• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.21-27
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• 2015

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were ${\alpha}$-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The $IC_{50}$ values for essential oil and methanolic extract was 8.4 and $28.9{\mu}g/ml$ against promastigotes, respectively. These values were 11.6 and $40.8{\mu}g/ml$ against amastigote forms, respectively. Glucantime as control drug also revealed $IC_{50}$ values of 88.3 and $44.6{\mu}g/ml$ for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

• Applied Microscopy
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• v.10 no.1_2
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• pp.27-32
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• 1980

A case of cutaneous leishmaniasis developed in a 48 year old Korean male who returned from middle east was studied by light and electron microscopic observations. Light microscopically, the lesion consisted of heavy chronic ill-defined granulomatous inflammation involving entire thickness of the dermis, composed of mainly histiocytic and small mononuclear cell infiltrations without evidence of necrosis or giant cell formation. Giemsa staining revealed numerous intracellular micro-organisms within histiocytes, showing dark stained central dot surrounded by light stained cytoplasm. Electron microscopically, the organisms were observed mostly ovoid in shape and frequently binary mitotic features within the host cells. follicle consisted of double unit membranes and microtubules, which are immediately below these membrnae. A long kinetoplast was noted within a very elongated mitochondrion at the center of the organisms and a flagella rose in front of the kineoplast but ended within the cytoplasm. Large numbers of free ribosomes, occasional Golgi complex and SER were also noted, but RER was seldom found. These ultrastructural features corresponded to promastigote stage of Leishmania tropica. In principle, leishmaniasis is a tropical disease and can not be found in temperate zone. However, travel to mideast by many Koreans may contract this disease while they are in endemic regions.

• Parasites, Hosts and Diseases
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• v.57 no.4
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• pp.359-368
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• 2019

In this study, we carried out extensive in vitro studies on various concentrations of tioxolone along with benzoxonium chloride and their niosomal forms against Leishmania tropica. Niosomes were prepared by the hydration method and were evaluated for morphology, size, release study, and encapsulation efficiency. This study measured leishmanicidal activity against promastigote and amastigote, apoptosis and gene expression levels of free solution and niosomal-encapsulated tioxolone along with benzoxonium chloride. Span/Tween 60 niosome had good physical stability and high encapsulation efficiency (more than 97%). The release profile of the entrapped compound showed that a gradual release rate. The combination of niosomal forms on promastigote and amastigote were more effective than glucantime. Also, the niosomal form of this compound was significantly less toxic than glucantime ($P{\leq}0.05$). The flowcytometric analysis on niosomal form of drugs showed that higher number of early apoptotic event as the principal mode of action (89.13% in $200{\mu}g/ml$). Also, the niosomal compound increased the expression level of IL-12 and metacaspase genes and decreased the expression level of the IL-10 gene, which further confirming the immunomodulatory role as the mechanism of action. We observed the synergistic effects of these 2 drugs that induced the apoptotic pathways and also up regulation of an immunomodulatory role against as the main mode of action. Also, niosomal form of this combination was safe and demonstrated strong anti-leishmaniasis effects highlights further therapeutic approaches against anthroponotic cutaneous leishmaniasis in future planning.

• Parasites, Hosts and Diseases
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• v.49 no.4
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• pp.357-364
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• 2011

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.696-704
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• 2021

Levan is an industrially important, functional biopolymer with considerable applications in the food and pharmaceutical fields owing to its safety and biocompatibility. Here, levan-type exopolysaccharide produced by Pantoea agglomerans ZMR7 was purified by cold ethanol precipitation and characterized using TLC, FTIR, ¹H, and ¹³C NMR spectroscopy. The maximum production of levan (28.4 g/l) was achieved when sucrose and ammonium chloride were used as carbon and nitrogen sources, respectively, at 35℃ and an initial pH of 8.0. Some biomedical applications of levan like antitumor, antiparasitic, and antioxidant activities were investigated in vitro. The results revealed the ability of levan at different concentrations to decrease the viability of rhabdomyosarcoma and breast cancer cells compared with untreated cancer cells. Levan appeared also to have high antiparasitic activity against the promastigote of Leishmania tropica. Furthermore, levan had strong DPPH radical scavenging (antioxidant) activity. These findings suggest that levan produced by P. agglomerans ZMR7 can serve as a natural biopolymer candidate for the pharmaceutical and medical fields.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.9-14
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• 2016

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 $50{\mu}g/ml$) 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 $IC_{50}$. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration ($IC_{50}$) of tamoxifen on promastigotes was $2.6{\mu}g/ml$ 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 $50{\mu}g/L$ 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.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.277-284
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• 1993

Leishmaniasis is one of the important tropical diseases in the world. Although it is not prevalent In Korea, imported cases have been recorded. The karyotype of Leishmcnic sp. has been observed to be variable by localities or by strains, but the karyotype of a strain is known to be stable. This study was performed to observe if the karyotype of a Leishmonio sp. would be changed under some stressful conditions. The karyotype, analyzed by pulsed Held gradient gel electrophoresis, was not grossly changed by heat shock, chemotherapeutics, UV illumination, and gamma irradiation. Radiation destroyed the chromosomes mechanically but subcultured organisms after irradiation showed unaffected karyotype. The present findings suggest that the karyotype of a Leishmnnia strain is so stable that it is not altered by temporary stimulation with heat, drugs, and radiation.

• Parasites, Hosts and Diseases
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• v.53 no.4
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• pp.385-394
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• 2015

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.

• Parasites, Hosts and Diseases
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• v.58 no.2
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• pp.173-179
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• 2020

Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune response in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its effect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mitochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was susceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis. In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aeruginosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.

• Parasites, Hosts and Diseases
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• v.35 no.1
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• pp.31-38
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• 1997

The skin ulcer in Leishmcnio mcior infection is known to be variable according to the inoculation sites even in a susceptible host. The present study traced the immunoblot patterns by the site of inoculation and duration of infection in BALB/c mice. L. mqior were subcutaneously inoculated on the nose, footpad, and back of the mice, in a dose of 3 × 106 promastigotes. Sera of the mice were collected every 10 days after inoculation. SDS-PAGE separated soluble protein bands of the promastigotes and immunoblot was carried out with the infection sera. The skin ulcer first appeared on the nose at 15 days, and on the footpad at 17 days after inoculation. The ulcer on the back appeared after 90 days. In the mice with ulcer on the nose or footpad, serum IgG antibody reacted to 202, 139, 98, 83, 81, 67, 65, 62, 59, 54, 52, 42, 26, and 23 kDa bands at 20 days after inoculation. In mice inoculated on the back, however, the immunoblot showed visible reactions with 202, 83, 81 74, 67, 65, 62, 59, 54, 52,20 and 17 kDa bands at 90 days after inoculation. The present result showed that the antigenic protein bands of L. mqior promastigotes were differed by the inoculation site and duration of infection. Since the skin ulcer and the serum antibodies to antigenic bands between 67-52 kDa appeared simultaneously, it is suggested that the serum IgG antibodies may play a role in formation of the skin ulcer in BALB/c mice.