Molecular and immunological tools for the evaluation of the cellular immune response in the neotropical monkey Saimiri sciureus, a non-human primate model for malaria research.
BACKGROUND
The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus.


METHODS
Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences.


RESULTS
The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-β, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture.


CONCLUSIONS
Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. ericcio@ioc.fiocruz.br.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. ericcio@ioc.fiocruz.br.
Evelyn K P
EK
Riccio
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. riccio@ioc.fiocruz.br.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. riccio@ioc.fiocruz.br.
Lilian R
LR
Pratt-Riccio
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. cesare@ioc.fiocruz.br.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. cesare@ioc.fiocruz.br.
Cesare
C
Bianco-Júnior
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. violette.sanchez@sanofipasteur.com.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. violette.sanchez@sanofipasteur.com.|Present address: Research Department, Sanofi Pasteur, Lyon, France. violette.sanchez@sanofipasteur.com.
Violette
V
Sanchez
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. prtotino@ioc.fiocruz.br.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. prtotino@ioc.fiocruz.br.
Paulo R R
PR
Totino
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. leojmc@ioc.fiocruz.br.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. leojmc@ioc.fiocruz.br.
Leonardo J M
LJ
Carvalho
Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, Rio de Janeiro, RJ, CEP: 21040-900, Brazil. malaria@fiocruz.br.|Centro de Pesquisa, Diagnóstico e Treinamento em Malária (CPD-Mal), Fiocruz, Reference Centre for Malaria in the Extra-Amazonian Region for the Secretary for Health Surveillance, Ministry of Health, Rio de Janeiro, RJ, Brazil. malaria@fiocruz.br.
Cláudio Tadeu
CT
Daniel-Ribeiro
D000818:Animals; D016207:Cytokines / Q000276:immunology*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D007111:Immunity, Cellular; D007963:Leukocytes, Mononuclear; D008288:Malaria / Q000276:immunology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012453:Saimiri / Q000276:immunology*
D016207:Cytokines