Cytokine assays

In the first part of the study, we tested plasma samples for IFN-γ, IL-8, and TGF-β; in the second part, we tested in addition for IL-10, IL-1β, TNF-α, IL-6, and MCP-1 in serum (see Additional file 1: Table S1).

The concentration of IL-8, IFN-γ, and TGF-β in plasma samples was measured by ELISA using commercially available kits (BD™OptEIA; BD Biosciences, Rockville, MD) according to the manufacturer’s instructions. The concentration of cytokine or chemokine was calculated based on standard curves provided with the kits, and results were expressed in pg/ml.

Concentrations of IL-1β, IL-6, TNF-α, IL-10, and MCP-1 were measured with multiplex analyte profiling technology (xMAP) using the Merck-Millipore, USA HSCYTOMAG-60 K Kit (Luminex®, magnetic beads Millipore, Billerica, MA) read on MAGPIX equipment (MILLIPLEX®, Millipore). The assay kits included a standard curve and two controls (high and lower concentration) for each cytokine. For both ELISA and xMAP platforms, all samples were tested in duplicate and the average values were used in the analysis.