IgE to wheat, prick test, and Patch test among children with celiac disease.

INTRODUCTION AND AIM: Celiac disease is one of the most common autoimmune disorders. This study aimed to evaluate the relationship between celiac disease and wheat sensitization. SUBJECTS AND METHODS: In the current study, children aged < 18 years with confirmed celiac disease were included. Data were analyzed using SPSS. RESULTS: Gastrointestinal problems were the most common indication for evaluation in terms of celiac disease. Prick and patch tests were positive in 43.4% and 34% respectively. CONCLUSION: Prick test and patch test for wheat sensitization were positive in about 30-45% of the children for celiac disease.

Assessment of Immune Responses in an Animal Model of Wheat Food Allergy via Epicutaneous Sensitization.

Wheat allergy is a pathological event involving immunocompetent cells against ingested wheat allergen and is clearly associated with transdermal sensitization. However, the molecular mechanisms involved in the disease etiology are not completely understood. A complex cellular and tissue network linking to food allergy makes it difficult to understand the molecular mechanism of allergenicity. Animal models are valuable tools to deduce basic principles of human disease without invasive intervention trials. A mouse model of wheat allergy has provided insights into effects of skin exposure to wheat protein; it is a plausible route of human sensitization for wheat anaphylaxis. Further investigation of this model will capture the essential occurrence and flow of events, bringing useful clues to develop effective treatment and control strategies against wheat allergy. Here, we describe a method for analyzing the expression of cell surface molecules in single cells isolated from lymphoid tissue with flow cytometry. Sensitization by wheat extracts significantly increases antigen-specific T cells in the spleen. Collecting information regarding the contribution of immune cells to allergic sensitization in the development of wheat allergy would be useful in preventing and treating food allergies.

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.

Gut microbiome alterations in patients with wheat-dependent exercise-induced anaphylaxis.

The intestinal microbiota plays a critical role in food allergy development. However, little is known regarding the structure and composition of the intestinal microbiota in patients with wheat-dependent exercise-induced anaphylaxis (WDEIA). We examined the gut microbiota alterations in patients with WDEIA and the microbiota's association with WDEIA. Fecal samples were collected from 25 patients with WDEIA and 25 healthy controls. Environmental exposure factors were obtained, serum total IgE, IgE specific to wheat, gluten, and ω-5 gliadin were measured. Fecal samples were profiled using 16S rRNA gene sequencing. The relative abundances of the bacterial genera Blautia (P < 0.05), Erysipelatoclostridium (P < 0.01), Akkermansia (P < 0.05) and Lachnospiraceae_NK4A136_group (P < 0.05) were significantly increased, while those of Lactobacillus (P = 0.001) and Dialister (P < 0.05) were significantly decreased in subjects with WDEIA. The microbial diversity did not differ between WDEIA patients and healthy controls. IgE specific to ω-5 gliadin was positively associated with the Oscillospira (r = 0.48, P < 0.05) and negatively associated with Leuconostoc (r = -0.49, P < 0.05). Total IgE levels were significantly negatively correlated with Bifidobacterium (P < 0.05). The gut microbiome compositions in WDEIA patients differed from those of healthy controls. We identified a potential association between the gut microbiome and WDEIA development. Our findings may suggest new methods for preventing and treating WDEIA.

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

INTRODUCTION AND OBJECTIVES: Wheat 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 METHODS: Patients 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. RESULTS: Ten 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. CONCLUSION: The 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.

Serum zonulin and its diagnostic performance in non-coeliac gluten sensitivity.

OBJECTIVE: Non-coeliac gluten sensitivity (NCGS) is characterised by intestinal and extraintestinal symptoms related to the ingestion of gluten-containing foods, in the absence of coeliac disease (CD) and wheat allergy. No biomarkers are available to diagnose NCGS and the gold standard double-blind placebo-controlled gluten challenge is clinically impractical. The aim of our work was to investigate the role of serum zonulin as a diagnostic biomarker of NCGS and to develop a diagnostic algorithm. DESIGN: In a multicentre study, we enrolled 86 patients with either self-reported or double-blind confirmed NCGS, 59 patients with diarrhoea-predominant IBS (IBS-D), 15 patients with CD and 25 asymptomatic controls (AC). Zonulin serum levels were assessed and the associated diagnostic power calculated. Clinical and symptomatic data were recorded. The effect of diet on zonulin levels was evaluated in a subgroup of patients with NCGS. RESULTS: Compared with ACs, the NCGS, irrespective of modality of diagnosis, and patients with CD had significantly increased levels of zonulin, as did both NCGS and patients with CD compared with participants with IBS-D. Self-reported NCGS showed increased zonulin levels compared with double-blind confirmed and not-confirmed NCGS. Six-month wheat avoidance significantly reduced zonulin levels only in HLA-DQ2/8-positive participants with NCGS. The diagnostic accuracy of zonulin levels in distinguishing NCGS from IBS-D was 81%. After exclusion of CD, a diagnostic algorithm combining zonulin levels, symptoms and gender improved the accuracy to 89%. CONCLUSION: Zonulin can be considered a diagnostic biomarker in NCGS and combined with demographic and clinical data differentiates NCGS from IBS-D with high accuracy. Wheat withdrawal was associated with a reduction in zonulin levels only in NCGS carrying HLA genotype.

Development and validation of a mouse-based primary screening method for testing relative allergenicity of proteins from different wheat genotypes.

BACKGROUND: Wheat allergy is a major food allergy that has reached significant levels of global public health concern. Potential variation in allergenicity among different wheat genotypes is not well studied at present largely due to the unavailability of validated methods. Here, we developed and validated a novel mouse-based primary screening method for this purpose. METHODS: Groups of Balb/c mice weaned on-to a plant protein-free diet were sensitized with salt-soluble protein (SSP) extracted from AABB genotype of wheat (durum, Carpio variety). After confirming clinical sensitization for anaphylaxis, mice were boosted 7 times over a 6-month period. Using a pooled-plasma mini bank, a wheat-specific IgE-inhibition (II)-ELISA was optimized. Then the relative allergenicity of SSPs from tetraploid (AABB), hexaploid (AABBDD) and diploid (DD) wheat genotypes were determined. The IC50/IC75 values were estimated using IgE inhibition curves. RESULTS: The optimized II-ELISA with an inhibition time of 2.5 h had a co-efficient of variation of <2%. Primary screening for relative allergenicity demonstrated that IgE binding to AABB-SSP was significantly abolished by the other two wheat genotypes. Compared to AABB, the relative allergenicity of SSPs of AABBDD and DD were significantly lower (p < .01). Furthermore, IgE inhibition curves showed significant differences in IC50 and IC75 values among the three wheat genotypes. CONCLUSION: We report a novel mouse-based primary screening method of testing relative allergenicity of wheat proteins from three different wheat genotypes for the first time. This method is expected to have broad applications in wheat allergy research.

An Animal Model for Wheat Allergy Skin Sensitisation: A Comparative Study in Naive versus Tolerant Brown Norway Rats.

BACKGROUND: Allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food, leading to the assumption that food allergy sensitisation may occur through the skin. Concerns have been raised regarding the safety of use of personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy through the skin, and even cause an abrogation of an already established oral tolerance. OBJECTIVE: To establish an animal model for food allergy skin sensitisation and compare the sensitising capacity of an unmodified and an acid-hydrolysed gluten product via slightly damaged skin in naïve versus tolerant rats. METHODS: Gluten products were applied on the slightly damaged skin of naïve or tolerant Brown Norway (BN) rats without adjuvant 3 times per week for 3 or 5 consecutive weeks. The effect of the skin applications was evaluated by means of different ELISAs and immunoblotting. RESULTS: A robust animal model was developed for food allergy skin sensitisation. In naïve rats, both gluten products were able to induce a statistically significant level of specific antibodies and sensitise through the skin, but in the wheat-tolerant rats, only the acid-hydrolysed gluten was able to sensitise through the skin, albeit at a level much lower than in the naïve rats. Results showed that new epitopes had been developed as a result of acid hydrolysis but original epitopes were maintained. This may explain why only the acid-hydrolysed gluten could induce specific antibody responses in the tolerant animals. CONCLUSIONS: This study showed that it is possible to sensitise BN rats through slightly damaged skin, and that the sensitising capacity is heavily influenced by the tolerance status of their immune system and the degree of modification of the wheat products.

Serum cytokines profile in treated celiac disease compared with non-celiac gluten sensitivity and control: a marker for differentiation.

BACKGROUND AND AIMS: There is increasing evidence regarding elevated serum levels of inflammatory cytokines in patients with celiac disease (CD), but little is known about their levels in patients with non-celiac gluten sensitivity (NCGS). The aim of this study was to evaluate the serum levels of inflammatory cytokines in Iranian patients with CD and NCGS and to compare them with those of healthy individuals. METHODS: A total of 110 treated CD, 15 with NCGS, and 46 healthy subjects were enrolled during 2016. Serum levels of IL-1, IL-6, IL-8, IL-15 and IFN-γ were measured using ELISA, and compared between groups. The correlation of the severity of mucosal damage and clinical symptoms with serum levels of cytokines was also assessed. RESULTS: The mean serum levels of IFN-γ (p = 0.04) and IL-6 (p = 0.007) were significantly different between the patients in the CD and control groups, and IL-8 was significantly higher in the CD group compared with patients in the NCGS group (p = 0.04). Statistically significant correlations were observed between the serum levels of IFN-γ and abortion (p = 0.01), IL-1 and weight loss (p = 0.043) and infertility (p = 0.0001) in CD patients, and between IFN-γ and abortion (p = 0.01) and infertility (p = 0.01) in the NCGS patients. Moreover, no significant relationship was observed between the severity of mucosal damage and the serum level of the studied cytokines. CONCLUSIONS: Inflammatory cytokines are implicated in the pathogenesis of CD, and their serum levels might help to identify a diagnostic marker to differentiate CD from NCGS. However, further studies with a larger sample size are recommended.

Autoantibodies in the Extraintestinal Manifestations of Celiac Disease.

Increased antibody reactivity towards self-antigens is often indicative of a disruption of homeostatic immune pathways in the body. In celiac disease, an autoimmune enteropathy triggered by the ingestion of gluten from wheat and related cereals in genetically predisposed individuals, autoantibody reactivity to transglutaminase 2 is reflective of the pathogenic role of the enzyme in driving the associated inflammatory immune response. Autoantibody reactivity to transglutaminase 2 closely corresponds with the gluten intake and clinical presentation in affected patients, serving as a highly useful biomarker in the diagnosis of celiac disease. In addition to gastrointestinal symptoms, celiac disease is associated with a number of extraintestinal manifestations, including those affecting skin, bones, and the nervous system. Investigations of these manifestations in celiac disease have identified a number of associated immune abnormalities, including B cell reactivity towards various autoantigens, such as transglutaminase 3, transglutaminase 6, synapsin I, gangliosides, and collagen. Clinical relevance, pathogenic potential, mechanism of development, and diagnostic and prognostic value of the various identified autoantibody reactivities continue to be subjects of investigation and will be reviewed here.

Wheat oral immunotherapy was moderately successful but was associated with very frequent adverse events in children aged 6-18 years.

AIM: This study investigated oral immunotherapy (OIT) for children aged 6-18 years with wheat allergies. METHODS: Well-cooked wheat spaghetti was given to 100 children with wheat allergies every day for 17 weeks, increasing from 0.3 to 2000 mg of wheat protein, followed by three- and nine-month maintenance phases. Blood samples were taken before therapy and at follow-up visits. The study was carried out in 2009-2015 in four Finnish paediatric allergology units. RESULTS: The children (67% male) had a mean age of 11.6 years (range 6.1-18.6), and 57 were using wheat daily 16 months after the initiation of therapy. Allergic symptoms occurred in 94/100 children: mild in 34, moderate in 36 and severe in 24. Specific immunoglobulin E (IgE) for ω-5-gliadin was significantly higher in patients who did not reach the target dose and were related to the intensity of reactions. CONCLUSION: The majority (57%) of children with wheat allergies could use wheat in their daily diet 16 months after the initiation of OIT, but 94/100 had adverse reactions and 60 were moderate or severe. Specific IgE to ω-5-gliadin may provide a biomarker for how much wheat can be tolerated and the intensity of the reactions to immunotherapy.

Non-coeliac gluten or wheat sensitivity: emerging disease or misdiagnosis?

Non-coeliac gluten or wheat sensitivity (NCG/WS) is a condition characterised by adverse gastrointestinal and/or extra-intestinal symptoms associated with the ingestion of gluten- or wheat-containing foods, in the absence of coeliac disease or wheat allergy. Up to one in 100 people in Australia may have coeliac disease but many more report adverse gastrointestinal and/or extra-intestinal symptoms after eating wheat products. In the absence of validated biomarkers, a diagnosis of NCG/WS can only be made by a double-blind, placebo-controlled, dietary crossover challenge with gluten, which is difficult to apply in clinical practice. Of people self-reporting gluten or wheat sensitivity, only a small proportion (16%) will have reproducible symptoms after a blinded gluten challenge of gluten versus placebo in a crossover dietary trial and fulfil the current consensus criteria for a diagnosis of NCG/WS. A wide range of symptoms are associated with NCG/WS, including gastrointestinal, neurological, psychiatric, rheumatological and dermatological complaints. The pathogenesis of NCG/WS is not well understood, but the innate immune system has been implicated, and there is overlap with coeliac disease and the functional gastrointestinal disorders (irritable bowel syndrome and functional dyspepsia). Identification of NCG/WS is important as gluten-free diets carry risks, are socially restricting and are costlier than regular diets.

Antigen-Specific Gut Inflammation and Systemic Immune Responses Induced by Prolonging Wheat Gluten Sensitization in BALB/c Murine Model.

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread around the globe to genetically predisposed individuals. The present study aims to develop a wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific antigliadin antibodies response and proinflammatory markers quantification. Ex vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1ß, IL-4, IL-15, IL-6, IFN-γ and TNF-α levels in mice sera. However, ex vivo primary cells of splenocytes (SPLs) and intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1ß, and IL-4 levels in G+ (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates enterocyte degeneration in the apical part of villi and damage of tight junctions in G+ (gliadin and gluten) sensitized murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G+ sensitized murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4, and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF-kBp50 signaling mechanism in G+ sensitized mice.

Responses of blood mononucleated cells and clinical outcome of non-celiac gluten sensitive pediatric patients to various cereal sources: a pilot study.

Non-celiac gluten sensitivity (NCGS) is a clinical entity recently documented by the scientific community in pediatric patients. Nevertheless, its triggering mechanisms remain largely unsettled. We studied 11 children with NCGS who were diagnosed based on a clear-cut relationship between wheat consumption and development of symptoms, after excluding celiac disease (CD) and wheat allergy, matched with 18 children with active CD. Sixteen pediatric patients were also enrolled as controls. Cultured peripheral blood mononucleated cells (PBMCs) obtained from NCGS, CD and control patients were cultured in the presence of wheat proteins extracted from ancient and modern cultivars. Results demonstrated that wheat proteins induced an overactivation of the proinflammatory chemokine CXCL10 in PBMC from NCGS pediatric patients and that this overexpression also depended on the wheat cultivar from which proteins were extracted. Proteins from modern wheat cultivar activated CXCL10 to a greater extent than those extracted from ancient wheat genotypes.

Lower Prevalence of Celiac Disease and Gluten-Related Disorders in Persons Living in Southern vs Northern Latitudes of the United States.

BACKGROUND & AIMS: The association between prevalence of celiac disease and geographic region is incompletely understood, but the occurrence of several autoimmune disorders has been found to vary along a North-South gradient. We examined geographic, demographic, and clinical factors associated with prevalence of celiac disease and gluten-free diet in the United States. METHODS: In a population-based study, we analyzed data on gluten-related conditions from the National Health and Nutrition Examination Survey, from 2009 through 2014, on 22,277 participants 6 years and older. We identified persons with celiac disease based on results of serum tests for IgA against tissue transglutaminase and endomysium or on both a health care provider diagnosis and adherence to a gluten-free diet. Gluten avoidance without celiac disease was defined as adherence to a gluten-free diet without a diagnosis of celiac disease. We compared mean serum levels of biochemical and nutritional markers based on status of gluten-related conditions. RESULTS: We found 0.7% of participants to have celiac disease and 1.1% of participants to avoid gluten without celiac disease. Celiac disease was more common among individuals who lived at latitudes of 35°-39° North (odds ratio, 3.2; 95% confidence interval, 1.4-7.1) or at latitudes of 40° North or more (odds ratio, 5.4; 95% CI, 2.6-11.3) than individuals who lived at latitudes below 35° North, independent of race or ethnicity, socioeconomic status, and body mass index. Gluten avoidance without celiac disease was more common among individuals who lived at latitudes of 40° North or more, independent of demographic factors and body mass index. Participants with undiagnosed celiac disease (identified by positive results from serologic tests) had lower mean levels of vitamin B-12 and folate (data collected from 2009 through 2012) than persons without celiac disease. Participants with a health care provider diagnosis of celiac disease had a lower mean level of hemoglobin than persons without celiac disease. Mean levels of albumin, calcium, iron, ferritin, cholesterol, vitamin B-6, and vitamin D (data collected from 2009 through 2010) did not differ between participants with gluten-related conditions and those without. CONCLUSIONS: In the US population, a higher proportion of persons living at latitudes of 35° North or greater have celiac disease or avoid gluten than persons living south of this latitude, independent of race or ethnicity, socioeconomic status, or body mass index. Mean levels of vitamin B-12 and folate are lower in individuals with undiagnosed celiac disease, and levels of hemoglobin are lower in participants with a diagnosis of celiac disease, compared with individuals without celiac disease.

Celiac Disease and Wheat Intolerance Syndrome: A Critical Update and Reappraisal.

Since the first description of celiac disease (CeD) by Samuel Gee in 1888 and the later "miraculous discovery" that bread was responsible for this condition following World War II in Europe, there has been an exponential growth of knowledge regarding CeD. Just when we thought that we knew everything there was to know about it, the disease is, however, offering new challenges, with its presentation having significantly morphed over the years from cases of overt gastrointestinal symptoms, malnutrition, and atrophic villi on duodenal biopsies to that of largely extraintestinal, subtle, or mild symptoms. Along with these changes, unexpectedly a new parallel entity appeared a few years ago and is gaining ground: the so-called nonceliac gluten sensitivity, an improper name because it should actually be referred to as wheat intolerance syndrome given that the role of gluten in all such cases is far from demonstrated and the implication of an immune involvement suggested by the term "sensitivity" is still unfounded. Lastly, wheat can be an offender also through an immunoglobulin E-mediated allergy, whose presence must also be evaluated and ruled out in selected cases.The practicing physician is therefore now challenged with the task of discerning which patients need to be assessed for one or the other of these disorders, and how.This review aims at providing an updated, critical reassessment of these 2 entities.

The role of IgG hypersensitivity in the pathogenesis and therapy of depressive disorders.

Depressive episodes are associated not only with changes in neurotransmission in the central nervous system, but also may lead to structural changes in the brain through neuroendocrine, inflammatory, and immunological mechanisms. The aim of this article is to present a new hypothesis connecting the inflammatory theory of depression with IgG food hypersensitivity and leaky gut syndrome. This new potential pathway that may mediate the pathogenesis of depression implies the existence of subsequent developmental stages. Overproduction of zonulin triggered, for example, by gliadin through activation of the epidermal growth factor receptor and protease-activated receptor causes loosening of the tight junction barrier and an increase in permeability of the gut wall ('leaky gut'). This results in a process allowing larger molecules that would normally stay in the gut to cross into the bloodstream and in the induction of IgG-dependent food sensitivity. This condition causes an increased immune response and consequently induces the release of proinflammatory cytokines, which in turn may lead to the development of depressive symptoms. It seems advisable to assess the intestinal permeability using as a marker, for example, zonulin and specific IgG concentrations against selected nutritional components in patients with depression. In the case of increased IgG concentrations, the implementation of an elimination-rotation diet may prove to be an effective method of reducing inflammation. This new paradigm in the pathogenesis of depressive disorders linking leaky gut, IgG-dependent food sensitivity, inflammation, and depression is promising, but still needs further studies to confirm this theory.