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J C Pagès

Publications and source records attributed to J C Pagès.

18 recordsLinked to original sources
PubMed2004

Toolbox for retrovectorologists.

Retroviral vectors have actively contributed to the advent of gene therapy as a realistic approach in human therapeutics. At the beginning, the use of retroviral vectors was thought to be as simple as the collection of a viral supernatant that was applied to the desired cell. Rapidly, target resistance to transduction appeared in various conditions, ex vivo as well as in vivo. At that time, retrovectorologists entered an active "back to the bench" era. This phase was thought to have reached its conclusion with the generation of theoretically safe lentiviral vectors and when, in 2000, a first clinical trial using retroviral vectors proved to be successful. Unfortunately, recent developments have shown that we still need to improve our knowledge of several steps in the retroviral life cycle before we can accurately adapt vectors to target specific cells. In this review we will first briefly detail key features of the life cycle of wild-type retroviruses. Thereafter, an overview of the minimal requirements needed to generate retroviral vectors will be followed by the relevant developments in this rapidly moving field. Of note, we have highlighted the crucial biosafety issues in a specific section.

Animals↗
PubMed2002

[Drainage after thyroid surgery: 264 patients].

OBJECTIVES: A prospective study was conducted in 1996-1997 in 100 patients who underwent thyroid surgery and who were randomly assigned to receive drainage or not. No statistical difference in complication rate was observed. The aim of the present retrospective study was to assess the consequences of this attitude in patients undergoing surgery since that time and to determine the number of postoperative complications, length of hospital stay, and type of thyroidectomy where cervical drains still appear to be indicated. PATIENTS AND METHODS: Total or partial thyroid surgery was performed in 264 patients between June 1997 and October 2000. Neck dissection was associated with 24 patients. RESULTS: Cervical drains were used in 29 patients (10.9%). Postoperative complications were comparable to those commonly reported. CONCLUSION: Except for neck dissection and mediastinal extension, thyroidectomy can be safely performed without drainage. This attitude reduces the overall hospital stay.

Adult↗
PubMed2001

Carbohydrate-deficient transferrin (CDT) determination by nephelometry using a commercial kit. Analytical and diagnostic aspects.

Carbohydrate-deficient transferrin (CDT) has been proposed as the most efficient marker of alcohol abuse. Absolute and relative concentrations of CDT were measured with a commercial assay (%CDTTIA from AXIS-Shield, Oslo, Norway) using rate nephelometry for transferrin determination. One hundred eighty-eight alcoholic patients (154 males, 34 females) and 132 control patients (113 males, 19 females) were included in the study. Within-run and day-to-day imprecision were 3.15% and 9.77%, respectively. The calibration curve was stable for more than 4 months with a shift below 5%. The commercial assay lacked sensitivity (Se = 0.48), but was highly specific (Sp = 0.98). Lowering the cut-off from 6% to 4.6% raised the sensitivity of the %CDTTIA test to 0.76 with a specificity of 0.90. We conclude that this adaptation to the Array Protein System (Beckman-Coulter) is suitable for routine use and offers precise results. It, however, requires an adaptation of the cut-off value for patients and of the target value for kit controls.

Adult↗
PubMed1999

LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock.

Experimental models of sepsis using endotoxin challenges, including studies with sensitized animals with D-galactosamine, have largely contributed to the basic rationale for innovative clinical trials in human septic shock, which have, to date, failed. The ability of these models to reproduce human disease has been highly discussed. We report here that the widely used D-galactosamine/LPS model does not account for septic shock. Treatment with YVAD-CMK, a potent tetrapeptide inhibitor of caspases of the interleukin (IL)-1beta converting enzyme (ICE) family, protects from LPS-induced liver apoptosis and mortality in D-galactosamine-sensitized mice when administered either before or up to 2 h after the lethal challenge. This curative effect is related to complete inhibition of caspase-3 activity in the liver. However, YVAD-CMK does not affect LPS-induced release of IL-1beta and does not protect from a lethal dose of LPS in unsensitized mice. These experiments demonstrate the difference between these two widely recognized experimental models of sepsis. LPS toxicity in D-galactosamine-treated mice, leading to blocked gene transcription, results from tumor necrosis factor (TNF)-alpha-induced caspase-3-dependent liver injury, not from the systemic inflammatory response. These results provide evidence that inhibitors of the ICE caspase family can prevent or even overcome the ongoing hepatic injury induced by TNF-alpha during sepsis, ischemia-reperfusion, or severe hepatitis.

Amino Acid Chloromethyl Ketones↗
PubMed1999

[Principles of physics and application of the laser in plastic surgery].

Recent advances in laser technology have expanded the surgeon's possibilities to treat various cutaneous lesions, as well as proposing a new tool for skin rejuvenation. Review of the basic physical principles of laser energy then overview of the different lasers used in plastic surgery as well as some of their clinical applications.

Biophysical Phenomena↗
PubMed1998

Amphotropic retroviral vectors displaying hepatocyte growth factor-envelope fusion proteins improve transduction efficiency of primary hepatocytes.

The development of retroviral vectors with cell-specific targeting capabilities will be an important step toward successful in vitro gene therapy. This article describes the generation of a retroviral vector with enhanced binding abilities for cells bearing the c-Met receptor: the Madin-Darby canine kidney (MDCK) cell line and primary hepatocytes. The human hepatocyte growth factor (HGF) was displayed on murine amphotropic retroviral vectors by fusion to the viral transmembrane envelope glycoprotein (TM). The resulting chimeric envelope HGF-TM was expressed in an amphotropic packaging cell line producing viral particles that display both HGF-TM and the wild-type envelope. These modified viral particles had a titer equivalent to that of unmodified particles. Modified particles infected MDCK cells more efficiently than did unmodified amphotropic retrovirus. Adding anti-HGF antibodies to the viral vector particle supernatant prior to infection confirmed that the increased infection was mediated by the HGF moiety. The chimeric viruses also infected primary mouse and nonhuman primate fetal hepatocytes more effectively. Furthermore, these cells could be induced to proliferate by the modified HGF-TM viruses. Since exogenous HGF is primarily taken up by the liver, these results may have implications for retroviral vector design for liver-directed human gene therapy.

3T3 Cells↗
PubMed1997

Preclinical studies for cell transplantation: isolation of primate fetal hepatocytes, their cryopreservation, and efficient retroviral transduction.

Fetal hepatocytes are an attractive target for in utero cellular transplantation. Their use could provide a very efficient way for implanting normal or transduced cells into the livers of affected fetuses. Marking cells with recombinant retroviruses is a powerful tool for evaluating the chimerism of grafted animals. The technique relies on the ex vivo transduction efficiency of the engrafted cells. We have isolated fetal primary hepatocytes from nonhuman primates. The cells were cultured and transduced with a retroviral vector carrying the Escherichia coli beta-galactosidase gene. Optimal gene transfer efficiency was obtained 48-60 hr after plating and was as high as 90%. Cryopreservation had little effect on cell viability and infectivity: The viability of thawed hepatocytes remained high (75-85%) and the infection efficiency was identical to that of freshly isolated cells. Efficient ex vivo retroviral gene transfer into fetal hepatocytes provides an appropriate system for testing allogenic grafting and for modifying immunogenicity of engrafted cells. These results open up new perspectives for cell transplantation through cell banking.

Animals↗
PubMed1996

ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis.

In liver, apoptosis is a physiological process involved in the clearance of injured cells and in homeostatic control [1]. However, in patients with viral fulminant hepatitis or with nonacute liver diseases [2], dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which express the cell-surface receptor Fas, by apoptosis. To date, treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Unravelling the molecular mechanisms that underlie acute liver failure could allow the design of an appropriate therapy. Ligand-bound Fas and tumour necrosis factor alpha (TNF-alpha) induce hepatic apoptosis in mice [3-6]. In various cell types, Fas- or TNF-alpha-induced apoptosis is blocked by viral proteins (such as p35 and CrmA) as well as by a decoy peptide (YVADcmk) [7-11], suggesting that these mechanisms of apoptosis involve ICE (interleukin-1 beta converting enzyme)-like proteases. Here, we report that, in vivo, pre-treatment of mice with YVADcmk protects them from the lethal effect of anti-Fas antibody and from liver failure induced by injection of TNF-alpha. Remarkably, YVADcmk administration is also highly effective in rescuing mice that have been pretreated with anti-Fas antibody from rapid death, despite extensive hepatic apoptosis. This dramatic curative effect could be of clinical benefit for the treatment of viral and inflammatory liver diseases.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Hepatocyte growth factor expressed by a retrovirus-producing cell line enhances retroviral transduction of primary hepatocytes: implications for in vivo gene transfer.

Hepatocyte Growth Factor (HGF) is the more potent mitogen of mature hepatocytes. We have examined the effect of human HGF expression by a recombinant retroviral cell line (MFG-LacZ) on retroviral transduction of primary mouse and human hepatocytes. The HGF in the supernatant of MFG-LacZ cell line was correctly processed and biologically active. Transduction of mouse and human hepatocytes with the supernatant of transfected cells was increased 5-fold, as determined by beta-galactosidase activity. The production of HGF was stable and did not interfere with the viral titers of the producer cells. This study provides evidence that expression of HGF within a retrovirus-producer cell line increases the transduction rate of primary hepatocytes. Since the number of corrected cells is a limiting step for phenotypic correction of liver deficiencies, our approach should improve hepatic gene therapy efficiency. Furthermore this cell line should be useful for in vivo liver gene therapy.

Animals↗
PubMed1996

[Towards gene therapy in familial hypercholesterolemia].

Familial hypercholesterolemia (FH) is an inherited disease caused by a defect in the gene encoding the Low Density Lipoprotein receptor (LDL-R). The ex vivo hepatic gene therapy which restore the expression of the normal protein in hepatocytes should correct the disease. Improved transduction efficiency and long lasting expression of the transduced gene remain the main goals of gene therapy research. We developed an efficient and reliable method for in vivo transduction of human, mouse and primate primary hepatocytes. A retroviral vector bearing the LDL-R cDNA driven by the liver-type pyruvate kinase promoter allows high and tissue specific expression of the gene in primary hepatocytes. A second vector with a housekeeping promoter corrects the LDL-R deficiency in fibroblasts from a FH patient. Ex vivo preclinical studies in non-human primates will provide new insight in transduced cells biology after reimplantation.

Animals↗
PubMed1993

Determination of urinary testosterone and epitestosterone during pubertal development: a cross-sectional study in 141 normal male subjects.

OBJECTIVES: Exogenous testosterone administration is classically detected by measuring the ratio of testosterone to epitestosterone in urine. Athletes are considered to be positive for drug abuse if the urinary testosterone to epitestosterone ratio is greater than 6. We aimed at investigating the urinary excretion of testosterone and epitestosterone during pubertal development. DESIGN: We performed a cross-sectional study of 141 normal male subjects between ages 8 and 26 years. PATIENTS: We studied 141 subjects: 32 at stage 1 of Tanner, 27 at stage 2, 30 at stage 3, 25 at stage 4 and 27 at stage 5. MEASUREMENTS: Subjects performed a 24-hour urine collection. Urinary testosterone and epitestosterone were measured by gas chromatography-mass spectrometry with selected ion monitoring. RESULTS: Urinary testosterone was 20.5 +/- 1.7 nmol/24 h (mean +/- SEM) at stage 1, 49 +/- 2.9 at stage 2, 98.8 +/- 3.4 at stage 3, 371.8 +/- 21.8 at stage 4 and 403.4 +/- 16.1 nmol/24h at stage 5. Urinary epitestosterone was 13.1 +/- 1.5 nmol/24h at stage 1, 29.1 +/- 3.3 at stage 2, 48.3 +/- 3.7 at stage 3, 156.3 +/- 14.8 at stage 4 and 221.1 +/- 18.6 nmol/24h at stage 5. The urinary excretions of both steroids increased significantly during puberty and were highly correlated with chronological age (P < 0.001). Comparison of the correlation slopes (P < 0.001) showed that the urinary profiles of testosterone and epitestosterone are not parallel during pubertal development. Two subjects presented a testosterone to epitestosterone ratio above 6, corresponding to a low urinary concentration of epitestosterone, without pathological explanation. CONCLUSION: Testosterone and epitestosterone do not present the same urinary profiles throughout puberty. Marked increases of the testosterone to epitestosterone ratio can be observed at this period and may interfere with doping tests.

Adolescent↗
PubMedDate not supplied

One step screening of retroviral producer clones by real time quantitative PCR.

BACKGROUND: Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. METHODS: We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. RESULTS: We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. CONCLUSION: We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.

Animals↗