Biopsy samples from patients with irritable bowel syndrome, but not from those with mastocytosis or unspecific gastrointestinal complaints reveal unique nerve activation in all gut regions independent of mast cell density, histamine content or specific gastrointestinal symptoms.

Introduction: We 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. Methods: Voltage-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. Results: The 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. Discussion: Nerve 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.

Clinical outcomes in patients receiving edoxaban or phenprocoumon for prevention of stroke in atrial fibrillation: a German real-world cohort study.

BACKGROUND: Appropriate and timely anticoagulant therapy with vitamin K antagonists (VKAs) or non-vitamin K oral antagonists (NOACs) is essential for stroke prevention in non-valvular atrial fibrillation (NVAF). Comparative data regarding effectiveness and safety for edoxaban vs phenprocoumon, the predominant VKA in Germany, are scarce. OBJECTIVES: The study evaluates effectiveness and safety of edoxaban vs phenprocoumon in NVAF patients in a German real-world setting. METHODS: German statutory health insurance claims data of the Institute for Applied Health Research Berlin (InGef) Research Database from 2014 until 2019 were analyzed. In NVAF patients, new users of edoxaban and phenprocoumon were compared to assess effectiveness (stroke/systemic embolism (SE)) and safety (bleeding) during therapy. Hazard ratios (HR) were estimated through multiple outcome-specific cox proportional hazard models adjusting for baseline characteristics. Outcomes of geriatric patients were analyzed in subgroup analyses. RESULTS: Between 2015 and 2018, 7,975 and 13,319 NVAF patients newly initiated treatment with edoxaban or phenprocoumon. After adjusting for baseline confounders, the risk of stroke/SE (HR: 0.85, 95% CI: 0.70-1.02) was numerically but not significantly lower, while the risk of major bleeding (HR: 0.69, 95% CI: 0.58-0.81) was significantly lower for edoxaban. In the geriatric subgroups, homogenous results compared to the main analysis were obtained. CONCLUSION: The results of this real-world analysis indicated better effectiveness and safety outcomes in patients with NVAF initiating edoxaban treatment compared to phenprocoumon. The findings confirm that the beneficial effects observed in the pivotal ENGAGE AF-TMI 48 trial can also be achieved in real-world use of edoxaban.

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

BACKGROUND & AIMS: The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). METHOD: Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. RESULTS: Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. CONCLUSION: Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS.

Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity.

Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca(2+) dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca(2+) transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca(2+)]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca(2+)]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatants.

Recordings from human myenteric neurons using voltage-sensitive dyes.

Voltage-sensitive dye (VSD) imaging became a powerful tool to detect neural activity in the enteric nervous system, including its routine use in submucous neurons in freshly dissected human tissue. However, VSD imaging of human myenteric neurons remained a challenge because of limited visibility of the ganglia and dye accessibility. We describe a protocol to apply VSD for recordings of human myenteric neurons in freshly dissected tissue and myenteric neurons in primary cultures. VSD imaging of guinea-pig myenteric neurons was used for reference. Electrical stimulation of interganglionic fiber tracts and exogenous application of nicotine or elevated KCl solution was used to evoke action potentials. Bath application of the VSDs Annine-6Plus, Di-4-ANEPPS, Di-8-ANEPPQ, Di-4-ANEPPDHQ or Di-8-ANEPPS revealed no neural signals in human tissue although most of these VSD worked in guinea-pig tissue. Unlike methylene blue and FM1-43, 4-Di-2-ASP did not influence spike discharge and was used in human tissue to visualize myenteric ganglia as a prerequisite for targeted intraganglionic VSD application. Of all VSDs, only intraganglionic injection of Di-8-ANEPPS by a volume controlled injector revealed neuronal signals in human tissue. Signal-to-noise ratio increased by addition of dipicrylamine to Di-8-ANEPPS (0.98±0.16 vs. 2.4±0.62). Establishing VSD imaging in primary cultures of human myenteric neurons led to a further improvement of signal-to-noise ratio. This allowed us to routinely record spike discharge after nicotine application. The described protocol enabled reliable VSD recordings from human myenteric neurons but may also be relevant for the use of other fluorescent dyes in human tissues.

Activation of human enteric neurons by supernatants of colonic biopsy specimens from patients with irritable bowel syndrome.

BACKGROUND & AIMS: Pathological features in irritable bowel syndrome (IBS) include alterations in mucosal cell content and mediator release that might alter signaling to nearby submucosal neurons. METHODS: Voltage sensitive dye imaging was used to record the effects of mediators, released from mucosal biopsies of IBS patients, on cell bodies of 1207 submucosal neurons from 76 human colonic tissue specimens. Supernatants, containing these mediators, were collected following incubation with colonic mucosal biopsies from 7 patients with diarrhea-predominant IBS (D-IBS), 4 with constipation-predominant IBS (C-IBS), and 4 healthy controls. Serotonin, histamine and tryptase concentrations in supernatants and lamina propria mast cell density were determined. RESULTS: In contrast to controls, IBS supernatants significantly increased the rate of spike discharge in 58% of human submucosal neurons. Neurons that responded to IBS supernatant had a median spike frequency of 2.4 Hz compared to 0 Hz for control supernatants. Supernatants from C-IBS and D-IBS evoked similar spike discharge. The activation induced by IBS supernatants was inhibited by histamine receptor (H1-H3) antagonists, 5-HT3 receptor antagonist, and protease inhibition. Serotonin, histamine and tryptase levels in supernatants correlated with the spike discharge induced by the supernatants. Mast cells density as well as histamine and tryptase levels in supernatants were higher in IBS than in controls. CONCLUSIONS: Mediators released from mucosal biopsies of IBS patients can activate human submucosal neurons. The activation required histamine, serotonin and proteases but was not associated with IBS subtype. Altered signaling between mucosa and the enteric nervous system might be involved in IBS pathogenesis.

Characterization of voltage-dependent sodium and calcium channels in mouse pancreatic A- and B-cells.

Insulin and glucagon are the major hormones of the islets of Langerhans that are stored and released from the B- and A-cells, respectively. Both hormones are secreted when the intracellular cytosolic Ca2+ concentration ([Ca2+]i) increases. The [Ca2+]i is modulated by mutual inhibition and activation of different voltage-gated ion channels. The precise interplay of these ion channels in either glucagon or insulin release is unknown, owing in part to the difficulties in distinguishing A- from B-cells in electrophysiological experiments. We have established a single-cell RT-PCR method to identify A- and B-cells from the mouse. A combination of PCR, RT-PCR, electrophysiology and pharmacology enabled us to characterize the different sodium and calcium channels in mouse islet cells. In both A- and B-cells, 60% of the inward calcium current (I(Ca)) is carried by L-type calcium channels. In B-cells, the predominant calcium channel is Ca(v)1.2, whereas Ca(v)1.2 and Ca(v)1.3 were identified in A-cells. These results were confirmed by using mice carrying A- or B-cell-specific inactivation of the Ca(v)1.2 gene. In B-cells, the remaining I(Ca) flows in equal amounts through Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3. In A-cells, 30 and 15% of I(Ca) is due to Ca(v)2.3 and Ca(v)2.1 activity, respectively, whereas Ca(v)2.2 current was not found in these cells. Low-voltage-activated T-type calcium channels could not be identified in A- and B-cells. Instead, two TTX-sensitive sodium currents were found: an early inactivating and a residual current. The residual current was only recovered in a subpopulation of B-cells. A putative genetic background for these currents is Na(v)1.7.