Monia Baldoni

PAR1 antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation

In fibroblasts, thrombin induces collagen deposition through activation of a G‐protein–coupled receptor, proteinase‐activated receptor 1 (PAR1). In the current study, we examined whether PAR1 antagonism inhibits hepatic stellate cell (HSC) activation in vitro and whether it protects against fibrosis development in a rodent model of cirrhosis. A rat HSC line was used for in vitro studies whereas cirrhosis was induced by bile duct ligation (BDL). The current results demonstrated that HSCs express PAR1, as well as proteinase‐activated receptors 2 (PAR2) and 4 (PAR4), and that all three PARs were up‐regulated in response to exposure to growth factor in vitro. Exposure to thrombin and to SFLLRN‐(SF)‐NH2, a PAR1 agonist, and GYPGKF (GY)‐NH2, a PAR4 agonist, triggered HSC proliferation and contraction, as well as monocyte chemotactic protein‐1 (MCP‐1) production and collagen I synthesis and release. These effects were inhibited by the PAR1 antagonist. Administration of this antagonist, 1.5 mg/kg/d, to BDL rats reduced liver type I collagen messenger RNA (mRNA) expression and surface collagen by 63%, as measured by quantitative morphometric analysis. Similarly, hepatic and urinary excretion of hydroxyproline was reduced significantly by the PAR1 antagonist. In conclusion, PARs regulates HSC activity; development of PAR antagonists might be a feasible therapeutic strategy for protecting against fibrosis in patients with chronic liver diseases. (HEPATOLOGY 2004;39:365–375.)

NCX-1000, a nitric oxide-releasing derivative of ursodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver

BACKGROUND/AIMS We studied whether acute administration of NCX-1000, a nitric oxide (NO)-releasing derivative of ursodeoxycholic acid (UDCA), to animals with established liver cirrhosis decreases intrahepatic resistance and modulates hepatic vascular hypereactivity to norepinephrine (NE). METHODS Four-week bile duct ligated (BDL) cirrhotic and control, sham-operated, rats were treated orally with 28 mg/kg per day NCX-1000 or 15 mg/kg per day UDCA for 5 days. Isolated normal and cirrhotic livers were perfused with NE, from 10 nM to 30 microM, in a recirculating system. RESULTS NCX-1000 administration to BDL cirrhotic rats decreased portal pressure (P<0.01) without affecting mean arterial pressure and heart rate. In the isolated perfused liver system, administration of NE resulted in a dose-dependent increase of intrahepatic resistance. Vasoconstriction caused by 30 microM NE was reduced by 60% in animals treated with NCX-1000 (P<0.001), while UDCA was uneffective. The same portal pressure lowering effect was documented in cirrhotic and sham operated rats. Administration of NCX-1000 to BDL and sham operated rats resulted in a similar increase of nitrite/nitrate and cGMP concentrations in the liver. CONCLUSIONS By selectively delivering NO to the liver, NCX-1000 increases cGMP concentrations and effectively counteracts the effect of endogenous vasoconstrictors on the hepatic vascular tone.

GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice

Background: Uncontrolled T cell activation and abnormal function of the innate immune system against normal enteric bacterial flora play a critical part in the pathogenesis of inflammatory bowel disease (IBD). Therefore, pharmacological strategies directed to restore the normal responsiveness of the immune system could be efficacious in the treatment of these pathological conditions. Glucocorticoid-induced tumour necrosis factor receptor (GITR)-related gene is a member of the tumour necrosis factor receptor superfamily that is constitutively expressed at high levels on regulatory T cells and at low levels on unstimulated T cells, B cells and macrophages. GITR triggering leads to activation of T effectors and reversal of suppressive function of regulatory T cells. Aim: To investigate the role of GITR in the development of experimental colitis in mice. Results: Using GITR−/− mice, GITR deletion protected against 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis by reducing innate immune responses and effector T cell activity. Effector T cells isolated from GITR−/− mice were less effective than T cells isolated from GITR+/+ mice to transfer colitis in immunodeficient mice. Blocking the GITR/ligand for GITR (GITRL) signal by giving soluble GITR prevented TNBS-induced colitis in normal GITR+/+ and also in lymphocyte-deficient SCID mice. Conclusions: Collectively, these data suggest that GITR plays a critical part in regulating both acquired and innate mucosal immune responses during the development of experimental colitis in mice. Therefore, targeting the GITR/GITRL system signalling may represent a potential pharmacological tool for the treatment of IBD.

Nitric Oxide Regulates Immune Cell Bioenergetic: A Mechanism to Understand Immunomodulatory Functions of Nitric Oxide-Releasing Anti-Inflammatory Drugs

The 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX-4016) is a NO-releasing derivative of aspirin. In this study, we provide evidence that NCX-4016 delivered to PMBC-derived T lymphocytes and monocytes causes a transitory inhibition of cell respiration and ≈50% reduction of cellular ATP, which translates in a time-reversible inhibition of cell proliferation and IL-2, IL-4, IL-5, and IFN-γ secretion. Exposure of lymphocytes and monocytes to aspirin, 2-(acetyloxy)benzoic acid 3-(hydroxymethyl)phenyl ester (NCX-4017), a non-NO-releasing analog of NCX-4016, and cyclooxygenase inhibitors, reduced PG formation, but has no effect on cytokine/chemokine release. In contrast, delivering NO with (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) reproduced most of the metabolic and anti-cytokine activities of NCX-4016. Scavenging NO with hemoglobin or adding selective substrates of complex II, III, and IV of the mitochondrial respiratory chain reverses NCX-4016′ inhibitory activities. Exposure to DETA-NO and NCX-4016 enhances glucose uptake, glycolytic rate, and lactate generation in CD3/CD28-costimulated lymphocytes, while reduced citric acid cycle intermediates. These effects were not reproduced by selective and nonselective cyclooxygenase 2 inhibitors. In summary, we demonstrated that exposure of lymphocytes to NCX-4016 causes a metabolic hypoxia that inhibits lymphocyte reactivity to costimulatory molecules, providing a potential counteregulatory mechanism to control activated immune system.

Intestinal superinfections in patients with inflammatory bowel diseases.

BACKGROUND Intestinal superinfections may occur in the setting of inflammatory bowel diseases (IBD), complicating the clinical picture and triggering flares of disease. AIMS To report our experience with intestinal superinfections in IBD patients over a three-year period. METHODS Charts of patients hospitalized for moderate-to-severe active disease during the observation period were reviewed, and data of patients with flares due to infections collected and analyzed. RESULTS Overall, 15 out of 113 IBD patients (13.3%) had flare-ups related to intestinal infections; 143 acute flare-ups were thus documented, with 17 episodes (12%) related to infective agents, represented by Campylobacter jejuni (3 infections), Clostridium difficile (7 infections), and Cytomegalovirus (7 infections). All but two infections occurred in ulcerative colitis patients, and all responded to appropriate treatment. CONCLUSIONS Intestinal superinfections may complicate the clinical picture of IBD patients, increasing the diagnostic and therapeutic burden. Appropriate early diagnostic and therapeutic measures are thus needed in these patients.

Colonic motility in ulcerative colitis.

Background Inflammatory conditions affecting the gut may cause motility disturbances, and ulcerative colitis – one of the main disorders among the inflammatory bowel diseases – may display abnormal colonic motility. Aim To review the abnormalities of the large bowel in ulcerative colitis, by considering the motility, laboratory (in vitro) and pathological studies dealing with this topic. Methods A comprehensive online search of Medline and the Science Citation Index was carried out. Results Patients with ulcerative colitis frequently display colonic motor abnormalities, including lack of contractility, an increase of propulsive contractile waves, an excessive production of nitric oxide, vasoactive intestinal polypeptide nerves, interleukin 1 beta, neurotensin, tachykinins levels and the weaker action of substance P, likely related to a neuromuscular dysfunction due to the inflammatory process. Conclusions A better understanding of the pathophysiological grounds of altered colonic motility in ulcerative colitis may lead to a more in-depth knowledge of the accompanying symptoms and to better and more targeted therapeutic approaches.

Small intestinal bacterial overgrowth and warfarin dose requirement variability

The dose of warfarin needed to obtain a therapeutic anticoagulation level varies widely among patients and can undergo abrupt changes for unknown reasons. Drug interactions and genetic factors may partially explain these differences. Intestinal flora produces vitamin K2 (VK2) and patients with small intestinal bacterial overgrowth (SIBO) rarely present reduced INR values due to insufficient dietary vitamin K. The present study was undertaken to investigate whether SIBO occurrence may affect warfarin dose requirements in anticoagulated patients. Based on their mean weekly dose of warfarin while on stable anticoagulation, 3 groups of 10 patients each were defined: low dose (LD, <or=17.5 mg/wk of warfarin); high dose (HD, from 35-70 mg/wk); and very high dose (VHD>or=70 mg/wk). Each patient underwent a lactulose breath test to diagnose SIBO. Plasma levels of warfarin and vitamin K-analogues were also assessed. Patients with an altered breath test were 50% in the VHD group, 10% in the HD group, and none in the LD group (P=0.01). Predisposing factors to SIBO were more frequent in the VHD group, while warfarin interfering variables were not. VHD patients were younger and had a higher plasma vitamin K1 (VK1) concentration (P>0.05). On the contrary, the plasma VK2 levels tended to be lower. This pilot study suggests that SIBO may increase a patients warfarin dose requirement by increasing dietary VK1 absorption through the potentially damaged intestinal mucosa rather than increasing intestinal VK2 biosynthesis. Larger studies are needed to confirm these preliminary data and to evaluate the effects of SIBO decontamination on warfarin dosage.

Ultrasonographic assessment of colonic wall in moderate–severe ulcerative colitis: Comparison with endoscopic findings

BACKGROUND Bowel ultrasound has been shown to be a useful tool to evaluate patients with inflammatory bowel disease, especially Crohns disease. However, such data are still scarce in ulcerative colitis patients. AIMS To establish the value of bowel ultrasound in moderate to severe ulcerative colitis patients, and compare these data with endoscopic findings. PATIENTS AND METHODS Endoscopic, ultrasound and C-reactive protein data from 51 patients with moderate to severe ulcerative colitis observed during a 3-year period were retrospectively obtained and analysed. RESULTS All patients displayed pathological thickness (>4 mm) of the colon wall. This value strongly correlated with C-reactive protein values (p=0.0001) and the endoscopic score (p<0.0001). Also, a strong correlation (p<0.0001) was found between CRP values and endoscopic score. CONCLUSIONS Bowel ultrasound, in expert hands, may represent a useful adjunctive (or first line) tool for the evaluation of patients with moderate to severe ulcerative colitis.

IL-9 and Mast Cells Are Key Players of Candida albicans Commensalism and Pathogenesis in the Gut

Summary Candida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-β in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut.

Duodenal submucosal tunnelization by fishbone.

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