A Pilot Study to Examine the Effect of Chronic Treatment with Immunosuppressive Drugs on Mucociliary Clearance in a Vagotomized Murine Model

Background

Previously, we have demonstrated that mucociliary clearance (MCC) is diminished within the first months after surgery in lung transplant patients and the explanation for the reduction in MCC is unknown. We hypothesized that chronic treatment with a commonly prescribed regimen of immunosuppressive drugs significantly impairs MCC. We tested this hypothesis in a murine model of lung transplantation.

Methods

Fifteen C57BL/6 mice underwent vagotomy on the right side to simulate denervation associated with lung transplantation in humans. For 6 days, seven mice (controls) were intraperitoneally injected with three 100 µL doses of phosphate buffered saline and eight mice (immunosuppressed) were injected with three 100 µL injections of tacrolimus (1 mg/kg), mycophenolate mofetil (30 mg/kg), and prednisone (2 mg/kg) once daily. Then, mice inhaled the radioisotope ^99mtechnetium and underwent gamma camera imaging of their lungs for 6.5 hrs. Counts in the right lung at 1–1.5 hrs and at 6–6.5 hrs were first background-corrected and then decay-corrected to time 0 counts. Decay-corrected counts were then divided by time 0 counts. Retention at each time point was subtracted from 1.00 and multiplied by 100% to obtain percent removed by mucociliary clearance.

Results

Although there was a slowing of MCC at 1–1.5 hrs for the immunosuppressed mice, there was no statistical difference in MCC measured at 1–1.5 hrs for the two groups of mice. At 6–6.5 hrs, MCC was significantly slower in the immunosuppressed mice, compared to controls, with 7.78±5.9% cleared versus 23.01±11.7% cleared, respectively (p = 0.006).

Conclusions

These preliminary results suggest that chronic treatment with immunosuppressive medications significantly slows MCC in vagotomized C57BL/6 mice. These findings could shed light on why MCC is reduced in lung transplant patients whose lungs are denervated during surgery and who are chronically treated with immunosuppressive drugs post surgery.     

Glucose effects on lung surfactant kinetics in conscious pigs

The primary goal of this study was to investigate the effects of glucose infusion on surfactant phosphatidylcholine (PC) metabolic kinetics in the lungs. A new stable isotope tracer model was used in which [1,2-(13)C(2)]acetate and uniformly labeled [U-(13)C(16)]palmitate were infused in 12 normal overnight-fasted pigs to quantify lung surfactant kinetics with or without glucose infusion (24 mg. kg(-1). min(-1)). With glucose infusion, the rate of surfactant PC incorporation from de novo synthesized palmitate increased from the control value of 2.1 +/- 0.2 to 15.5 +/- 1.9 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05), whereas the incorporation rate from plasma preformed palmitate decreased from the control value of 20.9 +/- 1.9 to 11.6 +/- 1.1 nmol palmitate. h(-1). g wet lung(-1) (P < 0.05). The palmitate composition in lamellar body surfactant PC increased from the control value of 61.7 +/- 2.1% to 75.9 +/- 0.6% (P < 0.05). The surfactant PC secretion rate decreased from the control value of 239.0 +/- 26.1 to 81.9 +/- 5.3 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05). We conclude that, whereas surfactant secretion was inhibited by glucose infusion, neither total surfactant PC synthesis nor the surfactant PC pool size was significantly affected due to an increased reliance on de novo synthesized fatty acids.     

Surfactant replacement attenuates the increase in alveolar permeability in hyperoxia

Rabbits exposed to hyperoxia develop surfactant deficiency, abnormal lung mechanics, and increased permeability to solute. We investigated whether replenishment of depleted alveolar surfactant by the intratracheal instillation of calf lung surfactant extract (CLSE) would mitigate the increase in alveolar permeability to solute. Twenty-eight rabbits were exposed to 100% O2 for 72 h and received intratracheal instillations of 125 mg CLSE (approximately 170 mumol dipalmitoyl phosphatidylcholine) at 24 and 48 h. The interlobar and intralobar distribution of CLSE was quantified by adding [14C]dipalmitoyl phosphatidylcholine liposes into the instillate and measuring the levels of activity in lung tissue. CLSE was nonuniformly distributed in the different lung lobes, the right lower lobe receiving more CLSE than the rest. Alveolar epithelial permeability to solute was assessed by instilling 10 ml isotonic saline, which contained a trace amount of [57Co]cyanocobalamin, in the right lower lobe and measuring the disappearance of the tracer from the alveolar saline and its appearance in the arterial blood during a 1-h period. CLSE treatment was associated with significantly increased 72-h survival in hyperoxia compared with saline-treated controls (number of survivors: 16/17 vs. 5/11, P less than 0.01). CLSE treatment significantly reduced the rate constant for the movement of cyanocobalamin out of the alveolar space (24 +/- 5 vs. 42 +/- 6 min-1 x 10(-3), P less than 0.01) and tracer appearance in the blood at the end of the study (7 +/- 1 vs. 34 +/- 13%, P less than 0.01) when compared with values in saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Physiologically-Motivated Compartment-Based Model of the Effect of Inhaled Hypertonic Saline on Mucociliary Clearance and Liquid Transport in Cystic Fibrosis

Background

Cystic Fibrosis (CF) lung disease is characterized by liquid hyperabsorption, airway surface dehydration, and impaired mucociliary clearance (MCC). Herein, we present a compartment-based mathematical model of the airway that extends the resolution of functional imaging data.

Methods

Using functional imaging data to inform our model, we developed a system of mechanism-motivated ordinary differential equations to describe the mucociliary clearance and absorption of aerosolized radiolabeled particle and small molecules probes from human subjects with and without CF. We also utilized a novel imaging metric in vitro to gauge the fraction of airway epithelial cells that have functional ciliary activity.

Results

This model, and its incorporated kinetic rate parameters, captures the MCC and liquid dynamics of the hyperabsorptive state in CF airways and the mitigation of that state by hypertonic saline treatment.

Conclusions

We postulate, based on the model structure and its ability to capture clinical patient data, that patients with CF have regions of airway with diminished MCC function that can be recruited with hypertonic saline treatment. In so doing, this model structure not only makes a case for durable osmotic agents used in lung-region specific treatments, but also may provide a possible clinical endpoint, the fraction of functional ciliated airway.     

Aerosolization of P2Y(2)-receptor agonists enhances mucociliary clearance in sheep.

The purpose of this study was to determine whether aerosolized INS316 (UTP) stimulates lung mucociliary clearance (MCC) in sheep and, if so, to compare its effects with INS365, a novel P2Y(2)-receptor agonist. In the first series of studies, we used a previously described roentgenographic technique to measure tracheal mucus velocity (TMV), an index of MCC, before and for 4 h after aerosolization of INS316 (10(-1) M and 10(-2) M) and INS365 (10(-1) M and 10(-2) M), or normal saline in a randomized crossover fashion (n = 6). In a second series of studies, we compared the ability of these agents to enhance total lung clearance. For these tests, the clearance of inhaled technetium-labeled human serum albumin was measured serially over a 2-h period after aerosolization of 10(-1) M concentration of each agent (n = 7). Aerosolization of both P2Y(2)-receptor agonists induced significant dose-related increases in TMV (P < 0.05) compared with saline. The greatest increase in TMV was observed between 15 and 30 min after drug treatment. The highest dose (10(-1) M) of INS316 produced a greater overall stimulation of TMV than did INS365 (10(-1) M). Both compounds, compared with saline, induced a significant increase in MCC (P < 0.05) within 20 min of treatment. This enhancement in MCC began to plateau at 60 min. Although the response to INS316 started earlier, there was no significant difference between the clearance curves for the two compounds. We conclude that inhaled P2Y(2)-receptor agonists can increase lung MCC in sheep and that for P2Y(2)-receptor stimulation TMV accurately reflects changes in whole lung MCC.     

Effect of surfactant concentration, compression ratio and compression rate on the surface activity and dynamic properties of a lung surfactant

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis-Constrained Sessile Drop (ADSA-CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression-Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM.     

Simulation of regional lung emptying during slow and forced expirations.

Regional lung emptying was simulated by means of a bialveolar lung model. The influence of bronchial asymmetry and the vertical pleural pressure gradient was evaluated. The model suggests that 1) in vivo the influence of the pleural pressure gradient prevails over that of the bronchial asymmetry; 2) in the presence of this gradient, the shape of phases III and IV of the single-breath washout curves obtained following inspiration of a tracer gas bolus at residual volume is determined by the recoil pressure-volume curve of the lung, by the vertical displacements of the alveoli, and,, at higher flow rates, by the elastic characteristics of the airways; 3) if the pleural pressure gradient is independent of lung volume and of flow rate, the factors mentioned in 2 suffice to produce single-breath washout curves (phases III and IV) and regional vs. overall lung volume relationships corresponding to those observed in vivo; 4) the configuration of the maximal expiratory flow-volume curve is relatively insensitive to pulmonary and bronchial asymmetry, at least in healthy individuals.     

Induction of ciliary orientation by matrix patterning and characterization of mucociliary transport

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ～75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.     

Effect of ventilation rate on instilled surfactant distribution in the pulmonary airways of rats.

Liquid can be instilled into the pulmonary airways during medical procedures such as surfactant replacement therapy, partial liquid ventilation, and pulmonary drug delivery. For all cases, understanding the dynamics of liquid distribution in the lung will increase the efficacy of treatment. A recently developed imaging technique for the study of real-time liquid transport dynamics in the pulmonary airways was used to investigate the effect of respiratory rate on the distribution of an instilled liquid, surfactant, in a rat lung. Twelve excised rat lungs were suspended vertically, and a single bolus (0.05 ml) of exogenous surfactant (Survanta, Ross Laboratories, Columbus, OH) mixed with radiopaque tracer was instilled as a plug into the trachea. The lungs were ventilated with a 4-ml tidal volume for 20 breaths at one of two respiratory rates: 20 or 60 breaths/min. The motion of radiodense surfactant was imaged at 30 frames/s with a microfocal X-ray source and an image intensifier. Dynamics of surfactant distribution were quantified for each image by use of distribution statistics and a homogeneity index. We found that the liquid distribution depended on the time to liquid plug rupture, which depends on ventilation rate. At 20 breaths/min, liquid was localized in the gravity-dependent region of the lung. At 60 breaths/min, the liquid coated the airways, providing a more vertically uniform liquid distribution.     

Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

Effects of ventilation on the surfactant system in sepsis-induced lung injury.

The present study examined the effects of mechanical ventilation, with or without positive end-expiratory pressure (PEEP), on the alveolar surfactant system in an animal model of sepsis-induced lung injury. Septic animals ventilated without PEEP had a significant deterioration in oxygenation compared with preventilated values (arterial PO(2)/inspired O(2) fraction 316 +/- 16 vs. 151 +/- 14 Torr; P < 0.05). This was associated with a significantly lower percentage of the functional large aggregates (59 +/- 3 vs. 72 +/- 4%) along with a significantly reduced function (minimum surface tension 17.7 +/- 1.8 vs. 11.8 +/- 3.8 mN/m) compared with nonventilated septic animals (P < 0.05). Sham animals similarly ventilated without PEEP maintained oxygenation, percent large aggregates and surfactant function. With the addition of PEEP, the deterioration in oxygenation was not observed in the septic animals and was associated with no alterations in the surfactant system. We conclude that animals with sepsis-induced lung injury are more susceptible to the harmful effects of mechanical ventilation, specifically lung collapse and reopening, and that alterations in alveolar surfactant may contribute to the development of lung dysfunction.     

Antimicrobial properties of fibrous cellulose and other polymeric materials modified with chlorhexidine]

Since fibrous cellulose and other polymeric materials are widely used as dressings, it was of interest to study the mechanism of the antimicrobial action of the products from such materials physically and chemically modified by chlorhexidine, a broad spectrum antiseptic. As vehicles and prolongation agents the following products were used: dressing gauze and cation exchange derivatives of cellulose and starch i.e. monocarboxycellulose (MCC), phosphate cellulose (PC), carboxymethyl cellulose (CMC), monocarboxyl starch (MCS) and alginic acid (AA), a natural compound. It was shown that chemical attachment of chlorhexidine provided a 2-4-fold increase in the antimicrobial effect of the preparations as compared to the use of physical sorption. The antimicrobial effect of the polymeric form of chlorhexidine based on MCC and PC was much higher than that based on MCC, MCS and AA.     

Influence of dexamethasone on the lipid distribution of newly synthesized fatty acids in fetal rat lung

There is a developmental increase in fatty acid biosynthesis and surfactant production in late-gestation fetal lung and both are accelerated by glucocorticoids. We have examined the distribution of the newly synthesized fatty acids to determine whether they are preferentially incorporated into surfactant. Explants of 18 day fetal rat lung were cultured with and without dexamethasone for 48 h and then with [3H]acetate for 4 h after which labeled fatty acids were measured. Incorporation of radioactivity from acetate was considered a measure of newly synthesized fatty acids. Phospholipids contained 86% of the newly synthesized fatty acids of which approx. 80% were in phosphatidylcholine. Phosphatidylcholine and disaturated phosphatidylcholine contained a much greater percentage of the labeled fatty acids than of the phospholipid mass determined by phosphorus assay while phosphatidylethanolamine, phosphatidylserine and sphingomyelin contained less. Dexamethasone increased the rate of acetate incorporation into total lipid fatty acids but it had little effect on fatty acid distribution, except that it increased the percentages in phosphatidylglycerol and disaturated phosphatidylcholine. The hormone also increased the mass of these two phospholipids to a greater extent than that of the total. These data suggested that the newly synthesized fatty acids are preferentially incorporated into surfactant phospholipids and that this process is accelerated by dexamethasone. However, since phosphatidylcholine and phosphatidylglycerol are not exclusive to surfactant, we compared isolated lamellar bodies with a residual fraction not enriched in surfactant. The rate of acetate incorporation into fatty acids in lamellar body phosphatidylcholine as well as its specific activity (radioactivity per unit phosphorus) were both increased by dexamethasone. Specific activity, however, was no greater in the lamellar bodies than in the residual fraction in both control and dexamethasone-treated cultures. Therefore, there is no preferential incorporation of newly synthesized fatty acids into phospholipids in surfactant as opposed to those in other components of the lung.     

Effects of high-frequency and conventional ventilation on the premature lamb lung

Twelve sets of twin lambs were delivered prematurely by cesarean section at 133-136 days gestational age and ventilated for 3 h with either high-frequency oscillation (HFO) or conventional mechanical ventilation (CMV). Blood gases and pH values were monitored at 30-min intervals, and ventilator settings were adjusted to maintain CO2 partial pressure (PCO2) values within the normal range. There were no differences in the sequential blood gas or pH values between the HFO or CMV lambs. Mean airway pressures (MAP) between 8.0 and 20.4 cmH2O were required, indicating lung disease of variable severity in the lambs. The bidirectional protein leak from the vascular space to the airways and alveoli and vice versa was measured with radiolabeled albumins given by intravascular injection and with fetal lung fluid at birth. The albumin leaks in both directions increased as MAP required to normalize PCO2 increased, but the degree of leak was independent of type of ventilation. Pathological findings of epithelial necrosis and hyaline membranes occurred to a similar extent in lung sections from both groups of lambs. In the HFO animals less phosphatidylcholine in the alveolar wash and more of a tracer dose of radiolabeled natural surfactant that had been given at birth became tissue associated. These results indicate a decrease in the initial secretion of surfactant and/or a stimulation of reuptake in the HFO animals. HFO did not protect the immature lung from the development of large protein leaks or the pathological changes of the respiratory distress syndrome.     

Application of ThinPrep Bronchial Brushing Cytology in the Early Diagnosis of Lung Cancer: A Retrospective Study

The majority of lung cancer patients are diagnosed at advanced stages of disease. This study evaluated the diagnostic value of ThinPrep (TP) bronchial brushing cytology in lung cancer. A total of 595 patients with suspicious lung cancer were enrolled in this study. The bronchial brushing samples were prepared by TP. The data were then compared to histology of lung tissue samples. Histologically, 479 of these 595 patients were diagnosed with lung cancer, including 223 cases of lung squamous cell carcinoma (SCC), 77 cases of lung adenocarcinoma (ADC), and 152 cases of small cell lung carcinoma (SCLC). The TP cytology revealed a total of 460 cases of lung cancer (including 232 SCCs, 91 ADCs, and 108 SCLCs). The TP cytological technique had 87.06% sensitivity and 62.93% specificity in the diagnosis of lung cancer. Specifically, TP cytology confirmed 195 of 223 SCCs, 47 of 77 ADCs, and 94 of 152 SCLCs. The TP cytology showed 87.44% sensitivity and 90.05% specificity for the diagnosis of SCC, with a Matthew''s correlation coefficient (MCC) of 0.820; while the sensitivity was reduced to 61.04% and the specificity was 90.93% for the diagnosis of ADC, with a MCC of 0.464. For the diagnosis of SCLC, the sensitivity was 61.84% and the specificity was 96.84%, with a MCC of 0.648. Thus, this study demonstrated the usefulness of TP bronchial brushing cytology in the early diagnosis of lung cancer, especially the early stage of lung SCC. A prospective clinical trial will verify these data before being translated into the clinic.     

“Ciliophagy”

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/?, map1lc3b^?/?, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/?, map1lc3b^?/?, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.     

Ventilation inhomogeneity during controlled ventilation. Which index should be used?

Six indexes for diagnosing uneven ventilation by tracer gas washout were studied. The indexes were lung clearance index, mixing ratio, Becklake index, multiple-breath alveolar mixing inefficiency, moment ratio, and pulmonary clearance delay, all of which increase with impaired pulmonary gas mixing. In model lung tests, indexes that compared the actual washout curve with a calculated ideal curve (mixing ratio, multiple-breath alveolar mixing inefficiency, and pulmonary clearance delay) were unaffected by changes in tidal volume and series dead space, whereas the others varied markedly. In both spontaneously breathing and mechanically ventilated patients all indexes showed a significant difference between smokers and nonsmokers (P less than 0.002), but the indexes were somewhat different in their assessment of different ventilatory patterns. However, the mean value for all indexes, with the exception of mixing ratio, was smallest with a fast insufflation followed by an end-inspiratory pause. Any of the indexes may be useful if its limitations are recognized, but mixing ratio, multiple-breath alveolar mixing inefficiency, and pulmonary clearance delay seem preferable, because they are not affected by changes in tidal volume and dead space fraction.     

“Ciliophagy”: The consumption of cilia components by autophagy

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/−, map1lc3b^−/−, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/−, map1lc3b^−/−, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.     

Metabolism of P2 receptor agonists in human airways: implications for mucociliary clearance and cystic fibrosis

Extracellular nucleotides are among the most potent mediators of mucociliary clearance (MCC) in human lungs. However, clinical trials revealed that aerosolized nucleotides provide only a transient improvement of MCC to patients diagnosed with cystic fibrosis (CF). In this study, we identified the mechanism that eliminates extracellular nucleotides from human airways. Polarized primary cultures of human bronchial epithelial cells were impermeable to extracellular nucleotides but rapidly dephosphorylated ATP into ADP, AMP, and adenosine. The half-life of a therapeutic ATP concentration (0.1 mm) was approximately 20 s within the periciliary liquid layer. The mucosal epithelial surface eliminated P2 receptor agonists (ATP = UTP > ADP > UDP) at 3-fold higher rates than the serosal surface. We also showed that mucosal (not serosal) ectoATPase activity increases toward areas most susceptible to airway obstruction (nose < bronchi < bronchioles). Bronchial cultures from patients with CF, primary ciliary dyskinesia, or alpha1-antitrypsin deficiency exhibited 3-fold higher mucosal (not serosal) ectoATPase activity than normal cultures. Time course experiments indicated that CF enhances ATP elimination and adenosine accumulation on the mucosal surface. Furthermore, nonspecific alkaline phosphatase was identified as the major regulator of airway nucleotide concentrations in CF, primary ciliary dyskinesia, and alpha1-antitrypsin deficiency. The ectoAT-Pase activity and mRNA expression of mucosally restricted nonspecific alkaline phosphatase were 3-fold higher on bronchial cultures from these patients than from healthy subjects. This study demonstrates that the duration of nucleotide-mediated MCC is limited by epithelial ectonucleotidases throughout human airways, with the efficiency of this mechanism enhanced in chronic inflammatory lung diseases, including CF.     

Effect of exogenous surfactant instillation on experimental acute lung injury

Pulmonary surfactant replacement has previously been shown to be effective in the human neonatal respiratory distress syndrome. The value of surfactant replacement in models of acute lung injury other than quantitative surfactant deficiency states is, however, uncertain. In this study an acute lung injury model using rats with chronic indwelling arterial catheters, injured with N-nitroso-N-methylurethane (NNNMU), has been developed. The NNNMU injury was found to produce hypoxia, increased mortality, an alveolitis, and alterations in the pulmonary surfactant system. Alterations of surfactant obtained by bronchoalveolar lavage included a reduction in the phospholipid-to-protein ratio, reduced surface activity, and alterations in the relative percentages of the individual phospholipids compared with controls. Treatment of the NNNMU-injured rats with instilled exogenous surfactant (Survanta) improved oxygenation; reduced mortality to control values; and returned the surfactant phospholipid-to-protein ratio, surface activity, and, with the exception of phosphatidylglycerol, the relative percentages of individual surfactant phospholipids to control values.