Diagnostic and Prognostic Biomarkers in Pneumonia

肺炎的诊断和预后生物标志物

• 批准号: 9154076
• 负责人: ANTHONY F. SUFFREDINI
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9154076
• 关键词: Acute; Acute Lung Injury; Adult Respiratory Distress Syndrome; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Aspergillosis; Aspergillus; Bacteria; Bacterial Infections; Biological Assay; Biological Markers; Biological Models; Blood; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Bronchoscopy; Canis familiaris; Cells; Childhood; Clinical; Clinical Microbiology; Communities; Complement; Cytolysis; Cytomegalovirus; Data; Databases; Diagnosis; Diagnostic; Diagnostic Procedure; Elements; Engraftment; Enrollment; Etiology; Excision; Genomics; Goals; Gram-Negative Bacteria; Heart; High Pressure Liquid Chromatography; Immune response; Immunity; Immunoassay; Individual; Infection; Inflammation; Informed Consent; Ions; Irrigation; Label; Laboratories; Leukocytes; Link; Lung; Lung Inflammation; Lung diseases; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular Profiling; Mycoses; Oryctolagus cuniculus; Outcome; Parasitic infection; Participant; Patients; Pattern; Peptides; Pneumonia; Population Study; Process; Prognostic Marker; Protein Analysis; Protein Databases; Proteins; Proteomics; Protocols documentation; Research; Research Personnel; Resolution; Sampling; Sensitivity and Specificity; Serum; Source; Specific qualifier value; Specificity; Specimen; Staining method; Stains; Staphylococcal Pneumonia; Sterility; Suspension substance; Suspensions; Syndrome; System; Techniques; Technology; Time; Two-Dimensional Gel Electrophoresis; Virus Diseases; base; candidate identification; clinical Diagnosis; clinical application; co-infection; expectation; interest; liquid chromatography mass spectrometry; microbial; microorganism; mycobacterial; novel diagnostics; pathogen; protein expression; response

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. Clinical application of these methods is ongoing. We have a total of 588 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 62, 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.

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。 这些特征图谱将基于质谱、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，我们研究了肺炎动物模型血液和灌洗液中的蛋白质生物标志物。我们已经研究了侵袭性肺曲霉病的兔模型（Proteomics 2010;10：4270-4280）和葡萄球菌肺炎的犬模型（Am J Physiol Heart Circ Physiol 2007;293;H2487-500）。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。 我们最近开发了新的基于质谱的方案，以检测肺炎患者支气管肺泡灌洗液中的细菌肽。鉴定对病原体特异性和独特的肽生物标志物提供了具有更高灵敏度的方法来检测BAL中的细菌的可能性。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，以快速识别革兰氏阴性病原体。前一种方法需要去除白细胞，使用高效液相色谱质谱法（LC/MS）和将所得离子解卷积到数据库（Biotyper）中，可以查询该数据库以鉴定微生物。自下而上的方法基于从基因组数据创建理论胰蛋白酶核心肽组，并与通过LC/MS-MS分析的从细菌的胰蛋白酶解物产生的肽进行比较。肽对特定菌株或细菌物种的特异性通过蛋白质组学数据库分析（Unipept，pBLAST）进行，然后用具有标记的靶向肽的裂解细菌进行实验验证。这些方法的临床应用正在进行中。 我们共有来自437个不同受试者的588个样本。 在437例受试者中，1例受试者说明其样本不能与外部研究者共享。 所有受试者均接受了有临床指征的支气管镜检查，未采集纯粹用于研究的样本。 迄今为止入组的儿科受试者总数为30例。 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。 到目前为止，感兴趣病例的入组包括：曲霉菌属58种、耶氏巴斯德菌62种、巨细胞病毒61种、分枝杆菌属41种和细菌（革兰氏阴性或革兰氏阳性）117种。 这些感染中约有50-60%发生在一种以上的微生物病原体（即曲霉菌属和巨细胞病毒）。 只有一种肺部病原体的感染比合并感染的情况少见。 目前正在分析新的支气管肺泡灌洗液和血液样本。