title: Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.
abstract: Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs 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 the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4(+) and CD8(+)) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-β-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72 h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient 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 could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4(+) and/or CD8(+) T cells.
authors.affiliation: Laboratório de Biologia das Interações Celulares, Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Laboratório de Biotecnologia, Departamento de Ciências Agrárias e Tecnológicas, Universidade Federal do Tocantins, Gurupi, Tocantins, Brazil; Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas/NUPEB, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Kelvinson Fernandes
authors.initials: KF
authors.lastname: Viana
authors.forename: Rodrigo Dian Oliveira
authors.initials: RD
authors.lastname: Aguiar-Soares
authors.forename: Bruno Mendes
authors.initials: BM
authors.lastname: Roatt
authors.forename: Lucilene Aparecida
authors.initials: LA
authors.lastname: Resende
authors.forename: Denise
authors.initials: D
authors.lastname: da Silveira-Lemos
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Corrêa-Oliveira
authors.forename: Olindo Assis
authors.initials: OA
authors.lastname: Martins-Filho
authors.forename: Sandra Lima
authors.initials: SL
authors.lastname: Moura
authors.forename: Marcos Santos
authors.initials: MS
authors.lastname: Zanini
authors.forename: Márcio Sobreira Silva
authors.initials: MS
authors.lastname: Araújo
authors.forename: Alexandre Barbosa
authors.initials: AB
authors.lastname: Reis
authors.forename: Rodolfo Cordeiro
authors.initials: RC
authors.lastname: Giunchetti
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology*; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004285:Dogs / Q000097:blood*; D005260:Female; D007891:Leishmania / Q000145:classification*; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D008297:Male; D009000:Monocytes / Q000166:cytology / Q000502:physiology*
keywords: CD4 and CD8 T cells; Canine visceral leishmaniasis; In vitro microbicidal analysis; Leishmania chagasi; Macrophage
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