Signal-amplifying biomarkers for the diagnosis of invasive fungal infections.

描述(由申请人提供)：我们建议开发生物分析技术来识别和表征信号放大生物标记物，以改善侵袭性肺曲霉菌病的诊断和监测。另一个目标是创建一种可靠的生物分析工具，用于严重真菌感染期间患者的预后。真菌蛋白水解酶在感染患者的肺中释放，导致肺部分泌物中所含蛋白质的蛋白降解。一个单一的蛋白酶分子可以切割多个底物分子，从而产生自然的信号放大效应。这将通过1)使用假说驱动的质谱学检测特定的蛋白质分解产物和2)通过生物化学方法，如体外裂解产生荧光的底物来利用。我们的策略将利用真菌蛋白水解酶和切割产物的免疫化学浓缩技术，使用含有单抗的浓缩基质。我们已经证明了一种细胞内真菌蛋白的免疫保护作用，这种蛋白在几丁质酶等肺酶降解细胞壁后可被人类免疫系统访问。基于这一发现，我们将检验一种假设，即人类几丁质酶可能在真菌感染期间被诱导，几丁质酶的水平(和其他裂解活性)因患者而异，并取决于遗传和病理生理条件。根据批准的临床方案，我们建议从疑似真菌感染的患者身上获取一套临床支气管肺泡灌洗液、血液和尿液样本。这些样本将用于开发和评估侵袭性肺曲霉菌病的信号放大生物标记物分析。这项研究的结果有望转化为新的临床应用，以更好地诊断、预测、监测和最终控制侵袭性真菌感染。 这里提出的研究旨在识别信号放大生物标志物，这些生物标志物将有助于侵袭性真菌感染的快速和敏感诊断。侵袭性真菌感染，如侵袭性肺曲霉菌病，在器官移植后的癌症治疗中是艾滋病患者和其他削弱人类免疫防御的严重疾病的主要问题。这项探索性研究的结果应该能够转化为临床应用，改善侵袭性真菌感染的诊断，这有可能挽救每年数千名美国人和世界各地数万名患者的生命。

DESCRIPTION (provided by applicant): We are proposing to develop bioanalytical techniques to identify and characterize signal- amplifying biomarkers that improve the diagnosis and monitoring of invasive pulmonary aspergillosis. An additional goal is the creation of a reliable bioanalytical tool for patient prognosis during severe fungal infections. Fungal proteases are released in the lungs of infected patients, causing the proteolytic degradation of proteins contained in pulmonary secretions. A single protease molecule can cleave multiple substrate molecules resulting in a natural signal amplification effect. This will be exploited by 1) detecting specific proteolytic products using hypothesis-driven mass spectrometry and 2) through biochemical methods such as in-vitro cleavage of fluorogenic substrates. Our strategy will utilize immunochemical enrichment techniques for both fungal proteases and cleavage products, using enrichment matrices containing monoclonal antibodies. We have demonstrated an immunoprotective role of an intracellular fungal protein that becomes accessible to the human immune system after cell-wall degradation by pulmonary enzymes such as chitinases. Based on this finding, we will test the hypothesis that human chitinases may be induced during fungal infections, and that the level of chitinase (and other lytic activity), varies from patient to patient and depends on genetic and pathophysiological conditions. In accordance with an approved clinical protocol, we propose to obtain a set of clinical bronchoalveolar lavage fluids, blood, and urine samples from patients with suspected fungal infections. These samples will be used for the development and evaluation of signal-amplifying biomarker assays for invasive pulmonary aspergillosis. The results of this research are expected to enable translation into novel clinical applications for better diagnosis, prognosis, monitoring and ultimately control of invasive fungal infections. The research proposed here is designed to identify signal-amplifying biomarkers that will be useful for the rapid and sensitive diagnosis of invasive fungal infections. Invasive fungal infections, such as invasive pulmonary aspergillosis, present a major problem during cancer therapy, following organ transplantation, for AIDS patients and other severe diseases that weaken the human immune defenses. The results of this exploratory study should enable translation into clinical applications that will improve the diagnosis of invasive fungal infections, which has the potential to save the lives of several thousand Americans and tens of thousands patients worldwide per year.