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Biomedical subjects

D Burnett

Publications and source records attributed to D Burnett.

At least 73 records · Page 4Linked to original sources
PubMed1992

Regulation of alpha 1-antitrypsin synthesis by granulocyte macrophage colony-stimulating factor in the U937 promonocytic cell line.

Undifferentiated U937 cells possess low numbers (approximately 1,100 per cell) of receptors for granulocyte macrophage colony-stimulating factor (GMCSF). The receptors present are of at least two types with different affinities. A small number (less than 200 per cell) are of higher affinity (approximate Kd 97pM), and a larger number (approximately 900 per cell) are of lower affinity (approximate Kd 680pM). Following differentiation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (PMA), the differentiated cells express a reduced number of receptors (approximately 680 per cell). These receptors are present as a single class with an approximate affinity of 230pM (Kd). The undifferentiated cells were responsive to GMCSF and this cytokine caused a seven-fold increase in accumulation of alpha 1-antitrypsin (alpha 1AT) compared to the control cells. This was accompanied by a similar increase in alpha 1ATmRNA, which suggests that the rate of alpha 1AT accumulation was regulated at the transcriptional level. After differentiation with PMA, alpha 1AT accumulation was not influenced by GMCSF, although the cells continued to bind the cytokine. These results imply that U937 cells do respond to GMCSF and this event involves binding of the ligand to a small number of specific cell surface receptors only present in the undifferentiated cells. Furthermore the response of these cells (in terms of alpha 1AT production) is associated with the type of GMCSF receptors expressed.

Cell Differentiation↗
PubMed1991

Transforming growth factor-beta induces human T lymphocyte migration in vitro.

In addition to its activities as a growth factor, recent studies suggest an immunoregulatory role for transforming growth factor-beta (TGF-beta). In this context we have demonstrated that TGF-beta is a potent chemotactic factor in vitro for human T lymphocytes at a concentration of 40 fM and for monocytes at a concentration of 0.4 fM but that it has no chemotactic activity for neutrophils. Furthermore, using an assay of lymphocyte subset chemotaxis we have been able to show that TGF-beta can induce migration of both CD4+ and CD8+ T lymphocytes in vitro. This study provides further evidence that TGF-beta acts as a cytokine, being able to attract T lymphocytes and monocytes to sites of inflammation. Its role in the pathogenesis of inflammatory reactions is likely to be complex.

Cells, Cultured↗
PubMed1991

Human sputum cathepsin B degrades proteoglycan, is inhibited by alpha 2-macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C.

The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.

Amino Acid Sequence↗
PubMed1991

Effects of neutrophil adherence on the characteristics of receptors for tumor necrosis factor-alpha.

Human recombinant [125I]TNF-alpha was incubated with non-adherent human neutrophils, cells adherent to fibronectin-coated plastic, or adherent cells scraped into suspension (post-adherent). Binding of TNF to all cells increased with doses of added TNF but adherent cells bound little TNF. Binding of TNF by post-adherent cells was greater than when adherent, but still significantly less than that of non-adhered neutrophils, suggesting that TNF receptors were relocated on the adherent surface of neutrophils. Scatchard analysis showed that adherent cells expressed significantly fewer TNF receptors, but of higher affinity, than non-adherent cells. The results suggest that altered expression of TNF receptors might contribute to the differential effects of TNF on adherent and non-adherent neutrophils.

Cell Adhesion↗
PubMed1991

Ethanol-induced changes in chloride flux are mediated by both GABA(A) and GABA(B) receptors.

Low concentrations of ethanol (10-30 mM) in the presence of a GABAB receptor agonist, baclofen, promoted 36Cl- uptake into membrane vesicles (microsacs) prepared from mouse cortex. Neither ethanol nor baclofen alone altered chloride influx. The GABAB antagonists, phaclofen and 2-hydroxy-saclofen, completely blocked the increase in chloride flux produced by ethanol in the presence of either baclofen or GABA. Ethanol increased the chloride conductance produced by the GABAA agonists muscimol, isoguvacine, imidazolacetic acid and amino-propane sulfonic acid and this action of ethanol was blocked by phaclofen. The specific GABAA antagonist, bicuculline, blocked ethanol-induced increase in chloride flux in the presence of either baclofen or GABA. GABA-activated chloride channels were also studied in Xenopus oocytes expressing mouse brain mRNA. In this preparation, GABA action was enhanced by ethanol, pentobarbital, and diazepam, and 2-hydroxy-saclofen partially antagonized the action of ethanol without altering the effects of pentobarbital or diazepam. These results suggest that ethanol enhancement of GABAA receptor-chloride channel function also requires activation of GABAB receptors.

Alcohol Drinking↗
PubMed1991

Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium.

It has been suggested that proteinase enzymes could play an important role in the pathogenesis of chronic bronchial infections including bronchiectasis and cystic fibrosis (CF). Because Pseudomonas aeruginosa frequently colonizes the respiratory tract in bronchiectasis and CF, we examined the in vitro effects of human neutrophil elastase (HNE) and proteinase enzymes produced by P. aeruginosa (elastase: PE; alkaline proteinase: PAP) on the ciliary beat frequency (CBF) and ultrastructure of human nasal ciliated respiratory epithelium. HNE (500 micrograms/ml) progressively reduced CBF and caused marked epithelial disruption; lower concentrations (100 and 20 micrograms/ml) also caused epithelial disruption but without slowing CBF. The effects of HNE (500 micrograms/ml) were completely abolished by adding alpha 1-antitrypsin (5 mg/ml). There was no synergy between HNE and pyocyanin, a product of P. aeruginosa which slows CBF. PE in phosphate-buffered saline also caused epithelial disruption without slowing CBF; however, PE in medium containing divalent metal ions caused CBF slowing as well as epithelial disruption at 100 micrograms/ml. PAP (500 micrograms/ml) had almost no effect on ciliated epithelium. The effects of HNE and PE on nasal and bronchial epithelium obtained from the same patient were similar. Light and transmission electron microscopy revealed that HNE and PE were cytotoxic and caused detachment of epithelial cells from neighboring cells and the basement membrane. There was cytoplasmic blebbing of the cell surface and mitochondrial damage; however, no increase of abnormalities in the ultrastructure of cilia on living cells was seen. These results support the hypothesis that HNE and PE contribute to the delayed mucociliary clearance and epithelial damage that is observed in patients with chronic bronchial infection.

Bronchi↗
PubMed1991

Extracellular proteolysis of fibronectin by neutrophils: characterization and the effects of recombinant cytokines.

We have used 125I-labeled fibronectin (FN) as an extracellular substrate for neutrophils (PMN) in order to investigate the mechanism responsible for FN solubilization by PMN and the effects of recombinant cytokines on this process. Pure active alpha 1-antitrypsin (alpha 1AT), when added to PMN before or during, but not after, adherence to FN, inhibited solubilization of the substrate in a dose-dependent manner, but alpha 1AT that had been inactivated by proteolysis or oxidation and alpha 1AT Pittsburgh (alpha 1AT 358Met-Arg) had no significant effect. The solubilization of FN was also inhibited by the PMN elastase inhibitor N-methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone but not by the chymotrypsin and cathepsin G inhibitor N-Cbz-glycyl-glycyl-phenylalanine-chloromethylketone, nor by catalase or superoxide dismutase. The products of solubilization of FN by PMN, analyzed by sodium dodecyl sulphate polyacrylamide electrophoresis, were similar to those produced by pure PMN elastase but not cathepsin G. These results suggest that FN solubilization by PMN is caused largely by the pericellular activity of PMN elastase. The solubilization of FN by PMN was increased significantly by adding tumor necrosis factor-alpha, interleukin-1 alpha, or interferon-gamma to the adherent cells but without a significant general release of elastase into the culture supernatants. Granulocyte/macrophage colony-stimulating factor (GM-CSF) had no significant effect. None of the cytokines had any effect when preincubated with the cells in suspension, and non increased FN solubilization by PMN incubated with the optimal (10(-6) mol/liter) or suboptimal dose (10(-8) mol/liter) of the peptide formylmethionylleucylphenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1991

Performance of techniques used to detect drugs of abuse in urine: study based on external quality assessment.

Twenty-five samples of lyophilized urine from the U.K. External Quality Assessment Scheme for Drugs of Abuse were analyzed by an average of 95 laboratories between April 1987 and December 1989. Samples contained mixtures of analytes and included replicated concentrations of morphine, methadone, amphetamine, and cocaine at 0, 1, 2, and 5 mg/L and of benzoylecgonine at 0, 0.4, 1, 2, and 4 mg/L. Some chromatographic techniques were inadequate for detecting morphine, amphetamine, cocaine, and benzoylecgonine at the lower concentrations of analytes studied: gas-liquid chromatography was least sensitive for morphine; in-house thin-layer chromatography (TLC) was least sensitive for the other analytes. Few significant differences in specificity were detected between techniques, although significant interference from structurally related compounds was demonstrated in assays of morphine, methadone, and amphetamine. Exceptions were the Boehringer (BCL) test for opiates and TLC applied to amphetamine and benzoylecgonine, which demonstrated considerable lack of specificity.

Amphetamine↗
PubMed1990

Changes in the expression of elastase and cathepsin B with differentiation of U937 promonocytes by GMCSF.

The human promonocytic cell line, U937, when treated for up to 72h with 12,O,tetradecanoyl-phorbol-13-acetate or granulocyte-macrophage colony-stimulating factor, exhibited increased phagocytic activity and expression of the marker p150/95. There was an associated increase in the monocyte proteinase cathepsin B and its mRNA but decreased cellular levels of neutrophil elastase and elastase mRNA. Granulocyte-macrophage colony-stimulating factor therefore causes differentiation of U937 cells, with appropriate effects on the synthesis of leukocyte proteinases.

Cathepsin B↗
PubMed1990

Developmental regulation of the human cathepsin G gene in myelomonocytic cells.

Cathepsin G is a neutral serine protease that is found in the azurophil granules of neutrophils and monocytes. Previous experiments had demonstrated that cathepsin G is actively produced by the promonocytic U937 cell line, and that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of these cells toward macrophages resulted in a reduction of cathepsin G activity. In this study, we have analyzed the mechanism of this TPA-induced down-regulatory event. Using in situ hybridization techniques, we demonstrated that cathepsin G mRNA is detectable only at the promyelocytic stage of myeloid development. Using U937 promonocytic cells as a model, we demonstrated; 1) cathepsin G protein levels decline in TPA-treated cells; 2) this decline was due to a nearly complete loss of cathepsin G mRNA in cells treated with TPA for 24 h; and 3) the rate of cathepsin G mRNA loss with TPA treatment was similar to that with actinomycin D. These results suggested that cathepsin G transcription was down-regulated within several hours of TPA addition. This was directly tested by performing nuclear run-off assays of TPA-treated U937 cells; cathepsin G transcription was shown to be strand-specific, and declined within 4 h of TPA addition. Cathepsin G transcription was essentially undetectable 8 or more hours after TPA treatment, suggesting that down-regulation is predominantly transcriptional. Cycloheximide treatment of U937 cells resulted in a partial block of TPA-mediated cathepsin G down-regulation, indicating that continuous protein synthesis is required for down-regulation to occur. A newly synthesized protein or proteins may therefore be required for the transcriptional down-regulation of cathepsin G during the normal development of promyelocytes or promonocytes.

Cathepsin G↗
PubMed1990

Inflammatory indices for chronic bronchitis and COAD. Proteases and antiproteases.

The study of lung secretion proteinases and their inhibitors, both functionally and quantitatively, provide information concerning the degree of inflammation in the lung. Interpretation of individual parameters may be difficult in isolation. However, a general pattern of response provides supportive evidence of a reduction in lung inflammation, including 1) reduction in plasma inhibitor concentrations, 2) increase in local inhibitor concentrations, 3) increase in enzyme inhibition, and 4) reduction in enzyme activity. Thus such changes provide an objective basis for the assessment of drug therapy.

Bronchitis↗
PubMed1990

IgA subclasses in sputum from patients with bronchiectasis.

The concentrations of IgA1 and IgA2 were measured in the serum and sputum from 27 clinically stable patients with bronchiectasis. Of the 27 patients, nine were regularly producing mucoid secretions, nine mucopurulent and nine purulent secretions. No significant differences were observed in serum IgA2 concentrations between the patient groups, although IgA1 concentrations were higher in the serum from the patients producing mucopurulent sputum than those producing mucoid or purulent sputum. The sputum concentrations of both IgA1 and IgA2 increased significantly with purulence. Furthermore, there was a significant increase in the proportion of IgA2 as the secretions became more purulent. The sputum concentrations of both IgA subclasses indicated significant local synthesis, with increased production (especially of IgA2) in the presence of inflammation due to infection.

Bronchiectasis↗
PubMed1990

Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation.

Sputum samples from 25 patients with bronchiectasis were assayed enzymatically for myeloperoxidase, neutrophil elastase and cathepsin B, and immunologically for cystatin A, cystatin B, cystatin C, cystatin S and kininogen. High myeloperoxidase and neutrophil elastase levels were found in those sputum samples that were assessed visually to be purulent. These samples were also found to contain high levels of cathepsin B activity and cystatin A, but low levels of cystatin S and of the most effective cathepsin B inhibitor, cystatin C. In contrast, sputum samples that were low in myeloperoxidase and neutrophil elastase activities had low levels of cathepsin B and cystatin A, but high cystatin C and S levels. It is concluded that cathepsin B activity in sputum is positively correlated with the degree of inflammation and neutrophil recruitment. Although this may be due in part to reduced amounts of cathepsin B inhibitors, particularly cystatin C, theoretical considerations suggest that factors other than the gross level of inhibitors must be involved in the control of cathepsin B activity.

Bronchiectasis↗
PubMed1990

Effect of non-steroidal anti-inflammatory drugs on neutrophil chemotaxis--an in vitro and in vivo study.

We have studied the effects of the two non-steroidal anti-inflammatory drugs (NSAIDs), nabumetone and indomethacin, on neutrophil chemotaxis in vitro and in vivo. When used in therapeutic concentrations in vitro, neither agent had any effect on the chemotactic response of neutrophils isolated from healthy volunteers. This was true for the three chemotactic agents studied: FMLP, zymosan activated serum and purulent sputum. Nabumetone and indomethacin decreased neutrophil chemotaxis over a period of 2 weeks in 12 normal subjects in vivo. The average chemotactic response to 10(-8) mol/l FMLP for all 12 during the control period was 42.1 +/- 6.1 cells per high power field and this fell to 26.1 +/- 4.9 (P less than 0.025) after 7 days and to 15.6 +/- 2.5 (P less than 0.005) after 14 days. The results were similar for both drugs analysed independently. The results suggest that NSAIDs have no effect on the chemotactic response of mature cells in vitro, but suppress chemotaxis progressively when given in vivo. This may be explained by an effect of NSAIDs on maturing cells prior to release into the circulation.

Adult↗
PubMed1990

Neutrophil activation--an important cause of tissue damage during liver allograft rejection?

Although neutrophils have been implicated as mediators of tissue damage in a variety of conditions, their role in graft rejection has not previously been studied. Peripheral neutrophil activation was assessed sequentially in 16 patients following liver transplantation by measuring the ability of neutrophils to respond to the chemotactic peptide FMLP, to release superoxide radicals, and to cause extracellular proteolysis. All three functions increased 1-2 days before episodes of acute rejection and only returned to normal after treatment with high-dose corticosteroids. Three patients who did not develop rejection showed no increase in neutrophil function. Lymphocytes isolated from patients with acute rejection produced factors that were both chemotactic for and capable of activating normal neutrophils. This study shows that neutrophil activation occurs during acute liver allograft rejection, possibly in response to lymphokine secretion, and suggests an important, and hitherto unrecognized, mechanism of graft damage during rejection.

Adult↗
PubMed1990

Effect of alpha-1-proteinase inhibitor on neutrophil chemotaxis.

Factors that modulate neutrophil migration into the lung are poorly understood. However, there is evidence that neutrophil activation by formylmethionylleucylphenylalanine (FMLP) depends upon a surface proteinase with chymotrypsin-like activity. This suggests that chymotrypsin inhibitors such as alpha-1-proteinase inhibitor (alpha 1PI) could modify neutrophil migration in response to FMLP. We have studied neutrophil chemotaxis using the multiple blind well assay system. This article presents evidence that alpha 1PI is an inhibitor of neutrophil migration in response to FMLP. The effect is related to the inhibitory function of the protein. Alpha-1-antichymotrypsin is more potent than alpha 1PI as an inhibitor of this movement, whereas antileukoprotease is less potent. The results suggest that a cell membrane-bound serine proteinase (perhaps cathepsin G) is necessary for the enhancement of cell movement after receptor binding of FMLP. Oxidized alpha 1PI or a 4,000-D peptide cleaved from alpha 1PI by porcine pancreatic elastase or human neutrophil elastase are capable of enhancing cell motility. The results suggest that alpha 1PI may play a role in cell migration into the lung during acute inflammatory process.

Animals↗