Leticia Tordesillas

Skin exposure promotes a Th2-dependent sensitization to peanut allergens

Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitis-associated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life.

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes : Peach Pru p 3 allergen as a model

Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Characterization of peach thaumatin‐like proteins and their identification as major peach allergens

Background Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross‐reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin‐like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen.

The Involvement of Thaumatin-Like Proteins in Plant Food Cross-Reactivity: A Multicenter Study Using a Specific Protein Microarray

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Plant Lipid Transfer Protein Allergens: No Cross-Reactivity between Those from Foods and Olive and Parietaria Pollen

Background: Cross-reactivity among plant food allergens belonging to the nonspecific lipid transfer protein (LTP) family is well known. In contrast, the relationship among these allergens and their putative homologs from olive (Ole e 7) and Parietaria (Par j 1) pollen has not been clarified. Methods: Sera with specific IgE to LTP allergens were obtained from peach-, mustard- and olive pollen-allergic patients. Purified LTP allergens from foods (peach, apple, mustard and wheat) and pollens (olive, mugwort and Parietaria) were tested by ELISA and ELISA-inhibition assays. Results: Plant food LTP-allergic patients showed a significantly higher number of sera (89–100 vs. 33–64%) with specific IgE and mean specific IgE levels (0.30–1.56 vs. 0.21–0.34 OD units) to the 4 food LTP allergens tested than to olive Ole e 7 and Parietaria Par j 1 pollen. ELISA-inhibition assays indicated cross-inhibition between food LTP allergens but no cross-reactivity between these allergens and Ole e 7 and Par j 1, or, even more, between the LTP allergens from olive and Parietaria pollen. Conclusions: LTP allergens from olive and Parietaria pollen cross-react neither with allergenic LTPs from plant foods nor between themselves. Therefore, both pollens do not seem to be related with the LTP syndrome.

Epicutaneous immunotherapy induces gastrointestinal LAP+ regulatory T cells and prevents food-induced anaphylaxis

Background: The attempt to induce oral tolerance as a treatment for food allergy has been hampered by a lack of sustained clinical protection. Immunotherapy by nonoral routes, such as the skin, may be more effective for the development of maintained tolerance to food allergens. Objective: We sought to determine the efficacy and mechanism of tolerance induced by epicutaneous immunotherapy (EPIT) in a model of food‐induced anaphylaxis. Methods: C3H/HeJ mice were sensitized to ovalbumin (OVA) orally or through the skin and treated with EPIT using OVA‐Viaskin patches or oral immunotherapy using OVA. Mice were orally challenged with OVA to induce anaphylaxis. Antigen‐specific regulatory T (Treg)‐cell induction was assessed by flow cytometry using a transgenic T‐cell transfer model. Results: By using an adjuvant‐free model of food allergy generated by epicutaneous sensitization and reactions triggered by oral allergen challenge, we found that EPIT induced sustained protection against anaphylaxis. We show that the gastrointestinal tract is deficient in de novo generation of Treg cells in allergic mice. This defect was tissue‐specific, and epicutaneous application of antigen generated a population of gastrointestinal‐homing LAP+Foxp3− Treg cells. The mechanism of protection was found to be a novel pathway of direct TGF‐&bgr;–dependent Treg‐cell suppression of mast cell activation, in the absence of modulation of T‐ or B‐cell responses. Conclusions: Our data highlight the immune communication between skin and gastrointestinal tract, and identifies novel mechanisms by which epicutaneous tolerance can suppress food‐induced anaphylaxis. GRAPHICAL ABSTRACT Figure. No caption available.

Molecular basis of allergen cross-reactivity: Non-specific lipid transfer proteins from wheat flour and peach fruit as models☆

Peach non-specific lipid transfer protein (Pru p 3; nsLTP) has been characterized as the major food allergen in the adult Mediterranean population. Its wheat homologous protein, Tri a 14 has a relevant inhalant allergen in occupational bakers asthma. Different sensitization patterns to these allergens have been found in patients with this latter disorder. The objective of the present study was to characterize IgE epitopes of Tri a 14 and to compare them with those of Pru p 3 using three complementary strategies: the analysis of IgE-binding capacity of decapeptides bound to membrane, the identification of mimotopes using a phage display random peptide library, and the analysis of the surface electrostatic potential of both allergens. Thus, synthetic overlapping decapeptides, covering the Pru p 3 and Tri a 14 amino acid sequences, were used to identify sequential regions involved in recognition of IgE from bakers asthma patients sensitized to both nsLTPs. A phage display library was screened with total IgE from the same patients, and positive clones sequentially selected using the purified allergens, allowed to identify mimotopes (conformational epitopes) of Tri a 14 and Pru p 3. Both sequential regions and mimotopes were localized in the corresponding 3D molecular surface and their electrostatic properties were analyzed. Common sequential regions with strong IgE-binding capacity (residues 31-40 and 71-80) were identified in Tri a 14 and Pru p 3, whereas regions Tri a 14(51-60) and Pru p 3(11-20) were found specific of each allergen. A major conformational epitope (mimotope), L34H35N36R39S40S42D43G74V75L77P78Y79T80, which comprised the two common sequential epitopes, was located in Tri a 14, and a very similar one in Pru p 3. However, differences were detected on the surface electrostatic potential of both mimotopes: a first part (around residues 31-45) showed similar positive features in both allergens, whereas a second part (around residues 74-80) was markedly negative in Tri a 14 but neutral-positive in Pru p 3. Tri a 14 and Pru p 3 have a similar conformational region involved in IgE-binding, although their electrostatic features are different. Additionally, common and specific sequential IgE-binding regions were mapped in both allergens. These findings could be instrumental in understanding the cross-reactivity and specificity of sensitization to both homologous allergens.

Characterization of IgE epitopes of Cuc m 2, the major melon allergen, and their role in cross-reactivity with pollen profilins

Background Plant profilins are described as minor allergens, although with some exceptions in foods such as melon, watermelon or orange. In fact, they could be responsible for many cross‐reactions among distantly related species. This is likely to be a consequence of the presence of common epitopes.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

Transport of Pru p 3 across gastrointestinal epithelium – an essential step towards the induction of food allergy?

Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated.