Examination of murine antibody response to secondary hydatidosis using ELISA and immunoelectrophoresis

Summary Antibody responses in mice with up to 64 weeks of secondary Echinococcus granulosus hydatidosis were examined by ELISA using hydatid protoscolex antigen (Px), Antigen 5 (Ag5) and Antigen B (AgB), and by immunoelectrophoresis (IEP) using sheep hydatid cyst fluid (SHCF). Anti-Px IgG antibodies, evident from 3–5 days post infection (p.i.), increased steadily until 16 weeks and maintained a high level afterwards. Anti-Ag5 IgG antibodies were negligible up to two weeks, but they showed a small increase around 2–3 weeks which was followed by a big increase around 16 weeks p.i. The high level of anti-Ag5 IgG antibodies persisted to the end of experiment. The level of anti-AgB IgG antibodies remained relatively low throughout infection. Anti-Px IgM antibodies appeared in the early period of infection, but became insignificant as the infection proceeded. Specific IgM antibodies to Ag5 and AgB showed two waves of increase, one between 3 days to 4 weeks p.i. and the other between 16 weeks to 46 weeks p.i. The level of IgA antibodies to Ag5 and AgB was low and only a moderate amount of anti-Px IgA antibodies was detected. Generally, a higher level of serum antibodies are associated with a larger number of mature cysts. Serum samples from 5 of 8 mice harbouring hydatid cysts formed 1–3 bands with SHCF in IEP, including Arc 5, but a precipitation arc with AgB was not observed. Analysis of hydatid cyst fluid from the infected mice (MHCF) in IEP also failed to demonstrate AgB. Despite the high levels of antihydatid antibodies generated in the infected mice, protoscoleces appeared to be unhindered in their growth to mature cysts.     

甘氨鹅脱氧胆酸对体外细粒棘球蚴原头节活力及ROS影响

目的 研究甘氨鹅脱氧胆酸(glycochenodeoxycholic acid, GCDCA)对体外细粒棘球蚴原头节活力及活性氧(Reactive oxygen species,ROS)水平的影响。方法 将细粒棘球蚴原头节分为对照组及GCDCA处理组(500、1 500、2 500 μ mol/ L),应用体外培养技术,通过伊红染色法每天定时在光学显微镜下观察原头节的活力并记录存活和死亡数目。使用试剂盒检测不同浓度GCDCA处理 24 h后各组原头节体内caspase-3酶活性变化;DCFH- DA染色法荧光显微镜观察 GCDCA作用24 h后原头节内 ROS水平变化。结果 500、1 500、2 500 μ mol/ L GCDCA处理后均可导致原头节活力减弱,且最高浓度组在第7 d时原头节全部死亡。500、1 500、2 500 μ mol/ L GCDCA组作用原头节24 h后 caspase-3酶活性与对照组相比明显升高(F=555.162, P<0.05)。500、1 500、2 500 μ mol/ L GCDCA作用原头节24 h后原头节内 ROS水平与对照组相比明显升高(F=216.901, P<0.05)。结论 GCDCA 能抑制体外原头节生长,促使原头节死亡,可能与活化caspase-3及提高原头节内ROS 水平相关,具体机制有待进一步研究。     

青海高原不同源原头蚴感染小鼠后继发性棘球蚴的超微结构观察

用透射电镜观察了青海高原藏羊和牦牛源原头蚴接种小鼠后继发性细粒棘球蚴的超微结构。二源继发性棘球蚴囊壁结构相同，由角质层和生发膜构成。角质层由纤维物质和不规则形颗粒形成层状结构，但近生发膜处藏羊源的结构致密，牦牛源的则较为疏松。生发膜可分为皮层区和细胞区。皮层区外缘的微毛藏羊源多而短粗，牦牛源的则呈细长，与各自原发性棘球蚴相同，提示微毛的形态结构稳定，不随宿主不同而改变；牦牛源皮层区基部可见线粒体和大量糖原，提示该区有活跃的生理生化代谢功能。细胞区由皮层细胞和其它成分构成，皮层细胞内细胞器丰富，线粒体形态藏羊源的与原发性相同，牦牛源的则与原发性的存在差异，提示牦牛源棘球蚴线粒体的形态结构不稳定，易受不同宿主的影响。皮层细胞胞质的近囊腔处存在巨大的囊泡，囊壁多为单层。内缘附有线粒体或异染色质样物质，可能与后期子囊的形成有关。本文结果提示该地区可能存在不同的细粒棘球绦虫虫株。     

3种药物体外抗细粒棘球蚴原头节作用的比较

目的　通过比较氧苯达唑、阿苯达唑和阿苯达唑脂质体体外抗细粒棘球蚴原头节作用 ,探讨氧苯达唑抗包虫病的作用。　方法　将氧苯达唑、阿苯达唑和阿苯达唑脂质体分别配成高、中、低浓度加入 RPMI16 4 0培养基中 ,体外培养细粒棘球蚴原头节 ,观察其每天的死亡率 ,直到对照组的头节全部死亡为止。　结果　将相同条件下 3种药物作用原头节的死亡率分别与对照组相比 ,阿苯达唑、氧苯达唑高、中、低浓度组与对照组相比 ,差异均有统计学意义 (P<0 .0 5 ) ;而阿苯达唑脂质体仅在高浓度时与对照组的差别有统计学意义 (P<0 .0 5 )。　结论　氧苯达唑、阿苯达唑和阿苯达唑脂质体均有显著的体外抗细粒棘球蚴原头节作用 ;氧苯达唑体外抗细粒棘球蚴原头节作用与阿苯达唑相当 ,可认为是一种新型抗包虫药 ;阿苯达唑脂质体剂型并未显示出特殊的体外抗原头节作用。     

抗包虫药物的体外药效对比实验研究

目的：包虫病是一种危害性很大的人畜共患病,除了甲苯咪唑（ＭＢＺ）和阿苯达唑（ＡＢＺ）这两种公认的有效药物以外,目前尚无新的更有效的药物出现。本实验通过体外进行了氧苯达唑（ＯＢＺ）、阿苯达唑和阿苯达唑脂质体（Ｌ－ＡＢＺ）的体外抗细粒棘球蚴原头节作用的比较,探讨氧苯达唑在抗包虫病方面的作用,为探索新的抗包虫病药物奠定基础。方法：将氧苯达唑、阿苯达唑和阿苯达唑脂质体分别加入ＲＰＭＩ１６４０培养液中,配成高（２５００ｎｇ／ｍｌ）、中（５００ｎｇ／ｍｌ）和低（１００ｎｇ／ｍｌ）３个浓度的加药培养液,将用无菌方法采集的自然感染的羊细粒棘球蚴原头节（活头节占９５％以上）加入上述加药培养基中进行培养,每天计数其死亡率,方法如下：取一滴培养液,０．１％伊红溶液染色５ｍｉｎ,在普通光镜下观察,死亡原头节红染着色,伴有结构破坏;活头     

Antibody response of Echinococcus granulosus infected mice: Protoscolex specific response during infection is associated with decreasing specific IgG1/IgG3 ratio as well as decreasing avidity

The antibody response was followed during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with dead PSC. Titres of antibodies recognizing peptidic and glucidic PSC epitopes, as well as their isotypic and avidity profiles were followed by ELISA. In addition, antigen recognition patterns were analysed by immunoblot. The response against carbohydrate epitopes was dominant in infected and immunized mice but stronger in the first group. Infected mice showed similar profiles of specific IgG and IgM with maximum titres from week 38 to 53. Although IgG1 and IgG3 were the predominant antibody subclasses, the ratio of IgG1/IgG3 antibody titres as well as antibody avidity decreased during the experiment, encompassing a decrease in recognition of peptidic epitopes. Immunized mice did not show significant levels of specific IgM and, after week 15, showed IgG titres lower than the infected mice. IgG1 was the predominant IgG subclass during all the experiment with background levels of IgG3. The mean Ab avidity was high and showed no significant changes during immunization. Different patterns of response were thus produced by dead and developing live parasites. Although high avidity IgG1 antibodies were early found in both cases, lower avidity IgG3 antibodies were increasingly produced afterwards only in infected animals. The isotype switch and avidity decrease observed only during infection are consistent with a possible parasitic mechanism to evade host immunity .     

伽马射线体外照射对细粒棘球蚴原头节的杀灭作用

目的 探讨细粒棘球蚴原头节体外照射γ-射线后导致的死亡、细胞内caspase-3的表达和显微结构的变化。方法 在体外培养的基础上,用不同剂量的γ-射线对细粒棘球蚴原头节进行照射。对照射后各组原头节细胞内的caspase-3蛋白酶活性进行测定,观察期原头节死亡率,并在倒置显微镜下动态观察原头节形态结构的变化。结果 原头节经过γ-射线体外照射后会出现死亡率升高,caspase-3表达增强现象,同时原头节形态结构出现肿胀、塌陷、碎裂的阶段性变化。结论 γ-射线体外照射可以直接杀灭原头节,且诱导原头节细胞发生凋亡。     

汉防己甲素体外抗泡球蚴作用的研究

目的　比较汉防己甲素、阿苯哒唑和汉防己甲素脂质体体外抑制原头节作用 ,探讨汉防己甲素抗泡型包虫病的作用。　方法　将汉防己甲素、阿苯哒唑和汉防己甲素脂质体分别配制成 2 0 μg/ml、40 μg/ml、80 μg/ml 3个浓度 ,并且选择低、中剂量的汉防己甲素和阿苯哒唑合用 ,加入RPMI164 0培养基中 ,体外培养泡球蚴原头节 ,观察计数每天的死亡率 ,直到所有的用药组头节全部死亡。　结果　汉防己甲素、阿苯哒唑低、中、高浓度组与相应的对照组比较 ,原头节死亡率的差异均有统计学意义 (P <0 .0 5 ) ;汉防己甲素脂质体在中、高浓度下与对照组的原头节死亡率差异有统计学意义(P <0 .0 5 ) ;联合用药组与单药组的原头节死亡率差异亦有统计学意义 (P <0 .0 5 )。　结论　汉防己甲素体外具有抑制泡球蚴原头节的作用 ,并且与阿苯哒唑联合使用时具有一定的协同作用。     

IGF-ⅠR抑制剂OSI-906体外抗细粒棘球绦虫原头蚴作用的研究

目的探讨胰岛素样生长因子I受体(insulin-like growth factor I receptor,IGF-ⅠR)抑制剂OSI-906体外抗细粒棘球绦虫原头蚴作用。方法体外培养细粒棘球绦虫原头蚴,用6个浓度梯度(100、50、25、12.5、6.25、3.125μmol/L)的OSI-906进行干预,分别于给药后1、2、3、4d用伊红染色,在倒置荧光显微镜下观察其活力(每组设置3个复孔),绘制原头蚴活力曲线;透射电镜下观察不同浓度OSI-906组原头节超微结构变化。结果 100和50μmol/L OSI-906作用不同时间原头蚴存活率与空白对照组组间和溶剂组相比差异有统计学意义(P<0.05);其余浓度组间比较原头蚴存活率差异无统计学意义(P>0.05)。第3d时100μmol/L的OSI-906组原头节存活率为(42.38±1.05)%;第4d时100μmol/L的OSI-906组原头蚴全部死亡。透射电镜观察OSI-906高浓度组原头蚴微毛结构消失,皮层细胞坏死,细胞膜严重破损,核形不规整,核仁大且边界不清,核基质密度极低,异染色质增多、边集。结论 IGF-ⅠR抑制剂OSI-906体外抗细粒棘球绦虫原头蚴作用显著,是一种潜在的抗包虫药物。     

白黎芦醇体外对多房棘球蚴原头节和微囊的作用研究

目的 研究白藜芦醇(Resveratrol,RES)体外对多房棘球蚴原头节(Protoscoleces,PSCs)和微囊的作用效果,为多房棘球蚴病的临床用药提供新的依据.方法 PSCs体外经不同浓度(5、10、20、40、80、100、200和400 μmol/L)的RES作用7 d,采用台盼蓝染色观察其活力与形态变化...