Captive Bubble Surfactometer Research Articles

Aerosol, chemical and physical properties of dry powder synthetic lung surfactant for noninvasive treatment of neonatal respiratory distress syndrome

Inhalation of dry powder synthetic lung surfactant may assist spontaneous breathing by providing noninvasive surfactant therapy for premature infants supported with nasal continuous positive airway pressure. Surfactant was formulated using spray-drying with different phospholipid compositions (70 or 80 total weight% and 7:3 or 4:1 DPPC:POPG ratios), a surfactant protein B peptide analog (KL4, Super Mini-B, or B-YL), and Lactose or Trehalose as excipient. KL4 surfactant underperformed on initial adsorption and surface activity at captive bubble surfactometry. Spray-drying had no effect on the chemical composition of Super Mini-B and B-YL peptides and surfactant with these peptides had excellent surface activity with particle sizes and fine particle fractions that were well within the margins for respiratory particles and similar solid-state properties. Prolonged exposure of the dry powder surfactants with lactose as excipient to 40 °C and 75% humidity negatively affected hysteresis during dynamic cycling in the captive bubble surfactometer. Dry powder synthetic lung surfactants with 70% phospholipids (DPPC and POPG at a 7:3 ratio), 25% trehalose and 3% of SMB or B-YL showed excellent surface activity and good short-term stability, thereby qualifying them for potential clinical use in premature infants.

Molecular and biophysical mechanisms behind the enhancement of lung surfactant function during controlled therapeutic hypothermia

Therapeutic hypothermia (TH) enhances pulmonary surfactant performance in vivo by molecular mechanisms still unknown. Here, the interfacial structure and the composition of lung surfactant films have been analysed in vitro under TH as well as the molecular basis of its improved performance both under physiological and inhibitory conditions. The biophysical activity of a purified porcine surfactant was tested under slow and breathing-like dynamics by constrained drop surfactometry (CDS) and in the captive bubble surfactometer (CBS) at both 33 and 37 °C. Additionally, the temperature-dependent surfactant activity was also analysed upon inhibition by plasma and subsequent restoration by further surfactant supplementation. Interfacial performance was correlated with lateral structure and lipid composition of films made of native surfactant. Lipid/protein mixtures designed as models to mimic different surfactant contexts were also studied. The capability of surfactant to drastically reduce surface tension was enhanced at 33 °C. Larger DPPC-enriched domains and lower percentages of less active lipids were detected in surfactant films exposed to TH-like conditions. Surfactant resistance to plasma inhibition was boosted and restoration therapies were more effective at 33 °C. This may explain the improved respiratory outcomes observed in cooled patients with acute respiratory distress syndrome and opens new opportunities in the treatment of acute lung injury.

Restoration of surfactant activity by polymyxin B in lipopolysaccharide-potentiated injury of immature rabbit lungs

During postnatal adaptation pulmonary surfactant may be inactivated by lipopolysaccharide (LPS). We evaluated the effect of surfactant therapy in combination with antibiotic polymyxin B (PxB) in double-hit model of neonatal lung injury. Surfactant (poractant alfa, Curosurf) was exposed to smooth (S) LPS without/with PxB and tested in captive bubble surfactometer. Preterm rabbits received intratracheally saline (control) or S-LPS and were ventilated with 100% oxygen. After 30 min, LPS-treated animals received no treatment, or surfactant (200 mg/kg) without/with 3% PxB; controls received the same dose of surfactant. Animals were ventilated for further 2 h. In vitro, addition of 5% S-LPS to surfactant increased minimum surface tension (γmin) and addition of 1–3% PxB to surfactant/S-LPS mixture restored γmin to low values. Animals only given S-LPS had lower lung compliance and lung gas volume (LGV) compared to surfactant groups. Treatment with surfactant/PxB, but not with surfactant only, restored LGV. Addition of PxB to the surfactant increased the alveolar expansion. S-LPS interferes with surface activity of the pulmonary surfactant and PxB improves the resistance of surfactant to LPS-induced inactivation. In our neonatal model of respiratory distress syndrome surfactant gives positive response even in simultaneous exposure to S-LPS, when enriched with PxB.

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

Mild Hypothermia Enhances Lung Surfactant Activity: Delving into the Molecular Mechanisms

Pulmonary surfactant (PS) is a lipid-protein complex that reduces the surface tension at the respiratory air-liquid interface of alveoli, minimizing the work of breathing. The composition and structure of PS are directly responsible for its mechanical properties in a healthy lung or under pathological conditions. We previously demonstrated that PS activity improves after 72h of Whole Body Hypothermia (WBH, 33 °C) in neonates with and without lung injury. PS performance is significantly better when samples are tested under cooling condition. Moreover, decreasing neonatal lung temperature, SP-C and poli-unsaturated phospholipids are significantly reduced in surfactant large aggregates, promoting the simultaneous increase in DPPC percentage. To understand the molecular mechanisms under this temperature-dependent improvement, we designed a surfactant in vitro model. We combined surfactant lipids at different percentages (DPPC, POPG, POPC and DOPC) with purified porcine surfactant proteins SP-B and SP-C. We tested the biophysical activity of the resulting synthetic models upon breathing-like dynamics in a Captive Bubble Surfactometer, at both 37 °C and 33 °C. We also analyzed the thermotropic profiles of these protein-lipid mixtures by Differential Scanning Calorimetry. Finally, to further confirm our results, we studied both the lateral structure and the lipid composition of porcine PS once subjected to compression at the air-liquid interface. To do so, we coupled Langmuir-Blodgett Balance experiments with Epifluorescence Microscopy and Lipidomic Analysis. Our in vitro data suggests that the improvement in PS activity at 33 °C depends on a better and preferential exclusion from the air-liquid interface of less active phospholipids. This process occurs during compression (at expiration) and SP-C may play a crucial role.

Mechanistic Insights in the Interaction of Chemicals with Surfactant Membrane Models in vitro

Every year, cases of respiratory distress are recorded in humans following exposure to chemicals. The underlying mechanisms of toxicity remain unclear. We have previously shown that pulmonary surfactant is the target of impregnation spray products, leading to lung damage in humans. Lung surfactant is a lipid-protein mixture whose main function is to reduce the surface tension in the alveoli at the end of expiration, minimizing the work of breathing. In this study, three chemicals (trimethoxyoctylsilane, 3-oxo-2-pentyl-cyclopentaneacetic acid and diisoamyl ether), classified as acute inhalation toxicants and of relevance for worker and consumer exposure, were studied with respect to their potential to perturb lung surfactant function. Two models were used: DPPC, the most abundant surface active lipid in surfactant, as well as native surfactant purified from porcine bronchoalveolar lavage. The constrained drop surfactometer and the captive bubble surfactometer were used to assess the effects of the chemicals on the biophysical activity of the two surfactant models under different exposure conditions. These two methodologies mimic the breathing dynamics of the lungs. In addition, the effect of the chemicals on the lung surfactant structure was explored by (i) Langmuir-Blodgett balance experiments coupled with epifluorescence microscopy, and (ii) cryogenic transmission electron microscopy. Lastly, the thermotropic behavior of surfactant after exposure to the chemicals was investigated using differential scanning calorimetry. The data provide evidences on the mechanism of toxicity of chemicals potentially harmful for the lungs. The identification of key event(s) for the acute inhalation toxicity pathway is crucial for the design of relevant toxicity assays in vitro, which will contribute to having safer chemicals in the workplace and on the market.

In Vitro Functional and Structural Characterization of A Synthetic Clinical Pulmonary Surfactant with Enhanced Resistance to Inhibition

CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purified from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fluorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 films formed in a Langmuir-Blodgett balance coupled with epifluorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than films made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by differential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defined composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung inflammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.

Inhibition and counterinhibition of Surfacen, a clinical lung surfactant of natural origin

Inactivation of pulmonary surfactant by different components such as serum, cholesterol or meconium contributes to severe respiratory pathologies through destabilization and collapse of airspaces. Recent studies have analyzed in detail how the interfacial properties of natural surfactant purified from animal lungs are altered as a consequence of its exposure to serum proteins or meconium-mobilized cholesterol. It has been also demonstrated that pre-exposure of surfactant to polymers such as hyaluronic acid provides resistance to inactivation by multiple inhibitory agents. In the current work, we have extended these studies to the analysis of Surfacen, a clinical surfactant currently in use to rescue premature babies suffering or at risk of respiratory distress due to congenital lack of surfactant. This surfactant is also strongly inhibited by both meconium and serum when tested in the captive bubble surfactometer (CBS) under conditions mimicking respiratory dynamics. As it occurs with native surfactant, Surfacen is markedly protected from inhibition by pre-exposure to hyaluronic acid, confirming that clinical surfactants can be improved to treat pathologies associated with strongly deactivating contexts, such as those associated with lung injury and inflammation. Remarkably, we found that, under physiologically-mimicking conditions, a cholesterol-free clinical surfactant such as Surfacen is less susceptible to inhibition by cholesterol-mobilizing environments than cholesterol-containing natural surfactant, as a consequence of a markedly reduced susceptibility to incorporation of exogenous cholesterol.

Dysfunction of pulmonary surfactant mediated by phospholipid oxidation is cholesterol-dependent

Pulmonary surfactant forms a cohesive film at the alveolar air-lung interface, lowering surface tension, and thus reducing the work of breathing and preventing atelectasis. Surfactant function becomes impaired during inflammation due to degradation of the surfactant lipids and proteins by free radicals. In this study, we examine the role of reactive nitrogen (RNS) and oxygen (ROS) species on surfactant function with and without physiological cholesterol levels (5–10%). Surface activity was assessed in vitro in a captive bubble surfactometer (CBS). Surfactant chemistry, monolayer fluidity and thermodynamic behavior were also recorded before and after oxidation. We report that physiologic amounts of cholesterol combined with oxidation results in severe impairment of surfactant function. We also show that surfactant polyunsaturated phospholipids are the most susceptible to oxidative alteration. Membrane thermodynamic experiments showed significant surfactant film stiffening after free radical exposure in the presence of cholesterol. These results point to a previously unappreciated role for cholesterol in amplifying defects in surface activity caused by oxidation of pulmonary surfactant, a finding that may have implications for treating several lung diseases.

The role of multilayers in preventing the premature buckling of the pulmonary surfactant

The pulmonary surfactant is a protein-lipid mixture that spreads into a film at the air-lung interface. The highly-compacted molecules of the film keep the interface from shrinking under the influence of otherwise high surface tension and thus prevent atelectasis. We have previously shown that for the film to withstand a high film pressure without collapsing it needs to assume a specific architecture of a molecular monolayer with islands of stacks of molecular multilayers scattered over the area. Surface activity was assessed in a captive bubble surfactometer (CBS) and the role of cholesterol and oxidation on surfactant function examined. The surfactant film was conceptualized as a plate under pressure. Finite element analysis was used to evaluate the role of the multilayer stacks in preventing buckling of the plate during compression. The model of film topography was constructed from atomic force microscope (AFM) scans of surfactant films and known physical properties of dipalmitoylphosphatidylcholine (DPPC), a major constituent of surfactant, using ANSYS structural-analysis software. We report that multilayer structures increase film stability. In simulation studies, the critical load required to induce surfactant film buckling increased about two-fold in the presence of multilayers. Our in vitro surfactant studies showed that surface topography varied between functional and dysfunctional films. However, the critical factor for film stability was the anchoring of the multilayers. Furthermore, the anchoring of multilayers and mechanical stability of the film was dependent on the presence of hydrophobic surfactant protein-C. The current study expands our understanding of the mechanism of surfactant inactivation in disease.

Functional and Structural Characterization of Pulmonary Surfactant Fractions Obtained from Bronchoalveolar Lavages

The alveolar surface of the lung is protected by a lipophilic material, the pulmonary surfactant, mainly composed by lipids and a few specific proteins: SP-A, SP-B, SP-C and SP-D. The main function of pulmonary surfactant is to reduce the surface tension of the thin aqueous layer covering the alveolar epithelium, avoiding the collapse of the smaller alveoli during the expiration. To achieve this function, surfactant lipids form an interfacial film at the air-liquid interface of this aqueous lining layer. Nevertheless, pulmonary surfactant is synthetized, packed and secreted by type II alveolar pneumocytes as tightly packed membranes assembled in specialized organelles, the lamellar bodies. Currently, the molecular mechanisms that let pulmonary surfactant be transformed from the bilayers of lamellar bodies into the functional interfacial film remain unclear. This is in part because most of the previous studies were made using surfactants obtained from bronchoalveolar lavages, a complex mixture that likely combines freshly secreted surfactant and surfactant that has been already exposed to respiratory dynamics and can be at least partially deactivated. In this work, we have characterized different fractions obtained by density gradient ultracentrifugation of pulmonary surfactant purified from bronchoalveolar lavages of porcine lungs. The analysis of these fractions by transmission electron microscopy showed a heterogeneous mixture of membranous structures. With regard to composition, each fraction showed important differences in cholesterol and the content of surfactant proteins. All fractions presented a relatively good surface activity when analyzed in the captive bubble surfactometer, except for some minority fraction. These results indicate that surfactant from bronchoalveolar lavage is composed by a heterogeneous mixture of membranes differing in composition and structure. We propose that some of these structures could be associated with different stages of surfactant dynamics, including freshly secreted material, interfacial structures and recycled membranes.

Human SP-A1 Enhances Interfacial Properties of Lung Surfactant and Restores a Proper Behavior in the Presence of Inhibitory Agents

Pulmonary surfactant is a lipid-protein complex secreted by type II alveolar cells. This surface-active agent prevents alveolar collapse by reducing surface tension at the respiratory air-liquid interface during expiration and contributes to the innate defense of the lung. Surfactant protein A (SP-A) is a hydrophilic collectin protein implicated mainly in the host defense. However, it has been shown that SP-A participates in the formation of surfactant films at the interface and helps to reestablish the biophysical properties of surfactant when this is impaired by the presence of some inhibitors. There are two SP-A genes in humans, SFTPA1 and SFTPA2, which products differ in structure and function. With the aim of investigating the differences in the biophysical properties of surfactant containing human SP-A1, SP-A2 or both, we have studied 1) pulmonary surfactant from individual humanized transgenic mice expressing human SP-A1, SP-A2, or both SP-A1/2, in a Captive Bubble Surfactometer (CBS), and 2) the capability of this two variants of the protein to restore the biophysical activity of a reconstituted organic extract (OE) from native porcine surfactant in the presence of serum as inhibitory agent, mimicking a situation of lung injury. The experiments revealed consistent functional differences in surfactant containing SP-A1 or SP-A2. In the presence of serum, surfactant from mice containing SP-A1 and the OE reconstituted upon addition of SP-A1, achieved lower surface tension after post-expansion interfacial adsorption and a more efficient reorganization of the film under interfacial compression-expansion cycling conditions, than surfactants containing no SP-A or only SP-A2. We conclude that the presence of hSP-A1 allows surfactant to adopt a particularly favorable structure with optimal biophysical properties and helps to restore a proper behavior in the presence of an inhibitory agent as serum.

Human Pulmonary Surfactant Protein SP-A1 Provides Maximal Efficiency of Lung Interfacial Films

Pulmonary surfactant is a lipoprotein complex that reduces surface tension to prevent alveolar collapse and contributes to the protection of the respiratory surface from the entry of pathogens. Surfactant protein A (SP-A) is a hydrophilic glycoprotein of the collectin family, and its main function is related to host defense. However, previous studies have shown that SP-A also aids in the formation and biophysical properties of pulmonary surfactant films at the air-water interface. Humans, unlike rodents, have two genes, SFTPA1 and SFTPA2. The encoded proteins, SP-A1 and SP-A2, differ quantitatively or qualitatively in function. It has been shown that both gene products are necessary for tubular myelin formation, an extracellular structural form of lung surfactant. The goal of this study was to investigate potential differences in the biophysical properties of surfactants containing human SP-A1, SP-A2, or both. For this purpose, we have studied for the first time, to our knowledge, the biophysical properties of pulmonary surfactant from individual humanized transgenic mice expressing human SP-A1, SP-A2, or both SP-A1 and SP-A2, in the captive bubble surfactometer. We observed that pulmonary surfactant containing SP-A1 reaches lower surface tension after postexpansion interfacial adsorption than surfactants containing no SP-A or only SP-A2. Under interfacial compression-expansion cycling conditions, surfactant films containing SP-A1 also performed better, particularly with respect to the reorganization of the films that takes place during compression. On the other hand, addition of recombinant SP-A1 to a surfactant preparation reconstituted from the hydrophobic fraction of a porcine surfactant made it more resistant to inhibition by serum than the addition of equivalent amounts of SP-A2. We conclude that the presence of SP-A1 allows pulmonary surfactant to adopt a particularly favorable structure with optimal biophysical properties.

Biophisical Evaluation of Drug Impact on Pulmonary Surfactant Performance

The respiratory surface of the mammalian's lung is covered by a thin aqueous layer, and on top of it, by a lipid-protein surface active material, the pulmonary surfactant (PS). It is synthesised by type II pneumocytes and secreted in the form of multilamellar structures. Its main function is to form a film to reduce the surface tension at the air-liquid interface to values below 2mN/m, to prevent pulmonary collapse during expiration and to minimize the work during inspiration. PS has unique biophysical properties to adsorb very rapidly (in few seconds) into the air-liquid interface and, once there, to spread efficiently along it. The pulmonary surfactant system, historically considered as a barrier to drug delivery, could therefore offer novel opportunities to vehiculize different drugs efficiently, while hiding and protecting them from clearence in the lung. Nevertheless, drug impact on pulmonary surfactant performance needs to be considered in a case by case basis when different molecular entities are combined with PS.In the present work we have investigated the effect of the interaction of an anti-inflammatory steroid and an anti-tuberculosis drug with different pulmonary surfactant preparations, and how drug effects depend on PS composition. After combining properly different surfactant systems with each drug at different drug/lipid ratios, we have evaluated their impact on surfactant function and mechanical properties of surfactant layers, as assessed in a captive bubble surfactometer. Furthermore, we have used the Langmuir-Blodgett technique to prepare supported films and analyse the structure of different drug-loaded surfactant layers in order to detect structural changes associated with the impact of drugs on surfactant activity.

Cholesterol-mediated surfactant dysfunction is mitigated by surfactant protein A

The ability of pulmonary surfactant to reduce surface tension at the alveolar surface is impaired in various lung diseases. Recent animal studies indicate that elevated levels of cholesterol within surfactant may contribute to its inhibition. It was hypothesized that elevated cholesterol levels within surfactant inhibit human surfactant biophysical function and that these effects can be reversed by surfactant protein A (SP-A). The initial experiment examined the function of surfactant from mechanically ventilated trauma patients in the presence and absence of a cholesterol sequestering agent, methyl-β-cyclodextrin. The results demonstrated improved surface activity when cholesterol was sequestered in vitro using a captive bubble surfactometer (CBS). These results were explored further by reconstitution of surfactant with various concentrations of cholesterol with and without SP-A, and testing of the functionality of these samples in vitro with the CBS and in vivo using surfactant depleted rats. Overall, the results consistently demonstrated that surfactant function was inhibited by levels of cholesterol of 10% (w/w phospholipid) but this inhibition was mitigated by the presence of SP-A. It is concluded that cholesterol-induced surfactant inhibition can actively contribute to physiological impairment of the lungs in mechanically ventilated patients and that SP-A levels may be important to maintain surfactant function in the presence of high cholesterol within surfactant.

Modeling nanoparticle-alveolar epithelial cell interactions under breathing conditions using captive bubble surfactometry.

Many advances have been made in recent years in cell culture models of the epithelial barrier of the lung from simple monolayers to complex 3-D systems employing different cell types. However, the vast majority of these models still present a static air-liquid interface which is unrealistic given the dynamic nature of breathing. We present here a method where epithelial lung cells are integrated into a system, the captive bubble surfactometer, which allows the cyclical compression and expansion of the surfactant film at the air-liquid interface, thus modeling the dynamics of breathing. We found that cellular uptake of deposited gold nanoparticles was significantly increased under the dynamic (breathing) conditions of compression and expansion as compared to static conditions. The method could be very useful for studying nanoparticle-alveolar lung cell interactions under breathing conditions for applications in nanomedicine and toxicology.

Surface tension of airway aspirates withdrawn during neonatal resuscitation reflects lung maturity

The indications for treatment of neonates with exogenous pulmonary surfactant are still discussed controversially. Some premature neonates are sufficiently treated by CPAP, others need conventional ventilation and/or surfactant. The available lung maturity tests have limitations. The captive bubble surfactometer (CBS) provides measurement of surface activity from rather small amounts of surfactant. This study aimed to determine surface activity from small volume aspirates of the upper airways of neonates by means of the CBS and to correlate the results with clinical data. Small upper airway aspirates from 159 neonates (gestational age 25-42 weeks) were withdrawn and concentrated 16.7-fold by ultracentrifugation and resuspension in saline. Surface activities after 5 min of adsorption were determined in the CBS and correlated to the perinatal data (e.g., gestational age, birth weight, gender), airway interventions (like CPAP, conventional ventilation) and surfactant treatment. Additionally, 27 samples were analyzed for surfactant specific phosphatidylcholine concentrations by using electrospray ionization tandem mass-spectroscopy. Surface activities show a significant correlation to gestational age, birth weight, and the need for airway interventions. Comparing the need for airway interventions versus surface activity, a receiver operating characteristic calculated a sensitivity of 0.77 and a specificity of 0.72 at a "cut off" of 44 mN/m. Surface activity correlates significantly with the phosphatidylcholine concentrations and the latter one correlates with the gestational age. Determination of surface activity from upper airway aspirates is feasible. Further clinical studies are needed to prove the predictive value of the method.

Improvement to the surface tension reducing capability of human pulmonary surfactant with elevated levels of cholesterol via addition of surfactant protein A

Pulmonary surfactant is composed of phospholipids, neutral lipids and proteins. Surfactant's composition is important for its ability to reduce surface tensions within alveoli. Previous studies showed that serum‐protein mediated surfactant dysfunction can be overcome by the addition of surfactant protein A (SP‐A) to the material. Elevated cholesterol levels in surfactant can also cause surfactant dysfunction, but it is unknown if SP‐A can reverse this form of impairment. It was hypothesized that the addition of SP‐A to surfactant with elevated levels of cholesterol will improve the material's ability to reduce surface tension. Human surfactant was depleted of cholesterol via repeated acetone precipitation and reconstituted with 0, 5, 10 or 20% cholesterol with or without 5% SP‐A. The minimum achievable surface tension of the samples was tested on a captive bubble surfactometer. Results showed that surfactant with 10 and 20% cholesterol had significantly higher minimum surface tensions than surfactant with 0 and 5% cholesterol. Addition of SP‐A to samples with 10 and 20% cholesterol resulted in significantly lower minimum surface tension as compared to analogous samples without SP‐A. In conclusion, cholesterol mediated surfactant dysfunction can be mitigated by SP‐A. Funding was provided by Canadian Institutes for Health Research.

Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology

Medical literature regularly reports on accidental poisoning in children after aspiration of combustibles such as lamp oils which usually contain hydrocarbons or rape methyl esters (RMEs). We aimed to analyze the toxic potential of alkanes and different combustible classes in vitro with regard to biologic responses and mechanisms mediating toxicity.Two different in vitro models were used, i.e. (i) a captive bubble surfactometer (CBS) to assess direct influence of combustibles on biophysical properties of surfactant film and (ii) cell cultures (BEAS-2B and R3/1 cells, primary macrophages, re-differentiated epithelia) closely mimicking the inner lung surface. Biological endpoints included cell viability, cytotoxicity and inflammatory mediator release. CBS measurements demonstrate that combustibles affect film dynamics, i.e. the surface tension/area characteristics during compression and expansion, in a dose and molecular chain length dependent manner.Cell culture results confirm the dose dependent toxicity. Generally, cytotoxicity and cytokine release are higher in short-chained alkanes and hydrocarbon-based combustibles than in long-chained substances, e.g. highest inducible cytotoxicity in BEAS-2B was for hexane 84.6%, decane 74% and hexadecane 30.8%. Effects of RME-based combustibles differed between the cell models. Our results confirm data from animal experiments and give new insights into the mechanisms underlying the adverse health effects observed.

Segregated ordered lipid phases and protein-promoted membrane cohesivity are required for pulmonary surfactant films to stabilize and protect the respiratory surface

Pulmonary surfactant is a lipid-protein complex essential to stabilize alveoli, by forming surface active films able to reach and sustain very low surface tensions (< 2 mN m(-1)) during the film compression that occurs at end-expiration. The particular lipid composition of surfactant, including a high proportion of dipalmitoylphosphatidylcholine (DPPC), induces segregation of fluid ordered and disordered phases in surfactant membranes and films at physiological temperatures. The segregation of DPPC-enriched ordered phase has been related with the ability of surfactant films to produce very low tensions, while the presence in surfactant of two specific hydrophobic polypeptides, SP-B and SP-C, is absolutely required to facilitate surfactant dynamics, including film formation and re-spreading during expansion at inspiration. In the present study, we have used X-ray scattering to analyze the structure of (1) whole native surfactant membranes purified from porcine lungs, (2) membranes reconstituted from the organic extract of surfactant containing the full lipid complement and the physiological proportion of SP-B and SP-C, and (3) membranes reconstituted from the lipid fraction of surfactant depleted of proteins. Small angle X-ray scattering data from whole surfactant or from membranes reconstituted from surfactant organic extract indicated the co-existence of two lamellar phases with different thicknesses. Such phase coexistence disappeared upon heating of the samples at temperatures above physiological values. When assessed in a captive bubble surfactometer, which mimics interfacial compression-expansion dynamics, the ability of surfactant films to produce very low tensions is only maintained at temperatures permitting the coexistence of the two lamellar phases. On the other hand, membranes reconstituted in the absence of proteins produced diffractograms indicative of the existence of a single dominant lamellar phase at all temperatures. These data suggest that SP-B and SP-C establish membrane-membrane interactions coupling the stacks of different segregated phases. The low compressibility of surfactant films that leads to the maximal pressures (minimal tensions) is supported on one hand by the highly packed solid-like character of segregated DPPC-enriched domains and, on the other hand, by a high cohesivity of multilayered structures promoted by hydrophoblic surfactant proteins, in particular SP-B, at the more dynamic disordered membrane regions, in which SP-B selectively partitions. Cryo-electron microscopy has shown that SP-B induces formation of tight membrane-membrane contacts, a finding that supports our inference concerning the role of these surfactant proteins.