The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 microM and 11 microM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 microM and 4.2 microM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.
Animals ; Apoptosis/*drug effects ; Cell Cycle/drug effects ; Cell Line ; DNA Fragmentation/drug effects ; Humans ; Leishmania major/cytology/*drug effects ; Leishmania tropica/cytology/*drug effects ; Leishmaniasis, Cutaneous/parasitology ; Mice ; Phosphorylcholine/*analogs & derivatives/pharmacology

Objective: To investigate Echinococcus (E.) granulosus genotypes as the causative agents of hydatidosis in humans in the southwest of Iran (Khuzestan province). Methods: In this study, isolates of 80 archived human paraffin embedded hydatid cysts were collected from pathology laboratories in Ahvaz city, Khuzestan province. DNA was extracted and examined by nested-PCR of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1), and PCR-RFLP. In addition, the sequences of fragments of genes coding for Cox space1 and NADH dehydrogenase 1 (ND1) were also examined. Results: Of the 80 paraffin samples, 44 (55.0%) were from the liver, 27 (33.8%) from the lung, and the rest from other organs. The amplified hydatid genomic DNA showed that the cysts were E. granulosus strains. The results of PCR-RFLP and sequencing analysis revealed the presence of G1 genotype (sheep strain) in all human isolates. Furthermore, no camel strain (G6) was detected among all samples in the regions studied. Conclusions: The molecular findings indicate that the predominant genotype involved in E. granulosus transmission in southwest of Iran is the common sheep strain (G1), which occurs in human populations. These results may have important implications for hydatid disease control in the studied areas.

Objective: To investigate the biochemical capacity, and in vitro inhibitory effects of hairy roots from two cultivars of Ficus carica L. (Sabz and Siah) on Leishmania major promastigotes and amastigotes. Methods: In the hairy roots, the activity of antioxidant enzymes compared to normal leaves and roots, and the presence of some phenolic compounds in comparison with fruits were investigated. The IC 50 values of hairy roots in promastigotes was determined by tetrazolium-dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and trypan blue assays. By calculating the infectivity index of peripheral blood mononuclear cells (PBMCs), the leishmanicidal activity (IC 50 values) of hairy roots for amastigotes was estimated. The effects of hairy roots (IC 50 values) treatment on the levels of IFN-γ and iNOS expression, intracellular reactive oxygen species, and iNOS protein expression in infected-PBMCs were determined. Results: Based on antioxidant enzyme assays and high performance liquid chromatography analysis, hairy roots exhibited high antioxidant capacity and contained high levels of phenolic compounds. According to the results of tetrazolium-dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and trypan blue assays, the hairy root extracts of both cultivars showed considerable dose-dependent inhibitory effects against Leishmania major promastigotes. Depending on the concentration and exposure time, treatment of infected-PBMCs with hairy root extracts caused the generation of a significant reactive oxygen species, up-regulation of IFN-γ and iNOS genes expression, and high value of iNOS protein compared to controls. Conclusions: The findings of this study suggest that the hairy roots of Ficus carica can be considered as a promising natural source of antileishmanial agents.