Lipidated Mycobacterium tuberculosis biomarkers in clinical specimens

描述（由申请人提供）：结核病（TB）仍然是传染病发病率和死亡率的主要原因。虽然基于痰液的结核病诊断测试已经取得了逐步进展，但在临床可获得的血液和尿液等标本中快速、高灵敏度地检测结核分枝杆菌抗原的能力将改变结核病诊断过程。结核分枝杆菌脂质是有吸引力的诊断靶点，虽然有几条证据表明结核患者的血液和尿液中存在某些结核分枝杆菌脂质，但生物学仍未被探索，诊断潜力尚未实现。我们的团队开发了一种基于波导的光学生物传感器平台，用于MTB脂arabinomannan （LAM）的灵敏检测。此外，我们基于MTB LAM的两亲性及其与脂质双分子层的相互作用，开发了一种新的血清中MTB LAM单体的超灵敏检测策略。少量临床标本的初步工作是有希望的。此外，体外研究表明，血清中的LAM与人脂蛋白，特别是高密度脂蛋白（HDL）一起包装在脂质组装体中，这提高了人脂蛋白作为两亲性MTB产物“水槽”的可能性。通过利用两亲性LAM的这种相互作用，我们开发了一种从血清中选择性拉下LAM的策略；这种新策略可以应用于其他生物标志物。这个R21项目的总体目标是：a)完善我们在临床标本中检测LAM的方法，使用现有的具有明确特征的TB疾病状态的成人血清和尿液标本；b)扩展LAM与人类脂蛋白相互作用的初步体外观察，以阐明LAM的生物分布，并确定其他人类脂蛋白是否与LAM相关。所提出的研究不仅可能在通过检测结核分枝杆菌脂质来诊断结核病的新型超灵敏方法方面取得突破，而且还可能在结核病和人类生物学方面取得突破，因为人类血浆脂蛋白在分枝杆菌脂质转运中的作用尚未被探索。提出的探索性研究有望为快速诊断测试平台的开发以及未来全面的基于假设的研究奠定基础，旨在阐明LAM和其他分枝杆菌病原体相关分子模式的生物学和治疗意义。现有用于研究的临床标本以及包括工程师、化学家和结核病临床医生在内的多学科团队增强了该项目的可行性。该项目直接源于NIAID/ dmid资助的结核病临床诊断研究联盟合同中研究人员寻求技术的活动。

DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a leading cause of infectious disease morbidity and mortality. While incremental advances in sputum-based TB diagnostic tests have been made, the ability to rapidly detect M. tuberculosis (MTB) antigens with high sensitivity in clinically accessible specimens such as blood and urine would transform the TB diagnostic process. MTB lipids are attractive diagnostic targets, and while several lines of evidence indicate that certain MTB lipids are present in blood and urine in TB patients, the biology remains unexplored and the diagnostic potential unrealized. Our team has developed a waveguide-based optical biosensor platform for sensitive detection of MTB lipoarabinomannan (LAM). Further, we have developed a novel ultrasensitive detection strategy for monomeric MTB LAM in serum, based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Preliminary work with a small number of clinical specimens is promising. Moreover, in vitro studies have shown that LAM in serum is packaged in lipid assemblies with human lipoproteins, especially high-density lipoprotein (HDL), raising the possibility that human lipoproteins serve as "sinks" for amphiphilic MTB products. By exploiting this interaction of amphiphilic LAM, we have developed a strategy for selective pull-down of LAM from serum; this novel strategy could be applied to other biomarkers. The overall goals of this R21 project are a) to refine our approach for detection of LAM in clinical specimens using existing serum and urine specimens from adults with well-characterized TB disease status, and b) to extend preliminary in vitro observations on the interaction of LAM with human lipoproteins to clarify the biodistribution of LAM and determine if other human lipoproteins are associated with LAM. The proposed studies could lead to a breakthrough not only with respect to novel ultrasensitive approaches for TB diagnosis through detection of MTB lipids, but also with respect to TB and human biology since the role of human plasma lipoproteins in mycobacterial lipid trafficking is unexplored. The proposed exploratory studies are expected to lay the groundwork for development of a rapid diagnostic testing platform as well as future comprehensive, hypothesis-based studies aimed at elucidating the biology and therapeutic implications of LAM and other mycobacterial pathogen-associated molecular patterns. The feasibility of this project is enhanced by the existing clinical specimens for study, as well as the multidisciplinary team including engineers, chemists, and TB clinicians. This project arose directly from technology-seeking activities of investigators in the NIAID/DMID-funded TB Clinical Diagnostics Research Consortium contract.