Diagnostic and Prognostic Biomarkers in Pneumonia

本研究(04-CC-0119)的目的是分析肺浸润性疾病患者的支气管肺泡灌洗液和血清，以发现与特定类型的肺部疾病相关的新的生物标志物和蛋白表达模式。支气管肺泡灌洗是获取下呼吸道样本以评估肺浸润物的标准方法，以诊断感染、恶性肿瘤或非感染性炎症。临床微生物学实验室收集灌洗液后，浓缩形成的要素(即病原体和细胞)进行染色和培养，并丢弃支气管肺泡灌洗液上清液。然而，上清液中含有丰富的蛋白质和其他分子。我们假设，支气管肺泡灌洗将是反映宿主-病原体相互作用的生物标记物的重要来源。分析支气管肺泡灌洗液和血清中的蛋白质质量分布和生物标志物有助于开发新的诊断方法，并扩大我们对感染性原因所致肺部炎症机制的理解。 研究人群将包括在临床中心和附属研究医院接受支气管镜检查以确定临床指征的所有患者。这将有助于获得反映与肺部疾病有关的一系列社区获得性和机会性病原体的BAL样本。此外，对一系列非感染性肺过程(如急性肺损伤、急性呼吸窘迫综合征和植入性综合征)的分析对于制定敏感性和特异性的衡量标准也很重要。 我们目前使用几种不同的平台来分析生物标本，包括BAL液或血清/血浆。赛弗吉蛋白质芯片阵列(美国加州帕洛阿尔托的赛弗吉生物系统公司)根据与专业表面的结合亲和力选择性地对样品进行分级，包括疏水、阳离子、阴离子和固定化金属亲和力捕捉。这种表面化学优化技术被称为表面增强激光解吸电离(SELDI)，当与质谱学相结合时，被称为SELDI-TOF。我们将用二维凝胶电泳和更高分辨率的质谱仪(Bruker Daltonics Ultraflex MS/MS)来补充我们的SELDI蛋白质表达谱。此外，我们还使用基于高强度抗体的蛋白质阵列来询问血液和BAL中的候选生物标记物。 通过开发与特定微生物学诊断相关的BAL液大型数据库，我们计划定义区分肺部感染和炎症的特定病因的蛋白质表达特征反应谱。这些标志性的图谱将基于质谱学、二维凝胶电泳法和悬浮阵列技术。由于宿主免疫力、采样时间效应以及抗生素或抗炎疗法等外部因素的差异，个体对感染的反应存在变异性，因此需要一个大型数据库。培养阴性的BAL液谱将有助于确定肺部炎症的非感染性病因。 第二个目标是对支气管镜检查时从患者身上收集的血清进行蛋白质组学分析。我们的目标是将血清蛋白质组图谱与BAL蛋白质组图谱联系起来，以确定一种侵入性较小的技术是否可以预测浸润性病因，其敏感性和特异性与BAL图谱相当。 为了补充患者研究，我们将研究肺炎动物模型的血液和灌洗液中的蛋白质生物标记物。我们目前正在研究侵袭性肺曲霉菌病的兔模型和葡萄球菌肺炎的犬模型。探索这些模型系统将有助于我们跨物种识别候选生物标记物。 到目前为止，共有490名患者参加了这项研究。大约一半的参与者有一种特殊的微生物学诊断，这是他们肺部浸润性病变的原因。目前正在对支气管肺泡灌洗和血液样本进行分析。

The objective of this study (04-CC-0119)is to analyze bronchoalveolar lavage and serum from patients with lung infiltrates in order to discover new biomarkers and protein expression patterns that are associated with specific types of pulmonary disease. Bronchoalveolar lavage is a standard method to obtain lower airway samples to evaluate pulmonary infiltrates in order to diagnose infection, malignancy or non-infectious inflammation. After collecting the lavage, the clinical microbiology laboratory concentrates the formed elements (i.e. pathogens and cells) for stains and culture and discards the bronchoalveolar lavage supernatant. The supernatant however is a rich source of proteins and other molecules. We hypothesize that bronchoalveolar lavage will be an important source of biomarkers that reflect host-pathogen interactions. The analysis of protein mass profiles and biomarker identification in bronchoalveolar lavage and serum may help develop new diagnostic methods and extend our understanding of mechanisms of lung inflammation due to infectious causes. The study population will include all patients undergoing bronchoscopy for clinical indications at the Clinical Center and affiliated study hospitals. This will facilitate the acquisition of BAL samples that reflect a spectrum of community-acquired and opportunistic pathogens associated with pulmonary disease. In addition analysis of a range of non-infectious pulmonary processes (e.g. acute lung injury, acute respiratory distress syndrome and engraftment syndrome) is important to develop measures of sensitivity and specificity. We currently use several different platforms for analysis of biologic specimens including BAL fluid or serum/plasma. The Ciphergen Protein Chip Arrays (Ciphergen Biosystems, Inc., Palo Alto, CA, USA) selectively fractionates samples based on binding affinity to specialized surfaced including hydrophobic, cation, anion and immobilized metal affinity capture. This technique of surface chemistry optimization is termed Surface Enhanced Laser Desorption-Ionization (SELDI) and when combined with mass spectrometry, termed SELDI-TOF. We will complement our SELDI protein expression profiles with two-dimensional gel electrophoresis and a higher resolution mass spectrometry system (Bruker Daltonics Ultraflex MS/MS).In addition we are using high intensity antibody based protein arrays to interrogate candidate biomarkers in blood and BAL. By developing a large database of BAL fluid linked to specific microbiologic diagnoses, we plan to define protein expression signature response profiles that distinguish specific etiologies of lung infection and inflammation. These signature profiles will be based on mass spectrometry, two-dimensional gel electrophoresis and suspension array technologies. Because of the variability associated with individual host responses to infection due to differences in host immunity, sampling time effects, and external factors such as antibiotic or anti-inflammatory therapies, a large database will be required. The profiles of culture-negative BAL fluid will be of similar interest to assist in defining non-infectious etiologies of lung inflammation. A secondary objective is to perform proteomic analysis on serum collected from patients at the time of bronchoscopy. The goal is to link serum proteomic profiles to BAL proteomic profiles to determine whether a less invasive technique can predict infiltrate etiology with comparable sensitivity and specificity to BAL profiles. To complement the patient studies we will investigate protein biomarkers in blood and lavage from animal models of pneumonia. We are currently studying a rabbit model of invasive pulmonary aspergillosis and a canine model of staphylococcal pneumonia. Exploring these model systems will facilitate our identification of candidate biomarkers across species. A total of 490 patients have been enrolled in this study to date. Approximately one half of the participants have a specific microbiologic diagnosis as a cause of their pulmonary infiltrates. The bronchoalveolar lavage and blood samples are currently being analyzed.