Navegando por Autor "Viana, Kelvinson Fernandes"
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Item Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.(2013) Viana, Kelvinson Fernandes; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Resende, Lucilene Aparecida; Lemos, Denise da Silveira; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Moura, Sandra Aparecida Lima de; Zanini, Marcos Santos; Araújo, Márcio Sobreira Silva; Reis, Alexandre Barbosa; Giunchetti, Rodolfo CordeiroCanine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, anddogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L.infantum). Biomarkers in the canine immune system is an important technique in thecourse of developing vaccines and treatment strategies against CVL. New methodologiesfor studying the immune response of dogs during Leishmania infection and after receivingvaccines and treatments against CVL would be useful. In this context, we used peripheralblood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i)establishment of in vitro conditions of monocytes differentiated into macrophages infectedwith L. chagasi and (ii) purification procedures of T-cell subsets (CD4+and CD8+) usingmicrobeads. Our data demonstrated that after 5 days of differentiation, macrophages wereable to induce significant phagocytic and microbicidal activity after L. chagasi infectionand also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl- _-d-glucosaminidase (NAG) levels presented similar levels of macrophage cultureand L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was ahallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells wereobtained on a magnetic separation column. We concluded that monocytes differentiatedinto macrophages at 5 days and displayed an intermediate frequency of parasitism andparasite load 72 h after L. chagasi infection. Furthermore, the purification system usingcanine T-lymphocyte subsets obtained after 5 days of monocyte differentiation provedefficient for CD4 or CD8 T-cell purification (≥90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that couldbe incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidalpotential induced by specific CD4+and/or CD8+T cells.Item Canine visceral leishmaniasis biomarkers and their employment in vaccines.(2019) Giunchetti, Rodolfo Cordeiro; Silveira, Patricia; Resende, Lucilene Aparecida; Leite, Jaqueline Costa; Melo Júnior, Otoni Alves de Oliveira; Alves, Marina Luiza Rodrigues; Costa, Laís Moreira; Lair, Daniel Ferreira; Chaves, Vinícius Rossi; Soares, Ingrid dos Santos; Mendonça, Ludmila Zanandreis de; Lanna, Mariana Ferreira; Ribeiro, Helen Silva; Gonçalves, Ana Alice Maia; Santos, Thaiza Aline Pereira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Souza, Juliana Vitoriano de; Moreira, Nádia das Dores; Siqueira, Fernando Augusto Mathias; Cardoso, Jamille Mirelle de Oliveira; Vital, Wendel Coura; Galdino, Alexsandro Sobreira; Viana, Kelvinson Fernandes; Martins Filho, Olindo Assis; Lemos, Denise da Silveira; Dutra, Walderez Ornelas; Reis, Alexandre BarbosaThe natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitro co-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-γ and TNF-α and decreased levels in IL-4, TGF-β and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products.Item Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.(2013) Resende, Lucilene Aparecida; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Viana, Kelvinson Fernandes; Mendonça, Ludmila Zanandreis de; Lanna, Mariana Ferreira; Lemos, Denise da Silveira; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Fujiwara, Ricardo Toshio; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa; Giunchetti, Rodolfo CordeiroIn the studies presented here, dogs were vaccinated against Leishmania (Leishmania) cha-gasi challenge infection using a preparation of Leishmania braziliensis promastigote proteinsand saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1immune response that was characterized by increased levels of interleukin (IL-) 12 andinterferon gamma (IFN- _) production by peripheral blood mononuclear cells (PBMC) uponstimulation with soluble vaccine antigen. Importantly, results showed that this type ofresponsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasichallenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN- _ andconcomitant nitric oxide (NO) when stimulated with Leishmania antigens as comparedto PBMCs from respective control groups (saponin, LB- treated, or non-treated controldogs). Moreover, transforming growth factor (TGF)- _ decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysisrevealed decreased frequency of parasitism in the presence of vaccine antigen. It is con-cluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immuneresponse against L. chagasi challenge infection.Item Leishmania (Viannia) braziliensis : immunoblotting analysis for the detection of IgG subclasses in the diagnosis of symptomatic and asymptomatic dogs.(2010) Zanini, Marcos Santos; Viana, Kelvinson Fernandes; Reis, Alexandre Barbosa; Campos, Diefrey Ribeiro; Mussi, Jamili Maria Suhet; Zanini, Surama; Lemos, Elenice MoreiraDuring a seroepidemiological survey 2004–2006 from areas in Brazil endemic for American cutaneous leishmaniasis (ACL), serum samples from 10 dogs with ulcerated cutaneous lesions (S-ACL) and 52 asymptomatic dogs (AS-ACL) of unknown age and breed living in areas endemic for ACL were monitored for 1 year for ulcerated cutaneous lesions and immunoblotting using peroxidase-conjugated secondary anti-IgG, anti-IgG1 and anti-IgG2 dog antibodies. We reported that antibodies against Leishmania (Viannia) braziliensis in the sera of 22/52 dogs with asymptomatic disease showed intense reactivity to pep tides larger than 66 kDa. We believe that dogs harboring subclinical amastigotes show an immunoblotting profile similar to that of symptomatic animals because a dog with self-healing presented antigens greater than 66 kDa. Such patterns can be exploited for diagnostic and epidemiological research for leishmaniasis.Item Metodologia para ensaios de candidatos vacinais contra leishmaniose visceral canina pelo co-cultivo de linfócitos e macrófagos infectados com Leishmania chagasi.(Programa de Pós-Graduação em Biotecnologia. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2012) Viana, Kelvinson Fernandes; Giunchetti, Rodolfo CordeiroNos últimos 30 anos, a leishmaniose visceral (LV) tem avançado para áreas urbanas e periurbanas, apontando o cão como principal reservatório da L. chagasi (sin. L. infantum). Neste contexto, pesquisadores consideram como consenso que uma vacina contra a leishmaniose visceral canina (LVC) poderia ser uma importante ferramenta no controle tanto da leishmaniose humana como canina. Por esta razão, foi desenvolvido um novo método para avaliar o sistema imune de cães que poderia ser empregado como uma alternativa para se analisar imunogenicidade e níveis de proteção em candidatos vacinais contra a LVC. Para tanto, foram empregadas células mononucleares do sangue periférico CMSP de cães saudáveis para padronização de: (i) condições in vitro de cultivo de monócitos diferenciados em macrófagos de cães (MD-MC) infectados previamente com L. chagasi e avaliados após 3, 24, 48, 72 e 96h da infecção nos tempos de 2, 3, 4 e 5 dias de diferenciação do MD-MC (n=5 cães saudáveis); (ii) purificação de subpopulações de células T (CD4+ e CD8+) considerando a razão de linfócitos:macrófagos variando de 1:5 a 2:1 (n=12 cães saudáveis). Em todos os experimentos foram utilizados sobrenadante de cultura para analisar diferentes biomarcadores imunológicos, tais como: níveis de óxido nítrico, a produção de mieloperoxidase (MPO) e N-acetilglicosaminidase (NAG); citocinas do tipo 1 (IFN-, TNF-, IL-12), tipo 2 (IL-4) e imunomodulatórias (IL-10); e avaliada a frequência do parasitismo e carga parasitária em MD-MC infectados com L. chagasi. Os resultados revelaram que após 5 dias da diferenciação do MD-MC e 72h da infecção por L. chagasi foi observado um melhor perfil morfológico e da habilidade microbicida. Apesar dos níveis de NAG permanecerem praticamente inalterados, a análise do MPO revelou redução significativa no tempo de cinco dias de diferenciação dos MD-MC, indicando a baixa contaminação por granulócitos neste período. Os experimentos de co-cultivo empregando MD-MC e subpopulações de células T mostrou um perfil marcante nos biomarcadores imunológicos e na atividade microbicida, considerando a razão de linfócitos (CD4+ ou CD8+) : macrófagos de 1:2 e 1:1:1 de linfócitos (CD4+ e CD8+) : macrófagos. Assim, nestas condições do co-cultivo foi observado associação entre a produção de óxido nítrico e os níveis de citocinas do tipo 1, tipo 2 e imunomodulatórias com a manutenção de uma frequência de parasitismo intermediária e estabilização da carga parasitária em MD-MC infectados por L. chagasi. Os dados obtidos com a realização deste trabalho estimulam a continuidade dos estudos empregando esta metodologia de MD-MC co-cultivados com células T CD4+ e/ou CD8+ provenientes de CMSP de cães imunizados com candidatos vacinais contra LVC.Item Neutrophil properties in healthy and Leishmania infantum-naturally infected dogs.(2019) Wardini, Amanda Brito; Silva, Lucia Helena Pinto da; Nadaes, Natalia Rocha; Nascimento, Michelle Tanny Cunha do; Roatt, Bruno Mendes; Reis, Alexandre Barbosa; Viana, Kelvinson Fernandes; Giunchetti, Rodolfo Cordeiro; Saraiva, Elvira MariaVisceral leishmaniasis is a chronic disease that afects humans and dogs as well. Dogs, the domestic reservoir of Leishmania, play a central role in the transmission of visceral leishmaniasis, the most severe form of this disease. Neutrophils are the most abundant leukocytes in blood and interact with the parasite after infection. Here, we evaluate the efector properties of neutrophils from healthy and naturally Leishmania infantum-infected dogs. Our results showed that the parasite induced neutrophil extracellular trap (NET) release from neutrophils in both groups. Additionally, phagocytosis and NETs contributed diferently to parasite killing by neutrophils from healthy and infected animals, and IFN-γ, IL-8, IL-4 and TNF-α production by neutrophils from both groups were diferentially modulated by the parasite. Our results contribute to a better understanding of the complex role played by neutrophils in canine visceral leishmaniasis, which may favor the development of more efective therapies.