Zorbozan Orçun, Harman Mehmet, Evren Vedat, Erdoğan Mümin Alper, Kılavuz Aslı, Tunalı Varol, Çavuş İbrahim, Yılmaz Özlem, Özbilgin Ahmet, Turgay Nevin
Ege University Faculty of Medicine, Department of Parasitology, Izmir, Turkey.
Dicle University Faculty of Medicine, Department of Dermatology, Diyarbakir, Turkey.
Mikrobiyol Bul. 2018 Jan;52(1):49-55. doi: 10.5578/mb.66350.
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.
利什曼病是一种媒介传播的人畜共患病，表现出不同的临床特征，如皮肤型、黏膜皮肤型、内脏型和内脏嗜性型。利什曼病治疗中使用的方案有毒性，且在给药过程中有许多局限性。治疗的局限性之一是在实际应用中对这些方案产生耐药性。此外，尤其对于耐药患者，还需要确定新的治疗选择。除了体外和体内研究外，使用原代细胞培养的体外模型可能是评估锑耐药患者新药选择的良好来源。在本研究中，旨在确定一种新的体外培养模型，以评估对治疗无反应的皮肤利什曼病患者的治疗选择。在我们的体外感染实验模型中，使用了从先前诊断为皮肤利什曼病的病例中分离出的热带利什曼原鞭毛虫前鞭毛体。用于体外模型的原代星形胶质细胞培养物是在无菌条件下，通过改良的麦卡锡方法，从2 - 3日龄的新生斯普拉格 - 道利大鼠脑中制备的。达到足够密度的星形胶质细胞用抗锑的热带利什曼原鞭毛虫前鞭毛体进行感染。孵育24小时后，收集细胞上的上清液，将细胞培养板在室温下干燥，然后用甲醇固定并用吉姆萨染色，以寻找热带利什曼无鞭毛体。在感染热带利什曼原鞭毛虫前鞭毛体的原代细胞培养物的胶质细胞中强烈观察到无鞭毛体。从感染平板中取出的液体培养基离心后的底部沉淀物制备的沉淀物的吉姆萨染色制剂上未见到前鞭毛体。在本研究中，来自一名对五价锑治疗无反应的皮肤利什曼病患者的前鞭毛体被证明可感染大鼠胶质细胞并转化为无鞭毛体形式。据我们所知，这种无鞭毛体胶质细胞模型是文献中第一个由热带利什曼原虫产生的模型。胶质细胞中出现热带利什曼无鞭毛体形式可能表明利什曼原虫物种感染中枢神经系统的能力。在对治疗无反应的利什曼病病例中，中枢神经系统可能是利什曼无鞭毛体逃避免疫系统的一个区域。我们的研究很重要，因为它是第一项显示热带利什曼无鞭毛体感染胶质细胞的研究。
