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

Malaria Journal

Research

Evelyn KP Riccio¹  Paulo RR Totino¹  Lilian R Pratt-Riccio¹  Leonardo JM Carvalho¹  Cláudio Tadeu Daniel-Ribeiro¹  Cesare Bianco-Júnior¹  Violette Sanchez² 

[1] Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, CEP: 21040-900, Rio de Janeiro, RJ, Brazil;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;Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Pavilhão Leônidas Deane, Salas 513-517, 5° andar Manguinhos, CEP: 21040-900, Rio de Janeiro, RJ, Brazil;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;Present address: Research Department, Sanofi Pasteur, Lyon, France;

关键词: Saimiri sciureus;    Immune response;    Malaria;    Cytokines;    Cellular response;   

DOI  :  10.1186/s12936-015-0688-1

 received in 2014-11-06, accepted in 2015-04-10,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundThe 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.MethodsBlood 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.ResultsThe 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.ConclusionsReal-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.

【 授权许可】

Unknown   
© Riccio et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.