Allergia inalatoria ed alimentare al frumento: uno studio allergomico

Wheat flour proteins are allergens involved in 60% to 70% of workplace-related respiratory-symptoms of bakers, furthermore wheat belongs to the six major food allergens inducing IgE-mediated hypersensitivity-reactions. In the case of wheat food-allergy the manifestations range from cutaneous, gastrointestinal, and respiratory symptoms to severe systematic reactions. Despite this allergological relevance the knowledge of wheat allergens at molecular level is scanty. It is nevertheless assumed that conformational and sequential epitopes might be both responsible for wheat allergic reactions. As a consequence of this limited knowledge the diagnosis of wheat allergy is sometime a difficult task. Skin prick tests (SPTs) play an important role in the allergological diagnosis but baker’s asthmatic patients result positives in a limited (from 5 to 15%) range. This scarce SPT predictability may be a consequence of the low solubility of numerous wheat proteins as well as an effect of differences in test-extracts standardization. OBJECTIVE The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy and baker’s asthma. In particular, the aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized Italian bakers and to identify the most frequently recognized allergens. Furthermore i) we tried to verify whether different epitopes are involved in the different forms of wheat allergy ii) we tested the real allergen content in SPT used in wheat allergy diagnosis in relationship to their performance in clinical practice. The employed strategy was the proteomic analysis of wheat followed by IgE blotting i.e. an “allergenomic” approach. METHODS To characterize wheat allergens, water/salt-soluble wheat flour proteins from the monocultivar Bolero were separated by using mono dimensional (1-DE) and 2-dimensional (2-DE) gel electrophoresis under reducing and non-reducing conditions. Furthermore, using 1-DE SPT-solutions, wholemeal wheat flour and flour extracts were separated. IgE-binding proteins were detected by immunoblotting using the sera of 43 patients with inhalant allergy and 9 with food allergy to wheat. After tryptic digestion, the peptides of some IgE-binding proteins, frequently recognized by IgE on 2-DE, were analyzed by nano HPLC–ESI–MS/MS. RESULTS The IgE immunoblots obtained with 43 different sera exhibited a remarkable inter-individual heterogeneity. Furthermore, the immunodetected profiles were very different under reducing and non-reducing condition. The analyzed SPT-solutions demonstrate to be only partially representative of the real allergenic content of wheat and different batch of these extracts show remarkable differences. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but with different isoelectric points. Five of the predominant IgE-binding protein spots were identified by using nano HPLC–ESI–MS/MS. In this study, we identified one already reported wheat inhalant allergen [monomeric alpha-amylase inhibitor, 0.28] by a database search. Moreover we identified two wheat inhalant allergens, rarely described in literature [triosephosphate-isomerase and thioredoxin peroxidase], and we found out two never reported wheat inhalant allergens [glucose and ribitol dehydrogenase homolog – barley and a heat shock protein]. In the case of food allergy, surprisingly, no specific IgEs for wheat proteins were detected in 8 out of 9 cases (88%). CONCLUSIONS. The clinical relevance of the identified 2 new allergens will be further investigated in the near future but certainly our findings could contribute to increase the specificity of diagnostic assays. IgE-blotting did not reveal food allergens for this reason, in the future, an alternative approach to allergomic must be developed.