CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

BACKGROUND: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. RESULTS: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS. CONCLUSION: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications

Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites

Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi's such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ∼370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa

Computational Reverse-Engineering of a Spider-Venom Derived Peptide Active Against Plasmodium falciparum SUB1

merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity. culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1.Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering

Centromeric Plasmids and Artificial Chromosomes: New Kids on the Plasmodium Transfection Block

International audienceIn this issue of Cell Host & Microbe, Iwanaga and colleagues (Iwanaga et al., 2010) report on the construction of plasmids and artificial chromosomes that are stably maintained throughout the Plasmodium life cycle. These new tools will have multiple applications, from episome-based genetic strategies to studies on telomere biology and antigenic variatio

METHODS FOR DETECTING VIRULENT PLASMODIUM, FOR EVALUATING PLASMODIUM VIRULENCE, AND FOR SCREENING NEW DRUGS EMPLOYING THE 3'UTR OF PLASMODIUM SUB2 AND THE PLASMODIUM SUB2 SERINE PROTEASE: WO2006/120579

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Screening and Evaluation of Inhibitors of Plasmodium falciparum Merozoite Egress and Invasion Using Cytometry

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Computational Design of Protein-Based Inhibitors of Plasmodium vivax Subtilisin-Like 1 Protease

International audienceBackground: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target.Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range.Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications

Data from: Computational design of protein-based inhibitors of Plasmodium vivax subtilisin-like 1 protease

Background: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target. Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range. Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications

Cytometric measurement of in vitro inhibition of Plasmodium falciparum field isolates by drugs: a new approach for re-invasion inhibition study.

International audienceBACKGROUND: A flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum. Standard [3H]-hypoxanthine incorporation assay gives only information on inhibition of maturation by drugs. This method is usable on field isolates and provides data on both inhibition of maturation and re-invasion. METHODS: The method is based on the staining of parasites with hydroethidine (HE) and thiazole orange (TO) which allow differential identification of early, trophozoite and late stage of the parasite by flow cytometry. Late stages of the parasites are obtained by incubation in culture for 24 hours. Reinvasion is followed by culturing parasitized red blood cells for 24 h more. RESULTS: Compared to the standard [3H]-hypoxanthine incorporation assay, it gave similar results as expressed by 50% inhibitory concentrations for chloroquine of laboratory strains and "field" isolates. The effect of quinine on the schizont-ring transition was also explored using this method. First data on the inhibition of re-invasion induced by quinine are presented for both P. falciparum-cultured strains and field isolates. DISCUSSION: This method is simple to use event for field isolate study. It is suitable to analyse effect of drugs on steps of the parasite life cycle different for the maturation one. Using this method quinine was found to have a inhibitory effect on re-invasion of red cells by Plasmodium

On the Knorr synthesis of 6-bromo-4-methylquinolin-2(1H)-one

International audienceIn the course of our work on infectious diseases, we were led to prepare 6-bromo-2-chloro-4-methylquinoline as a starting material. Since surprisingly little has been reported in the literature, the two synthetic steps to this compound were investigated. The synthesis involves a condensation between -ketoesters and 4-bromoaniline and the cyclization of the resulting anilides into 6-bromoquinolin-2(1H)-one otherwise known as the Knorr reaction. The 1H NMR monitoring of the first step allowed us to optimize the conditions leading specifically to the anilide without the occurrence of the alternative crotonate. To illustrate the scope of our finding, few additional anilides featuring electron attracting groups were prepared. The study of their cyclization revealed some unsuspected steric effect governing this second step. Aside from rectifying few claims in this chemistry, this study led to a three-step preparation of 6-bromo-2-chloro-4-methylquinoline in a 48 % overall yield from 4-bromoaniline