Departments of Molecular Parasitology, Bioinformatics and Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research (ICMR), Agamkuan, Patna, India.
J Antimicrob Chemother. 2012 Oct;67(10):2373-8. doi: 10.1093/jac/dks220. Epub 2012 Jul 3.
To evaluate the in vitro activity of antileishmanial drugs, paromomycin and miltefosine, to generate Th-1-biased immunomodulation in hosts against intracellular Leishmania donovani.
In silico protein-ligand interaction and in vitro drug-cell interaction assays were performed. Interaction assays of TLR4-deficient HEK293 cells and HEK293 cells engineered to express either TLR4 or TLR2 with different concentrations of miltefosine and/or paromomycin sulphate were performed for 48 h. Differentially transfected human peripheral blood monocyte-derived macrophages (PBMFs) were treated with the drugs, and nuclear factor (NF)-κB promoter activity was measured using a κB-luciferase reporter construct. PBMFs were infected with L. donovani. Cultures were incubated with miltefosine or paromomycin sulphate over different concentrations, as mono-treatment or combined. The in vitro antileishmanial effect of the drugs on macrophage-bound L. donovani amastigotes was measured in terms of parasite killing and production of tumour necrosis factor-α (TNF-α) and nitric oxide.
Computational studies reveal that paromomycin and miltefosine interact with TLR4. Both drugs, as monotherapy or in combination, induce release of TNF-α and nitric oxide in a TLR4-dependent manner. Interestingly, the TLR4-dependent action of the drugs leads to NF-κB promoter activation through MyD88. In vitro, both the drugs kill macrophage-bound L. donovani by inducing release of TNF-α and nitric oxide in a TLR4-dependent manner.
The in vitro activity of paromomycin and miltefosine against host cells is TLR4 dependent. This has implications for: (i) evaluation of in vitro activity of combinational antileishmanial therapy; (ii) the evaluation of drug susceptibility of clinical isolates; and (iii) the standardization of in vitro antileishmanial assays for determining toxicity in hosts.
评估抗利什曼原虫药物巴龙霉素和米替福新在宿主体内产生 Th1 偏向性免疫调节以对抗细胞内利什曼原虫 Donovan 的体外活性。
进行了计算机蛋白配体相互作用和体外药物细胞相互作用试验。用不同浓度的米替福新和/或硫酸巴龙霉素与 TLR4 缺陷型 HEK293 细胞和工程表达 TLR4 或 TLR2 的 HEK293 细胞进行 TLR4 相互作用试验 48 小时。用药物处理转染差异的人外周血单核细胞衍生的巨噬细胞（PBMF），并用κB-荧光素酶报告基因构建体测量核因子（NF）-κB 启动子活性。用 L. donovani 感染 PBMF。在不同浓度下，将米替福新或硫酸巴龙霉素单独或联合孵育进行单治疗或联合治疗。用药物对巨噬细胞结合的 L. donovani 无鞭毛体进行体外抗利什曼原虫作用测量，以寄生虫杀伤和肿瘤坏死因子-α（TNF-α）和一氧化氮的产生来衡量。
计算研究表明巴龙霉素和米替福新与 TLR4 相互作用。两种药物，无论是单独治疗还是联合治疗，都以 TLR4 依赖的方式诱导 TNF-α 和一氧化氮的释放。有趣的是，药物的 TLR4 依赖性作用通过 MyD88 导致 NF-κB 启动子激活。在体外，两种药物均通过诱导 TLR4 依赖性 TNF-α 和一氧化氮释放来杀死巨噬细胞结合的 L. donovani。
巴龙霉素和米替福新对宿主细胞的体外活性依赖于 TLR4。这对以下方面具有影响：（i）评估联合抗利什曼原虫治疗的体外活性；（ii）评估临床分离株的药物敏感性；（iii）标准化用于确定宿主毒性的体外抗利什曼原虫测定。
