Immune Basis of Allergic Reactions to Food.

Food allergies are diseases where the normal tolerance response to oral antigens is altered. Recent advances have begun to uncover mechanisms that mediate sensitization to food allergens and maintenance of the disease. Production of alarmins by epithelial cells triggers a cascade that leads to allergen-specific IgE synthesis. IL-9 has also been shown to play a role in mast cell recruitment and amplification of the allergic response. In recent years, increasing evidence suggests that sensitization to food allergens can be developed via nonoral routes, in particular the skin, thus leading to the "dual exposure hypothesis". Environmental factors such as diet or microbiota can shape the immune system to promote tolerance or sensitization to food antigens. While the mechanism of primary tolerance to food antigens is quite clear, that leading to permanent tolerance in food-allergic individuals through immunotherapy is still under study. Understanding the mechanisms by which oral tolerance is suppressed and sensitization develops will help to identify new targets to develop combined therapies for the treatment of food allergies.

Mechanisms underlying induction of allergic sensitization by Pru p 3.

BACKGROUND: Recently, the nature of the lipid-ligand of Pru p 3, one of the most common plant food allergens in southern Europe, has been identified as a derivative of the alkaloid camptothecin bound to phytosphingosine. However, the origin of its immunological activity is still unknown. OBJECTIVE: We sought to evaluate the role of the Pru p 3 lipid-ligand in the immunogenic activity of Pru p 3. METHODS: In vitro cultures of different cell types (monocyte-derived dendritic cells [moDCs], PBMCs [peripheral blood mononuclear cells] and epithelial and iNKT-hybridoma cell lines) have been used to determine the immunological capacity of the ligand, by measuring cell proliferation, maturation markers and cytokine production. To study the capacity of the lipid-ligand to promote sensitization to Pru p 3 in vivo, a mouse model of anaphylaxis to peach has been produced and changes in the humoral and basophil responses have been analysed. RESULTS: The lipid-ligand of Pru p 3 induced maturation of moDCsc and proliferation of PBMCs. Its immunological activity resided in the phytosphingosine tail of the ligand. The adjuvant activity of the ligand was also confirmed in vivo, where the complex of Pru p 3-ligand induced higher levels of IgE than Pru p 3 alone. The immunological capacity of the Pru p 3 ligand was mediated by CD1d, as maturation of moDCs was inhibited by anti-CD1d antibodies and Pru p 3-ligand co-localized with CD1d on epithelial cells. Finally, Pru p 3-ligand presented by CD1d was able to interact with iNKTs. CONCLUSIONS AND CLINICAL RELEVANCE: The Pru p 3 lipid-ligand could act as an adjuvant to promote sensitization to Pru p 3, through its recognition by CD1d receptors. This intrinsic adjuvant activity of the accompanying lipid cargo could be a general essential feature of the mechanism underlying the phenomenon of allergenicity.

Impact of glutathione on the allergenicity of the peach lipid transfer protein Pru p 3.

BACKGROUND AND OBJECTIVE: The allergenic potential of proteins can be altered under various physicochemical conditions. Glutathione (GSH) is a reducing agent that is used as an antioxidant in food products. We aimed to characterize the natural folding of peach proteins and test the allergenicity of reduced and natural Pru p 3, the major peach allergen. METHODS: Pru p 3 was purified from peach, and its conformation was analyzed by means of circular dichroism. Using a thiol fluorescent probe, reduced proteins were detected in fresh peach. GSH-reduced Pru p 3 was tested in vitro for T-cell proliferation and in vivo using skin prick testing. RESULTS: GSH-reduced Pru p 3 produced variable skin prick reactions in peach-allergic patients. The proliferative response of peripheral blood mononuclear cells from allergic patients to reduced Pru p 3 tended to be less intense, whereas secretion of the cytokines IFN-γ, IL-5, and IL-10 was comparable. In a pool of sera from peach-allergic patients, reduction hardly impaired IgE-binding. Moreover, the stability of reduced Pru p 3 to gastrointestinal digestion was similar to that of the natural form. CONCLUSIONS: GSH can at least transiently reduce Pru p 3. We found that the effect of reduction on the allergenicity of Pru p 3 varied. Therefore, as an additive, GSH does not seem to eliminate the risk of reactions for peach-allergic patients.

Allergenic characterization of new mutant forms of Pru p 3 as new immunotherapy vaccines.

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.

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

BACKGROUND: 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. OBJECTIVE: To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. METHODS: This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. RESULTS: The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. CONCLUSION: These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. CLINICAL RELEVANCE: The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

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. OBJECTIVE: To identify members of the TLP family in peach fruit and to characterize putative allergens. METHODS: Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N-terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (beta-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). RESULTS: Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N-asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed beta-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. CONCLUSION: Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area.

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. OBJECTIVE: To characterize the B epitopes of Cuc m 2, a model of plant food profilin, using phage display techniques and to compare with other profilins, such as those of timothy grass and birch pollen, and human I profilin, to understand the mechanism of cross-reaction among members of this family. METHODS: IgE of melon-allergic patients was used to select clones from a phage display 12 mer peptide library. After two rounds of screening, Cuc m 2-specific clones were eluted and the DNA insertion sequenced. The residues of each clone were mapped on the Cuc m 2 surface to define a mimotope, which was also localized on the three-dimensional surfaces of other profilins. RESULTS: Seventeen melon-allergic patients were selected. Sera from each of them recognized the melon profilin, Cuc m 2, but the majority also recognized Phl p 12 or Bet v 2, timothy grass-, and birch-pollen profilins, respectively. A Cuc m 2 mimotope was defined and mapped onto its surface giving the following sequence: S(2)W(3)A(5)Y(6)D(9)H(10)T(111)P(112)G(113)Q(114)N(116)M(117)R(121)L(122). The homologous residues in Phl p 12 and Bet v 2 had almost identical sequences. By contrast, the homologous sequence in human profilin showed many differences. CONCLUSIONS: The identified mimotope could be involved in cross-reactions among food and pollen profilins. Many of these cross-reactions observed in the clinical realm could be explained by the presence of a common epitope found in food and pollen allergens. A new strategy of immunotherapy based on this IgE region could be used in alternative immunotherapy strategies.

Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma.

BACKGROUND: Baker's asthma is an important occupational allergic disease. Wheat lipid transfer protein (LTP) Tri a 14 is a major allergen associated with wheat allergy. No panel of wheat recombinant allergens for component-resolved diagnosis of baker's asthma is currently available. OBJECTIVE: To evaluate the potential role of recombinant Tri a 14 as a novel tool for the diagnosis of baker's asthma, and to test the heat and proteolytic resistance of the wheat LTP allergen. METHODS: A cDNA encoding Tri a 14 was isolated and sequenced, the recombinant allergen produced in Pichia pastoris and purified by chromatographic methods. Physicochemical and immunological comparison of the natural and recombinant forms of Tri a 14 was carried out by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry, circular dichroism (CD) analysis, IgE immunodetection, and specific IgE determination and ELISA-inhibition assays using a pool or individual sera from 26 patients with baker's asthma. Thermal denaturation and simulated gastrointestinal digestion of both Tri a 14 forms were checked by spectroscopic and electrophoretic methods, respectively, and biological activity by basophil activation test (BAT). RESULTS: Natural and recombinant Tri a 14 were similarly folded, as indicated by their nearly identical CD spectra and heat denaturation profiles. A high interclass correlation coefficient (0.882) was found between specific IgE levels to both Tri a 14 proteins in individual sera from baker's asthma patients, but a slightly lower IgE-binding potency of rTri a 14 was detected by ELISA-inhibition assays. Natural and recombinant Tri a 14 elicited positive BAT in two and one out of three patients, respectively. Heat denaturation profiles and simulated gastrointestinal digestion assays indicated that Tri a 14 displayed a high heat and digestive proteolytic resistance, comparable to those of peach Pru p 3, the model food allergen of the LTP family. CONCLUSIONS: Recombinant Tri a 14 is a potential tool for baker's asthma diagnosis, based on its physicochemical and immunological similarity with its natural counterpart. Wheat Tri a 14 shows a high thermal stability and resistance to gastrointestinal digestion.