The individual PD20FEV1 × 10 was then used for the

subsequent Segmental Allergen Provocation (SAP). Inhaled and segmental allergen challenges were separated by at least 4 weeks. Bronchoscopy was performed as previously described [29, 42]. A volume of 2.5 ml of 0.9% saline was instilled into the anterior basal segment of the left lower lobe (B8 left) and one of the segments of the lingula (B4 or B5 left). Allergen diluted in 2.5 ml of saline was instilled into the anterior basal segment of the right lower lobe (B8 right) and the medial or lateral segment of the right middle lobe (B4 or B5 right). After 10 min, Erastin mouse bronchoalveolar lavage was performed in the anterior basal segments of the right and left lower lobes. Patients were re-bronchoscoped Panobinostat cost at different time points: In the first group, the second lavage was performed after 18 h in segments B4 or B5 right and left. Some of these patients also participated in the second part of the trial. This second group was lavaged 10 min and 42 h after segmental allergen challenge (Table 1). In the third arm of the trial, four patients were

lavaged 10 min and 162 h after allergen challenge. In patients who participated repeatedly the segmental allergen challenges were separated by at least six months. In all patients, peripheral blood was taken before bronchoscopy. From seven healthy subjects and seven patients with allergic asthma, 250-ml whole blood was drawn and mixed well with heparin. Cell subtypes were separated by Ficoll centrifugation. PBMC-CD14+ were harvested, and after washing and counting monocytes were separated via immunomagnetic BCKDHA separation by AutoMACS system (Miltenyi Biotec GmbH, Germany) after labelling with CD14 antibody (Miltenyi Biotec GmbH). CD14+

monocytes were washed; purification was controlled by flow-cytometry (94–98% purified monocytes, contamination with lymphocytes was <2%), and 5 × 105 cells per well were cultured in 1 ml RPMI 1640 medium (GIBCO, Paisley, Scotland, UK) + 10% FCS (Seromed, Berlin, Germany) + 1% penicillin/streptomycin (Biochrom AG, Berlin, Germany) at 37 °C and 5% CO2. Cells differentiated to macrophages in about 5 days. Medium was exchanged every 2 days. The above-mentioned PBMC-CD14+ cells (5 × 105 cells in 1 ml) were stimulated with either human IL-17 (50 ng/ml), LPS (10 ng/ml), leukotriene D4 (LTD4) (10−11 M) or a combination of LPS and LTD4 for a duration of 6, 12 and 24 h. Cells were also stimulated with LTD4 in the presence of the leukotriene antagonist Montelukast. LTD4 was added to cell cultures 30 min after stimulation with Montelukast (10−11 M), and cultures were incubated for 6, 12 and 24 h. Cell culture supernatants were stored at −20 °C until sCD14 measurement with an ELISA kit (IBL Hamburg, Germany) according to the manufacturer’s instructions. Data were analysed by SPSS software package. Results are reported as median (range) or as single values and median (Figs. 2–5).