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Biomedical subjects

D Burnett

Publications and source records attributed to D Burnett.

At least 109 records · Page 6Linked to original sources
PubMed1988

The assessment of alpha 1 proteinase inhibitor form and function in lung lavage fluid from healthy subjects.

The form and function of alpha 1 proteinase inhibitor in lung lavage fluid from healthy smoking and non smoking individuals has been accurately assessed using critically appraised techniques. The present study demonstrated that it is possible to accurately assess alpha 1 PI function in unconcentrated lavage fluid but that sample collection, storage and subsequent processing may all affect the results. Absolute levels of alpha 1 PI were elevated in subjects who smoke and a substantial quantity of inactive protein was found in both smokers and non smokers. The proportion of inactive alpha 1 PI was similar for both groups, which by inference implies that normal smoking subjects do not have decreased protection by this inhibitor at the bronchoalveolar level. Physicochemical analysis of the alpha 1 PI in these normal subjects showed that it was different from alpha 1 PI previously reported from patients with established disease and this may have important implications regarding the pathogenesis of their condition. Western immunoblotting of bronchoalveolar lavage fluid (BALF) showed that all of the alpha 1 PI was present in the native molecular mass form (54,000 Da). Pre-incubation of samples with methionine sulphoxide peptide reductase restored alpha 1 PI function only by approximately 10% suggesting the presence of little reversibly oxidised alpha 1 PI in either group. Anion exchange HPLC of BALF revealed the presence of two alpha 1 PI species, one of which co-eluted with native, oxidised or proteolyzed forms and the other which was more cationic and did not inhibit porcine pancreatic elastase. Finally, thirteen out of sixteen BALF samples inhibited more neutrophil elastase than could be accounted for by the amounts of functional alpha 1 PI present, suggesting that the presence of other inhibitors is a feature of normal lavage fluids.

Animals↗
PubMed1988

The response of patients with purulent bronchiectasis to antibiotics for four months.

Continuous treatment with antibiotics for 16 weeks was given to 10 patients with bronchiectasis who regularly produced purulent elastase positive secretions. Macroscopic clearance of secretions (from purulent to mucoid or mucopurulent) was achieved in all patients, and this was associated with a significant reduction in the average sputum elastase content by week 16 (p less than 0.001) when elastase was only detectable in three of the patients. Lung inflammation was also reduced as reflected in the sputum to serum albumin ratios. In addition patients noted a significant improvement in well being, and breathlessness as well as in sputum colour and volume and there was a significant improvement in peak expiratory flow. After cessation of treatment the time taken for the sputum to become purulent again ranged from 15 days to 10 months (median 2.5 months), which is longer than that seen previously following short courses of antibiotic treatment. Serum levels of the acute phase protein alpha 1 antichymotrypsin fell by week 16, although this failed to reach statistical significance. However, in eight patients where data were available the levels rose significantly after treatment had finished. The concentrations were consistently higher than those seen in healthy controls, and were also elevated in less severely affected patients suggesting that bronchiectasis is always associated with some systemic effect.

Adult↗
PubMed1988

Production of alpha 1-antichymotrypsin by the J111 cell line.

The J111 histiocytic cell line is capable of producing human alpha 1-antichymotrypsin (alpha 1 ACh) in culture. The protein is secreted rather than stored in the cell, has a similar size to plasma alpha 1 ACh (68 kDa) and can complex with human cathepsin G. Dexamethasone and culture supernatants from U937 monocyte-like cells and alveolar macrophages, but not bacterial lipopolysaccharide, stimulate alpha 1ACh secretion by J111 cells. The J111 cell line thus provides a useful tool for the study of factors controlling alpha 1ACh synthesis in vivo.

Autoradiography↗
PubMed1987

Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis.

Peripheral polymorphonuclear leucocytes (PMN) from subjects with emphysema or bronchiectasis digested significantly more iodine-125-labelled fibronectin (on average, 250% and 280%, respectively) than did those from control subjects. PMN from patients with bronchiectasis contained significantly more of the serine proteinase elastase than did the control cells, which may have contributed to their greater extracellular proteolysis. PMN from patients with emphysema, but not those with bronchiectasis, showed enhanced chemotaxis (on average 260%) in response to a chemotactic peptide compared with control cells. Thus, PMN from subjects with chronic obstructive lung diseases can digest more extracellular connective tissue protein than PMN from healthy subjects. This behaviour suggests a mechanism for the pathological tissue damage associated with these disorders. Furthermore, the sensitivity to chemotactic factors of PMN from emphysematous patients would contribute to the larger numbers of these cells in their lung tissues, thus increasing further the proteolytic burden in the lungs.

Aged↗
PubMed1987

Automated liquid chromatographic method for the determination of paracetamol and six metabolites in human urine.

An automated liquid chromatographic method, with a coefficient of variation for total imprecision of less than 4%, has been developed for the quantitative determination of paracetamol, paracetamol-4-glucuronide, paracetamol-4-sulphate, paracetamol-3-cysteine, paracetamol-3-mercapturate, 3-hydroxyparacetamol-3-sulphate and 3-methoxyparacetamol-4-sulphate in urine samples. The gradient elution system was based on 0.067 M phosphate buffer (pH 2.0) and acetonitrile on an octadecylsilica column. The on-column detection limit using an ultraviolet detector at 254 nm for each of the compounds using 3-hydroxyacetanilide as internal standard was of the order of 10-50 ng from urine and 2-10 ng from water. Application of the method to 24-h urine samples from subjects who had received a therapeutic dose of the drug confirmed the findings of previous studies for the importance of the glucuronide, sulphate, mercapturate and cysteine conjugates. 3-Hydroxyparacetamol-3-sulphate was shown to be present in the urine of all volunteers and to account for up to 5% of the dose. 3-Methoxyparacetamol-4-sulphate was not detected in any urine samples and if present as a metabolite must account for less than 0.1% of the dose.

Acetaminophen↗
PubMed1987

The effect of assay conditions on the measurement of anti-elastase function in lung secretions.

We have determined the effect of altering assay conditions on the observed neutrophil elastase inhibitory capacity in lung secretions from emphysematous patients with normal serum alpha 1 PI. alpha 1 PI, ALP and alpha 2-macroglobulin were detected in all samples. Measurement at low enzyme concentration (less than 33.6 nmol/l) caused a 43% reduction in observed neutrophil elastase inhibitory capacity of sputum sol-phase, while inhibition by secretions in buffer without added NaCl was 20% greater than in the presence of 0.2 mol/l NaCl. Increasing the concentration of the synthetic substrate Suc-[Ala]3-pNA in the assay from 0.45 mmol/l to 7.2 mmol/l reduced the observed inhibitory capacity by 53% and the use of elastin-fluorescein gave lower results for inhibitory capacity than the Suc-[Ala]3-pNA (median 0.26 mol neutrophil elastase/mol measured inhibitors (range 0-0.72) with elastin; 1.40 mol neutrophil elastase/mol measured inhibitors (0.80-3.21) with Suc-[Ala]3-pNA). Assay conditions therefore greatly influence the results. In addition these findings suggest the presence of an additional inhibitor of neutrophil elastase in these secretions.

Bronchitis↗
PubMed1987

A 13-week vapor inhalation study of 3,3-dimethyl-2-butanol in Sprague-Dawley rats.

Four groups of male and female Sprague-Dawley rats were exposed for 13 weeks to 3,3-dimethyl-2-butanol (PA) at concentrations of 0.00, 0.20, 1.00 or 5.00 mg/l (1 mg/l = 240 ppm). Exposures were for 6 hr per day, 5 days per week with sacrifices at 7 and 13 weeks of exposure, and at 4 weeks after exposure. The test animals were evaluated for abnormalities in physiology, behaviour, clinical laboratory parameters, and gross and microscopic morphology. No abnormalities were detected in electrocardiograms, respiratory indices, spontaneous activity, passive avoidance activity and open-field behaviour. Clinical signs related to PA exposure included alopecia, ataxia and lacrimation. There were no biologically significant between-group differences in body-weights during the study. The clinical laboratory data demonstrated a 30% increase in serum cholesterol and bilirubin at 7 weeks in high-dose males and an increase in urea nitrogen in intermediate and high-dose males at 13 weeks. There were no abnormalities in hematologic or coagulation parameters. At necropsy there were no significant gross abnormalities; however, examination of organ weights revealed enlarged kidneys in high-dose male rats at 13 weeks, enlarged ovaries in high-dose female rats at 13 weeks, and microscopic study of tissue sections revealed minimal to mild renal tubular injury in high and possibly intermediate dose males at several sacrifices. These findings suggest that the primary target organ of PA, when given by inhalation, is the kidney in male rats and possibly the ovary in female rats. The renal changes in the high-dose males were not fully reversible during the recovery period.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Pollutants, Occupational↗
PubMed1987

Lung lavage fluid from patients with alpha 1-proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function and cell profile.

The anti-elastase composition of bronchoalveolar lavage (BAL) fluid from alpha 1-proteinase inhibitor (alpha 1PI) deficient and bronchitic patients was determined by immunological and functional assays, together with the cell profile of the BAL fluid. alpha 1PI, anti-leucoprotease and alpha 2-macroglobulin were present in all the samples BAL fluid alpha 1PI concentrations were significantly lower in the group with serum alpha 1PI deficiency. Lavage fluid from alpha 1PI deficient subjects inhibited less porcine pancreatic elastase than bronchitic BAL fluid (P less than 0.005). However, the alpha 1PI was only about 50% active as an inhibitor in both groups. There was no difference in the amount of neutrophil elastase (NE) inhibited per ml of lavage fluid or per mol of the measured inhibitors in the secretions, but both groups inhibited more enzyme than would be expected for these inhibitors (alpha 1PI deficient: median 4.78 mol of NE/mol of known inhibitors, range 0.88-78.80; bronchitic: 1.14, 0.21-4.66), suggesting that an additional inhibitor is present. The total leucocyte and neutrophil counts were elevated (2P less than 0.01) in the lavages of alpha 1PI deficient patients, suggesting a greater potential elastase burden than subjects with normal alpha 1PI.

Adult↗
PubMed1987

Elastase inhibitors in sputum from bronchitic patients with and without alpha 1-proteinase inhibitor deficiency: partial characterization of a hitherto unquantified inhibitor of neutrophil elastase.

Anti-elastase function in sputum sol-phase from patients with alpha 1-proteinase inhibitor (alpha 1PI) deficiency was compared with sol-phase from patients with cigarette smoke-induced bronchitis and emphysema. Both alpha 1PI (2P less than 0.01) and anti-leucoprotease (ALP) (2P less than 0.01) concentrations were lower in sol-phase from the alpha 1PI-deficient group, although alpha 2-macroglobulin (alpha 2M) levels were similar. There was no difference in alpha 1PI function between the two groups, but the inhibitor was only congruent to 30% active. The absolute neutrophil elastase (NE) inhibitory capacity was similar in both groups (median 185 micrograms of NE inhibited/ml of sputum, range 80-480, for the alpha 1PI-deficient group; median 175, range 80-300, for the bronchitic group). A substantial proportion of NE inhibition in secretions could not be accounted for by the amount of alpha 1PI, ALP and alpha 2M present (median 74.8%, range 43.2-97.4, for alpha 1PI-deficient sol-phase; median 50.0%, range 0-80.8, for bronchitic sol-phase). Gel filtration of sol-phase demonstrated the presence of NE inhibition in the low molecular weight fractions which was markedly sensitive to changes in substrate concentration and ionic strength, in contrast to purified alpha 1PI and ALP. Sputum sol-phase from both groups failed to prevent hydrolysis of elastin-fluorescein or succinyltrialanyl-p-nitroanilide by NE completely during prolonged incubation in the presence of an excess of functional inhibitors. This was more apparent in secretions from subjects with alpha 1PI deficiency and may explain why such patients have a more rapidly progressive form of emphysema.

Adult↗
PubMed1987

Determination of elastase inhibitory activity of alpha 1-proteinase inhibitor and bronchial antileukoprotease: different results using insoluble elastin or synthetic low molecular weight substrates.

We have determined the effect of two elastase-specific synthetic low molecular weight substrates, L-pyroglutamylprolylvaline-paranitroanilide and succinyltrialanyl-paranitroanilide, together with insoluble elastin-fluorescein, on the determination of the neutrophil elastase (NE) inhibitory capacity of purified alpha 1-proteinase inhibitor (alpha 1-PI) and bronchial antileukoprotease (ALP). In addition, the inhibitory capacities of mixtures of alpha 1-proteinase inhibitor, antileukoprotease and alpha 2-macroglobulin prepared in ratios similar to that in lung secretions were determined. Purified inhibitors, alone or in combinations, inhibited about 1 mole neutrophil elastase per mole inhibitor when assessed using synthetic substrates. However, when elastin-fluorescein was used in the assay system, the purified inhibitors showed an inhibitory capacity that was 40-85% of the value obtained using synthetic substrates. Even less inhibition was observed when mixtures of inhibitors were assessed using elastin-fluorescein (23-44% of the value for synthetic substrates). Our data indicate that results of elastase inhibitor activity measurements depend on the type of substrate which has been used.

Blood Proteins↗
PubMed1987

Cells containing IgA subclasses in bronchi of subjects with and without chronic obstructive lung disease.

Necropsy specimens were obtained from the lungs of 10 subjects who had no history of lung disease, 10 who had died with chronic bronchitis, and 10 with bronchiectasis. Tissue sections were stained for IgA1 or IgA2 using the immunoperoxidase technique, and the number of cells in the bronchi stained for these proteins was counted. The total number of IgA positive cells was increased in bronchitic and bronchiectatic lungs compared with those from control subjects. The number of IgA2 positive cells was similar in those with bronchitis and bronchiectasis and significantly higher than in controls. Similarly, cells containing IgA1 were increased in the lungs of subjects with chest disease but were higher in those with bronchitis than in those with bronchiectasis. The proportion of IgA2:total IgA containing cells was similar in sections from controls (mean (SD) 25 (5.0)%) and those with bronchiectasis (mean (SD) 24 (4)%), but lower in those with bronchitis (mean (SD) 19 (5.0)%). The results show that cells containing IgA1 predominate in the major bronchi but that the proportion of cells containing IgA2 is higher than in non-mucosal lymphoid tissues. Bronchitis and bronchiectasis are associated with greater numbers of cells producing IgA in the bronchi, and this is consistent with increased local production of IgA in the lung secretions of bronchitic subjects.

Adult↗
PubMed1987

Studies of proteinases and their inhibitors in lung secretions.

The study of proteinases and their inhibitors in lung secretions may provide an understanding of the pathogenesis of many chronic lung diseases. However problems arise in the collection and assessment of the samples that leads to variability both within and between subjects. The purpose of the paper is to highlight these problems as well as the methods used to overcome them in secretions obtained from patients with chronic obstructive bronchitis. The results show that between subject variability is high; and may be up to 150% within an individual. Conventional means of reducing variability (standardisation for albumin) may not be appropriate for some proteins, leading to increased variability. The function of inhibitors is probably best expressed as enzyme inhibited/unit of inhibitor though even this shows daily variability within the same individual. An awareness of such variability is necessary for the interpretation of results obtained from patients.

Endopeptidases↗
PubMed1986

alpha 1-Antichymotrypsin in lung secretions is not an effective proteinase inhibitor.

This paper presents evidence that alpha 1-antichymotrypsin in lung secretions is not effective as an inhibitor of chymotrypsin-like enzymes. First, lung secretion samples inhibited more cathepsin G on a one-to-one molar basis than could be accounted for by the alpha 1-antichymotrypsin present. Second, the major cathepsin G inhibitory capacity of sputum was in gel filtration fractions that corresponded to a low molecular weight (10,000-15,000) and contained immunoreactive antileucoprotease. Third, although alpha 1-antichymotrypsin purified from plasma was almost fully active against cathepsin G, that purified from lung lavage retained less than 15% of its inhibitory function. Immunoblotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that alpha 1-antichymotrypsin in plasma and lung secretions are of similar molecular size and no enzyme-alpha 1-antichymotrypsin complexes could be detected in sputum or bronchoalveolar lavage fluids. However, in contrast to the alpha 1-antichymotrypsin purified from plasma, the lavage protein gave a broad elution profile following anion-exchange chromatography.

Cathepsin G↗