The MP pellets were resuspended in 100 ��L of RPMI medium (containing Ruxolitinib Hepes and L-glutamine, from Lonza, Verviers, Belgium) and stored at -80��C until further analysis. Supernatant, containing the corresponding plasma of subjects were also kept and stored at -80��C to serve as the negative control. MP protein content was assessed by Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, M��nchen, Germany) and adjusted to the same values for each subgroup in the experiments.Endothelial cell cultureHuman umbilical vein endothelial cells (HUVEC) were obtained from Promocell? (Heidelberg, Germany). The cells were cultured. For incubation with MPs, 104 cells per well were cultured in endothelial cell growth medium advanced (Provitro, Berlin, Germany) at 37��C in a 5% CO2 humidified atmosphere overnight.

The following day cells were subjected to Optimem medium (Gibco, Invitrogen, Karlsruhe, Germany) and incubated with isolated MPs from resuscitated patients, healthy subjects or their corresponding plasma. MPs were added to equal protein content per well and incubated with HUVECs for 24 hours.Ex vivo detection of apoptosis in cultured endothelial cells by ELISADetection and quantification of apoptosis in HUVECs after incubation with MPs or plasma of resuscitated and healthy probands was performed by the Cell Death Detection ELISA? (Boehringer, Mannheim, Germany), according to the manufacturer’s instructions. This is a colorimetric one-step sandwich ELISA for relative quantification of apoptosis by detection of histone-complexed DNA fragments (mono- and oligonucleosomes) after cell lysis.

Statistical analysisContinuous variables are given as means �� standard error of the mean. Data of CPR, control patients and healthy were compared by Mann-Whitney U- test, as appropriate. Comparisons between the different study populations and time points were performed using one-way repeated-measures analysis of variance, including an all-pair wise comparison. Correlations between selected variables were estimated by Spearman’s rank correlation coefficient. Basic data and outcome were compared by Fisher’s exact test, Chi-square test, and Student’s t-test, respectively. Statistical significance was defined as a two-tailed P < 0.05. Analyses were performed with SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).

ResultsBaseline characteristics of patientsIn the group of resuscitated patients, mean duration of mechanical resuscitation was 21.8 �� 2.5 minutes and estimated no-flow time was 4.5 �� 0.8 minutes. The presenting rhythm was ventricular fibrillation or ventricular tachycardia in 56% versus asystole or pulseless electrical activity in 44%. Twenty three patients (64%) presented a presumable cardiac cause for cardiac arrest and 14 patients Cilengitide (39%) survived until hospital discharge and mean GOS of survivors was 4.4 �� 0.2 at discharge.