Separate origins of hepatitis B virus surface antigen-negative foci and hepatocellular carcinomas in transgenic HBsAg (alb/psx) mice

We have examined the development and transgene expression in liver lesions of transgenic mice bearing the hepatitis B surface antigen (HBsAg) gene of hepatitis B virus under the control of the albumin promoter (alb/psx) to study liver regeneration and hepatocellular carcinoma (HCC) associated with hepatitis B virus infection. Storage of the HBsAg in the endoplasmic reticulum precedes loss of liver cells and regenerative hyperplastic nodules that do not express HBsAg. Histological analysis indicated that HBsAg-negative foci and nodules arose from liver progenitor cells in the portal zone and lacked mRNA expression. Genomic DNA from eight of nine HBsAg-negative laser capture-excised liver foci showed loss of part of the alb/psx gene, whereas no loss of the actin gene was observed. The alb/psx DNA was intact in adjacent HBsAg-positive tissue. Sequencing of polymerase chain reaction products suggested that alterations in the HBsAg transgene in HBsAg-negative foci occurred via large-scale deletions as opposed to single-site mutations. Southern blot analysis of HCC from 2-year-old transgenic HBsAg mice, however, revealed an intact alb/psx gene. Thus, HBsAg-negative progenitor cells with deletions in the transgene appear to be responsible for compensatory regeneration of the liver, whereas HCCs arise from clonal expansion of hepatocytes with intact alb/psx transgenes.     

Liver cell surface localization of hepatitis B antigen and of immunoglobulins in acute and chronic hepatitis and in liver cirrhosis.

This paper describes immunofluorescence studies on liver cell surface localization of hepatitis B surface antigen (HBsAg) and of IgG in acute and chronic hepatitis and in cirrhosis. In acute hepatitis B, HBsAg was found at the surface of hepatocytes in an early phase of the disease, but not during the recovery. This finding is consistent with the hypothesis that immune reactions to HBsAg may be responsible for the liver cell lysis. In HBsAg-positive chronic hepatitis and cirrhosis the antigen was found in the cytoplasm, but not on the surface of the hepatocytes, while in HBsAg-negative cases the antigen could not be detected in the liver cells. Both in HBsAg-positive and in HBsAg-negative chronic active hepatitis (CAH) and cryptogenic cirrhosis IgG bound to the membrane of the hepatocytes could be detected, suggesting a role of antibodies in the pathogenesis of the disease.     

Detection of a liver-membrane autoantibody in HBsAg-negative chronic active hepatitis.

Investigation of humoral immunity against hepatocellular membrane antigens in patients with chronic active hepatitis and other liver diseases showed two different immunofluorescence patterns of IgG on hepatocyte membranes. A linear pattern was seen in HBsAg-negative hepatitis, but HBsAg-positive cases and some of protracted, acute hepatitis B had a granular pattern. In patients with IgG bound to hepatocytes, continuing necrosis of parenchymal liver cells was seen. Conversely, hepatocytes without bound IgG were found in cases of chronic active hepatitis in remission, acute viral hepatitis without HBsAg and chronic persistent hepatitis, in "healthy" HBsAg-carriers and in patients with fatty liver or alcoholic cirrhosis. A liver-membrane autoantibody in serum, proved by fixation on membranes of isolated rabbit hepatocytes, could be demonstrated only in HBsAg-negative chronic active hepatitis with elevated IgG-concentrations. The results support the existence of different pathogenetic types of chronic active hepatitis, a so-called autoimmune type and a hepatitis virus-B-induced type.     

HBV-DNA hybridization in hepatocellular carcinoma associated with alcohol in Japan

To clarify the role of the hepatitis B virus (HBV) in hepatocellular carcinoma (HCC) associated with alcohol consumption, HBV-DNA in the liver of 19 patients with HCC were investigated. HBV-DNA was examined by Southern blot hybridization. HBV-DNA was integrated into tumor cells from five out of six (83%) patients with HCC associated with HBs antigen (HBsAg)-positive post-hepatitic liver cirrhosis (LC), but this was not related to the history of alcohol intake. In 13 HCC patients of HBsAg-negative alcoholic LC, HBV-DNA integration was not detected in any patient. These findings suggest that HBV does not play a major role in the pathogenesis of HCC in HBsAg-negative alcoholics in Japan.     

Localization of hepatitis B surface antigen in hepatocellular carcinoma

The author respectively examined the localization of hepatitis B surface antigen (HBsAg) in 150 autopsy cases of hepatocellular carcinoma (HCC) with particular attention to growth pattern of HCC. Varying numbers of orcein-positive cells were observed in 42 cases (28.0%) with various distribution patterns, and all the 42 cases were confirmed with immunoperoxidase technique for HBsAg. HBsAg-positive cells were detected in HCC tissue in 10 cases (6.6%) among the 150 cases. Regarding the growth pattern of HCC in the 10 cases, many HBsAg-positive cells were seen in HCCs of the sinusoidal and replacing types, in which the hepatocytes were frequently retained in the cancerous tissue, particularly around the tumor-nontumor boundary. On the other hand, there was no HBsAg-positive cell in the encapsulated type HCC which was the most common (approximately 50%) in the present study and in which the retained hepatocytes were hardly seen. Meticulous histological observation after decoloration of the positive reaction to HBsAg in the sections treated with immunoperoxidase technique and subsequent eosin stain disclosed that HBsAg-positive cells in HCC tissue were retained hepatocytes in 9 cases, and the possibility of HCC cell was not denied in one case. Moreover, HBsAg-positive cells were never detected in tumor thrombi of the portal vein branches and pulmonary metastases.     

Hepatitis B antigen in hepatocytes of chronic active liver disease

To study the morphologic interrelation of hepatocytes with the replication of hepatitis B vius (HBV) and immunocompetent cells in chronic active liver disease(CALD), organ cultures were prepared from liver biopsy specimens. Replication of hepatitis B core antigen (HBcAg) appears to occur in the nucleus of the hepatocyte in close association with intranuclear electron-dense strands and sometimes intranucleolar matrixes (likely HBcAg genomes), and cytoplasmic maturation of the HBcAg takes place in the preautolytic condition of host hepatocytes. Immunocompetent cells became progressively autolyzed in the early period of cultures. No difference in progression of hepatocyte injury in tissues from normal subjects and from hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients with CALD may suggest that intracellular synthesis of HBV alone is not cytopathic to host hepatocytes. This model is promising for the study of HBV replication and development, and also for testing the efficacy of new antiviral agents against the virus.     

Studies of lymphocyte subpopulations in the liver tissue and blood of patients with chronic active hepatitis (CAH)

Monoclonal antibodies to antigens on the surfaces of mononuclear cells (MNC) were used to characterize lymphocyte subpopulations infiltrating portal areas and parenchyma of livers in 31 patients with chronic active hepatitis (CAH). The distribution and numbers of infiltrating lymphocytes were determined in serial sections immunostained by the avidin-biotin-peroxidase complex method. T lymphocytes were the major component of inflammatory cells in the portal tracts. In the peripheral blood and portal areas, T helper-inducer (T4+) cells were the more numerous subpopulation. However, the hepatic lobules and areas of "piecemeal" necrosis always contained more T suppressor-cytotoxic (T8+) cells. The latter were demonstrated in contact with HBsAg-containing hepatocytes in tissues of patients with HBsAg-positive CAH. The mean numbers of T lymphocytes infiltrating the portal and periportal areas of livers from patients with HBsAg-negative and HBsAg-positive CAH were not different. Large numbers of B cells forming distinct follicles were seen in tissues from patients with HBsAg-positive CAH. The presence of increased numbers of portal T and B lymphocytes correlated with progressive liver damage as observed in two patients studied at yearly intervals.     

Status of hepatitis B virus DNA in hepatocellular carcinoma: a study based on paired tumor and nontumor liver tissues

To investigate the hepatitis B virus (HBV) DNA status in the liver when hepatocellular carcinoma (HCC) has developed, 35 paired nontumorous and tumorous liver tissues from 27 hepatitis B surface antigen (HBsAg)-seropositive and 8 HBsAg-negative patients with HCC were studied by Southern blot analysis. The hybridization patterns of HBV DNA were different in the nontumor and tumor parts in 26 (96.3%) of the 27 HBsAg-positive patients. In the nontumor parts, integration of HBV DNA into the host genome was significantly less when compared to the tumor parts (15/27 vs. 25/27, P less than 0.05), whereas free replicative viral forms were significantly more frequent (17/27 vs. 7/27). The integrated HBV DNA in the nontumor parts showed discrete band patterns in the majority of cases (13/15). Hepatitis B e antigen (HBeAg) was significantly associated with the expression of free replicative forms of HBV DNA in the tumor tissues. An integrated HBV DNA sequence was detected in the tumor part of one HBsAg-negative patient, but not in her nontumor counterpart. Our observation that discrete integrated HBV DNAs are present in the nontumor part, representing subclinical clonal expansion that precedes the development of HCC, suggests the risk of future new tumor growth from these cell clones.     

Anti-HBc IgM bei akuter und chronischer Hepatitis-B-Virus-Infektion

Summary Hepatitis B core antigen (HBcAg) synthesized in E. coli was used for determination of immunoglobulin M class-specific antibodies against HBcAg. It was found that 98% of cases with acute hepatitis B surface antigen (HBsAg) positive hepatitis type B were anti-HBc immunoglobulin M (IgM) positive. Atypical hepatitis B was detected in 33% of anti-HBc-positive HBsAg-negative cases with acute hepatitis. Anti-HBc IgM was positive for 6 months in acute resolving hepatitis type B, whereas cases resulting in chronic hepatitis B remained anti-HBc IgM-positive for up to 900 days. Chronic HBsAg carriers with severe liver disease had anti-HBc IgM more often than individuals with minor liver damage; 83% of HBsAg-positive liver cirrhoses, 63% of chronic aggressive hepatitis, 50% of HBsAg-positive liver carcinoma, but only 17% of chronic persistent hepatitis or 7% of healthy blood donors were anti-HBc IgM-positive. Determination of anti-HBc IgM is useful in detecting atypical hepatitis B virus infections without HBsAg in serum and, with some restrictions, in discriminating acute and chronic hepatitis type B.

     

Orcein-positive hepatitis B surface antigen and liver carcinoma: their association in an Eastern US population

We retrospectively examined the association of hepatitis B infection and hepatocellular carcinoma (HCC) in a US East Coast population using orcein staining of fixed liver tissue. Hepatitis B surface antigen (HBsAg) was present in non-neoplastic hepatocytes in eight of 53 cases of HCC, but in no cases of cholangiocarcinoma or metastatic tumor. In five of the eight positive cases, macronodular cirrhosis was present; in three positive cases, cirrhosis was absent. The rate of positivity in livers with both HCC and macronodular cirrhosis was 28%, compared with 4.7% in livers with macronodular cirrhosis but no carcinoma. The low, but significant association of HBsAg and HCC, both in the presence and absence of cirrhosis, suggests that HCC may develop in a subset of patients in the United States as a result of infection with hepatitis B virus.     

Methods for detection of hepatitis B surface antigen in paraffin sections of liver: a guideline for their use.

Methods for the localisation of hepatitis B surface antigen (HBsAg) in paraffin sections of the liver include the detection of ground-glass hepatocytes and the use of Shikata's orcein stain, and of immunoperoxidase and immunofluorescent techniques. A comparative study of the different methods on 20 livers shows the orcein stain to be the method of choice for routine use. The Shikata stain is not only specific but is relatively inexpensive, easily performed, and stains out distinct cytoplasmic inclusions even in stored formalin-fixed livers, old paraffin blocks, and autolysed livers. Since HBsAg is irregularly distributed in the liver, adequate sampling is necessary to prevent false negative; when sufficient tissue is available at least five blocks should be examined before a case is labelled as HBsAg-negative.     

Complexes between HBsAg and IgM in serum of patients with acute hepatitis

HBsAg bound to IgM was measured in the serum of HBsAg carriers with acute hepatitis using a radioimmunoassay based on selective absorption of IgM on solid phase coated with antiserum to human IgM. HBsAg/IgM was detected in 94 (100%) patients with acute type B hepatitis during the acute phase of infection and persisted after the fourth week only in 13 of them, who developed chronic liver disease. HBsAg/IgM was detected only in 1 patient out of 15 carriers of the HBsAg with superimposed non-B hepatitis. No activity was found in serum of 20 patients with acute HBsAg-negative hepatitis. The nature of the IgM component of the complex is uncertain, however, blocking experiments of the HBsAg/IgM reaction with polymerized human albumin suggest that the IgM component of the complex might represent antibody to the denatured protein. Persistent HBsAg/IgM complex detection in patients with acute type B hepatitis provides a useful tool to predict transition of HBV infection to chronicity. Its absence in patients with acute HBsAg-positive hepatitis is indicative of non-B hepatitis in chronic carriers of the HBsAg.     

Significance of anti-HBc IgM in the differential diagnosis of viral hepatitis

IgM antibody to hepatitis B core antigen (anti-HBc IgM) as determined by IgM capture immunoassay is generally present in high titer during acute hepatitis B infection. A strong positive reaction for anti-HBc IgM during acute hepatitis is indicative of an acute HBV infection even in hepatitis B surface antigen (HBsAg)-negative patients. With the help of anti-HBc IgM otherwise unidentified HBV infection can be diagnosed in HBsAg-negative patients and an optimal combination of diagnostic tests for acute hepatitis B infection would therefore include assays for both HBsAg and anti-HBc IgM. In the HBsAg carrier with or without chronic liver disease the presence and meaning of anti-HBc IgM is still a matter for discussion. Detection of a weak positive result for anti-HBc IgM in HBsAg-positive patients without a recent history of acute hepatitis cannot always be regarded as a definite marker of recent hepatitis B infection. However. quantitation of the anti-HBc IgM results seems to improve the clinical value of the test. Comparison of the available anti-HBc IgM assays is needed and may well establish a reliable cut-off level that would differentiate acute from chronic hepatitis B and ongoing from resolving hepatitis B in HBsAg-positive patients.     

Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53

Hepatitis B virus (HBV) genome was reported to be detected in serum or liver tissues in hepatocellular carcinoma (HCC) patients negative for hepatitis B surface antigen (HBsAg). Hepatitis B x (HBx) and p53 protein were reported to play an important role in HBV-related hepatocarcinogenesis. To clarify latent HBV infection in HBsAg- and anti-hepatitis C virus (anti-HCV)-negative HCC in a Japanese population and involvement of HBx and p53 protein in these patients, we performed the sensitive and specific nested polymerase chain reaction (PCR) and immunohistochemical analysis. Of 1,024 HCC patients we saw between 1974 and 1998, 66 (6.4%) were negative for HBsAg and anti-HCV. Serum DNA was amplified by nested PCR by using specific primers of surface (S), core (C) and X regions in 26 patients negative for HBsAg and anti-HCV. Eighteen (69%) patients were positive for either S, C, or X region and the results of PCR were confirmed by Southern blotting. Of 18 PCR-positive patients, 3 were positive for anti-HBs and 9 were positive for anti-HBc, however, one was negative for any HBV markers. In HBsAg-negative and PCR-positive patients, the positive rates of expression of HBx and p53 were 8/13 (62%) and 7/13 (54%), being comparable to those in HBsAg-positive HCC patients. The results of the present study suggest that high prevalence of HBV infection is observed in HBsAg-negative HCC in a Japanese population and expression of HBx and p53 is consistent with a role, in these patients, for the transforming ability of these proteins.     

LOCALIZATION OF HEPATITIS B SURFACE ANTIGEN IN HEPATOCELLULAR CARCINOMA

The author respectively examined the localization of hepatitis B surface antigen (HBsAg) in 150 autopsy cases of hepatocellular carcinoma (HCG) with particular attention to growth pattern of HCG. Varying numbers of orcein-positive cells were observed In 42 cases (28.0%) with various distribution patterns, and all the 42 cases were confirmed with immunoperoxidase technique for HBsAg. HBsAg-positive cells were detected in HCG tissue In 10 cases (6.6%) among the 150 cases. Regarding the growth pattern of HCG in the 10 cases, many HBsAg-positive cells were seen In HGGs of the sinusoidal and replacing types, in which the hepatocytes were frequently retained in the cancerous tissue, particularly around the tumor-nontumor boundary. On the other hand, there was no HBsAg-positive cell in the encapsulated type HGC which was the most common (approximately 50%) in the present study and in which the retained hepatocytes were hardly seen. Meticulous histological observation after decoloration of the positive reaction to HBsAg in the sections treated with immunoperoxidase technique and subsequent eosin stain disclosed that HBsAg-positive cells in HGC tissue were retained hepatocytes in 9 cases, and the possibility of HCC cell was not denied in one case. Moreover, HBsAg-positive cells were never detected in tumor thrombi of the portal vein branches and pulmonary metastases.     

HBsAg-negative chronic active hepatitis related to hepatitis B virus

Numerous cases of chronic hepatitis have been shown to be closely associated with persistent infection with hepatitis B virus (HBV).A group of 100 patients suffering from chronic active hepatitis (CAH) was investigated for HBV serologic markers. Of these, 35 patients were HbsA-gpositive; in 26 HBsAg-negative subjects, anti-HBc were detected using counterimmune electrophoresis and complement-fixation tests. These data suggest that chronic liver disease in patients who were only anti-HBc-positive might be related to a persistent infection with hepatitis B virus.Epidemiological, clinical and histopathological data were different when we compared CAH patients who were HBsAg-negative, but anti-HBc-positive, with HBsAg-positive CAH patients. A sequence is proposed leading from HBsAg-positive to HBsAg-negative CAH, cirrhosis, and hepatoma in temperate areas, according to a model similar to the one described in intertropical Africa.     

Expression of hepatitis B surface and core antigens and transforming growth factor-a in “oval cells” of the liver in patients with hepatocellular carcinoma

Recent studies have identified epithelial cell populations in human livers that are similar to the “oval cells” and “transitional cells” seen in rat livers during the early stages of chemical car-cinogenesis. It has been suggested that these cells might be precursors of hepatocytes and theoretically could be involved in hepatocarcino-genesis. The hepatitis B virus (HBV) also is believed to play a role in the etiology of hepatocellular carcinoma (HCC). Therefore, a study was conducted in nontumorous livers adjacent to HCCs obtained from 26 patients from China to determine whether HBV antigens could be identified in oval cells and transitional cells using an immunohistochemical technique. Hepatitis B surface antigen (HBsAg) was detected in the nontumorous livers of 22/26 (85%) patients. HBsAg was detected in oval cells in 18/26 (69%), in transitional cells in 21/26 (81%), and in mature hepatocytes in 22/26 (85%), but not in bile duct or ductule cells. Transforming growth factor-α (TGF-α) was expressed in oval cells, transitional cells, and bile duct cells in 24/26 (92%) patients, an in mature hepatocytes in 25/26 (96%). Coexpression of HBsAg and TGF-α was identified in the same cells in populations of oval cells and transitional cells of selected patients. Because of the possibility that oval cells could be a source of evolving HCC, these findings suggest that expression of TGF-α associated with HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. Thus, oval cells could be a site (or one of the sites) where HBV participates in the development of HCC. © 1994 Wiley-Liss, Inc.     

Molecular and serological aspects of HBsAg-negative hepatitis B virus infections in North America

A few hepatitis B virus (HBV) infections are characterized by the presence of HBV DNA in serum or liver tissue, or both, in the absence of detectable hepatitis B surface antigen (HBsAg) in serum. However, such infections have rarely been described previously in North American patients. In the present study, 31 hepatocellular carcinoma (HCC) patients from the United States and Canada who had no detectable HBsAg in their serum were studied. In these 31 HBsAg-negative HCC patients, HBV DNA was detected in HCC and/or in adjacent nontumorous liver tissue using nested polymerase chain reaction (PCR) in 5/9 (56%) patients from the United States and in 12/22 (55%) from Canada. The 17 HBV DNA-positive/HBsAg-negative patients from the United States and Canada included 9 without any serological markers for HBV and 8 with detectable antibodies to hepatitis B core antigen. In these patients, HBV genotype C was the most prevalent genotype (11/17; 64%). HBV genotypes have not been previously reported in HCC patients from North America. Replicative intermediate forms of HBV (covalently closed circular HBV DNA) were detected in 2/17 (12%) HBV DNA-positive/HBsAg-negative patients, indicating that at least two of these patients had actively replicating HBV infections. The use of tests to detect HBV DNA permitted the identification of HBV infections in HBsAg-negative HCC patients from North America. Among these patients, those with antibody to hepatitis C virus (HCV) would otherwise have been designated "HCV-associated HCCs" based on serological tests alone. These findings provide a new perspective on determining the possible viral etiologies of HCCs in North America.     

Different expression of hepatitis B surface antigen between hepatocellular carcinoma and its surrounding liver tissue,studied using a tissue microarray

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and is highly associated with chronic liver disease, including hepatitis B viral infection. In order to study the association between hepatitis B virus (HBV) infection and HCC development, tissue microarrays were used to detect the expression of hepatitis B surface antigen (HBsAg) in 194 HCCs and their surrounding liver tissues, using anti-HBsAg monoclonal antibody. The results showed that the expression of HBsAg is significantly lower in tumour tissue than in non-tumour tissue. Among the 138 cases with positive serum HBsAg, expression of HBsAg was more frequently detected in non-tumour tissue (103 cases, 75%) than in tumour tissue (11 cases, 8%). RT-PCR and Southern blot analysis were performed to explore the mechanism of the decreased expression of HBsAg in tumour cells. The RT-PCR results showed that absence or decreased expression of the HBV S gene was detected in 3/15 (20%) and 6/15 (40%) HCCs, respectively. Integration of HBV in 23 pairs of HCCs and their matched non-tumour liver tissues was studied by Southern blot. The results showed that the integrated HBV S gene sequence was detected in 19/23 tumours (83%) and 1/23 non-tumour tissues (4%), whereas the free replicative virus form was observed in 3/23 tumours (13%) and 14/23 non-tumour tissues (61%). These findings suggest that HBsAg-negative results in tumour tissues were directly related to HBV DNA insertion and provide new insights into the involvement of HBsAg in hepatocarcinogenesis.     

Hepatitis B virus antigens in liver resections from patients with hepatocellular carcinoma

A high rate of hepatitis B virus (HBV) antigen carriers among patients with hepatocellular carcinoma (HCC) has been recorded from areas of endemic HBV infections in Africa and Asia, but there are only rare and contradictory data for Europe. 12 specimens of resected liver tissue were immunohistologically investigated for hepatitis B surface antigen (HBsAg) as well as for hepatitis B core antigen (HBcAg). HBsAg was contained in non-tumorous liver tissue in 66 per cent of these cases. In two cases detection of HBcAg in the liver provided evidence to replication of the virus. HBcAg plus HBsAg were present in tumour tissue in one case. All of the HBV antigen carriers did not have chronic hepatitis. On the other hand, all patients with chronic hepatitis had HBV antigens in their liver tissue. HBV antigens were detectable in 7 non-cirrhotic livers, but were contained in only one of two cirrhotic livers. These results are likely to suggest a possible relevance of HBV infection to the aetiology of HCC even in central Europe without customary liver cirrhosis.