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Abstract：
BACKGROUND: Endothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines.
METHODS: Morphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ.
RESULTS: The four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of \"spiking pattern\" of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ.
CONCLUSIONS: ECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.
METHODS: Morphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ.
RESULTS: The four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of \"spiking pattern\" of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ.
CONCLUSIONS: ECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.
摘要：
背景：内皮细胞（ECs）在疟疾发病机理中起主要作用，作为寄生的红细胞与血管壁的直接接触点。ECs细胞骨架结构的研究，其主要功能是保持形状并为EC膜提供强度，对于确定恶性疟原虫疟疾的严重后遗症很重要。这项工作调查了细胞骨架的变化(微丝-肌动蛋白，疟疾血清（间日疟原虫，不复杂的恶性疟原虫和复杂的恶性疟原虫)，与促炎细胞因子的水平有关。
方法：使用免疫荧光技术评估了疟疾血清刺激的EC细胞骨架的形态和荧光强度。使用酶联免疫吸附测定（ELISA）测定肿瘤坏死因子（TNF）和干扰素（IFN）-γ（γ）的水平。对照实验组包括仅与培养基和非疟疾患者血清孵育的EC。实验组由与间日疟原虫的疟疾血清孵育的ECs组成，简单的恶性疟原虫和复杂的恶性疟原虫。比较了每个实验组的细胞骨架改变和荧光强度的形态学评分，并与TNF和IFN-γ相关。
结果：细胞骨架的四种形态变化包括（1）细胞骨架的收缩和皮质凝聚的EC，（2）偏心核的出现，(3)存在细胞骨架和EC膜的“尖峰模式”，(4)细胞骨架和ECs的断裂和不连续性。与复杂的恶性疟原虫疟疾血清刺激的ECs中的微管蛋白和波形蛋白丝相比，肌动蛋白丝有明显的损害。细胞骨架的形态损伤与荧光强度以及TNF和IFN-γ水平呈正相关。
结论：复杂的恶性疟原虫疟疾血清刺激的ECs显示细胞骨架改变，荧光强度增加，与高水平的TNF和IFN-γ相关。与复杂的恶性疟原虫疟疾血清孵育的EC的细胞骨架变化可导致EC交界改变和通透性变化。这是通过凋亡途径介导的。研究结果可为探索加强EC细胞骨架和减轻肺水肿和脑型疟疾等严重疟疾并发症的措施提供依据。此外,细胞骨架的免疫荧光强度可以作为疟疾严重程度的潜在预后指标。
方法：使用免疫荧光技术评估了疟疾血清刺激的EC细胞骨架的形态和荧光强度。使用酶联免疫吸附测定（ELISA）测定肿瘤坏死因子（TNF）和干扰素（IFN）-γ（γ）的水平。对照实验组包括仅与培养基和非疟疾患者血清孵育的EC。实验组由与间日疟原虫的疟疾血清孵育的ECs组成，简单的恶性疟原虫和复杂的恶性疟原虫。比较了每个实验组的细胞骨架改变和荧光强度的形态学评分，并与TNF和IFN-γ相关。
结果：细胞骨架的四种形态变化包括（1）细胞骨架的收缩和皮质凝聚的EC，（2）偏心核的出现，(3)存在细胞骨架和EC膜的“尖峰模式”，(4)细胞骨架和ECs的断裂和不连续性。与复杂的恶性疟原虫疟疾血清刺激的ECs中的微管蛋白和波形蛋白丝相比，肌动蛋白丝有明显的损害。细胞骨架的形态损伤与荧光强度以及TNF和IFN-γ水平呈正相关。
结论：复杂的恶性疟原虫疟疾血清刺激的ECs显示细胞骨架改变，荧光强度增加，与高水平的TNF和IFN-γ相关。与复杂的恶性疟原虫疟疾血清孵育的EC的细胞骨架变化可导致EC交界改变和通透性变化。这是通过凋亡途径介导的。研究结果可为探索加强EC细胞骨架和减轻肺水肿和脑型疟疾等严重疟疾并发症的措施提供依据。此外,细胞骨架的免疫荧光强度可以作为疟疾严重程度的潜在预后指标。
