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Biomedical subjects

J C Nicolas

Publications and source records attributed to J C Nicolas.

At least 91 records · Page 5Linked to original sources
PubMed1990

Dot ELISA for direct detection of BK virus in urine samples.

The dot ELISA technique was applied for direct detection of BK virus in clinical urine samples. The assay was performed on nitrocellulose paper dotted with the polyethylene glycol precipitated urine samples free of cellular debris. BK virus was detected with an anti-BK virus monoclonal antibody, and the complex was visualized by immunoperoxidase staining. Positive reaction appeared as well-defined dark blue spots. Of the 110 urine samples examined, 31 were positive in the dot ELISA and 79 proved negative. Comparing with the IIF results, the dot ELISA had a 88.46% of sensibility and 90.4% of specificity, and the results agreed completely in 99 samples. The simple dot ELISA technique can be recommended for detection of BK virus excretion in routinary diagnostic.

Animals↗
PubMed1990

Detection of Aujeszky's disease virus in nasal cells of fattening pigs by immunoassay.

Both conventional and specific pathogen free pigs were inoculated intranasally with a strain of Aujeszky's disease virus (ADV). Nasal cells were collected daily by swab, aspiration or wash. The nasal cells were examined for ADV by isolation on cell culture, direct or indirect immunofluorescence and immunoperoxidase staining by monoclonal antibodies. The infected pigs were studied for nasal shedding of infected cells until 30 days after infection. The study was also extended to naturally infected farm pigs. Swabbing, washing and aspiration proved effective methods of collecting between 10(5) and 10(8) pavement or columnar epithelial cells and non-epithelial cells. Macrophages and polymorphonuclear leucocytes were also identified. Infected nasal cells were detected by immunofluorescence and immunoperoxidase from one to 21 days after infection. The viral antigen was detected in both epithelial and non-epithelial cells, the fluorescence was nuclear and, or, 'cytoplasmic', in the latter case only the cell membrane was stained. ADV antigens were detected in nasal cavity cells in pigs infected with a virulent and a hypovirulent strain. Nasal swabs proved effective in confirming infection both by virus isolation and immunological assay, and the latter was shown to be a useful experimental tool for the rapid diagnosis of Aujeszky's disease virus infection in fattening pigs suffering from acute respiratory distress.

Animals↗
PubMed1990

[Herpes simplex virus in dental practice, longitudinal epidemiologic study].

The percentage of asymptomatic recurrences was measured using systematic salivary samples from 443 patients from January 1987 to April 1988. The study of symptomatic recurrences involved 11 patients having clinical lesions due to herpes simplex buccofacial infections. The analysis revealed 0.89% of the patients encountered (N = 443) had assymptomatic salivary excretion. The study of patients showing clinical signs of herpes simplex revealed that the virus was found in 0.37% of the patients seen in consultation (N = 6,379). The saliva carried the virus in 4 out of 5 cases of oral recurrence and in 12.5% of the cases (N = 32) of extraoral recurrence. The occurrence of the virus during the course of odontological practice exists in clinically detectable forms, but also, and in a non-negligible manner, may be in clinically undetectable forms. These data should be taken into consideration in the daily exercise of the profession by virtue of the risk of contamination--cross-over which may be brought about.

Adolescent↗
PubMed1990

A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules.

With the aim of quickly and easily characterizing new estrogen or anti-estrogen molecules, we developed a cellular model in which estrogenic action can be detected by bioluminescence. This model is based on MCF-7 cells stably transfected with a receptor gene which allows expression of the firefly luciferase enzyme under control of the estrogen regulatory element of the Xenopus vitellogenin A2 gene. A stably transfected cell line (cultured for more than eight months without loss of the chimeric estrogenic response) was established by cotransfection of a neomycin resistance gene and cloning under selective pressure. Subcloning luminescent clones was accomplished by using a single-photon detecting camera. This cellular model allowed the study of an estrogenic activity either in whole-cell or in cell-free experiments by detection of the induced luciferase. Estradiol induced the luciferase activity in a dose-dependent manner at subnanomolar concentrations. The induced luciferase activity reached a maximum level as early as 24 hours after the cells were incubated with estradiol. The antiestrogen 4-hydroxy-tamoxifen inhibited the luciferase activity induced by estradiol. The cross-reactivity of ligands, such as dexamethasone, progesterone, testosterone, aldosterone, calcitriol, oxysterol and retinoic acid, were also studied, showing an estradiol specificity for a 24-hour incubation time.

Animals↗
PubMed1990

[A bioluminescent solid phase for immunoassays and the detection of nucleic probes].

A luminescent adsorbent constituted of bacterial luciferase, FMN oxidoreductase and a protein, such as an antibody or an oligonucleotide coimmobilized on Sepharose, has been used to detect a label enzyme (Glucose 6 phosphate dehydrogenase). The label enzyme, bound to the solid phase, produces NADH and start an enzymatic chain reaction leading to light emission. The dehydrogenase, which is not bound to the solid phase, produces NADH in solution which is rapidly oxidized by a scavenger system (lactate dehydrogenase plus pyruvate) and thus does not participate in light emission. Using this solid phase, binding assays do not require separation of the excess of label, and the assay protocol is limited to the addition of sample, and luminescent reagents. The authors have used this solid phase for rapid immunoassays of haptens and proteins but also for the rapid quantitation of DNA sequences obtained by enzymatic amplification catalysed by a thermostable DNA polymerase.

FMN Reductase↗
PubMed1989

Complete characterization of the gene coding for the Epstein-Barr virus major membrane antigen gp 220/340 and selective expression of a secreted form of gp 220.

The gene which encodes the Epstein-Barr gp 220/340 was inserted into a eukaryotic expression vector. A cDNA clone corresponding to the mature mRNA coding for gp 220 was isolated from an Epstein-Barr virus cDNA library and inserted in the same expression vector, enabling us to identify the precise location of the intron within the gp 220/340 coding sequence. The recombinant plasmids direct the expression of membrane proteins detected by immunofluorescence experiments using an anti-gp 220/340 monoclonal antibody in transfected human cells. The region of the gp 220/340 gene encoding the domain for membrane anchorage was removed from the two recombinant plasmids and the sequence containing the intron produced secreted forms of both truncated gp 220 and gp 340 whereas only the former was obtained with the intronless sequence.

Antigens, Viral↗
PubMed1989

Use of bioluminescence in nucleic acid hybridization reactions.

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.

DNA↗
PubMed1989

Bioluminescent assays using glucose-6-phosphate dehydrogenase: application to biotin and streptavidin detection.

A streptavidin-glucose-6-phosphate dehydrogenase (G6PDH) conjugate was synthesized and its properties were studied, along with those of biotin-G6PDH conjugates. Two bioluminescent assays were used. Streptavidin was assayed in two steps: streptavidin samples were first incubated with a small amount of biotin-G6PDH and then with biotinylated rabbit gamma-globulins. The complex was immobilized on a bioluminescent immunoadsorbent. In the single-step biotin assay, free biotin was allowed to compete with biotin linked to rabbit gamma-globulins for binding to streptavidin-G6PDH in the presence of the bioluminescent immunoadsorbent. Neither assay required washing or separation steps and the sensitivity was 0.2 ng for streptavidin and 100 fg for biotin. Different applications are described: studies of biotin reactivity when linked to probes in solution or immobilized, and quantitation of biotin in biotinylated DNA probes and oligonucleotides.

Animals↗
PubMed1989

Use of glucose-6-phosphate dehydrogenase as a new label for nucleic acid hybridization reactions.

We describe here a sensitive new procedure for detecting DNA hybridization by dot blots. The method utilizes DNA or oligonucleotide probes labeled with biotin, sulfone, or haptens that can be detected by glucose-6-phosphate dehydrogenase (G6PDH) conjugates. Biotin labeling of DNA gave the best sensitivity. G6PDH activity was revealed by staining or by bioluminescence using an FMN oxidoreductase and a luciferase from Beneckea harveyi. Bioluminescent detection offered better sensitivity and faster revelation than the colorimetric assay and was found to be very useful in visualizing single mutations in human DNA after hybridization with an allele-specific biotinylated oligonucleotide probe. Revelation can be performed using a luminometer, photographic films, or a very sensitive video camera. The detection is limited by the nonspecific binding of the labeled reagent (streptavidin or antibodies). This limit is similar to that obtained with other nonisotopic labeling procedures, but our method is faster and several hybridization reactions can be performed on the same support.

Bacteriophage lambda↗
PubMed1989

A method of HLA class II typing using nonradioactive labelled oligonucleotides.

The typing of HLA class II genes using molecular biology techniques has brought undoubtedly new insights in the analysis of their polymorphism. Particularly interesting is the dot-blot analysis of enzymatically-amplified genomic DNA hybridized with sequence-specific oligonucleotides. In order to use this technique of typing on a routine basis, we established a non-radioactive detection method of enzymatically-amplified genomic DNA dot-blots. We could clearly demonstrate that, using biotin-labelled specific oligonucleotides, it was possible to specifically discriminate between DQB1 first domain DNA sequences displaying three, two or even only one base-pair difference at a given codon position. The very satisfactory sensitivity level reached by this non-radioactive detection method could safely allow its use for clinical applications of HLA typing at the DNA level.

DNA↗
PubMed1989

[Use of a technic of bioluminescence for determining estrogens and LH during static and dynamic study of the ovary].

Estrogens and LH are necessary among other assays for ovulation diagnostic and ovarian monitoring during stimulations. They can be both measured with a method based on a bioluminescent reaction. This method measures, with a standard luminometer, the reduced NAD produced and accumulated by the reaction of two dehydrogenase enzymes: the estradiol dehydrogenase for direct assay of estrogens (estrone + estradiol), the glucose-6-phosphate dehydrogenase for gonadotrophin determination. These techniques can be applied to all biological fluids. They are simple and straightforward, do not need extraction and can be automated. Urinary assays are very useful in clinical practice to appreciate ovarian function, either during a spontaneous cycle or under stimulation, and are mandatory to decide the timing of hCG injection. The reported studies can be listed as follows: determination of preovulatory LH rise (LH greater than 10 Ul/g of creatinine) prior to embryo transfer after cryopreservation and thawing, detection of LH surges during ovarian stimulation, either premature surges causing premature luteinization, either normal surges when follicular maturation is adequate (296 cycles), confirmation of pituitary desensitization when using GnRH agonists (43 cycles), study of the initial stimulatory effect of GnRH agonists (13 patients). This effect can be responsible for the inadequate results obtained with the so-called "short protocol" in this experience when compared with the "long protocol" in the author's experience with compared with the "long protocol" (8 p. cent pregnancy rate per stimulation cycle versus 20 p. cent respectively, intrafollicular LH in 129 follicular fluids (86 with GnRH agonists and 43 without) has no correlation with the fecundability of the ovum. These results lead to extend bioluminescent techniques to the study of other parameters, and in particular FSH.

Buserelin↗
PubMed1989

Restriction endonuclease analysis of human cytomegalovirus isolated in immunocompromised patients.

Cytomegalovirus is frequently isolated from immunosuppressed patients or from patients with acquired immunodeficiency syndrome. Twelve patients were studied for several months. In 7 patients more than 1 Cytomegalovirus was isolated; in one of these patients three viruses were isolated, two of them show small differences in their migration pattern and the third seems to be a different strain after restriction analysis of CMV genome. This could reflect variation in the genome of the CMV virus as it has been already shown for viruses from other families in immunocompromised patients and shows the occurrence of multiple strains infection in a single patient.

Acquired Immunodeficiency Syndrome↗
PubMed1989

[Bioluminescence assay of luteinizing hormone in plasma and urine].

The authors describe a bioluminescent immunoassay of LH in plasma and urine. It uses two monoclonal antibodies, one is labelled with glucose-6-phosphate-dehydrogenase, the other one is coimmobilized together with bioluminescent enzymes from marine bacteria on the same adsorbent (Sepharose). This assay can be performed directly on 20 microliters plasma or 10 microliters urine. The protocol is very fast, no separation step is required to remove the excess labeled antibodies. The inhibitory effect of the biological sample on luminescent reaction is determined by adding NADH to the assay tubes. The working range of this assay is 3 to 300 Ul/l, with a sensitivity of detection of 0.5 Ul/l. Recovery, linearity, within and between assay precision were evaluated and appeared to be satisfactory. The authors have observed a good correlation between results obtained with our method and with radioimmunoassay.

Female↗
PubMed1989

Characterization of a plasmid coding for resistance to broad-spectrum cephalosporins in Salmonella typhimurium.

Five strains of Salmonella typhimurium isolated from faeces of infants hospitalized at Trousseau Hospital in Paris have been found to be resistant to third-generation cephalosporins. This resistance is by a transferable plasmid-mediated mechanism. The beta-lactamase gene which encodes for an enzyme similar to SHV-2 has been cloned into plasmid pBR322 and expressed in Escherichia coli RR1.

Cefotaxime↗
PubMed1988

A 17-kDa protein induced in rat myometrium by hormonal treatment.

A 17-kDa protein mainly recovered in the myometrium fraction sedimenting at 10,000 g has been induced by estradiol treatment in spayed rats. Tamoxifen completely inhibited the effect of estradiol, and progesterone decreased it by 50%. The protein was scarce in unspayed animals and the level after estradiol treatment remained much lower than in spayed rats. Oral doses of the potent antiprogesterone RU 38486 increased the level of the 17-kDa protein in unspayed animals. It was not present in immature or gestating rats or in the endometrium or myometrial cell cytosol of any of the rats. Following electrophoretic purification, it bound calmodulin, but was not phosphorylated by cAMP protein kinases.

Animals↗
PubMed1988

Correlations between free plasma estradiol and estrogens determined by bioluminescence in saliva, plasma, and urine during spontaneous and FSH stimulated cycles in women.

Estrone and estradiol (E1 + E2) concentrations in saliva were compared with four other parameters of estrogen status in five normal ovulatory women and ten FSH stimulated women selected for an in vitro fertilization program. E1 + E2 in saliva, plasma, and urine were assessed by a rapid, specific and sensitive enzymatic assay using bioluminescence. The free fraction of plasma estradiol was determined by equilibrium dialysis and total plasma estradiol by conventional radioimmunoassay. The pattern of E1 + E2 variation in saliva was similar to that of free plasma estradiol and the two parameters were correlated in both spontaneous and stimulated cycles. However, the lower correlation coefficient (r = 0.52, P less than 0.001) in spontaneous cycles compared with the high (r = 0.96, P less than 0.001) in the stimulated cycles shows that salivary E1 + E2 could be representative of plasma free estradiol in stimulated cycles but not in normal cycles. The free fraction of plasma estradiol reproduced the variation of total plasma estradiol in spontaneous as well as in FSH stimulated cycles and both parameters were strongly correlated (r = 0.91, P less than 0.001 and r = 0.90, P less than 0.001), respectively. The data show that salivary E1 + E2 concentrations are highly representative of the free fraction of E2 in plasma and at a lesser extend (r = 0.72, P less than 0.001) of total plasma E2 in FSH stimulated cycles.

Adult↗
PubMed1988

Follow-up of subjects with isolated and persistent anti-core (anti-p24 or anti-p17) antibodies to HIV.

Systematic screening of blood donations by enzyme-linked immunosorbent assay (ELISA) for HIV antibodies carries a false-positive rate: the sera involved react in Western blot to core antigens (p24 or p17) but reactivity to envelope is absent. We studied 22 subjects with persistent and isolated anti-core reactivities; 75 HIV seropositive patients were controls. The epidemiological data and the follow-up and biological tests performed in these two populations argue that donors with persistent and isolated anti-core antibodies are not seroconverting for HIV. We conclude: (1) that verification of all anti-HIV ELISA-positive sera by Western blot is essential and that the presence of at least once anti-envelope (gp120 or gp41) antibody is indispensable for the diagnosis of HIV infection; (2) that the solitary anti-p24 or anti-p17 bands observed on Western blot are false-positive. There is no evidence that donors with such reactivities are HIV-infected.

Adult↗