Dengue infection of monocytic cells activates ER stress pathways, but apoptosis is induced through both extrinsic and intrinsic pathways

Monocytic cells are believed to be an important mediator of the pathology of dengue disease in cases of secondary infection where pre-existing antibodies from a first infection facilitate virus entry to Fc receptor bearing cells. In this study we assessed the induction of the ER stress in response to infection of monocytic U937 cells with all four DENV serotypes as well as the induction of apoptosis. Clear evidence of ER stress and the production of pro-apoptotic signals were observed, together with activation of caspase 9. Surprisingly, caspase 8 was also activated, independently of caspase 9, and this was accompanied by an increase in the expression of TNF-α, suggesting the simultaneous but independent activation of both extrinsic and intrinsic apoptosis pathways. Both the induction of ER stress and apoptosis were shown to be serotype independent.     

Autophagic machinery activated by dengue virus enhances virus replication

Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.     

Antioxidant defense is one of the mechanisms by which mosquito cells survive dengue 2 viral infection

Dengue viruses (DENVs) generally induce apoptosis in mammalian cells but cause only minor damage in mosquito cells. To find genes involved in determining the cell fate, datasets derived from expressed sequence tags (ESTs) of C6/36 cells with and without infection were established. Of overexpressed genes found in infected dataset, chaperone proteins were validated significantly upregulated in C6/36 cells at 24 hpi. It suggests that DENV-2 in mosquito cells activates the unfolded protein response to cope with endoplasmic reticular stress. Changes in the mitochondrial membrane potential and generation of superoxide provided further evidence that DENV-2 induces oxidative stress in both C6/36 and BHK-21 cells. Significant elevation of glutathione S-transferase (GST) activity was shown in infected C6/36, but not BHK-21, cells, while suppression of GST produced superoxide at 36 hpi and increased the cell death rate at 48 hpi. This indicates that mosquito cells protect themselves against viral infection through antioxidant defenses.

Research Highlights

? Elucidation on the response to DENV infection between mosquito and mammalian cells. ? Identification of ER-associated chaperones based on EST libraries of mosquito cells. ? Mosquito cells rescue themselves from DENV infection through antioxidant defenses. ? The first evidence of oxidative stress in mosquito cells against of DENV infection.     

CXCR4抑制剂AMD3100对2型登革热病毒诱导血管内皮细胞株Eahy926凋亡的影响

目的: 探讨趋化因子受体CXCR4抑制剂AMD3100对2型登革热病毒(DV2)诱导人脐静脉血管内皮细胞株 Eahy926凋亡的影响。方法: 免疫组织化学法检测Eahy926细胞的Ⅷ因子。Eahy926细胞分成未感染组和DV2感染组,流式细胞术检测两组细胞不同时点(24 h、36 h、48 h和60 h)CXCR4的表达水平。流式细胞术分析未感染组、DV2感染组及DV2+AMD3100组不同时点的细胞凋亡率。免疫荧光法检测细胞膜表面磷脂酰丝氨酸(PS)。结果: Eahy926细胞有Ⅷ因子表达。在DV2感染Eahy926后的4个时点中,CXCR4的表达均有上调,其中以48 h感染组最明显(66.13%±10.30%,P<0.05)。DV2感染能诱导Eahy926细胞凋亡,其中36 h感染组凋亡率出现高峰(29.85%±15.78%,P<0.05)。应用AMD3100后在各时点均能上调DV2感染组的凋亡率,免疫荧光观察到DV2感染组及DV2+AMD3100组绿色荧光标记的细胞增多。结论: DV2感染能诱导血管内皮细胞Eahy926凋亡并上调CXCR4的表达,CXCR4抑制剂AMD3100促进DV2诱导Eahy926细胞凋亡的发生。     

Functional implication of autophagy in steroid-secreting cells of the rat

Background: Autophagy, while frequently observed in embryonic cells undergoing differentiation and in pathologically altered cells, appears to occur less commonly in normal, fully differentiated cells. Our previous work revealed that the frequency of autophagic activity was rather high in the Leydig cells of rat testes, but the functional significance of autophagy in Leydig cells remains obscure. The purpose of the present study is to investigate the possible role of autophagy in steroid-secreting cells. Methods: The autophagic activity was investigated in two steroid-secreting cells, e.g., Leydig cells and adrenocortical fasciculata cells of rats. Cytidine monophosphatase (CMPase) cytochemistry was utilized to show the activity of lysosomal enzymes in autophagosomes. Electron microscopic morphometry was employed to analyze the frequencies of autophgy in the cells of the rats intact or treated with related hormones resulting in a hyper- or hypo-secretion of testosterone and corticosterone. Results: Autophagy took place in normal steroid-secreting cells with higher frequencies than in many other cells including the tubular cells of kidney and hepatocytes. The large number of autophagosomes or autophagic vacuoles allowed to outline the autophagic process in these cells. The C-shaped double-membrane profiles tending to demacate a portion of cytoplasm were referred to as pre-autophagosomes. So called early autophagosomes were the vacuoles enclosed completely by double delimiting membranes, containing normal-looking cellular components. The majority of sequestered organelles appeared to be mitochondria and smooth endoplasmic reticulum. The autophagosomes starting digestion were considered as late autophagosomes or autophagic vacuoles, the indications of which were the destruction of their contents or the presence of lysosomal enzymes demonstrated by a positive CMPase reaction. Residual bodies were frequently observed to be exocytosed. The quantitative assay revealed an alteration of autophagic activity in close relation with steroid-secreting states. The number of autophagosomes was one-fold higher in hyposecreting Leydig cells after 2 days testosterone administration, and three-fold higher in hyposecreting adrenocortical fasciculata cells after one dosage of dexamethasone administration. In addition, the autophagosomes showed a four-fold decrease in hypesecreting Leydig cells stimulated by LRH for 2 days. Conclusions: Considering that most of the autophagocytosed organelles were steroid-producing apparatus, we may conclude that, by removing part of steroid-producing organelles, autophagy might play a role in adapting to or even regulating the secretory activity. This hypothesis was strongly supported by the fact that the intensity of autophagy varied in company with the fluctuation of steroid secretion. © 1995 Wiley-Liss, Inc.     

登革2型病毒ZS01/01株E蛋白在真核细胞中的分泌表达研究

目的 对登革2型病毒(DENV-2)ZS01/01株E蛋白在哺乳动物细胞及昆虫细胞中的分泌表达进行研究.方法 RT-PCR扩增DENV-2 prM/E基因,通过融合PCR在prM基因前添加来自乙型脑炎病毒的信号肽序列,并将E基因羧基末端20%区域缺失或替换为乙型脑炎病毒SA14-14-2株E基因相应序列,将上述基因元件分别克隆入哺乳动物细胞表达载体pcDNA5/FRT及昆虫细胞表达载体pAcUW51-M中,将重组质粒转染293T细胞或S19细胞,利用间接免疫荧光(Immunofluoreseence assay,IFA)及Western Blot检测E蛋白的表达与分泌.结果 各重组质粒分别转染293T细胞或Sf9细胞后,E蛋白在细胞内均有效表达,而仅有携带乙脑信号肽且缺失或替换E基因羧基末端20%区域的重组质粒转染293T细胞后,上清中可检测到明显的E蛋白分泌.结论 信号肽及E基因羧基末端20%区域对登革病毒E蛋白的分泌至关重要,宿主细胞对其亦有一定影响.     

登革2型病毒ZS01/01株E蛋白在真核细胞中的分泌表达研究

目的 对登革2型病毒(DENV-2)ZS01/01株E蛋白在哺乳动物细胞及昆虫细胞中的分泌表达进行研究.方法 RT-PCR扩增DENV-2 prM/E基因,通过融合PCR在prM基因前添加来自乙型脑炎病毒的信号肽序列,并将E基因羧基末端20%区域缺失或替换为乙型脑炎病毒SA14-14-2株E基因相应序列,将上述基因元件分别克隆入哺乳动物细胞表达载体pcDNA5/FRT及昆虫细胞表达载体pAcUW51-M中,将重组质粒转染293T细胞或S19细胞,利用间接免疫荧光(Immunofluoreseence assay,IFA)及Western Blot检测E蛋白的表达与分泌.结果 各重组质粒分别转染293T细胞或Sf9细胞后,E蛋白在细胞内均有效表达,而仅有携带乙脑信号肽且缺失或替换E基因羧基末端20%区域的重组质粒转染293T细胞后,上清中可检测到明显的E蛋白分泌.结论 信号肽及E基因羧基末端20%区域对登革病毒E蛋白的分泌至关重要,宿主细胞对其亦有一定影响.     

Tumor necrosis factor induces enhanced responses to platelet-activating factor and differentiation in human monocytic Mono Mac 6 cells

Human Mono Mac 6 cells exhibit characterstics of mature blood monocytes. Treatment of these cells with human recombinant human tumor necrosis factor (TNF)resulted in an increase in phagocytosis and phorbol myristate acetate stimulated superoxide anion production at 12 h and growth retardation occurring at 24 h. Moreover, TNF induced a moderate increase of CD14 surface antigen expression, used as a phenotypic marker of monocyte/macrophage differentiation. Platelet-activating factor (PAF) stimulated a rapid rise in cytosolic free Ca⁺⁺ ([Ca⁺⁺]j) of 308 & 93 nM inTNF-treated cells compared to untreated cells (33 ± 8 nM, n = 4). The effect of TNF was dose and time dependent, evident after 12 h and maximal at 48 h. The enhanced PAF-induced [Ca⁺⁺]_i rise was inhibited by the PAF receptor antagonist L-659,989 and EGTA, indicating receptor-dependent Ca⁺⁺ influx. Furthermore, L-659,989 and PAF inhibited specific ³H-labeled PAF binding inTNF-treated, but not in untreated cells. Consistently, PAF stimulated arachidonic acid release only in TNF-treated cells. Preincubation of cells with anti-TNF monoclonal antibodies abolished TNF-induced effects, but failed to block lipopolysaccharide (LPS) effects. Distinct mechanisms of action by LPS were reflected by the different ability to induce surface antigen expression. In conclusion, the enhancement of PAF responses by TNF, associated with functional characteristics of differentiation in Mono Mac 6 cells, may represent a specific mechanism of cooperative interaction between PAF and TNF in inflammation, sepsis, immunoregulation and atherogenesis.     

HMGB1-induced autophagy in Schwann cells promotes neuroblastoma proliferation

Neuroblastoma inflicts mostly on children, and the pathogenesis remains elusive. Clinical diagnosis and therapeutic approaches are still on the incipient stage, so further understanding of the molecular and cellular mechanisms of the disease is necessary. Inflammation has been commonly regarded as a hallmark in tumorigenesis and development, and we identified a new inflammatory factor, HMGB1, is considerably increased in neuroblastoma. Our study shows that HMGB1 induces autophagy in Schwann cells through activation of TLR4, and knockdown of TLR4 obviates the HMGB1-induced autophagy. The HMGB1-induced autophagy is through classical pathway, as deficiency of Beclin 1 deprived autophagy in Schwann cells. Coculture of neuroblastoma with Schwann cells pretreated with HMGB1 promoted the proliferation of neuroblastoma cells, and if Beclin 1 is knocked down in Schwann cells, no promotion effects is observed. Taken together, our study demonstrates that HMGB1-induced autophagy in Schwann cells contributes to neuroblastoma cell proliferation, thus providing a potential therapeutic approach on neuroblastoma development.     

登革2型病毒ZSO1／01株E蛋白在真核细胞中的分泌表达研究

目的对登革2型病毒（DENV-2）ZSO1／01株E蛋白在哺乳动物细胞及昆虫细胞中的分泌表达进行研究。方法RT．PCR扩增DENV-2prM／E基因,通过融合PCR在prM基因前添加来自乙型脑炎病毒的信号肽序列,并将E基因羧基末端20％区域缺失或替换为乙型脑炎病毒SA14-2株E基因相应序列,将上述基因元件分别克隆人哺乳动物细胞表达载体pcDNA5／FRT及昆虫细胞表达载体pAcUW51-M中,将重组质粒转染293T细胞或Sf9细胞,利用间接免疫荧光（Immunofluoreseence assay,IFA）及Western Blot检测E蛋白的表达与分泌。结果各重组质粒分别转染293T细胞或Sit）细胞后,E蛋白在细胞内均有效表达,而仅有携带乙脑信号肽且缺失或替换E基因羧基末端20％区域的重组质粒转染293T细胞后,上清中可检测到明显的E蛋白分泌。结论信号肽及E基因羧基末端20％区域对登革病毒E蛋白的分泌至关重要,宿主细胞对其亦有一定影响。     

登革病毒促进人树突状细胞分泌TNF-α、IL-6、IFN-γ的动态观察

目的：研究登革病毒（DV）对人树突状细胞（DC）产生细胞因子的影响。 方法： 人外周新鲜血常规分离单核细胞，经细胞因子GM-CSF、IL-4诱导培养DC，形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型感染DC，于作用后6、12、24、48、72 h分别收集上清液和细胞，间接免疫荧光法检测细胞上病毒抗原表达，ELISA法检测登革病毒感染后细胞因子TNF-α、IL-6、IFN-γ水平的动态变化。 结果： 人外周血经GM-CSF、IL-4诱导培养1周即可得到典型树突状细胞。间接免疫荧光法证明感染的DC胞浆和胞膜上携带登革病毒抗原，DV感染使DC分泌TNF-α、IL-6能力显著大于对照组（P<0.01），但其分泌IFN-γ的能力无明显改变。 结论： 树突状细胞是登革病毒的靶细胞，登革病毒感染可促进树突状细胞分泌TNF-α、IL-6。树突状细胞可能参与机体抗登革病毒感染的免疫防御机制。     

细胞自噬对饥饿环境下椎间盘髓核细胞的保护作用

目的: 探讨饥饿条件下椎间盘髓核细胞是否发生自噬及自噬对髓核细胞的作用。方法: 原代培养SD大鼠髓核细胞,对细胞表型进行鉴定后,将细胞分为正常对照组、3-甲基腺嘌呤(3-methyladenine,3-MA)加DMEM培养基组、3-MA加EBSS培养基组和EBSS饥饿组,接着用单丹磺酰戊二胺(monodansylcadaverine,MDC)染色、透射电镜和Western blotting观察各组细胞的自噬差异,最后采用CCK-8法测定细胞生长抑制率以及TUNEL法测定细胞凋亡率。结果: 成功培养出SD大鼠髓核细胞。与对照组相比,电镜和荧光显微镜下观察到EBSS培养基诱导的髓核细胞出现自噬囊泡,但是3-MA 抑制后细胞自噬泡明显减少;Western blotting检测发现LC3-II/LC3-I和Beclin-1/β-actin在EBSS处理组明显大于3-MA+EBSS处理组和对照组(P<0.05), 而3-MA+EBSS处理组的细胞抑制率和凋亡率明显高于EBSS处理组(P<0.05)。结论: 饥饿可以诱导大鼠椎间盘髓核细胞发生自噬,并且3-MA能抑制自噬发生。自噬对饥饿环境下的髓核细胞可能具有一定的保护作用。     

Role of intracellular events in the pathogenesis of dengue; An overview

Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading in newer areas in the tropical and subtropical regions of the World. In last fifty years, in spite of intensive and extensive investigations, pathogenesis of dengue is still not clearly understood. Recently, the research focus is on studying the role of intracellular events in pathogenesis of viral infections. Entry of virion in the host cell is followed by quick succession of events, unfolded protein response, lipid bodies and lipophagy, endoplasmic reticulum stress and recent demonstration of autophagy. The turbulence caused by these events may result in clearance of the virus/enhanced replication and survival of the host cell/apoptosis. Both, increased virus load and apoptosis of host cell may have pathological effects on the host. In the present review, we have summed up the role of various intracellular events in viral infections with special emphasis on Dengue virus infection.     

自噬在熊果酸诱导的人脐静脉内皮细胞损伤中的作用

目的: 研究自噬在熊果酸诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤中的作用。方法: 体外培养HUVECs,运用不同浓度的熊果酸处理36 h,采用MTT法检测HUVECs增殖率的变化;透射电镜观察细胞超微结构的改变;单丹磺酰尸胺(MDC)荧光染色观察熊果酸对HUVECs自噬表达水平的影响;Western blotting检测微管相关蛋白轻链3(microtubule-associated protein light chain 3, LC3)及自噬相关蛋白Beclin-1的变化;RT-PCR检测LC3及Beclin-1 mRNA的变化;流式细胞术检测自噬抑制剂3-甲基腺嘌呤(3-methyladenine, 3-MA)抑制自噬前后经熊果酸处理的HUVECs凋亡率的变化。结果: 熊果酸抑制HUVECs增殖并呈剂量依赖性;熊果酸可诱导HUVECs产生自噬:透射电镜和MDC染色均显示HUVECs经熊果酸处理后自噬囊泡明显增加且自噬在基因及蛋白水平的表达均增加;3-MA与熊果酸联合作用于HUVECs时抑制了熊果酸诱导的HUVECs自噬空泡的积聚并且可明显促进HUVECs凋亡。结论: 熊果酸抑制HUVECs增殖,并诱导其发生自噬,自噬在此过程中对HUVECs起保护作用。自噬抑制剂3-MA能够增强熊果酸诱导的HUVECs的增殖抑制作用并且促进HUVECs发生凋亡。     

Induced proteins in cells infected with pseudorabies virus

Summary New proteins appearing after infection of cultured L 929 cells with pseudorabies virus (PRV) were analyzed by SDS polyacrylamide gel electrophoresis. Analysis was facilitated by using a virus-cell system with marked inhibition of host protein synthesis after infection. Infected cells were pulsed during successive two hour periods through the infectious cycle with³⁵S-methionine. Proteins were extracted with detergent and analyzed on high resolution reducing gels. Thirty-four protein bands were resolved on gels of different concentrations that varied from 7 to 15 percent. Calculated apparent molecular weights of the protein peaks were not dependent on gel concentration except for very large or small sized proteins. Eight glycoproteins were resolved after labeling with¹⁴C-glucosamine. The time course of incorporation of label was used as a measure of protein synthesis allowing the grouping of proteins according to the time of maximal synthesis. Several proteins shifted in MW during the course of infection, indicating possible post-translational cleavage or other minor modification.With 3 Figures     

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection

BackgroundDengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties.ObjectiveTo investigate plasma Gal-9 levels as a biomarker for DENV infection.Study designWe enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness.ResultsDuring the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P < 0.01).ConclusionGal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.     

CD147对前列腺癌PC-3细胞自噬的影响

 目的：研究白细胞分化抗原（CD）147在体外对前列腺癌PC-3细胞的自噬作用。方法：通过氨基酸饥饿法建立自噬模型,免疫印迹技术检测CD147的表达。利用RNA干扰CD147表达的细胞系,免疫印迹技术检测自噬蛋白LC3-I和LC3-II的表达;台盼蓝排斥实验检测细胞的死亡情况。结果：在PC-3细胞自噬模型中,随着饥饿诱导时间延长,CD147表达逐渐升高。用RNA干扰技术降低CD147表达后,与阴性对照组比较,在自噬模型中CD147干扰组自噬相关蛋白LC3-II表达增多;并且细胞死亡数量明显增加,阴性对照组细胞死亡率分别为（19.3±3.1）%和（22.3±3.5）%,而在CD147干涉组细胞死亡率为(38.4±3.1)%,差异有统计学意义（P<0.05）。结论：在前列腺癌PC-3细胞中CD147抑制饥饿诱导的自噬,减少自噬性细胞死亡的发生。     

登革病毒在白纹伊蚊细胞内免疫的研究

目的 研究白纹伊蚊对登革病毒产生细胞内免疫现象的分子机制。方法 扩增与克隆登革病毒NGC株的前膜蛋白基因prM，将prM基因以3种不同表达形式重组入昆虫表达载体pBh-spEGFP，以3种重组质粒分别转染白纹伊蚊C6／36细胞，测定转染后的细胞对登革病毒感染的免疫效果。结果 构建了包含prM基因的3种昆虫表达载体，Western blot和荧光显微镜观察证明在C6／36细胞中成功表达了prM-EGFP融合蛋白。MTT实验和空斑形成实验反映了C6／36细胞对登革病毒产生了细胞内免疫。结论 3种重组质粒均可诱导细胞内免疫现象，说明无论是否表达prM蛋白，只要有prM基因转录就可引起细胞内免疫现象；prM基因正义和反义转录形式均可诱导细胞内免疫；其形成机制可能是通过RNA干扰作用而产生。