BLR&D Research Career Scientist Award Application

BLR

• 批准号: 10451505
• 负责人: Melanie T Cushion
• 金额: --
• 依托单位: CINCINNATI VA MEDICAL CENTER RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10451505
• 关键词: 3-Dimensional; Age; American; Anidulafungin; Antifungal Agents; Area; Arthritis; Award; Biological; Biological Assay; Bone Marrow; Caring; Caspofungin; Cell Line; Cell Wall; Chemicals; Chloroquine; Chronic; Chronic Disease; Chronic Obstructive Pulmonary Disease; Clinical; Collaborations; Data; Development; Dihydrofolate Reductase Inhibitor; Disease; Dose; Drug Screening; Elderly; Evaluation; Female; Gene Expression; Gene Expression Profiling; General Population; Genes; Goals; HIV; HIV Seronegativity; HIV Seropositivity; Healthcare; Hematological Disease; Human; Immune; Immune system; Immunocompromised Host; Immunosuppression; Immunotherapy; In Vitro; Infection; Infection prevention; International; Interruption; Knowledge; Length; Life Cycle Stages; Lung; Malignant Neoplasms; Mammalian Cell; Methods; Micafungin; Microbiology; Microscopic; Midwestern United States; Mission; Modeling; Morbidity - disease rate; Mus; Mycoses; Ohio; Opportunistic Infections; Outcome; Partner in relationship; Patients; Pharmaceutical Preparations; Phase; Pneumocystis; Pneumocystis Infections; Pneumocystis carinii; Pneumocystis carinii Pneumonia; Pneumonia; Poland; Population; Portugal; Preclinical Drug Development; Proliferating; Property; Prophylactic treatment; Publications; Rattus; Reporting; Research; Rheumatoid Arthritis; Risk; Rodent Model; Role; Scientist; Seminal; Sexual Reproduction; Societies; Sphingolipids; Stress; Sulfamethoxazole; Therapeutic; Time; Toxic effect; Trimethoprim-sulfamethoxazole drug resistance; United States; Up-Regulation; Veterans; Withdrawal; analog; antimicrobial; asexual; base; cancer diagnosis; candidate identification; career; celecoxib; chronic inflammatory disease; clinically relevant; comorbidity; efficacy testing; experimental study; fungus; high risk; immunological status; immunosuppressed; infected vector rodent; knockout gene; male; metabolic abnormality assessment; military veteran; mortality; mouse model; new therapeutic target; novel therapeutics; pathogen; pathogenic fungus; polyglucosan; pre-clinical; prophylactic; response; screening; sex; targeted treatment; therapy duration; tool; transcriptome sequencing; transmission process; tumor necrosis factor-alpha inhibitor

Pneumocystis spp. are obligate fungal pathogens that cause a fatal pneumonia (PCP) in immunocompromised hosts. Few drugs are effective against PCP and there have been no new therapies for its treatment in decades. Typically, PCP has been associated with patients infected with HIV, however, the fulminate pneumonia, PCP, and colonization with Pneumocystis jirovecii (the species infecting humans) are emerging clinical problems in newly susceptible populations in the general and veterans’ populations including bone marrow recipients; patients receiving immunotherapy for rheumatoid arthritis and other chronic inflammatory diseases; and cancer chemo- and immunotherapies. The life cycle of Pneumocystis is suggested to contain both an asexual replication cycle and a sexual cycle involving mating with subsequent formation of asci containing 8 ascospores (1). During the previous Merit Review, we showed that echinocandin treatment of rodents infected with P. murina and P. carinii, which target β-1,3-D-glucan synthesis (BG), depleted the asci which contain BG but large numbers of non-BG expressing life cycle stages remained in the lungs and were unable to proliferate. We further demonstrated that anidulafungin and caspofungin could prevent infection in a prophylactic model, suggesting that formation of asci via the sexual cycle may be required for a productive infection (2). Analysis of gene expression profiles of P. murina in mice treated with anidulafungin, showed strong upregulation of genes associated with sexual replication, though the resulting infections were devoid of asci, the product of sexual reproduction, suggesting that P. murina attempted to undergo sexual replication, but could not due to a lack of BG. Based on these data, we posited that asci, and thus sexual replication, is required to facilitate progression through the life cycle leading to a productive infection. We further posited that presence of asci is required for transmission of Pneumocystis infection. In the present Merit Review, we will explore 2 critical but unanswered questions that will lead to a deeper knowledge of the life cycle of Pneumocystis, and also suggest potential vulnerabilities for targeted treatment concomitant with anidulafungin therapy: (1) Is sexual replication required for completion of the life cycle of Pneumocystis? Tracking of the replication status of P. murina during prolonged treatment with anidulafungin by global gene analysis, BG content, and microscopic methods will reveal whether the non-BG expressing forms numbers remain: 1) static over time, 2) increase, or 3) decrease; suggesting: 1) the lack of BG blocks replication; 2) that an asexual or alternative replication phase permits survival of the fungi; or 3) the lack of sexual replication results in elimination of the infection. (2) Can sexual replication rebound after cessation of prolonged anidulafungin treatment? Mice will be treated with anidulafungin for up to 8 weeks, with 2 cessation time points. Mice in the cessation groups will be tracked for microscopic, BG content, and gene expression evidence of asci formation and return of the pneumonia while remaining under immunosuppression. Mice in the treated and cessation groups will be evaluated for their ability to transmit the infection and the critical number of asci needed for transmission. All studies will be conducted in male and female mice, recognizing sex as a biological variable. The echinocandins are clinically available in the United States. Current monotherapy with any echinocandin for PCP is not warranted as withdrawal can result in return of the pneumonia. The results of the proposed studies will have immediate clinical relevance by determining the length of time viable Pneumocystis can remain in the lungs with concurrent anidulafungin treatment, providing a rationale for duration of therapy with eradication as the goal. These experiments will also identify whether immunosuppressed mice can transmit the infection after withdrawal of anidulafungin and if there is a critical number of asci needed. Finally, the studies will elaborate the life cycle of Pneumocystis and suggest new target strategies.

肺孢子虫属是导致免疫功能低下者致命性肺炎 (PCP) 的专性真菌病原体 主机。很少有药物能够有效对抗 PCP，而且几十年来也没有新的疗法可以治疗 PCP。 通常，PCP 与感染 HIV 的患者有关，但是，暴发性肺炎、PCP、 以及耶氏肺孢子菌（感染人类的​​物种）的定植正在出现的临床问题 普通人群和退伍军人群体中的新易感人群，包括骨髓接受者； 接受类风湿性关节炎和其他慢性炎症性疾病免疫治疗的患者；和癌症 化疗和免疫疗法。肺孢子虫的生命周期被认为包含无性复制 周期和性周期涉及交配以及随后形成含有 8 个子囊孢子的子囊 (1)。期间 在之前的优异评审中，我们展示了棘白菌素治疗感染鼠疟原虫和鼠疟原虫的啮齿动物。 carinii 以 β-1,3-D-葡聚糖合成 (BG) 为目标，耗尽了含有 BG 的子囊，但大量 非 BG 表达的生命周期阶段保留在肺部并且无法增殖。我们进一步 证明阿尼芬净和卡泊芬净可以在预防模型中预防感染，这表明 生产性感染可能需要通过性周期形成子囊 (2)。基因分析 用阿尼芬净处理的小鼠中 P. murina 的表达谱显示出强烈的基因上调 与性复制有关，尽管由此产生的感染缺乏子囊（性复制的产物） 繁殖，表明 P. murina 试图进行有性复制，但由于缺乏 BG。根据这些数据，我们假设 asci 以及性复制是促进进展所必需的 整个生命周期导致生产性感染。我们进一步假设，asci 的存在是必需的 肺孢子菌感染的传播。在本次优点评审中，我们将探讨 2 个关键但尚未得到解答的问题 这些问题将导致人们对肺孢子虫的生命周期有更深入的了解，并且还表明了潜在的潜力 阿尼芬净治疗同时进行靶向治疗的弱点：（1）是否需要有性复制 完成肺孢子虫的生命周期？追踪 P. murina 的复制状态 通过整体基因分析、BG 含量和显微方法，阿尼芬净长期治疗将揭示 非 BG 表达形式的数量是否保持：1) 随着时间的推移保持不变，2) 增加，或 3) 减少； 表明：1）BG 的缺乏会阻碍复制； 2）无性或替代复制阶段允许 真菌的生存；或 3) 缺乏有性复制导致感染消除。 (2) 能否发生性行为 停止长期阿尼芬净治疗后复制反弹？小鼠将被治疗 阿尼芬净最长 8 周，有 2 个停药时间点。将追踪戒烟组中的小鼠 子囊形成和肺炎复发的显微镜、BG 含量和基因表达证据 仍处于免疫抑制状态。将评估治疗组和戒烟组的小鼠的能力 传播感染以及传播所需的 Asci 临界数量。所有研究将在 雄性和雌性小鼠，将性别视为生物变量。棘白菌素类药物在临床上可用 美国。目前使用任何棘白菌素单一疗法治疗 PCP 都是不合理的，因为停药可能会导致 肺炎复发。拟议研究的结果将具有直接的临床意义 确定肺孢子菌与阿尼芬净同时使用时可在肺部停留的时间长度 治疗，为以根除为目标的治疗持续时间提供依据。这些实验也将 确定免疫抑制小鼠在阿尼芬净停药后是否会传播感染，以及是否存在 是需要的关键数量的asci。最后，研究将详细阐述肺孢子虫的生命周期并提出建议 新的目标策略。