急性脑血管病患者协同刺激分子表达的研究

目的：探讨急性脑血管病患者血液中协同刺激分子白细胞分化抗原簇80（chuster of differention80,CED80,B7-1)、白细胞分化抗原簇86（cluster of differention86,CD86,B7-2)、白细胞分化抗原簇28（cluster of differention28,CD28）,细胞毒性T淋巴细胞抗原－4（cytotoxic T lymphocyte antigen-4,CTLA-4)蛋白的表达和B7－1、B7－2信使核糖核酸（messenger ribonucleic acid,mRNA)的表达其与急性脑血管病病损的关系。方法：应用分子生物学技术,检测急性期脑梗死（26例）,脑出血患者（26例）和年龄匹配的26名健康对照者外周血淋巴细胞B7－1,B7－2,CD28和CTLA－4蛋白的表达和外周血淋巴细胞B7－1、B7－2mRAN的表达,各病列均经头部CT或MRI并计算病灶体积,采用改良爱丁堡＋斯堪的那维亚评分标准（SSS）对临床神经功能 缺损程度进行评分,分析其相互关系。结果：外周血淋巴细胞B7－1、CD28和CTLA－4蛋白的表达,脑梗死组高于脑出血组,两组均高于健康对照组;外周血淋巴细胞B7－2蛋白的表达,三组间无显著性差异,健康对照组外周血淋巴细胞未检测到CTLA－4蛋白的表达;外周血淋巴细胞B7－1mRNA的表达,脑梗死组高于脑出血组,两组均高于健康对照,外周血淋巴细胞B7－2mRNA的表达,三组间无显著性差异,脑梗死组B7－1及CD28与SSS呈正相关,脑出血组协同刺激分子的表达以及病灶的大小与SSS无相关性,脑梗死组病灶的大小与SSS呈正相关,结论：协同刺激分子B7－1、CD28和CTLA－4在急性脑血管病的病理机制中起着重要作用。     

共刺激分子CD28：CTLA4/B7在实验性自身免疫性重症肌无力中的作用

目的 研究共刺激分子CD28:CTLA4/B7在实验性自身免疫性重症肌无力(EAMG)发病中的作用.方法 将雌性Lewis鼠随机分为EAMC组和对照组;EAMG组采用人工合成的Ra97-116肽段3次法免疫Lewis鼠,对照组同期注入等量的PBS;3次免疫接种后采用流式细胞术检测CD28、B7-2、B7-1、CTLA4在外周血、淋巴细胞、单核细胞中的表达.结果 EAMG组大鼠成模率75%;与对照组比较,外周血CD28、B7-2B7-1、CTLA4的表达明显增加(P<0.05～0.01);EAMG组大鼠外周血CD28、CTLA4主要在淋巴细胞表达及B7-1、B7-2在淋巴细胞、单核细胞表达显著增加(P<0.05～0.01).结论 EAMG大鼠存在共刺激分子CD28:CTLA4/B7表达异常,共刺激分子CD28:CTLA4/B7可能参与了EAMG的发生、发展.     

协同刺激分子在格林-巴利综合征患者 脑脊液、血及周围神经中的表达

目的　探讨协同刺激分子在格林 巴利综合征 (GBS)患者脑脊液、血和周围神经组织中的表达及其作用。方法　用RT PCR法及原位杂交法观察GBS患者外周血、脑脊液和周围神经组织中协同刺激分子B7 1、B7 2、CD2 8、CTLA 4的表达。结果　GBS患者外周血、CSF和周围神经组织中协同刺激分子CD2 8、B7 1、B7 2在炎性细胞上的表达明显高于对照组 ;GBS患者CSF中CD2 8、B7 1、B7 2mRNA的表达明显高于外周血。结论　在GBS发病过程中协同刺激分子的表达对T细胞的活化 ,进而导致体液免疫和细胞免疫反应起重要作用。     

单纯多发性肌炎的临床、病理和CD28/CTLA-4:B7表达的研究

目的　研究单纯多发性肌炎 (SPM)的临床、病理特征和CD2 8/CTLA 4 :B7表达及其发病机制。方法　回顾性总结 14 1例SPM患者的临床资料 ,收集 6例治疗前症状高峰期PM病人的外周血 ,单色流式细胞术 (FCM)检测外周血淋巴细胞协同刺激分子CD2 8、CTLA 4、B7 1、BB 1和B7 2的表达 ,并与正常健康者对照。结果　本组主要表现肌无力、肌痛或肌捏痛 ,肌酸激酶 (CK)等血清肌酶谱增高 ,肌电图呈肌源性损害。肌肉病理主要表现为肌纤维变性坏死和再生 ,散在萎缩 ,肌内膜炎症细胞浸润。SPM组外周血淋巴细胞CD2 8、CTLA 4、B7 1、B7 2的表达增加 ,FCM显示CTLA 4及B7 1的平均荧光强度各自与对照组比较有显著性差异 (P <0 0 1) ,CD2 8及B7 2也有显著性差异 (P <0 0 5 ) ,BB 1在SPM组与对照组表达量均极少。结论　肌肉病理检查是诊断SPM的重要依据 ,协同刺激分子CD2 8/CTLA 4 :B7可能是SPM发病的重要环节。     

实验性自身免疫性重症肌无力大鼠CD28/CTLA-4:B7表达水平的研究

目的探讨实验性自身免疫性重症肌无力(EAMG)大鼠外周血单个核细胞(PBMC)CD28/CTLA4B7的表达水平。方法健康、雌性Lewis大鼠24只,随机分为正常组、EAMG组、完全福(氏)佐剂(CFA)对照组。EAMG组大鼠分别于足垫、腹部及背部皮下多点注射丁(氏)双鳍电鳐电器官乙酰胆碱受体蛋白乳剂1mL,第4周再次注射上述乳剂免疫大鼠。CFA对照组只接受等量的CFA皮下注射。初次免疫后7周分离PBMC,应用RTPCR和流式细胞术分析方法,分别进行CD28、CTLA4mRNA及B71、B72蛋白表达水平检测。结果(1)正常组大鼠PBMCCD28、CTLA4mRNA表达水平较低,尤其CTLA4mRNA仅有极少量表达;前二者在EAMG组大鼠表达水平均明显增加(P<0001),而正常组和CFA对照组之间表达水平差异无显著性(P>005)。(2)正常大鼠B71、B72在PBMC上仅少量表达而EAMG组表达明显增加(P<0001),正常组与CFA对照组比较差异均无显著性(P>005)。结论EAMG大鼠存在PBMCCD28/CTLA4B7协同刺激分子的表达异常,CD28/CTLA4B7共刺激通路可能参与了机体异常免疫反应的诱导与维持,在重症肌无力(MG)的发生过程中发挥重要作用。     

急性髓细胞性白血病患者外周血单个核细胞CD28 mRNA的表达

背景：大量研究显示,肿瘤患者外周血T细胞表面共刺激分子CD28蛋白表达存在差异,提示共刺激通路异常可能与恶性肿瘤的发生进展有关。 目的：观察急性髓细胞性白血病外周血单个核细胞共刺激信号分子CD28 mRNA在中的表达。 方法：急性髓细胞性白血病患者80例,其中M0型7例,M1型6例,M2型18例,M3型15例,M4型17例,M5型9例,M6型8例。并根据急性白血病疗效标准将80例患者分为完全治愈组、缓解组、未缓解组。采用Taqman探针实时荧光定量PCR检测80例患者及76名健康人群外周血单个核细胞CD28 mRNA的表达。 结果与结论：急性髓细胞性白血病外周血单个核细胞M1,M3和M4亚型中的CD28 mRNA表达量低于健康人群 (P < 0.05);急性髓细胞性白血病未缓解组中CD28 mRNA低于健康人群 (P < 0.05),完全治愈组和缓解组中CD28 mRNA表达与健康人群差异无显著性意义。说明急性髓细胞白血病患者外周血单个核细胞存在CD28 mRNA表达缺陷,并与临床分期、病情进展及预后有关。     

TGF-β1基因修饰的树突状细胞对实验性自身免疫性重症肌无力大鼠外周血单个核细胞CD28/CTLA-4:B7表达的影响

目的 探讨TGF-β1基因修饰的树突状细胞(DC)对实验性自身免疫性重症肌无力(EAMG)大鼠外周血单个核细胞(PBMC)CD28/CTLA-4:B7表达的影响.方法 近交系8～10周龄健康雌性Lewis大鼠30只,分为正常组、EAMG组、DC对照组、pcDNA3-TGF-β1-DC组、pcDNA3-DC对照组、生理盐水对照组.除正常组外,其余各组均采用丁氏双鳍电鳐电器官乙酰胆碱受体(AChR)蛋白二次免疫的方法复制EAMG大鼠模型.初次免疫后第5天分别皮下注射2×106的DC、pcDNA3-TGF-β1-DC、pcDNA3-DC及等体积的生理盐水,正常组和EAMG组不接受任何治疗.初次免疫后7周分离各组PBMC,应用RT-PCR和流式细胞术方法分别进行CD28、CTLA-4 mRNA及B7-1、B7-2蛋白表达水平的检测.结果 (1)正常组大鼠PBMC CD28、CTLA-4 mRNA的表达水平较低,尤其CTLA-4 mRNA极少量表达;EAMG组大鼠CD28、CTLA-4 mRNA的表达水平均明显增加;pcDNA3-TGF-β1-DC组CD28 mRNA的表达较EAMG组明显降低(P<0.01),而CTLA-4 mRNA的表达水平较EAMG组明显增加(P<0.05);EAMG组、DC治疗组、pcDNA3-DC对照组和生理盐水对照组之间CD28、CTLA-4 mRNA的表达水平无显著性差异(P＞0.05).(2)正常大鼠B7-1、B7-2在PBMC上少量表达;EAMG组B7-1、B7-2两者表达明显增加(P<0.001);pcDNA3-TGF-β1-DC治疗组B7-1、B7-2的表达较EAMG组均明显降低(P<0.01);DC对照组、pcDNA3-DC对照组、生理盐水对照组与EAMG组比较均无明显差异(P＞0.05).结论 EAMG大鼠存在PBMC CD28/CTLA-4:B7协同刺激分子的表达异常,主动调节机体的共刺激通路可能是TGF-β1基因修饰的DC治疗EAMG的机制之一.     

重症肌无力患者外周血T、B淋巴细胞Bcl-2的表达

目的　探讨重症肌无力 (MG)患者外周血单个核细胞Bcl 2蛋白表达及其临床意义。方法　以流式细胞仪双标记免疫荧光方法测定 4 7例临床确诊的MG患者外周血T、B淋巴细胞Bcl 2蛋白表达和CD3 T细胞Bcl 2蛋白表达的平均荧光强度 (MFI)。结果　 ( 1)MG组外周血CD3 、CD4 、CD8 T淋巴细胞和CD19 细胞Bcl 2蛋白表达明显高于对照组 (P <0 .0 1) ,CD3 T细胞蛋白表达Bcl 2的MFI( 0 .572± 0 .177)亦明显高于对照组 ( 0 .170± 0 .147) (P <0 .0 1)。 ( 2 )MG组外周血CD3 、CD4 、CD8 T淋巴细胞及CD19 细胞的Bcl 2蛋白表达与年龄无明显相关 ,而与临床严重程度绝对评分密切相关 (r=0 .63、0 .65、0 .61、0 .78,P <0 .0 5)。CD3 T细胞蛋白表达的MFI与MG患者病程相关密切 (r=0 .62 ,P <0 .0 1)。 ( 3)免疫抑制治疗后MG组临床严重程度绝对评分与淋巴细胞亚群Bcl 2蛋白表达、CD3 T细胞蛋白表达的MFI同步地较治疗前有明显下降 (P <0 .0 1)。结论　外周血淋巴细胞Bcl 2蛋白异常表达对MG发病及临床症状有重要作用。     

体外联合应用CD80和CD28/CpG ODN共刺激活化人外周血单个核细胞★

背景：启动维持并调节活化级联反应，决定了T细胞是活化增殖或转变为无反应状态甚至凋亡。B7分子与CD28的结合能有效协同T细胞受体途径活化T细胞，增强T细胞的增殖活性。 目的：初步分析CD80和CD28/含CpG的寡脱氧核苷酸(CpG containing oligodeoxynucleotides，CpG ODN)共刺激活化的人外周血单个核细胞对胃癌细胞株MKN45的杀伤作用。 方法：Ficoll密度梯度离心法分离人外周血单个核细胞，加入白细胞介素2及CD28，CpG ODN共刺激活化培养1~5 d。取MKN45细胞，设立4组：CD28/CpG ODN共刺激组、CD80联合CD28/CpG ODN共刺激组、单纯CD80组、空白对照组。以MTT法检测细胞杀伤率，电镜观察凋亡的MKN45细胞超微结构，流式细胞仪检测MKN45细胞凋亡率。 结果与结论：单纯CD80组对MKN45细胞无明显杀伤作用；与CD28/CpG ODN共刺激组比较，CD80联合CD28/CpG ODN共刺激组对MKN45细胞的杀伤作用显著增强(P < 0.05)，效应细胞与靶细胞之比达15︰1时即可达到半数杀伤率。电镜下效应细胞作用24 h，MKN45细胞就部分发生坏死，部分细胞可见凋亡。与空白对照组比较，单纯CD80组MKN45细胞凋亡率显著升高(P < 0.01)。提示CD80与共刺激活化的外周血单个核细胞合用，可提高活化的外周血单个核细胞对MKN45细胞的靶向性杀伤作用。     

小鼠骨髓来源树突状细胞吲哚胺2，3过氧化酶表达对T细胞增殖的影响*☆

目的：诱导树突状细胞产生吲哚胺2，3–过氧化酶，从而调节T细胞反应，可能是诱导器官移植免疫耐受的理想途径。观察小鼠骨髓来源的树突状细胞本身及其在多种因素刺激活化状态下吲哚胺2，3–过氧化酶的表达变化，分析其对T细胞增殖的影响。 方法：实验于2005-01/08在解放军第二军医大学免疫研究所完成，动物实验方法符合动物伦理学要求。①培养C57BL/6小鼠骨髓来源的树突状细胞，并分别利用脂多糖80 μg/L、CD40L500 μg/L、γ–干扰素50 μg/L及三者联合作用于培养7 d的上述细胞，利用反转录聚合酶链反应检测各组树突状细胞中吲哚胺2，3–过氧化酶mRNA的表达。②培养BALB/C小鼠脾脏来源的T淋巴细胞做为静息T淋巴细胞，并采用抗CD3单抗及抗CD28单抗各1 mg/L刺激T淋巴细胞作为活化T淋巴细胞。将树突状细胞及γ–干扰素作用下的树突状细胞与静息T淋巴细胞及活化T淋巴细胞共同培养，利用混合淋巴细胞反应法测定其刺激T细胞增殖的能力。 结果：①培养7 d的树突状细胞及在脂多糖、CD40L刺激下的树突状细胞均未见吲哚胺2，3–过氧化酶mRNA的表达，而γ–干扰素及γ–干扰素＋脂多糖＋CD40L三者联合刺激下的树突状细胞可检测到吲哚胺2，3–过氧化酶 mRNA的表达。②与树突状细胞相比，γ–干扰素活化的树突状细胞可明显抑制T细胞增殖（P < 0.01），加入色氨酸可使对T细胞增殖的抑制作用有所恢复，同树突状细胞比较差异亦具有显著性意义（P < 0.01）。 结论：活化的树突状细胞可以产生吲哚胺2，3–过氧化酶，通过降解色氨酸发挥抑制T细胞增殖的作用，可能在移植排斥反应中发挥效应。     

Resistance and susceptibility to experimental autoimmune neuritis in Sprague-Dawley and Lewis rats correlate with different levels of autoreactive T and B cell responses to myelin antigens

Experimental autoimmune neuritis (EAN) is a CD4⁺ T cell-mediated, inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barré syndrome (GBS) in humans. Various mouse and rat strains show different susceptibilities to EAN that can be induced by immunization with bovine PNS myelin (BPM) + Freund's complete adjuvant (FCA). We examined PNS-induced T and B cell responses and cytokine protein production as well as mRNA expression to study the mechanisms behind susceptibility to EAN in Lewis rats and resistance in Sprague-Dawley (SD) rats. Lewis rats with EAN have elevated PNS myelin-reactive interferon-γ (IFN-γ) production, TNF-α mRNA expression, and increased B cell responses to PNS myelin antigens, but low PNS myelin-reactive transforming growth factor-β (TGF-β) and interleukin (IL)-10 mRNA expression in lymph node mononuclear cells (MNC). In contrast, resistance to EAN in SD rats is associated with reduced BPM and P2 peptide-reactive IFN-γ production, TNF-α mRNA expression, and suppressed B cell responses to PNS myelin antigens as well as up-regulation of TGF-β and IL-10 mRNA expression. Resistance to EAN is also associated with low-grade inflammation or absence of histological evidence of EAN. These results suggest that differential autoreactive T and B cells responses to PNS myelin antigens are strain specific, and the susceptibility to EAN is related to quantitative rather than qualitative differences in distribution between pro-inflammatory and anti-inflammatory cytokines. J. Neurosci. Res. 54:373–381, 1998. © 1998 Wiley-Liss, Inc.     

CD28-B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28-/-) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28-/- mice manifested clinical signs of disease. Additionally, CD28-/- mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-gamma secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28-/- mice had milder infiltration of such inflammatory cells as macrophages, CD4+ T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180-199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.     

Targeting the CD80/CD86 costimulatory pathway with CTLA4‐Ig directs microglia toward a repair phenotype and promotes axonal outgrowth

Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T‐lymphocyte antigen‐4 (CTLA4)‐CD80/CD86 pathway, which critically controls the nature and duration of the T‐cell response. In the brain, up‐regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia‐T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4‐Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4‐Ig on brain cells were analyzed after intracerebral transplantation of CTLA4‐Ig‐expressing neurons or wild‐type neurons as control. This model provided in vivo evidence that CTLA4‐Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4‐Ig‐induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up‐regulated expression of the anti‐inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte‐dependent manner through the purinergic P2Y₁ receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4‐Ig‐derived compounds might represent new tools to manipulate CNS microglia. GLIA 2015;63:2298–2312     

B cells play a cooperative role via CD40L-CD40 interaction in T cell-mediated experimental autoimmune neuritis in Lewis rats

The expression of co-stimulatory molecules CD40 and CD40L was examined over the course of experimental autoimmune neuritis (EAN) induced in Lewis rats by immunization with bovine peripheral nerve myelin. In draining lymph nodes, highest level of CD40L expression was seen on day 7 post immunization (p.i.), i.e. before onset of clinical signs of EAN, while CD40 expression was increased on day 14 p.i., i.e. at peak of clinical disease. In contrast, both CD40 and CD40L expressing cells in sciatic nerves, a target organ of EAN, peaked on day 14 p.i., large numbers of both expressing cells were mainly detected on day 14-21 p.i. After co-culture with EAN rat B cells bearing CD40, P0 peptide 180-199-specific T cell line cells exhibited a rapid down-regulation of CD40L expression. Furthermore, EAN rats had enhanced P0 peptide 180-199-specific antibody responses on day 14 p.i., which might have contributed to their aggravated EAN and further demonstrated the role of antibodies in EAN. The results indicate that CD40L-CD40 interactions are involved in the initiation of the antigen-specific T cell responses associated with the generation and development of EAN, and may mediate autoantibody production in EAN. Evidently, B cells play a cooperative role via CD40L-CD40 interaction in T cell-mediated EAN of Lewis rats.