Plasma Serum Based Biomarkers in Sublingual Oral Immunotherapy for Milk Allergy

描述（由申请人提供）：牛奶过敏（CMA）是幼儿最常见的食物过敏，现在可能持续到青春期和成年期。严格避免牛奶仍然是这种过敏症的唯一治疗方法，但意外反应通常很严重，非常常见，通常是由于加工食品中隐藏的食物过敏原和食物的交叉污染。我们机构和其他地方最近的研究已经确定口服和舌下变应原免疫疗法（OIT和SLIT）是治疗CMA的有希望的方法。研究表明，大多数IgE介导的CMA患儿可以安全有效地对牛奶蛋白脱敏。然而，这种治疗似乎起作用的具体机制仍然未知，并且仍然存在问题，即治疗后对牛奶的耐受性增加是否是永久性的，或者更确切地说，代表一种短暂的脱敏，如果牛奶从饮食中停止，可能会使患者面临未来反应的重大风险。我们的长期目标是开发体外测试，帮助预测食物不良反应的风险以及OIT/SLIT的安全性/临床有效性。开发常规测试以进行此类预测的最佳机会最好是通过血清/血浆提供的多功能性来实现。我们的新方法，使用培养衍生的嗜碱性粒细胞（CDBA）从成人干细胞生长，应有助于血浆标记物的鉴定。因此，该提案包括2个目的：目的1涉及通过比较被动致敏CDBA的前后标本，分析接受SLIT和OIT的CMA儿童的血浆的“阻断活性”，以用于随后的牛奶过敏原激发。使用CDBA的优点是它们从未接触过免疫球蛋白（IgE/IgG），否则可能会混淆检测阻断IgG 4抗体的能力。类似的实验将测试OIT/SLIT前和后血浆样本对浆细胞样树突细胞（pDC）的影响，预测OIT/SLIT后血浆（具有更高的IgG 4水平）将增强先天免疫应答（TLR 9），其在过敏个体中似乎受损。目的2探索现有的假设，即循环过敏原-IgE复合物存在于CMA受试者的血浆中，并且这些复合物可以通过诱导CDBA和/或正常嗜碱性粒细胞中的表型/功能应答来检测。将在基于流式细胞仪的试验中检测CMA受试者的血浆标本诱导嗜碱性粒细胞自发组胺释放（SHR）的能力、与IgE依赖性活化相关的标志物（CD 63/CD 203 c）的表达以及随着延长的Fc 5 RI交联而下调的标志物（syk激酶）的表达。这些试验可能导致新的基于血浆的测试，用于预测是否有不良反应的风险，并预测成功的OIT/SLIT期间的临床疗效。食物特异性免疫疗法可能是食物过敏研究史上最令人兴奋的领域，该项目的临床和机制终点应该对食物过敏患者的护理产生直接影响。 公共卫生相关性：对牛奶过敏是幼儿最常见的食物过敏，这种过敏可能会持续到青春期和成年期。需要更好的测试来帮助预测不良反应和/或监测新治疗的安全性和有效性。我们相信，我们可以通过研究接受一种新疗法的牛奶过敏儿童的血浆（血液的液体部分）中的独特元素来开发此类测试。

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. PUBLIC HEALTH RELEVANCE: Allergy to cow's milk is the most common food allergy in young children, and this allergy is likely to persist into adolescence and adulthood. Better tests are needed in helping to predict adverse reactions and/or monitor the safety and effectiveness of new treatments. We believe we can develop such tests by investigating unique elements within the plasma (liquid part of blood) from milk allergic children undergoing a new kind of therapy.