Because intestinal absorption of food protein can trigger an allergic reaction, the effect of wheat proteins on intestinal epithelial cell permeability was evaluated and the abilities of these ...proteins in native or pepsin-hydrolyzed state to cross the epithelial cell monolayer were compared. Enterocytic monolayers were established by culturing Caco-2 cells, a model of enterocytes, on permeable supports that separate the apical and basal compartments. Proteins were added into the apical compartment, and the transepithelial resistance (TER) was measured; proteins that crossed the cell monolayer were detected in the basal medium by ELISA. Wheat proteins did not alter the cell monolayer. TER and Caco-2 cell viability were conserved, and the passage of dextran was prevented. Native and pepsin-hydrolyzed forms of ω5-gliadin and lipid transfer proteins were detected in the basal medium. The results suggest that these two major allergens in food allergy to wheat were able to cross the cell monolayer by the transcellular route. Keywords: Allergy; Caco-2; crossing; intestinal barrier; LTP; ω5-gliadins; wheat proteins
Background
Gluten proteins can be modified by deamidation to enhance their solubility and technological applications. However, severe allergic reactions have been reported after the consumption of ...food products containing deamidated gluten (DG) in subjects tolerant to wheat. This work aimed to characterize allergen profiles for these patients in comparison with those of patients allergic to wheat and to identify IgE‐binding epitopes.
Methods
Sera were obtained from 15 patients allergic to DG and from nine patients allergic to wheat proteins (WP). IgE‐binding profiles were characterized both in ELISA and in a humanized rat basophilic leukaemia (RBL) cell model. Epitopes were mapped on γ‐ and ω2‐gliadin sequences by Pepscan, and effect of glutamine/glutamic acid substitutions was studied.
Results
Compared to the heterogeneous pattern of allergens detected by IgE from patients allergic to WP, responses of patients allergic to DG were homogeneous. In ELISA, all the sera displayed IgE binding to deamidated γ‐ and ω2‐gliadins and deamidated total gliadins, frequently with high concentrations. These modified proteins induced RBL degranulation with most of the sera from DG‐allergic patients. A consensus epitope was found on native γ‐ and ω2‐gliadins (QPQQPFPQ); it was repeated several times in their sequences. The substitution of two or three glutamines of this epitope into glutamic acid at positions Q3 or Q4 and Q8 (QPEEPFPE) increased its recognition the best.
Conclusion
Allergy to DG is a separate entity from wheat allergy. It can be evidenced by strong IgE binding to deamidated gliadins or peptides of the type QPEEPFPE.
Antigenic profiles obtained by ELISA with IgE from patients with wheat food allergy (WFA) established that major allergens are albumins/globulins (AG) for children suffering from atopic ...eczema/dermatitis syndrome (AEDS), omega5-gliadins for adults suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA), anaphylaxis or urticaria and low-molecular-weight (LMW) glutenin subunits for patients with anaphylaxis. We aimed to characterize a new mast cell transfectant for its ability to degranulate with wheat proteins and patient sera and compare these results to those obtained by ELISA.
Thirty sera from patients with WFA were tested: 14 with AEDS (group 1) and 16 with WDEIA, anaphylaxis or urticaria (group 2). An IgE Fc receptor (FcepsilonRI) humanized rat RBL-2H3 line was established by transfection with cDNAs encoding alpha-, beta- and gamma-subunits for the human IgE receptor.
A humanized RBL clone was selected for its capacity to express mRNA alpha-, beta- and gamma-subunits of FcepsilonRI, to bind allergen-specific human IgE and to degranulate. In group 1, sera induced enhanced degranulation with AG extract, but rarely reacted with gliadins and glutenins. In group 2, half of the sera showed degranulation with LMW glutenins whereas the AG fraction and lipid transfer proteins were rarely positive. omega5-Gliadins did not appear as a major allergen in degranulation assays, although functional allergen-specific IgE was measurable in appreciable amounts.
Our data demonstrate that in wheat food allergen evaluation, correlation exists between mast cell degranulation and IgE measurements, depending on the type of allergen. Therefore, the biological activity of some allergen types may also be affected by other parameters.
At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. To characterize the epitopes of different wheat kernel allergens: ...α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit.The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations.
