乙型肝炎病毒感染与DNA甲基化

乙型肝炎病毒(HBV)感染是导致肝癌的重要病因,但HBV感染与肝癌中DNA甲基化改变的研究较少。此文综述HBV感染导致全基因组低甲基化和抑癌基因启动子高甲基化的研究现况,为进一步深入探讨 HBV相关性肝癌的发病机制提供新的思路。     

Hepatitis B virus DNA is enriched in polymorphonuclear leukocytes

DNA hybridization and cell separation techniques were used to determine which blood components contained hepatitis B viral DNA sequences. Free monomer-length hepatitis B virus was found in large amounts in the polymorphonuclear leukocyte cell fraction in two of five HBsAG-positive patients. In these two patients, viral DNA sequences were not detected in the plasma or platelet fraction, whereas the mononuclear cell DNA contained small amounts of a 7.2 kb size unintegrated hepatitis B genome. These studies indicate that the major reservoir of unit-length viral DNA in the asymptomatic hepatitis B carriers studied here was in the polymorphonuclear leukocyte fraction. The basis for the presence of the viral DNA within these cells is presently unknown, but may relate to viral replication within, or phagocytosis of virus by, these cells.     

Hepatitis B virus DNA in peripheral-blood mononuclear cells in chronic hepatitis B after HBsAg clearance.

In this study, peripheral-blood mononuclear cells from patients with chronic hepatitis B and spontaneous or therapy-induced disappearance of HBsAg were examined for HBV DNA. Samples were evaluated by in situ hybridization and polymerase chain reaction both before and after clearance of HBsAg. By in situ hybridization, positive signals were observed in 2 of 13 samples collected after HBsAg loss, in 8 of 15 samples before HBsAg loss and in 0 of 4 control patients without serological markers of active or prior HBV infection. When polymerase chain reaction analyses were performed, HBV DNA was detected in 5 of 12 HBsAg-negative samples and 10 of 15 HBsAg-positive samples from the study group. Testing of mononuclear cells after disappearance of HBsAg revealed that two of eight patients were HBV DNA positive by in situ hybridization and by polymerase chain reaction, whereas two additional patients were positive by polymerase chain reaction alone. Mononuclear cell-associated HBV DNA was detected between 2 and 9 mo after the disappearance of circulating HBsAg by in situ hybridization and as long as 4 yr later by polymerase chain reaction. These data indicate that patients who have undergone HBsAg seroconversion may nonetheless harbor HBV DNA in their peripheral-blood mononuclear cells for prolonged periods.     

Hepatitis B virus DNA in serum from patients with acute hepatitis B

Sera from 77 consecutive patients with acute type B hepatitis were examined for hepatitis B virus DNA (HBV DNA) by a spot hybridization method. The median follow-up time was 8 months (range, 1 week to 3 years). HBV DNA was detected in 26 (34%) patients on admission to the hospital. A significant positive correlation was found between short duration of symptoms and the presence of HBV DNA (p less than 0.025). Twenty-four (46%) of 52 HBeAg-positive patients were HBV DNA positive compared to 2 HBV DNA-positive patients of 25 HBeAg-negative patients (8%) (p less than 0.001). Four HBeAg-negative patients had serum HBV DNA initially or during follow-up; three had anti-HBe. Six of 77 patients with acute type B hepatitis (8%) became chronic HBsAg carriers, and HBV DNA was detectable from 5 months to more than 3 years after onset of symptoms. The presence of serum HBV DNA for more than 8 weeks after initial symptoms may predict development of a chronic HBsAg carrier state. In none of the chronic carriers was serum HBV DNA present after clearing of HBeAg.     

Hepatitis B virus DNA in the hepatocyte. A series of 160 biopsies

DNA-DNA hybridisation was used to examine 160 liver biopsies for the presence of the hepatitis B virus genome. HBeAg-positive HBsAg carriers were found to have replicating viral DNA in the hepatocytes and, very occasionally, HBV DNA was also integrated into the chromosomes. A high proportion of the anti-HBe-positive HBsAg carriers also have replicating HBV DNA and in the remainder integrated sequences are often, but not always, seen. No evidence was found, however, to implicate HBV in HBsAg-negative patients with alcoholic liver disease, nor in patients with a variety of other liver diseases including non-A, non-B hepatitis.     

Hepatocellular carcinoma with normal adjacent liver. Hepatitis B virus DNA status

We investigated hepatitis B virus (HBV) DNA status in the liver of 22 patients with hepatocellular carcinoma (HCC) developed on a non-cirrhotic, histologically normal liver tissue. HBV serological markers were present in 2 of the 22 subjects. HBV DNA sequences were identified in the liver of only 5 of the 22 patients with HCC. Evidence for clonal expansion of HBV-infected cells was found for one HBsAg-positive subject. This study indicates a much lower rate of HBV DNA positivity in the group of HCC developed on histologically normal livers as compared to that observed in HCC with liver cirrhosis.     

乙型肝炎病毒感染与肝细胞癌DNA甲基化研究进展

长期慢性乙型肝炎病毒（HBV）感染是肝细胞癌（HCC）发生的主要危险因子之一，近年来研究发现HCC的发生发展与表遗传学特别是抑癌基因启动子区CpG岛高甲基化关系密切，其发生可能是一个早期分子事件，具有基因特异性和肿瘤特异性，还可对肝癌的早期诊断和病情预后的监测及其防治具有重要意义。本文就HBV感染与HCC相关抑癌基因甲基化的研究现状作一综述。     

Hepatitis B virus

Recent developments in molecular biology have advanced our understanding of the pathogenesis of HBV-induced disease. New data derived from the molecular analysis of clinical material have begun to bridge the gap between bench research and the clinical arena. In this review, we consider topics that have relevance to clinical management and that have not been summarized in the recent literature. The recent advances that have been made in the areas of HBV variants,in vitro cell culture systems, and extrahepatic infection are discussed in greater detail.Supported by grants from the American College of Gastroenterology (B.Y.), Gulf Coast Regional Blood Center (C.N., B.Y.), and Schering Corporation (B.Y.).     

Multiple myeloma associated with Kaposi sarcoma.

A patient with multiple myeloma (MM) who developed Kaposi sarcoma (KS) is described. The KS appeared 18 months after the diagnosis of MM and 1 month after the treatment was changed from cyclophosphamide to melphalan. The treatment with melphalan was discontinued and the spread of the KS was arrested by irradiation and bleomycin. One month after the melphalan was restarted, the KS advanced. The patient died 28 months after the diagnosis of MM and 10 months after KS had developed. The association of KS and MM is discussed and the previously reported cases are reviewed. A possible connection between the treatment with melphalan and the development of KS is proposed.     

Hepatitis B virus DNA (HBV-DNA) in anti-HBe positive sera

HBV-DNA measured by the spot hybridization technique, was found in the sera of 28 of 106 (26.4%) anti-HBe positive carriers of HBsAg. Dane particle-associated HBeAg, HBcAg and HBV-specific DNA-polymerase activity were found in the sera of nine (8.5%), five (4.7%) and two (1.9%) of these patients, respectively. All carriers with serum HBV-DNA had chronic liver disease and 18 had intrahepatic delta-Ag and serum anti-delta at titers higher than 1/5000. Intrahepatic HBcAg was detected in the nuclei of 90% of delta negative individuals; 50% of them also had cytoplasmic fluorescence. Only two of the 18 patients with intrahepatic delta-Ag (11%) had HBcAg in the liver. Viral nucleic acid was not found in the sera of 15 other patients with chronic hepatitis, seven of whom had intrahepatic delta-Ag. Serum HBV-DNA was also negative in the remaining 63 symptomless carriers of HBsAg lacking markers of delta infection. Interestingly, although DNA-polymerase negative, some sera gave autoradiographic spots of high optical density. HBV-DNA was detected in them at concentrations typical of sera which are usually both DNA-polymerase and HBeAg positive. Detection of HBV-DNA in serum represents the most direct and sensitive in vitro assay for assessing HBV infectivity and characterizes HBsAg carriers with HBV-related liver damage and ongoing HBV replication independently from the state of HBeAg/anti-HBe system. In the Mediterranean area, the majority of anti-HBe positive carriers with serum HBV-DNA have chronic liver disease and delta infection.     

Hepatitis B virus DNA in human hepatocellular carcinoma: is the integration of hepatitis B virus DNA really carcinogenic?

Hepatitis B virus-specific DNA sequences (HBVDNA) in the liver were examined in 19 patients with hepatocellular carcinoma (HCC), 5 patients with liver cirrhosis (LC) and without HCC, 3 patients with chronic hepatitis (CH), 2 patients with metastatic liver cancer (MLC), 1 patient with primary biliary cirrhosis (PBC) and 4 patients with normal liver (NL) by the Southern blot hybridization procedure. Integration of HBVDNA was found in all 4 HCC patients with serum HBsAg, of whom one patient had HBVDNA only in the non-tumor (cirrhotic) region. Integration of HBVDNA was also detected in 4 of 8 HCC patients without serum HBsAg but with serum HBV-related antibodies, and in 2 HBsAg-positive patients of 5 LC patients. All 3 HBsAg-positive CH patients had only extrachromosomal HBVDNA. No HBVDNA was detected in the other 21 patients. Although integration of HBVDNA was observed in HBsAg-positive HCC patients with a higher frequency, integrated HBVDNA could also be detected in non-tumor regions of HCC patients and cirrhotic livers without HCC. It was concluded from these observations that integration of HBVDNA was frequently associated with HCC but might not have a direct causal effect on hepatocarcinogenesis even in HBV carriers.     

Hepatitis B virus DNA prediction rules for hepatitis B e antigen-negative chronic hepatitis B

After hepatitis B e antigen (HBeAg) seroconversion, hepatitis B may become inactive or progress to HBeAg-negative hepatitis with persistent or intermittent alanine aminotransferase (ALT) elevation. The aim of this study was to prospectively identify factors predictive of the clinical course in HBeAg-negative chronic hepatitis B (CHB). Patients were stratified by ALT and HBeAg status and followed every 3 months for up to 5 years. Kaplan-Meier and Cox regression analysis using the change from normal ALT to elevated ALT as endpoints were performed to determine factors associated with ALT elevation/normalization. Seventy-four HBeAg-negative and 32 HBeAg-positive patients were prospectively evaluated. For HBeAg-negative patients, hepatitis B virus (HBV) DNA was predictive of future ALT. Only 1 patient with normal ALT and an HBV DNA value lower than 10,000 copies/mL developed an elevated ALT within the subsequent year, whereas 67% with an HBV DNA value greater than 100,000 copies/mL had a rise in ALT above normal within 1 year. Patients with a previous history of ALT elevation and longer follow-up at all levels of HBV DNA were more likely to experience ALT elevations. For HBeAg-negative patients with elevated ALT and all HBeAg-positive patients, HBV DNA did not predict future ALT. Other viral and host factors were not predictive of future ALT. CONCLUSION: HBeAg-negative CHB has a fluctuating course. HBV DNA values lower than 10,000 copies/mL predict persistently normal ALT for at least 1 year. Patients with HBV DNA values between 10,000 and 100,000 copies/mL can safely be followed at 6 monthly intervals, whereas HBV DNA values greater than 100,000 copies/mL are highly predictive of future ALT elevation and should prompt regular follow-up.     

Solution structure of a Bcl-2 homolog from Kaposi sarcoma virus

Kaposi sarcoma-associated herpes virus (KSHV) contains a gene that has functional and sequence homology to the apoptotic Bcl-2 family of proteins [Sarid, R., Sato, T., Bohenzky, R. A., Russo, J. J. & Chang, Y. (1997) Nat. Med. 3, 293-298]. The viral Bcl-2 protein promotes survival of infected cells and may contribute to the development of Kaposi sarcoma tumors [Boshoff, C. & Chang, Y. (2001) Annu. Rev. Med. 52, 453-470]. Here we describe the solution structure of the viral Bcl-2 homolog from KSHV. Comparison of the KSHV Bcl-2 structure to that of Bcl-2 and Bcl-x(L) shows that although the overall fold is the same, there are key differences in the lengths of the helices and loops. Binding studies on peptides derived from the Bcl-2 homology region 3 of proapoptotic family members indicate that the specificity of the viral protein is very different from what was previously observed for Bcl-x(L) and Bcl-2, suggesting that the viral protein has evolved to have a different mechanism of action than the host proteins.