伯氏疟原虫氯喹敏感株和抗性株红内期虫体超微结构的比较

了解伯氏疟原虫 (Ｐｌａｓｍｏｄｉｕｍｂｅｒｇｈｅｉ)氯喹敏感 (Ｎ)株和抗性 (ＲＣ)株红内期虫体在抗药性、致病力和诱导免疫应答方面差异的形态学基础应用超薄切片和透射电镜技术 ,比较Ｎ株和ＲＣ株红内期虫体的超微结构。结果表明 ,Ｎ株和ＲＣ株滋养体食物胞膜为双层膜结构 ,膜上具有 1～ 2段 (大多为 1段 )电子密度沉着带的标志性结构。ＲＣ株滋养体对血红蛋白的摄取具有多种方式 ,疟色素形成明显少于Ｎ株 ,而且在裂体生殖中 ,未见疟色素中心化和形成融合泡。ＲＣ株滋养体核可出现类似核仁样结构。伯氏疟原虫氯喹抗性株长期在氯喹压力…     

酮替酚配伍周效磺胺对红内期约氏疟原虫超微结构的影响

酮替酚或周效磺胺单独使用治疗疟疾，红内期约氏疟原虫的超微结构均能受到一定的损伤，但配伍使用酮替酚和周效磺胺后虫体的结构损伤更为迅速，给药８小时后裂殖体形成受阻，滋养体除细胞结构逐渐消失外还出现伪食泡和逐渐增多增大的单膜或多膜空泡，给药２４小时查见结构完整的滋养体。     

酮替酚配伍周效磺胺对红内期约氏疟原虫超微结构的影响

酮替酚或周效磺胺单独使用治疗疟疾，红内期约氏疟原虫的超微结构均能受到一定的损伤，但配伍使用酮替酚和周效磺胺后虫体的结构损伤更为迅速，给药８小时后裂殖体形成受阻，滋养体除细胞结构逐渐消失外还出现伪食泡和逐渐增多增大的单膜或多膜空泡，给药２４小时未查见结构完整的滋养体。     

羟基喹哌对体外培养的恶性疟原虫红内期超微结构的影响

应用透射电镜观察羟基喹哌对体外培养的恶性疟原虫超微结构的影响。药物作用半小时起,疟原虫食物泡即发生肿胀。随着时间的推移,食物泡内色素凝聚成团,色素减少并且出现旋涡状膜小体及红细胞质团块。8h 后,细胞核、细胞质、线粒体、核糖体出现一系列变化。16h 以后,多数滋养体崩解仅残存大型空泡及团块状物。本结果提示羟基喹哌的靶细胞器为原虫食物泡。     

诺氏疟原虫的人体自然感染

云南省墨江县1例“间日疟”患者的血片,经回顾性镜检,发现疟原虫形态特别,早期滋养体多核,红细胞内有多个虫体寄生,晚期滋养体有形成带状趋势。裂殖体和配子体与间日疟原虫相似。经分子生物学鉴定为诺氏疟原虫。     

人结肠小袋纤毛虫滋养体的透射电镜观察

用透射电镜观察入结肠小袋纤毛虫滋养体,纵切面呈椭圆或卵圆形。体表有突起和小沟,纤毛从小沟内发出。虫体腹面有一胞口,胞口处有纤毛束,纤毛排列很有规律,其轴丝微管的“9 2”结构很清楚。虫体有大小核各一个,大核多为肾形,小核球形或三角形.位于大核的凹侧。大小核均有双层核膜和核孔。胞浆内含有大量线粒体、食物泡和多糖颗粒。线粒体及粗面内质网分布在虫体的外周。     

伯氏疟原虫的体外培养和透射电镜观察

用大鼠血清和盐酸苯肼诱导的大鼠网织红细胞,按常规的蜡烛缸法体外培养红内期的伯氏疟原虫,并在培养前和培养12、20和28h,对培养物作透射电镜观察。结果5d的培养除了第一个裂体周期疟原虫有明显增殖外,以后变性原虫渐进性增多。它表现为胞核凝固,核糖体和内质网减少,伪食物泡和食物泡缺损,自噬泡和残余泡形成,裂殖体不能正常成熟,游离裂殖子崩解,有嵴线粒体和微体样分泌泡的产生及多层膜结构的形成。     

约氏疟原虫红内期抗原定位的免疫电镜研究

应用LR White低温包埋法并结合胶体金标记免疫电镜细胞化学技术对保护性单克隆抗体M26-32识别的约氏疟原虫红内期抗原进行了超微结构定位。结果表明与单克隆抗体M26-32发生特异性免疫反应的抗原主要定位于早期滋养体、晚期滋养体、裂殖体和裂殖子的细胞质,为无性期不同发育阶段的共同抗原;在滋养体发育过程中该抗原有所增加,一部分可通过原虫的胞吐作用运出并定位于邻近虫体的感染红细胞细胞质。     

间日疟原虫红细胞内期电镜观察

间日疟原虫裂殖子钻入红细胞后,早期环状体呈哑铃形,其纳虫空泡内膜状物质通过红细胞中狭窄通道排出。滋养体形状不规则,或呈卵圆形,由单层表膜包绕,细胞质内具有无嵴线粒体,纳虫空泡中有电子致密颗粒。配子体形状规则,几乎充满被寄生的红细胞,由两层表膜包绕,细胞质内具有有嵴线粒体。雌配子体细胞质内核糖体、嗜锇小体和线粒体都比雄配子体丰富。被寄生的红细胞的3个主要变化为出现小泡、细胞质裂隙和凹窝小泡复合物。后者沿红细胞表膜分布,可能是薛氏小点。     

伯氏疟原虫氯喹敏感株和抗性株在疟色素形成和致病性上的差异

目的：研究伯氏疟原虫氯喹敏感株（Ｎ）和抗性株（ＲＣ）在疟色素形成和致病性上的差异。方法：用伯氏疟原虫氯喹敏感株和抗性株分别感染ＩＲＣ小鼠。实验分为５组：正常对照组（ＮＣ）,氯喹治疗对照组（ＣＣ）,Ｎ株感染组（Ｎ）,ＲＣ株感染组（ＲＣ）和ＲＣ株感染加氯喹治疗组（ＲＣＣ）,比较各组间末梢血中疟原虫的形态、原虫血症、肝脏组织学及超微结构的改变。结果：Ｎ组肝细胞损害较严重,细胞内线粒体肿胀融合,溶酶体增多,肝血窦内疟原虫少见。ＲＣ组炎症反应较突出,主要为单核细胞浸润以及枯否细胞活跃,大滋养体和裂殖体在肝血窦滞留;肝细胞损害较轻,表现为线粒体增生、肿胀及空泡化。Ｎ株疟原虫富含内食物泡,其内有疟色素颗粒,被寄生红细胞结构较完整;而ＲＣ株疟原虫外食物泡较多,位于疟原虫外围的红细胞胞质内,被寄生的红细胞呈蜂窝样改变,食物泡内无疟色素。结论：ＲＣ株疟原虫可能改变了原敏感株（Ｎ株）对血红蛋白的摄取和消化方式,从而阻碍了疟色素形成;Ｎ株和ＲＣ株疟原虫可能因诱导宿主免疫反应的明显差异,导致ＲＣ株较Ｎ株致病力为弱     

田鼠巴贝虫(Babesia mocroti)的超微结构观察

目的 观察田鼠巴贝虫的超微结构,了解田鼠巴贝虫在宿主红细胞中发育的形态变化。方法 运用扫描电镜观察田鼠巴贝虫侵入宿主红细胞的过程,运用透射电镜观察田鼠巴贝虫发育的形态变化过程及宿主红细胞形态变化情况。结果 扫描电镜下观察到田鼠巴贝虫裂殖子大小在406~981nm之间,虫体发育过程分为裂殖子、分裂中的裂殖子、滋养体3个阶段,裂殖子是通过红细胞膜上微孔进入宿主红细胞,分裂中的裂殖子可使红细胞变形,滋养体是通过溶解红细胞膜游离红细胞。透射电镜观察到田鼠巴贝虫裂殖子的核膜为双层膜结构,虫体中有核糖体、微管、内质网、线粒体和溶酶体等完整的细胞器,分裂中的裂殖子在侵入后期可见食物空泡,滋养体的外膜和核不规则,胞浆中含有颗粒和空泡,宿主红细胞的电子密度随着虫体的发育而逐渐变稀疏。结论 田鼠巴贝虫是含有完整细胞器的单细胞有机体,具有一般细胞所有的基本结构,它能完成多细胞动物所具有的生命机能,并以宿主红细胞中的血红蛋白作为生存氧料。     

Ultrastructural study on the erythrocytic schizogony of Plasmodium vivax

Erythrocytic schizogony is initiated by repeated division of the nucleus in rapid sequence. Another important feature of the developing schizont is the reappearance of segments of thick inner membrane and formation of lobes around these segments. These bulbous protuberances signal the start of budding process which ends in complete segmentation of the cell. The cytoplasmic organelles which de-differentiate at the beginning of trophozoite stage are formed again. Asynchrony during merozoite formation is observed in some schizonts where fully mature merozoites are seen lying in the parasitophorous vacuole while the mother cell is still in the process of giving rise to new merozoites.     

The role of lipids in Plasmodium falciparum invasion of erythrocytes: a coordinated biochemical and microscopic analysis

The role of lipids in Plasmodium falciparum invasion of erythrocytes was investigated by biochemical and fluorescent microscopic analysis. Metabolic incorporation of radioactive oleate or palmitate and fractionation of radiolabeled phospholipids by thin-layer chromatography revealed no difference in the major phospholipid classes of schizonts and early ring forms after merozoite invasion. Fluorescent anthroyloxy derivatives of oleate and palmitate were also metabolically incorporated into parasite phospholipids. By microscopic analysis, the fluorescent phospholipids were seen localized in the plasma membrane and, within the merozoite, concentrated near the apical end. During invasion fluorescent phospholipid appeared to be injected from the apical end of the merozoite into the host membrane, both within and outside the parasite-host membrane junctions. After invasion fluorescent lipid was only found in the parasite plasma membrane and/or parasitophorous vacuole membrane. Parallel experiments with a fluorescent cholesterol derivative, incorporated into parasite membranes by exchange, revealed neither heterogeneous distribution of label within the parasite nor evidence for cholesterol transfer from merozoite to host cell membrane. Results suggest that during invasion no major covalent alteration of parasite lipids, such as lysophospholipid formation, occurs. However, invasion and formation of the parasitophorous vacuolar membrane apparently involves insertion of parasite phospholipids into the host membrane.     

Effect of metalloprotease inhibitors on invasion of red blood cell by Plasmodium falciparum

For successful invasion, the malaria merozoite needs to attach to the red blood cell membrane, undergo reorientation, form a junction of the apical end with the host membrane, and internalize. Malaria proteases have been implicated in the invasion process, but their specific cellular functions remain unclear. To demonstrate the involvement of metalloprotease in the process of Plasmodium falciparum merozoite entry into host red blood cell, schizont-infected red blood cells and parasitophorous vacuolar membrane-enclosed merozoite structures were treated with 1,10-phenanthroline, a metal chelator, resulting in a reduction of invasion with IC50 value of 25 and 29 microM, respectively. Absence of an accumulation of schizont stages after treatment with 1,10-phenanthroline indicated that the inhibitory effect was not due to suppression of merozoite release from red blood cells, but on the invasion step. Although treatment with GM6001, a well-known inhibitor of the mammalian matrix and disintegrin metalloprotease family, was less effective, nevertheless this study points to the importance of metal-requiring protease in the process of invasion of host red blood cell by the malaria parasite.     

Isolation and characterization of parasite-inhibitory Plasmodium falciparum monoclonal antibodies

Three mouse hybridomas were isolated that produced IgM monoclonal antibodies (Mab) which reacted with erythrocytic stages of Plasmodium falciparum and inhibited the invasion of erythrocytes in vitro. Those Mab, initially identified by an ELISA screening of hybridoma culture medium, exhibited a strong binding to trophozoite and schizont but not to ring or merozoite stage parasites or to erythrocytes in an indirect immunofluorescence assay. All inhibited the parasite's ability to infect erythrocytes in an in vitro invasion inhibition assay. Western blot analysis of the binding of the Mab to SDS-PAGE-separated parasite antigens isolated from the ring, trophozoite, schizont or spontaneously released merozoite stages, indicated that two of the Mab bound to a Mr 105,000 antigen in trophozoites and schizonts while the third Mab did not. All three Mab also bound to Mr 30,000-40,000 antigens in all stages, however, in all instances binding to these antigens was enhanced in merozoites. It was further observed that the two Mabs that bound to a Mr 105,000 antigen: exhibited a markedly reduced binding to the Mr 105,000 antigen in merozoite preparations; exhibited different relative intensities of binding to the trophozoite and schizont antigens; both bound to the same Mr 105,000 antigen as demonstrated through Western blot analysis of antigens separated by two-dimensional gel electrophoresis. The findings suggest that the inhibitory Mab bound to different epitopes of the same antigen and that the antigen may either be processed or degraded at about the time of merozoite release and erythrocyte invasion.     

Malaria parasite invasion: interactions with the red cell membrane

The capacity to invade red cells is central to the biology of malaria parasites; both asexual multiplication and reinfection of the definitive mosquito host depend upon intraerythrocytic stages. The invasion process is complex. The briefly free merozoite specifically recognizes and adheres to ligands on the red cell surface, then alters the red cell membrane to produce an invagination into which it moves, and so becomes enclosed in a membrane-bound parasitophorous vacuole. Here we assess new evidence that bears on our understanding of this process. This has come from sources including biochemical and ultrastructural studies of the specialized surface and organelles of merozoites, from in vitro invasion studies using naturally refractory or artificially modified red cells, and from structural, chemical, and immunological analyses of the newly parasitized cell.     

Assay of erythrocyte components as inhibitors of Plasmodium falciparum merozoite invasion of erythrocytes

Three red blood cell membrane fractions (Band 3, macromolecules exhibiting I antigenic determinants, and a delipidated glycoprotein fraction) were separated from red blood cell membranes and tested for their ability to inhibit penetration of red blood cells by Plasmodium falciparum merozoites in an in vitro inhibition assay. The delipidated glycoprotein fraction (containing the major sialoglycoproteins and devoid of Band 3) was the only fraction that inhibited merozoite invasion. This fraction showed 73% and 70% inhibition at 1 mg/ml and 500 micrograms/ml, respectively, and slight inhibition below these levels.     

Pseudo-angiomatous growth pattern in recurrent plasma cell myeloma

Erythrocytes are remarkably dynamic structures, possessing multiple and complex pathways for regulating cell membrane properties to compensate for the absence of a nucleus and internal membranes. Unlike the invasion strategies of many viruses and bacteria into their eukaryotic hosts, however, the accepted model for malaria parasite entry into human erythrocytes casts the host cell in a largely passive role. This is in contrast to mounting evidence for a suite of dynamic alterations that the erythrocyte membrane undergoes during the rapid process of invasion by the blood stage malaria parasite – the merozoite. Here we review the cellular and molecular basis for merozoite invasion of the erythrocyte and explore the idea that radical changes in the erythrocyte membrane protein and lipid architecture probably accompany this key step in the establishment of human malaria disease.     

Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development

Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.