Understanding bacterial localization and protein dynamics as indicators during tuberculosis treatment efficacy

了解细菌定位和蛋白质动态作为结核病治疗效果的指标

• 批准号: 9761978
• 负责人: Nicole Ann Kruh-Garcia
• 金额: $ 7.6万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-10 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761978
• 关键词: Aftercare; Animal Experimentation; Animals; Bacillus (bacterium); Bacteria; Bacterial Proteins; Biological Assay; Biological Markers; Blood; Categories; Cells; Child; Clinic; Clinical; Clinical Treatment; Collaborations; Companions; Complement; Coughing; Data; Detection; Development; Diagnosis; Diagnostic; Drug Screening; Failure; Future; Goals; Gold; Growth; Human; In Vitro; Infection; Kinetics; Label; Liquid substance; Location; Logistic Regressions; Mass Spectrum Analysis; Measures; Methods; Modeling; Monitor; Mycobacterium tuberculosis; Mycobacterium tuberculosis antigens; Outcome Study; Pathogenesis; Patients; Pattern; Pharmaceutical Preparations; Pharmacotherapy; Phase; Phenotype; Production; Protein Dynamics; Proteins; Proteomics; Protocols documentation; Refractory; Regimen; Research; Sampling; Serum; Serum Proteins; Signal Transduction; Small RNA; Source; Sputum; Statistical Models; Time; Training; Translating; Treatment Efficacy; Treatment Protocols; Tuberculosis; Validation; Vesicle; base; biomarker validation; catalyst; clinically relevant; cohort; design; drug development; efficacy trial; exosome; extracellular; insight; macrophage; mycobacterial; novel; novel therapeutics; physical property; platform-independent; pre-clinical; protein complex; protein profiling; proteomic signature; response; success; treatment effect; treatment response; trend; tuberculosis diagnostics; tuberculosis treatment; vesicular release

Project Summary In order to complement the current sputum based methods of tuberculosis (TB) diagnosis and treatment monitoring, novel assays are required to detect the presence of live Mycobacterium tuberculosis (Mtb) bacilli in patients. The discovery of Mtb-specific protein encapsulation in exosomes released from Mtb-infected macrophage culture was the impetus for exploration of the phenomenon in the exosome fraction of serum; targeted mass spectrometry was applied to confirm the identification of over a dozen Mtb-antigens in exosomes isolated from sputum-confirmed tuberculosis patients. Exosomes are small vesicles released from nucleated cells and are a rich-source of biomarkers. To complement and extent the utility of serum exosomes as markers of treatment efficacy, this proposal focuses on the analysis of ultrapure exosomes using unbiased mass spectrometry (MS) for the discovery of Mtb proteins and their kinetic response in 25 TB patients over 4 points during anti-mycobacterial treatment. Targeted MS assays (MRM) will be used to confirm the proteomic differences. Statistical modeling will be used to determine a proteomic signature consistent with clearance of Mtb from the sputum and will be performed with a training set (n = 256, baseline/treatment pairs) and a validation set (n = 64, baseline/treatment pairs) of serum exosome samples. Ultimately, an Mtb protein signature in serum exosomes can be used in conjunction with sputum diagnostics or when sputum is not available (in children and during treatment when production/cough resolves) and will reflect the elimination of transmissible bacilli in the sputum. In order to translate discoveries to a monitoring assay relevant to treatment monitoring in the clinic our methods must be simplified to a platform independent of mass spectrometry, however for the purpose of pre- clinical animal research MRM-MS remains a feasible option for novel drug and treatment regimen efficacy trials.

项目概要 为了补充目前基于痰的结核病（TB）诊断和治疗方法 监测，需要新的检测方法来检测活的结核分枝杆菌 (Mtb) 杆菌的存在 患者。结核分枝杆菌感染释放的外泌体中发现了结核分枝杆菌特异性蛋白质封装 巨噬细胞培养是探索血清外泌体部分这一现象的动力； 应用靶向质谱法确认外泌体中十多种 Mtb 抗原的鉴定 从痰确诊的结核病患者中分离出来。外泌体是从有核细胞中释放出来的小囊泡 细胞，是生物标志物的丰富来源。补充和扩展血清外泌体作为标记物的效用 为了评估治疗效果，该提案重点关注使用无偏质量分析超纯外泌体 光谱法 (MS) 在 25 名结核病患者中发现 Mtb 蛋白及其动力学反应超过 4 个点 在抗分枝杆菌治疗期间。靶向 MS 检测 (MRM) 将用于确认蛋白质组学 差异。统计模型将用于确定与清除率一致的蛋白质组特征 来自痰液的 Mtb，将使用训练集（n = 256，基线/治疗对）和验证进行 血清外泌体样本组（n = 64，基线/治疗对）。最终，血清中的 Mtb 蛋白特征 外泌体可以与痰液诊断结合使用，或者当无法获得痰液时（儿童和儿童） 在治疗期间，当生产/咳嗽缓解时），并且将反映消除体内的传染性杆菌 痰。为了将发现转化为与临床治疗监测相关的监测测定，我们 方法必须简化为独立于质谱分析的平台，但是为了预 临床动物研究 MRM-MS 仍然是新药和治疗方案疗效试验的可行选择。