INACTIVATION OF PULMONARY SURFACTANT AND THE TREATMENT OF ACUTE LUNG INJURIES

Inactivation of pulmonary surfactant may be important in acute lung injury and acute respiratory distress syndrome. Treatment of surfactant dysfunction by instilling exogenous surfactants may improve gas exchange and pulmonary mechanics. Surfactants used for treatment vary in their attributes and effects, so when various surfactants are considered for therapy, resistance to inactivation is an important consideration. Animal models of acute lung injury exist in which the relative merits of surfactants can be compared. We hypothesize that the surfactants most resistant to inactivation in vitro will be the ones that are most effective in treatment of animal models of acute lung injury. Surfactants with higher concentrations of surfactant proteins (specifically A, B, and C) are more resistant to inactivation. Nonionic polymers mimic surfactant proteins in preventing surfactant inactivation under some conditions. Adding nonionic polymers to surfactant containing minimal amounts of SP-B and SP-C markedly improves lung function of animals with lung injury. Making surfactants more "inactivation-proof" may improve surfactant therapy of acute lung injuries.     

Phospholipase A2 is present in meconium and inhibits the activity of pulmonary surfactant: an in vitro study

Atelectasis, a major contributor to pulmonary dysfunction in meconium aspiration syndrome (MAS), is produced by bronchiolar obstruction and surfactant inactivation. It has been shown that substances in meconium, e.g. fatty acids, inhibit surfactant activity. However, the role of the enzyme phospholipase A2 (PLA2), which hydrolyses surfactant in adult respiratory distress syndrome (ARDS), has not yet been studied. Our objective was to investigate whether PLA2 is present in meconium and inhibits pulmonary surfactant activity in vitro. Therefore, the presence of PLA2 activity in meconium, collected from 10 newborns, was measured by the formation of lysophosphatidylcholine after incubation of meconium with radioactively labelled dipalmitoylphosphatidylcholine. Meconium was fractionated by Sephadex G-100 column chromatography and the fractions were assayed for PLA2 activity. Also, their effect on the surface tension of surfactant (Curosurf) was measured using a pulsating bubble surfactometer (PBS). PLA2 activity was present in all meconium samples. Addition of meconium to surfactant significantly increased surface tension (mean +/- SD: 1.7 +/- 1.6 mN/m to 24.3 +/- 6.7 mN/m, p = 0.0001) and only the addition of the PLA2 containing fraction from meconium to surfactant also significantly increased surface tension (mean 1.7 +/- 1.6 mN/m to 19.0 +/- 3.58 mN/m, p < 0.0001). Conclusion: PLA2 is present in meconium and inhibits the activity of pulmonary surfactant in vitro. Therefore, PLA2 in meconium may contribute to surfactant inactivation and alveolar atelectasis in MAS.     

Chitosan enhances the in vitro surface activity of dilute lung surfactant preparations and resists albumin-induced inactivation

Chitosan is a natural, cationic polysaccharide derived from fully or partially deacetylated chitin. Chitosan is capable of inducing large phospholipid aggregates, closely resembling the function of nonionic polymers tested previously as additives to therapeutic lung surfactants. The effects of chitosan on improving the surface activity of a dilute lung surfactant preparation, bovine lipid extract surfactant (BLES), and on resisting albumin-induced inactivation were studied using a constrained sessile drop (CSD) method. Also studied in parallel were the effects of polyethylene glycol (PEG, 10 kD) and hyaluronan (HA, 1240 kD). Both adsorption and dynamic cycling studies showed that chitosan is able to significantly enhance the surface activity of 0.5 mg/mL BLES and to resist albumin-induced inactivation at an extremely low concentration of 0.05 mg/mL, 1000 times smaller than the usual concentration of PEG and 20 times smaller than HA. Optical microscopy found that chitosan induced large surfactant aggregates even in the presence of albumin. Cytotoxicity tests confirmed that chitosan has no deleterious effect on the viability of lung epithelial cells. The experimental results suggest that chitosan may be a more effective polymeric additive to lung surfactant than the other polymers tested so far.     

Mechanisms to explain surfactant responses

Surfactant is now standard of care for infants with respiratory distress syndrome. Surfactant treatments are effective because of complex metabolic interactions between surfactant and the preterm lung. The large treatment dose functions as substrate; it is taken up by the preterm lung and is reprocessed and secreted with improved function. The components of the treatment surfactant remain in the preterm lung for days. If lung injury is avoided, then surfactant inhibition is minimized. Prenatal corticosteroids complement surfactant to further enhance lung function. The magic of surfactant therapy results from the multiple interactions between surfactant and the preterm lung.     

Inflammation and surfactant

An intact and well-functioning pulmonary surfactant system is critical for normal respiration and protection from lung infection. Surfactant is comprised of phospholipids that reduce surface tension and greatly reduce the work of breathing. The other major component consists of surfactant-associated proteins, which optimise the biophysical function of phospholipids and/or play an important role in host defence by acting as collectins. Alteration of surfactant composition and function occurs with various inflammatory disorders that affect the airways or the lung parenchyma including asthma, infant respiratory distress syndrome/bronchopulmonary dysplasia, cystic fibrosis, acute respiratory distress syndrome and interstitial lung disease. Although surfactant replacement therapy is indicated for infant respiratory distress syndrome, there is no well-proven role for exogenous surfactant in the treatment of inflammatory lung disorders at the present time.     

Surfactant protein-A : New insights into an old protein-II

Pulmonary surfactant is a lipoprotein substance that lines the lungs and helps reduce surface tension. Surfactant associated protein-A (SP-A) is the most abundant non-serum protein in pulmonary surfactant. This complex glycoprotein aids in the synthesis, secretion and recycling of surfactant phospholipids, and facilitates the reduction of surface tension by surfactant phospholipids. Recent evidence has highlighted the role of SP-A in the innate immune system present in the lung. SP-A may play a major role in defense against pathogens by interacting with both infectious agents and the immune system. Factors that affect fetal lung maturation, e.g. gestational age and hormones regulate SP-A gene expression. Mediators of immune function also regulate SP-A levels. A number of lung disorders, including infectious diseases and respiratory distress syndrome are associated with abnormal alveolar SP-A levels. SP-A can no longer be called a lung-specific protein, since it has recently been detected in other tissues. In most species, SP-A is encoded by a single gene, however in humans it is encoded by two, very similar genes. Models for the structure of the human SP-A protein molecule have been proposed, suggesting that the mature alveolar SP-A molecule is composed of both gene products. The study of SP-A may provide information helpful in understanding disease processes and formulating new treatment modalities.     

Surfactant protein-A : New insights into an old protein-part I

Pulmonary surfactant is a lipoprotein substance that lines the lungs and helps reduce surface tension Surfadtant associated protein -A (SP-A) is the most abundant non-serum protein in pulmonary surfactant. This complex glycoprotein aids in the synthesis, secretion and recycling of surfactant phospholipids, and facilitates the reduction of surface tension by surfactant phospholipids. Recent evidence has highlighted the role of SP-A in the innate immune system present in the lung. SP-A may play a major role in defense against pathogens by interacting with both infectious agents and the immune system. Factors that affect fetal lung maturation, e.g., gestational age and hormones, regulate SP-A gene expression. Mediators of immune function also regulate SP-A levels. A number of lung disorders, including infectious diseases and respiratory distress syndrome are associated with abnormal alveolar SP-A levels. SP-A can no longer be called a lung-specific protein, since it has recently been detected in other tissues. In most species, SP-A is encoded by a single gene, however in humans it is encoded by two, very similar genes. Models for the structure of the human SP-A protein molecule have been proposed, suggesting that the mature alveolar SP-A molecule is composed of both gene products. The study of SP-A may provide information helpful in understanding disease processes and formulating new treatment modalities.     

Disturbed surface properties in preterm infants with pneumonia

Congenital pneumonia in preterm infants is often associated with respiratory insufficiency requiring mechanical ventilation. This study was performed to show whether pneumonia in these infants is associated with an inhibition or deficiency of surfactant. The ratio of lecithin and sphingomyelin (L/S ratio) and minimal surface tension were determined in pharyngeal aspirates from 90 term born infants (healthy) and in tracheal aspirates from preterm infants with wet lung (n = 13), congenital pneumonia (n = 21) and respiratory distress syndrome (RDS) (n = 90). The L/S ratio was lower (p < 0.0001) in the RDS group (8.6) when compared with healthy (48.6), wet lung (42.9) and pneumonia (28.9). Surface tension was higher (p < 0.001) in RDS (37 mN/m) and pneumonia (33.7) when compared with healthy (22.9) or wet lung (21.2). For infants with RDS, L/S ratio <16.5 detects surfactant deficiency with 96% specificity and 70% sensitivity, surface tension >29 mN/m represents surfactant inhibition (specificity 97%, sensitivity 92%). Using these cut-off values in infants with pneumonia, 81% had a sufficient amount of surfactant but only 21% of infants with pneumonia had appropriate surface tension. Our study shows that lung effluent of respiratory insufficient infants with pneumonia, who need mechanical ventilation, has disturbed surface properties despite a sufficient amount of surfactant. In these infants, surfactant substitution could be beneficial.     

Inhibitory effect of meconium on pulmonary surfactant function tested in vitro using the stable microbubble test

Meconium aspiration syndrome is related to mechanical obstruction of the airways and subsequent chemical pneumonitis. It has also been suggested that meconium causes inhibition of surfactant function. To assess its inhibitory effect on surfactant function in vitro, we used a stable microbubble (SM) test that was thought to reflect the adequacy of pulmonary surfactant. The mixtures were prepared by adding serial dilutions of human meconium to various concentrations of Surfactant-TA (Surfacten^TM). The SM count at each concentration of surfactant significantly increased with the increasing concentration of surfactant. This shows that the SM test closely reflects the quantified function of surfactant. When various concentrations of meconium were added to the surfactant concentration of 0.05 mg/ml and 0.25 mg/ml, the SM test results were decreased even at low concentrations of meconium. Also the increase in the meconium concentration caused a decrease in the SM test result, which was dependent on the surfactant and the meconium concentration, accordingly. These results suggest that meconium inhibits surfactant function. Conclusion The stable microbubble test is an effective indirect method that tests the changes in surfactant quantity. In the in vitro experiment, we observed an inhibitory effect of meconium on the surfactant activity using the stable microbubble test. Received: 23 November 1999 and in revised form: 21 February 2000 / Accepted: 17 April 2000     

肺表面活性物质在儿童急性呼吸窘迫综合征的应用

肺表面活性物质(pulmonary surfactant,PS)是由Ⅱ型肺泡上皮细胞合成分泌的脂质蛋白混合物,主要功能是降低肺泡气-液界面表面张力.急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)时多种原因引起PS的量和质出现变化,导致其功能异常.外源性PS替代治疗可以改善儿童ARDS肺部气体交换,但提高存活率作用不肯定.这可能与ARDS病因、PS成分、给药方法、时机、剂量及次数等不同有关.目前不推荐PS作为儿童ARDS的常规治疗方法.     

Resistance of different surfactant preparations to inactivation by meconium.

A disease similar to acute respiratory distress syndrome may occur in neonates after aspiration of meconium. The aim of the study was to compare the inhibitory effects of human meconium on the following surfactant preparations suspended at a concentration of 2.5 mg/mL: Curosurf, Alveofact, Survanta, Exosurf, Pumactant, rabbit natural surfactant from bronchoalveolar lavage, and two synthetic surfactants based on recombinant surfactant protein-C (Venticute) or a leucine/lysine polypeptide. Minimum surface tension, determined with a pulsating bubble surfactometer, was increased >10 mN/m at meconium concentrations >or=0.04 mg/mL for Curosurf, Alveofact, or Survanta, >or=0.32 mg/mL for recombinant surfactant protein-C, >or=1.25 mg/mL for leucine/lysine polypeptide, and >or=20 mg/mL for rabbit natural surfactant. The protein-free synthetic surfactants Exosurf and Pumactant did not reach minimum surface tension <10 mN/m even in the absence of meconium. We conclude that surfactant activity is inhibited by meconium in a dose-dependent manner. Recombinant surfactant protein-C and leucine/lysine polypeptide surfactant were more resistant to inhibition than the modified natural surfactants Curosurf, Alveofact, or Survanta but less resistant than natural lavage surfactant containing surfactant protein-A. We speculate that recombinant hydrophobic surfactant proteins or synthetic analogs of these proteins can be used for the design of new surfactant preparations that are relatively resistant to inactivation and therefore suitable for treatment of acute respiratory distress syndrome.     

Pathophysiology of respiratory distress syndrome

Respiratory distress syndrome (RDS) is a major cause of neonatal mortality and morbidity, especially in preterm infants. Its aetiology includes developmental immaturity of the lungs, particularly of the surfactant synthesizing system. Surfactant is produced, stored and recycled by type II pneumocytes and is detectable from about 24 weeks’ gestation. It is a mixture of phospholipids, neutral lipids and proteins and is spread as a film over the alveolar surface to lower surface tension and to prevent alveolar collapse. The resulting clinical correlates of RDS can be predicted from the immature lung structure and atelectasis which occur due to surfactant deficiency. Various clinical factors are known to dysregulate surfactant production and function, leading to the development of RDS. Apart from preventing the incidence of prematurity, antenatal steroids and prophylactic surfactant are of proven benefit in reducing the incidence of RDS.     

Effect of fibrinogen degradation products and lung ground substance on surfactant function

Acute lung injury syndromes have many characteristics including protein-rich alveolar edema, hyaline membranes, and abnormal surface tension at the alveolar air-liquid interface. Increased surface tension can occur because of a relative surfactant deficiency and/or dysfunction. It has been previously demonstrated that surfactant dysfunction occurs when plasma protein inhibitors leak into the alveolar space during the induction of the lung injury and edema formation. The present study investigated whether inhibitors that would be generated during the stage of repair from lung injury could impair surfactant function. We determined whether fibrinogen degradation products (FDP) which would be released during lysis of the fibrin(ogen)-containing alveolar exudate and hyaline membranes, and components of the lungs' ground substance could inhibit the in vitro function of a lipid extract surfactant preparation. FDP were prepared by incubating human fibrinogen with plasmin or neutrophil elastase for 4 min to 60 h and were characterized by SDS-PAGE. Early (fragment X and Y) and late (fragment D and E) plasmin-derived FDP (MW greater than 40,000) inhibited surfactant function as assessed by a bubble surfactometer. The early elastase-derived FDP also inhibited surfactant, but the later and much smaller fragments (MW less than 15,000) did not affect surfactant function. Laminin also inhibited surfactant in a dose-dependent manner. Neither hyaluronic acid nor heparan sulfate affected surfactant performance in vitro. We conclude that plasmin-induced lysis of intraalveolar fibrinogen and hyaline membranes will result in prolonged generation (i.e. days) of surfactant inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyaluronan decreases surfactant inactivation in vitro

Hyaluronan (HA) is an anionic polymer and a constituent of alveolar fluid that can bind proteins, phospholipids, and water. Previous studies have established that nonionic polymers improve the surface activity of pulmonary surfactants by decreasing inactivation of surfactant. In this work, we investigate whether HA can also have beneficial effects when added to surfactants. We used a modified pulsating bubble surfactometer to measure mixtures of several commercially available pulmonary surfactants or native calf surfactant with and without serum inactivation. Surface properties such as equilibrium surface tension, minimum and maximum surface tensions on compression and expansion of a surface film, and degree of surface area reduction required to reach a surface tension of 10 mN/m were measured. In the presence of serum, addition of HA dramatically improved the surface activities of all four surfactants and in some cases in the absence of serum as well. These results indicate that HA reduces inactivation of surfactants caused by serum and add evidence that endogenous HAs may interact with alveolar surfactant under normal and abnormal conditions.     

Comparative randomized study: Administration of natural and synthetic surfactant to premature newborns with respiratory distress syndrome

BACKGROUND: The respiratory distress syndrome (RDS) in premature newborns has been etiologically correlated to immature lungs and specifically with surfactant deficiency. Exogenous administration of surfactant is nowadays considered to be the treatment of choice. In this paper we attempt a comparison of clinical results from the administration of natural Alveofact and synthetic Exosurf surfactants in premature newborns with respiratory distress syndrome. METHODS: The study subjects were 92 premature newborns who had been hospitalized in the Department of Neonatology, of the University of Crete. A total of 42 subjects received synthetic surfactant and 50 subjects received natural surfactant. The surfactant was administered in one to three doses, depending on respiratory support requirements. RESULTS: The time of administration was a little longer for the natural surfactant group. The duration of mechanical ventilatory support, requiring oxygen, the duration of hospitalization and the percentage of increase of arterial alveolar partial pressure oxygen ratio (a/APO2) were slightly higher for the synthetic surfactant group. The mortality rate during the neonatal period (28th day) was higher for the synthetic surfactant group than for the natural surfactant group (38.1 vs 24%). A similar tendency was noticed also as regards to complications, e.g. pneumothorax (11.2 vs 5.2%; relative risk (RR) 0.27) intraventricular hemorrhage (34.6 vs 21.1%; RR 0.61), septicemia (11.5 vs 5.2%; RR 0.46) and bronchopulmonary dysplasia (12.5 vs 2.8%; RR 0.22). CONCLUSION: The use of natural surfactant seems to offer more advantages in comparison with its synthetic counterpart.     

高分子聚合物逆转血浆所致肺表面活性物质失活

目的 包括血浆在内的许多物质能够抑制肺表面活性物质(PS)降低表面张力的能力,这是急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)的重要病理改变之一.本研究的目的 是探讨高分子聚合物是否可以防止或减轻血浆诱导的PS失活.方法 利用闭泡式表面张力仪分别测量葡聚糖(dextran)、聚乙二醇(PEG)和透明质酸(HA)、Ps(固尔苏,curosurf)以及加入了1%～3%血浆的混合物最大表面张力(γmax)和最小表面张力(γmin),并计算它们的稳定系数(SI).结果 不同高分子聚合物、血浆及它们的混合物γmin均>10mN/m,SI<0.8,而curosurf(1.25mg/ml)的γmin为(1.54±0.03)mN/m,SI为1.71±0.01.Curosurf与1%、2%或3%的血浆混合后γmin分别为(19.0±0.46)mN/m、(22.9±0.42)mN/m和(22.6±1.22)mN/m,SI为0.38±0.05、0.34±0.02和0.39±0.15.上述混合物与5%PEG、5%dextran或0.25%HA混合,γmin分别降至(1.59±0.22)mN/m、(1.43±0.24)mN/m、(17.2±0.72)mN/m;(0.88±0.06)mN/m、(1.18±0.06)mN/m、(1.46±0.22)mN/m及(1.22±0.10)mN/m、(1.33±0.07)mN/m、(1.42±0.18)mN/m,与curosurf同各浓度血浆混合物比较,差异均有统计学意义(P均<0.05).SI分别增加到1.72±0.04、1.73±0.04、0.43±0.04;1.82±0.01、1.82±0.01、1.79±0.03及1.79±0.02、1.79±0.01、1.79±0.04,与curosurf同各浓度血浆混合物比较,差异均有统计学意义(P均<0.05).不同浓度的高分子聚合物显著提高了血浆存在情况下curosurf降低γmin、增加SI的能力(rPEG=-0.718,P<0.01;rdextran=-0.682,P<0.01;rHA=-0.889,P<0.01).结论 高分子聚合物可以逆转血浆所致的curosurf失活,和PEG比较,dextran和HA与curosurf联合应用可以更好地对抗血浆的抑制.     

Composition and surface activity of normal and phosphatidylglycerol-deficient lung surfactant

The possibility that pulmonary surfactant, characterized by a phosphatidylglycerol deficiency, as in early fetal life, might have inferior surface properties was evaluated. We obtained this specific surfactant from adult rabbits by withholding glucose and giving them an excess of myoinositol by mouth and intravenously. Controls were given a similar quantity of glucose. The myoinositol resulted in a drastic reduction of surfactant phosphatidylglycerol, from 7.2 to 0.3% of phospholipids, and a corresponding increase in phosphatidylinositol from 4.8 to 11.3%. In addition, the myoinositol treatment increased the myoinositol that was disaturated from 18.5 to 27.3% (p less than 0.05). The corresponding figures for disaturated phosphatidyl-choline were 56.0 and 60.5%, respectively (NS). The myoinositol treatment for 4 days increased the pool size of alveolar surfactant by 32% (p less than 0.01). The surface activity was studied with modified Wilhelmy balance and the pulsating bubble surfactometer. Surfactant containing phosphatidylinositol rather than phosphatidylglycerol was not inferior, as compared to surfactant that contained phosphatidylglycerol (minimum surface tension: 2.0 versus 2.2 mN X m-1; collapse rate at 10 nM X m-1: 1.85 versus 1.95 min-1; rate of adsorption from subphase to surface: 32 versus 35 mN X m-1 X 30 s-1), nor was there a difference in the ability of the two surfactants to improve lung stability of 27-day-old rabbit fetuses (air retention at 35 cm H2O: 1.8 versus 1.8 ml/30 g; air retention at 0 cm H20: 0.8 versus 0.9 ml/30 g). We conclude that phosphatidylinositol surfactant does not have inferior surface properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Successful surfactant therapy of ARDS in an immunodepressed child

The acute respiratory distress syndrome in childhood is a rare disease, but as in the past still plagued with a high mortality rate. It is caused by severe pneumoniaes or infectious diseases with multiorgan failure, aspiration, trauma or immunodepression. There are no therapeutic guidelines based on controlled studies. Therefore different therapies i. e. high frequency oscillatory ventilation, nitric oxide application, surfactant therapy, extracorporal membrane oxygenation or a combination of these methods are used. We present the case of a 4 (3)/ 12 year old boy, who suffered from an acute lymphatic leukaemia. Caused by immunosuppressive therapy he got a severe broncho-pneumonia. During ventilation therapy an acute respiratory distress syndrome occurred. Due to a surfactant application over 7 days with a doses of 360 mg/kg body weight this RDS could be dominated. The extubation was possible after 17 days of ventilatory support. 3 weeks later the lung function was normalized and the chemotherapy resumed.     

Surfactant phospholipids and surface activity among preterm infants with respiratory distress syndrome who develop bronchopulmonary dysplasia

Our objective was to determine if preterm infants with respiratory distress syndrome who develop bronchopulmonary dysplasia have abnormalities in surfactant phospholipids and/or function. Tracheal aspirate samples obtained from preterm infants with respiratory distress syndrome on days 1, 3-5, 7-10, 14-17, 21-24 and 27-30 were analyzed for total phospholipids and phospholipids fractions by determination of total phospholipid phosphorus and thin layer chromatography, respectively. Surfactant properties were assessed with captive bubble surfactometer. Sixteen out of 56 (29%) infants died during the first 30 d of life. Infants who died were more immature, required more ventilatory support and had a surfactant with lower surface-tension-reducing properties than infants who survived (p < 0.05). Surviving infants were divided into group I (no bronchopulmonary dysplasia at 27-30 d, n = 25) and group II (with bronchopulmonary dysplasia at 27-30 d, n = 15). No significant differences in concentrations of surfactant phospholipids nor measurements of surface tension were noted among groups of infants. Surfactant therapy after birth was associated with a significant increase in concentrations of total phospholipids, lecithin, phosphatidylinositol and lower surface-tension measurements at 3-5 d of age among surviving infants (p < 0.01). Abnormalities in concentrations of surfactant phospholipids or surfactant function could not be demonstrated during the first month of life among preterm infants with respiratory distress syndrome who develop bronchopulmonary dysplasia.     

Secondary surfactant deficiencies.]

Preterm babies born before the 33rd week of gestation often exhibit primary surfactant deficiency responsible for the respiratory distress syndrome or hyaline membrane disease. In that situation, there is a limited and insufficient production of surfactant by type II alveolar cells of the lung due to immaturity. Secondary surfactant deficiencies occur in patients with prior normal surfactant synthesis and can be related to sepsis, hypoxia, ventilator induced lung injury or surfactant inhibition by a variety of substances reaching the alveolar spaces. They occur in full-term newborns with meconium aspiration syndrome, acute respiratory distress syndrome and congenital diaphragmatic hernia. In children and adults, acute respiratory distress syndrome and respiratory syncytial virus bronchiolitis can be responsible. In prematures they occur after the initial primary deficiency during pulmonary hemorrhage, pneumonia and bronchopulmonary dysplasia. Treatment with exogenous surfactant may be beneficial. There is a need for randomized controlled studies for evaluation of this treatment. Next generation of surfactants containing recombinant surfactant protein or synthetic peptides appear as promising agents in these situations of secondary surfactant deficiencies.