Lymphocyte cell death requires encapsulation of active caspase-1 with intact microvesicles.

<p>Whole blood was stimulated with LPS (1 µg/ml) for 1 h and microvesicles were isolated. Lymphocytes from healthy donors were then incubated overnight with microvesicles isolated from unstimulated blood, i.e. control microvesicles (CMV), or LPS (LMV), and LPS + YVAD (YVAD-LMV) treated whole blood. Intact microvesicles from LPS treated whole blood were also disrupted by mild homogenization (Ruptd. LMV) and analyzed for induction of lymphocyte apoptosis. Lymphocytes were also either left unstimulated (-LPS) or subjected to LPS directly and analyzed for cell death. Cell death was measured by LDH (<b>A</b>) and Annexin V/PI assays using flow cytometry (<b>B</b>) (n = 3). Representative data of apoptosis of lymphocytes by flow cytometry using Annexin V/PI assay (<b>C</b>).</p

Active caspase-1 is released in plasma microvesicles during sepsis.

<p>Plasma samples caspase-1 concentrations between critically ill control patients (n = 16) and septic patients (n = 34) (<b>A</b>). Microvesicles isolated on day 1 show a good correlation between plasma caspase-1 and MV caspase-1 (p<0.001) (<b>B</b>) There was no significant difference between caspase-1 concentrations in plasma and MVs between control patients and septic patients. However, when analyzed for caspase-1 activity there was significantly higher caspase-1 activity on day 1 (<b>C</b>) and on day 3 (<b>D</b>) between the septic patients and those that were critically ill (p<0.001 for both days).</p

Release of caspase-1 in microvesicles from endotoxin stimulated whole blood.

<p>Whole blood was stimulated with LPS for different times (0, 0.25, 0.5, 1 and 3 h) and microvesicles were isolated from the plasma of the stimulated blood. Caspase-1 was detected in the microvesicles by immunoblot (<b>A</b>) and ELISA (<b>B</b>). To show specificity, caspase-1 detection was also performed after blocking the antisera with excess recombinant caspase-1 before immunoblot (<b>C</b>).</p

Demographics Patient Cohort.

<p>All values presented as n (%) or median (IQ range) as appropriate.</p><p>Demographics of patients enrolled within 24 hours of initiation of mechanical ventilation and/or sepsis onset. Groups were compared by Wilcoxon Rank Sum for continuous variables or Pearson X² for dichotomous variables. Septic patients had a higher incidence of shock and subsequently higher SAPS II scores on the first hospital day. Control patient diagnosis included: seizure (2), stroke (1), lung cancer (1), drug overdose (1), carbon monoxide poisoning (1), pulmonary embolism (1), COPD without pneumonia (3), cirrhosis (1), and cardiovascular disease (5).</p