Endotoxin levels bound to PBMC and in plasmaThe levels of endotoxin in lysates of PBMC are presented in Figure Figure3.3. LPS was detected in PBMC lysates of 71.4% of AAS patients, with levels significantly higher than apply for it in CAS patients. Highest levels of LPS were observed after blood reperfusion (T4) in AAS patients and decreased progressively at POD1 and POD2. On the other hand, the CAS group showed no significant presence of LPS in their PBMC during the observational period. Plasma levels of LPS were also measured in AAS patients. Only 57.1% of the plasmas were positive and the detected levels were much lower than those found in PBMC (data not shown).Figure 3Assessment of endotoxin (LPS) associated with circulating peripheral blood mononuclear cells (PBMC) in AAS and CAS patients.

Endotoxin associated with patients’ PBMC was measured using a Limulus amebocyte assay. T1 (before anesthesia), T2 (before incision), …Comparison of the reliability of the assays for the detection of NOD2 agonist and endotoxinIn order to examine the reliability of detection of the NOD2 agonist and endotoxin tests, we compared the positive responses to the tests based on the detection limits. The detection limits of the LAL test in PBMC and plasma were 5 pg/ml and 1.6 pg/ml, respectively. The natural structure derived from PGN following translocation is unknown and may be different from that resulting from enzymatic digestion and purification performed in vitro. We avoided extrapolating the levels of luciferase activity to a standard curve of biochemically purified PGN.

Our test was expressed as fold increase as compared with the plasma before surgery, each patient being his own control. The detection limit of the NOD2 agonist test showed a 1.57-fold increase in luciferase activity as compared with the value at T1 (before anesthesia). This detection limit was calculated on the basis of the mean �� 2SD of the fold increase at T2 in control patients. For reference, the mean �� SEM and median fold increase for various PGN (5.0 ��g/ml) incubated in healthy plasma were 1.76 �� 0.19 and 1.68, respectively (Figure (Figure1b1b).In all AAS and CAS patients, we compared the detection reliability for the NOD2 agonist test in plasma with the endotoxin test in PBMC. In AAS patients, 90.5% (19 out of 21 patients) were positive in the NOD2 agonist test, and 71.4% (15 out of 21 patients) were positive in the LAL test (PBMC-associated LPS). In CAS patients, 23.8% (5 out of 21 patients) were positive in the NOD2 agonist test, and 19% (4 out of 21 patients) in the LAL test (Figure (Figure4).4). From Entinostat this result, we can conclude that the NOD2 agonist detection test was more reliable than the endotoxin test.