Bing-Fang Chen

Role of Hepatitis B Viral Load and Basal Core Promoter Mutation in Hepatocellular Carcinoma in Hepatitis B Carriers

BACKGROUND Several hepatitis B viral factors correlate with the progression of chronic liver disease. However, the independent and interactive effects of each known viral factor on the development of hepatocellular carcinoma (HCC) remain largely unknown. METHODS In a cross-sectional, retrospective, hospital-based setting, we comprehensively compared viral factors in 160 chronic hepatitis B virus (HBV) carriers and 200 patients with HCC, to clarify the independent and joint effect of each factor. RESULTS In univariate analysis, statistically significant odds ratios (ORs) were obtained for male sex (P < .001), advanced age (P < .001), HBV genotype C infection (P = .005), the precore A1896 mutation (P < .001), and the basal core promoter (BCP) T1762/A1764 mutation (P < .001). According to the results of multiple logistic-regression analysis, advanced age, male sex, the precore A1896 mutation, the BCP T1762/A1764 mutation, and an HBV load > or = 10(5) copies/mL were independently associated with the development of HCC. Compared with patients with an HBV load < 10(5) copies/mL and the BCP A1762/G1764 wild-type strain, the adjusted OR of developing HCC was > or = 30 in patients with an HBV load > or = 10(5) copies/mL and the BCP T1762/A1764 mutant, irrespective of the presence of the precore A1896 mutation and viral genotype. CONCLUSIONS HBV load and the BCP T1762/A1764 mutation are important in hepatocarcinogenesis.

Role of Hepatitis B Virus Precore/Core Promoter Mutations and Serum Viral Load on Noncirrhotic Hepatocellular Carcinoma: A Case-Control Study

BACKGROUND Apart from the presence of liver cirrhosis, hepatitis B virus (HBV) factors have also been shown to play a role in the development of hepatocellular carcinoma (HCC). Studying HBV-related noncirrhotic HCC may help clarify the effect of viral factors. METHODS In a hospital-based, age- and genotype-matched study, we aimed to determine the role played by basal core promoter (BCP) T1762/A1764 mutation, precore A1896 mutation, and serum viral load in noncirrhotic hepatocarcinogenesis by comparing 44 patients with HBV-related noncirrhotic HCC, 45 patients with chronic hepatitis B, and 42 patients with HBV-related cirrhotic HCC. HBV genotype, precore and BCP mutations, and viral load were determined by molecular assays. RESULTS In univariate analysis, statistically significant odds ratios were obtained for male sex (P=.005) and BCP T1762/A1764 mutation (P=.0003) in patients with noncirrhotic HCC, compared with patients with chronic hepatitis B. By multiple logistic regression analysis, male sex, BCP T1762/A1764 mutation, and viral load >or=10(5) copies/mL were independently associated with the risk of noncirrhotic HCC. The virologic characteristics were similar between patients with cirrhotic HCC and those with noncirrhotic HCC. CONCLUSIONS Our results suggest that BCP T1762/A1764 mutation and higher viral load may be involved in the carcinogenesis of cirrhotic and noncirrhotic HCC.

Application of Hepatitis B Virus Genotyping and Phylogenetic Analysis in Intrafamilial Transmission of Hepatitis B Virus

BACKGROUND Infection with hepatitis B virus (HBV) in early life frequently results in persistent infection, and clustering of the chronic infection within a family is common. However, the relative contribution of perinatal mother-to-infant transmission or early horizontal transmission to the intrafamilial clustering of HBV infection remains unclear. Therefore, we used HBV genotyping and phylogenetic analysis to elucidate the modes of intrafamilial HBV transmission in Taiwan. METHODS HBV genotypes and serological markers were determined for 103 individuals from 20 families with evidence of clustering HBV infection. RESULTS Three patterns of intrafamilial clustering of HBV infection were identified. Among the 20 families, 8 included a hepatitis B surface antigen (HBsAg)-positive mother (pattern I), 7 included an HBsAg-positive father (pattern II), and in the remaining 5, both parents were positive for HBsAg (pattern III). The rates of HBsAg positivity for children of the 3 representative groups of families were 85.7%, 65.4%, and 87.5%, respectively (P = .16). The identical genotyping results between index parent and carrier children indicated that pattern I clustering was caused by maternal transmission, whereas pattern II clustering was caused by paternal transmission. In pattern III clustering, a concordant HBV genotype between carrier children and mother or father was found in 3 and 2 families, respectively. The modes of transmission were confirmed by phylogenetic analysis in 1 family of each pattern. CONCLUSIONS In Taiwan, maternal and paternal transmissions are both important in the intrafamilial spread of HBV infection.

Fine mapping of hepatitis B virus pre-S deletion and its association with hepatocellular carcinoma.

Naturally occurring pre‐S deletion mutants have been identified in hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC).

Functional analysis of hepatitis B virus pre-s deletion variants associated with hepatocellular carcinoma

BackgroundNaturally occurring pre-S deletion mutants have been identified in hepatitis B carriers and shown to be associated with the development of hepatocellular carcinoma. The phenotypes of these pre-S deletion genomes remain unclear, and they were investigated in this study.MethodsThe pre-S deletion genomes: (1) pre-S1 deletion, (2) deletion spanning pre-S1 and pre-S2, (3) pre-S2 N-terminal deletion, and (4) pre-S2 internal deletion were constructed and analyzed by transfection into Huh-7 cells.ResultsFunctional analyses reveal that these mutants were divided into two groups: S promoter deletion and non-S promoter deletion variants. Compared with the wild-type genome, S promoter deletion variants led to an inverse ratio of pre-S1 mRNA and pre-S2/S mRNA, and intracellular accumulation of surface proteins. An interesting finding is that a small amount of L proteins was detected in the medium from S promoter deletion variant-transfected cells. Non-S promoter deletion variants conversely displayed a wild-type like mRNA and protein pattern. The secretion of surface proteins from non-S promoter deletion variants was inhibited less than from S promoter deletion variant. Immunofluorescence analysis showed mutant surface proteins colocalized with ER and exhibited an atypical distribution: granular staining pattern in the S-promoter deletion variants and perinuclear staining pattern in the non-S promoter deletion variants.ConclusionThis study shows that these pre-S deletion genomes exhibit two different phenotypes in mRNA transcription, surface protein expression and secretion. This diversity seems to result from the deletion of S promoter rather than result from the deletion of pre-S1 or pre-S2.

Potentiation of Lipopolysaccharide-Induced IL-6 Release by Uridine Triphosphate in Macrophages: Cross-Interaction with Cyclooxygenase-2-Dependent Prostaglandin E2 Production

Our previous study has demonstrated the potentiation by uridine triphosphate (UTP) of nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated murine J774 macrophages. In this study, we found that the amount of interleukin-6 (IL-6) release in response to LPS stimulation was greatly enhanced in the presence of UTP. This enhancement exhibited concentration dependence and occurred after 8 h of treatment with LPS. RT-PCR analysis indicated that the steady-state level of IL-6 mRNA induced by LPS was apparently increased upon co-addition of UTP. The potentiation by UTP was inhibited by the treatment with U73122 (a phosphatidylinositol-phospholipase C inhibitor), BAPTA/AM (an intracellular Ca2+ chelator), KN-93 (a selective inhibitor of calmodulin-dependent protein kinase) or PDTC (a nuclear factor κB inhibitor). To understand the cross-regulation among NO, PGE2 and IL-6, all of which are dramatically induced after LPS stimulation, the effects of L-NAME (a nitric oxide synthase inhibitor), indomethacin (a cyclooxygenase inhibitor), NS-398 (a cycloxygenase-2 inhibitor) and IL-6 antibody were tested. The results revealed the positive regulation between PGE2 and IL-6 synthesis because NS-398 and indomethacin inhibited LPS plus UTP-induced IL-6 release, and IL-6 antibody attenuated LPS plus UTP-induced PGE2 release. Taken together these results reinforce the role of UTP as a regulatory element in inflamed sites by demonstrating the capacity of this nucleotide to potentiate LPS-induced release of inflammatory mediators.

Naturally Occurring Hepatitis B Virus B-Cell and T-Cell Epitope Mutants in Hepatitis B Vaccinated Children

To control hepatitis B virus (HBV) infection, a universal HBV vaccination program for infants was launched in Taiwan in 1984. The aim of this study was to investigate the role of B-cell and T-cell epitope variations of HBsAg and polymerase in HBV infection in vaccinated children. One hundred sixty-three sera from vaccinated children were enrolled randomly. HBV serum markers, including hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) and core antigen (anti-HBc), were detected by ELISA. Nucleotide sequences encoding the S and the pre-S regions of HBsAg were analyzed in all HBsAg positive sera. Five children were HBsAg positive. Sequence analysis of S, pre-S, and overlapped polymerase (P) genes showed that HBV isolates of HBsAg-positive vaccinees were variants; no G145R but G145A and other substitutions were found in the “a” determinant. Fifteen, six, and eight amino acid substitutions within B-cell and T-cell epitopes of S, pre-S, and P regions were detected, respectively. Several immune-epitope mutants, such as S45T/A, N131T, I194V, and S207N in S, were detected in all isolates. In conclusion, our results suggested that these naturally occurring immunoepitope mutants, which changed their immunogenicity leading to escape from immune response, might cause HBV infection.

Beauvericin-induced cell apoptosis through the mitogen-activated protein kinase pathway in human nonsmall cell lung cancer A549 cells

Beauvericin (BEA) is a cyclic hexadepsipeptide that derives from Codyceps cicadae. Our previous study results indicated that the cytotoxic effects of BEA on human A549 lung cancer cells BEA occur through an apoptotic pathway, which involves the up-regulation of cytochrome c release from mitochondria, upregulation of caspase 3 activity, and cellular and morphological changes. In this study, we identified that the mitogen-activated protein kinase (MAPK) inhibitor U0126 inhibits the cytotoxic effects of BEA on A549 cells. After exposing human A549 cells to 10 μM BEA, we observed a significant and dose-dependent increase in the percentage of hypoploid (sub-G1) phase cells in the A549 population. Following the pretreatment of the A549 cells with 25 μM U0126, the distribution of A549 cells in the sub-G1 phase decreased significantly. The BEA treatment resulted in a significant increase apoptosis in A549 cells by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, the MEK1/2 (mitogen-activated protein kinase kinase)-ERK42/44 (extracellular signal-regulated kinases)-90RSK (ribosomal s6 kinase) signaling pathway was activated in BEA-induced apoptotic A549 cells. Furthermore, treatment with MEK1/2 inhibitor U0126 was capable to attenuate the BEA induced typical apoptotic morphological change, apoptotic cells, and MEK1/2-ERK42/44-90RSK signaling pathway. These results suggested that MEK1/2-ERK42/44-90RSK signaling pathway may play a important role in BEA-induced apoptosis in human NSCLC A549 cancer cells.