A New ABCA3 Gene Mutation c.3445G>A (p.Asp1149Asn) as a Causative Agent of Newborn Lethal Respiratory Distress Syndrome.

Mutations in adenosine triphosphate-binding cassette transporter A3 (ABCA3) (OMIM: 601615) gene constitute the most frequent genetic cause of severe neonatal respiratory distress syndrome (RDS) and interstitial lung disease (ILD) in children. Interstitial lung disease in children and especially in infants, in contrast to adults, is more likely to appear as a result of developmental deficits or is characterized by genetic aberrations of pulmonary surfactant homeostasis not responding to exogenous surfactant administration. The underlying ABCA3 gene mutations are commonly thought, regarding null mutations, to determine the clinical course of the disease while there exist mutation types, especially missense variants, whose effects on surfactant proteins are difficult to predict. In addition, clinical and radiological signs overlap with those of surfactant proteins B and C mutations making diagnosis challenging. We demonstrate a case of a one-term newborn male with lethal respiratory failure caused by homozygous missense ABCA3 gene mutation c.3445G>A (p.Asp1149Asn), which, to our knowledge, was not previously reported as a causative agent of newborn lethal RDS. Therapeutic strategies for patients with ABCA3 gene mutations are not sufficiently evidence-based. Therefore, the description of the clinical course and treatment of the disease in terms of a likely correlation between genotype and phenotype is crucial for the development of the optimal clinical approach for affected individuals.

Biophysical Activity of Impaired Lung Surfactant upon Exposure to Polymer Nanoparticles.

Colloidal drug carriers could improve the therapy of numerous airway diseases. However, it remains unclear to what extent nanoscale particulate matter affects the biophysical function of the essential surface-active lining layer of the lungs, especially under predisposed conditions of airway diseases. Accordingly, the current study investigated the impact of defined polymer nanoparticles on impaired lung surfactants. Admixtures of plasma proteins (albumin and fibrinogen) to Curosurf led to a controllable decrease in surface activity (i.e., adsorption and minimal surface tension of >25 and >5 mN/m, respectively), which served as models for dysfunctional lung surfactants. Next, Curosurf preincubated with plasma proteins was challenged with negatively- and positively charged poly(lactide) nanoparticles. Negatively charged nanoparticles significantly perturbed the biophysical function of impaired Curosurf in a dose-dependent manner, most-likely due to a binding of essential surfactant components. By contrast, addition of positively charged nanoparticles led to no further loss of surface activity, but a remarkable depletion of plasma protein content. Once adsorbed to the surface of polymer nanoparticles, plasma proteins were hindered to displace relevant surfactant components from the air/liquid interface. Overall, the current study indicated that, depending on their physicochemical properties, colloidal drug carriers could compromise the biophysical function of impaired lung surfactants. Notably, a positive surface charge represents a parameter for the rationale design of polymer nanomedicines causing negligible adverse events on an impaired surface-active lining layer in the lungs.

Overcoming inactivation of the lung surfactant by serum proteins: a potential role for fluorocarbons?

In many pulmonary conditions serum proteins interfere with the normal adsorption of components of the lung surfactant to the surface of the alveoli, resulting in lung surfactant inactivation, with potentially serious untoward consequences. Here, we review the strategies that have recently been designed in order to counteract the biophysical mechanisms of inactivation of the surfactant. One approach includes protein analogues or peptides that mimic the native proteins responsible for innate resistance to inactivation. Another perspective uses water-soluble additives, such as electrolytes and hydrophilic polymers that are prone to enhance adsorption of phospholipids. An alternative, more recent approach consists of using fluorocarbons, that is, highly hydrophobic inert compounds that were investigated for partial liquid ventilation, that modify interfacial properties and can act as carriers of exogenous lung surfactant. The latter approach that allows fluidisation of phospholipid monolayers while maintaining capacity to reach near-zero surface tension definitely warrants further investigation.

Exposure to polymers reverses inhibition of pulmonary surfactant by serum, meconium, or cholesterol in the captive bubble surfactometer.

Dysfunction of pulmonary surfactant in the lungs is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome. Serum, cholesterol, and meconium have been described as inhibitory agents of surfactant's interfacial activity once these substances appear in alveolar spaces during lung injury and inflammation. The deleterious action of these agents has been only partly evaluated under physiologically relevant conditions. We have optimized a protocol to assess surfactant inhibition by serum, cholesterol, or meconium in the captive bubble surfactometer. Specific measures of surface activity before and after native surfactant was exposed to inhibitors included i), film formation, ii), readsorption of material from surface-associated reservoirs, and iii), interfacial film dynamics during compression-expansion cycling. Results show that serum creates a steric barrier that impedes surfactant reaching the interface. A mechanical perturbation of this barrier allows native surfactant to compete efficiently with serum to form a highly surface-active film. Exposure of native surfactant to cholesterol or meconium, on the other hand, modifies the compressibility of surfactant films though optimal compressibility properties recover on repetitive compression-expansion cycling. Addition of polymers like dextran or hyaluronic acid to surfactant fully reverses inhibition by serum. These polymers also prevent surfactant inhibition by cholesterol or meconium, suggesting that the protective action of polymers goes beyond the mere enhancement of interfacial adsorption as described by depletion force theories.

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum.

Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries.

Inhibitors of inflammation and endogenous surfactant pool size as modulators of lung injury with initiation of ventilation in preterm sheep.

BACKGROUND: Increased pro-inflammatory cytokines in tracheal aspirates correlate with the development of BPD in preterm infants. Ventilation of preterm lambs increases pro-inflammatory cytokines and causes lung inflammation. OBJECTIVE: We tested the hypothesis that selective inhibitors of pro-inflammatory signaling would decrease lung inflammation induced by ventilation in preterm newborn lambs. We also examined if the variability in injury response was explained by variations in the endogenous surfactant pool size. METHODS: Date-mated preterm lambs (n = 28) were operatively delivered and mechanically ventilated to cause lung injury (tidal volume escalation to 15 mL/kg by 15 min at age). The lambs then were ventilated with 8 mL/kg tidal volume for 1 h 45 min. Groups of animals randomly received specific inhibitors for IL-8, IL-1, or NF-κB. Unventilated lambs (n = 7) were the controls. Bronchoalveolar lavage fluid (BALF) and lung samples were used to quantify inflammation. Saturated phosphatidylcholine (Sat PC) was measured in BALF fluid and the data were stratified based on a level of 5 µmol/kg (~8 mg/kg surfactant). RESULTS: The inhibitors did not decrease the cytokine levels or inflammatory response. The inflammation increased as Sat PC pool size in BALF decreased. Ventilated lambs with a Sat PC level > 5 µmol/kg had significantly decreased markers of injury and lung inflammation compared with those lambs with < 5 µmol/kg. CONCLUSION: Lung injury caused by high tidal volumes at birth were decreased when endogenous surfactant pool sizes were larger. Attempts to decrease inflammation by blocking IL-8, IL-1 or NF-κB were unsuccessful.

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.

A comparative study of mechanisms of surfactant inhibition.

Pulmonary surfactant spreads to the hydrated air-lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air-lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air-water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration.

Current perspectives in pulmonary surfactant--inhibition, enhancement and evaluation.

Pulmonary surfactant (PS) is a complicated mixture of approximately 90% lipids and 10% proteins. It plays an important role in maintaining normal respiratory mechanics by reducing alveolar surface tension to near-zero values. Supplementing exogenous surfactant to newborns suffering from respiratory distress syndrome (RDS), a leading cause of perinatal mortality, has completely altered neonatal care in industrialized countries. Surfactant therapy has also been applied to the acute respiratory distress syndrome (ARDS) but with only limited success. Biophysical studies suggest that surfactant inhibition is partially responsible for this unsatisfactory performance. This paper reviews the biophysical properties of functional and dysfunctional PS. The biophysical properties of PS are further limited to surface activity, i.e., properties related to highly dynamic and very low surface tensions. Three main perspectives are reviewed. (1) How does PS permit both rapid adsorption and the ability to reach very low surface tensions? (2) How is PS inactivated by different inhibitory substances and how can this inhibition be counteracted? A recent research focus of using water-soluble polymers as additives to enhance the surface activity of clinical PS and to overcome inhibition is extensively discussed. (3) Which in vivo, in situ, and in vitro methods are available for evaluating the surface activity of PS and what are their relative merits? A better understanding of the biophysical properties of functional and dysfunctional PS is important for the further development of surfactant therapy, especially for its potential application in ARDS.

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A.

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.

STAT3 regulates ABCA3 expression and influences lamellar body formation in alveolar type II cells.

ATP-Binding Cassette A3 (ABCA3) is a lamellar body associated lipid transport protein required for normal synthesis and storage of pulmonary surfactant in type II cells in the alveoli. In this study, we demonstrate that STAT3, activated by IL-6, regulates ABCA3 expression in vivo and in vitro. ABCA3 mRNA and immunostaining were decreased in adult mouse lungs in which STAT3 was deleted from the respiratory epithelium (Stat3(Delta/Delta) mice). Consistent with the role of STAT3, intratracheal IL-6 induced ABCA3 expression in vivo. Decreased ABCA3 and abnormalities in the formation of lamellar bodies, the intracellular site of surfactant lipid storage, were observed in Stat3(Delta/Delta) mice. Expression of SREBP1a and 1c, SCAP, ABCA3, and AKT mRNAs was inhibited by deletion of Stat3 in type II cells isolated from Stat3(Delta/Delta) mice. The activities of PI3K and AKT were required for normal Abca3 gene expression in vitro. AKT activation induced SREBP expression and increased the activity of the Abca3 promoter in vitro, consistent with the role of STAT3 signaling, at least in part via SREBP, in the regulation of ABCA3. ABCA3 expression is regulated by IL-6 in a pathway that includes STAT3, PI3K, AKT, SCAP, and SREBP. Activation of STAT3 after exposure to IL-6 enhances ABCA3 expression, which, in turn, influences pulmonary surfactant homeostasis.

Peripheral cell wall lipids of Mycobacterium tuberculosis are inhibitory to surfactant function.

The transmission of Mycobacterium tuberculosis (TB) requires extensive damage to the lungs to facilitate bacterial release into the airways, and it is therefore likely that the microorganism has evolved mechanisms to exacerbate its local pathology. This study examines the inhibitory effects of lipids extracted and purified chromatographically from TB on the surface-active function of lavaged bovine lung surfactant (LS) and a clinically relevant calf lung surfactant extract (CLSE). Total lipids from TB greatly inhibited the surface activity of LS and CLSE on the pulsating bubble surfactometer at physical conditions applicable for respiration in vivo (37 degrees C, 20 cycles/min, 50% area compression). Minimum surface tensions for LS (0.5 mg/ml) and CLSE (1 mg/ml) were raised from <1 mN/m to 15.7+/-1.2 and 18.7+/-1.3 mN/m after 5 min of bubble pulsation in the presence of total TB lipids (0.15 mg/ml). TB mixed waxes (0.15 mg/ml) and TB trehalose monomycolates (TMMs, 0.15 mg/ml) also significantly inhibited the surface activity of LS and CLSE (minimum surface tensions of 10-16 mN/m after 5 min of bubble pulsation), as did purified trehalose 6,6'-dimycolate (TDM, cord factor). Phosphatidylinositol mannosides (PIMs, 0.15 mg/ml) from TB had no inhibitory effect on the surface activity of LS or CLSE. Concentration dependence studies showed that LS was also inhibited significantly by total TB lipids at 0.075 mg/ml, with a smaller activity decrease apparent even at 0.00375 mg/ml. These findings document that TB contains multiple lipids that can directly impair the biophysical function of endogenous and exogenous lung surfactants. Direct inhibition by TB lipids could worsen surfactant dysfunction caused by plasma proteins or other endogenous substances induced by inflammatory injury in the infected lungs. TB lipids could also inhibit the effectiveness of exogenous surfactants used to treat severe acute respiratory failure in TB patients meeting criteria for clinical acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS).

Bile salt enhancers for inhalation: Correlation between in vitro and in vivo lung effects.

The lungs have potential as a means of systemic drug delivery of macromolecules. Systemic delivery requires crossing of the air-blood barrier, however with molecular size-dependent limitations in lung absorption of large molecules. Systemic availability after inhalation can be improved by absorption enhancers, such as bile salts. Enhancers may potentially interfere with the different constituents of the lungs, e.g. the lung surfactant lining the alveoli or the lung epithelium. We used two in vitro models to investigate the potential effects of bile salts on lung surfactant function (with the constrained drop surfactometer) and on the epithelium in the proximal airways (with the MucilAir™ cell system), respectively. In addition, we measured direct effects on respiration in mice inhaling bile salt aerosols. The bile salts inhibited lung surfactant function at different dose levels, however they did not affect the integrity of ciliated cells at the tested doses. Furthermore, the bile salt aerosols induced changes in the breathing pattern of mice indicative of pulmonary irritation. The bile salts were ranked according to potency in vitro for surfactant function disruption and in vivo for induction of pulmonary irritation. The ranking was the same, suggesting a correlation between the interference with lung surfactant and the respiratory response.

Dihydrotestosterone inhibits fetal rabbit pulmonary surfactant production.

Males have a higher morbidity and mortality for neonatal respiratory distress syndrome (RDS) than females, and respond less well to hormone therapy designed to prevent RDS by stimulating fetal pulmonary surfactant production. We have shown that male fetuses exhibit delayed production of pulmonary surfactant. We tested the hypothesis that the sex difference in fetal pulmonary surfactant production is under hormonal control. Pulmonary surfactant was measured as the saturated phosphatidylcholine/sphingomyelin ratio (SPC/S) in the lung lavage of fetal rabbits at 26 d gestation. There was an association between the sex of neighboring fetuses and the SPC/S ratio of the female fetuses, such that with one or two male neighbors, respectively, females had decreasing SPC/S ratios (P < 0.05). We injected dihydrotestosterone (DHT) into pregnant does from day 12 through day 26 of gestation in doses of 0.1, 1.0, 10, and 25 mg/d, and measured the SPC/S ratio in fetal lung lavage on day 26. In groups with the normal sex difference in fetal serum androgen levels (controls, 0.1 mg DHT/d) the normal sex difference in the SPC/S ratio was also present (females > males, P = 0.03). In the 1-mg/d group there was no sex difference in androgen levels and the sex difference in the SPC/S ratio was also eliminated as the female values were lowered to the male level. Higher doses of DHT (10, 25 mg/d) further reduced the SPC/S ratios. We injected the anti-androgen Flutamide (25 mg/d) from day 12 through day 26 of gestation. This treatment eliminated the normal sex difference in the lung lavage SPC/S ratio by increasing the male ratios to that of the females. We conclude that androgens inhibit fetal pulmonary surfactant production. An understanding of the mechanism of the sex difference in surfactant production may allow development of therapy that is as effective in males as in females for preventing RDS.

Pulmonary surfactant and bacterial lipopolysaccharide: the interaction and its functional consequences.

The respiratory system is constantly exposed to pathogens which enter the lungs by inhalation or via blood stream. Lipopolysaccharide (LPS), also named endotoxin, can reach the airspaces as the major component of the outer membrane of Gram-negative bacteria, and lead to local inflammation and systemic toxicity. LPS affects alveolar type II (ATII) cells and pulmonary surfactant and although surfactant molecule has the effective protective mechanisms, excessive amount of LPS interacts with surfactant film and leads to its inactivation. From immunological point of view, surfactant specific proteins (SPs) SP-A and SP-D are best characterized, however, there is increasing evidence on the involvement of SP-B and SP-C and certain phospholipids in immune reactions. In animal models, the instillation of LPS to the respiratory system induces acute lung injury (ALI). It is of clinical importance that endotoxin-induced lung injury can be favorably influenced by intratracheal instillation of exogenous surfactant. The beneficial effect of this treatment was confirmed for both natural porcine and synthetic surfactants. It is believed that the surfactant preparations have anti-inflammatory properties through regulating cytokine production by inflammatory cells. The mechanism by which LPS interferes with ATII cells and surfactant layer, and its consequences are discussed below.

Lysophospholipid and fatty acid inhibition of pulmonary surfactant: non-enzymatic models of phospholipase A2 surfactant hydrolysis.

Secretory A(2) phospholipases (sPLA(2)) hydrolyze surfactant phospholipids cause surfactant dysfunction and are elevated in lung inflammation. Phospholipase-mediated surfactant hydrolysis may disrupt surfactant function by generation of lysophospholipids and free fatty acids and/or depletion of native phospholipids. In this study, we quantitatively assessed multiple mechanisms of sPLA(2)-mediated surfactant dysfunction using non-enzymatic models including supplementation of surfactants with exogenous lysophospholipids and free fatty acids. Our data demonstrated lysophospholipids at levels >or=10 mol% of total phospholipid (i.e., >or=10% hydrolysis) led to a significant increase in minimum surface tension and increased the time to achieve a normal minimum surface tension. Lysophospholipid inhibition of surfactant function was independent of the lysophospholipid head group or total phospholipid concentration. Free fatty acids (palmitic acid, oleic acid) alone had little effect on minimum surface tension, but did increase the maximum surface tension and the time to achieve normal minimum surface tension. The combined effect of equimolar free fatty acids and lysophospholipids was not different from the effect of lysophospholipids alone for any measurement of surfactant function. Surfactant proteins did not change the percent lysophospholipids required to increase minimum surface tension. As a mechanism that causes surfactant dysfunction, depletion of native phospholipids required much greater change (equivalent to >80% hydrolysis) than generation of lysophospholipids. In summary, generation of lysophospholipids is the principal mechanism of phospholipase-mediated surfactant injury in our non-enzymatic models. These models and findings will assist in understanding more complex in vitro and in vivo studies of phospholipase-mediated surfactant injury.

Pulmonary surfactant function is abolished by an elevated proportion of cholesterol.

A molecular film of pulmonary surfactant strongly reduces the surface tension of the lung epithelium-air interface. Human pulmonary surfactant contains 5-10% cholesterol by mass, among other lipids and surfactant specific proteins. An elevated proportion of cholesterol is found in surfactant, recovered from acutely injured lungs (ALI). The functional role of cholesterol in pulmonary surfactant has remained controversial. Cholesterol is excluded from most pulmonary surfactant replacement formulations, used clinically to treat conditions of surfactant deficiency. This is because cholesterol has been shown in vitro to impair the surface activity of surfactant even at a physiological level. In the current study, the functional role of cholesterol has been re-evaluated using an improved method of evaluating surface activity in vitro, the captive bubble surfactometer (CBS). Cholesterol was added to one of the clinically used therapeutic surfactants, BLES, a bovine lipid extract surfactant, and the surface activity evaluated, including the adsorption rate of the substance to the air-water interface, its ability to produce a surface tension close to zero and the area compression needed to obtain that low surface tension. No differences in the surface activity were found for BLES samples containing either none, 5 or 10% cholesterol by mass with respect to the minimal surface tension. Our findings therefore suggest that the earlier-described deleterious effects of physiological amounts of cholesterol are related to the experimental methodology. However, at 20%, cholesterol effectively abolished surfactant function and a surface tension below 15 mN/m was not obtained. Inhibition of surface activity by cholesterol may therefore partially or fully explain the impaired lung function in the case of ALI. We discuss a molecular mechanism that could explain why cholesterol does not prevent low surface tension of surfactant films at physiological levels but abolishes surfactant function at higher levels.

Morphological alterations of exogenous surfactant inhibited by meconium can be prevented by dextran.

BACKGROUND: Surfactant dysfunction due to inhibition is involved in the pathophysiology of meconium aspiration syndrome. Dextran addition has been shown to reverse exogenous surfactant inactivation by meconium, but the precise mechanisms and the morphological correlate of this effect are yet unknown. Morphological surfactant analysis by transmission electron microscopy (TEM) and stereology allows the differentiation of active (large aggregates = LA) and inactive (small aggregates = SA) subtypes. METHODS: To determine the in vitro effects of meconium and dextran addition on the morphology of a modified porcine natural surfactant (Curosurf), Curosurf samples were either incubated alone or together with meconium or with meconium and dextran, fixed and processed for TEM. Volume fractions of surfactant subtypes [lamellar body-like forms (LBL), multilamellar vesicles (MV), unilamellar vesicles (UV)] were determined stereologically. RESULTS: All preparations contained LBL and MV (corresponding to LA) as well as UV (corresponding to SA). The volume fraction of UV increased with addition of meconium and decreased with further addition of dextran. Correspondingly, the UV/(LBL+MV) ratio (resembling the SA/LA ratio) increased when meconium was added and decreased when dextran was added to the surfactant-meconium mixture. CONCLUSION: Meconium causes alterations in the ultrastructural composition of Curosurf that can be visualized and analyzed by TEM and stereology. These alterations resemble an increase in the SA/LA ratio and are paralleled by an increase in minimum surface tension. Dextran prevents these effects and may therefore be a useful additive to exogenous surfactant preparations to preserve their structural and functional integrity, thereby improving their resistance to inactivation.

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C.

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.

Reversible and irreversible inactivation of preformed pulmonary surfactant surface films by changes in subphase constituents.

Several mechanisms for surfactant inactivation have been reported. In this study, we have measured the reversibility of surfactant inactivation caused by various lipid or protein constituents of plasma or by pH changes. A surfactant of bovine origin was studied in a pulsating surfactometer either when surfactants were premixed with different serum constituents (inactivators) or when inactivators were introduced into subphase fluid surrounding surfactant films formed at an air-liquid interface. Subphase exchanges with sodium bicarbonate or sodium borate raised pH and raised minimal surface tensions either when premixed with surfactant or when introduced with saline subphase beneath a preformed surfactant surface film. The pH effects on surfactant film function were reversible for sodium bicarbonate but not for sodium borate when the subphase with bicarbonate or borate was replaced with saline. Lipids (platelet-activating factor or lysophosphatidylcholine) had non-reversible effects on preformed films. Proteins (fibrinogen or C reactive protein) had reversible effects at low concentrations, but reversibility was less evident at high concentrations. Effects with whole serum were non-reversible at low protein concentrations (0.5 mg/ml). These results add evidence that surfactant inactivation can be caused by multiple mechanisms, both reversible and irreversible.