雷帕霉素纳米粒滴眼液联合环孢素A缓释膜治疗兔高危角膜移植术后免疫排斥反应的研究

目的 探讨局部联合应用雷帕霉素(RAPA)纳米粒滴眼液与环孢素A(CsA)缓释膜治疗兔高危角膜移植术后免疫排斥反应的效果和协同机制.方法 实验研究.(1)制备0.5%聚乳酸/胆固醇改性壳聚糖RAPA纳米粒滴眼液和聚乳酸/聚乙二醇CsA缓释膜.(2)设A组为同源对照组(17只兔),供受体均为新西兰白兔.建立102只(102只眼)新西兰白兔角膜新生血管模型,采用随机数字表法将其分为B、C、D、E、F、G6组,每组17只,供体为青紫兰兔,各组行穿透性角膜移植术.A组同源对照组;B组未治疗组;C组空白纳米粒滴眼液滴眼,每天2次共28 d;D组术中前房植入空白缓释膜;E组0.5%RAPA纳米粒滴眼液滴眼,每天2次共28 d;F组术中前房植入CsA缓释膜;G组术中前房植入CsA缓释膜并0.5%RAPA纳米粒滴眼液滴眼,每天2次共28 d.(3)术后观察100 d,隔天用显微镜观察角膜植片情况,记录免疫排斥反应发生时间和程度.(4)术后3、14、28及35 d用CD4和CD8单克隆抗体行免疫组织化学检查;用逆转录聚合酶链反应(RT-PCR)检测植片白细胞介素2(IL-2)和血管内皮生长因子(VEGF)表达.应用单因素方差分析和q检验,比较各组角膜植片平均存活时间.结果 各组兔眼角膜植片存活时间分别为A组(100.00±0.00)d、B组(8.44±1.24)d、C组(8.89±2.57)d、D组(8.56±2.30)d、E组(43.11±5.58)d、F组(43.67±9.54)d、G组(72.00±15.34)d.G组较E、F组,E、F组较B、C、D组,能显著延长角膜植片存活(qGE=11.42,qGF=11.24,qEB=13.64,qEC=13.38,qED=13.46,qFB=13.82,qFC=13.56,qFD=13.64;均P<0.01).免疫组织化学检查显示,B、C、D组术后14 d左右角膜植片中CD4+和CD8+T淋巴细胞大量聚集,呈进行性加重;E、F组术后35 d左右角膜植床与植片中CIM+和CD8+T淋巴细胞数开始出现;G组术后60 d以后,植片中CD4+和CD8+T淋巴细胞浸润.角膜植片RT-PCR检查显示,A组IL-2和VEGF始终未表达;F、G组IL-2表达在3 d开始被抑制;E、G组VEGF表达在14 d开始被抑制.结论 局部联合应用药物缓释剂治疗高危角膜移植术后免疫排斥反应较单独用药效果好,联合用药能减少单独用药的剂量和毒副作用.     

雷帕霉素缓释膜治疗兔高危角膜移植排斥反应实验研究

目的 探讨局部应用雷帕霉素(RAPA)缓释膜抑制兔高危角膜移植免疫排斥反应的机制.方法 将角膜新生血管白兔68只分为4组,每组17只,行穿透性角膜移植术,供体为健康灰兔.A组为未治疗组;B组术中前房植入空白缓释膜;C组术后每天1%RAPA滴眼液滴眼3次共28天;D组术中前房植入RAPA缓释膜.术后隔天用裂隙灯显微镜观察植片免疫排斥反应发生的时间;用CD.和CD8单克隆抗体行免疫组织化学染色,观察各组T淋巴细胞的迁移和数量. 结果兔角膜植片存活时间:D组(44.89±8.95)d,较A组(11.11±1.69)d、B组(11.56±2.70)d、C组(17.78±8.41)d明显延长(P＜0.01).免疫组化检查:术后14 d,B、c组植片中聚积了大量CD4和CD8 T淋巴细胞,并随时间延长而增加.D组药物治疗后期,植片仅见少量CD4 和CD8 细胞浸润;当RAPA缓释膜消失后,CD4 和CD8 T淋巴细胞开始聚集.结论 RAPA缓释膜能显著延长兔高危角膜移植植片存活时间,减轻全身用药可能引起的并发症.     

雷帕霉素不同剂型治疗兔高危角膜移植排斥反应疗效比较

目的探讨局部应用雷帕霉素(Rapamycin,RAPA)能否抑制兔高危角膜移植术后免疫排斥反应,比较不同给药方式和不同药物间的效果。方法用119只(119眼)新西兰大白兔制作角膜碱烧伤新生血管模型;分为A、B、C、D、E、F和G7组,每组17只,行穿透性角膜移植术,供体为健康灰色家兔。A组为同源对照组;B组为未治疗组;C组术后空白纳米粒滴眼液每天点眼2次共28天;D组术后1%RAPA滴眼液每天点眼3次共28天;E组术中前房植入RAPA缓释膜;F组术后0.5%RAPA纳米粒滴眼液每天点眼2次共28天;G组术中前房植入环孢素A(Cyclospoirne A,CsA)缓释膜。术后隔天用裂隙灯显微镜观察并记录角膜植片免疫排斥反应发生时间和程度。分别于术后7、14、28天应用高效液相法检查D、E、F组兔房水中RAPA药物浓度;3、14、28及35天行组织病理学及免疫组织化学检查。结果A组(100.0±0.0)d、E组(44.89±8.95)d、F组(45.22±5.52)d、G组(45.89±10.33)d兔角膜植片存活时间较B组(11.11±1.69)d、C组(11.78±3.19)d、D组(17.78±8.41)d明显延长(P<0.01)。房水中RAPA浓度:D组始终为0ng/mL;E组7、14、28天分别为(12.01±0.83)ng/mL、(11.14±1.04)ng/mL、(10.26±0.47)ng/mL;F组7、14、28天分别为(10.41±0.08)ng/mL、(10.87±0.40)ng/mL、(10.92±0.85)ng/mL。组织病理学和免疫组织化学检查:B、C组术后14d,角膜植片聚积了大量CD4 和CD8 T淋巴细胞,并随时间增加而增多;E、F、G组至术后28～35d,角膜植床及植片基质CD4 和CD8 T淋巴细胞数量开始出现并增加。结论RAPA纳米粒滴眼液组房水中RAPA浓度与前房植入RAPA缓释膜组相近且更恒定,能显著延长兔穿透性角膜移植植片的存活时间,与CsA局部应用效果相近;RAPA纳米滴眼液有较好的应用前景。     

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.     

国产环孢素A眼液治疗大鼠角膜移植术后免疫排斥反应的研究

目的　观察环孢素A(cyclosporine,CsA)滴眼液对鼠角膜移植术后免疫排斥反应的治疗效果。方法　随机对纯系大鼠LOU65只为受体及F344纯系大鼠33只为供体建立的角膜移植术后免疫排斥反应动物模型分为6组,各组分别于手术后滴用0.5%CsA、1.0%CsA、2.0%CsA、糖皮质激素、糖皮质激素和1.0%CsA及CsA基质滴眼液,观察角膜移植术后植片排斥反应指数(rejectionindex,RI),植片平均存活时间(meanssurvivaltime,MST),并对角膜植片进行病理学检查及免疫病理学分析。结果0.5%CsA、1.0%CsA、2.0%CsA、糖皮质激素及混合组的植片MST均较对照组延长1.63～8.46d,其中2.0%CsA组、糖皮质激素组及混合组的植片MST延长与其他组比较差异有显著性（t≥2.28,P<0.01）,混合组为（15.71±5.06）d。对照组的角膜植片排斥RI较其他组高,其中2.0%CsA组与糖皮质激素组的角膜植片排斥RI一致,分别为5.34±1.92和5.18±1.69;混合组治疗效果最好,RI为4.31±1.50,而CsA滴眼液治疗后大鼠血清中的浓度介于21.6～62.2mg/L。病理学检查各治疗组角膜植片淋巴细胞浸润减轻,新生血管减少;免疫病理显示,除0.5%CsA组外,其余各组的淋巴细胞相关抗原1（LFA-1,CD+11a）、ICAM-1表达和巨噬细胞浸润均明显减少。结论　环孢素A滴眼液能够抑制大鼠角膜移植术后免疫排斥反应的发生,与糖皮质激素联合应用能取得更佳的效果。     

雷帕霉素抑制大鼠角膜移植免疫排斥反应的实验研究

目的探讨雷帕霉素(RAPA)对大鼠角膜移植排斥反应的防治效果。方法以SD大鼠为供体,Wistar大鼠为受体建立角膜移植实验模型。采用完全随机分组设计方案,将68只Wistar鼠(68只眼)随机分为4组,A组为Wistar鼠自体原位角膜移植,B、C、D组为SDWistar鼠之间进行同种异体角膜移植。术后灌胃给药,A组和B组给予空白液,C组和D组分别给予RAPA(3mg·kg-1·d-1)和环孢霉素A(CsA)(10mg·kg-1·d-1),连续用药12d。根据Holland排斥反应评分系统,判断术后植片排斥情况。比较各组角膜植片的平均存活时间和植片存活率。采用广义估计方程对角膜的新生血管评分进行统计学分析。术后14d,对各组角膜进行组织学检查。结果RAPA组发生排斥反应的时间明显延迟,同种异体移植对照组植片平均存活时间(MST)为(11.0±1.5)d,RAPA组MST为(36.1±14.9)d,两组比较差异有统计学意义(P<0.05)。RAPA组角膜植片存活情况较CsA组好,但两组植片存活率比较差异无统计学意义(P>0.05)。RAPA组术后角膜新生血管的生长明显低于异体移植对照组(P<0.05)和CsA组(P<0.05)。组织学检查证实,术后14d,RAPA组角膜植片未见明显淋巴细胞浸润。结论口服RAPA能够延缓大鼠角膜移植排斥反应的发生,并能够抑制术后新生血管的生成。     

前房植入环孢素A缓释系统抑制鼠高危角膜移植术后的免疫排斥反应

目的　探讨前房内植入环孢素A缓释系统 (cyclosporineAdrugdeliverysystem ,CsADDS)抑制高危角膜移植术后免疫排斥反应的有效性和可行性。方法　 (1)对 6 0只Wistar大鼠 (6 0只眼 )用缝线法诱导角膜新生血管增生。 (2 )将发生角膜新生血管化的 4 0只Wistar大鼠 (40只眼 )随机分为4组 :对照组 ,1%CsA滴眼组 ,CsADDS结膜下植入组 ,CsADDS前房内植入组。每组均接受同种异系(Spregue Dawley大鼠 )角膜供体 ,行穿透性角膜移植术 ,术后比较各组大鼠免疫排斥反应发生的时间 ,并定期检测各组大鼠房水中CsA的浓度。 (3)正常Wistar大鼠 8只 (16只眼 ) ,随机分为 2组 ,分别在结膜下和前房内植入CsADDS ,术后 2和 4周行眼的组织病理学检查。结果　 (1) 5 1只Wistar大鼠 (5 1只眼 )经角膜基质缝线 ,成功诱导角膜新生血管增生。 (2 ) 4组共 4 0只Wistar大鼠角膜移植术后免疫排斥反应的发生时间分别为 :对照组 (8 2 0± 1 4 8)d ,1%CsA滴眼组 (10 6 0± 1 90 )d ,CsADDS结膜下植入组 (11 4 0± 2 5 0 )d ,CsADDS前房内植入组 (17 0 0± 6 0 5 )d。房水中CsA浓度均值分别为 :对照组 0 μg/L ;1%CsA滴眼组 (47 90± 3 4 8) μg/L ;CsADDS结膜下植入组术后 1、2、4周 ,房水中CsA浓度均值分别为 (5 9 0 0± 3 6 6 ) μg/     

FK506缓释系统前房植入抑制兔高危角膜移植术后的免疫排斥反应

目的探讨前房植入FK506药物缓释系统（DDS）对兔高危角膜移植术后免疫排斥反应的抑制作用和FK506房水药物浓度与免疫排斥反应的关系。方法107只新西兰白兔中随机数字法选取73只兔进行角膜新生血管化模型的制作,其中68只兔作为受体成功建立高危角膜移植动物模型,随机数字法分为对照组、空白DDS前房植入组、环孢素A（CsA）DDS前房植入组（含CsA 1mg）、0．1％FK506眼液滴眼组及FK506 DDS前房植入组（含FK5060．5mg）。角膜移植术后观察各组角膜植片排斥发生的时间,移植术后1周取各组实验兔眼房水和静脉血进行FK506药物浓度检测。0．1％FKS06眼液滴眼组和FKS06 DDS前房植入组在移植术后的不同时间点抽取实验兔眼房水和静脉血,进行FK506药物浓度的检测。观察各组兔移植术后4周和观察期结束时角膜植片的病理变化,同时应用原位杂交的方法检测各组角膜植片内白细胞介素2受体a（IL-2Bot）、单核细胞趋化蛋白1（MCP-1）、Fas及FasL mRNA的表达。结果FK506 DDS前房植入组角膜植片存活时间超过180d,明显优于其他各组（F=926．37,P=0．0000）,其房水和角膜组织中的FK506药物浓度明显高于FKS06眼液滴眼组（T=21．00,P=0．0022）。FKS06 DDS前房植入组在术后24周内均能在房水中检测出FK506。术后4周对照组和空白DDS前房植入组有大量的炎性细胞浸润,并有明显的IL．2Bet和MCP-1mRNA的表达,而CsADDS前房植入组、FK506眼液滴眼组及FK506 DDS植入组角膜未见明显的炎性细胞浸润,未见IL-2Pux和MCP-1mRNA的表达。各组均未见明显的Fas和FasL mRNA的表达。结论前房植入FK506 DDS可有效地抑制高危角膜移植术后免疫排斥反应的发生,房水中较高的FK506药物浓度是防治术后发生免疫排斥反应的重要因素。     

环孢素A缓释系统的眼内释药性和生物相容性的实验研究

目的研究前房植入环孢素A缓释系统(CsA DDS)的生物相容性和房水药物浓度变化。方法新西兰白兔36只,随机分为A、B、C、D四组,分别与A、B、C、D四型不同的CsA DDS相对应。每组9只兔随机编号为1～9号,其中1～3号兔各任选1只眼前房植入相应的CsA DDS,另1只眼植入不含CsA的空白缓释系统(EMDDS)为对照;4～6号兔各任选1只眼前房植入EM DDS,另1只眼行角膜切开及缝合术为对照;7～9号兔各任选1只眼前房植入CsA DDS,另1只眼行角膜切开及缝合术为对照。术后观察12周。结果四型CsA DDS均未见明显眼内毒性反应;A、B两型释药速率迅速,但维持时间短,C、D两型则缓慢而持久。结论前房植入CsA DDS对兔眼的生物相容性良好;4型CsA DDS中A、B型较适宜治疗排斥反应,C、D型较适宜预防排斥反应。     

小分子化合物J2抑制小鼠角膜移植术后排斥反应的研究

目的 探讨新型药物J2抗同种异基因小鼠角膜移植术后排斥反应的作用及其机制.方法 实验研究.采用随机分组设计的研究方法.建立以C57BL/6小鼠为供体,BALB/c小鼠为受体的角膜移植实验模型,其中供体38只,受体100只,随机分为A、B、C、D 四组,每组25只.A组为BALB/c小鼠白体原位角膜移植,B、C及D组均采用C57BL/6-BALB/c同种异体角膜移植,术后采用腹腔注射给药,自手术当日开始,A组、B组给予安慰剂,C组给予CsA 10 mg/kg体重,D组给予J215 mg/kg体重,每天1次,连续给药12 d.观察各组植片生存指数变化,苏木素-伊红染色及冰冻切片免疫组织化学观察角膜植片病理改变;分别在术后1、2、3及4周行逆转录聚合酶链反应(RT-PCR)检测角膜植片中细胞因子的表达.各组间植片存活时间差异采用One-Way ANOVO分析进行比较,两组之间比较采用SNK法.结果 B组角膜植片平均存活时间为(18.88±4.19)d,D组发生排斥时间明显延迟为(33.62±6.S0)d,两组比较差异有统计学意义(q=8.33,P=0.00),而D组与C组比较差异无统计学意义(q=0.85,P=0.60).组织学检查证实,术后21 d,B组见大量细胞浸润,而D组与C组植片未见明显的细胞浸润.免疫组织化学检测显示安慰剂组角膜植片大量CD4+和CD8+T淋巴细胞浸润,J2组与CsA组植片仅有少量的CD4+和CD8+T淋巴细胞浸润.RT-PCR结果显示,在正常小鼠角膜未见IL-10和IFN-γ基因表达;异基因移植组从第1周开始表达各种基因,第3周表达明显增强;而J2组与CsA组从第1周开始表达,第2、3周表达减弱,第4周表达强于前3周.结论 J2通过拮抗CD4与主要组织相容性抗原(MHC)Ⅱ分子结合抑制CD4+T淋巴细胞激活,从而具有抗小鼠角膜移植排斥作用.     

Prolongation of corneal allograft survival using cyclosporine in a polylactide-co-glycolide polymer

PURPOSE: To test for prolongation of corneal transplant survival with cyclosporine in a polymer placed in the anterior chamber of corneal allograft recipients. METHODS: Wistar inbred rats with vascularized corneas were recipients of corneal allografts from Sprague-Dawley donor rats. Grafted rats were randomized into six groups: untreated control animals, cyclosporine-polymer anterior chamber recipients, cyclosporine-polymer subconjunctival recipients, cyclosporine-olive oil drop recipients, polymer-only anterior chamber recipients, and autografted Wistar rats. Grafts were examined by slit lamp every 3 days and the clinical condition scored. The cyclosporine concentration in the aqueous humor was assayed at 1, 2, and 4 weeks. At 2 and 4 weeks after transplantation, the eyes were collected for histopathologic evaluation of the grafts. RESULTS: The median survival time of untreated corneal allografts was 8.2 +/- 1.48 days for grafts treated with topical cyclosporine, 8.5 +/- 1.50 days for polymer-only anterior chamber implants, 10.6 +/- 1.90 days for 1% cyclosporine drops, 11.4 +/- 2.50 days for grafts given subconjunctival cyclosporine-polymer, 17 +/- 3.05 days for grafts given cyclosporine-polymer implants in the anterior chamber, and more than 3 months in autografted rats. There was a statistically significant difference ( p < 0.05) between the survival time of the allografts in the animals treated with the cyclosporine-polymer in the anterior chamber compared with the other groups of graft recipients. Significantly higher concentrations of cyclosporine were found in the eyes given an anterior chamber implant of cyclosporine-polymer than in the other treatment groups or the untreated rats. The cyclosporine-polymer implants placed in the anterior chamber induced a transient inflammatory response in transplanted eyes. CONCLUSIONS: Cyclosporine-polymer placed in the anterior chamber significantly prolongs corneal allograft survival in a high-risk corneal graft rejection. This intraocular delivery system may be a valuable adjunct for the suppression of immune graft rejection in high-risk recipients of corneal transplants.     

ACAID induced by allogeneic corneal tissue promotes subsequent survival of orthotopic corneal grafts

PURPOSE: To determine whether immune deviation is induced by allogeneic corneal tissue implanted in the anterior chamber and whether survival of subsequent orthotopic corneal allografts is thereby enhanced. METHODS: Corneal tissue from C57BL/6 mice was implanted in the anterior chamber of eyes of BALB/c mice. The fate of these implants was assessed histologically, and the donor-specific immune response of recipient mice was tested for donor-specific delayed hypersensitivity and the capacity to accept or reject C57BL/6 corneas grafted orthotopically into the fellow eye. RESULTS: C57BL/6 cornea implants in the anterior chamber failed to induce donor-specific delayed hypersensitivity but impaired donor-specific delayed hypersensitivity in a proportion of recipients with implants in place for 2 weeks. Mice with donor-specific delayed hypersensitivity rejected the intraocular implants. Mice bearing C57BL/6 cornea implants in the anterior chamber for 2 (but not 4) weeks accepted the C57BL/6 corneas grafted orthotopically into the fellow eye at a high rate and with few rejection reactions. CONCLUSIONS: Implantation of allogeneic corneal tissue in the anterior chamber of the eye has the transient capacity to alter the recipient alloimmune response in a manner that promotes survival of subsequent orthotopic corneal allografts.     

Role of recipient epithelium in promoting survival of orthotopic corneal allografts in mice

PURPOSE. To determine whether epithelium-deprived corneal allografts covered with syngeneic epithelium display immune privilege in orthotopic transplantation and whether syngeneic epithelium containing antigen-presenting cells nullifies this effect. METHODS. Epithelium-deprived allogeneic corneas (C57BL/6) and epithelium-deprived allogeneic corneas reconstituted with syngeneic (BALB/c) epithelium (containing or deprived of Langerhans' cells) were transplanted orthotopically into normal eyes of BALB/c mice. Graft survival was assessed clinically and evaluated histologically. RESULTS. Epithelium-deprived corneal grafts survived in syngeneic recipients but were swiftly rejected in allogeneic recipients. These allografts incited intense stromal inflammation and neovascularization. Epithelium-deprived allografts that were resurfaced in vivo by syngeneic epithelium derived from immune-incompetent SCID mice also underwent intense acute rejection when placed in normal eyes of BALB/c mice. The epithelium of in vivo resurfaced grafts was replete with Langerhans' cells. By contrast, most of the epithelium-deprived allografts reconstituted in vitro with fresh, normal BALB/c corneal epithelium survived indefinitely when placed in eyes of BALB/c mice. Similar grafts reconstituted with BALB/c epithelium containing Langerhans' cells were swiftly rejected. CONCLUSIONS. Replacement of donor epithelium with Langerhans' cell-deficient syngeneic epithelium protects orthotopic allogeneic cornea grafts (stroma plus endothelium) from immune-mediated rejection. The presence of an intact, histocompatible layer of corneal epithelium has two important effects on orthotopic corneal allografts: It suppresses nonspecific inflammation and neovascularization within the graft, and it blunts the alloimmunogenicity of the histoincompatible stroma and endothelium.     

Role of CD4+ T cells in immunobiology of orthotopic corneal transplants in mice.

PURPOSE: To determine, with the use of mice genetically deficient in expression of CD4 or CD8 molecules, which T cells are responsible for rejection of orthotopic corneal allografts in mice. METHODS: Corneas were prepared from major histocompatibility complex (MHC)-only incompatible, minor histocompatibility (H)- only incompatible, and MHC-plus-minor H incompatible donors and grafted orthotopically to eyes of CD4 knockout (KO), CD8KO, and wild-type control mice. Graft survival patterns were assessed clinically and compared. Mice that retained healthy corneal allografts beyond 8 weeks were evaluated for evidence of donor-specific tolerance and anterior-chamber-associated immune deviation (ACAID) using local adoptive transfer reactions and challenge with orthotopic skin allografts. RESULTS: Corneas grafted to CD8KO mice were rejected with an incidence and tempo indistinguishable from that in wild-type control animals. By contrast, MHC-only, and minor-H-only incompatible corneal grafts survived indefinitely in eyes of CD4KO mice. Approximately 50% of corneal grafts that confronted CD4KO recipients with both MHC and minor H alloantigens experienced delayed rejection, whereas similar grafts in wild-type recipients were rejected acutely. CD4KO mice with long-accepted grafts displayed neither donor-specific ACAID nor allograft tolerance. CONCLUSIONS: CD8+ T cells play little or no role in acute rejection of orthotopic corneal allografts. Instead, acute rejection is mediated almost exclusively by CD4+ T cells. Moreover, when corneal allografts survive for 8 weeks without acute rejection, CD4+ T cells promote donor-specific ACAID thereby insuring long-term graft acceptance.     

Survival in high-risk eyes of epithelium-deprived orthotopic corneal allografts reconstituted in vitro with syngeneic epithelium

PURPOSE: In low-risk eyes of mice, most of the composite corneal grafts composed of syngeneic epithelium layered on allogeneic stroma and endothelium are accepted indefinitely. The study was undertaken to determine the fate of similar composite corneal grafts placed in high-risk mouse eyes. METHODS: Epithelium-deprived allogeneic corneas (C57BL/6) were reconstituted in vitro with BALB/c epithelium, and then transplanted orthotopically into high-risk eyes of BALB/c mice. Graft survival was assessed clinically and evaluated histologically. Acquisition of donor-specific delayed hypersensitivity (DH) was also assessed in recipient mice. Recipients bearing healthy composite grafts were immunized subcutaneously with injected C57BL/6 spleen cells at 2 or 8 weeks after grafting, after which the fate of the grafts was evaluated. RESULTS: Virtually all epithelium-deprived corneal allografts reconstituted in vitro with normal BALB/c corneal epithelium survived indefinitely when placed in high-risk eyes of BALB/c mice. Recipients of these composite grafts failed to acquire donor-specific DH when tested at both 2 and 8 weeks after grafting. Moreover, these recipients did not acquire the capacity to actively suppress donor-specific DH. Within 1 to 3 weeks of sensitization of recipient mice with spleen cells of donor origin, healthy composite grafts in residence for 2 or 8 weeks were rejected. CONCLUSIONS: Replacement of donor epithelium with syngeneic epithelium protects orthotopic allogeneic corneal grafts (stroma plus endothelium) placed in high-risk eyes from sensitizing their recipients and from immune-mediated rejection. Recipients of composite corneal grafts containing syngeneic epithelial layers act as though they are immunologically ignorant of the graft's presence.     

CD95 ligand expression on corneal epithelium and endothelium influences the fates of orthotopic and heterotopic corneal allografts in mice

PURPOSE: To determine the extent to which CD95 ligand (CD95L) expressed on corneal epithelium and endothelium influences survival of cornea grafts placed orthotopically and heterotopically in the anterior chamber (AC), an immune-privileged site. METHODS: Corneas from eyes of C57BL/6 (B6) and B6.gld (CD95L deficient) mice were (1) rendered into small full-thickness fragments, with or without an epithelial layer, and placed behind recipient corneas in the ACs of BALB/c eyes, while BALB/c corneas were similarly implanted in eyes of B6 and B6.lpr (CD95 deficient) mice; or (2) corneas were grafted orthotopically in BALB/c eyes as intact corneas or as composite corneas in which epithelium from one donor source was layered in vitro onto epithelium-deprived stroma+endothelium from another donor source before grafting. The fate of the grafts was assessed clinically and histologically, and the capacity of the grafts to sensitize recipient mice to donor alloantigens (delayed hypersensitivity, DH) was evaluated. RESULTS: Allogeneic, full-thickness B6.gld corneal fragment grafts placed in the AC of BALB/c mice were rejected and sensitized their recipients, whereas epithelium-deprived B6.gld cornea fragments survived indefinitely and failed to sensitize their recipients. BALB/c corneal fragment grafts placed in the AC of C57BL/6 or B6.lpr eyes were rejected. Orthotopic cornea grafts composed of B6.gld epithelium layered onto wild-type B6 stroma and endothelium were rejected at a tempo and incidence similar to full-thickness B6 grafts, whereas orthotopic composite cornea grafts containing B6 epithelium layered onto B6.gld stroma+endothelium were vigorously rejected. CONCLUSIONS: CD95L expression on epithelium of full-thickness cornea fragment grafts placed in the anterior chamber of BALB/c eyes protects these heterotopic grafts from rejection but has only a trivial role to play in determining the fate of orthotopic corneal grafts. In the latter type of corneal grafts, CD95L expression on the endothelium plays an essential role in preventing graft rejection.     

Role of tumor necrosis factor receptor expression in anterior chamber-associated immune deviation (ACAID) and corneal allograft survival

PURPOSE: To determine the role of tumor necrosis factor receptors (TNFRs) in corneal allograft rejection. METHODS: Corneal epithelial and endothelial cells were examined by flow cytometry for the expression of TNFRI and TNFRII and their susceptibility to TNF-alpha-induced apoptosis. Corneal allografts from normal and TNFRI and TNFRII knockout (KO) C57BL/6 mice were transplanted to BALB/c hosts, and the fate of the allografts was monitored. C57BL/6 spleen cells were injected into the anterior chamber (AC) of BALB/c mice to induce anterior chamber-associated immune deviation (ACAID) and promote corneal allograft survival. The presence of ACAID suppressor cells in corneal allograft recipients was tested using a local adoptive transfer (LAT) assay. RESULTS: Murine corneal epithelial and endothelial cells expressed TNFRI and TNFRII and were susceptible to TNF-alpha-induced apoptosis, yet corneal allografts from either TNFRI or TNFRII donors did not enjoy a lower incidence of rejection or a prolongation in survival time compared to corneal allografts from normal C57BL/6 donors. Moreover, all 31 of the TNFRII KO corneal grafts were rejected by na?ve BALB/c hosts. Rejection of TNFRII KO corneal grafts occurred even though suppressor cells developed in the hosts and inhibited the expression of delayed-type hypersensitivity to donor alloantigens. CONCLUSIONS: Expression of TNFRII on corneal cells conveys a degree of protection against immune rejection of corneal allografts by a mechanism that is independent of ACAID. Moreover, induction of ACAID before the application of TNFRII KO corneal allografts fails to improve survival and does not replace the TNFRII-dependent protective mechanism.     

Effect of allergic conjunctival inflammation on the allogeneic response to donor cornea

PURPOSE: Immunologic rejection is the most common cause of corneal allograft rejection. Ipsilateral ocular inflammation has been identified as a predictor of future corneal graft failure. This study investigates the effect of perioperative allergic conjunctivitis on corneal allograft survival. METHODS: C57BL6 donor corneas were transplanted into naive A/J mice, A/J mice sensitized to short ragweed (SRW) pollen by intraperitoneal injection and then challenged with topical SRW to induce allergic conjunctivitis (Sens(+)Chall(+)), and A/J mice sensitized to SRW and challenged with topical PBS (Sens(+)Chall(-)). Syngeneic grafts were also performed in eyes with allergic conjunctivitis. Graft survival and infiltrating cell phenotype in rejected grafts were compared between groups. RESULTS: Mice with allergic conjunctivitis (Sens(+)Chall(+)) rejected corneal allografts significantly more quickly than naive mice. Syngeneic grafts in allergic eyes survived indefinitely. The rate of rejection in Sens(+)Chall(-) mice was similar to that in naive mice. There were no significant differences, between groups, in the numbers of infiltrating CD4(+) cells, CD8(+) cells, and macrophages at the time of graft rejection. Eosinophils were seldom observed in rejected grafts in naive and Sens(+)Chall(-) mice but were observed consistently in Sens(+)Chall(+) eyes. Eosinophils were also found consistently in the ciliary body of Sens(+)Chall(+) eyes at the time of graft rejection. CONCLUSIONS: Active allergic conjunctivitis at the time of transplantation accelerates corneal allograft rejection. Local conjunctival inflammation is an important factor in accelerating rejection.