ASPERGILLUS FUMIGATUS COMPLEMENT INHIBITOR

烟曲霉补体抑制剂

• 批准号: 3454243
• 负责人: RONALD G WASHBURN
• 金额: $ 7.41万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3454243
• 关键词: Aspergillus; Shwartzman phenomenon; antifungal antibody; aspergillosis; autoimmune disorder; binding proteins; biological products; carbohydrate structure; chemical structure function; complement; complement inhibitors; complement pathway; disease /disorder model; electrofocusing; enzyme linked immunosorbent assay; fibrinolysis; fluorescence microscopy; fungal genetics; gel filtration chromatography; guinea pigs; high performance liquid chromatography; host organism interaction; human therapy evaluation; human tissue; immunoelectrophoresis; immunopathology; immunotherapy; laboratory mouse; laboratory rabbit; lung; microorganism immunology; molecular cloning; molecular pathology; mutant; mycotoxins; protein engineering; protein sequence; systemic lupus erythematosus; thrombosis; virulence

A complement inhibitor (CI) produced by Aspergillus fumigatus selectively inhibited the alternative pathway. This was the first observation of a CI produced by a fungus which causes human disease. Aspergillus CI might enhance pathogenicity of Aspergillus in the human host by impairing opsonization and diminishing generation of the chemoattractant for polymorphonuclear neutrophils and monocytes, C5a. CI will be chromatographically purified, and homogeneity of the final material will be judged using SDS-PAGE and isoelectric focusing. Following purification, aminoacid and monosaccharide content will be determined. The mechanism of alternative pathway inhibition will be clarified by adding graded concentrations of purified C3, Factor B, and Factor D to CI in a cryptococcal alternative pathway system. Binding between the relevant complement protein and CI will be assessed using a combination of gel filtration and immunoelectrophoresis. In vivo production of CI will be examined using labeled anti-CI IgG F(ab')2 in the cortisone-treated mouse model of invasive aspergillosis, and in available human tissues. ELISA will be employed to check for CI in the sera of infected mice and of patients with various forms of aspergillosis. A CI- mutant will be selected following ultraviolet irradiation of CI+ wild-type A.fumigatus. This mutant will allow an assessment of the in vivo significance of CI by comparison of LD50s for inhaled inocula of both isolates in the mouse model. The role of CI in inhibition of C3-mediated clot lysis will also be examined using an in vitro test. The potential usefulness of CI for treatment of human diseases in which complement becomes pathologically activated will be explored in mouse models of neutrophil-mediated lung injury and systemic lupus erythematosus, as well as the Shwartzman reaction. If these in vivo treatment and toxicity studies require larger quantities of CI than can be obtained chromatographically, Aspergillus CI DNA will eventually be cloned. This will be approached using the plasmid vector pBR322 first in E. coli K12; if necessary, the yeast Saccharomyces cerevisiae could be employed as an alternative to E. coli for production of this eukaryotic gene product.

烟曲霉产生的补体抑制剂 (CI) 选择性抑制替代途径。 这是第一个 观察一种真菌产生的 CI，它会导致人类 疾病。 曲霉菌 CI 可能增强其致病性 人类宿主中的曲霉通过损害调理作用和 化学引诱剂的产生减少 多形核中性粒细胞和单核细胞，C5a。 CI 将是 经色谱纯化，最终产物均质 将使用 SDS-PAGE 和等电聚焦来判断材料。 纯化后，氨基酸和单糖含量 将被确定。 替代途径的机制 通过添加梯度浓度的 隐球菌中纯化的 C3、因子 B 和因子 D 至 CI 替代途径系统。 相关之间的绑定 补体蛋白和 CI 将使用组合进行评估 凝胶过滤和免疫电泳。 体内产生 CI 将使用标记的抗 CI IgG F(ab')2 进行检查 可的松治疗的侵袭性曲霉病小鼠模型，以及 可用的人体组织。 ELISA 将用于检查 CI 在受感染小鼠和患有各种形式的患者的血清中 曲霉病。 CI-突变体将在紫外线照射后被选择 CI+野生型烟曲霉的照射。 这种突变体将允许 通过比较来评估 CI 的体内显着性 小鼠模型中两种分离株吸入接种物的 LD50。 CI 在抑制 C3 介导的血栓溶解中的作用也将是 使用体外测试进行检查。 CI 在治疗人类疾病方面的潜在用途 哪个补体被病理激活将是 在中性粒细胞介导的肺损伤小鼠模型中进行了探索 系统性红斑狼疮，以及施瓦茨曼 反应。 如果这些体内治疗和毒性研究需要 CI 的量比色谱法获得的量大， 曲霉 CI DNA 最终将被克隆。 这将是 首先在大肠杆菌 K12 中使用质粒载体 pBR322 进行接近； 如果需要，可以使用酿酒酵母 用作大肠杆菌的替代品来生产该产品 真核基因产物。