Figure 1

Experimental schedule [ 12 ]. (A) In a first set of experiments, mice were exposed to proteins on intact skin. Mice were anaesthetized and their abdomens were carefully shaved with an electric clipper. The remaining hairs were removed using a depilatory cream (Veet®) and the skin was then gently cleaned with water. Skin treatments were performed 4 to 6 days after depilation to be sure that no more lesions or inflammation are present [12]. Once a week for 6 weeks, 100 μg of purified BLG diluted in 30 μl of DPBS or 30 μl of milk containing the equivalent amount of BLG was then spread on the depilated skin of anaesthetized mice (n = 19-20/group). After 40 minutes, skin was gently cleaned with water. Control mice received 30 μl of PBS (n = 19/group). According to the same schedule, other groups of mice were exposed via the respiratory tract by intranasal administration of 50 μg of purified BLG diluted in 10 μl of DPBS or 10 μl of milk containing the equivalent amount of BLG (n = 20/group). Control mice received 10 μl of PBS. In both the experimental groups, after last administration, 6 mice per group were sacrificed. Spleen and ILN were removed and cells isolated. Systemic specific cytokine secretion was then assessed after ex vivo restimulation with 20 μg of BLG. (B) After the six cutaneous or intra-nasal exposures, the remaining mice from each of the experimental groups were separated into 2 subgroups (n = 7-8/subgroup). One group was experimentally sensitized by gavage with 6 mg of milk proteins mixed with Cholera toxin (10 μg/administration) [18]. The other mice received 6 mg of milk proteins in the absence of CT. Control mice consisted of mice pre-exposed with PBS then gavaged with milk +/− CT. Some mice were not exposed at all (naive mice; n = 10).