Donald D. Kasarda

Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens

Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.

N-terminal amino acid sequences of ω-gliadins and ω-secalins: Implications for the evolution of prolamin genes

Abstract Two new types of N-terminal amino acid sequence have been found for ω-gliadins from tetraploid and hexaploid wheats. A third type of sequence, which we have recently reported for an ω-gliadin from diploid wheat and for the ‘C’ hordeins of barley, has now been found for a group of components from rye (ω-secalins) and for ω-gliadins of tetraploid and hexaploid wheats. The sequences provide evidence that all these ω-type prolamins are related and derived in part from gene duplications, followed by divergence of the duplicated genes by point mutations and by insertion and deletion of portions of the gene DNA. Five-residue amino acid sequences that include mainly glutamine and proline occur a number of times in the N-terminal sequences of ω-type prolamins and may indicate that the genes for these prolamins have been formed through repeated duplication of short DNA sequences in which the codons for glutamine and proline predominated. The prolamins of wheat and related species seem to be unique to the grass family of flowering plants, which appeared late in the evolutionary process. The genes coding for these proteins may be the most recently evolved family of genes.

Evidence for the role of a human intestinal adenovirus in the pathogenesis of coeliac disease.

We previously noted a region of amino acid sequence homology between A-gliadin, a major alpha-gliadin component known to activate coeliac disease, and the early region E1b protein of human adenovirus serotype 12 (Ad12), an adenovirus isolated from the human intestinal tract. In the present study sera from coeliac disease patients from the United Kingdom and the United States were assayed for neutralising antibody to Ad12 as evidence of past exposure to that virus and for antibody to synthetic peptides of A-gliadin from the region of shared sequence with the Ad12 E1b protein. Eighty nine per cent of untreated coeliac disease patients had evidence of previous Ad12 infection. There was also a significant increase in the prevalence of neutralising antibody to Ad12 among treated adults (33.3%) and children (30.8%) with coeliac disease compared with controls (0-12.8%) in the western USA and in London. There was no evidence for an increased prevalence of infection with a closely related adenovirus, adenovirus 18, or another enteric virus, Echovirus 11, among coeliac disease subjects. Additional studies documented that a region of A-gliadin that shares amino acid sequence homology with the adenovirus 12 E1b protein could be recognised as an antigenic determinant in active coeliac disease patients. Taken together, these data are compatible with the hypothesis that a viral protein may play a role in the pathogenesis of coeliac disease, perhaps by virtue of immunological cross reactivity between antigenic determinants shared by the viral protein and alpha-gliadins.

In vitro (Organ culture) studies of the toxicity of specific A-gliadin peptides in celiac disease

Specific peptides of known amino acid sequence were prepared from alpha-gliadin (A-gliadin) by cleavage of the protein with cyanogen bromide and chymotrypsin and purification of the resulting peptides. The three peptides derived from the cyanogen bromide cleavage spanned the complete sequence of A-gliadin (266 residues). Four peptides derived from chymotryptic digestion covered the N-terminal sequence through residue 68. These peptides were tested for toxicity in celiac disease by organ culture of biopsied small intestinal tissues taken from patients with active celiac disease. Enterocyte height was used as a measure of peptide effect on cultured tissues. Five of seven peptides tested significantly inhibited increase of enterocyte height in the cultures and were considered toxic on this basis. The largest common sequences among the toxic peptides were -pro-ser-gln-gln- and -gln-gln-gln-pro-; these sequences were absent from the nontoxic peptides. The relationship of these sequences to the damaging effect of gliadins on the small intestinal mucosa in celiac disease remains to be investigated.

Absence of oats toxicity in adult coeliac disease.

Coeliac disease is a gluten-sensitive disorder characterised by malabsorption and a typical histological lesion. Treatment with a strict gluten-free diet results in complete clinical and histological recovery. The conventional gluten-free diet used to treat coeliac disease proscribes oats cereal as well as wheat, barley, and rye.1 However, the issue of oats toxicity has not been conclusively resolved, and the prohibition of this important cereal deprives patients of a valuable source of fibre and nutrients. The aim of this study was to examine the clinical, histological, and immunological responses of adult patients with coeliac disease to challenge with oats. Ten adult patients with coeliac disease in clinical and histological remission were recruited from the coeliac outpatient clinic in St Jamess Hospital, Dublin. Each patient …

Innate and adaptive immunity : the Yin and Yang of celiac disease

Summary:  Celiac disease is a multigenetic complex inflammatory disorder with an autoimmune component, induced by gluten, a protein found in wheat. It is a unique human disease model to dissect the innate and adaptive immune mechanisms underlying T‐cell‐mediated tissue destruction and the development of T‐cell lymphoma in conditions of chronic T‐cell activation.

Changes in polypeptide composition and grain quality due to sulfur deficiency in wheat

Qualitative and quantitative differences in protein composition were examined in 12 samples of flour milled from wheat (cultivar Olympic) grown under a wide range of sulfur and nitrogen inputs. Two-dimensional fractionation, comprising isoelectric focusing in one dimension and electrophoresis at pH 3 in the other, indicated that the changes in gliadin composition associated with sulfur deficiency involved the proportions of individual gliadins but not their charge, size or isoelectric point characteristics. Quantitative Polyacrylamide gel electrophoresis in the presence of SDS showed that sulfur deficiency resulted in increases in the proportions of polypeptides in the mol.wt. ranges 51,000 to 80,000 and above, but decreases in the proportions of lower mol.wt. polypeptides (8000 to 28,000, mainly albumins). The proportions of polypeptides in five size ranges were highly correlated with the sulfur to nitrogen ratios in the flour samples and also with their dough quality characteristics, particularly as measured with the Extensograph. Doughs produced from the variety, Olympic, usually are extensible but the increased proportion of high mol.wt. polypeptides, found in the sulfur-deficient samples, was associated with an increase in the toughness and a decrease in the extensibility of the dough.

Genetic aspects of wheat gliadin proteins

Inheritance of gliadin components unique to three different varieties of common wheat (Triticum aestivum L.) was studied in F1 and F2 seeds of intervarietal crosses using protein patterns obtained by polyacrylamide gel electrophoresis in aluminum lactate buffer (pH 3.2). The patterns of F1 seeds of the crosses Cheyenne × Justin and INIA 66R × Justin evidenced all the bands present in the patterns of the parents; band intensities reflected gene dosage levels dependent on whether the contributing parent was maternal or paternal in accordance with the triploid nature of endosperm tissue. Most of the gliadin components examined segregated in accordance with control by a single dominant gene, but in two instances single bands in the one-dimensional electrophoretic patterns segregated in the F2 as expected if controlled by two genes. A method of two-dimensional electrophoresis was developed that resolved these apparently single bands into two components each, which could segregate independently. Linkage analysis provided evidence of codominant alleles and closely linked genes coding for gliadin protein components in both coupling and repulsion situations. The gliadin protein components seem to be coded for by clusters of genes located on chromosomes of homoeologous groups 1 and 6 in hexaploid wheats.

Physical characterization of α-amylase inhibitors from wheat

Abstract Six albumins from the kernels of hexaploid wheat, designated 0.19, 0.28, 0.32, 0.35, 0.39 and 0.48 according to their electrophoretic mobilities, were characterized according to their molecular weights, circular dichroism spectra, fluorescence spectra, amino acid compositions, and their specificities in inhibiting α-amylases from human saliva, chick pancreas, yellow mealworm, Aspergillus oryzae , and Bacillus subtilis . The 0.19 and 0.28 proteins have been related to other proteins described in the recent literature for which, however, characterization had been inadequate for the evaluation of possible identities. The 0.28, 0.32, 0.35, 0.39 and 0.48 proteins have been shown to belong to a family of closely related proteins. For example, they had identical circular dichroism spectra in the near ultraviolet that differed considerably from that of 0.19 protein, and they were all strong inhibitors of the α-amylase from the yellow mealworm ( Tenebrio molitor L.) but were inactive against several other α-amylases, whereas the 0.19 protein was a strong inhibitor of α-amylases from human saliva and chick pancreas as well as that from the yellow mealworm.

Novel Immune Response to Gluten in Individuals with Schizophrenia

A link between celiac disease and schizophrenia has been postulated for several years, based primarily on reports of elevated levels of antibody to gliadin in patients. We sought to examine the proposed connection between schizophrenia and celiac disease by characterizing the molecular specificity and mechanism of the anti-gliadin immune response in a subset of individuals with schizophrenia. Blood samples from individuals with schizophrenia and elevated anti-gliadin antibody titer were examined for celiac disease-associated biomarkers, including antibodies to transglutaminase 2 (TG2) enzyme and deamidated gliadin peptides, as well as the HLA-DQ2 and -DQ8 MHC genes. The anti-gliadin antibody response was further characterized through examination of reactivity towards chromatographically separated gluten proteins. Target proteins of interest were identified by peptide mass mapping. In contrast to celiac disease patients, an association between the anti-gliadin immune response and anti-TG2 antibody or HLA-DQ2 and -DQ8 markers was not found in individuals with schizophrenia. In addition, the majority of individuals with schizophrenia and anti-gliadin antibody did not exhibit antibody reactivity to deamidated gliadin peptides. Further characterization of the antibody specificity revealed preferential reactivity towards different gluten proteins in the schizophrenia and celiac disease groups. These findings indicate that the anti-gliadin immune response in schizophrenia has a different antigenic specificity from that in celiac disease and is independent of the action of transglutaminase enzyme and HLA-DQ2/DQ8. Meanwhile, the presence of elevated levels of antibodies to specific gluten proteins points to shared immunologic abnormalities in a subset of schizophrenia patients. Further characterization and understanding of the immune response to gluten in schizophrenia may provide novel insights into the etiopathogenesis of specific disease phenotypes.