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Chris R Bleackley - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
Cheryl D Helgason - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
Bruna Gabriela Silva - One of the best experts on this subject based on the ideXlab platform.
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Avaliação de características cinéticas e metabólicas de dois hibridomas para produção de anticorpos monoclonais para tipagem sanguínea
Universidade Federal de São Carlos, 2012Co-Authors: Bruna Gabriela SilvaAbstract:A produção em larga escala de anticorpos monoclonais é o campo da Biotecnologia que tem conseguido os maiores avanços e realizações ao longo dos últimos 20 anos, especialmente na área de diagnóstico e de aplicações terapêuticas. O mercado mundial de produtos para diagnóstico clínico atingiu o valor de US$ 19,0 bilhões em 2008 e continua crescendo a um ritmo de 5% ao ano. O desenvolvimento de bioprocessos de alta produtividade em larga escala para produção de anticorpos monoclonais (AMs) depende da determinação e otimização de vários parâmetros cinéticos e metabólicos dos hibridomas. A finalidade deste trabalho foi analisar características cinéticas e metabólicas de hibridomas murinos para produção de imunoglobulinas para tipagem sanguinea pelo sistema ABO, de grande interesse para a saúde pública Brasileira. Três linhagens de hibridomas murinos denominadas ED7, ED9 e TAN obtidas num centro de pesquisa brasileiro para produzir os anticorpos anti-A, anti-B e anti-AB, respectivamente, foram avaliados com essa finalidade. Utilizando apenas as duas linhagens, ED7 e ED9, por apresentarem maior potencial e estabilidade para produção de AMs em larga escala foram realizados experimentos de cultivo em batelada em frasco spinner de 500 mL e em biorreator (Bioflo-110) de 2L, utilizando meios de cultura RPMI 1640 e Mab Quantum Yield-BD, ambos com adição de 10% v/v de soro fetal bovino. O primeiro sendo meio de origem dos clones híbridos, em alguns casos teve que ser suplementado com aminoácidos (Gln, Met, Cys, Ser, Lys e Pro) e, o segundo, um meio alternativo ao qual tiveram que ser adaptados os hibridomas sem suplementos antes dos experimentos de cultivo em regime de batelada. Os resultados mostraram a necessidade de balanceamento nutricional adicional dos meios, especialmente do RPMIS, para satisfazer as demandas nutricionais dos dois hibridomas. Mediante balanceamento nutricional e cultivos em condições melhor controladas em biorreator conseguiu-se incrementar a taxa especifíca máxima de crescimento μmax de 0,03040,0014 h-1 para 0,03290,002 h-1 e a produtividade de AM de 0,031 mg/L.h para 0,077 mg/L.h com a linhagem ED7. Com a linhagem ED9, o μmax diminuiu de 0,04120,0024 h-1 para 0,03730,0019 h-1 mas a produtividade de AM aumentou de 0,030 mg/L.h para 0,040 mg/L.h. Por outro lado, testes de aglutinação mostraram que os AMs estavam funcionais e com títulos estáveis e dentro dos limites aceitáveis. Apesar dos ganhos em produtividade dos dois AMs, os resultados permitiram constatar um problema típico encontrado no cultivo in vitro de hibridomas que se caracteriza por desregulação de seu metabolismo na presença de altas concentrações de glicose e glutamina, o que provoca produção exacerbada dos metabólitos lactato e amônia. Em função disto, pode-se prever que o futuro do desenvolvimento do bioprocesso de produção de anti-A e anti-AB tenha que passar por uma etapa de avaliação e otimização em sistema de cultivo de batelada alimentada.The large scale production of monoclonal antibodies is the Biotechnology field that has achieved the biggest advances and realizations over the last 20 years, especially in the area of diagnosis and therapeutic applications. The world market of products for clinical diagnosis reached the value of US$ 19.0 billions in 2008 and continues to grow at a rate of 5 % per year. The development of bioprocesses of high productivity in large scale production of monoclonal antibodies (MAbs) depends on the determination and optimization of various kinetic and metabolic parameters of Hybridomas. The aim of this study was to analyze kinetic and metabolic characteristics of murine Hybridomas for production of immunoglobulins for blood typing by the ABO system, of great interest to the Brazilian public health. Three strains of murine Hybridomas named ED7, ED9 and TAN generated in a brazilian research center to produce the antibodies anti-A, anti-B and anti-A,B, respectively, were evaluated for this purpose. Using only two lineage, ED7 and ED9, because of presenting bigger potential and stability for production of AMs in large scale, experiments of cultivation were carried out in spinner flask of 500 mL and in biorreator (Bioflo-110) of 2L, using culture media RPMI 1640 and MAb Quantum Yield-BD, both with addition of 10 % v/v of fetal bovine serum. The first one being the medium of origin of the hybrid clones, in some cases supplemented with amino acids (Gln, Met, Cys, Ser, Lys and Pro), and the second, a new alternative medium to which the Hybridomas had to be adapted before the cultivation experiments. The results showed the need for additional nutritional balance of the media, especially RPMIS, to meet the nutritional demands of the two Hybridomas. Through nutritional balance and cultivation in better controlled conditions in the bioreactor, it was possible to raise the maximum specific growth rate μmax from 0.0304 0.0014 h-1 to 0.0329 0.002 h-1 and the productivity of AM from 0.031 mg/L.h to 0.077 mg/L.h with the lineage ED7. With the lineage ED9 the μmax decreased from 0.0412 0.0024 h-1 to 0.0373 h-1 but increased the productivity of AM from 0.030 mg / L.h to 0.040 mg / L.h. On the other hand, agglutination tests showed that the antibodies were functional and with titles stable and within normal limits. Despite the gains in productivity of the two AMs, the results highlighted a typical problem found in vitro cultivation of Hybridomas, which is characterized by deregulation of its metabolism in the presence of high concentrations of glucose and glutamine, conduzing to overproction of the metabolites lactate and amonia. Because of this, one can predict that the future development of the bioprocess for the production of anti-A and anti-AB has to go through a stage of evaluation and system optimization in fed batch cultivation
Silva, Bruna Gabriela - One of the best experts on this subject based on the ideXlab platform.
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Avaliação de características cinéticas e metabólicas de dois hibridomas para produção de anticorpos monoclonais para tipagem sanguínea
Programa de Pós-graduação em Engenharia Química, 2012Co-Authors: Silva, Bruna GabrielaAbstract:The large scale production of monoclonal antibodies is the Biotechnology field that has achieved the biggest advances and realizations over the last 20 years, especially in the area of diagnosis and therapeutic applications. The world market of products for clinical diagnosis reached the value of US$ 19.0 billions in 2008 and continues to grow at a rate of 5 % per year. The development of bioprocesses of high productivity in large scale production of monoclonal antibodies (MAbs) depends on the determination and optimization of various kinetic and metabolic parameters of Hybridomas. The aim of this study was to analyze kinetic and metabolic characteristics of murine Hybridomas for production of immunoglobulins for blood typing by the ABO system, of great interest to the Brazilian public health. Three strains of murine Hybridomas named ED7, ED9 and TAN generated in a brazilian research center to produce the antibodies anti-A, anti-B and anti-A,B, respectively, were evaluated for this purpose. Using only two lineage, ED7 and ED9, because of presenting bigger potential and stability for production of AMs in large scale, experiments of cultivation were carried out in spinner flask of 500 mL and in biorreator (Bioflo-110) of 2L, using culture media RPMI 1640 and MAb Quantum Yield-BD, both with addition of 10 % v/v of fetal bovine serum. The first one being the medium of origin of the hybrid clones, in some cases supplemented with amino acids (Gln, Met, Cys, Ser, Lys and Pro), and the second, a new alternative medium to which the Hybridomas had to be adapted before the cultivation experiments. The results showed the need for additional nutritional balance of the media, especially RPMIS, to meet the nutritional demands of the two Hybridomas. Through nutritional balance and cultivation in better controlled conditions in the bioreactor, it was possible to raise the maximum specific growth rate μmax from 0.0304 ± 0.0014 h-1 to ±0.0329 0.002 h-1 and the productivity of AM from 0.031 mg/L.h to 0.077 mg/L.h with the lineage ED7. With the lineage ED9 the μmax decreased from 0.0412 ± 0.0024 h-1 to 0.0373± h-1 but increased the productivity of AM from 0.030 mg / L.h to 0.040 mg / L.h. On the other hand, agglutination tests showed that the antibodies were functional and with titles stable and within normal limits. Despite the gains in productivity of the two AMs, the results highlighted a typical problem found in vitro cultivation of Hybridomas, which is characterized by deregulation of its metabolism in the presence of high concentrations of glucose and glutamine, conduzing to overproction of the metabolites lactate and amonia. Because of this, one can predict that the future development of the bioprocess for the production of anti-A and anti-AB has to go through a stage of evaluation and system optimization in fed batch cultivation.Universidade Federal de Sao CarlosA produção em larga escala de anticorpos monoclonais é o campo da Biotecnologia que tem conseguido os maiores avanços e realizações ao longo dos últimos 20 anos, especialmente na área de diagnóstico e de aplicações terapêuticas. O mercado mundial de produtos para diagnóstico clínico atingiu o valor de US$ 19,0 bilhões em 2008 e continua crescendo a um ritmo de 5% ao ano. O desenvolvimento de bioprocessos de alta produtividade em larga escala para produção de anticorpos monoclonais (AMs) depende da determinação e otimização de vários parâmetros cinéticos e metabólicos dos hibridomas. A finalidade deste trabalho foi analisar características cinéticas e metabólicas de hibridomas murinos para produção de imunoglobulinas para tipagem sanguinea pelo sistema ABO, de grande interesse para a saúde pública Brasileira. Três linhagens de hibridomas murinos denominadas ED7, ED9 e TAN obtidas num centro de pesquisa brasileiro para produzir os anticorpos anti-A, anti-B e anti-AB, respectivamente, foram avaliados com essa finalidade. Utilizando apenas as duas linhagens, ED7 e ED9, por apresentarem maior potencial e estabilidade para produção de AMs em larga escala foram realizados experimentos de cultivo em batelada em frasco spinner de 500 mL e em biorreator (Bioflo-110) de 2L, utilizando meios de cultura RPMI 1640 e Mab Quantum Yield-BD, ambos com adição de 10% v/v de soro fetal bovino. O primeiro sendo meio de origem dos clones híbridos, em alguns casos teve que ser suplementado com aminoácidos (Gln, Met, Cys, Ser, Lys e Pro) e, o segundo, um meio alternativo ao qual tiveram que ser adaptados os hibridomas sem suplementos antes dos experimentos de cultivo em regime de batelada. Os resultados mostraram a necessidade de balanceamento nutricional adicional dos meios, especialmente do RPMIS, para satisfazer as demandas nutricionais dos dois hibridomas. Mediante balanceamento nutricional e cultivos em condições melhor controladas em biorreator conseguiu-se incrementar a taxa especifíca máxima de crescimento μmax de 0,0304±0,0014 h-1 para 0,0329±0,002 h-1 e a produtividade de AM de 0,031 mg/L.h para 0,077 mg/L.h com a linhagem ED7. Com a linhagem ED9, o μmax diminuiu de 0,0412±0,0024 h-1 para 0,0373±0,0019 h-1 mas a produtividade de AM aumentou de 0,030 mg/L.h para 0,040 mg/L.h. Por outro lado, testes de aglutinação mostraram que os AMs estavam funcionais e com títulos estáveis e dentro dos limites aceitáveis. Apesar dos ganhos em produtividade dos dois AMs, os resultados permitiram constatar um problema típico encontrado no cultivo in vitro de hibridomas que se caracteriza por desregulação de seu metabolismo na presença de altas concentrações de glicose e glutamina, o que provoca produção exacerbada dos metabólitos lactato e amônia. Em função disto, pode-se prever que o futuro do desenvolvimento do bioprocesso de produção de anti-A e anti-AB tenha que passar por uma etapa de avaliação e otimização em sistema de cultivo de batelada alimentada
John A Prendergast - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
