STUDY OF GAMMA GLOBULIN STRUCTURE AND HYPERSENSITIVITY

(改编自申请人的摘要)：现已确定IgE 分子以高亲和力与肥大细胞上的Fc Delta受体结合 细胞结合的IgE抗体通过抗原交联会引发 用于释放导致过敏性疾病的介质的细胞。这个 这项研究的目的是阐明免疫学和免疫学 依赖IgE的介质释放的生化机制。以前的工作 表明细胞结合的IgE抗体分子通过 多价配体诱导磷脂酶C(PLC)活化 肌醇磷脂的水解，这一过程可能起到 在介体释放的触发信号的转导中起主要作用。 在本建议书中，i)申请人须研究- 核苷酸结合蛋白(S)参与IgE介导的激活 PLC和组胺释放。由于调查人员已经确定了 大鼠肥大细胞和骨髓来源的小鼠肥大细胞的通透性方法 细胞(BMMC)，研究人员应引入非水解性GTP 类似物，如GTP伽马或GDPbetaS，进入渗透的细胞和 确定GTP类似物对抗原诱导的蛋白水解物的影响 肌醇磷脂及其介体释放。二)调查员的 最近的实验表明，一种新合成的抑制剂 磷脂酶A2抑制抗原诱导的组胺释放 和花生四烯酸从致敏的BMMC释放而不影响 肌醇磷酸盐的形成。调查人员应确认 该抑制剂不影响抗原诱导的PLC的激活，并且 确定该抑制物是否会影响与 磷脂酰肌醇周转率。调查人员预计， 实验将确定PLA2在IgE依赖中的可能作用 释放调解人。三)调查人员应努力建立文化 人嗜碱细胞选择性分化生长条件的研究 脐血细胞和骨骼中的粒细胞和肥大细胞 骨髓细胞。人T细胞或非T细胞来源的重组白介素系 将会被雇佣。成纤维细胞单层也将用于 肥大细胞的分化。四)调查人员还应继续 他们与分子生物学家合作确定了结合 人IgE分子上的Fc Delta受体与嗜碱性粒细胞上的Fc Delta受体结合。 代表小片段的重组多肽和合成多肽 将测试Delta Chain的FC部分的数据块能力 人嗜碱性粒细胞对IgE抗体的被动增敏 阻断125I标记的IgE与Fc Delta受体结合的能力 培养的人类嗜碱性粒细胞。

(Adapted from applicant's abstract): It has been established that IgE molecules bind to Fc delta receptors on mast cells with high affinity, and that cross linking of the cell bound IgE antibodies by antigen trigger the cells for the release of mediators which cause allergic diseases. The purpose of this research project is to elucidate the immunological and biochemical mechanisms of IgE-dependent mediator release. Previous work indicated that cross-linking of cell-bound IgE antibody molecules by multivalent ligand induced activation of phospholipase C (PLC) for hydrolysis of inositol phospholipids, and that this process may play a major role in the transduction of triggering signals for mediator release. In this proposal, i) the applicant shall study whether guanidine- nucleotide binding protein(s) is involved in the IgE-mediated activation of PLC and histamine release. As the investigators have established the method to permeabilize rat mast cells and bone marrow-derived mouse mast cells (BMMC), the investigators shall introduce non-hydrolyzable GTP analogue, such as GTP gammaS or GDPbetaS, into the permeabilized cells and determine the effect of the GTP analogue, on antigen-induced hydrolysis of inositol phospholipids and mediator release. ii) The investigator's recent experiment indicated that a newly synthesized inhibitor of phospholipase A2 (PLA2) inhibited the antigen-induced histamine release and arachidonic acid release from sensitized BMMC without affecting the formation of inositol phosphates. The investigators shall confirm that the inhibitor does not affect the antigen-induced activation of PLC, and determine whether the inhibitor may affect other enzymes involved in phosphatidylinositol turnover. The investigators expect that the experiments will establish a possible role of PLA2 in IgE-dependent mediator release. iii) The investigators shall try to establish culture conditions for selective differentiation and growth of human basophil granulocytes and mast cells from umbilical cord blood cells and bone marrow cells. Recombinant interleukins from human T-cells or non-T-cells will be employed. Fibroblast monolayers will also be employed for the differentiation of mast cells. iv) The investigators shall also continue their collaboration with molecular biologists to determine the binding site on human IgE molecules to Fc delta receptors on basophils. Recombinant peptides and synthetic peptides representing small segments of the Fc portion of delta chain will be tested for the ability to block passive sensitization of human basophils with IgE antibodies, and for the ability to block the binding of 125 I-labeled IgE to Fc delta receptors on cultured human basophils.