Development and validation of a chitin assay for environmental samples

描述(申请人提供)：哮喘是一种受遗传和环境影响的复杂疾病，但发病机制仍不清楚。最近在人类中发现的几丁质酶和几丁质酶样蛋白(CLP)被发现在炎症和哮喘中都发挥着重要作用。甲壳素是这些功能性和非功能性酶的配体，是一种病原体相关的分子模式，可诱导天然免疫途径，诱导Th2型嗜酸性炎症，并可作为佐剂对旁观者蛋白致敏。甲壳素是一种1-4-N-乙酰-D-氨基葡萄糖的不溶于水的长链聚合物，是常见室内生物的结构成分，如尘螨、蟑螂和真菌，这些都是已知的哮喘诱因。甲壳素在这些生物体丰富的环境中很常见。在几丁质酶基因(CHIT1)具有特定多态的个体中，暴露于真菌水平升高与哮喘症状的增加有关，这强烈地暗示了基因与环境的相互作用。越来越多的证据表明，几丁质酶和几丁质在哮喘中都起着重要的作用，因此评估环境中几丁质暴露对哮喘人群的影响至关重要。然而，目前还没有办法直接评估室内环境中的甲壳素负荷，因为没有可用的分析方法来这样做，就像室内过敏原一样。甲壳素特异性检测的缺乏是我们评估甲壳素暴露在多大程度上导致哮喘恶化和发展的关键障碍。这项研究的长期目标是了解空气中微生物暴露(及其成分，如甲壳素)在哮喘发生和恶化中的作用。我们的目标是建立一种用于从空气或粉尘中定量环境样品中甲壳素的ELISA法。我们将系统地研究目前可用的几丁质结合蛋白的特异性，选择 最优，并检测其特异性，以进一步发展的酶联免疫吸附试验。我们将制定一项测试环境样品的协议，以解决处理和检测不溶性甲壳素的独特问题。最后，我们将对以前队列研究的存档粉尘样本使用新的甲壳素检测方法，并将甲壳素含量与含有甲壳素的生物体(可培养真菌)及其过敏原的现有测量方法进行比较，如来自尘螨的Der p1和Der F1，来自蟑螂的Bla G1和来自交链格孢菌的Alt A1。这项研究的成功结果和具有良好特性的甲壳素环境测试的产生将对该领域产生相当大的影响，使其能够在探索甲壳素暴露对哮喘恶化和发展的影响程度以及任何基因-环境相互作用的程度方面取得进展。 公共卫生相关性：哮喘的患病率正在增加，并与各种环境暴露密切相关。该项目将开发一种新的甲壳素环境检测方法，使未来能够评估甲壳素在过敏性过敏和哮喘的发展和进展中的作用，如果意义重大，可能会导致新的公众 减少过敏性疾病的卫生干预措施。

DESCRIPTION (provided by applicant): Asthma is a complex disease influenced by both genetics and environmental exposures, but the pathogenesis still remains unclear. Chitinase and chitinase-like proteins (CLPs) recently identified in humans have been found to play a significant role in both inflammation and asthma. Chitin - the ligand of these functional and non-functional enzymes -is a pathogen-associated molecular pattern that induces innate immune pathways, induces a Th2-type eosinophilic inflammation, and can act as an adjuvant for sensitization to bystander proteins. Chitin is an insoluble, long chain polymer of ¿1-4-N-acetyl-D-glucosamine and is a structural component of common indoor organisms such as dust mites, cockroaches, and fungi that are known asthma triggers. Chitin will be common in the environment where these organisms are abundant. Exposure to elevated levels of fungi in individuals with specific polymorphisms in a chitinase gene (CHIT1) is associated with increased asthma symptoms, strongly implicating a gene by environment interaction. Increasing evidence suggests both chitinases and chitin play a significant role in asthma, hence it is critically important to evaluate the impact of environmental chitin exposure on asthmatic populations. However, there is currently no way to directly assess chitin loads in the indoor environment since there is no assay available to do so, as there is for indoor allergens. The lack of a chitin specific assay is a critical barrier to our ability to assess the extent to which chitin exposure i contributing to the exacerbation and development of asthma. The long-term goal of the research is to understand the role of airborne microbial exposures (and components of them e.g. chitin) in the development and exacerbation of asthma. Our objective is to develop an ELISA assay for quantifying chitin in environmental samples from air or dust. We will systematically examine the specificities of currently available chitin-binding proteins, choose the most optimal, and examine its specificity for further development in an ELISA assay. We will develop a protocol for testing environmental samples with issues unique to handling and detection of insoluble chitin. Finally we will use the new chitin assay on archived dust samples from previous cohort studies and will compare chitin content with existing measures of chitin-containing organisms (culturable fungi) and their allergens such as Der p1 and Der f1 from dust mites, Bla g1 from cockroach, and Alt a1 from Alternaria alternata. The successful outcome of this research and the production of a well characterized chitin environmental assay will have a considerable impact on the field, allowing it to advance in exploring the extent to which chitin exposure has an impact on asthma exacerbation and development and the extent of any gene - environment interaction. PUBLIC HEALTH RELEVANCE: Asthma is increasing in prevalence and is strongly associated with a variety of environmental exposures. This project will develop a new environmental assay for chitin that will allow future assessments of its role in the development and progression of allergic sensitization and asthma that, if significant, could lead to new public health interventions to reduce allergic disease.