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 5-year venom immunotherapy protocol with 50 µg maintenance dose: safety and efficacy in school children.

BACKGROUND: Venom immunotherapy (VIT) has been shown to be an effective and safe treatment for preventing sting-induced anaphylaxis in patients with systemic reactions to hymenoptera stings. A remaining problem is the relative effectiveness and safety of different immunotherapy protocols used with respect to maintenance dose, injection interval, and duration. OBJECTIVE: We aimed to describe a modified cluster VIT protocol with a maintenance dose of 50 µg lasting 5 yr and to evaluate retrospectively its safety and efficacy in children. PATIENTS AND METHODS: Fifty four children 9.5±3.2 yr old with a history of at least one anaphylactic reaction to hymenoptera stings underwent VIT between 1995 and 2006. The identification of the offending insect(s) was based on patient's report and documented with in-vivo (SPTs and IDs) and in-vitro (RAST/CAP) test results. A modified cluster outpatient protocol lasting 5 wks, reaching a maintenance dose of 50 µg was followed according to clinical history and test results. After the maintenance dose was achieved, the followed injection-intervals were 4 wks for the first year, 5 wks for the 2nd year and 3rd year, and 6 wks for the last 2 yr. RESULTS: Of the 54 children, 52 tolerated the 50 µg VIT protocol without side effects. Twenty one of them reported at least one field sting from at least one of the culprit, for their allergy, insects, 6±3.5 yr after they have started VIT treatment. In 11 of them, sting occurred 3.5±2.9 yr after the VIT was completed, whereas the other 10 of them during immunotherapy, 3.2±1.4 yr after they have started VIT. In the remaining two children, the maintenance dose was increased to 100 µg due to systemic reactions from the VIT. The data reflect outcomes 6-16 yr after the patients' initial allergic reaction. CONCLUSION: VIT with 50 µg maintenance dose lasting 5 yr appears to be safe and effective enough to induce tolerance in children with hymenoptera venom hypersensitivity.

Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential IgE epitopes indicates that sensitization to alpha-purothionin occurs via the gut. Both allergens can be used for in-vitro diagnosis of wheat food allergy. The induction of blocking IgG antibodies suggests the usefulness for immunotherapy.

Risk of allergic reactions to wine, in milk, egg and fish-allergic patients.

BACKGROUND: European legislators and wine producers still debate on the requirement for labeling of wines fined with potentially allergenic food proteins (casein, egg white or fish-derived isinglass). We investigated whether wines fined with known concentrations of these proteins have the potential to provoke clinical allergic reactions in relevant patients. METHODS: In-house wines were produced for the study, fined with different concentrations of casein (n = 7), egg albumin (n = 1) and isinglass (n = 3). ELISA and PCR kits specific for the respective proteins were used to identify the fining agents. Skin prick tests and basophil activation tests were performed in patients with confirmed IgE-mediated relevant food allergies (n = 24). A wine consumption questionnaire and detailed history on possible reactions to wine was obtained in a multinational cohort of milk, egg or fish allergic patients (n = 53) and patients allergic to irrelevant foods as controls (n = 13). RESULTS: Fining agents were not detectable in wines with the available laboratory methods. Nevertheless, positive skin prick test reactions and basophil activation to the relevant wines were observed in the majority of patients with allergy to milk, egg or fish, correlating with the concentration of the fining agent. Among patients consuming wine, reported reactions were few and mild and similar with the ones reported from the control group. CONCLUSION: Casein, isinglass or egg, remaining in traces in wine after fining, present a very low risk for the respective food allergic consumers. Physician and patient awareness campaigns may be more suitable than generalized labeling to address this issue, as the latter may have negative impact on both non-allergic and allergic consumers.

Asthma control in adolescents: role of leukotriene inhibitors.

Asthma is a chronic inflammatory disease of the airways and is a big burden worldwide. It affects both children and adults, but it is insufficiently studied in adolescents, although this age group has important peculiarities and is challenging to treat, due to, but not exclusively because of, lack of adherence to treatment instructions. Evidence-based guidelines for the treatment of asthma targeting specifically adolescents are lacking, due to the fact that most studies are conducted either on children or in adults. Exercise-induced asthma occurs commonly in adolescents, leading to impaired physical activity. This review describes current treatment options for asthma in adolescents, focusing on leukotriene receptor antagonists, both as a monotherapy and as an add-on therapy for optimal asthma control.