Enhancement of eosinophil-mediated cytotoxicity to schistosomula of Schistosoma mansoni by autologous mononuclear cells from patients

Adherent mononuclear cells (monolayer), when co-cultured with autologous peripheral blood eosinophils isolated from patients treated for Schistosoma mansoni infections, enhanced the eosinophil-mediated killing of antibody coated schistosomula. The monolayer increased the activity of the eosinophils by 225%, and was observed in 80% of the patients studied. Heat labile factors other than complement, present in immune serum, further enhanced the ability of eosinophils to kill schistosomula in the presence of the monolayer. On their own the adherent cells did not mediate obvious damage to the parasite. Eosinophils that had been pre-incubated with the monolayer (100 mins) and tested separately, killed equal numbers of schistosomula as in the co-culture assay; this excludes the possibility of concurrent schistosomula cytotoxicity by the two cell populations. The ability of the monolayer to activate eosinophils was not altered by inhibitors of protein synthesis. The monolayer was largely consistent of monocytes as demonstrated by an over 96% positive staining for non-specific esterases.     

Factors affecting the levels of antibody- and complement-dependent eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro

In experiments designed to test why high levels of antibody-dependent, eosinophil-mediated killing of schistosomula are routinely observed in this laboratory, several factors that may contribute to variations in eosinophil activity were examined. The most important factors were: (1) the source of eosinophils, with marked variation being demonstrated not only, as previously shown, between individuals, but also between different cell preparations from a single individual; (2) the serum used as a source of anti-schistosomulum antibodies and (3) the age of the schistosomula at the time of assay. In contrast, addition of fresh normal serum as a source of complement had a relatively slight effect when the killing assay was carried out in round bottomed tubes. A more marked enhancement was observed in flat bottomed microtitre plates, and it is suggested that this enhancement may be attributable to the release of chemotactic complement components. No difference was observed between a laboratory maintained and a recently derived isolate of Schistosoma mansoni, either in initial susceptibility or in loss of susceptibility after 3.5 h of culture. In contrast to the marked effects of eosinophils under most conditions tested, there was no evidence for extensive neutrophil-mediated damage under the same conditions.     

Temporal variation in the carbohydrate and peptide surface epitopes in antibody-dependent, eosinophil-mediated killing of Schistosoma mansoni schistosomula

Changes in the surface antigenicity and susceptibility to eosinophil-dependent killing during in vitro development of schistosomula of Schistosoma mansoni, were examined using sera from rabbits and mice immunized with antigens that are shed from the schistosomulum in vitro (shed antigen), a carbohydrate extract of shed antigen (SAg/CHO) or a periodate-insensitive fraction of shed antigen (SAg/PEP). Anti-SAg/CHO antisera recognised mainly carbohydrate epitopes on the parasite surface, whilst anti-SAg/PEP antisera bound to periodate-insensitive, putative peptide, surface epitopes. Anti-SAg/PEP antibodies failed to recognise the surface of newly transformed schistosomula unless the parasite was first treated with sodium periodate, suggesting that these epitopes may be masked by periodate sensitive (i.e., carbohydrate) epitopes. There was an increase in anti-SAg/PEP antibody binding to the larval surface with age of the parasite in vitro; five-day-old lung schistosomula were also recognised by anti-SAg/PEP antisera. In contrast, anti-SAg/CHO antibody binding declined with parasite age, and failed totally to recognise lung schistosomula. This change in epitope expression was reflected in eosinophil-dependent cytotoxicity assays, with anti-SAg/CHO antisera killing young larvae and anti-SAg/PEP antisera only killing older larvae. Lung worms were not killed by either antisera. The difference in epitopes recognised by the antisera was also reflected in the antigens identified by immunoprecipitation and SDS-PAGE.     

Antibodies are involved in the protective immunity induced in mice by Schistosoma mansoni schistosomula tegument (Smteg) immunization

The Schistosoma mansoni schistosomula tegument (Smteg) plays an important role in triggering the host immune response and mice immunization with Smteg formulated with Freund's adjuvant or alum + CpG induce partial protection against S. mansoni infection associated with an increased antibody production. In this study, we investigated the role of these antibodies in parasite killing both in vitro and in vivo. We demonstrated that these antibodies were able to bind to the surface of S. mansoni recently transformed schistosomula and that these antibodies significantly increase the percentage of schistosomula killed in vitro by complement activation. Passive transference of immune sera decreased the parasite burden and the number of eggs trapped in the organs of mice that received sera containing anti‐Smteg antibodies. These results demonstrate that antibodies specific to surface tegumental antigens are involved in parasite elimination in mice immunized with Smteg.     

Morphological studies on the killing of schistosomula of Schistosoma mansoni by human eosinophil and neutrophil cationic proteins in vitro

Purified eosinophil and neutrophil cationic proteins isolated from the lysosomal secretion granules of human granulocytes, evoke characteristic, dose-dependent morphological changes in young schistosomula of S. mansoni. The first sign of damage is seen within 15–30 min of incubation and involves the formation of surface microvilli and blebs. Subsequently, tegumental evaginations of varying size are developed, but these appear to explode with rapidity, so that lengths of expanded tegumental outer membrane are deposited over the severely damaged surface of the parasite. Both types of granulocyte proteins are able to effect comparable damage at equimolar concentration. Other cationic proteins such as protamine and poly-L-arginine also damage the parasite surface but the pathological changes differ from those induced by the granulocyte proteins and they take longer to develop. In contrast, lysozyme-treated parasites are virtually similar to control schistosomula incubated in medium alone. These findings are discussed in relation to published data concerning the interaction of intact granulocytes with young schistosomula both in vitro and in viva.     

Murine Schistosomiasis mansoni: anti-schistosomula antibodies and the IgG subclasses involved in the complement- and eosinophilmediated killing of schistosomula in vitro

Summary Protein A-sepharose affinity chromatography was used to isolate IgG subclasses from the serum of CBA mice chronically infected with Schistosoma mansoni. The subclasses were tested for the presence of two antibodies which are responsible for the death of young schistosomula in vitro; 'lethal antibody' (LA), which kills schistosomula in co-operation with complement and 'eosinophil adherence antibody' (EAA) which causes the death of schistosomula by promoting the adherence of eosinophils to the parasite. LA and EAA were detected only in the IgG fraction of the serum. LA was concentrated in the IgG_2a fraction and EAA in the IgG₁ fraction. The development of IgG subclasses specific for schistosomula was followed in mice exposed to twenty cercariae by the fluorescent antibody technique. IgG₁, IgG_2a and IgG_2b antibodies were detected 2 weeks after infection and their titres rose steadily to reach high levels by weeks 12 or 14. IgM antibody was not detected until week 6 and IgA until week 10; both were present at lower concentrations than the IgG₁ antibodies.     

Neutrophil-mediated cytotoxicity to schistosomula of Schistosoma mansoni in vitro: studies on the kinetics of complement and/or antibody-dependent adherence and killing

The capacity of rat peritoneal neutrophils to adhere to and kill schistosomula of Schistosoma mansoni in vitro has been investigated. Neutrophils adhere readily to schistosomula in the presence of antibody plus complement (C) (fresh immune rat serum), antibody alone (heat-inactivated immune rat serum) and C alone (fresh normal rat serum), but not with heat-inactivated normal rat serum. However, schistosomular killing is only achieved with neutrophils and fIRS or MRS. In the presence of hiIRS the cells detach after 6 h without producing a significant level of parasite death. The system involving neutrophils plus fIRS is the most efficient in terms of serum dilution and the rate of schistosomular killing. The complement-dependent antibody involved in this system belongs to the class IgG and occurs in rat serum at peak titres, 6–8 wk after a primary schistosome infection. Neutrophil adherence in the presence of MRS depends upon the generation of C3b molecules at the parasite surface via the alternative pathway of C activation. Studies on the antibody alone system indicate that the lack of significant schistosomular killing might result from the absence of factors which stimulate cell migration, since if a chemokinetic agent is introduced into the assay a 30% increase in mortality is recorded. The possible participation of neutrophils in the destruction of a primary and/or challenge infection in vivo is discussed.     

Antigenicity and immunogenicity of the tegumental outer membrane of adult Schistosoma mansoni

The tegumental membranes of adult Schistosoma mansoni have been isolated and purified and shown to function as potent immunogens; they elicit an essentially identical immune response in rabbits, rats and mice. Anti-membrane antisera harvested from these animals consistently recognized common antigens, of relative molecular weight (mol. wt) 32 000 and 20 000, on the surface of young schistosomula, 5 day old lung worms and adult worm purified membranes. An additional molecule of 25 000 mol. wt was present on the surface of lung worms and adult worm membranes and was specifically recognised by serum from chronically infected mice and by serum from rabbits inoculated with adult worm purified membranes. The concept of antigenic identity between developmental stages that parasitize the mammalian host was further substantiated by the observation that anti-membrane antiserum bound to live schistosomula, lung worms and adult parasites as measured by indirect immunofluorescence. In complement-mediated in vitro cytotoxicity assays, the sera from rabbits inoculated with either adult worm purified membranes, or the 32 000 mol. wt antigen partially purified from adult worm membranes, mediated levels of schistosomula killing as high as those obtained with sera from chronically infected mice. These rabbit antisera also promoted eosinophil adherence and killing of newly transformed schistosomula, but lung stage parasites, despite binding the anti-membrane antiserum, were refractory to both humoral and cellular cytotoxicity. The significance of antigenic identity is discussed in relation to the concept of concomitant immunity.     

Cytotoxic effects in vitro of human monocytes and macrophages on schistosomula of Schistosoma mansoni

Human peripheral blood monocytes from normal donors were isolated by differential centrifugation and cultured in vitro in hydrophobic Teflon-coated tissue culture bags. Cells were harvested between 0 and 10 days and tested for their ability to kill schistosomula of Schistosoma mansoni in an in-vitro cytotoxicity assay. Freshly isolated, unstimulated monocytes demonstrated minimal cytotoxic capability. However, this was increased if the cells were pretreated with human recombinant gamma interferon (IFN-gamma), or with specific anti-S. mansoni antiserum. As the monocytes matured in vitro there were marked increases in the levels of antibody-independent killing of schistosomula. Monocytes grown in vitro with IFN-gamma (10(4) u/ml) took 2-3 days to develop almost maximal cytotoxicity (mean 94% kill of schistosomula). In contrast, unstimulated monocytes (no IFN-gamma) took between 5 and 7 days to achieve comparable cytotoxicity (mean 99% kill). Killing of the schistosomula was dependent upon a high effector to target ratio, and was a relatively slow phenomenon in vitro, parasite attrition occurring between 17 and 36 h. Supernatants from cytotoxic macrophages were ineffective in mediating cytotoxicity of the parasite.     

Evidence that the reduced surface antigenicity of developing Schistosoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition

Antibody and lectin binding characteristics of Schistosoma mansoni schistosomula maturing in vivo and in vitro were quantitatively assessed and compared in order to investigate the basis of the reduced surface antigenicity of host derived larval schistosomes. Quantitative indirect immunofluorescence assays showed that schistosomula recovered from mice at 24 h and 5-10 days post infection bound low or insignificant amounts of a variety of anti-schistosome antibodies including those from chronically infected and radiation attenuated cercariae-vaccinated mice, a vaccinated rabbit and rabbits hyper-immunized with non-living larval and adult schistosome antigen preparations. In contrast, parasites maturing in vitro continued to bind highly significant levels of each of these antibody preparations until at least 10 days post transformation. To investigate the basis of the decreased surface antigenicity of parasites maturing in vivo, 6-day-cultured parasites were injected intravenously into mice and recovered from the lungs at various times thereafter and examined for their ability to bind both anti-parasite and anti-host antibodies. After 30 min in vivo, cultured schistosomula exhibited a significantly decreased capacity to bind anti-parasite antibodies and concanavalin A (Con A), and by 16 h had lost their binding sites for fucose binding protein (FBP) as well. That this reduction in antigenicity was due to shedding of surface antigens was suggested by the observation that the reduced ability of these parasites to bind anti-parasite antibodies coincided closely with the loss of 125I-labelled surface proteins. Furthermore unlike 6 day schistosomula which had developed wholly in vivo, 6-day-cultured parasites recovered after 30 min in vivo failed to bind anti-host antibodies suggesting that in these organisms parasite antigens were not masked by host molecules. These data argue that surface antigen shedding may explain the reduced surface antigenicity of schistosomula developing in vivo. While this surface modulation apparently occurs independently of host antigen uptake, it is dependent upon an as yet unidentified host factor.     

The response of mice immune to Schistosoma mansoni to a challenge infection which bypasses the skin: evidence for two mechanisms of immunity

Mice which had developed immunity to reinfection with Schistosoma mansoni following exposure to 20 cercariae and mice which had been immunized against S. mansoni by exposure to 400 highly irradiated (20 krad) cercariae, were tested for their ability to resist a percutaneous cercarial challenge and an intravenous challenge with 5-day-old lung-stage schistosomula derived from the same cercariae. Although both types of immune mice showed a marked resistance to a cercarial challenge, only the infected mice showed a comparable immunity to an intravenous challenge with lung schistosomula. These results confirm earlier studies which suggest that the major attrition of a cercarial challenge in infected mice occurs at the post-lung stage, whilst the attrition of a challenge infection in mice immunized with highly irradiated cercariae takes place in the skin. They provide further evidence for two separate mechanisms of immunity against S. mansoni in mice.     

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.     

小鼠感染日本血吸虫后iNOS的表达及NO介导的杀虫作用研究

目的研究小鼠在感染日本血吸虫后肺部诱导型一氧化氮合酶(iNOS)的动态表达及一氧化氮(NO)对童虫的杀伤作用。方法在感染日本血吸虫后不同时间点剖杀小鼠并取其肺组织,用半定量RTPCR的方法检测肺部iNOS的转录水平;用NOS的抑制剂AG(Aminoguanidine,氨基胍)处理感染小鼠,比较处理组与对照组虫荷的高低以确定NO对童虫的杀伤作用。结果未感染小鼠肺部无iNOS转录表达,感染后第4d肺部iNOS有较高水平的转录表达,第9d表达消失;AG处理小鼠的虫荷较未处理组高(P<0.05)。结论移行到肺部的童虫能诱导肺组织表达iNOS,由iNOS产生的NO能对童虫发挥杀灭作用。     

Ultrastructural studies of the killing of schistosomula of Schistosoma mansoni by activated macrophages in vitro

Immunologically activated murine macrophages have been shown elsewhere to kill skin stage schistosomula of Schistosoma mansoni in vitro, in a manner analogous to the extracellular killing of tumour cell targets. In this study, the kinetics of the interaction between activated macrophages and larval targets and the resultant ultrastructural changes in parasite morphology that culminated in death have been analysed in detail. Unlike granulocyte-mediated schistosomular killing, macrophage-mediated cytotoxicity did not appear to be directed against the surface tissues of the parasite. Macrophages adhered only transiently following initiation of the cultures, yet changes in the subtegumental mitochondria and muscle cells of the larva were detected within the first hour of incubation. Progressive internal disorganisation followed rapidly, but the tegument and tegumental outer membrane remained intact, to form a 'shell' that maintained the general shape of the parasite. Such changes were recognised irrespective of whether the effector cell population comprised peritoneal macrophages activated by lymphokine treatment in vitro, or by infection with Mycobacterium bovis (strain BCG), or S. mansoni in vivo. That macrophages rather than contaminating granulocytes or lymphocytes, had mediated the observed damage was demonstrated by the use of a lymphokine treated macrophage cell line, IC-21. The observation that macrophage cytotoxicity is directed against internal organelles rather than the tegumental outer membrane of this multicellular target, may help to elucidate the general mechanism of extracellular killing by these cells.     

The use of mouse/human chimaeric antibodies to investigate the roles of different antibody isotypes, including lgA2, in the killing of Schistosoma mansoni schistosomula by eosinophils

We report the use of a matched set of mice/human chimaeric antibodies, directed against the 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP) hapten, to investigate the roles of different human isotypes in antibody-mediated eosinophil-dependent killing of schistosomula. The chimaeric antibodies consist of mouse VH, VL and CL regions with human γ1, γ2, γ3 (2 allotypes), y4, α2, or e CH regions and were used in in vitro assays with human eosinophils and NIP-coated S. mansoni schistosomula. Some anti-NIP isotypes mediated high levels of killing, which was specific for NIP-coated larvae, and we suggest that these antibodies will be a valuable tool for studies on the role of antibody isotypes in anti-schistosome immune effector mechanisms. In particular, this method directly demonstrated, for the first time, that IgA is highly effective in mediating the killing of metazoan parasites by human eosinophils.     

蒿甲醚对曼氏血吸虫的作用:剂量与效应的关系和虫的形态学和组织病理学的变化

目的　应用感染曼氏血吸虫 (利比里亚株 )的小鼠观察蒿甲醚单剂量与效应的关系,虫体肝移及蒿甲醚所引起的虫的形态学和组织病理学变化。　方法　感染21d童虫的小鼠一次口服蒿甲醚12.5mg/kg至600mg/kg不同剂量 ,治后28d剖检观察各组虫数。感染46d或70d成虫的小鼠一次口服蒿甲醚40 0mg/kg后8～14d ,观察虫体肝移及其形态和组织病理学变化。　结果　蒿甲醚对21d童虫的最低有效剂量为200mg/kg ,减虫率为 81%。用蒿甲醚治疗后8h成虫开始肝移,3～7d全部肝移,14d有31%的虫返回肠系膜静脉。成虫虫体萎缩,咽部扩大,肠管膨胀及其色素减少。雌虫局部体表受损,白细胞附着,卵巢及卵黄腺变性退化,以及雄虫睾丸萎缩等。在肝内的虫体被嗜酸粒细胞为主的炎细胞包围和浸润。　结论　蒿甲醚对小鼠曼氏血吸虫21d童虫的最低有效剂量为200mg/kg ,可引起曼氏血吸虫成虫萎缩、退化或死亡。在肝内受损的虫体主要是被嗜酸粒细胞包围和侵袭所致。     

Schistosomiasis from S. mansoni in mice: the relationship between acquired immunity and serum levels of lethal antibody

Acquired immunity to Schistosoma mansoni induced by a primary infection with cercariae of one or both sexes was assessed in mice by the recovery of parasites of a challenge infection from the lungs or the liver. In addition, the antibody-dependent complement-mediated cytotoxicity against schistosomula in vitro was titrated in sera obtained from both groups of animals. The degree of immunity as detected by the lung recovery technique in mice infected with a mixture of male and female cercariae was variable and lower than 50%. In contrast, no immunity was observed in the group of animals infected with 40 ‘single-sex’cercariae. The titre of lethal antibody in a pool of sera from these animals was about 10, whereas it was about 640 in a pool of sera from bisexually infected animals. Lethal antibody titres ranged from 20 to 120 for unisexual infection and 240 to 1920 for bisexually infected mice. In some cases a significant degree of immunity was detected by liver perfusion in mice with a primary unisexual infection. However, the lethal antibody titre of sera from mice with a 'single-sex’infection remained low, even when the immunity was apparent.     

Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine,Sm23, following microseeding or gene gun delivery

Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0.03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31-34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique.     

The epidemiology of a recent focus of mixed Schistosoma haematobium and Schistosoma mansoni infections around the 'Lac de Guiers' in the Senegal River Basin, Senegal

A village with mixed Schistosoma mansoni and S. haematobium infections (probably in a early endemic phase) was identified around the Lac de Guiers in the Senegal River Basin. In documenting the epidemiology of both schistosomes, we focused on prevalence and intensity of infection, transmission patterns and the impact of treatment. S. mansoni prevalences (near 100%) and egg counts (overall geometric mean eggs per gram of faeces (epg) of 589 were high in all age groups, with 35% of individuals excreting > 1000 epg, and showing a slow decline in egg output only after the age of 30 years. The overall prevalence (28%) and egg counts (2% > 50 eggs/10 ml) of S. haematobium were low, with mean counts of 6.3 eggs/10 ml. Maximal mean S. mansoni egg counts were found in 5-9 year-old boys and in 15-19 year-old girls; S. haematobium maximal counts in 1-4 year-old boys and in girls aged 5-9. Extremely high Biomphalaria pfeifferi infection ratios were recorded over the whole year. Following a single treatment, re-infection was rapid with prevalences and mean egg counts of both Schistosoma species reaching pretreatment levels within 7 months.     

基于重组酶介导核酸等温扩增反应的曼氏血吸虫 基因检测方法的建立

目的　建立一种可用于曼氏血吸虫特异性基因片段检测的重组酶介导的核酸等温扩增方法（Recombinase?aided amplification, RAA）。方法　以曼氏血吸虫121 bp高重复基因片段作为靶序列,根据RAA反应原理设计、合成引物及荧光探针,建立并优化荧光RAA法反应体系。分别以不同拷贝数的含121 bp基因片段的重组质粒及不同浓度曼氏血吸虫基因组DNA为模板进行荧光RAA法扩增,评价该方法的敏感性;分别以日本和埃及血吸虫虫卵、十二指肠钩虫虫卵、华支睾吸虫囊蚴基因组DNA为模板进行荧光RAA法检测,评价其特异性。结果　建立的荧光RAA法可在39 ℃、20 min内特异性扩增曼氏血吸虫基因组DNA。以重组质粒为模板,荧光RAA法最低可检出的质粒拷贝数为10拷贝/μL;以基因组DNA为模板,荧光RAA法最低可检测浓度为0.1 fg/μL。以日本血吸虫虫卵、埃及血吸虫虫卵、十二指肠钩虫虫卵、华支睾吸虫囊蚴基因组DNA为模板进行荧光RAA法检测,结果均为阴性。结论　成功建立了一种可用于曼氏血吸虫DNA检测的荧光RAA法,该方法反应快捷、操作简便,敏感性和特异性均较好。