Weight loss and severe jaundice in a patient with hyperthyroidism.

BACKGROUND: Thyrotoxicosis may significantly alter hepatic function and is associated with autoimmune disorders of the liver. CASE REPORT: We report the case of a thyrotoxic patient with Graves' disease and histologically established cholestatic hepatitis. Medical treatment of hyperthyroidism normalized liver function tests. CONCLUSIONS: In patients with elevated liver function parameters and jaundice of unknown origin, thyroid function should generally be tested. Moreover, medical treatment of hyperthyroidism with thyrostatics may cause severe hepatitis whereas untreated hyperthyroid patients are at risk of developing chronic liver failure.

Ligand-mediated retargeting of recombinant adenovirus for gene transfer in vivo.

The development of efficient and safe methods for in vivo gene transfer is central to the success of gene therapy. Recombinant adenoviral vectors, although highly efficient, are limited by the host immune response, potential safety hazards due to obligatory cotransfer of viral proteins, and their broad tissue tropism. Here, we demonstrate in an animal model that host range and tissue tropism of a recombinant adenovirus from a distant species can be modified by complexing adenovirus with a cell-specific ligand. Thus, a replication-deficient lacZ recombinant human adenovirus, which naturally does not infect avian cells, allowed highly efficient and specific gene transfer to the liver of ducks in vivo when complexed with N-acetylglucosamine, a ligand for the chicken hepatic lectin. This combination of ligand-mediated receptor targeting with adenoviral uptake and intracellular processing of a given gene represents a novel approach to gene therapy of inherited and acquired liver diseases.

Antisense therapy of hepatitis B virus infection.

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is interferon-a with an efficacy of only 30-40% in highly selected patients. The discovery of animal viruses closely related to the HBV has contributed to active research on antiviral therapy of chronic hepatitis B. The animal model tested and described in this article are Peking ducks infected with the duck hepatitis B virus (DHBV). Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA. An antisense oligodeoxynucleotide directed against the 5'-region of the preS gene of DHBV inhibited viral replication and gene expression in vitro in primary duck hepatocytes and in vivo in Peking ducks. These results demonstrate the potential clinical use of antisense DNA as antiviral therapeutics.

Antiviral activities of penciclovir and famciclovir on duck hepatitis B virus in vitro and in vivo.

Chronic hepatitis B virus (HBV) infection is a major health problem worldwide. Antiviral strategies available at present, including interferon-alpha, have only limited efficacy, leading us to analyse the antiviral effects of penciclovir and famciclovir in the duck hepatitis B virus (DHBV) model of HBV infection in vitro and in vivo. In DHBV-infected duck hepatocytes, penciclovir effectively inhibited viral replication, with a concentration giving half-maximal inhibition of 0.25 microM. Furthermore, in vivo, penciclovir and its orally administered prodrug famciclovir strongly inhibited DHBV replication. These data demonstrate that penciclovir and famciclovir both have strong antiviral activities, and suggest that these agents might be useful for treating HBV infection in humans.

Inhibition of duck hepatitis B virus replication by 2',3'-dideoxy-3'-fluoroguanosine in vitro and in vivo.

The antiviral activity of 2',3'-dideoxy-3'-fluoroguanosine (FdG) or its triphosphate was evaluated in the duck hepatitis B virus (DHBV) system in vitro and in vivo. In primary DHBV-infected hepatocytes FdG results in a dose-dependent inhibition of viral replication with a nearly complete inhibition at a concentration of 1 microM. Also in vivo, FdG treatment of DHBV-infected ducklings reduces DHBV DNA replication by more than 90%. These data demonstrate that FdG is a strong inhibitor of DHBV replication in vitro and in vivo.

Antisense therapy of hepatitis B virus infection : in vivo analyses in the duck hepatitis B virus model.

Infectious diseases in general and viral infections in particular can be viewed as acquired genetic diseases (1, 2). At the molecular level, clinical signs and symptoms of viral infections are frequently caused by the expression or overexpression of the acquired genes. Based on this basic concept, such acquired genetic diseases should be amenable to treatment by a specific block of gene expression. Gene expression can be blocked at different levels by the following strategtes: sense strategy, antigene strategy, ribozymes, antisense strategy, and interfering peptrdes or proteins (Fig. 1). Fig. 1. Principle of gene expression and strategies aimed at block of gene expression (1) sense strategy based on the binding of regulatory proteins, e g., transcription factors, by oligonucleotides, resulting in a block of transcription; (2) antigene strategy based on triple helix formation between oligonucleotides and double-stranded DNA, resulting in a block of transcription; (3) ribozymes resulting in specifically targeted degradation of mRNA, resulting in a block of translation, (4) antisense strategy based on binding of oligonucleotides to mRNA, resulting in a block of translation; (5) functional inactivation of proteins by binding to other proteins or peptides synthesized intracellularly after transduction of the specific coding sequences.

Effects of anisotonic exposure on duck hepatitis B virus replication.

In cultured hepatocytes from in vivo duck hepatitis B virus-infected ducks the effect of medium osmolarity on viral replication was studied. A 10-day exposure to hypotonic media (277 mOsm/L due to removal of 26 mmol/L NaCl) lowered the duck hepatitis B virus DNA content of cells and of the medium by about 50%, whereas hyperosmotic exposure (421 mOsm/L by addition of 46 mmol/L NaCl) increased it about four-fold compared with normotonic standard incubation medium (329 mOsm/L). The tissue levels of viral RNA transcripts increased during the 10 days of hypertonic exposure but decreased only slightly after hypoosmotic treatment. Western-blot analysis for the production of viral pre-S/S proteins revealed a marked stimulation of viral protein synthesis in hypertonic media, whereas hypotonic exposure inhibited it. Conversely, total cellular protein synthesis as assessed from [3H]leucine incorporation into acid-precipitable material decreased during hyperosmotic exposure but increased during hypoosmotic exposure. We noted a comparable increase of duck hepatitis B virus DNA when raffinose (80 mmol/L) was added to hypotonic or normotonic media, without change in the NaCl concentrations. This suggests that the effects of anisotonicity on viral replication were not due to alterations of Na+ or Cl- activity in the incubation media, but might reflect changes of cellular volume. The effects of anisotonicity on viral replication were only seen after exposure of more than 8 hr of the cells to anisotonicity. The findings suggest that the cellular volume is an important determinant for duck hepatitis B virus replication, yet the underlying molecular mechanisms remain elusive.

Suramin prevents duck hepatitis B virus infection in vivo.

The effect of suramin on duck hepatitis B virus (DHBV) infection was investigated in vivo. Suramin pretreatment of Pekin ducklings completely prevented DHBV infection. In contrast, suramin given at the time of or after inoculation with DHBV did not inhibit viral infection, replication, or gene expression. These data indicate that suramin effectively blocks the early stages of DHBV infection in vivo.

In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotide strategies have been employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression. Pharmacologically, 'code blockers' are ideal agents for antitumour and antimicrobial treatments because of their specific mode of action. Here we report the inhibition of duck hepatitis B virus (DHBV) by antisense oligodeoxynucleotides in primary duck hepatocyte cultures in vitro as well as in DHBV-infected Pekin ducks in vivo. The most effective antisense oligodeoxynucleotide was directed against the 5' region of the pre-S gene and resulted in a complete inhibition of viral replication and gene expression in vitro and in vivo. These results demonstrate the application of antisense oligodeoxynucleotides in vivo and exemplify their potential as human antiviral therapeutics.

Human non-hepatocytes support hepadnaviral replication and virion production.

The competence of non-hepatocytes to support hepatitis B virus (HBV) gene expression and replication was studied by transient transfection of various human cell lines with a head-to-tail dimer of HBV DNA. Independent of their neuroectodermal, mesenchymal or epithelial origin, all non-hepatocyte cell lines tested synthesized and secreted hepatitis B surface antigen (HBsAg) and hepatitis B core/e antigen (HBc/eAg). Further analyses of two of these cell lines (LS 180 and COLO 320) identified the two major HBV transcripts of 3.6 and 2.2/2.4 kb length, respectively. LS 180 cells were permissive for HBV and duck hepatitis B virus (DHBV) DNA replication and secretion of infectious virions. COLO 320 cells also supported HBV DNA replication, but did not appear to export complete viral particles. These findings provide direct evidence that both HBV and DHBV can replicate in non-hepatic tumour cell lines, one of which is shown also to produce infectious virions.

Sulfated polyanions do not inhibit duck hepatitis B virus infection.

On the basis of the antiviral action of sulfated polyanions in human immunodeficiency virus and other viral infections, we studied the effect of dextran sulfate and heparin on duck hepatitis B virus infection. These agents do not affect viral uptake and replication in liver cells in vitro or in vivo. Sulfated polyanions, therefore, appear to have no potential for the treatment of hepadnavirus infections.

Inhibition of duck hepatitis B virus infection by lysosomotropic agents.

The early phases of hepadnaviral infection were studied in primary duck hepatocyte cultures. Incubation of duck hepatocytes in vitro with duck hepatitis B virus (DHBV) resulted in infection with high levels of viral replication. The lysosomotropic agents ammonium chloride and chloroquine effectively inhibited viral infection, indicating that DHBV infection, similar to infection with other enveloped viruses, depends on receptor-mediated endocytosis and involves membrane fusion triggered by low pH.

[Hepatology. New research results in its significance for the understanding of liver diseases]. / Hepatologie. Neuere Forschungsergebnisse in ihrer Bedeutung für das Verständnis von Leberkrankheiten.

By two exemplary clinical situations--acute viral hepatitis, acute-phase reaction of the liver--the significance of basic research for the understanding of clinical phenomena and for the development of new diagnostic and therapeutic procedures is demonstrated. The very different phenomena following infection with the hepatitis-B-virus can be explained by the variation in the interactions of virus and liver cell, by the immune reaction of the host, and by mutants of the virus. The reaction of the liver to an extrahepatic infection is mediated by interleukin-6, and characterized by an alteration in protein metabolism. The synthesis of acute-phase proteins is increased. The proteins confine the local injury and establish the homeostasis of the organism.

Duck hepatitis B virus infection of non-hepatocytes.

One hundred and seventeen ducklings, 42 inoculated with duck hepatitis B virus (DHBV) 2 days after hatching and 55 connatally infected, were studied over a 6-month period in parallel with 20 ducklings without DHBV infection. Using immunohistochemical, in situ and blot hybridization analyses, the natural course of hepatic and extrahepatic infection was examined. DHBV infection started in the liver 2-4 days post-inoculation. There, DHBV was found not only in hepatocytes, but also in bile duct epithelial cells. Further, DHBV infection occurred in exocrine and endocrine pancreas (beginning 6-10 days and 20 days post-inoculation, respectively) and in germinal centers of the spleen (beginning 8 weeks post-inoculation). Occasionally viral DNA was also found in kidney glomeruli. Using strand-specific RNA probes, viral DNA in pancreas and spleen was clearly demonstrated to be replicating intermediates. Hepatic and extrahepatic infection with DHBV was not associated with histologic inflammation or pathologic changes in these tissues or the liver. These data indicate that DHBV can infect cells other than hepatocytes. The biological significance of non-hepatocyte infection for the life-cycle of the virus and its potential significance for viral persistence remain to be determined.

[Chronic hepatitis B: diagnostic value of hepatitis-B-virus DNA analysis in serum and liver tissue]. / Chronische Hepatitis B: Stellenwert des Nachweises von Hepatitis-B-Virus-DNA in Serum und Lebergewebe.

The diagnostic value of hybridization analyses for the detection of hepatitis B virus (HBV) DNA in serum and liver tissue was investigated in 79 patients with chronic liver disease. Active viral replication was demonstrated by the detection of HBeAg or HBV-DNA in serum or liver tissue. However, HBV-DNA was also detected in the absence of HBeAg in serum. No correlation was found between the presence of HBV-DNA in serum or liver tissue and clinico-chemical or histological disease. In HBsAg-negative patients no HBV-DNA was detected in serum or liver tissue. These data indicate that serological markers are sufficient for the diagnosis of HBV infection. However, detection of HBV-DNA in serum or active viral replication in liver tissue is important in selecting patients for antiviral therapy (e.g. interferon) and monitoring its efficacy.

Duck hepatitis B virus: cloning and subcloning of the viral genome.

In the course of studies on the biology of hepadnavirus infections, duck hepatitis B virus (DHBV) DNA was isolated from the serum of a German Pekin duck. Viral DNA was cloned in E. coli using pBR 322 DNA as a vector. The cloned DHBV DNA F 1-6 was characterised by restriction enzyme analyses. DHBV DNA F 1-6 was subcloned in both orientations in plasmid pSP 65 to produce strand-specific RNA probes. These probes specifically identified asymmetrically replicating nascent minus-strand DHBV DNA species or plus-strand viral RNA transcripts.

Latent hepatitis B virus infection with full-length viral genome in a patient serologically immune to hepatitis B virus infection.

The presence, state, physical structure and cellular localization of hepatitis B virus (HBV) DNA were investigated in a patient with hepatitis B surface antigen (HBsAg)-negative chronic liver disease. HBV serology was positive for antibodies to hepatitis B core antigen (anti-HBc), to hepatitis B e antigen (anti-HBe) and to HBsAg (anti-HBs); no HBV DNA was detectable in serum. Southern blot analyses of DNA extracted from the liver demonstrated free monomeric HBV DNA as two distinct species: a predominant species of fully double-stranded relaxed circular molecules and a minor species of linear molecules of 3.2 kilobase pairs (kbp) length. Restriction enzyme analyses identified the HBV genome as HBsAg subtype adw2. Cell fractionation studies further revealed that the free viral DNA species were localized exclusively in liver cell nuclei. These findings in a patient serologically immune to HBV infection demonstrate that in hepatocytes HBV can establish a latent infection, characterized by the extrachromosomal presence of a full-length viral genome without production of infectious virus or synthesis of viral antigens.

Hepatocellular carcinoma in alcoholic liver disease: no evidence for a pathogenetic role of hepatitis B virus infection.

Hepatocellular carcinoma tissues from HBsAg-negative patients with chronic alcoholic liver disease were investigated for the presence of hepatitis B virus DNA. Southern blot analyses of DNA extracted from the hepatocellular carcinomas were negative for hepatitis B virus DNA in all 17 patients examined, at a level of sensitivity of less than 0.01 genome equivalent per cell. Similarly, in liver tissues from another 30 patients with alcoholic cirrhosis without hepatocellular carcinoma, no hepatitis B virus DNA was detectable. We conclude that in our patients there is no molecular evidence for a contribution of hepatitis B virus infection to the development of hepatocellular carcinoma in alcoholic liver disease.