Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties.

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).

IL-10-producing regulatory cells impact on celiac disease evolution.

Celiac Disease (CD) is a T-cell mediated disorder caused by immune response to gluten, although the mechanisms underlying CD progression are still elusive. We analyzed immune cell composition, plasma cytokines, and gliadin-specific T-cell responses in patients with positive serology and normal intestinal mucosa (potential-CD) or villous atrophy (acute-CD), and after gluten-free diet (GFD). We found: an inflammatory signature and the presence of circulating gliadin-specific IFN-γ+ T cells in CD patients regardless of mucosal damage; an increased frequency of IL-10-secreting dendritic cells (DC-10) in the gut and of circulating gliadin-specific IL-10-secreting T cells in potential-CD; IL-10 inhibition increased IFN-γ secretion by gliadin-specific intestinal T cells from acute- and potential-CD. On GFD, inflammatory cytokines normalized, while IL-10-producing T cells accumulated in the gut. We show that IL-10-producing cells are fundamental in controlling pathological T-cell responses to gluten: DC-10 protect the intestinal mucosa from damage and represent a marker of potential-CD.

Sulfated polysaccharides accelerate gliadin digestion and reduce its toxicity.

Celiac disease and other types of gluten intolerance significantly affect the life quality of patients making them restrict the diet removing all food produced from wheat, rye, oat, and barley flour, and some other products. These disorders arise from protease resistance of poorly soluble proteins prolamins, contained in gluten. Enhanced proteolytic digestion of gliadins might be considered as a prospective approach for the treatment of celiac disease and other types of gluten intolerance. Herein, we tested a range of sulfated polymers (kappa-carrageenan, dextran sulfate and different polysaccharides from brown seaweeds, and a synthetic polystyrene sulfonate) for the ability to activate gliadin digestion by human digestive proteases, pepsin and trypsin. Sulfated polysaccharide from Fucus evanescens enhanced proteolytic digestion of gliadins from wheat flour and reduced its cytotoxicity on intestinal epithelial Caco-2 cell culture. Regarding the non-toxic nature of fucoidans, the results provide a basis for polymer-based drugs or additives for the symptomatic treatment of gluten intolerance.

CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole-genome sequencing of an edited line showed mutation or deletion of nearly all ω-gliadin and half of the γ-gliadin gene copies and confirmed the lack of editing in the α/ß-gliadin genes. The estimated 75% and 64% reduction in ω- and γ-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to a non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the ω- and γ-gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

Intragastric administration of transamidated gliadin interferes with the systemic and intestinal immune responses to wheat gliadin in DQ8 transgenic mice.

We have previously shown the ability of transamidated gluten (spf) to modulate both innate and adaptive intestinal immunity elicited by wheat gliadin in HLA-DQ8 transgenic mice (DQ8 mice), a model of gluten sensitivity. Herein, we evaluated the influence of spf when administered intragastrically on the immune response to native gliadin in DQ8 mice. To address the issue, we analysed three regimens of antigen administration: before immunisation (pre-treatment), during immunisation (co-treatment) and through breast milk during the lactating phase (suckling treatment). Mice were immunised mucosally by intranasal delivery of digested wheat gliadin along with cholera toxin in multiple doses. After sacrifice, isolated spleen and mesenteric lymph node (MLN) cells were challenged in vitro and the cytokine profile of culture supernatants assessed by ELISA and multiparametric assay. We found that only pre-treatment with spf was effective in down-regulating the gliadin-specific IFN-γ response and only in spleen cells. Interestingly, spf pre-treatment also induced systemic IL-6, IL-17A and TNF-α. By contrast, we found that spf pre-treatment upregulated INF-γ in MLN but also significantly decreased IL-2. In conclusion, our data provide evidence that the preventive intragastric administration of transamidated gluten is able to interfere with the classical cytokine profile induced by gliadin via mucosal immunisation in a transgenic model expressing one of the HLA molecules associated with coeliac disease.

Diagnostic accuracy of a novel point-of-care test for simultaneous detection of anti-transglutaminase IgA and anti-deamidated gliadin IgG antibodies.

BACKGROUND: Point-of-care tests (POCTs) may have a role in detecting undiagnosed cases of Celiac disease (CD). We assessed the diagnostic accuracy of a novel POCT, compared with the conventional serological methods, for simultaneous anti-transglutaminase (tTG) IgA and anti-deamidated gliadin (DGP) IgG antibody detection. Furthermore, we evaluated the effect of different biological matrices (whole blood and serum) on test performance. METHODS: Serum and whole blood from celiac or suspected celiac patients who underwent duodenal biopsy were assayed for the presence of anti-tTG IgA and anti-DGP IgG both with the reference standard methods (Thermo Fisher Scientific, Uppsala, Sweden) and with the POCT (PRIMA Lab SA, Balerna, Switzerland). RESULTS: 266 sera (101 negative and 165 positive) and 60 whole blood samples (34 positive and 26 negative) were included in the study. POCT for anti-DGP IgG showed a sensitivity of 84.3% and a specificity of 90.1%, with positive (PPV) and negative predictive values (NPV) of 91.07% and 82.73%. POCT for anti-tTG IgA showed a sensitivity of 98.31% and a specificity of 98.02%, with a PPV and NPV of 98.31% and 98.02%. Test accuracies were 86.94% and 98.17%, respectively. The agreement of the results between the two different matrices showed a strong correlation rate: 95% for anti-DGP IgG and 100% for anti-tTG IgA. CONCLUSION: The anti-tTG IgA/anti-DGP IgG-based POCT showed good diagnostic accuracy with comparable sensitivities and specificities to reference standard methods in detecting CD in symptomatic patients and could be considered as a mass screening test before referring to conventional serology.

Purified Clinoptilolite-Tuff as an Efficient Sorbent for Food-Derived Peanut Allergens.

The avoidance of allergen intake is crucial for persons affected by peanut allergy; however, the cross-contamination of food is common and leads to unpredictable consequences after the consumption of supposedly "safe" food. The aim of the present study was to eliminate harmful traces of peanut allergens from food using purified clinoptilolite-tuff (PCT)-a specially processed zeolite material. Analyses were performed using a peanut ELISA and a Coomassie blue (Bradford) assay. Mimicking conditions of the human gastrointestinal tract demonstrated a higher efficacy of PCT in the intestine (pH 6.8) than in the stomach (pH 1.5). Adsorption rates were fast (<2 min) and indicated high capacities (23 µg and 40 µg per 1 mg of PCT at pH 1.5 and pH 6.8, respectively). Allergenically relevant peanut protein concentrations were sorbed in artificial fluids (32 µg/mL by 4 mg/mL of PCT at pH 1.5 and 80.8 µg/mL by 0.25 mg/mL of PCT at pH 6.8) when imitating a daily dose of 2 g of PCT in an average stomach volume of 500 mL. Experiments focusing on the bioavailability of peanut protein attached to PCT revealed sustained sorption at pH 1.5 and only minor desorption at pH 6.8. Accompanied by gluten, peanut proteins showed competing binding characteristics with PCT. This study therefore demonstrates the potential of PCT in binding relevant quantities of peanut allergens during the digestion of peanut-contaminated food.

Ultrasound-enhanced covalent reaction of gliadin: the inhibition of antigenicity and its potential mechanisms.

BACKGROUND: Wheat proteins can be divided into water/salt-soluble protein (albumin/globulin) and water/salt-insoluble protein (gliadins and glutenins (Glu)) according to solubility. Gliadins (Glia) are one of the major allergens in wheat. The inhibition of Glia antigenicity by conventional processing techniques was not satisfactory. RESULTS: In this study, free radical oxidation was used to induce covalent reactions. The effects of covalent reactions by high-intensity ultrasound (HIU) of different powers was compared. The enhancement of covalent grafting effectiveness between gliadin and (-)-epigallo-catechin 3-gallate (EGCG) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry and Folin-Ciocalteu tests. HIU caused protein deconvolution and disrupted the intrastrand disulfide bonds that maintain the tertiary structure, causing a shift in the side chain structure, as proved by Fourier, fluorescence and Raman spectroscopic analysis. Comparatively, the antigenic response of the conjugates formed in the sonication environment was significantly weaker, while these conjugates were more readily hydrolyzed and less antigenic during simulated gastrointestinal fluid digestion. CONCLUSION: HIU-enhanced free radical oxidation caused further transformation of the spatial structure of Glia, which hid or destroyed the antigenic epitope, effectively inhibiting protein antigenicity. This study widened the application of polyphenol modification in the inhibition of wheat allergens. © 2024 Society of Chemical Industry.

The changes induced by hydrodynamic cavitation treatment in wheat gliadin and celiac-toxic peptides.

Hydrodynamic cavitation (HC) is thought weaken the allergenicity of beer gluten proteins. However, the mechanism of action has not been thoroughly studied. In this study, an HC device was used to treat wheat gliadin and two specific celiac-toxic peptides, P1 and P2. FT-IR, MFS, HPLC, and CD were used to monitor the structural characteristics of gliadin and the two peptides. HC reduced the abundance of the coeliac toxic peptides P1 and P2 in solution and the contents of secondary structure ß-turns and PPII, which are related to reduced allergen immunoreactivity. This meant that both the primary and secondary structures of P1 and P2 were affected by HC, leading to fewer allergic reactions. This study was focused on the impact of HC on the secondary structures of allergens produced from gluten raw materials, and it has positive implications for reducing product allergenicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05973-7.

Exercise training restores weight gain and attenuates hepatic inflammation in a rat model of non-celiac gluten sensitivity.

Gluten intolerance is associated with several disorders in the body. Although research has grown in recent years, the understanding of its impact on different tissues and the effects of physical exercise in mitigating health problems in the condition of gluten intolerance are still limited. Therefore, our objective was to test whether gliadin would affect metabolism and inflammation in liver tissue and whether aerobic physical exercise would mitigate the negative impacts of gliadin administration in rodents. Wistar rats were divided into exercised gliadin, gliadin, and control groups. Gliadin was administered by gavage from birth to 60 days of age. The rats in the exercised gliadin group performed an aerobic running exercise training protocol for 15 days. At the end of the experiments, physiological, histological, and molecular analyzes were performed in the study. Compared to the control group, the gliadin group had impaired weight gain and increased gluconeogenesis, lipogenesis, and inflammatory biomarkers in the liver. On the other hand, compared to the gliadin group, animals in the exercise-gliadin group had a recovery in body weight, improved insulin sensitivity, and a reduction in some gluconeogenesis, lipogenesis, and inflammatory biomarkers in the liver. In conclusion, our results revealed that the administration of gliadin from birth impaired weight gain and induced an increase in hepatic inflammatory cytokines, which was associated with an impairment of glycemic homeostasis in the liver, all of which were attenuated by adding aerobic exercise training in the gliadin group.

A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease.

Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.

An elite γ-gliadin allele improves end-use quality in wheat.

Gluten is composed of glutenins and gliadins and determines the viscoelastic properties of dough and end-use quality in wheat (Triticum aestivum L.). Gliadins are important for wheat end-use traits, but the contribution of individual gliadin genes is unclear, since gliadins are encoded by a complex, multigenic family, including many pseudogenes. We used CRISPR/Cas9-mediated gene editing and map-based cloning to investigate the contribution of the γ-gliadin genes annotated in the wheat cultivar 'Fielder', showing that Gli-γ1-1D and Gli-γ2-1B account for most of the γ-gliadin accumulation. The impaired activity of only two γ-gliadin genes in knockout mutants improved end-use quality and reduced gluten epitopes associated with celiac disease (CD). Furthermore, we identified an elite haplotype of Gli-γ1-1D linked to higher end-use quality in a wheat germplasm collection and developed a molecular marker for this allele for marker-assisted selection. Our findings provide information and tools for biotechnology-based and classical breeding programs aimed at improving wheat end-use quality.

Alpha/Beta Gliadin MM1 Is a Novel Antigen for Wheat-Dependent Exercise-Induced Anaphylaxis.

INTRODUCTION: Screening for ω-5 gliadin specific IgE antibody (sIgE) has high diagnostic utility in cases of suspected wheat-dependent exercise-induced anaphylaxis (WDEIA); however, negative cases may require confirmatory tests, such as the oral challenge test. Thus, newly identified allergens that can be used for the serological diagnosis of WDEIA are needed. This study aimed to identify additional sIgE biomarkers of WDEIA. METHODS: Forty-two patients with WDEIA (5 negative/37 positive for ω-5 gliadin sIgE) were enrolled. For comparison, 8 patients with immediate-type wheat allergy without WDEIA and 20 healthy controls without wheat allergy were also enrolled. Extracted wheat proteins were separated by 2D-PAGE. Proteins that reacted with serum IgE antibody in 2D Western blotting (2D-WB) were identified using mass spectrometry. Recombinant proteins were synthesized in Escherichia coli, and the antigenicity was tested using ELISA and the basophil activation test. RESULTS: In 2D-WB, nine proteins reacted with the serum IgE antibody from at least 60% of patients with WDEIA (n ≥ 25/42). ELISA revealed that alpha/beta gliadin MM1 exhibited the highest positive immunoreactivity in 23 of 26 patients who were positive for ω-5 gliadin sIgE (88%) and in 5 of 5 patients who were negative for ω-5 gliadin sIgE (100%). Alpha/beta gliadin MM1 exhibited significantly higher basophil activation in 14 patients with WDEIA when compared to 5 individuals without a wheat allergy. CONCLUSIONS: Alpha/beta gliadin MM1 sIgE exhibited the highest seropositivity, even among patients who were negative for ω-5 gliadin sIgE. The inclusion of alpha/beta gliadin MM1 in allergen-sIgE tests may improve the sensitivity for diagnosing WDEIA.

Bromelain and ficin proteolytic effects on gliadin cytotoxicity and expression of genes involved in cell-tight junctions in Caco-2 cells.

Enzyme therapy for celiac disease (CeD), which digests gliadin into non-immunogenic and non-toxic peptides, can be an appropriate treatment option for CeD. Here, we have investigated the effectiveness of bromelain and ficin on gliadin digestion using in vitro, such as SDS-PAGE, HPLC, and circular dichroism (CD). Furthermore, the cytotoxicity of gliadin and 19-mer peptide before and after digestion with these enzymes was evaluated using the MTT assay in the Caco-2 cell line. Finally, we examined the effect of these treatments along with Larazotide Acetate on the expression of genes involved in cell-tight junctions, such as Occludin, Claudin 3, tight junction protein-1, and Zonulin in the Caco-2 cell line. Our study demonstrated bromelain and ficin digestion effects on the commercial and wheat-extracted gliadin by SDS-PAGE, HPLC, and CD. Also, the cytotoxicity results on Caco-2 showed that toxicity of the gliadin and synthetic 19-mer peptide was decreased by adding bromelain and ficin. Furthermore, the proteolytic effects of bromelain and ficin on gliadin indicated the expression of genes involved in cell-tight junctions was improved. This study confirms that bromelain and ficin mixture could be effective in improving the symptoms of CeD.

From celiac disease to coccidia infection and vice-versa: The polyQ peptide CXCR3-interaction axis.

Zonulin is a physiological modulator of intercellular tight junctions, which upregulation is involved in several diseases like celiac disease (CeD). The polyQ gliadin fragment binds to the CXCR3 chemokine receptor that activates zonulin upregulation, leading to increased intestinal permeability in humans. Here, we report a general hypothesis based on the structural connection between the polyQ sequence of the immunogenic CeD protein, gliadin, and enteric coccidian parasites proteins. Firstly, a novel interaction pathway between the parasites and the host is described based on the structural similarities between polyQ gliadin fragments and the parasite proteins. Secondly, a potential connection between coccidial infections as a novel environmental trigger of CeD is hypothesized. Therefore, this report represents a promising breakthrough for coccidian research and points out the potential role of coccidian parasites as a novel trigger of CeD that might define a preventive strategy for gluten-related disorders in general. Also see the video abstract here: https://youtu.be/oMaQasStcFI.

Specific Genetic Polymorphisms Contributing in Differential Binding of Gliadin Peptides to HLA-DQ and TCR to Elicit Immunogenicity in Celiac Disease.

Immunogenicity of gliadin peptides in celiac disease (CD) is majorly determined by the pattern of molecular interactions with HLA-DQ and T-cell receptors (TCR). Investigation of the interactions between immune-dominant gliadin peptides, DQ protein, and TCR are warranted to unravel the basis of immunogenicity and variability contributed by the genetic polymorphisms. Homology modeling of HLA and TCR done using Swiss Model and iTASSER, respectively. Molecular interactions of eight common deamidated immune-dominant gliadin with HLA-DQ allotypes and specific TCR gene pairs were evaluated. Docking of the three structures was performed with ClusPro2.0 and ProDiGY was used to predict binding energies. Effects of known allelic polymorphisms and reported susceptibility SNPs were predicted on protein-protein interactions. CD susceptible allele, HLA-DQ2.5 was shown to have considerable binding affinity to 33-mer gliadin (ΔG = - 13.9; Kd = 1.5E - 10) in the presence of TRAV26/TRBV7. Higher binding affinity was predicted (ΔG = - 14.3, Kd = 8.9E - 11) when TRBV28 was replaced with TRBV20 paired with TRAV4 suggesting its role in CD predisposition. SNP rs12722069 at HLA-DQ8 that codes Arg76α forms three H-bonds with Glu12 and two H-bonds with Asn13 of DQ2 restricted gliadin in the presence of TRAV8-3/TRBV6. None of the HLA-DQ polymorphisms was found to be in linkage disequilibrium with reported CD susceptibility markers. Haplotypic presentations of rs12722069-G, rs1130392-C, rs3188043-C and rs4193-A with CD reported SNPs were observed in sub-ethnic groups. Highly polymorphic sites of HLA alleles and TCR variable regions could be utilized for better risk prediction models in CD. Therapeutic strategies by identifying inhibitors or blockers targeting specific gliadin:HLA-DQ:TCR binding sites could be investigated.

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment.

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.

Analysis of Circulating Food Antigen-Specific T-Cells in Celiac Disease and Inflammatory Bowel Disease.

To demonstrate and analyze the specific T-cell response following barrier disruption and antigen translocation, circulating food antigen-specific effector T-cells isolated from peripheral blood were analyzed in patients suffering from celiac disease (CeD) as well as inflammatory bowel disease (IBD). We applied the antigen-reactive T-cell enrichment (ARTE) technique allowing for phenotypical and functional flow cytometric analyses of rare nutritional antigen-specific T-cells, including the celiac disease-causing gliadin (gluten). For CeD, patient groups, including treatment-refractory cases, differ significantly from healthy controls. Even symptom-free patients on a gluten-free diet were distinguishable from healthy controls, without being previously challenged with gluten. Moreover, frequency and phenotype of nutritional antigen-specific T-cells of IBD patients directly correlated to the presence of small intestinal inflammation. Specifically, the frequency of antigen specific T-cells as well as pro-inflammatory cytokines was increased in patients with active CeD or Crohn's disease, respectively. These results suggest active small intestinal inflammation as key for the development of a peripheral food antigen-specific T-cell response in Crohn's disease and celiac disease.

Inhibition of Transglutaminase 2 as a Therapeutic Strategy in Celiac Disease-In Vitro Studies in Intestinal Cells and Duodenal Biopsies.

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD.

Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease.

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1ß, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFß, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies.