Toxic Gliadin Peptide Research Articles

Thiol-disulfide homeostasis in children with celiac disease.

Toxic gliadin peptide damages enterocytes in celiac disease by causing oxidative stress. Thiols are organic compounds that defend against oxidative stress. This study aimed to investigate the changes in thiol-disulfide homeostasis in children with celiac disease. The study included patients with celiac disease, children diagnosed with functional gastrointestinal disorders, and healthy children. Patients' serumnative and total thiol-disulfide amounts, disulfide/total thiol percentage ratios, disulfide / native thiol percentage ratios, and native thiol/total thiol percentage ratios were measured. The study involved 172 children, of whom 90 (52.3%) were girls. The mean participant age was 8.6 ± 4.2years. A total of 59 (34.3%) children had celiac disease, 56 (32.6%) had functional gastrointestinal disorders, and 57 (33.1%) were healthy. The total thiol and disulfide levels of patients with celiac disease (305 ± 87μmol/L and 25 ± 15μmol/L, respectively) were significantly lower than those of healthy children (349 ± 82μmol/L and 40 ± 15μmol/L, respectively) (P = 0.006 and P < 0.001, respectively). Native and total thiol levels (226 ± 85μmol/L and 279 ± 99μmol/L, respectively) in patients with celiac disease who consumed a gluten-containing diet were significantly lower than those of patients who consumed a gluten-free diet (278 ± 64μmol/L and 327 ± 69μmol/L, respectively) (P = 0.017 and P = 0.041, respectively). Thiol-disulfide homeostasis, an important antioxidant defense component of the gastrointestinal system, is disrupted in children with celiac disease. A gluten-free diet helped partially ameliorate this decline.

A Peptide from Kiwifruit Exerts Anti-Inflammatory Effects in Celiac Disease Mucosa

Objective: Celiac disease is an immune-mediated disease of the intestine triggered by gluten. Gluten elicits, in genetically susceptible individuals, cytokine responses that are then transmitted to the immunocompetent cells. Vegetables and fruit have anti-inflammatory and antioxidant properties with a protective effect on intestinal epithelium. Kiwifruit is known to have beneficial effects on the intestinal tissues, and it is the only plant food containing the peptide kissper, with anti-inflammatory properties. The aim of this study was the evaluation of the kissper effect on the gluten-induced inflammation in celiac disease.Methods: We used an in vitro model of intestinal culture explant from celiac disease patients and non-celiac disease patients, cultured for 24 hours with the toxic gliadin peptide P31-43 and kissper preincubation.Results: Our data showed HLA-DR and TG2 reduction in the celiac disease mucosa pretreated with kissper, as well as a reduction of COX-2 in two patients. No differences we observed for the TGF-b1 and IL-15 levels in supernatants upon kissper pretreatment.Conclusions: The preliminary results suggest that kissper has a potential anti-inflammatory role in celiac disease.

The gliadin peptide 31-43 exacerbates kainate neurotoxicity in epilepsy models

Many neurological disorders of gluten-related diseases (GRD), not directly referable to the gastrointestinal tract, have been reported in association with celiac disease (CD), including ataxia, neuropathy and epilepsy. In particular, people with epilepsy diagnosed with CD seems to be characterized by intractable seizure. In these patients, gluten restriction diet has resulted in a reduction of both seizure frequency and antiepileptic medication. Many hypotheses have been suggested, however, molecular mechanisms that associates GRD and epileptogenesis are yet unknown. In this study, we examined the effects of the toxic gliadin peptide 31-43 in in vivo and in vitro models of kainate-induced-epilepsy. We observed that p31-43 exacerbates kainate neurotoxicity in epilepsy models, through the involvement of the enzymatic activity of transglutaminases. Moreover, electrophysiological recordings in CA3 pyramidal neurons of organotypic hippocampal slices show that p31-43 increases the inward current induced by kainate, the average sEPSC amplitude and the total number of evoked action potentials when applicated alone, thus suggesting that p31-43 is able to influence CA3-CA1 neurotransmission and can potentiate postsynaptic kainate receptors. Our results suggest a possible mechanism underlying the relationship between GRD and epilepsy through a potentiation of kainate-induced neurotoxicity and links the toxic effects of gluten to epilepsy.

Development and optimisation of a generic micro LC-ESI-MS method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour for food analysis.

We sometimes see manufactured bakery products on the market which are labelled as being gluten free. Why is the content of such gluten proteins of importance for the fabrication of bakery industry and for the products? The gluten proteins represent up to 80% of wheat proteins, and they are conventionally subdivided into gliadins and glutenins. Gliadins belong to the proline and glutamine-rich prolamin family. Its role in human gluten intolerance,as a consequence of its harmful effects, is well documented in the scientific literature. The only known therapy so far is a gluten-free diet, and hence, it is important to develop robust and reliable analytical methods to quantitatively assess the presence of the identified peptides causing the so-called coeliac disease. This work describes the development of a new, fast and robust micro ion pair-LC-MS analytical method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour. The use of RapiGest™ SF as a denaturation reagent prior to the enzymatic digestion showed to shorten the measuring time. During the optimisation of the enzymatic digestion step, the best 30-mer toxic peptide was identified from the maximum recovery after 3h of digestion time. The lower limit of quantification was determined to be 0.25ng/μL. The method has shown to be linear for the selected concentration range of 0.25-3.0ng/μL. The uncertainty related to reproducibility of measurement procedure, excluding the extraction step, has shown to be 5.0% (N = 12). Finally, this method was successfully applied to the quantification of 30-mer toxic peptides from commercial wheat flour with an overall uncertainty under reproducibility conditions of 6.4% including the extraction of the gliadin fraction. The results were always expressed as the average of the values from all standard concentrations. Subsequently, the final concentration of the 30-mer toxic peptide in the flour was calculated and expressed in milligrams per gram unit. The determined, calculated concentration of the 30-mer toxic peptide in the flour was found to be 1.29 ± 0.37μg/g in flour (N = 25, s y = 545,075, f = 25 - 2 (t = 2.069), P = 95%, two-sided).

Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study.

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridium sensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota. Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

Gliadin-dependent cytokine production in a bidimensional cellular model of celiac intestinal mucosa.

The downstream cascade of the inflammatory response to gliadin in celiac intestinal mucosa encompasses the early activation of the innate immunity that triggers the adaptive response. Therefore, the in vitro study of the pathogenic mechanism of celiac disease (CD) on enterocytes alone or mucosal T lymphocytes alone does not fully consider all the aspects of gliadin-dependent inflammation. Although the in vitro culture of specimens of intestinal mucosa obtained from celiac patients is the gold standard for the study of CD, this technique presents several technical challenges and the bioptic specimens are not easily available. So, in this paper, we described the gliadin-dependent cytokine production in a bidimensional cellular system, which is able to mimic both the innate and the adaptive steps of the mucosal immune response of CD. In the upper compartment, the intestinal epithelial cells are grown on a filter, and in the lower compartment, the mononuclear cells isolated from peripheral blood of celiac patients are cultured. Cells were apically exposed to the toxic gliadin peptide p31-43 for 3 h and then with the immunodominant gliadin fragment pα-9 for 21 h. The incubation with gliadin peptides resulted in increased levels of IL-15, INF-γ, IL-6, tumor necrosis factor (TNF)-α, IL-1β, and CCL 2, 3 and 4 in the basal supernatants, with respect to cells exposed to medium alone. The p31-43-driven epithelial priming of mucosal response consists of transglutaminase (TG2)-mediated deamidation of the immunostimulatory gliadin peptides, as demonstrated by the inhibition of pα-9 activity, when the system is exposed to blocking anti-TG2 antibody.

Epithelial transport of immunogenic and toxic gliadin peptides in vitro.

ScopeCeliac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31–43 and the immunogenic P56–68. As the size, and hence, the molecular weight of peptides might have an effect on the transport efficiency we chose two peptides of the same, rather short chain length.Methods and ResultsFluorescence labeled P31–43 and P56–68 were synthesized and studied in a transwell system with human enterocytes. Fluorometric measurements were done to reveal antigen translocation and flow cytometry as well as confocal microscopy were used to investigate cellular uptake of peptides. Structural changes of these peptides were analysed by MALDI-TOF-MS. According to fluorescence intensities, significantly more P31–43 compared to P56–68 was transported through the enterocyte layer after 24 h incubation. In contrast to previous reports, however, mass spectrometric data do not only show a time-dependent cleavage of the immunogenic P56–68, but we observed for the first time the degradation of the toxic peptide P31–43 at the apical side of epithelial cells.ConclusionConsidering the degradation of gliadin peptides by enterocytes, measurement of fluorescence signals do not completely represent translocated intact gliadin peptides. From our experiments it is obvious that even short peptides can be digested prior to the translocation across the epithelial barrier. Thus, the chain length and the sensibility to degradations of gliadin peptides as well as the integrity of the epithelial barrier seem to be critical for the uptake of gliadin peptides and the subsequent inflammatory immune response.

A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis.

Epithelial transport of immunogenic and toxic gliadin peptides in vitro

In contrast to immunogenic gliadin peptides (GP) like P56–68, the toxic P31–43 may mediate direct toxic effects by activation of the intestinal immune system in celiac disease (CD). Epithelial translocation of immunogenic and toxic GP is considered as an important step in the pathogenesis of CD. Thus, we studied transepithelial passage of fluorescence labeled GP in a transwell system using differentiated enterocytes (Caco‐2 cells). Quantification of GP translocation was done by fluorometric measurement. Structural changes of GP were analyzed by mass spectrometry (MALDI‐MS); flow cytometry and confocal microscopy were used to investigate intracellular uptake. P31–43 and P56–68 were able to cross the epithelial barrier into the basolateral compartment. Fluorometric analysis revealed a higher basolateral fluorescence intensity when cells were incubated with P31–43 compared to P56–68. Flow cytometric analysis and confocal microscopy showed a cellular uptake of both GP which was markedly reduced at 4°C and by the endocytosis inhibitor methyl‐β‐cyclodextrin. MALDI‐MS revealed cleavage of GP on the apical side of the cells and the translocation of intact P31–43 and P56–68 as well as fragments thereof depending on incubation time and concentration. In conclusion, toxic and immunogenic GP were shown to pass the epithelial barrier and may thereby be able to interact with the intestinal immune system.

Celiac disease: Prevalence, diagnosis, pathogenesis and treatment

Celiac disease (CD) is one of the most common diseases, resulting from both environmental (gluten) and genetic factors [human leukocyte antigen (HLA) and non-HLA genes]. The prevalence of CD has been estimated to approximate 0.5%-1% in different parts of the world. However, the population with diabetes, autoimmune disorder or relatives of CD individuals have even higher risk for the development of CD, at least in part, because of shared HLA typing. Gliadin gains access to the basal surface of the epithelium, and interact directly with the immune system, via both trans- and para-cellular routes. From a diagnostic perspective, symptoms may be viewed as either "typical" or "atypical". In both positive serological screening results suggestive of CD, should lead to small bowel biopsy followed by a favourable clinical and serological response to the gluten-free diet (GFD) to confirm the diagnosis. Positive anti-tissue transglutaminase antibody or anti-endomysial antibody during the clinical course helps to confirm the diagnosis of CD because of their over 99% specificities when small bowel villous atrophy is present on biopsy. Currently, the only treatment available for CD individuals is a strict life-long GFD. A greater understanding of the pathogenesis of CD allows alternative future CD treatments to hydrolyse toxic gliadin peptide, prevent toxic gliadin peptide absorption, blockage of selective deamidation of specific glutamine residues by tissue, restore immune tolerance towards gluten, modulation of immune response to dietary gliadin, and restoration of intestinal architecture.

Lysosomal accumulation of gliadin p31–43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa

BackgroundAn unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31–43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in...

Alteration in actin organisation and protein transport after treatment with gliadin peptides

Celiac disease is a chronic intestinal inflammatory disease characterized by villus atrophy and malabsorption in genetically predisposed individuals due to gliadin toxicity. Here, we analyze the effects of gliadin toxicity on the actin cytoskeleton and the actin-dependent transport of intestinal proteins in Caco-2 cells or in transfected COS-1 cells. For this cells were treated with peptic-tryptic gluten digest (Frazer’s Fraction, FF) as a source of gliadin peptides. Laser confocal microscopy demonstrated a significant effect of FF on the actin cytoskeleton as assessed by the reduced labeling density below the plasma membrane and around the nucleus. Concomitantly, the actin-dependent vesicular transport of the intestinal proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (ApN) to the cell surface was also disturbed. By contrast, the vesicular trafficking of lactase that follows an actin-independent pathway was not affected. Biosynthetic labeling experiments supported these confocal data and further revealed variations in the glycosylation patterns of SI, DPPIV and ApN. Altogether, our data demonstrate that toxic gliadin peptides alter the integrity of actin cytoskeleton leading to impaired trafficking, intracellular block of rafts-associated proteins to the cell surface and modulation of their glycosylation pattern. Grant support SFB 621, Bonn, Germany

In vitro presentation of gliadin-derived peptides by different cell lines

Background: Gliadin peptide presentation and T-cell activation are critical events in the pathogenesis of celiac disease. Several studies have been performed to identify the toxic gliadin peptides but the complexity of the antigenic fraction makes this analysis difficult. In this work, an in vitro model for the analysis of gliadin peptide presentation is studied. Methods: The human cell lines U937 and THP-1 (monocytic), DUCAF and VAVY (immortalised B cells) and HT-29 and Caco-2 (intestinal epithelial cells) were incubated with biotin-labelled gliadin (bG). FITC-labelled streptavidin was used to detect biotinylated peptides at the cell surface by flow cytometry. Results: All cell lines tested showed a fluorescence signal derived from bG, that was highest when cells were stimulated with IFN-γ for 48 h. Time course experiments performed using THP-1 cells showed that after 4-h incubation, almost a maximal signal can be reached. THP-1 cells incubated at 4 °C or after paraformaldehyde fixation showed a substantial signal reduction, suggesting that metabolic activity was necessary for the detection of the maximal fluorescence signal at the cell surface. The presence of HLA class II-bound biotinylated peptides was observed in cell lysates of THP-1 cells incubated with bG. Conclusions: In all cell lines tested, a specific biotin–peptide-derived signal was observed. This was increased after IFN-γ treatment and decreased after fixation or incubation at low temperature. The signal was higher in monocytic and B-cell lines than in the epithelial cell lines. The use of this procedure could be a useful tool to study the in vitro processing and presentation of naturally gliadin-derived peptides.

Relation between gliadin structure and coeliac toxicity.

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for coeliac patients. The numerous components of gliadin can be classified according to their primary structure into omega 5-, omega 1,2-, alpha- and gamma-type. Both omega-types have almost entirely repetitive amino acid sequences consisting of glutamine, proline and phenylalanine. alpha- and gamma-type gliadins contain four and five different domains, respectively, and are homologous within the domains III and V. Unique for each alpha- and gamma-type is domain I, which consists mostly of repetitive sequences rich in glutamine, proline and aromatic amino acids. Coeliac toxicity of gliadin is not destroyed by digestion with gastropancreatic enzymes. In vivo testing established the toxicity of alpha-type gliadins and in vitro testing of gliadin peptides revealed that domain I of alpha-type gliadins is involved in activating coeliac disease. The sequences -Pro-Ser-Gln-Gln- and -Gln-Gln-Gln-Pro- were demonstrated to be common for toxic gliadin peptides. Most of the in vivo and in vitro studies of synthetic peptides confirmed the importance of one or both of these sequences. Cultivated hexaploid, tetraploid and diploid wheat species do not differ significantly in potential toxic sequences of alpha-type gliadins.

In VitroSystems for the Study of Cereal Proteins Toxic in Coeliac Disease

For a number of years, we have been carrying out investigations intended to clarify the nature of the toxic gliadin peptides, and whether different species and/or cultivars of wheat are tolerated to varying extents by people suffering from coeliac disease. Our investigations have been carried out using three main biological systems: 1) cultures of biopsy specimens of human intestinal mucosa; 2) cultures of rat fetal intestine; and 3) cultures of erythroid K562(S) cells.Using these systems, we have tested several peptide mixtures prepared from gliadin and non-gliadin fractions extracted from wheats having various degrees of ploidy and from other cereals. Fraction preparation has been carried out by means of sequential digestion with pepsin and trypsin under experimental conditions simulating protein digestion in man. The results obtained indicate that the pathological symptoms associated with intolerance to gluten are largely due to the cytotoxic activity of wheat gliadin peptides toward immature enterocytes.