Wheat Allergic Patients Research Articles

A Narrative Mini Review on Current Status of Hypoallergenic Wheat Development for IgE-Mediated Wheat Allergy, Wheat-Dependent Exercise-Induced Anaphylaxis

Immunoglobulin E (IgE)-mediated food allergies to wheat that develop after school age typically shows a type of wheat-dependent exercise-induced anaphylaxis (WDEIA). At present, avoidance of wheat products or postprandial rest after ingesting wheat is recommended for patients with WDEIA, depending on the severity of the allergy symptoms. ω5-Gliadin has been identified as the major allergen in WDEIA. In addition, α/β-, γ-, and ω1,2-gliadins, high and low molecular weight-glutenins, and a few water-soluble wheat proteins have been identified as IgE-binding allergens in a small proportion of patients with IgE-mediated wheat allergies. A variety of approaches have been manufactured to develop hypoallergenic wheat products that can be consumed by patients with IgE-mediated wheat allergies. In order to analyze such approaches, and to contribute to the further improvement, this study outlined the current status of these hypoallergenic wheat productions, including wheat lines with a reduced allergenicity that are mostly constructed for the patients sensitized to ω5-gliadin, hypoallergenic wheat by enzymic degradation/ion exchanger deamidation, and hypoallergenic wheat by thioredoxin treatment. The wheat products obtained by these approaches significantly reduced the reactivity of Serum IgE in wheat-allergic patients. However, either these were not effective on some populations of the patients, or low-level IgE-reactivity to some allergens of the products was observed in the patients. These results highlight some of the difficulties faced in creating hypoallergenic wheat products or hypoallergenic wheat lines through either traditional breeding or biotechnology approaches in developing hypoallergenic wheat completely safe for all the patients allergic to wheat.

Clinical cross-reactivity of wheat and barley in children with wheat allergy.

Several studies have reported in vitro cross-reactivity between wheat and barley. However, evidence regarding the clinical cross-reactivity of wheat and barley is limited. This study examined the clinical cross-reactivity of barley and wheat among children with immediate-type wheat allergies. We examined the threshold dose of a wheat oral food challenge for wheat-allergic children. We examined the reactivity of barley, and the oral food challenges of barley tea and barley rice were implemented as needed. We measured the specific immunoglobulin E (sIgE) levels in wheat, ω-5 gliadin, and barley. We evaluated 53 children (39 [74%] boys) with a median age of 6.6 years. Among them, 39 (74%) patients had a history of anaphylaxis to wheat. The median wheat-, barley-, and ω-5 gliadin-sIgE levels were 57.3, 12.1, and 3.2 kUA /L, respectively. Twelve patients reacted to barley tea (1.8mg), 14 reacted to barley rice (220-440 mg), and 27 were tolerant to barley tea and barley rice. Barley-allergic patients had significantly higher wheat- and ω-5 gliadin- and barley-sIgE levels and significantly lower threshold doses of wheat than barley-tolerant patients. Omega-5 gliadin-sIgE was the most useful predictor of barley allergy among wheat-allergic patients; the ω-5 gliadin-sIgE 95% positive predictive value for barley allergy was 4.6 kUA /L. Half of wheat-allergic children reacted to barley. A lower threshold dose of wheat is related to cross-reactive barley allergies. Omega-5 gliadin-sIgE predicts cross-reactive barley allergy in children allergic to wheat. Clinical cross-reactivity to barley should be considered in the management of wheat-allergic children.

The Efficacy of a New Protocol of Oral Immunotherapy to Wheat for Desensitization and Induction of Tolerance.

Oral immunotherapy (OIT) is a novel approach to desensitization and tolerance induction in food allergy patients. This study aimed to design and implement a new wheat OIT protocol, evaluate its efficacy in tolerance induction, and assess specific immunoglobulin-E (IgE) and regulatory T cell changes. From 2015 to 2017, 26 patients with confirmed IgE-mediated hypersensitivity to wheat were treated via oral immunotherapy (OIT). Patients with prior anaphylactic episodes underwent OIT using the rush method. Specific IgE concentrations and the number of regulatory T cells (CD4+ CD25+ FOXP3+ T cells) were measured using Allergy Screen immunoblot assay and flow cytometry, respectively. This study was registered in the Iranian Registry of Clinical Trials (IRCT20181220042066N1). The results revealed success rates of 100% and 93.3% for desensitization and tolerance. Specific IgE was significantly reduced after 12 months of OIT. No significant change in regulatory T cell numbers was observed. In view of the promising findings of this study, the proposed OIT protocol could be viewed as an effective and valuable method to induce tolerance and desensitization in wheat allergic patients.

Evaluation of the discriminatory potential of antibodies created from synthetic peptides derived from wheat, barley, rye and oat gluten.

Celiac disease (CD) is triggered by ingestion of gluten-containing cereals such as wheat, barley, rye and in some cases oat. The only way for affected individuals to avoid symptoms of this condition is to adopt a gluten-free diet. Thus, gluten-free foodstuffs need to be monitored in order to ensure their innocuity. For this purpose, commercial immunoassays based on recognition of defined linear gluten sequences are currently used. These immunoassays are designed to detect or quantify total gluten regardless of the cereal, and often result in over or underestimation of the exact gluten content. In addition, Canadian regulations require a declaration of the source of gluten on the label of prepackaged foods, which cannot be done due to the limitations of existing methods. In this study, the development of new antibodies targeting discrimination of gluten sources was conducted using synthetic peptides as immunization strategy. Fourteen synthetic peptides selected from unique linear amino acid sequences of gluten were bioconjugated to Concholepas concholepas hemocyanin (CCH) as protein carrier, to elicit antibodies in rabbit. The resulting polyclonal antibodies (pAbs) successfully discriminated wheat, barley and oat prolamins during indirect ELISA assessments. pAbs raised against rye synthetic peptides cross-reacted evenly with wheat and rye prolamins but could still be useful to successfully discriminate gluten sources in combination with the other pAbs. Discrimination of gluten sources can be further refined and enhanced by raising monoclonal antibodies using a similar immunization strategy. A methodology capable of discriminating gluten sources, such as the one proposed in this study, could facilitate compliance with Canadian regulations on this matter. This type of discrimination could also complement current immunoassays by settling the issue of over and underestimation of gluten content, thus improving the safety of food intended to CD and wheat-allergic patients.

Omega-5 and Gamma Gliadin are the Major Allergens in Adult-Onset IgE-Mediated Wheat Allergy: Results from Thai Cohort with Oral Food Challenge.

BackgroundVarious clinical patterns based on routes of sensitization and sensitized allergens are reported in adult-onset IgE-mediated wheat allergy. There is still a paucity of data on IgE-bound wheat allergen profiles in wheat challenge-proven adult-onset wheat allergic cases. Therefore, we aim to identify the major sensitized allergens in Thai adult-onset wheat allergic patients whose first symptom occurred after the age of 18 years despite previous tolerance.MethodsThis cross-sectional pilot study recruited patients from the Thai Adult-onset IgE-mediated Wheat Allergy Cohort (TAWAC). The sera of patients with mostly challenge-proven cases were selected for allergen study, including ImmunoCAP and IgE-bound gliadins along with glutenins profiles. The IgE-bound proteins were identified by liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). Direct binding of IgE to recombinant gliadin and glutenin was performed to confirm the results of immunoblot and LC-MS/MS.ResultsEleven wheat-dependent exercise-induced anaphylaxis (WDEIA) and 4 typical wheat allergy (WA) patients were enrolled. Serum IgE from >50% of bound proteins had a molecular weight ranging from 35 to 55 kDa in both gliadin and glutenin extracts. Further, ELISA demonstrated that γ-gliadin and ω5-gliadin were the most important major allergens. Other major allergens include α/β-gliadin, HMW glutenin, and possibly α-amylase inhibitor or LWM glutenin. Gamma-gliadin sensitization was found in all WA patients (4/4), while ω-5 gliadin was found in all WDEIA patients (11/11) from ELISA.ConclusionWheat γ-gliadin and ω-5 gliadin are major wheat allergens among adult-onset wheat allergy patients in Thailand. Component-resolved diagnosis using γ-gliadin might be helpful in high suspicion of wheat allergy.

Extraction of total wheat (Triticum aestivum) protein fractions and cross-reactivity of wheat allergens with other cereals

A one-step mild extraction of total wheat protein fractions was developed in this study, and the allergic cross-reactivity among dietary cereals were assessed by SDS-PAGE, western blotting, indirect ELISA, and inhibition ELISA using sera from 12 wheat allergic patients. The fractions of albumin, globulin, gliadin and glutenins in wheat flour can be obtained by a one-step extraction with Na2CO3-NaHCO3 (20 mM, pH 9.6, 0.5 M NaCl, 40% ethanol, 1 mM PMSF) in comparison to sequential extractions. Results showed high cross-reactivity in wheat, barley and rye due to close resemblance and high sequence identity (>50%), whereas nearly negligible cross-reactivity among rice, buckwheat, and quinoa was observed. Our research findings suggest that people with wheat allergy should rely primarily on the use of rice, quinoa and non-grain buckwheat, which is an effective substitute for wheat, while those with hypersensitivity should avoid the use of barley and rye in their diet.

Clinical and in vitro cross-reactivity of cereal grains in children with IgE-mediated wheat allergy

Introduction and ObjectivesWheat and cereal grains have a broad range of cross-reactivity, but the clinical relevance of this cross-reactivity is uncertain. This study aimed to evaluate clinical and in vitro cross-reactivity with barley, oat, and Job’s tears among wheat-allergic patients. Materials and MethodsPatients aged 5 to 15 years with IgE-mediated wheat allergy were enrolled. Skin prick test (SPT) and specific IgE (sIgE) to wheat, barley, and oat, and SPT to Job’s tears were performed. Oral food challenge (OFC) was conducted if the SPT was ≤5 mm in size and there was no history of anaphylaxis to each grain. Profiles of sIgE bound allergens of wheat, barley, and oat, and inhibition ELISA of IgE binding to barley and oat with wheat were performed. ResultsTen patients with a median age of 8 years were enrolled. Nine of those patients had a history of wheat anaphylaxis. The median SPT size and sIgE level to wheat was 7.3 mm and 146.5 kUA/l, respectively. The cross-reactivity rate for barley, oat, and Job’s tears was 60.0%, 33.3%, and 20.0%, respectively. Significantly larger SPT size and higher sIgE level were observed in patients with positive cross-reactivity to barley and oat when compared to patients without cross-reactivity. Barley and oat extracts inhibited 59% and 16% of sIgE bound to wheat gliadins and glutenins, respectively. ConclusionThe cross-reactivity rate was quite low for oat and Job’s tears compared to that of barley; therefore, avoidance of all cereal grains may be unnecessary in patients with severe wheat allergy.

Wheat ATI CM3, CM16 and 0.28 Allergens Produced in Pichia Pastoris Display a Different Eliciting Potential in Food Allergy to Wheat ‡.

Although wheat is a staple food for most of the human population, some of its components trigger adverse reactions. Among wheat components, the alpha-amylase/trypsin inhibitors (ATI) are important triggers of several allergies and activators of innate immunity. ATI are a group of exogenous protease inhibitors and include several polypeptides. The three ATI polypeptides named CM3, CM16 and 0.28 are considered major allergens, and might also play a role in other common wheat-related pathologies, such as Non Celiac Wheat Sensitivity and even Celiac Disease. On this basis, we pointed to obtain high amounts of them in purity and to evaluate their allergenicity potential. We thus isolated the mRNA corresponding to the three ATI genes CM3, CM16 and 0.28 from 28 days post-anthesis wheat kernels and the corresponding cDNAs were used for heterologous expression in Pichia pastoris. The three purified proteins were tested in degranulation assay against human sera of patients with food allergy to wheat. A large range of degranulation values was observed for each protein according to the sera tested. All of the three purified proteins CM3, CM16 and 0.28 were active as allergens because they were able to induce basophils degranulation on wheat allergic patients’ sera, with the highest values of β-hexosaminidase release observed for CM3 protein.

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

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. To identify and assess the allergenicity of nsLTP from T.turgidum. 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 (invitro mediator release) and IgE cross-reactivity were investigated. 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. 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.

Safety evaluation of a β-amylase food enzyme obtained from wheat (Triticum spp.).

The food enzyme considered in this opinion is a β‐amylase (EC 3.2.1.2), obtained from the grain of wheat (Triticum spp.) by Roquette (France). The β‐amylase is intended to be used in starch processing for production of glucose syrups containing maltose to be used as a food ingredient. Since the presence of residual amounts of total organic solids (TOS) in glucose syrups after filtration and purification during starch processing is negligible, no dietary exposure was calculated. As the food enzyme is derived from edible parts of wheat, no toxicological tests are required. Wheat is known as a gluten‐containing cereal. However, the gluten content of the food enzyme was shown to be below the limit of quantification of the applied analytical method and well below the threshold value of 20 mg/kg for ‘gluten‐free’ products. Furthermore, the potential allergenicity was evaluated by searching for similarity between the amino acid sequence of the β‐amylase and the sequences of known food allergens; no match was found. Although β‐amylase from wheat is described as a potential occupational respiratory allergen, and oral wheat challenges in wheat allergic patients may result in clinical symptoms, the enzyme and the low levels of other wheat proteins will be removed from the final food ingredients through a downstream purification process. Based on the origin of the food enzyme from edible parts of grain, the manufacturing process, and the compositional and biochemical data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Oral Immunotherapy With Partially Hydrolyzed Wheat-Based Cereals: A Pilot Study.

To date, only few studies have assessed oral immunotherapy (OIT) for wheat allergy and often describe severe adverse reactions during therapy. We developed partially hydrolyzed wheat-based cereals (pHC), which were used in a multicenter, open-label, OIT pilot study, in immunoglobulin E–mediated wheat allergy children (NCT01332084). The primary objective of the study was to test whether wheat allergic patients tolerate pHC and primary end point was the presence or not of immediate adverse reactions to pHC during the 1-day initial escalation phase (stepwise increased doses of pHC), with evaluation of the maximum dose tolerated. Of the 9 patients enrolled in the trial, 4 discontinued OIT because of mild to severe reactions at the initial escalation phase. The 5 patients who passed the escalation phase consumed pHC daily for 1 to 6 months. One of these patients withdrew due to noncompliance, whereas the 4 others completed the study and successfully passed the wheat challenge test at the end of the study. About 60% of the adverse events were unrelated to the study product. Our study provides preliminary evidence that pHC is tolerated by a subset of wheat allergic patients. Further studies are warranted to test its efficacy as a potential therapeutic option for wheat allergic patients.

Wheat protein recognition pattern in tolerant and allergic children.

Wheat is one of the most common food allergens in early childhood. In contrast to other food allergies, wheat-specific IgE correlates badly with clinical symptoms and relevant components have been identified mostly for wheat-depended exercise-induced anaphylaxis. Moreover, a high percentage of patients present with immediate type symptoms but wheat-specific IgE cannot be detected with commercial available systems. We addressed the question whether the IgE recognition pattern between wheat allergic (WA) and clinically tolerant (WT) children differs in order to identify individual proteins useful for component-resolved diagnostics. Sera of 106 children with suspected wheat allergy, of whom 44 children had clinical relevant wheat allergy and 62 were tolerant upon oral food challenge, were analyzed for wheat-specific IgE using the ImmunoCap system as well as immunoblots against water and salt soluble, and water-insoluble protein fractions. 40 randomly selected sera were analyzed for specific IgE to ω5-gliadin. Sixty-three percent of the WT and 86% of the WA children were sensitized to wheat with >0.35 kUA /l in ImmunoCAP analysis. We could confirm the role of α-, ß-, γ-, and ω-gliadins, and LMW glutenin subunits as major allergens and found also IgE binding to a broad spectrum of water- and salt-soluble protein bands. It is of great importance that wheat allergic and tolerant patients showed IgE binding to the same protein bands. WT and WA did not significantly differ in levels of ω5-gliadin-specific IgE. Children with challenge proven clinical relevant food allergy and tolerant ones had a similar spectrum of IgE binding to the same protein bands. These findings imply that component-resolved diagnostics might not be helpful in the diagnostic work-up of wheat allergy.

A safe foodstuff for wheat allergic patients

Background Lipid Transfer Protein (LTP) allergy constitutes the first cause of food allergy to vegetables in Southern Europe. LTPs are highly stable proteins, resistant to both gastric digestion and high temperatures. Allergic reactions to multiples vegetables, not phylogenetically related, are produced based on the cross-reactivity among these proteins, a phenomenon that has been named LTP syndrome. Tri a 14, the wheat LTP, is involved in baker s asthma and anaphylactic reaction. In the present work, we studied Weetabix ® foodstuff tolerance in four patients with wheat allergy, characterized by oral food challenge (OFC) and skin tests, trying to identify the source of this tolerance in wheat-based foodstuff.

Evaluation of Allergenicity of Acid-Hydrolyzed Wheat Protein Using an in vitro Elicitation Test

Background: We performed an in vitro elicitation test to determine the ability of different types of wheat-allergic patients’ IgE to induce humanized mast cell activation after the addition of various time-treated acid-hydrolyzed wheat proteins (HWPs). Methods: The reactivity of heat- and various time-treated acid-hydrolyzed glutens (acid-HGs) and commercial acid-HWP (HWP1), using serum IgE from wheat allergy accompanied by skin and rhinoconjunctival sensitization to HWP1 in the facial soap, pediatric subjects with food allergy to native wheat, adult wheat-dependent exercise-induced anaphylaxis subjects, and nonatopic healthy subjects, was elucidated by dot blot and a luciferase assay-based in vitro elicitation test (EXiLE test). Results: Serum from subjects sensitized with HWP1 reacted only to acid-HGs (acid-HGs treated for 0.5–3 or 6 h), but not native gluten, in the results of the dot blot. In contrast, sera from pediatric subjects sensitized with native wheat reacted to native gluten more strongly and showed only slight reactions to 0.5- to 1-hour-treated acid-HGs. The results of the in vitro elicitation test showed that acid hydrolyzation of the gluten attenuated antigen-induced luciferase expression in a time-dependent manner for sera from native-wheat-sensitized pediatric subjects. On the other hand, in the sera from HWP1-sensitized subjects, acid hydrolyzation of the gluten for 0.5 h dramatically increased luciferase expression. Conclusions: Even after prolonged hydrolyzation, acid-HGs still retained the ability to activate mast cells in the case of HWP1-sensitized subjects.

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 Recombinant ω-Gliadin-like D-Type Glutenin and an α-Gliadin from Wheat (Triticum aestivum): Two Immunoglobulin E Binding Proteins, Useful for the Diagnosis of Wheat-Dependent Allergies

Among the wheat prolamins, D-type glutenins display a highly repetitive sequence similar to ω-gliadins, but they contain a cysteine, that allows them to be included in the gluten macropolymers. An ω-gliadin-like D-type glutenin, an α-gliadin, and an ω5-gliadin-like D-type glutenin were obtained as recombinant proteins and compared using synchrotron radiation circular dichroism. This technique evidenced the strong thermostability of the ω5-gliadin-like protein. The IgE reactivity of recombinant proteins was evaluated using 45 sera from wheat-allergic patients. The sera from patients diagnosed with cutaneous hypersensitivity to hydrolyzed wheat proteins often reacted with the ω-gliadin-like D-type glutenin and α-gliadin, whereas the IgE reaction was less frequent after dietary sensitization. So, these two proteins could be useful to diagnose these diseases. The sera from patients with exercise-induced anaphylaxis recognized the ω5-gliadin-like protein as a positive control and, less frequently, the other proteins tested. Only some sera from patients with baker's asthma reacted with the proteins tested.

Follow-up of the wheat allergy in children; consequences and outgrowing the allergy.

Allergy to wheat is a common food allergy. In spite of this fact, there is not enough literature regarding the features and outgrowing of this allergy. The objective of this study was to evaluate the manifestations of this allergy and to follow the patients to evaluate whether outgrowing allergy happens again and when it occurs.Eight wheat allergic patients diagnosed between 2000 and 2001 were re-evaluated together with 13 other new cases of wheat allergy referred to the Immunology and Allergy Pediatric Department from June 2004 to March 2006. For all cases, the demographic data along with a complete history regarding allergy to wheat and other types of allergy were collected in questionnaires. The specific IgE measurements (in vivo and in vitro) and oral food challenge (in the absence of a relevant history related to allergy to wheat) were performed. Severe anaphylaxis was seen after wheat ingestion in more than 90% of the patients. Oral tolerance to wheat developed in three patients (37.5%) out of 8 known previous cases who had been followed for eight years, the mean age of oral tolerance to wheat was 68 ± 6.36 (range; 36 months to 108 months).Clinical reactions in our wheat-allergic patients were more severe than those reported before. These patients were at risk for developing chronic allergic symptoms such as asthma. We also found that oral tolerance to wheat was happening in a minority of our patients.

Molecular characterization of wheat allergens specifically recognized by patients suffering from wheat‐induced respiratory allergy

Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour-Baker's asthma). The objective of this study was the molecular and immunological characterization of new recombinant wheat allergens and to evaluate their usefulness for the diagnosis of allergy to wheat. A T. aestivum cDNA library was constructed and screened with serum IgE from patients suffering from wheat allergy to identify cDNAs coding for new wheat allergens. The allergen-encoding cDNAs were expressed in Escherichia coli and purified to homogeneity. IgE reactivity of recombinant proteins was analysed in RAST-based, non-denaturing dot blot experiments and by ELISA with sera from wheat allergic patients and their allergenic activity was assessed in basophil degranulation experiments. We report the molecular characterization, recombinant expression and purification of five wheat allergens, a thioredoxin h isoform, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin. Homologous proteins were identified by sequence comparisons in various plants. 1-Cys-peroxiredoxin appeared to be the most relevant of the newly identified wheat allergens according to prevalence of IgE recognition and results from basophil degranulation experiments. It showed IgE cross-reactivity with seed proteins from barley, rye, rice, maize, soy, oat and spelt. 1-Cys-peroxiredoxin, glutathione transferase and dehydrin were mainly recognized by patients with baker's asthma but not wheat-induced food allergy. The characterized recombinant wheat allergens may be useful for the development of serological tests which allow the discrimination of different clinical manifestations of wheat allergy.

Immunoreactivity and Detection of Wheat Proteins by Commercial ELISA Kits

Wheat proteins are responsible for sensitivities, including baker's asthma, immunoglobulin E (IgE)-mediated allergic reaction, wheat-dependent, exercise-induced anaphylaxis, and celiac disease. The detection of gluten/wheat traces in foods is important to safeguard the health of wheat-sensitive individuals and comply with food labeling. Many immunoanalytical-based commercial kits are available for the quantification of gliadin/gluten/wheat proteins. We compared the immunoreactivity of wheat fractions with wheat-allergic human serum IgE and antibody conjugates used in six commercial immunoassay kits. Moreover, the performance of the kits was tested using corn flour spiked with gluten (5, 10, 25, and 50 ppm) and wheat flour (50, 100, 250, and 500 ppm). The albumin, globulin, gliadin, and glutenin fractions reacted with IgE from nine, eight, two, and eight patients' sera, respectively, out of nine wheat allergic patients tested. Among the antibodies from commercial kits, those from R-Biopharm, Morinaga, and Romer Labs reacted strongly with the gliadin fraction, whereas those from BioKits, ALLER-TEK, and ELISA Systems reacted strongly with the glutenin fraction. All kits showed minimal or no reactivity with albumin and globulin fractions. All kits detected the gluten and wheat flour in a corn flour matrix at the lowest spiked levels of 5 and 50 ppm, respectively. However, there was wide variation among the kits when comparing the recovery of gluten and wheat flour. The recovery was also dependent on the source material (gluten or wheat flour) used for spiking the corn flour matrix.

A Possible Hypoallergenic Cereal in Wheat Food Allergy and Baker’s Asthma

Background: Wheat is a potent allergen source and is one of the causes of baker’s asthma and food allergy. The best strategy for managing food hypersensitivity involves strict avoidance of the trigger. However, wheat is quite difficult to avoid. Several alternative strategies for the treatment of food allergy are under study. Spelt is a possible hypoallergenic crop that may be tried in patients with wheat allergy. Methods: We have evaluated the allergenic IgE hypersensitivity mediated by spelt in wheat allergic patients. Overall, 66 patients who suffered from baker’s asthma or food allergy (45 males and 21 females, mean age 28.6 ± 12.9 years) were included. We have also compared its reactivity with standard- ized extracts from wheat and with purified non-specific lipid transfer proteins from wheat (Tri a 14) and from peach (Pru p 3). Immunodetection with spelt and common bread wheat extracts (Triticum aestivum, cultivar Astral) was per- formed. Fresh wheat and spelt grain extracts were used both for oral and bronchial challenge and skin tests. Specific IgE detection to different cereals was performed using the Immuno CAP System (Phadia, Uppsala, Sweden). The bronchial challenge was positive with wheat Astral in 44 (67%) patients, all of them suffered from asthma. Thirteen (29.54%) of these 44 patients had negative the challenge with spelt. The oral challenge with wheat Astral was positive in 22 (33%) patients with wheat food allergy, and the same test was positive in only in 6 of them with spelt (27.3%). The diagnostic yield (sensitivity, specificity and predictive values) of routine tests in determining spelt allergy by specific positive challenge responses was determined. Prick tests for spelt versus positive challenge tests had a good sensitivity (94%, 86.5 - 99.4; 95%CI) and specificity (86%, 84 - 90; 95% CI) for the diagnosis of spelt allergy. Immunodetection detected minor differences among different extracts. Conclusion: In summary, the prick test and bronchial and oral challenges both efficiently detected sensitization to spelt and their levels were related to more severe clinical profiles, but the wheal area was significantly lower with spelt (p 0.001) and the percentage of positive challenge tests decreased. Our results suggest that spelt is an old crop that may be tried in patients with wheat allergy.