Effects of severe fever with thrombocytopenia syndrome virus infection on the expression of TLR2 and inflammatory cytokines in mouse peritoneal macrophages / 医学研究生学报

Objective The secretion level of inflammatory factors is closely related to the severity of severe fever with thrombocytopenia syndrome (SFTS). This paper mainly discussed the effect of SFTSV infection on the expression of Toll-like receptor 2（TLR2） and inflammatory factors in macrophages in mice. Methods The expression of TLR2 andTNF-α, IFN-β, IL-6, IL-10 and other inflammatory factors were observed at 0, 6, 12, 24, 36, 48, 72 hours after SFTSV infected mice macrophages by qPCR, Western blot and ELISA. Results QPCR results showed that the mRNA levels of TNF-α at 0, 6, 12, 24, 36, 48, 72 h were 0.85±0.14, 15.23±2.45, 28.67±1.59, 94.52±7.05, 55.86±1.82, 23.55±6.15, 9.76±2.03, and the mRNA levels of IFN-β were 0.93±0.21, 1.55±0.33, 14.51±1.98, 55.16±3.64, 26.57±1.49, 9.22±0.51, 5.18±1.06. QPCR results showed that the mRNA levels of TNF-α and IFN-β produced by abdominal macrophages in the infected mice showed a trend of first increasing (the highest at the 24 hour) and then decreasing, and the difference of TNF-α and IFN-β mRNA levels at other time points was statistically significant compared with that at the 0 hour (P<0.05). However, IL-6 and IL-10 mRNA levels continued to increase, and the difference at other time points was statistically significant (P<0.05). ELISA results showed that the expression of four inflammatory factors showed a trend of gradual increase: TNF-α 0 and 72 h were (38.31±4.25, 140.41±23.45) pg/mL; IFN-β 0th and 72th were (17.56±0.66, 1084.93±111.42) pg/mL; IL-6 protein 0 and 72 h were (113.30±0.07, 2302.32±134.09) pg/mL; IL-10 protein 0 and 72 h were (515.00±21.21, 2590.40±226.19) pg/mL, respectively, with significant increases at the 24, 36, 48 and 72 hours compared with that at the 0 hour (P<0.05). The expression of TLR2 mRNA generated by mouse peritoneal macrophages showed a trend of first increasing and then decreasing, and increased to the highest level at 24 h, and the difference between each time point and 0 h was statistically significant (P<0.01). The expression of TLR2 gradually increased after infection with time extension. Conclusion SFTSV infection can up-regulate the expression of TLR2 in macrophages, thereby leading to the increased secretion of the cytokines.

Comparison of Ex Vivo Expanded and Highly Purified NK Cell-Mediated Cytotoxicity Detected by 3 Different Staining Methods of Flow Cytometry / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To compare the cytotoxicity of ex vivo expanded NK cells detected by flow cytometry with 3 different staining methods.</p><p><b>METHODS</b>NK cells were collected from peripheral blood on the 17day after culture. The cultured cells were divided into 3 groups: group A , B, and C. The cells in group A were stained with CFSE/Annectin-V/7-AAD; the cells in group B were stained with Annectin-V/PI, and the cells in group C cells were stained with CFSE/PI. The E:T ratios in 3 groups were 10:1, 20:1 and 40:1, respectively, the K562 cells were incubated with NK cells for 4 hrs.</p><p><b>RESULTS</b>The purity of NK cells(CD3CD56) reached to (16.34±10.51)% on day 0 and to (83.63±10.63)% on the day 17 after incubation(P<0.05); the cytotoxicity of group A was significantly higher than thay of group B at different E:T ratio (P<0.05). The cytotoxicities in A, B, C groups at E:T ratio=10:1 were (36.56±3.69)%, (10.85±2.09)% and (22.35±2.71)% respectively; the cytotoxicities in A, B, C groups at E:T ratio=9:1 were (47.83±5.52)%, (39.07±5.55)% and (29.61±4.81)%; the cytotoxicities in A, B, C groups at E:T ratio=40:1 were (67.7±4.77)%, (51.51±4.43)% and (44.12±5.62)% respectively. Meanwhile, the cytotoxicity in group A was significantly higher than that in group C at different E:T ratio (P<0.05), the percentage of cytotoxicity was (36.56±3.69)% vs (10.85±2.09)%, (47.83±5.52)% vs (29.61±4.81)%, (67.7±4.77)% vs (44.12±5.62)%, respectively.</p><p><b>CONCLUSION</b>CFSE/Annectin-V/7-AAD is able to clearly show human NK cell cytotoxicity against human tumors. Moreover, this staining technique also allows to distinguish different stages of cytotoxic killing as early and late apoptotic phase.</p>

Hepatitis B e antigen from chronic hepatitis B patients induces Th1/Th2 cytokine imbalance in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs).</p><p><b>METHODS</b>PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis.</p><p><b>RESULTS</b>Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01).</p><p><b>CONCLUSION</b>HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.</p>

Dynamic changes and clinical significance of HBcAg18-27 specific cytotoxic T lymphocytes in acute hepatitis B patients / 中华肝脏病杂志

This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.

The influence of hepatitis B e antigen on the expression of toll-like receptor 2 on peripheral monocytes / 中华肝脏病杂志

<p><b>OBJECTIVES</b>In order to investigate the relationship among the toll-like receptor 2 (TLR2), hepatitis B e antigen and HBV DNA, the expression levels of TLR2 on peripheral blood monocytes of chronic hepatitis B (CHB) patients as well as on their monocytes stimulated by ligand of TLR2 (Pam3CSK4) and HBeAg were analyzed.</p><p><b>METHODS</b>Sixty-eight adults with CHB were enrolled, including 37 HBeAg-positive patients, 17 HBeAg-negative and HBV DNA negative patients, and 14 HBeAg-negative and HBV DNA positive patients. Sixteen healthy volunteers were also studied as controls. TLR2 expression levels on their peripheral blood monocytes stimulated with Pam3CSK4 or not stimulated were analyzed by FACS Caliber. The relationship of the expression levels of TLR2, HBeAg and HBV DNA were also analyzed. The level of TLR2 on peripheral blood monocytes of healthy volunteers and HBeAg-negative CHB patients stimulated by HBeAg was examined for six hours.</p><p><b>RESULTS</b>The TLR2 expression levels on CD14+ cells were significantly reduced in HBeAg-positive patients (47.57%+/-21.40 %) compared to both healthy volunteers (76.51%+/-7.46%) and HBeAg-negative patients (HBV DNA positive group 73.2%+/-14.2%, HBV DNA negative group 75.2%+/-11.3%); but there was no difference between those of the HBeAg-negative patients and the healthy volunteers. Expression levels of TLR2 on monocytes stimulated by TLR2 ligand in HBeAg-positive patients were obviously increased, and reached the basic levels of the healthy volunteers and the HBeAg-negative patients. The expression levels of TLR2 on monocytes stimulated by HBeAg of the healthy volunteers and the HBeAg-negative patients were markedly reduced.</p><p><b>CONCLUSIONS</b>In the presence of HBeAg, HBV down-regulates the expressions of TLR2 on CD14+ cells from peripheral blood, and there is no correlation between HBV-DNA and TLR2. Pam3CSK4 can boost the TLR2 expression in HBeAg-positive patients. The proposed interaction between HBV and TLR2 may provide an important clue to explain the reasons of the establishment of persistent HBV infection.</p>

The expression profile of Toll-like receptor on monocyte-derived dendritic cells in patients with chronic hepatitis B virus infection / 中华传染病杂志

Objective To evaluate the expression of Toll-like receptor(TLR)on the monocyte- derived dendritic cells(DC)from chronic hepatitis B(CHB)patients and to analyze the expression pro- file and significance of the TLR such as TLR3,TLR4,TLR?,TLR8 and TLRg,which are associat- ed with immune response to viral infection.Methods Peripheral blood mononuclear cell(PBMC) centrifugated by the hydroxyethyl starch(HES)centrifugation were cultured and induced into DC by granulocyte-maerophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4),and their mor- phology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus(HBV)-infected patients and 15 healthy volunteers.TLR3,TLR4,TLR7,TLRS,TLR9 expression on immature and mature DC were analyzed by FACS Calibur.DC was pulsed with HBcAg on day 3 and 5,then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated.Results Monocyte- derived DC developed different TLR expression patterns as they went through different maturation stages.TLR7,TLR8 expressions on immature DC and TLR3,TLR7 expressions on mature DC were lower in CHB than in control(for TLR7,TLR8 expression on immature DC:75.9%,1.0%vs 98.4%,15.4%,P

Quantitative detection of hepatitis B virus cccDNA in chronic hepatitis B patients / 中华检验医学杂志

Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.