Human whole blood model

The whole blood model was carried out according to the procedure described by Ison. Whole human venous blood was taken from a healthy volunteer with informed consent, collected into sterile Vacutainer tubes containing sodium heparin anti-coagulant (Becton Dickinson Systems, New Jersey) and processed within 1h. Whole blood (1ml) was dispensed into sterile 24 well tissue culture plates (Greiner bio-one, Germany) and triplicate wells were treated with various concentrations of live bacteria, isolated OM 1 or LPS from meningococci 2 (Nm-LPS) or E. coli (Ec-LPS), in the presence of varying concentrations of CyP. In addition, control wells consisted of blood alone and blood with CyP only. The plates were incubated at 37oC, initially on a rocking table (20 rpm) for the first 3h and then without rocking for the remainder of the experiment(s). Samples were taken at 6 and 24h, centrifuged (13,000g, 5 min) and the plasma layer removed and stored at –20oC for analysis.

Measurement of cytokine production in human plasma
The levels of pro-inflammatory (interleukin (IL)-1α, IL-1β, IL-6 and tumour necrosis factor (TNF)- α), chemoattractant (monocyte chemoattractant protein (MCP)-1, IL-8 and regulated upon activation, normal T cell expressed and secreted (RANTES), anti inflammatory (IL-10 ) and growth factor related (granulocyte-macrophage colony stimulating factor, GM-CSF) cytokine proteins were quantified by sandwich immuno-assay, as described. A two-sample t-Test was used to compare the mean levels of cytokine secretion following particular treatments, with p<0.05 as significant. Isolation and stimulation of human monocyte-derived dendritic cells (mo-DC). Buffy coats were obtained from the Swiss Blood Center, Basel and ethical permission for the use of human primary cells was obtained from the Federal Office of Public Health, Switzerland. Monocytes were purified from peripheral blood mononuclear cells by positive sorting using anti-CD14 conjugated magnetic microbeads (Miltenyi). Mo-DCs were generated as described previously by culturing monocytes in RPMI medium supplemented with 10% (v/v) foetal bovine serum, GM-CSF (Gentaur) and IL-4 (33). Mo-DC (0.5x106 cells/ml) were stimulated with Nm-LPS or Ec-LPS (100ng/ml) in the absence or presence of CyP (2.5-20 μg/ml). Production of human TNF-α and IL-6 cytokines was measured in mo- DC supernatant fluids after stimulation for 20 h, using DuoSet ELISA Development kits (R&D Systems). Cytokine data are from measurements with standard deviations below 5%.

Human Jurkat cells (1x1072 , expressing human MD-2) were transfected as described previously, by electroporation (250V, 975μF; Gene Pulser II, Bio-Rad) with 1.5μg of expression vector encoding human TLR4 together with 1μg of 3xNF-κB Luc reporter vector (kindly provided by G. Natoli, IFOM, Milano, Italy). After 24h, the cells were washed, resuspended in 12 ml of fresh medium and plated onto 12 well plates with 0.8 ml of cells for each stimulatory condition (100ng/ml of Nm-LPS or Ec-LPS, in the absence or presence of 5- 8 μg/ml of CyP). After 8h of stimulation, the cells were harvested, lysed and reporter gene activity was measured using the Luciferase Assay System (Promega) and Veritas luminometer (Turner BioSystems), according to the manufacturers’ instructions. Jurkat cells transfected with empty vector together with 3×NF-κBLuc reporter vector were used as a mock-control. Luciferase activity in the presence of CyP is shown as a mean percentage of the activity observed from cells stimulated with LPS alone (100%), with error bars denoting the standard deviation.