Phagocyte Contact Response in Aspergillosis Immunity

描述(由申请人提供)：烟曲霉是免疫受损人群中主要的空气传播真菌病原体。由于管理烟曲霉菌感染的困难，大多数患侵袭性曲霉病的人都会死亡。随着免疫受损人口的增加，烟曲霉菌的感染也将增加。虽然曲霉病的认识已经取得了重大进展，但相应的免疫细胞之间的关系还没有完全了解。这项工作的长期目标是鉴定寄主防御烟曲霉菌的分子基础。具体的焦点是确定吞噬细胞和烟曲霉菌之间的细胞和分子联系，以及杀微生物剂反应在相应免疫中的作用。1)用流式细胞术和免疫组织化学方法评价小鼠肺组织对烟曲霉分生孢子的特异性反应：在正常和免疫抑制的小鼠中，用特异性的单抗检测肺组织，以鉴定接种烟曲霉分生孢子后到达的细胞。可的松诱导的免疫抑制和通过敲除NADPH氧化酶而变得敏感的小鼠都将被检查。2)研究NADPH氧化酶在摄取分生孢子的免疫细胞亚细胞室中的作用：超氧化物产生试验将用于显示与含有摄取分生孢子的吞噬溶酶体相关的氧化酶的组装。组装的复合体将通过证明含有分生孢子的隔室中的超氧化物生成率以及免疫印迹法证明完整的氧化物组分来确认。一组单抗可用于检测氧化酶。3)通过噬菌体展示多肽文库分析，筛选与烟曲霉分生孢子和菌丝形态特异结合的多肽。利用多肽库对烟曲霉休眠分生孢子、膨大分生孢子和菌丝进行亲和力筛选。这一信息将被用来暗示这种宿主-病原体关系中的依附机制

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the leading airborne fungal pathogen in immune compromised people. Due to difficulties in managing A. fumigatus infections, most individuals that develop invasive aspergillosis will die. With an increasing immune compromised population, infections by A. fumigatus will also increase. While significant advancements in understanding aspergillosis have been made, the relationship among corresponding immune cells is not fully understood. The long-term goal of this work is to identify the molecular basis of host defense against A. fumigatus. The specific focus is to determine the cellular and molecular contact between phagocytes and A. fumigatus and the role of microbicidal responses in the corresponding immunity. Three aims are proposed to address this goal: 1) To evaluate the cell specific response to A. fumigatus conidia in the lungs of mice by flow cytometry and immunohistochemistry: Lung tissue will be probed using specific monoclonal antibodies to identify cells that arrive following inoculation of A. fumigatus conidia in normal and immune suppressed mice. Both cortisone-induced immune suppression and mice made susceptible to by knockout of the NADPH oxidase will be examined. 2) To investigate the role of the NADPH oxidase in subcellular compartments of immune cells which have ingested conidia: Superoxide generation assays will be used to show assembly of the oxidase associated with phagolysosomes containing ingested conidia. The assembled complex will be confirmed by demonstrating superoxide generation rates in compartments containing conidia, as well as demonstrating the complete set of oxidase components by immunoblot. A panel of monoclonal antibodies is available for oxidase detection. 3) To identify unique peptides that bind specifically to the A. fumigatus conidia and hyphal forms by phage-display peptide library analysis. Affinity selection of unique peptides will be carried out by probing the A. fumigatus resting conidia, swollen conidia, and hyphae with a peptide library. This information will be used to suggest mechanisms of attachment in this host-pathogen relationship