PubMed Health⌕ Search

Biomedical subjects

Jacques Robert

Publications and source records attributed to Jacques Robert.

36 records · Page 2Linked to original sources
PubMed2004

Xenopus as an experimental model for studying evolution of hsp--immune system interactions.

The frog Xenopus provides a unique model system for studying the evolutionary conservation of the immunological properties of heat shock proteins (hsps). General methods for maintaining and immunizing isogenetic clones of defined MHC genotypes are presented together with more recently developed protocols for exploring hsp-mediated immune responses in vitro (proliferative and cytotoxic assays) and in vivo (adoptive cell transfer and antibody treatment) in adults and in naturally MHC class I-deficient larvae. Finally, techniques to study modalities of expression of the endoplasmic reticulum resident gp96 at the cell surface of tumor and normal lymphocytes are considered.

Animals↗
PubMed2004

[Recent advances in pharmacogenomics in oncology].

Progress in understanding the molecular mechanisms of oncogenesis, together with the sequencing of the human genome and the availability of huge databases, bring new tools for the discovery and the evaluation of new potential targets for cancer therapy and for the individualisation of cancer chemotherapy as a function of the molecular characteristics of tumours. Targeting the genetic alterations of cancer cells appears feasible and the first successes of this approach allow to remain optimistic about the renewal of our therapeutic armamentarium. In addition, seeking for correlations between gene expression profiles and chemosensitivity has been performed on the in vitro models of the National Cancer Institute and may allow crucial improvements in the identification of patients who world best take advantage of a specific chemotherapy. Clinical trials, first on a retrospective basis, then performed prospectively, are implemented to validate this approach.

Antibodies↗
PubMed2004

MS-209 Schering.

MS-209, a quinolone-derived sphingomyelin synthase inhibitor that blocks P-glycoprotein and multidrug resistance-associated protein-1, is under development by Schering for the potential treatment of multidrug resistant tumors. By March 2003, phase II trials in breast cancer and non-small-cell lung cancer had been initiated.

Animals↗
PubMed2003

Development and characterization of a model system to study amphibian immune responses to iridoviruses.

The recent realization that viruses within the family Iridoviridae may contribute to the worldwide decline in amphibians makes it urgent to understand amphibian antiviral immune defenses. We present evidence that establishes the frog Xenopus laevis as an important model with which to study anti-iridovirus immunity. Adults resist high doses of FV3 infection, showing only transitory signs of pathology. By contrast, naturally MHC class-I-deficient tadpoles are highly susceptible to FV3 infection. Monitoring of viral DNA by PCR indicates a preferential localization of FV3 DNA in the kidney, with the inbred MHC homozygous J strain appearing to be more susceptible. Clearance of virus as measured by detection of FV3 DNA and also the disappearance of pathological and behavioral symptoms of infection, acceleration of viral clearance, and detection of IgY anti-FV3 antibodies after a second injection of FV3 are all consistent with the involvement of both cellular and humoral adaptive antiviral immune responses.

Animals↗
PubMed2003

Ontogeny of Xenopus NK cells in the absence of MHC class I antigens.

This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at approximately 7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3-4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3-4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRbeta V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity.

Animals↗
PubMed2003

Signaling FcRgamma and TCRzeta subunit homologs in the amphibian Xenopus laevis.

The genes encoding FcRgamma and TCRzeta homologs were identified using a bioinformatic approach in the amphibian Xenopus laevis. Deduced amino acid sequence of Xenopus TCRzeta is highly similar to the mammalian and avian counterparts, whereas that of FcRgamma differs by the presence of an additional ITAM-like motif. The presence of the negatively charged residue in the transmembrane regions of both subunits suggests their ability to serve as signal transducing modules in complex with activating receptors. The short extracellular regions contain characteristic cysteine residues responsible for dimerization in the mammalian subunits. According to Southern blot analysis, Xenopus laevis may possess two non-allelic genes for each subunit. Northern blots revealed FcRgamma transcripts of two sizes differentially expressed in thymus, spleen, intestine, liver and kidney. TCRzeta mRNA was predominantly expressed in the thymus and spleen. These data indicate that the amphibian immune system employs activating receptor complexes arranged in a mammalian-like way.

Amino Acid Sequence↗
PubMed2003

Bacterial stimulation upregulates the surface expression of the stress protein gp96 on B cells in the frog Xenopus.

The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherichia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.

Aeromonas hydrophila↗
PubMed2002

Alterations in the expression of cytochrome c oxidase subunits in doxorubicin-resistant leukemia K562 cells.

Doxorubicin (DOX), a widely used antitumoral drug, induces numerous modifications in sensitive cells, interacting with nuclear and mitochondrial DNA. In previous studies achieved in two K562 DOX-resistant sublines (K562/0.2R and K562/0.5R), we have shown stable mitochondrial damage comparatively with sensitive parental cells, such as decrease of cytochrome c oxidase activity (COX; EC 1.9.3.1) and cytochrome aa3 content. In order to explain these data, we have studied several COX genes and their expression, in relationship with altered COX activity and multidrug resistance (MDR) phenotype. We have observed a lower expression of the catalytic subunits COX I and II in MDR sublines, which was neither related to mutations in the corresponding mitochondrial genes, nor to a reduced transcription rate. In contrast, we have noticed an increase in both MDR K562 variants, in the mRNA expression of the catalytic subunit COX III, related to an increase in the half-life of these transcripts. Moreover, the doxorubicin resistance phenotype in K562 cells was accompanied by modifications of the expression and steady-state mRNA levels of several nuclear-encoded regulatory COX subunits. Thus, doxorubicin-resistant K562 cells represent an interesting model to study stable modifications concomitant to MDR phenotype. Our results seem to indicate compensatory mechanisms which highlight the complexity of regulatory systems of COX enzyme, involving coordinate regulation of both nuclear and mitochondrial subunit expression.

Catalysis↗
PubMed2002

Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins.

In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8(+) T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8(+) cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.

Animals↗
PubMed2002

Effects of paclitaxel, cyclophosphamide, ifosfamide, tamoxifen and cyclosporine on the metabolism of methoxymorpholinodoxorubicin in human liver microsomes.

The effects of paclitaxel, cyclosporine, cyclophosphamide, ifosfamide and tamoxifen on the metabolism of methoxymorpholinodoxorubicin (MMDx), a novel anticancer agent, were investigated using human liver microsomes. Paclitaxel, tamoxifen and cyclosporine dramatically inhibited MMDx metabolism, whereas ifosfamide had only a slight effect at high concentrations (200-300 microM) and cyclophosphamide had no effect. The inhibition was dependent on the concentrations of both MMDx and the coincubated drug. Thus, with 1 microM MMDx, paclitaxel (5 microM), tamoxifen (1 microM) and cyclosporine (1 microM) decreased the metabolic rate of MMDx by 36%, 53% and 62%, respectively. At higher concentrations (10, 5 and 5 microM, respectively, with paclitaxel, tamoxifen and cyclosporine) the inhibition was 52%, 91% and 91%, respectively. These three drugs preferentially inhibited the formation of three metabolites (M2, M3 and M6) among eight metabolites produced in liver microsomes. The inhibitory concentrations of paclitaxel, tamoxifen and cyclosporine on MMDx metabolism were in the range of those observed in patients upon administration of these drugs, which are known to be CYP3A4 substrates. These findings suggest that CYP3A4 drug substrates and MMDx in combination must be used with caution, particularly in view of the fact that MMDx is considered as a prodrug whose activation is entirely dependent upon metabolic transformation by CYP3A4.

Antibiotics, Antineoplastic↗
PubMed2002

Determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines.

PURPOSE: Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target. RESULTS: The cytotoxicity of irinotecan and SN-38 markedly differed in the two cell lines: irinotecan IC(50) values were 15.8 microM for LoVo cells and 5.17 microM for HT-29 cells; SN-38 IC(50) values were 8.25 n M for LoVo cells and 4.50 n M for HT-29 cells. Topoisomerase I expression (at the mRNA and the protein levels) and catalytic activity were similar in the two cell lines. Irinotecan induced similar amounts of cleavable complexes at its IC(50) in both cell lines. SN-38 induced a concentration-dependent formation of cleavable complexes, which was not significantly different in the two cell lines. Expression of the carboxylesterase CES1 was higher in HT-29 than in LoVo cells. Expression of the carboxylesterase gene CES2 was comparable in the two cell lines and much higher than CES1 gene expression. Carboxylesterase activity was extremely low using p-nitrophenylacetate as a substrate (1.45 and 1.84 pmol/min per mg proteins) and could not even be detected using irinotecan as a substrate. Cell accumulation of irinotecan was markedly different, reaching consistently higher levels in HT-29 cells than in LoVo cells. CONCLUSIONS: Our results indicate that (1) the cytotoxicity of irinotecan was likely due to the drug itself and not to its metabolite SN-38, and (2) that irinotecan uptake was more predictive of its cytotoxicity than topoisomerase I availability and activity in these two cell lines.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2002

Identification and characterization of Xenopus CD8+ T cells expressing an NK cell-associated molecule.

Growing evidence suggests that some immune responses are mediated not only by conventional and distinct NK cells and CTL, but also by T cell subsets expressing NK receptors and NK cell-associated molecules. Consistent with our previously published finding that the mAb 1F8 identifies non-T/non-B cells in Xenopus that effect NK-like killing in vitro, we now report that in vivo treatment with this mAb impairs rejection of transplanted MHC class I-negative tumor cells. However, we also find that the NK cell-associated molecule recognized by mAb 1F8 is expressed by a minor population of CD8+ T cells, in which fully rearranged TCRbeta mRNA of at least three different V families can be identified, by contrast, 1F8+/CD8- (NK) cells lack such TCRbeta message. Additionally, the expression of the NK cell-associated molecule can be induced in vitro by a transient submitogenic stimulation of naïve CD8+ T cells with PMA and ionomycin. Such induced expression of 1F8 also occurs in alloantigen-activated CTL and is coincident with a down-regulation of MHC-specific cytotoxicity. Taken together, these new data suggest that regulation of CD8+ T cell activity involving NK cell-associated molecules is a general and evolutionarily ancient phenomenon.

Animals↗
PubMed2002

Determination of drug interactions occurring with the metabolic pathways of irinotecan.

Irinotecan or CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine] is a derivative of camptothecine used in the treatment of advanced colorectal cancer. It requires activation to SN-38 (7-ethyl-10-hydroxycamptothecine) by carboxylesterase. Irinotecan and SN-38 are detoxified through two major metabolic pathways: the first one leads to oxidative degradation compounds, APC [7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine] and NPC [7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine], and involves cytochrome P450 (3A4 isoform); the second one leads to SN-38 glucuronide (SN-38G) and involves UDP-glucuronosyltransferase (UGT). Using human hepatic microsomes, we studied the interactions of 15 drugs of common use in colorectal cancer patients on these metabolic pathways. Only nifedipine had a significant effect on SN-38 formation, decreasing carboxylesterase activity by 50% at 100 microM and 35% at 10 microM. Three drugs had a significant effect on SN-38G formation: clonazepam increased UGT activity by 50% at 100 microM and 30% at 10 microM, and nifedipine and vinorelbine inhibited the activity by 65 and 55% at 100 microM, respectively, with no effect at 10 microM. Five drugs exerted a significant inhibition on SN-38 formation at 100 microM: clonazepam (70%), methylprednisolone (50%), nifedipine (80%), omeprazole (85%), and vinorelbine (100%). Only omeprazole and vinorelbine still exerted a significant inhibition at 10 microM (30 and 90%, respectively), whereas only vinorelbine had a significant effect at 2 and 0.5 microM (70 and 40%, respectively). In conclusion, potential clinical interactions with the metabolism of irinotecan are likely to be important for vinorelbine, which strongly inhibits irinotecan catabolism by CYP3A4 at clinically relevant concentrations, but not for the other drugs, which exert an effect at concentrations not achievable in patients.

Antineoplastic Agents, Phytogenic↗
PubMed2002

[New concepts for the study of anticancer drug resistance].

In the past decade, numerous mechanisms of resistance have been described, involving the availability of the drug at the target or the availability of the target itself, but resistance to cell death induction remains far from being understood. The involvement of p53, of Bcl2 and related proteins, of the Fas/Fas-L system and other membrane death receptor pathways, have especially been studied. However, conflicting results have been published concerning the impairment of apoptosis in resistance to cytotoxic drugs. This has shed important doubts on the currently accepted view, which presents apoptosis as a universal determinant of drug activity. These discrepancies are likely to be related to the cell-type specificity of apoptotic pathways and further research is warranted to get a complete picture of the role of cell death inhibition as a drug resistance mechanism. New genetic tools have been recently made available for the study of anticancer drug resistance. Differential or subtractive analyses of gene expression in drug-sensitive and drug-resistant cell lines or tumors have allowed the identification of genes which are potentially responsible for drug resistance, and which had not been recognized previously by the usual analytic approaches. The generation of genetic suppressor elements represents a more functional approach since they can be selected upon the actual resistance properties of the cell lines. Global transcriptome analysis can be performed through the use of cDNA microarrays, either for the comparison of drug-sensitive and resistant cell lines, or for the study of drug effects on gene expression. This may allow the identification of drug-response genes (whose expression is altered by the drug) and of signaling and metabolic pathways involved in drug activity.

Apoptosis↗
PubMedDate not supplied

Evolution of heat shock protein and immunity.

Heat shock proteins (hsps) are among the most abundant intracellular proteins. Their synthesis is rapidly up-regulated by various 'stressors' including temperature, glucose deprivation, infection and cancer. Certain hsps are able to: (i). associate and chaperone a large variety of cellular peptides; (ii). be efficiently internalized by antigen presenting cells (APC) through receptor-mediated endocytosis; (iii). channel antigenic peptides they chaperone in the APC's MHC class I presentation pathway; (iv). and stimulate inflammatory cytokines, chemokines and co-stimulatory molecules through the NFkappab signaling pathway. Extracellular release of hsps upon necrotic cell death and their modulated access at the surface of some cells, can be considered as a putative 'danger' signal. Based on the ancient origins and structural conservation of hsps, it has been proposed that, the role of hsps in immunity emerged early in evolution and to be widespread in extant organisms. Data from studies with the frog Xenopus support this proposition.

Animals↗
PubMedDate not supplied

Cellular parameters predictive of the clinical response of colorectal cancers to irinotecan. A preliminary study.

BACKGROUND: We have examined, in this study, the feasibility of determining cellular factors contributing to irinotecan activity in colorectal cancers. Irinotecan is a camptothecin derivative requiting carboxylesterase activation to SN-38, which interacts with its target enzyme, topoisomerase I. MATERIALS AND METHODS: In 9 surgical or biopsy samples of colorectal tumours and corresponding normal tissue, kept in a tumour bank, we evaluated topoisomerase I expression and activity, respectively by Western blotting and DNA relaxation assay, carboxylesterase activity using two different substrates and p53 status by immunohistochenistry. RESULTS: Topoisomerase I expression and activity were significantly correlated, as were the two types of determinations for carboxylesterase activity. Topoisomerase I was significantly more active in tumours than in corresponding normal tissue. The three samples presenting the highest topoisomerase I expression and activity originated from the patients who responded to irinotecan treatment. No such features were apparent for carboxylesterase activity and p53 staining. CONCLUSION: Topoisomerase I expression appeared as the parameter most likely to predict response to irinotecan therapy in the clinical setting.

Adult↗
PubMedDate not supplied

Quantification of the expression of multidrug resistance-related genes in human tumour cell lines grown with free doxorubicin or doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres.

Doxorubicin (dox) encapsulated in polyisohexylcyanoacrylate nanospheres (PIHCA-dox) can circumvent P-glycoprotein-mediated multidrug resistance (MDR). In order to investigate whether this drug formulation is able to select MDR cells in culture in the same way as free doxorubicin does, two human tumour cell lines, K562 and MCF7, were grown with increasing concentrations of either free dox or PIHCA-dox. For both drug formulations and for each selection level, the cell lines were more resistant to free dox than to PIHCA-dox. The MCF7 sublines selected with PIHCA-dox exhibited a higher level of resistance to both doxorubicin formulations than those selected with free doxorubicin. Different levels of overexpression of several genes involved in drug resistance (MDR1, MRP1, BCRP and TOP2alpha) occurred in the resistant variants. MDR1 gene overexpression was consistently higher in free dox-selected cells than in PIHCA-dox-selected cells, while this was the reverse for the BCRP gene. Overexpression of the MRP1 and TOP2alpha genes was also observed in the selected variants. Our results show that several mechanisms may be involved in the acquisition of drug resistance and that drug encapsulation markedly alters or delays these processes.

ATP Binding Cassette Transporter, Subfamily G, Mem↗