A. Provide supporting data showing that diabetic MPs contribute to the increases in the propagation of the virus compared to non-diabetic MP. A1. Demonstrate higher number of cellular internalizations of S-protein-bound DB MPs than that of S-protein-bound normal MPs. Experiments will be conducted in microvessels developed in microfluidics. Our preliminary data showed no significant differences in ACE2 expression and ACE2-mediated MP interaction with S-protein between normal and DB MPs. The main factors that contribute to DB MP-mediated increases in virus entry are their much higher quantity and more adhesive surface due to their largely externalized phosphatidylserine (PS) than that of normal MPs. GFPtagged S-protein will be incubated with MPs isolated from normal and DB plasma and flow cytometry will be used to confirm the S-protein-bound MPs. To demonstrate the quality differences between normal and DB MPs, equal amount of S-protein-bound DB and normal MPs will be perfused into the in vitro microvessels. Confocal images will be collected to compare the internalized S-protein-bound DB MPs with that of normal MPs by quantification of GFP fluorescence. To demonstrate the role of externalized PS on DB MPs in the MP adhesion and internalization, the S-protein-bound DB MPs will be pre-coated with a lipid binding protein, Annexin V, before perfusion into microvessels. We predict that precoating of DB MPs with Annexin V will prevent the DB MP adhesion to ECs and reduce their cellular internalizations. A2. Demonstrate DB MP-mediated increases in HCoV-NL63 infection and propagation when compared to normal MPs. HCoV-NL63 (BEI Resources) will be propagated in Caco-2 cells to produce a working virus stock. Cultured ECs will be incubated with HCoV-NL63 with serial dilutions (1:10-1000) in the presence of DB and normal plasma or equal number of isolated DB and normal MPs, respectively. The incubation of HCoV-NL63 alone serves as viral infection control. To demonstrate the specific role of DB MPs in facilitating viral infection from that of other components in DB plasma such as inflammatory mediators and cytokines, cultured ECs will be incubated with HCoV-NL63 in the presence of MP-free DB plasma and the results will be compared with DB plasma containing increased MPs. For rigor, efficiency of HCoVNL63 infection will be determined using three distinct methods. Total virus will be harvested from cells and supernatant at 96 hpi and quantified by both plaque assay and 50% tissue culture infectious dose (TCID 50) assay as previously published (PMID 19014487). Infection will also be quantified using a one-step, real-time, quantitative reverse transcription PCR assay on RNA harvested from cells at 96 hpi using a commercially available kit (Liferiver). Supernatant will be collected daily to examine the release of MPs and cytokines, serving as indicators of viral infection-induced EC activation. Results will be compared among groups (3-4 replicates per group for all p...