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Biomedical subjects

S E Wert

Publications and source records attributed to S E Wert.

At least 55 records · Page 3Linked to original sources
PubMed1996

Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells.

Thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3alpha (HNF-3alpha), and HNF-3beta regulate the transcription of genes expressed in the respiratory epithelium. To test whether members of the HNF-3/forkhead family influence TTF-1 gene expression, deletion constructs containing the 5' region of the human TTF-1 gene were transfected into immortalized mouse lung epithelial (MLE) cells. DNase I protection and electrophoretic mobility shift assays identified elements in the 5' region of the TTF-1 gene that bound MLE cell nuclear proteins consistent with the binding of HNF-3 to sites at positions -135 to -124 and -14 to -3. In MLE cells, TTF-1-luciferase reporter constructs were activated by cotransfection with HNF-3beta, activated to a lesser extent by HNF-3alpha, but not activated by HFH-8. HNF-3alpha. and HFH-8 inhibited the activation of TTF-1-luciferase by HNF-3beta. Site-specific mutagenesis of each of the HNF-3 binding sites in the human TTF-1 gene inhibited the binding of MLE cell nuclear proteins and inhibited transactivation of the TTF-1-luciferase constructs after cotransfection with HNF-3beta. Immunohistochemical staining demonstrated that both HNF-3beta and TTF-1 were detected in bronchiolar and alveolar type II cells in the human lung. Modulation of TTF-1 gene expression by members of the HNF-3/forkhead family members may provide a mechanism by which distinct HNF-3/forkhead family members influence respiratory epithelial cell gene expression and cell differentiation.

3T3 Cells↗
PubMed1996

Reversal of lung lesions in transgenic transforming growth factor alpha mice by expression of mutant epidermal growth factor receptor.

Transgenic mice expressing transforming growth factor alpha (TGF-alpha) in type II cells under control of the lung-specific surfactant protein-C (SP-C) promoter develop pulmonary fibrosis and marked airspace hypoplasia. To identify cellular signaling mechanisms involved in lesion formation, we generated transgenic mice expressing a mutant epidermal growth factor receptor lacking a portion of the intracytoplasmic domain (EGF-R-M) under control of the human SP-C promoter. Transcripts of the SP-C-EGF-R-M transgene were detected in distal bronchiolar and type II cells by in situ hybridization. The morphology of lungs from the SP-C-EGF-R-M transgenic mice was normal. Lung fibrosis was not detectable and airspace hypoplasia was significantly corrected in bitransgenic mice derived by breeding SP-C-TGF-alpha and SP-C-EGF-R-M mice. Correction of lung pathology in the bitransgenic mice occurred without altering the level of hTGF-alpha mRNA. To further demonstrate that reversal of TGF-alpha lesions required signaling through the EGF-R, SP-C-TGF-alpha transgenic mice were bred to mice homozygous for the wa-2 mutation which encodes a mutated EGF-R. TGF-alpha-induced lesions were reversed in homozygous wa-2 mice. Amelioration of TGF-alpha-dependent pulmonary lesions in SP-C-EGF-R-M mice or wa-2/wa-2 mice supports the concept that autocrine and paracrine signaling mediate fibrosis and airspace remodeling caused by TGF-alpha.

Animals↗
PubMed1995

Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development.

Animals↗
PubMed1995

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.

Animals↗
PubMed1995

Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice.

To evaluate the role of cell-mediated immunity during gene transfer to the respiratory epithelium, the time course of luciferase activity was assessed after intratracheal administration of Av1Luc1, an E1a-E3-deleted adenoviral (Ad5) vector expressing firefly luciferase, to FVB/N, BALB/c and BALB/c-nu/nu adult mice. Adenovirus-mediated luciferase activity was rapidly lost from the respiratory tract between 2 and 14 days after treatment of both FVB/N and BALB/c wild-type mice. In the wild-type mice, loss of luciferase activity was associated with an early inflammatory response consisting of infiltration with macrophages and polymorphonuclear leukocytes and a more prolonged response characterized by lymphocytic infiltration. In the immune-deficient nu/nu mice, luciferase activity was maintained at higher levels than in immune-competent mice after exposure to virus and was associated with a distinct pattern of inflammation, consisting primarily of macrophages and polymorphonuclear cells but lacking the lymphocytic infiltrates typical of the inflammation in wild-type mice. Adenoviral DNA was rapidly cleared from the lungs of both nu/nu and wild-type mice. Markedly increased expression of proliferating cell nuclear antigen (PCNA) was observed in bronchiolar and alveolar epithelial cells and in inflammatory cells after exposure to Av1LUc1. The proliferative response of the respiratory epithelium was more extensive and persistent in wild-type than in nu/nu mice. To assess further the impact of the immune system on adenovirus-mediated gene expression, cotton rats treated with cyclosporin A or dexamethasone were exposed to Av1Luc1. Both agents decreased lung inflammation and significantly increased lung luciferase activity. The loss of lung luciferase activity is dependent, in part, on the immune-mediated clearance of respiratory epithelial cells, which may limit the extent and duration of gene expression with recombinant adenoviral vectors.

Adenoviridae Infections↗
PubMed1995

Adenovirus-mediated gene transfer to the respiratory tract of fetal sheep in utero.

Many human genetic diseases, such as congenital surfactant protein B deficiency, manifest in the perinatal period. Prenatal gene therapy may be necessary to minimize morbidity in these diseases. We hypothesized that bacterial beta-galactosidase (beta-Gal) gene could be transferred to and expressed in the pulmonary epithelium of fetal sheep in utero using a replication-deficient adenovirus (Av1LacZ4). We instilled Av1LacZ4 (1.5 x 10(11) plaque-forming units, n = 10) or saline (n = 2) intratracheally to chronically instrumented fetal sheep at 112-134 days gestation (term = 145 days). Lung fluid was collected before and after Av1LacZ4 administration for cytological analysis. Lung tissue was examined for transgenic beta-Gal activity and evidence of toxicity. Transgenic beta-Gal activity was visualized as blue nuclear staining of tissue treated with X-Gal and was detected in the lungs of 5 animals for up to 14 days after administration. Transgenic beta-Gal activity was not detected in the lungs of animals analyzed beyond 14 days after treatment. Pulmonary histopathology was detected in most Av1LacZ4-treated animals and manifested as a mixed cellular infiltrate consisting of neutrophils, macrophages, and lymphocytes. Fetal lung fluid analysis revealed a predominantly lymphocytic response in most Av1LacZ4-treated animals within 3 days (2.88 x 10(6) vs. 4 x 10(3) total cells/ml in control animals). We have demonstrated that adenovirus vectors can direct gene transfer to the lungs of fetal sheep in utero. The transferred gene expression was transient and possibly limited by the induced inflammatory response.

Adenoviridae↗
PubMed1995

In situ localization of sodium-potassium ATPase mRNA in developing mouse lung epithelium.

The ontogeny of Na(+)-K(+)-adenosinetriphosphatase (ATPase) mRNA in the mouse lung was examined, using alpha- and beta-isoform-specific probes in Northern blot assays and for in situ hybridization analysis. Northern blot assays demonstrated an increase in Na(+)-K(+)-ATPase expression in the perinatal period, peaking at birth (D1), with alpha 1- and beta 1-isoform levels reaching six to eight times adult levels. In situ alpha 1-isoform hybridization signals were localized primarily to developing airway epithelium and were most intense on D1. Postnatally, alpha 1-isoform hybridization signals persisted in airway epithelium, although progressively diminishing in intensity relative to perinatal levels. In developing alveolar regions, alpha 1-isoform hybridization signals remained slightly above background during this period. beta 1-Isoform hybridization signals increased dramatically during the perinatal period in both developing airway and alveolar epithelia. Postnatally, beta 1-isoform hybridization signals declined slightly in airway epithelium and developed a punctate pattern in alveolar epithelium. These data indicate that the perinatal increase in Na(+)-K(+)-ATPase expression observed in the developing mouse lung is localized primarily to epithelial structures and is therefore likely to be related to the changes in transepithelial ion and fluid transport known to occur in the perinatal period.

Aging↗
PubMed1994

Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). A potential animal model of CF, the CFTR-/- mouse, has had limited utility because most mice die from intestinal obstruction during the first month of life. Human CFTR (hCFTR) was expressed in CFTR-/- mice under the control of the rat intestinal fatty acid-binding protein gene promoter. The mice survived and showed functional correction of ileal goblet cell and crypt cell hyperplasia and cyclic adenosine monophosphate-stimulated chloride secretion. These results support the concept that transfer of the hCFTR gene may be a useful strategy for correcting physiologic defects in patients with CF.

Animals↗
PubMed1994

Transgenic models for study of pulmonary development and disease.

This review summarizes progress in the application of transgenic mouse technology to the study of lung development and disease. Since advances in molecular genetics have greatly facilitated the isolation of cDNA and genes, our ability to readily assess roles of both normal and mutated genes in transgenic mouse in vivo represents a major advance, bridging molecular biology and whole animal physiology. Strategies have been developed in which lung epithelial cell promoter elements are used to drive normal or mutated genes into specific subsets of respiratory epithelial cells in the lungs of developing and mature transgenic mice. These mice have been used to elucidate the cis-acting elements controlling lung epithelial cell gene expression, to discern the role of specific polypeptides in lung morphogenesis and tumorigenesis, and to create animal models of pulmonary disease. The ability to mutate genes at their precise chromosomal locations through gene targeting in embryonic stem cells has lead to the production of animal models of lung diseases such as cystic fibrosis. Both gene insertion and gene targeting create permanent mouse lines that pass the modified gene to their progeny, providing animals for the study of the pathogenesis and treatment of pulmonary disorders.

Adenocarcinoma↗
PubMed1994

Adenoviral-mediated gene transfer of human surfactant protein B to respiratory epithelial cells.

Human surfactant protein B (SP-B) is a 79-amino acid, phospholipid-associated polypeptide expressed by respiratory epithelial cells of the lung. SP-B is essential for lung function, enhancing the spreading and stability of surfactant phospholipids that serve to reduce surface tension at the alveolar air-liquid interface. Congenital absence of SP-B results in neonatal respiratory failure and death. In the present work, we constructed a replication-deficient adenoviral vector, Av1SP-B1, in which the human SP-B cDNA is expressed under control of the Rous sarcoma virus (RSV) promoter in an E1-E3-deleted adenovirus type 5 (Ad5)-based vector system. Av1SP-B1 was produced in 293 kidney cells, directing the synthesis of the SP-B protein and SP-B peptides. Av1SP-B1 directed the synthesis of SP-B mRNA, precursor and active 8-9 kD polypeptide in immortalized mouse lung epithelial cells (MLE-12 cells), demonstrating complete processing to the human SP-B protein by these cells. Synthesis of human SP-B mRNA was detected as early as 12 h after infection and was maximal 48 h after infection in vitro. Northern blot analysis demonstrated that human SP-B mRNA was expressed in the lungs of cotton rats infected with Av1SP-B1 but not in those of uninfected animals or in animals infected with a reporter adenoviral vector, Av1LacZ4. In situ hybridization demonstrated the abundance and localization of the transferred human SP-B mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1994

Respiratory epithelial cell expression of human transforming growth factor-alpha induces lung fibrosis in transgenic mice.

Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung.

Animals↗
PubMed1993

Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.

Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10 expression in proximal regions of the developing bronchial tubules.

Animals↗
PubMed1992

cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

Adenocarcinoma↗
PubMed1992

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Animals↗
PubMed1992

Distinct effects of oxygen on surfactant protein B expression in bronchiolar and alveolar epithelium.

Hyperoxia causes severe lung injury in association with altered expression of surfactant proteins and lipids. To test whether oxygen induces surfactant protein B (SP-B) expression in specific respiratory epithelial cells, adult B6C3F1 and FVB/N mice were exposed to room air or 95% oxygen for 1-5 days. Northern blot analysis demonstrated an 8- to 10-fold increase in SP-B mRNA after 3 days that was maintained thereafter. In situ hybridization localized SP-B mRNA to bronchial, bronchiolar, and alveolar epithelial cells. Hyperoxia was associated with increased SP-B mRNA, noted primarily in the bronchiolar epithelium and decreased SP-B mRNA in the alveolar epithelium. After 5 days, central regions of lung parenchyma were nearly devoid of SP-B mRNA, while SP-B mRNA was maintained in alveolar cell populations close to vascular structures. To determine whether increased bronchiolar expression of SP-B mRNA during hyperoxia was a specific response, the abundance of CC10 mRNA (a Clara cell protein) was assessed. CC10 mRNA was detected in tracheal, bronchial, and bronchiolar, but not alveolar epithelium and was decreased upon exposure to hyperoxia. Immunocytochemistry demonstrated that SP-B proprotein was detected in bronchial, bronchiolar, and alveolar epithelial cells with staining increased in the bronchial and bronchiolar epithelium upon exposure to hyperoxia. SP-B gene expression in the respiratory epithelium is regulated at a pretranslational level and occurs in a cell specific manner during hyperoxic injury in the mouse.

Animals↗
PubMed1991

Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice.

Transgenic mice bearing chimeric genes consisting of 5'-sequences derived from the human surfactant protein C (SP-C) gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were generated. Analysis of CAT activity was utilized to demonstrate tissue-specific and developmental expression of chimeric genes containing 3.7 kb of sequences from the human SP-C gene. Lung-specific expression of the 3.7 SP-C-CAT transgene was observed in eight distinct transgenic mouse lines. Expression of the 3.7 SP-C-CAT transgene was first detected in fetal lung on day 11 of gestation and increased dramatically with advancing gestational age, reaching adult levels of activity before birth. In situ hybridization demonstrated that expression of 3.7 SP-C-CAT mRNA was confined to the distal respiratory epithelium. Antisense CAT hybridization was detected in bronchiolar and type II epithelial cells in the adult lung of the 3.7 SP-C-CAT transgenic mice. In situ hybridization of four distinct 3.7 SP-C-CAT transgenic mouse lines demonstrated bronchiolar-alveolar expression of the chimeric CAT gene, although the relative intensity of expression at each site varied within the lines studied. Glucocorticoids increased murine SP-C mRNA in fetal lung organ culture. Likewise, expression of 3.7 SP-C-CAT transgene increased during fetal lung organ or explant culture and was further enhanced by glucocorticoid in vitro. The 5'-regions of human SP-C conferred developmental, lung epithelial, and glucocorticoid-enhanced expression of bacterial CAT in transgenic mice. The increased expression of SP-C accompanying prenatal lung development and exposure to glucocorticoid is mediated, at least in part, at the transcriptional level, being influenced by cis-active elements contained within the 5'-flanking region of the human SP-C gene.

Animals↗
PubMed1990

Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass.

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.

Animals↗