Sodium chloride increases the ciliary transportability of cystic fibrosis and bronchiectasis sputum on the mucus-depleted bovine trachea.

Mucus retention in the lungs is an important feature of several respiratory diseases (Regnis, J.A., M. Robinson, D.L. Bailey, P. Cook, P. Hooper, H.K. Chan, I. Gonda, G. Bautovich, and P.T.P. Bye. 1994. Am. J. Respir. Crit. Care Med. 150:66-71 and Currie, D.C., D. Pavia, J.E. Agnew, M.T. Lopez-Vidriero, P.D. Diamond, P.J. Cole, and S.W. Clarke. 1987. Thorax. 42:126-130). On the mucus-depleted bovine trachea, the ciliary transport rate of sputum from patients with cystic fibrosis and bronchiectasis of other causes was slow, but the rate was doubled by increasing the sodium chloride content by 90 mM. Increasing the sputum osmolality by inspissation or by the addition of nonelectrolytes had a similar effect. The viscoelasticity of sputum, but not the bovine ciliary beat frequency, was markedly saline dependent over the pathophysiological range. This suggests that low mucus salinity, not (as is generally assumed) its under-hydration, contributes to its retention in bronchiectasis due to cystic fibrosis and other causes, probably by affecting its rheology. It also indicates how the genetic defect in cystic fibrosis might lead to impaired mucus clearance. Therapies that increase the osmolality of lung mucus might benefit patients with mucus retention.

Characterisation of Pseudomonas rhamnolipids.

The Gram negative organism, Pseudomonas aeruginosa, is often found in the lungs of patients with cystic fibrosis and other forms of severe bronchiectasis, where it secretes a number of extracellular toxins including the mono- and dirhamnolipids. The principal monorhamnolipid from P. aeruginosa has previously been identified as rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rh-C10.C10). A number of related mono- and dirhamnolipids have been purified from cultures of a clinical isolate of P. aeruginosa and identified by fast atom bombardment and electron impact mass spectrometry: these contain the 3-hydroxyoctanoyl-3-hydroxydecanoate (C8.C10) and 3-hydroxydecanoyl-3-hydroxydodecanoate (C10.C12) homologues. Structural isomers were also present where the order of the lipid linkage was transposed (Rh-C10.C8 and Rh-C12.C10). Unsaturated mono- and dirhamnolipids containing the 3-hydroxydecanoyl-3-hydroxydodec-5-enoate (C10.C12:1) lipid were also present.

Oxidant-mediated ciliary dysfunction in human respiratory epithelium.

Exposure of human nasal ciliated epithelium to reactive oxidants generated by the enzymatic xanthine-xanthine oxidase superoxide/hydrogen peroxide (H2O2) and glucose-glucose oxidase H2O2-generating systems, or to reagent H2O2 or hypochlorous acid (HOCl) resulted in significant alterations in ciliary beating. The earliest change noted was the presence of ciliary slowing, progressing eventually to complete ciliary stasis in some areas. Ciliary dyskinesia was seen within the first hour, often from as early as 15 min after exposure of the cells to reactive oxidants. Using peroxidases, various antioxidant enzymes, and oxidant scavengers, we confirmed that these detrimental effects on ciliary function were mediated primarily by H2O2 and HOCl. Moreover, 3-aminobenzamide (3-ABA), an inhibitor of the DNA repair enzyme poly ADP ribose polymerase, prevented H2O2-mediated inhibition of ciliary function, indicating that oxidant-mediated damage to DNA may well be the basis of the effects of H2O2 on ciliated epithelium. Acute and chronic inflammatory responses may therefore present the possible threat of H2O2- or HOCl-inflicted injury on bystander respiratory epithelium, leading to ciliary dyskinesia and slowing.

Anti-inflammatory, membrane-stabilizing interactions of salmeterol with human neutrophils in vitro.

1. We have investigated the effects of salmeterol (0.3-50 microM) on several pro-inflammatory activities of human neutrophils in vitro. 2. Oxidant production by FMLP- and calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (0.3-3 microM) of salmeterol, while the responses of phorbol myristate acetate- and opsonised zymosan-stimulated cells were affected only by higher concentrations (3-50 microM) of the drug. At these concentrations salmeterol is not cytotoxic, nor does it act as a scavenger of superoxide. 3. These anti-oxidative interactions of salmeterol with neutrophils were insensitive to propranolol but could be eliminated by washing the cells, or by pretreatment with low concentrations (1-2 microM) of the pro-oxidative, membrane-destabilizing phospholipids, lysophosphatidylcholine (LPC), platelet activating factor (PAF) and lysoPAF (LPAF). 4. At concentrations of 6.25-50 microM salmeterol interfered with several other activities of stimulated neutrophils, including intracellular calcium fluxes, phospholipase A2 activity and synthesis of PAF. 5. In an assay of membrane-stabilizing activity, salmeterol (25 and 50 microM) neutralized the haemolytic action of LPC, PAF and LPAF. 6. Of the other commonly used beta 2-adrenoceptor agonists, fenoterol, and formoterol, but not salbutamol, caused moderate inhibition of neutrophil oxidant generation by a superoxide-scavenging mechanism. However, unlike salmeterol, these agents possessed only weak membrane stabilizing properties. 7. We conclude that salmeterol antagonizes the pro-inflammatory, pro-oxidative activity of several bioactive lipids implicated in the pathogenesis of bronchial asthma, by a mechanism related to the membrane-stabilizing, rather than to the beta 2-agonist properties of this agent.

Effect of salmeterol on human nasal epithelial cell ciliary beating: inhibition of the ciliotoxin, pyocyanin.

1. Patients with airway infection by Pseudomonas aeruginosa have impaired mucociliary clearance. Pyocyanin is a phenazine pigment produced by P. aeruginosa which is present in the sputum of colonized patients, slows human ciliary beat frequency (CBF) in vitro and slows mucociliary transport in vivo in the guinea-pig. 2. We have investigated the effect of salmeterol, a long-acting beta 2-adrenoceptor agonist, on pyocyanin-induced slowing of human CBF in vitro. Salmeterol (2 x 10(-7) M) was found to reduce pyocycanin (20 micrograms ml-1)-induced slowing of CBF by 53% and the fall in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) by 26% and ATP by 29%. 3. Another beta 2-adrenoceptor agonist, isoprenaline (2 x 10(-7) M), also inhibited pyocyanin-induced slowing of CBF by 39%. 4. The effects of salmeterol (30 min preincubation) persisted after washing the cells. 5. Propranolol (10(-7) M) and the beta 2-specific antagonist, ICI 118551 (10(-6) M) blocked the protective effects of salmeterol completely, but atenolol (10(-6) M) was less effective. These results suggested that the effects of salmeterol on pyocyanin-induced effects were mediated primarily via the stimulation of beta 2-adrenoceptors. 6. Pyocyanin-induced ciliary slowing is associated with a substantial fall in intracellular cyclic AMP and ATP. Salmeterol reversed the effects of pyocyanin on cyclic AMP and ATP. 7. Mucociliary clearance is an important defence mechanism of the airways against bacterial infection. Salmeterol may benefit patients colonized by P. aeruginosa, not only by its bronchodilator action, but also by protecting epithelial cells from pyocyanin-induced slowing of CBF.

Human bronchoalveolar lavage cells and luminol-dependent chemiluminescence.

Human bronchoalveolar lavage cells from several different disease states were examined by the technique of zymosan-stimulated, luminol-dependent chemiluminescence. Light production correlated well with polymorphonuclear leucocyte contamination of the alveolar macrophage suspension but not with lymphocyte contamination. Regression analysis indicated that human alveolar macrophages produce little if any luminol-dependent chemiluminescence. Further investigation of metabolic activity, using measurements of superoxide release, oxygen consumption, and lucigenin-dependent chemiluminescence, showed that "respiratory burst" activity in alveolar macrophages was stimulated by opsonised zymosan.

Selective medium that distinguishes Haemophilus influenzae from Haemophilus parainfluenzae in clinical specimens: its value in investigating respiratory sepsis.

A medium is described, which is selective for the haemophilus genus and also distinguishes between the species Haemophilus influenzae and Haemophilus parainfluenzae isolated in primary culture from clinical material.

Early cellular responses to intradermal injection of Kveim suspension in normal subjects and those with sarcoidosis.

In a detailed controlled study of the cellular response to Kveim suspension in vivo we used immunohistological and histochemical methods to examine cryostat sections of immature Kveim biopsy specimens in subjects with sarcoidosis and normal controls. Changes seen at 48 hours, at which time papular reactions have sometimes been reported, are described. Eight cases of sarcoidosis previously confirmed by a positive Kveim test were studied, in five of whom the test remained positive; plus two subjects with sarcoidosis studied prospectively; and four healthy controls. There were two main features of the 48 hour response: collagen disruption with associated histiocytes, which showed increased acid phosphatase activity; and perivascular infiltrates of lymphocytes and small groups of dendritic cells. The T4:T8 ratios in the infiltrates were similar to those found in the peripheral blood of the subjects, and few lymphocytes showed evidence of activation. T lymphocytes were also seen free in the dermis and migrating to the epidermis. Small juxtacapillary clumps of dendritic cells, identified by NA1/34 (= OKT6; Langerhans' cells) and RFD1 (interdigitating cell) monoclonal antibodies, were found. The Langerhans' cells in the epidermis were, however, normal in number and distribution. These features, which were found in all groups, are not consistent with pre-existing hypersensitivity to Kveim suspension in sarcoidosis. Subsequent differences between sarcoid and normal subjects in the development of granulomas in the Kveim response may therefore relate to the different handling of the foreign material by the cells affected, rather than to differences in the early non-specific recruitment of the cells to the test site.

Simple method of monitoring colonising microbial load in chronic bronchial sepsis: pilot comparison of reduction in colonising microbial load with antibiotics given intermittently and continuously.

Aerobic and anaerobic culture of sputum on selective bacteriological media, combined with a new method of plating and plate reading, permitted rapid identification and quantitation of three genera of bacteria commonly associated with chronic bronchial sepsis (Haemophilus spp, Pseudomonas aeruginosa, and Staphylococcus aureus) and avoided time consuming serial dilution of sputum and subculture of organisms. The accuracy of this new technique was assessed in patients with chronic bronchial sepsis and was used to detect changes in the colonising microbial load of Haemophilus spp and Ps aeruginosa in patients with bronchiectasis receiving one of three different antibiotic regimens: intermittent seven day courses of amoxycillin for exacerbations; or a six month course of continuous oral or nebulised amoxycillin. The colonising microbial load of Haemophilus spp was reduced only temporarily (+++ to ++) after each intermittent course of antibiotic, but a sustained and greater reduction in the colonising microbial load of both Haemophilus spp (+++ to +) and antibiotic resistant P aeruginosa (+++ to +) was seen during both continuous treatments. Sputum purulence decreased in parallel with colonising microbial load, reflecting a reduction in host inflammatory response to the colonising microbial load.

Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth.

AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors. METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined. Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis. Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy. Antifungal activity against C albicans and A fumigatus was determined in a well plate assay. RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity. Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml. These phenazines were active against nine other yeast species pathogenic for man. Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin. CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa.

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases.

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pyocyanin and 1-hydroxyphenazine on in vivo tracheal mucus velocity.

Products of the bacterium Pseudomonas aeruginosa have been shown to slow the beating of human respiratory tract cilia in vitro. We have tested the effects of two of these compounds, pyocyanin and 1-hydroxyphenazine (given as a bolus dose dissolved in 2 microliters Ringer solution), on tracheal mucus velocity of radiolabeled erythrocytes in anesthetized guinea pigs. 1-Hydroxyphenazine (200 ng) caused a rapid slowing of tracheal mucus velocity (maximum fall 47% at 20 min) with recovery by 1 h. The effect of pyocyanin was slower in onset, 600 ng causing 60% reduction in tracheal mucus velocity at 3 h, and no recovery occurred. A combination of pyocyanin and 1-hydroxyphenazine produced an initial rapid slowing equivalent to the same dose of 1-hydroxyphenazine given alone, but the later slowing attributed to pyocyanin was greater than the same dose administered alone. This study demonstrates one mechanism by which products of P. aeruginosa may facilitate its colonization of the respiratory tract.

Interaction between Pseudomonas aeruginosa and Staphylococcus aureus: description of an anti-staphylococcal substance.

The presence of Pseudomonas aeruginosa in the sputum of 191 patients with cystic fibrosis was significantly related (p less than 0.0001) to the absence of Staphylococcus aureus. Cross-streaking tests showed that 40 of 50 clinical strains of P. aeruginosa produced substances that inhibited the growth of S. aureus. When incorporated into agar plates, this antibacterial substance(s) inhibited the growth of 177 of 189 strains of nine staphylococcal species, all of 16 methicillin-resistant S. aureus and 27 of 39 strains of six other gram-positive genera. The substance(s) did not inhibit 23 strains of seven gram-negative genera tested. The antibacterial activity was heat stable and could be extracted into chloroform; activity was retained on Sephadex G-15 (V/Vo approximately 2, Mr less than 500) and eluted as a single peak from high performance liquid chromatography, well separated from pseudomonic acid, pyocyanin and a number of other phenazines.

Microbial-host interactions in the airways in chronic respiratory infection.

The pathogenic events that take place in chronic respiratory infection highlight the successful microbial strategy of survival by persistence, or colonization. Microorganisms implement their strategy of persistence by two principal tactics: (1) sabotage of the host's bronchial defenses (ie, direct microbe-mediated damage to the host), and (2) subversion of the host's normally protective defenses into damaging host tissue itself (ie, indirect host-mediated damage provoked by the microbe). Among the various ways in which microorganisms directly damage host defenses and facilitate their own persistence in the respiratory tract are inhibition of ciliary function, inhibition of mucociliary transport, alteration of ion transport in respiratory epithelium, stimulation of mucus production, and damage to respiratory epithelium. Patients with chronic respiratory infection suffer a vicious circle of events leading to progressive lung damage and cardio-respiratory failure. Treatments to break this circle include antimicrobial therapy to reduce microbial colonization and anti-inflammatory/immunosuppressive therapy to modulate damaging host responses.

Radiological evidence of progression of bronchiectasis.

Radiological evidence of progression of bronchiectasis was sought in a group of 84 consecutive adult patients admitted to a tertiary unit with a particular interest in the disease. Methodical comparison for each patient of the earliest and most recent chest X-rays (n = 84), bronchograms (n = 1) and thoracic computed tomography (CT) scans (n = 32) was performed. Fifteen patients (18%) were considered to show radiological evidence of progression of bronchiectasis, 14 on chest X-ray, two of whom also showed progression on CT scans, and in one patient on bronchography alone. The likelihood of finding evidence of radiological progression increased the longer the interval between examinations. Serial radiology allows identification of patients with progression of disease and indicates the need to review management strategy.

Chest pain in chronic sputum production: a neglected symptom.

One hundred and fifteen patients (80 with bronchiectasis and 35 with mucus hypersecretion alone) who produced sputum on a daily basis were asked if they had suffered chest pain unassociated with an acute exacerbation of their chest symptoms during the last six months. Those with bronchiectasis complained of 28 separate pains of which 18 were considered to be of respiratory origin. Seventeen of these 18 pains were in an area associated with a bronchiectatic lobe(s). Only six chest pains (three considered to be of respiratory origin) were found among the 35 patients with mucus hypersecretion.

Relationship between psychological well-being and lung health status in patients with bronchiectasis.

Patients with bronchiectasis often complain of abnormal tiredness, difficulty in concentrating or low spirits. This study was carried out to examine levels of anxiety and depression in bronchiectasis and their relationship with other measures of lung health. One hundred and eleven patients with bronchiectasis determined by high-resolution computed tomography (CT) scan were studied using a range of physiological and psychological outcome measures. Patients completed anxiety and depression, health status (quality of life), fatigue and dypnoea questionnaires. Lung function was measured and exercise capacity was assessed using a shuttle walk test. Anxiety and depression scores formed a continuum. Moderate-severe anxiety was more frequent than equivalent levels of depression (17 vs 9% of patients). Anxiety and depression scores were associated with perceived health status (r=0.33 and 0.55). Neither anxiety nor depression was associated with the extent of bronchiectasis on CT scan. Depression was correlated with breathlessness and exercise performance (r=0.33 and 0.40), but anxiety was not. The correlation between depression and exercise performance was not simply due to the influence of somatic items in the depression questionnaire. We conclude that anxiety and depression are quite common in bronchiectasis in that 34% of patients had elevated scores for anxiety depression or both. The non-somatic components of depression were linked to dyspnoea and exercise performance, but anxiety was only related to perceived health. Therefore, treatment aimed at reducing symptoms and improving exercise capacity will not reduce levels of anxiety which need alternative therapy.

The reproducibility of bronchial circumference measurements using computed tomography.

Objective measurement of bronchial damage in patients with bronchiectasis is needed to identify progressive disease. This study evaluates the inter- and intraobserver variation in computed tomography (CT) measurements of bronchial wall circumference and examines the precision with which CT measurements of the bronchi can be repeated in patients with bronchiectasis. Twelve patients were scanned and the circumferences of 61 subsegmental bronchi were measured independently on two occasions, by three observers, using a tracing facility on the CT console. To determine the accuracy with which previously acquired sections could be repeated, five patients were scanned on two separate occasions. The mean bronchial circumference measured by the three observers was 16.1 +/- 8.3 mm. The standard deviations of differences between first and second measurements of bronchial circumference for the three observers (intraobserver variability) were: 0.60 mm, 0.67 mm and 0.40 mm. The standard deviation of readings for interobserver variability was 0.71 mm. The standard deviations of differences (and coefficients of variation) for measurements of bronchial circumference following rescanning were: 1.82 mm, 1.40 mm and 1.74 mm (9.9%, 7.6% and 9.3%, respectively). The reproducibility of measurements of wall circumference, between and within observers and between examinations, indicates that such measurements may be clinically useful in demonstrating the progression of bronchiectasis.

Roxithromycin, clarithromycin, and azithromycin attenuate the injurious effects of bioactive phospholipids on human respiratory epithelium in vitro.

The effects of the bioactive phospholipids (PL), platelet-activating factor (PAF), lyso-PAF, and lysophosphatidylcholine (LPC) on the beat frequency and structural integrity of human ciliated respiratory epithelium were studied in vitro, in the presence or absence of polymorphonuclear leukocytes (PMNL), the antimicrobial agents, roxithromycin, clarithromycin, and azithromycin and the antioxidative enzymes catalase and superoxide dismutase (SOD). All three PL caused dose-dependent slowing of ciliary beat frequency (CBF) and epithelial damage (ED) at concentrations > or = 1 microgram/ml, which were unaffected by inclusion of the antimicrobial agents and antioxidative enzymes. When epithelial strips were exposed to the combination of PMNL and PL, there was significant potentiation of ciliary dysfunction and ED, which was ameliorated by pretreatment of the PMNL with the antimicrobial agents or by inclusion of catalase, but not SOD. These results demonstrate that LPC, PAF, and lyso-PAF cause epithelial damage by direct mechanisms which are oxidant-independent, as well as by indirect mechanisms involving phagocyte-derived reactive oxidants. Macrolides and azalide antimicrobial agents may have beneficial effects on airway inflammation in asthma and microbial infections by protecting ciliated epithelium against oxidative damage inflicted by PL-sensitized phagocytes.

The role of nebulized antibiotics in treating serious respiratory infections.

The administration of a nebulized antibiotic in serious respiratory tract infections ensures high antibiotic concentrations at the site of infection, minimising systemic concentrations and their resultant risk of toxicity. Nebulized antibiotics have been used for the treatment of chronic infection with Pseudomonas aeruginosa, particularly in cystic fibrosis, but with variable clinical efficacy. Antibiotic delivery by nebulization is greatly influenced by the product formulation and the nebulizer. Use of intravenous formulations via a nebulizer can lead to exposure to potentially irritant or toxic additives and inappropriate pH or osmolality ranges, whilst the choice of nebulizer can greatly influence the drug deposition in the airway. Tobramycin Nebulizer Solution (TNS) is the first specific formulation for nebulization in cystic fibrosis using a designated nebulizer. The potential extrapolation of nebulized antibiotic therapy to other serious respiratory infections, in particular bronchiectasis and ventilator-associated pneumonia, is explored in this review.