Nitric oxide metabolites in cystic fibrosis lung disease.

Although the activity of nitric oxide (NO) synthases are increased in lung tissue of patients with cystic fibrosis, the concentrations of nasal and exhaled NO have recently been found to be decreased in cystic fibrosis. This could either be due to reduced NO formation or metabolism of NO within airway fluids. In this study, the stable NO metabolites, nitrate and nitrite, were determined in the saliva and sputum of 18 stable cystic fibrosis patients, 21 cystic fibrosis patients during a pulmonary exacerbation, and in saliva and endotracheal secretions of normal controls. Median saliva concentrations of NO metabolites (nitrate plus nitrite) were 704 mumol/l (95% confidence interval (CI) 419 to 1477) in stable cystic fibrosis patients, 629 mumol/l (95% CI 382 to 1392) in cystic fibrosis patients presenting with pulmonary exacerbation, and 313 mumol/l (95% CI 312 to 454) in controls. Median sputum NO metabolite concentration in stable cystic fibrosis was 346 mumol/l (95% CI 311 to 504). This was not significantly different from cystic fibrosis patients presenting with pulmonary exacerbation (median 184 mumol/l, 95% CI 249 to 572), but significantly higher than in endotracheal secretions of controls (median 144 mumol/l, 95% CI 96 to 260). Sputum NO metabolite concentration in cystic fibrosis pulmonary exacerbation significantly increased during antibiotic treatment. A positive correlation was observed between sputum NO metabolites and lung function in stable cystic fibrosis, suggesting less airway NO formation in cystic fibrosis patients with more severe lung disease. These data indicate that decreased exhaled NO concentrations in cystic fibrosis patients may be due to retention and metabolism of NO within the airway secretions. However, sputum NO metabolites are not a useful marker of airway inflammation in cystic fibrosis lung disease.

Rheology of cystic fibrosis sputum after in vitro treatment with hypertonic saline alone and in combination with recombinant human deoxyribonuclease I.

Treatment with recombinant human deoxyribonuclease I (rhDNase) is currently used as therapy for cystic fibrosis (CF) lung disease. Hypertonic saline (HS) acts as an expectorant promoting mucus secretion and augmenting the volume of sputum. We evaluated the individual and combined effects of HS and rhDNase in vitro on the viscoelasticity of CF sputum. Sputum samples were collected from nine CF patients to use for in vitro testing. Aliquots of CF sputum (0.20 to 0.40 g) were subjected to the following protocols: (1) negative control sample without any treatment; (2) positive control sample, adding 10% volume of normal saline (0.9% NaCl); (3) application of hypertonic saline (HS-3% NaCl); (4) combining approximately 100 nM concentration of rhDNase with protocols 2 and 3. The samples in protocols 2 through 4 were incubated for 30 min at 37 degrees C. For each protocol, CF sputum was analyzed at baseline and at 30 min for spinnability by filancemeter and viscoelasticity by magnetic microrheometry. Spinnability decreased for the sputum samples that were treated with rhDNase, in combination with either HS or normal saline. Treatment with HS alone and combined treatment with rhDNase and HS decreased log G* (the principal viscoelasticity index) to the same degree. Saline alone and rhDNase in normal saline both increased the predicted cough clearability of the sputum; however, the combined treatment with rhDNase and hypertonic saline had the best overall effect on cough clearability. The change in predicted mucociliary clearability, although greatest after HS, was not significant. These in vitro results suggest that combined treatment with rhDNase and HS should be evaluated further as a potential mucotropic approach to augment the clearance of purulent sputum in CF lung disease.

The effect of tasuldine, a bronchosecretolytic agent, on mucus rheology and clearability and the interaction with acetylcholine in ferrets.

Tasuldine (Ts) is an orally active bronchosecretolytic agent shown to be clinically effective in human studies. Tasuldine decreases the sialomucin content of the mucus and, in animal studies, this modulation of the glycopeptide correlates with decreased mucus viscosity. The aim of this study was to evaluate the effect of tasuldine on mucus viscoelasticity and correlate the rheological changes to mucociliary and cough clearability. Tracheal mucus samples were collected from anaesthetized adult ferrets by a modification of the cytology brush technique. Mucus was collected prior to and following administration of either vehicle (normal saline) or Ts (10 mg/kg i.v.), and followed by acetylcholine (ACH) challenge (ca. 4 ml of 10(-2)M i.v., slow infusion). The analysis included magnetic microrheometry to measure the viscosity and elasticity of microlitre quantities of mucus. Mucociliary transportability (NFPTR) was measured by means of the frog palate assay and mucus collection rates (mg/min) were used as an indirect measure of secretion rate. The principal index of mucus rigidity, log G*, decreased with tasuldine infusion (P = 0.014) and further decreased with ACH (P = 0.002). In simple terms, the mucus became less rigid or more deformable with tasuldine administration, thus benefiting clearability based on predictions from model studies. The changes observed with acetylcholine alone were consistent with a classic secretagogue response--the outputting of a large volume of watery mucus. NFPTR increased with tasuldine treatment, and even further with acetylcholine; however, the combination of Ts and ACH resulted in a decrease in NFPTR close to baseline, which was likely due to the fact that the resulting mucus was too liquid for maximal mucociliary efficiency. The index of mucus flux (mg/min) was very much elevated with ACH compared with control; this was not the case with Ts. This indicates that tasuldine, despite improving the rheological properties of the mucus, did not stimulate hypersecretion, as was the case for acetylcholine. The changes in mucus rheology with infused tasuldine can be considered beneficial with respect to their effects on predicted mucociliary and cough clearability, supporting the clinical effectiveness of this type of mucolytic therapy in airway diseases such as chronic bronchitis. The study also illustrates the potential danger of overliquification of mucus.

The surface and transport properties of meconium and reconstituted meconium solutions.

Passage of meconium in utero and subsequent pulmonary aspiration of meconium admixed with amniotic fluid is a major cause of neonatal respiratory distress. Airway clearance is the first defense of the lung, and clearance is dependent on the bulk physical (rheologic) as well as the surface properties of airway material. We therefore evaluated the surface adhesive properties and the transport properties of freshly passed meconium and of two dilutions of reconstituted, blended, meconium as used to mimic the effect of meconium passage into the amniotic fluid in animal models of meconium aspiration syndrome. Reconstituted and fresh meconium had similar physical and transport properties, including an extremely high interfacial (adhesion) tension and very poor transportability by either airflow or cilia. The similarities between the freshly passed and reconstituted meconium suggest that the latter is an adequate substitute for use in animal models of meconium aspiration syndrome. The high adhesiveness of meconium suggests a potential role for surfactant administration as an adhesive to improve airway clearance after meconium aspiration.

Ring distraction technique for measuring surface tension of sputum: relationship to sputum clearability.

Poor sputum clearance has been related to sputum adhesion tension. In this study, we describe a modified du Noüy ring method for measuring the surface tension (gamma) of small samples of sputum and for comparinge the calculated work of adhesion (Wad) for sputum specimens with the measured mucociliary transportability (MCTR) and cough transportability (CTR). The gamma, as measured by this method, correlates with gamma measured by sputum contact angle on a low-surface-energy solid (R2 = 0.368, P = 0.03). There is a small but significant difference in measurements made by these two methods (P = 0.03). Wad calculated from the surface tension ring method is inversely correlated with CTR (R2 = 0.181, P = 0.004) but has no correlation with MCTR in this study. The miniaturized ring method gives accurate and reproducible measurements of the surface tension of small amounts of respiratory secretions. Because sputum behaves enough like a liquid that the assumptions made in using the Young equation to calculate Wad appear valid, we also showed that the Neumann equation can be used to determine the surface tension of sputum by its contact angle on tetrafluoroethylene (Teflon).

A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine: airway epithelial function and mucus changes in dog.

A newly synthesized mucolytic agent, N-acetylcysteine L-lysinate (Nacystelyn) was studied. Tracheal mucus velocity (TMV), transepithelial potential difference (PD), rheological properties, and ion content of collected airway secretions were evaluated in six healthy mongrel dogs after placebo, Nacystelyn (NAL) and acetylcysteine (NAC) metered dose inhaler (MDI) aerosols. Although TMV was increased and viscoelasticity decreased after both treatments, the treatment effect with NAL was significantly greater. Furthermore, NAL increased the negative PD and CI- content of secretions in the trachea, an effect not observed after NAC. Both compounds increased ciliary beat frequency (CBF) on the frog palate at a concentration range similar to that approximated in dog airways. The increased mucociliary clearance could be partially explained by favourable rheological changes combined with stimulation of CBF. Since both compounds break disulfide bonds in mucus polymers, the greater change in mucus rheology and clearance rate after NAL, without change in water content, could be explained by the increase in CI- content. Nacystelyn appears to combine different modes of action which synergistically cause an increase in the clearance rate of airway secretions.

Effects of combined treatment with rhDNase and airflow oscillations on spinnability of cystic fibrosis sputum in vitro.

Treatment with either rhDNase or high-frequency oscillation has been shown to be effective in improving the physical and transport properties of airway secretions in cystic fibrosis (CF). The objects of this in vitro study was to examine whether combined treatment with oscillation and rhDNase results in greater change of CF sputum spinnability than either treatment by itself. Aliquots of sputum (0.4 g) from eight CF patients were subjected to the following protocols for 15 minutes and then followed for a total of 30 minutes: 1) incubation with 0.04 ml DNase 50 micrograms rhDNase/normal saline (10% dilution) at 37 degrees C to achieve 5 micrograms DNase/g of sputum final concentration; 2) airflow oscillation at 27 Hz similar to the airflow magnitude produced by a commercial high-frequency chest compression (HFCC) device; 3) negative control with no treatment; 4) positive (dilution) control, incubating with 10% saline by volume; 5) combination of DNase and oscillation, and 6) combination of saline and oscillation. For each protocol, sputum spinnability (in mm, mean +/- SD) was measured by means of a filancemeter at baseline, 15, and 30 minutes. Treatment with DNase decreased spinnability significantly more than either saline or oscillation at 15 and 30 minutes (P < 0.02 and P < 0.04, respectively). Incubation with saline or oscillation of CF sputum for 15 and 30 minutes decreased spinnability significantly compared with control. The combination of DNase and oscillation decreased spinnability significantly more than treatment with DNase alone (3.74 +/- 0.45 vs. 6.54 +/- 0.73 at 15 minutes, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Species differences in the physical and transport properties of airway secretions.

The clearance of airway secretions is vital in protecting the mammalian lung from pollution and infection. Diverse animal models have been used to study lung diseases associated with impaired secretion clearance. The extrapolation of data from animal models to humans is based on the assumption that there are structural and functional similarities in the airway epithelium and secretions. This manuscript reviews regulation of mucus secretion as well as the physical and transport properties of respiratory mucus. As tracheal size increases, the rigidity of airway secretions decreases, and rigidity is inversely correlated with mucociliary transportability. These differences are placed in the context of previously reported species and regional differences in transepithelial potential difference and the tracheobronchial epithelial cell population. Tracheal mucus transport velocity varies with the species studied and has been shown to positively correlate with tracheal surface area. A progressive increase in the rate of mucus transport from the small to the large airways has also been reported. The reduction in mucus rigidity from small to large airways could be one of the mechanisms responsible for velocity gradients, which facilitate mucociliary transport. Because airway dimensions, rather than anatomic level of the airway, may better predict epithelial secretory response, studies to assess the physiologic responses in human airways require the use of an animal model with a similar-sized airway.

Airway mucus and epithelial function in a canine model of single lung autotransplantation.

Impaired mucociliary function following lung transplantation has been reported in several human and animal studies. This could be a result of altered ciliary function or mucus properties or both. We assessed airway epithelial function by means of transepithelial potential difference (PD) measurements and physical analysis of mucus. Six mongrel dogs underwent single lung autologous transplantation. Measurements were performed preoperatively and 1, 2, 4, and 10 months postoperatively. At 1 and 2 months postoperatively, there was a significant fall in PD for the transplanted, left mainstem bronchus only (-13.5 +/- 1.7 mV at 1 month and -14.6 +/- 1.7 mV at 2 months postoperatively vs -18.6 +/- 2.3 mV preoperatively, baseline; p < 0.001 for both). The PD values in the small airways, right mainstem bronchus, and the trachea remained unchanged. At 2 months postoperation, the mucus collection rate on the left side was increased (p = 0.03), while the mucus viscoelasticity was decreased (p = 0.04). By 4 months postoperation, all epithelial parameters had returned to baseline, and there was no difference in radioaerosol clearance between the two lungs. The PD decrease and alterations in secretion rate and viscoelasticity reflect disturbed epithelial functional integrity at the site of anastomosis still present at 2 months postoperation. Recovery of bronchial epithelial function and clearance in canine studies of lung autotransplantation after healing of the anastomosis area suggest that persistent impairment of lung clearance observed in some long-term human lung transplantation survivors may be due to other mechanisms such as impaired healing or epithelial function or both, during immunosuppressive therapy. Mucociliary function in the anastomosis area is compromised until complete healing ensues; we speculate that chest physiotherapy may aid in overcoming this obstacle.

Effects of oscillating air flow on the rheological properties and clearability of mucous gel simulants.

This in vitro study addressed the question of clearance-related changes in the physical properties of mucous gel simulants (MGS) subjected to oscillating air flow. Delineating some of the possible mechanisms of action for the reported beneficial effects of high-frequency chest compression (HFCC) therapy constituted the rationale. The rheological variables measured were spinnability by filancemeter and viscoelasticity (mechanical impedance, G*, and loss tangent, tan delta) by magnetic microrheometry. Two derivative parameters, mucociliary clearability index (MCI) and cough clearability index (CCI), were computed from the rheological variables, based on relationships established from model studies of clearance. Two ranges of air flow oscillation frequencies used previously in animal and clinical studies, i.e., 12-13 Hz or 22-23 Hz, were applied. The measurements were made after application of oscillating air flow for 15, 30 and 60 minutes, and compared with those at baseline and negative control. A significant decrease in log G* with administration of oscillations was observed (p = 0.06 at 30 minutes, p < 0.01 at 60 minutes, for G* measured at 1 rad/s). Spinnability also decreased by 19.3% and 30.7% after 15 minutes; 32.9% and 41.1% after 30 minutes; 36.4% and 50.5% after 60 minutes, for 12 Hz and 22 Hz, respectively (all significantly different from baseline). There was a positive correlation between viscoelasticity and spinnability, and a negative correlation between spinnability and CCI, but no correlation between spinnability and MCI. Oscillating air flow seemed to act as a physical "mucolytic" that affected mostly the cough clearability of the mucus simulant.

Exogenous surfactant enhances mucociliary clearance in the anaesthetized dog.

Therapy with exogenous surfactants is currently used for the treatment of respiratory distress syndrome of the newborn (RDS) and is under investigation for treatments related to adult RDS. However, the possible use of exogenous surfactant as a means of enhancing mucus clearance in other respiratory diseases has not been addressed. We therefore studied the effects of an artificial surfactant (Curosurf) on in vivo tracheal mucus velocity in intubated pentobarbital-anaesthetized dogs. Five dogs were randomly administered, on separate occasions, either vehicle (saline) or 10 mg Curosurf by means of local instillation via a catheter into the right lung. Tracheal mucus was collected by inserting a soft-bristled cytology brush to the level of the carina, and analysed for viscoelasticity by microrheometry. Mucociliary clearability in vivo, tracheal mucus velocity (TMV) in mm.min-1, was determined by bronchoscopic observation of charcoal marker particle transit times. The initial placement of charcoal was at the same level of the lower trachea that mucus was collected from. The effect of ciliary beat frequency was assessed on the frog palate assay by a videoscopic technique. In the dog, TMV was significantly increased after administration of surfactant. The values of TMV in the vehicle- and surfactant-treated dogs were 6.3 +/- 4.0 vs 25.6 +/- 6.5 mm.min-1 (SD), respectively. There were no discernible differences between prevehicle and postvehicle TMV values, and no significant differences in any mucus viscoelastic parameter, as determined by magnetic rheometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucolytic treatment with N-acetylcysteine L-lysinate metered dose inhaler in dogs: airway epithelial function changes.

N-acetylcysteine L-lysinate Nacystelyn (L-NAC) is a newly synthesized mucolytic agent, of which the action in vivo has not been well defined. In six healthy mongrel dogs, the rheological properties of mucus, its mucociliary and cough clearability, and the transepithelial potential difference (PD) of the tracheobronchial epithelium were evaluated after placebo and L-NAC metered dose inhaler (MDI) aerosols. The principal index of mucus rigidity, log G*, decreased at all airway sites with L-NAC administration, i.e. the mucus became less rigid and more deformable (the overall change in G* was 0.29 log units, i.e. ca. twofold decrease). The viscoelasticity-derived mucus transportability parameters, mucociliary (MCI) and cough (CCI) clearability indices, increased with L-NAC MDI, particularly CCI, which predicts the effect of mucus rheology on cough clearability. PD increased significantly with L-NAC administration at all measurement sites, which appears to be a novel effect for a direct acting mucolytic agent. Tracheal mucus linear velocity (TMV) increased after L-NAC compared with placebo, as did the normalized frog palate transport rate (NFPTR). The increase in NFPTR was greater than that predicted from the mucus rheological properties alone, suggesting that L-NAC still resident in the collected mucus stimulated the frog palate cilia. The index of mucus flux, the collection rate in mg.min-1, was higher with L-NAC compared with placebo. From our results, we conclude that L-NAC shows potential benefit in terms of improving mucus rheological properties and clearability. It may act, in part, by stimulating the fresh secretion of mucus of lower viscoelasticity. The stimulation of mucociliary clearance could be related to ion flux changes, as indicated by the increase in PD.

Amiloride inhalation therapy in cystic fibrosis. Influence on ion content, hydration, and rheology of sputum.

Amiloride inhalation as treatment for cystic fibrosis (CF) lung disease has been shown in independent studies to increase mucus clearance by ciliary and/or cough action and to retard the decline in lung function. It is hypothesized that amiloride therapy decreases the excess sodium and water absorption that is a characteristic of CF airway epithelium and that it leads to an improvement in the rheologic properties of mucus favoring airway mucus clearance. The aim of this study was to investigate whether amiloride treatment (5 x 10(-3) M amiloride in one-third normal saline four times a day) would change sputum electrolyte composition in patients with CF after 25 wk of therapy as compared with placebo (one-third normal saline), and whether appropriate changes in sputum water content and rheologic properties would accompany any changes in electrolyte composition. Sputum samples were obtained from six patients with CF undergoing amiloride therapy, using the dental cotton protection technique to avoid salivary contamination. The samples were stored at -80 degrees C until analyzed. For electrolyte analyses an aliquot of the sputum (minimum, 30 mg) was analyzed with ion-selective electrodes for sodium and potassium, and a chloride meter was used to measure chloride content. Chronic (25-wk) amiloride therapy increased significantly the sputum sodium (94.8 +/- 16.4 to 121.4 +/- 15.4 mmol/L, p = 0.001) and chloride (64.4 +/- 11.8 to 77.2 +/- 8.0 mmol/L, p = 0.10) content when compared with 25 wk of saline treatment.(ABSTRACT TRUNCATED AT 250 WORDS)