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Notes: Background Characterization of fatal and non-fatal reactions to food indicates that the majority of reactions are due to the ingestion of prepared foods rather than the non-processed allergen. In an ongoing study that used a double-blind placebo-controlled food challenge to investigate peanut allergy and clinical symptoms, the observed reaction severity in four of the first six subjects was greater than anticipated. We hypothesized that this was due to differences in the composition of the challenge vehicle.Objective The aim was to investigate whether the severity of observed challenge reactions would be repeated on re-challenge with a lower fat challenge vehicle.Methods Peanut-allergic subjects were re-challenged with a lower fat recipe after reacting more severely than was anticipated to an initial peanut challenge. Similar challenge vehicle recipes were used, the only difference being the lower fat content (22.9% compared with 31.5%). The peanut content of the two recipes was analysed using RAST inhibition studies and ELISA tests.Results Three of four subjects reacted to much smaller doses of peanut protein on re-challenge (mean dose equivalence – 23 times less peanut) with the lower fat recipe. RAST inhibition showed that neither recipe altered epitope recognition. The higher fat recipe required twice as much peanut to cause 50% inhibition. ELISA detected far lower levels of peanut in the higher fat recipe (220 000 parts per million (p.p.m.)) than in the lower fat recipe (990 000 p.p.m.).Conclusion The fat content of a challenge vehicle has a profound effect on the reaction experienced after allergen ingestion. This is another factor to be considered in assessing the risk of certain foods to food-allergic consumers and adds another dimension to clinical, research and regulatory practice.

Notes: Background The risk for allergic reactions depends on the sensitivity of individuals and the quantities of offending food ingested. The sensitivity varies among allergic individuals, as does the threshold dose of a food allergen capable of inducing an allergic reaction.Objective This study aimed at determining the distribution of minimum provoking doses of hazelnut in a hazelnut-allergic population.Methods Thirty-one patients with a history of hazelnut-related allergic symptoms, a positive skin prick test to hazelnut and/or an elevated specific IgE level, were included. Double-blind, placebo-controlled food challenges (DBPCFC) were performed with seven increasing doses of dried hazelnut (1 mg to 1 g hazelnut protein) randomly interspersed with seven placebo doses.Results Twenty-nine patients had a positive challenge. Itching of the oral cavity and/or lips was the first symptom in all cases. Additional gastrointestinal symptoms were reported in five patients and difficulty in swallowing in one patient. Lip swelling was observed in two patients, followed by generalized urticaria in one of these. Threshold doses for eliciting subjective reactions varied from a dose of 1 mg up to 100 mg hazelnut protein (equivalent to 6.4–640 mg hazelnut meal). Extrapolation of the dose–response curve showed that 50% of our hazelnut-allergic population will suffer from an allergic reaction after ingestion of 6 mg (95% CI, 2–11 mg) of hazelnut protein. Objective symptoms were observed in two patients after 1 and 1000 mg, respectively.Conclusion DBPCFCs demonstrated threshold doses in half of the hazelnut-allergic patients similar to doses previously described to be hidden in consumer products. This stresses the need for careful labelling and strategies to prevent and detect contamination of food products with hazelnut residues.

Notes: Background IgE-binding peanut proteins smaller than 15 kDa were previously identified as potential allergens in the majority of our peanut allergic population.Objective To characterize the novel allergen in order to determine whether it was similar to one of the thus far identified recombinant peanut allergens (Ara h 1–7).Methods An IgE-binding protein of 〈15 kDa was purified and identified via N-terminal sequencing. Its IgE-binding properties were investigated using immunoblotting, basophil degranulation, and skin prick testing. Possible cross-reacting epitopes with other peanut allergens were studied using IgE-immunoblotting inhibition.Results The purified protein is a monomeric protein with a molecular weight of 14 981 Da as determined using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The amino acid sequence of the first 39 N-terminal residues is identical to that of Ara h 6, indicating that the allergen is Ara h 6. It is recognized by 20 out of 29 peanut-allergic patients on IgE-immunoblot, and its potent biological functionality is demonstrated by the degranulation of basophils, even at concentrations below 10 pg/mL, and by positive skin prick reactions. Ara h 6 has homology to Ara h 2, especially in the middle part and at the C-terminal part of the protein. Almost complete inhibition of IgE–Ara h 6 interaction with Ara h 2 demonstrates that at least part of the epitopes of Ara h 6 are cross-reactive with epitopes on Ara h 2.Conclusions Peanut-derived Ara h 6 is a biologically active allergen recognized by the majority of our peanut-allergic patient population and can be considered a clinically relevant peanut allergen.

Notes: Background While the ingestion of small amounts of an offending food can elicit adverse reactions in individuals with IgE-mediated food allergies, little information is known regarding these threshold doses for specific allergenic foods. While low-dose challenge trials have been conducted on an appreciable number of allergic individuals, a variety of different clinical protocols were used making the estimation of the threshold dose very difficult.Objective A roundtable conference was convened to develop a consensus clinical protocol for low-dose challenge trials for the estimation of threshold doses for specific allergenic foods.Methods In May 2002, 20 clinical allergists and other interested parties were invited to participate in a roundtable conference to develop consensus of the key elements of a clinical protocol for low-dose challenge trials.Results A consensus protocol was developed. Patients with convincing histories of food allergies and supporting diagnostic evidence including past challenge trials or high CAP–RAST scores can be enrolled in low-dose challenge trials. Care must be taken with younger patients to assure that they have not outgrown their food allergy. An approach was developed for the medication status of patients entering such trials. Challenge materials must be standardized, for example, partially defatted peanut flour composed of equal amounts of the three major varieties of peanuts (Florunner, Virginia, Spanish). Challenge materials must be appropriately blinded with sensory evaluation used to confirm the adequacy of blinding. A double-blind, placebo-controlled design should be used for low-dose challenge trials. Low-dose challenge trials would begin at doses of 10 μg of the allergenic food and would continue with doses of 100 μg and 1 mg followed by specific higher doses up to 100 mg depending upon the expert judgement of the physician; even higher doses might be applied to assure that the patient is indeed reactive to the particular food. A 30-min time interval would be used between doses, and reactive doses would be expressed as both discrete and cumulative doses. The goal of each challenge would be to develop objective symptoms; trials should not be discontinued on the basis of subjective symptoms only. Statistically, a minimum of 29 patients would be enrolled in low-dose challenge trials for each allergenic food because 0 reactors out of 29 patients at a particular dose allow the conclusion that there is 95% certainty that 90% of allergic individuals will not react to that dose.Conclusion A consensus protocol was developed. Using this protocol, it will be possible to estimate threshold doses for allergenic foods, the lowest amount that elicits mild, objective symptoms in highly sensitive individuals.

Notes: Background: Sunflower seed contains 2S albumins that in other crops have been associated with allergenicity. The sunflower seed methionine-rich 2S albumin (SSA) may be an IgE-binding protein responsible for anaphylactic reactions in some sunflower seed-sensitive subjects. The objective was to demonstrate that SSA is an IgE-binding protein. Methods: SSA was purified and the amino-acid sequence determined. The degree of purity of SSA was evaluated by silver staining, and its IgE-binding capacity by immunoblotting with serum from a subject with a convincing clinical history of anaphylaxis to sunflower seed. Results: The amino-acid sequence confirmed that the purified protein was the mature form of the methionine-rich storage protein SSA from sunflower seed (Helianthus annuus). The SSA was specifically recognized by IgE from the serum of the sunflower seed-allergic subject. Conclusions: SSA is an IgE-binding protein, and subjects allergic to sunflower seed whose IgE binds to SSA are at risk of developing allergic reactions if they consume SSA.

Notes: Background:  Peanut allergen Ara h 3 has been the subject of investigation for the last few years. The reported data strongly depend on recombinant Ara h 3, since a purification protocol for Ara h 3 from peanuts was not available.Methods:  Peanut allergen Ara h 3 (glycinin), was purified and its posttranslational processing was investigated. Its allergenic properties were determined by studying IgE binding characteristics of the purified protein.Results:  Ara h 3 consists of a series of polypeptides ranging from approximately 14 to 45 kDa that can be classified as acidic and basic subunits, similar to the subunit organization of soy glycinin. N-terminal sequences of the individual polypeptides were determined, and using the cDNA deduced amino-acid sequence, the organization into subunits was explained by revealing posttranslational processing of the different polypeptides. IgE-binding properties ofAra h 3 were investigated using direct elisa and Western blotting with sera from peanut-allergic individuals. The basic subunits, and to a lesser extent the acidic subunits, bind IgE and may act as allergenic peptides.Conclusions:  We conclude that peanut-derived Ara h 3, in contrast to earlier reported recombinant Ara h 3, resembles, to a large extent, the molecular organization typical for proteins from the glycinin family. Furthermore, posttranslational processing of Ara h 3 affects the IgE-binding properties and is therefore an essential subject of study for research on the allergenicity of Ara h 3.