Gene therapy of cystic fibrosis lung disease using E1 deleted adenoviruses: a phase I trial.
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Adenovirus E1 Proteins↗
Biomedical subjects
R H Simon
Publications and source records attributed to R H Simon.
Regulation of alveolar epithelial cell ICAM-1 expression by cell shape and cell-cell interactions.
In normal lung, intercellular adhesion molecule 1 (ICAM-1) is expressed at high levels on thin type I alveolar epithelial cells, but is minimally expressed on cuboidal type II cells. ICAM-1 is induced in primary culture on tissue culture-treated plastic as type II cells undergo transition toward a type I cell-like phenotype. We hypothesized that alveolar epithelial cell expression of ICAM-1 might be regulated in part by signals that influence the state of differentiation of these cells. We found that rat type II cells that were cultured as aggregates of cuboidal cells on a hydrated basement membrane gel (Matrigel) or on floating type I collagen gels, expressed markedly less ICAM-1 protein and mRNA compared with cells that had spread on plastic. In contrast, type II cells that had spread as monolayers on dishes coated with basement membrane proteins in planar configuration demonstrated ICAM-1 expression comparable to that of cells on plastic alone. Thus regulation of alveolar epithelial cell expression of this immunologically important adhesion molecule involves complex spatial interactions of the cells with the basement membrane and other epithelial cells.
Animals↗
Applications of lipid-coated microbubble ultrasonic contrast to tumor therapy.
Lipid-coated microbubbles (LCM) make an excellent diagnostic ultrasonic contrast agent in experimental tumor systems. LCM have been shown to aggregate in brain tumors and subcutaneous tumors after intravenous administration, and to provide persistent image enhancement for many minutes. In this work, experimental subcutaneous Walker Carcinosarcoma is insonated after the bubbles are given intravenously. Selective necrosis, lymphocyte proliferation and hemorrhage within the tumor can be demonstrated. Preliminary data are given to demonstrate this phenomenon. The mechanism of the effect is discussed in the context of both heating and cavitation.
Animals↗
Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study.
We have evaluated the biological efficacy of E1-deleted adenoviruses in baboons for lung-directed gene therapy of cystic fibrosis (CF). The experimental design attempted to simulate a phase I clinical trial with animals receiving a single dose of virus to an isolated pulmonary segment. A total of 14 animals divided into four groups, each of which received escalating doses of virus, were used. Individual animals were necropsied 4 and 21 days after gene transfer and tissues were carefully surveyed for gene expression. Expression of the transgene was localized primarily to the area into which it was infused; the efficiency of recombinant gene expression and the abundance of transgene sequences were proportional to dose and both diminished with time. Transgene expression was found predominantly in alveolar cells with patches of expression in the proximal and distal airway. Analysis of adenoviral protein expression within transgene-expressing cells revealed infrequent expression of the E2a gene and no detectable expression of late genes (i.e., fiber protein). These results suggest that recombinant adenovirus can be used to transfer genes efficiently to the lung of nonhuman primates and that therapeutic strategies of cystic fibrosis may require repetitive administration with current vectors.
Adenoviridae↗
Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study.
In preparation for human trials of gene therapy for cystic fibrosis (CF), we performed a preclinical study of gene transfer into the lungs of baboons. Recombinant adenovirus vectors containing expression cassettes for human cystic fibrosis transmembrane conductance regulator (CFTR) and Escherichia coli beta-galactosidase (lacZ) were instilled through a bronchoscope into limited regions of lung in 14 baboons. A detailed accounting of the extent, distribution, and duration of gene expression is contained in a companion article (Engelhardt et al., 1993b). In this article, we report the results of toxicity studies in which clinical laboratory tests, chest radiographs, and necropsy studies were used to detect adverse effects. The only adverse effect noted was a mononuclear cell inflammatory response within the alveolar compartment of animals receiving doses of virus that were required to induce detectable gene expression. Minimal inflammation was seen at 10(7) and 10(8) pfu/ml, but at 10(9) and more prominently at 10(10) pfu/ml, a perivascular lymphocytic and histiocytic infiltrate was seen. The intensity of inflammation increased between 4 and 21 days. At its greatest intensity, there was diffuse alveolar wall damage with intra-alveolar edema. Airways were relatively spared, despite the intensity of alveolar inflammation. Clinical tests did not accurately reflect the presence of lung inflammation, with the exception of chest radiographs which revealed alveolar infiltrates, but only in regions of lung having the greatest intensity inflammation. We conclude that adenovirus-mediated gene transfer into the lungs of baboons is associated with development of alveolar inflammation at high doses of virus.
Adenoviridae↗
Legionella pneumophila replicates within rat alveolar epithelial cells.
Legionella pneumophila replicates in the distal pulmonary airspace, causing legionnaires' pneumonia. Legionella organisms replicate within alveolar macrophages and recruited blood monocytes; however, when these cells are activated, they become potent inhibitors of L. pneumophila proliferation. L. pneumophila may replicate in other cells and thereby avoid the host defenses of macrophages. Experiments demonstrated that L. pneumophila replicate within primary cultures of rat pulmonary alveolar epithelial cells. Double-label immunofluorescent and electron microscopy demonstrated L. pneumophila within epithelial cells. Replication of L. pneumophila required similar numbers of alveolar epithelial cells or alveolar macrophages, required viable epithelial cells, and took place intracellularly. While replication of L. pneumophila occurred in both serum-free and serum-containing media, it was enhanced in the presence of serum. Pulmonary alveolar epithelial cells may represent an alternative site for replication of Legionella species in the terminal airspace and thus clarify some previously unexplained aspects of the pathogenesis of legionnaires' disease.
Animals↗
Detection of experimental rat liver tumors by contrast-assisted ultrasonography.
RATIONALE AND OBJECTIVES: The authors characterized the effect of intravenous lipid-coated microbubbles (LCMs) on the echogenicity of malignant liver tumors. METHODS: Novikoff hepatoma cells were inoculated into the livers of 16 anesthetized Sprague-Dawley rats. Sonograms were obtained weekly after tail-vein injection with either 0.2 mL/kg LCMs or saline control. RESULTS: A statistically significant difference in the signal-to-noise ratio (SNR) was observed between the group that received LCMs (10 rats) and the group that received saline (6 rats) (P < .01). The effect persisted for 30 minutes after contrast injection. Selective leakage and accumulation of LCMs into the tumor tissue itself was confirmed histologically using lipid-specific counterstains. CONCLUSIONS: Intravenous injection of the LCM contrast agent produces a rapid increase in the echogenicity of the experimental Novikoff tumor in the rat liver.
Animals↗
Differentiation-related expression of ICAM-1 by rat alveolar epithelial cells.
Local regulation of immune and inflammatory responses within the alveolar space is a critical aspect of normal pulmonary host defense. The type I and type II epithelial cells that line the alveolar space are in intimate contact with lymphocytes and macrophages within the alveolar space and are ideally situated to provide regulatory signals to these effector cells. The present studies were undertaken to investigate the expression by rat alveolar epithelial cells in vitro and in vivo of intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule that is involved in migration and activation of T cells and macrophages. An antibody specifically blocking rat ICAM-1 (mAb 1A29) inhibited the adherence of activated T lymphoblasts to monolayers of type II alveolar epithelial cells. The expression of ICAM-1 protein by alveolar epithelial cells in vitro was confirmed both by immunofluorescence microscopy and by Western blot analysis. However, in each instance, ICAM-1 was not detected in type II cells the day of isolation, but appeared at low levels after 1 day and in abundance throughout the monolayer after 2 days, with sustained expression thereafter. This suggested that ICAM-1 expression might be a type I cell feature, which was induced as isolated type II cells underwent transformation towards the type I cell-like phenotype in vitro. Using immunofluorescence microscopy on frozen sections of normal lung, ICAM-1 was found in a linear distribution along the alveolar space, consistent with expression on type I cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Type IV collagen production by rat pulmonary alveolar epithelial cells.
Maintenance and repair of the pulmonary alveolar basement membrane are critical processes for preserving normal alveolar structure and function. To elucidate the mechanisms that control type IV collagen production by pulmonary alveolar epithelial cells, type II pneumocytes from rat lungs were isolated and maintained in tissue culture as monolayers. Using Northern blot analysis and metabolic labeling, we found that the cultured epithelial cells express type IV collagen mRNA and incorporate [3H]proline into type IV collagen. To determine the effects of phenotype on control of type IV collagen synthesis, we took advantage of the well-described observation that isolated type II cells lose their distinctive type II features when cultured on plastic in serum-containing medium for 7 days and assume an appearance more like that of type I epithelial cells. We found that [3H]proline incorporation into type IV collagen increased from day 1 to day 2 and thereafter decreased gradually up to day 7. Despite this decrease in [3H]proline incorporation, type IV collagen mRNA increased over the same period. If the loss of type II cell characteristics was prevented by culturing the cells in EHS matrix, a basement membrane-like extracellular matrix, there was little increase in relative abundance of type IV collagen mRNA as compared with cells cultured on plastic. We therefore conclude that type IV collagen production by isolated pulmonary alveolar epithelial cells is controlled, at least in part, by translational or post-translational mechanisms. Furthermore, the full type II cell phenotype is not required for expression of type IV collagen genes.(ABSTRACT TRUNCATED AT 250 WORDS)
3T3 Cells↗
Quantitative assessment of tumor enhancement by ultrastable lipid-coated microbubbles as a sonographic contrast agent.
We have previously reported that ultrastable lipid-coated microbubbles make a suitable ultrasonic contrast agent in the brain, causing increased intensity of echoes that persists for many hours. We showed that intravenously administered lipid-coated microbubbles accumulate selectively in rat brain gliomas with echogenicity enhancement for up to 1 hour, allowing visualization of the growing lesions 40% (2 days) earlier than can be seen without contrast. This work is a detailed evaluation of the accumulation of the lipid-coated microbubbles in tumor and the effect of the bubbles on the echogenicity of insonified tumors. Using a lipid-specific stain, we measured and characterized the distribution of the bubbles in the brain and tumor. We showed that on the scan, the enhancement of the tumor is accompanied by a change in the signal-to-noise ratio of the echoes from the tumor. We identified characteristic textural changes associated with contrast-enhanced tumor using spectral analysis.
Animals↗
Fibrin degradation by rat pulmonary alveolar epithelial cells.
The persistence of intra-alveolar fibrin during acute and chronic inflammatory lung diseases indicates that the normally profibrinolytic environment of the alveolar space has been altered as part of the disease process. We have recently shown that alveolar epithelial cells may control fibrinolysis by expressing both urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1. In this study, monolayers of rat alveolar epithelial cells were used as a model of the alveolar surface and were found to lyse plasma-derived fibrin matrices by a process that was plasminogen and uPA dependent. Fibrinolysis was not achieved by fluid-phase epithelial products but required the presence of epithelial cells, optimally in close contact with the clot surface. Epithelial cell-mediated fibrinolytic activity was augmented 99% by endotoxin and suppressed 66% by dexamethasone. Fibrinolysis also increased 84% as cells aged in culture from day 1 to day 4, during which time the cells lose many type II cell characteristics and assume a type I cell-like phenotype. We conclude that alveolar epithelial cells actively participate in fibrin clearance through mechanisms that require close proximity between epithelial cell and clot surfaces. Alterations in these mechanisms may be partly responsible for the persistence of intraalveolar fibrin during lung inflammation.
Amiloride↗
Tissue factor procoagulant expression by rat alveolar epithelial cells.
Fibrin deposition in the alveolar space is characteristic of inflammatory lung injury. The formation of fibrin in the alveolus results from the coagulation of extravasated plasma. The cellular elements that promote intra-alveolar clotting have not been completely defined. We have investigated the capacity of alveolar epithelial cells (AEC) to promote coagulation through the expression of procoagulant activity (PCA) in tissue culture. Using a single-stage coagulation assay, rat AEC monolayers were found to contain 20,750 +/- 4,035 procoagulant units (PCU)/10(6) cells; 10- to 20-fold greater activity than that found in concomitantly isolated alveolar macrophages. The epithelial-derived procoagulant was shown to be tissue factor by a series of assays using clotting factor-deficient human plasmas. Freshly isolated AEC also possessed PCA (2,500 +/- 1,000 PCU/10(6) cells) and expressed a 2.1-kb mRNA that hybridized with a cDNA for murine tissue factor. Using a kinetic turbidometric assay of clot acceleration, PCA was found on the surface of unstimulated epithelial monolayers and could be increased to 170% of control by incubation with phorbol myristate acetate (PMA). This response to PMA was accompanied by a parallel increase in the relative abundance of tissue factor mRNA. AEC shed particulate PCA into the culture media that displayed a specific activity similar to that recovered from alveolar lining fluid. Therefore, by expressing both cell surface and particulate PCA, the alveolar epithelium likely contributes significantly to the modulation of intra-alveolar coagulation.
Animals↗
Lipid-coated uniform microbubbles for earlier sonographic detection of brain tumors.
Rapid technological improvements have fostered the continued clinical development of intraoperative neurosonology, despite the fact that no suitable contrast media have been available for ultrasound studies. Because they can be made of uniform size (99% are less than 4.5 mum in diameter) and are thought to cross disruptions in the tumor vessels, artificial lipid-coated microbubbles can fill this gap. Furthermore, these microbubbles are stable in vitro for at least 6 months, with an in vivo halflife of 20 hours or more. This study demonstrated that lipid-coated microbubbles injected intravascularly can intensify echoes from rat brain gliomas. Specifically, when this standardized microbubble contrast agent was injected intravenously daily in rats, the time to visual ultrasonic detection of developing brain tumors (C-6 gliomas) was 4.09 days (n = 11) after tumor inoculation, versus 6.67 days (n = 9) to detection without microbubble injection (Z = -3.71, p = 0.0004).
Air↗
Rat alveolar epithelial cells concomitantly express plasminogen activator inhibitor-1 and urokinase.
There is considerable evidence to suggest that intra-alveolar plasminogen activation is instrumental in many aspects of inflammatory lung injury and subsequent tissue repair. Rat alveolar epithelial cells produce large quantities of urokinase-type plasminogen activator (uPA) in vitro, and uPA expression is modulated in association with cellular differentiation and exposure to inflammatory mediators. We now report that these cells also secrete heat-stable PA inhibitory activity having the characteristics of PA inhibitor type 1 (PAI-1). In particular, immunoreactive PAI-1 was demonstrable in conditioned media, cell lysates, and extracellular matrix from epithelial cell cultures. As alveolar epithelial cells differentiated in vitro, secreted PA inhibitor activity increased significantly from 104 +/- PAI U/ml (n = 5, mean +/- SE) on day 2 to 442 +/- 150 on day 7 in parallel with increases in secreted and matrix-associated immunoreactive PAI-1. PAI-1 mRNA expression decreased over this same period suggesting posttranscriptional regulation. The levels of both newly synthesized antigen and PAI-1 mRNA were increased by exposure to lipopolysaccharide and tumor necrosis factor-alpha. Thus, by the coexpression of uPA and PAI-1, the alveolar epithelium may actively regulate the generation of plasmin in both the normal and injured alveolus.
Animals↗
Injury of rat pulmonary alveolar epithelial cells by H2O2: dependence on phenotype and catalase.
In a variety of inflammatory lung diseases, type I alveolar epithelial cells are more likely to be injured than are type II cells. Because oxidants have been implicated as a cause of injury in various inflammatory lung diseases, we evaluated the effects of differentiation on alveolar epithelial cell susceptibility to H2O2-induced injury. With the use of isolated rat type II cells in culture, we found that the cytotoxic effect of H2O2 increased between days 2 and 7, when type II cells are known to lose their distinctive type II properties and assume a more type I-like appearance. We previously reported that type II cells utilized both intracellular catalase and glutathione-dependent reactions to protect against H2O2. We therefore examined whether alterations in either of these protective mechanisms were responsible for the differentiation-dependent changes in sensitivity to H2O2. We found that catalase activity within alveolar epithelial cells decreased between 2 and 7 days in culture, whereas no changes were detected in glutathione-dependent systems. We then used a histochemical technique that detects catalase activity and found that type II cells within rat lungs possessed numerous catalase-containing peroxisomes, whereas very few were detected in type I cells. These findings demonstrate that as type II cells assume a type I-like phenotype, they become more susceptible to H2O2-induced injury. This increased susceptibility is associated with reductions in intracellular catalase activity, both in vitro and in vivo.
Animals↗
An application of fractal dimension to the detection of transients in the electroencephalogram.
It is sometimes desirable to identify a brief seizure, occasional spike, single evoked potential, or other transient in the EEG. Transient detection in an EEG can be a difficult task, often requiring prior knowledge of the characteristics of the transient. A fractal is a shape which retains structural detail despite magnification (scaling). The complexity of the structure of such a set, invariant under this scaling, can be characterized by a single number: the fractal dimension. Regarding the EEG as a fractal, we have shown that transient deterministic data in the EEG have a fractal dimension different from the quasirandom background. An extensive introduction to fractals is presented with the assumption that the reader is unfamiliar with the theory. In the preliminary results presented here, analysis by fractal dimension is shown to be a promising method of transient detection, requiring no prior knowledge of the characteristics of the transient. Possible applications of the technique to evoked potential technology and epilepsy surgery are discussed. Other applications to biology, neuroscience and medicine are reviewed.
Algorithms↗
Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts.
Hydrogen peroxide (H2O2) has been implicated in cardiac damage due to ischemia and reperfusion. We adapted an electron microscopic, histochemical method for demonstrating H2O2 produced by isolated cells to isolated, buffer-perfused rabbit hearts. The method involves formation of an electron-dense precipitate when H2O2 reacts with cerium chloride (CeCl3). We perfused hearts retrograde via the aorta with well-oxygenated bicarbonate-buffered solution, followed by one in which bicarbonate was replaced with imidazole (IPSS) to prevent precipitation of bicarbonate and CeCl3. Some hearts were made globally ischemic (30 min, 37 degrees C), reperfused 5 min with well-oxygenated IPSS containing 1 mM CeCl3, then processed for electron microscopy. Others were perfused with IPSS containing catalase (300 U/ml) or albumin before ischemia and upon reperfusion, followed by CeCl3 administration. Nonischemic control hearts perfused with IPSS (+/- catalase) were also studied. Electron micrographs were assessed visually and by computer for precipitate localization and amount. There was abundant precipitate on the luminal face of the coronary vascular endothelium in ischemic-reperfused, cerium-treated hearts, including those treated with albumin. There was significantly less in reperfused catalase-treated or nonischemic control hearts. X-ray microbeam analysis of the endothelial precipitate indicated the presence of Ce. This appears to be the first visual demonstration of a CeCl3-H2O2-dependent reaction product in intact isolated ischemic hearts. The data indicate that at the time of reperfusion some H2O2 is accessible to the vascular space, and that its amount can be reduced by perfused catalase. Further modifications this technique may be useful for assessing the sites and pathways by which H2O2 is generated by hearts or other buffer-perfused organs subjected to stresses such as ischemia or hypoxia.
Animals↗
Lipid-coated ultrastable microbubbles as a contrast agent in neurosonography.
Lipid-coated microbubbles can be synthesized from selected lipid monolayer systems for use as ultrasonic contrast. These microbubbles have the property of longevity of weeks in vitro (ultrastability) and longevity of hours in vivo. The bubbles can be manufactured with a mean diameter of approximately 2 microns in a tight diameter distribution; all are less than 6 microns and 99% are smaller than 4.5 microns. The current study compared the in vivo survival characteristics of these lipid-coated microbubbles with microbubbles produced by saline. The comparison was made in the rat brain using direct intraparenchymal injections and injections into a previously created cyst/coagulum. The echogenic enhancement by the lipid-coated microbubbles persisted in vivo for over 24 hours in both the intraparenchymal environment and in the cyst/coagulum. The saline bubble echos were not detectable by 3 hours in a cyst/coagulum, and not detectable in the parenchyma after 2 hours. The sonographic characteristics and longevity of lipid-coated microbubbles make this agent a potentially useful clinical contrast material for neurosonography.
Animals↗