Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation
Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation
复制标题
DOI：
10.1128/aac.50.4.1195-1201.2006
复制
发表时间：
2006-04-01
影响因子：
4.9
通讯作者：
Bolas-Fernández, F
中科院分区：
文献类型：
--
作者：
Iñiguez, SR;Dea-Ayuela, MA;Bolas-Fernández, F
建立了一种实时定量逆转录-PCR方法，用于定量感染婴儿利什曼原虫的金黄色叙利亚仓鼠中细胞因子mRNA的表达，并用人血清白蛋白（HSA）制成的微球中的阿替霉素B（AMB）治疗。在感染后（p.i.）用10（7）个代谢环前鞭毛体，以2和40 mg/kg AMB的剂量。到感染后第76天，未处理动物记录到高感染水平，肝脏中的寄生虫负荷总是比脾脏中高约2 log（10）/g。两种剂量的治疗都非常有效，但在40 mg/kg时，几乎完全消除了寄生虫。在未处理组的大多数动物中，脾细胞中γ干扰素（IFN-γ）以及较小程度的肿瘤坏死因子α（TNF-α）和转化生长因子β（TGF-β）的mRNA表达上调。白细胞介素-4的mRNA表达强烈下调，以及治疗感染的动物。用较低剂量的AMB-HSA处理下调IFN-γ和TNF-α的mRNA表达，对失活细胞因子TGF-β没有影响。相反，用较高剂量（40 mg/kg）的制剂治疗引起IFN-γ和TNF-α的中度上调和TGF-β的强烈抑制。未感染动物的治疗并没有改变细胞因子的表达模式与未经处理的控制。我们的结果表明，治疗L。用高效无毒剂量的AMB-HSA感染婴儿的叙利亚仓鼠引起抗炎细胞因子TGF-β的失活，这又导致Th 1细胞因子IFN-γ和TNF-α的上调。
A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 10(7) metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log(10) per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-gamma) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-gamma and TNF-alpha, with no effect on the deactivating cytokine TGF-beta. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-gamma and TNF-alpha and strong suppression of TGF-beta. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-beta, which in turn results in up-regulation of the Th1 cytokines IFN-gamma and TNF-alpha.