Ranking of immunodominant epitopes in celiac disease: Identification of reliable parameters for the safety assessment of innovative food proteins.

A ranking of gluten T-cell epitopes triggering celiac disease (CD) for its potential application in the safety assessment of innovative food proteins is developed. This ranking takes into account clinical relevance and information derived from key steps involved in the CD pathogenic pathway: enzymatic digestion, epitope binding to HLA-DQ receptors of the antigen-presenting cells and activation of pro-inflammatory CD4 T-cells, which recognizes the HLA-DQ-epitope complex and initiates the inflammatory response. In silico chymotrypsin digestion was the most discriminatory tool for the ranking of gluten T-cell epitopes among all digestive enzymes studied, classifying 81% and 60% of epitopes binding HLA-DQ2.5 and HLA-DQ8 molecules, respectively, with a high risk. A positive relationship between the number of prolines and the risk of gluten T-cell epitopes was identified. HLA-binding data analysis revealed the additional role played by the flanking regions of the 9-mer epitopes whereas the integration of T-cell activation data into the ranking strategy was incomplete because it was difficult to combine results from different studies. The overall ranking proposed in decreasing order of immunological relevance was: α-gliadins > ω-gliadins > hordeins > γ-gliadins âˆ¼ avenins âˆ¼ secalins > glutenins. This novel approach can be considered as a first step to reshape the risk assessment strategy of innovative proteins and their potential to trigger CD.

An integrated, accurate, rapid, and economical handheld consumer gluten detector.

Celiac disease, characterized by autoimmune reactions to dietary gluten, affects up to 3 million in the US and approximately 0.5%-1% globally. A strict, lifelong gluten-free diet is the only treatment. An economic, simple, accurate, rapid and portable gluten testing device would enable gluten-sensitive individuals to safeguard their food safety. We developed a novel solution, Nima™, a gluten sensor that integrates food processing, gluten detection, result interpretation and data transmission in a portable device, detecting gluten proteins at or below the accepted 20 ppm threshold. We developed specific monoclonal antibodies, an optimized lateral flow immunoassay strip, and one-step aqueous extraction. Compared with reference R5, NimaTM antibodies (13F6 and 14G11) had 35- and 6.6-fold higher gliadin affinities, respectively. We demonstrated device performance using a comprehensive list of foods, assessing detection sensitivity, reproducibility, and cross-reactivity. Nima™ presented a 99.0% true positive rate, with a 95% confidence interval of 97.8%-100%.

A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens.

BACKGROUND: Acid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found. OBJECTIVES: In this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients' IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs. METHODS: Sera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients' IgE. RESULTS: Acid-HWPs with medium (30%) and high (50-60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients' IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay. CONCLUSION: Increasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins.

Gluten-Free Diet Indications, Safety, Quality, Labels, and Challenges.

A gluten-free diet (GFD) is the safest treatment modality in patient with coeliac disease (CD) and other gluten-related disorders. Contamination and diet compliance are important factors behind persistent symptoms in patients with gluten related-disorders, in particular CD. How much gluten can be tolerated, how safe are the current gluten-free (GF) products, what are the benefits and side effects of GFD? Recent studies published in Nutrients on gluten-free products' quality, availability, safety, as well as challenges related to a GFD are discussed.

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.

Gluten and gluten-free: issues and considerations of labeling regulations, detection methods, and assay validation.

Gluten is a commonly used cereal derivative found in bakery products, among other items. In some susceptible individuals, however, it triggers immune responses of different kinds; there is, to a lesser extent, the wheat allergy that is immunoglobulin E (IgE)-mediated and leads to histamine release and typical allergic symptoms. In this case, other water-soluble proteins, like albumins, are also involved. On the other hand, there is, more frequently, celiac disease (CD), where the gluten causes immune reactions in the intestines of certain individuals, leading to degeneration of villi, which typically leads to malabsorption of nutrients and, consequently, malnutrition. The only currently effective health strategy for affected consumers is avoidance of gluten-containing products, based on clear labeling rules. However, despite unanimously accepted Codex definitions by all member jurisdictions, the national implementation of equivalent laws shows significant differences. In the context of CD and in support of the gluten-free statement, regulatory enforcement, as well as manufacturers' quality controls are mostly based on analytical results. However, numerous methods are available, some of which have been validated better than others, and many provide different results on identical samples. Reasons include detection of different gluten components and variability in extraction efficiency due to different buffer compositions, especially from processed foods. Last but not least, the lack of reference materials is hindering the process of generating comparable data across different ELISA kits, as well as other methods. How can such data still be used to support a gluten-free claim? New methodologies, in particular mass spectrometric analysis of gluten derived peptides, are being introduced in numerous laboratories. This methodology is not only capable of detecting gluten derived peptides but can also differentiate between and quantitate wheat, barley, rye, and oat. This paper presents analytical limitations, as well as promising new approaches in support of industry and enforcement activities to ensure compliance with the gluten-free claim under the current regulatory framework.

Assessment of allergenicity of diploid and hexaploid wheat genotypes: identification of allergens in the albumin/globulin fraction.

Wheat is an important part of the daily diet of millions of people. However, this staple food is also responsible for food allergies. Ancient cultivars of wheat are gaining interest today but nothing is known about their allergenicity. Many wheat proteins have been reported as causative food allergens, including some prolamin-type gluten proteins, and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work is to obtain information about the allergenicity of the salt soluble A/G fraction of an ancient diploid cultivar compared with a standard hexaploid bread wheat cultivar using 20 sera from patients with wheat allergy. Differences in the IgE reactivity of sera towards the two genotypes were quantified by ELISA. Qualitative differences in IgE-binding proteins were searched after 1D or 2D electrophoresis. For most of the sera, the concentration in A/G specific IgE was higher for the hexaploid T. aestivum (cv Récital) than for the diploid T. monococcum (cv Engrain). The analysis of 2D spots revealed by immunoblotting leads to the identification by mass spectrometry of 39 IgE-binding proteins, some of them unknown until now as wheat allergens. Numerous allergens were identified, differences observed between Engrain and Récital will be discussed.

New therapeutic strategies for celiac disease.

Celiac disease is an autoimmune condition affecting genetically susceptible individuals, characterized by inflammatory damage to the small intestine following ingestion of wheat gluten or barley and rye products. The only life-long treatment is strict gluten-free diet which is difficult personally and socially, affects quality of life, not widely available, more expensive, with lower palatability, resulting in low compliance. No doubt, there is therefore an urgent need for alternative therapeutic modalities. Based on the increasing knowledge on the sequential pathophysiological events driving the intestinal inflammatory cascade, new attractive and potential therapies were starting to immerge: selecting, changing, degrading, manipulating or binding the dietary toxic environmental factors, decreasing intestinal permeability toward gluten or blocking the deamination of gluten by inhibiting tissue transglutaminase or the HLA-DQ presenting groove by carefully designed false peptide, shifting the typical Th1 to Th2 inflammatory reaction or antagonizing major proinflammatory cytokines, enhancing regulatory immune function or developing preventive vaccines, blocking adhesion molecule, inducing gluten oral or intranasal tolerance or applying epithelial repairing mitogens to oppose the mucosal destruction. Safety, effectiveness, cost and affordability are prime issues to consider. Some modalities have shown promising results in vitro. Future will show who will win the race.

Celiac disease: risk assessment, diagnosis, and monitoring.

Celiac disease is an autoimmune disorder occurring in genetically susceptible individuals, triggered by gluten and related prolamins. Well identified haplotypes in the human leukocyte antigen (HLA) class II region (either DQ2 [DQA*0501-DQB*0201] or DQ8 [DQA*0301-DQB1*0302]) confer a large part of the genetic susceptibility to celiac disease.Celiac disease originates as a result of a combined action involving both adaptive and innate immunity. The adaptive immune response to gluten has been well described, with the identification of specific peptide sequences demonstrating HLA-DQ2 or -DQ8 restrictive binding motifs across various gluten proteins. As for innate immunity, through specific natural killer receptors expressed on their surface, intra-epithelial lymphocytes recognize nonclassical major histocompatibility complex (MHC)-I molecules such as MICA, which are induced on the surface of enterocytes by stress and inflammation, and this interaction leads to their activation to become lymphokine-activated killing cells. Four possible presentations of celiac disease are recognized: (i) typical, characterized mostly by gastrointestinal signs and symptoms; (ii) atypical or extraintestinal, where gastrointestinal signs/symptoms are minimal or absent and a number of other manifestations are present; (iii) silent, where the small intestinal mucosa is damaged and celiac disease autoimmunity can be detected by serology, but there are no symptoms; and, finally, (iv) latent, where individuals possess genetic compatibility with celiac disease and may also show positive autoimmune serology, that have a normal mucosa morphology and may or may not be symptomatic.The diagnosis of celiac disease still rests on the demonstration of changes in the histology of the small intestinal mucosa. The classic celiac lesion occurs in the proximal small intestine with histologic changes of villous atrophy, crypt hyperplasia, and increased intraepithelial lymphocytosis. Currently, serological screening tests are utilized primarily to identify those individuals in need of a diagnostic endoscopic biopsy. The serum levels of immunoglobulin (Ig)A anti-tissue transglutaminase (or TG2) are the first choice in screening for celiac disease, displaying the highest levels of sensitivity (up to 98%) and specificity (around 96%). Anti-endomysium antibodies-IgA (EMA), on the other hand, have close to 100% specificity and a sensitivity of greater than 90%. The interplay between gliadin peptides and TG2 is responsible for the generation of novel antigenic epitopes, the TG2-generated deamidated gliadin peptides. Such peptides represent much more celiac disease-specific epitopes than native peptides, and deamidated gliadin antibodies (DGP) have shown promising results as serological markers for celiac disease. Serology has also been employed in monitoring the response to a gluten-free diet.Despite the gluten-free diet being so effective, there is a growing demand for alternative treatment options. In the future, new forms of treatment may include the use of gluten-degrading enzymes to be ingested with meals, the development of alternative, gluten-free grains by genetic modification, the use of substrates regulating intestinal permeability to prevent gluten entry across the epithelium, and, finally, the availability of different forms of immunotherapy.

Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management.

Celiac disease (CD) is an inflammatory small intestinal disorder that can lead to severe villous atrophy, malabsorption, and malignancy. It is triggered by the gluten proteins of wheat, barley, and rye. All patients express the antigen-presenting molecules human leukocyte antigen-DQ2 (HLA-DQ2) and/or HLA-DQ8, which bind gluten peptides and thus activate destructive intestinal T cells. Patients with untreated CD have circulating IgA autoantibodies to the enzyme tissue transglutaminase (tTG), a component of endomysium. Testing for serum IgA tTG has a high predictive value. Therapy of CD is a lifelong gluten-free diet. Counseling by an expert dietitian and association with a celiac support group are important in helping the patient embark on a healthy gluten-free diet. Current research focuses on non-dietary therapies and treatment of refractory (diet-unresponsive) CD.