DETECTION AND CHARACTERIZATION OF EXTRACELLULAR VESICLES IN SPUTUM OF COPD PATIENTS AND HEALTHY CONTROL

<bold>Introduction:</bold> Only one study reported the presence of extracellular vesicles (EVs) in COPD patients’ sputum. <bold>Aims:</bold> To isolate and characterize EVs from COPD and healthy individuals' sputum and identify gene ontology annotations relative to biological pathways of the most expressed miRNAs in healthy lung/bronchus. <bold>Methods:</bold> 20 spontaneous sputum samples from COPD patients (m/f: 19/1) and three induced sputum samples from healthy controls (m/f: 2/1) were used for EVs isolation. The sputum supernatants were resuspended in PBS, precleared by centrifugation at 800g for 10 min at 4°C and passed through a 0.22-μm filter (Millipore). EVs were isolated by a standard membrane affinity spin column method (exoEasy maxi kit, Qiagen). EVs were characterized by assessing their morphology and size using Transmission Electron Microscopy (TEM) (Harvard University) and determining the CD9 and CD81 EV-markers with Western blot analysis (Tufts University). Applying the miTED database we retrieved the top 100 expressed miRNAs in healthy lung/bronchus. FunRich v3.1.3, GeneCodis and miEAA were used for the functional enrichment analysis (FEA) concerning BPs of the identified miRNAs. <bold>Results:</bold> EVs had a spherical shape ranging from 50-200 nm both in COPD and healthy individuals, according to TEM analysis. Enrichment of the EVs markers CD9 and CD81 both in healthy and COPD individuals was detected. Total EV-associated protein was increased in COPD patients compared to controls. FEA of the retrieved miRNAs was associated with response to bacterial and viral infections. <bold>Conclusions:</bold> EVs found in both COPD and control sputum, with higher EV-protein in COPD, suggest biomarker potential for infection studies.