Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells.

IMPORTANCE: Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Comparison of culture media for human intestinal organoids from the viewpoint of pharmacokinetic studies.

Human intestinal organoids are expected to be applied in pharmaceutical research. Various culture media for human intestinal organoids have been developed, but it remains unclear which media are preferable for pharmacokinetic studies. Here, we cultured human intestinal organoids with three major culture media that are already used widely around the world: the medium of Sato et al. (S-medium; reported in 2011), Fujii et al. (F-medium; 2018), and Miyoshi et al. (M-medium; 2013). The growth of human intestinal organoids cultured in S-medium was faster than that in F- or M-medium. The gene expression levels of most pharmacokinetic-related enzymes or transporters in human intestinal organoids cultured in M-medium were higher than those in S- or F-medium, and comparable to those in the adult human small intestine. The level of cytochrome P450 (CYP) 3A4 activity was also highest in human intestinal organoids cultured in M-medium. Collectively, the results underscored the importance of selection and optimization of culture medium for various applications using human intestinal organoids, including pharmacokinetic studies.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Paneth cells mediated the response of intestinal stem cells at the early stage of intestinal inflammation in the chicken.

The rapid renewal and repair of the intestinal mucosa are based on intestinal stem cells (ISC), which are located at the crypt bottom. Paneth cells are an essential component in the crypt, which served as the niche for ISC development. However, in the chicken, how the function of Paneth cells changes during intestinal inflammation is unclear and is the key to understand the mechanism of mucosal repair. In the present study, 36 HyLine White chickens (7 d of age, n = 6) were randomly divided into 1 control and 5 lipopolysaccharide (LPS) injection groups. The chickens were injected (i.p.) with PBS in the control group, however, were injected (i.p.) with LPS (10 mg/kg BW) in the LPS injection groups, which would be sampled at 5 time points (1 h postinjection [hpi], 2 hpi, 4 hpi, 6 hpi, and 8 hpi). Results showed that tumor necrosis factor-α mRNA transcription in duodenal tissue increased gradually since 1 hpi, peaked at 4 hpi, and then reduced remarkably, indicating that 4 hpi of LPS was the early stage of intestinal inflammation. Meanwhile, the MUC2 expression in duodenal tissue was dramatically reduced since 1 hpi of LPS. The ISC marker, Lgr5 and Bmi1, in the duodenal crypt were reduced from 1 hpi to 4 hpi and elevated later. Accordingly, the hydroethidine staining showed that the reactive oxygen species level, which drives the differentiation of ISC, in the duodenal crypt reduced obviously at 1 hpi and recovered gradually since 4 hpi. The analysis of Paneth cells showed that many swollen mitochondria appeared in Paneth cells at 4 hpi of LPS. Meanwhile, the Lysozyme transcription in the duodenal crypt was substantially decreased since 1 hpi of LPS. However, the Wnt3a and Dll1 in duodenal crypt decreased at 1 hpi of LPS, then increased gradually. In conclusion, Paneth cells were impaired at the early stage of intestinal inflammation, then recovered rapidly. Thus, the ISC activity was reduced at first and recovery soon.

Changes in the Neurochemical Characterization of Enteric Neurons in the Porcine Duodenum After Administration of Low-Dose Salmonella Enteritidis Lipopolysaccharides.

Lipopolysaccharides (LPS), also known as lipoglycans or endotoxins, form part of the outer membrane of Gram-negative bacteria. Previous studies have described the various harmful impacts of LPS on humans and animals. Nevertheless, many aspects of these effects are still not fully explained. One of them is the influence of endotoxins on the neurochemical characterization of neurons within the enteric nervous system (ENS), which is found in the intestinal wall and plays important adaptive roles during pathological processes and exposures. In this study, the impact of a low single dose of Salmonella Enteritidis LPS on the duodenal enteric neurons immunoreactive to substance P (SP), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating peptide (PACAP-27), and cocaine- and amphetamine-regulated transcript (CART) was studied using a double immunofluorescence technique. During the study, it was shown that even a low dose of LPS affects the number of enteric neurons containing the neuropeptides studied, and these changes were dependent on the type of the enteric plexus. The most visible changes concerned the SP-like immunoreactive (LI) neurons in the outer submucous plexus (LPS caused an increase in the percentage of these neurons from15.74 ± 0.61 to 21.72 ± 0.79%). Furthermore, the VIP-LI neurons in the inner submucous plexus were seen to decrease from 12.64 ± 0.83 to 5.96 ± 0.58%. The mechanisms behind these noted fluctuations are not clear, but it may be connected with the pro-inflammatory and neurotoxic activity of LPS.

Vitamin D receptor is overexpressed in the duodenum of patients with irritable bowel syndrome.

BACKGROUND AND AIM: Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders, and bile acids are thought to be associated with the pathogenesis of IBS. Bile acid receptors are expressed on intestinal epithelial cells. However, no study has assessed bile acid receptor proteins in IBS. Therefore, we examined the intestinal mucosal expression of bile acid receptors in patients with IBS. METHODS: Intestinal biopsies were performed in patients with IBS and controls. Mast cells, vitamin D receptor (VDR), and somatostatin were stained with specific antibodies. Levels of VDR, farnesoid X receptor (FXR), takeda-G-protein-receptor-5 (TGR5), claudins, and transient-receptor-potential-cation-channel-subfamily-V-member 6 (TRPV6) were assessed by western blotting. RESULTS: 3Mast cell counts in the second part of the duodenum were significantly higher in patients with IBS than in controls. VDR protein levels were significantly elevated in the duodenum and terminal ileum of patients with IBS compared with controls, although this difference was not seen in the cecum or rectum. FXR and TGR5 protein levels did not differ in any part of the intestine. VDR-positive cryptal epithelia in IBS were distributed not only at basal crypt but also along the upper part of the basal crypt epithelial cells. In contrast, the pattern of gut somatostatin-positive cells, claudins, and TRPV6 levels did not differ. CONCLUSIONS: The number of mast cells in the duodenum was significantly increased, and the protein expression levels of VDR, but not those of FXR or TGR5, were elevated in the duodenal epithelial crypt in patients with IBS.

Functional Dyspepsia and Duodenal Eosinophil Count and Degranulation: A Multiethnic US Veteran Cohort Study.

BACKGROUND: Duodenal eosinophilia may play a role in functional dyspepsia (FD), but existing study results are conflicted. We investigated the association between duodenal eosinophils (count and degranulation) and FD symptoms, accounting for atopic conditions, medications, and seasonal variations. METHODS: In a cross-sectional study conducted in the Michael E. DeBakey VA Medical Center in Houston, Texas, we analyzed duodenal histopathology of 436 patient samples from a prospective cohort with a validated symptom survey data and chart reviews. FD was defined using Rome II symptom criteria. Eosinophil count was number per 5 high-power fields (HPF), and eosinophil degranulation was eosinophilic granules in the stroma both determined by two independent investigators. RESULTS: The study cohort was predominantly male (87.4%) with a mean age of 59.3 (standard deviation (SD) ± 9.8). Mean and median eosinophil counts were 75.5 (± 47.8) and 63 (IQR: 43, 101) per five HPF, respectively. Duodenal eosinophilia (defined as ≥ 63 per 5 HPF) and eosinophil degranulation were present in 50.5% and 23.1% of patient samples, respectively. FD was observed in 178 patients (41.7%), but neither the mean eosinophil count nor duodenal eosinophilia was associated with FD. Eosinophil degranulation was independently associated with FD overall (OR 1.74; 95% CI 1.08, 2.78; p = 0.02) and early satiety (OR 2.04; 95% CI 1.26, 3.30; p = 0.004). CONCLUSIONS: In this large, ethnically diverse cohort of adult patients, we found no significant association between duodenal eosinophilia and FD. However, the presence of duodenal eosinophilic degranulation, an activated eosinophil marker, was significantly associated with FD, especially early satiety.

Effect of Crohn's Disease on Villous Length and CYP3A4 Expression in the Pediatric Small Intestine.

Changes in absorptive capacity and first-pass metabolism in the small intestine affect oral drug bioavailability. Characterization of such changes as a consequence of inflammation is important for developing physiologically-based pharmacokinetic (PBPK) models for inflammatory bowel disease. We sought to elucidate the impact of small intestinal Crohn's disease (CD) on villous length and CYP3A4 expression in children. Freshly frozen duodenal and terminal ileum (TI) biopsies from 107 children (1-19 years) with and without CD were evaluated for active inflammation. Villous length and CYP3A4 mRNA/protein expression were compared among regions of active and inactive inflammation in CD and controls. A twofold reduction in villous length was observed in inflamed duodena and ilia of children with CD, but in the absence of regional inflammation, villi in CD were comparable in length to controls. Expression of CYP3A4 mRNA correlated significantly with villous length in the TI (P = 0.0003), with a trend observed in the duodenum that did not reach statistical significance. In the presence of active inflammation, a significant decrease in CYP3A protein expression was confirmed in the duodenum, where protein expression also correlated significantly with villous length across diagnoses (P < 0.0001). Our findings suggest that previous observations of decreased CYP3A4 expression and function in inflamed intestine may not be due solely to downregulation by inflammatory cytokines, but also to villous blunting and subsequent loss of surface area for protein expression. This information is relevant for PBPK model development and could aid with dose adjustment decisions for oral CYP3A4 substrates administered during CD flare (e.g., budesonide).

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

BACKGROUND & AIMS: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODS: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction. RESULTS: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes. CONCLUSIONS: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.

Modeling age-dependent developmental changes in the expression of genes involved in citrulline synthesis using pig enteroids.

BACKGROUND: Age-dependent changes in the intestinal gene expression of enzymes involved in the metabolism of citrulline and arginine are well characterized. Enteroids, a novel ex-vivo model that recreates the three-dimensional structure of the intestinal crypt-villus unit, have shown to replicate molecular and physiological profiles of the intestinal segment from where they originated ("location memory"). OBJECTIVE: The present study tested the hypothesis that enteroids recapitulate the developmental changes observed in vivo regarding citrulline production in pigs ("developmental memory"). METHODS: Preterm (10- and 5-d preterm) and term pigs at birth, together with 7- and 35-d-old pigs were studied. Gene expression was measured in jejunal samples and in enteroids derived from this segment. Whole body citrulline production was measured by isotope dilution and enteroid citrulline production by accumulation in the media. RESULTS: With the exception of arginase I and inducible nitric oxide synthase, all the genes investigated expressed in jejunum were expressed by enteroids. In the jejunum, established markers of development (lactase and sucrase-isomaltase), as well as genes that code for enzymes involved in the production and utilization of citrulline and arginine, underwent the ontogenic changes described in the literature. However, enteroid expression of these genes, as well as citrulline production, failed to recapitulate the changes observed in vivo. CONCLUSIONS: Under culture conditions used in our study, enteroids derived from jejunal crypts of pigs at different ages failed to replicate the gene expression observed in whole tissue and whole body citrulline production. Additional extracellular cues may be needed to reproduce the age-dependent phenotype.

Development and characterization of rat duodenal organoids for ADME and toxicology applications.

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.

5HT expression in the stomach and duodenum of the Rhesus Monkey (Macaca mulatta).

This study aims to provide a histological description of different regions of the gastric and duodenal mucosa in Rhesus monkey, as well as to analyze the distribution and the relative frequency of 5-HT. The cardia region mucosa consists of simple columnar epithelium PAS + and AB + and the 5-HT cells were observed at the base of the gland (QA [5-HT cells]/mm²) = 8.72 ±â€¯4.98). The body region, has a smaller number of glands. The 5-HT cells were found predominant in the base of the gastric glands. QA= 6.96 ±â€¯3.81. When compared to body region, the stomach fundus has smaller gastric pits. The 5-HT cells are found at the base of the glands near the main cells. QA = 5.29 ±â€¯2.09. The pylorus region was found to have deep pits and well-developed gastric glands. The 5-HT cells are scarce, at the base of the pyloric gland. QA = 1.18 ±â€¯1.36. The duodenum presented goblet cells strong PAS + and AB +. 5-HT cells were found both in the lining epithelium and in the intestinal glands. QA = 8.16 ±â€¯2.59.

A cross-sectional study of nausea in functional abdominal pain: relation to mucosal mast cells and psychological functioning.

BACKGROUND: Nausea is a common symptom in youth with chronic abdominal pain. The aims of the current study were to assess: 1) the frequency of nausea in patients with functional dyspepsia (FD) and irritable bowel syndrome (IBS), respectively, as defined by Rome IV criteria; and, 2) relationships between nausea and mucosal inflammation as defined by antral and duodenal eosinophil and mast cell densities. A secondary aim was to assess relationships between nausea and other gastrointestinal symptoms, non-gastrointestinal somatic symptoms, and psychological dysfunction. METHODS: Records from patients with pain associated functional gastrointestinal disorders were retrospectively reviewed for gastrointestinal and somatic symptoms and anxiety, depression, and somatizations scores as assessed by the Behavior Assessment System for Children (BASC-2). In addition, previous gastric and mucosal biopsies were assessed for mast cell and eosinophil densities, respectively. RESULTS: 250 patients, ages 8 to 17 years, were assessed. Nausea was reported by 78% and was equally prevalent in those with FD alone, those with IBS alone, and those with both FD and IBS. Nausea was associated with increased mean (21.4 vs. 17.5) and peak (26.2 vs. 22.9) duodenal mast cell densities as compared those without nausea. Nausea was also associated with a wide variety of individual gastrointestinal symptoms, as well as headaches, fatigue, and dizziness. Lastly, nausea was associated with elevated self-report scores for anxiety (55.2 vs. 50.0), depression (50.2 vs. 46.1), and somatization (70.3 vs. 61.8). CONCLUSIONS: Nausea is common in children and adolescents with pain-associated FGIDs as defined by Rome IV and is not unique to either FD or IBS. Nausea is associated with increased mucosal mast cell density, non-gastrointestinal somatic symptoms, and psychologic dysfunction.

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

Molecular characterization of a potential receptor of Eimeria acervulina microneme protein 3 from chicken duodenal epithelial cells.

Eimeria acervulina is one of seven Eimeria spp. that can infect chicken duodenal epithelial cells. Eimeria microneme protein 3 (MIC3) plays a vital role in the invasion of host epithelial tissue by the parasite. In this study, we found that chicken (Gallus gallus) ubiquitin conjugating enzyme E2F (UBE2F) could bind to the MIC3 protein of E. acervulina (EaMIC3), as screened using the yeast two-hybrid system, and that it might be the putative receptor protein of EaMIC3. The UBE2F gene was cloned from chicken duodenal epithelial cells. The recombinant protein of UBE2F (rUBE2F) was expressed in E. coli and the reactogenicity of rUBE2F was analyzed by Western blot. Gene sequencing revealed that the opening reading frame (ORF) of UBE2F was 558 base pairs and encoded a protein of 186 amino acids with a molecular weight of 20.46 kDa. The predicted UBE2F protein did not contain signal peptides or a transmembrane region, but had multiple O-glycosylation and phosphorylation sites. A phylogenetic analysis showed that the chicken UBE2F protein is closely related to those of quail and pigeon (Coturnix japonica and Columba livia). A sporozoite invasion-blocking assay showed that antisera against rUBE2F significantly inhibited the invasion of E. acervulina sporozoites in vitro. Animal experiments indicated that the antisera could significantly enhance average body weight gains and reduce mean lesion scores following a challenge with E. acervulina. These results therefore imply that the chicken UBE2F protein might be the target receptor molecule of EaMIC3 that is involved in E. acervulina invasion.

TITLE: Caractérisation moléculaire d'un récepteur potentiel de la protéine 3 du micronème d'Eimeria acervulina dans les cellules épithéliales duodénales de poulet. ABSTRACT: Eimeria acervulina est l'une des sept Eimeria spp. qui peuvent infecter les cellules épithéliales duodénales de poulet. La protéine 3 du micronème d'Eimeria (MIC3) joue un rôle vital dans l'invasion du tissu épithélial de l'hôte par le parasite. Dans cette étude, nous avons constaté que l'enzyme de conjugaison de l'ubiquitine de poulet E2F (UBE2F) pouvait se lier à la protéine MIC3 d'E. acervulina (EaMIC3), telle que testé à l'aide du système de levure à deux hybrides, et qu'il pourrait s'agir de la protéine réceptrice putative d'EaMIC3. Le gène UBE2F a été cloné à partir de cellules épithéliales duodénales de poulet. La protéine recombinante d'UBE2F (rUBE2F) a été exprimée dans E. coli et la réactogénicité de rUBE2F a été analysée par Western blot. Le séquençage génétique a révélé que le cadre de lecture d'ouverture (ORF) d'UBE2F était de 558 paires de bases et codait une protéine de 186 acides aminés avec un poids moléculaire de 20,46 kDa. La protéine UBE2F prédite ne contenait pas de peptides signaux ni de région transmembranaire, mais avait plusieurs sites d'O-glycosylation et de phosphorylation. Une analyse phylogénétique a montré que la protéine UBE2F de poulet est étroitement liée à celles de la caille et du pigeon (Coturnix japonica et Columba livia). Un test de blocage des invasions de sporozoïtes a montré que les antisérums dirigés contre rUBE2F inhibaient de manière significative l'invasion des sporozoïtes d'E. acervulina in vitro. Les expériences sur les animaux ont indiqué que les antisérums pourraient améliorer de manière significative les gains de poids corporel moyens et réduire les scores moyens de lésions suite à une infection avec E. acervulina. Ces résultats impliquent donc que la protéine UBE2F de poulet pourrait être la molécule de récepteur cible d'EaMIC3 impliquée dans l'invasion d'E. acervulina.

The inhibitory activity of alginate against allergic reactions in an ovalbumin-induced mouse model.

Marine seaweed polysaccharides have been considered as a potential resource for antiallergic therapy. Alginate is an acidic linear polysaccharide and soluble dietary fiber that was extracted from brown algae, Laminaria japonica. The molecular weight of alginate was 108 kDa, and its water solution exhibited non-Newtonian characteristics, including viscoelasticity and shear-thinning behavior. The ability of alginate to inhibit allergic reactions was investigated in ovalbumin (OVA)-induced BALB/c mice, which have been widely used as a mouse model of egg allergy. The results showed that alginate could effectively attenuate the occurrence of allergic reactions, including improving the integrity of the intestinal epithelial villi and inhibition of mast cell degranulation in the jejunum, in OVA-induced mice. Moreover, after treatment with alginate, the levels of IgE, histamine and IL-4 in OVA-induced mice were remarkably decreased, and the levels of IFN-γ were markedly increased. In addition, the number of Treg cells in spleen tissues in OVA-induced mice was increased by alginate, and the OVA-induced differentiation of Th0 cells into Th2 cells was significantly inhibited. These results demonstrate that alginate possesses potential antiallergic activities in a mouse model of egg allergy, which might provide important evidence that alginate, extracted from Laminaria japonica, can be developed into a novel functional food for inhibiting egg allergy.

Gastrointestinal motility and morphology in mice: Strain-dependent differences.

BACKGROUND: BALB/c and C57BL/6 mice are widely used in biomedical research; however, the differences between strains are still underestimated. Our aims were to develop an experimental protocol to evaluate the duodenal contractility and gastrointestinal transit in mice using the Alternating Current Biosusceptometry (ACB) technique and to compare gastrointestinal motor function and morphology between BALB/c and C57BL/6 strains. METHODS: Male mice were used in experiments (a) duodenal contractility: animals which had a magnetic marker surgically fixed in the duodenum to determine the frequency and amplitude of contractions and (b) gastrointestinal transit: animals which ingested a magnetically marked chow to calculate the Oro-Anal Transit Time (OATT) and the Fecal Pellet Elimination Rate (FPER). The animals were killed after the experiments for organ collection and morphometric analysis. KEY RESULTS: BALB/c and C57BL/6 had two different duodenal frequencies (high and low) with similar amplitudes. After 10 hours of monitoring, BALB/c eliminated around 89% of the ingested marker and C57BL/6 eliminated 33%; OATT and FPER were slower for C57BL/6 compared with BALB/c. The OATT and amplitude of low frequency had a strong positive correlation in C57BL/6. For BALB/c, the gastric muscular layer was thicker compared to that measured for C57BL/6. CONCLUSIONS AND INFERENCES: The experimental protocol to evaluate duodenal contractility and fecal magnetic pellets output using the ACB technique in mice was successfully established. BALB/c strains had higher duodenal frequencies and a shorter time to eliminate the ingested marker. Our results showed differences in both motor function and gastrointestinal morphology between BALB/c and C57BL/6 strains.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

A lower duodenal immune response is associated with an increase of insulin resistance in patients with morbid obesity.

OBJECTIVE: The intestinal immune response could play an important role in obesity-related comorbidities. We aim to study the profile of duodenal cytokines and chemokines in patients with morbid obesity (MO), its relation with insulin resistance (IR) and the intake of metformin, and with the evolution of MO after sleeve gastrectomy (SG). RESEARCH DESIGN AND METHODS: Duodenal levels of 24 cytokines and 9 chemokines were analyzed in 14 nonobese and in 54 MO who underwent SG: with lower IR (MO-lower-IR), with higher IR (MO-higher-IR), and with type 2 diabetes treated with metformin (MO-metf-T2DM). RESULTS: MO-lower-IR had higher levels of cytokines related to Th1, Th2, Th9, Th17, Th22, M1 macrophages, and chemokines involved in the recruitment of macrophages and T-lymphocytes (p < 0.05), and total (CD68 expression) and M1 macrophages (ITGAX expression) (p < 0.05) when compared with nonobese patients, but with a decrease in M2 macrophages (MRC1 expression) (p < 0.05). In MO-higher-IR, these chemokines and cytokines decreased and were similar to those found in nonobese patients. In MO-metf-T2DM, only IL-4 (Th2) and IL-22 (Th22) increased their levels with regard to MO-higher-IR (p < 0.05). In MO-higher-IR and MO-metf-T2DM, there was a decrease of CD68 expression (p < 0.05) while ITGAX and MRC1 were similar with regard to MO-lower-IR. We found an association between CXCL8, TNFß and IL-2 with the evolution of body mass index (BMI) after SG (p < 0.05). CONCLUSIONS: There is an association between a higher IR and a lower duodenal immune response in MO, with a slight increase in those patients with metformin treatment. Intestinal immune response could be involved in the evolution of BMI after SG.

Long-term treatment with naproxen changes the chemical coding of the porcine intramural duodenum neurons.

Due to numerous therapeutic applications and high availability, non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs worldwide. However, long-term use of these drugs can lead to damage to the gastrointestinal mucosa. The enteric nervous system (ENS), which is part of the autonomic nervous system, controls most aspects of gastrointestinal activity. Enteric neurons are characterized by considerable chemical plasticity and the appearance of a pathological factor results in a change in the synthesis of neurotransmitters. The purpose of this study was to determine the effects of naproxen on expression of biologically active substances by intramural neurons supplying the porcine duodenum. The study was performed on eight immature pigs of the Pietrain x Duroc race (approximately 20kg of body weight). The animals were divided into two groups - a control (C group) and an experimental group (N group). Group C (n=4) consisted of animals which received empty gelatine capsules. Group N (n=4) was composed of pigs who received naproxen orally for 28 days, approximately one hour before feeding. After this time, animals from both groups were euthanized. Frozen sections (14µm thickness) were then prepared from the collected duodenum and subjected to double immunofluorescence staining. Antibodies against the neuronal marker PGP 9.5 and against vasoactive intestinal polypeptide (VIP), substance P (SP), neuronal nitric oxide synthase (nNOS), galanin (GAL), pituitary adenylate cyclase-activating polypeptide (PACAP) and cocaine- and amphetamine- regulated transcript peptide (CART) were used as primary antibodies. The polyclonal donkey anti-rabbit, anti-mouse and anti-guinea pig IgG antibodies - Alexa Fluor 488 and 546 - were also used for staining. Analysis of the results obtained with a fluorescence microscope showed a significant increase in the number of nNOS-, VIP-, GAL-, PACAP- and CART-immunoreactive ganglionated neurons and a decrease in the number of SP-positive neurons in the myenteric and submucosal plexuses of the porcine duodenum. The obtained results indicate the participation of enteric neurotransmitters in the neuronal duodenal response to naproxen-induced inflammation.