Transamidation Down-Regulates Intestinal Immunity of Recombinant α-Gliadin in HLA-DQ8 Transgenic Mice.

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.

Effects of Gliadin on Autoimmune Responses of Central Nervous System of C57BL/6 Mice.

Gluten sensitivity contributes to various degrees of neurological manifestations and neurodegenerative immunological changes. We investigated the experimental features of anti-gliadin immune responses in the central nervous system (CNS) of mice. Female C57BL6 mice were divided into three groups. Mice immunized with complete Freund's adjuvant (CFA) or gliadin emulsified in CFA, and the control group received phosphate-buffered saline (PBS). Immunohistochemistry, hematoxylin-eosin, and Luxol fast blue staining were performed on the sections. The serum levels of interleukin (IL)-17 and interferon-gamma (IFN-γ) were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the mRNA levels of chemokine (C-X-C motif) ligand-2 (CXCL-2), C-C motif chemokine ligand-2 (CCL-2), and CXCL-10.  In gliadin+CFA immunized mice, the microscopic lesions included perivascular edema, focal-microgliosis, and acute neuronal necrosis in the cortex, subcortical, Purkinje cell layer, and ventral horn of the spinal cord. While extravasation of anti-IgG antibodies and selective targeting of Purkinje cells were observed in gliadin+CFA immunized mice. A significant increase in serum IL-17 and IFN-g levels (p<0.05), as well as expression of CXCL-2, CCL-2, and CXCL-10 in mice immunized with gliadin+CFA, were monitored versus controls. Our findings indicated that the immune responses directed against gliadin peptides might contribute to blood-brain barrier breakdown, extravasation of serum anti-IgG, gliosis, and acute neuronal necrosis in the cortex and cerebellar Purkinje cells. Anti-IgG antibodies may cause extravasation of blood-born anti-gliadin antibodies and selective targeting of Purkinje cells observed in mice immunized with peptide tryptic (pt) -gliadin in CFA.

TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac Disease: A Randomized, Double-Blind, Placebo-Controlled Study.

BACKGROUND & AIMS: In celiac disease (CeD), gluten induces immune activation, leading to enteropathy. TAK-101, gluten protein (gliadin) encapsulated in negatively charged poly(dl-lactide-co-glycolic acid) nanoparticles, is designed to induce gluten-specific tolerance. METHODS: TAK-101 was evaluated in phase 1 dose escalation safety and phase 2a double-blind, randomized, placebo-controlled studies. Primary endpoints included pharmacokinetics, safety, and tolerability of TAK-101 (phase 1) and change from baseline in circulating gliadin-specific interferon-γ-producing cells at day 6 of gluten challenge, in patients with CeD (phase 2a). Secondary endpoints in the phase 2a study included changes from baseline in enteropathy (villus height to crypt depth ratio [Vh:Cd]), and frequency of intestinal intraepithelial lymphocytes and peripheral gut-homing T cells. RESULTS: In phase 2a, 33 randomized patients completed the 14-day gluten challenge. TAK-101 induced an 88% reduction in change from baseline in interferon-γ spot-forming units vs placebo (2.01 vs 17.58, P = .006). Vh:Cd deteriorated in the placebo group (-0.63, P = .002), but not in the TAK-101 group (-0.18, P = .110), although the intergroup change from baseline was not significant (P = .08). Intraepithelial lymphocyte numbers remained equal. TAK-101 reduced changes in circulating α4ß7+CD4+ (0.26 vs 1.05, P = .032), αEß7+CD8+ (0.69 vs 3.64, P = .003), and γδ (0.15 vs 1.59, P = .010) effector memory T cells. TAK-101 (up to 8 mg/kg) induced no clinically meaningful changes in vital signs or routine clinical laboratory evaluations. No serious adverse events occurred. CONCLUSIONS: TAK-101 was well tolerated and prevented gluten-induced immune activation in CeD. The findings from the present clinical trial suggest that antigen-specific tolerance was induced and represent a novel approach translatable to other immune-mediated diseases. ClinicalTrials.gov identifiers: NCT03486990 and NCT03738475.

Gliadin Nanoparticles Induce Immune Tolerance to Gliadin in Mouse Models of Celiac Disease.

BACKGROUND & AIMS: Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease. METHODS: We performed studies with C57BL/6; RAG1-/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG-/- mice were given intraperitoneal injections of CD4+CD62L-CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed. RESULTS: TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction. CONCLUSIONS: In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.

Development of antioxidant gliadin particle stabilized Pickering high internal phase emulsions (HIPEs) as oral delivery systems and the in vitro digestion fate.

In this paper, we demonstrate for the first time the use of gliadin particles to structure algal oil (rich in DHA) and to exert chemical stability against lipid oxidation via the Pickering high internal phase emulsion (HIPE) strategy. The gliadin/chitosan colloid particles (GCCPs) were effectively adsorbed and anchored at the algal oil-water interface. Concomitantly, the particle-coated droplets as building blocks constructed a percolating 3D-network framework, endowing Pickering HIPEs with viscoelastic and self-supporting attributes. In addition, Pickering HIPEs loaded with shell (HIP-curEs) or core curcumin (HIPEs-cur) were constructed to depress the oxidation of algal oil. The content of primary (lipid hydroperoxides) and secondary (malondialdehyde and hexanal) oxidation products in HIPEs was lower than that in bulk oil. The oxidative stability of HIPEs was further improved in shell and core curcumin. An in vitro gastrointestinal (GI) model was constructed to characterize the lipid digestion, lipid oxidation as well as curcumin bioaccessibility of the ingested Pickering HIPEs. Lipid oxidation in the Pickering HIPEs was retarded under GI fluids, especially in the presence of core curcumin. The free fatty acid (FFA) fraction released was below 30% for all HIPEs, reflecting that the Pickering HIPEs formed restrict the digestion of fat or oil and potentially help to fight obesity. Interestingly, this route enhanced the bioaccessibility of curcumin from only 2.13% (bulk algal oil) to 53.61% (core curcumin); in particular, it reached 76.82% for shell curcumin. These results help to fill the gap between the physicochemical performance of the gliadin particle stabilized Pickering HIPEs and their potential applications as oral delivery systems of nutraceuticals. This work opens concomitantly an attractive strategy to convert liquid oils into antioxidant soft solids without artificial trans fats, as a potential alternative for PHOs.

Effects of Gliadin consumption on the Intestinal Microbiota and Metabolic Homeostasis in Mice Fed a High-fat Diet.

Dietary gluten causes severe disorders like celiac disease in gluten-intolerant humans. However, currently understanding of its impact in tolerant individuals is limited. Our objective was to test whether gliadin, one of the detrimental parts of gluten, would impact the metabolic effects of an obesogenic diet. Mice were fed either a defined high-fat diet (HFD) containing 4% gliadin (n = 20), or a gliadin-free, isocaloric HFD (n = 20) for 23 weeks. Combined analysis of several parameters including insulin resistance, histology of liver and adipose tissue, intestinal microbiota in three gut compartments, gut barrier function, gene expression, urinary metabolites and immune profiles in intestinal, lymphoid, liver and adipose tissues was performed. Mice fed the gliadin-containing HFD displayed higher glycated hemoglobin and higher insulin resistance as evaluated by the homeostasis model assessment, more hepatic lipid accumulation and smaller adipocytes than mice fed the gliadin-free HFD. This was accompanied by alterations in the composition and activity of the gut microbiota, gut barrier function, urine metabolome, and immune phenotypes within liver and adipose tissue. Our results reveal that gliadin disturbs the intestinal environment and affects metabolic homeostasis in obese mice, suggesting a detrimental effect of gluten intake in gluten-tolerant subjects consuming a high-fat diet.

Clinical and laboratory features, and quality of life assessment in wheat dependent exercise-induced anaphylaxis patients from central China.

Wheat dependent exercise-induced anaphylaxis (WDEIA) is a rare but potentially severe food allergy caused by the combination of wheat ingestion and physical exercise. The impact of WDEIA on quality of life (QOL) is unclear. This study characterized the clinical and laboratory features and investigated the QOL in WDEIA patients from Central China. Twenty-eight WDEIA patients were analyzed, and QOL was measured by validated Chinese version Food Allergy Quality of Life Questionnaire-Adult Form (FAQLQ-AF) and Food Allergy Independent Measure (FAIM) after obtaining the diagnosis. The results showed that half of the patients were females. The median onset age was 37 years old. The symptoms occurred within 1 h after wheat ingestion (26/28). Symptoms of anaphylaxis included cutaneous (26/28), respiratory (11/28), gastro-intestinal (5/28) and cardiovascular manifestations (27/28). Skin prick tests were positive to salt soluble (89.3%) and salt insoluble wheat allergen extracts (100%). Positive rate to wheat, gluten and omega-5 gliadin specific IgE was 64.3%, 92.9% and 92.9% respectively. Specific IgE to omega-5 gliadin with a cut-off value 0.83 KU/L offered highly efficient diagnostic criterion for WDEIA (sensitivity: 89.3%; and specificity: 88.9%). The mean scores of FAQLQ-AF and FAIM were 4.70 and 4.98 respectively and level of anti-omega-5 gliadin IgE had positive correlations with FAQLQ scores. Thereby, WDEIA is commonly found in mid-age adults. In most cases, multi-organs especially skin and cardiovascular systems are involved. Salt insoluble wheat allergen skin test and serum specific IgE to gluten and omega-5 gliadin help to diagnose WDEIA. QOL in WDEIA patients is severely impaired.

Mechanisms of innate immune activation by gluten peptide p31-43 in mice.

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. Innate immunity contributes to the pathogenesis of CD, but the mechanisms remain poorly understood. Although previous in vitro work suggests that gliadin peptide p31-43 acts as an innate immune trigger, the underlying pathways are unclear and have not been explored in vivo. Here we show that intraluminal delivery of p31-43 induces morphological changes in the small intestinal mucosa of normal mice consistent with those seen in CD, including increased cell death and expression of inflammatory mediators. The effects of p31-43 were dependent on MyD88 and type I IFNs, but not Toll-like receptor 4 (TLR4), and were enhanced by coadministration of the TLR3 agonist polyinosinic:polycytidylic acid. Together, these results indicate that gliadin peptide p31-43 activates the innate immune pathways in vivo, such as IFN-dependent inflammation, relevant to CD. Our findings also suggest a common mechanism for the potential interaction between dietary gluten and viral infections in the pathogenesis of CD.

Identification of wheat sensitization using an in-house wheat extract in Coca-10% alcohol solution in children with wheat anaphylaxis.

BACKGROUND: Identification of wheat sensitization by a skin prick test (SPT) is essential for children with wheat-induced anaphylaxis, since oral food challenge can cause serious adverse effects. Wheat allergens are both water/salt and alcohol soluble. The preparation of wheat extract for SPT containing both water/salt and alcohol soluble allergen is needed. OBJECTIVE: To determine if a wheat extract using Coca's solution containing 10% alcohol (Coca-10% EtOH), prepared in-house, contians both water/salt and alcohol soluble allergens. METHODS: Serum of children with a history of anaphylaxis after wheat ingestion was used. Wheat flour was extracted in Coca-10% alcohol solution. An SPT with both commercial and in-house wheat extracts was performed as well as specific IgE (sIgE) for wheat and omega-5 gliadin. Direct and IgE inhibition immunoblots were performed to determine serum sIgE levels against water/salt as well as alcohol soluble (gliadins and glutenins) allergens in the extracts. RESULTS: Six children with history of wheat anaphylaxis had positive SPT to both commercial and in-house extracts. They also had different levels of sIgE against wheat and omega-5 gliadin allergens. The results of direct immunoblotting showed all tested sera had sIgE bound to ~35 kDa wheat protein. Further IgE inhibition immunoblotting identified the ~35 kDa wheat protein as gliadin but not gluten allergen. CONCLUSION: The in-house prepared Coca-10% EtOH solution could extract both water/salt and alcohol soluble allergens. The ~35 kDa gliadin appears to be a major wheat allergen among tested individuals.

Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice.

The induction of mucosal immunity requires efficient antigen delivery and adjuvant systems. Probiotic bacterial strains are considered to be very promising tools to address both of these needs. In particular, Bacillus subtilis spores are currently under investigation as a long-lived, protease-resistant adjuvant system for different antigens. Furthermore, a non-recombinant approach has been developed based on the stable adsorption of antigen on the spore surface. In the present study, we explored this strategy as a means of modulating the immune response to wheat gliadin, the triggering agent of celiac disease (CD), an enteropathy driven by inflammatory CD4(+) T cells. Gliadin adsorption was tested on untreated or autoclaved wild-type (wt) and mutant (cotH or cotE) spores. We found that gliadin was stably and maximally adsorbed by autoclaved wt spores. We then tested the immune properties of the spore-adsorbed gliadin in HLA-DQ8-transgenic mice, which express one of the two HLA heterodimers associated with CD. In vitro, spore-adsorbed gliadin was efficiently taken up by mouse dendritic cells (DCs). Interestingly, gliadin-pulsed DCs efficiently stimulated splenic CD4(+) T cells from mice immunised with spore-adsorbed gliadin. Nasal pre-dosing with spore-adsorbed gliadin failed to down-regulate the ongoing cellular response in gliadin-sensitised DQ8 mice. Notably, naïve mice inoculated intranasally with multiple doses of spore-adsorbed gliadin developed an intestinal antigen-specific CD4(+) T cell-mediated response. In conclusion, our data highlight the ability of spore-adsorbed gliadin to elicit a T-cell response in the gut that could be exploitable for developing immune strategies in CD.

Intraluminal administration of poly I:C causes an enteropathy that is exacerbated by administration of oral dietary antigen.

Systemic administration of polyinosinic:polycytidylic acid (poly I:C), mimics virally-induced activation of TLR3 signalling causing acute small intestine damage, but whether and how mucosal administration of poly I:C causes enteropathy is less clear. Our aim was to investigate the inflammatory pathways elicited after intraluminal administration of poly I:C and determine acute and delayed consequences of this locally induced immune activation. Intraluminal poly I:C induced rapid mucosal immune activation in C57BL/6 mice involving IFNß and the CXCL10/CXCR3 axis, that may drive inflammation towards a Th1 profile. Intraluminal poly I:C also caused enteropathy and gut dysfunction in gliadin-sensitive NOD-DQ8 mice, and this was prolonged by concomitant oral administration of gliadin. Our results indicate that small intestine pathology can be induced in mice by intraluminal administration of poly I:C and that this is exacerbated by subsequent oral delivery of a relevant dietary antigen.

Prevention or early cure of type 1 diabetes by intranasal administration of gliadin in NOD mice.

Induction of long-term tolerance to ß-cell autoantigens has been investigated both in animal models and in human type 1 diabetes (T1D) in order to prevent the disease. As regards external compounds, the dietary plant protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevent T1D in animal models. Herewith we investigated whether intranasal (i.n.) administration of gliadin or gluten may arrest the diabetogenic process. I.n. administration of gliadin to 4-week-old NOD mice significantly reduced the diabetes incidence. Similarly, the insulitis was lowered. Intranasal gliadin also rescued a fraction of prediabetic 13-week-old NOD mice from progressing to clinical onset of diabetes compared to OVA-treated controls. Vaccination with i.n. gliadin led to an induction of CD4(+)Foxp3(+) T cells and even more significant induction of γδ T cells in mucosal, but not in non-mucosal lymphoid compartments. This prevention strategy was characterized by an increased proportion of IL-10 and a decreased proportion of IL-2, IL-4 and IFN-γ-positive CD4(+)Foxp3(+) T cells, and IFN-γ-positive γδ T cells, preferentially in mucosal lymphoid organs. In conclusion, i.n. vaccination with gliadin, an environmental antigen with possible etiological influence in T1D, may represent a novel, safer strategy for prevention or even early cure of T1D.

Monitoring of daily gliadin intake in patients on gluten-free diets.

The aim of the study was to show patients suffering from the coeliac disease, their real gliadin daily intake, offer them very useful information concerning their diet and to find random possible mistakes. The monitoring was carried out within the context of their routine everyday diet regimen. The daily intake of gliadin in the diet was quantified on the basis of gliadin determination in their current daily food. The gluten-free diet was followed for 30 days. The patients were taking regular daily meals, drinks, and sometimes medicines or food supplements. The patients were provided with instructions, survey forms, digital scales, polyethylen bottles and sacks. The patients took out the stipulated amount, which served as a sample of each of their daily meals. The samples included both homemade meals as well as commercial products. The content of gliadin in daily meal was determined by the sandwich ELISA method. The daily gliadin intake was calculated on the base of the reported amount of meals ingested. 1,900 food samples were analyzed within the framework of this study. Several contaminated commercial foods were found; nevertheless this fact did not influence the otherwise satisfactory overall picture of the daily gliadin intake by the patients followed. The results in 14 patients revealed a satisfactory adherence to the gluten-free diet. It was proved that conscientiousness and awareness on the part of coeliac patients, or those taking care of them, is of paramount importance in determining the choice of foods comprising a gluten-free diet.

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.

Single domain antibodies are specially suited for quantitative determination of gliadins under denaturing conditions.

Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

Differential mucosal IL-17 expression in two gliadin-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease.

BACKGROUND: The immune-mediated enteropathy, celiac disease (CD), and gluten sensitivity (GS) are two distinct clinical conditions that are both triggered by the ingestion of wheat gliadin. CD, but not GS, is associated with and possibly mediated by an autoimmune process. Recent studies show that gliadin may induce the activation of IL-17-producing T cells and that IL-17 expression in the CD mucosa correlates with gluten intake. METHODS: The small-intestinal mucosa of patients with CD and GS and dyspeptic controls was analyzed for expression of IL-17A mRNA by quantitative RT-PCR. The number of CD3+ and TCR-gammadelta lymphocytes and the proportion of CD3+ cells coexpressing the Th17 marker CCR6 were examined by in situ small-intestinal immunohistochemistry. RESULTS: Mucosal expression of IL-17A was significantly increased in CD but not in GS patients, compared to controls. This difference was due to enhanced IL-17A levels in >50% of CD patients, with the remainder expressing levels similar to GS patients or controls, and was paralleled by a trend toward increased proportions of CD3+CCR6+ cells in intestinal mucosal specimens from these subjects. CONCLUSION: We conclude that GS, albeit gluten-induced, is different from CD not only with respect to the genetic makeup and clinical and functional parameters, but also with respect to the nature of the immune response. Our findings also suggest that two subgroups of CD, IL-17-dependent and IL-17-independent, may be identified based on differential mucosal expression of this cytokine.

Induction of antigen-specific tolerance by oral administration of Lactococcus lactis delivered immunodominant DQ8-restricted gliadin peptide in sensitized nonobese diabetic Abo Dq8 transgenic mice.

Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for the induction of tolerance. Celiac disease is associated with either HLA-DQ2- or HLA-DQ8-restricted responses to specific antigenic epitopes of gliadin, and may be treated by induction of Ag-specific tolerance. We investigated whether oral administration of LL-delivered DQ8-specific gliadin epitope induces Ag-specific tolerance. LL was engineered to secrete a deamidated DQ8 gliadin epitope (LL-eDQ8d) and the induction of Ag-specific tolerance was studied in NOD AB degrees DQ8 transgenic mice. Tolerance was assessed by delayed-type hypersensitivity reaction, cytokine measurements, eDQ8d-specific proliferation, and regulatory T cell analysis. Oral administration of LL-eDQ8d induced suppression of local and systemic DQ8-restricted T cell responses in NOD AB degrees DQ8 transgenic mice. Treatment resulted in an Ag-specific decrease of the proliferative capacity of inguinal lymph node (ILN) cells and lamina propria cells. Production of IL-10 and TGF-beta and a significant induction of Foxp3(+) regulatory T cells were associated with the eDQ8d-specific suppression induced by LL-eDQ8d. These data provide support for the development of effective therapeutic approaches for gluten-sensitive disorders using orally administered Ag-secreting LL. Such treatments may be effective even in the setting of established hypersensitivity.

Sensitization and elicitation of an allergic reaction to wheat gliadins in mice.

We developed a mouse model of allergy to wheat flour gliadins, a protein fraction containing major wheat allergens. We compared the antibody responses (i.e., specific IgE and IgG1) and the profiles of cytokines secreted by reactivated splenocytes induced after intraperitoneal injections of gliadins in three strains of mice, namely, Balb/cJ, B10.A, and C3H/HeJ. The intensities of the allergic reactions elicited by intranasal challenge were also compared. Both the sensitization and elicitation were the highest in Balb/cJ mice, whereas weak or no reaction was observed in the others strains. Interestingly, the specificity of the mouse IgE against the different gliadins (i.e., alpha-, beta-, gamma-, omega 1,2-, and omega 5-gliadin) was similar to that observed in children allergic to wheat flour. Balb/cJ mice may thus provide a relevant model for the study of sensitization and elicitation by wheat gliadins and for improving our understanding of the specific role and mechanisms of action of the different classes of gliadins.

Adjuvant effect of Lactobacillus casei in a mouse model of gluten sensitivity.

Probiotic strains have been reported to exert immunomodulatory activities in the gut-associated lymphoid tissue. In this study we explored the effect of Lactobacillus casei in transgenic mice expressing the human DQ8 heterodimer, a HLA molecule linked to Celiac Disease (CD). DQ8 mice, mucosally immunized with the gluten component gliadin, mounted an intestinal Th1-like response as observed in CD, without developing enteropathy. Co-administration of L. casei in sensitized mice specifically enhanced the gliadin-specific response mediated by CD4(+) T cells. Notably, both a strong increase of the gliadin-specific IFNgamma expression and a pro-inflammatory polarization of the cytokine milieu in the small intestinal mucosa were associated to the presence of the probiotic strain. However, this condition did not bring on any mucosal alteration. These findings suggest that the gliadin-specific enteropathy is not merely related to the HLA DQ8-restricted massive production of IFNgamma, but additional parameters are involved. Moreover, our data imply that the intrinsic adjuvanticity of L. casei can be exploited to further enhance both mucosal and systemic T cell-mediated responses.

Natural history of antibodies to deamidated gliadin peptides and transglutaminase in early childhood celiac disease.

INTRODUCTION: Gliadin proteins play a key role in the pathogenesis of celiac disease; however, as a screen for celiac disease, anti-gliadin antibody testing has been replaced by the more sensitive and specific serological assays for transglutaminase autoantibodies (TGAA). A new generation of anti-gliadin antibody assays has been developed to detect synthetic, deamidated homologous gliadin peptides (DGP) with high sensitivity and specificity. METHODS: Sera were collected prospectively from children with an increased risk for celiac disease as part of an ongoing study at Denver, and studied for the development of celiac autoimmunity. We investigated the high-performance DGP antibody assay in 50 TGAA-positive children both before the development of celiac autoimmunity and following the institution of a gluten-free diet to determine the relationship of DGP antibodies to TGAA. TGAA were measured by an in-house radioassay. RESULTS: DGP antibodies and TGAA parallel each other over the period of years children were studied. DGP antibodies resolved sooner than TGAA in subjects on a gluten-free diet. DGP antibodies appeared earlier than TGAA in 9 children. CONCLUSIONS: Measuring DGP antibodies may be more useful than TGAA in monitoring children on a gluten-free diet. DGP antibodies can precede the appearance of TGAA in some at-risk children.