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Four perennial wheat genotypes derived from crosses between Triticum aestivum and Thinopyrum elongatum, Th. intermedium or Th. ponticum were grown in Central Italy over 2 years of testing, and compared for their agronomical, biochemical, nutritional and technological traits with three commercial common wheat cultivars. Perennial wheat derivatives were characterized by post-harvest regrowth, small kernels, high number of tillers, high protein content and reduced sodium dodecyl sulphate sedimentation volume. Lines 11955 and OK72 exhibited soft kernel texture due to wild-type alleles at the puroindoline loci, whereas lines 235A and 280B produced medium-hard kernels for the presence of novel puroindolines A and B inherited from Th. elongatum and Th. intermedium, respectively. In addition, perennial wheat genotypes presented a high content of carotenoids and 5-n-alkylresorcinols compared with their annual counterparts. AR composition of line 235A, as determined by gas chromatography-mass spectrometry, was characterized by a high percentage (64.7 %) of long-chain (C21:0 + C23:0 + C25:0) homologues, which are claimed to prevent cardiovascular diseases and cancer. In addition, line OK72 was unique in having a C17/C21 homologue ratio as high as 0.34, likely inherited from Th. ponticum. This line along with line 280B also showed a high content of total dietary fiber. Finally, peculiar storage protein composition and kernel texture were observed in some perennial durum wheat derivatives obtained from crosses between T. turgidum subsp. durum and Th. junceiforme. This wheatgrass species was found to contain the 10-mer QQPQDAVQPF peptide, which is able to prevent prolamins from triggering inflammatory responses in celiac patients.

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.

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.

Seven cultivated Triticum species or subspecies ( Triticum monococcum , Triticum turgidum ssp dicoccum , T. turgidum ssp. durum , T. turgidum ssp turanicum , Triticum timopheevii , Triticum aestivum and Triticum zhukovskyi ) were compared for their contents of bioactive compounds, mainly 5-n-alkylresorcinols (ARs). Multivariate analysis of variance and principal component analysis were used to evaluate the differences in the phytochemical profiles and to establish the relationships among variables. Significant differences were observed for both total phenol (TP) and AR content. The highest AR level (377 μg/g) was observed in Triticum dicoccum , which also exhibited the highest variability for these compounds (298–436 μg/g). By contrast, the lowest AR content (286 μg/g) was found in Triticum durum . C21:0 was the main homologue chain in all the samples, its value ranging between 54.5% in T. durum and 41.2% in T. aestivum . The T. dicoccum and T. monococcum wheats had relatively low amounts of TP, whereas Triticum turanicum (215 mg/kg), T. timopheevii (250 mg/kg) and T. zhukovskyi (286 mg/kg) had approximately 3-fold higher TP levels. These results suggest that there are opportunities for breeding wheat varieties with superior health benefits and for promoting the use of ancient Triticum crops as novel sources of healthy food.

The effects of grain texture on pastamaking and breadmaking quality were studied in three F8 soft-textured durum wheat lines (SDLs) containing wild-type alleles Pina-D1a and Pinb-D1a as compared with their hard durum sister lines (HDLs). SDLs homozygous for a small 5DS segment, less than 14.4 cM in size, accumulated puroindolines A (Pin-A) and B (Pin-B) and showed SKCS values (19.9–23.6) significantly lower than those (72.6–76.8) of their hard-textured counterparts lacking Pin-A and Pin-B. In addition, SDLs exhibited approximately 24% higher flour extraction rates compared with HDLs. Reducing the kernel hardness decreased farinograph water absorption, dough tenacity (P) and, accordingly, alveograph P/L ratio, but increased farinograph stability, mixing tolerance and dough extensibility (L). Spaghetti cooking quality, as determined by the sensory judgment of firmness, stickiness and bulkiness, was unaffected by the kernel hardness, whereas the loaf volume exhibited a 10% increase associated with kernel softening. Flour and semolina, but not spaghetti, from SDLs showed a substantial reduction in yellowness (b*) and brownness (100 − L*) likely due to their finer particle size compared with HDLs. Alleles Pina-D1a and Pinb-D1a may offer new perspectives for breeding dual purpose (pasta and bread) durum wheat varieties.

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 transepithelial 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 g (IFN-g) 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 PTdigests, 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 genzianainduced IFN-g 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.

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.

Puroindoline a (Pin-a) and puroindoline b (Pin-b), two basic isoforms encoded by the Pina-D1 and Pinb-D1 loci respectively, involved in controlling grain texture in wheat, were isolated from starch granules of soft wheat cultivars using three different extraction procedures, and fractionated by acidic polyacrylamide gel electrophoresis (A-PAGE). Tris buffer containing 1% Triton X-114 extracted Pin-a and small amounts of Pin-b, whereas 1% SDS preferably extracted Pin-b. Large amounts of both puroindolines were isolated by a solution containing 50% propan-2-ol and 50 mM NaCl. This solution extracted reduced amounts of Pin-b and no traces of Pin-a from starch granules of 20 hard common wheats containing the null allele Pina-D1b. The absence of Pin-a was confirmed by immunostaining with an anti-Pin-a antiserum. With the exception of two cultivars, null Pin-a cultivars gave no PCR fragment with three primer pairs specific to either the coding region or the promoter region of Pina-D1a, suggesting that major changes had occurred at the Pina-D1 locus in these genotypes. Cultivars Fortuna and Glenman were unique in giving size-specific PCR fragments with all primer pairs for the allele Pina-D1a and showed a cytosine deletion at position 267 in the coding region of the Pin-a gene, which resulted in a TGA stop codon at position 361. However, there was no evidence of a mutated protein in the A-PAGE or SDS-PAGE patterns of Fortuna and Glenman. The novel gene, provisionally named Pina-D1c, is the first null allele due to a point mutation that has been identified at the Pina-D1 locus.

Among cereals, Avena sativa is characterized by an extremely soft endosperm texture, which leads to some negative agronomic and technological traits. On the basis of the well-known softening effect of puroindolines in wheat kernel texture, in this study, indolines and their encoding genes are investigated in Avena species at different ploidy levels. Three novel 14 kDa proteins, showing a central hydrophobic domain with four tryptophan residues and here named vromindoline (VIN)-1,2 and 3, were identified. Each VIN protein in diploid oat species was found to be synthesized by a single Vin gene whereas, in hexaploid A. sativa, three Vin-1, three Vin-2 and two Vin-3 genes coding for VIN-1, VIN-2 and VIN-3, respectively, were described and assigned to the A, C or D genomes based on similarity to their counterparts in diploid species. Expression of oat vromindoline transgenes in the extra-hard durum wheat led to accumulation of vromindolines in the endosperm and caused an approximate 50 % reduction of grain hardness, suggesting a central role for vromindolines in causing the extra-soft texture of oat grain. Further, hexaploid oats showed three orthologous genes coding for avenoindolines A and B, with five or three tryptophan residues, respectively, but very low amounts of avenoindolines were found in mature kernels. The present results identify a novel protein family affecting cereal kernel texture and would further elucidate the phylogenetic evolution of Avena genus. © 2014 Springer-Verlag Berlin Heidelberg.

A-PAGE fractionation of starch granule proteins from 63 bread wheat cultivars with contrasting grain texture characteristics revealed two prominent polypeptides and three minor ones, approximately 15 kDa in size. These proteins were found to be encoded by genes on the short arm of chromosome 5D. The two major friabilin components were assumed to correspond to puroindolines a and b (pinA and pinB), as suggested by PCR amplification of genes coding for pinA, glycine-type or serine-type pinB. Two electrophoretic patterns for pinA (presence vs absence) and three patterns for pinB were obtained by A-PAGE. In cultivars with pinA (allele Pina-D1a), pinB was found to be encoded by wild-type Pinb-D1a, serine-type Pinb-D1b or by the novel glycine-type b1 allele. Cultivars lacking pinA (allele Pina-D1b) were shown to contain eitherPinb-D1a or the novel b2 allele, both alleles coding for glycine-type pinB. The intensity of pinB in A-PAGE gels was found to be associated with grain hardness as determined by the SKCS method. Cultivars lacking pinA had the highest SKCS values, suggesting that both pinA and pinB may affect grain texture. In the presence of pinA, cultivars with wild-type allelePinb-D1a had soft grain texture, whereas those with alleles Pinb-D1b or b1 showed increased grain hardness. It is suggested that allele b1 affects the interaction of pinB with starch granules because of a sequence mutation different from the glycine-to-serine change.

Background Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and ex-vivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD. Methods We performed a single blind, cross-over study involving 12 CD patients who had been on a GFD for at least 12 months, challenged on day 0, 14 and 28 with a single fixed dose of 2.5 grams of the following (random order): Tm, rice (as reference atoxic protein) and Amygluten (as reference toxic protein) dispersed in a gluten-free pudding. The primary end-point of the study was the change in intestinal permeability, as assessed by changes in the urinary lactulose/rhamnose ratio (L/R ratio) measured by High Pressure Liquid Chromatography. We also assessed the occurrence of adverse gastrointestinal events, graded for intensity and duration according to the WHO scale. Variables were expressed as mean ± SD; paired t-test and χ2 test were used as appropriate. Results The urinary L/R ratio did not change significantly upon challenge with the 3 cereals, and was 0.055 ± 0.026 for Tm Vs 0.058 ± 0.035 for rice (p = 0.6736) and Vs 0.063 ± 0.054 with Amygluten (p = 0.6071). Adverse gastrointestinal events were 8 for Tm, Vs 11 for rice (p = 0.6321) and Vs 31 for Amygluten p = 0.0016), and, in all cases events were graded as “mild” or “moderate” with TM and rice, and as “severe” or “disabling” in 4 cases during Amygluten. Conclusions No definite conclusion can be drawn on the safety of Tm, based on no change in urinary L/R because even Amygluten, a toxic wheat protein, did not cause a significant change in urinary L/R indicating low sensitivity of this methodology in studies on acute toxicity. Tm was, however, well tolerated by all patients providing the rationale for further investigation on the safety of this cereal for CD patients. Trial registration EudraCT-AIFA n2008-000697-20

Hordoindolines, the tryptophan-rich polypeptides affecting grain hardness in barley, appeared as three pairs of polypeptides in the acidic polyacrylamide gel electrophoresis (A-PAGE) and two-dimensional A-PAGE × SDS-PAGE patterns of starch-granule proteins from 18 barley cultivars. On capillary RP-HPLC/nESI-MS/MS spectrometry, one pair of polypeptides was found to correspond to hordoindoline A (HINA), one to hordoindoline B1 (HINB1) and one to hordoindoline B2 (HINB2), the two polypeptides of each pair deriving from post-translational cleavage of a native hordoindoline at different positions at the N-terminus and/or C-terminus. Amongst the barley cultivars analyzed, cvs Hart and Sundance, which were claimed to be unique in lacking the Hina gene coding for HINA, revealed similar Hina coding sequences and accumulated hordoindoline HINA on their starch granules. The amount of total hordoindolines (HINA + HINB1 + HINB2) on the starch granules, as quantified by densitometric scanning of A-PAGE gels, was comparable with that of puroindolines (PINA + PINB) in soft-textured wheat. By contrast, the amount of B-type hordoindolines (HINB1 and HINB2 combined) was 50 % lower than that of PINB, suggesting that the absence of barley cultivars with soft kernels is likely due to the reduced amount of B-type hordoindolines accumulated on the starch granules. Approximately 22 and 27 % of the phenotypic variation for kernel hardness in 56 barley cultivars analyzed by the Single Kernel Characterization System (SKCS) were explained by differences in kernel weight and B-type hordoindoline level, respectively. By contrast, the outer husk of barley grain showed no effect on the SKCS index.

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-g 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-g 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. Am J Clin Nutr 2012;96:1339‐45.

Background Natural or induced variations in the noxiousness of gluten proteins for celiac disease (CD) patients are currently being investigated for their potential in breeding wheat crops with reduced toxicity. Aims We evaluated the bread wheat line C173 for its effects on the in vitro -grown duodenal mucosa of CD patients. Methods In vitro -grown duodenal mucosa biopsies of 19 CD patients on a gluten-free diet were exposed to peptic/tryptic-digested prolamins from bread wheat line C173 lacking gliadin–glutenin subunits, analyzed for morphology, cytokine and anti-tTG antibody production, and compared with mucosa biopsies exposed to prolamins from wild-type cv. San Pastore. Results Duodenal mucosa biopsies exposed to prolamins from C173 and San Pastore released higher amounts of IFN-γ, IL-2, IL-10 and anti-tTG antibodies in the culture medium than untreated controls. The line C173 differed from cv. San Pastore as it did not produce negative effects on enterocyte height, suggesting that manipulating prolamin composition can affect innate immune responses of CD mucosa to wheat gluten. Conclusions Our data demonstrated that this gliadin-deficient wheat has a lower direct toxicity but activates an immunologic reaction of the duodenal mucosa like that of the common wheat species.

Celiac disease (CD) is a permanent intolerance to wheat prolamins and related proteins displayed by genetically susceptible individuals. Blocking or modulation of CD-specific T cell response by altered prolamin peptides are currently considered as a potential alternative to the only effective therapy of CD based on a life-long gluten-free diet. Two prolamin peptides, the 9-mer ASRVAPGQQ and the 10-mer GTVGVAPGQQ sequences, were identified by mass spectrometry in the peptic/tryptic digest of prolamins (PTP) from durum wheat (Triticum turgidum ssp. durum) cv. Adamello, and investigated for their ability to preclude the stimulation of CD-specific mucosal T cells by gluten proteins.Gluten-specific polyclonal intestinal T cell lines from five CD children (mean age 5 years) were exposed to 50 microg/ml of a deamidated PTP from whole flour of common wheat (T. aestivum) cv. San Pastore, and tested for proliferation and production of interferon-gamma (INF-gamma) and interleukin 10 (IL-10). The same experiment was performed in the presence of 20 microg/ml of the 9-mer or the 10-mer peptide.T cells exposed to PTP showed a threefold increase in proliferation and INF-gamma production, and a significant (Por= 0.05) reduction in IL-10 secretion as compared with control cells incubated with the culture medium. Addition of either the 9-mer or the 10-mer peptide to PTP downregulated T cell proliferation and INF-gamma production, and caused a significant (Por= 0.05) increase in IL-10 secretion.The T cell reactivity elicited by PTP is precluded by both the 9-mer and the 10-mer sequence, suggesting that over-expression of these proteolytically stable peptides may result in a wheat flour with reduced toxicity for CD patients.

Spikes and seeds from diploid ‘einkorn’ wheat Triticum monococcum and two near-isogenic hard and soft common wheat (Triticum aestivum) lines were harvested at regular intervals from 7 days post-anthesis (dpa) and analysed by non-destructive magnetic resonance imaging (MRI) and time domain-nuclear magnetic resonance (TD-NMR). A large amount of free water occurred in rachises, glumes and awns of spikes collected at 7 dpa, and accumulated in the physiologically active cells of the endosperm at 21 dpa. In the final stages of kernel development, awns and seed embryos exhibited a high MR signal due to the presence of free water likely associated with biological activities. TD-NMR relaxation time distributions obtained by discrete exponential fitting, distributed exponential fitting and SLICING multivariate analysis offered detailed information on mobility behaviour of water molecules in developing seeds and were able to differentiate two soft and hard isolines from common wheat cv. Enesco at early stages of seed development.

Summary Background & aims Celiac disease is a gluten sensitive disorder that occurs in genetically susceptible individuals. The present study deals with variation in the immune response of mucosal T-cells from celiac children to prolamins extracted from nine landraces of farro wheat ( Triticum turgidum ssp. dicoccum ) with contrasting storage protein compositions. Methods The prolamin fraction from nine ‘dicoccum' wheat landraces was subjected to peptic–tryptic digestion and supplied to T-cells from mucosal explants of four celiac patients. Immune reactions in terms of cell proliferation and INF-γ secretion by intestinal T lymphocytes were then determined. Results T-cell lines exposed to digested prolamins from landraces L5563, L5558 and L5540 showed negligible proliferative responses and released INF-γ amounts similar to that of untreated control cells. By contrast, landraces Ersa 6, Ersa 8, Leonessa 4 and Leonessa 5 proved to be very active in triggering the immune responses, whereas landraces Filosini and Prometeo exhibited an intermediate behavior. One-dimensional fractionations by A-PAGE or SDS-PAGE revealed distinctive prolamin patterns amongst the landraces analysed. Conclusions ‘Dicoccum' wheat represents a heterogeneous species showing a wide variation in both prolamin composition and T-cell immunological activation, some ‘dicoccum' landraces being poor in or devoid of noxious gluten proteins.

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. Methods: Cytotoxicity was measured in terms of inhibition of cell growth, activation of apoptosis, release of nitric oxide (NO), detection of tissue transglutaminase (TG II) and alteration of transepithelial electrical resistance (TEER) on Caco-2/Tc7 and K562 (S) cell agglutination. Peptic-tryptic (PT) digest from bread wheat (T. aestivum S. Pastore) was used as a positive control. Results: PT digests of prolamins from spelt wheat (T. aestivum ssp. spelta) were found to exert toxic effects on Caco-2/TC7 cells and to agglutinate K562(S) cells. Increased amounts of NO and TG II expression were observed in Caco-2/TC7 cells exposed to 1 mg/mL of spelt prolamins, suggesting that spelt wheat can induce cellular mechanisms implicated in the pathogenesis of celiac disease. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro wheat (T. turgidum ssp. dicoccum) did not exhibit any negative effects on Caco-2/TC7 and K562(S) cells. Conclusions: The results have shown a constant and significant toxic effect of spelt wheat which is not shared by the two other ancient cereals. Future studies on celiac intestinal organ cultures are needed to increase the prospects of breeding programs aimed at developing wheat cultivars potentially tolerated by most celiac patients.

Background & Aims Nonceliac gluten-sensitive (NCGS) patients report intestinal and extra-intestinal symptoms shortly after ingesting gluten; these symptoms disappear on gluten-free diets, although these patients have no serologic markers of celiac disease or intestinal damage. In fact, there is no evidence for mucosal or serologic modifications in those individuals. We investigated immunologic responses of duodenal mucosa samples and peripheral blood basophils, isolated from NCGS patients, after exposure to gliadin. Methods Participants underwent a complete clinical evaluation to exclude celiac disease while on a gluten-containing diet, a skin prick test to exclude wheat allergy, and upper endoscopy (n = 119) at 2 tertiary medical centers in Italy. Patients were considered to have NCGS based on their symptoms and the current definition of the disorder. Subjects were assigned to the following groups: patients with celiac disease on gluten-free diets (n = 34), untreated patients with celiac disease (n = 35), patients with NCGS (n = 16), or controls (n = 34). Duodenal biopsy samples collected during endoscopy were incubated with gliadin peptides, and levels of inflammatory markers were assessed. Peripheral blood basophils were extracted and incubated with gliadin peptides or a mix of wheat proteins; activation was assessed based on levels of CD203c, CD63, and CD45. Results Duodenal mucosa samples collected from 69 patients with celiac disease showed markers of inflammation after incubation with gliadin. Some, but not all, markers of inflammation were detected weakly in biopsy samples from 3 controls and 3 NCGS patients ( P = .00 for all markers). There were no significant increases in the levels of CD63 and CD203c in NCGS patients. Conclusions Unlike the duodenal mucosa from patients with celiac disease, upon incubation with gliadin, mucosa from patients with NCGS does not express markers of inflammation, and their basophils are not activated by gliadin. The in vitro gliadin challenge therefore should not be used to diagnose NCGS.

In recent years it has become more frequent for physicians to come across types of gluten sensitivity other than celiac disease (CD). Regardless of the absence of any serological CD markers or intestinal damage, gluten sensitive (GS) patients often report intestinal and extraintestinal symptoms shortly after the ingestion of gluten and the disappearance of such symptoms on a gluten free diet (GFD). Regrettably, at present, there is no evidence of any mucosal or serological modifications in GS patients, or that gluten is really the trigger factor in gluten sensitivity. We aimed at evaluating the impact of gliadin challenge on two experimental settings, i.e. in vitro gliadin challenge of duodenal mucosa and peripheral blood basophils in GS patients compared to CD patients and healthy controls. We consecutively enrolled patients referred to tertiary centers for food intolerance and celiac disease. Participants underwent a complete clinical screening in order to rule out CD (serum a-tTG, EMA, total IgA and HLA status, where appropriate), an interview for evaluation of diet and symptoms, a skin prick test to exclude wheat allergy, upper endoscopy for duodenal biopsies used for both histological assessment and for the in vitro evaluation of the gliadin-induced mucosal expression of early and late inflammatory markers: PY99, epithelial HLA-DR, ICAM1, crypt HLA-DR, CD3, CD25 and CD69. In addition, a basophils activation assay, in which patients' basophils were challenged with a gliadin extract, was performed. Basophils were analyzed for the expression of hematopoietic membrane antigens CD203c, CD63 and CD45 by flow cytometry looking at differences in antigen expression. 119 subjects were screened for CD and GS and, according to the results, four groups were obtained: GS patients (no.16), CD on gluten free diet (no.34), CD on gluten containing diet (no.35) and healthy controls (no.34). As for the in vitro gliadin challenge, all CD patients, regardless of their dietetic status, showed a clear mucosal activation (increased immunofluorescence intensity both for early and delayed inflammation markers) when stimulated with gliadin, while only 3 controls and 3 GS patients showed an weak response for some, not all, inflammation markers. Also, there were no significant differences in the skin prick test results or in the level of CD63 and CD203c basophils expression, used as a marker of cell activation, among the groups. The present study shows that in GS patients, gliadin challenge test did not show any significant modifications in the expression of those mucosal inflammation markers found increased in CD patients. Therefore, this tool should not be used as a diagnostic instrument in case of GS. Moreover, the lack of differences in basophils activation and skin prick test seems to exclude this form of cellular sensitivity in our GS series also.

In 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.