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。     

FK-506抑制大鼠角膜移植免疫排斥反应的研究

目的建立近交系大鼠穿透性角膜移植动物模型,观察结膜下注射ＦＫ５０６对角膜移植免疫排斥反应的抑制作用。方法将近交系Ｌｏｕ大鼠２８只作受体,１４只Ｆ３４４大鼠作供体,分为３组,术后结膜下分别按每公斤体重注射０．１ｍｇＦＫ５０６、３ｍｇＣｓＡ及生理盐水,共２周。对角膜植片进行临床观察,以混浊、水肿和新生血管３项指标作为临床评估标准。结果３组角膜植片的存活时间分别为（２２１±５１７）、（１８４±１４）及（１２１±２１３）天,三者间差异有显著性（Ｐ＜０．０１）。结论ＦＫ５０６能显著延长大鼠角膜植片的存活时间,是一种有效的新型免疫抑制剂。     

FK506眼液防治同种异体角膜缘移植免疫排斥反应的实验研究

目的　探索同种异体角膜缘移植排斥反应规律及FK5 0 6眼液的免疫抑制作用。方法4 8只新西兰白兔随机均分成FK5 0 6组、环孢素A组及非治疗组 ,建立右眼角膜缘缺陷症动物模型 ,1个月后行同种异体角膜缘移植术 ,术后分别用 0 5 %FK5 0 6眼液、1%环孢素A眼液及生理盐水滴眼4周。角膜缘移植术前 1d ,术后 2、4周分别进行角膜中央印迹细胞学检查 ;术前 1d及术后 1～ 8周动态监测外周血T淋巴细胞CD2 5的表达 ;移植术后每日观察植片存活、角膜新生血管和上皮再生情况 ,共 10周 ;分别取术后第 4、8及 10周的角膜缘植片观测淋巴细胞浸润及T淋巴细胞上CD2 5的表达。结果　角膜缘创伤后 1个月 ,所有兔眼角膜中央印迹细胞学检查均为结膜表型 ;同种异体角膜缘移植术后 4周 ,FK5 0 6组和环孢素A组角膜中央保持角膜细胞表型 ,非治疗组呈结膜细胞表型。非治疗组最早出现上皮排斥反应 ,FK5 0 6与环孢素A均可抑制排斥反应的发生 ,停药后环孢素A组先于FK5 0 6组出现上皮排斥反应 (P <0 0 5 )。FK5 0 6组外周血、角膜缘植片T淋巴细胞CD2 5表达及角膜缘植片中淋巴细胞浸润程度低于环孢素A组 ,两组均低于非治疗组 (P <0 0 5 )。结论　同种异体角膜缘移植术后早期应用 0 5 %FK5 0 6眼液滴眼可抑制外周血及植片T淋巴细胞CD2 5     

FK506 in a biodegradable glycolide-co-clatide-co-caprolactone polymer for prolongation of corneal allograft survival

PURPOSE: FK506 has been extensively used in preventing immune rejection for human organ transplantation. This study aimed to evaluate the effects of a biodegradable FK506 drug delivery system (DDS) implanted into anterior chamber for the prolongation of corneal allograft survival in high-risk keratoplasty. METHODS: Biodegradable glycolide-co-clatide-co-caprolactone polymer (PGLC) was used as drug carrier to be incorporated with 0.5 mg of FK506 powder. The drug release from the FK506-PGLC DDS was evaluated in vitro and in vivo. The FK506-PGLC DDS was implanted into the anterior chamber of 12 high-risk keratoplasty rabbits. The graft survival time and clinical features of the FK506-PGLC DDS group were compared with the untreated, PGLC DDS, cyclosporin A-PGLC DDS, and 0.5% FK506 drops groups. The histopathological examination was performed to evaluate the safety of the FK506-PGLC DDS. RESULTS: The mean graft survival time was longest (> 180 days) in the FK506-PGLC DDS group. In vivo, the FK506 concentration in aqueous humor peaked on day 28 (17.9 +/- 2.3 ng/ml) and kept a sustained release for at least 168 days. No adverse reactions were observed in the FK506-PGLC DDS group. CONCLUSIONS: Biodegradable FK506-PGLC DDS implanted into anterior chamber can effectively prevent immune rejection in high-risk keratoplasty model, presenting a promising approach for the prolongation of corneal allograft survival.     

Immunotherapy in ocular diseases]

Basic and clinical studies on immunotherapy in immune-mediated ocular disorders, i.e. uveitis, allograft rejection in corneal transplantation and allergic conjunctivitis, were carried out using a variety of immunosuppressants, including immunophilin ligands (FK506 and cyclosporine). 1. In an animal model for uveitis, experimental autoimmune uveitis (EAU), immunophilin ligands were demonstrated in the rat and monkey to have unique immunological activities: (1) intense and prolonged suppression of EAU development, (2) therapeutic effects by treating animals only after disease onset, (3) selective suppression on cellular immune response to S-antigen, (4) induction of immunological tolerance and activation of antigen specific suppressor cells. Combination therapy with low doses of immunophilin ligand and other immunosuppressant was tested to achieve better effects with less side effects. A low dose of cyclosporine (2 mg/kg/day) with bucillamine (20 mg/kg/day) which suppresses antigen-presenting activity by macrophages caused much stronger suppression of EAU than the therapy with either cyclosporine or bucillamine alone. Similarly, a low dose of FK506 (0.1 mg/kg/day) with dexamethasone (0.01 mg/kg/day) caused stronger suppression of EAU. A multi-center clinical open trial of FK506 in refractory uveitis was carried out in Japan. A total of 40 cases of active uveitis in the posterior segment of the eye were treated with FK506 (0.05, 0.1 or 0.2 mg/kg/day) and the mean observation period was 26.2 +/- 12.4 weeks. FK506 therapy improved uveitis in 60% of all cases including 47% of patients resistant to previous therapy with cyclosporine. FK506 significantly suppressed the number of uveitis attacks in patients with Beh?et's disease. As for the side effects, 22.5% of patients showed abnormal values of renal function on FK506. The trough level of FK506 in whole blood correlated with adverse side effects as well as with therapeutic effect on uveitis, and it should be maintained between 15 and 25 ng/ml. 2. Effects of immunophilin ligands on the allograft rejection in corneal transplantation was examined in the rat. Fisher rat were used as donors and Lewis rats as recipients. This combination caused 100% rejection by 2-3 weeks after surgery as indicated by (1) edema, opacity and neovascularization in the graft, (2) infiltration of a variety of immune cells demonstrated by immunohistological examination and (3) high mixed leukocyte reaction (MLR). Systemic administration of cyclosporine (10 mg/kg/day) or FK506 (0.3, 1, 3 mg/kg/day) suppressed the allograft rejection. In addition, topical instillation of FK506 (0.3%) was effective to prolong the graft survival as long as the topical therapy continued.(ABSTRACT TRUNCATED AT 400 WORDS)     

雷公藤多甙和FK506滴眼防治大鼠角膜移植免疫排斥反应的比较

目的:观察雷公藤多甙(tripterygiitotorum,TⅡ)及FK506滴眼液局部应用对大鼠角膜移植免疫排斥反应的影响。方法:建立大鼠角膜移植模型,随机按局部滴用药物分组成A,B,C组,用裂隙灯记录及比较各组移植排斥指数,并比较角膜排斥发生时间。分别于术后15d及30d行常规病理学检查。并通过免疫组化检查CD25,CD5+4在角膜植片基质中的表达。结果:0.3g/LTⅡ及0.5g/LFK506抑制角膜移植免疫排斥反应的效应均有效,两者相比效果无显著性差异。HE染色及免疫组化分析可见15d时各有效组角膜植片厚度基本正常,基质中仅有少量CD25,CD5+4阳性细胞表达、淋巴细胞浸润,新生血管少见。结论:TⅡ与FK506疗效近似。具有抑制高危角膜移植免疫排斥反应发生的临床应用价值。     

FK506抑制兔同种异体角膜缘移植免疫排斥反应的实验研究

目的 探讨FK506滴眼液对同种异体角膜缘移植术后排斥反应的免疫抑制作用。方法 新西兰兔48只48眼建立角膜缘缺陷症动物模型。随机分为FK506组、环孢霉素A组及生理盐水对照组3组，每组16眼。1个月后实施同种异体角膜缘移植术，眼表观察10周。结果 术后10周，对照组、环孢霉素A组、FK506组角膜新生血管指数平均秩分别为15．50、8．00、5．00。对照组角膜新生血管指数较其他2组高（P＝0．00）；而FK506组与环孢霉素A组间无显著差异（P＝0．12）；对照组上皮排斥反应发生最早，环孢霉素A组次之，FK506组最晚；术后10周，对照组、环孢霉素A组、FK506组移植排斥反应指数平均秩分别为13．67、8．07、4．58，对照组排斥反应指数较FK506组（P＝0．02）和环孢霉素A组（P＝0．00）高；环孢霉素A组移植排斥反应指数显著高于FK506组（P＝0．04）。同种异体角膜缘移植术后眼局部应用FK506滴眼液可延迟排斥反应发生，其免疫抑制效果优于环孢霉素A。     

CD4和CD8基因敲除鼠行穿透性角膜移植术后免疫排斥特征的研究

目的　探讨CD4和CD8基因敲除小鼠行穿透性角膜移植术后免疫排斥反应发生的机制。方法　CD4、CD8基因敲除小鼠及C57BL/6小鼠各20只,分成3组,右眼接受穿透性角膜移植术,供体为BALB/c小鼠,术后裂隙灯显微镜评价角膜移植片情况,并详细记录免疫排斥的发生时间,在术后1、2、4周各取2只鼠术眼行免疫组织化学检查,观察眼前段CD+4 、CD+8 T细胞的变化。在术后2周, 3组小鼠各选其中10只接受皮肤移植,供体为BALB/c小鼠,监测皮肤移植后皮肤植片免疫排斥反应的时间和在皮肤发生排斥反应时角膜移植片的情况。结果　3组小鼠角膜移植术后免疫排斥发生时间明显不同,CD4基因敲除鼠角膜移植片保持透明,至少观察了90d未见免疫排斥反应发生;CD8基因敲除小鼠在(28±3)d时发生免疫排斥反应;C57BL/6小鼠发生免疫排斥反应的时间为(14±2)d(F=2034, P<0. 01)。移植皮肤后发生免疫排斥反应时间为:CD4基因敲除鼠(14±2)d,CD8基因敲除鼠(12±1)d,C57BL/6小鼠(10±1)d(F=42. 54, P<0. 01)。结论　小鼠行穿透性角膜移植术后免疫排斥反应可能是以T淋巴细胞,主要为CD+4 T细胞介导的免疫排斥反应,CD+8 T细胞参与了排斥反应过程。     

小分子化合物J2在抑制小鼠角膜移植排斥反应中作用的初步研究

目的 探讨小分子化合物J2在抑制小鼠角膜移植排斥反应中的作用。方法以23只C57BL／6小鼠作为供体,76只BALB／c小鼠作为受体建立角膜移植实验模型,随机数字法分为A、B、C及D组,A组为BALB／c小鼠自体原位角膜移植,B、C及D组为C57BL／6-BALB／c小鼠间同种异体角膜移植。术后灌胃给药,A组和B组给予不含药物的空白液,C组和D组分别给予环孢素A（CsA）和小分子化合物J2,连续灌胃12d,比较各组小鼠角膜植片的存活时间和存活率,术后21d对各组小鼠行外周血单核细胞行流式细胞学检查,并做角膜植片的组织学检查。结果A组观察期内角膜植片未发生排斥,B组角膜植片平均存活时间为（17．8±2．1）d,C组为（38．1±9．9）d,D组角膜植片存活时间为（40．6±8．3）d,D组与A组（P=0．04）及B组（P=0．00）比较存活时间差异均有统计学意义,与C组比较差异无统计学意义（P：0．99）。流式细胞学检查显示J2给药小鼠外周血CD^＋细胞、CD8^＋细胞未发生增殖,组织学检查证实术后21dD组角膜植片未见明显的淋巴细胞浸润。结论小分子化合物J2能够抑制排斥的发生,延长小鼠角膜植片存活时间。（中华胺群条峦,2007,43：608-612）     

Studies on the Effects of the Immunosuppressant FK—506 on the High—risk Corneal Graft Rejection

Purpose: To evaluate the clinical efficacy of FK-506 on suppressing high-risk cornea transplantation rejection.Methods: In a randomized controlled clinical trial, 56 eyes of 56 patients with high-risk keratoplasty (including total corneal transplantation TCT, total corneal transplantation with circular lamellar sclera CST, vascularization corneal transplantation and corneal retransplantation) were divided into the experimental group and the control group (each with 28 eyes). The experimental group was treated by FK-506 eyedrops (0. 5 mg/ml) and TobraDex eyedrops, compared with the control group that was treated by 1% CsA eyedrops and TobraDex eyedrops. In the average 8. 1-month follow-up period, the visual acuity, graft transparent duration and Rejection Index (RI) of grafts were observed. Results: In the follow-up period, the graft rejection rate of the experimental and the control group was 63. 6% and 95. 2% respectively (x2 =4. 72, P<0. 05) with significant difference.Conclusions: The local applicati     

FK506抑制大鼠角膜移植免疫排斥反应的免疫病理学研究

目的 研究角膜移植免疫排斥反应的免疫病理学变化及免疫细胞和某些相关免疫分子在角膜移植免疫排斥反应中的作用,阐明了ＦＫ５０６的免疫抑制机理。方法 应用免疫组织化学染色方法检测ＦＫ５０６,环胞霉素Ａ及对照组角膜植片中ＣＤ＾＋４,ＣＤ＾＋８,巨噬细胞,白细胞介素－２受体,Ⅱ类主要组织相容性抗原,细胞间粘附分子－１及淋巴细胞功能相关抗原－１的表达。结果 排斥的角膜组织大表达上述免疫细胞和分子,ＦＫ５０６可     

FK506滴眼液抑制兔同种异体角膜缘移植排斥反应的免疫病理学研究

目的 :观察同种异体角膜缘移植术后排斥反应的免疫病理学变化 ,探讨FK5 0 6滴眼液的免疫抑制作用。方法 :新西兰兔 48只 48眼建立角膜缘缺陷症动物模型。随机分为PK 5 0 6组、环孢霉素A组 (CsA )及生理盐水对照组 ,每组16眼。 1月后实施同种异体角膜缘移植术。应用免疫病理学方法检测术后 4、 8、 10周角膜缘植片CD2 5的表达和淋巴细胞浸润。结果 :术后 4、 8、 10周三组角膜缘植片CD2 5的表达和淋巴细胞浸润均有升高 ,对照组较FK5 0 6组和CsA组高 (P <0 0 5 ) ,CsA组显著高于FK5 0 6组 (P <0 0 5 )。结论 :PK 5 0 6滴眼液可抑制同种异体角膜缘移植术后植片CD2 5的表达 ,其免疫抑制作用优于CsA。     

FK-506抑制同种异体角膜缘移植术后免疫排斥反应的研究

目的：探讨ＦＫ－５０６抑制角膜缘干细胞移植术后免疫排斥反应的临床可行性与有效性。方法：应用前瞻性评估研究方法,将角膜缘移植术后病例６４只眼按随机原则分投药组及对照组各半,投药组应用０．５％ＦＫ－５０６滴眼液,对照组应用１％ＣｓＡ滴眼液。平均随访期半年,以术后角膜缘植片新生血管、水肿、混浊及溶解程度为临床主要评估指标。结果：随访期内在新生血管指数、上皮排斥发生率、移植排斥指数及假性胬肉指数四项指标投     

FK—506抑制全角膜移植术后免疫排斥反应的研究

目的 观察ＦＫ－５０６抑制全角膜移植术（包括带环形巩膜瓣的全角膜移植术）后免疫排斥反应的临床疗产。方法 对７６例（７６只眼）全角膜移植术患者按随机原则分试验组（ＦＫ－５０６组）及对照组（ＣｓＡ组）各３８例（３８只眼）,观察术后不同时期角膜免疫排斥反应的发生率,平均随访时间６个月。结果 全角膜移植术后,局部应用ＦＫ－５０６可有效抑制新生血管的发生;随访期内,治疗组（ＦＫ－５０６组）移植片排斥反应发生     

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.     

Long-term results of FK 506 eye drops following corneal transplantation

BACKGROUND: Immunological graft rejection is the main reason for graft failure following corneal transplantation despite the use of topical and systemic steroids. As steroids are associated with side effects, alternative therapeutic strategies are needed. PATIENTS AND METHODS: In this clinical trial patients undergoing corneal transplantation have been prospectively randomised to receive either prednisolone acetate 1 % eye drops 5 x /day, tapering off by one drop every month (n = 20), or to receive FK 506 eye drops 3 x /day for six months (n = 20). Patients in both groups received additionally systemic steroids for three weeks (fluocortolon 1 mg/kg body weight). Primary endpoints were the number of immune reactions and the clear graft survival, the secondary endpoint was the number of side effects. RESULTS: Three immune reactions in the steroid group and one immune reaction in the FK 506 group were seen within the follow-up time of three years. No irreversible graft rejections occurred in either group. Eight patients in the FK 506 group concluded the study early due to local side effects. CONCLUSIONS: In this long-term follow-up the use of FK 506 eye drops following corneal transplantation resulted in a lower number of immune reactions when compared to topical steroids. With a change in the galenic formulation FK 506 might be a powerful therapeutic option for preventing immunological graft rejection.     

A comparative investigation of FK506 and cyclosporin A in murine corneal transplantation

· Background: Acute rejection is the cause of over 50% of transplant opacifications in some immunological high-risk groups. More potent immunomodulating substances must be found in order to allow extended or individualised therapeutic options for combating rejection. · Methods: Rats of the inbred strains Brown Norway and Lewis were used as donors and recipients, respectively. FK506 (Prograf) was administered intraperitoneally for 14 days in a dosage of 0.3 mg/kg bw, and cyclosporin A (CSA; Sandimmun) was administered, likewise for 14 days, in an intramuscular dosage of 10 mg/kg bw. The transplants were examined every 3rd day by slit-lamp microscopy. Every transplant was subjected to histological or immunohistological evaluation. · Results: The average transplant survival period in the allogeneic strain combination was 7.9 days (SEM=1.1). Therapy with FK506 led to a statistically significant prolongation of transplant survival to 17.1 days (SEM=1.5, P<0.05). Therapy with CSA delayed transplant rejection to 21 days (SEM=0.0, P<0.05). No statistically significant difference was found between the two therapeutic regimens. There were no significant histomorphologic differences in rejected grafts in the FK506- and CSA-treated animals. · Conclusions: In this study we have shown that FK506 is able to delay corneal allograft rejection at a much lower dosage than CSA without a higher incidence of side effects related to toxicity or overimmunosuppression. Received: 8 September 1997 Revised version received: 16 December 1997 Accepted: 13 January 1998     

Effect of FK 506 on the cornea: use of topical FK 506 in corneal transplantation in a guinea pig-rat model

To evaluate the effect of different concentrations 0.03%, 0.1% and 0.3% of FK 506 on xenograft corneal rejection, guinea pig corneas were transplanted into rats. FK 506 was then applied topically four times a day for 21 days. The grafts were inspected and scored according to opacity, edema, and graft protrusion. All grafts in the control group were rejected by the 14th postoperative day, and grafts in the FK 506 treated groups began to be rejected by the 17th postoperative day. Inflammatory cell infiltration was less dense in the FK 506 treated grafts than in the control group. Higher concentrations of FK 506 appeared to be more effective in preventing and decreasing the severity of the graft rejection. Topical FK 506 can delay the development of xenograft corneal rejection and decrease its severity in this animal model.