HUMAN AUTOIMMUNITY TO A CORNEAL STROMAL ANTIGEN

人类对角膜基质抗原的自身免疫

• 批准号: 2415040
• 负责人: John D Gottsch
• 金额: $ 13.37万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415040
• 关键词: antibody specificity; antigen antibody reaction; autoantigens; autoimmunity; cellular pathology; clinical research; complementary DNA; cornea ulcer; corneal stroma; cytotoxic T lymphocyte; enzyme linked immunosorbent assay; epitope mapping; high performance liquid chromatography; human genetic material tag; human subject; immunocytochemistry; immunofluorescence technique; immunopathology; molecular cloning; northern blottings; nucleic acid hybridization; nucleic acid probes; peptide chemical synthesis; phenotype; protein sequence

Mooren's ulcer is a painful peripheral corneal ulceration that can progress relentlessly to blindness. The pathogenesis of this disease remains unknown and the diagnosis is one of exclusion. An autoimmune etiology has been suggested involving a cornea-specific antigen (CO-Ag) that incites both a humoral and cell-mediated immune response. Understanding the auto immune etiology of the disease could result in earlier recognition of the disease and be of immense value in the development of specific treatment regimens. Aim 1: To map pathogenic epitopes within the CO-Ag protein. The amino acid sequence of CO-Ag has been determined. Overlapping peptides corresponding to the entire CO-Ag protein will be synthesized. Each peptide fragment will be tested for reactivity against serum antibody from Mooren's ulcer patients and patients with other corneal peripheral ulcerative diseases. Identification of a disease-specific epitope will lead to the development of a diagnostic test to differentiate Mooren's ulcer from other peripheral corneal diseases. Aim 2: To test the hypothesis that an immune response mounted by Mooren's ulcer patient against a particular determinant of an infectious agent may cross-react with a sequence of a disease-specific epitope of CO- Ag leading to corneal tissue injury and melting disease by the mechanism of molecular mimicry. Aim 3: To isolate and characterize a human cDNA clone with coding sequences for CO-Ag autoantigen. Synthetic oligonucleotide probes will be created on the basis of the known amino acid sequence of CO-Ag. These probes will be used to screen a human corneal fibroblasts cDNA library constructed in the expression vector UniZapXR. The CO-Ag cDNA clone will be used a hybridization probe to determine the level of COAg mRNA expression in different tissues on Northern blot analysis. Aim 4: To analyze the phenotype of the infiltrating cells, and to-use anti-granzyme antibody for detection of activated cytotoxic T-cells in patient's corneal tissues by immunocytochemical staining in attempt to understand the role of cytotoxic T-cells in the pathogenesis of Mooren's ulcer.

蚕蚀性溃疡是一种疼痛的周边角膜溃疡，可导致 不断地进步直至失明。本病的发病机制 仍然未知，诊断是一种排除性诊断。自身免疫性 病因学已被认为涉及角膜特异性抗原（CO-Ag） 激发体液和细胞介导的免疫反应。 了解该疾病的自身免疫病因可能会导致 及早认识该疾病，对于疾病的治疗具有巨大的价值 制定具体的治疗方案。目标 1：绘制致病图谱 CO-Ag 蛋白内的表位。 CO-Ag的氨基酸序列有 已确定。对应于整个 CO-Ag 的重叠肽 将会合成蛋白质。每个肽片段都将被测试 对蚕蚀性溃疡患者血清抗体的反应性 患有其他角膜周边溃疡性疾病的患者。鉴别 疾病特异性表位的研究将导致诊断方法的开发 区分蚕蚀性溃疡和其他周边角膜的测试 疾病。目标 2：检验以下假设：免疫反应是由 蚕蚀性溃疡患者对抗传染病的特定决定因素 试剂可能与CO-的疾病特异性表位序列发生交叉反应 Ag导致角膜组织损伤和融化性疾病的机制 分子拟态。目标 3：分离并表征人类 cDNA 具有 CO-Ag 自身抗原编码序列的克隆。合成的 寡核苷酸探针将在已知的氨基的基础上创建 CO-Ag 的酸序列。这些探针将用于筛查人类 表达载体构建的角膜成纤维细胞cDNA文库 UniZapXR。 CO-Ag cDNA 克隆将使用杂交探针来 测定不同组织中 COAg mRNA 的表达水平 Northern印迹分析。 目标4：分析表型 浸润细胞，并使用抗颗粒酶抗体检测 激活患者角膜组织中的细胞毒性 T 细胞 免疫细胞化学染色试图了解细胞毒性的作用 T 细胞在蚕蚀性溃疡发病机制中的作用。