Selective matrix metalloproteinase inhibition increases breaking strength and reduces anastomotic leakage in experimentally obstructed colon.

PURPOSE: Colonic obstruction causes loss of collagen and impairment of anastomotic integrity by matrix metalloproteinases (MMPs). Unexpectedly, pharmacological MMP inhibition increased anastomotic leakage (AL) in obstructed colon possibly due to the non-selective nature of these compounds and the experimental model applied. We therefore studied the effects of selective MMP inhibition on the healing of anastomoses in colon obstructed by a novel laparoscopic technique. METHODS: Left colon was obstructed in 38 male Sprague-Dawley rats (226-284 g). After 12 h, stenoses were resected and end-to-end anastomoses constructed. Baseline breaking strength was determined in 6 animals on day 0. The remaining 32 rats were randomized to daily treatment with the selective MMP-8, MMP-9, and MMP-12 inhibitor AZD3342 (n = 16) or vehicle (n = 16). On day 3, anastomoses were evaluated for AL and breaking strength. Isolated anastomotic wound tissue was analyzed on total collagen and pepsin-insoluble and pepsin-soluble collagen by hydroxyproline. The soluble collagens were further differentiated into native, measured by Sircol, and fragmented forms. RESULTS: Baseline breaking strength was maintained with AZD3342 but decreased by 25% (P = 0.023) in the vehicle group. The anastomotic breaking strength of AZD3342-treated rats was 44% higher (P = 0.008) than the vehicle-treated rats. Furthermore, the AL rate was reduced (P = 0.037) with AZD3342 compared with vehicle treatment. AZD3342 treatment influenced neither the total or insoluble collagen concentrations nor the degree of fragmentation of the soluble collagen triple helices. CONCLUSION: Selective MMP inhibition increased anastomotic breaking strength and reduced AL after resection of colonic obstruction.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor.

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Roux-en-Y gastric bypass: hyperamylasemia is associated with small bowel obstruction.

BACKGROUND: Small bowel obstruction after Roux-en-Y gastric bypass (RYGB) can be difficult to diagnose, but usually requires surgical treatment; clinical presentation may be nonspecific. Delay in diagnosis can result in catastrophic outcomes. Patients who present with small bowel obstruction after gastric bypass occasionally have pancreatic enzyme elevation and have been misdiagnosed as having acute pancreatitis. The objective of this study was to determine if there was an association between small bowel obstruction and an elevated amylase or lipase after RYGB. METHODS: Ninety-nine cases of small bowel obstruction treated surgically were prospectively collected and retrospectively analyzed from a database of 4014 RYGB patients. Fifty-eight had a measurement of amylase or lipase at the time of operation. RESULTS: An elevated amylase or lipase was found in 48% of all patients. These elevated rates were higher in an acute obstruction compared to those presenting with chronic symptoms (64% versus 28%; P=.007) and in obstruction involving the biliopancreatic limb compared to those that did not involve that limb (65% versus 21%; P<.001). These elevated rates were most notable in acute biliopancreatic limb obstruction compared to an acute obstruction not in the biliopancreatic limb (94% versus 27%; P<.001). CONCLUSION: In RYGB patients, there is an association between small bowel obstruction and an elevated amylase or lipase. Acute obstruction of the biliopancreatic limb can be difficult to diagnose, and in these patients, the sensitivity of elevated amylase or lipase is very high. RYGB patients with abdominal pain should have their amylase and lipase measured. It is important to recognize that an elevation of these enzymes is not likely a result of acute pancreatitis.

Alterations of the interstitial cells of Cajal and the microstructure of the gastrointestinal tract in KIT distal kinase mutant mice.

The development and maintenance of interstitial cells of Cajal (ICC) are closely associated with SCF/KIT signal activity. In this study, we evaluate the distribution of ICC in KIT distal kinase domain mutant mice (Wads) and determine whether the loss-of-function mutations in KIT easily lead to gastrointestinal (GI) disorders. ICC were examined by anti-KIT immunohistochemistry and western blotting. The GI microstructure of wild-type (WT) and Wads mice in normal intestines and incomplete intestinal obstruction was evaluated by hematoxylin and eosin staining. The results in Wads(m/m) mice were as follows. Myenteric ICC were obviously decreased in the stomach and colon and were totally absent in the small intestine. Intramuscular ICC were nearly absent in the stomach and irregularly distributed in the colon. Moreover, the smooth muscle thickness of the small intestine was increased 1.3-fold in Wads(m/m), compared to WT and Wads(m/+) mice and the diameter of the intestinal lumen was also enlarged in Wads(m/m) mice. When constructing an incomplete intestinal obstruction model, the extent of distention involved was greater in Wads mice (1.6-fold in Wads(m/+) mice and 1.8-fold in Wads(m/m) mice vs. WT mice). Meanwhile, the intestinal lumen expansion and decrease in ICC were more pronounced in Wads mice than in WT mice. Our results suggest that the KIT distal kinase domain mutation leads to an ICC loss in a subtype and location-specific pattern in Wads(m/m) mice. The injury of the KIT signaling in mutant mice results in more serious pathological manifestations after being exposed to pathogenic factors.

A phase 1 open-label trial shows that smad7 antisense oligonucleotide (GED0301) does not increase the risk of small bowel strictures in Crohn's disease.

BACKGROUND: In Crohn's disease (CD), knockdown of Smad7, an inhibitor of Transforming Growth Factor (TGF)-ß1 activity, with a specific antisense oligonucleotide (GED0301) seems to be safe and tolerable and associates with TGF-ß1-mediated suppression of inflammatory pathways. AIM: Since TGF-ß1 has pro-fibrogenic effects in many organs, we evaluated whether GED0301 treatment associates with the formation of small bowel strictures. METHODS: Fifteen patients with active, inflammatory CD, receiving oral GED0301 once daily for 7 days, were monitored for the formation of small bowel strictures by Small Intestine Contrast Ultrasonography (SICUS). Serum basic fibroblast growth factor (bFGF) and human chitinase 3-like 1 (also known as YKL-40), two markers of CD-related intestinal strictures, and matrix metalloproteinases (MMP) and tissue inhibitor 1 of MMPs (TIMP1) were analysed at day 0 and day 180 by ELISA. Crohn's disease activity index (CDAI) changes were also monitored. RESULTS: Fourteen patients completed the 6-month study; the remaining underwent intestinal resection for a severe relapse not responsive to medical treatment. No patient developed small bowel stricture and none experienced obstructive symptoms during the study period. GED0301 treatment induced no significant change in the circulating levels of bFGF, YKL-40, MMPs and TIMP1. Seven of 12 patients who reached clinical remission following GED0301 treatment maintained a CDAI < 150 at day 180. CONCLUSION: Short-term treatment of patients with Crohn's disease using GED0301 is not associated with the development of small bowel stricture, thus reinforcing the concept that this drug is safe at least at early time points.

Histidine decarboxylase is identified as a potential biomarker of intestinal mucosal injury in patients with acute intestinal obstruction.

Various biomarkers currently used for the diagnosis of intestinal mucosal injury (IMI) in patients with acute intestinal obstruction have low sensitivity and specificity. In the present study, IMI, as indicated by the impaired expression of tight junction proteins, including zonula occludens-1, occludin and claudin-1, and inflammation were determined in colonic tissues of patients with 45 strangulated intestinal obstruction (STR-IO) and the adjacent "normal" colonic tissues of 35 patients with colon cancers by quantitative real-time polymerase chain reaction (QRT-PCR), Western blotting, immunohistochemistry and histological examination, respectively. Then, two-dimensional fluorescent difference gel electrophoresis coupled with linear trap quadrupole mass spectrometry was used to screen for potential biomarkers of IMI in the serum samples of 10 STR-IO, 10 simple intestinal obstruction (SIM-IO) and 10 normal healthy controls. A total of 35 protein spots were differentially expressed among the serum samples, and six of the proteins were identified as potential biomarkers. Among the six proteins, histidine decarboxylase (HDC) and ceruloplasmin (CP) were elevated significantly in patients with STR-IO, compared with patients with SIM-IO and healthy controls. Thus, HDC and CP were further validated by QRT-PCR, Western blotting, immunohistochemistry and enzyme-linked immunosorbent assay, respectively, in colonic tissues, serum and urine samples. Finally, the receiver operating characteristic curves were used to show the area under the curves of HDC, CP and several established biomarkers, followed by the determination of the appropriate cutoff values and their sensitivities and specificities. It was shown that for serum and urine, HDC levels achieved sensitivities and specificities compatible to or even greater than those of established biomarkers for the diagnosis of IMI in patients with acute intestinal obstruction, although further validation in a larger cohort is required.

Collagen levels are normalized after decompression of experimentally obstructed colon.

AIM: Our aim was to define the dynamics in collagen concentrations in the large bowel wall following decompression of experimental obstruction. METHOD: Colonic obstruction was created in 28 male rats by the placement of a silicone ring around the distal colon. The ring was removed after 4 days to mimic endoscopical decompression by stent deployment. Colon circumference and collagen concentration were measured proximal to the obstructed segment immediately and at 3 and 10 days after decompression. The corresponding colonic sites of 23 sham-operated and eight nonoperated control animals were subjected to identical analyses. RESULTS: Four days of obstruction resulted in a more than twofold increase in colonic circumference (20 vs 8 mm), with a concomitant 43% reduction (P = 0.001) in collagen concentration in the bowel wall proximal to the obstruction compared with sham animals. Three days after decompression, collagen concentrations remained reduced (P < 0.05), while there was no significant difference after 10 days with either sham-operated or nonoperated controls. Colonic circumference of the obstructed colon remained slightly distended (11 mm) on day 10 and tended to correlate (r(S) = 0.51, P = 0.053) with total matrix metalloproteinase activity. CONCLUSION: The marked reduction in collagen concentration in an experimentally obstructed colon is normalized 10 days after decompression. These findings may have clinical implications for the timing of surgical resection.

Epithelial inducible nitric oxide synthase causes bacterial translocation by impairment of enterocytic tight junctions via intracellular signals of Rho-associated kinase and protein kinase C zeta.

OBJECTIVE: Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C ζ in epithelial nitric oxide synthase-mediated barrier damage. DESIGN: Animal study and cell cultures. SETTING: Research laboratory. SUBJECTS: BALB/c mice. INTERVENTIONS: : Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated. MEASUREMENTS AND MAIN RESULTS: Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C ζ were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C ζ phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C ζ signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(-/-) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C ζ. Inhibitory protein kinase C ζ pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation. CONCLUSIONS: Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C ζ and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

Role of myosin light chain kinase in intestinal epithelial barrier defects in a rat model of bowel obstruction.

BACKGROUND: Bowel obstruction is a common cause of abdominal emergency, since the patients are at increased risk of septicemia resulting in high mortality rate. While the compartmentalized changes in enteric microfloral population and augmentation of bacterial translocation (BT) have already been reported using experimental obstruction models, alterations in epithelial permeability of the obstructed guts has not been studied in detail. Myosin light chain kinase (MLCK) is actively involved in the contraction of epithelial perijunctional actinomyosin ring and thereby increases paracellular permeability. In the current study we attempt to investigate the role of MLCK in epithelial barrier defects using a rat model of simple mechanical obstruction. METHODS: Wistar rats received intraperitoneal injection of ML-7 (a MLCK inhibitor) or vehicle at 24, 12 and 1 hrs before and 12 hrs after intestinal obstruction (IO). The distal small intestine was obstructed with a single ligature placed 10 cm proximal to the ileocecal junction in IO rats for 24 hrs. Sham-operated rats served as controls. RESULTS: Mucosal injury, such as villous blunting and increased crypt/villus ratio, was observed in the distal small intestine of IO rats. Despite massive enterocyte shedding, intestinal villi were covered with a contiguous epithelial layer without cell apoptosis. Increased transmural macromolecular flux was noticed in the distal small intestine and the proximal colon after IO. The bacterial colony forming units in the spleen and liver of IO rats were significantly higher than those of sham controls. Addition of ML-7 ameliorated the IO-triggered epithelial MLC phosphorylation, mucosal injury and macromolecular flux, but not the level of BT. CONCLUSIONS: The results suggest that IO-induced premature enterocytic sloughing and enhanced paracellular antigenic flux were mediated by epithelial MLCK activation. In addition, enteric bacteria may undergo transcytotic routes other than paracellular paths to cross the epithelium.

Activation of MAP kinase (ERK1/2) in human neonatal colonic enteric nervous system.

The aim of this study was to examine mitogen-activated protein kinase (ERK1/2) activation in the human neonatal colonic enteric nervous system. For this, we investigated by immunocytochemistry the cellular localization of phosphorylated ERK1/2 (P-ERK) in a series of normal human colon samples removed from newborns and in patients with intestinal obstruction such as Hirschsprung's disease (HSCR), stenosis and atresia. We checked the presence of P-ERK in the three distinct histological layers of normal colon. Phosphorylated ERK was detected in the colonic mucosa, in the enteric nervous system and in endothelial cells. In the mucosa from normal colon, P-ERK was detected at the upper part of the crypt, while P-ERK activation in epithelial cells is altered in HSCR, stenosis and atresia. In the normal colon, strong P-ERK staining was detected in myenteric and submucosal enteric plexuses. Using confocal microscopy analyses, we observed that P-ERK staining was localized in enteric glial cells and not in enteric neurons. Strong P-ERK staining was also observed in plexuses from stenosis and atresia whereas in HSCR, hypertrophic nerve fibres were not stained.

Phase 2 safety trial targeting amyloid beta production with a gamma-secretase inhibitor in Alzheimer disease.

OBJECTIVE: To evaluate the safety, tolerability, and amyloid beta (Abeta) response to the gamma-secretase inhibitor LY450139 in Alzheimer disease. DESIGN: Multicenter, randomized, double-blind, dose-escalation, placebo-controlled trial. SETTING: Community-based clinical research centers. Patients Fifty-one individuals with mild to moderate Alzheimer disease were randomized to receive placebo (n=15) or LY450139 (100 mg [n=22] or 140 mg [n=14]), with 43 completing the treatment phase. Intervention The LY450139 groups received 60 mg/d for 2 weeks, then 100 mg/d for 6 weeks, and then either 100 or 140 mg/d for 6 additional weeks. MAIN OUTCOME MEASURES: Primary outcome measures were adverse events, plasma and cerebrospinal fluid Abeta levels, vital signs, electrocardiographic data, and laboratory safety test results. Secondary outcome measures included the Alzheimer's Disease Assessment Scale cognitive subscale and the Alzheimer's Disease Cooperative Study Activities of Daily Living Scale. RESULTS: Group differences were seen in skin and subcutaneous tissue concerns (P=.05), including 3 possible drug rashes and 3 reports of hair color change in the treatment groups. There were 3 adverse event-related discontinuations, including 1 transient bowel obstruction. The plasma Abeta(40) concentration was reduced by 58.2% for the 100-mg group and 64.6% for the 140-mg group (P<.001). No significant reduction was seen in cerebrospinal fluid Abeta levels. No group differences were seen in cognitive or functional measures. CONCLUSIONS: LY450139 was generally well tolerated at doses of up to 140 mg/d for 14 weeks, with several findings indicating the need for close clinical monitoring in future studies. Decreases in plasma Abeta concentrations were consistent with inhibition of gamma-secretase. Trial Registration clinicaltrials.gov Identifier: NCT00244322.

Role of MutS homolog 2 (MSH2) in intestinal myofibroblast proliferation during Crohn's disease stricture formation.

Tissue remodeling and mesenchymal cell accumulation accompanies chronic inflammatory disorders involving joints, lung, vasculature, and bowel. Chronic inflammation may alter DNA-mismatch repair (MMR) systems in mesenchymal cells, but is not defined in Crohn's disease (CD) and its associated intestinal remodeling and stricture formation. We determined whether DNA-MMR alteration plays a role in the pathogenesis of CD tissue remodeling. Control and CD bowel tissues were used to generate primary cultures of muscularis mucosa myofibroblasts, which were assessed directly or following stimulation with TNF-alpha/LPS or H2O2. MutS homolog (MSH)2, MSH3, and MSH6 expression in tissues and myofibroblasts was determined. Immunohistochemical staining revealed an increased expression of MSH2 in CD muscularis mucosa and submucosal tissues compared with controls or uninvolved CD tissue, and MSH2 expression was increased in CD myofibroblasts compared with control cells. TNF-alpha/LPS and H2O2 further enhanced MSH2 expression in both control and CD cells, which were decreased by simvastatin. There were no significant changes in MSH3 and MSH6 expression. Proliferating cell nuclear antigen and Ki67 staining of CD tissue revealed increased proliferation in the muscularis mucosa and submucosa of chronically inflamed tissues, and enhanced proliferation was seen in CD myofibroblasts compared with controls. Simvastatin reversed the effects of inflammatory stress on the DNA-MMR and inhibited proliferation of control and CD myofibroblasts. Gene silencing with MSH2 siRNA selectively decreased CD myofibroblast proliferation. These data demonstrate a potential role for MSH2 in the pathogenesis of nonneoplastic mesenchymal cell accumulation and intestinal remodeling in CD chronic inflammation.

Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease.

Gastrointestinal disorders, especially strangulating intestinal obstructions, are still a major cause of illness and death in the horse. Circulating lipopolysaccharides may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO concentrations in the plasma and peritoneal fluid (PF) of horses with colic and to check the hypothesis that these concentrations would be higher in a case of strangulating obstruction than in cases of nonstrangulating disease. By using a specific enzyme-linked immunosorbent assay for equine MPO, we determined the MPO concentrations in horses admitted to a clinic for colic. Horses with nonstrangulating or strangulating obstruction of the large intestine (NSLI or SLI), strangulating obstruction of the small intestine (SSI), or inflammatory bowel disease (IBD) were compared with healthy horses. The horses with SLI, SSI, or IBD had significantly higher MPO levels in plasma and PF than did those in the other 2 groups. The mean plasma level was significantly higher in the horses with NSLI than in the healthy horses. High MPO values in PF indicated necrotic bowel. These results show that neutrophil activation occurs during nonstrangulating and strangulating intestinal obstruction in horses and that the plasma and PF MPO concentrations may be a marker of the severity of the disease.

Fatal gastrointestinal obstruction and hypertension in mice lacking nitric oxide-sensitive guanylyl cyclase.

The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for the beta1 subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3-4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor.

Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

OBJECTIVE: The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. MATERIAL AND METHODS: Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). RESULTS: In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. CONCLUSIONS: The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood.

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.

The C. elegans lethal gut-obstructed gob-1 gene is trehalose-6-phosphate phosphatase.

We identified the gob-1 (gut-obstructed) gene in a forward genetic screen for intestinal defects in the nematode Caenorhabditis elegans. gob-1 loss of function results in early larval lethality, at least in part because of a blocked intestinal lumen and consequent starvation. The gob-1 gene is first expressed in the 8E cell stage of the embryonic intestine, and the GATA factor ELT-2 is sufficient but not necessary for this early phase of gob-1 expression; gob-1 expression later becomes widespread in embryos, larvae, and adults. GOB-1 is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. Trehalose is a glucose disaccharide found in bacteria, fungi, plants, insects, and nematodes but not in mammals. Trehalose plays a number of critical roles such as providing flexible energy reserves and contributing to thermal and osmotic stress resistance. In budding yeast and in plants, the intermediate in trehalose synthesis, trehalose-6-phosphate, has additional critical but less well-defined roles in controlling glycolysis and carbohydrate metabolism. Strong loss-of-function mutants in the C. elegans tps-1 and tps-2 genes (which encode the two trehalose phosphate synthases responsible for trehalose-6-phosphate synthesis) completely suppress the lethality associated with gob-1 loss of function. The suppression of gob-1 lethality by ablation of TPS-1 and TPS-2, the upstream enzymes in the trehalose synthesis pathway, suggests that gob-1 lethality results from a toxic build-up of the intermediate trehalose-6-phosphate, not from an absence of trehalose. GOB-1 is the first trehalose-6-phosphate phosphatase to be identified in nematodes and, because of its associated lethality and distinctive sequence properties, provides a new and attractive target for anti-parasitic drugs.

[Dual effects of nitric oxide in acute colon obstruction]. / A nitrogen-monoxid kettos hatása a vastagbél elzáródás kórfolyamatában.

UNLABELLED: Nitric oxide (NO) plays central role in the pathophysiology of large bowel diseases. In the gastrointestinal tract the predominant form of nitric oxide synthase (NOS) isoenzymes is neuronal NOS (nNOS). The aims were to investigate the role of NO and the activation of NOS isoforms during acute colonic obstruction. Haemodynamic changes, large bowel motility and plasma levels of nitrate-nitrite (NOx) were observed for 7 hrs in anaesthetized dogs. Group 1 (n=6) served as sham-operated control. In groups 2 (n=8), 3 (n=6), and 4 (n=6) colon obstruction was initiated. Groups 3 and 4 were treated with non-selective NOS inhibitor N-nitro-L-arginine (NNA, 4 mg/kg) or with the selective nNOS inhibitor 7-nitroindazol (7-NI, 5 mg/kg) 3 hr after the obstruction. At the end of the experiments, tissue biopsies were taken from the oral and aboral parts of the colon to determine the constitutive and inducible NOS (cNOS and iNOS, respectively) activities. RESULTS: The cNOS activity of the colon was significantly higher orally then aborally in each group. After obstruction the characteristic features of hyperdynamic sepsis were observed. The obstruction caused significant increase in iNOS activity, which was significantly reduced by the NOS inhibitors. The obstruction increased the motility on both parts of the colon. The administration of NNA transiently inhibited, but later significantly increased the motility of the colon segments. Inhibition of nNOS by 7-NI treatment did not influence the hemodynamic parameters but decreased the motility. CONCLUSION: Neuronal NO increases colon motility at the early stage of large bowel obstruction, however, during a concomitant sepsis the excess of inducible NO will moderate this effect.

Changes of tissue endothelin-1 and nitric oxide synthase in a sheep model of large intestinal obstruction.

Large intestinal obstruction (LIO) in farm animals can cause a ischaemic necrosis of intestinal tissue, eventually leading to death. The roles of endothelin-1 (ET-1) and nitric oxide (NO) are not well understood in the process of LIO, but evidence suggests that endothelial-derived mediators may participate. In the present study, ET-1 concentration and total nitric oxide synthase (NOS) activity were measured in heart, liver, pancreas, lung and kidney in a model of LIO in sheep. Our data demonstrated that ET-1 concentration and NOS activity were altered, with significant increases of ET-1 in heart, lung and kidney and of NOS activity in pancreas and kidney, but a marked decline of NOS activity in liver (p < 0.05). It is postulated that these alterations in NOS activity and ET-1 concentration may contribute to the progressive loss of organ function, and finally lead to death in LIO in sheep.

Experimental colonic obstruction increases collagen degradation by matrix metalloproteinases in the bowel wall.

PURPOSE: Emergency resections for colonic obstruction are accompanied with increased risk of anastomotic dehiscence. Elevated local degradation of submucosal collagens by matrix metalloproteinases may predispose to anastomotic leakage. This study was designed to study the effect of colon obstruction and surgical trauma on matrix metalloproteinase activities and correlate these results to collagen concentration in the colon wall. METHODS: Colonic obstruction was induced in male, Sprague-Dawley rats (n = 58) by applying a constricting silicone ring around the left colon 3 cm above the peritoneal reflection. After four days of obstruction, 2-mm wide colonic segments were resected approximately 3 mm proximal and 3 mm distal to the stenosis for biochemical analyses. Colonic segments at corresponding locations were obtained from sham-operated rats (n = 5) without obstruction but with silicone ring placed adjacent to colon and from normal, nontraumatized rats (n = 10). Matrix metalloproteinase activity was determined by liberation of fragmented collagens from homogenized colonic tissue incubated ex vivo. Matrix metalloproteinase-2 specifically was analyzed by gelatin zymography. RESULTS: Endogenous collagenolysis by matrix metalloproteinases increased (P < 0.001) in colon as a consequence of obstruction (4.1-fold) and trauma (1.7-fold) compared with normal colon. In the proximity of the colon stenosis, total matrix metalloproteinase activity and matrix metalloproteinase-2 were significantly (P < 0.05) higher above than below the obstruction. Total activity was 22.9 (13.1-32.9) units/mg collagen proximal and 16.6 (12.7-18.4) units/mg collagen distal to the stenosis. Collagen concentration correlated inversely (r = -0.76; P < 0.001) with total matrix metalloproteinase activity. CONCLUSION: Colonic obstruction and trauma up-regulated matrix metalloproteinases and decreased collagen concentration in colonic wall.