Virological response to nucleos(t)ide analogues treatment in chronic hepatitis B patients is associated with Bacteroides-dominant gut microbiome.

BACKGROUND: Gut dysbiosis is present in chronic hepatitis B virus (HBV) infection. In this study, we integrated microbiome and metabolome analysis to investigate the role of gut microbiome in virological response to nucleos(t)ide analogues (NAs) treatment. METHODS: Chronic HBV patients were prospectively recruited for steatosis and fibrosis assessments via liver elastography, with full-length 16S sequencing performed to identify the compositional gut microbiota differences. Fasting plasma bile acids were quantified by liquid chromatography-tandem mass spectrometry. FINDINGS: All patients (n = 110) were characterized into three distinct microbial clusters by their dominant genus: c-Bacteroides, c-Blautia, and c-Prevotella. Patients with c-Bacteroides had a higher plasma ursodeoxycholic acids (UDCA) level and an increase in 7-alpha-hydroxysteroid dehydrogenase (secondary bile acid biotransformation) than other clusters. In NAs-treated patients (n = 84), c-Bacteroides was associated with higher odds of plasma HBV-DNA undetectability when compared with non-c-Bacteroides clusters (OR 3.49, 95% CI 1.43-8.96, p = 0.01). c-Blautia was positively associated with advanced fibrosis (OR 2.74, 95% CI 1.09-7.31, p = 0.04). No such associations were found in treatment-naïve patients. Increased Escherichia coli relative abundance (0.21% vs. 0.03%, p = 0.035) was found in on-treatment patients (median treatment duration 98.1 months) with advanced fibrosis despite HBV DNA undetectability. An enrichment in l-tryptophan biosynthesis was observed in patients with advanced fibrosis, which exhibited a positive correlation with Escherichia coli. INTERPRETATION: Collectively, unique bacterial signatures, including c-Bacteroides and c-Blautia, were associated with virological undetectability and fibrosis evolution during NAs therapy in chronic HBV, setting up intriguing possibilities in optimizing HBV treatment. FUNDING: This study was supported by the Guangdong Natural Science Fund (2019A1515012003).

FAT4 loss initiates hepatocarcinogenesis through the switching of canonical to noncanonical WNT signaling pathways.

BACKGROUND: Mutation and downregulation of FAT atypical cadherin 4 (FAT4) are frequently detected in HCC, suggesting a tumor suppressor role of FAT4. However, the underlying molecular mechanism remains elusive. METHODS: CRISPR-Cas9 system was used to knockout FAT4 (FAT4-KO) in a normal human hepatic cell line L02 to investigate the impact of FAT4 loss on the development of HCC. RNA-sequencing and xenograft mouse model were used to study gene expression and tumorigenesis, respectively. The mechanistic basis of FAT4 loss on hepatocarcinogenesis was elucidated using in vitro experiments. RESULTS: We found that FAT4-KO disrupted cell-cell adhesion, induced epithelial-mesenchymal transition, and increased expression of extracellular matrix components. FAT4-KO is sufficient for tumor initiation in a xenograft mouse model. RNA-sequencing of FAT4-KO cells identified PAK6-mediated WNT/ß-catenin signaling to promote tumor growth. Suppression of PAK6 led to ß-catenin shuttling out of the nucleus for ubiquitin-dependent degradation and constrained tumor growth. Further, RNA-sequencing of amassed FAT4-KO cells identified activation of WNT5A and ROR2. The noncanonical WNT5A/ROR2 signaling has no effect on ß-catenin and its target genes (CCND1 and c-Myc) expression. Instead, we observed downregulation of receptors for WNT/ß-catenin signaling, suggesting the shifting of ß-catenin-dependent to ß-catenin-independent pathways as tumor progression depends on its receptor expression. Both PAK6 and WNT5A could induce the expression of extracellular matrix glycoprotein, laminin subunit alpha 4. Laminin subunit alpha 4 upregulation in HCC correlated with poor patient survival. CONCLUSIONS: Our data show that FAT4 loss is sufficient to drive HCC development through the switching of canonical to noncanonical Wingless-type signaling pathways. The findings may provide a mechanistic basis for an in-depth study of the two pathways in the early and late stages of HCC for precise treatment.

Hepatitis B virus X protein promotes hepatocarcinogenesis via the activation of HMGA2/STC2 signaling to counteract oxidative stress-induced cell death.

Chronic hepatitis B virus (HBV) infection can cause oxidative stress and induce cell death. The mechanisms by which cells overcome oxidative stress to survive remain largely unknown. Here, we used human sera, liver tissues and cell lines to study how HBV modulates cellular pathways to counteract oxidative stress-induced cell death. We found high-mobility group AT-hook 2 (HMGA2), an architectural transcription factor is upregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Elevated serum HMGA2 is significantly associated with viral load in HBV carriers, and HBV-related HCC. We showed that HBV X protein (HBx) encoded by HBV-induced cell growth via HMGA2 activation. The growth-promoting effect is abolished when HMGA2 is suppressed. Ectopic HBx expression induced DNA damage and oxidative stress. HMGA2 silencing reduced oxidative stress in HBx-expressing cells. Cytoprotective stanniocalcin 2 (STC2) protein is a downstream target of HMGA2. Consistent with the findings in HMGA2, STC2 mRNA and protein expression are upregulated in HCC tissues. Elevated serum STC2 is also associated with viral load in HBV carriers, and HCC. STC2 is transcriptionally upregulated by HBx and HMGA2 to elicit cytoprotection against apoptosis. STC2 knockdown disrupted Bax/Bcl-2 balance that increased cytochrome c release, caspase 3/7 activity and apoptosis, and thus abolished the growth-promoting effect of HMGA2. Clinical relevance of HBx/HMGA2/STC2 signaling is evidenced by the significant correlation of serum HMGA2/STC2 in active HBV infection and HCC. These findings reveal a novel HBx regulatory HMGA2/STC2 pathway in counteracting reactive oxygen species-induced cell death. HMGA2 and STC2 may be therapeutic targets for prevention of hepatocarcinogenesis in chronic HBV infection.

Alleviation of Hepatic Steatosis: Dithizone-Related Gut Microbiome Restoration During Paneth Cell Dysfunction.

Non-alcoholic fatty liver disease (NAFLD), the world's most common chronic liver disease, is increasingly linked to gut dysbiosis. Paneth cells secrete antimicrobial peptides that regulate the gut microbiome, but their role in the pathogenesis of NAFLD remains unclear. Here, we determine the changes in NAFLD development and gut microbial composition and function via the injection of dithizone that can pharmacologically deplete the granules of Paneth cells. Eight-week-old C57BL/6J male mice (n = 31) were given a high-fat diet (HFD) or standard control diet for 12 weeks. Dithizone (10 mg/kg) was intravenously injected every 3 weeks during the period of diet feeding. Metagenomic DNA was extracted from fecal samples for PacBio Single-Molecule Real-Time sequencing to identify changes in microbial composition and predicted function. We observed dithizone-treated HFD mice, when compared to non-treated HFD mice, to have significant reductions in hepatic triglyceride content (28.98 vs. 53.52 mg/g, p = 0.0419); plasma insulin level (2.18 vs. 6.63 ng/ml, p = 0.0079); and relative mRNA levels of fatty acid synthase (0.52 vs. 1.57, p = 0.0428) and stearoyl-CoA desaturase-1 (0.43 vs. 1.20, p = 0.0121). Bacterial taxonomic profiling found dithizone-treated HFD mice, when compared to non-treated HFD mice, had a lower Firmicutes/Bacteroidetes ratio (2.53 vs. 5.26, p = 0.0541); a higher relative abundance of Bacteroides ASV21 and ASV42 (1.04 vs. 0.22%, p = 0.0277 and 0.96 vs. 0.09%, p = 0.0213); and a reduction in microbes belonging to Firmicutes (all p < 0.05). Bacteroides species correlated positively with predicted microbial functions such as L-methionine (r = 0.54, p = 0.0019) and tetrahydrofolate (r = 0.52, p = 0.0029) biosynthesis. Collectively, dithizone treatment was associated with alleviation in the severity of liver steatosis in HFD mice, possibly through gut microbiome modulation involving the increase in Bacteroides, suggesting microbiome-targeted therapies may have a role in the treatment of NAFLD.

Phenotypic Changes of PD-1 and GITR in T Cells Are Associated With Hepatitis B Surface Antigen Seroclearance.

BACKGROUND: Regulatory T cells (Tregs) possess hepatitis B virus (HBV)-specific immunoregulatory effects in chronic HBV infection. The role of Tregs in spontaneous seroclearance of hepatitis B surface antigen (HBsAg) remains to be determined. METHODS: We recruited treatment-naive chronic HBV patients achieving spontaneous HBsAg seroclearance (experimental group) and matched HBsAg-positive controls. Peripheral blood mononuclear cells were isolated using the Ficoll-Paque density gradient centrifugation method. The frequency of Tregs and inhibitory phenotypes and immunoregulatory cytokines of Tregs were detected by flow cytometry. RESULTS: Twenty-seven patients with HBsAg seroclearance (mean age: 52.40±6.00 y, 55.6% male) and 27 matched controls were recruited. Median HBsAg and HBV DNA levels in the control group were 2.80 (1.24 to 3.43) and 3.16 (1.68 to 3.85) log IU/mL, respectively. Mean frequencies of Tregs and expressions of FoxP3+ Tregs were comparable in both groups (both P>0.05). The mean expression of programmed death 1 (PD-1) and glucocorticoid-induced TNFR family-related gene (GITR) in total CD4+ T cells were significantly downregulated in the experimental group when compared with the control group (10.62% vs. 13.85%, P=0.003; 16.20% vs. 27.02%, P=0.002, respectively). When compared with the control group, PD-1+CD4+ Tregs expression in the experimental group was significantly downregulated (13.85% vs. 10.62%, P=0.003). A similar phenomenon was noted for GITR+CD8+ Tregs (20.16% vs. 14.08%, P=0.049). Intracellular cytokine productions showed no significant differences (all P>0.05). CONCLUSIONS: The reduced expression of PD-1 and GITR might attenuate the immunosuppressive capability of Tregs. Decreased expression on CD4+ T cells might represent an enhanced antiviral function, playing a role in initiating the "functional cure" of chronic HBV infection.

Tumor suppressive role of mitochondrial sirtuin 4 in induction of G2/M cell cycle arrest and apoptosis in hepatitis B virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is developed from uncontrolled cell growth after the malignant transformation of hepatocytes. The hepatitis B virus (HBV) X protein (HBx) has shown to induce cell cycle progression and hepatocarcinogenesis. A sub-fraction of HBx is localized in the mitochondria. Sirtuin 4 (SIRT4), a mitochondrial protein, has been demonstrated to play a tumor-suppressive role in many cancers, including HCC. However, little is known about the association between mitochondrial HBx and SIRT4 during hepatocarcinogenesis. We aimed to investigate the clinical significance and functional role of SIRT4 in HBV-related HCC. SIRT4 expression was significantly lower in the HCC tissues collected from 30 patients with HBV-related HCC than in normal liver tissues from control patients (p < 0.0001). TCGA data analysis indicated that SIRT4 expression was also lower in patients with HBV infection than in those without, and SIRT4 levels were positively associated with better patient survival. Similarly, HCC cell lines had lower SIRT4 expression than normal liver cell lines (all p < 0.01). Among the HCC cell lines, those harbored HBV had a lower SIRT4 expression than those without HBV (p < 0.0001). In vitro experiments revealed that stable HBx transfection suppressed SIRT4 expression in both HepG2 and Huh7 cells (both p < 0.001). Ectopic SIRT4 overexpression alone could induce cellular senescence through arresting cell-cycle progression at G2/M, and inducing cell apoptosis in HCC cells. Mechanistically, SIRT4 upregulated cell-cycle governing genes p16 and p21 protein expression, suppressed CyclinB1/Cdc2 and Cdc25c which normally induce cell-cycle progression, and suppressed survivin to induce apoptosis. Our findings demonstrate the interaction between HBV and SIRT4 in the context of HCC. SIRT4 involves in G2/M DNA damage checkpoint control and genomic stability in hepatocarcinogenesis, which could be targeted for future anticancer strategies.

HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA.

The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44-77% reduction) and secretory HBsAg (25-81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33-64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55-60 and 121-126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02-0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69-1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01-14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55-60 and 121-126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.

Targeted genomic profiling identifies frequent deleterious mutations in FAT4 and TP53 genes in HBV-associated hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV) is the major risk factor for hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV-associated HCC pathogenesis is still unclear. Genetic alterations in cancer-related genes have been linked to many human cancers. Here, we aimed to explore genetic alterations in selected cancer-related genes in patients with HBV-associated HCC. METHODS: Targeted sequencing was used to analyze six cancer-related genes (PIK3CA, TP53, FAT4, IRF2, HNF4α and ARID1A) in eight pairs of HBV-associated HCC tumors and their adjacent non-tumor tissues. Sanger sequencing, quantitative PCR, Western-blotting and RNAi-mediated gene knockdown were used to further validate findings. RESULTS: Targeted sequencing revealed thirteen non-synonymous mutations, of which 9 (69%) were found in FAT4 and 4 (31%) were found in TP53 genes. Non-synonymous mutations were not found in PIK3CA, IRF2, HNF4α and ARID1A. Among these 13 non-synonymous mutations, 12 (8 in FAT4 and 4 in TP53) were predicted to have deleterious effect on protein function by in silico analysis. For TP53, Y220S, R249S and P250R non-synonymous mutations were solely identified in tumor tissues. Further expression profiling of FAT4 and TP53 on twenty-eight pairs of HCC tumor and non-tumor tissues confirmed significant downregulation of both genes in HCC tumors compared with their non-tumor counterparts (P < 0.001 and P < 0.01, respectively). Functional analysis using RNAi-mediated knockdown of FAT4 revealed an increased cancer cell growth and proliferation, suggesting the putative tumor suppressor role of FAT4 in HCC. CONCLUSIONS: This study highlights the importance of FAT4 and TP53 in HCC pathogenesis and identifies new genetic variants that may have potentials for development of precise therapy for HCC.

Plasma Fibronectin Levels Identified via Quantitative Proteomics Profiling Predicts Hepatitis B Surface Antigen Seroclearance in Chronic Hepatitis B.

BACKGROUND: Seroclearance of hepatitis B surface antigen (HBsAg) is a potentially achievable target of chronic hepatitis B (CHB). Plasma proteins relevant to HBsAg seroclearance remain undetermined. METHODS: We prospectively recruited treatment-naive CHB patients with spontaneous HBsAg seroclearance and matched HBsAg-positive controls. Plasma protein profiling was performed using isobaric tags for relative and absolute quantitation-based proteomics, with the expression of candidate proteins validated in a separate cohort. The predictive value of fibronectin was assessed at 3 years, 1 year (Year -1) before, and at the time (Year 0) of HBsAg seroclearance. RESULTS: Four hundred eighty-seven plasma proteins were identified via proteomics, with 97 proteins showing altered expression. In the verification cohort (n = 90), median plasma fibronectin levels in patients with HBsAg seroclearance was higher than in controls (P = .009). In the longitudinal cohort (n = 164), patients with HBsAg seroclearance, compared with controls, had a higher median fibronectin levels at Year -1 (413.26 vs 227.95 µg/mL) and Year 0 (349.45 vs 208.72 µg/mL) (both P < .001). In patients with an annual HBsAg log reduction >0.5, Year -1 fibronectin level achieved an area under the receiving operator characteristic of 0.884 in predicting HBsAg seroclearance. CONCLUSIONS: Using proteomics-based technology, plasma fibronectin may be associated with HBsAg seroclearance and a potential predictor of "functional cure".

Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome.

The hepatitis B core protein (HBc) has been suggested to interact with covalently closed circular DNA (cccDNA) and regulate hepatitis B virus (HBV) transcription. However, direct evidence is lacking. We aimed to identify the specific HBc region(s) responsible for transcription regulation and its interaction with cccDNA. Seventeen mutants with mutations at the four arginine-rich clusters of the HBc carboxyl-terminal domain (CTD) were created. The effect of HBc mutations on the levels of HBV DNA, RNA, and hepatitis B surface antigen (HBsAg) were measured. The association of cccDNA with mutant HBc and histone acetyltransferases (HATs) was assessed by chromatin immunoprecipitation (ChIP). Compared with wild-type HBc, HBc mutants with mutations in clusters III and IV resulted in a significant reduction in HBV RNA levels (all P < 0.05). HBc arginine clusters III and IV mutants also had a significantly lower levels of intracellular HBV DNA (<5% of wild-type; P < 0.001) and HBsAg (<10% of wild-type; P < 0.0001). cccDNA-ChIP assay demonstrated that HBc clusters III and IV mutants had a smaller degree of association with cccDNA (P < 0.001). In the HBc mutants, the association between HATs with cccDNA were reduced. In conclusion, HBc-CTD arginine residues at clusters III and IV play an important role in the regulation of HBV transcription as well as subsequent replication steps, likely through the reduced interaction of HBc with cccDNA and reduced acetylation of cccDNA-bound histones. These findings may provide clues to the identification of novel therapeutic targets against HBV.

Deep sequencing analysis of quasispecies in the HBV pre-S region and its association with hepatocellular carcinoma.

BACKGROUND: The association between the evolution of hepatitis B virus (HBV) quasispecies and the development of hepatocellular carcinoma (HCC) is unknown. METHODS: We used deep sequencing to examine the dynamics of HBV quasispecies and their relationship to HCC development. Thirty-two chronic hepatitis B (CHB) patients with HCC (HCC group) and 32 matched CHB patients without HCC (controls) were recruited. Fourteen patients from each group had serial sera available up to 9 years before the time of the present study. Deep sequencing of the HBV pre-S regions was performed. HBV quasispecies complexity, diversity, and intrapatient prevalence of pre-S deletions/mutations were analyzed. RESULTS: Compared with control patients, HCC patients had a significant greater quasispecies complexity (p = 0.04 at the nucleotide level), greater diversity (p = 0.004 and 0.009 at the nucleotide level and the amino acid level respectively), and a trend of greater complexity at the amino acid level (p = 0.065). HCC patients had a higher intrapatient prevalence of pre-S deletions and point mutations (at codons 4, 27, and 167) compared with the control patients (all p < 0.05). Longitudinal observation in the sera of 14 HCC patients showed that quasispecies complexity (p = 0.027 and 0.024 at the nucleotide level and the amino acid level respectively) and diversity (p = 0.035 and 0.031 at the nucleotide level and the amino acid level respectively) increased as the disease progressed to HCC. CONCLUSIONS: Increased HBV quasispecies complexity and diversity in the pre-S region, probably reflecting enhanced virus-host interplay, was associated with disease progression from CHB to HCC.

Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a key to viral persistence in chronic hepatitis B infection. Serum hepatitis B core-related antigen (HBcrAg) is a novel marker for HBV disease. We aimed to determine whether HBcrAg could be a surrogate marker for intrahepatic cccDNA. METHODS: Three hundred and five liver biopsies and the corresponding sera collected from 138 nucleos(t)ide analogues-treated patients were analysed. 124 patients had paired liver biopsies at baseline and 1-year post-treatment, and 43 patients had a third biopsy after 6-12 years of treatment. Serum HBcrAg, HBV DNA and hepatitis B surface antigen (HBsAg), and intrahepatic HBV DNA and cccDNA were measured. RESULTS: HBcrAg strongly correlated with cccDNA (r=.70), intrahepatic total HBV DNA (r=.67) and serum HBV DNA (r=.69; all P<.0001). In the 130 samples with undetectable serum HBV DNA, HBcrAg was detectable in 101 (78%) samples, and HBcrAg levels still correlated positively with cccDNA (r=.42, P<.0001). At ≥6 years of therapy, the median logarithmic reduction in HBcrAg was 2.7 log kU/mL, which was comparable to the magnitude of reduction in cccDNA. Twenty-one patients had undetectable cccDNA after ≥6 years of treatment, in whom 15 (71%) had detectable HBcrAg (range: 1.2-537 kU/mL). CONCLUSIONS: Serum HBcrAg is a reliable surrogate marker for intrahepatic cccDNA. HBcrAg could be a very sensitive marker to reflect the cccDNA content and persistence of disease even with the cccDNA levels below the detection limit of assays.

Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), a mini-chromosome essential for HBV replication, is supposed to be resistant to nucleos(t)ide analogue treatment. We investigated the effect of long-term nucleos(t)ide analogue treatment on cccDNA. METHODS: Among 129 patients who had been enrolled in previous international nucleos(t)ide analogue clinical trials and had liver biopsies at baseline and one year after treatment, we recruited 43 patients on long-term continuous treatment for 72 to 145months for a third liver biopsy. Serum HBV DNA, hepatitis B surface antigen (HBsAg) levels, total intrahepatic HBV DNA (ihHBV DNA), cccDNA, HBV pregenomic RNA (pgRNA) as well as histologic changes were examined. RESULTS: At the time of the third biopsy, serum HBV DNA levels were undetectable in all but one patient. The median levels of HBsAg, ihHBV DNA, and cccDNA were 2.88logIU/ml, 0.03copies/cell, and 0.01copies/cell, respectively. Compared to baseline levels, there was reduction of HBsAg levels by 0.54log (71.46%), ihHBV DNA levels by 2.81log (99.84%), and cccDNA levels by 2.94log (99.89%), with 49% having cccDNA levels below the detection limit. One patient had undetectable HBsAg. The median pgRNA level, measured only in the third biopsy, was 0.021copies/cell, with 40% of patients having undetectable pgRNA. CONCLUSIONS: Long-term nucleos(t)ide analogue treatment induced marked depletion of cccDNA in the majority of patients while serum HBsAg levels, though reduced, were detectable in all but one patient. Whether cccDNA depletion is sustained and associated with better patient outcome requires further study. LAY SUMMARY: It is generally presumed that a form of hepatitis B virus DNA, called covalently closed circular DNA (cccDNA), which hides inside the nuclei of liver cells of patients with chronic hepatitis B, cannot be reduced by antiviral treatment. The present study showed that with prolonged treatment (median period 126months), cccDNA can be markedly reduced, with 49% of liver biopsies having undetectable cccDNA. This suggests that viral replication capacity would be very low after prolonged antiviral treatment.

Interleukin-1ß increases the risk of gastric cancer through induction of aberrant DNA methylation in a mouse model.

Interleukin-1ß (IL-1ß) has a significant role in chronic gastric inflammation and manifestations of gastric diseases. The present study aimed to elucidate the specific role of IL-1ß in induction of DNA methylation using IL-1 receptor type 1 knockout (IL-1R1-/-) mice. In the present study, wild-type (WT) and IL-1R1-/- mice were injected with IL-1ß (5 µg/kg/day). Serum levels of IL-1ß, interleukin-6 (IL-6) and nitric oxide (NO) were measured by enzyme-linked immunosorbent or NO assays. E-cadherin (E-cad) methylation status and messenger (m)RNA expression of IL-1ß, IL-6, E-cad and inducible nitric oxide synthase (iNOS) were analyzed. Results from the present study indicated significantly higher IL-1ß mRNA expression (P<0.001) in WT mice compared with IL-1R1-/- mice. IL-1ß and IL-6 release was significantly increased in treated WT mice compared with IL-1R1-/- mice at 1 h, 4 h and 8 h (all P<0.005). IL-1ß release was only detected in WT mice following a second dose measured at day 3, week 1 and week 2 when compared with IL-1R1-/- mice. Promoter methylation of E-cad and a decrease in gene expression was observed in treated WT mice. mRNA expression of iNOS in WT mice was significantly increased at week 1 compared with IL-1R1-/- mice (P=0.0411). Furthermore, a significantly increased level of NO production was observed in treated WT mice (P<0.005 at 8 h and week 1; P<0.001 at 4 h and day 3) when compared with IL-1R1-/- mice. The present results indicated that IL-1ß was able to directly induce DNA methylation, which may link inflammation-induced epigenetic changes and the development of gastric diseases.

Hepatitis B virus full-length genomic mutations and quasispecies in hepatocellular carcinoma.

BACKGROUND AND AIM: Hepatitis B virus (HBV) full-length genomic mutations and quasispecies characteristics in hepatocellular carcinoma (HCC) were investigated. METHODS: Hepatitis B virus DNA was extracted from the tumor and non-tumor tissues of 16 HCC patients. Overlapping DNA fragments covering the entire HBV genome were amplified and sequenced. To study HBV sequence at the quasispecies level, the preS region was amplified and clonally sequenced. HBV mutation profiles, quasispecies complexity and diversity, and phylogenetic characteristics were assessed. RESULTS: Fourteen patients had full-length HBV amplification. Hot-spot mutations at HBx aa130-131 and pre-S deletions were detected in 13 (93%) and 6 (43%) patients, respectively. Deletions in the X/preC/C regions were more frequently detected in the tumor than in the non-tumor tissues (P = 0.031). Compared with the non-tumor tissues, the tumor tissues had a lower quasispecies complexity (P = 0.014 and 0.043, at the nucleotide and amino acid levels, respectively) and diversity (P = 0.048 and 0.022, at the nucleotide and amino acid levels, respectively). Phylogenetic analysis showed that HBV sequences derived from tumor and non-tumor tissues were separately clustered, suggesting the occurrence of compartmentalization, which was confirmed by the correlation coefficient testing on both the number and length of branches of viral populations (all P < 0.02). CONCLUSIONS: Hepatitis B virus mutation patterns in HCC tumor tissues and non-tumor tissues were different. HBV quasispecies within the preS region were compartmentalized, and tumor tissues had a lower genome complexity and diversity. Our study suggests HBV evolution is conditioned by the differential host cellular environment in HCC tumors.

Hepatitis B surface antigen seroclearance during nucleoside analogue therapy: surface antigen kinetics, outcomes, and durability.

BACKGROUND: Hepatitis B surface antigen (HBsAg) seroclearance is the recommended treatment end point for nucleoside analogue (NA) therapy in chronic hepatitis B, yet the underlying kinetics and durability of HBsAg seroclearance in NA-treated patients have not been well described. METHODS: We compared the HBsAg kinetics and long-term serologic outcomes of 51 chronic hepatitis B patients achieving HBsAg seroclearance during NA therapy with those of 51 HBsAg-positive controls, matched for age, sex, hepatitis B e antigen status, NA type, and treatment duration. Viral profiles before and after HBsAg seroclearance during and after NA treatment cessation were determined. RESULTS: The median time to HBsAg seroclearance and the median follow-up duration after HBsAg seroclearance were 61.2 and 51.6 months respectively. Patients achieving HBsAg seroclearance maintained high median rates of HBsAg reduction throughout therapy (first 6 months, 0.40 IU/mL/year; after year 1, 0.39 IU/mL/year; p = 0.809). For controls, the median rate of HBsAg reduction was significantly slower with time (first 6 months and after year 1, 0.19 and 0.05 IU/mL/year; p = 0.006). The difference in the median HBsAg reduction rates after year 1 between the two groups was significant (p < 0.001). The cumulative rates of antibody to HBsAg development and HBsAg seroreversion 72 months after HBsAg seroclearance were 68.9 and 8.3% (one patient receiving immunosuppressive therapy; one patient with pre-S/S variant), respectively. Among 22 patients who discontinued therapy after HBsAg seroclearance, 21 remained HBsAg negative with undetectable hepatitis B virus DNA and one patient with reactivation had the pre-S/S variant. CONCLUSION: NA-treated patients achieving HBsAg seroclearance uniquely maintained high rates of HBsAg reduction throughout treatment, with HBsAg seroclearance durable in most of the patients after treatment cessation.

Evolutionary Changes of Hepatitis B Virus Pre-S Mutations Prior to Development of Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Deletions/mutations in the hepatitis B virus (HBV) pre-S region have been associated with hepatocellular carcinoma (HCC). We aimed to study the evolutionary changes of pre-S mutations prior to HCC development. METHODS: We studied the HBV pre-S sequences at 1 to 10 years preceding diagnosis of HCC in 74 patients with HBV-related HCC (HCC group). 148 chronic hepatitis B patients matched for sex and age in 2:1 ratio, who had been followed up for at least 3 years without HCC (HCC-free group) were recruited as controls. 56 and 47 patients of HCC and HCC-free groups respectively had serially stored sera for longitudinally examination at 1-3 years, 4-6 years, 7-9 years and ≥10 years prior to the recruitment of the study. RESULTS: Compared to the HCC-free group, higher frequencies of pre-S deletions and point mutations (at 11 codons) were observed in the HCC group (p<0.05). Multiple logistic regression analysis showed that pre-S deletions, point mutations at codon 51 and 167 were independent factors associated with HCC. Longitudinal observation showed that pre-S deletions and most of the 11 HCC-associated pre-S point mutations existed at least 10 years before HCC development, and were more prevalent preceding HCC development in patients from HCC groups than HCC-free group. The number of HCC-associated pre-S point mutations increased over time preceding HCC development, and correlated positively with the time to HCC diagnosis (r = 0.220, p = 0.005). CONCLUSIONS: High prevalence and cumulative evolution of pre-S mutations preceding HCC development suggested a possible carcinogenic role of pre-S mutations and their potential application in HCC risk prediction.

Estradiol induces apoptosis via activation of miRNA-23a and p53: implication for gender difference in liver cancer development.

Estrogen (E2) has been suggested to have a protective role in attenuating hepatocellular carcinoma (HCC) development. miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control gene expression. However, little is known about the mechanism underlying the protective role of E2 in hepatocarcinogenesis and the effects of E2 on apoptotic miRNAs expression. Using miRNA PCR array, we found more than 2-fold alteration was observed in 25 upregulated and 10 downregulated apoptotic miRNAs in E2-treated cells. Among these miRNAs, we found expression of miR-23a was related to p53 functional status in the male-derived liver cell-lines. We demonstrated that E2 via ERα transcriptionally activated miR-23a and p53 expression, and thus enhanced p53 activation of miR-23a expression. Moreover, miR-23a expression correlated inversely with the expression of target gene X-linked inhibitor of apoptosis protein (XIAP), but positively with the caspase-3/7 activity. Decreasing of XIAP might contribute to caspase-3 activity and cell apoptosis. Taken together, our findings reveal a novel E2-signaling mechanism in regulating miRNAs expression for controlling apoptosis in liver cells. Delineating the role of E2 in regulating the activation of p53 and miR-23a, expression in HCC is crucial to the understanding of the sex difference observed in HCC.

Application of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus.

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations.

Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection.

BACKGROUND: The underlying mechanism of HBsAg-negative hepatitis B virus (HBV) infection is notoriously difficult to elucidate because of the extremely low DNA levels which define the condition. We used a highly efficient amplification method to overcome this obstacle and achieved our aim which was to identify specific mutations or sequence variations associated with this entity. METHODS: A total of 185 sera and 60 liver biopsies from HBsAg-negative, HBV DNA-positive subjects or known chronic hepatitis B (CHB) subjects with HBsAg seroclearance were amplified by rolling circle amplification followed by full-length HBV genome sequencing. Eleven HBsAg-positive CHB subjects were included as controls. The effects of pivotal mutations identified on regulatory regions on promoter activities were analyzed. RESULTS: 22 and 11 full-length HBV genomes were amplified from HBsAg-negative and control subjects respectively. HBV genotype C was the dominant strain. A higher mutation frequency was observed in HBsAg-negative subjects than controls, irrespective of genotype. The nucleotide diversity over the entire HBV genome was significantly higher in HBsAg-negative subjects compared with controls (p = 0.008) and compared with 49 reference sequences from CHB patients (p = 0.025). In addition, HBsAg-negative subjects had significantly higher amino acid substitutions in the four viral genes than controls (all p<0.001). Many mutations were uniquely found in HBsAg-negative subjects, including deletions in promoter regions (13.6%), abolishment of pre-S2/S start codon (18.2%), disruption of pre-S2/S mRNA splicing site (4.5%), nucleotide duplications (9.1%), and missense mutations in "α" determinant region, contributing to defects in HBsAg production. CONCLUSIONS: These data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.