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Jacques Grassi

Publications and source records attributed to Jacques Grassi.

At least 37 records · Page 2Linked to original sources
PubMed2004

Sensitive nonradiometric method for determining thymidine kinase 1 activity.

BACKGROUND: Thymidine kinase 1 (TK1) is a cytoplasmic enzyme, produced only in the S-phase of proliferating cells, that has potential as a tumor marker. Specific determination of TK1 in serum is difficult, in part because of differences in the physical properties of serum TK1 compared with cytoplasmic TK1. METHODS: The first step in the new assay was phosphorylation of 3'-azido-2',3'-deoxythymidine (AZT) to AZT 5'-monophosphate (AZTMP) by TK1 present in patient material. The AZTMP formed was measured in a competitive immunoassay with specific anti-AZTMP antibodies and AZTMP-labeled peroxidase. Results were compared with those of a TK radioenzyme assay (REA) for 78 samples from patients suffering from hematologic diseases. RESULTS: The detection limit was 78 microIU/L, and within-run CVs <20% were seen for samples with TK1 down to 130 microIU/L. Cross-determination of the mitochondrial isoenzyme TK2 activity was <0.1%. Between-assay imprecision (CV) was 3.5-7.4%, and the within-assay imprecision was 4.1-9.1%. In studies of recovery and linearity on dilution, measured values ranged from 84% to 115% of expected at concentrations of 0.26-10.4 mIU/L. Results of the new assay (mIU/L) = 0.109 x TK REA (U/L) + 0.092. Heterophilic antibodies did not interfere in the assay. The upper 95th percentile, in 100 healthy individuals, was 0.94 mIU/L, and the median value was 0.43 mIU/L. CONCLUSION: The TK1 enzyme-labeled immunoassay uses a stable substrate, is precise, appears to be accurate, and is resistant to interferences. It may provide a practical tool in the management of hematologic malignancies.

Biomarkers, Tumor↗
PubMed2004

Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain.

Expression of the cellular prion protein (PrP(c)) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrP(c) is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrP(c) in the mouse brain, the immunocytochemical localization of PrP(c) was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrP(c) was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrP(c) antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrP(c) immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrP(c). Transgenic mice expressing PrP-GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases.

Animals↗
PubMed2004

Selective and efficient immunoprecipitation of the disease-associated form of the prion protein can be mediated by nonspecific interactions between monoclonal antibodies and scrapie-associated fibrils.

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads.

Animals↗
PubMed2004

Unexpected CD4 cell count decline in patients receiving didanosine and tenofovir-based regimens despite undetectable viral load.

BACKGROUND: We recently observed a significant CD4 cell count decline in patients receiving didanosine (ddI) 400 mg, tenofovir (TDF) and nevirapine (NVP), despite virological suppression. METHODS: We identified from our computerized patient database subjects who initiated combinations containing ddI and/or TDF for reasons other than virological failure, including simplification or intolerance. Changes in total, CD4+ and CD8+ lymphocyte counts since the initiation of therapy were analysed retrospectively. Plasma concentration of ddI was prospectively determined in eight of these patients receiving ddI 400 mg + TDF + NVP and 3 weeks after a ddI dosage reduction. RESULTS: A total of 302 patients were studied. A significant decrease in CD4 and CD8 and in total lymphocyte counts was only seen in subjects receiving ddI standard dose + TDF-containing regimens, despite the maintenance of viral suppression. More than 50% of these patients showed a decline of more than 100 CD4 cells at 48 weeks. In contrast, subjects not receiving ddI + TDF together experienced the expected progressive increase in CD4 T-cell counts. Plasma levels of ddI were elevated in all patients receiving the standard ddI dose + TDF. DdI plasma levels significantly decreased when patients weighting > 60 kg reduced ddI dose to 250 mg, achieving similar levels to those generated by ddI 400 mg without TDF. CONCLUSIONS: Co-administration of ddI at standard doses plus TDF appears to exert a deleterious effect on CD4 and CD8 counts. Although lymphocyte toxicity related to excessive ddI plasma levels could explain our findings, other mechanisms cannot be excluded. Pharmacokinetic data suggest ddI dose reduction when coadministered with TDF.

Adenine↗
PubMed2004

An enzyme immunoassay for the quantification of plasma and intracellular lopinavir in HIV-infected patients.

The protease inhibitor lopinavir (LPV; [1S-[1R*(R*), 3R*,4R*]]-N-[4-[[(2,6-dimethylphenoxy)-acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl) pentyl] tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H)-pyrimide acetamide) is widely used in anti-human immunodeficiency virus (HIV) therapy. Knowledge of the plasma and intracellular concentrations of the drug would be useful for a better understanding of LPV action and for therapeutic monitoring. The aim of this study was to develop a sensitive and specific immunoassay for LPV in plasma and cells. Anti-LPV polyclonal antibodies were raised in rabbits using a synthetic LPV derivative coupled to keyhole limpet hemocyanin (KLH) as immunogen. The enzyme tracer was prepared by chemically coupling the LPV derivative with acetylcholinesterase. These reagents were used to develop a competitive enzyme immunoassay (EIA) performed in microtitration plates. The assay was performed on a minimum of 50 microl of plasma or 2 x 10(6) cells. It showed good precision and efficiency in as much as recovery from human plasma and cell extracts spiked with LPV ranged between 87% and 104%, with coefficients of variation of less than 10%. The limit of detection (LOD) was 100 pg/ml, i.e., a value at least 10 times lower than those currently achieved using previously described techniques. Cross-validation with high-performance liquid chromatography (HPLC) revealed a good correlation between methods (r2=0.96). Intracellular concentrations of LPV were measured in cultured human T lymphoblastoid cells (CEM). A pharmacokinetic analysis of plasma and intracellular LPV was performed on a healthy volunteer, and measurements were done in patients infected with HIV. The results obtained indicated a high intracellular/extracellular concentration ratio in cultured cells (19.3) but not in cells from HIV patients (1.3). In contrast, in peripheral blood mononuclear cells (PBMC) the accumulation of ritonavir (RTV) was six times higher than LPV. To date, this is the first reported immunoassay for LPV, and this method is sensitive enough for monitoring plasma and intracellular LPV levels in HIV-infected patients and for intracellular studies.

Adult↗
PubMed2004

Immuno-quantitative polymerase chain reaction for detection and quantitation of prion protein.

Immuno-polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno-quantitative PCR (iqPCR), exploiting real-time PCR technology, in order to improve this immuno-detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post-mortem stage.

Animals↗
PubMed2004

Enhancement of antibody responses in DNA vaccination using a vector encoding a universal T-helper cell epitope.

DNA vaccination appears as a very promising approach to raise protective antibodies against a variety of proteins from pathogens or tumor cells, but is often hindered by the low immunogenicity of the genetic vectors used for the immunizations. To enhance the humoral response through improvement of the antigenic presentation of newly synthesized proteins upon vaccination, we engineered a plasmid coding for a low immunogenic protein (an scFv, i.e. the single-chain Fragment variable of a well-characterized antibody) fused to a small-size universal T-helper cell epitope derived from tetanus toxin, whose efficiency in classical protein-based immunization protocols has already been demonstrated. We found that immunization of C57Bl/6 mice using this vector greatly enhanced the production not only of specific antibodies recognizing essentially conformational epitopes on the undenatured scFv protein but also of antibodies against linear epitopes on the denatured protein. Since this T-epitope is known to be accommodated by several haplotypes of H-2 molecules in mice, as well as by various class II MHC molecules in humans, the results reported here allow us to conclude that this method could be of general interest for future applications of genetic immunization, including DNA-based vaccinations in humans.

Animals↗
PubMed2004

Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation.

The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein.

Animals↗
PubMed2004

Sensitive enzyme immunoassay for measuring plasma and intracellular nevirapine levels in human immunodeficiency virus-infected patients.

We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml(-1) limit of detection and thus approximately 100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml(-1) limit of detection was observed) on a minimum of 30 micro l of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.

Adult↗
PubMed2004

Comparative molecular analysis of the abnormal prion protein in field scrapie cases and experimental bovine spongiform encephalopathy in sheep by use of Western blotting and immunohistochemical methods.

Since the appearance of bovine spongiform encephalopathy (BSE) in cattle and its linkage with the human variant of Creutzfeldt-Jakob disease, the possible spread of this agent to sheep flocks has been of concern as a potential new source of contamination. Molecular analysis of the protease cleavage of the abnormal prion protein (PrP), by Western blotting (PrP(res)) or by immunohistochemical methods (PrP(d)), has shown some potential to distinguish BSE and scrapie in sheep. Using a newly developed enzyme-linked immunosorbent assay, we identified 18 infected sheep in which PrP(res) showed an increased sensitivity to proteinase K digestion. When analyzed by Western blotting, two of them showed a low molecular mass of unglycosylated PrP(res) as found in BSE-infected sheep, in contrast to other naturally infected sheep. A decrease of the labeling by P4 monoclonal antibody, which recognizes an epitope close to the protease cleavage site, was also found by Western blotting in the former two samples, but this was less marked than in BSE-infected sheep. These two samples, and all of the other natural scrapie cases studied, were clearly distinguishable from those from sheep inoculated with the BSE agent from either French or British cattle by immunohistochemical analysis of PrP(d) labeling in the brain and lymphoid tissues. Final characterization of the strain involved in these samples will require analysis of the features of the disease following infection of mice, but our data already emphasize the need to use the different available methods to define the molecular properties of abnormal PrP and its possible similarities with the BSE agent.

Animals↗
PubMed2004

ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages.

OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

A sensitive and specific enzyme immunoassay for the detection of methyl ether derivatives of cyclomaltoheptaose.

We have raised antibodies against two methylated derivatives of beta-CD, heptakis(2,6-di-O-methyl)cyclomaltoheptaose (Dimeb) and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose (Trimeb). These antibodies were used to develop two specific and sensitive enzyme immunoassays, presenting a detection limit close to 500 and 30 pg/mL for Trimeb and Dimeb, respectively. Cross reactivities of different linear and cyclic maltooligosaccharides were investigated, demonstrating a high specificity against the structural features of the secondary hydroxyls rim. Several commercial Dimeb samples, containing different mixtures of partially methylated beta-cyclodextrin derivatives including RAMEB, which contains only a few amount of pure Dimeb, could be easily evaluated by the Dimeb immunoassay. Both of these assays have been shown to allow accurate measurement in plasma and urine, thus appearing as useful tools for further applications in biological material.

Animals↗
PubMed2003

The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein.

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.

Amino Acid Sequence↗
PubMed2003

Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals.

This study was aimed to apply an LC-MS-MS method previously developed for intracellular nucleoside reverse transcriptase inhibitors-triphosphate (NRTI-TPs) to the determination of natural deoxyribonucleotides (dNTPs) in human peripheral blood mononuclear cells. The LC-MS-MS method was directly used in assay of dATP and dTTP. Interferences by ribonucleotides (rNTPs) prevented direct application to the two other analytes: dGTP and dCTP. A periodate oxidation procedure was therefore optimized to remove rNTPs from the cell medium in order to quantitate dCTP and dGTP. The determination of the intracellular ratio of NRTI-TP/dNTP in HIV-infected patients now involves use of the same chromatographic system for simultaneous assay of several analytes.

Chromatography, Liquid↗
PubMed2003

Pharmacological properties of peptides derived from an antibody against the tachykinin NK1 receptor for the neuropeptide substance P.

Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.

Animals↗
PubMed2003

A strategy for liquid chromatography/tandem mass spectrometric assays of intracellular drugs: application to the validation of the triphosphorylated anabolite of antiretrovirals in peripheral blood mononuclear cells.

The pharmacokinetics of intracellular drugs have recently aroused new interest because monitoring a drug's behaviour near the site of action can enhance knowledge of its efficacy and toxicity. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) is particularly attractive for intracellular analytes. Very few papers deal precisely with special features encountered in intracellular drug assay or with how closely the assay matches the actual recommendations. Particular problems are encountered mainly because the analytes are located intracellularly. This mainly concerns the handling of biological media, including provision of blank samples using Ficoll gradient separation, cell counts, optimisation of cell lysis, sample extraction, plotting standard curves using either fmol/10(6) cells or fmol/ml of extract or fmol/sample, the matrix effect as a function of the number of cells, stability before and during cell separation, as well as in storage conditions using clinical samples, biological matrix replacement and interference by endogenous compounds. This paper describes a strategy for the full validation and routine use of an LC/MS/MS assay applied to the simultaneous intracellular determination of the triphosphorylated anabolites of didanosine (2',3'-dideoxyadenosine triphosphate or ddA-TP) and stavudine (2',3'-didehydro-3'-deoxythymidine triphosphate or d4T-TP), two nucleoside reverse transcriptase inhibitors of HIV, in human peripheral blood mononuclear cells (PBMCs), as a guide for further LC/MS/MS assay of intracellular drugs.

Anti-HIV Agents↗
PubMed2003

A new sensitive cartridge-RIA method for determination of stavudine (D4T) triphosphate in human cells in vivo.

We describe a simple and sensitive method to determine stavudine triphosphate, the active intracellular anabolite of stavudine (D4T). Quantification of D4T triphosphate was performed with a combined cartridge-radioimmunoassay (cartridge-RIA) which enabled us to measure concentrations of D4T triphosphate as low as 0.5 ng/ml, or an intracellular concentration which corresponds to 20 fmol/10(6) cells if diluted like our previously published zidovudine (ZDV) assay. The only alternate methodology at present employs liquid chromatography mass spectroscopy (LC-MS/MS). The use of the cartridge-RIA methodology provides a cost-effective alternative for the determination of in vivo cellular pharmacokinetics studies of D4T in human immunodeficiency virus (HIV)-infected persons.

Humans↗