RESUMO
Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.
Assuntos
Fator VIII/biossíntese , Lentivirus/genética , Fragmentos de Peptídeos/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular , Modelos Animais de Doenças , Fator VIII/química , Fator VIII/genética , Fator VIII/farmacologia , Citometria de Fluxo , Vetores Genéticos/genética , Hemofilia A/tratamento farmacológico , Humanos , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Análise de SobrevidaRESUMO
293T and Sk-Hep-1 cells were transduced with a replication-defective self-inactivating HIV-1 derived vector carrying FVIII cDNA. The genomic DNA was sequenced to reveal LTR/human genome junctions and integration sites. One hundred and thirty-two sequences matched human sequences, with an identity of at least 98%. The integration sites in 293T-FVIIIDB and in Sk-Hep-FVIIIDB cells were preferentially located in gene regions. The integrations in both cell lines were distant from the CpG islands and from the transcription start sites. A comparison between the two cell lines showed that the lentiviral-transduced DNA had the same preferred regions in the two different cell lines.
Assuntos
Fator VIII/biossíntese , Fator VIII/genética , Vetores Genéticos , HIV-1/fisiologia , Integração Viral , Linhagem Celular , HIV-1/genética , Hepatócitos/virologia , Humanos , Rim/virologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Análise de Sequência de DNARESUMO
HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.
Assuntos
Clonagem Molecular , Produtos do Gene env/química , Vírus Linfotrópico T Tipo 1 Humano/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Vetores Genéticos , Anticorpos Anti-HTLV-I/genética , Anticorpos Anti-HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Immunoblotting , Reação em Cadeia da Polimerase , Produtos do Gene env do Vírus da Imunodeficiência Humana/isolamento & purificaçãoRESUMO
O HTLV-1 é o vírus causador da leucemia/linfoma de célula T no adulto e de uma desordem neurológica conhecida por mielopatia associada ao HTLV ou paraparesia espástica tropical. Um dos modos de transmissão é pelo sangue contaminado e seus subprodutos e, devido ao risco de infecções associadas ao HTLV sua pesquisa na triagem de doadores de sangue foi introduzida no Brasil a partir de 1993. Os kits diagnósticos utilizados nos bancos de sangue nacionais são na sua maioria comprados de empresas estrangeiras. O Brasil não detém a tecnologia para produção deste material e há a necessidade de produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, mostramos a expressão da gp21/HTLV-1 em Escherichia coli e sua reatividade frente a anticorpos monoclonais e de pacientes infectados. Expressar tais proteínas é o primeiro passo para obtenção de conjuntos diagnósticos com tecnologia brasileira.
HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.
Assuntos
Humanos , Clonagem Molecular , Produtos do Gene env/química , Vírus Linfotrópico T Tipo 1 Humano/química , Proteínas Oncogênicas de Retroviridae/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Anticorpos Anti-HTLV-I/genética , Anticorpos Anti-HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Immunoblotting , Reação em Cadeia da Polimerase , Proteínas Oncogênicas de Retroviridae/isolamento & purificaçãoRESUMO
Propylthiouracil and thiamazole are thionamides used in the treatment of hyperthyroidism. In addition to reducing thyroid hormone synthesis, these drugs have other activities that improve the hypermetabolic state of the patients as well as adverse and toxic effects. The capacity of these 2 drugs to interfere with the production of reactive oxygen species of human neutrophils exposed in vitro to these drugs was evaluated. The production of reactive oxygen species was assessed by chemiluminescence assays and the cells were stimulated with zymosan particles opsonized with a pool of normal human serum. No alteration was found in the chemiluminescence response of treated human neutrophils when compared to controls. The results show that these drugs, at the studied concentrations and with the experimental approach used, have no direct effect on the production of oxidative burst of neutrophils. We conclude that if these drugs have any action on the oxidative metabolism of neutrophils these might include some metabolization steps that do not take place in this in vitro model.
