RESUMO
Canine atopic dermatitis (CAD) is an immune system disorder that affects 10-15% of the canine population. Short ragweed (Ambrosia artemisiifolia) pollen represents one of the major seasonal sources of allergenic pollen proteins in Europe, particularly in the Pannonian valley of the Balkan region. In Serbia, about 66% of atopic dogs showed a positive intradermal skin test with its pollen extract, which is second to house dust mites. Therefore, characterization of Ambrosia artemisiifolia pollen components, in terms of defining major and minor allergens that induce clinically manifested allergic reaction in dogs, is important for valid diagnosis and efficient therapy. This study has, for the first time, characterized and identified major Ambrosia artemisiifolia allergens in CAD, using an immunoproteomic approach. To assess the prevalence of specific IgE in electrophoretically separated ragweed pollen proteins, individual reactivity of sera from dogs with CAD was analyzed and compared to the reactivity of sera from healthy dogs in the non-reducing conditions, which were found optimal for specific canine IgE detection. A specific IgE band (38 kDa) was recognized as the most dominant allergen in CAD, occurring in 81% of positive dog's sera. 2-D immunoblotting followed by a mass spectrometry peptide fingerprint analyses with pooled canine and human atopic sera, revealed that 38 kDa major Ambrosia atremisiifolia allergens in CAD were all five isoallergens of the Amb a 1 group (antigen E), including the previously named Amb a 2 (antigen K). In contrast to canine sera, human atopic sera also recognized lower mass allergens such as the ß fragment of Amb a 1 and profilins (Amb a 8 variants). The most prominent ragweed proteins in CAD, represent, as in humans, variants of all five isoallergens of the Amb a 1 group (pectate lyase): Amb a 1.0101 and its natural variant E1XUL2, Amb a 1.0202, 1.0304, 1.0402 and the natural variant of Amb a 1.0501, E1XUM0, as well as the α fragment of pollen allergen Amb a 1.0201.
Assuntos
Ambrosia/imunologia , Antígenos de Plantas/imunologia , Dermatite Atópica/veterinária , Doenças do Cão/imunologia , Hipersensibilidade Imediata/veterinária , Proteínas de Plantas/imunologia , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Ambrosia/genética , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Western Blotting , Dermatite Atópica/imunologia , Cães , Eletroforese em Gel Bidimensional , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/sangue , Dados de Sequência Molecular , Extratos Vegetais/química , Extratos Vegetais/genética , Extratos Vegetais/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteômica , Homologia de Sequência de Aminoácidos , Sérvia , Espectrometria de Massas em TandemRESUMO
Actinidin, an abundant cysteine protease from kiwifruit, is a specific biomarker of isolated allergy to kiwifruit. This study evaluates the IgE-binding properties of biologically active and thermally inactivated actinidin. Employing two different activity assays (caseinolytic assay and zymogram with gelatin) we showed that actinidin obtained from kiwifruit extract under native conditions represents a mixture of inactive and active enzyme. The structural integrity of actinidin was confirmed by SDS-PAGE, Edman degradation, mass fingerprint and Western blot with polyclonal antibodies. Although it was capable of inducing positive skin prick test reactions, we failed to detect IgE reactivity of active actinidin in Western blot with patient sera. Thermally inactivated actinidin exhibited IgE reactivity both in vivo and in vitro, indicating that heat processed kiwifruit products may induce clinical reactivity. These findings imply that apart from the allergenic epitopes on its surface, actinidin also contains hidden epitopes inside the protein which become accessible to IgE upon thermal treatment.