TeleThon ReseaRch PRoJecTs of the regulatory activity of sJW and hPf on cytokine signaling pathways in ins-1e cells; B) to assess their ability to counteract cytokinedriven changes in the expression of sTaT-1 and nf-kB target genes involved in inflammatory response and apoptosis; c) to screen microarray profiling of microRnas in beta cells exposed to cytokines and/or vegetal compounds; d) to test the immunomodulatory role of sJW and hPf on peripheral blood mononuclear cells (PBmc). methods: in ins-1e cells, exposed to cytokine mixtures for various time periods with/without sJW extract (1-2 microg/ml) or hPf (1-2 micromol/l), the phosphorylation state of various components of sTaT1, nf-kB and maPK pathways was evaluated by western blotting. Rna was extracted with the mirVana miRna isolation kit and used for RTqPcR gene expression analysis (large Rna) and miRna profiling. Results: aim a. cytokine-induced sTaT-1 phosphorylation in both tyrosine and serine residues was significantly reduced by sJW or hPf in a dose-dependent manner. These compounds prevented nfkB activation by hindering phosphorylations of the p65 subunit and the inhibitory subunit ikB activating kinase and also modulated maPK pathway through dose-dependent restriction of eRK1/2, p38 maPK and JnK phosphorylations. aim B. sJW and hPf abolished cytokine-induced mRna expression of pro-inflammatory genes, such as inos, cXcl9, cXcl10, icam-1, coX-2. The vegetal compounds were also able to partially correct the cytokine-induced unbalance between anti- and pro-apoptotic factors, mainly by preventing upregulation of pro-apoptotic proteins (e.g., dP5, Puma, BaK). aim c. several miRna were differentially expressed in cells exposed to cytokines as compared to control cells. some of these changes were reversed in the presence of the vegetal compounds (in particular sJW), but in most cases the function of these miRna in beta cells is unknown. aim d. in stimulated PBmc, expression of T-bet and cXcR3-a, markers of Th1 differentiation, was down-regulated by sJW and hPf after 4-h incubation, whereas that of gaTa-3 and cXcR3B, markers of Th2 polarization, was up-regulated or unchanged. conclusions: sJW extract and hPf are confirmed to represent promising pharmacological tools for prevention of beta cell loss in type 1 diabetes and to exert immunomodulatory effects in vitro. nKT cells (that is required to obtain the homotipic slam-slam interaction) or to in vitro culture conditions that bias the cytokine phenotype and function of nKT cells towards a predominant effector nKT17/nKT1 phenotype. specific aim 2. To assess the capacity of regulatory nKT2 cells and nKT2-inducing mdc to prevent and/or treat T1d in pre-clinical models. here we will assess the capacity of slam-migR1-transduced hematopoietic cells of nod mice to promote differentiation of regulatory nKT2 cells in vivo and to prevent autoimmune diabetes. We have started experiments of chimera generation to replace irradiated of nod mice with hematopoietic cells in vitro transduced with the slam-migR1 vector. specific aim 3. To analyze whether lack of co-stimulatory signals on mdc is responsible for defective nKT2 differentiation in T1d patients. We measured slam expression on mdc derived from blood monocytes of T1d patients (n=6) and controls (n=6). although we observed a reduced expression of slam on dcs of T1d patients, our results were not statistically significant. in the last year of the research project we will analyse a larger cohort of T1d and patients (n=20) to verify whether mdcs carry a genetic defect of slam expression that could affect regulatory nKT2 differentiation. ABSTRACT N. 215 Telethon Research Projects - other genetic diseases Principal Investigator Duration (yrs): 3 Starting year: 2008 federici massimo, casagrande Viviana, cavalera michele, mavilio maria, stohr Robert, menghini Rossella dipartimento di medicina dei sistemi, università degli studi di Roma Tor Vergata, Via montpellier 1, 00133 Roma. italia Principal Investigator FALCONE MARIKA MARIA CATERINA Telethon grant N. GJT08017 TimP3 regulates fatty acid metabolism, atherosclerosis progression and survival in apoe knockout mice. TimP3 is reduced in the muscle and carotid plaques from patients with Type 2 diabetes but if TimP3 alters glucose and lipid metabolism is unclear. We analyzed apoe-/(eKo) and apoe-/-Timp3-/- (eT3Ko) mice by monitoring the survival rate; lipids, metabolites and atherosclerotic plaques with lcms metabolomics and conventional staining; physiological values by calorimetric cages. eT3Ko mice revealed a significant higher rate of sudden death compared to eKo mice during a follow up of 12 months (n=18 per group; p<0.001); sudan-iV and oil-Red staining of eT3Ko aorta and aortic root displayed increased positive staining (40% increase, p<0.01) compared to eKo. moreover, eT3Ko mice showed higher levels of total cholesterol, hdl-cholesterol and triglycerides (p<0.05 for all). furthermore, eT3Ko mice exhibited lower heart rate (p<0.01), higher systolic and diastolic pressures (p=0.06; p<0.05), higher o2 consumption and co2 production, and lower respiratory exchange ratio compared to eKo mice (p<0.0001 for all). These data suggested a different use of metabolic substrates; specifically we found increased levels of c14:oh, c16:oh and c18:oh (p<0.01 for all) in the blood of eT3Ko mice, together with higher amount of arginine (p<0.05) and histidine (p<0.01). no differences in food and water intake were found. Western blot analysis showed reduced pThr172 amPK phosphorylation and increased pser473 aKT phosphorylation in skeletal muscle from eT3Ko compared to eKo mice (p<0.05 for all), suggesting that lipid oxidation may be dysfunctional in eT3Ko mice. loss of timp3 accelerates atherosclerosis and causes higher amount of atherosclerotic plaques, increased sudden death, plus defects in intermediary metabolism. The latter results in accumulation in the circulation of hydroxylated long-chain fatty acids that may complicate atherosclerotic plaque composition or perturb heart metabolism. 124.300 Duration (yrs): 3 263.000 TESTING TIMP3 AS A SWITCh TO BLOCK METABOLIC AND VASCuLAR COMPLICATIONS OF OBESITY Telethon Research Projects - other genetic diseases Total budget € GGP08065 Total budget € Centres: 1 ABSTRACT N. 214 Centres: 1 FEDERICI MASSIMO Telethon grant N. Starting year: 2009 EXPLOITING ThE ThERAPEuTIC POTENTIAL OF INVARIANT NKT CELLS IN TYPE 1 DIABETES di Pietro caterina (1), nichols Kim (2), caielli simone (1), falcone marika (1) (1) experimental diabetes unit, san Raffaele scientific institute, l30 lottoQ, Via olgettina 60, 20132, milan, italy, Tel. 02.2643.2317/2914, e-mail: falcone.marika@hsr.it (2) Pediatric oncology, children s hospital of Philadelphia, Philadelphia, usa Background: invariant nKT cells (inKT) represent a unique subset of innate lymphocytes that play a dual role and exert either pro-inflammatory or regulatory function crucial to maintain T cell tolerance and prevent autoimmune diseases like T1d. Both inKT cell functions are mediated through crosstalk between inKT cells and myeloid dendritic cells (mdcs). our research group demonstrated that genetic susceptibility to autoimmune diabetes in the nod mice is linked to a genetic defect of the slamf1 gene that impairs slam expression on myeloid dcs (mdcs) and negatively affects peripheral differentiation of regulatory nKT2 cells. objective: our goal is to restore mechanisms that drive differentiation of regulatory nKT cells in diabetic mice and to verify whether the same genetic nKT cell differentiation defect is present in patients affected by T1d. The final goal is to design new therapeutic approaches for T1d based on restoration of regulatory nKT cell function. specific aim 1. To restore nKT2 differentiation in autoimmuneprone nod mice by providing the necessary slam-induced co-stimulatory signals on myeloid dendritic cells (mdc). We successfully clone the murine slam gene into the migR1 retroviral vector that contains an internal ribosome entry site (iRes) linked to gfP transgene. We demonstrated that trasduction with the slam-migR1 vector completely restored the expression level of slam molecules on mdc of nod mice. our preliminary experiments indicate that slam-transduced dcs of nod mice were still unable to induce regulatory nKT2 differentiation. We are currently testing whether this defect is related to defective expression of slam molecules on nod ABSTRACT N. 216 Telethon Research Projects - other genetic diseases Principal Investigator BEGuINOT FRANCESCO Telethon grant N. GGP09012 Total budget € 245.400 Centres: 1 Duration (yrs): 3 Starting year: 2009 PREP1 GENE FuNCTION IN TYPE 2 DIABETES 123

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