Binding of gliadin to lymphoblastoid,myeloid and epithelial cell lines

The aim of our work was to investigate thein vitro reactivity of gliadin peptides of natural and synthetic origin with various cell lines. We have found that all tested cell lines of human, mouse and rat origin were agglutinated by enzymically digested gliadin (peptic-tryptic-and peptic-tryptic pancreatic digest of α-gliadin) in a concentration dependent manner. In order to test the specificity of binding, inhibition studies were performed using a panel of sugars as well as natural and synthetic peptides derived from gliadin. We have found that among twelve tested sugars only fetuin and phosphomannan were able to inhibition by gliadin peptides and most of the saccharides suggests that agglutinating activity of gliadin is the result of a nonspecific binding of gliadin to the cell membrane. Dedicated to Professor J. Šterzl on the occasion of his 70th birthday     

Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.     

Sourdough Bread Made from Wheat and Nontoxic Flours and Started with Selected Lactobacilli Is Tolerated in Celiac Sprue Patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.     

Separation of coeliac-active peptides from bread wheat with the aid of methylpyrrolidinone chitosan

Gliadin-derived peptides from haxaploid wheat, known to be toxic for in-vitro cultured small-intestinal mucosa from coeliac patients, are very active in agglutinating K562(S) cells. Chromatographic data obtained with the use of methylpyrrolidinone chitosan coupled to Sepharose-6B indicate that a minor (1·3%) fraction of the peptides binds very strongly to the support at nearly neutral pH values, and can be eluted at pH 2·85. This fraction is the one responsible for the agglutination of K562(S) cells, being one-hundred times more active than the digest from which it has been separated.     

Component plane presentation integrated self-organizing map for microarray data analysis

Wheat gliadin and other cereal prolamins have been said to be involved in the pathogenic damage of the small intestine in celiac disease via the apoptosis of epithelial cells. In the present work we investigated the mechanisms underlying the pro-apoptotic activity exerted by gliadin-derived peptides in Caco-2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that digested peptides from wheat gliadins (i) induce apoptosis by the CD95/Fas apoptotic pathway, (ii) induce increased Fas and FasL mRNA levels, (iii) determine increased FasL release in the medium, and (iv) that gliadin digest-induced apoptosis can be blocked by Fas cascade blocking agents, i.e. targeted neutralizing antibodies. This favors the hypothesis that gliadin could activate an autocrine/paracrine Fas-mediated cell death pathway. Finally, we found that (v) a small peptide (1157 Da) from durum wheat, previously proposed for clinical practice, exerted a powerful protective activity against gliadin digest cytotoxicity.     

The activity of wheat gliadin peptides in in vitro assays for coeliac disease

A fracation of a peptide-tryptic-pancreatinic digest of wheat gliadin (fraction 9), known to be toxic to individuals with coeliac disease, together with synthetic peptides containing key gliadin sequences, were tested for their effects on foetal chick intestine and on rat liverr lysosomes. Fraction 9 and a dodecapeptide corresponding to residues 75–86 of A-gliadin (RPQQPYPQPQPQ) were the only peptides to display appreciable activity in both assays. A synthetic hexapeptide corresponding to residues 76–85 was non-toxic in both assays. Two serine-containing peptides containing the key sequence PSQQ were also tested but were found to be non-toxic, as was the hexapeptide PSQQQP. The results suggest that the key sequences QQQP and PSQQ are not sufficient by themselves to cause activity. Further tests on synthetic peptides will be necessary to define the sequence of highest toxicity.     

Agglutinating Effects of Concanavalin A on Isolated Protoplasts of Daucus carota

The agglutinating effects of Concanavalin A (Con A) on protoplasts isolated from cell suspensions of Daucus carota were studied. Con A was shown to agglutinate the plant protoplasts in a manner similar to the way some animal cells are agglutinated. The agglutination process is dependent on the Concanavalin A concentration, protoplast density, treatment time, the temperature, and the membrane condition, α-D-Methylgluco-pyranosid completely inhibited Con A induced agglutination. The results are discussed in relation to membrane structure and morphology.     

In vitro toxicity testing of alcohol-soluble proteins from diploid wheat triticum monococcum in celiac disease

Peptic-tryptic digests of alcohol-soluble proteins from flours of 10 accessions of Triticum monococcum with contrasting storage protein compositions and bread-making characteristics were found unable to agglutinate K562(S) cells even at a peptide concentration as high as 14 g/L, agglutination being strongly correlated with toxicity in celiac disease. When fractionated by affinity chromatography on Sepharose-6B coupled with mannan, peptic-tryptic digests separated into three fractions. Fraction C peptides were shown to agglutinate K562(S) cells, whereas peptides in fractions A and B and in the mixed fraction B + C were inactive, suggesting that fraction B contains “protective” peptides that interfere with toxic peptides in fraction C in their agglutinating activity. These results offer an opportunity to study the biochemical and genetic bases of wheat toxicity at the diploid level. Moreover, the reduced toxicity, if any, of Triticum monococcum in the celiac disease, along with the good grain characteristics of some “monococcum” accessions, greatly increases the economical prospects of this wheat species. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 313–318, 1997.     

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.     

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10(9) cfu/ml) hydrolyzed completely the alpha2-gliadin-derived epitopes 62-75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of alpha2-gliadin-fragment 62-75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3(+) intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.     

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue probiotics and gluten intolerance

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10⁹ cfu/ml) hydrolyzed completely the α2-gliadin-derived epitopes 62–75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of α2-gliadin-fragment 62–75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3⁺ intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.     

A new agglutinating activity from wheat flour inhibited by tryptophan

A new compound endowed with agglutinating activity, designated the flour agglutinin, was extracted from wheat flour with water and purified by gel filtration and ion-exchange chromatography. The haptenic inhibitors of the plant agglutinis do not affect flour agglutinin activity which, on the other hand, is inhibited by d- and l-tryptophan.Flour agglutinin has a molecular weight of about 5 · 10⁴ as determined by gel filtration. It consists of a neutral heteropolysaccharide constituted of d-xylose and l-arabinose, and is homogeneous as judged by sedimentation analysis. Flour agglutinin activity is destroyed by treatment with Cellulase 2000 and periodate, but is not affected by α-amylase and proteolytic enzymes.Compared to germ agglutinin, flour agglutinin exhibits a peculiar range of cell specificity. It agglutinates several normal cell types, but has no effects on some neoplastic cells tested. Tryptic digestion of erythrocytes does not affect their susceptibility to flour agglutinin-induced agglutination.     

Gliadin Fragments and a Specific Gliadin 33-mer Peptide Close KATP Channels and Induce Insulin Secretion in INS-1E Cells and Rat Islets of Langerhans

In non-obese diabetic (NOD) mice, diabetes incidence is reduced by a gluten-free diet. Gluten peptides, such as the compound gliadin, can cross the intestinal barrier and may directly affect pancreatic beta cells. We investigated the effects of enzymatically-digested gliadin in NOD mice, INS-1E cells and rat islets. Six injections of gliadin digest in 6-week-old NOD mice did not affect diabetes development, but increased weight gain (20% increase by day 100). In INS-1E cells, incubation with gliadin digest induced a dose-dependent increase in insulin secretion, up to 2.5-fold after 24 hours. A similar effect was observed in isolated rat islets (1.6-fold increase). In INS-1E cells, diazoxide reduced the stimulatory effect of gliadin digest. Additionally, gliadin digest was shown to decrease current through K_ATP-channels. A specific gliadin 33-mer had a similar effect, both on current and insulin secretion. Finally, INS-1E incubation with gliadin digest potentiated palmitate-induced insulin secretion by 13% compared to controls. Our data suggest that gliadin fragments may contribute to the beta-cell hyperactivity observed prior to the development of type 1 diabetes.     

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.     

Agglutination of blastospores of Candida albicans by concanavalin A and its relationship with the distribution of mannan polymers and the ultrastructure of the cell wall.

Blastospores of Candida albicans were readily agglutinated by Concanavalin A (Con A) owing to the specific binding of this lectin to the mannan receptors of the cell surface. When mannan was extracted from the cell wall by neutral buffers, alkali and acid, the agglutination was decreased or lost depending on the degree of extraction. A relatively mild alkali treatment was sufficient to derange the multilayered wall organization and transform it into a uniform, medium-density structure having about the same thickness as the untreated wall. After a more drastic extraction, all the electron-dense components of the wall were lost, the residual, alkali-insoluble wall fabric being completely electron-transparent and of about the same thickness as the inner wall region of untreated cells. Thiol-reducing agents like mercaptoethanol or dithiothreitol also extracted wall materials, an effect which was enhanced by pronase. After dithiothreitol-pronase treatment, the outer wall layers were removed but the inner wall region was not apparently damaged and some electron-dense components remained. None of these treatments significantly affected blastospore agglutination by Con A--this was reduced (but not abolished) only by the sequential action of pronase and helicase, which led to sphaeroplast formation. These sphaeroplasts showed a varied amount of residual wall consisting of evenly distributed, fibrogranular components. Two main conclusions were drawn from these results: (i) mannan polymers extend throughout the wall of the blastospore of C. albicans; (ii) the layering of the wall, as seen by ordinary fixation and staining for electron microscopy, essentially reflects the distribution of the various alkali-soluble complexes, at different levels, both over and in the rigid, glucan-chitin matrix.     

Celiac sprue: correlation with murine T cell responses to wheat gliadin components

Celiac sprue is a disease in humans that is characterized by small intestinal mucosal injury and malabsorption. Dietary exposure to gliadin and similar proteins in rye and barley activates the disease in susceptible individuals. Celiac sprue appears to be the only disease with a marked HLA-association in which the proteins that activate the disease currently are well known. However, bread wheat gliadins are a complex mixture of proteins that contain at least 40 different components. In the present study we have purified the major gliadin components of Scout 66 wheat and used these proteins to examine murine T cell proliferative responses to gliadin. Differences in T cell proliferation stimulated by alpha-, beta-, gamma-, and omega-gliadins paralleled the known structural differences among these proteins. After priming with whole gliadin, the components that stimulated T cell proliferation were the same as those recognized to activate celiac sprue in humans. Studies with reduced and alkylated A-gliadin (i.e., S-methyl A-gliadin) suggested that epitopes determined by the native conformation of A-gliadin may be important in its interaction with T cells. By using three different A-gliadin peptides that span the entire molecule, T cell proliferative responses were shown to be stimulated predominantly by antigenic determinants on the NH2-terminal peptide.     

Synthesis of a spore-specific surface antigen during sporulation of Saccharomyces cerevisiae

Antisera raised against purified yeast ascospores caused agglutination of both ascospores and vegetative cells. A spore-specific activity was obtained by absorbing out anti-vegetative activity with vegetative cells. The anti-vegetative cell activity was directed against mannan, and was probably due to exposure of some spore coat mannan at the spore surface since concanavalin A and lentil lectin also caused agglutination of ascospores. The spore-specific activity was probably determined by a protein or proteins, since extraction of spores with a mixture of sodium dodecyl sulphate and dithiothreitol markedly affected their agglutination by the spore-specific serum. The spore-specific antigen was synthesized in a soluble form during sporulation several hours before the appearance of the spore surface and the pool of soluble antigen declined as the spore was assembled. Synthesis of the soluble antigen was inhibited by adding cycloheximide at all times up to its first appearance in the sporulating cell.     

Cell surface differentiation of Chlamydomonas during gametogenesis. I. Mating and concanavalin A agglutinability

Sexual agglutination of opposite gametes and agglutination of like gametes by concanavalin A (Con A) were studied in Chlamydomonas moewusii and C. reinhardtii for the possibility that the surface sites involved in these agglutinating phenomena may be the same. Our data show that the two agglutinating phenomena appeared at different times after the beginning of gametogenesis. Sucrose and mannose block agglutination of the gametes by Con A but do not affect mating ability. Trypsin eliminates mating ability, except in (?) gametes of C. moewusii, while Con A agglutinability remains. Monovalent Con A can selectively bind to Con A-binding sites to block agglutination of gametes by multivalent Con A while mating ability is unaffected. The data indicate that the mating agglutinin and the Con A-binding sites are two different flagellar surface agglutinins that occur coincidentally during gametic differentiation of both mating types of C. moewusii and C. reinhardtii. The function of the Con A-binding site on Chlamydomonas gametes is not known.     

Lectin-mediated sperm agglutination of bufo arenarum and leptodactylus chaquensis spermatozoa

Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.