A novel diagnostic immunoassay testing procedure for hepatitis B virus core antibody (anti-HBc) using homogeneous purified full-length hepatitis B virus core antigen (HBcAg) capsids obtained from Escherichia coli was compared with Abbott Architect anti-HBc chemiluminescent microparticle immunoassay (CMIA; indirect method) against a library of specimens. A monoclonal anti-HBc neutralization confirmatory assay was then used to determine the degree of discordance between specimens. The new assay was found to be superior in both sensitivity and specificity.Antibodies (anti-HBc) to hepatitis B virus (HBV) core antigen (HBcAg) are present in current and past HBV infections. Anti-HBc antibodies represent a long-term serological marker of HBV infection, initially appearing during the acute phase of the infection and generally persisting thereafter (16). Thus, as anti-HBc is a universal marker of HBV infection, routine blood donor screening for anti-HBc has been implemented in some countries with low endemicity. It is a cost-effective method of screening. Such screening procedures have resulted in a decrease in the risk of posttransfusion HBV infections (12). In some individuals, the only serological marker of HBV infection is the presence of anti-HBc antibodies (10, 24), and thus detection of "anti-HBcAg alone" could reflect unrecognized "occult" HBV infection and physicians should consider investigating such patients with HBV molecular tests (21). Additionally, isolated anti-HBc can be used as a marker to assess the risk of HBV reactivation in patients undergoing therapy that could result in immunosuppression or patients who are HIV positive (17) or hepatitis C virus (HCV) positive (25).With these uses in mind, having a more efficient and reliable assay for anti-HBc is desirable. Most current commercially available anti-HBc assays have poor sensitivity or specificity (2, 19) and can be attributed to the inferior performance of the competitive immunoassay, especially for detecting low-titer antiHBc-reactive samples. False-positive reactivity can partially be attributed to unspecific activation of premature B lymphocytes causing the production of IgM, IgA, or IgM-related molecules without previous exposure to HBV (18,19). The specificity of competitive assays for anti-HBc can be significantly improved by addition of mild reducing agents, but such modified procedures often lead to the loss of sensitivity, particularly for IgM anti-HBc (23). In this study, a novel immunoassay for anti-HBc based on the double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) method is compared with a commercial anti-HBc assay, the Architect chemiluminescent microparticle immunoassay (CMIA). MATERIALS AND METHODSExpression and purification of full-length rHBcAg in E. coli. Two full-length HBcAg genes were obtained by chemosynthesis: AB090269 (genotype D, named CpD) and AB014368 (genotype C, named CpC). The rare arginine codes in Escherichia coli (AGA and AGG) located in the HBcAg gene were changed to CGT. The two fragments were inse...

BackgroundCDCA5 plays an important role in the development of various human cancers, but the associated mechanisms have not been investigated in hepatocellular carcinoma (HCC).Materials and methodsWe evaluated expression levels and functions of CDCA5 in HCC and showed that CDCA5 is upregulated in HCC tissues compared with paired or unpaired normal liver tissues.ResultsIncreased CDCA5 expression in HCCs was significantly associated with shorter survival of patients. Knockdown of CDCA5 using lentivirus-mediated shRNA significantly inhibited cell proliferation and suppressed cell survival, as well as induced cell cycle arrest at the G2/M phase and cell apoptosis of HCC cells. The tumor suppression effects of CDCA5 knockdown were mediated by decreased expression of cyclin-dependent kinase 1 (CDK1) and CyclinB1, which were increased in HCC tissues comparing with adjacent normal liver tissues. Moreover, upregulation of CDCA5 was positively associated with increased CDK1 and CyclinB1 expression in HCC tissues.ConclusionThe present data warrant consideration of CDCA5 as a prognostic biomarker and therapeutic target for HCC.

Background and Aim The aim of this study is to evaluate the association of the regulatory T cells (Treg)/T‐helper (Th) 17 cells and transforming growth factor‐β1 (TGF‐β1)/interleukin‐17 (IL‐17) ratios with the survival and disease progression in patients with hepatitis B virus (HBV)‐associated liver cirrhosis (LC). Methods The frequencies of Treg and Th17 cells were analyzed in 28 patients with HBV‐LC, 70 patients with chronic hepatitis B (CHB) and 20 normal controls (NC) by flow cytometry. The levels of cytokines related to Treg/Th17 differentiation, including IL‐10, TGF‐β1, IL‐17, and IL‐23, were measured by ELISA. Results Compared with NC, Treg cells were significantly increased in CHB patients and slightly increased in HBV‐LC patients, whereas Th17 cells were markedly increased both in patients with CHB and HBV‐LC. HBV‐LC patients, especially the nonsurvival ones, manifested a profound decrease in the Treg/Th17 ratio, which was negatively correlated with Child‐Pugh and model of end‐stage liver disease scores. Serum IL‐10, TGF‐β1, IL‐17, and IL‐23 levels were all significantly higher in HBV‐LC patients than in NC. In addition, the TGF‐β1/IL‐17 ratio was also markedly increased in patients with HBV‐LC, especially in nonsurvival and decompensated liver cirrhosis patients, and positively correlated with total bilirubin, Child‐Pugh, and model of end‐stage liver disease scores. Conclusions The decreased Treg/Th17 ratio and increased TGF‐β1/IL‐17 ratio may be associated with the survival and disease progression in HBV‐LC patients, and both of the two ratios can be used independently to predict the prognosis and disease progression of HBV‐LC patients.

No abstract

The proinflammatory cytokines transforming growth factor beta 1 (TGF-β1) and interleukin (IL)-31 have been implicated in tissue injury. However, whether TGF-β1/IL-31 are stimulated and elevated in response to liver injury that leads to fibrogenesis in hepatitis B virus-related liver cirrhosis (HBV-LC) remains unclear. To investigate the association between TGF-β1/IL-31 and stages of chronic HBV infection, serum TGF-β1, IL-9, IL-10,IL-17, IL-22, IL-23, IL-31, IL-33, and IL-35 were determined among patients with chronic hepatitis B (CHB; n=19), HBV-LC (n=20), and a normal control population (NC; n=18). Disease severity in patients with HBV-LC was assessed using model for end-stage liver disease (MELD) scores. Serum TGF-β1 and IL-31 levels were strongly positively linked in all subjects, and both correlated positively with IL-22, IL-33, and IL-17. TGF-β1 and IL-31 levels in the blood were both significantly higher in CHB and HBV-LC patients than in NC subjects. Elevated serum TGF-β1 and IL-31 levels were positively associated with albumin, alpha-fetoprotein, creatinine, white blood cell count, and platelet levels. Serum TGF-β1 and IL-31 were markedly higher in HBV-LC patients who did not have esophageal varices, and IL-31 had the highest sensitivity and specificity (90.9% and 66.7%, respectively) for indicating the absence of this complication. In summary, TGF-β1 and IL-31 were linked to progression from CHB to LC, and correlated well with the severity of HBV-LC. These findings suggest possible roles of the TGF-β1/IL-31 pathway in the pathogenesis of liver fibrosis during chronic HBV infection.

The transforming growth factor ␤1/interleukin-31 (TGF-␤1/IL-31) pathway plays an important role in the process of cell injury and inflammation. The purpose of this work was to explore the role of the TGF-␤1/IL-31 pathway in the cytopathic process of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF). The quantitative serum levels of TGF-␤1, IL-9, IL-10, IL-17, IL-22, IL-23, IL-31, IL-33, and IL-35 were analyzed among chronic hepatitis B (CHB) patients (n ‫؍‬ 17), ACLF patients (n ‫؍‬ 18), and normal control (NC) subjects (n ‫؍‬ 18). Disease severity in patients with ACLF was assessed using the model for endstage liver disease (MELD) and Child-Pugh scores. Serum TGF-␤1 levels were strongly positively correlated with IL-31 in all subjects, and both of them were positively correlated with IL-17, IL-22, and IL-33. In CHB and ACLF patients, serum levels of TGF-␤1 and IL-31 were both increased significantly compared with those in NC subjects and positively correlated with total bilirubin (TBil) and alpha-fetoprotein (AFP) levels. ACLF patients showed the highest levels of TGF-␤1 and IL-31, which were positively correlated with Child-Pugh scores. Furthermore, the recovery from the liver injury in CHB was accompanied by decreased TGF-␤1 and IL-31 levels. More importantly, serum levels of TGF-␤1 and IL-31 were markedly upregulated in ACLF nonsurvivors, and IL-31 displayed the highest sensitivity and specificity (85.7% and 100.0%, respectively) in predicting nonsurvival of ACLF patients. Increasing activity of the TGF-␤1/IL-31 pathway is well correlated with the extent of liver injury, disease severity, and nonsurvival of ACLF patients, while reducing activity is detected along the recovery from liver injury in CHB, suggesting its potential role in the pathogenesis of liver injury during chronic HBV infection. Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) is triggered mainly by severe extensive liver injury, and the exact mechanisms of massive destruction of HBV-infected hepatocytes remain unclear. However, one of the current assumptions is that the imbalance of the cytokine network, the so-called cytokine storm theory (1), points to potential involvement of inflammatory cytokines in destroying the HBV-infected cells, which may provide an explanation for the aggravation of liver injury.Transforming growth factor-␤1 (TGF-␤1) is a 25-kDa homodimeric protein composed of two subunits linked by a disulfide bond and is a powerful inhibitor of DNA synthesis and cellular proliferation (2). It also mediates formation of extracellular matrix and facilitates cell differentiation (3). Previous studies have shown that TGF-␤1 plays a role in developing liver failure (LF). Miwa et al. found that the mRNA and protein expression of TGF-␤1 were significantly upregulated in both the plasma and liver tissue in patients with fulminant liver failure (FLF) (4). Yoshimoto et al. found that the overexpression of TGF-␤1 delayed liver regeneration and promoted perisinusoidal fibrosis and hepatocyte apoptos...

Background:The restoration of HBV-specific T-cell response during antiviral therapy is associated with CD4+T-cell activity. Treg cells and Th17 cells are subtypes of CD4+T cell. However, it has remained unknown how the Treg and Th17 cells and their associated cytokines affect nucleos(t)ide analogues (NA) antiviral efficacy.Objectives:The aim of the present study was to provide a new insight to evaluate the NA antiviral therapy for patients with chronic hepatitis B (CHB).Patients and Methods:Forty-four CHB patients hospitalized between July 2010 and August 2011 were enrolled in this study. They were received NA (entecavir, lamivudine and adefovir) treatment for 14.42 ± 13.08 weeks, and the peripheral blood was collected. The frequencies of Treg and Th17 cells were detected by flow cytometric analysis, and the levels of IL-10, TGF-β1, IL-17 and IL-23 were measured by enzyme-linked immunosorbent assay (ELISA).Results:In complete and partial-responders, Treg cells frequencies and IL-10, TGF-β1, IL-23 levels were all decreased significantly after NA therapy, while Th17 cells and the IL-17 levels were increased slightly. Treg/Th17 ratio was only dramatically declined in complete-responders. But there was no significant difference in non-responders. Either HBV DNA decreased by at least 2 log copies /mL or ALT turned to normal level, Treg cells frequencies and IL-10, TGF-β1, IL-23 levels were significantly reduced. Meanwhile, Treg cells were positively correlated with HBV DNA and ALT.Conclusions:The changes of Treg and Th17 cells and their associated cytokines were related to virological and biochemical responses.

CD4+ T helper (Th) cells are reported to be essential for initiating and maintaining an effective immune response to hepatitis B virus (HBV) infection. Th9 cells are a new subset of CD4+ Th cells that produce interleukin (IL)-9 and IL-10. The present study aimed to investigate the percentage of Th9 cells relative to the number of CD4+ cells in peripheral blood.We also measured serum IL-9 and IL-10 levels in different stages of HBV infection and their relationship with progress and prognosis of liver disease. Whole blood samples from 111 patients with HBV infection, including 39 chronic hepatitis B (CHB), 25 HBV-liver cirrhosis (HBV-LC), 21 acute-on-chronic liver failure (ACLF) patients, and 26 healthy controls were collected.The percentage of Th9 cells and serum IL-9 and IL-10 levels were determined. There was no significant difference in the percentage of Th9 cells and serum IL-9 and IL-10 levels among different groups, nor were these related to hepatitis B e antigen status, complications of cirrhosis, inflammation index, or prognosis indexes. There was no change in the percentage of Th9 cells before and after antiviral treatment in CHB patients. There was no correlation of Th9 cells with survival of ACLF patients. However, IL-9 and IL-10 levels were significantly higher in the nonsurvived ACLF patients compared to survived ACLF patients. Furthermore, baseline IL-9 level predicted the prognosis of ACLF patients with 87.5% sensitivity and 61.5% specificity.Thus, our data indicate that Th9 cells were unlikely involved in the pathogenesis of HBV infection, but elevation in IL-9 and IL-10 may signal poor prognosis for ACLF.