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Background: The immune responsiveness to gluten-containing grains represents a complex process whose establishment and maintenance are not completely elucidated. There are cases of gluten reaction defined as gluten sensitivity (GS) in which neither an allergic (wheat allergy) nor an autoimmune [celiac disease (CD)] mechanism can be advocated. Recent evidences suggest that early changes in intestinal permeability (IP) and activation of both innate and adaptive immune responses are involved in CD pathogenesis. Conversely, no data are currently available on the mechanisms leading to GS. Aims: To investigate the changes in IP, TJ protein genes expression, and innate and adaptive immune responses in GS. Methods:Biopsy samples were obtained from 28 GS patients(pts), 53 pts with active CD, 16 patients with CD in remission, and 37 healthy controls (age range: 5 years -50 years). Quantitative gene expression of TJ proteins Claudin (CL) 1, CL2, CL3, CL4, ZO-1, and TLR1, TLR2 and TLR4, FOXP3, and TGF-β were performed by Real-time PCR. IP was evaluated by means of the lactulose/mannitol test (LA/MA). The number of intraepithelial lymphocytes (IELs) were detected with CD3 and TCR-γδ immunostaining and examined by counting the peroxidase stained cells. ELISA analysis of IL6, IL8, TNFα, was conducted on PBMC of all patients. Results: Intestinal CL3 and CL4 expressions were significantly increased in GS subjects compared to CD patients (p

OBJECTIVE: Most of the recent studies suggest that oats are well tolerated by celiac disease (CD) patients. However, it is still possible that different oat cultivars may display different biological properties relevant for CD pathogenesis. We aimed to investigate biological and immunological properties of two oat varieties, Avena genziana and Avena potenza, in relation to their safety for CD patients. MATERIAL AND METHODS: Phosphorylation of extracellular signal-regulated kinase (ERK) and trans-epithelial electrical resistance (TEER) were evaluated in CaCo-2 cells treated with peptic-tryptic (PT) digests from the two oats and from gliadin (PTG). With the same PT-digests, duodenal biopsies from 22 CD patients were treated in vitro for 24 h and density of CD25+ cells in lamina propria and of intraepithelial CD3+ T cells was measured, as well as crypt cell proliferation and epithelial expression of interleukin 15. Finally, interferon ? (IFN-?) production was measured as evidence of gliadin-specific T-cell activation by PT-digests. RESULTS: In contrast to PTG, oats PT-digests were not able to induce significant increase in ERK phosphorylation and decrease in TEER in CaCo-2 cells. In the organ culture system, oats PT-digests, unlike PTG, did not induce significant increase in crypt enterocyte proliferation, increase in interleukin 15 expression or in lamina propria CD25+ cells. Nevertheless Avena potenza increased intraepithelial T-cell density, while Avena genziana-induced IFN-? 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

SCOPE: The involvement of oxidative stress in gluten-induced toxicity has been evidenced in vitro and in clinical studies but has never been examined in vivo. We recently demonstrated the protective activity of conjugated linoleic acid (CLA), which functions by the activation of nuclear factor erythroid 2-related factor2 (Nrf2), a key transcription factor for the synthesis of antioxidant and detoxifying enzymes (phase 2). Here, we evaluate the involvement of nuclear factor erythroid 2-related factor2 in gliadin-mediated toxicity in human Caco-2 intestinal cells and in gliadin-sensitive human leukocyte antigen-DQ8 transgenic mice (DQ8) and the protective activity of CLA. METHODS AND RESULTS: Gliadin effects in differentiated Caco-2 cells and in DQ8 mice, fed with a gliadin-containing diet with or without CLA supplementation, were evaluated by combining enzymatic, immunochemical, immunohistochemical, and quantitative real-time PCR (qRT-PCR) assays. Gliadin toxicity was accompanied by downregulation of phase 2 and elevates proteasome-acylpeptide hydrolase activities in vitro and in vivo. Notably, gliadin was unable to generate severe oxidative stress extent or pathological consequences in DQ8 mice intestine comparable to those found in celiac patients and the alterations produced were hampered by CLA. CONCLUSION: The beneficial effects of CLA against the depletion of crucial intestinal cytoprotective defenses indicates a novel nutritional approach for the treatment of intestinal disease associated with altered redox homeostasis.

Introduction: Celiac disease (CD), is the most common food-sensitive enteropathy in humans, caused by the lack of oral tolerance to wheat gluten (1). To study this disease we use a transgenic mouse model expressing the HLA-DQ8 molecule in the absence of endogenous class II genes (2). The adjuvant function Lactobacillus casei is well known (3). In this study we analyzed the effects of oral administration of L. casei in DQ8 transgenic mice following mucosal sensitization with gliadin along with cholera toxin. Methods: DQ8 transgenic mice were administered intragastrically with 500 mg of a chymotryptic digest of gliadin (ct-gliadin) along with 25 ?g of cholera toxin (CT) on days 0, 7 and 14. In someexperiments L. casei (1x1010/dose, four doses/week) was co-administered per os. On day 21 mice were sacrificed. Spleen and MLN were isolated for in vitro assessment of gliadin-specific immunity. Small intestine fragments were collected for RNA analysis and immunomorphometric measurements. Results: Co-administration of L.casei in gliadin-sensitive DQ8 transgenic mice caused a strong increase of the intestinal gliadin-specific cell mediated immune response (SI: 2.75 ± 0.1 vs 4.4 ± 0.03; control vs L. casei; p

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs.

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.

BACKGROUND & AIMS: The extensive infiltration of CD8(+) T cells in the intestinal mucosa of celiac disease (CD) patients is a hallmark of the disease. We identified a gliadin peptide (pA2) that is selectively recognized by CD8(+) T cells infiltrating intestinal mucosa of HLA-A2(+) CD patients. Herein, we investigated the phenotype, the tissue localization, and the effector mechanism of cells responsive to pA2 by using the organ culture of CD intestinal mucosa. The target of pA2-mediated cytotoxicity was also investigated by using the intestinal epithelial cell lines Caco2 and HT29, A2(+) and A2(-), respectively, as target cells. METHODS: Jejunal biopsy specimens from CD patients were cultured in vitro with pA2, and cellular activation was evaluated by immunohistochemistry and cytofluorimetric analysis. Cytotoxicity of pA2-specific, intestinal CD8(+) T cells was assayed by granzyme-B and interferon-gamma release and by apoptosis of target cells. RESULTS: pA2 challenge of A2(+) CD mucosa increased the percentage of CD8(+)CD25(+) and of CD80(+) cells in the lamina propria, the former mainly localized beneath the epithelium, as well as the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive cells (TUNEL(+)) in the epithelium. Intraepithelial CD3(+) cells and enterocyte expression of Fas were also increased. CD8(+)CD25(+) and CD8(+)FASL(+) T cells were significantly increased in cell preparations from biopsy specimens cultured with pA2. CD8(+) T-cell lines released both granzyme-B and interferon-gamma following recognition of pA2 when presented by Caco2 and not by HT29. CONCLUSIONS: These data indicate that gliadins contain peptides able to activate, through a TCR/HLA class I interaction, CD8-mediated response in intestinal CD mucosa and to induce the enterocyte apoptosis.

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 CD+ T cells. Notably, both a strong increase of the gliadin-specific IFN gamma 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 IFN gamma, 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. (C) 2008 Elsevier B.V. All rights reserved.

BACKGROUND: Studies on intestinal T cell clones from the mucosa of patients with coeliac disease have led to the identification of immunogenic gliadin epitopes. One is HLA-DQ8 restricted, its recognition by T cells being increased by introduction of negatively charged residues operated by tissue transglutaminase. AIM: To test HLA-DQ8 restricted epitope in both native (QYPSGQGSFQPSQQNPQA) and deamidated (QYPSGEGSFQPSQENPQA) forms in an organ culture system of treated coeliac mucosa from HLA-DQ8 positive and HLA-DQ8 negative patients. PATIENTS AND METHODS: Jejunal biopsies obtained from 10 patients with coeliac disease (six HLA-DQ8 positive and four HLA-DQ8 negative) were cultured in vitro with a peptic-tryptic digest (PT) of gliadin, or with the native (peptide A) or deamidated (peptide B) peptide. Intraepithelial CD3(+) and lamina propria total CD25(+) and CD3(+)CD25(+) cells were counted, lamina propria intercellular adhesion molecule 1 (ICAM-1) expression was evaluated, as well as that of Fas molecules on epithelial cells. RESULTS: In HLA-DQ8 positive, but not in HLA-DQ8 negative, coeliacs the density of intraepithelial CD3(+) cells, lamina propria total CD25(+), and CD3(+)CD25(+) cells, as well as expression of ICAM-1 and Fas molecules were significantly increased in biopsies cultured with PT, peptide A, or peptide B compared with biopsies cultured in medium alone. CONCLUSION: These data show that the DQ8 restricted gliadin peptide is immunogenic only in the intestinal mucosa of HLA-DQ8 positive coeliac patients in both native and deamidated forms.

Background Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten. Gluten-sensitive individuals (GS) cannot tolerate gluten and may develop gastrointestinal symptoms similar to those in CD, but the overall clinical picture is generally less severe and is not accompanied by the concurrence of tissue transglutaminase autoantibodies or autoimmune comorbidities. By studying and comparing mucosal expression of genes associated with intestinal barrier function, as well as innate and adaptive immunity in CD compared with GS, we sought to better understand the similarities and differences between these two gluten-associated disorders. Methods CD, GS and healthy, gluten-tolerant individuals were enrolled in this study. Intestinal permeability was evaluated using a lactulose and mannitol probe, and mucosal biopsy specimens were collected to study the expression of genes involved in barrier function and immunity. Results Unlike CD, GS is not associated with increased intestinal permeability. In fact, this was significantly reduced in GS compared with controls (P = 0.0308), paralleled by significantly increased expression of claudin (CLDN) 4 (P = 0.0286). Relative to controls, adaptive immunity markers interleukin (IL)-6 (P = 0.0124) and IL-21 (P = 0.0572) were expressed at higher levels in CD but not in GS, while expression of the innate immunity marker Toll-like receptor (TLR) 2 was increased in GS but not in CD (P = 0.0295). Finally, expression of the T-regulatory cell marker FOXP3 was significantly reduced in GS relative to controls (P = 0.0325) and CD patients (P = 0.0293). Conclusions This study shows that the two gluten-associated disorders, CD and GS, are different clinical entities, and it contributes to the characterization of GS as a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.

The biological effects of interferon (IFN)-gamma rely mainly on the activity of the transcription factor signal transducer and activator of transcription (STAT) 1 and the intracellular levels of suppressor of cytokine signaling (SOCS)-1, a negative regulator that controls the amplitude and duration of STAT-1 activation. IFN-gamma is a key mediator of the immunopathology in celiac disease (CD, gluten-sensitive enteropathy). Thus we have investigated STAT-1 signaling and SOCS-1 expression in this condition. As expected, high local concentrations of IFN-gamma were invariably seen in duodenal biopsies from CD patients in comparison to controls. On the basis of immunohistochemistry, STAT-1 phosphorylation, nuclear localization, and DNA-binding activity, STAT-1 activation was consistently more pronounced in CD compared with controls. Despite samples from CD patients containing abundant SOCS-1 mRNA, SOCS-1 protein was expressed at the same level in CD patients and controls. In explant cultures of CD biopsies, gliadin induced the activation of STAT-1 but not SOCS-1. Furthermore, inhibition of STAT-1 prevented the gliadin-mediated induction of ICAM-1 and B7-2. These data suggest that persistent STAT-1 activation can contribute to maintaining and expanding the local inflammatory response in CD.