重症肌无力患者末梢血Th1和Th2细胞内细胞因子的变化及意义

目的研究重症肌无力(MG)患者末梢血细胞因子及抗乙酰胆碱受体抗体(AchRab)水平,探讨细胞因子在MG发病中的作用。方法研究对象为17例MG患者,分为急性期组10例,非急性期组7例,设健康对照组15例。应用流式细胞术(FCM)测定末梢血产生各型细胞因子(CK)的CD4+T细胞%,采用酶联免疫吸附法(ELISA)测定血清中抗乙酰胆碱受体抗体(AchRab)。结果⑴MG患者急性期组和非急性期组IFN-γ+IL-4-CD4+T细胞%及AchRab的含量比健康对照组显著增多(P<0.05和P<0.001);⑵急性期组和非急性期组IFN-γ-IL-4+和IL-13+CD4+T细胞%比健康对照组显著减少(P<0.05);⑶IL-10+CD4+T细胞%各组之间无显著性差异;⑷各组IFN-γ+IL-4-CD4+T细胞%与AchRab均呈正相关。结论MG患者Th1和Th2细胞因子的平衡紊乱,Th1细胞因子IFN-γ对MG患者自身抗体的产生有促进作用。     

MG患者外周血Th17细胞与AChR抗体产生的关系研究

目的探讨重症肌无力(MG)患者外周血中Th17细胞及相关细胞因子白细胞介素17(IL-17)在MG发病中的作用。方法收集40例MG患者和10名健康人(对照组)外周血标本,采用流式细胞术检测外周血单个核细胞(PBMCs)中Th17细胞比例,反转录酶-聚合酶链锁反应(RT-PCR)检测PBMCs中维甲酸受体相关孤儿受体γt(RORγt)mRNA水平,ELISA检测血清中IL-17水平,放射免疫沉淀法检测血清中抗乙酰胆碱受体抗体(AChR-Ab)滴度;分离PBMCs中CD4~+T细胞和CD19~+B细胞与金黄色葡萄球菌肠毒素B(SEB)进行共培养,培养系统中加入人IL-17和(或)IL-21中和抗体,放射免疫测定法检测培养液中AChR-Ab滴度。采用MG评分(quantitative MG scoring system,QMGs)对MG的严重程度进行评估,并对MG患者的Th17细胞比例、RORγt mRNA和IL-17水平与病情QMGs的相关性,以及MG患者抗AChR-Ab滴度与PBMCs中Th17细胞比例的相关性进行分析。结果 MG患者PBMCs中Th17细胞比例[1.11%(0.90%,1.34%)]高于健康对照组Th17细胞比例[0.26%(0.08%,0.36%)](z=5.494,P0.001),且与疾病严重程度呈正相关(r=0.4394,P=0.0046);血清中IL-17水平和PBMCs中RORγt mRNA相对表达[分别71.46(53.91,104.76)pg/mL、2.63(1.94,3.12)]均较健康对照组[分别18.82(12.73,29.80)pg/mL、1.13(0.98,1.28)]显著增高(均P0.001);MG患者血清中抗AChR-Ab滴度[2.34(1.19,3.60)nmol/L]较健康对照组[-0.08(-0.24,-0.03)nmol/L]显著增高(z=4.662,P0.001),且与Th17细胞比例呈正相关(r=0.7066,P=0.0001)。MG患者外周血T、B细胞与SEB共培养后抗AChR-Ab水平高于未加入SEB时及健康对照(均P0.01);加入抗人IL-21或IL-17中和抗体后,两者AChR-Ab滴度与未加入抗体时AChR-Ab滴度比较均降低(均P0.05),且均仍高于MG患者未加入SEB时及健康对照(P0.01);在培养上清中同时加入抗人IL-21和IL-17中和抗体时AChR-Ab滴度明显低于加入单种抗体时,而与未加入SEB时及健康对照差异无统计学意义(均P0.05)。结论 MG患者外周血中Th17细胞可能通过IL-17促进AChR-Ab产生,参与疾病的病理过程。     

子痫前期女性胎盘及外周血中辅助T细胞及其细胞因子表达研究

目的探讨辅助T细胞及其细胞因子在子痫前期女性胎盘及外周血中表达的价值。方法选自本院于2015年6月至2017年6月期间收治的子痫前期患者79例(观察组);另正常妊娠孕妇42例作为对照组。采取胎盘组织和外周血清标本,采用免疫组织化法测定白介素-2(IL-2)、白介素-4(IL-4)、白介素-10(IL-10)及干扰素-γ(IFN-γ)蛋白表达,采用酶联免疫吸附法测定IL-2、IL-4、IL-10及IFN-γ含量。结果子痫前期重度组外周血T细胞辅助细胞1(Th1)高于子痫前期轻度组和对照组,而T细胞辅助细胞2(Th2)细胞频数表达低于子痫前期轻度组和对照组,且子痫前期轻度组外周血Th1细胞频数表达高于对照组,而Th2细胞频数表达低于对照组,差异具有统计学意义(P0.05)。子痫前期重度组血清IL-2和IFN-γ含量高于子痫前期轻度组和对照组,而血清IL-4和IL-10含量低于子痫前期轻度组和对照组,差异具有统计学意义(P0.05);子痫前期轻度组血清IL-2和IFN-γ含量高于对照组,而血清IL-4和IL-10含量低于对照组,差异具有统计学意义(P0.05)。子痫前期重度组IL-2、IL-4、IL-10及IFN-γ蛋白阳性表达率高于子痫前期轻度组和对照组,且子痫前期轻度组IL-2、IL-4、IL-10及IFN-γ蛋白阳性表达率高于对照组,差异具有统计学意义(P0.05)。结论辅助T细胞及其细胞因子在子痫前期女性胎盘及外周血中表达具有重要研究意义,Th1表达增加,Th2表达降低,Th1与Th2表达失衡可能是子痫前期发病的原因。     

他克莫司治疗重症肌无力作用机制的研究

目的：研究他克莫司治疗重症肌无力（MG）的免疫调节机制。方法：收集依据临床表现、实验室和电生理检查确诊的全身型MG患者外周血28份（来自22例患者）,健康对照外周血20份（来自20名健康者）。分离单核细胞,稀释后等量分为4份,分别加入他克莫司0、2、10及50ng·mL^-1,培养48hN收集上清液,以ELISA法检测上清液中IL-12、IL-2、IFN-γ、IL-4、IL-10、IL-13、TNF-α、sICAM-1的浓度。结果：对照组随他克莫司浓度的升高（0、2、10和50ng·mL^-1）,IL-2、IL-10的浓度显著升高（P〈0．05）,TNF-α水平有降低趋势,但差异无统计学意义（P〈0．05）。MG组IL-12、IL-2、IFN-Y、IL-10、IL-13和sICAM-1的水平均显著升高（P〈0．05）,而TNF-α水平明显降低（P〈0．05）。结论：他克莫司不是一个单纯的免疫抑制剂。它能抑制炎性因子TNF-α和黏附分子的分泌,诱导和影响Th1和Th2细胞因子产生,最终达到治疗MG的效果。     

Th1类细胞因子在评价高血压性脑出血手术疗效的作用

目的探讨Th1类细胞因子在评价高血压性脑出血手术疗效的作用。方法收集38例接受小骨窗开颅血肿清除术的患者作为观察组,采集其静脉血及术中吸附的血肿,采用ELISA检测其内Th1细胞因子白介素2(IL-2)及γ干扰素(IFN-γ)和Th2细胞因子白介素4(IL-4)及白介素10(IL-10)的含量。通过CT扫描测量血肿体积;通过NIHSS评分评价急诊时患者的神经功能缺损情况;检测急诊时中性粒细胞计数及C-反应蛋白水平及皮质醇以判断患者的应激程度。同时选取38例接受保守治疗的患者作为对照。结果①急诊时手术组的IL-2及IFN-γ均显著高于保守组,但IL-4及IL-10无统计学差异(P<0.05)。②治疗24h后,手术组患者的IL-2及IFN-γ显著低于急诊时(P<0.05),且IL-4及IL-10显著高于急诊时;治疗后24h保守组的IL-2及IFN-γ同样显著低于急诊时,但IL-4及IL-10与急诊时无统计学差异。③血清及血肿内IL-2及IFN-γ与血肿体积、皮质醇、中性粒细胞计数及C-反应蛋白均呈正相关性(r>0.05,P<0.05)。结论血清及血肿内Th1细胞因子IL-2及IFN-γ与出血量及应激存在显著正相关性,因此该类细胞因子可有效评价小骨窗开颅血肿清除术治疗高血压性脑出血的疗效。     

中枢神经系统炎性脱髓鞘患者血清AQP-4抗体与细胞因子的研究

目的研究中枢神经系统炎性脱髓鞘疾病患者血清水通道蛋白(AQP-4)抗体、细胞因子干扰素-γ(IFN-γ)及白细胞介素-4(IL-4)水平,以及AQP-4抗体与IFN-γ和IL-4间的相关性,探讨视神经脊髓炎(NMO)和多发性硬化(MS)的发病机制。方法入选患者共130例,其中NMO患者41例,MS患者59例,临床孤立综合征(CIS)患者15例,其他神经系统疾病患者15例。采用细胞转染的间接免疫荧光法检测患者血清AQP-4抗体滴度,ELISA法检测患者血清IFN-γ和IL-4水平,比较各组患者血清AQP-4抗体、IFN-γ和IL-4水平并分析AQP-4抗体与IFN-γ及IL-4的相关性。结果 NMO组血清AQP-4抗体滴度显著高于MS组、CIS组及其他疾病组(均P0.05),而IFN-γ和IL-4水平与另3组比较差异无统计学意义(均P0.05);MS组血清AQP-4抗体滴度与CIS组及其他疾病组比较差异无统计学意义(均P0.05)。NMO组血清AQP-4抗体滴度与IFN-γ和IL-4呈正相关(r分别为0.36、0.35,均P0.05),MS组血清AQP-4抗体滴度与IFN-γ及IL-4无相关性(r分别为0.03、-0.10,均P0.05)。结论血清AQP-4抗体可用于鉴别NMO和MS及其他炎性脱髓鞘病;NMO患者血清AQP-4抗体增高可能与细胞因子IFN-γ和IL-4有一定关系。     

髓鞘少突胶质细胞诱导实验性自身免疫性脑脊髓炎小鼠的中枢病理损伤与Th1、Th17细胞分泌炎症细胞因子的变化观察

目的：观察实验性自身免疫性脑脊髓炎（EAE）的中枢神经系统（CNS）病理损伤与Th1、Th17细胞分泌的相关炎症细胞因子的变化,探讨EAE致病的分子免疫学机制。方法：用髓鞘少突胶质细胞（MOG35-55）免疫诱导野生型C57BL／6小鼠制作EAE模型,记录小鼠行为学变化,苏木精-伊红染色观察CNS病理损害,RT-PCR法检测中枢Th1细胞因子γ-干扰素（IFN-γ）、Th17细胞因子IL、17和IL-6的mRNA表达水平。结果：MOG诱导的EAE模型组小鼠出现典型的EAE行为及病理学表现,大脑组织中INF-γ、IL-17和IL-6mRNA表达水平均较CFA对照组有明显升高。结论：在EAE炎症反应的过程中,Th1细胞和Th17细胞激活,各自分泌的炎症细胞因子（INF-γ／IL-6、IL-17）增加,它们可能参与了EAE发病的重要免疫病理机制。EAE的致病并不是单-Th1或者Th17细胞作用的结果,而是两类细胞因子都参与了作用。     

重症肌无力胸腺和外周血抗原特异性单个核细胞IFN-γ、IL-4的转录及表达

目的为了解重症肌无力(MG)胸腺细胞免疫状态,对10例行胸腺摘除(Tx)的MG患者及4例先天性心脏病行开胸手术患者的胸腺和外周血单个核细胞经特异性抗原乙酰胆碱受体(AChR)刺激后,其细胞IFN-γ和IL-4的mRNA转录和细胞培养上清液中的蛋白表达情况进行了检测.方法采用RT-PCR结合狭缝印迹杂交检测IFN-γ、IL-4 mRNA转录,采用ELISA检测IFN-γ和IL-4的表达情况.结果 10例MG患者的胸腺和外周血单个核细胞经AChR刺激后IFN-γ和IL-4的mRNA转录增高,其中8例胸腺增生者胸腺细胞和外周血细胞的IFN-γ mRNA转录、外周血细胞的IFN-γ表达均高于健康对照组(P＜0.05);胸腺细胞IL-4 mRNA转录和表达均高于健康对照,差异有显著性(分别为P＜0.01和P＜0.05),而外周血细胞IL-4 mRNA转录和表达与健康对照差异无显著性(P＞0.05);另2例伴胸腺瘤者,此两种细胞因子无论mRNA转录还是蛋白表达均很低.外周血细胞与胸腺细胞IL-4 mRNA转录有一定的相关性,基本反映胸腺的免疫学变化.结论胸腺增生MG患者的细胞免疫(Th1和Th2)高度活跃,PBMC的有些免疫学参数能反映MG患者胸腺的免疫学改变,但有待进一步累积更多资料.伴胸腺增生与伴胸腺瘤的MG发病机制可能不同,Tx是必要的.     

帕金森病患者外周血Th1/Th2类细胞因子的基因表达

目的 探讨细胞因子在帕金森病（PD）发病机制中的作用。方法 对26例初诊未经治疗的原发性PD患者（PD1）,27例经左旋多巴、美多巴治疗的PD患者（PD2）及30名年龄、性别相匹配的健康对照者,于清晨抽静脉血5ml,梯度离心分离外周血单核细胞（PBMC）后,采用逆转录聚合酶链反应（RT-PCR）技术,分别检测各组PBMC中白细胞介素-2（IL-2）、1干扰素（INF-1）、IL-4、IL-6、IL-10细胞因子信使核糖核酸（mRNA）的表达。结果 PD患者外周血单核细胞IL-6基因表达增强（P〈0.05）。PD组PMBC中IL-2、IFN-1、IL-4、IL-104种细胞因子基因的表达与对照组相比差异无显著性（均P〉0,05）。PD2组与PD1组比较,所测PMBC中5种细胞因子基因的表达虽均有所下降,但差异无显著性。结论 PD患者PBMC存在辅助性T细胞（Th2）漂移倾向,表明免疫机制在PD的发病中具有重要作用。     

丙戊酸对实验性自身免疫性神经炎大鼠保护作用的机制

目的观察丙戊酸(VAP)对实验性自身免疫性神经炎(EAN)大鼠的保护作用及其机制。方法实验大鼠随机分为VAP高剂量组、VAP低剂量组、EAN模型组、正常组,应用P2 57-81多肽与完全弗氏佐剂的混合液诱导EAN模型。VAP于免疫当天至第15d每天腹腔内注射。观察各组大鼠发病情况和坐骨神经组织病理学变化,检测外周血中Th17细胞和Foxp3+Treg细胞含量,检测淋巴结中TNF-α、IFN-γ、IL-17、TGF-βmRNA表达。结果 VAP高剂量组的最初发病时间迟于EAN组(P<0.05),其高峰期临床评分显著低于EAN组(P<0.05),坐骨神经炎性细胞浸润较EAN组明显减少;VAP高剂量组和低剂量组外周血中Th17细胞比例较EAN组显著减少(P<0.05),Foxp3+Treg细胞比例较EAN组显著增加(P<0.05),淋巴结中促炎细胞因子TNF-α、IFN-γ及IL-17mRNA表达与EAN组比较明显下降(P<0.05),VAP高剂量组抑炎细胞因子TGF-βmRNA表达与EAN组比较明显升高(P<0.05)。结论 VAP对EAN有治疗作用,这种作用可能与其能够增加Foxp3+Treg细胞和抑炎细胞因子TGF-β含量、减少TH17细胞含量和促炎细胞因子的表达有关。     

Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis, where disease is mediated by autoantigen-specific T cells. Although there is evidence linking CD4⁺ T cells that secrete IL-17, termed Th17 cells, and IFN-γ-secreting Th1 cells with the pathogenesis of EAE, the precise contribution of these T cell subtypes or their associated cytokines is still unclear. We have investigated the infiltration of CD4⁺ T cells that secrete IFN-γ, IL-17 or both cytokines into CNS during development of EAE and have examined the role of T cells in microglial activation. Our findings demonstrate that Th17 cells and CD4⁺ T cells that produce both IFN-γ and IL-17, which we have called Th1/Th17 cells, infiltrate the brain prior to the development of clinical symptoms of EAE and that this coincides with activation of CD11b⁺ microglia and local production of IL-1β, TNF-α and IL-6 in the CNS. In contrast, significant infiltration of Th1 cells was only detected after the development of clinical disease. Co-culture experiments, using mixed glia and MOG-specific T cells, revealed that T cells that secreted IFN-γ and IL-17 were potent activators of pro-inflammatory cytokines but T cells that secrete IFN-γ, but not IL-17, were less effective. In contrast both Th1 and Th1/Th17 cells enhanced MHC-class II and co-stimulatory molecule expression on microglia. Our findings suggest that T cells which secrete IL-17 or IL-17 and IFN-γ infiltrate the CNS prior to the onset of clinical symptoms of EAE, where they may mediate CNS inflammation, in part, through microglial activation.     

γ干扰素上调吉兰一巴雷综合征患者外周血中CD4^＋CD25^＋调节性T细胞的研究

目的探讨γ干扰素（IFN-γ）诱导吉兰-巴雷综合征（GBS）患者外周血CD4^＋CD25 T细胞转化为CD4^＋CD25^＋调节性T细胞的可行性。方法不同浓度IFN-γ刺激GBS患者外周血中的CD4^＋CD25T细胞。Real-timePCR检测诱导CD4^＋CD25^＋调节性T细胞中FoxP3的表达,共培养检测其抑制功能;结果与健康人中天然的CD4^＋CD25^＋调节性T细胞比较。结果IFN-γ可诱导CD4^＋CD25^＋T细胞转化为CD4^＋CD25^＋调节性T细胞。IFN-γ40ng.mL^-1诱导的CD4^＋CD25^＋调节性T细胞中FoxP3表达含量最高,但仍然低于天然的CD4^＋CD25^＋调节性T细胞;其抑制能力最强,与天然的CD4^＋CD25^＋调节性T细胞比较差异无统计学意义。结论IFN-γ可诱导GBS患者外周血CD4^＋CD25^＋T细胞转化为CD4^＋CD25^＋调节性T细胞,IFN-γ,40ng.mL^-1诱导的CD4^＋CD25^＋调节性T细胞表型和功能与天然的CD4^＋CD25^＋调节性T细胞相当。     

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.     

Cytokines and the pathogenesis of myasthenia gravis

Myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) are caused by autoantibodies against nicotinic acetylcholine receptor (AChR) in skeletal muscle. The production of anti-AChR antibodies is mediated by cytokines produced by CD4⁺ and CD8⁺ T helper (Th) cells. Emerging investigations of the roles of cytokines in MG and EAMG have revealed that the Th2 cell related cytokine interleukin 4 (IL-4), an efficient growth promoter for B-cell proliferation and differentiation, is important for anti-AChR antibody production. IL-6 and IL-10 have similar effects. The Th1 cytokine IFN-γ is important in inducing B-cell maturation and in helping anti-AChR antibody production and, thereby, for induction of clinical signs and symptoms. Results from studies of time kinetics of cytokines imply that IFN-γ is more agile at the onset of EAMG, probably being one of the initiating factors in the induction of the disease, and IL-4 may be mainly responsible for disease progression and persistance. Even though other Th1 cytokines like IL-2, tumor necrosis factor α (TNF-α), and TNF-β as well as the cytolytic compound perforin do not directly play a role in T-cell-mediated help for anti-AChR antibody production, they are actually involved in the development of both EAMG and MG, probably by acting in concert with other cytokines within the cytokine network. In contrast, transforming growth factor β (TGF-β) exerts immunosuppressive effects which include the down-regulation of both Th1 and Th2 cytokines in MG as well as EAMG. Suppressive effects are also exerted by interferon α (IFN-α). Based on elucidation of the role of cytokines in EAMG and MG, treatments that up-modulate TGF-β or IFN-α and/or suppress cytokines that help B-cell proliferation could be useful to improve the clinical outcome. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 543–551, 1997     

Increased cytokines/chemokines and hyponatremia as a possible cause of clinically mild encephalitis/encephalopathy with a reversible splenial lesion associated with acute focal bacterial nephritis

ObjectiveClinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), the second most common encephalopathy syndrome in Japan, is most often associated with viral infection. Bacterial MERS has been rarely reported but is mostly associated with acute focal bacterial nephritis (AFBN) for an unknown reason. We examined cytokines and chemokines in four MERS patients with AFBN to determine if they play an important role in the pathogenesis.MethodsWe examined the clinical charts and MRI results in four MERS patients with AFBN, and measured 10 cytokines and chemokines in serum and cerebrospinal fluid in the acute phase. These were analyzed using the Mann-Whitney U test, compared with the control group (cases with a non-inflammatory neurological disease). Longitudinal changes in the serum cytokine and chemokine levels were evaluated in two patients.ResultsHyponatremia was observed in all four patients with MERS associated with AFBN (128–134 mEq/L). CSF analysis revealed increased cytokines/chemokines associated with Th1 (CXCL10, TNF-α, IFN-γ), T reg (IL-10), Th17 (IL-6), and neutrophil (IL-8 and CXCL1). In serum, upregulation was observed in those associated with Th1 (CXCL10, TNF-α, IFN-γ), Th17 (IL-6), and inflammasome (IL-1ß). The increased serum cytokines/chemokines in the acute stage normalized within 2 weeks in patients 1 and 2, so examined, in accordance with their clinical improvement.ConclusionIncreased cytokines/chemokines and hyponatremia may be factors that explain why AFBN is likely to cause MERS.