Intragastric administration of transamidated gliadin interferes with the systemic and intestinal immune responses to wheat gliadin in DQ8 transgenic mice.

We have previously shown the ability of transamidated gluten (spf) to modulate both innate and adaptive intestinal immunity elicited by wheat gliadin in HLA-DQ8 transgenic mice (DQ8 mice), a model of gluten sensitivity. Herein, we evaluated the influence of spf when administered intragastrically on the immune response to native gliadin in DQ8 mice. To address the issue, we analysed three regimens of antigen administration: before immunisation (pre-treatment), during immunisation (co-treatment) and through breast milk during the lactating phase (suckling treatment). Mice were immunised mucosally by intranasal delivery of digested wheat gliadin along with cholera toxin in multiple doses. After sacrifice, isolated spleen and mesenteric lymph node (MLN) cells were challenged in vitro and the cytokine profile of culture supernatants assessed by ELISA and multiparametric assay. We found that only pre-treatment with spf was effective in down-regulating the gliadin-specific IFN-γ response and only in spleen cells. Interestingly, spf pre-treatment also induced systemic IL-6, IL-17A and TNF-α. By contrast, we found that spf pre-treatment upregulated INF-γ in MLN but also significantly decreased IL-2. In conclusion, our data provide evidence that the preventive intragastric administration of transamidated gluten is able to interfere with the classical cytokine profile induced by gliadin via mucosal immunisation in a transgenic model expressing one of the HLA molecules associated with coeliac disease.

Allergenic Biomarkers in the Molecular Diagnosis of IgE-Mediated Wheat Allergy.

IgE-mediated wheat allergy can take on various forms, including childhood food allergy to wheat, wheat-dependent exercise-induced anaphylaxis in young adults, baker's respiratory allergy/asthma in workers exposed to wheat flour inhalation, and contact urticaria that is caused by hydrolyzed wheat proteins in some cosmetics, and that is sometimes associated with a food allergy. Singleplex and multiplex immunoassays detect specific IgE antibodies to wheat allergenic molecular biomarkers such as omega-5 gliadin Tri a 19, lipid transfer protein Tri a 14, and alpha-amylase inhibitors. The fluorescence enzyme immunoassay with capsulated cellulose polymer solid-phase coupled allergens is a commonly used singleplex assay. Multiplex methods include the ELISA-based macroarray immunoassay using nano-bead technology and a microarray immunoassay on polymer-coated slides. Another promising diagnostic tool is the basophil activation test performed with omega-5 gliadin and other wheat protein types. Detailed comprehension of the structural and immunological features of the numerous wheat allergens significant in clinical settings is imperative for advancing diagnostic biomarkers for IgE-mediated wheat allergies.

Gluten Is Not Gluten.

Wheat gluten is responsible for the unique baking properties of wheat flour, but it also causes wheat-related disorders in predisposed individuals. Different commercially available gluten materials are commonly used for a variety of assays, but a detailed characterization of their composition is missing in many cases. This is why we aimed to provide an in-depth analysis of three commonly used gliadin and gluten materials from two different batches using gel electrophoretic and chromatographic techniques. The gliadin material did not show the typical qualitative and quantitative protein composition and does not appear to be representative of wheat gliadin. The two gluten materials had the expected protein composition, but both showed large batch-to-batch variability regarding total protein content. Since these variations result in different biochemical, immunological, and functional behaviors, it is important to analyze at least the total protein content of each material and each batch.

In Vitro Insights into the Dietary Role of Glucoraphanin and Its Metabolite Sulforaphane in Celiac Disease.

Sulforaphane is considered the bioactive metabolite of glucoraphanin after dietary consumption of broccoli sprouts. Although both molecules pass through the gut lumen to the large intestine in stable form, their biological impact on the first intestinal tract is poorly described. In celiac patients, the function of the small intestine is affected by celiac disease (CD), whose severe outcomes are controlled by gluten-free dietary protocols. Nevertheless, pathological signs of inflammation and oxidative stress may persist. The aim of this study was to compare the biological activity of sulforaphane with its precursor glucoraphanin in a cellular model of gliadin-induced inflammation. Human intestinal epithelial cells (CaCo-2) were stimulated with a pro-inflammatory combination of cytokines (IFN-γ, IL-1ß) and in-vitro-digested gliadin, while oxidative stress was induced by H2O2. LC-MS/MS analysis confirmed that sulforaphane from broccoli sprouts was stable after simulated gastrointestinal digestion. It inhibited the release of all chemokines selected as inflammatory read-outs, with a more potent effect against MCP-1 (IC50 = 7.81 µM). On the contrary, glucoraphanin (50 µM) was inactive. The molecules were unable to counteract the oxidative damage to DNA (γ-H2AX) and catalase levels; however, the activity of NF-κB and Nrf-2 was modulated by both molecules. The impact on epithelial permeability (TEER) was also evaluated in a Transwell® model. In the context of a pro-inflammatory combination including gliadin, TEER values were recovered by neither sulforaphane nor glucoraphanin. Conversely, in the context of co-culture with activated macrophages (THP-1), sulforaphane inhibited the release of MCP-1 (IC50 = 20.60 µM) and IL-1ß (IC50 = 1.50 µM) only, but both molecules restored epithelial integrity at 50 µM. Our work suggests that glucoraphanin should not merely be considered as just an inert precursor at the small intestine level, thus suggesting a potential interest in the framework of CD. Its biological activity might imply, at least in part, molecular mechanisms different from sulforaphane.

Gastrointestinal dysfunction in Parkinson's Disease: absence of anti-gliadin antibodies.

BACKGROUND AND OBJECTIVES: Parkinson disease (PD), which is a neurodegenerative disorder, includes several gastrointestinal symptoms that are similar to those of Celiac disease (CD). However, the presence of celiac antibodies in PD patients has not yet been studied. Our aim in this study is to compare anti-transglutaminase (ATA) and antigliadin antibodies (AGA) as well as gastrointestinal symptoms and nutrition habits between patients with Parkinson's disease (PD) and healthy controls. METHODS AND STUDY DESIGN: Serum AGA IgG and IgA and the ATA antibodies IgA and IgG were studied in 102 PD patients and 91 healthy controls. Gastrointestinal symptoms, specifically constipation, were investigated using the gastrointestinal system rating scale (GSRS) and the constipation rating scale (CRS). Dietary habits were also investigated and compared between the groups. RESULTS: No significant differences were found between the two groups in terms of celiac antibodies. As expected, the hypokinetic GSRS and CRS scores were significantly higher in the PD group (p<0.001). Dietary habits, especially carbohydrate-rich diets, had a negative impact on gastrointestinal symptoms in the PD patients. CONCLUSIONS: Studies have suggested a connection between PD and CD, which infers a probable non-celiac gluten intolerance and the need to offer PD patients an elimination diet. However, the results of our study did not support any link between celiac antibodies and PD. Notwithstanding, the negative impact of a carbohydrate-rich diet in PD patients still leaves a question regarding gluten sensitivity in these patients.

Quantitative proteomic analysis for characterization of protein components related to dough quality and celiac disease in wheat flour, dough, and heat-treated dough.

High-sensitivity 4D label-free proteomic technology was used to identify protein components related to gluten quality and celiac disease (CD) in strong-gluten wheat cultivar KX 3302 and medium-gluten wheat cultivar BN 207. The highly expressed storage protein components in KX3302 were high-molecular-weight-glutenin-subunits (HMW-GSs), α-gliadin, and globulin, whereas those in BN207 were γ-gliadin, low-molecular-weight-glutenin-subunits (LMW-GSs) and avenin-like proteins. In addition, BN207 had more upregulated metabolic proteins than KX3302. The abundance of storage proteins increased during dough formation. After heat treatment, the upregulated proteins accounted for 57.53 % of the total proteins, but the downregulated storage proteins accounted for 79.34 % of the total storage proteins. In cultivar KX3302, CD proteins mainly included α-gliadin and HMW-GSs, whereas in BN207, they were mainly γ-gliadin and LMW-GSs. Thermal treatment significantly reduces the expression levels of CD-related proteins. These findings provide a new perspective on reducing the content of CD-related proteins in wheat products.

How Important Are Genetic Diversity and Cultivar Uniformity in Wheat? The Case of Gliadins.

Improvements in self-pollinated crops rely on crosses between different genotypes. It has been suggested that the repeated use of "the best" genotypes may lead to the restriction of the genetic diversity of the crop. In wheat, the analysis of gliadin (storage protein) polymorphism has provided evidence that genetic diversity was high and stable throughout the 20th century. Moreover, a worldwide analysis of gliadin polymorphism shows that genetic diversity is structured spatially across countries and their regions. Therefore, the analysis of gliadin genotypes in a given grain sample can provide reliable information about the origin of grains in this sample. An unexpected finding is that many registered common wheat cultivars are genetically non-uniform and composed of authentic biotypes (genotypically related lines originated from the initial cross) in spite of current crop-registration rules that include a strict demand for each new cultivar to be genetically uniform (DUS rules). In summary, the results suggest that each cultivar is the fruit of joint effects of a breeder and of a region's environmental factors. We believe this finding will not be restricted to wheat and suggest there may be a need to re-evaluate relevant rules of cultivar registration for crop species in general.

Differential pattern of neurotoxicity induced by the gliadin peptides p31-43 and p57-68 in in vitro model of epilepsy.

Epilepsy is a central nervous system (CNS) disorder causing repeated seizures due to a transient excessive or synchronous alteration in the electrical activity of the brain. Several neurological disorders have been associated to gluten-related diseases (GRD), including epilepsy. However, the molecular mechanisms that associate GRD and epileptogenesis are still unknown. Our previous data have shown that the gliadin peptide 31-43 (p31-43) enhanced number and duration of seizures induced by kainate in mice and exacerbated CA3-kainate-induced neurotoxicity in organotypic hippocampal slices. Here, we investigated whether another important gliadin peptide p57-68 may exerts effects similar to p31-43 on kainate-induced neurotoxicity. We find that both peptides exacerbate kainate-induced damage in the CA3 region once simultaneously challenged. However, after pre-incubation, p31-43 additionally exacerbates neurotoxicity in the CA1 region, while p57-68 does not. These data suggested differential intracellular mechanisms activated by the peptides. Indeed, analysing intracellular signalling pathways we discover that p31-43 induces significant intracellular changes, including increased phosphorylation of Akt, Erk1/2, and p65, decreased p38 phosphorylation, and deacetylation of nuclear histone-3. Based on these observations, we demonstrate that p31-43 likely activates specific intracellular signaling pathways involved in neuronal excitability, inflammation, and epigenetic regulation, which may contribute to its exacerbation of kainate-induced neurotoxicity. In contrast, p57-68 appears to exert its effects through different mechanisms. Further research is necessary to elucidate the precise mechanisms by which these peptides influence neurotoxicity and understand their implications for neurological disorders.

In Vitro Protective Effect of Pea-Derived Peptides (PPs) via the Keap1/Nrf2 Signaling Pathway on Alpha-Gliadin-Sensitizing Peptide Induced Cacao-2 Cells.

SCOPE: Celiac disease (CD) is an allergic intestinal disease caused mainly by gliadin in wheat, which is widespread in the population and currently lacks effective treatment. α-Gliadin peptides cause cellular damage by substantially increasing cellular reactive oxygen species (ROS) levels. METHODS AND RESULTS: This study investigates the protective effect of 11 pea-derived peptides (PPs) on ɑ-gliadin peptide (P31-43) treated Caco-2 cells. Results show that cells treated with PP2, PP5, and PP6 peptides significantly reduce the cell mortality caused by P31-43. Three PPs significantly reduce the P31-43-induced decrease in ROS levels to control levels, and there is no difference between them and the vitamin C (Vc) group. The results in terms of antioxidant-related enzymes show that PPs significantly decrease superoxide dismutase activity (SOD), glutathione reductases (GR), and glutathione (GSH)/oxidized glutathione (GSSG) levels, thus significantly enhancing the antioxidant level of cells. By studying the key proteins of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway, it is found that PPs activate the Keap1/Nrf2 signaling pathway. CONCLUSION: The study finds that peptides from peas can effectively alleviate ɑ-gliadin peptide-induced cell damage. The discovery of these food-derived peptides provides novel potential solutions for the prevention and treatment of CD.

Wheat gliadin/xanthan gum intermolecular complexes: Interaction mechanism and structural characterization.

In this study, the interactions between wheat gliadin (GL) and xanthan gum (XG) were investigated to design new systems with potential applications as a gluten-free substitute product. Combining spectral with morphological and molecular docking methods allowed the establishment of the complexation mechanism between globular hydrophobic GL and the hydrophilic XG with an extended and partially disordered backbone. GL maintains intact its hydrophobic core even at high GL/XG ratios and organizes into small aggregate-type assemblies. The stable and uniform complexes have a low GL content, based on intermolecular hydrogen bonds and hydrophobic interactions. The GL/XG combining ratio influences the size, structure and interaction mechanism of the microparticles. The preferred sites of interaction and the binding affinities were determined by molecular docking on GL libraries and XG models. This research may provide significant knowledge for the development of low-GL wheat food products using a dietary fiber polysaccharide as a functional compound.

Parylene Double-Layer Coated Screen-Printed Carbon Electrode for Label-Free and Reagentless Capacitive Aptasensing of Gliadin.

Celiac patients are required to strictly adhere to a gluten-free diet because even trace amounts of gluten can damage their small intestine and leading to serious complications. Despite increased awareness, gluten can still be present in products due to cross-contamination or hidden ingredients, making regular monitoring essential. With the goal of guaranteeing food safety for consuming labeled gluten-free products, a capacitive aptasensor was constructed to target gliadin, the main allergic gluten protein for celiac disease. The success of capacitive aptasensing was primarily realized by coating a Parylene double-layer (1000 nm Parylene C at the bottom with 400 nm Parylene AM on top) on the electrode surface to ensure both high insulation quality and abundant reactive amino functionalities. Under the optimal concentration of aptamer (5 µM) used for immobilization, a strong linear relationship exists between the amount of gliadin (0.01-1.0 mg/mL) and the corresponding ΔC response (total capacitance decrease during a 20 min monitoring period after sample introduction), with an R2 of 0.9843. The detection limit is 0.007 mg/mL (S/N > 5), equivalent to 0.014 mg/mL (14 ppm) of gluten content. Spike recovery tests identified this system is free from interferences in corn and cassava flour matrices. The analytical results of 24 commercial wheat flour samples correlated well with a gliadin ELISA assay (R2 = 0.9754). The proposed label-free and reagentless capacitive aptasensor offers advantages of simplicity, cost-effectiveness, ease of production, and speediness, making it a promising tool for verifying products labeled as gluten-free (gluten content <20 ppm).

Control of the oil content of fried dough sticks through modulating structure change by reconstituted gluten fractions.

In our study, we explored how gluten's role during dough formation and thermal processing can mitigate the adverse effects of physical factors on product quality. We discovered that a gluten network with a gliadin/glutenin ratio of 5:5 effectively limits oil penetration into the dough's core. This particular ratio is found to reduce the exposure of hydrophobic groups due to the presence of hydrated ß-sheet structures. In contrast, gluten networks with higher gliadin proportions than typical wheat gluten tend to be looser, leading to increased chromophore exposure and facilitating more oil absorption. These observations highlighted the complex link between changes in gluten structure, varying protein compositions, and oil content in fried dough sticks. This research provided a foundation for developing specialized low-fat wheat flour and improving the quality of fried dough products.

Wheat gluten structure and (non-)covalent network formation during deep-fat frying.

The aim of this work was to investigate wheat gluten protein network structure throughout the deep-frying process and evaluate its contribution to frying-induced micro- and macrostructure development. Gluten polymerization, gluten-water interactions, and molecular mobility were assessed as a function of the deep-frying time (0 - 180 s) for gluten-water model systems of differing hydration levels (40 - 60 % moisture content). Results showed that gluten protein extractability decreased considerably upon deep frying (5 s) mainly due to glutenin polymerization by disulfide covalent cross-linking. Stronger gliadin and glutenin protein-protein interactions were attributed to the formation of covalent linkages and evaporation of water interacting with protein chains. Longer deep-frying (> 60 s) resulted in progressively lower protein extractabilities, mainly due to the loss in gliadin protein extractability, which was associated with gliadin co-polymerization with glutenin by thiol-disulfide exchange reactions. The mobility of gluten polymers was substantially reduced during deep-frying (based on the lower T2 relaxation time of the proton fraction representing the non-exchanging protons of gluten) and gluten proteins gradually transitioned from the rubbery to the glassy state (based on the increased area of said protons). The sample volume during deep-frying was strongly correlated to the reduced protein extractability (r = -0.792, p < 0.001) and T2 relaxation time of non-exchanging protons of gluten proteins (r = -0.866, p < 0.001) thus demonstrating that the extent of gluten structural expansion as a result of deep-frying is dictated both by the polymerization of proteins and the reduction in their molecular mobility.

Celiac disease prevalence in patients with idiopathic inflammatory myopathies, a cross-sectional study.

Up to 30% of patients with celiac disease (CD) suffer from concurrent autoimmune disease, compared to 3% of the general population. The association between CD and the current clinical phenotypes of inflammatory myopathies (IIM) patients has not been thoroughly addressed. Assess the CD features among patients with IIM and their relationship with the clinical phenotype and the myositis specific (MSA) and associated antibodies (MAA). For this cross-sectional study, we recruited 99 adult patients classified as IIM from a tertiary center in Mexico. We assessed serum MSA, MAA, and CD-associated autoantibodies (IgA anti-tissue transglutaminase (tTG) and both IgA and IgG anti-deaminated gliadin peptide (DGP)). Patients with highly suggestive serology for CD were then tested for IgG anti-endomysium antibodies, and a duodenal biopsy was performed. 70.7% of patients were positive for at least one antibody. Nine duodenal biopsies were taken, revealing findings compatible with celiac disease in two cases. Subjects with anti-MDA5 antibodies were more likely to have positive anti-tTG IgA antibodies (OR 6.76, 95% CI 1.85-24.62, P = 0.013) and suggestive CD serology (OR 6.41, 95% CI 1.62-25.29, P = 0.009). Patients with anti-Mi2 antibodies were more likely to have positive anti-DGP IgG antibodies (OR 3.35, 95% CI 1.12-9.96, P = 0.039), while positivity for these autoantibodies was less frequent in patients with anti-NXP2 antibodies (OR 0.22, 95% CI 0.06-0.80, P = 0.035). There is a higher prevalence of serologic and definite CD in patients with IIM compared to the general population. Identifying this subgroup of patients may have prognostic and therapeutic implications. Key points • The study estimated a serological celiac disease (CD) prevalence of 70.7% in patients with idiopathic inflammatory myopathies (IIM) and a biopsy-confirmed prevalence of 2%, suggesting that IIM patients should be considered a high-risk population for CD. • We identified a significant association between serological CD and the presence of anti-MDA5 and anti-Mi2 antibodies, suggesting a potential justification for celiac disease screening in this specific subgroup of patients. • The impact of gluten-free diets on IIM patients with serological markers of CD remains untested and warrants further investigation through prospective, randomized studies.

Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing.

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.

Pilot study indicates that a gluten-free diet lowers oxidative stress for gluten-sensitive persons with schizophrenia.

One-third of people with schizophrenia have elevated levels of anti-gliadin antibodies (AGA IgG). A 5-week randomized double-blind pilot study was performed in 2014-2017 in an inpatient setting to test the effect of a gluten-free diet (GFD) on participants with schizophrenia or schizoaffective disorder who also had elevated AGA IgG (≥ 20 U) but were negative for celiac disease. This earlier pilot study reported that the GFD-group showed improved gastrointestinal and psychiatric symptoms, and also improvements in TNF-α and the inflammatory cytokine IL-23. Here, we performed measurements of these banked plasma samples to detect levels of oxidative stress (OxSt) using a recently developed iridium (Ir)-reducing capacity assay. Triplicate measurements of these samples showed an Intraclass Correlation Coefficient of 0.84 which indicates good reproducibility. Further, a comparison of the OxSt measurements at the baseline and 5-week end-point for this small sample size shows that the GFD-group (N = 7) had lowered OxSt levels compared to the gluten-containing diet group (GCD; N = 9; p = 0.05). Finally, we showed that improvements in OxSt over these 5 weeks were correlated to improvements in gastrointestinal (r = +0.64, p = 0.0073) and psychiatric (r = +0.52, p = 0.039) symptoms. Also, we showed a possible association between the decrease in OxSt and the lowered levels of IL-23 (r = +0.44, p = 0.087), although without statistical significance. Thus, the Ir-reducing capacity assay provides a simple, objective measure of OxSt with the results providing further evidence that inflammation, redox dysregulation and OxSt may mediate interactions between the gut and brain.

The top 100 most cited articles in the past 30 years of wheat allergy: a bibliometric analysis.

Background: Wheat allergy (WA), characterized by immunological responses to wheat proteins, is a gluten-related disorder that has become increasingly recognized in recent years. Bibliometrics involves the quantitative assessment of publications within a specific academic domain. Objectives: We aimed to execute an extensive bibliometric study, focusing on the past 30 years of literature related to wheat allergy. Methods: We searched the Web of Science database on 5th Dec 2023. We used the keywords "wheat allergy or wheat anaphylaxis or wheat hypersensitivity," "gliadin allergy or gliadin anaphylaxis or gliadin hypersensitivity," "wheat-dependent exercise-induced anaphylaxis," and "baker's asthma" for our search. All items published between 1993 and 2023 were included. The top 100 most cited articles were identified and analyzed. Results: Our study conducted an in-depth bibliometric analysis of the 100 most-cited articles in the field of wheat allergy, published between 2002 and 2019. These articles originated from 20 different countries, predominantly Japan and Germany. The majority of these articles were centered on the pathogenesis and treatment of wheat allergy (WA). The Journal of Allergy and Clinical Immunology (JACI) was the most prolific contributor to this list, publishing 14 articles. The article with the highest citation count was published by Biomed Central (BMC) and garnered 748 citations. The peak citation year was 2015, with a total of 774 citations, while the years 1998, 2001, and 2005 saw the highest publication frequency, each with 7 articles. Conclusion: Our study aims to provide physicians and researchers with a historical perspective for the scientific progress of wheat allergy, and help clinicians effectively obtain useful articles that have a significant impact on the field of wheat allergy.

Amyloid-like Aggregation of Wheat Gluten and Its Components during Cooking: Mechanisms and Structural Characterization.

Amyloid-like aggregation widely occurs during the processing and production of natural proteins, with evidence indicating its presence following the thermal processing of wheat gluten. However, significant gaps remain in understanding the underlying fibrillation mechanisms and structural polymorphisms. In this study, the amyloid-like aggregation behavior of wheat gluten and its components (glutenin and gliadin) during cooking was systematically analyzed through physicochemical assessment and structural characterization. The presence of amyloid-like fibrils (AFs) was confirmed using X-ray diffraction and Congo red staining, while Thioflavin T fluorescence revealed different patterns and rates of AFs growth among wheat gluten, glutenin, and gliadin. AFs in gliadin exhibited linear growth curves, while those in gluten and glutenin showed S-shaped curves, with the shortest lag phase and fastest growth rate (t1/2 = 2.11 min) observed in glutenin. Molecular weight analyses revealed AFs primarily in the 10-15 kDa range, shifting to higher weights over time. Glutenin-derived AFs had the smallest ζ-potential value (-19.5 mV) and the most significant size increase post cooking (approximately 400 nm). AFs in gluten involve interchain reorganization, hydrophobic interactions, and conformational transitions, leading to additional cross ß-sheets. Atomic force microscopy depicted varying fibril structures during cooking, notably longer, taller, and stiffer AFs from glutenin.

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.

Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.