Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Otorhinolaryngological Manifestations and Esophageal Disorders in Celiac Disease: A Narrative Review.

Celiac disease (CeD) is a chronic gluten-sensitive immune-mediated enteropathy characterized by numerous intestinal and extra-intestinal signs and symptoms. Among extra-intestinal manifestations, otorhinolaryngological (ORL) complaints in CeD are relatively rare and their relation to CeD is frequently overlooked by physicians. Recent studies underlined that the prevalence of recurrent aphthous stomatitis, aphthous ulcers, geographic tongue, and xerostomia was significantly increased in CeD patients compared with healthy individuals. However, data about the other oral manifestations of CeD, such as atrophic glossitis, glossodynia, angular cheilitis, and salivary abnormalities, are scanty. Further ORL conditions associated with CeD include sensorineural hearing loss, nasal abnormalities, and obstructive sleep apnea. Moreover, several esophageal disorders such as gastroesophageal reflux disease and eosinophilic esophagitis have been associated with CeD. The pathophysiological link between both ORL and esophageal manifestations and CeD might be further investigated. In addition, also the role of gluten-free diet in improving these conditions is largely unclear. Certainly, otorhinolaryngologists can play an important role in identifying people with unrecognized CeD and may help prevent its long-term complications. The aim of this narrative review is to analyze the latest evidence on the association between CeD and ORL and esophageal manifestations.

Dental Manifestations and Celiac Disease-An Overview.

This review summarizes recent investigations on dental manifestations in celiac disease. Particular attention is paid to delayed dental eruption and maturity, dental enamel defects, molar incisor hypomineralization, dental caries, dental plaque, and periodontitis. Most studies confirmed a higher frequency of delayed dental eruption and maturation in children and dental enamel defects in children and adults with celiac disease compared to healthy individuals. The malabsorption of various micronutrients, especially calcium and vitamin D, as well as immunity, is considered the main cause of these conditions. An early diagnosis of celiac disease and introducing a gluten-free diet might prevent the development of these conditions. Otherwise, the damage has already been established, and it is irreversible. Dentists can play an important role in identifying people who may have unrecognized celiac disease and may help prevent its progress and long-term complications. Investigations on dental caries, plaque, and periodontitis in celiac disease are rare and inconsistent; these complaints need further examination.

Food Safety and Cross-Contamination of Gluten-Free Products: A Narrative Review.

A gluten-free diet (GFD) is currently the only effective treatment for celiac disease (CD); an individual's daily intake of gluten should not exceed 10 mg. However, it is difficult to maintain a strict oral diet for life and at least one-third of patients with CD are exposed to gluten, despite their best efforts at dietary modifications. It has been demonstrated that both natural and certified gluten-free foods can be heavily contaminated with gluten well above the commonly accepted threshold of 20 mg/kg. Moreover, meals from food services such as restaurants, workplaces, and schools remain a significant risk for inadvertent gluten exposure. Other possible sources of gluten are non-certified oat products, numerous composite foods, medications, and cosmetics that unexpectedly contain "hidden" vital gluten, a proteinaceous by-product of wheat starch production. A number of immunochemical assays are commercially available worldwide to detect gluten. Each method has specific features, such as format, sample extraction buffers, extraction time and temperature, characteristics of the antibodies, recognition epitope, and the reference material used for calibration. Due to these differences and a lack of official reference material, the results of gluten quantitation may deviate systematically. In conclusion, incorrect gluten quantitation, improper product labeling, and poor consumer awareness, which results in the inadvertent intake of relatively high amounts of gluten, can be factors that compromise the health of patients with CD.

Challenges of Monitoring the Gluten-Free Diet Adherence in the Management and Follow-Up of Patients with Celiac Disease.

Celiac disease (CD) is a chronic gluten-responsive immune mediated enteropathy and is treated with a gluten-free diet (GFD). However, a strict diet for life is not easy due to the ubiquitous nature of gluten. This review aims at examining available evidence on the degree of adherence to a GFD, the methods to assess it, and the barriers to its implementation. The methods for monitoring the adherence to a GFD are comprised of a dietary questionnaire, celiac serology, or clinical symptoms; however, none of these methods generate either a direct or an accurate measure of dietary adherence. A promising advancement is the development of tests that measure gluten immunogenic peptides in stools and urine. Causes of adherence/non-adherence to a GFD are numerous and multifactorial. Inadvertent dietary non-adherence is more frequent than intentional non-adherence. Cross-contamination of gluten-free products with gluten is a major cause of inadvertent non-adherence, while the limited availability, high costs, and poor quality of certified gluten-free products are responsible for intentionally breaking a GFD. Therefore, several studies in the last decade have indicated that many patients with CD who follow a GFD still have difficulty controlling their diet and, therefore, regularly consume enough gluten to trigger symptoms and damage the small intestine.

The Two Faces of Wheat.

Wheat-based foods have been staple foods since about 10,000 years and constitute a major source of energy, dietary fiber, and micronutrients for the world population. The role of wheat in our diet, however, has recently been scrutinized by pseudoscientific books and media reports promoting the overall impression that wheat consumption makes people sick, stupid, fat, and addicted. Consequently, numerous consumers in Western countries have started to question their dietary habits related to wheat consumption and voluntarily decided to adopt a wheat-free diet without a medical diagnosis of any wheat-related disorder (WRD), such as celiac disease, wheat allergy, or non-celiac gluten sensitivity. The aim of this review is to achieve an objective judgment of the positive aspects of wheat consumption as well as adverse effects for individuals suffering from WRDs. The first part presents wheat constituents and their positive nutritional value, in particular, the consumption of products from whole-grain flours. The second part is focused on WRDs that affect predisposed individuals and can be treated with a gluten-free or -reduced diet. Based on all available scientific knowledge, wheat consumption is safe and healthy for the vast majority of people. There is no scientific evidence to support that the general population would benefit from a wheat-free diet.

Preparation of a Defined Gluten Hydrolysate for Diagnosis and Clinical Investigations of Wheat Hypersensitivities.

Gluten is the trigger for celiac disease (CD), non-celiac gluten/wheat sensitivity (NCGS), and wheat allergy. An oral food challenge is often needed for diagnosis, but there are no standardized gluten challenge materials with known composition available. To fill this gap, two materials, commercially available gluten and a food-grade gluten hydrolysate (pepgluten), were extensively characterized. Pepgluten was prepared from gluten by incubation with a pepsin dietary supplement and acetic acid at 37 °C for 120 min. The components of pepgluten were crude protein (707 mg/g), starch (104 mg/g), water (59 mg/g), fat (47 mg/g), dietary fiber (41 mg/g) and ash (11 mg/g). The protein/peptide fraction of pepgluten (1 g) contained equivalents derived from 369 mg gliadins and 196 mg glutenins, resulting in 565 mg total gluten equivalents, 25 mg albumins/globulins, 22 mg α-amylase/trypsin inhibitors and 48 mg pepsin capsule proteins. The slightly acidic, dough-like smell and bitter taste of pepgluten could be completely camouflaged in multivitamin juice with bitter lemon, grapefruit juice, or vegetable and fruit smoothies. Thus, pepgluten met the criteria for placebo-controlled challenges (active and placebo materials are identical regarding appearance, taste, smell, and texture) and is appropriate as a standard preparation for the oral food challenge and clinical investigations to study wheat hypersensitivities.

Novel approaches for enzymatic gluten degradation to create high-quality gluten-free products.

Celiac disease (CD), a chronic enteropathy of the small intestine caused by ingestion of gluten, is one of the most prevalent food hypersensitivities worldwide. The essential treatment is a strict lifelong gluten-free diet based on the avoidance of gluten-containing products from wheat, rye, barley and, in rare cases, oats. Products made from naturally gluten-free raw materials often have inferior nutritional, textural and sensory properties compared to the corresponding gluten-containing products. Therefore, the incorporation of wheat, rye and barley flours after efficient removal of the harmful component gluten into gluten-free products would be beneficial. Gluten modification resulting in decreased CD-immunoreactivity may be achieved via the formation of crosslinks using microbial transglutaminase. To effectively eliminate CD-immunoreactivity, plant, fungal, bacterial, animal or engineered peptidases are capable of degrading gluten proteins and peptides into harmless fragments. The application of peptidases from germinated cereal grains, fungal peptidases and/or lactic acid bacteria during food processing yielded high-quality sourdough wheat breads, pasta, wheat starch and bran, rye products and beer, all with gluten contents below the Codex Alimentarius threshold of 20mg/kg for gluten-free products. As with all gluten-free products, the legislative compliance of such treated materials needs to be monitored closely. Provided that all safety requirements are met, gluten-containing raw materials treated in an adequate way to remove CD-active gluten fragments may be used together with naturally gluten-free ingredients to create an extended choice of high-quality gluten-free products.

Quantitation of the immunodominant 33-mer peptide from α-gliadin in wheat flours by liquid chromatography tandem mass spectrometry.

Coeliac disease (CD) is triggered by the ingestion of gluten proteins from wheat, rye, and barley. The 33-mer peptide from α2-gliadin has frequently been described as the most important CD-immunogenic sequence within gluten. However, from more than 890 published amino acid sequences of α-gliadins, only 19 sequences contain the 33-mer. In order to make a precise assessment of the importance of the 33-mer, it is necessary to elucidate which wheat species and cultivars contain the peptide and at which concentrations. This paper presents the development of a stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry to quantitate the 33-mer in flours of 23 hexaploid modern and 15 old common (bread) wheat as well as two spelt cultivars. All flours contained the 33-mer peptide at levels ranging from 91-603 µg/g flour. In contrast, the 33-mer was absent (

Improved Quantitation of Gluten in Wheat Starch for Celiac Disease Patients by Gel-Permeation High-Performance Liquid Chromatography with Fluorescence Detection (GP-HPLC-FLD).

Purified wheat starch (WSt) is commonly used in gluten-free products for celiac disease (CD) patients. It is mostly well-tolerated, but doubts about its safety for CD patients persist. One reason may be that most ELISA kits primarily recognize the alcohol-soluble gliadin fraction of gluten, but insufficiently target the alcohol-insoluble glutenin fraction. To address this problem, a new sensitive method based on the sequential extraction of gliadins, glutenins, and gluten from WSt followed by gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD) was developed. It revealed that considerable amounts of glutenins were present in most WSt. The gluten contents quantitated by GP-HPLC-FLD as sum of gliadins and glutenins were higher than those by R5 ELISA (gluten as gliadin content multiplied by a factor of 2) in 19 out of 26 WSt. Despite its limited selectivity, GP-HPLC-FLD may be applied as confirmatory method to ELISA to quantitate gluten in WSt.

Classification of spelt cultivars based on differences in storage protein compositions from wheat.

Wholemeal flours from 62 spelt and 13 wheat cultivars were studied. The quantitative protein compositions of the Osborne fractions determined by reversed-phase high-performance liquid chromatography, showed that the chromatograms of the reduced gliadin fractions were most suitable for the distinction of spelt from wheat and for the classification of spelt. The patterns of the reduced spelt gliadins showed one to three markers that were not present in wheat. Based on these markers, spelt cultivars were classified into three groups ranging from 'typical spelt' to 'similar to common wheat'. Marker 1 was identified as ω1,2-gliadin and markers 2, 3a and 3b were identified as γ-gliadins by means of N-terminal sequence analysis and determination of the relative molecular mass by mass spectrometry. As glutenin-bound ω-gliadins were present in wheat and absent in spelt, this protein type may be used to detect and quantitate small amounts of wheat in spelt products.

Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4.

Ingestion of wheat, barley, or rye triggers small intestinal inflammation in patients with celiac disease. Specifically, the storage proteins of these cereals (gluten) elicit an adaptive Th1-mediated immune response in individuals carrying HLA-DQ2 or HLA-DQ8 as major genetic predisposition. This well-defined role of adaptive immunity contrasts with an ill-defined component of innate immunity in celiac disease. We identify the α-amylase/trypsin inhibitors (ATIs) CM3 and 0.19, pest resistance molecules in wheat, as strong activators of innate immune responses in monocytes, macrophages, and dendritic cells. ATIs engage the TLR4-MD2-CD14 complex and lead to up-regulation of maturation markers and elicit release of proinflammatory cytokines in cells from celiac and nonceliac patients and in celiac patients' biopsies. Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs. These findings define cereal ATIs as novel contributors to celiac disease. Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.

Detoxification of gluten by means of enzymatic treatment.

Celiac disease (CD) is an inflammatory disease of the upper small intestine in genetically predisposed individuals caused by glutamine- and proline-rich peptides from cereal storage proteins (gluten) with a minimal length of nine amino acids. Such peptides are insufficiently degraded by gastrointestinal enzymes; they permeate the lymphatic tissue, are bound to celiac-specific, antigen-presenting cells, and stimulate intestinal T-cells. The typical clinical pattern is a flat small intestinal mucosa and malabsorption. Currently, the only therapy is a strict, lifelong gluten-free diet. Recent research has shown that gluten and gluten peptides can be degraded by prolyl endopeptidases from different sources. These peptidases can either be used to produce gluten-free foods from gluten-containing raw materials, or they have been suggested as an oral therapy for CD, in which dietary gluten is hydrolyzed by coingested peptidases already in the stomach, thus preventing CD-specific immune reactions in the small intestine. This would be an alternative for CD patients to the gluten-free diet. Furthermore, microbial transglutaminase could be used to detoxify gluten either by selectively modifying glutamine residues of intact gluten by transamidation with lysine methyl ester or by crosslinking gluten peptides in beverages via isopeptide bonds so that they can be removed by filtration.

Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation.

Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.

Endocytotic segregation of gliadin peptide 31-49 in enterocytes.

OBJECTIVE: Coeliac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in human leucocyte antigen (HLA)-DQ2/8-positive individuals. The molecular mechanisms relevant to oral tolerance induction towards toxic cereals such as gliadin remain poorly understood. Enterocytes, which express predominantly HLA-DR proteins, are capable of processing, transcytosing and presenting food antigens from the intestinal lumen to T lymphocytes of the lamina propria. METHODS: Epitope-specific monoclonal antigliadin antibodies are utilised to unravel the intraepithelial transport processes of gliadin peptides in human duodenal biopsy specimens from patients with CD and reconstitute the transepithelial and endocytic pathways of gliadin in intestinal epithelial HT29 cells. RESULTS: The gliadin peptide AA 31-49 is segregated from the peptides AA 56-68 and AA 229-246 along the endosomal pathway. Thus, AA 31-49 bypasses HLA-DR-positive late endosomes in intestinal cells and in biopsy specimens of patients with untreated CD. Further, it is localised in early endosomes and consequently escapes antigen presentation at the basolateral membrane, unlike peptides AA 56-68 and AA 229-246 that reach HLA-DR-positive late endosomes. Strikingly, forms of gliadin peptide AA 31-49 conjugated to cholera toxin B are sorted into late endosomes of HT29 cells. CONCLUSIONS: Endocytic segregation of gliadin peptide AA 31-49 seems to be a constitutive process. It explains why this peptide cannot stimulate gluten-sensitive T cells. Presentation of gliadin peptides by HLA-DR proteins via late endosomes within enterocytes might induce a tolerogenic effect and constitutes a potentially promising therapeutic approach for induction of tolerance towards gliadin.

Preparation and characterization of enzymatically hydrolyzed prolamins from wheat, rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten.

Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N x 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 microg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 microg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.

Quantitative protein composition and baking quality of winter wheat as affected by late sulfur fertilization.

Increasing prices for wheat products and fertilizers, as well as reduced sulfur (S) contributions from the atmosphere, call for an improvement of product quality and agricultural management. To detect the impact of a time-dependent S fertilization, the quantitative protein composition and the baking quality of two different wheat cultivars, Batis and Turkis, were evaluated. The glutathione concentration in grains serves as a reliable marker of the need for added S fertilizer. The quantitation of gliadins and glutenin subunits by reversed-phase high-performance liquid chromatography confirmed that S-rich proteins significantly increased with S fertilization, whereas the S-poor proteins significantly decreased. Proteome analysis by means of high-resolution protein profiles detected 55 and 37 proteins from Batis and Turkis changed by late S fertilization. A microscale baking test using wholemeal flour was implemented for the evaluation of baking quality, and late S fertilization was found to improve the composition of gluten proteins and baking quality.

Effects of elevated atmospheric CO2 concentrations on the quantitative protein composition of wheat grain.

The continuing increase in atmospheric CO 2 concentration is predicted to enhance biomass production and to alter biochemical composition of plant tissues. In the present study, winter wheat ( Triticum aestivum L. cv. 'Batis') was grown under ambient air (BLOW, CO 2 concentration: 385 muL L (-1)) and free-air CO 2 enrichment (FACE, CO 2 concentration: 550 muL l (-1)) and two different nitrogen (N) fertilization levels (normal N supply: N100, 50% of normal N supply: N50). Mature kernels were milled into white flour and analyzed for the contents of crude protein, Osborne fractions, single gluten protein types and glutenin macropolymer. Elevated CO 2 caused significant reductions in crude protein and all protein fractions and types ( p < 0.001) except albumins and globulins. Effects were more pronounced in wheat samples supplied with normal amounts of N fertilizer. Crude protein was reduced by 14% (N100) and 9% (N50), gliadins by 20% and 13%, glutenins by 15% and 15% and glutenin macropolymer by 19% and 16%, respectively. Within gliadins, omega5-gliadins (-35/-22%) and omega1,2-gliadins (-27/-14%) were more affected than alpha-gliadins (-21/-13%) and gamma-gliadins (-16/-12%). Within glutenins, HMW subunits (-23/-18%) were more affected than LMW subunits (-12/-15%). According to these results, flour from high CO 2 grown grain will have a diminished baking quality. To our knowledge, these are the first results of elevated CO 2 concentrations impacts on wheat grain protein composition gained under relevant growing conditions at least for Central Europe.

Concentrations of total glutathione and cysteine in wheat flour as affected by sulfur deficiency and correlation to quality parameters.

A method for the simultaneous quantitation of total glutathione and total cysteine in wheat flour by a stable isotope dilution assay using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) was developed. As internal standards, L-[(13)C3, (15)N]cysteine and L-gamma-glutamyl-L-[(13)C3, (15)N]cysteinyl-glycine were used. The method consisted of the extraction and reduction of flour with tris(2-carboxyethyl) phosphine after the addition of internal standards, protection of free thiol groups with iodoacetic acid, derivatization of free amino groups with dansyl chloride, and HPLC-MS/MS. The limits of detection and quantitation for glutathione were 0.75 nmol/g and 2.23 nmol/g flour, respectively. For cysteine, the limits of detection and quantitation were 0.72 nmol/g and 2.12 nmol/g flour, respectively. The developed method was found to be sensitive enough for quantitation of total glutathione and cysteine levels in wheat flour. This method was then utilized to investigate the effect of sulfur (S) deficiency on the amount of total glutathione and cysteine in flour. In S-deficient wheat, the concentrations of total glutathione and cysteine were proportional to the amount of S supplied during growth. The calculation of correlations revealed that GSH and Cys concentrations influenced the rheological dough properties and the baking performance at least as much as protein parameters. Thus, the low concentration of GSH and Cys in flour from S-deficient wheat had a similar effect on the technological properties as the altered composition of gluten proteins.

Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits.

BACKGROUND: In celiac disease gluten, the disease-inducing toxic component in wheat, induces the secretion of autoantibodies which are targeted against transglutaminase 2 (TG2). These autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels. In addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. Since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the pre-existence of mucosal TG2-specific IgA deposits in the cultured biopsy samples. RESULTS: In the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial CD25+ cells. However, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal TG2-specific IgA autoantibody deposits were still present. Furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge. CONCLUSION: Our results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal IgA deposits present. In addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method.