Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.     

Secretin and VIP-stimulated adenylate cyclase from rat heart

The exact positions of microelectrodes used to measure thePO₂ in the cerebral cortex of the rat were determined by staining the tissue with Alcian Blue. The measurement sites were subsequently located under a light microscope and correlated with the capillary and cellular arrangement of the cortex. The microelectrodes used for thePO₂ measurements were made of gold glass fibers; the Alcian Blue was injected hydrostatically through a micropipette attached to thePO₂ microelectrode. The sites where dye had been deposited were seen under a light microscope as green blue spots about 100 m in diameter. The capillaries were visualized by silver nitrate perfusion. Differences between the localPO₂ values in the neo- and the archeocortex were found. In the neocortex the meanPO₂ was 31 mm Hg, capillary volume 1.6%, capillary surface area 980/mm², capillary length 13.5/mm; whereas in the archeocortex these values where 21 mm Hg, 0.9%, 820/mm² and 9.4/mm respectively. These data indicate a relationship between the microcirculatory transport system and the local oxygen tension and provide further evidence that the meanPO₂ level tends to decrease when moving from the surface into the archeocortex.Supported by the Deutsche ForschungsgemeinschaftReported in part at the 3rd Symposium of ISOTT, Cambridge, GB, 1977; and at the 27th International Congress of Physiological Sciences, Paris, France, 1977     

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.     

A mammary gland adenomyoepithelioma in a C57BL/6 mouse

Mammary gland adenomyoepitheliomas are benign complex mammary gland tumors composed of neoplastic cells of epithelial and myoepithelial origins, described in many species (humans, dogs, cats, rats) and rarely in mice. We report here an adenomyoepithelioma in a C57BL/6 female mouse. Histologically, tubes and cords formed by neoplastic epithelial cells were separated by bundles of neoplastic myoepithelial cells in a clear and partially mucinous matrix. The tumor displayed characteristics of a benign neoplastic proliferation with a compressive growth pattern, and moderate cellular pleomorphism and mitotic index. At immunohistochemistry, the epithelial cells were strongly cytokeratin positive; the myoepithelial cells were weakly cytokeratin positive and strongly smooth muscle actin positive. This is to our knowledge, the first report of a mammary gland adenomyoepithelioma in a C57BL/6 mouse.     

Cell-specific localization of monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain revealed by double immunohistochemical labeling and confocal microscopy

Recent evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. This would imply the presence of specific transporters for lactate on both cell types. We have investigated the immunohistochemical localization of two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Using specific antibodies raised against MCT1 and MCT2, we found strong immunoreactivity for each transporter in glia limitans, ependymocytes and several microvessel-like elements. In addition, small processes distributed throughout the cerebral parenchyma were immunolabeled for monocarboxylate transporters. Double immunofluorescent labeling and confocal microscopy examination of these small processes revealed no co-localization between glial fibrillary acidic protein and monocarboxylate transporters, although many glial fibrillary acidic protein-positive processes were often in close apposition to elements labeled for monocarboxylate transporters. In contrast, several elements expressing the S100beta protein, another astrocytic marker found to be located in distinct parts of the same cell when compared with glial fibrillary acidic protein, were also strongly immunoreactive for MCT1, suggesting expression of this transporter by astrocytes. In contrast, MCT2 was expressed in a small subset of microtubule-associated protein-2-positive elements, indicating a neuronal localization.In conclusion, these observations are consistent with the possibility that lactate, produced and released by astrocytes (via MCT1), could be taken up (via MCT2) and used by neurons as an energy substrate.     

Early Bronchial Hyperresponsiveness Following Injection of Sephadex Beads in the Guinea Pig: Involvement of Platelet Activating Factor and Thromboxane A2

The effects of an intravenous injection (i.v.) of Sephadex beads (20 mg kg^–1) were examined on bronchial responsiveness to ACh (1–200 g kg^–1 i.v.) as well as on cell accumulation in guinea-pig lung. Bronchial hyperreactivity to ACh, measured as increase in pulmonary insufflation pressure (PIP), was observed 3 h following the i.v. injection of Sephadex beads. However, no significant increase in bronchial reactivity to ACh was measured at 6 and 12 h following Sephadex injection. A second later increase in bronchial hyperresponsiveness was observed at 24 h. Bronchoalveolar lavage performed at 3 h following Sephadex treatment showed that there was no significant increase in total or differential cell number. At 6 h and 12 h, a significant increase in total cell counts was observed. At 24 h, a greater than 5-fold increase in cell number was observed and was related to a marked eosinophil, neutrophil and macrophage infiltration. A platelet-activating factor (PAF) antagonist, CV-3988 (10 mg kg^–1 i.v.), and a thromboxane A₂ (TxA₂) antagonist, L655,240 (10 mg kg^–1 i.v.), significantly attenuated the Sephadex-induced bronchial hyperresponsiveness to ACh observed at 3 h. The results show that an i.v. injection of Sephadex beads in guinea pigs can induce an early bronchial hyperresponsiveness to ACh that is mediated by the release of both PAF and TxA₂ and is independent of airway cell infiltration.     

Three highly polymorphic microsatellites at the human myelin oligodendrocyte glycoprotein locus, 100 kb telomeric to HLA-F : Characterization and relation to HLA haplotypes

The MOG locus, located on chromosomal bands 6p21.3-p22 and mapped about 100 kb telomeric to HLA-F, was isolated from cosmid ICRFcl09A2434 and shown to contain three microsatellites. These CA-repeat polymorphic markers were characterized in a sample of 173 healthy unrelated individuals and 84 DNAs from the HLA Workshop reference panel, by a method combining fluorescence labeling of PCR products and use of an automated DNA sequencer. For the three markers, frequencies of heterozygotes are well predicted from allele frequencies by the Hardy—Weinberg rule, which suggests that problems of allele nonamplification are unlikely. Typing of cell lines homozygous in the HLA region allowed unambiguous definition of 81 HLA-MOG haplotypes and showed that several HLA ancestral haplotypes extended to the MOG region. The high degree of polymorphism (59%, 51%, and 81% at the three loci, respectively, and 87% at the haplotype level) makes these new markers informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis, and for studies aimed at precisely delineating the site of crossover in chromosomes in which recombination occurred in the distal part of the HLA class I region.     

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.     

Tomo-anatomie hépatique. Introduction à l'écho-anatomie du foie normal

Résumé Ce travail tente de définir une écho-anatomie du foie normal à partir de neuf plans tomographiques de référence, chacun étant caractérisé par la morphologie de la glande hépatique, par son contenu structural et par son environnement topographique.Les auteurs interprètent l'imagerie échographique directement à partir de l'observation macroscopique de coupes anatomiques correspondantes. Ils indiquent que la corrélation anatomo-échographique est satisfaisante et dégagent quelques indications spécifiques pour chacune des incidences.     

Ultrastructural Changes Associated with Peripheral Neuropathy in HIV/AIDS

Light and electron microscopic studies were performed on neuromuscular biopsy specimens from 12 neurologically affected seropositive patients, 7 with the acquired immune deficiency syndrome (AIDS), 2 with AIDS-related complex, and 3 with no symptoms except for neuropathy. All patients had an axonal injury associated or unassociated with demyelination and peripheral neurogenic atrophy. Capillary lesions were consistently present, which seems to be a new finding. Moreover, tubuloreticular inclusions (TRIs) were found in endomysial (9 of 12 cases) and endoneurial (7 of 12 cases) vessels. Statistical analysis showed that TRIs in more than 20% of endomysial vessels correlated with a survival time shorter than 12 months (P = 0.028). Thus the quantification of TRIs seems to be one of the vital prognostic elements in this patient population.