Epithelial Barrier: Protector and Trigger of Allergic Disorders.

The epithelial barrier has classically been considered as the only first line of defense against irritants, pathogens, and allergens. However, it is now known to play an essential role in the immune response to exogenous agents. In fact, recent reports postulate the epithelial barrier hypothesis as a possible explanation for the increasing incidence and severity of allergic diseases. The epithelial barrier preserves the isolation of internal tissues from potential external threats. Moreover, a coordinated interaction between epithelial and immune cells ensures the unique immune response taking place in mucosal tissues, which is reported to be dysregulated in allergic diseases. We and others have demonstrated that in severe allergic phenotypes, the epithelial barrier undergoes several histological modifications, with increased infiltration of immune cells, leading to dysfunction. This is common in atopic dermatitis, asthma, and food allergy. However, the precise role of the epithelial barrier in mucosal biology during progression of allergic diseases is not well understood. In this review, we aim to compile recent knowledge regarding the histological structure and immunological function of the epithelial barrier and to shed light on the role of this compartment in the onset and progression of allergic diseases.

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.

Immune Polarization in Allergic Patients: Role of the Innate Immune System.

Allergens come into contact with the immune system as components of a very diverse mixture. The most common sources are pollen grains, food, and waste. These sources contain a variety of immunomodulatory components that play a key role in the induction of allergic sensitization. The way allergen molecules bind to the cells of the immune system can determine the immune response. In order to better understand how allergic sensitization is triggered, we review the molecular mechanisms involved in the development of allergy and the role of immunomodulators in allergen recognition by innate cells.

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.

Bronchial Challenge With Tri a 14 as an Alternative Diagnostic Test for Baker's Asthma.

BACKGROUND: Baker's asthma (BA) is the most prevalent occupational respiratory disease in developed countries. It is caused by inhalation of wheat dust in the working environment and affects 1%-10% of workers in the baking industry. Diagnosis of BA is based on bronchial challenge with wheat, a technique that carries a high risk for patients. The wheat lipid transfer protein Tri a 14 is a major allergen in BA. OBJECTIVE: The aim of our study was to characterize Tri a 14 as a marker of BA in order to prevent patients from having to undergo bronchial challenge with wheat. METHODS: The study population comprised 55 patients selected at the Rio Hortega Hospital, Valladolid, Spain. Patients with BA were diagnosed using a skin prick test (SPT) with wheat and Tri a 14 and bronchial challenge test (BCT) with wheat. Patients with food allergy had a clear clinical history of allergy to peach confirmed by positive SPT to peach extract and Pru p 3. RESULTS: All patients in the BA group had a positive SPT result with wheat (100%), and most had positive results with Tri a 14 (95%). A positive BCT result with Tri a 14 was also observed in 22 of 27 of the patients with BA (82%). The response to Tri a 14 was specifically associated with BA. CONCLUSION: Tri a 14 is a good marker of BA and can be used in SPT and BCT as an alternative diagnostic method, thus avoiding bronchial challenge with wheat and reducing the risk associated with this technique.

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.