Venous blood was taken with parents agreement after clinical examination of the children in the study center. The heparin blood for the determination of cytokines and neuropeptides was processed after a maximum of 4 hours storage at room temperature; the blood samples were stored at –80 °C until further analysis was carried out. The neuropeptides VIP, SOM and SP were determined using ELISA (Phoenix Europe, Karlsruhe, Germany) and the cytokines (IFN-γ, IL-4, IL-5 and IL-9) using cytometric bead array (CBA, BD Bioscience, Heidelberg, Germany) according to the manufacturers instructions and as described by Herberth and colleagues [14 (link)].
The transcription factors (GATA3, Tbet, FOXP3) and the regulatory factors (SOCS1, SOCS3) were determined after RNA extraction from 1 ml of heparinized blood (MagNA Pure LC System and MagNA Pure LC mRNA Isolation Kit, Roche Applied Science, Mannheim, Germany) using real-time PCR (Lightcycler, LC search Primer Kits, Roche Applied Science, Mannheim, Germany) as described by Herberth and colleagues [14 (link)].
The transcription factors (GATA3, Tbet, FOXP3) and the regulatory factors (SOCS1, SOCS3) were determined after RNA extraction from 1 ml of heparinized blood (MagNA Pure LC System and MagNA Pure LC mRNA Isolation Kit, Roche Applied Science, Mannheim, Germany) using real-time PCR (Lightcycler, LC search Primer Kits, Roche Applied Science, Mannheim, Germany) as described by Herberth and colleagues [14 (link)].