A. Jake Demetris

Dilated Cardiomyopathy in Transgenic Mice With Cardiac-Specific Overexpression of Tumor Necrosis Factor-α

The failing human heart expresses tumor necrosis factor-alpha (TNF-alpha). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-alpha in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-alpha by preserving the destabilizing sequence in TNF-alpha cDNA. Expression was driven by the murine alpha-myosin heavy chain promoter. Use of this modified construct allowed to the establish a mutine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight-to-body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-alpha protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dtmax was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF-alpha recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.

Fibrosis correlates with a ductular reaction in hepatitis C: Roles of impaired replication, progenitor cells and steatosis

The mechanisms for progressive fibrosis and exacerbation by steatosis in patients with chronic hepatitis C (HCV) are still unknown. We hypothesized that proliferative blockade in HCV‐infected and steatotic hepatocytes results in the default activation of hepatic progenitor cells (HPC), capable of differentiating into both biliary and hepatocyte lineages, and that the resultant ductular reaction promotes portal fibrosis. To study this concept, 115 liver biopsy specimens from subjects with HCV were scored for steatosis, inflammation, and fibrosis. Biliary epithelium and HPC were decorated by cytokeratin 7 immunoperoxidase, and the replicative state of hepatocytes was assessed by p21 and Ki‐67 immunohistochemistry. A ductular reaction at the portal interface was common. There was a highly significant correlation between the area of ductular reaction and fibrosis stage (r = 0.453, P < .0001), which remained independently associated after multivariate analysis. HPC numbers also correlated with fibrosis (r = 0.544, P < .0001) and the ductular area (r = 0.624, P < .0001). Moreover, steatosis correlated with greater HPC proliferation (r = 0.372, P = .0004) and ductular reaction (r = 0.374, P < .0001) but was not an obligate feature. Impaired hepatocyte replication by p21 expression was independently associated with HPC expansion (P = .002) and increased with the body mass index (P < .001) and lobular inflammation (P = .005). In conclusion, the strong correlation between portal fibrosis and a periportal ductular reaction with HPC expansion, the exacerbation by steatosis, and the associations with impaired hepatocyte replication suggest that an altered regeneration pathway drives the ductular reaction. We believe this triggers fibrosis at the portal tract interface. This may be a stereotyped response of importance in other chronic liver diseases. (HEPATOLOGY 2005;41:809–818.)

Cardiac-specific overexpression of tumor necrosis factor-alpha causes lethal myocarditis in transgenic mice

BACKGROUND Tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine with negative inotropic effects, can be detected in myocardium with end-stage heart failure, after endotoxin administration, and during transplant rejection. Various studies suggest that TNF-alpha participates in the pathogenesis of cardiac dysfunction. To test this hypothesis, transgenic mice were made that selectively overexpress TNF-alpha in cardiomyocytes. METHODS AND RESULTS A transgene construct was made containing the murine alpha-myosin heavy chain promoter and the coding sequence of murine TNF-alpha, followed by the simian virus 40 T-antigen intron and polyadenylation signals. Injection of this construct into fertilized eggs yielded three transgenic mice, all of which died spontaneously before the completion of weaning. Gross pathologic analysis of these mice demonstrated a decrease in body weight with markedly increased heart weight. Histologic examination of the heart revealed a substantial, diffuse lymphohistiocytic inflammatory infiltrate, associated with interstitial edema. Reverse transcriptase polymerase chain reaction showed that the transgene was expressed in the heart. Enzyme-linked immunosorbent assay demonstrated a substantial amount of TNF-alpha protein in the transgenic heart. CONCLUSION Overexpression of TNF-alpha in the heart leads to severe myocarditis and cardiomegaly. These results support the hypothesis that myocardial expression of TNF-alpha can contribute to the pathogenesis of cardiac dysfunction.

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival.

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4+ Foxp3+ regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b+ cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4+ Foxp3+ Tregs, including an ST2L+ population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8+ IFN-γ+ cells. Also, despite reducing overall CD3+ cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3+ cells. Whereas control graft recipients displayed increases in systemic CD11b+ Gr-1hi cells, IL-33–treated recipients exhibited increased CD11b+ Gr-1int cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4+ Foxp3+ Tregs that underlie IL-33–mediated cardiac allograft survival.

Cyclooxygenase-2 Promotes Human Cholangiocarcinoma Growth: Evidence for Cyclooxygenase-2-Independent Mechanism in Celecoxib-Mediated Induction of p21waf1/cip1 and p27kip1 and Cell Cycle Arrest

The expression of cyclooxygenase-2 (COX-2) is increased in human cholangiocarcinoma. However, the biologic function and molecular mechanisms of COX-2 in the control of cholangiocarcinoma cell growth have not been well established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth of human intrahepatic cholangiocarcinoma cells. Overexpression of COX-2 or treatment with prostaglandin E2 (PGE2) enhanced human cholangiocarcinoma cell growth, whereas antisense depletion of COX-2 in these cells decreased PGE2 production and inhibited growth. These findings demonstrate a direct role of COX-2-mediated PGE2 in the growth regulation of human cholangiocarcinoma cells. Furthermore, the COX-2 inhibitor celecoxib induced a dose-dependent inhibition of cell growth, cell cycle arrest at the G1-S checkpoint, and induction of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. However, the high concentration of celecoxib (50 μm) required for inhibition of growth, the incomplete protection of celecoxib-induced inhibition of cell growth by PGE2 or COX-2 overexpression, and the fact that overexpression or antisense depletion of COX-2 failed to alter the level of p21waf1/cip1 and p27kip1 indicate the existence of a COX-2-independent mechanism in celecoxib-induced inhibition of cholangiocarcinoma cell growth.

A schema for histologic grading of small intestine allograft acute rejection

Background. Histologic evaluation of small bowel allograft biopsies is important for the diagnosis of acute rejection. However, a standard histologic schema to grade the severity of intestinal acute rejection is not currently available. The primary goal of this study was to develop a histologic grading system for the diagnosis of small bowel allograft acute rejection. Methods. We evaluated 3268 small bowel allograft biopsies obtained from adult patients who underwent small bowel transplantation at the University of Pittsburgh Medical Center between 1990 and 1999. A histologic grading system was proposed and validated by retrospective correlation with clinical outcomes. Results. Among the 3268 biopsies, 180 acute rejection episodes were diagnosed (88 indeterminate, 74 mild, 14 moderate, and 4 severe). All four histologically diagnosed, severe acute rejection episodes resulted in graft failure before resolution, despite aggressive immunosuppressive therapy. Four of the 14 moderate acute rejection episodes were associated with unfavorable clinical outcomes. In contrast, the 74 mild and 88 indeterminate acute rejection episodes were not associated with unfavorable clinical outcomes. Statistical analysis for trend revealed that grades indicating more severe acute rejection episodes were associated with a greater probability of unfavorable outcomes (P <0.01). In addition, there was good overall agreement among different pathologists regarding the diagnosis of acute rejection using the proposed schema, suggesting that this system is practical. Conclusions. This study provides a reliable predictive schema for assessment of the severity of human small bowel acute rejection.

Disease recurrence and rejection following liver transplantation for autoimmune chronic active liver disease

Autoimmune chronic active liver disease (ACALD), a major indication for liver transplantation, is associated strongly with antigenic determinants HLA-B8 and DR3. A retrospective analysis of 43 patients who underwent OLTx for putative ACALD and who, as well as their tissue organ donors, were typed, was performed. Disease recurrence and graft rejection episodes were determined by chart review and histopathological review of all material available. Disease recurrence was histologically documented in 11 (25.6%) of these 43 cases. Graft rejection episodes occurred in 24 (55.8%). All recurrences were in recipients of HLA-DR3-negative grafts. Nine of the recurrences were in HLA-DR3-positive recipients (odds ratio: 6.14, P<0.03). Two of 11 cases of disease recurrence were in recipients who were HLA-DR3-negative. Nine of these 11 had received HLA-DR3-negative grafts. Rejection occurred in 13 HLA-B8-positive recipients, 12 of whom received HLA-B8-negative grafts. Eleven HLA-B8-negative recipients experienced at least one rejection episode and 9 of these had received HLA-B8-negative grafts. Based upon these data we conclude: 1) that recurrence of putative ACALD is more likely to occur in HLA-DR3-positive recipients of HLA-DR3-negative grafts; (2) that recurrences were not seen in recipients of HLA-DR3-positive grafts; (3) that BXA-B8 status does not affect disease recurrence; and (4) that neither the HLA-B8 nor the DR3 status of the graft or recipient has an effect on the observed frequency of rejection.

The adverse impact on liver transplantation of using positive cytotoxic crossmatch donors

Because of the liver grafts ability to resist cytotoxic antibody-mediated rejection, it has become dogma that the conventional transplant crossmatch used to avoid hyperacute rejection of other organs is irrelevant to the liver. We examined this hypothesis in a consecutive series of adult primary liver recipients treated with FK506 and low-dose steroids. Twenty-five of 231 (10.8%) patients received a liver from a cytotoxic-positive crossmatch donor (more than 50% of donor T lymphocytes were killed by dithiothreitol-pretreated recipient serum). The outcome was compared with that of 50 negative crossmatch patients who had their transplantations just before and after the crossmatch positive cases. The one-year graft and patient survivals were 56% and 68%, for positive and 82% and 86% for negative crossmatch patients (P = 0.004, P = 0.03, respectively). The difference between patient and first graft survival was accounted for by retransplantation, which was 4 times more frequent in the positive-crossmatch cases. Histologically, failed allografts obtained at the time of retransplantation revealed a spectrum of pathologic findings related to vascular injury. This study showed a higher difficulty of intraoperative blood product management, a degraded prognosis, and a poorer average quality of ultimate graft function when liver transplantation was performed against positive cytotoxic crossmatches. In such patients for whom crossmatch-negative donors may never be found because of the broad extent and intensity of sensitization, special therapeutic strategies perioperatively must be evolved if results are to improve.

Incidence, prevalence, and clinical course of hepatitis C following liver transplantation

Hepatitis C virus (HCV) is the agent responsible for posttransfusion hepatitis. The incidence, timing, and clinical course of HCV positive hepatitis in liver transplant recipients are unknown. Three hundred and seventeen donor-recipient liver transplant pairs were grouped on the basis of their pretransplant HCV antibody status. The biopsy findings were examined. Four distinct groups were identified on the basis of HCV serology: group I, both were negative; group II, donor was negative and recipient was positive; group III, donor was positive and recipient was negative; group IV, both were positive. The prevalence of anti-HCV positivity in recipients was 13.6%. The rate of seroconversion was 9.2%. Histologic hepatitis not ascribable to any specific cause other than non-A, non-B (NANB) hepatitis occurred in 13.8%. The incidence of histologic chronic active hepatitis was 1.6%, and none progressed to cirrhosis. The concordance rate for a positive anti-HCV serology and NANB hepatitis was 2.8%. Of the 35 patients (group II and IV) with positive anti-HCV serology pretransplant, only 17 were positive posttransplantation. Based on these data it can be concluded that posttransplant NANB hepatitis occurred in 13.8% of liver recipients. Twenty percent of these were anti-HCV positive. Progression to histologic chronic active hepatitis occurs over a period of 1-5 years in 1.6% of cases.

Porcine partial liver transplantation: A novel model of the “small‐for‐size” liver graft

Increasing shortage of cadaveric grafts demands the utilization of living donor and split liver grafts. The purpose of this study was to 1) define the “small‐for‐size” graft in a pig liver transplant model 2) evaluate pathological changes associated with small‐for‐size liver transplantation. Pigs were divided into four groups based on the volume of transplanted liver: (a) control group (n=4), 100% liver volume (LV) (b) group I (n=8), 60% LV (c) group II (n=8), 30% LV (d) group III (n=15), 20% LV. Tacrolimus and methyl prednisone were administered as immunosuppression. Animals were followed for 5 days with daily serum biochemistry, liver biopsies on day 3 and 5 for light microscopy, and tissue levels of thymidine kinase (TK) and ornithine decarboxylase (ODC). Liver grafts were weighed pretransplant and at sacrifice. All the recipients of 100%, 60%, and 30% grafts survived. Transplantation of 20% grafts (group III) resulted in a 47% mortality rate. Group III animals showed significantly prolonged prothrombin times (p<0.05), elevated bilirubin levels (p<0.05), and ascites. The rate of regeneration, as indicated by TK activity and graft weight was inversely proportional to the size of the transplanted graft. The severity of the microvascular injury was inversely proportional to graft size and appeared to be the survival‐limiting injury. Frank rupture of the sinusoidal lining, parenchymal hemorrhage, and portal vein injury were prominent in group III animals 1 hour following reperfusion. This study established a reproducible large animal model of partial liver grafting; it defined the small‐for‐size syndrome in this model and described the associated microvascular injury. (Liver Transpl 2004;10:253–263.)