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Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days I and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60-80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60-100% oxygen, whereas levels of T1[alpha], a protein expressed by type I cells, were comparably increased in mice exposed to 40-100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to [greater than or equal to] 60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent. bronchopulmonary dysplasia; epithelium; hyperoxia; type II cells doi:10.1152/ajplung.00023.2009.

Byline: Daphne J. Janssen (1), Luc J. Zimmermann (2), Paola Cogo (3), Aaron Hamvas (4), Kajsa Bohlin (4,5), Ingrid H. Luijendijk (1), Darcos Wattimena (6), Virgilio P. Carnielli (7), Dick Tibboel (1) Keywords: Surfactant metabolism; Lung injury; Stable isotopes; Surfactant phosphatidylcholine; Congenital diaphragmatic hernia Abstract: Purpose Congenital diaphragmatic hernia (CDH) may result in severe respiratory insufficiency with a high morbidity. The role of a disturbed surfactant metabolism in the pathogenesis of CDH is unclear. We therefore studied endogenous surfactant metabolism in the most severe CDH patients who required extracorporeal membrane oxygenation (ECMO). Methods Eleven neonates with CDH who required ECMO and ten ventilated neonates without significant lung disease received a 24-h infusion of the stable isotope [U- 13.sup.C] glucose. The 13.sup.C-incorporation into palmitic acid in surfactant phosphatidylcholine (PC) isolated from serial tracheal aspirates was measured. Mean PC concentration in epithelial lining fluid (ELF) was measured during the first 4 days of the study. Results Fractional surfactant PC synthesis was decreased in CDH-ECMO patients compared to controls (2.4 +- 0.33 vs. 8.0 +- 2.4%/day, p = 0.04). The control group had a higher maximal enrichment (0.18 +- 0.03 vs. 0.09 +- 0.02 APE, p = 0.04) and reached this maximal enrichment earlier (46.7 +- 3.0 vs. 69.4 +- 6.6 h, p = 0.004) compared to the CDH-ECMO group, which reflects higher and faster precursor incorporation in the control group. Surfactant PC concentration in ELF was similar in both groups. Conclusion These results show that CDH patients who require ECMO have a decreased surfactant PC synthesis, which may be part of the pathogenesis of severe pulmonary insufficiency and has a negative impact on weaning from ECMO. Author Affiliation: (1) Department of Pediatrics and Pediatric Surgery, Intensive Care Erasmus MC-Sophia Children's Hospital, P.O. Box 2060, 3000 CB, Rotterdam, The Netherlands (2) Division of Neonatology, University Hospital Maastricht, Maastricht, The Netherlands (3) Department of Pediatrics, University of Padova, Padua, Italy (4) Department of Pediatrics, Washington University/St. Louis Children's Hospital, St. Louis, USA (5) Division of Pediatrics, Karolinska University Hospital Huddinge and the Karolinska Institute, Stockholm, Sweden (6) Internal Medicine, Erasmus MC-Dijkzigt, Rotterdam, The Netherlands (7) Division of Neonatology, Children's Hospital Ancona, Ancona, Italy Article History: Registration Date: 17/06/2009 Received Date: 04/06/2008 Accepted Date: 10/06/2009 Online Date: 07/07/2009

Deplefon of alveolar macrophages (AM) leads to an increase in endogenous surfactant that lasts several days beyond the repletion of AM. Furthermore, impairment to the endogenous pulmonary suffactant system contributes to ventilation-induced lung injury. The objective of the current study was to determine whether increased endogenous surfactant pools induced via AM depletion was protective against ventilation-induced lung injury. Adult rats were intratracheally instilled with either control or dichloromethylene diphosphonic acid (DMDP) containing liposomes to deplete AMs and thereby increase endogenous surfactant pools. Either 3 or 7 days following instillation, rats were exposed to 2 h of injurious ventilation using either an ex vivo or in vivo ventilation protocol and were compared with nonventilated controls. The measured outcomes were oxygenation, lung compliance, lavage protein, and inflammatory cytokine concentrations. Compared with controls, the DMDP-treated animals had significantly reduced AM numbers and increased surfactant pools 3 days after instillation. Seven days after instillation, AM numbers had returned to normal, but surfactant pools were still elevated. DMDP-treated animals at both time points exhibited protection against ventilation-induced lung injury, which included superior physiological parameters, lower protein leakage, and lower inflammatory mediator release into the air space, compared with animals not receiving DMDP. It is concluded that DMDP-liposome administration protects against ventilation-induced lung injury. This effect appears to be due to the presence of elevated endogenous surfactant pools. alveolar macrophages; inflammatory mediators

This study examines surfactant dysfunction in rats with inflammatory lung injury from intratracheal instillation of hydrochloric acid (ACID, pH 1.25), small nonacidified gastric particles (SNAP), or combined acid and small gastric particles (CASP). Rats given CASP had the most severe lung injury at 6, 24, and 48 h based on decreases in arterial oxygenation and increases in erythrocytes, total leukocytes, neutrophils, total protein, and albumin in bronchoalveolar lavage (BAL). The content of large surfactant aggregates in BAL was reduced in all forms of aspiration injury, but decreases were greatest in rats given CASP. Large aggregates from aspiration-injured rats also had decreased levels of phosphatidylcholine (PC) and increased levels of lyso-PC and total protein compared with saline controls (abnormalities for CASP were greater than for SNAP or ACID alone). The surface tension-lowering ability of large surfactant aggregates on a bubble surfactometer was impaired in rats with aspiration injury at 6, 24, and 48 h, with the largest activity reductions found in animals given CASP. There were strong statistical correlations between surfactant dysfunction (increased minimum surface tension and reduced large aggregate content) and the severity of lung injury based on arterial oxygenation and levels of albumin, protein, and erythrocytes in BAL (P < 0.0001). Surfactant dysfunction also correlated strongly with reduced lung volumes during inflation and deflation (P = 0.0004-0.005). These results indicate that surfactant abnormalities are functionally important in gastric aspiration lung injury and contribute significantly to the increased severity of injury found in CASP compared with ACID or SNAP alone. lung surfactant; surfactant dysfunction; gastric aspiration; acute lung injury

Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase [A.sub.2] ([sPLA.sub.2]) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human [sPLA.sub.2]s, including generation of lysophospholipids and the depletion of specific phospholipids. All five [sPLA.sub.2]s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X [sPLA.sub.2]s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group lid was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V [sPLA.sub.2] is less clear and may be the combined result of both mechanisms. lung injury; asthma; surface tension

Surface activity and sensitivity to inhibition from phospholipase [A.sub.2] (PL[A.sub.2]), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions exogenous surfactants; phospholipid analogs; phospholipase [A.sub.2]; phospholipase resistance; inhibition resistance; diether phosphonolipid; surfactant proteins

Expression of the pulmonary surfactant protein A (SP-A) gene is lung specific, developmentally regulated, and enhanced by hormones and factors that increase cAMP. We previously identified two E-box-like enhancers termed distal binding element (DBE) and proximal binding element (PBE) in the 5'-flanking region of the rabbit (r) SP-A gene that are essential for cAMP induction of rSP-A promoter activity (Gao E, Alcorn JL, and Mendelson CR. J Biol Chem 268: 19697-19709, 1993). We also found that DBE and PBE serve as binding sites for the basic helix-loop-helix-leucine zipper transcription factor, upstream stimulatory factor-1 (USF1) (Gao E, Wang Y, Alcorn JL, and Mendelson CR. J Biol Chem 272: 23398-23406, 1997). In the present study, PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert encoding the structurally related rabbit upstream stimulatory factor-2 (rUSF2) was isolated. The levels of rUSF2 mRNA reach peak levels in fetal rabbit lung at 28 days of gestation, in concert with the time of maximal induction of SP-A gene transcription. In yeast two-hybrid analysis, rUSF2 was found to preferentially form heterodimers, compared with homodimers, with rUSF1. Binding complexes of nuclear proteins isolated from fetal rabbit lung type II cells with the DBE and PBE were supershifted by anti-rUSF2 antibodies. Binding activity was enriched in nuclear proteins from type II cells compared with fibroblasts. Overexpression of rUSF2 in transfected lung A549 cells increased rSP-A promoter activity and acted synergistically with rUSF1. We suggest that heterodimers of USF2 and USF1 bound to two E-box elements in the SP-A gene 5'-flanking region serve a key role in developmental and hormonal regulation of SP-A gene expression in pulmonary type II cells. upstream stimulatory factor; E-box; surfactant protein A; gene expression; development; type II cells; regulation

Byline: G. Nakos (1), H. Tsangaris (1), S. Liokatis (2), E. Kitsiouli (2), M. E. Lekka (2) Keywords: Ventilator-associated pneumonia Atelectasis BAL Lung surfactant Platelet-activating factor (PAF) PAF-acetylhydrolase Abstract: Objective. Surfactant offers protection against alveolar collapse and contributes to the local defense mechanism, but it is unclear if surfactant alterations have a role in the development of atelectasis or ventilator-associated pneumonia (VAP). The present study was undertaken to monitor surfactant, as well as biochemical BAL fluid alterations, during the course of VAP and atelectasis in mechanically ventilated patients without primary cardiopulmonary disease, to elucidate the pathogenesis and to differentiate these two entities. Design. Prospective controlled study. Setting. 14-bed general ICU of a 750-bed University Hospital. Patients. Sixty-one ventilated patients, without primary cardiopulmonary disease--normal initial chest X-ray, satisfactory oxygenation (PaO.sub.2/FiO.sub.2>300 mmHg), and expected time of ventilation exceeding 2 weeks--were initially enrolled. Twelve of them developed VAP and eight lobar or segmental atelectasis during the 2-week study period. Interventions. An initial BAL was performed in all patients within 48 h from admission. Patients who developed VAP or atelectasis were subjected to a second and third BAL during and after the resolution of VAP or atelectasis, respectively. Measurements and results. VAP and atelectasis resulted in a significant increase of total protein and markers of inflammation, such as PAF and neutrophils, which partially remitted after their resolution. Large surfactant aggregates, which contribute to surface tension decrease, were significantly reduced during both entities and remained low even after their resolution. Conclusions. BAL alterations during VAP and atelectasis suggest increased alveolar-capillary permeability, severe surfactant abnormalities, and signs of local inflammatory reaction. These alterations are associated with the observed deteriorated gas exchange and lung mechanics and could predispose to further lung injury in ventilated patients. Author Affiliation: (1) Intensive Care Unit Department, University Hospital of Ioannina, University Street, 45500, Ioannina, Greece (2) Chemistry Department, University of Ioannina, Ioannina, Greece Article History: Received Date: 26/04/2002 Accepted Date: 17/01/2003 Article note: Electronic Publication

Byline: Jens Christian Moller (1) Keywords: Acute respiratory distress syndrome Surfactant Children Ventilation Oxygenation Pneumonia Abstract: Objective. To determine whether bovine surfactant given in cases of severe pediatric acute respiratory distress syndrome (ARDS) improves oxygenation. Design. Single-center study with 19 patients, followed by a multicenter randomized comparison of surfactant with a standardized treatment algorithm. Primary endpoint PaO.sub.2/FIO.sub.2 at 48 h, secondary endpoints: PaO.sub.2/FIO.sub.2 at 2, 4, 12, and 24 h, survival, survival without rescue, days on ventilator, subgroups analyzed by analysis of variance to identify patients who might benefit from surfactant Setting. Multicenter study in 19 reference centers for ARDS. Patients. Children after the 44th postconceptional week and under 14 years old, admitted for at least 4 h, ventilated for 12--120 h, and without heart failure or chronic lung disease. In the multicenter study 35 patients were recruited 20 were randomized to the surfactant group and 15 to the nonsurfactant group. Decreasing recruitment of patients led to a preliminary end of this study. Interventions. Administration of 100 mg/kg bovine surfactant intratracheally under continuous ventilation and PEEP, as soon as the PaO.sub.2/FIO.sub.2 ratio dropped to less than 100 for 2 h (in the pilot study increments of 50 mg/kg as long as the PaO.sub.2/FIO.sub.2 did not increase by 20%). A second equivalent dose within 48 h was permitted. Results. In the pilot study the PaO.sub.2/FIO.sub.2 increased by a mean of 100 at 48 h (n=19). A higher PaO.sub.2/FIO.sub.2 ratio was observed in the surfactant group 2 h after the first dose (58 from baseline vs. 9), at 48 h there was a trend towards a higher ratio (38 from baseline vs. 22). The rate of rescue therapy was significantly lower in the surfactant group. Outcome criteria were not affected by a second surfactant dose (n=11). A significant difference in PaO.sub.2/FIO.sub.2 in favor of surfactant at 48 h was found in the subgroup with an initial PaO.sub.2/FIO.sub.2 ratio higher than 65 and in patients without pneumonia. Conclusions. Surfactant therapy in severe ARDS improves oxygenation immediately after administration. This improvement is sustained only in the subgroup of patients without pneumonia and that with an initial PaO.sub.2/FIO.sub.2 ratio higher than 65 Author Affiliation: (1) , Department of Pediatrics, Winterberg 1, 66119, Saarbrucken, Germany Article History: Received Date: 26/02/2002 Accepted Date: 06/12/2002 Article note: Electronic Publication

Mutations in the genes for surfactant proteins B and C cause lung diseases due to an accumulation of abnormal proteins, a deficiency of the normal protein, or both mechanisms. These proteins combine with fats to lower the surface tension of fluid inside the lungs. This allows oxygen and other gases to enter and leave the body through the lungs.

Byline: Richard Plavka (1), Pavel Kopecky (1), Vaclav SebroA (1), Alena Leiska (2), Petr Svihovec (1), Josef Ruffer (1), Milena Dokoupilova (1), Blanka Zlatohlavkova (1), Vlastimil Janus (1), Martin Keszler (3) Keywords: Early surfactant High-frequency oscillatory ventilation Extreme prematurity Chronic lung disease Intraventricular hemorrhage Abstract: Abstract Objective. To determine whether early surfactant administration is superior to selective delayed treatment in terms of improving survival and/or reducing chronic lung disease in extremely premature neonates with respiratory distress syndrome (RDS) treated by high-frequency oscillatory ventilation (HFOV). Design. Prospective randomized clinical trial. Setting. Tertiary neonatal intensive care unit (NICU) in the Perinatology Center of Prague. Patients. Forty-three extremely premature infants who needed artificial ventilation within 3 h after delivery. Interventions. Patients were randomly assigned to either early (n=21) or delayed (n=22) administration of surfactant. All were ventilated by HFOV as the primary mode of ventilation using the high volume strategy aimed at optimizing lung volume. Curosurf at a dose of 100 mg/kg was given as a single bolus via the endotracheal tube within 1 min immediately after intubation in the early group (EARL), or during HFOV only when defined criteria were reached in the delayed (DEL) group. Measurements and results. No differences were noted in demographic data between the two groups. Fewer infants randomized to the EARL group required oxygen use or died at 36 weeks (combined outcome 29% vs 64%, p=0.021), and there was a lower incidence of any intraventricular hemorrhage in this group (43 vs 82%, p=0.008). Conclusions. When compared to delayed dosing, early administration of surfactant followed by HFOV facilitates and accelerates respiratory stabilization during the acute phase of severe RDS, may reduce the incidence of chronic lung disease or death and may positively influence the incidence of severe intracranial pathology in extremely premature infants with primary surfactant insufficiency. Author Affiliation: (1) Department of Obstetrics and Gynecology, Division of Neonatology, General Hospital of the 1st Medical Faculty, Charles University, 18 Apolinarska Street, 128 00 Prague, Czech Republic (2) Department of Radiology and Imaging, General Hospital of the 1st Medical Faculty, Charles University, 18 Apolinarska Street, 128 00 Prague, Czech Republic (3) Division of Neonatology, Georgetown University, Children's Medical Center, Washington, DC, USA Article History: Received Date: 16/08/2001 Accepted Date: 16/07/2002 Article note: Electronic Publication

An in vivo rat model was used to evaluate the effects of Escherichia coli pneumonia on lung function and surfactant in bronchoalveolar lavage (BAL). Total extracellular surfactant was increased in infected rats compared with controls. BAL phospholipid content in infected rats correlated with the severity of alveolar-capillary leak as reflected in lavage protein levels ([R.sup.2] = 0.908, P < 0.0001). Western blotting showed that levels of surfactant protein (SP)-A and SP-D in BAL were significantly increased in both large and small aggregate fractions at 2 and 6 h postinstillation of E. coli. SP-B was also increased at these times in the large aggregate fraction of BAL, whereas SP-C levels were increased at 2 h and decreased at 6 h relative to controls. The small-to-large (S/L) aggregate ratio (a marker inversely proportional to surfactant function) was increased in infected rats with >50 mg total BAL protein. There was a significant correlation ([R.sup.2] = 0.885, P < 0.0001) between increasing S/L ratio in BAL and pulmonary damage assessed by total protein. Pulmonary volumes, compliance, and oxygen exchange were significantly decreased in infected rats with >50 mg of total BAL protein, consistent with surfactant dysfunction. In vitro surface cycling studies with calf lung surfactant extract suggested that bacterially derived factors may have contributed in part to the surfactant alterations seen in vivo. Escherichia coli; pulmonary injury; phospholipid; surfactant dysfunction

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70-75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes. pneumocyte; lung; liposome; fluorescence

The purpose of this study was to identify culture conditions for maintenance of isolated mouse type II cells with intact surfactant protein (SP) and phospholipid production. Type II cells were isolated from 6-wk-old mice and cultured on Matrigel matrix-rat tail collagen (70:30 vol/ vol) in bronchial epithelial cell growth medium minus hydrocortisone plus 5% charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor. Under these conditions, type II cells actively produced surfactant phospholipids and proteins for at least 7 days. Synthesis and secretion of surfactant phospholipids and SP-A, -B, -C, and -D declined on day 1 of culture but recovered by day 3, reaching levels comparable to or exceeding freshly isolated cells by day 5. Abundant lamellar bodies were readily apparent in cells examined on days 5 and 7, and a surfactant pellet was recovered by centrifugation of media harvested on each day of culture. Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80. When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered surface tension to 5.2 [+ or -] 2.3 mN/m. This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture. surfactant; secretion; lung

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content. phosphatidylcholine; triglycerides; cytidylyltransferase

Adipocyte differentiation-related protein (ADrP) is an intrinsic lipid storage droplet protein that is highly expressed in lung. ADrP localizes to lipid storage droplets within lipofibroblasts, pulmonary cells characterized by high triacylglycerol, which is a precursor for surfactant phospholipid synthesis by alveolar type II epithelial (EPII) cells. The developmental pattern of ADrP mRNA and protein expression in lung tissue parallels triacylglycerol accumulation in rat lung. ADrP mRNA levels are relatively high in isolated lipofibroblasts, accounting for the high ADrP expression in lung. Isolated EPII cells, which do not store neutral lipids but derive them from lipofibroblasts, have low levels of ADrP mRNA expression. ADrP is found around lipid droplets in cultured lipofibroblasts, but not in EPII cells isolated from developing rat lung. After coculture with lipofibroblasts, EPII cells acquired ADrP, which associates with lipid droplets. Furthermore, [sup.3]H-labeled triolein in isolated ADrP-coated lipid droplets is a tenfold better substrate for surfactant phospholipid synthesis by cultured EPII cells than [sup.3]H-labeled synthetic triolein alone. Antibodies to ADrP block transfer of neutral lipid. These data suggest a role for ADrP in this novel mechanism for the transfer of lipid between lipofibroblasts and EPII cells. lipid storage droplets; neutral lipids; fetal lung development

Diesel exhaust particles (DEPs) have been shown in vivo as well as in vitro to affect the respiratory function and in particular the immune response to infection and allergens. In the current study, we investigated the effect of DEPs on the production of phosphatidylcholine (PC), a major constituent of surfactant, by rat alveolar type II (ATII) primary cells in vitro. Our results demonstrate that incubation of ATII cells with DEPs lead to a time- and dose-dependent increase in labeled PC release. This effect was mimicked by nitric oxide (NO) donors and cGMP and was abolished by inhibitors of NO synthase (NOS). In addition, a NOS inhibitor inhibits by itself the basal secretion of PC. We next examined the effects of DEPs on NOS gene expression and showed that DEPs increase NO production and upregulate both protein content and mRNA levels of the inducible NOS (NOS II). Together our data demonstrate that DEPs alter the production of surfactant by ATII cells through a NO-dependent signaling pathway. surfactant; nitric oxide

Although surfactant apoproteins are known to be mediators of innate responses, their relationship to adaptive responses has not been examined extensively. We investigated possible links between surfactant apoproteins and responses to allergens by studying alterations in surfactant apoproteins A, B, and D in a murine model of allergic pulmonary inflammation. Three murine strains (BALB/c, C57BL/6, and 129J) demonstrated increased immunostaining of surfactant apoproteins A and D in nonciliated epithelial cells of noncartilaginous airways after aerosolized challenge. In contrast, surfactant apoprotein B immunostaining was unchanged. Immunoblotting demonstrated increased surfactant A in bronchoalveolar lavage fluid after allergen sensitization and challenge. Surfactant apoprotein A and D induction required T and/or B lymphocyte responses to allergen, since the induction was absent in recombinase-activating gene-deficient mice, which lack functional lymphocytes. We conclude that increased immunoreactivity of two collectins, surfactant apoproteins A and D, occurs within the response to allergen. Our findings support a model in which surfactant apoproteins A and D are important to both innate immunity and adaptive immune responses to allergens. lung; T lymphocytes; murine models; innate

Mice lacking surfactant protein SP-A [SP-A(-/-)] and wild type SP-A(+/+) mice were infected with influenza A virus (IAV) by intranasal instillation. Decreased clearance of IAV was observed in SP-A(-/-) mice and was associated with increased pulmonary inflammation. Treatment of SP-A(-/-) mice with exogenous SP-A enhanced viral clearance and decreased lung inflammation. Uptake of IAV by alveolar macrophages was similar in SP-A(-/-) and SP-A(+/+) mice. Myeloperoxidase activity was reduced in isolated bronchoalveolar lavage neutrophils from SP-A(-/-) mice. B lymphocytes and activated T lymphocytes were increased in the lung and spleen, whereas T helper (Th) 1 responses were increased [interferon-[gamma] interleukin (IL)-2, and Ig[G.supb.2a]] and Th2 responses were decreased (IL-4, and IL-10, and Ig[G.sub.1]) in the lungs of SP-A(-/-) mice 7 days after IAV infection. In the absence of SP-A, impaired viral clearance was associated with increased lung inflammation, decreased neutrophil myeloperoxidase activity, and increased Th1 responses. Because the airway is the usual portal of entry for IAV and other respiratory pathogens, SP-A is likely to play a role in innate defense and adaptive immune responses to IAV. virus; lung; lectin; surfactant protein-A; surfactant protein-A-deficient mice

Oligohydramnios (OH) retards fetal lung growth by producing less lung distension than normal. To examine effects of decreased distension on fetal lung development, we produced OH in rats by puncture of uterus and fetal membranes at 16 days of gestation; fetuses were delivered at 21 or 22 days of gestation. Controls were position-matched littermates in the opposite uterine horn. OH lungs had lower weights and less DNA, protein, and water, but no differences in saturated phosphatidylcholine, surfactant proteins (SP)-A and -B, and mRNA for SP-A, -B, -C, and -D. To evaluate effects on epithelial differentiation, we used RT[I.sub.40] and RTI[I.sub.70], proteins specific in lung to luminal surfaces of alveolar type I and II cells, respectively. At 22 days of gestation, OH lungs had less RT[I.sub.40] mRNA (P < 0.05) and protein (P < 0.001), but RTI[I.sub.70] did not differ from controls. With OH, type I cells (in proportion to type II cells) covered less distal air space perimeter (P < 0.01). We conclude that OH, which retards lung growth, has little effect on surfactant and impedes formation of type I cells relative to type II cells. fetal lung development; lung distension; pulmonary epithelial differentiation; pulmonary hypoplasia; pulmonary surfactant

Gobran, Laurice I., and Seamus A. Rooney. Regulation of SP-B and SP-C secretion in rat type II cells in primary culture. Am J Physiol Lung Cell Mol Physiol 281: L1413-L1419, 2001.--Secretion of lung surfactant phospholipids is a highly regulated process. A variety of physiological and pharmacological agents stimulate surfactant phospholipid secretion in isolated type II cells. Although the lipid and hydrophobic protein components of surfactant are believed to be secreted together by exocytosis of lamellar body contents, regulation of surfactant protein (SP) B and SP-C secretion has not previously been examined. To address the question of whether secretion of SP-B and SP-C is stimulated by the same agonists that stimulate phospholipid secretion, we measured secretion of all four SPs under the same conditions used to measure phosphatidylcholine secretion. Freshly isolated rat type II cells were cultured overnight and exposed to known surfactant phospholipid secretagogues for 2.5 h, after which the amounts of SP-A, SP-B, SP-C, and SP-D in the medium were measured with immunoblotting. Secretion of SP-B and SP-C was stimulated three- to fivefold by terbutaline, 5'-(N-ethylcarboxyamido)adenosine, ATP, 12-O-tetradecanoylphorbol 13-acetate, and ionomycin. Similar to their effects on phospholipid secretion, the stimulatory effects of the agonists were abolished by Ro 31-8220. Secretion of SP-A and SP-D was not stimulated by the secretagogues tested. We conclude that secretion of the phospholipid and hydrophobic protein components of surfactant is similarly regulated, whereas secretion of the hydrophilic proteins is regulated differently. surfactant protein; lung surfactant; regulated exocytosis; surfactant secretagogues; P2[Y.sub.2] agonists; 12-O-tetradecanoylphorbol 13-acetate Received 13 June 2001; accepted in final form 2 August 2001

Research has been conducted on the surfactant protein-A involved in the biological functions such as biophysical activity, host defence and surfactant homeostasis. The use of the infrared transmission spectroscopy in investigating the bulk-phase interactions between surfactant protein-A, 1,2-dipalmitoylphosphatidylcholine and Ca (super)2+ ions is described.

Byline: Ilka Frerking (1), Andreas Gunther (2), Werner Seeger (2), Ulrich Pison (1) Abstract: The first successful clinical pilot studies of surfactant replacement were published about 20 years ago as a logical extension of experimental studies showing beneficial effects in pre-term animals. The efficacy of this therapy for immature new-borns has been confirmed in various controlled trials and surfactant therapy is now part of the routine management of the infant respiratory distress syndrome. During the last decade there has been growing insight into the functional role of surfactant components and the mechanisms by which exogenous surfactant exerts its therapeutic effects on lung mechanics, gas exchange and host defence. Of particular interest in this context is the essential role that surfactant-associated proteins play in the surface tension-limiting ability of surfactant, as well as their contribution to pulmonary defence. Indications for surfactant replacement have widened in recent years and promising results have been obtained for adult conditions such as the acute respiratory distress syndrome (ARDS), pneumonia, chronic obstructive and allergic lung diseases. This review outlines the complexity of the surfactant system and describes its basic biophysics, physiology and biochemistry. Problems related to the development of exogenous surfactant preparations, the exploration of clinical targets for surfactant therapy and pathophysiological mechanisms interfering with surfactant function in various forms of lung disease will be discussed. Author Affiliation: (1) Department of Anaesthesiology and Intensive Care Medicine, Charite, Campus Virchow-Klinikum, Humboldt University, 13353 Berlin, Germany (2) Department of Internal Medicine, Justus-Liebig University, 35385 Giessen, Germany Article History: Received Date: 27/08/2001 Accepted Date: 04/09/2001 Article note: Final revision received: 27 August 2001 Electronic Publication

Byline: M. Ragaller (1), J. Bleyl (2), U. Tscho (2), T. Winkler (3), M. Regner (3), S. Rasche (2), T. Koch (4), M. Albrecht (5) Keywords: Acute lung injury Perfluorocarbons Aerosol Jet nebuliser Prostacyclin Oleic acid Low surface tension Surfactant Abstract: Objective: To evaluate the effects of PFC aerosol compared to PGI.sub.2 aerosol and NaCl aerosol on gas exchange and lung mechanics in oleic acid-induced acute lung injury. Design: A prospective, controlled, randomised, in vivo animal laboratory study. Setting: Research laboratory at an university hospital. Subjects: Twenty one (n=21) adult sheep of either gender weighing 26.8+-6.4 kg. Interventions: The animals were randomised to three groups: PFC aerosol (perfluorooctane), PFC group prostacyclin aerosol (Flolan), PGI.sub.2 group and NaCl aerosol (0.9% sodium chloride solution), control group. After induction of anaesthesia and placement of vascular catheters, lung injury was induced with 0.12 ml*kg.sup.--1 oleic acid. Aerosols were continuously administered for 2 h using a jet nebuliser. Gas exchange, pulmonary mechanic, and haemodynamic parameters were obtained at regular intervals. Measurements and main results: PFC aerosol increased oxygenation (PaO.sub.2) 15 min after the initiation of treatment up to 120 min (P Author Affiliation: (1) Department of Anaesthesiology and Intensive Care Medicine, University Hospital Carl Gustav Carus, Medical Faculty, Technical University of Dresden, Fetscherstrasse 74, 01307 Dresden, Germany (2) Department of Anaesthesiology and Intensive Care Medicine, Technical University of Dresden, Dresden, Germany (3) Department of Anaesthesiology and Intensive Care Medicine, Technical University of Dresden, Dresden, Germany (4) Department of Anaesthesiology and Intensive Care Medicine, Technical University of Dresden, Dresden, Germany (5) Department of Anaesthesiology and Intensive Care Medicine, Technical University of Dresden, Dresden, Germany Article History: Received Date: 05/07/2000 Accepted Date: 23/01/2001 Article note: Final revision received: 19 January 2001 Electronic Publication

Byline: G.F. Vazquez de Anda (1), R.A. Lachmann (1), D. Gommers (1), S.J. Verbrugge (1), J. Haitsma (1), B. Lachmann (1) Keywords: Ventilation-induced lung injury Mechanical ventilation Pulmonary surfactant Animal Rat Abstract: Objective: It has been demonstrated that pulmonary surfactant plays a role in the pathophysiology of ventilation-induced lung injury (VILI). Therefore, we investigated whether exogenous surfactant might restore lung function and lung mechanics in an established model of VILI. Design: Prospective, randomized, animal study. Setting: Experimental laboratory of a university. Subjects: Twenty-four adult male Sprague-Dawley rats. Interventions: First, a group of six animals were killed immediately after induction of anesthesia and used as healthy controls. Then, in 18 rats, VILI was induced by increasing peak inspiratory pressure (PIP) to 45 cm[H.sub.2]O without positive end-expiratory pressure (PEEP) for 20 min. Thereafter, animals were randomly divided into three groups of six animals each: one group was killed immediately after VILI and served as VILI-control. In the other two groups, ventilator settings were changed to a PIP of 30 cm[H.sub.2]O and a PEEP of 10 cm[H.sub.2]O, and a respiratory rate of 40 bpm. One group received a bolus of surfactant and the other group received no treatment. Measurements and results: Blood gas tension and arterial blood pressures were recorded every 30 min for 2 h. After the study period, a pressure-volume curve was recorded. Then, a broncho-alveolar lavage (BAL) was performed to determine protein content, minimal surface tension, and surfactant composition in the BAL fluid. Oxygenation, lung mechanics, surfactant function and composition were significantly improved in the surfactant-treated group compared to the ventilated and non-ventilated control groups. Conclusion: We conclude that exogenous surfactant can be used to treat VILI. Author Affiliation: (1) Department of Anesthesiology, (Room Ee 2393), Erasmus University Rotterdam, P.O. Box 1738, 3000 Rotterdam, The Netherlands (2) Unidad de Cuidados Intensivos, Hospital de Especialidades 'Dr. Bernardo Sepulveda G.', del Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social. Av. Cuauhtemoc No. 330, C.P. 06725, Mexico City, Mexico Article History: Received Date: 19/05/2000 Accepted Date: 29/11/2000 Article note: Final revision received: 21 November 2000 Electronic Publication

Two rat surfactant protein A (SP-A) isoforms are the result of a novel mechanism that includes alternative translation initiation. One gene for rat surfactant protein A encodes the two isoforms. The rat SP-A isoforms mechanism has the alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.

Rat surfactant protein A (SP-A) trimers in association with phospholipid monolayers have been analyzed to determine their three-dimensional structure. The glycosylation site of SP-A was at the side of each subunit, which would suggest that the covalently linked carbohydrate moiety likely is in the spaces between the adjacent globular domains, which would not sterically interfere with ligand binding.

This review discusses the growth and differentiation of the fetal lung at molecular levels during the transition of fetus to an air-breathing environment. Data on the lung development processes and fetal capacity to synthesize the surfactant protein are presented.

Byline: G. F. Vazquez de Anda (1), A. Hartog (1), S. J. C. Verbrugge (1), D. Gommers (1), B. Lachmann (1) Keywords: Key words High frequency oscillatory ventilation; Pressure control ventilation; Surfactant deficiency; Alveolar recruitment; Open lung concept; Animal model Abstract: Objective: To demonstrate in experimental animals with respiratory insufficiency that under well-defined conditions, commercially available ventilators allow settings which are as effective as high frequency oscillatory ventilators (HFOV), with respect to the levels of gas exchange, protein infiltration, and lung stability. Design: Prospective, randomized, animal study. Setting: Experimental laboratory of a university. Subjects: 18 adult male Sprague-Dawley rats. Interventions: Lung injury was induced by repeated whole-lung lavage. Thereafter, the animals were assigned to pressure-controlled ventilation (PCV) plus The Open Lung Concept (OLC) or HFOV plus OLC (HFO.sub.OLC). In both groups, an opening maneuver was performed by increasing airway pressures to improve the arterial oxygen tension/fractional inspired oxygen (PaO.sub.2/FIO.sub.2) ratio to L 500 mm Hg thereafter, airway pressures were reduced to minimal values, which kept PaO.sub.2/FIO.sub.2 L 500 mm Hg. Pressure amplitude was adjusted to keep CO.sub.2 as close as possible in the normal range. Measurements and results: Airway pressure, blood gas tension, and arterial blood pressure were recorded every 30 min. At the end of the 3-h study period, a pressure-volume curve was recorded and bronchoalveolar lavage was performed to determine protein content. After the recruitment maneuver, the resulting mean airway pressure to keep a PaO.sub.2/FIO.sub.2 L 500 mm Hg was 25 +- 1.3 cm [H.sub.2]O during PCV.sub.OLC and 25 +- 0.5 cm [H.sub.2]O during HFOV.sub.OLC. Arterial oxygenation in both groups was above L 500 mm Hg and arterial carbon dioxide tension was kept close to the normal range. No differences in mean arterial pressure, lung mechanics and protein influx were found between the two groups. Conclusions: This study shows that in surfactant-deficient animals, PCV, in combination with a recruitment maneuver, opens atelectatic lung areas and keeps them open as effectively as HFOV. Author Affiliation: (1) Department of Anaesthesiology (Room Ee 2393), Erasmus University Rotterdam, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands Tel.: 31(10)4 08 73 12 Fax: 31(10)4 08 94 50, NL (2) Unidad de Cuidados Intensivos Hospital de Especialidades 'Dr. Bernardo Sepulveda G.', del Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social Av. Cuauhtemoc No. 330, C. P. 06725, Mexico, D. F. Article note: Received: 14 October 1998 Final revision received: 11 April 1999 Accepted: 25 June 1999

Electron microscopy was employed to investigate in situ lipid forms in alveoli of rat lungs after two ventilation strategies (15 min at pressures (cmH2O) of 20/0 or 20/10). Findings indicated that a ventilation pattern of 20/0 compared with that of 20/10 group was linked to a substantially larger vesicular form, suggesting that enhanced net production of these surfactant forms during a large tidal volume breathing pattern.

The protein kinase C (PKC) isomers that are translocated by ATP, UTP, 12-O-tetradecanoylphorbol 13-acetate (TPA) and dioctanoylglyerol were studied in cultured type II cells isolated from adult rats. Findings showed that direct activators and agents that generate endogenous diacylglycerols have different PKC activation patterns. Because it was activated by various kinds of secretagogues, PCK-mu may have an important role in the physiological regulation of surfactant secretion.

The role of plasma free fatty acids and de novo synthesized fatty acids in the synthesis of lung surfactant phosphatidylcholine (PC) has been studied. A new stable isotope tracer model was used in infusing (1,2-13C2)acetate and uniformly labeled (U-13C16)palmitate in nine overnight fasted pigs. Surfactant kinetics were measured during the basal state and during low-dose glucose infusion. Results indicate glucose does not affect PC synthesis. Plasma is also the main contributor of fatty acids for surfactant PC synthesis.

A series of transmission-electron-microscopic studies were conducted using purified surfactant protein-A and lipid vesicles made in vitro as well as native surfactant from bovine lung to elucidate the interaction of SP-A and various lipids commonly found in tubular myelin (TM). Dipalmitoylphosphatidylcholine-egg phosphatidylcholine bilayers formed corrugations, folds, and predominantly laticelike structures. These results offer evidence that SPs and lipid composition play crucial roles in the formation of the lipid membranes of TM and TM-like structures.

The effects of lipopolysaccharide on surfactant protein A (SP-A)- and D (SP-D)-mediated functions, in particular the production of nitric oxide metabolites by alveolar macrophages, were examined in rats. A correlation was found between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages. A procedure for reducing endotoxin contamination while maintaining optimal protein recovery was also developed.

The influence of two surfactant preparations, a bovine lung extract and a porcine lung extract, on the incorporation of fibronectin into fibrin was studied. Surfactant phospholipids were observed to be associated with insoluble fibrin, factor XIIIa-cross-linked fibrin, and cross-linked fibrin with incorporated fibronectin. It was concluded that factor XIIIa-cross-linked fibrin-fibronectin is able to incorporate surfactant phospholipids in amounts comparable to fibrin clots without fibronectin. It was also concluded that the binding of fibronectin to fibrin is partially inhibited in the presence of pulmonary surfactant.

Byline: R. K. L. Bouhafs (1), C. Jarstrand (1) Keywords: Key words: Bacteria--Surfactant--Reactive oxygen radicals--Lipid peroxidation. Abstract: The epithelium of the lung is lined with extracellular pulmonary surfactant. This is the surface that invading bacteria first come into contact with when they enter the alveoli. As bacteria become established and interact with this layer, various characteristics of surfactant may become altered. We studied free radical production by three bacterial species, group B streptococci, Escherichia coli, and Pseudomonas aeruginosa, as well as the effect of two concentrations of lung surfactant (Curosurf at 0.04 and 0.4 mg/ml) on this production estimated by the nitro blue tetrazolium reduction test. We also measured the lipid peroxidation of surfactant at various incubation times (0--20 h), using a LPO-586 test kit. In addition, the effect of vitamin E as an antioxidant in a concentration of 0.5 uM was determined by the lipid peroxidation test. We found that the nitro blue tetrazolium reduction by the three bacterial species and lipid peroxidation of lung surfactant increased with time. Vitamin E reduced the lipid peroxidation of this surfactant. By measuring bacterial growth at various incubation times we showed that lung surfactant was bactericidal to group B streptococcal and E. coli strains and that P. aeruginosa strains were resistant to surfactant. We conclude that bacteria, probably by their production of reactive oxygen species, cause lipid peroxidation of lung surfactant. Author Affiliation: (1) Department of Immunology, Microbiology, Pathology, and Infectious Diseases, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden, SE Article note: Accepted for publication: 5 October 1998

A study was conducted to show that recombinant surfactant protein D carbohydrate recognition domain is a chemoattractant for human neutrophils. Escherichia coli was prepared in Luria-Bertani broth supporting ampicillin at 37 degrees C. Plasmids determined from single colonies were then subjected to double-stranded sequencing using the T7 sequencing kit. Moreover, neutrophil chemotaxis was analyzed in modified Boyden chamber assays.

The binding of normal and nitrated surfactant protein A (SP-A) to Pneumocystis carinii and alveolar macrophages has been studied. The influence of SP-A on the interaction of P. carinii with the macrophages has also been examined. SP-A is the most abundant apoprotein and belongs to the C-type lectin group. Results indicate that SP-A can bind with P. carinii through its carbohydrate recognition domain. It has also been observed that the normal but not the nitrated type of SP-A facilitates the binding and phagocytosis of P. carinii to alveolar macrophages.

Byline: H. Nomori (1), H. Horio (1), G. Fuyuno (2), R. Kobayashi (2), S. Morinaga (3), K. Suemasu (1) Keywords: Key words: Surfactant protein--Smoking--Alveolar type 2 cells--Age. Abstract: Serum levels of surfactant protein A (SP-A) were studied in 237 healthy subjects in relation to sex, age, and smoking habits. SP-A values in male smokers were significantly higher than those in male nonsmokers (p &lt 0.001). The amount of cigarette smoking did not correlate significantly with SP-A values, however. SP-A values in young nonsmoking males and females were somewhat lower than those in older, but without significant difference. No significant difference in values was found between the sexes. We conclude that (1) smoking increases serum levels of SP-A, and (2) SP-A serum levels are not affected by age and sex. Author Affiliation: (1) Department of Surgery, Saiseikai Central Hospital, Tokyo, 108-0073, Japan, JP (2) Department of Medicine, Saiseikai Central Hospital, Tokyo, 108-0073, Japan, JP (3) Department of Pathology, Saiseikai Central Hospital, Tokyo, 108-0073, Japan, JP Article note: Accepted for publication: 6 March 1998

Retinoic acid (RA) enhanced the genetic transcription of surfactant protein-B (SP-B) messenger ribonucleic acid in H441 cells. The findings identified a direct activation of the SP-B mRNA transcription by RA, retinoid nuclear receptors (RAR) and retinoid X receptors (RXR). Apart from this, immunohistochemical analysis of the H441 cells revealed that RAR, RA and RXR contributed to mRNA accumulation and SP-B synthesis in vitro.

Experiments were performed to illustrate and compare the biophysical and functional properties of pulmonary surfactants from surfactant protein (SP) A deficient (-/-) mice to SP-A (+/+) mice. Differences in adsorption rate, structure, density and sensitivity to plasma protein inhibition were demonstrated by SP-A deficient mice in comparison to normo-SP-A mice. The findings suggested the sensitivity of SP-A(-/-) to inactivation following lung injury.

A study was conducted on surfactant protein (SP) C metabolism. In vivo measurements of SP-C clearance in two species were combined with in vitro measurements of the uptake of rSP-C by MLE-12 cells. The findings indicate a similarity between the responses of rSP-C uptake after the addition of the other SPs and nSP-C, suggesting that cellular uptake mechanisms seem to be similar for nSP-C and rSP-C. Further studies are recommended on the exact cellular pathways and mechanisms regulating this complex process of conservation and catabolism of surfactant components.

Tyrosine kinase enzyme inhibition experiments were undertaken to investigate the contribution of kinase signal transduction on the expression of surfactant protein in differentiating human fetal lung tissue cultures. Results reveal that inhibition of the cognate tyrosine kinase of the epidermal growth factor receptor resulted to the inhibition of surfactant protein synthesis indicating that tyrosine kinase subserves an important signal transduction function.