In this study, in an attempt to determine IL-17 could mediate the asthma allergic reaction associated with A. simplex infection and to characterize the mechanism of innate immune response, we analyzed the immune responses in an experimental airway

inflammation mouse model treated with A. simplex larva excretory–secretory (ES) selleck chemicals llc proteins. The Anisakis type I larvae were collected manually from the viscera, flesh and body cavities of naturally infected blue whiting (Micromesistius poutassou), and were thoroughly washed in sterile phosphate-buffered saline (PBS). After collection, to prevent any contamination with the host material, the worms were thoroughly and carefully washed several times over a 3 h period in PBS. Anisakis simplex larvae were classified on Quiazon’s criteria (21). They were then introduced into sterile flasks with serum-free RPMI 1640 medium supplemented with antibiotics (100 μg/mL penicillin/streptomycin; Gibco, Grand Island, NY, USA). The culture Gamma-secretase inhibitor was then maintained for seven

consecutive days at 37°C in 5% CO2. It was confirmed that, during this time, all of the larvae remained alive and evidenced good mobility. Following centrifugation (12 000 g for 30 min), the supernatants were concentrated by pressure applied in a concentrator (Amicon, Millipore Corporations, Billerica, MA, USA) with 3000 Da pore size membranes. Various proteins (3 kDa to above 100 kDa) were detected in SDS page gel electrophoresis. The find more unnecessary excessive salts were eliminated from collected medium using HiTrap Desalting™ (Amersham Bio-Sciences AB, Uppsala, Sweden) and dialyzed against PBS for 24 h with continuous agitation in a cold room to eliminate any antibiotic remnants. Lipopolysaccharide (LPS) was depleted (endotoxin levels <0·01 μg/mL) from ES proteins using Detoxi-Gel Affinity Pak prepacked columns (Pierce Biotechnology, Rockford, IL, USA), in accordance

with the manufacturer’s instructions. RNase I (6 mg) from bovine pancreas (EC 3·1·4·22; 50 Kunitz units/rag; BDH Chemicals Ltd, Poole, England), and RNase A type III (10 mg) (RNase C; Sigma-Aldrich, Saint Louis, MO, USA) were dissolved in 1 mL of PBS (pH 7·4). Then, 2 mL (10 μg/mL) of ES proteins and 0·1 mL of RNase A and C solution were mixed and incubated for 1 h at room temperature. Chicken egg OVA (Sigma-Aldrich) were reconstituted in sterile PBS at 1 mg/mL and stored at −20°C. For intranasal challenge, 10 μL (10 μg) of ES proteins was added to 40 μL (40 μg) of OVA immediately prior to intranasal administration. C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were induced with airway inflammation by ES proteins for six total challenges, as described previously (22,23). One day after the final challenge, the mice were killed for analysis of bronchoalveolar lavage fluid (BALF). At the time of lavage, the mice were killed with 200 μL of ketamine : lumpun : PBS (2 : 3 : 5).