Peptic-tryptic Digest Research Articles

A CELL SURFACE TISSUE TRANSGLUTAMINASE IS INVOLVED IN RECOGNITION OF GLIADIN PEPTIDES BY K 562 CELLS

Aim: Several studies have been demonstated that the agglutination activity of human chronic myelogenous leukemia K562 cells by prolamines peptictryptic digest (PT) is higly correlated with the toxicity of these prolamines in coeliac disease. The mechanism of the agglutination activity is unknown, but the presence of a binding site on cell surface of K562 cells has been supposed. In the latest years, a cell surface tissue transglutaminase has been identified in CD8 + T cell, in monocytes and in K562 cells. Tissue transglutaminase is known to play a pivotal role in pathogenesis of coeliac disease, binding the gliadin peptides in the small bowel mucosa of these patients. We hypothesize that the cell surface transglutaminase could have a role in the agglutination activity of K562 cells. Methods: K562 cells were grown in RPMI medium. For the agglutination test, cells were harvested, washed and resuspended in PBS at the final concentration of 108cells/ml. The agglutination test were carried out as follow: in each well of a 96 well plate 25 μl of PBS and 25 μl of cells were added. Then, mAb anti tTG CUB 7402 (Neomarker, USA) 1 μl of a solution at the concentration of 2 μg/ml and 25 μl of a 0,5 mg/ml PT solution were added. Finally, an incubation for 30 min occoured before evaluating the agglutination by optical microscope. Controls were performed with PT only and with mAB anti rabbit IgG. Results: The incubation of K562 cells with PT digest resulted in a massive agglutinationof the cells. No agglutination occoured when the mAb anti tTG was added prior to the PT digest. Summary: The ability of the mAb anti tTG of preventing the agglutination of K562 by gliadin peptides indicates that a cell surface tissue transglutaminase acts as binding site for the gliadin peptides. Conclusion: The results of these study suggest that tissue transglutaminase could play a role in recognition of gliadin peptides by the cells in coeliac disease and it could be involved in starting other responses than the activation of the adaptive immune system in the mucosa of the coeliac disease patients.

Pasta Made from Durum Wheat Semolina Fermented with Selected Lactobacilli as a Tool for a Potential Decrease of the Gluten Intolerance

A pool of selected lactic acid bacteria was used to ferment durum wheat semolina under liquid conditions. After fermentation, the dough was freeze-dried, mixed with buckwheat flour at a ratio of 3:7, and used to produce the "fusilli" type Italian pasta. Pasta without prefermentation was used as the control. Ingredients and pastas were characterized for compositional analysis. As shown by two-dimensional electrophoresis, 92 of the 130 durum wheat gliadin spots were hydrolyzed almost totally during fermentation by lactic acid bacteria. Mass spectrometry matrix-assisted laser desorption/ionization time-of-flight and reversed phase high-performance liquid chromatography analyses confirmed the hydrolysis of gliadins. As shown by immunological analysis by R5-Western blot, the concentration of gluten decreased from 6280 ppm in the control pasta to 1045 ppm in the pasta fermented with lactic acid bacteria. Gliadins were extracted from fermented and nonfermented durum wheat dough semolina and used to produce a peptic-tryptic (PT) digest for in vitro agglutination tests on cells of human origin. The whole PT digests did not cause agglutination. Affinity chromatography on Sepharose-6-B mannan column separated the PT digests in three fractions. Fraction C showed agglutination activity. The minimal agglutinating activity of fraction C from the PT digest of fermented durum wheat semolina was ca. 80 times higher than that of durum wheat semolina. Pasta was subjected to sensory analysis: The scores for stickiness and firmness were slightly lower than those found for the pasta control. Odor and flavor did not differ between the two types of pasta. These results showed that a pasta biotechnology that uses a prefermentation of durum wheat semolina by selected lactic acid bacteria and tolerated buckwheat flour could be considered as a novel tool to potentially decrease gluten intolerance and the risk of gluten contamination in gluten-free products.

In vitro-deranged intestinal immune response to gliadin in type 1 diabetes.

Dietary gluten has been associated with an increased risk of type 1 diabetes. We have evaluated inflammation and the mucosal immune response to gliadin in the jejunum of patients with type 1 diabetes. Small intestinal biopsies from 17 children with type 1 diabetes without serological markers of celiac disease and from 50 age-matched control subjects were examined by immunohistochemistry. In addition, biopsies from 12 type 1 diabetic patients and 8 control subjects were cultured with gliadin or ovalbumin peptic-tryptic digest and examined for epithelial infiltration and lamina propria T-cell activation. The density of intraepithelial CD3(+) and gammadelta(+) cells and of lamina propria CD25(+) mononuclear cells was higher in jejunal biopsies from type 1 diabetic patients versus control subjects. In the patients' biopsies cultured with peptic-tryptic gliadin, there was epithelial infiltration by CD3(+) cells, a significant increase in lamina propria CD25(+) and CD80(+) cells and enhanced expression of lamina propria CD54 and crypt HLA-DR. No such phenomena were observed in control subjects, even those with celiac disease-associated HLA haplotypes. In conclusion, signs of mucosal inflammation were present in jejunal biopsies from type 1 diabetic patients, and organ culture studies indicate a deranged mucosal immune response to gliadin.

Characterizing one of the DQ2 candidate epitopes in coeliac disease: A-gliadin 51-70 toxicity assessed using an organ culture system.

To investigate, using an organ culture system, in-vitro toxicity of region 51-70 of A-gliadin (SQQPYLQLQPFPQPQLPYSQ), a peptide overlapping some of the sequences recently characterized as DQ2-restricted T-cell epitopes in coeliac disease. Jejunal biopsies obtained from each of ten coeliac patients (eight treated, two untreated) and two non-coeliac patients were cultured in vitro for 18 h in the presence of A-gliadin amino acids 51-70 (200 microg/ml), organ culture medium only, peptic-tryptic digest of gliadin (1 mg/ml) or ovalbumin (1 mg/ml), the last two acting as positive and negative controls, respectively. Morphometric analysis involved measuring the cell height of 30 enterocytes, selected at random from the middle third of different villi for each section. Mean enterocyte cell heights (ECH) were compared with values for specimens cultured in medium alone. Levels of tissue transglutaminase antibody in biopsy supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). In eight of ten coeliac patients, A-gliadin 51-70 was significantly toxic, causing a 30% decrease in ECH when compared with medium alone. In two of ten subjects, the peptide did not show any toxic effect. In all ten cases, we found that both positive and negative controls worked as expected. The peptide was non-toxic in the non-coeliac individuals. Tissue transglutaminase antibody titre in the supernatant was not found to be related to mucosal damage. We showed that the peptide corresponding to amino acids 51-70 of A-gliadin is characterized by in-vitro toxicity to the jejunal coeliac mucosa, correlating with recent findings of an immunological role of similar peptides. The lack of response in two of ten subjects suggests that this epitope may not be relevant in all cases of coeliac disease.

Gliadin increases iNOS gene expression in interferon-gamma-stimulated RAW 264.7 cells through a mechanism involving NF-kappa B.

Nitric oxide (NO) plays an important role in the pathogenesis of the histological changes seen in coeliac disease. We have investigated the effect of peptic-tryptic digest of gliadin (Pt-G) and gliadin (G) on inducible nitric oxide synthase (iNOS) protein expression in RAW 264.7 macrophages stimulated with interferon-gamma (IFN-gamma). Pt-G and G enhanced in a concentration and time-dependent manner NO production by IFN-gamma-stimulated RAW 264.7 cells. The increase of iNOS protein expression was correlated with NF-kappa B/DNA binding activity and occurred at transcriptional level. Pyrrolidine dithiocarbamate and N-alpha-para-tosyl-L-lysine chloromethyl ketone, two known inhibitors of NF-kappa B activation, decreased significantly NO production and iNOS protein expression as well as NF-kappa B/DNA binding activity. Our results show that the effect of Pt-G and G on enhancement of iNOS protein expression in IFN-gamma-treated RAW 264.7 cells is mainly mediated through NF-kappa B and suggest that blockage of NF-kappa B activation reduces enhancing effect of gluten on NO production in inflamed mucosa of coeliac patients.

Nuclear factor kappa B is activated in small intestinal mucosa of celiac patients.

NF-kappa B regulates inflammatory and immune response by increasing the expression of specific genes. In celiac disease proinflammatory cytokines, adhesion molecules, and enzymes whose gene expression is known to be regulated by NF-kappa B are involved. This study investigated the activation of NF-kappa B in inflamed mucosa from patients with untreated celiac disease. Biopsy specimens from control, untreated, and treated patients were subjected to molecular biology analysis. NF-kappa B activation was evaluated by electrophoretic mobility shift assay. NF-kappa B related subunit protein level, and inducible nitric oxide synthase and cyclo-oxygenase 2 protein expression was analyzed by western blot. Both NF-kappa B/DNA binding activity and p50/p65 nuclear levels were higher in biopsy specimens from untreated patients than in those from treated patients and controls. The degradation of I kappa B beta in the cytosol and the reappearance in the nucleus indicated a persistent NF-kappa B activation in celiac disease. NF-kappa B activity was maintained in cultured biopsy specimens up to 6 h and decreased at 24 h, and then the addition of peptic-tryptic digest of gliadin caused the recovery of NF-kappa B activity at 6 h. NF-kappa B/DNA binding activity was correlated with inducible nitric oxide synthase and cyclo-oxygenase-2 protein expression. These results show for the first time that NF-kappa B is activated in the inflamed mucosa of celiac patients and suggest that it may represent a molecular target for the modulation of inflammatory response in celiac disease.

Gluten sensitivity in a subset of children with insulin dependent diabetes mellitus.

The association between celiac disease and insulin dependent diabetes mellitus (IDDM) is well established. Rectal gluten challenge has been used in patients with celiac disease and in first degree relatives as a tool to assess the mucosal immune response to gluten. The aim of this study was to assess the mucosal immune response to gluten in IDDM children by rectal gluten challenge. Rectal biopsy specimens were obtained from 19 children with IDDM before and 6 h after rectal challenge with 2 g of a peptic tryptic digest of gliadin. A total of 16 treated celiac patients and 10 control subjects were also investigated. Epithelium and lamina propria CD3(+) and gamma delta(+) lymphocytes were counted with reference to a standard reference area of muscularis mucosae (10(4) microm(2)). After a local instillation of gliadin, a significant (>mean + 1 SD) percentage increment of lamina propria and epithelium CD3(+) and of lamina propria and epithelium gamma delta(+) lymphocytes was observed in five IDDM children, as compared to 11 and 13 celiac patients and one and two controls, respectively. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis classified four of 19 IDDM children in the group of celiac patients. The positivity was associated with normal serology (antigliadin antibody, antiendomysial antibody, and antitissue transglutaminase antibodies) and a morphologically normal jejunal mucosa. All four patients had HLA-DQ alleles associated with celiac disease. Approximately 20% of IDDM children react to rectal instillation of gliadin. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.

Gluten sensitivity in a subset of children with insulin dependent diabetes mellitus

Objectives The association between celiac disease and insulin dependent diabetes mellitus (IDDM) is well established. Rectal gluten challenge has been used in patients with celiac disease and in first degree relatives as a tool to assess the mucosal immune response to gluten. The aim of this study was to assess the mucosal immune response to gluten in IDDM children by rectal gluten challenge. Methods Rectal biopsy specimens were obtained from 19 children with IDDM before and 6 h after rectal challenge with 2 g of a peptic tryptic digest of gliadin. A total of 16 treated celiac patients and 10 control subjects were also investigated. Epithelium and lamina propria CD3 + and γδ + lymphocytes were counted with reference to a standard reference area of muscolaris mucosae (10 4 μm 2). Results After a local instillation of gliadin, a significant (>mean + 1 SD) percentage increment of lamina propria and epithelium CD3 + and of lamina propria and epithelium γδ + lymphocytes was observed in five IDDM children, as compared to 11 and 13 celiac patients and one and two controls, respectively. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis classified four of 19 IDDM children in the group of celiac patients. The positivity was associated with normal serology (antigliadin antibody, antiendomysial antibody, and antitissue transglutaminase antibodies) and a morphologically normal jejunal mucosa. All four patients had HLA-DQ alleles associated with celiac disease. Conclusions Approximately 20% of IDDM children react to rectal instillation of gliadin. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.

Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance.

Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.

Forty-Eight Hours of Biopsy Culture Improve the Sensitivity of the in Vitro Gliadin Challenge in the Diagnosis of Celiac Disease

Celiac disease (CD) is a long-life intolerance to gliadin in genetically susceptible individuals (1)(2)(3)(4)(5)(6). Despite contrary views (7), diagnosis is still based on the histologic findings of intestinal mucosal atrophy with crypt hyperplasia in individuals on a gluten-containing diet and a return to normal after a gluten-free diet (GFD). The presence of circulating anti-endomysial antibodies (EMAs) and their disappearance after GFD confirm the diagnosis (8)(9)(10). It has recently been shown that EMAs are produced by intestinal mucosa of CD patients. EMAs disappear in treated CD patients but are newly produced after in vitro exposure of intestinal biopsy samples to gliadin (11). Culture of intestinal mucosa from treated CD patients in the presence of a peptic-tryptic (PT) digest of gliadin for 24 h frequently fails to produce detectable EMAs, but it has high specificity as well as high sensitivity in overt CD (12). Our aim was to increase the sensitivity of the in vitro culture by prolonging the duration to 48 h. We also tested a new culture method in batch to ease testing. We enrolled 11 untreated, EMA-positive CD patients (5 males and 6 females; mean age, 24.7 years; age range, 17–42 years) and 29 treated CD patients (11 males and 18 females; mean age, 32.0 years; age range, 11–70 years) after at least 12 months of GFD (median length of GFD, 730 days; range, 342–4015 days) and two monthly, consecutive serum EMA tests that were negative. As disease-controls we studied 67 patients (20 males and 47 females, mean age, 35.3 years; age range, 20–50 years) with gastrointestinal diseases other than CD (42 with gastroesophageal reflux disease, 8 with ulcer disease, 3 with ulcerative colitis, 3 with Crohn disease, 2 with lymphoma, 1 with small …

Keratinocyte growth factor and coeliac disease

BACKGROUNDCoeliac disease is characterised by increased epithelial renewal associated with a mucosal T cell response to gliadin. Keratinocyte growth factor (KGF) is produced by cytokine activated gut stromal cells and...

Immunologic evidence of no harmful effect of oats in celiac disease

It was recently shown that antiendomysial antibodies (EMAs), which are highly sensitive and specific for celiac disease, are produced by intestinal mucosa. Furthermore, EMAs were detected previously in supernatant fluid from cultured duodenal mucosa specimens collected from untreated celiac disease patients and in culture media of biopsy specimens collected from treated celiac disease patients after an in vitro challenge with gliadin. Moreover, it was recently shown in vivo that oats are not toxic to celiac disease patients, suggesting the safety of oats in a gluten free-diet. The objective was to better define the controversial role of oats in celiac disease to determine whether oats can be safely included in a gluten-free diet. We used an in vitro model to test whether oats induce EMA production in supernatant fluid from cultured duodenal mucosa specimens collected from 13 treated celiac disease patients. The biopsy specimens were cultured with and without peptic-tryptic digest (PT) of gliadin and avenin (from oats) and in medium alone. Samples from 5 of the 13 patients were cultured with the C fraction of PT-avenin. Indirect immunofluorescence was used to detect EMAs. EMAs were detected in specimens from all 13 patients after the challenge with gliadin but not after culture in medium alone. By contrast, no EMAs were detected in any of the specimens cultured with PT-avenin and its C fraction. Because the in vitro challenge with PT-avenin and its C fraction did not induce EMA production in treated celiac disease patients, it appears that oats have no harmful effect on celiac disease. Therefore, oats can be safely included in a gluten-free diet.

Induction of apoptosis in Caco-2 cells by wheat gliadin peptides

Recent experimental evidence suggests that enterocyte apoptosis is greater than hitherto assumed and may be responsible for villous atrophy in coeliac disease. We have previously demonstrated that a small peptide (M.W. 1157.5 Da), identified as the sequence H 2N–gln–gln–pro–gln–asp–ala–val–gln–pro–phe–COOH from durum wheat gliadin, is able to prevent K 562 (S) cell agglutination induced by the peptic-tryptic digests (PT) of prolamin fractions from the cereals which are not tolerated in coeliac disease (i.e. bread wheat, rye, barley and possibly oats), and toxic A-gliadin peptides in coeliac disease. In the present study we have investigated the effects of the bread wheat gliadin digest (PT) on apoptosis of Caco-2 cells and whether the ‘1157.5’ Da peptide may in any way interfere with them. We evaluated both earlier biochemical and later morphological nuclear apoptotic events in the human colon adenocarcinoma cell line Caco-2. After 48 h exposure to the PT gliadin digest and the ‘1157.5’ Da peptide, apoptosis was detected both for the early-stage apoptotic cells (adherent cells) and the late-stage apoptotic ones (detached cells which were floating in the culture medium). Exposure to the PT gliadin digest resulted in a high percentage of adherent cells that underwent cell death by apoptosis (about 30%), independent of the concentration range used; while the presence in the culture medium of peptide ‘1157.5’ Da determined complete inhibition of cell death. On the other hand, morphological nuclear modifications observed in the floating cells showed a difference in the rate of the apoptosis dependent on the PT concentration, with partial protection in the presence of the peptide. These findings show an action of bread wheat gliadin peptides leading to cell death by apoptosis in the Caco-2 cell line and that the ‘1157.5’ Da peptide is capable of preventing such an effect.

Enhanced peptide-binding capacities of small intestinal brush border membranes in celiac disease.

In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n = 49) and treated celiac patients on a gluten-free diet (n = 30), as well as control subjects (n = 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8-19 (G XIV) and 30-41 (G XI) of alpha-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/microg (95% confidence interval 116.0-128.9) vs 108.9 (102.1-115.7)] and of zein peptide [50.2 (38.4-61.9) vs 28.8 (13.4-44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6-70.5) vs 62.4 (56.3-68.5) and G XI [75.2 (69.8-80.6) vs 65.9 (55.2-76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells.

THE MAJORITY OF RELATIVES OF CELIAC PATIENTS WITH POSITIVE RECTAL GLUTEN CHALLENGE HAVE A LOW GRADE SMALL INTESTINAL ENTEROPATHY

111 A significant infiltration of CD3 and γδ+ lymphocytes is induced in the rectal mucosa of coeliac patients by a gliadin enema. Interestingly, a subset of siblings of coeliac children also react to rectal instillation of gliadin. Aim of this study has been to correlate in coeliac relatives the results of the rectal gluten challenge with the immunohistochemical features of the small intestinal mucosa, particularly with density of cells infiltrating the epithelium and signs of activated T cell mediated immunity. In 16 treated coeliac children, 10 disease-controls and 30 siblings of coeliacs, rectal biopsies were obtained before and 6 hours after rectal challenge with a peptic-tryptic digest of gliadin (Gastroenterology 1996; 111: 318-24 ). CD3+ and γδ+ lymphocytes were counted and a discriminant score was calculated. Jejunal biopsies were obtained 13 siblings and cryostat sections were incubated with antiCD3, antiTCRγδ, antiCD25 (IL2 receptor), antiCD54 (ICAM1) antibodies, and processed by immunohistochemistry (PAP or APAAP techniques). After local instillation of gliadin, in coeliac patients, but not in controls, immunohistochemistry showed a significant increase in CD3+ and γδ+ cells. The discriminant analysis allowed correct classification of 100% coeliacs and controls, while 11/30 (37%) siblings were classified as coeliacs. All of them had a negative serology (AGA, EMA) and a normal architecture of the jejunal mucosa. Nevertheless, the immunohistochemical analysis of the jejunal biopsies showed in 5/6 siblings with a positive response to rectal gluten challenge, but only in 1/7 of those with a negative response, increased density of CD3+ and/or γδ+ lymphocytes. In no case increased number of CD25+ cells or enhanced expression of ICAM 1 was observed. Our results demonstrate that in siblings of coeliac patients the positivity to rectal gluten challenge is often accompanied in the small intestine by a low-grade enteropathy characterized by a lymphocyte infiltration of the epithelium. The rectal gluten challenge is proposed as a useful tool to detect an abnormal mucosal immune response to gluten.

Agglutinating activity of alcohol-soluble proteins from quinoa seed flour in celiac disease.

The edible seeds of the quinoa plant contain small quantities of alcohol-soluble protein which, after peptic-tryptic digestion, are unable to agglutinate K562(s) cells. When separated by affinity chromatography on sepharose-6B coupled with mannan, peptic-tryptic digest separated in two fractions. Fraction B peptides (about 1% of total protein) were shown to agglutinate K562(s) cells at a very low concentration, whereas peptides in fraction A and in the mixed fraction A+B were inactive, suggesting that fraction A contains protective peptides that interfere with the agglutinating activity of toxic peptides in fraction B.

In vitro cytotoxic effect of wheat gliadin-derived peptides on the Caco-2 intestinal cell line is associated with intracellular oxidative imbalance: implications for coeliac disease

Coeliac disease (CD) is an inflammatory disorder of the upper small intestine in which gluten acts as an essential factor in its pathogenesis. Although it is generally accepted that cereal protein activation of the immune system is involved in CD progression, a non-immunomediated cytotoxic activity of gliadin-derived peptides on the jejunal/duodenal tract cannot be excluded. In this work, considering that (a) little has been reported about the intracellular metabolic events associated with gliadin toxicity, and (b) an important role for free radicals in a number of gastrointestinal disease has been demonstrated, we investigated the in vitro effects of gliadin-derived peptides on redox metabolism of Caco-2 intestinal cells during a kinetic study in which cells were exposed to peptic–tryptic digest of bread wheat up to 48 h. We found that the antiproliferative effects displayed by gliadin exposure was associated with intracellular oxidative imbalance, characterised by an increased presence of lipid peroxides, an augmented oxidised (GSSG)/reduced (GSH) glutathione ratio and a loss in protein-bound sulfhydryl groups. Significant structural perturbations of the cell plasma membrane were also detected. Additional experiments performed by using the specific GSH-depleting agent buthionine sulfoximine provide evidence that the extent of gliadin-induced cell growth arrest critically depends upon the ‘basal’ redox profile of the enterocytes. On the whole, these findings seem to suggest that, besides the adoption of a strictly gluten-free diet, the possibility for an adjuvant therapy with antioxidants may be considered for CD patients.

Gliadin activates mucosal cell mediated immunity in cultured rectal mucosa from coeliac patients and a subset of their siblings

Background—CD3 and γδ cells in the rectal mucosa increase after local instillation of gluten in children with coeliac disease and in half of their siblings.Aim—To establish an in vitro system...

Structural specificities and significance for coeliac disease of wheat gliadin peptides able to agglutinate or to prevent agglutination of K562(S) cells

Two peptides corresponding to bread wheat A-gliadin fragments 31–43 and 44–55, well known for their ability to damage the coeliac disease intestinal mucosa both in vitro and in vivo, have been confirmed to be very active in inducing in vitro agglutination of K 562 (S) cells. Removal of six amino acid residues from the carboxy-terminal end of the 31–43 peptide, or of five amino acid residues from the amino terminal end of the 44–55 peptide, resulted in a lower, but still very significant, cell agglutination activity. The peptide consisting of ten amino acid residues with a molecular mass of 1157.5 Da, isolated from durum wheat gliadin, was able to prevent agglutination of K 562 (S) cells induced not only by prolamine peptic–tryptic digests from all the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oats), but also by the 31–43 and 44–55 peptides. The ability to protect K 562 (S) cells from agglutination was exhibited to the fullest extent also by all the peptides derived from the 1157.5-Da peptide by five progressive deletions of the terminal carboxylic residue, whereas the sixth consecutive deletion yielded a completely inactive peptide. A similar total loss of activity was observed upon addition of a glycine residue to the amino terminal residue of the 1157.5-Da peptide and all the above-mentioned active peptides derived from it. The remarkable sequence homologies existing between peptides able to induce [Gln–Gln–Gln–Pro– and –Pro–Ser–Gln–Gln–] or to prevent [H 2N–Gln–Gln–Pro–Gln–Asp–COOH] induction of cell agglutination strongly suggest that all these peptides compete for identical or structurally related binding sites on the cell surface.

Majority of gliadin-specific T-cell clones from celiac small intestinal mucosa produce interferon-gamma and interleukin-4.

An abnormal mucosal cell-mediated immune response plays a fundamental role in the pathogenesis of celiac disease. To characterize locally infiltrating T cells, gliadin-specific T-cell clones were isolated from two treated celiac patients. Mucosal biopsies were cultured in vitro for 24 hr with a peptic-tryptic digest (PT) of gliadin. T-cell clones (TCC) were then isolated by limiting dilution. The production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) was evaluated by ELISA in culture supernatants obtained after a short incubation with anti-CD3 and PMA, or with antigen. Twenty-two TCC were specific for gliadin and/or PT. All were CD3+, CD4+, CD8-, TCR alphabeta+. In one such clone the PT-specific response was inhibited by an anti-DQ, but not by an anti-DR antibody. Of the five gliadin-specific TCC examined, four produced IL-4 and high levels of IFN-gamma; the remaining one initially produced only IL-4, but subsequently also IFN-gamma. All clones obtained from the celiac mucosa, including the gliadin-specific ones, produced high levels of IFN-gamma, in most cases with IL-4. This cytokine profile could explain most of the immunological features of the celiac mucosa.