Plasma Serum Based Biomarkers in Sublingual Oral Immunotherapy for Milk Allergy

描述(申请人提供)：牛奶过敏(CMA)是幼儿中最常见的食物过敏，现在可能会持续到青春期和成年期。严格避免牛奶仍然是治疗这种过敏的唯一方法，但通常严重的意外反应非常常见，通常是由于加工食品中隐藏的食物过敏原和食品的交叉污染造成的。最近来自我们机构和其他地方的研究已经确定口服和舌下变应原免疫疗法(OIT和SIT)是治疗CMA的有希望的方法。研究表明，大多数IgE介导的CMA儿童可以安全有效地对牛奶蛋白脱敏。然而，这种治疗的具体作用机制仍不清楚，问题仍然是，治疗后对牛奶的耐受性增加是永久性的，还是更确切地说，是暂时的脱敏，如果停止饮食，患者可能会面临未来反应的巨大风险。我们的长期目标是开发体外测试，帮助预测食品不良反应的风险以及OIT/SIT的安全性/临床疗效。开发一种常规检测来进行此类预测的最佳机会是通过血清/血浆提供的多功能性来实现的。我们使用成人干细胞培养的嗜碱性粒细胞(CDBA)的新方法应该有助于血浆标志物的鉴定。因此，该提案包括两个目标：目标1包括通过比较被动致敏CDBA的前后标本，分析接受SIT和OIT的CMA儿童的血浆“阻断活性”，以应对随后的牛奶过敏原的挑战。使用CDBA的好处是他们从未接触过免疫球蛋白(IgE/IgG)，否则可能会扰乱检测封闭的IgG4抗体的能力。类似的实验将测试卵巢/裂隙前后血浆标本对浆细胞样树突状细胞(PDCs)的影响，预测卵巢/裂隙后血浆(具有更高的IgG4水平)将增强似乎在过敏个体中受损的先天性免疫反应(TLR9)。目的2探索现有的假设，即循环变应原-IgE复合体存在于CMA受试者的血浆中，并可通过诱导CDBA和/或正常嗜碱性粒细胞的表型/功能反应来检测。来自CMA受试者的血浆样本将在基于流动的分析中被检测其诱导嗜碱性粒细胞自发组胺释放(SHR)的能力、与IgE依赖的激活相关的标志物(CD63/CD203c)的表达以及那些因Fc5RI长时间的交联而下调的标志物(SYK激酶)的表达。这些分析可能导致新的基于血浆的测试，有助于预测一个人是否有不良反应的风险，并预测成功的OIT/SIT的临床疗效。由于食物特异性免疫疗法可能是食物过敏研究历史上最令人兴奋的领域，这个具有临床和机械终点的项目应该会对食物过敏患者的护理产生立竿见影的影响。

DESCRIPTION (provided by applicant): Cow's milk allergy (CMA) is the most common food allergy in young children and is now likely to persist into adolescence and adulthood. Strict milk avoidance remains the only treatment for this allergy but accidental reactions, which are often severe, are extremely common, frequently due to the hidden presence of food allergens in processed foods and cross-contamination of foods. Recent studies from our institution and elsewhere have identified oral and sublingual allergen immunotherapy (OIT and SLIT) as promising approaches for treatment of CMA. Studies demonstrate that most children with IgE-mediated CMA can be desensitized to cow's milk protein, both safely and efficaciously. However, the specific mechanisms by which this treatment appears to work remain unknown, and questions remain to whether this increased tolerance to milk after treatment is permanent, or rather represents a transient desensitization which could place patients at significant risk of future reactions if milk was ever discontinued from their diet. Our long-range goal is to develop in vitro tests that help predict risks to adverse reactions to food as well as the safety/clinical efficacy of OIT/SLIT. The best chance of developing a routine test to make such predictions is best achieved with the versatility that serum/plasma provides. Our novel approach of using culture-derived basophils (CDBA) grown from adult stem cells should facilitate identification of plasma markers. Therefore, the proposal consists of 2 aims: Aim 1 involves analyzing plasma from CMA children undergoing SLIT and OIT for "blocking activity" by comparing pre and post specimens in passively sensitizing CDBA for subsequent challenge with milk allergen. The advantage of using CDBA is that they have never been exposed to immunoglobulins (IgE/IgG), which may otherwise confound the ability to detect blocking IgG4 antibody. Similar experiments will test pre- and post- OIT/SLIT plasma specimens for effects on plasmacytoid dendritic cells (pDCs), predicting that post-OIT/SLIT plasma (with greater IgG4 levels) will augment innate immune responses (TLR9) that are seemingly impaired in allergic individuals. Aim 2 explores the existing hypothesis that circulating allergen-IgE complexes are in plasma of CMA subjects and that these can be detected by inducing phenotypic/functional responses in CDBA and/or normal basophil. Plasma specimens from CMA subjects will be tested in flow-based assays for their ability to induce basophil spontaneous histamine release (SHR), the expression of markers (CD63/CD203c) linked to IgE-dependent activation as well as those (syk kinase) that are down regulated with prolonged Fc5RI cross-linking. These assays could result in new plasma-based tests useful in predicting whether one is at risk for adverse reactions and for predicting the clinical efficacy during successful OIT/SLIT. With food specific immunotherapy being potentially the most exciting area in the history of food allergy research, this project, with clinical and mechanistic endpoints should have immediate impact on the care of patients with food allergy.