Wheat allergy in children - new tools for diagnostics.

BACKGROUND: The detection of wheat-specific IgE in children often leads to a suspicion of wheat allergy, but little information is available on the most reliable wheat allergens for predicting clinical reactivity. OBJECTIVE: To evaluate the role of allergenic components of wheat in wheat allergy diagnostics. METHODS: One hundred and eight children (median age 1.5 years; range 0.6-17.3 years) with suspected wheat allergy underwent open or double-blinded, placebo-controlled oral wheat challenges. Responsiveness to different allergenic components of wheat was studied by skin prick tests and by determination of serum IgE antibodies using a semi-quantitative microarray assay. RESULTS: Thirty (28%) children reacted with immediate symptoms, and 27 (25%) with delayed symptoms to ingested wheat, whereas 51 (47%) children exhibited no reactions in oral wheat challenges. Positive IgE responses to any of the 12 allergenic components of wheat was seen in 93%, 41%, and 43% of those with immediate, delayed or no reactions to ingested wheat, respectively (P < 0.001 to P < 0.05 in every comparisons between those with immediate reactions and those with no reactions). Positive IgE responses to ≥5 different allergenic components improved significantly the diagnostic accuracy (with a positive likelihood ratio (LR+) of 5.10). Alpha-amylase inhibitors (AAI), in particular dimeric AAI 0.19 (LR+ 6.12), alpha-, beta-, and gamma-gliadins (LR+ from 3.57 to 4.53), and high-molecular-weight (HMW) glutenin subunits (LR+ 4.37) were the single allergenic components of wheat differentiating most effectively those with immediate symptoms from those who did not exhibit any reactions. CONCLUSIONS AND CLINICAL RELEVANCE: Wheat allergy diagnostics is difficult, even using sophisticated component methods. Our results confirm earlier findings about gliadins and identify the dimeric AAI 0.19, as a relevant allergen in clinically reactive patients when compared to non-reactive subjects. The accuracy of wheat allergy diagnosis may be improved by measuring IgE responses to several components of wheat.

Hev b 6.01 and Hev b 5 induce pro-inflammatory cytokines and chemokines from peripheral blood mononuclear cells in latex allergy.

BACKGROUND: Hev b 6.01 (prohevein) and Hev b 5 [acidic natural rubber latex (NRL) protein] are major IgE-binding allergens in NRL allergy. OBJECTIVE: To examine allergen-specific cytokine and chemokine responses in NRL-allergic patients. METHODS: Fourteen NRL-allergic patients and 10 healthy controls participated in the study. Hev b 6.01 and Hev b 5 were purified under non-denaturating conditions by chromatographic methods. Specific IgE antibodies were measured by ELISA and proliferation of peripheral blood mononuclear cells (PBMC) by (3)H-thymidine incorporation assay. Allergen-specific induction of cytokine and chemokine mRNA in PBMC was measured by real-time PCR and protein levels by ELISA. Surface expression of chemokine receptors was analysed by flow cytometry. RESULTS: Twelve (86%) NRL-allergic patients had positive skin prick test reactions and IgE antibodies against Hev b 6.01, but less than 30% responded to Hev b 5. Cell proliferation against Hev b 6.01, but not against Hev b 5, was significantly increased. Both allergens elicited significantly higher expression of pro-inflammatory and T-helper type 2 cytokines (TNF, IL-12p40, IL-13) and chemokines (CCL3, CCL4, CCL20) in the NRL-allergic patients than in controls. Interestingly, mRNA expression of the regulatory cytokine TGF-beta1 was reduced, whereas IL-10 expression was enhanced after allergen stimulations in patients with NRL allergy. Finally, the NRL-allergic patients showed increased CCR4 expression on CD3(+)CD8(-) T cells and decreased CXCR3 expression on CD3(+)CD8(+) T cells. CONCLUSION: Allergen-specific induction of cytokines and chemokines in PBMC and chemokine receptor expression on circulating T cells may contribute to the pathogenesis of NRL allergy.

IgE antibodies to omega-5 gliadin in children with wheat-induced anaphylaxis.

BACKGROUND: Wheat can cause severe immunoglobulin E (IgE)-mediated systemic reactions including anaphylaxis but knowledge on relevant wheat allergens at the molecular level is scanty. METHODS: Seven children (aged from 6 months to 13 years) experiencing from 2 to 10 anaphylactic reactions in a year after eating food-containing wheat were examined. Purified omega-5 gliadin was used as an allergen in IgE enzyme-linked immunosorbent assay (ELISA) and in skin prick testing (SPT). Wheat CAP radioallergosorbent test (RAST) and SPT were also examined. RESULTS: All seven anaphylactic children, but none of 15 control subjects had IgE antibodies to omega-5 gliadin in ELISA. Five of the six tested anaphylactic children showed positive SPT to omega-5 and crude gliadin, and all seven had positive wheat CAP RAST and SPT. One child was challenged with wheat, which caused anaphylaxis. After adherence to a wheat-free diet four children remained symptomless and three experienced one to two anaphylactic reactions. CONCLUSION: The present results show that wheat omega-5 gliadin is a major sensitizing allergen in children with wheat-induced anaphylaxis. They also suggest that omega-5 gliadin IgE ELISA could be used as a diagnostic test for this severe allergy.

Humoral and cellular responses to gliadin in wheat-dependent, exercise-induced anaphylaxis.

BACKGROUND: Wheat-dependent, exercise-induced anaphylaxis (WDEIA) is a severe allergy where wheat ingestion together with physical exercise induces anaphylaxis. We have previously shown that patients with WDEIA have IgE antibodies against gliadin proteins and identified omega-5 gliadin (Tri a 19) as a major allergen. OBJECTIVE: The aim of this study was to examine gliadin-specific IgG subclass, IgA and IgE antibodies, basophil histamine release and cell-mediated responses in WDEIA. METHODS: Sera and peripheral blood mononuclear cells (PBMC) were obtained from patients with WDEIA and from controls without wheat allergy. Serum antibodies to crude gliadin extract (CGE) and purified omega-5 gliadin were measured by ELISA and basophil reactivity by histamine-release test. Gliadin-induced cell-mediated responses were assessed by lymphocyte proliferation assay, and cytokine mRNA expression with real-time quantitative PCR. RESULTS: All patients with WDEIA, but none of the controls, had IgE antibodies to CGE and omega-5 gliadin. Both allergens released high levels of histamine from the basophils of patients with WDEIA. Levels of IgA antibodies to CGE and omega-5 gliadin were significantly elevated in the patients, but the distribution of IgG subclass antibodies showed no statistically significant differences between the two groups. Proliferative responses of PBMC to CGE were increased in patients with WDEIA, and stimulation of PBMC with CGE caused, both in patients and in controls, a clear induction of IL-10 mRNA. Compared with the controls, induction of IL-10 mRNA expression in patients with WDEIA was significantly (P < 0.01) suppressed. CONCLUSION: These results suggest that, in addition to IgE antibodies against omega-5 gliadin, specific IgA antibodies may be involved in the pathogenesis of WDEIA. Decreased expression of IL-10 mRNA in PBMC during gliadin stimulation may facilitate the development of gliadin-specific T cell responses.

Wheat omega-5 gliadin is a major allergen in children with immediate allergy to ingested wheat.

BACKGROUND: Sensitization to wheat by ingestion can lead to food allergy symptoms and wheat-dependent, exercise-induced anaphylaxis. Sensitization by inhalation causes bakers' asthma and rhinitis. Wheat allergens have been characterized at the molecular level in bakers' asthma and in wheat-dependent, exercise-induced anaphylaxis, in which omega-5 gliadin (Tri a 19) is a major allergen. However, little information is available regarding allergens responsible for hypersensitivity reactions to ingested wheat in children. OBJECTIVE: The aim of this study was to examine whether children with allergy to ingested wheat have IgE antibodies to omega-5 gliadin. METHODS: Sera were obtained from 40 children (mean age, 2.5 years; range, 0.7-8.2 years) with suspected wheat allergy who presented with atopic dermatitis and/or gastrointestinal and/or respiratory symptoms. Wheat allergy was diagnosed with open or double-blinded, placebo-controlled oral wheat challenge. Wheat omega-5 gliadin was purified by reversed-phase chromatography, and serum IgE antibodies to omega-5 gliadin were measured by means of ELISA. In vivo reactivity was studied by skin prick testing. Control sera were obtained from 22 children with no evidence of food allergies. RESULTS: In oral wheat challenge, 19 children (48%) reacted with immediate and 8 children (20%) with delayed hypersensitivity symptoms. Sixteen (84%) of the children with immediate symptoms had IgE antibodies to purified omega-5 gliadin in ELISA. In contrast, IgE antibodies to omega-5 gliadin were not detected in any of the children with delayed or negative challenge test results or in the control children. The diagnostic specificity and positive predictive value of omega-5 gliadin ELISA were each 100% for immediate challenge reactions. Skin prick testing with omega-5 gliadin was positive in 6 of 7 children with immediate challenge symptoms and negative in 2 children with delayed challenge symptoms. CONCLUSION: The results of this study show that omega-5 gliadin is a significant allergen in young children with immediate allergic reactions to ingested wheat. IgE testing with omega-5 gliadin could be used to reduce the need for oral wheat challenges in children.

Rye gamma-70 and gamma-35 secalins and barley gamma-3 hordein cross-react with omega-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis.

Patients with wheat-dependent, exercise-induced anaphylaxis experience severe allergic reactions when exercising after ingestion of wheat. The major wheat allergen associated with these reactions is a omega-5 gliadin, and patients following a gluten-free diet have remained free of symptoms. The aim of this study was to examine whether allergens cross-reacting with wheat omega-5 gliadin are present in rye, barley and oats. Sera from 23 adult patients with wheat-dependent, exercise-induced anaphylaxis were examined. Cereal allergens cross-reacting with wheat omega-5 gliadin were identified by immunoblot inhibition. The cross-reactive allergens were purified by gel filtration and reversed-phase chromatography and submitted to amino acid sequencing. Cross-reactivity was further studied by IgE ELISA and ELISA inhibition, and in vivo reactivity by skin prick testing. In immunoblotting rabbit anti-omega-5 gliadin antibodies bound to 70 kDa and 32 kDa proteins in rye and a 34-kDa protein in barley, but not to proteins in oats. N-terminal sequencing identified these proteins as rye gamma-70 secalin, rye gamma- 35 secalin and barley gamma-3 hordein, correspondingly. In ELISA 21/23 (91%) patients with wheat-dependent, exercise-induced anaphylaxis showed IgE antibodies to purified gamma-70 secalin, 19/23 (83%) to gamma-35 secalin and 21/23 (91%) to gamma-3 hordein. In ELISA inhibition omega-5 gliadin inhibited over 90% of the IgE binding of pooled patient sera to solid-phase gamma-secalins and gamma-3 hordein. Skin prick testing gave positive reactions to gamma-70 secalin in 10/15 (67%) patients, to gamma-35 secalin in 3/15 (20%) patients and to gamma-3 hordein in 7/15 (47%) patients. The results of this study show that gamma-70 and gamma-35 secalins in rye and gamma-3 hordein in barley cross-react with omega-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis. These findings suggest that also rye and barley may elicit symptoms in patients with wheat-dependent, exercise-induced anaphylaxis.

A novel wheat gliadin as a cause of exercise-induced anaphylaxis.

BACKGROUND: Food-dependent, exercise-induced anaphylaxis is a severe form of allergy; the reaction is caused by ingestion of a specific food before exercise. This disorder often escapes diagnosis because neither the ingested food nor the exercise alone induces the symptoms. OBJECTIVE: The aim of the study was to characterize the allergens involved in wheat-dependent, exercise-induced anaphylaxis and to describe the clinical outcome in a series of 18 adult patients. METHODS: All 18 patients had experienced recurrent episodes of generalized urticaria during exercise, 17 patients in association with collapse and 15 patients with an anaphylactic reaction. The symptoms appeared only when the patients had eaten food containing wheat before exercise. Wheat allergens were detected by immunoblotting, purified by gel filtration and reversed-phase chromatography, and subjected to N-terminal sequencing. The IgE-binding ability of the purified proteins was studied by ELISA, and their in vivo reactivity was studied by skin prick testing. RESULTS: IgE antibodies from pooled patient sera were bound to 65-kd and 40-kd wheat proteins in immunoblotting. The 65-kd allergen was a previously undescribed wheat protein, showing 61% sequence identity to gamma-gliadin, whereas the 40-kd allergen had 100% identity to alpha-gliadin. In ELISA, all 18 patients showed elevated IgE levels to the novel gamma-like gliadin, and 13 of the patients showed elevated IgE levels to the alpha-gliadin. None of the 54 control subjects with wheat allergy, urticaria, or coeliac disease had IgE antibodies to the gamma-like gliadin. The in vivo reactivity of the gamma-like gliadin was verified by positive skin prick test responses in all of the 15 patients who were tested. During the follow-up on a gluten-free or wheat-free diet, 3 patients experienced reactions after having unknowingly eaten wheat before exercise, but all the other patients who were adhering to the diet remained symptom-free. CONCLUSION: This study shows that wheat is a frequent cause of food-dependent, exercise-induced anaphylaxis and suggests that the major allergen is a previously undescribed gamma-like gliadin. For screening of this life-threatening allergy, we recommend skin prick testing with crude gliadin and we recommend a gluten-free diet for treatment.

Seasonal increase in human IgE and IgG4 antisaliva antibodies to Aedes mosquito bites.

BACKGROUND: Mosquito bite-sensitive subjects frequently have circulating IgE and IgG4 antibodies to Aedes mosquito saliva proteins. METHODS: In the present study we examined the antibody response during a mosquito season in 14 subjects living in Finnish Lapland. Immunoblotting was performed with Aedes communis saliva and the 22- and 36-kD antisaliva antibody bands were analyzed. RESULTS: The preseason sera showed IgE antibodies to the main saliva antigens in 12, IgG4 antibodies in all 14 and IgG1 antibodies in 12 subjects, and the postseason sera in all but 1 subject. The postseason sera showed significantly more intense IgE (p < 0.05), IgG4 (p < 0.001) and IgG1 (p < 0.01) antibody bands than the preseason sera. CONCLUSION: These results show that seasonal exposure to mosquito bites leads to an increased IgE, IgG4 and IgG1 antibody response, a phenomenon similar to that occurring e.g. in pollen allergy.

Detection of mosquito saliva-specific IgE antibodies by capture ELISA.

We developed an IgE-capture ELISA and measured mosquito saliva-specific IgE antibodies in 27 children sensitive to mosquito bites. Children with large 15-min bite wheals had significantly higher (P < 0.0005) mosquito saliva-specific IgE levels than children with small wheals. In the latter group, the saliva-specific IgE level was significantly higher (P = 0.031) than the levels of six infants never exposed to mosquitoes. A positive correlation (r = 0.65; P = 0.0002) was found between the size of the 15-min wheal and the mosquito saliva-specific IgE antibody levels. These results further support the role of mosquito saliva-specific IgE antibodies in the pathogenesis of mosquito-bite whealing. Compared to immunoblotting, IgE-capture ELISA provides a quantitative method to measure mosquito saliva-specific IgE antibodies.

Frequent occurrence of IgE and IgG4 antibodies against saliva of Aedes communis and Aedes aegypti mosquitoes in children.

We examined the prevalence of IgE and IgG4 class antibodies to the saliva of Aedes communis and Aedes aegypti mosquitoes in the sera of three groups of exposed children using a sensitive immunoblot method. The frequencies of IgE antibodies to the major 36-kD A. communis and A. aegypti saliva antigens ranged from 82 to 90% in the 20 Finnish, 17 Kenyan, and 20 Mexican children. The corresponding IgG4 antibody frequencies were 85, 41, and 20%, respectively. The nonexposed 20 Icelandic children did not show IgE or IgG4 antisaliva antibodies. Several of the Finnish children showed also IgE and IgG4 antibodies to a 22-kD A. communis saliva antigen. The Finnish children abnormally sensitive to mosquito bites had frequently IgE and IgG4 antibodies to the 22-kD A. communis saliva antigen, suggesting that these antibodies play a role in the pathogenesis of immediate cutaneous mosquito bite reactions. In contrast to this, no increase was found in the A. aegypti antibody frequencies in the Kenyan and Mexican children with papular urticaria, suggesting that humoral immune response to A. aegypti saliva is not involved in the development of this disorder. The present results show that humoral IgE and IgG4 immune responses to Aedes mosquito saliva antigens is common in children living both in temperature and tropical zones. The IgE antibodies seem to be involved in the immediate mosquito bite whealing, and the occurrence of the IgG4 subclass antisaliva antibodies might be an indicator of intense mosquito bite exposure.