, CA, USA) per immunization, while fenugreek immunized mice received 4.2 mg fenugreek protein with 10 μg CT per immunization. Blood samples before challenge were obtained from v. saphena lateralis on day 33/34. Challenges were performed with a large dose of one of the protein extracts (peanut, lupin, fenugreek and soy). Based on previous experience, the doses were 25 mg p.o. and 5 mg intraperitoneally (i.p.). Challenge

with the primary allergen was carried out by both routes in the two models. In the lupin model, challenge with cross-reactive legumes was performed both p.o. and i.p., but as the responses did not seem to differ with regards to anaphylactic reactions or mast cell responses between the two challenge routes in this model, only i.p. Selleck RAD001 challenges were performed with cross-reactive legumes in the fenugreek model. The p.o. dose was divided into two equal doses given 30 min apart. Some mice were not challenged and are referred to as immunized only. Control mice were either treated with CT only (sham immunized) or left untreated (naïve mice) (Table 1). check details Assessment of clinical anaphylactic reactions.  Anaphylactic symptoms were evaluated continuously from the start of the challenge until 30 min after the i.p. challenge or the second p.o. challenge. The scoring system described by Li et al. [27] was used: 0 – no symptoms; 1 – scratching and rubbing around the nose and head; 2 – puffiness

around the eyes and mouth, diarrhoea, pilar erecti, reduced activity and/or decreased activity with an increased respiratory rate; 3 – wheezing, laboured respiration, cyanosis around the mouth and tail; 4 – no activity after prodding or tremor and convulsion; 5 – death. The mice were exsanguinated immediately after the assessment of

the anaphylactic reactions. The clinical anaphylactic reactions were analysed by Ordinal Regression Oxalosuccinic acid using Statistical Package for Social Sciences (spss version 14.0; SPSS Inc., Chicago, IL, USA). Because of a quasi-complete separation in the data, contingency table analysis (Fisher exact test) was used to validate the statistics of the Ordinal Regression. Serum mouse mast cell protease-1 (MMCP-1) assay.  Serum levels of mouse mast cell protease-1 (MMCP-1) were determined at exsanguination with an ELISA kit (Moredun Scientific Ltd., Edinburgh, UK) and performed according to the manufacturer’s instructions. Results were analysed by one-way anova on log transformed data, and significant differences between the groups were determined by the Holm-Sidak method. Results are presented as box-plots showing the median, 25th–75th percentile, 10th–90th percentile and outliers. Total and allergen-specific IgE analyses.  Due to the inclusion of several sub-studies (Table 1), sera were analysed for total IgE before (49 mice), after (67 mice) or both before and after challenge (89 mice).