曼氏血吸虫表膜两型转糖蛋白在日本血吸虫表膜的免疫细胞化学定位

目的： 观察曼氏血吸虫表膜两型转糖蛋白（ Ｓ Ｇ Ｔ Ｐ１ 和 Ｓ Ｇ Ｔ Ｐ４） 分子在日本血吸虫成虫表膜上的分布,确定日本血吸虫和曼氏血吸虫的 Ｓ Ｇ Ｔ Ｐ 是否有同源性。方法： 采用超薄切片技术制备日本血吸虫超薄冷冻切片。在电镜下观察曼氏血吸虫的 Ｓ Ｇ Ｔ Ｐ１ 和 Ｓ Ｇ Ｔ Ｐ４ 抗体对日本血吸虫的相应抗原的免疫标记定位。结果： 免疫定位显示 Ｓ Ｇ Ｔ Ｐ１ 定位于日本血吸虫成虫表膜基底部质膜及其反褶的质膜上, Ｓ Ｇ Ｔ Ｐ４ 定位于成虫表膜顶部质膜及其内陷质膜上。结论： 两型转糖蛋白分子在曼氏血吸虫和日本血吸虫表膜上的定位相同。表明此两种血吸虫两型表膜的转糖蛋白分子有很大同源性。     

200 kD 糖蛋白和20 kD 蛋白分子在曼氏血吸虫不同发育阶段的表达与定位研究(英文)

采用间接荧光抗体技术对单克隆抗体 Ｍ４５ 所识别的２００ ｋ Ｄ及 Ｍ５０ 所识别的２０ ｋ Ｄ抗原在曼氏血吸虫不同发育阶段的表达和定位进行了研究。２００ ｋ Ｄ 抗原主要在虫卵、毛蚴和早期童虫阶段表达,主要位于毛蚴、尾蚴和早期童虫的表膜和分泌腺。２０ ｋ Ｄ 抗原则在所研究的发育阶段中,除虫卵阶段外都有表达,定位于童虫到成虫的表膜和尾蚴的实质中。由此可见,２０ ｋ Ｄ抗原主要表达于从童虫到成虫的终宿主体内寄生阶段,而２００ ｋ Ｄ 抗原主要表达于与淡水中自由生活相关的幼虫阶段。     

Effect of antisense-SGTPs on the glucose uptake of the blood fluke Schistosoma mansoni: observations in adult worms and schistosomula

AS-ODNs, complementary to Schistosoma mansoni glucose transporter proteins (SGTP1 and SGTP4), were chosen as potential therapeutic agents for schistosomiasis. AS-SGTP1 oligos lowered the glucose uptake of adult worms both in vitro and ex vivo. The most effective AS-ODN was that of 21 nucleotides complementary to the SGTP1 nucleotide sequence, including the initiation region of mRNA translation. This oligo was found to decrease glucose uptake in vitro by as much as 50% and at a concentration of 4.0 mg/ml, it killed all male worms within 24 hours. A significant decrease, up to 34%, in glucose uptake was also noted when 100 mg/kg x2 (with a 2 hours interval) of AS-ODN was administered ex vivo. Two out of six anti-SGTP4 oligos also decreased the glucose uptake of adult worms in vitro by 25-44%. Added to the culture of schistosomula, two AS-SGTP4 oligos were found to decrease glucose uptake by 20-43%.     

Localization of the A1.12/9 antigen family to the neurones, putative sensory receptors and tegument of Schistosoma mansoni

The A1.12/9 antigen family were localized to the periphery of vesicle-like structures in neurones and in the putative sensory receptors of cercariae by immuno-electron microscopy. After transformation into schistosomula, the antigens were rapidly lost until, after 18 h, no immunoreactivity could be detected. Expression resumed at approximately 36 h post-transformation and had returned to high levels by 4 days post-infection. This level was maintained through to the adult worm stage. In 4 day lung schistosomula and in 14 day and 21 day liver schistosomula immunolabelling was observed within the matrix of the tegument itself and also in association with the tegumental membrane and vesicles within the tegument. These properties suggest that the A1.12/9 antigens may be involved in neuropeptide processing pathways. In particular, this family may represent the schistosome homologue of the granin family with which they share common properties and some sequence homology.     

Schistosoma mansoni: localization of the 28 kDa secreted protease in cercaria

Summary Monospecific rabbit antibodies were utilized to localize the 28 kDa serine protease which is released from transforming schistosomula of Schistosoma mansoni in cercariae and freshly transformed schistosomula. This protease exerts two postulated activities, degradation of connective tissue proteins thus promoting skin penetration and release of the cercarial glycocalyx leading to accelerated schistosomular transformation. Upon immunogold labelling of cercarial cryosections, the 28 kDa protease was found stored in both the preacetabular and postacetabular glands. This enzyme was also detected in the cercarial glycocalyx by immunogold and immunofluorescence labelling and by its proteolytic activity. Following transformation and shedding of the glycocalyx, the same 28 kDa protease was found on the surface membrane of transformed schistosomula which are resistant to immune damage. It is suggested that the 28 kDa membrane protease which cleaves in vitro the complement proteins C3, C3b and C9, may promote in vivo immunoresistance of S. mansoni.     

Schistosoma mansoni: localization of the 28 kDa secreted protease in cercaria

Monospecific rabbit antibodies were utilized to localize the 28 kDa serine protease which is released from transforming schistosomula of Schistosoma mansoni in cercariae and freshly transformed schistosomula. This protease exerts two postulated activities, degradation of connective tissue proteins thus promoting skin penetration and release of the cercarial glycocalyx leading to accelerated schistosomular transformation. Upon immunogold labelling of cercarial cryosections, the 28 kDa protease was found stored in both the preacetabular and postacetabular glands. This enzyme was also detected in the cercarial glycocalyx by immunogold and immunofluorescence labelling and by its proteolytic activity. Following transformation and shedding of the glycocalyx, the same 28 kDa protease was found on the surface membrane of transformed schistosomula which are resistant to immune damage. It is suggested that the 28 kDa membrane protease which cleaves in vitro the complement proteins C3, C3b and C9, may promote in vivo immunoresistance of S. mansoni.     

Inhibition of surface membrane maturation in schistosomula of Schistosoma mansoni.

The surface membrane of the multicellular parasite Schistosoma mansoni is radically reorganized during the transformation of cercariae into schistosomula. The current study investigates factors involved in maturation of the surface from a trilaminate to a multilaminate membrane. When maturation was induced in the presence of puromycin (900 microM), the acquisition of a multilaminate surface and stainability with fluorescein-conjugated Con A were similar to that of control parasites. Similarly, although organisms treated with monensin (0.1 microM) for 3 hr showed large vacuoles in the perinuclear cytoplasm of the subtegumental cells, the surface membrane became multilaminate. In contrast, microtubule-active drugs interfered with maturation: the surface remained largely trilaminate and the percentage of organisms binding Con A to their surface was significantly reduced. Furthermore, large accumulations of multilaminate bodies were found in the subtegumental cells of colchicine-treated parasites, whereas few were seen in the controls. Colchicine-treated schistosomula failed to mature to adult worms upon injection into mice and, like cercariae, they were water tolerant. We therefore conclude that the components that constitute the schistosomula surface preexist in cercariae and suggest that they are stored in multilaminate bodies before being transported to the surface with the help of microtubules. The acquisition of the multilaminate membrane may be essential for survival of the parasites in vivo and in vitro.     

环孢素A体外抗AF18标记的曼氏血吸虫童虫的研究

目的　探讨环孢素A体外抗AF18标记的曼氏血吸虫童虫的作用以及虫体表膜流动性。　方法　制备童虫,环孢素A体外作用,AF18标记,荧光显微镜观察。　结果　童虫损伤或死亡,虫体逐渐从绿色变成黄色,虫体表面损伤。　结论　环孢素A增加童虫AF18的含量,减低童虫表膜流动性。     

环孢素A体外抗AF18标记的曼氏血吸虫童虫的研究

目的探讨环孢素A体外抗AF18标记的曼氏血吸虫童虫的作用以及虫体表膜流动性。方法制备童虫，环孢素A体外作用，AF18标记，荧光显微镜观察。结果童虫损伤或死亡，虫体逐渐从绿色变成黄色，虫体表面损伤。结论环孢素A增加童虫AF18的含量，减低童虫表膜流动性?     

Ultrastructural localization of a protective 68,000 molecular weight antigen in Schistosoma mansoni

Vaccination with SMW 68, an Mr 68,000 glycoprotein of Schistosoma mansoni, induces significant protection in mice against challenge schistosome infection. This resistance occurs without the use of adjuvants and without sensitizing animals to granuloma formation. Likewise, passive transfer of monoclonal antibody (MAb) 31-3B6 against SMW 68 confers partial protection against challenge infection. As a first step in understanding how the immune response to this molecule leads to resistance, SMW 68 was localized in three developmental stages of the parasite by immunoelectron microscopy using MAb 31-3B6 and polyclonal antisera raised against purified SMW 68. In cercariae and schistosomula, MAb 31-3B6 bound electron-dense granules within the head gland and similar granules in the preacetabular glands. In adult worms, SMW 68 or related antigens were found to be widely distributed in tissues. Binding of specific antisera was most pronounced in the gut and tegument of male worms, but less so in subtegumental muscles. We conclude that SMW 68 is presented to the immune system in various ways during parasite development. The protective protein or epitope is excreted, and presented on the surface and in the cytoplasm at various stages of the life cycle. The relationship of the location of this protein to its role in protective immunity is discussed.     

Protection of mice against Schistosoma mansoni infection by passive transfer of sera from infected rabbits

Sera from rabbits infected with unattenuated Schistosoma mansoni cercariae conferred significant levels of protection against S. mansoni challenge ( P  < 0.001) after passive transfer to mice. Infected rabbit sera were only effective in conferring protection when transferred during the first week of infection, and were not effective when administered against liver-stage worms. Immunoglobulins isolated from the infected rabbit sera with Protein A-Sepharose were shown to be responsible for the transfer of protection to mice. Immunofluorescence studies demonstrated that the sera were more reactive against the surface of three hour-old mechanically transformed schistosomula than against the surfaces of lung-stage schistosomula. The sera from infected rabbits reacted polyspecifically against antigens in cercaria, schistosomula, and the worm and egg stages of the S. mansoni life-cycle. The host parasite relationship of S. mansoni in the rabbit is discussed.     

Stimulation of peripheral blood mononuclear cells from patients with schistosomiasis mansoni by living and fixed schistosomula, and schistosomular membrane extracts and vesicles

Several antigenic preparations derived from Schistosoma mansoni schistosomula were compared as immunogens in proliferation assays utilizing peripheral blood mononuclear cells (PBMN) from patients with chronic intestinal schistosomiasis mansoni. Living and fixed schistosomula, either irradiated or nonirradiated, and freshly transformed or 24 hr cultured worms were found to induce very similar proliferative responses of patients' PBMN. Two schistosomulum surface membrane preparations were also compared. Membrane vesicles from schistosomula were found to elicit mean proliferative responses similar to those stimulated by a CaCl2-extracted tegument preparation and to do so at lower total protein concentrations. However, the responses to membrane vesicles were somewhat more variable. When saline extracts from schistosomular bodies submitted to these two treatments were used as stimulants in parallel PBMN cultures it was found that vesiculation more effectively removed antigens involved in the stimulation of PBMN. Broken vesicles were less stimulatory than intact ones. This suggests that the manner in which membrane antigens are presented to antigen processing cells is critical. Incubation of schistosomula at 37 degrees C, but not 4 degrees C in medium containing 10% fetal calf serum from 3 to 48 hr decreased the capacity of the membrane to produce vesicles when treated with high salt solution.     

Two heat-induced proteins are associated with transformation of Schistosoma mansoni cercariae to schistosomula.

Infection with Schistosoma mansoni, a parasitic fluke, is acquired when cercariae found in freshwater bodies at ambient temperature penetrate human skin. In this study the response of S. mansoni cercariae to change in temperature from ambient to that of mammalian hosts was compared to the response of other stages that do not experience such dramatic temperature shifts. In cercariae, temperature increases from 23 degrees C to either 37 degrees C or 42 degrees C primarily induced the synthesis of two proteins unique to this stage, of Mr 60,000 and 58,000. Neither protein could be induced by similar treatment in the other two stages of the parasite found in mammalian hosts. Instead, these stages predominantly synthesized a Mr 70,000 protein that crossreacted with monoclonal and polyclonal antibodies against the Mr 70,000 heat shock protein of Drosophila and chicken. No crossreactive material was detected in cercariae, suggesting that a protein homologous to the highly conserved heat shock protein of Mr 70,000 is absent or induced to very low levels by temperature increase at this stage. The induction of the Mr 60,000 and 58,000 proteins was confined to the initial 3-hr period during cercarial transformation to the schistosomula stage.     

Antibody isotype responses to the Schistosoma mansoni schistosomulum in the CBA/N mouse induced by different stages of the parasite life cycle

During infection with Schistosoma mansoni the extent and nature of immune reactions against schistosomula may be influenced by responses to cross-reactive antigens in eggs or adult worms, and there is now extensive evidence for cross-reactivity between the different stages of the parasite life cycle. In this study IgM and IgG subclass antibodies produced in (CBA/N x Balb/c) F1 male and female mice were measured over a period of time following exposure to a chronic infection, to unisexual male cercariae or to irradiated larvae. Antibody levels were also measured following immunization with antigen preparations derived from adult worms, schistosomula or eggs. (CBA/N x Balb/c) F1 male mice exhibit an X-linked immune deficiency which results in an inability to respond to T-independent (TI) type 2 polysaccharides. Isotype levels were measured by ELISA to detergent-soluble schistosomulum antigen. Results showed that antigens on the different stages of the parasite life cycle have a qualitative influence on the antibody response to the larval surface, and that T-independent type 2 polysaccharides, particularly abundant in egg, exhibit antigen-directed isotype restriction in the form of IgM and IgG3 antibodies.     

对硝基苯甲酰还原青蒿素对日本血吸虫童虫的作用

小鼠感染血吸虫尾蚴后第7d,1次灌服对硝基苯甲酰还原青蒿素300mg/kg。结果表明:给药后4h童虫皮层即有变化,8h已十分明显。主要表现为表皮水肿,皮层褶嵴融合,形成大量球状物或小泡。12h后皮层糜烂破溃剥落,并有白细胞附着于皮层损害处。从体表面积和体积的测量计算看,7～12d龄童虫生长基本停滞,而第13d又趋恢复。此结果为本药预防血吸虫病的最佳疗程的设计提供了科学依据。     

Antibody response against schistosomulum surface antigens and protective immunity following immunization with highly irradiated cercariae of Schistosoma mansoni

The production of antibodies against the schistosomulum surface antigens of Schistosoma mansoni in response to immunization with highly irradiated cercariae was followed. Four antigens were reproducibly identified by 125I surface labelling using Iodogen and immunoprecipitation; they had mol. wts of 38, 32, 20 and 15 kD. In addition a 92 kD antigen was also evident in most experiments. It was demonstrated that the 20 kD antigen was the same as that recognized by the monoclonal antibody NIMP/M.47 and that this antigen like the 38 and 32 kD antigens was thus identified during both chronic infection and following vaccination with irradiated cercariae. Two weeks following immunization with irradiated cercariae antibody was produced only against the 15 kD antigen but at 4 weeks the major response was against the 32 kD antigen. A second immunization with irradiated cercariae boosts the antibody response so that all four antigens were strongly precipitated. Further vaccinations did not lead to the identification of further antigens. Immunization of rats with highly irradiated cercariae also resulted in antibody production against the 38, 32, 20 and 15 kD antigens. Surface labelling of schistosomula transformed from irradiated cercariae resulted in the same four antigens being precipitated as from normal cercariae indicating that irradiation did not affect transformation nor antigen expression on 3h schistosomula. Furthermore, antibodies against the same surface antigens were detectable 4 weeks after immunization with equal numbers of cercariae irradiated with 0, 5, 25 or 50 krad. Vaccination of mice with irradiated, cloned cercariae resulted in identical antibody production and similar levels of immunity directed at both an homologous or heterogeneous challenge. Thus all parasites within our laboratory population appear to express the same antigens and there was no evidence for a genetically defined variation that could account for the partial resistance to reinfection exhibited by mice vaccinated with irradiated cercariae.     

Developmental expression of protein kinase C activity in Schistosoma mansoni.

The multicellular parasite Schistosoma mansoni undergoes complex physiologic changes during development from infective cercariae to adult worms in the mammalian host. The present study examined changes in protein kinase C (PKC) activity in S. mansoni during parasite maturation. Activation of PKC required Ca+2, phosphatidylserine, and either diacylglycerol or phorbol ester similar to mammalian PKC enzyme. A nine-fold increase in total PKC activity was found in adult worms as compared with larval parasites. Transformation of infective cercariae to parasitic schistosomula was associated with translocation of PKC activity from the cytosolic to membrane fraction. Tegumental extracts demonstrated significant PKC activity, suggesting a signal transduction system in the surface of the parasite. These data indicate that PKC activity is differentially expressed during parasite development and may have critical roles in regulation of cellular events in S. mansoni.     

Inhibition of human peripheral blood mononuclear cell proliferative responses by released materials from Schistosoma mansoni cercariae

During in vitro transformation of Schistosoma mansoni cercariae into schistosomula, surface and glandular materials are released into the culture medium. Extracts of these materials, termed cercarial released extracts 1 and 2 (CRE-1 and CRE-2), were analysed and found to consist primarily of protein and carbohydrate at ratios of 5:1 (CRE-1) and 7:1 (CRE-2). It was observed that inclusion of either CRE-1 or CRE-2 in cultures of human peripheral blood mononuclear cells (PBMN) led to decreased cell proliferation. This was true whether the cells were resting, control cultures or were stimulated with either phytohaemagglutinin or an antigenic preparation from adult S. mansoni worms. The inhibition was equally effective with PBMN of patients with active schistosomal infection or PBMN from uninfected individuals. Since these materials are released spontaneously during cercarial-to-schistosomular transformation they may have a putative immunosuppressive effect in decreasing antischistosomular activities early after cercarial penetration.     

Antibody isotype responses to the Schistosoma mansoni schistosomulum in the CBA/N mouse induced by different stages of the parasite life cycle

Summary During infection with Schistosoma mansoni the extent and nature of immune reactions against schistosomula may be influenced by responses to cross-reactive antigens in eggs or adult worms, and there is now extensive evidence for cross-reactivity between the different stages of the parasite life cycle. In this study IgM and IgG subclass antibodies produced in (CBA/N × Balb/c) Fl male and female mice were measured over a period of time following exposure to a chronic infection, to unisexual male cercariae or to irradiated larvae. Antibody levels were also measured following immunization with antigen preparations derived from adult worms, schistosomula or eggs. (CBA/N × Balb/c) Fl male mice exhibit an X-linked immune deficiency which results in an inability to respond to T-independent (TI) type 2 polysaccharides. Isotype levels were measured by ELISA to detergent-soluble schistosomulum antigen. Results showed that antigens on the different stages of the parasite life cycle have a qualitative influence on the antibody response to the larval surface, and that T-independent type 2 polysaccharides, particularly abundant in egg. exhibit antigen-directed isotype restriction in the form of IgM and IgG3 antibodies.     

环孢素A体外抗曼氏血吸虫雄虫的时间生物学研究

目的：探讨环孢素Ａ体外抗曼氏血吸虫的作用。方法：ＭＦ１小鼠实验感染曼氏血吸虫６ｗｋ后，经主动脉和门静脉灌注收虫。将雄虫放入含有１μｇ／ｍｌ、１０μｇ／ｍｌ、１５μｇ／ｍｌ、２０μｇ／ｍｌ和２５μｇ／ｍｌ环孢素Ａ的１９９培养液中体外培养。用扫描电镜对药物所致的虫体皮层损害作时间生物学观察。结果：在体外，雄虫经１μｇ／ｍｌ环孢素Ａ作用后，体嵴的结构疏松；经１０μｇ／ｍｌ环孢素Ａ作用２４ｈ后，雄虫皮层肿胀；雄虫经１５μｇ／ｍｌ环孢素Ａ作用８ｈ－２４ｈ后，虫体皮层破溃；虫体经２０μｇ／ｍｌ药物作用２４ｈ后，雄虫皮层明显破溃；雄虫经２５μｇ／ｍｌ环孢素Ａ作用８ｈ后，皮层褶嵴受损；作用１６ｈ后，皮层肿胀；作用２４ｈ后，皮层极度破溃，皮棘脱落。药物所致的虫体皮层损害与剂量和时间呈依赖关系。结论：环孢素Ａ具有直接杀曼氏血吸虫的作用。