Biomedical subjects

C Ménard

Publications and source records attributed to C Ménard.

At least 19 recordsLinked to original sources

PubMed2002

Strategies for the identification and analysis of viral immune-evasive genes--cytomegalovirus as an example.

Co-evolution of herpesviruses with their hosts has resulted in multiple interactions between viral genes and cellular functions. Some interactions control genomic maintenance and replication in specific tissues, other affect the immune control at various stages. Few immunomodulatory functions of genes can be predicted by sequence homology. The majority of genes with immunomodulatory properties only become apparent in functional assays. This chapter reviews procedures which have been used for successful identification of immunomodulatory genes in the past and deals with recent methods which may be applicable for the identification of additional immunomodulatory functions unknown so far.

Animals↗

PubMed2001

A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism.

Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.

3T3 Cells↗

PubMed2001

Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells.

OBJECTIVE: Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS: Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS: No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS: Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

Angioplasty, Balloon↗

PubMed2001

Porphyromonas gulae sp. nov., an anaerobic, gram-negative coccobacillus from the gingival sulcus of various animal hosts.

A new species, Porphyromonas gulae sp. nov., is proposed to include strains isolated from the gingival sulcus of various animal hosts which are distinct from related strains of Porphyromonas gingivalis of human origin. This bacterium exhibits the following characteristics: black-pigmented colonies; asaccharolytic, obligate anaerobic growth; and Gram-negative, non-motile and non-spore-forming, rod-shaped cells. Colonies do not fluoresce under UV light. Vitamin K1 and haemin are required for growth. Cells haemagglutinate sheep erythrocytes. Major fatty acid end products are butyric acid, isovaleric acid, succinic acid and phenylacetic acid. Strains are catalase-positive and indole is produced. Alkaline phosphatase, trypsin-like and N-acetyl-beta-glucosaminidase activities are strong. A beta-galactosidase and a glutamylglutamic acid arylamidase are also present. The G+C content of the chromosomal DNA is 51 mol%. DNA-DNA homology data and 16S rRNA gene sequence analysis provide strong evidence that strains from the animal biotype of P. gingivalis represent a Porphyromonas species that is distinct from P. gingivalis. The type strain of P. gulae is Loup 1T (= ATCC 51700T = NCTC 13180T).

Animals↗

PubMed2000

Rapid detection of group B streptococci in pregnant women at delivery.

BACKGROUND: Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates. METHODS: We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay. RESULTS: Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture. CONCLUSIONS: Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.

Anal Canal↗

PubMed2000

Novel genus-specific PCR-based assays for rapid identification of Neisseria species and Neisseria meningitidis.

This study presents the development of polymerase chain reaction (PCR)-based tests for the identification and detection of Neisseria species and Neisseria meningitidis. Currently, isolating and identifying these pathogens using conventional biochemical methods require 48-72 h. To improve speed and accuracy in diagnosing Neisseria infections, simple PCR-based tests that are specific for the genus Neisseria and the species Neisseria meningitidis have been developed. The genus-specific and species-specific DNA sequences were chosen by selecting and analyzing available database sequences. Neisseria-specific and Neisseria meningitidis-specific primer pairs were derived from the genes asd (coding for the aspartate beta-semialdehyde dehydrogenase) and ctrA (coding for a conserved outer membrane protein), respectively. Both the Neisseria-specific and Neisseria meningitidis-specific PCR assays were specific (they amplified only DNA from the target genus or species, out of 84 bacterial species tested). In addition, the Neisseria-specific assay amplified DNA from 321 of 322 strains tested representing 13 species of Neisseria, while the Neisseria meningitidis-specific assay amplified DNA from all 256 strains tested representing nine serogroups of Neisseria meningitidis. These PCR assays, which can be combined in multiplex, have been adapted to ensure that they are simple and can be performed within approximately 90 min. The tests provide new diagnostic tools for identifying Neisseria infections.

Aspartate-Semialdehyde Dehydrogenase↗

PubMed2000

Identification of mutans and other oral streptococci by random amplified polymorphic DNA analysis.

The identification and classification of the non-haemolytic or viridans group of streptococci have long been recognised as difficult and unsatisfactory. Phenotypic and genotypic heterogeneity have resulted in ambiguous speciation, particularly with mutans streptococci and other oral streptococci. This study was done to determine whether random amplified polymorphic DNA (RAPD) analysis is useful to identify and even classify oral and other streptococci. DNA was prepared and purified from 25 strains of mutans streptococci including 11 reference strains of Streptococcus mutans, seven of S. sobrinus, three of S. rattus and one each of the four other species of the mutans group, together with 20 other reference species, mostly streptococci, and from 49 fresh isolates of mutans streptococci and of S. mutans from human saliva and dental plaque. DNA amplification was primed with each of three arbitrarily selected primers nine or 10 nucleotides in length. The amplified DNA fragments (amplicons) obtained were compared by agarose gel electrophoresis. Species- and strain-specific RAPD fingerprints were obtained not only from pure genomic DNA, but also from the supernates of crude cellular or colony extracts. Pending the analysis of numerous other strains, the data suggest that RAPD may be of value: (i) to distinguish the species S. mutans and S. sobrinus from each other and potentially from other species of oral streptococci, (ii) to differentiate and possibly classify oral streptococci and (iii) as a valuable tool in mutans streptococci epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of streptococcal isolates.

Adult↗

PubMed2000

Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis.

Clinical isolates of Staphylococcus aureus (a total of 206) and S. epidermidis (a total of 188) from various countries were tested with multiplex PCR assays to detect clinically relevant antibiotic resistance genes associated with staphylococci. The targeted genes are implicated in resistance to oxacillin (mecA), gentamicin ¿aac(6')-aph(2"), and erythromycin (ermA, ermB, ermC, and msrA). We found a nearly perfect correlation between genotypic and phenotypic analysis for most of these 394 strains, showing the following correlations: 98% for oxacillin resistance, 100% for gentamicin resistance, and 98.5% for erythromycin resistance. The discrepant results were (i) eight strains found to be positive by PCR for mecA or ermC but susceptible to the corresponding antibiotic based on disk diffusion and (ii) six strains of S. aureus found to be negative by PCR for mecA or for the four erythromycin resistance genes targeted but resistant to the corresponding antibiotic. In order to demonstrate in vitro that the eight susceptible strains harboring the resistance gene may become resistant, we subcultured the susceptible strains on media with increasing gradients of the antibiotic. We were able to select cells demonstrating a resistant phenotype for all of these eight strains carrying the resistance gene based on disk diffusion and MIC determinations. The four oxacillin-resistant strains negative for mecA were PCR positive for blaZ and had the phenotype of beta-lactamase hyperproducers, which could explain their borderline oxacillin resistance phenotype. The erythromycin resistance for the two strains found to be negative by PCR is probably associated with a novel mechanism. This study reiterates the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections.

Anti-Bacterial Agents↗

PubMed2000

Osmotic stress-induced genetic rearrangements in Escherichia coli H10407 detected by randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA (RAPD) analysis is a DNA polymorphism assay commonly used for fingerprinting genomes. After optimizing the reaction conditions, samples of Escherichia coli H10407 DNA were assayed to determine the influence of osmotic and/or oligotrophic stress on variations in RAPD banding patterns. Genetic rearrangements or DNA topology variations could be detected as changes in agarose gel electrophoresis banding profiles. A new amplicon generated using DNA extracted from bacteria prestarved by an osmotic stress and resuscitated in rich medium was observed. Enrichment improved recovery of mutator cells and allowed them to be detected in samples, suggesting that DNA modifications, such as stress-induced alterations and supercoiling phenomena, should be taken into consideration before beginning RAPD analyses.

Base Sequence↗

PubMed2000

Development of a rapid PCR assay specific for Staphylococcus saprophyticus and application to direct detection from urine samples.

Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection of S. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).

DNA, Bacterial↗

PubMed2000

Development of conventional and real-time PCR assays for the rapid detection of group B streptococci.

BACKGROUND: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. METHODS: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCycler(TM). For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. RESULTS: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only approximately 30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. CONCLUSION: These assays provide promising tools for the rapid detection and identification of GBS.

Animals↗

PubMed1999

Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells.

The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca(2+) channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (alpha(1S)) and cardiac (alpha(1C)) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nM all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca(2+) channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca(2+) channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (alpha(1C)) L-type Ca(2+) channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca(2+) channels in mature cardiac cells.

Animals↗

PubMed1999

MHC class Ia molecules alone control NK-mediated bone marrow graft rejection.

Mice with functionally deleted genes encoding MHC class I heavy (H-2K(b), H-2D(b)) and light (beta2-microglobulin) chains were used in bone marrow cell transfer experiments to study the role of class Ia and Ib molecules in NK cell function. Absence of H-2K(b) and absence of H-2D(b) on bone marrow cells resulted in complete and in almost complete NK-mediated rejection, respectively. Absence of either H-2 class Ib (at least when expressed in H-2 class Ia-deficient mice) or cell surface class Ia free heavy chains did not result in bone marrow rejection. Thus, in C57BL/6 adult mice, the inactivation of NK cells required for bone marrow cell engraftment relies entirely upon-H-2 class Ia molecules. These results imply the existence of an inhibitory receptor which recognizes either directly or indirectly H-2D(b) molecules and further suggest that in C57BL/6 mice the NK cells which do not express a H-2K(b) specific inhibitory receptor necessarily express an H-2D(b)-specific one.

Animals↗

PubMed1999

Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis.

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.

Base Sequence↗

PubMed1999

Development of a PCR assay for rapid detection of enterococci.

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.

Bacterial Typing Techniques↗

PubMed1998

Beta2-microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition, but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains.

Mice lacking beta2-microglobulin (beta2m- mice) express greatly reduced levels of MHC class I molecules, and cells from beta2m- mice are therefore highly sensitive to NK cells. However, NK cells from beta2m- mice fail to kill beta2m- normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of beta2m- NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to beta2m- NK cells, we made the following observations: First, beta2m- NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to beta2m- concanavalin A (Con A)-activated T cell blasts. Secondly, beta2m- NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than beta2m+ NK cells. Thirdly beta2m- NK cells were not only tolerant to beta2m- Con A blasts but also to Con A blasts from H-2Kb-/Db- double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of beta2m- NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in beta2m- mice, which may instead operate through mechanisms involving triggering pathways.

Animals↗

PubMed1998

Adherence of Porphyromonas gingivalis to epithelial cells: analysis by flow cytometry.

Porphyromonas gingivalis, implicated in the pathogenesis of periodontitis, can adhere to epithelial cells and gingival fibroblasts. This study employed flow cytometry to evaluate the adherence of P. gingivalis to epithelial cells under various conditions. The cell lines SK-MES and KB were used in the first experiments. The P. gingivalis strains employed were ATCC 33277, ATCC 49417 and W83. Different adherence conditions were tested (contact time, bacteria/cell ratio, contact temperature). In later experiments, adherence of P. gingivalis to human gingival epithelial cells (GEC) obtained by explant was studied under various conditions. Results showed that P. gingivalis had a high affinity for buccal keratinocytes compared with SK-MES. Adherence showed a level of saturation. The number of receptors may be limited for each epithelial cell line, and there may be more receptors for gingival keratinocytes. Depending on contact time, P. gingivalis showed a higher affinity for GEC, compared with the other two lines. P. gingivalis thus showed specific adherence for a host cell type from a site associated with periodontal disease.

Bacterial Adhesion↗

PubMed1997

Determination of clenbuterol in beef liver and muscle tissue using immunoaffinity chromatographic cleanup and liquid chromatography with ultraviolet absorbance detection.

Clenbuterol, a beta-agonist, was determined in samples of beef liver and muscle. The method employed an acidic aqueous extraction followed by protein precipitation. The supernatant liquid was passed through a weak cation-exchange cartridge and then through a commercially available immunoaffinity cartridge. Clenbuterol was eluted from the immunoaffinity cartridge with 80% ethanol in water. The eluate was concentrated and analysed directly by reversed-phase liquid chromatography using gradient elution and UV detection at 245 nm. Detection limits were estimated to be 0.3 ng g-1 clenbuterol. A single immunoaffinity cartridge was used for ten sample extracts with no significant loss in capacity. No organic solvents other than ethanol and methanol were employed in the procedure. Recoveries of clenbuterol from samples of beef liver and muscle spiked at 2 and 5 ng g-1 carried through the entire procedure were 63 +/- 11% (range, 53-74%) compared to pure standards. Absolute recoveries of pure standards (30 ng clenbuterol) carried through the same analytical steps were 70 +/- 5% (n = 6), the losses being primarily due to the ion-exchange step.

Adrenergic beta-Agonists↗