T、B淋巴细胞的凋亡及其与疾病的关系

细胞凋亡是免疫系统的一个重要的事件 ,它调控着淋巴细胞的成熟、受体组分的选择及内环境的稳定。其中 T、B淋巴细胞在各自的发育过程中都发生大量的细胞凋亡。Fas/ Fas L 信号途径、Notch信号途径是免疫系统细胞凋亡的两条最主要的途径。此外 ,一些共刺激分子 (如 CD4 0 )在 T、B淋巴细胞存活及凋亡的选择上也发挥着重要作用。凋亡同细胞的生长、分化一样 ,对免疫细胞发挥正常功能是必不可少的 ,因此 ,免疫系统细胞凋亡的脱轨势必会给机体带来严重的影响 ,导致一系列相关疾病的发生     

腺病毒介导c-FLIPs基因诱导淋巴细胞抗凋亡作用

目的　探讨表达c FLIPs重组腺病毒诱导淋巴细胞抗凋亡作用。方法　采用RT PCR方法,从人T淋巴细胞株的总RNA中克隆c FLIPs基因;通过pAdeasy系统,构建表达c FLIPs的重组腺病毒Ad c FLIPs;Hochest染色法及PI染色流式细胞仪检测anti Apo 1诱导感染Ad c FLIPs后的H9细胞凋亡。结果　采用RT PCR方法从人T淋巴细胞株中扩增出c FLIPs基因;构建重组腺病毒Ad c FLIPs ;经Hoechst染色,荧光显微镜下观察细胞核的形态,结果发现,经anti Apo 1诱导凋亡处理2 4h后,未经Ad c FLIPs感染的H9细胞有大量的细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞;Ad c FLIPs感染的H9细胞中细胞核呈浓染致密的固缩形态或颗粒状荧光的细胞数明显减少,大部分细胞染色质呈弥漫均匀低强度荧光;流式细胞仪分析经PI染色后的H9细胞,未经Ad c FLIPs预处理的H9细胞在anti Apo 1作用2 4h后,细胞的凋亡率分别为4 8 33%±7 4 1% ;经Ad c FLIPs预处理1d的H9细胞,其细胞凋亡率分别下降到3 6 0 %±0 2 1%。结论　重组腺病毒Ad c FLIPs可有效地诱导淋巴细胞的抗凋亡作用。     

甲基强的松龙与脊髓保护

1　甲基强的松龙的一般特性甲基强的松龙 (MP)是一种人工合成的肾上糖皮质激素 ,它的糖皮质激素质作用 -抗炎作用是氢化可的松的 4倍 ,盐皮质激素作用是氢化可的松的 0 .8倍。血浆半衰期为2 .5 h,生物半衰期为 12～ 36 h。40 %～ 80 % MP以结合蛋白形式存在 ,意味着血浆中大部分 MP不能与细胞内的激素受体相互作用 ,不能通过细胞膜及血脑屏障。MP具有水溶性 ,脂溶性差 ,但实际 MP水中溶解度很低 ,大部分通过丁酸脂改变了结构。体内 ,MP被肝脂酶降解。2　 MP脊髓作用1981年 Means等〔1〕研究证实应用 MP(15 mg/ kg/ day,iv2 day;15 mg…     

体外联合应用IL-10和甲基强的松龙处理树突状细胞诱导移植的免疫耐受

目的探讨体外联合应用白细胞介素10(IL-10)和甲基强的松龙(Medron)处理供体树突状细胞(DC)对小鼠皮肤移植术后免疫耐受的诱导效果,为抗移植术后免疫排斥反应治疗提供依据。方法以健康成年C57BL/6小鼠为供体。BALB/c小鼠为受体,随机分为11组。除A组外,其余各组均于皮肤移植前3d自尾静脉输入对应的供体DC。具体对应关系如下A组为空白对照,尾静脉输入生理盐水;B组为输入未修饰的DC;C1组为10μg/LIL-10处理组;C2组为30μg/LIL-10处理组;D1组为10mg/LMedron处理组;D2组为20mg/LMedron处理组;E1~E4组分别为10μg/LIL-10 10mg/LMedron处理组、10μg/LIL-10 20mg/LMedron处理组、30μg/LIL-10 10mg/LMedron处理组、30μg/LIL-10 20mg/LMedron处理组。同时设立F组,为BALB/c对BALB/c的同种同基因皮片移植。各组行皮肤移植术,观察受体移植皮片存活情况。结果相对于生理盐水组,E3组(30μg/LIL-10 10mg/LMedron处理组)的移植皮片存活时间最长,经统计学分析两种药物之间具有交互作用(P<0.05)。结论用IL-10和修饰的供体树突状细胞对受体进行预处理,可明显延长移植皮片的存活时间。     

甲基强的松龙冲击疗法治疗脊髓炎疗效与护理

目的研究大剂量甲基强的松龙冲击疗法治疗脊髓炎比传统治疗方法的优势及护理特点. 方法急性脊髓炎甲基强的松龙冲击疗法34例;常规治疗31例. 结果早期使用甲基强的松龙冲击疗法治疗脊髓炎,能缩短疗程,提高治愈率,明显降低并发症.严格无菌操作、控制输液滴速是护理的关键. 结论治疗组的疗程及愈后明显优于常规组;并发症发生率明显低于常规组.     

甲基强的松龙冲击疗法治疗脊髓炎疗效与护理

目的：研究大剂量甲基强的松龙冲击疗法治疗脊髓炎比传统治疗的优势及护理特点。方法：急性脊髓炎甲基强的松龙冲击疗法34例；常规治疗31例。结果：早期使用甲基强的松龙冲击疗法治疗脊髓炎，能缩短疗程，提高治愈率，明显降低并发症。严格无菌操作、控制输液滴速是护理的关键。结论：治疗组的疗程及愈后明显优于常规组；并发症发生率明显低于常规组。     

甲基强的松龙对大鼠颅脑损伤后内皮素的影响

目的观察甲基强的松龙对大鼠颅脑损伤后脑组织中内皮素含量的影响,并探讨其作用机制。方法将45只SD大鼠随机分为3组,采用骨窗形成后硬膜外打击法造成鼠脑挫裂伤模型,正常组5只,麻醉后,只行开颅手术,不作头颅打击;治疗组大鼠致伤后即刻腹腔内注射30mg/kg甲基强的松龙,对照组则即刻腹腔内注射30mg/kg生理盐水。对照组和治疗组大鼠分别在伤后1h、6h、12h、24h时间点断头取脑,对大鼠脑外伤后脑组织中内皮素含量进行检测。结果大鼠脑皮质中的内皮素在伤后1h后较对照组升高显著(P<0.01),24h达到高峰。甲基强的松龙治疗组在伤后各时间点,内皮素较损伤组明显降低(P<0.05)。结论颅脑损伤后,受损脑组织中内皮素升高,甲基强的松龙可通过抑制损伤后内皮素活性,起到保护创伤神经元的作用。     

FTY720诱导小鼠外周淋巴细胞凋亡

ＦＴＹ72 0 (以下简称ＦＴＹ ) ,化学名 2 ａｍｉｎｏ 2 [2 (4 ｏｃｔｙｌｐｈｅｎｙｌ)ｅｔｈｙｌ] 1,3 ｐｒｏｐａｎｅｄｉｏｌｈｙｄｒｏｃｈｌｏｒｉｄｅ ,是一种新合成的免疫抑制剂。它是将冬虫夏草抽提物中具有免疫抑制作用的成分ＩＳＰ Ｉ进行结构改造而成。有报道大鼠脾细胞与ＦＴＹ共育后 ,出现典型的凋亡特征。但近来 ,有一些报道质疑ＦＴＹ诱导淋巴细胞凋亡的作用[1,2 ] ,为验证ＦＴＹ能否诱导小鼠外周淋巴细胞凋亡 ,进行了以下研究。1　材料与方法1 1　药物、动物　ＦＴＹ72 0由日本ＹｏｓｈｉｔｏｍｉＰｈａｒｍａｃｅｕｔ…     

大剂量甲基强的松龙治疗急性脊髓炎的临床观察

目的 观察大剂量甲基强的松龙治疗急性脊髓炎的临床疗效.方法 48例急性脊髓炎患者随机分为治疗组和对照组,分别用甲基强的松龙和氢化考的松治疗,观察其显效率及脊髓功能恢复情况.结果 治疗组显效率为75.15%,显著高于对照组的54.2%:治疗组患者在肌力改善时间、膀胱功能恢复时间及自行下地行走所需时间比对照组均显著缩短.结论 大剂量甲基强的松龙治疗急性脊髓炙有较好疗效.     

多发性硬化患者血和脑脊液淋巴细胞CD95和CD95L的变化

多发性硬化（multiple sclerosis,MS）是以中枢神经系统脱髓鞘和炎细胞浸润为特征的自身免疫性疾病。细胞凋亡在自身免疫性疾病的作用已引起重视,膜蛋白CD95通过与其配体CD95L的结合是触发凋亡的主要途径。本研究的目的是观察MS患者外周血（PB）和脑脊液（CSF）中淋巴细胞CD95和CD95L的表达水平。     

Anti-P-Glycoprotein Antibody-Induced Apoptosis of Activated Peripheral Blood Lymphocytes: A Possible Role of P-Glycoprotein in Lymphocyte Survival

P-glycoprotein (P-gp) is a 170-kDa glycoprotein encoded by the MDR-1 gene. In tumor cells overexpression of P-gp is associated with resistance to chemotherapy-induced apoptosis. P-gp is also expressed on cells of the immune system; however, its role in lymphocyte physiology remains unclear. Therefore, in this investigation, we examined a possible role of P-gp in the survival of in vitro activated peripheral blood mononuclear cells (MNCs). MNCs were activated with anti-CD3 monoclonal antibody (mAb) for 96 hr in the presence or absence of anti-P-gp mAb or isotype control and examined for apoptosis by TUNEL assay. Activation of caspase was determined by colorimetric assay. Activated lymphocytes (96 hr) are resistant to apoptosis. However, anti-P-gp mAb-induced apoptosis in anti-CD3 activated MNC. Induction of apoptosis was associated with increased expression of CD95L; activation of caspase 3, however, did not affect the expression of Bcl-2 and Bcl-x_L. Furthermore, both recombinant Fas-Fc fusion protein, a blocker of CD95-CD95L interactions, and Z-DEVD-FMK, a cell-permeable caspase 3 inhibitor, reversed anti-P-gp-induced apoptosis. These data demonstrate that anti-P-gp mAb promotes apoptosis in activated T lymphocytes by up-regulating CD95L expression and via CD95-CD95L interactions and suggest a possible role of P-gp in lymphocyte survival.     

The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies

The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue, but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N-glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N-glycosylation.     

系统性红斑狼疮病人T和B淋巴细胞凋亡的观察

为了研究SLE的T、B淋巴细胞及其亚类的凋亡情况,采用碘化丙锭染色,在流式细胞仪下观察计数:22例SLE病人淋巴细胞(包括总体淋巴细胞、CD3~+、CD4~+、CD8~+T细胞和CDl9~+B细胞)凋亡率在培养0、24、48h时均较正常组有显著增高,并以CD4~+T细胞和CDl9~+B细胞在活动期SLE病人中凋亡更突出。此外,SLE病人,自身抗体产生愈多,其细胞凋亡率愈高;疾病活动度增高,凋亡率也较高。提示;SLE病人的淋巴细胞凋亡在体外加速,与其自身抗体产生有密切关系,在其发病机制中起一定的作用。     

Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.     

Increased Spontaneous Ex Vivo Apoptosis and Subset Alterations in Peripheral Blood T Cells from Patients with Multiple Sclerosis

  In order to characterize the immunophenotype and the lymphocyte apoptosis in multiple sclerosis (MS) patients, the peripheral blood lymphocytes of 46 MS patients and 12 healthy volunteers were studied by flow cytometry. Immunophenotypic alterations included significant increases in T CD4+ lymphocytes and reductions in the percentages of CD3+ and CD8+ T cells. After 24 h of culture spontaneous apoptosis was increased in almost T lymphocyte subsets from MS patients and it was significantly higher in those patients who had suffered more than two relapses in the two previous years. The incidence of spontaneous apoptosis was not dependent on FasL-Fas interactions and correlated with the percentages of cells positive for active caspases but not with percentages of Fas+ cells. The increased susceptibility to apoptosis of peripheral blood T lymphocytes from MS patients is difficult to reconcile with the previously proposed role of a defective lymphocyte apoptosis in the pathophysiology of MS.Alfredo Prieto and David Díaz contributed equally to this work and are joint first authors.The GENIO-II Group is composed by Pablo Barreiro from Hospital La Paz, Madrid; Clara de Andrés and Ma Luisa Martínez from Hospital Gregorio Marañón, Madrid; Bonaventura Casanova from Hospital La Fe, Valencia; Juan Antonio García-Merino and Rosario Blanco from Hospital Puerta de Hierro, Madrid; Jesús Martín from Hospital Miguel Servet, Zaragoza; Francisco Coret from Hospital Clínico, Valencia; José Carlos Alvarez-Cermeño and Dr José Francisco Plaza from Hospital Ramón y Cajal, Madrid; Eugenia Vilar, Blanca Felgueroso and Julián Benito from Hospital de Móstoles; Asunción Morales Otal and M^a Cruz Gutiérrez del Olmo from Hospital 12 de Octubre, Madrid.     

SLE患者淋巴细胞凋亡和p53蛋白表达的相关性研究

研究SLE患者外周血淋巴细胞在体外培养下细胞凋亡和p5 3蛋白表达与SLE疾病活动相关性。AnnexinV联合PI染色定量法及免疫荧光染色法 ,分析了 4 4例SLE患者和 30例正常人外周血淋巴细胞在体外培养后凋亡发生率 ,凋亡相关基因p5 3蛋白的表达以及淋巴细胞凋亡发生与疾病活动的相关性。在体外培养作用下 ,活动期SLE患者淋巴细胞凋亡发生率较正常人显著增高 (P <0 0 1 ) ,而静止期则明显升高 (P <0 0 5 ) ,疾病活动指数与体外淋巴细胞凋亡率呈正相关 (P <0 0 1 )。p5 3蛋白表达在活动期SLE患者较正常人显著性下降 (P <0 0 1 ) ,静止期明显降低 (P <0 0 5 )。p5 3蛋白的表达与疾病活动指数SLEDAI,抗dsDNA抗体和C3补体之间有明显的相关性 (P <0 0 1 )。SLE患者外周淋巴细胞在体外培养凋亡率较正常人显著增高 ,表明SLE存在体内凋亡或凋亡相关性免疫机制失调     

Essential role for hematopoietic Fas ligand (FasL) in the suppression of melanoma lung metastasis revealed in bone marrow chimeric mice

The outcome of cancer metastasis depends on multiple interactions between the malignant cell and the host environment. Such interactions can influence primary cancer growth and metastasis by altering the balance between tumor cell proliferation and death. We have previously reported that the pro-apoptotic protein FasL could potently suppress spontaneous lung metastasis of the Fas-sensitive melanoma, K1735-P. In this report, we have constructed bone marrow chimeric mice using wt and FasL-deficient animals to delineate the source of FasL (hematopoietic or nonhematopoietic) required to control spontaneous metastatic spread from a subcutaneous tumor. Using FasL-deficient animals (gld) reconstituted with wt FasL bone marrow (wt --> gld), and wt animals reconstituted with FasL-derived bone marrow (gld --> wt), we show, for the first time, an essential role for hematopoietic-derived FasL in the suppression of K1735-P metastasis. When FasL was expressed only in the nonhematopoietic compartment (gld --> wt), K1735-P spread was ineffectively controlled with a metastatic phenotype similar to that observed in animals completely lacking FasL (gld --> gld or gld controls). These studies provide evidence for the indispensable role for FasL+ bone marrow-derived cells in the control of melanoma and offer a strategy to target FasL expression in the prevention or therapy of metastatic disease.     

Programmed Cell Death (Apoptosis) in Cord Blood Lymphocytes

Cord blood lymphocytes are functionally immature and have deficient immune responses. In order to determine whether the process of programmed cell death is distinct between cord blood and peripheral blood lymphocytes, we analyzed the expression of fas and bax (apoptosis promoting genes) and bcl-2 and bcl-x _L (apoptosis inhibiting genes) at protein or mRNA levels using flow cytometry and quantitative PCR methods, respectively. The susceptibility of T cell subsets from cord blood and adult peripheral blood to undergo apoptosis following restimulation with anti-CD3 or anti-Fas monoclonal antibodies was also studied. We observed that cord blood T cell subsets expressed lower levels of Fas and Bcl-2, a low bcl-2/bax ratio, and higher bcl-x _L compared to peripheral blood. Additionally, upon primary stimulation with anti-CD3, cord blood T cell subsets were more resistant to apoptosis compared to peripheral blood. In contrast, rechallenge of previously stimulated lymphocytes with anti-CD3 monoclonal antibody triggered apoptosis in a larger proportion of T cells from cord blood as compared to peripheral blood, whereas the number of T cells undergoing anti-Fas-induced programmed cell death were lower in cord blood compared to peripheral blood.     

Defective function of Fas in T cells from paediatric patients with autoimmune thyroid diseases

Triggering of the Fas receptor induces T cell apoptosis and is involved in shutting-off the immune response. Inherited defects impairing Fas function cause the autoimmune lymphoproliferative syndrome, and may play a role in other autoimmune diseases. The aim of this work was to analyse the Fas function in paediatric patients with thyroid autoimmunities. We found that T cells from 24/28 patients with Graves' disease (GD) and 12/35 patients with Hashimoto's thyroiditis (HT) displayed defective Fas function. In HT, the defect was more frequent in patients requiring replacement therapy (11/20) than in those not requiring (1/15); moreover, in untreated HT the highest defect was displayed by patients with the highest levels of autoantibodies. Fas was always expressed at normal levels and no Fas mutations were detected. Analysis of the healthy parents of seven Fas-resistant patients showed that several of them were Fas-resistant, which suggests a genetic component. Fusion of Fas-resistant T cells with the Fas-sensitive HUT78 T cell line generated Fas-resistant hybrid cells, which suggests the presence of molecules exerting a dominant negative effect on Fas function. Analysis of Fas-induced activation of caspase-8 and -9 showed decreased activity of both caspases in HT, whereas activity of caspase-9 was increased and that of caspase-8 was decreased in GD. These data suggest that heterogeneous inherited defects impairing Fas function favour the development of thyroid autoimmunities.     

Concentrations of Soluble CD95 and CD8 Antigens in the Plasma and Levels of CD8+CD95+, CD8+CD38+, and CD4+CD95+ T Cells Are Markers for HIV-1 Infection and Clinical Status

Apoptosis mediated via the CD95 (FAS/APO-1) receptor is thought to play a role in the depletion of CD4+ T cells in HIV infection. In the present study expression of the CD95 antigen on lymphocyte subsets and the plasma level of soluble CD95 (sCD95) were determined in HIV-1-infected adults. The expression of CD95 was increased on CD8 cells in all groups of HIV+ individuals, while increased expression of CD95+ cells on CD4 cells was limited to individuals with CD4 counts of <200 mm³. The proportion of CD4+ that expressed CD95 was inversely correlated with the percentage of CD4+ PBL. The concentration of sCD95 was significantly higher in the plasma of HIV-infected individuals than in normal controls. The level of sCD95 in HIV-infected subjects showed no correlation with the percentage of PBL expressing CD95, indicating that the increased level of sCD95 did not reflect release from CD95+ PBL. The plasma sCD95 concentration was significantly correlated with the percentage of CD8+ cells and, particularly, with CD8+CD38– cells. A striking inverse correlation was found between the sCD95 plasma concentration and the proportion of CD4+CD95+ cells out of the total CD4+ population. There was no correlation between the serum level of sCD95 and that of soluble CD8 (sCD8), both of which were increased in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals was correlated with the percentage of CD95+ and CD8+CD38+ cells. The present study indicates that plasma sCD95 may be one of the factors that regulate apoptotic death of lymphocytes in HIV infection.