Effects of plasma containing EV on leukocyte adhesion to endothelial cells from flow.

<p>HUVEC was pre-treated for 24h with plasma EV from healthy controls or trauma patients, with or without treatment with TNF (5U/ml) for the last 4h. Peripheral blood mononuclear cells (PBMC) or neutrophils (PMN) were perfused at a wall shear stress of 0.05 Pa for 4min followed by 5min washout, and adherent cells counted. Results for patient plasma were expressed relative to healthy control plasma tested on the same day with the same HUVEC and leukocytes. A, B. PBMC adhesion after the absence or presence TNF. C, D. Neutrophil adhesion after the absence or presence TNF. All data are mean ± SEM (number of patients tested at each time in brackets). In A, the ratio of adhesion for patient:control plasma was significantly >1 for all samples tested (p<0.01), but the increase was not statistically significant at any particular day.</p

Concentrations and size distributions of EV in plasma of healthy controls and patients analysed by nanoparticle tracking analysis (NTA) and flow cytometry (FC).

<p>A. NTA analysis of the concentration of EV in plasma for controls and patients from GHS (○) or SIRS (●) at different times after injury. The data are shown as median ± inter-quartile range (number of donors in brackets). Kruskal-Wallis test showed significant variation between samples (p<0.01); * = p<0.05, ** = p<0.01 compared to control by post-hoc Dunn test. B. The frequency distribution of EV diameter derived by NTA in plasma from controls and patients at different times after injury. Data are for controls (n = 13), Day 1 (pooled GHS and SIRS, n = 36) and Day 3 (pooled GHS and SIRS, n = 27). Friedman test showed significant variation with particle diameter and subject group both (p<0.01). Subsequent Kruskal-Wallis test for each particle diameter class showed signficant variation between subject groups for 50–100, 100–150 and 150-200nm; * = p<0.05, ** = p<0.01 for Day 1 and for Day 3 compared to control by post hoc Dunn test. C. Flow cytometry analysis of the concentration of EV in plasma for controls and patients at different times after injury. The data are shown as median ± inter-quartile range (number of donors in brackets). D. Correlation of EV concentration measured by FC with EV concentration at diameter >400nm measured by NTA. Linear regression showed a significant correlation between FC and NTA concentrations (R² = 0.24; P<0.01).</p

Concentrations of EV from different cellular origins in plasma of healthy controls and patients analysed by flow cytometry.

<p>EV in plasma from patients or controls were labelled with antibodies recognising markers for A. platelets (CD41⁺), B. leukocytes (CD45⁺) or C. endothelial cells (CD144⁺) and analysed by flow cytometry. In each case, Kruskal-Wallis test showed significant variation between samples but not with time post injury for samples from patients alone; * = p<0.05, ** = p<0.01 compared to control by post hoc Dunn test. D. Plot of numbers of CD41⁺ vs. CD42⁺ EV counts for all samples. Linear regression showed a significant correlation (R² = 0.1909, p<0.01) with equation: CD41⁺ = 2.1x10⁵ + 0.25 CD42b⁺.</p

Percentage of extracellular vesicles with diameter >400nm detected by NTA.

<p>Percentage of extracellular vesicles with diameter >400nm detected by NTA.</p

Characteristics of patients.

<p>Characteristics of patients.</p

Gates used for counting EV by flow cytometry.

<p>A. Side-scatter vs. forward scatter plot for megamix fluorescent polystyrene beads of sizes 500nm, 900nm and 3μm. R3 defines the gate used for counting EV. R1 incorporates 3μm beads and the approximate position of intact platelets. B. Side-scatter vs. forward scatter plot for plasma extracellular vesicles. Approximately 95% of detected particles with side scatter and forward scatter greater than the lower limits set by R3, fell within R3.</p