Molecular and immunological characterization of Tri a 36, a low molecular weight glutenin, as a novel major wheat food allergen.

Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ∼80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.

A combined biochemical, biophysical and immunological approach towards the identification of celiac disease-specific wheat antigens.

Celiac disease (CD) is an inflammatory affliction of the small bowel caused by an immunological hypersensitivity to ingested wheat antigens affecting almost 1 % of the population. The gliadin fraction of wheat has been shown to contain the pathogenic antigens which react with antibodies and T cells. However, there is only limited knowledge regarding the precise nature of the wheat antigens recognized by IgA antibodies from CD patients and diagnostic tests based on the gliadin fraction have been demonstrated to give frequently false positive results. The aim of this study was the characterization of wheat antigens specifically recognized by IgA antibodies of CD patients. We developed a combined biochemical, biophysical, and immunological approach for the identification of celiac disease-specific wheat antigens. It is based on sub-fractionation of the wheat gliadin fraction using two ion exchange chromatography steps, the localization of CD-specific antigens by immunoblotting with IgA antibodies from CD patients, subsequent digestion followed by electro spray ionization-liquid chromatography/mass spectrometry (LC-ESI-MS/MS) and N-terminal sequencing by Edman degradation. Through the sub-fractionation procedure it was possible to separate CD-specific IgA-reactive wheat antigens from other wheat antigens which were also recognized by IgA antibodies of individuals without CD or by CD patients on gluten-free diet. Analysis by LC-ESI-MS/MS and N-terminal sequencing of the sub-fractions and the proteins specifically recognized by CD patients identified certain γ-gliadins with molecular mass of 37,000 and 45,000 as CD-specific wheat antigens. The CD-specific γ-gliadins with the molecular mass of 37,000 and 45,000 should be useful to study pathomechanisms of the disease and to improve the specificity of diagnostic tests for CD.

Detection of antigens reactive to IgE and IgA during wheat seed maturation and in different wheat cultivars.

BACKGROUND: Dietary intake of wheat causes hypersensitivity reactions in patients suffering from IgE-mediated food allergy and coeliac disease. AIM: To study the expression of IgE- and IgA-reactive antigens during wheat seed maturation and in different wheat cultivars. METHODS: Summer wheat was grown in a glasshouse and seeds were harvested at defined maturation stages. Mature seeds were obtained from 13 different defined cultivars. Protein extracts were prepared from different maturation stages and cultivars with a standardized procedure based on seed weight and analysed by IgE and IgA immunoblotting using sera from clinically defined patients suffering from wheat allergy or coeliac disease. RESULTS: With a few exceptions the expression of IgE- and IgA-reactive wheat antigens increased during wheat seed maturation. Wheat cultivars could be identified in which the expression of certain IgE- and IgA-reactive components was strongly reduced or not detectable. CONCLUSIONS: The expression of IgE- and IgA-reactive antigens depends on wheat seed maturation and varies in different wheat cultivars.

Molecular and immunological characterization of a wheat serine proteinase inhibitor as a novel allergen in baker's asthma.

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly beta-sheets, random coils, and an alpha-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an approximately 50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.

Different profiles of wheat antigens are recognised by patients suffering from coeliac disease and IgE-mediated food allergy.

BACKGROUND: Dietary intake of wheat can cause two distinct immunologically mediated diseases with severe gastrointestinal manifestations, coeliac disease (CD) and IgE-mediated food allergy. The pathomechanisms underlying these diseases are different, but the profile of the target antigens in wheat has not been compared for the two diseases. METHODS: We compared IgA- and IgE-reactive antigens in wheat using sera from patients with coeliac disease (n = 35) and food allergy to wheat (n = 16) by one- and two-dimensional immunoblotting. Furthermore, the IgG subclass (IgG1-IgG4) reactivity to wheat antigens was studied by enzyme-linked immunosorbent assay. RESULTS: IgA antibodies from CD patients and IgE antibodies from allergic patients recognised distinct profiles of wheat antigens. Furthermore, the IgG subclass responses to wheat antigens were different in CD and wheat-allergic patients. CONCLUSION: This study thus demonstrates that wheat contains antigens/epitopes which are preferentially recognised by CD patients, whereas others elicit IgE-mediated food allergy. This finding suggests that the nature of a food antigen may influence the quality of the pathological immune response in the gut and has implications for the diagnosis and therapy of hypersensitivity to wheat.

Criancas asmaticas: a necessidade de controle ambulatorial / Asmathmatic children: the necessity of ambulatory control

O presente trabalho objetivou verificar a gravidade da asma, segundo o intervalo entreas crises, em criancas atendidas no Ambulatorio de Pneumologia Pediatrica do Hospital de Clinicas da Universidade Federal de Uberlandia (HC/UFU), nao submetidas anteriormente a controle ambulatorial. Foram avaliadas, no primeiro semestre de 1992,75 criancas com diagnostico previo de asma, em sua primeira consulta no Ambulatorio. A idade media dos pacientes foi de 6,5 anos, com desvio padrao (DP) de 4,0 e com relacao masculino-feminino de 2:1. Para se avaliar o intervalo entre as crises, utilizou-se a classificacao de LANDAU (1979) modificada, que objetiva o acompanhamento ambulatorial. O teste estatistico utilizado foi o Qui-quadrado. Observou-se 37 por cento de pacientes com asma episodica, 41 por cento com asma episodica frequente, 20 por cento com asma cronica e 1,3 por cento com asma cronica severa. As formas graves predominaram em escolares de modo estatisticamente significante (p< 0,01). Nao se observou relacao estatisticamente significante entre sexo e gravidade da asma. Nao houve concordancia com a classificacao de LANDAU (1979), sendo mais comuns as formas graves da doenca. Os autores concluem que a falta de controle ambulatorial, em um grupo onde os alergenos sao importantes precipitantes de crises, deve ser responsavel pela ocorrencia de formas mais graves de asma..