-European pricing policies for generics as well as interchangeablebrands in a class once generics are availableRaising awareness among interested parties about the deficiencies of the present EUpharmaceutical legislation and about our proposals to improve it in the public health interest.Critical review of drug documentation at the basis of marketing authorizations.Critical review of the criteria to assess pharmaceutical innovation and include new drugs in thenational reimbursement scheme.NATIONAL COLLABORATIONSItalian Drug Agency (AIFA)Istituto Superiore di SanitàDepartment of Health Lombardy RegionItalian Horizon Scanning Project203ANNUAL REPORT 2007

IRFMNEuropean Medicine Agency (EMEA)INTERNATIONAL COLLABORATIONSKarolinska Institutet, Clinical Pharmacology, Department of Laboratory Medicine, SEUniversity of Liverpool Management School, Prescribing Research Group, UKWorld Health Organisation (Department of Essential Drugs and Medicines Policy)Association of South East Asian Nations (ASEAN)EDITORIAL BOARD MEMBERSHIPRicerca & PraticaDialogo sui FarmaciNATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPTechnical Scientific Committee at the Italian Drug Agency (AIFA)Subcommittee of the Community Procedures at the Italian Drug Agency (AIFA)Scientific Committee of the Italian Horizon Scanning ProjectSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Garattini S, Bertele' V. Non-inferiority trials are unethical because they disregard patients' interests. Lancet 2007;370: 1875-1877Garattini S, Bertele' V. How can we regulate medicines better? BMJ 2007; 335: 803-805Barbui C, Cipriani A, Lintas C, Bertele' V, Garattini S. CNS drugs approved by the centralised European procedure:true innovation or dangerous stagnation? Psychopharmacology (Berl) 2007; 190: 265-268Bertele' V, Buonocore C, Michelacci F, Vitocolonna M, Garattini S. Efficacy and safety of immunosuppressive drugsapproved in EU through the centralised procedure. Eur J Clin Pharmacol 2007; 63: 707-712Bertele' V, Banzi R, Capasso F, Tafuri G, Trotta F, Apolone G, Garattini S. Haematological anticancer drugs inEurope: any added value at the time of approval? Eur J Clin Pharmacol 2007; 63: 713-719Bertele' V, Assisi A, Di Muzio V, Renzo D, Garattini S. New antirheumatic drugs: any real added value? A criticaloverview of regulatory criteria for their marketing approval. Eur J Clin Pharmacol 2007; 63: 879-889Godman B, Haycox A, Bertele' V, Schwabe U. UK OFT pricing proposal - a necessity to fund new drugs or newindications. BMJ online 9 May 2007. Available in URL from:http://www.bmj.com/cgi/eletters/334/7600/936#165136204ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESCritical appraisal of clinical methodologyRaising awareness about potential biases in clinical researchCritical evaluation of the EU pharmaceutical legislationRaising awareness among interested parties about the deficiencies of the present EUpharmaceutical legislation and about our proposals to improve it in the public healthinterest.Critical appraisal of ongoing reforms including pricing reforms in majorEuropean countriesEvaluation of reforms to enhance generic prescribing rates, drive down the costs of generics andcorresponding originator brands as well as potential prices of interchangeable brands oncestandards become available as generics.Development of Pan-European strategies for rational use of drugsEnhancing rational use in line with an approach that became known as the ‘five Es’, namely:evaluation; economics; enforcement; education and engineeringAssessment of drug dossiers for regulatory approvalsExpert support to the Rapporteurship for marketing authorisation applications and variations tothe conditions of marketing authorisationActivities for the Technical Scientific Committee at the AIFAConsultative activities for the Italian Drug Agency regarding regulatory duties with respect todrug quality, safety, efficacy, and cost.Activities for the sub-committee for the European ProceduresAssessment of the dossiers for marketing authorisation applications through mutual recognitionprocedures involving Italy as either Reference or Concerned Member State.205ANNUAL REPORT 2007

IRFMN206ANNUAL REPORT 2007

IRFMNLABORATORY FOR MOTHER ANDCHILD HEALTHSTAFFHeadMaurizio BONATI, M.D.207ANNUAL REPORT 2007

IRFMNCURRICULUM VITAEMaurizio Bonati has a Medical School degree at the University of Milan.Areas of interest: Monitoring and epidemiological evaluation of drug utilisation and effects of drugs andvaccines in motherhood and childhood. Research methodology in general hospital and paediatriccommunity practice. Transfer of information to the community. Epidemiology of paediatric and perinatalcare.Past and present roles both at the Mario Negri Institute and in other institutions: 1973-77 Research Fellowat the IRFMN, within the Neurochemistry Lab.; 1977-85 Research Assistant at the IRFMN, within theClinical Pharmacology Lab.; 1986-93 Chief of the Perinatal Clinical Pharmacology Unit at the IRFMN;Advisor to WHO for the Drug Utilization Research Group (pregnancy, paediatrics and breastfeeding);1987-92 coordinator of the International Cooperative Study of Drug Use in Pregnancy, under the auspicesof WHO and the support of EEC; 1992-93 co-editor of The Kangaroo; 2000-05 coordinator of theEuropean Cooperative Study: “Development of the European register of clinical trials on medicines forchildren” (DEC-net), under the 5 th Framework Programme’s Quality of life and Management of LivingResources; since 1989 he has been director of the Centre for Drug Information; since 1993 head of theLab. for Mother and Child Health; since 1997 teacher for the Lombardy region’s professional trainingcourses; since 2000 teacher for the Lombardy region’s professional training courses; since 2002 Editor ofthe Ricerca & Pratica scientific journal; since 2003 professor of the School of Specialisation inPaediatrics - University of Milan Bicocca; teacher at the annual European course “Evaluation ofMedicinal Products in Children” (promoted by ESDPPP and Eudipharm).Selected publications• Impicciatore P, Pandolfini C, Casella N, Bonati M. Reliability of public-oriented health care resources on the WorldWide Web the home management of fever in children. BMJ 1997;314: 1875-1881.• Conroy S, Choonara I, Impicciatore P, Mohn A, Arnell H, Rane A, Knoeppel C, Seyberth H, Pandolfini C, Raffaelli MP,Rocchi F, Bonati M, Jong G, de Hoog M, van den Anker J. Survey of unlicensed and off label drug use in paediatricwards in European countries. BMJ 2000;320: 79-82.• Cazzato T, Pandolfini C, Campi R, Bonati M, and the ACP Puglia-Basilicata Working Group. Drug prescribing in outpatientchildren in Southern Italy. Eur J Clin Pharmacol 2001;57:611-616.• Impicciatore P, Choonara I, Clarkson A, Provasi D, Pandolfini C, Bonati M. Incidence of adverse drug reactions inpediatric in/out-patients: a systematic review and meta-analysis of prospective studies. Br J Clin Pharmacol 2001;52: 77-83.• Clavenna A, Pandolfini C, Bonati M. Public disclosure of clinical trials in children. Curr Ther Res 2002;63:707-716.• Pandolfini C, Bonati M. A literature review on off-label drug use in children. Eur J Ped 2005;164: 552-558.• Bonati M, Clavenna A. The epidemiology of psychotropic drug use in children and adolescents. International Review ofPsychiatry 2005;17: 181-188.• Santoro E, Rossi V, Pandolfini C, Bonati M. DEC-net: the development of the European Register of Clinical Trials onMedicines for Children. Clinical Trials 2006;3:366-375.208ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE LABORATORY'S ACTIVITIESResearch, as a multidimensional approach to producing knowledge, characterises theLaboratory’s activity.Research provides the basis for planning and carrying out the Laboratory’s activity in a criticalway and involves the participation of health professionals, social workers, mothers, children,and parents.Special attention is given to activities involving countries in the north and south of the world.The main objective of the Laboratory for Mother and Child Health is to ensure a better motherand child well-being by undertaking interdisciplinary and collaborative work in the field.Four broad areas, or spheres, of research have been selected:- monitoring and epidemiological evaluation of utilisation and effects of drugs and vaccines;- research methodology in general hospital and paediatric community practice;- public health determinants of children’s well-being;- transfer of health information to the community.The Drug and Health Information Centre (Centro di Informazione sul Farmaco e la Salute),whose main activity entails promoting rational drug use during breastfeeding and in childhood,is part of the Laboratory.NATIONAL COLLABORATIONSAgenzia Italiana del Farmaco, (AIFA)Associazione Culturale Pediatri, (ACP)Centro Antiveleni-Unità di Tossicologia Clinica-Ospedali Riuniti di BergamoCentro per la Salute del Bambino, (CSB)Consorzio Interuniversitario, (CINECA)Federfarma LombardiaIstituto Superiore di Sanità (ISS)Osservatorio Italiano Salute Globale (OISG)Il Pensiero Scientifico EditoreUnità Operativa di Neuropsichiatria dell'Infanzia e dell'Adolescenza, Fondazione Policlinico diMilano, (UONPIA)Università degli Studi di Milano-Facoltà di Scienze PoliticheUniversità degli Studi di Milano, Bicocca-Facoltà di Medicina-Clinica Pediatrica209ANNUAL REPORT 2007

IRFMNINTERNATIONAL COLLABORATIONSAgenzia Europea per i Medicinali (EMEA)Centro de Epidemiologia Comunitaria y Medicina Tropical (CECOMET), EcuadorClinica Infantil Colsubsidio, Bogotà, ColombiaEuropean Network Drug Investigation Children (ENDIC)European Society for Developmental Perinatal & Paediatric Pharmacology (ESDPPP)Hôpital Robert Debré, Parigi, FranciaInternational Society of Drug Bulletins (ISDB)Organizzazione Mondiale della Sanità (OMS)Unione Europea (UE)Università di Nottingham - Derbyshire Children's Hospital, Derby, UKEDITORIAL BOARD MEMBERSHIPDr. Maurizio Bonati, head of the Laboratory, is a member of the following editorial boards:Dialogo sui Farmaci, Disturbi d’Attenzione e Iperattività, European Journal of ClinicalPharmacology, Paediatric & Perinatal Drug Therapy, Pediatria (Sao Paulo), Quaderni ACP,Quaderni di Farmacoeconomia, Ricerca & Pratica, Saludarte.PEER REVIEW ACTIVITIESAmerican Journal of Epidemiology, Acta Paediatrica, BMC Pregnancy and Childbirth, BritishMedical Journal, Dialogo sui Farmaci, Epidemilogia & Prevenzione, European Journal ofClinical Pharmacology, Giornale Italiano di Farmacia Clinica, Journal of AntimicrobialChemotherapy, Medico e Bambino, Paediatric and Perinatal Drug Therapy, Pediatric Drugs,Pediatric Health, Pediatrics, Prescrire, Quaderni ACP, The Italian Journal of Pediatrics,Vaccine.NATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPComitato scientifico ADHD – ISSCommissione Nazionale per le Vaccinazioni – Ministero della SaluteCommissione tecnico-scientifica per la programmazione e verifica delle vaccinazioni - RegioneLombardiaComitato Scientifico del Gruppo di Lavoro “Farmaci e Bambini” – AIFACommissione tecnica per l'elaborazione, gestione e aggiornamento del Prontuario TerapeuticoRegionale (P.T.R.) - Regione Autonoma Valle d'AostaGruppo di lavoro Network Italiano Promozione Acido Folico – ISSGruppo di Lavoro Pediatrico – AIFAGruppo di Lavoro "Promozione allattamento al seno" - Regione LombardiaPaediatric Expert Group (P.E.G.)- EMEA210ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE LABORATORY WASINVOLVEDJanuaryNECESSITA’ TERAPEUTICHE INSODDISFATE IN PEDIATRIA. 48° Seminario SIAR“Attualità Regolatorie: il progetto AIFA per la farmaceutica; l’informazione scientifica nelCodice comunitario; esperienze e suggerimenti per le procedure AIFA: il sistema trasparenza; ilRegolamento pediatrico. Società Italiana Attività Regolatorie (SIAR); Segrate (MI).EPIDEMIOLOGIA DELLE MALATTIE PSICHIATRICHE DELL’ETA’ EVOLUTIVAE DEI TRATTAMENTI FARMACOLOGICI. Congresso “Bambini e Farmaci”: traincertezza scientifica e diritto alla salute”. Istituto Superiore di Sanità (ISS) e Istituto diRicerche Farmacologiche Mario Negri (IRFMN); Roma.FebruaryLA FARMACOEPIDEMIOLOGÍA Y EL USO DE LAS MEDICINAS EN EL NIÑO. 2 doTaller International Salud Infanto Juvenil Camagüey 2007. Hospital Pediatrico ProvincialDocente Dr. Eduardo Agramonte Pina; Camagüey, Cuba.LETTURA CRITICA DELLA LETTERATURA SCIENTIFICA. BANCHE DATIBIBLIOGRAFICHE - PERCORSI DI RICERCA. I DATABASE DELLE AGENZIEINTERNAZIONALI: IL GIOCO DEGLI INDICATORI. Corso di perfezionamento inMedicina Tropicale e Salute Internazionale. Università degli Studi di Brescia; Brescia.MarchPERCORSO PER LA GESTIONE PSICOMOTORIA IN AREA CRITICA. Congresso“Linee guida tra EBM e pratica clinica. Il trattamento del disturbo bipolare e dell’agitazionepsicomotoria nell’adulto e in età evolutiva”. Azienda Ospedaliera Ospedale Riguarda Ca’Granda; Milano.FARMACI GENERICI IN PEDIATRIA: RISPARMIARE CONVIENE? IV Congressointerregionale della società italiana di pediatria “OSTIA 24”. Società italiana di pediatria (SIP);Ostia (Roma).LA SALUTE IN UN MONDO GLOBALE, TRA PROGRAMMI E DIRITTI NEGATI.Incontro “Pochi più ricchi, tanti sempre più poveri. E’ questo il mondo che vogliamo?”. CentroSocio Culturale COOP; Novate Milanese (MI).AprilPSICOFARMACI NEI BAMBINI. Corso “Orientarsi in salute & sanità per fare scelteconsapevoli”. @Partecipasalute, IRFMN; Milano.LO SCENARIO: EPIDEMIOLOGIA DELLA MALATTIA MENTALE. Seminario“Seminario di avvio della rete ADHD”. Istituto Superiore di Sanità (ISS); Roma.TRATTAMENTI PSICOFARMACOLOGICI IN GRAVIDANZA. Corso “Patologie eterapie psicofarmacologiche in gravidanza”. Azienda Ospedaliera di Melegnano; VizzoloPredibassi (MI).TRATTAMENTI PSICOFARMACOLOGICI IN GRAVIDANZA. Corso “Patologie eterapie psicofarmacologiche in gravidanza”. Azienda Ospedaliera di Melegnano; ZizzoloPredibassi (MI).MayBAMBINI E FARMACI. Corso. “Gli Argonauti VIII. In viaggio per Itaca” AssociazioneCulturale Pediatri (ACP); Messina.211ANNUAL REPORT 2007

IRFMNEPIDEMIOLOGIA DELLE MALATTIE PSICHIATRICHE DELL’ETA’ EVOLUTIVAE DEI TRATTAMENTI FARMACOLOGICI. . Corso “I percorsi diagnostici-terapeuticiassistenzialidei bambini e adolescenti con disturbi psichici nella azienda ULSS 20 di Verona”.Azienda ULSS 20 di Verona; Marzana (VR).USO DI FARMACI OFF-LABEL IN ETA’ PEDIATRICA. Conferenza. Ospedale deiBambini “V. Buzzi” Istituti Clinici di Perfezionamento; Milano.FARMACI E ALLATTAMENTO. Corso di aggiornamento PLS (Pediatri di Libera Scelta)Udine.REAZIONI AVVERSE IN GRAVIDANZA. REAZIONI AVVERSE IN PEDIATRIA.Master Farmacovigilanza. SEFAP, Milano.JuneCLINICAL TRIALS OF ANTIDEPRESSANTS IN CHILDREN, WHAT HAVE WELEARNT? - WHAT IS THE EVIDENCE OF EFFICACY? - WHAT IS THE EVIDENCEOF TOXICITY? WHY TRANSPARENCY IS ESSENTIAL. Corso “5th InternationalWorkshop on Paediatric Clinical Trial”. The Medical School, Derby City General Hospital;Derby, UK.PRESCRIZIONE FARMACOLOGICA IN PEDIATRIA DI FAMIGLIA E PERCORSIDIAGNOSTICO-TERAPEUTICI. Corso di aggiornamento per Pediatri di Famiglia.Formazione pediatrica ASL Pavia; Strabella (PV).JulyL’IMPORTANZA DEI DATI EPIDEMIOLOGICI NEI PROCESSI DECISIONALI E DIRICERCA IN SANITA’. (Tavola Rotonda). Convegno “Vent’anni di storiadell’epidemiologia del farmaco”. Osservatorio ARNO, Consorzio Interuniversitario CINECA;Bologna .SeptemberIL MONITORAGGIO CRITICO DELLE PRESCRIZIONI FARMACOLOHICHE PERMIGLIORARE LA QUALITA’ E L’APPROPRIATEZZA DELLA TERAPIA. 63°Congresso Nazionale della Società Italiana di Pediatria “Difendiamo i diritti dell’infanzia edell’adolescenza”. Società Italiana di Pediatria (SIP); Pisa.DISUGUAGLIANZE NELLA SALUTE MATERNO-INFANTILE. Corso “Health andHealth care services, as indicators of Human Rights”. Istituto Italiano per gli Studi Filosofici,Fondazione Lelio Basso-Sezione Internazionale; Napoli.Tavola Rotonda: l’in-formazione presente e futura. “Vaccinare per obbligo o per scelta?”.Associazione Culturale Pediatri (ACP); Senato della Repubblica, Roma.OctoberPSICOFARMACI & BAMBINI. Seminario “Psicofarmaci e bambini”. Gruppo ParlamentareForza Italia; Milano.ADHD E SALUTE DEL BAMBINO IN ITALIA. Corso: “Lo sviluppo delle Linee GuidaEuropee per le Terapie Farmacologiche dell’ADHD: Implementazione nel Mondo Reale edeffetti sulla qualità della vita”. Università degli Studi di Cagliari Dipartimento di Neuroscienze,Clinica di Neuropsichiatria Infantile; Cagliari.EPIDEMIOLOGIA. RICONOSCIMENTO DI UNA REAZIONE AVVERSA. REAZIONIAVVERSE PIU’ FREQUENTI IN ETA’ PEDIATRICA. Corso “la reazione avversa dafarmaco: come riconoscerla. L’importanza della segnalazione spontanea”. Istituti Clinici diPerfezionamento (ICO) Ospedale dei bambini V. Buzzi, Milano.212ANNUAL REPORT 2007

IRFMNACCESSO AI FARMACI NEI PAESI IN VIA DI SVILUPPO. XXVIII CongressoNazionale SIFO “Innovazione e Salute Pubblica. Efficacia a confronto con: Equità, Economia,Etica”. Società Italiana di Farmacia Ospedaliera (SIFO); Rimini, (RN).LO SCENARIO: EPIDEMIOLOGIA DEI DISTURBI PSICHICI DELL’ETA’EVOLUTIVA. 19° Congresso Nazionale ACP “Relazioni per crescere. Il bambino nelcontesto relazionale: basi biologiche ed implicazioni cliniche e sociali”. Associazione CulturalePediatri (ACP); Trani (BA).FARMACI E ALLATTAMENTO. Corso di aggiornamento PLS (Pediatri di Libera Scelta)Udine.NovemberLO SCENARIO: EPIDEMIOLOGIA DELLA SALUTE MENTALE IN ETA’EVOLUTIVA. Corso. UMANA_MENTE, Milano.INFORMAZIONE E DISINFORMAZIONE NELL’ERA DELLA COMUNICAZIONE.Seminario “Dialoghi di Bioetica e biodiritto 2007. Sintonie e interferenze: le dimensioni dellacomunicazione in ambito sanitario”. Provincia autonoma di Trento, Ordine dei MediciChirurghi e degli Odontoiatri della Provincia di Trento, Federazione Nazionale ColleghiInfermieri IPASVI, Ordine dei farmacisti della Provincia di Trento; Trento.DecemberMETILFENIDATO E ATOMOXETINA. Congresso “Confronti in pediatria 2007. LaPediatria attraverso i farmaci. Quelli facili e quelli difficili”.IRCCS Burlo Garofano; Trieste.IL REGISTRO NAZIONALE: UNO STRUMENTO DI SALUTE PUBBLICA. Convegno“Il Registro Nazionale dell’ADHD”. SINPIA, Milano.GRANTS AND CONTRACTSAIFA, Agenzia Italiana del FarmacoBoehringer IngelheimCINECA, Interuniversity ConsortiumEuropean UnionFondazione MonzinoIl Pensiero Scientifico EditoreProvincia of MilanLombardia Region - Assessorato alla SanitàValle d'Aosta Region - Assessorato alla Sanità, Salute e Politiche Sociali213ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Arias G, Palau M, Perez M, Bonati M. Philanthropic prize to remember that Latin American Children’s health is apriority. Pan American Journal of Public Health (Revista Panamericana de Salud Publica) 2007;22:294.Campi R, Bonati M. Italian child health statistic review: births and deaths. Ital J Ped 2007;33:67-73.Campi R, Bonati M. Italian child health statistic review: morbidity and social habits. Ital J Ped 2007;33:144-149.Choonara I, Bonati M. European legislation to improve medicines for children. Paedat Perin Drug Ther 2007;8:2-3.Clavenna A, Bonati M. Antidepressant prescriptions in paediatric outpatients in Europe. Paediatr Perin Drug Ther2007;8:103-8.Clavenna A, Rossi E, De Rosa M, Bonati M. Use of Psychotropic Medications in Italian Children and Adolescents.Eur J Pediatr 2007;166:339-47.Marchetti F, Bua J, Ventura A, Notarangelo LDE, Di Maio S, Migliore G, Bonati M. The awareness amongpaediatricians of off-label prescribing in children: a survey in Italian Hospitals. Eur J Clin Pharmacol 2007;63:81-85.Rossignoli A, Clavenna A, Bonati M. Antibiotic prescription and prevalence rate in the outpatient paediatricpopulation: analysis of surveys published during 2000-2005. Eur J Clin Pharmacol 2007;63:1099-106.214ANNUAL REPORT 2007

IRFMNLAY PRESS SELECTION PUBLISHED IN 2007Bianchi M, Clavenna A, Labate L, Bortolotti A, Fortino I, Locatelli GW, Giuliani G, Bonati M. Profilo prescrittivodei farmaci antiasmatici nella popolazione pediatrica della ASL di Lecco. Medico e Bambino 2007;26:653-659.Bonaccorsi A. All’origine delle disuguaglianze nella salute (traduzione). R&P 2007;23: 216-17.Bonati M. 2007: l’anno dei farmaci per i bambini? Quaderni acp 2007;14:241.Bonati M. Dalla letteratura...Pillole di buona pratica prescrittiva. L'utilizzo dei farmaci nell'infanzia: il programma delMinistero della Salute. Newsletter 2007;n.7:3.Bonati M. Disturbi mentali e farmaci: il caso dell’ADHD e degli psicostimolanti. Medico e Bambino 2007;XXVI:75-76.Bonati M. I disturbi mentali dell’età evolutiva: una distorsione tra richiesta e offerta di aiuto. Informazione suifarmaci 2007;3:57-59.Bonati M. I farmaci: una delle aree di intervento. R&P 2007;22:270.Bonati M. L’anestesia non fa la rete e neppure l’equità. Il Sole 24 Ore Sanità 2007;17-23 aprile:3.Bonati M. L’utilizzo dei farmaci nell’infanzia. Bollettino SIFO 2007;53:109.Bonati M. Quando è l’evidenza erariale a sopravanzare quella scientifica. R&P 2007; 23:97-99.Bonati M. Una distorsione tra richiesta e offerta di aiuto. SocialNews 2007; http://www.socialnews.it/ARTICOLIAPRILE2007/Aprile2007Bonati_1.htm.Bonati M, Campi R. Le disuguaglianze tra Nazioni nella salute infantile. Quaderni acp 2007;14:15-18.Bonati M, Campi R. La promozione della salute nei Paesi del Sud del mondo. Quaderni acp 2007;14:95-96.Bonati M, Gruppo di Lavoro sui Farmaci Pediatrici (Istituito presso AIFA) Decongestionanti nasali: nei bambini irischi superano i benefici. Medico e Bambino 2007;5:309-315.Briantea Paediatric Surveillance Unit. (Bonati M, Calati MG, Clavenna A). Il monitoraggio epidemiologico dellemalattie: l’esperienza pilota di otto ospedali sentinella. Quaderni acp 2007;14:242-244.Campi R, Bonati M. Bambini poveri e disuguali in Italia. R&P 2007;23:232-233.Centro di Informazione sul Farmaco e la Salute. Otite media acuta: meglio attendere prima dell’antibiotico. Quaderniacp 2007;14:45.Centro di informazione sul Farmaco e la Salute. Decongestionanti nasali: attenzione, pericolo di morte. Quaderni acp2007;14: 86.Cerzani M, Pasina L, Clavenna A, Nobili A, Garattini L. Revisione critica degli studi italiani di farmacoeconomiasull’uso dei farmaci antinfiammatori non steroidei in medicina generale. Quaderni di Farmacoeconomia 2007; 3:17-28.Clavenna A. In viaggio tra le evidenze. R&P 2007;23:119.Clavenna A. Società Italiana di Medicina delle Migrazioni. R&P 2007;23:74.Clavenna A, Bonati M. Dieta, Integratori, antibiotici e autismo. Medico e Bambino 2007; 26:421-22.Clavenna A, Bonati M, Campi R, Labate L, Nobili A, Pasina L,Tettamanti M, Monesi L, Marzona I, Roncaglioni C,Bortolotti A, Fortino I, Locatelli GW, Giuliani G. La prescrizione di farmaci per i bambini e gli anziani nella ASL diLecco. R&P 2007;23:100-112.Clavenna A, Fortinguerra F. Antibiotici: usare con cautela. Quaderni acp 2007;14:125.Clavenna A, Fortinguerra F. Desmopressina nel trattamento dell’enuresi: evitare gli spray. Quaderni acp2007;14:226.Clavenna A, Fortinguerra F. Farmaci antiraffreddore ancora nella bufera. Quaderni acp 2007;14:272.Clavenna A, partecipanti al corso “La risoluzione di scenari clinici con il supporto della EBM come strumento diformazione continua per il pediatra”. Esiste una terapia farmacologica per la sindrome delle apnee notturne?Quaderni acp 2007;14:73-74.Conti Nibali S, Bonati M, Font M, Toffol G. La prescrizione per DCI in Pediatria ambulatoriale Studio di fattibilità.215ANNUAL REPORT 2007

IRFMNQuaderni acp 2007;14:46-47.Conti Nibali S, Bonati M. DCI nella medicina generale: studio di fattibilità. Dialogo sui Farmaci 2007;2:78-80.De Rosa M, Rossi E, Bonati M, Clavenna A. L’85% sono antibiotici, antiasmatici e corticosteroidi. Il sole 24 oreSanità 2007; 30gen-5feb:3.Grandori L, Bonati M, Gangemi M. 8 passi di prevenzione a tutela della salute dei bambini. Medico e Bambino2007;25:643-644.Fortinguerra F, Clavenna A, Baviera M, Cattaneo A, Conti Nibali S, Labate L, Maschi S, Miselli M, Scurti V, ZanfiD, Zermiani G, Bonati M. Le inserzioni pubblicitarie sulle riviste italiane del medico pediatra. R&P 2007;23:236-251.Labate L. Dimensione campionaria. R&P 2007;22:256-261.Maschi S, Fortinguerra F, Clavenna A, Bonati M. Levotiroxina in età pediatrica. Dialogo sui farmaci 2007;5:133-135.OTHER PRODUCTS PUBLISHED IN 2007Book ChaptersBonati M, Campi R. Le disuguaglianze fra nazioni nella salute infantile. In: Oltre il DNA Scienza, società ecittadinanza. IBIS, Como; Pavia 2007:69-76.Clavenna A, Bonati M. Farmacologia e tossicologia in gravidanza e allattamento. In: Ostetrica e Ginecologia. Zanoio,Masson spa, Milano, 2007;Cap.23:497-512.Bonati M, Campi R e Gruppo di Lavoro per la Convenzione sui Diritti dell’Infanzia e dell’Adolescenza. I dirittidell’infanzia e dell’adolescenza in Italia. 3° rapporto di aggiornamento sul monitoraggio della Convenzione sui dirittidell’infanzia e dell’adolescenza in Italia 2006-2007. Gruppo di lavoro per la CRC c7o Save the Children, Roma,2007;7-118.BookCirillo B, Bonati M, Campi R, De Campora E, Siani P. Disuguguaglianze nella salute nell’infanzia e nell’adolescenzain Campagna. Phoebus Edizioni, Casalnuovo, Napoli 2007.Schiavetti B, Clavenna A, Fortinguerra F, Miglio D, Bonati M. Psicofarmaci in allattamento. Il Pensiero ScientificoEd., Roma 2007.International AbstractsBianchi M, Clavenna A, Labate L, Bonati M. Anti-asthmatic drug prescriptions in Italian children and adolescents.Paediatr Perinat Drug Ther 8:72;2007.Pandolfini C, Bonati M, Rossi V, Santoro E, Naylor C, Sammons H, Choonara I, Zarrabian S, Jacqz-Aigrain E,Castel JM, Danés I, Fuentes I, Arnau J. The DEC-net European register of paediatric drug therapy trials: its contentsand their context. Paediatr Perinat Drug Ther 8:72; 2007.Colombo C, Mosconi P, Bassi C, Bernardi F, Bosisio M, Copelli P, Crotti C, Erroi A, Magri M, Pandolfini C, PistottiV, Santoro E, Zucchetti E. Evaluating cancer pain websites. MedNet 2007 - 12 th World Congress on the Internet inMedicine, 7-10 October 2007, Leipzig, Germany 306;2007.216ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESPsychotropic drugs and breastfeedingThe major objective in creating this small formulary is to provide health professionals with atherapeutic guide that not only includes drug monographs, but also acts as an essential tool (thatis accurate, effective, and updated) for promoting the rational use of psychotropic drugs. Theformulary will be a source of information for current problems in daily clinical practice,especially for psychiatrists, family paediatricians, family physicians, and obstetricgynaecologists.It will be an important tool for psychiatrists for their specialistic pertinence, forfamily paediatricians for their role in caring for newborns, for family physicians for theirprescription activity, and for obstetric-gynaecologists for their role in the pre- and peri-natalperiods.The drug information reported in patient leaflets is limited, generic and generally vague in orderto protect manufacturers, since little safety (and pharmacokinetic) information is available onthe use of the majority of drugs during breastfeeding. In fact, under “special precautions” weoften read: “There is no evidence on drug excretion in milk. The benefits for the mother must beevaluated in relation with infant risks”. The documentation on real or potential risks, however,is often inexistent.For those drugs on which information is available (often only pharmacokinetic data with respectto the mother’s milk/plasma ratio and not with respect to safety) the patient leaflet may report:“During breastfeeding, the possibility of early weaning must be considered”. Or “If thetreatment is considered necessary for a breastfeeding mother, she must discontinuebreastfeeding to avoid having the breastfed infant ingest even minute drug amounts excreted inbreast-milk”. The usefulness of these sentences, which should be decisional for physicians andinformative for patients, is insufficient.Current medicine is founded on the basing of therapeutic choices on documented safety andefficacy evidence. There is therefore a need to systematically collect and evaluate the availableinformation on the risks of psychotropic drugs during breastfeeding and, briefly, also inpregnancy, as a part of a continuum that cannot be separated.This Guide is unique, and not only at the national level, although its structure reflects theworking experience of the Drugs and Human Lactation Working Group founded in 1985 by theEuropean section of WHO. The work behind the creation of this Guide is seen as an on-going,collegial review. It would be wonderful if the work could proceed with the readers’participation, including reporting any errors (we hope few) and suggesting any improvements.This would be fundamental for this product to become increasingly useful in clinical practiceand to possibly be extended to other drug classes and problems.Child and adolescent health inequalities in the Campania regionThis book is dedicated to those who provide care to children and families and who don’t acceptthese injustices and fight to reduce and eliminate them.It is dedicated to all those health care operators who believe reducing health inequalities is theright thing to do and who are aware of the change in the epidemiology of family and childhealth problems (reduction of childhood organic disorders , increase in chronic diseases and inrelative mortality, especially that which is determined socially, reduction of comunicablediseases, and increase in psycho-social problems), who want to have a comprehensive view ofthe causal chains in health and a longitudinal approach to the “path of life”.It is dedicated to all those who think that today’s children are tomorrow’s men and women, thatchildhood inequalities will become inequalities in later phases of life, and that being aware of,and fighting, them can contribute to society in general’s health and wellbeing.(Carlo Corchia,Dante Baronciani, Epidemiologia della disuguaglianza nell’Infanzia, 1995).217ANNUAL REPORT 2007

IRFMNThe book is divided into five fundamental parts that address the health determinants in order(from the distal ones—socioeconomic, social capital—to the proximal ones—behaviours andlifestyle habits), access to health services, mortality, and, in conclusion, strategies for reducinghealth inequalities. In testimony of Italy’s, and all industrialised countries’, persistence ofprofound inequalities in children’s health, it describes the regional and sub-regional differencesin the distal and proximal mortality determinants.Workgroup on the “Convention for child and adolescent rights”The Laboratory for Mother and Child Health is part of the Workgroup on the “Convention forchild and adolescent rights” (CRC) in Italy.The group was set up in December 2000 with the aim to prepare a report on the status ofchildren in Italy that would act as a supplementary (not alternative) report to that presented bythe Italian government, to be submitted to the United Nations Committee on child andadolescent rights. The first report was presented in Rome in 2001 and underwritten by 42associations and non-governmental organizations.On January 31, 2003, following a public meeting held in Geneva between the United NationsCommittee and a large governmental delegation a document was created. This documenthighlighted Italy’s progress in order to enact the Convention, but also reported on worryingmatters concerning the lack of adherence to certain principles, and precise recommendations arelisted for which the government will have to account in the next United Nations report in 2008(CRC/C/15/Add.198).While carrying out the monitoring activity, the workgroup decided on the creation of an annual,updated report on the status of children and adolescents in Italy and on the related UnitedNations Committee’s recommendations, focusing on priority issues.The report is not only intended to identify our health system’s faults, but also to be anopportunity to prompt a timely and constructive debate among the institutions responsible forthe status of children and adolescents in Italy, and, therefore, for the implementation of therights guaranteed by the convention.The third report, in addition to updating the monitoring of the issues already addressed in theprevious report, and to developing additional, detailed discussion, adds to the analysis with twonew chapters: the second one dedicated to the CRC’s general principles, starting from themonitoring of the “principle of participation” (art.12 CRC), and the third dedicated to the familyenvironment and alternative measures. The objective of the workgroup is, in fact, to expand onthe issues covered until it addresses all eight groupings into which the United NationsCommittee has divided the CRC’s articles, in the next supplementary United Nations Report.All this is carried out with the hope that it will stimulate, and contribute to, the development ofstandard procedures and legislative reforms that will bring about a tangible improvement in thecondition of all youths in Italy.ARNO paediatric projectFor over five years one million children and adolescents, and their families, have made up theARNO-paediatric project’s population. The project, a product of the collaboration between the“Mario Negri” Institute in Milan and the CINECA in Bologna, is unique for its size andduration at both the national and international levels.The initiative is part of the larger ARNO project that monitors out-hospital drug prescriptionsand that has been active at the CINECA since 1986 with the participation of numerous localhealth units across Italy.During 2006, 561,237 children aged less than 14 years (61% of total) were prescribed 620different drugs (active moieties), using 1,805,521 prescriptions, for a total of 2,697,979 boxes.Each child received an average of 3 prescriptions and 5 drug boxes.The prescription prevalence was highest in 1-4 year old children (76%), decreasingprogressively with increasing age and reaching 43% in 13 year olds. It was higher in males than218ANNUAL REPORT 2007

IRFMNfemales (62 vs 59%). Antibiotics (52%), antiasthmatics (26%), and systemic corticosteroids(8%) were the most prescribed drug classes and corresponded to 84% of prescriptions.The amoxicillin-clavulanic acid association was the most prescribed drug (461,755 boxes to217,720 children), followed by amoxicillin (256,892 boxes to 129,788 children), and bybeclometasone (195,443 boxes to 137,031 children). Beclometasone was the most prescribeddrug in children

IRFMNThe first evaluation of the outcomes will be made after 24 months of the register’sactivation. http://www.farmaco-iss.org/“FARMACI E BAMBINI” workgroupA paediatric group of the Italian Drug Agency (AIFA) was set up on April 4 th , 2006. TheLaboratory for Mother and Child Health, along with the Associazione Culturale Pediatri (ACP),Centro Salute del Bambino (CSB), Federazione Italiana Medici Pediatri (FIMP), SocietàItaliana Farmacia Ospedaliera (SIFO), Società Italiana di Pediatria (SIP), and experts in thesector and members of the AIFA, participate. The group’s function is to support the AIFA’sregulatory activities, informational initiatives, pharmacovigilance work, and promotion ofindependent clinical research concerning drug use in the paediatric population.The workgroup’s activities currently cover three broad areas:Pediatric research: the group’s role is to help define the paediatric population’s needs andpriorities in the research field. The group also aims to suggest the issues that should be reportedto the Commissione Ricerca e Sviluppo (CRS) to choose the new independent research grantoffers to finance at the Italian level and to eventually transfer to the European level.Pharmacovigilance: to help analyse the reports received by the spontaneous reporting network,thus helping to develop a more active, more efficient pharmacovigilance system.Information/training: to recommend the development of adequate tools for health care workers.More specifically, two of the workgroup’s main activities involve the assessment of the offlabeluse of drugs and of the adverse drug reactions received by the National PharmacovigilanceNetwork. During 2007, 3 dear doctor letters concerning the paediatric population were createdand distributed: a new contraindication to the use of nasal decongestants containingsympaticomimetics for topical use in children less than 12 years old; new safety informationconcerning the use of cefaclor and the updating of the Summary of Product Characteristics wasissued; and new, important information concerning the use of desmopressin in the nasal sprayformulation was issued. A formal article was published on a few national journals describing anevaluation of nasal decongestants. Furthermore, a systemic evaluation of reports concerning theuse of antiemetics, especially metoclopramide and domperidon, was carried out.Important safety information is available on the AIFA website http://www.agenziafarmaco.itVaccine commissionThe Laboratory for Mother and Child Health is part of the National Vaccine Commission andthe Lombardy Region’s Vaccine Commission.Ministero della Salute. The National Vaccine Commission was set up with a MinisterialLaw on February 20, 2007.The main goal of the commission is to update the National Vaccine Programme, a documentwhose main objectives are: to maintain a high vaccine coverage rate for the diseases for whichthe National Health Plans 1998-2000 and 2003-2005, as well as the WHO’s Regional EuropeanOffice, reached their goals; to promote appropriate vaccine interventions aimed at recuperatingoptimal coverage of those vaccines for which a basic working strategy has already been defined,but for which optimal coverage has still not been reached; to provide indications on the newobjectives, and on the startup of focused initiatives aimed at prevention through vaccinationfollowing the recent availability of new vaccines, as indicated in the National Health Plan 2003-2005; to increase the safety of immunisation practices; to increase the structural, organisational,training, and communication-based interventions in order to permit the evolution of vaccinepolicies from mandatory interventions to those characterised by an informed participation on thepart of families.Lombardia Region. At the regional level, the group is called the “Technical-scientificcommission for the planning and verification of vaccinations”, based on its assigned tasks. Itsmain goals are: the elaboration of vaccine strategies, based on epidemiologic profiles;evaluation of the ongoing interventions, in terms of protective efficacy and unwanted220ANNUAL REPORT 2007

IRFMNeffects;promotion of the qualitative improvement of vaccine management difficulties, includingthe vaccine registry.This commission developed a document entitled “Vaccines in children and adults: review andreorganisation of vaccine prophylaxis in the Lombardy Region”, approved with the 22/12/05law DGR VIII/1587. The aims of the document are to adapt the national and European vaccinestrategies and policies to the community and organisational contexts present in the region,providing guidelines for the improvement of the quality of the vaccination programme offered.Technical commission for the elaboration of the regional therapeuticformularyThe Valle d’Aosta Autonomous Region set up an agreement with the “Mario Negri”Pharmacological Research Institute, assigning the Laboratory for Mother and Child Health, astemporary representative of the “Mario Negri” Institute, the periodic task of providing technicaladvice on the fulfillment of the following activities: updating and revision of the regionaltherapeutic formulary (PTR); predisposing opinions on trial protocols on pharmaceutical drugproducts and other therapeutic remedies; creating documentation on topics on which thecollaboration is based.Paediatric pain guidelinesThe Presidio Ospedaliero degli Spedali Civili di Brescia’s health management committee set up3 work groups, selecting members from among physicians and nurses representing variouspaediatric wards, in order to create guidelines concerning:pain evaluation and treatment in a paediatric emergency ward; the use of analgesics duringvenipuncture procedures, positioning of the needle cannula, and capillary blood sampling inneonates; topical analgesic use during venipuncture procedures and positioning of the needlecannula.The scientific co-ordination, necessary for the creation and evaluation of the guidelines, hasbeen assigned to the Laboratory for Mother and Child Health.Health and Drug Information CentreThe Health and Drug Information Centre (HDIC), initially created to promote the policy onhospital drug formularies, progressively expanded to cover out-of-hospital practice and toinvolve different types of users, including professionals and members of the public. The core ofthe HDIC’s activities is the daily telephone service providing information, also to the lay public,and advice on drug therapy and needs during breastfeeding and in childhood. In January 2004,the centre’s activity concerning drug information in pregnancy was taken over by Bergamo’sClinical Toxicology Unit’s Poison Control Centre (Centro Antiveleni, Unità di TossicologiaClinica, Ospedali Riuniti di Bergamo). Together, the Poison Control Centre and the Laboratoryof Mother and Child Health form the Department of Clinical Pharmacy and Pharmacology.During 2007, 599 people contacted the Health and Drug Information Centre with a total of 1001questions. In all, 80% were from people who were directly involved (patients or familymembers), 13% from general/hospital paediatricians, 3% from general practitioners or othernon-paediatric specialists, and 4% from other health care workers. A total of 817 requests forinformation concerned drug use, 184 the use of parapharmaceuticals, general information, anddiagnostic exams. In all, 93% of the questions received by the centre concerned the safety ofdrugs during breastfeeding, while 7% concerned the administration of drugs to children.Although people called from all of Italy, most called from the north of the country (68%),especially from the Milan (228) and Brescia provinces (32). 21% of the requests were receivedfrom central Italy and 13% from the south.Drugs and breastfeeding.221ANNUAL REPORT 2007

IRFMNOf the 931 requests for information on breastfeeding, 79% came from 491mothers and/or relatives, while the remaining 21% from 144 health care workers, of whom 92were paediatricians. The mothers’ average age was 34 years (range 22-45) and that of thebreastfed babies was 5 months (range 0-42). Almost half (45%) of these babies was £3 months.In all, 81% of the newborns were exclusively breastfed and 12% were fed artificial formula inaddition to breast milk. The type of nutrition was still undecided for the remaining 7%.The drug classes for which information during breastfeeding was most often requested were:analgesics/antiinflammatories (11%), systemic antibacterials (9%) and psychotropic drugs (8%).More specifically, psychotropic drugs and antiepileptic agents were the classes for whichinformation was most often requested on the part of health care workers.The indication most frequently associated with drug use was symptomatic pain control (109queries), followed by allergy (64), and psychiatric disorders (66).A follow-up for 67 mothers whose child turned 6 months old was completed. Of these mothers,66% continued exclusive breastfeeding, 9% had introducedartificial milk in the baby’s diet, and 25% had stopped breastfeeding. In all, 52 mothers hadtaken the drug for which they had asked information and 15 did not take it. The reasons for nothaving taken the drug were: “because it was no longer necessary” (n=13) and “for fear” (n=2).In all, 48% of the newborns whose mothers were re-contacted for follow-up had been given oneor more drugs in the first 6 months of life: of the 83 drugs, 18 were anti-antinfectives, 17 wereantiasthmatics, and 10 antacids/proton pump inhibitors. Paracetamol was the most commonlyprescribed drug (8), followed by amoxicillin+clavulanic acid (6), beclomethasone (6), andsalbutamol+ipratropium bromide (6). The most common reason for prescribing drugs was“bronchitis” (n=15).Drugs and childrenThe requests for paediatric drug therapy information concerned 39 children aged 1 month to 12years. In all, 30 mothers/fathers, 7 family or hospital paediatricians, and 1 health care workercontacted the centre. Of the 70 queries received, 62 concerned general drug information, 7concerned side effects, 1 concerned dosage and 1 efficacy. The most common therapeuticgroups concerned psychotropic drugs (10), system antibiotics (8), and nasal products (5). Themost common reason for prescribing drugs was respiratory tract infection (16).Folic acid and SIDSAs part of the centre’s activity involving the monitoring of the generalpopulation’s level of health awareness, the information the mothers received on the use of folicacid in pregnancy to prevent neural tube defects and on the recommendations concerningsudden infant death syndrome (SIDS) was collected.Concerning folic acid, 91% of the mothers had taken it during pregnancy, but only 21% hadbegun to take it before conception.Of the 339 newborns for whom information could be collected, 286 were placed in thesupine sleep position, 46 on their side, and 7 face down. In 16% of the cases, the position wasincorrect for SIDS prevention. Over half of the mothers of those newborns had not received anyinformation concerning the safest sleep position to place their children in.Ricerca & PraticaRicerca & Pratica was born in January, 1985, as a manifestation of the “Mario Negri” Institutefor Pharmacological Research. Today, the journal is part of the International Society of DrugBulletins (ISDB), which represents independent journals.For more than twenty years, the journal has represented an arena for all those professionals whocollect data and carry out studies in general practice with the aim to increase their knowledgeand to improve their practice. Ricerca & Pratica is also appreciated for its ability to go beyondthe merely clinical aspect of medicine, without, however, forgetting that it is to this aspect thatthe readers dedicate most of their time and effort.222ANNUAL REPORT 2007

IRFMNThrough its activity, Ricerca & Pratica can therefore represent an exclusive, independentobservation point. It is also an area that promotes contemplation, evaluation, andinformation by applying “tools” such as data trustworthiness and importance, the balancebetween benefits and risks and between benefits and costs, independence from conflicts ofinterest, and the realistic objective to contribute to a progressive, equally distributedimprovement in the population’s health....and thenOffertasociale: Offertasociale runs several social services that protect the weaks within aconsortium of 29 counties in the Vimercate and Trezzo areas and entrusted the Laboratory forMother and Child Health with the carrying out of a course aimed at developing wellnessindicators for the young. In order to inform and assess public policies, it is necessary to havelocal indicators with which to measure community wellness. The population’s quality of life isincreasingly tied not only to per-capita income, but to a set of multidimensional factors. Amongthese are life expectancy at birth, literacy and education rates, school drop-out rates, etc.Learning to read the level of cohesion/social exclusion, also through the analysis of data ontroubled or hospitalised youth, those living in troubled youth centres, etc, is necessary.All this suggests that the communities, and the operators that provide services, learn the specifictools and methods related to a renewed cultural dimension based on concepts of “wellness”,“lifestyle quality”, “equal access to services”, etc. The course provided the tools for building acommunity level development index that took into consideration the indicators that could beused to represent the three dimensions of human development (income, education, and health)for the 29 counties in the area: the “community level development-wellness index” - HumanDevelopment Index for the 29 counties starting from the data received and from the proposedindicators.A report on a few determinants of human development of youths residing within the consortiumof 29 counties was produced during 2007.Nascere in casa: The Associazione Nazionale Culturale Ostetriche Parto a Domicilio(National Cultural Association of Home Birth Obstetricians) aims to increase home births,guaranteeing their quality and safety, verifying and comparing the national and internationalexperiences of care outside the hospital, and defining professional selection and assistancecriteria based on scientific evidence from the World Health Organization’s recommendationsand on women’s wishes. The Laboratory for Mother and Child Health collaborates with theassociation, analysing data collected by the obstetricians in order to improve the quality of careoffered to women and children through experience, but also through continuous study,continuing professional training, and debate. These improvements will help protect out-ofhospitalbirths and safeguard the obstetricians’ specific professional role.Co-operation with countries with limited resourcesAs an expression, test, and original method of expression of the choice to make the Laboratory’sresearch transferable and accessible to all populations, the Laboratory promoted and providedassistance to projects in, and for, the “South of the world”, also in collaboration with the WorldHealth Organization. The technical and organisational support for local groups and internationalnon-governmental organisations for carrying out socio-sanitary projects in countries withlimited resources, especially Colombia, Ecuador, Brazil, Bolivia, Cuba, Vietnam e the Balkans,continues.Bolivia:On April 20, 2007 the laboratory organised a one day meeting at the Mario Negri Institute,entitled:“Being born today in Potosì: From Bolivia to the Lombardy Region: a twodirectional,collaborative path”. The results of a project, supported by COOPI and theLombardy Region, for the improvement of the health of both women during pregnancy and of223ANNUAL REPORT 2007

IRFMNyoung mothers, by facilitating access to health centres though initiatives aimed at integrating theofficial medical system and the traditional, community level medical system. Reduction of thehigh maternal mortality in the city of Tinguipaya, Department of Potosí, through theintegration of the official and traditional, local medical systemsCuba:2do Taller International de Salud Infanto-Juvenil. Hospital Pediatrico Provincial DocenteDr. Eduardo Agramante Piña Camagüey 2007. The Laboratory actively supported thesecond International workshop on child health in Cuba, held in Camagüey, aimed atimplementing paediatric hospital care, evaluating it, and rendering it informed and up-to-date.The issues addressed highlighted the fact that the family practitioner in Cuba has, in the last 20years, gained increasing importance.Every practitioner assists about 125 families. The health care system is also supported by aseries of polyclinics that mostly manage emergencies. Change is also under way for thepaediatric hospitals, and involves continual, significant improvements.Medical students dedicate 15 weeks of study to paediatrics and an additional 6 during the lastacademic year. In Camagüey there are about 150 students enrolled in medicine, 80 of whomcome from Latin America and who have been selected from among the poorest countries. Theaim is to train competent staff in order to improve the health of the populations in countries withlimited resources. There are, today, 80,000 Cuban physicians, 20,000 of whom work abroad(10,000 just in Venezuela).Ecuador:The Laboratory for Mother and Child Health, as epidemiologic scientific advisor andorganisational partner, has participated since 2005 in the project entitled “Project for thereduction of maternal and perinatal mortality in the S.Lorenzo District - Esmeraldas -EcuadorRegion”. The project is co-ordinated by the Bussolengo’s (VR) local health unit as part of itswork in carrying out decentralised cooperation interventions for international development andsolidarity.The Laboratory has two other projects in Ecuador, in collaboration with Cecomet (Centro deEpidemiologia comunitaria y Medicina tropical) in Borbòn’s health district, situated north of theEsmeraldas province, Ecuador’s rural area.The names of the projects are:“Improving vaccine coverage along the Santiago and Cayapas rivers”. The aims of theproject are to: improve vaccine coverage in the under 5 population, in schoolchildren, in womenof child-bearing age, and in pregnant women in order to avoid vaccine-preventable morbidityand mortality; optimise community vigilance of vaccine-preventable diseases so that theprogramme’s advancement can be monitored; and improve the families’ knowledge of vaccinesand their importance.“Reducing child mortality through the supplementation of vitamins and nutritionalelements”. The integration of vitamin A reduces the risk of child mortality. Lack of this vitaminmakes children especially vulnerable to infections and leads to greater disease severity. It is alsothe main cause of blindness in children. According to UNICEF, only 25% of children under 5receive vitamin A supplementation, which is essential for development and for controllingmalaria. The study also involves zinc supplementation: lack of zinc in undernourisced childrenis among the factors leading to limited physical development and increases predisposition toinfections, lack of zinc in pregnant women increases the risk of mortality; iron supplementation:its deficiency leads to severe anemia both in pregnant women and in children; iodine; andvitamin D.224ANNUAL REPORT 2007

IRFMNCENTRE OF COMPUTER SCIENCEENGINEERINGSTAFFHeadCLIVIO LucaThe year 2007 has been for the Centre of Computer Science Engineering the first year of lifeand at the same time the most challenging from the point of view of works about the networkingand server-side infrastructures, that has been heavily revised.About the clinical trials handling software the year 2007 has been the first in witch many of theAIFA sponsored studies have been implemented with case report forms handled by internalmade software, while about the area of office automation some of the most important softwarefor administrative tasks have been completed. Also in the area of the multimediacommunication this has been an important year: the event of beginning working in the newInstitute has carried the necessity (and the opportunity) of preparing a lot of divulgativeelectronic publishing, such as DVDs, and the new institutional web site.Follows a short list of some of the principal collaborative activities done during the year.Clinical Trials and related eForms (E-CRF)• Lab.: Neurological Disorders (Neuroscience Dep.):• Extension European Register SLA• Trial L-ACETYLCARNITINE• Trial ANTIEPILETTICI• Trial EPILESSIA E STROKE• Trial EPO VS MP IN SPINAL SHOCK• Trial VALPROATO• Trial Immunoglobuline• Lab.: Clinical Trials (Oncology Dep.)• Trial FOLFOX• Trial MAPS• Trial STARPAN• Trial HEAD & NECK• Trial TOP• Trial TAILOR• Lab: Clinical Epidemilogy• Trial CADASIL• Lab.: Quality Assessment of Geriatric Therapies and Services (Neuroscience Dep.)• Trial GISAS• Registro dei Pazienti per lo studio delle Polipatologie e Politerapie – SIMI network• Italian Cochrane Centre225ANNUAL REPORT 2007

IRFMN• Trial ICEKUBE• SINPE (Società Italiana di Nutrizione Artificiale e Metabolismo)• DOMUS: Registro Italiano dei Pazienti nutriti per via artificiale• IEO (Istituto Europeo di Oncologia)• Trial THALDODEX• Implementation at IEO of a complete software platform for handling no-profit trialsWeb based applications related to other research projects− Realization of a software for clinical trials users administration− Implementation of a software for an assisted analysis of drugs interactions ( in collaborationwith Assessment of Geriatric Therapies and Services Lab.)− Implementation E-Form for the project PAINCARE-LUVI for an evaluation of the onlinesources of scientific informations (in collaboration with Medical Research and ConsumerInvolvement Lab., and LUVI foundation)− Implementation E-Form for the project “NO-GRAZIE” about the spots and promotions ofdrugs (in collaboration with Mother and Child Health Lab.)− E-Form for online registration to workshop SCARLET (in collaboration withEnvironmental Chemistry and Toxicology Lab.)− Electronic board Negri CommunityWeb based application for office automation− New booking system for meeting rooms in the Institute− New scientific event registration and reports− New employed (staff) registration handling system− New database for managing the scientific publications− New database for donors registration− Centralized registration and handling of physician and projects involved in ECMconvention− Centralized registration for research project related to Animal Care UnitMultimedia communication− Realization of networking and software infrastructure for managing the videoconferencemeeting and a remote assistance VPN between Mario Negri Milano and Mario NegriBergamo institutions− Realization of a DVD about Parkinson syndrome (in collaboration with GeriatricNeuropsychiatry and photographic unit).− Video editing and preparation of a DVD for presenting the new Institute (collaboration withphotographic unit).Web sites− New official institutional web site− Web site project “GISAS”− Web site Fondazione Mattioli− Web site MANGO group− New web site project “Rischio & Prevenzione”Teaching activities− Internal course on standard SQL language for database querying finalized to data analysis ofprescriptions in “Regione Lombardia”.− Internal course on E-CRF finalized to Case Report Form preparation for clinical trials.226ANNUAL REPORT 2007

IRFMNITALIAN COCHRANE CENTRESTAFFHeadAlessandro LIBERATI, M.D.227ANNUAL REPORT 2007

IRFMNCURRICULUM VITAEAlessandro Liberati obtained his MD Degree in 1978 at the University of Milano and his post doctoraldegree in Hygiene and Preventive Medicine in 1981 at the same university.Teaching activities: Primary responsibility of several academic and non academic training courses on themethodology of clinical research and systematic reviews/metanalyses. He is Director of the advancedMaster “Evidence based medicine e metodologia della ricerca sanitaria”, at the Università degli Studi diModena e Reggio Emilia.Areas of scientific expertise: methodology of clinical research with particular reference to controlledclinical trials, epidemiological methods for research synthesis (systematic reviews and metanalyses);methods of practice guidelines production and implementation, evaluation of ethical implications ofclinical research.Past and current roles at the Mario Negri Institute: 1980-1986 Junior researcher at the Laboratory ofClinical pharmacology; 1987-1989 Head, Unit of Clinical Epidemiology and Health Services Research;1990-1998 Head Laboratory of Clinical Epidemiology; since 1994 he is Director of the Italian CochraneCentre; since 1998 he is Associate Professor of Clinical Biostatistics and Epidemiology at the Universityof Modena and Reggio Emilia; since 1997 he is President of the Associazione per la Ricerca sullaEfficacia della Assistenza Sanitaria - Centro Cochrane Italiano (AREAS-CCI); since 2005 he is Presidentof the Local Ethics Committee of the Local Health Unit of Bologna; since 2004 he is Member of theCommissione Nazionale Ricerca Sanitaria; since 2005 he is Member of the Commissione Ricerca eSviluppo dell’Agenzia Italiana del Farmaco (AIFA).Selected publications• Liberati A, Moja LP, Moschetti I.The future of clinical research: why do we need an ecological approach? Recenti ProgMed. 2006; 97:604-10.• Vigna-Taglianti F, Vineis P, Liberati A, Faggiano F. Quality of systematic reviews used in guidelines for oncologypractice. Ann Oncol. 2006 ;17:691-701.• Clinical Evidence edizione italiana, IV edizione. 2006 Zadig, Milano.• Moja L., Moschetti I., Liberati A., Gensini G.F., Gusinu R. Understanding systematic reviews: the meta-analysis graph(also called ‘forest plot’). Intern Emerg Med 2007;2:140-142• Moja L., Virgili G., Liberati A., Gensini G.F., Gusinu R., Conti A.A. Scale to climb borderline personalities: whenscience goes nowhere. Intern Emerg Med 2007;2:315-317• Moja L., Minozzi S., Liberati A., Gusinu R., Gensini G.F. The drama of cancer pain: when the research abandonspatients and reason. Intern Emerg Med 2007;2:226-228• Moja L., Moschetti I., Liberati A., Gensini G.F., Gusinu R. Systematic reviews highlight the complex balance betweengood and harm from screening studies. Intern Emerg Med 2007;2(1):57-9• Moja L., Moschetti I., Liberati A., Manfrini R., Deligant C., Satolli R., Addis A., Martini N., Dri P. Using ClinicalEvidence in a national continuing medical education program in Italy. PLoS Medicine 2007 4 (5)e113.doi:10.1371/Journal• Mosconi P., Colombo C., Satolli R., Liberati A. PartecipaSalute, an Italian project to involve lay people, patients'associations and scientific-medical representatives on the health debate. Health Expect 2007 Jun;10(2):194-204• Clinical Evidence edizione italiana, V edizione. 2007 Zadig, Milano.228ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE CENTRE'S ACTIVITIESThe Italian Cochrane Centre (ICC) (http://www.cochrane.it) was founded in 1994 and isaffiliated to the Cochrane Collaboration (CC). The CC is an international non profitorganization that prepares, maintains and promotes systematic reviews of the effects of healthcare interventions. The main product of the Cochrane Collaboration is the Cochrane Library, aquarterly publication containing Cochrane systematic reviews and other relevant databases ofother siblings international organizations.The objectives of the ICC are centered around supporting various activities of the CochraneCollaboration within Italy. In particular:a) to disseminate the knowledge of CC and CC activities throughout Italy;b) to provide methodological and practical support to all individuals and groups who areinterested in collaborating with the CC;c) to contribute to the adoption and dissemination of Evidence-based Medicine in Italy.FINDINGS/MAIN RESULTSThe ICC has created a national network with researchers and health care providers who areproducing systematic reviews for the Cochrane Collaboration and are actively involved in otheractivities related to the dissemination of evidence based medicine.NATIONAL COLLABORATIONSAgenzia Italiana del Farmaco (AIFA), RomaIstituto Superiore di Sanità, RomaMinistero della Salute, RomaCentro Valutazione Efficacia Assistenza Sanitaria (CeVEAS), ModenaDipartimento di Epidemiologia della ASL Roma EIstituto Neurologico "Carlo Besta", MilanoAgenzia Sanitaria Regionale, Regione Emilia Romagna, BolognaUniversità degli Studi di MilanoUniversità di Modena e Reggio EmiliaAzienda Sanitaria Locale 20, AlessandriaINTERNATIONAL COLLABORATIONSThe Cochrane Collaboration, Oxford, UKCentre for Reviews and Dissemination, University of York, York, UKBritish Medical Journal Publishing Group, London, UKCentre for Statistics in Medicine, Oxford, UKThomas Chalmers Centre for Systematic Reviews, Ottawa, CanadaThe Campbell Collaboration , Philadelphia, USA229ANNUAL REPORT 2007

IRFMNEDITORIAL BOARD MEMBERSHIPClinical Evidence (Alessandro Liberati)Evidence Based Health Policy and Research (Alessandro Liberati)Journal of Clinical Epidemiology (Alessandro Liberati)Journal of Health Services Research (Alessandro Liberati)PEER REVIEW ACTIVITIESAnnals of Internal Medicine (Alessandro Liberati)British Medical Journal (Alessandro Liberati)JAMA (Alessandro Liberati)Evidence Based Health Policy and Research (Alessandro Liberati)Canadian Medical Association Journal (Alessandro Liberati)EVENT ORGANIZATIONV Edition Master on “Evidence Based Medicine e metodologia della ricerca sanitaria”, March2007 - April 2008, Università degli Studi di Modena e Reggio Emilia1-day Cochrane Workshop in Evidence Based Medicine, MilanFocus in: Cardiology 28 September 2007, Ophthalmology 5 October 2007Gastroenterology 12 October 2007, General Medicine 23 e 26 November 2007Workshop on 29 November 2007, Milan:- Identifying and publishing uncertainties about the effects of treatments in DUETs - theDatabase of Uncertainties about the Effects of Treatments- Transparency in the publication and reporting of research. The Equator project and theimportance of reporting guidelines- What’s' behind the scene of medical journals?- Transparency in the production of guidelines and recommendation: the WHO experience.XII Annual Meeting of the Italian Cochrane Network "La trasparenza nella produzione edisseminazione delle informazioni scientifiche" 29-30 November 2007, Milan230ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE CENTRE WASINVOLVEDV Edition Master on “Evidence Based Medicine e metodologia della ricerca sanitaria”, March2007 - April 2008, Università degli Studi di Modena e Reggio Emilia“Prove di efficacia e pratica terapeutica: EBM e Cochrane Collaboration”, Master perCoordinatori di Unità Operativa e/o di Dipartimento per le professioni infermieristiche eostetriche, Università degli Studi di Milano, anno 2006-2007. 15-22-23 October 2007.Symposium for Centre and Branch Directors, Amsterdam 9-11 April 2007Workshop on “Evidence-based Medicine e revisioni sistematiche”, Ischia 8-10 April 2007Master Universitario di II livello MeS Management e Sanità-II edizione “Elementi diepidemiologia per l’analisi dei bisogni sanitari e valutazione sanitaria. Metodi quantitativi eelementi di statistica”, Pisa 22 November 2007Meeting Centri Cochrane e Gruppi di Revisione Europei " Cochrane Continental EuropeanEntities" 13-14 June 2007, Oslo, NorvegiaXV Cochrane Colloquium, 23-27 October 2007, San Paolo, BrasileGRANTS AND CONTRACTSIstituto di Ricerche Farmacologiche Mario Negri, MilanoAIFA - Agenzia Italiana del Farmaco, Roma231ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Moja L., Moschetti I., Liberati A., Gensini G.F., Gusinu R. Understanding systematic reviews: the meta-analysisgraph (also called ‘forest plot’). Intern Emerg Med 2007;2:140-142Moja L., Virgili G., Liberati A., Gensini G.F., Gusinu R., Conti A.A. Scale to climb borderline personalities: whenscience goes nowhere. Intern Emerg Med 2007;2:315-317Moja L., Minozzi S., Liberati A., Gusinu R., Gensini G.F. The drama of cancer pain: when the research abandonspatients and reason. Intern Emerg Med 2007;2:226-228Moja L., Moschetti I., Liberati A., Gensini G.F., Gusinu R. Systematic reviews highlight the complex balancebetween good and harm from screening studies. Intern Emerg Med 2007;2(1):57-9Moja L., Moschetti I., Liberati A., Manfrini R., Deligant C., Satolli R., Addis A., Martini N., Dri P. Using ClinicalEvidence in a national continuing medical education program in Italy. PLoS Medicine 2007 4 (5)e113.doi:10.1371/JournalMosconi P., Colombo C., Satolli R., Liberati A. PartecipaSalute, an Italian project to involve lay people, patients'associations and scientific-medical representatives on the health debate. Health Expect 2007 Jun;10(2):194-204LAY PRESS SELECTION PUBLISHED IN 2007.Liberati A, Mosconi P, Colombo C. Che cosa vuol dire Informati bene PartecipaSalute 2007Mosconi P, Colombo C, Satolli R, Liberati A. La ricerca clinica risponde ai bisogni dei pazienti? Ricerca & Pratica2007;137:192-203Mosconi P, Colombo C, Satolli R, Liberati A. Il corso di PartecipaSalute 2007 allo specchio: i commenti di chi haorganizzato. PartecipaSalute 2007Liberati A. Meno ricerca ma di migliore qualità. La dispensa di PartecipaSalute 2007;59-68Liberati A, Marsico G. Comitati etici: partecipare alla pari. La dispensa di PartecipaSalute 2007;91-100232ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESEducational and dissemination activitiesIn 2007 the Italian Cochrane Centre (ICC) participated in the organization of fifth year of theMaster’s program in Evidence Based Medicine (EBM) and health research methodology incollaboration with the University of Modena and Reggio Emilia in Italy. ICC organized andconducted a series of workshops about basic EBM concepts for healthcare decision-makingdirected toward general practitioners, specialists and doctors in training. These workshops wereheld at the Mario Negri Institute in Milan.In collaboration with AIFA and Zadig (a scientific publishing group), ICC has made asignificant contributions toward the ECCE project (continuing education centred on evidencebased medicine). The ECCE project was established in 2005 and promoted by AIFA as a part ofa large educational project for continuing medical education (CME) of all Italian doctors.Within the first year of the project, ECCE attracted 17,000 doctors (2005); by the end of 2007that number rose to 33000.AIFA and Zadig have also been partners of the ICC in the translation, adaptation, distributionand evaluation of Clinical Evidence - an international source of the best available evidence foreffective healthcare (published by BMJ publishing group). This past year we have been workingon the 6 th Italian edition of Clinical Evidence which corresponds to the 17th English edition.In collaboration with PartecipaSalute project, ICC began to translate and disseminate theCochrane Collaborations' press releases (plain language summaries of Cochrane SystematicReviews) to scientific journalists, doctors and consumersResearch activitiesICC researchers have collaborated in the production of 7 Cochrane Systematic Reviews and 7protocols currently available in the Cochrane Library.http://www3.interscience.wiley.com/cgibin/mrwhome/106568753/HOME?CRETRY=1&SRETRY=0ICC researchers are involved in methodological projects focused on the study of the quality ofSystematic Reviews.In 2007 ICC has collaborated with PartecipaSalute project. PartecipaSalute aims to involveconsumers and their disease-related associations with follow objectives:PartecipaSalute ("Participate in Health Care") is a project initiated at September 2003 to fostera strategic alliance between patients’ groups and professional societies with the common goalof promoting better health and shared decision-making. The project’s main aims are:- to increase patients’ associations’ involvement in healthcare assistance and in decisionmaking processes;- to promote a partnership between patients’ associations and medical societies solicitingmedical societies’ attention to patients’ need and perspectives- to develop a partnership between citizens, patients and healthcare system.The project developed a website - http://www.partecipasalute.it/cms/ - in order to offer criticaltools to read and understand medical and healthcare information, for better healthcaredecisions.233ANNUAL REPORT 2007

IRFMN234ANNUAL REPORT 2007

IRFMNTHE CATULLO AND DANIELABORGOMAINERIO CENTEROne of the buildings on the Mario Negri Institute campus is The Catullo and DanielaBorgomainerio Center built in 1987 thanks to a donation from Mrs. Angela MarchegianoBorgomainerio. This is a Center for the study of rare childhood diseases and even today some ofthe laboratories housed in the building still conduct this research. For example, the study of newtherapies used to treat a very rare form of acute myeloid leukemia, know as acute promyelocyticleukemia. A number of new studies are being done to identify new drugs having differentmechanisms able to synergize with trans retinoic acid.Research on epidemiological childhood leukemia is also done at the Borgomainerio and asimilar line of research involves testicular cancer in adolescents and young adults.Paediatric research activities done at the Borgomainerio Center are also performed incollaboration with groups located at other Institute locations including, The Aldo and CeleDaccò Center for Clinical Research on Rare Diseases at Ranica in Bergamo, the RegionalCentre for Drug Information (CRIF) and the Laboratory for Mother and Child Health which areboth located in Milan.235ANNUAL REPORT 2007

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IRFMNG.A. PFEIFFER MEMORIAL LIBRARYSTAFFHead LibrarianVanna Pistotti237ANNUAL REPORT 2007

IRFMNThe Library, specialised in pharmacology and clinical epidemiology, was founded in 1963thanks to a generous donation from the Gustavus and Louise Pfeiffer Research Foundation, inDenville, New Jersey, USA.Numerous public and private organisations help keep it operative, through donations in moneyor books, and subscriptions to periodicals.STAFFOne Head and two Assistants plus a clerical workerWHAT THE LIBRARY OFFERSThe library has a collection of about 5000 textbooks, monographs and congressionalproceedings, and 200 periodicals of which a major part are in an electronic format. The booksare classified according to the US National Library of Medicine Classification and the MedicalSubject headings of Medline (MeSH). Besides the internal collection, the Library has access tothe National Periodical Catalogue and to other Library systems (SBBL, GIDIF, RBM).DATABESES AND ELECTRONIC JOURNALSFrom every computer in the Institute it is now possible to have access to more than 2000electronic journals and to five of the most important databases: Medline, Cinhal, the CochraneLibrary and Embase.SPECIAL PROJECTSThe Library cooperates to the realisation of the Italian Information Specialists’ (GIDIF, RBM)journal catalog which is updated annually.It collaborates to the Institute web site, particularly taking care of the Publications section, bothscientific and lay press.TRAININGEvery year courses on the use of the database and electronic journals are organised. Thesecourses are designed for use by those working at the Institute but outsiders who are interestedmay attend.238ANNUAL REPORT 2007

IRFMN2006 PUBLICATIONSPistotti V, Colombo CNavigare sulla rete alla ricerca della buona informazionePartecipa Salute 2007Pistotti V, Santoro ENavigare sulla rete alla ricerca di informazioni di saluteIn ; La dispensa di Partecipasalute , 2007; 85-89Pistotti VPubMed: ma è davvero difficile usarlo al meglio?Ricerca & Pratica 2007 n.138 : 270-271CONTRACTSSince 1994 the library has been part of the Lombard Biomedical Library System. Sixteenuniversity and research organisation libraries in Lombardy take part in this project, whichallows easy, free access to scientific information to over 140 centres and institutions theLombardy Region.239ANNUAL REPORT 2007

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IRFMNNegri Bergamo LaboratoriesANNUALREPORT 2007departments and laboratories241ANNUAL REPORT 2007

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IRFMNDEPARTMENT OF MOLECULARMEDICINESTAFFHeadAriela BENIGNI, Biol.Sci.D., Ph.D.Unit of Gene TherapyHeadSusanna TOMASONI, Biol.Sci.D., Ph.D.Laboratory of Cell Biology and XenotransplantationHeadMarina MORIGI, Biol.Sci.D., Ph.D.Unit of Platelet-Endothelial Cell InteractionHeadMiriam GALBUSERA, Biol.Sci.D.Laboratory of Immunology and Genetics of Organ Transplantation andRare DiseasesHeadMarina NORIS, Chem.Farm.D., Ph.D.Unit of Cellular biology of Autoimmunity and Transplant RejectionHeadSistiana AIELLO, Biol.Sci.DUnit of Genetics of Rare DiseasesHeadJessica CAPRIOLI, Biol.Sci.DUnit of Cellular and Molecular Biology of Transplantation ToleranceHeadFederica CASIRAGHI, ChemistLaboratory of Experimental Models of Kidney DiseasesHeadCarla ZOJA, Biol.Sci.D., Ph.D.Unit of Pathology and ImmunophatologyHeadMauro ABBATE, M.D.243ANNUAL REPORT 2007

IRFMNCURRICULA VITAEAriela Benigni got the Biol.Sci. degree in 1979 at the University of Milano, Italy, and the Ph.D. atMaastricht University, Netherlands, in 2001.Educational training: in 1979 Post Doctoral Fellow, Istituto di Ricerche Farmacologiche Mario Negri(IRFMN), Laboratory of Cancer Chemotherapy, Milan, Italy; in 1980-1981 Post Doctoral Fellow,Associazione Bergamasca per lo Studio delle Malattie Renali, Laboratory of the Division of Nephrologyand Dialysis, Ospedali Riuniti di Bergamo, Italy; in 1982 Post Doctoral Fellow, Centre Regional deTransfusion Sanguigne de Strasbourg, France.Areas of interest: vasoactive and inflammatory mediators of progressive renal injury with a particularemphasis on endothelin-1; combined treatment of antipertensive and renoprotective drugs to haltprogressive renal injury; use of stem cells for tissue regeneration in acute renal failure in vivo e in vitrogene transfer; prevention of acute graft rejection through gene therapy; induction of kidney transplanttolerance by gene therapy; correction of genetic deficiency in rare diseases.Employement: in 1983 Scientist, IRFMN, Laboratory of Kidney Disease, Bergamo, Italy; in 1990-1994 Head Laboratory of Prostaglandin and Leukotriene Metabolism, IRFMN, Bergamo, Italy;from January 1991 Scientific Secretary, IRFMN, Bergamo, Italy; in 1994-1999 Head Laboratory ofVasoactive and Inflammatory Mediators of Tissue damage, IRFMN, Bergamo, Italy; from January2000 Head, Department of Molecular Medicine, IRFMN, Bergamo, Italy; from March 2007Consultant World Health Organization (WHO) for the multicentre observational study “Screeningfor Pre-eclampsia: evaluation of the predictive ability of angiogenic factors for Pre-eclampsia”;from September 2007 Senior Fellow at the University of Oxford, Nuffield Department of Obstetrics& Gynaecology.Selected publications:• G. Remuzzi, A. Benigni, A. Remuzzi. Mechanisms of progression and regression of renal lesions of chronicnephropathies and diabetes. J Clin Invest 2006;116:288-296.• Azzollini, M. Noris, S. Conti, M. Abbate, M. Giacca, G. Remuzzi. Adeno-Associated Virus-mediated CTLA4Ig genetransfer protects MHC-mismatched renal allografts from chronic rejection. J Am Soc Nephrol 2006;17:1665-1672.• B. Imberti, M. Morigi, S. Tomasoni, C. Rota, D. Corna, L. Longaretti, D. Rottoli, F. Valsecchi, A. Benigni, J. Wang, M.Abbate, C. Zoja, G. Remuzzi. Insulin-like growth factor-1 sustains stem cell-mediated renal repair. J Am Soc Nephrol2007;18:2921-2928.• C. Zoja, F. Casiraghi, S. Conti, D. Corna, D. Rottoli, R.A. Cavinato, G. Remuzzi, A. Benigni. Cyclin-dependent kinaseinhibition limits glomerulonephritis and extends lifespan of mice with systemic lupus. Arthritis & Rheum 2007;56:1629-37.• Benigni, C. Caroli, L. Longaretti, E. Gagliardini, C. Zoja, M. Galbusera, D. Moioli, P. Romagnani, A. Tincani, L.Andreoli, G. Remuzzi. Involvement of renal tubular toll-like receptor 9 in the development of tubulointerstitial injury insystemic lupus. Arthritis & Rheum 2007;56:1569-78.Marina Morigi got her Biol.Sci. degree in 1987 at the University of Milano, Milano, Italy and the Ph.D.at Maastricht University, Netherlands, in 2005.Educational training: in 1984-1987 Research training, IRFMN, Bergamo, Italy; in 1987 1995 PostDoctoral Fellow, IRFMN, Bergamo, Italy; in 1991 Stage at Brigham and Women’s Hospital, Laboratoryof Dr. P. Marsden, Boston, USA.Employement: since 1995 Scientist, IRFMN, Bergamo, Italy; in 1996-1999 Head, Unit of Renal andEndothelial Cell Biology; since 2000 Head, Laboratory of Cell Biology and Xenotransplantation,IRFMN, Bergamo, Italy.Areas of interest: role of Shigatoxin in the pathogenesis of endothelial dysfunction and microvascularthrombosis in Hemolytic Uremic Syndrome; in vitro model of hyperacute xenograft rejection (porcineendothelium exposed to human serum as a source of xenoreactive natural antibodies and complement);renal toxicity of the proteins filtered through the capillary barrier. In vitro model to study intracellularsignals, gene expression and production of inflammatory mediators in cultured proximal tubular cellsand glomerular epithelial cells; cell therapy and tissue regeneration: Capability of adult stem cells todifferentiate and to regenerate renal tissue in acute and chronic experimental models of renal disease.Murine embryonic stem cell therapy to correct the genetic defect characteristic of Fabry disease in anexperimental mouse model.244ANNUAL REPORT 2007

IRFMNSelected publications• M. Morigi, B. Imberti, C. Zoja, D. Corna, S. Tomasoni, M. Abbate, D. Rottoli, S. Angioletti, A. Benigni, N. Perico, M.Alison, G. Remuzzi. Mesenchymal Stem Cells Are Renotropic, Helping to Repair the Kidney and Improve Function inAcute Renal Failure. J Am Soc Nephrol 2004;15:1794-1804.• A. Remuzzi, S. Mantero, M. Colombo, M. Morigi, E. Binda, D. Camozzi, B. Imberti. Vascular smooth muscle cells onhyaluronic acid: culture and mechanical characterization of an engineered vascular construct. Tissue Eng 2004;10:699-710.• M. Morigi, S. Buelli, S. Angioletti, C. Zanchi, L. Longaretti, C. Zoja, M. Galbusera, S. Gastoldi, P. Mundel, G. Remuzzi,A. Benigni. In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implicationfor permselective dysfunction of chronic nephropathies. Am J Pathol 2005;166:1309-1320.• Morigi M, Buelli S, Zanchi C, Longaretti L, Macconi D, Benigni A, Moioli D, Remuzzi G, Zoja C. Shigatoxin-inducedendothelin-1 expression in cultured podocytes autocrinally mediates actin remodeling. Am J Pathol. 2006 Dec;169(6):1965-75.• Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, Rottoli D, Valsecchi F, Benigni A, Wang J, AbbateM, Zoja C, Remuzzi G. Insulin-like growth factor-1 sustains stem cell mediated renal repair. J Am Soc Nephrol. 2007Nov;18(11):2921-8.Marina Noris got her degree in Pharmaceutical Chemistry and Technologies in 1986 at the Universityof Rome “La Sapienza) and the Ph.D. at Maastricht University, Netherlands, in 2005.Educational training: in 1984-1986 Fellow, Istituto di Chimica Farmaceutica e Tossicologica, Universityof Rome, Italy; in 1986-1987 Post Doctoral Fellow, Istituto di Chimica Farmaceutica e Tossicologica,University of Rome, Italy; in September 1987-March 1994 Post Doctoral Fellow, IRFMN, Unit ofMediators of Inflammation and Tissue Damage, Laboratory of Kidney Disease, Bergamo, Italy.Areas of interest: immunology of transplantation, tolerance induction; genetics of hemolytic uremicsyndrome, thrombotic thrombocytopenic purpura, focal segmental glomerulosclerosis, diabeticnephropathy, role of nitric oxide and arginine dysfunctions in uremia and in pre-eclampsia.Employment: in 1994-1996 Head, Unit of Endothelial Cell Pathophysiology, IRFMN, Bergamo, Italy;1996-1999 Head, Laboratory of Cellular and Molecular Biology of the immune response andautoimmunity, IRFMN, Italy; from January 2000: Head, Laboratory of Immunology and Genetics of RareDiseases and Organ Transplantation, Department of Molecular Medicine, IRFMN, Bergamo, Italy.Selected publications• Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L, Gamba S, Porrati F,Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP, Remuzzi G. Genetics of HUS: the impactof MCP, CFH and IF mutations on clinical presentation, response to treatment, and outcome. Blood, 2006; 108(4):1267-79.• Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, Aiello S, Cassis L, Gotti E, Gaspari F, CattaneoD, Perico N, Remuzzi G. Regulatory T Cells and T Cell Depletion: Role of Immunosuppressive Drugs. J Am Soc Nephrol.2007; 18 (3):1007-1018.• Bresin E, Daina E, Noris M, Castelletti F, Stefanov R, Hill P, Goodship TH, Remuzzi G; International Registry of Recurrentand Familial HUS/TTP. Outcome of renal transplantation in patients with non-Shiga toxin-associated hemolytic uremicsyndrome: prognostic significance of genetic background. Clin J Am Soc Nephrol. 2006 Jan;1(1):88-99• Noris M, Bucchioni s, Galbusera M, Donadelli R, Bresin E, Castelletti F, Caprioli J, Brioschi S, Scheiflinger F, Remuzzi Gand the International Registry of Recurrent and Familial HUS/TTP. Complement factor H mutation in familial thromboticthrombocytopenic purpura with ADAMTS13 deficency and renal involvement. J Am Soc Nephrol 2005; 16:1177-1183• Manuelian T, Hellwage J, Meri S, Caprioli J, Noris M, Heinen S, Jozsi M, Neumann HP, Remuzzi G, Zipfel PF. Mutations infactor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in haemolytic uremicsyndrome. J Clin Invest 2003; 111: 1181-1190.• Noris M, Brioschi S, Caprioli J, Todeschini M, Bresin E, Porrati F, Gamba S, Remuzzi G, on behalf of the InternationalRegistry of Familial and Recurrent HUS/TTP. Familial haemolytic uraemic syndrome and an MCP mutation. Lancet 2003;362:1542-1547.Carlamaria Zoja got her Biol.Sci. degree at the University of Milano, Italy, in 1979 and the Ph.D. atthe University of Maastricht, The Netherlands in 2001.Educational Training: in 1979-1981 Post Doctoral Fellow, ‘Associazione Bergamasca per lo studio delleMalattie Renali’, Laboratory of the Division of Nephrology and Dialysis, Ospedali Riuniti di Bergamo,Italy; in 1981-1983 Post Doctoral Fellow, Center for Thrombosis and Vascular Research, Department ofResearch Katholieke Universiteit, Leuven, Belgium; in 1983-1985: Post Doctoral Fellow, IRFMN,Laboratory of Kidney Disease, Bergamo, Italy.Areas of interest: experimental models of kidney diseases of immunological and non immunologicalorigin; vasoactive and inflammatory mediators of renal disease progression; role of proteinuria inprogressive kidney damage; protection of renal disease progression by a multidrug approach; novel245ANNUAL REPORT 2007

IRFMNimmunosuppressive and anti-inflammatory strategies for the treatment of lupus nephritis; role ofShigatoxin in the pathogenesis of endothelial dysfunction in Hemolytic Uremic Syndrome.Employement: since 1985 Scientist, IRFMN, Bergamo, Italy; in 1990-1994: Head, Unit of ExperimentalModelling for Human Renal Diseases, Laboratory of Kidney Diseases, IRFMN, Bergamo, Italy; since1995: Head, Laboratory of Experimental Models of Kidney Diseases, IRFMN, Bergamo, Italy.Selected publications• C.Zoja, S. Angioletti, R. Donadelli, C. Zanchi, S. Tomasoni, E. Binda, B. Imberti, M. te Loo, L. Monnens, G.Remuzzi,M. Morigi. Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-kB dependent up-regulationof IL-8 and MCP-1. Kidney Int 2002;62:846-856.• C. Zoja, D. Corna, D. Camozzi, D. Cattaneo, D. Rottoli, C. Batani, C. Zanchi, M. Abbate, G. Remuzzi. How to fullyprotect the kidney in a severe model of progressive nephropathy: a multidrug approach. J Am Soc Nephrol2002;13:2898-2908.• A. Benigni, C. Zoja, D. Corna, C. Zatelli, S. Conti, M. Campana, E. Gagliardini, D. Rottoli, C. Zanchi, M. Abbate,S. Ledbetter, G. Remuzzi. Add-on anti-TGF-b antibody to ACE inhibitor arrests progressive diabetic nephropathy in therat. J Am Soc Nephrol 2003;14:1816-1824.• R. Donadelli, C. Zanchi, M. Morigi, S. Buelli, C. Batani, S. Tomasoni, D. Corna, D. Rottoli, A. Benigni, M. Abbate, G.Remuzzi, C. Zoja. Protein overload induces fractalkine upregulation in proximal tubular cells through NF-kB and p38MAPK dependent pathways. J Am Soc Nephrol 2003; 14:2436-2446.• C.Zoja, D. Corna, D. Rottoli, C. Zanchi, M. Abbate and G. Remuzzi. Imatinib ameliorates renal diseases and survivalin murine lupus autoimmune diseases. Kidney International 2006; 70; 97-103.• C.Zoja, F.Casiraghi, S.Conti, D.Corna, D.Rottoli, R.A.Cavinato, G.Remuzzi, A.Benigni. Cyclin-Dependent kinaseinhibition limits glomerulonephritis and extends lifespan of mice with systemic lupus. Arthritis & Rheumatism 2007;56; 1629-1637.Mauro Abbate obtained his M.D. degree in 1988 at the University of Brescia, Italy.Educational training: in 1984-1988 Graduate Student, IRFMN, Bergamo, Italy; in 1988-1992 PostDoctoral Fellow, IRFMN, Bergamo, Italy; in 1992-1994 Research Fellow, The Renal Unit,Massachusetts General Hospital, HMS, Boston, USA.Areas of interest: renal disease progression: the role of proteinuria, complement, and mediators of injuryin progressive kidney damage; mechanisms of glomerular injury; anti-GBM glomerulonephritis;mechanisms of tubular injury; kidney fibrosis; the renal biopsy; membranous nephropathy.Employement: in 1996 - 2000: Scientist, IRFMN, Bergamo, Italy; from 2000 Head, Unit of RenalPathology and Immunopathology, IRFMN, Bergamo, Italy.Selected publications• Abbate M, Zoja C, Rottoli D, Corna D, Tomasoni S, Remuzzi G: Proximal tubular cells promote fibrogenesis by TGFβ1-mediatedinduction of peritubular myofibroblasts. Kidney International 2002;61,2066-2077.• Abbate M, Zoja C, Morigi M, Rottoli D, Angioletti S, Tomasoni S, Zanchi C, Longaretti L, Donadelli R, Remuzzi G:Transforming Growth Factor-β1 Is Up-Regulated by Podocytes in Response to Excess Intraglomerular Passage ofProteins: A Central Pathway in Progressive Glomerulosclerosis. Am J Pathol.2002;161,2179-93.• Ruggenenti P, Chiurchiu C, Brusegan V, Abbate M, Perna A, Filippi C, Remuzzi G: Rituximab for idiopathicmembranous nephropathy: a one year prospective study. J Am Soc Nephrol. 2003; 14,1851-1857.• Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, Rottoli D, Angioletti S, Benigni A, Perico N, Alison M,Remuzzi G.:Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renalfailure. J Am Soc Nephrol. 2004;15:1794-804.• Macconi D, Abbate M, Morigi M, Angioletti S, Mister M, Buelli S, Bonomelli M, Mundel P, Endlich K, Remuzzi A,Remuzzi G.: Permselective dysfunction of podocyte-podocyte contact upon angiotensin II unravels the molecular targetfor renoprotective intervention. Am J Pathol. 2006 Apr;168(4):1073-85.• Abbate M, Zoja C, Remuzzi G.: How does proteinuria cause progressive renal damage? J Am Soc Nephrol. 2006;11:2974-84.Sistiana Aiello got the Biol.Sci. degree in 1993 at the University of Milano, Italy, and the Specializationin Pharmacology Research in 1996, at IRFMN, Bergamo, Italy.Educational training: in 1990-1993 research training, IRFMN, Bergamo; in 1993-2000 post doctoralfellow, IRFMN, Bergamo.Areas of interest: transplant immunology with a particular interest on dendritic cell biology andmechanisms by which T regulatory cells arise and work; in vitro and in vivo studies on new compoundswith immunosuppressive capacity or capable to prevent ischemia/reperfusion tissue injury; vasoactive andinflammatory mediators of progressive renal injury with a particular emphasis on platelet activating factor(PAF) and nitric oxide (NO).Employement: since 2000 Scientist within Laboratory of Immunology and Genetics of Rare disease and246ANNUAL REPORT 2007

IRFMNOrgan Transplantation; IRFMN, Bergamo; since 2006 Head, Unit of Cellular Biology of Autoimmunityand Transplant Rejection, IRFMN, Transplant Research Center “Chiara Cucchi de Alessandri e GilbertoCrespi”, Ranica.Selected publications:• S. Aiello, P. Cassis, L. Cassis, S. Tomasoni, A. Benigni, A. Pezzotta, R.A. Cavinato, D. Cugini, N. Azzollini, M. Mister,L. Longaretti, A.W. Thomson, G. Remuzzi, M. Noris. DnIKK2-transfected dendritic cells induce a novel population ofiNOS-expressing CD4 + CD25 - cells with tolerogenic properties. Transplantation 2007; 83:474-484.• S. Tomasoni, S. Aiello, L. Cassis, M. Noris, L. Longaretti, R.A. Cavinato, N. Azzollini, A. Pezzotta, G. Remuzzi, A.Benigni. Dendritic cells genetically engineered with adenoviral vector encoding dnIKK2 induce the formation of potentCD4 + T regulatory cells. Transplantation 2005;79:1056-1061.• L. Cassis, S. Aiello, M. Noris. Natural versus adaptive regulatory T cells. Contrib Nephrol 2005; 146: 121-131.• M. Mister, M. Noris, J. Szymczuk, N. Azzollini, S. Aiello, A. Arduini, L. Trochimowicz, E. Gagliardini, M. Abbate, N.Perico, G. Remuzzi. Propionyl-L-carnitine prevents renal function deterioration due to ischemia/reperfusion in isolatedperfused rat kidney and in kidney graft. Kidney Int 2002; 61:1064-1078.• S. Aiello, M. Noris, G. Piccinini, S. Tomasoni, F. Casiraghi, S. Bonazzola, M. Mister, M.H. Sayegh, G. Remuzzi.Thymic dendritic cells express inducible nitric oxide synthase and generate nitric oxide in response to self- andalloantigens. J Immunol 2000;164:4649-4658.Jessica Caprioli got the Biological Science degree in 1994 at the University of Pavia, Italy, and theSpecialization in Medical Genetics in 2001 at the University of Milano, Italy.Educational training: stage at St. John’s College in Cambridge for studies at the Department of Genetics,1994. Medical Research Assistant, Lundbeck Italia S.p.A, 1995. Training for Biological ScientistProfessional Qualification at the Laboratory of cellular and molecular biology of the immune responseand autoimmunity, IRFMN, Negri Bergamo Laboratories (qualification obtained in 1997). School ofProfessional Education in Pharmacological Research of Regione Lombardia (Specialization obtained in1998). Specialization School in Medical Genetics (University of Milano), obtained in 2001.Areas of interest: Role of the complement system in the pathogenesis of hemolytic uremic sindrome andthrombotic thrombocytopenic purpura. Research of the genes involved in the predisposition to focalsegmental glomerulosclerosis. Study of the genes that determine the onset of proteinuria in anexperimental animal model. Research of the genes involved in the pathogenesis of uric acidnephrolithiasis.Employement: 1995-2001 grant recipient at the Laboratory of cellular and molecular biology of theimmune response and autoimmunity, IRFMN, Negri Bergamo Laboratories. 2002-2006 researcher at theLaboratory of immunology and genetics of rare diseases and transplantation, IRFMN, Negri BergamoLaboratories. Since 2006 head of the Unit of genetics of renal diseases.Selected publications:• M. Galbusera, M. Noris, C. Rossi, S. Orisio, J. Caprioli, Z.M. Ruggeri, B. Amadei, P. Ruggenenti, B. Vasile, G. Casariand G. Remuzzi on behalf of the Italian Registry of Familial and Recurrent HUS/TTP. Increased fragmentation of vonWillebrand factor, due to abnormal cleavage of the subunit, parallels disease activity in recurrent hemolytic uremicsyndrome and thrombotic thrombocytopenic purpura (HUS/TTP) and discloses genetic predisposition in families. Blood,1999; 94: 1-12.• J. Caprioli, P. Bettinaglio, PF Zipfel, B. Amadei, E. Daina, S. Gamba, C. Skerka, N. Marziliano, G. Remuzzi and M.Noris on behalf of the Italian Registry of Familial and Recurrent HUS/TTP. The molecular basis of familial hemolyticuremic syndrome: mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. Journal of theAmerican Society of Nephrology, 2001; 12:297-307.• J. Caprioli, F. Castelletti, S. Bucchioni, P. Bettinaglio, E. Bresin, G. Pianetti, S. Gamba, S. Brioschi, E. Daina, G.Remuzzi and M. Noris. For the International Registry of Recurrent and Familial HUS/TTP. Complement Factor Hmutations and gene polymorphisms in hemolytic uraemic syndrome: the C-257T, the A2089G and the G2881Tpolymorphisms are strongly associated with the disease Human molecular genetics, 2003; 12:1-11.• M. Noris, S. Brioschi, J. Caprioli, M. Todeschini, E. Bresin, F. Porrati, S. Gamba and G. RemuzziFor the InternationalRegistry of Recurrent and Familial HUS/TTP. Mutation in membrane cofactor protein of the complement system infamilial hemolytic uraemic syndrome. Identification of a second disease-associated gene The Lancet, 2003; 362:1542-1547.• Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L, Gamba S, Porrati F,Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP, Remuzzi G. Genetics of HUS: theimpact of MCP, CFH and IF mutations on clinical presentation, response to treatment, and outcome. Blood, 2006;108(4):1267-79.247ANNUAL REPORT 2007

IRFMNFederica Casiraghi has obtained his degree in Industrial Chemistry in 1988, and the degree in ClinicalMonitoring and in Biochemical Research in 1993-1994 at IRFMN, Bergamo, Italy.Educational Training: 1989-1994 research fellow, IRFMN, Bergamo.Areas of interest: Transplant immunology with particular focus on pharmacological and cellular therapiesfor induction and maintenance of transplantation tolerance. Characterization of regulatory T cells in renaltransplant patients and in experimental models of allograft tolerance. Impact of differentimmunosuppressive drugs on T cell function in renal transplant patients. Vasoactive and inflammatorymediators of progressive renal injury with a particular emphasis on arachidonic acid metabolites.Employment: since 1994 Scientist within Laboratory of Immunology and Genetics of Rare Disease andOrgan Transplantation, IRFM, Bergamo; since 2006 Head, Unit of Cellular and Molecolar Biology ofTransplantation Tolerance, Transplant Research Center “Chiara Cucchi de Alessandri e Gilberto Crespi”,Ranica.Selected Publications:• Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, Aiello S, Cassis L, Gotti E, Gaspari F,Cattaneo D, Perico N, Remuzzi G. Regulatory T Cells and T Cell Depletion: Role of Immunosuppressive Drugs. J AmSoc Nephrol. 2007; 18 (3):1007-1018.• Cavinato RA, Casiraghi F, Azzollini N, Cassis P, Cugini D, Mister M, Pezzotta A, Aiello S, Remuzzi G, Noris M.Pretransplant donor peripheral blood mononuclear cells infusion induces transplantation tolerance by generatingregulatory T cells. Transplantation, 2005;79(9):1034-9.• Zoja C, Benigni A, Noris M, Corna D, Casiraghi F, Pagnoncelli M, Rottoli D, Abbate M, Remuzzi G. Mycophenolatemofetil combined with a cyclooxygenase-2 inhibitor ameliorates murine lupus nephritis. Kidney Int. 2001; 60(2): 653-63.• Tomasoni S, Noris M, Zappella S, Gotti E, Casiraghi F, Bonazzola S, Benigni A, Remuzzi G. Upregulation of renal andsystemic cyclooxygenase-2 in patients with active lupus nephritis.J Am Soc Nephrol.1998; 9(7): 1202-12.• Casiraghi F, Ruggenenti P, Noris M, Locatelli G, Perico N, Perna A, Remuzzi G. Sequential monitoring of urinesolubleinterleukin 2 receptor and interleukin 6 predicts acute rejection of human renal allografts before clinical orlaboratory signs of renal dysfunction. Transplantation 1997; 63(10): 1508-14.Miriam Galbusera got her Biol.Sci. degree in 1981 at the Università degli Studi di Milano.Educational training: in 1981-1983 Post Doctoral Fellow, Istituto di Patologia Speciale Medicadell'Università degli Studi di Milano, Italy; in 1985 - 1989 Post Doctoral Fellow, IRFMN, Bergamo,Italy; in 1989-1991 Post Doctoral Fellow at Scripps Clinic and Research Foundation, Laboratory ofThrombosis and Hemostasis, La Jolla, CA, USA; in 1991-1995 Post Doctoral Fellow, IRFMN, Bergamo,Italy.Areas of interest: ADAMTS-13 and VWF in thrombotic microangiopathies, VWF biochemistry,xenotransplantation, platelet-endothelial cell interaction under flow condition, platelet pathophysiology inuremia, receptor studies in kidney and platelets.Employement: 1995 - 1999: Scientist, IRFMN, Bergamo, Italy; from 2000 Head, Unit of Platelet-Endothelial Cell Interaction, IRFMN, Bergamo, Italy.Selected publications• Tripodi, A., Chantarangkul, V., Bohm, M., Budde, U., Dong, J.-F., Friedman, K.D., Galbusera, M., Girma, J.-P., Moake,J., Rick, M.E., Studt, J.-D., Turecek, P.L., Mannucci, P.M.: Measurement of von Willebrand factor cleaving protease(ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of codedplasmas. J Thromb Haemost 2004; 2:1601-1609.• Galbusera, M., Morigi, M., Buelli, S., Gastoldi, S., Macconi, D., Testa, C., Angioletti, S., Remuzzi, G.: Xenogeneicserum-induced thrombus formation on porcine endothelium is mediated by vitronectin receptor and P-selectin: role ofreactive oxygen species. Xenotransplantation 2005;12:110-120.• Ruiz-Torres, M.P., Casiraghi, F., Galbusera, M., Macconi, D., Gastoldi, S., Todeschini, M., Porrati, F., Belotti, D.,Pogliani, E.M., Noris, M., Remuzzi, G.: Complement activation: the missing link between ADAMTS13 deficiency andmicrovascular thrombosis of thrombotic microangiopathies. Thromb Haemost 2005; 93:443-452.• Noris, M., Bucchioni, S., Galbusera, M., Donadelli, R., Bresin, E., Castelletti, F., Caprioli, J., Brioschi, S., Scheiflinger,F., Remuzzi, G.: Complement factor H mutation in familial thrombotic thrombocytopenic purpura with ADAMTS13deficiency and renal involvement. J Am Soc Nephrol 2005; 16:1177-1183.• Galbusera, M., Bresin, E., Noris, M., Gastoldi, S., Belotti, D., Capoferri, C., Daina, E., Perseghin, P., Scheiflinger, F.,Fakhouri, F., Grunfeld, J-P., Pogliani, E., Remuzzi, G.: Rituximab prevents recurrence of thrombotic thrombocytopenicpurpura: a case report. Blood 2005;106:925-928.• Rieger, M., Mannucci, P.M., Kremer Hovinga, J.A., Herzog, A., Gerstenbauer, G., Konetschny, C., Zimmermann, K.,Scharrer, I., Peyvandi, F., Galbusera, M., Remuzzi, G., Böhm, M., Plaimauer, B., Lämmle, B., Scheiflinger, F.:ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases. Blood2005;106:1262-1267.248ANNUAL REPORT 2007

IRFMNSusanna Tomasoni got her Biological Science degree in 1991 at the University of Milan.Educational training: in 1989-1991 Graduate student, University of Milan; in 1991-1994 PhD student,University of Milan; in 1994 Research Fellow, Renal Division, Brigham & Women’s Hospital, HarvardMedical School, Boston, USA; in 1995 PhD degree in Physiological Science, University of Bologna;1995-1998: Post Doctoral Fellow, IRFMN, Bergamo, Italy.Areas of interest: construction of adenoviral vectors for gene therapy; gene transfer to the kidney in thecontext of transplantation; transfection of dendritic cell for cell therapy; progression of renal disease.Employement: in 1998-2000 Scientist, IRFMN, Bergamo, Italy; from 2000 Head, Unit of Gene Therapy,IRFMN, Bergamo, ItalySelected publications• Tomasoni S, Azzollini N, Casiraghi F, Capogrossi M C, Remuzzi G, Benigni A. CTLA4Ig gene transfer prolongssurvival and induces donor-specific tolerance in a rat renal allograft. J Am Soc Nephrol 2000; 11: 747-752.• Tomasoni S, Benigni A. Gene therapy: How to target the kidney. Promises and pitfalls. Curr Gene Ther 2004; 4: 115-122.• Tomasoni S, Longaretti L, Azzollini N, Gagliardini E, Mister M, Buehler T, Remuzzi G, Benigni A. Favorable effect ofcotransfection with TGF-beta and CTLA4Ig of the donor kidney on allograft survival. Am J Nephrol 2004; 24: 275-283• Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, Rottoli D, Angioletti S, Benigni A, Perico N, AlisonM, Remuzzi G. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renalfailure. J Am Soc Nephrol 2004; 15: 1794-1804• Tomasoni S, Aiello S, Cassis L, Noris M, Longaretti L, Cavinato R, Azzollini N, Pezzotta A, Remuzzi G, Benigni A.Dendritic cells genetically engineered with adenoviral vector encoding dnIKK2 induce the formation of potent CD4+ T-regulatory cells. Transplantation 2005; 79: 1056-1061.• Benigni A, Tomasoni S, Turka LA, Longaretti L, Zentilin L, Mister M, Pezzotta A, Azzollini N, Noris M, Conti S,Abbate M, Giacca M, Remuzzi G, Adeno-associated virus-mediated CTLA4Ig gene transfer protects MHC-mismatchedrenal allografts from chronic rejection. J Am Soc Nephrol, 2006, 17: 1665-72.249ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe Department of Molecular Medicine was established in 1999 at the Negri Bergamolaboratories to coordinate the work of three laboratories and three units. The activities of theDepartment of Molecular Medicine are strictly interrelated with those of the Department ofRenal Medicine of the Clinical Research Center for Rare Diseases Aldo e Cele Daccò.The following major objectives have been pursued:1) identification of mediators and mechanisms responsible for the relentless decline of renalfunction in kidney diseases and development of therapeutic interventions to slow or even haltthe disease progression to end-stage renal failure;2) understanding the mechanisms underlying endothelial cell dysfunction in thromboticmicroangiopathies and hyperacute rejection of xenograft3) finding new strategies for modulating the immune response and preventing acute and chronicrejection of kidney allograft as well as exploration of immunological pathways leading to donorspecific unresponsiveness and tolerance of the graft;4) investigation of the molecular and genetic basis of rare diseases such as hemolytic uremicsyndrome/thrombotic thrombocytopenic purpura and pre-eclampsia and search for diseasesusceptibilitygenes or gene polymorphisms predicting the patient's response to drug therapy inmore common and complex polygenic disorders.Such goals have been pursued using various approaches: 1) experimental models of kidneydiseases of immunological and non-immunological origin mimicking human renal diseases tostudy vasoactive and inflammatory mediators and to test novel antiproteinuric andrenoprotective drugs; 2) in vitro cultures of renal cells to address the toxicity of protein overloadreproducing the condition of exaggerated protein traffic of proteinuric progressivenephropathies; 3) in vitro models to assess the interaction of vascular endothelial cells withleukocytes and platelets under controlled flow conditions; 4) experimental models of kidneyallotransplant to study immunological processes responsible for acute and chronic rejection, thenephrotoxicity of immunosuppressor drugs as well as to explore pathways responsible foraccomodation; 5) gene transfer of viral constructs carrying genes encoding immunomodulatorymolecules to overcome acute rejection of allotransplantation avoiding immunosuppression; 6)identification of candidate genes with linkage analysis and search for mutations as well asassessment of gene polymorphisms.FINDINGS/MAIN RESULTSInsulin growth factor -1 produced by mesenchymal stem cells induces regeneration of proximaltubular cells in experimental acute kidney injuryBone marrow-derived human mesenchymal stem cells cure acute kidney injury and prolongsurvival in miceEndothelin -1 mediates renal cell damage induced by Shigatoxin-2, the bacterial toxinimplicated in the epidemic form of Hemolytic Uremic SyndromeComplement is responsible for trombus formation on microvascular endothelial cells inresponse to Shigatoxin250ANNUAL REPORT 2007

IRFMNA multidrug approach that simultaneously interrupts pathways of injury induces regression ofrenal lesions in experimental diabetesDNA contained in circulating immunocomplexes induces renal Toll-like receptor 9 expressionresponsible for tubulo-interstitial damage in experimental lupus nephritisThymic- and graft-dependent mechanisms in tolerance induction to kidneyallotransplantation in rats have been identifiedSeliciclib, an inhibitor of cell cycle, reduces lymphocyte alloreactivity and prolongskidney allograft survival in ratsThe R1210C mutation of complement factor H, highly prevalent in atypical HUS patients, isalso present as a rare polymorphism in different human populations and predisposes to thedevelopment of atypical HUSThe characterization of mutants in factor I gene has allowed to better understand the atypicalHUS pathogenesis in patients carrying this genetic defectCorrection of a protein deficiency by a gene therapy approach in geneticallymodified animalsNATIONAL COLLABORATIONSDipartimento di Scienze Farmacologiche, Università di MilanoDipartimento di Scienze Biomediche e Tecnologia, Università di MilanoLaboratorio di Terapia genica e cellulare, G. Lanzani, Divisione di Ematologia, Ospedali Riunitidi BergamoLaboratorio di Tecnologie riproduttive, Istituto Sperimentale Lazzaro Spallanzani, CremonaCentro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore, MilanoDipartimento di Istologia Microbiologia e Biotecnologie Mediche, Università di PadovaInternational Centre for Genetic Engineering and Biotechnology, Molecular Medicine Group,TriesteU.O. di Ostetricia e Ginecologia, Ospedale San Gerardo di MonzaU.O. di Ostetricia e Ginecologia, Azienda Ospedaliera Ospedali Riuniti di BergamoU.O. di Ostetricia e Ginecologia, Azienda Ospedaliera Spedali Civili di BresciaI.R.C.C.S. Policlinico San Matteo, PaviaAzienda Sanitaria Ospedaliera O.I.R.M. - S. Anna, TorinoIstituto Gaslini, Laboratorio di Nefrologia, GenovaINTERNATIONAL COLLABORATIONSAcademisch Ziekenhuis Maastricht, Interne Geneeskunde, Maastricht, NetherlandBeth Israel Deaconess Medical Center and Harvard Medical School, Boston, USACentro de Investigaciones Biologicas and Centro de Investigacion Biomedica en EnfermedadesRaras, Madrid, SpainChildren's Hospital and Regional Medical Center, University of Washington, Seattle, USADepartements of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, USA251ANNUAL REPORT 2007

IRFMNDeparment of Medicine, Division of Rheumatology, Washington University School ofMedicine, St. Louis, USAErasmus University of Rotterdam, NetherlandFlanders Interuniversity Institute for Biotechnology (VIB), University of Lund,SwedenHans-Knoll Institute for Natural Products Research, Jena, GermaniaInselspital, University of Bern, SwitzerlandINSERM, Paris, FranceInsitute of Human Genetics, University of Newcastle upon Tyne, UKMax Delbruck Center for Molecular Medicine, Berlin, GermanyMolecular Genetics and Rheumatology Section, Division of Medicine, Imperial College,London, UKMonash Medical Center, Melbourne, AustraliaNational Institute of Health, Bethesda, USAOsaka University School of Medicine, Osaka, JapanPediatric Nephrology and Hypertension, University of Utah, USARenal Transplant Unit, Hospital de Bellvitge, Barcelona, SpainRosalind Franklin University of Medicine and Science, Chicago, USAService d’Immunologie Biologique, Hopital Europeen Georges Pompidou, Paris, FranceThe Scripps Research Institute, La Jolla, USAUniversitaet Hamburg, Institut fur Molekulare Neuropathobiologie, Hamburg, GermanyUniversity of Colorado Cardiovascular Institute, Denver, USAUniversity of Groningen, NetherlandUniversity of Iowa, Department of Internal Medicine and Pediatrics, Iowa City, USAUniversity of Liverpool, School of Biological Sciences, UKUniversity of Oxford, UKUniversity of Pittsburgh School of Medicine, Pittsburgh, USAVanderbilt University School of Medicine, Nashville, USAWeizman Institute of Science, Rehovot, IsraelWorld Health Organization, Geneva, SwitzerlandEDITORIAL BOARD MEMBERSHIPJournal of American Society of Nephrology (Carla Zoja, Marina Noris)PEER REVIEW ACTIVITIESAmerican Journal of PathologyAmerican Journal of PhysiologyAmerican Journal of Physiology-Renal PhysiologyBloodBritish Journal of HaematologyCirculationDiabetesFebs LettersHypertensionKidney InternationalJournal of American Society of Nephrology252ANNUAL REPORT 2007

IRFMNJournal of Clinical InvestigationJournal of ImmunologyJournal of Molecular MedicineJournal of NephrologyNature GeneticsNature MedicineNephrologyNephrology, Dialysis and TransplantationNew England Journal of MedicinePediatric NephrologyPlosPARTICIPATION IN EVENTS IN WHICH THE DEPARTMENT WASINVOLVEDISN-Nature Genetics Forefronts Symposium on Nephrogenetics: from development tophysiology, Danver, MA, (USA), March 8-11 2007International Conference on Rare Diseases and Orphan Drugs, Roma, November 5-8 2007World Congress of Nephrology, Rio de Janeiro (Brazil), April 22-25 2007European Meeting on Complement in human disease, Cardiff (UK), September 8-11 2007Workshop on Thrombotic Microangiopathies, Jena (Germany), October 4-6 2007VIP meeting, Lovanio (Belgium), December 3, 2007Congresso della Società Italiana di Pediatria, Salerno, November 16-17 2007GENECURE Meetings, Stoccolma (Sweden), September 18-19 2007; Naarden (Netherland),January 26-27, 2007Meeting Update Meeting on the Relation of Immunity, its evolution and its effect on Solid,Organ Transplantation, Barcelona (Spain), July 6 2007.Meeting Proteinuria in Renal Transplantation pathophysiology, Diagnosis, Treatment, Firenze,March 21-22 2007.Tenth International Conference on Endothelin (ET-10), Bergamo, September 16-19 2007.3rd International Workshop on Thrombotic Microangiopathies, Jena (Germany), October 4-62007.XIV Telethon Scientific Convention. Salsomaggiore Terme, Italy, March 12-14, 2007.Annual meeting of the American Society of Nephrology, San Francisco, October 31-November5, 2007253ANNUAL REPORT 2007

IRFMN10 th International Symposium of Nephrology at Montecatini, Pistoia, March 22-24, 200733° Congresso SIF, Cagliari, June 6-9, 2007Corso Monografico “Cellule staminali in medicina rigenerativa e oncologia”, Collegio Ghisleri,Pavia, 15 February 2007Annual EuReGene Meeting, Bruxelles, February 1-4, 2007WCN 2007, Satellite Symposium, Stem Cells and renal diseases: potential use in kidney injury,“Potential of mesenchymal stem cells in the repair of tubular injury”, Foz do Iguaço, Paranà,Brazil, April 25-27, 2007GRANTS AND CONTRACTSAssociazione Progetto Alice ONLUSComitato Telethon Fondazione ONLUSCommissione Europea FP6Fondazione Aiuti per la Ricerca sulle Malattie Rare (ARMR)Fondazione ART per la Ricerca sui Trapianti ONLUSFondazione CariploFondazione La Nuova Speranza – Lotta alla Sclerosi focale ONLUSFondazione ROTRF/JDRFIstituto Superiore di SanitàACRAF (Aziende Chimiche Riunite Angelini Francesco Spa)EncysiveFarmaceutici Damor SpaGenzyme CorporationGiuliani SpaNovartis Farma SpaSanofi-Aventis SpaSpeedel Pharma LtdSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007G. Monteferrante, S. Brioschi, J. Caprioli, G. Pianetti, P. Bettinaglio, E. Bresin, G. Remuzzi, M. Noris. Geneticanalysis of the complement factor H related 5 gene in haemolytic uraemic syndrome. Mol Immunol 2007;44:1704-1708.S. Heinen, M. Jozsi, A. Hartmann, M. Noris, G. Remuzzi, C. Skerka, P.F. Zipfel. Hemolytic uremic syndrome: afactor H mutation (E1172Stop) causes defective complement control at the surface of endothelial cells. J Am SocNephrol 2007;18:506-514.M. Noris, F. Casiraghi, M. Todeschini, P. Cravedi, D. Cugini, G. Monteferrante, S. Aiello, L. Cassis, E. Gotti, F.Gaspari, D. Cattaneo, N. Perico, G. Remuzzi. Regulatory T cells and T cell depletion: role of immunosuppressivedrugs. J Am Soc Nephrol 2007;18:1007-1018.S. Aiello, P. Cassis, L. Cassis, S. Tomasoni, A. Benigni, A. Pezzotta, R.A. Cavinato, D. Cugini, N. Azzollini, M.Mister, L. Longaretti, A.W. Thomson, G. Remuzzi, M. Noris. DnIKK2-transfected dendritic cells induce a novel254ANNUAL REPORT 2007

IRFMNpopulation of inducible nitric oxide synthase-expressing CD4 + CD25- cells with tolerogenic properties.Transplantation 2007;83:474-484.A. Gianella, E. Nobili, M. Abbate, C. Zoja, P. Gelosa, L. Mussoni, S. Bellosta, M. Canavesi, D. Rottoli, U. Guerrini,M. Brioschi, C. Banfi, E. Tremoli, G. Remuzzi, L. Sironi. Rosuvastatin treatment prevents progressive kidneyinflammation and fibrosis in stroke-prone rats. Am J Pathol 2007;170:1165-1177.C. Zoja, F. Casiraghi, S. Conti, D. Corna, D. Rottoli, R.A. Cavinato, G. Remuzzi, A. Benigni. Cyclin-dependentkinase inhibition limits glomerulonephritis and extends lifespan of mice with systemic lupus. Arthritis & Rheum2007;56:1629-1637.A. Benigni, C. Caroli, L. Longaretti, E. Gagliardini, C. Zoja, M. Galbusera, D. Moioli, P. Romagnani, A. Tincani, L.Andreoli, G. Remuzzi. Involvement of renal tubular toll-like receptor 9 in the development of tubulointerstitial injuryin systemic lupus. Arthritis & Rheum 2007;56:1569-1578.N. Perico, M. Abbate, G. Remuzzi. More on renal disease progression: is interstitial inflammation truly protective?(Editorial) J Am Soc Nephrol 2007;18:1630-1632.P. Cravedi, M. Noris, G. Remuzzi. Report of the first World Transplant Congress. (Special Feature) Clin J Am SocNephrol 2007;2:393-400.D. Corna, F. Sangalli, D. Cattaneo, F. Carrara, F. Gaspari, A. Remuzzi, C. Zoja, A. Benigni, N. Perico, G. Remuzzi.Effects of rosuvastatin on glomerular capillary size-selectivity function in rats with renal mass ablation. Am JNephrol 2007;27:630-638.R.A. Cavinato, F. Casiraghi, N. Azzollini, M. Mister, A. Pezzotta, P. Cassis, D. Cugini, N. Perico, G. Remuzzi, M.Noris. Role of thymic- and graft-dependent mechanisms in tolerance induction to rat kidney transplant by donorPBMC infusion. Kidney Int 2007;71:1132-1141.D. Kavanagh, A. Richards, V. Fremeaux-Bacchi, M. Noris, T. Goodship, G. Remuzzi, J.P. Atkinson. Screening forcomplement system abnormalities in patients with atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol2007;2:591-596.J. Caprioli, G. Remuzzi. Complement hyperactivation may cause atypical haemolytic uraemic syndrome-gain-offunctionmutations in factor B. (Editorial) Nephrol Dial Transplant 2007;22:2452-2454.P. Ruggenenti, N. Perico, E. Gotti, P. Cravedi, V. D’Agati, E. Gagliardini, M. Abbate, F. Gaspari, D. Cattaneo, M.Noris, F. Casiraghi, M. Todeschini, D. Cugini, S. Conti, G. Remuzzi. Sirolimus versus cyclosporine therapyincreases circulating regulatory T cells, but does not protect renal tranplant patients given alemtuzumbab inductionfrom chronic allograft injury. Transplantation 2007;84:956-964.B. Imberti, M. Morigi, S. Tomasoni, C. Rota, D. Corna, L. Longaretti, D. Rottoli, F. Valsecchi, A. Benigni, J. Wang,M. Abbate, C. Zoja, G. Remuzzi. Insulin-like growth factor-1 sustains stem cell-mediated renal repair. J Am SocNephrol 2007;18:2921-2928.E. Gagliardini, A. Benigni. Therapeutic potential of TGF-β inhibition in chronic renalfailure. Expert Opin Biol Ther 2007;7:293-304.255ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Cell Biology and XenotransplantationInsulin-like growth factor-1 sustains stem cell-mediated renal recoveryfollowing an acute injuryIn collaboration with the Laboratory of Experimental Models of Kidney Diseases and with theUnit of Gene TherapyBone marrow-derived mesenchymal stem cells (MSC) can contribute to renal remodeling. Inmice with acute renal failure (ARF) induced by cisplatin, MSC administration restored renaltubular structure and improved renal function, but the underlying mechanism was unclear. Weexamined the process of kidney cell repair in a setting of MSC co-cultured with cisplatininjuredproximal tubular cells (PTEC). Exposure of PTEC to cisplatin led to a marked reductionin cell viability at 4 days. MSC protected PTEC from cisplatin-induced damage by promotingtubular cell proliferation , as assessed by BrdU labeling that marks cells during mitosis, andPKH26 red fluorescence cell linker. This effect was mediated by insulin-like growth factor-1(IGF-1), a mitogenic and anti-apoptotic factor, highly expressed by MSC as mRNA and protein,because functional blocking of the growth factor by specific antibody blocked cell proliferation.At variance, TGF- or IL-10 were not involved in cell mitosis. Knocking down IGF-1expression in MSC by small interfering(si)-RNA resulted in a significant decrease in PTECproliferation and increased apoptosis. Consistently, in the murine model of cisplatin-inducedARF infusion of MSC transfected with siRNA -IGF-1 limited their protective effect on renalfunction and tubular structure. These findings indicate that MSC exert beneficial effects ontubular cell repair in ARF by virtue of their capability of producing the mitogenic and prosurvivalfactor IGF-1.Life-saving effect of human bone marrow-mesenchymal stromal cells inexperimental acute renal injuryIn collaboration with the Laboratory of Experimental Models of Kidney Diseases and with theUnit of Gene TherapyTransplantation of bone marrow-mesenchymal stromal/stem cells (BM-MSC) from rodents hasbeen identified as a strategy for renal repair in experimental models of acute kidney injury(AKI), a highly life-threatening clinical setting. The therapeutic potential of BM-MSC of humanorigin has not been evaluated so far. The aim of the present study was to test whether humanBM-MSC treatment could prevent AKI induced by the nephrotoxic anticancer drug cisplatin,and prolong survival in an immunodeficient mouse model. Two groups of NOD-SCID micewere subcutaneously injected with cisplatin (12.7 mg/kg). Twenty-four hours later, the firstgroup was intravenously injected with saline, the second group with 5x10 5 BM-MSC. Resultsshowed that human BM-MSC infusion decreased proximal tubular epithelial cell injury andameliorated renal function resulting in a reduced recipient mortality. Here, finding PKH-26–fluorescent human BM-MSC in the proximity of tubular profiles and, less frequently, within thecontext of tubular epithelium supports the concept that MSC contributed locally to tubularregeneration via a paracrine mechanisms. Infused BM-MSC acted to significantly reduce renalapoptotic cells and to increase peritubular capillary diameter and both peritubular endothelialand lumen volume density, as documented by morphometric analysis. These findings indicatethat infusion of human MSC of bone marrow origin by preserving the integrity of the tubularepithelium and peritubular microvessels prolongs survival in AKI mice. Human BM-MSCrepresent an ideal cell population for future cell therapy and hold potential for successfulapplication in human AKI.256ANNUAL REPORT 2007

IRFMNShigatoxin-2 reduces nephrin gene expression via endothelin (ET)-1/ETAreceptor mechanism in cultured glomerular podocytesIn collaboration with the Laboratory of Experimental Models of Kidney Diseases and with theUnit of Gene TherapyShigatoxin (Stx) is the offending agent of post-diarrheal Hemolytic Uremic Syndrome (HUS), adisease characterized by microvascular thrombosis and glomerular ischemic changes thatcontribute to acute kidney injury. We have previously documented that glomerular podocytesare a functionally relevant target of Stx-2 that, via up-regulation of ET-1 gene and protein,promotes cytoskeletal changes and glomerular permeability dysfunction in an autocrine manner.Here, we studied whether Stx-2 altered in podocytes the expression of nephrin, key componentof the slit diaphragm, through an ET-1-dependent pathway. Exposure of murine differentiatedpodocytes to Stx-2 50 pM and 1 nM for 24 hours significantly reduced nephrin mRNA levels ina dose-dependent manner in respect to control cells (Stx-2 50pM: 0.6±0.04, Stx-2 1nM:0.3±0.06-fold vs control:1). Blockade of ETA receptor with LU302146 prevented Stx-2-dependent decline of nephrin expression, indicating the involvement of ET-1 via ETA receptor(Stx-2 50pM+LU: 0.9±0.1, Stx-2 1nM+LU: 0.9±0.1-fold vs control: 1). Consistent with Stx-2effect, ET-1 (100nM) added to podocytes decreased by 58% nephrin mRNA levels. Inhibitionof Rho kinase -crucial for the formation of cytoskeletal stress fibers- partially recovered nephrinmRNA levels in respect to Stx-2-treated cells. Injection of Stx-2 in mice caused podocytechanges including focal foot process effacement and increased expression of ET-1 associatedwith 30% reduction of nephrin protein expression in respect to control mice. In summary, ourdata show that ET-1 produced by podocytes in response to Stx, is an important mediator of Stxinducedreduction of nephrin expression, and might play a pivotal role in dysfunction of thefiltration barrier in HUS.Shiga toxin induces complement deposition on human microvascularendothelial cells.Shiga toxin (Stx) produced by E.coli is the main cause of post-diarrheal HUS (D+ HUS), adisorder of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure thatmainly affects children. We previously documented that Stx directly affected endothelial antithrombogenicproperties promoting thrombus formation on human microvascular endothelial cellsat high shear stress via upregulation of beta3 integrin and P-selectin. Evidence are availableshowing that P-selectin expressed on activated cells interacts with complement C3 component.In this study we investigated whether local activation of endothelial cells by Stx might promotecomplement deposition through P-selectin dependent pathway.Our results showed a marked increase of C3 deposits on cultured human microvascularendothelial cells (HMEC-1) incubated with Stx1 for 24 h and than perfused at high shear stress(60 dynes/cm 2 ) with human serum as a source of complement. Both Stx1 and Stx2 at theconcentration of 50 pM induced a strong granular C3 deposits uniformely distributed onendothelial surface. To exclude any possible effect of lipolysaccharide contamination of Stxpreparation, the effect of purified Stx1 (Stx1p) on C3 deposits was tested. Results showed thatStx1p induced similar increase in C3 deposits as compared to not purified Stx1.Since Stx induced the upregulation on endothelial surface of P-selectin, an adhesive protein thatmay directly interact with C3, we studied the role of this ligand in mediating endothelial C3deposition in response to Stx. Functional blocking of P-selectin with a specific antibody markedlyreduced C3 deposits induced by Stx on endothelial surface, indicating that Stx1 induced C3deposits via P-selectin expression on endothelial cells. C3 deposits induced by Stx on HMEC-1was completely abolished by treating human serum with the soluble complement receptor 1 (sCR-1), an inhibitor of both the classical and the alternative pathway of complement. Treatment withEGTA, an inhibitor of the classical pathway, did not prevent C3 deposition indicating that C257ANNUAL REPORT 2007

IRFMNactivation induced by Stx occurred via the alternative pathway. Further studies will be performedto establish whether there is a causative link between the activation of the alternative complementpathway and microvascular thrombosis in D+ HUS.Laboratory of Experimental Models of Kidney DiseasesInvolvement of renal tubular Toll-like receptor 9 in the development oftubulointerstitial injury in systemic lupusIn collaboration with the Unit of Gene TherapyToll-like receptor 9 (TLR-9), a receptor for CpG DNA, has been implicated in the activation ofimmune cells in lupus. We undertook this study to determine whether the expression of TLR-9in resident renal cells in lupus nephritis is related to the development of tubulointerstitial injury.TLR-9 was analyzed in selectively retrieved renal tissue from (NZB/W) F1 mice at differentstages of the disease by laser capture microdissection combined with real-time quantitativereverse transcriptase-polymerase chain reaction, and in renal biopsy specimens from lupusnephritis patients by immunohistochemistry. We investigated for the molecular componentresponsible for TLR-9 activation by cultured proximal tubular cells in serum from patients withlupus.Renal tissue from NZB/W mice displayed robust TLR-9 expression localized to proximaltubular cells. TLR-9 levels correlated with proteinuria and tubulo-interstitial injury to the extentthat seliciclib, a cyclin-dependent kinase inhibitor, while reducing proteinuria and renalstructural damage, prevented tubular TLR-9 generation in lupus mice. Consistently, exaggeratedTLR-9 staining was found in proximal tubular cells of lupus patients, which correlated withtubulo-interstitial damage. DNA-containing immune complexes purified from sera of patientswith lupus induced TLR-9 in cultured proximal tubular cells. This was prevented by CCGG-richshort oligonucleotides, specific antagonists of CpG DNA, indicating that the DNA componentof immune complexes was required for TLR9 stimulation.These findings suggest that tubular TLR-9 activation has a pathogenetic role in tubulointerstitialinflammation and damage in experimental and human lupus nephritis, and indicate anovel target for future therapies.Regression of podocyte lesions by a multidrug approach in experimentaldiabetesThe degree of renoprotection afforded by renin-angiotensin system (RAS) inhibitors inprogressive nephropathies crucially depends on the time at which therapy is started, with loweffectiveness provided by late treatment. Here, we used a rat model of diabetic nephropathy toassess the effect of maximal RAS inhibition by angiotensin converting enzyme inhibitor (ACEi)plus angiotensin II receptor antagonist (ATIIra) combined with statin that displays pleiotropicproperties complementing cholesterol lowering effects. Diabetes was induced inuninephrectomized rats by streptozotocin. Animals were orally treated from 4 to 8 months afterdisease induction with vehicle, lisinopril plus candesartan, lisinopril+candesartan+rosuvastatinor rosuvastatin. Six healthy rats served as control. Blood pressure increased during time indiabetic rats and was significantly reduced by treatment with RAS inhibitors and triple therapy.ACEi+ATIIra significantly reduced proteinuria in respect to vehicle; remission of proteinuria tocontrol levels was achieved by the addition of rosuvastatin. Glomerulosclerosis was amelioratedby ACEi+ATIIra, but totally prevented by triple therapy. Lower podocyte number perglomerulus in diabetic rats on vehicle was partially restored by ACEi+ATIIra while normalizedby combining the three drugs. Staining with nephrin, key functional component of the slitdiaphragm, was significantly decreased in diabetic rats as compared to controls and normalizedby triple therapy.258ANNUAL REPORT 2007

IRFMNThis multidrug approach might represent a strategy to induce remission of proteinuria andregression of renal lesions in diabetic patients who do not fully benefit RAS inhibition.Laboratory of Immunology and Genetic of Rare Diseases andOrgan TransplantationRole of thymic- and graft-dependent mechanisms in tolerance inductionto rat kidney transplant by donor PBMC infusionWe previously demonstrated the presence of regulatory T cells (Treg) in lymph nodes (LN)from rats made tolerant to a kidney allograft by donor PBMC infusion. Here we investigatedthe origin of Treg and characterized their phenotype and mechanisms underlying theirsuppressive effect. At different points after PBMC infusion, thymus, LN and graft-infiltratinglymphocytes(GIL) alloreactivity was evaluated in MLR, coculture and transwell experiments.GIL phenotype (by FACS and immunohistochemistry) and cytokines mRNA expression wereanalyzed. Before transplantation, CD4 + thymocytes and LN cells from donor PBMC-infusedrats showed a reduced anti-donor but a normal anti-third-party proliferation. Anti-donorhyporesponsiveness was reverted by IL-2. CD4 + thymocytes had no regulatory activity on anaive MLR. Treg appeared in LN at 60 days post-transplant. CD4 + -GIL isolated early (5 days)and late post-transplant (days 60-80) were hyporesponsive and suppressed a naïve MLR. IL-10mRNA was up-regulated in GIL and an anti-IL-10 mAb reverted their inhibitory effect. Cellto-cellcontact potentiated the suppressive activity of CD4 + -GIL. We suppose that allografttolerance in this model is mediated by pre-transplant generation of anergic cells in the thymus,which may have a permissive role to prevent early graft disruption. The healed graft is a sourceof donor antigens which lead to early selection of Treg. In the late phase tolerance ismaintained by appearance of Treg in LN.Effect of seliciclib (CYC202, R-roscovitine) on lymphocyte alloreactivityand acute kidney allograft rejection in ratT cell stimulation by alloantigens is followed by cell cycle progression, an event that iscritically dependent on cyclin-dependent kinases (CDKs). We conducted a study to evaluatewhether the CDK inhibitor seliciclib affected rat lymph node cell (LNc) activation andproliferation induced by either concanavalin A or allogeneic splenocytes in vitro and studiedthe mechanisms underlying the suppressive effect. We also investigated theimmunosuppressive properties of seliciclib in vivo.Seliciclib completely inhibited in vitro proliferation of LNc and CD8 + Tcells, in response toeither concanavalin A or allogeneic splenocytes. The percentage of activated LNc was lower inMLR added with seliciclib than in MLR added with vehicle. The percentages of viable andapoptotic cells at the end of MLR with seliciclib were comparable to those of MLR withvehicle. LNc pre-exposed in MLR to seliciclib did not respond to further stimulation withalloantigens, and neither IL-2 nor IL-15 restored proliferation. These data indicate that theinhibitory effect of seliciclib on T cell alloreactivity is not due to cytotoxic effect but isassociated with induction of profound T cell anergy. LNc harvested at the end of the primaryMLR with seliciclib did not suppress the proliferation of syngeneic LNc cells toward allogeneicsplenocytes, thus excluding that seliciclib induced the formation of regulatory cells. Finally,seliciclib partially prolonged grafted animal survival in a rat model of fully MHC-mismatchedkidney transplantation.Altogether these results document that seliciclib regulates lymphocyte reactivity and may exertan immunosuppressive effect in vivo in the setting of transplantation.259ANNUAL REPORT 2007

IRFMNCharacterisation of mutations in complement Factor I (CFI) associatedwith Hemolytic Uremic SyndromeThe hemolytic uraemic syndrome (HUS) is characterized by the triad of thrombocytopenia,microangiopathic haemolytic anaemia and acute renal failure. Cases not associated with apreceding Shiga-like toxin producing E.coli are described as atypical HUS (aHUS). Recentstudies have identified mutations in the complement regulatory proteins factor H (CFH),membrane cofactor protein (MCP) and factor-I (CFI) that predispose to aHUS.CFI is a two chain serine protease in which the light chain carries the catalytic domain whilethe heavy chain’s (55 kDa) function is unclear. It downregulates the alternative and classicalcomplement pathways by cleaving the α chains of C3b and C4b in the presence of cofactors(known as cofactor activity). Many CFI mutations in aHUS result in low CFI levels with aconsequent quantitative defect in complement regulation. In others, the mutant protein ispresent in normal amounts but the presumed functional deficiency has not yet been defined. Inthis report we examine the nature of the functional defect in HUS-associated CFI mutations.The D501N and D506V mutations reside in the serine protease domain of CFI and result insecreted proteins which lack C3b and C4b cofactor activity. The delTTCAC (1446-1450)mutant, also in the serine protease domain, leads to a protein which is not secreted. The R299Wmutant lies in a region of the CFI heavy chain of no known function. Our assessmentsdemonstrate decreased C3b and C4b cofactor activity, providing evidence that this region isimportant for cofactor activity. In two other heavy chain mutants, no function deficiency wasfound.These defective mutant proteins will result in an inability to appropriately control thecomplement cascade at sites of endothelial cell injury. The excessive complement activation fora given degree of damage may result in generation of a pro-coagulant state and aHUS.R1210C, a prevalent mutation in complement factor h associated withatypical Haemolytic Uremic SyndromeMutations in the gene encoding complement factor H that alter the C3b/polyanions-binding siteat the C-terminal region of the protein impair the capacity of factor H to protect host cells andare strongly associated with atypical haemolytic uremic syndrome (aHUS). Most of theseaHUS-associated CFH mutations are ‘unique mutations’ normally found in only a singlepatient or family. One exception is the CFH mutation that has been found in severalR1210Cunrelated aHUS patients from distinct geographical origins. Here, 5 aHUS pedigrees and 7individual aHUS patients (represented by 13 affected and 16 healthy CFH R1210C mutationcarriers) were analyzed to identify potential correlations of the CFH R1210C mutation withparticular clinical phenotypes and to characterize the origins of this mutation. Our resultsshowed that the clinical phenotype of aHUS patients carrying the CFH R1210C mutation washeterogeneous. Interestingly, 12 out of 13 patients carry at least one additional genetic factorconferring predisposition to aHUS. These data are in accord with the low penetrance of aHUS(30%) in the CFH R1210C mutation carriers, indicating that the presence of other genetic orenvironmental risk factors significantly contribute to the manifestation and severity of aHUS inCFH R1210C mutation carriers. Genotype analysis of CFH and CFHR3 SNPs in the 12unrelated carriers suggests that the CFH R1210C mutation has a single origin. In conclusion,CFH R1210C is a prevalent and prototypic aHUS mutation that may be present, as a rarepolymorphism, in many human populations.260ANNUAL REPORT 2007

IRFMNUnit of Gene TherapyGene therapy to correct Thrombotic Thrombocytopenic PurpuraThrombotic Thrombocitopoenic Purpura (TTP) is a rare disease with an incidence of 2:100000per year. TTP is a thrombotic microangiopathy, characterized by anemia and thrombocytopeniacaused respectively by red blood cells fragmentation in the microcirculation and bymicrovascular thrombi formation. TTP patients show prevalent, but not exclusive, neurologicalsymptoms and renal dysfunction. They have plasma accumulation of von Willebrand multimerswith a molecular weight higher than normal due to the lack of activity, acquired or familiar, ofADAMTS13, a plasma protein that cleaves vWF secreted by endothelial cells. vWF ultralargemultimers are more adhesive for platelets than the normal counterpart, causing their depositionand thrombi formation. TTP treatment is based on plasma exchange. Although this therapy hasincreased considerably the survival (from 10% to 80-90%) it may expose patients to the risk ofinfections or major complications such as hemorrhage due to the insertion of the central venouscatheter, venous thrombosis and hypotension equiring dopamine. In patients with TTP,recurrencies of the disease are common and relapsing TTP is a debilitating and life-threateningdisease. For these reasons researchers are looking for alternative therapies. Gene therapy couldbe useful since ADAMTS13 activity levels of 5-6% are protective.In order to transfer genes we took advantage of viruses unable to replicate and to induce diseasebut still able to infect cells. We constructed two different viral vectors, adenoviral (Ad) andadeno-associated (AAV), containing ADAMTS13 gene. RT-PCR and Western Blot analysisshowed that they infect cells efficiently and induce ADAMTS13 mRNA and protein expression.Adenoviral vector induces the secretion of the functional active protein as revealed by aCollagen Binding Assay.Recently ADAMTS13 knock out mice were generated. These mice are healthy but, if crossedwith mice expressing high levels of vWF and exposed to Shiga-toxin produced by E.Coli, showa syndrome similar to TTP. Studies are now in progress in order to establish the best vector ableto induce in vivo expression of appreciable levels of ADAMTS13 (5-6% at least) functionallyactive and to correct the disease.261ANNUAL REPORT 2007

IRFMN262ANNUAL REPORT 2007

IRFMNDEPARTMENT OF BIOMEDICALENGINEERINGSTAFFHead Andrea REMUZZI, Eng. D.Laboratory of Renal BiophysicsHeadDaniela MACCONI, Biol.Sci.D.Laboratory of Biomedical TechnologiesHeadBogdan ENE-IORDACHE, Eng.D.Unit of Tissue EngineeringHeadMarina FIGLIUZZI, Biol.Sci.D.Unit of Medical ImagingHeadLuca ANTIGA, Ph.D.263ANNUAL REPORT 2007

IRFMNCURRICULA VITAEAndrea Remuzzi got his degree in Mechanical (Biomedical) Engineering in 1979, Politecnico di Milano.Research experience: 1980 Politecnico di Milano, Dipartimento di Ingegneria Biomedica; 1981 IstitutoMario Negri (Milano), Laboratorio di Farmacologia Cardiovascolare; 1982-83 Massachusetts Institute ofTechnology, Mechanical Engineering Department, Cambridge, USA.Areas of interest: biological transport phenomena, mathematical models, renal pathophysiology, cellularresponse to mechanical stimulation, tissue engineering, pancreatic islet transplantation, clinical databases,computational fluid dynamics.Chronology of appointment: From 1984 to 1986 Ricercatore Istituto Mario Negri (Bergamo), Laboratoriodi malattie renali, 1986-1989 Head, Unità di Bioingegneria, Istituto Mario Negri, 1989-1993 Head,Laboratorio di Bioingegneria, Istituto Mario Negri, 1993-1999 Head, Dipartimento di Ricerca Renale,Istituto Mario Negri, from 2000 Head, Dipartimento di Bioingegneria, Istituto Mario Negri. Since 1998contract professor of Bioengineering, Politecnico di Milano.Selected publications• Davies PF, Remuzzi A, Gordon EJ, Dewey CF Jr, Gimbrone MA Jr. Turbulent fluid shear stress induces vascularendothelial cell turnover in vitro. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2114-7. PMID: 3457378• Remuzzi A, Puntorieri S, Battaglia C, Bertani T, Remuzzi G. Angiotensin converting enzyme inhibition amelioratesglomerular filtration of macromolecules and water and lessens glomerular injury in the rat. J Clin Invest. 1990Feb;85(2):541-9. PMID: 1688888• Morigi M, Micheletti G, Figliuzzi M, Imberti B, Karmali MA, Remuzzi A, Remuzzi G, Zoja C. Verotoxin-1 promotesleukocyte adhesion to cultured endothelial cells under physiologic flow conditions. Blood. 1995 Dec 15;86(12):4553-8.PMID: 8541545• Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, Orisio S, Remuzzi G, Remuzzi A. Nitric oxidesynthesis by cultured endothelial cells is modulated by flow conditions. Circ Res. 1995 Apr;76(4):536-43. PMID: 7534657• Giavazzi R, Foppolo M, Dossi R, Remuzzi A. Rolling and adhesion of human tumor cells on vascular endothelium underphysiological flow conditions. J Clin Invest. 1993 Dec;92(6):3038-44. PMID: 7504697• Antiga L, Ene-Iordache B, Remuzzi A. Computational geometry for patient-specific reconstruction and meshing of bloodvessels from MR and CT angiography. IEEE Trans Med Imaging. 2003 May;22(5):674-84. PMID: 12846436• Remuzzi A, Gagliardini E, Sangalli F, Bonomelli M, Piccinelli M, Benigni A, Remuzzi G. ACE inhibition reducesglomerulosclerosis and regenerates glomerular tissue in a model of progressive renal disease. Kidney Int. 2006 Jan 4;PMID: 16395266Daniela Macconi got her Biol.Sci.D. degree in Milan in the 1983.Research experience: 1977-81 CNR Institute of Neuroscience - Cell Mol Pharmacology - and Departmentof Medical Pharmacology, University of Milan, Milan, Italy;1982-83 Laboratory of the Division ofNephrology and Dialysis, Ospedali Riuniti di Bergamo, Bergamo, Italy; 1984-85 University of Michigan,Medical School, Department of Pathology, Medical Science I, Ann Arbor Michigan, USA; 1985-89Mario Negri Institute for Pharmacological Research, Laboratory of Kidney Disease, Bergamo, Italy.Areas of interest: glomerular permeability, renal disease progression, podocytes, angiotensin II, reactiveoxygen speciesChronology of appointment: From 2000 Head Laboratory of Renal Biophisics, Department ofBiomedical Engineering; 1994-2000 Head, Unit of Inflammatory Mediator of Leucocyte Origin; 1989- 94Scientist, 1985-89 post-doctoral fellow Mario Negri Institute for Pharmacological Research, Bergamo,Italy; 1982-83 fellow Laboratory of the Division of Nefrology e Dialysis, Ospedali Riuniti di Bergamo,Bergamo, ItalySelected publications• Macconi D, Abbate M, Morigi M, Angioletti S, Mister M, Buelli S, Bonomelli M, Mundel P, Endlich K, Remuzzi A,Remuzzi G: Permselective dysfunction of podocyte-podocyte contact upon angiotensin II unravels the molecular target forrenoprotective intervention. Am J Pathol.168:1073-85, 2006.• Macconi D, Bonomelli M, Benigni A, Plati T, Sangalli F, Longaretti L, Conti S, Kawachi H, Hill P, Remuzzi G, RemuzziA. Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury. Am JPathol.168:42-54, 2006.• Ruiz-Torres MP, Casiraghi F, Galbusera M, Macconi D, Gastoldi S, Todeschini M, Porrati F, Belotti D, Pogliani EM, NorisM, Remuzzi G: Complement activation: the missing link between ADAMTS-13 deficiency and microvascular thrombosisof thrombotic microangiopathies. Thromb Haemost. 93:443-52, 2005.• Galbusera M, Buelli S, Gastoldi S, Macconi D, Angioletti S, Testa C, Remuzzi G, Morigi M: Activation of porcineendothelium in response to xenogeneic serum causes thrombosis independently of platelet activation. Xenotransplantation.12:110-20, 2005.264ANNUAL REPORT 2007

IRFMN• Morigi M, Macconi D, Zoja C, Donadelli R, Buelli S, Zanchi C, Ghilardi M, Remuzzi G: Protein overload-induced NFkappaBactivation in proximal tubular cells requires H(2)O(2) through a PKC-dependent pathway. J Am Soc Nephrol.13:1179-89, 2002• Macconi D, Ghilardi M, Bonassi ME, Mohamed EI, Abbate M, Colombi F, Remuzzi G, Remuzzi A: Effect of angiotensinconvertingenzyme inhibition on glomerular basement membrane permeability and distribution of zonula occludens-1 inMWF rats. J Am Soc Nephrol 11:477-89, 2000Bogdan Ene-Iordache got MSc in Mechanical Engineering in 1990 at the Oil & Gas Institute in Ploiesti(Romania). In 1992 he joined the Bioengineering Laboratory at NegriBERGAMO Laboratories.Main interests: renal research (hemodynamics and remodeling of arteriovenous fistula for vascular access,morfometrical analysis of glomerular capillaries) and controlled clinical trials (data management and dataanalysis for controlled clinical studies); coordination of IT activities and web sites in the ClinicalResearch Center for Rare Diseases ‘Aldo e Cele Daccò’; applied clinical informatics (Nephrology,Diabetology and Hematology Units, Bergamo Hospital).Roles: since January 2000 is head of the Biomedical Technologies Laboratory, Department of BiomedicalEngineering.Selected publications• Ene-Iordache B, Imberti O, Foglieni O, Remuzzi G, Bertani T and Remuzzi A. Effects of angiotensin-converting enzymeinhibition on glomerular capillary wall ultrastructure in MWF/Ztm rats. J Am Soc Nephrol 5: 1378-1384, 1994.• Ene-Iordache B and Remuzzi A. Numerical analysis of blood flow in reconstructed glomerular capillary segments.Microvasc Res 49: 1-11, 1995.• Remuzzi A and Ene-Iordache B. Capillary network structure does not affect theoretical analysis of glomerular sizeselectivity. Am J Physiol 268: F972-F979, 1995.• Ene-Iordache B, Mosconi L, Remuzzi G, Remuzzi A. Computational fluid dynamics of a vascular access case forhemodialysis. J Biomech Eng 123(3): 284-292, 2001.• Ene-Iordache B, Mosconi L, Antiga L, Bruno S, Anghileri A, Remuzzi G, Remuzzi A. Radial artery remodeling in responseto shear stress increase within arteriovenous fistula for hemodialysis access. Endothelium 10(2): 95-102, 2003.• Ruggenenti P, Fassi A, Ilieva AP, Bruno S, Iliev IP, Brusegan V, Rubis N, Gherardi G, Arnoldi F, Ganeva M, Ene-IordacheB, Gaspari F, Perna A, Bossi A, Trevisan R, Dodesini AR, Remuzzi G for the Bergamo Nephrologic DiabetesComplications Trial (BENEDICT) Investigators. Preventing microalbuminuria in type 2 diabetes. NEJM 351(19): 1941-1951, 2004.Marina Figliuzzi got her Biol.Sci.D. degree in Milan in the 1991.Research experience :1991-94 Mario Negri Institute for Pharmacological Research, Bergamo, Italy.Areas of interest: isolation of pancreatic islets from human, bovine , pig and rat pancreas, cell culture,immunoisolation devices for pancreatic islets, differentiation of progenitor pancreatic cells in insulincontaining cells, immunhistochemistry.Chronology of appointment: From 2000 Head Unit of Tissue Engineering, Department of BiomedicalEngineering; 1991-2000 fellow laboratory of Renal research, Mario Negri Institute for PharmacologicalResearch, Bergamo, Italy.Selected publications• Figliuzzi M, Cornolti R, plati T, Rajan N, Adobati F, Remuzzi G, Remuzzi A: Subcutaneous xenotransplantation of bovinepancreatic islets. Biomaterials. 26:5640-47, 2005.• Figliuzzi M, Zappella S, morigi M, Rossi P, marchetti P, Remuzzi A: Influence of donor age on bovine pancreatic isletisolation. Transplantation. 70:1032-37, 2000• Morigi M, Micheletti G, Figliuzzi M, Imberti B, Karmali MA, Remuzzi A, Remuzzi G, Zoja C. Verotoxin-1 promotesleukocyte adhesion to cultured endothelial cells under physiologic flow conditions. Blood.: 86 4553-58, 1995.• Zoja C, Morigi M, Figliuzzi M, Bruzzi I, Oldroyd S, Benigni A, Ronco P, Remuzzi G. Proximal tubular cell synthesis andsecretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis.26: 934-41, 1995.• Morigi M, Zoja C, Figliuzzi M, Foppolo M, Micheletti G, Bontempelli M, Saronni M, Remuzzi G, Remuzzi A. Fluid shearstress modulates surface expression of adhesion molecules by endothelial cells. Blood. 85:1696-703, 1995.• Morigi M, Zoja C, Figliuzzi M, Remuzzi G, Remuzzi A. Supernatant of endothelial cells exposed to laminar flow inhibitsmesangial cell proliferation. Am J Physiol. 264:C1080-3, 1993.Luke Antiga graduated in 1999 in Biomedical Engineering and got his PhD in Bioengineering in 2003,Politecnico di Milano, having worked at the research laboratory of Biomedical Technology, Departmentof Bioengineering Institute Mario Negri.Training activities: 2003 Post-doctoral fellow at Imaging Research Laboratories, Robarts ResearchInstitute, London, Ontario.265ANNUAL REPORT 2007

IRFMNAreas of interest: acquisition and image processing for medical and microscopy applications, numericalmodeling of transport phenomena.Roles: from 2000 to 2002 Doctoral Student at the Laboratory of Biomedical Technology, Department ofBioengineering, from 2002 to 2003 visiting scientist Robarts Institute for medical Imaging, London,Ontario, Canada, from 2004 to 2006 resercher at the Laboratory of Biomedical Technology, Departmentof Bioengineering, from 2007 Head Unit of Medical Imaging, Department of Bioengineering.Selected publications• Lee SW, Antiga L, Spence JD and Steinman DA. Geometry of the carotid bifurcation predicts its exposure to disturbedflow. Stroke, in press, 2008.• Antiga L, Piccinelli M, Fasolini G, Ene-Iordache B, Ondei P, Ruggenenti P, Remuzzi G and Remuzzi A. Computedtomography evaluation of ADPKD progression: a progress report. Clinical Journal of the American Society of Nephrology(CJASN), 1(4): 754-760, Jul 2006.• Thomas JB, Antiga L, Che S, Milner JS, Hangan Steinman DA, Spence JD, Rutt BK and Steinman DA. Variation in thecarotid bifurcation geometry of young vs. older adults: Implications for "geometric risk" of atherosclerosis. Stroke, 36(11):2450-2456, Nov 2005.• Antiga L, Steinman DA. Robust and objective decomposition and mapping of bifurcating vessels. IEEE Transactions onMedical Imaging, 23(6): 704-713, June 2004.• Antiga L, Ene-Iordache B and Remuzzi A. Computational geometry for patient-specific reconstruction and meshing ofblood vessels from MR and CT angiography. IEEE Transactions on Medical Imaging, 22(5): 674-684, May 2003.• Antiga L, Ene-Iordache B, Remuzzi G and Remuzzi A. Automatic generation of glomerular capillary topologicalorganization. Microvascular Research, 62: 346-354, June 2001.266ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe research activity of the Department of Biomedical Engineering is aimed to the applicationof engineering techniques to the study of biological processes, pathological conditions and forthe development of innovative therapeutic strategies. Research activities in progress, in basicand applied research, are based on theoretical models, digital histological analysis, medicalimage processing for the quantification of three-dimensional structures at macro- andmicroscopic level, direct measure of physical-chemical parameters at experimental and clinicallevel, cell culture and computer based data management. There are mainly four areas of researchat the moment: the study of renal disease progression at experimental and clinical level,investigation of vascular flow dynamics, cell culture for in vitro tissue engineering and thedevelopment of information systems for clinical data management in clinical trials and inconventional medical activities.FINDINGS/MAIN RESULTSEvidence has been produced showing that the arterial vessels geometry and haemodynamicconditions influence the localization of cerebral aneurysms. These results open new ground inthe understanding of the mechanisms responsible for this vascular disease.Demonstration of a significant relationship between morfometrical parameters of renalparenchyma and the loss of renal function in patients with polycystic kidney disease.Demonstration that the use of mesenchymal cells improves the function of transplanted islandsunder the kidney capsule.Demonstration that drugs that inhibit angiotensin II allow to regenerate the glomerular capillarywith mechanisms that involves podocytes proliferation and endothelial cells remodeling.Implementation of systems for data management of clinical information generated duringconduction of controlled clinical trials.Development of an electronic case report form (e-CRF) for data handling in randomized clinicaltrials.NATIONAL COLLABORATIONSDipartimento di Bioingegneria, Politecnico di Milano, Milano.Fidia Advanced Biopolymers, Abano Terme, Padova.Istituto di Fisiologia Clinica CNR, Pisa.Unità di Diabetologia, Ospedali Riuniti, Bergamo.STMicroelectronics , Agrate Brianza, Milano267ANNUAL REPORT 2007

IRFMNINTERNATIONAL COLLABORATIONSMassachussetts Institute of Technology, Cambridge MA, USA.National Alliance for Medical Imaging Computing, USA.University of Toronto, Ontario, Canada.Ghent University, Ghent, Belgium.Technical University, Eindhoven, The Netherlands.University Hospital, Maastricht, The Netherlands.Centre Européen d’Etudes du Diabètes, Strasburg, FranceEDITORIAL BOARD MEMBERSHIPDrugs of Today, (Andrea Remuzzi)International Journal of Artificial Organs, (Andrea Remuzzi)PEER REVIEW ACTIVITIESKidney InternationalJournal of the American Society of NephrologyAmerican Journal of Physiology – Renal PhysiologyPhysiological ReviewesMedical & Biological Engineering & ComputingIEEE Transactions on Medical ImagingIEEE Transactions on Biomedical IngeneeringMedical PhysicsJournal of BiomechanicsMedical Engineering and PhysicsArtificial OrgansInternational Journal of Artificial OrgansBiomaterialsContemporary Clinical Trials268ANNUAL REPORT 2007

IRFMNEVENT ORGANIZATIONSeminar: “Imaging vascular access: a pre- and post- operative approach”, 2 February, Ranica,Bergamo, Italy.Seminar: “Openclinica: a web-based software platform for managing multi-site clinicalresearch” 12 Luglio, Ranica, Bergamo, Italy.Worshop: “Worshop nuove tecnologie: La microscopica elettronica a trasmissione e ascansione", 26 November, Milan, Italy.Seminar: Bogdan Ene-Iordache - Master di Primo Livello in Ricerca Clinica - Gestione dei datidi uno studio clinico: l'informatizzazione. 16 May 2007, Villa Camozzi, Ranica, Italy.PARTICIPATION IN EVENTS IN WHICH THE DEPARTMENT WASINVOLVEDNational Alliance for Medical Image Computing, Third All Hands Meeting, Salt Lake City,January 2007.National Alliance for Medical Image Computing, Summer Project Week, MIT, Boston, June2007.L'emodinamica nell'evoluzione e nell'instabilità della placca aterotrombotica, CongressoRegionale SIAPAV, Crema, October 2007.ESAO, European Society for Artificial Organs, Krems, Austria, September 2007. Experimentalstudy of patient-specific kink in arterio-venous graft using particle image velocimetry.ASME Summer Bioengineering Conference, Keystone, CO, June 2007. Variation in thehemodynamics of young adult carotid bifurcations.13 th Symposium on Intracranial Pressure and Brain Monitoring, Mechanism and Treatment, SanFrancisco, CA, USA, July 22-26, 2007.World Congress of Nephrology, Rio de Janeiro, Brazil, April 21-25, 2007.Nefropatie croniche e complicanze cardiovascolari: nuove prospettive di prevenzione etrattamento alla luce dei più recenti trials clinici. 30 November 2007. Ranica26 th Workshop of the AIDIPIT Study Group, Montpellier, France, February 2007.“Characterization of the immunereaction in a bioartificial pancreas in a model of xeno-, alloandsyngenic transplantation”.26 th Workshop of the AIDIPIT Study Group, Montpellier, France, February 2007. “Theinfluence of encapsulation on oxygen consumption rate by bovine pancreatic islets”.269ANNUAL REPORT 2007

IRFMNGRANTS AND CONTRACTSResearch grant from PKD foundation - Effect of long-acting somatostatin on diseaseprogression in ADPKD: a long-term, three year, follow-up study.Research project – FP6 UE-STEPS – "A system approach to tissue engineering products andprocesses". FP6-500465DEMAND study: a prospective, randomized, double-blind, parallel-group study aimed atassessing whether the ACE inhibitor delapril alone or in combination with the dCCBmanidipine slow the rate of GFR decline as compared with placebo plus conventionalantihypertensive therapy.Research Project – ROTRF/JDRF – “Mesenchymal stem cells to protect islet allograft indiabetic rats” Contract n. ID 678864716Reserch Projects: AIFA for contreolled clinical studies (VARIETY, VALID, ATHENA).ISN Research grant for the Kidney Disesease Data Center (COMGAN).Grant from Lombardia Region: development and maintenance of the web-site of “Centro diCoordinamento per le Malattie Rare – Regione Lombardia”SELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Remuzzi A, Sangalli F, Fassi A, Remuzzi G. Albumin concentration in the Bowman's capsule: multiphotonmicroscopy vs micropuncture technique. Kidney Int. 2007 Dec;72(11):1410-1;Corna D, Sangalli F, Cattaneo D, Carrara F, Gaspari F, Remuzzi A, Zoja C, Benigni A, Perico N, Remuzzi G. Effectsof rosuvastatin on glomerular capillary size-selectivity function in rats with renal mass ablation. Am J Nephrol.2007;27(6):630-8.Remuzzi A. Vitamin D, insulin resistance, and renal disease. Kidney Int. 2007 Jan;71(2):96-8.Luciani D, Cavuto S, Antiga L, Miniati M, Monti S, Pistolesi M, Bertolini G. Bayes pulmonary embolism assisteddiagnosis: a new expert system for clinical use. Emerg Med J. 2007 Mar;24(3):157-64.Remuzzi G, Cravedi P, Costantini M, Lesti M, Ganeva M, Gherardi G, Ene-Iordache B, Gotti E, Donati D, SalvadoriM, Sandrini S, Segoloni G, Federico S, Rigotti P, Sparacino V, Ruggenenti P. Mycophenolate mofetil versusazathioprine for prevention of chronic allograft dysfunction in renal transplantation: the MYSS follow-uprandomized, controlled clinical trial. J Am Soc Nephrol. 2007 Jun;18(6):1973-85.270ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESLaboratory of Renal BiophysicsThree dimensional reconstruction of the glomerular capillary network.We have recently documented that angiotensin II blockade not only retards the progression ofrenal diseases, but also induces partial regression of the glomerular lesions. To quantify theextent of the regression of sclerotic lesions and the potential regeneration of the glomerularcapillary induced by a therapy with angiotensin converting enzyme (ACE) inhibitors we aredeveloping three dimensional (3D) reconstruction of tissues. The glomerular capillary structureallows to concentrate in a small space a capillary network that filters high volume of waterwhile retains plasmatic proteins in the circulation. We are setting up a new technique to studythe morphology of the glomerular capillary and its wall. The aim is to examine specimens ofrenal tissue by a new electron microscopy technique, based on a combination of an electronbeam with an ionic one, in order to achieve sectioning of samples and acquisition of serialimages for 3D reconstruction of the ultrastructure by mathematical algorithms.Role of glomerular podocytes in the regression of glomerular damageinduced by angiotensin converting enzyme (ACE) inhibitors.Proteinuria is a key predictor of renal disease progression. Clinical studies have documentedthat lowering proteinuria by pharmacological interventions retards renal disease progression. Ascompared to other anti-hypertensive drugs, ACE inhibitors or AII type 1 receptor (AT1R)blockers are more effective in slowing down the decline of glomerular filtration rate and inreducing by 50% the risk of death, dialysis or renal transplantation in both diabetic and nondiabetic patients. Prolonged treatment (4 to 7 years) with these drugs can induce remission andin some patients even improve renal function. Three-dimensional reconstruction of the capillarytuft by serial section analysis allowed us to document that administration of a high dose of anACE inhibitor in MWF rats with advanced nephropathy, not only remarkably reduced sclerosisvolume in most glomeruli, but also increased the volume of glomerular tuft occupied by intactcapillary, indicating consistent glomerular tuft repair.So far, the therapeutic effect of AII blockade has been mainly attributed to its capability tocontrol extracellular matrix deposition. However the possibility that the treatment can modulateglomerular cell survival and repair is not well explored yet. We have recently show that theACE inhibitor induces a selective increase in the number of glomerular epithelial cells(podocytes), highly differentiated cells with a limited capability to proliferate. On this basis, wethen studied the effect of lisinopril, on the proliferation of podocytes and documented that anincrease of it. Our results indicate that the ACE inhibitor allows the glomerular capillary topartially repair the damage in a coordinate way to maintain the podocyte number required forthe ultrafiltration process. Studies are in progress to evaluate whether the restoration ofpodocyte number within the capillary is dependent on progenitor/stem cells of renal or extrarenalorigin. We have also studied the role of parietal epithelial cells of the Bowman’s capsulein tissue repair process and hypothesized that cells from Bowman’s capsule can replace orincrease visceral epithelial cells. Our studies on ACE inhibition-induced effects are instrumentalfor the understanding of the mechanisms responsible for glomerular damage regression andopen new therapeutic prospects for chronic progressive nephropathies.271ANNUAL REPORT 2007

IRFMNApplication of confocal microscopy to the study cells and tissues.Fluorescent probes to investigate the distribution and co-localization of proteins and otherspecific antigens in cell monolayers and tissue specimens are commonly used in the majority ofexperimental applications. Bidimensional images are not of high resolution to provide correctinformation and the use of 3D reconstruction of fluorescent signal is sometimes mandatory.These problems can not be solved by the classic florescent microscopy that has somelimitations. For these reasons we have developed the laser confocal microscopy by using theLSM510 META microscope from Zeiss (Germany). The microscopy is equipped with four laserlines that allow to excite the sample within the whole visible spectrum plus the characteristic“META scan head” that allow to analyze the emission spectrum of different fluorophores tooptimally separate them. The instrument is characterized by and used for its high specificity andfor the excellence of the revealed and elaborated signal.Do peptides derived from albumin degradation have a role in thedevelopment of renal damage?In proteinuric nephropathies loss of the permselective properties of the glomerular membraneleads to abnormal filtration of proteins, mainly albumin, that damage the proximal tubular cellinducing the release into the interstitium of chemokines responsible for the recruitment andactivation of inflammatory cells. The interstitial infiltrates are also characterized by the presenceof immune competent cells including dendritic cells (DCs) and T lymphocytes that accumulatewithin renal parenchyma even in absence of an immune insult. As antigen presenting cells, DCscan initiate immune response or tolerance. Within the kidney, DCs are in close contact with thetubular epithelium and work as immune sentinels to probe renal interstitium in search forforeign antigens. It is conceivable that upon injury, the inflammatory stimuli released from thetubular cell make the DC antigenic favoring the presentation of normally ignored self-antigensand triggering an immune response. To verify our hypothesis we have performed in vitro studiesexposing renal proximal tubular cells to albumin concentrations mimicking those present in theultrafiltrate in proteinuric conditions. We have identified peptides derived from albumindegradation by mass spectrometry and we have evaluated their potential antigenicity, insyngeneic conditions, in mixed leukocyte reaction , using bone marrow-derived DCs (incollaboration with the Department of Molecular Medicine and the Laboratory of AnalyticBiochemistry-Department of Environmental Health Sciences).Laboratory of Biomedical TechnologiesDevelopment of computerized systems for controlled clinical trias.Numerous clinical trials are conducted in the Clinical Research Center for Rare Diseases “Aldoe Celè Daccò”. These studies must be carried out with respect to the GCP (Good ClinicalPractice) guidelines and require high quality in data handling. Every clinical study requires apaper case report form (CRF) for collection of patients’ clinical observations. These data mustbe checked for inconsistency by dedicated monitoring staff, and then recorded electronically. Inour laboratory we developed applications tailored for data management of clinical studies usingrelational databases systems (RDBMS) and specific programs aimed to data elaboration,validation and extraction for subsequent statistic analyses.REIN, BENEDICT MYSS REIN2, DKG are only some of the trials concluded successfully andof which results were published in prestigious medical journals. This accomplishment is, in asmall part, due to the contribution of our Laboratory. Recently, we have developed aninnovative system of data handling using electronic CRFs instead of the usual paper for theDEMAND study. This system is based on the use of laptop computers by the clinicalinvestigators working in eight diabetes outpatient clinics.272ANNUAL REPORT 2007

IRFMNManagement of the Registry for Rare Disease in Lombardy.Together with our colleagues from the Information Center for Rare Diseases we are involved inthe conduction of the Regional Center for Rare Diseases of Lombardia, Italy. We are directlyinvolved for the development and maintenance of the web site of the center and management ofa regional Registry for Rare Diseases. We have set-up the databases and developed related webpagesfor among centers, associations of patients and congenital rare diseases. These are hostedon the homepage of the center (http://malattierare.marionegri.it/).The Registry for Rare Diseases was born in 2007 as collaboration between Mario NegriInstitute, Lombardia Informatica (LI) and Regione Lombardia. The main idea is to create aregional registry for rare diseases where all medical staff from Lomardia could registerinformation regarding rare diseases. The application (called Sistema Malattie Rare - SMR) wasdeveloped by LI in collaboration with our group. SMR application is using web-basedtechnology and can be used jointly with the health patient card. It is actually in use in almost allcenters dedicated for rare diseases in Lombardia. Our laboratory is involved in the maintenanceof SMR, statistical analyses of registry data and creation of a minimal set of data that are sharedat national level by the Istituto Superiore di Sanità (ISS). File download from LI is securedbecause data are encrypted using an asymmetrical algorithm with public and private key at 2048bit. Also data transfer at ISS is performed securely by using the htpp Internet protocol.KDDC – a coordinating center for data collection and surveillance ofprevention programs on non-communicable chronic diseases in emergingcountries.Chronic kidney diseases are emerging as a global threat to human health. Prevalence andincidence of renal diseases in developing countries are not known, and this is an obstacle to theadoption of preventive measures. Prevention is the only hope for these countries wheretreatment options for end stage renal failure are simply not available to the vast majority of thepopulation because of their costs. The International Society of Nephrology (ISN), through theCommission for Global Advancement of Nephrology (COMGAN), has established a researchcommittee in order to face the problems about prevention of kidney diseases in developingcountries. The coordination of the team and intervention programs was committed to the MarioNegri Institute for Pharmacological Research at the Clinical Research Center “Aldo e CeleDaccò”. The general aim of the project is to define programs in developing countries to identifythose subjects who are at risk of developing a renal disease later in life, in order to design aprevention strategy on national basis by means of interventions of the local ministries of healthto governmental and financial level.The Kidney Disease Data Center (KDDC), headquartered in our Laboratory, is dedicated to datamanagement for the prevention programs underway in emerging countries. We have set up aninstrument to collect clinical data from different Centres located world-wide. Data are stored ina dedicated server in our Laboratory. Results of our epidemiological analyses, shared also withmedical staff of the center, allow us to have a general overview on the health of populationunder study. The prevention program is underway and we have already collected data fromparticipating centers from Moldova, Bolivia, Egypt, Nepal, China and Mongolia. First results onscreening on several thousands of subjects, confirm the need to proceed with preventionprograms in theses countries. Other countries are willing to start their programs, as for exampleMorocco, Mozambique, Mexico and Argentina. Through the activity of KDDC it will bepossible to monitor the course of the prevention programs and to tailor them to fit the needs ofeach participating country.273ANNUAL REPORT 2007

IRFMNThree-dimensional reconstruction and hemodynamic simulation ofvascular segments from CT and MR imaging.Evidence that atherosclerotic plaques form mainly at bifurcations of the arterial system led tothe hypothesis that impaired hemodynamic conditions may favour the initiation and progressionof atherosclerosis. Furthermore, it has been demonstrated how intimal hyperplasia, cause offailure of grafts and arterovenous fistulas, is also localized at sites of complex hemodynamics.Analogously, recent studies on formation, localization and growth of cerebral aneurysms haveunderlined the importance of hemodynamics on the development of this pathology. Given theinfluence of vessel shape on hemodynamics, it is important to dispose of accurate and fastmethods for three-dimensional reconstruction of vascular geometry. Modern angiographictechniques, such as computed tomography (CT) and magnetic resonance (MR), allow to obtaindetailed information on vascular structures. In our laboratory, we developed techniques forthree-dimensional reconstruction, geometric analysis, computational mesh generation and fluiddynamicsimulation of vascular tracts from CT and MR angiography. CT and MR images aretransferred from clinical scanners on local workstations and are then processed with modelingalgorithms to generate three-dimensional surfaces representing the interface between thevascular wall and its lumen. The accuracy of the reconstruction has been experimentallyvalidated by us. Accurate measurements are then performed on the generated model usingcomputational geometry algorithms. These techniques have been applied to several vascularsegments, mainly cerebral aneurysms, arterovenous fistulas and grafts for hemodialysis access.The pathologies studied include atherosclerosis, intimal hyperplasia, development of cerebralaneurysms and Takayasu's arteritis, a rare disease affecting the arterial system.Quantification of anatomical structures from CT and MR imaging.The introduction of non-invasive three-dimensional imaging techniques, such as computedtomography (CT) and magnetic resonance (MR), in the clinical setting has opened thepossibility to analyze the anatomical structures of organs in high detail. The non-invasiveness ofsuch techniques allows to extend the observation to populations of patients followed over time.The evaluation of natural evolution of diseases or of treatment efficacy often requires a detailedmorphological quantification which cannot be uniquely performed by visual inspection. In thiscontext, we developed image-based quantification techniques characterized by high accuracyand reproducibility. Wevare applying these tools to a study on patients affected by autosomaldominant polycystic disease (ADPKD) with the aim of testing the efficacy and safety of sometreatments. Volumes of kidneys and their main tissue components (cysts and parenchyma) havebeen quantified from CT o RM images.Development of devices for the transplantation of immunoisolated islets.The project’s main objective was to develop an immunisolation device for pancreatic islets thatcan be implanted in diabetic patients permitting allo-islet transplantation without the use ofpharmacological immunosuppression and avoiding allosensitization of the patient. The studystarted from the design and characterization of the semi-permeable membrane used for thedevice construction. The device that we are developing can be implanted with minimallyinvasive surgical procedures and easy to retry. This system is a device made using polysulfonehollow fibers. The aims of our studies in the next months will be to improve functionality usingnanotechnologies for materials characterization. Moreover we will develop new kinds of deviceand we will test new implantation sites.Differentiation of pancreatic precursor cells.Type 1 diabetes mellitus is an autoimmune disease characterized by distruction of insulinproducingbeta cells in pancreatic islets. The damage leads to a deficiency of insulin resulting ina complete relance on infusion of exogenous insulin. The replacement of the beta cell mass is a274ANNUAL REPORT 2007

IRFMNpotential cure for type I diabetes, but this procedure suffer from a shortage of available donortissue in comparison to the number of potential recipients. In our laboratoris we have studiedthe method to obtain beta cells from progenitor cells extracted by pancreatic tissue. Actually, itis possible to obtain insulin-producing cell in vitro, but the level of insulin is much lower thanthat contained in pancreatic islet. Our research is oriented towards the development of newstrategies to detect and to select progenitors of beta cells in the pancreas. To this purpose wedeveloped a method to incorporate, in vitro, into the pancreatic islets two thymidine analogous(IdU and CldU). This technique permits to identify stem cells into the islets and to evaluate theirproliferation capacity.Co-transplantation of mesenchymal stem cells and pancreatic islets toinduce immunotolerance.Several recent studies have suggested that pancreatic islet transplantation offers an alternativeto pancreas transplantation. In the last year in our laboratories we have defined the optimalconditions for isolation of rat islets using an automated method. Rat pancreatic islets have beentransplanted under the kidney capsule of diabetic rats. Diabetes was induced by single injectionof streptozotocin. Two models of islet transplantation have been used, syngenic- and allotransplantation.In syngenic transplantation rat islets have been implanted into inbreed rats andnormoglycemia conditions were maintained for several months. Different strains of rats wereused as donor and recipient in allogenic transplantation. However, immunosuppression isrequest to prevent islet graft rejection. To avoid the use of immunosuppressive drug therapy, wehave planned to study the immunomodulatory properties of mesenchymal stem cells (MSCs).Preliminare experiments have been done using Wistar rats as donors and Lewis rata asrecipients. To evaluate if MSCs can suppress the immune response toward allograft , LewisMSCs were transplanted simultaneously to pancreatic islets.Vascular tissue engineering.Artificial vascular prostheses can be used in patients only if their caliber is larger than 6 mm. Alower caliber prostheses implies a high trombotic risk. For this reason, several investigators aredeveloping bioartificial biological vascular substitutes using tissue engineering technique. In ourlaboratory we used a biodegradable hialuronic acid matrix as a scaffold to produce cellularizedvascular constructs. We set up culture and seeding conditions both for smooth muscle cells andmesenchymal bone marrow cells. To obtain physiological mechanical conditioning of thetubular constructs we developed a new type of rotating wall bioreactor that allows to keep theculture of vascular constructs in dynamic conditions. This simple method allowed to decreaseapoptosis incidence and to increase cell matrix depositions. More recently we tested, incollaboration with IFC-CNR in Massa, the possibilityto use innovative elastomeric material toconstruct cellularized vascular substitutes.275ANNUAL REPORT 2007

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IRFMNLABORATORY OF BIOLOGY ANDTHERAPY OF METASTASIS*STAFFHeadTumor Angiogenesis UnitHeadMolecular Cancer Therapeutics UnitHeadRaffaella GIAVAZZI, Biol.Sci.D., Ph.D.Giulia TARABOLETTI, Biol.Sci.D.Maria Rosa BANI, Biol.Sci.D., Ph.D.* Research activities of this Laboratory are listed in the Department of Oncology section (pag. 7)277ANNUAL REPORT 2007

IRFMN278ANNUAL REPORT 2007

IRFMNAldo and Cele Daccò CenterRanica (Bg)ANNUALREPORT 2007departments and laboratories279ANNUAL REPORT 2007

IRFMN280ANNUAL REPORT 2007

IRFMNDEPARTMENT OF RENAL MEDICINESTAFFHeadPiero RUGGENENTI, M.D.Laboratory of BiostatisticsHeadAnnalisa PERNA, Stat.Sci.D.Unit of Drug MonitoringHeadGiulia GHERARDILaboratory of Clinical ChemistryHeadFlavio GASPARI, Chem.D.Laboratory of Advanced Development of DrugsHeadNorberto PERICO, M.D.Unit of Pharmacology and PharmacogeneticsHeadDario CATTANEO, Chem.Pharm. D., Ph.DUnit of Early Clinical Evaluation of DrugsHeadAneliya PARVANOVA, M.D.281ANNUAL REPORT 2007

IRFMNCURRICULA VITAEPiero Ruggenenti got his Medicine degree in 1983 at the University of Milan, Italy; he got hisspecialization in Cardiology in 1985 and in Clinical Nephrology in 1989 at the same University; hespecialized in Pharmacological Research in 1988 at IRFMN.Educational training: in 1980-1983 researcher at "Centro di Fisiologia Clinica ed Ipertensione, ClinicaMedica IV", Università degli Studi di Milano; in 1984 Researcher at IRFMN, Bergamo, Italy in 1987-1988 Honorary Registrar of the Unit for Metabolic Medicine, Division of Medicine (University ofLondon) of Guy's and St. Thomas's Hospitals, London; in 1988-1989 Assistant Professor of the Divisionof Nephrology and Dialysis of the Ospedali Riuniti di Bergamo.Areas of interest: mechanisms of chronic renal disease progression, diabetes and diabetic complications,clinical transplantation, thrombotic microangiopathies, cardiovascular complications of chronic renaldisease, clinical trials, clinical pharmacology.Employment: from 1990 Assistant Professor of the Division of Nephrology and Dialysis of theOspedali Riuniti di Bergamo; in 1994-1999 Head, Unit of Advanced Development of Drugs, DaccòCenter, Ranica, Bergamo, Italy; since 2000 Head, Department of Renal Medicine, Daccò Center,Bergamo, Italy.Selected publications• P. Ruggenenti, A. Perna, L. Mosconi, M. Matalone, G. Garini, M. Salvadori, C. Zoccali, F. Scolari, Q. Maggiore, G.Tognoni, G. Remuzzi (for The GISEN Group). Randomised placebo-controlled trial of effect of ramipril on decline inglomerular filtration rate and risk of terminal renal failure in proteinuric, non-diabetic nephropathy. Lancet1997;349:1857-1863• P. Ruggenenti, A. Perna, G. Gherardi, F. Gaspari, R. Benini, G. Remuzzi, on behalf of GISEN. Renal function andrequirement for dialysis in chronic nephropathy patients on long-term ramipril: REIN follow-up trial. Lancet1998;352:1252-1256.• P. Ruggenenti, A. Perna, G. Gherardi, G. Garini, C. Zoccali, M. Salvadori, F. Scolari, F.P. Schena, G. Remuzzi.Renoprotective properties of ACE-inhibition in non-diabetic nephropathies with non-nephrotic proteinuria. Lancet1999;354:359-364.• G. Remuzzi, C. Chiurchiu, M. Abbate, V. Brusegan, M. Bontempelli, P. Ruggenenti. Rituximab for idiopathicmembranous nephropathy. Research Letter. Lancet 2002;360:923-924.• P. Ruggenenti, A. Fassi, A. Parvanova, S. Bruno, I. Iliev, V. Brusegan, N. Rubis, G. Gherardi, F. Arnoldi, M. Ganeva, B.Ene-Iordache, F. Gaspari, A. Perna, A. Bossi, R. Trevisan, A.R. Dodesini, G. Remuzzi for the Bergamo NephrologicDiabetes Complications Trial (BENEDICT) Investigators. Preventing microalbuminuria in type 2 diabetes. N Engl JMed 2004;351:1941-1951• G. Remuzzi, P. Cravedi, A. Perna, B.D. Dimitrov, M. Turturro, G. Locatelli, P. Rigotti, N. Baldan, M. Beatini, U.Valente, M. Scalamogna, P. Ruggenenti Dual Kidney Transplant Group. Long-term outcome of renal transplantationfrom older donors. N Engl J Med 2006;354:343-352.Flavio Gaspari got his Chemistry degree in 1977 at the University of Milano, Italy, and thespecialization in the same University in 1979.Educational training: in 1981-1985 Fellow and Researcher at IRFMN, Milan; in 1985-1991 at IRFMN,Bergamo, Italy.Areas of interest: pharmacokinetics and the metabolism of xanthines in different animal species; drugpharmacokinetics in uremic patients and in subjects with different degrees of renal function; analyticalmethods to measure the most important immunosuppressive drugs to determine their pharmacokinetics inkidney, heart, and liver transplant recipients; evaluation of the renal function by using differentapproaches, in the study of renal disease progression, and in the comparison of different methods foralbuminuria determination.Employement: He is Chief of Laboratory of Pharmacokinetics and Clinical Chemistry since January2000 and he was Chief of this Unit since 1991.Selected publications• Gotti E, Perico N, Gaspari F, Cattaneo D, Lesti MD, Ruggenenti P, Segoloni G, Salvadori M, Rigotti P, Valente U,Donati D, Sandrini S, Federico S, Sparacino V, Mourad G, Bosmans JL, Dimitrov BD, Iordache BE, Remuzzi G. Bloodcyclosporine level soon after kidney transplantation is a major determinant of rejection: insights from the MycophenolateSteroid-Sparing Trial. Transplant Proc. 2005 Jun;37(5):2037-40.• Perico N, Gaspari F, Remuzzi G. Assessing renal function by GFR prediction equations in kidney transplantation. Am JTransplant. 2005 Jun;5(6):1175-6.282ANNUAL REPORT 2007

IRFMN• D. Cattaneo, F. Gaspari, S. Zanoni, S. Baldelli, E.Gotti, A. Perna, N. Perico, G. Remuzzi. Two-hour post-dosecyclosporine monitoring does not fit all in kidney transplantation. Therapy 2005;2:95-105.• Ruggenenti P, Fassi A, Ilieva AP, Bruno S, Iliev IP, Brusegan V, Rubis N, Gherardi G, Arnoldi F, Ganeva M, Ene-Iordache B, Gaspari F, Perna A, Bossi A, Trevisan R, Dodesini AR, Remuzzi G; Bergamo Nephrologic DiabetesComplications Trial (BENEDICT) Investigators. Preventing microalbuminuria in type 2 diabetes. N Engl J Med. 2004Nov 4;351(19):1941-51. Epub 2004 Oct 31.• Gaspari F, Ferrari S, Stucchi N, Centemeri E, Carrara F, Pellegrino M, Gherardi G, Gotti E, Segoloni G, Salvadori M,Rigotti P, Valente U, Donati D, Sandrini S, Sparacino V, Remuzzi G, Perico N; MY.S.S. Study Investigators.• Performance of different prediction equations for estimating renal function in kidney transplantation. Am J Transplant.2004 Nov;4(11):1826-35.• Cattaneo D, Merlini S, Pellegrino M, Carrara F, Zenoni S, Murgia S, Baldelli S, Gaspari F, Remuzzi G, Perico N.Related Articles, Links Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy andoptimization of drug dosing. Am J Transplant. 2004 Aug;4(8):1345-51.Norberto Perico got his Medicine degree in 1983 at the University of Milano, Italy. He got hisspecialization in Pharmacological Research in 1986 at IRFMN, Bergamo and in Clinical Nephrology in1989 at the University of Verona, Italy.Educational training: in 1982 Fellow, Department of Pharmacology, New York Medical College,Valhalla, New York, USA; in 1984-1988 Post Doctoral Fellow, Laboratory of Kidney Diseases, IRFMN,Bergamo, Italy; in 1988-1989 Researcher in the same laboratory.Areas of interest: pathophysiology and pharmacology of cyclosporine nephrotoxicity; newimmunosuppressive strategies to prevent renal graft rejection; innovative approach to induce tolerance toorgan transplantation; mechanism(s) and management of progression of chronic renal diseases.Employment: in 1990-1994 Head, Renal Physiology Unit, Laboratory of Kidney Diseases, IRFMN,Bergamo, Italy; in 1990-2000 Assistant Professor, Division of Nephrology and Dialysis, Ospedali Riunitidi Bergamo, Italy; in 1994 –1999 Head, Laboratory of Transplant Immunology, IRFMN, Bergamo, Italy;from January 2000 Head, Laboratory of Drug Development, Department of Renal Medicine, IRFMN,Bergamo, Italy; from September 2000 Health Director, Daccò Center, IRFMN, Bergamo, Italy. FromOctober 2002 he’s Member, ISN-COMGAN Research Committee of the International Society ofNephrology.Selected publications:• E. Gotti, N. Perico, A. Perna, F. Gaspari, D. Cattaneo, R. Caruso, S. Ferrari, N. Stucchi, G. Marchetti, M. Abbate, G.Remuzzi. Renal transplantation: can we reduce calcineurin inhibitor/stop steroids? Evidence based on protocol biopsyfindings. J Am Soc Nephrol 2003;14:755-766.• N. Perico, P. Ruggenenti, G. Remuzzi. Losartan in diabetic nephropathy. Expert Rev. Cardiovasc. Ther. 2004; 2(4): 473-483.• Remuzzi G, Lesti M, Gotti E, Ganeva M, Dimitrov BD, Ene-Iordache B, Gherardi G, Donati D, Salvadori M, Sandrini S,Valente U, Segoloni G, Mourad G, Federico S, Rigotti P, Sparacino V, Bosmans JL, Perico N, Ruggenenti P. mofetilversus azathioprine for prevention of acute rejection in renal transplantation (MYSS): a randomised trial. Lancet. 2004Aug 7;364(9433):503-12.• Gaspari F, Ferrari S, Stucchi N, Centemeri E, Carrara F, Pellegrino M, Gherardi G, Gotti E, Segoloni G, Salvadori M,Rigotti P, Valente U, Donati D, Sandrini S, Sparacino V, Remuzzi G, Perico N; MY.S.S. Study Investigators. of differentprediction equations for estimating renal function in kidney transplantation. Am J Transplant. 2004 Nov;4(11):1826-35.• Perico N, Cattaneo D, Sayegh MH, Remuzzi G. Delayed graft function in kidney transplantation. Lancet. 2004 Nov13;364(9447):1814-27.• Cattaneo D, Merlini S, Zenoni S, Baldelli S, Gotti E, Remuzzi G, Perico N. Influence of co-medication with sirolimus orcyclosporine on mycophenolic acid pharmacokinetics in kidney transplantation. Am J Transplant. 2005 Dec;5(12):2937-44.283ANNUAL REPORT 2007

IRFMNAnnalisa Perna got her Statistical Sciences degree in 1984 at the University of Bologna, Italy.Educational training: She completed her research training at IRFMN, Bergamo Labs. and at the DaccòCenter.Areas of interest: statistical methodology of long-term randomised clinical trials in nephrology,statistical methods for calculating sample size and for meta-analytic techniques. She is also involved inperforming systematic reviews for the Cochrane Collaboration – Renal Review Group.Employment: she is Head of the Laboratory of Biostatistics - Department of Renal Medicine at DaccòCenter, Ranica (Bergamo).Principal publications:• G. Remuzzi, P. Cravedi, A. Perna, B.D. Dimitrov, M. Turturro, G. Locatelli, P. Rigotti, N. Baldan, M.Beatini, U. Valente, M. Scalamogna, P. Ruggenenti. Dual Kidney Transplant Group. Long-term outcome ofrenal transplantation from older donors. N Engl J Med 2006;354:343-352.• Ruggenenti P, Perna A, Loriga G, Ganeva M, Ene-Iordache B, Turturro M, Lesti M, Perticucci E, Chakarski IN,Leonardis D, Garini G, Sessa A, Basile C, Alpa M, Scanziani R, Sorba G, Zoccali C, Remuzzi G for the REIN-2 Studygroup. Blood-pressure control for renoprotection in patients with non-diabetic chronic renal disease (REIN-2):multicentre, randomised controlled trial. Lancet 365:939-946, 2005.• Schieppati A, Perna A, Zamora J, Giuliano AG, Braun N, Remuzzi G. Immunosuppressive treatment for idiopathicmembranous nephropathy in adults with nephrotic syndrome. Cochrane Database Syst Rev. Oct 18(4):CD004293, 2004• Ruggenenti P, Fassi A, Ilieva AP, Bruno S, Iliev IP, Brusegan V, Rubis N, Gherardi G, Arnoldi F, Ganeva M, Ene-Iordache B, Gaspari F, Perna A, Bossi A, Trevisan R, Dodesini AR, Remuzzi G. Preventing microalbuminuria in type 2diabetes. N Engl J Med 351(19)1941-51, 2004.• The BENEDICT Group. The Bergamo NEphrologic Diabetes Complications Trial (BENEDICT): design and baselinecharacteristics. Control Clin Trials 24(4): 442-461, 2003.• Plata R, Cornejo A, Arratia C, Anabaja A, Perna A, Dimitrov BD, Remuzzi G, Ruggenenti P for the Commission onGlobal Advancement of Nephrology (COMGAN), Research Subcommittee of the International Society of Nephrology.Angiotensin-converting-enzyme inhibition therapy in altitude polycythaemia: a prospective randomised trial. Lancet 359:663-66, 2002.Dario Cattaneo got the Pharmacy degree in 1996 at the University of Milan, and the specialisation inPharmacology (2001) awarded by the same University. In 2000 he got the specialization in“Pharmacological Research” at the Mario Negri Institute for Pharmacological Research (IRFMN) and in2005 he has been awarded the PhD degree by the Open University of London, UK.Educational Training: in 1997 Post Doctoral Fellow, IRFMN, Laboratory of Pharmacokinetics andClinical Chemistry; in 2000 beneficiary of the Fellowship “Girola” and from 2001 to 2005 recipient ofthe “Monzino” Fellowship for his research activity done at the IRFMN.Areas of interest: pharmacology (pharmacokinetics, pharmacodynamics and pharmacogenetics) ofimmunosuppressants, antiviral agents and hypolipidemic drugs; study of secondary forms ofdyslipidemia; polypharmacological approaches for the treatment of chronic kidney diseases; assessmentof humoral response as predictor of acute or chronic rejection after organ transplantation.Employement: in 1997 researcher, IRFMN, Laboratory of Pharmacokinetics and Clinical Chemistry,.Since 2006, Head of the Unit of Pharmacology and Pharmacogenetics, IRFMN, Ranica Bergamo.Component of the Ethical Committee (2003) of the Hospital “Bolognini” (Seriate, Italy) and theHospital “E.Medea” (Bosisio Parini, Italy) since 2006. Member of the editorial board of CurrentClinical Pharmacology and affiliate of the International Association of Therapeutic Drug Monitoringand Clinical Toxicology (IATDMCT) since 2005.Selected publications• Cattaneo D, Merlini S, Zenoni S, Baldelli S, Gotti E, Remuzzi G, Perico N. Influence of co-medication with sirolimus orcyclosporine on mycophenolic acid pharmacokinetics in kidney transplantation. Am J Transplant. 2005 Dec;5(12):2937-44.• Cattaneo D, Gotti E, Perico N, Bertolini G, Kainer G, Remuzzi G. Cyclosporine formulation and Kaposi's sarcoma afterrenal transplantation. Transplantation. 2005 Sep 27;80(6):743-8.• Perico N, Cattaneo D, Sayegh MH, Remuzzi G. Delayed graft function in kidney transplantation. Lancet. 2004 Nov 13-19;364(9447):1814-27.• Cattaneo D, Merlini S, Pellegrino M, Carrara F, Zenoni S, Murgia S, Baldelli S, Gaspari F, Remuzzi G, Perico N.Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy and optimization of drugdosing. Am J Transplant. 2004 Aug;4(8):1345-51.• Cattaneo D, Perico N, Gaspari F, Gotti E, Remuzzi G. Glucocorticoids interfere with mycophenolate mofetilbioavailability in kidney transplantation. Kidney Int. 2002 Sep;62(3):1060-7.284ANNUAL REPORT 2007

IRFMNGiulia Gherardi got her Scientific High School Diploma on 1989 at the Liceo Scientifico Marie Curie inZogno (Bergamo), the Nurse Diploma on 1995 at the Scuola per Infermieri Professionali, OspedaliRiuniti, Bergamo.Educational training: Clinical Research Nurse Diploma on 1997 at IRFMN –Daccò Center.Areas of interest: statistical methodology of long-term randomised clinical trials in nephrology,diabetology; the organisation and the monitoring of clinical trials.Emplyment: In1997-2003 involved as co-organazing, speaker, co-speaker and tutor for the ClinicalResearch Course for Nurse at IRFMN – Daccò Center (Ranica – Bergamo). Several training activities forNurses in Clinical Research area. In 1997-1999, Clinical Research Monitor at IRFMN – Daccò Center;since 2000 Monitoring Unit Chief.Selected pubblications• Gaspari F, Ferrari S, Stucchi N, Centemeri E, Carrara F, Pellegrino M, Gherardi G, Gotti E, Segoloni G, Salvadori M,Rigotti P, Valente U, Donati D, Sandrini S, Sparacino V, Remuzzi G and Perico N on the behalf of the MY.S.S. studyinvestigators. Performance of different prediction equations for estimating renal function in kidney transplantation.American Journal of Transplantation. 2004; 4: 1826-1835.• Ruggenenti P, Fassi A, Parvanova Ilieva A, Bruno S, Petro Iliev I, Brusegan V, Rubis N, Gherardi G, Arnoldi A, GanevaM, Ene-Iordache B, Gaspari F, Perna A, Bossi A, Trevisan R, Dodesini AR, Remuzzi G, for the Bergamo NephrologicDiabetes Complications Trial (BENEDICT) Investigators. Preventing microalbuminuria in type 2 diabetes. N Engl JMed. 2004; 351: 1941-51.• Remuzzi G, Lesti M, Gotti E, Ganeva M, Dimitrov BD, Ene-Iordache B, Gherardi G, Donati D, Salvadori M, Sandrini S,Valente U, Segoloni G, Mourad G, Federico S, Rigotti P, Sparacino V, Bosmans JL, Perico N, Ruggenenti P, for theMY.S.S. Group. Mycophenolate mofetil versus azathioprine for prevention of acute rejection in renal transplantation(MYSS): a randomized trial. Lancet. 2004 Aug 7; 364: 503 – 12.• Ruggenenti P, Perna A, Gherardi G, Benini R, Remuzzi G. Chronic proteinuric nephropathies: outcomes and responseto treatment in a prospective cohort of 352 patients with different patterns of renal injury. Am J Kidney Dis. 2000 Jan;35 (6): 1155 – 65.• Ruggenenti P, Perna A, Zoccali C, Gherardi G, Benini R, Testa A, Remuzzi G. Chronic proteinuric nephropathies. II.Outcomes and response to treatment in a prospective cohort of 352 patients: differences between women and men inrelation to the ACE polymorphism. Gruppo Italiano di Studi Epidemiologici in Nefrologia. J Am Soc Nephrol. 2000Jan; 11 (1): 88 – 96.• Ruggenenti P, Perna A, Gherardi G, Garini G, Zoccali C, Salvadori M, Scolari F, Schena FP, Remuzzi G.Renoprotective properties of ACE-inhibition in non-diabetic nephropathies with non-nephrotic proteinuria. Lancet.1999 Jul 31; 354 (9176): 359 – 64.Aneliya Parvanova Ilieva got her Medical Doctor degree at the Faculty of Medicine, ThracianUniversity (former Higher Medical Institute), Stara Zagora, Bulgaria in 1988, and the specialisation inPharmacology in Department of Pharmacology, Faculty of Medicine, of the same university in 1992.Educational training: 1989-1998: Teaching of 3 rd , 4 th and 5 th -year medical students and 2 nd and 3 rd -yearclinical nurses in a general pharmacology and clinical pharmacology, Thracian University, Stara Zagora,Bulgaria. Examiner of these students in theoretical and practical, oral and written exams and tests. 1993:Course on investigation of isolated organs – Bulgarian Academy of Sciences, Sofia. 1998: Visitingscientist, IRFMN, Ranica, Bergamo, Italy. 1998: Proficiency in the methods for insulin sensitivityevaluation (hyperinsulinemic euglicaemic clamp technique) and glomerular filtration rate evaluation(plasma clearance of iohexol).Areas of interest: primary and secondary prevention of the chronic microvascular diabetic complications(diabetic nephropathy, diabetic retinopathy and diabetic neuropathy); role of insulin resistance andhyperhomocysteinemia in these pathologies.Employment: She participated as investigator in several clinical studies. She is Chief Unit of EarlyClinical Evaluation of Drugs at IRFMN since 2000. She is a member of the Union of Bulgarian Doctors(since 1989), of the Union of Pharmacologists in Bulgaria (since 1990), and member of the Union ofScientists in Bulgaria (since 1991).Selected publications• Parvanova A, Chiurchiu C, Ruggenenti P, Remuzzi G. Inhibition of the renin-angiotensin system and cardio-renalprotection: focus on losartan and angiotensin receptor blockade. Expert Opinion on Pharmacotherapy 2005 Sep; 6(11):1931-1942.• Ruggenenti P, Fassi A, Parvanova A, Bruno S, Iliev I, Brusegan V, Rubis N, Gherardi G, Arnoldi F, Ganeva M, Ene-Iordache, Gaspari F, Perna A, Bossi A, Trevisan R, Dodesini A, Remuzzi G. Preventing Microalbuminuria in Type 2Diabetes. NEJM 2004;351(19):1941-51.• Parvanova A, Iliev I, Filipponi M, Dimitrov BD, Vedovato M, Tiengo A, Trevisan R, Remuzzi G, Ruggenenti P. Insulinresistance and proliferative retinopathy: a cross-sectional, case-control study in 115 patients with type 2 diabetes. J ClinEndocrinol Metab 2004 Sep; 89(9):4371-6.285ANNUAL REPORT 2007

IRFMN• The BENEDICT Group. The Bergamo Nephrologic DIabetes Complications Trial (BENEDICT): design and baselinecharacteristics. Controlled Clinical Trials 2003; 24:442-461.• Parvanova A, Iliev I, Dimitrov BD, Arnoldi F, Zaletel J, Remuzzi G, Ruggenenti P. Hyperhomocysteinemia andincreased risk of retinopathy: a cross-sectional, case-control study in patients with type 2 diabetes. Diabetes Care 2002;25 (12): 2361.286ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE DEPARTMENT'S ACTIVITIESThe Department of Renal Medicine was established on 1999 at the Clinical Research Center forRare Diseases “Aldo e Cele Daccò” – Villa Camozzi, Ranica to coordinate the activities of threeLaboratories and two Units.The activities of the Department are mainly focused on the study of the mechanisms ofprogression of chronic nephropathies, of new prevention and intervention strategies for diabeticnephropathy, non diabetic chronic nephropathies, chronic allograft dysfunction, ofcardiovascular complications of diabetes, chronic renal disease, dialysis and transplantation andof thrombotic microangiopathies.The main aims of these activities are:1. To identify screening and intervention strategies aimed to prevent the onset of nephropathyand of other chronic complications of diabetes and/or hypertension.2. To define intervention strategies to prevent or slow the progression of chronic nephropathiesand eventually obtain remission/regression of renal dysfunction.3. To optimize immunosuppressive protocols in kidney transplantation and to define new donorselection criteria in order to expand the pool of available organs.These aims will be pursued through the following modalities:1. Pilot pathophysiology and clinical pharmacology studies fully finalized at the ClinicalResearch Center to test new pathogenetic hypotheses and new treatment modalities.2. National and international networks and multicenter trials aimed to verify the efficacy oftreatments of potential interest identified as described at point 1.3. Meta-analyses and probabilistic models to test new risk factors and treatments in largesamples of patients and to transfer this information at individual level.Many of these activities rest on the possibility of a tight cooperation with the Department ofMolecolar Medicine, the Department of Bioengineering and the Public-Private Department ofSpecialist and Transplant Medicine. This cooperation allows to plan the research activities ofthe Department on the basis of new information derived from basic research and of problems ofmajor clinical relevance emerging from routine clinical activities.FINDINGS/MAIN RESULTSDefinition and validation of specific treatments aimed to prevent the developing ofnephropathies in subjects with type 2 diabetesDefinition and validation of new integrated treatment protocols aimed to slow the progressionand/or to achieve remission/regression of diabetic and non-diabetic chronic nephropathiesInstitution of a standardized protocol “on line” (The “Remission Clinics”) finalized to achieveregression/remission of chronic nephropathies in hospital practice in the setting of a multicenterNetworkCharacterization of the antiproteinuric, nephroprotective and cardioprotective effect ofmaximized and polypharmacologic renin-angiotensin system inhibitionIdentification of acquired or congenital risk factors for chronic complications of diabetesDefinition and validation of new, specific treatments for idiopathic membranous nephropathyand for HUS forms associated with genetic defect of complement factors287ANNUAL REPORT 2007

IRFMNDefinition and validation of new laboratory procedures and predictive models to helpmonitoring and optimizing immunosuppressive therapy in clinical transplantationDefinition and validation of selection and allocation criteria of kidneys from marginal donors toincrease the donor pool and the transplant activity Optimization of Doppler ultrasoundtechniques for the diagnosis and monitoring of vascular complication of renal transplant andchronic dialysis patientsFinalization and activation of multicenter clinical trials aimed to prevent diabetic nephropathy,the progression of chronic nephropathies, acute and chronic allograft rejection and to identifypredictors and promoters of target organ damageComputerization of data acquisition and monitoring procedures for the conduction of controlledclinical trialsNATIONAL COLLABORATIONSLombardia- Ospedale C. Cantù, Abbiategrasso (MI)- Ospedale Civile di Asola, Asola (MN)- Ospedale Fenaroli, Alzano Lombardo (BG)- Azienda Ospedaliera OO.RR., Bergamo- Ospedale Caduti Bollatesi, Bollate (MI)- Azienda Ospedaliera Spedali Civili, Brescia- Ospedale San Biagio, Clusone (BG)- Ospedale S. Anna, Como- Azienda Ospedaliera Istituti Ospedalieri, Cremona- Ospedale di Desio (MI)- Ospedale Briolini, Gazzaniga (BG)- Azienda Ospedaliera di Melegnano, Melegnano – Vizzolo Predabissi (MI)- Ospedale San Leopoldo Mandic, Merate (LC)- Ospedale Maggiore Policlinico, Milano- Ospedale Provinciale San Carlo Borromeo, Milano- Azienda Ospedaliera - Polo Universitario L. Sacco, Milano- Ospedale Fatebenefratelli, Milano- Ospedale Niguarda Ca' Granda, Milano- Clinica Pediatrica “G. e D. De Marchi’, Milano- Ospedale San Raffaele, Milano- Ospedale Pediatrico di Montichiari, Montichiari (BS)- Ospedale San Gerardo, Monza (MI)- Istituti Clinici Zucchi, Monza (MI)- Università degli Studi di Pavia, Dipartimento di Medicina Interna e Terapia Medica, Pavia- Centro Antidiabetico, Ponte San Pietro (BG)- Azienda Ospedaliera Ospedale Treviglio Caravaggio, Romano di Lombardia (BG)- Istituto clinico Humanitas, Rozzano (MI)- Ospedale Bolognini, Seriate (BG)- Azienda Ospedaliera Ospedale Treviglio Caravaggio, Treviglio (BG)- Ospedale Regionale di Circolo Fondazione Macchi, Varese288ANNUAL REPORT 2007

IRFMN- USL 60, Unità Operativa di Nefrologia e Dialisi, Vimercate (LC)Piemonte- Azienda Ospedaliera Santa Croce e Carli, Cuneo- Ospedale Civile, Ivrea- A.S.O. Maggiore della Carità, Novara- Azienda Ospedaliera San Giovanni Battista, Torino- Ospedale Mauriziano Umberto I, Torino- Ospedale Regina Margherita, Torino- Ospedale Martini, Torino- Ospedale Luigi Einaudi, TorinoVeneto, Trentino Alto-Adige e Friuli Venezia Giulia- Casa di Cura Abano Terme, Abano Terme (PD)- Ospedale Civile, Belluno- Ospedale S. Giacomo Apostolo, Castelfranco Veneto, Treviso- Ospedale Provinciale Umberto I, Mestre (VE)- Ospedale Giustinianeo, Padova- Università degli Studi di Padova, Istituto di Anatomia Patologica, Padova- Ospedale Civile, Padova- Ospedale S. Camillo dé Lellis, Schio (VI)- Ospedale Regionale Santa Maria dei Battuti, Treviso- Ospedale Ca’ Fondello, Divisione Nefrologia e Dialisi, Treviso- Ospedale Civile Maggiore Borgo Trento, Verona- Ospedale Policlinico Borgo Roma, Verona- Ospedale Civile San Bortolo, Vicenza- Ospedale Santa Chiara, Trento- Istituto Scientifico per l'infanzia Burlo Garofalo, Trieste- Ospedale S. Antonio, S. Daniele del Friuli, Udine- Università degli Studi di Udine, Centro Trapianti Fegato-Rene-Pancreas, UdineLiguria, Emilia Romagna e Toscana- Azienda Ospedaliera San Martino, Genova- Istituto “G. Gaslini”, Genova- Ospedale S. Orsola Malpighi, Bologna- Ospedale Policlinico, Modena- Istituto di Clinica Medica e Nefrologia, Parma- Ospedale Santa Maria delle Croci, Ravenna- Arcispedale Santa Maria Nuova, Reggio Emilia- Ospedale Santa Maria Annunziata, Bagno a Ripoli, Firenze- Azienda Ospedaliera Careggi-Monna Tessa, Firenze- Ospedale Nuovo “S. Giovanni di Dio”, Firenze- Azienda Ospedaliera Meyer, Firenze- Ospedale di S. Miniato, S. Miniato (FI)- Azienda Ospedaliera Cisanello, Pisa- Ospedale di Pistoia, PistoiaMarche- Ospedale Regionale Torrette, Torrette di Ancona, Ancona- Ospedale I.N.R.C.A., Ancona- Azienda Ospedaliera S. Salvatore, Pesaro289ANNUAL REPORT 2007

IRFMNLazio, Basilicata e Campania- Ospedale Polispecializzato, Anzio, Roma- Ospedale Fatebenefratelli, Roma- Ospedale Pediatrico Bambino Gesù, Roma- Policlinico Gemelli, Roma- Ospedale Policlinico Umberto I, Roma- Ospedale San Camillo Forlanini, Roma- Università Cattolica del Sacro Cuore, Roma- Dipartimento di Biopatologia Umana, Università La Sapienza, Roma- Ospedale Grande degli Infermi, Viterbo- Ospedale Riuniti, Matera- Azienda Ospedaliera Ospedale Civile, Caserta (NA)- Università Federico II di Napoli, Cattedra di Nefrologia, Napoli- Università di Napoli, Policlinico Nuovo, Napoli- Azienda Ospedaliera “S. G. di Dio e Ruggi d’Aragona”, SalernoAbruzzo- Ospedale G. Bernabeo, Ortona, Chieti- Presidio Ospedaliero “San Massimo”, Penne (PE)- Presidio Ospedaliero "G.Mazzini", TeramoPuglia, Calabria, Sicilia e Sardegna- Ospedale Regionale “Miulli”, Acquaviva delle Fonti, Bari- Ospedale Pediatrico “Giovanni XXIII”, Bari- Ospedale Policlinico, Bari- Azienda Ospedaliera V.Fazzi, Lecce- Ospedale Casa Sollievo dalla Sofferenza, S.Giovanni Rotondo (FG)- Presidio Ospedaliero di Martina Franca, Martina Franca, Taranto- A.U.S.L. TA/1 - Presidio Ospedaliero, Taranto- Azienda Ospedaliera Ospedale Pugliese Ciaccio, Catanzaro- Ospedale dell'Annunziata, Cosenza- Centro di Fisiologia Clinica del CNR, Divisione di Nefrologia, Reggio Calabria- Azienda Ospedaliera “Bianchi-Melacrino-Morelli”, Reggio Calabria- Ospedale “N. Giannettasio”, Rossano Calabro, Cosenza- Nuovo Presidio Ospedaliero, Acireale, Catania- Azienda Ospedaliera "Ferrarotto", Catania- Ospedale Zonale Maggiore, Modica (RG)- Ospedale Civico, Palermo- Ospedale “V. Cervello”, Palermo- Azienda Ospedaliera "Umberto I", Siracusa- Azienda Sanitaria G. Brotzu, Ospedale San Michele, Cagliari- Istituto di Clinica e Biologia dell’Età Evolutiva, Cagliari- Ospedale A. Segni, Ozieri, Sassari- Ospedale SS. Annunziata, Sassari- Istituto di Patologia Speciale Medica dell'Università degli Studi di Sassari- Ospedale Policlinico, SassariUmbria- Azienda Ospedaliera di Perugia, Perugia290ANNUAL REPORT 2007

IRFMNINTERNATIONAL COLLABORATIONS- University Medical Center, Ljubljana Slovenia- University Hospital Ziekenhuius, Edegem Antwerpen, Belgio- Clinique de Nephrologie-Dialyse Chu Brugmann, Bruxelles, Belgio- University Ziekenhuius Gent, Gent, Belgio- U.Z. Gasthuisberg, Leuven, Belgio- General Hospital Maria Middelares, Sint Niklaas, Belgio- University of Groningen, AV Groningen, Olanda- Academisch Ziekenhuis, Maastricht, Olanda- Thracian University, Stara Zagora, Bulgaria- The Birmingham Children's Hospital, Birmingham, Inghilterra- Guy’s Hospital, London, Inghilterra- Manchester Children's Hospital, Manchester, Inghilterra- Nottingham City Hospital, Nottingham, Inghilterra- Aalborg Hospital, Aalborg, Denmark- Nephrological Department, University of Copenaghen, Copenaghen, Danimarca- Steno Diabetes Center, Gentofte, Danimarca- Department of Nephrology, Odense University Hospital, Odense, Danimarca- Department of Nephrology, Sahlgrenska University Hospital, Goteborg, Svezia- Ospedale San Giovanni, Bellinzona Svizzera- Department of Nephrology, University of Wien, Wien, Austria- Carl Thiem Klinikum, Cottbus, Germania- Klinikum der Johann Wolfgang, Frankfurt am Main, Germania- Arbeitsgruppe fyr Biomolekulare Medizin, Hamburg, Germania- Univeristatklinik Heidelberg, Heidelberg, Germania- Medizinische Klinik, Mannheim, Germania- Luitpold Krankenhaus Med. Universitatklinik/Dialyse, Wurzburg, Germania- Hospital Ntra Sra. de Sonsoles, Avila, Spagna- Hospitalet de Llobregat, Institut Català de la Salut, Barcellona, Spagna- Fundacion Jimenez Diaz, Madrid, Spagna- Hospital Clinico Martin Logas, Madrid, Spagna- Hospital 12 de Octubre, Madrid, Spagna- Hospital Gregorio Maranon, Madrid, Spagna- Hospital La Paz, Madrid, Spagna- Hospital Puerta de Hierro, Madrid, Spagna- Hospital Ramon y Cajal, Madrid, Spagna- Hospital Severo Ochoa, Leganes, Madrid, Spagna- Hospital Universitario de Tarragona Joan XXIII, Tarragona, Spagna- Hospital Garcia de Orta, Almada, Portogallo- Brigham & Women's Hospital, Boston, USA- Hennepin County Medical Center, Minneapolis, USA- SIU School of Medicine, Springfield, USA- The Toronto Hospital, Toronto, Canada291ANNUAL REPORT 2007

IRFMN- INCUCAI, Buenos Aires, Argentina- Hospital Italiano de Buenos Aires, Buenos Aires, Argentina- Hospital Regional de Valdivia, Valdivia, Cile- Soroka Medical Center, Beer Sheva, IsraeleEDITORIAL BOARD MEMBERSHIPJournal of the American Society of Nephrology (Piero Ruggenenti)Journal of Nephrology (Piero Ruggenenti)Current Diabetes Reviews (Piero Ruggenenti)Clinical Journal of the American Society of Nephrology (Piero Ruggenenti)Current Pharmacology Reviews (Dario Cattaneo)PEER REVIEW ACTIVITIESAmerican Journal of Physiology-Renal PhysiologyAmerican Journal of Kidney DiseasesAmerican Journal of TransplantationBritish Journal of HaematologyJournal of the American Society of Nephrology (JASN)Clinica Chmica ACTAClinical BiochemistryClinical ChemistryClinical Journal of the American Society of Nephrology (CJASN)Clinical Pharmacology & TherapeuticsCurrent Clinical PharmacologyCurrent Diabetes ReviewsDiabetesDiabetic MedicineDrugsExpert Opinion on PharmacotherapyFuture MedicineHypertensionJournal of Clinical InvestigationJournal of Chromatographic SciencesJournal of Clinical PharmacologyJournal of ImmunologyJournal of NephrologyJournal of PostGraduate MedicineKidney InternationalNature MedicineLancet292ANNUAL REPORT 2007

IRFMNLupusNew England Journal of MedicineNephrology Dialysis and TransplantationPediatric NephrologyPediatric TransplantationPlosTalantaTherapyTransplant ImmunologyTransplantationWHO BullettinPARTICIPATION IN EVENTS IN WHICH THE DEPARTMENT WASINVOLVEDC-440T/T-331C polymorphisms in the UGT-1A9 gene affect the pharmacokinetics ofmycophenolic acid in renal transplant recipients”. All’interno dell’ American TransplantCongress, San Francisco, May, 5-9 2007.“Pharmacokinetics of mycophenolic acid after the conversion from mycophenolate sodium tothe mofetil formulation in kidney transplant recipients”. All’interno dell’ IATDMCT Meeting,Nizza, September 2007.“Blood cell count and lipid lowering therapy affect sirolimus exposure in kidney transplantrecipients on calcineurin inhibitor-free regimen”. All’interno dell’IATDMCT Meeting, Nizza,September 2007.“Farmacocinetica degli immunosoppressori: update su acido micofenolico”. All’interno delcorso di aggiornamento Roche, Isola di Capo Rizzuto, 19 September 2007.“Statine, dislipidemia e nuovi ipolipemizzanti”. All’interno del convegno: Nefropatie croniche ecomplicanze cardiovascolari. Ranica 1 December 2007.Conference “Clinical management of hyperglicemia and metabolic syndrome”, Treviglio, Italy,17 November 2007Tenth International Conference on Endothelin, Bergamo, Italy, 16-19 September 2007International Workshop ”Endocannabinoids in Endocrinology, Metabolism and CardiovascularDisease”, Padua, Italy, July, 5-7 2007Retraining course of BLS-D “BASIC LIFE SUPPORT - EARLY DEFIBRILATION”. 28 March 2007Investigator meeting – Clinical trial PLANET, Barcelona, March, 9-12 2007Society for Clinical trials, Montreal Canada, May 20-23 2007“Progression of chronic renal disease – The state of the art and an update”10 th International Symposium of Nephrology at Montecatini – Montecatini Terme, Italy, March22-24, 2007Roche Aleglitazar Investigator Meeting, Istanbul, Turkey, April 17-20, 2007293ANNUAL REPORT 2007

IRFMN“Il laboratorio in nefrologia” - 5° Incontro Nefrologico a Viterbo, 8 June 2007VAM Almirall 2007. Vascular Disease: A multidisciplinary approach, “Nephroprotection”,Barcellona, January 26 th -27 th , 2007.Aspreva’s Renal Strategy Meeting, Amsterdam, February 23 rd , 2007.CME Course. Proteinuria in renal transplantation: Pathophysiology, Diagnosis, Treatment,“Post transplant HUS: new ways to prevention and treatment”, Firenze, March 21 st -22 nd , 2007.XLIV ERA-EDTA Congress, Literature Update “Diabetic and non-diabetic progressivenephropathies”, Barcellona, June 21-24, 2007.48° Congresso Nazionale SIN, “Il rene policistico dell’adulto: stiamo uscendo dal tunnel?”,Fiera del Levante – Bari, October 7-10, 2007.Cardio Kidney Diabetes (CKD) Global Consensus Conference, Chicago, IL, October 17-19,2007.39° Corso di Aggiornamento in Nefrologia e Metodiche Dialitiche, “Role and Organization ofRemission Clinics”, Milano, 6 - 9, 2007GRANTS AND CONTRACTSAIFA (Agenzia Italiana del Farmaco)PKD FoundationAbbott GmbH & Co.Astrazeneca SPAACRAF Spa (Aziende Chimiche Riunite Angelini Francesco)Chiesi FarmaceuticiDompè SpaFarmaceutici Damor SpAKing Pharmaceuticals Inc.Novartis FarmaRoche SpASanofi-Aventis SpaSigma Tau SpaSolvay PharmaceuticalsSpeedel Pharma Ltd294ANNUAL REPORT 2007

IRFMNSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, Aiello S, Cassis L, Gotti E, Gaspari F,Cattaneo D, Perico N, Remuzzi G. “Lymphopenia and calcineurin signalling expand T regulatory cells in humanrecipients of organ transplantation”. JASN 2007, 18:1007-1018.Cattaneo D. Does IgA nephropathy affect long-term graft outcome after kidney transplantation? J PostGrad Med,2007, 53:84.Corna D, Sangalli F, Cattaneo D, Carrara F, Gaspari F, Remuzzi A, Zoja C, Benigni A, Perico N, Remuzzi G.“Effects of rosuvastatin on glomerular capillary size-selectivity function in rats with renal mass ablation”. Am JNephrol 2007, 27:630-638.Baldelli S, Merlini S, Cattaneo D, Nicastri A, Bartolini B, Perico N, Remuzzi G. “C-440T/T-331C polymorphisms inthe UGT-1A9 gene affect the pharmacokinetics of mycophenolic acid in renal transplant recipients”.Pharmacogenomics, 2007, 8:1127-1141.Carlucci F, Anzini M, Rovini M, Cattaneo D, S. Merlini S, Tabucchi A .”Development of a capillary electrophoresismethod for the determination of mycophenolic acid in human plasma: a comparison with HPLC”. Electrophoresis2007, 28:3908-3914.Cattaneo D, Cortinovis M, Baldelli S, Bitto A, Gotti E, Remuzzi G, Perico N. Pharmacokinetics of mycophenolatesodium and comparison with the mofetil formulation in stable kidney transplant recipients. CJASN 2007, 2: 1147-1155.Ripamonti D, Cattaneo D, Airoldi M, Frigerio L, Bertuletti P, Ruggeri M, Suter F, Maggiolo F. Atazanavir plus lowdoseritonavir in pregnancy: pharmacokinetics and placental transfer. AIDS 2007; 21:2409-2415.Cravedi P, Ruggenenti P, Remuzzi G. Intensified inhibition of renin-angiotensin system: a way to improve renalprotection? Curr Hypertens Rep. 2007;9(5):430-6.Ruggenenti P, Cravedi P, Remuzzi G. Latest treatment strategies for membranous nephropathy. Expert OpinPharmacother. 2007;8(18):3159-71.Ruggenenti P, Perico N, Gotti E, Cravedi P, D'Agati V, Gagliardini E, Abbate M, Gaspari F, Cattaneo D, Noris M,Casiraghi F, Todeschini M, Cugini D, Conti S, Remuzzi G. Sirolimus versus cyclosporine therapy increasescirculating regulatory T cells, but does not protect renal transplant patients given alemtuzumab induction fromchronic allograft injury. Transplantation. 2007;84(8):956-64.Cravedi P, Noris M, Remuzzi G. Report of the first World Transplant Congress. Clin J Am Soc Nephrol.2007;2(2):393-400.Cravedi P, Ruggenenti P, Sghirlanzoni MC, Remuzzi G. Titrating rituximab to circulating B cells to optimizelymphocytolytic therapy in idiopathic membranous nephropathy. Clin J Am Soc Nephrol. 2007;2(5):932-7.Remuzzi G, Cravedi P, Costantini M, Lesti M, Ganeva M, Gherardi G, Ene-Iordache B, Gotti E, Donati D, SalvadoriM, Sandrini S, Segoloni G, Federico S, Rigotti P, Sparacino V, Ruggenenti P. Mycophenolate mofetil versusazathioprine for prevention of chronic allograft dysfunction in renal transplantation: the MYSS follow-uprandomized, controlled clinical trial. J Am Soc Nephrol. 2007;18(6):1973-85.Cravedi P, Ruggenenti P, Remuzzi G. Does remission of renal disease associated with antihypertensive treatmentexist? Curr Hypertens Rep. 2007;9(2):160-5.Sanna-Cherchi S, Carnevali ML, Martorana D, Cravedi P, Maggiore U, Alinovi R, Bovino A, Mattei S, Orlandini G,Gatti R, Savi M, Sado Y, Neri TM, Allegri L. Alterations of type IV collagen alpha chains in patients with chronicacquired glomerulopathies: mRNA levels, protein expression and urinary loss. Am J Nephrol. 2007;27(2):129-37.Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, Aiello S, Cassis L, Gotti E, Gaspari F,Cattaneo D, Perico N, Remuzzi G. Regulatory T cells and T cell depletion: role of immunosuppressive drugs. J AmSoc Nephrol. 2007;18(3):1007-18.295ANNUAL REPORT 2007

IRFMNCravedi P, Ruggenenti P, Remuzzi G. Kidney Failure Stabilizes After An Increase over 2 Decades. EDTNA/ERCAJournal 3.2007.M. Alpa, B. Ferrero, R. Cavallo, A. Perna, C. Naretto, M. Gennaro, D. Di Simone, L. Bellizia, M. Mansouri, D.Rossi, V. Modena, O. Giachino, L.M. Sena, D. Roccatello. Anti-GM1 and anti-sulfatide antibodies in patients withsystemic lupus erythematosus, Sjogren sindrome, mixed cryoglobulinemia and idiopathic systemic vasculitis. Clinand Experimental Rheumatology 2007, 25(4): 556-562.A. Perna, G.A. Giuliano, A. Schieppati, M.Costantini, M. Ganeva, E. Daina, R. Stevanov, G. Remuzzi. Controlledtrials in rare diseases: how many? How informative? Adequate? 28 th Annual Meeting of the Society for ClinicalTrials, Montreal, Canada, May 20-23, 2007.RESEARCH ACTIVITIESLaboratory of BiostatisticsMycophenolate Steroid Sparing Study (MYSS) Follow-Up extension study:final analysesMYSS study results did not show any significant difference between Mycophenolate Mofetil(MMF) and Azathioprine (AZA), combined with Cyclosporine Neoral and steroids, in reducingacute rejection in cadaveric kidney allograft recipients. No difference was found even withinpatients who, after 6 months from transplant, progressively tapered and interrupted steroidtreatment. Due to the relatively limited follow-up, however, the above study could not assess theeffects of MMF and AZA on the onset and progression of chronic allograft dysfunction, asyndrome of proteinuria and worsening renal function with progressive nephron loss andscarring of the graft (chronic allograft nephropathy). This is a key issue since, after recipientdeath, chronic allograft dysfunction represents the major cause of graft loss in the long-tem.Moreover, results from registry analyses showed that continued treatment with MMF versusAZA was associated with a protective effect against renal function deterioration beyond 1 yearafter transplantation and superior graft survival at 4 years. Despite the limitations of theretrospective design of the above analyses, these data further limited the possibility to useresults of the MYSS trial to change the practice of most transplant centres to regard MMF as akey component of immunosuppressive drug regimens based on ciclosporine microemulsion. Toaddress this issue, we designed the MYSS follow-up study. This was an extension of the MYSSstudy, prospectively comparing long-term outcomes of the two cohorts of MYSS patients in thesetting of a similar immunosuppressive regimen based on the microemulsion Neoral, accordingto their original randomization to MMF or AZA.Results showed that long-term outcomes in the two groups were comparable, independentlyfrom residual immunosuppression including steroid or not. Thus, in kidney transplantation, thelong-term risk/benefit profile of MMF and AZA therapy in combination with cyclosporineNeoral is similar. In view of the cost, standard immunosuppression regimens for kidneytransplantation should perhaps include AZA rather than MMF.Remission Clinic Program: final analysesFrom January 1999 to November 2004, patients referred to the Unit of Nephrology of theAzienda Ospedaliera, ‘Ospedali Riuniti di Bergamo’ who had a 24 h urinary protein excretionrate of 3 grams or more for at least six months entered the Remission Clinic program. Theprogram included patients who were treated and monitored according to a sequential, stepwise,multimodal protocol titrated to urinary protein excretion. After a period of up-titration during296ANNUAL REPORT 2007

IRFMNthe first month, patients were maintained on ramipril (dose range: 2.5-20 mg/day) and losartan(dose range: 50-200 mg/day) doses allowed as deemed appropriate according to blood pressurecontrol and tolerability. At 3 months after inclusion into the Remission Clinic program, thosepatients who had an heart rate > 60 beats/min and were not receiving a beta-blocker for aspecific indication were prescribed a fixed dose (80 mg/day) of a ndCCB (verapamil) that, iftolerated, was up-titrated to 120 mg/day. Three months later, a fixed dose (10 mg/day) of astatin (atorvastatin) was prescribed and, if tolerated, was up-titrated to 20 mg/day. Then patientswere seen every 3-6 months up to study end. Stopping rules for safety/tolerability reasons wereapplied. Results of the 56 consecutive patients included in the Remission Clinc program werecompared with those of a matched cohort of 56 proteinuric patients who received ACE inhibitortherapy alone. The primary target of treatment was twenty-four hour urinary protein excretionrate. Three categories of response to treatment were a priori defined on the basis of 24 h urinaryprotein excretion achieved during the follow-up period: 1. Residual 24 h proteinuria in clinicalrange (i.e. persistently more than 1.0 g); 2. Reduction of 24 h proteinuria to sub-clinical range(1.0 g or less, but more than 0.3 g in 2 consecutive visits); 3. Reduction of 24 h proteinuria tonormal range (0.3 g or less in 2 consecutive visits). The primary efficacy variable was the rateof estimated GFR decline (eGFR). Hard end-points were all-cause and cardiovascular deaths,non-fatal cardiovascular events and ESRD, considered either individually, either as a compositeend point including all the eventsPreliminary data showed that the Remission Clin approach was more effective in reducingproteinuria than a therapy based only on ACE inhibitor. Reduction of proteinuria translated intoimproved renal function and decreased cardiovascular events.Preprocessing of hospital clinical data for comparing differentimmunosuppressive therapies in renal transplanted patients.Since August 2005 the Unit of Nephrology of the Azienda Ospedaliera ‘Ospedali Riuniti diBergamo’ outlined an immunosuppressive regimen based on low doses of Rabbit Anti-humanThymocyte Globulin (RATG) in association to basiliximab. This induction therapy attempted tominimize or eliminate steroids, with aim to reduce acute rejections. In order to facilitatestatistical evaluation of acute rejection, of adverse drug reactions due to immunosuppressivetherapies and of renal and patient survival hospital clinical data were preprocessed, extractedand elaborated by means of dedicated software. For data retrieval we used a relational database,Microsoft Access ® , where the records of all subjects referring to the Unit of Nephrology arestored.. An ‘ad hoc’ program coding previous and concomitant diseases was built up. By meansof SQL and Visual Basic we facilitated the choice and description of the disease within a predefinedlist. For previously archived data, an algorithm for an automatic research of key wordswas implemented, adding the identified code to the corresponding diseases. Finallydemographic and clinical data at baseline and on follow up were extracted for data analysis. Forfollow up visits an ad hoc algorithm searching the nearest visit to a pre-defined time windowwas implemented. The above data were extracted and subsequently imported in SAS System ®and submitted to statistical analysis.We compared the outcomes of kidney transplant patients (n=40) who received combinedinduction with low-dose RATG plus basiliximab with those of a matched-cohort (n=40) ofpatients who did not receive induction therapy. Beside induction, the first cohort of patientsreceived maintenance immunosuppression with low-dose mycophenolate mofetil andcyclosporine. Methylprednisolone was infused on day 0, 1 and 2 post transplant. Thereafter,patients were free of steroids. Reference patients received standard-dose MMF and CsA In thisgroup, steroids were progressively tapered and withdrawn from month 6 to month 9 aftertransplantation.Preliminary results showed that the induction strategy with low-dose RATG combined withbasiliximab allowed to prevent acute rejection without steroids, and to reduce the doses ofmaintenance immunosuppression with MMF and CsA. Steroid avoidance improved the blood297ANNUAL REPORT 2007

IRFMNpressure levels and the lipidic profile. Moreover, the use of lower than conventional doses ofCsA in patients who received combined induction was associated with a better renal functionBENEDICT Phase B: final analysesPhase B of the multicenter double-blind, randomized Bergamo Nephrologic DiabetesComplications Trial (BENEDICT) assessed the effects of angiotensin-converting-enzyme(ACE) inhibitors alone or combined to non-dihydropyridine calcium-channel blockers onregression to normoalbuminuria or progression to macroalbuminuria in type 2 diabetics withmicroalbuminuria, and evaluated whether these effects were modulated by the ACEinsertion(I)/deletion(D) polymorphism. Two-hundred-eighty-one subjects were randomized toreceive 4.5 years of treatment with trandolapril (2 mg per day) plus verapamil (180 mg per day)or trandolapril alone (2 mg per day). Target blood pressure was 120/80 mmHg. Main efficacyvariables were regression to normoalbuminuria (overnight albuminuria

IRFMNSurvey on the quality of published randomized controlled trials in rarediseasesWe conducted a descriptive survey in 71 rare diseases with MESH term selected from a list of1608 orphan indications in order to evaluate the main characteristics and quality of randomizedcontrolled trials (RCTs) in this neglected area. Through PubMed (1994-2005) we found 23459titles, retrieving 351 abstracts of RCTs, from which we selected 168 full published articles withparallel group design. In the 2007 we completed data collection and data analysis.Laboratory of Clinical ChemistryReliability of neutrophil gelatinase-associated lipocalin (NGAL) as an earlymarker in predicting acute renal dysfunction in patients with solid organcancer given cisplatin as chemotherapeutic agentAcute renal failure represents a very important and potentially devastating disorder in clinicalmedicine. Despite substantial technical improvements in treatments, mortality and morbidityassociated with acute renal failure remain dismally high. Ischemic and nephrotoxic insults to thekidney are the leading cause of acute renal failure and most often manifest as acute tubularnecrosis. While several anti-neoplastic agents frequently exhibit nephrotoxicity, the platinumderivatives are among the most frequent compounds leading to renal injury. Since itsintroduction into clinical trials, cisplatin has had a major impact in cancer medicine, changingthe course of therapeutic management of several tumors, such as those of ovary, testes, and thehead and neck. Unfortunately, approximately 25-35% of patients develop evidence ofnephrotoxicity following an initial dose of cisplatin. Overall these findings indicate that there isa pressing need for way to protect the kidney while administering effective chemotherapeuticagents such as cisplatin. Unfortunately, creatinine is an unreliable indicator during acutechanges in kidney function. Among compounds that might serve as a novel biomarkers for theinitiation phase of acute renal failure, neutrophil gelatinase-associated lipocalin (NGAL) hasbeen identified as one of the most strikingly upregulated genes and overexpressed proteins inthe kidney after ischemia. Markedly increased NGAL concentrations were easily detected inurine early after renal ischemia in mouse and rat models. Recent studies have demonstrated thatNGAL is a useful early predictor of acute renal failure also in humans. Thus, it has been shownthat the concentration of NGAL in urine and serum is strikingly raised in children with acuterenal failure after cardiopulmonary bypass. Interestingly, urine and serum concentrations ofNGAL markedly increased 2 h after surgery.Taken altogether these findings indicate that urinary and/or serum NGAL concentrationmonitoring may represent a sensitive, specific, and highly predictive early biomarker for acuterenal failure. So far, however, no data are available on the reliability of urine and/or serumNGAL to predict the development of acute renal dysfunction in cancer patients receivingcisplatin treatment.A study in 46 adult patients with solid tumors is in progress and is aimed to evaluate thesuitability of early changes increase in urinary and/or serum NGAL concentrations (determinedby ELISA assay) soon after cisplatin infusion, to predict development of subsequent acute renaldysfunction. The study is also aimed to determine, by means of receiver-operatingcharacteristics curve (ROC) analysis, the accuracy of NGAL urinary and/or serumconcentrations to predict acute renal injury; to define a cut-off level of NGAL in both urine andserum samples to predict subsequent acute renal dysfunction; and to evaluate a possiblecorrelation between urinary and/or serum NGAL concentrations and the degree of renal functionimpairment, as the peak of serum creatinine concentration and the nadir of GFR estimated bythe Calvert equation.299ANNUAL REPORT 2007

IRFMNComparison between different analytical methods for albumindetermination in urineAlthough the limit of urinary albumin excretion rate (UAE) of 20 g/min was chosen as adefinition of microalbuminuria because 95% of normal individuals had excretion rates belowthat limit, it is recognized that the risk of cardiovascular events and of progression to overtnephropathy is elevated also in subjects in the “high normal” range (10 – 20 g/min). Mountingevidence indicates a continuous relationship between UAE and risk since epidemiological datasuggest an inconsistency with the concept of threshold level. Thus, both reliability andsensitivity of the chosen method for urinary albumin determination may play an important rolein patients monitoring.Immunochemical assays are the most widely used methods for microalbuminuria measurement.Recent works have demonstrated that intact albumin in urine may exist in two forms,immunoreactive and immuno-unreactive, but only the immunoreactive form can be detected byconventional immunochemical methods. With the purpose of measuring both forms of intactalbumin, we recently developed a high performance size-exclusion liquid chromatography(HPLC) method which is capable to determine both immunoreactive and immuno-unreactivealbumin moieties. On the other hand, HPLC methods to measure albumin, are not commonlyavailable in clinical laboratories. Other immunochemical methods that use less demandinginstruments as been recently proposed as point-of-care testing (POCT) to assess albumin lossresulting from diabetes or other chronic kidney disease states. These instruments offer rapidresults and are also good candidates to become the instrument of choice for prevention programsin developing countries due to no need of technical assistance, minimal performance requiredfor the personnel and relatively low cost per test.In a study aimed to evaluate the performance of different analytical methods, we compared thedetermination of albumin urinary levels in diabetic patients as measured by conventional(nephelometric) and the chromatographic method developed by our laboratory as well as byHemoCue 201, a commercially available POCT instrument, which performs a highly specificturbidymetric assay for human albumin excreted in the urine.Findings indicate that especially for albuminuria

IRFMNReliability of 13 different GFR prediction equations in type 2 diabeticpatientsDiabetic nephropathy affects 25-40% of diabetic patients, and diabetes is the leading cause ofend-stage renal disease. Evaluation of glomerular filtration rate (GFR) is therefore of criticalimportance in the clinical management of both clinic and outclinic diabetic patients. However,the measurement of the true GFR either by the renal clearance of the gold standard inulin or byplasma clearance of contrast agents is time consuming, difficult to perform and cannot be easilyimplemented in clinical daily practice. Urinary creatinine clearance is widely used to measureGFR, but it is inconvenient and sometimes inaccurate due to variable creatinine metabolism andtubular secretion. To circumvent these drawbacks, a number of equation has been developed toprovide an estimate of renal function from serum creatinine and demographic characteristics.Despite being widely used, their performance and suitability to monitor renal function inpatients with type 2 diabetes have not been assessed so far.In 279 normoalbuminuric patients and 134 microalbuminuric subjects enrolled in theBENEDICT and in the DEMAND study we compared with the “gold standard” plasma iohexolclearance the performance in monitoring renal function of 13 different GFR equations.Data analysis showed that GFR was markedly underestimated in both groups of patients. TheRule and Ibrahim formulas performed slightly better than the other models with about 50% theestimated GFR within +10% error as compared to the reference iohexol.To better investigate the performance of the 13 prediction equations in dependence of thekidney function, the diabetic patients were stratified according in the following GFR ranges:from 60 to 89 ml/min/1.73m 2 , from 90 to 119 ml/min/1.73m 2 , and > 120 ml/min/1.73m 2 .Almost all the tested model underestimated GFR and this behavior was more evident at higherrange of renal function, however both Rule and Ibrahim showed a marked overestimation in thelow range of GFR. These findings showed that, irrespectively of albumin excretion rate, none ofthe 13 GFR models seems to safely substitute for the direct measurement of the renal functionby means of an affordable and suitable reference methods, such as iohexol plasma clearance.The development of a new robust GFR prediction equation in type 2 diabetic patients is stillneeded.Development of a sensitive and specific HPLC method for thedetermination of arginine in plasma samples of patients with preeclampsiaExperimental data strongly suggest that nitric oxide (NO), a potent endothelial-derivedvasodilator, might be implicated in gestational vasodilatation. NO is synthesized from theaminoacid L-arginine by a family of enzymes, the NO synthases (NOS). In the normal placenta,adequate concentration of L-arginine selectively orients the endothelial isoform of nitric oxidesynthase (ecNOS) toward NO, which is vital for the low-resistance placenta circulation and alsoexerts a facilitating role on cytotrophoblast invasion. In the pre-eclamptic placenta, instead, alower than normal L-arginine concentration re-directs ecNOS toward peroxynitrite which isgenerated in exuberant amounts at the expense of NO. This favors microvascular damage andimpairs cytotrophoblast invasion.To investigate whether increasing L-arginine bioavailability after oral administration of thecompound might restore physiological NO production in pre-eclamptic placenta we performed,together with the Obstetrics and Gynecology units of the Brescia and Bergamo hospitals, a pilotstudy aimed to explore the impact of the above treatment on pregnancy outcome. For thispurpose we developed a high performance liquid chromatographic (HPLC) method toaccurately determine arginine in both pregnant women with pre-eclampsia and healthy controlsubjects.The developed HPLC method has a good performance and sensitivity and accurately quantifiesarginine in plasma. After a simple plasma proteins precipitation step by means of trichloroacetic301ANNUAL REPORT 2007

IRFMNacid, 10 l are injected onto a 3 cm reversed-phase column. A sodium hydroxide solution ispumped and mixed with the column effluent maintained at 75 °C to allow post-columderivatization of eluted arginine with ninhydrin in mobile phase. The reaction is highly specificfor guadininic compounds, such as arginine, and the derivative is easily quantified by means ofa fluorescence detector.The simplicity and rapidity of sample handling and the analytical specificity for arginineallowed to accurately evaluate the pharmacokinetic profiles of arginine after oral administrationin both pregnant women with pre-eclampsia and in healthy pregnant controls.Laboratory of Drug DevelopmentIndications for rituximab therapy in patients with membranousnephropathyRituximab, a chimeric monoclonal antibody able to deplete CD20 positive B cells effectivelyreduces proteinuria in patients with idiopathic membranous nephropathy (IMN), but response totreatment may vary from patient to patient. Retrospective analyses of heterogeneous patientpopulations given different immunosuppressive regimens failed to identify determinants ofresponse. Aim of the present study was to assess the possible determinants of response torituximab, in order to identify those patients who may benefit the best of this therapyTo this purpose, we retrospectically evaluated by multivariate analyses the association betweenbaseline clinical, laboratory and histology covariates and proteinuria reduction achieved after 4weekly rituximab infusions (375 mg/m 2 ) in IMN patients with proteinuria >=3.5 g/24h while onACE-inhibition for at least 6 months and no previous remissions. Glomerular and tubulointerstitial(TI) scores at baseline biopsy predicted the outcome. Among glomerular and TI scorecomponents, tubular atrophy and interstitial fibrosis were significantly associated with threemonthproteinuria. Urinary protein excretion decreased from 9.1±4.0 g/24h to 4.6±3.5 g/24h(p

IRFMNdepletion of B cells was achieved for a maximum of 4 doses. To assess the cost/effectiveness ofthis novel regimen, we designed a matched-cohort analysis comparing the outcome of 12 newincident patients who received a B cell-driven treatment with that of 24 historical referencepatients who were given the standard protocol of four weekly doses of 375 mg/m 2 .Only one patient needed a second dose to achieve full CD20 cell depletion. At 1 yr, time courseof the components of nephrotic syndrome and the proportion of patients who achieved diseaseremission (25%) was identical in both groups. Persistent CD20 cell depletion was achieved inall patients. Costs for rituximab treatment and hospitalizations totalled 3770.90 euros($4902.20) and 13,977.60 euros ($18,170.80) with the B cell-driven and the four-dose protocol,respectively. One patient on standard protocol had a severe adverse reaction at second rituximabdose. Thus, B cell titrated as effectively as standard rituximab treatment achieves B celldepletion and idiopathic membranous nephropathy remission but is fourfold less expensive,allowing for more than 10,000 euros, approximately $13,000 in savings per patient.Thus, avoiding unnecessary reexposure to rituximab is extremely cost-saving and may limit theproduction of antichimeric antibodies that may increase the risk for adverse reactions andprevent re-treatment of disease recurrences.Pharmacokinetics of mycophenolic acid from mycophenolate sodium andcomparison with that from mofetil formulation in kidney transplantrecipientsThe introduction of mycophenolate mofetil (MMF) has improved long-term graft survival afterorgan transplantation. However the use of this agent may be limited by gastrointestinal andhematological side-effects. To overcome these problems, an enteric-coated formulation ofmycophenolate sodium (EC-MPS) has been recently developed. Detailed data on thepharmacokinetics of MPA released from EC-MPS are lacking. To compare the profiles ofmycophenolic acid (MPA) derived from the two formulations, we have performedpharmacokinetic studies in 20 kidney transplant recipients given EC-MPS (n=10) or MMF(n=10) as a part of their immunosuppressive regimes. At month 6 and 12 post-surgery, despiteno differences in mean MPA Cmax and AUC between the two formulations, aberrant andextremely variable pharmacokinetic curves were found in all patients given EC-MPS.Additionally, these patients presented MPA Tmax ranging from 0 to 480 minutes and doseadjustedMPA trough (C0) 4.7-fold higher compared to patients given MMF. Given theemerging strong support for the clinical outcome benefit of MPA monitoring in transplantsetting, the manufacturer should face with all the potential problems related with the entericcoating of EC-MPS that limit the application of therapeutic drug monitoring, before promotingan extensive use of this novel formulation. Results of this research have been presented at theAmerican Transplant Congress 2007 (S. Francisco, May 2007) and published in the ClinicalJournal of the American Society of Nephrology.Blood cell count and lipid lowering therapy affect sirolimus exposure inkidney transplant recipients on calcineurin inhibitor-free regimenSirolimus (SRL) is an immunosuppressive agent characterized by a narrow therapeutic index.Studies in organ transplant recipients given this drug in combination with cyclosporine (CsA)have shown that SRL exposure is extremely variable. However, no data are available on thepharmacokinetics of SRL in calcineurin inhibitor-free protocols.Fifty-five pharmacokinetics profiles were collected from 20 kidney transplant patients givenSRL over a 36 months follow-up. None of them were on CsA or tacrolimus. Large interindividualvariability in the daily distribution of SRL concentrations was documented. Patientswith low blood cell count showed a significant decrease in daily SRL exposure, requiring 35%increments in the daily dose to reach drug concentration targets. At variance, concomitantadministration of omega-3 polyunsaturated fatty acids increased SRL AUC 0-24 by 25%303ANNUAL REPORT 2007

IRFMNcompared to values found in normolipidemic patients. Altogether, these information can be usedto optimize SRL dose-adjustments in the routine clinical practice. These results have beenpresented at the meeting of the International Association of Therapeutic Drug Monitoring andClinical Toxicology (Nice, September 2007).Genetics partly explains patient variability of mycophenolic acidpharmacokinetics in renal transplant recipientsMycophenolic acid (MPA) is widely used as immunosuppressive agent in renal transplantation.It is mainly metabolized by uridine diphosphate glucuronosyltransferase 1A9 (UGT1A9) to 7-O-glucuronide (MPAG). The UGT1A9 gene that encodes for the UGT protein is localized onchromosome 2q37 and several single nucleotide polymorphisms (SNPs) in the gene have beendescribed. Moreover, excretion in the bile of MPAG occurs through membrane drug effluxprotein, the more relevant being MRP2. This protein is encoded by a gene located on thechromosome 10q24 from whom different SNPs have been so far identified that could also affectthe expression of the protein. The present study was designed to study the impact of thepolymorphisms of UGT1A9 and to investigate the role of polymorphisms in MRP2 in renaltransplant patients MPA pharmacokinetics.Thirty-four patients were enrolled in the pharmacogenetic study. Patients had clinicalevaluations and laboratory tests at 6 month after transplantation, as well as the assessment of thecomplete MPA pharmacokinetic profiles and MPAG measurement. They were genotyped forsingle nucleotide polymorphism (SNPs) in UGT1A9 C-1252T, T-1887G, C-665T, C-440T, T-331C, T-275A, T98C and for MRP2 SNPs C-24T and G1249A. Association of polymorphismswith MPA and MPAG pharmacokinetic parameters was studied.Great MPA pharmacokinetic variability was found, confirmed by values of MPA/MPAGmetabolic ratio ranging from 0.012 to 0.195 among the study patients. In particular, amultimodal frequency distribution was documented that highlighted the presence of at least twodifferent phenotypes.. Significant higher MPA trough levels were found in patients carrier of C-665T polymorphism. MPA and MPAG AUC were significantly associated with the presence ofUGT1A9 -440/-331 genotype. The presence of MRP2 promoter C-24T and exon 10 SNPsG1294A did not cause any significant variation in any MPA and MPAG pharmacokineticparameters.The study has demonstrated a significant impact on MPA pharmacokinetics in renal allograftrecipients of different polymorphisms in the promoter region of the UGT1A9 in particular C-440T/T-331C and C-665T. these results were presented at the American Transplant Congress2007 (S. Francisco, May 2007) and published in Pharmacogenomics.304ANNUAL REPORT 2007

IRFMNLABORATORY OF CLINICALEPIDEMIOLOGYSTAFFHeadGuido BERTOLINI, M.D.Clinical Knowledge Engineering UnitHeadDavide LUCIANI, M.D.305ANNUAL REPORT 2007

IRFMNCURRICULA VITAEGuido Bertolini got his Medical degree in 1989 at the University of Bologna, and the specialization inPharmacological Research in 1993 at the “Mario Negri” Institute and in Gastroenterology in 1994 at theUniversity of Pavia.He founded and chaired from 1997 to 2000 the School of Clinical Methodology and Quality of CareImprovement at the Ospedali Riuniti di Bergamo and the Istituto di Ricerche Farmacologiche MarioNegri. From 1999 to 2003 he was contract professor at the post-doctoral schools in Anaesthesia andIntensive Care, University of Brescia and Milano; from 2002 to 2005 he has been contract professor ofEducational Science at the Faculty of Lettere e Filosofia, University of Bergamo.Current research interests: Clinical Research Methodology, Continuous Quality of Care Assessment andImprovement, Health services research and outcome, Medical decision making, Medical Education.These interests are mainly developed within the fields of Intensive Care Medicine and Rare Diseases.Since 1997 he chairs the GiViTI Coordinating Center for research in intensive care medicine. He has beenHead of the Unit of Epidemiology and Education for Clinical Practice at the “Mario Negri” Institute andsince 2001 he is the Head of the Laboratory of Clinical Epidemiology. Since 2001 he is Vice-chairman ofthe Research Group on Cost-effectiveness, Section on Health Services Research and Outcomes –European Society of Intensive Care Medicine and, since 2004, he is President of the Scientific Committeeof the “Ospedale maggiore” in Crema.Selected publications• Bertolini G, Rossi C, Anghileri A, Livigni S, Addis A, Poole D. Use of drotrecogin alfa (activated) in Italian intensivecare units: the results of a nationwide survey. Intensive Care Med 2007; 33: 426-434.• Rossi C, Simini B, Brazzi L, Rossi G, Radrizzani D, Iapichino G, Bertolini G. Variable costs of ICU patients: amulticenter prospective study. Intensive Care Med. 2006;32:545-52• Vanoli M, Daina E, Salvarani C, Sabbadini MG, Rossi C, Bacchiani G, Schieppati A, Baldissera E, Bertolini G.Takayasu's arteritis: A study of 104 Italian patients. Arthritis Rheum 2005; 15;53: 100-107. IF: 7.421.• Malacarne P, Rossi C, Bertolini G; GiViTI Group. Antibiotic usage in intensive care units: a pharmaco-epidemiologicalmulticentre study. J Antimicrob Chemother. 2004 Jul; 54:221-4• Galli M, Luciani D, Bertolini G, Barbui T. Anti-beta 2-glycoprotein I, antiprothrombin antibodies, and the risk ofthrombosis in the antiphospholipid syndrome. Blood 2003;102:2717-2723.• Simini B, Bertolini G. Should same anesthetist do preoperative anesthetic visit and give subsequent anesthetic?Questionnaire survey of anesthetists. BMJ 2003; 327: 79-80.Davide Luciani got his Medical Degree at the University of Bologna in 1995, and the Diploma in"Tropical Medicine and Hygiene" at the University of Liverpool in 1997. In 2001, he spent one year atthe Department of Statistical Science (University College London). Bayesian probabilistic applications,decision theory and the graphical approach to pathophysiological modeling represent his main interests.Within his research activity, these skills are meant as the main methodological ingredients in theformalization of clinical reasoning, in order to improve its effectiveness and to exploit its educationalvalue.Since 2005 he is responsible of the Unit of Clinical Knowledge Engineering.Selected publications• Luciani D, Cavuto S, Antiga L, Miniati M, Monti S, Pistolesi M, Bertolini G Bayes pulmonary embolism assisteddiagnosis: a new expert system for clinical use Emerg Med J 2007 ; 24 : 157-164• M.Cesana, R.Cerutti, E.Grossi, E.Fagiuoli, M.Stabilini, F.Stella, D Luciani.Bayesian Data Mining Techniques: TheEvidence Provided by Signals Detected in Single-Company Spontaneous Reports Databases. Drug Information Journal,vol. 41, pp. 11-21, 2007• Latronico N, Bertolini G, Guarneri B, Botteri M, Peli E, Andreoletti S, Bera P, Luciani D, Nardella A, Vittorielli E,Simini B, Candiani A Simplified electrophysiological evaluation of peripheral nerves in critically ill patients: the Italianmulti-centre CRIMYNE study Crit Care. 2007;11(1):R11.• Bertolini G, Luciani D, Biolo G Immunonutrition in septic patients: A philosophical view of the current situation ClinNutr 2007 ; 26 : 25-29• Luciani D, Marchesi M, Bertolini G. The role of Bayesian Network in the diagnosis of pulmonary embolism. J ThrombHaemost 2003; 1: 698-707• Galli M, Luciani D, Bertolini G, Barbui T. Anti-beta 2-glycoprotein I, antiprothrombin antibodies, and the risk ofthrombosis in the antiphospholipid syndrome. Blood ,2003 Oct 15;102(8):2717-23Haemostasis, 2003 Apr;1(4):698-707306ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE LABORATORY'S ACTIVITIESThe general aim of the Laboratory of Clinical Epidemiology is to contribute to the improvementof health care in different medical fields. The guiding principles are mainly two: to helpphysicians use the available knowledge and resources at their best; to play a role in the growthof useful knowledge for clinical practice. The Laboratory operates particularly in the field ofIntensive Care Medicine and Rare Diseases.Within the Laboratory, the Unit of Clinical Knowledge Engineering aims to bring the value ofclinical reasoning out, through the implementation of probabilistic models for its formalization,thus favoring the evaluation and the continuous improvement of complex clinical activities.FINDINGS/MAIN RESULTSThe participation to the Margherita project on the quality of care assessment in intensive careunit (ICU), which started in 2002, has further increased during this year. The patients recruitedin 2007 were more than 60.000. This allowed the development of a highly accurate model forthe prediction of hospital mortality in critically ill patients. Each participating ICU has receivedboth a general and an individual report. In the latter, in addition to a detailed description of thecase-mix, we supplied each ICU with the comparison of its own performance with that ofothers.We monitored, on behalf of the Italian Ministry of Health, the clinical use of Drotrecogin alfa(activated) in ICU. This is a new drug for critically ill patients with severe sepsis or septicshock. In the framework of a general appropriate utilization, we found some problems and, thus,the possibility to further increase the quality of care in this field. We provided each ICU with adetailed report on the results of the project.We realized one of the biggest continuous infection surveillance program in ICU. We identifiedthe major problems of each unit in the diagnosis and treatment of infections.We developed the first version of an electronic clinical record for the ICU: a clinical recordshared by many units that will allow to compare their process of care in the framework of acontinuous quality of care improvementWe realized a survey on end-of-life decisions in ICU which involved 90 centres. Results will beavailable early in 2007.We further developed an expert system to assist physicians in the complex diagnosis ofPulmonary Embolism. The new version showed excellent validity.We eventually contributed to the set-up of a European network for the study of patients withparaneoplastic syndrome.NATIONAL COLLABORATIONSDipartimento di Neurologia, Ospedale Regionale S. Maria dei Battuti Cà Foncello, Treviso.Dipartimento di Specialità Chirurgiche, Scienze Radiologiche e Medico Forensi, Cattedra diAnestesia dell’Università degli Studi di Brescia.Servizio Anestesia e Rianimazione, Osp. San Giovanni Bosco, Torino.Divisione di Medicina Interna, Università di Trieste.Divisione di Fisiopatologia Respiratoria, Università di Firenze.Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, Palermo.307ANNUAL REPORT 2007

IRFMNIstituto di Anestesia e Rianimazione, Università di Milano.Servizio Anestesia e Rianimazione, Osp. Civile, Belluno.INTERNATIONAL COLLABORATIONSBloomsbury Institute of Intensive Care Medicine, Institute of Biomedical Research, UniversityCollege London, UK.Department of Statistical Science, University College London, UK.Klink fur Anaesthesiologie und Intensivtherapie, Friedrich-Schiller-Universitat, Jena, GermanyMachine Intelligence Group, University of Aalborg, Denmark.American Board of Family Medicine, Kentucky, USEDITORIAL BOARD MEMBERSHIPRicerca & Pratica (Guido Bertolini)Dedalo. Gestire i sistemi complessi in sanità (Guido Bertolini)British Medical JournalIntensive Care MedicineCriticale Care MedicineCanadian Medical Association JournalRicerca & PraticaPEER REVIEW ACTIVITIESNATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPScientific Committee, azienda ospedaliera “Ospedale maggiore” di Crema.EVENT ORGANIZATIONEducational course, Statistica multivariata per la ricerca biomedica, February-May, Ranica(BG).Workshop, Le decisioni di fine-vita in rianimazione, September 10, Ranica (BG).Workshop, Startup Meeting Compact, September 12, Ranica (BG).Educational course, Utilizzo delle banche dati in medicina, September 21-28, Ranica (BG).Congress, GiViTI Annual Meeting, October 18-20, Pesaro.Educational course, Statistica per medici, November 7-14, Ranica (BG).308ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE LABORATORY WASINVOLVEDMeeting, PNS, Additional Outcomes from the Statistical Analysis, February 24, TrevisoWorkshop, Come raccogliere i dati in medicina, L’esperienza del GiViTI: il progetto MargheritaDue, March 16, FirenzeCongress, BRAIN06, Hot Topic, April 27, BresciaCongress, SMART, Alternative ai trial controllati randomizzati nella ricerca clinica, May 11,MilanoCongress, III Meeting di Formazione e Aggiornamento sugli Interventi in TI, Gli aspettimetodologici della valutazione degli interventi nutrizionali nel paziente critico, May 30,CalampisoMeeting, Antibiotici: attualità, riflessioni, Farmacoepidemiologia degli antibiotici: il casomodellodelle terapie intensive, June 19, SeriateMeeting, POR Sardegna 2000-2006 - Seminario finale misura 5.3, La valutazione dellaperformance della TI: quali dati raccogliere e come utilizzarli, July 30, OristanoMeeting, II Meeting GiViTI Regione Veneto, Il SAPS III e i nuovi progetti GiViTI, Comeestrarre i dati dalla propria Terapia Intensiva, Presentazione di Margherita, September 29,TrevisoCongress, 60° SIAARTI, Le eterne dicotomie della sperimentazione clinica, October 11,PerugiaWorkshop, “Salute e malattia nell’era della tecnica”, November 10, Spiazzi di Gromo (BG)Meeting, “Farmaci: usarli bene per stare meglio. Presentazione di un progetto integrato traOspedale e Territorio, Il ruolo tra formazione ed informazione”, November 18, BergamoMeeting, Registro scompenso cardiaco, La metodologia dei registri epidemiologici, November25, PalermoWorkshop, Formazione degli operatori, Presentazione del software Margherita Tre, December11-12, LivornoMeeting, Progetto Sorveglianza Infezioni, Lettura dei dati della TI di Cesena, December 12,Cesena309ANNUAL REPORT 2007

IRFMNGRANTS AND CONTRACTSRegione LombardiaRegione ToscanaRegione VenetoRegione PiemonteARS ToscanaAstraZeneca ItaliaSanofi-Synthelabo ItaliaDraeger ItaliaBellco SpAMinistero della SaluteAzienda ULSS 16, Padova - ItaliaIstituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, PalermoAzienda ASL4 PiemontePrivate donationSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Luciani D, Cavuto S, Antiga L, Miniati M, Monti S, Pistolesi M, Bertolini G. Bayes pulmonary embolism assisteddiagnosis: a new expert system for clinical use. Emerg Med J 2007; 24: 157-164. IF:Bertolini G, Rossi C, Anghileri A, Livigni S, Addis A, Poole D. Use of drotrecogin alfa (activated) in Italianintensive care units: the results of a nationwide survey. Intensive Care Med 2007; 33: 426-434. IF:Latronico N, Bertolini G, Guarneri B, Botteri M, Peli E, Andreoletti S, Bera P, Luciani D, Nardella A, Vittorielli E,Simini B, Candiani A. Simplified electrophysiological evaluation of peripheral nerves in critically ill patients: theItalian multi-centre CRIMYNE study. Crit Care 2007; 11(1): R11. IF:Bertolini G, Luciani D, Biolo G. Immunonutrition in septic patients: A philosophical view of the current situation.Clin Nutr 2007; 26(1): 25-29. IF:Bertolini G, Rossi C, Anghileri A, Livigni S, Addis A, Poole D. Response to the letter by Williams et al. IntensiveCare Med 2007; 33: 1490-1491. IF:Iapichino G, Radrizzani D, Rossi C, Pezzi A, Anghileri A, Boffelli S, Giardino M, Mistraletti G, Bertolini G.Proposal of a flexible structural-organizing model for the intensive care units. Minerva Anestesiol 2007; 73: 501-506.Luca A, Angermayr B, Bertolini G, Koenig F, Vizzini G, Ploner M, Peck-Radosavljevic M, Gridelli B, Bosch J. Anintegrated MELD model including serum sodium and age improves the prediction of early mortality in patients withcirrhosis. Liver Transpl 2007;13:1174-1180. IF:310ANNUAL REPORT 2007

IRFMNOTHER PRODUCTS PUBLISHED IN 2007Bertolini G (a cura di) Scelte sulla vita. L’esperienza di cura nei reparti di terapia intensiva. Milano: Guerini Studio,2007.Boffelli S, Rossi C, Bertolini G. Progetto Margherita. Promuovere la ricerca e la valutazione in Terapia Intensiva.Rapporto 2006. Bergamo: Ed. Sestante, 2007.RESEARCH ACTIVITIESAppropriateness in Intensive Care UnitsThe main purpose of these research activities is the assessment and improvement of the qualityof care in Italian Intensive Care Units (ICUs). It is a multi-annual project promoted on behalf ofGiViTI, a collaborative network composed by half of the Italian ICUs and coordinated by theLaboratory. The main focus is the “Margherita” project. Its aim is the continuous evaluation ofthe quality of care and it is based on a free software developed by the Laboratory and distributedto all the ICUs adhering to the GiViTI group. The software has been realized on a modularstructure, which enables to easily integrate the basic data collection (the “core” of Margherita)with the data collection of specific research projects (the “petals” of Margherita).Additionally, in the current year, a software based on a probabilistic model (bayesian network)covering a large set of clinical variables, has been implemented. Such an activity, lead by theUnit of Clinical Knowledge Engineering, and also involving the Department of StatisticalScience (University College London) and the Machine Intelligence Group of the University ofAalborg (Denmark), pursues the realization of tools for the retrospective evaluation of specifictreatments in ICU. By means of this model, it is meant to overcome the current limits of anoverall evaluation of ICU, likewise it happens when the traditional models for mortalityprediction are adopted. Within this activity, a bayesian system to monitor the StandardisedMortality Ratio of the single ICU. This system, which resembles the bayesianpharamcovigilance system currently in use at the WHO and FDA, will identify periods duringwhich some problems occurred, allowing the physicians of the Center to promptly and efficacyreact.Studies on Multiple Organ Failure pathological processesThe Laboratory of Clinical Epidemiology has lead several investigations to clarify whichpathophysiological mechanisms induce multiple organ failure, a condition still burdened withhigh mortality. Among these, the investigation on the neuromuscular impairment in criticalpatients (the observational study 'Crimyne'), the study on the impact of enteral feeding, and thenew treatments proposed for severe sepsis (Xigris and the removal of inflammation mediatorsthrough specific filter applied to circuit of plasma-filtration).The value of a strict glycemic control of critical patients has been recently emphasized, given itsconnection to a drug like insulin that, in spite of its large availability and low cost, induces arelevant reduction of mortality in ICU. The Unit of Clinical Knowledge Engineering hasdeveloped a model based on a differential equations system whose aim is to support thephysician in dosing both insulin and glucose infusions, in order to extend the possibility of astrict control even to patients with a high risk of hypoglycaemia. This model represents aprecious opportunity to investigate the pathophysiological mechanisms behind the benefits of311ANNUAL REPORT 2007

IRFMNinsulin already demonstrated at an empirical level, allowing the explanation of the dynamicbehaviour of glycemic fluctuations on the basis of the patient's metabolic profile.The reconstruction of clinical reasoning in the medical practice andeducationThis area represents the main concern of the Unit of Clinical Knowledge Engineering, whoseobjective is the valorization of clinical reasoning in solving complex clinical problems.The diagnosis of pulmonary embolism still represents a relevant clinical challenge, due to thecomplexity of the patient's clinical presentation and the variability of diagnostic resourcesamong Centres. In this regards, we are conducting an Italian multicenter study, involvingmainly Emergency Units, with the aim of prospectively validating the diagnostic softwareBayPAD (Bayes Pulmonary embolism Assisted Diagnosis). Such a tool, relying on aprobabilistic model covering 72 clinical variables and doing without the need to input all thecontemplated observations, would overcome the main reasons which prevented ordinary clinicalguidelines to be largely accepted. Moreover, the results of the retrospective validation of thesystem have been obtained.The Unit started a project for the realization of a software assisting the physician in tracing backthe basis of his clinical decisions before the description provided by clinical reports, amongthose that are typical of particular medical specialty. The software has the double target to createspecific applications based on probabilistic models representing complex clinical decisionproblems, and to involve physicians in their construction. The last target is achievable given thestrong analogy between the causal structure of the exploited models (bayesian networks) and thepathophysiological structure of medical knowledge. By this, it will be given the chance to adoptthis system within medical training projects, with a special attention to e-learning programs.Clinical Epidemiology of rare diseases and orphan medicineOur purpose is to find out and describe clinical and research problems related either to rarediseases or to neglected aspects of well-known diseases. We also focus on the needs of thepatients with rare diseases. A specific project is connected with this research activity: “PNS –Euronetwork”. It is a European project on paraneoplastic neurological syndromes that isfinanced by the Fifth and Sixth Framework Program of the European Community. Its purposesare various: to develop a network of reference centers for these pathologies all sharing acommon database; to organize a sample bank of biological fluids and cerebrospinal liquid topoint out the best antibody indicators for the diagnosis and prognosis of these patients; to realizesome research projects on the treatment of this syndrome.312ANNUAL REPORT 2007

IRFMNLABORATORY OF COORDINATION OFDIAGNOSIS AND INFORMATION ONRARE DISEASESSTAFFHeadArrigo SCHIEPPATI, M.D.Information Centre for Rare DiseasesHeadErica DAINA, M.D.313ANNUAL REPORT 2007

IRFMNCURRICULA VITAEArrigo Schieppati got his degree in Medicine at the University of Milan in 1978 and the specialisation inMedical Nephrology in 1984 at the same University.He performed his training at the Mario Negri Bergamo Laboratories with Dr. Remuzzi, and completed itwith stages at the laboratories of prof. Patrono (Catholic University in Rome), prof. John Gordon(Cambridge, GB), and at the Division of Renal Diseases - University of Colorado Medical School,directed by Dr. Schrier (Denver, USA). Since 1982 he works at the Division of Nephrology and Dialysis– Riuniti Hospital – Bergamo, where he is in charge of Outpatients Clinic and Day Hospital. From 1992to 1995 he was Head of the Information Center for Rare Diseases and since 1996 he is Head of theLaboratory for Coordination of Information and Diagnosis of Rare Disease at the Clinical ResearchCenter for Rare Diseases Aldo e Cele Daccò of the Mario Negri Institute.Areas of interest: diagnosis and therapy of chronic renal diseases, hypertension and rare kidney diseases.Affiliations: ethical committee Riuniti Hospital - Bergamo; member of the working group of the regionalnetwork for rare diseases in Lombardy; scientific committee Bolognini Hospital – Seriate (BG); memberof the Task Force on Rare Diseases (DG Health and Consumer Protection); International Society ofNephrology; American Society of Nephrology; Editorial Board Journal of Nephrology, Editorial Board ofJASN - Nephrology Self Assessment Program.Selected publications• Schieppati A, Remuzzi G. Chronic renal diseases as a public health problem: epidemiology, social, and economicimplications. Kidney Int Suppl. 2005 Sep;(98):S7-S10.• Remuzzi G, Schieppati A, Boissel JP, Garattini S, Horton R. Independent clinical research in Europe. Lancet. 2004 Nov6-12;364(9446):1723-6.• Schieppati A, Perna A, Zamora J, Giuliano GA, Braun N, Remuzzi G. Immunosuppressive treatment for idiopathicmembranous nephropathy in adults with nephrotic syndrome. Cochrane Database Syst Rev. 2004 Oct 18;(4):CD004293.• Remuzzi G, Schieppati A, Ruggenenti P. Clinical practice. Nephropathy in patients with type 2 diabetes. N Engl J Med.2002 Apr 11;346(15):1145-51.• Schieppati A, Remuzzi G, Garattini S. Modulating the profit motive to meet needs of the less-developed world. Lancet.2001 Nov 10;358(9293):1638-41.• Ruggenenti P, Schieppati A, Remuzzi G. Progression, remission, regression of chronic renal diseases. Lancet. 2001 May19;357(9268):1601-8.Erica Daina got his degree in Medicine at the University of Milan in 1987 and the specialisation inMedical Nephrology in 1990 at the same University.She performed her training at the II° Medical Division - San Raffaele Hospital - Milan, and at theDivision of Nephrology and Dialysis - Riuniti Hospital - Bergamo.In March 1988 she started her collaboration with the Mario Negri Institute and since June 1993 she worksas full-time clinical researcher at the Clinical Research Center for Rare Diseases Aldo e Cele Daccò.Since 1996 she is Unit Head – Information Center for Rare Diseases. Areas of interest: rare diseases,Takayasu arteritis, Hemolytic Uremic Syndrome/Thrombotic Thrombocytopenic Purpura, Fabry’sdisease, Alport’s syndrome. Since January 2002 she is representative of Coordinating Centre - RegionalNetwork for Rare Diseases - and collaborates to didactics activity at the University of Turin (ClinicalPathology of Rare Diseases - School of Clinical Pathology Specialization).Selected publications• Vanoli M, Daina E, Salvarani C, Sabbadini MG, Rossi C, Bacchiani G, Schieppati A, Baldissera E, Bertolini G; ItakaStudy Group. Takayasu's arteritis: A study of 104 Italian patients. Arthritis Rheum. 2005 Feb 15;53(1):100-7.• Remuzzi G, Galbusera M, Noris M, Canciani MT, Daina E, Bresin E, Contaretti S, Caprioli J, Gamba S, Ruggenenti P,Perico N, Mannucci PM; Italian Registry of Recurrent and Familial HUS/TTP. von Willebrand factor cleaving protease(ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremicsyndrome. Blood. 2002 Aug 1;100(3):778-85.• Noris M, Daina E, Gamba S, Bonazzola S, Remuzzi G. Interleukin-6 and RANTES in Takayasu arteritis: a guide fortherapeutic decisions? Circulation. 1999 Jul 6;100(1):55-60.• Daina E, Schieppati A, Remuzzi G. Mycophenolate mofetil for the treatment of Takayasu arteritis: report of three cases.Ann Intern Med. 1999 Mar 2;130(5):422-6.• Bresin E, Daina E, Noris M, Castelletti F, Stefanov R, Hill P, Goodship T H J, Remuzzi G, International RegistryRecurrent Familial HUS/TTP. Outcome of renal transplantation in patients with non-Shiga toxin-associated HemolyticUremic Syndrome: Prognostic significance of genetic background. Clinical Journal American Society Nephrology 2006;1: 88-99314ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE LABORATORY’S ACTIVITIESRare Diseases (RD) represent about ten percent of all human medical illnesses and infirmities. Itis difficult to define what exactly is intended as a RD. The US Congress in the Orphan Drug Acthas given the first definition in 1983. Under this law it is considered rare a disease that affectsless than 200 000 Americans (prevalence 0.75 per 1 000).Recently, the European Parliament adopted a more strict definition; they consider rare acondition that affects not more than five individuals per 10 000 in the European Community(prevalence 0.5 per 1 000). Besides the epidemiological parameter, RD have certaincharacteristics in common: 1) most of them are of genetic origin; 2) rarity often brings adifficult and/or late diagnosis; 3) generally, RD are heavy social burdens both to the family andcommunity and cause early mortality.The greatest barrier to prevention, diagnosis and treatment of RD is inadequate knowledge.Once a diagnosis of RD is put, a major complaint of patients and those involved in their care isthe difficulty to obtain pertinent information about causes, symptoms and either established orexperimental treatments. Often, patients with RD are willing to participate in clinical studies,but they do not know where and how, and physicians or health authorities are seldom able tohelp them.RD is not a very attracting field for basic and clinical investigators for several reasons: it isdifficult to find adequate animal models for many rare disorders; clinical trials may requiremore patients than available; financial support is insufficient.Few countries have a central body or system to disseminate information on RD. Accurateinformation on the incidence and prevalence of RD is extremely important for both basic andclinical investigators. Invaluable help to research advances in RD would come from theavailability of registries and databases containing diagnostic, clinical and biological data ofpatients with rare disorders.A pilot experience has been established at the Clinical Research Centre for Rare Diseases “Aldoand Cele Daccò” since 1992. This Centre is unique with its integration of an Information Centrefor Rare Diseases, clinical facilities for the implementation and development of clinical studies,educational activities for physicians, nurses and patients.In 2001 it has been nominated Coordinating Centre of the Regional Network for Rare Diseasesin the Lombardy Region, an area of 9 million people in Northern Italy. As Coordinating Centreis also working with the National Centre of Rare Diseases at Istituto Superiore di Sanità. All theup-to-date information regarding the activities of the Coordinating Centre are available at theweb site: http://malattierare.marionegri.it315ANNUAL REPORT 2007

IRFMNFINDINGS/MAIN RESULTSThe database of the Information Centre for Rare Diseases contains data about 10403 patientsaffected by 782 different rare disorders.In the Bank of biological materials, samples from 1241 patients with rare conditions and theirfamilies have been collected.The Centre has established contacts with more than 310 Italian Associations for rare diseases. Itwas even possible that patients with more than 81 different rare diseases - for which noAssociations have been established in Italy yet - to meet among themselves.In July 2000, the European Commission recognized the Laboratory as a site for "Postgraduatetraining on rare diseases" (contract No. QLK4-1999-50547).In December 2001 (Delibera della Giunta Lombarda N. 7328), the Centre was identified as"Coordinating Centre of the Regional Network for Rare Diseases".The Laboratory coordinates the International Registry of Recurrent and Familial HemolyticUremic Syndrome (HUS) and Thrombotic Thrombocytopenic Purpura (TTP), since 1996. Theresearch projects developed in collaboration with the Laboratory of Immunology and Genetic ofRare Diseases and Organ Transplantation have allowed to better comprehend the pathogenesisof these diseases and to identify some genetically determined forms of HUS, associated todefects of complement regulatory factors, and of TTP, associated to congenital deficiency ofADAMTS13 enzyme.NATIONAL COLLABORATIONSItalian National Institute of HealthG. Bosco Hospital, Turin, Regional Coordinating Center for Rare DiseasesRiuniti Hospital, BergamoItalian Takayasu's Arteritis Study Group - ITAKA GroupBiotechnology Laboratory, IRCCS Policlinico San Matteo, PaviaAssessorato alla Sanità, Lombardia RegionUniversity of Torino, School of Clinical Pathology, Faculty of Medicine and SurgeryItalian Network for Promotion of Folic Acid to Prevent Birth DefectsUniversity of Turin, Department of Experimental Medicine and Oncology, 2 nd Level Master inRare DiseasesItalian Society of Neonatology (Lombary section), Rare Congenital Respiratory Diseases StudyGroupUniversity of Milan, 1 st Level Master in Clinical ResearchDepartment of Molecular Biology, Unit of Medical Genetics, Policlinico “Le Scotte”, SienaUnit of Cytogenetics and Genetics, Careggi Hospital, FirenzeLaboratory of Metabolic Diseases, National Neurological Institute “Carlo Besta”, MilanoLaboratory of Biochemistry and Genetics, National Neurological Institute “Carlo Besta”,Milano“BergamoScienza” Association316ANNUAL REPORT 2007

IRFMNINTERNATIONAL COLLABORATIONSEuropean CommunityInternational Registry of Recurrent and Familial Hemolytic Uremic Syndrome and ThromboticThrombocytopenic PurpuraInformation Centre for Rare Diseases and Orphan Drugs – ICRDOD, BulgariaEDITORIAL COMMITTEE MEMBERSHIPJournal of American Society of Nephrology - Nephrology Self Assessment Program (ArrigoSchieppati)Journal of Nephrology (Arrigo Schieppati)Quaderni di Farmacoeconomia (Erica Daina)NATIONAL AND INTERNATIONAL COMMITTEE MEMBERSHIPNetwork for Rare Diseases – Lombardy Region (Delibera Regione Lombardia N°7328,11/12/2001).Task Force on Rare Diseases (established by DG Health and Consumer Protection on 21January 2004).Scientific Committee A.O. Bologni di Seriate.Ethical Committe, A.O. Ospedali Riuniti di BergamoEVENT ORGANIZATION“Consensus Conference: Kidney and Liver Transplantation in Hemolitic Uremic Syndrome”Clinical Research Centre for Rare Diseases “Aldo and Cele Daccò” – Mario Negri InstituteRanica (Bergamo), December 15 th 2007PARTICIPATION IN EVENTS IN WHICH THE LABORATORY WASINVOLVED10° Convegno “Patologia Immune e Malattie Orfane”Torino, January 25-27, 2007Workshop: Epidemiological methods to define incidence, prevalence anda mortality or rarediseases affecting “the young”Rome, February 16, 2007Clinical and Experimental RheumatologyII Corso teorico-pratico. La Settimana delle malattie rare in ReumatologiaPisa/Buti, march 14-20, 2007Diagnosticare le malattie rare in Reumatologia317ANNUAL REPORT 2007

IRFMNMilan, March 17, 2007Workshop: Progetto RAPSODYRome, March 24, 2007Malattie metaboliche rare: la diagnosi e la gestione del pazienteMonza, may 12 2007Rare Diseases Task Force: Working Group on Coding and ClassificationParis, May 2 2007Partecipasalute: Orientarsi in salute e sanità per fare scelte consapevoliMilan, May 17, 2007La malattia di Anderson FabryBergamo, May 28, 20072° Convegno Nazionale: Medicina del trapianto: responsabilità e nuove opportunità per gliinfermieriBergamo, June 8-9, 2007INSIEME Giornate di socializzazione per malatti affetti da patologie rareMilan, June 29-july 1, 2007CAPOIRA Seminari di formazione: Comprendere i protocolli di ricercaMilan, July 2, 2007 and Rome, September 20, 2007I Test Genetici nella pratica clinicaTivoli Terme (Rome), July 5-6, 200711th European Meeting Complement in Human DiseaseCardiff UK, September 8-11, 2007Progetto di formazione sul campo: Registro Regionale Malattie RarePresidi Rete Regionale, 2007Comunicazione e Malattie Rare – Focus GroupRome, October 4, 20073rd International Workshop on Thrombotic MicroangiopathiesJena, October 4-6, 2007Tavola Rotonda: Geni, proteine, malattieGenoa, October 26, 2007La gestione pratica della malattia di Pompe: aspetti diagnostici e terapiaPavia, November 21, 2007La Federazione Lombarda Malattie Rare si presenta alle Associazioni di Malattie RareMilan, November 24, 2007European Conference on Rare Diseases318ANNUAL REPORT 2007

IRFMNLisbon, November 27-28, 2007European Commission"Fondazione Ricerca Malattie Rare", BergamoLombardy RegionGRANTS AND CONTRACTSSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Monteferrante G, Brioschi S, Caprioli J, Pianetti G, Bettinaglio P, Bresin E, Remuzzi G, Noris MGenetic analysis of the complement factor H related 5 gene in haemolytic uraemic syndromeMol Immunol; 2007; 44: 1704-1708OTHER PRODUCTS PUBLISHED IN 2007Noris M, Bresin E, Mele C, Remuzzi G, Caprioli J (November 2007) Atypical Hemolytic-Uremic Syndrome in:GeneReviews at GeneTests: Medical Genetics Information Resource [database online]. Copyright, University ofWashington, Seattle, 1997-2007. Available at http://www.genetests.org.319ANNUAL REPORT 2007

IRFMNRESEARCH ACTIVITIESInformation Centre for Rare DiseasesIn 1992, in the frame of the Clinical Research Centre for Rare Diseases “Aldo e Cele Daccò”was established an Information Centre for Rare Diseases.The Information Service is available to patients with rare diseases, their families and doctors. Itoffers information, update and useful addresses free of charge with particular emphasis onetiology, pathogenesis, genetics, treatments and availability of referral research centres.One of the most difficult issues to address when studying rare diseases are those, related to therecruitment of a sufficient number of patients with a given disease. The database of theInformation Centre for Rare Diseases has become a useful tool for identification of potentiallyeligible patients for clinical studies. Till now, the Information Centre for Rare Diseases hascollected patients’ clinical data for about 200 different rare conditions with 10 or more cases.Furthermore, intensive collaboration with groups with the same interest from Italy and abroadprovides many possibilities for starting of clinical projects with a multicentral design.Bank of biological samples and description of hereditary nephropathiesThe aim of this project is to collect clinical data and biological samples from patients and theirfamilies with rare genetic conditions. A database with clinical data and a Bank for biologicalsamples collection and preservation has been created.The availability of clinical data and biological samples is useful to perform new biochemicaland genetic tests within specific research projects aimed to better reveal the mechanisms of thediseases, their manifestation and therapeutic opportunities.In particular, the attention is focused on rare genetic disorders of the kidney. A thorough clinicalevaluation, including clinical data collection, medical physical examination, renalultrasonography, laboratory tests of blood and urine is offered to patients, affected by hereditarynephropathies (Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis,Glomerulopathy with Fibronectin deposits, Membranoproliferative glomerulonephritis,Medullary Cystic Kidney disease, Cystinuria), who are addressing our Centre. After obtaining awritten informed consent, biological samples from patients and their relatives are collected,labelled with specific codes to assure the anonymity and conserved in the Bank for biologicalsamples. In case the responsible gene for a hereditary nephropathy is known (Alport syndrome,Fabry disease, Cystinuria), the blood samples are redirected to the relevant Laboratory ofreference. For other nephropathies, where the identification of the gene mutation is unknown orstill in course, the blood samples are conserved with the aim to be used in specific futureresearch projects.Evaluation of the long term efficacy of enzyme replacement therapy inFabry diseaseFabry disease is an X-linked disorder of the glycosphingolipid catabolism caused by thedeficient activity of the lysosomal hydrolase alfa-galactosidase A (A-gal A) in tissues and fluidsof affected hemizygous males. Most heterozygous females have an intermediate level ofenzymatic activity.The enzymatic defect leads to a systemic deposition of glycosphingolipid in the heart, kidneys,eyes and other organs and tissues. Preliminary studies of enzyme replacement therapydemonstrate that periodic infusions of recombinant human Alfa-galactosidase A are safe (interms of infusion reactions) and effective in patients with Fabry disease.The purpose of this study is to evaluate the long term efficacy of enzyme replacement therapy inpatients with Fabry disease and renal involvement.Twenty patients (males and females) referred to the SMIMAF (Studio Multicentrico Italianosulla Malattia di Anderson-Fabry) will be enrolled.320ANNUAL REPORT 2007

IRFMNInternational Registry of Recurrent and Familial Hemolytic UremicSyndrome and Thrombotic Thrombocytopenic PurpuraHemolytic Uremic Syndrome (HUS) and Thrombotic Thrombocytopenic Purpura (TTP) arerare diseases of microangiopathic haemolytic anemia and thrombocytopenia with signs of renal(most prevalent in HUS) and cerebral (most prevalent in TTP) damage. There are extremely rareforms of HUS and TTP, often occurring in families, in which the patients relapse even aftercomplete recovery of the first episode with permanent renal and neurological sequelae. In thefamilial and recurrent forms of HUS and TTP, the attention is concentrated mainly on thegenetic predisposition to the disease.The Laboratory coordinates the International Registry of Recurrent and Familial HUS/TTP,since 1996. The Registry has collected more than 460 cases of HUS/TTP referred from 95Italian and 73 European and extra-European Centres. Clinical and laboratory data of all patientsreferred to the Registry are collected by a uniform data extraction form. The family history andalso the personal data of the unaffected relatives are collected, when possible. Biologicalsamples are collected from all patients and available relatives, for the biochemical and geneticanalyses. Many research projects in collaboration with the Laboratory of Immunology andGenetic of Rare Diseases and Organ Transplantation and the Laboratory of Cellular Biology –Negri Bergamo are developed and have still allowed to identify some genetically determinedforms of HUS and TTP. The maintenance of a centralised bank of biological samples ensure theavailability of clinical material for new investigative approaches as they will be developed.Rituximab in relapsing and chronic thrombotic thrombocytopenic purpuraThrombotic thrombocytopenic purpura (TTP) is a rare disease characterized bythrombocytopenia, microangiopathic hemolytic anemia and neurological signs. In most cases,TTP is related to an acquired deficiency of ADAMTS13 activity, due to the presence ofautoantibodies anti-ADAMTS13. The absence of ADAMTS13 activity leads to theaccumulation of von Willebrand Factor ultralarge multimers (normally cleaved to smallermolecular forms by ADAMTS13 enzyme), which probably induces platelets aggregation andthe ensuing process of thrombotic microangiopathy.The purpose of the project is to evaluate the use of a monoclonal anti-CD20 antibody(Rituximab) in patients with relapsing and chronic thrombotic thrombocytopenic purpura,related to the presence of anti-ADAMTS13 antibodies. Rituximab is a humanized monoclonalantibody that targets the CD20 molecule on B cells (white cells producing antibodies).Administration of Rituximab leads to a selective B cell depletion that usually lasts 6 to 9months. Inhibition of B cell activation or growth by rituximab treatment limit the uncontrolledsynthesis of autoantibodies, that may predispose to chronic relapsing TTP.Rituximab is infused intravenously once weekly for a total of four infusions. After Rituximabtreatment, the patients are periodically controlled with medical examination, blood and urineexams, and with ADAMTS13 plasma activity assay and research of anti-ADAMTS13autoantibodies, in collaboration with the Laboratory of Cellular Biology – Negri Bergamo.Observational period lasts 12 months.Evaluation of urinary podocyte excretionRecent evidence recognize an important role for podocytes in the progression of renal diseases.Podocytes contribute to glomerular permeability and are important target cell for renal diseaseprogression. Glomerular injury is usually associated with the leakage of protein across the filterinto the urine and with the disappearance of podocyte foot process. Injuried podocytes eitherundergo apoptosis or detach from the glomerular basement membrane, with subsequentappearance in the urine. Urinary excretion of podocytes has been reported as possible predictorof disease progression in a number of progressive glomerular diseases, such as IgA321ANNUAL REPORT 2007

IRFMNnephropathy, focal segmental glomerulosclerosis, diabetic nephropathy. During the past yearsthe Clinical Research Center for Rare Diseases, in collaboration with the Department ofMolecular Medicine at Mario Negri Institute, has established an indirect immunohistochemistrymethod for the detection of podocytes in urinary sediments. This method has allowed to identifyand quantitate the extent of urinary podocyte loss in patients with glomerular disease. Thepresent study has the aim to evaluate excretion of urinary podocytes in rare genetic renaldiseases. This methodology, applied on a larger number of patients, could provide a noninvasive way of indexing the extent of glomerular damage and a useful instrument to evaluatethe response to treatment.Effects of an intensified treatment with ACE-inhibitors, Angiotensin IIreceptor antagonists and Statins in Alport syndromeAlport syndrome (AS) represents a form of progressive hereditary nephritis in which the geneticdefect resides in the synthesis of one of several subunits of type IV collagen, the predominantconstituent of basement membranes in renal glomeruli. Renal impairment occurs with time andsevere renal failure with hypertension and uremia represent the end stage of the disease, even ifa high variability in the rate of progression is described. The prognosis is variable. Males areusually affected by a progressive form of the disease. Affected females with X-linked syndromeusually have a good prognosis with a mild renal impairment. Other clinical manifestations areanterior lenticonus which occur in about one third of patients. Other findings are myopia, lensopacities and retinal pigment abnormalities and corneal vesicles or erosions. The disease is alsoassociated to a sensor neural deafness which can occur in approximately half of the patientaffected and usually correlates with renal impairment.No definite treatment exists in order to delay the time of dialysis or a kidney transplant. Manystudies showed that Angiotensin converting enzyme (ACE) inhibitors slow glomerular filtrationrate (GFR) decline and limit progression to end stage renal disease (ERDS) and dialysis inseveral chronic nephropathies associated with proteinuria. The combination of ACE-I withAngiotensin II receptor antagonists may reduce proteinuria more effectively than the two drugsalone. Moreover the addition of Statins may synergize the antiproteinuric effects of ACE-I andATAII antagonists in experimental models of chronic renal diseases.The purpose of this study is to evaluate the effect of a standardized multimodalnephroprotection intervention (Remission Clinic) in Alport patients with renal involvement.Nine patients with Alport syndrome and macroalbuminuria will be enrolled in this study.Recruitment of normo or microalbuminuric patients will be stopped and the analysis will beperformed on the patients that will be available when the recruitment phase ofmacroalbuminuric patients will be completed.Update of the Congenital Respiratory Malformations databaseThis Project originated from the collaboration between the Study Group on Respiratory Diseaseof the newborn and the Coordinating Centre for Rare Diseases.The Congenital Respiratory malformations database was conceived to facilitate the diagnosticand assistance procedures for paediatricians facing with more frequent respiratorymalformations. The update consisted of a review of the lists of malformation categories and ofall syndromes previously included; more syndromes have been added.Furthermore, in the brief description, more detailed than the previous version, the Code for RareDisease was added, allowing to address the patient to the Referral Centres for Lombardy.A link with the OMIM database is provided for each syndrome for a better description of thedisease.322ANNUAL REPORT 2007

IRFMNIdentification of new genes associated to the Autosomal DominantFamilial form of Focal Segmental GlomerulosclerosisFocal segmental glomerulosclerosis (FSGS) is a pathological entity, and is a significant cause ofend-stage renal disease (ESRD). Glomerular disease is the third leading cause of ESRD, andFSGS comprises a significant proportion of this subgroup: up to 5% of adults and 20% ofchildren with ESRD.The diagnosis of FSGS is based on renal pathology. The clinical hallmarks include proteinuria,nephrotic syndrome, occasional hematuria and frequent progression to ESRD. Hypertension isalso a common finding. At present, there are no consistently reliable treatments for FSGS andresponse rates to available treatments have been estimated at

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IRFMNCESAVA. and A. Valenti Health Economics CenterSTAFFHeadLivio GARATTINI, Econ.D.325ANNUAL REPORT 2007

IRFMNCURRICULUM VITAELivio Garattini: got his degree in Economics in March 1983 at the Bocconi University in Milan.Educational activities: “King’s Fund College”, London: courses of health care management; “Centre forHealth Economics”, York: review of publications on the English NHS; “Ecole Nationale de la SantéPublique”, Rennes: courses of health policy.Areas of interest: Health Economics and health Policy Analysis.At present he is the Director of CESAV (Centre of Health Economics A. e A. Valenti - M. NegriInstitute);1981-1983: researcher at M. Negri Institute;1983-1984: clerk at Banca Commerciale Italiana in Milan;1984- 1985: junior consultant at “Sogess srl” in Milan;1985-1990: researcher at Bocconi University in Milan.Selected publications:• Garattini L, De Compadri P, Clemente R, Cornago D (2003) “Economic Evaluations in Italy: a Review of the Literature”International Journal of Technology Assessment in Health Care 19(4): 685-697.• Garattini L, Ghislandi S (2006) “Off-patent drugs in Italy- A short-sighted view?” The European Journal of HealthEconomics 7(1): 79-83.• Garattini L, Ghislandi S (2007) “Should we really worry about “launch delays” of new drugs in OECD countries?”(Editoriale) The European Journal of Health Economics 8(1): 1-3.• Cornago D, Li Bassi L, De Compadri P, Garattini L (2007) “Pharmacoeconomic studies in Italy: a critical review of theliterature” The European Journal of Health Economics 8(2):89-95.• Garattini L, Cornago D De Compadri P (2007) “Pricing and reimbursement of in-patent drugs in seven Europeancountries: A comparative analysis” Health Policy 82:330-339.• Koleva D, Motterlini N, Banfi P, Garattini L (2007) “Healthcare costs of COPD in Italian referral centres: A prospectivestudy” Respiratory Medicine 101:2312-2320.326ANNUAL REPORT 2007

IRFMNINTRODUCTION TO THE CENTER'S ACTIVITIESThe "Angelo e Angela Valenti" Centre for Health Economics (CESAV) was established in 1992at the "M. Negri Institute" and based at Villa Camozzi- Ranica (Bergamo)-Italy. CESAV isprimarily a research centre, but also does educational work. The centre is involved in healtheconomics and health policy research. The main areas of research are: Economic Evaluation ofHealth Care Programs (i.e. assessment of costs and benefits of alternative health care treatmentsand services) and Comparative Health Policy Analysis (i.e. study of foreign health care systems,in particular aimed at identifying possible innovations for European countries).FINDINGS/MAIN RESULTSIn 2007 economic evaluations regarded the costs of the following pathologies: glaucoma,multiple myeloma and therapies for hepatitis C. The studies of comparative analysis werefocused on the management of vaccination campaigns in health authorities in Lombardy and onthe management of drug expenditure in a National sample of health authorities.NATIONAL COLLABORATIONSPublic and private institutions, other health care organizations (Ministry of Health, Regional andLocal Health Authorities, Hospital Trusts).INTERNATIONAL COLLABORATIONSCES (Collège des Economistes de la Santé) of ParisCorvinus University of BudapestGlobal Fund of GenevaWidO of BonnUniversity Carlos III of MadridUniversity of HannoverUniversity of YorkUniversity Pompeu Fabra of BarcelonaUniversity Erasmus of Rotterdam327ANNUAL REPORT 2007

IRFMNEDITORIAL BOARD MEMBERSHIPActa Bio Medica (Livio Garattini)Biomedical Statistics and Clinical Epidemiology (Livio Garattini)Economia e Politica del Farmaco (Livio Garattini)FarmacoEconomia News (Livio Garattini)Farmeconomia e Percorsi Terapeutici (Livio Garattini)Health Policy (Livio Garattini)L'Internista (Livio Garattini)Journal of Medical Economics (Livio Garattini)PharmacoEconomics Italian Research Articles (Livio Garattini)Quaderni di FarmacoEconomia (Livio Garattini)The European Journal of Health Economics (Livio Garattini)PEER REVIEW ACTIVITIESBJCP (British Journal of Clinical Pharmacology)Health PolicyPharmacoEconomicsSocial Science & MedicineThe European Journal of Health EconomicsEVENT ORGANIZATIONCongress: Convegno Nazionale CESAV di Farmacoeconomia:“Economia del farmaco: frasoluzioni tecniche e decisioni politiche”.“Prezzi e rimborsabilità dei farmaci innovativi”.“Educazione Continua Medica”:“L’ECM in Europa: esperienze a confronto”.“Gli aspetti economici del glaucoma”.“Informazione Medico-Scientifica sui farmaci”:“La normativa sull’informazione medico scientifica in Italia: la situazione a livellonazionale”.“La normativa sull’informazione medico scientifica in Italia: i risultati di un’inchiesta alivello regionale”.May 29-30, Ranica (BG)Congress: “La farmacoeconomia nelle terapie per il CCR”.“Gli studi di farmacoeconomia sui trattamenti chemioterapici nel CCR”:“Una revisione critica degli studi di farmacoeconomia”.“Metodologie di analisi a confronto: i risultati di una simulazione”.Opinioni a confronto: “Gli studi di farmacoeconomia: l’utilità percepita in oncologia”.December 11, Ranica (BG).328ANNUAL REPORT 2007

IRFMNPARTICIPATION IN EVENTS IN WHICH THE CENTER WASINVOLVEDCongress: “Global Pricing and Reimbursement Congress 2007”. “European Pricing andReimbursement for Generic Medicines”Country By Country Pricing And Reimbursement Policies“Current pricing policies in Italy”.“Reimbursement processes and timelines”.“Identifying critical cost containment measures”.(Global Pricing and Reimbursement-Congress 2007)Pricing Models and Systems-A Country by Country Breakdown“The current pricing policies in Italy and the impact on the European generic industry”.“Reimbursement processes and timelines for generic medicines”.“Reference pricing and pharmacists incentive schemes”.(European Pricing and Reimbursement for Generic Medicines).30-31 January, Prague.Congress: “Congreso Europeo de Oficina de Farmacia”. “Los genéricos en Europa: visionfarmacoeconomica”. 28 February-2 March, Barcellona.Course: “Le maculopatie e la degenerazione maculare legata all’età: gestione clinico-terapeuticaed economica”.“L’impatto socioeconomico delle maculopatie:farmacoeconomia e Qualità di Vita.Discussione”.15 May, Milan.Workshop: “European Advisory Panel meeting to discuss the positioning of temsirolimus foruse in the treatment of renal cell carcinoma”.13 July, London.Workshop: “ADHD (Attention Deficit and Hyperactivity Disorder) Advisory Board Meeting”.23-24 July, Frankfurt on Maine.Congress: “Acomplia Focus Meeting”.Round Table on “Knowing our customers”.4 October, Paris.Congress: “Le maculopatie e la degenerazione maculare legata all’età: gestione clinicoterapeuticaed economica”.“L’impatto socioeconomico delle maculopatie: farmacoeconomia e Qualità di Vita”.10 October, Genoa.Congress: “Il corretto management terapeutico ed assistenziale nella terapia del dolore”.“Impatto economico della terapia del dolore”.7 November, Milan.Congress: “Le maculopatie e la degenerazione maculare legata all’età: gestione clinicoterapeuticaed economica”.“Analisi farmaco-economiche applicate alla CNV”.7 December, Naples.329ANNUAL REPORT 2007

IRFMNGRANTS AND CONTRACTSAbbottEGGrunenthal-Prodotti FormentiJannsen CilagNovartis FarmRatiopharmSanofi AventisSanofi Pasteur MSDSchering PloughVivisolSELECTION OF SCIENTIFIC PUBLICATIONS FROM 2007Garattini L, Ghislandi S “Should we really worry about “launch delays” of new drugs in OECD countries?” (Editorial) TheEuropean Journal of Health Economics 8(1): 1-3.Cornago D, Li Bassi L, De Compadri P, Garattini L “Pharmacoeconomic studies in Italy: a critical review of the literature”The European Journal of Health Economics 8(2): 89-95.Garattini L, Koleva D, Motterlini N, Cornago D “Medical Costs of Chronic Musculoskeletal Pain in Italy” Clinical DrugInvestigation 27(2): 139-148.Garattini L, Cornago D, De Compadri P “Pricing and reimbursement of in-patent drugs in seven European countries: Acomparative analysis” Health Policy 82: 330-339.Koleva D, Motterlini N, Schiavone M, Garattini L “Medical Costs of Glaucoma and Ocular Hypertension in Italian ReferralCentres: A Prospective Study” Ophthalmologica 221(5): 340-347.Koleva D, Motterlini N, Banfi P, Garattini L “Healthcare costs of COPD in Italian referral centres: A prospective study”Respiratory Medicine 101: 2312-2320.Garattini L “Pricing and Reimbursement Policies in Italy: Current and Future Trends” in Garau M, Mestre-Ferrandiz J(edited by) European Medicines Pricing and Reimbursement. OHE. Oxon, Radcliffe Publishing.330ANNUAL REPORT 2007

IRFMNLAY PRESS SELECTION PUBLISHED IN 2007Motterlini N, Garattini L “I costi di struttura dei reparti di pneumologia in Italia” FarmacoEconomia News 1: 3-7.Cerzani M, Barbui C, Garattini L “Le valutazioni economiche del trattamento farmacologico con antipsicotici nellaschizofrenia: una rassegna degli studi italiani” Quaderni di Farmaco Economia 2: 17-28.Mc Nee, England S (Translation by: De Compadri P, Garattini L, Li Bassi L) “Pharmac: un esempio di sfide esuccessi” Quaderni di Farmaco Economia 3:6-15.Cerzani M, Pasina L; Clavenna A, Nobili A, Garattini L “Revisione critica degli studi italiani di farmacoeconomiasull’uso dei farmaci antinfiammatori non steroidei in medicina generale” Quaderni di Farmaco Economia 3:17-28.Gritti S, De Compadri P, Garattini L “L’informazione medico scientifica fra Stato e Regioni” Quaderni di FarmacoEconomia 4: 7-15.De Compadri P, Koleva D, Zaniboni A, Garattini L “Modalità di somministrazione per terapie del CCR a confronto:un esercizio di valutazione economica” Quaderni di Farmaco Economia 4: 16-27.Garattini L “Finanziaria 2007: vere e false novità” Dialogo sui farmaci 1:33-34.RESEARCH ACTIVITIESEducational activityEducational activities are developed only if related to research studies, in order to offer originalcontributions which naturally reinforce the research aims.Economic Evaluation of Health Care ProgramsThe aim of this research area is to assess the costs of illnesses and the cost-effectiveness ratiosof the diagnostic/therapeutic existing alternatives. In general, analyses can be classified into twogroups: partial economic evaluations (e.g. observational studies on the costs of pathologies) andfull economic evaluations (e.g. cost-effectiveness analyses).Comparative Health Policy AnalysisThe aim of this research area is to study the organization of health care systems, in order todraw lessons from international comparisons. This is particularly important in a "market" likehealth care where economic competition lacks by definition and therefore public regulationplays a crucial role.331ANNUAL REPORT 2007

IRFMN332ANNUAL REPORT 2007

IRFMNThe Transplant Research CenterChiara Cucchi De Alessandri eGilberto CrespiThe Transplant Research Center (CRT) was set up in 2002 to support and promote the work ofoutstanding research scientists throughout the world and to carry out major organ transplantresearch programs.The Center is housed in the Villa Camozzi, at Ranica, under the same roof as the Mario NegriInstitute in Bergamo and is managed in collaboration with the Institute.The Center’s staff is mainly made up of senior and junior researchers that were trained in thelaboratories of the Mario Negri Institute in Bergamo, focusing on transplant immunology,research for less toxic immunosuppressant drugs, and new gene therapy techniques to preventacute rejection of transplanted organs.Information on the Center’s activities can be found in the sections addressed to the Departmentof Molecular Medicine (Laboratory of Immunology and Genetics of Organ Transplantation andRare Diseases) and the Department of Renal Medicine (Laboratory of Pharmacokinetics andClinical Chemistry).333ANNUAL REPORT 2007

IRFMN334ANNUAL REPORT 2007

IRFMNEDUCATIONAL ACTIVITIESDean, Mario Salmona, PH.D.The Institute's training programs fall under the heading of biomedical science, and are part ofthe Lombardy Region's professional training schemes. There are courses for specializedlaboratory technicians, and for graduates intending to do research. Special training for clinicaltrial nurses is provided at the Aldo and Cele Daccò Clinical Research Center for Rare Diseases,at Ranica, near Bergamo. In 1999 the Institute has set up a Ph.D. course, in collaboration withthe Open University of London. This degree is recognised throughout Europe and in the USA.In 2006 the Institute also set up a First Level master Course in Clinical Research, incollaboration with the Milan University and a Second Level master Course in rare Diseases, incollaboration with the University of Torino.Students enrolled in formal courses receive three months' preparatory training, after which theyare assigned study grants. Between 1963 and 2004 the Mario Negri Institute awarded 6,123grants, 668 of them to foreign researchers who came to the Institute for special training.Everything possible is done to help students find work once they finish the course.The main feature of these courses is that technicians and researchers receive their training "onsite". They work full-time in research programs of a high scientific standard, using advancedequipment and learning the latest methods, in regular contact with colleagues in differentcountries. Besides its scientific value, this approach provides an excellent preparation on thehuman and personal scale.Students are usually assigned to one of the Institute’s Laboratories, where they gradually gainspecialized skills by working on specific research projects. They are expected to attend lessons,seminars, courses and congresses and learn to make full use of the Institute’s well-stockedlibrary. Students all have access to the internet and to biomedical database and can print outcopies of articles they need to consult, from major international journals. Should the opportunityarise, students are expected to be available for trips abroad, to participate in conferences orcourses.The Pharmacological Research Specialists and Biochemical Research Technicians will receivediplomas issued officially by the Lombardy Region and the Mario Negri Institute forPharmacological Research. These have legal value throughout Italy, and are recognised incompetitions for public posts, where they are worth a certain number of points. Mario NegriInstitute diplomas are widely considered a guarantee of an excellent theoretical and practicaltraining. The Open University of London Ph.D. degree earned at the Institute has legal valuethroughout Europe and in the USA.Once they have their diploma or Phd., graduates who want to continue doing research at theInstitute may be offered a chance to spend a year or two abroad.At the moment these courses are available:335ANNUAL REPORT 2007

IRFMNThree-year course for graduates, in Milan or Bergamo, leading to a diploma asPharmacological Research Specialist.Three-year course for diploma-holders, in Milan or Bergamo, leading to a diploma asBiochemical Research Technician.Research doctorates (Ph.D.), run under an agreement with the Open University of London.Two-year courses are run at the Daccò Center, in Ranica, near Bergamo, for nurses intending towork in clinical trials. These give a diploma as Professional Clinical Trial Nurse.Other training opportunitiesPREPARING A THESIS FOR A DEGREE:Students can prepare their thesis in scientific subjects at the Institute, with the approval of theiruniversity faculty. These students must work at the Institute for at least two years.SUMMER STUDENTSIn June and July each year the Institute accepts a certain number of students in their last twoyears at high school, to give them experience as part of school/work programs.336ANNUAL REPORT 2007

IRFMNSTAFFExecutive OfficesServices and Offices337ANNUAL REPORT 2007

IRFMN338ANNUAL REPORT 2007

IRFMNProf. Silvio GarattiniSilvio Garattini was born in Bergamo (Italy) on 12th Nov 1928. Diploma in Chemistry. Degree inMedicine. Lecturer in Chemotherapy and Pharmacology. Assistant then Deputy Professor at the MilanUniversity Institute of Pharmacology until 1962.Founder in 1963 and director of the Mario Negri Institute for Pharmacological Research. The Institute hasnow four locations (Milan, Bergamo, Ranica (Bg), S. Maria Imbaro (Ch)) and more than 850 people.He is a member of the Gruppo 2003 (a group of the most cited Italian scientists in international scientificliterature). Garattini's publications in Italian and in English in international scientific journals, and textson pharmacology, run into the hundreds. Founder of the European Organisation for Researchand Treatment of Cancer (EORTC).During the last ten years professor Garattini has been a member of various organizations, among which:the Italian National Research Council (CNR) - Committee on Biology and Medicine; the National HealthCouncil, the Committee for Italian Research Policy, set up by the Presidency of the Council of Ministers;the Commissione Unica del Farmaco (CUF) of the Ministry of Health.He has held the following posts: President of the UICC Committee on Antitumoral Chemotherapy,President of the European Organisation for Research and Treatment of Cancer (EORTC), Consultant tothe World Health Organisation.He was President of the European Society of Biochemical Pharmacology; member of the Committee forProprietary Medicinal Products (CPMP) of the European Agency for the Evaluation of MedicinalProducts (EMEA), Member of the CEPR (Committee of Experts of Research Policy) at the Ministry forUniversity and Scientific and Technological Research; Member Scientific Committee of the Lega Italianaper la Lotta Contro i Tumori; member of the Board of the Istituto Superiore di Sanità.Vice-president of the Consiglio Superiore di Sanità; President of the research and DevelopmentCommission of the Agenzia Italiana del Farmaco (AIFA); President of the Angelo and Angela ValentiFoundation and of the "Via di Natale" Foundation; President of the Technical Commission forPharmaceutical Assistance of the Regione Autonoma of Sardegna; Member of the Strategic Committeefor Welfare, Regione Lombardia. Member of the Consiglio Superiore di Sanità and the ComitatoNazionale di Bioetica.Silvio Garattini is a Fellow of the New York Academy of Sciences, the American Association for theAdvancement of Science, Honorary Fellow of the Royal College of Physicians (PharmaceuticalMedicine), London, and a member of numerous other Italian and international scientific societies.He has received many awards for his work, including the French Legion d'Honneur for scientific merit,and the Grand Ufficiale della Repubblica Italiana, and holds honorary degrees from the Universities ofBialystok in Poland, and Barcelona in Spain. Most recent prizes: Prize Ippocrate, 2003; Prize Mens Sanain Corpore Sano; Prize Nuova Spoleto, 2003; Prize Angelo dell’anno; Alkmeon International Prize;International Prize Sant’Agostino Città di Bergamo; Prize Il Campione della Scienza; Medal Natta; PrizeCoppola.In its 40-plus years, the Mario Negri Institute for Pharmacological Research, under Professor Garattini'sleadership, has published more than 10000 scientific papers and about 200 books, on topics ranging fromcancer and its treatment to tumour immunology, neuropsychopharmacology, and cardiovascular and renalpharmacology. More than 4000 young Italian and 600 foreigner graduates and technicians have obtainedspecialist qualifications at the Institute.339ANNUAL REPORT 2007

IRFMN340ANNUAL REPORT 2007

IRFMNProf. Giuseppe RemuzziGiuseppe Remuzzi was born in Bergamo, Italy in 1949. Upon completion of his medical training at theUniversity of Pavia in 1974, he received specialty training in Hematology and Nephrology at theUniversity of Milan.Since 1975, he has pursued his academic career at the Ospedali Riuniti of Bergamo, where he wasappointed Professor of Nephrology and Director of the Department of Medicine and Transplantation in1996. Since 1999, he is Director of the Department of Nephrology and Dialysis of the same hospital. TheNegri Bergamo Laboratories, which he has directed since 1984, is a group of basic scientists,physiologists, pharmacologists, molecular and cellular biologists, pathologists and clinicians devoted tothe study of renal disease. As Research Coordinator of the Negri Bergamo Laboratories and the affiliatedClinical Research Center for Rare Diseases "Aldo e Cele Dacco", both divisions of the Mario NegriInstitute for Pharmacological Research, he conduct a diverse team of researchers studying human renaldiseases and their corresponding experimental models from the perspective of pathophysiology andtherapeutic intervention. He touched major advances in many areas of nephrology, with particular focuson platelet endothelial interactions, vascular prostaglandin biology, coagulation and renal disease,progression of renal disease, experimental models of glomerular damage, and transplant immunology andtolerance. Particularly his contributions to the understanding of the pathophysiology of hemolytic uremicsyndrome, prostaglandin metabolism in pregnancy, renal vascular biology in uremia, the role of proteintrafficking in renal disease progression, the induction of graft tolerance by intrathymic injection of donorantigens, the role of the co-stimulatory CD28-B67 pathway in transplant rejection and the prevention ofrenal and cardiovascular damage in diabetes.Prof. Remuzzi serves on editorial boards of numerous journals including the prestigious New EnglandJournal of Medicine and is member of the International Advisory Board of The Lancet.In recognition of his achievements, he has been awarded in 1998 honorary memberships of theAssociation of American Physicians and the British Royal College of Physicians. In 2001 he wasnominated Chairman of the Research Committee of the COMGAN (Commission on Global Advancementof Nephrology) of the International Society of Nephrology (ISN). He received an Honorary Professorshipat the University of Maastricht in 2003.In 2005 during the World Congress of Nephrology in Singapore he received the ISN Jean HamburgerAward and in November 2007 he received during the annual congress of the American Society ofNephrology the precious John P. Peters Award.Prof. Remuzzi has authored and co-authored more than 885 scientific articles, reviews and monographs.341ANNUAL REPORT 2007

IRFMNMario Negri Institute MilanExecutive OfficeDirectorProf. Silvio GARATTINI, M.D.Administrative OfficeMaria Grazia PEZZONI, ChiefTechnical OfficeFabio BRIGHENTI, D. Arch.General MaintenanceEmanuele SINELLIStudies OfficeArmanda JORI, Pharm.D.Press OfficeIsabella BORDOGNA, Phil. D.Public Relations OfficeClaudio PANTAROTTO, ChemistPrevention and Safety OfficeEmilio BENFENATI, Chem.D.Annamaria SEGALINI, Phys.D.English Style EditorJudi BAGGOTTPhotography and Audio-Visual ServiceFelice DE CEGLIEPurchasing OfficeEufrasia COVIELLODirector’s OfficeRosanna MAPELLI, ChiefGeneral SecretariatElena POZZOLI342ANNUAL REPORT 2007

IRFMN343ANNUAL REPORT 2007

IRFMNNegri Bergamo LaboratoriesExecutive OfficesDirectorResearch CoordinatorScientific SecretariatProf. Silvio GARATTINI, M.D.Giuseppe REMUZZI, M.D.Ariela BENIGNI, Biol.Sci.D., Ph.D.Administration of Research Projects/Admn. AssistanceDaniela MELACINI, Biol.Sci.D.Press and Communication OfficeFrancesca Di Fronzo, Mod. Lit. D.Audio-Visual ServiceAntonella PICCINELLI, Biol.Sci.D.Prevention and Safety OfficeChiefAnnamaria SEGALINI, Phys.D.LibraryChiefAnna BOZZALEValeria MIGLIOLIGeneral MaintenanceGiancarlo GASPARIDirector’s OfficeAntoinette van ENGELEN, Chief344ANNUAL REPORT 2007

IRFMNCentro Aldo e Cele Daccò Ranica (Bg)Executive OfficesDirectorResearch CoordinatorScientific SecretariatHealth DirectorProf. Silvio GARATTINI, M.D.Giuseppe REMUZZI, M.D.Ariela BENIGNI, Biol.Sci.D., Ph.D.Norberto PERICO,M.D.Press and Communication OfficeFrancesca Di Fronzo, Mod. Lit. D.Prevention and Protection OfficeChiefAnnamaria SEGALINI, Phys.D.Paola BOCCARDO, Biol.Sci.D.Library ‘Mansueto Astori’ChiefAnna BOZZALEMonica MINALIDirector’s OfficeDaniela RICEPUTI, ChiefClinical Trials OfficePaola BOCCARDO, Biol.Sci.D.Custodian/general maintenaceGiampiero CUGUSI345ANNUAL REPORT 2007