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Characterization of Pulmonary Microvesicles Bryan Prahl and Dr. James Burritt University of Wisconsin-Stout Menomonie, Wisconsin Methods: Fig. 1: Microscopy of pulmonary microvesicles using a Zeiss Axioscope 2-Plus microscope. Fig. 2: Measure of superoxide production using a Veritas luminometer . Fig. 3: Protein analysis of pulmonary microvesicle samples at different time points from BALF of C57 mice, using a 4-15% Tris-HCl gel electrophoresis. X, XO A 1500000 X, XO X X, XO, SOD MCLA Rgt 1000000 RLU •Pulmonary exosomes have reported to regulate the immune system through several mechanisms. •Pulmonary microvesicles are similar to exosomes, but twenty times larger on average. •Microvesicles are unreported to the best of our knowledge. •Our data suggest pulmonary microvesicles do not produce antimicrobial reactive oxygen species, but does upregulate a specific protein when exposed to a pathogen such as Aspergillus fumigatus. •We are collecting further data to better describe these unreported pulmonary vesicles. Proteins Associated with Microvesicles Superoxide Production Morphology of Microvesicles 500000 0 0 10 20 30 Time (min) B BALF - MCLA 75000 MV 2 Hours MV 7 Hours MCLA 50000 RLU Overview: 25000 A A A 250 150 100 75 B C D E F 50 37 25 20 15 10 0 0 25 50 75 100 Summary •Pulmonary microvesicles have not been described before in BALF to the best of our knowledge. •Microvesicles appear to lack the ability to produce superoxide for immune defense. Time Figure 1 key points: Figure 2 key points: Figure 3 key points: • Pulmonary microvesicles are typically 2 microns in size. • They are roughly 20X larger than previously described pulmonary exosomes, suggesting they are an entirely different particle. • Xanthine and Xanthine Oxidase are positive controls for superoxide production. • Microvesicles at both the 2 and 7 hour time points do not show the ability to produce superoxide. • A: Protein Standards • B: 7 hour time point • C: 4 hour time point • D: 2 hour time point • E: 0 hour time point • F: Naïve • Upregulation of a 67kD protein is seen as time increases in mice inoculated with Aspergillus fumigatus •Proteins associated with microvesicles are upregulated as time increases when the host has been inoculated with Aspergillus fumigatus. •Future analysis will involve studying recruitment of microvesicles and further characterization.