Correction to: Piezo proteins: incidence and abundance in the enteric nervous system. Is there a link with mechanosensitivity?

The Authors regret forgetting in the original version of this article to mention that this work was also supported by the US National Institute of Health (NIH) (1OT2OD024899-01).

Piezo proteins: incidence and abundance in the enteric nervous system. Is there a link with mechanosensitivity?

Piezo channels play fundamental roles in many physiological processes. Their presence and functional role in the enteric nervous system is still not known. We hypothesize that they play a role in mechanotransduction in enteric neurons. Our aims are to quantify the presence of both Piezo1 and 2 in enteric neurons throughout the gastrointestinal tract using immunohistochemistry and analyze their function(s) using neuroimaging techniques and pharmacological investigations. In order to perform a systematic and comparative study, we performed our experiments in gastrointestinal tissue from guinea pigs, mice and humans. Piezo1 (20-70%) is expressed by both enteric neuronal cell bodies and fibers in the myenteric and submucosal plexi of all the species investigated. Generally, Piezo1 expressing somata are more numerous in the submucosal plexus (50-80%) than in the myenteric plexus (15-35%) apart from the stomach where Piezo1 is expressed in up to 60% of cell bodies. Myenteric Piezo1 neurons mainly (60-100%) but not exclusively, also express nitric oxide synthase, a minority express choline acetyltransferase. In the submucosal plexus, Piezo1 neurons co-express vasoactive intestinal peptide (40-90%). Conversely, expression of Piezo2 is extremely rare in the somata of enteric neurons and is present in few neurites. In functional experiments, 38-76% of the mechanosensitive neurons expressed Piezo1 channels. Statistical analysis showed a positive significant correlation between mechanosensitive and Piezo1 positive neurons. However, pharmacological experiments using an activator and an inhibitor of Piezo channels did not demonstrate changes in mechanotransduction. A major role of Piezo1 in the mechanosensitivity of enteric neurons can be excluded.

Neural influences on human intestinal epithelium in vitro.

KEY POINTS: We present the first systematic and, up to now, most comprehensive evaluation of the basic features of epithelial functions, such as basal and nerve-evoked secretion, as well as tissue resistance, in over 2200 surgical specimens of human small and large intestine. We found no evidence for impaired nerve-evoked epithelial secretion or tissue resistance with age or disease pathologies (stomach, pancreas or colon cancer, polyps, diverticulitis, stoma reversal). This indicates the validity of future studies on epithelial secretion or resistance that are based on data from a variety of surgical specimens. ACh mainly mediated nerve-evoked and basal secretion in the small intestine, whereas vasoactive intestinal peptide and nitric oxide were the primary pro-secretory transmitters in the large intestine. The results of the present study revealed novel insights into regional differences in nerve-mediated secretion in the human intestine and comprise the basis by which to more specifically target impaired epithelial functions in the diseased gut. ABSTRACT: Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R-basal) and short circuit currents (ISC; secretion) under basal conditions (ISC-basal) and after electrical field stimulation (ISC-EFS) of nerves in 2221 resectates from 435 patients. ISC-EFS was TTX-sensitive and of comparable magnitude in the small and large intestine. ISC-EFS or R-basal were not influenced by the patients' age, sex or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC-EFS depended on epithelial cAMP-driven chloride and bicarbonate secretion but not on amiloride-sensitive sodium absorption. Although atropine-sensitive cholinergic components prevailed for ISC-EFS of the duodenum, jejunum and ileum, PG97-269-sensitive [vasoactive intestinal peptide (VIP) receptor 1 antagonist] VIPergic together with L-NAME-sensitive nitrergic components dominated the ISC-EFS in colonic preparations. Differences in numbers of cholinergic or VIPergic neurons, sensitivity of epithelial muscarinic or VIP receptors, or stimulus frequency-dependent transmitter release were not responsible for the region-specific transmitter contribution to ISC-EFS. Instead, the low atropine-sensitivity of ISC-EFS in the colon was the result of high cholinesterase activity because neostigmine revealed cholinergic components. Colonic ISC-EFS remained unchanged after tachykinin, P2X, P2Y or A1 and A2 receptor blockade. R-basal was smaller and ISC-basal was higher in the small intestine. TTX and bumetanide decreased ISC-basal in all regions, suggesting nerve-dependent secretory tone. ISC-basal was atropine-sensitive in the small intestine and PG97-269-sensitive in the large intestine. This comprehensive study reveals novel insights into region-specific nerve-mediated secretion in the human small and large intestine.

Multicenter double-blinded randomized controlled trial of standard abdominal wound edge protection with surgical dressings versus coverage with a sterile circular polyethylene drape for prevention of surgical site infections: a CHIR-Net trial (BaFO; NCT01181206).

OBJECTIVE: To determine whether circular plastic wound edge protectors (CWEPs) significantly reduce the rate of surgical site infections (SSIs) in comparison to standard surgical towels in patients undergoing laparotomy. BACKGROUND: SSIs cause substantial morbidity, prolonged hospitalization, and costs and remain one of the most frequent surgical complications. CWEPs have been proposed as a measure to reduce the incidence of SSIs. METHODS: In this randomized controlled, multicenter, 2-arm, parallel-group design, patient- and observer-blinded trial patients undergoing open elective abdominal surgery were assigned to either intraoperative wound coverage with a CWEP or standard coverage with surgical towels. Primary endpoint was superiority of intervention over control in terms of the incidence of SSIs within a 30-day postoperative period. RESULTS: Between September 2010 and November 2012, 608 patients undergoing laparotomy were randomized at 16 centers across Germany. Three patients in the device group and 11 patients in the control group did not undergo laparotomy. Patients' and procedural characteristics were well balanced between the 2 groups. Forty-eight patients discontinued the study prematurely, mainly because of relaparotomy (control, n=9; intervention, n=9) and death (control, n=4; intervention, n=7). A total of 79 patients experienced SSIs within 30 days of surgery, 27 of 274 (9.9%) in the device group and 52 of 272 (19.1%) in the control group (odds ratio=0.462, 95% confidence interval: 0.281-0.762; P=0.002). Subgroup analyses indicate that the effect could be more pronounced in colorectal surgery, and in clean-contaminated/contaminated surgeries. CONCLUSIONS: Our trial shows that CWEPs are effective at reducing the incidence of SSIs in elective and clean or clean-contaminated open abdominal surgery.

Allergen-free extracts from birch, ragweed, and hazel pollen activate human and guinea-pig submucous and spinal sensory neurons.

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.

Distension evoked mucosal secretion in human and porcine colon in vitro.

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 (Pser) or mucosal (Pmuc) pressure application (2-60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, Pser or Pmuc caused secretion due to Cl- and, in human colon, also HCO3- fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, Pmuc evoked larger responses than Pser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. Pser and Pmuc 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.

Activity of protease-activated receptors in the human submucous plexus.

BACKGROUND & AIMS: Protease-activated receptors (PARs) are expressed in the enteric nervous system. Excessive release of proteases has been reported in functional and inflammatory bowel diseases. Studies in several animal models indicate the involvement of neural PARs. We studied the actions of different PAR-activating peptides (AP) in the human submucous plexus and performed comparative studies in guinea pig submucous neurons. METHODS: We used voltage- and calcium-sensitive dye recordings to study the effects of PAR1-AP, PAR2-AP, PAR4-AP, the PAR1 activator thrombin, and the PAR2 activator tryptase on neurons and glia in human and guinea pig submucous plexus. Human preparations were derived from surgical resections. Levels of mucosal secretion evoked by PAR-APs were measured in Ussing chambers. RESULTS: PAR1-AP and thrombin evoked a prominent spike discharge and intracellular Ca(2+) concentration ([Ca](i)) transients in most human submucous neurons and glia. PAR2-AP, tryptase, and PAR4-AP caused significantly weaker responses in a minor population. In contrast, PAR2-AP evoked much stronger responses in enteric neurons and glia of guinea pigs than did PAR1-AP or PAR4-AP. PAR1-AP, but not PAR2-AP or PAR4-AP, evoked a nerve-mediated secretion in human epithelium. The PAR1 antagonist SCH79797 inhibited the PAR1-AP, and thrombin evoked responses on neurons, glia, and epithelial secretion. In the submucous layer of human intestine, but not guinea pig intestine, PAR2-AP evoked [Ca](i) signals in CD68(+) macrophages. CONCLUSIONS: In the human submucous plexus, PAR1, rather than PAR2 or PAR4, activates nerves and glia. These findings indicate that PAR1 should be the focus of future studies on neural PAR-mediated actions in the human intestine; PAR1 might be developed as a therapeutic target for gastrointestinal disorders associated with increased levels of proteases.

How big is the little brain in the gut? Neuronal numbers in the enteric nervous system of mice, Guinea pig, and human.

BACKGROUND: 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. METHODS: We used standardized preparation techniques and immunohistochemistry with validated panneuronal markers (human or mouse anti-HuD/C) to determine neuronal densities in specimen from the entire gastrointestinal tract of mice, guinea pig, and humans. In parallel, we measured the dimensions of the gastrointestinal regions in mouse and guinea pig. For humans, we had to rely on literature data. KEY RESULTS: The average neuronal densities along the gastrointestinal tract were 35,011 ± 25,017 1/cm2 for the myenteric and 16,685 ± 9098 1/cm2 for the submucous plexus in mice, 24,315 ± 16,627 and 11,850 ± 6122 1/cm2 for guinea pig myenteric and submucous plexus, respectively, and 21,698 ± 9492 and 16,367 ± 5655 1/cm2 for human myenteric and submucous plexus, respectively. The total number of neurons in the ENS was 2.6 million for mice, 14.6 million for guinea pig, and 168 million for human. CONCLUSIONS & INFERENCES: This study reports the first comprehensive nerve cell count in mice, guinea pig, and human ENS. Neuronal densities were comparable between the three species and the differences in the total numbers of enteric neurons are likely due to body size and intestinal length. The number of enteric neurons is comparable to the number of neurons in the spinal cord for all three species.

Metabotropic 5-HT receptor-mediated effects in the human submucous plexus.

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.

The beta3-adrenoceptor agonist GW427353 (Solabegron) decreases excitability of human enteric neurons via release of somatostatin.

BACKGROUND & AIMS: beta3 Adrenoceptor (beta3-AR) is expressed on adipocytes and enteric neurons. GW427353 is a human selective beta3-AR agonist with visceral analgesic effects. Some of its effects may involve release of somatostatin (SST) and actions on enteric neurons. The aim of this study was to investigate the mode of action of GW427353 in human submucous neurons. METHODS: Voltage sensitive dye imaging was used to record from human submucous neurons. SST release from human primary adipocytes was measured with enzyme-linked immunoabsorbent assay. Immunohistochemistry was used to detect adiponectin, beta3-AR, SST, SST2 receptors, tyrosine hydroxylase (TH), and protein gene product 9.5. RESULTS: Confocal imaging showed cytoplasmic beta3-AR labeling in somata of submucous neurons and nerve varicosities. GW427353 had no direct postsynaptic actions but decreased fast synaptic input to submucous neurons. Tissue perfusion with GW427353 reduced nicotine-evoked neuronal spike frequency, an effect prevented by the beta3-AR antagonist SR-59230 and the SST2-receptor antagonist CYN154806 and mimicked by the SST2 receptor agonist octreotide. Adipocytes expressed adiponectin, beta3-AR, and SST. TH-positive fibers were in close proximity to adipocytes. Submucous neurons expressed SST2 receptors. Human primary adipocytes released SST in response to GW427353 in a concentration-dependent manner, an effect abolished by SR-59230. CONCLUSIONS: Inhibitory action of GW427353 involves release of SST which stimulates inhibitory SST2 receptors on human submucous neurons. Adipocytes are a potential source for SST. beta3-AR activation may be a promising approach to reduce enteric neuron hyperexcitability. The action of GW427353 may be the neurophysiologic correlate of its beneficial effect in patients with irritable bowel syndrome.

Fast synaptic excitatory neurotransmission in the human submucosal plexus.

BACKGROUND: Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. METHODS: We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. KEY RESULTS: Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect. CONCLUSION & INFERENCE: Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

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.

Higher-order processing and change-to-automaticity as explanations of the item-position effect in reasoning tests.

Higher-order processing and learning are two alternative explanations of the item-position effect. Whereas higher-order processing as explanation emphasizes the recruitment of executive processes, learning as explanation highlights the improvement in performance due to repetition and is specified as change-to-automaticity. In a sample of 287 participants the item-position effect was captured by means of Advanced Progressive Matrices. Higher-order processing was inferred from rule acquisition, and change-to-automaticity was derived from sustained attention data. The results of independent investigations revealed that both higher-order processing and change-to-automaticity contributed to the occurrence of the item-position effect.

Compression and stretch sensitive submucosal neurons of the porcine and human colon.

The pig is commonly believed to be a relevant model for human gut functions-however, there are only a few comparative studies and none on neural control mechanisms. To address this lack we identified as one central aspect mechanosensitive enteric neurons (MEN) in porcine and human colon. We used neuroimaging techniques to record responses to tensile or compressive forces in submucous neurons. Compression and stretch caused Ca-transients and immediate spike discharge in 5-11% of porcine and 15-24% of human enteric neurons. The majority of these MEN exclusively responded to either stimulus quality but about 9% responded to both. Most of the MEN expressed choline acetyltransferase and substance P; nitric oxide synthase-positive MEN primarily occurred in distal colon. The findings reveal common features of MEN in human and pig colon which we interpret as a result of species-independent evolutionary conservation rather than a specific functional proximity between the two species.

Peppermint and caraway oils have muscle inhibitory and pro-secretory activity in the human intestine in vitro.

BACKGROUND: Herbal medicinal products with a broad activity spectrum may be promising alternatives to treat functional gastrointestinal disorders (FGD). Menthacarin® is a drug with a fixed combination of peppermint and caraway oils, which is clinically used to treat FGD-associated symptoms. MATERIALS: We studied the effects of peppermint and caraway oils on contractile and secretory activity in 255 human small and large intestinal preparations derived from surgical resections (73 patients). Motility was recorded in circular smooth muscle strips and secretion with the Ussing chamber-voltage clamp technique. Electrical field stimulation evoked nerve induced contractile responses. KEY RESULTS: Peppermint and caraway oil concentrations dependently inhibited muscle contractility as indicated by sustained muscle relaxation and decrease in phasic contractility. These effects occurred in small and large intestinal preparations with IC50 values ranging between 17 and 90 µg/mL for peppermint oil and between 7 and 127 µg/mL for caraway oil. Neither peppermint nor caraway oil influenced the nerve evoked contractile response. The inhibition of contractile activity, but not the muscle relaxation, was prevented by the L-type calcium channel activator Bay K8644 but not by the neurotoxin tetrodotoxin. Both peppermint oil and caraway oil increased epithelial secretion, which remained in tetrodotoxin. CONCLUSION & INTERFERENCE: The findings revealed a strong muscle inhibitory and pro-secretory action of peppermint and caraway oils at clinically relevant concentrations. Both actions were nerve-independent. The inhibition of contractility was mediated by inhibition of L-type calcium channels. The effects on muscle and epithelial activity may contribute to the beneficial effects observed in patients with FGD.

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.

Do Adaptive Representations of the Item-Position Effect in APM Improve Model Fit? A Simulation Study.

The item-position effect describes how an item's position within a test, that is, the number of previous completed items, affects the response to this item. Previously, this effect was represented by constraints reflecting simple courses, for example, a linear increase. Due to the inflexibility of these representations our aim was to examine whether adapted representations are more appropriate than the existing ones. Models of confirmatory factor analysis were used for testing the different representations. Analyses were conducted by means of simulated data that followed the covariance pattern of Raven's Advanced Progressive Matrices (APM) items. Since the item-position effect has been demonstrated repeatedly for the APM, it is a very suitable measure for our investigations. Results revealed no remarkable improvement by using an adapted representation. Possible reasons causing these results are discussed.

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine.

Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations. Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F) in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%). Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39) peaking 90 s (64/144) after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9). Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS) in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15) in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1-3. Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare.

Anti-Hu antibodies activate enteric and sensory neurons.

IgG of type 1 anti-neuronal nuclear antibody (ANNA-1, anti-Hu) specificity is a serological marker of paraneoplastic neurological autoimmunity (including enteric/autonomic) usually related to small-cell lung carcinoma. We show here that IgG isolated from such sera and also affinity-purified anti-HuD label enteric neurons and cause an immediate spike discharge in enteric and visceral sensory neurons. Both labelling and activation of enteric neurons was prevented by preincubation with the HuD antigen. Activation of enteric neurons was inhibited by the nicotinic receptor antagonists hexamethonium and dihydro-ß-erythroidine and reduced by the P2X antagonist pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid (PPADS) but not by the 5-HT3 antagonist tropisetron or the N-type Ca-channel blocker ω-Conotoxin GVIA. Ca++ imaging experiments confirmed activation of enteric neurons but not enteric glia. These findings demonstrate a direct excitatory action of ANNA-1, in particular anti-HuD, on visceral sensory and enteric neurons, which involves nicotinic and P2X receptors. The results provide evidence for a novel link between nerve activation and symptom generation in patients with antibody-mediated gut dysfunction.

Extracts from peppermint leaves, lemon balm leaves and in particular angelica roots mimic the pro-secretory action of the herbal preparation STW 5 in the human intestine.

AIM: The herbal preparation STW 5 contains fresh plant extracts from bitter candytuft whole plant, extracts from greater celandine herb, angelica root, lemon balm leaves, peppermint leaves, caraway fruit, liquorice root, chamomile flower and milk thistle fruit. We recently reported that STW 5 increased intestinal chloride secretion and proposed that this action may be involved in its clinical efficacy in the treatment of irritable bowel syndrome. The aim of this study was to identify the extracts responsible for the secretory action in order to provide the basis to develop novel target oriented herbal combinations. METHODS: We used the Ussing chamber voltage clamp technique to study the effects of individual extracts of STW 5 on short circuit current (Isc, reflecting electrogenic ion transport across epithelial cells) in mucosal/submucosal preparations of human small or large intestinal specimens and the human epithelial cell line T84. RESULTS: STW 5 at concentrations of 512 µg/ml and 5120 µg/ml evoked an increase in Isc. The increase at the lower concentration was due to pro-secretory effects of angelica which were nerve mediated. The increase at the higher concentration was additionally mimicked by peppermint and lemon balm. The remaining extracts did not influence ISC in the large intestine. The results were similar in T84 cells except that angelica had no effect while chamomile induced secretion. These pro-secretory effects were reduced by adenylate cyclase inhibitor MDL-12330A, cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 and calcium activated chloride channels blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS). Liquorice decreased ISC only in small intestine which was reversed by the epithelial sodium channel blocker amiloride. CONCLUSIONS: Results suggested that the pro-secretory action of STW 5 is mainly due to angelica with lesser contribution of peppermint and lemon balm. Their effects involve activation of cAMP- and Ca(++)-activated Cl(-) channels. We suggest that peppermint, lemon balm and in particular angelica may be the basis to develop novel herbal preparations to specifically treat secretory disorder based on impaired epithelial secretion, such as constipation.