Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24h of septic onset, Interleukins (IL)-8, -1beta, -6, -10, and -12p70, tumor necrosis factor-alpha (TNF-alpha), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p<0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p<0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p<0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR=1.3, p=0.01 for CD64 and OR=1.26, p=0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

Soluble guanylyl cyclase activation by HMR-1766 (ataciguat) in cells exposed to oxidative stress

Many vascular diseases are characterized by increased levels of ROS that destroy the biological activity of nitric oxide and limit cGMP formation. In the present study, we investigated the cGMP-forming ability of HMR-1766 in cells exposed to oxidative stress. Pretreatment of smooth muscle cells with H(2)O(2) reduced cGMP production stimulated by sodium nitroprusside (SNP) or BAY 41-2272. However, pretreatment with H(2)O(2) significantly increased HMR-1766 responses. Similar results were obtained with SIN-1, menadione, and rotenone. In addition, HMR-1766 was more effective in stimulating heme-free sGC compared with the wild-type enzyme. Interestingly, in cells expressing heme-free sGC, H(2)O(2) inhibited instead of potentiated HMR-1766 responses, suggesting that the ROS-induced enhancement of cGMP formation was heme dependent. Moreover, using truncated forms of sGC, we observed that the NH(2)-terminus of the beta(1)-subunit is required for the action of HMR-1766. Finally, to study tolerance development to HMR-1766, cells were pretreated with this sGC activator and reexposed to HMR-1766 or SNP. Results from these experiments demonstrated lack of tolerance development to HMR-1766 as well as lack of cross-tolerance with SNP. We conclude that HMR-1766 is an improved sGC activator as it has the ability to activate oxidized/heme-free sGC and is resistant to the development of tolerance; these observations make HMR-1766 a promising agent for treating diseases associated with increased vascular tone combined with enhanced ROS production.     

Regulation of Ang2 release by PTEN/PI3‐kinase/Akt in lung microvascular endothelial cells

Angiopoietin‐2 (Ang2) is a Tie‐2 ligand that destabilizes vascular structures, allowing for neovascularization or vessel regression depending on local vascular endothelial cell growth factor (VEGF) concentrations. Although various stimuli have been shown to affect Ang2 expression, information on the underlying mechanisms involved in Ang2 production in endothelial cells (EC) is just beginning to emerge. In the present study, we have used adenovirus‐mediated gene transfer and pharmacological inhibitors to examine the role of the PTEN/PI3‐K/Akt pathway on Ang2 release. Inhibition of PI3‐kinase with wortmannin led to a stimulation of basal Ang2 release in EC, while overexpression of an active form of Akt reduced Ang2. In addition, adenovirus‐mediated gene transfer of the phosphatase PTEN stimulated Ang2 release. Incubation of the cells with Ang1, an agent that activates the PI3‐K/Akt pathway in EC, reduced Ang2 release. This effect of Ang1 could be prevented by wortmannin and LY‐294002 pretreatment. Similarly, in VEGF‐treated EC the increase in Ang2 production observed was greater in the presence of a PI3‐K inhibitor. Our observations that PTEN acts as a positive modulator of Ang2 release, while activation of the PI3‐K/Akt pathway downregulates Ang2, reveal an additional mechanism through which the PTEN/PI3‐K/Akt pathway could affect the angiogenic process. J. Cell. Physiol. 209: 239, 2006. © 2006 Wiley‐Liss, Inc.     

Plasma pro- and anti-inflammatory cytokine levels and outcome prediction in unselected critically ill patients

Purpose. To determine the inter-relationships between cytokine levels and physiological scores in predicting outcome in unselected, critically ill patients. Methods. To this end, 127 patients (96 men), having a mean ± SD age of 45 ± 20 years, with a wide range in admission diagnoses (medical, surgical, and multiple trauma patients) were prospectively investigated. Severity of critical illness and organ dysfunction were graded by acute physiology and chronic health evaluation (APACHE II) and sequential organ failure assessment (SOFA) scores, respectively. Blood samples were drawn on admission in the ICU to determine pro- and anti-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and IL-10. The main outcome measure was 28-day mortality. Results. Overall, 88 patients survived and 39 patients died. Univariate logistic regression analysis showed that SOFA, APACHE II, IL-8, IL-6, and IL-10 on admission in the ICU were related to mortality. Multiple logistic regression analysis in the entire cohort of critically ill patients revealed that SOFA (OR = 1.341, p < 0.001) and IL-6 (OR = 1.075, p = 0.01) constituted independent outcome predictors. receiver operator characteristics curve analysis showed that SOFA, APACHE II, and IL-6 had the highest area under the curve values. IL-6 correlated with APACHE II (r_s = 0.44, p < 0.0001) and SOFA (r_s = 0.40, p < 0.0001) scores. Conclusions. In mixed ICU patients cytokine concentrations on admission in the ICU represent independent outcome predictors in the presence of disease severity scores.     

Εx-vivo effect of dexamethasone on cytokine production from whole blood of septic patients: Correlation with disease severity

Background: Controversial findings of former clinical trials on the effect of low dose hydrocortisone in patients with septic shock led to investigate the effect of corticosteroids on the production of cytokines from endotoxin (LPS)-stimulated whole blood. Methods: Whole blood from 33 septic patients was sampled within 24 h alter diagnosis. Hydrocortisone was not administered during follow-up. Whole blood was stimulated with 30 ng/ml of LPS in the presence of 0.01, 0.1, 1 and 10 μM of dexamethasone. Concentrations of cytokines and of sTREM-1 were estimated in supernatants after six hours of incubation. Results: Dexamethasone inhibited LPS-stimulated release of ΤΝFα, of IL-6, of IL-8 and of IL-10 in dose-dependent manner. A dual effect on the kinetics of release of IL-1β and of sTREM-1 was shown. Release of IL-1β was either decreased, what was connected with unfavorable outcome, or it was unaffected what was connected with a favorable outcome. Release of sTREM-1 was either increased, what was connected with unfavorable outcome, or it was decreased what was connected with a favorable outcome. Conclusions: Part of the beneficiary effect of corticosteroids in sepsis may be due to an effect on the release of IL-1β and of sTREM-1. This effect does not seem to be homogeneous for all septic patients.     

Inhibition of HMGCoA reductase by simvastatin protects mice from injurious mechanical ventilation

Background

Mortality from severe acute respiratory distress syndrome exceeds 40% and there is no available pharmacologic treatment. Mechanical ventilation contributes to lung dysfunction and mortality by causing ventilator-induced lung injury. We explored the utility of simvastatin in a mouse model of severe ventilator-induced lung injury.

Methods

Male C57BL6 mice (n = 7/group) were pretreated with simvastatin or saline and received protective (8 mL/kg) or injurious (25 mL/kg) ventilation for four hours. Three doses of simvastatin (20 mg/kg) or saline were injected intraperitoneally on days −2, −1 and 0 of the experiment. Lung mechanics, (respiratory system elastance, tissue damping and airway resistance), were evaluated by forced oscillation technique, while respiratory system compliance was measured with quasi-static pressure-volume curves. A pathologist blinded to treatment allocation scored hematoxylin-eosin-stained lung sections for the presence of lung injury. Pulmonary endothelial dysfunction was ascertained by bronchoalveolar lavage protein content and lung tissue expression of endothelial junctional protein Vascular Endothelial cadherin by immunoblotting. To assess the inflammatory response in the lung, we determined bronchoalveolar lavage fluid total cell content and neutrophil fraction by microscopy and staining in addition to Matrix-Metalloprotease-9 by ELISA. For the systemic response, we obtained plasma levels of Tumor Necrosis Factor-α, Interleukin-6 and Matrix-Metalloprotease-9 by ELISA. Statistical hypothesis testing was undertaken using one-way analysis of variance and Tukey’s post hoc tests.

Results

Ventilation with high tidal volume (HVt) resulted in significantly increased lung elastance by 3-fold and decreased lung compliance by 45% compared to low tidal volume (LVt) but simvastatin abrogated lung mechanical alterations of HVt. Histologic lung injury score increased four-fold by HVt but not in simvastatin-pretreated mice. Lavage pleocytosis and neutrophilia were induced by HVt but were significantly attenuated by simvastatin. Microvascular protein permeability increase 20-fold by injurious ventilation but only 4-fold with simvastatin. There was a 3-fold increase in plasma Tumor Necrosis Factor-α, a 7-fold increase in plasma Interleukin-6 and a 20-fold increase in lavage fluid Matrix-Metalloprotease-9 by HVt but simvastatin reduced these levels to control. Lung tissue vascular endothelial cadherin expression was significantly reduced by injurious ventilation but remained preserved by simvastatin.

Conclusion

High-dose simvastatin prevents experimental hyperinflation lung injury by angioprotective and anti-inflammatory effects.     

Soluble guanylyl cyclase expression is reduced in LPS-induced lung injury

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC alpha1 and beta1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the beta1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in beta1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased alpha1 and beta1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-alpha was required for the LPS-triggered reduction in sGC protein levels, as no change in alpha1 and beta1 levels was observed in TNF-alpha knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.     

Regulation of Ang2 release by PTEN/PI3-kinase/Akt in lung microvascular endothelial cells

Angiopoietin-2 (Ang2) is a Tie-2 ligand that destabilizes vascular structures, allowing for neovascularization or vessel regression depending on local vascular endothelial cell growth factor (VEGF) concentrations. Although various stimuli have been shown to affect Ang2 expression, information on the underlying mechanisms involved in Ang2 production in endothelial cells (EC) is just beginning to emerge. In the present study, we have used adenovirus-mediated gene transfer and pharmacological inhibitors to examine the role of the PTEN/PI3-K/Akt pathway on Ang2 release. Inhibition of PI3-kinase with wortmannin led to a stimulation of basal Ang2 release in EC, while overexpression of an active form of Akt reduced Ang2. In addition, adenovirus-mediated gene transfer of the phosphatase PTEN stimulated Ang2 release. Incubation of the cells with Ang1, an agent that activates the PI3-K/Akt pathway in EC, reduced Ang2 release. This effect of Ang1 could be prevented by wortmannin and LY-294002 pretreatment. Similarly, in VEGF-treated EC the increase in Ang2 production observed was greater in the presence of a PI3-K inhibitor. Our observations that PTEN acts as a positive modulator of Ang2 release, while activation of the PI3-K/Akt pathway downregulates Ang2, reveal an additional mechanism through which the PTEN/PI3-K/Akt pathway could affect the angiogenic process.     

Genetic polymorphisms within tumor necrosis factor gene promoter region: a role for susceptibility to ventilator-associated pneumonia

Debatable findings exist among various studies regarding the impact of single nucleotide polymorphisms (SNPs) within the promoter region of the tumor necrosis factor (TNF) gene for susceptibility to infections. Their impact was investigated in a cohort of mechanically ventilated patients who developed ventilator-associated pneumonia (VAP). Two-hundred and thirteen mechanically ventilated patients who developed VAP were enrolled. Genomic DNA was extracted and SNPs at the -376, -308 and -238 position of the promoter region of the TNF gene were assessed by restriction fragment length polymorphisms. Monocytes were isolated from 47 patients when they developed sepsis and stimulated by bacterial endotoxin for the production of TNFα and of interleukin-6 (IL-6). Patients were divided into two groups; 166 patients bearing only wild-type alleles of all three studied polymorphisms; and 47 patients carrying at least one A allele of the three studied SNPs. Time between start of mechanical ventilation and advent of VAP was significantly shorter in the second group than in the first group (log-rank: 4.416, p: 0.041). When VAP supervened, disease severity did not differ between groups. Stimulation of TNFα and of IL-6 was much greater by monocytes for patients carrying A alleles. Carriage of at least one A allele of the three studied SNPs at the promoter region of the TNF-gene is associated with shorter time to development of VAP but it is not associated with disease severity. Findings may be related with a role of the studied SNPs in the production of pro-inflammatory cytokines.     

Urethan anesthesia protects rats against lethal endotoxemia and reduces TNF-alpha release

Kotanidou, Anastasia, Augustine M. K. Choi, Richard A. Winchurch, Leo Otterbein, and Henry E. Fessler. Urethan anesthesia protects rats against lethal endotoxemia and reduces TNF- release. J. Appl. Physiol. 81(5):2304-2311, 1996.Urethan is a commonly used animalanesthetic for nonrecovery laboratory surgery. However, urethan hasdiverse biological effects that may complicate the interpretation ofexperimental findings. This study examined the effect of urethan on theresponse to an intravenous bolus of lipopolysaccharide (LPS; 30 mg/kg)in rats. In instrumented rats, urethan (1.2 gm/kg ip) completelyprevented the fall in arterial pressure immediately after LPSadministration but did not prevent late cardiovascular collapse. Inuninstrumented rats, urethan also attenuated indexes of organ injurymeasured 4 h after LPS administration, including mural bowelhemorrhage, hemoconcentration, hypoglycemia, metabolic acidosis, andlung myeloperoxidase activity, a measure of neutrophil sequestration.The peak increase in tumor necrosis factor- (TNF-) 90 min afterLPS administration was reduced 88% by urethan (2,060 ± 316 vs.16,934 ± 847 pg/ml; P < 0.001).In uninstrumented animals, urethan at 1.2 gm/kg reduced the 90%mortality rate of a lethal dose of LPS to 0-10% whengiven up to 24 h before LPS administration but did not reduce mortalitywhen given 2 h after LPS. Urethan neither directly bound LPS byLimulus assay nor inhibitedLPS-stimulated TNF- mRNA expression in cultured mouse peritonealmacrophages, but TNF- mRNA expression was suppressed by serum from aurethan-treated rat. Moreover, rauwolscine, which shares₂-adrenoceptor-blocking activity with urethan, also prevented death from a subsequent 90% lethal dose LPS bolus. We conclude that urethan or its metabolites protect against LPS, in part, by reducing TNF- release andspeculate that this may be mediated by₂-adrenoceptors. These actionsof urethan make it an undesirable anesthetic agent for in vivo studies of sepsis or LPS.