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Biomedical subjects

D Burnett

Publications and source records attributed to D Burnett.

At least 55 records · Page 3Linked to original sources
PubMed1995

Quality and accreditation systems in clinical biochemistry in the European Union.

The European Community Confederation of Clinical Chemistry (EC4) formed in Nice in April 1993 has established a working group on laboratory accreditation. The aim of the group is to explore the possibilities for harmonisation of accreditation and quality systems in clinical laboratories in the European Community (EC). It is felt essential that professions should play a key role in the process, and that the principle of subsidiarity should be observed in relation to implementation and organisation in individual member states. The first task has been to collect information concerning such systems. In September 1993 a questionnaire was distributed to the twelve IFCC related societies for clinical chemistry in the EC. By December 1994 eleven societies had responded. The questionnaire related to the existence or planned introduction of quality and accreditation systems, the basis of the standards used and requirements for analytical aspects and qualifications of staff as well as professional aspects. Questions also addressed the way in which inspection were organised, the selection and training of inspectors, the organisation of systems and what interest there was in harmonisation. The results of this study are presented in this paper.

Accreditation↗
PubMed1994

Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.

The macrolide FK506 inhibited, by up to 50%, neutrophil migration and the production of the superoxide radical in response to the formyl peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The production of the superoxide radical in response to phorbol 12-myristate 13-acetate (PMA) was unaffected by FK506. The inhibition of neutrophil functions was accompanied by a partial reversal of FMLP-induced synthesis of cellular proteins, despite a rise in intracellular Ca2+. Neutrophils treated with FK506 demonstrated a small (average 23%) though significant decrease in formyl-peptide receptor numbers but receptor binding affinity was unaffected. The effects of FK506 on neutrophil activation appear to be analogous to those in T-lymphocytes. The incomplete inhibition, by FK506, of neutrophil responses suggests further that activation by FMLP is mediated via distinct multiple signalling pathways, including protein kinase activation and protein synthesis. The inability of FK506 to reduce FMLP-induced rises in cellular Ca2+ or PMA-induced activation of neutrophils suggests that its action is distal to Ca2+ mobilization and distinct from pathways relying on PKC activation. Thus the immunosuppressive effects of FK506 in vivo might be mediated through the inhibition of inflammatory cells other than lymphocytes and the drug therefore has therapeutic potential in a variety of inflammatory conditions. The drug also has potential in vitro for the characterization of signalling pathways from the plasma membrane to the nucleus.

Calcium↗
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

External quality assessment of techniques for the detection of drugs of abuse in urine.

Five chromatographic and six immunoassay techniques were compared using data reported by 131 participants in the UK National External Quality Assessment Scheme for Drugs of Abuse in Urine. Twenty five samples were studied containing none or one of three concentrations of amphetamine, barbiturates, benzodiazepines, benzoylecgonine, methadone and morphine. Technique sensitivity and specificity achieved with realistic clinical samples of 25 mL vol were assessed as the percentage of true positive and true negative tests, respectively. Thin-layer chromatography was inadequate for the detection of several analytes, the sensitivity for 0.5 mg/L of benzoylecgonine being < 30%, and for 1.5 mg/L of amphetamine < 86%. Gas chromatography with mass spectrometry was significantly less sensitive than other techniques for the detection of 0.5 mg/L of benzoylecgonine (71%) and 1.5 mg/L of morphine (88%). High-performance liquid chromatography was the most sensitive for amphetamine. Immunoassays performed well when operating above their specified cut-off concentrations but, because they are directed to quinalbarbitone showed reduced cross-reactivity with amylobarbitone, the barbiturate more commonly prescribed in the UK.

Chromatography↗
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

GABAA receptor subunit expression and sensitivity to ethanol.

Ethanol has been demonstrated to potentiate GABAergic mechanisms in several different cell types and also appears to be active on GABAA receptors in specific brain regions. By expressing brain mRNA and cloned GABA receptor subunits in Xenopus oocytes, we have investigated the requirements for ethanol modulation of the GABAA receptor. Using hybrid arrest techniques, the GABA receptor subunits alpha 1, beta 1, gamma 1, gamma 2S+ gamma 2L, gamma 2L and gamma 3 were individually prevented from expressing. None of these treatments altered potentiation by pentobarbital, the gamma 2S+ gamma 2L and gamma 2L reduced diazepam potentiation, however only the gamma 2L hybridization reduced ethanol sensitivity. By expression of cloned GABA subunits the effects could be reproduced by comparing ethanol sensitivity of alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L. The gamma 2L is an alternatively spliced variant of gamma 2 containing an extra eight amino acids bearing a consensus site for phosphorylation by protein kinase C. By using in vitro mutagenesis techniques, alteration of key residues in the phosphorylation site prevented ethanol modulation of the receptors containing mutant gamma 2 subunits. These results suggest that phosphorylation of a site on the gamma 2L subunit can alter the modulatory effects of ethanol on GABAA receptors containing this subunit.

Amino Acid Sequence↗
PubMed1993

Secretion of soluble chemotactic factors, including interleukin-6: a mechanism for the recruitment of CD8-positive T lymphocytes to human liver allografts during rejection.

In 35 patients receiving first liver transplants we assessed the chemotactic responsiveness of peripheral blood lymphocytes to bile, the subset composition of the responding population and the ability of peripheral blood lymphocytes to produce chemotactic factors. In 13 patients in whom acute rejection developed, lymphocyte chemotaxis in vitro was significantly greater in bile sampled 1 or 2 days after transplantation and before episodes of acute rejection than in bile sampled when rejection was clinically apparent or after steroid therapy during stable graft function. The chemotactic factors present showed preferential activity for CD8-positive T cells. Bile sampled during the same posttransplant periods from 11 patients who did not exhibit rejection showed significantly less chemotactic activity. In vitro cultured peripheral blood lymphocytes from patients in whom rejection developed produced lymphocyte chemotactic factors that induced a similar pattern of chemotactic responsiveness with preferential activity for CD8-positive T cells. Separate culture of purified CD4-positive and CD8-positive T cells obtained from patients in whom rejection developed showed that CD4-positive cells produced the factor(s). Analysis of the subset responses of normal peripheral blood lymphocytes to a variety of chemotactic cytokines showed interleukin-6 to have specificity similar to that observed with chemotactic bile, mixed peripheral blood lymphocyte culture supernatants and supernatants of CD4-positive T cells. Chemotactic activity was reduced by 45% to 90% in five chemotactic bile samples and three peripheral blood lymphocyte culture supernatants by treatment with affinity purified interleukin-6 monoclonal antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
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↗
PubMed1992

The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils.

Apoptosis of neutrophils at sites of inflammation in vivo is thought to lead to their recognition and safe elimination by macrophages. Little is known, however, about the regulation of apoptosis in myeloid cells. We report here that the human promonocytic leukemic cell line, U937, and mature human neutrophils can be induced to become apoptotic when cultured with interleukin-6. Apoptosis of U937 cells, assessed morphologically and by the presence of DNA fragmentation, was increased significantly in a dose-dependent fashion by concentrations of 0.5-100 ng/ml interleukin-6. Apoptosis of U937 cells was evident after 48 h of incubation with 20 ng/ml interleukin-6, and the effect was eliminated by adsorption of interleukin-6 with a specific monoclonal antibody. Apoptosis was not evident in the presence of the differentiating agent phorbol 12-myristate 13 acetate; the induction of apoptosis in U937 cells was not therefore a consequence of differentiation. Apoptosis of mature neutrophils was enhanced after 24 h in culture with interleukin-6. Interleukin-6 might be an important factor in the normal resolution of inflammation through the induction of apoptosis of neutrophils.

Antibodies, Monoclonal↗
PubMed1992

Use of an enzyme-linked immunosorbent assay to assess penetration of amoxicillin into lung secretions.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure total amoxicillin concentrations penetrating lung secretions, which were compared with "active" concentrations measured by conventional bioassay. An antibody was raised in rabbits to amoxicillin conjugated to bovine serum albumin and used in a competitive binding ELISA (sensitivity, 10 ng/ml; precision [coefficient of variation], 9%). The measurement of amoxicillin in lung secretions by using the ELISA method was verified by high-performance liquid chromatography. Amoxicillin concentrations were found to be similar in both whole sonicated sputum and sol-phase sputum obtained by ultracentrifugation following single oral doses of 3 g (4.6 mg/liter for sonicated and 4.7 mg/liter for sol-phase preparations) and 250 mg (0.23 mg/liter for both preparations). Eight patients with bronchiectasis received 500 mg of amoxicillin three times daily. On the second day of therapy (4 h after the morning dose), the mean concentration of amoxicillin in sputum was 0.88 mg/liter (standard error of the mean [SEM], 0.11) by ELISA and 0.40 mg/liter (SEM, 0.05) by bioassay, suggesting a significant degree of local inactivation. This difference between total and active amoxicillin levels was found to correlate significantly (r = 0.693; P less than 0.05) with beta-lactamase levels (mean, 29.5 mU/ml; SEM, 9.4). A pharmacokinetic study on day 3 revealed maximum levels in secretions 2 to 4 h after dosing (mean, 1.36 mg/liter; SEM, 0.26). At the end of successful therapy (day 14), total and active levels were lower (mean, 0.48 mg/liter; SEM, 0.11 [total]; mean, 0.21 mg/liter; SEM, 0.06 [active]); this result was associated with a reduction in lung inflammation (decreased serum-derived albumin in the lung secretions). In conclusion, antibiotic penetration is partly dependent on the degree of lung inflammation. The differences observed in total and active levels of amoxicillin and the relationship to beta-lactamase activity in sputum suggest why higher doses of antibiotic may be required to produce a therapeutic response in some patients.

Administration, Oral↗