Characterization of fungal exposures

真菌暴露的表征

• 批准号: 8543856
• 金额: $ 16万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8543856
• 关键词: Address; Adverse effects; Aerosols; Agreement; Agriculture; Air; Allergens; Allergic; Alternaria; Animal Model; Animals; Antibodies; Antigens; Area; Aspergillus; Asthma; Basidiomycota; Biological Assay; Biological Markers; Blood Circulation; Breathing; Caliber; Candida albicans; Carbon Dioxide; Cell surface; Cells; Chaetomium; Chemicals; Child; Chronic berylliosis; Cities; Cladosporium; Clinical; Collaborations; Cryptococcus; Cytolysis; DNA; Data; Dermatophagoides farinae antigen f 1; Development; Disease; Dust; Environment; Environmental Exposure; Enzyme-Linked Immunosorbent Assay; Evaluation; Experimental Animal Model; Exposure to; Family; Farming environment; Fungal Antigens; Fungal DNA; Fungal Genes; Fungal Spores; General Population; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Genome; Genomics; Goals; Growth; Hazard Assessment; Hazard Identification; Health; Home environment; Hospitals; Housing; Human; Humidity; Hypersensitivity; Hyphae; IgE; Immune; Immune Response Genes; Immune response; Immune system; Immunoassay; Immunoglobulin G; Immunologic Techniques; Immunologics; Individual; Indoor Air Quality; Inflammatory; Kansas; Light; Liquid substance; Location; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methodology; Methods; Minority; Mitogens; Modeling; Molds; Monoclonal Antibodies; Morphology; Mus; National Institute of Environmental Health Sciences; National Toxicology Program; Nose; Occupational; Occupational Asthma; Occupational Diseases; Occupational Exposure; Outcome; Paecilomyces; Participant; Penicillium; Performance; Peripheral Blood Mononuclear Cell; Play; Population; Prevalence; Principal Investigator; Probability; Process; Protein Databases; Proteins; Public Health; RNA Sequences; Reagent; Recombinants; Relative (related person); Reporting; Reproduction spores; Research; Rhinitis; Ribosomal RNA; Rice; Risk; Risk Assessment; Rodent; Role; Sampling; Scanning Electron Microscopy; Scientist; Serum; Silicosis; Simulate; Site; Source; Specificity; Stachybotrys; System; Technology; Temperature; Testing; Toxicology; United States; Universities; Vacuum; Water; Woman; Work; Workplace; Xenobiotics; aerosolized; age group; air sampling; animal data; base; chemical addition; cohort; cooperative study; cross reactivity; cytokine; deoxynivalenol; design; disorder prevention; enolase; exposed human population; farmer; field study; fungus; immune function; improved; insight; interest; member; particle; pathogen; pesticide exposure; rRNA Genes; respiratory; response; ultraviolet irradiation

The goal of this interagency agreement is to provide support of National Toxicology Program (NTP) hazard identification (or hazard assessment) activities targeted toward the prevention of diseases or adverse effects caused by environmental exposure to chemical or physical agents. These cooperative studies continue to improve the risk assessment process by determining quantitatively what constitutes an adverse health effect on the immune system in humans. These studies evaluate unique cohorts of individuals from professions associated with immune-mediated occupational diseases including asthma, respiratory and contact allergy, chronic beryllium disease, rhinitis, and silicosis. Occupational cohorts are being studied for a number of endpoints including, impact of genetic polymorphisms on inflammatory disease development and clinical outcomes, the role that genetic variations play in environmental and workplace related diseases and identification of unique immunological biomarkers for disease. Traditional methods for assessing fungal exposure have well-documented limitations. For example, fungal assessment based on viable culture generates biases against slow-growing fungi, and several fungi are not capable of growth on artificial culture. Non-viable methods of assessment are also limited to the identification of fungi to the Order level due to the morphologically indiscernible spore morphologies shared by many fungi (ex. Aspergillus, Penicillium, and Paecilomyces). To more completely assess the fungal species to which people are exposed, a fungal ribosomal RNA (rRNA) gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States indoor built environments are much more diverse than previously estimated using traditional methods of analysis. In collaboration with Children¿s Mercy Hospital (Kansas City, MO) NIOSH will isolate mold specific ribosomal RNA genes from air and dust samples derived from homes participating in the Kansas City Safe and Healthy Homes Partnership Project with the aim of characterizing the complete diversity of fungi. The routine sampling performed in these homes included on-site measurements for specific indoor air quality parameters such as temperature, relative humidity, and carbon dioxide concentration. Measurements were also made to determine air circulation in the target rooms. A composite dust sample from the subject's bedroom and the family vacuum were collected and analyzed by monoclonal immunoassay for the allergens Der f1, Der p1, Fel d1, Can f1, Mus m1 and Bla g2. Total antigen content of Alternaria, Aspergillus, Cladosporium and Penicillium species has also be determined using a previously developed polyclonal assay. Air sampling from at least 4 areas of the home have been examined microscopically for fungal conidia. Two additional samples have been collected using the size-selective NIOSH BC 251 sampler and the Biostage viable sampler. The sampling locations for these samplers included the child's bedroom and a more heavily contaminated area of the house (usually the basement). A Biostage viable sampler was also utilized to collect viable fungal bioaerosols using the same sampling interval and location. Methods have been optimized to extract DNA from fungal particles derived from both dust and air samples. Fungal gene sequences have been obtained for 31 dust samples and 10 aerosol samples. To date, data from dust samples indicate a prevalence of species within the Order Pleosporales (genera commonly detected: Phoma, Pithomyces, Curvularia, Alternaria, Stagnospora, and Epicoccum). Other basidiomycete fungi are also common and detected in dust samples and include species within the genera Cryptococcus, Rhodoturula, Wallemia, and Puccinia (rusts). Interestingly, aerosol samples have yielded many rRNA sequences highly similar to Aspergillus and Penicillium species. Although these genera are typically encountered in viable culture studies, they have been less prevalent in the few fungal rRNA sequencing studies that have been conducted thus far. The genomic DNA extraction method that was optimized during the preliminary study may be more efficient at extracting DNA from the spores of these genera, which are typically small and recalcitrant to cell lysis. A second project is developing species-specific monoclonal antibodies (mAbs) to recombinant fungal antigens. These mAbs will be important for the quantification of species-specific biomarkers of fungal exposure, particularly to those fungi that are being studied by the NTP. Aspergillus terreus has been used as a model organism as its genome has been sequenced. This has made it possible to develop fungal specific proteins using recombinant technologies. A. terreus is an emerging and poorly characterized opportunistic pathogen and an important source of occupational exposure in agricultural and other water-damaged environments. To date, A. terreus terrelysin, has been identified, characterized and based on the sequence reported by NIOSH, annotated in several protein databases. Twelve mAbs were developed and characterized. Native terrelysin was measured in growing hyphae in the first four days of liquid culture, suggesting that terrelysin may be expressed during hyphal aggregration and may function as a biomarker of A. terreus exposure. Cross-reactivity of the terrelysin mAbs was also tested with other closely related species belonging to the genera Aspergillus, Penicillium and Paecilomyces and identified to be specific. NIOSH continues to work with these A. terreus reagents with the aim of developing a standardized immunoassay. Recombinant Chaetomium globosum enolase (rec-CgEnol) has been cloned, expressed, purified and confirmed using mass spectrometry. Three rec-CgEnol specific mAbs have been produced. The specificity of each rec-CgEnol mAb is currently being examined in cross reactivity studies. In addition, conserved fungal allergens derived from the fungal order, Pleosporales, are currently being identified as part of a collaborative study with Johns Hopkins University. Preliminary data demonstrate several candidate allergens that are shared between members of this occupationally relevant fungal order. mAbs to hyphal exoantigens derived from the occupational contaminant, Paecilomyces variotii have also been produced. Current research efforts are aimed at identifying and characterizing these hyphal exoantigens and developing immunoassays that will be used to quantify this occupational contaminant in a collaborative exposure assessment study of Norwegian sawmill workers. A third project is developing an animal model for dry fungal aerosol exposures to identify and study potential human health effects. Current exposure methods rely on liquid installation of fungal spores or extracts, which may not simulate human exposures in mold contaminated indoor environments. An acoustical generator and nose only chamber to be used for the delivery of dry fungal spores and hyphal fragments has been developed and characterized. In characterization studies using A. fumigatus grown on rice, airborne concentrations exceeding 1 X 106 spores could be aerosolized using the chamber. Field emission scanning electron microscopy analysis confirmed that particles aerosolized were amerospores and no hyphal material was observed. Recently, the system has been optimized for the acoustical generation of hyphal fragments and preliminary data demonstrated that hyphal particles of equal aerodynamic diameter could be aerosolized. Aerosols from A. fumigatus, A. terreus, A. vericolor, Paecilomyces variotii, and Stachybotrys chartarum be utilized and viable and non-viable (UV irradiated) fungal aerosols will be compared using the model. These studies will provide unique insight into the innate and adaptive immune responses following inhalation of dry fungal spores and hyphae aerosolized from contaminated building materials. Finally, NIOSH has evaluated allergic sensitization in a cohort of sera from 677 farmers with or without pesticide exposures as part of the NIEHS Agricultural Health Study. Total IgE (tIgE) and mold mix specific IgE (sIgE) was determined using the Phadia ImmunoCap assay. Total IgE greater than 100 kU/L was quantified in 21% of the farmer population. Allergic sensitization to mold Mx2 was identified in 4% (28/677) of this cohort. Mold Mx2 positive sera were then tested for specific IgE to a panel of potential farm-specific mold allergens using the commercially available Phadia ImmunoCap test. Allergic sensitization in the atopic farmer population (n=144) was greatest to common fungal contaminants including A. alternata (10%), P. betae (9%), E. purpurascens (9%), C. albicans (9%), P. chrysogenum (8%), and A. fumigatus (8%), and not to common crop contaminating species. Because of NTP interest in additional fungal contaminants, all 677 sera were screened for deoxynivalenol (DON) levels in the serum. DON levels in the serum were evaluated using a modified commercially competitive ELISA produced by NEOGEN with a quantification range between 0.6-10 ng/mL. No DON has been found in the farmers¿ serum.

该机构间协议的目标是为国家毒理学计划 (NTP) 危害识别（或危害评估）活动提供支持，旨在预防因环境接触化学或物理制剂而引起的疾病或不利影响。这些合作研究不断得到改进。这些研究评估了与免疫介导的职业病相关的专业人士的独特群体，包括哮喘、呼吸道和接触性过敏、慢性铍病、鼻炎、职业群体正在研究许多终点，包括遗传多态性对炎症性疾病发展和临床结果的影响、遗传变异在环境和工作场所相关疾病中的作用以及识别疾病的独特免疫生物标志物。例如，基于活菌培养的真菌评估会对生长缓慢的真菌产生偏差，并且一些真菌不能在人工培养物上生长。由于许多真菌（例如曲霉属、青霉属和拟青霉属）共有孢子形态，因此也仅限于真菌目级别的鉴定。为了更全面地评估人们接触的真菌物种，真菌核糖体 RNA。 (rRNA) 基因测序研究旨在检验以下假设：美国室内建筑环境中的真菌生物气溶胶比之前使用传统方法估计的要多样化得多。与儿童合作进行分析。 NIOSH 将从参与堪萨斯城安全与健康家庭合作项目的家庭的空气和灰尘样本中分离出霉菌特异性核糖体 RNA 基因，目的是表征真菌的完整多样性。在这些家庭中进行的测试包括对特定室内空气质量参数（例如温度、相对湿度和二氧化碳浓度）的现场测量，还进行了测量以确定目标房间和卧室的复合灰尘样本。收集并通过单克隆免疫测定法对家族真空中的过敏原 Der f1、Der p1、Fel d1、Can f1、Mus m1 和 Bla g2 进行分析，还使用先前开发的方法测定了链格孢属、曲霉属、枝孢属和青霉属物种的总抗原含量。已使用显微镜检查了家中至少 4 个区域的空气样本中的真菌分生孢子。尺寸选择性 NIOSH BC 251 采样器和 Biostage 活菌采样器 这些采样器的采样位置包括孩子的卧室和房屋中污染较严重的区域（通常是地下室）。使用相同的采样间隔和位置对真菌生物气溶胶进行了优化，以从灰尘和空气样本中提取真菌颗粒中提取 DNA，获得了 31 个灰尘样本和 10 个气溶胶的真菌基因序列。迄今为止，来自灰尘样本的数据表明，Pleosporales 中的物种普遍存在（常见的属：茎点霉属、曲霉菌属、链格孢属、Stagnospora 和附球菌属）其他担子菌属真菌也很常见，并在灰尘样本中检测到，包括。有意地，气溶胶样本中产生了许多高度相似的 rRNA 序列。尽管这些属通常在活培养研究中遇到，但在迄今为止进行的少数真菌 rRNA 测序研究中并不常见。在初步研究期间优化的基因组 DNA 提取方法可能是这样的。更有效地从这些属的孢子中提取 DNA，这些孢子通常很小且难以细胞裂解。第二个项目是开发针对重组真菌抗原的物种特异性单克隆抗体 (mAb)。这些单克隆抗体对于真菌暴露的物种特异性生物标志物的量化非常重要，特别是对于 NTP 正在研究的真菌，土曲霉已被用作模型生物，因为其基因组已被测序。使用重组技术开发真菌特异性蛋白质。土曲霉是一种新兴的、特征不明的机会性病原体，也是农业和其他受水破坏的环境中职业接触的重要来源。 terreus terrelysin 已被鉴定、表征，并基于 NIOSH 报告的序列，开发并表征了 12 种单克隆抗体，在液体培养的前四天中，在生长的菌丝中测量了 terrelysin。在菌丝聚集过程中表达，可作为土曲霉暴露的生物标志物。 mAb 还与属于曲霉属、青霉属和拟青霉属的其他密切相关的物种进行了测试，并被鉴定为具有特异性。NIOSH 继续与这些土曲霉试剂合作，旨在开发标准化的免疫测定法。 CgEnol) 已通过质谱法进行克隆、表达、纯化和确认，已获得三种 rec-CgEnol 特异性单克隆抗体。每种rec-CgEnol mAb 的特异性目前正在交叉反应性研究中进行检查。此外，作为与约翰·霍普金斯大学合作研究的一部分，目前正在鉴定来自真菌目的保守的真菌过敏原。初步数据表明，该职业相关真菌目成员之间共有几种候选过敏原，这些单克隆抗体针对源自职业污染物拟青霉的菌丝外抗原。目前的研究工作旨在识别和表征这些菌丝外抗原，并开发免疫测定法，用于在挪威锯木厂工人的合作暴露评估研究中量化这种职业污染物。干燥真菌气溶胶暴露来识别和研究潜在的人类健康影响当前的暴露方法依赖于真菌孢子或提取物的液体安装，这可能无法模拟人类在霉菌污染的室内环境中的暴露。已经开发出用于输送干燥真菌孢子和菌丝碎片的鼻腔室，并对其进行了表征。在使用水稻上生长的烟曲霉的特性研究中，可以使用该腔室将空气中浓度超过 1 X 106 的孢子雾化。扫描电子显微镜分析证实，雾化的颗粒是无小孢子，并且没有观察到菌丝物质。最近，该系统已针对菌丝的声学产生进行了优化。碎片和初步数据表明，可以利用来自烟曲霉、土曲霉、毛曲霉、拟青霉和黑葡萄穗霉的气溶胶，以及活的和非活的（紫外线照射）真菌气溶胶。将使用该模型进行比较，这些研究将为吸入干燥后的先天性和适应性免疫反应提供独特的见解。最后，作为 NIEHS 农业健康研究的一部分，NIOSH 评估了 677 名接触或未接触农药的农民的血清过敏情况。 sIgE）使用 Phadia 免疫帽测定法测定总 IgE 大于 100 kU/L。在 21% 的农民群体中，4% (28/677) 的农民对霉菌 Mx2 过敏，然后使用市售 Phadia 免疫帽测试 特应性农民群体 (n=144) 对常见真菌的过敏反应最为严重。污染物包括 A. alternata (10%)、P. betae (9%)、E. purpurascens (9%)、C. albicans (9%)、P. chrysogenum (8%) 和 A. fumigatus (8%) ，而不是常见的农作物污染物种，因为 NTP 对其他真菌污染物感兴趣，所以对所有 677 份血清进行了脱氧雪腐镰刀菌烯醇 (DON) 筛查。使用 NEOGEN 生产的改良商业竞争性 ELISA 评估血清中的 DON 水平，定量范围为 0.6-10 ng/mL。血清。