Artificial Surfactant Research Articles

Surfactant therapy of newborn rabbits impairs lung macrophage bactericidal activity.

Because in vitro studies indicate that pulmonary alveolar macrophages (PAM's) filled with phospholipid vesicles have depressed microbicidal capacity, we tested the intrapulmonary bactericidal activity of newborn PAM's after surfactant treatment. Term newborn rabbits received intratracheally either homologous surfactant or one of two artificial phospholipid vesicle preparations followed by pulmonary aerosol infection with group B streptococci (GBS). Four hours after lung infection, phagocytic killing of GBS was reduced by 70-90% in animals treated with the homologous and one of the artificial surfactants compared with untreated animals or animals that received intrapulmonary injections of the surfactant vehicle (P less than 0.02). The other artificial phospholipid preparation decreased intrapulmonary inactivation of GBS by 30-40% compared with the controls. The phospholipid vesicles in the three preparations were avidly ingested and processed by newborn PAM's. The diminished in vivo killing of GBS was not attributed to decreased viability or phagocytic behavior of the PAM's toward GBS. The bactericidal defect that was evident in the newborn PAM's appeared related to the uptake of large phospholipid vesicles in the preparations rather than to the phospholipid content of the surfactants themselves. When in vitro conditions that stimulated the alveolar environment were used, the natural surfactant preparation promoted GBS proliferation, whereas the artificial preparations did not. Our findings indicate that surfactant administration reduces the bactericidal activity of neonatal PAM's. We conclude that additional investigations are needed to ascertain the effect of surfactant replacement therapy on lost defenses of the lung.

Prolonged ventilation of the premature newborn rabbit after treatment with natural or apoprotein-based artificial surfactant.

Premature rabbit neonates (gestational age 27 days) were treated at birth with natural surfactant purified from chloroform extracts of porcine lung lipids either by acetone precipitation (Surfactant CK, n = 10) or liquid gel chromatography (Curosurf, n = 22). Another group of animals received artificial surfactant "reconstituted" from isolated low molecular weight (less than or equal to 15 K) apoproteins and synthetic dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) (Aposurf, n = 10). The phospholipid concentrations of the preparations were adjusted to provide the same individual dose of DPPC for each group of treated animals (3 or 4 mg). In comparison with untreated controls from the same litters, there was a 4-7-fold enhancement of lung-thorax compliance in all groups of surfactant-treated animals during a 3-h period of artificial ventilation. The average initial (20 min) compliance value was lower in the Aposurf-treated group than in animals receiving natural surfactant preparations, but the difference between the groups gradually diminished and was no longer statistically significant during the 2nd and 3rd h of artificial ventilation. Judged from the fall in tidal volume during ventilation with a short expiration phase (0.17 instead of 0.75 s), the apoprotein-based artificial surfactant was also less effective in stabilizing the lungs. A similar conclusion could be drawn from data on alveolar expansion in histological sections, evaluated by automated image analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Early and Late Surfactant Treatments in Baboon Model of Hyaline Membrane Disease

Because the issue of optimal time for artificial surfactant therapy for hyaline membrane disease has not been established, the effects of treatment with a reconstituted bovine surfactant (surfactant TA) were compared at two time periods in a hyaline membrane disease model in a premature baboon. The baboons were delivered by cesarean section at 75% of gestation (139.5 +/- 1.5 days, mean +/- SD). One group was treated with surfactant TA within ten minutes after birth (ultraearly), another group was treated at two hours of age (late) and a third (comparison group) did not receive the surfactant. Both treatment groups had significantly higher compliance and ratio of arterial to alveolar Po2 ratio and lower mean airway pressure and oxygen requirement (Fio2) than the comparison group. At autopsy, the largest residual volume and hysteresis in pulmonary pressure-volume curves were noted in the ultraearly group, intermediate values were found in the late group, and least values were found in the comparison group. These data indicate that early surfactant therapy for hyaline membrane disease results in greater improvement in lung mechanics than delaying treatment, even for two hours. Delivery room treatment with surfactant of infants at risk for hyaline membrane disease is perhaps better than therapy for established hyaline membrane disease.

Effect of reconstituted pulmonary surfactant containing the 6000-dalton hydrophobic protein on lung compliance of prematurely delivered rabbit fetuses.

Chloroform:methanol extracts of bovine pulmonary surfactant contain small hydrophobic proteins, designated surfactant-associated apoproteins 6,000 (SAP-6), but do not contain the major surfactant-associated 35,000-dalton glycoprotein, designated SAP-35. Examination of lipid extract surfactant on sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed hydrophobic proteins with apparent molecular masses of 15,000, 7,000, and 3,5000 daltons prior to reduction. After reduction, the 15,000-dalton species largely disappeared and was replaced by a 5,000-dalton species. In addition, the 7000- and 3500-dalton species exhibited a slightly enhanced mobility. Amino acid analysis demonstrated that SAP-6 possesses a more highly hydrophobic profile than SAP-35. Combining the protein-containing fractions from silicic acid chromatography of lipid extract with synthetic dipalmitoylphosphatidylcholine produced a reconstituted surfactant preparation which was just as active as lipid extract surfactant on a pulsating bubble surfactometer. The reconstituted surfactant contained SAP-6 but not SAP-35. Pressure-volume studies revealed that, at the optimal dose, reconstituted surfactant containing half the SAP-6 concentration of lipid extract exhibited similar effectiveness to lipid extract surfactant in promoting lung expansion with prematurely delivered rabbit fetuses of 27 days gestation. Reconstituted surfactant with an identical SAP-6 protein concentration as lipid extract possessed the same biological properties as the preparation with 1% SAP-6 protein. These studies support the view that an artificial surfactant composed of synthetic or semisynthetic lipids plus human SAP-6 produced via biotechnology could be useful for prevention and/or treatment of the neonatal respiratory distress syndrome.

Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant. Characterization and biophysical activity.

Hydrophobic low-molecular-mass proteins were isolated from minced pig lungs and separated into two fractions. Electrophoresis of protein fraction 1 showed two major bands. Calculations of molecular masses from the electrophoretic mobilities are unreliable because of the extreme hydrophobicity of the peptides. However, the two bands were at positions corresponding to apparent molecular masses of about 3 kDa and 14 kDa, while sequence degradation disclosed only one major structure. Electrophoretic separation of protein fraction 2 revealed one band, at an apparent molecular mass of about 6 kDa. Microheterogeneities at the N terminus of both fractions were observed. However, the two fractions had different N-terminal structures and amino acid compositions. Consequently they are concluded to represent different polypeptides without common segments. Bronchoalveolar lavage from humans also contains surfactant polypeptides and at least the fraction 2 peptide is highly similar in human and porcine surfactants. Artificial surfactant preparations, obtained by recombination of protein fraction 1 or 2 with a mixture of synthetic phospholipids, were evaluated with the pulsating bubble method and in experiments on artificially ventilated premature newborn rabbits. The addition of protein fraction 1 to the phospholipid mixture improved surface adsorption from more than 300 s to about 2 s and reduced minimum surface tension from more than 20 mN/m to nearly 0 as measured with a pulsating bubble. When this surfactant preparation was instilled into the airways of newborn rabbits, the tidal volumes at insufflation pressure 25 cm H2O was increased about twentyfold compared to the volumes obtained in non-treated controls. Preparations based on protein fraction 1 had better in vitro and in vivo properties than those based on protein fraction 2. Both these protein-based preparations were decidedly more effective than phospholipids alone.

Late hypertriglyceridemia in very low birth weight infants fed human milk exclusively

efficacy in respiratory distress syndrome. Pediatrics 1983; 71:473-82. 4. Wilkinson A, ,lenkins PA, Jeffrey JA. Two controlled trials of dry artificial surfactant: early effects and later outcome in babies with surfactant deficiency. Lancet 1985;2:287-90. 5. Enhorning G, Grossman G, Robertson B. Tracheal deposition of surfactant before the first breath. Am Rev Respir Dis 1973;107:921. 6. Weibel ER, Stereological methods, vol 1. Practical methods for biological morphometry. New York: Academic Press, 1979. 7. Enhorning G, Robertson B. Lung expansion in the premature rabbit fetus after tracheal deposition of surfactant. Pediatrics 1972;50:58-62. 8. Egan EA, Notter RH, Kwong MS, et al. Natural and artificial lung surfactant replacement therapy in premature lambs. ,l Appl Physiol 1983;55:875-83. 9. Robertson B, Enhorning G. The alveolar lining of the premature newborn rabbit after pharyngeal deposition of surfactant. Lab Invest 1974;31:54-9. 10..lobe A, Ikegami M, Jacobs H, et al. Surfactant and pulmonary blood flow distribution following treatment of premature lambs with natural surfactant. J Clin Invest 1984;73:84856. 11. Hulbert WC, Forster BB, Laird W, et al. An improved method for fixation of the respiratory epithelial surface with the mucous and surfactant layers. Lab Invest 1982;47:35463.

Artificial surfactant: an artificial lung expanding compound significantly reduces mortality in premature babies

Artificial surfactant: an artificial lung expanding compound significantly reduces mortality in premature babies

Nonselective squeeze-out of dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol from binary mixed monolayers with dipalmitoylphosphatidylcholine

In order to enable the possible use of dipalmitoylphosphatidylcholine as an artificial lung surfactant, the addition of dioleoylphosphatidylcholine or dioleoylphosphatidylglycerol has been suggested. A preferential loss of molecules of the second component during compression of the interfacial layer was proposed. In this study two types of measurement were carried out in order to verify such a preferential squeeze-out. In the first type, electron micrographs of a pure dipalmitoylphosphatidylcholine monolayer and of mixed monolayers of dipalmitoylphosphatidylcholine and egg phosphatidylglycerol were taken in order to study the nature of the structures formed during compression of the monolayer. The electron microscopy photos show horizontally stacked layers in the collapse phase of dipalmitoylphosphatidylcholine, and long vertical ridges in the mixed monolayers up to 20% second component. At higher concentrations of the second component no such structures can be detected. The second type involved monolayer studies with binary mixtures of dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine or dioleoylphosphatidylglycerol, one of the pair being always radioactively labelled. Counting the radioactivities in bulk phase and monolayer after compression revealed nonselective squeeze-out of either component.

Artificial pulmonary surfactant inhibited by proteins.

With a pulsating bubble surfactometer we assessed the ability of various agents, fibrinogen, human serum, albumin, and a 55,000-dalton serum protein, to interfere with the surface activity of Surfactant TA. From a highest final protein concentration of 4 mg/ml the potential inhibitors were diluted down to 0.125 mg/ml in six steps, and each concentration was evaluated together with two final phospholipid concentrations, 6.25 and 1.25 mg/ml, of the surfactant preparation. The strongest inhibiting action was exerted by fibrinogen, followed by human serum and the 55,000-dalton serum protein; the weakest inhibitor was albumin. Bilirubin, when added in an amount of 1.73 mg/100 ml dissolved in human serum, significantly (P less than 0.001) augmented the inhibition over that exerted by human serum alone. Adsorption rate, as reflected in the mean value of surface tension 2 and 10 s after creation of a bubble, not pulsating, was seriously affected by each of the protein-containing inhibitors in concentrations exceeding 1 mg/ml. Surface tension was raised significantly when the pulsating bubble was at maximal and minimal size. The effect was dose dependent. At maximal size it showed no tendency to disappear during the 10-min recording, but at minimal bubble size the inhibition gradually diminished. We conclude that proteins present in the airways may seriously interfere with the activity of Surfactant TA.

Ten centre randomized trial of artificial surfactant for the prophylaxis of the complications of prematurity : C.J. Morley, Paediatric Department, University of Cambridge, Cambridge, U.K. (For the trial collaborators)

Ten centre randomized trial of artificial surfactant for the prophylaxis of the complications of prematurity : C.J. Morley, Paediatric Department, University of Cambridge, Cambridge, U.K. (For the trial collaborators)

Growth and development two years after artificial surfactant replacement at birth

Two year follow-up of 80 surviving preterm babies from a controlled trial of surfactant replacement is reported. There were no statistically significant differences in rates of hospital admission, respiratory infections or antibiotic treatment. Wheezing, skin rashes and food intolerance were reported by parents with similar frequency in each group. Weight, length and occipito-frontal circumference were similar at all ages up to and including 2 years. Median developmental quotient (Griffiths scales) was 100 in the treated group and 95 in the control group (P = 0.053). Rates of cerebral palsy were similar in each group although milder forms predominated in the treated group. It is concluded that artificial surfactant (dipalmitoylphosphati-dylcholine and high-density lipoprotein) replacement at birth is not associated with adverse long term effects on preterm babies.

Two Controlled Trials of Dry Artificial Surfactant

Neonatal Unit, Department of Paediatrics, University of Oxford, John Radcliffe Maternity Hospital, Oxford, England

Normal surface properties of phosphatidylglycerol-deficient surfactant from dog after acute lung injury

Lung surfactant was isolated from bronchoalveolar lavage of dogs during the late phase of recovery (15 days) from acute alveolar injury induced by subcutaneous injection of N-nitroso-N-methylurethane. This surfactant was compared with surfactant from control dogs in terms of in vitro surface properties, phospholipid composition and protein content, and those of its subfractions. Phospholipid composition and protein content were similar in the two groups, except that phosphatidylglycerol (PG) was markedly reduced and phosphatidylinositol (PI) was increased in the experimental group. In both, isopycnic densities of their subfractions in continuous sucrose density gradient were identical. The time course of surfactant adsorption was similar in both groups. Minimum surface tension (gamma min) was 4.1 +/- 1.5 dynes/cm in the experimental dogs and 3.8 +/- 1.3 dynes/cm in the controls. Surface compressibility (SC), stability index (SI), and dynamic respreadability (DR) of the surfactants from the two groups were nearly identical. When compared to an artificial surfactant composed of dipalmitoyl phosphatidylcholine (DPPC) and PG in 9:1 molar ratio a mixture of DPPC-PI 9:1 prepared identically showed similar gamma min, SC, SI, and DR, and a much higher surface adsorption rate. These results suggest that PG is not essential for normal in vitro surfactant function and that its role may be assumed by PI.

1371 NEONATAL LUNG DEFENSES AFTER SURFACTANT THERAPY

Artificial surfactant has been used to treat Hyaline Membrane Disease (HMD), but its effect on phagocytic function in the neonatal lung is poorly defined. A mixture of 70% phosphatidylcholine/30% phosphatidylglycerol w/w was diluted in sterile phosphate-buffered saline (PBS) and sonicated. Either 4 mg/Kg of this mixture or an equal volume (100 μl) of PBS was instilled into the surgically exposed trachea of less than 12 hr old rabbits. One hr after instillation, in vivo bacterial clearance was studied by exposing the groups to a Streptococcus agalactiae type Ia (GBS) aerosol and the numbers of viable GBS-compared in the left lung at 0 and 4 hrs after infection. Results follow: Intrapulmonary killing of GBS was significantly lower in surfactant vs control (p<0.05). Additional litters were divided and treated with either surfactant or PBS, and alveolar macrophages (AM) recovered 1 hr later by lavage. Phagocytosis was ascertained using FITC-labeled GBS opsonized in newborn rabbit serum (50 GBS:AM) by a fluorescence quenching method. The percentage of phagocytic AM (x = 91.4 vs 93.0) and GBS/phagocytic AM (x = 16.9 vs 18.6) were similar in surfactant vs PBS animals. Surfactant therapy may impair bacterial killing by newborn AM.

Liposome phospholipid substitution and lung function in surfactant deprived rats

In vivo activity of an artificial surfactant was studied in surfactant depleted rats. After tenfold alveolar lavage, PaO2, tidal volume, and compliance of the respiratory system fell to one third of initial value. Substitution of large unilamellar vesicles containing 90% Dipalmitoylphosphatidylcholine and 10% unsaturated phosphatidylglycerol largely restored oxygenation and lung mechanics in most animals. Complete normalization with weaning from the ventilator, however, was achieved neither with liposomes nor with natural surfactant concentrate.

Pulmonary surfactant.

The mammalian lung is stabilized by a specialized material, the pulmonary surfactant, which acts by reversibly reducing the surface tension at the air-liquid interface of the lung during breathing. Pulmonary surfactant contains approximately 90% lipid and 10% proteins. Dipalmitoyl phosphatidylcholine, the major lipid component, appears to be primarily responsible for the ability to reduce surface tension to near 0 dyn/cm (1 dyn = 10 microN). The other components of pulmonary surfactant promote the adsorption and spreading of this disaturated lecithin at the air-liquid interface. Surfactant activity can be accessed by physical and biological assays. Apparent discrepancies between the results obtained with the Wilhelmy plate surface balance and the pulsating bubble surfactometer have led to the suggestion that separate "protein-facilitated" (catalytic type) and "protein-mediated" (chemical type) processes may be involved in adsorption and (or) spreading at the different surfactant concentrations used with these two techniques. Artificial surfactants, which mimic the essential properties of the natural product with the pulsating bubble surfactometer, can be produced with synthetic lipids. Treatment of prematurely delivered infants suffering from the neonatal respiratory distress syndrome with lipid extracts of pulmonary surfactant leads to a marked improvement in gaseous exchange.

Artificial pulmonary surfactant: Potential role for hexagonal H II phase in the formation of a surface-active monolayer

Natural surfactant possesses the ability to rapidly reduce the surface tension of a bubble pulsating at 20 cycles per min at 37°C to less than 30 dyn/cm at maximum radius and to less than 1 dyn/cm at minimum radius. The preparation of two artificial surfactant systems, containing either dipalmitoylphosphatidylcholine (DPPC) and phosphatidylethanolamine (PE) (5:5), or DPPC plus PE and phosphatidylglycerol (5:4:1 or 6:3:1) , is described. Formation of artificial surfactants which mimic the essential properties of natural surfactant was correlated with the appearance of particles of aggregated lipids. The effects of lipid composition, calcium ion concentration, pH, temperature and mechanical agitation were determined. It is proposed that these artificial surfactant systems may produce a surface-active monolayer through the involvement of nonbilayer structures with properties similar to hexagonal H II phase.

Inhibition of bacterial adhesion by an artificial surfactant

Non-cytotoxic and non-antimicrobial concentrations of an artificial surfactant inhibited the adhesion of 14C-radiolabelled Klebsiella pneumoniae WG14 to embryonic lung epithelial cells L-132. The minimal antiadhesive concentration was 200 ng · ml−1. In a dynamic adhesion test model, already adhering bacteria were removed from the epithelial cell surface by the surfactant. The antiadhesive action might be based on protection of the epithelial cells.

Inhibition of bacterial adhesion by an artificial surfactant

Non-cytotoxic and non-antimicrobial concentrations of an artificial surfactant inhibited the adhesion of 14C-radiolabelled Klebsiella pneumoniae WG14 to embryonic lung epithelial cells L-132. The minimal antiadhesive concentration was 200 ng · ml−1. In a dynamic adhesion test model, already adhering bacteria were removed from the epithelial cell surface by the surfactant. The antiadhesive action might be based on protection of the epithelial cells.

CONTROLLED TRIAL OF ARTIFICIAL SURFACTANT TO PREVENT RESPIRATORY DISTRESS SYNDROME

In a randomised, controlled trial of the effectiveness of artificial surfactant therapy 100 babies of <34 weeks' gestation were intubated at birth and received manual ventilation. The 49 babies in the treated group also received 30 mg dipalmitoylphosphatidylcholine and 3 mg high-density lipoprotein in a 5 ml suspension. There were no significant differences in mortality or in the incidence or severity of respiratory distress syndrome between the surfactant-treated and control groups as a whole or between subgroups divided on the basis of sex or pulmonary maturity (as assessed by the lecithinsphingomyelin ratio). However, there appeared to be a trend towards improved survival in treated babies of 27-29 weeks' gestation.