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Coeliac disease is an increasingly recognised pathology, induced by the ingestion of gluten in genetically predisposed patients. Undigested gliadin peptide can induce adaptive and innate immune response that unleash the typical intestinal mucosal alterations. A growing attention is paid to alternative therapeutic approaches to the gluten-free diet: one of these approaches is the use of probiotics and/or postbiotics. We performed lactic fermentation of rice flour with and without pH control, using Lactobacillus paracasei CBA L74 as fermenting strain. We evaluated bacterial growth, lactic acid production during fermentation and gliadin peptide P31-43 entrance in CaCo-2 cells with and without pH control. When pH control was applied no differences were observed in terms of bacterial growth; on the contrary, lactic acid production was greater, as expected. Both samples could inhibit the P31-43 entrance in CaCo-2 cells but the effect was significantly greater for samples obtained when the pH control was applied.

Coeliac disease (CD) is the prototype of an inflammatory chronic disease induced by food. In this context, gliadin p31-43 peptide comes into the spotlight as an important player of the inflammatory/innate immune response to gliadin in CD. The p31-43 peptide is part of the p31-55 peptide from α-gliadins that remains undigested for a long time, and can be present in the small intestine after ingestion of a gluten-containing diet. Different biophysical methods and molecular dynamic simulations have shown that p31-43 spontaneously forms oligomeric nanostructures, whereas experimental approaches using in vitro assays, mouse models, and human duodenal tissues have shown that p31-43 is able to induce different forms of cellular stress by driving multiple inflammatory pathways. Increased proliferative activity of the epithelial cells in the crypts, enterocyte stress, activation of TG2, induction of Ca

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls’ DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31&ndash, 43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31&ndash, 43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31&ndash, 43-HLA-DQ binding.

Prostate cancer represents the major cause of cancer-related death in men and patients frequently develop drug-resistance and metastatic disease. Most studies focus on hormone-resistance mechanisms related to androgen receptor mutations or to the acquired property of prostate cancer cells to over-activate signaling pathways. Tumor microenvironment plays a critical role in prostate cancer progression. However, the mechanism involving androgen/androgen receptor signaling in cancer associated fibroblasts and consequences for prostate cancer progression still remains elusive. We now report that prostate cancer associated fibroblasts express a transcriptional-incompetent androgen receptor. Upon androgen challenging, the receptor co-localizes with the scaffold protein filamin A in the extra-nuclear compartment of fibroblasts, thus mediating their migration and invasiveness. Cancer-associated fibroblasts move towards epithelial prostate cancer cells in 2D and 3D cultures, thereby inducing an increase of the prostate cancer organoid size. Androgen enhances both these effects through androgen receptor/filamin A complex assembly in cancer-associated fibroblasts. An androgen receptor-derived stapled peptide, which disrupts the androgen receptor/filamin A complex assembly, abolishes the androgen-dependent migration and invasiveness of cancer associated fibroblasts. Notably, the peptide impairs the androgen-induced invasiveness of CAFs in 2D models and reduces the overall tumor area in androgen-treated 3D co-culture. The androgen receptor in association with β1 integrin and membrane type-matrix metalloproteinase 1 activates a protease cascade triggering extracellular matrix remodeling. The peptide also impairs the androgen activation of this cascade. This study offers a potential new marker, the androgen receptor/filamin A complex, and a new therapeutic approach targeting intracellular pathways activated by the androgen/androgen receptor axis in prostate cancer-associated fibroblasts. Such a strategy, alone or in combination with conventional therapies, may allow a more efficient treatment of prostate cancer.

Ataxia-Telangiectasia (A-T) is characterized by cerebellar neurodegeneration and immunodeficiency. Recent studies suggest that very low glucocorticoids (GCs) doses may help improve A-T neurological phenotype in some patients. Interestingly, in GCs studies an unexpected improvement of lymphocytes proliferation in some A-T patients has been observed. GCs are able to upregulate IL-7 Rα expression and rescue it from the recycling. In this study, we compared several immunological functions, including PBMC proliferative responses, cell activation events and IL-7/IL-7 Rα axis functionality, with the neurological behavior during an in-vivo GCs treatment between the most Responder patient to GC and the Non-Responder at all. During in-vivo GC treatment, we observed an increase of lymphocyte proliferation upon stimulation with PHA or IL-7 only in the Responder. This finding paralleled the increase in the surface expression of IL-7 R and up-regulation of the CD69 T-cell activation marker. Internalization and recycling of IL-7 R occurred properly only in the Responder. Microarray analysis revealed a remarkable difference in the DE-genes levels among Responder and Non-Responder, mostly concerning miRNAs and Multiple Complex families. Our findings suggest that the improvement of lymphocyte functionality, which correlates to the neurological behavior, is mediated through an effect of GCs on the IL-7/IL-7 Rα axis.

Celiac Disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides (e.g., A-gliadin P57-68) induce an adaptive Th1 pro-inflammatory response. Other gliadin peptides (e.g., A-gliadin P31-43) induce a stress/innate immune response involving interleukin 15 (IL15) and interferon α (IFN-α). In the present study, we describe a stressed/inflamed celiac cellular phenotype in enterocytes and fibroblasts probably due to an alteration in the early-recycling endosomal system. Celiac cells are more sensitive to the gliadin peptide P31-43 and IL15 than controls. This phenotype is reproduced in control cells by inducing a delay in early vesicular trafficking. This constitutive lesion might mediate the stress/innate immune response to gliadin, which can be one of the triggers of the gliadin-specific T-cell response.

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the &alpha, gliadin peptide 31&ndash, 43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31&ndash, 43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31&ndash, 43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31&ndash, 43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.

Celiac Disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides (e.g., A-gliadin P57-68) induce an adaptive Th1 pro-inflammatory response. Other gliadin peptides (e.g., A-gliadin P31-43) induce a stress/innate immune response involving interleukin 15 (IL15) and interferon α (IFN-α). In the present study, we describe a stressed/inflamed celiac cellular phenotype in enterocytes and fibroblasts probably due to an alteration in the early-recycling endosomal system. Celiac cells are more sensitive to the gliadin peptide P31-43 and IL15 than controls. This phenotype is reproduced in control cells by inducing a delay in early vesicular trafficking. This constitutive lesion might mediate the stress/innate immune response to gliadin, which can be one of the triggers of the gliadin-specific T-cell response., Giuliana Lania, Merlin Nanayakkara et al. show that cells from celiac disease patients have delays in early endocytic trafficking and an increase sensitivity to gliadin peptide P31-43. Delaying early vesicular trafficking in control cells induces a celiac-like cellular phenotype, highlighting a role for the early-recycling endosomal system in celiac disease.

Coeliac Disease, a model of food-induced inflammatory disease Celiachia, un modello di patologia infiammatoria indo:a da alimen; M. Vi&oria Barone Background/aim. Epidemiological studies, have associated the increase in chronic inflammatory diseases, such as diabetes, atherosclerosis, asthma, chronic liver disease, autoimmune diseases, degenerative diseases and inflammatory bowel diseases to the spread of the so-called " Western diet”. The Mediterranean diet has increasingly been regarded as the gold-stantard diet for human health. The nutrients exert their effects on tissue inflammation, either by direct action on the cells, or because they regulate the composition of the intestinal microbiota. The enterocyte and the intestinal immunocompetent cells, are equipped with complex systems to "feel" the food and to respond to them. Even cereals (and in particular wheat) can cause intestinal inflammation, for example in celiac disease. The intestinal damage from gluten (the complex of soluble alcohol proteins in wheat) in celiac disease consists of inflammation and remodelling of the mucosa, with flattening of the villi and hypertrophy of the crypts. There are two main types of inflammatory response to gluten peptides in celiac disease (1): there is the adaptive response mediated by CD4 + T cells to some peptides: the 33 mer of A-Gliadin, resistant to gastric digestion, is a prototype endoluminal and parietal intestinal. The peptide is deamidated by tissue transglutaminase (tTG) and presented by class II histocompatibility antigens, DQ2 and DQ8, to CD4 + T cells, with Th1-type response, mediated by gamma interferon and other proinflammatory cytokines. Then there is the response, not mediated by T cells, to other peptides: the peptide 31-43 of A-gliadin (P 31-43) is the prototype. P 31-43, which is part of peptide 31-55, is resistant to gastric and intestinal digestion and causes inflammation with multiple mechanisms, the best known of which consists of a stress / innate and proliferative response, mediated by EGF and IL15 (2). Peptide P31-43 is also a growth factor for various cell lines and for the enterocyte of celiac, as it is able to activate the EGF-EGFR system, the most powerful mitogen present in our body. The gliadin-induced proliferation of celiac crypt is not only dependent on EGF but also on IL15. The proliferation of crypt enterocytes and the innate immune response to celiac gliadin are regulated by a cooperation between EGF and IL15. P31-43 induce also a stress/innate immune response involving interferon α (IFN-α) (3). Thus a double action of gliadin is delineated in the induction of the mucosal damage of celiac subjects. On the one hand, gliadin can activate the T-mediated response and on the other it can induce an innate/ inflammatory response. The combined effects of this double action induce the typical lesion of the celiac mucosa. But what causes the sensitivity to gliadin peptides in celiac cells is not really clear. In this study, we describe a stressed/inflamed celiac cellular phenotype in enterocytes and fibroblasts probably due to an alteration in the early-recycling endosomal system. Moreover we show that celiac cells are more sensitive to the gliadin peptide P31-43 and IL15 than controls. This phenotype is reproduced in control cells by inducing a delay in early vesicular trafficking. Methods. For organ culture studies, biopsy fragments from duodenum were obtained from CD patients with villous atrophy controls, affected by gastroesophageal reflux, and CD patients on GFD (Gluten free diet). Fibroblasts were cultured from skin and intestinal biopsies obtained from CD patients, (GFD, GCD) and controls. We used double immunofluorescence staining, western blotting and immunoprecipitation to evaluate EGFR and EEA1 levels and co- localization in fibroblasts and biopsy fragment from patients with CD and controls. We transfected cells with siHRS and mRNA analysis was perfomed to evaluate the levels of EEA1. Results. We found in CD biopsies and fibroblasts an increase of markers of the innate immune response (EGFR, IL15-R α, MXA) and of the inflammatory response (NFkB). CD cells (enterocytes and fibroblasts) presented a constitutive alteration in the intracellular vesicular system at the level of the early-recycling compartment. In fact, in CD enterocytes and fibroblasts, the numbers of early vesicles were increased, and the EGF/EGFR trafficking was delayed in the early endocytic vesicles; moreover, the decay of the EGFR was prolonged, and TfR levels were increased. We induced a delay in early endocytic trafficking by transfecting cells with siHRS in control cells and rendered them more sensitive to gliadin treatment using as read out STAT5 and NFkB levels. Conclusions. In the present study we show that cells from celiac disease patients present a delay of the endocytic trafficking and are more sensitive to the wheat gliadin peptide P31-43 than control cells. We also found that inducing delays in early vesicular trafficking leads to a celiac-like cellular phenotype, carachterized by inflammation and activation of innate immunity, thus implicating the early-recycling endosomal system in celiac disease. This constitutive lesion might mediate the stress/innate immune response to gliadin, which in turn triggers the gliadin-specific T-cell response. 1) Barone MV, Troncone R, Auricchio S.Gliadin peptides as triggers of the proliferative and stress/ innate immune response of the celiac small intestinal mucosa. Int J Mol Sci. 2014 15(11):20518-37. 2) Barone MV, Zimmer KP. Endocytosis and transcytosis of gliadin peptides. Mol Cell Pediatr. 2016 3:8. 3) Nanayakkara M, Lania G, Maglio M, Auricchio R, De Musis C, Discepolo V, Miele E, Jabri B, Troncone R, Auricchio S, Barone MV.P31-43, an undigested gliadin peptide, mimics and enhances the innate immune response to viruses and interferes with endocytic trafficking: a role in celiac disease.Sci Rep. 2018 8:10821

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L.paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N.flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L.paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N.flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N.flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N.flavescens with early vesicles. Mitochondrial respiration was lower (P&nbsp

Androgens stimulate neuronal differentiation of PC12 cells by nontranscriptional action of the endogenous androgen receptor (AR). AR is also required for NGF-induced neuritogenesis of PC12. Androgens or NGF trigger AR association with filamin and TrkA, TrkA interaction with PI3-K δ, and activation of PI3-K δ and Rac., Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

A Correction to this paper has been published: https://doi.org/10.1038/s41419-021-03483-4

Endocytosis is a fundamental cell biological process, which carries out essential functions in a polarized epithelial cell such as enterocytes provided with a huge surface area of the brush border membrane. Major tasks of enterocytes, which are regulated by endocytic signals, are digestion and absorption of nutrients and drugs/pharmacological agents, barrier permeability to microorganism, toxins and antigens, and transcytotic crosstalk between intestinal lumen and lamina propria cells with access to the circulation.Investigations on inflammatory bowel diseases such as food allergy, celiac disease, Crohn's disease, and ulcerative colitis focus on immune processes originating within enterocytes as antigen presenting cells. Thus the initiation of oral tolerance, that is, the binding of food antigens to MHC class II proteins, might be localized within late endosomes of enterocytes. Furthermore, the late endosomal compartment of enterocytes seems to be involved in the processing of luminal antigens during the pathogenesis of celiac disease and inflammatory bowel diseases. Investigations of inherited diseases such as microvillus inclusion disease have revealed a pathogenetic defect in the autophagocytotic and/or recycling pathway of enterocytes.Our progress in the cell and molecular biological understanding of the endocytosis and the methodical opportunities of translational research offer now new therapeutic options for patients suffering from endocytosis-related diseases of enterocytes.

Celiac disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides are resistant to intestinal digestion (e.g., A-gliadin P31–43) and induce a stress/innate immune response, but the reason why they are dangerous in the intestines of patients with CD is unknown. In the present study, P31–43 activated IFN-α, a mediator of the innate immune response in CD, in the intestine of subjects with CD and an enterocyte cell line, CaCo-2. P31–43 cooperated with a viral ligand to activate the TLR7 pathway by interfering with endocytic trafficking. Based on these results, the vesicular pathway regulates the innate/inflammatory response to viral ligands and bioactive dietary peptides. Suggesting that together with viral infections, alimentary proteins able to mimic and potentiate the innate immune response to viruses, can trigger an autoimmune disease such as CD.

Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides induce innate and adaptive T cell-mediated immune responses. The major mediator of the stress and innate immune response to gliadin peptides (i.e., peptide 31–43, P31–43) is the cytokine interleukin-15 (IL-15). The role of epithelial growth factor (EGF) as a mediator of enterocyte proliferation and the innate immune response has been described. In this paper, we review the most recent literature on the mechanisms responsible for triggering the up-regulation of these mediators in CD by gliadin peptides. We will discuss the role of P31–43 in enterocyte proliferation, structural changes and the innate immune response in CD mucosa in cooperation with EGF and IL-15, and the mechanism of up-regulation of these mediators related to vesicular trafficking. We will also review the literature that focuses on constitutive alterations of the structure, signalling/proliferation and stress/innate immunity pathways of CD cells. Finally, we will discuss how these pathways can be triggered by gliadin peptide P31–43 in controls, mimicking the celiac cellular phenotype.

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

Background In vivo assays cannot always be conducted because of ethical reasons, technical constraints or costs, but a better understanding of the digestive process, especially in infants, could be of great help in preventing food-related pathologies and in developing new formulas with health benefits. In this context, in vitro dynamic systems to simulate human digestion and, in particular, infant digestion could become increasingly valuable. Objective To simulate the digestive process through the use of a dynamic model of the infant gastroenteric apparatus to study the digestibility of starch-based infant foods. Design Using M.I.D.A (Model of an Infant Digestive Apparatus), the oral, gastric and intestinal digestibility of two starch-based products were measured: 1) rice starch mixed with distilled water and treated using two different sterilization methods (the classical method with a holding temperature of 121°C for 37 min and the HTST method with a holding temperature of 137°C for 70 sec) and 2) a rice cream with (premium product) or without (basic product) an aliquot of rice flour fermented by Lactobacillus paracasei CBA L74. After the digestion the foods were analyzed for the starch concentration, the amount of D-glucose released and the percentage of hydrolyzed starch. Results An in vitro dynamic system, which was referred to as M.I.D.A., was obtained. Using this system, the starch digestion occurred only during the oral and intestinal phase, as expected. The D-glucose released during the intestinal phase was different between the classical and HTST methods (0.795 grams for the HTST versus 0.512 for the classical product). The same analysis was performed for the basic and premium products. In this case, the premium product had a significant difference in terms of the starch hydrolysis percentage during the entire process. Conclusions The M.I.D.A. system was able to digest simple starches and a more complex food in the correct compartments. In this study, better digestibility of the premium product was revealed.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

Several recent reports describe a role of probiotics as a therapeutic approach for celiac disease (CD). Two undigested A-gliadin peptides, P31-43 and P57-68, are central to CD pathogenesis, inducing an innate and an adaptive immune response, respectively. They enter enterocytes and localize to vesicular compartment to induce their toxic/immunogenics effects. In this article, we tested the effect of probiotic Lactobacillus paracasei (LP) CBA L74 (International Depository Accession Number LMG P-24778), its supernatant and LP-fermented cereals on gliadin peptides, P31-43 and P57-68, entrance in Caco-2 cells. Both LP CBA L74 and its supernatant inhibit P31-43 (intensity of fluorescence; FI: 75%) and P57-68 (FI: 50%) entrance in Caco2 cells, indicating that this biological effect is due to some product included in LP CBA L74 supernatant. This effect was present also after fermentation of cereals. This study describes a novel effect of probiotics in the prevention of undigested gliadin peptides toxic effects.

Inflammation of intestinal tissue in patients affected by celiac disease (CD) originates from the adaptive and innate immune responses elicited by the undigested gliadin fragments through molecular mechanisms not yet completely described. Undigested A-gliadin peptide P31-43 is central to CD pathogenesis, entering enterocytes in vesicular compartments by endocytosis and inducing an innate immune response in CD intestinal mucosa. This study focused on the reasons why P31-43 does not behave as adaptive immunogenic agent. Once obtained by NMR analysis, the three-dimensional model of P31-43 was used to implement a series of in silico experiments aimed to explore the ability of the peptide to interact with HLA-DQ2 and the corresponding receptor onto T cells. Our results show that P31-43 is a poor ligand for DQ2 and/or T-cell receptor. This study was also aimed to investigate, from a structural point of view, the previous experimental findings by which P31-43 is able to enhance the phosphorylation level of the protein ERK2, while some P31-43 Ala-mutants decrease or totally inhibit that process. The molecular models of P31-43, P31-43 P36A, and F37A mutants were used for in silico docking experiments onto the ERK2 structure. The experiments support the hypothesis that P31-43 F37A works as an ERK2 phosphorylation inhibitor because it binds to the ERK2 phosphorylation site. This study reports on the structural properties of so far never NMR characterized gliadin peptides relevant in CD and explores details about their mechanisms of action.

Background— The sphingosine-1-phosphate receptor 1 (S1PR1) and β 1 -adrenergic receptor (β1AR) are G-protein–coupled receptors expressed in the heart. These 2 receptors have opposing actions on adenylyl cyclase because of differential G-protein coupling. Importantly, both of these receptors can be regulated by the actions of G-protein–coupled receptor kinase-2, which triggers desensitization and downregulation processes. Although classic signaling paradigms suggest that simultaneous activation of β1ARs and S1PR1s in a myocyte would simply result in opposing action on cAMP production, in this report we have uncovered a direct interaction between these 2 receptors, with regulatory involvement of G-protein–coupled receptor kinase-2. Methods and Results— In HEK (human embryonic kidney) 293 cells overexpressing both β1AR and S1PR1, we demonstrated that β1AR downregulation can occur after stimulation with sphingosine-1-phosphate (an S1PR1 agonist), whereas S1PR1 downregulation can be triggered by isoproterenol (a β-adrenergic receptor agonist) treatment. This cross talk between these 2 distinct G-protein–coupled receptors appears to have physiological significance, because they interact and show reciprocal regulation in mouse hearts undergoing chronic β-adrenergic receptor stimulation and in a rat model of postischemic heart failure. Conclusions— We demonstrate that restoration of cardiac plasma membrane levels of S1PR1 produces beneficial effects that counterbalance the deleterious β1AR overstimulation in heart failure.

Background: On ingestion of gliadin, the major protein component of wheat and other cereals, the celiac intestine is characterized by the proliferation of crypt enterocytes with an inversion of the differentiation/proliferation program. Gliadins and A-gliadin peptide P31-43, in particular, act as growth factors for crypt enterocytes in patients with celiac disease (CD). The effects of gliadin on crypt enterocyte proliferation and activation of innate immunity are mediated by epidermal growth factors (EGFs) and innate immunity mediators [interleukin 15 (IL15)]. Objective: The aim of this study was to determine the molecular basis of proliferation and innate immune response to gliadin peptides in enterocytes. Design: The CaCo-2 cell line was used to study EGF-, IL15-, and P31-43–induced proliferation. Silencing messenger RNAs and blocking EGF receptor and IL15 antibodies have been used to study proliferation in CaCo-2 cells and intestinal biopsy samples from patients with CD and control subjects. Results: In the CaCo-2 cell model, IL15 and EGF cooperated to induce proliferation in intestinal epithelial cells at both the transcriptional and posttranscriptional levels, and the respective receptors interacted to activate each other’s signaling. In addition, the effects of the P31-43 peptide on CaCo-2 cell proliferation and downstream signaling were mediated by cooperation between EGF and IL15. The increased crypt enterocyte proliferation in intestinal biopsy samples from patients with CD was reduced by EGF receptor and IL15 blocking antibodies only when used in combination. Conclusions: EGF receptor/IL15R-a cooperation regulates intestinal epithelial cell proliferation induced by EGF, IL15, and the gliadin peptide P31-43. Increased proliferation of crypt enterocytes in the intestine of CD patients is mediated by EGF/IL15 cooperation. Am J Clin Nutr 2013;98:1123–35.