Myt3 suppression sensitizes islet cells to high glucose-induced cell death via Bim induction.

Diabetes is a chronic disease that results from the body's inability to properly control circulating blood glucose levels. The loss of glucose homoeostasis can arise from a loss of ß-cell mass because of immune-cell-mediated attack, as in type 1 diabetes, and/or from dysfunction of individual ß-cells (in conjunction with target organ insulin resistance), as in type 2 diabetes. A better understanding of the transcriptional pathways regulating islet-cell survival is of great importance for the development of therapeutic strategies that target ß-cells for diabetes. To this end, we previously identified the transcription factor Myt3 as a pro-survival factor in islets following acute suppression of Myt3 in vitro. To determine the effects of Myt3 suppression on islet-cell survival in vivo, we used an adenovirus to express an shRNA targeting Myt3 in syngeneic optimal and marginal mass islet transplants, and demonstrate that suppression of Myt3 impairs the function of marginal mass grafts. Analysis of grafts 5 weeks post-transplant revealed that grafts transduced with the shMyt3 adenovirus contained ~20% the number of transduced cells as grafts transduced with a control adenovirus. In fact, increased apoptosis and significant cell loss in the shMyt3-transduced grafts was evident after only 5 days, suggesting that Myt3 suppression sensitizes islet cells to stresses present in the early post-transplant period. Specifically, we find that Myt3 suppression sensitizes islet cells to high glucose-induced cell death via upregulation of the pro-apoptotic Bcl2 family member Bim. Taken together these data suggest that Myt3 may be an important link between glucotoxic and immune signalling pathways.

Identification and analysis of murine pancreatic islet enhancers.

AIMS/HYPOTHESIS: The paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression. METHODS: We combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets. RESULTS: We identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs). CONCLUSIONS/INTERPRETATIONS: Our results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.

Interferon-gamma-associated responses to woodchuck hepatitis virus infection in neonatal woodchucks and virus-infected hepatocytes.

Acute hepatitis and recovery from woodchuck hepatitis virus (WHV) infection involves increased intrahepatic expression of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) mRNAs. In the present study, recovery correlated with increased intrahepatic expression of mRNAs for major histocompatibility complex class 1 (MHC1), beta(2)-microglobulin, 2'5'-oligoadenylate synthetase (2'5'-OAS), and indoleamine dioxygenase (IDO). By comparison, acute WHV infection progressing to chronicity was associated with diminished expression of these IFN-gamma-associated mRNAs in liver. Transfection of WHV-infected primary hepatocytes (WPH) from WHV carriers with an IFN-gamma-expressing plasmid (pIFN-gamma) resulted in dose-dependent accumulations of MHC1, TNF-alpha, 2'5'-OAS, and IDO mRNAs within 96 h. Markers of T cells and immune-mediated cytotoxicity that accumulate in recovering liver were not apparent in WPH based on the relative lack of CD3, CD4, Fas ligand, perforin, and granzyme B mRNAs. Expression of pIFN-gamma, and TNF-alpha-expressing plasmid (pTNF-alpha), did not affect total WHV RNA, or fully double-stranded WHV DNA in WPH, but each reduced some of the replicative intermediate (RI) species of WHV DNA synthesis. WPH treated with recombinant IFN-alpha protein had a higher fold induction of 2'5'-OAS mRNA associated with partial reductions in WHV RNAs and the major RI species. Thus, IFN-gamma expression in carrier WPH induced several host responses often observed in liver of recovering woodchucks, and impaired a stage of WHV DNA synthesis by a non-cytolytic mechanism mediated by TNF-alpha. Local enhancement of IFN-gamma-associated responses in chronic WHV-infected hepatocytes may promote therapeutic antiviral effects, but additional effector mechanisms evident during recovery appear necessary for more complete clearance of WHV infection.

Hepatocellular carcinoma in black-tailed prairie dogs (Cynomys ludivicianus): tumor morphology and immunohistochemistry for hepadnavirus core and surface antigens.

From 1994 to 2002, tissues from 61 prairie dogs were submitted to Northwest ZooPath for histopathology. Of these, 12 (20%) had hepatocellular carcinoma (HCC). Three were pets submitted from private veterinary practices. The others were submitted from zoos in the United States. All were adults, ranging from young adult to 7 years of age, with average age of 5.1 years. The most common clinical signs were weight loss, lethargy, palpable abdominal mass, and respiratory difficulty. All tumors were well-differentiated HCCs in which four histologic patterns were recognized. The trabecular pattern was predominant in nine tumors, and the pseudoglandular pattern was predominant in two tumors. The pelioid pattern was also represented in eight tumors. A papillary pattern was present in one case. In seven cases vacuolar change resembling lipidosis was present in the neoplastic hepatocytes of both primary and metastatic tumors. Anaplasia was mild to moderate in most tumors, but a marked degree of anaplasia was noted in the metastatic foci of the case with papillary differentiation. Metastasis to lung was noted in five cases. One of these also had metastasis to the spleen, and another had metastasis to heart and mediastinum. In two cases there was concurrent hepatitis and in two cases, cirrhosis. All tumors and nonneoplastic liver stained negatively for woodchuck hepatitis virus surface and core antigens, and orcein and Victoria blue positive staining of hepatocytes typical of hepadnavirus infection in humans and woodchucks was not present. HCC is apparently common in captive prairie dogs. The hepatic neoplasia observed in prairie dogs was similar to that associated with hepadnaviral infection in humans, woodchucks, and ground squirrels, but no direct evidence of hepadnaviral infection was detected. The rate of metastasis in captive prairie dogs was higher than that reported in woodchucks.

Long-term entecavir treatment results in sustained antiviral efficacy and prolonged life span in the woodchuck model of chronic hepatitis infection.

Entecavir (ETV) is a guanosine nucleoside analogue with potent antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus. To explore the consequences of prolonged virus suppression, woodchucks received ETV orally for 8 weeks and then weekly for 12 months. Of the 6 animals withdrawn from therapy and monitored for an additional 28 months, 3 had a sustained antiviral response and had no evidence of hepatocellular carcinoma (HCC). Of the 6 animals that continued on a weekly ETV regimen for an additional 22 months, 4 exhibited serum viral DNA levels near the lower limit of detection for >2 years and had no evidence of HCC. Viral antigens and covalently closed circular DNA levels in liver samples were significantly reduced in all animals. ETV was well tolerated, and there was no evidence of resistant variants. On the basis of historical data, long-term ETV treatment appeared to significantly prolong the life of treated animals and delay the emergence of HCC.

Antiviral beta-L-nucleosides specific for hepatitis B virus infection.

Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.

Anti-HBV specific beta-L-2'-deoxynucleosides.

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Seasonal changes in serum leptin, food intake, and body weight in photoentrained woodchucks.

Male woodchucks (Marmota monax) were maintained in northern vs. southern hemisphere photoperiods, provided feed and water ad libitum, and evaluated every 2 wk for 23 mo for body weight, absolute and relative food intake, body temperature, serum testosterone, and serum concentrations of leptin measured using an anti-mouse leptin enzyme-linked immunoassay. During late spring and summer, body weight increased 56 +/- 4% above winter nadirs, and during the autumn and early winter weights decreased 27 to 43% below midsummer maxima. Serum leptin initially increased during increases in body weight, in the late spring, reached peak values (490 +/- 32 pg/ml) in summer during the initial decline in body weight, and later decreased along with body weight to reach basal values (20 +/- 5 pg/ml) in late winter. Spontaneous declines in food intakes in summer began 2-6 wk before resulting declines in body weight and occurred during increases in leptin >100 pg/ml. The rate of decline in food intakes was greatest when serum leptin was at or near peak values. Food intake increased in late winter when leptin was low and 7-10 wk before resulting increases in body weight. Testis recrudescence occurred when leptin was declining to near basal levels. The results suggest that leptin is involved in the hormonal regulation of the circannual cycle in the drive for voluntary food intake in this species.

The woodchuck model of hepatitis B virus infection.

The woodchuck hepatitis virus (WHV) was the first of the mammalian and avian hepadnaviruses described after discovery of the virus of hepatitis B (HBV). Woodchucks chronically infected with WHV develop progressively severe hepatitis and hepatocellular carcinoma, which present as lesions that are remarkably similar to those associated with HBV infection in humans. The initial virological studies and studies of pathogenesis utilized woodchucks that had been trapped in the wild and had acquired WHV infection naturally. Research with wild woodchucks was complicated by lack of knowledge of their backgrounds (e.g., dietary history, exposure to parasites or environmental toxins, and source and duration of WHV infection). Breeding colonies of woodchucks have been established and maintained in laboratory animal facilities, and laboratory-reared woodchucks are superior for experimental studies of pathogenesis or hepatocarcinogenesis. It is possible to infect neonatal woodchucks born in the laboratory with standardized inocula and produce a high rate of chronic WHV carriers that are useful for controlled investigations. WHV has been shown experimentally to cause hepatocellular carcinoma, supporting conclusions based on epidemiological and molecular virological studies that HBV is an important etiological factor in human hepatocarcinogenesis. Chronic WHV carrier woodchucks have become a valuable animal model for the preclinical evaluation of antiviral therapy for HBV infection, providing useful pharmacokinetic and pharmacodynamic results in a relevant animal disease model. It also has been shown that the pattern of toxicity and hepatic injury observed in woodchucks treated with certain fluorinated pyrimidines is remarkably similar to that observed in humans that were treated with the same drugs, suggesting the woodchuck has significant potential for the preclincial assessment of antiviral drug toxicity.

Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck.

Effective incorporation of tritiated thymidine ([(3)H]TdR) into proliferating lymphocytes is important because [(3)H]TdR is a standard label to study proliferate T-cell responses. We analyzed the thymidine utilization of woodchuck peripheral blood lymphocytes (PBL) since the [(3)H]TdR incorporation assay was not applicable to measure proliferative immune responses in the woodchuck, a current major virus/host model for human hepatitis B virus infection. Incorporation of [(3)H]TdR into DNA as well as the activity of the salvage pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [(3)H]TdR incorporation of proliferating woodchuck PBL remained residual regardless of the use of methotrexate, an inhibitor of the competitive deoxythymidine monophosphate de novo synthesis pathway. Using a human probe, specific for the proliferation-associated TK1, we proved the genomic presence and transcription of TK1 sequences in various species. TK1 sequences were detected in the genome of human, mouse, woodchuck, and chicken specimens. In contrast to proliferating human PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in proliferating woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the ability of woodchuck cells to transcribe TK1 and to express functional TK1 proteins. Our study characterizes the unique failure of sufficient [(3)H]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in the woodchuck.

Large nontransplanted hepatocellular carcinoma in woodchucks: treatment with adenovirus-mediated delivery of interleukin 12/B7.1 genes.

BACKGROUND: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. METHODS: Large (2-5 cm), established intrahepatic tumors were given an injection once with 1 x 10(9) plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. RESULTS: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1-2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P =.006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4(+) and CD8(+) T cells and interferon gamma (IFN gamma). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4(+) and CD8(+) T cells and IFN gamma) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (> or = 95%) 7 weeks after treatment. CONCLUSION: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans.

Pathogenesis of experimental neonatal woodchuck hepatitis virus infection: chronicity as an outcome of infection is associated with a diminished acute hepatitis that is temporally deficient for the expression of interferon gamma and tumor necrosis factor-alpha messenger RNAs.

Surgical biopsies of the liver were obtained from woodchuck hepatitis virus (WHV)-infected neonatal woodchucks at 2 time points before the self-limited or chronic outcomes became obvious by serologic criteria. Following segregation of outcomes, livers were analyzed for intrahepatic type 1 cytokine messenger RNAs (mRNAs) (interleukin 2 [IL-2], interferon gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha]) and leukocyte inflammatory phenotype (IgG+ plasma cells, lysozyme+ macrophages, CD3+ T cells). Baselines were assessed using age-matched uninfected control livers. At week 8 (early acute phase), intrahepatic type 1 cytokine mRNAs were similarly low in both outcome settings and no different from age-matched uninfected controls. This was consistent with the minimal initial viral loads and lack of histologic inflammation at this time. At week 14 (mid-acute phase), changes in viral load between outcome groups related inversely to the intrahepatic inflammatory responses. Animals that eventually became resolved had increased intrahepatic expression of IFN-gamma and TNF-alpha mRNAs and robust inflammation by CD3+ T cells, plasma cells, and macrophages. At the same time point of infection, animals that eventually became chronic carriers had an acute hepatitis involving the same cell types, but at diminished levels, and markedly deficient intrahepatic expression of IFN-gamma and TNF-alpha mRNAs. IL-2 mRNA remained at baseline control levels in both outcome groups. These cotemporal comparisons map a critical deviation in host response to the acute stage of an evolving chronic infection. They strongly suggest that increasing viral load and chronicity as an outcome of neonatal WHV infection result from a temporal deficiency in the acute intrahepatic effector mechanisms mediated by IFN-gamma and TNF-alpha.

Animal models of hepadnavirus-associated hepatocellular carcinoma.

Animal models of hepatitis B virus infection have been valuable for determining the mechanisms of hepadnavirus replication, for studies of pathogenesis, and for investigations of viral hepatocarcinogenesis. The woodchuck model also seems to be useful in the discovery and development of antiviral drugs to treat HBV infection and for testing new forms of immunotherapy. In particular, the woodchuck seems to be ideal for studying the effect of antiviral treatment and immunotherapy on the outcome of hepadnavirus infection and on survival. The median life expectancy of experimentally infected, chronic WHV carriers is approximately 29 months, and almost all develop HCC. New types of prophylaxis or therapy can be evaluated under controlled experimental conditions, in a relevant animal model, and within a reasonable time frame.

Antiviral L-nucleosides specific for hepatitis B virus infection.

A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides beta-L-2'-deoxycytidine, beta-L-thymidine, and beta-L-2'-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (alpha, beta, and gamma) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10(8) genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Antiviral activity of clevudine [L-FMAU, (1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl) uracil)] against woodchuck hepatitis virus replication and gene expression in chronically infected woodchucks (Marmota monax).

L: -FMAU [1-(2-fluoro-5-methyl-beta,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of hepatitis B virus (HBV) and duck hepatitis B virus replication in cell culture and duck hepatitis B virus replication in acutely infected Peking ducks. The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of woodchuck hepatitis virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection.

Binding of ouabain and human ouabainlike substance to different Na+, K+-ATPase isoforms.

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual alpha-isoforms of Na+,K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney alpha1, rabbit kidney alpha1 and porcine cerebral cortex alpha3 Na+,K+-ATPase. OLS was initially isolated by solid phase extraction from human serum using C18 columns. The extract was further purified by reverse phase HPLC in an acetonitrile/water (containing 0.1% TFA) step-up gradient (16-80%). In this system, two distinct ouabain immunoreactive peaks were resolved. Peak I demonstrated a polarity identical with that of authentic ouabain. In contrast, peak II was relatively non-polar and eluted later in the run. The final step in the purification of OLS involved immuno-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100 nmol/l) for ouabain with canine alpha1 and porcine alpha3 Na+,K+-ATPase showed similar inhibitory profiles (IC50=15 nmol/l), whilst rabbit alpha1 Na+,K+-ATPase was relatively insensitive to ouabain and purified peak I OLS. Two fold serial dilution of Peak I OLS, with subsequent analysis by canine and porcine Na+,K+-ATPase inhibition assays and RIA, demonstrated strong positive correlations between OLS determined by RIA and both canine (y=0.945x-2.532, r2=0.977) and porcine (y=0.428x-1.685; r2=0.993) Na+,K+-ATPase assays. The difference in the respective slopes suggests, however, that peak I OLS has a greater affinity for the canine derived enzyme compared to the porcine. In conclusion, these data suggest that like authentic ouabain, peak I OLS is a-isoform and species selective.

Temporal pathogenesis of experimental neonatal woodchuck hepatitis virus infection: increased initial viral load and decreased severity of acute hepatitis during the development of chronic viral infection.

Acute hepatitis B virus (HBV) infections either resolve or progress to chronicity. Identification of early deviations in host-virus responses associated with these outcomes can further differentiate cause-effect mechanisms that initiate and maintain chronicity. Neonatal woodchucks were infected experimentally with the woodchuck hepatitis virus (WHV) at 3 days of age. At 8 or 14 weeks of age (i.e. , the early- or mid-acute stage of infection), whole blood and large surgical biopsies of the liver were obtained from infected animals and uninfected controls. These were stored for later correlating histopathologic responses and viral load with the subsequently determined outcome of infection. As of 1 year postinfection, half of the surgically treated infected woodchucks had developed self-limited infections, while the other half developed chronic infections. The self-limited outcome was characterized by decreased viral load in acute-phase liver and plasma and a generally robust acute hepatic inflammatory response. Comparisons at the same early time points revealed that the chronic outcome was characterized by increasing initial viral load in liver and plasma, and a detectable, but diminished, acute hepatic inflammation. These cotemporal comparisons indicate that there is an early host-response deviation during the acute phase of a developing chronic infection. Continued analysis of the tissues banked from this study will facilitate further temporal characterization of acute-phase mechanisms that determine resolution versus chronicity in WHV infection. Understanding such mechanisms may be useful in the rational design of therapy for established chronic HBV infection.

Correct binding of viral X protein to UVDDB-p127 cellular protein is critical for efficient infection by hepatitis B viruses.

A fully effective treatment of chronic human hepatitis B virus (HBV) infection is still missing and HBV remains the first etiological agent of liver cancer. Although the viral regulatory X protein is essential for infection, its mode of action remains obscure, due the lack of an in vitro infection system. In the accompanying study, we showed the functional importance of interaction between X and the host protein UVDDB-p127, in the transactivation and apoptotic properties of the viral protein. Here, we addressed the biological role of X-UVDDB interaction in the infectious process using a genetic approach in the woodchuck virus closely related to HBV. We show that (i) mutations in X, which markedly affect UVDDB-binding, also abolished productive infection in woodchucks, (ii) in the few cases where mutant viruses led to infection, compensatory mutations had occurred in the X gene of the viral progeny, which restored correct UVDDB-binding. We conclude that efficient viral replication in vivo requires proper X-UVDDB interaction. The interaction may thus provide a novel therapeutic target for the treatment of hepatitis

Enhanced antiviral benefit of combination therapy with lamivudine and alpha interferon against WHV replication in chronic carrier woodchucks.

Cell culture studies in our laboratory previously demonstrated synergistic antiviral activity for the combinations of lamivudine and a novel recombinant hybrid human alpha B/D interferon (rHu alpha B/D IFN) against hepatitis B virus (HBV) replication. Based on these results, a study was designed to determine if an enhanced antiviral effect with this drug combination could be demonstrated in vivo using the woodchuck hepatitis virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers during previous studies by our laboratories. Two combination treatment regimens were compared to matched monotherapies in a placebo-controlled trial. The first used simultaneous administration of rHu alpha B/D IFN and lamivudine for 24 weeks. The other combination treatment regimen used a staggered dosing schedule of 12 weeks of administration of lamivudine alone, followed by 12 weeks of simultaneous dosing with both drugs, followed by 12 weeks of therapy with rHu alpha B/D IFN alone. Both treatment regimens with combinations of lamivudine and rHu alpha B/D IFN were more effective at reducing WHV replication in chronically infected wood-chucks than the corresponding monotherapies. Both combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. The staggered treatment regimen reduced viraemia and intrahepatic WHV replication significantly more than that expected for additive interactions, indicating synergistic antiviral effects. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.