Interactive effects of mechanical ventilation, inhaled nitric oxide and oxidative stress in acute lung injury

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interactions of inhaled nanoparticles with the alveolar-capillary barrier of the human respiratory tract

In dieser Arbeit wurden zytotoxische Effekte sowie die inflammatorische Reaktionen des distalen respiratorischen Traktes nach Nanopartikelexposition untersucht. Besondere Aufmerksamkeit lag auch auf der Untersuchung unterschiedlicher zellulärer Aufnahmewege von Nanopartikeln wie z.B. Clathrin- oder Caveolae-vermittelte Endozytose oder auch Clathrin- und Caveolae-unabhängige Endozytose (mit möglicher Beteiligung von Flotillinen). Drei unterschiedliche Nanopartikel wurden hierbei gewählt: amorphes Silica (aSNP), Organosiloxan (AmorSil) und Poly(ethyleneimin) (PEI). Alle unterschiedlichen Materialien gewinnen zunehmend an Interesse für biomedizinische Forschungsrichtungen (drug and gene delivery). Insbesondere finden aSNPs auch in der Industrie vermehrt Anwendung, und stellen somit ein ernstzunehmendes Gesundheitsrisiko dar. Dieser wird dadurch zu einem begehrten Angriffsziel für pharmazeutische Verabreichungen von Medikamenten über Nanopartikel als Vehikel aber bietet zugleich auch eine Angriffsfläche für gesundheitsschädliche Nanomaterialien. Aus diesem Grund sollten die gesundheitsschädigenden Risiken, sowie das Schicksal von zellulär aufgenommenen NPs sorgfältig untersucht werden. In vivo Studien an der alveolaren-kapillaren Barriere sind recht umständlich. Aus diesem Grund wurde in dieser Arbeit ein Kokulturmodel benutzt, dass die Alveolar-Kapillare Barrier in vivo nachstellt. Das Model besteht aus dem humanen Lungenepithelzelltyp (z.B. NCI H441) und einem humanen microvasculären Endothelzelltyp (z.B. ISO-HAS-1), die auf entgegengesetzten Seiten eines Transwell-Filters ausgesät werden und eine dichte Barriere ausbilden. Die NP Interaktion mit Zellen in Kokultur wurde mit denen in konventioneller Monokultur verglichen, in der Zellen 24h vor dem Experiment ausgesät werden. Diese Studie zeigt, dass nicht nur die polarisierte Eigenschaft der Zellen in Kokultur sondern auch die unmittelbare Nähe von Epithel und Endothelzelle ausschlaggebend für durch aSNPs verursachte Effekte ist. Im Hinblick auf inflammatorische Marker (sICAM, IL-6, IL8-Ausschüttung), reagiert die Kokultur auf aSNPs empfindlicher als die konventionelle Monokultur, wohingegen die Epithelzellen in der Kokultur auf zytotoxikologischer Ebene (LDH-Ausschüttung) unempfindlicher auf aSNPs reagierten als die Zellen in Monokultur. Aufnahmestudien haben gezeigt, dass die Epithelzellen in Kokultur entschieden weniger NPs aufnehmen. Somit zeigen die H441 in der Kokultur ähnliche epitheliale Eigenschaften einer schützenden Barriere, wie sie auch in vivo zu finden sind. Obwohl eine ausreichende Aufnahme von NPs in H441 in Kokultur erreicht werden konnte, konnte ein Transport von NPs durch die epitheliale Schicht und eine Aufnahme in die endotheliale Schicht mit den gewählten Inkubationszeiten nicht gezeigt werden. Eine Clathrin- oder Caveolae-vermittelte Endozytose von NPs konnte mittels Immunfluoreszenz weder in der Mono- noch in der Kokultur nachgewiesen werden. Jedoch zeigte sich eine Akkumulation von NPs in Flotillin-1 und-2 enthaltende Vesikel in Epithelzellen aus beiden Kultursystemen. Ergebnisse mit Flotillin-inhibierten (siRNA) Epithelzellen, zeigten eine deutlich geringere Aufnahme von aSNPs. Zudem zeigte sich eine eine reduzierte Viabilität (MTS) von aSNP-behandelten Zellen. Dies deutet auf eine Beteiligung von Flotillinen an unbekannten (Clathrin oder Caveolae -unabhängig) Endozytosemechanismen und (oder) endosomaler Speicherung. Zusammenfassend waren die Aufnahmemechanismen für alle untesuchten NPs in konventioneller Monokultur und Kokultur vergleichbar, obwohl sich die Barriereeigenschaften deutlich unterscheiden. Diese Arbeit zeigt deutlich, dass sich die Zellen in Kokultur anders verhalten. Die Zellen erreichen hierbei einen höheren Differenzierungsgrad und eine Zellkommunikation mit anderen relevanten Zelltypen wird ermöglicht. Durch das Einbringen eines dritten relevanten Zelltyps in die Kokultur, des Alveolarmakrophagen (Zelllinie THP-1), welcher die erste Verteidigungsfront im Alveolus bildet, wird diese Aussage weiter bekräftigt. Erste Versuche haben gezeigt, dass die Triplekultur bezüglich ihrer Barriereeigenschaften und IL-8-Ausschüttung sensitiver auf z.B. TNF- oder LPS-Stimulation reagiert als die Kokultur. Verglichen mit konventionellen Monokulturen imitieren gut ausgebildete, multizelluräre Kokulturmodelle viel präziser das zelluläre Zusammenspiel im Körper. Darum liefern Nanopartikelinteraktionen mit dem in vitro-Triplekulturmodel aufschlussreichere Ergebnisse bezüglich umweltbedingter oder pharmazeutischer NP-Exposition in der distalen Lung als es uns bisher möglich war.

Variability of lung function predicts loss of asthma control following withdrawal of inhaled corticosteroid treatment

BACKGROUND One aspect of a multidimensional approach to understanding asthma as a complex dynamic disease is to study how lung function varies with time. Variability measures of lung function have been shown to predict response to beta(2)-agonist treatment. An investigation was conducted to determine whether mean, coefficient of variation (CV) or autocorrelation, a measure of short-term memory, of peak expiratory flow (PEF) could predict loss of asthma control following withdrawal of regular inhaled corticosteroid (ICS) treatment, using data from a previous study. METHODS 87 adult patients with mild to moderate asthma who had been taking ICS at a constant dose for at least 6 months were monitored for 2-4 weeks. ICS was then withdrawn and monitoring continued until loss of control occurred as per predefined criteria. Twice-daily PEF was recorded during monitoring. Associations between loss of control and mean, CV and autocorrelation of morning PEF within 2 weeks pre- and post-ICS withdrawal were assessed using Cox regression analysis. Predictive utility was assessed using receiver operator characteristics. RESULTS 53 out of 87 patients had sufficient PEF data over the required analysis period. The mean (389 vs 370 l/min, p<0.0001) and CV (4.5% vs 5.6%, p=0.007) but not autocorrelation of PEF changed significantly from prewithdrawal to postwithdrawal in subjects who subsequently lost control, and were unaltered in those who did not. These changes were related to time to loss of control. CV was the most consistent predictor, with similar sensitivity and sensitivity to exhaled nitric oxide. CONCLUSION A simple, easy to obtain variability measure of daily lung function such as the CV may predict loss of asthma control within the first 2 weeks of ICS withdrawal.

Deposition and biokinetics of inhaled nanoparticles

Particle biokinetics is important in hazard identification and characterization of inhaled particles. Such studies intend to convert external to internal exposure or biologically effective dose, and may help to set limits in that way. Here we focus on the biokinetics of inhaled nanometer sized particles in comparison to micrometer sized ones.The presented approach ranges from inhaled particle deposition probability and retention in the respiratory tract to biokinetics and clearance of particles out of the respiratory tract. Particle transport into the blood circulation (translocation), towards secondary target organs and tissues (accumulation), and out of the body (clearance) is considered. The macroscopically assessed amount of particles in the respiratory tract and secondary target organs provides dose estimates for toxicological studies on the level of the whole organism. Complementary, microscopic analyses at the individual particle level provide detailed information about which cells and subcellular components are the target of inhaled particles. These studies contribute to shed light on mechanisms and modes of action eventually leading to adverse health effects by inhaled nanoparticles.We review current methods for macroscopic and microscopic analyses of particle deposition, retention and clearance. Existing macroscopic knowledge on particle biokinetics and microscopic views on particle organ interactions are discussed comparing nanometer and micrometer sized particles. We emphasize the importance for quantitative analyses and the use of particle doses derived from real world exposures.

Update on macrophage clearance of inhaled micro- and nanoparticles

Lung macrophages, that is, the intravascular, interstitial, pleural, and surface macrophages, are part of the mononuclear phagocyte system. They are derived from the hematopoietic stem cell in the bone marrow with the monocytes as their putative precursors. Macrophages residing on the inner surfaces of the lungs and immersed within the lung lining layer, that is, the alveolar and the airway macrophages, are constantly exposed to the environment; it is those cells that are recognized as first line of cellular host defense.

The simultaneous role of an alveolus as flow mixer and flow feeder for the deposition of inhaled submicron particles

In an effort to understand the fate of inhaled submicron particles in the small sacs, or alveoli, comprising the gas-exchange region of the lung, we calculated the flow in three-dimensional (3D) rhythmically expanding models of alveolated ducts. Since convection toward the alveolar walls is a precursor to particle deposition, it was the goal of this paper to investigate the streamline maps' dependence upon alveoli location along the acinar tree. On the alveolar midplane, the recirculating flow pattern exhibited closed streamlines with a stagnation saddle point. Off the midplane we found no closed streamlines but nested, funnel-like, spiral, structures (reminiscent of Russian nesting dolls) that were directed towards the expanding walls in inspiration, and away from the contracting walls in expiration. These nested, funnel-like, structures were surrounded by air that flowed into the cavity from the central channel over inspiration and flowed from the cavity to the central channel over expiration. We also found that fluid particle tracks exhibited similar nested funnel-like spiral structures. We conclude that these unique alveolar flow structures may be of importance in enhancing deposition. In addition, due to inertia, the nested, funnel-like, structures change shape and position slightly during a breathing cycle, resulting in flow mixing. Also, each inspiration feeds a fresh supply of particle-laden air from the central channel to the region surrounding the mixing region. Thus, this combination of flow mixer and flow feeder makes each individual alveolus an effective mixing unit, which is likely to play an important role in determining the overall efficiency of convective mixing in the acinus.

Regular treatment with salmeterol and inhaled steroids for chronic asthma: serious adverse events

Epidemiological evidence has suggested a link between beta2-agonists and increased asthma mortality. There has been much debate about possible causal links for this association, and whether regular (daily) long-acting beta2-agonists are safe. This is an updated systematic review.

Fate of inhaled particles after interaction with the lung surface

Inhaled particles may cause increased pulmonary and cardiovascular morbidity and mortality. The wall structures of airways and alveoli act as a series of structural and functional barriers against inhaled particles. Deposited particles are displaced and come into close association with epithelial cells, macrophages and dendritic cells. The cellular interplay after particle deposition in a triple cell co-culture model of the human airway wall was investigated by laser scanning microscopy. Furthermore, the cellular response was determined by measurement of TNF-alpha. Dendritic cells gained access to the apical side of the epithelium where they sampled particles and interacted with macrophages.

The role of macrophages in the clearance of inhaled ultrafine titanium dioxide particles

The role of macrophages in the clearance of particles with diameters less than 100 nm (ultrafine or nanoparticles) is not well established, although these particles deposit highly efficiently in peripheral lungs, where particle phagocytosis by macrophages is the primary clearance mechanism. To investigate the uptake of nanoparticles by lung phagocytes, we analyzed the distribution of titanium dioxide particles of 20 nm count median diameter in macrophages obtained by bronchoalveolar lavage at 1 hour and 24 hours after a 1-hour aerosol inhalation. Differential cell counts revealing greater than 96% macrophages and less than 1% neutrophils and lymphocytes excluded inflammatory cell responses. Employing energy-filtering transmission electron microscopy (EFTEM) for elemental microanalysis, we examined 1,594 macrophage profiles in the 1-hour group (n = 6) and 1,609 in the 24-hour group (n = 6). We found 4 particles in 3 macrophage profiles at 1 hour and 47 particles in 27 macrophage profiles at 24 hours. Model-based data analysis revealed an uptake of 0.06 to 0.12% ultrafine titanium-dioxide particles by lung-surface macrophages within 24 hours. Mean (SD) particle diameters were 31 (8) nm at 1 hour and 34 (10) nm at 24 hours. Particles were localized adjacent (within 13-83 nm) to the membrane in vesicles with mean (SD) diameters of 592 (375) nm at 1 hour and 414 (309) nm at 24 hours, containing other material like surfactant. Additional screening of macrophage profiles by conventional TEM revealed no evidence for agglomerated nanoparticles. These results give evidence for a sporadic and rather unspecific uptake of TiO(2)-nanoparticles by lung-surface macrophages within 24 hours after their deposition, and hence for an insufficient role of the key clearance mechanism in peripheral lungs.

Preischemic iloprost application for improvement of graft preservation: which route is superior in experimental pig lung transplantation: inhaled or intravenous?

BACKGROUND: Optimal allograft protection is essential in lung transplantation to reduce postoperative organ dysfunction. Although intravenous prostanoids are routinely used to ameliorate reperfusion injury, the latest evidence suggests a similar efficacy of inhaled prostacyclin. Therefore, we compared donor lung-pretreatment using inhaled lioprost (Ventavis) with the commonly used intravenous technique. METHODS: Five pig lungs were each preserved with Perfadex and stored for 27 hours without (group 1) or with (group-2, 100 prior aerosolized of iloprost were (group 3) or iloprost (IV). Following left lung transplantation, hemodynamics, Po(2)/F(i)o(2), compliance, and wet-to-dry ratio were monitored for 6 hours and compared to sham controls using ANOVA analysis with repeated measures. RESULTS: The mortality was 100% in group 3. All other animals survived (P < .001). Dynamic compliance and PVR were superior in the endobronchially pretreated iloprost group as compared with untreated organs (P < .05), whereas oxygenation was comparable overall W/D-ratio revealed significantly lower lung water in group 2 (P = .027) compared with group 3. CONCLUSION: Preischemic alveolar deposition of iloprost is superior to IV pretreatment as reflected by significantly improved allograft function. This strategy offers technique to optimize pulmonary preservation.

Comparison of magnetic resonance imaging of inhaled SF6 with respiratory gas analysis

Magnetic resonance imaging of inhaled fluorinated inert gases ((19)F-MRI) such as sulfur hexafluoride (SF(6)) allows for analysis of ventilated air spaces. In this study, the possibility of using this technique to image lung function was assessed. For this, (19)F-MRI of inhaled SF(6) was compared with respiratory gas analysis, which is a global but reliable measure of alveolar gas fraction. Five anesthetized pigs underwent multiple-breath wash-in procedures with a gas mixture of 70% SF(6) and 30% oxygen. Two-dimensional (19)F-MRI and end-expiratory gas fraction analysis were performed after 4 to 24 inhaled breaths. Signal intensity of (19)F-MRI and end-expiratory SF(6) fraction were evaluated with respect to linear correlation and reproducibility. Time constants were estimated by both MRI and respiratory gas analysis data and compared for agreement. A good linear correlation between signal intensity and end-expiratory gas fraction was found (correlation coefficient 0.99+/-0.01). The data were reproducible (standard error of signal intensity 8% vs. that of gas fraction 5%) and the comparison of time constants yielded a sufficient agreement. According to the good linear correlation and the acceptable reproducibility, we suggest the (19)F-MRI to be a valuable tool for quantification of intrapulmonary SF(6) and hence lung function.

Association between inflammatory mediators and response to inhaled nitric oxide in a model of endotoxin-induced lung injury

INTRODUCTION: Inhaled nitric oxide (INO) allows selective pulmonary vasodilation in acute respiratory distress syndrome and improves PaO2 by redistribution of pulmonary blood flow towards better ventilated parenchyma. One-third of patients are nonresponders to INO, however, and it is difficult to predict who will respond. The aim of the present study was to identify, within a panel of inflammatory mediators released during endotoxin-induced lung injury, specific mediators that are associated with a PaO2 response to INO. METHODS: After animal ethics committee approval, pigs were anesthetized and exposed to 2 hours of endotoxin infusion. Levels of cytokines, prostanoid, leucotriene and endothelin-1 (ET-1) were sampled prior to endotoxin exposure and hourly thereafter. All animals were exposed to 40 ppm INO: 28 animals were exposed at either 4 hours or 6 hours and a subgroup of nine animals was exposed both at 4 hours and 6 hours after onset of endotoxin infusion. RESULTS: Based on the response to INO, the animals were retrospectively placed into a responder group (increase in PaO2 > or = 20%) or a nonresponder group. All mediators increased with endotoxin infusion although no significant differences were seen between responders and nonresponders. There was a mean difference in ET-1, however, with lower levels in the nonresponder group than in the responder group, 0.1 pg/ml versus 3.0 pg/ml. Moreover, five animals in the group exposed twice to INO switched from responder to nonresponder and had decreased ET-1 levels (3.0 (2.5 to 7.5) pg/ml versus 0.1 (0.1 to 2.1) pg/ml, P < 0.05). The pulmonary artery pressure and ET-1 level were higher in future responders to INO. CONCLUSIONS: ET-1 may therefore be involved in mediating the response to INO.