Hepatitis B virus DNA in primary hepatocellular carcinoma tissue.

Tumour, cirrhotic, and metastatic tissues from four patients with primary hepatocellular carcinoma have been investigated for the presence of hepatitis B viral DNA by nucleic acid hybridization. Tumours from two of three patients with a current HBV infection contained 1--2 genomes per cell of unintegrated viral DNA, while tumours from the third HBs antigen-positive patient contained less than one genome equivalent per ten cells. A tumour from one patient with anti-HBs contained no detectable HBV DNA. A variety of models involving HBV as an etiologic agent may be advanced to explain the statistical correlation of HBV infection with primary hepatocellular carcinoma (PHC). The data presented here argue against the model that HBV DNA integrated into every cell is required to maintain the oncogenic transformation of hepatocytes, but they do not rule out other models.     

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.     

Hepatitis B virus infection and primary hepatocellular carcinoma.

For many years, epidemiological studies have demonstrated a strong link between chronic hepatitis B virus (HBV) infection and the development of primary hepatocellular carcinoma (PHC). Other hepatocarcinogens such as hepatitis C virus and aflatoxin also contribute to hepatocarcinogenesis either in conjunction with HBV infection or alone. Cellular and molecular biological studies are providing explanations for the HBV-PHC relationship, and models are now being formulated to further test the relative importance of various factors such as viral DNA integration, activation of oncogenes, genetic instability, loss of tumor suppressor genes, and trans-activating properties of HBV to the pathogenesis of PHC. Further research will probably define more than a single mechanism whereby chronic HBV infection results in PHC.     

Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus.

We report the temporal patterns of various immunohistological and serological parameters of acute self-limited hepatitis B virus infection of two chimpanzees, and we provide evidence that the synthesis of hepatitis B core antigen precedes that of hepatitis B surface antigen. Our data suggest that existence of a biphasic hepatitis B virus infection involving a hematogenous reinfection of the liver and indicate that recruitment of liver cells to produce hepatitis B virus may occur in a pattern consistent with a replicative cycle of about 8 days.     

Hepatitis B and primary cancer of the liver: recent data on the role of B virus in oncogenesis]

Although epidemiologic studies have clearly demonstrated the importance of the hepatitis B virus in the genesis of hepatocellular carcinoma, the molecular basis for this tumorigenic effect is still under debate. Studies of woodchucks infected with a virus closely related to the human hepatitis B virus suggest that integration of the viral DNA in the host genome often plays a direct role by activating myc cellular oncogenes through insertional mutagenesis. A similar mechanism involving other cell genes has been found less frequently in human hepatocellular carcinomas. The human hepatitis B virus may contribute to tumorigenesis in a more indirect fashion, by inducing preneoplastic liver lesions which gradually become malignant.     

Comparison of direct immunofluorescent staining of clinical specimens for respiratory virus antigens with conventional isolation techniques.

Staining of clinical respiratory specimens obtained by nasopharyngeal and throat swabs with direct fluorescein isothiocyanate-conjugated antisera to para-influenza virus types 1, 2, and 3, influenza A virus, respiratory syncytial virus, adenovirus, mumps virus, and measles virus was compared with isolation procedures in routine tissue culture systems. Direct staining of clinical specimens with the commercially available conjugated offered a rapid means of diagnosing respiratory infections on a routine clinical basis when used in conjunction with isolation in tissue culture systems. Of 292 patients who were culture positive for these viruses, 259 were diagnosed by detection of the viral antigen in clinical specimens by direct immunofluorescence. No specimens that subsequently yielded a different respiratory virus by culture were positive by the direct immunofluorescence method. Use of selected antisera based on clinical history of the patient reduced the cost of rapid viral diagnosis.     

Hepatitis B core and surface antigens in liver tissue. Light and electron microscopic localization by the peroxidase-labeled antibody method.

Hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) were localized in human liver tissues by the peroxidase-labeled antibody method at the light and electron microscopic levels. Several methods of fixation, staining, and inhibition of endogenous peroxidase activity were studied. The periodate-lysine-paraformaldehyde fixative effectively preserved the tissue structure and the antigenicity of both antigens, and the peroxidase-labeled Fab' fraction of IgG penetrated well into hepatocytes. HBcAg was present in nuclei, or cytoplasm of hepatic cells, or both. In nuclei, the antigen was found both in virus-like particles of approximately 20 nm. diameter and in nuclear ground substance. In the cytoplasm, the antigen was found on membrane-bound ribosomes and free polysomes, and also in the ground substance of the cytosol near ribosomes and around nuclear membranes, especially near nuclear pores. HBcAg-positive virus-like particles were also demonstrated sparsely or in clusters in the cytoplasm. HBsAg was not present in nuclei but was found in the perinuclear space and in cisternae of endoplasmic reticulum, and on nuclear, endoplasmic reticulum, and cell membranes of hepatic cells. HBsAg-positive 25- to 30-nm. wide tubular forms, round particles (probably cross-sections of tubular forms), and a few large particles of 40 to 50 nm. diameter were seen in cisternae. Such HBsAg-positive particles were also present in the intercellular space and in Disse's space. These findings suggest that HBcAg produced on the cytoplasmic ribosomes migrates through nuclear pores to the nucleus and is assembled into core particles there. These particles may then move through nuclear pores to the cytoplasm where they are invested with HBsAg-positive membrane in cisternae of endoplasmic reticulum or as they enter the endoplasmic reticulum. These virus particles are then released together with other HBsAg-positive forms into the intercellular space by reversed phagocytosis.     

Hepatitis B antigen in the liver cells in cirrhosis and hepatocellular carcinoma.

The demonstration of hepatitis B antigen in the liver cells in formalin fixed paraffin embedded tissues by Shikata's orcein staining method affords an opportunity to conduct retrospective studies on necrospy materials. Such a study in Singapore showed orcein-positive liver cells in 22 out of 52 (42.3%) and 37 out of 50 (74.0%) cases of cirrhosis of the liver and hepatocellular carcinoma respectively, while only 5 out of 113 (4.4%) 'normal' livers gave positive results. There is a significant difference in the frequency of hepatocellular carcinoma in orcein-positive and orcein-negative cirrhotic livers (28 out of 50, 10 out of 40 respectively). These results suggest a possible aetiological relationship between hepatitis B antigen and hepatocellular carcinoma.     

Hepatitis B virus core-related antigens as markers for monitoring chronic hepatitis B infection

A sensitive chemiluminescence enzyme immunoassay has been developed for hepatitis B virus (HBV) core-related antigen (HBcrAg) detection. We aimed to investigate the usefulness of HBcrAg measurement for monitoring chronic hepatitis B disease. HBcrAg levels were measured by a chemiluminescence enzyme immunoassay in 54 untreated patients and 39 patients treated with either entecavir or lamivudine. The HBcrAg concentration correlated positively with the levels of serum HBV DNA (r = 0.820), intrahepatic total HBV DNA (r = 0.700), and covalently closed circular DNA (cccDNA) (r = 0.664; for all, P values were <0.001). A higher HBcrAg concentration was associated with a greater proportion of hepatitis B core antigen immunostaining. Although the differences were not statistically significant, patients with higher Knodell necroinflammation and fibrosis scores tended to have higher serum HBcrAg concentration levels. In the treated patients, the logarithmic reduction in HBcrAg at week 48 correlated positively with the logarithmic reduction of serum HBV DNA, intrahepatic total HBV DNA, and cccDNA. Of the 31 patients with undetectable serum HBV DNA (<300 copies/ml) at the end of treatment, 20 (65%) still had detectable HBcrAg. A greater reduction in posttreatment HBcrAg concentration was associated with histological improvement and a decrease in hepatitis B core antigen immunostaining. HBcrAg concentrations of <40,000 kU/ml at baseline and <200 kU/ml at week 24 were associated with a higher chance of having undetectable HBV DNA at week 48. In conclusion, serum HBcrAg levels correlated with HBV virological markers and reflected the chronic hepatitis B disease activity in the liver.     

Assessment of virus infectivity by the immunofluorescent and immunoperoxidase techniques.

The immunofluorescent and immunoperoxidase cell-counting techniques were comparable in precision and reproducibility for the quantitative assessment of influenza virus infectivity. The dose-response function was linear with each procedure, and comparable results were obtained for estimating neutralizing antibodies in antiviral serum.     

Hepatitis B virus infection in dentists.

To evaluate viral hepatitis as a hazard in general dentistry, we surveyed participants in an annual health-screening program at the 1972 American Dental Association session. Of 1245 practitioners, 0.9 per cent were positive for hepatitis B surface antigen, and 12.7 per cent were antibody positive. Of those who had had clinical hepatitis while studying or practicing dentistry, 43 per cent were seropositive. The frequency of evidence for prior infection with hepatitis B virus increased uniformly with increasing years of professional experience. The proportion of seropositive dentists did not vary with geographic region of the United States, or size of community. Only 10.5 per cent recognized illicit self-injection among patients, and their infection rate was not increased. These data indicate an increased frequency of infection with hepatitis B virus among general dentists, and are compatible with relatively uniform endemicity of subtype/ad strans of that agent in the general population for several decades.     

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.     

HBV体外感染卵巢颗粒细胞

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。