Comparative study of clinical pulmonary surfactants using atomic force microscopy

Clinical pulmonary surfactant is routinely used to treat premature newborns with respiratory distress syndrome, and has shown great potential in alleviating a number of neonatal and adult respiratory diseases. Despite extensive study of chemical composition, surface activity, and clinical performance of various surfactant preparations, a direct comparison of surfactant films is still lacking. In this study, we use atomic force microscopy to characterize and compare four animal-derived clinical surfactants currently used throughout the world, i.e., Survanta, Curosurf, Infasurf and BLES. These modified-natural surfactants are further compared to dipalmitoyl phosphatidylcholine (DPPC), a synthetic model surfactant of DPPC:palmitoyl-oleoyl phosphatidylglycerol (POPG) (7:3), and endogenous bovine natural surfactant. Atomic force microscopy reveals significant differences in the lateral structure and molecular organization of these surfactant preparations. These differences are discussed in terms of DPPC and cholesterol contents. We conclude that all animal-derived clinical surfactants assume a similar structure of multilayers of fluid phospholipids closely attached to an interfacial monolayer enriched in DPPC, at physiologically relevant surface pressures. This study provides the first comprehensive survey of the lateral structure of clinical surfactants at various surface pressures. It may have clinical implications on future application and development of surfactant preparations.     

A concentration-dependent mechanism by which serum albumin inactivates replacement lung surfactants.

Endogenous lung surfactant, and lung surfactant replacements used to treat respiratory distress syndrome, can be inactivated during lung edema, most likely by serum proteins. Serum albumin shows a concentration-dependent surface pressure that can exceed the respreading pressure of collapsed monolayers in vitro. Under these conditions, the collapsed surfactant monolayer can not respread to cover the interface, leading to higher minimum surface tensions and alterations in isotherms and morphology. This is an unusual example of a blocked phase transition (collapsed to monolayer form) inhibiting bioactivity. The concentration-dependent surface activity of other common surfactant inhibitors including fibrinogen and lysolipids correlates well with their effectiveness as inhibitors. These results show that respreading pressure may be as important as the minimum surface tension in the design of replacement surfactants for respiratory distress syndrome.     

A natural surfactant from pig lungs

An exogenous natural lung surfactant obtained from minced pig lungs can be produced by a technology using a low cost, DEAE-cellulose adsorbent. This surfactant is composed mainly with phospholipids and the two hydrophobic polypeptides, SP-B and SP-C, both of which are necessary for optimal function of surfactants used for treatment of respiratory distress syndrome.     

Surfactant in respiratory distress syndrome and lung injury

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.     

Pulmonary surfactant therapy.

Surfactant replacement therapy is now an integral part of the care of neonates since several clinical trials of natural surfactant extracts and synthetic preparations have shown efficacy in the treatment of infants with hyaline membrane disease. In these studies, early treatment with exogenous surfactant substantially reduced mortality and the incidence of air leak, although it did not appear to reduce the incidence of other complications, in particular bronchopulmonary dysplasia. Early reports of exogenous surfactant therapy in patients with the adult respiratory distress syndrome, although promising, remain limited in number. More research is needed to improve on current modes of therapy and to investigate the possible role of surfactant in other lung diseases of both newborns and adults.     

高频振荡呼吸机治疗呼吸窘迫综合征致呼吸机相关性肺损伤的疗效观察

目的:观察高频振荡呼吸机在治疗呼吸窘迫综合征(NRDS)致呼吸机相关性肺损伤(VALI)患儿的临床疗效。方法:选择我院2012年6月~2014年10月收治的NRDS致VALI患儿83例为研究对象,采用随机数字表法将患者随机分为研究组(45例)和对照组(38例)。两组均进行一般治疗,在此基础上对照组采用常规机械通气,研究组采用高频振荡呼吸机行高频通气。观察两组患者治疗24 h后p H值、二氧化碳分压(Pa CO2)、氧分压(Pa O2)、血压(BP)、心率(HR)及并发症情况。结果:两组患者治疗24 h后p H值、Pa CO2、Pa O2及BP比较,差异均无统计学意义(P0.05),研究组HR心率低于对照组,差异存在统计学意义(P0.05);研究组纵膈气肿、肺损伤、间质气肿及气胸的发生率均低于对照组,差异有统计学意义(P0.05)。结论:高频振荡呼吸机治疗NRDS致VALI患儿能够明显改善其症状及减少并发症的发生率,是治疗NRDS致VALI的有效的方式。     

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

Role of alveolar type II cells and of surfactant-associated protein C mRNA levels in the pathogenesis of respiratory distress in mink kits infected with Aleutian mink disease parvovirus.

Neonatal mink kits infected with Aleutian mink disease parvovirus (ADV) develop an acute interstitial pneumonia with clinical symptoms and pathological lesions that resemble those seen in preterm human infants with respiratory distress syndrome and in human adults with adult respiratory distress syndrome. We have previously suggested that ADV replicates in the alveolar type II epithelial cells of the lung. By using double in situ hybridization, with the simultaneous use of a probe to detect ADV replication and a probe to demonstrate alveolar type II cells, we now confirm this hypothesis. Furthermore, Northern (RNA) blot hybridization showed that the infection caused a significant decrease of surfactant-associated protein C mRNA produced by the alveolar type II cells. We therefore suggest that the severe clinical symptoms and pathological changes characterized by hyaline membrane formation observed in ADV-infected mink kits are caused by a dysfunction of alveolar surfactant similar to that observed in respiratory distress syndrome in preterm infants. However, in the infected mink kits the dysfunction is due to the replication of ADV in the lungs, whereas the dysfunction of surfactant in preterm infants is due to lung immaturity.     

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

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

Glycolipid accumulation in bronchoalveolar space in adult respiratory distress syndrome

Surfactant lipids in the alveolar space are believed to play an important role in normal respiratory function. Although the surface-active phospholipids have been extensively studied, the possible role of glycolipids in the surfactant remains to be explored. We have studied the glycolipid composition of cell-free bronchoalveolar lavage from healthy subjects and from adult patients with respiratory distress syndrome. Glycolipids were barely detectable in bronchoalveolar lavage from healthy subjects. However, in adult respiratory distress syndrome, the amount of glycolipid relative to phospholipid was increased by more than twenty times. These lipids, identified as lactosylceramide (galactose-glucose-ceramide) and paragloboside (galactose-N-acetylglucosamine-galactose-glucose-ceramide), may prove to be sensitive markers of lung injury. Since the glycolipids decreased the surface activity of surfactant in vitro, their potential role in the pathogenesis of adult respiratory distress syndrome should be considered.     

Current perspectives in pulmonary surfactant — Inhibition, enhancement and evaluation

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

Acid aspiration increases sensitivity to increased ambient oxygen concentrations

Previously we have demonstrated that prolonged exposure to 100% ambient oxygen leads to a marked loss in functional lung volume and lung compliance, hypoxemia, and surfactant system abnormalities similar to acute respiratory distress syndrome (ARDS). However, 50% oxygen administration is believed to be safe in most clinical settings. In the present study, we have evaluated the effects of a 24-h exposure to 50% oxygen in rabbits immediately following experimental gastric acid aspiration. Mild hypoxemia, but no changes in mortality, lung volume, lung compliance, surfactant metabolism, or edema formation occurred after 24 h of normoxia postacid aspiration. Conversely, a relatively short (24-h) exposure to 50% oxygen after acid aspiration results in increased pulmonary edema, physical signs of respiratory distress, and mortality, as well as decreased arterial oxygenation, lung volume, lung compliance, and type II alveolar cell surfactant synthesis. These results suggest that acid aspiration alters the "set point" for oxygen toxicity, possibly by "priming" cells through activation of inflammatory pathways. This pathogenic mechanism may contribute to the progression of aspiration pneumonia to ARDS.     

Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome.

Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.     

肺表面活性物质救治近足月儿呼吸窘迫综合征的临床疗效观察

目的:观察肺表面活性物质在近足月儿呼吸窘迫综合征救治中的作用。方法:按照家属自愿的原则将41例确诊为新生儿呼吸窘迫综合征且胎龄介于34周-36周之间的近足月儿分为治疗组及对照组,治疗组23例,对照组18例,比较两组患儿血气分析结果、呼吸机参数、气管插管率、上机时间、存活率及住院时间。结果:治疗组患儿,用药后无论临床表现还是胸片均有不同程度的改善。用药后6小时,治疗组PaO2及PaCO2均优于对照组(P<0.05);比较两组中机械通气患儿呼吸机参数,治疗组明显低于对照组(P<0.05);治疗组气管插管率明显低于对照组,且上机时间(nCPAP、nIPPV、气管插管机械通气)也较对照组明显缩短(P<0.05),但两组患儿存活率及住院时间差异无统计学意义。结论:对于近足月呼吸窘迫综合征患儿,尽早明确诊断并在发病早期给予PS替代治疗,可提高此类患儿的生存率及预后,对降低早产儿病死率有重要意义!     

Stimulation of lung maturity: investigation of ambroxol in various animal models

The surfactant or phospholipid lining of the alveolar surface is essential for lung function and airway stability. In premature neonates, the idiopathic respiratory distress syndrome (IRDS) is usually due to a prenatal deficiency in pulmonary surfactant. Experimental and clinical investigations with corticosteroids and other drugs were conducted to study various aspects of lung maturation. The present study of Ambroxol, a new compound with surfactant stimulating properties, was carried out on adult and foetal animals. In adult experimental animals an increase in the activity of Type II pneumocytes was shown by measuring various biochemical parameters. In models of premature animals comparable to clinical IRDS conditions, the antenatal treatment of foetal lambs and rabbits with Ambroxol enhanced lung maturation.     

Current status and prospects of basic research and clinical application of mesenchymal stem cells in acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a common and clinically devastating disease that causes respiratory failure. Morbidity and mortality of patients in intensive care units are stubbornly high, and various complications severely affect the quality of life of survivors. The pathophysiology of ARDS includes increased alveolar–capillary membrane permeability, an influx of protein-rich pulmonary edema fluid, and surfactant dysfunction leading to severe hypoxemia. At present, the main treatment for ARDS is mechanical treatment combined with diuretics to reduce pulmonary edema, which primarily improves symptoms, but the prognosis of patients with ARDS is still very poor. Mesenchymal stem cells (MSCs) are stromal cells that possess the capacity to self-renew and also exhibit multilineage differentiation. MSCs can be isolated from a variety of tissues, such as the umbilical cord, endometrial polyps, menstrual blood, bone marrow, and adipose tissues. Studies have confirmed the critical healing and immunomodulatory properties of MSCs in the treatment of a variety of diseases. Recently, the potential of stem cells in treating ARDS has been explored via basic research and clinical trials. The efficacy of MSCs has been shown in a variety of in vivo models of ARDS, reducing bacterial pneumonia and ischemia-reperfusion injury while promoting the repair of ventilator-induced lung injury. This article reviews the current basic research findings and clinical applications of MSCs in the treatment of ARDS in order to emphasize the clinical prospects of MSCs.     

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum

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

Improvement of pulmonary surfactant activity by introducing D-amino acids into highly hydrophobic amphiphilic α-peptide Hel 13-5

The high costs of artificial pulmonary surfactants, ranging in hundreds per kilogram of body weight, used for treating the respiratory distress syndrome (RDS) premature babies have limited their applications. We have extensively studied soy lecithins and higher alcohols as lipid alternatives to expensive phospholipids such as DPPC and PG. As a substitute for the proteins, we have synthesized the peptide Hel 13-5D3 by introducing D-amino acids into a highly lipid-soluble, basic amphiphilic peptide, Hel 13-5, composed of 18 amino acid residues. Analysis of the surfactant activities of lipid-amphiphilic artificial peptide mixtures using lung-irrigated rat models revealed that a mixture (Murosurf SLPD3) of dehydrogenated soy lecithin, fractionated soy lecithin, palmitic acid (PA), and peptide Hel 13-5D3 (40:40:17.5:2.5, by weight) superior pulmonary surfactant activity than a commercially available pulmonary surfactant (beractant, Surfacten®). Experiments using ovalbumin-sensitized model animals revealed that the lipid-amphiphilic artificial peptide mixtures provided significant control over an increase in the pulmonary resistance induced by premature allergy reaction and reduced the number of acidocytes and neutrophils in lung-irrigated solution. The newly developed low-cost pulmonary surfactant system may be used for treatment of a wide variety of respiratory diseases.     

肺脏超声对新生儿呼吸窘迫综合征的诊断价值及肺超声评分的评估价值分析

目的:探讨肺脏超声对新生儿呼吸窘迫综合征(NRDS)的诊断价值,并分析肺超声评分的临床应用价值。方法:本研究选择2017年5月至2018年5月于我院确诊的NRDS患儿45例作为观察组,选择同期于我院就诊的非肺病患儿45例作为对照组,所有患儿均行肺脏超声检查。分析NRDS患儿肺脏超声特征性征象,比较肺脏超声对两组患儿各种征象的检出率,分析肺脏超声对NRDS的诊断价值,比较两组肺超声评分。结果:NRDS患儿全部存在肺实质征象,超声下肺组织回声呈肝样伴支气管充气征,轻度的NRDS患儿于肺脏超声下表现为局灶性的肺实质,且支气管充气征不明显;重度的NRDS患儿于肺脏超声下表现为肺实质范围的进一步扩大,且支气管充气征随病情的加重而愈发明显。观察组肺实质、胸膜线异常、A线消失、弥漫性肺水肿、支气管充气征等征象的检出率显著高于对照组(P0.05),两组B线存在征象的检出率比较无统计学差异(P0.05)。肺实质、胸膜线异常和A线消失三种特征征象同时存在时对NRDS诊断的灵敏度和特异性均为100.00%,肺实质、胸膜线异常和支气管充气征三种特征征象同时存在时对NRDS诊断的灵敏度为80.00%,特异性为100.00%。观察组双肺、左肺、右肺、双侧肺、双肺底肺超声评分均高于对照组(P0.05)。结论:肺脏超声对NRDS的诊断价值较高,且肺超声评分可以评估NRDS患儿的病情严重程度,有助于指导患儿的治疗。