Measurement of inflammatory markers
According to the study protocol, 100 mg of tissue was diluted in 1 ml of
0.9% NaCl solution containing protease inhibitor cocktail (cOmplete™
Protease Inhibitor Cocktail, Roche Diagnostics, Mannheim, Germany).
Next, the tissue was homogenized (1000 rpm, 5 minutes, 4°C) and
centrifuged at 1500 g for 10 minutes at 4oC. The
protein concentration was measured in each sample using Coomassie Plus
(Bradford) assay kit (ThermoFischer Scientific, Darmstadt, Germany).
The chemokines Eotaxin/ CCL11, MIP-1α/ CCL3, MCP-3/ CCL7, ENA-78/ CXCL5,
TARC/ CCL17, PARC/ CCL18 and IP-10/ CXCL10 (Table 1 ) were
investigated. The chemokine concentrations were measured by using
commercially available LEGENDplex Multiple Chemokine Assay (Biolegend
San Diego USA).
The IL-5- and TNF-α concentrations were measured with the Luminex
100-system (Luminex, Austin, Texas, USA). The concentration of the
additional cytokines (IL-22, IL-17, and IFN-γ) was measured by using
commercially available ELISA kits from R&D Systems (Minneapolis, USA).
The concentrations of tissue eosinophil cationic protein (ECP) and total
IgE and IgE directed against a mixture of enterotoxins from
Staphylococcus aureus (S. aureus enterotoxin A, C, and toxic shock
syndrome toxin-1) were examined using UniCAP-system (Phadia, Uppsala,
Sweden). Myeloperoxidase (MPO) was measured with ELISA (BioCheck, Foster
City, Calif., USA). The parameters measured and detection limits are
presented in Table 2 . Values below the limit of detection were
considered to be negative.
In agreement with the general study protocol (12), the
IL-5 levels were retrospectively defined as follows: negative IL-5
level when under the concentration of 12.98 pg/mL (assay detection
limit), low IL-5 level (IL-5 <
100 pg/mL), moderate IL-5 level (IL-5 100 to 151 pg/mL)
or high IL-5 level (IL-5 > 151 pg/mL).