Geng-lin Zhang

HGF and Direct Mesenchymal Stem Cells Contact Synergize to Inhibit Hepatic Stellate Cells Activation through TLR4/NF-kB Pathway

Aims Bone marrow-derived mesenchymal stem cells (BMSCs) can reduce liver fibrosis. Apart from the paracrine mechanism by which the antifibrotic effects of BMSCs inhibit activated hepatic stellate cells (HSCs), the effects of direct interplay and juxtacrine signaling between the two cell types are poorly understood. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated HSCs. Methods We used BMSCs directly and indirectly co-culture system with HSCs to evaluate the anti-fibrosis effect of BMSCs. Cell proliferation and activation were examined in the presence of BMSCs and HGF. c-met was knockdown in HSCs to evaluate the effect of HGF secreted by BMSCs. The TLR4 and Myeloid differentiation primary response gene 88(MyD88) mRNA levels and the NF-kB pathway activation were determined by real-time PCR and western blotting analyses. The effect of BMSCs on HSCs activation was investigated in vitro in either MyD88 silencing or overexpression in HSCs. Liver fibrosis in rats fed CCl4 with and without BMSCs supplementation was compared. Histopathological examinations and serum biochemical tests were compared between the two groups. Results BMSCs remarkably inhibited the proliferation and activation of HSCs by interfering with LPS-TLR4 pathway through a cell–cell contact mode that was partially mediated by HGF secretion. The NF-kB pathway is involved in HSCs activation inhibition by BMSCs. MyD88 over expression reduced the BMSC inhibition of NF-kB luciferase activation. BMSCs protected liver fibrosis in vivo. Conclusion BMSCs modulate HSCs in vitro via TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secreting HGF. BMSCs have therapeutic effects on cirrhosis rats. Our results provide new insights into the treatment of hepatic fibrosis with BMSCs.

Imbalance of interleukin-17-producing CD4 T cells/regulatory T cells axis occurs in remission stage of patients with hepatitis B virus-related acute-on-chronic liver failure

Although regulatory T cells (Treg) and interleukin‐17‐producing CD4 T cells (Th17) have been demonstrated to play opposing roles in inflammation‐associated diseases, their frequency and balance in different stages of hepatitis B virus (HBV)‐related acute‐on‐chronic liver failure (ACLF) remain unknown.

High level of IL-27 positively correlated with Th17 cells may indicate liver injury in patients infected with HBV

Interleukin‐6/IL‐12 family cytokines play a key role in inflammatory diseases via their effects on the differentiation or regulation of T helper cells.

Plasma Interleukin-10: A Likely Predictive Marker for Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

Background: The pathogenesis of HBV-related acute-on-chronic liver failure (HBV-ACLF) is mainly based on a heightened immune-inflammatory reaction; however, the intimate underlying mechanism remains unclear. Objectives: The aim of the study was to explore potential key immune molecular targets that could serve as early predictive markers for HBV-ACLF. Patients and Methods: Twenty-seven patients with acute exacerbation of chronic hepatitis B (CHB) (defined by: alanine transaminase ≥ 20 ULN, total bilirubin ≥ 5 ULN, 40% < prothrombin time activity ≤ 60%) and without cirrhosis were divided into 18 cases which did not progress to HBV-ACLF (defined by: prothrombin time activity < 40% and development within four weeks of hepatic encephalopathy and/or ascites) and nine cases that developed HBV-ACLF. Nine healthy people defined the normal control group (NC). Interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, TNF-α and IFN-γ protein levels were assayed by Cytometric Bead Array (CBA) in blood plasma. The ELISA method was applied to confirm IL-10 detection using the CBA method. Results: IL-4, IL-12p70 and IFN-γ were undetectable; IL-1β, IL-6, IL-8, IL-10 and TNF-α levels were significantly higher than in NC. Moreover, cytokines reached the highest levels in acute exacerbation of CHB, with the exception of IL-2 and IL-8. When comparing the HBV-ACLF patients prior to and at the time of ACLF diagnosis, IL-10 was the only cytokine that exhibited a significant decrease (P = 0.008). IL-10 concentrations were positively correlated to ALT levels (r = 0.711, P < 0.001). Conclusions: The assessment of plasma IL-10 levels in chronic hepatitis B acute exacerbation may provide an early predictive marker for progression to HBV-ACLF.

Expression and clinical significance of myeloid derived suppressor cells in chronic hepatitis B patients.

We here document discovery of expression profile of myeloid derived suppressor cells (MDSCs) in chronic hepatitis B (CHB) patients and changes in the course of disease. The study population was composed of 75 outpatient HBV cases and 15 healthy control cases. Peripheral blood samples were collected for separation of mononuclear cells. Levels of MDSCs labeled with Lin-DR-CD11b+CD33+ obtained from peripheral blood mononuclear cells (PBMC), were revealed to have significant differences between the CHB and other groups. They were 0.414% for health control cases and 0.226% for CHB cases (Z=-2.356, p=0.0189). It also observed that the group of HBeAg positive cases had significant difference in MDSCs/ PBMC median (X(2)=11.877, p=0.003), compared with group of HBeAg negative cases and the healthy control group. It suggested considerable MDSCs might be involved in HBeAg immune tolerance. In addition, negative correlations between MDSCs/PBMC and parameters of ALT, AST and TBil, while positive correlation between MDSCs/ PBMC and ALB parameter were found. Multiple comparisons between the four phases and health control phase again, there was a statistically sifnificant difference (X(2)=17.198, p=0.002). Taken together, these findings may provide a new immunotherapy strategy for reduced the expression levels of MDSCs in CHB patients, through induction of an autoimmune response to virus removal.

Complement Factor 3 Could Be an Independent Risk Factor for Mortality in Patients with HBV Related Acute-on-Chronic Liver Failure

The complement is thought to be involved in the pathogenesis of multiple liver disorders. However, its role in patients with HBV related acute-on-chronic liver failure (HBV-ACLF) remains unclear. Serum levels of the third and fourth complement components (C3, C4) and complement function (CH50) were examined in this prospective, observational study. Associations between their expression and disease activity were analyzed. Survival was analyzed by Kaplan-Meier curves. Predictors of clinical outcome were determined by Cox regression analysis. C3, C4, and CH50 levels were significantly lower in HBV-ACLF patients compared to controls. C3, C4, and CH50 levels were negatively correlated with Tbil levels but positively associated with PTA levels. C3 levels were negatively associated with MELD-Na. C3 levels were significantly lower in HBV-ACLF patients who died compared to patients who survived. In a median hospital stay of 39 days, mortality occurred in 41 patients with a progressive increase based on C3 grade (P = 0.008). The actuarial probability of developing mortality was significantly higher in patients with low C3 grade compared to those with high C3 grade (P < 0.001). Multivariate Cox regression analysis showed that C3 levels were an independent predictor of mortality. Complement played a pathogenic role in HBV-ACLF patients and C3 was an independent predictor of mortality.

The paradoxical changes of membrane and soluble herpes virus entry mediator in hepatocellular carcinoma patients

The herpes virus entry mediator (HVEM) network has become new directions in targeting novel checkpoint inhibitors for cancer therapy. However, the changes of membrane‐bound HVEM (mHVEM) and soluble HVEM (sHVEM) in hepatocellular carcinoma (HCC) are not fully understood. This study aims to study the changes of mHVEM and sHVEM in HCC patients.

Favorable Outcomes of Chinese HCV-Related Cirrhotic Patients with Sustained Virological Response after Pegylated Interferon Plus Ribavirin Treatment

Few studies have conducted follow-up investigations of the clinical course in HCV-related cirrhotic patients who achieved a sustained virological response (SVR) with pegylated interferon plus ribavirin treatment (PegIFN + RBV). We investigated the clinical course and laboratory data in a prospective cohort study enrolling HCV-related cirrhotic patients who received PegIFN + RBV between August 2008 and July 2013 in China. Complete blood counts, liver function tests, and HCV-RNA were serially examined. Liver-related complications were recorded. To detect hepatocellular carcinoma (HCC), alpha-fetoprotein assays, and ultrasound scans were repeated at 6-month intervals. Twenty-five patients were enrolled, including 8 patients with decompensation events before treatment. Eighteen patients achieved SVR with a mean follow-up period of 25.78 months. During the follow-up period, only one patient exhibited HCV-RNA positivity and no decompensation events were detected, but 4 patients developed HCC after SVR. APRI decreased more in patients with SVR than in patients with non-SVR (median, −1.33 versus 0.86, P < 0.001). The albumin levels and platelet counts significantly increased during the follow-up period after SVR (44.27 ± 4.09 versus 42.63 ± 4.37, P = 0.037 and 173.89 ± 87.36 versus 160.11 ± 77.97, P = 0.047). These data indicated that HCV-related cirrhotic patients with SVR after PegIFN + RBV may have a favorable clinical course and improvements in laboratory data. Moreover, HCC should be monitored.

IFNA2 p.Ala120Thr impairs the inhibitory activity of Interferon-α2 against the hepatitis B virus through altering its binding to the receptor.

Background Our previous study found that a rare genetic mutation IFNA2p.Ala120Thr affects the structure of IFN‐&agr;2 and contributes to increased host susceptibility to CHB. However, the way in which the single amino acid residue mutation affects IFN‐&agr;2 activity is unclear. The purpose of this research was to investigate the effects and mechanisms of IFNA2p.Ala120Thr on IFN‐&agr;2 activity. Methods Plasmid transfection of BL‐21 was used to construct both wild type IFNA2 (wt) and p.Ala120Thr IFNA2 (mut) proteins. The HepG2‐NTCP model was established using a lentiviral vector (LV003). Anti‐HBV activity of wt and mut were tested on HepG2‐NTCP infected cells with HBV, through the detection of HBsAg and HBcAg using immunohistochemistry and by detecting HBV DNA with RT PCR. IF and Co‐IP were performed in order to investigate the binding of the IFNA2 protein and its receptor. The changes in IFNAR density and signal molecule phosphorylation were measured with western blotting. We used qPCR to further explore anti‐HBV protein expression including APOBEC3, MxA, OAS1, and PKR. Results Cell model experiments confirmed that IFNA2p.Ala120Thr impairs anti‐HBV activity of IFN‐&agr;2. Co‐IP tests indicated that the binding of mut‐IFN&agr; to IFNR was weaker in the mut‐treated group. IFNR density on the cells surface increased after treatment with wt‐IFN‐&agr;2. Obvious differences in the STAT phosphorylation profiles were seen between the mut‐treated and wt‐treated groups. The expression of four main kinds of anti‐HBV proteins induced by mut was higher in the HepG2‐NTCP cells. Thus, IFNA2p.Ala120Thr affects anti‐HBV activity of IFN‐&agr;2. Conclusion IFNA2p.Ala120Thr impairs the anti‐HBV ability of IFN‐a2, mainly by reducing its binding to the IFN receptor. Mut IFN‐a2 has a very weak binding, barely inducing STAT phosphorylation, and induces the expression of only a low level of related anti‐HBV ISG. This is quite different from the effects of wt IFN‐a2, implying that modifying the key structural position of IFNa may lead to the modulation of targeted gene expression. HighlightsWe analyse the negative effects of genetic mutation IFNA2 p.Ala120Thr on interferon‐&agr;2 activity against hepatitis B virus.IFN‐a2 carrying this mutation bind loosely to IFNR.Wild type IFN‐a2 and mutant IFN‐a2 have distinct signaling pathway phosphorylation patterns.Mutant IFN‐a2 induced lower levels of Anti‐HBV associated ISG expressions in cell culture.