Short communicationQuantifying Aotus monkey cytokines by real-time quantitative RT-PCR
Introduction
Aotus spp. monkeys have been successfully used as experimental models in malaria for studying potential pharmacological substances, disease progression and vaccine efficacy [1].
Despite malaria being the world's most important tropical parasitic disease [2], there is a lack of knowledge regarding specific immune mechanisms leading to protection against malarial infection and protective mechanisms elicited after immunization with potential vaccine candidates. Establishing the correlation between a given Th1/Th2 profile for dissecting the mechanisms of anti-malarial immunity is of major importance in obtaining a successful vaccine against malaria [3] and understanding the progress and pathogenesis of natural or induced infection [4]. The use of a recently developed PCR technique [5], [6] for quantifying Aotus monkey cytokine mRNAs might allow a correlation to be established between Th1 (IFN-γ and TNF-β) and Th2 (IL-4 and IL-10) profiles and protection after vaccination with anti-malarial vaccine candidates.
Several semi-quantitative techniques have been described which are time-consuming, difficult to perform and require post-reaction PCR product manipulation [7]. Real-time quantitative RT-PCR is considered the most sensitive and accurate technique for cytokine quantification [8], [9], measuring PCR product accumulation during the reaction's exponential phase.
Accurate and sensitive quantification of these cytokines in Aotus monkeys will provide greater insight into the immunological processes underlying protection after immunization with malaria vaccine candidates.
Section snippets
Results and discussion
Primer and probes which performed best in real-time amplification were: IFN-γ Forward (F) 5′TGGAGACCATCAAGGAAGACA3′, Reverse (R) 5′ACAGTTCAGCCATCACTTGGA3′ and Taqman Probe (P) 5′TTGAATGTCCAGCGCAAAG CAATACA3′; TNF-β Forward 5′GCTATCCACCCACACAGATGG3′, Reverse 5′CGAAGGCTCCAAAGAAGACA3′ and Probe 5′CATCCACCACCTAGTCCTC AGCCCTAGT3′; IL-4 Forward 5′AGGGCTGCGACTGTGCTC3′, Reverse 5′CGTACTCTGGTTGGCTTCCTTCA3′ and Probe 5′GGACACTCGCTGCCTGGGTGC3′; IL-10 Forward 5′CCCTGTGAAAACAAGAGCAAGG3′, Reverse
Aotus monkeys
Aotus nancymaae monkeys from our experimental primate station in Leticia, Amazonas, Colombia, were used for this study. Monkey maintenance and care complied with the National Institute of Health guidelines for the use of laboratory animals under the supervision of the Colombian Wildlife Corporation (CORPOAMAZONIA).
PCR primer design
Reported human and Aotus IL-4, IL-10, IFN-γ [12] and TNF-β gene sequences (GenBank accession number AY373461) were aligned; Primer Express Software (Applied Biosystems, Foster City,
Acknowledgements
This work was supported by the Presidency of the Republic of Colombia, the Colombian Ministry of Health and the Spanish International Cooperation Agency (AECI). We would like to thank Verónika Franco, Josefa Rodriguez, Pilar Martinez and Jason Garry for their invaluable help and collaboration with some aspects of this work.
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