Development and validation of a chitin assay for environmental samples

描述（由申请人提供）：哮喘是一种复杂的疾病，受遗传和环境暴露的影响，但发病机制尚不清楚。最近在人类中发现的几丁质酶和几丁质酶样蛋白（CLPs）在炎症和哮喘中都起着重要作用。几丁质-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，可诱导先天免疫途径，诱导th2型嗜酸性粒细胞炎症，并可作为对旁观者蛋白致敏的佐剂。几丁质是一种不溶性的长链聚合物，由1-4- n -乙酰- d -氨基葡萄糖组成，是常见室内生物（如尘螨、蟑螂和真菌）的结构成分，这些生物都是已知的哮喘诱因。甲壳素在这些生物丰富的环境中很常见。几丁质酶基因（CHIT1）特异性多态性个体暴露于真菌水平升高与哮喘症状增加相关，强烈暗示基因与环境相互作用有关。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作用，因此评估环境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几丁质负荷，因为没有可用的分析方法，就像室内过敏原一样。缺乏几丁质特异性检测是我们评估几丁质暴露对哮喘恶化和发展的影响程度的关键障碍。这项研究的长期目标是了解空气中微生物暴露（及其成分，如几丁质）在哮喘发生和恶化中的作用。我们的目标是开发一种ELISA法，用于定量空气或粉尘中环境样品中的几丁质。我们将系统地检查目前可用的几丁质结合蛋白的特异性，选择

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.