Assessment of laboratory and field assays of sunlight-induced killing of mosquito larvae by photosensitizers

The effectiveness of light-induced killing of mosquito larvae in the presence of photosensitizers was studied with larvae of Aedes aegypti (L.), Anopheles stephensi (Liston), and Culex quinquefasciatus Say grown in the laboratory and of Cx. quinquefasciatus grown under field conditions. Tested photosensitizers included xanthene, chlorin, and porphyrin derivatives. All the larvae were treated at the fourth instar. Preliminary laboratory experiments showed a light-induced lethal effect of Rose Bengal (RB) on three species of mosquito larvae. Compared with other photosensitizers, RB seemed to be more efficient at even lower concentration than chlorin (e6) and chlorophyllin on Ae. aegypti larvae. Among the four porphyrin derivatives, i.e., chloroquinoline tetraphenyl propioamidoporphine, tetraphenyl porphine tetrasulfonate, hematoporphyrin (HP), and tetraphenylporphinepropionic acid porphine, HP was the only effective photosensitizer on Ae. aegypti larvae. The best conditions for field tests using RB were conducted on Cx. quinquefasciatus in Bobo-Dioulasso, Burkina Faso. The mortality induced by RB varied from 80 to 96% obtained with unfiltered cesspit water to 0.4 to 6.7% in cesspits with a heavy load of organic materials, thus providing the basis for further developments of this technique under field conditions.     

Virus-inhibiting surgical glove to reduce the risk of infection by enveloped viruses

Needle puncture and other accidents that occur during surgery and other procedures may lead to viral infections of medical personnel, notably by hepatitis C (HCV) and human immunodeficiency virus (HIV), now that hepatitis B can be prevented by vaccination. A new surgical glove called G-VIR, which contains a disinfecting agent for enveloped viruses, has been developed. Herpes simplex type 1 (HSV) was used as a standard enveloped virus in both in vitro and in vivo tests of the virucidal capacity of the glove. Bovine viral diarrhea virus (BVDV) and feline immunodeficiency virus (FIV) were used as models for HCV and HIV, respectively. For in vitro study, a contaminated needle was passed through a glove and residual virus was titrated; for in vivo studies, animals were stuck with a contaminated needle through a glove. Despite variation in virus enumeration inherent in the puncture technique, statistical evaluation showed that infection was reproducibly and substantially reduced by passage through the virucidal layer. For BVDV, the amount of virus passing through the virucidal glove was reduced in 82% of pairwise comparisons with control gloves that lacked the virucidal agent; when plaque counts were adjusted to a common dilution, the median count for the virucidal glove was on the average reduced >10-fold. In experiments in which the proportion of wells infected with FIV was measured, the ratio of TCID(50) values (control glove to G-VIR) was >15, and probably much higher. For HSV, the amount of virus passing through the virucidal glove was reduced in 81% of comparisons with control gloves; the median of adjusted plaque counts was reduced on the average approximately eightfold or ninefold. In vivo tests with FIV and HSV in cats and mice, respectively, found smaller percentage reductions in infection than the in vitro tests but confirmed the virucidal effect of the gloves.     

Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone

To study the epidemiology of Pseudomonas aeruginosa colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental P. aeruginosa isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with P. aeruginosa, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal P. aeruginosa colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD(r)). The MDR strain was found at a higher frequency in sputum samples than the SSD(r) strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (oprI, oprL, and oprD) and the pyoverdine type revealed that all predominant strains except the SSD(r) strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.     

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.     

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.