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Pierre Charneau

Publications and source records attributed to Pierre Charneau.

At least 19 recordsLinked to original sources
PubMed2006

Wild-type and central DNA flap defective HIV-1 lentiviral vector genomes: intracellular visualization at ultrastructural resolution levels.

HIV-1 and other lentiviruses have the unique ability among retroviruses to efficiently replicate in non-dividing cells as a result of the active nuclear import of their DNA genome across an interphasic nuclear membrane. Previous work has shown that a three-stranded DNA structure synthesized during HIV-1 reverse transcription, called the central DNA flap, acts as a cis-determinant of HIV-1 genome nuclear import. Concordantly, DNA Flap re-insertion in lentiviral-derived gene therapy vectors stimulates gene transfer efficiencies and complements the level of nuclear import to wild-type levels quantitatively indistinguishable from wild-type virus in all cell types and tissues examined so far. In order to define the precise nature of the replicative defect of DNA flap mutant viruses, we carried out in situ DNA hybridization experiments with electron microscopy to determine the subcellular localization of DNA flap mutant and wild-type HIV-1 genomes. We found that Flap defective DNA genomes accumulate at the cytoplasmic face of the nuclear membrane with no overlap across the nuclear membrane, whereas wild-type genomes localize throughout the nuclear compartment. These data provide an unequivocal confirmation of the role of the DNA flap in HIV-1 nuclear import and further establish that the DNA flap controls a step that immediately precedes translocation through the nuclear pore. Further, the widespread distribution of wild-type genomes within the open chromatin confirms the recent genome-wide mapping of HIV-1 cDNA integration sites and points to an as-yet poorly understood step of intranuclear transport of HIV-1 pre-integration complexes.

Active Transport, Cell Nucleus↗
PubMed2006

Immunogenic HLA-B*0702-restricted epitopes derived from human telomerase reverse transcriptase that elicit antitumor cytotoxic T-cell responses.

PURPOSE: The human telomerase reverse transcriptase (hTERT) is considered as a potential target for cancer immunotherapy because it is preferentially expressed in tumor cells. To increase the applicability of hTERT-based immunotherapy, we set out to identify CTL epitopes in hTERT restricted by HLA-B*0702 molecule, a common MHC class I allele. EXPERIMENTAL DESIGN: HLA-B*0702-restricted peptides from hTERT were selected by using a method of epitope prediction and tested for their immunogenicity in human (in vitro) and HLA-B*0702 transgenic mice (in vivo). RESULTS: All the six hTERT peptides that were predicted to bind to HLA-B*0702 molecule were found to induce primary human CTL responses in vitro. The peptide-specific CD8+ CTL lines were tested against various hTERT+ tumor cells. Although differences were observed according to the tumor origin, only three CTL lines specific for p277, p342, and p351 peptides exhibited cytotoxicity against tumor cells in a HLA-B*0702-restricted manner. In addition, this cytotoxicity was inhibited by the addition of peptide-loaded cold target cells and indicated that these epitopes are naturally processed and presented on the tumor cells. Further, in vivo studies using humanized HLA-B*0702 transgenic mice showed that all the candidate peptides were able to induce CTL responses after peptide immunization. Furthermore, vaccination with a plasmid DNA encoding full-length hTERT elicited peptide-specific CTL responses, indicating that these epitopes are efficiently processed in vivo. CONCLUSIONS: Together with previously reported hTERT epitopes, the identification of new CTL epitopes presented by HLA-B*0702 increases the applicability of hTERT-based immunotherapy to treating cancer.

Animals↗
PubMed2006

A single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against West Nile virus.

BACKGROUND: Lentiviral vectors, due to their capacity to transduce non-dividing cells, have become precious and worldwide used gene transfer systems. Their ability to efficiently and stably transduce dendritic cells (DCs) has led to their successful use as vaccination vectors for eliciting strong, specific and protective cellular immune responses mostly in anti-tumoral but also in anti-viral applications. However, the ability of lentiviral vectors to elicit an antibody-based protective immunity has, to date, not been evaluated. In the present study, we evaluated the potential of a lentiviral vector-based vaccine to elicit humoral immunity against West Nile virus (WNV). WNV is a mosquito-borne flavivirus that emerged in North America and causes encephalitis in humans, birds and horses. Neutralizing anti-WNV antibodies have been shown to be crucial for protection against WNV encephalitis. METHODS: The ability of lentiviral vector TRIP/sE(WNV), expressing the secreted soluble form of the envelope E-glycoprotein (sE(WNV)) from the highly virulent IS-98-ST1 strain of WNV, to induce a specific humoral response and protection against WNV infection was assessed in a mouse model of WNV encephalitis. RESULTS: Remarkably, a single immunization with a minute dose of TRIP/sE(WNV) was efficient at eliciting a long-lasting, protective and sterilizing humoral immunity, only 1 week after priming. CONCLUSIONS: This study broadens the applicability of lentiviral vectors as efficient non-replicating vaccines against pathogens for which a neutralizing humoral response is one active arm of the protective immunity. The TRIP/sE(WNV) lentiviral vector appears to be a promising tool for veterinary vaccination against zoonotic WNV.

Animals↗
PubMed2006

Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes.

Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein derivative FlAsH. This labeling allowed us to image both intracytoplasmic and intranuclear HIV-1 complexes in three dimensions over time (4D) in human cells and enabled us to analyze HIV-1 kinetics by automated 4D quantitative particle tracking. In the cytoplasm, HIV-1 complexes underwent directed movements toward the nuclear compartment, kinetically characteristic of both microtubule- and actin-dependent transport. The complexes then adopted smaller movements in a very confined volume once associated with the nuclear membrane and more diffuse movements once inside the nucleus. This work contributes new insight into the various movements of HIV-1 complexes within infected cells and provides a useful tool for the study of virus-host cell interactions during infection.

Biological Transport↗
PubMed2006

A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells.

As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2K(k)] through the (Gal4)(5)-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2K(k) as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.

Carrier Proteins↗
PubMed2006

Nuclear import defect of human immunodeficiency virus type 1 DNA flap mutants is not dependent on the viral strain or target cell type.

We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.

Active Transport, Cell Nucleus↗
PubMed2006

Plasticity of hepatic cell differentiation: bipotential adult mouse liver clonal cell lines competent to differentiate in vitro and in vivo.

In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells (cholangiocytes). The persistence of such progenitor cells in adult mouse liver is still debated. In damaged liver of adult murine animals, when hepatocyte proliferation is compromised, bipotential oval cells emerge, probably from bile ducts, proliferate, and differentiate to regenerate the liver. However, treatment to elicit oval cell proliferation is not necessary to obtain bipotential stem cells from adult mouse liver. Here, we have isolated bipotential clonal cell lines from healthy liver of 8-10-week-old C57BL/6 mice. Primary cultures established from hepatocyte-enriched suspensions were characterized by time-lapse image acquisition, immunocytology, and RNA transcript analysis. Although hepatocytes dedifferentiated with loss of apical polarity and other hepatocyte markers, they rapidly activated expression of bile duct/oval cell markers. Reversibility of these processes was achieved in part by culture under dilute Matrigel or by aging of confluent cultures. Cell lines were obtained at high frequency from mass cultures, from isolated colonies, and by primary cloning of the hepatocyte-enriched suspension. Cells of the clonal cell lines do not grow in soft agar and are nontumorigenic, and they express cytokeratin 19, A6 antigen, and alpha6 integrin, as well as a large panel of hepatocyte functions. Furthermore, they can participate in liver regeneration in albumin-urokinase-type plasminogen activator/severe combined immune-deficient mice, where they differentiate in clusters of hepatocytes and occasionally bile ducts. These results demonstrate the existence, in normal adult mouse liver, of a significant pool of clonogenic cells that are (or can become) bipotential.

Animals↗
PubMed2005

Direct recognition by alphabeta cytolytic T cells of Hfe, a MHC class Ib molecule without antigen-presenting function.

Crystallographic analysis of human Hfe has documented an overall structure similar to classical (class Ia) MHC molecules with a peptide binding groove deprived of ligand. Thus, to address the question of whether alphabeta T cells could recognize MHC molecules independently of bound ligands, we studied human and mouse Hfe interactions with T lymphocytes. We provide formal evidence of direct cytolytic recognition of human Hfe by mouse alphabeta T cell receptors (TCR) in HLA-A*0201 transgenic mice and that this interaction results in ZAP-70 phosphorylation. Furthermore, direct recognition of mouse Hfe molecules by cytotoxic T lymphocytes (CTLs) was demonstrated in DBA/2 Hfe knockout mice. These CTLs express predominantly two T cell antigen receptor alpha variable gene segments (AV6.1 and AV6.6). Interestingly, in wild-type mice we identified a subset of CD8+ T cells positively selected by Hfe that expresses the AV6.1/AV6.6 gene segments. T cell antigen receptor recognition of MHC molecules independently of bound ligand has potential general implications in alloreactivity and identifies in the Hfe case a cognitive link supporting the concept that the immune system could be involved in the control of iron metabolism.

Animals↗
PubMed2005

Activity-dependent adjustments of the inhibitory network in the olfactory bulb following early postnatal deprivation.

The first-order sensory relay for olfactory processing, the main olfactory bulb (MOB), retains the ability to acquire new interneurons throughout life. It is therefore a particularly appropriate region for studying the role of experience in sculpting neuronal networks. We found that nostril closure decreased the number of newborn granule cells in the MOB, the complexity of their dendritic arborization, and their spine density, without affecting the preexisting population of granule cells. Accordingly, the frequency of miniature synaptic inhibitory events received by mitral cells was reduced. However, due to a compensatory increase in newborn granule cell excitability, action potential-dependent GABA release was dramatically enhanced, thus counteracting the reduction in spine density and leading to an unaltered synchronization of mitral cell firing activity. Together, this study reveals a unique form of adaptive response brought about exclusively by the cohort of newborn cells and used to maintain normal functioning of the MOB.

Animals↗
PubMed2004

The structure of HIV-1 genomic RNA in the gp120 gene determines a recombination hot spot in vivo.

By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.

Base Sequence↗
PubMed2004

Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes.

Cell lines have many advantages: they can be manipulated genetically, expanded, and stockpiled for organ transplantation. Freshly isolated hepatocytes, oval cells, pancreatic cells, and hematopoietic stem cells have been shown to repopulate the damaged liver. Here we show that bipotential mouse embryonic liver (BMEL) stem cell lines participate in liver regeneration in albumin-urokinase plasminogen activator/severe combined immunodeficiency disease (Alb-uPA/SCID) transgenic mice. In the liver, BMEL-GFP cells proliferate and differentiate into both hepatocytes and bile ducts, forming small to large clusters detected throughout the 3-8 weeks analyzed after transplantation. Moreover, they respond like host cells to signals for growth, differentiation, and even zonal expression of metabolic enzymes, showing regulated expression of cytokeratins and liver-enriched transcription factors. Immunostaining for MHC class I molecules revealed that cells do not coexpress donor and recipient H-2 haplotypes, as would be the case had cell fusion occurred. This report shows that immortalized stem cell lines not only are competent to participate in the repair of a damaged tissue but also can differentiate into the two major epithelial cell types of a complex organ, hepatocytes and bile ducts.

Animals↗
PubMed2004

Interclade neutralization and enhancement of human immunodeficiency virus type 1 identified by an assay using HeLa cells expressing both CD4 receptor and CXCR4/CCR5 coreceptors.

We report on the development and evaluation of a human immunodeficiency virus (HIV) neutralization assay that uses P4P cells, which are CD4+CXCR4+CCR5+ HeLa cells that carry the lacZ gene under the control of the HIV-1 long terminal repeat. The results of the present study suggest that the P4P assay can be used for the extensive study of both neutralizing and enhancing activity in serum samples from HIV-1-infected patients and from vaccinated individuals.

CD4 Antigens↗
PubMed2004

Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Amino Acid Sequence↗
PubMed2004

Histopathological and cognitive defects induced by Nef in the brain.

Complex mechanisms of human immunodeficiency virus type-1 (HIV-1) brain pathogenesis suggest the contribution of individual HIV-1 gene products. Among them, the Nef protein has been reported to harbor a major determinant of pathogenicity in AIDS-like disease. The goal of the present study was to determine whether Nef protein expressed in vivo by primary macrophages could induce a brain toxicity also affecting the behavior of the rat. To achieve this goal we grafted Nef-transduced macrophages into the rat hippocampus. Two months post-transplantation, we observed that Nef induces monocyte/macrophage recruitment, expression of TNF-alpha, and astrogliosis. No apoptotic event was detected. We further demonstrated that Nef neurotoxicity is associated with cognitive deficits.

Animals↗
PubMed2004

A single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (HIV) type 1 clade B envelope glycoproteins induces neutralizing antibodies and cellular immune responses to HIV.

The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.

AIDS Vaccines↗
PubMed2003

Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein.

Expansion of human hematopoietic stem cells (HSCs) is a major challenge in cellular therapy, and currently relies on the use of recombinant cytokines or on gene transfer of transcription factors. Of these, the HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes. Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. This method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

Animals↗
PubMed2003

Transplantation of human adult astrocytes: efficiency and safety requirements for an autologous gene therapy.

Ex vivo gene therapy is emerging as a promising approach for the treatment of neurodegenerative diseases and central nervous system (CNS) trauma. We have shown previously that human adult astrocytes can be expanded in vitro and can express various therapeutic transgenes (Ridet et al. [1999] Hum. Gene Ther. 10:271-280; Serguera et al. [ 2001] Mol. Ther. 3:875-881). Here, we grafted normal and lentivirally-modified human adult astrocytes into the striatum and spinal cord of nude mice to test whether they are suitable candidates for ex vivo CNS gene therapy. Transplanted cells survived for at least 2 months (longest time analyzed) and sustained transgene expression. Importantly, the absence of proliferating cell nuclear antigen (PCNA) staining, a hallmark of cell division, ascertains the safety of these cells. Thus, adult human astrocytes are a promising tool for human CNS repair; they may make autologous ex vivo gene transfer feasible, thereby avoiding the problems of immunological rejection and the side effects of immunosuppressors.

Adult↗