Major HBV splice variant encoding a novel protein important for infection.

BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.

The emergence and successful elimination of SARS-CoV-2 dominant strains with increasing epidemic potential in Taiwan's 2021 outbreak.

Taiwan's experience with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 guided its development of strategies to defend against SARS-CoV-2 in 2020, which enabled the successful control of Coronavirus disease 2019 (COVID-19) cases from 2020 through March 2021. However, in late-April 2021, the imported Alpha variant began to cause COVID-19 outbreaks at an exceptional rate in Taiwan. In this study, we aimed to determine what epidemiological conditions enabled the SARS-CoV-2 Alpha variant strains to become dominant and decline later during a surge in the outbreak. In conjunction with contact-tracing investigations, we used our bioinformatics software, CoVConvert and IniCoV, to analyze whole-genome sequences of 101 Taiwan Alpha strains. Univariate and multivariable regression analyses revealed the epidemiological factors associated with viral dominance. Univariate analysis showed the dominant Alpha strains were preferentially selected in the surge's epicenter (p = 0.0024) through intensive human-to-human contact and maintained their dominance for 1.5 months until the Zero-COVID Policy was implemented. Multivariable regression found that the epidemic periods (p = 0.007) and epicenter (p = 0.001) were two significant factors associated with the dominant virus strains spread in the community. These dominant virus strains emerged at the outbreak's epicenter with frequent human-to-human contact and low vaccination coverage. The Level 3 Restrictions and Zero-COVID policy successfully controlled the outbreak in the community without city lockdowns. Our integrated method can identify the epidemiological conditions for emerging dominant virus with increasing epidemiological potential and support decision makers in rapidly containing outbreaks using public health measures that target fast-spreading virus strains.

Isolation of murine hepatic myeloid cells with high yield and purity using immunomagnetic beads for subset analysis.

There are numerous established techniques for isolating hepatic myeloid cells; however, preserving their phenotypic and functional characteristics can be challenging. We present a straightforward and efficient method to isolate hepatic myeloid cells, including Kupffer cells and lymphocyte antigen 6 complex, locus C+ (Ly6C+) monocytes/macrophages. The procedure involves perfusion of the liver with collagenase and purification with immunomagnetic particles. This protocol ensures the isolation of large quantities of purified, viable, and functional cells without influencing their physiological characteristics. For complete details on the use and execution of this protocol, please refer to Wu et al. (2019).1.

[Dihydromyricetin improves Parkinson's disease-like lesions in T2DM rats by activating AMPK/ULK1 pathway].

The purpose of this study was to explore the effect and mechanism of dihydromyricetin (DHM) on Parkinson's disease (PD)-like lesions in type 2 diabetes mellitus (T2DM) rats. The T2DM model was established by feeding Sprague Dawley (SD) rats with high-fat diet and intraperitoneal injection of streptozocin (STZ). The rats were intragastrically administered with DHM (125 or 250 mg/kg per day) for 24 weeks. The motor ability of the rats was measured by balance beam experiment, the changes of dopaminergic (DA) neurons and the expression of autophagy initiation related protein ULK1 in the midbrains of the rats were detected by immunohistochemistry, and the protein expression levels of α-synuclein (α-syn), tyrosine hydroxylase (TH), as well as AMPK activation level, in the midbrains of the rats were detected by Western blot. The results showed that, compared with normal control, the rats with long-term T2DM exhibited motor dysfunction, increased α-syn aggregation, down-regulated TH protein expression, decreased number of DA neurons, declined activation level of AMPK, and significantly down-regulated ULK1 expression in the midbrain. DHM (250 mg/kg per day) treatment for 24 weeks significantly improved the above PD-like lesions, increased AMPK activity, and up-regulated ULK1 protein expression in T2DM rats. These results suggest that DHM may improve PD-like lesions in T2DM rats by activating AMPK/ULK1 pathway.

Metastatic tumor antigen 1 contributes to hepatocarcinogenesis posttranscriptionally through RNA-binding function.

BACKGROUND AND AIMS: Both nuclear and cytoplasmic overexpression of metastatic tumor antigen 1 (MTA1) contributes to tumorigenesis of HCC. Most studies have focused on nuclear MTA1 whose function is mainly a chromatin modifier regulating the expression of various cancer-promoting genes. By contrast, the molecular mechanisms of cytoplasmic MTA1 in carcinogenesis remain elusive. Here, we reveal a role of MTA1 in posttranscriptional gene regulation. APPROACH AND RESULTS: We conducted the in vitro and in vivo RNA-protein interaction assays indicating that MTA1 could bind directly to the 3'-untranslated region of MYC RNA. Mutation at the first glycine of the conserved GXXG loop within a K-homology II domain-like structure in MTA1 (G78D) resulted in the loss of RNA-binding activity. We used gain- and loss-of-function strategy showing that MTA1, but not the G78D mutant, extended the half-life of MYC and protected it from the lethal -7-mediated degradation. The G78D mutant exhibited lower activity in promoting tumorigenesis than wild-type in vitro and in vivo. Furthermore, RNA-immunoprecipitation sequencing analysis demonstrated that MTA1 binds various oncogenesis-related mRNAs besides MYC . The clinical relevance of cytoplasmic MTA1 and its interaction with MYC were investigated using HBV-HCC cohorts with or without early recurrence. The results showed that higher cytoplasmic MTA1 level and MTA1- MYC interaction were associated with early recurrence. CONCLUSIONS: MTA1 is a generic RNA-binding protein. Cytoplasmic MTA1 and its binding to MYC is associated with early recurrence in patients with HBV-HCC. This function enables it to regulate gene expression posttranscriptionally and contributes to hepatocarcinogenesis.

Molecular Mechanisms and Animal Models of HBV-Related Hepatocellular Carcinoma: With Emphasis on Metastatic Tumor Antigen 1.

Hepatocellular carcinoma (HCC) is an important cause of cancer death worldwide, and hepatitis B virus (HBV) infection is a major etiology, particularly in the Asia-Pacific region. Lack of sensitive biomarkers for early diagnosis of HCC and lack of effective therapeutics for patients with advanced HCC are the main reasons for high HCC mortality; these clinical needs are linked to the molecular heterogeneity of hepatocarcinogenesis. Animal models are the basis of preclinical and translational research in HBV-related HCC (HBV-HCC). Recent advances in methodology have allowed the development of several animal models to address various aspects of chronic liver disease, including HCC, which HBV causes in humans. Currently, multiple HBV-HCC animal models, including conventional, hydrodynamics-transfection-based, viral vector-mediated transgenic, and xenograft mice models, as well as the hepadnavirus-infected tree shrew and woodchuck models, are available. This review provides an overview of molecular mechanisms and animal models of HBV-HCC. Additionally, the metastatic tumor antigen 1 (MTA1), a cancer-promoting molecule, was introduced as an example to address the importance of a suitable animal model for studying HBV-related hepatocarcinogenesis.

The ESCRT-III molecules regulate the apical targeting of bile salt export pump.

BACKGROUND: The bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting. METHODS: The human BSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP. RESULTS: We identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. Cholestatic BSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP. CONCLUSIONS: Our results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.

Identification of novel avian and mammalian deltaviruses provides new insights into deltavirus evolution.

Hepatitis delta virus (HDV) is a satellite virus that requires hepadnavirus envelope proteins for its transmission. Although recent studies identified HDV-related deltaviruses in certain animals, the evolution of deltaviruses, such as the origin of HDV and the mechanism of its coevolution with its helper viruses, is unknown, mainly because of the phylogenetic gaps among deltaviruses. Here, we identified novel deltaviruses of passerine birds, woodchucks, and white-tailed deer by extensive database searches and molecular surveillance. Phylogenetic and molecular epidemiological analyses suggest that HDV originated from mammalian deltaviruses and the past interspecies transmission of mammalian and passerine deltaviruses. Further, metaviromic and experimental analyses suggest that the satellite-helper relationship between HDV and hepadnavirus was established after the divergence of the HDV lineage from non-HDV mammalian deltaviruses. Our findings enhance our understanding of deltavirus evolution, diversity, and transmission, indicating the importance of further surveillance for deltaviruses.

Expression of Metastatic Tumor Antigen 1 Splice Variant Correlates With Early Recurrence and Aggressive Features of Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Overexpression of metastatic tumor antigen 1 (MTA1) was correlated with poor prognosis of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HBV-HCC). The aim of this study was to examine the clinical significance of the expression of MTA1 and its exon 4-excluded form (MTA1dE4), the most abundant spliced variant of MTA1, in patients receiving curative resection for HBV-HCC. We collected 102 patients with HBV-HCC and received curative resection retrospectively and examined the expressions level of total MTA1/MTA1dE4 in their paired nontumor and tumor liver tissues by using RT-qPCR. The association between MTA1/MTA1dE4 expression and various tumor features as well as tumor recurrence was analyzed. During the median follow-up period of 4 years, 25 patients (24.5%) showed early recurrence (within 12 months postresection) and 42 (54.5%) showed late recurrence. In Kaplan-Meier analysis, MTA1dE4 overexpression in tumor, but not MTA1, was associated with early recurrence (P = 0.0365), but not late recurrence. In multivariate analysis, only alpha-fetoprotein (AFP) ≥200 ng/mL (P = 0.006) and large tumor size (P = 0.027) were correlated with early recurrence. In the subgroup of patients with AFP <200 ng/mL, high MTA1dE4, but not total MTA1, expression could help predict early recurrence (P = 0.0195). In vitro, wound healing and invasion assays were performed in HCC cells, and MTA1dE4 was found to exhibit a higher ability in promoting migration and invasion of hepatoma cells than full-length MTA1. Conclusion: MTA1dE4 expression is correlated with more aggressive tumor characteristics and might serve as a more sensitive marker for early recurrence of HBV-HCC, especially for low-AFP patients.

Lymphocyte Antigen 6 Complex, Locus C+ Monocytes and Kupffer Cells Orchestrate Liver Immune Responses Against Hepatitis B Virus in Mice.

To understand the mechanism(s) of age-dependent outcomes of hepatitis B virus (HBV) infection in humans, we previously established an age-related HBV mouse model in which 6-week-old (N6W) C3H/HeN mice exhibited virus tolerance whereas 12-week-old (N12W) counterparts presented virus clearance. By investigating the hepatic myeloid cell dynamics in mice of these two ages, we aim to identify factors associated with HBV clearance. C3H/HeN mice were transfected with an HBV plasmid by hydrodynamic injection. Serum HBV markers were monitored weekly. Hepatic leucocyte populations and their cytokine/chemokine productions were examined at baseline, day 3 (D3), day 7 (D7), and day 14 after injection. C-C chemokine receptor type 2 (CCR2) antagonist and clodronate (CLD) were respectively administered to N12W and N6W mice to study the roles of lymphocyte antigen 6 complex, locus C (Ly6C)+ monocytes and Kupffer cells (KCs) in viral clearance. N12W mice had a significantly higher number of TNF-α-secreting Ly6C+ monocytes and fewer IL-10-secreting KCs at D3 in the liver than their younger N6W counterparts after HBV transfection. In addition, the elevated number of interferon-γ+ TNF-α+ CD8+ T cells at D7 was only seen in the older cohort. The enhanced Ly6C+ monocyte induction in N12W mice resulted from elevated C-C motif chemokine ligand 2 (CCL2) secretion by hepatocytes. CCR2 antagonist administration hampered Ly6C+ monocyte recruitment and degree of KC reduction and delayed HBV clearance in N12W animals. Depletion of KCs by CLD liposomes enhanced Ly6C+ monocyte recruitment and accelerated HBV clearance in N6W mice. Conclusions: Ly6C+ monocytes and KCs may, respectively, represent the resistance and tolerance arms of host defenses. These two cell types play an essential role in determining HBV clearance/tolerance. Manipulation of these cells is a promising avenue for immunotherapy of HBV-related liver diseases.

Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis.

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Liver Gene Expression Profiles Correlate with Virus Infection and Response to Interferon Therapy in Chronic Hepatitis B Patients.

The natural course of chronic hepatitis B (CHB) infection and treatment response are determined mainly by the genomic characteristics of the individual. We investigated liver gene expression profiles to reveal the molecular basis associated with chronic hepatitis B and IFN-alpha (IFNα) treatment response in CHB patients. Expression profiles were compared between seven paired liver biopsy samples taken before and 6 months after successful IFNα treatment or between pretreatment biopsy samples of 11 IFNα responders and 11 non-responders. A total of 132 differentially up-regulated and 39 down-regulated genes were identified in the pretreated livers of CHB patients. The up-regulated genes were mainly related to cell proliferation and immune response, with IFNγ and B cell signatures significantly enriched. Lower intrahepatic HBV pregenomic RNA levels and 25 predictive genes were identified in IFNα responders. The predictive gene set in responders significantly overlapped with the up-regulated genes associated with the pretreated livers of CHB patients. The mechanisms responsible for IFNα treatment responses are different between HBV and HCV patients. HBV infection evokes significant immune responses even in chronic infection. The up-regulated genes are predictive of responsiveness to IFNα therapy, as are lower intrahepatic levels of HBV pregenomic RNA and pre-activated host immune responses.

Characterization of metastatic tumor antigen 1 and its interaction with hepatitis B virus X protein in NF-κB signaling and tumor progression in a woodchuck hepatocellular carcinoma model.

The metastatic tumor antigen 1 (MTA1) protein is associated with tumor invasiveness and poor prognosis in patients with hepatocellular carcinoma (HCC), particularly in those with hepatitis B virus (HBV)-related HCC. Chronically woodchuck hepatitis virus (WHV)-infected woodchuck is an ideal animal model for studying the pathogenesis of HBV-associated liver diseases, including HCC. To investigate the roles of MTA1 in HBV-associated hepatocarcinogenesis in the woodchuck model, we cloned the woodchuck MTA1 (wk-MTA1) complementary (c)DNA and characterized its molecular functions. The sequence and organization of the wk-MTA1 protein were highly conserved among different species. Similar to its expression in human HCC, wk-MTA1 was upregulated in woodchuck HCC, as determined at RNA and protein levels. Furthermore, an MTA1-spliced variant, wk-MTA1dE4, was overexpressed in woodchuck HCC, and it was attributed to approximately 50% of the total transcripts. The percentage of wk-MTA1dE4-overexpressed woodchuck HCCs was higher than that of the total wk-MTA1-overexpressed HCCs (77.8% vs 61.1%) and wk-MTA1dE4 may represent a more sensitive marker than the total wk-MTA1 in woodchuck HCC. We overexpressed or knocked down wk-MTA1 in a woodchuck HCC cell line and demonstrated that wk-MTA1 could interact with the WHV X protein (WHx) and play indispensable roles in WHx-mediated NF-κB activation and tumor cell migration- and invasion-promoting activities. In conclusion, our results support the hypothesis that woodchuck HCC recapitulates HBV-associated HCC with respect to the molecular characteristics of MTA1 and provides new clues for conducting mechanistic studies of MTA1 in HBV-associated hepatocarcinogenesis, including the possible clinical significance of wk-MTA1dE4.

Clinical significance of circulating miR-122 in patients with dual chronic hepatitis B and C virus infection.

BACKGROUND: The clinical significance of serum microRNA-122 (miR-122) has been shown in viral hepatitis B and C, respectively. Specifically, miR-122 stimulates hepatitis C virus (HCV) replication but suppresses hepatitis B virus (HBV) replication. The profile and clinical significance of serum miR-122 in patients with dual chronic hepatitis B and C would be an interesting and important clinical issue. METHODS: A total of 76 patients with HBV/HCV dual infection, 105 with HCV monoinfection and 39 with HBV monoinfection were enrolled. All patients received peginterferon alfa-2a (PEG-IFN)-based treatment. Serum miR-122 levels were quantified by using a sensitive hybridization-based assay. RESULTS: At baseline, the serum miR-122 level was lower in HCV-monoinfected patients than in HBV-monoinfected patients, whereas HBV coinfection increased the expression of miR-122. In multivariate analysis, the serum miR-122 level was positively correlated with the serum HBsAg level in patients with HBV/HCV dual infection and those with HBV monoinfection. In dually infected patients who received PEG-IFN-based treatment, a high baseline miR-122 level was positively correlated with a greater reduction of the posttreatment serum HBsAg level. CONCLUSION: In summary, the serum miR-122 level is highly correlated with the HBsAg level in HBV/HCV dually infected patients and may serve as a biomarker to predict posttreatment HBsAg decline.

Serum cytokine/chemokine profiles in acute exacerbation of chronic hepatitis B: clinical and mechanistic implications.

BACKGROUND AND AIMS: Acute exacerbation (AE) of chronic hepatitis B virus (HBV) infection is common and negatively impacts the clinical outcome. Although upsurge of viral load always precedes or coincides with AE, the underlying immunological mechanisms remain unclear and were investigated. METHODS: We prospectively followed the serum cytokine/chemokine profiles, viral load, and alanine aminotransferase (ALT) levels in 250 patients and identified 44 consecutive patients (male: 72.7%; age: 40.4 ± 9.7 years; hepatitis B e antigen [HBeAg] positivity: 63.6%; genotype B/C: 75%/25%) who developed AE during the follow-up in a medical center. The impact of clinical characteristics (age, gender, HBeAg, ALT, HBV genotype), cytokines (tumor necrosis factor-alpha, interferon gamma, interleukin [IL]-2, IL-4, IL-6, and IL-10), and chemokines (CXCL10/interferon gamma-induced protein [IP]-10, CCL2/MCP-1, CXCL9/MIG, CCL5/RANTES, and CXCL8/IL-8) on the serum HBV DNA dynamics at different time points (baseline, peak of serum HBV DNA level, peak of serum ALT level, and after AE) were analyzed. RESULTS: Of 44 patients, serum HBV DNA level surged before the peak of serum ALT level in 23 (52.3%), and coincided with the peak of ALT in 21 (47.7%). The upsurge of serum viral load significantly correlated with the increase of IL-10 (P = 0.0037) and CXCL10/IP-10 (P = 0.0044). Upsurge of serum viral load was preceded by an increase in serum IL-4 (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05). Combination of HBV genotype, IL-6 level at baseline, and ALT level at the peak of serum HBV DNA reliably predicted subsequent AE pattern (P = 0.0116). CONCLUSIONS: Enhanced Th2 activity is likely involved in the surge of HBV DNA level before hepatitis exacerbation.

Clinical and virological features of occult hepatitis B in patients with HBsAg seroclearance post-treatment or spontaneously.

BACKGROUND: Occult hepatitis B virus (HBV) infection (OHB) may exist in patients experiencing hepatitis B surface antigen (HBsAg) seroclearance. AIMS: We examined the clinical and virological features of OHB in patients who lost HBsAg post-treatment or spontaneously. METHODS: We collected 44 patients with HBsAg seroclearance: 15 patients with dual HBV/hepatitis C virus (HCV) infection who lost HBsAg after peginterferon alfa-2a (PEG-IFN) plus ribavirin therapy; 13 HBV mono-infected patients who lost HBsAg after various oral antiviral therapies; and 16 patients who lost HBsAg spontaneously. OHB was defined as detectable serum HBV DNA in the absence of HBsAg. Viral mutations associated with OHB were identified by comparison with matched controls that remained positive for HBsAg, and further characterized in vitro. RESULTS: The prevalence of OHB was 34.1% (15/44) in all patients, which was not significantly different among three groups. One mutation in surface promoter/polymerase region, C3050T (preS1T68I), was identified to be associated with the seroclearance of HBsAg in six cases. This mutation does not change the amino acid sequence of the polymerase protein. The S promoter activity was significantly lower in the construct containing C3050T mutation as compared with the wild-type (P = 0.0008). However, this mutation did not affect HBV replication, transcription and translation in the context of the full-length HBV genome. OHB was not rare in patients with HBsAg seroclearance. CONCLUSIONS: One mutation, C3050T (preS1T68I), decreased S promoter activity; nevertheless, other factors may play more important role in the clearance of HBsAg in these OHB patients.

Areca nut-induced buccal mucosa fibroblast contraction and its signaling: a potential role in oral submucous fibrosis--a precancer condition.

Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200-1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine-two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca(2+)/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-L-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca(2+)/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.

Hepatitis B virus nucleocapsid but not free core antigen controls viral clearance in mice.

We have recently shown that hepatitis B virus (HBV) core antigen (HBcAg) is the major viral factor for HBV clearance using a hydrodynamics-based mouse model. Knockout of HBcAg hampers the development of antiviral immune responses and thus promotes HBV persistence. Here, we further demonstrated that only in the capsid form, but not the free or dimer form, can HBcAg exert its contributory role in HBV clearance. HBcAg is the main structural protein of HBV icosahedral nucleocapsid. A mutant HBV DNA which expresses an assembly-defective HBcAg, HBcAgY132A, surprisingly prolonged HBV surface antigenemia in both C57BL/6 and BALB/c mice without affecting viral transcription and translation. This result was not due to a loss of the possible immune epitope caused by the single-amino-acid substitution of HBcAg. Moreover, the particular HBV mutant failed to induce robust humoral and cellular immunity against HBV. These data revealed the requirement of capsid structure for inducing adequate immunity that leads to HBV clearance in mice.

Persistent hepatitis B viral replication in a FVB/N mouse model: impact of host and viral factors.

The mechanism underlying the chronicity of hepatitis B virus (HBV) infection has long been an interesting question. However, this mechanism remains unclear largely due to the lack of an animal model that can support persistent HBV replication and allow for the investigation of the relevant immune responses. In this study, we used hydrodynamic injection to introduce HBV replicon DNA into the livers of three different mouse strains: BALB/c, C57BL/6, and FVB/N. Interestingly, we found that an HBV clone persistently replicated in the livers of FVB/N mice for up to 50 weeks but was rapidly cleared from the livers of BALB/c and C57BL/6 mice. Flow cytometric analysis and quantitative reverse transcription PCR analysis of the mouse livers indicated that after DNA injection, FVB/N mice had few intrahepatic activated cytotoxic T lymphocytes (CTLs) and produced low levels of alanine aminotransferase, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and the CXCL9 and CXCL10 chemokines. These findings were in sharp contrast with those observed in BALB/c and C57BL/6 mice, reflecting a strong correlation between the degree of liver inflammation and viral clearance. Mutational analysis further demonstrated that a change of Asn-214 to Ser-214 in the HBV surface antigen rendered the persistent HBV clone clearable in FVB/N mice, which was accompanied by increased levels of activated CTL and upregulated expression of IFN-γ, CXCL9, and CXCL10 in the livers. These results indicate that the heterogeneity of the host factors and viral sequences may influence the immune responses against HBV. An inadequate activation of immune or inflammatory responses can lead to persistent HBV replication in vivo.

Serum interleukin 6 level correlates with outcomes of acute exacerbation of chronic hepatitis B.

PURPOSE: Acute exacerbation (AE) of chronic hepatitis B virus (HBV) infection is common and negatively impacts the clinical outcome. Factors predicting outcomes after exacerbations were only partly clarified. We investigated the host immune parameters associated with long-term outcomes. METHODS: We prospectively examined the profiles of serum cytokines and chemokines in 36 consecutive hepatitis B e antigen (HBeAg)-positive patients (male 72%, age 40.8 ± 9.9 years, genotype B/C 75%/25%) who developed AE in a medical center. The patients were followed up for a median of 4 years (range 2-6 years) post-AE. The impact of six cytokines (tumor necrosis factor alfa, interferon gamma, IL-2, IL-4, IL-6, and IL-10) and five chemokines (CXCL10/IP-10, CCL2/MCP-1, CXCL9/MIG, CCL5/RANTES, and CXCL8/IL-8) at the onset of AE activity on the long-term outcomes were analyzed. RESULTS: Of 36 patients, 22 (61.1%) developed HBeAg seroconversion during follow-up (Group I), and the remaining 14 patients did not obtain HBeAg seroconversion (Group II). Baseline characteristics were generally similar between two groups of patients. In Group I patients, the frequency of undetectable serum IL-6 level (<3 pg/mL) at the onset of AE was significantly higher in comparison with Group II patients in multivariate analysis (86.4 vs. 42.9%, P = 0.016). CONCLUSIONS: Our findings indicate that undetectable serum IL-6 level at the early stage of AE correlated with the long-term outcomes and may serve as a useful clinical predictor.