SP-B mRNA Research Articles

Effect of exogenous surfactant on the development of surfactant synthesis in premature rabbit lung.

Surfactant replacement is an effective therapy for neonatal respiratory distress syndrome. Full recovery from respiratory distress syndrome requires development of endogenous surfactant synthesis and metabolism. The influence of exogenous surfactant on the development of surfactant synthesis in premature lungs is not known. We hypothesized that different exogenous surfactants have different effects on the development of endogenous surfactant production in the premature lung. We treated organ cultures of d 25 fetal rabbit lung for 3 d with 100 mg/kg body weight of natural rabbit surfactant, Survanta, and Exosurf and measured their effects on the development of surfactant synthesis. Additional experiments tested how these surfactants and Curosurf affected surfactant protein (SP) SP-A, SP-B, and SP-C mRNA expression. Surfactant synthesis was measured as the incorporation of 3H-choline and 14C-glycerol into disaturated phosphatidylcholine recovered from lamellar bodies. Randomized-block ANOVA showed significant differences among treatments for incorporation of both labels (p < 0.01), with natural rabbit surfactant less than control, Survanta greater than control, and Exosurf unchanged. Additional experiments with natural rabbit surfactant alone showed no significant effects in doses up to 1000 mg/kg. Survanta stimulated disaturated phosphatidylcholine synthesis (173 +/- 41% of control; p = 0.01), increased total lamellar body disaturated phosphatidylcholine by 22% (p < 0.05), and increased 14C-disat-PC specific activity by 35% (p < 0.05). The response to Survanta was dose-dependent up to 1000 mg/kg. Survanta did not affect surfactant release. No surfactant altered the expression of mRNA for SP-A, SP-B, or SP-C. We conclude that surfactant replacement therapy can enhance the maturation of surfactant synthesis, but this potential benefit differs with different surfactant preparations.

Gene Expression of Surfactant Protein B and C in Endotoxin and Thiourea Treated Rats

Background :T he surfactant specific proteins, SP-B and SP-C are believed to be important regulators of the surfactant function and homeostasis. Since acute respiratory distress syndrome(ARDS) is usually viewed as the functional and morphological expression of a similar underlying lung injury caused by a variety of insults, and since abnormalities in the surfactant function have been described in ARDS, the authors investigated the different effects of endotoxin and thiourea on the accumulation of mRNA encoding SP-B and SP-C. Methods : Sprague-Dawley rats were given 5 mg/kg of an intraperitoneal endotoxin from Salmonella enteritidis and 3.5 mg/kg intraperitoneal thiourea and were sacrificed at different time periods. Results : 1. The SP-B mRNA levels 6 and 24 hours after the 5 ㎎/㎏ endotoxin treatment was significantly reduced by 26.1% and 50%, respectively(P<0.01, P<0.001). 2. The SP-B mRNA levels 24 hours after the 3.5 ㎎/㎏ thiourea treatment was reduced by 9.8% and 12.5%, respectively. 3. The SP-C mRNA levels 6 and 24 hours after the 5 ㎎/㎏ endotoxin treatment was significantly reduced by 38.7% and 53.6%, respectively(P<0.01, P<0.001). 4. The SP-C mRNA level 6 hours after the 3.5 ㎎/㎏ thiourea treatment was reduced by 22.8%(P<0.05).

1α,25-Dihydroxy-3-epi-vitamin D 3, a natural metabolite of 1α,25-dihydroxy vitamin D 3: production and biological activity studies in pulmonary alveolar type II cells

Pulmonary alveolar type II cells have been shown to be a possible target for the secosteroid hormone, 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3], during perinatal transition. At present, there is great interest to isolate and identify the metabolites of 1α,25(OH) 2D 3 produced in its target tissues and to determine the contribution of each individual metabolite of 1α,25(OH) 2D 3 to the final expression of the pleiotropic actions attributed to 1α,25(OH) 2D 3. Of all the known metabolites of 1α,25(OH) 2D 3, 1α,25(OH) 2-3-epi-D 3 has gained most attention as it is produced only in specific tissues and possesses significant activity in tissues in which it is produced. Furthermore, in vivo studies indicate that this metabolite when compared to 1α,25(OH) 2D 3 is less calcemic. Therefore, we performed the present study to identify production of 1α,25(OH) 2-3-epi-D 3 in alveolar type II cells, and to evaluate its effect on surfactant synthesis. We incubated NCI-H441 cells, an alveolar type II cell line, with 1α,25(OH) 2D 3 and demonstrated that these cells metabolize 1α,25(OH) 2D 3 to various previously well-characterized polar metabolites, and to a less polar metabolite which was unequivocally identified as 1α,25(OH) 2-3-epi-D 3 by GC/MS and HPLC analysis. Further, biological activity studies in H441 cells indicated that 1α,25(OH) 2-3-epi-D3 possesses significant activity in terms of its ability: (i) to increase surfactant phospholipid synthesis, (ii) to induce surfactant SP-B mRNA gene expression, and (iii) to increase surfactant SP-B protein synthesis. However, the activity of 1α,25(OH) 2-3-epi-D 3 when compared to 1α,25(OH) 2D 3 in generating VDR-mediated transcriptional activity in ROS 17/2.8 cells transfected with human osteocalcin VDRE/growth hormone gene construct, was significantly reduced. The high metabolic stability of 1α,25(OH) 2-3-epi-D 3, as previously proposed by us, may be a possible explanation for the high in vitro activity in spite of the reduced VDR-mediated transcriptional activity. In summary, we report for the first time the pathways of 1α,25(OH) 2D 3 metabolism in pulmonary alveolar type II cells and indicate that 1α,25(OH) 2-3-epi-D 3, a natural intermediary metabolite of 1α,25(OH) 2D 3 possesses significant activity in stimulating surfactant synthesis in alveolar type II cells.

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro.

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.

Nitrate and nitrite in exhaled breath condensate are derived from the lower airway

Nitrate and nitrite in exhaled breath condensate are derived from the lower airway

Mice lacking CCAAt/enhancer-binding protein-alpha show hyperproliferation of alveolar type II cells and increased surfactant protein mRNAs.

The lung-specific surfactant proteins (SP) are essential for normal respiratory function. Transcription factors may play an important role in the regulation of surfactant proteins. The CCAAT/enhancer-binding protein (C/EBP) family consists of transcription factors that can stimulate expression of genes in lipid-metabolizing epithelial cells. C/EBPalpha-deficient mice have been shown to exhibit abnormal pulmonary histopathology. Recently, we demonstrated that C/EBP family members are differentially expressed in alveolar type II cell proliferation and in pulmonary fibrosis. In the present study, to investigate whether the C/EBP family would be involved in the regulation of surfactant proteins, we examined the protein expression of SP-A, and SP-C, and mRNA expression of SP-A, SP-B, and SP-C in the lungs from newborn C/EBPalpha-deficient mice. Using immunohistochemistry, we demonstrated that positive cells for SP-C, specific to alveolar type II cells, in the lungs were more abundant in the newborn C/EBPalpha-deficient mice than in control mice, which suggests the hyperproliferation of alveolar type II cells in the lungs of the C/EBPalpha-deficient mice. In situ hybridization analysis revealed that expression of SP-A, SP-B, and SP-C mRNAs were increased in the lungs of newborn C/EBPalpha-deficient mice. Northern blot analysis revealed that surfactant protein mRNAs were also increased. Thus, these results suggest that C/EBPalpha may play a key role in the proliferation of alveolar type II cells and the regulation of genes of surfactant protein.

VEGF induces airway epithelial cell proliferation in human fetal lung in vitro.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [(3)H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C.

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.

Mechanical stretch promotes alveolar epithelial type II cell differentiation.

Functional maturation of pulmonary alveolar epithelial cells is crucial for extrauterine survival. Mechanical distension and mesenchymal-epithelial interactions play important roles in this process. We hypothesized that mechanical stretch simulating fetal breathing movements is an important regulator of pulmonary epithelial cell differentiation. Using a Flexercell Strain Unit, we analyzed effects of stretch on primary cultures of type II cells and cocultures of epithelial and mesenchymal cells isolated from fetal rat lungs during late development. Cyclic stretch of isolated type II cells increased surfactant protein (SP) C mRNA expression by 150 +/- 30% over controls (P < 0.02) on gestational day 18 and by 130 +/- 30% on day 19 (P < 0.03). Stretch of cocultures with fibroblasts increased SP-C expression on days 18 and 19 by 170 +/- 40 and 270 +/- 40%, respectively, compared with unstretched cocultures. On day 19, stretch of isolated type II cells increased SP-B mRNA expression by 50% (P < 0.003). Unlike SP-C, addition of fibroblasts did not produce significant additional effects on SP-B mRNA levels. Under these conditions, we observed only modest increases in cellular immunoreactive SP-B, but secreted saturated phosphatidylcholine rose by 40% (P < 0.002). These results indicate that cyclic stretch promotes developmentally timed differentiation of fetal type II cells, as a direct effect on epithelial cell function and via mesenchymal-epithelial interactions. Expression of the SP-C gene appears to be highly responsive to mechanical stimulation.

Decreased surfactant protein-B expression and surfactant dysfunction in a murine model of acute lung injury.

This study examines the relationships between inflammation, surfactant protein (SP) expression, surfactant function, and lung physiology in a murine model of acute lung injury (ALI). 129/J mice received aerosolized endotoxin lipopolysaccharide [LPS] daily for up to 96 h to simulate the cytokine release and acute inflammation of ALI. Lung elastance (E(L)) and resistance, lavage fluid cell counts, cytokine levels, phospholipid and protein content, and surfactant function were measured. Lavage and lung tissue SP content were determined by Western blot and immunohistochemistry, and tissue messenger RNA (mRNA) levels were assessed by Northern blot and in situ hybridization. Tumor necrosis factor-alpha and neutrophil counts in bronchoalveolar lavage fluid increased within 2 h of LPS exposure, followed by increases in total protein, interleukin (IL)-1beta, IL-6, and interferon-gamma. E(L) increased within 24 h of LPS exposure and remained abnormal up to 96 h. SP-B protein and mRNA levels were decreased at 24, 48, and 96 h. By contrast, SP-A protein and mRNA levels and SP-C mRNA levels were not reduced. Surfactant dysfunction occurred coincident with changes in SP-B levels. This study demonstrates that lung dysfunction in mice with LPS-ALI corresponds closely with abnormal surfactant function and reduced SP-B expression.

Effect of reactive oxygen species on fetal lung maturation

Objectives: Fetal lung maturation in preterm infants with chorioamnionitis is known to be accelerated. However, the molecular basis of this pathological acceleration has not been elucidated. We investigated whether reactive oxygen species play a role in the acceleration of fetal lung maturation. Study Design: On the 16th day of gestation, xanthine (1 mM) and xanthine oxidase solution (0.1–100 mU/ml) were injected into the intrauterine cavity of pregnant rats. On the 19th day of gestation, we examined the expression of the mRNA of surfactant associated proteins A, B and C (sp-A, sp-B and sp-C) by reverse transcription-polymerase chain reaction. Results: sp-A, sp-B and sp-C mRNAs were observed in lung tissue from fetal rats stimulated by xanthine–xanthine oxidase in contrast to the control. Conclusion: Reactive oxygen species in amniotic fluid might be an important factor in accelerated fetal lung maturation associated with chorioamnionitis in the rat experimental model.

Expression and localization of lung surfactant protein B in Eustachian tube epithelium.

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.

Re-expression of pulmonary surfactant proteins following tracheal obstruction in fetal sheep.

Increased fetal lung expansion, induced by tracheal obstruction (TO), is a potent stimulus for fetal lung growth, but rapidly reduces surfactant protein (SP) mRNA levels. Our aim was to determine the time course for the re-expression of the surfactant proteins in fetal lung tissue following the release of a TO and to relate these to the changes in lung liquid volume. Fetal sheep were exposed to either: (1) no treatment (controls); (2) 4 days of TO; (3) 4 days of TO, followed by release of the obstruction for 24 h; (4) 4 days of TO followed by release of the obstruction for 3 days. Four days of TO increased lung liquid volumes from 26.8 +/- 1.9 to 72.0 +/- 5.6 ml kg(-1) and reduced SP-A, SP-B and SP-C mRNA levels to 38.5 +/- 10.7, 56.8 +/- 10.3 and 18.3 +/- 5.3 % of control values, respectively. One day after TO release, lung liquid volumes were reduced to 17.4 +/- 5.3 ml kg(-1) (control 128 days, 31.0 +/- 3.8 ml kg(-1)) and SP-A and SP-B mRNA levels were not different from control levels. In contrast, SP-C mRNA levels only increased to 45.4 +/- 17.3 % of control. Three days after TO release, lung liquid volumes increased to 48.0 +/- 8.5 ml kg(-1) and SP-A and SP-B mRNA levels were reduced to 48.8 +/- 10.2 % and 71.5 +/- 19.8 % of control, respectively; SP-C mRNA levels remained at 35.3 +/- 12.3 % of control. Following the release of a TO, SP-A, SP-B and SP-C mRNA levels were closely and inversely related to the volume of lung liquid. Based on these relationships, the lung liquid volumes that equate to 100 % expression were considerably less than control lung volumes (< 10 vs. 30-40 ml kg(-1)) in fetuses of this age. Thus, the changes in fetal lung SP-A, SP-B and SP-C mRNA levels following the release of a TO are variable, differ between the proteins and are closely related to the changes in lung liquid volumes. We conclude that the re-expression of surfactant proteins following TO is variable and that the change in lung liquid volume is potentially a good indicator for surfactant protein re-expression. Experimental Physiology (2001) 86.1, 55-63.

Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

Intra-amniotic endotoxin accelerates lung maturation in fetal rabbits.

The hypothesis that endotoxin in amniotic fluid accelerates fetal lung maturation was tested. On day 25 of gestation, LPS (5 microg/fetus) was injected intra-amniotically into one uterine horn of eight New Zealand white rabbits, whereas the contralateral amniotic sacs were injected with saline vehicle. The fetuses were delivered 48 h after LPS administration and their lungs were studied. One dam went into premature labor prior to the 48 h time point and was excluded from the study. Mean white cell counts in amniotic fluid and bronchoalveolar lavage fluid from LPS-treated fetuses were increased 3.2-fold (p = 0.04) and 9.9-fold (p = 0.04), respectively. Fetal weights and lung weights were not affected by LPS. Surfactant protein SP-A and SP-B mRNA expressions in LPS-treated fetuses were increased 2.3-fold (p = 0.03) and 1.4-fold (p = 0.04), respectively. Static lung compliance was increased in animals treated with LPS (p = 0.001). Lungs from LPS-treated animals had better aeration than those of controls. Mean volume of inflation-fixed lungs of LPS-treated fetuses was 1.7 times greater than that of controls (p = 0.03). Intra-uterine exposure to LPS increases surfactant protein expression and improves lung stability and aeration in preterm animals.

Intra‐amniotic endotoxin accelerates lung maturation in fetal rabbits

The hypothesis that endotoxin in amniotic fluid accelerates fetal lung maturation was tested. On day 25 of gestation, LPS (5 μg/fetus) was injected intra‐amniotically into one uterine horn of eight New Zealand white rabbits, whereas the contralateral amniotic sacs were injected with saline vehicle. The fetuses were delivered 48 h after LPS administration and their lungs were studied. One dam went into premature labor prior to the 48 h time point and was excluded from the study. Mean white cell counts in amniotic fluid and bronchoalveolar lavage fluid from LPS‐treated fetuses were increased 3.2‐fold (p= 0.04) and 9.9‐fold (p= 0.04), respectively. Fetal weights and lung weights were not affected by LPS. Surfactant protein SP‐A and SP‐B mRNA expressions in LPS‐treated fetuses were increased 2.3‐fold (p= 0.03) and 1.4‐fold (p= 0.04), respectively. Static lung compliance was increased in animals treated with LPS (p= 0.001). Lungs from LPS‐treated animals had better aeration than those of controls. Mean volume of inflation‐fixed lungs of LPS‐treated fetuses was 1.7 times greater than that of controls (p= 0.03). Conclusion: Intra‐uterine exposure to LPS increases surfactant protein expression and improves lung stability and aeration in preterm animals.

Effect of hyperoxia on surfactant protein gene expression in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats

The hypoplastic lung in congenital diaphragmatic hernia (CDH) has both a quantitative and qualitative reduction in surfactant. Recently, the role of oxygen (O2) as a regulator of pulmonary surfactant-associated protein (SP) gene expression has been reported. The mRNA level of SP has been demonstrated to be increased in the lungs of animals exposed to hyperoxia. The aim of this study was to investigate SP mRNA expression in hypoplastic CDH lung in rats during mechanical ventilation in order to determine the effect of O2 on SP synthesis in CDH. A CDH model was induced in pregnant rats following administration of nitrofen. The newborn rats with CDH and controls were intubated and ventilated. Ventilation was continued for 6 h under 100% oxygen. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amounts of mRNA expression of SP-A, SP-B, SP-C, and SP-D. Relative amounts of SP-A, SP-B, and SP-D mRNA expression in CDH lung were significantly decreased compared to controls at birth and 6 h after ventilation. There was no significant difference in SP-C mRNA expression between CDH animals and controls. Upregulated mRNA expression of SP-A, SP-B, and SP-D in lungs of control animals at 6 h after ventilation suggests that oxygenation accelerates postnatal SP synthesis in normal lungs. The inability of O2 to increase SP mRNA expression in hypoplastic CDH lung suggests that the hypoplastic lung is not responsive to increased oxygenation for the synthesis of SP.

Antenatal dexamethasone enhances surfactant protein synthesis in the hypoplastic lung of nitrofen-induced diaphragmatic hernia in rats

Background/Purpose: Pulmonary hypoplasia is one of the main causes for the high mortality rate in patients with congenital diaphragmatic hernia (CDH). The expression of surfactant protein A in the hypoplastic CDH lung is reduced, and its concentration is decreased in the amniotic fluid of pregnancies complicated by CDH. In a CDH experimental model, prenatal glucocorticoid treatment has proved its efficacy in correcting the parameters of pulmonary biochemical and morphologic immaturity. The aim of this study was to investigate whether maternal administration of dexamethasone has any effect on the expression of surfactant protein A and surfactant protein B in nitrofen-induced experimental CDH rat model. Methods: CDH was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (Dex, 0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 of gestation. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: group I, control (n = 16); group II, nitrofen-induced CDH (n = 16); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 16). Indirect immunohistochemistry was performed using alkaline-phosphatase-coagulated streptavidin using anti-SP-A and anti-SP-B polyclonal antibodies. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate relative amount of SP-A and SP-B mRNA expression. Results: In the CDH lung (group II) we observed a markedly reduced number of type II pneumocytes positive for SP-A, and SP-B was increased to a level close to that of the control group. The relative amount of SP-A and SP-B was reduced significantly in group II compared with controls (P <.05) and significantly increased in group III compared with group II animals (P <.01). Conclusion: These results suggest that antenatal glucocorticoid treatment increases the production of surfactant proteins in the CDH hypoplastic lung. J Pediatr Surg 35:1468-1473. Copyright © 2000 by W.B. Saunders Company.

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

Down-regulation of thyroid transcription factor-1 gene expression in fetal lung hypoplasia is restored by glucocorticoids.

The thyroid transcription factor (TTF)-1 has an essential role in lung morphogenesis and development. It is involved in the transcription of surfactant proteins (SP), which are critical in respiratory function. Neonates with congenital diaphragmatic hernia die of respiratory failure caused by pulmonary hypoplasia with associated biochemical immaturity. To gain new insights into the causes of this disorder and the effect of prenatal hormonal treatment on reducing mortality in these infants, we evaluated the expression of TTF-1 as marker of lung morphogenesis and SP-B as marker of lung maturity. Using a rat model of lung immaturity, we show that TTF-1 and SP-B messenger RNA (mRNA) levels are drastically reduced in congenital lung hypoplasia. Interestingly, prenatal dexamethasone (Dex) treatment increased both TTF-1 and SP-B mRNAs over control levels when administered to rats with lung hypoplasia, but it had no effect on TTF-1 or a moderate effect on SP-B mRNA when administered to control rats. TRH alone also increases TTF-1 and SP-B mRNA levels but to a lesser extent than Dex. When administered together with Dex, TRH counteracts the induction observed with the glucocorticoid. The decrease in TTF-1 mRNA levels in lung hypoplasia is paralleled by a down-regulation of TTF-1 protein levels, as well as by a decrease in the TTF-1/DNA complex when the TTF-1-binding site of the SP-B promoter was used as a probe. Both parameters were reestablished after glucocorticoid treatment. Moreover, the regulation of TTF-1 gene expression described in this report is accompanied by the same regulation in its promoter activity, as demonstrated in transfection experiments performed in H-441 human lung-derived adenocarcinoma cells. In conclusion, our data demonstrate, for the first time, that lung hypoplasia and the associated respiratory dysfunction caused by SP-B deficiency are caused, in part, by down-regulation of TTF-1 gene expression. The observations that prenatal glucocorticoid treatment induces the expression of TTF-1 supports routine in utero glucocorticoid treatment of patients expected to have lung hypoplasia.