程序性死亡因子1及其配体在糖尿病视网膜病变患者外周血单个核细胞中的表达

目的 观察程序性死亡因子1(PD1)及其配体PD-L1和PD-L2在糖尿病视网膜病变(DR)患者外周血单个核细胞(PBMCs)中的表达.方法 临床检查确诊为DR的40例患者(DR组)以及同期行体检的健康志愿者20名(对照组)纳入研究.DR组40例患者中,非增生型DR(NPDR)20例,增生型DR(PDR) 20例.抽取两组受检者晨起空腹静脉血2 ml,采用荧光定量聚合酶链式反应(PCR)检测PD-1、PD-L1、PD-L2 mRNA的表达水平.对比分析DR、对照组之间,PDR与NPDR组之间各指标表达水平的差异.结果 荧光定量PCR检测发现,DR组患者PBMCs中PD-1、PD-L1 mRNA相对表达量显著低于对照组,差异有统计学意义(t=-2.060,-2.562;P=0.043,0.013);PD-L2 mRNA相对表达量较对照组无明显变化,差异无统计学意义(t=-0.857,P=0.395).PDR组患者PBMCs中PD-1 mRNA相对表达量较NPDR组有降低趋势,PD-L1、PD-L2 mRNA相对表达量有升高趋势,但差异均无统计学意义(t=-1.335,0.987,0.131;P=0.190,0.334,0.897).结论 DR患者PBMCs中PD-1、PD-L1 mRNA相对表达较正常者低,PD-L2 mRNA相对表达较正常者无明显变化.     

大鼠全层巩膜切除眼外滤过术后前房相关性免疫偏离的研究

目的 研究大鼠全层巩膜切除眼外滤过术后前房相关性免疫偏离(ACAID)诱导能力的变化及其与术后时间的关系.方法 对照实验研究.50只近交系Wistar大鼠,右眼为实验眼,建立全层巩膜切除眼外滤过术模型后,采用计算机随机数字表法分为5组,每组10只鼠.另外5只SD大鼠,1只Wistar大鼠,提供诱导ACAID所需去上皮角膜片.A组为阴性对照组,在全层巩膜切除眼外滤过术后1周,前房植入Wistar大鼠角膜片;B组为阳性对照组,在全层巩膜切除眼外滤过术后1周,颈部皮下注射SD大鼠脾细胞,前房不植入角膜片;C、D、E组为实验组,分别在全层巩膜切除眼外滤过术后1、4、8周,前房植入SD大鼠角膜片.B组在颈部皮下注射SD大鼠脾细胞悬液,1周后右耳廓皮下注射SD大鼠脾细胞悬液,诱导迟发型超敏反应.其他各组均在前房植入角膜片,2周右耳廓皮下注射SD大鼠脾细胞悬液,诱导迟发型超敏反应.耳廓膨胀指数反映诱导迟发型超敏反应的程度.同时各组取心脏血,检测血清中白细胞介素(IL)4和IL-10浓度,取脾脏组织检测细胞转录因子GATA-3 mRNA表达水平.通过诱导迟发型超敏反应,检测Th2的GATA-3和Th2细胞来源细胞因子IL-4及IL-10,分析机体免疫状态,并进而评价ACAID.此外,各组鼠取心脏血和脾脏组织后,再摘除眼球,进行组织病理学检查,观察前房炎性反应变化.采用SPSS 13.0统计学软件进行数据分析.多组间诱导迟发型超敏反应、IL-4、IL-10及逆转录聚合酶链反应(RT-PCR)检测结果比较采用单因素方差分析,进一步两两比较采用LSD法,各组数据与阳性对照组或阴性对照组比较采用t检验.结果 (1)诱导迟发型超敏反应检测,结果显示全层巩膜切除眼外滤过术后,B、C、D、E组鼠均诱发出迟发型超敏反应.(2)血清IL-4和IL-10浓度检测,结果表明全层巩膜切除眼外滤过术后,3个实验组间血清IL-4及IL-10浓度差异均有统计学意义(F=49.124,6.336;均P<0.01);其中D组鼠血清IL-4及IL-10浓度明显低于C组和E组;血清IL-4及IL-10浓度呈现随术后短时间内上升,而后下降,并随时间推移再次增高的趋势.(3)脾脏组织GATA-3表达检测,结果显示D组和E组鼠脾脏组织中GATA-3表达明显上调.(4)眼球组织病理学检查,可见C组鼠前房内有少量炎性渗出和炎性细胞,D组和E组鼠前房内未见明显炎性渗出和炎性细胞.结论 大鼠全层巩膜切除和眼外滤过术后,将暂时失去诱导ACAID的能力.但随术后前房炎性反应的消退,血清IL-10及IL-4浓度再次增高,脾脏GATA-3表达明显上调,表明机体ACMD的诱导能力有逐渐恢复的趋势.(中华眼科杂志,2009,45:32-37)     

前房相关免疫偏离形成中IDO的表达及意义

目的探讨吲哚胺2,3-二氧化酶(IDO)在前房相关免疫偏离(ACAID)形成中的作用。方法分别将卯白蛋白(OVA)及其与完全弗氏佐剂的混合液注入BALB/C小鼠前房及后足垫内,皮内注射OVA观察迟发型超敏反应,评价ACAID的形成。分别于小鼠前房注射OVA后第3、7、10和14d处死动物,取其脾脏,采用免疫组织化学和Westernblot法检测小鼠脾脏组织中IDO的表达及其动态变化。结果正常小鼠脾脏组织中有少量IDO表达;前房注射OVA后第7、10和14dIDO表达量明显增加(P<0.05)。免疫组织化学显示的阳性细胞数量变化与Western-blot检测的蛋白动态变化相一致。结论在ACAID形成过程中IDO表达显著性增加,提示它可能参与了ACAID的形成。     

哌仑西平影响豚鼠实验性近视眼的研究

目的 探讨玻璃体腔注射选择性M_1受体拮抗剂哌仑西平对豚鼠形觉剥夺性近视眼视网膜、脉络膜、巩膜和虹膜-睫状体组织中M_1和M_4受体mRNA表达的影响.方法 采用随机分组设计的实验研究.1～2周龄的三色豚鼠24只,随机分为4组:正常对照组(N)6只,单纯形觉剥夺组(FDM)6只,药物对照组(S)6只,哌仑西平组(P)6只,均以左眼为实验眼,P组隔日玻璃体腔注射500μg哌仑西平;S组隔日玻璃体腔注射生理盐水.21 d后结束实验.提取各组眼视网膜、脉络膜、巩膜和虹膜-睫状体组织总RNA,半定量RT-PCR检测M_1和M_4亚型mRNA表达变化.3组间数据的比较采用单因素方差分析和Tukey post hoc检验.结果 21d时,FDM与N组相比较,FDM组眼轴相对延长0.29mm,产生相对近视-4.92 D,差异有统计学意义(P<0.001).P组与S组相比较,眼轴相对减少了0.30 mm,产生+0.88 D的相对远视,差异均有统计学意义(P<0.001).S组与FDM之间屈光度数的差异无统计学意义;而眼轴变化有统计学意义(S组相对延长0.08 mm,而FDM组相对延长0.29/mm).半定量PCR结果显示:P组与S组相比较,其视网膜、脉络膜和虹膜睫状体组织M_1和M_4亚型mRNA的表达差异无统计学意义(P>0.05).而在后极部巩膜组织,M_1和M_4亚型mRNA的表达较药物对照组显著性增加(P<0.05),其中M_1受体表达增加19.16%,M_4受体表达增加64.29%.结论 哌仑西平能够有效抑制豚鼠形觉剥夺近视的发展.巩膜组织M_1和M_4亚型及其胆碱能通路可能参与M受体拮抗剂抑制近视发展.     

真菌性小鼠角膜炎中炎性因子的表达

目的 探讨4种炎性因子在真菌性小鼠角膜炎发病中的表达和作用.方法 实验研究.240只BALB/c小鼠采用随机数字表法分为4组,分别为茄病镰刀菌感染组、烟曲霉菌感染组、白色念珠菌感染组及损伤对照组,每组60只.建立小鼠真菌性角膜炎模型.分别于术后1、3、5及7 d,于裂隙灯显微镜下观察角膜疾病的发展,病理学检查病变角膜的炎症反应和菌丝生长情况,反转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测巨噬细胞炎性蛋白2(MIP-2)、细胞因子诱生性中性粒细胞趋化物(KC)、白细胞介素(IL)1β和IL-6炎性因子的mRNA和蛋白质表达水平.对临床评分、RT-PCR和ELISA结果比较应用参数检验中方差分析(one-way ANOVA),进一步两两比较采用LSD检验法.结果 随时间变化各感染组小鼠角膜病变和炎症反应出现先增强到术后7 d开始减弱的趋势.RT-PCR结果显示,MIP-2和IL-1β的表达在烟曲霉菌感染组最强,KC在白色念珠菌感染组最强,IL-6在茄病镰刀菌感染组最强.各感染组中IL-1β和KC的表达强于IL-6和MIP-2.MIP-2、KC及IL-1β在茄病镰刀菌感染组、烟曲霉菌感染组及白色念珠菌感染组的表达高峰分别为术后5、1及3 d,IL-6在茄病镰刀菌和烟曲霉菌感染组的高峰为术后1和7 d,在白色念珠菌感染组的高峰为术后1和5 d.对各实验组同一因子mRNA的表达强度的相对值进行相互比较分析显示,各组间差异均有统计学意义(MIP-2:F=1675.339,P<0.01;KC:F=730.267,P<0.01;IL-1β:F=297.106,P<0.01;IL-6:F=174.513,P<0.01).ELISA结果显示,各感染组的4种因子蛋白表达量从高到低依次为IL-1β、MIP-2、KC及IL-6.不同实验组间和相同实验组中每种因子的蛋白表达随时间变化规律与4种因子的mRNA表达规律吻合.对各实验组同一因子的蛋白表达水平进行相互比较分析表明,各组间差异均有统计学意义(MIP-2:F=2871.736,P<0.01;KC:F=886.598,P<0.01;IL-1β:F=3595.488,P<0.01;IL-6:F=89.225,P<0.01).结论 MIP-2、KC和IL-1β的mRNA和蛋白表达水平与真菌性角膜炎的病变程度和炎症反应密切相关,其中MIP-2和IL-1β的持续高水平表达可能在真菌性角膜炎病变损害中起着重要的作用.     

B7-H3在小鼠角膜移植免疫赦免中的作用探讨

目的 探讨共刺激分子B7-H3在小鼠角膜中的表达情况以及在角膜移植免疫赦免中的作用.方法 实验研究.以C57BL/6小鼠为供体、BALB/c小鼠为受体建立角膜移植实验模型,采用简单随机抽样方法,取8只未行角膜移植的BALB/c小鼠作为正常对照组,另取8只BALB/c小鼠作为自体角膜移植组,最后30只BALB/c小鼠进行同种异体角膜移植;按Sonoda法观察小鼠角膜移植后的存活率,8周内植片混浊评分≥2分判定为排斥组,8周时未达到2分的认为是存活组;各组各取3只眼球,HE染色后行组织病理学观察并行B7-H3分子的免疫组织化学检测;各组各取5只眼角膜,行荧光定量PCR检测B7-H3分子mRNA的表达情况.各组角膜组织中的B7-H3 mRNA表达差异倍数比较采用设计单因素的方差分析,组间的多重比较采用LSD-t检验.结果 同种异体角膜移植组并未完全排斥,30只小鼠中有9只存活,21只排斥,移植存活率为30％;自体角膜移植组移植存活率为100％;免疫组织化学表明B7-H3分子在正常对照组和自体角膜移植组的角膜上皮、内皮细胞和虹膜睫状体中均有表达,在存活组中的表达明显增加,而在排斥组中的表达明显减少;qPCR检测在正常对照组(4.30±0.023)和自体角膜移植组(4.33±0.031)中均可以检测出B7-H3分子mRNA的表达;同种异体角膜移植排斥组B7-H3分子的mRNA表达程度(3.89±0.037)明显下降;但在同种异体角膜移植存活组中B7-H3分子的mRNA表达(5.04±0.058)明显增加;将各组B7-H3 mRNA的表达差异进行统计学分析,先进行方差齐性检验(F =429.546),再采用LSD法进行两两比较,除了正常对照组与自体角膜移植组之间差异无统计学意义(P =0.387)之外,正常对照组与存活组及排斥组比较,差异均有统计学意义(P =0.001,0.003)结论 B7-H3分子在促进角膜移植的免疫赦免中发挥一定的作用,可能是维持移植角膜免疫耐受状态的重要因子之一.     

缺氧诱导因子1α和血管内皮生长因子干扰性RNA抑制鼠视网膜新生血管的研究

目的 探讨缺氧诱导因子1α(HIF-1α)和血管内皮生长因子(VEGF)小片段干扰性RNA(siRNA)对小鼠视网膜新生血管的抑制作用.方法 本研究采用随机对照方法.构建HIF-1αsiRNA重组质粒.C57BL/6J小鼠玻璃体腔注射增强型绿色荧光蛋白(EGFP)表达质粒pEGFP-N1,1 d后视网膜铺片观察绿色荧光蛋白(GFP)的表达.选7天龄C57BL/6J小鼠90只,17只为正常组,73只建立氧诱导的视网膜新生血管模型,分为模型对照组、空载体组和基因治疗组(HIF-1α siRNA组、VEGF siRNA组及共转染组).于出氧舱前1 d,向空载体组小鼠玻璃体腔注射空载体质粒;HIF-1α siRNA组注射HIF-1α siRNA,VEGF siRNA组注射VEGF165 siRNA,共转染组注射HIF-1αsiRNA+VEGF165 siRNA.采用荧光造影视网膜铺片方法观察血管形态变化;制作组织切片计算突破视网膜内界膜的血管内皮细胞核数;采用逆转录聚合酶链反应和Western blot检测视网膜HIF-1α和VEGF的表达.采用单因素方差分析和组间最小显著差法进行统计学分析.结果 pEGFP-N1经脂质体LF2000介导转染视网膜细胞后1 d即可见GFP表达.基因治疗组较模型对照组新生血管丛明显减少,荧光渗漏明显减轻,其中共转染组效果最明显.基因治疗组[HIF-1α siRNA组为(27.73±2.33)个,VEGF siRNA组为(15.43±3.23)个,共转染组为(8.70 ±2.88)个]较其他3组突破视网膜内界膜的细胞核数量减少,差异均具有统计学意义(F=3016.527,P＜0.01).视网膜HIF-1α mRNA及蛋白表达水平:模型对照组(1.08±0.06,0.383±0.009)和空载体组(1.09±0.05,0.386 ±0.010)较正常组(0.81 ±0.07,0.035±0.003)上调,而HIF-1α siRNA组(0.46±0.06,0.182±0.008)较模型对照组下调,抑制效率分别为57.4%和52.5%,差异均有统计学意义(F=139.804,2686.001;P＜0.01).VEGF mRNA及蛋白表达水平:模型对照组(1.53 ±0.07,0.340±0.004)和空载体组(1.59±0.06,0.337±0.009)较正常组(0.27±0.08,0,051±0.008)明显上调,而基因治疗组较模型对照组明显下调,差异均有统计学意义(F=421.423,2513.583;P＜0.01),其中共转染组下调最明显,抑制效率分别为85.6%和80.9%.结论 HIF-1α siRNA和VEGF165siRNA均可有效抑制小鼠视网膜新生血管形成,两种siRNA共转染抑制效果最明显.     

细胞因子信号传导抑制因子在实验性自身免疫性葡萄膜视网膜炎视网膜内的表达

目的 探讨细胞因子信号传导抑制因子(SOCS)在实验性自身免疫性葡萄膜视网膜炎(EAU)视网膜内的表达与意义.方法 随机对照实验设计方法.90只Lewis大鼠采用分层随机抽样进行分组,分别为未治疗组40只大鼠、治疗组40只大鼠及正常对照组10只大鼠.用光感受器间维生素A类结合蛋白(IRBP)诱导EAU动物模型,治疗组从免疫后1～28 d,给予环孢素(CsA)灌胃,每天20 mg/kg体重,而未治疗组给予等量生理盐水,正常对照组未给予免疫和治疗.分别于免疫后7、14、21、28 d按分层随机号抽取治疗组与未治疗组各10只大鼠进行相关枪测,观察大鼠眼部炎性变化,双眼取材,分别用实时荧光定量聚合酶链反应和蛋白免疫印迹法检测大鼠视网膜内SOCS mRNA和蛋白的表达,酶联免疫吸附试验方法检测血清细胞因子白细胞介素4(IL-4)、IL-12、干扰素γ(IFN-γ)的水平.利用单因素方差分析对治疗组与未治疗组大鼠血清中细胞因子的浓度和视网膜内SOCS蛋白表达量进行对比分析,多重比较采用最小显著差异法;用Mann-Whitney两样本比较秩和检验进行治疗组与未治疗组大鼠炎性反应分值比较.结果 免疫后14 d,未治疗组可见明显的虹膜睫状体炎,房水闪辉阳性,虹膜后粘连,瞳孔不规则甚至前房积脓,炎性反应分值是1.5±0.5;而治疗组眼部炎性反应不明显,炎性反应分值是0.5±0.3.未治疗组的IL-12、IFN-γ及IL-4免疫后14 d达到最高峰(均P＜0.05),免疫后28 d IL-12、IFN-γ下降到正常水平(均P＞0.05),IL-4仍高于正常对照组(P＜0.05);治疗组中IL-12、IFN-γ的动态变化不明显(均P＞0.05),IL-4在整个病程中呈上升趋势(均P＜0.05).SOCS1、含SH2结构的细胞因子诱导蛋白(CIS)mRNA免疫后14 d表达最高,未治疗组分别是正常对照组的3.41倍和3.36倍,治疗组分别为1.44倍和1.73倍;SOCS3和SOCS5mRNA在整个病程中逐渐升高,免疫后28 d表达最高,未治疗组中分别为正常对照组的1.95倍和3.16倍,治疗组中分别为正常对照组的1.59倍和3.58倍.未治疗组SOCS1和CIS蛋白在免疫后7、14、21 d时显著高于正常对照组(均P＜0.05),治疗组二者免疫后14 d显著高于正常对照组(均P＜0.05);两组中SOCS3和SOCS5蛋白在免疫后均显著高于正常对照组(均P＜0.05).结论 EAU发生过程中视网膜SOCS1升高与Th1型反应增强有关;在发病的不同阶段中CIS与SOCS3升高和Th2细胞反应增强以对抗Th1细胞反应,与缓解葡萄膜炎的发病强度有关;SOCS5水平升高在眼局部炎性反应发生过程中可能起保护作用.     

CD86在排斥反应和前房相关免疫偏离过程中的作用

目的检测大鼠角膜共刺激分子CD86（B7—2）的原位表达,探讨CD86分子与角膜移植排斥反应和前房相关免疫偏离（ACAID）过程的关系。方法制作穿透角膜移植排斥反应和同一供体前房内注射脾细胞诱导ACAID的大鼠模型;角膜移植组进行排斥反应指数（RI）评分;ACAID组进行迟发型超敏反应（DTH）评价;采用免疫组织化学的方法检测CD86在角膜中的原位表达（以脾脏的表达为阳性对照）。结果角膜移植组植片均出现不同程度的新生血管、角膜水肿、混浊、增厚;ACAID组角膜透明,房水清,DTH评价术后2周及4周诱导成功率100％;免疫组织化学检测CD86在正常大鼠角膜组织全层无阳性表达,在移植后出现急性排斥反应的大鼠角膜上皮层中有大量阳性细胞表达,在ACAID诱导成功的大鼠角膜中未见阳性细胞表达。结论共刺激分子CD86参与移植后的急性排斥反应,但可能不参与免疫赦免过程。     

细胞毒性T淋巴细胞相关抗原4-凋亡相关蛋白配体双功能蛋白抑制小鼠角膜移植免疫排斥反应的研究

目的 探讨利用基因重组技术合成的细胞毒性T淋巴细胞相关抗原4(CTLA4)-凋亡相关蛋白配体(FasL)双功能蛋白预防小鼠角膜移植术后免疫排斥反应发生的疗效及其作用机制.方法 为实验研究.建立小鼠穿透性角膜移植动物模型,供体为C57BL/6小鼠(45只),受体为BALB/c小鼠(90只).利用随机分组法将实验动物分为3组,每组30只,A组即对照组(不进行任何治疗),B组即环孢素A缓释系统(CsA DDS)前房植入组,C组即10 μg/ml CTLA4-FasL双功能蛋白浸泡角膜植片组.术后比较3组间角膜植片的存活时间,并分别利用免疫组织化学检测CD4+T细胞,逆转录PCR(RT-PCR)检测CD80、CD86、白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)mRNA,DNA原位末端标记(TUNEL)检查凋亡的发生情况.结果 A、B、C组角膜植片的存活时间分别为(14.3±1.3)、(58.0±2.8)、(106.3±17.5)d.C与A组、C与B组、B与A组进行组间两两比较,差异均有统计学意义(均P=0.000).术后A组角膜植片中炎性细胞数量不断增加,以CD4+T细胞浸润为主;B组角膜植片中未见明显炎性细胞浸润;C组角膜植片中浸润的CD4+T细胞数量在术后7 d达到最多,之后发生骤减.RT-PCR检查可见在术后3 d A组和B组角膜和虹膜组织表达CD86 mRNA,术后7 d表达CD80 mRNA,而在相同时间点上C组均减弱表达CD86和CD80mRNA.术后14 d,A组发生排斥的角膜中可检测到IL-10、TNF-α、IFN-γ mRNA,B组和C组则检测不到上述细胞因子的表达.TUNEL检查显示术后7 d C组角膜和虹膜组织中分布着大量发生凋亡的单核细胞,而A、B组均未观察到细胞凋亡现象.结论 CTLA4-FasL双功能蛋白既能阻断T细胞活化所需的CD28-CD80/CD86共刺激信号途径,又能诱导T细胞凋亡,通过双重作用机制发挥免疫抑制作用,从而有效地延长角膜植片的存活时间.     

CD4+PD-1+ T cells acting as regulatory cells during the induction of anterior chamber-associated immune deviation

PURPOSE: To study the expression and functional characteristics of programmed death-1 (PD-1) and its ligands in the spleens of mice undergoing anterior chamber-associated immune deviation (ACAID). METHODS: ACAID was induced in BALB/c mice by intracameral injection of ovalbumin (OVA). The expression of PD-1 and its ligands in the spleens of ACAID mice was determined by quantitative real-time PCR, Western blotting, and flow cytometry. In vitro proliferation assays, enzyme-linked immunosorbent assays, and adoptive transfer assays were used to investigate the functional characteristics of splenic CD4+PD-1+ T cells of ACAID mice. RESULTS: Both mRNA and protein of PD-1, PD-L1, and PD-L2 were markedly upregulated in the spleens of ACAID mice compared with controls. CD4+PD-1+ T cells from ACAID mice produced large amounts of IL-10 and exhibited in vitro antigen-specific suppressive activity. CD4+PD-1+ T cells from ACAID mice were able to significantly inhibit the antigen-specific, delayed-type hypersensitivity response when adoptively transferred to naive mice. CONCLUSIONS: CD4+PD-1+ T cells from ACAID mice, as regulatory cells, are involved in the induction of antigen-specific suppression in association with enhanced expression of IL-10. CD4+PD-1+ T cells in the murine spleen may represent a substantial population of regulatory T cells possibly responsible for the induction of ACAID after intracameral injection of antigen.     

Effect of CD4~+CD25~+ regulatory T cells in the development of anterior chamber-associated immune deviation

AIM:To investigate whether CD4+CD25+ regulatory T(Treg) cells play a role in the development of anterior chamber-associated immune deviation(ACAID).· METHODS:The dynamic changes in the frequency of CD4+CD25+ T cells,CD4+CD25+ FoxP3+ T cells and CD4+CD25+ PD-1+ T cells from spleens of mice with ACAID were analyzed by flow cytometry.Foxp3 mRNA expression in purified CD4+CD25+ T cells was analyzed using real-time PCR.The suppressive effect of purified CD4+CD25+ T cells on the proliferation of CD4+CD25-T cells was evaluated by [3H] thymidine incorporation.A blocking experiment was performed to further address the role of CD4+CD25+ T cells in ACAID.The expression of IL-10 in purified CD4+CD25+ T cells was evaluated by ELISA.· RESULTS:Increased frequencies of CD4+CD25+ T cells,CD4+CD25+ Foxp3+ T cells and CD4+CD25+ PD-1+ T cells were observed in ACAID.The CD4+CD25+ T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4+CD25-T cells.Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID.Furthermore,purified CD4+CD25+ T cells from ACAID mice secreted IL-10.· CONCLUSION:Our results demonstrate that Treg cells are induced in the mice undergoing ACAID.These Treg cells may play a role in the development of ACAID.     

OVA-specific CD8+T cells do not express granzyme B during anterior chamber associated immune deviation

Purpose To examine antigen (Ag)-specific CTL response during anterior chamber associated immune deviation (ACAID).Methods OVA or OVA257-264 peptide was injected into the anterior chamber (AC) of C57BL/6 mice. There were 16 mice in each ACAID group induced with OVA or OVA257-264 peptide. The mice were primed by SC injection with OVA or OVA 257-264 peptide in complete Freund’s adjuvant (CFA) on day 7. Ag-specific CD8⁺T cells in spleens were analyzed on day 14 using Pentamer H-2K^b-SIINFEKL(OVA257-264 peptide). IFN-γ ELISPOT and intracellular granzyme B staining were used to characterize the CTL response. Twelve mice in each group immunized with OVA or OVA257-264 peptide in CFA served as positive controls. Twelve normal mice served as negative controls and 12 receiving injection of CFA as CFA controls for studying the influence of CFA on the Ag-specific CTL response.Result The results showed that anterior chamber inoculation of OVA or OVA257-264 peptide could induce ACAID as evidenced by an impaired DTH response. The frequency of Ag-specific CD8⁺T cells in ACAID mice was not different from that in mice challenged with Ags in CFA only (positive controls). IFN-γ production by these cells in ACAID mice was not different compared to positive controls. However, Ag-specific CD8⁺T cells in ACAID mice failed to secrete granzyme B. Mice challenged only with OVA peptide and CFA also showed a granzyme B negative CD8⁺T cell response. Ag-specific CTL response induced by CFA alone was similar with the negative control.Conclusion These results show that the frequency of Ag-specific CD8⁺T cells is not altered during ACAID. The Ag-specific CTL response during ACAID is characterized by the absence of granzyme B expression.This study was supported by the Fund for Innovative Research Groups of China (30321004), National Natural Science Foundation (30572004) and Natural Science Foundation for Research Groups of Guangdong Province (2005-04).     

IL-6 antagonizes TGF-beta and abolishes immune privilege in eyes with endotoxin-induced uveitis

PURPOSE: To determine the immunosuppressive status of aqueous humor (AqH) from mouse eyes afflicted with endotoxin-induced uveitis (EIU) and to identify the relevant cytokines responsible for immunomodulatory activity within EIU AqH. METHODS: Bacterial lipopolysaccharide (LPS) was injected into hind footpads of C3H/HeN mice; and AqH, collected at 6, 12, 24, and 48 hours, was evaluated for content of transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and interferon (IFN)-gamma and capacity to suppress anti-CD3-driven T-cell proliferation. Cytokine mRNA expression in iris-ciliary body (I/CB) was analyzed by RNase protection assays. RESULTS: During 6 to 24 hours after LPS injection, total TGF-beta levels in AqH increased even though the fluid lost its capacity to suppress T-cell activation. At this time, AqH contained high levels of IL-6, and I/CB contained high levels of IL-6 mRNA. When IL-6 was neutralized with specific antibodies, inflamed AqH reacquired its capacity to suppress T-cell activation, which correlated with high levels of TGF-beta. Coinjection of IL-6 plus antigen into the anterior chamber of the eye of normal mice prevented antigen-specific anterior chamber-associated immune deviation (ACAID). CONCLUSIONS: LPS-induced intraocular inflammation is associated with local production of IL-6, which robs AqH of its immunosuppressive activity, perhaps by antagonizing TGF-beta. The fact that IL-6 antagonized ACAID induction in normal eyes suggests that strategies to suppress the intraocular synthesis of IL-6 may reduce inflammation and restore ocular immune privilege.     

Expression of Tim-3 is transiently increased before development of anterior chamber-associated immune deviation

PURPOSE: To assess the expression of T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) in the spleens of BALB/c mice undergoing anterior chamber-associated immune deviation (ACAID). METHODS: ACAID was generated after intracameral (i.c.) injection of ovalbumin (OVA) into BALB/c mice and evaluated by assessing the delayed-type hypersensitivity (DTH) response following a subsequent subcutaneous (s.c.) injection of OVA emulsified in complete Freund's adjuvant (CFA) on Days 0, 3, 7, 14, 21 and 28. Tim-3 mRNA levels were detected using real-time RT-PCR, and the frequency of CD4+Tim-3+ T cells in splenocytes as well as the coexpression of Tim-3 with CD25 on CD4+ T cells were assessed by flow cytometry. RESULTS: A significantly suppressed DTH response was observed on Days 7, 14, 21, and 28, but not on Days 0 and 3 during the development of ACAID. The levels of Tim-3 mRNA and the frequency of CD4+CD25+Tim-3+ T cells in the splenocytes reached a peak on Day 3, declined on Day 7, and returned to basal levels thereafter. CONCLUSIONS: A transient upregulation of Tim-3 expression was observed in the early stage of ACAID, suggesting its possible involvement in the development of ACAID.     

程序性死亡分子-1及其配体通路在眼科领域的研究进展

共刺激信号已经成为免疫学研究的一个热点。程序性死亡分子-1（PD-1）及其配体属于抑制性共刺激分子,其在自身免疫性疾病、器官移植排斥反应、病原微生物感染、肿瘤免疫逃逸等方面都发挥着重要作用。近年来,PD—1及其配体（PD-1／PD—L）通路在眼科方面的研究也取得了一定进展。眼部多种疾病和现象,如眼部免疫性疾病、眼部感染性疾病、眼部肿瘤、眼组织的免疫赦免等均发现有PD—1／PD—L通路的改变。这些发现不仅进一步阐明了一些眼部疾病的发病机制,而且为其预防和治疗提供了新的方向。就PD—1／PD—L通路的生物学特性及其在眼科生理病理中的研究进展进行综述。     

Expression of Tim-3 Is Transiently Increased before Development of Anterior Chamber-Associated Immune Deviation

Purpose: To assess the expression of T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) in the spleens of BALB/c mice undergoing anterior chamber-associated immune deviation (ACAID). Methods: ACAID was generated after intracameral (i.c.) injection of ovalbumin (OVA) into BALB/c mice and evaluated by assessing the delayed-type hypersensitivity (DTH) response following a subsequent subcutaneous (s.c.) injection of OVA emulsified in complete Freund's adjuvant (CFA) on Days 0, 3, 7, 14, 21 and 28. Tim-3 mRNA levels were detected using real-time RT-PCR, and the frequency of CD4⁺Tim-3⁺ T cells in splenocytes as well as the coexpression of Tim-3 with CD25 on CD4⁺ T cells were assessed by flow cytometry. Results: A significantly suppressed DTH response was observed on Days 7, 14, 21, and 28, but not on Days 0 and 3 during the development of ACAID. The levels of Tim-3 mRNA and the frequency of CD4⁺CD25⁺Tim-3⁺ T cells in the splenocytes reached a peak on Day 3, declined on Day 7, and returned to basal levels thereafter. Conclusions: A transient upregulation of Tim-3 expression was observed in the early stage of ACAID, suggesting its possible involvement in the development of ACAID.