Human alveolar long-term clearance of ferromagnetic iron oxide microparticles in healthy and diseased subjects.

Monodisperse ferrimagnetic microparticles (Fe3O4) with 1.3 microm geometric diameter were inhaled to study alveolar long-term clearance in healthy and diseased human subjects. Nineteen younger (age 20 to 39 years) and 20 older (age 40 to 65 years) healthy volunteers participated in the study as well as 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 15 patients with chronic obstructive bronchitis (COB). In each group the subjects were divided into never smokers (NS) and active smokers (S). Clearance was measured by magnetopneumography (MPG) for 300 days after inhalation. In COB, 50% of the deposited particles were removed from the lungs after 2 days, indicating high bronchial deposits due to bronchial obstructions. In healthy NS, only 10% of the particles were removed after 2 days and cigarette smoking enhanced the fraction of fast-cleared particles. In subjects who smoked, slow clearance was significantly impaired (P < . 02). Clearance half-lives (in days) for younger, healthy, NS were 124 +/- 66 (mean +/- SD) compared to 220 +/- 74 for S. Similarly for older subjects, the timeswere 162 +/- 120 for NS and 459 +/- 334 for S. The impairment of alveolar clearance due to cigarette smoking increases by 5.7 +/- 1.3 days/pack-year (P < .01). Alveolar clearance was impaired in SAR and in IPF; half-lives were 275 +/- 109 days (P < .05) and 756 +/- 345 days (P < .02), respectively, compared to healthy NS. Most COB patients were ex-smokers, their long-term clearance was 240 +/- 74 days, which is more than healthy NS (P < .01), but less than healthy S and might indicate a recovery of alveolar clearance. In view of studies using totally inert particles like Teflon, we conclude that the lung clearance measured with iron oxide tracer particles primarily reflects clearance by intraphagosomal particle dissolution within alveolar macrophages, which is impaired by cigarette smoke consumption and in patients.

A new methodology for controlled particle inhalation by small rodents.

In order to investigate the deposition, retention, and clearance mechanisms implicated in particle inhalation under standardized conditions, we developed a continuous negative-pressure ventilation system, whereby the breathing pattern in small rodents could be controlled during exposure to aerosols. Using an on-line open-flow set-up, 19 anesthetized, intubated, and paralyzed Syrian golden hamsters, individually contained within a whole-body box, were artificially ventilated under the said continuous negative-pressure conditions, 1 of 5 different combinations of breathing frequency and tidal volume being established. The animals were then exposed to aerosols containing 6-micron diameter polystyrene spheres, and the deposition of particles in the conducting airways was monitored photometrically. During exposure, the level of respiration (mean lung inflation) was stabilized by means of a negative-pressure vent. Breathing frequency and tidal volume, as well as the compliance of the system, remained virtually unchanged during the course of a single experiment, and in each case, a reproducible deposition of particles was achieved. Our findings indicate that tidal volume, but not breathing frequency, has a marked influence on the particle deposition ratio. Breathing frequency exerts opposing and counterbalancing effects on this latter parameter by enhancing the impaction of particles on the one hand, and by decreasing sedimentation on the other.

Regional deposition and retention of particles in shallow, inhaled boluses: effect of lung volume.

The regional deposition of particles in boluses delivered to shallow lung depths and their subsequent retention in the airways may depend on the lung volume at which the boluses are delivered. To evaluate the effect of end-inspiratory lung volume on aerosol bolus delivery, we had healthy subjects inhale radiolabeled, monodisperse aerosol (99mTc-iron oxide, 3.5-microm mass median aerodynamic diameter) boluses (40 ml) to a volumetric front depth of 70 ml into the lung at lung volumes of 50, 70, and 85% of total lung capacity (TLC) end inhalation. By gamma camera analysis, we found significantly greater deposition in the left (L) vs. right (R) lungs at the 70 and 85% TLC end inhalation; ratio of deposition in L to R lung, normalized to L-to-R ratio of lung volume (mean L/R), was 1.60 +/- 0.45 (SD) and 1. 96 +/- 0.72, respectively (P < 0.001 for comparison to 1.0) for posterior images. However, at 50% TLC, L/R was 1.23 +/- 0.37, not significantly different from 1.0. These data suggest that the L and R lungs may be expanding nonuniformly at higher lung volumes. On the other hand, subsequent retention of deposited particles at 2 and 24 h postdeposition was independent of L/R at the various lung volumes. Thus asymmetric bolus ventilation for these very shallow boluses does not lead to significant increases in peripheral alveolar deposition. These data may prove useful for 1) designing aerosol delivery techniques to target bronchial airways and 2) understanding airway retention of inhaled particles.

Bronchial airway deposition and retention of particles in inhaled boluses: effect of anatomic dead space.

The fractional deposition of particles in boluses delivered to shallow lung depths and their subsequent retention in the airways may depend on the relative volume and size of an individual's airways. To evaluate the effect of variable anatomic dead space (ADS) on aerosol bolus delivery we had healthy subjects inhale radiolabeled, monodisperse aerosol (99mTc-iron oxide, 3.5 micron mean mondispersed aerosol diameter) boluses (40 ml) to a volumetric front depth of 70 ml into the lung at a lung volume of 70% total lung capacity end inhalation. By using filter techniques, aerosol photometry, and gamma camera analysis, we estimated the fraction of the inhaled boluses deposited in intrathoracic airways (IDF). ADS by single-breath N2 washout was also measured from 70% total lung capacity. Results showed that among all subjects IDF was variable (range = 0.04-0.43, coefficient of variation = 0.54) and increased with decreasing ADS (r = -0.76, P = 0.001, n = 16). We found significantly greater deposition in the left (L) vs. right (R) lungs; mean L/R (ratio of deposition in L lung to R lung, normalized to ratio of L-to-R lung volume) was 1.58 +/- 0.42 (SD; P < 0.001 for comparison with 1.0). Retention of deposited particles at 2 h was independent of ADS or IDF. There was significant retention of particles at 24 h postdeposition (0.27 +/- 0.05) and slow clearance of these particles continued through 48 h postdeposition. Finally, analysis of central-to-peripheral ratios of initial deposition and 24-h-retention gamma-camera images suggest significant retention of insoluble particles in large bronchial airways at 24 h postdeposition (i.e., 24 h central-to-peripheral ratio = 1.40 +/- 0. 44 and 1.82 +/- 0.54 in the R and L lung, respectively; P < 0.02 for comparison with 1.0). These data may prove useful for 1) designing aerosol delivery techniques to target bronchial airways and 2) understanding airway retention of inhaled particles.

Probing mechanical properties of living cells by magnetopneumography.

Magnetopneumography (MPG) has been used to study long-term particle clearance from human lungs as well as cellular motility of pulmonary macrophages (PMs). This study describes an extension of the method enabling the measurement of mechanical properties of PM cells in vivo. Ferromagnetic microparticles are inhaled and then retained in the alveolar region of the lungs, where they are phagocytized within hours by PMs. The magnetic particles can be rotated in weak magnetic fields, and the response to this twisting shear (force) is detected as a macroscopic magnetic field producing a measure of cytoskeletal mechanics. Cytoplasmic viscosity is very high compared with that of water and is strongly non-Newtonian. Under rotational stresses from 0.4 to 6.4 Pa, it acts like a pseudoplastic fluid showing a characteristic shear rate dependence. The viscosity as well as the stiffness of the cytoskeleton increases with increasing shear stress as seems typical for living tissue and evidence for an intact cytoskeletal matrix. The particle recoil as measured by the amount of recoverable strain following a short twisting force describes a cytoplasmic elasticity that depends on both level and duration of stress. These investigations on the mechanical properties of living human cells are promising and should lead to better understanding of cellular dysfunction in disease as well as pathways for drug administration.

Smoking impairs alveolar macrophage activation after inert dust exposure.

Magnetopneumography was applied to investigate intracellular phagosome motion in alveolar macrophage cells of healthy subjects (non-smokers and smokers). Ingested magnetic microparticles are inhaled and phagocytized by alveolar macrophages within hours. Thereby the particles are transferred into phagolysosomes. After magnetization the particles produce a macroscopic magnetic field of the lungs. Cellular motility causes a decay of the field (relaxation) by stochastic disorientation of the dipole particles (phagolysosomes) in the cells. Our studies have shown that the deposition of magnetite test particles induces a non-specific activation of the macrophage cells with a faster relaxation. This activation vanishes within the first day after particle deposition. This macrophage activation due to dust exposure was not present in smokers. It follows that cigarette smoking either causes a damage of the cellular defense or causes an adaptation of the macrophage cells to the permanent cigarette smoke inhalation.

Thirty month variability of aerosol pulse dispersion and conventional lung function parameters in healthy middle aged smokers and nonsmokers.

Chronic cigarette consumption is a generally accepted reason for the development of chronic obstructive pulmonary disease (COPD). COPD correlates to histomorphological parameters of lung structure as well as pulmonary function tests (PFT). COPD related changes affect PFT determined by conventional methods (bodyplethysmography, spirometry) as well as parameters of convective gas mixing. This study evaluates the diagnostic potential of a non-invasive aerosol method for the discrimination between healthy smokers and nonsmokers in comparison to conventional PFT. The aerosol method is based on the inhalation of small aerosol pulses suspended in particle free air and determines their changes during the breathing maneuver. Changes of aerosol pulse parameters (APP) are used to describe the convective component of gas mixing during ventilation. PFT and APP were determined in 40 healthy subjects (nonsmoker: 51.1 +/- 1.5 years; smoker: 49.6 +/- 1.5 years, 39.1 +/- 2.2 pack years) before and after a time interval of 30 months. Conventional PFT in smokers and nonsmokers showed no relevant differences between the values at the beginning and the end of the observation period. Thirty months later, at the end of the observation interval, a very similar behavior of the APP was obtained, which strongly confirmed the prior observed differences between smokers and nonsmokers. The data suggest that cigarette smoke-induced variations of lung function are also detectable in clinically asymptomatic smokers. Even in cases of normal PFT, most APP are able to discriminate between healthy smokers and nonsmokers. Since PFT showed only minor differences between both groups, it is indicated that APP are superior to PFT in the detection of early disturbances of lung ventilation in healthy smokers. Mean values of PFT and APP in smokers and nonsmokers showed a high reproducibility of the data obtained at the beginning of the study as well as at the end of the observation period. The data of our study further confirm that parameters of pulmonary gas exchange and gas mixing are affected by cigarette smoke at an earlier time than parameters of breathing mechanics.

An approach to deposition and clearance measurements in human airways.

By using the aerosol bolus inhalation technique, aerosol particles can be delivered into the airways of the human respiratory tract. For that purpose the aerosol bolus is injected near the end of a clean air inhalation. It could be shown experimentally and theoretically that the particles were only deposited in the airways. Radioactive labeled particles were deposited with this technique and clearance from the airways was determined. It could be shown that the mucociliary clearance from the airways was particle size dependent. The clearance efficiency from the airways increased with increasing particle size.

Retention of particles inhaled in boli with and without induced bronchoconstriction.

Large lung retentions (up to 50%) of particles < or = 4 microns inhaled with a bolus technique at a penetration depth less than dead space have been reported to occur after 24 h. This retention may be due to retarded clearance of particles deposited in the airways of the tracheobronchial tract; an alternative explanation could be that particles are deposited in the alveolar region. The purpose of the present study was to confirm the occurrence of retained fractions and to study the influence of a cholinergic drug, which is assumed to give a more central particle deposition, on these retentions in human lungs after shallow aerosol bolus inhalation. Twelve healthy subjects inhaled, with a bolus technique, monodisperse Teflon particles (2.4 microns geometric diameter, 3.5 microns aerodynamic diameter), labeled with 111In. The volumetric lung depth of the inspired bolus was around 60 mL and flow rate was about 300 mL/s. Six subjects inhaled the test particles after a provocation with a cholinergic aerosol, which induced a threefold increase in airway resistance. The other six subjects inhaled a cholinergic aerosol after inhalation of the test particles or inhaled no cholinergic aerosol at all. Radioactivity in the body was measured after 0.5, 24, 48, and 72 h with a whole-body scanner with three 127 x 101-mm Nal detectors. The investigation confirmed results obtained earlier by a group in Frankfurt claiming that great retentions occur after 24 h. The retentions tended to be lower in the group receiving a bronchoconstricting drug before the bolus inhalations. There was a significant lung clearance of particles between 24 and 72 h, in contrast to the findings in earlier studies in healthy subjects and asthmatics who inhaled Teflon particles in large volumes. On the other hand, the clearance agreed well with the clearance in healthy subjects with extensive deposition of Teflon particles in the small ciliated airways, obtained by means of an extremely low inhalation flow rate. The results suggest that a considerable fraction of the particles in the bolus inhalation have been deposited in small ciliated airways in which the mucociliary transport is less efficient or in the alveolar region.

Effect of flow rate on aerosol bolus penetration in a hollow canine lung airway cast.

A cast of dog tracheobronchial airways, which is complete to airways of 1 mm in diameter, was used for experimental studies of the effect of flow rate on aerosol bolus penetration during a tidal breath. Aerosol boli of 35 cm3 were injected during a 1-L inhalation of air. The recoveries of aerosol particles in expired air were measured after various breathholding times. The results show that the penetration depth of an aerosol bolus increases with decreasing flow rate, especially for flow in transition from a flat profile to a parabolic profile.

Dispersion of aerosol particles in an airway cast of a dog.

Aerosol bolus dispersion was measured in the first branching generations of a replicate hollow cast. The cast was made from a solid cast of a beagle-dog tracheobronchial tree. The dispersion was measured after penetration of boluses into different volumetric depths, with different particle sizes and flow rates into the cast. An enhanced flow rate led to a decreasing dispersion in the cast. Particle size up to about 4 microns has no significant effect on dispersion.

In vivo and in vitro studies of the cellular defense system of the human lung.

Magnetic microparticles were used to investigate the defence system of the human lungs against foreign material. About 0.5 mg of spherical monodisperse magnetite particles were deposited in the alveolar region of the human lung by voluntary inhalation. After primary magnetization a remanent magnetic field (RMF) of the lung can be measured that allows estimation of the amount of dust retained in the lung. The decay of this RMF, called relaxation, results from a misalignment of the dipole particles due to the activity of pulmonary macrophages. This macrophage activity was characterized by a cell energy Ez. With a secondary magnetization the lung can be remagnetized by rotation of the dipole particles. This allows estimation of the intracellular viscosity and the motility of the alveolar macrophages in vivo. The macrophage cell-line J774 was used to verify the dynamic processes of the magnetic particles within the cells in vitro. In vitro and in vivo relaxation curves of polydisperse and of spherical monodisperse magnetite particles are presented. Thermal relaxation of mono-disperse and polydisperse particles within a viscous standard could be verified with the Brownian rotary diffusion model. Relaxation with monodisperse particles was double exponential in vivo as well as in vitro, suggesting that 2 different viscous compartments of the cytoplasm should be considered. Relaxation in the macrophage cell-line J774 was particle-size-dependent.

[Long-term effect of the beta 2-sympathomimetic formoterol in young smokers--study of duration and site of effect of bronchodilatation using conventional methods of lung function and monodispersed aerosols]. / Langzeitwirkung des beta 2-Sympathomimetikums Formoterol bei jungen Rauchern--Untersuchung von Wirkdauer und Wirkort der Bronchodilatation mittels konventioneller Lungenfunktionsmethoden und monodisperser Modellaerosole.

Formoterol is a novel selective beta 2-sympathomimetic inducing bronchodilatation after inhalation or oral application. Compared to other beta 2-sympathicomimetics the substance begins to act very rapidly even at a much lower dosage level, while it remains effective for at least 12 hours. In the present study the bronchodilatory effect of 24 micrograms MDI formoterol was investigated in 18 healthy smokers between 20 and 30 years of age means. Measurement was effected by means of conventional lung function diagnostics (body plethysmography, spirometry) and an biophysical aerosol measurement method for determining the effective airways dimensions (EAD). This method is based on the gravitational losses of a previously inspired monodisperse model aerosol during apnoea periods of different duration. It enables determination of the EAD as a function of the volumetric lung depth (VLT). A marked and universally measurable bronchodilatation is detectable directly after formoterol has been inhaled. The longterm action of formoterol was confirmed for more than 15 hours after application, using the conventional lung function test and the EAD method. Over and above this the EAD determination showed that the bronchodilatory effect was much more marked in the central airways than in the periphery of the lungs, thus confirming the effect generally described for beta 2-sympathomimetics as being mainly directed towards the central airways region.

[Granulometry and measurement of a aerosol drug deposit (fusafungine) in normal and pathological airways]. / Granulométrie et mesure du dépôt d'un aérosol médicamenteux (fusafungine) dans les voies aériennes normales et pathologiques.

We measured with a laser velocimeter granulometric deposit of an aerosol anti-infectious agent, fusafungin, administered with a controlled inhalator. Total drug deposit was determined on the basis of a granulometric spectrum of the polydispered aerosol (mass mean aerodynamic diameter (MMAD) = 2.8 +/- 1.7 microns) and dispersion in the airways was estimated using the Stahlhofen model. We first compared deposits obtained with oral inhalation in 19 normal subjects and 20 patients with chronic obstructive lung disease. Total deposit in the airways of patients with chronic obstructive lung disease (82%) was not significantly different from that in normal subjects (85%). Estimated dispersion in normal airways was 27% in the alveoles, 8.4% in the tracheobronchic region and 23.5% in the extrathoracic regions. We then compared deposits after nasal inhalation in 22 normal subjects and 21 patients with rhinitis: nasal deposit was significantly greater in patients with rhinitis (54.5%) than in controls (44.7%). We conclude that such an inhalator can be adapted for local treatment of ENT infections and upper respiratory infections. Deposit is not modified in case of obstructive bronchopathy.

Behaviour of magnetic micro-particles in the human lung.

Magnetic micro-particles were used to investigate the defence system of the human lungs against foreign material. After primary magnetisation a remanent magnetic field (RMF) of the lung can be measured that allows estimation of the amount of dust retained in the lung. After calibration of the system with a lung phantom the magnetic contamination retained in the lungs of dental technicians and welders was estimated at mean values of 22 and 500 mg respectively. In normal controls only 0.3 mg was found. About 0.5 mg of spherical monodisperse magnetite particles was deposited in the alveolar region of the lung by voluntary inhalation. The decay of the RMF, called relaxation, results from a misalignment of the dipole particles due to the activity of pulmonary macrophages. This macrophage activity is characterised by a cellular energy Ez. With a secondary magnetisation the lung can be remagnetised by rotation of the dipole particles. This allows an estimation of the intracellular viscoelasticity and the motility of the alveolar macrophages in vivo. Secondary magnetisation and relaxation curves of spherical monodisperse magnetic particles are presented. Intracellular viscosity was estimated to be n approximately equal to 100 Pa.s at shear rates near 0.01 s-1, the rigidity modulus being v approximately equal to 4-8 Pa. Macrophage activity was described by a cellular energy EZ approximately 5 x 10(-18) J. Additionally, non-magnetic aerosol exposure resulted in a faster relaxation, which was interpreted to be due to activation of the macrophages. The magnetite particles were cleared with a half-time of approximately 110 days.

Investigation of the defense system of the human lungs with ferrimagnetic particles.

Magnetic microparticles were used to investigate the defense system of the human lungs against foreign material. About 0.5 mg of spherical monodisperse magnetite particles were deposited in the alveolar region of the human lung by voluntary inhalation. After primary magnetization a remanent magnetic field (rmf) of the lung can be measured that allows estimation of the amount of dust retained in the lung. The decay of this rmf, called relaxation, results from a misalignment of the dipole particles due to the activity of pulmonary macrophages. This macrophage activity was characterized by a cell energy E z. With a secondary magnetization the lung can be remagnetized by rotation of the dipole particles. This allows estimation of the intracellular viscosity and motility of the alveolar macrophages in vivo. Secondary magnetization and relaxation curves of spherical monodisperse magnetite particles are presented. Intracelluar viscosity was estimated to be n approximately equal to 100 Paxs by a shear-rate near 0.01 s -1, macrophage activity was E z approximately equal to 5x10 -18 J. Aerosol exposure resulted in a faster relaxation, which was interpreted to be due to activation of the macrophages. The magnetite particles were cleared with a half-time of approximately equal to 110 days.

Deposition and dispersion of aerosols in the airways of the human respiratory tract: the effect of particle size.

Small volumes of aerosols (boluses) were inspired predominantly into the conducting airways of human lungs with a fast operating valve system, injecting preselected aerosol volumes near the end of a clean air inhalation. Particle recovery and bolus dispersion in the exhaled air after various periods of breathholding were investigated by measuring aerosol number concentration directly in front of the mouth with a laser photometer. Inspired and expired flow rates were measured with a pneumotachograph. The effect of particle size on these measurements has been investigated using aerosol particles with aerodynamic diameters (dae) between 0.9 and 5 microns. For aerosol particles smaller than 2 microns, bolus dispersion increases with increasing periods of breathholding (tb). After reaching a maximum, dispersion decreases with even longer tb. An increase in particle size yields a smaller increase in dispersion during the first seconds of breathholding while it is not changed significantly without breathhold. Particle losses during inhalation and exhalation increases with particle size. However, with increasing periods of breathholding, the losses of the smaller particles (less than 1.5 microns) were found to be much higher than expected theoretically, implying particle losses by sedimentation in the same airway structures. The small aerosol particles are deposited in smaller airways than bigger particles. These observations can be explained by cardiogenic mixing during periods of breathholding by pulsatile flow oscillations and confirm measurements with enhanced heart rate as described in an earlier paper. Small particles with restricted settling velocities remained longer in an airborne state in the airways and this leads to a more efficient cardiogenic mixing.

Effect of heart rate on aerosol recovery and dispersion in human conducting airways after periods of breathholding.

With a newly developed aerosol inhalation device, small volumes of aerosols ("boluses") can be inspired predominantly into the conducting airways of the human lungs. The aerosol is injected by a fast-operating valve system using preselected volumes near the end of a clean air inhalation of 1000 cm3. Particle behavior in upper human airways was investigated by measuring particle recovery and bolus dispersion in exhaled air with a laser photometer positioned directly in front of the mouth after various periods of breathholding. The effect of physical motion of the heart on these measurements has been investigated by increasing the heart rate of a subject by more than a factor of 2. Monodisperse sebacate aerosols were used with droplets in the aerodynamic size (dac) range between 0.8 and 1.1 micron to minimize particle losses by diffusion and by inertial forces. It was shown that motion of the heart considerably influences both particle recovery and dispersion of such boluses during postinhalation periods of breathholding. For a twofold enhancement in heart frequency the standard deviation of the expired aerosol bolus was increased by up to 60% after certain breathholding periods. Particle recovery from shallow volumetric lung depths was significantly decreased.

[Measuring human mucociliary clearance]. / Messung der menschlichen Mukoziliarclearance.

Using a new inhalation technique, it is possible to inject aerosol boluses of defined volume at any moment during inspiration into the inhaled air. Deposition of particles in different lung depths was achieved by holding the breath at end-inspiration. Particle clearance of the inhaled radioactively labelled particles was determined by measuring the radioactivity retained in the lungs as a function of time after inhalation. Even for shallow volumetric lung depths as small as 35 cm3 slow clearance of a significant fraction of the deposit has been observed.