Oral myeloid cells uptake allergoids coupled to mannan driving Th1/Treg responses upon sublingual delivery in mice.

BACKGROUND: Polymerized allergoids coupled to nonoxidized mannan (PM-allergoids) may represent novel vaccines targeting dendritic cells (DCs). PM-allergoids are better captured by DCs than native allergens and favor Th1/Treg cell responses upon subcutaneous injection. Herein we have studied in mice the in vivo immunogenicity of PM-allergoids administered sublingually in comparison with native allergens. METHODS: Three immunization protocols (4-8 weeks long) were used in Balb/c mice. Serum antibody levels were tested by ELISA. Cell responses (proliferation, cytokines, and Tregs) were assayed by flow cytometry in spleen and lymph nodes (LNs). Allergen uptake was measured by flow cytometry in myeloid sublingual cells. RESULTS: A quick antibody response and higher IgG2a/IgE ratio were observed with PM-allergoids. Moreover, stronger specific proliferative responses were seen in both submandibular LNs and spleen cells assayed in vitro. This was accompanied by a higher IFNγ/IL-4 ratio with a quick IL-10 production by submandibular LN cells. An increase in CD4+ CD25high FOXP3+ Treg cells was detected in LNs and spleen of mice treated with PM-allergoids. These allergoids were better captured than native allergens by antigen-presenting (CD45+ MHC-II+ ) cells obtained from the sublingual mucosa, including DCs (CD11b+ ) and macrophages (CD64+ ). Importantly, all the differential effects induced by PM-allergoids were abolished when using oxidized instead of nonoxidized PM-allergoids. CONCLUSION: Our results demonstrate for the first time that PM-allergoids administered through the sublingual route promote the generation of Th1 and FOXP3+ Treg cells in a greater extent than native allergens by mechanisms that might well involve their better uptake by oral antigen-presenting cells.

The allergenic structure of the thaumatin-like protein ole e 13 is degraded by processing of raw olive fruits

Background: The thaumatin-like protein (TLP) Ole e 13 in raw olive fruit is responsible for occupational allergy in olive oil mill workers. However, these workers do not experience allergic symptoms after ingestion of edible olive. Objectives: To analyze the presence of IgE-reactive TLP in raw and edible olive fruit and to assess the allergenic potency of both sources. Methods: The content of TLP in raw and edible olive fruit protein extracts was analyzed using immunoblotting with sera from allergic patients and with olive TLP-specific IgG. The structural and immunological stability of TLP were assayed using immunoblotting after treatment of both raw olive and purified TLP with 0.25 M NaOH solution for 24 hours. Olive pollen extract was investigated by immunoblotting for TLP content. Results: The TLP contained in raw olive fruit was not present in edible olives as a result of maceration before human consumption. No TLP was detected in olive pollen using specific IgG or sera from patients allergic to olive fruit. Sera from patients allergic to olive pollen did not react with purified TLP. Conclusions: IgE-reactive TLP is not present in edible olive, thus explaining the low number of patients allergic to this highly consumed fruit. Patients allergic to olive pollen are not sensitized to TLP and, therefore, not expected to be at risk of food allergy to olive fruit or TLP plant sources (AU)

Introducción: La aceituna natural contiene una proteína de la familia de las taumatinas (TLP) que es responsable de la alergia ocupacional en trabajadores de molinos de aceite. Sin embargo, éstos no presentan síntomas cuando ingieren aceitunas comestibles. Objetivos: Analizar la presencia de TLP en aceituna natural y comestible, y correlacionar sus niveles con la potencia alergénica de ambos productos. Métodos: El contenido de TLP en los extractos proteicos de las aceitunas fue analizado por inmunotransferencia y tinción con sueros de pacientes alérgicos así como con antisuero específico para TLP de olivo. La estabilidad estructural e inmunológica de la TLP se ensayó mediante inmunotinción después del tratamiento del extracto de aceituna natural y de la TLP purificada con NaOH 0.25 M durante 24 h. También se analizó la presencia de TLP en el polen de olivo por inmunotinción. Resultados: La TLP presente en la aceituna natural no se detecta en la comestible como consecuencia del tratamiento de maceración al que es sometida para obtener el producto apto para el consumo humano. No se observó TLP reactiva en el polen de olivo, ni con anticuerpos específicos ni con sueros de pacientes alérgicos a aceituna. Sueros de pacientes alérgicos al polen de olivo no reaccionan con la TLP purificada de aceituna. Conclusiones: La TLP de olivo no está presente en las aceitunas comestibles lo que explica el escaso número de pacientes alérgicos a la aceituna. Además, los pacientes alérgicos al polen de olivo no están sensibilizados a TLP, por lo que no tendrían riesgo de sufrir alergia alimentaria a aceitunas o a fuentes vegetales de TLPs (AU)

Olive pollen recombinant allergens: value in diagnosis and immunotherapy.

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity.

BACKGROUND: 1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens. OBJECTIVE: The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods. METHODS: The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used. RESULTS: The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex. CONCLUSION: rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.