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

项目总结 IgE分子在过敏性疾病的发病机制中处于中心地位。在致敏个体中，再次暴露于 导致免疫球蛋白E结合，导致Fcε受体交联化和激活肥大 细胞和嗜碱性粒细胞。这触发了介质向局部组织的释放，导致了大量的 与过敏疾病有关的症状，包括过敏性休克。人类免疫球蛋白E分子的研究 和它的靶向过敏原，一直非常有限。我们几乎所有关于这一过程的知识都来自于 研究使用了过敏性患者血清，这种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；因此对分子的研究 IgE与靶变应原的相互作用存在很大缺陷。研究这一过程的理想方法是自然地使用- 从过敏性受试者身上分离出的人类免疫球蛋白E单抗。不幸的是，由于许多 无法克服的内在技术障碍以前从未制造过这样的抗体。我们已经确立了 人B细胞杂交瘤细胞培养、鉴定及永生化的方法 过敏者的外周血液。在这个提议中，我们开发了第一个自然产生的阿尔法- 红肉过敏受试者的GAL特异性人IgE单抗。我们打算定义精确的分子 从过敏原和抗体的角度来看，对红肉的IgE介导反应的基础。我们将开始 通过从几个高度特征化的研究对象的外周血液B细胞中产生人类杂交瘤。 然后，纯化的IgE单抗将被用来精确定义人抗α-Gal IgE的关键分子细节 有约束力的。我们将在各种分子检测中使用这组抗体来帮助定位、鉴定、 并最终确定了扁虱中发现的过敏原(S)的特征，这种过敏原负责使人类致敏。因为这些IgE 单抗代表被诱导进行类转换重组的B细胞，我们将使用它们的变量 用于询问来自mAbs研究对象的Illumina测序数据集的基因序列 得到了。这将使人们对这种过敏性疾病背后的B细胞群(S)有一个敏锐的了解 并可能提供预测个体敏感化风险所需的见解。总之，这项提案将开始 确定人类IgE B细胞反应的确切糖蛋白靶点以及B细胞的来源的研究 本身，这将提供敏感认识和发展基础上亟需的细节 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.