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GOALS/BACKGROUND: Animal studies have highlighted how the microbiota acts in a sex-specific manner with sex hormones demonstrating an association with the composition and diversity of the microbiota. This systematic review aimed to gather the available scientific evidence to explore the association between sex hormones and gut microbiota composition and diversity, in humans. STUDY: Four bibliographic databases were searched in July 2020 using terms related to “microbiota”, “microflora”, “sex hormones”, “testosterone”, and “estrogen”. Human studies that investigated the correlation between sex hormones and the microbiota composition or diversity using next generation sequencing (NGS) were included. RESULTS: 10,468 records were screened with thirteen studies included in this review. In healthy women, higher estrogen levels were found to be associated with a higher abundance of Bacteroidetes, a lower abundance of Firmicutes, the Ruminococcaceae family and increased diversity. In healthy men, raised testosterone levels positively correlated with Ruminococcus, Acinetobacter, and an increased microbial diversity. Escherichia and Shigella spp. were correlated with raised testosterone in healthy women whereas Ruminococcus spp. was negatively associated with elevated testosterone levels. Women with altered testosterone/oestrogen profiles (such as in PCOS), had a differing gut microbiota compared to healthy women. CONCLUSIONS: The findings gathered highlight an association between sex hormones and the gut microbiota composition/diversity and may contribute to the sex-based variations observed in disease pathogenesis. Factors such as age and medical conditions are implicated in the associations observed and should be accounted for in future studies. As the understanding of the complex symbiotic relationship between humans and their gut microbiota increases, microbiota modulation could be an attractive option for the prevention and treatment of gastrointestinal disorders.

Monocytes are mononuclear phagocytes that can differentiate to a variety of cell fates under the influence of their microenvironment and hardwired commitment. We found that inhibition of TRPM8 in human blood CD14+ monocytes during a critical 3-h window at the beginning of their differentiation into macrophages led to enhanced survival and LPS-driven TNFα production after 24 h. TRPM8 antagonism also promoted LPS-driven TNFα production in CD14+ monocytes derived from the intestinal mucosa. Macrophages that had been derived for 6 days under blockade of TRPM8 had impaired phagocytic capacity and were transcriptionally distinct. Most of the affected genes were altered in a way that opposed normal monocyte to macrophage differentiation indicating that TRPM8 activity promotes aspects of this differentiation programme. Thus, we reveal a novel role for TRPM8 in regulating human CD14+ monocyte fate and function.

The underlying causes of heartburn, characteristic symptom of gastroesophageal reflux disease (GERD), remain incompletely understood. Superficial afferent innervation of the esophageal mucosa in nonerosive reflux disease (NERD) may drive nociceptive reflux perception, but its acid-sensing role has not yet been established. Transient receptor potential vanilloid subfamily member-1 (TRPV1), transient receptor potential melastatin 8 (TRPM8), and acid-sensing ion channel 3 (ASIC3) are regulators of sensory nerve activity and could be important reflux-sensing receptors within the esophageal mucosa. We characterized TRPV1, TRPM8, and ASIC3 expression in esophageal mucosa of patients with GERD. We studied 10 patients with NERD, 10 with erosive reflux disease (ERD), 7 with functional heartburn (FH), and 8 with Barrett's esophagus (BE). Biopsies obtained from the distal esophageal mucosa were costained with TRPV1, TRPM8, or ASIC3, and CGRP, CD45, or E-cadherin. RNA expression of

Patients with inflammatory bowel disease often experience ongoing pain even after achieving mucosal healing (i.e., post-inflammatory pain). Factors related to the brain–gut axis, such as peripheral and central sensitization, altered sympatho-vagal balance, hypothalamic–pituitary–adrenal axis activation, and psychosocial factors, play a significant role in the development of post-inflammatory pain. A comprehensive study investigating the interaction between multiple predisposing factors, including clinical psycho-physiological phenotypes, molecular mechanisms, and multi-omics data, is still needed to fully understand the complex mechanism of post-inflammatory pain. Furthermore, current treatment options are limited and new treatments consistent with the underlying pathophysiology are needed to improve clinical outcomes.

Background Chronic and recurring pain is a characteristic symptom in irritable bowel syndrome (IBS). Altered signaling between immune cells and sensory neurons within the gut may promote generation of pain symptoms. As transient receptor potential melastatin 8 (TRPM8) agonists, such as L-menthol in peppermint oil, have shown to attenuate IBS pain symptoms, we began investigating potential molecular mechanisms.Methods Colonic biopsy tissues were collected from patients with IBS and controls, in two separate cohorts. Immunohistochemistry was performed to identify TRPM8 localization. Quantitative PCR was performed to measure mucosal mRNA levels of TRPM8. In addition, functional experiments with the TRPM8 agonist icilin were performed ex vivo to examine cytokine release from biopsies. Daily diaries were collected to ascertain pain symptoms.Results In biopsy tissue from IBS patients, we showed that TRPM8 immunoreactivity is colocalized with immune cells predominantly of the dendritic cell lineage, in close approximation to nerve endings, and TRPM8 protein and mRNA expression was increased in IBS patients compared to controls (p < 0.001). TRPM8 mRNA expression showed a significant positive association with abdominal pain scores (p = 0.015). Treatment of IBS patient biopsies with icilin reduced release of inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha (p < 0.05).Conclusions and inferences These data indicate TRPM8 may have important anti-inflammatory properties and by this virtue can impact neuro-immune disease mechanisms in IBS.

Background: Receptors located on enteroendocrine cells (EECs) of the colon can detect nutrients in the lumen. These receptors regulate appetite through a variety of mechanisms, including hormonal and neuronal signals. We assessed the effect of obesity on the expression of these G-protein coupled receptors (GPCRs) and hormones at both mRNA and protein level. Methods: qPCR and immunohistochemistry were used to examine colonic tissue from cohorts of patients from the Netherlands (proximal and sigmoid tissue) and the United Kingdom (tissue from across the colon) and patients were grouped by body mass index (BMI) value (BMI &lt, 25 and BMI ≥ 25). Results: The mRNA expression of the hormones/signaling molecules serotonin, glucagon, peptide YY (PYY), CCK and somatostatin were not significantly different between BMI groups. GPR40 mRNA expression was significantly increased in sigmoid colon samples in the BMI ≥ 25 group, but not proximal colon. GPR41, GPR109a, GPR43, GPR120, GPRC6A, and CaSR mRNA expression were unaltered between low and high BMI. At the protein level, serotonin and PYY containing cell numbers were similar in high and low BMI groups. Enterochromaffin cells (EC) showed high degree of co-expression with amino acid sensing receptor, CaSR while co-expression with PYY containing L-cells was limited, regardless of BMI. Conclusions: While expression of medium/long chain fatty acid receptor GPR40 was increased in the sigmoid colon of the high BMI group, expression of other nutrient sensing GPCRs, and expression profiles of EECs involved in peripheral mechanisms of appetite regulation were unchanged. Collectively, these data suggest that in human colonic tissue, EEC and nutrient-sensing receptor expression profiles are not affected despite changes to BMI.

ObjectiveColonic enteroendocrine cells (EECs) store and release potent anorectic hormones that are key regulators of satiety. EECs express multiple nutrient sensing receptors, particularly for medium-chain fatty acids (MCFAs): GPR84 and FFAR4. Here we show a non-surgical approach with targeted colonic delivery of MCFA, which induces EEC and neuronal activation leading to anorectic effects.DesignA randomised, double-blind, placebo-controlled, cross-over study was performed in obese adults given combined GPR84 and FFAR4 agonists in colonic release capsules before meals. We measured serum hormones, energy intake and appetite perception. Cell type, activation by agonists and hormone/serotonin release were determined in human colonic explants. Mouse colonic afferent nerve responses to nutrients/mediators were recorded electrophysiologically.ResultsSubjects receiving GPR84 and FFAR4 agonists had reduced overall calorific intake and increased postprandial levels of PYY versus placebo. Receptors including GPR84 and FFAR4 were coexpressed on human colonic EEC. Activation of GPR84 exclusively induced intracellular pERK, whereas FFAR4 selectively activated pCaMKII. Coactivation of GPR84 and FFAR4 induced both phosphoproteins, and superadditive release of GLP-1 and PYY. Nutrients and hormones convergently activated murine colonic afterent nerves via GLP-1, Y2 and 5-HT3 receptors.ConclusionsColonic GPR84 and FFAR4 agonists reduce energy intake and increase postprandial PYY in obese adults. Human colonic EECs coexpress these receptors, which activate cells via parallel intracellular pathways and synergistically evoke hormone release. Further synergism occurs in sensory nerve responses to MCFA and EEC mediators. Thus, synergistic activation of colonic endocrine cells via nutrient receptors is an important target for metabolic regulation.Trail registration numberNCT04292236.

Introduction The enteric nervous system (ENS) is located throughout the length of the gastrointestinal (GI) tract as a series of interconnected ganglionated plexi. It has the unique ability to orchestrate GI behavior independently of the central nervous system. The ENS has two major cell populations: Enteric neurons and enteric glial cells. While enteric neurons regulate gut reflexes with the help of neuropeptides such as substance P, it is becoming evident that enteric glial cells also actively participate in this process via bidirectional signaling with neurons and other cells in the gut wall. Enteric glial cells are also critical for maintaining epithelial barrier function (Yu, 2014). Given the proximity of the intestinal microbiota to the ENS, it is perhaps not surprising that the function of the ENS is under partial control of gut microbes and the host immune system through complex mechanistic processes. The complex signaling between microbes and their human hosts, and the resulting effect on normal physiologic functions and GI anatomy, is still poorly understood. Given the number of bacterial species inhabiting the gut, identifying specific contributions by individual species is a major challenge. Mono-colonization of Germ Free (GF) mice allows identification of strain specific influences on GI anatomy. We set out to investigate the role of Bacteroides Thetaiotaomicron (Bt), a major and stable endosymbiont of the human gut (Wexler and Goodman, 2017), in influencing the components of the ENS. Our aims were to find out whether Bt alters; 1) Neuronal budding using neuronal marker GAP-43, 2) Glial cell density using glial cell marker S100β, and 3) Substance P expression using substance P antibody. Methods 3 mice groups were used: Germ-free (GF) N=5, Conventional (CONV) N=5 and germ-free mice recolonized with Bt (Bt-RECOL) N=5. Colonic sections from each mouse were mounted onto slides. 5 slides from each mice group were stained via indirect immunofluorescence to investigate the expression of biomarkers GAP43, S100β and substance P separately. 5 fields of view of the mucosa and myenteric plexus per slide for every marker used were observed under an epifluorescence microscope. 150 digital images per marker were captured and saved [Mucosa: GF (n=25), CONV (n=25) and Bt-RECOL (n=25); Myenteric plexus: GF (n=25), CONV (n=25) and Bt-RECOL (n=25)]. Quantitative assessment of the digital images was carried out using imageJ software. Positive staining was represented as number of pixels per image and this data was saved in MS Excel. PRISM 7 (GraphPad software) was used to calculate statistical significance. Comparison between groups was performed using One-Way ANOVA. All values are mean ± standard error of mean (SEM). p Results Neuronal budding, measured by GAP-43, displayed no significant changes in GF vs. CONV group within the mucosa (GF: 3696.96 ± 258.06 vs. CONV: 4202.63 ± 336.34 pixels/image). However, a significant increase in GAP-43 immunostaining within the myenteric plexus was observed in GF vs. CONV (GF: 11519.42 ± 1547.95 vs. CONV: 7280.97 ± 894.46 pixels/image, p = 0.0218). Bt-RECOL significantly increased mucosal GAP-43 (Bt-RECOL: 4975.71 ± 447.65 vs. GF: 3696.96 ± 258.06 pixels/image, p = 0.0169) and myenteric plexus GAP-43 immunostaining (Bt-RECOL: 13958.66 ± 1931.47 vs. CONV: 7280.97 ± 894.46 pixels/image, p = 0.0029). Glial cell density measured by S100β, was significantly reduced in the mucosa of GF mice compared to CONV mice (GF: 3031.12 ± 437.99 vs. CONV: 5508.13 ± 507.96 pixels/image, p = 0.0006). No significant difference was observed within the myenteric plexus (GF: 6562.18 ± 803.85 vs. CONV: 7919.89 ± 659.24 pixels/image). Bt-RECOL restored mucosal (GF: 3031.12 ± 437.99 vs. Bt-RECOL: 5820.71 ± 356.06 pixels/image, p = The expression of substance P was non-significant between CONV and GF mice groups within the mucosa. A significant increase in SP immunoreactivity was observed in Bt-RECOL group when compared to both GF and CONV groups (Bt-RECOL: 1629.46 ± 151.95 vs. GF: 546.96 ± 94.15 pixels/image, p Conclusion Conventionalization with Bacteroides Thetaiotaomicron (Bt) restored glial cell population in addition to increased neuronal budding. We also show that Bt enhances substance P expression. Our data shows that Bt is required for normal neuronal innervation in the colon suggesting its critical role in maintaining normal colonic function. We hypothesize that functional activity of the ENS can be regulated by Bt through direct or indirect mechanisms. Future studies on how Bt interacts with the enteric nervous system (pathway interactions and key molecules involved) can help us develop tools to greatly impact intestinal health.

Background: Nutrient-sensing receptors located on enteroendocrine (EEC) cells modulate appetite via detection of luminal contents. Colonic &lsquo, tasting&rsquo, of luminal contents may influence changes to appetite observed in obesity and after weight loss induced by bariatric surgery. We assessed the effects of obesity and gastric bypass-induced weight loss on expression of nutrient-sensing G-protein coupled receptors (GPCRs), EEC and enterochromaffin (EC) cells and mucosal innervation. Methods: qPCR and immunohistochemistry were used to study colonic tissue from (a) chow-fed/lean, (b) high-fat fed/obese, (c) Roux-en-Y gastric bypass surgery (RYGB), and (d) calorie restriction-induced weight loss mice. Results: Expression of GPR41, GPR43, GPR40, GPR120, GPR84, GPR119, GPR93 and T1R3 was increased in obese mice. Obesity-induced overexpression of GPR41, 40, 84, and 119 further increased after RYGB whereas GPR120 and T1R3 decreased. RYGB increased TGR5 expression. L-cells, but not EC cells, were increased after RYGB. No differences in mucosal innervation by protein gene product (PGP) 9.5 and GLP-1R-positive nerve fibers were observed. Stimulation of colonic mucosa with GPR41, GPR40, GPR85, GPR119, and TGR5 agonists increased cell activation marker expression. Conclusions: Several nutrient-sensing receptors induced activation of colonic EEC. Profound adaptive changes to the expression of these receptors occur in response to diet and weight loss induced by RYGB or calorie restriction.

Key points Tenascin X (TNX) functions in the extracellular matrix of skin and joints where it maintains correct intercellular connections and tissue architectureTNX is associated exclusively with vagal‐afferent endings and some myenteric neurones in mouse and human stomach, respectively.TNX‐deficient mice have accelerated gastric emptying and hypersensitivity of gastric vagal mechanoreceptors that can be normalized by an inhibitor of vagal‐afferent sensitivity.Cultured nodose ganglion neurones showed no changes in response to capsaicin, cholecystokinin and potassium chloride in TNX‐deficient mice.TNX‐deficient patients have upper gastric dysfunction consistent with those in a mouse model. Our translational studies suggest that abnormal gastric sensory function may explain the upper gut symptoms present in TNX deficient patients, thus making it important to study gastric physiology.TNX deficiency should be evaluated routinely in patients with connective tissue abnormalities, which will enable a better understanding of its role and allow targeted treatment. For example, inhibitors of vagal afferents‐baclofen could be beneficial in patients. These hypotheses need confirmation via targeted clinical trials. Abstract Tenascin‐X (TNX) is a glycoprotein that regulates tissue structure via anti‐adhesive interactions with collagen in the extracellular matrix. TNX deficiency causes a phenotype similar to hypermobility Ehlers–Danlos syndrome involving joint hypermobility, skin hyperelasticity, pain and gastrointestinal dysfunction. Previously, we have shown that TNX is required for neural control of the bowel by a specific subtype of mainly cholinergic enteric neurones and regulates sprouting and sensitivity of nociceptive sensory endings in mouse colon. These findings correlate with symptoms shown by TNX‐deficient patients and mice. We aimed to identify whether TNX is similarly present in neural structures found in mouse and human gastric tissue. We then determined whether TNX has a functional role, specifically in gastric motor and sensory function and nodose ganglia neurones. We report that TNX was present in calretinin‐immunoreactive extrinsic nerve endings in mouse and human stomach. TNX deficient mice had accelerated gastric emptying and markedly increased vagal afferent responses to gastric distension that could be rescued with GABAB receptor agonist. There were no changes in nodose ganglia excitability in TNX deficient mice, suggesting that vagal afferent responses are probably the result of altered peripheral mechanosensitivity. In TNXB‐deficient patients, significantly greater symptoms of reflux, indigestion and abdominal pain were reported. In the present study, we report the first role for TNX in gastric function. Further studies are required in TNX deficient patients to determine whether symptoms can be relieved using GABAB agonists., Key points Tenascin X (TNX) functions in the extracellular matrix of skin and joints where it maintains correct intercellular connections and tissue architectureTNX is associated exclusively with vagal‐afferent endings and some myenteric neurones in mouse and human stomach, respectively.TNX‐deficient mice have accelerated gastric emptying and hypersensitivity of gastric vagal mechanoreceptors that can be normalized by an inhibitor of vagal‐afferent sensitivity.Cultured nodose ganglion neurones showed no changes in response to capsaicin, cholecystokinin and potassium chloride in TNX‐deficient mice.TNX‐deficient patients have upper gastric dysfunction consistent with those in a mouse model. Our translational studies suggest that abnormal gastric sensory function may explain the upper gut symptoms present in TNX deficient patients, thus making it important to study gastric physiology.TNX deficiency should be evaluated routinely in patients with connective tissue abnormalities, which will enable a better understanding of its role and allow targeted treatment. For example, inhibitors of vagal afferents‐baclofen could be beneficial in patients. These hypotheses need confirmation via targeted clinical trials.

IntroductionDiet may modulate immune responses in health and disease. We hypothesised a role for nutrient sensing pathways. Nutrient sensing enteroendocrine cells (EEC) within intestinal epithelium release local neuroendocrine peptides.1 We examined their effects on immune responses in intestinal mu

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus ( P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis.

Background Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The KV7 family (KV7.1–KV7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral KV7 channels to visceral nociception. Results Immunohistochemical staining of mouse colon revealed labelling of KV7 subtypes (KV7.3 and KV7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the KV7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B2 bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0–80 mmHg) in a concentration-dependent manner, whereas the KV7 blocker XE991 potentiated such responses. In human bowel tissues, KV7.3 and KV7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. Conclusions We show that KV7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted KV7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.

Little is known about the causes of heartburn in patients with gastro-esophageal reflux disease. Visible epithelial damage is seldom associated with symptom severity, evidenced by the significant symptom burden in patients with nonerosive reflux disease (NERD) compared with patients with erosive reflux disease (ERD) or Barrett's esophagus (BE). We studied the distribution of mucosal nerve fibers in patients with NERD, ERD, and BE, and compared the results with those of healthy subjects.We performed a prospective study of 13 patients with NERD, 11 patients with ERD, and 16 patients with BE undergoing endoscopic evaluation in the United Kingdom or Greece. Biopsies were obtained from the proximal and distal esophageal mucosa of patients with NERD, from the distal esophageal mucosa of patients with ERD, and the distal-most squamous epithelium of patients with BE. These were examined for the presence and location of nerve fibers that reacted with a labeled antibody against calcitonin gene-related peptide (CGRP), a marker of nociceptive sensory nerves. The results were compared with those from 10 healthy volunteers (controls).The distribution of CGRP-positive nerves did not differ significantly between the distal esophageal mucosa of controls (median, 25.5 cell layers to surface; interquartile range [IQR], 21.4-28.8) vs patients with ERD (median, 23 cell layers to surface; IQR, 16-27.5), or patients with BE (median, 21.5 cell layers to surface; IQR, 16.1-27.5). However, CGRP-positive nerves were significantly more superficial in mucosa from patients with NERD-both distal (median, 9.5 cell layers to surface; IQR, 1.5-13.3; P.0001 vs ERD, BE, and controls) and proximal (median, 5.0 cell layers to surface; IQR, 2.5-9.3 vs median 10.4 cell layers to surface; IQR, 8.0-16.9; P = .0098 vs controls).Proximal and distal esophageal mucosa of patients with NERD have more superficial afferent nerves compared with controls or patients with ERD or BE. Acid hypersensitivity in patients with NERD might be partially explained by the increased proximity of their afferent nerves to the esophageal lumen, and therefore greater exposure to noxious substances in refluxate.

Interest in pharmacological treatments for obesity that act in the brain to reduce appetite has increased exponentially over recent years, but failures of clinical trials and withdrawals due to adverse effects have so far precluded any success. Treatments that do not act within the brain are, in contrast, a neglected area of research and development. This is despite the fact that a vast wealth of molecular mechanisms exists within the gut epithelium and vagal afferent system that could be manipulated to increase satiety. Here we discuss mechano- and chemosensory pathways from the gut involved in appetite suppression, and distinguish between gastric and intestinal vagal afferent pathways in terms of their basic physiology and activation by enteroendocrine factors. Gastric bypass surgery makes use of this system by exposing areas of the intestine to greater nutrient loads resulting in greater satiety hormone release and reduced food intake. A non-surgical approach to this system is preferable for many reasons. This review details where the opportunities may lie for such approaches by describing nutrient-sensing mechanisms throughout the gastrointestinal tract.

Conditions characterised by chronic visceral pain represent a significant healthcare burden with limited treatment options. While animal models have provided insights into potential mechanisms of visceral nociception and identified candidate drug targets, these have not translated into successful treatments in humans.To develop an in vitro afferent nerve preparation using surgically excised freshly isolated human colon and vermiform appendix-mesentery tissues.Non-inflamed appendix (n=18) and colon (n=9) were collected from patients undergoing right and left hemicolectomy. Electrophysiological recordings were made from mesenteric nerves and the tissue stimulated chemically and mechanically.Ongoing neuronal activity was sparse and where units occurred peak firing rates were: colon (2.0±0.4 spikes/s, n=4) and appendix (2.4±0.6 spikes/s, n=9). Afferent nerves innervating the appendix responded with a significant increase in activity following stimulation with inflammatory mediators (73±10.6 vs 3.0±0.3 spikes/s, n=6, p0.001, inflammatory mediator vs baseline) and capsaicin (63±15.8 vs 2±0.3 spikes/s, n=3, p0.001, capsaicin vs buffer). Afferent nerves innervating the colon responded with increased activity to blunt probing of the serosal surface.This first-in-human study demonstrates afferent nerve recordings from human gut tissue ex vivo and shows that tissue may be stimulated both chemically and mechanically to study neuronal responses. Collectively, the results provide preliminary evidence to validate this in vitro human tissue model as one that may aid future disease mechanistic studies and candidate drug testing.

Objective The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation. Design Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses. Results Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (−20.8±6.9%, n=6, p

Objective Inhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that respond to dietary sugars, amino acids and fatty acids. Bypass surgery for obesity and type II diabetes works by shunting nutrients to the distal gut, where it increases activation of nutrient receptors and mediator release, but cellular mechanisms of activation are largely unknown. We determined which nutrient receptors are expressed in which gut regions and in which cells in mouse and human, how they are associated with different types of EEC, how they are activated leading to hormone and 5-HT release. Design and results mRNA expression of 17 nutrient receptors and EEC mediators was assessed by quantitative PCR and found throughout mouse and human gut epithelium. Many species similarities emerged, in particular the dense expression of several receptors in the distal gut. Immunolabelling showed specific colocalisation of receptors with EEC mediators PYY and GLP-1 (L-cells) or 5-HT (enterochromaffin cells). We exposed isolated proximal colonic mucosa to specific nutrients, which recruited signalling pathways within specific EEC extracellular receptor-regulated kinase (p-ERK) and calmodulin kinase II (pCAMKII), as shown by subsequent immunolabelling, and activated release of these mediators. Aromatic amino acids activated both pathways in mouse, but in humans they induced only pCAMKII, which was colocalised mainly with 5-HT expression. Activation was pertussis toxin-sensitive. Fatty acid (C12) potently activated p-ERK in human in all EEC types and evoked potent release of all three mediators. Conclusions Specific nutrient receptors associate with distinct activation pathways within EEC. These may provide discrete, complementary pharmacological targets for intervention in obesity and type II diabetes.

This work was supported by an EFIC-Grunenthal grant awarded to Madusha Peiris, CAG/CIHR/CCFR Fellowship to David E Reed, Rosetrees Postdoctoral Grant (A1296) to James RF Hockley/Ewan St. John Smith, Medical Research Council Grant (G0900907) to David C Bulmer and a Wellcome Trust University Award to L Ashley Blackshaw.

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the μ opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the μ opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the μ opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for finetuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

TRPM8 is the molecular sensor for cold; however, the physiological role of TRPM8+ neurons at mucosal surfaces is unclear. Here we evaluated the distribution and peptidergic properties of TRPM8+ fibers in naive and inflamed colons, as well as their role in mucosal inflammation. We found that Trpm8(-/-) mice were hypersusceptible to dextran sodium sulfate (DSS)-induced colitis, and that Trpm8(-/-) CD11c+ DCs (dendritic cells) showed hyperinflammatory responses to toll-like receptor (TLR) stimulation. This was phenocopied in calcitonin gene-related peptide (CGRP) receptor-deficient mice, but not in substance P receptor-deficient mice, suggesting a functional link between TRPM8 and CGRP. The DSS phenotype of CGRP receptor-deficient mice could be adoptively transferred to wild-type (WT) mice, suggesting that CGRP suppresses the colitogenic activity of bone marrow-derived cells. TRPM8+ mucosal fibers expressed CGRP in human and mouse colon. Furthermore, neuronal CGRP contents were increased in colons from naive and DSS-treated Trpm8(-/-) mice, suggesting deficient CGRP release in the absence of TRPM8 triggering. Finally, treatment of Trpm8(-/-) mice with CGRP reversed their hyperinflammatory phenotype. These results suggest that TRPM8 signaling in mucosal sensory neurons is indispensable for the regulation of innate inflammatory responses via the neuropeptide CGRP.

Multiple sclerosis (MS) and its different forms are studied in the animal model experimental autoimmune encephalomyelitis (EAE). Relapsing-remitting MS, the most common form of the disease can be induced in mice where clinical symptoms fluctuate in severity over time. However, the animal model does not experience periods of recovery where clinical signs are absent, unlike the human disease. We have developed a novel model of relapsing-remitting EAE in C57BL/6 mice immunised with myelin oligodendrocyte glycoprotein (MOG) peptide and Quil A as adjuvant. These animals have relapses that are followed by periods of recovery, during which time the animals do not exhibit illness. Furthermore, administration of the PPARgamma agonist pioglitazone prior to a predicted relapse prevents the expected development of symptoms in a dose-dependent fashion. Immune cell infiltration into white matter of the CNS and decreased production of inflammatory cytokine IFN-gamma in treated animals were also observed. Our model will be a valuable tool in assessing intervention therapies for RR-MS sufferers.

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G A A b st ra ct s in inflamed colons compared to WT + 2% DSS mice suggesting greater degree of vasculogenesis under inflammatory conditions. Conclusion: These results show that lymphatic dysregulation through genetic ablation of FOXC2 significantly exacerbates the onset, progression and severity of experimental IBD. These data suggest that alterations in lymphatic patterning and valve formation (under FOXC2 control) may contribute to IBD progression. Although abnormal lymphatic patterning is not clearly linked with IBD development, therapies which preserve postnatal valve integrity may help to resist gut injury or recover gut integrity in IBD associated gut injury. With lymphatics playing such an important potential role in the pathophysiology of IBD, research focusing on factors interfering with lymphatic damage and therapy to restore lymphatic functioning may open new doors for treatment modalities targeting this area. This work supported by Department of Defense Grant ‘Lymphatic Vascular Based Therapy in IBD' (W81XWH-11-1-0577)

Background: Despite the importance of gastric inhibitory polypeptide (GIP) as a physiological incretin, postprandial circulating glucose levels in GIP receptor null (GIPR-/-) and wild-type (wt) mice are similar. However, unlike wt mice, GIPR-/mice do not develop diet-induced obesity, suggesting a potential role for GIPR antagonists in the treatment of obesity. While specific GIPR antagonists, such as N-terminal truncated peptides and peptides with a Glu to Pro substitution at position 3 (Pro3-GIP), have been identified, their short circulating T1/2 has hampered their development. We hypothesize that the T1/2 of these antagonists can be increased significantly by fusing them to the Fc-fragment of IgG, a strategy that has been employed previously for this purpose. Aim: To develop long-acting antagonists to GIPR by fusion to the IgG Fc-fragment. Methods: PCR and molecular cloning were used to produce chimeric genes encoding GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG. After expression in HEK 293 cells, protein A agarose chromatography was used to purify protein from conditioned media. Fc-fusions were identified by Western analysis using a specific GIP antibody, and biological activity was assessed in the presence and absence of 1 nM synthetic GIP(1-42) using a GIP-specific reporter cell line. Results: Transgenic HEK 293 cells produced protein with the predicted molecular size for the Fc fusion and that possessed GIP-specific immunoreactivity. Purified Pro3-GIP-Fc-IgG induced reporter expression with an EC50 of 0.5 μM, whereas GIP(6-42)-Fc-IgG did not. Both GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG antagonized report gene expression induced by 1 nM GIP, with IC50 values of 4 μM and 20 μM, respectively. Summary and Conclusion: Pro3-GIP-Fc-IgG, but not GIP(6-42)-FcIgG, induced reporter gene expression, while both fusion products inhibited reporter gene expression induced by 1 nM GIP, indicating that the former is a partial agonist, while only the latter functions as an antagonist. The IC50 values for GIP(6-42)-Fc-IgG and Pro3-GIPFc-IgG were higher than the corresponding values reported for synthetic GIP(6-42) and Pro3-(GIP), indicating the possibility of partial interference of ligand binding by the Fc fragment. Nevertheless, the increase in T1/2 imparted by the fusion of N-terminal truncated GIP peptides the Fc fragment may thereby provide viable candidates for treating obesity and related disorders.

Introduction Increasing evidence from animal studies show that apical nutrient sensing receptors, expressed in gut enteroendocrine cells, play a key role in the release of satiety peptides {1,2} . Early human studies indicate a similar expression pattern of these receptors and role in peptide release {3} . In this study the anatomical relationship between amino acid sensing (CaSR), carbohydrate sensing (T1R1), and short chain fatty acid sensing (GPR43) receptors and appetite regulating peptides GLP-1, PYY, 5-HT was investigated in the human gut. Methods Healthy full thickness human gut sections were incubated with primary and fluorescent secondary antibodies and they were viewed under the fluoroscopic microscope to investigate co-localization of the CaSR, T1R1 and GPR43 with the GLP1, PYY and 5HT. Results The co-localization rate between CaSR and PYY, GLP1 and 5HT was 0%, Conclusion CaSR is expressed at protein level and is colocalized with PYY, 5HT and GLP1 in the human antrum, terminal ileum and ascending colon. T1R1 expression at protein level is very limited in all the tested tissues. GPR43 expression was not observed. The results suggest that CaSR is linked to PYY, GLP1 and 5HT release in the human gut, with data being stronger for the 5HT release. Disclosure of Interest None Declared References Sternini, et al. 2008. Enteroendocrine cells: a site of ‘taste’ in gastro- intestinal chemosensing. Curr Opin Endocrinol Diabetes Obes 15(1): 73–78. Frenchet et al. 2000. The effects of intestinal infusion of long-chain fatty acids on food intake in humans. Gastroenterology 2000; 119: 943–8. Page et al. 2012. Peripheral neural targets in obesity. British Journal of Pharmacology, 1568-RC.R1.