Ficus carica hairy roots: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

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.

Ficus carica hairy roots: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

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.

Molecular characterization of Echinococcus granulosus in paraffin-embedded human tissues from Southwest Iran

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.

[Serological evaluation of anti-toxoplasma IgM and IgG antibodies in renal transplant recipient's before and after transplant by ELFA, ELISA and ISAGA methods]

Toxoplasmosis is a shared human-animal disease with worldwide distribution caused by Toxoplasma gondii. More than half of the world's population is seropositive for toxoplasmosis. The possibility of reactivation of the old infection or acquisition of infection from donor's tissue increases in the transplant recipient patients who receive immunosuppressive therapy. In this study, IgM and IgG antitoxoplasma immunoglobulins seroconversion in renal transplant recipients was evaluated before and after transplantation. This was a prospective cohort study on a total of 102 recipients. Two serum samples were obtained from each patient. The first sample was taken before administration of any immunosuppressive drugs and second sample was taken 3 months after transplantation. The IgM and IgG anti-toxoplasma antibodies were assayed by ELFA and ELISA techniques. IgM/ISAGA method was also used. ELFA identified 65 [63.7%] pre-transplantation samples as IgG+ and did not detect any positive IgM samples. However, IgM was detected in 3 [2.9%] post-transplantation samples by this method. Forty nine [48%] pre-transplantation samples were reported IgG+ by ELISA and no IgM positive sample was identified by this method. ELISA method detected 2 [1.9%] IgM positive reactions in post-transplantation samples. By IgM/ISAGA method, we detected no IgM positive reactions in pre-transplantation samples whereas 3 months later [second sampling] IgM antibody was detected in 3 [2.9%] cases. Secondary toxoplasmosis infection was observed in 30 cases per 1000 recipients, which indicates that screening for toxoplasmosis infection should be performed in developed countries for these patients. On the other hand, as the risk of re-active toxoplasmosis infection exists in developing nations, they should consider the necessary preventive measures to control this condition. In addition, we suggest that ELFA is the best method because of the most valuable results

Comparison of traditional methods and pcr for diagnosis of cutaneous leishmaniasis in South-West of Iran

The usual method for laboratory diagnosis of cutaneous leishmaniasis was the direct observation of parasites under a light microscope. Although this method has high specificity, it has low sensitivity. The purpose of this study is to compare three methods of direct observation, culture and Mini-exon-PCR to diagnose cutaneous leishmaniasis in Khuzestan province. This study intends to compare sensitivity of PCR approach with sensitivity of the existing traditional methods to diagnose cutaneous leishmaniasis using Mini-exon gene. A total 216 skin biopsies prepared from patients with cutaneous leishmaniasis were studied though direct method, culture in NNN, culture in RPMI 1640 and Mini-exon-PCR and the sensitivity of these methods were compared with each other. In this study Mini-exon-PCR was considered as the gold standard method. Results showed that 46.7% with direct method, 35.1% with culture method in RPMI 1640,57.8% with culture methodin NNNand 70.3% withPCRwere positive.Sensitivity wasobtained 66.4% for microscopic observation, 50% for culture in RPMI1640, and 82.2% for culturein NNNand 100% forPCR. This study showed that PCR on samples stored in normal saline has higher sensitivity and specificity than other traditional methods [p<0.05]. Thus, Mini-exon-PCR on samples in normal saline is a reliable method to diagnose cutaneous leishmaniasis, especially in cases where the diagnosis is negative with the other methods.

Miltefosine-Induced Apoptotic Cell Death on Leishmania major and L. tropica Strains

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.