Intestinal Organ Culture Research Articles

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Addition of α-synuclein aggregates to the intestinal environment recapitulates Parkinsonian symptoms in model systems.

The gut-brain axis plays a vital role in Parkinson's disease (PD). The mechanisms of gut-brain transmission mainly focus on α-synuclein deposition, intestinal inflammation and microbiota function. A few studies have shown the trigger of PD pathology in the gut. α-Synuclein is highly conserved in food products, which was able to form β-folded aggregates and to infect the intestinal mucosa. In this study we investigated whether α-synuclein-preformed fibril (PFF) exposure could modulate the intestinal environment and induce rodent models replicating PD pathology. We first showed that PFF could be internalized into co-cultured Caco-2/HT29/Raji b cells in vitro. Furthermore, we demonstrated that PFF perfusion caused the intestinal inflammation and activation of enteric glial cells in an ex vivo intestinal organ culture and in an in vivo intestinal mouse coloclysis model. Moreover, we found that PFF exposure through regular coloclysis induced PD pathology in wild-type (WT) and A53T α-synuclein transgenic mice with various phenotypes. Particularly in A53T mice, PFF induced significant behavioral disorders, intestinal inflammation, α-synuclein deposition, microbiota dysbiosis, glial activation as well as degeneration of dopaminergic neurons in the substantia nigra. In WT mice, however, the PFF induced only mild behavioral abnormalities, intestinal inflammation, α-synuclein deposition, and glial activation, without significant changes in microbiota and dopaminergic neurons. Our results reveal the possibility of α-synuclein aggregates binding to the intestinal mucosa and modeling PD in mice. This study may shed light on the investigation and early intervention of the gut-origin hypothesis in neurodegenerative diseases.

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

Antigen-Presenting Human γδ T Cells Promote Intestinal CD4+ T Cell Expression of IL-22 and Mucosal Release of Calprotectin.

The cytokine IL-22 plays a critical role in mucosal barrier defense, but the mechanisms that promote IL-22 expression in the human intestine remain poorly understood. As human microbe–responsive Vγ9/Vδ2 T cells are abundant in the gut and recognize microbiota-associated metabolites, we assessed their potential to induce IL-22 expression by intestinal CD4+ T cells. Vγ9/Vδ2 T cells with characteristics of APCs were generated from human blood and intestinal organ cultures, then cocultured with naive and memory CD4+ T cells obtained from human blood or the colon. The potency of blood and intestinal γδ T-APCs was compared with that of monocytes and dendritic cells, by assessing CD4+ T cell phenotypes and proliferation as well as cytokine and transcription factor profiles. Vγ9/Vδ2 T cells in human blood, colon, and terminal ileum acquired APC functions upon microbial activation in the presence of microenvironmental signals including IL-15, and were capable of polarizing both blood and colonic CD4+ T cells toward distinct effector fates. Unlike monocytes or dendritic cells, gut-homing γδ T-APCs employed an IL-6 independent mechanism to stimulate CD4+ T cell expression of IL-22 without upregulating IL-17. In human intestinal organ cultures, microbial activation of Vγ9/Vδ2 T cells promoted mucosal secretion of IL-22 and ICOSL/TNF-α–dependent release of the IL-22 inducible antimicrobial protein calprotectin without modulating IL-17 expression. In conclusion, human γδ T-APCs stimulate CD4+ T cell responses distinct from those induced by myeloid APCs to promote local barrier defense via mucosal release of IL-22 and calprotectin. Targeting of γδ T-APC functions may lead to the development of novel gut-directed immunotherapies and vaccines.

Anti-inflammatory effects of Bifidobacterium longum subsp infantis secretions on fetal human enterocytes are mediated by TLR-4 receptors.

The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR-4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR-4 but also use the TLR-4 to modulate the immune response. Probiotics may also use the TLR-4 in immature enterocytes for anti-inflammation. Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1β by using TLR-4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues, and primary NEC enterocytes. Furthermore, we show that PCM, using TLR-4, downregulates the mRNA expression of interleukin-1 receptor-associated kinase 2 (IRAK-2), a common adapter protein shared by IL-1β and TLR-4 signaling. PCM also reduces the phosphorylation of the activator-protein 1 (AP-1) transcription factors c-Jun and c-Fos in response to IL-1β stimulation in a TLR-4-dependent manner. This study suggests that PCM may use TLR-4 through IRAK-2 and via AP-1 to prevent IL-1β-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR-4 therapy to prevent NEC may not be a good strategy.

Hydrocortisone-induced anti-inflammatory effects in immature human enterocytes depend on the timing of exposure.

The immature human gut has a propensity to exaggerated inflammatory responses that are thought to play a role in the pathogenesis of necrotizing enterocolitis (NEC). Prenatal exposure to corticosteroids has been reported to reduce the risk of NEC, while postnatal dexamethasone treatment is associated with adverse neurodevelopmental outcomes in preterm infants. The aim of this study was to investigate the direct role of hydrocortisone in gene expression patterns and inflammatory responses in immature human enterocytes. Time-dependent hydrocortisone effects in nontransformed primary human fetal intestinal epithelial cell line H4 were investigated by cDNA microarray. Fetal intestinal organ culture and cell culture experiments were conducted. Inflammatory responses were induced by stimulation with IL-1β and TNF-α with and without hydrocortisone. IL-8 and IL-6 expression and secretion were measured as functional readout. Here we report time-dependent hydrocortisone-induced changes in gene expression patterns detected by cDNA microarray. Hydrocortisone significantly attenuated IL-1β-induced inflammatory responses in the immature human gut when administered at the time of the proinflammatory insult: IL-1β-induced IL-8 and IL-6 secretion in the fetal ileum as well as H4 cells were significantly reduced. Hydrocortisone also inhibited IL-8 secretion in response to TNF-α. In contrast, TNF-α-induced IL-8 secretion was not reduced in cells treated with hydrocortisone for 48 h before stimulation. Our observations provide a physiological basis for understanding the differential clinical effects of corticosteroids in the immature human gut depending on the timing of treatment.

Human milk oligosaccharides and galactosyloligosaccharides attenuate inflammation in human intestine

The immature intestinal mucosa is hyperresponsive to exogenous and endogenous inflammatory insults. Human milk provides immune protection to infants, and its oligosaccharides (HMOS) are putative immunomodulators. Of these, galactosyloligosaccharides (GOS) are high in colostrum, but not mature milk. The objective is to determine the ability of GOS to directly attenuate inflammation, and potential mechanisms investigated. Human models of immature and mature intestine were used to investigate the ability of a mixture of HMOS or synthetic GOS to attenuate inflammation induced by proinflammatory stimuli and enteric pathogens. These results were confirmed in the ex vivo immature human intestinal organ cultures. The ability of TNFα and of pathogens to elicit pro‐inflammatory neutrophil chemoattractants IL‐8, MIP‐3α and MCP1 and their mRNA levels was significantly attenuated by HMOS and GOS. TNFα‐induced nuclear translocation of NF‐κB was mitigated by HMOS and GOS. Consistent with previous indications of bioactivity of galactosyllactose from human colostrum, trimeric galactosyloligosaccharides interact with cell receptors to inhibit inflammatory response. HMOS and GOS suppress NF‐κB activation, thereby directly attenuating TNFα‐ and pathogen‐induced inflammation in human intestine. This provides a novel mechanism whereby specific galactosyloligosaccharides found in human milk may prove a novel therapeutic agent against gut inflammation.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

Standardization of a human organ culture model of intestinal inflammation and its application for drug testing

Targeting early molecular events in intestinal inflammation may represent a useful therapeutic strategy for maintaining remission in inflammatory bowel disease. Recently, we established an intestinal organ culture model (LEL model), which allows to study the initiation of an intestinal inflammatory response in human tissue. In this model, EDTA-mediated depletion of epithelial cells of colonic mucosa results in an instantaneous inflammatory response in resident lamina propria cells, which shows features of intestinal inflammation in vivo. Furthermore, activated immune cells emigrate from the lamina propria onto the luminal side of the basement membrane.Here, we standardize the LEL model and explore its suitability for drug testing. To this end, human mucosal punches of defined surface area were prepared, depleted of epithelial cells, and cultured at an optimized ratio of medium volume/punch area. The intra-assay variability of measurements of inflammatory parameters ranged from 13% for cell migration to 19% for secretion and 30% for tissue gene expression, respectively, of the inflammatory mediators IL-8 and IL-6. Importantly, known suppressive effects of dexamethasone, a drug employed for the treatment of inflammatory bowel diseases, on leucocyte migration, IL8, IL6, and TNF-α production as well as CD86 surface expression by myeloid cells were observed in this model.In conclusion, the present results suggest that the LEL model may represent a useful human experimental system not only for studying initial activation mechanisms in intestinal inflammation but also for evaluating drug compounds for the treatment of mucosal inflammation.

INFLUENCE OF AGE ON THE SUSCEPTIBILITY OF PIGLETS TO ESCHERICHIA COLI O138:F18

The ex vivo and in vivo studies were undertaken to examine age-effect on the susceptibility of pigsto E. coli F18-diarrhea. The ex vivo experiment was conducted with 2 and 4 weeks old pigs based on theporcine intestinal organ culture (PIOC) model. The in vivo experiment was performed with 2 and 3weeks old pigs fed milk replacer and inoculated with E. coli F18. E. coli F18 inoculation to the intestinalcultures resulted in higher (P<0.01) counts of E. coli associated to the intestinal tissue, but suchdifference was not observed between the 2 and 4 weeks old pigs. Faecal dry matter (DM) decreased(P=0.01), whereas the proportion of pigs with diarrhea and faecal haemolytic E. coli counts increased(P<0.05) following inoculation, however, the difference between the ages was not observed in the invivo study. The interaction (P=0.01) between age and sampling day was observed for the number of totalwhite blood cells (WBC). The proportion of neutrophils decreased (P=0.01), whereas the lymphocytes,red blood cells (RBC), hemoglobin (Hb) and hematocrit (Hct) increased (P<0.01) after inoculation, butthe differences between age groups were not observed regarding these variables. In conclusion, age (2versus 3 or 4 weeks) did not influence the susceptibility of piglet to E. coli F18-diarrhea.

Initiation of an inflammatory response in resident intestinal lamina propria cells -use of a human organ culture model.

Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL1B, IL6, IL8, IL23A, TNFA, CXCL2, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.

Molecular Cloning, Characterization, and mRNA Expression of Intestinal Fatty Acid Binding Protein (&lt;i&gt;I-FABP&lt;/i&gt;) in &lt;i&gt;Columba Livia&lt;/i&gt;

Intestinal fatty acid binding protein (I-FABP) is involved in fatty acid transportation in mammals. To verify its role in fatty acid metabolism in birds, the model of pigeon intestinal organ culture was established, and a full-length cDNA of I-FABP was cloned from the intestine of Columba livia by rapid amplification of cDNA ends (RACE) method for the first time. The full length cDNA of C. livia I-FABP was 855 bp, including a 5′untranslated region (UTR) of 34 bp, a 3′UTR of 422 bp and an open reading frame (ORF) of 399 bp encoding a protein of 132 amino acids with the predicted molecular weight of 15.13 kDa. Sequence comparison indicated that I-FABP of C. livia had high identity with other avian I-FABP and belonged to the first subfamily of FABPs. Using quantitative real-time PCR, pigeon I-FABP mRNA in duodenum and jejunum increased progressively with development stage. I-FABP mRNA was expressed at the highest level at 28 day post hatch in the duodenum, while in ileum, it reached the maximum at the day of hatch, and decreased subsequently. The effects of fatty acids on pigeon I-FABP expression were also investigated in vitro. The results showed that significant increases in the pigeon I-FABP mRNA level were induced by linoleic acid and arachidonic acid, whereas I-FABP gene expression appeared to be unaffected by oleic acid and α-linolenic acid. The results indicate that I-FABP may be indicative of intestine development in pigeon, and it could be regulated by n-6 polyunsaturated fatty acids.

Usefulness of the organ culture system when villous height/crypt depth ratio, intraepithelial lymphocyte count, or serum antibody tests are not diagnostic for celiac disease

The existence of mild forms of celiac disease (CD) can make the histology-based diagnosis difficult to reach. Since anti-endomysium (EMA) and anti-tissue transglutaminase (anti-tTG) are detectable in culture supernatants of duodenal biopsies from CD patients, our aim was to assess if this system can support the histology in the diagnostic work-up. A total of 559 suspected CD patients underwent serum EMA/anti-tTG detection, upper endoscopy with duodenal biopsy sampling, histologic analysis, and organ culture to detect EMA/anti-tTG in supernatants. A subgroup of 30 patients with organ culture positive results were put on a gluten-free diet (GFD). Their gluten-dependency was evaluated by the psychological general well-being and beck depression inventory indexes. Statistical analysis was performed by Cohen k inter-test, Friedman test, and Dunn multiple comparison. Two hundred forty-one out of 559 (43.1%) patients showed intestinal villous atrophy, whereas serum and organ culture EMA/anti-tTG were positive in 293/559 (52.4%) and 334/559 (59.7%) patients, respectively. The strength of agreement resulted good for serology vs histology (k = 0.730), good for organ culture vs histology (k = 0.662), and very good for serology vs organ culture (k = 0.852). After 12 months of GFD, psychological general well-being index significantly increased, and beck depression inventory index significantly decreased (P < 0.001 for each one). Data highlight the organ culture system as a useful tool to assist the histology in diagnosing CD, mainly in cases without villous atrophy or in seronegative patients. The marked improvement in quality of life after a GFD further supports the reliability of this system in diagnosing CD.

In vitro model for IgE mediated food allergy

Background. In intestinal food allergy, the non-specificity of gastrointestinal symptoms and the limited access to the reacting organ are the reasons for the limited understanding of the pathophysiology of this disease and the difficulties in establishing an appropriate diagnosis in the individual patient. Objective. To develop an in vitro model reproducing pathophysiological mechanisms of IgE mediated food allergy. Methods. Distal duodenum biopsies of nine patients with food allergy and 10 control subjects were cultured for 3 h with medium alone and with 1mg/ml of peptic-tryptic digest of wheat gliadin, wheat albumins, and apple proteins. Each biopsy was used for conventional histological examination and for immunohistochemical detection of IgE-positive cells. We have also analyzed the expression of tight junction proteins, occludin, claudin-1, and ZO-1 by immunoconfocal microscopy. Histamine and tryptase release were measured in the culture medium and collected at 0, 30 min, and 3 h of culture using an enzyme and radio immunoassay, respectively. Results. Exposure of small intestinal biopsy specimens of patients with food allergy to food allergens led to a significative increase of IgE-positive cells with a significative increase of histamine and tryptase release and an altered expression of tight junction proteins. No differences were found in intestinal biopsies of controls, cultured with or without food antigens. Conclusions. Small intestinal organ culture is a functional model of food allergy and could be considered as an in vitro oral food challenge, with evident reduction of costs and risks for the patients.

Breast Milk-Transforming Growth Factor-β2 Specifically Attenuates IL-1β-Induced Inflammatory Responses in the Immature Human Intestine via an SMAD6- and ERK-Dependent Mechanism

Background: Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. Objectives: We hypothesized that transforming growth factor-β₂ (TGF-β₂), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs). Methods: Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1β (IL-1β) with or without TGF-β₂. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA. Results: TGF-β₂ significantly attenuated IL-1β-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-β₂ reduced the IL-1β-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-β₂ markedly inhibited IL-1β-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-β₂ on IL-1β-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. Conclusions: TGF-β₂ attenuates IL-1β-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-β₂ is dependent on SMAD6. Breast milk TGF-β₂ may provide the neonate with important immunoregulatory support. TGF-β₂ might provide a novel means to improve intestinal immunophysiology in premature neonates.

Establishment of a Primary Culture Method for Mouse Intestinal Epithelial Cells by Organ Culture of Fetal Small Intestine

Studies of the physiological functions of intestinal epithelial cells (IECs) have been limited by the difficulty of primary culture of IEC. We established a method for primary culture of mouse IEC by culturing fragments of fetal small intestines pretreated with EDTA. This method reproducibly resulted in the expansion of cytokeratin-positive epithelial cells, and vigorous expansion of the epithelial cells was observed only from intestinal fragments of embryonic days 15-16. These cells expressed alkaline phosphatase activity and major histocompatibility complex (MHC) class II molecules, indicating the mature phenotype of IEC in a small intestine. The cells also presented antigens to CD4(+) T cells. Furthermore, the cells expressed various cytokines and chemokines, and the expression was enhanced by bacterial stimulation. These results indicate that the primary-cultured mouse IEC prepared by the method established here can be a beneficial tool in study of the functions of IECs, especially in mucosal immunity.

Enzymatic detoxification of gluten by germinating wheat proteases: Implications for new treatment of celiac disease

Introduction. Currently the only treatment for celiac disease is a lifelong gluten-free diet. The diet is, however, often burdensome, and thus new treatment options are warranted. We isolated proteases from germinating wheat grain naturally meant for total digestion of wheat storage proteins and investigated whether these enzymes can diminish toxic effects of gluten in vitro and ex vivo.Methods. Pepsin and trypsin digested (PT) gliadin was pretreated with proteases from germinating wheat, whereafter the degradation was analyzed by HPLC-MS (high-performance liquid chromatography and mass spectroscopy) and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The toxicity of cleaved PT-gliadin products was assessed in Caco-2 epithelial cells, celiac patient-derived T cells, and in human small intestinal mucosal organ culture biopsies.Results. Proteases from germinating wheat degraded gliadin into small peptide fragments, which, unlike unprocessed PT-gliadin, did not increase epithelial permeability, induce cytoskeletal rearrangement or changes in ZO-1 expression in Caco-2 cells. Pretreated gliadin did not stimulate T cell proliferation in vitro or enhance the production of autoantibodies to culture supernatants and the activation of CD25+ lymphocytes in the organ culture to the same extent as unprocessed PT-gliadin.Discussion. Germinating wheat enzymes reduce the toxicity of wheat gliadin in vitro and ex vivo. Further studies are justified to develop an alternative therapy for celiac disease.

The cytokine interleukin 21: a new player in coeliac disease?

In this issue of Gut ,1 Fina et al demonstrate that interleukin 21 (IL21) expression is increased in the intestinal mucosa of patients with active coeliac disease (CD) but not...

Environmental factors of celiac disease: Cytotoxicity of hulled wheat species Triticum monococcum, T. turgidum ssp. dicoccum and T. aestivum ssp. spelta

In the present paper, the toxicity of prolamines derived from three cereals with a different genome was investigated in human colon cancer Caco-2/TC7 and human myelogenous leukemia K562(S) cells. The purpose of this study was to investigate if species from ancient wheat could be considered as healthy food crops devoid or poor in cytotoxic prolamines for celiac disease. Cytotoxicity was measured in terms of inhibition of cell growth, activation of apoptosis, release of nitric oxide (NO), detection of tissue transglutaminase (TG II) and alteration of transepithelial electrical resistance (TEER) on Caco-2/Tc7 and K562 (S) cell agglutination. Peptic-tryptic (PT) digest from bread wheat (T. aestivum S. Pastore) was used as a positive control. PT digests of prolamins from spelt wheat (T. aestivum ssp. spelta) were found to exert toxic effects on Caco-2/TC7 cells and to agglutinate K562(S) cells. Increased amounts of NO and TG II expression were observed in Caco-2/TC7 cells exposed to 1 mg/mL of spelt prolamins, suggesting that spelt wheat can induce cellular mechanisms implicated in the pathogenesis of celiac disease. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro wheat (T. turgidum ssp. dicoccum) did not exhibit any negative effects on Caco-2/TC7 and K562(S) cells. The results have shown a constant and significant toxic effect of spelt wheat which is not shared by the two other ancient cereals. Future studies on celiac intestinal organ cultures are needed to increase the prospects of breeding programs aimed at developing wheat cultivars potentially tolerated by most celiac patients.

Adherence of Enterohemorrhagic Escherichia coli O157, O26, and O111 Strains to Bovine Intestinal Explants Ex Vivo

We used bovine intestinal organ culture to study infection by enterohemorrhagic Escherichia coli serogroups O157, O26, and O111. We show colonization and attaching and effacing lesion formation on explants derived from the ileum, colon, and rectum. Intimin and Tir were detected at the sites of adherent bacteria; Tir was essential for colonization.