Thermal treatment reduces gliadin recognition by IgE, but a subsequent digestion and epithelial crossing permits recovery.

Wheat is one of the most important crops in the world in terms of human nutrition. With regards to health, some individuals exhibit wheat-related disorders such as food allergy to wheat (FAW). In this disorder, gluten is involved, particularly the gliadins which are among the main proteins responsible for FAW. Food processing, as well as digestibility and intestinal transport are key factors to consider since they may affect the allergenic potential of food allergens. Wheat is always consumed after heat processing and this step may impact epitope accessibility by inducing aggregation and may irreversibly destroy conformational epitopes. Our aim was to investigate the effects of heating and digestion on the structure of well-known allergens (total gliadins and α-gliadins) and their capacity to maintain their allergenic potential after crossing an intestinal barrier. The sizes of the processed (heated and heated/digested) proteins were characterized by laser light scattering and chromatographic reverse phase. The IgE-binding capacities of native and processed proteins were checked using a dot blot with sera from wheat allergenic patients. Furthermore, the abilities of these samples to cross the intestinal barrier and to induce mast cell degranulation were investigated by combining two in vitro cellular models, Caco-2 and RBL-SX38. The heat treatment of total gliadins and α-gliadins induced the production of large aggregates that were hardly recognized by IgE of patients in dot-blot. However, after limited pepsin hydrolysis, the epitopes were unmasked, and they were able to bind IgE again. Native proteins (gliadins and α-type) and processed forms were able to cross the Caco-2 cells in small amount. Permeability studies revealed the capacity of α-gliadins to increase paracellular permeability. In the RBL assay, the total native gliadins were able to trigger cell degranulation, but none of their processed forms. However after crossing the CaCo-2 monolayer, processed gliadins recovered their degranulation capacity to a certain extent. Total native gliadins remained the best allergenic form compared to α-type.

The thermal aggregation of ovalbumin as large particles decreases its allergenicity for egg allergic patients and in a murine model.

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.

How much does transgenesis affect wheat allergenicity?: Assessment in two GM lines over-expressing endogenous genes.

Wheat kernel albumins/globulins (A/G) and gluten proteins are responsible for baker's asthma and food allergy in atopic subjects. Although no commercial genetically modified wheats are currently being grown, they are under study and the allergenicity of GM products is a major concern. In order to establish the expected and unexpected effects of genetic transformation on allergenicity and also to carry out a safety assessment of genetic transformation, two GM wheat lines (bread and pasta wheat) transformed with endogenous genes were compared to their untransformed counterparts (wt), first by an allergenomic approach, and second, using ELISA with sera from patients suffering from food allergy to wheat and baker's asthma. The 2D immunoblots performed on sera from patients suffering from food allergy and baker's asthma on the A/G fraction of the four lines (two GM and two wt) revealed comparable IgE-binding profiles. A total of 109 IgE-binding spots were analyzed by mass spectrometry, and most of the proteins identified had already been described as allergens or potential allergens. Only two IgE-binding proteins were specific to one GM line. The concentration of specific IgE against the A/G fractions of GM wheat lines and their wt genotypes differed for some sera. BIOLOGICAL SIGNIFICANCE: The originality of our paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera, such focus has never been done before in wheat and should be of interest to the researches working in this field. Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy. In this paper we used a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis. Concerning the LC-MS/MS analyses classical software and parameters were used as described in Material and methods. We worked on wheat which is actually not fully sequenced that was a difficulty; we therefore searched against two databanks (proteins and ESTs) in order to compare the results. Moreover all proteins reported in our paper were identified with at least three unique peptides. The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP). This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity.

Allergy to deamidated gluten in patients tolerant to wheat: specific epitopes linked to deamidation.

BACKGROUND: Gluten proteins can be modified by deamidation to enhance their solubility and technological applications. However, severe allergic reactions have been reported after the consumption of food products containing deamidated gluten (DG) in subjects tolerant to wheat. This work aimed to characterize allergen profiles for these patients in comparison with those of patients allergic to wheat and to identify IgE-binding epitopes. METHODS: Sera were obtained from 15 patients allergic to DG and from nine patients allergic to wheat proteins (WP). IgE-binding profiles were characterized both in ELISA and in a humanized rat basophilic leukaemia (RBL) cell model. Epitopes were mapped on γ- and ω2-gliadin sequences by Pepscan, and effect of glutamine/glutamic acid substitutions was studied. RESULTS: Compared to the heterogeneous pattern of allergens detected by IgE from patients allergic to WP, responses of patients allergic to DG were homogeneous. In ELISA, all the sera displayed IgE binding to deamidated γ- and ω2-gliadins and deamidated total gliadins, frequently with high concentrations. These modified proteins induced RBL degranulation with most of the sera from DG-allergic patients. A consensus epitope was found on native γ- and ω2-gliadins (QPQQPFPQ); it was repeated several times in their sequences. The substitution of two or three glutamines of this epitope into glutamic acid at positions Q3 or Q4 and Q8 (QPEEPFPE) increased its recognition the best. CONCLUSION: Allergy to DG is a separate entity from wheat allergy. It can be evidenced by strong IgE binding to deamidated gliadins or peptides of the type QPEEPFPE.

Specific IgG levels to wheat in wheat tolerant professional cyclists may depend on a homeostatic immune response to a high consumption of wheat.

BACKGROUND: Implication of IgG antibodies to wheat has been alleged in gastrointestinal symptoms. Precise data on the specific IgG levels in healthy subjects are lacking. Our objectives are to compare levels of IgG antibodies to wheat protein fractions in healthy non atopic or atopic subjects, and in healthy professional cyclist subjects, taking into account the quantitative consumption of wheat. METHODS: 24 control subjects and 26 professional cyclist subjects were selected. ELISA was performed to 2 wheat commercial solutions and to 3 wheat protein fractions. RESULTS: No significant difference was observed between non atopic and atopic subjects. For wheat flour extract, physiological norm determined was 3.27 mg/L sIgG concentration +/- 1.25 CI (95% confidence intervals) for the professional cyclists (vs 1.56 mg/L +/- 0.91 CI in control subjects, p-value: 0.040). For gluten solution, physiological norm was 1.42 mg/L +/- 0.60 CI (vs 0.50 +/- 0.24 CI in control subjects, p-value: 0.010). CONCLUSION: Atopic and non atopic healthy adults have a similar level of sIgG to wheat. Increased levels of sIgG are observed correlatively with an excessive consumption, and could contribute to homeostasis of tolerance. Studies searching for a pathogenic role of sIgG in certain pathologies should take into account the quantitative consumption.

Immunoglobulin-E-binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins.

BACKGROUND: At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. OBJECTIVE: To characterize the epitopes of different wheat kernel allergens: α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. METHODS: Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. RESULTS: Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit. CONCLUSION AND CLINICAL RELEVANCE: The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations.

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

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

Morpho-functional modifications of human neutrophils induced by aqueous cigarette smoke extract: comparison with chemiluminescence activity.

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke-related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles.

Brachypodium distachyon grain: identification and subcellular localization of storage proteins.

Seed storage proteins are of great importance in nutrition and in industrial transformation because of their functional properties. Brachypodium distachyon has been proposed as a new model plant to study temperate cereals. The protein composition of Brachypodium grain was investigated by separating the proteins on the basis of their solubility combined with a proteomic approach. Salt-soluble proteins as well as salt-insoluble proteins separated by two-dimensional gel electrophoresis revealed 284 and 120 spots, respectively. Proteins from the major spots were sequenced by mass spectrometry and identified by searching against a Brachypodium putative protein database. Our analysis detected globulins and prolamins but no albumins. Globulins were represented mainly by the 11S type and their solubility properties corresponded to the glutelin found in rice. An in silico search for storage proteins returned more translated genes than expressed products identified by mass spectrometry, particularly in the case of prolamin type proteins, reflecting a strong expression of globulins at the expense of prolamins. Microscopic examination of endosperm cells revealed scarce small-size starch granules surrounded by protein bodies containing 11S globulins. The presence of protein bodies containing glutelins makes B. distachyon closer to rice or oat than to wheat endosperm.

Sensitization and elicitation of an allergic reaction to wheat gliadins in mice.

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.

Interest of ImmunoCAP system to recombinant omega-5 gliadin for the diagnosis of exercise-induced wheat allergy.

BACKGROUND: omega-5 gliadin is a major allergen in exercise-induced wheat allergy (EIWA), but it is also implicated in immediate-type reactions to wheat. An ImmunoCAP assay to measure omega-5 gliadin-specific IgE has become available. This study aimed to evaluate this new biological test in wheat allergy diagnosis and to also determine if it was able to discriminate EIWA from other types of wheat allergy. METHODS: Sixty-one patients with wheat allergy were divided into 3 groups as a function of their symptoms (EIWA, immediate-type reactions and atopic dermatitis). These patients underwent skin prick tests with purified omega gliadins and ImmunoCAP to wheat flour, gluten and recombinant omega-5 gliadin. RESULTS: The experimental data showed that 78% of EIWA patients had a positive skin prick test to natural omega-5 gliadin and the same proportion had detectable specific IgE to recombinant omega-5 gliadin, indicating that omega-5 gliadin is the main allergen, but not the only one, in our population. Additionally, we showed that this detection was not EIWA specific since omega-5 gliadin-specific IgE was detected in 30% of other patients who had a wheat allergy. These results lead to a positive predictive value of 37.5% and to a negative predictive value of 91%. CONCLUSIONS: Although not specific to EIWA, the new ImmunoCAP omega-5 gliadin is an important biological test because of its negative predictive value. In case of food-dependent exercise-induced allergy, the absence of omega-5 gliadin-specific IgE will almost completely exclude the implication of wheat.

Evaluation of an in vitro mast cell degranulation test in the context of food allergy to wheat.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Wheat grain allergies: an update on wheat allergens.

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.

Epithelial stem cells of the eye surface.

OBJECTIVES: Epithelial stem cells of the eye surface, of the cornea and of the conjunctiva, have the ability to give rise to self renewal and progeny production of differentiated cells with no apparent limit. The two epithelia are separated from each other by the transition zone of the limbus. The mechanisms adopted by stem cells of the two epithelia to accomplish their different characteristics, and how their survival, replacement and unequal division that generates differentiated progeny formation are controlled, are complex and still poorly understood. They can be learned only by understanding how stem cells/progenitors are regulated by their neighbouring cells, that may themselves be differently unspecialised, forming particular microenvironments, known as 'niches'. Stem cells operate by signals and a variety of intercellular interactions and extracellular substrates with adjacent cells in the niche. Technical advances are now making it possible to identify zones in the corneal limbus and conjunctiva that can house stem cells, to isolate and expand them ex vivo and to control their behaviour creating optimal niche conditions. With improvements in biotechnology, regenerative cornea and conjunctiva transplantation using adult epithelial stem cells becomes now a reality. RESULTS AND CONCLUSIONS: Here we review our current understanding of stem cell niches and illustrate recent significant progress for identification and characterization of adult epithelial stem cells/progenitors at cellular, molecular and mechanistic levels, improvement in cell culture techniques for their selective expansion ex vivo and prospects for a variety of therapeutic applications.