Diagnostic and Prognostic Biomarkers in Pneumonia

Clinical Center

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Abstract

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. 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. 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 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. A total of 568 cases 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. Enrollment thus far in the cases of interest includes: Aspergillus species 57, P. jiroveci 60, Cytomegalovirus 58, Mycobacterial species 37, and Bacteria (gram negative or gram positive) 111. 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.