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严重过敏反应和死亡的最常见食物相关原因，与其他食物过敏不同，通常持续到成年期。迄今为止的研究表明，至少有两个花生的特点，可能有助于其过敏性：耐消化和能力的主要花生过敏原，阿糖胞苷h1促进Th 2反应结合ICAM-1-抓取非整联蛋白的树突状细胞。我们的研究现在证明了可能导致强过敏性的其他作用：水溶性花生提取物（PE）在给予小鼠时会导致休克和炎症细胞因子的产生。具体地说，我们证明：1）补体，更具体地说，C3 a，并在较小程度上，刺激性免疫球蛋白（IG）受体有助于PE诱导的休克：2）PE诱导的休克更多地依赖于巨噬细胞而不是肥大细胞，但伴随着肥大细胞的激活：3）血小板活化因子（PAF），并在较小程度上，组胺有助于PE诱导的休克; 4）TLR 2、TLR 4、MyD 88和适应性免疫系统不是PE诱导的休克所必需的; 5）PE与IgE介导的肥大细胞脱粒协同作用诱导休克; 6）PE通过C3非依赖性机制诱导TNF产生和低水平Th 2细胞因子产生，但不刺激IFN-？生产这些观察结果提出了三个有趣的假设，它们一起为花生如此致敏性提供了一个潜在的解释：1）花生具有主要通过引起补体衍生的过敏毒素的产生来诱导休克的组分，所述过敏毒素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，诱导一些花生过敏性疾病患者经历严重的过敏反应。过敏性患者;和3）由花生组分刺激的炎症反应作为佐剂，促进对主要花生过敏原的Th 2反应的诱导。我们提出了三个目标来验证这些假设，并确定花生中促进过敏性的分子。目标1中的研究将使用针对PE产生的单克隆抗体，通过亲和色谱法、质谱法纯化单个花生大分子以鉴定这些分子，并进行体内研究以测试其诱导休克和炎性细胞因子产生的能力。目标2中的研究将确定PE是否会导致体外小鼠和人血浆中C3 a的生成，并将使用补体因子B和C4缺陷小鼠来鉴定PE激活C3的途径。目标3的研究将：1）免疫小鼠以确定从PE纯化的诱导TNF产生和/或补体激活的蛋白质是否具有佐剂活性; 2）确定TNF产生是否被作为toll样受体配体、氧化剂和/或凝集素的花生蛋白质刺激;和3）使用流式细胞术方法和技术用于体内消耗特定细胞类型以鉴定由PE诱导产生TNF的细胞。RFA-AI-07-032/Finkelman，Fred D.鉴定负责花生致敏性的蛋白质本申请是对食物过敏RFA探索性研究的响应，通过使用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，现在引起过敏的0.8%的美国人，是如此过敏。阐明花生和树坚果的特殊特性，有助于其过敏性可能提供线索，一般食物过敏的发病机制，建议如何预防和治疗这种疾病，并建议如何对花生和树坚果进行遗传修饰，使其过敏性降低。

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.