Long-term clearance from small airways in patients with cystic fibrosis.

Impaired mucociliary clearance is a hallmark of cystic fibrosis (CF). Early morphological changes first appear in the small airways. Lung clearance was investigated in 11 young CF adults with mild-to-moderate lung disease using a method depositing particles mainly in the small airways. Radiolabelled Teflon particles (6 microm) were inhaled with an extremely slow inhalation flow, 0.05 L x s(-1). Lung retention was measured immediately following inhalations and, on four occasions up to 21 days. The results were compared with data from healthy subjects. The lung retention at 24 h in % of deposition was 67% (95% confidence interval 58-76) in the CF patients, compared to 48% (42-53) in the healthy subjects. Clearance on days 1-7 was larger in the CF patients, 22% (15-29) compared to the healthy subjects, 14% (12-16). No difference was observed between the CF patients and the healthy subjects in the slow clearance phase at day 7 to day 21, representing small airway clearance. Impaired mucociliary clearance in CF patients results in increased 24-h retention and a prolonged rapid clearance phase. The results of the study do not support the current authors' hypothesis that clearance from small airways is slower in cystic fibrosis patients compared to healthy subjects. Furthermore, the data suggest that mucociliary transport is not the dominant clearance mechanism in small airways.

RBDATA-EULEP: providing information to improve internal dosimetry.

The overall aim of the concerted action RBDATA-EULEP is to provide information to improve the assessments of intakes of radionuclides and of the resulting doses. This involves a review of the behaviour of radionuclides following intake, and the transfer of expertise on methodology by organising small training workshops. The main activity is the development of an electronic database, effectively an annotated bibliography, but the electronic format used facilitates extension, updating and information retrieval. It consists of linked tables of references and experiments, with details and comments on the materials, procedures and results. By June 2002 it contained information on 524 inhalation, 282 ingestion and 164 injection experiments from 391 references. It will be extended, and Internet access provided. Prospective users include groups developing standards for internal dosimetry, scientists conducting research on radionuclide biokinetics and health physicists assessing the consequences of accidental intakes.

Increased airway responsiveness of a common fragrance component, 3-carene, after skin sensitisation--a study in isolated guinea pig lungs.

Lungs from skin-sensitised and non-sensitised guinea pigs were exposed via the airways to 3-carene (1900 mg/m3) and perfused with buffer containing either autologous plasma or lymphocytes. The experiments were performed in order to investigate the importance of blood components for the increased lung responsiveness seen in skin-sensitised animals. A reduction in lung function was noted in all lungs during 3-carene exposure. There was no difference in the 3-carene response between lungs from skin-sensitised animals versus lungs from non-sensitised animals when the perfusion buffer contained lymphocytes. However, when plasma diluted with buffer was used as perfusion medium, there was a significant enhancement in the response in lungs from sensitised versus lungs from non-sensitised animals. This implies that skin sensitisation increases lung responses to inhaled 3-carene and those components in plasma, and not the lymphocyte fraction, contributes to the observed increased lung responsiveness.

Lung clearance in children with Duchenne muscular dystrophy or spinal muscular atrophy with and without CPAP (continuous positive airway pressure).

Bronchiolar clearance was studied in 7 boys in the age range of 8 to 17 years, 6 with Duchenne muscular dystrophy (DMD) and 1 with spinal muscular atrophy type II (SMA-II). These boys had healthy lungs but a severely reduced muscular strength (wheelchair dependent). In 6 of the boys, clearance was studied twice, at one occasion as a control and at the other occasion following treatment with continuous positive airway pressure (CPAP). A control group of healthy adults was used. In the clearance examinations, 6-microm Teflon particles, labeled with III In was inhaled extremely slowly, 0.05 L/s. This gives a deposition mainly in the bronchioles. Lung retention was measured after 0,24,48, and 72 hours. A model for deposition of particles in the adult lung was scaled down to represent the children in this study. Deposition in various airway generations was calculated to be similar in children and adults. Also the measured retentions were similar in the boys and the adults. In the clearance experiments during CPAP treatment, there was a significantly lower retention after 72 hours (but not after 24 and 48 hours) than in the control experiments. Theresults indicate that a severe reduction of muscular strength, and thereby a reduction of mechanical movement of the lung, does not affect clearance from large and small airways. However, some effect of clearance from small airways cannot be excluded due to the short measuring period. The small but significant effect of the CPAP treatment might have potential clinical importance and suggest that bronchiolar clearance can be affected by some form of mechanical force.

Human alveolar macrophage phagocytic function is impaired by aggregates of ultrafine carbon particles.

Alveolar macrophages (AM) were collected by bronchoalveolar lavage from healthy volunteers. The AM were loaded with small masses (0.03-3 microg/10(6) AM) of ultrafine carbon particle aggregates. The phagocytic activity of the cells was studied 20 h after the loading. Fluorescein-labeled silica particles (3 microm) were used as test particles and the attachment and ingestion processes were studied separately. In some experiments, AM were incubated with interferon-gamma (IFN-gamma) for 20 h before and during the test of phagocytic activity and during measurement of oxidative metabolism. The ingested carbon particles induced a dose-related impairment of both the attachment and the ingestion processes with a marked impairment down to a carbon particle dose around 0.2 microg/10(6) AM. Such levels should reasonably occur after inhalation of existing concentrations of urban air particles, which to a considerable extent consist of aggregates of ultrafine particles with a carbon skeleton. Incubation with IFN-gamma (12.5 U/ml) also induced significant impairments in both the attachment and the ingestion processes. Loading with carbon further aggravated the effect of IFN-gamma. In contrast to earlier studies in rat AM, IFN-gamma did not impair the oxidative metabolism at rest in these human AM; instead the oxidative metabolism was increased. This difference was due to a difference between rat and human AM and not between rat and human IFN-gamma. Our results suggest that ingested environmental particles in AM, e.g., after an episode of high particle concentration, may impair phagocytic capacity of the cells, especially after infections that induce an increased production of IFN-gamma. Consequently, there might be a risk for additional infections. Moreover, inhaled particles not phagocytized by AM might damage the lung tissue.

Comparison of clearance of particles inhaled with bolus and extremely slow inhalation techniques.

Ten healthy nonsmokers inhaled 6-microm (aerodynamic diameter) Teflon particles labelled with 111In twice, once with the shallow bolus technique (volumetic lung depth 76+/-20 mL ([+/- SD]) and once with the extremely slow inhalation technique (0.05 L/s). The radioactivity in the lungs was measured at 1 and 24 hours as well as at 1, 2, and 3 weeks after both inhalations. The 24-hour lung retention a percentage of lung deposition was significantly lower for the bolus inhalation, 46%+/-9% (+/- SD) than for the extremely slow inhalation, 56%+/-11%. The retention after 21 days as a percentage of the 24-hour retention was 55%+/-9% for the shallow bolus inhalation and 56%+/-10% for the extremely slow inhalation. Also within the subjects, clearance was similar for the 2 modes of inhalation. Deposition of particles inhaled with the 2 modes of inhalation was calculated with 2 model, one being based on Monte (Carlo particle transport together with an asymmetric lung model. Deposition predicted with this model agreed well with the experimental data under the assumption that there are large retained fractions only in small ciliated airways (bronchioli) and not in large ones. For the bolus inhalation, the model predicted 43% to 50% deposition in the bronchial (BB) region of initial lung deposition, 33% to 38% in the bronchiolar (bb) region, and 16% to 22% in the alveolar region. For the extremely slow inhalation, the model predicted 31% to 34% deposition in the BB region, 45% to 47% in the bb region, and 21% to 22% in the alveolar region. In addition, it predicted about the same ratio between bb and alveolar depositions for the 2 modes of inhalation. Thus, both the experimental and theoretical data indicate that the shallow bolus particles to a considerable extent reach both the bb and the alveolar regions and that they do that at about the same extent as the particles inhaled extremely slow. This conclusion is concerning the experimental data based on the assumption that there are no large retained fractions in the BB region. Another interpretation of the similar clearance for the two modes of inhalation is that there are large retained fractions in both the BB and the bb regions and that individual charactristics of clearance of these fractions are of importance rather than the site of deposition.

Functional and morphological differences between human alveolar and interstitial macrophages.

Macrophages play an essential role in pulmonary host defense. They are, however, a heterogeneous cell population located in different lung compartments. This study was designed to elucidate differences between two macrophage populations obtained from the human lung, i.e., alveolar macrophages (AM) and interstitial macrophages (IM). Macroscopically tumor-free lung segments from nine patients undergoing lobectomy or pulmectomy were studied. All patients had a diagnosis of primary lung cancer. AM were recovered by bronchoalveolar lavage and IM were isolated by mechanical fragmentation of the lavaged lung segments followed by enzymatic treatment. The cell fractions were analyzed with respect to morphology (transmission electron microscopy) and function (phagocytosis). The cells in the IM fraction were smaller (7.6 +/- 1.8 microm (mean +/- SD) compared with 16.0 +/- 4.1 microm) and morphologically more heterogeneous than those in the AM fraction. Interestingly, a considerable portion of the cells in the IM fraction had a typical AM-like appearance. Despite this, the AM fraction had a higher phagocytic activity compared to IM, with faster attachment and ingestion processes (P <0.001 for both). We conclude that the heterogeneity of human lung macrophages must be taken into consideration when their role in the inflammatory response is studied.

Does lung retention of inhaled particles depend on their geometric diameter?

Experiments with a bolus technique suggest that retained fractions in the airways are dependent on the geometric diameter of the particles. This view has been adopted by the International Commission on Radiological Protection (ICRP) in its new human respiratory tract model (HRTM). The aim of the present study was to test this view by the use of an inhalation technique, in which particles with an aerodynamic diameter of about 6 microns are inhaled extremely (0.05 l/s) and as a result, the particles are deposited mainly in small ciliated airways. Nine healthy subjects inhaled on one occasion monodisperse 111In-labelled polystyrene particles (geometric diameter 6.05 microns, aerodynamic diameter 6.2 microns) and on another occasion monodisperse 111In-labelled Teflon particles (geometric diameter 4.47 microns, aerodynamic diameter 6.5 microns). Both particles were inhaled at 0.045 L/s and radioactivity in the body was measured after 0, 24, 48, and 72 hours as well as after 1, 2, and, for some subjects, also 3 weeks. The retention in the lungs at 24 hours was slightly lower for the Teflon particles (47%) than for the polystyrene particles (51%). From earlier experimental data with different particle sizes as well as from predictions with theoretical lung models, this difference is reasonably explained by the somewhat larger aerodynamic diameter of the Teflon particles. Clearance of the 2 particle types between 1 day and 2 weeks was similar within each individual as well as in the whole group. The differences between the clearance of 4.5 microns and 6 microns geometric diameter particles observed in the present experiment are significantly different (P < .01) from the differences seen in earlier shallow bolus experiments as well as from the differences for such particles calculated with the HRTM, i.e., our experiment does not support the hypothesis that the fraction retained after 1 day is dependent on the geometric diameter in the size range studied.

Rat alveolar and interstitial macrophages in the fibrosing stage following quartz exposure.

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.

Increased airway responsiveness after skin sensitisation to 3-carene, studied in isolated guinea pig lungs.

Inhalation of 3-carene has been shown to induce bronchoconstriction in concentrations not far from the threshold limit value. In this study, one group of guinea-pigs were sensitised by dermal exposure to 3-carene according to the modified Cumulative Contact Enhancement Test protocol and another group of animals was used as controls. Lungs from the skin-sensitised and control guinea-pigs were perfused with diluted autologous blood (13 ml blood/87 ml buffer) and exposed to 3-carene at an air concentration of 3000 mg/m(3). In both groups there was a reduction in compliance and conductance but this reduction was significantly (P<0.05) more pronounced (2.5-3 times) in lungs obtained from sensitised animals than from control animals. In a previous study with similar design, but with plain buffer instead of diluted autologous blood as perfusate, we found no statistically significant difference in lung bronchoconstriction. Thus, it is concluded that skin sensitisation can increase lung reactivity to 3-carene and that important mediators of this effect seem to be present in the blood.

Lipid peroxidation by alveolar macrophages challenged with Cryptococcus neoformans, Candida albicans or Aspergillus fumigatus.

Increased formation of oxygen radicals has previously been shown for alveolar macrophages (AM) challenged with Cryptococcus neoformans cells opsonized with fresh serum or polyclonal immunoglobulin G. AM show similar responses to Candida albicans or Aspergillus fumigatus. Oxygen radicals are capable of damaging various macromolecules, including lipids. In the present study, lipid peroxidation (LPO) caused by AM incubated with the fungi was examined in the presence and absence of lung surfactant. The level of malonaldehyde was used as an indicator of LPO. AM damage was examined by electron microscopy (EM), by trypan blue exclusion and by counting the AM loss from culture dish to supernatant. Stimulation of AM by each fungus increased cellular LPO but did not affect AM viability. A slight surfactant LPO induced by AM alone was shown with significantly increased values after addition of each fungus. EM studies showed that dense lipid droplets, presumably consisting of oxidized lipids, were ingested in high amounts together with C. neoformans cells that had been opsonized in fresh serum, and in low amounts in combination with C. albicans. These processes were accompanied by increased numbers of AM in the supernatants. LPO and detachment of AM were counteracted by vitamin E. In the lungs, AM exposed to one of these fungal pathogens might promote peroxidation of surfactant lipids.

Ingested aggregates of ultrafine carbon particles and interferon-gamma impair rat alveolar macrophage function.

Alveolar macrophages (AM), obtained by lavage from the rat lung, were allowed to ingest aggregated ultrafine carbon particles, about 1 microgram/10(6) AM, which is a realistic result of long-term exposure to ambient air. The effects of the ingested carbon on the phagocytosis of test particles and oxidative metabolism of the AM were studied. In addition, the effects of short-term (40 min or 2 h) and long-term (28 or 44 h) incubation with interferon gamma (IFN-gamma) on AM loaded and unloaded with carbon were investigated. Phagocytic activity was studied using fluorescein-labeled 3.2-microgram silica particles. The attachment and ingestion processes were evaluated separately. The ingested carbon markedly impaired the phagocytosis of silica particles; the accumulated attachment (sum of attached and ingested particles per AM) decreased from 5.0 to 4.2 particles/AM and the ingested fraction (number of ingested particles per AM divided with accumulated attachment) from 0.42 to 0.27. The short-term incubation with IFN-gamma tended to increase the accumulated attachment (from 5.0 to 5.7 particles/AM) and decreased the ingested fraction (from 0.42 to 0.34) in unloaded AM. Long-term incubation with IFN-gamma markedly impaired both the accumulated attachment (to 3.8 particles/AM) and the ingested fraction (to 0.24) in unloaded AM and the carbon load further decreased the accumulated attachment to 2.8 particles/AM, and the ingested fraction to 0.21. The oxidative metabolism was not effected by the ingested carbon or the short-term incubation with IFN-gamma, but the long-term incubation with IFN-gamma increased it with a factor of almost 3. Our results suggest that ingested environmental particles in AM may markedly impair their phagocytic capacity, especially during long-term exposure to IFN-gamma as after infections, and there might be an increased risk for additional infections. Moreover, during an episode of high ambient particle concentration the inhaled particles will not be efficiently phagocytized and may thereby damage the Lung tissue.

Assessment of long-term bronchiolar clearance of particles from measurements of lung retention and theoretical estimates of regional deposition.

Twelve healthy nonsmokers inhaled monodisperse Teflon particles labelled with 51Cr (half-life 27.8 days) with an aerodynamic diameter (dae) of 6.1 microns, 5 at a normal flow, 0.5 L/s, and 7 at an extremely slow flow, 0.05 L/s. Lung retention after 24 hours was measured for about 6 months and could be well described by a 2-component exponential function. After the normal inhalation, 14% of the particles retained after 24 hours cleared with a half-time of 3.7 days and 86% with a half-time of 217 days. After the slow inhalation, 35% of the particles retained after 24 hours cleared with a half-time of 3.6 days and 65% with a half-time of 170 days. Deposition was calculated using 3 different models including the recent Human Respiratory Tract Model (HRTM), adopted by the International Commission on Radiological Protection (ICRP), and a model based on Monte Carlo particle transport, together with an asymmetric lung model. Generally, the 3 models agreed fairly well and predicted a considerably higher deposition in the bronchiolar region (generations 9-15) at the slow flow than at the normal flow. Together, the experimental data and the predictions of the deposition models indicate that about 40% of the particles deposited in the conducting airways during the slow inhalation were retained after 24 hours. They also strongly indicate that the particles which cleared with a half-time of about 4 days were mainly deposited in the bronchiolar region, and that about 25% of the particles deposited in the bronchiolar region cleared in this phase. The experimental data agreed quite well with the HRTM predictions made using its default parameter values for slow clearance in the bronchial tree.

Production of nitric oxide by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus.

Cryptococcus neoformans and Aspergillus fumigatus are airborne fungi and the alveolar macrophages (AM) constitute a first line of host defence against both pathogens. We investigated the ability of rat AM to produce nitric oxide (NO) when challenged in vitro with C. neoformans, A. fumigatus conidia or inert silica particles alone and together with interferon gamma (IFN-Gamma). The role of NO in the killing of C. neoformans as well as the relationship between phagocytosis of the yeast or A. fumigatus conidia and NO production by AM were studied. Both fungi, but not the inert particles induced a small but significant increase in NO production by AM. A synergistically enhanced NO production by AM was observed when each fungus, but not silica particles, were incubated together with IFN-Gamma. AM treated with IFN-Gamma and challenged with C. neoformans showed higher killing activity than untreated AM, a finding that correlated with increased NO production by AM. Both effects were reduced by an inhibitor of NO synthesis. Increased NO production by IFN-Gamma activated AM was found together with an increased accumulated attachment of A. fumigatus conidia and serum opsonized, but not unopsonized C. neoformans. The IFN-Gamma dependent increase in accumulated attachment of the fungi might be responsible for the synergistic effect of the fungi and IFN-Gamma on the NO production. Our data suggest that activated rat AM might efficiently use the antimicrobial nitric oxide system in the defence against these pathogens in the normal host.

Human tracheobronchial deposition and effect of a cholinergic aerosol inhaled by extremely slow inhalations.

Ten subjects inhaled the same amounts of cholinergic aerosol of a mass median diameter (MMD) of 7.7 microns in a normal provocation test and in a test with extremely slow inhalations (ESI). This new technique using ESI and large droplets/particles gives a high degree of deposition in small ciliated airways which cannot be obtained by using small particles. The purpose was to compare measured effects with calculated doses of the aerosol in large and small ciliated airways. The effect on large airways was measured by airway resistance (R(aw)), and the effect on small airways was measured by the phase III slope of single breath nitrogen test (N2-delta). Mouth and throat deposition was calculated from human experimental data, and deposition of the cholinergic aerosol into large and small airways was calculated, using a computerized lung model. The study showed that the extremely slow inhalation caused a larger effect on R(aw) and tendency to a larger effect on N2-delta compared to the effect in the normal provocation. Deposited dose in the large airways, in percent of inhaled dose, was calculated to be 25-33% for normal inhalation and 20-24% for ESI. Calculated deposited dose in the small airways (bronchioles; generations 12-16) was 1.8-3.4% for normal inhalation and 18-25% for ESI. For large airways a stronger effect was induced by ESI, perhaps by the more uniform distribution of particles within each generation, compared to normal inhalations when particles deposit near the bifurcations. Concerning the small airways, N2-delta did not differ significantly between normal and ESI provocations, indicating that they did not react much on cholinergic exposure. We believe that our approach using ESI for small airway deposition of a nebulized aerosol can be of value for estimating the effects of various substances on large and small airways.

Lung deposition and extremely slow inhalations of particles. Limited effect of induced airway obstruction.

Studies of lung deposition and clearance have focused on the large airways. Still, lung diseases affect also the small airways. We have developed a method for selective particle deposition in the smallest ciliated airways. Eight healthy subject inhaled 6-micron radiolabelled test particles on 3 occasions at 0.05 L/s and retention was measured for 72 hours. At one occasion, the subjects inhaled the particles at a normal airway resistance. At a second occasion, a 2-3-fold increase in airway resistance was induced by a cholinergic provocation before inhalation of the particles. At a third occasion, a corresponding provocation was induced after inhalation of the particles. The percentage lung depositions were 76 +/- 7, 68 +/- 7, and 73 +/- 8 (mean +/- SD) for "normal airway resistance," "provocation before," and "provocation after" exposures, respectively. The lower value for the "provocation before" exposure was probably a result of increased mucociliary clearance, due to cholinergic stimulation, before the first measurements of radioactivity. The retentions at 24 hours were 51 +/- 7, 52 +/- 9, and 51 +/- 8 in percent of initial lung deposition for "normal airway resistance," "provocation before," and "provocation after" exposures, respectively. We conclude that our inhalation technique is useful in studying conditions in the bronchioles, as deposition is rather independent of airway resistance.

Clearance in small ciliated airways in allergic asthmatics after bronchial allergen provocation.

BACKGROUND: Asthma tends to affect mucociliary clearance, as assessed from measurements in large airways. However, there is no knowledge about clearance in the smallest airways of the tracheobronchial region in acute exacerbation of asthma. OBJECTIVE: The aim of the study was to investigate clearance from the bronchiolar region in patients with allergic asthma in a situation resembling a mild acute exacerbation of the disease. We also aimed to compare clearance data with corresponding data found for healthy subjects and asthmatics on therapy. METHODS: Tracheobronchial clearance was studied twice in 9 patients with mild asthma of the allergic type after inhalation of 6 microm (aerodynamic diameter) monodisperse Teflon particles labelled with 111In. At one exposure, inhalation was performed 4 h after bronchial provocation with an allergen the patients were allergic to. The second exposure was a control measurement. The particles were inhaled at an extremely slow flow, 0.05 liter/s, which gives deposition mainly in the small ciliated airways (bronchioles). Lung retention was measured at 0, 24, 48 and 72 h. RESULTS: All patients demonstrated an early asthmatic reaction of varying degree after bronchial provocation. There was significant clearance of radioaerosol in each 24-hour period for both exposures, with the possible exception of the period between 24 and 48 h for the provocation exposure, with similar fractions of retained particles at all points of time. The retained fractions were significantly larger compared to a group of healthy subjects and asthmatics on regular treatment with anti-inflammatory drugs. CONCLUSIONS: Our results indicate that in allergic asthmatics a bronchial allergen provocation with an early asthmatic reaction does not significantly influence overall clearance from the bronchiolar region. However, in the present group of patients, retention in small ciliated airways was significantly higher compared to healthy subjects and asthmatics on regular treatment.