Background. Evidence is emerging that the endoplasmic reticulum (ER) participates in initiation Of apoptosis induced by the unfolded protein response and by aberrant Ca++ signaling during cellular stress such as ischemia/reperfusion injury (I/R injury). ER-induced apoptosis involves the activation of caspase-12 and C/EBP homologous protein (CHOP), and the shutdown of translation initiated by phosplonylation of eIF2 alpha. Sodium 4-phenylbutyrate (PBA) is a low molecular weight fatty acid that acts as a chemical chaperone reducing the load of mutant or unfolded proteins retained in the ER during cellular stress and also exerting anti-inflammatory activity. It has been used succesfully for treatment of area cycle disorders and sickle cell disease. Thus, we hypothesized that PBA may reduce ER-induced apoptosis triggered by I/R injury to the liver.Methods. Groups of male C57BL/6 mice were subjected to warm ischemia (70% of the liver mass, 45 minutes). Serum aspartate aminotransferase was assessed 6 hours after reperfusion,- apoptosis was evaluated by enzyme-linked immunosorbent assays of caspase-12 and plasma tumor necrosis factor alpha, Western blot analyses of eIF2 alpha, and reverse transcriptase-polymerase chain reaction of CHOP expression.Results. A dose dependent decrease in aspartate aminotransferase was demonstrated in mice given intraperitoneal PBA (I hour before and 12 hours after reperfusion), compared with vehicle-treated controls,- this effect was associated with reduced pyknosis, parenchymal hemorrhages, and neutrophil infiltrates in PBA-treated mice, compared with controls. In a lethal model of total liver I/R injury, all vehicle-treated controls died within 3 days after reperfusion. In contrast, 50 % survival (> 30 days) was observed in animals given PBA. The beneficial effects of PBA were associated with a greater than 45 % reduction in apoptosis, decreased ER-mediated apoptosis characterized by significant reduction in caspase-12 activation, and reduced levels of both phosphorylated eIF2 alpha and CHOP. Significant reductions in plasma levels of tumor necrosis factor alpha and liver myeloperoxidase content were demonstrated after PBA treatment.Conclusions. Reduction in ER stress-induced hepatocellular injury was achieved by the administration of PBA. Targeting the ER-associated cell death pathway might offer a novel approach to reduce I/R injury to the liver.

End-stage liver disease is being treated by liver transplantation since more than 20 years. Despite social and legislative efforts, the number of cadaveric organs suitable for liver transplantation has not grown to match the surplus of patients with end-stage liver disease. With the growing discrepancy between donors and recipients, the median waiting time for liver transplantation has increased dramatically. As a result, the number of patients who die while waiting is increasing. To attempt to meet the growing needs of recipients, surgeons are developing innovative techniques to increase the number of donated livers. These include: split liver transplantation and transplantation of a part of the liver from living donors. This review will focus on adult-to-adult transplantation of the right lobe from a living donor.

In the last few years, there have been developments in many aspects of liver transplantation. Improvements in surgical techniques and immunosuppression markedly increased the success rates of liver transplantation. This success has lead to increasing numbers of recipients. However, the availability of cadaveric organs for transplantation has not been changed in the last 10 years, resulting in a growing discrepancy between donors and recipients. Thus, it is necessary to properly select the best candidates for a successful liver transplant. This article will review the indications and contraindications for liver transplantation in the Model for End Stage Liver Disease (MELD) score era.

Functional poly(ethylene glycol) (PEG) derivatives, including monosuccinimidyl PEG (MSPEG) with molecular weight (MW) of 2000 (2kDa) as well as 5kDa and disuccinimidyl PEG (DSPEG) with MW of 3 and 6kDa, were synthesized and characterized. They were used to modify the surface of adult porcine islets for cytoprotection. The islets were isolated, purified and modified with functional PEG. Untreated porcine islets were used as control. An in vitro human antibody/complement-mediated cytotoxicity test based on the release of intracellular lactate dehydrogenase was used to evaluate cytotoxicity of human serum to the modified islets. In vitro cell viability was assessed using membrane-integrity straining and islet metabolism in culture. In vitro islet functionality was evaluated by glucose-stimulated insulin release of islets in static incubation with human serum. In vivo islet functionality was evaluated by monitoring non-fasting blood glucose level in streptozotocin-induced diabetic (SCID) immunocompromized mice after intraportal transplantation of porcine islets. Results show that all the PEG derivatives used in the study showed significant in vitro and in vivo cytoprotections against cytotoxic effects elicited by human serum and diabetic SCID mice, respectively, to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to MSPEG ones, due to the capacity of the succinimidyl groups to selectively react with amino groups of the albumin under physiological conditions. The effects of both MW and concentration of the PEG derivatives on cytoprotection were significant. It appears that this novel biotechnology will be an attractive approach for improved xenotransplantation of islets.

Insulin-dependent diabetes mellitus is an autoimmune disease that causes a progressive destruction of the pancreatic beta cells. As a result, the patient requires exogenous insulin to maintain normal blood glucose levels. Both the pancreas and the islets of Langerhans have been transplanted successfully in humans and in animal models, resulting in full normalization of glucose homeostasis. However, insulin independence, transient or persistent, was documented in only a small fraction of cases until recently. The chronic immunosuppression required to avoid immunological rejection appears to be toxic to the islets and adds the risk of lymphoproliferative disease reported earlier. For islet transplantation to become the method of choice, it is essential first to identify islet-friendly immunosuppressive regimens and/or to develop methods that induce donor-specific tolerance and improve islet isolation and transplantation protocols. Indeed, researchers have already successfully allografted islets in the presence of nonsteroidal immunosuppression in a process known as the Edmonton protocol. An alternative method, gene therapy, could replace these other methods and better meet the insulin requirement of an individual without requiring pancreatic or islet transplantation. This alternative, however, requires animal models to develop and test clinical protocols and to demonstrate the feasibility of preclinical trials. Nonhuman primates are ideally suited to achieve these goals. The efforts toward developing a nonhuman primate diabetic model with demonstrable insulin dependence are discussed and include pancreatic and islet transplant trials to reverse the diabetic state and achieve insulin independence. Also described are the various protocols that have been tested in primates to circumvent immunosuppression by using tolerance induction strategies in lieu of immunosuppression, thus exploring the field of donor-specific tolerance that extends beyond islet transplantation.

BACKGROUND:Patient and graft survival after liver transplantation are adversely affected by early posttransplant renal dysfunction. Therefore, our immunosuppressive strategies should be as "renal sparing" as possible. This is the largest published series to date using daclizumab induction therapy in a renal-sparing regimen.METHODS:This is a retrospective, nonrandomized study comparing 209 adult liver transplants with daclizumab induction to 115 transplants with no induction.RESULTS:Patient and graft survival were similar, despite higher pretransplant acuity of illness and older age in the induction group. Acute rejection within the first 6 months occurred less commonly in the induction group (25.4% vs. 39.1%, P=0.01), despite significantly delayed initiation and lower doses of a calcineurin inhibitor. Mycophenolate mofetil was used more commonly in induction patients, but the efficacy of daclizumab in preventing rejection was independent of this. Patients with a pretransplant creatinine concentration 1.5 mg/dL or less had less rejection if they received induction. Renal function worsened in noninduction patients but showed sustained improvement throughout follow-up in induction patients with a pretransplant creatinine concentration greater than 1.5 mg/dL. Induction therapy provided better rejection prophylaxis among those requiring temporary calcineurin inhibitor cessation because of renal dysfunction. The incidences of histologic hepatitis C recurrence and cytomegalovirus infection were similar in each group.CONCLUSIONS:Liver recipients with and without pretransplant renal dysfunction have less acute rejection with daclizumab induction therapy. This is not associated with an increased risk of over-immunosuppression. Sustained renal improvement in recipients with pretransplant renal dysfunction is possible with daclizumab induction.

Background. Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly [ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2.Methods. Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining: Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SLID mice reconstituted with human lymphocytes (5 X 10(8)/intraperitoneally/15 days before PIT).Results. No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end" capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P < .05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P < .001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100 % of the animals given islets modified with DSPEG + AdBcl-2 during the observation time.Conclusions. Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.

BACKGROUND:Current isolation techniques recover only 20% to 50% of the pancreatic islets. Brain death (BD) is characterized by activation of proinflammatory cytokines (PICs) with reduced islet yields and functionality. We previously reported that 17beta-estradiol (E2) induces cytoprotection to human islets exposed to PICs. Furthermore, inhibition of PIC release has been demonstrated after E2 treatment. In the present study, we evaluated if E2 treatment to BD donors would improve pancreatic islet recovery and functionality.METHODS:BD was induced in male, 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. Rats were mechanically ventilated for 6 hours. Only rats with mean arterial blood pressure > 75 mm Hg were used. Animals (n = 6) received E2 (1 mg/kg/iv immediately after BD induction), vehicle (V), or the combination of 17beta-estradiol and a selective estrogen receptor antagonist ICI 182,780 (ICI, 3 mg/kg/ip/1 hour before BD induction). Islet viability was determined by ethidium bromide-acridine orange. PICs were assessed by ELISA. Islet functionality was determined by static incubation and glucose disposal rate (Kg) after intraportal transplantation (3000 islet equivalent[IEQ]/syngeneic streptozotocin-induced diabetic rat).RESULTS:A 2- to 3-fold reduction in TNF-alpha, IL-1beta, and IL-6 was demonstrated in BD donors given E2; this effect reversed by ICI 182,780. Pancreatic sections from control BD donors presented 26.5% +/- 4% TUNEL-positive beta-cells compared with 15.1% +/- 3% in 17beta-estradio-treated animals. Islet recovery was enhanced in E2-treated donors (1233.4 +/- 123 IEQ/pancreas) compared with controls (725 +/- 224 IEQ, P < .05). Islet viability was significantly enhanced by E2. Higher islet functionality was demonstrated in vitro and in vivo after transplantation in islets recovered from E2-treated BD donors.CONCLUSIONS:Islet recovery and functionality in vitro and in vivo were significantly improved by 17beta-estradiol treatment to BD donors. These observations may lead to strategies to reduce the effects of BD on isolated islets and improve the results in clinical islet transplantation.

Clinical studies indicate that significant loss of functional islet mass occurs in the peritransplant period. Islets are injured as a result of detrimental effects of brain death, pancreas preservation, islet isolation, hypoxia, hyperglycemia, and immune-mediated events. In addition, recent studies demonstrated that islets are injured as a result of their exposure to blood and of activation of intrahepatic endothelial and Kupffer cells, resulting in inflammation and thrombosis. Activated protein C (APC) is an anticoagulant enzyme that also exerts anti-inflammatory and antiapoptotic activities by acting directly on cells. Here, we report that exogenous administration of recombinant murine APC (mAPC) significantly reduced loss of functional islet mass after intraportal transplantation in diabetic mice. Animals given mAPC exhibited better glucose control, higher glucose disposal rates, and higher arginine-stimulated acute insulin release. These effects were associated with reduced plasma proinsulin, intrahepatic fibrin deposition, and islet apoptosis early after the transplant. In vitro and in vivo data demonstrated that mAPC treatment was associated with a significant reduction of proinflammatory cytokine release after exposure of hepatic endothelial cells to islets. mAPC treatment also prevented endothelial cell activation and dysfunction elicited by intrahepatic embolization of isolated islets inherent to pancreatic islet transplantation (PIT). This study demonstrates multiple remarkable beneficial effects of mAPC for PIT and suggests that APC therapy may enhance the therapeutic efficacy of PIT in diabetic patients.

BACKGROUND:Ischemia/reperfusion injury (I/R injury) of the liver remains a significant problem during liver surgery and transplantation. I/R injury is associated with liver apoptosis, which is mediated by death receptors such as Fas and tumor necrosis factor alpha (TNF-alpha), and/or mitochondrial dysfunction induced by cellular stress. Caspase-8 is presumed to be the apex of the death-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" proteases in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8 and caspase-3 siRNA in a murine model of liver I/R injury.METHODS:In C57BL/6 mice, 45% or 70% of the liver mass was clamped for 90 minutes. For survival analysis, total hepatic ischemia was induced for 45 minutes. In vivo delivery of siRNA was performed via the portal vein by high-volume injection (0.5 nmol of siRNA in 1 mL containing 10% lipiodol) 60 minutes before ischemia. As a control, animals received either vehicle or non-sense siRNA (siRNA-scrambled).RESULTS:Liver uptake of siRNA was analyzed in transgenic mice who express beta-galactosidase (beta-gal) (C57BL/6J-TgN(MTn-LacZ)204Bri) after administration of siRNA-LacZ. A 3- to 4-fold decrease in beta-gal activity was accomplished at 0.5 nmol. No significant change in beta-gal activity was demonstrated in mice receiving non-sense siRNA. Immunohistochemical studies found that 60% of the liver cells efficiently took up siRNA. Significant reduction in serum aspartate transaminase was found in animals treated with siRNA caspase-8 or caspase-3 compared with siRNA-scrambed or vehicle-treated controls. More than a 60% reduction in caspase-8 and caspase-3 gene expression and activities was accomplished after siRNA administration. Animals treated with siRNA presented lower infiltration of polymorphonuclear leukocytes and better preservation of the liver architecture compared with controls. All of the control mice subjected to total liver ischemia died within 5 days. In contrast, 30% of the animals given siRNA caspase-8 and 50% of those treated with siRNA caspase-3 survived indefinitely (>30 days).CONCLUSIONS:Small interfering RNA targeted to caspase-8 and caspase-3 provided significant protection against I/R injury to the liver. This approach could be therapeutic in liver transplantation and other conditions associated with I/R injury to the liver.