Edwina S. Baskin-Bey

Clinical Trial of the Pan‐Caspase Inhibitor, IDN‐6556, in Human Liver Preservation Injury

Cold ischemia/warm reperfusion (CI/WR) injury remains a problem in liver transplantation. The aim of the current study was to assess the utility of the pan‐caspase inhibitor IDN‐6556 on CI/WR injury during human liver transplantation. This report is a post hoc analysis of a Phase II, multi‐center, randomized, placebo‐controlled, double‐blinded, parallel group study. Subjects were assigned to four treatment groups: Group 1 (Organ storage/flush: Placebo—Recipient: Placebo); Group 2 (Organ storage/flush: 15 μg/mL—Recipient: Placebo); Group 3 (Organ storage/flush: 5 μg/mL—Recipient: 0.5 mg/kg); and Group 4 (Organ storage/flush: 15 μg/mL—Recipient: 0.5 mg/kg). Liver cell apoptosis was assessed by serum concentrations of the apoptosis‐associated CK18Asp396 (‘M30’) neo‐epitope, TUNEL assay and caspase 3/7 immunohistochemistry. Liver injury was assessed by serum AST/ALT determinations. Serum markers of liver cell apoptosis were reduced in all groups receiving drug as compared to placebo. However, TUNEL, caspase 3/7 positive cells and serum AST/ALT levels were only consistently reduced in Group 2 (drug exposed to organ only). This reduction in serum transaminases was significant and observed across the study. In conclusion, IDN‐6556 when administered in cold storage and flush solutions during liver transplantation offers local therapeutic protection against CI/WR‐mediated apoptosis and injury. However, larger studies are required to confirm these observations.

Liver transplantation for gastroenteropancreatic neuroendocrine cancers: Defining selection criteria to improve survival

Liver transplantation for gastroenteropancreatic neuroendocrine cancer (GEP) is controversial. The aim of this study was to assess patient outcomes after liver transplantation for hepatic metastases from GEP. Medical records of patients who underwent liver transplantation for GEP were reviewed. Immunohistochemistry for assessing the Ki67 proliferation index was performed on explanted liver tissue. Nineteen patients who underwent liver transplantation had a mean follow‐up of 22 months with a range of 0 to 84 months. There was 1 intraoperative death, and 3 patients had disease recurrence after liver transplantation leading to death in 1 patient. Overall estimated 1‐year survival for 17 patients included in the treatment protocol (mean follow‐up, 15 months) was 87% with an estimated 1‐year recurrence‐free rate (conditional on survival) of 77%. Three of 11 patients with pancreatic islet cell GEP developed disease recurrence, whereas all 8 patients with carcinoid GEP remain free of disease. Analysis of the Ki67 proliferation index in 18 patients did not differentiate those with recurrence from those without disease recurrence. In conclusion, liver transplantation for patients with hepatic metastases from GEP is a viable therapeutic option in highly selected patients. Liver Transpl 12:448–456, 2006.

Proteasome inhibition induces hepatic stellate cell apoptosis

Induction of hepatic stellate cell (HSC) apoptosis attenuates hepatic fibrosis, and, therefore, mechanisms to induce HSC cell death are of therapeutic interest. Proteasome inhibitors induce apoptosis in transformed cells, especially those cells dependent upon nuclear factor kappa B (NF‐κB) activation. Because stimulated HSCs also trigger NF‐κB activation, the aim of this study was to determine if proteasome inhibitors induce HSC apoptosis. The immortalized human HSC line, LX‐2, and primary rat HSCs were treated with the proteasome inhibitors bortezomib and MG132. Both proteasome inhibitors induced HSC apoptosis. Proteasome inhibition blocked NF‐κB activation and, more importantly, NF‐κB inhibition by Bay11‐7082–triggered HSC apoptosis. Activated HSC survival is dependent upon the NF‐κB target gene A1, an anti‐apoptotic Bcl‐2 family member, as siRNA targeted knockdown of A1‐induced HSC apoptosis. In contrast, proteasome inhibition–induced alterations in TRAIL, death receptor 5, and Bim could not be implicated in the apoptotic response. The relevance of these findings was confirmed in the bile‐duct–ligated mouse where bortezomib reduced hepatic markers of stellate cell activation and fibrosis. In conclusion, proteasome inhibition is a potential therapeutic strategy for inducing HSC apoptosis and inhibiting liver fibrogenesis. (HEPATOLOGY 2006;43:335–344.)

Improving the prediction of donor kidney quality: deceased donor score and resistive indices.

Background. The deceased donor score (DDS), expanded criteria donor (ECD) definition, and resistive index (RI) were developed for pretransplant evaluation of donors. DDS and ECD are determined by a calculation of risk from donor variables, while RI is determined from flow characteristics of kidneys during machine preservation (MP). This study was designed to compare DDS, ECD status, and RI as predictors of outcome after deceased donor transplantation. We were also interested to see if DDS or ECD could identify kidneys most likely to benefit from MP. Methods. We retrospectively reviewed 48,952 deceased donor renal transplants reported to UNOS from 1997–2002. DDS (0-39 pts.), ECD status (+ or −), and preservation technique (MP vs. cold storage [CS]) were determined in all cases. RI during MP was studied in a single-center cohort of 425 transplants. Results. DDS was superior to ECD status and RI in its correlation with early and late renal function after transplantation. DDS identified a subgroup of ECD- kidneys, those with DDS ≥20 pts, that functioned significantly below expectation and similar to ECD+ kidneys. Benefits of MP, which include improved early graft function and a trend towards longer graft survival, were greatest in the group of kidneys with DDS ≥20 pts. Conclusions. DDS was the best predictor of outcome after deceased donor renal transplantation and may be useful in identifying kidneys most likely to benefit from MP.

United Network for Organ Sharing's expanded criteria donors: is stratification useful?*

Abstract:  The United Network for Organ Sharing (UNOS) Expanded Criteria Donor (ECD) system utilizes pre‐transplant variables to identify deceased donor kidneys with an increased risk of graft loss. The aim of this study was to compare the ECD system with a quantitative approach, the deceased donor score (DDS), in predicting outcome after kidney transplantation. We retrospectively reviewed 49 111 deceased donor renal transplants from the UNOS database between 1984 and 2002. DDS: 0–39 points; ≥20 points defined as marginal. Recipient outcome variables were analyzed by ANOVA or Kaplan–Meier method. There was a 90% agreement between the DDS and ECD systems as predictors of renal function and graft survival. However, DDS identified ECD− kidneys (10.7%) with a significantly poorer outcome than expected (DDS 20–29 points, n = 5,252). Stratification of ECD+ kidneys identified a group with the poorest outcome (DDS ≥30 points). Predictability of early post‐transplant events (i.e. need for hemodialysis, decline of serum creatinine and length of hospital stay) was also improved by DDS. DDS predicted outcome of deceased donor renal transplantation better than the ECD system. Knowledge obtained by stratification of deceased donor kidneys can allow for improved utilization of marginal kidneys which is not achieved by the UNOS ECD definition alone.

Constitutive androstane receptor (CAR) ligand, TCPOBOP, attenuates Fas-induced murine liver injury by altering Bcl-2 proteins

The constitutive androstane receptor (CAR) modulates xeno‐ and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild‐type (CAR+/+) and CAR knockout (CAR−/−) mice were treated with the CAR agonist 1,4‐bis[2‐(3,5‐dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP‐treated CAR+/+ but not in CAR−/− mice. Likewise, acute and chronic ConA‐mediated liver injury and fibrosis were also reduced in wild‐type versus CAR−/− TCPOBOP‐treated mice. The proapoptotic proteins Bak (Bcl‐2 antagonistic killer) and Bax (Bcl‐2‐associated X protein) were depleted in livers from TCPOBOP‐treated CAR+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor‐1 (Mcl‐1) was increased fourfold. Mcl‐1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl‐1 transcription. Indeed, site‐directed mutagenesis of a putative CAR consensus binding sequence on the Mcl‐1 promoter decreased Mcl‐1 promoter activity. Mcl‐1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl‐1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas‐mediated hepatocyte injury via a mechanism involving upregulation of Mcl‐1 and, likely, downregulation of Bax and Bak. (HEPATOLOGY 2006;44:252–262.)

Cytotoxic immune response to a xenogeneic bioartificial liver.

Prior studies have suggested the possibility of immune-mediated death of xenogeneic hepatocytes in a bioartificial liver (BAL) during hemoperfusion. This study was designed to elucidate how immunity may cause death of xenogeneic hepatocytes in the BAL. Healthy dogs were treated with a BAL containing hollow fiber membranes with large pores (200 nm) or small pores (400 kDa). The immune response of recipient dogs to BAL therapy was monitored over 3 h of treatment. We observed significantly greater loss of viability of hepatocytes in the 200 nm group compared with the 400 kDa group (p < 0.001). Low viability after treatment with the large pore membrane was associated with positive staining for dog IgG, dog IgM, and dog complement on dead hepatocytes. Significant levels of dog antibody were detected in samples of BAL medium from the 200 nm group. These canine antibodies were cytotoxic to porcine hepatocytes. In contrast, medium from the 400 kDa group contained only trace levels of dog IgG and were noncytotoxic. We conclude that antibody-mediated cytotoxicity contributed to the death of hepatocytes during treatment with a xenogeneic BAL. Immune-mediated death of hepatocytes was reduced by increasing selectivity of the BAL membrane.

Improving utilization of deceased donor kidneys by matching recipient and graft survival.

Younger renal transplant recipients often outlive their allografts, whereas older recipients often die before their allograft fails. Thus, our aim was to assess the utility of matching recipient and graft survival to improve allocation of deceased donor kidneys. We reviewed the records of 49,206 patients (United Network for Organ Sharing, 1995–2002). Donor grafts were stratified by Deceased Donor Score (DDS). We observed a disparity between recipient survival and renal graft survival which contributed to an annual gap between supply and demand of renal transplants. Utilization of DDS and distribution of marginal kidneys to older recipients would improve allocation.

Matching graft to recipient by predicted survival: can this be an acceptable strategy to improve utilization of deceased donor kidneys?

A new kidney allocation scheme is needed to address the current shortage of deceased donor kidneys for transplantation in the United States. With this goal in mind, we have derived a novel utility-based system to balance supply and demand. Our system uses a North American-based recipient risk score and the deceased donor score to maximize the total number of years of renal allograft function as a means to improve allocation of kidneys from deceased donors. Essentially, donor renal allografts are matched to a wait-listed candidate with similar predicted survival, which poses an ethical issue. However, this novel utility-based system is practical and could improve deceased donor renal allocation by minimizing both waste and need for retransplantation.

Idiopathic benign biliary strictures in surgically resected patients with presumed cholangiocarcinoma.

OBJECTIVE Distinguishing between malignant and benign biliary strictures remains problematic. The aim of this study was to compare and contrast the clinical features of patients with benign and malignant biliary strictures. METHODS Medical records of patients who underwent surgical resection for presumed cholangiocarcinoma were reviewed. Immunohistochemistry for hypoxia inducible factor-1-alpha (HIF-1-alpha) was performed on all bile ductule samples. RESULTS Twelve patients with benign strictures (group I) were compared to 26 patients with cholangiocarcinoma (group II). Group I was predominantly female (ratio 2: 1), (p<0.01), whereas the gender ratio was 1: 1 in patients in group II. Bismuth-Corlette type strictures in group I were more likely to be type I/II, whereas type III strictures predominated in group II. The CA 19-9 was <100 U/ml in 6 and >100 U/ml in 1 patient of group I and <100 in 13 and >100 in 11 patients in group II. Half of the patients in group I had positive immunoreactivity for HIF-1-alpha in bile ductules. CONCLUSION Benign biliary strictures masquerading as cholangiocarcinomas occur more often in women, are less often Bismuth-Corlette type III, have serum CA 19-9 values <100 U/ml, and hypoxia may play a role in a subset of these strictures.