Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma

Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB-adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC.     

Hepatitis B viral factors and clinical outcomes of chronic hepatitis B

Hepatitis B virus (HBV) infection is an important health problem and the major cause of chronic hepatitis, cirrhosis as well as hepatocellular carcinoma (HCC) worldwide. The natural history of chronic HBV infection can be divided into 4 dynamic phases in HBV carriers who acquire the virus early in life. In general, the frequency and severity of hepatitis flares in the immune clearance or reactivation phase predict disease progression in HBV carriers, and early HBeAg seroconversion typically confers a favorable outcome. In contrast, late or absent HBeAg seroconversion after multiple hepatitis flares accelerates the progression of chronic hepatitis to cirrhosis. Recently, several hepatitis B viral factors predictive of clinical outcomes have been identified. For example, serum HBV DNA level at enrollment is the best predictor of adverse outcomes (cirrhosis, HCC and death from liver disease) in adults with chronic HBV infection. In addition, HBV genotype C, basal core promoter (BCP) mutant and pre-S deletion mutant are associated with increased risk of HCC development. In conclusion, hepatitis B viral factors such as serum HBV DNA level, genotype and mutants have already been clarified to influence disease progression of chronic hepatitis B. Further studies are needed to investigate the pathogenic mechanism of each viral factor.     

Identification and extensive analysis of inverted-duplicated HBV integration in a human hepatocellular carcinoma cell line

Hepatitis B virus (HBV) DNA is often integrated into hepatocellular carcinoma (HCC). Although the relationship between HBV integration and HCC development has been widely studied, the role of HBV integration in HCC development is still not completely understood. In the present study, we constructed a pooled BAC library of 9 established cell lines derived from HCC patients with HBV infections. By amplifying viral genes and superpooling of BAC clones, we identified 2 clones harboring integrated HBV DNA. Screening of host-virus junctions by repeated sequencing revealed an HBV DNA integration site on chromosome 11q13 in the SNU-886 cell line. The structure and rearrangement of integrated HBV DNA were extensively analyzed. An inverted duplicated structure, with fusion of at least 2 HBV DNA molecules in opposite orientations, was identified in the region. The gene expression of cancer-related genes increased near the viral integration site in HCC cell line SNU-886.     

Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.     

Serum ficolin-2 concentrations are significantly changed in patients with hepatitis B virus infection and liver diseases

Human ficolin-2 is an important lectin complement pathway activator that is secreted from liver cells and has been implicated as an anti-infection innate immune molecule. However, the role of ficolin-2 protein and its dynamic changes over the course of and in the prognosis of chronic hepatitis B(CHB) and hepatocellular carcinoma(HCC) remain unclear. In this study, we analyzed ficolin-2 protein expression in a cohort of individuals with CHB infection, HCC and cirrhosis. A sandwich enzyme-linked immunosorbent assay(ELISA) method was used to measure serum ficolin-2 concentrations. Ficolin-2 expression in liver tissues was detected by immunohistochemical staining. Serum ficolin-2 concentrations in CHB patients were significantly higher than in healthy controls and HBV carriers. After 48 weeks of routine amelioration liver function treatment, serum ficolin-2 concentrations decreased and were positively correlated with favorable alanine aminotransferase(ALT), HBV DNA and HBe Ag-seroconversion outcomes. Interestingly, we observed much lower expression of serum and intrahepatic ficolin-2 in HCC and cirrhosis compared with healthy controls. Our findings suggest that serum and intrahepatic ficolin-2 levels may be considered one of the indicators for the response of chronic HBV infection, HCC and cirrhosis.     

Replication of hepatitis B virus which carries foreign DNA in vitro.

Targeting a specific DNA sequence to the desired tissues is an important step in gene therapy. The hepatitis B virus (HBV) is the only DNA virus that has hepatocyte specificity. We attempted to construct an HBV-based vector for targeting the liver. We observed the replication and secretion of virus particles in an HBV construct that lacks X gene and carries an extra 63 bp DNA fragment in vitro. Replication was observed in the cell line HuH-7 but not HepG2. From this construct, we designed an HBV-based vector that could carry foreign DNA. HBV based vectors provide for the possibilities of generating therapeutic agents for individual patients. Our host vector system may be used to clear out the HBV from the HBV carrier or chronic hepatitis B patients by introducing a genetically engineered HBV into these patients.     

Hepatitis B virus DNA integration and expression of an erb B-like gene in human hepatocellular carcinoma.

Southern blot studies on Hepatitis B Virus (HBV) DNA integration in 13 human hepatocellular carcinomas (HCCs) patients revealed the presence of several distinct HBV integration sites in different human liver disease patients. In one HCC patient the DNA fragment containing the HBV integration also hybridized to an erb B probe. The erb B/HBV co-migrating DNA fragment was cloned and sequenced, and showed that HBV DNA is integrated next to a cellular DNA fragment which is homologous to the tyrosine protein kinase domain of the human epidermal growth factor receptor gene and other cell surface receptor genes. The virus-integrated cellular DNA sequence is expressed in this HCC patient, suggesting a possible role for this gene in hepatocarcinogenesis.     

A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA.

BALB/c nude mice were injected intrahepatically with hepatitis B virus (HBV) DNA prepared from recombinant plasmids. Hepatitis B surface antigen appeared in the circulation in 19 of 23 mice (82%) 3 to 20 weeks postinjection and persisted for more than 6 months in most animals. Hepatitis B e antigen appeared transiently in the circulation in 12 of the 23 mice (52%) within a few weeks after the appearance of hepatitis B surface antigen. Antibodies to the core, X, and/or polymerase gene products of HBV have also been observed in 14 (61%) of the mice. Histopathological examination of the livers at 7 months postinjection demonstrated that nearly half had characteristics consistent with chronic hepatitis. HBV DNA appeared to be integrated into host liver DNA. No evidence of viral replication was observed in sera or livers from these mice at 7 months postinjection. These results demonstrate that an HBV chronic carrierlike state can be established in mice and that such a model could be used to study host and virus factors important in the establishment and maintenance of HBV-associated chronic liver disease.     

Mutation analyses of integrated HBV genome in hepatitis B patients

Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers （HBF） and their children. In this study, we used nest polymerase chain reaction （PCR）, fluorescence in situ hybridization （FISH）, and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells （PBMC） from 74 cases of HBFs and their children who were born after their fathers＇ HBV infection （caHBF）. We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B.     

Structural rearrangement of integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically infected hepatic tissues.

Cellular DNAs from human livers chronically infected with hepatitis B virus (HBV) were analyzed by Southern blot hybridization for the presence of integrated HBV DNA. In 15 of 16 chronically infected hepatic tissues, random HBV DNA integration was evident. By molecular cloning and structural analyses of 19 integrants from three chronically infected hepatic tissues, deletion of cellular flanking DNA in all cases and rearrangement of HBV DNA with inverted duplication or translocation of cellular flanking DNA at the virus-cell junction in some cases were noted. Thus, the rearrangement of HBV DNA or cellular flanking DNA is not a specific incident of hepatocellular carcinoma formation. Detailed analyses of various integrants bearing rearranged viral DNA failed to indicate any gross structural alteration in cellular DNA, except for a small deletion at the integration site, indicating that viral DNA rearrangement with inverted duplication possibly occurs before integration of HBV DNA. Based on nucleotide sequencing analyses of virus-virus junctions, a one- to three-nucleotide identity was found. A mechanism for this inverted duplication of HBV DNA is proposed in which illegitimate recombination between two complementary viral strands may take place by means of a nucleotide identity at the junction site in a weakly homologous region (patchy homology) on one side of adjoining viral sequences. For virus-cell junctions, the mechanism may be basically similar to that for virus-virus junctions.     

IL-23 production of liver inflammatory macrophages to damaged hepatocytes promotes hepatocellular carcinoma development after chronic hepatitis B virus infection

Liver inflammation after chronic hepatitis B virus (HBV) infection is essential for hepatocellular carcinoma (HCC) development. We did a nested case-control study based on QBC chronic HBV infection cohort to identify HCC-related inflammatory cytokines. Serum levels of distinct Th-cell representative cytokines at varied periods before HCC diagnosis were determined in 50 HCC cases and 150 age- and gender-matched controls who did not develop HCC in 8–10?years. The individuals with HCC outcome had statistically higher serum levels of IL-23 than controls (P?<?0.01). Further analysis in HCC tissues showed that CD14⁺ inflammatory macrophages were the major IL-23 producers. Monocytes-derived macrophages generated more amount of IL-23 after being stimulated with cell-associated HBV core antigen from damaged HBV-infected hepatocytes than the cells being stimulated with HBV-S and HBV e antigen, which are secreted from infected hepatocytes. IL-23 upregulated IL-23 receptor expressions on macrophages, enhanced macrophage-mediated angiogenesis. In HBV-transgenic (Alb1HBV) mice, administration of diethylnitrosamine induced more liver tumors than in wild-type mice. The livers of Alb1HBV mice had higher concentrations of IL-23 and vascular endothelial growth factor (VEGF) than the wild-type mice. Neutralizing IL-23 activity, diethylnitrosamine-treated Alb1HBV mice developed significantly less tumors and produced less VEGF, tumor angiogenesis was inhibited with dramatically decreased CD31⁺ cells within tumor mass (all P?<?0.01).

Distinct Distribution Pattern of Hepatitis B Virus Genotype C and D in Liver Tissue and Serum of Dual Genotype Infected Liver Cirrhosis and Hepatocellular Carcinoma Patients

Aims

The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.

Methods

The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.

Results

HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.

Conclusions

Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.     

Integration of HBV-DNA into liver and hepatocellular carcinoma cells during persistent HBV infection

The hepatitis B virus carrier state (persistent HBV infection) is characterized by the presence of viral surface antigen (HBsAg) and virion particles (Dane particles) in the blood. From 1% to 10% of carriers develop chronic liver disease and/or hepatocellular carcinoma. Recent studies have demonstrated integrated HBV-DNA in hepatocellular carcinomas and in several human hepatoma cell lines. In hepatoma patients, integrated HBV-DNA has been found in all HBsAg carriers. Nontumorous liver also revealed integrated HBV-DNA with the same or a different hybridization pattern from that observed in the tumor. To explore when integration occurs, carriers of short-term (less than 2 years) or long-term (greater than 8-10 years) were evaluated. DNA extracts from percutaneous (needle) liver biopsies showed free viral DNA with no specific integration bands in short-term carriers. In long-term carriers, HBV-DNA was integrated into the host genome with either a diffuse or a unique hybridization pattern. HBV-DNA integration correlated with the duration of the carrier state and absence of virions in the serum but did not correlate with histologic evidence of chronic hepatitis. These studies suggest that integration of HBV-DNA occurs during persistent HBV infection irrespective of liver disease and precedes development of hepatocellular carcinoma.     

乙型肝炎、肝硬变和肝癌中HBxAg的表达及其在肝癌发生中的作用

对３７９例良、恶性肝组织进行的免疫组织化学研究显示,３３％的慢性迁延性肝炎（６／１８）、７６％的慢性活动性肝炎（２６／３４）、９２％的肝硬变（５７／６２）和９７％的肝细胞性肝癌（ＨＣＣ）（５８／６０）中有ＨＢｘＡｇ表达,阳性率高于ＨＢｓＡｇ或ＨＢｃＡｇ。癌周肝中的ＨＢｘＡｇ阳性率显著高于非癌周肝。与其它２种ＨＢＶ抗原不同,ＨＢｘＡｇ表达在细胞类型上有较明显的选择性,在肝小多角细胞（ＳＰＬＣ）、小细胞性不典型增生（ＳＣＤ）及ＨＣＣ中较强。与ＩＧＦⅡ、ｃ－ｅｒｂＢ－２、ｃ－ｍｙｃ和ＥＧＦ－Ｒ表达进行的对照研究表明ＨＢｘＡｇ与ＩＧＦⅡ和ｃ－ｅｒｂＢ－２这２种ＨＣＣ发生相关基因的表达关系密切。ＰＣＮＡ染色结果显示ＨＢｘＡｇ阳性组织的细胞增殖活性显著高于ＨＢｘＡｇ阴性组织。我们的结果还表明ＨＢｘＡｇ表达与肝细胞不典型增生的发生和进展有关、提出ＨＢＶＸ基因可能通过其表达产物（ＨＢｘＡｇ）首先激活ＩＧＦⅡ、ｃ－ｅｒｂＢ－２基因,继而引起显著的ＳＰＬＣ增生和ＳＣＤ而参与ＨＣＣ发生的．     

Low frequency of precore mutants in anti-hepatitis B e antigen positive subjects with chronic hepatitis B virus infection in Chennai, Southern India

The natural course of chronic hepatitis B (CH-B) virus infection is reportedly variable, and the long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B infection are distinct from HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in the south Indian setting remain largely unclear. We prospectively studied 679 consecutive patients for HBsAg, HBeAg, anti-HBeAg, and HBV DNA by qualitative PCR. Randomly selected samples were subjected to bidirectional sequencing to reveal core/precore variants. Of the total 679 chronic HBV cases investigated, 23% (154/679) were replicative HBV carriers. Furthermore, amongst the 560 HBV DNA samples analyzed, 26% (146/560) were viremic. Among the 154 HBeAg positive cases, HBV DNA was positive in 118 cases (77%), significantly (p<0.001) higher than the anti-HBe positive (7%) (28/406) cases. Significant increase in liver disease (p<0.01) with ALT enzyme elevation (p<0.001) was observed in both HBe and anti-HBe viremic cases. Interestingly, low frequencies of mutations were seen in the precore region of the HBV strains studied. HBV precore and core promoter variants were less often detected in subjects with "e" negative chronic HBV infection and, therefore, may not have a prognostic role in determining liver disease sequelae in this part of tropical India.