[Advances in colonic manometry in adults with colonic motility disorders].

In recent years, colonic manometry has been gradually introduced into clinical practice. It helps clinicians to gain a better understanding of the physiology and pathophysiology of colonic contractile activity in healthy adults and patients with colonic dysfunction. More and more patterns of colonic motility are being discovered with the help of colonic manometry. However, the clinical significance of these findings still needs to be further investigated. This review enhances our understanding of colonic motility and the current state of development and application of colonic manometry, as well as the limitations, future directions and potential of the technique in assessing the impact of treatment on colonic motility patterns, by analyzing and summarizing the literature related to colonic manometry.

Microbial experience through housing in a farmyard-type environment alters intestinal barrier properties in mouse colons.

To close the gap between ultra-hygienic research mouse models and the much more environmentally exposed conditions of humans, we have established a system where laboratory mice are raised under a full set of environmental factors present in a naturalistic, farmyard-type habitat-a process we have called feralization. In previous studies we have shown that feralized (Fer) mice were protected against colorectal cancer when compared to conventionally reared laboratory mice (Lab). However, the protective mechanisms remain to be elucidated. Disruption of the protective intestinal barrier is an acknowledged player in colorectal carcinogenesis, and in the current study we assessed colonic mucosal barrier properties in healthy, feralized C57BL/6JRj male mice. While we found no effect of feralization on mucus layer properties, higher expression of genes encoding the mucus components Fcgbp and Clca1 still suggested mucus enforcement due to feralization. Genes encoding other proteins known to be involved in bacterial defense (Itln1, Ang1, Retnlb) and inflammatory mechanisms (Zbp1, Gsdmc2) were also higher expressed in feralized mice, further suggesting that the Fer mice have an altered intestinal mucosal barrier. These findings demonstrate that microbial experience conferred by housing in a farmyard-type environment alters the intestinal barrier properties in mice possibly leading to a more robust protection against disease. Future studies to unravel regulatory roles of feralization on intestinal barrier should aim to conduct proteomic analyses and in vivo performance of the feralized mice intestinal barrier.

Propulsive colonic contractions are mediated by inhibition-driven poststimulus responses that originate in interstitial cells of Cajal.

The peristaltic reflex is a fundamental behavior of the gastrointestinal (GI) tract in which mucosal stimulation activates propulsive contractions. The reflex occurs by stimulation of intrinsic primary afferent neurons with cell bodies in the myenteric plexus and projections to the lamina propria, distribution of information by interneurons, and activation of muscle motor neurons. The current concept is that excitatory cholinergic motor neurons are activated proximal to and inhibitory neurons are activated distal to the stimulus site. We found that atropine reduced, but did not block, colonic migrating motor complexes (CMMCs) in mouse, monkey, and human colons, suggesting a mechanism other than one activated by cholinergic neurons is involved in the generation/propagation of CMMCs. CMMCs were activated after a period of nerve stimulation in colons of each species, suggesting that the propulsive contractions of CMMCs may be due to the poststimulus excitation that follows inhibitory neural responses. Blocking nitrergic neurotransmission inhibited poststimulus excitation in muscle strips and blocked CMMCs in intact colons. Our data demonstrate that poststimulus excitation is due to increased Ca2+ transients in colonic interstitial cells of Cajal (ICC) following cessation of nitrergic, cyclic guanosine monophosphate (cGMP)-dependent inhibitory responses. The increase in Ca2+ transients after nitrergic responses activates a Ca2+-activated Cl− conductance, encoded by Ano1, in ICC. Antagonists of ANO1 channels inhibit poststimulus depolarizations in colonic muscles and CMMCs in intact colons. The poststimulus excitatory responses in ICC are linked to cGMP-inhibited cyclic adenosine monophosphate (cAMP) phosphodiesterase 3a and cAMP-dependent effects. These data suggest alternative mechanisms for generation and propagation of CMMCs in the colon.

Mucosal Integrity Testing Can Detect Differences in the Rectums of Patients with Inflammatory Bowel Disease Compared to Controls: A Pilot Study.

BACKGROUND: While the pathogenesis of inflammatory bowel disease (IBD) is incompletely understood, disruption of epithelial integrity is suspected to play a prominent role in disease initiation and progression. Currently, there is no convenient way to measure this in vivo. AIMS: Our aim is to determine whether a mucosal integrity (MI) testing device that has been used to measure MI in the esophagus can also be used to measure barrier function in the colon during colonoscopy. METHODS: Mucosal integrity testing was measured in patients with IBD (n = 17) and controls (n = 7) during colonoscopy. During the procedure, an MI catheter was passed down the working channel of the colonoscope and placed along the mucosal wall to measure MI in the rectum, left, transverse, and right colon. In patients with IBD, MI measurements and biopsies were taken in areas which appeared inflamed when present. We then determined if there was a significant difference in MI between patients with IBD and controls. RESULTS: MI was significantly higher in the rectum of patients with IBD (CD and UC combined) versus control colons [767 (618-991) vs. 531 (418-604) ohms, P < 0.01]. There were no significant differences in MI among patients with IBD versus controls in the right, transverse, or left colon. Within the IBD group, there were no significant differences in MI between inflamed versus non-inflamed rectums. There was no correlation between quality of life scores or endoscopic severity with MI, though this study was likely underpowered to detect these differences. CONCLUSION: Rectal MI is significantly higher in patients with IBD versus controls. Future studies are needed to determine how this information can be used clinically.

Colorectal keratins: Integrating nutrition, metabolism and colorectal health.

The colon mucosa is lined with crypts of circa 300 cells, forming a continuous barrier whose roles include absorption of water, recovery of metabolic energy sources (notably short chain fatty acids), secretion of a protective mucus barrier, and physiological signalling. There is high turnover and replenishment of cells in the mucosa, disruption of this may lead to bowel pathologies including cancer and inflammatory bowel disease. Keratins have been implicated in the processes of cell death, epithelial integrity, response to inflammation and as a result are often described as guardians of the colonic epithelium. Keratin proteins carry extensive post-translational modifications, the cofactors for kinases, acetyl transferases and other modification-regulating enzymes are themselves products of metabolism. A cluster of studies has begun to reveal a bidirectional relationship between keratin form and function and metabolism. In this paper we hypothesise a mechanistic interaction between keratins and metabolism is governed through regulation of post-translational modifications and may contribute significantly to the normal functioning of the colon, placing keratins at the centre of a nutrition-metabolism-health triangle.

High-resolution impedance manometry characterizes the functional role of distal colonic motility in gas transit.

BACKGROUND: The colonic motor patterns associated with gas transit are poorly understood. This study describes the application of high-resolution impedance manometry (HRiM) in the human colon in vivo to characterize distal colonic motility and gas transit; (a) after a meal and (b) after intraluminal gas insufflation into the sigmoid colon. METHODS: HRiM recordings were performed in 19 healthy volunteers, with sensors positioned from the distal descending colon to the proximal rectum. Protocol 1 (n = 10) compared pressure and impedance prior to and after a meal. Protocol 2 (n = 9) compared pressure and impedance before and after gas insufflation into the sigmoid colon (60 mL total volume). KEY RESULTS: Both the meal and gas insufflation resulted in an increase in the prevalence of the 2-8/minute "cyclic motor pattern" (meal: (t(9) = -6.42, P<0.001); gas insufflation (t(8) = -3.13, P = 0.01)), and an increase in the number of antegrade and retrograde propagating impedance events (meal: Z = -2.80, P = 0.005; gas insufflation Z = -2.67, P = 0.008). Propagating impedance events temporally preceded antegrade and retrograde propagating contractions, representing a column of luminal gas being displaced ahead of a propagating contraction. Three participants reported an urge to pass flatus and/or flatus during the studies. CONCLUSIONS AND INFERENCES: Initiation of the 2-8/minute cyclic motor pattern in the distal colon occurs both following a meal and/or as a localized sensorimotor response to gas. The near-absence of a flatal urge and the temporal association between propagating contractions and gas transit supports the hypothesis that the 2-8/minute cyclic motor pattern acts as a physiological "brake" modulating rectal filling.

Ex Vivo Human Colon Tissue Exposure to Pristine Graphene Activates Genes Involved in the Binding, Adhesion and Proliferation of Epithelial Cells.

Toxicology studies on pristine graphene are limited and lack significant correlations with actual human response. The goal of the current study was to determine the response of total colonic human tissue to pristine graphene exposure. Biopsy punches of colon tissues from healthy human were used to assess the biological response after ex vivo exposure to graphene at three different concentrations (1, 10, and 100 µg/mL). mRNA expression of specific genes or intestinal cytokine abundance was assessed using real-time PCR or multiplex immunoassays, respectively. Pristine graphene-activated genes that are related to binding and adhesion (GTPase and KRAS) within 2 h of exposure. Furthermore, the PCNA (proliferating cell nuclear antigen) gene was upregulated after exposure to graphene at all concentrations. Ingenuity pathway analysis revealed that STAT3 and VEGF signaling pathways (known to be involved in cell proliferation and growth) were upregulated. Graphene exposure (10 µg/mL) for 24 h significantly increased levels of pro-inflammatory cytokines IFNγ, IL-8, IL-17, IL-6, IL-9, MIP-1α, and Eotaxin. Collectively, these results indicated that graphene may activate the STAT3-IL23-IL17 response axis. The findings in this study provide information on toxicity evaluation using a human-relevant ex vivo colon model and serve as a basis for further exploration of its bio-applications.

Optogenetic activation of the distal colon epithelium engages enteric nervous system circuits to initiate motility patterns.

Digestive functions of the colon depend on sensory-motor reflexes in the enteric nervous system (ENS), initiated by intrinsic primary afferent neurons (IPANs). IPAN terminals project to the mucosal layer of the colon, allowing communication with epithelial cells comprising the colon lining. The chemical nature and functional significance of this epithelial-neural communication in regard to secretion and colon motility are of high interest. Colon epithelial cells can produce and release neuroactive substances such as ATP and 5-hydroxytryptamine (5-HT), which can activate receptors on adjacent nerve fibers, including IPAN subtypes. In this study, we examined if stimulation of epithelial cells alone is sufficient to activate neural circuits that control colon motility. Optogenetics and calcium imaging were used in ex vivo preparations of the mouse colon to selectively stimulate the colon epithelium, measure changes in motility, and record activity of neurons within the myenteric plexus. Light-mediated activation of epithelial cells lining the distal, but not proximal, colon caused local contractions and increased the rate of colonic migrating motor complexes. Epithelial-evoked local contractions in the distal colon were reduced by both ATP and 5-HT receptor antagonists. Our findings indicate that colon epithelial cells likely use purinergic and serotonergic signaling to initiate activity in myenteric neurons, produce local contractions, and facilitate large-scale coordination of ENS activity responsible for whole colon motility patterns.NEW & NOTEWORTHY Using an all-optical approach to measure real-time cell-to-cell communication responsible for colon functions, we show that selective optogenetic stimulation of distal colon epithelium produced activity in myenteric neurons, as measured with red genetically encoded calcium indicators. The epithelial-induced neural response led to local contractions, mediated by both purinergic and serotonergic signaling, and facilitated colonic motor complexes that propagate from proximal to distal colon.

Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures.

Enteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6-8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

Identifying causal models between genetically regulated methylation patterns and gene expression in healthy colon tissue.

BACKGROUND: DNA methylation is involved in the regulation of gene expression and phenotypic variation, but the inter-relationship between genetic variation, DNA methylation and gene expression remains poorly understood. Here we combine the analysis of genetic variants related to methylation markers (methylation quantitative trait loci: mQTLs) and gene expression (expression quantitative trait loci: eQTLs) with methylation markers related to gene expression (expression quantitative trait methylation: eQTMs), to provide novel insights into the genetic/epigenetic architecture of colocalizing molecular markers. RESULTS: Normal mucosa from 100 patients with colon cancer and 50 healthy donors included in the Colonomics project have been analyzed. Linear models have been used to find mQTLs and eQTMs within 1 Mb of the target gene. From 32,446 eQTLs previously detected, we found a total of 6850 SNPs, 114 CpGs and 52 genes interrelated, generating 13,987 significant combinations of co-occurring associations (meQTLs) after Bonferromi correction. Non-redundant meQTLs were 54, enriched in genes involved in metabolism of glucose and xenobiotics and immune system. SNPs in meQTLs were enriched in regulatory elements (enhancers and promoters) compared to random SNPs within 1 Mb of genes. Three colorectal cancer GWAS SNPs were related to methylation changes, and four SNPs were related to chemerin levels. Bayesian networks have been used to identify putative causal relationships among associated SNPs, CpG and gene expression triads. We identified that most of these combinations showed the canonical pathway of methylation markers causes gene expression variation (60.1%) or non-causal relationship between methylation and gene expression (33.9%); however, in up to 6% of these combinations, gene expression was causing variation in methylation markers. CONCLUSIONS: In this study we provided a characterization of the regulation between genetic variants and inter-dependent methylation markers and gene expression in a set of 150 healthy colon tissue samples. This is an important finding for the understanding of molecular susceptibility on colon-related complex diseases.

High-fat diet-induced colonocyte dysfunction escalates microbiota-derived trimethylamine N-oxide.

A Western-style, high-fat diet promotes cardiovascular disease, in part because it is rich in choline, which is converted to trimethylamine (TMA) by the gut microbiota. However, whether diet-induced changes in intestinal physiology can alter the metabolic capacity of the microbiota remains unknown. Using a mouse model of diet-induced obesity, we show that chronic exposure to a high-fat diet escalates Escherichia coli choline catabolism by altering intestinal epithelial physiology. A high-fat diet impaired the bioenergetics of mitochondria in the colonic epithelium to increase the luminal bioavailability of oxygen and nitrate, thereby intensifying respiration-dependent choline catabolism of E. coli In turn, E. coli choline catabolism increased levels of circulating trimethlamine N-oxide, which is a potentially harmful metabolite generated by gut microbiota.

A Novel Microphysiological Colon Platform to Decipher Mechanisms Driving Human Intestinal Permeability.

BACKGROUND & AIMS: The limited availability of organoid systems that mimic the molecular signatures and architecture of human intestinal epithelium has been an impediment to allowing them to be harnessed for the development of therapeutics as well as physiological insights. We developed a microphysiological Organ-on-Chip (Emulate, Inc, Boston, MA) platform designed to mimic properties of human intestinal epithelium leading to insights into barrier integrity. METHODS: We combined the human biopsy-derived leucine-rich repeat-containing G-protein-coupled receptor 5-positive organoids and Organ-on-Chip technologies to establish a micro-engineered human Colon Intestine-Chip (Emulate, Inc, Boston, MA). We characterized the proximity of the model to human tissue and organoids maintained in suspension by RNA sequencing analysis, and their differentiation to intestinal epithelial cells on the Colon Intestine-Chip under variable conditions. Furthermore, organoids from different donors were evaluated to understand variability in the system. Our system was applied to understanding the epithelial barrier and characterizing mechanisms driving the cytokine-induced barrier disruption. RESULTS: Our data highlight the importance of the endothelium and the in vivo tissue-relevant dynamic microenvironment in the Colon Intestine-Chip in the establishment of a tight monolayer of differentiated, polarized, organoid-derived intestinal epithelial cells. We confirmed the effect of interferon-γ on the colonic barrier and identified reorganization of apical junctional complexes, and induction of apoptosis in the intestinal epithelial cells as mediating mechanisms. We show that in the human Colon Intestine-Chip exposure to interleukin 22 induces disruption of the barrier, unlike its described protective role in experimental colitis in mice. CONCLUSIONS: We developed a human Colon Intestine-Chip platform and showed its value in the characterization of the mechanism of action of interleukin 22 in the human epithelial barrier. This system can be used to elucidate, in a time- and challenge-dependent manner, the mechanism driving the development of leaky gut in human beings and to identify associated biomarkers.

An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.

Identification, isolation and analysis of human gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) comprise key intestinal immune inductive sites, including the Peyer's patches of the small intestine and different types of isolated lymphoid follicle (ILF) found along the length of the gut. Our understanding of human GALT is limited due to a lack of protocols for their isolation. Here we describe a technique that, uniquely among intestinal cell isolation protocols, allows identification and isolation of all human GALT, as well as GALT-free intestinal lamina propria (LP). The technique involves the mechanical separation of intestinal mucosa from the submucosa, allowing the identification and isolation of submucosal ILF (SM-ILF), LP-embedded mucosal ILF (M-ILF) and LP free of contaminating lymphoid tissue. Individual SM-ILF, M-ILF and Peyer's patch follicles can be subsequently digested for downstream cellular and molecular characterization. The technique, which takes 4-10 h, will be useful for researchers interested in intestinal immune development and function in health and disease.

Macrophages: Checking Toxicity of Fungal Metabolites in the Colon.

It is well known that the intestine absorbs nutrients, electrolytes, and water. Chikina et al. recently demonstrated that it is also able to sense, recognize, and block the absorption of toxins through a very sophisticated interactive cellular cooperation between novel subpopulations of macrophages and epithelial cells.

Does Bowel Function Change After Colectomy for Colon Malignancy?

BACKGROUND: Colon cancer patients often ask how surgery will affect bowel function. Current understanding is informed by conflicting data, making preoperative patient counseling difficult. We aimed to evaluate patient-reported bowel function changes after colectomy for colon malignancy. MATERIAL AND METHODS: This was a retrospective analysis of a prospectively collected institutional database from July 2015 to June 2019. The included patients underwent colectomy for adenocarcinoma of the colon, and completed the Colorectal Functional Outcome (COREFO) questionnaire at preoperative presentation and postoperative followup. Preoperative and postoperative scores were compared using paired t-tests. Multivariable analysis was performed using domains demonstrating statistical significance on bivariate analysis, assessing the factors that were associated with symptomatic bowel function. RESULTS: We identified 117 patients with a mean age of 64 ± 13 y. The median time between preoperative and postoperative questionnaire completion was 52 d (interquartile range 45-70). Bowel movement frequency increased significantly from a mean preoperative score of 9.72 to a mean postoperative score of 14.2 (P = 0.003). There were no significant differences in the remaining four domains of bowel function or global function. Multivariable analysis demonstrated higher likelihood of symptomatic postoperative frequency scores in male patients (OR 3.85, 95% CI 1.44-11.11, P = 0.007) and patients with symptomatic preoperative frequency (OR 5.56, 95% CI 1.62-19.02, P = 0.006). CONCLUSIONS: Patient-reported bowel movement frequency worsens at postoperative follow-up after colectomy for colon cancer, while overall bowel function does not change. Men and patients with preoperative symptomatic frequency have an increased likelihood of reporting symptomatic postoperative frequency. These findings should guide more personalized and evidence-based preoperative patient counseling.

1907-2020: more than one century of colonic mass movements in humans.

Motility of the large bowel may be grossly subdivided in two types of contractile activity: low-amplitude single or cyclic propagated waves and high-amplitude propagated activity. The latter is mainly apt to shift relatively large amounts of colonic contents, and it is related to defecation. The main component of this propagated activity is represented by the radiologically identified mass movements that have a manometric equivalent known as high-amplitude propagated contractions (HAPC). The present article reviews origins and characterization of HAPC in the time course of colonic motility investigations, and correlates it with technological advancements in recent years, putting into perspective the future possible options to better detect and investigate these important physiological events.

Distinct contributions of cathelin-related antimicrobial peptide (CRAMP) derived from epithelial cells and macrophages to colon mucosal homeostasis.

The cathelin-related antimicrobial peptide CRAMP protects the mouse colon from inflammation, inflammation-associated carcinogenesis, and disrupted microbiome balance, as shown in systemic Cnlp-/- mice (also known as Camp-/- mice). However, the mechanistic basis for the role and the cellular source of CRAMP in colon pathophysiology are ill defined. This study, using either epithelial or myeloid conditional Cnlp-/- mice, demonstrated that epithelial cell-derived CRAMP played a major role in supporting normal development of colon crypts, mucus production, and repair of injured mucosa. On the other hand, myeloid cell-derived CRAMP potently supported colon epithelial resistance to bacterial invasion during acute inflammation with exacerbated mucosal damage and higher rate of mouse mortality. Therefore, a well concerted cooperation of epithelial- and myeloid-derived CRAMP is essential for colon mucosal homeostasis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis.

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.