From the organ bath to the whole person: a review of human colonic motility.

Motor function of the colon is essential for health. Our current understanding of the mechanisms that underlie colonic motility are based upon a range of experimental techniques, including molecular biology, single cell studies, recordings from muscle strips, analysis of part or whole organ ex vivo through to in vivo human recordings. For the surgeon involved in the clinical management of colonic conditions this amounts to a formidable volume of material. Here, we synthesize the key findings from these various experimental approaches so that surgeons can be better armed to deal with the complexities of the colon.

Alterations in GLP-1 and PYY release with aging and body mass in the human gut.

The lining of our intestinal surface contains an array of hormone-producing cells that are collectively our bodies' largest endocrine cell reservoir. These "enteroendocrine" (EE) cells reside amongst the billions of absorptive epithelial and other cell types that line our gastrointestinal tract and can sense and respond to the ever-changing internal environment in our gut. EE cells release an array of important signalling molecules that can act as hormones, including glucagon-like peptide (GLP-1) and peptide YY (PYY) which are co-secreted from L cells. While much is known about the effects of these hormones on metabolism, insulin secretion and food intake, less is understood about their secretion from human intestinal tissue. In this study we assess whether GLP-1 and PYY release differs across human small and large intestinal tissue locations within the gastrointestinal tract, and/or by sex, body weight and the age of an individual. We identify that the release of both hormones is greater in more distal regions of the human colon, but is not different between sexes. We observe a negative correlation of GLP-1 and BMI in the small, but not large, intestine. Increased aging correlates with declining secretion of both GLP-1 and PYY in human large, but not small, intestine. When the data for large intestine is isolated by region, this relationship with age remains significant for GLP-1 in the ascending and descending colon and in the descending colon for PYY. This is the first demonstration that site-specific differences in GLP-1 and PYY release occur in human gut, as do site-specific relationships of L cell secretion with aging and body mass.

Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding.

BACKGROUND AND AIMS: Gut functions including motility, secretion, and blood flow are largely controlled by the enteric nervous system. Characterizing the different classes of enteric neurons in the human gut is an important step to understand how its circuitry is organized and how it is affected by disease. METHODS: Using multiplexed immunohistochemistry, 12 discriminating antisera were applied to distinguish different classes of myenteric neurons in the human colon (2596 neurons, 12 patients) according to their chemical coding. All antisera were applied to every neuron, in multiple layers, separated by elutions. RESULTS: A total of 164 combinations of immunohistochemical markers were present among the 2596 neurons, which could be divided into 20 classes, with statistical validation. Putative functions were ascribed for 4 classes of putative excitatory motor neurons (EMN1-4), 4 inhibitory motor neurons (IMN1-4), 3 ascending interneurons (AIN1-3), 6 descending interneurons (DIN1-6), 2 classes of multiaxonal sensory neurons (SN1-2), and a small, miscellaneous group (1.8% of total). Soma-dendritic morphology was analyzed, revealing 5 common shapes distributed differentially between the 20 classes. Distinctive baskets of axonal varicosities surrounded 45% of myenteric nerve cell bodies and were associated with close appositions, suggesting possible connectivity. Baskets of cholinergic terminals and several other types of baskets selectively targeted ascending interneurons and excitatory motor neurons but were significantly sparser around inhibitory motor neurons. CONCLUSIONS: Using a simple immunohistochemical method, human myenteric neurons were shown to comprise multiple classes based on chemical coding and morphology and dense clusters of axonal varicosities were selectively associated with some classes.

Electrophysiological and morphological features of myenteric neurons of human colon revealed by intracellular recording and dye fills.

BACKGROUND: Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals. METHODS: Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia. KEY RESULTS: Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n = 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs. CONCLUSIONS: Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.

Stimulation of extrinsic sympathetic nerves differentially affects neurogenic motor activity in guinea pig distal colon.

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5 Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10 Hz and greater. Phentolamine (3.6 µM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits.

Characterization of viscerofugal neurons in human colon by retrograde tracing and multi-layer immunohistochemistry.

Background and Aims: Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic motility reflexes and may modulate glucose metabolism and feeding. We used rapid retrograde tracing from colonic nerves to identify VFNs in human colon for the first time, using ex vivo preparations with multi-layer immunohistochemistry. Methods: Colonic nerves were identified in isolated preparations of human colon and set up for axonal tracing with biotinamide. After fixation, labeled VFN cell bodies were subjected to multiplexed immunohistochemistry for 12 established nerve cell body markers. Results: Biotinamide tracing filled 903 viscerofugal nerve cell bodies (n = 23), most of which (85%) had axons projecting orally before entering colonic nerves. Morphologically, 97% of VFNs were uni-axonal. Of 215 VFNs studied in detail, 89% expressed ChAT, 13% NOS, 13% calbindin, 9% enkephalin, 7% substance P and 0 of 123 VFNs expressed CART. Few VFNs contained calretinin, VIP, 5HT, CGRP, or NPY. VFNs were often surrounded by dense baskets of axonal varicosities, probably reflecting patterns of connectivity; VAChT+ (cholinergic), SP+ and ENK+ varicosities were most abundant around them. Human VFNs were diverse; showing 27 combinations of immunohistochemical markers, 4 morphological types and a wide range of cell body sizes. However, 69% showed chemical coding, axonal projections, soma-dendritic morphology and connectivity similar to enteric excitatory motor neurons. Conclusion: Viscerofugal neurons are present in human colon and show very diverse combinations of features. High proportions express ChAT, consistent with cholinergic synaptic outputs onto postganglionic sympathetic neurons in prevertebral ganglia.

Characterisation of parasympathetic ascending nerves in human colon.

Background: In the human large bowel, sacral parasympathetic nerves arise from S2 to S4, project to the pelvic plexus ("hypogastric plexus") and have post-ganglionic axons entering the large bowel near the rectosigmoid junction. They then run long distances orally or aborally within the bowel wall forming "ascending nerves" or "shunt fascicles" running in the plane of the myenteric plexus. They form bundles of nerve fibres that can be distinguished from the myenteric plexus by their straight orientation, tendency not to merge with myenteric ganglia and greater width. Aim: To identify reliable marker(s) to distinguish these bundles of ascending nerves from other extrinsic and intrinsic nerves in human colon. Methods: Human colonic segments were obtained with informed consent, from adult patients undergoing elective surgery (n = 21). Multi-layer immunohistochemical labelling with neurofilament-H (NF200), myelin basic protein (MBP), von Willebrand factor (vWF), and glucose transporter 1 (GLUT1), and rapid anterograde tracing with biotinamide, were used to compare ascending nerves and lumbar colonic nerves. Results: The rectosigmoid and rectal specimens had 6-11 ascending nerves spaced around their circumference. Distal colon specimens typically had 1-3 ascending nerves, with one located near the mesenteric taenia coli. No ascending nerves were observed in ascending colon specimens. GLUT1 antisera labelled both sympathetic lumbar colonic nerves and ascending nerves in the gut wall. Lumbar colonic nerves joined the myenteric plexus and quickly lost GLUT1 labelling, whereas GLUT1 staining labelled parasympathetic ascending nerves over many centimetres. Conclusion: Ascending nerves can be distinguished in the colorectum of humans using GLUT1 labelling combined with NF200.

Identifying Types of Neurons in the Human Colonic Enteric Nervous System.

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

The effects of loperamide on excitatory and inhibitory neuromuscular function in the human colon.

BACKGROUND: In most animal species, opioids alter colonic motility via the inhibition of excitatory enteric motor neurons. The mechanisms by which opioids alter human colonic motility are unclear. The aim of this study was to describe the effects of loperamide on neuromuscular function in the human colon. METHODS: Tissue specimens of human colon from 10 patients undergoing an anterior resection were divided into three inter-taenial circular muscle strips. Separate organ baths were used to assess: (1) excitatory transmission (selective blockade of inhibitory transmission: L-NOARG/MRS2179); (2) inhibitory transmission (selective blockade of excitatory transmission: hyoscine hydrobromide); and (3) a control bath (no drug additions). Neuromuscular function was assessed using force transducer recordings and electrical field stimulation (EFS; 20 V, 10 Hz, 0.5 ms, 10 s) prior to and following loperamide and naloxone. KEY RESULTS: In human preparations with L-NOARG/MRS2179, loperamide had no significant effects on isometric contractions. In preparations with hyoscine hydrobromide, loperamide reduced isometric relaxation during EFS (median difference + 0.60 g post-loperamide, Z = -2.35, p = 0.019). CONCLUSIONS AND INFERENCES: Loperamide had no effect on excitatory neuromuscular function in human colonic circular muscle. These findings suggest that loperamide alters colonic function by acting primarily on inhibitory motor neurons, premotor enteric neurons, or via alternative non-opioid receptor pathways.

Sympathetic Pathways Target Cholinergic Neurons in the Human Colonic Myenteric Plexus.

Background: The sympathetic nervous system inhibits human colonic motility largely by effects on enteric neurons. Noradrenergic axons, which branch extensively in the myenteric plexus, are integral to this modulatory role, but whether they contact specific types of enteric neurons is unknown. The purpose of this study was to determine the association of noradrenergic varicosities with types of enteric neurons. Methods: Human colonic tissue from seven patients was fixed and dissected prior to multi-layer immunohistochemistry for human RNA binding proteins C and D (HuC/D) (pan-neuronal cell body labelling), tyrosine hydroxylase (TH, catecholaminergic labelling), Enkephalin (ENK), choline acetyltransferase (ChAT, cholinergic labelling) and/or nitric oxide synthase (NOS, nitrergic labelling) and imaged using confocal microscopy. TH-immunoreactive varicose nerve endings and myenteric cell bodies were reconstructed as three dimensional digital images. Data was exported to a purpose-built software package which quantified the density of varicosities close to the surface of each myenteric cell body. Results: TH-immunoreactive varicosities had a greater mean density within 1 µm of the surface of ChAT +/NOS- nerve cell bodies compared with ChAT-/NOS + cell bodies. Similarly, ENK-immunoreactive varicosities also had a greater mean density close to ChAT +/NOS- cell bodies compared with ChAT-/NOS + cells. Conclusion: A method for quantifying close associations between varicosities and nerve cell bodies was developed. Sympathetic axons in the myenteric plexus preferentially target cholinergic excitatory cells compared to nitrergic neurons (which are largely inhibitory). This connectivity is likely to be involved in inhibitory modulation of human colonic motility by the sympathetic nervous system.

The human enteric nervous system. Historical and modern advances. Collaboration between science and surgery.

BACKGROUND: There are considerable advantages and opportunities for surgeons and trainee surgeons in conducting a period of research allied with basic scientists. Such clinicians are well placed to define relevant clinical questions, provide human material (tissue, biopsy and blood) and translate the techniques derived in experimental animals to human subjects. METHODS: This small review explores research conducted on the nervous system of the intestines, with an emphasis on the translation of findings from animal to human. RESULTS: This work shows that new techniques of immunohistochemistry and retrograde tracing, developed in animal tissue, have greatly expanded our knowledge of the structure of the human enteric nervous system. CONCLUSIONS: Such findings have sparked therapeutic trials for the treatment of gastrointestinal disorders in patients.

A longitudinal cohort study of watch and wait in complete clinical responders after chemo-radiotherapy for localised rectal cancer: study protocol.

BACKGROUND: Rectal Cancer is a common malignancy. The current treatment approach for patients with locally advanced rectal cancer involves neoadjuvant chemoradiotherapy followed by surgical resection of the rectum. The resection can lead to complications and long-term consequences. A clinical complete response is observed in some patients after chemoradiotherapy. A number of recent studies have shown that patients can be observed safely after completing chemoradiotherapy (without surgery), provided clinical complete response has been achieved. In this approach, resection is reserved for cases of regrowth. This is called the watch and wait approach. This approach potentially avoids unnecessary surgical resection of the rectum and the resulting complications. In this study, we will prospectively investigate this approach. METHODS: Adult patients with a diagnosis of rectal cancer planned to receive neoadjuvant long course chemoradiotherapy (± subsequent combination chemotherapy) will be consented into the study prior to commencing treatment. After completing the chemoradiotherapy (± subsequent combination chemotherapy), based on the clinical response, subjects will be allocated to one of the following arms: subjects who achieved a clinical complete response will be allocated to the watch and wait arm and others to the standard management arm (which includes resection). The aim of the study is to determine the rate of local failure and other safety and efficacy outcomes in the watch and wait arm. Patient reported outcome measures and the use of biomarkers as part of the clinical monitoring will be studied in both arms of the study. DISCUSSION: This study will prospectively investigate the safety of the watch and wait approach. We will investigate predictive biomarkers (molecular biomarkers and imaging biomarkers) and patient reported outcome measures in the study population and the cost effectiveness of the watch and wait approach. This study will also help evaluate a defined monitoring schedule for patients managed with the watch and wait approach. This protocol covers the first two years of follow up, we are planning a subsequent study which covers year 3-5 follow up for the study population. TRIAL REGISTRATION: Name of the registry: Australia and New Zealand Clinical Trials Registry (ANZCTR). TRIAL REGISTRATION NUMBER: Trial ID: ACTRN12619000207112 Registered 13 February 2019, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376810.

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.

Long range synchronization within the enteric nervous system underlies propulsion along the large intestine in mice.

How the Enteric Nervous System (ENS) coordinates propulsion of content along the gastrointestinal (GI)-tract has been a major unresolved issue. We reveal a mechanism that explains how ENS activity underlies propulsion of content along the colon. We used a recently developed high-resolution video imaging approach with concurrent electrophysiological recordings from smooth muscle, during fluid propulsion. Recordings showed pulsatile firing of excitatory and inhibitory neuromuscular inputs not only in proximal colon, but also distal colon, long before the propagating contraction invades the distal region. During propulsion, wavelet analysis revealed increased coherence at ~2 Hz over large distances between the proximal and distal regions. Therefore, during propulsion, synchronous firing of descending inhibitory nerve pathways over long ranges aborally acts to suppress smooth muscle from contracting, counteracting the excitatory nerve pathways over this same region of colon. This delays muscle contraction downstream, ahead of the advancing contraction. The mechanism identified is more complex than expected and vastly different from fluid propulsion along other hollow smooth muscle organs; like lymphatic vessels, portal vein, or ureters, that evolved without intrinsic neurons.

Dynamin regulates L cell secretion in human gut.

BACKGROUND: The mechanochemical enzyme dynamin mediates endocytosis and regulates neuroendocrine cell exocytosis. Enteroendocrine L cells co-secrete the anorectic gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) postprandially and is a potential therapeutic target for metabolic diseases. In the present study, we aimed to determine if dynamin is implicated in human L cell secretion. METHODS: Western blot was performed on the murine L cell line GLUTag. Static incubation of human colonic mucosae with activators and inhibitors of dynamin was carried out. GLP-1 and PYY contents of the secretion supernatants were assayed using ELISA. RESULTS AND CONCLUSION: s: Both dynamin I and II are expressed in GLUTag cells. The dynamin activator Ryngo 1-23 evoked significant GLP-1 and PYY release from human colonic mucosae while the dynamin inhibitor Dynole 3-42 significantly inhibited release triggered by known L cell secretagogues. Thus, the cell signaling regulator dynamin is able to bi-directionally regulate L cell hormone secretion in the human gut and may represent a novel target for gastrointestinal-targeted metabolic drug development.

A Gut-Intrinsic Melanocortin Signaling Complex Augments L-Cell Secretion in Humans.

OBJECTIVE: Hypothalamic melanocortin 4 receptors (MC4R) are a key regulator of energy homeostasis. Brain-penetrant MC4R agonists have failed, as concentrations required to suppress food intake also increase blood pressure. However, peripherally located MC4R may also mediate metabolic benefits of MC4R activation. Mc4r transcript is enriched in mouse enteroendocrine L cells and peripheral administration of the endogenous MC4R agonist, α-melanocyte stimulating hormone (α-MSH), triggers the release of the anorectic hormones Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in mice. This study aimed to determine whether pathways linking MC4R and L-cell secretion exist in humans. DESIGN: GLP-1 and PYY levels were assessed in body mass index-matched individuals with or without loss-of-function MC4R mutations following an oral glucose tolerance test. Immunohistochemistry was performed on human intestinal sections to characterize the mucosal MC4R system. Static incubations with MC4R agonists were carried out on human intestinal epithelia, GLP-1 and PYY contents of secretion supernatants were assayed. RESULTS: Fasting PYY levels and oral glucose-induced GLP-1 secretion were reduced in humans carrying a total loss-of-function MC4R mutation. MC4R was localized to L cells and regulates GLP-1 and PYY secretion from ex vivo human intestine. α-MSH immunoreactivity in the human intestinal epithelia was predominantly localized to L cells. Glucose-sensitive mucosal pro-opiomelanocortin cells provide a local source of α-MSH that is essential for glucose-induced GLP-1 secretion in small intestine. CONCLUSION: Our findings describe a previously unidentified signaling nexus in the human gastrointestinal tract involving α-MSH release and MC4R activation on L cells in an autocrine and paracrine fashion. Outcomes from this study have direct implications for targeting mucosal MC4R to treat human metabolic disorders.

Evidence for Glucagon Secretion and Function Within the Human Gut.

Glucagon is secreted by pancreatic α cells in response to hypoglycemia and increases hepatic glucose output through hepatic glucagon receptors (GCGRs). There is evidence supporting the notion of extrapancreatic glucagon but its source and physiological functions remain elusive. Intestinal tissue samples were obtained from patients undergoing surgical resection of cancer. Mass spectrometry analysis was used to detect glucagon from mucosal lysate. Static incubations of mucosal tissue were performed to assess glucagon secretory response. Glucagon concentration was quantitated using a highly specific sandwich enzyme-linked immunosorbent assay. A cholesterol uptake assay and an isolated murine colonic motility assay were used to assess the physiological functions of intestinal GCGRs. Fully processed glucagon was detected by mass spectrometry in human intestinal mucosal lysate. High glucose evoked significant glucagon secretion from human ileal tissue independent of sodium glucose cotransporter and KATP channels, contrasting glucose-induced glucagon-like peptide 1 (GLP-1) secretion. The GLP-1 receptor agonist Exendin-4 attenuated glucose-induced glucagon secretion from the human ileum. GCGR blockade significantly increased cholesterol uptake in human ileal crypt culture and markedly slowed ex vivo colonic motility. Our findings describe the human gut as a potential source of extrapancreatic glucagon and demonstrate a novel enteric glucagon/GCGR circuit with important physiological functions beyond glycemic regulation.

Young-onset colorectal cancer is associated with a personal history of type 2 diabetes.

BACKGROUND: Colorectal cancer (CRC) is rising in incidence in young adults, and this observation is currently unexplained. We investigated whether having a personal history of type 2 diabetes mellitus (T2D) was a potential risk factor for young-onset colorectal cancer (YOCRC). METHODS: The South Australian Young Onset (SAYO) CRC study is a series of young adults with CRC below age 55. Ninety unrelated YOCRC cases were recruited to the study. Personal history and detailed family history of T2D were obtained at face-to-face interview and confirmed from medical records. Whole exome sequencing was conducted on germline DNA from each CRC case. Controls for personal history studies of T2D were 240 patients with proven clear colonoscopies and no known CRC predispositions. RESULTS: The median age of YOCRC cases was 44 years (18-54) and of controls was 45 years (18-54), and 53% of both cases and controls were females (P = 0.99). Left-sided (distal) CRC was seen in 67/89 (75%) of cases. A personal history of T2D was confirmed in 17/90 (19%) YOCRC patients compared with controls (12/240, 5%; P < 0.001; odds ratio = 4.4; 95% confidence interval, 2.0-9.7). YOCRC patients frequently reported at least one first-degree relative with T2D (32/85, 38%). Ten of 87 (12%) of YOCRC cases had CRC-related pathogenic germline variants, however, no pathogenic variants in familial diabetes-associated genes were seen. CONCLUSIONS: Though the mechanism remains unclear, our observations suggest that there is enrichment for personal history of T2D in YOCRC patients. IMPACT: A diagnosis of T2D could therefore potentially identify a subset of young adults at increased risk for CRC and in whom early screening might be appropriate.

Characterization of putative interneurons in the myenteric plexus of human colon.

BACKGROUND: The enteric nervous system contains multiple classes of neurons, distinguishable by morphology, immunohistochemical markers, and projections; however, specific combinations differ between species. Here, types of enteric neurons in human colon were characterized immunohistochemically, using retrograde tracing combined with multiple labeling immunohistochemistry, focussing on non-motor neurons. METHODS: The fluorescent carbocyanine tracer, DiI, was applied to the myenteric plexus in ex vivo preparations, filling neurons projecting within the plexus. Limits of projection lengths of motor neurons were established, allowing them to be excluded from the analysis. Long ascending and descending interneurons were then distinguished by labeling for discriminating immunohistochemical markers: calbindin, calretinin, enkephalin, 5-hydroxytryptamine, nitric oxide synthase, and substance P. These results were combined with a previous published study in which nitric oxide synthase and choline acetyltransferase immunoreactivities were established. KEY RESULTS: Long ascending neurons (with projections longer than 8 mm, which excludes more than 95% motor neurons) formed four types, in descending order of abundance, defined by immunoreactivity for: (a) ChAT+/ENK+, (b) ChAT+/ENK+/SP+, (c) ChAT+/Calb+, and (d) ChAT+/ENK+/Calb+. Long descending neurons, up to 70 mm long also formed at least four types, distinguished by immunoreactivity for (a) NOS + cells (without ChAT), (b) ChAT+/NOS+, (c) ChAT+/Calret+, and (d) ChAT+/5HT + cells (with or without NOS). CONCLUSIONS AND INFERENCES: Long interneurons, which do not innervate muscularis externa, are likely to coordinate neural activity over distances of many centimeters along the colon. Characterizing their neurochemical coding provides a basis for understanding their roles, investigating their connectivity, and building a comprehensive account of human colonic enteric neurons.

Postoperative ileus-An ongoing conundrum.

BACKGROUND: Postoperative ileus is common and is a major clinical problem. It has been widely studied in patients and in experimental models in laboratory animals. A wide variety of treatments have been tested to prevent or modify the course of this disorder. PURPOSE: This review draws together information on animal studies of ileus with studies on human patients. It summarizes some of the conceptual advances made in understanding the mechanisms that underlie paralytic ileus. The treatments that have been tested in human subjects (both pharmacological and non-pharmacological) and their efficacy are summarized and graded consistent with current clinical guidelines. The review is not intended to provide a comprehensive overview of ileus, but rather a general understanding of the major clinical problems associated with it, how animal models have been useful to elucidate key mechanisms and, finally, some perspectives from both scientists and clinicians as to how we may move forward with this debilitating yet common condition.