Progression Of Chronic Hepatitis B Virus Infection Research Articles

Perspectives on the Underlying Etiology of HCC and Its Effects on Treatment Outcomes.

Hepatocellular carcinoma (HCC) continues to be a serious medical problem with poor prognosis worldwide. The distribution of the major etiologies of HCC is changing due to the progress of anti-viral treatments, including hepatitis B virus (HBV) suppression by nucleoside/nucleotide analogues (NAs) and increased sustained virologic response (SVR) rates by direct-acting antivirals (DAAs) for hepatitis C virus (HCV), as well as the rising trend of nonviral liver disease. Although viral hepatitis remains the most common cause of HCC, non-alcoholic liver disease (NAFLD) with metabolic syndrome and alcohol-associated liver disease (ALD) are increasing. Effective and well-tolerated NAs treatment can slow the disease progression of chronic HBV infection to cirrhosis, end-stage liver disease, and reduce HCC risk. Treatment with NAs is also associated with significant improvement in the long-term survival of patients with HBV infection who already have HCC. DAAs have achieved viral elimination in almost all patients with HCV without significant adverse events, even in patients with decompensated liver cirrhosis and HCC. Similarly, DAA therapy can reduce disease progression, liver and non-liver complications, and improve the long-term survival of patients with chronic HCV infection with or without HCC. Meanwhile, NAFLD is a rapidly increasing cause of HCC along with the epidemics of obesity and type 2 diabetes globally. NAFLD-related HCC can occur in patients without cirrhosis and is known to have a lower survival rate than viral hepatitis-related HCC. Since there is currently no specific pharmacotherapy effective for NAFLD, lifestyle modification and prevention of complications are important to improve prognosis. Additionally, ALD is the second fastest-growing cause of HCC-related deaths, especially with an accelerated trend since the COVID-19 pandemic. This review provides an overview of the epidemiologic trends in the etiologies of HCC, and the progress of treatments for each etiology and the impact on outcome in the patients with HCC.

Circulating soluble CD36 as a novel biomarker for progression and prognosis of HBV-related liver diseases.

Our previous study suggested CD36 may be a positive regulator of hepatitis B virus (HBV) replication in vitro. Therefore, the present study aimed to investigate whether circulating soluble CD36 (sCD36) could serve as a diagnostic and prognostic biomarker for HBV-related liver diseases based on the clinic collected data. A total of 282 subjects were divided into healthy controls (HC, n = 47), chronic hepatitis B (CHB, n = 68), HBV-related liver cirrhosis (HBV-LC, n = 167). Soluble CD36 in plasma was measured by ELISA, and monocyte or platelet CD36 expression was determined by flow cytometry. There was a step-wise increase of sCD36 with the progression of chronic HBV infection, and it was the highest in the HBV- LC group with liver failure (1.50, IQR:1.04-2.00) as compared with HC (0.38, IQR:0.27-0.38), CHB (0.75, IQR:0.40-1.13), and HBV-LC without liver failure (1.02, IQR,0.61-1.35) group. Circulating sCD36 was not correlated with serum HBV DNA levels, but correlated with liver function parameters. Additionally, ROC analysis confirmed sCD36 could be used to predict liver failure for HBV-LC patients, which yielded an AUC of 0.775 with 71.0% sensitivity and 72.2% specificity. Multivariate logistic regression analysis revealed sCD36 is an independent risk factor in predicting liver failure. Moreover, plasma sCD36 in HBV-LC patients was significantly correlated with prognostic indices, including MELD, MELD-Na and CHILD-PUGH scores. On the other hand, CD36 expression on monocytes or platelets was positively correlated with plasma sCD36 levels, whereas they were not strongly associated with the disease severity. Circulating sCD36 could be used as a novel noninvasive biomarker for predicting liver failure and prognosis in chronic HBV infected patients.

Clinical Significance and Properties of IFN-γ+IL-17+ Th17 Cells in Liver Injury Associated with Chronic Hepatitis B Virus Infection

Introduction: Our previous study demonstrated that intrahepatic Th17 cells exacerbated the progression of chronic hepatitis B virus (HBV) infection. Meanwhile, we found a small group of IFN-γ and IL-17 double-positive Th17 cells (IFN-γ⁺IL-17⁺ Th17 cells) in liver tissues. This study aimed to investigate the clinical significance and properties of IFN-γ⁺IL-17⁺ Th17 cells in liver injury associated with chronic HBV infection. Methods: The frequencies of CD4⁺ Th cells, Tregs, and CD4⁺ T cells expressing specific chemokine receptors in the blood and liver tissues were detected using flow cytometry. The chemotaxis of C C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3) toward IFN-γ⁺IL-17⁺ Th17 cells and Tregs was evaluated by transwell chemotactic assay. Analyses of different variables were performed using GraphPad Prism v 5.01 and IBM SPSS Statistics 23.0. HBV-specific IFN-γ⁺IL-17⁺ Th17 cells were investigated using a cell stimulation assay with HBV antigens in vitro. Results: The frequencies of IFN-γ⁺IL-17⁺ Th17, Th17 cells, and Tregs in the blood were increased from normal controls to chronic hepatitis B (CHB) and acute-on-chronic liver failure (ACLF). The same trend could also be observed in CHB liver tissues compared to those in CHB blood specimens. Furthermore, the frequencies of IFN-γ⁺IL-17⁺ Th17 cells were positively associated with Th17 cells, Th17 cell-related cytokines (IL-17 and IL-6), HBV DNA load, and the levels of HBsAg, HBeAg, and ALT. The ratios of IFN-γ⁺IL-17⁺ Th17 cells to Tregs extremely decreased in ACLF blood specimens compared with those in CHB blood specimens. Additionally, CCR5 and CXCR3 were conducive to the recruitment of IFN-γ⁺IL-17⁺ Th17 cells and Tregs to liver tissue. Conclusions: IFN-γ⁺IL-17⁺ Th17 cells have Th17 cell-like properties in the progression of chronic HBV infection. CCR5 and CXCR3 facilitated the recruitment of IFN-γ⁺IL-17⁺ Th17 cells and Tregs to the liver. Importantly, the ratio of IFN-γ⁺IL-17⁺ Th17 cells to Tregs might be an effective assessment indicator of the severity of liver injury.

Effect of microbiota metabolites on the progression of chronic hepatitis B virus infection.

Accumulating evidence shows that the intestinal microbiota is closely related to the pathophysiology and the disease progression of chronic hepatitis B virus (HBV) infection. The intestinal microbiota acts on the host through its metabolites. This review aimed to discuss the effects of gut microbiota metabolites on the disease progression of chronic HBV infection. A literature search on PubMed database and Wiley Online Library with pre-specified criteria yielded 96 unique results. After consensus by all authors, the contents from 86 original publications were extracted and included in this review. In liver disease with HBV infection, the intestinal microbiota changed in different stages and affected the production of bacterial metabolites. The abundance of bacteria producing short-chain fatty acids such as butyrate reduced, which was associated with bacterial translocation and the progression of liver disease. The intestinal microbiota-bile acid-host axis was destroyed, affecting the progression of the disease. Under the control of intestinal microbiota, tryptophan affected the gut-liver axis through three main metabolic pathways, among which the kynurenine pathway was closely related to the immune response of hepatitis B. The level of trimethylamine-N-oxide decreased in liver cancer with HBV infection and were used as a potential biomarker of liver cancer. Vitamin deficiencies, including those of vitamin D and vitamin A related to microbiota, were common and associated with survival. Hydrogen sulfide regulated by the intestinal microbiota was also closely related to the gut-liver axis. In liver disease with hepatitis B infection, the intestinal microbiota is imbalanced, and a variety of intestinal microbiota metabolites participate in the occurrence and development of the disease.

Clinical Value of Plasma Secreted Frizzled-Related Protein 4 in the Chronic Hepatitis B Virus Infection: A Cross-sectional Study

Background: Secreted frizzled-related protein 4 (sFRP4) is elevated in hepatocellular carcinoma (HCC) patients, suggesting that it can be served as a candidate marker for diagnosing HCC. However, little is known about its role in the different stages of chronic hepatitis B virus (HBV) infection. Objectives: This study was conducted to explore the clinical value of plasma sFRP4 in the different stages of chronic HBV infection. Methods: A total of 303 patients with chronic HBV infection were enrolled in this cross-sectional study. They were classified into the chronic hepatitis B (CHB), liver cirrhosis (LC), HCC, and acute-on-chronic liver failure (ACLF) groups on admission. Additionally, 30 healthy subjects were included in the healthy control (HC) group. The clinical value of plasma sFRP4 in the different stages of chronic HBV infection was analyzed. Results: There were 54, 85, 105, 59, and 30 cases in the CHB, LC, HCC, ACLF, and HC groups, respectively. ACLF group had the highest plasma sFRP4 levels compared to the CHB, LC, and HCC groups (all P < 0.001), followed by the HCC and LC groups. LC and HCC groups were found with up-regulated sFRP4 than the CHB group (all P < 0.05). High levels of plasma sFRP4 were recognized as an independent risk factor for distinguishing patients with ACLF from patients with CHB and LC [adjusted odds ratio (OR):1.005, 95% confidence interval (CI): 1.000 - 1.010, P = 0.043], with the area under the receiver operating characteristic curve (AUC) of 0.790 (95% CI: 0.726 - 0.844, P < 0.001). However, in patients with ACLF, plasma sFRP4 levels in the deteriorated group were higher than in the improved group, with a marginally significant difference (P = 0.071). The AUC for predicting the 90 days prognosis in patients with ACLF was 0.640 (P = 0.064). Conclusions: Plasma sFRP4 might be a biomarker to reflect the progression of chronic HBV infection. However, it was not significantly related to the prognosis in patients with ACLF; we did not find this, which may be due to the small sample size.

Integrated Analysis of Long Noncoding RNA Expression Profiles in Acute-on-Chronic Liver Failure.

People infected with chronic hepatitis B virus (HBV) might progress to acute-on-chronic liver failure (ACLF) with a high fatality rate. Long noncoding RNAs (lncRNAs) are involved in human diseases, but it is unknown whether lncRNAs are involved in the progression of chronic HBV infection to ACLF. Hence, this study is aimed at systemically identifying and characterizing the landscape and the molecular mechanism of lncRNAs in the pathogenesis of chronic HBV infection progress to ACLF. RNA sequencing (RNA-Seq) of peripheral blood samples from 5 ACLF and 5 HBV infection patients was performed. We detected 9733 lncRNAs, including 406 annotated lncRNAs and 9327 novel lncRNAs. A total of 407 lncRNAs were found to be significantly dysregulated in the patients with ACLF as compared with those in the chronic HBV infection patients. The flanking protein-coding genes of differentially expressed lncRNAs were enriched with pathways that might contribute to the pathogenesis of ACLF, such as the WNT signaling pathway. Furthermore, 9 selected differentially expressed lncRNAs validated by the qRT-PCR, showing that the expression patterns of these 9 lncRNAs were consistent with the RNA-Seq data. Four selected differentially expressed lncRNAs were also validated in another patient cohort comprising 80 patients with ACLF and 65 patients with chronic HBV infection. Aberrant lncRNAs might be used to develop novel diagnostic biomarkers or drug targets for ACLF.

IL-35: A Novel Immunomodulator in Hepatitis B Virus-Related Liver Diseases.

Chronic hepatitis B virus (HBV) infection is a risk factor for liver cirrhosis (LC) and hepatocellular carcinoma (HCC), however, little is known about the mechanisms involved in the progression of HBV-related diseases. It has been well acknowledged that host immune response was closely related to the clinical outcomes of patients with HBV infection. As the factors closely related to the immunomodulatory process, cytokines are crucial in the cell-cell communication and the host responses to HBV infection. Recently, a newly discovered cytokine, designated as interleukin-35 (IL-35), has been proved to be essential for the progression of chronic HBV infection, the development of cirrhosis, the transformation of cirrhosis to HCC, and the metastasis of HCC. Specifically, it showed various biological activities such as inhibiting the HBV-specific cytotoxic T lymphocyte (CTL) proliferation and cytotoxicity, deactivating the immature effector T-cells (Teffs), as well as delaying the proliferation of dendritic cells. It regulated the immune responses by acting as a “brake” on the activation of Teffs, which subsequently played important roles in the pathogenesis of various inflammatory diseases and malignancies. In this review, we focused on the most recent data on the relationship between IL-35 and chronic HBV infection, LC and HCC.

Risk factors associated with the progression of chronic HBV infection in patients with normal ALT

Hepatitis B virus (HBV) infection is one of the common causes of chronic hepatitis in China. Active antiviral therapy can effectively reduce the occurrence risk of end-stage liver diseases, such as decompensated cirrhosis, liver failure and hepatocellular carcinoma. The existing international and domestic guidelines for the prevention and treatment of chronic hepatitis B recommends regular follow-up for patients with normal alanine aminotransferase (ALT). Studies have shown that a high percentage (25.4%-88.9%) of patients with chronic HBV infection have normal ALT levels; however, hepatic histopathology revels obvious inflammation, fibrosis (G≥2 and/or S≥2), cirrhosis and liver cancer. In this paper, we reviewed the relatively insidious risk factors associated with the progression of chronic HBV infection in patients with normal ALT.

Differential expression profile of long noncoding RNAs in chronic HBV infection: New insights into pathogenesis.

Increasing studies have revealed that long noncoding RNAs (lncRNAs) might play vital roles in the development and progression of various diseases including viral infectious diseases. However, the expression and biological functions of lncRNAs in chronic hepatitis B virus (HBV) infection remain largely unknown. Therefore, lncRNA microarray was performed to analyze the lncRNAs' and messenger RNAs' (mRNAs) expression profiles in liver tissues from patients with chronic HBV infection. Subsequently, a comprehensive bioinformatics analysis was conducted to investigate the potential functions of the differentially expressed genes. As a result, a total of 203 differentially expressed lncRNAs and 180 mRNAs were identified in chronic HBV infection. The expressions of five differentially expressed lncRNAs were further validated using quantitative real-time polymerase chain reaction. Gene ontology, pathway analysis, and gene set enrichment analysis revealed that differentially expressed lncRNAs might be mainly be involved in cytokine-cytokine receptor interaction and varied biotransformation processes, including fatty acid metabolism, amino acid metabolism, carbon metabolism, and drug metabolism. Additionally, coexpression networks between differentially expressed lncRNAs and mRNAs were constructed to reveal the hub regulator and analyze the functional pathways. This study provided an overview of lncRNA and mRNA expression in liver tissues from patients with chronic HBV infection. These differentially expressed lncRNAs might play crucial roles in the pathogenesis and progression of chronic HBV infection, which deserve further investigation.

Factors associated with disease progression and viral replication in patients with chronic hepatitis B virus infection.

Hepatitis B virus (HBV) infection remains a severe clinical concern in China. Of note, the progression of HBV infection varies between different populations. To identify the factors that influence the disease progression and prognosis, a total of 478 chronic HBV-infected patients were enrolled, and liver function parameters, HBV DNA levels and hepatic fibrosis indices were analyzed. First, the results demonstrated a significant difference in hepatitis B e antigen (HBeAg) expression between male and female patients (χ2=4.061, P=0.044). Furthermore, when comparing either HBeAg-negative or -positive male and female patients, males exhibited a greater variation in HBV DNA levels. Although significant differences between male and female patients in certain abnormal ratios of liver function parameters were identified, a trend in the differences was observed in the HBeAg-negative and -positive groups. When considering age, the results of the present study confirmed that HBV DNA levels decreased with advanced age, and the values of the majority of biomarkers exhibited an evident decreasing trend with increasing age. In addition, it was demonstrated that all HBeAg seropositive patients had higher levels of hepatic fibrosis indexes and higher abnormal ratios of hepatic fibrosis values in their serum when compared with those of HBeAg seronegative patients, particularly with regard to serum IV collagen. The present results revealed that HBV DNA replication was closely associated with liver function; however, it was notable that in HBeAg-negative patients, the association between HBV DNA levels and liver function was particularly significant among subjects aged <61. Furthermore, this result was not observed in HBeAg-positive patients. In conclusion, the present study indicated the importance of host factors (including sex and age) and viral factors (including HBeAg expression pattern and HBV DNA levels) in the progression of chronic HBV infection, and its influence regarding prognosis and treatment. The present results provide a foundation for clinical management strategies for chronic HBV infection, particularly in individual schemes.

Hepatitis B virus large surface protein is priming for hepatocellular carcinoma development via induction of cytokinesis failure.

Chronic hepatitis B virus (HBV) infection is a main risk factor for development of liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV vaccination and antiviral therapy lead to substantial risk reduction for HCC development, it is evident that both can reduce, but not completely eliminate the risk. High serum levels of HBV surface antigen (HBsAg) were shown to predict disease progression of chronic HBV infection in patients harboring low viral load, and in line with this, HBV surface proteins were shown to exert oncogenic functions. As HBsAg seroclearance is infrequently achieved in patients who have undergone antiviral therapy, it is necessary to gain further insights into molecular mechanisms of HBsAg seroclearance failure after antiviral therapy and HCC development mediated by HBV surface proteins. A recent study published in this journal has shown that the HBsAg large surface protein (LHBs) contributes to HCC development by inducing cytokinesis failure and consequent aneuploidy via induction of DNA damage and polo-like kinase 1 (PLK1)-mediated G2/M checkpoint failure in hepatocytes. Inhibition of PLK1 by a PLK1-specific small molecule inhibitor was shown to restore G2/M checkpoint in vitro and to reduce tumor burden in vivo. The initial LHBs-induced hepatocyte aneuploidy may give rise to further aneuploidy and thereby lead to self-propagating cycles of chromosomal instability driving intra-tumor heterogeneity and clonal cancer evolution. Thus, LHBs-induced cytokinesis failure may be a priming event for HCC development. In conclusion, the study not only provides further mechanistic insights into the oncogenic role of LHBs, but also identifies a potential target to interfere with the vicious circle of LHBs-induced aneuploidy, which may be especially useful in patients showing failure of HBsAg seroclearance after antiviral therapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Programmed cell death-1 3'-untranslated region polymorphism is associated with spontaneous clearance of hepatitis B virus infection.

Hepatitis B virus (HBV)-specific CD8+ T cells play an important role in the clearance of HBV infection. Programmed cell death-1 (PD-1), an immunosuppressive molecule that regulates T-cell activation and peripheral immune tolerance, is increasingly shown to influence the outcome of HBV infection. rs10204525, a single-nucleotide polymorphism in the 3'-untranslated region (3'-UTR) of PD-1, has been associated with susceptibility and disease progression of chronic HBV infection in far-eastern patients. The aim of our study was to assess the impact of rs10204525 variation on HBV infection in Moroccan patients. A total of 236 patients with chronic HBV infection and 134 individuals with spontaneous HBV resolution were genotyped using a Taqman assay. In addition, PD-1 mRNA expression in peripheral blood nuclear cells was determined by quantitativereverse-transcription polymerase chain reaction. We found that the AA genotype is protective (odds ratio, 0.43; 95% confidence interval, 0.19 to 0.97; P = 0.038) against HBV infection. Interestingly, PD-1 messenger RNA (mRNA) expression analysis has revealed that chronic HBV carriers with GG and GA displayed higher levels of PD-1 mRNA compared with corresponding genotypes in resolved subjects (P = 0.031 and0.014, respectively). Our data suggest that Mediterranean HBV-infected patients carrying PD-1 GG and GA genotypes at rs10204525 have high PD-1 mRNA expression and may be more prone to installation of chronicity.

Omega-6-derived oxylipin changes in serum of patients with hepatitis B virus-related liver diseases.

Chronic hepatitis B virus (HBV) infection is the main etiologic risk factor for hepatocellular carcinoma (HCC). Early studies indicated that the increase of omega-6-derived oxylipins may be involved in the pathogenesis of HBV-related HCC, yet their changes during the distinct clinical phases of chronic HBV infection remain unclear. To fill this gap, in this study we investigated the omega-6-derived oxylipin profiles in patients with three major clinical stages of chronic HBV infection (chronic hepatitis B, liver cirrhosis, and HCC). Eighteen omega-6-derived oxylipins were quantified in serum samples of 34 patients with chronic hepatitis B, 46 patients with HBV-related liver cirrhosis, 38 patients with HBV-related HCC, and 50 healthy controls using liquid chromatography tandem mass spectrometry. Seven oxylipins were found to be altered in patients with HBV-related liver diseases, including 9,10-dihydroxyoctadecenoic acid (9,10-DiHOME), 12,13-DiHOME, 14,15-dihydroxyeicosatrienoic acid (14,15-DiHETrE), 13-hydroxyoctadecadienoic acid (13-HODE), 12-hydroxyeicosatetraenoic acid (12-HETE), 11-HETE, and thromboxane B2 (TXB2). Of these, three oxylipins derived from the cytochrome P450 (CYP450) pathways including 9,10-DiHOME, 12,13-DiHOME, and 14,15-DiHETrE were found to be associated with the levels of α-fetoprotein (AFP), a tumor marker. In combination with AFP, age, and gender, a combination of these seven differential oxylipins could significantly enhance the prediction of HBV-related liver diseases, particularly for liver cirrhosis (p < 0.05). This study for the first time shows the correlations between CYP450-derived oxylipins and the progression of chronic HBV infection, and sheds a new light on the surveillance of HBV-related live diseases using oxylipins.

The Influence of B and T Lymphocyte Attenuator Genetic Variants on Susceptibility to Chronic Hepatitis B Virus Infection

Background/Aims: B and T lymphocyte attenuator (BTLA) is an immune inhibitory receptor involved in the pathogenesis of chronic viral infections. Little is known about the effects of BTLA gene polymorphisms on chronic hepatitis B virus (HBV) infections. In this study, we investigated whether the polymorphisms of BTLA are associated with the progression of chronic HBV infection. Methods: A total of 382 chronic HBV carriers and 170 healthy individuals in the same region were recruited for this study. The chronic HBV carriers were divided into three groups: asymptomatic HBV carriers (ASC), moderate chronic hepatitis B group (MCHB), and severe chronic hepatitis B group (SCHB). Two BTLA functional single nucleotide polymorphisms (SNPs; rs76844316 and rs9288952) were genotyped by polymerase chain reaction and sequenced directly. Results: The results showed that the frequency of the G allele of rs76844316 was significantly lower in the SCHB group than in the other three groups. Subjects bearing at least one G allele (TG or GG genotype) at rs76844316 had decreased susceptibility to severe chronic hepatitis B compared with those bearing the TT genotype. Haplotype analysis of the two SNPs revealed that the frequency of the G-G haplotype was significantly lower in SCHB patients than in controls. Moreover, in the SCHB group, patients carrying the G allele of rs76844316 tended to have lower ALT levels than those without it. Conclusion: Our findings suggest that the genetic variants of rs76844316 in BTLA influence the susceptibility to severe chronic hepatitis B and might play a protective role against the progression of chronic hepatitis B.

Expression and role of interleukin 35-producing B cells and interleukin 35 from peripheral blood of patients with chronic hepatitis B

Objective To explore the level of interleukin 35-producing B cells (i35-Breg) as well as its effect factors, interleukin-35 (IL-35), in peripheral blood of patients with chronic hepatitis B (CHB), and their relationship with hepatitis B virus(HBV) DNA and liver inflammatory degree. Methods A total of 35 treatment-naive CHB patients, 17 interferon (IFN)-treated HBeAg-positive CHB patients and 15 healthy controls (HC) were enrolled. The levels of i35-Breg and IL-35 in peripheral blood were tested by flow cytometry and enzyme-linked immunosorption assay (ELISA). Kruskal-Wallis test, Wilcox rank sum test and two variables correlation analysis were used for statistical analysis. Results The percentage of i35-Breg cells as well as IL-35 level in peripheral blood of naive CHB patients were 3.05% (0.89%, 4.97%) and 2.81 μg/L (0.30 μg/L, 12.33 μg/L), respectively, which were both significantly higher than those in HC group, which were 0.17% (0.13%, 0.45%) and 0.17 μg/L(0, 1.93 μg/L), respectively. The difference were statistical significant (Z=-3.309 and -2.419, respectively, P=0.001 and 0.016, respectively). The peripheral level of i35-Breg was negatively correlated with the viral load in treatment-naive CHB patients (r=-0.529, P=0.008), while there was no correlation between the peripheral level of IL-35 and the viral load in treatment-naive CHB patients (r=0.11, P=0.54). The levels of i35-Breg and IL-35 in HBeAg positive CHB patients were 3.16% (1.34%, 5.62%) and 4.58 μg/L (0.79 μg/L, 22.37 μg/L), respectively, which were both higher than those in HC group. The difference was statistically significant (F=3.39 and 3.37, respectively, both P<0.01). Compared to HC group, the IL-35 levels in peripheral blood of CHB patients with ALT and AST levels less than 300 U/L were 3.03 μg/L (0.74 μg/L, 22.37 μg/L) and 3.25 μg/L(0.83 μg/L, 22.35 μg/L), respectively, with statistically significant difference (F=2.868 and 3.114, respectively, both P<0.01). Compared to HC group, the peripheral level of i35-Breg in treatment-naive CHB patients with ALT levels less than 300 U/L was 3.14% (1.03%, 4.65%), with statistically significant difference (F=3.219, P=0.004). The IL-35 level showed a decreased trend in CHB who received IFN therapy, but there was no statistically significant difference (χ2=1.45, P=0.48). Furthermore, the baseline IL-35 level in patients who developed sustained viral response (SVR) was 0 (0, 13.33 g/L), which was lower than that in patients who developed partial or primary no response 0.61 μg/L (0, 24.72 μg/L). However, there was no statistical difference (F=0.75, P=0.68). Conclusions i35-Breg as well as its effect factor, IL-35, are involved in the progression of chronic HBV infection. The percentage of i35-Breg cells as well as IL-35 level in peripheral blood of treatment-naive CHB patients are increased. The peripheral level of i35-Breg is negatively correlated with the viral load, while there is no correlation between the peripheral level of IL-35 and the viral load. The percentage of i35-Breg cells as well as IL-35 level in CHB patients with low inflammatory degree are increased. Key words: Hepatitis B, chronic; Interleukin-35; IL-35-producing B cells

Altered expression of A20 gene in peripheral blood mononuclear cells is associated with the progression of chronic hepatitis B virus infection.

A20 is an important negative immune regulator but its role in chronic hepatitis B virus (HBV) infection is still unknown. This present study was to investigate the potential role of A20 gene in the progression of chronic HBV infection. A total of 236 chronic HBV patients were included and consisted of 63 hepatocellular carcinoma (HCC), 87 liver cirrhosis (LC) and 86 chronic hepatitis B (CHB). The mRNA level of A20 gene in peripheral blood mononuclear cells was determined using quantitative real-time polymerase chain reaction. Receptor operating characteristic curve (ROC) was performed to determine the diagnostic value of A20 mRNA in different stages of chronic HBV infection. A20 mRNA levels in all HBV patients were significantly higher than healthy controls (n=30), of whom HCC and LC patients showed higher A20 mRNA level than CHB patients. In CHB patients, A20 mRNA was closely associated with alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin. In LC patients, A20 mRNA was significantly associated with ALT, AST, albumin, haemoglobin and platelet. In HCC patients, elevated A20mRNA was also observed in patients with vascular invasion, liver cirrhosis and ascites, compared with those without. ROC analysis revealed that A20 mRNA could effectively discriminate LC from CHB, decompensated LC from compensated LC, and HCC from CHB. In conclusion, A20 mRNA expression in peripheral blood mononuclear cells was associated with dynamic progression of chronic HBV infection. A20 gene might be a potential biomarker to determine the different stages of chronic HBV infection.

Hepatitis B virus genotypes A and D in Uganda

BackgroundThe prevalence of hepatitis B virus (HBV) infection in Uganda is 10%. Hepatitis B virus genotypes impact on treatment response, rate of spontaneous recovery and progression of chronic HBV infection and hepatocellular carcinoma. There is little information on the HBV genotypic distribution in Uganda. ObjectivesTo determine HBV genotypes in Uganda. MethodsThe MBN clinical laboratory performs HBV viral load and genotype testing in Uganda. It receives hepatitis B surface antigen (HBsAg)-positive samples from all over the country for additional HBV testing. Samples are stored for 6 months before being discarded. Our study used delinked stored samples. PCR-positive samples had DNA extracted and used as template for HBV genome amplification by nested PCR. Reverse hybridisation was performed and genotypes were determined by the line probe assay method (INNO-LiPA). ResultsOne hundred stored HBsAg-positive plasma samples with detectable viral loads were analysed. Of these, 93 samples showed PCR amplification products and gave genotype-specific probe lines on the INNO-LiPA assay. Of the patients, where gender was recorded, 60.9% were female, and the overall median age (IQR) was 25 (2–60) years. There was a predominance of HBV genotype D (47 patients; 50.5%), followed by genotype A, (16 patients; 17.2%). One patient (1.1%) had genotype E. In 28% of the samples mixed infections were detected with genotypes A/E (9.7%) and A/D (6.5%) being most common. Genotypes B, C, E and H only occurred as part of mixed infections. ConclusionHepatitis B genotypes D and A were predominant in our study population.

Association of CD40 -1C/T Polymorphism in the 5′-Untranslated Region with Chronic HBV Infection

Background: CD40 is an important costimulatory molecule in both celluar and humoral immune responses, involved in the pathogenic processes of chronic inflammatory diseases. Few studies were performed on the association of CD40 single nucleotide polymorphism (SNP) with chronic hepatitis B virus (HBV) infection. In this study, we studied whether the CD40-1C/T polymorphism had any effect on the progression of chronic HBV infection in Chinese population. Methods: CD40 -1C/T polymorphism in the 5′-untranslated region was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 453 chronic HBV carriers, who were divided into asymptomatic HBV carriers (ASC), moderate chronic hepatitis B group (MCHB) and severe chronic hepatitis B group (SCHB). 202 healthy individuals in the same region were enrolled in this study as the controls. The CD40 expression on B lymphocytes was detected by flow cytometry. The concentrations of soluble CD40 (sCD40) in sera were assayed by a commercial ELISA kit. Results: Our results showed the frequencies of TT genotype and T allele of CD40-1C/T polymorphism were higher significantly in ASC than those in controls (P< 0.05), while this result was not found in either MCHB or SCHB. On the surface of B lymphocytes, the CD40 expression levels in the individuals with TT genotype were significantly lower than those with CC and CT genotypes in either ASC group or healthy controls (P<0.001). The sCD40 levels in the sera of ASC, MCHB and SCHB groups were significantly higher than the controls (P<0.001). Conclusions: The CD40 -1C/T polymorphism may contribute to the susceptibility of asymptomatic HBV carriers through its effect on cell-surface CD40 expression, which indicated CD40 signaling was involved in immune tolerance of chronic HBV infection.

NK cell receptor imbalance and NK cell dysfunction in HBV infection and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer mortality and a common poor-prognosis malignancy due to postoperative recurrence and metastasis. There is a significant correlation between chronic hepatitis B virus (HBV) infection and hepatocarcinogenesis. As the first line of host defense against viral infections and tumors, natural killer (NK) cells express a large number of immune recognition receptors (NK receptors (NKRs)) to recognize ligands on hepatocytes, liver sinusoidal endothelial cells, stellate cells and Kupffer cells, which maintain the balance between immune response and immune tolerance of NK cells. Unfortunately, the percentage and absolute number of liver NK cells decrease significantly during the development and progression of HCC. The abnormal expression of NK cell receptors and dysfunction of liver NK cells contribute to the progression of chronic HBV infection and HCC and are significantly associated with poor prognosis for liver cancer. In this review, we focus on the role of NK cell receptors in anti-tumor immune responses in HCC, particularly HBV-related HCC. We discuss specifically how tumor cells evade attack from NK cells and how emerging understanding of NKRs may aid the development of novel treatments for HCC. Novel mono- and combination therapeutic strategies that target the NK cell receptor-ligand system may potentially lead to successful and effective immunotherapy in HCC.Cellular & Molecular Immunology advance online publication, 6 October 2014; doi:10.1038/cmi.2014.91.

Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein

Chronic hepatitis B virus (HBV) infection is a major risk factor for developing liver cancer, and the HBV X protein (pX) has been implicated as a cofactor in hepatocyte transformation. We have shown that HBV replication as well as in vitro transformation by pX are associated with induction of the mitotic polo-like kinase 1 (Plk1) and down-regulation of the chromatin remodeling components Suz12 and Znf198. Herein, we demonstrate the same inverse relationship between Plk1 and Suz12/Znf198 in liver tumors from X/c-myc bitransgenic mice and woodchuck hepatitis virus (WHV)-infected woodchucks. Employing these animal models and the HBV replicating HepAD38 cells we examined the effect of Suz12/Znf198 down-regulation on gene expression. Genes analyzed include hepatic cancer stem cell markers BAMBI, DKK1,2, DLK1, EpCAM, MYC, and proliferation genes CCNA1, CCND2, IGFII, MCM4-6, PLK1, RPA2, and TYMS. Suz12 occupancy at the promoters of BAMBI, CCND2, DKK2, DLK1, EpCAM, and IGFII was demonstrated by chromatin immunoprecipitation in untransformed hepatocytes, but was markedly reduced in pX-transformed and Suz12 knockdown cells. Accordingly, we refer to these genes as "Suz12 repressed" genes in untransformed hepatocytes. The Suz12 repressed genes and proliferation genes were induced in HBV-replicating HepAD38 cells and, interestingly, they exhibited distinct expression profiles during hepatocellular carcinoma (HCC) progression in X/c-myc bitransgenics. Specifically, CCND2, EpCAM, and IGFII expression was elevated at the proliferative and preneoplastic stages in X/c-myc bitransgenic livers, whereas BAMBI and PLK1 were overexpressed in hepatic tumors from X/c-myc bitransgenics and WHV-infected woodchucks. Importantly, most of these genes were selectively up-regulated in HBV-induced HCCs. The distinct expression profile of the identified Suz12 repressed genes in combination with the proliferation genes hold promise as biomarkers for progression of chronic HBV infection to HCC.