Quantifying Aotus monkey cytokines by real-time quantitative RT-PCR

Abstract

Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-β and IFN-γ). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/μl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.

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.