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Non-celiac wheat sensitivity (NCWS) is a clinical entity induced by the ingestion of gluten that leads to intestinal and/or extraintestinal symptoms, and is diagnosed when celiac disease and wheat allergy have been ruled out. In addition to gluten, other grains’ components, including amylase trypsin inhibitors (ATIs) and fermentable short-chain carbohydrates (FODMAPs), may trigger symptoms in NCWS subjects. Several studies suggest that, compared with tetraploid and hexaploid modern wheats, ancient diploid wheats species could possess a lower immunogenicity for subjects suffering from NCWS. This review aims to discuss available evidence related to the immunological features of diploid wheats compared to common wheats, and at outlining new dietary opportunities for NCWS subjects.

Alternative or complementary treatments to a gluten-free diet are urgently needed for Celiac Disease. By exploiting the health-promoting properties of polyphenols on a transgenic mouse model of Celiac Disease enteropathy, this study provides the first in vivo evidence regarding the ability of 1 mg day-1 doses of green tea catechins and grape seed procyanidins to ameliorate some of the most characteristic histological changes of gliadin-treated DQ8 mice, including villus flattening, crypt hyperplasia, and infiltration of intraepithelial lymphocytes. Mechanistically, polyphenols were found to increase the intestinal nucleophilic tone of DQ8 mice by orchestrating an adaptive antioxidant response characterized by enhanced GSR enzyme activity and GSH content. Taken together, this work constitutes a highly relevant breakthrough as it provides the fundamental basis concerning the significance of natural polyphenols to be used in, for instance, the development of innovative functional foods aimed at CD individuals.

Immuno-laser capture microdissection (Immuno-LCM) has been used to analyze cell-specific gene expression profiles. However, the usefulness of such a technique is frequently limited by RNA degradation. We, therefore, developed a rapid protocol of LCM on mirror sections, which allows for preserving RNA integrity. With such a procedure, we investigated cell-type-specific gene expression of γδ intraepithelial lymphocytes (IELs) in untreated celiac disease (CD). An increase in TGF-β mRNA expression levels was observed in γδ + IELs compared to intestinal enterocytes (IEs), whereas anti-inflammatory IL-10 mRNA production from γδ + IELs was lower compared to IEs. In untreated CD patients, the production of anti-inflammatory cytokines by γδ + IELs is suggestive of a regulatory function, thus playing a critical role in limiting inflammation. This work underscores the importance of LCM on mirror sections as a valuable tool to perform cell-type-specific molecular analysis in tissue.

Enzymatic transamidation of wheat gliadin by microbial transglutaminase inhibits IFN-? secretion by intestinal T cell lines from celiac disease (CD) patients. Here, we analysed its effects on intestinal biopsies from CD patients and studied the underlying mechanisms in HLA-DQ8 transgenic (tg) mice, a model of T-cell mediated gluten sensitivity. In vitro challenge with a soluble form of transamidated gliadin (spf) upregulated IL-10 transcript levels in human biopsy samples. Furthermore, the ratio of IL-10/IFN-? transcripts was significantly increased following treatment with spf. In DQ8 tg mice, recall responses in vitro in the presence of dendritic cells pulsed with transamidated gliadin showed that gliadin-specific CD4+ T cells did not produce IFN-? at any tested dose. On the contrary, spf-specific CD4+ T cells still secreted IFN-?, but they also produced significant levels of IL-10 with both native and transamidated gliadin. Interestingly, this anti-inflammatory activity was restricted to a specific reverse-phase high-pressure liquid chromatography (RP-HPLC) fraction encompassing ?-gliadins. These findings suggested an ability of transamidated gliadin to revert, as well as to prevent, the inflammatory phenotype triggered by native gliadin. This property was intrinsically associated with specific components of the ?-gliadin fraction.

Celiac disease (CD) is a food enteropathy that occurs in genetically susceptible individuals following the ingestion of gluten. Both gluten cytotoxicity and immunity activation play a role in CD pathogenesis; however, the chronological assessment of the different pathogenic mechanisms remains elusive. The models developed so far have only partially addressed this issue. Herein, Ab°DQ8 transgenic mice were administered wheat gliadin and indomethacin for 10 days to induce enteropathy. Gliadin-induced alteration of the small intestinal architecture was associated with increased expression of tissue transglutaminase in the lamina propria and a marked hypoxic environment. Enteropathic mice showed activation of innate immunity, featuring an increase of pro-inflammatory IFN-γ and IL-15 mRNAs, as well as CD11c+CD103+, CD11b+CD11c+, and CD11b+CD103+ dendritic cell subsets. However, the temporal assessment of examined parameters indicated that the induction of innate immunity during the generation of the mucosal lesion, occurred belatedly, highlighting a major role of gliadin intrinsic cytotoxicity in the pathogenic mechanism of this model. These results have important implications for the use of this model to test the impact of biotechnological interventions to reduce the cytotoxicity of gliadin.

Scope Several studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. Methods and Results ATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-α were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-γ production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. Conclusion TM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

Laser capture microdissection (LCM) is a powerful tool for selection and isolation of single cells or compartments from complex primary tissues to perform molecular analyses. Celiac disease is a genetic autoimmune disorder where the ingestion of gluten leads to damage in the small intestine. Increased intraepithelial lymphocytes and the presence of the lamina propria inflammatory infiltrate of the duodenal mucosa is a common part of the disease. These cells promote inflammatory processes through the release of cytokines. Here, we describe the use of LCM and real-time quantitative PCR (RT-qPCR) to analyze cytokine profile information in distinct duodenal mucosa tissue compartments of celiac patients.

Celiac disease (CD) is an enteropathy caused by the ingestion of wheat gluten in genetically susceptible individuals. A complete understanding of the pathogenic mechanisms in CD has been hindered because of the lack of adequate in vivo models. In the present study, we explored the events after the intragastric administration of gliadin and of the albumin/ globulin fraction from wheat in human leukocyte antigen-DQ8 transgenic mice (DQ8 mice) treated with indomethacin, an inhibitor of cyclooxygenases (COXs). After 10 days of treatment, mice showed a significant reduction of villus height, increased crypt depth, increased number of lamina propria-activated macrophages, and high basal interferon-? secretion in mesenteric lymph nodes, all of which were specifically related to gliadin intake, whereas the albumin/globulin fraction of wheat was unable to induce similar changes. Cotreatment with NS-398, a specific inhibitor of COX-2, also induced the intestinal lesion. Enteropathy onset was further characterized by high levels of oxidative stress markers, similar to CD. Biochemical assessment of the small intestine revealed the specific activation of matrix metalloproteinases 2 and 9, high caspase-3 activity, and a significant increase of tissue transglutaminase protein levels associated with the intestinal lesion. Notably, after 30 days of treatment, enteropathic mice developed serum antibodies toward gliadin (IgA) and tissue transglutaminase (IgG). We concluded that gliadin intake in combination with COX inhibition caused a basal inflammatory status and an oxidative stress condition in the small intestine of DQ8 mice, thus triggering the mucosal lesion and, subsequently, an antigen-specific immunity. © 2014 the American Physiological Society.

mTGase-mediated transamidation of wheat flour reverses the T cell mediated inflammatory response in models of gliadin sensitivity. CD is an immune-mediated enteropathy caused by the ingestion of wheat gluten. The modification of gluten by intestinal tTGase plays a crucial role in CD pathogenesis. In this study, we observed that extensive transamidation of wheat flour with K-C2H5 by mTGase yielded spf and K-gliadins fractions. By Western blot, we found that these modifications were associated with strongly reduced immune cross-reactivity. With the use of DQ8 tg mice as a model of gluten sensitivity, we observed a dramatic reduction in IFNγ production in gliadin-specific spleen cells challenged with spf and K-gliadins in vitro (n=12; median values: 813 vs. 29 and 99; control vs. spf and K-gliadins, P=0.012 for spf, and P=0.003 for K-gliadins). For spf, we also observed an increase in the IL-10/IFNγ protein ratio (n=12; median values: 0.3 vs. 4.7; control vs. spf, P=0.005). In intestinal biopsies from CD patients challenged in vitro with gliadins (n=10), we demonstrated further that K-gliadins dramatically reduced the levels of antigen-specific IFNγ mRNA in all specimens responsive to native gliadins (four of 10; P

Different approaches have been used to study the pattern of cytokines in celiac disease (CD). Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of CD patients, dissected by LCM, analyzing cytokine expression profile. Frozen section of jejunum was obtained from 15 untreated CD and 15 control. Surface epithelium and lamina propria compartment were isolated by LCM. RNA from each LCM sample was extracted and, after a retrotranscription step, messenger RNA levels for MxA, IL-15, TNF-α, IFN-γ, IL-17α, IL-21, IL-10, and TGF-β were determined by quantitative reverse transcriptase-PCR. Increased gene expression levels of MxA, IL-15, TNF-α, IL-10, and TGF-β was observed in the surface epithelium of untreated CD with respect to control. Furthermore, all the cytokines investigated were upregulated in the lamina propria of untreated CD as compared to control. Within the untreated CD group the expression of IL-15 was higher, in the surface epithelium than in the lamina propria, whereas the expression levels of IL-17 and IL-21 were higher in the lamina propria than in the surface epithelium. Finally, high levels of IL-10 and TGF-β were detected in both compartments of untreated CD biopsies. In CD, surface epithelium and lamina propria compartments, play a prominent role in determining innate and adaptive immunity, respectively. Conversely, surface epithelium and lamina propria produce high levels of anti-inflammatory cytokines, suggesting that both compartments are involved in the immunoregulatory response.

Non-celiac gluten sensitivity (NCGS) is a clinical entity triggered by the ingestion of gluten-containing grains leading to intestinal and/or extraintestinal symptoms that resolve once the gluten-containing foodstuff is eliminated from the diet, and it is diagnosed when celiac disease (CD) and wheat allergy (WA) have been ruled out. The limited knowledge about the pathophysiology of NCGS and the lack of validated biomarkers are still major limitations for clinical studies, making it difficult to differentiate NCGS from other gluten-related disorders (GRD). In the absence of clear-cut diagnostic criteria, NCGS is still mainly a diagnosis of exclusion. Several studies suggest that NCGS is an immune-mediated disease that likely activates an innate immune response. Moreover, it has recently been hypothesized that in addition to gluten, other components of wheat may be responsible for the symptoms observed in individuals without CD. This review aims at discussing available evidence related to the histological and immunological features in the gut mucosa of patients with NCGS and at outlining new dietary opportunities for these patients.

CYTOKINE PROFILE IN COELIAC DISEASE: A LASER CAPTURE MICRODISSECTION APPROACH Background and Aim: Different approaches have been used to study the pattern of cytokines in celiac disease (CD), which have led sometimes to contradictory results. Laser capture microdissection (LCM) is a powerful tool for the isolation of specific cell populations from stained sections. The aim of this study was to characterize the pattern of cytokines within the tissue compartments of the small intestinal mucosa of CD patients, dissected by LCM. Methods: Frozen section of jejunal biopsies were obtained from 7 untreated patients and 7 controls. Epithelium and lamina propria were isolated separately by LCM, and the cytokine messenger RNA (mRNA) levels for IFN-?, IL-21, IL-15, IL-10 and TGF-?, were determined by using real-time reverse transcription PCR. Results: The mRNA levels of all the cytokines analyzed were significantly higher, in both compartments, in untreated CD than from controls. Active celiac disease was characterized by higher expression of IFN-g and IL-21 mRNA in lamina propria than in epithelium and by higher expression of IL-15 mRNA in epithelium than in lamina propria. Furthermore, same levels of IL-10 and TGF-b mRNA expression was seen in both compartments of untreated CD biopsies. Finally no differential mRNA expression was observed for cytokines, in controls between the two compartments. Conclusion: Our results suggest that an imbalance in the relative amounts of cytokine mRNAs production, between epithelium and lamina propria, is an important feature of active CD. Particularly, the epithelium seems mostly produce cytokines involved in the innate immunity whereas lamina propria may produce cytokines involved in the adaptive immune response. Finally, both compartment produce cytokines involved in the suppressive activity. This work underlines the importance of LCM as a valuable tool to determine potential inflammatory components in epithelial and lamina propria cells that may enhance understanding to CD pathogenesis.