Evaluation of retroviral vector encoding bicistônico of endostatin and interleukin-2 for use in anti-tumor gene therapy

Gene therapy has been used in preclinical studies and clinical trials in order to assuage or cure a disease. Retroviral vectors are tools for gene transfer are widely used. Bicistronic vectors are attractive alternatives for treatment of complex disease. Endostatin (ES), the C-terminal fragment of collagen XVIII, is a potent angiogenesis inhibitor. At present, ES has been widely used in a variety of experimental tumor models and clinical trial. Immunotherapy, with interleukin-2 (IL-2), has been used as adjuvant treatment for tumors in several preclinical studies and clinical trials. The objective of this project was evaluated an IRES-based bicistronic retroviral vector encoding ES and IL-2, in anti-tumor gene therapy. In this study, the potential of Bicistronic therapy on activation of tumorinfiltrating lymphocytes in metastatic renal cell carcinoma (mRCC) was examined, using an orthotopic metastatic mouse model. Renca cells were injected into the renal subcapsule of BALB/C mice. After seven days, the nefrectomy was performed. Then, the animals were randomly divided into two groups (10 mice/group): control and the second group of mice received a subcutaneous inoculation of NIH/3T3-LendSN-IRES-IL-2 cells. Ten days after the nefrectomy, the animals were exsanguinated and killed. In the survival studies, daily mice were monitored daily, until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and number of nodule mestastatic, nodule area, microvascular area (MVA), proliferation of RenCa cells infiltrating tumor cells, plus tumors-infiltrating lymphocytes were evaluated. There was a significant decrease in the number of metastatic lung nodules, wet weight, lung nodule area, MVA and proliferation of Renca cells in the Bicistronic-treated group compared to the control group. These significant differences revealed an antitumor effect on the bicistronic (ES+IL-2) treatment. Subcutaneous inoculation of NIH/3T3- LendSNIRES-IL-2 cells resulted in an increase in ES and IL-2 levels and in the infiltration of CD4, CD4 producer IFNg, CD8, CD8 producer IFNg and NK (CD49b) cells in the treated group. The Bicistronic therapy Kaplan Meier survival curves showed that the probability of survival was significantly higher for mice treated (log-rank test, P=0.0016). Concluding, retroviral ES and IL-2 gene transfer led to secretion of functional ES and IL-2 that were sufficiently active to inhibit tumor angiogenesis and tumor growth, increasing the infiltration of immune cells.A terapia gênica tem sido empregada em estudos pré-clínicos e clínicos, com o intuito de amenizar ou curar uma doença. Vetores retrovirais são ferramentas de transferências gênica largamente utilizadas. Vetores retrovirais bicistrônico é uma alternativa interessante para o tratamento de doenças complexas. A endostatina (ES), fragmento do colágeno XVIII, tem sido utilizada na terapia anti-angiogênica, devido sua ação inibitória no crescimento de células endoteliais. A imunoterapia tem sido utilizada como tratamento coadjuvante de tumores. Dentre as citocinas utilizadas, a interleucina-2 (IL-2) promovendo a proliferação de linfócitos T, tem sido utilizada em diversos estudos pré-clínicos e clínicos. O objetivo deste projeto foi avaliar um vetor retroviral bicistrônico codificando ES e IL-2, utilizando a sequência IRES, na terapia gênica anti-tumoral. Neste estudo, foi avaliado o potencial do vetor bicistrônico na ativação da infiltração linfocitária em carcinoma renal metastático, utilizando um modelo ortotópico metastático. Células Renca foram injetadas na subcápsula renal de camundongos Balb/C. Após 7 dias, foi realizado a nefrectomia nesses animais. Os animais foram foram divididos igualmente em dois grupos. Um grupo foi o controle e o segundo grupo recebeu a inoculação subcutânea de células NIH/3T3-LendSN-IRES-IL-2. Dez dias após à nefrectomia, os animais foram exsanguinados e sacrificados para a retirada dos pulmões. No estudo de sobrevivência, os animais foram monitorados até a morte. No final dos experimentos in vivo, os níveis séricos de ES e de IL-2 circulantes foram avaliados, o pulmão foi pesado e os números dos nódulos metastático foram quantificados, assim como, a área nodular e a área microvascular intranodais. A proliferação das Células Renca, a infiltração e ativação linfocitária dentro dos nódulos metastáticos foram avaliados. Houve uma diminuição significativa no número de nódulos metastáticos, no peso pulmonar, na área nodular e microvascular e na proliferação das células Renca dentro dos nódulos metastáticos, no grupo tratado com o vetor bicistrônico quando comparado com o grupo controle. Essas significantes deiferenças revelam o efeito anti-tumoral no tratamento com ES+IL-2. A inoculação subcutânea das células NIH/3T3-LendSN-IRES-IL-2 promoveu um significativo aumento nos níveis séricos de ES e IL-2 circulantes, como também, na infiltração de células CD4, CD4 secretoras de IFN-g, CD8, CD8 secretoras de IFN-g e de células Natural Killer (NK). A curva de sobrevivência mostrou que a probabilidade de sobrevida foi significantemente maior nos animais tratados. Em conclusão, a transferência gênica retroviral de ES e IL-2, resultou em uma produção de ES e IL-2, as quais apresentaram suficientemente ativas para a inibição da angiogênese e do crescimento tumoral, com aumento da infiltração das células do sistema imune.TEDEBV UNIFESP: Teses e dissertaçõe

Endostatin gene therapy enhances the efficacy of IL-2 in suppressing metastatic renal cell carcinoma in mice

We investigated whether the administration of IL-2 combined with endostatin gene therapy was able to produce additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma. Renca cells were injected into the tail vein of BALB/c mice. After 24 h, the animals were randomly divided into four groups (5 mice/group). One group of mice was the control, the second group received treatment with 100,000 UI of Recombinant IL-2 (Proleukin, Chiron) twice a day, 1 day per week during 2 weeks (IL-2), the third group received treatment with a subcutaneous inoculation of 3.6 x 10(6) endostatin-producing cells, and the fourth group received both therapies (IL-2 + ES). Mice were treated for 2 weeks. in the survival studies, 10 mice/group daily, mice were monitored daily until they died. the presence of metastases led to a twofold increase in endostatin levels. Subcutaneous inoculation of NIH/3T3-LendSN cells resulted in a 2.75 and 2.78-fold increase in endostatin levels in the ES and IL-2 + ES group, respectively. At the end of the study, there was a significant decrease in lung wet weight, lung nodules area, and microvascular area (MVA) in all treated groups compared with the control group (P < 0.001). the significant difference in lung wet weight and lung nodules area between groups IL-2 and IL-2 + ES revealed a synergistic antitumor effect of the combined treatment (P < 0.05). the IL-2 + ES therapy Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice treated with the combined therapy (log-rank test, P = 0.0028). Conjugated therapy caused an increase in the infiltration of CD4, CD8 and CD49b lymphocytes. An increase in the amount of CD8 cells (P < 0.01) was observed when animals received both ES and IL-2, suggesting an additive effect of ES over IL-2 treatment. A synergistic effect of ES on the infiltration of CD4 (P < 0.001) and CD49b cells (P < 0.01) was also observed over the effect of IL-2. Here, we show that ES led to an increase in CD4 T helper cells as well as cytotoxic lymphocytes, such as NK cells and CD8 cells, within tumors of IL-2 treated mice. This means that ES plays a role in supporting the actions of T cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, BrazilUniv São Paulo, Dept Radiol, São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Immunol, São Paulo, BrazilAlbert Einstein Jewish Inst Educ & Res, São Paulo, BrazilIPEN CNEN, Dept Biotechnol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, BrazilFAPESP: 2007/54253-6Web of Scienc

Endostatin- and interleukin-2-expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma

Background Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. Methods Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. Results Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)gamma-producing, CD8, CD8 IFN gamma-producing and natural killer (CD49b) cells. Conclusions Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells (C) Copyright. 2011 John Wiley & Sons, Ltd.FAPESP[2007/54253-6]CNPq[481888/2008]CAPES/PNPD[0188085

Chikungunya virus outbreak in the Amazon region: replacement of the Asian genotype by an ECSA lineage

Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Laboratório de Ecologia de Doenças Transmissíveis na Amazônia. Manaus, AM, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Medicina Tropical. São Paulo, SP, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brazil / Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Patologia Experimental. Salvador, BA, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Patologia Experimental. Salvador, BA, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil / Fundação Ezequiel Dias. Instituto Octávio Magalhães. Laboratório Central de Saúde Pública. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Laboratório de Ecologia de Doenças Transmissíveis na Amazônia. Manaus, AM, Brazil.Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Laboratório de Ecologia de Doenças Transmissíveis na Amazônia. Manaus, AM, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Biologia. Departamento de Genética Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brazil.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom.Harvard Medical School. Department of Pediatrics. Boston, MA, USA / Boston Children’s Hospital. Computational Health Informatics Program. Boston, MA, USA.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom / Boston Children’s Hospital. Computational Epidemiology Lab. Boston, MA, USA.University of Birmingham. Institute of Microbiology and Infection. Birmingham, United Kingdom.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Medicina Tropical. São Paulo, SP, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro de Inovações Tecnológicas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro de Inovações Tecnológicas. Ananindeua, PA, Brasil.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil.Laboratório Central de Saúde Pública. Boa Vista, RR, Brazil.Laboratório Central de Saúde Pública. Boa Vista, RR, Brazil.Laboratório Central de Saúde Pública. Boa Vista, RR, Brazil.Secretaria Municipal de Saúde de Boa Vista. Superintendência de Vigilância em Saúde. Boa Vista, RR, Brazil.Fundação de Medicina Tropical Doutor Heitor Vieira. Departamento de Virologia. Manaus, AM, Brazil.Secretaria Municipal de Saúde de Boa Vista. Superintendência de Vigilância em Saúde. Boa Vista, RR, Brazil.Laboratório Central de Saúde Pública do Amazonas. Manaus, AM, Brazil.Organização Pan - Americana da Saúde/Organização Mundial da Saúde. Brasília, DF, BrazilMinistério da Saúde. Secretaria de Vigilância em Saúde. Brasília, DF, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Brasília, DF, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Brasília, DF, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Brasília, DF, Brazil.Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Laboratório de Ecologia de Doenças Transmissíveis na Amazônia. Manaus, AM, Brazil.University of Birmingham. Institute of Microbiology and Infection. Birmingham, United Kingdom.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom.Universidade de São Paulo. Faculdade de Medicina. Instituto de Medicina Tropical. São Paulo, SP, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Brasília, DF, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brazil / Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Laboratório de Genética Celular e Molecular. Belo Horizonte, MG, Brazil.University of Oxford. Department of Zoology. South Parks Road, Oxford, United Kingdom.Background Since its first detection in the Caribbean in late 2013, chikungunya virus (CHIKV) has affected 51 countries in the Americas. The CHIKV epidemic in the Americas was caused by the CHIKV-Asian genotype. In August 2014, local transmission of the CHIKV-Asian genotype was detected in the Brazilian Amazon region. However, a distinct lineage, the CHIKV-East-Central-South-America (ECSA)-genotype, was detected nearly simultaneously in Feira de Santana, Bahia state, northeast Brazil. The genomic diversity and the dynamics of CHIKV in the Brazilian Amazon region remains poorly understood despite its importance to better understand the epidemiological spread and public health impact of CHIKV in the country. Methodology/Principal Findings We report a large CHIKV outbreak (5,928 notified cases between August 2014 and August 2018) in Boa vista municipality, capital city of Roraima’s state, located in the Brazilian Amazon region. In just 48 hours, we generated 20 novel CHIKV-ECSA genomes from the Brazilian Amazon region using MinION portable genome sequencing. Phylogenetic analyses revealed that despite an early introduction of the Asian genotype in 2015 in Roraima, the large CHIKV outbreak in 2017 in Boa Vista was caused by an ECSA-lineage most likely introduced from northeastern Brazil. Epidemiological analyses suggest a basic reproductive number of R0 of 1.66, which translates in an estimated 39 (95% CI: 36 to 45) % of Roraima’s population infected with CHIKV-ECSA. Finally, we find a strong association between Google search activity and the local laboratory-confirmed CHIKV cases in Roraima. Conclusions/Significance This study highlights the potential of combining traditional surveillance with portable genome sequencing technologies and digital epidemiology to inform public health surveillance in the Amazon region. Our data reveal a large CHIKV-ECSA outbreak in Boa Vista, limited potential for future CHIKV outbreaks, and indicate a replacement of the Asian genotype by the ECSA genotype in the Amazon region

Genomic, epidemiological and digital surveillance of Chikungunya virus in the Brazilian Amazon.

BackgroundSince its first detection in the Caribbean in late 2013, chikungunya virus (CHIKV) has affected 51 countries in the Americas. The CHIKV epidemic in the Americas was caused by the CHIKV-Asian genotype. In August 2014, local transmission of the CHIKV-Asian genotype was detected in the Brazilian Amazon region. However, a distinct lineage, the CHIKV-East-Central-South-America (ECSA)-genotype, was detected nearly simultaneously in Feira de Santana, Bahia state, northeast Brazil. The genomic diversity and the dynamics of CHIKV in the Brazilian Amazon region remains poorly understood despite its importance to better understand the epidemiological spread and public health impact of CHIKV in the country.Methodology/principal findingsWe report a large CHIKV outbreak (5,928 notified cases between August 2014 and August 2018) in Boa vista municipality, capital city of Roraima's state, located in the Brazilian Amazon region. We generated 20 novel CHIKV-ECSA genomes from the Brazilian Amazon region using MinION portable genome sequencing. Phylogenetic analyses revealed that despite an early introduction of the Asian genotype in 2015 in Roraima, the large CHIKV outbreak in 2017 in Boa Vista was caused by an ECSA-lineage most likely introduced from northeastern Brazil. Epidemiological analyses suggest a basic reproductive number of R0 of 1.66, which translates in an estimated 39 (95% CI: 36 to 45) % of Roraima's population infected with CHIKV-ECSA. Finally, we find a strong association between Google search activity and the local laboratory-confirmed CHIKV cases in Roraima.Conclusions/significanceThis study highlights the potential of combining traditional surveillance with portable genome sequencing technologies and digital epidemiology to inform public health surveillance in the Amazon region. Our data reveal a large CHIKV-ECSA outbreak in Boa Vista, limited potential for future CHIKV outbreaks, and indicate a replacement of the Asian genotype by the ECSA genotype in the Amazon region

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data