RESUMEN
BACKGROUND: Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. OBJECTIVE: We show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. METHODS: We used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. RESULTS: NsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. CONCLUSIONS: Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.
RESUMEN
BACKGROUND: Poppy seed (PS) can be a cause of severe allergic reactions, especially in individuals with concurrent allergy to tree nuts and other seeds, but diagnostic criteria and sensitization patterns are lacking. OBJECTIVE: To assess the role of PS extract and individual allergens in diagnosing PS allergy and their cross-reactivities with tree nuts and buckwheat. METHODS: Our retrospective study included 36 PS-sensitized patients; 10 with a positive and 26 with a negative oral food challenge (OFC). We identified individual PS allergens and compared the diagnostic performance of specific IgE (sIgE) to PS extract with its allergens. Cross-reactivities between PS and related allergens from other seeds were assessed by a competitive enzyme-linked immunosorbent assay. RESULTS: We identified 4 novel PS allergens: Pap s 1 (vicilin), Pap s 1 (27-424) (α-hairpinin), Pap s 2 (legumin), and Pap s 3 (small hydrophilic seed protein). A positive OFC correlated with higher PS-sIgE levels and elevated sIgE levels for the PS allergens, except for Pap s 3. PS and α-hairpinin-sIgE effectively differentiated allergic from tolerant patients, with area under the curve values of 0.95 and 0.94. PS-sIgE >10.00 kUA/L exhibited 90% sensitivity and 73% specificity, whereas α-hairpinin-sIgE >2.60 kUA/L showed 100% sensitivity and 77% specificity. PS vicilin and legumin highly cross-reacted with hazelnut and buckwheat homologs, whereas α-hairpinin-sIgE cross-reacted with the related almond allergen. CONCLUSIONS: This is the most extensive study on PS allergy to date. PS and α-hairpinin-sIgE are highly sensitive indicators of clinical reactivity to PS, whereas vicilin and legumin-sIgE contribute to concurrent sensitization to hazelnut and buckwheat.