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Biomedical subjects

R A Stockley

Publications and source records attributed to R A Stockley.

At least 145 records · Page 8Linked 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

Chronic bronchitis: the antiproteinase/proteinase balance and the effect of infection and corticosteroids.

Proteolytic enzymes have the potential to destroy many of the lung structures, provided that their activity exceeds the capacity of their natural inhibitors. Neutrophil elastase has been implicated in the pathogenesis of chronic bronchitis and is regularly present in secretions from these patients. Several factors, including infection and inhibitor deficiency, influence the balance between enzymes and inhibitors and may play a role in disease progression. Many enzymes and inhibitors are found in lung secretions and further study of their interactions and effect on lung tissues will clarify the nature and clinical effect of chronic bronchitis.

Adrenal Cortex Hormones↗
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

Deoxyribonucleic acid (DNA) polymorphism of the alpha 1-antitrypsin gene in chronic lung disease.

Specific gene probes were used to study restriction fragment length polymorphisms of the human alpha 1-antitrypsin gene. A polymorphism due to loss of a recognition site for the restriction enzyme Taq I was identified in eight of 42 patients with bronchiectasis and nine of 49 patients with pulmonary emphysema, none of whom had alpha 1-antitrypsin deficiency. Among a control group without lung disease the polymorphism was significantly less frequent, being found in only five of 101 apparently healthy blood donors. The deoxyribonucleic acid (DNA) polymorphism was also present in two of 14 unrelated patients with alpha 1-antitrypsin deficiency, indicating a lack of association with any specific alpha 1-antitrypsin protein phenotype. The polymorphism identified in this study may be a new marker for genetic predisposition to chronic lung disease.

Adolescent↗
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

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

Bronchiectasis--new therapeutic approaches based on pathogenesis.

This article has highlighted several new concepts in the pathogenesis of bronchiectasis and proposed a hypothesis that could explain the deterioration seen in some of the patients. Modification of our current antibiotic policy has been shown to be beneficial in both the short and long term. The results of recent studies would suggest that patients with purulent sputum benefit from antibiotic therapy whether this represents an acute exacerbation or the usual clinical state. Exacerbations respond well to broad-spectrum antibiotics effective against H. influenzae and S. pneumoniae in conventional doses. However failure to respond, particularly when the usual secretions are purulent, often indicates that dosage is inadequate and a trial of a higher dosage of an agent such as amoxicillin may well prove effective. Treatment of patients who are also penicillin sensitive needs to be clarified, although no orally effective alternative has yet been identified. Relapse is rapid in patients whose usual secretions are purulent, and long-term therapy improves well-being and several biochemical indices of important pathogenic mechanisms. However, as we understand the reasons for bacterial colonization and persistence better, it should prove possible to modify other mechanisms therapeutically, thus negating the need for long-term antibiotic therapy and the associated potential risks of resistance.

Acute-Phase Proteins↗
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↗
PubMed1986

Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds.

Cathepsin B synthesis by the human HL60 promyelocyte cell line was investigated by immunohistochemistry and by the assay of the enzyme in cell lysates using a fluorimetric substrate. HL60 cells were shown to produce cathepsin B in response to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Intracellular levels of cathepsin B and immunohistochemical staining of the enzyme were related to time in culture with increasing concentrations of TPA from 1 nmol/1 to 8.0 nmol/1. Synthesis of cathepsin B was associated with TPA-induced phagocytic activity of cells in culture, expression of alpha-naphthyl acetate esterase and reduced cell division. Cathepsin B production was, therefore, related to differentiation of the HL60 promyelocytes into mature macrophage-like cells. Cathepsin B activity in HL60 cell lysates was significantly increased by incubation of the cells with 10 micrograms/ml endotoxin (lipopolysaccharide) from Escherichia coli, but not carrageenan. The production of cathepsin B by TPA-induced HL60 cells was significantly reduced by 0.25 mumol/1 dexamethasone and the non-steroidal anti-inflammatory compound 4-(6-methoxy-2-naphthyl)-butan-2-one but not by indomethacin. The HL60 promyelocytic cell line is a useful model for the study of factors affecting proteinase synthesis by human mononuclear phagocytes.

Anti-Inflammatory Agents↗
PubMed1986

Effect of cigarette smoking, pulmonary inflammation, and lung disease on concentrations of carcinoembryonic antigen in serum and secretions.

A specific radioimmunoassay for carcinoembryonic antigen was used to investigate aspects of its measurement in lung disease. The results confirm that serum carcinoembryonic antigen concentrations are higher in healthy smokers and patients with chronic obstructive bronchitis than in healthy non smokers (p less than 0.01). Corticosteroid treatment reduced the concentration in nine patients with bronchitis (p less than 0.05). Other inflammatory lung diseases (bronchiectasis, pneumonia, fibrosing alveolitis) are not associated with a raised serum carcinoembryonic antigen concentration. The sputum concentrations were about 100 times those found in serum and there was a positive correlation (r = 0.611 2p less than 0.01) between the concentrations in sputum and serum in patients with bronchitis. No preferential rise in sputum concentration was found in current smokers or patients with lung carcinoma (n = 16). A higher ratio of carcinoembryonic antigen to albumin concentration (p less than 0.05) was, however, found in lavage fluid obtained from the tumour site than in fluid from "normal" lung in the same patients, suggesting an increase in carcinoembryonic antigen secretion in the vicinity of the tumour. Despite this "local" effect the sputum concentration does not, however, appear to be a useful marker of lung carcinoma and the measurement could not be used as a screening test.

Bronchitis↗