BACKGROUND:Luminal proteases have been implicated in epithelial barrier dysfunction and visceral hypersensitivity in irritable bowel syndrome (IBS), yet their impact on the enteric nervous system (ENS), the principal regulator of gastrointestinal function, remains unknown. OBJECTIVE:To investigate whether faecal mediators differentially activate enteric neurons across IBS subtypes and whether proteolytic and proteomic profiles explain neuronal phenotypes. DESIGN:The effects of faecal supernatants (FSN) from 21 IBS-D (diarrhoea-predominant), 9 IBS-C (constipation-predominant) and 18 healthy control (HC) patients recruited across centres in three countries on guinea pig distal colon submucous plexus neurons were assessed using a neuroimaging technique. Faecal proteolytic activities and proteomic profiles were analysed. RESULTS:IBS-D and IBS-C supernatants evoked significantly stronger neuronal activation than HC, demonstrating that FSN directly modulate ENS. In IBS-D, but not IBS-C, effects were mediated by serine and cysteine proteases and PAR-1. Proteome analysis revealed a significant difference in 47 proteins between IBS-D and HC, including several immunoglobulin components, underlying the role of microinflammation in IBS-D. A combination of amylases, trypsin-2 and an immunoglobulin protein demonstrated high diagnostic performance to distinguish IBS-D from HC. CONCLUSION:These findings uncover a previously unrecognised luminal-ENS axis in IBS and reveal fundamentally different pathological mechanisms between IBS-D and IBS-C. IBS-D is characterised by proteases and PAR-1-dependent neuronal activation and a distinct immune-enriched faecal proteome, whereas mediators in IBS-C act independently of these factors. These findings establish a functional link between faecal protease activity, ENS signalling and molecular biomarkers, highlighting new therapeutic and diagnostic avenues for subtype-specific management of IBS.

BACKGROUND:Functional dyspepsia (FD) and irritable bowel syndrome (IBS) are disorders of gut-brain interaction (DGBIs). Patients with these disorders experience abdominal symptoms, frequently in relation to meal intake, and often are treated using pharmacological approaches that offer limited symptom relief. In addition to various pharmacotherapies, established treatment options include lifestyle modifications (such as diet) and, in certain patients, psychological interventions. Because of the limitations of the currently available treatments, many patients look for alternative options, including herbal preparations. PURPOSE:In this review, we summarize the preclinical and clinical evidence informing the use of the herbal preparation, STW 5-II, for the treatment of patients with FD and IBS. Data from clinical trials provide evidence that STW 5-II is superior to placebo in offering symptom relief. Moreover, a substantial body of preclinical data on the mechanisms of action of STW 5-II suggests that its ingredients target multiple mechanisms relevant to pathophysiology and symptom generation that may underlie its beneficial clinical effects in patients with DGBIs.

Fennel tea has a region-specific effect on the stomach motility. It relaxes the fundus and the corpus and acts pro-motility in the antrum. The store operated Ca2+ entry blocker SKF-96365 hampers the spasmolytic effect of fennel tea in the fundus and corpus. The image was created using Biorender.

INTRODUCTION:The herbal preparation STW 5 ameliorates functional dyspepsia partly by relaxing smooth muscle of the proximal stomach, thus improving gastric accommodation. We explored the unknown pathways responsible for this effect by testing targets known to modulate gastric smooth muscle relaxation. METHODS:STW 5-induced relaxation of smooth muscle strips from guinea pig gastric corpus before and after pharmacological interventions were recorded with force transducers in an organ bath. ORAI1 mRNA expression was tested in the proximal stomach. KEY RESULTS:Blockade of Ca2+-activated K+ and Cl- channels, voltage-gated L- or T-type Ca2+ channels, TRPA1-, TRPV1-, adenosine or 5-HT4 receptors, antagonizing ryanodine receptors, inhibiting cyclooxygenase or sarcoplasmic reticulum calcium ATPase did not affect STW 5-evoked relaxation. Likewise, protein-kinase A or G were not involved. However, the relaxation evoked by STW 5 was significantly reduced by phorbol-12-myristat-13-acetat, an activator of protein-kinase C, by 2- aminoethyldiphenylborinate, an inhibitor of the IP3 receptor-mediated Ca2+ release from the sarcoplasmic reticulum or by SKF-96365, a nonselective store-operated calcium entry (SOCE) blocker. Furthermore, the mixed TRPC3/SOCE inhibitor Pyr3, but not the selective TRPC3 blocker Pyr10, reduced the effect of STW 5. Finally, BTP2, a potent blocker of ORAI-coupled SOCE, almost abolished STW 5-evoked relaxation. Expression of ORAI1 could be demonstrated in the corpus/fundus. CONCLUSIONS & INFERENCES:STW 5 inhibited SOCE, most likely ORAI channels, which are modulated by IP3- and PKC-dependent mechanisms. Our findings impact on the design of drugs to induce muscle relaxation and help identify phytochemicals with similar modes of actions to treat gastrointestinal disturbances.

IntroductionWe previously showed enteric nerve activation after application of colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS). The question remains whether this is a region-specific or a generalized sensitization. We tested the nerve-activating properties of supernatants from large and small intestinal regions of IBS patients with diarrhea (IBS-D) in comparison to those from mastocytosis patients with diarrhea (MC-D) or non-IBS/non-MC patients with GI-complaints. MC-D patients were included to test samples from patients with an established, severe mast cell disorder, because mast cells are suggested to play a role in IBS.MethodsVoltage-sensitive dye imaging was used to record the effects of mucosal biopsy supernatants from IBS-D, MC-D, and non-IBS/non-MC on guinea pig submucous neurons. Mast cell density and histamine concentrations were measured in all samples.ResultsThe median neuroindex (spike frequency × % responding neurons in Hz × %) was significantly (all p < 0.001) increased for IBS-D (duodenum and colon, proximal and distal each, 49.3; 50.5; 63.7; 71.9, respectively) compared to non-IBS/non-MC (duodenum and colon, proximal and distal each, 8.7; 4.9; 6.9; 5.4, respectively) or MC-D supernatants (duodenum and colon, proximal and distal each, 9.4; 11.9; 0.0; 7.9, respectively). Nerve activation by MC-D and non-IBS/non-MC supernatants was comparable (p>0.05). Mast cell density or histamine concentrations were not different between IBS-D, MC-D, and non-IBS/non-MC samples.DiscussionNerve activation by biopsy supernatants is an IBS hallmark that occurs throughout the gut, unrelated to mast cell density or histamine concentration. At least as important is our finding that GI complaints per se were not associated with biopsy supernatant-induced nerve activation, which further stresses the relevance of altered nerve behavior in IBS.

Anti-neuronal autoantibodies can lead to subacute gastrointestinal dysmotility, presenting with various symptoms typical of intestinal pseudoobstruction, achalasia, gastroparesis, or slow intestinal transit, among others. Such autoantibodies may be produced in response to a remote tumor and accelerate the diagnosis of malignancy, but in other cases they appear without an identifiable underlying cause. One example is the type I anti-neuronal nuclear antibody (ANNA-1 otherwise known as anti-Hu), which is usually linked to small cell-lung carcinoma. Anti-Hu can directly activate enteric neurons and visceral sensory nerve fibers and has a cytotoxic effect. Various other anti-neuronal antibodies have been described, targeting different ion channels or receptors on nerve cells of the central or the enteric nervous system. Autoimmune processes targeting enteric neurons may also play a role in more common disorders such as esophageal achalasia, celiac disease, or multiple sclerosis. Furthermore, anti-enteric neuronal antibodies have been found more abundant in the common functional gastrointestinal disorder, irritable bowel syndrome (IBS), than in controls. The pathogenesis of IBS is very complex, involving the release of various mediators from immune cells in the gut wall. Products of mast cells, such as histamine and tryptase, excite visceral afferents and enteric neurons, which may contribute to symptoms like abdominal pain and disturbed motility. Elevated serine- and cysteine-protease activity in stool of IBS-D and IBS-C patients, respectively, can be a factor leading to leaky gut and visceral hypersensitivity. More knowledge on these mediators in IBS may facilitate the development of novel diagnostic methods or therapies.

Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings. A well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments. The structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.

The blockade or deletion of the pro-inflammatory P2X7 receptor channel has been shown to reduce tissue damage and symptoms in models of inflammatory bowel disease, and P2X7 receptors on enteric neurons were suggested to mediate neuronal death and associated motility changes. Here, we used P2X7-specific antibodies and nanobodies, as well as a bacterial artificial chromosome transgenic P2X7-EGFP reporter mouse model and P2rx7-/- controls to perform a detailed analysis of cell type-specific P2X7 expression and possible overexpression effects in the enteric nervous system of the distal colon. In contrast to previous studies, we did not detect P2X7 in neurons but found dominant expression in glia and macrophages, which closely interact with the neurons. The overexpression of P2X7 per se did not induce significant pathological effects. Our data indicate that macrophages and/or glia account for P2X7-mediated neuronal damage in inflammatory bowel disease and provide a refined basis for the exploration of P2X7-based therapeutic strategies.

BACKGROUND:Non-allergenic, low molecular weight components of pollen grains are suspected to trigger changes in gut functions, sometimes leading to inflammatory conditions. Based on extensive neuroimmune communication in the gut wall, we investigated the effects of aqueous pollen extracts (APE) on enteric and spinal sensory neurons.METHODS:Using Ca2+ and fast potentiometric imaging, we recorded the responses of guinea-pig and human submucous and guinea-pig dorsal root ganglion (DRG) neurons to microejection of low (<3 kDa) and high (≥3 kDa) molecular weight APEs of birch, ragweed, and hazel. Histamine was determined pharmacologically and by mass spectrometry (LC-MS/MS).KEY RESULTS:Birch APE<3kDa evoked strong [Ca+2 ]i signals in the vast majority of guinea-pig DRG neurons, and in guinea-pig and human enteric neurons. The effect of birch APE≥3kDa was much weaker. Fast neuroimaging in human enteric neurons revealed an instantaneous spike discharge after microejection of birch, ragweed, and hazel APE<3kDa [median (interquartile range) at 7.0 Hz (6.2/9.8), 5.7 Hz (4.4/7.1), and 8.4 Hz (4.3/12.5), respectively]. The percentage of responding neurons per ganglion were similar [birch 40.0% (33.3/100.0), ragweed 50.8% (34.4/85.6), and hazel 83.3% (57.1/100.0)]. A mixture of histamine receptor (H1-H3) blockers significantly reduced nerve activation evoked by birch and ragweed APEs<3kDa , but was ineffective on hazel. Histamine concentrations in ragweed, birch and hazel APE's < 3 kDa were 0.764, 0.047, and 0.013 μM, respectively.CONCLUSIONS:Allergen-free APEs from birch, ragweed, and hazel evoked strong nerve activation. Altered nerve-immune signaling as a result of severe pollen exposure could be a pathophysiological feature of allergic and non-allergic gut inflammation.

It was suggested that intestinal mucosal secretion is enhanced during muscle relaxation and contraction. Mechanisms of mechanically induced secretion have been studied in rodent species. We used voltage clamp Ussing technique to investigate, in human and porcine colonic tissue, secretion evoked by serosal (P ser ) or mucosal (P muc ) pressure application (2–60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, P ser or P muc caused secretion due to Cl - and, in human colon, also HCO 3 - fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, P muc evoked larger responses than P ser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. P ser and P muc induced secretion was tetrodotoxin (TTX) sensitive in porcine colon. In human colon, a TTX sensitive component was only revealed after piroxicam. However, synaptic blockade by ω-conotoxin GVIA reduced the response to mechanical stimuli. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion. In conclusion, in both species, distension induced secretion was predominantly mediated by PGs and a rather small nerve dependent response involving mechanosensitive somata and synapses.

Abkürzungsverzeichnis AST: Aspartat-Aminotransferase ALT: Alanin-Aminotransferase BFI: bowel function index CACP: Chirurgische Arbeitsgemeinschaft für Coloproktologie CAS: constipation assessment Skala (scale) CSBM: komplette spontane Stuhlentleerung (complete spontaneous bowel movement) DEGAM: Deutsche Gesellschaft für Allgemeinmedizin und Familienmedizin DGAV: Deutsche Gesellschaft für Allgemein- und Viszeralchirugie DGEM: Deutsche Gesellschaft für Ernährungsmedizin DGG: Deutsche Gesellschaft für Geriatrie DGIM: Deutsche Gesellschaft für Innere Medizin DGK: Deutsche Gesellschaft für Koloproktologie DGNHK: Deutsche Gesellschaft für Naturheilkunde DGNM: Deutsche Gesellschaft für Neurogastroenterologie & Motilität DGP: Deutsche Gesellschaft für Palliativmedizin DGVS: Deutsche Gesellschaft für Verdauungs- und Stoffwechselkrankheiten EFSA: Europäische Behörde für Lebensmittelsicherheit 5-HT4-Antagonisten: 5-Hydroxytryptamin-Rezeptor-Antagonisten ICC: Interstitielle Cajal-Zelle GINMP: gastrointestinale neuromuskuläre Pathologien GIT: Gastrointestinaltrakt KHK: koronare Herzkrankheit MACE: Malone antegrade continence enema MAGDA: Informationsforum für Magen-Darm-Erkrankungen MRT: Magnetresonanztomographie NPV: negativer prädiktiver Wert NTC: Normaltransit-Obstipation (normal transit constipation) OD: obstruktive Defäkation ODS: obstruktives Defäkations-Syndrom OIC: Opoid-/Opiat-induzierte Obstipation (opoid-induced constipation) PAC-QOL: Patient Assessment of Constipation Quality of Life PAMORA: peripher wirksamer µ-Opioid-Rezeptorantagonist (peripherally acting µ-opioid receptor antagonist) PEG: Polyethylenglycol PNE: perkutane Nervenevaluation RDS-O: obstipationsdominantes Reizdarmsyndrom RCT: randomisiert kontrollierte Studie (randomized controlled trial) scFOS: Fructooligosaccharide SNS: sakrale Nervenstimulation STC: Obstipation mit verzögertem Colontransit (slow transit constipation) TCM: traditionelle chinesische Medizin TES: transcutaneous electrical stimulation TSH: Thyreoidea-stimulierendes Hormon SBM: spontane Stuhlentleerung (spontaneous bowel movement)

Despite numerous studies on the enteric nervous system (ENS), we lack fundamental knowledge on neuronal densities or total neuron numbers in different species. There are more anecdotal than actual figures on nerve counts.

P2X7 receptors play an important role in cytokine release and immune cell regulation. Their upregulation has been described in inflammatory and degenerative processes and P2X7 blockade or deletion has been shown to reduce tissue damage and severity of symptoms in animal models of inflammatory bowel disease (IBD). Several studies have found that P2X7 receptors are present on enteric neurons and glia and it was proposed that they mediate neuronal death during IBD. However, the cell type-specific localization of P2X7 receptors has been a matter of debate, since some antibodies have been found to be unspecific. Here we describe the preparation of whole-mount myenteric plexus from the colon of BAC transgenic P2X7-EGFP reporter mice and subsequent immunofluorescence staining of P2X7 receptors together with cell type-specific marker proteins.

BACKGROUND:Serotonin (5-HT) is an important mediator in the gastrointestinal tract, acting on different neuronal 5-HT receptors. The ionotropic 5-HT3 receptor mediates immediate but transient spike discharge in human enteric neurons. We studied the role of the metabotropic 5-HT1P , 5-HT4 , and 5-HT7 receptors to activate human submucous neurons. METHODS:Neuroimaging using the voltage sensitive dye Di-8-ANEPPS was performed in submucous plexus preparations from human surgical specimens of the small and large intestine. We synthesized a new, stable 5-HT1P agonist, 5-benzyloxyhydrazonoindalpine (5-BOHIP). KEY RESULTS:5-HT evoked a fast and late-onset spike discharge in enteric neurons. The fast component was blocked by the 5-HT3 receptor antagonist cilansetron, while the remaining sustained response was significantly reduced by the 5-HT1P receptor antagonist 5-hydroxytryptophanyl-5-hydroxytryptophan amide (5-HTP-DP). The newly synthesized 5-HT1P agonist 5-BOHIP induced a slowly developing, long-lasting activation of submucous neurons, which was blocked by 5-HTP-DP. We could not demonstrate any 5-HT7 receptor-induced spike discharge based on the lack of response to 5-carboxamidotryptamine. Similarly, the 5-HT4 agonists 5-methoxytryptamine and prucalopride evoked no immediate or late-onset spike discharge. CONCLUSIONS & INFERENCES:Our work demonstrated for the first time the presence of functional 5-HT1P receptors on human submucous neurons. Furthermore, we found no evidence for a role of 5-HT4 or 5-HT7 receptors in the postsynaptic activation of human submucous neurons by 5-HT.

Autoren Jutta Keller1, Thilo Wedel2, Holger Seidl3, Martin E. Kreis4, Ivo van der Voort5, Maximilian Gebhard6, Jost Langhorst7, Petra Lynen Jansen8, Oliver Schwandner9, Martin Storr10, Pia van Leeuwen8, Viola Andresen1, Jan C. Preiß11, Peter Layer1 Collaborators: H. Allescher, T. Andus, S. C. Bischoff, S. Buderus, M. Claßen, U. Ehlert, S. Elsenbruch, M. Engel, A. Enninger, W. Fischbach, M. Freitag, T. Frieling, A. Gillessen, M. Goebel-Stengel, J. Gschossmann, F. Gundling, S. Haag, W. Häuser, U. Helwig, S. Hollerbach, G. Holtmann, M. Karaus, M. Katschinski, H. Krammer, W. Kruis, R. Kuhlbusch-Zicklam, P. Lynen Jansen, A. Madisch, H. Matthes, S. Miehlke, H. Mönnikes, S. Müller-Lissner, B. Niesler, C. Pehl, D. Pohl, C. Posovszky, M. Raithel, G. Röhrig-Herzog, R. Schäfert, M. Schemann, A. Schmidt-Choudhury, S. Schmiedel, A. Schweinlin, J. Schwille-Kiuntke, A. Stengel, J. Tesarz, W. Voderholzer, G. von Boyen, J. von Schönfeld in Zusammenarbeit mit: Deutsche Gesellschaft für Allgemeinmedizin und Familienmedizin (DEGAM), Deutsche Gesellschaft für Allergologie und Klinische Immunologie (DGAKI), Deutsche Gesellschaft für Allgemeinund Viszeralchirurgie (DGAV), Deutsche Gesellschaft für Ernährungsmedizin (DGEM), Deutsche Gesellschaft für Geriatrie (DGG), Deutsche Gesellschaft für Innere Medizin (DGIM), Deutsche Gesellschaft für Naturheilkunde (DGNHK), Deutsche Gesellschaft für Pathologie und Bundesverband deutscher Pathologen e. V. (DGP/BDP), Deutsche Gesellschaft für Psychosomatische Medizin und Ärztliche Psychotherapie (DGPM), Deutsche Gesellschaft für Verhaltensmedizin und Verhaltensmodifikation (DGVM), Deutsche Schmerzgesellschaft e. V., Deutsches Kollegium für Psychosomatische Medizin (DKPM), Deutsche Gesellschaft für Tropenmedizin und Internationale Gesundheit (DTG), Gesellschaft für Pädiatrische Gastroenterologie und Ernährung (GPGE), Deutsche Gesellschaft für Kinderund Jugendmedizin (DGKJ), Swiss Society of Neurogastroenterology and Motility (SwissNGM), Informationsforum für Patient:innen mit Magen-Darm-Erkrankungen (MAGDA)

This protocol is to investigate the presence and localization of neurochemical markers in the submucosal and myenteric plexus of the porcine colon using immunhistochemical staining techniques.