ABSTRACT
BACKGROUND: Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. METHODOLOGY/PRINCIPLE FINDINGS: We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. CONCLUSIONS/SIGNIFICANCE: In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level.
Subject(s)
Scrub Typhus , Humans , CD4-Positive T-Lymphocytes , Leukocytes, Mononuclear , CD8-Positive T-Lymphocytes , CytokinesABSTRACT
Antimalarials targeting the ubiquinol-oxidation (Qo) site of the Plasmodium falciparum bc1 complex, such as atovaquone, have become less effective due to the rapid emergence of resistance linked to point mutations in the Qo site. Recent findings showed a series of 2-aryl quinolones mediate inhibitions of this complex by binding to the ubiquinone-reduction (Qi) site, which offers a potential advantage in circumventing drug resistance. Since it is essential to understand how 2-aryl quinolone lead compounds bind within the Qi site, here we describe the co-crystallization and structure elucidation of the bovine cytochrome bc1 complex with three different antimalarial 4(1H)-quinolone sub-types, including two 2-aryl quinolone derivatives and a 3-aryl quinolone analogue for comparison. Currently, no structural information is available for Plasmodial cytochrome bc1. Our crystallographic studies have enabled comparison of an in-silico homology docking model of P. falciparum with the mammalian's equivalent, enabling an examination of how binding compares for the 2- versus 3-aryl analogues. Based on crystallographic and computational modeling, key differences in human and P. falciparum Qi sites have been mapped that provide new insights that can be exploited for the development of next-generation antimalarials with greater selective inhibitory activity against the parasite bc1 with improved antimalarial properties.
ABSTRACT
BACKGROUND: The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate. METHODS: Native PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized. RESULTS: PfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4-9, optimal salt concentration of 100-200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner. CONCLUSION: PfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.
