Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte-endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice.

OBJECTIVE: P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to play a significant role in septic lung injury. However, the detailed role of PSGL-1 in the pulmonary leukocyte recruitment remains elusive. We have developed a method based on intravital fluorescence microscopy of the lung microcirculation to examine the role of PSGL-1 in the extravasation process of leukocytes in septic lung damage. METHODS: Male C57BL/6 mice were treated with a control antibody or an anti-PSGL-1 antibody prior to cecal ligation and puncture (CLP). Leukocyte-endothelium interactions and microvascular hemodynamics were studied in pulmonary arterioles, capillaries and venules 4 h after CLP. RESULTS: Immunoneutralization of PSGL-1 decreased CLP-induced leukocyte rolling in pulmonary arterioles and venules significantly. Inhibition of PSGL-1 had no effect on leukocyte adhesion in venules, whereas the number of adherent leukocytes in lung arterioles and the number of trapped leukocytes in capillaries were markedly decreased. Moreover, immunoneutralization of PSGL-1 improved microvascular perfusion in the lung of septic animals. CONCLUSIONS: Taken together, these results document that PSGL-1 mediates leukocyte rolling in arterioles and venules. However, inhibition of PSGL-1 only decreases leukocyte adhesion in arterioles, suggesting that leukocyte rolling is not a prerequisite for pulmonary venular adhesion of leukocytes in sepsis. In addition, our data show that capillary trapping of leukocytes is dependent on PSGL-1 function.

P-selectin mediates neutrophil rolling and recruitment in acute pancreatitis.

BACKGROUND: The adhesive mechanisms regulating leucocyte-endothelium interactions in the pancreas remain elusive, but selectins may play a role. This study examined the molecular mechanisms mediating leucocyte rolling along the endothelium in the pancreas and the therapeutic potential of targeting the rolling adhesive interaction in acute pancreatitis (AP). METHODS: Pancreatitis was induced by retrograde infusion of 5 per cent sodium taurocholate into the pancreatic duct, repeated intraperitoneal administration of caerulein (50 µg/kg) or intraperitoneal administration of L-arginine (4 g/kg) in C57BL/6 mice. A control and a monoclonal antibody against P-selectin were administered before and after induction of AP. Serum and tissue were sampled to assess the severity of pancreatitis, and intravital microscopy was used to study leucocyte rolling. RESULTS: Taurocholate infusion into the pancreatic duct increased the serum level of trypsinogen, trypsinogen activation, pancreatic neutrophil infiltration, macrophage inflammatory protein (MIP) 2 formation and tissue damage. Immunoneutralization of P-selectin decreased the taurocholate-induced increase in serum trypsinogen (median (range) 17·35 (12·20-30·00) versus 1·55 (0·60-15·70) µg/l; P = 0·017), neutrophil accumulation (4·00 (0·75-4·00) versus 0·63 (0-3·25); P = 0·002) and tissue damage, but had no effect on MIP-2 production (14·08 (1·68-33·38) versus 3·70 (0·55-51·80) pg/mg; P = 0·195) or serum trypsinogen activating peptide level (1·10 (0·60-1·60) versus 0·45 (0-1·80) µg/l; P = 0·069). Intravital fluorescence microscopy revealed that anti-P-selectin antibody inhibited leucocyte rolling completely in postcapillary venules of the inflamed pancreas. CONCLUSION: Inhibition of P-selectin protected against pancreatic tissue injury in experimental pancreatitis. Targeting P-selectin may be an effective strategy to ameliorate inflammation in AP.

A unique 3D endoanal ultrasound feature of perianal Crohn's fistula: the 'Crohn ultrasound fistula sign'.

AIM: Using a high-resolution 3D endoanal ultrasound, we have observed that some perianal fistulas show a hypoechogenic fistula tract surrounded by a well-defined hyperechogenic area with a thin hypoechogenic edge in patients with Crohn's disease ['Crohn's Ultrasound Fistula Sign' (CUFS)], unlike conventional fistula tracks. The study aimed to determine the prevalence of CUFS in a consecutive series of patients with anal fistula. METHOD: Of 157 patients (median age 45, range 14-86 years, 100 males) with perianal fistula were examined with 3D endoanal ultrasound. All 3D volumes were stored and analysed retrospectively by two independent observers blinded to the clinical information of the patients. RESULTS: There were 29 patients with Crohn's disease of whom 20 (69%) showed CUFS. CUFS was absent in 125 (98%) of 128 patients without Crohn's disease. The positive and negative predictive value of CUFS for Crohn's disease was 87% and 93%, respectively. The kappa value of the two independent observers was 0.77, indicating a substantial interobserver agreement. CONCLUSION: This study provides a new 3D endoanal ultrasound criterion, CUFS, of perianal fistula in patients with Crohn's disease. The sign can be used to discriminate a Crohn's from other types of fistula, which may be useful in the management of patients with anal fistula.

Rho kinase signalling mediates radiation-induced inflammation and intestinal barrier dysfunction.

BACKGROUND: Radiotherapy is important in the management of pelvic malignancies, but radiation-induced intestinal damage is a dose-limiting factor. Microvascular injury and epithelial barrier dysfunction are considered to be rate-limiting aspects in radiation-induced enteropathy. This study investigated the role of Rho kinase signalling in radiation-induced inflammation and intestinal barrier dysfunction. METHODS: The specific Rho kinase inhibitor Y-27632 (1 and 10 mg/kg) was given to C57BL/6J mice before challenge with 20 Gy radiation. Leucocyte- and platelet-endothelium interactions in the colonic microcirculation were assessed by intravital microscopy. Levels of myeloperoxidase (MPO) and CXC chemokines (macrophage inflammatory protein 2 and cytokine-induced neutrophil chemoattractant), and intestinal leakage were quantified after 16 h. RESULTS: Radiation increased leucocyte and platelet recruitment, MPO activity, CXC chemokine production and intestinal leakage. Y-27632 significantly reduced radiation-induced leucocyte rolling and abolished adhesion; it also decreased platelet rolling and adhesion by 55 and 74 per cent respectively (P < 0·050). Inhibition of Rho kinase signalling significantly decreased radiation-provoked formation of CXC chemokines, MPO activity by 52 per cent, and intestinal leakage by 67 per cent (P < 0·050). CONCLUSION: Rho kinase activity constitutes an important signalling mechanism in radiation-induced inflammation and intestinal barrier dysfunction.

Role of platelets in experimental acute pancreatitis.

BACKGROUND: Platelets not only control thrombosis and haemostasis but may also regulate inflammatory processes. Acute pancreatitis (AP) is characterized by changes in both coagulation and proinflammatory activities. The role of platelets in AP is not yet known. METHODS: AP was induced in C57BL/6 mice by repeated caerulein administration (50 µg/kg intraperitoneally). Mice received a platelet-depleting or control antibody before caerulein challenge. Neutrophil infiltration, myeloperoxidase (MPO) and macrophage inflammatory protein (MIP) 2 levels, acinar cell necrosis and haemorrhage in the pancreas, as well as serum amylase activity, were determined 24 h after caerulein injection. In an alternative model of pancreatitis, L-arginine (4 g/kg intraperitoneally) was given twice with an interval of 1 h and tissue samples were taken after 72 h [Correction added after online publication 29 September 2010: in the preceding sentence, 4 mg/kg was corrected to 4 g/kg]. RESULTS: Caerulein administration increased acinar cell necrosis, neutrophil infiltration, focal haemorrhage and serum amylase levels. Platelet depletion reduced acinar cell necrosis, haemorrhage and serum amylase levels in AP. Depletion of platelets decreased caerulein-induced MPO levels and neutrophil recruitment in the pancreas. Platelet depletion abolished caerulein-induced MIP-2 generation in the pancreas and circulation. The effects of platelet depletion on necrosis, neutrophils and MPO levels were confirmed in L-arginine-induced pancreatitis. CONCLUSION: Platelets play a crucial role in AP by regulating neutrophil infiltration, most likely mediated by MIP-2 production in the pancreas.

CD44/CD70 blockade and anti-CD154/LFA-1 treatment synergistically suppress accelerated rejection and prolong cardiac allograft survival in mice.

Current treatments that are efficient in controlling effector T cell responses to allografts have limited efficacy on the accelerated rejection mediated by memory T cells. Effective targeting of alloreactive memory T cells may therefore be explored to improve therapeutic approaches towards solving this problem. In this study, we investigated the synergistic effect of CD44/CD70 blockade and anti-CD154/LFA-1 treatment on the accelerated rejection mediated by memory T cells. While CD44/CD70 blockade had limited effects on the alloresponses of effector T cells in vivo, it diminished the expansion of both CD4(+) and CD8(+) memory T cells in recipients adoptively transferred with donor-sensitized T cells. In combination with anti-CD154/LFA-1 treatment, CD44/CD70 blockade significantly prolonged cardiac allograft survival in adoptive transfer recipients. We demonstrated that treatment with the combination of all four antibodies (anti-CD154/LFA-1/CD44/CD70) inhibited accelerated rejection by markedly suppressing the alloresponses of effector and memory T cells and reducing the number of graft-infiltration lymphocytes in adoptive transfer recipients. Meanwhile, CD44/CD70 blockade and anti-CD154/LFA-1 treatment synergically enhanced regulatory T cells (Tregs) by increasing the proportion of splenic Tregs and the expression of IL-10 in these recipients. Our findings contribute to the potential design of therapies for accelerated allograft rejection.

The major histocompatibility complex (MHC) of the secondary transplant tissue donor influences the cross-reactivity of alloreactive memory cells.

Memory cells are currently thought to be a major barrier to tolerance induction in transplantation. However, whether alloreactive memory cells resulting from a primary transplant have cross-reactivity in a second transplant is unclear. Here, we used skin transplantation from BALB/c mice donors to presensitize C57 BL/6 (B6) mice. One month later, several strains of mice (including BALB/c, DBA/2, NOD, C3H and B6 mice) were chosen as donors to construct a memory model of heterotopic cardiac transplantation. The higher degree of major histocompatibility complex (MHC) mismatch to sensitizing MHC resulted in longer median survival times (MSTs, BALB/c 3.63 days versus C3H 6.08 days). After 3.5 days of cardiac transplantation, compared with the BALB/c and DBA/2 groups, in the groups of NOD and C3H, the infiltration of inflammatory cells in the grafts, the proportion and proliferation of memory cells in spleens and the function of allogeneic antibodies decreased significantly. The varying degrees of MHC mismatch between the primary and secondary donors influenced the intensity of alloreactive memory cell function, the higher degree of MHC mismatch resulted in better tolerance during secondary transplantation, and these may be related to the changed activation, proliferation and function of the alloreactive memory cells.

Long-term survival after self-expanding metallic stent or stoma decompression as bridge to surgery in acute malignant large bowel obstruction.

AIM: Self-expanding metallic stents (SEMS) as bridge to surgery have been questioned due to the fear of perforation and tumour spread. This study aimed to compare SEMS and stoma as bridge to surgery in acute malignant large bowel obstruction in the Swedish population. METHOD: Medical records of patients identified via the Swedish Colorectal Cancer Register 2007-2009 were collected and scrutinized. The inclusion criterion was decompression intended as bridge to surgery due to acute malignant large bowel obstruction. Patients who underwent decompression for other causes or had bowel perforation were excluded. Primary endpoints were 5-year overall survival and 3-year disease-free survival. Secondary endpoints were 30-day morbidity and mortality rates. RESULTS: A total of 196 patients fulfilled the inclusion criterion (SEMS, 71, and stoma, 125 patients). There was no significant difference in sex, age, ASA score, TNM stage and adjuvant chemotherapy between the SEMS and stoma groups. No patient was treated with biological agents. Five-year overall survival was comparable in SEMS, 56 per cent (40 patients), and stoma groups, 48 per cent (60 patients), P = 0.260. Likewise, 3-year disease-free survival did not differ statistically significant, SEMS 73 per cent (43 of 59 patients), stoma 65 per cent (62 of 95 patients), P = 0.32. In the SEMS group, 1.4 per cent (one patient) did not fulfil resection surgery compared to 8.8 per cent (11 patients) in the stoma group (P = 0.040). Postoperative complication and 30-day postoperative mortality rates did not differ, whereas the duration of hospital stay and proportion of permanent stoma were lower in the SEMS group. CONCLUSION: This nationwide registry-based study showed that long-term survival in patients with either SEMS or stoma as bridge to surgery in acute malignant large bowel obstruction were comparable. SEMS were associated with a lower rate of permanent stoma, higher rate of resection surgery and shorter duration of hospital stay.

p38 Mitogen-activated protein kinase signalling regulates vascular inflammation and epithelial barrier dysfunction in an experimental model of radiation-induced colitis.

BACKGROUND: : Microvascular injury and epithelial barrier dysfunction are rate-limiting aspects in radiation enteropathy. This study examined the role of p38 mitogen-activated protein kinase (p38 MAPK) signalling in radiation-induced colitis in an experimental model. METHODS: : The p38 MAPK inhibitor SB239063 was administered to mice immediately before exposure to 20 Gy radiation. Leucocyte- and platelet-endothelium interactions in the colonic microcirculation were assessed by intravital microscopy. Levels of myeloperoxidase (MPO) and CXC chemokines (macrophage inflammatory protein (MIP) 2 and cytokine-induced neutrophil chemoattractant (KC)), and albumin leakage were quantified 16 h after irradiation. RESULTS: : Irradiation induced an increase in leucocyte and platelet recruitment, MPO activity, CXC chemokine levels and intestinal leakage. Inhibition of p38 MAPK by SB239063 decreased radiation-induced leucocyte and platelet recruitment (leucocyte rolling and adhesion by 70 and 90 per cent, both P < 0.001; that of platelets by 70 and 74 per cent, both P < 0.001). It also reduced radiation-provoked increases in colonic MPO activity by 88 per cent (P < 0.001), formation of MIP-2 and KC by 72 and 74 per cent respectively (P = 0.003 and P < 0.001), and intestinal leakage by 81 per cent (P < 0.001). CONCLUSION: : p38 MAPK is an important signalling pathway in radiation-induced colitis.

Soluble CD40L (CD154) is increased in patients with shock.

OBJECTIVE: Recent data suggest that soluble CD40L (sCD40L) plays an important role in murine sepsis. The aim of the present study was to determine plasma levels of CD40L in critically ill patients with systemic inflammatory response syndrome (SIRS) and shock, with and without sepsis. DESIGN: A prospective observational one-centre cohort study in a mixed-bed ICU of an university hospital. Fifty-three consecutive patients fulfilling the criteria for SIRS with shock as well as seven age-matched controls were included. ELISA was used to determine sCD40L in the plasma. RESULTS: The level of sCD40L in plasma from healthy controls was 0.18 ± 0.03 ng/ml. It was found that sCD40L levels were significantly higher in patients with non-septic shock (0.72 ± 0.18 ng/ml) and septic shock (0.50 ± 0.1 ng/ml). However, the levels of sCD40L were not different between these two groups of patients, or in those with low and high APACHE scores. CONCLUSION: Our data show that sCD40L is increased in patients with shock from septic and non-septic etiologies. However, further studies are needed to delineate the functional significance of sCD40L in the clinical outcome in shock patients.

Capture of platelets to the endothelium of the femoral vein is mediated by CD62P and CD162.

Platelets contribute to blood coagulation at sites of vascular injury and to the recruitment of leukocytes at sites of inflammation. Under pathological conditions, platelets are involved in numerous diseases and clinical complications, such as deep venous thrombosis, embolism and atherosclerosis. But so far, little is known about the mechanisms of inflammation in large veins and the role of platelets in inflamed large veins. For this purpose, we investigated primary and secondary interactions between platelets, leukocytes and endothelial cells in the femoral vein in vivo with special regard to the role of CD62P (P-selectin) and CD162 (PSGL-1). Mice were challenged with lipopolysaccharide (LPS)/D-galactosamine (D-gal) and either CD162 or CD62P was blocked by intravenous administration of a corresponding antibody at the time point of LPS/D-gal injection. Four hours after LPS/gal injection, intravital fluorescence microscopy of the femoral vein was performed and primary and secondary platelet-leukocyte-endothelial cell-interactions were visualized after in vivo platelet and leukocyte staining with rhodamine 6G. Analysis of intravital fluorescence microscopy revealed that LPS/D-gal caused a strong inflammatory reaction of the venous endothelium with significant induction of platelet and leukocyte tethering, rolling and adhesion. Secondary interactions of platelets to adherent or rolling platelets or leukocytes were also increased after LPS/D-gal-injection. Immunoneutralization of either CD162 or CD62P significantly decreased platelet primary and secondary capture as well as leukocyte rolling and adhesion. CD162 and CD62P play a central role in mediating inflammatory primary and secondary interactions of platelets and leukocytes to the endothelium in inflamed large veins in vivo. Thus, blocking CD162 or CD62P might be an attractive tool for preventing platelet and leukocyte-driven venous diseases.

Platelet-dependent accumulation of leukocytes in sinusoids mediates hepatocellular damage in bile duct ligation-induced cholestasis.

BACKGROUND: Although it is well known that extrahepatic cholestasis induces liver damage, the mechanisms are still not completely understood. The aim of the present study was to evaluate the role of platelets and P-selectin in cholestasis-induced liver injury. EXPERIMENTAL APPROACH: C57BL/6 mice underwent bile duct ligation (BDL) and pretreatment with an anti-GP1balpha antibody, which depletes platelets, an anti-P-selectin antibody or a control antibody. Hepatic platelet and leukocyte recruitment as well as microvascular perfusion were determined by intravital fluorescence microscopy. KEY RESULTS: BDL caused significant liver damage and sinusoidal perfusion failure. BDL further induced hepatic platelet accumulation with widespread intravascular platelet aggregates, increased platelet adhesion in postsinusoidal venules and massive platelet accumulation in liver sinusoids. Administration of the anti-GP1balpha antibody reduced systemic platelet count by 90%. Depletion of platelets in BDL mice not only abolished accumulation and adhesion of platelets in sinusoids and venules but also restored sinusoidal perfusion and reduced liver enzymes by more than 83%. Platelet depletion further reduced BDL-associated sinusoidal leukocyte accumulation by 48% although leukocyte-endothelium interactions in venules were not affected. Immunoneutralization of P-selectin also inhibited hepatic microvascular accumulation of platelets and leukocytes, and protected against cholestasis-provoked hepatocellular damage. CONCLUSIONS AND IMPLICATIONS: Platelets play an important role in BDL-induced liver injury by promoting leukocyte recruitment and deteriorating microvascular perfusion. Moreover, our findings demonstrate that cholestasis-induced accumulation of platelets is mediated by P-selectin. Thus, targeting platelet accumulation may be a useful strategy against liver damage associated with obstructive jaundice.

Rho-kinase signalling mediates endotoxin hypersensitivity after partial hepatectomy.

BACKGROUND: Excessive loss of functional liver mass results in hepatic dysfunction and is associated with an increased sensitivity to infection. This experimental study investigated the role of Rho-kinase in hepatectomy-induced sensitization to endotoxin. METHODS: Male C57BL/6J mice underwent 68 per cent hepatectomy and were injected 24 h later with 100 microg Escherichia coli lipopolysaccharide (LPS). Simultaneously, animals received either fasudil or Y-27632 for Rho-kinase inhibition, or phosphate-buffered saline. Untreated hepatectomized animals served as positive controls and sham-operated animals as negative controls. Liver injury and inflammatory parameters were assessed 6 h after LPS challenge by serum alanine aminotransferase (ALT) levels, histomorphology and enzyme-linked immunosorbent assay. RESULTS: Hepatectomy resulted in a significant susceptibility to LPS, as indicated by inflammatory leucocyte recruitment (mean(s.e.m.) 10(1) leucocytes per high-power field), hepatocellular disintegration (ALT 22.4(3.1) microkat/l) and apoptotic cell death (3.8(0.2) per cent). Rho-kinase inhibition reduced leucocytic infiltration by more than 33 per cent, abolished hepatocellular apoptosis entirely, and reduced tumour necrosis factor alpha expression by more than 48 per cent and CXC chemokine expression by more than 36 per cent. CONCLUSION: Hepatectomy increased susceptibility to LPS by Rho-kinase-dependent mechanisms. Blocking Rho-kinase signalling decreased LPS-induced liver injury in hepatectomized mice.