Plasma Serum Based Biomarkers in Sublingual Oral Immunotherapy for Milk Allergy

描述（由申请人提供）：牛奶过敏（CMA）是幼儿中最常见的食物过敏，现在很可能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。我们机构和其他机构最近的研究已经确定口服和舌下过敏原免疫疗法（OIT和SLIT）是治疗CMA的有希望的方法。研究表明，大多数患有ige介导的CMA的儿童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尚不清楚，并且问题仍然存在，即治疗后对牛奶的耐受性增加是永久性的，还是更确切地说代表一种短暂的脱敏，这种脱敏可能会使患者在未来的重大风险中产生反应，如果牛奶从他们的饮食中停止。我们的长期目标是开发体外试验，以帮助预测食品不良反应的风险以及OIT/SLIT的安全性/临床疗效。利用血清/血浆提供的多功能性，开发常规测试来进行此类预测的最佳机会是最好的。我们使用从成体干细胞中培养的嗜碱性粒细胞（CDBA）的新方法应该有助于鉴定血浆标志物。因此，该提案包括两个目标：目标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“阻断活性”，以应对随后的牛奶过敏原挑战。使用CDBA的优点是它们从未暴露于免疫球蛋白（IgE/IgG），否则可能会混淆检测阻断IgG4抗体的能力。类似的实验将测试OIT/SLIT前后血浆标本对浆细胞样树突状细胞（pDCs）的影响，预测OIT/SLIT后血浆（具有更高的IgG4水平）将增强过敏个体似乎受损的先天免疫反应（TLR9）。目的2探讨了现有的假设，即循环过敏原- ige复合物存在于CMA受试者的血浆中，这些复合物可以通过诱导CDBA和/或正常嗜碱性粒细胞的表型/功能反应来检测。来自CMA受试者的血浆标本将通过基于流动的分析来测试其诱导嗜碱性粒细胞自发组胺释放（SHR）的能力，与ige依赖性激活相关的标记物（CD63/CD203c）的表达以及那些随着Fc5RI交联时间延长而下调的标记物（syk激酶）的表达。这些试验可能产生新的基于血浆的试验，可用于预测患者是否有不良反应的风险，以及预测成功的OIT/SLIT期间的临床疗效。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，该项目具有临床和机制终点，应该对食物过敏患者的护理产生直接影响。

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.