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Biomedical subjects

R A Stockley

Publications and source records attributed to R A Stockley.

At least 91 records · Page 5Linked to original sources
PubMed1994

1 alpha,25-Dihydroxyvitamin D3 promotes monocytopoiesis and suppresses granulocytopoiesis in cultures of normal human myeloid blast cells.

Primitive myeloid blast cells (2-10 x 10(6)) were purified from 18-22-week fetal liver-derived mononuclear cell preparations by negative selection followed by counterflow cell elutriation. The cells, when maintained in liquid culture in the presence of 100 U/ml interleukin-3 (IL-3) for the first 5 days and 10 U/ml IL-3 and 30 ng/ml granulocyte colony-stimulating factor thereafter, underwent considerable proliferation resulting in an approximately 30-fold increase in cell number by day 14. Analyses of cell morphology and of the numbers of cells that expressed the neutrophil-associated antigen CD15, the monocyte-associated antigen 61D3, and enzymes alpha-naphthyl acetate esterase (ANAE), human leukocyte elastase, and cathepsin G revealed that proliferation of the cells was associated with their concomitant differentiation toward neutrophils and monocytes. The cultures generated predominantly neutrophils; by day 14, wells seeded with 2 x 10(5) cells produced approximately 5 x 10(6) neutrophils as opposed to only approximately 3.5 x 10(5) cells with a monocytoid morphology. This predominance of granulocytopoiesis over monocytopoiesis was confirmed by the numbers of cells that had acquired expression of the CD15 antigen and ANAE, which were approximately 2 x 10(6) and 1 x 10(5), respectively. By contrast, parallel cultures containing 100 nM 1 alpha,25-dihydroxyvitamin D3 (VitD3) generated more monocytes than neutrophils. At day 14, VitD3-treated cultures contained approximately 2 x 10(6) cells with morphologies consistent with their differentiation toward monocytes and approximately 1 x 10(6) ANAE-positive cells, compared with approximately 9.5 x 10(5) cells having morphologies of granulocyte-series cells and approximately 4.5 x 10(4) CD15-positive cells. In both control and VitD3-treated cultures, the enzymes cathepsin G and human leukocyte elastase were expressed almost exclusively by cells that were differentiating toward neutrophils. These data reveal that VitD3 promotes monocytopoiesis and suppresses granulocytopoiesis of primitive blast cells.

Calcitriol↗
PubMed1994

Sputum and serum pharmacokinetics of loracarbef (LY163892) in patients with chronic bronchial sepsis.

Sputum and serum pharmacokinetics of loracarbef (LY163892) were performed in 19 patients with purulent bronchiectasis. Nine of the patients received 200 mg twice daily and ten patients, 400 mg twice daily, for a total of 14 days. beta-Lactamase activity was measurable in the lung secretions of all 19 patients at the start of therapy. Mean peak serum concentrations of 11.7 mg/L (S.E.M. 1.7) were recorded at 1 h after administration of 200 mg doses on day 2 of therapy and were 18.5 mg/L (S.E.M. 1.9) at 1.5 h in the 400 mg group. Loracarbef was shown to penetrate lung secretions even in the presence of beta-lactamase activity. Mean peak sputum concentrations were achieved between 2 and 4 h following dosing and were 0.2 mg/L (S.E.M. 0.05) in the 200 mg group and 0.4 mg/L (S.E.M. 0.08) in the 400 mg group. On days 7 and 14 of therapy, sputum loracarbef concentrations were similar 4 h after the morning dose (0.23 mg/L following 200 mg; 0.35 mg/L after 400 mg). These concentrations were approximately 2% of the peak serum concentration and penetration into lung secretions is similar to other beta-lactams.

Adult↗
PubMed1994

The significance of anti-streptokinase antibodies.

Antibodies to streptokinase (SK) are widespread in the population, but reports of their effect on the action of SK are conflicting. Specific anti-SK IgG was purified from the sera of 10 patients, five with low titres of anti-SK IgG and five with high titres. The effect of increasing specific anti-SK IgG antibodies on the action of SK was evaluated in vitro using a fluorimetric assay for plasmin and by a fibrin plate lysis assay. The inhibition of SK by whole plasma from a further group of patients was also assessed by the fibrin plate assay. There was a positive correlation between the serum antibody concentration and the quantity of specific anti-SK eluted (r = 0.797; P < 0.005). The addition of specific anti-SK IgG caused a dose-related decrease in SK activity (fluorimetric assay r = -0.93; P = 0.02; fibrin plate assay r = -0.98; P < 0.001). The addition of patient plasma to the fibrin plate assay also resulted in decreased lysis, which was dependent upon antibody titre (r = -0.95; P < 0.0001). Significant in vitro reduction of the activity of SK by specific antibody was demonstrated, and this was similar with plasma containing comparable amounts of antibody. The findings suggest that treatment with SK would be unlikely to induce an effective thrombolytic state when antibody titres are high (such as those seen within 2 years of an initial dose of SK).

Amino Acid Sequence↗
PubMed1994

Effect of fluticasone propionate on neutrophil chemotaxis, superoxide generation, and extracellular proteolytic activity in vitro.

BACKGROUND: Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. METHODS: The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. RESULTS: Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. CONCLUSIONS: These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue.

Administration, Topical↗
PubMed1994

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

BACKGROUND: Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS: The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. RESULTS: When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. CONCLUSIONS: It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Fibronectins↗
PubMed1994

A discrete subpopulation of human monocytes expresses a neutrophil-like proinflammatory (P) phenotype.

We have demonstrated that a discrete and naturally occurring subpopulation of human monocytes expresses a neutrophil-like proinflammatory (P) phenotype. P monocytes constitute 20-30% of the circulating monocyte pool and are characterized by 1) avid adherence to extracellular matrix through high-level cell-surface expression of alpha 5-, beta 1-, and beta 2-integrins; 2) high capacity to produce reactive oxygen species; 3) high content of serine proteinases and alpha 1-proteinase inhibitor; and 4) proteolytic activity against a soluble peptide human leukocyte elastase substrate, [3H]elastin, and solid-phase fibronectin, even in the presence of proteinase inhibitors. However, P monocytes express little or no cell-surface HLA-DR antigen, suggesting that they are unable to participate in specific immune responses. In contrast, the remainder of circulating monocytes have a low proinflammatory potential but contain the population of monocytes with high-level expression of HLA-DR antigen. P monocytes can readily be separated from the remainder of monocytes on the basis of 1) their capacity to adhere to fibronectin; and 2) their absent expression of HLA-DR antigen when flow cytometry or immunomagnetic beads are used. Our data indicate that, when recruited to sites of inflammation, P monocytes can either promote resolution of inflammation or contribute to tissue injury.

Cell Separation↗
PubMed1994

Neutrophil formyl-peptide receptors. Relationship to peptide-induced responses and emphysema.

A reproducible assay was established to assess the number of formyl-peptide receptors expressed on the surface of human polymorphonuclear leukocytes (PMN). Using this assay the number of receptors was shown to demonstrate wide within- and between-subject variability. However, the receptor numbers were related to the chemotactic response (r = 0.572) and degranulation response (r = 0.512) to the peptide formyl-methionyl-leucyl-phenylalanine. Subsequent studies showed increased receptor numbers on PMN from patients with emphysema (median, 459 x 10(3)/cell; range, 207 to 1,080) as compared with age-matched control subjects (median, 288; range, 168 to 519; p < 0.02), which may explain the increased chemotactic response of the PMN to formyl peptides. This difference was not observed in patients with bronchiectasis, suggesting that the increased receptor number is a feature of emphysema. Furthermore, the increase was largely a feature of smokers with emphysema (median, 463; range, 362 to 1,080), whereas age-matched smokers without emphysema had lower numbers of receptors (p < 0.001; median, 332; range, 243 to 411). This observation suggests a mechanism that may explain the susceptibility of some smokers to the development of emphysema.

Adult↗
PubMed1994

The role of proteinases in the pathogenesis of chronic bronchitis.

Proteinases have been implicated in the pathogenesis of many acute and chronic destructive lung diseases. Their role has been implied by a combination of genetic deficiency of their inhibitors and/or their ability to induce lung disease in animal models that resemble the human counterpart. However, identification of proteinases in lung tissue has proven difficult, with the exception of airway secretions, which often contain excessive and varied enzyme activity. Recent studies have focused on the role of these enzymes in the pathogenesis of airway diseases. This article reviews the evidence for their role in chronic bronchitis, the implications of this role, and new approaches to therapy based on the pathogenic mechanisms involved.

Animals↗
PubMed1994

The effects of beta 2-agonists and methylxanthines on neutrophil function in vitro.

Therapeutic agents which affect polymorphonuclear neutrophil (PMN) functions have the potential to reduce or increase PMN activation and, hence, influence the progression of lung inflammation. We have assessed the effects of the beta 2-agonist, terbutaline, and the methylxanthine, aminophylline, on PMN functions in vitro at both therapeutic and higher concentrations. At therapeutic levels, both agents increased PMN chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP) in a dose-dependent manner from a control value of 22.5 +/- 3.58 cells.field-1 to 26.1 +/- 4.73 cells.field-1 with 4 mg.l-1 terbutaline, and to 26.3 +/- 4.49 cells.field-1 with 20 mg.l-1 aminophylline. When the cells were preincubated with higher doses of the agents in separate experiments there was inhibition of chemotaxis from a control value of 31.1 +/- 2.06 cells.field-1 to 18.3 +/- 0.82 cells.field-1 at 160 mg.l-1 terbutaline, and to 16.1 +/- 0.77 cells.field-1 at 400 mg.l-1 aminophylline. A similar effect was seen when the PMNs were preincubated with terbutaline and aminophylline prior to assessment of superoxide anion generation, with stimulation of superoxide release at therapeutic levels of the drugs and inhibition at higher doses (19% increase from resting control cells at terbutaline 4 mg.l-1 and 53% reduction at 160 mg.l-1; 28% increase with aminophylline 20 mg.l-1 and 22% reduction at 400 mg.l-1). Both terbutaline and aminophylline had no effect on PMN degranulation, as assessed by the degradation of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminophylline↗
PubMed1994

Indomethacin potentiates the induction of HL60 differentiation to neutrophils, by retinoic acid and granulocyte colony-stimulating factor, and to monocytes, by vitamin D3.

We have confirmed previous observations that HL60 cells treated with a combination of 10 nM retinoic acid (RA), and 30 ng/ml granulocyte colony-stimulating factor (G-CSF) differentiate efficiently towards neutrophils, as characterized by their growth arrest and acquisition of phagocytic ability. Such low concentrations of RA alone provoked only a small proportion of HL60 cells to differentiate, and G-CSF alone provoked no differentiation. In the presence of 30 microM indomethacin (an inhibitor of the enzyme cyclooxygenase that catalyses the first step of prostanoid synthesis), the onset of differentiation provoked by RA plus G-CSF was more rapid, but the final proportion of mature cells was unchanged. Indomethacin also potentiated the growth arrest and differentiation of cells in response to 10 nM RA alone. Although the potentiating effect of indomethacin on RA-induced differentiation occurred at several indomethacin and RA concentrations, it was only apparent when the RA concentration used was alone sufficient to induce a small proportion of cells to differentiate. Indomethacin shifted the G-CSF dose-response curve of cells treated with 10 nM RA to lower G-CSF concentrations. 1 alpha,25-dihydroxy vitamin D3 (VitD3) induces HL60 cells to differentiate to monocytes and indomethacin also potentiated the differentiation of HL60 cells in response to low doses of VitD3 5,8,11-eicosatriynoic acid, an inhibitor of 5-lipoxygenase and 12-lipoxygenase, neither potentiated neutrophil differentiation of HL60 cells, nor prevented indomethacin potentiation of the differentiation of RA-primed cells. Treatment of cells with dexamethasone, a steroid whose effects include inhibition of arachidonate mobilization by phospholipase A2, potentiated RA-primed neutrophil differentiation in a manner similar to indomethacin. These observations suggest that an arachidonate metabolite formed downstream of cyclooxygenase suppresses differentiation of HL60 cells both to neutrophils and monocytes, probably by inhibiting some event essential to commitment to differentiation.

Arachidonic Acid↗
PubMed1993

Chemotactic activity of urethral secretions in men with urethritis and the effect of treatment.

The chemotactic activity of urethral exudate and the effect of treatment were assessed in 67 patients with nongonococcal urethritis (NGU) or gonorrhea. All samples demonstrated chemotactic activity related to the number of neutrophils seen on Gram's staining of the urethral smear. Chemotactic activity decreased after treatment in patients with Chlamydia-positive NGU (mean +/- SE, 21.1 +/- 2.7 cells/high-power field [hpf] before treatment; 8.7 +/- 2.2 after treatment; P < .004) and gonorrhea (25.6 +/- 5.2 cells/hpf before treatment; 1.6 +/- 0.7 after treatment; P < .015). Chemotactic activity increased again in Chlamydia-positive NGU patients to 21.0 +/- 3.3 cells/hpf (P < .05) 2-3 weeks after cessation of therapy in the absence of demonstrable infection or further intercourse. There was no significant decrease in chemotactic activity after therapy in the Chlamydia-negative NGU or persistent urethritis groups. These data suggest a previously unrecognized persistent chemotactic stimulus in urethral exudate from patients with urethritis.

Chemotaxis, Leukocyte↗
PubMed1993

Why do patients develop reactions to streptokinase?

Minor reactions to streptokinase are not uncommon, although the etiology is unknown. It is widely presumed, however, that these, like the more serious immune reactions, are antibody-mediated. We measured specific anti-streptokinase IgG, subclasses IgG1, IgG2, IgG3, IgG4 and IgE by ELISAs, haemagglutination, indirect Coombs' test and immunoblotting in six patients who developed reactions to streptokinase. Evidence of complement activation by streptokinase was sought by a haemolytic complement assay and by measurement of C3, C4 and C3d. The patients who reacted to streptokinase presented with low titres of anti-streptokinase IgG (median = 5; range 0-32) and IgG1 (median = 3; range 0-14). No evidence of any other IgG subclass was found, nor of specific anti-streptokinase IgE. Anti-streptokinase IgG1 was found to fix complement; patients who reacted to streptokinase were found to have low levels of total complement 1 year post reaction. Probable aggregates and fragments of human albumin (added stabilizer) were found in the streptokinase preparation and proved to be antigenic in some patients, but were not found to be related to the development of reactions. The findings suggest that patients who develop reactions to streptokinase cannot be predicted on the basis of antibody titres at presentation. Minor reactions to streptokinase would not appear to be antibody-mediated, although complement activation may be involved.

Aged↗
PubMed1993

Effects of recombinant GM-CSF and IgA opsonisation on neutrophil phagocytosis of latex beads coated with P6 outer membrane protein from Haemophilus influenzae.

BACKGROUND: IgA is the major antibody class in mucosal secretions, yet its biological functions remain poorly understood and its role as an opsonin for neutrophils has been the subject of controversy. It has been reported that treatment of neutrophils with granulocyte-macrophage colony stimulating factor (GM-CSF) induces the cells to phagocytose particles opsonised with IgA. A study was performed to investigate the effects of GM-CSF and IgA opsonisation on the ability of human neutrophils to recognise and phagocytose latex beads coated with the P6 outer membrane protein of Haemophilus influenzae. METHODS: Human neutrophils with and without preincubation with 100 pmol/l GM-CSF, were incubated with non-opsonised P6-coated latex beads or beads opsonised with IgA purified from the blood of a bronchiectatic patient with high titres of IgA anti-P6. Phagocytosis was measured by counting internalised beads during microscopic examination. RESULTS: The phagocytosis of IgA opsonised beads by untreated neutrophils (mean (SE) 2.1 (0.43) beads/cell) was significantly greater than that of non-opsonised beads (mean (SE) 1.3 (0.30) beads/cell). Treatment of neutrophils with GM-CSF resulted in increased phagocytosis of non-opsonised beads (mean (SE) 2.1 (0.39) beads/cell) but opsonisation with IgA increased this further (mean (SE) 3.4 (0.53) beads/cell). CONCLUSIONS: Human neutrophils recognise and phagocytose non-opsonised particles coated with bacterial antigen. Antibodies of the IgA isotype opsonise for neutrophil phagocytosis of particles coated with bacterial antigen but this behaviour is enhanced, in an additive fashion, by treatment of the cells with GM-CSF. The results suggest that IgA and GM-CSF are important cofactors for neutrophil recognition and elimination of bacterial pathogens.

Bacterial Outer Membrane Proteins↗
PubMed1993

Bronchoalveolar lavage and the study of proteinases and antiproteinases in the pathogenesis of chronic obstructive lung disease.

Bronchoalveolar lavage has been used for 15 yrs to investigate the role of proteinases and antiproteinases in the pathogenesis of emphysema, but the results are confused by numerous technical factors, many of which may prove insurmountable. Even if the problems can be overcome, the technique will probably not prove sensitive enough to provide a true insight into the pathogenesis of emphysema in man. Nevertheless, the studies with this technique have provided important information and methodologies that have advanced our scientific, if not pathological, knowledge. Perhaps further applications of the knowledge obtained, to cellular and genetic studies, will eventually establish the true mechanisms involved in determining whether a smoker remains "healthy" or develops disabling disease. Lavage may have played a major role in the study of emphysema, if for no other reasons than to establish the fact that the pathogenesis of the disease is far from clear.

Bronchoalveolar Lavage Fluid↗