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    Evaluating the glucantime concentration for the ex vivo glial cell model of antimony-resistant leishmania tropica amastigotes
    (Galenos Publishing House, 2021) Zorbozan, Orçun; Evren, Vedat; Harman, Mehmet; Özbilgin, Ahmet; Yılmaz, Özlem Alkan; Turgay, Nevin
    Objective: Because the protocols used in the treatment of leishmaniasis can be toxic and have many limitations, such as the development of resistance against such protocols, new treatment options are needed, especially against resistant patients. Ex vivo models may be a good source for evaluating new drug options for patients with antimony-resistant parasites. This study aimed to evaluate the Glucantime concentration for our ex vivo glial cell amastigote model we had defined in previous work. Methods: We prepared the astroglial cell culture from brains of 2 to 3 day old neonatal Sprague-Dawley rats under sterile conditions by modifying McCarthy’s method. Four plates of cells were infected with antimony-resistant Leishmania tropica promastigotes. After 24 h of incubation, we added Glucantime to 3 plates with different concentrations. After 72 h, we removed the supernatant and then dried, fixed, and stained the plates with Giemsa to count the amastigotes in the glial cells. Results: We observed the amastigotes in glial cells in the control flask. Glial cells were ruined in flasks, which include 75 µg/ mL and 37.5 µg/mL Glucantime. The number of amastigotes per 100 glial cells was 116 for the flask with 7.5 µg/mL Glucantime concentration, while 487 for the control flask. Conclusion: We found that while high concentrations of Glucantime were toxic for glial cells, 7.5 µg/mL Glucantime concentration managed to reduce the number of Leishmania tropica amastigotes in glial cells.
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    Infecting Glial Cells with Antimony Resistant Leishmania tropica: A New ex-vivo Model
    (Ankara Microbiology Soc, 2018) Zorbozan, Orcun; Harman, Mehmet; Evren, Vedat; Erdogan, Mumin Alper; Kilavuz, Asli; Tunali, Varol; Cavus, Ibrahim
    Leishmaniasis is a vector-borne zoonotic disease that shows different clinical features like cutaneous, mucocutaneous, visceral and viscerotropic forms. The protocols used in the treatment of leishmaniasis are toxic and have many limitations during administration. One of the limitations of treatment is the resistance against the protocols in practice. There is also a need to define new treatment options especially for resistant patients. Ex-vivo models using primary cell cultures may be a good source for evaluating new drug options in patients with antimony resistance, in addition to in-vitro and in-vivo studies. In this study, it was aimed to define a new ex-vivo culture model to evaluate treatment options in patients with cutaneous leishmaniasis who did not respond to treatment. In our experimental model of ex-vivo infection, Leishmania tropica promastigotes isolated from a case previously diagnosed with cutaneous leishmaniasis were used. The primary astroglial cell culture used for the ex-vivo model was prepared from 2-3 days old neonatal Sprague Dawley rat brains under sterile conditions by the modification McCarthy's method. The astroglia cells, which reached sufficient density, were infected with antimony resistant L.tropica promastigotes. After 24 hours of incubation, the supernatant on the cells were collected, the cell culture plate was dried at room temperature, then fixed with methyl alcohol and stained with Giemsa to search for L.tropica amastigotes. Amastigotes were intensely observed in glia cells in primary cell cultures infected with L.tropica promastigotes. No promastigotes were seen on Giemsa stained preparations of the precipitates prepared from the bottom sediment after the centrifugation of the liquid medium removed from the infected plates. In this study, promastigotes from a cutaneous leishmaniasis patient unable to respond to pentavalent antimony therapy were shown to infect rat glia cells and converted to amastigote form. This amastigote glial cell model, as far as we know, is the first model in the literature produced by L.tropica. The occurrence of L.tropica amastigote forms in glia cells may be indicative of the ability of Leishmania species to infect the central nervous system. The central nervous system may be an area for the Leishmania amastigotes to escape from the immune system in cases of leishmaniasis without a treatment response. Our study is important because it is the first study to show the infection of glia cells with L.tropica amastigotes.