Antigen In Liver Tissue Research Articles

Distribution of HLA-DR, HLA-DP, HLA-DQ Antigens in Liver Tissue from Patients with Primary Sclerosing Cholangitis

This study describes the distribution of the major histocompatibility class II antigens HLA-DR, HLA-DP, and HLA-DQ in liver tissue from eight patients with primary sclerosing cholangitis, by means of the immunoperoxidase technique on cut cryostat sections. HLA-DR was expressed on the bile duct epithelium and vascular endothelium from all patients. HLA-DP showed an expression much like HLA-DR on the bile duct epithelium, but the vascular endothelium mostly did not stain for HLA-DP. HLA-DQ was generally more weakly expressed, and only three of the patients expressed HLA-DQ on the bile ducts. None of the hepatocytes expressed any of the MHC class II antigens.

Cancer occurrence in Vanuatu in the South Pacific, 1980-86.

A total of 269 pathologically confirmed carcinoma cases (118 male, 151 female) were recorded in the seven year period 1980 to 1986 in Vanuatu, an island nation within the Melanesia region in the South Pacific. Cervical cancer was numerically the most important malignancy in females (25% of all female cancers). In males, liver cancer was the most commonly observed (14.4% of all male cancers). Almost one-half (44.4%) of the available paraffin blocks from liver carcinoma cases (18 cases) demonstrated positivity of HBV antigens in liver tissue. The most interesting feature was the high proportion of thyroid cancers, especially in females. It represented 12.1 percent of all cancers in female and 5.2 percent in male Melanesians in Vanuatu. These percentages were found to be even higher than among Hawaiians for whom the highest incidence rates in the world have been recorded. Since our study was based solely on pathologically diagnosed cases, the findings should be regarded as minimum estimates.

Cytoplasmic localization of hepatitis B core antigen in hepatitis B virus infected livers

Routine use of commercially available antisera against hepatitis B core antigen (HBcAg) obtained from Escherichia coli transfected with HBV-DNA, has permitted a re-evaluation of the histochemical distribution of the antigen in liver tissue. HBcAg, classically described almost exclusively in the nucleus, was found with a very high frequency in the cytoplasm of liver cells. Our data indicate, however, that formalin fixation and paraffin embedding destroy part of HBcAg antigenicity and eliminate most of its cytoplasmic expression. HBcAg was found in 6/7 (85.7%) of HBsAg/HBeAg positive subjects, and in 2/12 (16/6%) of HBsAg/anti-HBe positive subjects; in both subgroups the cytoplasmic expression of the antigen correlated with the presence of circulating hepatitis B virus-deoxyribonucleic acid (HBV-DNA).

Immunohistochemical techniques for the demonstration of viral antigens in liver tissue.

Liver biopsy specimens from patients positive for serum HBsAg reveal various expression patterns when properly stained by immunohistochemical techniques for the demonstration of HBsAg, HBcAg and HDAg. A negative staining for these three antigens seems to be associated with two conditions, i.e., self-limiting acute lobular hepatitis (ALH) or low amounts of intrahepatic antigens. The discrepancy between serum positivity and tissue negativity for HBsAg can be explained either by sampling error or by the higher sensitivity of the radioimmunoassay as compared with immunohistochemical methods. The use of amplification systems such as the avidin-biotin-peroxidase complex enhances the sensitivity of immunohistochemical peroxidase-antiperoxidase (PAP) techniques and makes it possible to detect very small amounts of both HBsAg and HBcAg in liver cells from paraffin-embedded tissue sections which had been negative with the conventional PAP technique. In cases with a positive staining for viral antigens, two main expression patterns (non-aggressive and aggressive) can be distinguished. The non-aggressive pattern is reflected in the HBcAg-free HBsAg-positive type (HBsAg carrier) or the generalized type of nuclear core (HBcAg carrier), while the aggressive pattern is reflected in the presence of HDAg, the presence of HDAg and HBcAg, the focal type of nuclear core or the cytoplasmic HBcAg. Superinfection of HBsAg carriers or switching from generalized to focal core, with or without cytoplasmic expression of HBcAg, results in transition from non-aggressive to aggressive pattern. The aggressive pattern occurs in association with histological features of chronic active hepatitis (CAH). When it occurs in ALH cases or in milder forms of chronic hepatitis, an evolution into CAH has to be expected. Features of severe CAH, eventually with cirrhosis, are found in association with two new expression patterns: the cytoplasmic core and the simultaneous presence of HBcAg and HDAg. When features of CAH are observed in liver tissue specimens with HBcAg-free HBsAg-positive type, the liver disease may be due to viral superinfection or to non-viral etiology, e.g., alpha 1-antitrypsin deficiency.

Significance of Serum and Hepatic Markers of Hepatitis B Viral Infection in Hbsag–Positive and Hbsag–Negative Chronic Active Hepatitis

The correlation between serum and hepatic markers of hepatitis B virus (HBV) has been studied in 70 subjects with chronic active hepatitis of whom 18 were HBsAg+ and 52 were HBsAg-. In HBsAg+ subjects, sera were tested for HBeAg/anti-HBe status and for HBV DNA sequences using a DNA dot hybridization technique. Anti-HBs and anti-HBc were measured in serum in the HBsAg- group. Immunoperoxidase staining was used to detect HBsAg, HBcAg and delta antigen in liver tissue. Of the 18 HBsAg+ patients, 13 were HBeAg+ and 5 were anti-HBe+. A good correlation was shown between HBeAg and HBV DNA in serum and HBcAg expression in liver tissue. Neither HBV DNA in serum nor HBcAg in liver tissue was detected in any of the anti-HBe+ patients. HBsAg and/or HBcAg were detected in liver tissue in 17 of 18 HBsAg+ subjects (95%). However, neither HBsAg nor HBcAg were detected in liver tissue in 52 HBsAg- patients. This group included 11 patients with antibody markers in serum of past HBV infection. Thus, in contrast to previous studies, a good correlation was demonstrated between the serum and hepatic markers of viral replication, and no evidence was obtained to implicate the HBV as an etiological agent in HBsAg- chronic active hepatitis.

Production of monoclonal antibodies to hepatitis B surface and core antigens, and use in the detection of viral antigens in liver biopsies.

Hybridomas secreting monoclonal antibodies to HBsAg and HBcAg were prepared from immunized mice. An antibody capture radioimmunoassay was used to detect and select appropriate hybrids for propagation and cloning. The advantages of this assay were discussed. The resulting monoclonal antibodies were compared with conventional polyclonal antisera for the detection of virus antigens in liver tissue and found to give excellent results.

Hepatitis B virus antigens in liver tissue in hepatocellular carcinoma and advanced chronic liver disease, relationship to liver cell dysplasia

Hepatitis B surface (HBs) and core (HBc) antigens (Ag) were studied in liver tissue in HBsAg seropositive patients with chronic liver disease complicated (n = 32) and not complicated (n = 36) by hepatocellular carcinoma. Both groups were matched by age, sex and underlying disease. There was no qualitative and quantitative difference in tissue HBsAg between the two groups. However, HBcAg was significantly less in quantity in hepatocytes of patients with hepatocellular carcinoma compared to chronic liver disease without cancer. Serum hepatitis B e antigen tested by radioimmunoassay was also less frequently positive in patients with hepatocellular carcinoma. These findings seem to suggest that hepatitis B virus replication becomes less active in the process of hepatocarcinogenesis. The relationship between intrahepatic hepatitis B antigens and liver cell dysplasia was also studied. In hepatocellular carcinoma, tissue hepatitis B antigens often coexisted in the same liver having liver cell dysplasia, but no such association was observed in chronic liver disease without cancer. However, no indication was obtained that the dysplastic cells harbor HBsAg more frequently than non-dysplastic cells.

Clinical Application of Interferon

Human α and β interferon was evaluated as treatment for viral and malignant diseases.Lympocytes from patients with chronic hepatitis B have decreased activity to produce interferon stimulating in vitro. Five patients with chronic hepatitis B were injected daily with human α interferon, in doses ranging from 100, 000 to 1, 000, 000 units/day. Total amounts injected were from 15, 400, 000 to 96, 000, 000 units. DNA polymerase activity was measured in three from these five patients. After interferon treatment, DNA activity decreased in all these three cases. Hepatitis B antigen was assayed using RPHA method. In only one case, HBs Antigen became negative. HBc antigen in liver tissue became negative in one case examined immunofluorescence technique. Disappearance of e antigen was observed in two of five cases. Before and after treatment, liver biopsy was done. In two of these five cases, marked improvement (disappearance of cell necrosis) could be recognized. But suppressive effect wa transient. Interferon therapy was tolerated in all patients. Low grade fever developed during course of therapy. All cases had no elevation of transaminase by therapy. All five patients did not show bone marrow suppresion.Phase I study of human β interferon against malignant tumors (collaborative study) started recently. Eighteen cases already are checking about effect of interferon, only one case injected topically showed very remarkable diminution of size of tumor.

Evidence for hepatitis B and other virus infection in HBsAg-negative chronic active hepatitis.

Antibody to the hepatitis B core antigen (anti-HBc) was detected in sera from 6 (19%) of 32 patients with HBsAg-negative chronic active hepatitis. In three cases with the highest anti-HBc titers, core antigen was detected in liver cell nuclei and in one case HBsAg was also present in hepatocytes, suggesting continuing hepatitis B virus infection. During follow-up, anti-HBc titers fell slowly in those with no viral antigens in liver tissue, and in two cases with virus in the liver at presentation, viral antigens were no longer demonstrable four and eight years later. In one case, clearance of virus was preceded by the appearance of HBsAg in liver and serum, suggesting reactivation of viral replication. In three of the anti-HBc-negative cases a nuclear antigen unrelated to the hepatitis B virus was detected by immunofluorescence, and it is possible that liver disease in these patients may be related to persistent non-A, non-B virus infection.

Localization of Hepatitis A Antigen in Liver Tissue by Peroxidase-Conjugated Antibody Method: Light and Electron Microscopic Studies

Abstract Hepatitis A antigen (HAAg) was localized in liver tissue from marmosets inoculated with human hepatitis A virus (HAV) by light and electron microscopy by using a peroxidase-conjugated antibody method. The fine granular peroxidase staining was scattered throughout the cytoplasm of liver cells when viewed with the light microscope. The distribution of HAAg-positive cells was focal. Virus-like particles, 24 to 27 nm in diameter, were observed in the cytoplasm of hepatocytes and smaller cells, resembling Kupffer cells, by standard thin-section electron microscopy (thin section EM). By immunoperoxidase electron microscopy (immunoperoxidase EM), HAAg was detected on the particles, which were aggregated within cytoplasmic vesicles of the hepatocyte. The surrounding membrane of the vesicles was also HAAg-positive. Similar HAAg particles were observed in the cytoplasm of smaller cells adjacent to hepatocytes as well. Thus, immunoperoxidase EM revealed that the 24- to 27-nm virus-like particles in the cytoplasma of liver cells obtained from marmosets were infected with HAV contained HAAg.

Orcein staining of hepatitis B antigen in paraffin sections of liver biopsies.

Liver biopsies from 97 hepatitis B antigen (HBsAG)-positive patients were stained by a modified orcein method described by Shikata et al (1974) in order to detect the antigen in liver tissue. The results were consitently negative in acute hepatitis, but positive in nearly two-thirds of biopsies from 53 patients with chronic liver disease. The distribution of positive staining was frequently irregular so that there is a problem of sampling error in needle biopsies. The deposits were seen in the cytoplasm of liver cells and occasionally in Kupffer cells, but never in nuclei. There was an inverse relationship between staining and parenchymal necrosis. Biopsies from asymptomatic HB(s)Ag carriers were often strongly positive, as were "ground-glass" hepatocytes in carriers and patients with chronic liver disease. The mechanism of staining is unclear but may be related to the presence of disulphide bonds in (HBsAG. The technique is simple and of use both in fresh and stored material.

Evaluation of a modified fluorescent technique for the detection of Australia antigen in liver tissue

A modified fluorescent method is described for the detection of Australia antigen (AuAg, hepatitis associated antigen, hepatitis B antigen) in liver tissue and the results are compared with those obtained with the conventional technique. Of 84 unselected biopsies tested, 40 were positive by the conventional as well as by the modified method. Seven additional biopsies, negative by the conventional method, became positive by the latter technique. Moreover, the intensity of the fluorescence and the number of positive cells were clearly increased in 85% of the specimens which were positive by the conventional technique. The modified method proves to be more sensitive than the conventional immunofluorescent procedure.

The in vivo stability of antibody.

When rabbits were fed S(35)-labelled amino acids and simultaneously injected with antigen at the peak of antibody production, the circulating antibody became rapidly labelled within a few hours with the isotope. The specific radioactivity of antibody was measured as antibody was allowed to decline in the absence of antigenic stimulation. At various times, in different animals, antigen was reinjected and circulating antibody was measured for specific radioactivity. The initial antibody which appeared after the antigenic stimulus always had a higher specific activity than antibody circulating just prior to the reinjection. The appearance of antibody of higher specific activity was demonstrated to be a specific response to the antigen which was reinjected. It was concluded that there is a reservoir of antibody which is stabilized in tissue and which is not in equilibrium with that in circulation. A mechanism for this stabilization is suggested and discussed from previous investigations demonstrating the long retention of antigen in liver tissue.