Unique microbial-derived volatile organic compounds in portal venous circulation in murine non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease is now the leading liver disease in North America. The progression of non-alcoholic fatty liver disease to the inflammatory condition, non-alcoholic steatohepatitis is complex and currently not well understood. Intestinal microbial dysbiosis has been implicated in the development of non-alcoholic fatty liver disease and progression of non-alcoholic steatohepatitis. Volatile organic compounds are byproducts of microbial metabolism in the gut that may enter portal circulation and have hepatotoxic effects contributing to the pathogenesis of non-alcoholic steatohepatitis. To test this hypothesis, we measured volatile organic compounds in cecal luminal contents and portal venous blood in a mouse model of non-alcoholic steatohepatitis. METHODS: Gas chromatography-mass spectrometry analysis was conducted on cecal content and portal vein blood for volatile organic compound detection from mice fed a methionine and choline deficient diet, which induces non-alcoholic steatohepatitis. The colonic microbiome was studied by 16S rRNA gene amplification using the Illumina MiSeq platform. RESULTS: Sixty-eight volatile organic compounds were detected in cecal luminal content, a subset of which was also present in portal venous blood. Importantly, differences in portal venous volatile organic compounds were associated with diet-induced steatohepatitis establishing a biochemical link between gut microbiota-derived volatile organic compounds and increased susceptibility to non-alcoholic steatohepatitis. CONCLUSION: Our model creates a novel tool to further study the role of gut-derived volatile organic compounds in the pathogenesis of non-alcoholic steatohepatitis.

Mesalazine (5-aminosalicylic acid) alters faecal bacterial profiles, but not mucosal proteolytic activity in diarrhoea-predominant irritable bowel syndrome.

BACKGROUND: Imbalances in gut luminal bacteria may contribute to the pathogenesis of irritable bowel syndrome (IBS). AIM: To explore select bacteriological and anti-inflammatory effects of mesalazine (mesalamine; 5-aminosalicylic acid or 5ASA) and their relation to potential therapeutic effects in IBS. METHODS: Prospective pilot study of 12 women with diarrhoea-predominant IBS. Patients received oral mesalazine (1.5 g b.d.) for 4 weeks followed by a 4-week washout phase. Molecular profiling of stool bacterial communities and IBS symptoms were assessed before, during and after mesalazine treatment. Colonic mucosal biopsies were assessed for proteolytic activity. Qualitative and quantitative effects of mesalazine on stool microbiota, mucosal proteolytic activity and IBS symptoms were assessed. RESULTS: Faecal bacteria decreased by 46% on mesalazine treatment (P = 0.014), but returned to baseline during washout. Firmicutes and Bacteroidetes represented 95% of identified phylotypes, with a trend towards an increase in the proportion of Firmicutes at week 4 in symptomatic responders [median (IQR) 14% (49) increase] compared with nonresponders [median 5% (11) decrease, P = 0.088]. Rectosigmoid mucosal proteolytic activity did not change between baseline and treatment [median 23.2 (17.9) vs. 19.5 (46.7) mU activity/mg tissue, P = 0.433]. Eight of 12 (67%) patients responded favourably to mesalazine based on a global relief questionnaire, with significant decreases in days with discomfort and increases in bowel movement satisfaction. CONCLUSIONS: Mesalazine treatment is associated with a decrease in faecal bacteria abundance and rebalancing of the major constituents of the microbiota. Further study of the bacteriological and anti-inflammatory properties of mesalazine in IBS is warranted.

In vitro anaerobic biofilms of human colonic microbiota.

The human gastrointestinal tract hosts a complex community of microorganisms that grow as biofilms on the intestinal mucosa. These bacterial communities are not well characterized, although they are known to play an important role in human health. This study aimed to develop a model for culturing biofilms (surface-adherent communities) of intestinal microbiota. The model utilizes adherent mucosal bacteria recovered from colonic biopsies to create multi-species biofilms. Culture on selective media and confocal microscopy indicated the biofilms were composed of a diverse community of bacteria. Molecular analyses confirmed that several phyla were represented in the model, and demonstrated stability of the community over 96 h when cultured in the device. This model is novel in its use of a multi-species community of mucosal bacteria grown in a biofilm mode of growth.

Cost-effectiveness in Canada of intravenous proton pump inhibitors for all patients presenting with acute upper gastrointestinal bleeding.

BACKGROUND: The administration of proton pump inhibitors intravenously after endoscopic treatment of peptic ulcers significantly reduces the recurrence of bleeding. AIM: To evaluate the incremental cost-effectiveness in Canada of intravenous proton pump inhibitor before endoscopic therapy to patients presenting with acute upper gastrointestinal bleeding, compared with endoscopic treatment alone. METHODS: From a third-party payer perspective, we modelled the costs and effectiveness over 60 days of the two approaches using decision analysis. The probabilities of various outcomes, such as re-bleeding and the need for surgery, were taken from the published literature. We included the costs of intravenous proton pump inhibitor, therapeutic endoscopy, surgical procedures and hospitalizations, all expressed in 2001 Canadian dollars. RESULTS: In a hypothetical cohort of 1000 patients, the intravenous proton pump inhibitor approach resulted in mean savings of 20,700 Canadian dollars with 37 re-bleeding episodes averted. The investigation of uncertainty resulted in a likelihood of intravenous proton pump inhibitor being cost-effective of at least 0.73. CONCLUSION: It is common in Canada to administer intravenous proton pump inhibitors to patients with upper gastrointestinal bleeding even before endoscopic confirmation of bleeding peptic ulcers. Our results suggest that this approach has a high likelihood of being cost-effective.

Decreased orexigenic response to neuropeptide Y in rats with obstructive cholestasis.

Neuropeptide Y (NPY) is a key factor in the neurochemical control of food intake, and obstructive cholestasis can be associated with disturbances in food intake. Our aim in this study was to determine whether obstructive cholestasis in the rat is associated with defective central responsiveness to NPY. Cholestasis was induced in rats by surgical bile duct resection. Rats with obstructive cholestasis exhibited a 20% reduction in food intake 2 days after laparotomy (compared with sham-resected controls) that had resolved by 4 days after surgery. Responsiveness to the orexigenic action of NPY was tested by measuring food intake after intracerebroventricular injection of NPY. In sham-resected rats, NPY infusion strikingly increased food intake, whereas bile duct-resected (BDR) rats showed a consistent significantly impaired feeding response to NPY at postlaparotomy days 2, 4, and 7. Separate experiments measured specific binding of [(3)H]NPY to hypothalamic receptors. Fos protein expression was measured in the hypothalamic paraventricular nucleus (PVN) as a marker of NPY-induced neuronal activation. The decreased orexigenic responsiveness to NPY was not caused by altered NPY binding at hypothalamic receptors or its ability to activate neurons in the PVN. Therefore, cholestatic rats demonstrate an attenuated NPY-induced orexigenic drive that occurs early after biliary obstruction, when cholestatic rats exhibit reduced food intake, and persists despite the return of food intake to normal levels and the presence of intact central NPY-related neuronal pathways.

Differential leptin responses to acute and chronic biliary obstruction in rats.

BACKGROUND/AIMS: Recently leptin, a protein released from adipocytes, has been identified as a potent circulating satiety factor. We therefore undertook this series of experiments to examine leptin's role in the anorexia associated with biliary obstruction. METHODS: Rats underwent either surgical bile duct resection (BDR) or sham resection (sham). Body weight, and food and water intake were measured during a baseline period and for 8 days after surgery. At 4, 8 and 16 h as well as on days 2, 4, 6, and 8 postsurgery, sham and BDR rats were sacrificed and sera collected for subsequent measurement of leptin hormone concentration by RIA. White adipose tissue was collected on days 2, 4, 6 and 8 for leptin mRNA determination by Northern blot. RESULTS: Obstructive cholestasis in BDR rats caused significant anorexia for up to 7 days post-surgery, whereas in sham rats, a significant decrease in food intake was only observed in the first 24-h period following surgery. In both sham and BDR rats, water intake was significantly decreased during the first 24-h period after surgery, but had recovered to baseline levels by day 2 in both groups. Fat pad mass corrected to body weight was not significantly different between the two experimental groups. Serum leptin levels were significantly increased 4 and 8 h after surgery, had normalized by 16 h post-surgery, and were then decreased in BDR rats on days 2, 4, 6 and 8 compared with controls. Leptin mRNA levels in epididymal fat pads were decreased by approximately 2-fold in BDR rats compared with sham rats on days 2, 4, 6 and 8. Furthermore, day 5 BDR and sham rats demonstrated similar anorectic responses to centrally administered leptin. CONCLUSIONS: Leptin production is significantly increased early after biliary obstruction but is reduced after prolonged biliary obstruction. Increased circulating leptin levels may contribute to the profound anorexia observed early after biliary obstruction but appear not to mediate the anorexia observed during more chronic biliary obstruction.

Colonic and jejunal motor disturbances after colonic antigen challenge of sensitized rat.

BACKGROUND & AIMS: Inflammation in the colon may alter motility in the proximal gut and potentiate clinical symptoms. The aim of this study was to characterize the effect of colonic anaphylaxis on local (colonic) and remote (small intestinal) motility and identify the mechanism and mediators involved. METHODS: Rats were sensitized by intraperitoneal injection of 10 microg egg albumin and surgically prepared with electrodes in jejunum and colon and a colostomy tube. Colonic and jejunal myoelectric activity were recorded in fasted animals before and after colonic antigen challenge without and then after pretreatment with specific antagonists. RESULTS: Colonic antigen challenge of sensitized rats was associated with significant (1) increase in colonic myoelectric spike activity, (2) disruption of fasting jejunal motility and initiation of aborally propagating spike complexes, and (3) increase in plasma rat mast cell protease II levels with a decrease in granulated mast cells in colon but not jejunum. The myoelectric disturbance in both colon and jejunum was inhibited significantly by pretreatment with atropine and hexamethonium, doxantrazole, cyclooxygenase, and lipoxygenase inhibitors. Methysergide inhibited only the jejunal disturbance. CONCLUSIONS: Colonic antigen challenge of sensitized animals results in local mast cell activation and the release of mediators that modulate neural pathways to initiate both a local colonic and a remote jejunal myoelectric disturbance.

Long-term antigen challenge results in progressively diminished mucosal mast cell degranulation in rats.

BACKGROUND & AIMS: The effects of short-term antigenic activation of mast cells on the gastrointestinal tract have been well characterized, but little is known about the effects of long-term exposure to antigen on mucosal mast cell reactivity. The aim of this study was to determine the effects of long-term antigen exposure on mucosal mast cell reactivity in the gastrointestinal mucosa. METHODS: Rats sensitized to chicken ovalbumin were orally challenged (short-term or long-term) with antigen. Rat mast cell protease II (RMCP-II) content was measured in serum as an index of mucosal mast cell degranulation. RESULTS: Short-term oral antigen challenge caused a 30-fold increase in serum RMCP-II levels. RMCP-II release was markedly diminished in long term-challenged rats (P < 0.001), despite increased tissue RMCP-II levels in stomach and jejunum. Although short-term antigen challenge significantly increased gastric acid secretion, no such response was observed after the long-term antigen challenge. In rats undergoing long-term challenge, a significant release of RMCP-II in response to intravenous antigen was not observed; however, mucosal mast cells remained responsive to intravenous anti-immunoglobulin E. CONCLUSIONS: Repeated activation of mucosal mast cells results in a progressive diminution of RMCP-II release not attributable to depletion of this mediator. This may represent an adaptive response aimed at minimizing the potentially deleterious effects of repeated exposure to an antigen.

Mast cells do not contribute to nonsteroidal anti-inflammatory drug-induced gastric mucosal injury in rodents.

BACKGROUND: By releasing pro-ulcerogenic mediators, mast cells may contribute to the mucosal injury associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs). METHODS: To study this, rat and mouse models of NSAID-induced gastric damage were used in which administration of indomethacin causes haemorrhagic injury in the corpus region of the stomach, and the "re-feeding" model in which penetrating antral ulcers are induced in the rat by naproxen. Mast cell degranulation was determined histologically and by measurement of tissue and serum levels of rat mast cell protease-II, a mediator specific to mucosal mast cells. The effects of either increasing or decreasing the number of gastric mucosal mast cells on the susceptibility of the stomach to injury induced by indomethacin were also studied. RESULTS: Gastric injury induced by indomethacin was not accompanied by significant mast cell degranulation. Moreover, neither increasing nor decreasing the number of gastric mucosal mast cells had a significant effect on the susceptibility of the gastric mucosa to damage induced by indomethacin. In the re-feeding model, prior depletion of gastric mucosal mast cells did not significantly affect the severity of antral ulceration induced by naproxen, nor the ability of prostaglandin E2 to prevent this damage. Finally, indomethacin-induced damage was similar in severity in mice with a genetic defect resulting in the complete absence of mast cells as it was in normal, congenic littermates. CONCLUSION: Mast cells do not play a significant role in the development of gastric injury induced by acute NSAID administration in the rat or mouse.

Hepatic mucosal mast cell hyperplasia in rats with secondary biliary cirrhosis.

Mast cells have been shown to play a role in many chronic inflammatory and fibrotic disorders. However, their possible contribution to the pathological changes that occur in liver cirrhosis is unknown. To explore this, we examined whether changes in hepatic mast cell number and mediator content were associated with fibrotic changes in experimental biliary cirrhosis. Rats were studied 7, 14, or 21 days after bile duct resection (BDR). Hepatic mast cells were identified by histochemical and immunohistochemical stains. Rat mast cell protease II (RMCP-II), a marker of mast cell degranulation, was measured in liver by enzyme-linked immunosorbent assay. Hepatic collagen deposition was assessed by Sirius Red F3BA staining. In day 21 BDR rats, there was a one- to twofold increase (P < .001) in the number of hepatic mast cells, but this was not observed in day 7 or 14 BDR rats. Mild fibrotic changes were noted in BDR rat livers as early as 7 days after induction of cholestasis. Significant expansion and organization of fibrous tissue had occurred in day 14 BDR rats which progressed to bridging fibrosis by day 21. Liver RMCP-II levels were decreased by 50 percent (P < .05) and mast cell degranulation was apparent as shown by histamine immunostaining. These results suggest that hepatic mast cell hyperplasia and degranulation occur during prolonged cholestasis in the rat. Although these changes do not correlate with the onset of hepatic fibrosis, they do occur at a time during which there is significant deposition and organization extracellular matrix elements. Hepatic mast cells, by releasing profibrogenic mediators, may contribute to fibrotic changes in biliary cirrhosis.

Mast cell activation augments gastric mucosal injury through a leukotriene-dependent mechanism.

Several lines of evidence suggest a role for mast cells as modulators of gastric mucosal integrity, but the effect of antigenic mast cell activation on mucosal resistance to injury has not previously been examined. In this study, rats were sensitized to the nematode Nippostrongylus brasiliensis and were studied 35-42 days later. With use of an ex vivo gastric chamber preparation, the stomach was exposed for 10 min to 20% ethanol. In some rats, antigen was administered intra-arterially 10 min before application of ethanol. Sensitized rats exhibited similar levels of ethanol-induced gastric injury as control rats, despite having significantly greater numbers of mucosal mast cells. However, antigen administration, which did not in itself produce mucosal injury, significantly augmented (approximately 3-fold) the extent of injury in sensitized but not control rats. Prior treatment with dexamethasone depleted mucosal mast cells in control and sensitized rats. Moreover, this treatment abolished the increase in mucosal injury observed in sensitized rats treated with antigen and topical ethanol. Pretreatment with a leukotriene D4-receptor antagonist, but not a platelet-activating factor-receptor antagonist or a cyclooxygenase inhibitor, abolished the increased susceptibility of sensitized rats to gastric damage induced by antigen and topical ethanol. These results suggest that mucosal mast cell number per se does not influence mucosal susceptibility to injury; however, activation of mast cells markedly increases the susceptibility to injury through a peptidoleukotriene-dependent mechanism.

Hemoprotein-dependent production of a neutrophil-activating factor from arachidonic acid.

Hemoproteins have been suggested to contribute to various forms of tissue injury by catalyzing the peroxidation of lipids. In this study, the ability of hemoglobin to catalyze the production of a neutrophil-activating factor from arachidonic acid was examined. Incubation of arachidonic acid, hydrogen peroxide, and hemoglobin at 37 degrees C for 30 min resulted in the production of a lipid-extractable substance that was chemotactic for neutrophils in vitro and could stimulate leukocyte adherence in vivo. These actions could be inhibited by two leukotriene B4 (LTB4) receptor antagonists. The peroxidation product cross-reacted significantly with an antibody directed against LTB4, but not with an antibody directed against LTC4. The production of this factor was hemoprotein dependent. Immunoreactive LTB4 and biological activity were produced only when hemoglobin, or another hemoprotein, cytochrome c, was present in the reaction mixture. The amount of the factor produced could be increased in a concentration-dependent manner by increasing the amounts of arachidonic acid or hydrogen peroxide in the reaction mixture. The production of this factor could be inhibited by 5-aminosalicylic acid, catalase, or deferoxamine. Separation of the lipid-extractable products of the peroxidation of arachidonic acid on high-performance liquid chromatography revealed that the immunoreactive (with anti-LTB4) and chemotactic substance had a retention time distinct from that of LTB4 and the hydroxyeicosatetraenoic acids. A lipid-extractable substance with significant cross-reactivity to anti-LTB4 could also be produced if plasma was substituted for arachidonic acid in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of Acyl Lipids Containing Very-Long Chain Fatty Acids in Microspore-Derived and Zygotic Embryos of Brassica napus L. cv Reston.

Biosynthesis of very long chain (>C(18)) fatty acids (VLCFAs) and the pathway for their incorporation into acyl lipids was studied in microspore-derived (MD) and zygotic embryos of Brassica napus L. cv Reston. In the presence of [1-(14)C]oleoyl-coenzyme A or [1-(14)C] eicosenoyl-coenzyme A, malonyl-coenzyme A, and reducing equivalents, maximal in vitro elongation activity was expressed in protein preparations from early-mid cotyledonary stage MD embryos (17-20 days in culture), when endogenous eicosenoic (20:1) and erucic (22:1) acids were just beginning to accumulate (approximately 1.5 milligrams per gram dry weight). The biosynthesis of VLCFAs and their incorporation into glycerolipids in vitro in the MD embryo system occurred at rates comparable to those measured in developing zygotic Reston embryos at about 20 days postanthesis. When glycerol-3-phosphate was supplied as acyl acceptor in time-course experiments using homogenates prepared from 18-day MD embryos, newly synthesized [(14)C]20:1 and [(14)C]22:1 were incorporated primarily into triacylglycerols (TAGs) and, to a lesser extent, into lyso-phosphatidic/phosphatidic acids, diacylglycerols, and phosphatidylcholines as well as the acyl-coenzyme A and free fatty acid pools. [(14)C]24:1 was not detected in any acyl lipid. Stereospecific analyses of the radiolabeled TAGs indicated that [(14)C]20:1 and [(14)C]22:1 moieties were esterified predominantly at the sn-3 position, but were also found at the sn-1 position. [(14)C]20:1, but not [(14)C]22:1, was detected at the sn-2 position. Similar patterns of (14)C-labeled VLCFA distribution were obtained in experiments conducted using a 15,000g pellet fraction from 18-day MD embryos. All trends observed in the formation of TAGs containing VLCFAs in the Reston MD embryo system were also confirmed in studies of zygotic embryos of the same cultivar. The data support the biosynthesis of 20:1 and then 22:1 via successive condensations of malonyl-coenzyme A with oleoyl-coenzyme A and, for the first time in B. napus, demonstrate the incorporation of newly synthesized VLCFAs into TAGs via the Kennedy pathway.