大鼠自身免疫性葡萄膜视网膜炎眼部细胞因子信号抑制因子的表达

目的:大鼠自身免疫性葡萄膜视网膜炎(experimentalautoimmune uveoretinitis,EAU)大鼠模型眼部研究细胞因子信号抑制因子(suppressor of cytokine signaling,SOCS)的表达。方法:用光感受器间维生素A类结合蛋白(interphotoreceptorretinoid-binding protein,IRBP)免疫Lewis鼠160只后,在眼组织切片上应用SOCS多克隆抗体进行免疫组织化学染色,观察其在眼组织中的表达并与正常大鼠40只做对照。结果:用IRBP免疫Lewis鼠后,免疫组织化学染色发现在虹膜、睫状体和视网膜部位可见SOCS-1和SOCS-5蛋白表达;而在正常Lewis鼠未见SOCS蛋白表达。统计学结果显示,SOCS-1和SOCS-5蛋白表达与EAU严重程度呈正相关(r1=0.954,r2=0.963,P<0.01)。结论:自身免疫性葡萄膜视网膜炎Lewis鼠出现SOCS阳性表达细胞在EAU的发病过程中起了一定的作用。     

溴隐亭对大鼠实验性自身免疫性葡萄膜视网膜炎的影响

目的 观察泌乳素(prolactin, PRL)释放抑制剂——溴隐亭(bromo criptine, BRC)阻断PRL对大鼠实验性自身免疫性葡萄膜视网膜炎（experimental autoimmune uveoretinitis，EAU）的治疗效果。 方法 24只Wistar大鼠用牛视网膜可溶性抗原免疫后，随机数字表法随机分为治疗组和对照组。治疗组每日给予BRC葡萄糖溶液 5 mg/(kg·d)经口灌服，对照组每日葡萄糖溶液50 g/L经口灌服，观察2组EAU发病情况。免疫15 d后评价大鼠迟发型变态反应（delayed type hypersensitivity，DTH）；免疫23 d后眼球摘除，行组织学检查。 结果 治疗组EAU发病率和组织学炎症评分均低于对照组，其差异均有显著性的意义(P＜0.05,P＜0.001)；2组之间DTH比较差异无显著性的意义 (P＞0.05)。 结论 在整体水平上，BRC干预能够抑制大鼠EAU的发生和减轻EAU的病情。 （中华眼底病杂志，2003，19：34-37）     

用血管活性肠肽预防实验性自身免疫性葡萄膜视网膜炎

背景：血管活性肠肽（VIP）是一种神经肽类物质，目前所知存在于淋巴组织的微循环中，具有显著的抗炎作用。 目的：检测VIP对实验性自身免疫性葡萄膜视网膜炎（EAU）发展的作用。     

大鼠自身免疫性葡萄膜视网膜炎辅助T细胞的类细胞因子水平表达

目的观察实验性自身免疫性葡萄膜视网膜炎（experimental autoimmune uveoretinitis，EAU）大鼠血清以及脾细胞培养上清液中辅助T细胞1／辅助T细胞2（helper T cell 1／helper T cell2，Th1／Th2）类细胞因子水平。方法用Fmoc法合成光感受器间维生素A类结合蛋白R16多肽片段，联合免疫佐剂诱导EAU动物模型。在EAU高峰期取大鼠血清，分离脾细胞，培养后取上清液，应用酶联免疫吸附法检测血清以及脾细胞培养上清液中Th1类细胞因子（interferon-γ，IFN-γ、interleukin-2，IL-2）和Th2类细胞因子（IL-4、IL-10）水平。结果EAU组大鼠血清中IFN-1和IL-2的浓度分别为（33．8±5．2）μg／L，（52．5±7．9）μg/L，显著高于空白对照组[（6．2±1．4）μg/L，（3．7±0．8）μg/L和弗氏完全佐剂对照组[（complete Freund＇s adjuvant，CFA）；（9．2±1．9）μg/L，（5．1±1．1）μg/L]（P〈0．05）；而IL-4、IL-10的浓度与空白对照组和CFA组相比差异无统计学意义。EAU组大鼠脾细胞培养上清液中IFN-γ、IL-2的浓度分别为（1105．3±197．5）μg/L，（45．0±16．2）μg/L，显著高于空白对照组（5．24±1．7）μg/L，（4．1±1．3）μg/L和CFA组（25．14±5．9）μg/L，（5．1±1．9）μg/L（P〈0．01），IL4、IL-10的浓度与空白对照组和CFA组相比差异无统计学意义；CFA组大鼠脾细胞培养上清液中IFN-1的浓度明显高于空白对照组（P〈0.05），IL-2、IL-4、IL-10的浓度与空白对照组相比差异无统计学意义。结论在EAU高峰期，Th1类细胞因子水平显著升高，提示EAU是Th1细胞诱导的疾病（中国眼耳鼻喉科杂志，2008，8：280-282）     

CD4＋CD25＋T细胞在实验性自身免疫性葡萄膜视网膜炎中的作用

背景实验性自身免疫性葡萄膜视网膜炎（EAU）已被证明是一种由T淋巴细胞介导的器官特异性自限性疾病。研究表明 CD4＋CD25＋T细胞可能参与了EAu的调控,但其作用机制尚有待研究。目的探讨EAU中 CD4＋CD25＋T细胞的表达变化。方法按照Caspi的方法提纯牛视网膜S抗原,与弗氏完全佐剂混合后,于24只Lewis大鼠右后足底部注射0．1ml制作EAU模型,4只未处理大鼠作为正常对照组。免疫后每日州裂隙灯显微镜观察大鼠眼部变化。造模后7、12、15、21d处死动物,取大鼠视网膜、引流淋巴结、脾脏,对大鼠眼组织切片进行苏木精一伊红染色前进行病理评分。对各时间点收集的大鼠视网膜、引流淋巴结、脾脏分别制备单细胞悬液,流式细胞仪检测各时间点3种组织中 CD4＋CD25＋T细胞的表达情7兄。结果造模7d后大鼠睫状充血,虹膜血管扩张;15d后炎症达高峰,前房大量炎性渗出;21d后炎症消退。眼部病理评分结果与临床所见一致,造模15d组病理评分与其他各组比较差异均有统计学意义（P=0．000）。正常对照组大鼠脾脏和淋巴结中有2．0％CD4＋T细胞表达CD25,造模组 CD4＋CD25＋T细胞表达增加,亓在EAU高峰期达最高,EAU消退期轻微下降,与正常对照组比较差异有统计学意义（P=0．000）。结论 CD4＋CD25＋T细胞在EAU动物模型炎症组织中表达的动态变化与炎症反应有关,提示 CD4＋CD25＋T细胞参与EAU的发生发展和消退过程。     

实验性自身免疫性葡萄膜视网膜炎中可诱导的共刺激分子的表达及意义

目的探讨可诱导的共刺激分子（ICOS）在实验性自身免疫性葡萄膜视网膜炎(EAU)中的表达及意义。方法Lewis大鼠28只，用视网膜S抗原（50 μg）和福完全佐剂免疫24只大鼠以诱导EAU模型（免疫组），另外4只作为正常对照。裂隙灯显微镜每日观察大鼠眼部变化。免疫组大鼠分别于免疫后第7、12、15、21天被处死，摘除其脾脏后制作冰冻连续切片并提取组织蛋白。使用多克隆抗ICOS抗体，用免疫组织化学细菌蛋白过氧化物酶（SP）法在上述组织切片上进行免疫组织化学染色，并对提取的蛋白进行Western blotting检测。对照组大鼠在相应各时间点做同样处理。结果正常脾组织中可见少量ICOS阳性细胞；分别在免疫后第7、12 d，脾脏中ICOS阳性细胞数量明显增加，免疫后第15天时阳性细胞数量最多，21 d时阳性细胞数量减少，但仍显著高于正常组；Western blotting检测结果显示，ICOS蛋白的动态变化与免疫组织化学显示的阳性细胞数量变化一致。结论脾脏ICOS表达升高出现于EAU发生之前，随炎症的出现和加重而升高，在EAU消退期其表达有所降低，提示ICOS参与EAU的形成、发展和消退，在EAU发病中有重要意义。(中华眼底病杂志,2005,21:114-117)     

口服耐受对自身免疫性葡萄膜视网膜炎影响的实验研究

目的 观察口服牛视网膜S抗原对人S抗原多肽-35诱发的实验性自身免疫性葡萄膜视网膜炎(EAU)的影响.设计实验性对照研究.研究对象20只雌性纯种Lewis大鼠.方法 用人S抗原多肽-35诱发EAU,提纯牛视网膜S抗原诱导口服耐受,分高剂量组(2 mg/次)和低剂量组(0.2 mg/次),同时设实验对照组(胎牛血清蛋白).主要指标EAU发病情况和脾组织白介素-4(IL-4)和γ-干扰素(IFN-γ)的表达水平.结果 口服高剂量组的Lewis大鼠的EAU发病情况较实验对照组有显著减轻(P＜0.05),而且口服高剂量组Lewis大鼠脾组织IL-4的表达水平则高于实验对照组(F=4.214,P=0.017).结论 口服高剂量牛视网膜S抗原诱导的免疫耐受町以成功抑制人S抗原多肽-35诱发的实验性自身免疫性葡萄膜视网膜炎.(眼科,2008,17:250-253)     

细胞因子信号抑制因子在实验性自身免疫性葡萄膜视网膜炎外周血单个核细胞内的表达变化

目的 观察和探讨细胞因子信号抑制因子（SOCS)在实验性自身免疫性葡萄膜视网膜炎（EAU）外周血单个核细胞（PBMC）内的表达及意义。方法 光感受器间维生素A 类结合蛋白(IRBP)免疫100只Lewis大鼠诱导EAU动物模型，随机分为对照组和治疗组。治疗组从免疫后第1天至第28天，给予环孢霉素A（CSA）灌胃，20 mg/（kg·d）；对照组给予等量生理盐水。免疫前、免疫后7、14、21、28 d采用裂隙灯显微镜观察大鼠眼部变化并抽取其心脏血，酶联免疫吸附试验（ELISA）方法检测血清白细胞介素（IL）-4、IL-12、干扰素（IFN）-γ的含量；实时荧光定量聚合酶链反应（PCR）和蛋白免疫印迹法检测PBMC内SOCS mRNA和蛋白表达。结果免疫后14 d 时炎症最明显。对照组可见明显的虹膜睫状体炎，治疗组前房轻微炎性渗出，但无虹膜后粘连及前房积脓。对照组血清中 IL-12、IFN-γ免疫后14 d达到最高峰，28 d下降到基础水平，治疗组在免疫后14 d达到高峰，但是升高程度明显低于对照组，其他时间点与免疫前比较无差异；IL-4在对照组中下降不明显，治疗组在整个病程中呈上升趋势。SOCS1、SOCS5 mRNA免疫后14 d表达最高，对照组分别是免疫前的4.05和3.83倍，治疗组分别为1.15和1.16倍；两组中含SH2结构的细胞因子诱导蛋白（CIS）与SOCS3 mRNA轻度升高，对照组较治疗组略明显。对照组中SOCS1、SOCS5蛋白在免疫后7、14、21 d时显著高于免疫前，CIS、SOCS3蛋白在免疫后14、21 d显著高于免疫前；治疗组中仅SOCS1蛋白在免疫后14 d显著高于免疫前，其他时间点较免疫前无明显改变。 结论 SOCS1、SOCS5水平升高与EAU发生过程中的Th1型反应增强有关，CIS、SOCS3水平轻度升高可能是存在着Th2细胞反应的增强以对抗Th1细胞反应，实现动态免疫平衡。     

局部使用作用于肿瘤坏死因子α的反义寡核苷酸对实验性小鼠单疱病毒性脉络膜视网膜炎的影响

目的 观察局部使用作用于肿瘤坏死因子α（TNF-α）的反义寡核苷酸对实验性单疱病毒性脉络膜视网膜炎病理过程的影响。 方法 将50只BALB/c小鼠随机分为实验组和对照组，每组25只。将单纯疱疹病毒-Ⅰ型（HSV-Ⅰ）注入所有小鼠右眼（注射眼）前房内，建立单疱病毒性脉络膜视网膜炎的小鼠模型。实验组中分别于HSV-Ⅰ型感染前1 d，感染后1、4 d，将异硫氰酸荧光素标记的作用于TNF-α的反义寡核苷酸2 μl注射于感染小鼠的左眼（非注射眼）球结膜下;对照组则于相同的时间段内注射等量的磷酸盐缓冲液于感染小鼠的左眼（非注射眼）球结膜下，观察两组小鼠眼部的炎症改变并根据有无前房炎症反应、瞳孔和虹膜血管扩张、白内障形成、玻璃体混浊等行临床评分。于病毒感染后10 d处死所有小鼠，观察其组织学变化并采用酶联免疫吸附试验（ELISA）测定小鼠脉络膜视网膜的TNF α含量。 结果 感染后，两组小鼠右眼均表现为急性炎症改变;实验组小鼠左眼炎症反应临床评分明显小于对照组。组织学观察结果:对照组有12只小鼠左眼表现程度不等的坏死性脉络膜视网膜炎，实验组有2只小鼠左眼表现为轻度的脉络膜视网膜炎，两组间视网膜、脉络膜及睫状体部位炎性细胞计数差异有统计学意义（P＜0.05），而前房、玻璃体腔及虹膜部位无明显差别（P＞0.05）。ELISA测定结果:实验组脉络膜视网膜TNF-α含量为（60±1.25） pg，对照组脉络膜视网膜TNF-α含量为（305±1.03）pg，两者相比差异有统计学意义（P＜0.05）。 结论 采用作用于TNF-α的反义寡核苷酸局部治疗小鼠单疱病毒性脉络膜视网膜炎，能明显降低感染小鼠眼内细胞因子TNF-α的含量，减轻眼内脉络膜视网膜炎症反应。 (中华眼底病杂志, 2006, 22: 245-248)     

反义bcl-2寡核苷酸对体外培养的葡萄膜黑色素瘤细胞多药耐药性的影响

目的：探讨体外培养的葡萄膜黑色素瘤细胞对不同化疗药物的敏感性,并通过反义bcl-2寡核苷酸（bcl-2 antisense oligodeoxynucleotide,bcl-2 AS-ODN）特异阻断bcl-2基因的表达逆转肿瘤耐药性。方法：原代培养葡萄膜黑色素瘤细胞,采用噻唑蓝染色法测定肿瘤细胞对不同浓度的5-氟尿嘧啶、噻替哌、顺铂、阿霉素、长春新碱和氮烯咪胺体外敏感性;通过阳离子脂质体导入bcl-2 AS-ODN,阻断bcl-2基因的表达,利用免疫组化及Western blot法测定肿瘤细胞bcl-2的表达情况,并根据多药相互作用原理测定肿瘤细胞对化疗药物敏感性。结果：葡萄膜黑色素瘤对不同种类的化疗药物均有不同程度的抗药性。Bcl-2 AS-ODN可明显抑制bcl-2基因的表达,并随浓度升高抑制作用增强。Bcl-2AS-ODN与各种化疗药物呈协同作用,能够增加化疗药物对肿瘤细胞的杀伤作用。结论：所选6种化疗药物在临床常用剂量范围对葡萄膜黑色素瘤细胞毒性作用较小,葡萄膜黑色素瘤细胞的多药耐药性与bcl-2基因的高表达有关,bcl-2 AS-ODN能够部分逆转肿瘤细胞的多药耐药性。     

实验性自体免疫性葡萄膜炎的治疗研究进展

葡萄膜炎是眼科的一种常见致盲性眼病,目前治疗主要以皮质类固醇激素为主,但激素具有副作用大等缺点。自成功建立实验性自体免疫性葡萄膜炎模型以来,多种新型药物以及治疗方法均已得到实验应用,为临床治疗提供了可靠的依据。本文对近年来实验性自体免疫性葡萄膜炎的治疗研究进展进行了综述。     

Treatment of experimental autoimmune uveoretinitis with atorvastatin and lovastatin

Statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are approved for cholesterol reduction and are commonly used to treat atherosclerosis and coronary artery disease. Statins may also be potent immunomodulatory agents and may be beneficial in the treatment of autoimmune diseases. In this study, we investigated therapeutic effects of atorvastatin and lovastatin on experimental autoimmune uveoretinitis (EAU). EAU was induced in Lewis rats using bovine S-antigen (S-Ag) peptide. Atorvastatin was suspended in 0.5% aqueous methylcellulose and was administered orally at a dose of 10 mg/kg and at a low-dose of 1 mg/kg. Lovastatin was dissolved in DMSO:PBS (1:1) and was administered by intraperitoneal (i.p.) injection at a dose of 2 mg/kg. Both statin treatments were initiated after the clinical onset once daily for 14 days. The rats were examined every other day for clinical signs of EAU. The histological scores and delayed-type hypersensitivity (DTH) were evaluated on day 28 post-immunization. Morphologic and immunohistochemical examinations were performed with light and confocal microscopy, respectively. Lymphocyte proliferation was measured by [(3)H]thymidine incorporation into antigen-stimulated T cells from inguinal lymph nodes. After 72 h, supernatants were collected and assayed for IFN-gamma by ELISA. Clinical and histological scores of EAU were decreased in both the atorvastatin (10 mg/kg)- and lovastatin (2 mg/kg)-treated groups. The invasion of T cells and macrophages, and Müller cell proliferation, were inhibited in both atorvastatin- and lovastatin-treated groups. DTH was significantly inhibited in both groups, compared with vehicle-treated groups (controls). Lymphocyte proliferation assay demonstrated decreased proliferation in the presence of 25 microg/ml S-Ag peptide in both groups, compared with controls. In the supernatants of lymph node cells stimulated with S-Ag peptide (5 microg/ml), 77 or 87% inhibition of IFN-gamma production was observed in rats treated with atorvastatin or lovastatin, respectively, compared with controls. The current results indicate that atorvastatin administrated orally following the clinical onset has therapeutic effect in EAU as well as lovastatin administrated intraperitoneally. Statins may be useful for treating intraocular inflammation.     

IL-17A-OVA疫苗对实验性自身免疫性葡萄膜炎的影响

 目的 研究IL-17A-OVA多肽疫苗对于实验性自身免疫性葡萄膜炎（EAU）的影响。设计 实验研究。 研究对象  30只C57BL/6小鼠。方法  制备IL-17A-OVA疫苗。将30只C57BL/6小鼠随机分为疫苗组、佐剂组及环孢素组3组,每组10只,佐剂组及疫苗组提前免疫小鼠,第10周时三组小鼠同时用光感受器间维生素A类结合蛋白（IRBP）诱发EAU动物模型。环孢素组造模成功后环孢素连续灌胃2周。HE染色观察小鼠眼球活组织切片,ELISA方法检测小鼠血清中IL-17A、IL-17A特异性抗体、IL-6及IL-23水平。主要指标 小鼠血清中IL-17A、IL-17A特异性抗体、IL-6及IL-23水平。结果 组织病理学显示疫苗组、环孢素组与佐剂组组织病理学评分均有显著性差异（佐剂组评分较高）,疫苗组与环孢素组组织病理学评分无显著性差异;血清中IL-17A水平疫苗组（25.785±4.677 pg/ml）与环孢素组（23.129±3.940 pg/ml）均较佐剂组（33.774±6.478 pg/ml）显著降低（F=8.125,P=0.003）;IL-17A特异性抗体水平疫苗组（106.379±12.603 pg/ml）较环孢素组（59.431±8.626 pg/ml）及佐剂组（56.712±4.214 pg/ml）显著升高（F=63.008,P=0.000）,环孢素组与佐剂组无显著性差异;血清中IL-6水平疫苗组（41.124±4.959 pg/ml）与环孢素组（29.418±3.34 7 pg/ml）均较佐剂组（49.329±8.768 pg/ml）显著降低（F=18.603,P=0.000）;IL-23水平环孢素组（73.492±12.324 pg/ml）较佐剂组（109.770±20.848 pg/ml）及疫苗组（100.893±13.467 pg/ml）显著降低,疫苗组与佐剂组无显著性差异（F=4.916,P=0.018）。结论 IL-17A-OVA疫苗可通过产生IL-17A特异性抗体在减轻EAU的炎性反应中起到一定的作用,该疫苗可能成为自身免疫性葡萄膜炎治疗的新方法。（眼科,2017,26： 34-38）     

Effects of heme oxygenase-1 inducer and inhibitor on experimental autoimmune uveoretinitis

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is an animal model of posterior uveitis and heme oxygenase-1 (HO-1) is a well-known anti-oxidant factor. However, there is no report a protective role of HO-1 on EAU in vivo. To verify that HO-1 is induced in EAU by interphotoreceptor retinoid-binding protein (IRBP), that an HO-1 inducers ameliorates the associated inflammation, and that an HO-1 inhibitor exacerbates this inflammation. METHODS: Forty four Lewis rats were given either 40 mol/kg hemin or 40 mol/kg SnPP (tin protoporphyrin IX) by intraperitoneal injection and twenty two uveitis control rats were injected with 0.5 mL of saline once daily 5-20 days after IRBP immunization inducing EAU. Three normal control rats were used for Western blotting and ELISA assay of HO-1. The clinical uveitis signs of inflammation were scored in the three groups from 0 to 4 on alternate three days. To confirm the clinical results, histological and immunohistochemical stain of HO-1 were performed on the day of peak inflammation and Western blotting and ELISA assay of HO-1 were performed on 6th, 12th and 18th day after IRBP immunization. RESULTS: Hemin, an inducer of HO-1, ameliorated the clinical signs of EAU. In contrast, SnPP-treated rats show that the severity of the clinical sign were exacerbated at the peak period of the disease. These results are roughly compatible with histological, immunoblotting, and immunohistochemical evaluations and an ELISA assay of HO-1. CONCLUSIONS: We suggest that HO-1 plays an important protective role in EAU.     

雷帕霉素对大鼠实验性自身免疫性葡萄膜炎的治疗作用

背景雷帕霉素不仅具有抗菌作用,而且是一种良好的免疫抑制剂,可用于多种自身免疫性疾病的治疗．对实验性自身免疫性葡萄膜炎（EAU）的治疗作用是目前研究的热点之一。目的研究雷帕霉素对EAU的治疗作用,并观察雷帕霉素对EAU各免疫细胞群炎性因子表达的影响。方法25只Lewis大鼠采用随机数字表法分为EAU组（20只）和正常对照组（5只）。光感受器间维生素A类结合蛋白（IRBP）R16肽段与完全氟氏佐剂充分乳化后于Lewis大鼠后足皮下注射以建立EAU模型,EAU模型鼠再按分层随机的原则分为模型对照组和雷帕霉素组,每组10只大鼠。雷帕霉素组造模后即应用O．2mg／（kg·d）雷帕霉素（0．4m1）腹腔内连续注射7d,模型对照组及正常对照组大鼠采用等体积的生理盐水进行腹腔内注射。造模后第4天开始每日裂隙灯下观察大鼠EAU的症状,造模后第14天制备大鼠视网膜切片,以苏木精-伊红染色法进行组织病理学观察,参照Caspi的标准对EAU症状及组织病理学分级进行评分。应用免疫组织化学染色法检测各组大鼠视网膜中炎性因子干扰素-γ（IFN-γ）、白细胞介素-17（IL-17）的表达情况。结果造模后6d模型对照组大鼠EAU炎症评分逐渐升高,12d达到高峰,然后逐渐下降。雷帕霉素组大鼠EAU炎症评分变化呈现相同的趋势,但各时间点EAU炎症评分均明显低于模型对照组,差异均有统计学意义（P〈0．01）。视网膜组织病理学研究表明,模型对照组大鼠视网膜结构紊乱,大量炎性细胞浸润,组织病理学评分为3．30±0．48,而雷帕霉素组视网膜结构接近正常,组织学评分为0．90±0．45,差异有统计学意义（t=16．541,P〈0．01）。雷帕霉素组IFN-γ、IL-17在大鼠视网膜中的表达量（A值）分别为21．16±4．23和49．86±6．59,明显低于模型对照组的62．14±7．32和124．85±6．33,差异均有统计学意义（q=33．334、q=56．923,P〈0．01）。结论雷帕霉素通过抑制EAU视网膜中IFN-γ、IL-17等炎性因子的表达而对EAU发挥治疗作用,其机制可能是通过抑制Th1、Th17细胞群来实现的。     

A clinical grading system for retinal inflammation in the chronic model of experimental autoimmune uveoretinitis using digital fundus images

Experimental autoimmune uveoretinitis (EAU), a widely used animal model of human posterior/pan-uveitis, is extremely valuable in allowing understanding of the pathogenesis of uveitis as well as in developing new treatments. Depending on the animal strain and immunization protocol used, the clinical course of EAU can be acute, severe and involving the anterior and posterior part of the eye, or chronic, mild and involving only the posterior part of the eye. Clinical signs of EAU can be examined by bio-microscopy. Using appropriate criteria EAU can be quantitatively evaluated clinically in living animals. However, correlation of research within different laboratories is difficult since clinical grading systems are subjective and susceptible to considerable variability. In this study, we have developed a recordable, image-based clinical grading system for the chronic models of EAU. Fundus images were taken from EAU mice using an endoscopic imaging system. Fundus changes were classified as (1) inflammatory changes (including optic disc inflammation, vasculitis and retinal tissue inflammation) and (2) retinal structural damage. Each element was scored separately based on the severity of the lesions, and the average score of the three inflammatory elements was used as the overall EAU clinical inflammation grade of the eye. The validity and reproducibility of the grading system was tested using a set of images scored independently in a masked manner by 5 individuals. The grading system proved robust, easy to use and reliable. We offer this image-based EAU clinical grading system as a useful quantitative evaluation method for clinical grading of the severity of inflammation in the chronic EAU model, in which the inflammation can be mild and mainly involves posterior part of the eye.     

Inducible co-stimulator (ICOS) is upregulated in experimental autoimmune uveoretinitis

Purpose To study the expression of inducible co-stimulator (ICOS) and its association with T cell effector function in experimental autoimmune uveoretinitis (EAU).Methods Eighteen Lewis rats were immunized by retinal S-antigen (50 μg) emulsified in complete Freund’s adjuvant (CFA). Twelve normal rats served as normal controls and 18 receiving injection of CFA and PBS as CFA controls for studying the influence of CFA on the expression of ICOS in CD4⁺CD25⁺ T cells. ICOS expression on cells from the spleens, inguinal nodes and retinae on day 0 (normal rats), 7, 13 and 21 was investigated using fluorescent quantitative real-time-PCR and Western blot. Expression of B7RP-1, an ICOS ligand, was also studied by Western blot. The phenotype of the cells from the aforementioned three tissues was identified with flow cytometry using antibodies to ICOS, CD4 and CD25. ICOS⁺ cells from the lymph nodes, and spleens on day 13 were magnetically sorted and cultured with S-antigen to study the cytokines production with enzyme-linked immunosorbent assay.Result An obvious uveitis was induced in all the immunized rats on day 13 after S-antigen immunization. The mRNA and protein of ICOS were scarcely detectable in normal rat spleens. In EAU rats, an up-regulation of ICOS could be observed on day 7 and was very pronounced on day 13, followed by a decrease on day 21 in the spleens, draining nodes and retinae. Similarly, B7RP-1 expression seemed to be up-regulated during EAU. Flow cytometry showed that ICOS⁺ cells were mostly CD4 positive. Kinetics of ICOS⁺CD4⁺CD25⁺ T cells was similar to that of ICOS⁺ cells. CFA alone was also able to induce increased expression of ICOS in CD4⁺CD25⁺ T cells. IFN-γ was secreted predominantly by ICOS⁺ T cells.Conclusion ICOS expression is upregulated in association with T cell effector capacity in EAU. It is presumed that the ICOS/B7RP-1 costimulatory pathway may play a role in the development of EAU.This study is supported by the Fund for Innovative Research Groups of China (30321004), Specialized Research Fund for the Doctoral Program of Higher Education in China (20030558077).     

Genetic deficiency of C3 as well as CNS-targeted expression of the complement inhibitor sCrry ameliorates experimental autoimmune uveoretinitis

In this report, we describe the effect of complement deficiency and inhibition on experimental autoimmune uveoretinitis (EAU). C57BL/6 mice genetically deficient in C3 (C3-/-) or expressing a soluble complement activation inhibitor (soluble complement receptor related protein Y or sCrry) in a CNS-targeted fashion, (sCrry/GFAP) were induced for EAU via peripheral immunisation with a peptide of amino acids 1-20 of human interphotoreceptor retinoid binding protein in complete Freund's adjuvant with concurrent intraperitoneal pertussis toxin. The incidence and severity of EAU in the mutant mice was compared with that in simultaneously induced C57BL/6 wild type mice. The sCrry protein was detected in retinal extracts from transgenic but not wild type mice by western blot. C3-/- mice had a significant reduction in the incidence of EAU compared with wild type mice (incidence 44 versus 89%, respectively, p=0.0417) and a significant reduction in the severity of EAU (median disease score values 0 versus 1.3, respectively, p=0.0253). Similarly, sCrry mice had a significant reduction in the incidence of EAU compared with wild type mice (incidence 57 versus 100% respectively, p=0.0033) and a significant reduction in the severity of EAU (median disease score values 0.18 versus 1.85, respectively, p=0.0054). A genetic deficiency of C3 and production of a soluble complement inhibitor targeted to the CNS and eye are protective against EAU.     

间充质干细胞对大鼠实验性自身免疫性葡萄膜炎视网膜中白细胞介素17表达的影响

背景 研究显示Th17细胞是实验性自身免疫性葡萄膜炎(EAU)发病的重要致炎细胞群,而白细胞介素17( IL-17)作为Th17细胞的标志性因子参与了EAU的发生与发展.间充质干细胞(MSCs)作为一种免疫调节剂在多种自身免疫性疾病中对Th17具有抑制作用. 目的 研究MSCs对EAU的治疗作用以及对IL-17在大鼠视网膜表达的影响.方法 从10只SPF级Wistar大鼠股骨骨髓中分离、培养MSCs并进行传代,第3～5代细胞用于实验.54只SPF级6～8周龄Lewis大鼠按随机数字表法分为实验组48只和正常对照组6只.光感受器间维牛素A类结合蛋白(IRBP)与含有结核杆菌素HR37a的2.5 g/L弗氏完全佐剂(CFA)等体积混合后行实验组大鼠单后足垫皮下注射建立EAU动物模型,然后按照分层随机原则分为模型对照组和MSCs治疗组,每组24只大鼠.MSCs治疗组于造模的同时行1 ml MSCs尾静脉注射(5×106个／ml),连续注射3d,模型对照组以同法注射等体积PBS.注射后每日在裂隙灯显微镜下观察大鼠眼部炎症反应,并根据Caspi临床分级进行评分.分别于造模后9、12、15、20 d随机选取模型对照组和MSCs治疗组各6只大鼠制备视网膜切片,采用组织病理学方法观察大鼠视网膜形态学改变和炎症反应,参照Caspi组织学分级法进行评分.采用免疫组织化学染色法检测造模后9、12、15、20d大鼠视网膜中IL-17的表达.结果 培养的细胞呈梭形生长,漩涡状排列,经流式细胞术鉴定CD29、CD44表达阳性,CD45、CD34表达阴性.MSCs治疗组造模后9、12、15、20 d眼前节炎症反应评分均低于模型对照组(U=2.815,P=0.005;U=2.768,P=0.006;U=2.900,P=0.004;U=2.855,P=0.004),视网膜组织学检测炎症评分均低于模型对照组,差异均有统计学意义( U=2.345,P=0.019:U=2.559,P=0.011;U=2.166,P=0.030;U=2.373,P=0.018).免疫组织化学染色显示,MSCs治疗组造模后9、12、15、20d大鼠视网膜IL-17蛋白阳性表达的平均吸光度(A)值分别为26.47±5.68、77.78±9.65、47.02±6.68和26.59±5.94,明显低于模型对照组的45.34±4.63、105.95±10.74、64.11±9.76和37.02±6.51,差异均有统计学意义(t=6.305,P=0.000:t=4.799,P=0.001:t=3.540,P=0.005;t=2.900,P=0.016).结论 MSCs能减轻EAU的程度并抑制EAU的进展,降低了1L-17在眼组织中的表达.     

Metabolic profile analysis of free amino acids in experimental autoimmune uveoretinitis rat plasma

AIM: To determine the differences of amino acid (AA) levels in experimental autoimmune uveoretinitis (EAU). METHODS: AA analysis of the plasma samples in EAU rats induced by interphotoreceptor retinoid-binding protein emulsion were performed with high performance liquid chromatography (HPLC) and phenylisothiocyanate (PITC) pre-column derivation methods were performed. Using partial least squares discriminant analysis (PLS-DA), the potential biomarkers were identified in EAU rat plasma, and the metabolic pathways related to EAU were further analyzed. RESULTS: The method results showed that linear (r≥0.9957), intra-day reproducible [relative standard deviation (RSD)=0.04%-1.33%], inter-day reproducible (RSD=0.06%-2.07%), repeatability (RSD=0.03%-0.89%), stability (RSD=0.05%-2.48%) and recovery (RSD=1.98%-4.39%), with detection limits of 0.853-11.4 ng/mL. The metabolic profile in EAU rats was different from that in the control groups five AAs concentrations were increased and nine AAs were reduced. Moreover, five metabolic pathways were related to the development of EAU. CONCLUSION: The developed method is a simple, rapid and convenient for determination of AAs in EAU rat plasma, and these findings will provide a comprehensive insight on the metabolic profiling of the pathological changes in EAU.