Blood samples were collected from a peripheral vein and kept on ice. Plasma was collected by centrifugation at 800 g for 15 min at 4°C, aliquoted, and stored at -70°C until the analysis day. A multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (Bio-Plex Human Cytokine Assay; Bio-Rad Inc., Hercules, CA, USA). Cytokines measured were: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, CXCL8 (IL-8), IL-10, IL-12 (p70), IL-13, IL-17, granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), monocyte chemoattractive protein (MCP-1/CCL2), macrophage inflammatory protein (MIP-1β/CCL4), and TNF-α. Briefly, 20 μl plasma samples were diluted 1:4 and incubated with antibodycoupled beads. Complexes were washed, then incubated with biotinylated detection antibody and, finally, with streptavidin-phycoerythrin prior to assessing cytokine concentration titres. Concentrated human recombinant cytokine was provided by the vendor (Bio-Rad Laboratories). A range of 1.95-32,000 pg/ml recombinant cytokines was used to establish standard curves and to maximize the sensitivity and the assay dynamic range. Cytokine levels were determined using a multiplex array reader from Luminex™ Instrumentation System (Bio-Plex Workstation from Bio-Rad Laboratories). The analyte concentration was calculated using software provided by the manufacturer (Bio-Plex Manager Software).