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Background and Aim. Little information is available about the assessment and optimal use of the gamma-glutamyl transpeptidase-to-platelet ratio (GPR) and transient elastography (TE) in predicting liver cirrhosis in patients with chronic hepatitis B (CHB) and concurrent nonalcoholic fatty liver disease (NAFLD). This study is aimed at comparing their diagnostic performances and developing an optimal approach for predicting liver cirrhosis in CHB patients with NAFLD. Methods. Consecutive CHB patients with NAFLD were enrolled. The GPR was calculated, and TE was performed using liver biopsy as a reference standard. The accuracy of predicting liver cirrhosis using GPR and TE was assessed and compared, and an optimal approach was developed. Results. Both TE and GPR correlated significantly with the histological fibrosis stage. TE and GPR had excellent performance in predicting liver cirrhosis, and the comparison of areas under the receiver operating characteristic curves revealed that TE was superior to GPR (0.95 vs. 0.85, P=0.039). Moreover, the dual cutoffs established by the likelihood ratio showed that GPR had a similar misclassification but higher indeterminate rate than TE (54.5% vs. 11.7%, P<0.001). Additionally, a 2-step approach using GPR followed by TE had comparable performance to that of both GPR and TE tests for all patients (misclassification: 8.9% vs. 8.3%, P=0.866; indeterminate rate: 15.2% vs. 17.2%, P=0.750) but could reduce TE scans by approximately one-third. Conclusions. Both TE and GPR show excellent performance in predicting liver cirrhosis in CHB patients with NAFLD. The 2-step approach using GPR followed by TE may be optimal for the assessment of cirrhosis in CHB patients with NAFLD.

Background: Since the systemic inflammation has been found to be associated with disease progression and mortality in patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (HBV-ACLF), the objective of this study was to detect inflammatory factors in ACLF patients by a Luminex-based multiplex immunoassay system for high throughput screening of the cytokine with the most prognostic value.Methods: Luminex-based multiplex immunoassay technology was used to determine the concentrations of 48 cytokines in total at once in serum samples from 40 patients with HBV-ACLF, 30 patients with chronic hepatitis B (CHB) and 25 healthy volunteers as normal controls (NC). Then, the receiver operating characteristic (ROC) curve analysis was applied to evaluate the prognostic prediction accuracy. Besides, Kaplan–Meier curves was used to analyze survival, while the Cox regression analysis to determine the mortality predictors.Results: The level of IL-6, IL-10, IL-15, IL-18, M-CSF, IP-10 and CXCL9 were significantly higher in patients with HBV-ACLF than in either patients with CHB or NC subjects, while the level of EGF, PDGF-AA, PDGF-AB/BB, MDC and sCD40L were significantly lower. The concentrations of IL-6, CXCL9, and IL-15 was higher in non-surviving patients with HBV-ACLF than in surviving patients while MDC was lower. Increased serum IL-6 was positively correlated with disease severity. The ROC curve analysis showed that IL-6 and CXCL-9 accurately predicted 90-day survival in patients with HBV-ACLF, with an accuracy equivalent to those of the Model for End-Stage Liver Disease (MELD), MELD-Na. Kaplan–Meier analysis showed an association between the increase in serum concentration of IL-6 as well as CXCL9 and poor overall survival in patients with HBV-ACLF. Moreover, the multivariate Cox regression analysis showed that only serum IL-6 was an independent predictor of overall survival in patients with HBV-ACLF.Conclusion: Although HBV-related ACLF patients have significantly increased serum levels of multiple cytokines, only serum IL-6 levels could be an independent prognostic biomarker in patients with HBV-ACLF.

Background : Tumor mutation burden (TMB) was associated with prognosis in various malignancies, but it remains to be elucidated in hepatocellular carcinoma (HCC). We aimed to investigate the prognostic effects of TMB and its relationship with immune infiltration so as to establish a panel model capable of predicting prognosis. Methods : TMB was calculated in all 374 HCC patients from the Cancer Genome Atlas, and high TMB and low TMB groups were determined based on propensity score matching and X-tile analysis. WCGNA was utilized to screen out genes related to immune cells and TMB, followed by multivariate analysis to generated the final TMB-Infiltration (TMB-IF) panel. 453 HCC patients from ICGC-LIRI-JP, GSE76427 and GSE20017 verified the robustness and extensibility of TMB-IF. Additionally, whole-exome sequencing (WES) was performed in 8 follow-up patients to investigate the relationship between HCC recurrence and TMB. Results : Patients in high TMB group had worse prognosis than those in low TMB group, with a cutoff TMB value of 4.9. Enrichment analysis demonstrated that differentially expressed genes were mainly related to T cell activation, cell membrane localization and matrix composition. Tumor immune infiltration analysis revealed the infiltrations of Th2, Th17, and Tgd were up-regulated in high TMB group, while those of Tr1, MAIT, and DC were up-regulated in low TMB group. And TMB-IF fit well with the actual survival observation, with a C-index 0.785 (0.700-0.870), which verified in ICGC-LIRI-JP was 0.670 (0.573-0.767), and 0.774 (0.670-0.877) in GSE76427. In addition, TMB-IF showed a good performance in predicting tumor vascular invasion with a C-index of 0.847 (0.778-0.916). Conclusions : Higher TMB means worse prognosis in HCC patients. Patients with higher frequency of immune-related gene mutations and TMB are prone to relapse after radical treatment.

Cadherin epidermal growth factor (EGF) laminin G (LAG) seven-pass G-type receptor 1 (CELSR1) is a member of a special subgroup of adhesion G protein-coupled receptors. Although Celsr1 has been reported to be a sensitive gene for stroke, the effect of CELSR1 in ischemic stroke is still not known. Here, we investigated the effect of CELSR1 on neuroprotection, neurogenesis and angiogenesis in middle cerebral artery occlusion (MCAO) rats. The mRNA expression of Celsr1 was upregulated in the subventricular zone (SVZ), hippocampus and ischemic penumbra after cerebral ischemic injury. Knocking down the expression of Celsr1 in the SVZ with a lentivirus significantly reduced the proliferation of neuroblasts, the number of CD31-positive cells, motor function and rat survival and increased cell apoptosis and the infarct volume in MCAO rats. In addition, the expression of p-PKC in the SVZ and peri-infarct tissue was downregulated after ischemia/ reperfusion. Meanwhile, in the dentate gyrus of the hippocampus, knocking down the expression of Celsr1 significantly reduced the proliferation of neuroblasts, however, it had no influence on motor function, cell apoptosis or angiogenesis. These data indicate that CELSR1 has a neuroprotective effect on cerebral ischemia injury by reducing cell apoptosis in the peri-infarct cerebral cortex and promoting neurogenesis and angiogenesis, mainly through the Wnt/PKC pathway.

Background Our previous study found that a rare genetic mutation IFNA2p.Ala120Thr affects the structure of IFN-α2 and contributes to increased host susceptibility to CHB. However, the way in which the single amino acid residue mutation affects IFN-α2 activity is unclear. The purpose of this research was to investigate the effects and mechanisms of IFNA2p.Ala120Thr on IFN-α2 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-α2. Co-IP tests indicated that the binding of mut-IFNα to IFNR was weaker in the mut-treated group. IFNR density on the cells surface increased after treatment with wt-IFN-α2. 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-α2. 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.

Background & Aim 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. Methods Serum samples were collected from 65 HCC patients, from which sHVEM levels were examined by enzyme-linked immunosorbent assay. Expressions of mHVEM on peripheral lymphocytes from 20 HCC patients were determined using flow cytometry and associations between mHVEM on T and B cells were analyzed. Results The levels of mHVEM were down-regulated on peripheral lymphocytes in HCC patients, with a strong positive correlation between mHVEM expression on T and B cells. In contrast, the levels of soluble HVEM were up-regulated in the serum of HCC patients. Furthermore, we found that the increase of sHVEM level was correlated with advanced stages HCC. Conclusion Our data demonstrated paradoxical changes of membrane and soluble HVEM in the peripheral blood of HCC patients for the first time. These data supported the notion that roles of HVEM are likely to be immunosuppressive rather than activating tumor immunity. Future studies are warranted to further explore the translational values of mHVEM and sHVEM in peripheral blood as diagnostic markers and therapeutic targets.

Background and Aims. Serum immunoglobulins are frequently increased in patients with chronic liver disease, but little is known about the role of serum immunoglobulins and their correlations with interleukin-27 (IL-27) in patients with HBV-related acute-on-chronic liver failure (HBV-ACLF). This study was aimed at determining the role of serum immunoglobulin (IgG, IgA, and IgM) levels and their associations with IL-27 in noncirrhotic patients with HBV-ACLF. Methods. Samples were assessed from thirty patients with HBV-ACLF, twenty-four chronic hepatitis B (CHB) subjects, and eighteen normal controls. Disease severity of HBV-ACLF was evaluated. Serum IL-27 levels were examined by enzyme-linked immunosorbent assay. Immunoglobulin levels were assessed using immunoturbidimetric assay. Correlations between immunoglobulin levels and IL-27 were analyzed. Receiver operating characteristic (ROC) curves were used to predict the 3-month mortality. Results. 25 (83.3%) HBV-ACLF patients had elevated serum IgG levels (>1 ULN), 14 (46.7%) patients had elevated IgA, and 15 (50%) had raised IgM. IgG, IgA, and IgM levels were higher in HBV-ACLF patients than in CHB patients and normal controls. Moreover, IgG, IgA, and IgM levels were positively correlated with Tbil levels but negatively correlated with prothrombin time activity (PTA) levels. Additionally, IgG levels were significantly increased in nonsurviving patients than in surviving HBV-ACLF patients (P=0.007) and positively correlated with MELD score (r=0.401, P=0.028). Also, IgG levels were positively correlated with IL-27 levels in HBV-ACLF patients (r=0.398, P=0.029). Furthermore, ROC curve showed that IgG levels could predict the 3-month mortality in HBV-ACLF patients (the area under the ROC curve: 0.752, P=0.005). Conclusions. Our findings demonstrated that serum immunoglobulins were preferentially elevated in HBV-ACLF patients. IgG levels were positively correlated with IL-27 and may predict prognosis in HBV-ACLF patients.

A novel organic crystal was prepared from l -lysine (Lly) and p-toluenesulfonic acid (pTS), which was grown from an aqueous solution by slow cooling method. The crystal system and the lattice parameters have been confirmed by single crystal X-ray diffraction studies. The FT-IR, FT-Raman, 1H-NMR and 13C-NMR spectral of the crystal have been recorded and analyzed. The spectral analyses confirmed the presence of various functional groups and the molecular configurations in LLTS crystal. The UV-Vis-NIR transmittance spectrum has been carried out which shows the cutoff wavelength around 280 nm. The thermal properties of crystal have been evaluated from thermogravimetric (TG) and differential thermal analysis (DTA). The melting point of grown crystal is fairly high, at around 259 °C. The nonlinear optical (NLO) properties of LLTS crystal were demonstrated by powder SHG experiment and also by quantum chemical calculations. The powder SHG efficiency of LLTS crystal is relatively low and very different from theoretical calculation results.

Geng-lin Zhang,1,2,* Ting Zhang,3,* Qi-yi Zhao,1,2 Chan Xie,1,2 Chao-shuang Lin,1,2 Zhi-liang Gao1,2,4 1Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 2Guangdong Provincial Key Laboratory of Liver Disease, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 3Department of Ultrasound, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 4Key Laboratory of Tropical Disease Control, Sun Yat-sen University, Ministry of Education, Guangzhou, China *These authors contributed equally tothis work Background: IL-17-producing CD8+ T (Tc17) cells promote inflammation and have been identified in chronic hepatitis. However, the role of Tc17 cells in patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (HBV-ACLF) remains unclear.Methods: The frequency of Tc17 cells in blood samples from 66 patients with HBV-ACLF was determined by flow cytometry. The levels of Tc17 cell-related cytokines were measured by FlowCytomix assays. The prognostic prediction accuracy was evaluated by the receiver operating characteristic (ROC) curve analysis. Survival was analyzed using Kaplan–Meier curves. Mortality predictors were determined by the Cox regression analysis.Results: The frequency of Tc17 cells was markedly higher in patients with HBV-ACLF than in those with chronic hepatitis B and normal control subjects. Increased frequencies of Tc17 cells may indicate liver injury and were positively correlated with disease severity. The Tc17 cell frequency was significantly higher in non-surviving patients with HBV-ACLF than in surviving patients. The ROC curve analysis showed that Tc17 cell frequency accurately predicted 90-day survival in patients with HBV-ACLF, with an accuracy equivalent to those of the Model for End-Stage Liver Disease (MELD), MELD-Na, and Chronic Liver Failure Consortium ACLF scores. Kaplan–Meier analysis showed an association between the increase in circulating Tc17 cells and poor overall survival in patients with HBV-ACLF. Moreover, the multivariate Cox regression analysis showed that Tc17 cell frequency was an independent predictor of overall survival in patients with HBV-ACLF.Conclusion: Tc17 cells may play a proinflammatory role in HBV-ACLF pathogenesis. Furthermore, the increased frequency of circulating Tc17 cells could be an independent prognostic biomarker in patients with HBV-ACLF. Keywords: liver disease, inflammation, prognosis, T cells

Our previous study demonstrated that Th17 cells increased significantly in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). However, their prognostic role in HBV-ACLF patients remains unknown. Sixty-eight consecutive HBV-ACLF patients were enrolled in this cohort study. Th17 cells were examined using flow cytometry. Disease severity scores were assessed. ROC curves were used to evaluate the value in predicting prognosis. Survival was analyzed using Kaplan–Meier curves. Predictors of mortality were determined by regression analysis. Th17 cells were significantly higher in HBV-ACLF patients compared to patients with chronic hepatitis B and normal controls (both P

Background/Aims: MicroRNA-197 (miR-197) has been shown to play roles in epithelialmesenchymal transition (EMT) and metastasis. The Wnt/β-catenin pathway is associated with EMT, but whether miR-197 regulatesWnt/β-catenin remains unclear. This study was to demonstrate the role of miR-197 on the Wnt/β-catenin pathway in hepatocellular carcinoma (HCC). Methods: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-197 in 105 HCC specimens and 15 HCC cell lines. We tested the predicted target gene of miR-197 using a genetic report system. The role of miR-197 in HCC cell invasion and migration (wound healingand cell invasion and migrationby Transwell assays) and in an HCC xenograft modelwas analyzed. Results: Using a miRNA microarray analysis of HCC specimens and compared with non-metastatic HCC, miR-197 was identified as one of the most upregulated miRNAs in metastatic HCC. miR-197 expression was positively associated with the invasiveness of HCC cell lines. Metastatic HCC cells with high miR-197 expression had Wnt/β-catenin signaling activation. High levels of miR-197 expression also promoted EMT and invasionHCC cells in vitro and in vivo. miR-197 directly targeted Axin-2, Naked cuticle 1 (NKD1), and Dickkopf-related protein 2 (DKK2), leading to inhibition of Wnt/β-catenin signaling. High miR-197 expression was found in HCC specimens from patients with portal vein metastasis;high miR-197 expression correlated to the expression of Axin2, NKD1, and DKK2. Conclusion: miR-197 promotes HCC invasion and metastasis by activating Wnt/β-catenin signaling. miR-197 could possibly be used as a prognostic marker and therapeutic target for HCC.

BTLA/HVEM (B and T lymphocyte attenuator/herpes virus entry mediator) pathways play a critical role in T cell suppression in tumor. However, its dynamic changes in different T cell subsets in peripheral blood and their clinical significance are largely unclear in cancer patients. In the current study, we showed distinct changes of BTLA and HVEM expressions on peripheral blood CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC); BTLA expression were significantly upregulated on circulating CD4+ but not CD8+ T cells. In sharp contrast, the levels of HVEM expression were significantly downregulated on circulating CD8+ but not CD4+ T cells. A strong positive correlation between BTLA expression on circulating CD4+ T cells and BTLA expression on autologous CD8+ counterparts was observed in healthy donors but absent in HCC patients. More importantly, we found that blockade of the BTLA/HVEM pathway increased IFN-γ production in both circulating CD4+ and CD8+ T cells. Collectively, our data suggested that the BTLA/HVEM pathway contributes to peripheral T cell suppression in HCC patients, and BTLA/HVEM may serve as attractive targets for HCC immunotherapy.

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±4.09versus42.63±4.37,P=0.037and173.89±87.36versus160.11±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.

A novel l -arginine salt nonlinear optical single crystal, l -arginine p-nitrobenzoate monohydrate (LANB) has been grown by slow cooling method from aqueous solution. Its solubility at different temperatures in water was measured. The grown crystal was characterized by the elemental analyses, X-ray single crystal and powder diffractions, Fourier transform infrared and Raman spectra. The structure analysis revealed that LANB belongs to the monoclinic crystallographic system, space group P21, with unit cell parameters: a = 8.566(3), b = 5.817(2), c = 17.131(7) A, β = 101.223(5)°, Z = 2 and V = 837.2(6) A3. The proton and carbon configurations of l -arginine were confirmed through 1H NMR and 13C NMR spectra analyses. The linear and nonlinear optical properties of LANB crystal were studied by the use of transmission spectrum and second harmonic generation (SHG). The thermal properties were investigated by using thermo gravimetric (TG) and differential thermal analysis (DTA).

Objective: This study aims to investigate the using of bone marrow mesenchymal stem cells (BMSCs) genetically engineered to produce interferon-β (IFN-β) as a gene delivery system to treat hepatocellular carcinoma (HCC) in vitro and in vivo. Methods: To measure the effects on tumour cell growth in vitro, IFN-β-producing BMSCs (BMSC/IFN-β) were co-cultured with the HCC cell line HepG2 and Huh7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the IFN-β secretion in the BMSC culture condition medium (CM). The effect of BMSC/IFN-β on HCC cells proliferation was examined both in vitro and in vivo by using MTT, colony formation assay, BrdU staining, cell cycle analysis, and xenografted NOD/SCID mouse tumour model. To examine the impact of BMSC/IFN-β on the AKT/FOXO3a signalling, RT–PCR and western blotting were performed. Results: The BMSC/IFN-β cells can stably secrete high levels of IFN-β. Both MTT and colony forming assay showed that HCC cells had a lower growth rate when cultured in BMSC/IFN-β-CM as compared with that in BMSC/vector-CM or DMEM culture group. Co-culture with BMSC/IFN-β-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-β-CM-treated HCC cells, the proportion of G1-phase cells increased but it decreased in the S phase of the cell. The BMSC/IFN-β inhibited HCC growth in NOD/SCID mice and proved the survival period of these mice. Compared with the control group, p21 and p27 expression of hepatoma cells increased, whereas cyclin D1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-β-CM. It was associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a. Conclusion: The IFN-β gene-modified BMSCs can effectively inhibit the proliferation of HCC cells in vitro and in vivo through inhibiting AKT/FOXO3a pathway. These results indicate that BMSC/IFN-β are a powerful anticancer cytotherapeutic tool for HCC.

Background and Aim 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. Methods Fourteen patients with HBV-associated ACLF were studied and defined into different stages according to disease activity. Circulating Th17 cells and Treg cells were analyzed by flow cytometry, and the cytokines were quantitated by enzyme-linked immunosorbent assay. Results were correlated with temporal changes in viral load, disease progression and compared with 30 chronic hepatitis B (CHB) subjects and 18 healthy subjects. Results We showed a significantly higher frequency of circulating Th17 cells in the remission stage of ACLF when compared with the progression stage, the CHB group, or normal controls. However, the frequency of circulating Treg cells was significantly lower in the remission stage of ACLF when compared with the progression stage or the CHB group. The increase in Th17 cells and concomitant decrease in Treg cells created an imbalance in the remission stage of ACLF patients, which negatively correlated with disease progression. In addition, we showed that ACLF patients in the remission stage had an altered profile of cytokines that regulated the induction of Th17 cells and Treg cells. Conclusions ACLF patients in the remission stage had an imbalance of Th17 to Treg cells, which could be used as a prognostic marker to predict disease progression. This imbalance could play a role in the immunopathogenesis of HBV-related ACLF.

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.Serum samples were collected from 65 HCC patients, from which sHVEM levels were examined by enzyme-linked immunosorbent assay. Expressions of mHVEM on peripheral lymphocytes from 20 HCC patients were determined using flow cytometry, and associations between mHVEM on T and B cells were analyzed.The levels of mHVEM were downregulated on peripheral lymphocytes in HCC patients, with a strong positive correlation between mHVEM expression on T and B cells. In contrast, the levels of soluble HVEM were upregulated in the serum of HCC patients. Furthermore, we found that the increase in sHVEM level was correlated with advanced stages HCC.Our data demonstrated paradoxical changes of membrane and soluble HVEM in the peripheral blood of HCC patients for the first time. These data supported the notion that roles of HVEM are likely to be immunosuppressive rather than activating tumor immunity. Future studies are warranted to further explore the translational values of mHVEM and sHVEM in peripheral blood as diagnostic markers and therapeutic targets.

Depression in patients with chronic hepatitis B (CHB) can affect the quality of life, disease diagnosis and case fatality rate. The aim of this study was to explore depression in patients with CHB and cirrhosis, and the effect of the severity of liver cirrhosis on the depressive emotional state. The depressive emotional state was investigated using the Hamilton Depression Scale (HAMD) and Hamilton Anxiety Scale (HAMA) in 114 patients with CHB and cirrhosis, comprising 42 cases classified as Child-Pugh grade (CPG)-A, 38 cases classified as CPG-B and 34 cases classified as CPG-C at a single hepatology center. Patients with mood disorders accounted for 33.33% of the 114 cases with CHB and liver cirrhosis and comprised 10 cases of CPG-A, 12 cases of CPG-B and 16 cases of CPG-C classification. The results shows that HAMA and HAMD scores of patients in the CPG-C group were significantly higher than those in the CPG-A group (P0.05). The incidence rate of mood disorders in the CPG-C group was significantly higher than that in the CPG-B group (P=0.0336), and the incidence rate of mood disorders was higher in the CPG-B group compared with the CPG-A group, but the difference was not statistically significant (P=0.4370). The incidence rate of mood disorders in patients in the CPG-A group was significantly lower than that in the CPG-C group (P=0.0078). The study shows that a considerable proportion of patients with liver cirrhosis have mood disorders, and the depression rates of CHB-infected patients with liver cirrhosis are closely associated with the severity of the cirrhosis.

As a potential alternative treatment for terminal liver diseases, amniotic fluid derived mesenchymal stem cells (AFMSCs) have many advantages over other stem cells: avoiding much ethical controversy and decrease in both quantity and differentiation potential with age. However, the therapeutic role of AFMSC for fulminant hepatic failure (FHF) has not yet been clearly elucidated. Therefore, we investigated the reparation effects of transplanted AFMSCs in rats with FHF. AFMSCs were transplanted into injured liver via the portal vein in the rat FHF model. Therapeutic effect was evaluated after cell transfusion by histologic pathology, hepatic enzyme levels and animal survival. Cryostat sections were prepared and directly assessed for green fluorescent protein (GFP) expression and localization, and in vivo differentiation of AFMSC was confirmed by double-immunostaining analyses. Our results show that AFMSCs prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and animals survival after injection with AFMSCs using GFP, we demonstrated that the engrafted cells and their progeny incorporated into injured livers and produced albumin. We found that AFMSCs transplantation modestly promoted the repair of FHF in rats. AFMSCs implanted in the injured liver may be a novel therapeutic approach in the treatment of FHF. Key words : Amniotic fluid-derived mesenchymal stem cells, fulminant hepatic failure, cell transplantation, treatment, hepatogenic differentiation.

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.

Cerebral dopamine neurotrophic factor (CDNF), previously known as the conserved dopamine neurotrophic factor, belongs to the evolutionarily conserved CDNF/mesencephalic astrocyte-derived neurotrophic factor MANF family of neurotrophic factors that demonstrate neurotrophic activities in dopaminergic neurons. The function of CDNF during brain ischemia is still not known. MANF is identified as an endoplasmic reticulum (ER) stress protein, however, the role of CDNF in ER stress remains to be fully elucidated. Here, we test the neuroprotective effect of CDNF on middle cerebral artery occlusion (MCAO) rats and neurons and astrocytes treated with oxygen&ndash, glucose depletion (OGD). We also investigate the expression of CDNF in cerebral ischemia and in primary neurons treated with ER stress-inducing agents. Our results show that CDNF can significantly reduce infarct volume, reduce apoptotic cells and improve motor function in MCAO rats, while CDNF can increase the cell viability of neurons and astrocytes treated by OGD. The expression of CDNF was upregulated in the peri-infarct tissue at 2 h of ischemia/24 h reperfusion. ER stress inducer can induce CDNF expression in primary cultured neurons. Our data indicate that CDNF has neuroprotective effects on cerebral ischemia and the OGD cell model and the protective mechanism of CDNF may occur through ER stress pathways.

A DNA fragment (MA4-CA) encoding HIV CA viral like particles (VLPs) was transferred intoLycium barbarum L. byAgrobacterium tumefaciens-mediated transformation. PCR amplification analysis of DNA from transgenic plants confirmed the presence of the fusion gene under the control of promoter p35S. CA protein was identified in transformed leaf extracts by enzyme-linked immunosorbent assay (ELISA), indicating that a transgenicLycium barbarum L. expression system can produce immunogenic CA.

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.

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.

Many studies on T helper (Th)1, Th2, T regulatory and Th17 cells have been carried out in acute-on-chronic liver failure (ACLF). However, CD8+ T cell, as a main participant in immune-mediated injuries and defense against microorganisms, has seldom been studied in ACLF. The purpose of this study was to investigate the CD8+ T cell function, and the outcomes of patients with severe hepatitis [SH; serum bilirubin (SB) ≥10 mg/dl and prothrombin activity (PTA)

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.Fourteen patients with HBV-associated ACLF were studied and defined into different stages according to disease activity. Circulating Th17 cells and Treg cells were analyzed by flow cytometry, and the cytokines were quantitated by enzyme-linked immunosorbent assay. Results were correlated with temporal changes in viral load, disease progression and compared with 30 chronic hepatitis B (CHB) subjects and 18 healthy subjects.We showed a significantly higher frequency of circulating Th17 cells in the remission stage of ACLF when compared with the progression stage, the CHB group, or normal controls. However, the frequency of circulating Treg cells was significantly lower in the remission stage of ACLF when compared with the progression stage or the CHB group. The increase in Th17 cells and concomitant decrease in Treg cells created an imbalance in the remission stage of ACLF patients, which negatively correlated with disease progression. In addition, we showed that ACLF patients in the remission stage had an altered profile of cytokines that regulated the induction of Th17 cells and Treg cells.ACLF patients in the remission stage had an imbalance of Th17 to Treg cells, which could be used as a prognostic marker to predict disease progression. This imbalance could play a role in the immunopathogenesis of HBV-related ACLF.

Background 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. Aims The aim of this study was to determine the role of interleukin-27 (IL-27) and its association with helper T cells in hepatitis B virus (HBV)-infected patients. Methods Samples were assessed from 51 HBV-infected patients [28 chronic hepatitis B (CHB) subjects and 23 acute-on-chronic liver failure (ACLF) subjects] and 18 normal controls (NC). Serum IL-27 levels were examined by enzyme-linked immunosorbent assay. Circulating helper T cells were determined using flow cytometry and associations between IL-27 expression and helper T cells were analysed. Results Serum IL-27 levels rose in HBV-infected patients (502.88 ± 23.35 pg/ml) compared to (NC, 277.14 ± 23.96 pg/ml, P

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.