PubMed Health⌕ Search

Biomedical subjects

P L Ballard

Publications and source records attributed to P L Ballard.

At least 19 recordsLinked to original sources
PubMed1996

Transcriptional regulation of human pulmonary surfactant proteins SP-B and SP-C by glucocorticoids.

Expression of the pulmonary surfactant-associated proteins SP-B and SP-C is under both developmental and hormonal regulation. We used human fetal lung to investigate developmental changes and the mechanism of glucocorticoid stimulation of SP-B and SP-C gene expression. There were similar approximately 3-fold increases in SP-B cytoplasmic mRNA content and transcription rate comparing lung samples of 24 wk versus 16 wk gestation. During 5 days of lung explant culture without hormones, the transcription rate increased for SP-B and decreased for SP-C, paralleling changes in mRNA content. Treatment with 100 nM dexamethasone maximally increased transcription of the SP-B gene (approximately 3-fold) and SP-C gene (approximately 11-fold) after 2 and 8 h, respectively, similar to changes in mRNA content. In dose-response studies, the maximal increase in transcription rate occurred at approximately 10 nM dexamethasone for SP-B and at > or = 100 nM for SP-C. Induction of SP-B mRNA content and transcription rate were not affected by prior cycloheximide exposure, whereas induction of SP-C mRNA was decreased by as little as 1 h exposure to inhibitor. We conclude that glucocorticoids, acting directly in type II cells, regulate the SP-B and SP-C genes primarily at the level of transcription. Induction of SP-C, but not SP-B, requires ongoing protein synthesis which likely reflects involvement of a labile transcription factor. The difference in glucocorticoid sensitivity may indicate that the two surfactant protein genes contain glucocorticoid response elements with different affinities for receptor.

Anti-Inflammatory Agents↗
PubMed1995

Characterization of the promoter of human pulmonary surfactant protein B gene.

Pulmonary surfactant protein B (SP-B) is required for normal surfactant function and for survival at birth. To further study SP-B gene expression, we sequenced genomic clones and examined promoter activity of SP-B DNA fragments by transient transfection. A plasmid construct containing human SP-B fragment -1039/+431 linked to chloramphenicol acetyltransferase (CAT) reporter gene was readily expressed in H441 cells, which are derived from a human lung adenocarcinoma, but was < 4% as active in Hep G2, HeLa, and Calu 6 cell lines. SP-B promoter activity in H441 cells was orientation dependent and increased by linked Rous sarcoma virus (RSV) enhancer and was stronger than for thymidine kinase (tk) and RSV promoters. Deletional mapping of the 5' flanking region with exonuclease III suggested nonspecific negative (-811/-1039)- and positive (-453/-641)-control regions and a cell-specific enhancer region at -208 to -54. When a fragment from -403 to -35 base pairs (bp) was placed upstream or downstream of tkCAT, in either orientation, expression in H441 cells but not other cell lines was increased 4- to 28-fold relative to tkCAT. Deletional analysis of the 3' terminus indicated a requirement for at least 7 bp 3' of the transcription start site. Promoter activity was strongly inhibited in a dose-dependent fashion by phorbol ester, with responsiveness mapped to bp -208/-54, but was not responsive to glucocorticoid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Adenovirus-mediated gene transfer to human fetal lung ex vivo.

Gene therapy of the fetus or newborn infant is a potentially useful approach for prevention or treatment of specific lung diseases. To begin to address issues such as efficiency, duration, and cellular distribution of transgene expression, we studied transduction of human lung cells by recombinant, replication-deficient adenovirus containing the lacZ gene driven by the beta-actin promoter and cytomegalovirus enhancer (H5.010CBlacZ). Human fetal lungs of 20- to 24-wk gestation received approximately 10(11) viral particles by instillation into a major bronchus, and the tissue was cultured as explants in serum-free Waymouth's medium. beta-galactosidase staining (X-gal) was detected by 24 h in defined regions of treated tissue and localized to epithelial cells of airways and terminal saccules. beta-galactosidase activity in homogenate of treated tissue was maximal 3-5 days after exposure to virus, ranging from 0.2 to 1.5 A420.min-1.mg protein-1 in four experiments (control values were approximately 0.001). When virus was added directly to lung explants in culture, beta-galactosidase was expressed in most of the peripheral cells and rarely in interior cells, the level of activity was dose dependent between 10(8) and 10(11) viral particles/ml, and transgene expression was sustained for at least 28 days. Treatment of isolated cultured cells with virus resulted in equivalent staining of both epithelial cells and fibroblasts. We conclude that fetal lung cells are efficiently transduced by recombinant adenovirus, indicating the feasibility of gene therapy in the infant or fetus. Cultured fetal lung may be useful for testing gene constructs being considered for therapy.

Adenoviridae↗
PubMed1995

Elemental composition of lamellar bodies from fetal and adult human lung.

Previous studies in the rat using electron probe microanalysis have suggested that most of the Ca2+ in alveolar space is probably derived from secreted lamellar bodies (LB). However, the LB Ca2+ content in cultured rat type II cells is low and unaltered by dexamethasone supplementation. In this study, we examined LB Ca2+ content in adult human lung and cultured explants of fetal lung treated with hormones to promote type II cell differentiation. Lung tissue of 20 to 24 wk of gestation was cultured for 4 to 6 days without hormone and with dexamethasone (10 nM), triiodothyronine (2 nM), 8-bromo-cyclic adenosine monophosphate (0.1 mM), or their combinations. The cultured tissue samples were processed for light and electron microscopy or rapidly frozen and stored in freon-22 under liquid nitrogen. Thin cryosections from the frozen samples were prepared and examined using the electron probe microanalysis. Human lung tissue up to 24 wk of gestational age had no detectable LB before explant culture. Explants cultured for 4 days without hormone supplementation (control) had no detectable LB, single-hormone treatments resulted in small LB, and combination treatments resulted in the formation of many large LB in explant type II cells. Despite such morphologic changes, LB Ca2+ in both control and hormone-treated explants was low (overall mean, 4.1 +/- 0.6; P < 0.05) compared with LB of in situ rat and adult human lung (30 +/- 2 and 27 +/- 1.5 mmol/kg dry wt, respectively) and was similar to that found in cultured rat type II cells. LB phosphorus and sulfur under all explant culture conditions were comparable. These observations indicate that human and rat LB elemental compositions are similar and that optimal in vitro conditions with respect to LB Ca2+ metabolism have not been established.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation.

Pulmonary surfactant protein B (SP-B) enhances phospholipid film formation in vitro and is essential for normal surfactant function in vivo. We examined human fetal lung before and during explant culture for content and cellular localization of SP-B mRNA and protein. SP-B mRNA was low in preculture specimens (18-20 wk) but hybridization signal increased over epithelial cells during culture and was enhanced by dexamethasone treatment (10 nM). SP-B immunofluorescence was very low in preculture specimens, increased during culture, and was uniformly intense in epithelial cells of dexamethasone-treated tissue. With a newly developed immunoassay, SP-B protein was undetectable in preculture lung (< 2% of adult), appeared during culture (26% of adult), and was further increased approximately 3-fold by dexamethasone treatment (86% of adult); lung tissue of two newborn infants contained 7-9-fold more SP-B than is found in the adult. Using Western blot with enhanced chemiluminescence, mature SP-B was undetectable in 16-wk specimens but was present in 19-24-wk preculture tissue at 0.2-2.9% of the adult level. By comparison, SP-B mRNA content is 14 and 50% of adult level in 19- and 24-wk lung tissue, respectively; levels increase 3-fold during culture and a further 3-fold with dexamethasone. Based on these observed differences between mRNA and protein content, we conclude that basal SP-B gene expression in epithelial cells of human fetal lung is regulated primarily at the level of translation or protein stability, whereas glucocorticoids act transcriptionally. We speculate that SP-B protein accumulates only as type II cells differentiate and acquire lamellar bodies for processing and storage of SP-B.

Adult↗
PubMed1995

Partial deficiency of surfactant protein B in an infant with chronic lung disease.

OBJECTIVE: To evaluate components of pulmonary surfactant and identify mutations in the surfactant protein B gene (SP-B) of a term infant with severe respiratory distress and chronic lung disease. PATIENT AND TESTING: Respiratory distress developed in an infant delivered at term, and he required extracorporeal bypass support for 2 weeks. Until his unexpected death at 9.5 months, he was ventilator and oxygen dependent and required continual dexamethasone therapy. Tracheobronchial lavage samples were analyzed for content of surfactant proteins (SPs), and DNA from blood samples were sequenced and analyzed by polymerase chain reaction restriction analysis for the presence of SP-B gene mutations. Surfactant lipid composition and function, the contents of SPs and their messenger RNAs (mRNAs), and the immunostaining pattern for SPs were determined in postmortem lung tissue. RESULTS: The lavage sample contained SP-A but not SP-B, and DNA restriction analysis indicated that the patient and his mother were heterozygous for the previously described 121ins2 mutation of SP-B. Postmortem lung tissue contained normal levels of SP-A and its mRNA, a low but detectable level of SP-B, and near normal content of SP-B mRNA. SP-C was abundant on staining, and some 6-kd precursor was present in tissue. A surfactant fraction was deficient in phosphatidylglycerol and was not surface active. On DNA sequencing, a point mutation was found in exon 7 of the patient's SP-B gene allele without the 121ins2 mutation, resulting in a cysteine for arginine substitution, and the father was a carrier for the same mutation. CONCLUSIONS: We describe a patient who is a compound heterozygote with a new mutation and only a partial deficiency of SP-B. Some forms of inherited SP-B deficiency may have low expression of immunoreactive and possibly functional SP-B with milder lung disease and longer survival. These infants may benefit from glucocorticoid therapy and may not develop antibodies to SP-B after either lung transplant or gene therapy.

Alleles↗
PubMed1994

Glucocorticoid and cAMP increase fatty acid synthetase mRNA in human fetal lung explants.

During late fetal development, synthesis of surfactant phospholipid requires a large supply of fatty acid precursor. Fatty acid synthetase is a regulatory enzyme for de novo fatty acid synthesis in lung as well as other lipogenic tissues. In this study, we report hormonal induction of FAS mRNA in human fetal lung explants (16-23 week gestation) cultured up to 7 days in Waymouth's medium (no serum) supplemented with dexamethasone (Dex, 10 nM) or agents that increase cAMP (8-Br-cAMP, 0.1 mM; isobutylmethylxanthine, 0.1 mM; forskolin, 0.01 mM; PGE1, 0.01 mM). Exposure of explants to Dex or cAMP agents increased FAS mRNA content by 6 h and maximal stimulation occurred at 72 h for Dex (approx. 3-fold increase) and 24 h for cAMP (approx. 2-fold increase). In the presence of both Dex and cAMP there was a synergistic increase in FAS mRNA content at all times (approx. 11-fold increase at 72 h). Induction of FAS mRNA was specific for steroids with glucocorticoid activity, reversible on removal of hormone, and was half-maximal at 2-3 nM Dex consistent with receptor mediation. Actinomycin D blocked induction by Dex but not by cAMP suggesting a transcriptional effect by glucocorticoid but not by cAMP. T3, which increases phosphatidylcholine synthesis, did not induce FAS mRNA. The findings indicate that both glucocorticoid and cAMP increase FAS gene expression consistent with an important role for FAS in regulating the supply of fatty acid for surfactant phospholipid synthesis.

Cyclic AMP↗
PubMed1993

Hormonal regulation of cholinephosphate cytidylyltransferase in human fetal lung.

Cytidylyltransferase (CTP: cholinephosphate cytidylyltransferase, EC 2.7.7.15, CYT) is a regulatory enzyme for synthesis of pulmonary surfactant phosphatidylcholine (PC). The effects of glucocorticoid, T3, and cAMP on CYT activity were studied in explants of human fetal lung (18-22 weeks gestation) cultured for 1-6 days in serum-free medium. Dexamethasone (Dex, 10 nM) treatment for 5 days increased homogenate CYT activity (+115%, P < 0.02) when assayed in the presence of added lipid co-factor (L-alpha-phosphatidylglycerol, PG, 1.1 mM) and tended to increase activity in its absence (+77%, P = 0.12). Cytosolic activity was also significantly elevated in the presence of added co-factor (+124%, P < 0.01), but there was no effect of Dex on microsomal specific activity. Dex increased the recovery of CYT activity in the cytosolic fraction (75% vs. 43% (control) of the homogenate activity), but not in the microsomal, nuclear or mitochondrial fractions. Assayed in the presence of added co-factor, stimulation of CYT by Dex was apparent after 48 h exposure and maximal by 5-6 days exposure to < or = 30 nM concentration. T3 or agents that increase endogenous cAMP stimulated cytosolic activity by 40% and 36-74%, respectively, after 4-6 days exposure, but none produced an additive increase in the presence of Dex. We conclude that stimulation of CYT activity contributes to hormonal induction of surfactant lipids by each of these hormones. Glucocorticoids may increase the amount of CYT enzyme as well as activate the enzyme via increased synthesis of lipid co-factor.

Choline-Phosphate Cytidylyltransferase↗
PubMed1993

Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.

Pulmonary surfactant, which is necessary for normal lung function, is under both developmental and hormonal regulation. Glucocorticoids induce all components of surfactant and have a unique biphasic effect on surfactant protein A (SP-A), either stimulating or inhibiting accumulation in cultured fetal lung depending on dose and time of exposure. In this study we further characterized glucocorticoid inhibition of SP-A in cultured explants of human fetal lung. Decreased content of SP-A mRNA was the dominant response to dexamethasone added either early or later during culture. Inhibition occurred at < or = 1 nM dexamethasone on prolonged exposure, was blocked by RU 486, and was observed with other glucocorticoids but not sex steroids. When cortisol was removed from the culture medium, inhibition was rapidly reversed. The immediate inhibitory effect of 100 nM dexamethasone on SP-A mRNA content was completely blocked in the presence of cycloheximide. SP-A gene transcription, measured by nuclear elongation assay, was decreased by 60% after 4- to 8-h exposure to 100 nM dexamethasone. Stability of SP-A mRNA, determined both by addition of actinomycin D and by label-chase experiments, was transiently decreased immediately after adding dexamethasone (t1/2 approximately 3 h). In tissue treated with dexamethasone for > or = 8 h the stability of SP-A mRNA in control and treated explants was not different (t1/2 approximately 8 h). Our findings indicate that inhibition of SP-A is the dominant response to glucocorticoid. This effect is receptor mediated and apparently involves induction of a labile protein(s) that decreases gene transcription and transiently reduces mRNA stability.

Cells, Cultured↗
PubMed1993

Differential glucocorticoid regulation of the pulmonary hydrophobic surfactant proteins SP-B and SP-C.

Glucocorticoids increase expression of the genes for the pulmonary surfactant-associated proteins SP-B and SP-C in fetal lung both in vivo and in vitro. To examine the mechanism of these effects, we studied induction of SP-B and SP-C mRNAs in human fetal lung cultured as explants. Both mRNA levels rose rapidly in response to 100 nM dexamethasone (Dex), with a faster response for SP-B: maximal levels of induction were achieved in < or = 12 h for SP-B (3.5-fold versus control) versus approximately 24 h for SP-C mRNA (35-fold versus control). Cycloheximide (2.5 micrograms/ml) did not affect glucocorticoid induction of SP-B mRNA but markedly decreased induction of SP-C mRNA. In control cultures, cycloheximide did not significantly reduce levels of either transcript. In nuclear run-on assays, Dex increased the rate of gene transcription for both SP-B (2.8 +/- 0.3-fold versus control, n = 4) and SP-C (10- to 30-fold). Using actinomycin D to assess mRNA stability, the t1/2 of SP-B mRNA was increased from 7.5 +/- 0.4 h to 18.8 +/- 2.9 h by Dex treatment (P < 0.05), whereas the t1/2 of SP-C mRNA was not affected (9.3 +/- 1.7 h versus 8.1 +/- 1.2 h; NS). A similar increase in SP-B mRNA t1/2 with Dex (from 6 h to 19 h) was observed in label-chase studies with [3H]uridine. We conclude that glucocorticoids regulate the hydrophobic surfactant proteins of alveolar type II cells by different mechanisms: induction of SP-B is a primary response and includes an increase in both transcription rate and mRNA stability, whereas induction of SP-C is a secondary process, requiring ongoing protein synthesis, involving increased transcription rate without a change in mRNA stability.

Culture Techniques↗
PubMed1993

Secretion of surfactant protein A and phosphatidylcholine from type II cells of human fetal lung.

Surfactant protein A (SP-A) appears to have a role in lung immune defense as well as generation and metabolism of the alveolar surface-active film. Previous studies indicated that lamellar bodies isolated from lung tissue had a relatively low content of SP-A and that exogenous SP-A was needed for rapid formation of a surface-active film in vitro. We therefore tested the hypothesis that SP-A was secreted from type II cells primarily by a pathway separate from lamellar bodies. Cells were isolated from explants of human fetal lung that had been cultured with hormones to promote differentiation of type II cells, and secretion of surfactant lipid and SP-A were compared. Cultured cells secreted labeled phosphatidylcholine in a nearly linear fashion for 48 h. Basal secretion of SP-A, assayed by enzyme-linked immunosorbent assay, was linear for only 12 h after plating of cells; during this time, there was no accumulation of intracellular SP-A. Addition of secretagogues (phorbol ester, calcium ionophore, and beta-adrenergic agonist) stimulated phosphatidylcholine secretion approximately 4-fold. In contrast, the secretion rate of SP-A was not significantly affected by secretagogues. These findings indicate that a relatively small amount of secreted SP-A (approximately 10%) is released with lamellar bodies. Most SP-A is released by constitutive secretion and may be important for both surfactant- and nonsurfactant-related functions.

Cells, Cultured↗
PubMed1992

Respiratory disease in very-low-birthweight infants after prenatal thyrotropin-releasing hormone and glucocorticoid. TRH Study Group.

Although prenatal glucocorticoid treatment reduces neonatal respiratory morbidity, respiratory distress syndrome and chronic lung disease (CLD) develop in many very-low-birthweight infants despite therapy. To investigate the effect of additional prenatal treatment with thyrotropin-releasing hormone (TRH), we did a multicentre, blinded, randomised trial. 404 women with threatened preterm delivery at less than 32 weeks' gestation received betamethasone plus TRH (4 doses of 400 micrograms 8-hourly) or betamethasone plus placebo. 103 infants who were fully treated and of less than 1500 g birthweight were evaluated during the neonatal period. TRH treatment (55 infants) did not affect the total incidence of respiratory distress syndrome (47% vs 58% in controls) or of severe respiratory distress syndrome (13% vs 25% in controls, p = 0.11). CLD (defined as requirement for supplemental oxygen at 28 days after birth) developed in significantly fewer TRH-treated infants (18% vs 44% of controls, p less than 0.01). The unadjusted relative risk of CLD with TRH therapy was 0.40 (95% CI 0.26-0.80, p less than 0.05), and this was not materially changed after adjustment for potentially modifying variables. There were significantly fewer adverse outcomes, defined as death or continuing oxygen requirement, in the TRH group than in the steroid-alone group both at 28 days and when infants reached 36 weeks' postconceptional age. The incidence of other complications of prematurity was similar in the two groups. Prenatal TRH reduces the incidence of chronic lung disease among betamethasone-treated infants.

Betamethasone↗
PubMed1992

Plasma thyroid hormones and prolactin in premature infants and their mothers after prenatal treatment with thyrotropin-releasing hormone.

We assayed TSH, triiodothyronine, free thyroxine, and prolactin (PRL) in plasma of women and infants participating in a trial of prenatal thyrotropin-releasing hormone (TRH) treatment for prevention of newborn lung disease. Women in labor at 26-34 wk of gestation received 400 micrograms of TRH i.v. every 8 h (one to four doses) plus 12 mg betamethasone (one or two doses); controls received saline plus betamethasone. Mean cord concentrations in control infants were TSH 9.7 mU/L, triiodothyronine 0.6 nmol/L (40.2 ng/dL), free thyroxine 14.4 pmol/L (1.13 ng/dL), and PRL 67.6 micrograms/L. TRH increased maternal plasma TSH by 100% at 2-4 h after treatment and decreased levels by 28-34% at 5-36 h. In cord blood of treated infants delivered at 2-6 h, TSH, triiodothyronine, and PRL were all increased about 2-fold versus control, and free thyroxine was increased 19%; the response was similar after one, two, three, or four doses of TRH. In treated infants delivered at 13-36 h, cord TSH and triiodothyronine levels were decreased 62 and 54%, respectively, and all thyroid hormones were lower after birth at 2 h of age versus control. We conclude that prenatal TRH administration increases thyroid hormones and PRL in preterm fetuses to levels similar to those normally occurring at term. Pituitary-thyroid function is transiently suppressed after treatment to a greater extent in fetus than mother, and infants born during the early phase of suppression do not have the normal postnatal surge in thyroid hormones.

Betamethasone↗
PubMed1992

Cyclic adenosine 3',5'-monophosphate increases beta-adrenergic receptor concentration in cultured human fetal lung explants and type II cells.

cAMP regulates the maturation of many biochemical processes that occur during normal lung development, including the changing levels of surfactant proteins and phospholipids. We examined the effect of cAMP on the beta-adrenergic receptor concentration in the developing human lung. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, increased both the tissue cAMP content and beta-adrenergic receptor concentration in treated explants above those in untreated explants. 8-Bromo-cAMP treatment also elevated the beta-adrenergic receptor concentration of lung explants compared to that in untreated controls. These data indicate the ability of elevated cAMP to increase the beta-adrenergic receptor concentration. Both lung cAMP and beta-adrenergic receptor concentrations increase spontaneously in culture. To test for a possible causal relationship, we cultured explants with protein kinase inhibitors. We found that H-8, a preferential inhibitor of the cAMP-dependent protein kinase [protein kinase-A (PKA)], but not H-7, which inhibits PKA and protein kinase-C with similar potency, blocked the spontaneous rise in beta-adrenergic receptor concentration in human fetal lung explants, indicating that PKA activity is required for this rise in beta-adrenergic receptor concentration. Type II cells isolated from cultured lung treated with H-8 had fewer beta-adrenergic receptors than cells isolated from untreated explants. These studies show that cAMP increases the beta-adrenergic receptor concentration in human fetal lung and specifically in type II cells through a PKA-dependent mechanism, consistent with a role for cAMP in beta-adrenergic receptor regulation during normal lung development.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Differentiation of type II cells during explant culture of human fetal lung is accelerated by endogenous prostanoids and adenosine 3',5'-monophosphate.

Explant culture of the fetal lung, in the absence of serum or hormones, results in precocious biochemical and morphological differentiation of alveolar epithelial cells. In this study we tested the hypothesis that these maturational events are induced by endogenous cAMP. During culture of human fetal lung the content of tissue cAMP increased 140% from days 3-6. Treatment with isobutylmethylxanthine caused a further doubling of cAMP content, and indomethacin blocked most of the increase in cAMP. Isobutylmethylxanthine accelerated and indomethacin inhibited the increases during culture in surfactant protein-A (SP-A), SP-A mRNA, SP-B mRNA, phosphatidylcholine content, and activity of fatty acid synthetase. There was no effect of these treatments on the content of SP-C mRNA, which did not increase during culture. Increasing concentrations of prostaglandins E1 and E2 in the presence of indomethacin produced a parallel stimulation of cAMP content, SP-A, and fatty acid synthetase activity. The temporal increase in SP-A was also blocked by inhibitors of protein kinase-A. In morphological studies, indomethacin-treated explants appeared less mature, with decreased intralumenal volume and more columnar epithelial cells. We conclude that increased tissue cAMP levels, stimulated by endogenous prostaglandins, accelerate differentiation of alveolar epithelial cells during explant culture.

1-Methyl-3-isobutylxanthine↗