Cardiolipin cl

Human LL-37 (molecular mass, 4.5 kDa, pI: 10.6) was obtained from Sigma-Aldrich (St. Louis, MO, USA). LL-37 was resuspended in milliQ water to a final concentration of 1 mg/ml and aliquots were stored at -20°C. Rough lipopolysaccharide (Re 595, Re-LPS), from Salmonella enterica serotype Minnesota, and cardiolipin (CL) were obtained from Sigma-Aldrich. Palmitoyloleoyl-phosphatidylethanolamine (POPE) and palmitoyloleoylphosphatidylglycerol (POPG) were obtained from Avanti Polar Lipids (Birmingham, AL, USA). The organic solvents used to dissolve the lipids were of HPLC grade from Scharlau (Barcelona, Spain). Chocolate agar plates for the growth of nontypeable Haemophilus influenzae strains were from bioMérieux (Marcy l’Etoile, France). The fluorescent dye Sytox Green was from Molecular Probes (Eugene, OR, USA). Propidium podide (PI) and the P2X7 inhibitor, A-438079, were from Sigma-Aldrich. Human IL-8 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). All other reagents were of analytical grade and were from Sigma-Aldrich.

To detect IgM and IgG antibodies to lipids we used a method published previously (25 (link)) with minimal modifications (described below). We incubated the wells with one of the following lipids: L-α-phosphatidylcholine (PC), 3-sn-phosphatidylethanolamine (PE), L-α-phosphatidylinositol (PI), 3-sn-phosphatidyl-L-serine (PS), N-Acyl-4-sphingenyl-1-O-phosphorylcholine (SM), 3-O-suphohexylceramide (SUL) and diphosphatidylglycerol, or cardiolipin (CL) (Sigma-Aldrich, St. Louis, MO, USA). Samples were diluted 1/100 in blocking solution and added to the wells in triplicate. We detected the presence of IgM or IgG to lipids in serum samples using the secondary antibodies anti human IgM (Jackson ImmunoResearch) or anti human IgG (Jackson ImmunoResearch), respectively. Positive sera were defined when the optic density (OD) was higher than the third quartile 3 (Q3) of the control group.

An aCL ELISA was performed according to our previous publication [19 ] and was sensitive to β2GP1-independent and β2GP1-dependent aCLa. Polystyrene plates (polySorp, Nunc, Roskilde, Denmark) were covered with either 20 µL/well of 50 µg/mL cardiolipin (CL) (Sigma-Aldrich, Steinheim, Germany) in ethanol or ethanol alone (as a control well). After that, the wells were blocked by 1.0% bovine serum albumin (BSA) in 0.05 M of Trizma buffer pH-8.5. The serum samples were instilled in the wells for an hour after diluting to 1:50 in RPMI medium with 0.5% BSA and 0.01% Tween-20. The amount of bounded aCLAb was determined by incubation with horseradish peroxidase-conjugated goat anti-human IgG (λ chain specific) and a reaction of peroxidase with TMB. The optical density (OD) was read at λ = 450 nm using a Multiscan device (LabSystems, Finland). Values below 10 GPL for CL were considered as negative (>20 GPL were considered as positive) and were excluded from the investigation.