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To study the antigens secteted by promastigote and amastigote forms of Leishmania infantumwhich are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitrocultured parasites, metabolically labelled with [35S] methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitroculture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secteted antigens of the amastigote forms are immunoprecipitated with eithet human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitrolife cycle.

ABSTRACTWe previously documented the induction of Leishmaniaamastigote apoptosis by trivalent antimony (SbIII) and nitric oxide (NO). We demonstrate here that SbIII-resistant amastigotes were resistant to NO toxicity when delivered extracellularly by NO donors or intracellularly via macrophage activation. Shared biochemical targets for SbIII and NO resistance in Leishmaniaare discussed.

Immunopathology of Chagas' disease in Balb/c mice infected with 2 Trypanosoma cruzi clones, belonging to the T. cruzi I lineage and presenting different in vitro virulence (P/209 cl1>SO34 cl4) was compared. In the acute phase, evading mechanisms such as parasite-induced lymphocyte polyclonal activation and T cell immunosuppression were higher in mice infected with the clone giving a higher parasitaemia (P/209 cl1). A similar increase of non-specific isotypes was observed in both infections with IgG2a prevalence. Interestingly, CD8+ cell hypercellularity and lymphocyte immunosuppression were observed during the chronic phase (245 days post-infection) in mice infected by the most virulent clone. In the same way, the parasite-specific antibody response was more intense in P/209 cl1-infected mice over the acute phase. During the chronic phase this response remarkably dropped down in SO34 cl4-infected mice exclusively. Finally, P/209 cl1-infected mice presented a more severe inflammation and tissue damage in heart and quadriceps than SO34 cl4-infected mice. This comparative study showed differences between the two clones: a higher virulence in vivo being clearly associated with a greater ability to induce evasion mechanisms and severe tissue damage.

ABSTRACTOur study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B3. A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmaniaamastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania majorSIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantumwere adapted to survive in culture medium containing 4, 20, 30 and 120 μg/ml of potassium antimonyl tartrate Sb(III). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 μg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 μM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

ABSTRACTThe mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmaniaspecies, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantumcultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC50s], about 1.8 mg/ml and 22 μg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes ofL. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plusm-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantumamastigotes (IC50s, of 0.54 and 1.32 μg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to theLeishmaniaamastigote stage.

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microassay, previously described as a means of quantifying Leishmania amazonensis in vitro at the amastigote stage (D. Sereno and J. L. Lemesre, Parisitol. Res., in press), we have compared the activities of seven drugs, including those currently used to treat leishmaniasis, against axenically grown amastigote and promastigote forms of three Leishmania species (L. amazonensis, L. mexicana, and L. infantum, responsible for diffuse cutaneous, cutaneous, and visceral leishmaniasis, respectively). The ability of axenically cultured amastigote organisms to be used in an investigation of antileishmanial agents was first evaluated. We have confirmed the toxicities of sodium stibogluconate (Pentostam), pentamidine, and amphotericin B to active and dividing populations of axenically cultured amastigotes. The toxicity of potassium antimonyl tartrate trihydrate, which is generally higher than that of Pentostam, seemed to indicate that pentavalent antimony can be metabolized in vivo to compounds, possibly trivalent in nature, which are more active against the amastigote organisms. When the drug susceptibilities of parasites at both stages were compared, great variations were found for all the drugs studied. These major differences, which show the specific chemosusceptibility of the parasite at the mammalian stage, demonstrate the potential of using cultured amastigotes instead of promastigotes in a drug-screening procedure for early detection. This in vitro model may help in the isolation of active compounds, particularly those with low-grade activities, against the mammalian stage of the parasite.

ABSTRACTThe in vitro growth of promastigote cells of Leishmania amazonensiswas found to strongly depend on interactions among strains that differed in their pentamidine resistance. In particular, the growth of resistant strains was reduced when they shared the same environment with a less-resistant strain.