Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Av Universidad 3000, Col Copilco Universidad, 04510 Mexico Distrito Federal, Mexico.
Parasitol Res. 2010 May;106(6):1327-37. doi: 10.1007/s00436-010-1803-4. Epub 2010 Mar 18.
The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.
已报道过克氏锥虫前鞭毛体在补体依赖反应中对正常或免疫血清裂解的易感性。耗尽补体的鼠免疫血清可减轻前鞭毛体的诱导损伤，其特征为形态学改变和死亡。本工作旨在研究暴露于去补体鼠免疫血清的前鞭毛体死亡的机制。前鞭毛体在 RPMI 培养基中维持。免疫血清通过用全粗前鞭毛体提取物免疫小鼠制备。活的前鞭毛体在 37°C 下与去补体的正常或免疫血清孵育。电镜下，聚集的寄生虫显示出两个或多个寄生虫之间的膜融合的特征模式；这种融合还产生了亚膜微管的交错。通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）和膜联蛋白 V 测定法，通过流式细胞术确定细胞凋亡。通过 4'，6-二脒基-2'-苯基吲哚二盐酸（DAPI）细胞化学检测核特征，该方法显示凋亡核浓缩。还测量了半胱天冬酶活性。与用去补体正常血清孵育的寄生虫相比，用去补体免疫血清孵育的寄生虫摄取了 26％的特异性荧光，而 TUNEL 结果显示了这一点。膜联蛋白 V-Fluos 染色试剂盒显示，与用去补体正常血清孵育的寄生虫相比，用去补体免疫血清孵育的前鞭毛体将磷脂酰丝氨酸暴露在质膜的外叶。寄生虫与免疫血清的孵育显示半胱天冬酶 3 活性。我们得出结论，尽管途径尚未阐明，但特异性抗体能够诱导前鞭毛体的聚集和凋亡。
