克氏锥虫肌纤蛋白等位基因的比较和转录产物的分析

克氏锥虫(Tryponosoma cruzi)是恰加斯病(Chagas disease)的致病因子。克氏锥虫与它所感染的脊椎动物细胞相反，具有非典型的细胞支架。这种细胞支架由主要成分为肌纤蛋白的表膜下微管构成。肌纤蛋白是一种保守蛋白，在大多数真核细胞的构成和功能中占重要地位，但可提供的相关资料     

整合素β1在自身免疫性心肌炎小鼠心肌的表达

目的:观察整合素β1在自身免疫性心肌炎小鼠心肌的表达变化。方法:猪肌凝蛋白皮下注射免疫Balb/c小鼠,建立慢性自身免疫性心肌炎模型,于初次免疫后14、30和60d对心肌组织进行病理学检查,同时用激光共聚焦技术和间接免疫荧光法检测整合素β1在心肌的表达变化。结果:在初次免疫后14d,小鼠心脏有局部炎症出现,整合素β1在心脏血管周围的表达高于正常组（P<0.05）;在第30天,小鼠心脏炎性浸润更加明显,整合素β1在心肌的表达增强,显著高于正常组（P<0.05）;第60天,小鼠心脏炎症细胞减少,而纤维化则开始出现,整合素β1在心肌的表达最强（P<0.05）,且排列紊乱。结论:整合素β1在自身免疫性心肌炎的发生及发展过程中起着不同重要作用,整合素β可能参与自身免疫反应及心肌的重构。     

脂多糖提高巨噬细胞可溶性肿瘤坏死因子相关凋亡诱导配体的表达并诱导肝细胞凋亡

目的探讨脂多糖(LPS)、巨噬细胞表达肿瘤坏死因子相关凋亡诱导配体(TRAIL)和肝细胞凋亡的相互关系。方法LPS刺激巨噬细胞后,以流式细胞仪测定细胞表面的TRAIL表达,用酶联免疫吸附法(ELISA)法检测培养上清中可溶性TRAIL的表达;以(?)铬释放试验测定可溶性TRAIL和膜性TRAIL 对HepG2细胞的毒性作用,并用Annexin V染色法验证凋亡细胞的产生。结果经100 ng/ml LPS刺激后, 只有9.8%的巨噬细胞有TRAIL的表达,而培养上清中可溶性TRAIL达(67.4±5.1)ng/ml,有明显增加。巨噬细胞培养上清液中可溶性TRAIL能溶解HepG2细胞,经Annexin V染色法证实为细胞凋亡,此作用可被特异性TRAIL抗体阻断。结论LPS能增加巨噬细胞可溶性TRAIL表达,并诱导肝细胞凋亡,提示TRAIL在病毒性肝炎发病机制中有重要作用。     

脂多糖诱导小胶质细胞激活与多巴胺能神经元毒性作用关系的研究

目的探讨脂多糖(LPS)诱导小胶质细胞(Mic)激活规律与黑质多巴胺(DA)能神经元变性的关系。方法分别于LPS注入大鼠脑黑质后7d、14d、30d观察大鼠行为学改变,并采用免疫组织化学及原位杂交等方法观察Mic的激活情况以及酪氨酸羟化酶(TH)神经元的表达。结果行为学改变表现为7d仅引起轻度的旋转行为〔(85±13)圈/30min〕,至14d时旋转次数增多〔(121±17)圈/30min〕,30d时达高峰〔(295±21)圈/30min〕;7dMic部分激活,14d时大部分激活,30d时Mic已全部活化;TH阳性细胞数7d时轻度减少〔(135.22±12.11)PU〕,14d时进一步减少〔(21.54±4.89)PU〕,30d时已罕见〔(11.31±2.56)PU〕。THmRNA的表达规律与其一致,分别为(60.69±8.47)PU、(39.87±7.03)PU、(14.23±3.51)PU。结论Mic的激活是构成PD炎症损伤机制的重要环节。     

花青素调节JAK2/STAT3信号通路对自身免疫性心肌炎大鼠Th17/Treg平衡的影响

目的:探讨花青素对自身免疫性心肌炎(AE)大鼠T细胞17/调节性T细胞(Th17/Treg)平衡及Janus激酶2/信号转导和转录激活因子3(JAK2/STAT3)信号通路的影响。方法:将60只SD大鼠分为对照(NC)组、模型(Model)组、花青素组(50 mg/kg花青素)、STAT3激活剂Colivelin组(1 mg/kg Colivelin)、花青素+Colivelin组(50 mg/kg花青素+1 mg/kg Colivelin),每组12只。采用酶联免疫吸附实验(ELISA)检测血清炎性因子水平;苏木素-伊红(HE)染色检测心肌病理变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)染色检测心肌细胞凋亡;实时荧光定量聚合酶链式反应(RT-qPCR)检测脾脏维甲酸相关孤儿受体γt(RORγt)、叉头蛋白p3(Foxp3)mRNA表达情况;蛋白质免疫印迹法(Western Blot)检测心肌JAK2/STAT3通路相关蛋白表达。结果:Model组心肌明显坏死和水肿,大量炎性细胞浸润;花青素组心肌无明显的坏死和细胞肿胀;Colivelin组心肌损伤加重;与花青素组...     

肠侵袭性大肠埃希菌O抗原多糖的致病作用

目的研究肠侵袭性大肠埃希菌(EIEC)O抗原多糖(OPS)的特异性致病作用。方法提取并纯化大肠埃希菌O29——EIEC的脂多糖(LPS)亚单位分子——OPS进行体内外试验，观察其毒性作用并与LPS作对比。结果EIEC OPS可引起Hela细胞病变；致兔回肠袢肠黏膜出血，但不引起肠腔积液；电镜观察OPS致病变的Hela细胞，明显可见细胞的超微病理损害。结论EIEC OPS有特异的致病作用，与其LPS的内毒素作用不同；大肠埃希菌O29(致病菌株)OPS毒性作用比大肠埃希菌HB101(非致病菌株)OPS毒性强烈。     

双氢青蒿素治疗对卡氏肺孢子虫肺炎大鼠脾细胞上清液一氧化氮水平的影响

目的研究双氢青蒿素治疗对卡氏肺孢子虫肺炎(PCD)大鼠脾细胞上清液一氧化氮(N0)水平的影响。方法以醋酸可的松皮下注射Wistar大鼠，建立卡氏肺孢子虫肺炎动物模型，用60mg／kg双氢青蒿素治疗实验大鼠，杀鼠取肺，胶原酶消化法分离脾细胞，脂多糖(LPS)刺激培养72h，用NO试剂盒检测大鼠脾细胞上清液NO活性，同时设有感染组和正常对照组。结果感染组和治疗组大鼠NO水平高于正常对照组，治疗组NO水平低于感染组。结论卡氏肺孢子虫(PC)感染可能引起大鼠脾细胞分泌高水平NO，发挥杀伤PC作用，同时加重宿主组织炎症反应，抑制宿主的免疫应答；经双氢青蒿素治疗后，PCP大鼠脾细胞分泌NO降低，组织炎症反应减轻，使宿主的免疫应答接近正常状态。     

Specific suppression of delayed hypersensitivity expression to Trypanosoma cruzi in mice

The absence of cutaneous delayed type hypersensitivity (DTH) expression was investigated in Trypanosoma cruzi infected mice. Neither spleen cells nor peritoneal exudate cells from infected mice transferred DTH to normal recipients in local or systemic adoptive transfer experiments. Expression of DTH in T. cruzi immunized mice was suppressed specifically by Thy-1 negative spleen cells from acutely and subacutely infected animals. Suppression was observed only upon systemic transfer, but not when infected mice spleen cells were added to DTH effector cells and transferred to normal recipients. These results suggest that cutaneous DTH expression in acutely infected mice, might be blocked by mechanisms other than those described for suppression of lymphocyte proliferation and of DTH induction to T. cruzi.     

Susceptibility to acute Trypanosoma cruzi infection in autoimmune strains of mice

We previously observed that mice bearing the autoimmune associated lpr gene exhibit increased susceptibility to challenge with Trypanosoma cruzi, the causative agent of Chagas' disease. We have now tested two other autoimmune prone strains of mice, BXSB and NZB, and found that these animals also show increased sensitivity to acute T. cruzi infection. When challenged with a standard dose (10(2)) of Y strain trypomastigotes, BXSB males (BXSB-YSB), which develop early onset autoimmune disease, suffered high mortality, while late onset autoimmune BXSB females and minimally autoimmune male BXSB mice whose Y chromosome was derived from C57BL/6J mice (BXSB-YB/6) also recover. NZB mice were found to be highly susceptible to challenge while NZW and NZB/W were resistant. A finding common to all groups of susceptible autoimmune mice was increased plasma levels of T. cruzi specific antibody, especially IgM. The data indicate that in two of the three autoimmune prone strains examined, increased T. cruzi susceptibility appears to be linked to restricted genetic elements (i.e. lpr gene and the YSB associated factor) which also influence the rapidity of onset of autoimmunity.     

Specific cutaneous hypersensitivity responses in mice infected or immunized with Trypanosoma cruzi

Summary The appearance and development of cutaneous hypersensitivity to epimastigote antigen was followed during the early phase of Trypanosoma cruzi infection in mice. The effect of living BCG on the kinetics of skin reactivity was studied in animals infected with T. cruzi blood trypomastigotes or artificially immunized with disrupted epimastigotes. BCG stimulated the immediate response in infected animals while preferentially stimulating delayed responses in immunized animals. Infection with living blood trypomastigotes depressed already existing delayed-type responses. The development of delayed-type responses was also impaired in animals immunized in the course of the infection.     

Trypanosoma cruzi variants with reduced virulence obtained by mutagenesis

Summary Previous reports have indicated that by mutagen treatment of mouse tumour cells in vitro it is possible to obtain at high frequency stable tumour cell variants that fail to form tumours in syngeneic mice because of increased immunogenicity. By analogy with these tumour cell variants, we examined whether variants with reduced virulence could be obtained by mutagen treatment of trypomastigotes derived from a Trypanosoma cruzi strain that was adapted to culture and produced lethal infections in DBA/2 mice at a dose of 5 × 10⁴ parasites. A very large frequency of T. cruzi clones were obtained that failed to provoke an acute lethal infection after injection of 5 × 10⁵ parasites. Most of these variants with reduced virulence (vir^-) multiplied actively in normal mice until day 8 after injection. After that time the parasitaemia decreased gradually. For most variants a low level of residual parasitaemia persisted for more than 100 days. Unlike the situation encountered with mouse tumour cell variants it was not possible to demonstrate the presence of new antigens on the T. cruzi vir^- variants. However, these variants seemed to have acquired an increased immunogenicity since they provoked the rejection of virulent parasites injected concomitantly. Mice that had been immunized with living vir^- clones were protected against a challenge with a virulent clone derived from the original parasite population.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal oulbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B- lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., ³H-thymidine incorporation) and cell membrane levels of interleukin-2 receptors (H.-2R; determined by flow cytomet.y) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

Autoimmune myocarditis induced by Trypanosoma cruzi

Antiheart immune reactions have been reported in patients with Chagas' disease, and we have postulated that the observed cardiac lesions are mediated by autoimmune antiheart reactions elicited by the etiologic agent Trypanosoma cruzi. In this report, BALB/c mice infected with a low inoculum of T. cruzi developed splenic lymphocyte cytotoxicity against normal syngeneic neonatal cardiac myofibers in vitro 150 days after infection, whereas splenic lymphocytes obtained from mice at 15, 45, 90, or 120 days after infection or from matched controls did not. No antiheart antibody or antibody-directed cellular cytotoxicity was observed, nor was there an increase in natural killer cell activity. Hearts from mice studied at 150 days after infection showed mononuclear cell myocarditis with myocytolysis in the absence of intracellular T. cruzi forms. Hearts from the other mice did not exhibit any histologic changes. Other reports from our laboratory have identified a cross-reacting antigen (SRA) shared by T. cruzi and striated muscle. Immunization of BALB/c mice with SRA produced immunopathogenic dynamics similar to those seen with long-term T. cruzi infection. Collectively these data indicate that the cardiac lesions seen in patients with Chagas' disease may be attributed to autoimmune reactions elicited by cross-reacting antigens of T. cruzi and striated muscle.     

Antibody-dependent cell cytotoxicity against Trypanosoma cruzi is only mediated by protective antibodies

The antibody-dependent cell cytotoxicity (ADCC) to Trypanosoma cruzi blood forms (Btry) using non-adherent spleen cells is only mediated by sera from chronic chagasic patients or mice. Both display 'lytic antibodies' (LA), which are immunoglobulins directed against epitopes only present in living BTry, and 'conventional serology antibodies' (CSA), which are responsible for the positive diagnostic tests in Chagas disease. Sera from mice immunized with different T. cruzi antigens or from treated patients (displaying only CSA but not LA) are unable to mediate ADCC. These data confirm the central role played by LA in the host resistance against T. cruzi. Moreover, they probably explain why most immunizing agents used as vaccines in Chagas' disease and which elicit CSA but not LA, do not display significant protection against T. cruzi. We also demonstrate that trypsinization of BTry increases significantly the rate of parasite destruction by ADCC, suggesting that enzyme sensitive membrane components may help BTry to evade from this immune effector mechanism.     

Peanut agglutinin affinity chromatography of Trypanosoma cruzi glycoproteins

Glycoproteins from Trypanosoma cruzi epimastigotes have been extracted by diiodosalycilic acid and lithium salts, and phenol-water biphasic partition. Peanut agglutinin has been used in a one step preparative method for fractionating the total extract in order to separate the so-called galactose-terminal glycoproteins. The different fractions have been studied by SDS electrophoresis, ultracentrifugation and immunoelectrophoresis techniques. The experimental immunogenicity, antigenicity and specificity of the PNA affinity fractions has been evaluated.     

Antibody-dependent cytotoxicity of Trypanosoma cruzi antigen-coated mouse cell lines by eosinophils and neutrophils

Eosinophils and neutrophils are shown to be cytotoxic against two syngeneic mouse cell lines cells when these are coated with T. cruzi antigen and anti-T. cruzi antibody. Activity is detected within 5 h of incubation. Highest levels of cytotoxicity are obtained at antibody dilutions of 1:100 and 1:1000, while antiserum at 1:10 is shown to be inhibitory. Eosinophils show significant activity at an effector to target ratio of 5:1. No cytotoxicity occurs in the absence of either antigen, antibody or effector cells. This phenomenon may be a model for the tissue destruction in acute T. cruzi infection, where the lysis of trypanosomes may lead to antigen coating of host cells, followed by antibody-dependent granulocyte-mediated cytotoxicity of the host cells.