Objective To study programmed death-1 (PD-1) and T-cell immunoglobulin mucin3 (Tim-3) co-expression on peripheral blood mononuclear cells (PBMC) and hepatitis B virus (HBV)-specific CD8+ T cells in patients with chronic HBV infection and its correlation with liver inflammatory activities.Methods One hundred and sixty subjects with chronic HBV infection who visited the outpatient and inpatient Department of Infectious Diseases in the Third Affiliated Hospital of Sun Yatsen University were enrolled,including 63 cases of mild or moderate CHB (MCHB),31 of severe CHB (SCHB),55 of acute-on-chronic liver failure (ACLF) and 11 inactive HBsAg carriers.Twenty healthy subjects were enrolled as controls.Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect HBV DNA,enzyme-linked immunosorbent assay (ELISA) to measure HBV serological markers,and flow cytometry to detect human leukocyte antigen (HLA)-A2 and determine the expression of PD-1 and Tim-3 on PBMC and HBV-specific CD8+ T cells.Cell counts of Tim-3+,PD-1+,and Tim-3+/PD1+ PBMC and HBV-specific CD8+ T cells were compared among different groups,and their correlation with commonly used inflammatory activity indicators were studied.Analysis of variance and Kruskal-Wallis test were used for measurement data with gaussian distribution and skewed distribution,respectively.Spearman correlation analysis was used to assess the association between Tim-3/PD-1 expression and inflammatory activity indicators.Results The frequency of Tim-3+/PD-1+ PBMC was 0.25 ％ in ACLF group (P=0.0049),0.24 ％ in SCHB group (P-0.0025) and 0.13％ in MCHB group (P=0.0006),which were significantly higher than that in control group (0.03％),and the frequency of Tim-3 /PD-1-PBMC in the three groups were significantly lower than that in control group (P =0.0000),but the differences between HBsAg carriers (0.10％) and controls (0.03％) were not statistically significant (P=0.28).Among 160 subjects,78 were HLA-A2 positive.The frequency of Tim-3+/PD-1+ HBV-specific CD8+ T cells was 68.72％±17.21％ in ACLF group,and 59.66％± 19.25％ in SCHB group,which were significantly higher than that in HBsAg carrier group (33.93％ ± 10.80％,P=0.0000,P=0.0054).The frequency of Tim-3 /PD-1-HBV-specific CD8+ T cells in ACLF group was 2.80％,which was significantly lower than that in HBsAg carrier group (27.24％,P=0.0004).The frequency of Tim3+/PD-1+ HBV-specific CD8+ T cells was positively correlated with alanine aminotransferase (r=0.26,P=0.022),aspartate aminotransferase (r=0.28,P=0.012) and total bilirubin levels (r=0.36,P=0.001); and inversely correlated with albumin level (r=-0.35,P=0.002) in serum.Furthermore,it was positively correlated with international normalized ratio (INR,r=0.34,P =0.045) and model for end-stage liver disease score (r=0.43,P=0.027) in ACLF group.Conclusions Co-expressions of Tim-3 and PD-1 on PBMC and HBV-specific CD8+ T cells are significantly upregulated in patients with CHB,and correlated with liver inflammatory activities.These findings indicate that Tim-3 and PD-1 co-expression may involve in disease activity and liver failure in CHB.

Objective:To investigate a potential role of Toll-like receptor 4(TLR4) ,a pathogen pattern recognition receptor, in the early phase of severely burned rats. Methods: Rats burn model(30% of total body surface area[TBSA],Ⅲgrade) were established with vapor at 108 C for 8 seconds. Rats were sacrificed before and 2,5,12,24,48 and 72 h after burning, and the spleen specimens and peripheral blood samples were harvested. TLR4 mRNA and TNF-?mRNA expression in splenocyte was measured by reverse-transcription PCR(RT-PCR); the expression of TLR4 protein were measured by Western bloting; the endothelial toxicity concentration in plasma was detected by limulus lysate test. Results: It was found that the expression of TLR4 mRNA.TNF-?mRNA,TLR4 protein,and the level of ET were significantly increased in burned group compared with normal control group. The expression of TLR4 mRNA and protein peaked at 8 h after burning, the expession of TNF-?mRNA peaked at 12 h.and the level of ET peaked at 8 h after burnings the peak values of them were (3. 66?0. 51),(2. 27?0. 19), (1.65?0. 23),and (11. 68?2. 63) Eu/ml, respectively, all significantly higher than those of the control group(P

Objective To evaluate the effects of nucleoside/nucleotide analogue treatment on immunoglobulin and complement in patients with chronic hepatitis B (CHB).MethodsA total of 157 CHB patients were recruited and divided into CHB group,liver cirrhosis (LC) group and severe hepatitis B (SHB) group.There were 50 patients who received oral antiviral treatment (lamivudine 100 mg/d,or entecavir 0.5 mg/d,or telbivudine 600 mg/d).Serum levels of complement 3 and 4 (C3,C4),C-reaction protein (CRP),hemolytic complement (CH50),immunoglobulin G,M,A (IgG,IgM,IgA),hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were detected by enzyme-linked immunosorbent assay (ELISA) or immunoturbidimetry.Hepatitis B virus (HBV) DNA was quantified by real-time polymerase chain reaction (RT-PCR) before and 1,2,3 and 4 weeks after nucleoside antiviral therapy.Comparison of means was done by t test and Mann-Whitney test.The correlation was analyzed by Pearson correlation coefficient test.ResultsSerum IgA and IgM levels of SHB and LC patients were significantly higher than those of CHB patients (P＜0.01).Levels of C3,C4,CH50 and CRP were significantly different among three groups.Levels of C3,IgM,IgG and HBV DNA in HBeAg positive patients were significantly different from those in HBeAg negative patients.There was a statistically significant difference of IgA,IgM,C3 and CH50 levels between patients with high HBV DNA level and low HBV DNA level in HBeAg-positive patients.While in the HBeAg-negative patients,only the IgA level was significantly different with HBV DNA levels.After anti-viral treatment,immunoglobulin and HBV DNA levels were all decreased in three groups,while the serum complement level was increased compared to baseline,and the differences became significant at week 4 of treatment. HBV DNA level was negatively correlated with C3 (r=-0.78,P=0.021) and HBeAg titer was positively correlated with C3 (r=0.87,P=0.015).ConclusionsThe immunoglobulin,CRP,C3,C4,and C H50 could reflect the inflammatory activity in liver.The changes of C3 level can predict the efficacy of antiviral therapy.

Objective To investigate the susceptibility of bone marrow mesenchymal stem cell (BMSC) to hepatitis B virus (HBV) infection during induction toward hepatocyte and the role of asialoglycoprotein receptor (ASGPR) in BMSC HBV infection. Methods BMSC obtained from hepatitis B patients were tested for HBV infection and then cultured with HBV infectious serum in vitro and induced to differentiate into hepatocyte through exposure to hepatocyte growth factor (HGF), fibroblast growth factor-4(FGF-4), and epidermal growth factor(EGF). Subsequently these cells were determined for the presence of hepatitis B virus e antigen( HBeAg), hepatitis B virus surface antigen(HBsAg) and ASGPR. All experiments were repeated for 3 times in 5 different samples. The results were analyzed by non-parametric test. Results After 6 days of exposure, BMSC-derived hepatocyte-like cells expressed hepatic special genes and proteins, including alpha fetoprotein(AFP),cytokeratin18 (CK18), albumin (Alb), and manifested hepatocyte functions, including glycogen synthesis, urea secretion and albumin synthesis. Expressions of CK18 and Alb were increased, and AFP was decreased with time of induction. The BMSC were resistant to HBV infection both in vitro and in vivo or after induction toward hepatocyte. ASGPR expression level was low in BMSC, which was increased in the induced BMSC but still lower than that of the control HepG2 cells. Conclusions BMSC are resistant to HBV infection both in vitro and in vivo. The low level expression of ASGPR may be a reason for this.

Objective To evaluate the therapeutic efficacy and its related factors of entecavir treatment for patients with acute on chronic hepatitis B liver failure (ACHBLF).Methods One hundred and eight patients with ACHBLF were enrolled and divided into entecavir group (n=53) and control group (n=55).HBV DNA level, liver function and 48-week survival rate were observed, and C ox regression model was established to identify the factors which may affect the efficacy of entecavir treatment.Results Totally 70 patients died in the study and 66 died within 12 weeks.The statistical difference on cumulative survival rate between two groups was observed from the third week on (χ2=5.357, P ＜ 0.05).The 48-weekcumulative survival rate in entecavir group was 47.2% (25/53), while that in the control group was 23.6%(13/55) (χ2=7.432, P ＜ 0.01).In entecavir group, for patients aged ＜ 40 with serum bilirubin level ＜513 μnol/L and international normalized ratio (INR) ＜ 2.5, the fatality rates decreased 74.9%, 75.3%and 76.0%, respectively.Conclusions Entecavir may improve the survival rate of patients with ACHBLF.Age, serum bilirubin level and INR are major factors related to the therapeutic efficacy.

Objective To explore the association between interleukin (IL)-28B variation and spontaneous clearance of hepatitis C virus (HCV) infection.MethodsA total of 280 HCV infected patients including 200 chronic hepatitis C (CHC) patients and 80 spontaneous clearance patients of HCV infection were enrolled in the study.The rs8099917 single nucleotide polymorphyism (SNP) of IL-28B gene was detected and the association between IL-28B variation and spontaneous clearance of HCV infection was analyzed.The data were compared using chi square test.ResultsThe genotype frequencies of TT and non TT (TG and GG) of IL-28B rs8099917 between CHC patients and spontaneous clearance patients were not significantly different ( x2 =15.874,P ＜ 0.01 ). The prevalence of TT genotype in the spontaneous clearance group was 86.2％,which was higher than that in CHC group (62.0％).T and G allele frequencies in CHC group were 78.0％ and 22.0％,respectively,and those in spontaneous clearance group were 92.5％ and 7.5％,respectively,which indicated that T allele was dominant in spontaneous clearance patients. The allele frequency of rs8099917 was statistically different between the two groups (P＜0.01).The spontaneous HCV clear rate in rs8099917 TT patients was 2.84 folds of non-TT patients,while the risk of chronicity of non-TT (TG+GG) patients was 1.36 folds of TT patients.Conclusion The IL-28B (rs8099917) TT genotype is associated with spontaneous clearance of HCV infection,which can be an important factor to predict the spontaneous clearance of HCV.

Objective To study the affect and the related molecular mechanism of glycogen synthase kinase-3β in the proliferation of osteosarcomaand its value in the target therapy of osteosarcoma.Methods The expression level of p-GSK-3β(Ser9)and GSK-3β were detected in human osteoblast cell and osteosarcoma cells by western blot.Observe the effect of GSK-3β inhibitors and siRNA interference on the GSK-3β regulate osteosarcoma cells using apoptosis protein chip.Evaluate the valueof GSK-3β target therapy on osteosarcoma in vivo.Results The expression level of p-GSK-3β (Ser9)was lower in osteosarcoma cells.LiCL,GSK inhibitor Ⅸ,siRNA knockdown could inhibit the cell viability and up-regulated the apoptosis-related protein cleaved-caspase3.The results of the protein array showed that downstream proteins of NF-κB downregulated significantly.The results were validated by western blot,while the downregulation of p-Iκ-Bα and nuclear NF-κB p65 were also observed after LiCL treatment.Inhibition of GSK-3β by either LiCl or specific siRNA resulted in a significant reduction of NF-κB luciferase reporter activity.Furthermore,the NF-κB luciferase reporter activity was significantly increased in CA cell lines,but not in KD cell lines.By contrast,NF-κB-luciferase reporter activity was significantly decreased in stably GSK-3β knockdown cells.GSK3β inhibitor LiCL and shRNA knock down demonstrated a strong cytotoxicity effect on osteosarcoma cells in vivo.Conclusion GSK-3β is in the state of relative active in osteosarcoma in osteosarcoma and important in cell proliferation.GSK-3β regulates cell survival partially through the NF-κB pathway.It is a promising therapeutic target in osteosarcoma.

Objective To study the growth inhibition,apoptosis induction effects of cinobufagin(CB)on human osteosarcoma(OS) cell line U2OS,MG63 and SaO2 in vitro and the underlying mechanism of action of cinobufagin in OS cells.Methods Cell viability was assessed by MTT assay.Cell-cycle status,apoptosis-inducing effects were evaluated by flow cytometry,fluorescent staining and DNA fragmentation assays.Inhibitors of apoptosis proteins (IAPs) and Bcl-2 family proteins including Bax,cleaved-PARP,xIAP,cIAP-1,survivin and p65 were tested by Western blot.Results MTT assay showed that CB could inhibited the growth of U2OS,MG63 and SaO2 cells in a dose-and time-dependent manner.The 48 h IC50 of CB on U2OS,MG63 and SaO2 cells were (104.83± 16.96) nmol/L,(47.07±7.5) nmol/L,and (136.72±10.08) nmol/L respectively.The induction of G2/M cell-cycle arrest was seen in the cells treated with CB.After cells were cultured for 12 h in the presence of 100 nmol/L CB,the percentages of cells in the G0/G1 phase were decreased,while G2/M phase were increased in U2OS,MG63 and SaOS2 cells,respectively.The results showed CB inhibited the proliferation of osteosarcoma cells through blocking the cell cycle in G2/M phase.Induction of apoptosis was confirmed by Hoechst 33258 and Annexin V/PI staining.After treating with 100 nmol/L CB for 48 h,the extents of apoptosis were 33.6％±6.4％,36.4％±7.8％ and 29.3％±5.1％,respectively.These results indicate that the anti-tumor activity of cinobufagin in osteosarcoma cells was due to a G2/M cell cycle arrest and apoptosis inducing effect.Western blot showed that CB could induce the apoptosis related family proteins Bax,cleaved-PARP up-regulation,xIAP,cIAP-1,survivin and p65 downregulation in OS cells.Conclusion CB can inhibit the cell viability and induce G2/M cell cycle arrest and apoptosis in U2OS,MG63 and SaO2 cells.The apoptosis-inducing effect of CB is confirmed by the regulation of apoptosis related proteins IAPs and Bcl-2 in vitro.

ObjectiveTo evaluate the reliahility and validity of the chronic HBV-infections related stigma scale.MethodsThe initial items and construct of the scale were developed according to theoretical analysis and interviews of experts and patients.A total of 151 patients with chronic HBV-infection were administered by convenient sampling method in this pilot study. The reliability and the validity of the scale were then evaluated.ResultsThe response rate of the scale was 94.5％.The Cronbach α coefficients of all dimeusions ranged from 0.75-0.87.The results of correlation analysis showed that there were higher correlation coefficients ( r ranged from 0.62-0.86) between items and their hypothesized subscales than those with other subscales ( r ranged from 0.14-0.55).The scale distinguished between patients with low subscale scores ( the subscale scores were ( 1.89 ±0.30 ),( 1.86 ± 0.29 ),( 1.96 ± 0.23 ),( 2.29 ± 0.45 ),( 1.59 ± 0.42 ) independently) and those with high subscale scores(the subscalc scores were (3.62 ±0.44),(3.99 ±0.41 ),(3.79 ±0.37),(4.13 ±0.34),(3.10 ±0.53 ) independently) (P ＜ 0.01 ).Confirmatory factor analysis showed that the main indices of goodness of fit CFI was 0.94,NNFI 0.92,RMSEA 0.087.ConclusionThe chronic HBV-infections related stigma has good psychometric properties regarding to reliability and validity.

Objective To evaluate the long following-up outcome of the medial gastrocnemius muscle transferring reconstruction the patella tendon after the wide resection of aggressive bone tumors in the proximal tibia. Methods With the 69 patients of the osteogenetic sarcoma in the proximal tibia were treated with the wide resection and reconstruction the patella tendon. After the long following up the knee extensor,function and complications were evaluated. Results With the 69 patients, the 45 survival patients were followed up for the average 68.6 (24-128) months. The local recurrence rate was 8.7%(6/69). The strength of knee extending was in the average of grade 4.2(3.6-5.0), the degree of knee flexion was in the average of 95°(75°-135°), the degree of knee extension was in the average of-2°(0°-12°), the knees of five patients cannot fully extension. The MSTS functional score was in the average of 77% (23.1/30). Conclusion During the limb salvage of the proximal tibial aggressive bone tumors, the medial gastrocnemius muscle transferring reconstruction the patella tendon could offer the knee extension strength; improve the soft tissue coverage and functional results.