antimony-potassium-tartrate and miltefosine

The parasite

Topics: Animals; Antimony Potassium Tartrate; Cytosol; Drug Resistance; Female; Host-Parasite Interactions; Leishmania donovani; Leishmaniasis, Visceral; Macrophages, Peritoneal; Male; Mice, Inbred BALB C; Mitochondrial Proteins; Oxidative Stress; Peroxidases; Phosphorylcholine; Protozoan Proteins; Reactive Oxygen Species; Trypanocidal Agents

Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 μM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 μM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

Topics: Adolescent; Adult; Aged; Animals; Antimony Potassium Tartrate; Antiprotozoal Agents; Benzoates; Female; Flow Cytometry; Fluorescein; Humans; Imidazoles; In Vitro Techniques; Leishmania; Leishmaniasis, Visceral; Macrophages, Peritoneal; Male; Mice; Mice, Inbred BALB C; Middle Aged; Monocytes; Nitric Oxide; Phosphorylcholine; Species Specificity; Young Adult

In the present study, we selected in vitro populations of Leishmania Viannia guyanensis, L.V. braziliensis, L. Leishmania amazonensis and L.L. infantum chagasi that were resistant to potassium antimony tartrate (SbIII). The resistance index of these populations varied from 4- to 20-fold higher than that of their wild-type counterparts. To evaluate the stability of the resistance phenotype, these four resistant populations were passaged 37 to 47 times in a culture medium without SbIII. No change was observed in the resistance indexes of L.V. guyanensis (19-fold) and L.L. infantum chagasi (4-fold). In contrast, a decrease in the resistance index was observed for L.V. braziliensis (from 20- to 10-fold) and L.L. amazonensis (from 6- to 3-fold). None of the antimony-resistant populations exhibited cross-resistance to amphotericin B and miltefosine. However, the resistant populations of L.V. braziliensis, L.L. amazonensis and L.L. infantum chagasi were also resistant to paromomycin. A drastic reduction was observed in the infectivity in mice for the resistant L.V. guyanensis, L.L. amazonensis and L.V. braziliensis populations. The SbIII-resistant phenotype of L.V. braziliensis was stable after one passage in mice. Although the protocol of induction was the same, the SbIII-resistant populations showed different degrees of tolerance, stability, infectivity in mice and cross-resistance to antileishmanial drugs, depending on the Leishmania species.

Topics: Amphotericin B; Animals; Antimony Potassium Tartrate; Antiprotozoal Agents; Culture Media; Drug Resistance; Inhibitory Concentration 50; Leishmania; Leishmaniasis; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Paromomycin; Phenotype; Phosphorylcholine; Selection, Genetic; Serial Passage; Spleen; Virulence

Resistance to treatment is a growing problem in efforts to control Old World leishmaniasis. Parasites resistant to new therapeutics such as miltefosine have not been reported from the field yet but based on experimental evidence, may appear soon. Therefore, we attempted to identify genetic markers that may correlate with miltefosine resistance. Using a functional cloning approach, we have isolated a gene from Leishmania infantum that, upon over-expression, confers protection not only against miltefosine, but also against Sb(III), the active principle of anti-leishmanial antimonials. The gene encodes a very large putative polypeptide of 299 kDa that shows no similarities to known proteins or functional motifs. Database mining and karyotyping experiments suggest that in L. infantum this gene is part of a 44-kbp duplicated region that is found on two separate chromosomes, CHR08 and CHR29.

Topics: Animals; Antimony Potassium Tartrate; Cosmids; Drug Resistance; Leishmania infantum; Leishmaniasis, Visceral; Phosphorylcholine; Protozoan Proteins; Reverse Transcriptase Polymerase Chain Reaction