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Colorectal cancer (CRC) is the third most common cancer and a significant cause of cancer-related deaths worldwide. Metastasis is the worst prognostic factor for patients with CRC. HuR (ELAVL1) is overexpressed in CRC and has been reported to promote colon cancer growth by targeting RNA in the cell cytoplasm. Herein, the role of miR-155-5p in regulating HuR expression and cell migration was examined in colon cancer cells. MiR-155-5p knockdown in serum-starved colon cancer cells decreased both colon cancer cell chemotaxis and cytoplasmic expression of HuR. Bioinformatics analysis predicted two putative binding sites in the AU-rich elements (AREs) at the 3'-UTR of HuR mRNA. MiR-155-5p binding to HuR was verified using specific target site blockers and functionally validated by use of RNA immunoprecipitation assays, showing that miR-155-5p-dependent regulation of HuR expression is mediated by AREs. Targeting AREs with a specific blocker inhibited colon cancer cell migration. Taken together, these novel findings demonstrate that AREs mediate miR-155-5p positive regulation of HuR mRNA levels and translation as well as migration in colon cancer cells, suggesting that targeting miR-155-5p and/or Hur might be useful therapeutic strategies against colon cancer metastasis.

Dysfunctional coagulation aggravates clinical outcome in patients with sepsis. The aim of this study was to define the role of Rac-1 in the formation of platelet-derived microparticles (PMPs) and thrombin generation (TG) in abdominal sepsis. Male C57BL/6 mice underwent cecal ligation and puncture (CLP). Scanning electron microscopy and flow cytometry were used to quantify PMPs. TG was determined by use of a fluorimetric assay. It was found that CLP increased Rac1 activity in platelets, which was abolished by administration of the Rac1 inhibitor NSC23766. Sepsis-induced TG in vivo was reflected by reduced capacity of plasma from septic animals to generate thrombin ex vivo. Administration of NSC23766 increased peak and total TG in plasma from CLP mice indicating that Rac-1 regulates sepsis-induced formation of thrombin. The number of circulating PMPs was markedly elevated in animals with abdominal sepsis. Treatment with NSC23766 significantly decreased formation of PMPs in septic mice. Platelet activation in vitro caused release of numerous MPs. Notably, NSC23766 abolished PMP formation in activated platelets in vitro. These findings suggest that Rac-1 regulates PMP formation and TG in sepsis and that inhibition of Rac1 activity could be a useful target to inhibit dysfunctional coagulation in abdominal sepsis.

Platelets are known to play an important role in acute pancreatitis (AP) via promotion of neutrophil accumulation, although mechanisms behind platelet-dependent accumulation of neutrophils in the pancreas remain elusive. Platelets contain a wide spectrum of different pro-inflammatory compounds, such as chemokines. CXCL4 (platelet factor 4) is one of the most abundant chemokine in platelets, and we hypothesized that CXCL4 might be involved in platelet-dependent accumulation of neutrophils in the inflamed pancreas. The aim of this study was to examine the role of CXCL4 in severe AP. Pancreatitis was provoked by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine in C57BL/6 mice. Animals were treated with an antibody against platelets or CXCL4 before induction of pancreatitis. Plasma and lung levels of CXCL2, CXCL4, and interleukin (IL)-6 were determined by use of enzyme-linked immunosorbent assay. Flow cytometry was used to examine surface expression of macrophage-1 (Mac-1) on neutrophils. Plasma was obtained from healthy individuals (controls) and patients with AP. Challenge with taurocholate increased plasma levels of CXCL4, and depletion of platelets markedly reduced plasma levels of CXCL4 indicating that circulating levels of CXCL4 are mainly derived from platelets in AP. Inhibition of CXCL4 reduced taurocholate-induced neutrophil recruitment, IL-6 secretion, edema formation, amylase release, and tissue damage in the pancreas. However, immunoneutralization of CXCL4 had no effect on CXCL2-evoked neutrophil expression of Mac-1 or chemotaxis in vitro, suggesting an indirect effect of CXCL4 on neutrophil recruitment in AP. Targeting CXCL4 significantly attenuated plasma and lung levels of CXCL2, which is a potent neutrophil chemoattractant, and inhibition of the CXCL2 receptor attenuated neutrophil infiltration and tissue damage in the inflamed pancreas. A significant role of CXCL4 was confirmed in an alternate model of AP induced by L-arginine challenge. Moreover, patients with AP had significantly increased plasma levels of CXCL4 compared with healthy controls. These findings' results suggest that platelet-derived CXCL4 is a potent stimulator of neutrophil accumulation in AP and that this is mediated via generation of CXCL2 in the inflamed pancreas. We conclude that CXCL4 plays an important role in pancreatic inflammation and that targeting CXCL4 might be a useful way to ameliorate tissue damage in AP.

Excessive neutrophil recruitment is a major feature in septic lung damage although the signaling mechanisms behind pulmonary infiltration of neutrophils in sepsis remain elusive. In the present study, we hypothesized that Rac1 might play an important role in pulmonary neutrophil accumulation and tissue injury in abdominal sepsis. Male C57BL/6 mice were treated with Rac1 inhibitor NSC23766 (5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were collected for the quantification of neutrophil recruitment and edema and CXC chemokine formation. Blood was collected for the determination of Mac-1 on neutrophils and proinflammatory compounds in plasma. Gene expression of CXC chemokines and tumor necrosis factor alpha was determined by quantitative reverse transcription-polymerase chain reaction in alveolar macrophages. Rac1 activity was increased in lungs from septic animals, and NSC23766 significantly decreased pulmonary activity of Rac1 induced by CLP. Administration of NSC23766 markedly reduced CLP-triggered neutrophil infiltration, edema formation, and tissue damage in the lung. Inhibition of Rac1 decreased CLP-induced neutrophil expression of Mac-1 and pulmonary formation of CXC chemokines. Moreover, NSC23766 abolished the sepsis-evoked elevation of messenger RNA levels of CXC chemokines and tumor necrosis factor alpha in alveolar macrophages. Rac1 inhibition decreased the CLP-induced increase in plasma levels of high mobility group protein B1 and interleukin 6, indicating a role of Rac1 in systemic inflammation. In conclusion, our results demonstrate that Rac1 signaling plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis. Thus, targeting Rac1 activity might be a useful strategy to protect the lung in abdominal sepsis.

Background and objectives: Platelet-derived CD40L is known to regulate neutrophil recruitment and lung damage in sepsis. However, the mechanism regulating shedding of CD40L from activated platelets is not known. We hypothesized that matrix metalloproteinase-9 might cleave surface expressed CD40L and regulate pulmonary accumulation of neutrophils in sepsis. Methods: Abdominal sepsis was induced by cecal ligation and puncture (CLP) in wild-type and MMP-9-deficient mice. Edema formation, CXC chemokine, myeloperoxidase levels, neutrophils in the lung as well as plasma levels of CD40L and MMP-9 were quantified. Results: CLP increased plasma levels of MMP-9 but not MMP-2. The CLP-induced decrease of platelet surface CD40L and increase of soluble CD40L levels were significantly attenuated in MMP-9 gene-deficient mice. Moreover, pulmonary MPO activity and neutrophil infiltration in the alveolar space as well as edema formation and lung injury were markedly decreased in septic animals lacking MMP-9. In vitro studies revealed that inhibition of MMP-9 decreased platelet shedding of CD40L. Moreover, recombinant MMP-9 was capable of cleaving surface expressed CD40L on activated platelets. In human studies, plasma levels of MMP-9 were significantly increased in patients with septic shock compared to healthy controls although MMP-9 levels did not correlate with organ injury score. Conclusions: Our novel data propose a role of MMP-9 in regulating platelet-dependent infiltration of neutrophils and tissue damage in septic lung injury by controlling CD40L shedding from platelets. We conclude that targeting MMP-9 may be a useful strategy to limit acute lung injury in abdominal sepsis. This article is protected by copyright. All rights reserved. (Less)

Leukocyte accumulation is a rate-limiting step in inflammatory lung injury. The aim of this study was to define the role of CD11a/CD18 and CD11b/CD18 in sepsis-induced leukocyte rolling and adhesion in lung arterioles, capillaries and venules in male C57BL/6 mice using intravital fluorescence microscopy. Cecal ligation and puncture (CLP) markedly increased leukocyte rolling in arterioles and venules but not in capillaries in the lung. Immunoneutralization of CD11a, but not CD11b, decreased CLP-provoked leukocyte rolling in lung arterioles. Inhibition of CD11a or CD11b abolished CLP-induced arteriolar and venular leukocyte adhesion. Immunoneutralization of CD11a and CD11b reduced sepsis-induced leukocyte sequestration in pulmonary capillaries. Moreover, blocking CD11a or CD11b function improved microvascular blood flow in the lung of CLP animals. Considered together, our novel findings show that CD11a and CD11b mediate leukocyte adhesion in both arterioles and venules as well as trapping in capillaries in the lung. In addition, our data demonstrate that CD11a but not CD11b supports leukocyte rolling in pulmonary arterioles. Thus, these findings elucidate the molecular mechanisms behind leukocyte-endothelium interactions in the lung during systemic inflammation.

Recent data suggest that platelets regulate inflammatory changes and tissue damage in acute pancreatitis although the role of platelets in leukocyte-endothelium interactions in the pancreatic microcirculation is not known. The aim of this study was to define the impact of platelets on leukocyte rolling and adhesion in acute pancreatitis. Acute pancreatitis was induced in C57BL/6 mice by caerulein challenge. Mice were treated with an a anti-GP1b alpha (CD42b) antibody, which depletes platelets, or a control antibody before caerulein. Leukocyte rolling and adhesion were determined by the use of intravital fluorescence microscopy 18 h after the last dose of caerulein. In separate experiments, leukocyte-endothelium interactions were determined before and after administration of an anti-P-selectin, anti-PSGL-1 and a control antibody in mice with caerulein pancreatitis. Circulating platelet-neutrophil aggregates and pancreatic P-selectin mRNA were quantified 1 and 6 h respectively after caerulein challenge. Caerulein administration increased leukocyte and platelet interactions in the pancreatic microvasculature, increased tissue damage and expression of P-selectin mRNA in the pancreas as well as platelet-neutrophil complexes in the circulation. Notably, platelet depletion markedly reduced caerulein-provoked leukocyte rolling and adhesion in postcapillary venules. Interestingly, depletion of platelets significantly decreased caerulein-induced gene expression of P-selectin in the pancreas. Moreover, immunoneutralization of P-selectin and PSGL-1 abolished leukocyte rolling in the pancreatic venules triggered by caerulein. Our novel findings demonstrate that platelets regulate leukocyte rolling in acute pancreatitis via induction of P-selectin, which was critical in supporting leukocyte rolling in inflamed venules of the pancreas. (C) 2012 Elsevier B.V. All rights reserved. (Less)

Nanotechnology is a rapidly developing field in science and industry. The exposure to nanoparticles (NPs) will steadily grow in the future and there is thus an urgent need to study potential impacts of the interaction between NPs and the human body. The respiratory tract is the route of entry for all accidentally inhaled NPs. Moreover, NPs may intentionally be delivered into the lung as contrast agents and drug delivery systems. The present review provides an overview of currently used techniques for the in vivo imaging of NPs in the lung, including x-ray imaging, computed tomography, gamma camera imaging, positron emission tomography, magnetic resonance imaging, near-infrared imaging, and intravital fluorescence microscopy. Studies based on these techniques may contribute to the development of novel NP-based drug delivery systems and contrast agents. In addition, they may provide completely new insights into nanotoxicological processes. From the Clinical Editor Nanoparticles are rapidly gaining ground in various therapeutic and diagnostic applications. This review provides an overview of current in vivo imaging techniques of NPs in the lung, including x-ray, CT, gamma camera imaging, PET, MRI, near-infrared imaging, and intravital fluorescence microscopy, aiding the development of novel NP-based techniques and nanotoxicology.

Recent data suggest that platelets not only control thrombosis and hemostasis but may also regulate inflammatory processes such as acute pancreatitis. However, the specific role of platelet-derived mediators in the pathophysiology of acute pancreatitis is not known. Herein, we examined the role of CD40 ligand (CD40L, CD154) in different models of acute pancreatitis. Acute pancreatitis was induced by repetitive caerulein administration (50 mu g/kg, i.p.) or infusion of sodium taurocholate (5%-10 mu l) into the pancreatic duct in wild-type C578L/6 and CD40L-deficient mice. Neutrophil infiltration, myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2) levels, acinar cell necrosis, edema and hemorrhage in the pancreas as well as serum amylase activity and lung levels of MPO were quantified 24 h after induction of acute pancreatitis. Caerulein and taurocholate challenge caused a clear-cut pancreatic damage characterized by increased acinar cell necrosis, neutrophil infiltration, focal hemorrhage, edema formation as well as increased levels of serum amylase and MIP-2 in the pancreas and lung MPO and histological damage. Notably, CD40L gene-deficient animals exhibited a similar phenotype as wild-type mice after challenge with caerulein and taurocholate. Similarly, administration of an antibody directed against CD40L had no effect against acute pancreatitis. Our data suggest that CD40L does not play a functional role in experimental acute pancreatitis. Thus, other candidates than CD40L needs to be explored in order to identify platelet-derived mediators in the pathophysiology of acute pancreatitis. (C) 2011 Elsevier B.V. All rights reserved. (Less)

Bile duct obstruction is associated with hepatic accumulation of leukocytes and liver injury. Emerging data suggest that platelets may play an important role in tissue damage and inflammation. Herein, we characterized the platelet response in cholestatic liver injury and evaluated the role of Rho-kinase signaling. For this purpose, C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 (10 mg/kg) and vehicle before undergoing bile duct ligation (BDL) for 12 h. Platelet rolling and adhesion, formation of platelet aggregates as well as microvascular perfusion in the liver were analyzed using intravital fluorescence microscopy. Liver damage was monitored by measuring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Administration of Y-27632 reduced the BDL-associated increase of ALT and AST by 95% and 89%, respectively. The inhibition of Rho-kinase also reduced cholestasis-induced platelet rolling and adhesion by more than 46% and 73% in postsinusoidal venules and platelet adhesion in sinusoids by 60%. In addition, Y-27632 decreased platelet aggregation in hepatic sinusoids and postsinusoidal venules by 69% and 81%. BDL caused a significant reduction of hepatic microvascular perfusion. Importantly, pretreatment with Y-27632 restored sinusoidal perfusion in cholestatic animals. Our findings demonstrate that Rho-kinase regulates multiple aspects of platelet interaction in the microcirculation of cholestatic animals. Moreover, inhibition of Rho-kinase signaling not only attenuates platelet responses but also maintains microvascular perfusion and protects against hepatocellular injury in cholestasis. Thus, targeting Rho-kinase signaling may be an effective way to protect against platelet-mediated liver injury in obstructive jaundice.

Background The role of specific adhesion molecules in cholestasis-induced leukocyte recruitment in the liver is not known. Therefore, the aim of our experimental study was to evaluate the role of lymphocyte function antigen-1 (LFA-1) in cholestatic liver injury. Methods C57BL/6 mice underwent bile duct ligation for 12 hours. Mice were pretreated with an anti–LFA-1 antibody or control antibody. Subsequently, hepatic accumulation of leukocytes and sinusoidal perfusion were determined by means of intravital fluorescence microscopy. Hepatocellular damage was monitored by measuring serum levels of alanine aminotransferase and aspartate aminotransferase. CXC chemokines in the liver were determined by enzyme-linked immunosorbent assay. Results Bile duct ligation provoked clear-cut recruitment of leukocytes and liver damage, as indicated by increased serum activities of liver enzymes and sinusoidal perfusion failure. Neutrophils expressed greater levels of LFA-1 and inhibition of LFA-1 significantly decreased serum activity of alanine aminotransferase and aspartate aminotransferase levels in cholestatic mice. Immunoneutralization of LFA-1 reduced leukocyte adhesion in postsinusoidal venules that had been induced by bile duct ligation, whereas leukocyte rolling and sinusoidal accumulation were not changed. Moreover, blocking LFA-1 function restored sinusoidal perfusion in cholestatic animals. Conclusion These findings demonstrate an important role of LFA-1 in supporting cholestasis-induced leukocyte recruitment in the liver. Thus, targeting LFA-1 may help to protect against pathologic inflammation and liver damage in cholestatic liver diseases.

Platelets constitute a key role in vascular injuries, however, the detailed mechanisms behind platelet-endothelial cell and platelet-leukocyte interactions in the femoral artery are not yet fully elucidated. We used intravital fluorescence microscopy of the femoral artery in C57BL/6 mice to study primary and secondary capture of platelets onto endothelial cells as well as onto adherent platelets and leukocytes in vivo. By use of monoclonal antibodies, the role of P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) in these adhesive interactions in mice exposed to endotoxin was determined. Intravenous injection of endotoxin significantly increased gene expression of P-selectin as well as platelet tethering, rolling and adhesion in the femoral artery. Pretreatment with the anti-PSGL-1 antibody decreased platelet tethering by 85%, platelet rolling by 88% and platelet adhesion by 96%. Immunoneutralization of P-selectin reduced platelet tethering by 91%, platelet rolling by 98%, and platelet adhesion by 97%. In addition, inhibition of P-selectin and PSGL-1 completely abolished secondary capture of platelets onto adherent platelets and leukocytes. Our data show that P-selectin and PSGL-1 mediate early interactions between platelets and other cells, including endothelial cells and leukocytes, in inflamed arteries. These novel results suggest that interference with P-selectin and PSGL-1 may be a useful target in strategies aiming to protect the vascular wall during arterial inflammation.

Dietary polyunsaturated fatty acids, such as docosahexaenoic acid, may inhibit pathological processes involving endothelial cell activation. Herein, it was found that treatment of endothelial cells with docosahexaenoic acid dose dependently reduced neutrophil adhesion provoked by tumor necrosis factor-alpha (TNF-alpha). In fact, pretreatment with 100 microM of docosahexaenoic acid for 24 h decreased TNF-alpha-induced neutrophil adhesion by 50%. Moreover, this pretreatment with docosahexaenoic acid (100 microM, 24 h) down-regulated TNF-alpha-induced endothelial cell surface expression of P-selection by 75%. Importantly, immunoneutralization of P-selectin reduced neutrophil adhesion to TNF-alpha-activated endothelial cells by more than 50%, indicating a significant role of P-selectin in this model. On the other hand, CXC chemokines, i.e. macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), are also important regulators of neutrophil activation and adhesion. However, pretreatment with docosahexaenoic acid had no effect on TNF-alpha-provoked production of MIP-2 and KC in endothelial cells. Our study provide evidence that docosahexaenoic acid inhibits expression of P-selectin and subsequent adhesion of neutrophils to endothelial cells in response TNF-alpha, which may help explain the anti-inflammatory effects exerted by docosahexaenoic acid.

BACKGROUND: Markedly decreased or absent gastric acid production is associated with a number of clinically significant conditions, and identification of patients with hypo/achlorhydria may be important. However, current methods of assessing impaired acid secretion are unreliable, time-consuming, and/or complex. The aim of this prospective study was to evaluate a modified endoscopic Congo red test for the diagnosis of hypo/achlorhydria by correlation with a standard gastric acid secretory test. METHODS: One hundred six consecutive outpatients with or without dyspeptic symptoms referred for endoscopy were evaluated by using a modified endoscopic Congo red test and a standard test of gastric acid secretion. The modified endoscopic Congo red test suggested hypo/achlorhydria when there was no color shift or a shift of small extent (less than one third of fundic mucosa). Hypo/achlorhydria by the standard gastric acid secretory test was defined as a maximal acid output of less than 6.9 mmol/hour in men and 5.0 mmol/hour in women. RESULTS: The accuracy of the modified endoscopic Congo red test for the diagnosis of hypo/achlorhydria was 0.98 (95% CI [0.93, 0.99]). The sensitivity was 1.0 (95% CI [0.92, 1.00]) and specificity 0.96 (95% CI [0.88, 0.99]). All patients tolerated the modified endoscopic Congo red test well. CONCLUSION: The modified endoscopic Congo red is an accurate, simple, fast, inexpensive, and well-tolerated chromoendoscopic method for identification of patients with hypo/achlorhydria during routine upper endoscopy.

Background/Aims : This study was designed to examine the role of selectins and CD18 in leukocyte recruitment in hepatic injury induced by tumor necrosis factor- α (TNF- α ) and galactosamine (Gal) in vivo. Methods : Intravital fluorescence microscopy of the hepatic microcirculation was used to quantify leukocyte–endothelium interactions provoked by 24 h of systemic TNF- α /Gal challenge in rats. Hepatic injury was evaluated with liver enzymes. Results : When administered after 24 h of TNF- α /Gal challenge, fucoidan, a selectin-function inhibitor, reduced leukocyte rolling by 69%, whereas firm adhesion was unaltered. In contrast, passive immunization against CD18 decreased leukocyte adhesion by 60%, whereas rolling remained unchanged. Notably, when administered prior to TNF- α /Gal, fucoidan attenuated both leukocyte rolling and adhesion, by 57 and 69%, respectively. Pretreatment with an anti-CD18 antibody decreased TNF- α /Gal-induced rolling and firm adhesion by 25 and 90%, respectively. Moreover, pretreatment with fucoidan and the anti-CD18 antibody both protected against TNF- α /Gal-induced increases in liver enzymes. For example, the pretreatments reduced alanine aminotransferase by 59 and 87%, respectively. Conclusions : Our data suggest that TNF- α /Gal-induced leukocyte rolling is selectin-mediated and a precondition for CD18-dependent firm adhesion in hepatic venules. Thus, reducing leukocyte recruitment by inhibition of selectins or CD18 may be useful to control TNF- α -induced liver injury.

Macrophage inflammatory protein-2 (MIP-2) belongs to the C-X-C subfamily of chemokines and appears to play an important role in cytokine-induced inflammatory and immune cell-mediated responses. We found that tumor necrosis factor-alpha (TNF-alpha) time- and dose-dependently increased gene and protein expression of MIP-2 in endothelial cells. Moreover, it was observed that dexamethasone treatment inhibited endothelial cell expression of MIP-2 in response to TNF-alpha stimulation and markedly reduced the number of adherent neutrophils. Moreover, we found that a monoclonal antibody against murine MIP-2 abolished neutrophil adhesion to TNF-alpha-activated endothelial cells. These data demonstrate that TNF-alpha induces expression of MIP-2 in endothelial cells and support the hypothesis that the anti-inflammatory action of dexamethasone may, at least in part, be attributable to an inhibition of MIP-2 induction on cytokine-activated endothelial cells.

Background: Surface cooling is frequently used in a number of conditions, especially traumatic, ischemic, burn, and neurologic injury to reduce the tissue damage. However, the protective mechanisms of cold therapy on traumatized tissues remain unclear. Tumor necrosis factor-α (TNF-α) is a fundamental mediator in inflammatory reactions and trauma-induced tissue injury. In the present study, we examined the microvascular response to TNF-α challenge and the effects of local cooling on the TNF-α–induced changes in the striated muscle of hamsters. Methods: By the use of the dorsal skinfold chamber preparation and in vivo fluorescence microscopy in combination with computer-based image analysis, we determined TNF-α–induced leukocyte rolling and adhesion to microvascular endothelium, capillary perfusion, venular leakage,and cellular apoptosis with and without surface cooling. Results: We found that topical administration of 2000 units TNF-α caused a progressive impairment of microvascular perfusion and increased leukocyte recruitment and vascular macromolecular leakage. Local cooling to 10°C for 60 minutes markedly (P

Atherogenesis and vascular injury after angioplasty are both characterized by infiltration of leukocytes and smooth muscle cells into the vessel wall intima (Tanaka et al., 1993; Ross, 1995; O'Brien et al., 1996). Tumor necrosis factor-α (TNF-α) is a pluripotent cytokine that has been implicated as a key mediator in the development of atherosclerotic lesions and angioplasty-induced restenosis (Ross, 1995; Tanaka et al., 1996). Previous studies have generally concerned leukocyte-endothelium interactions in the venular section of the microcirculation. Lawrence and Springer (1991) have reported that P-selectin-mediated rolling is a prerequisite for ICAM-1-dependent adhesion in vitro. Notably, it has been demonstrated that TNF-α stimulates leukocyte rolling in arterioles which is mainly dependent on P-selectin function (Thorlacius et al. 1997; Kunkel et al., 1997). However, the mechanisms of leukocyte adhesion in arterioles remain elusive. In this study, we examined the role of P-selectin-dependent rolling and ICAM-1 function for TNF-α-induced firm leukocyte adhesion in arterioles. To investigate this, we used intravital microscopy of the mouse cremaster muscle stimulated with TNF-α.

Background & Aims Neutrophils are involved in the development of acute pancreatitis (AP), but it is not clear how neutrophil-induced tissue damage is regulated. In addition to secreting antimicrobial compounds, activated neutrophils eliminate invading microorganisms by expelling nuclear DNA and histones to form extracellular web-like structures called neutrophil extracellular traps (NETs). However, NETs have been reported to contribute to organ dysfunction in patients with infectious diseases. We investigated whether NETs contribute to the development of AP in mice. Methods AP was induced in C57BL/6 mice by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine. Pancreata were collected and extracellular DNA was detected by Sytox green staining, levels of CXC chemokines, histones, and cytokines also were measured. Cell-free DNA was quantified in plasma samples. Signal transducer and activator of transcription 3 phosphorylation and trypsin activation were analyzed in isolated acinar cells. NETs were depleted by administration of DNase I to mice. Plasma was obtained from healthy individuals (controls) and patients with severe AP. Results Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice and increased levels of cell-free DNA in plasma. Neutrophil depletion prevented taurocholate-induced deposition of NETs in the pancreas. Administration of DNase I to mice reduced neutrophil infiltration and tissue damage in the inflamed pancreas and lung, and decreased levels of blood amylase, macrophage inflammatory protein-2, interleukin 6, and high-mobility groups protein 1. In mice given taurocholate, DNase I administration also reduced expression of integrin α M (macrophage-1 antigen) on circulating neutrophils. Similar results occurred in mice with L-arginine–induced AP. Addition of NETs and histones to acinar cells induced formation of trypsin and activation of signal transducer and activator of transcription 3; these processes were blocked by polysialic acid. Patients with severe AP had increased plasma levels of NET components compared with controls. Conclusions NETs form in the pancreata of mice during the development of AP, and NET levels are increased in plasma from patients with AP, compared with controls. NETs regulate organ inflammation and injury in mice with AP, and might be targeted to reduce pancreatic tissue damage and inflammation in patients.

We have examined the effect of ropivacaine, a local anesthetic, on leukocyte-endothelium interactions induced by tumor necrosis factor-alpha (TNF-alpha) in vivo by the use of intravital microscopy in the mouse cremaster microcirculation. It was found that ropivacaine markedly reduced venular leukocyte adhesion and tissue recruitment in response to TNF-alpha challenge, whereas leukocyte rolling was unchanged. Thus, treatment with ropivacaine may be a useful pharmacological tool to control acute inflammation.

Background & Aims The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. Methods We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. Results Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine–induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. Conclusions NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.

Mo1309 Intraoperative ERCP With Guidewire Assisted Rendezvous Cannulation; A Laparo-Endoscopic Way to Avoid Post ERCP Pancreatitis Fredrik Swahn*, Sara Regner, Lars Enochsson, Lars R. Lundell, Henrik Thorlacius, Urban Arnelo Gastrocentrum Surgery, CLINTEC, Stockholm, Sweden; Department of Surgery, Clinical Sciences, Malmo Skane University Hospital, Malmo, Sweden Background: Laparoscopic cholecystectomy (LC) can be combined with endoscopic retrograde cholangiopancreatography (ERCP) in conjunction with transcystic guidewire assisted rendezvous (RV) cannulation in the management of common bile duct stones (CBDS). The aim of this study was to examine whether this approach is associated with a lower pancreatic proenzyme leakage rate than conventional (CV) ERCP cannulation technique. Methods: Patients (n 122) with symptomatic gallstone disease, without ongoing pancreatitis, cholangitis, cholecystitis or previous sphincterotomy were prospectively enrolled in this case control designed study. Forty of these patients had an LC where the per-operative cholangiogram suggested CBDS including a laparo-endoscopic (LE) RV. Another 41 patients with retained CBDS cholecystolithiasis underwent CV ERCP. Patients (n 41) submitted to LC and cholangiographie with negative findings served as a control group. Post-procedural pancreas amylase, trypsinogen-2 and procarboxy-peptidase B (proCAPB) blood concentrations were analyzed in all patients as well as perand post-procedural complications and CBDS clearance rates. Results: Post ERCP pancreatitis (PEP) occurred in 3 of the 41 (7%) patients submitted to CV ERCP as compared to none in the RV ERCP group (p 0.11, n.s.). Laboratory values suggestive of acute pancreatic injury such as pancreas amylase 4 hours after ERCP were 2.0 4.2 [SD] cat/L in the CV ERCP group, 0.5 0.33 cat/L in the RV ERCP group (P-value 0.0015). At 8 hours the corresponding values were 3.5 9.0 cat/l and 0.6 0.4 cat/l, respectively (p 0.03). ProCAPB after 4 hours was 90.7 237.7 nmol/L compared to 7.2 8.0 nmol/L after RV ERCP (p 0.0001) and after another 4 hours of observation corresponding values were 111.7 287.3 nmol/L and 7.9 7.6 nmol/L (p 0.0001) respectively (Fig.1). Trypsinogen-2 levels at 24 hours after procedure were 317.8 547.1 g/L in the CV ERCP group, 145.6 67.2 g/L (p 0.03) after RV ERCP and 134.0 80.3 g/L among the controls. No major per-or post procedural complications were observed in either groups. Post procedural concentrations of pancreas amylase (p 0.003) and proCAPB (p 0.002) were significantly correlated with pancreatic duct cannulation and opacification. Albeit that the final outcome in both study groups eventually reach complete stone clearance, there was a significant (p 0.01) advantage for the RVgroup (n 38/40, 95%) during the index procedure, compared with the CV-group (29/41, 71%). Conclusion: One-stage LE ERCP in conjunction with guidewire assisted RV cannulation minimizes pancreatic enzyme leakage compared with CV cannulation technique. These results suggests that RV technique is an important LE therapeutic method that can be employed to prevent PEP and thereby should be the preferred technique for transpapillary CBDS extraction in conjunction with LC.