Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 10 4 parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of Leishmania spp .Leishmania spp. are intracellular protozoa that affect humans and dogs worldwide and are transmitted by the bite of hematophagous sand flies. They cause a large spectrum of diseases, ranging from spontaneously healing skin lesions to fatal visceral symptoms, if left untreated. Two million new human cases arise every year, and at least 350 million people are exposed to the risk of the Leishmania parasite infection (2, 9). Experimental hosts, such as laboratory mice, are largely used to study the immunobiology of these parasites and to screen the efficacy of newly developed drugs and vaccines (4,11,20,27). Most of those studies require detection and quantitation of the Leishmania burdens in different mouse tissues. This is still routinely performed by culture-based techniques (6, 28), which have several limitations, in particular, the amount of time required and the putative presence of viable but noncultivable parasites, such as persistent parasites (1). PCR-based methods for detecting Leishmania species have been developed to amplify rRNA genes, miniexon genes, kinetoplast DNA (kDNA), and repetitive nuclear sequences (15,18,24,26). Recently, we have developed a PCR-based assay with which to quantify the parasite load in mice infected with Leishmania major (23) by using primers from the conserved sequences of kDNA. However, this technique is still cumbersome as it requests agarose gel image analysis.A more rapid alternative is real-time quantitative PCR, which quantifies DNA...

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e Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the diseaserelevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.

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In their mammalian hosts, Leishmania are obligate intracellular parasites that mainly reside in macrophages. They are also phagocytosed by dendritic cells (DCs), which play decisive roles in the induction and shaping of T cell-dependent immune responses. Little is known about the role of DCs in the Leishmania life cycle. Here, we examined the ability of mouse bone marrow-derived DCs to serve as hosts for L. amazonensis. Both infective stages of Leishmania (metacyclic promastigotes and amastigotes) could be phagocytosed by DCs, regardless of whether they had previously been experimentally opsonized with either the complement C3 component or specific antibodies. Parasites could survive and even multiply in these cells for at least 72 hours, within parasitophorous vacuoles displaying phagolysosomal characteristics and MHC class II and H-2M molecules. We then studied the degree of maturation reached by infected DCs according to the parasite stage internalised and the type of opsonin used. The cell surface expression of CD24, CD40, CD54, CD80, CD86, OX40L and MHC class II molecules was barely altered following infection with unopsonized promastigotes or amastigotes from nude mice or with C3-coated promastigotes. Even 69 hours post-phagocytosis, a large proportion of infected DCs remained phenotypically immature. In contrast, internalisation of antibody-opsonized promastigotes or amastigotes induced DCs to mature rapidly, as shown by the over-expression of costimulatory, adhesion and MHC class II molecules. Thus, in the absence of specific antibodies (e.g. shortly after infecting naive mammals), infected DCs may remain immature or semi-mature, meaning that they are unable to elicit an efficient anti-Leishmania T cell response. Absence of DC maturation or delayed/incomplete DC maturation could thus be beneficial for the parasites, allowing their establishment and amplification before the onset of immune responses.