Lung Surfactant and Neonatal Respiratory Distress Syndrome

JOHN A. CLEMENTS and MARY ELLEN AVERY

Cardiovascular Research Institute and Department of Pediatrics, University of California, San Francisco, San Francisco, California; and Department of Pediatrics, Harvard Medical School, and The Children's Hospital, Boston, Massachusetts

	INTRODUCTION

TOP INTRODUCTION REFERENCES

I would rather discover a single fact, even a small one, than debate the great issues at length without discovering anything at all.

Galileo Galilei

Galileo Galilei may not have been the first reductionist in science, but he had a good grasp of the concept. His words have been ratified many times throughout the subsequent years, and the story of lung surfactant is but another example of the power of a "small" fact. In Jere Mead's words, "Sometimes the answer to a question is far more important than the question itself." Such a question was, "Is alveolar surface tension high or low?" Driven by curiosity alone, the research that was intended to answer this question fanned out into multiple disciplines and spun off significant therapeutic advances. To those persuaded by the Galilean pragma, this was no surprise, but if it had not coincided with an era of growing largesse from the National Heart, Lung, and Blood Institute (NHLBI), the story might have turned out quite differently. Fortunately, Claude Lenfant and Suzanne Hurd acceded during the critical time to positions of authority at NHLBI, and they and their associates pushed for programs in laboratory research and its practical applications that accelerated the development of the surfactant field (as well as others). The outcome has been a revolution in the treatment of a major disease of the newborn and the promise of deeper understanding and better management of other lung problems. It is hard to think of a more compelling example of the progress that can be made when enlightened public officials and academic researchers work together with mutual respect and willing cooperation. In the bibliography of a recent review of the surfactant field (1), we identified 31 key papers by American authors; 24 of these were supported by NHLBI. What a splendid record! It is a privilege to tell briefly here how all this came about.

	LABORATORY INVESTIGATIONS OF THE SURFACTANT SYSTEM

The original observations of surface tension and surface films antedated the founding of NHLBI by many centuries. This early history has fascinating aspects for which we do not have space here. Interested readers can find an excellent summary and references in Molecular Theory of Capillarity by Rowlinson and Widom (2). The omnipresent manifestations of surface tension (capillarity) were well known to the ancients as were the calming effects of films on "troubled waters." Many qualitative observations were made in later years, but quantitative experiments and theories did not flourish until the 18th century, when capillary phenomena were much discussed by natural philosophers. It was on this considerable foundation that Laplace and Young at last formulated the laws correctly and established a firm basis for mathematical analysis of surface mechanics. Their laws have survived essentially intact up to the present, despite profound advances in the molecular understanding of surface phenomena, and still provide the framework for current explanations of the role of surface tension in the stress-strain properties of the lungs.

The observation of surface films on water likewise goes back to antiquity, but the understanding and quantification of their behavior is more recent. Benjamin Franklin did his famous experiment calming the waves on the pond at Clapham Common with a teaspoonful of oil in 1765, but it was not until 1890 that Lord Rayleigh reported his use of surface films to estimate size and mass of a molecule. His paper was read by a Miss Agnes Pockels, who lived in the university town of Braunschweig, Germany. Though she had no formal training in science and no academic appointment, she carried out beautiful experiments in her kitchen and drew insightful conclusions from them. She wrote Rayleigh a letter about her methods and results, and he realized that they were better than his. (Her baking dish trough with barriers and her disk-detachment force method were precursors of the widely used apparatus known to surface chemists as the PLAWM balance, named for its developers, Pockels, Langmuir, Adam, Wilhelmy, and McBain.) With his wife's help Rayleigh translated the letter into English and sent it to Nature with a request that it be published (3). Pockels continued to make important contributions until the 1930s. At the age of 69 she was awarded the Laura-Leonard Prize of the Kolloid-Gesellschaft. On her 70th birthday she became the first woman to receive an honorary doctorate from the Technical University of Braunschweig. After her death at age 73, the university street was renamed Pockelsstrasse in her honor. Although she had no grants from NHLBI, she helped lay the foundation for work on surface films in the alveoli.

Effects of Surface Tension

Kurt von Neergaard appears to have been the first to apply the surface tension laws to the lungs, and his 1929 work (4), though mostly ignored for 25 yr after it was published, is now cited regularly as the initiating event in this field. Though Neergaard did not reveal how he came to think about alveolar surface tension in the first place, he did show how to demonstrate its existence by comparing the recoil of lungs filled with air and with aqueous solutions. This elegantly simple and convincing experiment has been repeated many times since the 1950s, even reaching the physiology student laboratory. Neergaard also tried to see if the alveoli contain a surface active material, but he chose the wrong method and failed to find what today is called lung surfactant. That discovery fell to others.

In about 1949 Mead and his coworkers began thinking about the effects of surface tension in the airspaces of the lungs, prompted by their studies of pulmonary edema (cited in reference 5). They were not aware of Neergaard's paper until their experiments were well along and a literature search turned it up, but they had already made a characteristically insightful analysis of their data. Radford, a member of Mead's team, saw the possibility of applying a thermodynamic treatment to the pressure-volume data in order to calculate the internal surface area of the lungs (6). To do this he had to assume an alveolar surface tension and, as Neergaard had done in his calculations, he chose the value for serum as the most likely. This choice led to area values about one-tenth of prevailing histological estimates. Radford knew that overestimating surface tension would cause him to underestimate area, but he justified his choice by citing Neergaard's failure to find surface active material in lung extracts.

Unbeknownst to American physiologists, the English investigator Pattle had begun to think about surface tension in the lungs, stimulated like Mead and coworkers by the effects of edema on pulmonary mechanics. Trained as a physicist, Pattle had been interested in surface tension since childhood. He was amazed by the stability of bubbles in the foam that come out of the airways during acute edema, and he concluded that they had brought with them "a protein layer that can abolish the tension of the alveolar surface" (7).

In the meantime Clements had also started to wonder about alveolar surface tension. He had had casual conversations with Mead and Radford since 1950 about their work, and when a pre-publication copy of Radford's report became available, he began a serious analysis of it. Soon it appeared that the data were compatible with two possible explanations. Either the surface tension was large and the area was small, as Radford concluded, or the area was large and the tension was small. If the latter was true, the lungs would contain a surfactant, as Pattle's results now implied. In 1955 Brown and Clements joined forces and decided that Brown would repeat and reanalyze Radford's experiments and Clements would try to extract surfactant. Brown confirmed Radford's data and his new calculations suggested that lung area was large, like the histologic estimates, and that alveolar surface tension fell to low values during deflation (8). Because of the unusual properties of lung surfactant, Clement's initial results were inconclusive. By modifying the PLAWM surface balance, he was able to directly demonstrate the postulated activity in lung tissue but not in serum or other tissues (9). All of these results were duly published, but they stimulated little interest among physiologists and physicians.

Then, in 1959, Avery and Mead (10) reported that they could not find surfactant in extracts of the lungs of infants who had died of hyaline membrane disease and hypothesized that prematurity and lack of surfactant were responsible. This paper made the clinical relevance of lung surface tension and surfactant obvious to all. It attracted worldwide interest to the subject and stimulated the development of a whole field of laboratory and clinical studies. From this time onward, more and more of the publications began to acknowledge funding from NIH, and especially NHLBI. The literature has become far too large to permit our attempting a complete tabulation, but a random sampling of papers suggests that NHLBI support has been a major factor in advancing this field.

Once the existence of lung surfactant was proven and its potential role in pulmonary function was apparent, a host of questions urged themselves upon interested investigators and shaped the development of the field. In this cameo review we cannot do justice to such a large body of literature, and we apologize to the many researchers for whose work we do not have space. For the most part we try to refer only to the initial publication of a given observation.

The first question that demanded an answer was whether the low surface tension predicted by theory and shown by extracts actually exists at alveolar surfaces. Twenty years went by before Schürch and colleagues (11) proved this by direct micropuncture measurements in the alveoli.

Chemical Composition

Another urgent issue was the chemical nature of the surfactant. This proved to be highly complex, engaging the attention of many workers, and in fact is still under investigation. After some false preconceptions were discarded, it appeared that the surfactant was built somewhat like a cell membrane, rich in phospholipids, and containing protein (12). The saturated lipid dipalmitoyl phosphatidylcholine (DP PC) turned out to be the most abundant component, with monoenoic PC, cholesterol, and anionic phospholipids runners-up. Four apoproteins have been purified from surfactant and studied extensively, using new techniques of biochemistry and molecular biology. Two of them, SP-A and SP-D, are large, water-soluble proteins belonging to the collectin family (15, 16). They have collagen-like N-terminal regions and calcium ion and carbohydrate binding domains in their C-terminal regions. SP-B and SP-C are small, highly hydrophobic proteins that bind strongly to lipids and, in fact, dissolve in organic solvents (17).

The complexity of the surfactant composition demands explanation. What do all the components do? Many suggestions have been put forward, but the picture is not yet complete. The most important mechanical functions of the surfactant are probably to move rapidly from the aqueous phase into the air- water interface, to form a stable film that can sustain low surface tension for long intervals, and if film collapse is forced by extreme reduction in alveolar surface area, to respread rapidly from the collapsed material. In early studies DP PC was found to give a low compressibility, stable film, but when it was suspended in buffer, it moved into the interface too slowly to meet physiologic criteria (22). Lipid extracts worked faster but not as fast as the complete protein-containing preparation (22). After evidence became available suggesting that the alveolar surface film could be nearly pure DP PC (23), it appeared that the other components might be carriers that promoted DP PC entry. Soon it became clear that SP-B and SP-C significantly accelerate film formation (24) and that SP-A greatly enhances their effect (19). These and other observations (25, 26) imply interactions between the lipids and the apoproteins that determine the structure of the complex, speed film formation, and possibly select DP PC for the interface (27, 28). The regulation of lipid composition to maintain appropriate fluidity may be critical to correct functioning of the complex (29).

The morphology of the surfactant and its relationship to the functions of the material have intrigued investigators throughout the history of the subject. True to its chemical complexity, it can take many forms (30). Stored in the lamellar bodies of alveolar epithelial type II cells, it presents as stacked bilayers. Upon secretion into alveolar liquid, it takes on water and rearranges into the fascinating tubular myelin form (31). Amazingly, this form can be prepared in vitro from DP PC, anionic phospholipid, SP-A, SP-B, calcium ions, and water (32). Sometimes an osmiophilic layer in the tubular myelin seems to continue into the interface (33), as though a surface film could be generated directly. Freeze fracture micrographs of alveolar lining liquid show many small unilamellar vesicles (34), which may be surfactant components that have left the surface film and are ready for uptake by type II cells and alveolar macrophages. Subfractionation of surfactant obtained by lavage gives results that are consistent with this picture (35).

It is obvious that the surfactant cannot last forever in the alveoli, and therefore it must be renewed by secretion of nascent and clearance of rundown material. It has been difficult to demonstrate these processes unambiguously because of the multicomponent, polymorphous nature of surfactant and because of the cellular heterogeneity of lung parenchyma. Over many years, however, convincing evidence has emerged from a variety of studies on intact animals, isolated lungs, and purified alveolar cells for a surfactant life cycle in which type II cells, macrophages, and alveolar lining play the major roles in turnover. Type II cells and their lamellar inclusions were associated with surfactant in early studies (36, 37). Later autoradiographic investigation with labeled precursors of surfactant proteins and lipids suggested that they are synthesized in type II cell endoplasmic reticulum and transported via Golgi apparatus and multivesicular bodies to the lamellar bodies for storage (38). Several recent studies have shown that the apoproteins are synthesized as proproteins and are processed in these organelles to the secreted mature forms (see review by Weaver in reference 39). SP-A appears to be secreted at least in part by a constitutive pathway bypassing lamellar bodies (40). The relative flux through the two pathways in different developmental and physiologic states is not known. Deficiency of SP-B in hereditary respiratory distress syndrome (41) and knockout mice (42) is associated with abnormal lamellar bodies, an alveolar proteinosis-like picture, and failure to process SP-C to the mature form. SP-B may have several roles that remain to be elucidated in transport, processing, assembly, secretion, and clearance of surfactant components.

Regulation of Surfactant

Mechanical strain and drugs such as beta-adrenergic agonists stimulate surfactant secretion in intact animals and in isolated type II cells (43). These results suggest that turnover may be keyed to the level of lung ventilation, and indeed, exercise- induced hyperventilation is associated with much faster transport (46). Presumably this response reflects both lung stretch and increased plasma catecholamine concentrations. In addition, cyclic changes in alveolar surface area appear to promote conversion of newly secreted, apoprotein-rich, active surfactant aggregates into protein-poor, inactive forms (47, 48) that are ready for clearance. How the physical events and possible enzymatic reactions may work together in the conversion process remains to be clarified, as does the role of alveolar phospholipid transfer protein (49). The removal of surfactant components from airspaces seems to occur mainly through uptake by type II cells and alveolar macrophages (50), with the macrophages normally accounting for perhaps 10 to 20% of the clearance. How the ratios of fluxes of the several components through these pathways may change with physiologic and disease states is not yet clear. The differences in composition of lavage surfactant in syndromes like alveolar proteinosis and adult respiratory distress syndrome may in part reflect such changes. In vitro SP-A inhibits secretion (53) and enhances uptake (51) of surfactant lipids. SP-B may also influence both intra- and extracellular transport (42, 54). Serum proteins can inhibit surface activity (55) and conversion of large to small aggregate forms (56). It is obvious that regulation of surfactant turnover and composition is multifactorial and will need far more study before we can understand it well under either normal or disease conditions. We can expect the resulting information to improve management of the lungs in established diseases and both before and after transplantation (57, 58).

Another persistent theme in research on surfactant is its development and control in the fetus. Naturally, the broad foundation for this work antedated NHLBI by many years, going back to Macklin, Barcroft, and their predecessors, but the first paper relating surfactant to a specific morphologic event in the fetal lung was supported by the institute. This was the demonstration that the detectibility of surface activity in fetal mouse lungs coincided with the appearance of lamellar inclusion bodies in the type II cells late in gestation (36). This led to studies correlating changes in morphology, mechanical stability, phospholipid content, and surfactant in fetal lamb lungs (59), relating the patterns of phospholipid composition in amniotic fluid to lung maturity (60), and quantifying the increase in flux of surfactant into lung fluid during development (61). Later, when SP-A was isolated and purified and an antibody against it became available, radioimmunoassay showed that SP-A appeared in small amounts in lamb tracheal fluid as early as 112 d gestation, leveled off until about 135 d, and then surged near term (62). Analysis of mRNAs for SP-B and C detected them at 13 wk in human lung, and at 24 wk they had increased to 50% and 15% of adult levels, respectively. In contrast, SP-A was low (63, 64). These and other results suggest that the expression of the apoproteins is independently controlled. But how?

Hormonal Control

That hormones may play an important role in such control was foreshadowed by reports such as the 1912 paper of Gudernatsch (65), which showed that horse thyroid would push tadpoles into rapid metamorphosis, and the 1953 paper of Moog (66), in which she reported that injection of cortisone accelerated by up to 6 d the elaboration of alkaline phosphatase and the differentiation of the epithelim in the duodenum of the fetal mouse. The latter paper fired the imagination of Buckingham and stimulated her and her colleagues to see whether similar events occurred spontaneously in the lungs of fetal rabbits. They did occur, and the changes were correlated with the appearance of surface activity, leading the authors to speculate that fetal steroids might also be causing maturation of the lung (67). Because of her unfortunate early death, Buckingham did not carry these studies to completion.

For several years previously, Liggins had been exploring the role of fetal pituitary and adrenal hormones in initiating parturition. In a paper submitted for publication in February 1968 he reported that ACTH infusion into the fetus could greatly accelerate delivery and noted, "Lambs delivered by Caesarean section were alive at delivery as were the more mature lambs born spontaneously. Less mature foetuses were dead when found but appeared to be fresh, some having aerated lungs; autolysis of liver and kidneys was absent." (68). The following year he achieved the same results with dexamethasone and suggested that steroids might be accelerating appearance of surfactant activity (69). Soon thereafter, Avery's group confirmed and extended the results with physiologic, morphologic, and biochemical measurements (70). These pathfinding studies opened the way for a large number of investigations, too numerous to cite here, that have greatly clarified the mechanisms of action of steroids on the fetal lung (71).

In 1973, 61 yr after Gudernatsch demonstrated the maturational effects of thyroid on tadpoles, Wu and colleagues (72) found that thyroxine could accelerate type II cell differentiation in the fetal rabbit, increasing lamellar body content and decreasing glycogen. Because thyroid hormones cross the placenta poorly, Rooney and coworkers (73) employed thyroid-releasing hormone (TRH), which crosses well, to stimulate the fetal pituitary to secrete TSH. This in turn stimulates T₃ production and accelerates maturation of the lung (73). TRH stimulates release of prolactin, which may also play a role. Thyroid hormone was found to stimulate PC synthesis in cultured fetal rabbit lung (74). Glucocorticoids and thyroid hormones are synergistic in enhancing lung distensibility and PC synthesis (75, 76) but not apoprotein formation (77). Taken as a whole, the evidence from laboratory investigations suggests that glucocorticoid and thyroid, and perhaps other hormones, regulate the maturation of the fetal lung and that administration of exogenous hormones to the immature fetus can accelerate morphologic differentiation and functional development.

In the last 15 yr the advent of methods and concepts of molecular biology (partly due to the urging of NHLBI officials) has brought about noticeable changes in research on lung surfactant. The availability, for example, of amino acid and nucleotide sequencing, gene cloning, polymerase chain reaction, oligonucleotide probes, monoclonal antibodies, and a rapidly growing database of sequences has made commonplace experiments that one could only dream of a few years ago. As a result, the structures and properties of the major proteins of the surfactant system are known (15), and a great deal has been learned about the structures and control of the genes that code for them and about how their expression is regulated during fetal life (78). The identification of hereditary gene defects (41) and the study of transgenic animals (42) are important advances that are helping to define the cellular physiology of the system. The new methods will also facilitate investigation of the roles of surfactant components in alveolar host defense processes (79, 80).

	RESEARCH ON HYALINE MEMBRANE DISEASE: RESPIRATORY DISTRESS SYNDROME

Pathologic Descriptions

The attention given by pathologists to the causes of death of newborn infants preceded by several decades the awareness of pediatricians that the leading finding at autopsy of live-born premature infants was airless lungs. The clinical course was remarkable in that death, if it was to occur before the ventilator era, usually did so within the first few days after birth. With the advent of metabolic and ventilatory support, deaths sometimes came at a later age, although the illness was almost always manifest within the first hours or days after birth. As the specialty of neonatology emerged in the 1950s and 1960s, more attention was given to the supportive care of the infants, particularly with appropriate ventilators, after which survival with late consequences of the disease became more common. Termed bronchopulmonary dysplasia, or chronic lung disease of prematurity, the condition was presumably related to lack of knowledge of the appropriate volume-pressure relationships and oxygen requirements of these infants. (Indeed, the condition was once called respirator lung disease.) Prolongation of life for very low-birth-weight infants was not possible until the late 1960s and early 1970s, when microdeterminations of blood gases and more appropriate ventilators became available.

Among the pathologists whose work was most apposite for neonatologists was Peter Gruenwald. When he was working at the Margaret Hague Maternity Hospital in Jersey City, New Jersey, Gruenwald presented a paper to the New York Pathological Society entitled "Pathological Aspects of Lung Expansion in Mature and Immature Newborn Infants." He wrote, "It is therefore suggested that three factors contribute to the development of the pattern of atelectasis of premature infants, which is characterized by distension of respiratory bronchioles and collapse of alveoli: 1) a low ratio of capacity of alveoli versus bronchi in premature infants favors the loss of air from the alveoli when only part of the total volume of air leaves the lungs; 2) in conditions of collapse following air breathing the respiratory surfaces have an increased adhesiveness, tending to perpetuate atelectasis caused by the first factor; 3) surface tension favors the formation of large air bubbles in the bronchioles, rather than smaller ones in the alveoli. Hyaline membranes do not occur in all instances of atelectasis of premature infants, but only in those in which air in the respiratory bronchioles is replaced by fluid. These membranes are therefore not the cause of atelectasis. They probably consist of inspissated edema fluid after most of its water has been absorbed." (81)

Clinical Observations

This prescient observation of Gruenwald's was not taken very seriously by the community of perinatal physiologists and neonatologists who were attracted to the study of hyaline membrane disease, which was then estimated to affect 25,000 babies, with at least 10,000 deaths per year in the United States alone (82). The number of deaths varied from center to center, but there was general agreement on a linear decline of mortality with increasing gestational age from 26 to 36 wk, with rare cases over 37 wk. Risk factors were identified as prematurity (implying short gestation), aggravated by perinatal asphyxia; cesarean section when carried out on premature infants before onset of labor; and diabetes in the mother. Intrauterine infection was sometimes, but not always, associated with prematurity. Inflammation was not seen before the appearance of hyaline membranes after a few hours of air breathing.

It was also generally agreed that the risk of male infants having respiratory distress syndrome was about one and one-half times that of female infants, and that the risk for the second-born of twins was somewhat greater. Non-white infants in general had a lower likelihood of having respiratory distress syndrome at a given gestational age in comparison with white infants. Occasional familial occurrences of respiratory distress syndrome suggested a genetic predisposition that was subsequently supported by evidence that some individuals had surfactant-associated protein A allelic differences, but no clear pattern of inheritance was established (83). Deficiency of surfactant-associated protein B has been found in the uniformly fatal congenital familial alveolar proteinosis (41). As of late 1997, only mutations of proteins A and B have been linked to respiratory disease.

Surfactant Replacement Therapy

After attempts to administer the principal phospholipid component of natural surfactants, dipalmitoyl phosphatidylcholine, by aerosol were unsatisfactory (84, 85), there followed a decade of further animal investigations and biochemical studies. Encouraged by Enhorning and Robertson's successful treatment of immature rabbits by tracheal instillation of surfactant from adult rabbits (86) and by their own studies, Fujiwara and colleagues (87) reported in 1980 the use of a material from cow lungs that they called artificial surfactant. This landmark paper was not immediately acclaimed, in part because the published experience was not in the form of randomized, controlled trials. Nonetheless, the article stimulated many investigators, including the Fujiwara group, to publish prospective, controlled, clinical trials of surfactant replacement therapy (88, 89).

By 1990 it was estimated that approximately 30,000 infants in 500 hospitals in North America, Europe, and Japan had been enrolled in clinical trials to evaluate safety, effectiveness, optimal time of administration, and appropriate amount of surfactants. At least 35 randomized, controlled trials that involved over 6,000 infants have been reported with the following results: mortality and incidence of pneumothoraces were significantly reduced by both natural and synthetic surfactants. In general the best responses occurred when surfactants were given early, i.e., within the first 12 h after birth. No added gains came from attempts to instill the surfactants before the first breath, possibly because the materials spread promptly and widely throughout the lungs within minutes of administration and initiation of ventilation.

The 17 years since the original reports of efficacy of surfactant replacement have been characterized by studies from around the industrialized world with either surfactants derived from bovine or porcine lungs, with or without supplementation of phospholipids, or synthetic mixtures of phospholipids and additives (90). A pilot study of surfactant from human amniotic fluid was promising but was not pursued for various reasons (94). The most widely used preparations at present are fortified extracts from animal lungs.

The impact on infant survival has been reflected in United States national statistics, with only 1,460 deaths attributed to respiratory distress syndrome in 1995 compared to 5,498 in 1979 (95). The deaths since 1995 have been largely among infants under 1,000 g birth weight, and many among those of 400-600 g birth weight whose deaths in 1979 could have been attributed to extreme prematurity. Although many other interventions to support life of low-birth-weight infants were mobilized during that era, surfactant replacement therapy has been a significant life-saver, especially for infants over 28 wks' gestational age. Notable events in the understanding, prevention and treatment of respiratory distress syndrome are summarized in Table 1.

	SUMMARY AND ACKNOWLEDGMENT

This brief history of the observations that led to improvements in clinical management, and in many instances, prevention of respiratory distress syndrome, illustrates the power of multidisciplinary and multicenter approaches to medical problem-solving.

The initial observations on surfactant deficiency in lungs of infants who died with atelectasis and hyaline membranes were supported from 1957 to 1959 by a special fellowship from the National Institute of Neurological Disease and Blindness to Avery, who was working as a research fellow with Mead at the Harvard School of Public Health. Through subsequent years the NHLBI supported the research at many centers that led to surfactant replacement therapy. Among the beneficiaries of this support were Clements and his colleagues at the University of California, San Francisco. Most of the later clinical trials in the United States were supported by Abbott-Ross Laboratories (Survanta), Burroughs Wellcome Company (Exosurf), and ONY and Forest Laboratories (Infasurf).

In retrospect, the development of this field has been an exemplary story of cooperation among clinicians and scientists in government, academia, and industry. The benefits in knowledge gained, in lives saved, in suffering avoided, and in health care costs abated will last far into the future. Much credit for these advances belongs to the current and former members of the staff of the Lung Division of the NHLBI. On this celebration of the founding of the Pulmonary Program and the Lung Division, we salute them!

	Footnotes

Correspondence and requests for reprints should be addressed to John A. Clements, Cardiovascular Research Institute, Moffitt Hospital, Rm. 1315, University of California, San Francisco, 505 Parnassus, San Francisco, CA 94143-0130.

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