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Biomedical subjects

P Lemarchand

Publications and source records attributed to P Lemarchand.

At least 37 records · Page 2Linked to original sources
PubMed1995

Effects of smoking on bronchial clearance of technetium-99m-DTPA and indium-113m-DTPA.

UNLABELLED: In asymptomatic smokers, epithelial permeability in the distal lung regions is increased. To date, the effect of smoking on the epithelial permeability in proximal lung regions is still debated. The measurement of bronchial clearance of inhaled radiolabeled diethylene-triaminepentaacetic acid (BC-DTPA) can be used to assess epithelial permeability of proximal bronchi, but there are two potential limitations to this method: in vivo breakdown of 99mTc-DTPA in smokers and mucociliary transport of DTPA. METHODS: Eight nonsmokers and eight asymptomatic smokers were studied. We used a spinning disk system to generate an aerosol of large particles of 99mTc-DTPA or 113mIn-DTPA (MMAD 6.3 microns). To measure the bronchial clearance of 99mTc-DTPA and 113mIn-DTPA, we analyzed the perihilar regions of the lung. To determine the contribution of mucociliary transport, we measured the activity over a tracheal region of interest (ROI) in eight nonsmokers. RESULTS: Technetium-99m-DTPA bronchial clearance did not differ in smokers (1.16 +/- 0.54%/min; mean +/- s.d.) or nonsmokers (1.29 +/- 0.51%/min; ns). The 113mIn-DTPA bronchial clearances in nonsmokers (1.24 +/- 0.51%/min) and in smokers (1.01 +/- 0.66%/min) were similar to the 99mTc-DTPA bronchial clearances (ns). In the tracheal ROI, we found no increase in activity. CONCLUSION: In smokers, BC-DTPA was not increased compared to nonsmokers. In contrast to distal lung regions, there was no evidence of breakdown of the 99mTc-DTPA complex in the proximal regions of smokers' lungs. Mucociliary clearance does not significantly contribute to BC-DTPA.

Adult↗
PubMed1994

In vivo adenovirus-mediated gene transfer to lungs via pulmonary artery.

On the basis of the knowledge that the pulmonary and bronchial circulations have extensive anastomoses, we hypothesized that gene transfer to the endothelium of both pulmonary and bronchial circulations might be achieved with replication-deficient recombinant adenovirus (Ad) vectors administered to the pulmonary circulation. To evaluate this concept, the right upper lobe branches of the sheep pulmonary artery and vein were temporarily occluded and a replication-deficient recombinant Ad vector containing the Escherichia coli lacZ reporter gene coding for beta-galactosidase (beta-Gal) was infused into the lumen of the occluded pulmonary artery. After 15 min, the pulmonary circulation was restored, and 1 or 4 days later the lungs were evaluated by histochemical analysis for beta-Gal activity. Gene transfer and expression were positive in 13 of 17 evaluated sheep. No beta-Gal activity was detected in any category of cells of uninfected lobes. As hypothesized, beta-Gal activity was detected in endothelial cells of the right upper lobe pulmonary and bronchial circulations. Unexpectedly, gene transfer was also observed in epithelial cells of the alveoli and the airways (bronchi and bronchioles) as well as in the epithelium of submucosal glands. These studies demonstrate that it is possible to use Ad vectors for transfer and expression of genes to lung parenchymal cells served by both the pulmonary and bronchial circulations. Furthermore, whereas administration of such vectors via the airways results in gene transfer only to the epithelium, pulmonary artery administration permits gene transfer to both endothelium and epithelium, thus expanding the target range of Ad gene transfer to the lungs.

Adenoviridae↗
PubMed1994

Vulnerability of the human airway epithelium to hyperoxia. Constitutive expression of the catalase gene in human bronchial epithelial cells despite oxidant stress.

Although catalase is a major intracellular antioxidant, the expression of the human catalase gene appears to be limited in the airway epithelium, making these cells vulnerable to oxidant stress. The basis for this limited gene expression was examined by evaluation of the expression of the endogenous gene in human bronchial epithelial cells in response to hyperoxia. Hyperoxia failed to upregulate endogenous catalase gene expression, in contrast to a marked increase in expression of the heat shock protein gene. Sequence analysis of 1.7 kb of the 5'-flanking region of the human catalase gene showed features of a "house-keeping" gene (no TATA box, high GC content, multiple CCAAT boxes, and transcription start sites). Transfection of human bronchial epithelial cells with fusion genes composed of various lengths of the catalase 5'-flanking region and luciferase as a reporter gene showed low level constitutive promoter activity that did not change after exposure to hyperoxia. Importantly, using a replication-deficient recombinant adenoviral vector containing the human catalase cDNA, levels of catalase were significantly increased in human airway epithelial cells and this was associated with increased survival of the cells when exposed to hyperoxia. These observations provide a basis for understanding the sensitivity of the human airway epithelium to oxidant stress and a strategy for protecting the epithelium from such injury.

Base Sequence↗
PubMed1993

Protection of human endothelial cells from oxidant injury by adenovirus-mediated transfer of the human catalase cDNA.

In a variety of disorders, endothelial cells are exposed to high levels of oxidants, generated within the cells and/or consequent to local inflammation. In the context of the sensitivity of endothelial cells to oxidant stress, particularly related to H2O2, we have designed a replication deficient recombinant adenovirus containing the human catalase cDNA (AdCL) to transfer the catalase cDNA to the endothelial cells, in order to augment intracellular anti-H2O2 protection. Human umbilical vein endothelial cells that were not infected or infected with control adenovirus maintained low levels of catalase mRNA. Endothelial cells infected with AdCL expressed AdCL-driven exogenous catalase mRNA, as early as 24 hr and at least for 7 days. Catalase protein levels were increased significantly over controls in cells infected with AdCL, as were catalase activity levels, with catalase activity correlated closely with levels of catalase protein. Importantly, when the endothelial cells were exposed to 500 microM H2O2, all the AdCL infected endothelial cells survived, compared to only 37% of the control cells. Thus, a recombinant adenovirus containing the human catalase cDNA is able to infect human endothelial cells in vitro and express high levels of functional intracellular catalase, protecting the cells against H2O2-mediated oxidant stress. These observations support the feasibility of the transfer of catalase cDNA to human endothelium to protect against oxidant injury.

Adenoviridae↗
PubMed1993

Efficient and selective adenovirus-mediated gene transfer into vascular neointima.

BACKGROUND: Previous attempts to target arterial smooth muscle cells (SMCs) for gene delivery using liposomal or retroviral methods were limited by low transfection efficiency. We therefore evaluated the efficiency of adenovirus-mediated gene delivery in cultured vascular SMCs and in an in vivo model of balloon injury-induced SMC cell proliferation. METHODS AND RESULTS: We used a recombinant adenovirus, Ad.RSV beta gal, which contained the beta-galactosidase (beta-gal) histochemical marker gene. For in vitro studies, rat aortic SMCs were incubated in media containing Ad.RSV beta gal for 5 to 120 minutes. The proportion of SMCs expressing the beta-gal gene product increased from 25% (5-minute exposure) to 80% (120-minute exposure). For in vivo studies, uninjured and injured rat carotid segments were incubated with 0.5 to 1.0 x 10(9) pfu Ad.RSV beta gal for 45 minutes. Uninjured arteries showed adenovirus-mediated gene transfer limited to the endothelium. Injured arteries were exposed to adenovirus 0, 3, 7, or 12 days after injury. In these segments, beta-gal expression was minimal with infection at 0 or 3 days after injury but marked when infection was delayed until 7 or 12 days after injury. Neointimal cells constituted the dominant target of adenovirus gene transfer, with efficiency of gene transfer ranging from 10% to > 75%. Medial SMCs, whether covered or uncovered by neointimal cells, were minimally infected. Infection with a control adenovirus vector showed no beta-gal staining. CONCLUSIONS: Recombinant adenovirus selectively targets neointimal cells with high-efficiency gene transfer. This suggests that adenovirus vectors should be useful in targeting cells for the delivery of genes whose products may be relevant to the treatment of restenosis.

Adenoviridae↗
PubMed1993

In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors.

Replication-deficient recombinant adenovirus vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in the endothelium. To evaluate the feasibility of adenovirus-mediated gene transfer in vivo in normal intact blood vessels, adenovirus vectors containing the Escherichia coli lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA were injected in vivo into the lumen of an occluded vessel segment of sheep jugular vein and/or carotid artery. After 15 minutes of incubation, circulation was restored; the vessels were harvested 1-28 days later and evaluated for gene transfer and expression. Three days after in vivo exposure to the lacZ adenovirus vector, the endothelium of jugular veins and carotid arteries expressed beta-galactosidase. Exposure of jugular veins and carotid arteries in vivo to the alpha 1AT adenovirus vector resulted in the expression of alpha 1AT mRNA transcripts detected by Northern analysis and in the synthesis and secretion of alpha 1AT detected by ex vivo [35S]methionine labeling. Expression with the adenovirus vectors was efficient and easily detectable 1-14 days after injection, with maximum expression at 7 days. Expression was no longer evident at 28 days. Thus, adenovirus vectors are capable of transferring exogenous genes to the endothelium of normal arteries and veins with expression for at least 2 weeks, suggesting that these vectors have the potential for a variety of cardiovascular experimental and clinical applications.

Adenoviridae↗
PubMed1993

Efficient gene transfer into myocardium by direct injection of adenovirus vectors.

Previous studies have established that gene transfer into myocardial cells in vivo is detectable after direct injection of plasmid DNA. Recently, adenovirus vectors have been shown to provide an efficient method for gene transfer into a wide range of tissues. Therefore, this study sought to assess the efficiency and stability of adenovirus-mediated gene transfer into myocardium and to compare this method with that using plasmid-based gene transfer techniques. Adult rats underwent myocardial injection via a subdiaphragmatic approach. Gene transfer efficiency was compared using direct injection of an adenovirus vector encoding for the marker gene beta-galactosidase (beta-gal), a control adenovirus vector encoding for the cystic fibrosis transmembrane conductance regulator gene, a plasmid encoding for beta-gal, or a control plasmid. Hearts infected with an adenovirus vector containing the beta-gal gene showed significantly increased beta-gal enzymatic activity compared with hearts injected with beta-gal plasmid. Histological examination revealed that cardiac myocytes were the target of adenovirus-mediated gene transfer. A time course of gene expression showed that beta-gal enzymatic activity peaked during the first week following injection. Adenovirus vectors provide an efficient but transient method for in vivo gene expression in myocardium.

Adenoviridae↗
PubMed1992

Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation.

To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation.

Amino Acid Sequence↗
PubMed1992

Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells.

To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors.

Adenoviruses, Human↗
PubMed1992

Bronchial clearance of DTPA is increased in acute asthma but not in chronic asthma.

To investigate bronchial permeability in asthma, we measured the bronchial clearance of 113mIn-DTPA in seven asthmatics during and after an acute attack of asthma, seven asthmatics with chronic airflow limitation, and seven asthmatics without airflow limitation but with bronchial hyperresponsiveness to methacholine. We compared these results with those from seven normal subjects, seven patients with chronic bronchitis and bronchial infection, and seven patients with emphysema. An aerosol of 113mIn-DTPA was produced with a spinning disc to ensure a predominantly bronchial deposition of inhaled particles (6.3 microns MMAD). Radioactivity over the chest was recorded with a gamma-camera for 10 min after the subject inhaled the aerosol. Central regions of interest were selected, and the logarithm of the radioactivity was plotted against time; bronchial clearance of 113mIn-DTPA was calculated as the negative slope of the regression line. Clearance was substantially higher in asthmatics during their acute attacks than in all other groups (p less than 0.0001), and it decreased toward normal levels after recovery from the acute episode. The bronchial clearance of 113mIn-DTPA in all other groups did not differ from normal. We conclude that the bronchial clearance of 113mIn-DTPA is increased in asthmatics during attacks of asthma but in the stable state is not related either to bronchial hyperresponsiveness or to airflow limitation. Our findings are best explained by an increase in permeability of the bronchial mucosa of asthmatics during acute attacks.

Acute Disease↗
PubMed1990

[A case of primary osteosarcoma of the liver].

The authors report a case of primary osteosarcoma of the liver in a 75 year-old man with post hepatitic B cirrhosis. The hepatic tumor was discovered on ultrasound and computed tomography. Angiography showed a hypervascularized tumor. The patient died rapidly. Diagnosis of primary osteosarcoma of the liver was established through histopathological examination of post mortem specimens and was confirmed by immunohistochemical study. From this and two other previously reported cases the authors describe the different primary hepatic tumors mimicking osteosarcoma.

Aged↗