RESUMO
A technique is described for the injection of live virus into early- and mid-gestation mouse embryos in utero. The procedure is quick, easy, harmless to the embryos, and does not require specialized surgical or microinjection equipment. Since the developing embryo contains most different cell types in a very wide range of differentiation states, the procedure permits a rapid and near complete characterization of the host cell type range in a single experimental system. Under anaesthesia, a simple laparotomy was used to reveal the uterine horns of 9.5 or 12.5 days post-conception(dpc) females. One uterine horn was deflected onto the ventral abdominal surface. Embryos were injected through the uterine wall and the uterine horn replaced into the abdominal cavity. The entire operation could be completed in 10-15 min without distinguishable pain to the mother or adverse effect on the pregnancy. The procedure is presented in sufficient detail to permit its ready adoption in situations where a more complete characterization of host cell type range is sought.
Assuntos
Embrião de Mamíferos/virologia , Vírus Miúdo do Camundongo/fisiologia , Infecções por Parvoviridae/virologia , Útero , Animais , Feminino , Laparotomia , Camundongos , GravidezRESUMO
The cell wall sulfated polysaccharide of the red microalga Porphyridium sp. exhibited impressive antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and -2) both in vitro (cell culture) and in vivo (rats and rabbits). Depending on the concentration, this polysaccharide completely inhibited or slowed down the development of the cytopathic effect in HSV-infected cells, but did not show any cytotoxic effects on vero cells even when a concentration as high as 250 microg/ml was used. There was indirect evidence for a strong interaction between the polysaccharide and HSV and a weak interaction with the cell surface. When tested in vivo, Porphyridium sp. polysaccharide conferred significant and efficient protection against HSV-1 infection: at a concentration as low as 100 microg/ml, it prevented the appearance and development of symptoms of HSV-1 infection in rats and rabbits. The polysaccharide did not exhibit any cytotoxic effects at a concentration of 2 mg/ml in vivo.
Assuntos
Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Polissacarídeos/farmacologia , Rodófitas/química , Adsorção/efeitos dos fármacos , Animais , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/patogenicidade , Técnicas In Vitro , Ceratite Herpética/tratamento farmacológico , Masculino , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Coelhos , Ratos , Células VeroRESUMO
Parvoviruses infect and kill tumor cells in vivo and in vitro more efficiently than normal cells. Infection of transformed cells by the parvovirus minute virus of mice (MVM) results in high expression of the major non-structural cytolytic viral protein NS1, which induces a cell death modulated by cellular factors. In this work, we show that MVMp infection and/or NS1 protein expression in permissive transformed rat fibroblast cells leads to apoptosis in wild type and p53(-/-) cells. Apoptotic cell morphology correlates with mitochondrial membrane permeabilization and activation of caspases 9 and 3 but not caspase 8. Thus, further characterization of the antitumor activity of MVMp and its NS1 protein may contribute to the eradication of tumors, including those lacking p53.
Assuntos
Apoptose , Fibroblastos/fisiologia , Fibroblastos/virologia , Vírus Miúdo do Camundongo/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Células Cultivadas , Camundongos , Camundongos Knockout , Ratos , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.
Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/virologia , Vírus Miúdo do Camundongo/fisiologia , Animais , Proteínas do Capsídeo/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/patologia , Expressão Gênica , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Vírus Miúdo do Camundongo/classificação , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/patogenicidade , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Especificidade da EspécieRESUMO
Activation of the minute virus of mice (MVM) P4 promoter is a key step in the life cycle of the virus and is completely dependent on host transcription factors. Since transcription-factor composition varies widely in different cell types, there is the possibility that only some cell types in the host organism have the capacity to initiate expression from the P4 promoter and therefore that the promoter may be a factor in determining the tropism of MVM. In this study, the ability of various cell types to activate P4, independent of the other virus-host interactions, was examined in transgenic mouse lines bearing a beta-galactosidase reporter sequence driven by the P4 promoter. It was found that lacZ was expressed during embryogenesis and in the adult in a cell-type-specific and differentiation-dependent pattern. The data are consistent with cell-type and stage-specific activation of the P4 promoter having a role in determining the host cell-type range of MVM. The ability of some parvoviruses to replicate in, and kill oncogenically transformed cells, and to destroy induced tumors in laboratory animals is the basis of recent approaches to use MVM-based vectors in cancer gene therapy. Since these vectors rely on the activation of the P4 promoter by the target tissues, understanding the promoter dependence on cell-type and differentiation status is important for their design and potential use.