不同毒力疟原虫感染早期根治性治疗对免疫记忆形成影响的研究

为探讨不同毒力疟原虫感染早期根治性治疗对免疫记忆形成的影响,用致死型和非致死型约氏疟原虫感染DBA/2小鼠,感染后3 d进行根治性治疗,并于初次感染后90 d进行再感染。通过计数红细胞感染率和检测再感染前后不同时间点脾细胞中记忆性T、B细胞的百分含量后发现,再感染后2组小鼠均出现了短暂的低水平虫体血症;再感染前小鼠脾细胞中记忆性T、B细胞百分率均略高于正常鼠;但再感染后均出现了有意义的升高;在每一相同检测时间点,2组小鼠的虫体血症水平和记忆性T、B细胞百分率均无显著差异。这表明,不同毒力疟原虫感染早期的根治性治疗可使宿主产生水平相近的免疫记忆,再感染可促进免疫记忆的形成。     

左旋精氨酸上调约氏疟原虫感染DBA/2小鼠的抗体应答效应

探讨左旋精氨酸（L-Arginine,L-Arg）对约氏疟原虫（Plasmodium yoelii17XL,P.y17XL）感染DBA/2小鼠体液免疫应答的调节效应。于P.y17XL感染前7 d,连续每日给予DBA/2小鼠L-Arg（1.5 g/kg体重）灌胃预处理,动态观察各组感染小鼠原虫血症水平和生存率;于感染后第0、8和10天分别提取小鼠脾细胞,流式细胞术检测DBA/2小鼠浆细胞分泌IgG1和IgG2a的水平。与NC组比较,L-Arg能够显著降低感染小鼠的原虫血症水平,自愈时间提前。感染后8 d和10 d,L-Arg组CD138＋B220＋IgG1＋细胞数量出现有意义的增加,感染后10 d,L-Arg组CD138＋B220＋IgG2a＋细胞数量明显升高。L-Arg处理可诱导DBA/2小鼠建立更加有效的抗体免疫应答,明显加速DBA/2感染小鼠清除疟原虫。     

TLR9激动剂对约氏疟原虫体液免疫记忆的影响

为探讨TLR9激动剂对疟疾体液免疫记忆的影响,用非致死型约氏疟原虫感染BALB/c小鼠,感染前2 d注射TLR9激动剂CpGl826,90 d后进行二次感染。薄血膜染色法观察红细胞感染率,流式细胞术检测脾细胞悬液中记忆性和活化性B细胞百分比,双夹心ELISA法检测特异性抗体水平。结果显示,二次感染前,TLR9激动剂处理鼠记忆性和活化性B细胞以及抗体水平略高于对照组;二次感染后,其再感染发生率和虫血症水平均略低于对照组;活化性B细胞和抗体以及记忆性B细胞也分别于二次感染后1 d和3 d出现了有意义的升高,且升高幅度均略高于对照组。表明TLR9激动剂对约氏疟原虫感染后体液免疫记忆的建立和维持有一定促进作用。     

致死型夏氏疟原虫感染BALB/c小鼠CD4~＋ CD25~＋ Foxp3~＋调节性T细胞在Th2免疫应答极化中的作用研究

为探讨CD4＋ CD25＋ Foxp3＋调节性T细胞（Treg细胞）在疟疾感染过程中对Th2极化的调控作用,利用Treg细胞消除的致死型夏氏疟原虫（Plasmodium chabaudi chabaudi AS,P.c chabaudi AS）感染鼠疟模型进行研究。结果显示,对照组小鼠在感染后8 d原虫血症达到峰值40.5%,随后迅速下降,于感染后18 d小鼠自愈。相比,Treg细胞消除组于感染后10 d,原虫血症水平迅速上升至32%,随后小鼠相继死亡。在感染后8～10 d,Treg细胞消除小鼠脾脏CD4＋ CD25＋ Foxp3＋细胞占CD4＋细胞百分比含量明显低于对照组。同时,血清疟原虫特异性抗体IgG1和IgG2a水平均明显降低。结果提示,P.c chabaudi AS感染中CD4＋ CD25＋ Foxp3＋细胞参与调控Th2型免疫应答的极化,进而干预疟原虫清除。     

TLR9激动剂对约氏疟原虫记忆性T细胞的影响

为探讨TLR9激动剂对疟疾记忆性T细胞的影响,用非致死型约氏疟原虫感染BALB/c小鼠,感染前2 d注射TLR9激动剂Cp Gl826,90 d后进行二次再感染。薄血膜染色法观察虫血症,流式细胞术检测脾细胞悬液中记忆性和活化性T细胞百分率,双夹心ELISA法检测脾细胞培养上清中细胞因子水平。结果发现,再感染前,TLR9激动剂注射组记忆性T细胞百分率(12.98%)高于对照组(10.16%);再感染后,其再感染发生率(50%)和峰值血症水平(0.64%)均低于对照组(70%和0.82%);而活化性T细胞和IFN-γ水平于再感染后1 d即出现了显著升高,分别达到6.62%和372.74 pg/m L,其升高幅度高于对照组(5.04%和216.17 pg/m L)。这表明,TLR9激动剂对约氏疟原虫记忆性T细胞的产生和维持有一定促进作用。     

BALB/c小鼠感染不同疟原虫CD4＋ Th应答和凋亡特点的比较研究

探讨不同疟原虫感染BALB/c小鼠的免疫应答特点。BALB/c小鼠经腹腔注射致死型约氏疟原虫（P.y17XL）和夏氏疟原虫（P.cAS）感染的红细胞,计数红细胞感染率;ELISA动态检测感染小鼠脾细胞培养上清中IFN-γ和IL-4水平;流式细胞术检测脾中CD4＋T细胞凋亡数量。约氏疟原虫（P.y17XL）感染早期,小鼠原虫血症持续上升;IFN-γ和IL-4分泌水平仅在感染后第3天出现一过性有意义的升高,而且峰值较低;脾中CD4＋T细胞大量凋亡,小鼠全部死亡;而夏氏疟原虫（P.cAS）感染小鼠,原虫血症上升缓慢;IFN-γ分泌水平在感染后第5天达峰值;IL-4分泌水平在感染后第10天达峰值,且峰值较高维持时间较长;脾中CD4＋T细胞凋亡细胞于感染后8 d出现有意义升高,小鼠全部存活。抗疟保护性免疫有赖于Th1和Th2型免疫应答的有效建立和协调过渡,感染期间CD4＋T细胞凋亡的时相和数量可能是影响免疫应答的强度或引起宿主免疫抑制的原因,从而影响宿主疟原虫感染的结局。     

Tim-1分子在约氏疟原虫感染模型中的表达分析

Tim-1分子表达在T细胞和调节性B细胞表面,具有促进Th2应答的作用。为探讨Tim-1分子在抗疟疾免疫应答中的作用,利用致死型约氏疟原虫(Plasmodium yoelii)感染鼠疟模型研究了Tim-1分子表达与小鼠感染结局和免疫应答模式的关系。结果显示,BALB/c小鼠对P.yoelii易感,在感染后6～7 d全部死亡,感染后第3、5天脾内细胞因子IFN-γmRNA水平明显低于对P.yoelii抵抗的DBA2小鼠,而脾IL-10 mRNA水平显著高于DBA2小鼠。BALB/c小鼠脾内Tim-1+T、B细胞百分比在感染后第3、5天显著升高,而DBA2小鼠感染前后脾内Tim-1+T、B细胞百分比没有显著变化。结果提示,Tim-1分子参与抗P.yoelii免疫应答的调节,可能与易感鼠产生高水平Th2型细胞因子有关。     

特异性IgG抗体在异种/同种异株疟原虫感染治愈后P.y17XL再感染中的保护作用

为探讨特异性IgG抗体在异种/同种异株疟原虫治愈后再感染过程中的作用,用不同种/株疟原虫感染BALB/c小鼠,经治愈后用P.y17XL再感染。通过计数红细胞感染率和检测再感染后血清中特异性IgG抗体的水平变化,发现P.y17XNL感染治愈组小鼠几乎不出现原虫血症,其余异种疟原虫感染治愈组小鼠出现了不同程度的虫血症发生时相延迟和水平降低,部分生存率有所延长;不同虫种/株感染治愈后P.y17XL再感染的小鼠IFN-γ水平均明显低于同时间点P.y17XL初次感染的小鼠;P.v、P.y17XNL感染治愈小鼠血清中P.y17XL特异性IgG抗体水平出现显著增加(P<0.05),且以IgG1亚类升高为主。表明特异性IgG抗体可在宿主抗同源疟原虫再感染中发挥着重要作用,而对异种疟原虫再感染保护性有限。     

大蒜素对致死型约氏疟原虫鼠疟模型CD4+活化/凋亡T细胞的影响

探讨大蒜素对致死型约氏疟原虫(Plasmodium yoelii 17XL,P.y 17XL)感染BALB/c小鼠T细胞免疫应答的影响。P.y 17XL感染后第0~2天每日口服给予不同浓度大蒜素(3 mg/kg或9 mg/kg)处理;动态监测各组小鼠原虫血症水平和生存期;感染后第0天、第3天和第5天无菌提取小鼠脾细胞,FACS检测小鼠活化T细胞(CD4~+CD69~+)、凋亡CD4~+T细胞(7AAD~-Annexin V~+)数量变化;ELISA检测脾细胞培养上清中IFN-γ和TNF-α的水平。结果显示:与NC组相比,大蒜素处理组能降低感染小鼠的原虫血症水平,延长生存期。大蒜素处理能提高感染小鼠活化T细胞的数量,降低凋亡T细胞数量,并能提高脾细胞培养上清中IFN-γ和TNF-α的水平。大蒜素能在感染早期促使宿主建立有效的Th1免疫应答,从而抑制红内期P.y 17XL疟原虫感染进程。     

PD-1对伯氏疟原虫感染小鼠体液免疫应答的影响

利用伯氏疟原虫Plasmodium berghei ANKA(P.b ANKA)感染BALB/c小鼠,PD-1单抗阻断后,流式细胞术检测脾脏浆细胞、滤泡辅助性T细胞(Tfh)数量。qRT-PCR检测IL-21、IL-10和IL-6 mRNA水平,ELISA检测血清抗体,以探讨程序性死亡受体-1(programmed cell death-1, PD-1)在疟原虫初次感染中对体液免疫应答的影响。结果发现,PD-1单抗阻断加速了P.b ANKA感染小鼠的死亡。与对照组相比,PD-1阻断组感染后第12天短寿浆细胞(CD138~+CD44~+)数量明显降低(P0.05),长寿浆细胞(CD138~+CD44~-、CD138~-CD44~+)和Tfh(CD4~+CXCR5~+)细胞数量无差异性改变,脾细胞IL-21的mRNA水平明显下降(P0.05),血清抗裂殖子表面蛋白(merozoite surface protein, MSP)-1特异性IgG无明显改变。P.b ANKA感染中PD-1通路可能通过影响Tfh分泌IL-21进而干扰浆细胞数量影响体液免疫应答。     

Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii

Plasmodium yoelii 17XL was used to investigate the mechanism of Plasmodium falciparum-caused cerebral malaria, although its histological effect on other mouse organs is still unclear. Here, histological examination was performed on mice infected with P. yoelii 17XL; the effect of P. yoelii 17XL infection on anemia and body weight loss, as well as its lesions in the brain, liver, kidney, lung, and spleen, also was investigated. Plasmodium yoelii 17XL-infected red blood cells were sequestered in the microcirculation of the brain and in the kidney. Compared with the nonlethal P. yoelii 17XNL strain, infection by P. yoelii 17XL caused substantial pulmonary edema, severe anemia, and significant body weight loss. Although P. yoelii 17XNL and 17XL produced a similar focal necrosis in the mouse liver, infection of P. yoelii 17XL induced coalescing of red and white pulp. Mortality caused by P. yoelii 17XL may be due to cerebral malaria, as well as respiratory distress syndrome and severe anemia. Plasmodium yoelii 17XL-infected rodent malaria seems to be a useful model for investigating severe malaria caused by P. falciparum.     

Plasmodium yoelii: influence of antimalarial treatment on acquisition of immunity in BALB/c and DBA/2 mice

The effect of antimalarial drugs on immune responses to the malaria infection is evaluated in vivo using two experimental self-cured rodent models. BALB/c and DBA/2 mice were infected by Plasmodium yoelii 17XNL and 17XL strains, respectively, and then treated with different doses of antimalarial drugs: chloroquine (228mg/kg or 114mg/kg of the body weight) or artesunate (78mg/kg or 39mg/kg). The effect of antimalarial drugs on host immune responses was evaluated by parasitemia, splenocyte IFN-gamma production level, and parasite-specific IgG level in the serum, however, no significant differences were observed between drug-treated and untreated groups. Moreover, most of the infected mice of all groups showed the ability to resist homologous reinfection (challenged on day 60 post-infection), only a few mice experienced transient, low parasitemia. The rechallenged mice were accompanied by high level of parasite-specific IgG. Therefore, this research implicated that, for BALB/c and DBA/2 mice, chloroquine or artesunate treatment of blood-stage P. yoelii infections does not compromise acquired immunity to malaria in either primary infection or upon rechallenge.     

约氏疟原虫Pys48核酸疫苗传播阻断效应的实验研究

探讨约氏疟原虫（Plasmodium yoelii17XL）Pys48核酸疫苗免疫BALB/c小鼠的特异性抗体产生特点及其效应。将Pys48核酸疫苗肌肉内注射免疫BALB/c小鼠,并以空质粒注射组作为对照,3次免疫后通过P.y17XL攻击小鼠;采用ELISA检测免疫后小鼠血清中特异性抗体水平;通过P.y17XL感染小鼠,取其感染后第3天含有配子体的血液进行体外培养,观察合子、动合子的形成数量。ELISA结果显示疫苗免疫组小鼠血清特异性抗体滴度明显高于对照组;而合子、动合子数量明显低于对照组。提示Pys48核酸疫苗具有良好的免疫原性,免疫小鼠后可以建立起有效的传播阻断效应。     

不同维生素免疫调节作用在鼠疟模型中的实验观察

探讨不同维生素对P. y17XL感染BALB/c小鼠的免疫调节作用。将6~8周龄,雌性BALB/c小鼠,随机分为维生素（ V）处理组和对照组。 V处理组小鼠分别经50 mg/kg VA、600 mg/kg VE 或1 g/只VC连续10 d灌胃,0.2 mL/只;对照组小鼠分别给予相同剂量的溶剂（大豆油或生理盐水）处理。之后,各组小鼠分别经腹腔接种1＆#215;106 P. y17XL寄生的红细胞,动态观察感染小鼠原虫血症水平和生存率;流式细胞术检测感染后第0、3和5天小鼠脾细胞树突状细胞（ DCs）亚群（ pDCs与mDCs）百分比及功能分子（ TLR9和MHC域）的表达。与对照组相比,VA和VE处理组小鼠原虫血症水平升高,生存率降低;感染后第5天脾细胞中pDCs与mDCs亚群水平以及DCs表面受体TLR9和表面分子MHC域的表达水平显著下降。相反,VC处理组小鼠原虫血症水平降低,生存率延长,pDCs与mDCs亚群以及TLR9和MHC域的表达水平显著升高。结果表明,不同维生素对疟疾感染产生不同的调控作用,VA和VE通过抑制DCs数量和功能,加重疟疾感染,而VC则促进DCs数量和功能,推迟感染进程。     

Passive immunization against murine malaria with an IgG3 monoclonal antibody

Spleen cells of BALB/c mice that were immune to the 17X strain of P. yoelii were fused with P3X63Ag8 myeloma cells. Two hundred fifty-three of 1053 hybrid cells produced antibodies reactive with disrupted 17X parasites in a solid phase radioimmunoassay. One of these antibodies, McAb 302, reacted with the merozoites of the 17X (nonlethal) and 17XL (lethal) variants of P. yoelii. Of greater significance, McAb 302 passively protected mice against challenge infection with the lethal variant. Mice treated with this antibody before infection developed low-grade parasitemia (less than 0.3%) of short duration when challenged with P. yoelii 17XL . In contrast, control mice that had been untreated or injected with ascites fluid lacking McAb 302 uniformly died with fulminating malaria upon challenge with the same parasite. In other experiments, McAb 302 was shown capable of controlling blood parasite levels when administered to mice with patent P. yoelii 17XL infections. Although all control mice died, mice protected with a single dose of McAb 302 ultimately cleared their infections. Regardless of how passive immunization was performed, mice given McAb 302 were resistant to subsequent challenge with P. yoelii 17XL , indicating they had developed significant immunity during their initial controlled infections. McAb 302 also showed pronounced passive protective activity against the nonlethal 17X strain of P. yoelii, which is a parasite of reticulocytes. The protection afforded by McAb 302 was specific, because mice passively immunized with this antibody died when challenged with the unrelated P. vinckei. McAb 302 was shown to possess the IgG3 isotype and precipitated a 230-kd protein plus several smaller polypeptides from metabolically labeled parasite antigen preparation derived from both variants of P. yoelii. It did not react with similar preparations of other murine plasmodial species.