Fine characterization of immunological mechanisms mediated by the major allergens ofParietaria judaica and hypoallergenic hybrid, rPjEDcys

Risultato della ricerca: Other

Abstract

Purpose: Allergy is a hypersensitivity disease IgE-mediated, affecting more than 25% of the population. Thesymptoms of IgE-mediated allergies reactions can be transiently ameliorated pharmacologically, but the onlycurative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenicallergen derivatives with reduced allergenic activity have been engineered to reduce side effects during SIT.Parietaria judaica (Pj) pollen contains two major allergens belonging to the family of Lipid Tranfer Proteins(Par j 1 and Par j 2). By means of DNA recombinant technology, a hybrid hypoallergenic (PjEDcys),expressing disulphide bond variants of Par j 1 and Par j 2, was generated. The aim of this research project isto study the immunological mechanisms activated by the major allergens of Parietaria judaica, Par j 1 andPar j 2, and hypoallergenic hybrid rPjEDcys. Moreover, the project I am involved is trying to address thequestion whether this engineered hypoallergenic derivative can be a potential products for safer AllergenSpecific Immunotherapy (SIT).Methods: Par j 1, Par j 2 and PjEDcys were produced as recombinant proteins. Human Peripheral BloodMononuclear Cell (PBMC) from P. judaica allergic patients were stimulated in vitro with wild-typerecombinant allergens and hybrid. PBMC proliferation assay, cytokine secretion assay, magnetic cell sortingof different subset of regulatory T cells, multiparametric flow cytometric analysis and molecularcharacterization using Real Time-PCR on sorted cells allow to study the biological properties of wild-typerecombinant allergens and hybrid hypoallergenic derivate.Results: In vitro analysis suggested that PjEDcys have a reduced allergenity and maintained T cellsreactivity. PBMC of P. judaica allergic patients stimulated in vitro with the hybrid and the wild-typerecombinant allergens scored a percentage of proliferating CD4+ and CD56+ cell higher than unstimulatedsample. Consistent with these data, cytokine secretion assay on CD4+ cells demonstrated that PBMCstimulation with rPjEDcys showed a percentage of IL-5 and IL-13 secreting T CD4+ cells lower than thewild-type allergens. Both rPjEDcys and wild-type stimulation promote the secretion of IFN- γ and IL-10 byT CD4+ cells. Finally whit the aim to study which subset of regulatory cells respond to wild-tipe allergensand hypoallergenic hybrid new experiment are setting.Discussion: In this experimental setting, the use of the major allergens of Pj and the hybrid polypeptides,rPjEDcys allows me to study the immunological mechanisms activated by the two different antigenstimulation and to investigate differences between the wild-type allergen and the hypoallergenic mutantrPjEDcys. Our data showed that CD4+ cells are clearly the predominant cell population proliferating inresponse to mixture of Par j 1 and Par j 2 allergens. The hypoallergenic derivate rPjEDcys retain the abilityto stimulate CD4+ cells proliferation like the mixture of allergens (rPar j 1 and rPar j 2). Moreover theseresults highlighted a particular interesting datum; the mixture of allergens and the rPjEDcys hybrid showedthe ability to stimulate an innate immune response, inducing CD56+ cells proliferative response. Cytokinesecretion assay demonstrate that rPjEDcys reduce the secretion of IL-5 and IL-13, Th2 cytokines with acritical role in the development of allergy, compared to wild-type allergens. This may reflect the differentbiological function exerted by rPjEDcys.Conclusion: Collectivelly, our findings demonstrate that PjEDcys show a reduced allergenicity butmaint
Lingua originaleEnglish
Numero di pagine1
Stato di pubblicazionePublished - 2016

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