Current Papers in Liver Disease - April, 1996

By Howard J. Worman, M. D.
Columbia University

This is a past issue of Current Papers in Liver Disease.

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Polson, A. G., Bass, B. L., and Casey, J. L. 1996. RNA editing of hepatitis delta virus antigenome by the dsRNA-adenosine deaminase. Nature. 380:454-456.

Hepatitis delta or hepatitis D virus (HDV) is a replication-defective human pathogen that requires hepatitis B infection for propagation. HDV can super-infect or co-infect patients with hepatitis B and cause worsening liver disease. HDV contains a single-stranded, closed, circular RNA genome. For replication, a complementary RNA antigenome is first synthesized from which the genomic strand is replicated. Both the genome and antigenome can form highly base-paired, rod-shaped structures. A single open reading frame encodes two isoforms of the only HDV polypeptide, hepatitis D antigen (HDAg). The short form of this protein is required for replication while the longer form, with 19 additional carboxyl-terminal amino acids, inhibits replication and is necessary for packaging. The two isoforms of HDAg arise by RNA editing. In this study, the authors show that RNA editing takes place at a location called the amber/W site in the HDV antigenome, with conversion of an adenosine to an inosine and a resulting change from an amber stop codon to a tryptophan codon in the genomic strand. They demonstrate that the RNA editing enzyme double-stranded RNA adenosine deaminase (dsRAD), a ubiquitous enzyme in most multicellular organisms, edits the antigenome in vitro. Mutations in viral RNA that alter the level of editing in transfected cells correspondingly inhibits RNA editing in vitro by dsRAD. These results show that dsRAD or a highly related enzyme is responsible for RNA editing of the antigenome of HDV to produce the two HDAg isoforms.

Kobayashi, M., Tanaka, E., Sodeyama, T., Urushihara, A., Matsumoto, A., and Kiyosawa, K. 1996. The natural course of chronic hepatitis C: a comparison between patients with genotypes 1 and 2 hepatitis C viruses. Hepatology. 23:695-699.

Several studies have demonstrated that different hepatitis C virus (HCV) genotypes cause different severity of liver disease. In this study, the authors studied 140 patients in Japan. They found that 96 patients were infected with genotype 1b, 4 with either genotype 1a or 1b and 36 with either genotype 2a or 2b (genotype could not be determined in 4 patients). Patients infected with genotype 1 had higher circulating levels of HCV RNA than those infected with genotype 2. Deterioration in the grade and stage of liver histology, determined by follow-up biopsy 5 to 20 years after the initial biopsy, was seen more frequently in patients infected with genotype 1 (68.0% grade, 63.0% stage) than in those infected with genotype 2 (41.7% grade, 38.9% stage). These results suggest, as have previous studies, that more severe progression of chronic hepatitis C is seen in patients infected with genotype 1b than with genotype 2.

Gane, E. J., Portmann, B. C., Naoumov, N. V., Smith, H. M., Underhill, J. A., Donaldson, P. T., Maertens, G., and Williams, R. 1996. Long-term outcome of hepatitis C infection after liver transplantation. New England Journal of Medicine. 334:815-820.

Cirrhosis caused by chronic hepatitis C virus (HCV) infection is a common indication for orthotopic liver transplantation. HCV viremia usually persists after transplantation and recurrent hepatitis C can occur in the allograft. In this retrospective study, the outcomes of 149 patients with HCV infection who received orthotopic liver transplantation were compared to those of 623 patients in a control group who were transplanted without HCV infection. There was no difference in the five year cumulative survival in the patients who were transplanted with (70%) and without (69%) HCV infection. Of the 149 patients with HCV infection, 130 survived more than six months after transplantation. On their most recent liver biopsies, 12% had no evidence of chronic hepatitis (20 months median follow-up), 54% had mild chronic hepatitis (35 months median follow-up), 27% had moderate chronic hepatitis (35 months median follow-up) and 8% had cirrhosis (51 months median follow-up). Serum ALT activity was a poor indicator of the degree of inflammation. Graft loss occurred in 27 of the HCV infected patients after a median of 303 days, however, chronic rejection was diagnosed in the same percent as in the control group. The immunosuppressive regimen used or the extent of HLA mismatching between donors and recipients had no effect on the severity of graft injury. HCV genotype 1b was the type most commonly present in the infected patients and it was associated with an increased incidence of moderate hepatitis and cirrhosis. These data show that, although 5-year survival is the same as in control patients transplanted for other reasons, moderate chronic hepatitis and cirrhosis occur in a significant number of patients who are infected with HCV and undergo orthotopic liver transplantation. The optimal methods of diagnosis and treatment remain to be determined.

Mazzarerro, V., Regalia, E., Doci, R., Andreola, S., Pulvirenti, A., Bozzetti, F., Montalto, F., Ammatuna, M., Morabito, A., and Gennari, L. 1996. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. New England Journal of Medicine. 334:693-699.

The utility of orthotopic liver transplantation for hepatocellular carcinoma is unclear. In this prospective cohort study, 48 patients with cirrhosis and small hepatocellular carcinomas received orthotopic liver transplantation. In 94% of patients, cirrhosis was related to infection with hepatitis B virus and/or C virus. Eligibility criteria were either a single tumor less than 5 cm in diameter or no more than 3 tumor nodules each less that 3 cm in diameter. Twenty-eight patients with adequate liver function underwent treatment, primarily chemoembolization, prior to transplantation. Median follow-up after transplantation was 26 months. After 4 years, the actuarial survival rate was 75% and the recurrence-free survival rate was 83%. These rates were significantly lower in the patients whose tumors exceeded the eligibility criteria on pathological examination of the explanted liver. The results show that orthotopic liver transplantation may be an effective treatment for small, unresectable hepatocellular carcinomas.

Pontoglio, M., Barra, J., Hadchouel, M., Doyen, A., Kress, C., Bach, J. P., Babinet, C., and Yaniv, M. 1996. Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome. Cell. 84:575-585.

Hepatocyte nuclear factor 1 (HNF1) is a transcription factor that activates many genes, including several involved in hepatocyte function such as those encoding albumin and alpha-1-antitrypsin. A related factor vHNF1 has also been characterized. HNF1 and vHNF1 are primarily expressed in liver, kidney, intestine, pancreas and stomach. These investigators used embryonic stem cell technology to create a strain of knockout mice lacking HNF1. Homozygous knockout mice undergo apparently normal embryonic development but fail to thrive and die around weaning after a progressive wasting syndrome with marked liver enlargement and evidence of hepatocellular damage. Transcription from the albumin and alpha-1-antitrypsin genes is reduced and the gene encoding phenylalanine hydroxylase is completely silent leading to phenylketonuria. These mice also suffer from severe Fanconi syndrome cuased by renal proximal tubule dysfunction. HNF1 appears to be essential for normal liver and kidney function but the exact nature of the hepatic disease that occurs in its absence remains to be determined.

Paulusma, C. C., Bosma, P. J., Zaman, G. J. R., Bakker, C. T. M., Otter, M., Scheffer, G. L., Scheper, R. J., Borst, P., and Oude Elferink, R. P. J. 1996. Congenital jaundice in rats with a mutation in a multidrug-resistance-associated protein gene. Science. 271:1126-1128.

Dubin-Johnson syndrome is an autosomal recessive congenital disorder characterized by chronic conjugated hyperbilirubinemia. The TR- rat has a similar phenotype with a recessive defect in the hepatobiliary secretion of bilirubin glucuronides and other multivalent organic anions. Secretion of these components from the hepatocyte into the bile is mediated by an ATP-dependent transport system in the canalicular membrane called cMOAT that has been functionally characterized but not cloned. The human multidrug resistance-associated protein 1 (hMRP1) transports some of the same substrates as cMOAT, and for this reason, the authors of this paper looked for a protein homologous to hMRP1 in rat liver. They first isolated partial cDNA clones for rat MRP1 and then used these as probes to screen rat liver cDNA libraries. They identified a cDNA that encoded a member of the ABC transporter family with 47.6% sequence identity to hMRP1. The RNA for this protein was predominantly expressed in liver and antibodies raised against it reacted with a polypeptide of approximately 200 kDa in normal rat hepatocyte canalicular membranes. The RNA for this protein was significantly reduced and the protein could not be detected in hepatocytes from TR- rats. Sequencing of the cDNA from TR- rats showed that it contained a frameshit mutation which resulted in the introduction of a premature stop codon. These results show that a rat homologue of hMRP1 encodes cMOAT and that a mutation in its gene, resulting in a truncated, non-viable protein, is responsible for the impaired transport of organic compounds into the bile in TR- rats. Future work should establish if mutations in the human homologue of rat cMOAT are responsible for Dubin-Johnson syndrome.

Harpaz, R., von Seidlein, L., Averhoff, F. M., Tormey, M. P., Sinha, S. D., Kotsopoulou, D., Lambert, S. B., Robertson, B. H., Cherry, J. D., and Shapiro, C. N. 1996. Transmission of hepatitis B virus to multiple patients from a surgeon without evidence of inadequate infection control. New England Journal of Medicine. 334:549-554.

Transmission of hepatitis B virus (HBV) from surgeons to patients has been known to occur but is generally uncommon. In this study, a 47 year-old woman was identified who became infected with HBV after undergoing a thymectomy in which a thoracic surgery resident, who contracted hepatitis B six months earlier, assisted. In one year, 19 of 144 (13%) susceptible patients undergoing procedures in which this surgical resident assisted had evidence of recent HBV infection. None of 124 susceptible patients of other thoracic surgeons at one of the hospitals where this resident worked developed recent HBV infection. The HBsAg subtype and the partial HBV DNA sequences from the surgical resident and the infected patients were identical. The surgical resident was HBeAg positive and had high serum concentrations of HBV DNA. No deficiencies in infection-control practices were noted during operations. This study confirms the transmission of HBV from heath care providers to patients. As noted in an accompanying editorial (Gerberding, J. L. 1996. New England Journal of Medicine. 334:594-595), however, the risk of death from HIV or HBV infection acquired during an invasive procedure performed by an infected surgeon is only between 2.4 and 24 in one million. Nonetheless, as the authors point out at the end of the paper, pre-employment vaccination of the resident would have prevented this outbreak.

Esteban, J. I., Gomez, J., Martell, M., Cabot, B., Quer, J., Camps, J., Gonzalez, A., Otero, T., Moya, A., Esteban, R., and Guardia, J. 1996. Transmission of hepatitis C virus by a cardiac surgeon. New England Journal of Medicine. 334:555-560.

Two patients were identified in Spain who developed hepatitis C after open heart surgery despite the apparent absence of hepatitis C virus (HCV) from transfused blood. Both of these cases were linked to a cardiac surgeon known to have chronic hepatitis C. Of 222 patients operated upon by this surgeon who participated in studies of post-transfusion hepatitis between 1988 and 1994, six contracted postoperative hepatitis C despite transfusion of only seronegative blood. Each of these patients underwent valve-replacement surgery. Five of these six patients were infected with HCV genotype 3, as was the surgeon, whereas 13 other patients of the surgeon who had transfusion-associated hepatitis were infected with HCV genotype 1. Nucleotide sequencing revealed that the HCV genotype 3 isolates from the five patients and the surgeon were highly related and likely of the same origin. This report provides evidence that HCV may be transmitted from a surgeon to patients during open heart surgery.

Meraz, M. A., White, J. M., Sheehan, K. C. F., Bach, E. A., Rodig, S. J., Dighe, A. S., Kaplan, D. H., Riley, J. K., Greenlund, A. C., Campbell, D., Carfer-Moore, K., DuBois, R. N., Clark, R., Aguet, M., and Schreiber, R. D. 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiolgic specificity in the JAK-STAT signaling pathway. Cell. 84:431-442.

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Durbin, J. E., Hackenmiller, R., Simon, M. C., and Levy, D. E. 1996. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell. 84:443-450.

Interferon-alpha is widely used in the treatment of viral hepatitis B and C, however, its mechanism of action is not completely understood. Interferons alpha and gamma, upon stimulation of their respective receptors, induce phosphoryation of STAT1 by Janus protein tyrosine kinases (JAKs) that are associated with the receptors. Tyrosine-phosphorylated STAT1 forms complexes with other STATs (at least six mammalian STATs have been identified) and other proteins which are transported to the nucleus to stimulate transcription of genes that mediate the interferon response. It has been unclear if STAT1 participates in all interferon-dependent biological responses and if other cytokines rely on this pathway for their cellular responses. These two groups used embryonal stem cell technology to disrupt the Stat1 gene in mice. The knockout mice showed no overt developmental abnormalities but failed to thrive and were susceptible to infections by viruses and microbial pathogens. Their cells were unresponsive to alpha and gamma interferons but responded normally to many other cytokines. These results show that interferons alpha and gamma exert their cellular effects via the STAT1 signaling pathway.

Shimizu, Y. K., Feinstone, S. M., Kohara, M., Purcell, R. H., and Yoshikura, H. 1996. Hepatitis C virus: detection of intracellular virus particles by electron microscopy. Hepatology. 23:205-209.

Microscopic detection of hepatitis C virus (HCV) particles in the infected liver is extremely rare. Particles of 55 nm to 65 nm in diameter, presumed to be HCV, have been visualized in serum samples from infected monkeys and humans, however, serum can contain many different viruses. Intracellular HCV particles have not been previously visualized. These authors used human T cell (HPBMa10-2) and B cell (Daudi) lines that were infected with HCV and chimpanzee liver from the acute phase of hepatitis C infection to detect viral particles by electron microscopy. Viruslike particles of approximately 50 nm diameter were seen in cytoplasmic vesicles and immunoperoxidase electron microscopy using antibodies against HCV core and envelope polypeptides confirmed that the particles likely contained viral antigens. Although studies of this type are frequently confounded by contamination with different viruses, the paper provides reasonable evidence for the direct visualization of HCV in cells.

Linnen, J., Wages, J., Jr., Zhang-Keck, Z.-Y., Fry, K. E., Krawczynski, K. Z., Alter, H., Koonin, E., Gallagher, M., Alter, M., Hadziyannis, S., Karayiannis, P., Fung, K., Nakatsuji, Y., Shih, W.-K., Young, L., Piatak, M., Jr., Hoover, C., Fernandez, J., Chen, S., Zou, J.-C., Morris, T., Hyams, K. C., Ismay, S., Lifson, J. D., Hess, G., Foung, S. K. H., Thomas, H., Bradley, D., Margolis, H., and Kim, J. P. 1996. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science. 271:505-508.

In this paper, the authors identify a RNA virus associated with acute and chronic hepatitis similar to the previously identified GB-C virus and distantly related to the hepatitis C, GB-B and GB-A viruses. A cDNA expression library was constructed from the plasma of a patient with chronic hepatitis C. Immunoscreening of the expression library with the patient's serum identified several hepatitis C virus sequences and several other sequences that were unique. From these unique sequences, an anchored polymerase chain reaction method was used to amplify overlapping clones for the entire viral genome. The virus was termed the hepatitis G virus (HGV). Using these sequences, overlapping cDNAs for HGV were also isolated from the plasma of another patient. The polyprotein sequence identities between HGV and GB-A, GB-B and a hepatitis C virus (HCV) isolate were 43.8%, 28.4% and 26.8%, respectively. HGV was 85.5% identical in nucleotide sequence and 100% identical in amino acid sequence to the corresponding portion of GB-C that has been characterized. Using reverse transcription-polymerase chain reaction, HGV sequences were identified in 13% of 38 patients with non-A-E hepatitis in the U. S. It was also identified in about 18% of patients with hepatitis C. HGV was detected in serum samples from patients in the U. S., Australia, South America and Europe. HGV was also implicated in two prospectively studied patients with post-transfusion hepatitis who were negative for HGV prior to transfusion. These findings demonstrate that HGV, a virus the same as or very similar to GB-C, is associated with acute and chronic hepatitis worldwide.

Enomoto, N., Sakuma, I., Asahina, Y., Kurosaki, M., Murakami, T., Yamamoto, C., Ogura, Y., Izumi, N., Marumo, F., and Sato, C. 1996. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. New England Journal of Medicine. 334:77-81.

Different genotypes of the hepatitis C virus (HCV) can produce different clinical outcomes. Infection with HCV genotype 1b generally causes more active liver disease and progression to cirrhosis and hepatocellular carcinoma. This genotype is also more resistant to treatment with interferon. These investigators previously identified a region from amino acid 2209 to amino acid 2248 in the nonstructural protein 5A (NS5A) of HCV genotype 1b strain HCV-J that is associated with interferon sensitivity. In this study, 84 patients chronically infected with HCV genotype 1b, who had received interferon-alpha for six months, were retrospectively analyzed for response. The amino acid sequences of NS5A between amino acids 2209 and 2248 were also determined in the patients. Response to interferon-alpha, as indicated by the absence of HCV RNA after six months of therapy, did not occur in any of the 30 patients whose NS5A sequences were identical to wild type strain HCV-J. Of 38 patients with 1 to 3 amino acid substitutions in this region, 5 (13%) had a response to interferon-alpha treatment, as did all 16 patients with 4 to 11 amino acid substitutions in this region. In patients with chronic HCV genotype 1b infection, there is a correlation between response to interferon-alpha therapy and mutations in the NS5A gene. How mutations in this gene lead to interferon sensitivity remain to be determined, however, if they are deciphered, important therapeutic advancements may be realized.