Frederick A. Nunes

Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis.

Although first generation recombinant adenoviruses, deleted of sequences spanning E1a and E1b, have been useful for in vivo applications of gene therapy, expression of the recombinant gene has been transient and often associated with the development of inflammation. We show that with first generation adenovirus–mediated gene transfer to the mouse lung, viral proteins are expressed leading to destructive cellular immune responses and repopulation of the lung with nontransgene containing cells. Second generation E1 deleted viruses further crippled by a temperature sensitive mutation in the E2a gene were associated with substantially longer recombinant gene expression and less inflammation. Stable expression of human CF transmembrane conductance regulator has been achieved in lungs of CF mice instilled with a second generation virus.

Gene Transfer into the Liver of Nonhuman Primates with E1-Deleted Recombinant Adenoviral Vectors: Safety of Readministration

Preclinical studies were designed to investigate the safety of recombinant adenoviruses infused into the portal vein of adult rhesus monkeys, as well as the safety and efficacy of readministration of these agents. The vectors used were recombinant adenoviruses, the E1 region of which was replaced with a marker gene expression cassette. Four 3- to 5-kg rhesus monkeys underwent portal vein cannulation, and infusion of escalating doses of recombinant first-generation vector. Serial sequential liver biopsies were performed, and necropsies were performed out to 14 months. X-Gal histochemical analysis of the liver showed evidence of dose-dependent increased gene transfer throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was also present in transduced livers, and occurred in a dose-dependent manner. Severe toxicity, including mortality, was noted at the highest dose of vector. Readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response. The data suggest that intraportal administration and readministration of recombinant adenoviral E1-deleted vectors are feasible and safe. Vector administration at the highest dose (1 x 10(13) particles/kg) was associated with severe clinical and biochemical toxicity, and significant gene expression was associated with transaminitis. Readministration of vector is safe, but gene transfer is limited by the presence of neutralizing antibody.

The outcome of liver grafts procured from hepatitis C-positive donors

BACKGROUND The growing prevalence of hepatitis C virus (HCV) infection in the general population has resulted in an increased frequency of potential organ donors that carry the virus. The survival of grafts from HCV+ donors has not been studied in detail. METHODS Two study populations were examined retrospectively to assess the survival of liver grafts procured from HCV+ donors. First, we evaluated the survival of all 13 HCV+ and 103 HCV- grafts that were transplanted at our institution to HCV+ recipients from January 1, 1995 to December 31, 1999. In parallel, we analyzed a subset of the United Network for Organ Sharing (UNOS) liver transplant database from the same 5-year time period that was comprised of 14,195 adult patients for whom donor and recipient HCV serologies were known. Kaplan-Meier graft survival for both patient populations was calculated based on donor and recipient HCV serologic status. A Cox proportional hazards analysis was performed on UNOS data to identify variables independently predicting graft survival. RESULTS For transplants performed at our institution, we found no statistically significant difference in the Kaplan-Meier graft survival of HCV+ and HCV- grafts transplanted to HCV+ recipients (P=0.68). The incidence of biopsy-proven, recurrent HCV posttransplant was similar in recipients receiving either HCV+ or HCV- grafts (4/13 vs. 18/103, chi-square P=0.211). Analysis of UNOS data revealed that the survival of HCV+ grafts in HCV+ recipients was equivalent to the survival of HCV- grafts in HCV+ recipients. Unexpectedly, the survival of grafts in HCV+ recipients in general was significantly inferior to that of grafts in HCV- recipients. Multivariate analysis of all patients found recipient but not donor HCV status to be independently predictive of graft survival. CONCLUSIONS Analysis of data from a single center and the national UNOS database suggests that transplantation of liver allografts from HCV+ donors to HCV+ recipients results in graft survival comparable to HCV- grafts transplanted to HCV+ recipients. In contrast, recipient HCV positivity is an independent predictor of graft failure compared with patients transplanted for other causes of liver disease.

Peripheral virus-specific T-cell interleukin-10 responses develop early in acute hepatitis C infection and become dominant in chronic hepatitis.

BACKGROUND/AIMS Interleukin-10 (IL-10) has been ascribed pro-viral but anti-fibrotic properties in chronic hepatitis C virus (HCV) infection. In this study, we examined the role of HCV-specific T-cell IL-10 response in patients with acute and chronic HCV infection. METHODS Peripheral HCV-specific T-cell IL-10 and IFNgamma responses were measured in cytokine Elispot assay using overlapping HCV-derived peptides in patients with chronic (n=61), resolved (n=15) and acute (n=8) hepatitis C, looking for their onset, quantity, breadth and durability relative to clinical and virological outcomes. The source and effect of HCV-specific IL-10 response were determined in depletion and IL-10 neutralization experiments. RESULTS Both HCV-specific IL-10 and IFNgamma responses were detected early within 1-2 months of acute clinical hepatitis C. However, only HCV-specific IL-10 response correlated with elevated liver enzymes, increased viremia and suppressed HCV-specific CD4(+) T-cell proliferation in acute infection. While these associations were lost in established chronic infection, HCV-specific IL-10 responses were increased in patients without cirrhosis while IL-10 blockade enhanced antiviral effector IFNgamma responses. CONCLUSIONS HCV-specific IL-10 Tr1 responses may play a dual role in HCV infection, dampening effector T-cells to promote viral persistence in acute infection but also protecting against progressive fibrosis in chronic infection.

Fatal Hyperammonemia after Orthotopic Lung Transplantation

Hyperammonemia has been reported in association with a variety of disorders causing hepatic dysfunction: portosystemic encephalopathy (1), inborn errors of the urea cycle (2), the Reye syndrome (3), transient hyperammonemia of the newborn (4), Jamaican vomiting sickness (5), Udorn encephalopathy (6), valproic acid therapy (7), asparaginase therapy (8), and urinary tract infection from urea-splitting organisms (9). Idiopathic hyperammonemia has also been described in patients after bone marrow transplantation (10) and chemotherapy for leukemia (11). In a previous report, we described a case of fatal hyperammonemia occurring after orthotopic lung transplantation in a patient without evidence of significant biochemical hepatic dysfunction (12). In addition, we reported on hepatic glutamine synthetase enzyme deficiency in the livers of two patients with hyperammonemia after orthotopic lung transplantation (13). In the current study, we measured the incidence of post-orthotopic lung transplantation hyperammonemia; herein, we summarize and analyze its associated clinical features. Methods We collected clinical data from 145 patients who underwent orthotopic lung transplantation at the University of Pennsylvania Medical Center between November 1991 and November 1996. The first patient who had had orthotopic lung transplantation at our center died of hyperammonemia. Subsequently, 142 of 144 patients routinely had plasma ammonium levels measured at the time of transplantation and if their mental status changed. Hyperammonemia was diagnosed if the plasma ammonium level was greater than twice the normal level (9 to 33 mol/L) and had exceeded 200 mol/L on at least one occasiona standard definition in common practice (10). The standard immunosuppressive regimen was intravenous methylprednisolone (a 500-mg to 1000-mg bolus given intraoperatively, followed by 0.5 mg/kg of body weight per day), azathioprine (2 mg/kg per day) and cyclosporine (titrated to therapeutic trough levels between 250 and 350 g/L through high-performance liquid chromatography). Antilymphocyte -globulin was administered (5 to 10 mg/kg per day) for 3 to 7 days. Acute rejection was diagnosed according to standard clinical and histologic criteria (14) and was treated with a 3-day regimen of high-dose intravenous methylprednisolone (15 mg/kg per day). Major gastrointestinal complications were defined as diseases of the gastrointestinal tract requiring surgical intervention or resulting in hemodynamic changes. Total parenteral nutrition was composed of 10% Travesol amino acids (Travesol Laboratories, Inc., Deerfield, Illinois), 10% glucose, and 20% Intralipid (Cutter Laboratories, Inc., Berkeley, California). To treat hyperammonemia, patients received sodium benzoate, sodium phenylacetate, or both, with a loading dose of 250 mg/kg intravenously followed by 250 mg/kg per day intravenously. The dosage of intravenous arginine hydrochloride was 210 mg/kg per day. Comparisons of data between the patients with hyperammonemia and the remaining patients who had had orthotopic lung transplantation were performed by using the Student t-test or Fisher exact test when appropriate (15). The study was approved by the University of Pennsylvanias institutional review board. Results Incidence Hyperammonemia was identified in 6 of the 145 (4.1%) patients who had had orthotopic lung transplantation at the University of Pennsylvania Medical Center between November 1991 and November 1996. The median number of ammonium levels checked in these patients was three. Most patients had multiple measurements. Transplantation Characteristics of Patients with Hyperammonemia None of the 145 patients had significant liver-associated laboratory abnormalities before transplantation (aminotransferase levels<2 times the upper limit of normal) or a history of inborn errors of metabolism. Patient 6 had the Ellis van Creveld syndrome, which is not known to be associated with hyperammonemia. The Table shows the peritransplantation clinical variables of patients with and those without hyperammonemia. Development of major gastrointestinal complications, use of total parenteral nutrition, and lung transplantation for primary pulmonary hypertension were associated with hyperammonemia. Table. Comparison of Peritransplantation Clinical Features of Patients with and Those without Hyperammonemia Presentation Hyperammonemia occurred between postoperative day 5 and day 26 (mean SD, 15.2 8.8 days). All patients with hyperammonemia had symptoms of metabolic encephalopathy, lethargy (n=4), agitation (n=2), seizure (n=3), or tremor (n=1); they eventually became unresponsive and comatose. Evidence of cerebral edema was identified in all patients by intracranial pressure monitoring or computed tomography of the head. Patients 2 and 3 developed peritonitis secondary to colonic perforation 48 and 72 hours before the onset of hyperammonemia, respectively. This complication required emergency laparotomy in both cases. Patient 3 also had a candidal lung abscess in the left lower lobe 5 days before onset of encephalopathy. Patients 1 and 4 developed acute graft rejection and received pulse methylprednisolone 24 to 48 hours before the onset of hyperammonemia. In patient 4, hemodynamically significant upper gastrointestinal bleeding occurred 1 day before a sharp increase in the plasma ammonium level. Primary graft dysfunction preceded the onset of neurologic symptoms in patient 5. Patient 6 developed sepsis 1 day before the diagnosis of hyperammonemia. Laboratory Studies The postoperative daily maximum ammonium levels for all patients with hyperammonemia are shown in the Figure. These patients had only mildly abnormal results on liver-associated laboratory tests, if any, at the onset of hyperammonemia (aminotransferase levels<4 times the upper limit of normal). As described previously, levels of urinary orotic acid and plasma amino acids in patients 1 and 2 showed no abnormalities that would suggest congenital urea-cycle enzyme defects, whereas the hepatic glutamine synthetase levels were deficient (14). Figure. Maximum daily plasma ammonium levels, total parenteral nutrition use, concurrent medical stressors, and plasma ammonium - decreasing therapies in patients with hyperammonemia. Treatment Upon detection of hyperammonemia, total parenteral nutrition was withheld from all patients except patient 3. Patient 5 died within several hours of hyperammonemia detection and therefore received no therapy to reduce the plasma ammonium level; all other patients with hyperammonemia received lactulose, neomycin, or both. The varied use of dialysis and plasma ammonia-trapping in the patients with hyperammonemia is illustrated in the Figure. Outcome The mortality rate among the patients with hyperammonemia was clearly higher than that among patients without hyperammonemia (Table). Four of the six patients with hyperammonemia died during the initial episode of hyperammonemia, 0 to 7 days after diagnosis (mean, 3.3 2.5 days), on postoperative days 4 through 29 (mean, 17.5 11.3 days). Patient 2 survived the initial episode on postoperative day 9, with normalization of plasma ammonium level and mental status, but died 3 days after the onset of the second episode on postoperative day 34. The plasma ammonium level in patient 6 returned to normal by day 4 of therapy. She regained consciousness by day 10 of therapy and subsequently had near complete recovery of baseline physical and mental function. Autopsy Findings Family consent permitted autopsies on three of the five patients who died of hyperammonemia. Patients 2 and 3 had mild cholestasis, severe microvesicular steatosis, small bowel ischemia, and colonic perforations. Patient 1 had lipofuscin deposits in pericentral hepatocytes. Discussion We describe severe hyperammonemia in a cohort of patients who had undergone orthotopic lung transplantation. The presentation of hyperammonemia in these patients was invariably fulminant, with associated cerebral edema; most patients died. Risk factors for hyperammonemia are unknown; however, the development of hyperammonemia in these patients appears to be multifactorial. All patients with hyperammonemia had received immunosuppressive agents and total parenteral nutrition. In addition, the occurrence of hyperammonemia was uniformly preceded by at least one major medical stressor, especially major gastrointestinal complications. However, under similar conditions, not every patient who had had orthotopic lung transplantation developed hyperammonemia. Therefore, these factors may be necessary but not sufficient for the development of hyperammonemia. Hepatic glutamine synthetase deficiency may play a role in the pathogenesis of hyperammonemia under these conditions, as we have demonstrated in two of the patients. Finally, further studies are necessary to determine the significance of a statistical association between lung transplantation for primary pulmonary hypertension and the occurrence of hyperammonemia. Although the exact relation between these medical stressors and hyperammonemia is not clear, all patients with hyperammonemia developed critically increased intracranial pressure and other severe neurologic symptoms, such as status epilepticus, which are clearly the main cause of the high mortality rate. In our previous report on two patients with hyperammonemia who underwent liver biopsies before their deaths, we demonstrated a significantly reduced hepatic glutamine synthetase activity (13). It is unclear whether this activity is an acquired deficiency or a carrier state with an acquired component. Hepatic glutamine synthetase activity may be the crucial factor in the development of hyperammonemia in patients who have had orthotopic lung transplantation and may account for the variability in the development of hyperammonemia among patients after transplantation under similar stressors. However, measurement of hepatic glutamine synthetase activ

Strain-Specific T-Cell Suppression and Protective Immunity in Patients with Chronic Hepatitis C Virus Infection

ABSTRACT Hepatitis C virus (HCV) frequently persists with an apparently ineffective antiviral T-cell response. We hypothesized that some patients may be exposed to multiple HCV subtypes and that strain-specific T cells could contribute to the viral dynamics in this setting. To test this hypothesis, CD4 T-cell responses to three genotype 1a-derived HCV antigens and HCV antibody serotype were examined in chronically HCV infected (genotypes 1a, 1b, 2, 3, and 4) and spontaneously HCV recovered subjects. Consistent with multiple HCV exposure, 63% of patients infected with genotypes 2 to 4 (genotypes 2-4) and 36% of those infected with genotype 1b displayed CD4 T-cell responses to 1a-derived HCV antigens, while 29% of genotype 2-4-infected patients showed serotype responses to genotype 1. Detection of 1a-specific T cells in patients without active 1a infection suggested prior self-limited 1a infection with T-cell-mediated protection from 1a but not from non-1a viruses. Remarkably, CD4 T-cell responses to 1a-derived HCV antigens were weakest in patients with homologous 1a infection and greater in non-1a-infected patients: proportions of patients responding were 19% (1a), 36% (1b), and 63% (2-4) (P = 0.0006). Increased 1a-specific CD4 T-cell responsiveness in non-1a-infected patients was not due to increased immunogenicity or cross-reactivity of non-1a viruses but directly related to sequence divergence. We conclude that the T-cell response to the circulating virus is either suppressed or not induced in a strain-specific manner in chronically HCV infected patients and that, despite their ability to clear one HCV strain, patients may be reinfected with a heterologous strain that can then persist. These findings provide new insights into host-virus interactions in HCV infection that have implications for vaccine development.

Racial Differences in Hepatitis C Treatment Eligibility

Black Americans are disproportionally infected with hepatitis C virus (HCV) and are less likely than whites to respond to treatment with peginterferon (PEG‐IFN) plus ribavirin (RBV). The impact of race on HCV treatment eligibility is unknown. We therefore performed a retrospective analysis of a phase 3B multicenter clinical trial conducted at 118 United States community and academic medical centers to evaluate the rates of and reasons for HCV treatment ineligibility according to self‐reported race. In all, 4,469 patients were screened, of whom 1,038 (23.2%) were treatment ineligible. Although blacks represented 19% of treated patients, they were more likely not to be treated due to ineligibility and/or failure to complete required evaluations (40.2%) than were nonblack patients (28.5%; P < 0.001). After the exclusion of persons not treated due to undetectable HCV RNA or nongenotype 1 infection, blacks were 65% less likely than nonblacks to be eligible for treatment (28.1% > 17.0%; relative risk, 1.65; 95% confidence interval, 1.46‐1.87; P < 0.001). Blacks were more likely to be ineligible due to neutropenia (14% versus 3%, P < 0.001), anemia (7% versus 4%, P = 0.02), elevated glucose (8% versus 3%, P < 0.001), and elevated creatinine (5% versus 1%, P < 0.001). Conclusion: Largely due to a higher prevalence of neutropenia and uncontrolled medical conditions, blacks were significantly less likely to be eligible for HCV treatment. Increased access to treatment may be facilitated by less conservative neutrophil requirements and more effective care for chronic diseases, namely, diabetes and renal insufficiency. (HEPATOLOGY 2011;)

Chemiluminescent measurement of increased free radical formation after ischemia/reperfusion : mechanisms of free radical formation in the liver

It has been proposed that xanthine oxidase-derived superoxide mediates reperfusion injury in the liver; however, there is a little direct evidence to support this hypothesis. In this paper we describe a model system to directly and noninvasively measure oxyradical formation and hepatic injury in isolated perfused rat liver. Using this sensitive chemiluminescent technique, we clearly demonstrate the theorized burst in oxygen radical production upon reperfusion of previously ischemic liver, without perturbing the system with chemical luminescence enhancers. This increase in chemiluminescence (CL) upon reperfusion was diminished by the free radical scavengers trolox and ascorbate, as well as N-2-mercaptoproprionyl-glycine (MPG), thereby confirming the oxyradical nature of this signal. Additionally, superoxide dismutase and the xanthine oxidase inhibitor allopurinol, but not catalase, attenuated the reperfusion effect, providing the most direct evidence so far that XOD derived superoxide anion is formed during liver reperfusion. Hepatic injury (AST release) did not appear to relate to increased CL, supporting the notion that the oxyradical flux may serve as a signal for other events leading to tissue injury. Further studies using this sensitive chemiluminescent technique should aid in delineating the detailed mechanism(s) of reperfusion injury.

Combination Therapy with Lamivudine and Adenovirus Causes Transient Suppression of Chronic Woodchuck Hepatitis Virus Infections

ABSTRACT Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with ∼1013 particles of an adenovirus type 5 vector expressing β-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.

Distinct features in natural history and outcomes of acute hepatitis C.

Background: Acute hepatitis C (AHCV) provides a diagnostic challenge with diverse clinical presentations. Goals: This study was aimed to examine the clinical and demographic features as well as outcomes in AHCV patients identified from inpatient and outpatient hospital settings. Study: Patients with suspected AHCV were recruited from Philadelphia VA Medical Center, Hospital of University of Pennsylvania and Brooklyn VA Medical Center between 2000 and 2010. AHCV was diagnosed by acute serum alanine aminotransferase elevation with anti-hepatitis C virus (HCV) seroconversion, HCV-RNA fluctuations above 1 log, and/or recent high-risk exposure without prior HCV infection, excluding those with human immunodeficiency virus infection. Clinical and therapeutic outcomes were monitored for at least 6 months. Results: A total of 40 AHCV patients were enrolled with a median follow-up of 129 weeks. They were mostly men (68%) and whites (73%) with median age of 43 years, diverse risk factors (33% injection drugs, 20% health care–associated, 3% sexual, and 45% unknown), and wide variations in peak alanine aminotransferase (143 to 3435 U/L) and total bilirubin levels (0.4 to 19.3 mg/dL). Viremia resolved spontaneously in 23% and persisted without therapy in 27%, whereas 50% received interferon &agr;-based therapy with 90% cure (18/20). Distinct clinical scenarios included: (1) wide viremic fluctuations >1 log (65%) and intermittent HCV-RNA negativity; (2) autoantibodies (25% antinuclear antibodies, 69% antismooth muscle antibodies) or autoimmune features; (3) delayed spontaneous viral clearance in 2 patients; (4) rapid cirrhosis progression in 2 patients. Conclusions: AHCV is a heterogenous disease that requires careful monitoring. The lack of apparent risk factor in high proportion of patients and its diverse presentations warrant diagnostic vigilance.