Kunie Kohno

Specific IgE Determination to Epitope Peptides of ω-5 Gliadin and High Molecular Weight Glutenin Subunit Is a Useful Tool for Diagnosis of Wheat-Dependent Exercise-Induced Anaphylaxis

Wheat ω-5 gliadin and a high m.w. glutenin subunit (HMW-glutenin) have been reported as major allergens in wheat-dependent exercise-induced anaphylaxis. A simultaneous detection of specific IgE to epitope sequences of both proteins is considered to be a reliable method for diagnosis of wheat-dependent exercise-induced anaphylaxis. However, the IgE-binding epitope of HMW-glutenin remains unknown. The aim of this study was to determine the IgE-binding epitopes of HMW-glutenin to establish a useful system of identifying patients with wheat-dependent exercise-induced anaphylaxis. For determination of IgE-binding epitopes of HMW-glutenin overlapping peptides were synthesized and reactivities of IgE Abs in the sera of patients to those peptides were analyzed. Three IgE-binding epitopes, QQPGQ, QQPGQGQQ, and QQSGQGQ, were identified within primary sequence of HMW-glutenin. Epitope peptides, which include IgE-binding sequences of ω-5 gliadin and a HMW-glutenin, were synthesized and peptide-specific IgE Abs were measured by CAP-System fluorescent enzyme immunoassay. Twenty-nine of 30 patients with wheat-dependent exercise-induced anaphylaxis had specific IgE Abs to these epitope peptides. None of the 25 sera from healthy subjects reacted to both epitope peptides. Twenty-five patients with atopic dermatitis who had specific IgE to wheat and/or gluten had very low or nonexistent levels of epitope peptide-specific IgE Abs. These results indicated that measurement of IgE levels specific to epitope peptides of ω-5 gliadin and HMW-glutenin is useful as an in vitro diagnostic method for the assessment of patients with wheat-dependent exercise-induced anaphylaxis.

Identification of the IgE-binding epitope in omega-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a severe IgE-mediated allergic reaction provoked by the combination of wheat-ingestion with intensive physical exercise over the next few hours. Among wheat proteins, ω-5 gliadin, which is one of the components of fast ω-gliadin, has been reported as a major allergen in the anaphylaxis. In this study, we detected IgE-binding epitopes within the primary sequence of ω-5 gliadin using arrays of overlapping peptides synthesized on derivatized cellulose membranes. Sera from four patients with WDEIA having specific IgE to the fast ω-gliadin were used to probe the membrane. Seven epitopes, QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ, and PYPP, were detected within the primary sequence of ω-5 gliadin. By using sera of 15 patients, 4 of them, QQIPQQQ, QQFPQQQ, QQSPEQQ, and QQSPQQQ, were found to be dominant epitopes. Mutational analysis of the QQIPQQQ and QQFPQQQ indicated that amino acids at positions Gln1, Pro4, Gln5, Gln6, and Gln7 were critical for IgE binding. These results will provide a useful tool for developing safer wheat products in addition to diagnostic and immunotherapy techniques for WDEIA.

Sensitivity and specificity of recombinant omega-5 gliadin-specific IgE measurement for the diagnosis of wheat-dependent exercise-induced anaphylaxis.

Background:  A recent study has shown that the measurement of specific IgE antibodies to B‐cell epitope peptides of wheat ω‐5 gliadin (Pep A) and high molecular weight glutenin subunit (Pep B) are useful to diagnose wheat‐dependent exercise‐induced anaphylaxis (WDEIA).

Molecular cloning, recombinant expression and IgE‐binding epitope of ω‐5 gliadin, a major allergen in wheat‐dependent exercise‐induced anaphylaxis

Wheatω‐5 gliadin has been identified as a major allergen in wheat‐dependent exercise‐induced anaphylaxis. We have detected seven IgE‐binding epitopes in primary sequence of the protein. We newly identified four additional IgE‐binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat‐dependent exercise‐induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because ω‐5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the ω‐5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR‐based strategy we isolated two full‐length ω‐5 gliadin genes, designated ω‐5 and ω‐5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by ω‐5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the ω‐5b gene would encode a 393 amino acid, but it contains two stop codons indicating that ω‐5b is pseudogene. The C‐terminal half (178 amino acids) of the ω‐5a gliadin protein, including all 11 IgE‐binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP‐HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native ω‐5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE‐binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat‐dependent exercise‐induced anaphylaxis in vitro.

IgE antibodies to ω‐5 gliadin associate with immediate symptoms on oral wheat challenge in Japanese children

Background:  Gliadins have been implicated in immunoglobulin E (IgE)‐mediated allergy to ingested wheat and ω‐5‐gliadin is known to represent a major allergen in wheat‐dependent exercise‐induced anaphylaxis. Less known is whether ω‐5‐gliadin is a clinically relevant allergen in children with immediate allergy to ingested wheat. This study investigates whether specific IgE antibodies to ω‐5‐gliadin (sIgE‐ω‐5‐gliadin‐ab) could be used as a marker for oral wheat challenge outcome in wheat‐sensitized children. A secondary objective was to study whether the level of sIgE‐ω‐5‐gliadin was related to symptom severity in children with a positive challenge test.

Recombinant high molecular weight‐glutenin subunit‐specific IgE detection is useful in identifying wheat‐dependent exercise‐induced anaphylaxis complementary to recombinant omega‐5 gliadin‐specific IgE test

Wheat‐dependent exercise‐induced anaphylaxis (WDEIA) is a special form of food allergy typically induced by exercise after ingestion of wheat products. We identified wheat omega‐5 gliadin and high molecular weight‐glutenin subunit (HMW‐glutenin) as major allergens for WDEIA and clarified that simultaneous detection of serum IgE binding to synthetic epitope peptides of these allergens identifies more than 90% of WDEIA patients. However, the short synthetic peptides are not suitable for CAP‐fluorescent enzyme‐immunoassay (CAP‐FEIA), which is widely utilized for detecting allergen‐specific IgE.

Serum Gliadin Monitoring Extracts Patients with False Negative Results in Challenge Tests for the Diagnosis of Wheat-Dependent Exercise-Induced Anaphylaxis*

BACKGROUND Challenge testing with wheat plus exercise and/or aspirin is a gold standard for the diagnosis of wheat-dependent exercise-induced anaphylaxis (WDEIA); however, the test may often yield false-negative results. Our previous study suggested that an increase in serum wheat gliadin levels is required to induce allergic symptoms in patients with WDEIA. Based on this knowledge, we sought to extract the patients with false negative results in the challenge tests of WDEIA. METHODS Thirty-six patients with suspected WDEIA were enrolled. First, group categorizations-Group I, challenge tests were positive; Group II, challenge tests were negative and serum gliadin were undetectable; Group III, challenge tests were negative and serum gliadin were detectable-were given according to the results of wheat plus exercise and/or aspirin challenge testing and serum gliadin levels. Second, diagnoses were made using retests and/or dietary management in Group II and III. RESULTS Positive results for wheat plus exercise and/or aspirin challenge tests gave a diagnosis of definite WDEIA in 17 of 36 patients (Group I). Of the remaining 19 challenge negative patients, serum gliadin was undetectable in ten patients (Group II). Of the ten patients (Group II), three of them were diagnosed as definite WDEIA by retesting and six of them were diagnosed as probable WDEIA using a wheat elimination diet, whereas one patient was non-WDEIA. In the rest of the nine challenge negative patients, serum gliadin was detectable (Group III). No allergic episodes with a normal diet provided a diagnosis of non-WDEIA in seven of the nine patients, whereas the remaining two patients were probable WDEIA or had another food allergy because of repeated episodes. CONCLUSIONS Our study revealed that serum gliadin monitoring during challenge testing is useful.

Pre-treatment with misoprostol prevents food-dependent exercise-induced anaphylaxis (FDEIA).

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HLA-B*58:01 strongly associates with allopurinol-induced adverse drug reactions in a Japanese sample population.

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Altitudes of residential areas affect salt intake in a rural area in Japan: a Shimane CoHRE Study.

There is increasing evidence of an association between residential environments and hypertension. As shown in our previous study, the inconvenience of the locations of residential areas may be one of the factors influencing the blood pressures of inhabitants. Salt intake is one of the likely mediators between inconvenience and hypertension. Therefore, in this study, we evaluated the association between the altitudes of residential areas and salt intake in a rural Japanese region because altitude may be one of the proxies for inconvenience. In this cross-sectional study, 1016 participants living in a mountainous region in Japan were recruited during health examinations. The altitude of each participant’s residence was estimated using a geographic information system. Subjects were divided into quartile groups according to the altitudes of their residences. To evaluate salt intake, we employed the 24-h salt intake estimation of Kawano et al. (e24-h salt intake) and the urinary sodium-to-potassium ratio (uNa/K). Linear regression analyses indicated that altitude was an independent factor influencing both e24-h salt intake and uNa/K after adjustments for age, sex, body mass index, physical activity, alcohol consumption, triglycerides and county of residence. The same result was observed when the subjects who did not take antihypertensive medications were analyzed (N=633). The present study indicated that altitude of residence had a significant positive influence on salt intake in a rural area of Japan.