Venous blood sample
inflammation, wheeze
BLOOD, OXIDATIVE STRESS, STERILE, INFLAMMATION
Six millilitres of venous blood will be sampled at baseline visit and at the end visit. One to two hours prior to the blood puncture, lidocaine 1% gel with 4 × 4 cm plaster will be applied. This blood will be used for.allergy testing: total immunoglobulin E (IgE) and determination of specific IgE antibodies to inhalant allergens (ImmunoCAP allergens gx3 (grass pollen), tx9 (tree pollen), wx3 (weed pollen), mx1 (moulds), d1 (house dust mite), e1 (cat dander), e5 (dog dander) (Phadiatop test; Phadia, Uppsala, Sweden)white blood cell count and absolute number of eosinophils will be determinedleukocyte subset analysis by flow cytometry: Extended phenotyping will be performed to evaluate B-cell maturation and differentiation (CD19, CD27, CD38, and IgD), and T-cell maturation and differentiation (CD3, CD4, CD8, CD28, CD45RA and CD127). These panels enable the distinction of pro- and anti-inflammatory lymphocyte subsets, the degree of memory formation as a marker for pathogen exposure, and early senescence. In addition, monocyte subsets (classical and non-classical) and dendritic cell subsets (myeloid and plasmacytoid dendritic cells) will be evaluated (CD11c, CD14, CD16, CD123, HLA-Dr, BDCA-2, and BDCA-3)isolation of peripheral blood mononuclear cells (PBMC’s): PBMC’s will be stored in liquid nitrogen for in vitro activation with distinct stimuli followed by analyses of the produced cytokine repertoire.isolation of eosinophils: In a subset of children (80 children with wheeze, 40 healthy controls) peripheral blood eosinophils will be isolated by FACS sorting. Blood eosinophils will be isolated from 2 ml of EDTA blood using an adapted FACS sorting strategy based on Mori et al. within 24 h of blood sampling, sorting Siglec8 + and CD193 + cells [whole transcriptome RNA sequencing for gene expression of markers of inflammation and oxidative stress. One millilitre of venous blood will be transferred into sterile National Lab Cryovials. Invitrogen™ RNA