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Abstract

Wheat gluten proteins such as gliadins constitute major food allergens. Gluten can be modified industrially by deamidation which increases its solubility and enhances its use as a food ingredient. Sensitization to deamidated gluten has been reported to cause severe allergic reactions with anaphylaxis. The aim of this study was therefore to compare the sensitization and elicitation potentials of native (NG) and deamidated (DG) gliadins. The reactivity pattern of mice IgE was also compared with that of DG-allergic patients. The ability of DG to sensitize Balb/c mice using intra-peritoneal administration with aluminium hydroxide as an adjuvant, and to elicit an allergic response after a challenge, was tested in comparison with NG. Mice sensitized with DG secreted higher levels of total IgE, IL-4, gliadin-specific IgE and IgG1 than mice sensitized with NG. By contrast, mice sensitized with NG produced higher levels of gliadin-specific IgG2a and INFγ. After a challenge, histamine levels were higher in mice sensitised with DG. DG can sensitize mice much more efficiently than NG. Moreover, this mouse model of allergy to DG revealed an IgE reactivity pattern against purified gliadins which was very similar to that of DG-allergic patients.

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... Although used at less extent, the SPT and food challenges have been carried out to characterise the allergenicity of proteins as affected by different properties (e.g. denaturation, digestion) [90,104,107,108,110,111,117,120,121,123,124,[126][127][128][129][130][131][132]. The in vivo food challenges, SPT, and even more important, oral food challenges (OFC, DBPCFC) are of high value because of the true human clinical read-out. ...
... In most cases, in vitro IgE-binding properties are investigated for the ease of testing, although they usually do not allow for extrapolation to clinical reactivity. Likewise, the in vivo assays using animal models are also less applied to evaluate the allergenic potential of proteins [109,115,123,[134][135][136][137][138]. ...
... Accordingly, ATI were considered key sensitisers of wheat allergy and that these proteins can be used in nutritional therapeutic strategies to address allergen-and gluten-induced intestinal and extraintestinal inflammation [172]. Likewise, Denery-Papini et al. [121] and Gourbeyre et al. [123] reported that, despite different sensitisation paths (oral and intraperitoneal), deamidated gliadins were much more competent than native gliadins in inducing allergic sensitisation in mice, and subsequently, in triggering a more severe elicitation phase. Mirotti et al. [135] also described that lipids were necessary for the sensitisation of mice to Ber e 1 (Brazil nut). ...
Article
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This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.
... In food allergy animal studies, both adjuvant-based models as well as adjuvant-free (AF) models have been reported [23]. For wheat allergy studies, a number of animal models have been developed using rats, mice and dogs as experimental species [23,[33][34][35][36][37][38][39][40][41]. Most of these models typically use adjuvants, such as alum, to enhance an allergenic response to the wheat proteins. ...
... It is reported that while the use of an adjuvant provides a convenient way to enhance the readouts of allergenicity such as IgE responses to food proteins, adjuvant-related effects may mask or exaggerate the intrinsic allergenic potential of food proteins [23]. The mechanism of how alum might enhance or mask the intrinsic allergenicity of wheat proteins is unknown at present [23,32,[36][37][38][39]41,42]. Several studies suggest that a novel AF model, that uses the transdermal sensitization method, may be useful in the assessment of the intrinsic allergenic potential of food proteins [43][44][45][46][47]. Furthermore, it is unknown whether the mechanisms of wheat allergenicity are similar or different in these two different types of models. ...
... These include rat, mouse and dog species [33,36,69,70]. Here, we focused on using Balb/c mice because of their wide application in food allergenicity studies [23,[38][39][40][41][43][44][45][47][48][49]70]. Bodinier et al. (2009) [70] reported ex vivo spleen IL-4/IL-5 responses to gliadins in mice. ...
Article
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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.
... Several wheat allergens have been identified in different fractions of wheat grain, amongst which gliadins. They are monomeric and ethanolsoluble proteins (Gourbeyre et al., 2012). According to their mobility in polyacrylamide gels, gliadins are classified into α-and β-, γ-, and ωgliadins, which represent 44-60%, 30-45% and 6-20% respectively of total gliadins (Wieser et al., 1994;Wieser, 2007;Banc et al., 2009;Barak et al., 2015). ...
... Secondary, our results demonstrated that NGsensitized mice displayed higher concentrations of specific IgG and IgE as determined by ELISA method. These results corroborate those of (Gourbeyre et al., 2012) who reported that mice sensitized with NG and deamidated gliadins (DG) displayed higher concentration of NG and DG specific IgE antibodies. However, levels of anti-DG IgE were higher. ...
... Our results showed that in almost all of experimental mice, systemic anaphylaxis symptoms appears within 30 min after IP challenge with either native or hydrolyzed gliadins, these symptoms include scratching and rubbing around the nose and head, puffiness around the eyes and mouth, pilar erecti, reduced activity, and/or decreased activity with increased respiratory rate, wheezing, labored respiratory, and cyanosis around the mouth and the tail, as well as increased vascular leakage. However less intense symptoms were observed in mice challenged with corolase 7089, these results concord with the study of Gourbeyre et al., (2012) who showed that regarding the severity of clinical signs following IP challenge with native and hydrolyzed gliadins, there were no significant differences between groups used for sensitization in the anaphylaxis scores. We noted also, that IP challenge of native and hydrolyzed gliadins induced a more pronounced temperature drop in sensitized mice. ...
Article
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Enzymatic hydrolysis of wheat proteins is an effective mean to improve various functional proprieties. This study examines the effect of enzymatic hydrolysis of gliadins and the ability of these hydrolysates to elicit anaphylactic reactions in mice allergic to native gliadins (NG).Two groups of female Balb/c mice were established: naïve group sensitized with aluminum hydroxide (Alum) and NG group sensitized with 10 µg of NG. NG-specific IgG and IgE antibodies level were determined in serum by ELISA. Symptom scores, body temperature and vascular leakage were determined after in vivo challenge to NG and hydrolyzed gliadins. Jejunums were used for histological analysis and for the assessment of local anaphylactic response by an ex vivo study in Ussing chamber (measurement of electrophysiological parameters; short-circuit current (Isc) (µA/cm²) and conductance (G) (mmho/cm²)).NG-sensitized mice secreted higher levels of NG-specific IgG and IgE antibodies and showed significantly higher Isc (µA/cm²) and G (mmho/cm²) values as well as alterations of the intestinal barrier and villous atrophy. in vivo IP challenge of NG-sensitized mice with corolase 7089 hydrolysates caused less severe clinical manifestations than those observed after challenge with peptic and tryptic hydrolysates, the latter being similar to those observed with NG. The same results were observed for body temperature and vascular permeability.NG and digestive enzymes hydrolysates can elicit severe anaphylaxis symptoms, whereas, the industrial enzyme hydrolysates induce less severe symptoms.
... Several wheat allergens have been identified in different fractions of wheat grain, amongst which gliadins. They are monomeric and ethanolsoluble proteins (Gourbeyre et al., 2012). According to their mobility in polyacrylamide gels, gliadins are classified into α-and β-, γ-, and ωgliadins, which represent 44-60%, 30-45% and 6-20% respectively of total gliadins (Wieser et al., 1994;Wieser, 2007;Banc et al., 2009;Barak et al., 2015). ...
... Secondary, our results demonstrated that NGsensitized mice displayed higher concentrations of specific IgG and IgE as determined by ELISA method. These results corroborate those of (Gourbeyre et al., 2012) who reported that mice sensitized with NG and deamidated gliadins (DG) displayed higher concentration of NG and DG specific IgE antibodies. However, levels of anti-DG IgE were higher. ...
... Our results showed that in almost all of experimental mice, systemic anaphylaxis symptoms appears within 30 min after IP challenge with either native or hydrolyzed gliadins, these symptoms include scratching and rubbing around the nose and head, puffiness around the eyes and mouth, pilar erecti, reduced activity, and/or decreased activity with increased respiratory rate, wheezing, labored respiratory, and cyanosis around the mouth and the tail, as well as increased vascular leakage. However less intense symptoms were observed in mice challenged with corolase 7089, these results concord with the study of Gourbeyre et al., (2012) who showed that regarding the severity of clinical signs following IP challenge with native and hydrolyzed gliadins, there were no significant differences between groups used for sensitization in the anaphylaxis scores. We noted also, that IP challenge of native and hydrolyzed gliadins induced a more pronounced temperature drop in sensitized mice. ...
Article
Full-text available
Enzymatic hydrolysis of wheat proteins is an effective mean to improve various functional proprieties. This study examines the effect of enzymatic hydrolysis of gliadins and the ability of these hydrolysates to elicit anaphylactic reactions in mice allergic to native gliadins (NG).Two groups of female Balb/c mice were established: naïve group sensitized with aluminum hydroxide (Alum) and NG group sensitized with 10 µg of NG. NG-specific IgG and IgE antibodies level were determined in serum by ELISA. Symptom scores, body temperature and vascular leakage were determined after in vivo challenge to NG and hydrolyzed gliadins. Jejunums were used for histological analysis and for the assessment of local anaphylactic response by an ex vivo study in Ussing chamber (measurement of electrophysiological parameters; short-circuit current (Isc) (µA/cm²) and conductance (G) (mmho/cm²)).NG-sensitized mice secreted higher levels of NG-specific IgG and IgE antibodies and showed significantly higher Isc (µA/cm²) and G (mmho/cm²) values as well as alterations of the intestinal barrier and villous atrophy. in vivo IP challenge of NG-sensitized mice with corolase 7089 hydrolysates caused less severe clinical manifestations than those observed after challenge with peptic and tryptic hydrolysates, the latter being similar to those observed with NG. The same results were observed for body temperature and vascular permeability.NG and digestive enzymes hydrolysates can elicit severe anaphylaxis symptoms, whereas, the industrial enzyme hydrolysates induce less severe symptoms.
... The gluten sensitivity for BALB/c mice for was carried out according to Chen et al. (2011); Gourbeyre et al. (2012); Vijaykrishnaraj et al. (2017) (Chen et al., 2011;Gourbeyre et al., 2012;Vijaykrishnaraj et al., 2017) with slight modifications. The mice were sensitized through intraperitoneal (IP) means; 0.2 mg/0.025 ...
... The gluten sensitivity for BALB/c mice for was carried out according to Chen et al. (2011); Gourbeyre et al. (2012); Vijaykrishnaraj et al. (2017) (Chen et al., 2011;Gourbeyre et al., 2012;Vijaykrishnaraj et al., 2017) with slight modifications. The mice were sensitized through intraperitoneal (IP) means; 0.2 mg/0.025 ...
... A significant T H 2 response was found in WF challenged animals whereas, the HF group did not show T H 2 response after oral administration. These study results complement the earlier study report of deamidated gliadins sensitized BALB/c mice (Gourbeyre et al., 2012), which states that deamidated and native gliadin sensitized mice show higher concentrations of specific IgG and IgE levels in the sera. It also says that the WF orally challenged mice showed a significantly high level of IFNγ (T H 2 response) as compared with the control and HF animal groups. ...
Article
Gluten is responsible for adverse immune effects and causes wheat allergy, celiac disease, and other wheat-related disorders to genetically predisposed individuals. Wheat flour protein immunogenicity can be reduced by proteolytic degradation enzymes. The present study aims to utilize Aspergillus niger prolyl endoprotease (EC 3.4.21.26) to specifically cleave the proline-rich sequences and degradation of gluten proteins in the wheat flour. The Triticum aestivum (HD-2851, NIAW-917), Triticum durum (UAS-428), Triticum dicoccum (DDK- 1025) were taken for proteolytic degradation using AN-PEP. The enzymatic modification was carried out in an optimum condition (pH-4.0, at 40 °C) for 5 h. The wheat flour gliadin content was reduced up to 90–95% in HD-2851, DDK-1025, NIAW-917 and UAS-428 shown up to 74% reduction of gliadins. The complete hydrolysis of gliadins (α, β, ω, ϒ) and glutenin subunits were confirmed by using immunoblot, ELISA, RP-HPLC chromatograph, anti-PEP-I (PSQQQQQPK), anti-PEP-II (LGQQQPFPPQQPYPQPQPFK) and anti-HMW-GS antibodies. The modified wheat flour was taken for the processing of pasta with the replacement of 30% chickpea flour. The gluten content of MWF pasta was found to be 48 ppm which is 99.95% lesser than the control pasta. Sensory evaluation revealed that the pasta was acceptable with overall quality when compared with control pasta.
... Interestingly, wheat contains not only salt-soluble allergens but also alcohol-soluble and acid-soluble protein allergens that can trigger IgE-mediated reactions in humans [8,[1][2][3] . Previously, elegant mouse models of wheat hypersensitivity to alcohol-soluble wheat gluten proteins were developed [10][11][12][13][14] . One study evaluated the immune response to purified lipid transfer protein (LTP) in mice [11] . ...
... They did not study anaphylaxis, mast cell responses, dermatitis, or cytokine/chemokine responses. Gourbeyre et al. [12] used native and deamidated gliadins plus alum and used IP sensitization and IP challenge methods in BALB/c mice [12] . They studied IgE responses and symptom scores and histamine [13] also used deamidated gliadins plus alum and BALB/c mice, but used IP sensitization followed by intragastric challenges [13] . ...
... They did not study anaphylaxis, mast cell responses, dermatitis, or cytokine/chemokine responses. Gourbeyre et al. [12] used native and deamidated gliadins plus alum and used IP sensitization and IP challenge methods in BALB/c mice [12] . They studied IgE responses and symptom scores and histamine [13] also used deamidated gliadins plus alum and BALB/c mice, but used IP sensitization followed by intragastric challenges [13] . ...
Article
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Background: Wheat allergy and other immune-mediated disorders triggered by wheat proteins are growing at an alarming rate for reasons not well understood. A mouse model to study hypersensitivity responses to salt-soluble wheat protein (SSWP) extract is currently unavailable. Here we tested the hypothesis that SSWP extract from wheat will induce sensitization as well as allergic disease in mice. Methods: Female BALB/cJ mice were weaned onto a plant protein-free diet. The mice were injected a total of 4 times with an SSWP (0.01 mg/mouse) fraction extracted from durum wheat along with alum as an adjuvant. Blood was collected biweekly and SSWP-specific IgE (SIgE) and total IgE (TIgE) levels were measured using ELISA. Systemic anaphylaxis upon intraperitoneal injection with SSWP was quantified by hypothermia shock response (HSR). Mucosal mast cell degranulation was measured by the elevation of mMCP-1 in the blood. The mice were monitored for dermatitis. Skin tissues were used in histopathology and for measuring cytokine/chemokine/adhesion molecule levels using a protein microarray system. Results: Injection with SSWP resulted in time-dependent SIgE antibody responses associated with the elevation of TIgE concentration. Challenge with SSWP elicited severe HSR that correlated with a significant elevation of plasma mMCP-1 levels. Sensitized mice developed facial dermatitis associated with mast cell degranulation. Lesions expressed significant elevation of Th2/Th17/Th1 cytokines and chemokines and E-selectin adhesion molecule. Conclusion: Here we report a mouse model of anaphylaxis and atopic dermatitis to SSWP extract that may be used for further basic and applied research on wheat allergy.
... Body temperature was measured with a rectal probe before and 30 min after the oral challenge. The clinical score was evaluated 1 h after the oral challenge according to the symptomatic scale described previously (20). ...
... Blood samples were collected at the end of the protocol and centrifuged at 2000 g for 20 min. The quantifications of specific IgE, IgG1, IgG2a, and IgA and histamine levels were assayed on serum samples by indirect F-ELISA (20). ...
... Pooled cell suspension was then specifically stimulated with DG at 10 lg/ml. After 5 days of growth, supernatants were collected and the concentrations of cytokines were measured by ELISA as described (20). ...
Article
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Background: Food allergies affect 4 to 8% of children and are constantly on the rise, thus making allergies a timely issue. Most importantly, prevention strategies are non-existent, and current therapeutic strategies have limited efficacy and need to be improved. One alternative to prevent or reduce allergies, particularly during infancy, could consist of modulating maternal immunity and microbiota using non-digestible food ingredients, such as prebiotics. For this purpose, we studied the preventive effects of Balb/c mothers' consuming prebiotics during pregnancy and breastfeeding on food allergy development in offspring mice. Methods: After weaning, the offspring from mothers who were exposed to GOS/inulin mixture or fed a control diet were intraperitoneally sensitized to wheat proteins to induce a systemic allergic response and orally exposed to the same allergen. Immunological, physiological and microbial parameters were analyzed. Results: GOS/Inulin mixturediet modified the microbiota of mothers and their offspring. Offspring from mothers who received GOS/Inulin prebiotics were protected against food allergies and displayed lower clinical scores, specifically of IgE and histamine levels, compared to offspring from mothers fed a control diet. Moreover, GOS/Inulin supplementation for the mother resulted in stronger intestinal permeability in the offspring. Enhancement of the regulatory response to allergic inflammation and changes in the Th2/Th1 balance toward a dampened Th2 response were observed in mice from GOS/Inulin mixture-exposed mothers. Conclusion: The treatment of pregnant and lactating mice with non-digestible GOS/Inulin prebiotics promotes a long-term protective effect against food allergies in the offspring. This article is protected by copyright. All rights reserved.
... Gluten proteins are by definition not soluble in water but enzymatic or acid hydrolysis can alter their structure and size and thereby resulting in soluble protein hydrolysates. Acid hydrolyses also result in partial deamidation of the proteins [6]. Acid hydrolyzed wheat gluten obtain emulsifying properties which make them useful in food products such as coffee creamers and shake-and-bake products but also in cosmetic products for hair and body [6,7]. ...
... Acid hydrolyses also result in partial deamidation of the proteins [6]. Acid hydrolyzed wheat gluten obtain emulsifying properties which make them useful in food products such as coffee creamers and shake-and-bake products but also in cosmetic products for hair and body [6,7]. ...
... Gourbeyre et al. [6] have studied the sensitizing capacity of acid hydrolyzed gluten proteins. In this study, mice were dosed four times i.p. with 10 mg of native or deamidated gliadin together with adjuvant (Al(OH) 3 ) on day 0, 10, 20 and 30. ...
Article
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Background: Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis. Objectives: To examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten. Methods: High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay. Results: Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level of specific IgE but with a low functionality. Orally all three gluten products induced specific IgG1 and IgE but with different dose-response relations. Sensitizing rats i.p. or orally with unmodified or enzymatic hydrolyzed gluten induced specific IgG1 responses with similar binding capacity which was different from that of acid hydrolyzed gluten indicating that acid hydrolysis of gluten proteins induces formation of 'new' epitopes. Conclusions: In rats not tolerant to gluten acid hydrolysis of gluten enhances the sensitizing capacity by the i.p. but not by the oral route. In addition, acid hydrolysis induces formation of new epitopes. This is in contrast to the enzymatic hydrolyzed gluten having an epitope pattern similar to unmodified gluten.
... To further explore its allergenicity in vivo, model mice were sensitized with HCl-deamidated gliadins using intraperitoneal administration with aluminum hydroxide as an adjuvant. Unexpectedly, deamidated gliadins were more effective than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens (Gourbeyre et al., 2012). This can be explained by the drastic conditions of sensitization (systemic injection) that enabled a direct interaction between the allergen and the immune system. ...
... This can be explained by the drastic conditions of sensitization (systemic injection) that enabled a direct interaction between the allergen and the immune system. When deamidated gliadins were injected via the intraperitoneal route, they may spread more easily in the blood and thus come into contact with the immune system (Gourbeyre et al., 2012). However, the impact of the digestive tract was not considered in this system. ...
Article
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With the ever‐increasing demands for functional and sustainable foods from the general public, there is currently a paradigm shift in the food industry toward the production of novel protein‐based diet. Food scientists are therefore motivated to search for natural protein sources and innovative technologies to modify their chemical structure for desirable functionality and thus utilization. Deamidation is a viable, efficient, and attractive approach for modifying proteins owing to its ease of operating, specificity, and cost‐effective processes. Over the past three decades, the knowledge of protein deamidation for food applications has evolved drastically, including the development of novel approaches for deamidation, such as protein‐glutaminase and ion exchange resin, and their practices in new protein substrate. Thanks to deamidation, enhanced functionalities of food proteins from cereals, legumes, milk, oil seeds and others, and thereby their processabilities as food ingredients have been achieved. Moreover, deamidated proteins have been used to fabricate engineered food colloids, including self‐assembled protein particles, protein–metallic complexes, and protein–carbohydrate complexes, which have demonstrated tailored physicochemical properties to modulate oral perception, improve gastrointestinal digestion and bioavailability, and protect and/or deliver bioactive nutrients. Novel bioactivity, altered digestibility, and varied allergenicity of deamidated proteins are increasingly recognized. Therefore, deamidated proteins with novel techno‐functional and biological properties hold both promise and challenges for future food applications, and a comprehensive review on this area is critically needed to update our knowledge and provide a better understanding on the protein deamidation and its emerging applications.
... Two mouse model studies evaluated this elegantly as discussed below. Gourbeyre et al. (2012) used the Balb/c mouse model to test this hypothesis (Tables 2 and 4) [32]. They found that: i) Native gliadin elicited a higher T helper 1 type of immune response and the deamidated gliadin (DG) elicited a higher T helper 2 or allergic immune response and histamine response. ...
... Two mouse model studies evaluated this elegantly as discussed below. Gourbeyre et al. (2012) used the Balb/c mouse model to test this hypothesis (Tables 2 and 4) [32]. They found that: i) Native gliadin elicited a higher T helper 1 type of immune response and the deamidated gliadin (DG) elicited a higher T helper 2 or allergic immune response and histamine response. ...
Article
Full-text available
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.
... According to previous studies [25][26][27], several pairs of 8-week-old BALB/c mice provided by the Animal Experimental Center of Wuhan University were cohabited and inbred for at least three generations. These mice were maintained on a strict gluten-free diet purchased from the Trophic Animal Feed High-Tech Co., Ltd, Nantong, Jiangsu, China. ...
... All data present the results of more than three independent experiments and are shown as mean ± SD. *P \ 0.05; **P \ 0.01; ***P \ 0.001. VD3 1,25-Dihydroxy vitamin D3, PT-G pepsintrypsin-resistant gliadin, the effects of VD3 on PT-G-induced TJ injuries in vivo, we established a gluten-sensitized mouse model according to the previous studies[25][26][27]. First, we assessed intestinal mucosal barrier function by measuring serum FD-4 concentrations. ...
Article
Full-text available
Background: Tight junction (TJ) injuries induced by pepsin-trypsin-resistant gliadin (PT-G) play an important role in the pathogenesis of celiac disease. Previously, 1,25-dihydroxy vitamin D3 (VD3) was reported to be a TJ regulator that attenuates lipopolysaccharide- and alcohol-induced TJ injuries. However, whether VD3 can attenuate PT-G-induced TJ injuries is unknown. Aim: The aim of this study was to evaluate the effects of VD3 on PT-G-induced TJ injuries. Methods: Caco-2 monolayers were used as in vitro models. After being cultured for 21 days, the monolayers were treated with PT-G plus different concentrations of VD3. Then, the changes in trans-epithelial electrical resistance and FITC-dextran 4000 (FD-4) flux were determined to evaluate the monolayer barrier function. TJ protein levels were measured to assess TJ injury severity, and myeloid differentiation factor 88 (MyD88) expression and zonulin release levels were determined to estimate zonulin release signaling pathway activity. Additionally, a gluten-sensitized mouse model was established as an in vivo model. After the mice were treated with VD3 for 7 days, we measured serum FD-4 concentrations, TJ protein levels, MyD88 expression, and zonulin release levels to confirm the effect of VD3. Results: Both in vitro and in vivo, VD3 significantly attenuated the TJ injury-related increase in intestinal mucosa barrier permeability. Moreover, VD3 treatment up-regulated TJ protein expression levels and significantly decreased MyD88 expression and zonulin release levels. Conclusions: VD3 has protective effects against PT-G-induced TJ injuries both in vitro and in vivo, which may correlate with the disturbance of the MyD88-dependent zonulin release signaling pathway.
... 47−49 Hydrochloric acid treatment has generally been used for deamidation of wheat protein. 50,51,47 However, hydrochloric acid simultaneously causes hydrolysis of peptide bonds, which produces bitter-tasting peptides and reduces the processing properties of wheat such as extensibility and elasticity. The cation-exchange resin treatment used in this study does not cause peptide-bond hydrolysis and rather improves the processing properties. ...
... Similarly, gliadin with 52% deamidation generated by treatment with hydrochloric acid showed low reactivity with IgE antibody. 51 Yong et al. 23 reported that wheat gluten deamidated by protein glutaminase had low allergenicity when the deamidation degree reached 72%. These findings indicate that more than 50% deamidation is required to reduce the reactivity of gliadin deamidated by acid or enzyme, probably because of the exposure of inner epitope structure to the surface during acidic hydrolysis and enzymatic reaction. ...
Article
Gliadin is the principal allergen of wheat-dependent, exercise-induced anaphylaxis (WDEIA). The primary structure of IgE-binding epitopes in wheat gliadin includes tandem sequencing sites of glutamine residues. Therefore, deamidation would be an effective approach to reduce the allergenicity of wheat proteins. In our previous study, we deamidated wheat gliadin without causing peptide-bond hydrolysis nor polymerization by the carboxylated cation-exchange resins, and we found that the deamidated gliadin scarcely reacted with the sera of patients RAST positive to wheat. In this study, we examined the allergenicity of the deamidated gliadin in a mouse model of wheat-gliadin allergy. The oral administration of the deamidated gliadin to gliadin-sensitized mice suppressed the enhancement in the intestinal permeability, serum allergen level, serum allergen-specific IgE level, mast-cell-surface expression of FcεRI and serum and intestinal histamine levels. Our results indicate that gliadin deamidated with no peptide-bond hydrolysis by cation-exchange resins has low allergenicity even in in vivo conditions.
... Several animal models (dog, rat, mice) have been used to study wheat allergenicity using gluten and non-gluten proteins [17][18][19][20][21][22][23]. Wheat gliadin has been used in most gluten allergy mouse model studies [20,22,24,25]. There are two mouse models reported for wheat glutenin hypersensitivity [19,26]. ...
Article
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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.
... Several studies have reported on mouse models used to study gluten hypersensitivity (59, 64,[73][74][75][76]; all except one used alum adjuvant to elicit IgE responses to gluten upon intraperitoneal injections, the exception used detergent as adjuvant to elicit sensitization upon application over damaged skin by tapestripping of stratum corneum. Key endpoints used in these studies were as follows: specific IgE and total IgE, HSR, and symptom scoring. ...
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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.
... Nevertheless, we found that the acid hydrolyzed gluten products exhibited the greatest sensitizing capacity compared with Un Glu and E Glu, which is in line with previous studies of skin-mediated and intraperitoneal sensitization. 9,15,19,24,[28][29][30] Thus, skin sensitization in BN rats may be a potential tool for evaluating the sensitizing potential of food proteins in food allergy risk assessment. ...
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Background: Adverse reactions to wheat-containing skin care products have been linked to food allergy development. Objectives: To determine the role of skin barrier dysfunction and inflammation in sensitization to gluten-derived hydrolysates via the skin in Brown Norway rats with and without oral tolerance to wheat. Methods: Skin barrier defect was induced by mechanical disruption, and skin inflammation was induced by topical application of SLS or MC903. Unmodified, enzyme hydrolyzed, or acid hydrolyzed gluten products were applied to the skin 3 times per week for 5 weeks. Subsequently, rats were orally gavaged with unmodified gluten. Results: Wheat-naïve rats were readily sensitized to gluten hydrolysates via the skin. Skin barrier defect and skin inflammation had little effect on the skin sensitization and hydrolysate-specific IgE levels. Oral administration of unmodified gluten promoted the production of unmodified gluten-specific IgE in rats sensitized via the skin. Sensitization through intact skin, disrupted skin barrier, or inflamed skin was unable to break tolerance to unmodified gluten in rats on a wheat-containing diet. Conclusions: Mechanical skin barrier disruption and skin inflammation play a limited role in experimental skin sensitization to gluten-derived hydrolysates. This article is protected by copyright. All rights reserved.
... Blood was collected via cardiac puncture after the oral challenge and centrifuged at 1500 g for 15 min. The assay for the quantification of gliadin-specific Igs was performed in serum samples via indirect ELISA, as previously described (Gourbeyre et al. 2012). Specific Ig binding is expressed by the ratio of the mean fluorescence intensity measured with wheat native gliadins (IF) to the mean fluorescence intensity measured with carbonate buffer (IF0) as the background signal (Lupi et al. 2013). ...
Article
Allergic diseases are increasing worldwide, and their precise causes are not fully understood. However, this observation can be correlated with growing chemical pollution of the environment. Bisphenol A (BPA) alters the immune system, microbiota and barrier functions. Here, we studied the effect of oral BPA at levels equivalent to human exposure to understand the mechanisms of immunological, physiological and microbial action on food allergies. In a murine model of allergy, we evaluated the effect of direct oral exposure to BPA at 4 µg/kg bw/d corresponding to tolerable daily intake (TDI). We studied symptoms, intestinal physiology and humorall and cellular immune responses during food allergy. We explored the relationship between oral exposure to BPA and changes in the gut microenvironment. Markers of food allergy and intestinal permeability were increased following exposure to BPA. We also observed a modulated humorall and T-cell response with aggravation of food allergy inflammation. Moreover, BPA exposure induced gut dysbiosis and decreased microbial diversity induced by food allergy. Altogether, these results suggest that the 2015 European Food Safety Authority (EFSA) TDI should be reviewed to consider the immunotoxicity of BPA.
... It also produces weight loss and chronic diarrhea. Gourbeyre et al. (2012) used the Balb/c mouse model to test this hypothesis. They found that: i) Native gliadin elicited a higher T helper 1 type of immune response and the deamidated gliadin (DG) elicited a higher T helper 2 or allergic immune response and histamine response. ...
Article
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ARTICLE INFO ABSTRACT The prevalence of wheat allergy has reached significant levels in many countries. Immunoglobulin E (IgE)-mediated food allergy is the most common type of adverse reaction towards wheat proteins. Increased intestinal permeability after gluten exposure occurs in all individuals. Both genetic and environmental factors influence the nature of reactivity to gluten and phenotypic expression of enteropathy. Gluten can trigger adverse inflammatory, immunological and autoimmune reactions in some people. It can produce abroad spectrum of gluten related disorders. This comprehensive review explored to identify the strengths, challenges, and future prospects of animal models in basic and applied wheat protein allergy evidenced from clinical trials and its role as potential pathogenic co-factor.
... Wheat protein hydrolysates could also be used in food products as a protein supplement (Flambeau et al., 2016). Acid hydrolysed wheat gluten acquires emulsifying properties which make them useful in food products such as coffee creamers and also in cosmetic products (Gourbeyre et al., 2012). Garg et al. have investigated the effect of heating and acidic pH on the characteristics of wheat gluten (Garg et al., 2019). ...
Article
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In this work, wheat gluten was hydrolysed with pineapple crude extract, obtained from pineapple core which is a waste from the pineapple processing industry. The efficacy of crude pineapple extract was at par with the commercial bromelain in terms of hydrolysing wheat gluten. The hydrolysis proceeded at a rapid rate during the first 60 min, then slowed down till 120 min, followed by a further steep rise until 180 min and a very slow reaction up to the 24‐h incubation period. The resulting hydrolysates had a fair solubility (>40%) over a pH range of 2–10. The molecular masses of the identified mixture of peptides ranged between 500 and 5000 Da, with 16% and 79% of the identified peptides in the range 0.5–1.0 and 1.0–2.0 kDa, respectively. The functional properties of the hydrolysates were remarkably better than the original gluten.
... However, it can cause allergic reactions in animals. Ingestion of gluten protein contained in wheat or other grains can influence immune responses (BATTAIS et al., 2008;GOURBEYRE et al., 2012). In the present study, wheat gluten protein ingestion significantly decreased feed intake and body weight gain of broilers. ...
... In the present study, no statistically significant differences could be found between specific IgG1 or IgE responses to G, EHG and AHG. This is in contrast to a study by [21] where mice were i.p. immunization with native or deamidated gliadin (corresponding to AHG) and alum as adjuvant. They found that deamidated gliadin induced a higher IgE response and higher histamine release after challenge than native gliadin. ...
Article
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Background There are several reports describing allergy to hydrolyzed wheat products. After a large outbreak in Japan it was established that sensitization was caused by skin contact with acid hydrolyzed gluten in soap. It is still not clear if other forms of hydrolyzed gluten may sensitize, and if the skin is the only relevant route of sensitization in humans and to what extent oral tolerance to wheat play a role. Objectives The aim of the present study was to examine if wheat-tolerant rats may be sensitized via the oral or i.p. route when exposed to gluten, enzymatic or acid hydrolyzed gluten. Methods Brown Norway rats, tolerant to wheat, were dosed by three i.p. injections without adjuvant or by oral gavage daily for 35 days with the three gluten products, respectively. Sera were analyzed by ELISA for specific IgG1 and IgE. In addition inhibition and avidity ELISAs were performed. Results were compared to a similar study in rats naïve to wheat. Results More than half the animals had measurable IgG1 at the start of the dosing period. I.p. immunization resulted in significant specific IgG1 and IgE to the antigen used for immunization but significantly lower than in naïve rats. The results of inhibition and avidity ELISA’s indicate that the underlying tolerance to epitopes common to the three products influences the immune response. Oral dosing did not induce significant changes in response to either gluten or the hydrolyzed gluten product used for dosing. Conclusions The study shows that i.p. immunization with the three products can break the underlying tolerance to wheat. Exposure by the oral route to enzymatic or acid hydrolyzed gluten is very unlikely to break an already established tolerance to gluten and induce sensitization.
... The carboxylic acid deami- dation is a promising method to improve the functional properties of wheat gluten by converting the amide groups to carboxyl groups ( Glu & Asp, respectively). Deamidation dissociates protein polymers by in- creasing the electrostatic repulsion among protein molecular chains and increases the surface hydrophobicity (Liao, , which can enhance the molecular flexibility (Jekle, Mühlberger, & Becker, 2016), nutrition, function (Lei, Zhao, Selomulya, & Xiong, 2015;Qiu, Sun, Zhao, Cui, & Zhao, 2013), and celiac-related characteristics ( Gourbeyre et al., 2012;Malalgoda & Simsek, 2017) of proteins. However, even after the deamidation of wheat gluten, about a total of 10% insoluble fractions still remain, among which 10% is starch (Roels, Grobet, & Delcour, 1998). ...
... Therefore, deamidation by cation-exchange resins would be effective for retaining T-cell epitopes because the reaction is moderate. Although the sequences of T-cell epitopes in wheat gliadin has yet to be determined, Gourbeyre et al. (2012) found that the addition of gliadin deamidated by HCl to T cells from UG-sensitized mice activated T cells and increased the production of IL-4. As for other food allergens, oral administration of T-cell epitopes of ovomucoid and ovalbumin in egg white induced the oral tolerance (Rupa and Mine, 2012;Yang et al., 2010). ...
Article
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Wheat allergy is a serious disease because it is difficult to be outgrown and sometimes induces anaphylaxis. Gliadin, a major allergen in wheat, deamideted by cation-exchange resins (DG) had low allergenicity not only in vitro but also in vivo. As the allergen-specific immunotherapy, a treatment for allergies, hold a risk of severe allergic reaction, the deamidated gliadin that has low allergenicity would provide an effective tool for treatment of wheat allergy. Thus, we examined if DG would induce oral tolerance using a mouse model of wheat allergy. Mice sensitized with untreated gliadin (UG) by intraperitoneal injection were orally administered with DG every other day for four weeks, and then UG was orally challenged to induce systemic anaphylaxis. Repeated oral administration of DG suppressed allergic reaction induced by UG challenge, and dramatically increased the number of regulatory T cells indicating deamidated gliadin induced oral tolerance.
... Studies conducted on animal models (mice and rats) can provide some answers. In 2012, Gourbeyre et al. sensitised mice intraperitoneally and noticed that, compared to native gliadins, gliadins deamidated by acid hydrolysis induced a higher production of IgE antibodies to deamidated gliadins but also to native gliadins (Gourbeyre et al. 2012). In 2018, Castan et al. confirmed this last point by stating that sensitisation with deamidated gliadins induced in mice a stronger allergic reaction than sensitisation with native gliadins, with in particular earlier IgE production and immune response (Castan et al. 2018). ...
Article
Wheat gluten can be chemically or enzymatically hydrolysed to produce functional ingredients useful in food and cosmetics. However severe allergies to hydrolysed wheat proteins (HWP) have been described in Europe and Japan since the early 2000's. Triggering proteins and IgE epitopes were described both for French and Japanese cohorts and appeared remarkably similar leading to define a new wheat allergic entity. Deamidation induced by functionalisation generate neo-allergens responsible for this particular allergy. This article aims to review the processes leading to deamidation and the clinical features of the patients suffering from this allergy. Then the molecular determinants involved in HWP-allergy were exhaustively described and hypothesis regarding the sensitizing mechanism of HWP-allergy are discussed. Finally, current regulation and tools aiming at managing this risk associated with HWP are presented.
... Successful development and validation of this mouse-based method here, suggest that a similar approach could be used to develop and validate methods for allergenicity assessment of alcohol-soluble and acid-soluble proteins from wheat or any other allergenic food. For example, there are five useful mouse models that already have been developed using alcohol-soluble gluten proteins (gliadins) earlier (Bodinier et al., 2009;Denery-Papini et al., 2011;Gourbeyre et al., 2012;Abe et al., 2014;Adachi et al., 2012). These animal model protocols may be employed for developing and validating II-ELISA using the same approach that is described here in this paper. ...
Article
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.
... Briefly, Gliadins (10 mg) were solubilized in 1 mL of 50% (v/v) ethanol, 0.1 N hydrochloric acid, and heated at 90˚C for 40, 90, and 120 min. Deamidated Gluten (D-Gluten) was prepared by acid hydrolysis of gluten according to Gourbeyre et al. [27]: Gluten (20 mg) was solubilized in 1 mL of 0.1 N hydrochloric acid, and heated at 90˚C for 1h. The reaction was stopped by neutralization with 1 mL 0.1 N NaOH. ...
Article
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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.
... The results of previous human serum experiments have shown that IgE reactivity to acid-HWG caused a peak in acidic hydrolysis for 0.5 h, and the antigenicity of acid-HWG disappeared following acidic hydrolysis for 24 h (Nakamura et al. 2013a). GP19S is composed of WG and de-amidated by acidic hydrolysis, and its antigenicity is higher than that of WG because of the development of a new epitope (Gourbeyre et al. 2012;Nakamura et al. 2013b,c;Yokooji et al. 2013). The findings of the present investigation suggested that GP19S increased the expression of loTSLP due to de-amidation and/or developing new epitopes by acidic hydrolysis. ...
Article
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A growing body of evidence suggests that epicutaneous sensitization of protein allergens induces immediate-type hypersensitivity (IHS) following induction of Type 2 immune responses in animals and humans. Thymic stromal lymphopoietin (TSLP) derived from keratinocytes is a cytokine that can activate dendritic cells and has been implicated in development of inflammatory Type 2 helper T-cells. However, there is no direct evidence that allergens directly regulate TSLP expression in keratinocytes. This study aimed to evaluate the response of TSLP to protein allergens in cultured human keratinocytes and to identify appropriate endpoints for IHS. The transcription of long-form TSLP (loTSLP) was strongly induced by ovalbumin, wheat gluten (WG), acid-hydrolyzed WG (acid-HWG), and extracts from feces of Dermatophagoides pteronyssinus and D. farina, and trypsin, but not by rare allergens, human serum albumin (HSA), or extracts of mite bodies. In acid-HWG, loTSLP mRNA was significantly augmented by acid hydrolysis of WG for 0.5 h compared to WG. However, prolonged acid hydrolysis attenuated this induction similarly to that reported in previous animal studies. These results suggested that intense loTSLP transcriptional induction was a characteristic of a high-allergenic protein. Additionally, TSLP production was induced by exposure to ovalbumin, WG, and acid-HWG in combination with a trio of cytokines, i.e. interleukin (IL)-4, IL-13, and tumor necrosis factor (TNF)-α. However, no TSLP protein was detected following exposure to HSA, even in the presence of these cytokines. With acid-HWG, TSLP protein release was consistent with loTSLP transcription. Thus, intense loTSLP transcriptional induction and TSLP protein expression are each effective indicators that can be used for in vitro screening of IHS.
... There are multiple routes used to induce allergic sensitization to food allergens including i.p., oral, intranasal (i.n.) and cutaneous administration [20,21]. However, the route of administration may alter the resulting immune response. ...
Article
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Food allergy is a major health problem of increasing concern. The insufficiency of protein sources for human nutrition in a world with a growing population is also a significant problem. The introduction of new protein sources into the diet, such as newly developed innovative foods or foods produced using new technologies and production processes, insects, algae, duckweed, or agricultural products from third countries, creates the opportunity for development of new food allergies, and this in turn has driven the need to develop test methods capable of characterizing the allergenic potential of novel food proteins. There is no doubt that robust and reliable animal models for the identification and characterization of food allergens would be valuable tools for safety assessment. However, although various animal models have been proposed for this purpose, to date, none have been formally validated as predictive and none are currently suitable to test the allergenic potential of new foods. Here, the design of various animal models are reviewed, including among others considerations of species and strain, diet, route of administration, dose and formulation of the test protein, relevant controls and endpoints measured.
... The level of glutamine (Gln) residues was evaluated from the DABA/norleucine ratio, whereas the level of Glu was determined from the Glu (not converted in DABA)/norleucine ratio. Percentage deamidation was calculated as Glu/(Gln + Glu)/100 as described in ref 20. ...
Article
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Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food processing measures can impact on epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report on the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, rye, and deamidated gluten down to 2 ppm in food as well as its performance in foods testing.
... Il a également été montré que des individus tolérant au blé pouvaient développer des allergies sévères au gluten si celui-ci est modifié par désamidation, un procédé technologique utilisé par les industriels pour augmenter sa solubilité et ainsi élargir son champ d'utilisation (utilisation dans des viandes cuisinées, pâtés, charcuterie…). Il s'est avéré que les protéines de blé désamidées sont beaucoup plus allergisantes et que l'allergie au gluten désamidé, étudiée grâce à un modèle souris, résulte de mécanismes différents de ceux observés dans le cas de l'allergie à la farine de blé ( Gourbeyre et al., 2012). ...
Article
Rapport rédigé par une douzaine de membres du GROUPE FILIERE CEREALES de l'INRA, dont l'animateur est Gilles Charmet. Le public visé ne se réduit pas à une catégorie dans celles proposées par PRODINRA, il comprend aussi le public scientifique et le professionnel. Le rapport lui-même et l'annexe 1 sont à diffusion publique, mais l'annexe 2 est confidentielle.
... Purified α-, ω2-, and ω5-gliadins, total gliadins, and LMW-GS were deamidated by acidic treatment as previously described. 24 A set of total gliadin fractions deamidated to different extents was also prepared. Gliadins (10 mg) were solubilized in 1 mL of 50% (v/v) ethanol, 0.1 N hydrochloric acid, and heated at 90°C for 40, 60, 90, and 120 min. ...
Article
Diversification of gluten applications in food and cosmetics industries was achieved through the production of water-soluble gluten that can be obtained by deamidation. Current analytical methods dedicated to gluten detection failed to detect deamidated gluten. After immunizing mice with the peptide LQPEEPFPE conjugated to keyhole limpet hemocyanin, five mouse monoclonal antibodies (mAbs) were produced and sequences of bound epitopes were determined as XPXEPFPE, where X is Q or E. The mAbs exhibited high specificity for deamidated gliadins and low molecular weight glutenin subunits. A competitive ELISA based on INRA-DG1 mAb was developed with an IC50% of 85 ng/mL and a limit of detection of 25 ng/mL. The intra and inter assay coefficients of variation (CV) were < 10 % except for the inter-assay CV of the low-level control (40 ng/ml), which was 20 %. This assay was capable of detecting three of the four deamidated gluten samples spiked in rice flour at 20 mg/kg.
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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.
Article
Scope: Personal care products containing hydrolysed gluten have been linked to spontaneous sensitisation through the skin, however the impact of the hydrolysate characteristics on the sensitising capacity is generally unknown. Methods and results: The physicochemical properties of five different wheat-derived gluten products (1 unmodified, 1 enzyme hydrolysed, and 3 acid hydrolysed) were investigated, and the skin sensitising capacity was determined in allergy-prone Brown Norway rats. Acid hydrolysed gluten products exhibited the strongest intrinsic sensitising capacity via the skin. All hydrolysed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolysed products was associated with product-specific sensitisation in wheat tolerant rats. Sensitisation to acid hydrolysed gluten products was associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitisation induced by unmodified gluten or enzyme hydrolysed gluten. Conclusion: Acid hydrolysis enhances the skin sensitising capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolysed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals. This article is protected by copyright. All rights reserved.
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Background Regulatory T cells (Tregs) are known to protect against allergies. Moreover, the decrease in the frequency and efficiency of Tregs amplifies allergic symptoms. Aim This study investigated whether expanding Tregs in vivo with an IL‐2/IL‐2 antibody complex could be safe, well tolerated and efficient in a therapeutic setting in allergies. Methods We produced an anti‐IL‐2 antibody (1C6) and demonstrated that when it is complexed to human IL‐2, it increases IL‐2 efficiency to induce Tregs in vivo without any detectable side effects. Furthermore, the IL‐2/1C6 complex induces an increase in Helios expression by Tregs, suggesting that it not only elevated Treg numbers but also boosted their functions. Using mouse models of house‐dust‐mite‐induced airway inflammation and wheat‐gliadin‐induced food allergies, we investigated the therapeutic potential of the IL‐2/1C6 complex in allergies. Results IL‐2/1C6 treatment significantly reduced allergic symptoms, specific IgE production, the adaptive immune response and tissue damage. Interestingly, IL‐2/1C6 treatment modulated innate lymphoid cells by increasing ILC2s in asthma and decreasing ILC3s in food allergies. Conclusion In conclusion,complexed IL‐2/anti‐IL‐2 may restore Treg numbers and function in respiratory and food allergies, thereby improving allergic markers and symptoms. Our IL‐2/anti‐IL‐2 complex offers new hope for reestablishing immune tolerance in patients with allergies.
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Gliadins are the main cause of wheat allergies, and the prevalence of gliadin allergy has increased in many countries. l-Arabinose, a kind of plant-specific five-carbon aldose, possesses beneficial effects on food allergy to gliadins. This study investigated the antiallergic activities and underlying mechanisms of l-arabinose in a wheat gliadin-sensitized mouse model. BALB/c mice were sensitized to gliadin by intraperitoneal injections with gliadin followed by being given a gliadin challenge. l-arabinose-treated mice exhibited a marked reduction in the productions of total immunoglobulin E (IgE), gliadin-specific IgE, gliadin-specific IgG1, and histamine, with an increase in IgG2a level as compared with gliadin-sensitized mice. Beside that, a significant decrease in Th2-related cytokine level, IL-4, and an increase in Th1-related cytokine level, IFN-γ, in the serum and splenocytes were observed after treatment with l-arabinose. l-Arabinose treatment also improved the imbalance of Th1/Th2 immune response on the basis of the expression levels of related cytokines and key transcription factors in the small intestine and spleen of sensitized mice. In addition, gliadin-induced intestinal barrier impairment was blocked by l-arabinose treatment via regulation of TJ proteins and suppression of p38 MAPK and p65 NF-κB inflammation signaling pathways. Notably, the results confirmed that l-arabinose treatment increased CD4+ Foxp3+ T cell populations and Treg-related factors associated with increased expression of IL-2 and activation of STAT5 in gliadin-sensitized mice. In conclusion, l-arabinose attenuated the gliadin-induced allergic symptoms via maintenance of Th1/Th2 immune balance and regulation of Treg cells in a gliadin-induced mouse model, suggesting l-arabinose could be used as a promising agent to alleviate gliadin allergy.
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Assessment of human health risk requires an understanding of antigen dose metrics associated with toxicity. Whereas assessment of the human health risk for delayed‐type hypersensitivity is understood, the metrics remain unclear for percutaneous immediate‐type hypersensitivity (ITH) mediated by IgE/IgG1. In this work, we aimed to investigate the dose metric for percutaneous ITH mediated by IgE/IgG1 responses. Papain, which causes ITH via percutaneous sensitization in humans, was used to sensitize guinea pigs and mice. The total dose per animal or dose per unit area was adjusted to understand the drivers of sensitization. Passive cutaneous anaphylaxis (PCA) and enzyme‐linked immunosorbent assay (ELISA) for papain‐specific IgG1 enabled quantification of the response in guinea pigs. In mice, the number of antigen‐bearing B cells in the draining lymph nodes (DLN) was calculated using flow cytometry, papain‐specific IgG1 and IgE levels were quantified by ELISA. PCA positive test rates and the amounts of antigen‐specific antibody corresponded with total dose per animal, not dose per unit area. Furthermore, the number of B cells taking up antigen within DLN also correlated with total dose. These findings indicate that the total antigen dose is the important metric for percutaneous IgE/IgG1‐mediated ITH. Assessment of human health risk requires an understanding of antigen dose metrics associated with toxicity. Whereas assessment of the human health risk for delayed‐type hypersensitivity is understood, the metrics remain unclear for percutaneous immediate‐type hypersensitivity (ITH) mediated by IgE/IgG1. The results obtained in mice and guinea pigs demonstrated that the incidence of papain‐evoked percutaneous IgE/IgG1‐mediated ITH correlates with the amount of total antigen dosage. Therefore, it is suggested that the total dose will dictate the effectiveness and extent of percutaneous ITH.
Article
Background: Wheat is known as the most widely consumed food all over the world. Although many types of wheat allergy have been recognized, their treatment still has a long way to go due to the complex pathogenesis. Oral immunotherapy (OIT) is under investigation for the treatment of wheat allergies. Previous studies have demonstrated that OIT using intact wheat allergens can induce tolerance, but is accompanied by a high risk of anaphylactic reactions. Objectives: Our objective was to prepare modified wheat allergens with hypoallergenic and tolerance-inducing properties to reduce adverse effects during immunotherapy. Methods: Wheat gliadin was degraded by hydrolysis with pepsin and trypsin, and then the hydrolysate was deamidated with hydrochloric acid. The IgE-binding capacity and T cell reactivity of the degraded gliadins were evaluated in vitro. Pepsin-digested gliadin (peptic-GLI) was applied in a mouse model to investigate whether it would induce oral tolerance. Results: Degradation with pepsin decreased IgE-binding capacity and maintained T cell reactivity. Oral administration of peptic-GLI to mice before sensitization and challenge with gliadin could significantly suppress the production of IgE, IgG1, and type 2 T helper cytokines. Moreover, the development of anaphylactic reactions and allergic responses of the small intestine induced by gliadin challenge were inhibited by oral administration of peptic-GLI. Conclusions: The findings of this study indicate that peptic-GLI with low allergenicity and potential for tolerance induction may become useful in wheat immunotherapy with less adverse effects.
Article
Gliadins are major wheat allergens. Their treatment by acid or enzymatic hydrolysis has been shown to modify their allergenic potential. As the interaction of food proteins with dendritic cells (DCs) is a key event in allergic sensitization, we wished to investigate whether deamidation and enzymatic hydrolysis influence gliadin processing by DC, and to examine the capacity of gliadins to activate DCs. We compared the uptake and degradation of native and modified gliadins by DCs using mouse bone marrow-derived DCs. We also analyzed the effects of these interactions on the phenotypes of DCs and Th lymphocytes. Modifying gliadins induced a change in physicochemical properties (molecular weight, hydrophobicity, sequence) and also in peptide size. These alterations in turn led to increased uptake and intracellular degradation of the proteins by DCs. Native gliadins (100 µg/mL), but not modified gliadins, increased the frequency of DC expressing CD80 (15.41±2.36 % vs 6.81±1.10 %), CCR7 (28.53±8.17 % vs 17.88±2.53 %), CXCR4 (70.14±4.63 % vs 42.82±1.96 %) and CCR7-dependent migration (2.46±1.45 vs 1.00±0.22) compared with native gliadins. This was accompanied by a Th lymphocyte activation (30.37±3.87 % vs 21.53±3.14 %) and proliferation (16.39±3.97 % vs 9.31±2.80 %). Moreover hydrolysis decrease peptide size induce a an increase in gliadin uptake and degradation. Deamidation and extensive enzymatic hydrolysis of gliadins modify their interaction with DCs, leading to alteration of their immunostimulatory capacity. These findings demonstrate the strong relationship between the biochemical characteristics of proteins and immune cell interactions.
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Microbial transglutaminase is heavily used in the food processing industries to improve food qualities. Being a protein's glue, by cross-linking it creates neoepitope complexes that are immunogenic and potentially pathogenic in celiac disease. Despite low sequence identity, it imitates functionally its family member, the endogenous tissue transglutaminase, which is the autoantigen of celiac disease. The present comprehensive review highlights the enzyme characteristics, endogenous and exogenous intestinal sources, its cross-talks with gluten and gliadin, its immunogenicity and potential pathogenicity and risks for the gluten induced conditions. If substantiated, it might represent a new environmental inducer of celiac disease. The present findings might affect nutritional product labeling, processed food additive policies and consumer health education.
Article
Scope Food allergies result from a complex immune response involving both innate and adaptive immune cells. Major proteins of wheat flour, gliadins, appear to be important allergens, and their characteristics can influence the allergic response. Our study investigates the immune reaction when developing a food allergy to gliadins in native, deamidated or hydrolyzed forms. Methods We analyzed the immune response after one or two intraperitoneal sensitizations and after oral challenge with each gliadin form. Results We demonstrate that deamidated gliadins induce a stronger allergic reaction compared to native gliadins. Moreover, deamidation induces an earlier increase in intestinal permeability associated with more pronounced Th2 and Th17 polarizations together with an influx of antigen presenting cells especially cDC2. Conclusion Altogether, our data indicate that industrial processes such as deamidation or hydrolysis influence food allergenicity through immune modulation and help us to develop tools to determine how these processes can influence this reaction and encourage or decrease allergic reactions. This article is protected by copyright. All rights reserved
Article
Wheat allergy is an IgE-mediated disorder. Polyphenols, which are known to interact with certain proteins, could be used to reduce allergic reactions. In this study, we screened several polyphenol sources for their ability to interact with gliadins, mask epitopes and impact basophil degranulation. Polyphenol extracts from artichoke leaves, cranberries, apples and green tea leaves were examined. Of these extracts, the first three formed insoluble complexes with gliadins. Only the cranberry and apple extracts masked epitopes in dot blot assays using anti-gliadin IgG and IgE antibodies from patients with wheat allergies. The cranberry and artichoke extracts limited cellular degranulation by reducing mouse anti-gliadin IgE recognition. In conclusion, the cranberry extract is the most effective polyphenol source at reducing the immunogenicity and allergenicity of wheat gliadins.
Article
As one of the eight foods that account for 90 % of food allergies, wheat must be excluded from the diet in patients suffering from wheat allergies. From studies of wheat-dependent exercise-induced anaphylaxis (WDEIA), which has been used as a model to develop hypoallergenic wheat, we now know that the gluten fraction of wheat protein, particularly ω-5 gliadin and high-molecular-weight (HMW)-glutenin, is responsible for the allergic response. However, studies of allergic responses with WDEIA have been performed with a single wheat cultivar. Thus, in an effort to provide more information for the development of hypoallergenic wheat, we compared various cultivars with different countries of origin and characteristics. For the first step, we compared the allergen contents (ω-5 gliadin and HMW-glutenin) in each cultivar and the allergic response caused by each cultivar. Domestic wheat cultivars had lower contents of ω-5 gliadin and HMW-glutenin than those of imported wheat cultivars. Additionally, some cultivars caused varying allergic responses due to their allergen components. From regression analysis of allergen contents and allergic responses in vivo, we suggest a prediction model to estimate the extent of allergic response based on the ω-5 gliadin and HMW-glutenin contents. Further studies are needed to analyze the biological interactions between allergens from various cultivars and allergic response factors.
Article
Most egg-allergic children can tolerate extensively cooked eggs. Ovalbumin, a major allergen in egg whites, is prone to aggregate upon heating. This study compares ovalbumin’s allergenicity when it is aggregated as large particles to ovalbumin in its native form. Immunoglobulins (Ig)-binding and the degranulation capacities of native and aggregated ovalbumin were measured with sera from egg-allergic children and from mice sensitized to native or aggregated ovalbumin. The influence of ovalbumin structure on Ig production upon sensitization and elicitation potency by challenge was also studied. We showed that heat aggregation of ovalbumin as large particles enhances IgG production and promotes IgG2a production (a shift toward the T helper 1 profile). Aggregated ovalbumin displayed lower Ig-binding and basophil-activation capacities for sera from both allergic patients and mice. This work illustrates the links between ovalbumin structure after heating and allergenicity potential using parameters from both the sensitization and elicitation phases of the allergic reaction.
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Recent reports suggest that hydrolyzed wheat protein (HWP) variants such as Glupearl® 19S (GP19S) induce immediate-type hypersensitivity via epicutaneous (EC) sensitization. The identification of strong allergens is a key step in product assessment before commercial launch. However, few reports have described the estimation of actual and potential anaphylactic sensitizing capacity. In this study we assessed the strength of both the actual and potential anaphylactic sensitizing capacity by investigating the immediate-type hypersensitivity inducing potential of HWP compared with gluten. We assessed these strengths via the EC route using an EC or intradermal (ID) sensitization method. We quantified the strength of immediate-type hypersensitivity by evaluating the titer of serum antibodies isolated from sensitized subjects using passive cutaneous anaphylaxis (PCA) reactions. We also evaluated the cross-reactivity between GP19S and gluten. GP19S and gluten applied by both the sensitization methods induced obvious IgG1-mediated PCA reactions. GP19S had stronger sensitizing potential than gluten, according to the serum titers and dye spot diameters. The difference in antibody titers between GP19S and gluten was 16-fold for the EC method versus 2-fold for the ID method. GP19S cross-reacted with gluten. Acid hydrolysis of gluten increased anaphylactic sensitizing capacity in the EC method. To our knowledge, our study is the first to quantitatively confirm that HWP and gluten can induce immediate-type hypersensitivity through an intact skin. These findings suggest that acid-HWP imposes a higher risk of EC sensitization than gluten because of the ease with which the former confers a sensitizing effect through the intact skin.
Article
Epidemiological data suggest a link between food allergies and the subsequent development of asthma. Although this progression may result from the additional effects of exposure to multiple allergens, whether both allergies amplify each other's effects remains unknown. This study investigated whether oral exposure to food allergens influences the outcomes of subsequent respiratory exposure to an asthma-inducing allergen. Mice were sensitized and orally challenged with wheat (FA) and then exposed to house dust mite (HDM) extract (RA). Immunoglobulin (Ig), histamine, and cytokine levels were assayed by ELISA. Intestinal and lung physiology was assessed. Ig levels, histamine release, and cytokine secretion were higher after exposure to both allergens than after separate exposure to each. Intestinal permeability was higher, although airway hyper-responsiveness and lung inflammation remained unchanged. Exposure to food and respiratory allergens amplifies systemic and gut allergy-related immune responses without any additional effect on lung function and inflammation.
Article
Background Recent studies have highlighted the importance of extra-intestinal routes of sensitization to food-related allergens as the cause of epidemics of food allergy. Instances of Japanese women developing food allergy to wheat after exposure to hydrolyzed wheat protein (HWP) present in facial soap have been reported. However, the epidemiologic impact of these ingredients as a cause of food allergy has not been well studied.Methods To clarify the epidemiological relationship between food allergy to wheat and contact exposure to HWP, a case-control study of Japanese women aged 20-54 yrs. with self-reported wheat allergy (cases, n=157) and age-matched control subjects without wheat allergy (controls, n=449) was performed using a large-scale web-based research panel. Subjects answered a web-based questionnaire regarding the use of skin and hair care products, as well as other possible risk factors.ResultsCurrent use of an HWP-containing facial soap (Cha no Shizuku R, Yuka, Japan) was significantly associated with an increased risk of wheat allergy (adjusted odds ratio, 2.6; 95% confidence interval, 1.2-5.7; frequencies of current use in cases and controls; 11 and 6%, respectively). Use of Cha no Shizuku was more common in subjects with more recent-onset wheat allergy, implying that this soap may have contributed to the recent epidemic of wheat allergy.Conclusions An epidemiological relationship between wheat allergy and contact exposure to HWP has been documented. This study implicates a possible role of contact exposure to food-derived protein hydrolysates as a risk factor for the development of food allergy manifesting itself as anaphylaxis.This article is protected by copyright. All rights reserved.
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Wheat grain is a major staple of our diet. However, proteins derived from wheat grain have been implicated in both respiratory and food allergies, as well as in contact hypersensitivity. Numerous wheat allergens are present in the different fractions of wheat grain: a-amylase/trypsin inhibitor and lipid transfer protein are found in the water/salt soluble fraction, and omega5-gliadins and LMW-glutenins have been detected in the gluten fraction. This review discusses what is currently known about wheat grain proteins and allergens. The type of IgE-binding profiles (allergens or even epitopes) in patients with wheat food allergy as a function of age, symptoms, or genetic variability of wheat cultivars provides interesting and useful data for developing hypoallergenic foods as well as new tools for diagnostic and therapeutic methods.
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Celiac disease is a multifactorial disorder and provides a privileged model to decipher how the interplay between environmental and genetic factors can alter mucosal tolerance to a food antigen, lead to chronic intestinal inflammation, and ultimately promote T-cell lymphomagenesis. Here we summarize how HLA-DQ2/8 molecules, the main genetic risk factor for this disease can orchestrate a CD4(+) T-cell adaptive immune response against gluten, and discuss recent data which shed light on the innate and adaptive immune stimuli that collaborate to induce a proinflammatory TH1 response, a massive expansion of intraepithelial lymphocytes, and a cytolytic attack of the epithelium. The intestinal immune response driven in genetically predisposed patients by chronic exposure to gluten emerges as the pathological counterpart of normal acute intestinal responses to intracellular pathogens.
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With the exception of rice, the major cereals falls into two groups. The temperate cereals comprise barley, wheat, rye and oats, and the tropical cereals maize, sorghum and millets. Only the prolamins of wheat, barley and maize have been studied in detail at the molecular and physicochemical levels, and we will therefore focus on these. We will, however, also draw comparisons with the prolamins of other species where appropriate.
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Although allergen-specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti-IgE or allergen using a rat mast cell line (RBL) transfected with the alpha-chain of the human high-affinity IgE receptor (FcepsilonRI). Purified IgE specific for 4-hydroxy-3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a beta-hexosaminidase release assay after anti-IgE or allergen-specific challenge. For purified monoclonal IgE a significant correlation (r = 0.97) was found between the proportion of bound allergen-specific IgE and the strength of the degranulation response. In contrast, no correlation (r = 0.27) was detected after sensitization with serum IgE. Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen-specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.
Article
Gluten hydrolysates were prepared by limited enzymatic hydrolysis with a protease having a chymotryptic activity in the presence or in the absence of cysteine during the dispersion phase of the process. The hydrolysates were fractionated by ultrafiltration, using two inorganic membranes with different molecular weight cut-off (MWCO), 50 kg/mol and 150 kg/mol. The retentates were enriched in hydrophobic peptides and the permeates were enriched in hydrophilic peptides. The foaming and emulsifying properties of hydrolysates, retentates and permeates were analysed at two pHs (4 and 6·5) and two salt concentrations (0·2 and 2% NaCl). Hydrolysates displayed a foaming capacity, but the foams were not stable. Permeates generated foams at pH 6·5 only, and these foams had a very short life-time. Permeates displayed no emulsifying properties. Retentates yielded foams with a good stability and were more efficient than whole hydrolysates to stabilise emulsions. They provided a strong resistance to coalescence. The functional properties of retentates were only sightly influenced by pH and ionic strength. Neither cysteine addition, which helps gluten dispersion and increases the yield of soluble hydrolysate, nor the MWCO of the ultrafiltration membranes influenced the functionality of the hydrolysate fractions.
Article
Background: Wheat-dependent exercise-induced anaphylaxis is an anaphylaxy induced by physical exercise after ingestion of wheat. An immediate-type hypersensitivity to water/salt-insoluble fraction of wheat proteins (gluten) has been considered to underlie in this disease. Objective: The aim of the study is to determine the major allergen in Japanese patients with wheat-dependent exercise-induced anaphylaxis by using a panel of purified wheat gliadins and glutenins. Methods: Water/salt-insoluble wheat proteins, α-gliadin, β-gliadin, γ-gliadin, fast ω-gliadin, slow ω-gliadin, high molecular weight glutenin and low molecular weight glutenin, were purified, and five patients with wheat-dependent exercise-induced anaphylaxis, whose diagnose had been determined by positive-challenge test, were evaluated for skin prick test, dot-blotting test and CAP–RAST inhibition test by using these purified wheat proteins. Results: The fast ω-gliadin was the most potent allergen among these water/salt-insoluble proteins when evaluated by skin prick test and dot-blotting test. Fast and slow ω-gliadin, and γ-gliadin caused dose-dependent inhibition of the serum IgE-binding to solid-phase gluten in the patients. The incubation with fast ω-gliadin of the patient's serum caused dose-dependent inhibition in the IgE-binding to γ-gliadin as well as slow ω-gliadin, indicating a cross-reactivity of these proteins in IgE-binding. Conclusion: We concluded that fast ω-gliadin is a major allergen among these water/salt-insoluble proteins for wheat-dependent exercise-induced anaphylaxis in Japanese patients, and IgE against fast ω-gliadin cross-reacts to γ-gliadin and slow ω-gliadin.
Article
Six γ-gliadin components were separated by a procedure that included ion-exchange chromatography on sulphopropyl-trisacryl M and hydrophobic interaction chromatography on phenyl sepharose CL-4B. It was possible to prepare relatively large quantities of purified gliadin components by this procedure. Each purified gliadin had a distinct mobility on Polyacrylamide gel electrophoresis (PAGE) at acid pH and on two-dimensional Polyacrylamide gel electrophoresis. They also had characteristic properties of adsorption onto the hydrophobic gel. Four components γ46, γ48 and γ50 + 51, had apparent molecular weights of 36,000 and two others, γ44 and γ45, had apparent molecular weights of 41,000 (estimated by PAGE in the presence of sodium dodecyl sulphate). These differences in molecular size were confirmed by gel filtration chromatography in the presence of 6 m guanidine hydrochloride. The amino acid composition of γ50 + 51 was more typical of α- and β-gliadins than of γ-gliadins. γ44, γ45, γ46 and γ48 had amino acid compositions typical of the γ-gliadins, such as their low tyrosine and high tryptophan contents: they migrated in two-dimensional PAGE (pH 3·2/9/·2) as spots having distinct mobilities at basic pH. A relationship was observed between retention of γ-gliadins on hydrophobic gel at basic pH and their net charges at pH 9·2.
Article
An easy and rapid procedure for the determination of glutamine in synthetic peptides and isolated proteins is developed. It involves a prehydrolysis reaction of glutamine residues with bis(1,1-trifluoroacetoxy)iodobenzene (BTI), protein hydrolysis using a microwave technique, and high-performance liquid chromatography (HPLC) after precolumn derivatization of amino acids with dansyl chloride. Optimum conditions for the BTI-mediated conversion of glutamine to the corresponding diaminobutyric acid (DABA) were established. DABA and the proteic amino acids could be simultaneously measured with high sensitivity (2.0 pmol/injection; S/N = 3:1) and good reproducibility (CV 2.8%). The linearity between DABA formation and glutamine concentration was excellent (r = 0.9996). Recovery of DABA (mean recovery 93 ± 4%) from the selected test peptides and proteins (cathepsin G peptide, whey proteins, ovalbumin) was in good agreement with data derived from sequence analyses. Keywords: Glutamine; content in protein/peptides; analysis; BTI; microwave hydrolysis; HPLC
Article
Wheat storage proteins are responsible for the viscoelastic properties of dough. Their effect on dough rheology depends on the glutenin components and the proportion of gliadins, high and low molecular weight glutenin subunits (HMW-GS, LMW-GS). A prediction of dough behaviour is possible when the concentration of each prolamin group is known. A method of sequential extraction and quantification of wheat flour proteins was developed. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to quantify gliadins and HMW-GS and LMW-GS. Non-prolamin proteins and lipids were extracted with phosphate buffer and phosphate buffer containing Triton X114 respectively and 70% (v/v) ethanol was used to extract gliadins. Several solvent systems were tested to exhaustively extract glutenins and a buffer containing 0·05 M tetraborate pH 8·5, 2% (v/v) 2-mercaptoethanol, 1 g litre−1 glycine, and 8 M urea was selected. It facilitated the most complete extraction and was compatible with RP-HPLC analysis of extracts. The quantities of extracted prolamins were estimated from RP-HPLC profiles using peak area determination. The concentration of different classes of prolamins obtained by RP-HPLC was compared with their determination by nitrogen (N) content. The average yield of extractions performed on 45 cultivars was 0·99 (coefficient of variation (CV)=7·6%) for gliadins and 0·89 (CV=9·1%) for glutenins. Quantifications of proteins by N determination and RP-HPLC were strongly correlated: regression coefficients were 0·86 for total prolamins and gliadins, and 0·71 for glutenins with the gradient of regression of approximately 1. Therefore, RP-HPLC could be used to quantify prolamin groups without using N determination. © 1998 SCI.
Article
Wheat is cultivated as a source of flour. Gluten, a major reserve protein of wheat, is used industrially in both natural and modified forms. Gluten-derived products are used as emulsifiers and stabilizers in foods and cosmetics. These hydrolysed wheat proteins can induce allergic reactions such as allergic contact dermatitis and, most often, type I hypersensitivity. Among the latter are contact urticarias after cosmetic exposure, and anaphylaxis or generalised urticaria after food intake. The new allergens in these products must be identified and characterised.
Article
Wheat gliadins, that are glutamine rich and lysine poor proteins, are good substrates for transglutaminases reactions. This study was conducted to determine the efficiency with which guinea pig liver transglutaminase catalyzes transfer and hydrolysis reactions of native and acylated gliadins. In all reactions, 35% of the total glutaminyl residues were modified. Neutral pH simultaneously enhanced glutaminyl residue hydrolysis and protein cross-linking, while acidic pH reduced the cross-linking reaction. Functional properties of two enzymatically modified gliadins and a chemically deamidated one were tested at neutral pH. A deamidation level of 25–27% appeared to be an optimum for the emulsification properties. Enzymatically modified gliadin showed better resistance to coalescence than the chemically deamidated one; a result that probably is related to the presence of high molecular weight polymers.
Article
Wheat is one of the major crops grown, processed and consumed by humankind and is associated with both intolerances (notably coeliac disease) and allergies. Two types of allergy are particularly well characterized. The first is bakers' asthma, which results from the inhalation of flour and dust during grain processing. Although a number of wheat proteins have been shown to bind IgE from patients with bakers' asthma, there is no doubt a well‐characterized group of inhibitors of α‐amylase (also called chloroform methanol soluble, or CM, proteins) are the major components responsible for this syndrome. The second well‐characterized form of allergy to wheat proteins is wheat‐dependent exercise‐induced anaphylaxis (WDEIA), with the ω 5 ‐gliadins (part of the gluten protein fraction) being the major group of proteins which are responsible. Other forms of food allergy have also been reported, with the proteins responsible including gluten proteins, CM proteins and non‐specific lipid transfer proteins. Processing of wheat and of related cereals (barley and rye, which may contain related allergens) may lead to decreased allergenicity while genetic engineering technology offers opportunities to eliminate allergens by suppressing gene expression.
Article
Wheat isolates, used as binders and emulsifiers in the food industry, mainly in meat products, are neo-allergens resulting from deamidation of wheat gluten. In this paper, we describe cases of anaphylactic reactions caused by a wheat isolate occurring after eating pasta. Sensitivity to wheat isolate was demonstrated by positive skin tests and specific IgE reactivity to wheat isolate extract (20.1 KU/l, class 4), and also by detection and quantification of wheat isolate in pasta by immunoblotting and ELISA inhibition.
Article
The clinical manifestations of allergy to wheat flour are similar to those of allergies to other foods. In adults, food-dependent exercise-induced anaphylaxis, chronic urticaria, and gastrointestinal food allergies (that is, irritable bowel syndrome, eosinophilic colitis, ulcerative colitis) are the most frequently described clinical manifestations of allergy to wheat. Wheat isolates, used as binders and emulsifiers in the food industry, are neo-allergens resulting from chemically induced desamidation of wheat gluten (heating at high temperature in an acidic medium). Wheat isolate allergens can induce severe systemic reactions (e.g., urticaria) and anaphylactic shock. Diagnosis consists of three steps: a suspicion based on the patient's history, identification of the allergen by skin testing and by laboratory tests, and confirmation by oral challenge or by an avoidance regime.
Article
Gluten, the dough-forming protein of wheat flour, is the key to the unique ability of wheat to suit the production of leavened products. The past five decades have seen the rise of gluten as a commodity in its own right, through the large-scale industrial separation of wheat starch from gluten, plus the controlled drying of the gluten so as to retain its functional properties. The resulting Vital Dry Gluten is most widely used in bakery products. However, gluten (vital, de-vital or modified) is finding increasing use as a food ingredient to provide a range of functional properties at a more modest price than competitors such as milk and soy proteins.
Article
Wheat is the dominant crop in temperate countries being used for human food and livestock feed. Its success depends partly on its adaptability and high yield potential but also on the gluten protein fraction which confers the viscoelastic properties that allow dough to be processed into bread, pasta, noodles, and other food products. Wheat also contributes essential amino acids, minerals, and vitamins, and beneficial phytochemicals and dietary fibre components to the human diet, and these are particularly enriched in whole-grain products. However, wheat products are also known or suggested to be responsible for a number of adverse reactions in humans, including intolerances (notably coeliac disease) and allergies (respiratory and food). Current and future concerns include sustaining wheat production and quality with reduced inputs of agrochemicals and developing lines with enhanced quality for specific end-uses, notably for biofuels and human nutrition.
Article
We developed a mouse model of allergy to wheat flour gliadins, a protein fraction containing major wheat allergens. We compared the antibody responses (i.e., specific IgE and IgG1) and the profiles of cytokines secreted by reactivated splenocytes induced after intraperitoneal injections of gliadins in three strains of mice, namely, Balb/cJ, B10.A, and C3H/HeJ. The intensities of the allergic reactions elicited by intranasal challenge were also compared. Both the sensitization and elicitation were the highest in Balb/cJ mice, whereas weak or no reaction was observed in the others strains. Interestingly, the specificity of the mouse IgE against the different gliadins (i.e., alpha-, beta-, gamma-, omega 1,2-, and omega 5-gliadin) was similar to that observed in children allergic to wheat flour. Balb/cJ mice may thus provide a relevant model for the study of sensitization and elicitation by wheat gliadins and for improving our understanding of the specific role and mechanisms of action of the different classes of gliadins.
Article
The actual dilemma in studying the binding and triggering capacity of IgE from allergic patients is the lack of cultured basophils or mast cell analogs of human origin. Human IgE binds with exquisite species specificity to the high affinity IgE receptor (Fc epsilon RI) expressed on the surface of these cells. In rodents this receptor has been characterized as a tetrameric plasma membrane protein composed of an IgE-binding alpha chain, a beta chain and two disulfide-linked gamma chains. In order to establish a cell line expressing the alpha chain of human Fc epsilon RI which can be triggered with IgE from human patients and specific allergen, we transfected the cDNA coding for the human alpha subunit into rat basophilic leukemia cells. The resulting transfectants express the human alpha chain on the cell surface in the form of a hybrid complex associated with endogenous rat gamma chains. After sensitization with human IgE from mite-specific patients, the transfectant produces a calcium response upon incubation with allergen. The established cell line can be used as a model system to study the mechanism of mast cell triggering through IgE from allergic patients.
Article
Cow's milk allergy (CMA) is one of the leading causes of food allergy in children. Understanding the mechanisms involved in the development of CMA has been hampered by the lack of suitable animal models. We sought to develop a mouse model of IgE-mediated cow's milk hypersensitivity (CMH) that mimics the clinical features of immediate CMA in humans. Three-week-old C3H/HeJ mice were sensitized by intragastric administration of cow's milk (CM) plus cholera toxin and boosted 5 times at weekly intervals. CM-specific IgE antibody levels were significantly increased at 3 weeks and peaked at 6 weeks after the initial feeding. Intragastric challenge with CM at week 6 elicited systemic anaphylaxis accompanied by vascular leakage, significantly increased plasma histamine, and increased intestinal permeability to casein. Histologic examination of intestinal tissue revealed marked vascular congestion, edema, and sloughing of enterocytes. The role of IgE in mediating CMH was confirmed by abrogation of passive cutaneous anaphylaxis reactions by heat inactivation of immune sera. Development of IgE-mediated CMH in this model is likely to be TH2 cell mediated because in vitro stimulation of spleen cells from mice allergic to CM induced significant increases in the levels of IL-4 and IL-5, but not IFN-gamma. This model should provide a useful tool for evaluating the immunopathogenic mechanisms involved in CMA and for exploring new therapeutic approaches.
Article
Solubility, foaming capacity/stability, water holding and fat absorption capacities, and emulsifying capacity/stability of a solubilized wheat protein isolate (SWPI) were compared with those of commercial protein, that is, sodium caseinate (NaCAS), dried egg white (DEW), nonfat dry milk (NFDM), and soy protein isolate (SPI). SWPI was highly soluble at pH 6.5-8.5. Foaming capacity of SWPI was superior to those of SPI, NFDM, and DEW, and its foaming stability was similar to those of the commercial proteins. Foaming properties of SWPI were greatly improved in the presence of 0.5% (w/v) CaCl(2). Water holding capacity of SWPI was greater than that of NaCAS, NFDM, and DEW, whereas its fat absorption capacity was comparable to that of SPI, NaCAS, and DEW. SWPI exhibited emulsifying properties similar to those of SPI. SWPI was incorporated at 5, 10, 15, or 20% into ice cream, chocolate chip cookies, banana nut muffins, and hamburger patties. Products containing <5% SWPI were acceptable to consumers.
Article
An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.
Article
Immediate contact allergy to cosmetics seems to be rare, since only a few case reports on it have been published. We report on a case of IgE-mediated allergic contact urticaria caused by hydrolyzed wheat in a body cream.
Article
Wheat-dependent exercise-induced anaphylaxis is an anaphylaxy induced by physical exercise after ingestion of wheat. An immediate-type hypersensitivity to water/salt-insoluble fraction of wheat proteins (gluten) has been considered to underlie in this disease. The aim of the study is to determine the major allergen in Japanese patients with wheat-dependent exercise-induced anaphylaxis by using a panel of purified wheat gliadins and glutenins. Water/salt-insoluble wheat proteins, alpha-gliadin, beta-gliadin, gamma-gliadin, fast omega-gliadin, slow omega-gliadin, high molecular weight glutenin and low molecular weight glutenin, were purified, and five patients with wheat-dependent exercise-induced anaphylaxis, whose diagnose had been determined by positive-challenge test, were evaluated for skin prick test, dot-blotting test and CAP-RAST inhibition test by using these purified wheat proteins. The fast omega-gliadin was the most potent allergen among these water/salt-insoluble proteins when evaluated by skin prick test and dot-blotting test. Fast and slow omega-gliadin, and gamma-gliadin caused dose-dependent inhibition of the serum IgE-binding to solid-phase gluten in the patients. The incubation with fast omega-gliadin of the patient's serum caused dose-dependent inhibition in the IgE-binding to gamma-gliadin as well as slow omega-gliadin, indicating a cross-reactivity of these proteins in IgE-binding. We concluded that fast omega-gliadin is a major allergen among these water/salt-insoluble proteins for wheat-dependent exercise-induced anaphylaxis in Japanese patients, and IgE against fast omega-gliadin cross-reacts to gamma-gliadin and slow omega-gliadin.
Article
Food allergy to wheat induces different symptoms as atopic eczema/dermatitis syndrome (AEDS), urticaria and more severe reactions as wheat-dependent exercise-induced anaphylaxis (WDEIA). Different gliadin classes are involved in this allergy but IgE-binding epitopes are known only on omega5-gliadins and for WDEIA cases. The aim of the study was to identify IgE-binding epitopes on several gliadin classes and for several patients with different symptoms and ages. Eleven sera were analysed by pepscan with overlapping synthetic peptides. Sera from five patients with anaphylaxis, urticaria or WDEIA, displayed strong IgE-binding to sequential epitopes of the repetitive domains of alphabeta, gamma, omega2 or omega5-gliadins with two immunodominant epitopes on omega5-gliadin and a consensus motif of the type QQX1PX2QQ (X1 being L, F, S or I and X2 Q, E or G). One patient allergic to deamidated wheat proteins also had IgE to a repetitive peptide of gamma and omega2-gliadins of the type QPQQPFP. Sera from four patients with AEDS detected no linear epitopes on gliadins, despite the fact that they contained specific IgE to alpha, beta, gamma or omega-gliadins. One child with AEDS recognized cysteine-containing sequences in the nonrepetitive domains of alphabeta and gamma-gliadins. B epitopes in wheat allergy were different from B epitopes of coeliac disease. Differences exist in IgE-binding epitopes between patients with food allergy to wheat. IgE from those suffering from WDEIA, anaphylaxis and urticaria detected sequential epitopes in the repetitive domain of gliadins whereas IgE from AEDS patients probably recognized conformational epitopes.
Article
Various foods may be associated with food-dependent exercise-induced anaphylaxis (FDEIAn). However, although the most frequently reported cause of FDEIAn has been wheat, the mechanism of FDEIAn for wheat has remained largely uninvestigated. To investigate the effect of wheat-fractionated proteins on FDEIAn, female B10.A mice (16-20 g) were divided into four groups; i.e. salt-soluble (S-group), gliadin-rich (GLI-group), and glutenin-rich (GLU-group)-sensitized mice, and unsensitized mice. The three sensitized groups were run on a treadmill after oral intake of each wheat-fractionated protein. The mice showed a significant increase in serum IgE, especially in the GLI- and GLU-group. After oral administration of each wheat-fractionated protein, the running time until exhaustion was remarkably shorter for the GLI- and GLU-group than for the S-group and unsensitized mice. The level of intestinal erosion was higher in all the sensitized mice than that in the unsensitized ones after exhaustive running. Furthermore, moderate exercise for 30 min after oral ingestion of each wheat-fractionated protein also induced intestinal erosion in the GLI- and GLU-group. In addition, we observed leaking of gliadin and glutenin proteins out of the intestine into the liver. These results indicated that the main factor involved in wheat-dependent exercise-induced anaphylaxis might be the gliadin and glutenin in wheat proteins.
Article
Cosmetics containing hydrolysed wheat proteins (HWP) can induce rare but severe allergic reactions. 9 patients, all females without common wheat allergy, but with contact urticaria to such cosmetics, were studied. 6 of them also experienced generalized urticaria or anaphylaxis to foods containing HWP. All patients had low to moderate levels of immunoglobulin (Ig)E specific of wheat flour (f4) or gluten (f79). Their sensitivity to HWP and their tolerance to unmodified wheat proteins extracted from grains were confirmed using skin tests. Immunoblotting analyses showed that IgE from all patients reacted with almost all HWP tested. Reactions generally occurred with large random peptide aggregates. IgE reacted also with unmodified grain proteins, which contrasted with skin tests results. They reacted always with salt soluble proteins but variably with gluten proteins. No reaction occurred with gliadins in patients without associated immediate hypersensitivity to food containing HWP. These results show the role of hydrolysis on the allergenicity of wheat proteins, both through skin or digestive routes. At least part of the epitopes involved is pre-existing in unmodified wheat proteins. The aggregation of peptide bearing these epitopes and others created by hydrolysis, along with the increased solubility and the route of exposure, are possible factors of the allergenicity of HWP.
Article
Wheat gliadin was deamidated by using a cation-exchange resin in the presence or absence of added cysteine, with the change in digestibility being measured. The allergenicity of the gliadin was evaluated by using sera from patients RAST-positive to wheat. Gliadin-specific IgE was measured after the gliadin had been orally administered to rats. The addition of cysteine before the treatment with a cation exchanger effectively increased the deamidation level of gliadin. Deamidated gliadin showed higher solubility than the undeamidated form. There was no difference in the peptic digestibility of the gliadin, whereas deamidation enhanced the pancreatic digestibility in vitro and the digestibility in the mouse stomach in vivo. Deamidation of gliadin reduced its reactivity toward the sera of patients with wheat allergy. Rats administered with deamidated gliadin showed suppressed elevation of the gliadin-specific IgE level.
Article
Antigenic profiles obtained by ELISA with IgE from patients with wheat food allergy (WFA) established that major allergens are albumins/globulins (AG) for children suffering from atopic eczema/dermatitis syndrome (AEDS), omega5-gliadins for adults suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA), anaphylaxis or urticaria and low-molecular-weight (LMW) glutenin subunits for patients with anaphylaxis. We aimed to characterize a new mast cell transfectant for its ability to degranulate with wheat proteins and patient sera and compare these results to those obtained by ELISA. Thirty sera from patients with WFA were tested: 14 with AEDS (group 1) and 16 with WDEIA, anaphylaxis or urticaria (group 2). An IgE Fc receptor (FcepsilonRI) humanized rat RBL-2H3 line was established by transfection with cDNAs encoding alpha-, beta- and gamma-subunits for the human IgE receptor. A humanized RBL clone was selected for its capacity to express mRNA alpha-, beta- and gamma-subunits of FcepsilonRI, to bind allergen-specific human IgE and to degranulate. In group 1, sera induced enhanced degranulation with AG extract, but rarely reacted with gliadins and glutenins. In group 2, half of the sera showed degranulation with LMW glutenins whereas the AG fraction and lipid transfer proteins were rarely positive. omega5-Gliadins did not appear as a major allergen in degranulation assays, although functional allergen-specific IgE was measurable in appreciable amounts. Our data demonstrate that in wheat food allergen evaluation, correlation exists between mast cell degranulation and IgE measurements, depending on the type of allergen. Therefore, the biological activity of some allergen types may also be affected by other parameters.
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