Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and spread by sandflies. The standard therapy for this ailment is the first-line medication of pentavalent antimonial and the second drug line of pentamidine amphotericin B. All are practiced over the years and exhibit adverse toxicity effects. Herbal product-derived medicine is a promising potential source for treating parasitic diseases. Xanthatin, a xanthanolide sesquiterpene lactone, is isolated from Xanthium strumarium L. treats several ailments in many countries. In the present study, we investigated the leishmanicidal activity of the xanthatin by using a metabolomicsbased analysis in J774 macrophages and AMASTIGOTES phases in Leishmania major. Xanthatin was isolated and identified by NMR spectroscopy. Macrophage toxicity of xanthatin performed by MTT assay. Macrophages infected by the L. major’ s promastigote stationary phase, the infection rate (IR), and multiplication index (MI) were calculated. Axenic AMASTIGOTES were treated with xanthatin. Cell quenching and metabolite extraction were performed, and the metabolome profile was analyzed with NMR spectroscopy. Outliers were classified by using multivariate statistical analysis software, and relevant metabolites and pathways were worked out. The xanthatin IC50 rate defined 0. 75 μ g/mL base on macrophages viability and also in-vitro activity of xanthatin on AMASTIGOTES showed the best leishmanicidal activity in IR and MI values of 53% and 62. 5%, respectively. Xanthatin altered amino sugars and nucleotide sugars metabolism, starch and sucrose metabolism, cyanoamino acid, and galactose metabolism. Our finding revealed that the main target of xanthatin is carbon metabolism, which is an essential step for AMASTIGOTES virulence.

Introduction: Leishmania infantum is the causative agent of visceral leishmaniasis (VL). Cell-mediated immunity (CMI) is required to control leishmaniases. Therefore, simple tests that can evaluate the cellular immunity of the target populations can help to understand the immune status of the human subjects, their immunity to the re-infection and the evaluation of the effectiveness of the potential vaccines. Here, we compared antigens based on single clones of L. infantum promastigotes and axenic AMASTIGOTES by in vitro and in vivo tests. Methods: Using serial dilutions, L. infantum promastigotes were selected as single clones (PSC) or were grown under axenic conditions with succinate-tris to prepare amastigote-like single clones (ASC). Antigens prepared from PSC and ASC were then compared with typical Leishmania major and L. infantum AMASTIGOTES by SDS-PAGE, Western-blotting and proliferation tests as well as an in vivo delayed-type hypersensitivity test on guinea pigs. Results Both PSC and ASC exhibited a distinctive ~50-kDa band could be detected by Western-blotting. The proliferation tests results indicated that both PSC and ASC could cause higher lymphocyte proliferation compared to typical L. infantum and L. major promastigotes; however the differences were not significant. Moreover, both PSC and ASC had an ability to induce comparable DTH and hence CMI. Conclusion: Similar proliferation or delayed-type hypersensitivity could be caused with antigens based on PSC, ASC or the typical promastigotes and any of these reagents could potentially be used for in vivo detection of CMI in VL epidemiological or vaccine studies.

This study has been carried out on mononuclear cells of peripheral blood to identify that L.major and L.tropica parasites have life cycle inside macrophage similar to the host. The in-vitro dynamic proliferation of the obligate intracellular protozoan parasites of L.tropica and L.major on peripheral mononuclear cells of five groups was investigated as follows: 1- control group(ten individuals) 2- Infected with L.tropica and sensitive to the treatment(five individuals) 3- Infected with L.major and sensitive to the treatment(five individuals) 4- Infected with L.tropica and resistant to the treatment (five individuals) 5- Infected with L.major and resistant to treatment (five individuals). Separated mononuclear cells of peripheral blood were infected with L.major and L.tropica and they were cultivated for 14 days. In the days 1, 3, 5, 7, 10, 14 after infection, the number of infected macrophages and the number of amastigots/macrophage have been counted.Using statistical trials t.test and paired t.test, no significant differences observed between two healty groups. On the other hand, significant differences observed between resistant and control groups as well as sensitive and resistant groups (P<0.05). Severe infection with L.major and L.tropica in both resistant groups has been shown in this study.The identification of the dynamic of proliferative cycle of L.tropica and L.major in human macrophages can help us for successful treatment of cutaneous leismaniasis.

Background: Leishmaniasis consisted of a set of parasitic disease with a spectrum of clinical symptoms appear, including coetaneous Leishmaniasis, mucosal and visceral all symptoms. Recently, there has been considerable progress in the use of herbal medicines for the treatment of Leishmaniasis. The purpose of this study was to evaluate the effect of thyme essential oils of four plant species compared with Amphotericin B on Leishmania AMASTIGOTES in invitro. Methods: Leishmania (Leishmania major) strains were cultured with medium RPMI-1640 containing 10% fetal calf serum (FCS) and antibiotics and transported at a temperature of 25 ± 2 ° C. The stationary phase of growth were studied in different concentrations of essential oils in comparison with the control drug, Amphotericin B on Leishmania AMASTIGOTES by using colorimetric meth. The absorbance was measured (Optical density) by means of (ELISA reader) and was calculated as the IC 50. The mean absorbance of the groups studied showed no significant difference (P <0/001), the Duncan test showed significant differences between the concentrations of the test and control groups. Results: The results showed that optical absorption and IC 50 species of thyme essential oils affect drug compared with controls. Conclusions: Our results showed that the herb thyme is effective for the treatment of coetaneous Leishmaniasis. According to these results suggest that the efficacy was evaluated alone or in combination against human coetaneous Leishmaniasis as a randomized clinical trial.

Introduction. Leishmaniasisi is a group of diseases that caused by protozoa of the genus- Leishmania related to trypanosomatidae family. Promastigote forms or leptomunas of the parasite is transfered to vertebrate host including human by the bite of infectious sandfly and sheltered in reticuloendothelial cells which multiplies and changes to immobile amastigote form. It is feasible to culture and mass production of promastigotes in semi solid, biphasic and liquid media. For the reason that there are many differences between promastigote form and amastigote form in metabolical, Biochemical and enzymatical characters. Data obtained from studies on AMASTIGOTES which are the main of the parasite in human body can resulted in detail data about the parasite. Feasible access to this from of parasite can help very much for successful control of Leishmaniasis, especially designing ways for therapeutic methods.Method. L. major (MRHOIIR/75/ER) from previously infected Balb/c mice was transfered to modified N.N.N medium with overlay of liquid RH.LR After isolation and growth at 26±1°C promastigote from of parasites were transfered to liquid cell culture medium, RPMI 1640. Culture media with PH: 3.5, 4.5, 5.5 (±0.1) were provided and 5xl06 parasites were added to each medium. All cultures were incubated for 24 in 25 and then were 28°c, 32°c and 37°c. The above procedure was repeated with needed changes many times unitl finally amastigote forms were resulted from fellagelated forms of the parasite.Results. The most suitable media was media with PH: 3.5 and most suitable temperature was 37°c which caused the most number of amastigote in vitro.Discussion. There are other methods that can obtain AMASTIGOTES in vitro. For example culture of parasite in a media containing macrophages from peritoneal cavity of hamsters or other cell sources lik human and cells. But this methods have draw back as extratime consuming, high price lab equipments, high expenses and contamination by host materials, so that the benefit of peresent study to the mentioned methods is really cleared.

Background: Leishmaniasis is a vector-borne disease caused by Leishmania species. The transcription factor (NF-κ B) activates and the innate immune system starts working when this parasite attacks macrophages. In addition, glucocorticoids increase nitric oxide (NO) and INFγ leads to the apoptosis of the cell by inhibiting the NF-κ B activity. The aim of this study was the in vitro investigation of the effects of the glucocorticoids on Leishmania major AMASTIGOTES. Methods: Leishmania major was produced in a massive volume. Then, promastigotes penetrated into macrophages and converted to AMASTIGOTES by adding promastigotes to the J774 mouse macrophage cell line and providing appropriate conditions. Next, the infected macrophages with L. major parasite were treated with different concentrations of prednisolone and mometasone. After 24 and 48 hours, the effect of the drugs was evaluated based on the average number of AMASTIGOTES in the infected macrophages. In addition, the amount of NO and the mean interleukin 12 (IL-12) level secreted by the infected macrophages treated with different concentrations of drugs were measured by Griess reagent and ELISA reader, respectively. Results: The average number of AMASTIGOTES in the infected macrophages treated with different concentrations of the drug was significantly different from the control group. Further, the amount of NO and the mean level of IL-12 secreted by infected macrophages had a direct and significant relationship with different concentrations of drugs, but the results of Tukey post hoc test showed that the reduction in the number of promastigotes was not time-dependent. Conclusions: In general, prednisolone and mometasone stimulated macrophages increased the IL-12 levels and NO secretion and finally decreased the number of parasites in infected macrophages.

Background: The first line treatment against cutaneous leishmaniasis is meglumine antimoniate. This drug is expensive and has serious side effects, including development of drug resistance. Objectives: In this research, because of paucity of information, the apoptotic and leishmanicidal effects of ketotifen and cromolyn sodium, as cell membrane stabilizer drugs, were investigated on standard strain of Leishmania major. Methods: In this experimental study, L. major parasites were first cultured in RPM1 1640 media, supplemented with 10% fetal bovine serum (FBS) and antibiotics at 24  1º C. Drug concentrations of 5, 10, 15, and 20  g/mL were then added to L. major culture at 24-, 48-and 72-hour intervals. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) tetrazolium assays were performed to determine parasite viability and drug toxicity. Leishmania major promastigotes were augmented to the in vitro cultured macrophages (J774 cells) and then incubated for 72 hours. Halfmaximal inhibitory concentration (IC50) were ascertained by counting the parasites. The inhibitory effect of the drugs were compared with that of glucantime. Flow cytometry was performed in the next step, to evaluate apoptosis. Each test was repeated three times. Results: IC50 values of ketotifen and cromolyn sodium after 72 hours were calculated to be 2. 04 and 17. 67  g/mL for promastigotes and 0. 12 and 14. 79  g/mL for AMASTIGOTES, respectively. The results of MTT assays showed 20% and 35% promastigote viability after 72 hours of exposure to ketotifen and cromolyn sodium at 20  g/mL concentration. Apoptosis in ketotifen and cromolyn sodium was quantified to be 11. 52% and 9. 96% in promastigotes and 99. 5% and 98. 6% in amastigote-infected macrophages, respectively. The results indicated that the drugs induce early and late apoptosis in parasites. All treatments produced results, which differed significantly from the control groups (P < 0. 05). Conclusions: Drugs used in this study, especially Ketotifen, showed lower toxicity yet similar anti-leishmanial effectsonboth forms, as cromolyn sodium did. It could be suggested that further investigations about the in vivo effects of these drugs, as candidates for cutaneous leishmaniasis treatment, are required.