蛋白激酶Cθ信号途径在结核分枝杆菌抗原激活人γδT细胞增殖和分化中的作用

目的 研究蛋白激酶Cθ(protein kinase Cθ,PKCθ)信号途径在结核分枝杆菌抗原(Mycobacterium tuberculosis antigen,Mtb-Ag)激活人γδT细胞增殖和分化中的作用.方法 健康人外周血单个核细胞(PBMC)用Mtb-Ag和IL-2优势刺激和扩增γδT细胞,或预先用5.0μmol/L Rottlerin(楸毒素)预处理,培养不同时间后,用流式细胞术(FCM)检测γδT细胞表面活化分子和细胞因子表达;同时采用活体染料羧基荧光素乙酰乙酸(CFSE)标记细胞,流式细胞术分析Mtb-Ag刺激γδT细胞后的增殖和各子代细胞百分率.结果 PBMC经Mtb-Ag刺激后3d,γδT细胞CD69和CD25表达分别为46.2%和45.6%,而Rotderin预处理显著地抑制了CD69和CD25表达(P＜0.01);PBMC经Mtb-Ag激活培养5、10和15d,培养扩增细胞中的γδT细胞比例分别为9.6%、54.6%和82.4%,其中第5天已有少部分γδT细胞发生增殖,第10天和第15天时几乎全部γδT细胞分裂都在6代以上,用Rottlerin预处理,显著抑制了γδT细胞增殖反应,但在培养第10天后仍有少部分γδT细胞发生增殖反应;同时在培养第7天、14天和21天,用PMA(佛波酯)+Ionomycin(离子霉素)再刺激后,产生IFN-γ的γδT细胞均在80%左右;培养21d时,有2.6%的γδT细胞表达IL-4.在Rottlerin预处理组产生TH1型细胞因子IFN-γ的γδT细胞均显著减少(P＜0.05),而γδT细胞表达TH2型细胞因子IL-4则几乎完全抑制(P＜0.01).结论 PKCθ信号途径在Mtb-Ag刺激γδT细胞的增殖和分化中均起重要作用.     

Coxsackievirus-mediated hyperglycemia is enhanced by reinfection and this occurs independent of T cells

The induction of autoimmunity by viruses has been hypothesized to occur by a number of mechanisms. Coxsackievirus B4 (CB4) induces hyperglycemia in SJL mice resembling diabetes in humans. While virus is effectively cleared within 2 weeks, hyperglycemia does not appear until about 8-12 weeks postinfection at a time when replicative virus is no longer detectable. In SJL mice, reinfection with CB4 enhanced the development of hyperglycemia. As predicted, the immune system responded more rapidly to the second infection and virus was cleared more swiftly. However, while infiltrating T cells were found within the pancreas, depletion of the CD4 T cell population prior to secondary infection or use of CD8 knock-out mice had no effect on the development of virus-mediated hyperglycemia. In conclusion, enhanced hyperglycemia induced by CB4 occurs independent of the T cell response.     

表皮生长因子和甲硫氨酸脑啡肽诱导人白细胞c—fos基因表达的研究

由健康人外周血分离淋巴细胞及溶血处理后的全白细胞，以外源性表皮生长因子和甲硫氨酸脑啡肽培育后取细胞液帛晟滴片和滴膜，用生物素标记的ｃ－ｆｏｓｃＤＮＡ探针进行原位杂交和斑点印迹杂交。结果显示表皮生升因子和甲硫氨酸脑啡肽两组ｃ－ｆｏｓ原癌基因的表达对照组增。     

A simple technique for evaluation of methods of cell separation

A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.     

The renal cell carcinoma lysis by a specific cytotoxic T cell clone is independent of the Fas/Fas-L cytotoxic pathway

The expression of Fas antigen at the surface of renal cell carcinoma and the susceptibility to Fas-mediated lysis by a tumor specific CTL clone were investigated. Renal cell carcinoma cell lines expressed Fas antigen and were susceptible to apoptosis mediated by antibodies to Fas/APO1. Using RT-PCR, we further showed that these cell lines expressed mRNA for Fas deleted transmembrane region, corresponding to a soluble form of Fas/APO-1. To investigate the role of the Fas/FasL pathway in the cytotoxic response against RCC cells, we analyzed the induction of Fas-L on a tumor specific T cell clone (CTL 8C2), previously generated against one RCC cell line. Fas-L expression on CTL 8C2 was detected by RT-PCR after stimulation with autologous tumor cells. However, the cytotoxic activity of CTL 8C2 was completely abolished when EGTA was added, suggesting that the cytolysis was mainly mediated by a Ca++-dependent pathway, perforin/granzyme-based.     

Influence of FK506 and cyclosporin A on alloantibody production and lymphocyte activation following blood transfusion.

The effect of administration of cyclosporin A (CyA) or the novel macrolide FK506 was investigated in AO rats given DA blood transfusions. CyA (10 mg/kg, orally) or FK506 (1 mg/kg, intramuscularly) administered for 14 days from the time of transfusion effectively inhibited primary anti-MHC class I alloantibody production. This profound inhibitory effect persisted throughout the 2-month investigation period, with little increase in 'secondary' alloantibody production following a challenge injection 28 days after drug withdrawal. Flow cytometric analysis revealed no significant differences in the absolute numbers of W3/25+ (CD4+), OX-8+ (CD8+) or OX-12+ (B lymphocytes), in either the spleen or peripheral blood of transfused compared with normal, untreated animals. However, a small but significant increase in the numbers of splenocytes expressing the activation marker OX-40 (activated CD4+ cells) was observed in transfused animals. Either CyA or FK506 significantly reduced the number of cells expressing OX-39 (interleukin-2 receptors) and OX-40. Treatment of transfused animals with CyA, but not FK506 for 14 days resulted in minor, transient reduction in peripheral blood OX-19+ and W3/25+ cells, while 'sparing' the OX-8+ cells; these changes were not observed in spleens. In contrast, the absolute spleen cell numbers of OX-19+, W3/25+ and OX-8+ cells were significantly reduced in transfused animals given 14 days of FK506 treatment, while the corresponding blood cells were unaffected. Induction of splenic lymphoproliferative responses by the T cell mitogen concanavalin A remained normal in animals receiving transfusion alone or with CyA. In contrast, profound inhibition of mitogenic responses was observed in FK506-treated animals and this inhibitory effect declined gradually following drug withdrawal. No non-specific suppressor cell activity was detected in the spleens of rats given transfusion alone or in CyA or FK506-treated transfused animals.     

肺癌组织培养液和病人血清对自身T细胞集落形成能力的影响

本文观察了10例肺癌病人围术期的肺癌组织、正常肺组织培养液和血清对自身外周血T细胞集落形成能力的影响,并以14例正常人的血清作为对照。结果显示:肺癌人病在去肿瘤负荷前T细胞集落产率为160.73±124.02/10~5,故低于正常人的397.81±133.89/10~5(P<0.001),也低于去肿瘤负荷后的(P<0.001)306.53±79.86/10~5(P<0.001)。术前病人的血清对T细胞集落的抑制率为46.97±21.42%,术后则下降至27.63±23.25%(P<0.01)。加进肺癌组织培养液时,病人的T细胞集落产率为224.83±104.05/10~5,正常肺组织培养液则为323.12±125.27/10~5(P<0.001),肺癌组织培养液和病人血清两者对T细胞集落产率的抑制性呈正相关关系(r=0.817,P<0.005)。本研究结果认为,肺癌组织能产生细胞免疫抑制物质进入血循环,导致病人T细胞集落形成能力明显下降。手术根治性切除肺癌组织,能有效地解除T细胞集落形成能力受抑制的情况。     

Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment

The availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8+ T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8+ T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferon-gamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8+ T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0.005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0.005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.     

热休克蛋白70诱导抗肿瘤免疫的机制研究

目的　研究肿瘤热休克蛋白 70 (HSP70 )诱导的抗肿瘤免疫产生的机制。方法　用液相色谱法提纯小鼠肿瘤细胞株中的HSP70。通过动物实验观察HSP70的抗肿瘤作用 ,并用流式细胞技术测定HSP70免疫后小鼠外周血中T细胞亚群的变化。用ELISA法测定HSP70免疫后小鼠体内细胞因子的水平。结果　用HSP70免疫后 ,小鼠外周血中CD8+T细胞及几种主要Th1型细胞因子 (IL 2、TNF α、TNF β和IFN γ)均升高 ,与对照组相比较 ,差异显著 (P <0 .0 1)。结论　HSP70免疫后 ,小鼠外周血中CD8+ T细胞及Th1型细胞因子均有明显升高。此作用可能是其诱导特异性抗肿瘤免疫的重要机制     

Induction of immunoglobulin-producing human peripheral blood lymphocytes in serum-free medium

Induction of immunoglobulin-secreting cells from human peripheral blood lymphocytes in a serum-free culture medium was studied. Albumin, transferrin, insulin and fibronectin can replace serum entirely for support of pokeweed mitogen (PWM)-stimulated B lymphocytes, measured by a reverse hemolytic plaque assay using protein A-coated red cells. In this serum-free system, growth and maturation to IgM and IgG secretion occur at the same or higher efficiency as in conventional serum-containing medium, with maximum numbers of plaque-forming cells on day 6 at optimal dose of PWM, 0.5 ～ 5 μg/ml. This system can be used to avoid the interference from undefined serum components.