Molecular basis for anaphylaxis due to galactose-alpha-1,3-galactose (alpha-gal)

项目摘要 IgE分子位于过敏性疾病发病机制的中心。在致敏个体中， 致敏变应原导致IgE接合，引起Fcε受体交联和肥大细胞活化。 细胞和嗜碱性粒细胞。这触发了介质释放到局部组织中，导致大量的 与过敏性疾病相关的症状，包括过敏性休克。人类IgE分子的研究， 和它的目标过敏原，已经非常有限。我们对这一过程的几乎所有知识都来自于 使用过敏患者血清的研究，该血清含有许多抗体的混合物，具有许多特异性， 针对许多不同的表位，并具有许多不同的亲和力;因此，分子的研究， IgE与目标过敏原的相互作用存在很大缺陷。研究这个过程的理想方法是自然地使用- 从过敏受试者中分离的人IgE单克隆抗体（mAb）。不幸的是，由于许多 不可逾越的内在技术障碍，以前从未制造过这样的抗体。我们建立 一种通过从人源性免疫球蛋白B细胞制备人杂交瘤来生长、鉴定和永生化编码IgE的B细胞的方法， 过敏个体的外周血。在这个提议中，我们开发了第一组自然发生的α- 来自红肉过敏受试者的gal特异性人IgE mAb。我们打算精确定义 从过敏原和抗体的角度来看，IgE介导的红肉反应的基础。我们将开始 通过从少数高度表征的研究对象的外周血B细胞产生人杂交瘤。 纯化的IgE单克隆抗体将用于精确定义人抗α-gal IgE的关键分子细节 约束力我们将在各种分子测定中使用这组抗体，以帮助定位，鉴定， 并最终表征在蜱中发现的导致人类致敏的过敏原。这些IgE mAb代表被诱导进行类别转换重组的B细胞，我们将使用它们的可变 基因序列，以询问来自研究受试者的Illumina测序数据集， 获得了这将使我们能够敏锐地了解这种过敏性疾病背后的B细胞群 并且可以提供预测个体的致敏风险所需的见解。我们一起开始 确定人IgE B细胞应答的确切糖蛋白靶点和B细胞来源的研究 这将提供非常需要的细节基本的敏感性和发展， 对这种常见的低聚糖过敏。

PROJECT SUMMARY The IgE molecule lies at the center of the pathogenesis of allergic diseases. In sensitized individuals, re-exposure to the offending allergen results in IgE engagement, causing Fcε receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, resulting in the vast array of symptoms associated with allergic diseases, including anaphylactic shock. Studies of the human IgE molecule, and its targeted allergens, have been very limited. Nearly all of our knowledge of this process has come from studies using allergic patient serum, which contains a mixture of many antibodies, with many specificities, directed toward many different epitopes, and having many different affinities; thus studies of the molecular interactions of IgE with target allergens are greatly flawed. The ideal way to study this process is to use naturally- occurring human IgE monoclonal antibodies (mAbs), isolated from allergic subjects. Unfortunately, due to many impassible intrinsic technical hurdles no such antibodies have previously ever been made. We have established a method to grow, identify and immortalize IgE encoding B cells by making human hybridomas from the peripheral blood of allergic individuals. In this proposal, we develop the first panel of naturally-occurring alpha- gal-specific human IgE mAbs from subjects with red meat allergy. We intend to define the precise molecular basis for IgE-mediated reactions to red meat, from both the allergen and the antibody perspective. We will begin by generating human hybridomas from peripheral blood B cells of a few highly characterized research subjects. Purified IgE mAbs then will be used to precisely define critical molecular details of human anti-alpha-gal IgE binding. We will employ this panel of antibodies in various molecular assays to assist in localizing, identifying, and ultimately characterizing the allergen(s) found in ticks, responsible for sensitization of humans. As these IgE mAbs represent the B cells which are induced to undergo class-switch recombination, we will use their variable gene sequences to interrogate Illumina sequencing datasets from the research subjects from which the mAbs were obtained. This will allow for a keen understanding of the B cell population(s) underlying this allergic disease and may provide insights needed to predict individuals’ risk of sensitization. Together, this proposal will begin studies to define the exact glycoprotein targets of the human IgE B cell response, and the origin of the B cells themselves, which will provide much needed details underlying the sensitization and development of hypersensitivity to this common oligosaccharide.