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Background & Aims Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology. Methods In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence. Results All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose–response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot, gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2–restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or near significantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure. Conclusions Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796.

BACKGROUND & AIMS: Diarrhea is a major cause of illness and death in children and young animals. The aim of this study was to investigate the possible therapeutic effect of bromelain, a proteolytic extract obtained from pineapple stems on bacterial toxin and second-messenger agonist-induced intestinal secretion. METHODS: The effect of bromelain pretreatment on short-circuit responses to Escherichia coli heat-labile enterotoxin, heat-stable enterotoxin, and Vibrio cholerae cholera toxin was evaluated in rabbit ileum mounted in Ussing chambers. RESULTS: Bromelain was 62% effective in preventing heat-labile enterotoxin- induced secretion, 51% effective against cholera toxin, and 35% effective against heat-stable enterotoxin [corrected]. Bromelain also prevented secretory changes caused by prostaglandin E2, theophylline, calcium-ionophore A23187, 8-bromoadenosine 3':5'-cyclic monophosphate, and 8-bromoguanosine 3':5'-cyclic monophosphate, well-known intracellular mediators of ion secretion. The efficacy of bromelain was not caused by reduced tissue viability resulting from its proteolytic effects on enterocytes, indicated by experiments measuring uptakes of nutrients into intestinal cells and experiments measuring short-circuit responses to glucose. CONCLUSIONS: Bromelain prevents intestinal fluid secretion mediated by secretagogues that act via adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate, and calcium- dependent signaling cascades. It may be clinically useful as an antidiarrheal drug. (Gastroenterology 1997 Jul;113(1):175-84)

Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of gluten-containing grains (including wheat, rye, and barley) in genetically susceptible individuals. CD is associated with HLA molecules DQ2 (90%-95%) and DQ8 (5%-10%), and in the continued presence of gluten the disease is self-perpetuating. CD is one of the most common lifelong disorders worldwide and is characterized by a variety of clinical presentations. These include the typical malabsorption syndrome (classic symptoms) and a spectrum of symptoms potentially affecting any organ or body system (nonclassic symptoms). Because CD often is atypical or even clinically silent, many cases go undiagnosed and are exposed to the risk of long-term complications. There is growing interest in the social aspects of CD because the burden of illness related to this condition is doubtless higher than previously thought.

Background & Aims: Enteric infections have been implicated in the pathogenesis of both food intolerance and autoimmune diseases secondaryto the impairment of the intestinal barrier. On the basis of our recent discoveryof zonulin, a modulator of small-intestinal tight junctions, we asked whether microorganisms might induce zonulin secretion and increased small-intestinal permeability. Methods: Both ex vivo mammalian small intestines and intestinal cell monolayers were exposed to either pathogenic or nonpathogenic enterobacteria. Zonulin production and changes in paracellular permeabilitywere monitored in Ussing chambers and microsnapwells. Zonula occludens 1 protein redistribution after bacteria colonization was evaluated on cell monolayers. Results: Small intestines exposed to enteric bacteria secreted zonulin. This secretion was independent of either the species of the small intestines or the virulence of the microorganisms tested, occurred only on the luminal aspect of the bacteria-exposed smallintestinal mucosa, and was followed bya decrease in small-intestinal tissue resistance (transepithelial electrical resistance). The transepithelial electrical resistance decrement was secondaryto the zonulin-induced tight junction disassembly, as also shown by the disengagement of the protein zonula occludens 1 protein from the tight junctional complex. Conclusions: This zonulindriven opening of the paracellular pathwaymayrepresent a defensive mechanism, which flushes out microorganisms and contributes to the host response against bacterial colonization of the small intestine.

The engineering of multicellular organotypic models of the human intestinal mucosa has wide-ranging potential as a tool for discovery in both health and disease, including interaction with pathogens, antigen trafficking, inflammatory and physiological processes. However, models reported to date remain relatively simple composed of few cell types. Here, we report the development of an organotypic model with close structural and functional resemblance to the human intestinal mucosa; it is comprised of fibroblasts, lymphocytes, epithelial cells, and endothelial cells. Moreover, epithelial cells in our 3-D system can differentiate into multiple lineages (e.g., goblet cells, M cells and differentiated enterocytes). We hypothesized that under both microgravity and proper culture conditions, aggregates of randomly distributed cells will self-organize into a spatial configuration resembling those in native tissues. Our hypothesis was based on recent developments showing that: (1) the culture of epithelial cells as well as endothelial cells under microgravity can be accompanied by a commensurate increase in the cell differentiation1–3 and (2) gelled collagen-I can constitute a flexible extracellular matrix (ECM) to be used under microgravity.4 It is known that collagen-I possesses most of the properties of an ideal scaffold, such as specific gravity similar to the culture medium and the capability to incorporate other relevant ECM proteins.4 In our model, fibroblasts and endothelial cells were embedded in a collagen-I matrix enriched with additional gut basement membrane proteins (i.e., laminin, collagen IV, fibronectin and heparin sulfate proteoglycan) and added to Rotating Wall Vessel (RWV) bioreactors containing epithelial cells (Supplementary Methods). These epithelial cells consisted of a human enterocyte cell line (i.e., HCT-8) that was originally derived from the junction of the small and large bowel.5 The choice of this cell line was based on its ability to successfully differentiate in a low shear microgravity environment1 provided by the RWV bioreactor.6 Activated lymphocytes were added twice to the culture at days 4 (± 1 day) and 9 (± 1 day) (Supplementary Methods). After 10 to 15 days, histological staining and electron microscopy demonstrated the presence of villus-like structures in the constructs. About 60–80% of these epithelial cells were organized as a monolayer of polarized cells with nuclei located in a basal position near the ECM, a major feature of well-differentiated cells (Figure 1A). H&E staining also demonstrated the presence of a brush border, or microvilli, seen collectively as a fuzzy fringe in the apical surface of the epithelium (Figure 1B). The presence of microvilli was further evidenced by immunochemical staining using an anti-villin mouse monoclonal antibody (mAb) or by electron microscopy (Figure 1C and D, respectively). Assessment by immunostaining for zonula occludens (ZO)-1, a protein involved in the formation of tight junctions, and electron microscopy demonstrated that our 3-D organotypic model recapitulates the formation of tight junctions and desmosomes (Figure 1E and F). In addition, because immortalized epithelial cells in 3-D systems may lose the growth pattern found in the normal epithelium, we assessed the expression of E-cadherin and the Ki-67 proliferation marker in our 3-D model. As expected, HCT-8 cell lines expressed E-cadherin but only negligible amount of Ki-67 antigen, a pattern consistent with normal highly-differentiated epithelium (Figures 1G–I). Finally, these HCT-8 cells also expressed numerous markers for multi-lineage differentiation to globet (mucin 2, MUC 2) and M (Sialyl Lewis Antigen) cell lineages (Figures 1J and K). Goblet and M cells were distributed throughout the epithelial layer (Figures 1J and K, respectively). We also investigated the presence of the machinery for the specific digestion and transfer of glucose. We observed apical expression of dissacharidases (i.e., sucrase-isomaltase, SI) and sugar transporters (i.e., energy-dependent sodium-glucose co-transporter 1, SGLT1) which strongly suggests that epithelial cells in our 3-D model are able to absorb and transport glucose (data not shown). Taken together, these results demonstrate a remarkable degree of differentiation of HCT-8 epithelial cells in our 3-D model. Figure 1 Epithelial cell differentiation. Hematoxylin and eosin staining of 14 day-cells cultured in the 3-D microgravity model: tissues were stained purple and scaffold stained pink. Lower (A) and higher magnification (B) shows the presence of polarized epithelial ... We also examined the presence and persistence of other cell types by monitoring the presence of defined cellular markers for each individual cell type (i.e., CD45 for hematopoietic-derived cells, including lymphocytes; CD31 for endothelial cells and Vimentin for fibroblasts). These studies were performed by immunochemistry. We observed that the lymphocytes migrated through the epithelial cell layer and localized to the ECM. We were able to detect lymphocytes for at least 7 days (last day examined) (Figure 1L). As in the native gut tissue, in our 3-D model fibroblasts and endothelial cells remained dispersed throughout the ECM. Interestingly, we observed that individual endothelial cells exhibited cytoplasmic extensions that form bridges between them, suggesting that these endothelial cells were activated and underwent a remodeling phase (Figures 1M and N). However, the vast majority of these endothelial cells do not appear to differentiate towards the formation of vessel-like conduits. Additional studies are needed to determine the proper conditions that will further enable vessel-like conduits to form. Interestingly, fibroblasts showed different shapes, ranging from classical spindle shaped to rounded-fat cell-like shaped with the nucleus pushed to one side (Figures 1O and P). These results suggest that cells were activated and remain able to both receive and respond to differentiation signals. Finally, several studies have demonstrated that good viability for primary cells lasting more than 10 days in culture systems is difficult to achieve.7 Thus, we assessed cell viability by immunochemical staining using antibodies to cleaved Caspase-3. Up to 20 days after the initiation of the cultures, few cleaved Caspase-3-positive cells were observed in the ECM, the major location of primary cells including lymphocytes, fibroblasts and endothelial cells (data not shown). Although the combination of the features described above give some insights into the similarities of our 3-D organotypic model to in vivo tissues, we further assessed its functionality by observing its response to exposure to a human enteric bacterial pathogen. Specifically, we examined the changes in the epithelial cell morphology, as well as cytokine production, following exposure to wild-type Salmonella enterica serovar Typhi (S. Typhi) strain ISP1820, to evaluate whether it mimicked the reported stages of the infection. The identification and characterization of S. Typhi-infected cells was accomplished using a polyclonal antibody that specifically detects common Salmonella antigens (CSA-1). A visible feature of the Salmonella entry process in vivo, i.e., membrane rearrangements or ruffles, were observed in our system as early as 1 hour after infection (Supplementary Figure 1B) and increased over time (Supplementary Figures 1C and D). Of note, these ruffles were absent in the control (not-infected cells, Supplementary Figure 1A). One day after infection, the ruffles fade away and bacteria, when present, was mostly observed on the cell surface/brush border of healthy looking epithelial cells or intracellular in epithelial cells sloughed from the monolayer which likely died as a result of the infection (Supplementary Figures 1E and F). We also observed that the exposure to Salmonella resulted in production of cytokines, including IL-1β, IL-6, IL-8, IL-11, IL-12p70, IL-17a, IL-21 and TNF-α (Supplementary Figures 1G, H and I). These results are in agreement, and extend previous studies showing the importance of these cytokines in the control of Salmonella infection8, 9 and further confirming the functional capacity of this 3-D organotypic model to properly react to the presence of an enteric pathogen. To summarize, although similar approaches have been attempted by other investigators with moderate success,1, 10 our system provides major innovations and advances such as a matrix enriched with gut basement membrane proteins. Also, to our knowledge, except for our system described above, attempts to integrate multiple cell types in 3-D constructs grown under microgravity have been unsuccessful. Finally, we provided evidence that our 3-D model might be a helpful tool to investigate the early events of the host interaction with gastrointestinal pathogens, including invasion, pathogenesis and immune responses.

Celiac disease (CD) is a syndrome characterized by damage of the small intestinal mucosa caused by the gliadin fraction of wheat gluten and similar alcohol-soluble proteins (prolamines) of barley and rye in genetically susceptible subjects. The presence of gluten in these subjects leads to self-perpetuating mucosal damage, whereas elimination of gluten results in full mucosal recovery. The clinical manifestations of CD are protean in nature and vary markedly with the age of the patient, the duration and extent of disease, and the presence of extraintestinal pathologic conditions. In addition to the classical gastrointestinal form, a variety of other clinical manifestations of the disease have been described, including atypical and asymptomatic forms. Therefore, diagnosis of CD is extremely challenging and relies on a sensitive and specific algorithm that allows the identification of different manifestations of the disease. Serologic tests developed in the last decade provide a noninvasive tool to screen both individuals at risk for the disease and the general population. However, the current gold standard for the diagnosis of CD remains histologic confirmation of the intestinal damage in serologically positive individuals. The keystone treatment of CD patients is a lifelong elimination diet in which food products containing gluten are avoided.

BACKGROUND & AIMS: Zonula occludens toxin is a novel toxin elaborated by Vibrio cholerae that modulates intestinal tight junctions. The aim of this study was to establish whether the permeabilizing effect of the toxin leads to intestinal secretion. METHODS: Rabbit intestine was mounted in Ussing chambers and exposed to increasing concentrations of purified toxin. The tissues were also fixed, exposed to zonula occludens toxin, and processed for fluorescence microscopy to determine the distribution of the toxin receptor within the intestine. Then purified toxin was simultaneously perfused in three distinct rabbit intestinal segments in vivo, and water and electrolyte absorption were measured. RESULTS: Zonula occludens toxin induced a time- and dose- dependent decrease of tissue resistance starting at a toxin concentration of 1.1 x 10(-13) mol/L. When tested in vivo, the toxin induced a secretion of water and chloride and the passage of polyethylene glycol 4000 in the bloodstream. Both the in vitro and in vivo effects of the toxin were observed only in the small intestine but not in the colon and paralleled the distribution of the toxin receptor within the intestine. CONCLUSIONS: The intestinal secretion induced by zonula occludens toxin follows the opening of tight junctions caused by the toxin, possibly representing a novel mechanism of intestinal secretion. (Gastroenterology 1997 Mar;112(3):839-46)