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1.
J Fr Ophtalmol ; 31(1): 80-6, 2008 Jan.
Article in French | MEDLINE | ID: mdl-18401305

ABSTRACT

The public health law passed in 2004 is the implementation into French law of the European Clinical Trial directive 2001/20/EC on the implementation of good clinical practice in conducting clinical trials on medicinal products for human use. This law goes further than the European directive since it reconsiders all the components related to all fields of biomedical research. It has (i) standardized communication with the authorities (French Drug Agency/French Health Authority and the Ethics Committee); (ii) reinforced protection to participants in clinical trials and modified the information delivered to participants and how their informed consent is obtained; (iii) added risk-benefit assessment; and (iv) increased transparency for the public.


Subject(s)
Biomedical Research/trends , Biomedical Research/history , Ethics, Medical , France , History, 21st Century , Humans , Public Health/legislation & jurisprudence , Public Health/standards , Public Health/trends
2.
J Gene Med ; 3(1): 42-50, 2001.
Article in English | MEDLINE | ID: mdl-11269335

ABSTRACT

BACKGROUND: Gene delivery to the myocardium using blood-borne adenoviral vectors is hindered by the endothelium, which represents a barrier limiting the infection rate of underlying myocytes. However, endothelial permeability may be modulated by pharmacological agents. METHODS: In the present study, we modeled the endothelial barrier in vitro using a human umbilical vein endothelial cell (HUVEC) monolayer seeded on a Transwell membrane as a support and diffusion of fluorescent dextrans as a permeability index. We used alpha-thrombin (100 nM) as a pharmacological agent known to increase endothelial permeability and tested the barrier function of the endothelial cell monolayer on adenovector-mediated luciferase gene transfer to underlying isolated cardiac myocytes. RESULTS: A confluent HUVEC monolayer represented a considerable physical barrier to dextran diffusion; it reduced the permeability of the micropore membrane alone to fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights 4, 70, 150 and 2000 kDa by approximately 54, 78, 88 and 98%, respectively. Alpha-thrombin (100 nM) increased the permeability coefficients (P(EC)) by 276, 264, 562 and 4166% for the same dextrans, respectively. A confluent HUVEC monolayer represented a major impediment to adenovector-mediated luciferase gene transfer to cardiac myocytes, largely reducing gene transfer efficiency. However thrombin induced a nine-fold increase in myocyte infection. CONCLUSION: In our model, the endothelial cell monolayer represents a major impediment to myocyte adenovector-mediated gene transfer which can be partially improved by pharmacologically increasing endothelial permeability. The Transwell model is therefore particularly useful for testing the efficiency of pharmacological agents in modulating adenovector passage through the endothelial barrier.


Subject(s)
Adenoviridae/genetics , Endothelium, Vascular/metabolism , Genetic Vectors , Muscle, Smooth/metabolism , Animals , Capillary Permeability/drug effects , Cells, Cultured , Endothelium, Vascular/cytology , Gene Transfer Techniques , Humans , Male , Muscle, Smooth/cytology , Rats , Rats, Wistar , Thrombin/pharmacology
3.
Cardiovasc Res ; 49(2): 288-97, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11164839

ABSTRACT

OBJECTIVE: We examined the functional consequences of expressing adult rabbit fast skeletal sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA1a) in isolated adult rat ventricular myocytes. METHODS: Myocytes were infected with a recombinant adenovirus harboring SERCA1a. Then 2 days after myocyte infection, protein expression was estimated using Western blot and SDS-PAGE analysis. We also measured the ATP-dependent oxalate-facilitated Ca(2+) uptake of myocyte homogenates and monitored Ca(2+) transient in myocytes loaded with the Ca(2+) dye, indo-1. RESULTS: SERCA1a gene expression resulted in a 36% increase in the total SERCA protein level in infected myocytes compared to controls (P<0.01), while SERCA2 and phospholamban levels did not change. This increase was associated with a 42% rise in SR Ca(2+) uptake (P<0.01), while tau (the time constant of Ca(2+) transient decay), and the time to peak fell by 32% (P<0.01) and 38% (P<0.001), respectively. Increasing the frequency of stimulation from 0.2 to 2 Hz decreased tau in both cell types (P<0.01). However, the decrease was much smaller in infected (P<0.01) than in uninfected cells (P<0.001). Isoproterenol (1 microM) further decreased tau in infected myocytes by 23% (P<0.05). In these cells, the diastolic [Ca(2+)](i) decreased by 50% (P<0.05) while the systolic [Ca(2+)](i) increased by 19% (P<0.05). No difference was found in the speed of SR Ca(2+) reloading after caffeine washout between the two cell types. CONCLUSION: Adenovirus-mediated SERCA1a gene transfer to adult rat ventricular myocytes enhances SR Ca(2+) handling to a degree similar to that observed following physiological stimulation.


Subject(s)
Adenoviridae/genetics , Calcium-Transporting ATPases/genetics , Calcium/metabolism , Genetic Vectors/administration & dosage , Myocardium/metabolism , Sarcoplasmic Reticulum/enzymology , Analysis of Variance , Animals , Blotting, Western , Calcium-Transporting ATPases/analysis , Calcium-Transporting ATPases/metabolism , Cells, Cultured , Gene Transfer Techniques , Male , Microscopy, Confocal , Myocardium/enzymology , Rats , Rats, Wistar , Sarcoplasmic Reticulum Calcium-Transporting ATPases
4.
Hum Gene Ther ; 11(7): 1015-22, 2000 May 01.
Article in English | MEDLINE | ID: mdl-10811230

ABSTRACT

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.


Subject(s)
Adenoviridae/genetics , Coronary Vessels , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Heart/virology , Myocardium/cytology , Animals , Buffers , Calcium/metabolism , Cardiomyopathies/chemically induced , Coronary Circulation , Edema/chemically induced , Heart/drug effects , Hemodynamics , Histamine/pharmacology , In Vitro Techniques , Male , Nitroprusside/pharmacology , Norepinephrine/pharmacology , Rats , Rats, Wistar , Vasodilation/drug effects , Vasodilator Agents/pharmacology
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