The use of microbial transglutaminase in a bread system: A study of gluten protein structure, deamidation state and protein digestion.

Microbial transglutaminase (mTG) catalyses the formation of protein crosslinks, deamidating glutamine in a side-reaction. Gluten deamidation by human tissue transglutaminase is critical to activate celiac disease pathogenesis making the addition of mTG to wheat-based products controversial. The ability of mTG (0-2000 U.kg-1) to alter gluten's structure, digestibility and the deamidation state of six immunogenic gluten peptides within bread was investigated. Gluten's structure was altered when mTG exceeded 100 U.kg-1, determined by confocal microscopy, extractability and free sulfhydryl assays. The effect of mTG on six immunogenic peptides was investigated by in vitro digestion (INFOGEST) and mass spectrometry. The addition of mTG to bread (0-2000 U.kg-1) did not alter the deamidation state or digestibility of the immunogenic peptides investigated. Overall, this investigation indicated that the addition of mTG to bread does not create activated gluten peptides. This analysis provides evidence for risk assessments of mTG as a food processing aid.

Exposure sources, amounts and time course of gluten ingestion and excretion in patients with coeliac disease on a gluten-free diet.

BACKGROUND: A major deficit in understanding and improving treatment in coeliac disease (CD) is the lack of empiric data on real world gluten exposure. AIMS: To estimate gluten exposure on a gluten-free diet (GFD) using immunoassays for gluten immunogenic peptides (GIP) and to examine relationships among GIP detection, symptoms and suspected gluten exposures METHODS: Adults with biopsy-confirmed CD on a GFD for 24 months were recruited from a population-based inception cohort. Participants kept a diary and collected urine samples for 10 days and stools on days 4-10. 'Doggie bags' containing » portions of foods consumed were saved during the first 7 days. Gluten in food, stool and urine was quantified using A1/G12 ELISA. RESULTS: Eighteen participants with CD (12 female; age 21-70 years) and three participants on a gluten-containing diet enrolled and completed the study. Twelve out of 18 CD participants had a median 2.1 mg gluten per exposure (range 0.2 to >80 mg). Most exposures were asymptomatic and unsuspected. There was high intra-individual variability in the interval between gluten ingestion and excretion. Participants were generally unable to identify the food. CONCLUSIONS: Gluten exposure on a GFD is common, intermittent, and usually silent. Excretion kinetics are highly variable among individuals. The amount of gluten varied widely, but was typically in the milligram range, which was 10-100 times less than consumed by those on an unrestricted diet. These findings suggest that a strict GFD is difficult to attain, and specific exposures are difficult to detect due to variable time course of excretion.

Randomised clinical trial: a placebo-controlled study of subcutaneous or intradermal NEXVAX2, an investigational immunomodulatory peptide therapy for coeliac disease.

BACKGROUND: Nexvax2 contains three gluten-derived peptides, intended to tolerize coeliac disease patients to gluten. Sequences cover six epitopes that trigger immune activation in human leucocyte antigen-DQ2.5-positive patients, most notably after an initial dose. Patients experience gastrointestinal symptoms with increases in serum interleukin-2. Consistent with Nexvax2's induction of non-responsiveness, reactivity disappears after repeated doses, or is avoided with gradual dose escalation. Early clinical trials used intradermal dosing, but pharmacokinetics and rapid onset of effect suggest that subcutaneous delivery may also be effective. AIMS: To document the relative bioavailability of Nevax2 peptides after subcutaneous and intradermal dosing, and the tolerability and ability of subcutaneous dosing to induce non-responsiveness to Nexvax2 peptides. METHODS: A randomised, double-blind, placebo-controlled study was conducted to assess plasma pharmacokinetics after subcutaneous and intradermal Nexvax2 dosing in HLA DQ2.5-positive patients, who had symptoms after an oral gluten challenge. Randomisation was to semi-weekly Nexvax2 (n = 12) or placebo (n = 2) injections, over a 5-week subcutaneous dose escalation and 2-week maintenance period, the latter with four doses of 900 µg, two subcutaneous and two intradermal. Post-dose circulating peptide and interleukin-2 levels were assessed. Investigators recorded adverse events experienced by patients. RESULTS: Subcutaneous dosing resulted in slightly greater exposure. Interleukin-2 responses were seen with the gluten challenge but not after subcutaneous or intradermal dosing of 900 µg. Adverse events were generally mild and self-limited. CONCLUSIONS: Subcutaneous and intradermal dosing of Nexvax2 yield similar bioavailability of constituent peptides; subcutaneous dose escalation avoids an immune response to dominant gluten epitopes.

Changes in digestibility of proteins from chickpeas (Cicer arietinum L.) germinated in presence of selenium and antioxidant capacity of hydrolysates.

Germination in the presence of selenium (Se) is an alternative to increase the healthy properties of seeds. This study aimed to compare the Se accumulation in different protein fractions from germinated chickpea (Cicer arietinum L.) and the effect on digestibility and cellular antioxidant activity (CAA) of protein hydrolysates. Chickpeas were germinated during four days after soaking with sodium selenite (0, 1, or 2 mg/100 g seeds). Total protein (TP) and glutelin (Glu), albumin (Alb) and globulin (Glo) fractions were digested and ultrafiltrated through a 10 kDa membrane. Se accumulated in the order of Glu > Alb > Glo. Ultrafiltrated Glu hydrolysate of four days germinated chickpeas treated with 2 mg Na2SeO3/100 g increased CAA (51.47%), demonstrating the potential health benefits of selenization. The intensity of vicilin bands (34-37 kDa) increased from the second to the fourth day compared with the control samples. Glo digestibility was higher in selenized chickpea sprouts.

In Situ Gluten-Chitosan Interlocked Self-Assembled Supramolecular Architecture Reduces T-Cell-Mediated Immune Response to Gluten in Celiac Disease.

SCOPE: The prevalence of celiac disease has increased since the last half of the 20th century and is now about 1% in most western populations. At present, people who suffer from celiac disease have to follow a gluten-exclusion diet throughout their lives. Compliance to this restrictive diet is demanding and the development of alternative strategies has become urgent. METHODS AND RESULTS: In this context, it is found that the biocompatible aminopolysaccharide chitosan imposes a different gluten reorganization after gluten redox reaction producing in situ mechanically interlocked supramolecular assemblies between gluten and chitosan. These new structures result in the decrease of gluten digestibility, tissue transglutaminase deamidation activity, and interferon-γ production in intestinal T cell lines generated from biopsy specimens of celiac disease patients. CONCLUSION: Overall, the results demonstrate the potential of this research avenue to celiac disease is problematic, as the reorganization of gluten proteins to a novel supramolecular architecture shows a positive impact on known pathogenesis mechanisms of the disease. At present, the only therapy for celiac disease is adherence to a gluten-free diet. Here, it is shown that chitosan-imposed gluten reorganization to an interlocked self-assembled supramolecular architecture reduces gluten digestibility, R5-reactivity, tissue transglutaminase deamidation activity, and its capacity to stimulate a T-cell-mediated immune response in celiac disease.

Gluten peptides drive healthy and celiac monocytes toward an M2-like polarization.

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPß transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD.

Digestibility of gluten proteins is reduced by baking and enhanced by starch digestion.

SCOPE: Resistance of proteins to gastrointestinal digestion may play a role in determining immune-mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood. METHODS AND RESULTS: Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS-PAGE and immunoblotting using monoclonal antibodies specific for celiac-toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases. CONCLUSION: The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten-starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices.

Diseño de un genosensor electroquímico para la detección indirecta de gluten en alimentos / Electrochemical genosensor for indirect wheat gluten detection in food

Se propone un nuevo genosensor electroquímico para la detección de una secuencia específica de ADN que codifica un fragmento inmunogénico de la Alfa-2-gliadina, proteína del gluten de trigo responsable de la celiaquía. El diseño del genosensor se basa en la formación de una monocapa autoensamblada de sonda de captura y un agente bloqueante, mercaptohexanol, sobre electrodos de oro serigrafiados. Se eligió un ensayo tipo sándwich, utilizando una sonda indicadora marcada con biotina y el conjugado estreptavidina-fosfatasa alcalina como molécula de marcaje. La detección del analito se basó en la medida de la corriente de oxidación del 1-naftol, producto formado por la hidrólisis enzimática del 1-naftil-fosfato, mediante voltamperometría de pulso diferencial. Se investigaron y optimizaron los parámetros implicados en la composición de la fase sensora mediante voltametría cíclica, encontrándose como relación óptima sonda de captura: mercaptohexanol 2 microM:9 mM. Con el objetivo de minimizar las adsorciones inespecíficas, se optimizaron las concentraciones de sonda indicadora y conjugado enzima-estreptavidina, especies involucradas en la fase de medida, obteniéndose como valores óptimos 1 microM y 1,075x10-3 g/L respectivamente. El genosensor propuesto presentó una respuesta lineal entre 20 y 250 nM(AU)

A new electrochemical genosensor has been developed for the detection of a specific DNA sequence that encodes for an immunogenic fragment of Alpha-2-gliadin, protein of gluten wheat that plays an important role in celiac disease. The genosensor is based on a mixed self-assembled monolayer consisting on a capture probe and a diluent molecule, mercaptohexanol, both immobilized on screen-printed gold electrodes. A sandwich-type hybridization assay was selected, using a signaling-DNA probe labeled with biotin and streptavidin-alkaline phosphatase as a reporter molecule. Detection of DNA gluten is based on the measurement of the oxidization current of 1-naphthol, product formed by Alpha-naphthyl phosphate enzymatic hydrolysis, by differential pulse voltammetry. Parameters involved in the sensing phase were investigated and optimized by cyclic voltammetry. The optimal capture probe to mercaptohexanol ratio was found to be 2 micreM:9 mM. In order to minimize unspecific adsorptions, both signaling probe and enzyme-streptavidin conjugate concentrations (measurement phase parameters) were optimized (1 micreM and 1.075·10-3 g/L respectively). A linear response from 20 nM to 250 nM is obtained with the proposed genosensor(AU)

Novel therapies for coeliac disease.

Coeliac disease is a widespread, lifelong disorder for which dietary control represents the only accepted form of therapy. There is an unmet need for nondietary therapies to treat this condition. Most ongoing and emerging drug-discovery programmes are based on the understanding that coeliac disease is caused by an inappropriate T-cell-mediated immune response to dietary gluten proteins. Recent genome-wide association studies lend further support to this pathogenic model. The central role of human leucocyte antigen genes has been validated, and a number of new risk loci have been identified, most of which are related to the biology of T cells and antigen-presenting cells. Here, we review the status of potential nondietary therapies under consideration for coeliac disease. We conclude that future development of novel therapies will be aided considerably by the identification of new, preferably noninvasive, surrogate markers for coeliac disease activity.

Interferon-gamma released by gluten-stimulated celiac disease-specific intestinal T cells enhances the transepithelial flux of gluten peptides.

Celiac sprue is a T-cell-mediated enteropathy elicited in genetically susceptible individuals by dietary gluten proteins. To initiate and propagate inflammation, proteolytically resistant gluten peptides must be translocated across the small intestinal epithelium and presented to DQ2-restricted T cells, but the effectors enabling this translocation under normal and inflammatory conditions are not well understood. We demonstrate that a fluorescently labeled antigenic 33-mer gluten peptide is translocated intact across a T84 cultured epithelial cell monolayer and that preincubation of the monolayer with media from gluten-stimulated, celiac patient-derived intestinal T cells enhances the apical-to-basolateral flux of this peptide in a dose-dependent, saturable manner. The permeability-enhancing activity of activated T-cell media is inhibited by blocking antibodies against either interferon-gamma or its receptor and is recapitulated using recombinant interferon-gamma. At saturating levels of interferon-gamma, activated T-cell media does not further increase transepithelial peptide flux, indicating the primacy of interferon-gamma as an effector of increased epithelial permeability during inflammation. Reducing the assay temperature to 4 degrees C reverses the effect of interferon-gamma but does not reduce basal peptide flux occurring in the absence of interferon-gamma, suggesting active transcellular transport of intact peptides is increased during inflammation. A panel of disease-relevant gluten peptides exhibited an inverse correlation between size and transepithelial flux but no apparent sequence constraints. Anti-interferon-gamma therapy may mitigate the vicious cycle of gluten-induced interferon-gamma secretion and interferon-gamma-mediated enhancement of gluten peptide flux but is unlikely to prevent translocation of gluten peptides in the absence of inflammatory conditions.

Intestinal translocation capabilities of wheat allergens using the Caco-2 cell line.

Because intestinal absorption of food protein can trigger an allergic reaction, the effect of wheat proteins on intestinal epithelial cell permeability was evaluated and the abilities of these proteins in native or pepsin-hydrolyzed state to cross the epithelial cell monolayer were compared. Enterocytic monolayers were established by culturing Caco-2 cells, a model of enterocytes, on permeable supports that separate the apical and basal compartments. Proteins were added into the apical compartment, and the transepithelial resistance (TER) was measured; proteins that crossed the cell monolayer were detected in the basal medium by ELISA. Wheat proteins did not alter the cell monolayer. TER and Caco-2 cell viability were conserved, and the passage of dextran was prevented. Native and pepsin-hydrolyzed forms of omega5-gliadin and lipid transfer proteins were detected in the basal medium. The results suggest that these two major allergens in food allergy to wheat were able to cross the cell monolayer by the transcellular route.

Kinetics of mucosal granulocyte activation after gluten challenge in coeliac disease.

OBJECTIVE: To elucidate the dynamics of the rectal inflammatory response to rectal gluten challenge in coeliac disease by measuring inflammatory mediators released by activated neutrophils, eosinophils and mast cells/basophils. MATERIAL AND METHODS: The release of myeloperoxidase (MPO), eosinophilic cationic protein (ECP) and histamine was measured continuously during the early challenge period (3-6 h after gluten challenge) in coeliac patients (n = 9) and healthy controls (n = 5). A segmental perfusion technique was used to carry out this part of the study. Another method, the mucosal patch technique, was used to enable studies of the late challenge period (5-48 h after gluten challenge) in coeliac patients (n = 10) and healthy controls (n = 15). RESULTS: During the early challenge period the MPO levels began to increase as early as 3 h after challenge and increased progressively (p < 0.001) during the next 3 h. A decline in MPO levels was seen 15 h after challenge and another phase of increasing levels at 24 h. The MPO values declined 48 h after challenge but still remained significantly increased (p < 0.05). The ECP levels started to increase 4 h after challenge and increased progressively during the next 2 h (p < 0.05). The ECP kinetics during the late challenge period was similar as for MPO but the relative increase in ECP was more modest. No increase in histamine was found except in one patient who had a transient, early increase of histamine (3-5 h after challenge). No signs of inflammatory reaction to gluten were seen in the controls. CONCLUSIONS: There is a pronounced neutrophil activation in coeliac patients after rectal gluten challenge. This activation is apparent 4 h after challenge and remains for at least 48 h. A more modest eosinophil activation defined by ECP levels starts 1-2 h later and also remains for at least 48 h. The biphasic pattern of MPO and ECP after challenge suggests a biphasic inflammatory reaction.

Transglutaminases: a meeting point for wheat allergy, celiac disease, and food safety.

Wheat is the staple cereal in many countries and its uses in manufactured foods are ever growing due to the technological qualities of gluten proteins. Transglutaminases (TG) are ubiquitous enzymes with many functions. They are able to transform proteins by deamidation and/or transamidation. This last reaction can cross-link proteins together. Intestinal tissue TG has been shown to play an important role in two kinds of immune reactions to wheat: celiac disease and wheat-dependent exercise-induced anaphylaxis. In addition, new epitopes have been suspected in cases of anaphylaxis to wheat isolates, a food ingredient consisting mainly of deamidated gluten proteins. As a microbial TG is included in many food technological processes, its safe use should be checked. This assessment must cover not only the safety of the TG itself but also that of the deamidated/cross-linked proteins generated by this enzyme. This article aims at discussing the possible consequences of using TG in food industry in the light of today knowledge about immune reactions to wheat.

Coeliac disease.

In the last few decades, the comprehension of epidemiological, pathogenic and clinical aspects of coeliac disease has increasingly improved. Serological screening studies on the general population have shown that the true coeliac disease prevalence in Europe is higher than previously reported. It has become clear that tissue transglutaminase has a crucial role in the pathogenesis of coeliac disease pathogenesis, and there is evidence that substitution of deamidated amino acidic residues at a critical position along the gliadin sequence dramatically increases immunological activation. The toxicity of many gluten epitopes has been investigated, so far, but recent studies have indicated the region 57-75 of alpha gliadin as a possible candidate epitope in the pathogenesis of coeliac disease. However, the wide heterogeneity of gliadin and glutenin molecules complicate any attempts to identify the toxic epitope, and the fascinating idea to produce detoxified grains will represent a great challenge in the near future. From a clinical point of view, there is now evidence of a broad spectrum of gluten conditions. Extra-intestinal signs, i.e., alopecia, unexplained neurological disorders, cryptic hypertransaminasaemia, increased red cell width, frequently constitute the only clinical manifestation at the diagnosis.

Intestinal permeability of Irish setter puppies challenged with a controlled oral dose of gluten.

A combined test of intestinal permeability using lactulose (L) and rhamnose (R), and absorptive function using xylose (X) and 3-O-methylglucose (G), was carried out at four, six, eight and 16 weeks of age in 22 healthy control and six gluten-sensitive Irish setter (IS) dogs fed a diet containing a controlled dose of gluten from weaning. Comparisons were made with two groups of 12 healthy control dogs of breeds other than IS, one fed the same diet as the setters and the other fed a gluten-free diet. Gluten-sensitive IS showed a rise in permeability (mean [SEM] urinary L/R) from 0.23 (0.07) at four weeks to 0.39 (0.05) at eight weeks, remaining at 0.36 (0.04) at 16 weeks. These results were significantly higher in gluten-sensitive than control IS at six, eight and 16 weeks, compatible with jejunal biopsy lesions characteristic of gluten-sensitive enteropathy demonstrated in affected dogs at 16 weeks. Urinary L/R ratios of control dogs of breeds other than IS peaked at six weeks 0.27 (0.02), and were significantly higher than those of control IS at six and eight weeks, demonstrating differences in permeability between Irish setter dogs and other breeds at this age. There were no significant differences in urinary X/G ratios at six, eight and 16 weeks of age between any of the groups of dogs challenged with gluten. Urinary L/R and X/G ratios were similar in the control dogs of breeds other than IS fed gluten-containing and gluten-free diets. These findings indicate that intestinal permeability testing of puppies during controlled oral gluten challenge provides a practical screening test for gluten sensitivity in Irish setter dogs at an early age.

Lymphocytic enterocolitis in patients with 'refractory sprue'.

We describe a patient with refractory sprue with malabsorption, a flat small-bowel biopsy specimen unresponsive to a gluten-free diet, and colonic biopsy specimens consistent with lymphocytic (microscopic) colitis. To investigate further the relation between celiac disease and lymphocytic or collagenous colitis (a similar and possibly related entity), we examined colorectal and small-bowel biopsy specimens in patients indexed histologically as having celiac disease who have been seen at The Johns Hopkins Hospital since 1958. Of 135 indexed patients, 21 had colorectal biopsies. Colorectal biopsy specimens were abnormal in 7 of the 21 patients. Four patients had biopsy specimens resembling lymphocytic colitis, 2 patients had acute colitis, and another patient had both lymphocytic and acute colitis. No patients had collagenous colitis. Three of the patients with lymphocytic colitis and celiac-like changes of the small bowel never responded to a gluten-free diet and may represent a distinctive panintestinal disease for which the term "lymphocytic enterocolitis" with malabsorption is proposed.