Claudia Constantin

Micro‐arrayed wheat seed and grass pollen allergens for component‐resolved diagnosis

Background:  Wheat is a potent allergen source and can cause baker’s asthma, food and pollen allergy. The aim of the study was to develop an allergen micro‐array for differential diagnosis of baker’s asthma, wheat‐induced food allergy and grass pollen allergy.

Molecular characterization of wheat allergens specifically recognized by patients suffering from wheat-induced respiratory allergy

Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour‐Bakers asthma).

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 bakers 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 β-sheets, random coils, and an α-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 bakers 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 ∼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 bakers 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.

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.

The high molecular weight glutenin subunit Bx7 allergen from wheat contains repetitive IgE epitopes

Wheat is one of the most common food allergen sources for children and adults. The aim of this study was to characterize new wheat allergens using an IgE discovery approach and to investigate their IgE epitopes.

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.

Identification of wheat proteins involved in active stage of celiac disease: are gamma gliadins the major disease-specific antigens?

Methods We developed a method wherein the alcohol extracted gliadins was fractionated in two steps of ion-exchange chromatography, Sulphopropyl (SP) was used for the first step and the flow through (FT) fraction obtained was further fractionated using DEAE. Each generated fraction’s reactivity to serum IgA, from clinically well defined CD (active/diet) patients and non-CD patients was analyzed. Identification of disease specific antigens in the fractions was attempted using mass spectrometry and N-terminal sequencing.

Evidence for higher sensitivity of recombinant Tri a 36 compared to omega-5-gliadin for diagnosis of wheat food allergy

Background Wheat is one of the most important food allergen sources. Using natural wheat allergen extracts for serological diagnosis of wheat-induced food allergy false positive test results are frequently obtained, in particular in grass pollen allergic patients. Therefore, Tri a 19, an omega-5-gliadin, which is known as a major allergen in wheat dependent exercise induced anaphylaxis (WDEIA) and in wheat food allergy in children, is widely used for the serological diagnosis of wheat-induced food allergy.

Isolation, characterization and expression in Escherichia coli of a cDNA coding for a novel major wheat food allergen

Wheat (Triticum aestivum) is a main component of the daily diet but can cause three distinct forms of wheat allergy: Bakers asthma, wheat food allergy and wheat pollen allergy. The panel of wheat allergens is still incomplete. The aim of the study was to identify and characterize wheat allergens for the development of improved diagnostic tests and allergen-specific forms of treatment.