Isolation of Alveolar Type II Cells from Adult Bovine Lung.

Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during epithelial regeneration. Their secretion of surfactant protein helps maintain homeostasis and exerts protective, antimicrobial properties. ATII cells remain difficult to study, partly due to inefficient and expensive isolation methods, a propensity to differentiate into ATI cells, and susceptibility to fibroblast contamination. Published methods of isolation often require specialized technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis. Presented here is a simple and cost-effective method for generation of bovine primary ATII cells. These cells exhibit an ATII phenotype in 2D and 3D culture and are conducive to further study of the role of ATII cells in bovine respiratory diseases. © 2019 by John Wiley & Sons, Inc.

Protective role of glucocorticosteroid prior to endotoxin exposure in cultured neonatal type II alveolar epithelial cells.

BACKGROUND: Dexamethasone (DEX) is widely used for antenatal lung maturation and has been investigated to prevent premature lung injury by inhibiting postnatal inflammation. Its pharmacological mechanisms in the treatment of bacterial infection-induced injury of neonatal lung parenchymal cells remain to be clarified. We hypothesized that DEX pretreatment may attenuate endotoxin-induced growth suppression and regulate cytokine mRNA expression in cultured neonatal type II alveolar epithelial cells (AEC-II). METHODS: AEC-II of newborn piglets were freshly isolated and cultured. After pretreatment of 0.01, 0.1, 1.0 and 10 µmol/l DEX (E0.01, E0.1, E1.0 and E10 group, respectively) for 24 h, the cells were cultured with 1 µg/ml lipopolysaccharides (LPS) for 7 days with medium replacement every 24 h. Messenger RNA expression of surfactant proteins (SPs), pro-inflammatory cytokines and multiple growth factors (GF) were determined by RT-PCR, along with the cell growth and apoptosis measurements. RESULTS: LPS without DEX pretreatment suppressed cell proliferation, enhanced expression of pro-inflammatory cytokine mRNA and apoptosis, which was ameliorated in all DEX-pretreated groups on day 3. On day 3 and 5, only cells pretreated by E1.0 and E10 showed a 20-fold increase in insulin-like GF-1 mRNA expression whereas the expression of other GFs was down-regulated. LPS exposure reduced the expression of SP-A, B, C and Aquaporin-5 mRNA on day 3-7. However, the expression of SP-C mRNA was increased in E1.0 on day 3, which was supported by in situ expression of pro-SP-C with immunocytochemical assay. CONCLUSION: LPS-induced in vitro AEC-II injury was partially prevented by DEX pretreatment, with 1.0 µmol/l being the potentially optimal concentration. (253 words).

Hyperoxia treatment of TREK-1/TREK-2/TRAAK-deficient mice is associated with a reduction in surfactant proteins.

We previously proposed a role for the two-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy among the three TREK isoforms (TREK-1, TREK-2, and TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all three TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient [triple knockout (KO)] mice to either room air, 72-h HO, MV [high and low tidal volume (TV)], or a combination of HO + MV and measured quasistatic lung compliance, bronchoalveolar lavage (BAL) protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression and attempted to prevent HO-induced lung injury by prophylactically administering an exogenous surfactant (Curosurf). HO treatment increased lung injury in triple KO but not WT mice, including an elevated LIS, BAL protein concentration, and markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO + MV (low TV) further decreased lung compliance in triple KO but not WT mice, and HO + MV (high TV) was lethal for triple KO mice. In triple KO mice, the HO-induced lung injury was associated with decreased surfactant protein (SP) A and SPC but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors nuclear factor-1 (NF-1), NKX2.1/thyroid transcription factor-1 (TTF-1) or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple KO mice.

Effect of tobacco extract on surfactant synthesis and its reversal by retinoic acid-role of cell-cell interactions in vitro.

Tobacco induces oxidative stress in the alveolar epithelium and causes its damage. Retinoic acid (RA) has a cardinal role in alveolar cell growth, differentiation, and maturation. The aim of the study was to investigate the role of cell-cell interactions and whether RA could reverse the effect of tobacco extract on epithelial function as expressed by surfactant synthesis. For this, an in vitro model, which provides multiple cell type interactions, as seen in vivo, was used. We had used the major lung cell types, alveolar epithelial and mesenchymal cells represented by the cell lines A549 (human lung adenocarcinoma cell line), and human fetal lung fibroblast-1 (HFL-1) for developing the monoculture and co-culture systems and studied the effect of tobacco extract and retinoic acid. The effect of tobacco and retinoic acid both singly and in combination on proliferation and surfactant synthesis was analyzed. Retinoic acid induced proliferation and upregulated surfactant synthesis in monocultures and co-cultures. Tobacco extract at 100 µg/ml concentration decreased A549 proliferation and upregulated surfactant protein mRNA expression. In co-cultures treated with tobacco extract (100 µg/ml), retinoic acid (1 µM), regulated cell proliferation, and surfactant protein mRNA expression vis-à-vis the monoculture system. This clearly points to the fact that cell-cell interactions modulate the effect of additives or stimulants and help in assessing the in vivo combinatorial responses in vitro and that the retinoic acid effect is regenerative.

Staphylococcus aureus and Pseudomonas aeruginosa express and secrete human surfactant proteins.

Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express 'human-like' surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment.

Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells.

The alveolar surface is covered by an epithelium composed of 2 main cell types: type I and type II cells. Alveolar type II (ATII) cells have a distinct morphology with apical microvilli and characteristic lamellar bodies, which are the intracellular storage form of pulmonary surfactant. ATII cells play an important role in innate immunity and produce and secrete pulmonary surfactant. They proliferate to restore the epithelium after damage to the more sensitive type I cells. We developed an efficient and rapid method to isolate and purify ATII cells from mice. Alveolar epithelial cells were dissociated in the murine lung with dispase and lung tissue was gently minced with a GentleMACS Dissociator. ATII cell purification was performed using negative depletion with CD45 MicroBeads and positive selection for the epithelial-cell adhesion molecule (Ep-CAM) by magnetic labeling with Streptavidin MicroBeads in MACS LS columns. The purity of these cells as measured by flow cytometry was up to 92.1% and 91.1% for co-staining with Ep-CAM and cytokeratin and co-staining with Ep-CAM and SP-A, respectively. The resulting ATII cell population has a high purity, viability, and yield. The phenotype of isolated and cultured ATII cells was confirmed by electron micrographs, expression of surfactant proteins (SP-A, proSP-B, mature SP-B, proSP-C, SP-D), and lysophosphatidylcholine acyltransferase (LPCAT) by western blotting and immunocytofluorescence. This protocol is based on surface antigens and our data demonstrated that murine ATII cells can be rapidly isolated, efficiently purified, and effectively cultured.

Prostaglandins mediate the fetal pulmonary response to intrauterine inflammation.

Intra-amniotic (IA) lipopolysaccharide (LPS) induces intrauterine and fetal lung inflammation and increases lung surfactant and compliance in preterm sheep; however, the mechanisms are unknown. Prostaglandins (PGs) are inflammatory mediators, and PGE(2) has established roles in fetal lung surfactant production. The aim of our first study was to determine PGE(2) concentrations in response to IA LPS and pulmonary gene expression for PG synthetic [prostaglandin H synthase-2 (PGHS-2) and PGE synthase (PGES)] and PG-metabolizing [prostaglandin dehydrogenase (PGDH)] enzymes and PGE(2) receptors. Our second study aimed to block LPS-induced increases in PGE(2) with a PGHS-2 inhibitor (nimesulide) and determine lung inflammation and surfactant protein mRNA expression. Pregnant ewes received an IA saline or LPS injection at 118 days of gestation. In study 1, fetal plasma and amniotic fluid were sampled before and at 2, 4, 6, 12, and 24 h after injection and then daily, and fetuses were delivered 2 or 7 days later. Amniotic fluid PGE(2) concentrations increased (P < 0.05) 12 h and 3-6 days after LPS. Fetal lung PGHS-2 mRNA and PGES mRNA increased 2 (P = 0.0084) and 7 (P = 0.014) days after LPS, respectively. In study 2, maternal intravenous nimesulide or vehicle infusion began immediately before LPS or saline injection and continued until delivery 2 days later. Nimesulide inhibited LPS-induced increases in PGE(2) and decreased fetal lung IL-1ß and IL-8 mRNA (P ≤ 0.002) without altering lung inflammatory cell infiltration. Nimesulide decreased surfactant protein (SP)-A (P = 0.05), -B (P = 0.05), and -D (P = 0.0015) but increased SP-C mRNA (P = 0.023). Thus PGHS-2 mediates, at least in part, fetal pulmonary responses to inflammation.

Leptin does not influence surfactant synthesis in fetal sheep and mice lungs.

In the fetus, leptin in the circulation increases at late gestation and likely influences fetal organ development. Increased surfactant by leptin was previously demonstrated in vitro using fetal lung explant. We hypothesized that leptin treatment given to fetal sheep and pregnant mice might increase surfactant synthesis in the fetal lung in vivo. At 122-124 days gestational age (term: 150 days), fetal sheep were injected with 5 mg of leptin or vehicle using ultrasound guidance. Three and a half days after injection, preterm lambs were delivered, and lung function was studied during 30-min ventilation, followed by pulmonary surfactant components analyses. Pregnant A/J mice were given 30 or 300 mg of leptin or vehicle by intraperitoneal injection according to five study protocols with different doses, number of treatments, and gestational ages to treat. Surfactant components were analyzed in fetal lung 24 h after the last maternal treatment. Leptin injection given to fetal sheep increased fetal body weight. Control and leptin-treated groups were similar in lung function (preterm newborn lamb), surfactant components pool sizes (lamb and fetal mice), and expression of genes related to surfactant synthesis in the lung (fetal mice). Likewise, saturated phosphatidylcholine and phospholipid were normal in mice lungs with absence of circulating leptin (ob/ob mice) at all ages. These studies coincided in findings that neither exogenously given leptin nor deficiency of leptin influenced fetal lung maturation or surfactant pool sizes in vivo. Furthermore, the key genes critically required for surfactant synthesis were not affected by leptin treatment.

Decreased surfactant phosphatidylcholine synthesis in neonates with congenital diaphragmatic hernia during extracorporeal membrane oxygenation.

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)C] glucose. The (13)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.

ErbB4 regulates surfactant synthesis and proliferation in adult rat pulmonary epithelial cells.

ErbB4 is a predominant heterodimer for other ErbB receptors in late fetal lung development where it participates in regulating type II cell surfactant synthesis. To further elucidate the role of ErbB4 in pulmonary alveolar epithelial cell function, the authors hypothesized that ErbB4 participates in maintaining adult lung type II cell homeostasis. The authors used small interfering RNA (siRNA) to down-regulate endogenous, ErbB4 receptors in the adult rat lung epithelial L2 cell line and measured neuregulin 1beta (NRG1beta)-, and fibroblast conditioned medium (FCM)-induced effects on L2 cell surfactant phospholipid synthesis and proliferation. Under control conditions, total and phosphorylated ErbB4 were significantly increased after both NRG1beta and FCM treatment, as were surfactant phospholipids synthesis and cell proliferation. Down-regulation of ErbB4 with siRNA reduced stimulation of NRG1beta- and FCM-induced ErbB4 phosphorylation, decreased endogenous surfactant phospholipid synthesis, and blocked NRG1beta- and FCM-stimulated surfactant phospholipid synthesis. NRG1beta- and FCM-induced cell proliferation was not affected. The authors conclude that ErbB4 participates in maintaining adult lung alveolar epithelial cell surfactant synthesis and proliferation with development-specific functions.

Diesel exhaust enhanced susceptibility to influenza infection is associated with decreased surfactant protein expression.

We have previously shown that exposure of respiratory epithelial cells to diesel exhaust (DE) enhances susceptibility to influenza infection and increases the production of interleukin (IL)-6 and interferon (IFN)-beta. The purpose of this study was to confirm and expand upon these in vitro results by assessing the effects of DE exposure on the progression of influenza infection and on development of associated pulmonary immune and inflammatory responses in vivo. BALB/c mice were exposed to air or to DE containing particulate matter at concentrations of 0.5 or 2 mg/m(3) for 4 h/day for 5 days and subsequently instilled with influenza A/Bangkok/1/79 virus. Exposure to 0.5 mg/m(3) (but not the higher 2-mg/m(3) dose) of DE increased susceptibility to influenza infection as demonstrated by a significant increase in hemagglutinin (HA) mRNA levels, a marker of influenza copies, and greater immunohistochemical staining for influenza virus protein in the lung. The enhanced susceptibility to infection observed in mice exposed to 0.5 mg/m(3) of DE was associated with a significant increase in the expression of IL-6, while antiviral lung IFN levels were unaffected. Analysis of the expression and production of surfactant proteins A and D, which are components of the interferon-independent antiviral defenses, showed that these factors were decreased following exposure to 0.5 mg/m(3) of DE but not to the higher 2-mg/m(3) concentration. Taken together, the results demonstrate that exposure to DE enhances the susceptibility to respiratory viral infections by reducing the expression and production of antimicrobial surfactant proteins.

Surfactant and its role in chronic sinusitis.

Although numerous studies have focused on the nature and defensive role of surfactant in the lower airways, relatively little is known about its role in the upper airways. Decreased levels of the main component of surfactant--phospholipids--have been implicated in atrophic rhinitis. The lamellar body arrangement of phospholipids has now been demonstrated in both normal and diseased sinus tissue, resulting in the implication that these structures may play a crucial role in mucociliary clearance against inhaled pathogens, as well as in the regulation of mucous viscosity. Furthermore, they may be secreted from sinonasal ciliated epithelium. Surfactant proteins (SPs) make up a relatively smaller proportion of surfactant, but appear to have an important role in innate immunity. Altered levels of SPs have been observed in a number of respiratory tract diseases. These SPs may prove to play a significant role in chronic sinusitis. Demonstrated expression of SP-A and SP-D in diseased and normal sinus tissue may mean that these SPs are excreted into the airway-lining fluid of the sinuses. Additionally, initial contact and interaction between pathogens and SP-A and SP-D may occur relatively early after inhalation and deposition into the mucus of the respiratory tract. These findings may lead to potential therapeutic options for difficult-to-treat sinus disease in the future.

Tracheal occlusion in fetal rats alters expression of mesenchymal nuclear transcription factors without affecting surfactant protein expression.

BACKGROUND/PURPOSE: Mesenchymal nuclear transcription factors (MNTF) are involved in lung development and maturation and regulate surfactant protein (SP) expression. Prolonged (>2 weeks) fetal tracheal occlusion (TO) has been shown to accelerate lung growth and inhibit pulmonary surfactant synthesis. The effects of TO on SP expression and MNTF, however, have not been formally assessed. The objectives of this study were to evaluate the effects of short-term (3 days) TO on normal lung growth and protein expression of pulmonary MNTF involved in SP synthesis. METHODS: At E19 (term, 22 days), 2 fetuses per time-dated Sprague-Dawley rats underwent either TO (n = 23) or a sham (n = 22) operation. Lungs were harvested 72 hours post surgery. Pulmonary SP-A; SP-B; SP-C messenger RNA (mRNA) expression; and SP-A and SP-B, Hoxb5, thyroid transcription factor 1, and retinoic X receptor-alpha protein expression were analyzed. RESULTS: Lung weight was significantly increased by TO (TO 0.32 +/- 0.02g vs SHAM 0.14 +/- 0.01 g; P < .001), resulting in 123% increase of the lung-to-body-weight ratio. No difference of SP-A-mRNA (177 +/- 4.3 TO vs 169 +/- 4.4 SHAM; P = .25), SP-B-mRNA (87.7 +/- 0.2 TO vs 87.4 +/- 0.02 SHAM; P = .33), and SP-C-mRNA (186.5 +/- 3.2 TO vs 183.2 +/- 2.7 SHAM; P = .45) expression was found. Surfactant protein A (175.6 +/- 25.3 TO vs 192.5 +/- 19.8 SHAM; P = .59) and SP-B (163.4 +/- 5.2 TO vs 166.8 +/- 9.3 SHAM; P = .75) protein expression were similar in both groups; however, Hoxb5 (70.3 +/- 18.9 TO vs 130.6 +/- 5.1 SHAM; P = .02) and thyroid transcription factor 1 (102.6 +/- 19 TO vs 181.1 +/- 6.3 SHAM; P = .007) expression were significantly decreased. Retinoic X receptor-alpha expression tended to be increased by TO (171.9 +/- 6.0 TO vs 155.4 +/- 6.7 SHAM; P = .06). CONCLUSIONS: Short-term TO late in gestation induces rapid lung growth. Surfactant protein-mRNA and protein expression are not significantly altered. Thyroid transcription factor 1 and Hoxb5 are down-regulated by TO, suggesting that duration and timing of occlusion are important in balancing the effects of TO on lung growth vs lung maturation.

Glucocorticoid metabolism in the human fetal lung: implications for lung development and the pulmonary surfactant system.

It has been nearly 35 years since Liggins and Howie first reported the benefits of antenatal glucocorticoid (GC) treatment to promote the maturation of the human fetal lung, and nearly that long since Pasqualini and colleagues demonstrated that the human fetal lung actively metabolizes GCs. Since that time, our understanding of the effects of GCs on fetal lung maturation and pulmonary surfactant production has increased dramatically. Similarly, characterization of the enzymes involved in GC metabolism has greatly expanded our understanding of GC signaling in target tissues. In man, the biologically active GC (cortisol) and the biologically inactive GC (cortisone) are interconverted by the tissue-specific expression of the type 1 and type 2 11beta-hydroxysteroid dehydrogenase enzymes (HSD1 and HSD2). Much of the research on GC metabolism in peripheral target tissues has focused on the role of HSD1 in amplifying the effects of GCs in liver and adipose tissue or on the role of HSD2 in blocking the effects of GCs in the kidney and placenta. In contrast, the role of GC metabolism in modulating the effects of GCs on fetal lung maturation and the pulmonary surfactant system in humans is less understood. The goal of this review article is to present a brief overview of the role of GCs in human fetal lung maturation and pulmonary surfactant production, and to familiarize the reader with the biochemistry of the metabolism of natural and synthetic GCs by the HSD enzymes. In addition, we will review data concerning the expression and activity of the HSD enzymes in the human fetal lung and contrast this to what is known about the HSD enzymes in the fetal rodent lung. Although rodents, rabbits, sheep, and several primates have been invaluable model systems for the study of fetal lung development, we have chosen to largely focus this review on human lung, since there are significant differences in GC metabolism between humans and other species.

The effect of repeated umbilical cord occlusions on pulmonary surfactant protein mRNA levels in the ovine fetus.

OBJECTIVES: In this study we sought to determine the effect of brief repeated umbilical cord occlusions (rUCO) on surfactant protein (SP) mRNA levels in the fetal sheep lung at two different gestational ages. METHODS: Fourteen fetuses at 112 to 115 days' gestation (control n = 7, rUCO n = 7) and 15 fetuses at 130 to 133 days' gestation (control n = 7, rUCO n = 8) were studied over 4 successive days with rUCO of 90 seconds duration performed every 30 minutes for 3 to 5 hours each day in the rUCO animals. Blood samples were collected for corticotrophin (ACTH) and cortisol measurements. Animals were killed within 1 hour of the final cord occlusion. SP-A, -B, -C, and -D mRNA levels were determined in lung tissue using a ribonuclease protection assay. RESULTS: Cord occlusions resulted in temporary increases in circulating ACTH on day 1 with both gestational ages, but the elevations were blunted by day 4. Plasma cortisol levels increased transiently with the larger effect being observed on day 4, in particular with the near-term group. With advancing gestational age there was a significant (P < .05) increase in the level of SP-A (control 112-115 days: 0.01 +/- 0.01 vs control 130-133 days: 0.07 +/- 0.02 fmol/mg RNA), SP -B (control 112-115 days: 0.02 +/- 0.01 vs control 130-133 days: 0.07 +/- 0.01 fmol/mg RNA) and SP-C (control 112-115 days: 0.13 +/- 0.09 vs control 130-133 days: 0.51 +/- 0.10 fmol/mg RNA), but not SP-D mRNA levels (control 112-115 days: 0.002 +/- 0.002 vs control 130-133 days: 0.01 +/- 0.002 fmol/mg RNA). At 112 to 115 days, there was no significant change in any of the SP mRNA levels following rUCO compared to controls. However, the same regime of rUCO at 130 to 133 days resulted in an 85% reduction in SP-A and SP-B mRNA content and a 66% reduction in SP-C mRNA levels compared to controls. CONCLUSION: The surprising decrease in SP-A and SP-B mRNA levels, which contrasts with other studies, suggests intermittent asphyxial episodes impact differently on surfactant apoprotein mRNA expression than does prolonged hypoxia.

Distribution of surfactant proteins in type II pneumocytes of newborn, 14-day old, and adult rats: an immunoelectron microscopic and stereological study.

Surfactant proteins (SP) have an important impact on the function of the pulmonary surfactant. In contrast to humans, rat lungs are immature at birth. Alveolarization starts on postnatal day 4. Little is known about the distribution of SP during postnatal alveolarization. By immunoelectron microscopy, we studied the distribution of SP-A, SP-D, SP-B, and precursors of SP-C in type II pneumocytes before, near the end and after alveolarization and in mature lungs. We determined the subcellular volume fractions and the relative labeling index to obtain information about preferential labeling of compartments and non-randomness of labeling. Independently of alveolarization, the overall cellular distribution of SP was non-random. A preferential labeling for SP-A and SP-D was found in small vesicles and multivesicular bodies (mvb). SP-B and precursors of SP-C were localized in mvb and lamellar bodies (lb). There are no postnatal changes in labeling for all three SP in these compartments. Labeling intensity for SP-B in lb increased in close correlation with a significant increase in the volume fractions of lb during alveolarization. Our results support the concept that postnatal alveolarization in rat lungs is associated with significant increases in the SP-B content in lb and volume fraction of lb in type II pneumocytes. The postnatal compartment-specific distribution of SP-A, precursors of SP-C and SP-D does not change.

Combined status of MUC1 mucin and surfactant apoprotein A expression can predict the outcome of patients with small-size lung adenocarcinoma.

AIM: Lung cancer is still a disease of high mortality, despite advanced diagnostic techniques. Here, we aim to report a unique method to predict the recurrence and outcome of patients with pulmonary adenocarcinomas. METHODS AND RESULTS: Immunohistochemical expression of MUC1 mucin and surfactant apoprotein A (SP-A) was examined in 185 cases of surgically removed lung adenocarcinomas of non-bronchioloalveolar type smaller than 30 mm. Staining results were evaluated semiquantitatively, and the expression of MUC1 and SP-A was compared in each case. There were 140/185 (76%) cases showing MUC1 expression higher than SP-A expression (MUC1>SP-A), and 45/185 (24%) cases showing MUC1 expression lower than or equal to SP-A expression (MUC1SP-A pattern, but in 7% (3/45) of the patients with a MUC1< or =SP-A pattern after the median observation period of 41 months (1-99 months) (P < 0.01). The MUC1>SP-A group showed higher recurrence and worse survival than the MUC1

Preservation of the characteristics of the cultured human type II alveolar epithelial cells.

The human type II alveolar epithelial cells lost their specific characteristics during cultivation. We examined the ultrastructural and biochemical nature of the human type II cells cultured by two culture systems. To make a physiological alveoli model, the epithelial cells were seeded onto the cell culture insert and allowed contact with the air directly. The cells exposed to the air expressed polarity and immature lamellar bodies in their cytoplasm. Separately, the alveolar epithelial cells were cultured as spheroids to construct the three-dimensional condition. These cells expressed mature morphological characteristics as epithelial cells and lamellar bodies. The expression of the surfactant apoprotein-A (SP-A) and -C (SP-C) mRNA was compared in the cells cultured as a monolayer, the air exposed and the spheroids. SP-A mRNA was detected in all the cultured epithelial cells, but SP-C mRNA, a specific protein for the type II cells, was expressed only in the cells forming spheroids. The expression of uPA, one of the fibrinolytic enzymes, its receptor (uPAR) and its inhibitor-1 (PAI-1) were also examined. The epithelial cells exposed to the air and formed spheroids expressed a larger amount of uPA mRNA than the monolayer, although the amount of uPAR mRNA were comparable in these cells. The amount of PAI-1 mRNA significantly increased when the epithelial cells were exposed to the air. These results indicate that the type II alveolar epithelial cells induced and preserved their specific characteristics by taking the physiological three-dimensional structure, and these characteristics were partially restored by exposure to the air. Those findings suggest that the alveolar epithelial cells should be cultivated in three-dimensional form with contact to the air to regenerate an appropriate alveolar tissue.