Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes.

OBJECTIVES: This study focuses on the importance of sterols in the action of miltefosine (hexadecylphosphocholine, HePC) against Leishmania donovani. METHODS: Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-beta-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC-lipid interactions. RESULTS: CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased. CONCLUSIONS: Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, (1)H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.

Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids.

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.

Miltefosine affects lipid metabolism in Leishmania donovani promastigotes.

Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active antileishmanial drug. Transient HePC treatment of Leishmania donovani promastigotes at 10 microM significantly reduced the phosphatidylcholine content and enhanced the phosphatidylethanolamine (PE) content in parasite membranes, suggesting a partial inactivation of PE-N-methyltransferase. Phospholipase D activity did not seem to be affected by HePC. In addition, the enhancement of the lysophosphatidylcholine content could be ascribed to phospholipase A2 activation. Moreover, transient HePC treatment had no effect on the fatty acid alkyl chain length or the fatty acid unsaturation rate. Concerning sterols, we found a strong reduction of the C24 alkylated sterol content, and the enhancement of the cholesterol content could be the result of the HePC condensation effect with sterols. Because some of the effects observed after transient HePC treatment were different from those previously observed in HePC-resistant parasites, it could be hypothesized that continuous in vitro drug pressure induces the mechanisms of regulation in Leishmania lipid metabolism.

Alteration of fatty acid and sterol metabolism in miltefosine-resistant Leishmania donovani promastigotes and consequences for drug-membrane interactions.

Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active drug approved for the treatment of visceral leishmaniasis. In order to investigate the biochemical modifications occurring in HePC-resistant (HePC-R) Leishmania donovani promastigotes, taking into account the lipid nature of HePC, we investigated their fatty acid and sterol metabolisms. We found that the content of unsaturated phospholipid alkyl chains was lower in HePC-R parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of HePC-R parasite membranes. We also demonstrated that HePC insertion within an external monolayer was more difficult when the proportion of unsaturated phospholipids decreased, rendering the HePC interaction with the external monolayer of HePC-R parasites more difficult. Furthermore, HePC-R parasite membranes displayed a higher content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in HePC-R parasites. Sterol biosynthesis was found to be modified in HePC-R parasites, since the 24-alkylated sterol content was halved in HePC-R parasites; however, this modification was not related to HePC sensitivity. In conclusion, HePC resistance affects three lipid biochemical pathways: fatty acid elongation, the desaturase system responsible for fatty acid alkyl chain unsaturation, and the C-24-alkylation of sterols.

Antiplasmodial activity of acetogenins and inhibitory effect on Plasmodium falciparum adenylate translocase.

Three Annonaceous acetogenins exhibited in vitro antimalarial activities on a chloroquine-resistant Plasmodium falciparum strain, with IC50s ranging from 5 to 10 microM. Structure-activity relationships showed that maximal antimalarial activity occurred in the presence of at least one tetrahydrofuran moiety and a synergistic action with chloroquine was observed. These acetogenins partially inhibited the P. falciparum adenylate translocase.

[Hemodialysis in pregnancy]. / Hémodialyse et grossesse: à propos d'une observation.

Pregnancy brought to term is rare in patients undergoing haemodialysis. Both mother and foetus are exposed to lethal from complications frequently caused by hypertension and anaemia. We are reporting the first case of pregnancy observed in an haemodialysis centre in Antananarivo, Madagascar. In spite of the modest means of the centre (use of acetate, lack of recombinant erythropoietin, limited biological controls) the foetus could be kept by intensification of the dialysis. No complication occurred to the mother when in the literature its frequency is 16%. The baby was premature and low weighted. His mental and physical development was normal. This success is the consequence of heavy management especially for the patient.