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Biomedical subjects

M Denis

Publications and source records attributed to M Denis.

At least 55 records · Page 3Linked to original sources
PubMed1997

Development of a semiquantitative PCR assay using internal standard and colorimetric detection on microwell plate for pseudorabies virus.

We have developed a semiquantitative PCR assay on microtitre plates for quantitation of pseudorabies virus (PRV). The test is based on co-amplification with an internal control (IC) of the target viral DNA, followed by hybridization of the biotin-amplified products on a capture probe covalently immobilized to a Covalink-NH MicroWells plate and then visualization with colorimetric enzymatic reactions. PCR was performed in the presence of uracil-N-glycolsylase (UNG) with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. Our colorimetric test had a 3.5 log dynamic range with a detection level of 30 DNA copies per PCR reaction. A standard curve for quantitation of pseudorabies virus was established from co-amplification of 10 to 10(5) PRV molecules with 1000 IC molecules. Ratios of viral optical density/IC optical density were plotted against the number of PRV DNA target molecules in the PCR amplification. Integration of 96-well formats and automation using robots at different steps of the test ensured a good repeatability. Calibration of the quantitative test using samples from experimentally-infected pigs is in progress.

Automation↗
PubMed1997

Development of a RT-PCR test coupled with a microplate colorimetric assay for the detection of a swine Arterivirus (PRRSV) in boar semen.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) through boar semen has been demonstrated, stressing the need for a reliable semen PRRSV detection test. A diagnostic assay was developed based on amplification of the PRRSV RNA by reverse transcription and polymerase chain reaction (RT-PCR) followed by detection of the amplification products by hybridization and colorimetric assay in microwell plates. A highly reproducible and efficient method of viral RNA isolation from semen samples was set up. A combined RT-PCR procedure was performed, incorporating the use of uracil-N-glycosylase (UNG) in combination with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. An RNA internal control was added during the RNA isolation procedure to detect false negative results. The colorimetric detection in microwell plates of amplification products from either PRRSV or IC RNA gave specific and objective results and was automated. A cut-off value of 1000 RNA copies or 10 TCID50 of PRRSV per ml of semen samples could be detected with this assay. Semen samples collected from experimentally-infected boars were tested with this assay and showed PRRSV excretion early after infection and for an extended period.

Animals↗
PubMed1997

The terminal oxidase in the marine bacterium Pseudomonas nautica 617.

The molecular properties of a novel membrane quinol oxidase from the marine bacterium Pseudomonas nautica 617 are presented. The protein contains 2b hemes/mole which may be distinguished by EPR spectroscopy but not by optical spectroscopy and electrochemistry. Respiration, though being cyanide insensitive, is not inhibited by carbon monoxide and oxygen reduction is carried out only half-way with production of hydrogen peroxide. The terminal oxidase represents, therefore, a unique example in the large family of terminal oxidases known up to date.

Hydrogen Peroxide↗
PubMed1997

Association of nitric oxide production by kidney proximal tubular cells in response to lipopolysaccharide and cytokines with cellular damage.

Recent findings suggest that nitric oxide (NO) is an important biologic mediator which exerts a wide variety of effects on numerous physiological and pathophysiological processes. L-Arginine is oxidized to L-citrulline with concomitant NO production; as a result, nitrate and nitrite accumulates. This study was conducted to determine the potential NO production by proximal tubular cells (PTC) in response to bacterial lipopolysac-charides (LPS) and cytokines and to evaluate the cytotoxic effect associated with NO release. After a 7-day stimulation with LPS (100 micrograms/ml), interleukin-1 beta (IL-1 beta) (10 ng/ml), and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml), the nitrate and nitrite levels were determined by a spectrophotometric method based on the Griess reaction. Moreover, alpha-methylglucopyranoside phosphate and lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay served as indicators of sodium-dependent hexose transport integrity and cell death, respectively. IL-1 beta and TNF-alpha used alone or together or combined with LPS led to a significant generation of NO by PTC. Our results also demonstrate that NO induced by LPS and cytokines could inhibit sodium-dependent transport and could induce PTC damage.

Animals↗
PubMed1996

Functional anatomy of spatial mental imagery generated from verbal instructions.

Positron emission tomography (PET) was used to monitor regional cerebral blood flow variations while subjects were constructing mental images of objects made of three-dimensional cube assemblies from auditorily presented instructions. This spatial mental imagery task was contrasted with both passive listening (LIST) of phonetically matched nonspatial word lists and a silent rest (REST) condition. All three tasks were performed in total darkness. Mental construction (CONS) specifically activated a bilateral occipitoparietal-frontal network, including the superior occipital cortex, the inferior parietal cortex, and the premotor cortex. The right inferior temporal cortex also was activated specifically during this condition, and no activation of the primary visual areas was observed. Bilateral superior and middle temporal cortex activations were common to CONS and LIST tasks when both were compared with the REST condition. These results provide evidence that the so-called dorsal route known to process visuospatial features can be recruited by auditory verbal stimuli. They also confirm previous reports indicating that some mental imagery tasks may not involve any significant participation of early visual areas.

Acoustic Stimulation↗
PubMed1996

The role of glycoproteins gC, gE, gI, and gG in the induction of cell-mediated immune responses to bovine herpesvirus 1.

Mutant viruses with deletions in genes encoding non-essential glycoproteins are considered as promising bovine herpesvirus 1 (BHV1) vaccine candidates. The present study compared the influence of various gene deletions (gC, gE, gI, gG) on the induction of cell-mediated immune responses against the virus. The highest BHV1 specific lymphoproliferative response was observed in the group of calves inoculated with the gC- mutant. However, in all groups of inoculated calves, limiting dilution analysis showed marked individual variability in the number of BHV1 specific T lymphocytes that were stimulated. The same animals were then challenged with wild-type BHV1. In these animals, limiting dilution analysis did not reveal gE, gI nor gG as a major T lymphocyte antigen. However, further analysis suggested the T cell antigenicity of gE in a low number of BHV1 hyperimmunized calves. Stimulation of MHC unrestricted cytotoxicity was also evaluated after inoculation with the various deletion mutants. Cytotoxicity in gC- inoculated calves was as high as in BHV1 inoculated calves. In conclusion, among the BHV1 deletion mutants that were tested, the gC- mutant stimulated the best cell-mediated immune responses.

Animals↗
PubMed1995

Antigen-induced alveolitis: cytokine production in a mouse model.

Mice of the C57BL/6 strain were injected intraperitoneally with 10(8) sheep red blood cells (SRBC), then instilled intratracheally with 10(8) SRBC two to three weeks later. After a single intratracheal exposure, a significant cellular infiltrate occurred, composed mostly of macrophages and lymphocytes. Lymphocytes proliferated significantly in response to SRBC antigen in vitro and released interleukin-2 (IL-2). Alveolar macrophages isolated from mice challenged with SRBC released higher levels of IL-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) upon in vitro lipopolysaccharide (LPS) stimulation compared to unprimed, challenged mice or mice receiving intraperitoneal SRBC alone. Lymphocytes from primed mice challenged three times with SRBC proliferated significantly less in response to the antigen than mice receiving one SRBC challenge and released significant levels of interferon gamma (IFN-gamma). Bronchoalveolar macrophages isolated from primed mice given three SRBC challenges released slightly higher levels of TNF-alpha and IL-6 in response to LPS than those from unprimed mice. After the third instillation, levels of hydroxyproline in the lungs increased significantly, indicative of a fibrotic reaction. Neutralization of IL-1 (by anti-mouse type 1 IL-1 receptor) or TNF-alpha resulted in the partial abrogation of the initial neutrophil influx, with some effect on the subsequent lymphocyte and macrophage influx. Blocking IL-1 or IL-2 but not TNF-alpha also resulted in a significant decrease in lung hydroxyproline increase, as well as lung granulomatous response and fibrosis. Overall, these results suggest that lymphoproliferation in the lungs in response to an antigen can result in fibrosis, mediated in part by IL-2 and IL-1.

Alveolitis, Extrinsic Allergic↗
PubMed1995

Antioxidant therapy partially blocks immune-induced lung fibrosis.

A mouse model of hypersensitivity pneumonitis was generated by challenge with a thermophilic actinomycete. Oxygen radical scavengers were administered to challenged mice: vitamin E at 1000 units daily, polyethylene glycol-superoxide dismutase (SOD) at 500 units daily, polyethylene glycol-catalase at 10,000 units daily, 1,3,dimethyl-2-thiourea (DMTU) at 2 mg daily, and the biomimetic SOD, copper(II) [diisopropyl salicylate]2 (CuDIPS) at 1 mg daily. At three weeks after actinomycete challenge, a 10-fold increase in bronchoalveolar (BAL) cell number was observed. Treatments with catalase or DMTU were without effect on the BAL cell number in challenged mice. However, infusion of vitamin E was associated with an increased BAL cell influx (15-fold increase at two and three weeks). Similarly, treatment with PEG-SOD and CuDIPS resulted in an increase in cell number at two and three weeks. PEG-SOD or CuDIPS treatment resulted in a strong neutrophilia, whereas control challenged mice had a cellular influx mostly of macrophages and lymphocytes. Vitamin E treatment of challenged mice led to an increased T lymphocyte recruitment at two and three weeks. In vitro studies showed that actinomycete challenge was associated with an enhancement of alveolar macrophage O2- release, which was blocked by PEG-SOD, vitamin E, or DSC treatment but was unaffected by catalase or DMTU treatment. In control challenged mice, there was a 25-fold increase in the BAL albumin concentration at two weeks. PEG-SOD, vitamin E, or CuDIPS treatment all decreased the albumin concentration; the three modulators also diminished lung fibrosis at two or three weeks, as seen by a decrease in lung hydroxyproline and collagen synthesis by lung fibroblasts. Examination of sections from lungs of challenged animals showed evidence of cellular infiltrates around the bronchi and the blood vessels. Challenged mice given continuous infusions of vitamin E, SOD, or CuDIPS had lung histological scores that were significantly lower than control challenged mice or challenged mice treated with catalase or DMTU. Thus, therapies based on O2- scavenging or treatment with a general antioxidant such as vitamin E may hold some promise in the treatment of hypersensitivity pneumonitis.

Animals↗
PubMed1995

Nitric oxide and interleukin-8 as inflammatory components of cystic fibrosis.

We examined the production of reactive nitrogen intermediates in the tracheo-bronchial tree of patients with cystic fibrosis (CF). Examination of the soluble phase of sputa from 17 CF patients revealed the presence of high levels of NO2-/NO3- assayed by the Greiss reaction. We also examined the presence of the chemotactic cytokine interleukin-8 (IL-8) in these samples so as to assess another important inflammatory marker; high levels of IL-8 were present in the sputa of cystic fibrosis subjects. The elevated nitrite was not produced by the presence of Pseudomonas bacteria in the sputa, inasmuch as bacteria in culture released undetectable amounts of nitrite in culture media. Neutrophils from the sputa of CF patients with disease exacerbation released higher amounts of nitrite and IL-8. Neutrophils from the sputa were also shown to spontaneously release substantial amounts of nitrite in the supernatants, and this release was partly blocked by the antagonist NG-mono-methyl-L-arginine (L-NMMA). Blood neutrophils were shown to release nitrite only in response to challenge with CF-associated strains of Pseudomonas, and not exposure to cytokines. There was no significant differences in nitrite release between normal and CF blood polymorphonuclear leucocytes (PMNs). A study of upper airway epithelial cell lines showed that these cells released low amounts of nitrite after infection with CF-associated strains of Pseudomonas but not after cytokine exposure. Epithelial cell lines with CF or normal phenotypes were shown to release similar quantities of nitrite, upon stimulation with Pseudomonas. These data demonstrate that elevated levels of reactive nitrogen intermediates and IL-8 are produced in the tracheo-bronchial tree of subjects with CF. Levels of IL-8 and nitrite were higher in the secretions of CF subjects with disease exacerbation. The involvement of nitric oxide and other reactive nitrogen intermediates produced by neutrophils and other cells in the tissue damaging processes in CF deserves further investigation.

Adolescent↗
PubMed1995

The involvement of nitric oxide in a mouse model of adult respiratory distress syndrome.

The release of free radicals and pro-inflammatory cytokines such as nitric oxide (NO) and tumor necrosis factor alpha (TNF alpha) is commonly observed in adult respiratory distress syndrome (ARDS) following infection or exposure to microbial products. The aim of this study was to scrutinize the involvement of NO in ARDS in a mouse model determined by the sequential exposure to lipopolysaccharide (LPS) and formyl-norleucyl-phenylalanine (FNLP). Nitrite measurements in bronchoalveolar lavage fluids (BALF) and sera demonstrated that exposure to microbial products elicits large amounts of NO in LPS/FNLP-challenged mice. This release was significantly inhibited by infusion with the inducible NO synthase antagonist, aminoguanidine (AG). Our results show that LPS/FNLP exposure induces lung damage as demonstrated by protein and lactate dehydrogenase (LDH) increases in BALF. Liver damage was also detected in LPS/FNLP-challenged mice with increases in serum ornithine-carbamoyltransferase (OCT) levels. LPS/FNLP infusion led to elevated levels of the cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) in the sera. LPS/FNLP also led to neutrophil adhesion in the lung vasculature, as seen by increased levels of myeloperoxydase. Interestingly, inhibition of NO release in challenged mice led to an important increase in markers of tissue damage in the lungs and livers, but a decrease in neutrophil recruitment. Infusion of AG in LPS/FNLP-challenged mice led to a much increased level of sera TNF alpha. These data suggest that after exposure to microbial products, NO generated as a result of activation of the inducible NO synthase blocks the full expression of tissue damage in the lungs.

Animals↗
PubMed1995

Mental scanning of visual images generated from verbal descriptions: towards a model of image accuracy.

Recent mental scanning experiments have shown that subjects are not only able to construct mental images from verbal descriptions, but that these images have structural properties similar to those of images derived from perception. In addition, the specific sequencing of a description can affect the internal structure of images of described objects, in particular their metric properties. Discontinuous descriptions require additional exposure to achieve the structural coherence of images constructed from continuous descriptions. Thus, the capacity of images to reflect accurately the objects they refer to is not an all-or-nothing property, but rather results from stepwise elaboration. This study describes a quantitative model designed to account for the gradual process of image elaboration and the progressive increase in image accuracy. The model posits that the location of a landmark mentioned in a description is not represented as a sharp point in the mental image, but is instead associated with a region around this point. Learning the description essentially consists of progressively narrowing each 'region of uncertainty' associated with a landmark to its exact location. Additional experimental data were collected to provide a more finegrained understanding of image elaboration. Computations of the regions of uncertainty associated with each landmark were used to develop a computer program simulating the whole mental scanning protocol, which provided support for our account of image accuracy.

Adult↗
PubMed1995

Proinflammatory cytokines in hypersensitivity pneumonitis.

We examined the synthesis and release of the proinflammatory cytokines macrophage inflammatory protein-1 alpha (MIP-1 alpha) and interleukin-8 (IL-8) in the context of subjects with hypersensitivity pneumonitis (HP). Subjects with acute HP were found to have alveolar macrophages that released high levels of MIP-1 alpha and IL-8, whereas control subjects had cells that released low levels of these factors. Bronchoalveolar lavage fluid from HP subjects contained low but significant levels of MIP-1 alpha and IL-8. Immunohistochemistry revealed that macrophages from acute HP subjects expressed MIP-1 alpha and IL-8 at high levels. Treatment of acute HP subjects by contact avoidance or by corticosteroids reduced the synthesis of both cytokines. Supernatants of alveolar macrophages from subjects with HP were shown to attract activated CD8+ T lymphocytes, and this activity was significantly inhibited by anti-MIP-1 alpha. MIP-1 alpha and IL-8 may be important inflammatory cytokines in the development of HP via their capacity to attract inflammatory cells (activated CD8+ T lymphocytes and neutrophils, respectively) into the airways of subjects with HP.

Acute Disease↗
PubMed1995

Environmental safety assessment of vaccines derived from biotechnology.

Products derived from biotechnology are generally safer than previous products obtained by conventional, more empirical methods. Given the great variety of biotechnology-derived products and use situations, risk assessment procedures must be based on a case-by-case analysis. It is therefore difficult to propose a generic, well-defined risk assessment procedure. In the case of vectored vaccines, two main risks must clearly be assessed: the possible risk of tissue or species tropism modification, with special regard to non-target species, the risk of recombination with wild counterparts of the vector. This paper begins with a classification and description of the main biotechnology-derived products to be used in veterinary medicine, including deoxyribonucleic acid (DNA) vaccines, and then attempts to define general rules for the risk assessment of each category of products.

Animals↗
PubMed1995

[Still disease in adults revealed by a digestive manifestation].

Adult Still's disease is characterized by typical spiking fever, oligopolyarthritis, neutrophilic leukocytosis and involvement of various organs. We report a case which illustrated typical digestive features of Still's disease as dysphagia, peritonitis and manifests the hitherto unreported complication of gastric ulcerations. Treatment with prednisone was started in order to control arthritis, resulting in improvement of both gastric ulcerations (partially resistant to omeprazole treatment) and arthralgia. After seven years of follow-up, the patient remains clinically and biochemically stable with steroid and methotrexate treatment.

Anti-Inflammatory Agents, Non-Steroidal↗