Current Papers in Liver Disease - July, 1999

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

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

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Lohmann, V., Korner, F., Koch, J.-O., Herian, U., Theilmann, L., and Bartenschlager, R. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 285:110-113.

A robust cell culture system for the hepatitis C virus (HCV) has not been established. For this reason, it is extremely difficult to study how HCV infects cells and to test anti-viral drugs in a model system (the only animals that can be infected are humans and chimpanzees). In this very exciting paper, Lohmann et al. make a major initial step in devising a culture system for HCV. The were able to obtain replication of subgenomic HCV RNAs in cultured cells. These subgenomic replicons are composed of only the part of the HCV genome that encodes the non-structural proteins but are competent to be replicated in cells and synthesize viral proteins. The replicons described in this paper will allow for future studies of HCV replication, pathogenesis and evolution in cell culture. They may also allow for cell-based testing of certain types of anti-viral drugs.

Taylor, D. R., Shi, S. T., Romano, R. R., Barber, G. N., and Lai, M. M. C. 1999. Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science. 285:107-110.

Treatment of hepatitis C virus (HCV) infection with interferon-alpha is effective in only a minority of individuals. This suggests that the virus may use various tricks to be resistant to interferon. PKR is a host cell protein kinase that is activated by interferon and inhibits protein synthesis, in turn decreasing viral replication in infected cells. Previous studies have shown that one of the HCV non-structural proteins, NS5A, binds to and inactivates PKR. In this study, the authors show that another HCV protein, E2, also interacts with and inactivates PKR. This provides another mechanism by which HCV may achieve resistance to interferon.

Mamiya, N., and Worman, H. J. 1999. Hepatitis C virus core protein binds to a DEAD box RNA helicase. Journal of Biological Chemistry. 274:15751-15756.

The core protein of hepatitis C virus (HCV) is expressed in infected cells. In this study, the investigators show that HCV core protein binds to a cellular protein called DBX. DBX is a member of the DEAD-box family of proteins and functions as a RNA helicase that unwinds host cell RNA and helps it become translated into protein. Using two model systems, the authors provide evidence that core protein may inhibit the function of DBX. They also show that DBX and HCV core protein localize together in cells. These results suggest that HCV core protein may inactivate a factor involved in the translation of cellular RNA to protein. This may cause damage to infected cells and also possibly help the viral RNA gain access to the host cell protein synthesis machinery. [Note regarding possible conflict of interest: Dr. Worman, an author on this paper, is also the author of Current Papers in Liver Disease.]

Ono-Nita, S. K., Kato, N., Shiratori, Y., Lan, K.-H., Yoshida, H., Carrilho, F. J., and Omata, M. 1999. Susceptibility of lamivudine-resistant hepatitis B virus to other reverse transcriptase inhibitors. Journal of Clinical Investigation. 103:1635-1640.

Lamivudine is a reverse transcriptase inhibitor that is active against the DNA polymerase of the hepatitis B virus. Lamivudine inhibits viral replication in patients with hepatitis B and, in the United States, is approved for the treatment of patients with HBeAg (a serological marker of viral replication) in their serum. However, about 15% to 40% of patients treated with lamivudine develop drug-resistant virus. Resistance occurs as a result of mutations in a portion of the viral polymerase with the sequence tyrosine-methionine-aspartic acic-aspartic acid (YMDD using one letter amino acid codes). In this study, Ono-Nita et al. examined the effects of lamivudine and other reverse transcriptase inhibitors (adefovir, lobucavir, penciclovir and nevirapine) on hepatitis B virus replication in cells. They transfected cells with hepatitis B virus DNA without (wild-type) and with YMDD mutations (lamivudine-resistant) and measured replication. Lamivudine, adefovir and lobucavir inhibited the replication of wild-type viral DNA. Adefovir and lobucavir also inhibited the replication of lamivudine-resistant YMDD mutants. Penciclovir and nevirapine did not inhibit viral DNA replication. These results suggest that lobucavir and adefovir may effective in treating patients with hepatitis B virus infection that becomes resistant to lamivudine. They also suggest that combination therapies with lamivudine and lobucavir or lamivudine and adefovir are rational treatment strategies that should be studied in human subjects.

Cacciola, I., Pollicino, T., Squadrito, G., Cerenzia, G., Orlando, M. E., and Raimondo, G. 1999. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. New England Journal of Medicine. 341:22-26.

In some individuals without evidence of infection based on standard blood testing (detectable hepatitis B surface antigen [HBsAg]), the hepatitis B virus can be detected in liver or serum by very sensitive methods such as polymerase chain reaction. This is called occult hepatitis B virus infection. The clinical significance of occult hepatitis B virus infection is not entirely clear. In this study from Italy, Cacciola et al. show, using the polymerase chain reaction, that 33% of 200 patients with chronic hepatitis C virus infection had concurrent occult hepatitis B virus infection. In contrast, only 14% of 50 patients with liver diseases not related to hepatitis C had occult hepatitis B virus infection. Patients with chronic hepatitis C and occult hepatitis B virus infection were more likely to have cirrhosis (33%) than those who did not have concurrent occult hepatitis B virus infection (14%). Although the clinical significance of these findings remains unclear, it appears that very low levels of hepatitis B virus can be detected in patients with chronic hepatitis C in Italy.

Bacon, B. R., Olynyk, J. K., Brunt, E. M., Britton, R. S., and Wolff, R. K. 1999. HFE genotype in patients with hemochromatosis and other liver diseases. Annals of Internal Medicine. 130:953-962.

In 1996, Feder et al. identified the gene responsible for hereditary hemochromatosis (Nature Genetics. 1996;13:399-408.; see September, 1996 Current Papers in Liver Disease). This gene is now called HFE. Two different mutations in HFE have been found in patients with hereditary hemochromatosis. The first results in a cysteine residue at position 282 in the protein being converted to a tyrosine (C282Y). The second results in a histidine residue at position 63 being converted to an aspartic acid (H63D). Since the initial discovery of HFE, several studies have shown that between 64% and 100% of patients with hereditary hemochromatosis are homozygous for the C282Y mutation. In this study, Bacon et al. show that 60 of 66 individuals (91%) with a clinical diagnosis of hereditary hemochromatosis were homozygous for the C282Y mutation. The remainder had various combinations of the C282Y and H63D mutations and one had neither of these mutations. Of 132 patients with other liver diseases, 5% were also found to be C282Y heterozygotes. All 66 patients heterozygous for the C282Y mutation, with and without a clinical diagnosis of hereditary hemochromatosis, had elevated liver iron content, although about 15% of them did not have concentrations high enough to meet a previous diagnostic criteria for the disease. There results indicate that genetic testing for the C282Y mutation in HFE is useful in patients with liver disease and suspected iron overload and may lead to a diagnosis of hereditary hemochromatosis in individuals without apparent clinical criteria. They also suggest that other mutations in HFE and possibly different genes, either alone or in combination with the C282Y and H63D mutations in HFE, can cause hereditary hemochromatosis.

Ibdah, J. A., Bennett, M. J., Rinaldo, P., Zhao, Y., Gibson, B., Sims, H. F., and Strauss, A. W. 1999. A fetal fatty-acid oxidation disorder as a cause of liver disease in pregnant women. New England Journal of Medicine. 340:1723-1731.

Acute fatty liver of pregnancy is a potentially fatal disorder that occurs during the third trimester. HELLP (hemolysis, elevated liver enzyme levels, low platelet count) syndrome also occurs late in pregnancy and shares some similarities with acute fatty liver but is more somewhat common and usually less severe. The causes of these two disorders are not known but previous studies have suggested that some cases may occur in mothers of fetuses with inherited disorders of fatty acid oxidation. In this study, Ibdah et al. identified 24 children with 3-hydroxyacyl-CoA dehydrogenase deficiency. Most of these children who became ill several months after birth. Molecular analysis showed that 19 had a deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase, a single enzyme activity that is part of the mitochondrial trifunctional protein that catalyzes several steps in fatty acid beta-oxidation. Eight of these children had homozygous mutations in which glutamic acid at residue 474 was changed to a glutamine. Eleven were compound heterozygotes with the glutamic acid 474 to glutamine mutation in one gene and another mutation in the other. While carrying a fetus with a glutamic acid 474 to glutamine mutation in at least one gene, 79% of mothers had either acute fatty liver of pregnancy or HELLP syndrome. In other pregnancies in which the fetus did not carry this mutation, the mothers did not suffer from liver disease during pregnancy. Five other children had mutations that caused deficiency of the entire mitochondrial trifunctional protein (none had the glutamic acid to glutamine mutation at residue 474). None of their mothers suffered from liver disease during pregnancy. This study shows that a certain mutation in the long chain 3-hydroxyacyl-CoA dehydrogenase in the fetus may cause acute liver disease during pregnancy in the mother. It is not clear how abnormalities in fetal fatty acid metabolism cause liver disease in the mother. It is also not clear if other abnormalities in fetal fatty acid oxidation are responsible for additional cases of acute liver disease during pregnancy.

Makishima, M., Okamoto, A. Y., Repa, J. J., Tu, H., Learned, M., Luk, A., Hull, M. V., Lustig, K. D., Mangelsdorf, D. J., and Shan, B. 1999. Identification of a nuclear receptor for bile acids. Science. 284:1362-1365.

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Parks, D. J., Blanchard, S. G., Bledsoe, R. K., Chandra, G., Consler, T. G., Kliewer, S. A., Stimmel, J. B., Willson, T. M., Zavacki, A. M., Moore, D. D., and Lehmann, J. M. 1999. Bile acids: natural ligands for an orphan nuclear receptor. Science.284:1365-1368.

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Wang, H., Chen, J., Hollister, K., Sowers, L. C., and Forman, B. M. 1999. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Molecular Cell.3:543-553

Nuclear receptor are proteins that bind to various hormones and other chemicals and regulate gene expression. Well known examples are thyroid hormone receptor and retinoic acid receptor. The molecules that bind to and activate these receptors are known as ligands. There are many so-called orphan receptor for which the ligands are not known. Now, three groups have now shown that the bile acid chenodeoxycholic acid is the ligand for an orphan nuclear receptor known as farnesoid X receptor (FXR). Chenodeoxycholic acid is made in the liver by the oxidation of cholesterol, which is catalyzed by the enzyme 7-alpha-hydroxylase. It is then secreted into the bile and a portion is ultimately reabsorbed from the intestine back into the circulation and returned to the liver. These studies show that, by binding to FXR, chenodeoxycholic acid decreases the expression of the gene encoding 7-alpha-hydroxylase and stimulates expression of the gene encoding an intestinal transporter for the uptake of bile acids. These results show that bile acids can regulate their own synthesis and transport by binding to the nuclear receptor FXR. They have important implications in understanding the regulation of bile acid synthesis in the liver and, in turn, the levels of cholesterol in the blood.

Petersen, B. E., Bowen, W. C., Patrene, K. D., Mars, W. M.,Sullivan, A. K., Murase, N., Boggs, S. S., Greenberger, J. S., and Goff, J. P. 1999. Bone marrow as a potential source of hepatic oval cells. Science. 284:1168-1170.

Some data suggest that the liver may have "stem cells" which are are hypothesized to be undifferentiated cells that can divide and differentiate into hepatocytes and bile duct cells. Hepatic oval cells, which have been partially characterized, may be these "stem cells" or intermediates in the differentiation of stem cells to hepatocytes and bile duct cells. Oval cells divide under certain conditions, mainly when hepatocytes are prevented from dividing in response to liver damage. The paper by Petersen et al. suggests that hepatic oval cells may originate in the bone marrow. To test this hypothesis, the authors performed bone marrow transplantation in mice whose livers were then treated with 2-acetylaminofluorene, which prevents hepatocyte division, and damaged by carbon tetrachloride. They then showed that certain genetic and biochemical markers, present only in the donor bone marrow cells, were present in some hepatocytes and bile duct cells. For example, female mice with damaged livers transplanted with bone marrow from male mice had Y chromosomes in some of their hepatocytes. They also showed that when mice with damaged livers received liver transplants from mice lacking a certain biochemical marker, some hepatocytes contained the marker that potentially came from cells in the recipient's bone marrow. These results suggest that cells originating in the bone marrow may be able to differentiate into liver cells. In the long-term, this knowledge may be used to devise ways to regenerate damaged livers.

Belay, E. D., Bresee, J. S.,Holman, R. C., Khan, A. S., Shahriari, A., and Schonberger, L. B. 1999. Reye's syndrome in the United States from 1981 through 1997. New England Journal of Medicine. 340:1377-1382.

Reye's syndrome is an acute illness with fatty infiltration of the liver and hepatic encephalopathy. It occurs almost exclusively in children and the mortality rate is about 30%. In 1980, Reye's syndrome was reported to be associated with the use of aspirin during varicella or influenza-like illness. That year, the United States Centers for Disease Control and Prevention cautioned physicians and parents not to use aspirin in such instances. In 1982, the surgeon general of the United States issued an advisory on this matter and in 1986 warning labels were required on all aspirin-containing medications. This study reports surveillance data collected from December 1980 through 1987 of reported cases of Reye's syndrome. During this time period, 1,207 cases were reported in the United States in patients less than 18 years old. A peak of 555 cases were reported in 1980 and the incidence rapidly declined in subsequent years as physicians and the public were made aware of the association of the illness with aspirin consumption. Since 1987, there have been no more than 36 cases a year of Reye's syndrome. These data show that the number of reported cases of Reye's syndrome in infants and children has declined rapidly since an association with aspirin use during varicella or influenza-like illnesses was first reported. As a result, Reye's syndrome is now a rare disease.

Guidotti, L. G., Rochford, R., Chung, J., Shapiro, M., Purcell, R., and Chisari, F. V. 1999. Viral clearance without destruction of infected cells during acute HBV infection. Science. 284:825-829.

It has long been thought that the hepatitis B virus (HBV) is cleared from infected liver cells by a cytopathic mechanism (a mechanism in which infected cells are killed) mediated primarily by CD8+ T lymphocytes. Recent work using transgenic mice that express HBV in their livers, however, suggested that HBV replication and gene expression can be abolished by a noncytopathic (without killing of infected cells) mechanism. One drawback of the transgenic mice studies was that these mice do not express a form of viral DNA in infected cells that is normally expressed in natural HBV infection. In this study, Guidotti et al. examined the disappearance of HBV DNA from infected liver and blood of chimpanzees with acute hepatitis B. Two healthy chimpanzees were infected with HBV obtained from the sera of transgenic mice that express the virus. Both chimpanzees developed acute hepatitis B. The investigators found that 90% of HBV DNA disappeared from their livers and sera long before the peak of T cell infiltration of the liver and the onset of most of the liver disease. These results show that the body can eliminate most HBV from infected livers without destroying infected cells. This may represent a general mechanism that has evolved in primates to preserve vital organs that are acutely infected with viruses. Deciphering the details of this mechanism by which HBV is eliminated from liver cells without killing them may potentially lead to the development of novel drugs for the treatment of hepatitis B in man.

Copyright, 1999, Howard J. Worman, M. D. All rights reserved. Printing or other reproduction is prohibited without the written authorization of Howard J. Worman.