Identification Of Proteins Responsible For Peanut Allergenicity

描述（由申请人提供）：对花生和树坚果过敏是最常见的与食物相关的严重过敏反应和死亡原因，与其他食物过敏不同，通常会持续到成年。迄今为止的研究表明，至少有两个花生特性可能有助于其致敏性：抗消化和花生主要过敏原Ara h1通过与树突状细胞上的icam -1抓取非整合素结合来促进Th2反应的能力。我们的研究现在证明了可能导致强致敏性的其他影响：水溶性花生提取物（PE）在给小鼠施用时引起休克和炎症细胞因子的产生。具体来说，我们证明：1)补体，更具体地说，C3a，以及在较小程度上，刺激性免疫球蛋白（Ig）受体有助于pe诱导的休克；2) pe诱导休克对巨噬细胞的依赖性大于对肥大细胞的依赖性，但伴随肥大细胞活化；3)血小板活化因子（PAF）和组胺在较小程度上参与pe诱导的休克；4) pe致休克不需要TLR2、TLR4、MyD88和适应性免疫系统；5) PE与ige介导的肥大细胞脱颗粒作用协同诱导休克；6) PE通过不依赖c3的机制诱导TNF的产生和低水平Th2细胞因子的产生，但不刺激IFN-？生产。这些观察结果提出了三个有趣的假设，它们共同为花生如此易致敏提供了一个潜在的解释：1)花生含有的成分主要通过引起补体衍生的过敏毒素的产生而诱发休克，这种过敏毒素会诱导巨噬细胞和肥大细胞产生PAF和组胺；2) C3相关的类过敏反应可能与花生诱导的ige介导的肥大细胞脱颗粒协同作用，导致部分花生过敏患者出现严重的过敏反应；3)花生成分刺激的炎症反应作为佐剂，促进对主要花生过敏原的Th2反应的诱导。我们提出三个目标来验证这些假设，并确定花生中促进过敏原的分子或分子。Aim 1的研究将使用针对PE产生的单克隆抗体纯化单个花生大分子，通过亲和色谱，质谱来鉴定这些分子，并在体内研究以测试其诱导休克和炎症细胞因子产生的能力。Aim 2的研究将确定PE是否导致体外小鼠和人血浆中C3a的生成，并将使用补体因子B-和c4缺陷小鼠来确定PE激活C3的途径。Aim 3的研究将：1)免疫小鼠，以确定从PE中纯化的诱导TNF产生和/或补体活化的蛋白是否具有佐剂活性；2)确定花生蛋白（toll样受体配体、氧化剂和/或凝集素）是否刺激TNF的产生；3)使用流式细胞术方法和技术在体内消耗特定的细胞类型，以鉴定由PE诱导产生TNF的细胞。本应用响应了食物过敏的探索性研究，通过离体研究和体内小鼠模型研究来研究食物过敏的发病机制。具体来说，我们正在研究为什么花生会引起过敏，现在有0.8%的美国人会引起过敏。阐明花生和树坚果致敏性的特殊特性可能为了解食物过敏的一般发病机制提供线索，为预防和治疗这种疾病提供方法，并为花生和树坚果的基因改造提供方法，使其降低致敏性。

DESCRIPTION (provided by applicant): Allergy to peanuts and tree nuts is the most common food-related cause of severe anaphylaxis and death and, unlike other food allergies, typically persists into adulthood. Studies to date demonstrate at least two peanut characteristics that may contribute to its allergenicity: resistance to digestion and the ability of the major peanut allergen, Ara h1 to promote a Th2 response by binding to ICAM-1-grabbing non-integrin on dendritic cells. Our studies now demonstrate additional effects that are likely to contribute to strong allergenicity: water-soluble peanut extract (PE) causes shock and inflammatory cytokine production when administered to mice. Specifically, we demonstrate that: 1) complement, more specifically, C3a, and, to a lesser extent, stimulatory immunoglobulin (Ig) receptors contribute to PE-induced shock; 2) PE-induced shock depends more on macrophages than on mast cells but is accompanied by mast cell activation; 3) platelet activating factor (PAF) and, to a lesser extent, histamine contribute to PE-induced shock; 4) TLR2, TLR4, MyD88, and the adaptive immune system are not required for PE-induced shock; 5) PE acts synergistically with IgE-mediated mast cell degranulation to induce shock; and 6) PE acts through a C3-independent mechanism to induce TNF production and low level Th2 cytokine production but does not stimulate IFN-? production. These observations suggest three intriguing hypotheses that, together, offer a potential explanation for why peanuts are so allergenic: 1) peanuts have components that induce shock primarily by causing the production of complement- derived anaphylatoxins that induce macrophages and mast cells to produce PAF and histamine; 2) the C3- related anaphylactoid response may act synergistically with peanut-induced IgE-mediated mast cell degranulation to induce the severe anaphylaxis experienced by some peanut-allergic patients; and 3) the inflammatory response stimulated by peanut components acts as an adjuvant that promotes the induction of a Th2 response to the major peanut allergens. We propose three aims to test these hypotheses and identify the molecule or molecules in peanuts that promote allergenicity. Studies in Aim 1 will use monoclonal antibodies generated against PE are to purify individual peanut macromolecules by affinity chromatography, mass spectroscopy to identify these molecules and in vivo studies to test their ability to induce shock and inflammatory cytokine production. Studies in Aim 2 will determine whether PE causes the generation of C3a in mouse and human plasma in vitro and will use complement factor B- and C4-deficient mice to identify the pathway by which PE activates C3. Studies in Aim 3 will: 1) immunize mice to determine whether proteins purified from PE that induce TNF production and/or complement activation have adjuvant activity; 2) determine whether TNF production is stimulated by peanut proteins that are toll-like receptor ligands, oxidants, and/or lectins; and 3) use flow cytometric approaches and techniques for depleting specific cell types in vivo to identify the cells that are induced by PE to produce TNF. RFA-AI-07-032/Finkelman, Fred D. Identification of Proteins Responsible For Peanut Allergenicity This application is responsive to the Exploratory Investigations in Food Allergy RFA by using ex vivo studies and in vivo mouse model studies to investigate the pathogenesis of food allergy. Specifically, we are studying why peanuts, which now cause allergy in 0.8% of Americans, are so allergenic. Elucidation of the special characteristics of peanuts and tree nuts that contribute to their allergenicity may provide clues to food allergy pathogenesis in general, suggest ways to prevent and treat this disorder, and suggest ways to genetically modify peanuts and tree nuts to make them less allergenic.