In vitro antileishmanial activity of three saponins isolated from ivy, alpha-hederin, beta-hederin and hederacolchiside A(1), in association with pentamidine and amphotericin B.

The in vitro antileishmanial activity of three saponins isolated from ivy, alpha-hederin, beta-hederin and hederacolchiside A(1), was investigated on parasites of the species Leishmania mexicana, in their promastigote and amastigote forms compared with their toxicity versus human monocytes. The results showed that saponins exhibited a strong antiproliferative activity on all stages of development of the parasite but demonstrated a strong toxicity versus human cells. Association of subtoxic concentrations of saponins with antileishmanial drugs such as pentamidine and amphotericin B demonstrated that saponins could enhance the efficiency of conventional drugs on both the promastigote and the amastigote stages of development of the parasite. The results demonstrated moreover that the action of saponins on promastigote membrane was cumulative with those of amphotericin B.

Flow cytometric detection of Leishmania parasites in human monocyte-derived macrophages: application to antileishmanial-drug testing.

A flow cytometric technique was developed for detection of amastigotes of the protozoan Leishmania infantum in human nonadherent monocyte-derived macrophages. The cells were fixed and permeabilized with paraformaldehyde-ethanol, and intracellular amastigotes were labeled with Leishmania lipophosphoglycan-specific monoclonal antibody. Results showed that flow cytometry provided accurate quantification of the infection rates in human macrophages compared to the rates obtained by the conventional microscopic technique, with the advantage that a large number of cells could be analyzed rapidly. The results demonstrated, moreover, that labeling of intracellular amastigotes could reliably be used to evaluate the antileishmanial activities of conventional drugs such as meglumine antimoniate, amphotericin B, pentamidine, and allopurinol. They also established that various Leishmania species (L. mexicana, L. donovani) could be detected by this technique in other host-cell models such as mouse peritoneal macrophages and suggested that the flow cytometric method could be a valid alternative to the conventional method.

Lack of in vitro antimicrosporidian activity of thalidomide.

Thalidomide was evaluated for its in vitro activity against Encephalitozoon species by using the MRC-5 cell system. A cytotoxic effect was observed for concentrations of 10(1) microg/ml (P < 10(5)) and 5 microg/ml (P < 10(5)). Thalidomide did not significantly inhibit the growth of any of the microsporidia under study. These data suggest that thalidomide is not an etiologic treatment in microsporidial enteritis.

In vitro susceptibilities of the microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis to albendazole and its sulfoxide and sulfone metabolites.

In vitro comparisons demonstrated that the efficacy of albendazole, albendazole-sulfoxide, and albendazole-sulfone against pathogenic Encephalitozoon species was proportional to the degree of oxidation at a concentration of >10(-3) microgram/ml. Furthermore, at a concentration of <10(-2) microgram/ml, benzimidazoles were more effective against Encephalitozoon cuniculi and Encephalitozoon hellem than against Encephalitozoon intestinalis.

Purification of Encephalitozoon cultures contaminated by mycoplasmas by murine intraperitoneal inoculation.

Encephalitozoon species are strict intracellular microsporidia. Cocultures with eukaryotic cell lines can become accidently contaminated by mycoplasmas. We propose a decontamination protocol based on differential cell targeting after intraperitoneal inoculation in mice. Mycoplasma-free microsporidia were isolated from the brains and spleens of inoculated mice 24 h postinoculation by using the centrifugation shell vial system. Identification was confirmed by direct sequencing of PCR-amplified 16S rRNA.