Stimulation of endothelial adenosine Al receptors enhances adhesion of neutrophils in the intact guinea pig coronary system

Objective: The primary aim was to determine the action of pathophysiologically relevant adenosine concentrations (0.1-1 μM) on adhesion of neutrophils to coronary endothelium. Further aims were to evaluate the nature and localisation of the adenosine receptor involved. and to assess the effect of endogenous adenosine. Methods: Adhesion was studied in isolated perfused guinea pig hearts by determining the number of cells emerging in the coronary effluent after intracoronary bolus injections of 600 000 neutrophils prepared from guinea pig or human blood. The system was characterised by the use of the proadhesive stimulus thrombin. Results: A 5 rnin infusion of adenosine (0.1-0.3 μM) or the A1 receptor agonist N6-cyclopentyladenosine (CPA, 0.01 μM) significantly increased adhesion from about 20% (control) to 30%. This effect was prevented by the A1 receptor antagonist dipropyl-8-cyclopentylxanthine (DPCPX. 0.1 μM). It was not diminished by cessation of adenosine infusion 90 s prior to neutrophil injection. At a higher concentration of adenosine (1 μM), adhesion did not seem to be enhanced. However, coinfusion of the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX. 0.1 μM) with 1 μM adenosine unmasked the A1 action, adhesion rising to 39%. Adenosine had a quantitatively identical effect on adhesion of human neutrophils. Total ischaemia of 15 min duration raised adhesion of subsequently applied neutrophils to 35%. This effect was completely blocked by DPCPX, as well as by ischaemic preconditioning (3 X 3 min). Preconditioning raised initial postischaemic coronary effluent adenosine from about 0.8 μM to 1.5 μM. Conclusions: The findings suggest a bimodal participation of adenosine in the development of postischaemic dysfunction by an endothelium dependent modulation of neutrophil adhesion. Stimulation occurs via endothelial A1 receptors at submicromolar adenosine levels, whereas cardioprotection by adenosine may in part relate to the use of pharmacologically high concentrations of adenosine or enhanced endogenous production after preconditioning

Application of Peptides Containing the Cleavage Sequence of Pro-TNFα in Assessing TACE Activity of Whole Cells

Tumor necrosis factor-α (TNFα) is presumably shed from cell membranes by TNFα-cleaving enzyme (TACE). The peptides SPLAQAVRSSSR and Dabcyl-LAQAVRSSSR-Edans, each encompassing the cleavage sequence of pro-TNFα recognized by TACE, were applied to intact umbilical vein endothelium (HUVEC), peripheral blood leukocytes (PBL) and the mast cell line HMC-1, which express TACE, to homogenates of rat heart tissue and to membrane and cytoplasmic extracts of PBL. Formation of SPLAQA (specific cleavage) was determined by HPLC, while cleavage (specific plus non-specific) of Dabcyl-TNFα-Edans was followed over time by measuring fluorescence. Participation of TACE was assessed from inhibition due to the drug TAPI-2. Incubation with recombinant human TACE gave specific cleavage, fully inhibitable by TAPI-2 (IC50<0.1 μM). HUVEC rapidly degraded TNFα-peptide, but in a non-specific manner (no SPLAQA detectable) and 50 μM TAPI-2 was without effect. Fluorescence was evoked when Dabcyl- LAQAVRSSSR-Edans was incubated with HMC-1 or PBL and also with cytoplasmic and membrane fractions of lysed PBL, but in no case was there significant inhibition by TAPI-2. However, marginal (10%) inhibition of fluorescence by 50 μM TAPI-2 was observed with homogenized heart tissue. This contained TACE, about 75% of which was without the inhibitory cysteine switch (Western blot). In conclusion, simple peptide analogs of pro-TNFα cannot be employed as substrates for measuring membrane TACE activity, largely due to extensive non-specific proteolytic cleavage by whole cells and cell extracts

Release of TNF-α during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers

Objectives: Our study sought to elucidate the role of oxidative stress for shedding of tumor necrosis factor-α (TNF-α) and for activating TNF-α-converting enzyme (TACE). Background: TNF-α, a central inflammatory cytokine, is discussed as one of the mediators of reperfusion injury. Shedding of membrane-bound pro-TNF-α is thought to be largely due to TNF-α-converting enzyme (TACE). Methods: Release of TNF-α and TACE dependency were studied in isolated rat hearts and in the human mast cell line HMC-1. Results: In reperfused hearts, interstitial release of TNF-α occurred in two phases (2–10 and >45 min). It depended on the presence of oxygen during reperfusion and was attenuated by reduced glutathione. Infusion of the oxidants H2O2 or HOCl elicited release in non-ischemic hearts. TNF-α release was inhibited in hearts treated with degranulation inhibitors ketotifen or cromoglycate, suggesting mast cells as major source for myocardial TNF-α. This was confirmed by tissue staining. Post-ischemic release of histamine, however, did not parallel that of TNF-α. Heart tissue contained mainly mature TACE. HMC-1 expressed abundant pro-TACE and cleaved the pro-TNF-α-peptide Ac-SPLAQAVRSSSR-NH2. However, cleavage was nonspecific and only partly inhibited by TACE inhibitor TAPI-2 (10–100 μmol/l), while it was stimulated by H2O2 and HOCl and fully blocked by the nonspecific metalloprotease inhibitor o-phenanthroline. Conclusions: The mechanism underlying TNF-α release from post-ischemic myocardium is oxidation-dependent but largely independent of activation of TACE. Mast cell stabilizers may be useful in preventing TNF-α release during reperfusion

On The External And Iterative Coupling Of Multiple Open Channel Flow Models With OpenMI

For water management questions on the national scale, cross-border studies or for water authorities it can be necessary to couple models of the same type, i. e. models that represent the same flow processes, but different modelling areas, at shared boundaries. With the help of a simple test case how two open channel flow models (Sobek) can be coupled externally or iteratively according to the OpenMI standard. We discuss the simulation results from external coupling and iterative coupling against the simultaneous solution in one equation system. Conclusions on the choice of a coupling method for practical applications are made and technical recommendations are given

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

Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation

BACKGROUND: Several strains of bacteria have sequenced and annotated genomes, which have been used in conjunction with biochemical and physiological data to reconstruct genome-scale metabolic networks. Such reconstruction amounts to a two-dimensional annotation of the genome. These networks have been analyzed with a constraint-based formalism and a variety of biologically meaningful results have emerged. Staphylococcus aureus is a pathogenic bacterium that has evolved resistance to many antibiotics, representing a significant health care concern. We present the first manually curated elementally and charge balanced genome-scale reconstruction and model of S. aureus' metabolic networks and compute some of its properties. RESULTS: We reconstructed a genome-scale metabolic network of S. aureus strain N315. This reconstruction, termed iSB619, consists of 619 genes that catalyze 640 metabolic reactions. For 91% of the reactions, open reading frames are explicitly linked to proteins and to the reaction. All but three of the metabolic reactions are both charge and elementally balanced. The reaction list is the most complete to date for this pathogen. When the capabilities of the reconstructed network were analyzed in the context of maximal growth, we formed hypotheses regarding growth requirements, the efficiency of growth on different carbon sources, and potential drug targets. These hypotheses can be tested experimentally and the data gathered can be used to improve subsequent versions of the reconstruction. CONCLUSION: iSB619 represents comprehensive biochemically and genetically structured information about the metabolism of S. aureus to date. The reconstructed metabolic network can be used to predict cellular phenotypes and thus advance our understanding of a troublesome pathogen

ACE-inhibition prevents postischemic coronary leukocyte adhesion and leukocyte-dependent reperfusion injury

Objective: Polymorphonuclear leukocytes (PMN), retained in the microvascular bed, can contribute to postischemic myocardial reperfusion injury. Since a beneficial effect of ACE-inhibition on reperfusion injury has been reported, we investigated the impact of cilazaprilat on PMN dependent reperfusion injury in isolated guinea pig hearts. Methods: Hearts (n=5 per group) were subjected to 15 min of ischemia. Immediately thereafter, a bolus of PMN was injected into the coronary system. External heart work (EHW) and total cardiac nitric oxide release were measured. For microscopic evaluation, hearts received rhodamine 6G labelled PMN after ischemia, were arrested 5 min later and further perfused with FITC dextran (0.1%). Localization of retained PMN was assessed by fluorescence microscopy. Leukocyte activation was studied by FACS analysis of the adhesion molecule CD11b before and after coronary passage of the PMN. The ACE-inhibitor cilazaprilat (Cila, 2 μM) and the NO-synthase inhibitor nitro-L-arginine (NOLAG, 10 μM) were used to modulate nitric oxide formation of the heart. Results: Postischemic EHW recovered to 67±5% (controls) and 64±6% (Cila) of the preischemic value. Addition of PMN severely depressed recovery of EHW (39±2%) and NO release (39±6% of the preischemic value). Simultaneously, ischemia led to a substantial increase in postcapillary PMN adhesion (from 21±5 to 172±27 PMN/mm² surface) and CD11b-expression of the recovered PMN (3-fold). Cila attenuated postischemic PMN adhesion (83±52 PMN/mm²) and activation of PMN, whereas it improved recovery of work performance (64±4%) and NO release (65±4%) in the presence of PMN. Conversely, NOLAG increased PMN adhesion (284±40 PMN/mm²) and myocardial injury. We conclude that ACE-inhibition prevents leukocyte dependent reperfusion injury mainly by inhibition of postcapillary leukocyte adhesion. The effect may be mediated by NO, given the proadhesive effect of NOLAG

Heparinase selectively sheds heparan sulphate from the endothelial glycocalyx

A healthy vascular endothelium is coated by the endothelial glycocalyx. Its main constituents are transmembrane syndecans and bound heparan sulphates. This structure maintains the physiological endothelial permeability barrier and prevents leukocyte and platelet adhesion, thereby mitigating inflammation and tissue oedema. Heparinase, a bacteria] analogue to heparanase, is known to attack the glycocalyx. However, the exact extent and specificity of degradation is unresolved. We show by electron microscopy, immunohistological staining and quantitative measurements of the constituent parts, that heparinase selectively sheds heparan sulphate from the glycocalyx, but not the synclecans

Integrated planning of the partially automated Banji coal mine in China

AbstractThe chinese mining company SDIC Xinji currently develops in the province of Anhui, south of Beijing, the coal mine Banji, pursuing the aim to realize an exemplary high-performance mine with the highest degree of production efficiency and a high safety standard, using state of the art technology. Remarkable with this assignment of task is the fact, that power supply technology, high voltage switch technology, automation and communication were being projected from one supplier, as an integrated complete system, based on a three- dimensional mine layout model