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Biomedical subjects

J C Nicolas

Publications and source records attributed to J C Nicolas.

At least 199 records · Page 11Linked to original sources
PubMedDate not supplied

Applications of low-light imaging to life sciences.

Photon imaging is a new technique for the quantitative analysis of bioluminescence and chemiluminescence and can be performed both at the macro and micro levels. The high sensitivity and spatial resolution of photon-counting cameras have resulted in the development of new applications in the life sciences. At the macro level, imaging is a valuable tool for the rapid identification of biological samples emitting long-lasting glows in assays using microtitre plates or filter formats (immunoassays, DNA probes, phagocytosis, gene expression, metabolite and drug analysis) and also for in vivo studies of promoter activity. At the micro level, low-light imaging can be used for analysing multiple analytes on micro sensors) and for advanced cell analysis (immunocytology, in situ hybridization, identification of cells or tissues expressing the luciferase gene, intracellular or intercellular protein traffic, metabolite analysis and imaging of Ca2+ flux and phosphorylation reactions). Two-dimensional photon-counting instrumentation is a versatile and powerful research tool for imaging and is complementary to conventional luminometers. The main applications to the life sciences involve many types of luminescence assays and can be performed on multiple samples in standard and non-standard formats. Photon-counting coupled to imaging is very helpful in selecting microorganisms or cells expressing bioluminescent genes. Measurements can be made in vitro and in vivo with a sensitivity comparable to that of phototube luminometers.

Animals↗
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Imaging reactive oxygen species in asthma.

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.

Acridines↗
PubMedDate not supplied

Stable luciferase transfected cells for studying steroid receptor biological activity.

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.

Animals↗
PubMedDate not supplied

Chimeric receptors as a tool for luminescent measurement of biological activities of steroid hormones.

Some applications of chimeric cellular models are presented to study the biological activities of steroid hormones. We have used several chimeric constructs encoding the DNA binding domain of Gal4 yeast protein fused to the hormone binding domain of various steroid receptors (MR, PR, GR and ER). Interactions of these chimeric receptors with a 17-mer DNA sequence, specific for Gal-4, control expression of the firefly luciferase as a reporter gene. Stable transfected cell lines expressing the firefly luciferase under the control of different steroids were established and an efficient and easy sub-cloning was allowed with the help of an imaging system using a single-photon-counting camera. In the cell lines obtained, the bioluminescent response can be easily measured and thus used to measure specific biological activities of steroid agonists or antagonists. We observed that the responses are effector-concentration-dependent and their biological activities will be compared to those of native receptors.

Animals↗
PubMedDate not supplied

Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands.

To characterize the specificity of synthetic compounds for nuclear receptors, we established stable cell lines expressing the luciferase gene and different wild-type or chimaeric receptors. MCF-7 cells, which express the oestrogen receptor alpha (ER alpha), and HeLa cells, which do not express the oestrogen receptor, were transfected with a plasmid containing the luciferase gene downstream from a minimum promoter (beta-globin) and an oestrogen-responsive element, generating the MELN and the HELN cell lines, respectively. MELN cells enabled the detection of compounds that bind to the ER alpha or interfere with its pathway. HELN cells were used to establish stable transfectants expressing different nuclear receptors containing the DNA-binding domain of the oestrogen receptors. We thus established ER alpha or ER beta reporter cell lines by transfecting ER alpha or ER beta expression plasmids, and also retinoic acid receptor alpha, beta or gamma reporter cell lines by transfecting the chimaeric RAR gene, in which the DNA-binding domain was replaced by the ER alpha DNA-binding domain.

Cell Line↗
PubMedDate not supplied

DNA hybridization for detection of human cytomegalovirus in bronchoalveolar lavage and pharynx biopsy.

A simplified hybridization procedure was used for detection of cytomegalovirus (CMV) in human specimens. The probe was a 32P-labelled cloned DNA fragment of CMV strain AD169. This probe did not hybridize to DNA from uninfected cells or other herpesviruses (herpes simplex virus, Epstein-Barr virus). Specific hybridization was obtained with unselected bronchoalveolar lavage specimens, but the sensitivity of the test (33%) was lower than that of culture (80%) and immunofluorescence (60%) assays which are routinely performed in our laboratory. The detection procedure was also carried out with pharynx biopsy specimens which had been kept frozen at -70 degrees C. CMV DNA was detected in 14% of tumour specimens and only in 1.7% of control specimens (p less than 0.05). The indications of DNA hybridization for CMV diagnosis are discussed.

Bronchoalveolar Lavage Fluid↗
PubMedDate not supplied

Enzyme-linked immunosorbent assay for antibodies to ZEBRA, an Epstein-Barr trans-activator.

Patients with AIDS and AIDS-related complex often show symptoms of Epstein-Barr virus (EBV) reactivation. Several EBV-encoded trans-acting factors activate the EBV lytic cycle, and one, called ZEBRA (BamHI Z EBV replication activator), controls the switch of EBV from a latent to a productive cycle. We describe here a simple ELISA test using a bacterially expressed ZEBRA protein as antigen. Utilizing this technique, we evaluated the humoral response to ZEBRA antigen in 38 patients with nasopharyngeal carcinoma and 134 subjects with an asymptomatic HIV 1 infection. The control group consisted of 40 healthy adult blood donors.

Antibodies, Viral↗
PubMedDate not supplied

BK virus (BKV) detection in urine specimens of immunocompromised patients: comparison between the DNA-DNA hybridization assay, the immunofluorescence test and the dot enzyme immunoassay.

Three methods used for the detection of BK virus in urine specimens, the indirect immunofluorescence test, the dot enzyme immunoassay and the DNA-DNA hybridization assay, were compared by testing specimens from 49 immunocompromised patients. All three assays were effective in detecting BK virus. The technical advantage of each of them was discussed. The immunofluorescence test was found to be the simplest one to perform; the DNA-DNA hybridization assay displayed exquisite sensitivity; and the easy reading of the dot enzyme immunoassay did not require the specialized training inherent to immunofluorescence assays. The dot enzyme immunoassay might therefore be the most practical method for screening urine specimens of immunocompromised patients, especially when the sediment is poor in cells. Conversely, the indirect immunofluorescence test might be the method of choice for checking patients with haemorragic cystitis whose urine samples usually contain large amounts of cells.

BK Virus↗
PubMedDate not supplied

Detection of human cytomegalovirus DNA in liver biopsies from patients with cytomegalovirus-related liver disease.

Infection with human cytomegalovirus (HCMV) has been associated with severe diseases in immunologically impaired patients. Cytomegalovirus hepatitis has been frequently described in this population, but this diagnosis is still difficult. Molecular hybridization with the V EcoRI restriction fragment of human cytomegalovirus strain AD 169 has been tested upon DNA extracted from liver samples to assess the usefulness of this technique for cytomegalovirus hepatitis diagnosis. This probe was shown by the Southern technique not to hybridize with DNA extracted from cells infected with other herpesviruses or with DNA of non-infected normal liver. The sensitivity was estimated to be 2 x 10(5) genomes. Twenty-five renal transplant recipients under immunosuppressive therapy and three patients having the acquired immunodeficiency syndrome were studied. In 9 out of 10 renal transplant recipients with normal liver, previous exposure to cytomegalovirus, as defined by serological tests, was not sufficient to allow positive detection by the probe. Out of 11 patients with abnormal liver, cytomegalovirus DNA sequences were shown in 5. In 2 patients with histological evidence of cytomegalovirus hepatitis, a very strong signal showed the presence of viral genomes. These results show that the Southern technique with the V EcoRI probe can be useful for the diagnosis of HCMV hepatitis and might be proposed for the detection of this viral genome in human tissues.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Disorders linked to insufficient androgen action in male children.

Virilization of the external genitalia in the male fetus requires testosterone and dihydrotestosterone (DHT), which is formed from testosterone by the action of the enzyme, 5alpha-reductase type 2 (5alphaR-2). Mediation of the effects of both testosterone and DHT requires a functional androgen receptor (AR) located in the cytoplasmic compartment of target cells. DHT (or testosterone) binding induces a conformational change which facilitates AR nuclear transport, phosphorylation and dimerization, ultimately regulating of the rate of transcription of androgen-dependent genes. Any event which impairs DHT formation (mutation within the 5alphaR-2 gene or 5alphaR-2 inhibitors) or normal function of the AR (mutation in the AR gene, antiandrogens) may result in insufficient androgen action in the male fetus and in subsequent undervirilization in the newborn. Hypospadias may be due to a defect in androgen action due to mutation of the 5alphaR-2 or of the AR gene. Mutation of unidentified genes is likely to underlie this displacement of the urethral meatus from the tip to the ventral side of the phallus. An aetiological role for environmental chemical products has been postulated, since ethnic as well as geographical differences in the incidence of hypospadias have been noted. Increasing evidence has been gathered indicating that widely used industrial and agricultural chemicals have deleterious effects on normal male sexual differentiation. Cryptorchidism and micropenis may represent an intersex phenotype, even if they are isolated. Aetiological factors include 5alphaR-2 gene mutation, AR gene mutation or environmental hormonal disruptors. In conclusion, several phenotypes have been attributed to insufficient androgen action during fetal life. Whereas mutations in the 5alphaR-2 gene and AR gene are natural, attention should be focused on environmental endocrine disruptors that are able to mimic steroid 5alpha-reductase deficiency or partial androgen insensitivity syndrome.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMedDate not supplied

[Determination of plasma and urinary estrogens by use of a new enzyme method].

The transhydrogenase function of the oestradiol dehydrogenase of human placenta is used in such a condition as the transhydrogenase activity is directly related to oestrone and oestradiol concentration. The method is highly specific. Reacting oestrone with hydrazin before the assay allows specific determination of oestradiol. The limit of sensitivity is 10 picograms (precision 10%) in plasma and 1 microgram in urine (precision 10%). The determination is done directly on hydrolyzed urine without extraction and is rapid and highly specific. The method seems to be competitive for oestrogen radioimmuno assay in plasma and offers great advantages towards conventional chemical methods in urine. Repetitive profiles of urinary oestrogen excretion can be obtained easely.

17-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

[Importance of measurement of plasma viral load for the therapeutic follow-up of children infected with HIV-1. Study of its evolution according to modifications of treatment in 55 children].

Since 1987 we started to treat all vertically HIV-1 infected newborns by an antiretroviral therapy as soon as the diagnosis was confirmed. From the study of 55 HIV-1 infected children followed at Armand Trousseau Hospital (Paris, France) we retrospectively evaluated the use of the plasma viral load as a marker of the treatment efficiency. This parameter and CD4 counts were measured the day of any modification in antiviral therapy (Day 0), 1 and 3 months later (M1 and M3) and every 3 months as long as possible. In the 3 groups of treatment modifications a decrease in plasma viral load was observed. A statistically significant increase in CD4 counts was only observed in the group treated with the association of 2 nucleoside analogues and 1 proteinase inhibitor. This group demonstrated the most important decrease in plasma viral load (> 0.7 log 10 Eq Cop/ml). This leads us to propose to the "ANRS" a protocol study for the treatment of all vertically HIV-1 infected children by the association of 3 nucleoside analogues and 1 proteinase inhibitor as early as 8 weeks post natal when the HIV infection is confirmed.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Epstein-Barr virus].

Epstein-Barr Virus (EBV) is the prototype member of the human herpesvirus subfamily Gammaherpesviridae. EBV establishes a latent infection in lymphoïd cell and replicates in epithelial cells. Infection of individuals with EBV is widespread in all human populations, as shown by the high proportion of individual with specific antibodies in their serum. EBV is the etiological agent of the infectious mononucleosis and has been implicated in the pathogenesis of an increasing number of human malignancies, the best characterized being endemic Burkitt's lymphomas, nasopharyngeal carcinomas (NPC), and polyclonal lymphomas in immunocompromised patients. EBV infection in vivo is a complex mixture of latent, reactivated, transforming, or replicative type of infection. EBV infection is characterized by sporadic viral excretion in the oropharynx and persistent latent infection in the bone marrow and peripheral blood lymphocytes. EBV infection includes an intense immune response. During primary infection, the humoral response is primarily directed toward antigens of the lytic cycle, membrane antigen complex, early antigen (EA), viral capsid antigen (VCA) while the antibodies response to EBNAs is delayed. The cell mediated response controls the proliferation of EBV-infected lymphocytes through two classes of cytotoxic cells, namely, natural killer and T8 cytotoxic cells. Laboratory diagnosis of EBV infection is recently based upon molecular biology techniques which provides a useful tool for direct identification of EBV and may allow to better understand the role of the virus in the pathogenesis of EBV associated disorders.

Antibody Formation↗
PubMedDate not supplied

[Value of the fractionation of serum immunoglobulins for the diagnosis of rubella infection in pregnant women. Critical study].

The authors present the results of fractionation by ultracentrifugation in the identification of rubella antibodies in 20 vaccinated children and 389 pregnant women suspected of having rubella. Testing for specific IgM was constantly positive when an exanthematous disease was seen in association with a significant increase an antibody level as measured by standard techniques, making questionable the reed of fractionation under such circumstances. By contrast, however, the shortest delay in collection of the first sample of serum renders fractionation essential. Alone, it made possible the diagnostic of rubella in patients in whom the first serum, although collected within 3 to 5 days after the rash, had the same antibody level as a second serum collected later. Whilst fractionisation requested for contact only without rash did not reveal specific IgM, positive results were obtained rarely in sera with a high antibody level on routine examination. The very uncertain significance of these last results, the frequency of cases of recent rubella with very modest antibody levels and the occasional finding of duodtful results, render the authors highly sceptical as to the value of fractionisation in the presence of a high serum level on routine examination in a pregnant woman.

Female↗