Evaluation elements forming Z-DNA as potential regulators of the expression of biopharmaceuticals in CH0-K1 cells

Dissertação (mestrado)—Universidade de Brasília, Programa de Pós-graduação em Patologia Molecular, 2013.Introdução: o grupo de Imunologia Molecular/UnB já produziu anticorpos recombinantes e humanizados de interesse comercial como o anti-CD18, anti-CD3, ou acadêmico como o anti-Z22,assim como fatores plasmáticos humanos. Os vetores de expressão utilizados contêm o promotor de CMV-IA. O objetivo desse trabalho foi explorar regiões formadoras de Z-DNA, e testar diretamente o papel do anticorpo estabilizador de Z-DNA (antiZ22NLS - Z22) no efeito transcricional do promotor CMV modificado (zCMV-IA). Material e Métodos: O primeiro passo foi reconstruir o cassete de expressão dos vetores tradicionais utilizados nesse trabalho, pCO e pCO?600. Para isso, fusionamos o gene GFP ao anticorpo recombinante aCD3. Os passos seguintes foram a introdução de sequências formadoras de Z-DNA (Z1, Z3 e Z4) e controles não formadores de Z-DNA (Z2 e Z5) a montante do promotor CMV-IA. Resultados e Discussão: foram construídos 10 vetores de expressão: 5 construções pCO (pZ1, pZ2, pZ3, pZ4 e pZ5) e 5 construções pCO?600 (?Z1, ?Z2, ?Z3, ?Z4 e ?Z5); transfecção de células CHO-K1 com as construções utilizando Lipofectamina LTX na proporção 1:1 de DNA e reagente LTX; Por fim co-transfecção de células com cada vetor de expressão e vetor pMacZ22NLS e como controle de cada vetor de expressão também foi feita transfecção com vetor pMac vazio. Em comparação aos controles em todas as construções a presença do pMacZ22 NLS aumentou a eficiência de transfecção. Através de citometria de fluxo se verificou aumento da MFI no gate das células GFP+ em até 25 % quando foi co-transfectado vetor com anti-Z-DNA. Conclusão: foi mostrado o papel de anti Z22 como indutor e estabilizador de Z-DNA e seu efeito facilitador da transcrição. _______________________________________________________________________________________ ABSTRACTIntroduction: Recombinant antibodies are now a reality in therapeutics. But its production is still a challenging issue. We have been developing expression vectors for heterologous expression of recombinant antibodies based on the CMV promotor. We had previously shown that a Z-DNA forming region upstream the promotor/enhancer enhances luciferase expression in a repórter vector. The aim of this study was to explore Z-DNA forming regions, and directly test the role of a Z-DNA stabilizing antibody on a modified CMV promoter (zCMV). Material and Methods: Initially, the GFP gene was fused with a recombinant antibody anti-CD3 and the fusion cassette was introduced in both pCO and pCO?6OO. Then we introduced the Z-DNA forming sequences (Z1, Z3 and Z4) and control sequences (Z2 and Z5) upstream of the CMV promoter. CHO cells were transfected using Lipofectamine LTX 1:1, DNA and LTX reagent. To provide the trans acting anti-Z-DNA antibody, we cotransfect cells with the vector pMACZ22NLS, that produces a scFv of the anti-Z-DNA mAb Z22 fused to a nuclear localization signal (Intrabody). Flow cytometry was use to follow GFP expression. Results and Discussion: 10 expression vectors were constructed: 5 pCO constructs (pZ1, pZ2, pZ3, pZ4 and pZ5) and 5 pCO?600 constructs (AZ1, AZ2, AZ3, AZ4 and AZ5); The constructions with Z-DNA forming sequences showed an improvement of expression of the reporter gene when compared to the control sequences. Moreover the co-transfection with the anti-Z-DNA vector enhances the MFI of gated GFP+ cells by 25%. Conclusion: The Z-DNA forming sequences improve heterologous gene expression and the presence of anti-Z-DNA in trans was shown to enhance the Z-DNA effect probably due to a stabilization effect

Faster cryptococcus melanization increases virulence in experimental and human cryptococcosis

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host–pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host–pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence