Peptic-tryptic Digest Research Articles

A small peptide from durum wheat gliadin prevents cell agglutination induced by prolamin-peptides toxic in coeliac disease

A peptide (m.w. 1157.5 Da) able to prevent the agglutination of K562(S) cells induced by the peptic-tryptic prolamine digests of the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oat) was characterized as one of the components of the peptic-tryptic digest of durum wheat gliadin. This peptide was synthesized in a high degree of purity with the solid phase method with the Applied Biosystem 431 A. An amino acid sequence was identified in the 1157.5 Da peptide as being related to the largest common sequences previously detected in a series of bread wheat toxic peptides by other authors.

GLIADIN ACTIVATES MUCOSAL CELL-MEDIATED IMMUNITY IN IN VITRO CULTURED RECTAL MUCOSA FROM COELIAC PATIENTS AND A SUBSET OF THEIR SIBLINGS

An increment in CD3+ and γδ+ cells in the rectal mucosa has been reported after local instillation of gluten in children with coeliac disease and in half of their siblings. Aim of this study was to establish an in vitro system to fully assess immunological changes induced by gluten in the rectal mucosa. Rectal biopsy specimens were obtained from 11 children with treated coeliac disease, 9 controls and 8 siblings of coeliac children, and in vitro cultured for 24 hours in the presence of 1 mg/ml of a peptic-tryptic digest of gliadin (PT) or ovalbumin (OVA) as control protein. At the end of the culture, biopsies were snap frozen and an immunohistochemical analysis carried out. CD3+ and CD25+ cells were counted and the expression of adhesion molecules ICAM1, ELAM1, VCAM1 also evaluated. In the lamina propria of coeliac biopsies cultured in the presence of PT, but not in those from controls, the expression of VCAM1 molecules was enhanced, and the number of CD25+ cells (mean±SD: 413±142/mm2) significantly (p<0.001; paired t test) higher than in biopsies cultured in medium alone (206±88); also the density of intraepithelial CD3+ cells was significantly higher (19.6±10.2/100 epithelial cells vs 10.8±5.8; p<0.05). No differences were noted in coeliac biopsies cultured with OVA. A discriminant analysis of immunohistochemical data allowed correct classification of 100% coeliacs. 4 out of 8 siblings (50%) were also allocated in the coeliac group. In conclusion, our data confirm that gliadin is able to activate mucosal cell-mediated immunity in the rectal mucosa in coeliac patients, and in a subset of their first-degree relatives. The ease of access and the validation of such in vitro system render the rectal mucosa particularly suitable to study immunogenicity of cereal proteins and peptides and to screen gluten-sensitive subjects.

THE IN VITRO-INFLUENCE OF GLIADIN PEPTIDES ON THE HYDROLASES OF THE DUODENAL BRUSH BORDER MEMBRANE IN COELIAC DISEASE IN REMISSION

Aims: The molecular mechanism inducing the mucosal damage in coeliac disease (CD) is still unknown. Gliadin causes villus atrophy in CD, and patients on a gluten-free diet recover completely. On the basis of the organ culture of intestinal biopsies we developed an in vitro-system for CD in remission. We used this model to verify our hypothesis postulating a correlation between the mucosal damage caused by gliadin and the biosynthesis of the intestinal hydrolases. Besides we measured the enzyme activity to prove whether this level is afflicted additionally or alternatively. Methods: Intestinal biopsies from 14 coeliac patients in remission (following a gluten-free diet for at least 2 years) and 23 healthy children (control group) were organ cultured for 24 hours in presence or absence of gliadin peptides. A peptictryptic digest of maize prolamine operated as the non-toxic control. The effect of these additives on the enzyme activity of the disaccharidases was determined by a modificated method of Dahlqvist. The influence on the biosynthesis of several hydrolases (LPH, SI, MGA, ACE, APN, DPP IV) was studied by biosynthetical protein-labelling during organ culture, followed by immunoprecipitation, SDS-PAGE, autoradiography and quantified by densitometric scanning. Results: In our model we demonstrated a rapid and specific gliadin-induced reduction of the biosynthesis of all studied hydrolases in biopsies from coeliac patients on gluten-free diet. This phenomenon was neither seen after incubation with maize prolamine nor in healthy children. We could not show an effect of gliadin on the disaccharidase activities after 24 hours of incubation. Because of beginning necrosis in the biopsies a longer incubation was not possible, so we were not able to examine later effects. Conclusions: Our model is suitable for studying the in vitro-situation of CD in remission. Moreover, our results indicate a fast effect of gliadin peptides on inhibiting the biosynthesis of hydrolases of duodenal brush border membranes. It remains unclear whether the gliadin-induced reduction primarily influences the protein biosynthesis or has to be interpreted as a secondary effect of an impaired mRNA synthesis. We suppose that the effect occurs at the translational or transcriptional level. The early modifications within 24 hours of incubation are hardly to explain by activities of the immune system. More likely the results suggest a toxic effect of gliadin peptides on the intestinal mucosa preceeding immunological changes mediated by cytokines.

Hydrophobic interactions between gliadin and proteins and celiac disease.

Gliadin-protein interaction and its relationship to the pathogenesis hypotheses of celiac disease was investigated. Wheat germ agglutinin was not immunodetected in gliadin preparations. Peptic-tryptic gliadin digest was used to study the gliadin-protein interactions by crossed immunoelectrophoresis and affinity blotting. Biotinylated gliadin digest interacted with IgG and bovine serum albumin but not with several glycoproteins. Since albumin and IgG light chains are not glycosylated, this interaction is not lectin-like, neither completely immunological because of recognition of the IgG Fc fraction. Immobilized and boiled IgG was not recognized by gliadin digest as a lectin. Gliadin digest fractions from T-gel chromatography reduced the fluorescence intensity of cis-parinaric acid bound to albumin. The gliadin-protein interaction is not lectin-like or completely immunological but hydrophobic. Hydrophobicity of gliadins may contribute to the pathogenic events that result in celiac disease.

Inhibition of the cellular metabolism of Caco-2 cells by prolamin peptides from cereals toxic for coeliacs

Peptic-tryptic (PT) digests of prolamins derived from several cereals were tested on differentiated Caco-2 cells to study the effect on cellular metabolism, particularly on DNA, RNA, protein and glycoprotein synthesis. Cell viability was evaluated after treatment with the same cereals. Whereas PT digests from bovine serum albumin and both durum wheat types (diploid and tetraploid) did not exert any effect, bread wheat, oats, barley and rye exerted an inhibitory effect close to 80% for DNA and RNA synthesis and close to 60% for (glyco)protein synthesis. Cell viability evaluated by MTT tests did not show any differences between treated and untreated cells. These observations, and previous results, suggest that, whereas prolamin-derived peptides from bread wheat, barley, rye and oats did not cause an immediate cytotoxic effect, they, were however, responsible for cell damage impairing cell metabolism.

In siblings of celiac children, rectal gluten challenge reveals gluten sensitization not restricted to celiac HLA

BACKGROUND & AIMS: Inflammatory changes in the rectum of patients with celiac disease after local instillation of gluten have been reported. The aim of this study was to examine rectal mucosa after local gluten challenge in children with celiac disease and their siblings. METHODS: Rectal biopsy specimens were obtained before and 6 hours after rectal challenge with a peptictryptic digest of gliadin in 33 children with treated celiac disease, 12 controls, and 19 siblings of children with celiac disease. Epithelium and lamina propria volumes were determined, and CD3+ and gamma delta + lymphocytes were counted. RESULTS: After local instillation of gliadin, a significant increment in the absolute number of intraepithelial lymphocytes was noted in patients with celiac disease but not in controls. Immunohistochemical analysis showed a significant increase in CD3+ and gamma delta + cells, with the gamma delta/CD3 ratio remaining unchanged after challenge. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis was used to classify 6 of 13 siblings as having celiac disease. The positivity was not associated with the presence of the heterodimer encoded by the DQA*0501 DQB1*0201 alleles in any of the siblings. CONCLUSIONS: All patients with celiac disease were identified by rectal gluten challenge. Approximately half of the siblings reacted to rectal instillation of gluten. The genetic background of such sensitization to gluten remains to be elucidated. (Gastroenterology 1996 Aug;111(2):318-24)

Peptic-tryptic digests of gliadin: contaminating trypsin but not pepsin interferes with gastrointestinal protein binding characteristics

For many years, peptic-tryptic digests of gliadin, known as Frazer's fraction III, have been used in investigations of gliadin effects. Potential contamination by the proteases pepsin and trypsin, however, was not considered. To investigate the influence of contaminating proteases on binding of gliadin peptides to rat small intestinal brush border membranes we compared binding characteristics of different gliadin digests. Binding of biotinylated probes was studied in dot blots and Western blots with an enhanced chemiluminescence system. In gliadin peptide preparations only contaminating trypsin, but not pepsin, was detectable by specific antisera. Digestion with insoluble proteases attached to cross-linked beaded agarose yielded gliadin peptides free of contaminating pepsin and trypsin. These peptides bound 30% less to brush border membranes. Using these peptides, there was no trypsin-typical binding pattern to low molecular mass membrane proteins in contrast to peptide preparations which contained contaminating trypsin. In conclusion, contaminating trypsin might alter gliadin peptide binding characteristics by direct binding to brush border membranes and by interfering with interactions between gliadin peptides and brush border membranes.

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 Trificum 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.

Toxicity of Bread Wheat Lines Lacking Prolamins Encoded by theGli-B1/Gli-B5/Glu-B3andGli-D1/Glu-D3Loci in Coeliac Disease as Determined by their Agglutinating Activity

Peptic-tryptic (PT) digests of alcohol-soluble proteins from the flour of three mutant lines of bread wheat, lacking γ-gliadins, γ-gliadins and low molecular-weight glutenin subunits encoded by the Gli-B1/Gli-B5/Glu-B3 loci (line S. Pastore 4A), the Gli-D1/Glu-D3 loci (line Alpe 1I-) or both groups of loci (line DM 22166), were compared with those of the normal cultivars S. Pastore and Alpe 1 I for their agglutinating activities on human myelogenous leukemia K562(S) cells, agglutination being strongly associated with toxicity for the coeliac intestine. All of the genotypes tested contained A-type α-gliadins, which constituted about 19% of the gliadins in the S. Pastore and Alpe 1I cultivars, 24.5% in the S. Pastore 4A and Alpe 1I null lines, and 34.8% in the double mutant line, DM 22166, as determined by densitometric scanning of their acid polyacrylamide gel electrophoresis patterns. The minimal concentrations of PT digest required to agglutinate 100% of K562(S) cells were 73mg/l and 96mg/l, in the S. Pastore and Alpe 1I cultivars, respectively, compared with 146mg/l, 138mg/l and 200mg/l in the “null” lines, S. Pastore 4A, Alpe 1 I-and DM 22166, respectively. The results indicated that proteins other than α-gliadins are involved in the gluten-sensitive enteropathy.

In vitro assessment of acetic-acid-soluble proteins (glutenin) toxicity in celiac disease.

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 A controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 10(7) Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and alpha/beta- and gamma-gliadins.

In vitro assessment of acetic‐acid‐soluble proteins (glutenin) toxicity in celiac disease

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 Å controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 107 Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and α/β-and γ-gliadins. © 1997 John Wiley & Sons, Inc.

Characterization of human, mouse and rabbit anti-gliadin antibodies by ELISA and western blotting.

Monoclonal, hyperimmune rabbit and human serum anti-gliadin antibodies were analyzed by ELISA and immunoblotting techniques. In Western blotting the difference in reactivity between monoclonal and human antibodies was quantitative rather than qualitative. Rabbit antisera differed in reactivity according to the protein used for immunization. The rabbits immunized by the peptic-tryptic pancreatic digest of gliadin reacted similarly to the patients. In ELISA, significantly higher reactivity with crude, A-, glyc-gli, alpha-, beta- and omega-gliadins was found in the patients' sera than in controls.

Binding of gliadin to lymphoblastoid, myeloid and epithelial cell lines.

The aim of our work was to investigate the in 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 alpha-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 inhibit the agglutination of K562 cells with peptic-tryptic- but not with peptic-tryptic pancreatic digest of alpha-gliadin. The lack of 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.

Absence of gluten-specific T lymphocytes in the skin of patients with dermatitis herpetiformis.

Dermatitis Herpetiformis (DH) is an immunobullous skin disease with an associated gluten-sensitive enteropathy. Withdrawal of gluten from the diet leads to the resolution of both the skin lesions and the enteropathy. A T cell-mediated immune response to gluten has been implicated in the damage to the gut; the possible gluten specificity of the T cell infiltrate in DH skin lesions has not, however, been investigated. T cell lines (TCL) were therefore established from the skin lesions of eight patients with DH by culturing skin fragments for 11-17 days with a medium supplemented with 20 U/ml of IL-2. In three cases, gliadin (fraction of gluten toxic to the DH gut) and irradiated, autologous peripheral blood mononuclear cells were also added. The TCL were stained for CD3, CD4, CD8, TCR alpha beta and gamma delta expression by indirect immunofluorescence, and their proliferative responses to mitogens and gluten fraction III (a peptic-tryptic digest of gluten) investigated. Of the eight CD3+ TCL, four were predominantly CD4+ (82.1-98.8%), three predominantly CD8+ (92.6-98.6%) and one TCL contained both 87.6% CD4+ and 95.2% CD8+ T cells, a substantial proportion of which were presumably double-labelled CD4+, CD8+ T cells. All eight TCL, which were almost exclusively TCR alpha beta +, proliferated in response to PHA whilst six out of the eight were stimulated by Concanavalin A. None of the TCL proliferated to gluten fraction III alone; however, two TCL showed increased proliferation to the antigen in the presence of exogenous IL-2 or IL-4 (10 U/ml) compared to cytokine alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Gluten Stimulation of Coeliac Mucosa In Vitro Induces Activation (CD25) of Lamina Propria CD4H T cells and Macrophages but no Crypt‐Cell Hyperplasia

Jejunal biopsy specimens from 10 patients with treated coeliac disease and seven non-coeliac controls were challenged in vitro with peptic-tryptic gluten digest. Mucosal T cells were examined in situ by three-colour immunofluorescence staining for expression of the activation marker CD25 (the p55 alpha-chain of interleukin-2 receptor) and the nuclear proliferation marker revealed by monoclonal antibody Ki-67. Intraepithelial T cells expressed CD25 rarely whereas the proportion of activated lamina propria T cells increased (P < 0.002) from median 2.8% (cultured with 20% fetal calf serum alone for 24-48 h) to 10.0% after 24 h with gluten (n = 10; range 1.1-17.4%) and to 10.4% after 48 h (n = 7; range 1.4-17.5%). Such gluten-induced increase of CD25+ T cells was not observed in specimens from non-coeliac control subjects. Crypt-cell hyperplasia and T-cell proliferation (Ki-67+) were observed neither in the coeliac nor in the control mucosae after gluten stimulation. Three-colour staining combining a polyclonal antibody reagent to CD3 and a monoclonal antibody to CD25 with a monoclonal antibody to CD45R0, CD4, CD8, the p75 beta-chain of interleukin-2 receptor, integrin alpha E beta 7, or HLA-DR showed that most of the CD25+ T cells (> 90%) were CD4+CD8-, co-expressed CD45R0 and the p75 beta-chain, and often also the integrin alpha E beta 7 but not HLA-DR. In addition to these activated T cells, a dominating population of CD25+CD3-CD4+ subepithelial pan-HLA-class II+ macrophages (CD68+) with variable expression of the p75 beta-chain was often induced by gluten challenge.

Coeliac disease: characterisation of monoclonal antibodies raised against a synthetic peptide corresponding to amino acid residues 206-217 of A-gliadin.

A dodecapeptide of A-gliadin, which shares amino acid homologies with the E1b protein of adenovirus 12, was used to produce murine monoclonal antibodies. Five monoclonal antibodies were produced and were screened by enzyme linked immunosorbant assay, immunodot assay, and immunoblotting. The antibodies were tested against whole wheat gliadin and its alpha, beta, gamma, and omega subfractions, and the prolamins of rye, barley, oats, maize, millet, rice, and sorghum. Four of the five antibodies cross reacted with one or more of the coeliac non-toxic cereals--maize, millet, sorghum, and rice. The monoclonal antibody that did not cross react with these non-toxic cereals, did not recognize Frazer's fraction III, a peptic-tryptic digest of wheat gluten which is known to be toxic. The results suggest that the A-gliadin dodecapeptide shares a region of homology with cereals that do not exacerbate coeliac disease. This study does not support the hypothesis that prior infection with adenovirus 12 is a precipitating factor in coeliac disease.

Gliadin induced changes in the expression of MHC-class II antigens by human small intestinal epithelium. Organ culture studies with coeliac disease mucosa.

Jejunal biopsies from 16 treated coeliac disease patients and from nine controls were cultured with and without a peptic-tryptic digest of gliadin. Cultures with a peptic-tryptic digest of maize prolamins were also undertaken. Frozen sections of baseline and cultured mucosa were stained by immunofluorescence with an anti-HLA-DR monoclonal antibody. Before culture the villous epithelium from both controls and treated coeliac disease expressed DR molecules while the crypt epithelium did not. When biopsies from treated coeliac disease were cultured with gliadin the expression of DR was enhanced in the crypt epithelium in eight of 14 cultures and in 11 of 14 was reduced or absent on the villous epithelium. No change was observed in control cultures. We conclude that gliadin is capable of inducing HLA-DR on the crypt epithelium of in vitro cultured coeliac disease mucosa, providing indirect evidence that gliadin may activate cell mediated immune mechanisms within the small bowel mucosa. This model could prove useful in identifying the immunogenic sequence(s) of gliadins and related prolamins.

Coeliac active peptides from gliadin: large-scale preparation and characterization.

Larger amounts of coeliac active peptides are required for pathogenetic investigations. Therefore, a simplified preparative procedure by means of gel-permeation chromatography and reversed-phase HPLC was developed for the isolation of the peptides B3141-B3146, which are present in peptic tryptic digests of gliadin [this journal (1983) 176:85-94]. The peptides are derived from the N-terminal part of alpha-gliadins and are closely related. The amino acid sequence of B3143 is VPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQQFPPQQPYPQPQPFPSQQPYL. B3144 has proline instead of glutamine in position 34. The previously described peptide B3142 [this journal (1984) 179:371-376] corresponds to B3144 except for the missing C-terminal leucine.

Rectal gluten challenge and diagnosis of coeliac disease

44 patients referred consecutively for jejunal biopsy underwent rectal gluten challenge with 2 g peptic-tryptic digest (Frazer's fraction III; FF3). Rectal biopsy was done before the challenge and 6 h afterwards. Total intraepithelial lymphocytes (IEL) overlying a 10 4 μm 2 test area of muscularis mucosae were quantified by computerised image analysis. The subjects comprised 21 controls with disorders other than coeliac disease and 23 patients (14 treated, 9 untreated) with coeliac disease diagnosed by strict jejunal biopsy gold standard criteria. There was no difference between the groups in IEL numbers before challenge. Coeliac disease patients but not controls responded to FF3 with a rise in mucosal IEL (median 60·5% rise for treated, 63·0% for untreated). There was no response to challenge with β-lactoglobulin in coeliac disease or control subjects. When a predefined, post-challenge IEL "predictive index" of more than 10% above baseline was used to indicate a diagnosis of coeliac disease, it gave a sensitivity of 90% and specificity of 91% (95% confidence intervals 78-93%). Rectal gluten challenge is a simple, safe, and reliable test of gluten sensitivity, both as a screening test for untreated coeliac disease and as a confirmatory test in patients with treated coeliac disease.

Studies of intestinal lymphoid tissue. XII. Epithelial lymphocyte and mucosal responses to rectal gluten challenge in celiac sprue

The immunopathologic, structural, and functional changes within rectal mucosa of known celiac sprue subjects were quantitated during local challenge with a peptic-tryptic digest of gluten. In the celiac sprue patients challenged with 2 g of digest, major effects occurred in lamina propria, submucosa, and local microvasculature. The lamina propria swelling was biphasic, starting 1–2 h after challenge with widespread extravascular deposition of fibrinogen, indicative of increased microvascular permeability, receding by 24 h postchallenge. A rapid fall in mast cells together with granule discharge suggested their involvement in this response. The late-phase swelling (48–72 h) was preceded by a rapid influx of neutrophils and basophils, the latter showing evidence of degranulation beyond 72 h. Reestablishment of vessel lumina, a rise in mast cells, and loss of neutrophils indicated tapering of the inflammatory cellular cascade by 96 h. Lymphocytes, first seen to enter the lamina by 2 h postchallenge, increased progressively, thereby resulting in substantial infiltration between 36 and 96 h. A marked rise in epithelial lymphocytes, maximal at 6–8 h, waned by 24 h. Volumes of surface and crypt epithelium remained constant throughout. In another challenge series with 4 g of gluten digest, electrical potential difference across rectal mucosa decreased significantly 12 h postchallenge, but the associated decreases in net sodium and chloride absorptive fluxes were insignificant. It is concluded that rectal mucosa is sensitized to gluten in celiac sprue disease and thus offers a promising and convenient in vivo substrate for investigative and diagnostic purposes.