Diagnostic and Prognostic Biomarkers in Pneumonia

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，我们研究了肺炎动物模型的血液和灌洗液中的蛋白质生物标记物。我们研究了侵袭性肺曲霉病的兔模型(蛋白质组学2010；10：4270-4280)和葡萄球菌肺炎的犬模型(Am J Physiol心脏环Physiol 2007；293；H2487-500)。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。 我们最近开发了新的基于质谱学的方案来检测肺炎患者的支气管肺泡灌洗液中的细菌肽。识别对病原体具有特异性和唯一性的多肽生物标志物为检测BAL中的细菌提供了一种灵敏度更高的方法。我们已经开发了两种方法(自上而下和自下而上的方法)，可以应用于临床样本，以快速鉴定革兰氏阴性病原体。前一种方法需要去除白细胞，使用高效液-质联用(LC/MS)，并将生成的离子去卷积到数据库(Biotyper)，该数据库可用于微生物的鉴定。自下而上的方法是基于从基因组数据创建理论上的胰酶核心多肽组，并与通过LC/MS-MS分析的细菌胰酶产生的多肽进行比较。通过蛋白质组数据库分析(Unipept，pBLAST)完成对特定菌株或细菌物种的多肽的特异性，然后用标记的靶标多肽与裂解细菌进行实验验证。我们最近使用这种方法基于LC-MS/MS分析消化肽(Clin Chem)来快速鉴定鲍曼不动杆菌的菌株特异性多肽标记。2016年6月；62(6)：866-75。)这些方法的临床应用正在进行中。 我们总共有来自437个不同受试者的588个临床样本。在437名受试者中，有1名受试者明确表示，他们的样本不能与外部调查人员分享。所有受试者都接受了临床指示的支气管镜检查，没有纯粹收集样本进行研究。到目前为止，注册的儿科科目总数为30个。 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。到目前为止，在感兴趣的病例中登记的包括：曲霉58种、P.jirovei 63、巨细胞病毒61、分枝杆菌41和细菌(革兰氏阴性或革兰氏阳性)117。大约50%-60%的这些感染是由一种以上的微生物病原体(即曲霉菌和巨细胞病毒)引起的。只有一种肺部病原体的感染比合并感染的情况要少一些。目前正在分析新的支气管肺泡灌洗液和血液样本。

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 have investigated protein biomarkers in blood and lavage from animal models of pneumonia. We have studied a rabbit model of invasive pulmonary aspergillosis (Proteomics 2010;10: 4270-4280) and a canine model of staphylococcal pneumonia (Am J Physiol Heart Circ Physiol 2007;293;H2487-500). Exploring these model systems will facilitate our identification of candidate biomarkers across species. We have recently developed new mass spectrometry-based protocols to detect bacterial peptides in bronchoalveolar lavage from patients with pneumonia. Identifying peptide biomarkers that are specific and unique for a pathogen offers the possibility of a method with higher sensitivity to detect bacteria in BAL. We have developed two methods (top down and bottom up approaches) that can be applied to clinical samples in order to rapidly identify Gram-negative pathogens. The former approach requires the removal of leukocytes, use of high performance liquid chromatography mass spectrometry (LC/MS) and deconvolution of the resultant ions to a database (Biotyper) that can be interrogated for the identification of the microorganism. The bottom up approach is based upon the creation of a theoretical tryptic core peptidome from genomic data and comparison with peptides generated from tryptic digests of bacteria that are analyzed by LC/MS-MS. The specificity of the peptides for a particular strain or species of bacteria is done by proteomic database analysis (Unipept, pBLAST) and then validated experimentally with lysed bacteria with labeled targeted peptides. We have recently used this approach to rapidly identify strain-specific peptide markers of Acinteobacter baumannii based on LC-MS/MS profiling of digested peptides (Clin Chem. 2016 Jun;62(6):866-75.). Clinical application of these methods is ongoing. We have a total of 588 clinical samples from 437 different subjects. Of the 437 subjects, 1 subject specified that their samples could not be shared with outside investigators. All subjects underwent the clinically indicated bronchoscopies and no samples were collected purely for research. The total number of pediatric subjects enrolled to date is 30. Approximately one half of the participants have a specific microbiologic diagnosis as a cause of their pulmonary infiltrates. Enrollment thus far in the cases of interest includes: Aspergillus species 58, P. jiroveci 63, Cytomegalovirus 61, Mycobacterial species 41, and Bacteria (gram negative or gram positive) 117. Approximately 50-60% of these infections occur with more than one microbial pathogen (i.e. Aspergillus species and cytomegalovirus). Infections with only a single pulmonary pathogen are somewhat less common than co-infection states. New samples of the bronchoalveolar lavage and blood are currently being analyzed.