RESUMEN
A plasmid DNA that encodes chicken interleukin-2 (pCI-ChIL-2-EGFP) was investigated for its distribution and expression after intramuscular (i.m.) injection in chickens. After the i.m. injection, serum distribution was detectable from 2 h post inoculation (p.i.), peaked at 8 h p.i., and disappeared at 7 days p.i. The plasmid DNA was also observed in several organs including heart, liver, lung, spleen, bursa and inoculated muscle at different time points, but at 19 days p.i. the plasmid DNA was not found in any organ except inoculated muscle. Fluorescence of enhanced green fluorescent protein (EGFP) was found in cytoplasm and nucleus of cultured Vero cells, chicken embryo fibroblasts and peripheral blood lymphocytes, which were transfected in vitro with the plasmid DNA or in vivo with Lipofectamine. The expression profile of the fusion gene (ChIL-2-EGFP) in vivo was measured by RT-PCR, ELISA and fluorescence microscopy. The EGFP expression was detected from 8 h p.i. to 14 days p.i. and peaked at 5 days p.i., when the number of EGFP-expression myocytes was about 5% in the injected site. These results demonstrate that intramuscular administration of plasmid DNA leads to widespread distribution and long-term expression in vivo.
Asunto(s)
Pollos/metabolismo , ADN Recombinante/administración & dosificación , Interleucina-2/genética , Plásmidos/farmacocinética , Animales , Embrión de Pollo , Pollos/inmunología , Chlorocebus aethiops , Clonación Molecular , ADN Recombinante/genética , ADN Recombinante/inmunología , ADN Recombinante/farmacocinética , Ensayo de Inmunoadsorción Enzimática/veterinaria , Interleucina-2/biosíntesis , Interleucina-2/sangre , Interleucina-2/inmunología , Microscopía Fluorescente/veterinaria , Plásmidos/genética , Plásmidos/metabolismo , Distribución Aleatoria , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Organismos Libres de Patógenos Específicos , Transfección/veterinaria , Células VeroRESUMEN
In this study, total blood lymphocytes were prepared from patients with hemorrhagic fever with renal syndrome (HFRS) by a density gradient on Ficoll-Hypaque. T and B cells were then purified by passing the total lymphocytes over a nylon wool column. The purities of total lymphocytes, T cells and B cells were 97.8 +/- 2.3%, 91.6 +/- 4.5% and 74.2 +/- 12.1%, respectively. Also, after modification of cell fixation and smear drying, the number of cells were increased and the time needed for slide preparation was shortened. Detection of viral antigen by immunofluorescence assay using monoclonal antibodies to Hantavirus (HTNV) showed that the total lymphocytes. T cells and B cells were infected by HTNV during the early stages of HFRS and no specific fluorescence was seen in the cells from the late diuretic phase to convalescent phase. The results suggest that virus replication in blood lymphocytes may partly contribute in the early stages to the impairment of cell immune response and in vivo spread of HTNV to its target sites.
Asunto(s)
Antígenos Virales/análisis , Fiebre Hemorrágica con Síndrome Renal/inmunología , Orthohantavirus/inmunología , Linfocitos B/inmunología , Separación Celular , Técnica del Anticuerpo Fluorescente , Humanos , Linfocitos T/inmunologíaRESUMEN
Total lymphocytes in peripheral blood were prepared from patients with hemorrhagic fever with renal syndrome by a density gradient on Ficoll-Hypaque, and then T and B cells purified by passing the lymphocytes over a nylon column with modifications. Also, after modification of cell fixation and use of a spinner for smear drying, the number of cells were increased and the duration for slide preparation was shortened, thus resulting in quality slides. Detection of viral antigen by immunofluorescence assay using monoclonal antibodies to Hantavirus (HTNV) showed that the total lymphocytes, T cells and B cells were infected with HTNV during the early stages of the illness and no specific fluorescence was seen in the cells from the late diuretic phase to convalescent phase.