1,720,979 research outputs found
Human mast cell tryptase stimulates the release of an IL-8-dependent neutrophil chemotactic activity from human umbilical vein endothelial cells (HUVEC)
No full textTryptase, the major product of human mast cell activation, is a potent stimulus of vascular leakage and neutrophil accumulation in vivo in animal studies, but the mechanisms of action remain unclear. Using HUVEC cultures we have sought to investigate the potential of tryptase to alter monolayer permeability or induce the release of neutrophil chemotactic activity. Tryptase (1–100 mU/ml) failed to alter the permeability of endothelial cell monolayers as assessed by albumin flux over 1 h. However, supernatants from endothelial cells treated with tryptase (1–50 mU/ml) for a 24-h period induced neutrophil migration across Transwell filters, with maximal migration observed at 10 mU/ml tryptase. Pretreatment of tryptase with the protease inhibitor leupeptin abolished the chemotactic activity, indicating a dependence on the catalytic site. Moreover, this effect was abolished by addition of an IL-8 neutralizing antibody, suggesting that IL-8 release makes an important contribution to the chemotactic activity. The interaction of mast cell tryptase with endothelial cells could be important in stimulating the ingress of neutrophils following mast cell activation in inflammatory disease
The heterogeneity of mast cell tryptase from human lung and skin
No full textThere has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase-specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9–12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono-antennary or bi-antennary complex-type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein-N-glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis–Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues
Elevated serum concentrations of beta-tryptase, but not alpha-tryptase, in Sudden Infant Death Syndrome (SIDS). An investigation of anaphylactic mechanisms
No full textBackground
Sudden Infant Death Syndrome, (SIDS) or cot death, remains the most common category of post-perinatal death in the UK. By definition, the cause of death is unknown, but a long-standing theory is that some of these deaths could be the result of anaphylaxis.Objective
To investigate the potential contribution of anaphylactic mechanisms to deaths in infancy by determining relative levels of ?- and ?-tryptases and both total and allergen-specific IgE in sera from groups of infants whose deaths were attributed to SIDS or to other causes.Methods
Serum samples were collected at the time of post-mortem examination from infants whose death was classed as SIDS (n = 40) and from a comparison group in which cause of death had been established (n = 32). Serum tryptase concentrations were measured with a radioimmunoassay with monoclonal antibody G5 which detects primarily ?-tryptase or an ELISA with antibody AA5 which has equal sensitivity for ?- and ?-tryptases. Levels of total IgE and IgE specific for casein, ?-lactoglobulin, house dust mite and moulds were determined.Results
Analysis of the results of the two assays for tryptase indicated that levels of the ?-like tryptase (the form secreted on anaphylactic degranulation) were significantly higher in serum from infants with SIDS compared with those whose death was explained. There was no evidence for an increase in serum levels of ?-tryptase (the variant secreted constitutively from mast cells). Total levels of serum IgE did not differ between the two groups and, reflecting the low circulating IgE concentrations in infancy, an elevation in IgE specific for the panel of allergens was not detected.Conclusions
In a proportion of SIDS victims there may be increased serum levels of ?-like tryptase, a marker for anaphylaxis. The failure to detect an increase in ?-tryptase would suggest that mast cell hyperplasia is not a feature of cot death. The nature of the inciting agents remains unclear, but anaphylaxis deserves serious consideration as a possible cause of sudden death in infancy
The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors
No full textMast cells have been implicated as having pivotal roles in arthritis, but little is known of the processes leading to the activation of synovial mast cells or their potential for pharmacological control. We have investigated the ability of tryptase and chymase, and inhibitors of these major mast cell proteases to modulate the activation of mast cells from human synovial tissue. The tryptase inhibitor drug N-(1-hydroxy-2-naphthoyl)--arginyl--prolinamide hydrochloride (APC366) inhibited immunoglobulin E (IgE)-dependent histamine release in a dose-dependent manner, with about 70% inhibition being achieved at a dose of 300 ?M. Histamine release stimulated by calcium ionophore A23187 was also inhibited by this compound. The chymase inhibitor chymostatin inhibited IgE-dependent histamine release by approximately 60% at 1 ?g/ml. Tryptase at concentrations of 3.0 ?g/ml and greater stimulated histamine release from synovial cells, which was dependent on catalytic activity, whereas chymase had little effect on these cells. The activation of mast cells by tryptase may represent an amplification process in the synovium. The mast cell stabilising properties of inhibitors of tryptase and chymase could be of therapeutic value in arthritis
The long-acting beta2-agonist salmeterol xinafoate: effects on airway inflammation in asthma
No full textSalmeterol xinafoate is an inhaled long-acting beta2-adrenoceptor agonist recently introduced for the treatment of asthma. Both in vitro and animal studies suggest that it may have anti-inflammatory activities of benefit in this disease. To assess this directly, the effects of 6 weeks' treatment with salmeterol on indices of clinical activity, airway dysfunction and inflammation in subjects with stable atopic asthma were investigated. In a double blind study, asthmatic patients were randomized to 6 weeks' treatment with either salmeterol 50 microg twice daily (n=14) or placebo (n=12). They underwent bronchoscopy with bronchoalveolar lavage (BAL) and bronchial biopsy immediately before starting treatment and again after 6 weeks. Treatment with salmeterol improved clinical indices of asthma activity, but there were no changes in BAL differential cell counts or mediator levels, and no change in T-cell numbers or activation status. In the biopsy specimens there were no changes in numbers of inflammatory cells, sub-basement membrane collagen deposition or mast cell degranulation. Regular treatment with salmeterol improves clinical indices of asthma but has no effect on the underlying inflammatory process. These findings strengthen guideline recommendations that long-acting beta2-agonists should not be prescribed as sole antiasthma medication
Airway inflammation and atopic asthma: a comparative bronchoscopic investigation
No full textFlexible fibre-optic bronchoscopy under local anaesthesia has been used to investigate the cellular airway events in atopic asthma. The findings have been compared to those from atopic individuals without asthma and non-atopic healthy controls, in an attempt to discern those changes relevant to clinical disease expression. Immunohistochemical and electron-microscopic analyses of airway biopsies identified that an atopic diathesis is associated with tissue eosinophil infiltration and mast cell degranulation. The eosinophilia was greatest in those atopic individuals with asthma. Flow-cytometric analysis of airway lavage revealed significantly enhanced T lymphocyte activation in clinical asthma. These findings are consistent with the hypothesis that T lymphocyte activation, through cytokine release, amplifies the tissue eosinophilia in asthma and that this combination is associated with clinical disease expression
Quantitation of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry
No full textWe have used fiberoptic bronchoscopy to obtain endobronchial biopsies in which mast cells and eosinophils were enumerated using monoclonal antibodies directed against mast cell tryptase (AA1) and the eosinophil cationic protein (EG2). Eleven symptomatic atopic asthmatics treated with beta 2-agonists alone and six normal subjects were studied. Over a period of 2 wk prior to bronchoscopy, patients recorded asthma symptom scores, bronchodilator usage, and twice-daily peak expiratory flow. Five days before bronchoscopy, methacholine responsiveness was assessed. Two biopsies were taken from the subcarinae, one of which was processed into araldite for immunostaining by the streptavidin biotin immunoperoxidase method and the other into Spurr resin for electron microscopy. The number of AA1 staining mast cells present in the bronchial mucosa was not significantly different in the epithelium or submucosa between the asthmatic and the normal subjects. However, in the biopsies from asthmatics, there were significantly greater numbers of EG2-staining eosinophils in the epithelium (median, 1.2/mm versus zero; p less than 0.005) and in the submucosa (median, 50/mm2 versus 1/mm2; p less than 0.001). Electron microscopy showed morphologic features of mast cell and eosinophil degranulation in the asthmatics. No correlation could be established between mast cell or eosinophil numbers and indices of disease activity of PC20 methacholine, which points to the complexity of mechanisms responsible for the symptoms and the airway hyperresponsiveness of asthma
Mast cell tryptase as a stimulus for up-regulation of adhesion molecule expression and cytokine release from endothelial cells
No full textRationaleMast cell tryptase can be released in substantial quantities at sites of allergic inflammation. Pro-inflammatory actions mediated through protease activated receptor 2 (PAR-2) have been proposed, but the precise contribution of tryptase to disease processes remains unclear. We have investigated alterations in the whole genome expression profile of endothelial cells in response to tryptase.MethodsHuman umbilical vein endothelial cells (HUVECs) were isolated from umbilical vein tissue and grown to confluence. Following addition of tryptase or other agents, quantitative polymerase chain reactions (qPCR) followed by whole genome microarray analysis were performed. Translation of certain genes was investigated by immunocytochemistry or specific ELISA. In parallel studies, calcium flux was measured using a fluorescence based microplate procedure.ResultsMicroarray analysis indicated that the genes whose expression was most up-regulated following tryptase treatment of endothelial cells were those for the adhesion molecules ICAM-1, VCAM-1, EPCAM and ITGAL, and the cytokines IL-2, IL-3, IL-6 and CXCL10. Increased expression was seen also for genes for the cell signalling molecules TNFAIP3, TLL1 and SMAD2. None of these were up-regulated in response to a peptide agonist (SLIGKV-NH2) of PAR-2. Microarray data was confirmed by separate qPCR experiments, immunocytochemistry and by the measurement of cytokines in cell supernatants. The actions of tryptase were inhibited using selective protease inhibitors indicating a requirement for an intact catalytic site. Addition of tryptase to endothelial cells stimulated concentration-dependent increases in intracellular calcium.ConclusionsThe pro-inflammatory actions of tryptase may be mediated through increased expression of adhesion molecules and production of inflammatory cytokines in endothelial cells
Antimicrobial activity of simvastatin against chronic rhinosinusitis-related <i>Staphylococcus aureus</i>: an in vitro study
No full textIntroduction: Staphylococcus aureus (S. aureus) in chronic rhinosinusitis (CRS), particularly when localised intracellularly, is linked to disease recalcitrance and poor post-surgical outcomes. Antibiotics frequently fail to penetrate the mammalian cell membrane, resulting in an inability to address the intracellular component of S. aureus. This contributes to treatment failure and development of antimicrobial resistance. We investigated the antimicrobial effects of simvastatin, a widely used, inexpensive medication with extracellular and intracellular antimicrobial properties, against CRS-related S. aureus.Methods: simvastatin’s antimicrobial activity, in prodrug and hydroxylated forms, was assessed against S. aureus using the broth dilution method to determine the minimal inhibitory concentration (MIC). Intracellular activity of simvastatin was evaluated by pre-treating S. aureus -infected LAD2 mast cells with simvastatin and performing colony forming unit (CFU) enumeration and confocal microscopy. Cell viability was assessed using lactate dehydrogenase (LDH) assays.Results: Simvastatin exhibited an extracellular MIC of 40 mmol/l against S. aureus. Intracellularly, it significantly reduced the bacterial burden by 46-fold in a dose-dependent manner between concentrations of 0.1-100 mmol/l. Toxicity to LAD2 cells was observed at 100 µmol/l. Confocal microscopy revealed a lower percentage of infected cells in the group pretreated with 30 µmol/l simvastatin (15.3%) compared to untreated cells (32.8%). Hydroxylated simvastatin demonstrated no antimicrobial activity against S. aureus.Conclusions: simvastatin demonstrates in vitro antimicrobial activity against CRS-related S. aureus with the potential for repurposing as a novel antibiotic-sparing topical agent for the treatment of refractory CRS. This could improve surgical outcomes and reduce the risk of antimicrobial resistance.<br/
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