Nasal lavage procedure and processing
Nasal lavage samples were collected at baseline, and 1, 6, 24, 48 and 72 h post-challenge as described by Naclerio et al . (16). Briefly, 5 ml of a prewarmed (37°C) saline solution (0.9% w/v NaCl) was instilled into each nostril with a 10 ml syringe, attached by tubing to a nasal adapter. The subjects were seated in a forward-flexed neck position to prevent fluid from reaching the nasopharynx. The fluid was withdrawn into the syringe and flushed back into the nasal cavity 3 times. Mean [SD] recovery of nasal lavage fluid (10 ml for each washing) was 6.9 ml [0.9] for allergen and 6.8 ml [0.9] for placebo. The nasal lavage samples were kept on ice and then immediately centrifuged at 4°C for 10 min at 300 x g to generate a supernatant which was stocked at -80° C for later analysis. The cell pellet was resuspended in Dulbecco’s phosphate-buffered saline (DPPS) (Lonza). Total numbers of leukocytes were counted by using an improved Neubauer haemocytometer and cell numbers were normalized to nasal lavage volume.