Allergen, adjuvant and anaesthetics.  Chicken egg ovalbumin (OVA), grade VII, was from Sigma-Aldrich, St. Louis, MO, USA. The Al(OH)3 adjuvant (Alhydrogel) was from Brenntag Biosector, Denmark. Two different types of anaesthetics were used; Isoflurane (Isoba vet; Intervet/Schering-Plough Animal Health, Lysaker, Norway) and a cocktail named ZRF, consisting of Zoletil Forte (Virbac International, Carros Cedex, France), Rompun (Bayer Animal Health GmbH, Leverkusen, Germany) and Leptanal (Janssen-Cilag International NV, Beerse, Belgium) and isotonic saline. Isoflurane gas was administered as a 3.5% mixture with

medical O2 in a coaxially ventilated open mask to effect. learn more The ZRF cocktail contains 18.7 mg PKC inhibitor Zolazepam, 18.7 mg Tiletamine, 0.45 mg Xylazine and 2.6 μg fentanyl per ml and was administered to effect with a nominal dose of 0.1 ml/10 g i.p. Intraperitoneal sensitization study.  Groups of mice received first sensitization at ages 1, 6 and 20 weeks and are hereafter referred to as 1-, 6- and 20-week-old mice. The mice were sensitized by i.p. administration of 0, 0.1, 10 or 1000 μg OVA in 1 mg Al(OH)3 in Hank’s balanced salt solution (HBSS) in a 0.1-ml bolus. Two weeks later, they were boosted i.p. with the corresponding dose, but without Al(OH)3 in 0.1 ml. All mice in the

1000-μg groups suffered from severe anaphylactic chock and died or were killed upon booster administration. One week later, a blood sample much was taken from the remaining groups, which

were then anaesthetized with isoflurane and challenged by i.n. instillation of 10 μg OVA in 35 μl HBSS per day for 3 days. Three days after the last challenge, the mice were anaesthetized with ZRF before the chest was opened and blood drawn by heart puncture. Lung-draining mediastinal lymph nodes (MLNs) were collected, lungs lavaged and the lymph nodes and bronchoalveolar lavage fluid (BALF) kept on ice. Intranasal sensitization study.  Groups of 1-, 6- and 20-week-old mice were sensitized i.n. [13] with 10 μg OVA with 120 μg Al(OH)3 in HBSS on days 1, 2 and 3 (Table 1). On days 22, 23 and 24, they were boosted i.n. with 10 μg OVA in HBSS. All i.n. exposures were performed under isoflurane anaesthesia. On day 27, blood was drawn by heart puncture. Nose- and lung-draining lymph nodes [superficial cervical (SLNs) and MLNs, respectively [14]] were collected and kept on ice; lungs were lavaged and thereafter collected for histopathology. The BALF was also kept on ice. In a concurrent study, control groups of age- and sex-matched mice were immunized i.n. with OVA alone without Al(OH)3 (Table 1). This OVA-only exposure did not induce sensitization or any significant responses, when compared with OVA + Al(OH)3-treated mice. For clarity, the OVA-only groups are not presented, except for a few observations. Determination of instillation volumes in the intranasal sensitization study.  The mice of the different age groups were exposed according to Table 1.