Serum hepatitis B core-related antigen as a surrogate marker of hepatitis B e antigen seroconversion in chronic hepatitis B.

BACKGROUND: Quantitative hepatitis B core-related antigen (qHBcrAg) has a better correlation with intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) than HBV DNA or hepatitis B e antigen (HBeAg), but data are still lacking for its clinical application. AIM: The aim was to investigate serum qHBcrAg levels in patients with chronic hepatitis B and assess the correlation of serum qHBcrAg with pregenomic RNA (pgRNA), cccDNA, and HBeAg seroconversion. METHODS: This study was a secondary analysis of patients who underwent percutaneous liver biopsy between July 2014 and June 2019 in two multicenter randomized controlled clinical trials of peginterferon vs nucleos(t)ide analog (NUC)-based therapy (NCT03509688 and NCT03546530). Serum qHBcrAg, pgRNA, HBV DNA, hepatitis B core antigen, HBeAg, liver cccDNA, and HBV DNA were measured. The correlations of serum qHBcrAg with other biomarkers were analyzed. RESULTS: A total of 139 patients were included. The mean qHBcrAg levels were 5.32 ± 1.18 log10 U/mL at baseline and decreased during treatment (all P < 0.0001). Serum qHBcrAg levels were positively correlated with pgRNA (r = 0.597, P < 0.0001) and cccDNA (r = 0.527, P < 0.0001) levels. The correlation of serum qHBcrAg level and intrahepatic HBV DNA levels at baseline was weak but significant (r = 0.399, P < 0.0001). HBcrAg predicted HBeAg seroconversion, with areas under the receiver operating characteristics curve of 0.788 at 24 wk and 0.825 at 48 wk. Log HBcrAg at wk 24 and 48 was independently associated with HBeAg seroconversion [odds ratio (OR) = 2.402, 95% confidence interval (CI): 1.314-4.391, P = 0.004; OR = 3.587, 95%CI: 1.315-9.784, P = 0.013]. CONCLUSION: Serum HBcrAg levels were correlated with HBV virological markers and could be used to predict HBeAg seroconversion.

Validation of the GALAD Model and Establishment of GAAP Model for Diagnosis of Hepatocellular Carcinoma in Chinese Patients.

PURPOSE: GALAD is a statistical model for estimating the likelihood of having hepatocellular carcinoma (HCC) based on gender, age, AFP, AFP-L3, and PIVKA-II. We aimed to assess its performance and build new models in China, where hepatitis B virus (HBV) is the leading etiology of HCC. PATIENTS AND METHODS: We built the GALAD-C model with the same five variables in GALAD, and the GAAP model with gender, age, AFP, and PIVKA-II, using logistic regression based on 242 patients with HCC and 283 patients with chronic liver disease (CLD). We also collected 50 patients with other malignant liver tumors (OMTs) and 50 healthy controls (HCs). A test dataset (169 patients with HCC and 139 with CLD) was used to test the performance of GAAP. RESULTS: The GALAD-C and GAAP models achieved comparable performance (area under the receiver operating characteristic curve [AUC], 0.922 vs 0.914), and both were superior to GALAD, PIVKA-II, AFP, and AFP-L3% (AUCs, 0.891, 0.869, 0.750, and 0.711) for discrimination of HCC from CLD for the entire dataset. The AUCs of the GALAD, GALAD-C and GAAP models were excellent for the hepatitis C virus (HCV) subgroup (0.939, 0.958 and 0.954), and for discrimination HCC from HCs (0.988, 0.982, and 0.979), but were relatively lower for the HBV subgroup (0.855, 0.904, and 0.894), and for HCC within Milan Criteria (0.810, 0.841, and 0.840). They were not superior to AFP (0.873) for discrimination of HCC from OMT (0.873, 0.809, and 0.823). GAAP achieved an AUC of 0.922 in the test dataset. CONCLUSION: GALAD was excellent for discrimination of HCC from CLD in the HCV subgroup of a cohort of Chinese patients. The GAAP and GALAD-C models achieved better performance compared with GALAD. These three models exhibited better performance in patients with an HCV etiology than those with HBV.

High prevalence of Clonorchis sinensis infections and coinfection with hepatitis virus in riverside villages in northeast China.

In China, the prevalence of Clonorchis sinensis (C. sinensis) infections is only evaluated at the provincial level by national sampling surveys, and data from villages and counties are still lacking. In this study, we conducted a cross-sectional survey in 10 villages located along the Lalin River in northeast China. Clonorchiasis was diagnosed using a modified Kato-Katz method that detects the C. sinensis egg in stools. A total of 3,068 persons were screened and 2,911 were recruited for the study. Overall, the prevalence of C. sinensis infection was 29.3%. Among 175 participants who were cured after antiparasitic treatment, 54 (30.86%) were re-infected in this survey. After calibration of potential confounders, male gender, occupation as a farmer, smoking, and occasionally or frequently eating raw fish were independent risk factors for C. sinensis infection. The results of laboratory examinations in the C. sinensis/hepatitis B or C virus co-infection group were similar to those in the hepatitis B or C virus mono-infection groups. In conclusion, C. sinensis is highly endemic in villages along the Lalin River, and the primary route of infection is the consumption of raw freshwater fish. Co-infection with C. sinensis did't aggravate the clinical manifestations of viral hepatitis in this cross-sectional study.

Detection of residual HCV-RNA in patients who have achieved sustained virological response is associated with persistent histological abnormality.

BACKGROUND: Whether achieving sustained virological response (SVR) in patients with hepatitis C attains complete elimination of hepatitis C virus (HCV) is unknown, because occult HCV infection (OCI), defined as the detection of HCV-RNA in hepatocytes or peripheral blood mononuclear cells (PBMC) in absence of serum HCV-RNA, may occur. We thus investigated the prevalence and clinical relevance of OCI. METHODS: Subjects from three hospitals who had achieved serum HCV clearance, including 60 of Direct-acting antiviral agents (DAAs) induced SVR, 50 of pegylated interferon plus ribavirin (PR) induced SVR, and 30 of spontaneous resolution, were subjected to detect HCV-RNA in liver by robust RNAscope assay and PBMC by qPCR. Paired liver biopsies at baseline and at SVR24 were analyzed. RESULTS: OCI was detected in 16 of 140 subjects (11.4%), with 15.0% in DAA-based group, 10.0% in PR group and 6.7% in spontaneously resolved group. In DAA-based subgroups, the incidence of OCI was gradually increased in group of solely DAA(s) therapy, combining DAA and PR therapy and combining DAA and ribavirin therapy. OCI is more frequent in patients with genotype 3. No correlation between baseline viral load, interleukin-28B genotype, baseline transaminases, post-SVR transaminases and OCI were found. However, OCI was significantly linked with severity of fibrosis and active inflammation at post-SVR, even considering basal fibrosis status. In addition, both the magnitude and the frequency of fibrosis regression were lower in patients with OCI than in those without OCI. In the multivariate analysis, PR therapy was identified an independent negative prognostic factor for both hepatic inflammation (P = .022) and fibrosis regression (P = .015). Importantly, we found HCV relapse in one of the OCI patients at 48 weeks after the end of PR treatment. CONCLUSIONS: HCV-RNA can persist in hepatocytes and/or PBMC in a certain of patients who achieved spontaneous or treatment-induced HCV RNA clearance from serum and associated with persistent histological abnormality. Our findings provide new insights into cure of HCV and could influence the following-up scenario after SVR.

Hepatitis B Virus-Induced Imbalance of Inflammatory and Antiviral Signaling by Differential Phosphorylation of STAT1 in Human Monocytes.

It is not clear how hepatitis B virus (HBV) modulates host immunity during chronic infection. In addition to the key mediators of inflammatory response in viral infection, monocytes also express a high-level IFN-stimulated gene, CH25H, upon response to IFN-α exerting an antiviral effect. In this study, the mechanism by which HBV manipulates IFN signaling in human monocytes was investigated. We observed that monocytes from chronic hepatitis B patients express lower levels of IFN signaling/stimulated genes and higher levels of inflammatory cytokines compared with healthy donors. HBV induces monocyte production of inflammatory cytokines via TLR2/MyD88/NF-κB signaling and STAT1-Ser727 phosphorylation and inhibits IFN-α-induced stat1, stat2, and ch25h expression through the inhibition of STAT1-Tyr701 phosphorylation and in an IL-10-dependent, partially autocrine manner. Further, we found that enhancement of STAT1 activity with a small molecule (2-NP) rescued HBV-mediated inhibition of IFN signaling and counteracted the induction of inflammatory cytokines. In conclusion, HBV contributes to the monocyte inflammatory response but inhibits their IFN-α/ß responsiveness to impair antiviral innate immunity. These effects are mediated via differential phosphorylation of Tyr701 and Ser727 of STAT1.

High level of serum Cripto-1 in hepatocellular carcinoma, especially with hepatitis B virus infection.

BACKGROUND: Human Cripto-1 (CR-1), a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic protein family (EGF-CFC), is highly expressed in a variety of human cancers. We aimed to detect serum CR-1 level in liver diseases especially in hepatocellular carcinoma (HCC) patients. METHODS: Serum CR-1 level was Sandwich-type enzyme-linked immuno sorbent assay (ELISA) detected in 330 patients with liver diseases including HCC, cirrhosis, and chronic hepatitis and 50 volunteers without hepatitis B virus (HBV) or hepatitis C virus (HCV) infection as control. RESULTS: The serum CR-1 level was significantly higher in HCC patients than volunteer controls and it was also significantly higher in HBV-related HCC than HCV-related HCC. In addition, serum CR-1 level was correlated with serum alpha-feto-protein (AFP) in HBV-related HCC patients. The serum CR-1 was also higher in cirrhosis and chronic hepatitis than volunteer controls. The serum CR-1 in HBV-related cirrhosis was higher than chronic hepatitis B, but there was no significant difference between HCV-related cirrhosis and chronic hepatitis C. CONCLUSIONS: Serum CR-1 was higher in HCC patients and might serve as a complementary biomarker to clinical diagnosis of HBV-related HCC. The high level of serum CR-1 in HBV-related liver disease might be partly attributed to HBV infection.

Role of HDAC9-FoxO1 Axis in the Transcriptional Program Associated with Hepatic Gluconeogenesis.

Histone deacetylase 9 (HDAC9) regulates hepatic gluconeogenesis by deacetylating Forkhead box O 1 (FoxO1). HDAC9 upregulation is involved in hepatitis C virus (HCV)-associated exaggerated gluconeogenesis. Herein, we found in addition to FoxO1, HDAC9 also regulates other gluconeogenic transcription factors, including peroxisomeproliferator-activated receptor-γ coactivator-1α (PGC-1α), cyclic AMP-responsive element-binding protein (CREB), and glucocorticoid receptor (GR). Unlike FoxO1, which is regulated by post-translational modification responses to HDAC9, HDAC9 regulates PGC-1α, CREB and GR by altering gene expression. Similar to PGC-1α, CREB and GR were found to be novel regulatory targets of FoxO1 by examination of the FoxO1 binding site in their promoter. PGC-1α, CREB and GR were upregulated in response to HDAC9 via FoxO1 deacetylation. These findings indicate that HDAC9-FoxO1 signalling contributes to gluconeogenesis by modulating the expression of gluconeogenic transcription factors. In particular, metabolic profiling demonstrated a clear shift towards gluconeogenesis metabolism, and HDAC9-FoxO1 signalling can be strongly induced to upregulate gluconeogenic transcription factors following HCV infection. The positive correlation between HDAC9 and gluconeogenic transcription factor expression levels in the livers of both HCV-infected patients and normal individuals further emphasizes the clinical relevance of these results. Thus, HDAC9-FoxO1 signalling axis is involved in regulating gluconeogenic transcription factors, gluconeogenesis, and HCV-induced type 2 diabetes.

Accuracy of M2BPGi, compared with Fibro Scan®, in analysis of liver fibrosis in patients with hepatitis C.

BACKGROUND: Mac-2 Binding Protein Glycosylation isomer (M2BPGi) is a novel serological glyco-biomarker for staging liver fibrosis. Here, we aimed to evaluate the efficiency of serum M2BPGi in identifying liver fibrosis stages in Chinese patients with chronic hepatitis C infection. METHODS: Serum M2BPGi levels were evaluated in 680 patients with chronic hepatitis C and 164 healthy controls who underwent the Fibro Scan® test of liver fibrosis. The diagnostic accuracy of serum M2BPGi values was compared to that of other fibrosis markers, including Fibro Scan®, the aspartate transaminase to platelet ratio index (APRI), the fibrosis index based on four factors (FIB4), and the gamma-glutamyltranspeptidase to platelet ratio (GPR). RESULTS: Among the chronic hepatitis C patients, the median serum M2BPGi level increased with increasing fibrosis score as follows: 0.88 (≤F2), 1.70 (F2/F3), and 5.68 (cirrhosis). M2BPGi concentrations could also distinguish between healthy controls (0.38 ± 0.24) and hepatitis C patients (1.57 ± 2.28). After adjusting for potential confounders, M2BPGi was the most significant factor associated with the liver stiffness measurement (effect size = 0.275, P < 0.001). The optimum cutoff values of serum M2BPGi for patients with F2 and F4 were 0.945 and 1.355, respectively. The area under the curve of serum M2BPGi for prediction of significant fibrosis (F ≥ 4) using was comparable to that of APRI (0.892 vs. 0.873), while it was superior to that of other alternative markers, including FIB4 (0.818) and GPR (0.851). Compared with other non-invasive markers, M2BPGi had the greatest specificity for diagnosing cirrhosis and cirrhosis in hepatitis C patients. CONCLUSIONS: Our results suggest that the level of serum M2BPGi would be a simple and reliable diagnostic tool for identifying liver fibrosis stage in Chinese patients with chronic hepatitis.

Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection. IMPORTANCE: In this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A.

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22's targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.

The Metabolic Regulator Histone Deacetylase 9 Contributes to Glucose Homeostasis Abnormality Induced by Hepatitis C Virus Infection.

Class IIa histone deacetylases (HDACs), such as HDAC4, HDAC5, and HDAC7, provide critical mechanisms for regulating glucose homeostasis. Here we report that HDAC9, another class IIa HDAC, regulates hepatic gluconeogenesis via deacetylation of a Forkhead box O (FoxO) family transcription factor, FoxO1, together with HDAC3. Specifically, HDAC9 expression can be strongly induced upon hepatitis C virus (HCV) infection. HCV-induced HDAC9 upregulation enhances gluconeogenesis by promoting the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, indicating a major role for HDAC9 in the development of HCV-associated exaggerated gluconeogenic responses. Moreover, HDAC9 expression levels and gluconeogenic activities were elevated in livers from HCV-infected patients and persistent HCV-infected mice, emphasizing the clinical relevance of these results. Our results suggest HDAC9 is involved in glucose metabolism, HCV-induced abnormal glucose homeostasis, and type 2 diabetes.

Use of parenteral caffeinum natrio-benzoicum: an underestimated risk factor for HCV transmission in China.

BACKGROUND: Fuyu city in China has a high prevalence of hepatitis C virus (HCV) infection resulting in a high morbidity and mortality from chronic liver disease and hepatocellular carcinoma. This study was conducted to identify the risk factors for HCV infection in Fuyu city. METHODS: Recruitment of study subjects involved a cross-sectional survey using non-random, convenience sampling. Information on demographic variables, risk factors for HCV infection, clinical manifestations, behavioral practices and family history was collected by administering a questionnaire. Anti-HCV antibody was detected using Abbott ARCHITECT i2000SR. HCV infection was confirmed by HCV-RNA testing by the Roche Taqman HCV test. Univariate and multivariate analyses were performed to identify the factors associated with HCV infection. RESULTS: Out of 3,228 persons that participated in the survey, 3,219 were enrolled in the study. The prevalence of HCV infection was 42.1 % (1355/3219). Among 734 patients with chronic HCV infection whose HCV-RNA genotyping was performed, genotype 1b was the most common (58.0 %), followed by genotype 2a (40.2 %), while co-infection with genotypes 1b and 2a was detected in 1.8 % of the subjects. On univariate analysis, male gender, older age, parenteral caffeinum natrio-benzoicum and share syringes (PCNBSS), and nine other factors were significantly associated with HCV infection. After adjusting for potential confounders, male gender, old age, cigarette smoking, lower education level, history of blood transfusion, blood donation, prior dental surgery, and PCNBSS were found to be independently associated with HCV infection. CONCLUSIONS: The prevalence of HCV infection is likely to be high among residents in Fuyu and we observed that genotypes 1b and 2a dominated in the city. Our findings support the hypothesis that PCNBSS which became endemic in Fuyu city during 1970s-1980s is strongly associated with HCV positivity.

Natural Killer p46 Controls Hepatitis B Virus Replication and Modulates Liver Inflammation.

Natural killer (NK) cells play an important role in hepatitis B virus (HBV) infection control, and are regulated by a complex network of activating and inhibitory receptors. However, NK cell activity in HBV patients remains poorly understood. The objective of this study was to investigate the phenotypic and functional characteristics of circulating NK cells in patients during different chronic hepatitis B (CHB) infection stages. We investigated NK cell phenotypes, receptor expression and function in 86 CHB patients and 20 healthy controls. NK cells were purified and NK cell subsets were characterized by flow cytometry. Cytotoxic activity (CD107a) and interferon-gamma (IFN-γ) secretion were examined, and Natural Killer p46 (NKP46) blockade and spontaneous NK cell cytolytic activity against K562, HepG2 and HepG2.215 cell lines was studied. Activating NKp46 receptor expression was higher in inactive HBsAg carriers when compared with other groups (p = 0.008). NKp46 expression negatively correlated with HBV DNA (R = -0.253, p = 0.049) and ALT (R = -0.256, p = 0.045) levels. CD107a was higher in immune-activated groups when compared with immune-tolerant groups (p = 0.039). CD107a expression was related to viral load (p = 0.02) and HBeAg status (p = 0.024). In vitro NKp46 blockade reduced NK cell cytolytic activity against HepG2 and HepG2.215 cell lines (p = 0.02; p = 0.039). Furthermore, NK cells from high viral load CHB patients displayed significantly lower specific cytolytic activity against anti-NKp46-loaded K562 targets (p = 0.0321). No significant differences were observed in IFN-γ secretion (p > 0.05). In conclusion, NKp46 expression regulates NK cell cytolytic function. NKp46 may moderate NK cell activity during HBV replication suppression and HBV-associated liver damage and may be critical for NK cell activity during CHB infection.

Association of serum level of growth differentiation factor 15 with liver cirrhosis and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) and liver cirrhosis are associated with high mortality worldwide. Currently, alpha-fetoprotein (AFP) is used as a standard serum marker for the detection of HCC, but its sensitivity and specificity are unsatisfactory, and optimal diagnostic markers for cirrhosis are lacking. We previously reported that growth differentiation factor 15 (GDF15) was significantly induced in HCV-infected hepatocytes. This study aimed to investigate GDF15 expression and its correlation with hepatitis virus-related liver diseases. A total of 412 patients with various liver diseases were studied. Healthy and Mycobacterium tuberculosis-infected subjects were included as controls. Serum and tissue GDF15 levels were measured. Serum GDF15 levels were significantly increased in patients with HCC (6.66±0.67 ng/mL, p<0.0001) and cirrhosis (6.51±1.47 ng/mL, p<0.0001) compared with healthy controls (0.31±0.01 ng/mL), though the GDF15 levels in HBV and HCV carriers were moderately elevated (1.34±0.19 ng/mL and 2.13±0.53 ng/mL, respectively). Compared with HBV or HCV carriers, GDF15 had a sensitivity of 63.1% and a specificity of 86.6% at the optimal cut-off point of 2.463 ng/mL in patients with liver cirrhosis or HCC. In HCC patients, the area under the receiver operating curve was 0.84 for GDF15 and 0.76 for AFP, but 0.91 for the combined GDF15 and AFP. Serum GDF15 levels did not significantly differ between the high-AFP and low-AFP groups. GDF15 protein expression in HCC was significantly higher than that in the corresponding adjacent paracarcinomatous tissue and normal liver. Using a combination of GDF15 and AFP will improve the sensitivity and specificity of HCC diagnosis. Further research and the clinical implementation of serum GDF15 measurement as a biomarker for HCC and cirrhosis are recommended.

IL28B is associated with outcomes of chronic HBV infection.

PURPOSE: The role of IL28B gene variants and expression in hepatitis B virus (HBV) infections are not well understood. Here, we evaluated whether IL28B gene expression and rs12979860 variations are associated with HBV outcomes. MATERIALS AND METHODS: IL28B genetic variations (rs12979860) were genotyped by pyrosequencing of DNA samples from 137 individuals with chronic HBV infection [50 inactive carriers (IC), 34 chronic hepatitis B (CHB), 27 cirrhosis, 26 hepatocellular carcinoma (HCC)], and 19 healthy controls. IL28A/B mRNA expression in peripheral blood mononuclear cells was determined by qRT-PCR, and serum IL28B protein was measured by ELISA. RESULTS: Patients with IL28B C/C genotype had greater IL28A/B mRNA expression and higher IL28B protein levels than C/T patients. Within the various disease stages, compared to IC and healthy controls, IL28B expression was reduced in the CHB, cirrhosis, and HCC cohorts (CHB vs. IC, p=0.02; cirrhosis vs. IC, p=0.01; HCC vs. IC, p=0.001; CHB vs. controls, p<0.01; cirrhosis vs. controls, p<0.01; HCC vs. controls, p<0.01). When stratified with respect to serum HBV markers in the IC and CHB cohorts, IL28B mRNA and protein levels were higher in HBeAg-positive than negative individuals (p=0.01). Logistic regression analysis revealed that factors associated with high IL28B protein levels were C/C versus C/T genotype [p=0.016, odds ratio (OR)=0.25, 95% confidence interval (CI)=0.08-0.78], high alanine aminotransferase values (p<0.001, OR=8.02, 95% CI=2.64-24.4), and the IC stage of HBV infection (p<0.001). CONCLUSION: Our data suggest that IL28B genetic variations may play an important role in long-term development of disease in chronic HBV infections.

Hepatitis C virus core protein triggers expansion and activation of CD4(+)CD25(+) regulatory T cells in chronic hepatitis C patients.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) are increased in patients with chronic hepatitis C, which may contribute to the sustained suppression of hepatitis C virus (HCV)-specific T-cell responses and viral persistence in HCV-infected individuals. We postulated that HCV core protein (HCVc) directly contributes to the expansion of Tregs in HCV-infected patients, and we provide evidence to support this hypothesis in the report. Peripheral blood mononuclear cells (PBMCs) and sera were collected from 87 treatment-naïve chronic HCV-infected patients, CD4(+)CD25(+) Tregs were measured by flow cytometry, and HCV RNA and HCVc levels were detected using qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. CD4(+), CD8(+), CD4(+)CD25(+) and CD4(+)CD25(-) T cells were purified from healthy donors and cultured with recombinant HCVc and Toll-like receptor (TLR) ligands. Flow cytometry was used to analyze cell proliferation, and ELISA was performed to measure cytokine production. In the 87 chronic HCV-infected patients, HCVc showed a significant correlation with HCV RNA and CD4(+)CD25(+) Tregs. Mechanistic studies showed that HCVc, together with anti-CD3 antibody, augmented CD4(+)CD25(+) Treg proliferation, but inhibited CD4(+)CD25(-) T-cell proliferation and IFN-γ production, in a dose-dependent and Treg-dependent manner. Moreover, unlike the TLR3 ligand (poly I:C) and the TLR4 ligand (lipopolysaccharide, LPS), the TLR2 ligand (lipoteichoic acid, LTA) and HCVc both inhibited TCR-induced CD4(+) T-cell proliferation and IFN-γ secretion in a Treg-dependent manner. These data indicate that HCVc, like other TLR2 ligands, triggers CD4(+)CD25(+) Treg activation and expansion to inhibit host immune responses, which may play a critical role in viral persistence in HCV-infected patients.

Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice.

The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O(Tg)) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O(Tg) mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT.

The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3'UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.