A. Coito

Blockade of very late antigen-4 integrin binding to fibronectin in allograft recipients: I. Treatment with connecting segment-1 peptides prevents acute rejection by suppressing intragraft mononuclear cell accumulation, endothelial activation, and cytokine expression.

BACKGROUND Allograft rejection is associated with infiltration of inflammatory cells and local deposition of fibronectin (FN). This study was carried out to examine the hypothesis that peptides known to specifically block adhesive interactions between the connecting segment-1 (CS1)-binding domain of FN and alpha4beta1 integrin on circulating cells may interfere with the immune cascade, which would lead to acute rejection in transplant recipients. METHODS AND RESULTS Cardiac allografts from Lewis x Brown Norway F1 hybrids were rejected in 7+/-1 days in Lewis rats. Treatment with bioactive CS1 peptides (4 mg/kg/day i.v. for 7 days) abrogated acute rejection and prolonged cardiac allograft survival to 13+/-1 days (P<0.001). This effect correlated with decreased expression of total fibronectin and cell adhesion molecules, such as alpha4beta1, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, as well as reduced infiltration by CD4+ and CD8+ T cells at the graft site. Treatment with CS1 peptides decreased alloantigen activation, as evidenced by decreased intragraft infiltration by CD25+ cells, and diminished expression of mRNA coding for Th1 (interleukin [IL]-2, interferon-gamma)- and Th2 (IL-4, IL-5, IL-6)-type cytokines. CS1-mediated immunosuppressive effects could be reversed and acute rejection recreated after adjunctive treatment of rats with recombinant IL-2. CONCLUSION Our data are consistent with the model in which in vivo interaction between the alpha4beta1 integrin receptor and the cell-associated CS1 motif of FN is critical for rejection cascade. The novel therapeutic approach of selectively blocking the alpha4beta1-FN activation pathway with CS1 peptides prevents acute allograft rejection by inhibiting expansion of antigen-specific T cells and inducing a transient state of cytokine-responsive anergy in the residual T-cell population.

Liver Regeneration After Ischemia Reperfusion Injury in High-Fat-Diet Induced Hepatic Microsteatosis and Macrosteatosis.: Abstract# C1646

C1646 Liver Regeneration After Ischemia Reperfusion Injury in HighFat-Diet Induced Hepatic Microsteatosis and Macrosteatosis. N. Kuriyama,1,2 S. Isaji,2 S. Duarte,1 R. Busuttil,1 A. Coito.1 1The DumontUCLA Transplant Center, Los Angeles, CA; 2Hepatobiliary Pancreatic and Transplant Surgery, Mie University School of Medicine, Tsu, Mie, Japan. Fatty livers have low tolerance to IRI and high risks of organ failure after transplantation. Impaired liver regeneration, often attributed to fatty livers, is a frequent feature in acute liver failure. The present study unveils mechanisms of hepatic regeneration in clinically relevant models of lean and steatotic hepatic IRI. Methods and Results: C57BL6 mice were fed with standard or high-fat (HF; 60% of total kilocalories) diets for up to 8 weeks. Mice fed with a standard diet had virtually no liver fat inclusions, whereas HF-diet-fed mice with about 29-33g and 40-45g of body weight were characterized by prevalence of liver microsteatosis (MiS) and macrosteatosis (MaS), respectively (assessed by H&E, Oil-red O staining, and triglyceride levels). Mice were submitted to partial warm ischemia for 60or 90-min ischemia followed by reperfusion. In 60-min IRI, all lean, MiS, and MaS mice survived post-reperfusion. MaS mice had signifi cantly higher serum AST (11632±3238 vs. 2123±1424 vs. 380±269) levels (U/L) compared with MiS (p<0.05) and lean (p<0.05) mice, respectively, at 24h after IRI. Cyclin D1 is one of the key regulatory proteins of the cell cycle; its expression is required for transition from the G1 into the S phase. The relatively well-preserved lean livers showed signifi cantly increased Cyclin D1 expression (1.18±0.26 vs. 0.24±0.13 vs. 0.04±0.01), but reduced PCNA levels (0.03±0.003 vs. 0.86±0.14 vs. 0.29±0.03), compared with MiSand MaS livers (respectively, p<0.05) at 48h post-IRI. In 90-min IRI, all lean and MiS mice survived surgery, despite signifi cant liver damage, while only 40% of the MaS mice were alive after the fi rst two postoperative days. Here, lean mice showed signifi cantly higher expression levels of both Cyclin D1 (0.47±0.01vs. 0.31±0.02 vs. 0.10±0.02) and PCNA (0.40±0.05 vs. 0.23±0.05 vs. 0.13±0.04), compared with MiS and MaS mice (respectively, p<0.05) at 48h post-IRI. PCNA protein expression levels correlated with the hepatocyte proliferation index in all evaluated tissues. Conclusion: This thorough study demonstrates distinct expression patterns of cell-cycle regulators in macrosteatotic, microsteatotic and lean livers after IRI. Moreover, it suggests that while Cyclin D1 levels refl ect the capability of livers to regenerate post-reperfusion, PCNA levels represent their regenerative activity. Abstract# C1647 Hepatocyte Gsk3 Inactivation Via Phosphorylations Is Essential for Ischemia Reperfusion Induced Infl ammatory Cell Death. J. Zhu,1,2 S. Yue,1 M. Zhang,2 R. Busuttil,1 Q. Xia,2 J. Kupiec-Weglinski,1 Y. Zhai.1 1Surgery, David Geffen School of Medicine-UCLA, Los Angeles, CA; 2Liver Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. Background: Glycogen synthase 3 α and β are ubiquitously expressed and play roles in various aspects of cellular responses, such as proliferation, apoptosis, and immune activation. We have documented previously that IR induced liver Gsk3b phosphorylation and Gsk3 inhibition protected livers from IRI via an IL-10 mediated immune regulatory mechanism. As Gsk3 is generally constitutively active and inhibited via phosphorylation (at serine 21 and 9 in Gsk3α and β respectively) upon stimulation, the question of what the biological signifi cance of this inhibitory phosphorylation process is in liver cells has yet to be defi ned. Methods: In a murine liver partial warm ischemia model, we investigated the effect of phosphorylation-resistant Gsk3αβ in both immune activation and hepatocyte death against IR by using dual Gsk3αS21A and Gsk3βS9A knock-in (KI) mice. Macrophages and hepatocytes isolated from the KI animals were studied in vitro. Results: Dual Gsk3 KI mice were protected from liver IRI, as measured by sALT levels and liver histological examination at both 6 and 24h post reperfusion. However, liver infl ammatory gene inductions by IR, including TNF-α, IL-6, IL-10, IL-12p40, and CXCL10, measured by qRT-PCR, were signifi cantly enhanced in these KI mice, as compared with those in WT ones. This was in parallel with our in vitro fi nding that bone marrow derived macrophages from these KI mice responded to TLR stimulation more vigorously that infl ammatory cytokine productions, TNF-α, IL-12p40, as well as IL-10, were all increased, measured by ELISA, as compared with cells from WT mice. Hepatocytes Gsk3 became phosphorylated in response to infl ammatory stimuli, including TNF-α. Although Gsk3 inhibitor SB216763 had no signifi cant effect in regulating WT hepatocyte death in their response to TNF-α, Gsk3 KI hepatocytes became signifi cantly more resistant to TNF-α induced cell death (in combination with either Actinomycin D or Tunicamycin), as measured by LDH assay, and the Gsk3 inhibitor SB216763 readily reversed their resistance. Conclusions: Gsk3 has distinctive roles in different liver cell types. Its inhibitory phosphorylation limits infl ammatory immune activation in macrophages, while promotes infl ammatory cell death in hepatocytes in their response to IR. C1647 Hepatocyte Gsk3 Inactivation Via Phosphorylations Is Essential for Ischemia Reperfusion Induced Infl ammatory Cell Death. J. Zhu,1,2 S. Yue,1 M. Zhang,2 R. Busuttil,1 Q. Xia,2 J. Kupiec-Weglinski,1 Y. Zhai.1 1Surgery, David Geffen School of Medicine-UCLA, Los Angeles, CA; 2Liver Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. Background: Glycogen synthase 3 α and β are ubiquitously expressed and play roles in various aspects of cellular responses, such as proliferation, apoptosis, and immune activation. We have documented previously that IR induced liver Gsk3b phosphorylation and Gsk3 inhibition protected livers from IRI via an IL-10 mediated immune regulatory mechanism. As Gsk3 is generally constitutively active and inhibited via phosphorylation (at serine 21 and 9 in Gsk3α and β respectively) upon stimulation, the question of what the biological signifi cance of this inhibitory phosphorylation process is in liver cells has yet to be defi ned. Methods: In a murine liver partial warm ischemia model, we investigated the effect of phosphorylation-resistant Gsk3αβ in both immune activation and hepatocyte death against IR by using dual Gsk3αS21A and Gsk3βS9A knock-in (KI) mice. Macrophages and hepatocytes isolated from the KI animals were studied in vitro. Results: Dual Gsk3 KI mice were protected from liver IRI, as measured by sALT levels and liver histological examination at both 6 and 24h post reperfusion. However, liver infl ammatory gene inductions by IR, including TNF-α, IL-6, IL-10, IL-12p40, and CXCL10, measured by qRT-PCR, were signifi cantly enhanced in these KI mice, as compared with those in WT ones. This was in parallel with our in vitro fi nding that bone marrow derived macrophages from these KI mice responded to TLR stimulation more vigorously that infl ammatory cytokine productions, TNF-α, IL-12p40, as well as IL-10, were all increased, measured by ELISA, as compared with cells from WT mice. Hepatocytes Gsk3 became phosphorylated in response to infl ammatory stimuli, including TNF-α. Although Gsk3 inhibitor SB216763 had no signifi cant effect in regulating WT hepatocyte death in their response to TNF-α, Gsk3 KI hepatocytes became signifi cantly more resistant to TNF-α induced cell death (in combination with either Actinomycin D or Tunicamycin), as measured by LDH assay, and the Gsk3 inhibitor SB216763 readily reversed their resistance. Conclusions: Gsk3 has distinctive roles in different liver cell types. Its inhibitory phosphorylation limits infl ammatory immune activation in macrophages, while promotes infl ammatory cell death in hepatocytes in their response to IR. Abstract# C1648 The MicroRNA Expression Profi le in Donation After Cardiac Death (DCD) Liver Grafts and Its Ability to Identify Primary Non Function. C1648 The MicroRNA Expression Profi le in Donation After Cardiac Death (DCD) Liver Grafts and Its Ability to Identify Primary Non Function. S. Khorsandi, S. Salehi, W. Jassem, H. Vilca-Melendez, A. Prachalias, P. Srinivasan, N. Heaton. Institute of Liver Studies, King’s College Hospital, London, United Kingdom. Purpose DCD grafts are a marginal organ and usage has to be balanced against the risk of primary non function (PNF) or initial poor graft function (IPGF). To give insight into pathophysiology and potentially rationalise DCD usage, the aim of this work was to determine if a microRNA expression profi le was able to discriminate between DCD grafts of varying functional quality,. Methods Three DCD groups were studied. PNF group (n=8) retransplanted within a week, good group (n=7) AST ≤ 1000 IU/L and poor group/IPGF (n=8) AST ≥ 2500 IU/L. miRNA was isolated from FFPE trucut post perfusion biopsies. Affymetrix GeneChip® miRNA 2.0 Arrays were used. Expression data analysis was performed using Qlucore Omics Explorer 3.0. A statistical signifi cance level of p<0.05 was used to identify differentially expressed miRNA in comparison of the three groups. To validate microarray results real time quantitative PCR (RT-qPCR) was performed on all samples for miRNA species identifi ed as being signifi cant. Ct (threshold cycle) values were normalised using the average Ct of the endogenous control to generate ΔCT and fold-change to reference sample of normal liver (2ΔΔCT) for relative expression analysis. Data was then analysed by one way non parametric ANOVA to detect signifi cance (p<0.05) between the three groups. If signifi cance was found the Bonferroni correction for multiple comparisons was applied. Results From the array analysis 16 miRNA species (miR-10