Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 µg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 µg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-gamma/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

Anti-leishmanial Effects of Trinitroglycerin in BALB/C Mice Infected with Leishmania major via Nitric Oxide Pathway

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.

Effects of combined therapy with thalidomide and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice

For treating Leishmania major infection in BALB/c mice, we used thalidomide in conjunction with glucantime. Groups of mice were challenged with 5 x 10(3) metacyclic promastigotes of L. major subcutaneously. A week after the challenge, drug treatment was started and continued for 12 days. Thalidomide was orally administrated 30 mg/kg/day and glucantime was administrated intraperitoneally (200 mg/kg/day). It was shown that the combined therapy is more effective than single therapies with each one of the drugs since the foot pad swelling in the group of mice received thalidomide and glucantime was significantly decreased (0.9 +/- 0.2 mm) compared to mice treated with either glucantime, thalidomide, or carrier alone (1.2 +/- 0.25, 1.4 +/- 0.3, and 1.7 +/- 0.27 mm, respectively). Cytokine study showed that the effect of thalidomide was not dependent on IL-12; however, it up-regulated IFN-gamma and down-regulated IL-10 production. Conclusively, thalidomide seems promising as a conjunctive therapy with antimony in murine model of visceral leishmaniasis.

Childhood cutaneous leishmaniasis: report of 117 cases from Iran

Cutaneous leishmaniasis (CL), due to Leishmania major, is endemic in different parts of Iran and has long been recognized in most provinces of Iran. This study was conducted to determine the prevalence of childhood leishmaniasis in 3 areas at the southeast of Kashan. A descriptive study was carried out on all children referred to central laboratories during a 3-year period. Initial information including age, sex, sites of ulcer on the body, number of lesions, address, and the place of the disease was obtained. The study gathered 117 children, and the results showed a prevalence of 7.2% in patients with lesions among the population and 4.2% of people displayed lesion and scar. The ages of subjects were from 6 to 15 years (average 9.75 years). The boy: girl ratio was 1.2. All of our patients lived in an endemic area. The face was affected in 47.0% of cases. The encountered forms of leishmaniasis are as follows: papulonodular 27.4%, ulcer 60.7%, sporotrichoid 6%, impetiginous 2.5%, and erysipeloid 3.4%. Treatment with intramuscular meglumine antimoniate 20-30 mg/kg/day was done for 93 patients. Meglumine antimoniate treatment was tolerated with no side effects. All leishmaniasis lesions healed within an average period of 2-14 months. Hyperpigmented scars were formed in 25.6% of the patients, atrophic scars in 4.3%, and hypopigmented scars were in 3.4%, respectively. The findings of this study indicate increased prevalence of CL in the villages at the area of Kashan and Aran-Bidgol. The clinical finding patterns belonged to different endemic strains of L. major in Isfahan, which indicates the possible transmission of infection from Isfahan to this area.

Zinc sulphate in the treatment of cutaneous leishmaniasis: in vitro and animal study

This study was designed to evaluate the effectiveness of zinc sulphate both in vitro and in an animal model against both strains of old world cutaneous leshmaniasis. The in vitro sensitivities of promastigotes and axenic amastigotes of both Leishmania major and L. tropica to zinc suphate was determined, the LD50 calculated and compared to the standard treatment for cutaneous leishmaniais pentavalent antimony compounds. The results show that the two forms of both strains were sensitive to zinc sulphate and their respective LD50 were lower compared to the pentavalent antinomy compound. Furthermore the sensitivities of the forms of both strains were tested using a simple slide method and compared to results of the standard method. To confirm this result, zinc sulphate was administered orally to mice infected with cutaneous leishmaniasis both therapeutically and prophylactically. Results showed that oral zinc sulphate was effective in both treatment and prophylaxis for cutaneous leishmaniasis. These results encourage the use of oral zinc sulphate in the treatment of cutaneous leishmaniasis clinically.

Efficacy of ivermectin on the infectivity of leishmania major promastigotes

Ivermectin is widely used as an antiparasitic drug for livestock and recently used for some human parasites. The effect of ivermectin on the infectivity of Leishmania major promastigotes was studied in Syrian Golden hamsters. The results showed that hamsters infected with promastigotes treated for one day with 100 mug/ml ivermectin in culture medium did not develop skin lesion at the site of infection. However, amastigotes were demonstrated in Giemsa stained splenic smears. On the other hand, promastigotes treated for 2 days with 90 mug/ml ivermectin did not acquire the infection, i.e. the promastigotes died or lost their infectivity

Leishmanicidal activity of nystatin [mycostatin]: a potent polyene compound

The susceptibility of promastigote of Leishmania major to Nystatin in vitro was examined. L. major [MHOM/PK/88/DESTO] promastigote were cultured in medium 199 supplemented with 10% heat inactivated foetal bovine serum and 2% urine. The growth of the promastigote was monitored in the absence and presence of the experimental compound [Nystatin] for upto 5 days post-inoculation. The EC50 value [the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value] obtained for Nystatin against the promastigote of L. major was less than 9.76 iu ml. Certain polyene compounds like Amphotericin-B and Nystatin [mycostatin] are familiar for their fungicidal activity. Amphotericin-B is used since long as antileishmanial drug as well. Results obtained suggest that Nystatin has a very good anti leishmanial activity in vitro. The mode of action proposed for this drug is same as for Amphotericin-B as both of these polyene compounds interact with the various sterols present on the surface of the parasite, thus unusual gaps and pores are formed on the surface that results in the leakage of the ions. This leakage finally leads to the destruction of the parasite

Possible roles of nitric oxide and peroxynitrite in murine leishmaniasis

Activated macrophages simultaneously synthesize nitric oxide and superoxide anion which can react with each other producing peroxynitrite. Consequently, it has been difficult to assess the precise contribution of each of the formed reactive oxygen- and nitrogenderived species to the microbicidal activities of macrophages, particularly in vivo. To explore this problem, we are examining the formation and potential roles of nitrogen-derived intermediates in Leishmania amazonensis murine infection. Thus far, our results have demonstrated that peroxynitrite is a potent leishmanicidal agent in vitro and that both nitric oxide and peroxynitrite are formed during infection of susceptible BALB/c mouse strain. Nitric oxide was detected as the nitrosyl-hemoglobin complex by electron paramagnetic resonance analysis of blood drawn from mice at different times of infection, and it was shown to increase with the evolution of the disease. These results will be discussed in the context of the dual physiological role of nitric oxide either as a signaling molecule or as a deleterious agent.

efficacy of different dosage regimens of pentavalent antimony on cutaneous leishmaniasis in mice

The efficacy of different dosage regimens of sodium stibogluconate [SSG], a pentavalent antimony [sb] agent in the treatment of cutaneous leishmaniasis [CL] in male Balb/c mice was investigated. Mice with CL were divided into four groups. Mice in group 1, were injected intramuscularly with saline [control] and those in groups 2, 3 and 4 were injected with a total daily dose of 300 mg Sb/kg :300 once 150 twice and 100 thrice mg sb/kg respectively daily for 10 days. None of the mice in groups 1 or 4 was cured, however the cure rates in groups 2 and 3 were 50% and 33%, respectively. Lesion size was reduced by 89% in group 2 as compared to 56% and 16% in groups 3 and 4 respectively. Peak plasma antimony level [C[max]]was determined in another 3 groups of mice that were given 300, 150 and 100 mg sb/kg. The C[max] were: 34.3, 15.9 and 8.4 micro g /ml in groups 1.2 and 3 respectively. Those of groups 1 and 2 but not 3 were within the in vitro ED50 range [12-20 mg/1] of SSG. In conclusion, higher cure and healing rates as well as a more effective antimony plasma level are obtained when the total daily requirements of SSG is given as a single dose. This regimen should, thus be used instead of divided doses when treating CL