PubMed Health⌕ Search

Biomedical subjects

C Drouet

Publications and source records attributed to C Drouet.

34 records · Page 2Linked to original sources
PubMed1996

In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

Apoptosis↗
PubMed1995

Contribution of apoptosis to the phenotypic changes of adrenocortical cells in primary culture.

The aim of this study was to evaluate the occurrence and physiological consequences of apoptosis in primary cultures of bovine adrenocortical cells (of fasciculata-reticularis origin). Under ACTH-free culture conditions, we observed apoptotic cells in the cell layer and the accumulation of apoptotic bodies in the culture medium. These were hardly detectable in ACTH-supplemented cultures. Under ACTH-free conditions, the DNA content of apoptotic bodies collected over 48 h represented up to 10-15% of that of the cell layer at the onset of the culture (as compared to 3% in ACTH-supplemented cultures). Past the fourth day of culture in the absence of ACTh, most cells lacked several markers of their originating fasciculata-reticularis phenotype and progressively evolved to an undifferentiated phenotype. The vast majority of the apoptotic bodies released during the first 4 days of culture were immunoreactive for P450 17 alpha. Inversely, during the same period of time, the proliferating cells (PCNA-positive) did not appear to express P450 17 alpha. Therefore, apoptosis could contribute, together with dedifferentiation, to the phenotype shift observed in ACTH-depleted cultures of adrenal fasciculata-reticularis cells. These observations also characterize this endocrine cell system as an in vitro model for the study of hormone-repressed apoptosis.

Adrenal Cortex↗
PubMed1994

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells.

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigen-presenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,kappa and IgG2a,kappa murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and purified in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of kappa light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigen-presenting cells.

Animals↗
PubMed1994

Non-coordinated biosynthesis of early complement components in a deficiency of complement proteins C1r and C1s.

We report on a 60-year-old woman with systemic lupus erythematosus and a total (95%) C1r and a partial (36%) C1s deficiency. The patient complained about cutaneous lesions on forearms and legs without other systemic involvement. Elevated anti-nuclear, anti-native DNA and anti-SSA antibodies were present. The finding of persistently depressed levels of haemolytic complement activity (CH50) on both serum and plasma, associated with normal levels of C3, C4 and C2 components, and normal alternative pathway haemolytic activity showed a deficiency of an early component of the classical pathway. Indeed C1r component was below the limits of detection whereas C1s component was lowered (36%). The depressed CH50 was only corrected by purified C1r. Biosynthesis of C1r and C1s by patient's monocytes was spontaneously normal but not up-regulated by interferon-gamma for C1r alone, whereas the biosynthesis of C1s, but also of interleukin-6, was increased, indicating a specific disregulation of C1r. The deficiency was associated with a lupus syndrome and a fatal assumed septic shock. This is in agreement with other reported cases.

Cells, Cultured↗
PubMed1993

The interaction of antigenic peptides with the H-2Kd MHC class I molecule.

An important event in the recognition of antigen by T cells is the selective interaction of peptides with major histocompatibility complex (MHC) molecules. We have defined several critical structural features that promote the efficient interaction of antigenic peptides with the MHC class I molecule, H-2Kd. For four unrelated antigens, we found that optimal synthetic peptides were short, only 9 or 10 amino acids long. These and other H-2Kd-restricted peptides were found to share a distinct 2-residue binding motif. Two regions in the H-2Kd antigen binding site that might accommodate the motif residues were identified by analysis of Ala-substituted H-2Kd molecules. A molecular model showing the possible interaction of one antigenic peptide with the H-2Kd molecule is presented.

Amino Acid Sequence↗
PubMed1993

OH. treatment of tetanus toxin reduces its susceptibility to limited proteolysis with more efficient presentation to specific T cells.

At inflammatory sites, before their processing, antigens are exposed to oxygen free radicals released by activated cells. The effect of hydroxyl radicals (OH.) on the structure of a protein antigen, tetanus toxin (TT) was investigated, as well as the consequences on processing and presentation. A chemical system composed of Fe-EDTA, ascorbate and H2O2 was used to produce physiological amounts of OH. radicals. TT exposed to OH. radicals presented a marked decrease of its intrinsic fluorescence with a concomitant increase of the content of bityrosine, but no fragmentation of the protein was detected by SDS-PAGE. Processing of the modified TT was analysed, by incubating TT at acidic pH with fractions enriched in plasma membranes and lysosomes obtained from a lymphoblastoid cell line (LCL). Proteolysis of OH.-treated TT was less important than proteolysis of native TT, especially upon prolonged incubations. Oxidized TT presented by LCL cells induced a greater proliferation of three different TT specific T cell clones, compared to native TT. When proteolytic digests of TT were presented by fixed LCL cells to a homologous T cell line, the proliferative response obtained in the presence of digests of OH.-treated TT was sustained, even in the case of prolonged proteolysis, whereas the response to digests of native TT fell rapidly. The relative resistance of OH.-treated TT to proteolysis appears thus responsible for its greater presentation to specific T cells, probably by protecting epitopes.

Animals↗
PubMed1991

Conversion of a self peptide sequence into a Kd-restricted neo-antigen by a Tyr substitution.

We have previously found that a Tyr residue was critical for the interaction of peptides with the Kd molecule, and therefore may be acting as an anchor residue. In the present report we show that it is possible to convert a self peptide sequence into a Kd-restricted neo-antigen by a single Tyr substitution at position 2 of the peptide. This supports the idea that Tyr is a critical element in the binding motif of Kd-restricted peptides and is a finding that could also prove useful for vaccine development.

Amino Acid Sequence↗
PubMed1991

Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-alpha promoter in primary macrophages.

In macrophages, the TNF-alpha promoter is specifically induced by bacterial endotoxin, and provides a good model for gene regulation during bacterial infections. We have analyzed the protein-binding characteristics and enhancer activity of four kappa B-like enhancers and of a MHC class II-like Y box found in the mouse TNF-alpha promoter. In addition to members of the NF-kappa B/rel transcription factor family, at least two of the kappa B sites also bound a nuclear protein identified as NF-GMa, a factor that binds to promoter sequences from many cytokines. When inserted upstream of an enhancer-less promoter, two of the kappa B sites were active as LPS-inducible enhancers in primary macrophages, whereas the other two were not. Mutations in nucleotides known to contact nuclear factors severely reduced affinity of the kappa B sites for NF-kappa B. Introduction of the same mutations into a construct containing 1059 bp of the TNF-alpha promoter coupled to a CAT reporter gene resulted in a stepwise reduction in inducibility by LPS; mutation of all four sites (11 bp of 1059) reduced inducibility by 90%, providing compelling evidence for the role of transcription factors belonging to the NF-kappa B/rel family in the activation of the TNF-alpha promoter. The TNF-alpha Y box bound an abundant nuclear factor, but had no detectable activity in our assays, either as an enhancer or as a mutation-sensitive controlling element.

Animals↗
PubMed1990

[Evaluation of the bactericidal effect of the membrane attack complex of serum complement].

The bactericidal activity of serum complement and particularly of the membrane attack complex (MAC-C5b-C9) was studied on E. coli C600 with a simple functional test. The test evaluates the in vitro kinetics of the bactericidal effect and the subsequent counting of surviving germs. Homozygous deficiency of a particular membrane attack complex protein was easily detected from a total loss of bactericidal activity. These results were confirmed on Neisseriae meningitidis A and C, but in this case a more complex protocol was required. Deficits of proteins of the membrane attack complex sequence of complement are often found in sera of patients suffering from recurrent Neisseriae infections. This simple test, adapted to family studies, appears, thus, as a valuable basis for a detection of relatives at risk.

Blood Bactericidal Activity↗
PubMed1989

Identification of a human non-interferon lymphokine activating monocyte complement biosynthesis.

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon.

Amino Acids↗
PubMed1989

Biosynthesis of C1r and C1s subcomponents.

Biosynthesis of C1r and C1s subcomponents has been studied using monocytes and macrophages, hepatocytes and hepatoma cell lines or fibroblasts. Both proteins have been detected in supernatants and cell lysates as proenzymic monocatenar molecules. C1r and C1s were secreted by stimulated monocytes and by Hep G2 cells, according to a 1:1 stoichiometry. Monocyte C1s secretion was enhanced by lymphokines, such as alpha- or gamma-interferon or by placental soluble factors. Expression of both proteins was coordinately modulated by a newly purified 14 kDa lymphokine at a pretranslational level. Data from in vitro RNA translation are discussed.

Animals↗
PubMed1988

A sensitive method to assay blood complement C1- inhibitor activity.

Hereditary angioneurotic edema results from deficiency of complement protein C1- inhibitor. Using a new spectrophotometric assay for C1-s esterase activity on the N-alpha-benzoyl-L-arginine ethyl ester, we describe a routinely available method for quantifying low C1- Inhibitor functional activities in EDTA-treated plasma of hereditary angioneurotic edema patients. C1- Inhibitor activity is deduced from the residual esterase activity of C1-s incubated with 20-80 microliters plasma samples. Arbitrary units (volume of sample inhibiting 50% of C1-s activity) were used to express C1- Inhibitor normal activity which was estimated as 22,500 +/- 5,000 (SD) U/l in 45 healthy individuals. The correlation with C1- Inhibitor antigen in these healthy individuals and 89 patients with varying concentrations of C1 Inhibitor ranging from 0.05-1.05 g/l was r = 0.91. Levels down to 2,000 U/l could be estimated. Specific inhibitory activity is an absolute requirement to distinguish between type I and type II hereditary angioneurotic edema.

Complement C1 Inactivator Proteins↗
PubMed1987

Validation of a radioreceptor assay technique for monitoring pharmacological active material during intensive diazepam tetanus therapy.

The present study's aim was to monitor the daily plasma levels of diazepam and its metabolites in two tetanic patients who were receiving very strong doses of diazepam in continuous infusion. The kinetics of high doses of diazepam were estimated. These plasma levels were determined selectively using gas-liquid chromatography (GLC), then correlated with a global estimation by radioreceptor-assay (RRA). At the end of the infusion, the kinetics of diazepam and of RRA active material were calculated using a linear, open, two compartment model. Muscle-relaxant and residual sedative effects were clinically monitored. The mean concentrations of diazepam (2.33, 0.60 micrograms/ml), desmethyldiazepam (3.27, 1.00 micrograms/ml) and oxazepam (0.73, 0.47 micrograms/ml) estimated by GLC and of RRA active material (5.62, 1.08 micrograms/ml) correlated better when active metabolites were considered (r = 0.898) than when diazepam alone was taken into account (r = 0.730), indicating that these metabolites influence the overall activity of the parent compound. Half-lives of RRA results (52.6 h for patient 1 and 82.5 h for patient 2) were higher than those of diazepam estimated using GLC results (21.2 h for patient 1 and 42.3 h for patient 2). This could be related to the contribution of the active metabolites and might explain the residual sedative effect observed for at least seven days following cessation of the diazepam (DZP) infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation↗
PubMed1982

[Rapid method for the determination of urinary oxalic acid by gas liquid chromatography (author's transl)].

A rapid method for the determination of urinary oxalic acid by gas-liquid chromatography is described. The procedure involves extraction of oxalate from urine by tetrahydrofuran followed by evaporation to dryness and subsequent with diesterification with the boron-trifluoride propanol. The derivative is extracted with hexane and is detected by FID gas chromatography. Malonic acid is used as internal standard. Analytical recovery ranged from 94 to 105%. The coefficient of variation in replicate aliquots over the entire range is less than 6%. The expected range for our method is calculated to be 44 to 577 mumol of oxalate per 24-h urine.

Chromatography, Gas↗
PubMed1982

[Study of the extraction of organic acids from urine. Preliminary step to their gas chromatographic separation (author's transl)].

Ten organic acids are extracted from urine. Two extraction methods are used: anion exchange on DEAE-Sephadex columns and organic solvent extraction with five different solvents: diethyl ether, ethyl acetate, isopropyl chloride, light petroleum, and tetrahydrofuran. In order to quantify the extractions, the corresponding 14C-labeled acids are added to standard acid solutions and extraction rates are measured by a liquid scintillation counting system. The results show that: (1) The efficiency of anion exchange is generally good for all tested acids. (2) The extraction efficiency is not identical for the different solvents, one solvent being more efficient for a certain acid than another: tetrahydrofuran, which is generally a good solvent, is too hygroscopic to be usable. Isopropyl chloride and light petroleum are too specific with the most apolar molecules. Ethyl acetate and diethyl ether are similar and usable because of their acceptable solubilisation power as to the most polar molecules, their good solubilisation reproducibility and their readiness of use. (3) The solvent extraction method is not as time-consuming as the anion-exchange method which generally requires lengthy elution and extraction.

Carboxylic Acids↗