• Parasites, Hosts and Diseases
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• 제53권1호
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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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• 제10권1_2호
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• pp.27-32
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• 1980

중동지방에 다녀온 48세의 한국인 남자 피부에 발생한 leishmania증 1예에 대하여 광학 및 전자현미경적 관찰을 하여 문헌 고찰과 아울러 보고하였다. 광학현미경적으로는 조직구 침윤을 주로 하는 만성 육아종성염증반응을 보였고 Giemsa염색상 다수의 leishmania충체를 조직구내에서 관찰할 수 있었다. 전자현미경적으로는 난원형의 단세포 또는 분열중인 충체가 조직구내에 존재하였고 충체는 이중막으로 싸였으며 그 바로밑에 microtubule이 배열되고 세포질내에는 신장된 mitochondrion내에 kinetoplast가 존재하고 그 전방에 flagella가 위치하였으며 기타 다수의 ribosome과 드물게 Golgi complex등을 관찰할 수 있었다. 이와같은 특징은 Leishmania tropica의 promastigote stage와 일치하였다. Leishmania증은 원칙적으로 열대병이나 열대지방과의 접촉이 빈번함에 따라 앞으로 우리나라에서도 보다 많은 증예가 발생할 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제57권4호
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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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• 제49권4호
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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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• 제31권5호
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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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• 제54권1호
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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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• 제31권3호
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• pp.277-284
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• 1993

세계적으로 중요한 열대풍토병 병원체의 하나인 열대리슈마니아의 핵형에 영향을 줄 것으로 기대되는 인자와 그 효과를 관찰하고자 하였다. 토기의 혈액을 포함한 N.N.N. 배지에서 유지하고 있는 열대리슈마니아(Leishmania major)의 promastigote를 열쇽. 약제 첨가 자외선 조사 및 감마선을 이용한 방사선 조사를 여러 가지 방법으로 시행하고 주기변동전기영동(pulsed field gradient gel electrophoresis)을 이용하여 핵형의 변동 여부를 관찰하였다 그 결과 여러 방식에 의한 열쇽과 약제 처리 및 자외선 조사에 따른 핵형의 변화가 없었다 그러나 방사선 조사군에서는 50 Gy 이상 조사한 군에서 1 mesa base pair(Hb) 크기에 있는 염색체부터 소실되기 시작하여 방사선 조사량이 증가함에 따라서 250-500 Kb의 작은 염색체도 파괴되어 500 Gy 이상 군에서는 뚜렷한 염색체 분획이 없이 젤 하단 200 Kb 크기 아래 부분에 몰려 있었다. 이러한 소견은 방사선에 의하여 염색체가 불규칙하게 파손된 것을 의미한다고 하겠다. 방사선을 300 Gy까지 조사한 충체는 계대 배양이 가능하였고. 이들은 원래의 핵형을 유지하였다. 방사선조사 후에 배양된 충체는 염색체가 파괴되지 않았거나 부분적인 손상 후에 DNA 재결합에 의해 원상회복된 것으로 판단된다. 열대리슈마니아의 핵형은 일시적인 자극에 의하여 쉽게 변형되지 않는 안정된 것임을 확인하였다.

• Parasites, Hosts and Diseases
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• 제53권4호
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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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• 제58권2호
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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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• 제35권1호
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• pp.31-38
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• 1997

BALB/c 마우스에서 감염부위와 감염기간에 따른 숙주 체액 면역반응의 변화를 알아보고자 하였다. 배양한 큰리슈만편모충의 전편모형(promastigote)을 BALB/c 마우스의 코. 등 발바닥 부위로 나누어 각각 $3{\;}{\times}{\;}10^6$마리씩 피하 감염 후 10-100일 동안에 궤양의 형성과정을 관찰하고 채혈하여 SDS-PAGE와 면역이적법을 시행하여 각 부위별로 나타나는 항체 반응을 관찰하였다. 외관상으로는 감염 15일부터 코에 감염시킨 마우스에서 먼저 궤양이 형성되기 시작하였고. 코에 궤양이 나타난 후 2-3일 뒤에 발에서 궤양이 형성되었으며 등에서는 감염시킨 후 90일이 되어서야 궤양이 관찰되었다. 감염후 20일에 실시한 면역이적법에 의하면 코 감염군에서는 202, 139, 98, 83, 81, 67, 65, 62, 59, 54, 52, 42, 26, 23 kDa의 항원성 분획이 관찰되었고 발 감염군에서의 항원 분획양상도 코 감염군과 같았으나 등감염군에서는 202, 83, 81, 65 kDa의 희미한 항원성 분획이 관찰되었다. 그러나 감염 후 90일이 경과한 등 감염군에서는 202, 83, 81, 74, 67, 65, 62, 59, 54, 52, 20, 17 kDa의 항원 분획이 관찰되었다 이상의 결과로부터 감염부위와 감염기간에 따라 큰리슈만편모충에 대한 혈청반응이 항원 분획에 따라 다르게 나타남을 관찰하였다. 이 차이는 세 감염부위의 온도차에 의한 결과일 가능성도 있으나 다른 부위에 감염될 경우 한 숙주 내에서도 다른 면역반응이 유발되어 나타날 수도 있다고 추측하였다. 특히 궤양 형성 시기와 혈청 내 67-52 kDa 분획에 대한 항체 출현 시기가 일치하는 것으로 보아 궤양 형성에 이 항체가 관여할 가능성이 있음을 시사한다.