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Objective To investigate the population and role of IL-10+ CD19+ regulatory B cell (Breg) in patients with chronic hepatitis B.Methods Patients with acute hepatitis B (AHB) (n =28),chronic hepatitis B (CHB) (n =31) and normal subjects (n =25) were collected from Changshu No.2 People's Hospital between 2011 June and 2012 October.Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with CpG ODN 2006 and PMA.Flow cytometry was used to analyze the population of IL-10-CD19 + Breg,CD4 + CD25high Treg,and ELISA was used to analyze the concentration of IL-10 in culture supernatant.Results The population of Breg in Peripheral blood of the CHB group [1.28％ (1.05％-2.20％)] was higher than that in the AHB group [0.87％(0.55％-1.22％)] and the HCs group [0.89％ (0.51％-1.37％)] (P =0.001,0.006),and the difference between the AHB group and the HCs group was not statistically significant (P=0.669).Breg in the CHB group [14.30％ (10.70％-16.70％)] was higher than that in the AHB group [10.30％ (7.05％-13.30％)] and the HCs group [10.40％(6.85％-12.60％)] (P =0.003,0.001),treg in the CHB group [5.80％ (4.20％-9.10％)] was also higher than that in the AHB group [4.05％ (2.53％-5.40％)] and the HCs group [4.50％ (2.55％-5.50％)] (P ＜0.001,P =0.005),and there was no significantly difference between the AHB group and the HCs group (Breg:P =0.796 ; Treg:P =0.227).Spearman correlation analysis showed that Breg was positively correlated with Treg in the CHB group (r =0.50,P =0.004),however there was no significantly correlation in the AHB group and the HCs group (r =-0.15,P =0.462; r =0.09,P =0.669).The concentration of IL-10 in the CHB group was higher than that in the AHB group and the HCs group (P ＜ 0.001),and the difference between the AHB group and the HCs group was not statistically significant (P=0.341).Spearman correlation analysis showed that IL-10 were positively correlated with the population of Breg in the CHB group (r =0.409,P =0.022).Conclusion The poluations of regulatory B cell and regulatory T cell increased in patients with chronic hepatitis B,and Breg cell might play the immune regulation role through secreting IL-10 in chronic HBV infection.
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Objective To investigate the correlation between immune inflammation and overactivity of T helper cells in childhood asthma by cell proliferation assay and activation induced cell death in vitro.Methods Th1/Th2/Th17 cytokines were determined by cytometric bead array.Cell proliferation and activation induced cell death were detected when CD4+ T cells were purified by magnetic beads and stimulated by PHA and antiCD3.At last,mRNA of Fas,FasL and Bcl-2 were mesured by real-time PCR.Results Cytokines of IL-4(2.451± 1.052ng/L vs 1.796 ±0.615 ng/L,P =0.018),IL-10( 1.920 ±0.813ng/L vs 1.390 ±0.162ng/L,P =0.006)and TNF(5.112 ±5.842 ng/L vs 1.506 ±0.551 ng/L,P =0.009) in sera of asthma group were higher than those in control group.Compared to control group,proliferation ability of CD4 + T cells in asthma group was greater ( OD450:0.498 ± 0.052 vs 0.274 ± 0.032,P ＜ 0.001 ) and apoptosis rate was lower( 35.62 ± 0.05 ％ vs 65.28±3.85％,P ＜0.001 ).mRNA expression of Fas in asthma group was lower but Bcl-2 was higher than those in control group.Conclusion It is implicated that defective expression of Fas and over expression of Bcl-2 in CD4+ T cells may contribute to apoptosis inhibition and cell proliferation,which could explain overeactivity of CD4 + T cells and lvmphocvte infiltration in childhood asthma.
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Objective To investigate the roles of TLR3 pathway activiated by polyI:C in proliferation and apoptosis of multiple myeloma (MM) RPMI8226 cell line.Methods RPMI8226 cells were cultured in RPMI 1640 with different dose of polyl:C.Cells were collected in different time.Proliferation and apoptosis were detected by CCK-8 kit and flow cytometry,separately.Results The proliferation of RPM18226 was inhibited by polyI:C,and it was dose and time dependent,24 h:12.30％ ±2.04％,22.50％±2.20％,37.90％ ±1.30％ ; 48 h:17.80％ ±1.52％,29.60％ ±0.85％,45.80％ ±1.68％ ;72 h:25.10％±1.01％,34.60％±1.27％,60.50％±2.08％,P＜0.05.RPMI8226 cells were incubated with 50 μg/ml,100 μg/ml and 200 μg/ml polyI:C for 48 h.Apoptotic rate were 5.60％ ±1.06％,8.71％ ±1.06％ and 13.93％ ±1.17％,P＜0.05.TLR3 and TRIF mRNA expression increased obviously and dose dependent,TLR3:1.41±0.10,2.24±0.16,4.08±0.13; TRIF:1.07±0.16,1.97±0.13,3.56±0.19,P＜0.05.Conclusion The proliferation of MM cells were inhibited by TLR3 pathway obviously,and apoptosis was induced by polyI:C.
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ObjectiveTo investigate mechanisms for IL-27 induced proliferation and differentiation of peripheral blood CD4+ T cells in primary biliary cirrhosis (PBC).MethodsPeriperal blood CD4+ T cells were isolated from patients with PBC,chonic hepatitis B (CHB) and health controls (HCs).After IL-27 stimulation,proliferation ability of CD4+ T cells was evaluated by CCK-8 kit,and cytokines were analyzed by ELISA.Real-time PCR was employed to assay mRNA expression of T-bet and GATA3 in CD4+ T cells.p-STAT-1 and pSTAT-3 expression in CD4+ T cells were detected by Western blot.ResultsEnhanced proliferation of CD4+ T cells was found in all subjects after IL-27 stimulation.However,the proliferation ability in patients with PBC was greater than that in CHB and HCs ( P＜0.001 ).Levels of IL-2 and IFN-γ in supernatant from IL-27-incubated PBC blood CD4+ T cells were higher than that from CHB and HCs (P＜0.001 ).In normal situation,T-bet mRNA of CD4+ T cells in PBC group was higher than that in CHB group (P=0.007).Furthermore,after IL-27 stimulation,elevated T-bet mRNA expression and GATA3 inhibition were found in patients with PBC.High expression of p-STAT-1 and p-STAT-3 in blood CD4+ T cells were found in PBC,CHB and HCs after stimulation by IL-27.But their expression in patients with PBC were higher than those in patients with CHB and HCs.ConclusionProliferation of blood CD4+ T cells could be induced by IL-27 in patients with PBC.The signaling pathways of p-STAT-1,p-STAT-3 were involved to induce Th1 immune response and related cytokines expression.This study implicated that IL-27 may play important roles in early inflammation damage in PBC.
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Objective To investigate a new therapeutic pathway for primary biliary cirrhosis (PBC) by immune tolerance reestablishment in a PBC mouse model. Methods Spleenic cells from naive mice were incubated with M2 in the presence of ECDI and the ceils were injected into caudal vein of the mice which would be used for development of PBC model. Spleenic cells incubated with bovine serum albumin (BSA) were injected as controls. 16 weeks later, anti-mitoehondrial antibody (AMA) , alkaline phosphatase(AKP) and portal inflammation were assayed for evaluating the prevention effect. Results AMA positive rate in tolerance group was lower than that in BSA and PBC groups ( P = 0. 007, P = 0. 003 ). The difference between BSA and PBC was not significantly. Serum AKP levels in tolerance, BSA and PBC group were (80.5 ±9.8) U/L, (93.8 ±15.7) U/L and (92.5 ±17.7) U/L, separately. The level in tolerance group was lower than that in BSA and PBC groups (P =0.0095, P =0.029). The rates of portal areas with cell infiltration were 42. 67% ± 12. 30% , 57. 07% ± 11. 35% and 51. 53% ± 9. 96% , separately. The number of infiltrated portal tracts in tolerance group was less than that in PBC group (P = 0.039) and BSA group (P = 0. 0024). Conclusion PBC was prevented to some extent by reestablishing immune tolerance to M2 autoantigen. This provides clues for finding a better treatment proposal.
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Objective To investigate the immune tolerance in animal models of primary biliary cirrhosis (PBC) by determining the cell proliferation and activation induced cell death (AICD).Methods C57BL/6 mice were injected with 5 mg/kg of polyI:C to develope PBC models. The lymphocytes and CD4~+ T cells were separated from spleens and livers 16 weeks later and were stimulated by M2, conA and anti-CD3 for cell proliferation and AICD. Expression of apoptosis related genes and proteins were detected by real time polymerase chain reaction (PCR) and Western blotting, respectively. Results ① The lymphocyte proliferation was 0.1988 ± 0.0111 in blank controls and 0. 2068±0. 0115 in PBS treated mice with no significant difference (P>0.05). However, an abundant lymphocyte proliferation was found in PBC mice (0. 358 ± 0. 022), which was higher than that in controls and PBS treated mice. The proliferation of lymphocyte from liver was greater than that from spleen in PBC mice (P<0.01). ② The apoptotic rate in blank controls (74.70%±4.58%) and PBS treated mice (74.20%±4.44%) was higher than that in PBC mice (44.85%±6.47%,P<0.01),but no difference was found between blank controls and PBS treated mice (P>0.05). Furthermore, the apoptosis rate of T cells from livers were significantly lower than that from spleens in PBC mice (P<0.01). ③ The expressions of FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in PBC mice were lower than those in PBS treated mice (P<0. 01), but there was no change in expression of Fas was found. ④ The expression of Fas-associated death domain-like interleukin-1-β-converting enzyme-inhibitory protein (FLIP_L) in PBC mice was higher than that in blank controls. Moreover, the expression of FLIP_L in livers was higher than that in spleens in PBC mice (P<0. 01). Conclusios The elevated expression of FLIP_L may inhibit AICD. Besides, the decreased expressions of FasL and TRAIL may also help in the enhancement of the anti-apoptotic ability in lymphocytes and in the aggravation of portal area inflammation.
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OBJECTIVE To study the existence of integron and metallo-?-lactamase in Stenotrophomonas maltophilia in our hospital.METHODS The antibiotic susceptibility was tested by K-B method.The genomic DNA and their plasmids were extracted.The integron and metallo-?-lactamase gene were amplified by polymerase chain reaction(PCR).RESULTS L1 and L2 genes were amplified in chromosomes and plasmids.Integrons Ⅰ and Ⅱ were detected in genomic DNA.CONCLUSIONS Existence of metallo-?-lactamase is one of reasons of multi-drug resistance in S.maltophilia.S.maltophilia can receive integrons and gain its multi-drug resistance from other strains.