concanavalin-a and Celiac-Disease

In an approach to examine the lectin-hypothesis in the pathogenesis of coeliac disease, the presence of lectin-like components in three wheat gluten preparations known to induce coeliac disease, gliadin, Frazer fraction III and an acetic acid/ethanol extract of gluten, was investigated. Lectin-like components in these wheat gluten preparations were traced in binding studies employing a variety of model glycoproteins glycosylated with the different types of N-linked oligosaccharides, i.e., those of the high mannose-, complex- and hybrid-type. Binding affinity of wheat proteins to these glycoproteins was analyzed by affinity dotting and blotting techniques and was compared to that of the well characterized lectins Galanthus nivalis agglutinin, Concanavalin A and wheat germ agglutinin. Though the three wheat gluten preparations exhibited binding reactivity for distinct model glycoproteins, no correlation was found between the type of N-glycosylation of the model glycoproteins and their binding capability to the different wheat gluten preparations. Moreover, binding of the three gluten preparations to the model glycoproteins could not be inhibited by competitive saccharides (methyl-alpha-D-mannopyranoside, N-acetyl-D-glucosamine, mannan). Enzymatic deglycosylation of the ligand glycoproteins with endo-beta-N-acetylglucosaminidase H (Endo H, EC 3.2.1.96) or peptide N-glycosidase F (PNGase F, EC 3.5.1.52) abolished their binding reactivity for the plant lectins, but did not affect binding of the wheat gluten preparations. These results give no evidence for the presence of lectin-like components in wheat gluten preparations and do question the 'lectin hypothesis' of coeliac disease.

Topics: Amidohydrolases; Celiac Disease; Concanavalin A; Galanthus; Gliadin; Glutens; Glycoproteins; Glycosylation; Hexosaminidases; Humans; Intestinal Mucosa; Lectins; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Oligosaccharides; Pepsin A; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Plant Lectins; Triticum; Trypsin

Lymphocyte activation was examined by interleukin-2 (IL-2) receptor expression on peripheral blood mononuclear cells from coeliac and control subjects. Purified T cells were incubated with gluten fraction 111 (a known toxic peptide for coeliac subjects), soyabean hydrolysate (an unrelated hydrolysed food antigen), and Concanavalin-A (Con-A, a non-specific mitogen). After 1-5 days incubation, expression of IL-2 receptors was assessed using a cellular enzyme-linked immunosorbent assay (ELISA). Gluten fraction 111 induced expression of IL-2 receptors on T lymphocytes from coeliac but not from normal subjects (P = 0.0005), whereas soyabean hydrolysate did not induce IL-2 receptor expression. Lymphocytes from both coeliac and normal subjects had similar increased IL-2 receptor expression after incubation with Con-A. Flow cytometry was also used to confirm specific expression of IL-2 receptor expression of lymphocytes from coeliac subjects. Interleukin-2 receptor expression increased from 0 to 5.4% of cultured mononuclear cells after 7 days incubation with gluten fraction III. These cells were CD3-positive and CD4-positive. We conclude that peripheral blood lymphocytes from coeliac subjects are sensitized specifically to gluten fraction III.

Topics: Analysis of Variance; Celiac Disease; Cells, Cultured; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glutens; Humans; Immunophenotyping; Lymphocyte Activation; Plant Proteins, Dietary; Receptors, Interleukin-2; Soybean Proteins; T-Lymphocytes

Simultaneous feeding of gliadin and swainsonine, an inhibitor of alpha-D-mannosidases, in rats disturbed enterocytic maturation as shown by a marked loss of activities of alkaline phosphatase and gamma-glutamyltransferase. Morphologically, simultaneous treatment with gliadin and swainsonine caused destruction and decreased density of microvilli, as shown by electron microscopy. Neither gliadin nor swainsonine when given alone had significant effects on enterocytic enzyme activities or enterocytic morphology. Binding of enterocytic glycoproteins to both gliadin-Sepharose and concanavalin A-Sepharose was significantly increased in rats treated with swainsonine. Because swainsonine causes the formation of hybrid-type oligosaccharides with a high binding affinity to mannose-specific lectins, the observed alterations of enterocytic maturation and morphology are presumably caused by the increased binding of gliadin to enterocytic glycoproteins. A possible analogy in the etiology of celiac disease is discussed.

Topics: Alkaline Phosphatase; Alkaloids; Animals; Celiac Disease; Concanavalin A; Disease Models, Animal; Gliadin; Glycoproteins; Intestine, Small; L-Lactate Dehydrogenase; Male; Mannosidases; Microvilli; Plant Proteins; Protein Binding; Rats; Rats, Inbred Strains; Sepharose; Swainsonine

Different immune functions of 10 patients with glutein-sensitive enteropathy (GSE), 9 with Crohn's disease (CD), 11 with ulcerative colitis (UC) and 13 healthy controls were characterized. The numbers of suppressor T cells in GSE were comparable to those of the controls; otherwise, the lymphocyte subpopulations were decreased in these bowel diseases. In the whole-blood cultures, the lymphocyte proliferative responses to PHA were normal in the bowel diseases, but the responses to Con A were decreased in CD. In cultures with D-penicillamine, the inhibition of the helper effect of CD patients was more pronounced in PHA-stimulated cultures than in Con A-stimulated cultures. The total Ig and IgA production did not markedly differ among the groups. PWM-induced IgM secretion was significantly decreased in GSE, CD and UC, and IgG secretion in CD and UC, as compared to controls. In GSE, an increased Con A inducible suppressor cell activity was observed in the IgM production. Altogether, no clear-cut immunological imbalance was detected in any of the bowel diseases; this in agreement with previous works. However, there are some differences in the regulatory cell balance among the patients with GSE, CD and UC. The determination of lymphocyte proliferative responses to PHA and Con A together with D-penicillamine seems to provide a new immunological criterium for distinguishing between Chrohn's disease and ulcerative colitis.

Topics: Adult; Aged; Antibody Formation; Antigens, Differentiation; Celiac Disease; Colitis, Ulcerative; Concanavalin A; Copper; Copper Sulfate; Crohn Disease; Female; Humans; Immunoglobulins; Leukocyte Count; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Penicillamine; Phytohemagglutinins

Binding of 125I-crude gluten digest (Frazer's fraction III. FF-III) and 125I-concanavalin A (Con A) to isolated rat enterocytes and of 125I-FF-III to human enterocytes was investigated. Specific binding of 125I-FF-III to rat enterocytes was observed but binding was not inhibited by any of a range of simple and complex saccharides. although casein and bovine serum albumin displaced FF-III at high concentrations. Con A also bound to enterocytes in a specific manner and was inhibited by alpha-methyl-D-mannoside, confirming a lectin-mediated interaction. 125I-FF-III exhibited quantitatively similar specific binding to both normal human and coeliac enterocytes. The primary interaction of gliadin peptides with the enterocyte surface membrane is not lectin-mediated and unlikely to be of fundamental importance in the pathogenesis of coeliac disease.

Topics: Animals; Celiac Disease; Cell Membrane; Concanavalin A; Gliadin; Humans; Intestinal Mucosa; Male; Plant Proteins; Rats; Rats, Inbred Strains

The agglutinating properties of a crude gluten digest, purified gliadin fractions and established plant lectins were investigated using mammalian erythrocytes, rat enterocytes and normal and coeliac human enterocytes as the target systems. Gliadin preparations failed to cause agglutination of any of the cells tested, whereas established pure plant lectins were active cell agglutinins. These studies indicate that gliadin peptides do not interact with intestinal cells in a polyvalent, lectin-like manner and as such cannot be regarded as true lectins. Mucosal damage in coeliac disease is unlikely therefore to be related to lectin-like activity of gliadin.

Topics: Agglutination; Alkaline Phosphatase; Animals; Cattle; Celiac Disease; Cells, Cultured; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Gliadin; Guinea Pigs; Horses; Humans; Intestinal Mucosa; Lectins; Peanut Agglutinin; Phytohemagglutinins; Plant Proteins; Rabbits; Rats; Rats, Inbred Strains; Sucrase

Dietary lectins of gluten origin have been suggested to play an important role in the mechanisms leading to the characteristic morphology of the intestine found in patients with celiac disease. To further explore this issue we have used Wheat Germ Agglutinin (WGA) or Concanavalin A (Con A) to challenge rat small intestine and study the ultrastructural changes of such a treatment. Both lectins affected the enterocytes at the base of the villi more than those at the top. The morphological findings included disarrangement of the cytoskeleton, increased endocytosis and shortening of the microvilli. The interrelationship between the observed changes, and their relevance for similar morphological alterations found in patients with celiac disease are discussed. In conclusion, the morphological findings in our rat model resemble early changes in patients with celiac disease, thus supporting the idea that lectins or lectin-like substances are involved in the pathogenesis of this disease.

Topics: Animals; Celiac Disease; Concanavalin A; Cytoskeleton; Female; Intestine, Small; Microscopy, Electron; Microvilli; Rats; Rats, Inbred Strains; Time Factors; Wheat Germ Agglutinins

Immunoregulatory cells were enumerated in 19 coeliac disease children on a gluten free diet by means of monoclonal antibodies that define total T lymphocytes (T3), helper/inducer T cells (T4), suppressor/cytotoxic T cells (T8) and monocytes (M1), as well as by means of surface receptors for Fc fragments of IgM and IgG (T mu and T gamma, respectively). In addition, suppressor cell function was assessed in 17 coeliac disease patients by examining the ability of concanavalin-A (Con-A)-activated suppressor cells to inhibit autologous cell response to mitogenic stimulus as compared with age-matched controls. No statistically significant differences were found in the percentages of subsets defined by monoclonal antibodies between coeliac disease patients and age-matched controls, whereas coeliac disease patients had a significant decrease of the subpopulation bearing membrane receptor for Fc fragment of IgG. Mean value was 8.5% in coeliac patients versus 13.4% in age-matched controls. In the functional assay, mononuclear cells from 10 out of 17 coeliac disease patients either totally or partially failed to suppress responder cells after Con-A-activation. This defect is not related to HLA-DR status, because no difference was found between patients-HLA-matched and unmatched normal individuals. In this assay, mononuclear cells of three coeliac disease patients with low suppressor activity were able to inhibit responder cells to the same extent as controls, when indomethacin was used to block prostaglandin production in the induction phase of Con-A-activated suppressor cells. Our results suggest that an abnormality in immunoregulation may play a role in the pathogenesis of coeliac disease.

Topics: Adult; Antibodies, Monoclonal; Celiac Disease; Child; Child, Preschool; Concanavalin A; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Immunoglobulin G; Immunoglobulin M; Indomethacin; Receptors, Fc; T-Lymphocytes; T-Lymphocytes, Regulatory

The lectins wheat germ agglutinin (WGA) and Concanavalin A (ConA) were perfused into an isolated small intestinal segment alone or after prior perfusion with neuraminidase for a 10 day period in the rat. Intestinal morphometry, intraepithelial Lymphocyte (IEL) and round cell content as well as digestive capacity was measured in the loop and in the adjacent segments. Both lectins induce a mucosal transformation in all segments but ConA is more effective than WGA. Pre-incubation with neuraminidase enhances the action of ConA throughout, whereas only a partial enhancement of the effect of WGA is observed. The mucosal transformation after long term perfusion with the lectins resembles the hyperregenerative adaptation of the mucosa due to gluten in coeliac disease and may thus serve as an animal model for this disease.

Topics: Animals; Celiac Disease; Cell Survival; Concanavalin A; Female; Intestinal Absorption; Intestinal Mucosa; Jejunum; Lectins; Lymphocytes; Neuraminidase; Rats; Rats, Inbred Strains; Time Factors; Wheat Germ Agglutinins

The generation of suppression by concanavalin A in peripheral blood mononuclear cells in treated and untreated coeliac subjects using an in vitro assay was found to be significantly increased when compared with controls. The response of peripheral blood mononuclear cells to the plant mitogen concanavalin A (con A) was also significantly depressed in both groups of coeliac patients. It is proposed that the depressed cell mediated immunity found in this and other studies in coeliac patients is because of increased suppression. The possible connection between these findings and the increased incidence of malignancy also found in coeliac disease is discussed.

Topics: Adolescent; Adult; Aged; Celiac Disease; Cells, Cultured; Concanavalin A; Female; Humans; Immune Tolerance; Immunity, Cellular; Lymphocyte Activation; Male; Middle Aged; T-Lymphocytes, Regulatory

The PT-digest of bread wheat gliadin was very active in agglutinating undifferentiated human K562(S) cells. This activity was quantitatively, but not qualitatively, similar to that of Con A or WGA. Moreover, Con A-induced cell agglutination was inhibited by mannan and mannose, WGA-induced agglutination by NAG only, and cell agglutination induced by bread wheat gliadin peptides was inhibited by each of these three saccharides. Not only was mannan the most active saccharide in preventing cell agglutination induced by bread wheat gliadin peptides, but it was also able to dissociate agglutinated cells. As compared to the PT- digest of whole bread wheat gliadin, the digest obtained from purified A-gliadin was tenfold more active. The PT-digest of durum wheat gliadin did not show any agglutinating activity.

Topics: Agglutination Tests; Celiac Disease; Cell Aggregation; Cell Differentiation; Cells, Cultured; Concanavalin A; Flour; Gliadin; Humans; Lectins; Peptide Fragments; Plant Lectins; Plant Proteins; Triticum; Wheat Germ Agglutinins

Chromatographically separated fractions of a proteolytic digest of wheat gliadin were assayed for cytotoxic properties using cultured human embryonic intestinal, lung, kidney, adrenal, and HEp-2 cells. In all cell types noxious effects were observed microscopically over a 24 h period. The most active fraction was that previously shown to produce xylose malabsorption in subjects with coeliac disease, disruption of lysosomes, and inhibition of morphological recovery of cultured mucosa from a patient on a gluten-free diet.

Topics: Adrenal Glands; Celiac Disease; Cell Line; Chromatography, Gel; Concanavalin A; Dietary Proteins; Embryo, Mammalian; Gliadin; Humans; In Vitro Techniques; Intestinal Mucosa; Kidney; Lectins; Lung; Plant Lectins; Plant Proteins; Triticum; Xylose