Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Disulfide bonds in the SAPA domain of the pulmonary surfactant protein B precursor.

The disulfide bonds formed in the SAPA domain of a recombinant version of the NH2-terminal propeptide (SP-BN) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through sequential digestion of SP-BN with GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cys32-Cys49 and Cys40-Cys55, and we propose a disulfide connectivity pattern of 1-3 and 2-4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z âˆ¼ 2136 and âˆ¼ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptides 24AWTTSSLACAQGPE37 and 45QALQCR50 linked by Cys32-Cys49 and 38FWCQSLE44 and 51ALGHCLQE58 linked by Cys40-Cys55 respectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1-3/2-4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-BN propeptide, the MS analysis of both digests identified the bond Cys100-Cys112, numbered 7-8, which is coincident with the bond position in the kringle motif. SIGNIFICANCE: The SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.

Silica nanoparticle exposure inhibits surfactant protein A and B in A549 cells through ROS-mediated JNK/c-Jun signaling pathway.

Exposure to silica nanoparticles (SiNPs) is related to the dysregulation of pulmonary surfactant that maintains lung stability and function. Nevertheless, there are limited studies concerning the interaction and influence between SiNPs and pulmonary surfactant, and the damage and mechanism are still unclear. Herein, we used A549 cells to develop an in vitro model, with which we investigated the effect of SiNPs exposure on the expression of pulmonary surfactant and the potential regulatory mechanism. The results showed that SiNPs were of cytotoxicity in regarding of reduced cell viability and promoted the production of excessive reactive oxygen species (ROS). Additionally, the JNK/c-Jun signaling pathway was activated, and the expression of surfactant protein A (SP-A) and surfactant protein B (SP-B) was decreased. After the cells being treated with N-acetyl-L-cysteine (NAC), we found that the ROS content was effectively downregulated, and the expression of proteins related to JNK and c-Jun signaling pathways was suppressed. In contrast, the expression of SP-A and SP-B was enhanced. Furthermore, we treated the cells with JNK inhibitor and c-Jun-siRNA and found that the expression of protein related to JNK and c-Jun signaling pathways, as well as SP-A and SP-B, changed in line with that of NAC treatment. These findings suggest that SiNPs exposure can upregulate ROS and activate the JNK/c-Jun signaling pathway in A549 cells, thereby inhibiting the expression of SP-A and SP-B proteins.

Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes.

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). We found that only one HDL protein-SFTPB (pulmonary surfactant protein B)-predicted incident CVD in all the models tested. In a fully adjusted model that controlled for lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12-4.21, P = 0.022). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker-and perhaps mediator-of CVD risk in patients with T1DM.

A first-in-human clinical study of a new SP-B and SP-C enriched synthetic surfactant (CHF5633) in preterm babies with respiratory distress syndrome: two-year outcomes.

OBJECTIVE: To assess at 24 months corrected age (CA) the neurological, respiratory, and general health status of children born prematurely from 27+0 to 33+6 weeks' gestation who were treated in a first-in-human study with a new fully synthetic surfactant (CHF5633) enriched with SP-B and SP-C proteins. OUTCOME MEASURES: Children were assessed using Bayley Scales of Infant Development (BSID), with a score below normal defined as BSID-II Mental Development Index score <70, or BSID-III cognitive composite score <85. In addition, a health status questionnaire was used to check for functional disability including respiratory problems and related treatments, sensory and neurodevelopment assessments, communication skills as well as the number of hospitalizations. RESULTS: 35 of 39 survivors had a neurodevelopmental assessment, 24 infants being evaluated by Bayley's Scales and 11 by health status questionnaires only. 23 children had scores within normal limits and one had BSID-III <85. The remaining 11 were judged clinically to have normal development. Health status questionnaires detected only issues that would normally be expected in preterm-born children. CONCLUSIONS: This assessment offers reassurance that treatment with CHF5633 surfactant was not associated with adverse neurodevelopmental, respiratory, or health outcomes by two years corrected age.

Impacts of lipopolysaccharide on fetal lung developmental maturity and surfactant protein B and surfactant protein C protein expression in gestational diabetes mellitus rats.

The rise of bioinformatics based on computer medicine provides a new method to reveal the complex biological data. This experiment is to explore the impacts of lipopolysaccharide on fetal lung developmental maturity and expressions of lung surfactant protein B (SP-B) and lung surfactant protein C (SP-C) in rats with gestational diabetes mellitus (GDM), thereby discussing the mechanism of developmental disorders in rats. Forty-eight conceived female rats were experimental subjects. Twenty-eight rats were randomly selected to construct the GDM models. All conceived rats underwent section on the 21st day of pregnancy. The ultrastructure of alveolar type II epithelial cells and the morphology of lung tissue were observed under a microscope. The protein localization and expression of SP-B and SP-C were determined by immunohistochemistry; the protein levels of SP-B and SP-C were determined by Western blot. Blood glucose and body weight of the GDM group were higher than those of the control group; the number of alveoli and alveolar area in the GDM group was lower than those in the control group; the alveolar interval in the GDM group was significantly higher than that in the control group (P < 0.05). The average absorbance of SP-B and SP-C in fetal lung tissue was significantly lower in the GDM group than that in the control group (P < 0.01). Changes in fetal lung tissue structure of rats were related to SP-B and SP-C, which was one of the main factors that affected the maturation of fetal lung tissue.

Treatment of Respiratory Distress Syndrome with Single Recombinant Polypeptides that Combine Features of SP-B and SP-C.

Treatment of respiratory distress syndrome (RDS) with surfactant replacement therapy in prematurely born infants was introduced more than 30 years ago; however, the surfactant preparations currently in clinical use are extracts from animal lungs. A synthetic surfactant that matches the currently used nature-derived surfactant preparations and can be produced in a cost-efficient manner would enable worldwide treatment of neonatal RDS and could also be tested against lung diseases in adults. The major challenge in developing fully functional synthetic surfactant preparations is to recapitulate the properties of the hydrophobic lung surfactant proteins B (SP-B) and SP-C. Here, we have designed single polypeptides that combine properties of SP-B and SP-C and produced them recombinantly using a novel solubility tag based on spider silk production. These Combo peptides mixed with phospholipids are as efficient as nature-derived surfactant preparations against neonatal RDS in premature rabbit fetuses.

Structural and functional stability of the sulfur-free surfactant protein B peptide mimic B-YL in synthetic surfactant lipids.

BACKGROUND: Optimal functionality of synthetic lung surfactant for treatment of respiratory distress syndrome in preterm infants largely depends on the quality and quantity of the surfactant protein B (SP-B) peptide mimic and the lipid mixture. B-YL peptide is a 41-residue sulfur-free SP-B mimic with its cysteine and methionine residues replaced by tyrosine and leucine, respectively, to enhance its oxidation resistance. AIM: Testing the structural and functional stability of the B-YL peptide in synthetic surfactant lipids after long-term storage. METHODS: The structural and functional properties of B-YL peptide in surfactant lipids were studied using three production runs of B-YL peptides in synthetic surfactant lipids. Each run was held at 5 °C ambient temperature for three years and analyzed with structural and computational techniques, i.e., MALDI-TOF mass spectrometry, ATR-Fourier Transform Infrared Spectroscopy (ATR-FTIR), secondary homology modeling of a preliminary B-YL structure, and tertiary Molecular Dynamic simulations of B-YL in surfactant lipids, and with functional methods, i.e., captive bubble surfactometry (CBS) and retesting in vivo surface activity in surfactant-deficient young adult rabbits. RESULTS: MALDI-TOF mass spectrometry showed no degradation of the B-YL peptide as a function of stored time. ATR-FTIR studies demonstrated that the B-YL peptide still assumed stable alpha-helical conformations in synthetic surfactant lipids. These structural findings correlated with excellent in vitro surface activity during both quasi-static and dynamic cycling on CBS after three years of cold storage and in vivo surface activity of the aged formulations with improvements in oxygenation and dynamic lung compliance approaching those of the positive control surfactant Curosurf®. CONCLUSIONS: The structure of the B-YL peptide and the in vitro and in vivo functions of the B-YL surfactant were each maintained after three years of refrigeration storage.

Immature Circulating SP-B, Bound to HDL, Represents an Early Sign of Smoke-Induced Pathophysiological Alterations.

Cigarette smoking is a major independent risk factor for cardiovascular diseases (CVD). The underlying mechanisms, however, are not clearly understood. Lungs are the primary route of exposure to smoke, with pulmonary cells and surfactant being the first structures directly exposed, resulting in the leakage of the immature proteoform of surfactant protein B (proSP-B). Herein, we evaluated whether proSP-B joined the cargo of high-density lipoprotein (HDL) proteins in healthy young subjects (n = 106) without any CVD risk factor other than smoking, and if HDL-associated proSP-B (HDL-SPB) correlated with pulmonary function parameters, systemic inflammation, and oxidative stress. At univariable analysis, HDL-SPB resulted significantly higher in smokers (2.2-fold, p < 0.001) than in non-smokers. No significant differences have been detected between smokers and non-smokers for inflammation, oxidation variables, and alveolar-capillary diffusion markers. In a multivariable model, HDL-SPB was independently associated with smoking. In conclusion, HDL-SPB is not only a precocious and sensitive index of the acute effects of smoke, but it might be also a potential causal factor in the onset of the vascular damage induced by modified HDL. These findings contribute to the emerging concept that the quality of the HDL proteome, rather than the quantity of particles, plays a central role in CVD risk protection.

Fusion Expression and Fibrinolytic Activity of rPA/SP-B.

BACKGROUND: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). OBJECTIVE: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. METHODS: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. RESULTS: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. CONCLUSION: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

Pulmonary surfactant-associated protein B regulates prostaglandin-endoperoxide synthase-2 and inflammation in chronic obstructive pulmonary disease.

NEW FINDINGS: What is the central question of this study? It is reported that polymorphism of the gene for pulmonary surfactant-associated protein B (SFTPB) is associated with chronic obstructive pulmonary disease (COPD): what are the function and mechanism of action of SFTPB in COPD? What is the main finding and its importance? Under stimulation of the risk factors of COPD, SFTPB expression is decreased, which may be involved in the formation of COPD. The progress of COPD induces an inflammatory response and reduces SFTPB expression. Levels of prostaglandin-endoperoxide synthase-2 (PTGS2) and inflammatory responses are changed by SFTPB, which indicates that SFTPB promotes the progression of COPD by PTGS2 and inflammation. ABSTRACT: Pulmonary surfactant-associated protein B (SFTPB) is a critical protein for lung homeostasis, and polymorphism of its gene is associated with chronic obstructive pulmonary disease (COPD). However, few studies have so far confirmed the functional involvement of SFTPB in COPD. Serum SFTPB and inflammatory cytokine levels were measured in 54 patients with acute exacerbation of COPD and 29 healthy controls. A549 cells were induced using 10% cigarette smoke extract (CSE) and treated with dexamethasone to investigate the effect of inflammation on SFTPB expression, and the effect of SFTPB overexpression and silencing on inflammatory cytokines was measured using real-time PCR and enzyme-linked immunosorbent assay. SFTPB expression was assessed in mouse lung tissues using immunofluorescence. Serum levels of SFTPB were significantly lower in COPD patients than in controls (P = 0.009). Conversely, levels of interleukin (IL)-6 and prostaglandin-endoperoxide synthase-2 (PTGS2) were increased in COPD patients (IL-6: P = 0.006; PTGS2: P = 0.043). After CSE treatment, SFTPB mRNA and protein levels were significantly decreased compared to controls (mRNA: P = 0.002; protein: P = 0.011), while IL-6, IL-8 and PTGS2 were elevated. Dexamethasone treatment increased SFTPB levels. Following overexpression of SFTPB in A549 cells, mRNA and protein levels of IL-6, IL-8 and PTGS2 were significantly reduced, while gene silencing induced the opposite effect. SFTPB levels were significantly reduced in the lung tissue of a mouse model of COPD compared to controls. Reduced SFTPB levels may induce PTGS2 and inflammatory responses in COPD and SFTPB could be a key protein for evaluation of COPD progression.

Surfactant Protein B Promotes Cytosolic SiRNA Delivery by Adopting a Virus-like Mechanism of Action.

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an in vitro non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy.

Impact of synthetic surfactant CHF5633 with SP-B and SP-C analogues on lung function and inflammation in rabbit model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with diffuse inflammation, alveolar epithelial damage, and leakage of plasma proteins into the alveolar space, which together contribute to inactivation of pulmonary surfactant and respiratory failure. Exogenous surfactant delivery is therefore considered to hold potential for ARDS treatment, but clinical trials with natural derived surfactant or synthetic surfactant containing a surfactant protein C (SP-C) analogue have been negative. Synthetic surfactant CHF5633, containing analogues of SP-B and SP-C, may be effective against ARDS. The aim here was to compare treatment effects of CHF5633 and animal-derived surfactant poractant alfa in animal model of ARDS. ARDS was induced in adult New Zealand rabbits by mild lung lavages followed by injurious ventilation until respiratory failure (P/F ratio <26.7 kPa). The animals were then treated with intratracheal bolus of 200 mg/kg CHF5633 or poractant alfa (Curosurf® ), or air as control. The animals were subsequently ventilated for an additional 4 hr and respiratory parameters were recorded regularly. Postmortem, histological analysis, degree of lung edema, and levels of the cytokines TNFα, IL-6, and IL-8 in lung homogenates were evaluated. Both surfactant preparations improved lung function, reduced the levels of pro-inflammatory cytokines, and degree of lung edema to very similar degrees versus the controls. No significant differences in any of the analyzed parameters were observed between the CHF5633- and poractant alfa-treated groups. This study indicates that single dose of CHF5633 improves lung function and attenuates inflammation as effectively as poractant alfa in experimental ARDS caused by injurious ventilation.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Synthetic Surfactant CHF5633 Compared with Poractant Alfa in the Treatment of Neonatal Respiratory Distress Syndrome: A Multicenter, Double-Blind, Randomized, Controlled Clinical Trial.

OBJECTIVE: To compare efficacy and safety of a new synthetic surfactant, CHF5633, enriched with surfactant proteins, SP-B and SP-C peptide analogues, with porcine surfactant, poractant alfa, for the treatment of respiratory distress syndrome in infants born preterm. STUDY DESIGN: Neonates born preterm on respiratory support requiring fraction of inspired oxygen (FiO2) ≥0.30 from 240/7 to 266/7 weeks and FiO2 ≥0.35 from 270/7 to 296/7 weeks of gestation to maintain 88%-95% oxygen saturation were randomized to receive 200 mg/kg of CHF5633 or poractant alfa. If necessary, redosing was given at 100 mg/kg. Efficacy end points were oxygen requirement (FiO2, respiratory severity score [FiO2 × mean airway pressure]) in the first 24 hours, 7 and 28 days, discharge home, and/or 36 weeks of postmenstrual age; mortality and bronchopulmonary dysplasia at 28 days and 36 weeks of PMA. Adverse events and immunogenicity were monitored for safety. RESULTS: Of the 123 randomized neonates, 113 were treated (56 and 57 in CHF5633 and poractant alfa groups, respectively). In both arms, FiO2 and respiratory severity score decreased from baseline at all time points (P < .001) with no statistically significant differences between groups. Rescue surfactant use (19 [33.9%] vs 17 [29.8%]), bronchopulmonary dysplasia (31 [55.4%] and 32 [56.1%]), and mortality at day 28 (4 [7.1%] and 3 [5.3%]) were similar in the CHF5633 and poractant alfa groups, respectively. In 2 (3.4%) and 1 (1.7%) neonates, adverse drug reactions were reported in CHF5633 and poractant alfa groups, respectively. No immunogenicity was detected. CONCLUSIONS: Treatment with CHF5633 showed similar efficacy and safety as poractant alfa in neonates born preterm with moderate-to-severe respiratory distress syndrome. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02452476.

Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy.

BACKGROUND: Administration of antenatal steroids is standard of care for women assessed to be at imminent risk of preterm delivery. There is a marked variation in antenatal steroid dosing strategy, selection for treatment criteria, and agent choice worldwide. This, combined with very limited optimization of antenatal steroid use per se, means that treatment efficacy is highly variable, and the rate of respiratory distress syndrome is decreased to perhaps as low as 40%. In some cases, antenatal steroid use is associated with limited benefit and potential harm. OBJECTIVE: We hypothesized that individual differences in maternofetal steroid exposure would contribute to observed variability in antenatal steroid treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between maternofetal steroid exposure and antenatal steroid treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. STUDY DESIGN: Ewes carrying a single fetus underwent surgery to catheterize a fetal and maternal jugular vein at 119 days' gestation. Animals recovered for 24 hours before being randomized to either (1) a single maternal intramuscular injection of 2 mL saline (negative control group, n=10) or (2) a single maternal intramuscular injection of 0.25 mg/kg betamethasone phosphate plus acetate (antenatal steroid group, n=20). Serial maternal and fetal plasma samples were collected from each animal after 48 hours before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. RESULTS: One animal from the control group and one animal from the antenatal steroid group did not complete their treatment protocol and were removed from analyses. Animals in the antenatal steroid group were divided into a responder subgroup (n=12/19) and a nonresponder subgroup (n=7/19) using a cutoff of partial pressure of arterial CO2 at 30-minute ventilation within 2 standard deviations of the mean value from saline-treated negative control group animals. Although antenatal steroid improved fetal lung maturation in the undivided antenatal steroid group and in the responder subgroup both physiologically (blood gas- and ventilation-related data) and biochemically (messenger ribonucleic acid expression related to fetal lung maturation), these values did not improve relative to saline-treated control group animals in the antenatal steroid nonresponder subgroup. No differences in betamethasone distribution, clearance, or protein binding were identified between the antenatal steroid responder and nonresponder subgroups. CONCLUSION: This study correlated individual maternofetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation that was not significantly different to saline-treated control group animals. These nonresponsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of antenatal steroid therapy is not solely determined by maternofetal drug levels and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid signaling.

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 novel in vitro model of primary human pediatric lung epithelial cells.

BACKGROUND: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. METHODS: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. RESULTS: PHLE cells can be expanded in culture up to passage 6, with a doubling time of ~4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B, and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. CONCLUSION: Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.