Prospective study of device-related complications in intensive care unit detected by virtual autopsy.

BACKGROUND: There has been increasing use of invasive techniques, such as extracorporeal organ support, in intensive care units (ICU), and declining autopsy rates. Thus, new measures are needed to maintain high-quality standards. We investigated the potential of computed tomography (CT)-based virtual autopsy to substitute for medical autopsy in this setting. METHODS: We investigated the potential of virtual autopsy by post-mortem CT to identify complications associated with medical devices in a prospective study of patients who had died in the ICU. Clinical records were reviewed to determine the number and types of medical devices used, and findings from medical and virtual autopsies, related and unrelated to the medical devices, were compared. RESULTS: Medical and virtual autopsies could be performed in 61 patients (Group M/V), and virtual autopsy only in 101 patients (Group V). In Group M/V, 41 device-related complications and 30 device malpositions were identified, but only with a low inter-method agreement. Major findings unrelated to a device were identified in about 25% of patients with a high level of agreement between methods. In Group V, 8 device complications and 36 device malpositions were identified. CONCLUSIONS: Device-related complications are frequent in ICU patients. Virtual and medical autopsies showed clear differences in the detection of complications and device malpositions. Both methods should supplement each other rather than one alone for quality control of medical devices in the ICU. Further studies should focus on the identification of special patient populations in which virtual autopsy might be of particular benefit. CLINICAL TRIAL REGISTRATION: NCT01541982.

Anti-angiogenic effects of novel cyclin-dependent kinase inhibitors with a pyrazolo[4,3-d]pyrimidine scaffold.

BACKGROUND AND PURPOSE: Cyclin-dependent kinase 5 (CDK5) has recently emerged as an attractive target in several tumour entities. Inhibition of CDK5 has been shown to have anti-angiogenic effects in vitro and in vivo. However, potent inhibitors of CDK5, which can be applied in vivo, are still scarce. We have recently developed a new series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines that show a preference for inhibiting CDK5 and tested them in vitro and in vivo in a murine model of hepatocellular carcinoma. EXPERIMENTAL APPROACH: All compounds were initially examined for effects on proliferation of HUVECs. The most potent compounds were then tested on migration, and one of them, LGR2674, was selected for assessing effects on nuclear fragmentation, cell cycle, cell viability and metabolic activity. Furthermore, LGR2674 was tested in a tube formation assay and in vivo in a murine model of hepatocellular carcinoma, induced by s.c. injection of HUH7 cells (measurement of in vivo toxicity, tumour vascularization, tumour cell proliferation and tumour size). KEY RESULTS: LGR2674 showed an EC50 in the low nanomolar range in the proliferation and migration assays. Cytotoxic effects started at 50 nM, a concentration that did not influence the cell cycle. In vivo, LGR2674 was well tolerated and caused a clear reduction in vessel density in the tumours; also tumour cell proliferation was inhibited and tumour growth retarded. CONCLUSIONS AND IMPLICATIONS: Pyrazolo[4,3-d]pyrimidine is a novel scaffold for the development of potent CDK inhibitors with in vivo potential. Such structures are good candidates for broadening our pharmacological arsenal against various tumours.

In vitro study of 2,3-dehydrosilybin and its galloyl esters as potential inhibitors of angiogenesis.

2,3-Dehydrosilybin exhibits substantial anticancer and antiangiogenic effects, which can be potentially improved by semi-synthetic modification such as esterification with gallic acid. The aim of this study was to examine the potential antiangiogenic effect of 2,3-dehydrosilybin and its galloyl esters (3-O-galloyl-2,3-dehydrosilybin; 7-O-galloyl-2,3-dehydrosilybin; 20-O-galloyl-2,3-dehydrosilybin and 23-O-galloyl-2,3-dehydrosilybin) and to determine which molecular mechanism could be responsible for their activity. The effect on cell proliferation, tube formation, signal transduction pathways (PI3K/Akt and ERK) and the cell cycle was studied in human microvascular endothelial cells (HMEC). The results showed that all compounds decreased the growth of HMEC, but the strongest effect was observed for 20-O-galloyl-2,3-dehydrosilybin at 5 µmol/l. In addition, at 5 and 10 µmol/l, this was the only compound that significantly inhibited HMEC tube formation. Based on an assessment of Akt and ERK1/2 expression, we suggest that 20-O-galloyl-2,3-dehydrosilybin influences the angiogenic process through the Akt pathway.

In vitro anti-cancer effects of the actin-binding natural compound rhizopodin.

Several natural compound interfere with microtubules or the actin cytoskeleton. Compounds interfering with the microtubules like Vinca-alkaloids or taxanes, are extensively used for cancer therapy. In contrast, knowledge about pharmacological properties of actin binding drugs is poor and drugs interfering with actin are far from clinical use. Rhizopodin is a natural compound that strongly affects the actin cytoskeleton at nanomolar concentrations. Initial work revealed interesting anti-bacterial and cytotoxic effects, but the cellular effects and pharmacological properties of rhizopodin have not been characterized. We hypothesized that rhizopodin might exert anti-cancer activity. Therefore, the aim of this study was to characterize the cellular and pharmacological effects of rhizopodin in cancer. Effects of rhizopodin demonstrated prominent effects on the actin cytoskeleton as shown in the actin-pyrene assay and by immunostaining of cancer cells. To investigate cellular effects of rhizopodin, we analyzed cell proliferation, cell death induction by propidium iodide exclusion and western blot, as well as migration by impedance measurement using the xCELLligence device in MDA-MB-231 breast cancer and T24 bladder cancer cell lines. Rhizopodin inhibited proliferation and induced cell death of MDA-MB-231 and T24 cells at nanomolar concentrations. PARP cleavage by rhizopodin suggests caspase-dependent cell death induction. Importantly, rhizopodin potently inhibited MDA-MB-231 and T24 cancer cell migration at subtoxic doses where no actin aggregation was observed, indicating a specific underlying signaling of rhizopodin. In summary, our study elucidates rhizopodin as actin-binding natural compound that exerts potent anti-cancer effects. Therefore, our work provides the basis for further in depth characterization of rhizopodin as an antitumoral agent.

Anti-angiogenic effects of the tubulysin precursor pretubulysin and of simplified pretubulysin derivatives.

BACKGROUND AND PURPOSE: The use of tubulin-binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti-angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA). EXPERIMENTAL APPROACH: The compounds were tested in cellular angiogenesis assays (proliferation, cytotoxicity, cell cycle, migration, chemotaxis, tube formation) and in vitro (tubulin polymerization). The efficacy of Prt was also tested in vivo in a murine subcutaneous tumour model induced with HUH7 cells; tumour size and vascularization were measured. KEY RESULTS: The anti-angiogenic potency of all the compounds tested ran parallel to their inhibition of tubulin polymerization in vitro. Prt showed nearly the same efficacy as TubA (EC(50) in low nanomolar range in all cellular assays). Some modifications in the Prt molecule caused only a moderate drop in potency, while others resulted in a dramatic loss of action, providing initial insight into structure-activity relations. In vivo, Prt completely prevented tumour growth and reduced vascular density to 30%. CONCLUSIONS AND IMPLICATIONS: Prt, a chemically accessible precursor of some tubulysins is a highly attractive anti-angiogenic compound both in vitro and in vivo. Even more simplified derivatives of this compound still retain high anti-angiogenic efficacy.

Ginkgo biloba extract EGb 761 increases endothelial nitric oxide production in vitro and in vivo.

Beneficial effects of Ginkgo biloba on peripheral arterial occlusive disease have been repeatedly shown in clinical trials, especially after use of EGb 761, a standardized special extract. Since the underlying mechanisms are widely unknown, we aimed to elucidate the molecular basis on which EGb 761 protects against endothelial dysfunction in vitro and in vivo. Application of therapeutically feasible doses of EGb 761 for 48 h caused endothelial nitric oxide (NO) production by increasing endothelial nitric oxide synthase (eNOS) promoter activity and eNOS expression in vitro. Phosphorylation of eNOS at a site typical for Akt (Ser 1177) was acutely enhanced by treatment with EGb 761, as was Akt phosphorylation at Ser 478. Furthermore, the extract caused acute relaxation of isolated aortic rings and NO-dependent reduction of blood pressure in vivo in rats. These influences on eNOS represent a putative molecular basis for the protective cardiovascular properties of EGb 761.

Insights from knock-out models concerning postischemic release of TNFalpha from isolated mouse hearts.

The inflammatory cytokine tumor necrosis factor alpha (TNFalpha) is controversially discussed in ischemia/reperfusion damage of the heart. Purpose of this study was to elucidate cellular sources of TNFalpha and parameters which possibly influence its release in the heart following ischemia. Isolated hearts of mice were subjected to 15 min of global ischemia and 90 min of reperfusion. We employed hearts of various mice knock-out strains (interleukin-6(-/-), matrix metalloprotease-7(-/-), mast-cell deficient WBB6F1-Kit(W)/Kit(W-v), TNF-R1(-/-)) and wildtype mice, the latter perfused without and with infusion of cycloheximide or TNFalpha-cleaving-enzyme inhibitor (TAPI-2). Normoxic control hearts showed basal release of TNFalpha during the whole experiment. Immunohistology identified cardiac mast cells, macrophages and endothelial cells as main sources. TNFalpha release was stimulated during postischemic reperfusion, occurring in a two-peak pattern: directly after ischemia (0-10 min) and again after 60-90 min. The first peak mainly reflects tissue washout of TNFalpha accumulated during ischemia. The second, protracted peak arose continuously from the basal level and was abolished by protein synthesis inhibitor cycloheximide. Both properties are characteristic for de novo synthesis of TNFalpha, e.g., in cardiac muscle cells. However, immunohistological staining for TNFalpha failed in cardiomyocytes after 90 min of reperfusion. In contrast to hearts of TNF-R1(-/-) and Kit(W/W-v)-mice, those of IL-6(-/-) and MMP-7(-/-) mice lacked the late TNFalpha peak. TAPI did not suppress release of TNFalpha. While autostimulation via TNF-R1 also does not seem obligatory and mast cell can be ignored as source of the second peak, IL-6 may support de novo synthesis of TNFalpha. Additionally, TNFalpha release may essentially involve cleavage of membrane bound TNFalpha by MMP-7.

Dynamic analysis of metabolic effects of chloroacetaldehyde and cytochalasin B on tumor cells using bioelectronic sensor chips.

PURPOSE: To study the interplay of drugs and energy metabolism of tumor cells, metabolic changes induced by chloroacetaldehyde and cytochalasin B were analyzed in colon carcinoma cells LS174T. METHODS: O(2)-consumption and extracellular acidification were recorded using a bioelectronic sensor-chip system, which monitors these parameters in a culture continuously for at least 24 h. In parallel cultures cell number, cellular ATP-content, mitochondrial transmembrane potential, and the content of reactive oxygen species (ROS) were determined. RESULTS: When cell death was induced by chloroacetaldehyde (50 muM), the rate of acidification declined gradually for the next 15 h, while O(2)-consumption decreased rapidly within 30 min. This correlated with a loss in mitochondrial potential. However, cellular ATP-level showed a transient increase at 2 h; also ROS levels increased up to 6 h. In cells treated with cytochalasin B (2 muM), which inhibits glucose uptake, the rate of O(2)-consumption increased and the acidification activity dropped, even upon glutamine depletion. Mitochondrial membrane potential transiently increased after 1 h, while ATP-content decreased; there was no change in the level of ROS. CONCLUSION: The pattern of changes in basic energy metabolism differs with the type of cell death and growth inhibition involved in the cytotoxic action of two different drugs.

Evidence for inflammatory responses of the lungs during coronary artery bypass grafting with cardiopulmonary bypass.

OBJECTIVE: The occurrence of a systemic inflammatory reaction during cardiac surgery with cardiopulmonary bypass (CPB) has been well established, and the heart itself has been shown to release inflammatory mediators after ischemia. The hypothesis of the present study was that the lungs are also a site of inflammatory responses during early reperfusion. METHODS: In 20 consecutive patients undergoing coronary artery bypass grafting, blood was simultaneously drawn from the right atrium (RA) and the pulmonary vein (PV) before CPB and at 1 min, 10 min, and 20 min of reperfusion. The levels of interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor (TNF)-alpha were determined, as well as the adhesion molecules CD41 and CD62 on platelets and CD11b and CD41 on leukocytes. As a measure of the pulmonary release, ratios of PV and RA levels were calculated. RESULTS: Before CPB, the concentrations of cytokines tended to be lower in the PV compared with the RA. At 1 min of reperfusion, no significant concentration increases were found in the PV. At 10 min of reperfusion, the PV/RA ratio (mean +/- SEM) for IL-6 was 2.06 +/- 0.37 and 1.24 +/- 0.15 for IL-8 (p = 0.02 and p = 0.04, respectively, compared with the pre-CPB ratios of 0.89 +/- 0.4 and 0.99 +/- 0.2). At 20 min of reperfusion, PV/RA ratios for IL-6 (1.95 +/- 0.37) and IL-10 (0.99 +/- 0.4) were higher than before CPB (0.89 +/- 0.04, p = 0.05 and 0.85 +/- 0.06, p = 0.03, respectively). Adhesion molecule counts on platelets and polymorphonuclear neutrophils (PMNs) tended to be higher in the PV than in the RA before CPB. At 1 min of reperfusion, the PV/RA ratio of CD41 on monocytes (0.89 +/- 0.04) and of CD41 on PMNs (1.05 +/- 0.05) was less than before CPB (1.24 +/- 0.08, p = 0.0002 and 1.55 +/- 0.14, p = 0.0002). At 10 min and 20 min of reperfusion, similar changes were found. CONCLUSIONS: The observed changes indicate an inflammatory response of the lungs. Proinflammatory cytokines are increased in pulmonary venous blood. At the same time, activated blood cells are retained in the pulmonary circulation. This may contribute to pulmonary dysfunction almost routinely observed after CPB.

Reduction of pro-inflammatory cytokine levels and cellular adhesion in CABG procedures with separated pulmonary and systemic extracorporeal circulation without an oxygenator.

OBJECTIVE: We have recently shown that a considerable amount of pro-inflammatory cytokines is released during pulmonary passage after aortic declamping in patients undergoing coronary artery bypass grafting. The present study was performed to investigate whether bilateral extracorporeal circulation with the lungs as oxygenators can reduce the inflammatory responses of the lungs. METHODS: Eighteen consecutive patients undergoing coronary artery bypass grafting were randomly assigned to routine extracorporeal circulation with cannulation of right atrium and aorta (routine circulation, ten patients) or to a bilateral extracorporeal circulation with additional cannulation of left atrium and pulmonary artery (bilateral circulation, eight patients). Blood was simultaneously drawn from right atrium and pulmonary vein at 1, 10 and 20 min reperfusion. The levels of interleukin (IL)-6 and IL-8 and the adhesion molecules CD41 and CD62 on platelets and CD11b and CD41 on leukocytes were determined. Because of considerable interindividual scatter, the pulmonary venous levels are normalized to percent of the respective right atrial value at each time point. RESULTS: At 1 min reperfusion pulmonary venous levels of IL-6 and IL-8 in routine circulation were +44+/-15% and +43+/-28% of the respective right atrial values. The respective values in bilateral circulation were -3+/-4% and -6+/-7% (P=0.02 and P=0.05 vs. respective right atrium). Similar increments were found after 10 and 20 min. Platelet-monocyte coaggregates were retained during pulmonary passage at 1 min reperfusion in routine circulation (-21+/-6%), but washed out in bilateral circulation (+5+/-8%, P=0. 007). At 20 min reperfusion, activated polymorphonuclear neutrophils (PMN) were retained in routine circulation (-16+/-9%) but washed out in bilateral circulation (+19+/-29%, P=0.05; all data given as mean+/-SEM). CONCLUSIONS: Bilateral extracorporeal circulation without an artificial oxygenator significantly reduces the inflammatory responses during pulmonary passage after aortic declamping.

Endothelial function and hemostasis.

The vascular endothelium influences not only the three classically interacting components of hemostasis: the vessel, the blood platelets and the clotting and fibrinolytic systems of plasma, but also the natural sequelae: inflammation and tissue repair. Two principal modes of endothelial behaviour may be differentiated, best defined as an anti- and a prothrombotic state. Under physiological conditions endothelium mediates vascular dilatation (formation of NO, PGI2, adenosine, hyperpolarizing factor), prevents platelet adhesion and activation (production of adenosine, NO and PGI2, removal of ADP), blocks thrombin formation (tissue factor pathway inhibitor, activation of protein C via thrombomodulin, activation of antithrombin III) and mitigates fibrin deposition (t- and scuplasminogen activator production). Adhesion and transmigration of inflammatory leukocytes are attenuated, e.g. by NO and IL-10, and oxygen radicals are efficiently scavenged (urate, NO, glutathione, SOD). When the endothelium is physically disrupted or functionally perturbed by postischemic reperfusion, acute and chronic inflammation, atherosclerosis, diabetes and chronic arterial hypertension, then completely opposing actions pertain. This prothrombotic, proinflammatory state is characterised by vaso-constriction, platelet and leukocyte activation and adhesion (externalization, expression and upregulation of von Willebrand factor, platelet activating factor, P-selectin, ICAM-1, IL-8, MCP-1, TNF alpha, etc.), promotion of thrombin formation, coagulation and fibrin deposition at the vascular wall (expression of tissue factor, PAI-1, phosphatidyl serine, etc.) and, in platelet-leukocyte coaggregates, additional inflammatory interactions via attachment of platelet CD40-ligand to endothelial, monocyte and B-cell CD40. Since thrombin formation and inflammatory stimulation set the stage for later tissue repair, complete abolition of such endothelial responses cannot be the goal of clinical interventions aimed at limiting procoagulatory, prothrombotic actions of a dysfunctional vascular endothelium.

Sodium nitroprusside in patients with compromised left ventricular function undergoing coronary bypass: reduction of cardiac proinflammatory substances.

OBJECTIVE: The aim of the present study was to investigate whether the nitric oxide donor sodium nitroprusside can reduce the cardiac inflammatory response during coronary artery bypass grafting in patients with severely compromised left ventricular function. METHODS: Patients (n = 30) were assigned to receive placebo or sodium nitroprusside (0.5 microg. kg(-1). min(-1)) for the first 60 minutes of reperfusion. Interleukin 6, interleukin 8, and tumor necrosis factor alpha levels; platelet adhesion molecule CD41 and CD62 levels; and CD11b on leukocytes were determined in the radial artery and coronary sinus before cardiopulmonary bypass and during reperfusion (1, 5, 10, 35, and 75 minutes). RESULTS: At 1 minute of reperfusion, coronary venous levels of CD41-positive polymorphonuclear leukocytes were 8% lower than arterial levels in the placebo group and 18% higher in the sodium nitroprusside group (P =.021). At 5 minutes of reperfusion, the respective levels were 29% and 1% for interleukin 6 (P =.015), -5% and 20% for CD41-positive monocytes (P =.032), and -2% and 16% for CD11b-positive monocytes (P =.038). At 10 minutes of reperfusion, these levels were -14% and 21% for CD41-positive monocytes (P =.006). At 35 minutes of reperfusion, these levels were -13% and 7% for CD41-positive monocytes (P =.017), -41% and 23% for CD11b-positive monocytes (P =.001), and 7% and 25% for CD62-positive platelets (P =. 041). At 75 minutes of reperfusion, the levels were 15% and -7% for tumor necrosis factor alpha (P =.025) and -10% and 10% for CD62-positive platelets (P =.041). CONCLUSIONS: Transcardiac production of proinflammatory cytokines is reduced in patients undergoing coronary artery bypass grafting treated with the nitric oxide donor sodium nitroprusside. At the same time, less activated leukocytes and platelets are retained in the coronary circulation.

Endothelial preconditioning by transient oxidative stress reduces inflammatory responses of cultured endothelial cells to TNF-alpha.

Brief episodes of ischemia can render an organ resistant to subsequent severe ischemia. This 'ischemic preconditioning' is ascribed to various mechanisms, including oxidative stress. We investigated whether preconditioning exists on an endothelial level. Human umbilical vein endothelial cells (HUVECs) were transiently confronted with oxidative stress (1 mM H(2)O(2), 5 min). Adhesion molecules ICAM-1 and E-selectin and release of cytokines IL-6 and IL-8 to subsequent stimulation with TNF-alpha (2.5 ng/ml, 4 h) were measured (flow cytometry and immunoassay), as were nuclear translocation of the transcription factor NFkappaB (Western blotting, confocal microscopy) and redox status of HUVECs (quantification of glutathione by HPLC). TNF-alpha elevated IL-6 in the cell supernatant from 8.8 +/- 1 to 41 +/- 3 pg/ml and IL-8 from 0.5 +/- 0. 03 to 3 +/- 0.2 ng/ml. ICAM-1 was increased threefold and E-selectin rose eightfold. Oxidative stress (decrease of glutathione by 50%) reduced post-TNF-alpha levels of IL-6 to 14 +/- 3 and IL-8 to 1 +/- 0.2; the rise of ICAM-1 was completely blocked and E-selectin was only doubled. The anti-inflammatory effects of preconditioning via oxidative stress were paralleled by reduction of the translocation of NFkappaB on stimulation with TNF-alpha, and antagonized by the intracellular radical scavenger N-acetylcysteine. 'Anti-inflammatory preconditioning' of endothelial cells by oxidative stress may account for the inhibitory effects of preconditioning on leukocyte adhesion in vivo.

Reactive oxygen species and nitric oxide in myocardial ischemia and reperfusion.

Oxygen radicals and reactive oxygen species (ROS) are known to be generated in large amounts under inflammatory conditions and in the first few minutes of postischemic organ reperfusion. Due to the interaction of ROS with nitric oxide (NO), formed constitutively by endothelial cells, two alternatives are feasible. On the one hand, reaction with superoxide radicals may induce toxification (formation of peroxynitrite), and, on the other hand, by reacting with superoxide and hydroxyl radicals, NO can serve as a radical scavenger (formation of the innocuous anions, nitrate and nitrite, respectively). However, NO is considered to play a pivotal role in numerous physiological and pathophysiological processes, with effects arising from both lack and surfeit of this easily diffusible and chemically very reactive molecule. Physiologic contributions to vascular dilatation and inhibition of platelet and leukocyte activation, e.g., are infringed by enhanced inactivation of NO. Such inactivation occurs readily due to spontaneous reaction of NO with the superoxide radical, formed, e.g., by stressed endothelial cells and activated leukocytes. Conversely, overproduction of NO by induced NO synthase (iNOS) may lead to circulatory shock, cell apoptosis or even cell necrosis. Caution would, thus, seem to be warranted when attempting to interfere with homeostasis of NO. We have investigated the ability of NO to act as a radical scavenger during myocardial reperfusion in experimental and clinical settings. In the former, inhibition of angiotensin converting enzyme was employed to generate more endogenous NO (via bradykinin), in the latter, low-dose sodium nitroprusside was used as the donor of exogenous NO in patients undergoing coronary bypass grafting. Inhibition of leukocyte adhesion, attenuation of platelet activation and mitigation of redox-stress and inflammation were observed in both instances. Accordingly, modest enhancement of NO levels should afford cardioprotection during reperfusion.

Ketamine does not inhibit inflammatory responses of cultured human endothelial cells but reduces chemotactic activation of neutrophils.

BACKGROUND: Ketamine is a widely used general anaesthetic, which has been reported to inhibit neutrophil function and neutrophil-endothelial interaction. To date, however, it is unknown whether ketamine has any direct effects on endothelial cells with respect to inflammation. Therefore, we investigated the influence of varying concentrations of ketamine (0.5, 1, and 3 microM) on the endothelial expression of cytokines and adhesion molecules with relevance for inflammation. METHODS: Cultured human umbilical vein endothelial cells were stimulated with tumor necrosis factor alpha (TNFalpha, 2.5 ng/ml) for 4 h in the absence or presence of ketamine. The adhesion molecules ICAM-1 and E-selectin on the endothelial cells were measured by flow cytometry. Release of the proinflammatory cytokines IL-6 and IL-8 by endothelial cells was quantified by ELISA. The acute effect of ketamine on leukocyte activation by the supernatant of endothelial cells pre-stimulated with TNFalpha (4 h) was tested by flow cytometric measurement of CD11b, a leukocyte activation marker, after 15 min of coincubation. RESULTS: TNFalpha caused dramatic upregulation of both adhesion molecules (15-fold and 5-fold vs. control for ELAM-1 and ICAM-1, respectively) and of both cytokines (500-fold and 1.8-fold for IL-6 and IL-8, respectively). No concentration of ketamine employed in our study had any effect on these inflammatory parameters. However, activation of leukocytes by supernatant of TNFalpha-conditioned endothelial cells (70% increase of CD11b) was attenuated by coincubation of the PMN with 0.5 and 5 microM ketamine (47% and 44% increase, respectively). CONCLUSION: These data suggest that ketamine exerts its antiinflammatory actions primarily via inhibition of leukocyte reactivity. Indeed, no inhibition of endothelial responses was detectable in our study.

Indirect enhancement of neutrophil activity and adhesion to cultured human umbilical vein endothelial cells by isoprostanes (iPF2alpha-III and iPE2-III).

Isoprostanes are metabolites of arachidonic acid found in blood under various conditions of oxidative stress. Because arachidonic acid derivatives are major mediators of inflammation, we investigated the potential inflammatory effects of iPF2alpha-III (previously 8-isoPGF2alpha) and iPE2-III (8-isoPGE2) on human polymorphonuclear granulocytes (PMN), as well as on human umbilical vein endothelial cells (HUVECs). The early activation marker CD11b on PMN and the adhesion molecules ICAM-1, E-selectin, and P-selectin on HUVECs were quantified by flow cytometry. Levels of the cytokines interleukin (IL)-6 and IL-8 were measured in the culture supernatant by enzyme-linked immunosorbent assay. Furthermore, adhesion of PMN to HUVECs was assessed. Neither isoprostane showed any direct stimulatory effects on PMN or HUVECs at concentrations of 0.1 or 1 microM: there was no acute elevation in expression of CD11b or P-selectin and no change of ICAM-1 or E-selectin after 4 or 24 h of incubation, respectively. The levels of interleukin IL-6 and IL-8 were also unaltered. However, PMN adhesion was significantly enhanced both after 4 and 24 h of incubation of HUVECs with iPF2alpha-III, and CD11b expression on PMN was elevated by contact of these cells with the supernatant of pre-exposed HUVECs. Neither of these actions were inhibited by an endothelin receptor antagonist (bosentan) or a combined thromboxane A2/isoprostane-receptor antagonist (SQ29548). Thus, although not having a direct pro-inflammatory potential, isoprostanes might indirectly accentuate PMN stimulation. This seems to occur via a receptor-independent mechanism, perhaps the production of an active metabolite of isoprostanes by endothelial cells.

Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass.

OBJECTIVES: The investigation centers on whether there is a reperfusion-induced specific cardiac inflammatory reaction after bypass surgery. BACKGROUND: Cardiopulmonary bypass (CPB) leads to systemic inflammation. Additionally, cardiac inflammation due to reperfusion could occur. Knowledge about nature and time course of this reaction might help to develop cardioprotective interventions. METHODS: In 12 patients receiving coronary bypass grafts, arterial and coronary venous blood was obtained before onset of CPB, and 1, 5, 10, 25, 35 and 75 min after cardiac reperfusion. Plasma levels of IL6 and IL8 were measured by immunoassay. CD11b, CD41, and CD62 on blood cells were quantified by flow cytometry. Measurement of CD41, a platelet marker, on neutrophils and monocytes allowed detection of leukocyte-platelet microaggregates. RESULTS: Transcardiac veno-arterial difference of IL6 rose in the 10th and 25th min of reperfusion (from 0 to 7 pg/ml; p < 0.05), and after 75 min (15 pg/ml). IL8 did not change. CD11b on neutrophils (PMN) decreased transcardially to 95, 88 and 82% of the initial level in the 5th, 10th, and 75th min, respectively, suggesting sequestration of activated neutrophils. CD62 on platelets rose about 30% in the 75th min. Initially, leukocyte-platelet microaggregates were formed during coronary passage (+31% of the arterial level for PMN, +23% for monocytes). During reperfusion, coaggregates were retained (PMN: -1% and -7% in the 5th and 10th min, monocytes: -22%, -13% and -12% in the 1st, 5th and 10th min. CONCLUSIONS: During early reperfusion after aortic declamping, the coronary bed is already a source of proinflammatory stimuli and target for activated leukocytes, partly in conjunction with platelets. Mitigation of these phenomena might help to improve cardiac function after CPB especially in patients at risk.

Significant leukocyte and platelet retention during pulmonary passage after declamping of the aorta in CABG patients.

During reperfusion of the heart and the lungs in patients undergoing coronary artery bypass grafting, these organs have been shown to release inflammatory mediators. The present study was performed to quantitatively determine cellular retention or washout during pulmonary passage in early reperfusion. In 14 consecutive patients undergoing coronary artery bypass grafting blood was simultaneously drawn from right atrium and pulmonary vein at 1, 10 and 20 min reperfusion. The counts for platelets, leukocytes and the leukocyte subsets polymorphonuclear neutrophils (PMN), lymphocytes and monocytes were determined. Pulmonary veno-right atrial (transpulmonary) differences are given in percent with respective right atrial values being considered as 100%. Before CPB leukocyte counts were 4.7 +/- 0.5 in right atrium and 4.2 +/- 0.4 in pulmonary vein, x10(9)/l, resp. (transpulmonary difference of -8 +/- 3%). During reperfusion, pulmonary retention was in the range of 20-23% (p <0.01 vs. right atrial value). The basal values for PMN were 2.4 +/- 0.3 in right atrium and 1.9 +/- 0.3 in pulmonary vein, x10(9)/l, resp. (transpulmonary difference -15 +/- 8%). Thereafter, retention was in the range of 25-30% (p <0.01 vs. right atrium). Basal values for lymphocytes were 1.5 +/- 0.2 in right atrium and 1.6+/-0.3 in pulmonary vein, x10(9)/l, resp. (transpulmonary difference +6 +/- 10%). A tendency towards a washout of lymphocytes at 1 min reperfusion (+1 +/- 12%) was followed by retention of these cells at 10 and 20 min reperfusion (-14 +/- 12% and -10 +/- 5%, p <0.05 vs right atrium). Before ischemia monocyte counts were 0.7 +/- 0.2 in right atrium and 0.6 +/- 0.2 in pulmonary vein, x10(9)/l, resp. (transpulmonary difference -10 +/- 4%) and -9 +/- 9%, -27 +/- 12% (p <0.05 vs right atrium) and -22 +/- 14% at 1, 10 and 20 min reperfusion. During early reperfusion of the lungs after declamping of the aorta, significant amounts of leukocytes, platelets and the leukocyte subsets are retained in the pulmonary vascular bed. These retained cells may be responsible for the previously described pulmonary release of cytokines.

Sodium nitroprusside during coronary artery bypass grafting: evidence for an antiinflammatory action.

BACKGROUND: It was the aim of the present study to investigate whether a nitric oxide donor can reduce systemic inflammation and the cardiac inflammatory response during coronary artery bypass grafting with cardiopulmonary bypass. METHODS: Patients undergoing elective coronary artery bypass grafting (n = 22) were randomly assigned to treatment with either sodium nitroprusside (0.5 microg x kg(-1) x min(-1)) or placebo (controls), both for the first 20 minutes of reperfusion. Interleukin-6 and interleukin-8 levels, the adhesion molecules CD41 and CD62 on platelets and CD41 on monocytes and PMN (as markers for coaggregate formation), CD11b on monocytes and PMN, as well as platelet and leukocyte counts were determined in radial artery and coronary sinus blood before cardiopulmonary bypass and during reperfusion (1, 5, 10, 25, and 35 minutes). RESULTS: A reduction of systemic interleukin-6 levels (15.4+/-3.5 pg/mL, 36.7+/-5.9 pg/mL, and 46.8+/-8.0 pg/mL versus 33.4+/-7.7 pg/mL, 76.7+/-13.2 pg/mL, and 106.0+/-26.5 pg/mL, respectively, at 1, 25, and 35 minutes of reperfusion) and interleukin-8 (29.6+/-4.5 pg/mL versus 54.0+/-9.4, pg/mL, resp., at 35 minutes of reperfusion) resulted from treatment with sodium nitroprusside. No intracardiac production of interleukin-8 in sodium nitroprusside-treated patients (-1.1+/-0.4 pg/mL and -2.8+/-2.2 pg/mL, resp., for the coronary sinus-radial artery difference at 5 and 25 minutes of reperfusion) was observed, whereas cardiac production of interleukin-8 was present in controls (2.5+/-1.5 pg/mL and 5.5+/-2.8 pg/mL, resp.). Retention of platelet/leukocyte coaggregates occurred during coronary passage in controls (coronary sinus-radial artery difference for CD41-positive monocytes at 1 and 10 minutes of reperfusion, -16.3%+/-8.5% and -8.8%+/-2.6%, resp.). This was reduced in sodium nitroprusside-treated patients (with 5.8%+/-5.2% and 0.0%+/-3.2%). Retention of platelets in controls (ratio of coronary sinus to radial artery platelet count at 5 and 10 minutes of reperfusion, 88%+/-6% and 91%+/-5%) was compared to washout in treated patients (108%+/-6% and 113%+/-7%). CONCLUSIONS: In patients undergoing routine coronary artery bypass grafting, administration of sodium nitroprusside during early reperfusion alleviates systemic inflammation and the cardiac inflammatory response.