Detection of Sensitization Profiles with Cellular In Vitro Tests in Wheat Allergy Dependent on Augmentation Factors (WALDA).

Wheat allergy dependent on augmentation factors (WALDA) is the most common gluten allergy in adults. IgE-mediated sensitizations are directed towards ω5-gliadin but also to other wheat allergens. The value of the different in vitro cellular tests, namely the basophil activation test (BAT) and the active (aBHRA) and passive basophil histamine-release assays (pBHRA), in the detection of sensitization profiles beyond ω5-gliadin has not been compared. Therefore, 13 patients with challenge-confirmed, ω5-gliadin-positive WALDA and 11 healthy controls were enrolled. Specific IgE (sIgE), skin prick tests, BATs, aBHRA, and pBHRA were performed with allergen test solutions derived from wheat and other cereals, and results were analyzed and compared. This study reveals a distinct and highly individual reactivity of ω5-gliadin-positive WALDA patients to a range of wheat allergens beyond ω5-gliadin in cellular in vitro tests and SPT. In the BAT, for all tested allergens (gluten, high-molecular-weight glutenin subunits, α-amylase/trypsin inhibitors (ATIs), alcohol-free wheat beer, hydrolyzed wheat proteins (HWPs), rye gluten and secalins), basophil activation in patients was significantly higher than in controls (p = 0.004-p < 0.001). Similarly, significant histamine release was detected in the aBHRA for all test substances, exceeding the cut-off of 10 ng/mL in all tested allergens in 50% of patients. The dependency of tests on sIgE levels against ω5-gliadin differed; in the pBHRA, histamine release to any test substances could only be detected in patients with sIgE against ω5-gliadin ≥ 7.7 kU/L, whereas aBHRA also showed high reactivity in less sensitized patients. In most patients, reactivity to HWPs, ATIs, and rye allergens was observed. Additionally, alcohol-free wheat beer was first described as a promising test substance in ω5-gliadin-positive WALDA. Thus, BAT and aBHRA are valuable tools for the identification of sensitization profiles in WALDA.

Identification of RNAi hypoallergic bread wheat lines for wheat-dependent exercise-induced anaphylaxis patients.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is one of the most severe forms of wheat allergy. It occurs in patients when they exercise after ingesting wheat-containing foods. Nowadays, the only possible alternative for WDEIA patients is to avoid such foods. This study investigated the potential of six RNA of interference (RNAi) wheat lines with low-prolamin content as alternatives for WDEIA patients. For that purpose, a high performance-liquid chromatography (HPLC) analysis was performed to evaluate differences in gluten protein fractions among these lines. Next, western blots were conducted to measure the immunoglobulin E (IgE) reactivity to wheat proteins in sera from five WDEIA patients. Additionally, monoclonal antibodies (moAb) recognition sites and the IgE binding sites were searched in all peptides identified by LC-MS/MS after protein digestion. The results showed a 61.4%-81.2% reduction in the gliadin content of the RNAi lines, accompanied by an increase in their high-molecular weight (HMW) glutenin content compared to the wild type bread wheat line (WT). In all cases, the reduction in gliadin content correlated with a decrease in IgE reactivity observed in the sera of WDEIA patients, highlighting the E82 and H320 lines. These two RNAi lines exhibited a ≤90% reduction in IgE reactivity. This reduction could be attributed to an absence of IgE binding sites associated with α- and ω5-gliadins, which were present in the WT. Overall, these lines offer a potential alternative for foodstuff for individuals with WDEIA.

Phenotypic Distinctions Between Omega-5-Gliadin Allergy and Peanut Allergy: Clinical Profile, Reaction Rates and Triggers, and Quality of Life.

Background: Different phenotypes of food allergy may exist, exhibiting distinct clinical features, and driven by different pathogenic mechanisms. We compared omega-5-gliadin (O5G) allergy to peanut allergy, focusing on clinical features, reaction rates and triggers, and quality of life (QOL). Methods: We surveyed adults with O5G allergy and peanut allergy regarding their diagnosis, co-morbidities, allergic reactions, and QOL measured by the FAQLQ-AF. Results: We received responses from 43/80 (54%) individuals with O5G allergy and 43/130 (33%) with peanut allergy. Compared to peanut allergic individuals, those with O5G allergy were older at age of onset (37.2 vs 2.5 years, p < 0.001), had fewer additional atopic conditions (0.88 vs 2.93, p < 0.001) or food allergies (0.15 vs 1.86, p < 0.001), and more frequent reactions before diagnosis (1.085 vs 0.29 per month, p < 0.05) Reaction rates improved in both groups following diagnosis. Reactions to peanut were more often triggered by accidental exposure (84% vs 26%, p < 0.001) and being away from home (65% vs 28%, p < 0.001), while reactions to O5G were more often due to deliberate ingestion (30% vs 9%, p < 0.05) or unexpected exercise (35% vs 2%, p < 0.001). Overall QOL score was similar between groups (4.2 in O5G allergy, 4.7 in peanut allergy, p = 0.12), but worse among women and those with additional food allergies. Conclusion: Phenotypic differences between O5G and peanut allergy support the development of different clinical approaches and the possibility of targeting distinct pathogenic mechanisms for prevention and treatment. Quality of life was impaired to a similar degree between groups.

The basophil activation test differentiates between patients with wheat-dependent exercise-induced anaphylaxis and control subjects using gluten and isolated gluten protein types.

BACKGROUND: Oral food challenge using gluten and cofactors is the gold standard to diagnose wheat-dependent exercise-induced anaphylaxis (WDEIA), but this procedure puts patients at risk of an anaphylactic reaction. Specific IgE to ω5-gliadins as major allergens and skin prick tests to wheat may yield negative results. Thus, we designed a proof-of-principle study to investigate the utility of the basophil activation test (BAT) for WDEIA diagnosis. METHODS: Different gluten protein types (GPT; α-, γ-, ω1,2- and ω5-gliadins, high-molecular-weight glutenin subunits [HMW-GS] and low-molecular-weight glutenin subunits [LMW-GS]) and gluten were used in different concentrations to measure basophil activation in 12 challenge-confirmed WDEIA patients and 10 control subjects. The results were compared to routine allergy diagnostics. Parameters analyzed include the percentage of CD63+ basophils, the ratio of %CD63+ basophils induced by GPT/gluten to %CD63+ basophils induced by anti-FcεRI antibody, area under the dose-response curve and test sensitivity and specificity. RESULTS: GPT and gluten induced strong basophil activation for %CD63+ basophils and for %CD63+/anti-FcɛRI ratio in a dose-dependent manner in patients, but not in controls (p < 0.001, respectively). BAT performance differed from acceptable (0.73 for LMW-GS) to excellent (0.91 for ω5-gliadins) depending on the specific GPT as evaluated by the area under the receiver operating characteristic curve. Patients showed individual sensitization profiles. After determination of the best cut-off points, ω5-gliadins and HMW-GS showed the best discrimination between patients and controls with a sensitivity/specificity of 100/70 and 75/100, respectively. CONCLUSION: This study shows the alternative role of BAT in better defining WDEIA and the causative wheat allergens. The best BAT parameters to distinguish WDEIA patients from controls were %CD63+ basophil values for ω5-gliadins and HMW-GS.

Evaluation of Diagnosis and Management of Omega-5-Gliadin Allergy: A Retrospective Survey.

BACKGROUND: Allergy to the omega-5-gliadin component of gluten (O-5-G allergy) often manifests when wheat ingestion is followed by a co-factor, usually exercise. There is no established best approach to management. OBJECTIVE: We sought to identify the beneficial effects, firstly of establishing a firm diagnosis, and secondly of stringent management, either by avoiding gluten ingestion altogether or separating it temporally from exercise by at least 4 hours. We also determined how frequently patients adhered to their physicians' clinical recommendations. METHODS: We undertook a survey of individuals diagnosed with O-5-G allergy at our institution over 8 years, who had a consistent clinical history and confirmatory laboratory evidence. RESULTS: Of 80 eligible individuals, 43 responded (54%). Symptoms began in adulthood for all bar one, and concurrent asthma and eczema was uncommon (9% prevalence, respectively). Median time to diagnosis was 2 years. Achieving a diagnosis reduced the rate of reactions (0.35 per month vs 1.085 reactions per month, p=0.029). Many patients (10/43) did not adhere to the recommended stringent approach, to either avoid wheat/gluten or separate food and exercise by 4 hours. However, those adopting a stringent approach had a substantially lower risk of recurrent allergic reaction (0.22 per month vs 0.74 per month, p=0.004). CONCLUSION: The epidemiology of O-5-G allergy implies pathogenic mechanisms potentially distinct from those of childhood-onset food allergy. Accurate diagnosis improves the clinical trajectory, primarily through the adoption of a stringent management approach.

Wheat Anaphylaxis in Adults Differs from Reactions to Other Types of Food.

BACKGROUND: Wheat is one of the most commonly consumed foods and a known elicitor of anaphylaxis in children and adults. Reactions in adults are often cofactor dependent and characterized by a prolonged time between food intake and the onset of symptoms making the diagnosis of wheat anaphylaxis challenging. OBJECTIVE: To characterize a cohort of patients with the history of wheat anaphylaxis to better understand this atypical phenotype of anaphylaxis. METHODS: Data from the European Anaphylaxis Registry from 2007 to 2019 (n = 10,636) including 250 patients (213 adults and 37 children) with a history of anaphylaxis caused by wheat were analyzed. RESULTS: Wheat was the most common food elicitor of anaphylaxis in adults in the registry in Central Europe. Reactions to wheat in adults were frequently associated with exercise as a cofactor (82.8%) and partially delayed (57.5%). Only 36.9% of patients had atopic comorbidities, which was uncommonly low for adult patients allergic to other kinds of foods (63.2%). Anaphylaxis to wheat presented frequently with cardiovascular symptoms (86.7%) including severe symptoms such as loss of consciousness (41%) and less often with respiratory symptoms (53.6%). The reactions to wheat were more severe than reactions to other foods (odds ratio [OR] = 4.33), venom (OR = 1.58), or drugs (OR = 2.11). CONCLUSIONS: Wheat is a relevant elicitor of anaphylaxis in adults in Central Europe. Wheat anaphylaxis is highly dependent on the presence of cofactors and less frequently associated with atopic diseases compared with other food allergies. More data on mechanisms of wheat-induced anaphylaxis are required to develop preventive measures for this potentially life-threatening disease.

Evaluation of the allergenicity of ω5-gliadin-deficient Hokushin wheat (1BS-18) in a wheat allergy rat model.

We previously developed Hokushin wheat line as a hypoallergenic wheat lacking ω5-gliadin (1BS-18), a major allergen for wheat-dependent exercise-induced anaphylaxis. However, the allergenicity of 1BS-18 has not been understood completely. In this study, we evaluated the allergenicity of 1BS-18 such as anaphylactic elicitation ability and sensitization ability using rats sensitized with ω5-gliadin or glutens prepared from Hokushin (Hokushin gluten) or 1BS-18 (1BS-18 gluten). Rats were sensitized by intraperitoneal administration of ω5-gliadin, Hokushin gluten or 1BS-18 gluten. Immunoglobulin E-mediated systemic anaphylaxis was evaluated by measuring changes in rectal temperature for 30 min after intravenous challenge with ω5-gliadin or the test glutens in unsensitized rats or rats sensitized with ω5-gliadin or the test glutens. In ω5-gliadin-sensitized rats, intravenous challenge with ω5-gliadin or Hokushin gluten significantly decreased the rectal temperature at 30 min after challenge while challenge with 1BS-18 gluten did not reduce the rectal temperature. Furthermore, intravenous challenge with ω5-gliadin significantly decreased the rectal temperature in rats sensitized with Hokushin gluten or 1BS-18 gluten. However, the reduced degree observed in 1BS-18 gluten-sensitized rats was smaller than that in Hokushin gluten-sensitized rats. In conclusion, 1BS-18 elicited no allergic reaction in ω5-gliadin-sensitized rats and had less sensitization ability for ω5-gliadin than that of Hokushin wheat.

Cofactors of wheat-dependent exercise-induced anaphylaxis do not increase highly individual gliadin absorption in healthy volunteers.

BACKGROUND: In wheat-dependent exercise-induced anaphylaxis (WDEIA), cofactors such as exercise, acetylsalicylic acid (ASA), alcohol or unfavorable climatic conditions are required to elicit a reaction to wheat products. The mechanism of action of these cofactors is unknown, but an increase of gliadin absorption has been speculated. Our objectives were to study gliadin absorption with and without cofactors and to correlate plasma gliadin levels with factors influencing protein absorption in healthy volunteers. METHODS: Twelve healthy probands (six males, six females; aged 20-56 years) ingested 32 g of gluten without any cofactor or in combination with cofactors aerobic and anaerobic exercise, ASA, alcohol and pantoprazole. Gliadin serum levels were measured up to 120 min afterwards and the intestinal barrier function protein zonulin in stool was collected before and after the procedure; both were measured by ELISA. Stool microbiota profile was obtained by 16S gene sequencing. RESULTS: Within 15 min after gluten intake, gliadin concentrations in blood serum increased from baseline in all subjects reaching highly variable peak levels after 15-90 min. Addition of cofactors did not lead to substantially higher gliadin levels, although variability of levels was higher with differences between individuals (p < 0.001) and increased levels at later time points. Zonulin levels in stool were associated neither with addition of cofactors nor with peak gliadin concentrations. There were no differences in gut microbiota between the different interventions, although the composition of microbiota (p < 0.001) and the redundancy discriminant analysis (p < 0.007) differed in probands with low versus high stool zonulin levels. CONCLUSION: The adsorption of gliadin in the gut in healthy volunteers is less dependent on cofactors than has been hypothesized. Patients with WDEIA may have a predisposition needed for the additional effect of cofactors, e.g., hyperresponsive or damaged intestinal epithelium. Alternatively, other mechanisms, such as cofactor-induced blood flow redistribution, increased activity of tissue transglutaminase, or increases in plasma osmolality and acidosis inducing basophil and mast cell histamine release may play the major role in WDEIA.

Wheat allergy in patients with recurrent urticaria.

BACKGROUND: Clinical observation revealed that most of wheat-induced anaphylaxis (WIA)/wheat-dependent exercise-induced anaphylaxis (WDEIA) patients showed a history of recurrent urticaria. We aim to determine the association between recurrent urticaria and anaphylaxis in wheat allergy. METHODS: Population-based cohort study involved patients with WIA (n = 193, including WDEIA n = 104), recurrent urticaria (n = 177), non-wheat-related anaphylaxis (n = 584), atopic disease (excluding anaphylaxis, n = 221) and healthy control (n = 95) from 2009 to 2016. Detailed course of urticaria and anaphylaxis were obtained from medical records and following-up questionnaire. Serum IgE specific to wheat, gluten and ω-5 gliadin and skin prick test to wheat were examined. Clinical and laboratory data were statistically analyzed. RESULTS: In recurrent urticaria patients, wheat allergy was not rare, and 6.8% (n = 12) was diagnosed as wheat-induced urticaria. Patients with WIA/WDEIA had higher prevalence of recurrent urticaria history than those with non-wheat-related anaphylaxis (164/193, 84.9% vs 85/584, 14.5%), and 70.4% of them (136/193) had recurrent urticaria prior to their first anaphylactic attack. Among patients with WIA/WDEIA and previous urticaria, 46.3% experienced an exacerbation of urticaria. The value of serum specific IgE to ω-5 gliadin was significantly higher in patients with WIA/WDEIA than those with wheat-induced urticaria. CONCLUSIONS: We recommend screening wheat allergy in recurrent urticaria to identify patients who have a potential risk to develop severe reactions earlier.

Identification and molecular characterization of allergenic non-specific lipid-transfer protein from durum wheat (Triticum turgidum).

BACKGROUND: Common wheat (Triticum aestivum) and durum wheat (T. turgidum) are both involved in Baker's asthma (BA) and food allergy (FA) including wheat-dependent exercise-induced asthma (WDEIA). However, allergens in durum wheat have not been described, and the over-expression of T. turgidum non-specific lipid-transfer protein (nsLTPs) is considered to increase resistance to phytopathogens. OBJECTIVE: To identify and assess the allergenicity of nsLTP from T. turgidum. METHODS: Recombinant T. turgidum nsLTP Tri tu 14 was generated and tested for structural integrity (circular dichroism-spectroscopy) and purity (SDS-PAGE). Thirty-two wheat allergic patients were enrolled: 20 Spanish patients (BA) with positive bronchial challenge to wheat flour, and 12 Italian patients (wheat FA/WDEIA) with positive double-blind placebo-controlled food challenge/open food challenge (OFC) to pasta. IgE values to wheat, Tri tu 14, Tri a 14 (T. aestivum) and Pru p 3 (P. persica) were determined by ImmunoCAP testing. Allergenic potency (in vitro mediator release) and IgE cross-reactivity were investigated. RESULTS: Tri tu 14 was found to share 49% and 52% amino acid identity with Tri a 14 and Pru p 3, respectively. Among 25 Tri a 14 CAP positive sera, 23 (92%) were reactive to wheat extract, 22 (88%) to Tri tu 14 and 20 (80%) to Pru p 3. The correlation between Tri a 14 and Tri tu 14 specific IgE levels was r = 0.97 (BA) and r = 0.93 (FA/WDEIA), respectively. FA/WDEIA patients showed higher specific IgE values to Tri tu 14 and Pru p 3 than BA patients. Tri tu 14 displayed allergenic activity by mediator release from effector cells and IgE cross-reactivity with Pru p 3. The degree of IgE cross-reactivity between the two wheat nsLTPs varied between individual patients. CONCLUSIONS AND CLINICAL RELEVANCE: Sensitization to Tri tu 14 likely appears to be more important in wheat FA/WDEIA than in BA. Over-expression of Tri tu 14 in wheat would represent a risk for patients with nsLTP-mediated FA.

Assessment of the Allergenic Potential of Transgenic Wheat (Triticum aestivum) with Reduced Levels of ω5-Gliadins, the Major Sensitizing Allergen in Wheat-Dependent Exercise-Induced Anaphylaxis.

The ω5-gliadins are the major sensitizing allergens in wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, two-dimensional immunoblot analysis was used to assess the allergenic potential of two transgenic wheat lines in which ω5-gliadin genes were silenced by RNA interference. Sera from 7 of 11 WDEIA patients showed greatly reduced levels of immunoglobulin E (IgE) reactivity to ω5-gliadins in both transgenic lines. However, these sera also showed low levels of reactivity to other gluten proteins. Sera from three patients showed the greatest reactivity to proteins other than ω5-gliadins, either high-molecular-weight glutenin subunits (HMW-GSs), α-gliadins, or non-gluten proteins. The complexity of immunological responses among these patients suggests that flour from the transgenic lines would not be suitable for individuals already diagnosed with WDEIA. However, the introduction of wheat lacking ω5-gliadins could reduce the number of people sensitized to these proteins and thereby decrease the overall incidence of this serious food allergy.

Study on the Immunoreactivity of Triticum monococcum (Einkorn) Wheat in Patients with Wheat-Dependent Exercise-Induced Anaphylaxis for the Production of Hypoallergenic Foods.

Wheat [Triticum aestivum (T.a.)] ingestion can cause a specific allergic reaction, which is called wheat-dependent exercise-induced anaphylaxis (WDEIA). The major allergen involved is ω-5 gliadin, a gluten protein coded by genes located on the B genome. Our aim was to study the immunoreactivity of proteins in Triticum monococcum (einkorn, T.m.), a diploid ancestral wheat lacking B chromosomes, for possible use in the production of hypoallergenic foods. A total of 14 patients with a clear history of WDEIA and specific immunoglobulin E (IgE) to ω-5 gliadin were enrolled. Skin prick test (SPT) with a commercial wheat extract and an in-house T.a. gluten diagnostic solution tested positive for 43 and 100% of the cases, respectively. No reactivity in patients tested with solutions prepared from four T.m. accessions was observed. The immunoblotting of T.m. gluten proteins performed with the sera of patients showed different IgE-binding profiles with respect to T.a., confirming the absence of ω-5 gliadin. A general lower immunoreactivity of T.m. gluten proteins with scarce cross-reactivity to ω-5 gliadin epitopes was assessed by an enzyme-linked immunosorbent assay (ELISA). Given the absence of reactivity by SPT and the limited cross-reactivity with ω-5 gliadin, T.m. might represent a potential candidate in the production of hypoallergenic bakery products for patients sensitized to ω-5 gliadin. Further analyses need to be carried out regarding its safety.

Advances in in vitro diagnostics in allergy, asthma, and immunology in 2012.

Laboratory tests play an increasing role in risk assessment, diagnostics, and disease monitoring. Great advances have been achieved lately, particularly in the field of clinical immunology and allergy. These include neonatal screening of immunodeficiencies and asthma biomarkers and investigation into the role of recombinant allergens in in vitro testing. The latter area has implications for the diagnostics of food allergy, pollen-induced allergies, asthma, and insect allergies.