脲溶性色素相关抗原诱导实验性葡萄膜炎研究

目的 探讨脲溶性牛黑素相关抗原能否在不同大鼠身上诱导实验性自身免疫性葡萄膜炎。 方法 将脲溶性牛黑素相关抗原加弗氏完全佐剂和百日咳杆菌分别免疫Lewis、F344及Wistar大鼠，通过临床观察和组织病理学对动物模型作动态观察比较。 结果 脲溶性牛黑素相关抗原可在Lewis、F344及Wistar大鼠身上诱导出实验性自身免疫性葡萄膜炎，其表现为双眼同时发病，以眼前段为主，个别严重者可伴有局灶性脉络膜炎，不伴视网膜炎和松果体炎，与牛黑素相关抗原所诱发的实验性自身免疫性前葡萄膜炎相类似。Lewis和F344大鼠发病时间及炎症严重程度相似，而Wistar大鼠发病时间较迟，炎症较轻。 结论 脲溶性牛黑素相关抗原可在Lewis、F344和Wistar大鼠身上诱导出实验性自身免疫性前葡萄膜炎。Wistar大鼠与Lewis和F344大鼠相比，发病时间较迟，炎症改变较轻，这种差异可能与遗传背景不同有关。(中华眼底病杂志,1998,14:149-152)     

黑素相关抗原诱发的实验性自身免疫性葡萄膜炎

黑素相关抗原诱发的实验性自身免疫性葡萄膜炎动物模型的建立 ,对于开展葡萄膜炎基础和临床研究具有重要意义。该动物模型以前葡萄膜炎为主 ,且可复发 ,不伴松果体炎及视网膜炎 ,更能代表人类葡萄膜炎中最常见的类型—前葡萄膜炎。本文就有关黑素相关抗原诱导的动物模型特征及其在葡萄膜炎研究中的应用作一综述     

黑素相关抗原诱发的实验性自身免疫性葡萄膜炎

黑素相关抗原诱发的实验性自身免疫性葡萄膜炎动物模型的建立，对于开展葡萄膜炎基础和临床研究具有重要意义。该动物模型以前葡萄膜炎为主，且可复发，不伴松果体炎及视网膜炎，更能代表人类葡萄膜炎中最常见的类型－前葡萄膜炎。本就有关黑素相关抗原诱导的动物模型特征及其在葡萄膜炎研究中的应用作一综述。     

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)     

光感受器间维生素A类结合蛋白诱发的色素膜视网膜炎…

光感受器间维生素Ａ类结合蛋白具有强烈的致色素膜视网膜炎和松果体炎活性。我们用不同剂量ＩＲＢＰ免疫Ｌｅｗｉｓ大鼠，发现２５μｇ和５０μｇＩＲＢＰ可诱发出实验性自身免疫性色素膜炎和实验性自身免疫性松果体炎。ＩＲＢＰ诱发的ＥＡＵ和ＥＡＰ，为人类色素膜视网膜炎及其伴随的神经系统受累的发生机制及内在联系等方面的研究提供了很好的动物模型。     

IRBPR16多肽的合成及其致葡萄膜视网膜炎活性的探讨

目的 探讨光感受器间维生素A类结合蛋白（IRBP）的R16多肽片段的致葡萄膜视网膜炎活性。设计实验研究。研究对象36只Lewis大鼠。方法应用Fmoc法合成并纯化牛IRBPR16多肽片段,以诱导实验性自身免疫性葡萄膜视网膜炎（EAU）模型,并对该模型进行临床观察和组织学检查。培养EAU大鼠的引流淋巴结细胞,测定淋巴细胞增殖反应。各实验同时建立单纯弗式完全佐剂（CFA）免疫组和空白对照组。主要指标多肽分析,视网膜形态学,淋巴细胞增殖反应。结果合成的IRBPR16多肽片段纯度为95．6％。应用IRBPR16多肽片段作为抗原免疫Lewis大鼠,可成功诱导出EAU模型。EAU的临床分级为（3．33±0．52）级,病理分级为（3．67±0．92）级;CFA组和空白对照组大鼠眼部均无异常改变。EAU组大鼠引流淋巴结中抗原特异性淋巴细胞增殖反应增强,为（33．27±7．24）×10^cpm,显著高于CFA组[（1．91±1．16）×10^3cpm]和空白对照组[（1．23±0．51）×10^3cpm]（P〈0．05）。结论IRBPR16多肽片段具有较强的致葡萄膜视网膜炎活性,引流淋巴结抗原特异性淋巴细胞增殖反应增强。IRBPR16多肽诱导的EAU为研究人类葡萄膜视网膜炎提供了一个重要的动物模型。     

视网膜S抗原的制备和提纯方法的改进

实验性自身免疫性葡萄膜炎(EAU)是由CD4+T淋巴细胞介导的器官特异性炎症,可由多种视网膜蛋白诱发,主要有视网膜可溶性抗原(S-Ag)、光感受器间维生素A类结合蛋白(IRBP)和视紫红质等,其中以S抗原诱发的葡萄膜炎更具有临床意义[1]。由于S抗原所致葡萄膜炎的活性与其纯度呈正相关,所以S抗原的纯化尤为重要。我们将Al-Mahdawi等[2]的方法进行改进简化,获得了较高纯度的牛视网膜S抗原,并免疫Lewis大鼠成功地复制出EAU模型。现将结果报告如下。1材料和方法冰浴下暗室解剖新鲜牛眼,沿角膜缘环行切开眼球,挤出晶状体和玻璃体,用毛笔刷轻取…     

经鼻黏膜诱导免疫耐受治疗实验性自身免疫性葡萄膜炎

目的探讨经鼻腔滴注牛视网膜S抗原诱导免疫耐受对Lewis大鼠自身免疫性葡萄膜视网膜炎(EAU)的影响。方法利用盐析和离子交换层析方法纯化牛视网膜S抗原,然后用其诱导Lewis大鼠鼻黏膜耐受,再用视网膜S抗原诱发EAU,观察EAU的发病情况、眼部临床表现、组织学改变、皮肤迟发型超敏反应、由视网膜S抗原和刀豆蛋白A刺激的淋巴细胞增殖反应,同时观察环磷酰胺对免疫耐受的影响。结果用牛视网膜S抗原鼻腔滴注诱导免疫耐受后再诱导EAU,耐受组8只大鼠仅有2只发病(25%),对照组6只(100%)全部发病,耐受组大鼠发病比例明显低于对照组(P=0·0097)。耐受组大鼠的发病开始时间平均为16·5d,对照组为10·3d,两者之间差异有统计学意义(F=26·32,P=0·000;q=9·723,P<0·01);耐受组大鼠EAU病变的临床评分为0·89,对照组为3·94,差异有统计学意义(F=12·48,P=0·000;q=7·904,P<0·01);耐受组大鼠的组织学分级为1·21,对照组为4·12,差异有统计学意义(F=11·80,P=0·000;q=7·510,P<0·01)。耐受组大鼠的EAU表现为虹膜血管轻度扩张,前房和玻璃体内少量炎性渗出,视网膜轻度肿胀,视网膜感光细胞损害均较轻。耐受组大鼠的皮肤迟发型超敏反应和由视网膜S抗原刺激的淋巴细胞增殖反应也明显轻于对照组;腹腔注射环磷酰胺可轻度增强S抗原诱导的免疫耐受作用,仅用环磷酰胺对EAU的炎性反应无明显影响。结论视网膜S抗原诱导的黏膜耐受可有效地预防由视网膜S抗原诱发的EAU。     

葡萄膜炎的诊断及相关问题

葡萄膜炎是累及葡萄膜、视网膜、视网膜血管及玻璃体的一组炎症性疾病 ,也称为眼内炎症。由于炎症的初始部位及累及的组织不同 ,临床上有多种类型 ,如虹膜睫状体炎、脉络膜炎、脉络膜视网膜炎、视网膜炎及视网膜血管炎等 ;按病因可分为感染性、自身免疫性、外伤性及特发性葡萄膜炎等多种类型 ;根据炎症发生的部位将葡萄膜炎分为前、中间、后及全葡萄膜炎 4类 ;根据葡萄膜炎的临床特点及与全身性疾病的关联性 ,又将其分为Ｂｅｈｃｅｔ病、Ｖｏｇｔ Ｋｏｙａｎａｇｉ Ｈａｒａｄａ(ＶＫＨ)综合征及Ｆｕｃｈｓ综合征等类型。这些众多的名称不仅…     

大鼠的实验性自身免疫性葡萄膜视网膜炎的免疫致病机理

为了了解实验性自身免疫性葡萄膜视网膜炎(EAU)免疫致病机理,用Lewis鼠研究T淋巴细胞和肥大细胞的作用。用致敏的淋巴细胞特别是辅助性/诱异性T细胞亚群成功地转移给首次用来作实验的同系大鼠。接受了对S抗原致敏了的辅助性/诱导性T细胞的实验性自身免疫性葡萄膜视网膜炎(EAU)的大鼠充分表现了对此抗原的迟发型皮肤超敏反应但极轻微的Arthus反应。分析了环孢霉素、环磷酰胺,地塞米松等免疫抑制剂药物对S抗原免疫的鼠产生EAU的作用。通过选择性抑制对S抗原的迟发型皮肤超敏反应,说明仅有环孢霉素能完全抑制EAU的发生。这些资料说明T淋巴细胞在EAU致病机理中起主要的作用。根据在疾病发作以前,脉络膜的肥大细胞发生脱颗粒作用,说明除了淋巴细胞参与以外,脉络膜的肥大细胞也起了附带作用。     

Xenopus IRBP, a phylogenetically remote protein, is uveitogenic in Lewis rats

Mammalian interphotoreceptor retinoid-binding proteins (IRBPs) are highly uveitogenic in Lewis rats. Xenopus laevis IRBP resembles mammalian IRBP in its four-fold structure, and has approximately 70% amino acid sequence identity with the bovine protein. This study investigated the uveitogenicity of recombinant Xenopus IRBP and two of its derived peptides in Lewis rats. Rats immunized with Xenopus IRBP developed uveoretinitis as well as pineal inflammation. The Xenopus molecule was, however, less immunopathogenic than the bovine IRBP. Of the two Xenopus IRBP peptides tested, 1180-1191 was remarkably uveitogenic, whereas sequence 521-540 exhibited low activity. It is assumed, therefore, that as with bovine IRBP, peptide 1180-1191 is the major uveitogenic sequence in Xenopus IRBP. The role individual residues of these peptides play in the immunopathogenic process is discussed. Our data thus demonstrate that despite its being phylogenetically remote, Xenopus IRBP is uveitogenic in Lewis rats Copyright 2000 Academic Press.     

Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium.

Retinal pigment epithelial cell fractions have been investigated for their capacity to induce experimental uveitis. Cells of the dark (melanotic) and light areas of the bovine RPE have subsequently been extracted by buffer, Triton X-100, sodium dodecyl sulfate (SDS), and treated with various reagents in order to study some characteristics of the antigen. The SDS-insoluble melanotic fraction, consisting of spindle-shaped, mature melanin granules, proved to be the most uveitogenic preparation. Using pertussis toxin as coadjuvant, 1 microgram of melanin-protein (3.4 x 10(6) granules) was able to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats. The pathogenic activity of the responsible pathogen (PEP-X) was not diminished by SDS, nor eliminated by mildly alkaline SDS or formic acid treatment. However, HCl-deproteinized granules were not uveitogenic. The results show that PEP-X is a highly stable melano-antigen that is probably covalently bound to the granule surface. This is the first time that a melanin-bound antigen has been demonstrated to evoke specific autoaggressive activity. EAAU could adoptively be transferred by sensitized and in vitro stimulated CD4 T-lymphocytes. The evoked inflammation started 3-4 days after injection, was similar to those induced by immunization, and consisted mainly of severe iridocyclitis accompanied by dense flare and cells in the anterior chamber. Choroiditis developed in severe cases of EAAU but no inflammation was detected in the retina, pineal gland or other organs of these rats. EAAU could not be transferred by serum. Immunized PVG rats and guinea-pigs did not develop ocular inflammation. In monkeys a high dose of antigen evoked a very mild EAAU accompanied by choroiditis. In view of its characteristics, EAAU may be a new model for human anterior uveitis.     

A new procedure for experimental autoimmune uveitis with small uveitogenic peptides

PURPOSE: Demonstration of experimental autoimmune uveitis (EAU) with extremely small, fragmented peptides (12-30 amino acid residues) of interphotoreceptor retinoid-binding protein (IRPB). METHOD: Very small fragmented peptides (no. 854, 888, 907, and 1057) were conjugated to heat-killed Group A Streptococcus cells and administered as a single intravenous injection to Lewis rats. A non-uveitogenic peptide 950 was also conjugated to heat-killed Streptococcus and administered. Administration of a mixture of small peptides and Streptococcus was a control for the peptides conjugated with Streptococcus. RESULTS: The uveitogenic peptide/Streptococcus conjugates produced uveitis inflammatory responses in the uvea, retina and pineal gland. Administration of mixtures of small peptides and Streptococcus cells, and a non-uveitogenic peptide 950 conjugated with Streptococcus did not produce autoimmune uveitis. CONCLUSIONS: Since mixtures of small uveitogenic peptides and Streptococcal cells did not develop autoimmune uveitis, conjugated Streptococcal cells provided a vehicle for macrophage phagocytosos of very small uveitogenic IRBP peptides. Subsequent antigen presentation from macrophages to lymphocytes developed autoimmune uveitis. Peptide 888, one of four IRBP peptides that encompass the major uveitogenic domain, proved to be the most effective in development of uveitis.     

Multiple recurrences in melanin-protein-induced uveitis in the rat

The purpose of the present study was to investigate the occurrence and sequelae of multiple recurrences in experimental melanin-protein-induced uveitis (EMIU). Lewis rats were immunized with purified bovine choroidal melanin granules, and the development of EMIU was studied during six months. Multiple recurrences were observed in virtually all rats that developed primary EMIU. The spontaneous recurrences exhibited an increasingly mild character and a decreasing frequency over time. They occurred one to four times per eye with intervals of five to six weeks. If the inflammations were more severe or chronic the uveal tissues were more seriously damaged. The anterior uvea became slender by loss of cells and stroma during the process suggesting a role as target. Unlike in primary EMIU, the retina finally exhibited areas with damage of the photoreceptor and pigment epithelial cells. Mononuclear cells were the predominant inflammatory cell type in the entire uvea in eyes with serious recurrences or chronic uveitis. The number of recurrences per se did not correlate with the extent of tissue damage but the overall severity of the disease over six months did. In rats recovered from mild recurrences, a single injection of pertussis toxin, or melanin granules emulsified in Freund's incomplete adjuvant was sufficient to reinduce severe EMIU with extensive damage of the uvea. Hence, specific as well as aspecific stimulation of the immune system caused severe recurrences of this type of uveitis. EMIU resembles non-infectious human anterior uveitis in several respects, even in its multiple recurrences.     

Anti-rat ICAM-1 antibody does not influence the course of experimental melanin-induced uveitis

PURPOSE: Experimental melanin-induced uveitis (EMIU) is a T cell-mediated rat model of acute anterior uveitis. We investigated the possibility of preventing the inflammation with monoclonal antibody directed against rat intercellular adhesion molecule 1 (ICAM-1). METHODS: To induce EMIU, Lewis rats were immunized with bovine ocular melanin extract (250-500 microg). Each day from day 6 post-immunization, rats were injected intraperitoneally with anti-ICAM-1 monoclonal antibody (IA29) or normal mouse serum, and examined with a slit-lamp biomicroscope. On the first day of clinical inflammation, intravital microscopy of iris vasculature was performed on each animal following intraperitoneal injection of rhodamine 6G. At the peak of clinical inflammation, rats were killed, and eyes were examined histologically. Binding potency of IA29 was tested by flow cytometry using concanavalin A-stimulated rat T cells. Immunohistochemical staining was used to detect IA29 on rat uveal vascular endothelium. RESULTS: The ability of IA29 to bind T cell blasts was present to a 1:2000 dilution, and IA29 was readily detectable on uveal vascular endothelium following systemic administration. However, there was no significant difference (p > 0.05) in incidence, time of onset, or severity or histological appearance of EMIU for the rats treated with IA29 when compared with the control rats. Intravital microscopy revealed sticking of leukocytes in the iris vasculature in both groups. CONCLUSIONS: We were unable to demonstrate an inhibitory effect of anti-rat ICAM-1 antibody on the outcome of EMIU. Our observations may reflect a redundancy in the adhesion molecule profile responsible for this uveitis.     

Rabbit ocular and pineal autoimmune response to retina antigens

Different forms of experimental autoimmune uveitis (EAU) can be produced by varying protocols to present different autoantigens to several species of experimental animal. We have studied the clinical, histological and serological responses of rabbits to footpad injection of various fractions of retina extract. Rabbits injected with retina extract or S antigen developed posterior uveitis. However, rabbits injected with retina extract, also developed an anterior uveitis and pinealitis not seen in rabbits receiving S antigen. The Serological response of rabbits to retina extract was different than that to purified S antigen. Antisera of rabbits receiving retina extract reacted with rabbit retina and pineal gland as well as with guinea pig retina but not with guinea pig pineal gland. In contrast anti-S antigen sera reacted with rabbit retina and guinea pig retina and pineal gland but not with rabbit pineal gland. Gel filtration chromatography of the ammonium sulfate supernate of retina extract was used to differentiate the antigens with which these two sera reacted. An analysis of these experiments gives preliminary evidence of an autoantigen(s) of rabbit retina and pineal gland that is not S antigen. The existence of multiple autoantigens common to retina and pineal gland in various species is significant in that it further underscores the relationship of these tissues. Furthermore, it is not unrealistic to expect more than one autoantigen of retina or uvea to be involved in autoimmune uveitis.     

A New Procedure for Experimental Autoimmune Uveitis with Small Uveitogenic Peptides

Purpose: Demonstration of experimental autoimmune uveitis (EAU) with extremely small, fragmented peptides (12–30 amino acid residues) of interphotoreceptor retinoid-binding protein (IRPB). Method: Very small fragmented peptides (no. 854, 888, 907, and 1057) were conjugated to heat-killed Group A Streptococcus cells and administered as a single intravenous injection to Lewis rats. A non-uveitogenic peptide 950 was also conjugated to heat-killed Streptococcus and administered. Administration of a mixture of small peptides and Streptococcus was a control for the peptides conjugated with Streptococcus. Results: The uveitogenic peptide/Streptococcus conjugates produced uveitis inflammatory responses in the uvea, retina and pineal gland. Administration of mixtures of small peptides and Streptococcus cells, and a non-uveitogenic peptide 950 conjugated with Streptococcus did not produce autoimmune uveitis. Conclusions: Since mixtures of small uveitogenic peptides and Streptococcal cells did not develop autoimmune uveitis, conjugated Streptococcal cells provided a vehicle for macrophage phagocytosos of very small uveitogenic IRBP peptides. Subsequent antigen presentation from macrophages to lymphocytes developed autoimmune uveitis. Peptide 888, one of four IRBP peptides that encompass the major uveitogenic domain, proved to be the most effective in development of uveitis.     

Production of anti-33kDa protein monoclonal antibody and organ-specificity of 33kDa protein]

We produced monoclonal antibody TS-SC6 specific for 33kDa protein (33K) from bovine retina and studied the localization of 33K in mammalian retinas (rat, mouse, bovine, guinea pig and human) and pineal gland of rat. An immunohistochemical study showed that TS-SC6 reacted with the outer plexiform layer (OPL), outer nuclear layer (ONL) and rod inner segments (IS) in rat, ONL, IS and IS in mouse and guinea pig. It reacted with ONL and rod outer segments (OS) in bovine, ONL and OS in human. Immunoreactivity was seen in the pineal gland, but there was no immunoreactivity in the central nervous system surrounding the pineal gland. Immunoblot analysis was carried out with soluble fractions prepared from the retina, pineal gland, cerebrum, liver, kidney and intestine of rats. TS-SC6 reacted with 33K of the retina and pineal gland, respectively. However, it did not react with soluble fractions from the other tissues.     

Apoptosis is a prominent feature of acute anterior uveitis in the Fischer 344 rat

AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models. METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin. Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide. Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells. Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS). RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points. However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined. Many infiltrating neutrophils expressed iNOS. CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases. In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis.