sepharose and Celiac-Disease

Tissue transglutaminase (tTG) autoantibodies are serological markers for celiac disease. The aim was to study the efficacy of the pTnT-rhtTG plasmid and subsequent diagnostic accuracy of tTG autoantibodies for childhood celiac disease using radioligand binding assays.. Coupled in vitro transcription and translation of tTG were performed by pTnT-rhtTG as well as by the pGEMt Easy-rhtTG vectors using the TNT SP6 Coupled Reticulocyte Lysate System in the presence of [³⁵S] methionine. Sera from 190 celiac disease children and 74 controls were measured for tTG autoantibodies in two separate radioligand binding assays using anti-human IgA agarose and protein A sepharose beads for the detection of IgA-tTG and IgG-tTG, respectively.. Median incorporation of [³⁵S] methionine into the pTnT-rhtTG was 26% compared to 16% for the pGEMt Easy-rhtTG plasmid (p = 0.0016). Using pTnT-rhtTG (as compared to pGEMt Easy-rhtTG), sensitivities were IgA-tTG = 96.3% (95.7%) and IgG-tTG = 95.8% (97.3%) and specificities were IgA-tTG = 91.9% (90.5%) and IgG-tTG = 94.6% (98.4%). According to receiver operator characteristics for the pTnT (pGEMt Easy) assays, area under the curves were IgA-tTG = 98.4% (98.4%) and IgG-tTG = 97.7% (97.2%), respectively.. The pTnT-rhtTG plasmid increased the efficacy of tTG antigen usage without reducing the diagnostic accuracy of IgA-tTG and IgG-tTG for childhood celiac disease. The pTnT-rhtTG plasmid is therefore recommended over the pGEMt Easy-rhtTG for the assessment of IgA-tTG and IgG-tTG using radioligand binding assays.

Topics: Adolescent; Antibodies, Anti-Idiotypic; Antigens, Neoplasm; Autoantibodies; Biomarkers; Case-Control Studies; Celiac Disease; Cell-Free System; Child; Child, Preschool; Female; Humans; Infant; Male; Methionine; Plasmids; Protein Binding; Radioligand Assay; Reticulocytes; ROC Curve; Sepharose; Sulfur Radioisotopes; Sweden; Transglutaminases; Young Adult

The K 562 (S) cell agglutinating activity of peptides obtained from in vitro digestion of bread wheat gliadins has been shown to be associated with a small fraction (coded as Fraction C), that can be easily separated by affinity chromatography of the whole digest on a sepharose 6-B-mannan or sepharose 6-B-oligomers of N-acetyl-glucosamine. Although the whole gliadin digests from 12 durum wheat varieties were unable to agglutinate K 562 (S) cells, all these digests were found to contain an active Fraction C. The lack of agglutinating activity of the whole durum wheat gliadin digests has been shown to be associated with the presence in these digests of another peptide fraction (coded as Fraction B) that is eluted much earlier from the sepharose 6-B-mannan column and is able to inhibit the cell agglutinating activity of Fraction C. Such an active Fraction B is not present in bread wheat gliadin peptides, although peptides with the same elution profile as Fraction B have been detected.

Topics: Acetylglucosamine; Agglutination; Agglutination Tests; Binding Sites; Bread; Celiac Disease; Cell Differentiation; Cells, Cultured; Chemical Fractionation; Chromatography, Affinity; Gliadin; Humans; Mannans; Peptide Fragments; Plant Lectins; Sepharose; Triticum; Wheat Germ Agglutinins

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

Antibodies against gluten and gliadin were determined by the indirect immunofluorescence technique, using frozen wheat grain sections or gliadin-coated Sepharose beads. The methodological characteristics and diagnostic usefulness of the two techniques were evaluated. The reproducibility of both was improved by introducing a fluorometric reading-off procedure. Antibody quantitations were preferably performed as end-point titrations. The two techniques had different dose-response relationships. The grain section technique was more discriminative for small variations in antibody concentration than the bead technique. The latter was, however, more reproducible. Besides antibodies against gliadin, a number of patients with gluten enteropathy had antibodies against the main septa of wheat grains. Fluorescence intensity was preferably expressed in multiples of background intensity. A reaction was visually perceived when the fluorometrically-measured fluorescence intensity reached 2.5 times the background intensity. Using this value as the limit for positive reactions, antibodies were demonstrated in 81% of the cases with verified gluten intolerance, compared with 28% in cases with other intestinal allergies and 8% in normals. The diagnostic specificity of both the grain section and Sepharose bead technique for gluten enteropathy increased with increasing antibody concentration and was apparently 100% when the fluorescence intensity produced by a 1/10 serum dilution reached a value 7 to 8 times that of the background. Antibodies against reticulin were demonstrated in 1/4 of the cases having anti-gluten antibodies but in none of those with non-gluten-induced gastro-intestinal symptoms. Antibodies of the IgG class against cow's milk were demonstrated more often and in higher titre in cases with anti-gluten antibodies than in those without them.

Topics: Adolescent; Adult; Antibodies; Celiac Disease; Child; Dose-Response Relationship, Immunologic; Female; Fluorescent Antibody Technique; Gliadin; Glutens; Humans; Male; Microspheres; Plant Proteins; Reticulin; Sepharose

Production of leucocyte-migration-inhibition factor (L.I.F.) by peripheral-blood lymphocytes in response to challenge with gluten fractions was studied in 55 patients with coeliac disease and in 32 controls. 96% of the patients with coeliac disease demonstrated significant L.I.F. reaction in response to gluten fractions irrespective of their dietary status. Only 2 out of 32 controls had a positive reaction. This was in response to the B2 or B3 fraction, but never to both. The agarose microdroplet method of L.I.F. assay is reliable and technically simple enough for use in most clinical laboratories. The assay of L.I.F. production by peripheral-blood lymphocytes in response to gluten fractions, would be a useful adjunct in the diagnosis of gluten-sensitive enteropathy.

Topics: Adolescent; Allergens; Celiac Disease; Cell Migration Inhibition; Child; Child, Preschool; Female; Glutens; Humans; In Vitro Techniques; Infant; Lectins; Leukocytes; Macrophage Migration-Inhibitory Factors; Male; Sepharose