Advances in Gluten Hypersensitivity: Novel Dietary-Based Therapeutics in Research and Development.

Gluten hypersensitivity is characterized by the production of IgE antibodies against specific wheat proteins (allergens) and a myriad of clinical allergic symptoms including life-threatening anaphylaxis. Currently, the only recommended treatment for gluten hypersensitivity is the complete avoidance of gluten. There have been extensive efforts to develop dietary-based novel therapeutics for combating this disorder. There were four objectives for this study: (i) to compile the current understanding of the mechanism of gluten hypersensitivity; (ii) to critically evaluate the outcome from preclinical testing of novel therapeutics in animal models; (iii) to determine the potential of novel dietary-based therapeutic approaches under development in humans; and (iv) to synthesize the outcomes from these studies and identify the gaps in research to inform future translational research. We used Google Scholar and PubMed databases with appropriate keywords to retrieve published papers. All material was thoroughly checked to obtain the relevant data to address the objectives. Our findings collectively demonstrate that there are at least five promising dietary-based therapeutic approaches for mitigating gluten hypersensitivity in development. Of these, two have advanced to a limited human clinical trial, and the others are at the preclinical testing level. Further translational research is expected to offer novel dietary-based therapeutic options for patients with gluten hypersensitivity in the future.

Is Wheat Glutenin Extract Intrinsically Allergenic? Evaluation Using a Novel Adjuvant-Free Mouse Model of Systemic Anaphylaxis.

Wheat is a prominent allergenic food that can trigger life-threatening anaphylaxis. Presently, it remains unclear whether wheat glutenin (WG) extract possesses inherent sensitization potential independently, without the use of adjuvants, and whether it can sensitize mice to the extent of inducing life-threatening systemic anaphylaxis. In this study, we tested the hypothesis that repeated skin exposures to WG extract without adjuvant will sensitize mice with the resultant anaphylactic reaction upon systemic WG challenge. Balb/c mice were bred and maintained on a strict plant protein-free diet and were repeatedly exposed to a WG extract or vehicle once a week for 9 weeks. WG-specific (s)IgE and total (t)IgE levels were quantified. Mice were challenged with WG extract to induce anaphylactic reactions as measured by hypothermic shock response (HSR) and mucosal mast cell degranulation response (MMCR). We also conducted proteomic analysis of 120 spleen immune markers. These skin-sensitized mice exhibited exposure-dependent IgE responses and near-fatal anaphylaxis upon challenge. Proteomic analysis identified seven dramatically elevated immune biomarkers in anaphylactic mice. These data reveal that WG is intrinsically allergenic, and that chronic skin exposure to WG extract can prime the mice for potentially fatal anaphylaxis.

Chronic application of alcohol-soluble gluten extract over undamaged skin causes clinical sensitization for life-threatening anaphylaxis via activation of systemic Th2 immune responses in mice.

Introduction: Gluten allergy is a major public health problem that is growing at an alarming rate. Specific mechanisms underlying sensitization to gluten remain incompletely understood. Currently, it is unclear whether chronic exposure to alcohol-soluble gluten extract via undamaged skin has the capacity to clinically sensitize mice for life-threatening anaphylaxis. Using an adjuvant-free mouse model, here we tested the hypothesis that chronic application of alcohol-soluble durum gluten (ASDG) extract will clinically sensitize mice for life-threatening anaphylaxis. Methods: This study was conducted in a gluten-free Balb/c mouse colony that was established and maintained on a plant protein-free diet. Groups of adult female mice were exposed dermally to ASDG extract or vehicle once a week for 9-weeks. Specific (s) and total (t) IgE levels were quantified. Mice were challenged systemically with ASDG to measure symptoms of systemic anaphylaxis. Hypothermic shock response (HSR) and mucosal mast cell degranulation response (MMCR) were determined upon challenge. Spleen Th1, Th2, and other immune markers were quantified. Results: We found that chronic exposure to ASDG elicited robust elevation of sIgE and tIgE. Systemic challenge with ASDG, but not vehicle, elicited life-threatening anaphylaxis associated with dramatic HSR and MMCR. Correlation analysis demonstrated direct positive inter-relationships among IgE, HSR, and MMCR. Anaphylaxis was associated with significant elevation of prototypic Th2 but not Th1 immune markers in the spleen. Discussion/Conclusion: Our study collectively demonstrates that ASDG is intrinsically allergenic; and chronic exposure to ASDG via undamaged skin can clinically sensitize mice for life-threatening anaphylaxis via activating the systemic Th2 immune responses.

Intrinsic Allergenicity Potential of Salt-Soluble Protein Extracts from the Diploid, Tetraploid and Hexaploid Wheats: Validation Using an Adjuvant-Free Mouse Model.

Wheat allergies are potentially life-threatening and, therefore, have become a major health concern at the global level. It is largely unknown at present whether genetic variation in allergenicity potential exists among hexaploid, tetraploid and diploid wheat species. Such information is critical in establishing a baseline allergenicity map to inform breeding efforts to identify hyper-, hypo- and non-allergenic varieties. We recently reported a novel mouse model of intrinsic allergenicity using the salt-soluble protein extract (SSPE) from durum, a tetraploid wheat (Triticum durum). Here, we validated the model for three other wheat species [hexaploid common wheat (Triticum aestivum), diploid einkorn wheat (Triticum monococcum), and the ancient diploid wheat progenitor, Aegilops tauschii], and then tested the hypothesis that the SSPEs from wheat species will exhibit differences in relative allergenicities. Balb/c mice were repeatedly exposed to SSPEs via the skin. Allergic sensitization potential was assessed by specific (s) IgE antibody responses. Oral anaphylaxis was quantified by the hypothermic shock response (HSR). The mucosal mast cell response (MMCR) was determined by measuring mast cell protease in the blood. While T. monococcum elicited the least, but significant, sensitization, others were comparable. Whereas Ae. taushcii elicited the least HSR, the other three elicited much higher HSRs. Similarly, while Ae. tauschii elicited the least MMCR, the other wheats elicited much higher MMCR as well. In conclusion, this pre-clinical comparative mapping strategy may be used to identify potentially hyper-, hypo- and non-allergenic wheat varieties via crossbreeding and genetic engineering methods.

An Adjuvant-Free Mouse Model Using Skin Sensitization Without Tape-Stripping Followed by Oral Elicitation of Anaphylaxis: A Novel Pre-Clinical Tool for Testing Intrinsic Wheat Allergenicity.

Wheat is a major food allergen per the regulatory bodies of various nations. Hypersensitivity reactions to wheat have been steadily increasing for reasons that are not completely understood. Wheat-allergy models typically use adjuvants to induce sensitization to wheat proteins followed by an intraperitoneal challenge to elicit anaphylaxis. Although these models are very useful, they lack the ability to reveal the intrinsic allergenicity potential of wheat. To improve the mouse model of wheat allergy, we tested the hypothesis that repeated skin application of salt-soluble protein extract (SSPE) from durum wheat will clinically sensitize the mice to oral anaphylaxis to SSPE. Balb/c mice were bred and maintained on a plant-protein-free diet and used in the experiments. Adult female mice were exposed to SSPE once a week for 9 weeks via a solution on intact skin. Sensitization was measured by SSPE-specific IgE (sIgE) antibody and total IgE (tIgE) levels. Oral anaphylaxis was quantified by hypothermic shock response (HSR), and mucosal mast cell response (MMCR) was quantified by measuring MMCP-1 after oral challenge. Using single mouse data, correlation analyses were performed to determine the relationship among the allergenicity readouts. Spleen cytokines were quantified using a protein microarray method. Our results show that (i) repeated skin exposures to SSPE elicited robust increases in the sIgE and tIgE levels; (ii) skin exposure to SSPE was sufficient to sensitize mice for oral anaphylaxis and MMCR; (iii) both HSR and MMCR showed a strong correlation with each other, as well as with sIgE, and a modest correlation with tIgE levels; (iv) selected Th2/Th17/Th1 cytokines were elevated in skin-sensitized mice; and (v) oral allergen-challenged mice showed selective elevation of IL-6 and a panel of chemokines compared to saline-challenged mice. Together, we report the development and characterization of a novel adjuvant-free wheat-allergy mouse model that uses skin sensitization without tape-stripping followed by oral elicitation of anaphylaxis. Furthermore, validation of quantifiable wheat allergenicity readouts makes this model particularly suitable as a pre-clinical testing tool to assess the intrinsic sensitization/oral-anaphylaxis elicitation potential of novel wheat proteins (e.g., processed wheat) and to develop hypo/non-allergenic wheat products.

A Mouse-Based Method to Monitor Wheat Allergens in Novel Wheat Lines and Varieties Created by Crossbreeding: Proof-of-Concept Using Durum and A. tauschii Wheats.

Wheat allergies are potentially life-threatening because of the high risk of anaphylaxis. Wheats belong to four genotypes represented in thousands of lines and varieties. Monitoring changes to wheat allergens is critical to prevent inadvertent ntroduction of hyper-allergenic varieties via breeding. However, validated methods for this purpose are unavailable at present. As a proof-of-concept study, we tested the hypothesis that salt-soluble wheat allergens in our mouse model will be identical to those reported for humans. Groups of Balb/cJ mice were rendered allergic to durum wheat salt-soluble protein extract (SSPE). Using blood from allergic mice, a mini hyper-IgE plasma bank was created and used in optimizing an IgE Western blotting (IEWB) to identify IgE binding allergens. The LC-MS/MS was used to sequence the allergenic bands. An ancient Aegilops tauschii wheat was grown in our greenhouse and extracted SSPE. Using the optimized IEWB method followed by sequencing, the cross-reacting allergens in A. tauschii wheat were identified. Database analysis showed all but 2 of the durum wheat allergens and all A. tauschii wheat allergens identified in this model had been reported as human allergens. Thus, this model may be used to identify and monitor potential changes to salt-soluble wheat allergens caused by breeding.

Creating hypo-/nonallergenic wheat products using processing methods: Fact or fiction?

Wheat allergy is a potentiallylife-threatening disease that affects millions of people around the world. Food processing has been shown to influence the allergenicity of wheat and other major foods. However, a comprehensive review evaluating whether or not food processing can be used to develop hypo-/nonallergenic wheat products is unavailable. There were three objectives for this study: (1) to critically evaluate the evidence on the effect of fermentation, thermal processing, and enzyme or acid hydrolysis on wheat allergenicity so as to identify the potential for and challenges of using these methods to produce hypo-/nonallergenic wheat products; (2) to identify the molecular effects of food processing needed to create such products; and (3) to map the concept questions for future research and development to produce hypo-/nonallergenic wheat products. We performed literature research using PubMed and Google Scholar databases with various combinations of keywords to generate the data to accomplish these objectives. We found that: (1) food processing significantly modulates wheat allergenicity; while some methods can reduce or even abolish the allergenicity, others can create mega allergens; and (2) fermentation and enzymatic hydrolysis hold the most potential to create novel hypo-/nonallergenic wheat products; however, preclinical validation and human clinical trials are currently lacking. We also identify five specific research concepts to advance the research to enable the creation of hypo-/nonallergenic wheat products for application in food, medical, and cosmetic industries.

Mechanisms of Wheat Allergenicity in Mice: Comparison of Adjuvant-Free vs. Alum-Adjuvant Models.

Wheat protein is considered a major type of food allergen in many countries including the USA. The mechanisms of allergenicity of wheat proteins are not well understood at present. Both adjuvant-based and adjuvant-free mouse models are reported for this food allergy. However, it is unclear whether the mechanisms underlying wheat allergenicity in these two types of models are similar or different. Therefore, we compared the molecular mechanisms in a novel adjuvant-free (AF) model vs. a conventional alum-adjuvant (AA) model of wheat allergy using salt-soluble wheat protein (SSWP). In the AF model, Balb/cJ mice were sensitized with SSWP via skin exposure. In the AA model, mice were sensitized by an intraperitoneal injection of SSWP with alum. In both models, allergic reactions were elicited using an identical protocol. Robust IgE as well as mucosal mast cell protein-1 responses were elicited similarly in both models. However, an analysis of the spleen immune markers identified strikingly different molecular activation patterns in these two models. Furthermore, a number of immune markers associated with intrinsic allergenicity were also identified in both models. Since the AF model uses skin exposure without an adjuvant, the mechanisms in the AF model may more closely simulate the human wheat allergenicity mechanisms from skin exposure in occupational settings such as in the baking industry.

Advances in Molecular Mechanisms of Wheat Allergenicity in Animal Models: A Comprehensive Review.

The prevalence of wheat allergy has reached significant levels in many countries. Therefore, wheat is a major global food safety and public health issue. Animal models serve as critical tools to advance the understanding of the mechanisms of wheat allergenicity to develop preventive and control methods. A comprehensive review on the molecular mechanisms of wheat allergenicity using animal models is unavailable at present. There were two major objectives of this study: To identify the lessons that animal models have taught us regarding the molecular mechanisms of wheat allergenicity and to identify the strengths, challenges, and future prospects of animal models in basic and applied wheat allergy research. Using the PubMed and Google Scholar databases, we retrieved and critically analyzed the relevant articles and excluded celiac disease and non-celiac gluten sensitivity. Our analysis shows that animal models can provide insight into the IgE epitope structure of wheat allergens, effects of detergents and other chemicals on wheat allergenicity, and the role of genetics, microbiome, and food processing in wheat allergy. Although animal models have inherent limitations, they are critical to advance knowledge on the molecular mechanisms of wheat allergenicity. They can also serve as highly useful pre-clinical testing tools to develop safer genetically modified wheat, hypoallergenic wheat products, novel pharmaceuticals, and vaccines.

The effects of an intensive lifestyle modification program on carotid artery intima-media thickness: a randomized trial.

PURPOSE: This study evaluated the effect of the Dr. Dean Ornish Program for Reversing Heart Disease on cardiovascular disease as measured by the intima-media thickness of the common carotid artery and compared this effect to outcomes from patients participating in traditional cardiac rehabilitation. DESIGN: Randomized clinical trial. SETTING: Swedish American Health System. SUBJECTS: Ninety three patients with clinically confirmed coronary artery disease were randomly assigned to the intervention (n = 46) or traditional cardiac rehabilitation (n = 47). INTERVENTION: Dr. Dean Ornish Program for Reversing Heart Disease. MEASURES: Ultrasound of the carotid artery and other cardiovascular risk factors were measured at baseline, 6, and 12 months. ANALYSIS: Intent-to-treat analysis. RESULTS: There was no significant reduction in the carotid intima-media thickness of the carotid artery in the Ornish group or the cardiac rehabilitation group. Ornish Program participants had significantly improved dietary habits (p < .001), weight (p < .001), and body mass index (p < .001) as compared with the rehabilitation group. The decrease in the number of patients with angina from baseline to 12 months was 44% in Ornish and 12% in cardiac rehabilitation. CONCLUSIONS: The Ornish Program appears to causes improvements in cardiovascular risk factors but does not appear to change the atherosclerotic process as it affects the carotid artery.