Sequential Immunization Strategies to Elicit HIV-1 bNAbs in Animal Models With a Polyclonal B Cell Repertoire

Background: Immunization regimens that can elicit broadly neutralizing antibodies (bNAbs) in humans would be an effective vaccine against HIV-1. Our previous work showed that an immunization strategy involving a sequence of Env-based germline targeting immunogens that were gradually engineered to resemble the native Env protein, successfully elicited bNAb-like antibodies in a knock-in mouse carrying the inferred germline PGT121/10-1074 antibody. Despite this achievement, immunization protocols that elicit bNAbs in systems with a polyclonal B cell repertoire have not been reported to date. The low frequencies of germline bNAb precursors in polyclonal systems hinder their activation by immunization which therefore requires high affinity immunogens. In addition, competition between different epitope-specific B cells in polyclonal germinal centers may frustrate bNAb development. Methods: Based on our previous results in knock-in mice, we have aimed to optimize sequential immunization strategies to elicit bNAbs in animal models with polyclonal B cell repertoires. Results: The results of immunization experiments in several animal models will be presented

Sequential Immunization Strategies to Elicit HIV-1 bNAbs in Animal Models With a Polyclonal B Cell Repertoire

Background: Immunization regimens that can elicit broadly neutralizing antibodies (bNAbs) in humans would be an effective vaccine against HIV-1. Our previous work showed that an immunization strategy involving a sequence of Env-based germline targeting immunogens that were gradually engineered to resemble the native Env protein, successfully elicited bNAb-like antibodies in a knock-in mouse carrying the inferred germline PGT121/10-1074 antibody. Despite this achievement, immunization protocols that elicit bNAbs in systems with a polyclonal B cell repertoire have not been reported to date. The low frequencies of germline bNAb precursors in polyclonal systems hinder their activation by immunization which therefore requires high affinity immunogens. In addition, competition between different epitope-specific B cells in polyclonal germinal centers may frustrate bNAb development. Methods: Based on our previous results in knock-in mice, we have aimed to optimize sequential immunization strategies to elicit bNAbs in animal models with polyclonal B cell repertoires. Results: The results of immunization experiments in several animal models will be presented

Molecular aspects of DARC gene evolution in primates

Genes envolvidos com a interação hospedeiro-patógeno são fortemente afetados pela seleção natural positiva. O gene codificante do antígeno sangüíneo Duffy, também conhecido como DARC (Duffy Antigen Receptor for Chemokines), tem um importante papel na invasão dos eritrócitos pelos parasitas causadores da malária, Plasmodium vivax em humanos e Plasmodium knowlesi em outros primatas. A estrutura do gene DARC já é conhecida, estando este presente na região 1q22q23 do cromossomo 1, e sendo composto por dois éxons separados por um grande íntron. Em uma população africana a deleção de um nucleotídeo no domínio GATA-1 da região promotora do gene é responsável pela não expressão de DARC nos eritrócitos e pela resistência à invasão pelo P. Vivax. Além disso, o antígeno DARC age como um receptor promíscuo de quimiocinas, sendo expresso nos eritrócitos, células endoteliais de vênulas e outros tecidos. Devido a esse papel dual, no presente estudo seqüenciou-se regiões homólogas do gene DARC em macacos do Novo e Velho Mundo e utilizando métodos estatísticos procurou-se indícios da seleção natural positiva na sua história evolutiva. Nenhuma nova mutação foi encontrada no promotor ou na região codificante. As árvores filogenéticas pelos métodos de máxima parcimônia, máxima verossimilhança e neighbor-join apresentaram topologias semelhantes com três grande clados monofiléticos reconhecíveis e com a espécie Macaca fascicularis apresentando um perfil polifilético. O teste de seleção positiva pelos métodos de Nei-Gojobori, máxima verossimilhança por ramos e máxima verossimilhança por sítios não demonstraram, estatisticamente, à ação da seleção positiva sobre o gene DARC. Porém, o teste de máxima verossimilhança por sítio em domínios demonstrou que existem regiões do gene DARC sujeitas à diferentes pressões seletivas, mas também falhou em detectar a assinatura da seleção positiva. Os resultados indicam a presença da seleção darwiniana sobre a região de ligação do P. vivax, porém os testes de máxima verossimilhança utilizados, aparentemente, não possuem poder suficiente para detectar a sua assinatura. Além disso, os resultados sugerem que a região de ligação do P. vivax está sob influência de duas pressões seletivas antagônicas (seleção positiva exercida pelo parasita e seleção purificadora exercida pelas quimiocinas) o que pode, também, explicar a não detecção da seleção positiva.Genes involved in pathogen-host interactions are strongly affected by positive natural selection. The gene of blood Duffy antigen, also known as DARC (Duffy Antigen Receptor for Chemokines), has an important role in the invasion of red blood cells by the parasites that cause malaria, Plasmodium vivax in humans and Plasmodium knowlesi in other primates. The structure of the DARC gene is known, it was mapped in 1q22-q23 region of chromosome 1, and is composed by two exons separated by a large intron. In an African population a nucleic acid deletion in GATA-1 of the gene promoter is responsible for the non-expression of DARC on red blood cells and the resistance to invasion by P. vivax. Moreover, the DARC antigen acts as a promiscuous receptor for chemokines and is expressed in red blood cells, endothelial venules cells and other tissues. Because of this dual role, in this study we sequenced homologous regions of the DARC gene in monkeys of the New and Old World and using statistical methods we tried to detect positive natural selection in their evolutionary history. New mutations were not found at promoter or in coding region. The phylogenetic trees by the methods of maximum parsimony, maximum likelihood and neighbor-join showed similar topologies with three large monophyletic clades recognizable and with the Macaca fascicularis showing a poliphyletic profile. The test of positive selection by the methods of Nei-Gojobori, maximum likelihood by branchs and maximum likelihood by sites not shown, statistically, the action of positive selection on the DARC gene. But the maximum likelihood test using sites divided in domains showed that some regions of the DARC gene are subject to different selective pressures, but also failed to detect the signature of positive selection. The results indicate the presence of darwinian selection on P. vivax binding region, but the maximum likelihood tests used, apparently, do not have enough power to detect its signature. Moreover, the results suggest that P. vivax binding region is under the influence of two opposing selective pressures (positive selection exerted by the parasite and purifying selection exerced by chemokines) that can also explain the non-detection of positive selection

Molecular evolution of a malaria resistance gene (DARC) in primates

Genes involved in host-pathogen interactions are often strongly affected by positive natural selection. The Duffy antigen, coded by the Duffy antigen receptor for chemokines (DARC) gene, serves as a receptor for Plasmodium vivax in humans and for Plasmodium knowlesi in some nonhuman primates. In the majority of sub-Saharan Africans, a nucleic acid variant in GATA-1 of the gene promoter is responsible for the nonexpression of the Duffy antigen on red blood cells and consequently resistance to invasion by P. vivax. The Duffy antigen also acts as a receptor for chemokines and is expressed in red blood cells and many other tissues of the body. Because of this dual role, we sequenced a 3,000-bp region encompassing the entire DARC gene as well as part of its 5' and 3' flanking regions in a phylogenetic sample of primates and used statistical methods to evaluate the nature of selection pressures acting on the gene during its evolution. We analyzed both coding and regulatory regions of the DARC gene. The regulatory analysis showed accelerated rates of substitution at several sites near known motifs. Our tests of positive selection in the coding region using maximum likelihood by branch sites and maximum likelihood by codon sites did not yield statistically significant evidence for the action of positive selection. However, the maximum likelihood test in which the gene was subdivided into different structural regions showed that the known binding region for P. vivax/P. knowlesi is under very different selective pressures than the remainder of the gene. In fact, most of the gene appears to be under strong purifying selection, but this is not evident in the binding region. We suggest that the binding region is under the influence of two opposing selective pressures, positive selection possibly exerted by the parasite and purifying selection exerted by chemokines.PSC CUNYPSC CUNYC3IRG from City University of New YorkC3IRG from City University of New YorkINCTC/CNPqINCTC/CNPqCTC/CEPID/FAPESPCTC/CEPID/FAPESPRegional Blood Center of Ribeirao PretoRegional Blood Center of Ribeirao PretoMillennium Institute/CNPqMillennium Institute/CNP

Biochemical responses in armored catfish (Pterygoplichthys anisitsi) after short-term exposure to diesel oil, pure biodiesel and biodiesel blends

Biodiesel fuel is gradually replacing petroleum-based diesel oil use. Despite the biodiesel being considered friendlier to the environment, little is known about its effects in aquatic organisms. In this work we evaluated whether biodiesel exposure can affect oxidative stress parameters and biotransformation enzymes in armored catfish (Pterygoplichthys anisitsi, Loricariidae), a South American endemic species. Thus, fish were exposed for 2 and 7d to 0.01mLL-1 and 0.1mLL-1 of pure diesel, pure biodiesel (B100) and blends of diesel with 5% (B5) and 20% (B20) biodiesel. Lipid peroxidation (malondialdehyde) levels and the activities of the enzymes glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase were measured in liver and gills. Also, DNA damage (8-oxo-7, 8-dihydro-2'-deoxyguanosine) levels in gills and 7-ethoxyresorufin-O-deethylase activity in liver were assessed. Pure diesel, B5 and B20 blends changed most of the enzymes tested and in some cases, B5 and B20 induced a higher enzyme activity than pure diesel. Antioxidant system activation in P. anisitsi was effective to counteract reactive oxygen species effects, since DNA damage and lipid peroxidation levels were maintained at basal levels after all treatments. However, fish gills exposed to B20 and B100 presented increased lipid peroxidation. Despite biodiesel being more biodegradable fuel that emits less greenhouse gases, the increased lipid peroxidation showed that biofuel and its blends also represent hazards to aquatic biota. © 2013 Elsevier Ltd

Biochemical responses in mussels Perna perna exposed to diesel B5

In Brazil B5 blend (5% biodiesel and 95% diesel oil) has been adopted as mandatory fuel since 2010 for automotive vehicles. Since little is known about the effects of B5 exposure can promote on antioxidant system of marine biota this study aimed to assess if B5 can generate modifications in antioxidant parameters of mussels Perna perna. To address this question mussels were exposed to two concentrations of B5 (0.01 mL L(-1) and 0.1 mL L(-1)) for 6h, 12h, 48 h and 168 h. Then the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were evaluated in gills and digestive gland as well as the contents of glutathione (GSH) and lipid peroxidation by measuring the malondialdehyde concentration (MDA). In the gills, GST activity decreased after 48 h and GR after 12h of exposure to B5. In digestive glands, the activities of SOD, GPx and GR were changed due to treatments. GSH concentration increased in digestive gland after 6h and 12h and in gills after 48 h for B5 0.1 mL L(-1) and after 168 h in the digestive gland for B5 0.01 mL L(-1) treatment. No lipid peroxidation was detected. The integrated biomarker response index (IBR) evidenced a B5 effect in the digestive gland after 168 h of exposure. Regarding the experimental conditions and species used in this study, long-term exposure to B5 is apparently more likely to affect the parameters tested in P. perna mussels.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Osteoblastic differentiation of bone marrow mesenchymal stromal cells in Bruck Syndrome

Zanette, D. L. Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-04-11T16:41:40Z No. of bitstreams: 2 Kaneto CM Osteoblastic differentiation....pdf: 1456492 bytes, checksum: 51e5345c47756e1ab4f1bd7a700f9e95 (MD5) Kaneto CM Osteoblastic differentiation....pdf: 1456492 bytes, checksum: 51e5345c47756e1ab4f1bd7a700f9e95 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-04-11T17:12:03Z (GMT) No. of bitstreams: 2 Kaneto CM Osteoblastic differentiation....pdf: 1456492 bytes, checksum: 51e5345c47756e1ab4f1bd7a700f9e95 (MD5) Kaneto CM Osteoblastic differentiation....pdf: 1456492 bytes, checksum: 51e5345c47756e1ab4f1bd7a700f9e95 (MD5)Made available in DSpace on 2017-04-11T17:12:03Z (GMT). No. of bitstreams: 2 Kaneto CM Osteoblastic differentiation....pdf: 1456492 bytes, checksum: 51e5345c47756e1ab4f1bd7a700f9e95 (MD5) Kaneto CM Osteoblastic differentiation....pdf: 1456492 bytes, checksum: 51e5345c47756e1ab4f1bd7a700f9e95 (MD5) Previous issue date: 2016FAPESP, CNPq, and Center for Cell-based Therapy.Universidade de São Paulo. Escola Medica de Ribeirão Preto. Departamento de Genetica. Ribeirão Preto, SP, Brasil / Universidade Estadual de Santa Cruz. Biological Science. Ilheus, BA, BrazilUniversidade Estadual do Sudoeste da Bahia. Department of Natural Science. Vitória da Conquista, BA, BrazilUniversidade de São Paulo. Escola Medica de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / National Institute of Science and Technology in Cell Therapy. Regional Blood Center of Ribeirão Preto. Ribeirão Preto, SP, BrazilThe Rockefeller University. Laboratory of Molecular Immunology. New York, NY, USAUniversidade Federal de Sergipe. Department of Internal Medicine and Pathology. Aracaju, SE, BrazilThe Rockefeller University. Laboratory of Molecular Immunology. New York, NY, USAUniversidade Estadual de Santa Cruz. Biological Science. Ilheus, BA, BrazilNational Institute of Science and Technology in Cell Therapy. Regional Blood Center of Ribeirão Preto. Ribeirão Preto, SP, BrazilUniversidade de São Paulo. Escola Medica de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Department of Clinical Medicine. Ribeirão Preto, SP, BrazilUniversidade de São Paulo. Escola Medica de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Universidade Estadual de Santa Cruz. Biological Science. Ilheus, BA, BrazilOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus. Methods: We analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. Results: No mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different. Conclusions: Our work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patient