Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections.

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 approximately 10(13) particles of an adenovirus type 5 vector expressing beta-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.

The fate of liver grafts declined for subjective reasons and transplanted out of a local organ procurement organization.

BACKGROUND: Decisions made by transplant surgeons to decline liver grafts for local use are based on both objective and ill-defined subjective parameters. These livers may be offered and subsequently transplanted at non-local centers. We analyzed the fate of these exported livers, focusing on the outcome of grafts declined for subjective reasons. The aim is to determine whether local surgeons' concerns about inferior graft function are justified. METHODS: Over a 3-year period, 13.3% of 555 livers in our organ procurement organization (OPO) were exported and transplanted out of the local area. Donor data and reason for decline were obtained from an extensive OPO database. Objective reasons for decline were based on no appropriate matched recipient due to donor size, serologies, or malignancy with potential for spread. Subjective parameters were related to the procuring surgeon's assessment and included variables such as medical and social history, abnormal liver enzymes, older age, organ visualization, and biopsy. Recipient data were obtained from questionnaires sent to outside transplant centers. RESULTS: There was a significantly higher rate of nonfunction in the subjective group (17.1%), compared to the objective group (0%). One-year graft and patient survival were 79 and 83% for the objective group and 59 and 68% for the subjective group (P=NS). When donors declined for medical/social history were excluded from the subjective group, leaving only grafts declined based solely on the surgeon's assessment of graft quality, there is a significant difference in graft survival (79% for objective and 46% for this subjective subgroup, P=0.03). CONCLUSIONS: Livers declined for local use based on subjective assessment by the procuring surgeon have a high nonfunction rate, associated with a high morbidity. Therefore, the use of these grafts should be restricted to recipients at the most urgent status.

Fatal hyperammonemia after orthotopic lung transplantation.

BACKGROUND: A case of fatal hyperammonemia complicating orthotopic lung transplantation was previously reported. OBJECTIVE: To describe the incidence, clinical features, and treatment of hyperammonemia associated with orthotopic lung transplantation. DESIGN: Retrospective cohort analysis. SETTING: Academic medical center and lung transplantation center in Philadelphia, Pennsylvania. PATIENTS: 145 sequential adult patients who underwent orthotopic lung transplantation. MEASUREMENTS: Plasma ammonium levels. RESULTS: Six of the 145 patients who had had orthotopic lung transplantation developed hyperammonemia, all within the first 26 days after transplantation. The 30-day post-transplantation mortality rate was 67% for patients with hyperammonemia compared with 17% for those without hyperammonemia (P = 0.01). Development of major gastrointestinal complications (P = 0.03), use of total parenteral nutrition (P < 0.001), and lung transplantation for primary pulmonary hypertension (P = 0.045) were associated with hyperammonemia. CONCLUSIONS: Hyperammonemia is a potentially fatal event occurring after orthotopic lung transplantation. It is associated with high nitrogen load, concurrent medical stressors, primary pulmonary hypertension, and hepatic glutamine synthetase deficiency.

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.

Gastrointestinal complications of immunosuppression.

The gastrointestinal manifestations of drug-induced immunosuppression may result from direct drug effects, from infectious complications, or both. Graft-versus-host disease (GVHD) is a third mechanism whereby immunosuppressive agents are linked with gastrointestinal injury. This article reviews individual immuno-suppressive medications, first concentrating on their reported gastrointestinal side effects, then reviewing other gastrointestinal phenomena, which may represent side effects of immunosuppressive agents but have not been reported yet.

Liver transplantation at the University of Pennsylvania and the Children's Hospital of Philadelphia.

The liver transplant program at the University of Pennsylvania and the Children's Hospital of Philadelphia experienced healthy growth in its clinical activity in the past 5 years. Patterns of referral and patient evaluation were established, care of patients while waiting on the list or being followed after transplantation was streamlined. We are now achieving excellent outcomes while transplanting relatively sicker patients. Innovative surgical procedures are implemented resulting in more efficient utilization of cadaveric and living-donor liver grafts. The protocols that are used for patient care are more standard, yet flexible and accommodate recent advancement in transplantation immunobiology. This progress of the clinical program was enhanced by careful preservation of the academic mission of the institution, which encourages the liver transplant faculty to be involved in NIH-supported clinical and basic science research.

Assessment of liver function: pre- and peritransplant evaluation.

Liver transplantation has been demonstrated to be a successful therapeutic modality for patients with end-stage liver disease. The high rate of survival for an otherwise terminal condition has resulted in significant expansion of the indications and diseases treated by this procedure, and is hampered only by the limited numbers of organs available for transplantation. Efforts in clinical and laboratory medicine should be directed to identify candidates who would benefit most from this procedure, to provide better means for accurate assessment of liver reserve and the appropriate timing for transplantation, to identify quality liver grafts that would have the potential to tolerate cold preservation and reperfusion injury, and to assist in accurate monitoring of graft function immediately after transplantation. The aim of this manuscript is to describe the existing pathways for clinical and laboratory assessment of pretransplant residual liver function, the donor liver graft, and immediate posttransplantation function.

Liver-directed gene therapy.

Liver-directed gene therapy represents a promising new modality for the treatment of inherited and acquired liver diseases. Clinical trials of liver-directed gene therapy are underway for diseases such as FH, OTC deficiency, and cancer. The main obstacles to effective gene therapy are the limitations of present gene delivery technology to express a desired gene safely and stably at therapeutic levels. With improved gene delivery technology and refinements in the ex vivo and in vivo approaches, a truly useful clinical tool will emerge.

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.

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.

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.

Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse.

The analgesic potency and toxicity (lethality) of morphine were increased 2.5 times after implantation of 7.5-mg s.c. naltrexone pellets in the mouse for 8 days. Implantation for 8 days also resulted in a 41% [3H][D-Ala2-D-Leu5]enkephalin and 55% [3H] [D-Ala2-MePhe4-Gly(ol)5]enkephalin increase in radiolabeled opioid binding in mouse brain relative to placebo-implanted controls. Treatment for 1 day did not produce any significant increases in binding or morphine's analgesic potency. Brain morphine concentrations did not differ after a dose of morphine (8 mg/kg) that produced analgesia in 86% of 8-day naltrexone-treated mice vs. 39% of placebo-treated mice. The increase in the analgesic potency of morphine by chronic naltrexone treatment in the mouse is particularly striking as it is approximately 3 times greater than that observed for the rat. The decrease in the LD50 of morphine after 8 days of naltrexone treatment raises the possibility that the toxicity of opiates may be increased in patients who discontinue naltrexone maintenance treatment and resume opiate abuse.