Assuntos
Células Apresentadoras de Antígenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/imunologia , Adenoviridae/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Apoptose , Proteína Ligante Fas , Vetores Genéticos/genética , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Ratos , Ratos Endogâmicos Lew , Linfócitos T/citologiaAssuntos
Células Dendríticas/imunologia , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/genética , Tolerância ao Transplante/imunologia , Adenoviridae , Linhagem Celular , Células Dendríticas/transplante , Células Dendríticas/virologia , HumanosRESUMO
Recent reports have pointed out the difficulties in generating recombinant adenoviral vectors expressing FasL using eukaryotic cells. In the present study we cloned the murine FasL (mFasL) gene into the pCA14 or pShuttle vectors and recombined them with the adeno-5 virus backbone pBHG10 or pAdEasy-1 in eukaryotic (911 cells) or prokaryotic (E. coli) cells, respectively. Recombination of pCA14-mFasL with pBHG10 in Fas-carrying 911 cells leads to rapid expression of mFasL and cell death by apoptosis before virus replication is initiated. The same effect is observed when 911 cells are transfected with the pCA14-mFasL shuttle vector only. If recombination (pShuttle-mFasL with pAdEasy-1) is performed first in bacteria and 911 cells are transfected thereafter with recombined AdEasy-mFasL, virus production starts immediately and the cells survive longer. The resulting AdEasy-mFasL viruses are able to infect other eukaryotic cells and induce expression of functional mFasL. This study describes a method for efficient generation of recombinant FasL adeno-5 viruses in eukaryotic cells. The method may be generally suitable for producing viruses carrying deleterious genes. Gene Therapy (2000) 7, 70-74.
Assuntos
Adenoviridae/genética , Glicoproteínas de Membrana/genética , Apoptose/genética , Proteína Ligante Fas , Vetores Genéticos/genética , Humanos , Retina/citologia , Retina/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosAssuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Extratos Vegetais/farmacologia , Linfócitos T/imunologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Concanavalina A , Sinergismo Farmacológico , Humanos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosAssuntos
Linfócitos T CD4-Positivos/imunologia , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Extratos Vegetais/farmacologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Biomarcadores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Antígenos HLA-DR/análise , Humanos , Imunossupressores/isolamento & purificação , Ionomicina/farmacologia , Lectinas Tipo C , Mitógenos , Extratos Vegetais/isolamento & purificação , Receptores de Interleucina-2/análise , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We have defined factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model and analyzed the influence of CTLA4Ig treatment on indirect alloactivation induced by donor MHC I peptides in a DA-->LEW heart allograft model. Indirect allorecognition of MHC I led to accelerated graft rejection and was accompanied by the induction of anti-peptide antibodies and donor peptide-activated T cells. In an attempt to block the B7-induced costimulatory signal of T cell activation, CTLA4Ig was administered to graft recipients in addition to MHC I peptide treatment. CTLA4Ig therapy, however, was not effective in preventing the humoral or cellular anti-donor immune response, nor did it prevent accelerated graft rejection.
