Current Papers in Liver Disease - May, 1998

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

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

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Chayam, K., Suzuki, Y., Kobayashi, M., Kobayashi, M., Tsubota, A., Hashimoto, M., Miyano, Y., Koike, H., Kobayashi, M., Koida, I., Arase, Y., Saitoh, S., Murashima, N., Ikeda, K., and Kumada, H. 1998. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology. 27:1711-1716.

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Allen, M. I., Deslauriers, M., Andrews, C. W., Tipples, G. A., Walters, K.-A., Tyrrell, D. L. J., Brown, N., and Condreay, L. D. 1998. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Hepatology. 27:1670-1677.

Lamivudine is an inhibitor of hepatitis B virus (HBV) RNA-dependent DNA polymerase that has shown considerable promise in the treatment of patients with hepatitis B. One factor that may limit its use as a therapeutic agent is the development of resistance, which is known to occur as a result of mutations in a particular amino acid motif of tyrosine-methione-aspartate-aspartate (YMDD) in the polymerase. These two studies in Hepatology further examined the phenomenon of lamivudine resistance. The study by Allen et al. examined the genomic sequences of HBV from 20 patients who developed resistance to lamivudine. The authors showed that the 20 mutations at the YMDD motif comprise two "groups." Group I had a substitution of valine for methionine (YVDD) and group II had a substitution of isoleucine for methionine (YIDD). Group I mutants always had a second mutation that resulted of leucine for another methionine at a position upstream of the YMDD motif. The authors further showed that these mutations inhibited polymerase sensitivity to lamivudine in vitro and then used "molecular modeling" methods to determine the effects that these mutations had on polymerase structure. The structural information obtained from such modeling methods may allow the development of agents for "follow-up therapy" of lamivudine-resistant strains. In the paper by Chayama et al., 20 patients on long-term lamivudine were studied. The authors found mutations in the YMDD motif in five subjects who showed clinical evidence of resistance (so-called "DNA breakthrough" or the ability to detect HBV DNA in serum) 9 to 14 months after starting lamivudine therapy. Four of five mutants were YIDD (Group II of Allen et al.) and one was YVDD (Group I of Allen et al.). Mutants were detected in serum 1 to 4 months before "DNA breakthrough" and were predominant at "breakthrough." However, the mutant forms were replaced by wild-type virus within 3 to 4 months after cessation of lamivudine therapy in two patients. One of these two patients had an exacerbation of hepatitis after stopping lamivudine and developing primarily wild-type virus in serum. The authors conclude that "the replication of YMDD mutant viruses is less than wild-type and is re-overtaken by wild type after cessation of therapy." This suggests that retreatment with lamivudine, possibly in combination with other drugs, might be useful in some patients experiencing hepatitis with lamivudine-resistant variants.

Peet, D. J., Turley, S. D., Ma, W., Janowski, B. A., Lobaccaro, J.-M. A., Hammer, R. E., and Mangelsdorf, D. J. 1998. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR-alpha. Cell. 93:693-704.

The conversion of cholesterol to bile acids takes place in the liver. Abnormalities in the metabolism of cholesterol to bile acids can result in elevations of serum cholesterol and low density lipoprotein (LDL) concentrations. LXRs are nuclear receptors that activate the expression of certain genes when bound to oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol and 24,25(S)-epoxycholesterol that are monooxidized metabolites of cholesterol. LXR-alpha is a LXR nuclear receptor expressed predominantly in the liver. In this study, the authors examined mice from which the gene encoding LXR-alpha was knocked out. These mice appeared generally normal when fed a usual diet. When fed a diet high in cholesterol, in contrast to normal mice, the LXR-alpha knockout mice failed to induce the expression of cholesterol-7-alpha-hydroxylase, the rate-limiting enzyme in bile acid synthesis. This defect in cholesterol-7-alpha-hydroxylase gene expression was associated with decreased bile acid secretion, increased serum cholesterol and LDL concentrations and massive accumulation of cholesterol in the liver that led to hepatic dysfunction. These results demonstrate that LXR-alpha is a major sensor of dietary cholesterol that activates a regulatory pathway for sterol metabolism, including the conversion of cholesterol to bile acids in the liver. The findings in this study also suggest that individuals with mutations in LXR-alpha may have inherited defects in cholesterol metabolism associated with fat accumulation in the liver.

Wu, C. H., and Wu, G. Y. 1998. Targeted inhibition of hepatitis C virus-directed gene expression in human hepatoma cell lines. Gastroenterology. 114:1304-1312.

The hepatitis C virus (HCV) has a positive-stranded RNA genome that encodes a single polyprotein. Upstream from the start of the protein-coding sequence is a 5'-untranslated region (5'-UTR) that is necessary for viral protein synthesis. This 5'-UTR allows the viral RNA to enter the ribosome (cell protein synthesis machinery) by a different mechanism that that used by virtually all cellular mRNAs. In this paper, Wu and Wu show, as has been shown previously by several groups, that antisense oligonucleotides directed against a portion of the HCV 5'-UTR inhibit the synthesis of proteins downstream from the HCV 5'-UTR. They further show, as they have previously in other systems, that oligonucleotides can be delivered to human hepatoma cell lines as asialoglycoprotein-polylysine complexes (human hepatocytes express asialoglycoprotein receptors on their surface). Although there is really nothing novel in this paper, it again demonstrates that the 5'-UTR of HCV is a potential target for the development of specific antiviral drugs. The results also suggest a system to deliver such drugs to the liver.

Feray, C., Gigou, M., Samuel, D., Ducot, B., Maisonneuve, P., Reynes, M., Bismuth, A, and Bismuth, H. 1998. Incidence of hepatitis C in patients receiving different preparations of hepatitis B immunglobulins after liver transplantation. Annals of Internal Medicine. 128:810-816.

A few studies have suggested that antibodies against the hepatitis C virus (HCV) developed by infected humans and monkeys may be protective against re-infection. In contrast, antibodies against hepatitis B virus from sera of infected individuals (so-called HBIG) have been repeatedly demonstrated to prevent infection and recurrence of hepatitis B following orthotopic liver transplantation. In this study, the authors retrospectively looked at HCV infection in subjects who received liver transplantation for cirrhosis secondary to hepatitis B and other causes. Those who were transplanted for cirrhosis caused by hepatitis B received long-term HBIG after transplantation. Of 218 patients retrospectively found to have had HCV infection before transplantation, the incidence of HCV in blood after transplantation was 54% (25 of 46 patients) in those who were transplanted for concurrent hepatitis B and received HBIG and 94% (162 of 172 patients) in those transplanted for other reasons who did not receive HBIG (P < 0.001). Among 210 patients not found to have HCV infection prior to transplantation, 26% (18 of 68 patients) who received HBIG acquired HCV infection after transplantation compared to 47% (40 of 86 patients) who did not receive HBIG (P < 0.001). The apparent protection against HCV infection was only observed in patients who received HBIG prior to March 1990, the time when the blood bank started destroying batches of HBIG prepared from the plasma of donors found to be infected with HCV. These results suggest that HBIG prepared before March 1990, that may have been made from individuals co-infected with HCV, were protective against HCV infection after orthotopic liver transplantation. The authors speculate that HBIG preparations from that time may have had anti-HCV antibodies that were protective. This hypothesis remains to be confirmed experimentally or in prospective, controlled trials.

Fox, I. J., Chowdhury, J. R., Kaufman, S. S., Goertzen, T. C., Chowdhury, N. R., Warkentin, P. I., Dorko, K., Sauter, B. V., and Strom, S. C. 1998. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. New England Journal of Medicine. 338:1422-1426.

Crigler-Najjar syndrome type I is an autosomal recessive inherited disorder in which the enzyme that converts bilirubin to bilirubin glucuronides (bilirubin UDP-glucuronosyltransferase) is lacking from hepatocytes, the major cell type of the liver. Lack of this enzyme results in severe jaundice and kernicterus (bilirubin deposits in the brain). As liver structure and other functions are normal in subjects with Crigler-Najjar syndrome type I, replacement of only hepatocytes with normal enzyme activity can theoretically cure the disorder. In this case report, hepatocyte transplantation was performed in a 10 year-old girl with Crigler-Najjar syndrome type I. Hepatocytes from a normal donor were infused into the portal vein (the major vein that leads to the liver) of the recipient who received immunosuppresive therapy with tacrolimus and corticosteroids. The s serum bilirubin concentration of the recipient decreased from greater than 25 mg/dl to between 10 mg/dl and 14 mg/dl. Prior to hepatocyte transplantation, essentially only unconjugated bilirubin was present in the bile. Afterwards, 33% of the bilirubin pigments in bile were glucuronides. These improvements lasted for more than 11 months. This case report shows that hepatocyte transplantation is safe in humans and can partially correct the defect in Crigler-Najjar syndrome type I.

Sokal, E. M., Conjeevaram, H. S., Roberts, E. A., Alvarez, F., Bern, E. M., Goyens, P., Rosenthal, P., Lachaux, A., Shelton, M., Sarles, J., and Hoofnagle, J. 1998. Interferon alfa therapy for chronic hepatitis B in children: a multinational randomized controlled trial. Gastroenterology. 114:988-995.

Interferon alpha is effective in the treatment of chronic hepatitis B in adults and is approved for this indication in the United States and several other countries. Its safety and efficacy has not been as carefully evaluated in children. This trial at several centers in Canada, Europe and the United States examined the use of interferon alpha in children between the ages of 1 and 17 years with chronic hepatitis B. Subjects were randomized to either 24 weeks of treatment with interferon alpha-2b (6 million units per square meter three times a week) or to observation (there was no placebo control). All subjects had detectable serum hepatitis Be antigen (HBeAg) and viral DNA which are indicative or virus replication. No children had cirrhosis and only a few had significant fibrosis on liver biopsy. Treated subjects were evaluated 24 weeks after stopping interferon alpha and control subjects after 48 weeks of observation. Of 149 patients enrolled, 144 were ultimately available for evaluation (70 treated and 74 observed). Serum HBeAg and viral DNA became negative in 26% of treated and 11% of observed children (P < 0.05). Loss of HBeAg has been associated with a significantly improved prognosis in adults. Hepatitis B surface antigen became negative (most likely indicative of viral clearance and cure) in 10% of treated children and 1% of controls. Pretreatment and posttreatment liver biopsies were only available from 10 subjects and suggested improvement in histology. Most adverse events were described as mild or moderate and dose reductions were required in 24% of the treated children. These results show that interferon alpha treatment of children promotes clearance of markers of virus replication and hepatitis B surface antigen.

Kasahara, A., Hayashi, N., Mochizuki, K., Takayanagi, M., Yoshioka, K., Kakumu, S., Iijima, J., Urushihara, A., Kiyosawa, K., Okuda, M., Hino, K., Okita, K., and the Osaka Liver Disease Study Group. 1998. Risk factors for hepatocellular carcinoma and its incidence after interferon treatment in patients with chronic hepatitis C. Hepatology. 27:1394-1402.

Individuals with chronic hepatitis C can develop cirrhosis and hepatocellular carcinoma. This study prospectively followed 1,022 subjects in Japan with chronic hepatitis C for the development of hepatocellular carcinoma after treatment with interferon. After treatment with an average cumulative dose of interferon of approximately 500 million units, 313 subjects had sustained normal serum alanine aminotransferase (ALT) activities (sustained responders), 304 subjects had normal serum ALT activities during treatment but elevated activities after stopping interferon (transient responders) and 405 had no response to interferon treatment (non-responders). Subjects were followed every 3 to 6 months by ultrasonography after stopping interferon. After a median follow-up of 36 months (13 to 97 month range), hepatocellular carcinoma was detected in 5 sustained responders, 9 transient responders and 32 non-responders. Cox regression analysis showed that the risk of developing hepatocellular carcinoma was 7.90-fold in sustained responders compared to non-responders. Older age (greater than or equal to 55 years) and male sex were also significantly associated with the development of hepatocellular carcinoma. This study suggests that non-response to interferon treatment, older age and male sex are risk factors for the development of hepatocellular carcinoma in patients with chronic hepatitis C. Prior to treatment, only about 3% of subjects in this study had cirrhosis on liver biopsy and it is likely that carcinoma developed mostly in those who advanced to cirrhosis over the next several years. A somewhat reassuring result is that hepatocellular carcinoma developed in only 4.5% of individuals with chronic hepatitis C who were followed in this study.

Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, A. F., and Meier, P. J. 1998. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. Journal of Biological Chemistry. 273:10046-10050.

Bile salts are secreted from hepatocytes, the principal cell type in the liver, into the small intrahepatic bile ducts. An active transport protein for this process is presumably located on the apical or canalicular membrane of the hepatocytes. This paper describes the cDNA cloning of sister of P-glycoprotein from rat liver. By injection of RNA into frog oocytes, the investigators show that sister of P-glycoprotein functions as an ATP-dependent bile salt transporter. Sister of P-glycoprotein is predominantly expressed in the liver where the authors showed by immunofluorescence and immunoelectron microscopy that it is localized to canalicular microvilli and subcanalicular vesicles of hepatocytes. This study demonstrates that sister of P-glycoprotein is very likely a liver canalicular bile salt export pump. Recently, a gene encoding a different (P-type) ATPase called FIC1 was shown to be mutated in individuals with benign recurrent intrahepatic cholestasis and progressive familial intrahepatic cholestasis type 1 (see Bull et al. Nature Genetics. 1998;18:219-224 in the March, 1998 Current Papers in Liver Disease). The exact roles played by sister of P-glycoprotein, FIC1 and possibly other molecules in the secretion of bile acids and the pathophysiology of various inherited cholestatic disorders remain to be established.

Copyright, 1998, Howard J. Worman, M. D. All rights reserved. Printing or other reproduction is prohibited without the written authorization of Howard J. Worman.