Inhibition of glutathione synthesis as a chemotherapeutic strategy for leishmaniasis.
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This study focuses on the use of buthionine sulphoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, on Leishmania donovani growth. The effect of BSO on amastigote multiplication within macrophages showed that 5 mM BSO decreased infectivity by about 50% and the mean number of amastigotes per 100 infected macrophages by 21%. The mechanism may be that BSO resulted in enhanced nitric oxide (NO) levels within macrophages, probably due to inhibition of GSH content since GSH (10 mm) given after BSO treatment led to a decrease in NO compared to macrophages treated with BSO alone which were preexposed to the Leishmania surface molecule lipophosphoglycan. (+info)
Susceptible hosts: a resort for parasites right in the eye of the immune response.
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Trypanosomatid protozoan parasites express an aggressive strategy of parasitism by infecting host macrophages and inducing extensive T-lymphocyte activation. One goal of such strategy is to drive the immune response of genetically susceptible hosts to a state of unresponsiveness regarding parasite killing. Unresponsiveness is achieved through different mechanisms, depending on the parasite species. In this brief review, recent findings on the molecular and cellular bases of the parasites' exploitation of host immune responses are discussed. (+info)
Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.
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The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species. (+info)
Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis.
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BACKGROUND: Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. MATERIALS AND METHODS: Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apoptosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V. RESULTS: Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-II-mediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 microM and 45.5 microM, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. CONCLUSION: Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design. (+info)
Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages.
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Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites. (+info)
Identification of vaccine candidates for experimental visceral leishmaniasis by immunization with sequential fractions of a cDNA expression library.
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Visceral leishmaniasis caused by the intracellular parasite Leishmania donovani is a significant public health problem in many regions of the world. Because of its large genome and complex biology, developing a vaccine for this pathogen has proved to be a challenging task and, to date, protective recombinant vaccine candidates have not been identified. To tackle this difficult problem, we adopted a reductionist approach with the intention of identifying cDNA sequences in an L. donovani amastigote cDNA library that collectively or singly conferred protection against parasite challenge in a murine model of visceral leishmaniasis. We immunized BALB/c mice with plasmid DNA isolated and pooled from 15 cDNA sublibraries ( approximately 2,000 cDNAs/sublibrary). Following systemic challenge with L. donovani, mice immunized with 6 of these 15 sublibraries showed a significantly reduced (35- to 1,000-fold) hepatic parasite burden. Because of the complexity and magnitude of the sequential fractionation-immunization-challenge approach, we restricted our attention to the two sublibraries that conferred the greatest in vivo protection. From one of these two sublibraries, we identified several groups of cDNAs that afforded protection, including a set of nine novel cDNAs and, surprisingly, a group of five cDNAs that encoded L. donovani histone proteins. At each fractionation step, the cDNA sublibraries or the smaller DNA fractions that afforded in vivo protection against the parasite also induced in vitro parasite-specific T helper 1 immune responses. Our studies demonstrate that immunization with sequential fractions of a cDNA library is a powerful strategy for identifying anti-infective vaccine candidates. (+info)
Cyclooxygenase-2 expression in macrophages: modulation by protein kinase C-alpha.
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Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-alpha in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-gamma, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-alpha-overexpressing clones, IFN-gamma-induced COX-2 expression was up-regulated in DN PKC-alpha-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-gamma-induced COX-2 mRNA and protein levels in DN PKC-alpha-overexpressing clones. Taken together, these results suggest a role for PKC-alpha in the modulation of LPS- and IFN-gamma-induced COX-2 expression, as well as in IFN-gamma-induced PGE2 secretion. (+info)
Visceral leishmaniasis in mice devoid of tumor necrosis factor and response to treatment.
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Tumor necrosis factor (TNF)-deficient mice were challenged with Leishmania donovani to characterize TNF in the response of visceral intracellular infection to antileishmanial chemotherapy. In wild-type controls (i) liver infection peaked at week 2 and resolved, (ii) discrete liver granulomas developed at weeks 2 to 4 and involuted, and (iii) leishmanicidal responses to antimony (Sb), amphotericin B (AmB), and miltefosine were intact. In TNF knockout (KO) mice (i) initial liver infection was unrestrained, plateaued, and then declined somewhat by week 6, (ii) an absent early granulomatous reaction abruptly accelerated with striking tissue inflammation, widespread hepatic necrosis, and 100% mortality by week 10, and (iii) while the initial response to AmB and miltefosine was intact, killing induced by Sb therapy was reduced by >50%. Although initial AmB treatment during weeks 2 to 3 killed 98% of liver parasites, 75% of AmB-treated KO mice subsequently relapsed and died by week 12; however, additional maintenance AmB preserved long-term survival. These results for a model of visceral infection indicate that endogenous TNF is required early on to control intracellular L. donovani, support granuloma development, and mediate optimal initial effects of Sb and prevent relapse after ordinarily curative AmB treatment. A compensatory, TNF-independent antileishmanial mechanism developed in TNF KO mice; however, its effect was uncontrolled fatal inflammation. Chemotherapeutic elimination of the parasite stimulus reversed the hyperinflammatory response and preserved survival. (+info)