经口感染弓形虫诱导小鼠黏膜免疫动物模型的建立

目的建立经口感染弓形虫速殖子诱导黏膜免疫的动物模型。方法BALB/c小鼠分别灌胃接种5×103、5×104、5×105、5×106个RH株速殖子,观察小鼠的体况、病理变化;检测肠道分泌型IgA(SIgA)和Peyer’spatches(PP)淋巴细胞及小肠上皮内淋巴细胞(IEL)T细胞亚群的变化。结果经口接种5×104个弓形虫RH株速殖子可使小鼠出现临床症状和病理改变;SIgA水平升高;黏膜诱导部位的CD4 T亚群及效应部位的CD8 T亚群水平升高。结论5×104个弓形虫RH株速殖子灌胃接种小鼠可以诱导机体黏膜免疫应答。     

霍乱毒素联合弓形虫排泄-分泌抗原鼻内免疫小鼠诱导的GALT和NALT黏膜免疫应答

目的观察霍乱毒素(CT)联合弓形虫排泄-分泌抗原(ESA)鼻内免疫小鼠诱导的肠相关淋巴组织(GALT)和鼻相关淋巴组织(NALT)黏膜部位的免疫应答。方法将48只5~6周龄BABL/c小鼠随机均分为3组,分别用PBS、ESA和CT+ESA滴鼻免疫小鼠2次,间隔2周。末次免疫后14 d,处死小鼠,ELISA测定小鼠粪便和鼻咽冲洗液sIgA抗体水平;计数派伊尔淋巴集结(PP)、肠上皮淋巴细胞(IEL)、NALT和鼻通道(NC)淋巴细胞数,免疫细胞化学法检测CD4+、CD8+T细胞亚群水平。结果免疫后14 d,CT+ESA组小鼠鼻咽冲洗液和粪便sIgA水平显著高于ESA组和PBS组(P<0.01);CT+ESA和ESA组小鼠GALT部位的PP淋巴细胞显著增生,主要以CD4+T细胞为主;而IEL以CD8+T细胞增生为主,CD4+/CD8+比值降低(P<0.05)。CT+ESA NALT内淋巴细胞明显增生。CT+ESA组NC淋巴细胞显著升高(P<0.01),以CD4+T细胞增生为主。结论 CT佐剂联合ESA滴鼻免疫BALB/c小鼠可诱导GALT和NALT黏膜部位免疫应答。     

经口灌胃感染弓形虫诱导的BALB/c小鼠肠道粘膜免疫应答

目的 研究BALB/c小鼠经口感染(灌胃)弓形虫速殖子后肠液IgA抗体分泌的动态变化及肠道粘膜组织诱导与效应部位T淋巴细胞亚群的变化,探讨肠道粘膜免疫应答机制.方法 将6～8周龄BALB/c小鼠100只随机分为对照组和感染组.感染组经口感染(灌胃)弓形虫RH株速殖子5×104个/只,对照组给予等量PBS.于感染后第2、4、6、8、10、13、16、19、22、25 d处死小鼠,每次5只.收集各时间点肠道冲洗液,用ELISA法测定肠液IgA水平;分离第2、4、6、8、10 d Peyer's Patches(PP)、肠系膜淋巴结(MLN)及小肠上皮内淋巴细胞(IEL),制备悬液并涂片,用免疫细胞化学方法测定CD4+、CD8+T细胞亚群水平.结果 在感染后第4、6、8、13 d实验组的肠液IgA抗体分泌显著高于对照组(P＜0.01).实验组PP结CD4+T细胞水平在6、8、10 d显著高于对照组(P＜0.05);CD8+T细胞水平与对照组无显著性差异,CD4+/CD8+比值在6、8、10 d显著升高(P＜0.05).肠系膜淋巴结的CD4+、CD8+及CD4+/CD8+比值各时间点均无显著变化.感染后第6、8、10 d,效应部位小肠上皮内淋巴细胞CD8+T细胞增高显著(P＜0.01)、CD4+/CD8+比值倒置(P＜0.05).结论 经口感染弓形虫BALB/c小鼠的肠道产生高水平的IgA抗体,作为局部首道屏障,发挥着重要的抗虫作用.诱导部位PP CD4+T细胞水平明显增高,诱导对弓形虫抗原的处理提呈作用;效应部位IEL CD8+T细胞亚群增殖明显,在清除虫体的过程中起主导作用.     

经口感染弓形虫诱导的小鼠肠道粘膜T细胞亚群变化

目的 研究经口感染弓形虫速殖子小鼠肠道粘膜组织诱导部位与效应部位T细胞亚群的变化,探讨肠道粘膜免疫应答机制。 方法 将6~7周龄BALB／c小鼠100只随机分为对照组和感染组。感染组灌胃接种RH株弓形虫速殖子5×104个／只,对照组给予等体积PBS。于感染后第2、4、6、8、10 d分别处死小鼠,分离Peyer's patches(PP结)、肠系膜淋巴结(MLN)淋巴细胞、小肠上皮内淋巴细胞(IEL),用免疫细胞化学法检测CD4 、CD8 T细胞亚群。 结果感染组小鼠PP结CD4 T细胞有随感染天数增加呈升高趋势,第6、8、10 d显著高于对照组(P<0.01);CD8 T细胞亦有增高趋势,但与对照组相比差异无显著性(P>0.05);CD4 ／CD8 比值第6 d起增高(P<0.05),第8 d最大(P<0.01),第10d回落(P<0.05)。MLN中T细胞亚群有较弱增高趋势,无统计学意义(P>0.05)。IEL CD4 、CD8 T细胞在第6、8、10 d均增高,其中CD8 T细胞增高显著(P<0.01);CD4 ／CD8 比值第6 d开始下降,两组间差异有显著性(P<0.05)。 结论 诱导部位PP结CD4 T细胞明显增高,诱导对弓形虫抗原的处理、提呈作用;效应部位IELCD8 T细胞增殖明显,在清除虫体的过程中起主导作用。     

IL-10为弓形虫经口感染后防止具有抗性遗传的BALB/c小鼠以及易感遗传的C57BL/6小鼠小肠坏死并降低病死率所必需

为了控制急性刚地弓形虫感染,免疫发挥着重要的作用。曾有过报道:近交系小鼠在急性感染后死亡率不同取决于感染途径。经口感染是一种自然的感染途径。在此过程中,弓形虫首先侵入的是小肠。因此,弄清调控肠道对此寄生虫免疫应答的调节因子,将对急性感染和疾病有一个更精确的认识。     

可溶性弓形虫速殖子抗原联合蜂胶和IFN-γ鼻内免疫小鼠诱导的细胞免疫应答

目的比较蜂胶、IFN-γ佐剂以及两种佐剂混合鼻内免疫辅助STAg增强机体细胞免疫应答的水平,探讨两种佐剂联合应用的免疫效果。方法将5~6周龄雌性BALB/c小鼠60只随机分为4组:20μg STAg组,20μg STAg+40μg蜂胶组,20μg STAg+1 000 U IFN-γ组和20μg STAg+40μg蜂胶+1 000 U IFN-γ组。将抗原和佐剂溶于20μl PBS中,双侧鼻孔(10μl/鼻孔)滴鼻免疫。免疫2次,间隔14 d,末次免疫后第10 d用RH株弓形虫速殖子4×104个/只灌胃攻击。逐日观察小鼠存活情况。攻击后第43 d处死全部存活小鼠,免疫细胞化学(ICC)法检测小鼠PP结、IEL和脾T淋巴细胞亚群水平。结果与STAg组相比,各佐剂组小鼠T淋巴细胞亚群比例有升高的趋势,其中IFN-γ、蜂胶+IFN-γ佐剂组小鼠PP结CD4+、CD8+T淋巴细胞,IEL、脾CD8+T淋巴细胞数显著高于STAg组,CD4+/CD8+T淋巴细胞比值倒置。结论在抗弓形虫感染中,IFN-γ的佐剂作用优于蜂胶,有效激发机体细胞免疫应答,可用作鼻内免疫抗弓形虫感染的粘膜佐剂。IFN-γ+蜂胶佐剂鼻内免疫效果优于单独蜂胶、IFN-γ佐剂,应用联合佐剂是抗弓形虫感染免疫的一个新思路。     

TSo联合IFN-γ鼻内免疫小鼠诱导的抗弓形虫脾淋巴细胞免疫应答

目的研究速殖子超声裂解物(TSo)和IFN-γ鼻内免疫小鼠经口感染弓形虫速殖子后,脾T细胞亚群的动态变化。方法将6~7周龄BALB/c小鼠100只随机分为4组,每组25只。分别用10μlPBS、20μgTSo、500UIFN-γ和20μgTSo 500UIFN-γ鼻内免疫小鼠2次,间隔2周。末次免疫后第10天,用RH株弓形虫速殖子4×104个/只灌胃攻击。分别于攻击后第7、10、13、16、19天处死小鼠,制备脾淋巴细胞悬液并涂片,免疫细胞化学法检测脾CD4 、CD8 T细胞亚群。结果攻击后第16、19天TSo IFN-γ组脾CD4 T细胞比率较第10天升高(P<0.05);脾CD8 T细胞第10、13、16、19天呈现较高水平;CD4 /CD8 比值第10、13天出现倒置。结论TSo联合IFN-γ鼻内免疫小鼠可激发系统免疫反应,提高其细胞免疫应答水平,增强脾CD8 T细胞的细胞毒作用,抵抗弓形虫感染。鼻黏膜免疫是一种安全有效的免疫接种途径。     

大鼠心脏移植术后环孢素A对弓形虫感染及淋巴细胞亚群的作用

目的　　观察大鼠心脏移植受体经不同途径感染弓形虫及术后使用环孢素A (CyclosporinA ,CsA)对免疫功能和弓形虫感染的影响。　方法　用ELISA法检测大鼠心脏移植术后受体弓形虫特异性抗体IgM、IgG及循环抗原 ,流式细胞术测定移植术后受体的T淋巴细胞亚群的变化。　结果　移植术后使用CsA可使弓形虫感染危险度提高 ,供体携带弓形虫病原体引起的感染大于受体隐性感染的复发的发生率 ,CD8+ T淋巴细胞升高更快。感染发作时CD8+ T淋巴细胞明显升高 ,CD4+ CD8+ 比值降低或倒置。　结论　移植术后使用CsA导致免疫抑制是弓形虫感染的重要原因 ,感染发作时CD8+ 是主要的细胞毒T淋巴细胞。     

胃肠道免疫学

过去1～1.5年在胃肠道免疫学的某些成就,引人注目.这包括:进一步确定了T淋巴细胞在调节肠道IgA抗体应答的作用;证实了肠道淋巴组织(GALT)内对牛痘病毒的细胞毒T     

原虫感染与艾滋病

原虫感染主要引起T细胞介导的免疫应答反应。艾滋病患者的原虫感染,如利什曼原虫、弓形虫和隐孢子虫,比免疫机能健全的病人危害性更为严重,尤其是弓形虫和隐孢子虫在艾滋病患者中特别普遍。艾滋病患者的大脑弓形虫病通常是隐性感染的复发,患者T辅助细胞(CD4~+)的耗竭使包囊中释放出来的缓殖子在脑中存活。在免疫功能丧失的情况下,缓殖子转变为速殖子进而分裂增殖,迅速导致组织坏死性脑炎。艾滋病患者坏死性脑炎中多数是由于弓形虫感染,表现为大脑局部脓肿或弥漫性脑膜脑炎。大约10～80%的艾滋病患者患弓形虫脑炎;相比之下其他免疫功能低下的人群,如皮肤移植者等,产生弓形虫脑炎情况就相当少。另外免疫功能低下病人弓形虫感染并不     

Cytomegalovirus-induced reactivation of Toxoplasma gondii pneumonia in mice: lung lymphocyte phenotypes and suppressor function.

Active cytomegalovirus (CMV) infection is associated with immunosuppression and predisposes to the development of life-threatening superinfections in immunocompromised patients. In a mouse model of virus-induced immunosuppression, acute murine CMV (MCMV) infection induced reactivation of dormant Toxoplasma gondii infection, producing Toxoplasma pneumonia. Changes in lung lymphocyte numbers and phenotypes appeared to be integral to the pathogenesis of MCMV-induced reactivation of T. gondii pneumonia. Numbers of lung CD4+ cells decreased during acute MCMV infection in mice with dormant T. gondii infection as well as in previously uninfected mice. Dually infected mice subsequently developed reactivation of Toxoplasma pneumonia. The pneumonia was characterized by a large influx of T lymphocytes, predominantly CD8+ cells, into the lungs. These lung lymphocytes markedly suppressed the ability of immune splenocytes to proliferate in response to T. gondii antigens and concanavalin A in vitro. These results suggested that the initial fall in the numbers of lung CD4+ cells observed after MCMV infection may have induced reactivation of T. gondii infection in the lungs. The subsequent pneumonia appeared to be a manifestation of a massive influx of T lymphocytes, especially CD8+ cells, into the lungs.     

Phenotypes, proliferative responses, and suppressor function of lung lymphocytes during Toxoplasma gondii pneumonia in mice.

Toxoplasma gondii pneumonia has emerged as an important problem in immunocompromised patients, especially those with AIDS. The characteristics of lung lymphocytes, including their phenotypes, proliferative responses, and suppressor function during T. gondii pneumonia were studied. During primary acute T. gondii infection, the numbers of T lymphocytes in the lungs increased even though mice lacked histologic evidence of pneumonia. As mice recovered from acute toxoplasmosis, numbers of lung CD4+ cells and the ratio of CD4+ to CD8+ cells decreased. Subsequently, T. gondii infection reactivated, manifested as pneumonia. During pneumonia, lung T lymphocytes, especially CD8+ cells, increased even more. Lung lymphocytes from mice with T. gondii pneumonia decreased the proliferative responses of splenocytes from T. gondii-immune mice to both concanavalin A and T. gondii lysate antigens in vitro. The striking increase in lung CD8+ cells and suppressor activity appear to be integral to the pathogenesis of T. gondii pneumonia.     

Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes

BACKGROUND & AIMS: Acute inflammatory ileitis occurs in susceptible (C57BL/6) mice after oral infection with Toxoplasma gondii. Overproduction of interferon (IFN)-gamma and synthesis of nitric oxide mediate the inflammation. We evaluated the role of transforming growth factor (TGF)-beta produced by intraepithelial lymphocytes (IELs) in this process. METHODS: We analyzed the histologic and immunologic consequences of adoptive transfer of antigen-primed IELs into susceptible mice treated with anti-TGF-beta before oral challenge with T. gondii cysts. An in vitro coculture of enterocytes and IELs assessed the production of chemokines and cytokines in the presence of anti-TGF-beta. RESULTS: Antigen-primed IELs prevent acute ileitis in susceptible mice that is reversed with anti-TGF-beta. Resistant mice (CBA/J) develop ileitis after treatment with anti-TGF-beta. Antigen-primed IELs can induce systemic immunosuppression as measured by depressed IFN-gamma production. In vitro, primed IELs reduce the production of inflammatory chemokines by infected enterocytes and IFN-gamma by splenocytes. CONCLUSIONS: Regulation of the ileal inflammatory process resulting from T. gondii is dependent on TGF-beta-producing IELs. The IELs are an essential component in gut homeostasis after oral infection with this parasite.     

Major role for CD8+ T cells in the protection against Toxoplasma gondii following dendritic cell vaccination

Toxoplasma gondii is the causative agent of toxoplasmosis, a worldwide zoonosis for which an effective vaccine is needed. Vaccination with pulsed dendritic cells is very efficient but their use in a vaccination protocol is unconceivable. Nevertheless, unravelling the induced effector mechanisms is crucial to design new vaccine strategies. We vaccinated CBA/J mice with parasite extract-pulsed dendritic cells, challenged them with T. gondii cysts and carried out in vivo depletion of CD4⁺ or CD8⁺ T lymphocytes to study the subsequent cellular immune response and protective mechanisms. CD4⁺ lymphocytes were poorly implicated either in spleen and mesenteric lymph node (MLN) cytokine secretion or in mice protection. By contrast, the increasing number of intracerebral cysts and depletion of CD8⁺ cells were strongly correlated, revealing a prominent role for CD8⁺ lymphocytes in the protection of mice. Splenic CD8⁺ lymphocytes induce a strong Th1 response controlled by a Th2 response whereas CD8⁺ cells from MLNs inhibit both Th1 and Th2 responses. CD8⁺ cells are the main effectors following dendritic cell vaccination and Toxoplasma infection while CD4⁺ T cells only play a minor role. This contrasts with T. gondii infection which elicits the generation of CD4⁺ and CD8⁺ T cells that provide protective immunity.     

Role of gamma interferon and T cells in congenital Toxoplasma transmission

In the BALB/c mouse model, primary infection with Toxoplasma gondii during the second third of gestation leads to a high percentage of infected foetuses. However, immunity induced by infection contracted before pregnancy prevents parasites from crossing the placenta and completely protects the foetuses, as well as the pregnant women. In order to clarify the roles of CD4+, CD8+ T lymphocytes and IFN-gamma in this protection, pregnant BALB/c mice were treated with depleting monoclonal antibodies against CD4, CD8, IFN-gamma, or control antibody. Only the foetuses of the groups treated with anti-CD8 and anti-IFN-gamma antibodies developed congenital toxoplasmosis. The maternal production of IFN-gamma was depressed in the mice depleted of CD4 and CD8 cells (P < 0.001). Determination of the blood parasite load demonstrated that materno-foetal transmission of T. gondii correlates with maternal parasitaemia. Together, these results show that CD8+ T lymphocytes and IFN-gamma play an important role in protection against congenital toxoplasmosis during reinfection.     

弓形虫缓殖子期相关蛋白的研究进展

弓形虫是一种机会致病性原虫,专性细胞内寄生,能导致人兽共患的弓形虫病.近年来,国内外学者对弓形虫主要抗原及其免疫特性进行了多方面的研究,含缓殖子的组织包囊作为弓形虫慢性感染阶段,该时期的相关蛋白是弓形虫病致病机制研究的重要分子.本文就弓形虫缓殖子期相关蛋白研究进展进行总结,阐述其在弓形虫慢性感染致病机制中的作用.     

弓形虫病的诊断技术及其研究进展

弓形虫人体感染越来越多,刚地弓形虫感染诊断的快速和特异是发现和治疗此病的关键。本文对病原学、免疫学和核酸等弓形虫检测技术及其最新的研究进展进行了综述。     

Peripheral blood lymphocytes T gammadelta in children with acute acquired toxoplasmosis

The aim of the study was to determine the percentage participation of lymphocytes CD3TCRgammadelta in peripheral blood of children with acute toxoplasmosis. The study compromised 30 children aged 2-14 years. Increase of the percentage of CD3TCR gammadelta cells was observed in the group of children with clinical symptoms of toxoplasmosis (mean value 5.7%) in relation to the comparative group (mean 4.4%), but in 12 children (40%) the mean value was 8.1%. Toxoplasma gondii infection produces a strong signal for activation of cells with gammadelta receptors. Examination of CD3TCRgammadelta cells may be helpful in the diagnosis of the acute phase of T. gondii infection.     

弓形虫感染对大鼠脑组织神经丝mRNA表达的影响

目的探讨弓形虫感染对大鼠脑组织神经丝（NF）mRNA表达和细胞免疫水平的影响。方法将4周龄雄性SD大鼠随机分成2组：弓形虫感染组（A组）腹腔感染弓形虫速殖子2×10^2／ml悬液2ml;正常对照组（B组）腹腔注射灭菌生理盐水2ml。感染弓形虫速殖子9周后,应用逆转录聚合酶链反应检测大鼠大脑组织中高分子量NF（NF—H）、中分子量NF（NF—M）和低分子量NF（NF—L）mRNA表达水平;流式细胞术检测大鼠外周血CD3^＋、CD4^＋、CD8^＋T淋巴细胞;ELISA测定其血清IFN-γ、TNF-α、IL-4等细胞因子。结果大鼠弓形虫感染9周后,大脑组织NF．LmRNA下降为正常对照组的64％（P〈0．01）;NF—M为96％（P〉0．05）;NF—H为89％（P〈0．05）。感染组大鼠的CD4^＋和CD8^＋T淋巴细胞与正常对照组相比,差异均无统计学意义（P均〉0．05）;感染组大鼠血清中IFN-γ、TNF-α、IL-4等细胞因子的水平均高于正常对照组大鼠水平（P〈0．05）。结论弓形虫感染可导致大鼠脑组织中NF亚单位mRNA表达水平的下降,血清IFN-γ、TNF-α、IL-4水平的升高。