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D S Strayer

Publications and source records attributed to D S Strayer.

At least 19 recordsLinked to original sources
PubMed1999

SP-A as a cytokine: surfactant protein-A-regulated transcription of surfactant proteins and other genes.

Pulmonary surfactant is a mixture of phospholipids and surfactant-associated proteins made by alveolar type II cells that is necessary for normal lung function. Surfactant secretion and reuptake by type II cells are regulated in part by interaction of surfactant protein-A (SP-A) with a specific receptor (SPAR) on type 11 cells. Several chemicals and hormones affect both surfactant secretion and also surfactant gene expression, but consequences of SP-A-SPAR interaction beyond regulating surfactant secretion and reuptake are unknown. Accordingly, we studied the effects of SP-A on surfactant protein gene transcription, mRNA levels, and transcript stability. SP-A elicited new transcription of surfactant proteins SP-A, SP-B, and SP-C and SPAR and c-Jun but had no effect on beta-actin or c-fos transcription. Antibody against SP-A receptor blocked SP-A-induced transcription, confirming that these actions of SP-A were receptor-mediated. SP-A effects on overall transcript levels were more complex. However, SP-A, SP-B, and SP-C mRNA levels doubled in SP-A-treated cells compared to controls. SP-A is known to stabilize surfactant, control its secretion and reuptake by type II cells, and augment host antimicrobial defenses. These data indicate that SP-A also acts as an autocrine cytokine: it binds its receptor and specifically regulates transcription of surfactant proteins and other genes.

Actins↗
PubMed1998

Effects of overexpression of Ran/TC4 mammalian cells in vitro.

We investigated the effect of overexpression of Ran/TC4 on cell cycle progression. Ran/TC4 (ras-related nuclear protein) is a highly conserved 25-kDa GTP-binding protein that, in concert with its guanine-nucleotide-exchange factor RCC1, is involved in signal transduction. Ran and RCC1 act on nuclear transport of RNA and protein, cell cycle regulation at the G1/S interphase, chromatin decondensation after mitosis, and chromosome stability. These two proteins are essential for the coupling of DNA synthesis with the onset of mitosis. The cDNA for rabbit Ran/TC4 was identified in a cDNA library using degenerate oligonucleotide probes devised on the basis of deduced protein sequence data. This cDNA was cloned into pCDM8 expression vector to yield a plasmid, pTC4, in which Ran/TC4 expression is driven by the cytomegalovirus intermediate early promoter. Both a human tumor cell line, MCF7, and a normal rabbit fibroblast line, RK-13, were tested. Following transfection with pTC4 we observed an increase in Ran/TC4 transcript levels. Transfection with pTC4 prolonged the duration of S phase in both MCF7 and RK-13 cells and led to reduced cell proliferation and decreased total cell numbers. DNA fragmentation was seen in pTC4-transfected cultures but not in control cultures. These findings underscore the function of Ran/TC4 as a molecular switch that guides the cell to completion of DNA synthesis before it enters mitosis and suggest that its overexpression may greatly alter cell cycle kinetics and cell viability.

Amino Acid Sequence↗
PubMed1998

Lung function and bacterial proliferation in experimental neonatal pneumonia in ventilated rabbits exposed to monoclonal antibody to surfactant protein A.

Surfactant protein A (SP-A) increases the resistance of surfactant to inhibition by plasma and other proteins. In a previous study we found that a monoclonal anti-SP-A antibody (R 5) increased the sensitivity of surfactant to inhibition by fibrinogen in vivo and in vitro. SP-A has been shown to stimulate microbial phagocytosis and killing by alveolar macrophages. We hypothesized that using R 5 to inactivate SP-A in an animal model mimicking congenital group B streptococcal (GBS) pneumonia might result in increased bacterial proliferation and a deterioration in lung function. Newborn near term rabbits were delivered by Cesarean section, anesthetized, tracheotomized, and ventilated for 5 h in a plethysmograph system allowing measurement of dynamic lung-thorax compliance. Postnatally the animals received one intratracheal injection (5 ml/kg) of R 5, nonspecific IgG, or normal saline. At 30 min all animals received a standard dose of an encapsulated GBS strain by intratracheal injection. The number of bacteria (mean log10 CFU/g lung +/- S.D.; CFU = colony forming unit) was evaluated in lung homogenates. Histologic lung sections were judged by light microscopy. Bacterial proliferation was similar in rabbits treated with the monoclonal antibody (9.33 +/- 0.39; n = 14) and in control animals receiving saline (9.16 +/- 0.35; n = 14) or nonspecific IgG (9.26 +/- 0.31; n = 11). No significant differences were noted on the histologic analysis or in measurements of lung function. We conclude that intratracheal instillation of a monoclonal anti-SP-A antibody did not increase bacterial proliferation in GBS-infected newborn rabbits. These findings suggest that SP-A does not play an important role in protection against encapsulated GBS strains in the neonatal period.

Animals↗
PubMed1998

Use of SV40 to immunize against hepatitis B surface antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent.

We have described a novel gene transfer system, in which replication-incompetent, T antigen-deleted simian virus-40 (SV40) is used as the transduction vehicle. We report here successful immunization using such an SV40-derived viral vector. Hepatitis B surface antigen (HBsAg) cDNA was cloned downstream of two tandem SV40 early promoters to yield a T antigen-deficient SV40 derivative, SV(HBS). Cultured TC7 cells were exposed to SV(HBS), and expression of HBsAg was detected 24 h later by Northern blot and RT-PCR analysis. Immunochemistry and Western blot analysis were also performed 24 h after infection to detect expression of HBsAg. Once it was ascertained that we could express HBsAg in this way, we used SV(HBS) to elicit anti-HBs. SV(HBS) was injected intraperitoneally or subcutaneously into mice every 4 weeks. These mice were bled every 2 weeks and their sera assayed for antibody activity against HBsAg and SV40. Production of anti-HBs was measured by ELISA and confirmed by Western blot analysis, both of which demonstrated significant levels of anti-HBs after the second injection. We also tested production of anti-SV40 antibodies by the ability of sera to neutralize SV(HBS) infectivity. We found no evidence of neutralization of SV(HBS) infectivity even after eight inoculations. Thus, replication-incompetent SV40 is itself not a strong antigen. Our data suggest that SV40-based transduction systems may be a useful vehicle for immunization and for other gene transfer applications when a need for multiple inoculations is anticipated.

Animals↗
PubMed1998

Mechanism for secretagogue-induced surfactant protein A binding to lung epithelial cells.

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of 125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of 125I-A2R and 125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of approximately 32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was approximately 210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Recognition of normal, neoplastic, and fetal airway epithelial cell membranes by two monoclonal antibodies.

The reactivity of two rat monoclonal antibodies was studied. These antibodies, A2R and A2C, bind a 32 kDa alveolar type II cell membrane receptor for surfactant protein A. A2R and A2C also bind apical cell membranes of ciliated and nonciliated cells of the conducting airways. Because this reactivity suggested possible utility in targeting those cells for therapeutic gene transfer, the binding activity of these two antibodies was examined in human tissues. In conducting airways, A2R and A2C bound apical epithelial cell membranes throughout the embryologic period studied: from 15 weeks of gestation, through maturity. Reactivity was more restricted to ciliated cells of the airways as maturation progressed. In the peripheral lung, A2C and A2R only bound most cells in the early developing lung, but mainly type II cells in mature lungs. Other normal tissues recognized by these antibodies included crypt lining cells of the adult and fetal stomach, large bile duct epithelium, and pancreatic acinar cells. All of these cells derive from embryonic foregut endoderm. Other normal tissues, both of endodermal and nonendodermal origin, were negative. Pulmonary carcinomas were studied. A2C and A2R recognized all non-small cell carcinomas of the lung tested. In contrast, none of the small cell carcinomas or carcinoid tumors of the lung were recognized by these antibodies. The function of p32 in these diverse cell types is not clear, but whatever its role in these tissues, antibodies versus p32 may potentially be used to target gene or drug therapy to the normal or malignant cells they recognize.

Adult↗
PubMed1997

Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells.

Stable, efficient gene transfer to normal human peripheral blood mononuclear cells (PBMC) is a prerequisite for therapy of a number of diseases, both hereditary and acquired, affecting these cells. Current approaches to gene transfer to PBMC entail ex vivo mitogenic stimulation and multiple transduction steps followed by selection, usually of progenitor populations. Thus, the ability to transfer gene expression to normal, resting PBMC could complement gene transfer strategies that target dividing precursor cells. We report successful short-term transduction of human PBMC using two different SV40-derived viral vectors SV40-derivative viruses were constructed by cloning cDNAs for firefly luciferase (luc), or hepatitis B surface antigen (HBSAg), into shuttle plasmids to create the SV40 derivative viruses SVluc and SV(HBS) respectively. Both genes were cloned downstream from SV40 early promoter. Normal, resting, human PBMC were exposed to these viruses, and unselected cultured cells were assayed 24 to 48 h later for expression of transduced genes by immunochemistry and Northern blot analysis. Expression of both luciferase and HBSAg was detected using both approaches. Levels of expression of luciferase were slightly higher in PBMC which were stimulated with concanavalin A (con A). Conversely, expression of HBSAg was less in con A-stimulation did not alter infectivity of PBMC by SV40-derivative virus. While longevity and stability of expression in vitro are as yet unknown, this demonstration of successful gene transfer to resting, normal human PBMC, assayed on unselected cells, suggests that SV40-based transduction systems may be potential candidates for use in transient gene transfer to mononuclear blood cells.

Blotting, Northern↗
PubMed1997

Surfactant protein-A receptor-mediated inhibition of calcium signaling in alveolar type II cells.

Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveolar cells secrete surfactant in response to such secretagogs as terbutaline, calcium (Ca) ionophores (e.g., ionomycin [Io]), and adenosine triphosphate (ATP). A cell membrane receptor for SP-A, one of the surfactant proteins, regulates secretion by negative feedback. We used quantitative fluorescence microscopy to study the effects of SP-A on alterations in cytosolic Ca2+ ([Ca2+]i) elicited by surfactant secretagogs. Freshly isolated type II cells were loaded with Fura-2, then treated with secretagog, in the presence or absence of SP-A. Io and ATP produced biphasic increases in cytosol [Ca2+]i, reflecting first Ca2+ release from intracellular stores, and then influx through the cell membrane. Thapsigargin (TG) and Io directly initiate Ca2+ release; ATP elicits Ca2+ release via receptor-mediated mechanisms. Ca2+ release causes cell membrane Ca channels to open by as yet poorly understood mechanisms. Io itself acts as an additional Ca2+ channel. SP-A blocks much of the Ca2+ release and some of the Ca2+ influx elicited by these secretagogs. Antibody against SP-A receptor restores secretagog-induced Ca2+ fluxes from inhibition by SP-A, confirming that the inhibitory activity of SP-A is mediated through its receptor. Type II cells incubated in Ca2+-free medium plus SP-A show diminished Ca2+ release responses to TG or ATP, suggesting that the action of SP-A to prevent secretagog initiated increases in [Ca2+]i may reflect its ability to block Ca2+ release from cytoplasmic Ca stores. The feedback inhibition of surfactant secretion by SP-A may, correspondingly, be a manifestation of this effect. Because recent work suggests that TGF-beta also inhibits Ca2+ fluxes, SP-A and TGF-beta could be representative of a group of physiologic regulators that act by modulating intracellular Ca signaling.

Adenosine Triphosphate↗
PubMed1996

SV40 as an effective gene transfer vector in vivo.

SV40 was used to transduce gene expression in vitro and in vivo. Using cloned SV40 genome, we replaced large T antigen gene (Tag) with a polylinker, and inserted firefly luciferase, controlled by SV40 early promoter. Transfection into Tag-expressing cells yielded Tag-deficient virus, SVluc. SVluc was Tag-deficient and therefore replication-deficient in cells that did not supply Tag. SVluc transduced functional luciferase expression in vitro. BALB/c mice were inoculated with SVluc, and their tissues were assayed 3-21 days post-inoculation (dpi) for luciferase protein production and enzyme activity. Luciferase protein was detected by immunohistochemistry throughout the experiment, from 3 to 21 dpi. There was no inflammatory reaction against SVluc-infected cells at any time, in any tissue studied. Luciferase activity was first detected by luminometry 14 dpi, and remained level through day 21. Thus, replication-deficient recombinant SV40 can mediate gene transfer in vitro and in vivo.

Animals↗
PubMed1996

Regulation of p53 gene expression by a poxviral transcription factor.

Identification of regulators of p53 expression is a crucial step in understanding the diverse functions of p53 and its role in cellular homeostasis and responsiveness to insult. Several viral proteins inactivate p53 as a modulator of cell cycle progression and apoptosis. Here, we report that a unique leporipoxviral transcription factor greatly increases levels of p53 mRNA. C7, an early transcription factor from malignant rabbit fibroma virus (MV), is an important determinant of MV virulence. Its effects on cellular gene expression were studied both during MV infection and in isolation, with C7 DNA cloned into a pKC4 expression plasmid. In both settings, C7 caused increased p53 mRNA levels. The increased p53 mRNA reflected new transcription. C7-induced increased transcription was selective: mRNAs for some cellular genes increased but those for many other genes (e.g., Bc12) were unchanged. Immunoblot and immunohistochemical analysis of pKC7-transfected and MV-infected cells showed that increased transcription led to an increase in p53 protein. EMSA analysis suggested that C7 bound the human p53 promoter between -240 and -614 bp. These studies document the direct effects of a viral transcription factor on cellular gene expression, specifically that it upregulates p53 transcription.

Animals↗
PubMed1996

Receptor-mediated regulation of pulmonary surfactant secretion.

Surfactant protein A (SP-A) regulates surfactant secretion via an SP-A specific type II cell membrane receptor (SPAR). We report here that two anti-SPAR monoclonal antibodies can modulate the secretory inhibition caused by SP-A. A2C and A2R are rat monoclonal antibodies raised independently and recognize a 32-kDa protein on rat alveolar type II cell membranes. Immunocytochemical studies show that these antibodies bind to isolated type II cells. Scatchard analysis confirms that SP-A binds alveolar type II cells through a single affinity receptor and shows that A2C and A2R recognize that same receptor. Both antibodies inhibit the binding of 125I-SP-A to isolated type II cells. The functional activity of this 32-kDa protein was studied by examining surfactant secretion in isolated type II cells. Surfactant phospholipid secretion was measured in cells that were exposed to various surfactant phospholipid secretagogues (ATP, dibutyryl cAMP, terbutaline, or ionomycin), +/-SP-A (100 ng/ml), +/-A2C or A2R. Both antibodies block the negative feedback loop by which SP-A inhibits surfactant secretion. This activity of A2C and A2R is dose-dependent and is independent of the secretagogue used. Thus, the 32-kDa type II cell membrane protein bound by A2C and A2R is the functional receptor on alveolar type II cell membranes and regulates type II cell surfactant secretion.

Animals↗
PubMed1996

Antibody to surfactant protein A increases sensitivity of pulmonary surfactant to inactivation by fibrinogen in vivo.

It has been suggested that surfactant protein-A (SP-A) protects surfactant activity from inhibitors such as fibrinogen. Substantial evidence indicates that inhibition of surfactant activity is often important in the pathogenesis of acute respiratory failure. Studies on surfactant function in the pulsating bubble surfactometer imply that SP-A helps to maintain low surface tension in the presence of inhibitors such as fibrinogen. We tested whether SP-A acts in this way in vivo. Rabbit pups, 29 d gestational age, were treated with a monoclonal antibody to rabbit SP-A (R5) followed by fibrinogen, or with control preparations (normal IgG and saline, respectively). Lung compliance was measured during ventilation throughout these experiments. Air-space volume and pulmonary edema were quantitated morphometrically. Animals receiving anti-SP-A antibody + fibrinogen showed substantial and significant impairment in lung compliance compared with control littermates receiving normal IgG and/or saline. Lungs from these animals showed decreased pulmonary air-space volume and increased alveolar edema. We conclude that SP-A protects pulmonary surfactant from inhibition by fibrinogen in vivo. This protective activity may be important in the pathogenesis of both adult and neonatal respiratory distress syndromes, and it may also be useful in devising therapies for these diseases.

Animals↗
PubMed1996

SV40 mediates stable gene transfer in vivo.

Gene transfer in vivo requires an efficient, nonreplicating, transfer agent. We report here the efficacy of recombinant, replication-deficient SV40 in transferring firefly luciferase (luc) production to murine hematopoietic cells and selected internal organs in vivo. Replication-deficient SV40 was made by replacing the large T antigen gene (Tag) with a polylinker, into which luc cDNA (luc) was cloned. Luc expression was controlled by SV40 early promoter. Tag-, luc+ SV40 DNA was transfected into Tag-expressing cells to yield a replication-deficient SV40-derivative virus containing luc (SVluc). The ability of SVluc to transfer luc production in vivo was tested in two ways: SVluc was inoculated into BALB/C mice intravenously; also bone marrow cells treated with SVluc were infused into syngeneic hosts. Luc production was followed for 105 days by immunochemical analysis of peripheral blood and selected internal organs using anti-luciferase antibody, and by assay of luc enzyme activity in peripheral blood. Luc was found in 20-25% of peripheral blood nucleated cells from day 20 until > or = 105 days. Luc-producing cells were also identified in liver, spleen, brain, kidney, skin and colon from day 20, also until > or = 105 days. Analysis of whole blood showed fluctuating levels of functionally active luc enzyme beginning on day 21, and remaining substantially and significantly greater than control values to day 105. Thus, SV40 may transfer sustained expression of foreign genes to bone marrow and other organs, for at least 3 months.

Animals↗
PubMed1995

The upstream region of the SP-B gene: intrinsic promoter activity and glucocorticoid responsiveness related to a new DNA-binding protein.

We identified and cloned the rabbit SP-B gene, encoding the pulmonary surfactant-associated protein, and sequenced its upstream region from -2635 to +428, including a much larger fragment of the upstream region than has previously been reported for an SP-B for any species. Rabbit SP-B showed substantial homology to its human counterpart in the coding and noncoding regions immediately upstream from the TATAA box. Using a luciferase (Luc) reporter gene (luc) construct we measured promoter activity with a 212-bp fragment (SPB212) from nucleotides (nt) -41 to -252, inclusive. SPB212 functioned as an active promoter in this assay. Further, we identified, cloned and sequenced the cDNA encoding a unique DNA-binding protein, N, that bound SPB212 at approx. -195. When the N cDNA was cloned into the expression vector pKC4 and cotransfected with the luc reporter construct, N significantly enhanced Luc production, but only in the presence of dexamethasone. Therefore, we identified and sequenced a functional promoter region upstream from rabbit SP-B, and isolated and characterized a DNA-binding protein that confers enhanced glucocorticoid responsiveness on this promoter.

Amino Acid Sequence↗
PubMed1995

DNA binding proteins that amplify surfactant protein B gene expression: isolation and characterization.

We identified and characterized two proteins that bind the promoter of surfactant protein B (SP-B) and affect its expression. Proteins A2 and B were identified and their cDNAs cloned and sequenced. Both were novel. They bound a 212-bp functional promoter region at an NF1 site, located between -184 and -198. Effects of these DNAbp on SP-B promoter activity were studied by contransfecting a reporter construct of this 212-bp sequence + luciferase, together with expression constructs for A2 and B into H441 cells. Alone, A2 and B expression elicited modest but statistically significant increases in SP-B promoter activity. When dexamethasone was added, B further increased SP-B promoter activity. For SP-B, basal expression and glucocorticoid responsiveness may involve a number of hitherto unknown gene activators.

Adult↗
PubMed1995

Levels of SP-A-anti-SP-A immune complexes in neonatal respiratory distress syndrome correlate with subsequent development of bronchopulmonary dysplasia.

As part of a double-blind, randomized, placebo-controlled study of human surfactant therapy for neonatal respiratory distress syndrome (NRDS), we measured circulating immune complexes between surfactant protein-A and anti-surfactant protein-A antibodies (SAS). Plasma from almost all infants contained detectable immune complexes. Immune complex levels in surfactant-treated infants were comparable with those of placebo-treated controls. Despite the relatively small sample size, maximum SAS immune complex values between 2 and 4 weeks after birth correlated significantly with subsequent development of BPD. Levels of these immune complexes correlated with eventual BPD independently of, and more strongly than, gestational age and birth weight. Thus, plasma SAS immune complex measurements may be useful in analyzing the course and outcome of NRDS, in particular the likelihood of subsequent development of BPD. This assay may also help to identify infants at risk for BPD and to target preventative therapy to them.

Antigen-Antibody Complex↗