Development of a mouse model of cashew anaphylaxis.
First, our cashew protein extraction method was characterized by analyzing subsequent protein extracts on SDS-PAGE (Figure S1 ). The obtained electrophoretic profile was identical to that of the initial cashew flour as well to that of commercial cashew protein extracts. Moreover, major allergens (Ana o 1/2/3) were retrieved at molecular weights and proportions identical to what has been described previously.10,11 Using this extract, two routes of sensitization were investigated: oral and skin. The evaluation of skin as a sensitization route was prompted by previous data suggesting that skin is a proposed portal of entry for peanut allergens, especially in patients suffering from atopic dermatitis.12 Here, mice were treated by laser microporation to mimic eczematous skin before topical application of allergens. Both routes of sensitization induced cashew-specific IgE, IgG1 and IgG2a (Figure 1A ). However, IgE titers were significantly higher in mice sensitized orally. Conversely, IgG2a titers were significantly higher in mice sensitized cutaneously. Following oral challenge, anaphylactic reactions were similar between the two routes (Figures 1B and 1C ). Importantly, both routes were able to promote mast-cell degranulation following oral challenge, as evidenced by a significant increase in mMCP-1 and mMCP-7 levels. However, the greatest increases were observed in orally sensitized mice (Figures 1D and 1E ). There is strong evidence for IgE-dependent anaphylaxis and little evidence for IgG-dependent anaphylaxis in humans. Conversely, a consistent role for IgG in anaphylaxis has been demonstrated in mice.13 Thus, we considered the oral route as the most clinically relevant since it gave the surest indicators of IgE-mediated anaphylaxis (highest IgE titers and strongest mast-cell activation). To validate this, orally sensitized mice received anti-IgE or anti-IgG blocking antibodies 24 hours prior to oral challenge. As expected, anti-IgE was able to limit anaphylactic symptoms and significantly reduced mast-cell degranulation (Figure 2 ). Although anti-IgG was also able to reduce temperature drop (Figure 2A ), it had no impact on diarrhea nor mast-cell activation (Figures 2B, 2C and 2D ). Overall, these data confirmed that IgE is the main mediator of anaphylaxis in orally sensitized mice, but that IgG is also involved, albeit to a more modest level.