弓形虫影响前列腺素E2合成的细胞内信号调节途径

目的　探讨弓形虫感染巨噬细胞过程中花生四烯酸 (AA)及前列腺素E₂ (PGE₂ )的产生及其相关的细胞内信号调节途径。　方法　构建刚地弓形虫 小鼠巨噬细胞侵染模型 ,应用气相色谱、酶联免疫吸附测定 (ELISA)、逆转录 聚合酶链反应 (RT-PCR)和蛋白质印迹法 (Westernblotting)检测钙离子调节剂乙二醇双 ( 2-氨基乙醚 )四乙酸 (EG-TA)、钙离子螯合剂 (BAPTA/AM )、钙调蛋白抑制剂三氟拉嗪 (TFP)及蛋白激酶C (PKC)抑制剂 1-( 5-磺酰基 ) 异喹啉-2-甲基-哌嗪盐酸盐 (H7)对弓形虫诱导的AA、PGE₂ 含量及促细胞分裂剂诱导性环加氧酶-2 (COX-2 )mRNA和蛋白质表达水平的影响。　结果　EGTA、BAPTA/AM及TFP均可抑制弓形虫诱导的巨噬细胞AA和PGE₂ 合成 ;PKC抑制剂H7可明显抑制弓形虫和脂多糖 (LPS)诱导的巨噬细胞PGE₂ 合成 ,并伴随着COX-2mRNA和蛋白质表达呈剂量依赖性减少。　结论　弓形虫侵染巨噬细胞过程可能通过钙信号转导途径调节COX-2底物AA的含量和PKC依赖的COX-2代谢途径调节PGE₂ 的合成。     

弓形虫诱导巨噬细胞COX-2的表达增加PGE2合成

目的 探讨弓形虫侵染巨噬细胞过程中前列腺素E2(PGE2)的产生途径。方法 用LPS及弓形虫作用于小鼠RAW264．7巨噬细胞系。用气相色谱、ELISA方法分别检测培养上清液中PGE2及花生四烯酸(AA)含量；用RT—PCR及Western blot方法分别检测环加氧酶—l(COX—1)、环加氧酶—2(COX—2)的mRNA及蛋白质表达水平；特异性抑制剂阻断作用于细胞后检测PGE2含量，COX—l／COX—2的mRNA及蛋白质表达。结果 PGE2的合成在弓形虫侵染巨噬细胞4—8h开始升高，12--l6h后达到饱和水平；COX—2mRNA表达在4—8h出现最高峰，在特异性COX—2抑制剂Nimesulide及Indomethacin作用下其表达水平下降，而蛋白质表达水平不受影响。COX—l的mRNA及蛋白质表达在抑制剂处理前后及不同的时间点都末见明显变化。结论 弓形虫可能通过诱导巨噬细胞表达COX—2增加PGE2的合成。     

Toxoplasma gondii infection can regulate the expression of tumour necrosis factor-alpha receptors on human cells in vitro

The in vitro regulation of tumour necrosis factor (TNF)-alpha receptors during Toxoplasma gondii infection of human MRC5 fibroblasts and human myelomonocytic THP-1 cells was investigated. Cells were infected with the virulent RH of T. gondii. TNFR membrane receptors were analysed by flow cytometry with biotinylated TNF-alpha. Shedding of the soluble form of TNFR1 and TNFR2 in cell culture supernatants was measured by enzyme-linked immunosorbent assay, and expression of mRNA production of TNFR1 and TNFR2 was analysed by quantitative real-time polymerase chain reaction, 1 h after infection. In the MRC5 cell line, T. gondii infection did not induce any up- or down-regulation of membrane TNFRs, soluble TNFRs or mRNA of TNFRs. However, THP-1 cell infection with living parasites induced a significant soluble TNFR1 release by THP-1 cells after 1 h. We detected an approximately 50% up-regulation (P < 0.01) of soluble TNFR1 in infected THP-1 cells compared to controls. No change in soluble TNFR2 levels was observed in the same conditions. Moreover, infection decreased the level of TNF membrane receptors, but had no effect on TNFR1 and TNFR2 mRNA levels. TNFR modulation by T. gondii infection, in vitro, depends on the cell type. Furthermore, our data suggest that living parasites control the shedding of the soluble form of TNFR1. This mechanism may influence the role of TNF-alpha in toxoplasmosis.     

弓形虫急性感染小鼠睾丸的病理学及发病机理的研究

目的　观察弓形虫急性感染小鼠睾丸的病理学变化及探讨其发病机理。方法　将弓形虫急性感染和正常对照NIH小鼠睾丸做印片及切片 ,观察生精细胞的病理变化及弓形虫侵入细胞的情况 ;应用免疫组化S -P法进行弓形虫抗原及bcl 2、c myc、P53、bax的检测。结果　弓形虫急性感染组小鼠睾丸印片中见生精细胞胞质及核内弓形虫速殖子 ;睾丸切片病理变化为生精停滞 ,精原细胞胞质空泡性变。免疫组化染色显示弓形虫 ;bcl 2及c myc在感染组和正常组间的表达无统计学意义 (P >0 .0 5 ) ;P53及bax在两组间均未表达。结论　弓形虫急性感染小鼠睾丸生精停滞等病理变化可能是弓形虫及其分泌的毒素直接损害、干扰生精细胞生成及分裂发生障碍和死亡的结果 ,与凋亡基因无明显关系     

急性弓形虫感染小鼠脑蛋白质二维电泳图谱初步分析

目的应用二维电泳（2-DE）技术研究急性弓形虫感染对小鼠脑蛋白质组的改变。方法分别提取纯化急性弓形虫感染和正常同窝配对的C57BL/6J小鼠脑组织蛋白,以固相pH3～10梯度聚焦为第一向,SDS-PAGE垂直电泳为第二向进行双向凝胶电泳,应用PDQuest 1.0软件分析蛋白差异。结果急性弓形虫感染与正常同窝配对的C57BL/6J小鼠脑组织2-DE图谱分别检测出（132±10）个和（170±13）个蛋白斑点,对两组电泳图谱进行匹配后,有19个蛋白点仅在急性弓形虫感染小鼠脑蛋白2-DE图谱中表达;另有37个蛋白斑点仅在正常同窝配对小鼠中表达。部分蛋白在两组小鼠脑组织中含量也发生明显变化。结论急性弓形虫感染小鼠脑蛋白质有明显改变,为研究弓形虫感染致脑组织损害及筛选治疗新药提供了有益的线索。     

Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection

Infection with Toxoplasma gondii induces chemokine up-regulation in several cell types. Here, we investigated the role of stress products (norepinephrine, NE) on chemokine production in mouse intestinal epithelial cells (IECs). Purified IECs were used to determine the expression levels of chemokines by real-time PCR. There was significantly increased expression in CCL2, CCL3, CCL5, CXCL2, CXCL9 and CXCL10 in IECs following peroral infection with T. gondii (INF) on day eight post-infection (PI) compared to infected mice subjected to cold-water stress (INF+CWS). In vitro studies using the MODE-K cell line showed increased chemokine mRNA and protein expression in infected but not in cells exposed to parasite antigen. Down-regulation of chemokine expression was more pronounced when active infection was used in combination with NE. Chemokine receptor expression was increased in IECs isolated from INF and decreased in the INF+CWS group. In MODE-K cells, there was decreased mRNA expression of chemokine receptors when incubated with β-adrenergic antagonists. Neither, adrenergic antagonists blocked the effect of infection on chemokine receptor expression. Cold-water stress was able to decrease expression of chemokines and their receptors in IECs in vivo and in vitro. Cold-water stress-mediated modulation of innate intestinal responses are beneficial in C57BL/6 mice during T. gondii infection.     

刚地弓形虫入侵细胞过程中细胞骨架和Ca2+ 浓度变化

目的 探讨弓形虫入侵不同类型细胞过程中胞内游离Ca²⁺ 浓度及细胞骨架的变化。 方法 常规方法制备刚地弓形虫RH株速殖子悬液，分别感染吞噬性细胞（小鼠单核巨噬样细胞J774A.1）和非吞噬性细胞（人脐静脉内皮细胞HUVEC）。光学显微镜观察弓形虫感染情况及细胞骨架抑制剂秋水仙素、松胞菌素D对感染率的影响。用荧光显微镜观察弓形虫速殖子入侵J774A.1、HUVEC过程中细胞微丝和微管变化。用激光共聚焦显微镜检测弓形虫速殖子入侵过程中宿主细胞游离Ca²⁺ 浓度变化。 结果 正常J774A.1胞内游离Ca²⁺ 浓度为102.0%±6.2%。弓形虫感染后 2 min游离Ca²⁺ 浓度升高，测得荧光强度为 305.2%±21.5%，感染后30～40 min最高荧光强度为1219.7%±58.4%，显著高于基础值（P<0.01）。而经磷脂酶C抑制剂U73122预处理的J774A.1，Ca²⁺ 浓度无明显变化(P>0.05）；虫体入侵过程中J774A.1微丝结构发生凝集。微丝结构抑制剂松胞菌素D（P<0.01）和微管结构抑制剂秋水仙素（P<0.05）均可明显降低弓形虫感染率。弓形虫入侵HUVEC过程中Ca²⁺ 浓度变化不明显（P>0.05），宿主细胞微丝、微管结构亦无明显变化。松胞菌素D和秋水仙素对弓形虫入侵HUVEC的能力影响均较小(P>0.05）。 结论 弓形虫入侵吞噬性细胞J774A.1过程中胞内游离Ca²⁺ 浓度显著升高，细胞骨架微丝结构发生凝聚，而入侵非吞噬性细胞HUVEC过程中胞内游离Ca²⁺ 浓度和细胞骨架均无明显变化。     

弓形虫感染对早孕期人绒毛膜滋养层细胞人类白细胞抗原G表达的影响

目的 研究弓形虫感染早孕期绒毛膜滋养层细胞中人类白细胞抗原G(human leukocyte antigen G,HLA-C)表达水平的变化,探讨弓形虫感染致不良妊娠的分子免疫机制.方法 选取正常妊娠6～12周妇女行人工流产术后的绒毛组织,分离滋养层细胞.将绒毛膜滋养层细胞分为实验组和对照组,实验组加入1×104个弓形虫,对照组加入等量生理盐水,培养48 h后,采用实时荧光定量PCR检测各组人绒毛膜滋养层细胞HLA-G mRNA表达水平,流式细胞技术检测各组人绒毛膜滋养层细胞中HLA-G阳性细胞亚群数量.结果 实验组和对照组HLA-G mRNA平均表达水平分别为0.15±0.04和0.29±0.07,两组之间的差异具有统计学意义(P＜0.01);实验组绒毛膜滋养层细胞HLA-G阳性细胞亚群的数量为(12.19±2.04)%,对照组为(24.24±3.07)%,两组之间的差异具有统计学意义(P＜0.01).结论 早孕期弓形虫感染可下调绒毛膜滋养层细胞HLA-G的表达,减少绒毛膜滋养层细胞中HLA-G阳性细胞亚群数量.     

Detection of Toxoplasma gondii in human placenta by PCR and placental histologic findings

Since isolation of Toxoplasma gondii from human placenta strongly correlates with fetal infection, the aims of the study were: to detect fragments of T. gondii B1 gene in human placentae by PCR and to evaluate their pathology. 36 placentae included in three groups were obtained: group I (n = 7) from pregnancies with prenatal diagnosis of fetal toxoplasmosis; II (n = 17) from women with serologic features of primary infection during pregnancy; III (n and 13) from pregnancies with fetal T. gondii infection based on clinical signs. T. gondii DNA was found in 2/4 samples from the I group and in 1/14 from the II group. Villitis was identified in 3/15 other placentae from the II group. In the III group we did not recognize neither T. gondii DNA nor villitis. We consider PCR and pathologic evaluations of placentae as the two complementary methods. PCR can be especially helpful in pregnancies not screened against T. gondii as positive result in placenta can confirm mother's primary infection.     

microRNA?155在弓形虫感染调节巨噬细胞极化中的作用

目的 探讨弓形虫感染对宿主细胞内microRNA?155（miR?155）表达的影响及其对巨噬细胞极化的作用。 方法 利用miRNAs基因芯片检测弓形虫感染宿主细胞内miRNAs表达水平,应用实时定量聚合酶链反应技术（qPCR）检测miR?155表达水平。采用脂质体转染法将pEGFP?miR?155导入人巨噬细胞,利用流式细胞术检测转染效率。采用流式细胞术、qPCR、酶联免疫法检测弓形虫感染组、pEGFP?miR?155过表达组以及miR?155抑制组诱导巨噬细胞表面分子CD86及诱导型一氧化氮合酶（iNOS）和白细胞介素（IL）?12表达水平。结果 基因芯片和qPCR检测结果显示,随着弓形虫感染时间的延长,miR?155表达量上升;pEGFP?miR?155转染宿主细胞的转染效率可达82.6%。弓形虫感染组和pEGFP?miR?155过表达组CD86表达水平显著高于对照组和miR?155抑制组,iNOS和IL?12基因表达量显著升高。结论 弓形虫感染能够通过上调miR?155表达参与驱动人源巨噬细胞向M1型偏移。     

PCR技术检测生殖道弓形虫感染的临床实验分析

目的　通过对人类生殖道弓形虫感染状况的检测 ,探讨弓形虫感染与不孕不育的关系 ,以及弓形虫感染生殖道的可能方式。方法　实验设置不孕不育组和正常生育组夫妇 ,用PCR方法检测生殖道分泌物中弓形虫DNA ,把检测结果输入计算机进行SPSS统计学处理 ,分析弓形虫感染的概率。结果　不孕不育组弓形虫感染率为 18 15 % ,而正常生育组为6 2 9% ,P <0 0 1,两组间感染率有显著差异。夫妻之间感染率无显著性差异。结论　弓形虫感染与不孕不育有一定关系 ,夫妻间性行为可能是弓形虫传播的一种途径。     

弓形虫既往感染在孕期内对胎儿的影响

目的探讨既往感染者孕期内弓形虫的活动情况及对胎儿的影响。方法对68例抗弓形虫抗体IgG阳性、IgM阴性孕妇的血清、脐血采用酶联免疫吸附试验检测弓形虫抗体IgG、IgM、弓形虫循环抗原（CAg）,PCR法检测虫体DNA;胎盘样本采用直接涂片、匀浆涂片及PCR法观察弓形虫感染情况。结果68例弓形虫抗体IgG阳性孕妇中脐血弓形虫抗体IgG阳性28例,IgG胎盘垂直传播率41.2%;脐血弓形虫DNA阳性6例,宫内感染发生率8.8%;胎盘组织中虫体DNA阳性9例,宫内感染发生率13.2%。结论既往弓形虫感染孕期内仍可导致垂直传播。     

弓形虫慢性感染大鼠脑组织中Toll样受体4基因表达及其对脑损害影响

目的 目的 探讨Toll 样受体4 （TLR4） 在弓形虫慢性感染大鼠脑组织中的表达, 及其对脑部损害的作用。 方法 方法 将10 只SD雄性大鼠随机分为对照组和模型组, 每组5只。感染组每鼠腹腔感染1×107 /ml弓形虫速殖子2 ml, 对照组每鼠腹腔注射灭菌生理盐水2 ml。弓形虫感染后10周, 用ELISA法检测大鼠血清炎症因子IL?1β和IL?4水平; 采用逆转录多聚酶链反应 （RT?PCR） 法检测大鼠脑组织TLR4 mRNA的表达。 结果 结果 与正常对照组相比, 弓形虫慢性感染组大鼠脑TLR4 mRNA 表达显著升高 （P＜0.05）, 血清IL?1β含量显著上升 （P＜0.05）, IL?4含量也有所升高, 但差异无统计学意义 （P＞0.05）。 结 结论 论 TLR4在弓形虫慢性感染大鼠的脑部损害中具有一定作用。     

Infection of placental trophoblasts by Toxoplasma gondii

How the intracellular parasite Toxoplasma gondii causes placental inflammation and infects the fetus is unknown. By use of a culture model of primary human trophoblasts, we examined the consequences of infection by a virulent strain of T. gondii. Infection fractions (parasitophorous vacuoles per trophoblast nuclei) < or =0.9 were observed 1 day after challenge at an inoculum ratio of T. gondii to nuclei of 10. The culture content of infectious T. gondii increased 45-fold in 48 h. Two days after infection, almost 30% of trophoblast nuclei became apoptotic, and 30%-35% of nuclei were lost. Almost 90% of apoptotic nuclei were not adjacent to a parasitophorous vacuole, suggesting infection protected against apoptosis. However, there was no T. gondii-dependent accumulation of putative cytotoxic factors, such as tumor necrosis factor-alpha, that could mediate paracrine killing. Both mature and immature trophoblasts can be productively infected, and uninfected, but not infected, cells undergo apoptosis.     

弓形虫体外感染大鼠心肌细胞的实验研究

目的　观察弓形虫速殖子体外感染大鼠心肌细胞及在细胞内的增殖。方法　用弓形虫RH株速殖子感染体外培养的大鼠心肌细胞,观察虫体在不同的孵育时间对心肌细胞的侵袭率及每个感染细胞内的平均虫数。结果　虫体最早感染心肌细胞的时间为10min ,对心肌细胞的侵袭率随着感染时间的延长而增大。弓形虫侵入心肌细胞后的迟滞期约为8h ,虫体在细胞内增殖一倍的时间约8h。结论　弓形虫感染大鼠心肌细胞及其在细胞内的增殖,为阐述弓形虫对心肌细胞的易感性提供了重要的基础资料。     

Interferon-γ signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii

We assayed mitogen-activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii . In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii . The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN-γ. However, IFN-γ treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN-γ altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN-γ directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN-γ may be involved in mediating their protective signals .     

弓形虫入侵的宿主细胞骨架重塑触发线粒体重新分布

目的探讨宿主细胞骨架重塑在弓形虫侵入HFF细胞以及触发细胞线粒体重新分布中的作用。方法体外培养HFF细胞,预先用1 μg/mL细胞松驰素D(CD)处理30 min,接种弓形虫速殖子分别培养1 h和20 h,Western blotting检测弓形虫表面抗原(surface antigen,SAG)1蛋白表达。同时用MitoTracker^􀆿 Red CMXRos荧光探针标记细胞线粒体,激光共聚焦显微镜下观察HFF细胞线粒体在弓形虫侵入前后和CD处理前后聚集和分布情况。结果感染后1 h,CD处理组HFF细胞内虫体量与CD未处理组差异不大,20 h时CD未处理组细胞内虫体量显著多于CD处理组。此时可见HFF细胞线粒体明显聚集成明亮点状且分布于纳虫泡周围,而未感染组细胞线粒体未见明显聚集分布。CD可以显著抑制弓形虫侵入HFF细胞后引起的线粒体聚集。结论触发宿主细胞骨架重塑是弓形虫侵入宿主细胞并引起细胞线粒体向纳虫泡聚集所必须的。     

Pathogen-specific induction of CD154 is impaired in CD4+ T cells from human immunodeficiency virus-infected patients

The pathogenesis of immunodeficiency associated with human immunodeficiency virus (HIV) infection remains incompletely understood. CD154, a molecule that is expressed primarily on activated CD4(+) T cells, is pivotal for regulation of cell-mediated and humoral immunity and is crucial for control of many opportunistic infections. We investigated whether CD4(+) T cells from HIV-infected patients exhibit defective induction of CD154 in response to opportunistic pathogens. Incubation of purified human CD4(+) T cells with monocytes plus antigenic preparations of either Candida albicans, cytomegalovirus, or Toxoplasma gondii resulted in induction of CD154. Expression of CD154 in response to these pathogens was impaired in CD4(+) T cells from HIV-infected patients. This defect correlated with decreased production of interleukin (IL)-12 and interferon (IFN)-gamma in response to T. gondii. Recombinant CD154 partially restored secretion of IL-12 and IFN-gamma in response to T. gondii in cells from HIV-infected patients. Together, defective induction of CD154 is likely to contribute to impaired cell-mediated immunity against opportunistic pathogens in HIV-infected patients.     

Role of CD40 ligand signaling in defective type 1 cytokine response in human immunodeficiency virus infection

The pathogenesis of defective interleukin (IL)-12 and interferon (IFN)-gamma production in human immunodeficiency virus (HIV)-infected patients remains to be elucidated. This study investigated the possibility that perturbations in CD40 ligand signaling are involved in this defect. CD40 ligand trimer (CD40LT) stimulated peripheral blood mononuclear cell (PBMC) production of IL-12 in response to Toxoplasma gondii and cytomegalovirus (CMV). Regardless of the CD4 cell count, CD40LT restored IL-12 secretion in response to T. gondii in HIV-infected patients. In the presence of CD40LT, PBMC from both HIV-infected patients and control subjects produced high levels of IL-12 in response to CMV. CD40LT restored T. gondii- and CMV-triggered IFN-gamma secretion by T cells and PBMC from HIV-infected patients with a CD4 cell count >200 cells/microL. CD4 cells from HIV-infected patients, even those with a CD4 cell count >500 cells/microL, had defective CD40L induction after T cell stimulation mediated by antigen-presenting cells. Together, impaired CD40L induction is likely to contribute to defective IL-12 and IFN-gamma production in HIV infection.