Search

Background Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. Methods ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. Results We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10–30 kDa ACE effector which is deficient in Gaucher spleen. Conclusions The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease.

Background: Angiotensin-converting enzyme (ACE) inhibitors (ACEI) are widely used in the management of cardiovascular diseases but with significant interindividual variability in the patient’s response. Objectives: To investigate whether interindividual variability in the response to ACE inhibitors is explained by the “ACE phenotype”—for example, variability in plasma ACE concentration, activity, and conformation and/or the degree of ACE inhibition in each individual. Methods: The ACE phenotype was determined in plasma of 14 patients with hypertension treated chronically for 4 weeks with 40 mg enalapril (E) or 20 mg E + 16 mg candesartan (EC) and in 20 patients with hypertension treated acutely with a single dose (20 mg) of E with or without pretreatment with hydrochlorothiazide. The ACE phenotyping included (1) plasma ACE concentration; (2) ACE activity (with 2 substrates: Hip-His-Leu and Z-Phe-His-Leu and calculation of their ratio); (3) detection of ACE inhibitors in patient’s blood (indicator of patient compliance) and the degree of ACE inhibition (ie, adherence); and (4) ACE conformation. Results: Enalapril reduced systolic and diastolic blood pressure in most patients; however, 20% of patients were considered nonresponders. Chronic treatment results in 40% increase in serum ACE concentrations, with the exception of 1 patient. There was a trend toward better response to ACEI among patients who had a higher plasma ACE concentration. Conclusion: Due to the fact that “20% of patients do not respond to ACEI by blood pressure drop,” the initial blood ACE level could not be a predictor of blood pressure reduction in an individual patient. However, ACE phenotyping provides important information about conformational and kinetic changes in ACE of individual patients, and this could be a reason for resistance to ACE inhibitors in some nonresponders.

Background Matrix-metalloproteinases (MMP) and cancer cell invasion are crucial for solid tumour metastasis. Important signalling events triggered by inflammatory cytokines, such as tumour necrosis factor α (TNFα), include Src-kinase-dependent activation of Akt and focal adhesion kinase (FAK) and phosphorylation of caveolin-1. Based on previous studies where we demonstrated amide-type local anaesthetics block TNFα-induced Src activation in malignant cells, we hypothesized that local anaesthetics might also inhibit the activation and/or phosphorylation of Akt, FAK and caveolin-1, thus attenuating MMP release and invasion of malignant cells. Methods NCI-H838 lung adenocarcinoma cells were incubated with ropivacaine or lidocaine (1 nM-100 µM) in absence/presence of TNFα (20 ng ml−1) for 20 min or 4 h, respectively. Activation/phosphorylation of Akt, FAK and caveolin-1 were evaluated by Western blot, and MMP-9 secretion was determined by enzyme-linked immunosorbent assay. Tumour cell migration (electrical wound-healing assay) and invasion were also assessed. Results Ropivacaine (1 nM–100 μM) and lidocaine (1–100 µM) significantly reduced TNFα-induced activation/phosphorylation of Akt, FAK and caveolin-1 in NCI-H838 cells. MMP-9 secretion triggered by TNFα was significantly attenuated by both lidocaine and ropivacaine (half-maximal inhibitory concentration [IC50]=3.29×10−6 M for lidocaine; IC50=1.52×10−10 M for ropivacaine). The TNFα-induced increase in invasion was completely blocked by both lidocaine (10 µM) and ropivacaine (1 µM). Conclusions At clinically relevant concentrations both ropivacaine and lidocaine blocked tumour cell invasion and MMP-9 secretion by attenuating Src-dependent inflammatory signalling events. Although determined entirely in vitro, these findings provide significant insight into the potential mechanism by which local anaesthetics might diminish metastasis.

Triglyceride micro-emulsions such as Intralipid® have been used to reverse cardiac toxicity induced by a number of drugs but reservations about their broad-spectrum applicability remain because of the poorly understood mechanism of action. Herein we report an integrated mechanism of reversal of bupivacaine toxicity that includes both transient drug scavenging and a cardiotonic effect that couple to accelerate movement of the toxin away from sites of toxicity. We thus propose a multi-modal therapeutic paradigm for colloidal bio-detoxification whereby a micro-emulsion both improves cardiac output and rapidly ferries the drug away from organs subject to toxicity. In vivo and in silico models of toxicity were combined to test the contribution of individual mechanisms and reveal the multi-modal role played by the cardiotonic and scavenging actions of the triglyceride suspension. These results suggest a method to predict which drug toxicities are most amenable to treatment and inform the design of next-generation therapeutics for drug overdose.

Background: Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase–Akt–nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability. Methods: Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed. Results: Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10−10 M for ropivacaine; IC50 = 5.864 × 10−10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10−10 M for ropivacaine; IC50 = 6.377 × 10−10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated. Conclusions: Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory “side-effect” of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

Mechanical ventilation of lungs is capable of activating the innate immune system and inducing sterile inflammatory response. The proinflammatory cytokine IL-1β is among the definitive markers for accurately identifying ventilator-induced lung inflammation. However, mechanisms of IL-1β release during mechanical ventilation are unknown. In this study, we show that cyclic stretch activates the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasomes and induces the release of IL-1β in mouse alveolar macrophages via caspase-1– and TLR4-dependent mechanisms. We also observed that NADPH oxidase subunit gp91phox was dispensable for stretch-induced cytokine production, whereas mitochondrial generation of reactive oxygen species was required for stretch-induced NLRP3 inflammasome activation and IL-1β release. Further, mechanical ventilation activated the NLRP3 inflammasomes in mouse alveolar macrophages and increased the production of IL-1β in vivo. IL-1β neutralization significantly reduced mechanical ventilation-induced inflammatory lung injury. These findings suggest that the alveolar macrophage NLRP3 inflammasome may sense lung alveolar stretch to induce the release of IL-1β and hence may contribute to the mechanism of lung inflammatory injury during mechanical ventilation.

Caveolin-1 is a key regulator of pulmonary endothelial barrier function. Here, we tested the hypothesis that caveolin-1 expression is required for ventilator-induced lung injury (VILI). Caveolin-1 gene-disrupted (Cav-1(-/-)) and age-, sex-, and strain-matched wild-type (WT) control mice were ventilated using two protocols: volume-controlled with protective (8 mL/kg) versus injurious (21 mL/Kg) tidal volume for up to 6 hours; and pressure-controlled with protective (airway pressure = 12 cm H(2)O) versus injurious (30 cm H(2)O) ventilation to induce lung injury. Lung microvascular permeability (whole-lung (125)I-albumin accumulation, lung capillary filtration coefficient [K(f, c)]) and inflammatory markers (bronchoalveolar lavage [BAL] cytokine levels and neutrophil counts) were measured. We also evaluated histologic sections from lungs, and the time course of Src kinase activation and caveolin-1 phosphorylation. VILI induced a 1.7-fold increase in lung (125)I-albumin accumulation, fourfold increase in K(f, c), significantly increased levels of cytokines CXCL1 and interleukin-6, and promoted BAL neutrophilia in WT mice. Lung injury by these criteria was significantly reduced in Cav-1(-/-) mice but fully restored by i.v. injection of liposome/Cav-1 cDNA complexes that rescued expression of Cav-1 in lung microvessels. As thrombin is known to play a significant role in mediating stretch-induced vascular injury, we observed in cultured mouse lung microvascular endothelial cells (MLECs) thrombin-induced albumin hyperpermeability and phosphorylation of p44/42 MAP kinase in WT but not in Cav-1(-/-) MLECs. Thus, caveolin-1 expression is required for mechanical stretch-induced lung inflammation and endothelial hyperpermeability in vitro and in vivo.

Background Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have antiinflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells. Methods NCI-H838 lung cancer cells were incubated with tumor necrosis factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1 nM-100 μM). Cell migration and total cell lysate Src-activation and intercellular adhesion molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated. Results Ropivacaine treatment (100 μM) of H838 cells for 20 min decreased basal Src activity by 62% (P=0.003), and both ropivacaine and lidocaine coadministered with tumor necrosis factor-α statistically significantly decreased Src-activation and intercellular adhesion molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 h was inhibited by 26% (P=0.005) in presence of 1 μM ropivacaine and 21% by 1 μM lidocaine (P=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition. Conclusions This study indicates that amide-, but not ester-linked, local anesthetics may provide beneficial antimetastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of tumor necrosis factor-α-induced Src-activation and intercellular adhesion molecule-1 phosphorylation, providing the first evidence of a molecular mechanism that appears to be independent of their known role as sodium-channel blockers.

Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.

Lung hyperinflation is known to be an important contributing factor in the pathogenesis of ventilator-induced lung injury. Mechanical stretch causes epithelial barrier dysfunction and an increase in alveolar permeability, although the precise mechanisms have not been completely elucidated. p120-catenin is an adherens junction-associated protein that regulates cell-cell adhesion. In this study, we determined the role of p120-catenin in cyclic stretch-induced alveolar epithelial barrier dysfunction. Cultured alveolar epithelial cells (MLE-12) were subjected to uniform cyclic (0.5 Hz) biaxial stretch from 0 to 8 or 20% change in surface area for 0, 1, 2, or 4 h. At the end of the experiments, cells were lysed to determine p120-catenin expression by Western blot analysis. Immunofluorescence staining of p120-catenin and F-actin was performed to assess the integrity of monolayers and interepithelial gap formation. Compared with unstretched control cells, 20% stretch caused a significant loss in p120-catenin expression, which was coupled to interepithelial gap formation. p120-Catenin knockdown with small interfering RNA (siRNA) dose dependently increased stretch-induced gap formation, whereas overexpression of p120-catenin abolished stretch-induced gap formation. Furthermore, pharmacological calpain inhibition or depletion of calpain-1 with a specific siRNA prevented p120-catenin loss and subsequent stretch-induced gap formation. Our findings demonstrate that p120-catenin plays a critical protective role in cyclic stretch-induced alveolar barrier dysfunction, and, thus, maintenance of p120-catenin expression may be a novel therapeutic strategy for the prevention and treatment of ventilator-induced lung injury.

Reduced lung capillary expression of angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, and of caveolin-1, an important regulator of endothelial cell signalling, has been demonstrated in various models of pulmonary arterial hypertension (PAH). We addressed the relationship between PAH and ACE expression in caveolin-1 knockout mice (Cav1(-/-)), which have moderate PAH. Tissue ACE activity was reduced by 50% in lungs from 3-month-old Cav1(-/-) mice compared to wild type (WT). A similar reduction in lung endothelial ACE expression was observed by measuring the lung uptake of (125)I-labeled monoclonal anti-ACE antibody and by quantitative immunohistochemistry. These alterations in ACE are limited to capillary segments of the pulmonary circulation. Functionally, the increase in pulmonary artery pressure (PAP) in response to ACE conversion of angiotensin I to angiotensin II in isolated, perfused mouse lungs was reduced significantly in Cav1(-/-) mice compared to WT. Thus, these complementary approaches demonstrate the dependence of lung microvascular endothelial cell ACE protein expression on caveolin-1 expression and underscore the vital role of caveolin-1-regulated pulmonary vascular homeostasis on endothelial ACE expression and activity. In summary, we have revealed a novel role of caveolin-1 in the regulation of ACE expression in pulmonary capillary endothelial cells. Further understanding of the mechanism by which reduced caveolin-1 expression leads altered pulmonary vascular development, PAH, and reduced ACE expression may have important clinical implications in patients with these severe lung diseases.

Background Lipid emulsion infusion reverses cardiovascular compromise due to local anesthetic overdose in laboratory and clinical settings. The authors compared resuscitation with lipid, epinephrine, and saline control in a rat model of bupivacaine-induced cardiac toxicity to determine whether lipid provides a benefit over epinephrine. Methods Bupivacaine, 20 mg/kg, was infused in rats anesthetized with isoflurane, producing asystole in all subjects. Ventilation with 100% oxygen and chest compressions were begun immediately, along with intravenous treatment with 30% lipid emulsion or saline (5-ml/kg bolus plus continuous infusion at 0.5 ml . kg . min) or epinephrine (30 microg/kg). Chest compressions were continued and boluses were repeated at 2.5 and 5 min until the native rate-pressure product was greater than 20% baseline. Electrocardiogram and arterial pressure were monitored continuously and at 10 min, arterial blood gas, central venous oxygen saturation, and blood lactate were measured. Effect size (Cohen d) was determined for comparisons at 10 min. Results Lipid infusion resulted in higher rate-pressure product (P < 0.001, d = 3.84), pH (P < 0.01, d = 3.78), arterial oxygen tension (P < 0.05, d = 2.8), and central venous oxygen saturation (P < 0.001, d = 4.9) at 10 min than did epinephrine. Epinephrine treatment caused higher lactate (P < 0.01, d = 1.48), persistent ventricular ectopy in all subjects, pulmonary edema in four of five rats, hypoxemia, and a mixed metabolic and respiratory acidosis by 10 min. Conclusions Hemodynamic and metabolic metrics during resuscitation with lipid surpassed those with epinephrine, which were no better than those seen in the saline control group. Further studies are required to optimize the clinical management of systemic local anesthetic toxicity.

Objectives Cocaine intoxication leads to over 500,000 emergency department visits annually in the United States and ethanol cointoxication occurs in 34% of those cases. Cardiotoxicity is an ominous complication of cocaine and cocaethylene overdose for which no specific antidote exists. Because infusion of lipid emulsion (Intralipid) can treat lipophilic local anesthetic toxicity and cocaine is an amphipathic local anesthetic, the authors tested whether lipid emulsion could attenuate cocaine cardiotoxicity in vivo. The effects of lipid emulsion were compared with the metabolically inert sulfobutylether-β-cyclodextrin (SBE-β-CD; Captisol) in an isolated heart model of cocaine and cocaethylene toxicity to determine if capture alone could exert similar benefit as lipid emulsion, which exhibits multimodal effects. The authors then tested if cocaine and cocaethylene, like bupivacaine, inhibit lipid-based metabolism in isolated cardiac mitochondria. Methods For whole animal experiments, Sprague-Dawley rats were anesthetized, instrumented, and pretreated with lipid emulsion followed by a continuous infusion of cocaine to assess time of onset of cocaine toxicity. For ex vivo experiments, rat hearts were placed onto a nonrecirculating Langendorff system perfused with Krebs-Henseleit solution. Heart rate, left ventricle maximum developed pressure (LVdevP), left ventricle diastolic pressure, maximum rate of contraction (+dP/dtmax), maximum rate of relaxation (–dP/dtmax), rate-pressure product (RPP = heart rate × LVdevP), and line pressure were monitored continuously during the experiment. A dose response to cocaine (10, 30, 50, and 100 μmol/L) and cocaethylene (10, 30, and 50 μmol/L) was generated in the absence or presence of either 0.25% lipid emulsion or SBE-β-CD. Substrate-specific rates of oxygen consumption were measured in interfibrillar cardiac mitochondria in the presence of cocaine, cocaethylene, ecgonine, and benzoylecgonine. Results Treatment with lipid emulsion delayed onset of hypotension (140 seconds vs. 279 seconds; p = 0.008) and asystole (369 seconds vs. 607 seconds; p = 0.02) in whole animals. Cocaine and cocaethylene induced dose-dependent decreases in RPP, +dP/dtmax, and –dP/dtmaxabs (p

Background: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions: Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distinguishing tissue-specific ACEs.

Background Caveolae mediated transendothelial transport of albumin has recently been shown to be the primary mechanism regulating microvascular endothelial albumin permeability. The authors investigated the effects of isoflurane and sevoflurane on pulmonary endothelial albumin permeability and assessed the potential role of the caveolae scaffold protein, caveolin-1, in these effects. Methods Isolated rat lungs and cultured rat lung microvessel endothelial cells (RLMVECs) were exposed to 1.0 or 2.0 minimum alveolar concentration (MAC) isoflurane or sevoflurane for 30 min. I-albumin permeability-surface area product and capillary filtration coefficient were determined in the isolated lungs. In RLMVECs, uptake and transendothelial transport of I-albumin were measured in the absence and presence of pretreatment with 2 mm methyl-beta-cyclodextrin, a caveolae-disrupting agent. Uptake of fluorescent-labeled albumin, as well as phosphorylation of Src kinase and caveolin-1, was also determined. In Y14F-caveolin-1 mutant (nonphosphorylatable) expressing RLMVECs, uptake of I-albumin and phosphorylation of caveolin-1 were evaluated. Results In the isolated lungs, 2.0 MAC isoflurane increased I-albumin permeability-surface area product by 48% without affecting capillary filtration coefficient. In RLMVECs, isoflurane more than doubled the uptake of I-albumin and caused a 54% increase in the transendothelial transport of I-albumin. These effects were blocked by pretreatment with methyl-beta-cyclodextrin. The isoflurane-induced increase in uptake of I-albumin in wild-type RLMVECs was abolished in the Y14F-caveolin-1 mutant expressing cells. Isoflurane also caused a twofold increase in Src and caveolin-1 phosphorylation. Neither 1.0 MAC isoflurane nor 1.0 or 2.0 MAC sevoflurane affected any index of albumin transport or phosphorylation of caveolin-1. Conclusion Isoflurane, but not sevoflurane, increased lung transendothelial albumin permeability through enhancement of caveolae-mediated albumin uptake and transport in the isolated lung. This effect may involve an enhanced phosphorylation of caveolin-1.

A patient's recovery from lung inflammatory injury or development of multi-system organ failure is determined by the host's ability to resolve inflammation and repair tissue damage, both of which require the clearance of apoptotic neutrophils by macrophages (efferocytosis). Here, we investigated the effects of isoflurane on macrophage efferocytosis and resolution of lung inflammatory injury. Treatment of murine bone marrow-derived macrophages (BMDMs) or alveolar macrophages with isoflurane dramatically enhanced phagocytosis of apoptotic neutrophils. Isoflurane significantly increased the surface expression of the receptor tyrosine kinase Mer in macrophages, but markedly decreased the levels of a soluble form of Mer protein in the medium. Isoflurane treatment also caused a decrease in a disintegrin and metalloproteinase 17 (ADAM17) on the cell surface and a concomitant increase in its cytoplasmic fraction. These responses induced by isoflurane were completely reversed by a pharmacological inhibitor or genetic deletion of AMP-activated protein kinase (AMPK). In a mouse model of lipopolysaccharide-induced lung injury, isoflurane accelerated the recovery of lung inflammation and injury that was coupled with an increase in the number of alveolar macrophages containing apoptotic bodies. In alveolar macrophage-depleted mice, administration of isoflurane-pretreated BMDMs facilitated resolution of lung inflammation following lipopolysaccharide challenge. Thus, isoflurane promoted resolution of lipopolysaccharide-induced lung inflammatory injury via enhancement of macrophage efferocytosis. Increased macrophage efferocytosis following isoflurane treatment correlates with upregulation of Mer surface expression through AMPK-mediated blockade of ADAM17 trafficking to the cell membrane.

The inflammatory response is a primary mechanism in the pathogenesis of ventilator-induced lung injury. Autophagy is an essential, homeostatic process by which cells break down their own components. We explored the role of autophagy in the mechanisms of mechanical ventilation-induced lung inflammatory injury. Mice were subjected to low (7 ml/kg) or high (28 ml/kg) tidal volume ventilation for 2 h. Bone marrow-derived macrophages transfected with a scrambled or autophagy-related protein 5 small interfering RNA were administered to alveolar macrophage-depleted mice via a jugular venous cannula 30 min before the start of the ventilation protocol. In some experiments, mice were ventilated in the absence and presence of autophagy inhibitors 3-methyladenine (15 mg/kg ip) or trichostatin A (1 mg/kg ip). Mechanical ventilation with a high tidal volume caused rapid (within minutes) activation of autophagy in the lung. Conventional transmission electron microscopic examination of lung sections showed that mechanical ventilation-induced autophagy activation mainly occurred in lung macrophages. Autophagy activation in the lungs during mechanical ventilation was dramatically attenuated in alveolar macrophage-depleted mice. Selective silencing of autophagy-related protein 5 in lung macrophages abolished mechanical ventilation-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and lung inflammatory injury. Pharmacological inhibition of autophagy also significantly attenuated the inflammatory responses caused by lung hyperinflation. The activation of autophagy in macrophages mediates early lung inflammation during mechanical ventilation via NLRP3 inflammasome signaling. Inhibition of autophagy activation in lung macrophages may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.

Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase-Akt-nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability.Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed.Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10 M for ropivacaine; IC50 = 5.864 × 10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10 M for ropivacaine; IC50 = 6.377 × 10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated.Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory "side-effect" of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

Background Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. Results We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.

Sepsis and septic shock remain the leading causes of death in intensive care units worldwide.1 Complex pathophysiology together with heterogeneous disease patterns are key features making the treatment of sepsis extremely challenging.2–5 Numerous approaches have been undertaken to attenuate the harmful host response to infection, with a mostly unsuccessful translation into clinical outcome. Despite new pathophysiological insights and major efforts in developing goal-directed therapies, mortality in septic patients remains considerably high, and the treatment of late stage diagnosed patients is generally associated with bad outcome.6 Volatile anesthetics such as desflurane, isoflurane, and sevoflurane have been identified as effective modifiers of the inflammatory response in various states of tissue injury, exerting beneficial effects on organ function and overall outcome in both animals7–12 and patients.13–16 Potential benefits of the application of volatile anesthetics in in vivo models of experimental sepsis have not been systematically explored, and their effect on survival remains unclear. Previous studies elucidating the protective potential of volatile anesthetics have traditionally focused on ischemia–reperfusion injury and not on sepsis. In addition, biomarkers of organ injury were determined, but outcome parameters were not.17 In this study, we investigated in a model of severe murine sepsis with intraabdominal focus (peritonitis) whether the volatile anesthetics desflurane, isoflurane, and sevoflurane impact on overall survival of septic animals. The cecal ligation and puncture (CLP) model is considered the “gold standard” in sepsis research with a disease profile similar to that in human sepsis in that it encompasses more of the clinical features and drug responses of human sepsis, than, for example, the lipopolysaccharide model,18,19 despite the absence of some key features (e.g., kidney and lung injury). In this study, mice were exposed to desflurane, isoflurane, or sevoflurane during induction of sepsis (conditioning). To investigate the effect in a postconditioning setting, desflurane and sevoflurane were applied 24 h after the CLP procedure, when the mice showed pronounced symptoms of inflammation. Survival as well as markers of renal and hepatic organ function was compared with the CLP group without intervention. Being aware of the limitations of animal models of sepsis, the current study illustrates the beneficial effects of the volatile anesthetics desflurane and sevoflurane in states of severe inflammation leading to a remarkably improved 7-day survival and reduced end-organ damage.

Background Hospitalized patients outside of the operating room frequently require emergency airway management. This study investigates complications of emergency airway management in critically ill adults, including: (1) the incidence of difficult and failed intubation; (2) the frequency of esophageal intubation; (3) the incidence of pneumothorax and pulmonary aspiration; (4) the hemodynamic consequences of emergent intubation, including death, during and immediately following intubation; and (5) the relationship, if any, between the occurrence of complications and supervision of the intubation by an attending physician. Methods Data were collected on consecutive tracheal intubations carried out by the intensive care unit team over a 10-month period. Non-anesthesia residents were supervised by anesthesia residents, critical care attending physicians, or anesthesia attending physicians. Results Two hundred ninety-seven consecutive intubations were carried out in 238 adult patients. Translaryngeal tracheal intubation was accomplished in all patients. Intubation was difficult in 8% of cases (requiring more than two attempts at laryngoscopy by a physician skilled in airway management). Esophageal intubation occurred in 25 (8%) of the attempts but all were recognized before any adverse sequelae resulted. New infiltrates suggestive of pulmonary aspiration were present on chest radiography after 4% of intubations. Seven patients (3%) died during or within 30 min of the procedure. Five of the seven patients had systemic hypotension (systolic blood pressure < or = 90 mmHg), and four of the five were receiving vasopressors to support systolic blood pressure. Patients with systolic hypotension were more likely to die after intubation than were normotensive patients (P < 0.001). There was no relationship between supervision by an attending physician and the occurrence of complications. Conclusions In critically ill patients, emergency tracheal intubation is associated with a significant frequency of major complications. In this study, complications were not increased when intubations were accomplished without the supervision of an attending physician as long as the intubation was carried out or supervised by an individual skilled in airway management. Mortality associated with emergent tracheal intubation is highest in patients who are hemodynamically unstable and receiving vasopressor therapy before intubation.

Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have antiinflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells.NCI-H838 lung cancer cells were incubated with tumor necrosis factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1 nM-100 μM). Cell migration and total cell lysate Src-activation and intercellular adhesion molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated.Ropivacaine treatment (100 μM) of H838 cells for 20 min decreased basal Src activity by 62% (P=0.003), and both ropivacaine and lidocaine coadministered with tumor necrosis factor-α statistically significantly decreased Src-activation and intercellular adhesion molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 h was inhibited by 26% (P=0.005) in presence of 1 μM ropivacaine and 21% by 1 μM lidocaine (P=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition.This study indicates that amide-, but not ester-linked, local anesthetics may provide beneficial antimetastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of tumor necrosis factor-α-induced Src-activation and intercellular adhesion molecule-1 phosphorylation, providing the first evidence of a molecular mechanism that appears to be independent of their known role as sodium-channel blockers.

Background: The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. Hypothesis: Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. Methodology/Principal Findings: The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The ‘‘short’’ ACE inhibitor enalaprilat (tripeptide analog) and ‘‘long’’ inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. Conclusions/Significance: The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon antihypertensive therapy.

BACKGROUND: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. METHODOLOGY/PRINCIPAL FINDINGS: HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. Conclusions/Significance The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).

Background Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death. Methodology/Principal Findings Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10–20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold. Conclusions/Significance Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.

Background Angiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots. Methodology/Principal Findings We have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension. Conclusions/Significance We have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease.

We investigated the role of caveolae in the mechanism of increased pulmonary vascular permeability and edema formation induced by the activation of polymorphonuclear neutrophils (PMNs). We observed that the increase in lung vascular permeability induced by the activation of PMNs required caveolin-1, the caveolae scaffold protein. The permeability increase induced by PMN activation was blocked in caveolin-1 knockout mice and by suppressing caveolin-1 expression in rats. The response was also dependent on Src phosphorylation of caveolin-1 known to activate caveolae-mediated endocytosis in endothelial cells. To address the role of PMN interaction with endothelial cells, we used an intercellular adhesion molecule (ICAM)-1 blocking monoclonal antibody. Preventing the ICAM-1–mediated PMN binding to endothelial cells abrogated Src phosphorylation of caveolin-1, as well as the increase in endothelial permeability. Direct ICAM-1 activation by crosslinking recapitulated these responses, suggesting that ICAM-1 activates caveolin-1 signaling responsible for caveolae-mediated endothelial hyperpermeability. Our results provide support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of PMNs adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury.

Caveolae mediated transendothelial transport of albumin has recently been shown to be the primary mechanism regulating microvascular endothelial albumin permeability. The authors investigated the effects of isoflurane and sevoflurane on pulmonary endothelial albumin permeability and assessed the potential role of the caveolae scaffold protein, caveolin-1, in these effects.Isolated rat lungs and cultured rat lung microvessel endothelial cells (RLMVECs) were exposed to 1.0 or 2.0 minimum alveolar concentration (MAC) isoflurane or sevoflurane for 30 min. I-albumin permeability-surface area product and capillary filtration coefficient were determined in the isolated lungs. In RLMVECs, uptake and transendothelial transport of I-albumin were measured in the absence and presence of pretreatment with 2 mm methyl-beta-cyclodextrin, a caveolae-disrupting agent. Uptake of fluorescent-labeled albumin, as well as phosphorylation of Src kinase and caveolin-1, was also determined. In Y14F-caveolin-1 mutant (nonphosphorylatable) expressing RLMVECs, uptake of I-albumin and phosphorylation of caveolin-1 were evaluated.In the isolated lungs, 2.0 MAC isoflurane increased I-albumin permeability-surface area product by 48% without affecting capillary filtration coefficient. In RLMVECs, isoflurane more than doubled the uptake of I-albumin and caused a 54% increase in the transendothelial transport of I-albumin. These effects were blocked by pretreatment with methyl-beta-cyclodextrin. The isoflurane-induced increase in uptake of I-albumin in wild-type RLMVECs was abolished in the Y14F-caveolin-1 mutant expressing cells. Isoflurane also caused a twofold increase in Src and caveolin-1 phosphorylation. Neither 1.0 MAC isoflurane nor 1.0 or 2.0 MAC sevoflurane affected any index of albumin transport or phosphorylation of caveolin-1.Isoflurane, but not sevoflurane, increased lung transendothelial albumin permeability through enhancement of caveolae-mediated albumin uptake and transport in the isolated lung. This effect may involve an enhanced phosphorylation of caveolin-1.

In humans, the sole endogenous source of carbon monoxide is heme degradation. We report the development of prolonged carboxyhemoglobinemia in a critically ill mechanically ventilated patient who required massive transfusion because of retroperitoneal hemorrhage secondary to pheochromocytoma. After the transfusion of 27 U of packed red blood cells, the maximum carboxyhemoglobin level was 6.4%. Although ventilation was controlled with a fraction of inspired oxygen of 0.35- 0.5 and external drainage of blood occurred, the concentration of carboxyhemoglobin remained at 1.7%-5.6% for days. Red blood cells for transfusion may be contaminated with carbon monoxide and may have carboxyhemoglobin levels of up to 12%; this may also have contributed to carboxyhemoglobinemia in our patient. If significantly increased concentrations of carboxyhemoglobin develop, therapy to decrease the concentration of carboxyhemoglobin (such as fraction of inspired oxygen of 1.0 and/or minute ventilation or hyperbaric oxygen) or removal of the source should be considered.

IMPLICATIONS The elimination of potassium in patients with end-stage kidney failure is limited. An increase in potassium concentrations can lead to lethal arrhythmias. In the described case, a large potassium concentration was treated during a liver transplantation using a new technical approach.

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.

Cerebral venous sinus thrombosis (CVST) can present with a headache similar to that after a dural puncture. We report on a patient who developed postural headache after epidural anesthesia for delivery. The headache became more intense during the following 6 days, and the patient had a tonic clonic seizure. A magnetic resonance angiogram demonstrated CSVT, and anticoagulation therapy was started, with resolution of the symptoms over 2 wk. Any postdural-puncture headache that loses its positional character, becomes persistent, or does not improve with a properly performed blood patch should raise the suspicion of CVST.

Volatile anesthetics are known to have immunomodulatory effects in conditions of organ injury. A recent study in an experimental sepsis model has shown remarkably improved survival when mice were exposed to volatile anesthetics. In the present study, we show that hexafluoroisopropanol – a water-soluble primary sevoflurane metabolite – has beneficial effects on the overall survival in a murine model of cecal ligation and puncture. Seven-day survival as well as tissue damage markers including transaminases and high mobility group box protein-1 were assessed as measures of end organ damage. In animals undergoing cecal ligation and puncture procedure hexafluoroisopropanol conditioning - but not late postconditioning 24 hours after sepsis induction - significantly increased survival rate (17% vs. 77%, p = 0.037) and attenuated secretion of organ damage markers. This study shows survival benefits by administration of the metabolite of a volatile anesthetic. If successfully translated, hexafluoroisopropanol might offer interesting therapeutic opportunities in the future treatment of abdominal sepsis.

Background Inhalational anesthetics have been shown to influence T cell functions both in vitro and in vivo, in many cases inducing T cell death, suggesting that exposure to these drugs could modify the course of an autoimmune disease. We tested the hypothesis that in mice immunized to develop experimental autoimmune encephalomyelitis (EAE), a well established model of multiple sclerosis (MS), treatment with the commonly used inhalational anesthetic sevoflurane would attenuate disease symptoms. Methods C57Bl6 female mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide residues 35 to 55 to induce a chronic demyelinating disease. At day 10 after immunization, the mice were subjected to 2 h of 2.5% sevoflurane in 100% oxygen, or 100% oxygen, alone. Following treatment, clinical scores were monitored up to 4 weeks, after which brain histology was performed to measure the effects on astrocyte activation and lymphocyte infiltration. Effects of sevoflurane on T cell activation were studied using splenic T cells isolated from MOG peptide-immunized mice, restimulated ex vivo with MOG peptide or with antibodies to CD3 and CD28, and in the presence of different concentrations of sevoflurane. T cell responses were assessed 1 day later by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for proliferation, lactate dehydrogenase (LDH) release for cell death, and inflammatory activation by production of interleukin (IL)-17 and interferon (IFN)γ. Results Clinical scores in the oxygen-treated group increased until day 28 at which time they showed moderate to severe disease (average clinical score of 2.9). In contrast, disease progression in the sevoflurane-treated group increased to 2.1 at day 25, after which it remained unchanged until the end of the study. Immunohistochemical analysis revealed reduced numbers of infiltrating leukocytes and CD4+ cells in the CNS of the sevoflurane-treated mice, as well as reduced glial cell activation. In splenic T cells, low doses of sevoflurane reduced IFNγ production, cell proliferation, and increased LDH release. Conclusions These results are the first to show attenuation of EAE disease by an inhaled anesthetic and are consistent with previous reports that inhaled anesthetics, including sevoflurane, can suppress T cell activation that, in the context of autoimmune diseases such as MS, could lead to reduced clinical progression.

The pathophysiologic role of high levels of circulating catecholamines in patients with congestive heart failure remains unclear. To assess the hemodynamic contribution of circulating catecholamines, metyrosine (alpha-methyl-p-tyrosine), an inhibitor of catecholamine synthesis, was administered to nine patients with acutely decompensated chronic congestive heart failure. Baseline left ventricular ejection fraction averaged 23.3 +/- 9.9%, whereas cardiac output averaged 3.69 +/- 1.03 liters/min, with a pulmonary wedge pressure of 27.4 +/- 8.5 mm Hg. After 48 h of metyrosine administration, plasma norepinephrine concentration decreased from 919.4 +/- 810.6 to 335.4 +/- 143.1 pg/ml (p less than 0.05). Plasma epinephrine concentration averaged 176.4 +/- 166.0 pg/ml at baseline, and was unchanged during metyrosine administration. Despite the significant decrease in circulating norepinephrine, no significant hemodynamic changes were observed during metyrosine administration. These results suggest that high levels of circulating norepinephrine may be more a marker of severe congestive heart failure than an important contributor to the underlying pathophysiology at this advanced stage of the disease process.