Perfil de anticorpos específicos ao extrato de pólen de Lolium multiflorum (azevém italiano) e suas frações em indivíduos alérgicos e não-alérgicos

Abstract

Allergic diseases are usually derived from a strong immune response mediated by IgE antibodies to different exogenous proteins and other components. These allergens can come from various sources such as pollens, mites, fungi, animal epithelia, body elements and insect pellets, food and medicines. The polens of Lolium multiflorum (Lm), a grass widely cultivated in southern Brazil, are recognized as an important allergenic source, and every day grows the number of individuals sensitized to the pollen of this plant. Although well known in the field of agronomic engineering as a forage plant, there are very few studies evaluating the role of this grass in allergenic sensitization. Studies that isolate and characterize Lm polen allergens may be useful in the treatment and diagnosis of allergic diseases, revealing which proteins have greater ability to trigger symptoms. That is why it is so necessary to carry out studies in this field in order to improve the quality of life of patients allergic to Lm. The present thesis consists of three chapters referring to the allergenic capacity of the crude extract of Lm polens and their fractions isolated by chromatography techniques. In Chapter I, ion exchange chromatography effectively isolated the S2 fraction, containing clinically relevant components for patients sensitized to Lm pollen. Both extract S1 and fraction S2 were effective in separating atopic and non-atopic individuals using ELISA and Immunoblotting techniques. The sensitivity and specificity of the ELISA tests were similar for S1 and S2. The 32 kDa band was the most important immunodominant component, reacting with L. multiflorum-specific IgE and IgG4 antibodies. Mass spectrometry revealed group 1 and 5 allergens in the 32 kDa band and a hypothetical Oryza sativa protein in the 58 kDa band. The S2 fraction is a potential alternative to the total extract for diagnosis and specific immunotherapy. In Chapter II, two fractions (unbound and linked to the ConA spine) with clinically relevant components for patients sensitized to L. multiflorum pollen were isolated. By observing the electrophoretic profiles, distinct protein components were isolated in each fraction. The fractions and the crude extract were effective in separating atopic and non-atopic individuals using ELISA and Immunoblotting. The reactivity of IgE was evidenced by specific bands in the serum of atopic individuals, which did not occur in non-atopic individuals. Specific reactive bands were observed in the crude extract as well as fractions linked and not linked to the ConA, demonstrating a possible approach for diagnosis and treatment of respiratory allergies caused by L. multiflorum. Finally, in chapter III, the fractions S2 and S3 were obtained, important for patients sensitized to Lm pollen. Differences were observed in the reactivity of specific IgG antibodies between the groups, especially in the fractions S2 and S3. The level of high avidity IgG exceeded 50% in all subjects tested. Significant differences in the ELISA index were found between different subclasses of IgG in the fractions S2 and S3. Correlations between IgG subclasses varied between fractions, suggesting that S2 and S3 are promising for the treatment of allergic conditions induced by L. multiflorum pollen.