Norberto Pogna

Environmental factors of celiac disease: Cytotoxicity of hulled wheat species Triticum monococcum, T. turgidum ssp. dicoccum and T. aestivum ssp. spelta

Background and Aim:  In the present paper, the toxicity of prolamines derived from three cereals with a different genome was investigated in human colon cancer Caco‐2/TC7 and human myelogenous leukemia K562(S) cells. The purpose of this study was to investigate if species from ancient wheat could be considered as healthy food crops devoid or poor in cytotoxic prolamines for celiac disease.

On-chip detection of multiple serum antibodies against epitopes of celiac disease by an array of amorphous silicon sensors

In this paper, we present the preliminary results of an ELISA-on-chip device, intended as a technological demonstrator of a novel analytical system suitable for the diagnosis and follow-up of celiac disease. The idea of the work is to combine an array of amorphous silicon photosensors with a pattern of a poly(2-hydroxyethyl methacrylate) polymer brush film, which acts as anchor for the immobilization of gliadin peptides containing the celiac disease epitopes. Recognition relies on a sandwich immunoassay between antibodies against the peptides and secondary antibodies marked with horseradish peroxidase to obtain a chemiluminescent signal. Detection is based on the measurement of photocurrent induced in the array of amorphous silicon photosensors by the chemiluminescent signal. An ad-hoc procedure has been developed in order to enable the fabrication of the photodiode array and the polymer brush pattern on the two sides of the same glass substrate ensuring the compatibility of the different technological steps. The sensitivity and the selectivity of the chip for multiplex immunoassays were demonstrated using two gliadin peptides (VEA and DEC). In particular, we found that the average amount of the bound HRP revealed by our analytical protocol is 3.5(±0.3) × 10−6 pg μm−2 and 0.85(±0.3) × 10−6 pg μm−2 for specific and non-specific interactions, respectively.

Immunogenicity of monococcum wheat in celiac patients

BACKGROUND Research is intense to find wheat of low or null toxicity for patients with celiac disease (CD). Among candidates, there are diploid wheat species. OBJECTIVE We compared the immunological properties of 2 lines of diploid monococcum wheat (Triticum monococcum ssp. monococcum), Monlis and ID331, with those of common wheat (Triticum aestivum). DESIGN Interferon-γ production and the proliferation of intestinal gliadin-specific T cell lines and clones were measured as evidence of T cell activation by peptic and tryptic (PT) digests of gliadins from 2 monococcum lines. Furthermore, organ cultures of jejunal biopsies from 28 CD patients were set up to assess the effects of PT gliadin on innate and adaptive immune response by using immunohistochemistry. RESULTS Monlis and ID331 induced interferon-γ production and proliferation in celiac mucosal T cells. In organ cultures, Monlis PT digest induced a significant increase of IL-15 epithelial expression and crypt enterocyte proliferation, whereas ID331 had no effect. Both monococcum lines caused intraepithelial T cell infiltration and lamina propria T cell activation. CONCLUSIONS Our data show that the monococcum lines Monlis and ID331 activate the CD T cell response and suggest that these lines are toxic for celiac patients. However, ID331 is likely to be less effective in inducing CD because of its inability to activate the innate immune pathways.

Chromosomal localization of zein genes by in situ hybridization in Zea mays.

SummaryIn order to localize the genes coding for zein, the major storage protein of maize endosperm, zein 125I-mRNA and 3H-cDNA labelled at high specific activity were used for in situ hybridization on heterozygous interchanges and paracentric inversions of the KYS strain of Zea mays. The analysis of the diplotene-metaphase I microsporocytes indicated the presence of zein structural genes on the long arm of chromosomes 4 and 5, the short arm of chromosome 7 and the distal segment of the long arm of chromosome 10. The two hybridization sites on chromosomes 7 and 10 are found near opaque-2 and opaque-7 loci which are known to regulate zein synthesis. The present data are discussed in relation to results obtained by other authors using genetical mapping of zein genes.

Immunogenicity of two oat varieties, in relation to their safety for celiac patients

Abstract Objective. Most of the recent studies suggest that oats are well tolerated by celiac disease (CD) patients. However, it is still possible that different oat cultivars may display different biological properties relevant for CD pathogenesis. We aimed to investigate biological and immunological properties of two oat varieties, Avena genziana and Avena potenza, in relation to their safety for CD patients. Material and Methods. Phosphorylation of extracellular signal-regulated kinase (ERK) and trans-epithelial electrical resistance (TEER) were evaluated in CaCo-2 cells treated with peptic–tryptic (PT) digests from the two oats and from gliadin (PTG). With the same PT-digests, duodenal biopsies from 22 CD patients were treated in vitro for 24 h and density of CD25+ cells in lamina propria and of intraepithelial CD3+ T cells was measured, as well as crypt cell proliferation and epithelial expression of interleukin 15. Finally, interferon γ (IFN-γ) production was measured as evidence of gliadin-specific T-cell activation by PT-digests. Results. In contrast to PTG, oats PT-digests were not able to induce significant increase in ERK phosphorylation and decrease in TEER in CaCo-2 cells. In the organ culture system, oats PT-digests, unlike PTG, did not induce significant increase in crypt enterocyte proliferation, increase in interleukin 15 expression or in lamina propria CD25+ cells. Nevertheless Avena potenza increased intraepithelial T-cell density, while Avena genziana-induced IFN-γ production in 3/8 CD intestinal T cell lines. Conclusions. Our data show that Avena genziana and Avena potenza do not display in vitro activities related to CD pathogenesis. Some T-cell reactivity could be below the threshold for clinical relevance.

Extensive in vitro gastrointestinal digestion markedly reduces the immune-toxicity of Triticum monococcum wheat: implication for celiac disease.

SCOPE The ancient diploid Triticum monococcum is of special interest as a candidate low-toxic wheat species for celiac disease patients. Here, we investigated how an in vitro gastro-intestinal digestion, affected the immune toxic properties of gliadin from diploid compared to hexaploid wheat. METHODS AND RESULTS Gliadins from Triticum monococcum, and Triticum aestivum cultivars were digested using either a partial proteolysis with pepsin-chymotrypsin, or an extensive degradation that used gastrointestinal enzymes including the brush border membrane enzymes. The immune stimulatory properties of the digested samples were investigated on T-cell lines and jejunal biopsies from celiac disease patients. The T-cell response profile to the Triticum monococcum gliadin was comparable to that obtained with Triticum aestivum gliadin after the partial pepsin-chymotrypsin digestion. In contrast, the extensive gastrointestinal hydrolysis drastically reduced the immune stimulatory properties of Triticum monococcum gliadin. MS-based analysis showed that several Triticum monococcum peptides, including known T-cell epitopes, were degraded during the gastrointestinal treatment, whereas many of Triticum aestivum gliadin survived the gastrointestinal digestion. CONCLUSION The pattern of Triticum monococcum gliadin proteins is sufficiently different from those of common hexaploid wheat to determine a lower toxicity in celiac disease patients following in vitro simulation of human digestion.

Variation in noxiousness of different wheat species for celiac patients

Abstract Peptic-tryptic (PT) digested prolamins from spelt wheat Triticum aestivum ssp. spelta and landraces ERSA 6 and ERSA 8 of farro wheat T. turgidum ssp. dicoccum were found to agglutinate K562(S) cells, and exert strong toxic effects on Caco-2/TC7 cells. Cytotoxicity of spelt prolamins against Caco-2/TC7 cells was greatly reduced by 10-mer peptide QQPQDAVQPF. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro landraces Prometeo, L5563, L5540 and L5558 did not exhibit any negative effects on K562(S) and Caco-2/TC7 cells. Toxic genotypes ERSA 6 and ERSA 8 were found to share the same gliadin pattern, which was absent in inactive landraces. Monococcum, farro and spelt wheats differed from each other in their responses to antibodies specific for 13-mer cytotoxic sequence FPGQQQPFPPQQP and 10-mer peptide QQPQDAVQPF. This latter sequence was found to occur in high amounts in common wheat line FG, Phaseoulus vulgaris, Ph. coccineus and Lens culinaria.

Gene sequences of vromindolines in Avena species

Vromindolines, a family of oat starch-bound proteins responsible for the extremely soft endosperm of this cereal, are characterized by a tryptophan-rich domain and ten conserved cysteine residues, as it was observed in the starch-bound proteins from other cereals, and proved to be specific of the genus Avena. Two-dimensional electrophoresis of vromindolines from different oat species, both cultivated and wild, revealed that they are present in all genomes and ploidy levels, with no major differences in their molecular weight and electrophoretic mobility. PCR amplification and sequencing of genes coding for VIN-2 and VIN-3 indicated that both proteins are strongly conserved across the species analysed. Some differences were found between diploid and polyploid accessions at specific amino acid positions along the protein. In particular the C-genome accessions (A. clauda, A. eriantha, A. ventricosa) differed from the A-genome diploids (A. canariensis, A. damascena, A. longiglumis, A. nuda, A. strigosa) for some peculiar sequences. Tetraploids (A. barbata, A. magna and A. insularis) showed two sequences each for both proteins; the variability observed in them was similar to that found in A. sativa. The characteristics of these genes could help to clarify the genetic relationship among the various species of the genus Avena. Moreover, the knowledge of the genetic control of these proteins represent an important tool for the modulation of oat endosperm texture.