RELATION OF GAMMA GLOBULIN STRUCTURE TO HYPERSENSITIVITY

丙种球蛋白结构与超敏反应的关系

• 批准号: 3124626
• 负责人: KIMISHIGE ISHIZAKA
• 金额: $ 26.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1974
• 资助国家: 美国
• 起止时间: 1974-09-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124626
• 关键词: allergens; antibody receptor; antigen antibody reaction; complement fixation tests; density gradient ultracentrifugation; disulfide bond; electron microscopy; enzyme structure; histamine release; human tissue; hypersensitivity; immunochemistry; immunoglobulin E; laboratory mouse; laboratory rabbit; laboratory rat; mast cell; tissue /cell culture

The purpose of this research project is to elucidate the biochemical mechanisms of IgE-dependent mediator release from mast cells, which cause allergic diseases. Our efforts will be focused to analyze biochemical events which are caused by the bridging of IgE-receptors on mast cells. Recently, we established a culture system to obtain human basophilis in the culture of the mononuclear cells in umbilical cord blood. Cultured basophils could be sensitized with human IgE for anti-IgE-induced histamine release. Thus, i) we shall identify growth factor(s) for human basophils and characterize the cultured human basophils. ii) Experiments will be carried out to determine that membrane-associated enzymes, such as serine protease and methyltransferase, are involved in the activation of cultured basophils, and that this process will lead to Ca2+ uptake and mediator release. iii) The affinity of human IgE and rodent IgE for IgE-receptors on cultured human basophilis will be determined, and iv) IgE-receptors on the cells will be identified by SDS gel electrophoresis. v) We shall determine the substrate specificity of membrane-associated serine protease, which is activated upon bridging of IgE receptors. vi) Possible relationship between the proteolytic enzyme and Alpha or Beta subunits of IgE-receptors will be determined. vii) The possibility is considered that the activation of the protease results in the formation of a kinin-like substance, which in turn activates the other membrane-associated enzymes for mediator release. We shall determine whether kallikrein-like enzymes and/or bradykinin may induce the activation of methyltransferases and adenylate cyclase in rodent mast cells and human basophils. viii) The possible role of phospholipid methylation in the enhancement of turnover of phosphatidylinositol will be investigated by using rodent mast cells and human cultured basophils. ix) We shall analyze intracellular Ca2+ movement and Ca++ influx into mast cells (basophils) induced by bridging of IgE-receptors. A fluorescent quinoline Ca++ indicator, Quin 2, will be employed for the anlysis. x) Finally, biochemical mechanisms of desensitization will be investigated in mouse mast cells. We shall test the hypothesis that desensitization is due to activation of membrane-associated enzymes, and that different enzymes are involved in the activation of mast cells for mediator release and desensitization. Similar experiments on desensitization will be carried out with cultured human basophils in future years.

该研究项目的目的是阐明生物化学 肥大细胞 IgE 依赖性介质释放机制，导致 过敏性疾病。 我们的努力将集中于分析生化 由肥大细胞上 IgE 受体桥接引起的事件。 最近，我们建立了一个培养系统来获得人类嗜碱性粒细胞 脐带血中单核细胞的培养。 有文化的 嗜碱性粒细胞可被人类 IgE 敏化，对抗 IgE 诱导的组胺 发布。 因此，i) 我们将确定人类嗜碱性粒细胞的生长因子 并对培养的人类嗜碱性粒细胞进行表征。 ii) 实验将是 进行以确定膜相关酶，例如丝氨酸 蛋白酶和甲基转移酶，参与培养物的激活 嗜碱性粒细胞，这个过程将导致 Ca2+ 的吸收和介体 发布。 iii) 人类 IgE 和啮齿动物 IgE 对 IgE 受体的亲和力 将测定培养的人类嗜碱性粒细胞，以及 iv) IgE 受体 细胞将通过 SDS 凝胶电泳进行鉴定。 v) 我们将 确定膜相关丝氨酸蛋白酶的底物特异性， 它在 IgE 受体桥接时被激活。 六）可能 蛋白水解酶与α或β亚基之间的关系 IgE 受体将被确定。 vii) 认为有可能 蛋白酶的激活导致激肽样物质的形成 物质，进而激活其他膜相关酶 用于调解员释放。 我们将确定激肽释放酶样酶是否 和/或缓激肽可能诱导甲基转移酶的激活和 啮齿动物肥大细胞和人类嗜碱性粒细胞中的腺苷酸环化酶。 八） 磷脂甲基化在增强细胞周转中的可能作用 将使用啮齿动物肥大细胞研究磷脂酰肌醇 人类培养的嗜碱性粒细胞。 ix) 我们将分析细胞内 Ca2+ 运动 和 Ca++ 通过桥接诱导流入肥大细胞（嗜碱性粒细胞） IgE 受体。 荧光喹啉 Ca++ 指示剂 Quin 2 将 用于分析。 x) 最后，生化机制 将在小鼠肥大细胞中研究脱敏作用。 我们将测试 假设脱敏是由于激活 膜相关酶，并且不同的酶参与 激活肥大细胞以释放介质和脱敏。 相似的 将在培养人身上进行脱敏实验 未来几年的嗜碱性粒细胞。