Diagnostic and Prognostic Biomarkers in Pneumonia

本研究（04-CC-0119）的目的是分析肺浸润患者的支气管肺泡灌洗液和血清，以发现与特定类型肺部疾病相关的新生物标志物和蛋白质表达模式。支气管肺泡灌洗是获得下呼吸道样本以评估肺浸润以诊断感染、恶性肿瘤或非感染性炎症的标准方法。收集灌洗液后，临床微生物实验室将有形成分（即病原体和细胞）浓缩用于染色和培养，并丢弃支气管肺泡灌洗液上清液。然而，上清液是蛋白质和其他分子的丰富来源。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志物的重要来源。对支气管肺泡灌洗液和血清中蛋白质质量谱和生物标志物鉴定的分析可能有助于开发新的诊断方法，并扩展我们对感染性原因引起的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治疗临床适应症的所有患者。这将有助于采集反映与肺部疾病相关的社区获得性和机会性病原体谱的BAL样本。此外，对一系列非感染性肺部过程（例如急性肺损伤、急性呼吸窘迫综合征和植入综合征）的分析对于制定灵敏度和特异性指标非常重要。 我们目前使用两种不同的质谱平台来分析生物样本，包括BAL液或血清/血浆。Ciphergen蛋白质芯片阵列（Ciphergen Biosystems，Inc.，Palo Alto，CA，USA）基于对特定表面的结合亲和力选择性分级样品，所述特定表面包括疏水性、阳离子、阴离子和固定化金属亲和捕获。这种表面化学优化技术被称为表面增强激光解吸电离（SELDI），当与质谱法结合时，称为SELDI-TOF。我们将用二维凝胶电泳和更高分辨率的质谱系统（Bruker Daltonics Ultraflex MS/MS）来补充我们的SELDI蛋白表达谱。 通过开发与特定微生物诊断相关的BAL液大型数据库，我们计划定义蛋白质表达特征响应谱，以区分肺部感染和炎症的特定病因。这些特征谱将基于质谱、二维凝胶电泳和悬浮阵列技术。由于宿主免疫力、采样时间效应和外部因素（如抗生素或抗炎治疗）的差异，个体宿主对感染的反应存在变异性，因此需要大型数据库。培养阴性BAL液的特征将具有类似的意义，以帮助确定肺部炎症的非感染性病因。 次要目的是对在支气管镜检查时从患者采集的血清进行蛋白质组学分析。目的是将血清蛋白质组学谱与BAL蛋白质组学谱联系起来，以确定侵入性较低的技术是否可以预测浸润病因，其灵敏度和特异性与BAL谱相当。 为了补充患者研究，我们将研究肺炎动物模型血液和灌洗液中的蛋白质生物标志物。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识别跨物种的候选生物标志物。 迄今为止，共有240例患者入组本研究。大约一半的参与者有特定的微生物诊断作为其肺浸润的原因。目前正在分析支气管肺泡灌洗和血液样本。

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 two different mass spectrometry 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 electrophores and a higher resolution mass spectrometry system (Bruker Daltonics Ultraflex MS/MS). 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 240 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.