雷公藤甲素对病毒性心肌炎细胞凋亡及Fas/FasL 蛋白表达的研究

目的:研究雷公藤甲素对柯萨奇病毒B3病毒(CVB3)感染的病毒性心肌炎小鼠心肌细胞凋亡和Fas/FasL蛋白表达的抑制作用,探讨TP治疗病毒性心肌炎的作用机制。方法:将Balb/c小鼠随机分成4组作为动物模型,分别为对照组、模型组、利巴韦林组和TP组。对照组腹腔注射生理盐水,其余三组腹腔注射CVB3,利巴韦林组和TP组小鼠分别予以相应的药物治疗后,测定各组小鼠存活率及心肌病变积分,采用末端转移酶标记技术(TUNEL法)检测小鼠心肌细胞凋亡,免疫组化法检测Fas/FasL蛋白阳性表达。结果:空白对照组心肌无病变,利巴韦林组、TP组与模型组相比有显著性差异(P<0.01)。正常组鲜见心肌细胞凋亡,模型组细胞凋亡率较正常组显著增加(P<0.01),治疗组利巴韦林组和TP组凋亡率比模型组明显降低(P<0.05,P<0.01)。模型组Fas/FasL表达比正常组显著增多(P<0.01),治疗组利巴韦林组和TP组较模型组显著降低(P<0.01)。结论:雷公藤甲素具有通过抑制Fas/FasL蛋白的表达,减缓心肌细胞凋亡,达到抑制病毒性心肌炎从而保护心肌细胞的作用。     

病毒性心肌炎所致小鼠心力衰竭心肌组织内质网应激相关的凋亡关系的研究

目的：探讨病毒性心肌炎心力衰竭小鼠心肌组织内质网应激介导的凋亡途径。方法：40只雄性Balb／c小鼠分为病毒性心肌炎组和正常对照组（n=20）,病毒性心肌炎组应用柯萨奇B3病毒制作BALB／c小鼠病毒性心肌炎模型,观察小鼠的一般情况,7d行血流动力学检查后处死取心脏标本,用TUNEL法检测心肌细胞凋亡,RT-PCR检测心肌细胞内质网伴侣蛋白葡萄糖调节蛋白（GAP）78和GRP04的mRNA表达水平。结果：①与正常对照组相比,病毒性心肌炎组小鼠血流动力学指标明显降低（P〈0．01）;②TUNEL染色显示病毒性心肌炎心力衰竭小鼠心肌组织凋亡明显增多（P〈0．01）;③病毒性心肌炎组小鼠内质网伴侣蛋白GRP78和GRP94的mRNA表达水平均明显高于对照组（P〈0．01）。结论：病毒性心肌炎心力衰竭小鼠内质网应激可能介导了心肌细胞凋亡。     

桂皮醛对小鼠柯萨奇病毒诱发病毒性心肌炎的作用(英文)

已知Toll样受体4(Toll-like receptors 4,TLR4)及其下游信号组分在柯萨奇病毒(CoxsackievirusB,CVB)诱发的病毒性心肌炎中扮演重要的角色,其在治疗中的作用仍不明确。桂皮醛具有抗病毒以及成剂量依赖性抑制由TLR4诱导的核因子活性的作用,而其对病毒性心肌炎的作用机制尚不明确。我们的实验结果显示:在体外,桂皮醛对正常心肌细胞的IC50为15μM;100-1000μM桂皮醛能显著抑制心肌细胞中的病毒滴度(P0.01),而细胞存活率与CVB组无统计学差异(P0.01)。而在病毒性心肌炎小鼠体内,与模型组比较,20和40mg/kg桂皮醛i.p.使第7 d血清中NO的含量以及心肌中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS),肿瘤坏死因子(tumor necrosis factor,TNF-α),核因子κB P65(nuclear factor-κB P65,NF-κB P65)和TLR4蛋白质表达显著降低(P0.05)。降低第21 d心脏体重比(Heat Weight/Body Weight,Hw/Bw)比值,提高小鼠生存率,减轻病理损伤的作用。这些结果显示桂皮醛虽在体外无抗病毒活性,但其在体内具有降低病毒滴度和抑制TLR-4-NF-κB信号传导的作用,对病毒性心肌炎小鼠具有治疗作用。桂皮醛可能通过对TLR-4-NF-κB信号传导抑制作用,作为一种新的方法治疗病毒性心肌炎。     

基于PI3K/PKB通路研究EGCG对CVB3感染致病毒性心肌炎小鼠细胞凋亡的影响

细胞凋亡是造成病毒性心肌炎(VMC)发病过程中心肌损伤的重要因素;表没食子儿茶素没食子酸酯(EGCG)对缺血再灌注引起的细胞凋亡具有抑制作用,但是否抑制VMC发病过程中的心肌细胞凋亡尚不明确.因此,本研究将分析EGCG对VMC小鼠细胞凋亡的影响及分子机制.BALB/c小鼠随机分为对照组、VMC组(腹腔注射CVB3悬液造模)、VMC+EGCG组(腹腔注射CVB3悬液造模后腹腔注射EGCG)、VMC+EGCG+LY组(腹腔注射CVB3悬液造模后腹腔注射EGCG及P13K抑制剂LY294002).检测血清心肌损伤标志物肌钙蛋白I(cTnI)及乳酸脱氢酶(LDH),心肌中CVB3滴度及RNA表达、HE染色、凋亡基因及p-PI3K、p-PKB表达.结果显示,与对照组比较,VMC组血清cTnI、LDH含量、心肌中CVB3滴度及RNA表达、细胞凋亡率、cleaved caspase-3表达升高,心肌中Survivin、p-PI3K、p-PKB表达降低(P＜0.05);与VMC组比较,VMC+EGCG组血清cTnI、LDH含量及心肌中细胞凋亡率、cleaved caspase-3表达降低,心肌中Survivin、p-PI3K、p-PKB表达升高(P＜0.05),心肌中CVB3滴度及RNA表达无明显变化(P＞0.05);与VMC+EGCG组比较,VMC+EGCG+LY组血清cTnI、LDH含量、心肌中细胞凋亡率、cleaved caspase-3表达升高,心肌中Survivin、p-PI3K、p-PKB表达降低(P＜0.05),心肌中CVB3滴度及RNA表达无明显变化(P＞0.05).以上结果表明EGCG对VMC小鼠心肌细胞凋亡具有抑制作用且该作用与激活PI3K/PKB通路有关.     

CVB3诱导的急性心肌炎C57BL/6小鼠模型转录组分析

研究柯萨奇病毒B3(CVB3)感染小鼠所引起的心肌组织转录组变化规律.C57BL/6小鼠腹腔接种浓度为104TCID50的CVB3,建立C57BL/6急性病毒性心肌炎小鼠模型,逐日测量休重.接种第3、6、9、11和14d分别取心脏,计算心脏指数,并取部分心肌组织进行HE染色分析病理学改变;病毒接种后的第3、第6和第9d,取部分组织匀浆进行转录组测序,分析三个时间点的共同差异表达基因,对其进行GO和KEGG信号通路的富集,并对其中12个基因进行了 qRT-PCR的验证.在CVB3感染后,小鼠体重下降至对照组的80％,心脏指数在第3d明显升高,随后逐渐下降.通过转录组分析找到100个共同差异基因,从中选出的12个基因,经qRT-PCR验证与转录组表达趋势一致.GO和KEGG信号通路富集发现,CVB3感染后,小鼠心肌组织出现病毒性心肌炎、NK细胞通路,T、B细胞激活及先天性免疫反应通路的改变.差异基因的蛋白与蛋白互作网络分析显示,先天性免疫中MHC-Ⅰ型分子蛋白基因H2-Q7、H2-Kl、H2-D1等,NK细胞毒作用通路中的Gzmb、Gzma,以及蛋白酶体信号通路基因Psmb8、 Psmb9﹑Psmb10和lfit3位于相互作用中心.CVB3感染C57BL/6小鼠心肌组织的转录组变化涉及病毒性心肌炎、NK细胞和T、B细胞激活及先天性免疫反应等通路.CVB3感染引起的急性病毒性心肌炎是多通路和多基因综合作用的结果.     

柯萨奇病毒B3型诱导的免疫偏离与心肌炎发病关系的研究

目的 研究2种近交系小鼠在柯萨奇病毒B3型(CVB3)感染后辅助性T细胞(Th)免疫偏离对心肌炎发病的影响。方法 用CVB3腹腔感染BALB/c和C57BL/62种近交系小鼠,感染后7d通过检测小鼠血清肌酸激酶(CK)活性,观察心脏外观变化以及心脏石蜡切片H.E染色观察心脏病理改变,比较2种小鼠心肌炎的发病情况;通过体外感染心肌细胞观察病毒复制情况以及体内心脏组织病毒载量的分析,比较2种小鼠对病毒感染和复制的差异;通过检测感染小鼠细胞因子白细胞介素-4(IL-4)、IL-12和γ干扰素(IFN-γ)的表达,抗CVB3VP1抗体的亚型以及T-bet和Gata-3的表达,比较2种小鼠Th免疫偏离的情况。结果 CVB3在体外和体内都可以感染BALB/c和C57BL/6小鼠心肌细胞,但仅BALB/c小鼠感染后可发生明显的病毒性心肌炎,C57BL/6小鼠则不能;BALB/c小鼠感染后表现为Th1型免疫反应而C57BL/6小鼠则偏向于Th2型免疫反应。结论 CVB3感染2种品系小鼠表现为不同的心肌炎发生率,与其诱导了不同类型的免疫偏离密切相关。     

淫羊藿苷对CVB3诱导的幼龄大鼠心肌炎的作用

目的探究淫羊藿苷(Icariin)对柯萨奇B3病毒(coxsackievirus B3,CVB3)诱导的幼龄大鼠心肌炎的作用及作用机制。方法将幼龄SD大鼠随机分为对照组(Ctrl组)、Icariin组、CVB3组和CVB3+Icariin组,CVB3组和CVB3+Icariin组大鼠腹腔注射CVB3复制心肌炎模型。HE染色检测心肌组织病理改变,Western blot检测Caspase-3和Caspase-9的表达,试剂盒检测心脏功能指标肌红蛋白(myoglobin,Mb)、肌酸激酶同工酶(Creatine kinase MB,CK-MB)、肌钙蛋白I(Cardiac troponin I,c Tn I)、氧化应激指标超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的浓度,ELISA检测炎症因子白介素-6(Interleukin-6,IL-6)和IL-1β的浓度。结果与Ctrl组相比,CVB3组大鼠心肌组织病理损伤加重;与CVB3组相比,CVB3+Icariin组大鼠心肌组织病理损伤明显减轻;同时,CVB3组Caspase-3和Caspase-9的表达水平明显高于Ctrl组,差异有统计学意义(P0.01),CVB3+Icariin组大鼠Caspase-3和Caspase-9表达水平均低于CVB3组,差异有统计学意义(P0.01); CVB3能明显升高大鼠血清Mb、CK-MB和c Tn I的浓度(P0.01),Icariin能降低模型大鼠Mb、CK-MB和c Tn I的浓度,差异有统计学意义(P0.01);与Ctrl组相比,CVB3组大鼠血清SOD浓度明显降低(P0.01),MDA浓度明显升高(P0.01),而Icariin能减弱CVB3对SOD和MDA的调控作用,差异有统计学意义(P0.01);此外,CVB3能升高模型大鼠血清中IL-6和IL-1β的浓度(P0.01),Icariin能降低IL-6和IL-1β的浓度(P0.01)。结论 Icariin能通过抑制氧化应激和炎症反应减轻CVB3诱导的幼龄大鼠心肌炎。     

RNA干扰在柯萨奇病毒致心肌炎小鼠模型中的抗病毒研究

为了研究慢病毒介导的shRNA(Short hairpin RNA,shRNA)在柯萨奇B组3型病毒(Coxsackievirus B3,CVB3)导致的心肌炎小鼠模型中的抗病毒作用,合成针对CVB3基因组3753～3771区域的慢病毒Lenti-sh3753,感染HeLa细胞后感染CVB3病毒,通过荧光显微镜观测shRNA的表达和病毒致细胞病变效应,并测定培养上清中的病毒滴度,将慢病毒Lenti-sh3753感染BALB/c小鼠后感染CVB3病毒,观察小鼠的存活率,心脏组织中的病毒滴度和病理变化。结果发现Lenti-sh3753能在HeLa细胞中表达shRNA,并能有效抑制细胞中病毒RNA的复制。在小鼠模型上,Lenti-sh3753能提高小鼠的存活率,降低心脏中的病毒含量,从而减轻病理反应。这些结果提示,Lenti-sh3753在细胞和动物模型中能针对性地降解CVB3病毒RNA,明显降低病毒滴度,有效控制病毒感染。     

病毒性心肌炎与支原体肺炎患儿心肌损伤标志物水平的检验意义

目的:探讨病毒性心肌炎与支原体肺炎患者心肌损伤标志物水平检测意义。方法:回顾性分析医院收治的病毒性心肌炎患儿53例和肺炎支原体肺炎患儿49例分别作为病毒性心肌炎组和支原体肺炎组,选取同期体检正常儿童50例作为对照组,分别检测心肌酶指标和心肌蛋白指标。结果:病毒性心肌炎组心肌肌钙蛋白I(c Tnl)、肌红蛋白(MYO)显著高于支原体肺炎组、对照组,差异显著(P0.05);支原体肺炎组和对照组组间差异显著,具有统计学意义(P0.05)。病毒性心肌炎组肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、门冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)均显著高于支原体肺炎组、对照组,差异显著(P0.05);支原体肺炎组、对照组组间对比差异显著,具有统计学意义(P0.05)。2组入院10 d c Tnl、MYO均低于入院第1 d,具有统计学意义(P0.05);病毒性心肌炎组入院第10 d c Tnl、MYO显著高于支原体肺炎组,具有统计学意义(P0.05)。2组入院10 d CK、CK-MB、AST、LDH均低于入院第1 d,具有统计学意义(P0.05);病毒性心肌炎组入院第10 d CK、CK-MB、AST显著高于支原体肺炎组,差异具有统计学意义(P0.05)。根据ROC曲线分析临床性能,c Tnl、MYO、CK、CK-MB、AST、LDH的临界值分别为0.38μg/L、56.2μg/L、236.58 U/L、32.8 U/L、71.6 U/L、232.8 U/L,灵敏度分别为82.7%、85.4%、84.8%、89.6%、90.2、79.8%。结论:心肌损伤标志物可作为诊断病毒性心肌炎和支原体肺炎的重要指标,应用ROC回归曲线确定各指标的临界值,还可对两种疾病进行鉴别诊断。     

替米沙坦对CVB3诱导的病毒性心肌炎小鼠的保护作用

该文探讨了替米沙坦对柯萨奇B3(Coxsackie B3,CVB3)病毒诱导的病毒性心肌炎小鼠的保护作用。该研究将60只小鼠随机分为对照组、模型组、观察组,每组20只。将CVB3病毒溶解后腹腔注射制作模型,观察组小鼠给予替米沙坦喂食,7天后处死。观察比较3组小鼠心肌组织病理情况,使用试剂盒检查各组超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)及谷胱甘肽过氧化物酶(glutathion peroxidase,GSH-Px)的水平;使用酶联免疫检测白细胞介素-1β(interleukin-1β,IL-1β)、γ干扰素(interferon-γ,IFN-γ)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)。结果显示,观察组小鼠心肌组织细胞排列趋近于规律,且细胞间缝隙较小,同时炎症细胞较少。模型组小鼠心肌组织中氧化应激指标MDA较对照组显著升高,GSH-Px以及SOD较对照组显著降低(P0.01);观察组小鼠心肌组织中MDA较模型组显著降低,GSH-Px以及SOD较模型组显著升高(P0.01)。模型组小鼠心肌组织中IFN-γ、TNF-α以及IL-1β含量较对照组显著升高(P0.01);观察组小鼠心肌组织中炎症因子含量较模型组显著降低(P0.01)。模型组小鼠心肌细胞iNOS、p-p65、TLR4蛋白表达水平较对照组均显著升高(P0.01);观察组小鼠心肌细胞iNOS、p-p65、TLR4蛋白表达水平较模型组均显著降低(P0.01)。模型组小鼠心肌细胞中Nrf2相关蛋白表达水平较对照组显著降低(P0.01);观察组小鼠心肌细胞中Nrf2相关蛋白表达水平较模型组显著升高(P0.01)。该研究得出结论:针对病毒性心肌炎的小鼠模型,早期使用替米沙坦后可以通过参与氧化应激以及炎症反应过程来达到减轻心肌受损的目的。     

金黄色葡萄球菌LDH多克隆抗体制备及其交叉反应性

目的:在原核载体中表达、纯化金黄色葡萄球菌乳酸脱氢酶,免疫小鼠获得多克隆抗体,使用多克隆抗体分析与其它菌种的交叉反应性。方法:复苏p ET28a-ldh/Bl21重组菌,IPTG诱导重组融合蛋白表达、以抗HIS标签的单克隆抗体进行western-blot鉴定重组蛋白。使用纯化的重组蛋白以及相应的佐剂免疫小鼠,利用ELISA测定血清抗体效价,并使用小鼠抗血清进行免疫印迹法鉴定重组蛋白的反应原性及其与金黄色葡萄球菌、表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌的交叉反应性。结果:SDS-PAGE电泳在39 KDa处可见目的条带,免疫印迹法验证了重组LDH的表达,纯化后获得2.8 mg重组蛋白。纯化蛋白免疫小鼠能诱导产生特异性体液免疫应答,ELISA检测特异性Ig G效价为1:50000,western-blot鉴定显示所制备的多克隆抗血清能分别识别金黄色葡萄球菌重组及天然乳酸脱氢酶,但不识别表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌中天然乳酸脱氢酶。结论:纯化的LDH具有良好的免疫活性,免疫小鼠获得高滴度的多克隆抗体。使用多克隆抗体western-blot显示与其它菌种LDH不存在交叉反应性,为后续使用该重组蛋白进行金黄色葡萄球菌感染的诊断研究奠定基础。     

Passive immunization reduces murine cytomegalovirus-induced brain pathology in newborn mice

Human cytomegalovirus (HCMV) is the most frequent cause of congenital viral infections in humans and frequently leads to long-term central nervous system (CNS) abnormalities that include learning disabilities, microcephaly, and hearing loss. The pathogenesis of the CNS infection has not been fully elucidated and may arise as a result of direct damage of CMV-infected neurons or indirectly secondary to inflammatory response to infection. We used a recently established model of mouse CMV (MCMV) infection in newborn mice to analyze the contribution of humoral immunity to virus clearance from the brain. In brains of MCMV-infected newborn mice treated with immune serum, the titer of infectious virus was reduced below detection limit, whereas in the brains of mice receiving control (nonimmune) serum significant amounts of virus were recovered. Moreover, histopathological and immunohistological analyses revealed significantly less CNS inflammation in mice treated with immune serum. Treatment with MCMV-specific monoclonal antibodies also resulted in the reduction of virus titer in the brain. Recipients of control serum or irrelevant antibodies had more viral foci, marked mononuclear cell infiltrates, and prominent glial nodules in their brains than mice treated with immune serum or MCMV-specific antibodies. In conclusion, our data indicate that virus-specific antibodies have a protective role in the development of CNS pathology in MCMV-infected newborn mice, suggesting that antiviral antibodies may be an important component of protective immunological responses during CMV infection of the developing CNS.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

盐酸阿比朵尔抗柯萨奇病毒B3的实验研究

为观察盐酸阿比朵尔对柯萨奇病毒的抑制作用,本实验以利巴韦林为阳性对照药物,采用细胞培养技术、细胞病变效应(CPE)抑制法、活细胞染色计数法(MTT)和培养上清中病毒滴度测定观察盐酸阿比朵尔抗柯萨奇病毒的作用。结果表明盐酸阿比朵尔的半数中毒浓度(TD50)为896.54μg/mL,药物抗病毒生物合成组、药物直接作用组、药物抗病毒吸附2h组、药物抗病毒有吸附8h组的病毒抑制率分别能达到74.48%、45.68%、28.90%和48.27%,在抗生物合成组,盐酸阿比朵尔能明显抑制柯萨奇病毒所致的CPE效应,降低培养上清中的病毒滴度,病毒抑制率随药物浓度增加而增高,存在量效关系。这提示盐酸阿比朵尔在细胞内对柯萨奇病毒有一定的抑制作用。     

Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.     

Maternal Antibody-Mediated Disease Enhancement in Type I Interferon-Deficient Mice Leads to Lethal Disease Associated with Liver Damage

Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals’ death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and therapeutic candidates.     

不同佐剂对人乳头瘤病毒16型L2E7E6蛋白免疫效果的影响

目的:研究CpG佐剂、弗氏佐剂、聚肌胞苷酸佐剂及左旋咪唑、西米替丁作为佐剂对人乳头瘤病毒16型L2E7E6融合蛋白在小鼠体内产生的免疫效果的影响。方法:以单独蛋白组、蛋白加各佐剂组分别肌肉注射免疫C57BL/6小鼠,检测不同佐剂诱发小鼠产生的体液免疫和细胞免疫应答水平,并观察其对小鼠肿瘤生长的抑制作用。结果:各免疫组均能检测到高滴度的抗L2、E7、E6蛋白IgG抗体(以IgG1为主),其中弗氏佐剂能显著提高E6蛋白的IgG和IgG1抗体水平和E7蛋白的IgG1抗体水平(P<0.05),CpG佐剂明显提高了E7蛋白的IgG2a抗体水平(P<0.01);而西米替丁佐剂则降低了E7抗原的IgG抗体水平(P<0.05);同时可以检测到CpG佐剂组能诱发小鼠产生针对E7、E6较强的细胞免疫反应,且能抑制70%的荷瘤小鼠肿瘤生长;此外弗氏佐剂与聚肌胞苷酸佐剂可产生较弱的针对E7肽的细胞免疫反应,能延缓荷瘤小鼠肿瘤形成时间,与单纯蛋白组相比差异显著(P<0.05)。结论:CpG佐剂、弗氏佐剂和聚肌胞苷酸佐剂都能提高人乳头瘤病毒16型L2E7E6融合蛋白的细胞免疫反应水平和抑制肿瘤生长能力,其中CpG佐剂效果较好,为促进该蛋白作为疫苗的研发提供了实验依据。