Bovine Pulmonary Surfactant Research Articles

Bovine pulmonary surfactant alleviates inflammation and epithelial cell apoptosis in the early phase of lipopolysaccharide-induced acute lung injury in rats

ABSTRACT We investigate the impact of bovine pulmonary surfactant (PS) on LPS-induced ALI in vitro and in vivo to improve recognition and prevent mortality in sepsis-induced ALI. Primary alveolar type II (AT2) cells were treated with LPS alone or in combination with PS. Cell morphology observation, CCK-8 proliferation assay, flow cytometry apoptosis assay, and ELISA for inflammatory cytokine levels were performed at different time points after treatment. An LPS-induced ALI rat model was established and treated with vehicle or PS. Lung wet/dry weight ratio, histopathological changes, lung function parameters, and serum inflammatory cytokine levels were examined 6 h after PS treatment. Survival analysis by Kaplan-Meier method. RNA sequencing was conducted to identify LPS-induced differentially expressed genes in rat lungs. Proapoptotic gene expression in rat lungs was determined by Western blot. LPS significantly inhibited cell proliferation while promoting apoptosis of AT2 cells starting 2 h after treatment, accompanied by a significant increase in inflammatory cytokine production; PS reversed these effects. PS decreased the lung wet/dry ratio in septic rats, histological abnormalities, alterations in lung function parameters, and inflammatory cytokines production; while improving the overall survival of rats. LPS-induced differentially expressed genes were closely associated with apoptosis. PS attenuated LPS-induced upregulation of proapoptotic gene expression starting 2 h after treatment in AT2 cells while restoring lung ATPase activity in vivo. Bovine PS alleviates LPS-induced ALI in the early phase, possibly by suppressing inflammation and AT2 cell apoptosis, as a preemptive therapeutic agent for managing sepsis-induced ALI.

1044. In vitro Activity of Tebipenem Against Relevant Clinical Isolates in the Presence of Pulmonary Surfactant

BackgroundTebipenem (TBP) is an orally administered broad-spectrum carbapenem antibiotic under development for the treatment of acute pyelonephritis and complicated urinary tract infections. This study evaluated the effect of bovine pulmonary surfactant (BPS) on the in vitro activity of TBP and ertapenem (ETP) against a recent collection of clinical isolates.MethodsA total of 10 isolates recovered from patients with infections in 2018 were tested for antimicrobial susceptibility to TBP and ETP in the absence or presence of 1%, 5%, or 10% BPS (Infasurf; ONY Biotech). These isolates included the following species: C. freundii, E. cloacae, E. coli, H. influenzae, H. parainfluenzae, K. pneumoniae, methicillin-susceptible S. aureus, M. catarrhalis, S. pneumoniae, and S. pyogenes. Isolates were tested with the appropriate broth microdilution method for each organism as specified by CLSI. For most organisms, MICs were determined in cation-adjusted Mueller-Hinton broth (CAMHB). CAMHB was supplemented with 2.5-5% lysed horse blood for streptococci and Haemophilus Test Medium broth for Haemophilus spp. Daptomycin (DAP) was tested against S. aureus ATCC 29213 as a positive control.ResultsAll isolates displayed TBP MIC values ranging from ≤0.004 to 0.06 mg/L in media without BPS. There were no observed MIC increases >2-fold in the presence of BPS. 4 of the 10 isolates displayed slightly higher (≥4-fold) ETP than TBP MIC values. The ETP MIC values ranged from 0.015-0.25 mg/L in media without BPS. Similarly, there were no observed instances of a >2-fold shift toward lower potency in the presence of BPS. For both TBP and ETP, MIC endpoint values were easily determined, except for in the case of the 2 Haemophilus strains growing in the presence of 5% or 10% BPS. For these conditions, resazurin was added to establish an MIC value. The MIC values found with this method did not differ from the MIC values found in either HTM media or HTM media with 1% BPS. As expected, the addition of BPS shifted DAP S. aureus MIC values to >8 mg/L for all 3 BPS concentrations.ConclusionTBP displayed potent activity against all isolates tested, as all observed MIC values were ≤0.06 mg/L. The addition of BPS to the testing medium did not affect the in vitro MIC values of TBP or ETP against these species.Disclosures S J Ryan Arends, PhD, AbbVie (formerly Allergan) (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Abby L. Klauer, n/a, Cidara Therapeutics, Inc. (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Nicole Cotroneo, Spero Therapeutics (Employee, Shareholder) Ian A. Critchley, Ph.D., Spero Therapeutics (Employee, Shareholder) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)

Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes.

Influenza and RSV are human viruses responsible for outbreaks in hospitals, long-term care facilities and nursing homes. The present study assessed an air treatment using ozone at two relative humidity conditions (RHs) in order to reduce the infectivity of airborne influenza. Bovine pulmonary surfactant (BPS) and synthetic tracheal mucus (STM) were used as aerosols protectants to better reflect the human aerosol composition. Residual ozone concentration inside the aerosol chamber was also measured. RSV's sensitivity resulted in testing its resistance to aerosolization and sampling processes instead of ozone exposure. The results showed that without supplement and with STM, a reduction in influenza A infectivity of four orders of magnitude was obtained with an exposure to 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min. Consequently, ozone could be considered as a virucidal disinfectant for airborne influenza A. RSV did not withstand the aerosolization and sampling processes required for the use of the experimental setup. Therefore, ozone exposure could not be performed for this virus. Nonetheless, this study provides great insight for the efficacy of ozone as an air treatment for the control of nosocomial influenza A outbreaks.

Exebacase Is Active In Vitro in Pulmonary Surfactant and Is Efficacious Alone and Synergistic with Daptomycin in a Mouse Model of Lethal Staphylococcus aureus Lung Infection.

ABSTRACTExebacase (CF-301) is a novel antistaphylococcal lysin (cell wall hydrolase) in phase 3 of clinical development for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis, used in addition to standard-of-care antibiotics. In the current study, the potential for exebacase to treat S. aureus pneumonia was explored in vitro using bovine pulmonary surfactant (Survanta) and in vivo using a lethal murine pneumonia model. Exebacase was active against a set of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains, with an MIC90 of 2 μg/ml (n = 18 strains), in the presence of a surfactant concentration (7.5%) inhibitory to the antistaphylococcal antibiotic daptomycin, which is inactive in pulmonary environments due to specific inhibition by surfactant. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, exebacase synergized with daptomycin in the presence of surfactant in vitro, resulting in daptomycin MIC reductions of up to 64-fold against 9 MRSA and 9 MSSA strains. Exebacase was also observed to facilitate the binding of daptomycin to S. aureus and the elimination of biofilm-like structures formed in the presence of surfactant. Exebacase (5 mg/kg of body weight 1 time every 24 h [q24h], administered intravenously for 3 days) was efficacious in a murine model of staphylococcal pneumonia, resulting in 50% survival, compared to 0% survival with the vehicle control; exebacase in addition to daptomycin (50 mg/kg q24h for 3 days) resulted in 70% survival, compared to 0% survival in the daptomycin-alone control group. Overall, exebacase is active in pulmonary environments and may be appropriate for development as a treatment for staphylococcal pneumonia.

The Anionic Phospholipids of Bovine Pulmonary Surfactant.

The only known compositional change in the phospholipids (PL) of pulmonary surfactant in response to a physiologic stimulus occurs around the time of birth. In most species, the predominant anionic PL changes from phosphatidylinositol (PtdIns) to phosphatidylglycerol (PtdGro). Because prior studies have shown that the change in the headgroup itself is functionally insignificant, we tested the hypothesis that the PtdIns and PtdGro contain different diacyl pairs. Experiments used electrospray-ionization mass spectrometry to determine the molecular species in PtdIns, PtdGro, and phosphatidylcholine (PtdCho) in surfactant from newborn calves and cows. The profiles for the two anionic PL were distinct. The PtdIns contained long, unsaturated fatty acid chains and no disaturated species. The PtdGro more closely resembled the profile from PtdCho. For each headgroup, the molecular species for calf and cow were similar. The differences between the two anionic PL indicate that the switch from PtdIns to PtdGro during maturation involves more than simple substitution of the headgroup, and suggest that the functional significance of the shift may reflect the different pool of diacyl pairs.

Risk factors for minimally invasive surfactant administration failure in preterm infants with respiratory distress syndrome

To identify risk factors for minimally invasive surfactant administration (MISA) failure in the treatment of preterm infants with respiratory distress syndrome (RDS) and the influence of MISA failure on neonatal outcome. A retrospective analysis was performed for the clinical data of 148 preterm infants with a gestational age of ≤32 weeks and a clinical diagnosis of RDS, who were admitted to the neonatal intensive care unit of eight tertiary hospitals in Beijing, Tianjin and Hebei Province from July 1, 2017 to December 31, 2018 and were treated with MISA (bovine pulmonary surfactant, PS). According to whether MISA failure (defined as the need for mechanical ventilation within 72 hours after MISA) was observed, the infants were divided into two groups: MISA failure group (n=16) and MISA success (n=132). A logistic regression analysis was used to investigate the risk factors for MISA failure and its influence on neonatal outcome. The MISA failure rate was 10.8% (16/148). The logistic regression analysis showed that a high incidence rate of grade >II RDS before PS administration, low mean arterial pressure and high pulse pressure before administration, a low dose of initial PS administration, and long injection time and operation time were the risk factors for MISA failure (OR=5.983, 1.210, 1.183, 1.055, 1.036, and 1.058 respectively, P<0.05). After the control for the above risk factors, the logistic regression analysis showed that the MISA failure group had a significantly higher incidence rate of bronchopulmonary dysplasia (BPD) (OR=8.537, P<0.05). A high grade of RDS, a low mean arterial pressure, and a high pulse pressure before administration are independent risk factors for MISA failure, and a low dose of initial PS administration, a long injection time, and a long operation time may increase the risk of MISA failure. MISA failure may increase the incidence rate of BPD in preterm infants.

Microbiological interaction studies between ceftazidime-avibactam and pulmonary surfactant and between ceftazidime-avibactam and antibacterial agents of other classes

For an antibacterial agent to be considered for clinical studies in nosocomial pneumonia (NP), it should be active in the presence of pulmonary surfactant. Furthermore, owing to the common practice of treating such infections with more than one antibacterial agent, it should be free of antagonistic interactions with agents of other classes. The aim of this study was to demonstrate the effect of pulmonary surfactant on the activity of ceftazidime and ceftazidime-avibactam and to determine the interaction (if any) of ceftazidime-avibactam and six antimicrobial agents common in the treatment of NP. Minimum inhibitory concentration (MIC) determination for ceftazidime and ceftazidime-avibactam was performed with and without the presence of four concentrations of bovine pulmonary surfactant, and a chequerboard assay was used to determine any interaction between ceftazidime and ceftazidime-avibactam with tobramycin, levofloxacin, linezolid, vancomycin, tigecycline and colistin. Here we report that the in vitro antimicrobial activity of ceftazidime-avibactam against β-lactamase-producing Gram-negative bacteria remained unaltered in the presence of pulmonary surfactant at concentrations that antagonised the antimicrobial activity of daptomycin. Furthermore, in chequerboard interaction studies, an absence of antagonism was demonstrated between ceftazidime-avibactam and six antimicrobial agents of different classes when tested against aerobic species frequently isolated from NP. The results support the further investigation of ceftazidime-avibactam as a potential treatment for NP caused by susceptible bacteria.

Surfactant protein C is an essential constituent for mucosal adjuvanticity of Surfacten, acting as an antigen delivery vehicle and inducing both local and systemic immunity

We have reported that Surfacten® (St), a bovine pulmonary surfactant free of antigenic c-type lectins, is a useful mucosal adjuvant for nasal vaccination. To prepare ample supplies a synthetic adjuvant that mimics St, we analyzed essential constituents of St for mucosal adjuvanticity. Intranasal inoculation of influenza virus hemagglutinin (HA) vaccine combined with St free of surfactant protein (SP)-C resulted in failure of HA vaccine delivery to dendritic cells and loss of local and systemic immune responses. Naïve bovine SP-C, synthetic human or bovine SP-C peptide reconstituted with three major St lipids restored delivery activity and local and systemic immune responses to levels similar to those of St and provided almost complete protection against lethal doses of influenza virus challenge in mice. The delivery of fluoresceinated HA vaccine to cultured dendritic cells was significantly enhanced by co-administration of St or synthetic adjuvant, and moderately stimulated the expression of MHC class II and CD86. In addition, both St and synthetic adjuvant markedly sustained HA vaccine and achieved a wide antigen distribution in murine nasal cavity. These results suggest that synthetic mucosal adjuvant reconstituted with SP-C peptide and major St lipids is useful for ample supply of the potent mucosal adjuvant as an antigen delivery vehicle for intranasal vaccination.

Effect of Nebulized Bovine Surfactant for Experimental Otitis Media with Effusion

ObjectivesIn this study, we evaluated the efficacy of nebulized bovine pulmonary surfactant on experimentally induced otitis media with effusion (OME) in guinea pigs.MethodsTwenty guinea pigs were divided into three groups. Four untreated animals served as normal controls. Experimental OME was established in both ears of the remaining 16 animals by a transbullar injection of 10 µL of Pseudomonas aeruginosa lipopolysaccharide in saline. Thereafter, the guinea pigs received nebulized phosphate buffered saline (n=8) or nebulized bovine pulmonary surfactant (n=8). Nebulization was given daily for 7 days. On day 8, all the animals' passive opening pressure (POP) of the Eustachian tube was measured and histopathological observations of the bulla were made by light microscopy.ResultsNebulized bovine pulmonary surfactant significantly reduced the POP compared to that of saline nebulization. The bovine pulmonary surfactant improved the tubal patency and produced less histopathologcally-evident edematous bullar mucosa.ConclusionNebulization of bovine pulmonary surfactant plays an important role in treating otitis media with effusion in guinea pigs. Our results suggest that the chosen nebulized bovine pulmonary surfactant can be of good clinical benefit for treating OME in the future.

Influenza vaccine with Surfacten, a modified pulmonary surfactant, induces systemic and mucosal immune responses without side effects in minipigs

Immune responses and side effects of intranasally administered flu vaccine with the commercial product Surfacten, a modified bovine pulmonary surfactant, were investigated in minipigs. The use of minipigs was based on the anatomical resemblance of nasal lymph nodes, the principal antigen uptake site of respiratory mucosal immunity, between pig and human. Intranasal instillation of HA vaccine adjuvanted with Surfacten elicited significantly higher serum hemagglutination inhibition titers than the antigen alone, with wide cross-neutralizing activities of secretory IgA in nasal washes. No significant induction of inflammatory cytokines or migration of inflammatory cells was observed at the site of immunization or serum after the first immunization. These data suggest the potential usefulness of Surfacten for mucosal vaccination.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Deacylated Pulmonary Surfactant Protein SP-C Transforms From α-Helical to Amyloid Fibril Structure via a pH-Dependent Mechanism: An Infrared Structural Investigation

Bovine pulmonary surfactant protein C (SP-C) is a hydrophobic, α-helical membrane-associated lipoprotein in which cysteines C4 and C5 are acylated with palmitoyl chains. Recently, it has been found that the α-helix form of SP-C is metastable, and under certain circumstances may transform from an α-helix to a β-strand conformation that resembles amyloid fibrils. This transformation is accelerated when the protein is in its deacylated form (dSP-C). We have used infrared spectroscopy to study the structure of dSP-C in solution and at membrane interfaces. Our results show that dSP-C transforms from an α-helical to a β-type amyloid fibril structure via a pH-dependent mechanism. In solution at low pH, dSP-C is α-helical in nature, but converts to an amyloid fibril structure composed of short β-strands or β-hairpins at neutral pH. The α-helix structure of dSP-C is fully recoverable from the amyloid β-structure when the pH is once again lowered. Attenuated total reflectance infrared spectroscopy of lipid-protein monomolecular films showed that the fibril β-form of dSP-C is not surface-associated at the air-water interface. In addition, the lipid-associated α-helix form of dSP-C is only retained at the surface at low surface pressures and dissociates from the membrane at higher surface pressures. In situ polarization modulation infrared spectroscopy of protein and lipid-protein monolayers at the air-water interface confirmed that the residual dSP-C helix conformation observed in the attenuated total reflectance infrared spectra of transferred films is randomly or isotropically oriented before exclusion from the membrane interface. This work identifies pH as one of the mechanistic causes of amyloid fibril formation for dSP-C, and a possible contributor to the pathogenesis of pulmonary alveolar proteinosis.

Stimulation of early embryonic development in cattle by coculture with surfactant.

Our purpose was to determine the efficacy of Surfacten, a bovine pulmonary surfactant, on the maturation of in vitro bovine ova. We used Surfacten as a supplement to the coculture media both at the onset of coculture and after cleavage in bovine ova had been determined. The controls received no Surfacten. The maturation rate in bovine embryos to the blastocyst stage statistically improved (P < 0.05) in the series in which Surfacten was added to the media at the onset of coculture, compared with the controls and the series in which Surfacten was added after cleavage had been determined. Surfacten, a commercially available surfactant which is a naturally occurring phospholipid that dramatically increases in the cervical mucus and the ampullaris of the oviduct at or near the time of ovulation, improves the maturation of bovine embryos in vitro by making the coculture medium approach the conditions found in the oviducts.

Exogenous pulmonary surfactant as a drug delivering agent: influence of antibiotics on surfactant activity.

1. It has been proposed to use exogenous pulmonary surfactant as a drug delivery system for antibiotics to the alveolar compartment of the lung. Little, however, is known about interactions between pulmonary surfactant and antimicrobial agents. This study investigated the activity of a bovine pulmonary surfactant after mixture with amphotericin B, amoxicillin, ceftazidime, pentamidine or tobramycin. 2. Surfactant (1 mg ml-1 in vitro and 40 mg ml-1 in vivo) was mixed with 0.375 mg ml-1 amphotericin B, 50 mg ml-1 amoxicillin, 37.5 mg ml-1 ceftazidime, 1 mg ml-1 pentamidine and 2.5 mg ml-1 tobramycin. Minimal surface tension of 50 microliters of the mixtures was measured in vitro by use of the Wilhelmy balance. In vivo surfactant activity was evaluated by its capacity to restore gas exchange in an established rat model for surfactant deficiency. 3. Surfactant deficiency was induced in ventilated rats by repeated lavage of the lung with warm saline until PaO2 dropped below 80 cmH2O with 100% inspired oxygen at standard ventilation settings. Subsequently an antibiotic-surfactant mixture, saline, air, or surfactant alone was instilled intratracheally (4 ml kg-1 volume, n = 6 per treatment) and blood gas values were measured 5, 30, 60, 90 and 120 min after instillation. 4. The results showed that minimal surface tensions of the mixtures were comparable to that of surfactant alone. In vivo PaO2 levels in the animals receiving ceftazidime-surfactant or pentamidine-surfactant were unchanged when compared to the surfactant group. PaO2 levels in animals receiving amphotericin B-surfactant, amoxicillin-surfactant or tobramycin-surfactant were significantly decreased compared to the surfactant group. For tobramycin it was further found that PaO2 levels were not affected when 0.2 M NaHCO3 (pH = 8.3) buffer was used for suspending surfactant instead of saline. 5. It is concluded that some antibiotics affect the in vivo activity of a bovine pulmonary surfactant. Therefore, before using surfactant-antibiotic mixtures in clinical trials, interactions between the two agents should be carefully evaluated.

Effect of pulmonary surfactant protein A and neutral lipid on accretion and organization of dipalmitoylphosphatidylcholine in surface films.

The effects of surfactant-associated protein A (SP-A) on lipid adsorption to the air-water interface and accumulation of dipalmitoylphosphatidylcholine (DPPC) in the surface region were investigated at 37 degrees C. Dispersions used were bovine pulmonary lipid extract surfactant with or without neutral lipid (NL). Lipid adsorption was examined with the Wilhelmy plate technique and DPPC accumulation by monitoring surface radioactivity from [14C]DPPC with a scintillation probe. SP-A enhanced the rate of lipid adsorption, while both SP-A and NL increased the extent of DPPC accumulation. At the specific radioactivity used [14C]DPPC monolayers were undetectable: the surface radioactivity arose from surface-associated DPPC beneath the monolayer. At the highest concentration studied (0.3 mg lipid/ml), NL greatly enhanced DPPC accumulation and SP-A attenuated this effect. Langmuir-Blodgett (L-B) films were prepared from [14C]DPPC-labeled dispersions (0.3 mg lipid/ml) at equilibrium surface tension. Autoradiographs of L-B films from lipid extract surfactant exhibited a diffuse misty appearance, while NL promoted formation of heterogeneous DPPC aggregates. Addition of SP-A to lipid extracts without NL generated DPPC aggregates; more uniform larger aggregates appeared in the presence of SP-A and NL. Radiation measurements confirmed that the L-B films were composed of more than monolayers. SP-A did not increase DPPC levels in films deposited from lipid extracts unless NL was present. These results indicate that neutral lipid cooperates with surfactant-associated protein A to organize dipalmitoylphosphatidylcholine in the surface films and enhance formation of a DPPC-rich reservoir below the air-water interface.

Effect of short‐term exogenous pulmonary surfactant treatment on acute lung damage associated with the intratracheal instillation of silica

The objective of our study was to investigate whether coating the surface of silica with Survanta, a commercially available, bovine pulmonary surfactant, would reduce the in vitro cytotoxicity to alveolar macrophages (AMs), as well as attenuate lung damage in vivo following intratracheal instillation of silica. In the in vitro studies, alveolar macrophages from male Fischer 344 rats were incubated for 1 and 24 h with native or Survanta-treated silica (0.5 mg/ml). At both time points, the native, uncoated silica caused a dramatic loss of AM viability. The AMs were protected, however, when the silica was treated with the Survanta surfactant. This protective effect was significantly greater after 1 h when compared with 24 h. In the in vivo studies, a high dose of silica (10 mg/100 g body weight) was suspended in Survanta and intratracheally instilled into the lungs of male Fischer 344 rats. A number of biochemical and cellular parameters were measured within the bronchoalveolar lavage fluid (BALF) 1 and 14 d after the instillation exposures to assess lung damage. One day after the instillations, the suspension of silica in Survanta resulted in significant reductions in the silica-induced increases in total protein, beta-glucuronidase activity, and influx of neutrophils (PMNs) into the airspaces of the lungs. Fourteen days after the instillation exposures, this protective effect was lost. When Survanta was instilled into the lungs 15 min after the intratracheal instillation of silica, a significant reduction also was demonstrated in the silica-induced elevations in BALF total protein, beta-glucuronidase activity, and influx of PMNs 1 d after the instillation exposures. In an attempt to protect silica-exposed lungs over a longer period of time, Survanta was instilled into the lung 15 min after the silica instillation, and then every other day over a 7-d treatment period. Twenty-four hours after the last Survanta instillation, slight but significant decreases in the silica-induced elevations in BALF total protein and beta-glucuronidase activity were observed. The Survanta treatment, however, had no effect in preventing the infiltration of PMNs into the airspaces of the lungs. The results of this study indicate that artificially coating the silica with surfactant phospholipid offers short-term protection against its toxicity under both in vitro and in vivo conditions.

Effect of a Bovine Lung Surfactant Protein Isolate (SP-B/C) on Egg Phosphatidylglycerol Acyl Chain Order in a Lipid Mixture with Dipalmitoylphosphatidylcholine and Palmitic Acid

Dynamic surface tension measurements of films of a d621,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine:L-α-phosphatidyl-DL-glycerol:d31palmitic acid (d62-DPPC:EggPG:d31-PA) lipid matrix in the presence of a bovine pulmonary surfactant protein isolate (SP-B/C) demonstrate the improved surface activity over that of the lipids alone. Thus, significant interaction of the proteins with the lipid matrix is demonstrated. The effect of SP-B/C on the acyl chain order of the negatively charged EggPG within a d62-DPPC:EggPG:d31-PA lipid matrix in D2O saline was investigated in thermal perturbation Fourier transform IR spectroscopic studies. The EggPG thermotropic phase behavior was determined independently of the other lipid components with perdeuterated lipids and D2O. The data demonstrate the high degree of EggPG acyl chain disorder in the absence of the protein isolate. A broad transition occurs between 30 and 40 °C. The addition of the protein isolate did not alter the acyl chain order at 0.281 and 1.46 mg/mL of protein. However, alterations in the lipid carbonyl vibrational mode were observed.

Ovine pulmonary surfactant induces killing of Pasteurella haemolytica, Escherichia coli, and Klebsiella pneumoniae by normal serum.

Pulmonary surfactant has been shown to play an increasingly important role in bacterial clearance at the alveolar surface in the lung. This study describes a bactericidal mechanism in which ovine pulmonary surfactant induces killing of Pasteurella haemolytica by normal serum. To demonstrate killing, six bacterial species were incubated first with pulmonary surfactant for 60 min at 37 degrees C and then with serum for an additional 60 min at 37 degrees C. P. haemolytica type A1 strains 82-25 and L101, a P. haemolytica type 2 strain, Escherichia coli, and Klebsiella pneumoniae were susceptible and Pasteurella multocida, Serratia marcescens, and Pseudomonas aeruginosa were not susceptible to killing by ovine pulmonary surfactant and normal serum. No bacteria incubated with bovine pulmonary surfactant were killed by normal serum. Although the species origin of pulmonary surfactant was selective, the species origin of serum was not. P. haemolytica incubated with ovine pulmonary surfactant was killed by fetal calf serum, gnotobiotic calf serum, pooled normal sheep serum, pooled normal rabbit serum, and pooled guinea pig serum. Ultrastructurally, killed P. haemolytica suspensions contained dead cells and cells distorted with vacuoles between the cytoplasmic membrane and the cytoplasm. The mechanism of killing did not correlate with concentrations of complement or lysozyme or titers of residual antibody in either the pulmonary surfactant or the serum, and killing was reduced by preincubation of surfactant with P. haemolytica lipopolysaccharide. Preliminary characterization of both surfactant and serum implicate a low-molecular-weight proteinaceous component in the surfactant and serum albumin in the serum. This mechanism may help clear certain gram-negative bacteria from the lungs of sheep as a part of the pulmonary innate defense system.

Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures

The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N 2 by (1) evaporation at room temperature; (2) evaporation at 45° C; or (3) incubation at 45° C overnight prior to evaporation at 45°C. In all cases, 45°C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipids from the monolayer. While SP-B was not as effective in facilitating phospholipid adsorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.

Low-molecular-weight hydrophobic proteins from bovine pulmonary surfactant

Pulmonary surfactant stabilizes the lung by reducing the surface tension in the terminal air spaces. Lipid extract surfactant contains approx. 1% (w/w) low-molecular-weight hydrophobic proteins SP-B (15 kDa: nonreduced) and SP-C (3.5 kDa) and with the remainder being mainly phospholipids. The hydrophobic proteins were purified from bovine lipid extract surfactant using delipidation by phospholipase C digestion followed by hydroxyapatite chromatography. The phospholipase C step removed most of phosphatidylcholine resulting in a 10-fold enrichment of hydrophobic proteins relative to phospholipid. Chromatography of this preparation on a hydroxyapatite column resulted in the elution of phospholipids followed by SP-C and then SP-B. The column chromatography was repeated to remove residual phospholipids and yield purified SP-B and SP-C. The final recovery of SP-B from the lipid extracts was about 15–20% and that of SP-C was 5–10%. The bovine surfactant proteins were reconstituted with phospholipids and examined for their ability to lower the surface tension with a pulsating bubble surfactometer. Reconstituted surfactant preparations containing SP-B and dipalmitoylphosphatidylcholine plus dioleoylphosphatidylglycerol were capable of reducing the surface tension to near zero values at minimum bubble radius while the reconstitutes with SP-C only lowered the surface tension to approx. 20 mN/m. A more rapid decrease in surface tension was observed with reconstituted samples containing both hydrophobic proteins. These results indicate that both SP-B and SP-C can promote the adsorption and spreading of surfactant lipids at the air/liquid interface. In addition, SP-B appears to facilitate the squeeze-out of unsaturated phospholipids leading to an enrichment of dipalmitoylphosphatidylcholine in the monolayer.