Toll样受体在约氏疟原虫感染早期树突状细胞应答中的作用地位

目的探讨致死型约氏疟原虫(Plasmodium yoelii17XL,P.y17XL)感染早期,Toll样受体(Toll like receptor,TLR)在树突状细胞(dendritic cells,DCs)活化中的作用地位。方法用P.y17XL感染易感的BALB/c和抵抗的DBA/2小鼠,计数红细胞感染率;制备感染前和感染后第3d、5d小鼠脾细胞悬液,采用流式细胞分析技术检测两种小鼠感染不同时间脾细胞悬液中细胞内表达TLR9(Toll like receptor 9,TLR9)的DCs和细胞表面表达TLR4(Toll like receptor 4,TLR4)的DCs的百分含量。结果两种小鼠脾DCs细胞内TLR9的表达水平均于感染后第3d开始明显升高(P<0.01),在第5d达到最高水平(P<0.01),但两种小鼠相比无统计学意义。同时,两种小鼠DCs表面TLR4的表达水平均未见明显变化。结论在P.y17XL感染早期,TLR9可能是介导DCs活化的模式识别受体。     

T细胞亚群在感染约氏疟原虫小鼠中的保护性免疫作用

对约氏疟原虫有保护性免疫的BALB/c小鼠,体内注射单抗去除CD~(4 )或CD~(8 )T细胞后,对保护性免疫无明显影响。但将它们脾细胞中CD~(8 )T细胞注入裸鼠,可以转移部分免疫力,而注入CD~(4 )T细胞则不能。在约氏疟原虫感染小鼠模型中,由于在感染早期原虫侵入网织红细胞,CD~(8 )T细胞可被激活而有杀伤作用。在晚期,抗体在免疫中起主要作用。     

影响约氏疟原虫红细胞外期发育因素的研究

采用正交试验设计,观察环境温度、光照周期、脾脏切除和雌激素水平对大鼠体内约氏疟原虫红细胞外期生长发育的影响。结果显示:脾脏在清除子孢子方面有重要作用,切脾组的红外期原虫密度显著高于对照组;雌激素水平的升高则明显抑制红外期的发育。低温环境对红外期的密度虽无明显影响,但使之发育变缓,不同步更为显著。光照周期的长短似乎对红外期的生长发育无明显影响。两种因素交互作用时,脾脏切除和雌激素水平升高的影响相互抵消,其他两两因素的作用,经方差分析,均未见显著性差异。     

枯否氏细胞,肝细胞对疟原虫子孢子感染和发育的影响

１次接种９００万约氏疟原虫子孢子悬液，３０ｍｉｎ后的肝脏超微结构显示，枯否氏细胞吞噬复合体，邻近肝细胞线粒体空泡化，嵴突消失；枯否氏细胞内可见形态退变的子孢子；线粒体、内质网、核糖体丰富的肝细胞内子孢子结构清晰；线粒体空泡化、嵴突消失的肝细胞内子孢子退变；子孢子进入枯否氏细胞或肝细胞的时间不玫，１个肝细胞内可见２个子孢子。     

枯否氏细胞、肝细胞对疟原虫子孢子感染和发育的影响

１次接种９００万约氏疟原虫子孢子悬液，３０ｍｉｎ后的肝脏超微结构显示，枯否氏细胞吞噬复合体，邻近肝细胞线粒体空泡化，嵴突消失；枯否氏细胞内可见形态退变的子孢子；线粒体、内质网、核糖体丰富的肝细胞内子孢子结构清晰；线粒体空泡化、嵴突消失的肝细胞内子孢子退变；子孢子进入枯否氏细胞或肝细胞的时间不一，１个肝细胞内可见２个子孢子。     

IL-6对大鼠体内约氏疟原虫红外期发育的影响

目的观察ＩＬ－６对大鼠体内约氏疟原虫红外期发育的影响。方法子孢子（Ｓｐ）接种前２ｈ给Ｗｉｓｔａｒ大鼠腹腔注射１００００ＵｒＨｕＩＬ－６,４２ｈ后活杀、常规制肝切片,观察Ｓｐ发育率,外期原虫（ＥＥＦ）直径。大鼠在接种Ｓｐ前３ｈ、０．５ｈ分别经尾静脉注射１００００Ｕ、３００００ＵｒＨｕＩＬ－６,经肠系膜静脉灌注２８万Ｓｐ,１ｈ后制备肝脏样品,用电镜观察结果。结果与对照组比较,１００００ＵｒＨｕＩＬ－６对约氏疟原虫Ｓｐ发育率没有显著影响,但ＥＥＦ直径明显变小。Ｓｐ接种１ｈ后肝细胞内可见形态退变的Ｓｐ。结论ＩＬ－６作为一种免疫效应分子可抑制大鼠体内约氏疟原虫ＥＥＦ的发育。     

调节性T细胞抑制约氏疟原虫感染小鼠早期Th1应答的建立

目的探讨调节性T细胞(Tregs)参与约氏疟原虫感染早期调控Th1型免疫应答的相关机制。方法用约氏疟原虫(致死型)感染对照组和anti-CD25 mAb注射组BALB/c小鼠,计数红细胞感染率;感染后第0、3和5d制备脾细胞悬液,磁珠分选、纯化树突状细胞(DCs)并体外培养;ELISA方法检测脾细胞培养上清中IFN-γ和DCs培养上清中IL-12的水平,Griess方法检测脾细胞培养上清中NO含量。结果两组小鼠脾细胞培养上清中IFN-γ和NO水平在感染后第3～5d均明显升高,但对照组小鼠IFN-γ和NO水平明显低于anti-CD25 mAb注射组小鼠。anti-CD25 mAb注射组小鼠DCs培养上清中IL-12的水平于感染后第3d达峰值,并于感染后第5d仍维持较高水平。相比,对照组小鼠DCs培养上清中IL-12的水平仅于感染后第3d出现有意义的升高。结论Tregs在致死型约氏疟原虫感染BALB/c小鼠早期可能通过抑制DCs分泌细胞因子IL-12来抑制Th1型免疫应答的有效建立。     

双氢青蒿对约氏疟原虫在蚊体内发育的影响

目的：观察双氢青蒿素（dihydroqinghaosu，DQHS）对约氏疟原虫在斯氏按蚊体内发育的影响。方法：受染疟原虫小鼠一次性经口灌喂不同浓度DQHS药液后，供蚊虫吸血，应用光镜和电镜观察对照组和用药组的疟原虫在蚊体内的发育情况。结果：DQHS对疟原虫配子体有一定的抑制作用，其作用强度与配子体成熟程度和用药剂量不同有关。未成熟配子体对药物较敏感；随着药物剂量的增加，卵囊和子孢子的阳性率和密度随之逐渐下降；但180mg或240mg／kg用药组对子孢子密度的影响的差别无显著性意义。电镜观察60mg／kg作用16h后，用药组的蚊胃上卵囊（12d－13d解剖蚊），出现膜受损，甚至胞质空泡化。120mg／kg药量对蚊体内3日龄卵囊作用16h，卵囊仍继续发育，比较对照组和用药组的卵囊和子孢子密度, 两组间差别无显著意义(P > 0. 05) ; 两组卵囊超微结构形态亦无明显差异。结论: DQHS 影响约氏疟原虫配子体感染性而减少蚊媒传播, 但对蚊体内子孢子增殖期不起直接的抑制作用。     

Inhibitory role of toll-like receptors agonists in Plasmodium yoelii liver stage development

It is well known that innate immune plays an important role in controlling the development of Plasmodium liver stage. However, little is known about the role of toll-like receptors (TLR) signalling in the pre-erythrocytic immunity against Plasmodium. Here, we found that pre-treatment with individual TLR agonist pam3CSK4 (TLR2), poly(I:C) (TLR3), LPS (TLR4) and CpG (TLR9) could decrease significantly the liver malaria parasite load in mice for 58%, 63%, 75% and 88% respectively. Moreover, no parasitaemia was observed within 14 days in CpG group mice challenged with 100 sporozoites. At 24 h prior to CpG injection, administration of gadolinium chloride (GdCl₃) led to the rebound of liver Plasmodium load through inhibiting selectively Kupffer cells (KC) phagocytosis capacity but failed to neutralize completely CpG-induced immunity against malaria liver stage. Compared with the control, pre-treatment of CpG up-regulated hepatic pro-inflammatory cytokines IL-12, IFN-γ and TNF-α, but down-regulated anti-inflammatory cytokines IL-10 and TGF-β. Hence, our data demonstrated the inhibitory role of diverse TLR agonists in the Plasmodium development during pre-erythrocytic stage. As the most robust agonist, CpG might inhibit the development of Plasmodium liver stage through regulation of intrahepatic inflammatory cytokines and enhancement of KC cells phagocytosis capacity.     

CD4~+CD25~+调节性T细胞在约氏疟原虫感染小鼠应答早期中的作用

目的探讨CD4+CD25+调节性T细胞在约氏疟原虫感染早期的作用及意义。方法用约氏疟原虫(致死型)感染DBA/2和BALB/c小鼠,计数红细胞感染率;在感染后第0d、3d、4d、5d和6d提取脾细胞应为,流式细胞术检测两种小鼠感染不同时间脾细胞悬液中CD4+T细胞和CD4+CD25+T细胞百分含量;ELISA法检测脾细胞培养上清IFNγ、IL10和TGFβ1水平。结果DBA/2小鼠的IFNγ水平在感染后第3d迅速升高后缓慢下降(P<0.01),CD4+CD25+T细胞数仅在感染后第5d出现有意义的升高(P<0.05),而IL10水平和CD4+T细胞数量都无明显改变。BALB/c小鼠的IFNγ水平在感染后第3d出现有意义的升高后迅速下降(P<0.01),CD4+CD25+T细胞数在感染后第3d开始明显升高,于感染后第5d出现下降(P<0.05),而IL10水平自感染后第3～6d持续维持高水平(P<0.05),CD4+T细胞数量于感染后第6天明显下降(P<0.05)。结论CD4+CD25+调节性T细胞在致死型约氏疟原虫感染BALB/c小鼠早期可能通过抑制性细胞因子IL10发挥免疫抑制作用。     

Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells

Irradiation-attenuated sporozoite vaccinations confer sterile protection against malaria infection in animal models and humans. Persistent, nonreplicating parasite forms in the liver are presumably necessary for the maintenance of sterile immunity. A novel vaccine approach uses genetically attenuated parasites (GAPs) that undergo arrested development during liver infection. The fate of GAPs after immunization, their persistence in vaccinated animals, and the immune mechanisms that mediate protection are unknown. To examine the developmental defects of genetically attenuated liver stages in vivo, we created deletions of the UIS3 and UIS4 loci in the Plasmodium yoelii rodent malaria model (Pyuis3[-] and Pyuis4[-]). The low 50% infectious dose of P. yoelii in BALB/c mice provides the most sensitive infectivity model. We show that P. yoelii GAPs reach the liver, invade hepatocytes, and develop a parasitophorous vacuole but do not significantly persist 40 h after infection. A single dose of Pyuis4(-) sporozoites conferred complete protection, but full protection by Pyuis3(-) sporozoites required at least 2 immunizations. CD8(+) T cells were essential for protection, but CD4(+) T cells were not. Our results show that genetically distinct GAPs confer different degrees of protective efficacy and that live vaccine persistence in the liver is not necessary to sustain long-lasting protection. These findings have important implications for the development of a P. falciparum GAP malaria vaccine.     

Strain specificity in the liver-stage development of Plasmodium falciparum in primary cultures of new world monkey hepatocytes

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.     

Scanning electron microscopic observations of ookinete formation of Plasmodium yoelii yoelii in vitro

The ookinete formation and mature ookinete of Plasmodium yoelii yoelii were described. During the development from zygote to ookinete, at first a finger-shaped or a stick-shaped projection protruded from one side of zygote. The anterior end of the projection assumed a truncated cone shape. Following enlargement of the projection, the body of zygote gradually wrinkled and shrank and became smaller. An annular structure developed between the newly formed ookinete and the remaining body of zygote, which ultimately separated from the ookinete. The ookinete was banana-shaped. The surface of mature ookinetes was smooth. On the tip there was an apical complex showing truncated cone shape. Behind the apical end there were a few pellicular folds. Through the course of development of the ookinete the parasite constantly showed obvious movement. As the parasites protruding forward, longitudinal and spiral wrinkles appeared on their body surface.