P0007 : Hepatic T cells derived from normothemic machine perfused grafts contained more regulatory T cells and fewer proinflammatory cytokines producing T cells than those from cold storage grafts

POSTERS P0006 HEPATIC ISCHEMIA/REPERFUSION INDUCES CHANGES IN TISSUE AND BILIARY LEVELS OF ARGININE AND ITS METHYLATED DERIVATIVES L.G. Di Pasqua1 , V. Rizzo2 , C. Berardo1 , P. Richelmi1 , M. Vairetti1 , A. Ferrigno1 . 1 Internal Medicine and Therapeutics, University of Pavia, 2 Molecular Medicine, University of Pavia and IRCCS S. Matteo, Pavia, Italy E-mail: [email protected] Background and Aims: Arginine (Arg), the substrate for nitric oxide synthase (NOS), can be methylated to form symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA), the last an endogenous inhibitor of NOS. SDMA is mostly excreted in the urine, while ADMA is mainly subjected to degradation in the liver. We recently reported that ADMA is also secreted in bile (Ferrigno et al., 2014). Arg competes with ADMA and SDMA for cellular transport across cationic amino-acid transporters (CATs) (Closs et al., 1997). Here we evaluate the changes in serum, tissue and biliary levels of Arg, citrulline (Cit), ADMA and SDMA as well as the modifications in CATs after ischemia-reperfusion (I/R) injury. Methods: Male Wistar rats were subjected to 30-min partial hepatic ischemia by clamping the hepatic artery and the portal vein (n = 8) or sham-operated (n = 7). After 60-min reperfusion, serum, tissue and bile samples were collected and the concentrations of ADMA, SDMA, Arg and Cit were measured. AST, ALT and Alkaline Phosphatase (AP) levels in serum and bile were also determined. Using RT-PCR analysis, mRNA of cationic transporters, 2A (CAT-2A) and 2B (CAT-2B), for Arg and its methylated derivatives were also quantified. Results: A decrease in serum Arg and an increase of Cit were detected after I/R; no changes in ADMA and SDMA were found. Both forms of methylarginenes, Arg and Cit were also detected in bile: a significant increase in ADMA and Cit and a decrease in SDMA and Arg were observed in bile at the end of reperfusion. On the contrary, lower ADMA levels and higher SDMA levels were quantified in liver biopsy after I/R when compared with shamoperated rats. A marked increase in AST, ALT and AP levels in serum and bile confirmed both hepatocyte and cholangiocyte damage. A decrease in mRNA transporter CAT-2A but not in CAT-2B was detected. Conclusions: This study supported our previous results on the biliary ADMA clearance and demonstrated, for the first time, that the liver is also responsible for the biliary excretion of SDMA: the clearing of SDMA is not only confined to the kidney, but the liver could also take up SDMA from the portal and systemic circulation. These data also suggested that tissue and biliary changes in ADMA and SDMA should be CAT-2B transporter-dependent representing a step forward in the understanding of the mechanisms involved in the control of Arg and its methylated derivatives during hepatic I/R. Supported by Fondazione Cariplo, grant no. 2011-0439. P0007 HEPATIC T CELLS DERIVED FROM NORMOTHEMIC MACHINE PERFUSED GRAFTS CONTAINED MORE REGULATORY T CELLS AND FEWER PROINFLAMMATORY CYTOKINES PRODUCING T CELLS THAN THOSE FROM COLD STORAGE GRAFTS X. Huang1 , R. Ravikumar2 , C. Coussios2 , P. Srinivasan1 , N. Heaton1 , P. Friend2 , W. Jassem1 , Y. Ma1 . 1 Liver Institute, King’s College, London, 2 Surgical Sciences, Oxford University, Oxford, United Kingdom E-mail: [email protected] Background and Aims: In liver transplantation, ischemia reperfusion (I/R) injury is associated with an inflammatory response that has an impact on graft and patient outcomes. Ex-vivo normothermic machine perfusion (NMP) of donor allografts prior

to transplantation has the potential to attenuate I/R injury, leading to improved liver function following implantation and increased utilization of grafts from marginal donors. Livers contain a significant amount of mononuclear cells that are involved in hepatic inflammatory processes. To assess the influence of NMP on hepatic inflammation, the phenotype and function of intra-hepatic lymphocytes derived from NMP- versus cold storage (CS)-preserved livers were compared. Methods: All samples were obtained from liver allografts used for the phase I clinical trial of the Organanox NMP device. Second passage perfusates were collected from 12 grafts donated after brain death (DBD) immediately after NMP and compared to that of 21 CS grafts. Hepatic mononuclear cells (HMC) contained in the perfusate were isolated by density gradient centrifugation. Flow cytometry was used to define the phenotypes and the proportions of activated cytokine producing HMC by utilizing staining with various leukocyte lineage and activation markers. Results: The mean preservation time was 11.5 hours for NMP and 9.5 hours for CS. Although the frequency of CD4+ T cells, NK and NKT cells were comparable between NMP and CS grafts, the frequency of CD8+ T cells (p = 0.01) and CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) (p = 0.03) was significantly higher in NMP than in CS liver grafts. The proportions of both CD4+ and CD8+ T cells producing the pro-inflammatory cytokines IFNg (p = 0.008 and p = 0.006, respectively) and IL-17 (p < 0.001 and p < 0.001, respectively) was significantly lower in NMP than in CS grafts. Additionally, there were fewer CD4+ T cells producing the T cell trophic factor IL-2 in NMP versus CS allografts. There were no significant differences in T cell expression of immunosuppressive cytokines IL-10 or TGF-b. Conclusions: These data demonstrate more Tregs and less proinflammatory T cells in NMP allografts, as compared to their CS counterparts. Brain death has been previously characterized as a pro-inflammatory state and the utilization of NMP rather than standard cold storage techniques modulates the allograft inflammatory response. Further studies are warranted to determine if these findings are associated with the improved clinical outcomes. P0008 A MULTIDRUG STRATEGY TO ATTENUATE ISCHEMIA REPERFUSION INJURY IN EXPERIMENTAL LIVER TRANSPLANTATION K. Yamanaka1 , P. Houben1 , H. Bruns1 , D. Schultze1 , E. Hatano2 , P. Schemmer1 . 1 Dept. of General, Visceral and Transplant Surgery, University Hospital Heidelberg, Heidelberg, Germany; 2 Dept. of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan E-mail: [email protected] Background and Aims: To develop a multidrug strategy designed to target various pathophysiological ischemia reperfusion injury (IRI) mechanisms in liver transplantation (LT), focusing on high-mobility group box 1 (HMGB1) release as a molecular marker. Methods: Orthotropic liver transplantation with arterialization was performed in rats after 8 hours cold storage in HTK solution. Donors underwent i.v. multidrug preconditioning with glycine, taurine, alanine, arginine and prednisolone. Controls received the same volume normal saline i.v. prior to organ donation. Results: Trypan blue staining and transmission electron microscopy revealed that necrotic cell death of sinusoidal endothelial cells and ischemic changing of hepatocytes were attenuated significantly after multidrug preconditioning. Further HMGB1 was decreased in rinsed solution compared to controls, preconditioning with glycine alone and after preconditioning with a combination of glycine and taurine (P = 0.0308). The examination at 1, 3 and 5 hours after reperfusion showed that multidrug preconditioning significantly decreased time-dependent necrotic changing and inflammatory response, and decreased the risk of early death after transplantation

Journal of Hepatology 2015 vol. 62 | S263–S864

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