Probabilistic tsunami forecasting for early warning.

Tsunami warning centres face the challenging task of rapidly forecasting tsunami threat immediately after an earthquake, when there is high uncertainty due to data deficiency. Here we introduce Probabilistic Tsunami Forecasting (PTF) for tsunami early warning. PTF explicitly treats data- and forecast-uncertainties, enabling alert level definitions according to any predefined level of conservatism, which is connected to the average balance of missed-vs-false-alarms. Impact forecasts and resulting recommendations become progressively less uncertain as new data become available. Here we report an implementation for near-source early warning and test it systematically by hindcasting the great 2010 M8.8 Maule (Chile) and the well-studied 2003 M6.8 Zemmouri-Boumerdes (Algeria) tsunamis, as well as all the Mediterranean earthquakes that triggered alert messages at the Italian Tsunami Warning Centre since its inception in 2015, demonstrating forecasting accuracy over a wide range of magnitudes and earthquake types.

Expression of RAD21 immunoreactivity in myenteric neurons of the human and mouse small intestine.

BACKGROUND: RAD21 is a double-strand-break repair protein and component of the cohesin complex with key roles in cellular functions. A RAD21 loss-of-function mutation was found in cases of chronic intestinal pseudo-obstruction (CIPO) with associated enteric neuronal loss. Analysis of RAD21 expression in the enteric nervous system is lacking, thus we aimed to characterize RAD21 immunoreactivity (IR) in myenteric ganglia. METHODS: Double labeling immunofluorescence in mouse and human jejunum was used to determine colocalization of RAD21 with HuC/D, PGP9.5, neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), choline acetyl transferase (ChAT), Kit, platelet-derived growth factor receptor-α (PDGFRα), and glial fibrillary acid protein (GFAP) IRs. RESULTS: A subset of PGP9.5- and HuC/D-IR neuronal cell bodies and nerve fibers in the myenteric plexus of human and mouse small intestine also displayed cytoplasmic RAD21-IR Cytoplasmic RAD21-IR was found in 43% of HuC/D-IR neurons in adult and neonatal mice but did not colocalize with nNOS. A subset of ChAT-positive neurons had cytoplasmic RAD21-IR Punctate RAD21-IR was restricted to the nucleus in most cell types consistent with labeling of the cohesin complex. Cytoplasmic RAD21-IR was not detected in interstitial cells of Cajal, fibroblast-like cells or glia. Subsets of neurons in primary culture exhibited cytoplasmic RAD21-IR Suppression of RAD21 expression by shRNA knockdown abolished RAD21-IR in cultured neurons. CONCLUSIONS: Our data showing cytoplasmic RAD21 expression in enteric neurons provide a basis toward understanding how mutations of this gene may contribute to altered neuronal function/survival thus leading to gut-motor abnormalities.

High temporal resolution gastric emptying breath tests in mice.

BACKGROUND: Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. METHODS: We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [13 C]-octanoic acid and off axis- integrated cavity output spectroscopy (OA-ICOS). Stretched gamma variate and 2-component stretched gamma variate models fit measured breath excretion data. KEY RESULTS: These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin-induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two-phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two-phase gastric emptying curves. A greater proportion of two-phase gastric emptying was induced in Balb/c mice with the administration of PYY (8-80 nmol) 60 min following meal ingestion. CONCLUSIONS AND INFERENCES: Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two-phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying.

Diabetic and idiopathic gastroparesis is associated with loss of CD206-positive macrophages in the gastric antrum.

BACKGROUND: Animal studies have increasingly highlighted the role of macrophages in the development of delayed gastric emptying. However, their role in the pathophysiology of human gastroparesis is unclear. Our aim was to determine changes in macrophages and other cell types in the gastric antrum muscularis propria of patients with diabetic and idiopathic gastroparesis. METHODS: Full thickness gastric antrum biopsies were obtained from patients enrolled in the Gastroparesis Clinical Research Consortium (11 diabetic, 6 idiopathic) and 5 controls. Immunolabeling and quantitative assessment was done for interstitial cells of Cajal (ICC) (Kit), enteric nerves protein gene product 9.5, neuronal nitric oxide synthase, vasoactive intestinal peptide, substance P, tyrosine hydroxylase), overall immune cells (CD45) and anti-inflammatory macrophages (CD206). Gastric emptying was assessed using nuclear medicine scintigraphy and symptom severity using the Gastroparesis Cardinal Symptom Index. RESULTS: Both diabetic and idiopathic gastroparesis patients showed loss of ICC as compared to controls (Mean [standard error of mean]/hpf: diabetic, 2.28 [0.16]; idiopathic, 2.53 [0.47]; controls, 6.05 [0.62]; P=.004). Overall immune cell population (CD45) was unchanged but there was a loss of anti-inflammatory macrophages (CD206) in circular muscle (diabetic, 3.87 [0.32]; idiopathic, 4.16 [0.52]; controls, 6.59 [1.09]; P=.04) and myenteric plexus (diabetic, 3.83 [0.27]; idiopathic, 3.59 [0.68]; controls, 7.46 [0.51]; P=.004). There was correlation between the number of ICC and CD206-positive cells (r=.55, P=.008). Enteric nerves (PGP9.5) were unchanged: diabetic, 33.64 (3.45); idiopathic, 41.26 (6.40); controls, 46.80 (6.04). CONCLUSION: Loss of antral CD206-positive anti-inflammatory macrophages is a key feature in human gastroparesis and it is associates with ICC loss.

Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.

BACKGROUND: Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. METHODS: Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow-derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high-powered field. KEY RESULTS: Bone marrow-derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT-PCR. Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2-CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1-CM, including tumor necrosis factor alpha (TNF-α). ELISA detected 0.697±0.03 ng mL-1 TNF-α in M1-CM. Recombinant mouse TNF-α reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50 = 0.817 ng mL-1 ). Blocking M1-CM TNF-α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z-VAD.fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z-VAD.fmk, n=6, P<.05). CONCLUSIONS & INFERENCES: This work demonstrates that TNF-α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase-dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis.

Effects of hemin on heme oxygenase-1, gastric emptying, and symptoms in diabetic gastroparesis.

BACKGROUND: Therapeutic options for management of diabetic gastroparesis are limited. Failure to maintain upregulation of heme oxygenase (HO1) leads to loss of interstitial cells of Cajal and delayed gastric emptying (GE) in non-obese diabetic mice. Our hypothesis was that hemin upregulation of HO1 would restore normal GE in humans with gastroparesis. AIMS: To compare effects of hemin and placebo infusions on HO1 activity and protein, GE, autonomic function, and gastrointestinal symptoms in diabetic gastroparesis. METHODS: In a single-center, double-blind, placebo-controlled, randomized clinical trial, we compared intravenous hemin, prepared in albumin, or albumin alone (placebo) in 20 patients, aged 41 ± 5 (SEM) years with diabetic gastroparesis. After infusions on days 1, 3, and 7, weekly infusions were administered for 7 additional weeks. Assessments included blood tests for HO1 protein and enzyme activity levels, GE with 13 C-spirulina breath test, autonomic functions (baseline and end), and gastrointestinal symptoms every 2 weeks. KEY RESULTS: Nine of 11 patients randomized to hemin completed all study procedures. Compared to placebo, hemin increased HO1 protein on days 3 (p = 0.0002) and 7 (p = 0.008) and HO1 activity on day 3 (p = 0.0003) but not after. Gastric emptying, autonomic functions, and symptoms did not differ significantly in the hemin group relative to placebo. CONCLUSIONS & INFERENCES: Hemin failed to sustain increased HO1 levels beyond a week and did not improve GE or symptoms in diabetic gastroparesis. Further studies are necessary to ascertain whether more frequent hemin infusions or other drugs would have a more sustained effect on HO1 and improve GE.

Expression and function of the Scn5a-encoded voltage-gated sodium channel NaV 1.5 in the rat jejunum.

BACKGROUND: The SCN5A-encoded voltage-gated sodium channel NaV 1.5 is expressed in human jejunum and colon. Mutations in NaV 1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage-gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown. METHODS: The presence and distribution of Scn5a-encoded NaV 1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral-mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT-PCR. KEY RESULTS: We found NaV 1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies of different portions of NaV 1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage-clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage-dependent inward current that was eliminated by Na(+) replacement by NMDG(+) . Constitutive rat Scn5a knockout resulted in death in utero. NaV 1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half-width, and 7 mV hyperpolarization of the membrane potential at peak amplitude. CONCLUSIONS & INFERENCES: Scn5a-encoded NaV 1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology.

Changes in nitrergic and tachykininergic pathways in rat proximal colon in response to chronic treatment with otilonium bromide.

BACKGROUND: Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. METHODS: Adult Sprague-Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. KEY RESULTS: Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. CONCLUSIONS & INFERENCES: Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

Macrophages in diabetic gastroparesis--the missing link?

BACKGROUND: Diabetic gastroparesis results in significant morbidity for patients and major economic burden for society. Treatment options for diabetic gastroparesis are currently directed at symptom control rather than the underlying disease and are limited. The pathophysiology of diabetic gastroparesis includes damage to intrinsic and extrinsic neurons, smooth muscle, and interstitial cells of Cajal (ICC). Oxidative damage in diabetes appears to be one of the primary insults involved in the pathogenesis of several complications of diabetes, including gastroparesis. Recent studies have highlighted the potential role of macrophages as key cellular elements in the pathogenesis of diabetic gastroparesis. Macrophages are important for both homeostasis and defense against a variety of pathogens. Heme oxygenase 1 (HO1), an enzyme expressed in a subset of macrophages has emerged as a major protective mechanism against oxidative stress. Activation of macrophages with high levels of HO1 expression protects against development of delayed gastric emptying in animal models of diabetes, while activation of macrophages that do not express HO1 are linked to neuromuscular cell injury. Targeting macrophages and HO1 may therefore be a therapeutic option in diabetic gastroparesis. PURPOSE: This report briefly reviews the pathophysiology of diabetic gastroparesis with a focus on oxidative damage and how activation and polarization of different subtypes of macrophages in the muscularis propria determines development of delay in gastric emptying or protects against its development.

Association of low numbers of CD206-positive cells with loss of ICC in the gastric body of patients with diabetic gastroparesis.

BACKGROUND: There is increasing evidence for specific cellular changes in the stomach of patients with diabetic (DG) and idiopathic (IG) gastroparesis. The most significant findings are loss of interstitial cells of Cajal (ICC), neuronal abnormalities, and an immune cellular infiltrate. Studies done in diabetic mice have shown a cytoprotective effect of CD206+ M2 macrophages. To quantify overall immune cellular infiltrate, identify macrophage populations, and quantify CD206+ and iNOS+ cells. To investigate associations between cellular phenotypes and ICC. METHODS: Full thickness gastric body biopsies were obtained from non-diabetic controls (C), diabetic controls (DC), DG, and IG patients. Sections were labeled for CD45, CD206, Kit, iNOS, and putative human macrophage markers (HAM56, CD68, and EMR1). Immunoreactive cells were quantified from the circular muscle layer. KEY RESULTS: Significantly fewer ICC were detected in DG and IG tissues, but there were no differences in the numbers of cells immunoreactive for other markers between patient groups. There was a significant correlation between the number of CD206+ cells and ICC in DG and DC patients, but not in C and IG and a significant correlation between iNOS+ cells and ICC in the DC group, but not the other groups. CD68 and HAM56 reliably labeled the same cell populations, but EMR1 labeled other cell types. CONCLUSIONS & INFERENCES: Depletion of ICC and correlation with changes in CD206+ cell numbers in DC and DG patients suggests that in humans, like mice, CD206+ macrophages may play a cytoprotective role in diabetes. These findings may lead to novel therapeutic options, targeting alternatively activated macrophages.

Review article: carbon monoxide in gastrointestinal physiology and its potential in therapeutics.

BACKGROUND: While carbon monoxide (CO) is a known toxin, it is now recognised that CO is also an important signalling molecule involved in physiology and pathophysiology. AIMS: To summarise our current understanding of the role of endogenous CO in the regulation of gastrointestinal physiology and pathophysiology, and to potential therapeutic applications of modulating CO. METHODS: This review is based on a comprehensive search of the Ovid Medline comprehensive database and supplemented by our ongoing studies evaluating the role of CO in gastrointestinal physiology and pathophysiology. RESULTS: Carbon monoxide derived from haem oxygenase (HO)-2 is predominantly involved in neuromodulation and in setting the smooth muscle membrane potential, while CO derived from HO-1 has anti-inflammatory and antioxidative properties, which protect gastrointestinal smooth muscle from damage caused by injury or inflammation. Exogenous CO is being explored as a therapeutic agent in a variety of gastrointestinal disorders, including diabetic gastroparesis, post-operative ileus, organ transplantation, inflammatory bowel disease and sepsis. However, identifying the appropriate mechanism for safely delivering CO in humans is a major challenge. CONCLUSIONS: Carbon monoxide is an important regulator of gastrointestinal function and protects the gastrointestinal tract against noxious injury. CO is a promising therapeutic target in conditions associated with gastrointestinal injury and inflammation. Elucidating the mechanisms by which CO works and developing safe CO delivery mechanisms are necessary to refine therapeutic strategies.

Distribution of TMEM100 in the mouse and human gastrointestinal tract--a novel marker of enteric nerves.

Identification of markers of enteric neurons has contributed substantially to our understanding of the development, normal physiology, and pathology of the gut. Previously identified markers of the enteric nervous system can be used to label all or most neuronal structures or for examining individual cells by labeling just the nucleus or cell body. Most of these markers are excellent but have some limitations. Transmembrane protein 100 (TMEM100) is a gene at locus 17q32 encoding a 134-amino acid protein with two hypothetical transmembrane domains. TMEM100 expression has not been reported in adult mammalian tissues but does appear in the ventral neural tube of embryonic mice and plays a role in signaling pathways associated with development of the enteric nervous system. We showed that TMEM100 messenger RNA is expressed in the gastrointestinal tract and demonstrated that TMEM100 is a membrane-associated protein. Furthermore TMEM100 immunoreactivity was restricted to enteric neurons and vascular tissue in the muscularis propria of all regions of the mouse and human gastrointestinal tract. TMEM100 immunoreactivity colocalized with labeling for the pan-neuronal marker protein gene product 9.5 (PGP9.5) but not with the glial marker S100ß or Kit, a marker of interstitial cells of Cajal. The signaling molecule, bone morphogenetic protein (BMP) 4, was also expressed in enteric neurons of the human colon and co-localized with TMEM100. TMEM100 is also expressed in neuronal cell bodies and fibers in the mouse brain and dorsal root ganglia. We conclude that TMEM100 is a novel, membrane-associated marker for enteric nerves and is as effective as PGP9.5 for identifying neuronal structures in the gastrointestinal tract. The expression of TMEM100 in the enteric nervous system may reflect a role in the development and differentiation of cells through a transforming growth factor ß, BMP or related signaling pathway.

Gastrointestinal hypomotility with loss of enteric nicotinic acetylcholine receptors: active immunization model in mice.

BACKGROUND: Autoimmune gastrointestinal dysmotility (AGID) is a limited form of dysautonomia. The only proven effector to date is IgG specific for ganglionic nicotinic-acetylcholine receptors containing α3 subunits [α3*- nicotinic acetylcholine receptor (nAChR)]. Rabbits immunized with recombinant α3-polypeptide produce α3*-nAChR autoantibodies, and profound AGID ensues. Human and rabbit α3*-nAChR-specific-IgGs induce transient hypomotility when injected into mice. Here, we describe success and problems encountered inducing gastrointestinal hypomotility in mice by active immunization. METHODS: We repeatedly injected young adult mice of seven different strains susceptible to autoimmunity (spontaneous diabetes or neural antigen immunization-induced myasthenia gravis or encephalomyelitis) with: (i) α3-polypeptide, intradermally or (ii) live α3*-nAChR-expressing xenogeneic cells, intraperitoneally. We measured serum α3*-nAChR-IgG twice monthly, and terminally assessed blue dye gastrointestinal transit, total small intestinal α3*-nAChR content (radiochemically) and myenteric plexus neuron numbers (immunohistochemically, ileal-jejunal whole-mount preparations). KEY RESULTS: Standard cutaneous inoculation with α3-polypeptide was minimally immunogenic, regardless of dose. Intraperitoneally injected live cells were potently immunogenic. Self-reactive α3*-nAChR-IgG was induced only by rodent immunogen; small intestinal transit slowing and enteric α3*-nAChR loss required high serum levels. Ganglionic neurons were not lost. CONCLUSIONS & INFERENCES: Autoimmune gastrointestinal dysmotility is inducible in mice by active immunization. Accompanying enteric α3*-nAChR reduction without neuronal death is consistent with an IgG-mediated rather than T cell-mediated pathogenesis, as is improvement of symptoms in patients receiving antibody-depleting therapies.

Immunoreactivity for Ano1 detects depletion of Kit-positive interstitial cells of Cajal in patients with slow transit constipation.

BACKGROUND: Depletion of interstitial cells of Cajal (ICC) is associated with several gastrointestinal (GI) motility disorders. Changes in ICC networks are usually detected by immunolabeling for the receptor tyrosine kinase Kit. Ano1 (DOG1 or TMEM16A) was recently described as a marker of ICC in GI tract. Our aim was to determine whether Ano1 immunoreactivity can be used as a reliable marker for ICC in tissues from patients with motility disorders. METHODS: Four tissues from patients with normal ICC numbers and four tissues from patients with slow transit constipation and loss of Kit-positive ICC were studied. Interstitial cells of Cajal were detected by double labeling using antisera to Kit and Ano1. KEY RESULTS: Both the processes and cell bodies of ICC in tissue from controls and slow transit constipation were immunoreactive for Ano1. There was a near complete overlap between Kit and Ano1 immunoreactivity. Tissues from patients with slow transit constipation contained significantly fewer Ano1-positive ICC than control tissues. The numbers of ICC identified by Ano1 and Kit immunoreactivity were nearly identical across the range of ICC numbers from an average of 1.64 to 7.05 cells per field and correlated with an R(2) value of 0.99. CONCLUSIONS & INFERENCES: Ano1 is a reliable and sensitive marker for detecting changes in ICC networks in humans. Labeling with antibodies selective for Ano1 reproducibly detects depletion of Kit-positive ICC in tissues from patients with slow transit constipation.

Changes in interstitial cells of cajal with age in the human stomach and colon.

BACKGROUND: Aging produces inevitable changes in the function of most organs including the gastrointestinal tract. Together with enteric nerves and smooth muscle cells, interstitial cells of Cajal (ICC) play a key role in the control of gastrointestinal motility, yet little is known about the effect of aging on ICC. The aim of this study was to determine the effect of aging on ICC number and volume in the human stomach and colon. METHODS: Gastric and colonic tissues from patients aged 25-70 and 36-92 years old, respectively, and with no co-existent motility disorders were immunolabeled with an anti-Kit antibody and ICC were counted in the circular muscle and myenteric regions. Network volumes were measured using 3D reconstructions of confocal stacks. The effects of aging were determined by testing for linear trends using regression analysis. KEY RESULTS: In both stomach and colon, the number of ICC bodies and volume significantly decreased with age at a rate of 13% per decade. ICC size was only affected in the myenteric plexus in the colon. The changes associated with age were not differentially affected by sex or colonic region. CONCLUSIONS & INFERENCES: The number and volume of ICC networks in the normal human stomach and colon decline with age. This decrease in ICC likely reduces the functional capacity of the gastrointestinal motor apparatus, may contribute to changes in gastrointestinal motility with aging and may influence intestinal responses to insults such as disease, operative interventions and medications in older patients. Tissue specimens must be carefully age-matched when studying ICC in disease.

Lack of serotonin 5-HT2B receptor alters proliferation and network volume of interstitial cells of Cajal in vivo.

BACKGROUND: Normal gastrointestinal motility requires intact networks of interstitial cells of Cajal (ICC). Interstitial cells of Cajal numbers are maintained by a balance between cell loss factors and survival/trophic/growth factors. Activation of 5-HT(2B) receptors expressed on ICC increases ICC proliferation in vitro. It is not known whether 5-HT(2B) receptors on ICC are activated in vivo. The aims of this study were to investigate if adult ICC proliferate, whether the proliferation of ICC in vivo is affected by knocking out the 5-HT(2B) receptor, and if alterations in proliferation affect ICC networks. METHODS: Proliferating ICC were identified by immunoreactivity for Ki67 in both the myenteric and deep muscular plexus regions of the jejunum in mice with a targeted insertion of a neomycin resistance cassette into the second coding exon of the htr2b receptor gene. KEY RESULTS: Adult ICC do proliferate. The number of proliferating ICC was lower in the myenteric plexus region of Htr2b(-/-) compared to Htr2b(+/+) mice. The volume of Kit-positive ICC was 30% lower in the myenteric plexus region and 40% lower in the deep muscular plexus region in Htr2b(-/-) mice where the number of ICC was also reduced. CONCLUSIONS & INFERENCES: Interstitial cells of Cajal proliferate in adult mice and activation of 5-HT(2B) receptors results in increased proliferation of ICC in vivo. Furthermore, lack of 5-HT(2B) receptor signaling reduces the density of ICC networks in mature mice. These data suggest that 5-HT(2B) receptor signaling is required for maintenance of ICC networks, adding 5-HT to the growing number of factors shown to regulate ICC networks.

First-in-human study demonstrating pharmacological activation of heme oxygenase-1 in humans.

Heme oxygenase (HO)-1 degrades heme and protects against oxidative stress, but it has not been pharmacologically induced in humans. In this randomized study of 10 healthy volunteers, hemin (3 mg/kg intravenously in 25% albumin) was shown to increase plasma HO-1 protein concentration four- to fivefold and HO-1 activity ~15-fold relative to baseline at 24 and 48 h (placebo -56.41 +/- 6.31 (baseline), 69.79 +/- 13.00 (24 h), 77.44 +/- 10.62 (48 h) vs. hemin -71.70 +/- 9.20 (baseline), 1,126.20 +/- 293.30 (24 h), 1,192.20 +/- 333.30 (48 h)) in four of five subjects as compared with albumin alone (P

Cellular pathogenesis of diabetic gastroenteropathy.

Gastroenteropathy manifesting in upper gastrointestinal symptoms, delayed gastric emptying, constipation, diarrhea and fecal incontinence occurs frequently in patients with diabetes mellitus and represents a significant health care burden. Current treatments are largely symptomatic and ineffective. Better understanding of the cellular and molecular pathogenesis of these disorders is required for the development of more effective therapies. Recent advances in our understanding of the inherent, high-level complexities of the control systems that execute and regulate gastrointestinal motility, together with the utilization of new experimental models and sophisticated physiological, morphological and molecular techniques have lead to the realization that diabetic gastroenteropathies cannot be ascribed to any singular defect or dysfunction. In fact, these disorders are multifactorial and involve a spectrum of metabolic and dystrophic changes that can potentially affect all key components of motor control including the systemic autonomic and enteric nervous systems, interstitial cells of Cajal and smooth muscle cells. Candidate pathomechanisms are also varied and include imbalance between pro- and anti-oxidative factors, altered trophic stimuli to mature cells and their progenitors, and, possibly, autoimmune factors. The goal of this paper is to review the cellular changes underlying diabetic gastroenteropathies and their potential causes, with particular focus on functional interactions between various cell types. It is proposed that diabetic gastroenteropathies should be considered a form of gastrointestinal neuromuscular dystrophy rather than a "functional" disorder. Future research should identify ways to block cytotoxic factors, support the regeneration of damaged cells and translate the experimental findings into new treatment modalities.

Effect of age on the enteric nervous system of the human colon.

The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age-related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33-99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu-positive neurons declined with age (slope = -1.3 neurons/mm/10 years, P = 0.03). The number of ChAT-positive neurons also declined with age (slope = -1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS-positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = -0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS-positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.

Apoptotic cell death of human interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are specialized mesenchyme-derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c-Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase-3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double-labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 +/- 0.2% of ICC were positive for activated caspase-3 and in the circular muscle layer 2.1 +/- 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.