High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice

We studied the development of liver tumors in male HBx gene transgenic mice. Of two lineages studied, in the lineage with the lowest HBx gene expression liver tumors developed only in an incidence comparable with that in normal CD-1 strain, whereas 84% of male mice with a high level of the HBx gene product succumbed to liver neoplasia, indicating that continued HBx gene expression higher than a certain threshold level may be necessary for the development of hepatic neoplasia. Sixty-five mice from a lineage with a high level of HBx expression were then followed throughout their 24-mo lifespan. The livers of transgenic mice showed foci of cellular alteration with cytoplasmic vacuolations around the central veins from the age of 2 mo, but these foci did not expand progressively by the age of 12 mo. Immunostaining demonstrated such hepatocytes had higher expression of HBx protein than surrounding cells. Neoplastic lesions including liver cell adenomas and hepatocellular carcinomas developed from the age of 13 mo. By bromodeoxyuridine labeling analysis, hepatocytes in altered foci were found to have increased DNA synthesis, whereas no labeling was observed in age- and sex-matched nontransgenic littermate controls. Furthermore, DNA content analysis revealed the existence of several small aneuploid peaks in the transgenic liver before the age of tumor development. These results suggest that the continued expression of HBx gene may initiate a complex process to hepatocellular carcinoma by inducing DNA synthesis and placing large numbers of hepatocytes subjective to secondary events for transformation.     

Regulation of hepatitis B virus mRNA expression in a hepatitis B surface antigen transgenic mouse model by IFN-gamma-secreting T cells after DNA-based immunization

The immunotherapeutic effect of DNA-mediated immunization against chronic hepatitis B virus (HBV) infection has been evaluated in transgenic mice expressing the sequences that code for the envelope proteins of HBV in the liver. In this model of HBV chronic carriers, a single i.m. injection of plasmid DNA encoding HBV envelope proteins is sufficient to generate specific immune responses leading to the clearance of the transgene expression product and the control of HBV mRNA. The relative contributions of the T cell subpopulations induced by DNA immunization were examined using adoptive transfer experiments. It was shown that either CD8+ or CD4+ T lymphocytes from immunocompetent DNA-immunized animals were sufficient to control viral gene expression in the livers of the recipient transgenic mice. This effect was mediated by a cytokine-dependent mechanism common to both T cell subpopulations; this mechanism did not require cell lysis, but did involve the production of IFN-gamma by the activated T cells.     

Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in transgenic mice

We have previously demonstrated that hepatitis B virus (HBV) replication and gene expression are abolished in the livers of HBV transgenic mice by cytotoxic T lymphocytes (CTLs) and during lymphocytic choriomeningitis virus (LCMV) infection, stimuli that trigger the production of alpha/beta interferon, gamma interferon, and tumor necrosis factor alpha in the liver. We now report that hepatic HBV replication and gene expression are inhibited by the local induction of these cytokines during adenovirus- and murine cytomegalovirus (MCMV)-induced hepatitis. Further, we show that MCMV also blocks HBV replication and gene expression in the proximal convoluted tubules of the kidney by causing interstitial nephritis and inducing the same cytokines in the renal parenchyma. These results suggest that inflammatory cytokines probably contribute to viral clearance during acute viral hepatitis in humans, and they imply that induction of these cytokines in the liver and other infected tissues of chronically infected patients might have therapeutic value.     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.     

Proteins of hepatitis B surface antigen

Purified 22-nm forms of hepatitis B surface antigen (Hbsag) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might by glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBSAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBSAg yielded patterns identical to those obtained with protein stain. Comparison of HBSAg labeled by the chloramine-T and lactoperoxide procedures indicated that there was no distinction between internal or external components within the 22-nm structure.     

Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.     

Identification of vertical transmission of hepatitis B virus from mother to children by direct sequencing a segment of surface gene of hepatitis B virus

Intermittent catheterization (ICP) is a well-proven effective means of urologic management for spinal cord diseased (SCD) persons who meet the following criteria: adequate low pressure bladder capacity (350-400 cc minimum), adequate hand function, unobstructed urethra and compliant, understanding, continent, cooperative patients. Time-directed (Q4 H-Q6 H), ICP-obtained volumes on twenty-one patients revealed a majority of early, unnecessary as well as some late over-distended bladder catheterizations. The PCI 5000 or "Bladder Manager", a miniaturized ultrasonic bladder volume measuring device developed by Diagnostic Ultrasound of Seattle, was evaluated. It allowed the patients to perform volume-directed ICP which results in less frequent catheterizations and prevents bladder overdistension.     

Transmission of hepatitis B virus from hepatitis B core antibody-positive donors in living related liver transplants

A factor fundamental to bone formation has been identified. Gene targeting shows that core-binding factor alpha 1 (Cbfa1) plays an essential role in bone formation and osteoblast differentiation. Thus, it is now possible to begin examining the molecular mechanism of bone formation--especially osteoblast differentiation.     

Molecular biology of hepatitis B virus e antigen

Hepatitis B virus (HBV) e antigen (HBeAg) was discovered in 1972 as one of the serological markers of HBV infection. Although 25 years have passed since its initial discovery, the function of this antigen in the life cycle of HBV has remained elusive. Mutations in the HBV genome that prevent the expression of HBeAg do not abolish the replication of HBV, indicating that this antigen is not essential for HBV replication. In contrast, the conservation of the HBeAg gene in the genomes of related animal viruses, including the distantly related duck HBV, argues for an important function of this antigen. The purpose of the present article is to review the molecular biology of HBeAg and to examine its possible functions in the life cycle of HBV.     

Inhibition of hepatocellular carcinoma development in hepatitis B virus transfected mice by low dietary casein

In a comprehensive human ecological study, primary liver cancer has been shown to be highly significantly associated with 1) the prevalence of persistent infection with hepatitis B virus (HBV) and 2) plasma cholesterol concentrations that are, in turn, associated with the consumption of animal based foods. In rat studies, aflatoxin-induced hepatocellular carcinoma is substantially prevented by decreasing the intake of animal based protein (casein), a hypercholesterolemic nutrient. Thus the development of primary liver cancer associated with persistent HBV infection or with aflatoxin exposure may be controlled by reduced intake of animal-based proteins. Transgenic mice transfected with an HBV gene fragment containing the viral transactivator of hepatis B virus, HBx, which induces the formation of hepatocellular carcinoma, were used to examine the ability of dietary casein to modify tumor formation. Reducing the concentration of dietary casein to 6% from the traditional level of 22% markedly inhibited (by 75%) hepatic tumor formation in these transgenic mice. Tumor development also was substantially altered by interchanging dietary casein concentration well after tumor development had begun (at 8 months), increasing by 173% from the expected yield when casein intake was increased and decreasing by 99% when casein was reduced. These findings suggest that the development of liver tumor formation among individuals persistently infected with HBV may be controlled by minimizing or eliminating the intake of animal protein-based foods.     

DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice

Hepatitis B virus (HBV) remains a serious worldwide health problem and the possibility to control it will depend on the availability of safe, effective and affordable vaccines. Recombinant protein or plasma-derived vaccines containing HBV surface antigen (HBsAg) are safe and generally efficacious, however, they are too expensive for widespread use in areas of HBV endemicity and are only partially effective for treatment of HBV chronic carriers. Immunization of mice by injection of HBsAg-expressing plasmid DNA results in rapid induction of strong and long-lasting humoral and cell-mediated immune responses. Here we report optimization of the humoral response with the use of necrotizing agents, co-expression of cytokines or co-stimulatory molecules and formulation of the DNA with cationic liposomes. DNA-based immunization of HBsAg-transgenic mice can also overcome non-response to HBsAg. Thus, DNA vaccines against HBV may be useful for both prophylactic and therapeutic purposes.     

Establishment of gastric surface mucous cell lines from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene

Brown adipose tissue (BAT) is the major thermogenic organ of the human neonate. To determine whether it is also active in the peripheral conversion of T4 to T3, as shown in several animal species, interscapular BAT from 13 newborns of 25-40 weeks gestational age who survived 4 days, at most, was investigated. BAT was found to contain significant amounts of the mitochondrial uncoupling protein (UCP), the rate-limiting component of heat production. The specific content of UCP increased from 29.4 +/- 3.3 to 62.5 +/- 10.2 pmol/mg protein between 25 and 40 weeks of gestation, respectively, and the UCP/F1-ATPase molar ratio, a sensitive marker of brown fat differentiation, increased similarly. BAT was also found to contain iodothyronine 5'-deiodinase (5'D), which appears to be a type II enzyme, based on high affinity for T4 (Km, 2.9 nmol/L) and insensitivity to propylthiouracil (10% inhibition by 1 nmol/L). 5'D was active by 25 weeks gestation, and the specific activity increased from 116 +/- 15 to 417 +/- 46 fmol/h.mg protein during the period examined. The development of 5'D activity was similar to the changes in UCP content; both exhibited a major increase before 32 weeks gestation. The results indicate that thermogenic function and 5'D activity develop in human BAT rather early, during the first half of the last trimester of gestation. The activities of 5'D in human BAT are comparable with 5'D activities found in animal BAT stimulated during the perinatal period, by cold exposure, or by increased cAMP levels.     

Frequent detection of hepatitis B virus X-gene DNA in hepatocellular carcinoma and adjacent liver tissue in hepatitis B surface antigen-negative patients

Hepatitis B virus is associated with human hepatocellular carcinoma. We performed polymerase chain reaction for the X, C, S, and preS2/S regions of the viral genome in 23 hepatitis B surface antigen-negative hepatocellular carcinomas and adjacent liver. Hepatitis B viral genomes were detected in 17 of 23 tumors and adjacent tissues (73.9%). Among recognized transactivators, the X gene was present in 16 (69.6%) cases of hepatocellular carcinoma, but preS2/S was detected in only 7 (30.4%). Hepatitis B virus C and S regions were detected in 3 (13.0%) and 9 (39.1%) hepatocellular carcinomas, respectively. Serologic study revealed antibodies to hepatitis B surface antigen, hepatitis B core antigen, and hepatitis B e antigen in 14 patients; among these, X-gene DNA was detected in 12 of 14 tumors (85.7%). The X gene was also detected in 4 of 9 tumors of seronegative patients. The X gene, present in many hepatocellular carcinomas, may promote hepatocellular carcinoma in hepatitis B surface antigen-negative patients.     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.