探究获得高成熟率伯氏疟原虫裂殖体的体外培养条件?

为获得大量有活力的伯氏疟原虫裂殖体,本研究对伯氏疟原虫ANKA虫株的体外培养条件主要从培养基的用量、培养基胎牛血清的含量、培养的细胞浓度、培养时间及培养的气体环境等方面进行了优化。当小鼠体内虫血率达到1％～3％,在红内期疟原虫处于环期或早期滋养体阶段时取血分组培养,观察在不同培养条件下裂殖体的状态并检测其活力。与既往的体外培养方法相比,优化后的培养方法,可将裂殖体的成熟率提高到80％,每个裂殖体含12～16个裂殖子,裂殖体尾静脉注射重新入侵红细胞4 h后的虫血率为1?57％,与对照组相比裂殖体的活力提高3?4倍。优化的方法可以提高裂殖体的得量和活力,为伯氏疟原虫的转染奠定基础。     

Plasmodium berghei: susceptibility and growth characteristics of hepatoma cells as hosts for malaria schizonts.

Three different human hepatoma cell lines (Hep G2, London; Hep G2, Brussels; Mahlavu) have been compared with respect to susceptibility for in vitro infection with Plasmodium berghei sporozoites and subsequent development of mature schizonts and infectious merozoites. The results were very different, even with host cells derived from the same parent line. Both Hep G2 lines were able to function as host cells, but the Mahlavu line was completely refractory. Hep G2 (London) was even more susceptible than the Hep G2-A16 clone. Hepatoma cells harbouring young exoerythrocytic forms permitted their development to mature schizonts and production of merozoites inside the intraperitoneal cavity of OF1 mice. Pronounced differences in growth parameters of subpopulations of Hep G2 cells were stated as well. Optimal conditions for in vitro culture of the most suitable cell line were identified. Finally, the seeding, plating and cloning efficiencies were determined.     

Purification of a Plasmodium berghei neutral endopeptidase and its localization in merozoite

A Plasmodium berghei neutral endopeptidase specific for the fluorogenic substrates valyl-leucyl-glycyl-arginyl/lysyl-aminoethyl-carbazole was purified by Fast Protein Liquid Chromatography. The enzyme was a Mr 68,000 polypeptide. Immunization of mice with the purified enzyme gave a specific antiserum, as demonstrated by immunoblotting. Immunofluorescence with this antiserum showed a strong labelling of P. berghei merozoites in mature segmented schizonts and of merozoites released from schizont-infected red blood cell. This labelling was mainly associated with the merozoite apex. It is possible that this endopeptidase is involved in the reinvasion.     

Role of host cellular response in differential susceptibility of nonimmunized BALB/c mice to Plasmodium berghei and Plasmodium yoelii sporozoites.

We found BALB/c mice to be on the order of 2,000 times more susceptible to Plasmodium yoelii than Plasmodium berghei sporozoites, as measured by the ability of these sporozoites to differentiate into microscopically detectable hepatic schizonts in the livers of immunologically naive mice. One of the factors that determine the relative insusceptibility of mice to P. berghei sporozoites is the innate cellular inflammatory response that the mice mount after injection with sporozoites. The cellular inflammatory response against P. berghei is initiated soon after sporozoite injection; by 24 h, substantial histopathological changes have developed within the liver. There is considerably less of a cellular inflammatory response against P. yoelii; significant histopathological changes within the liver are not observed until well after hepatic schizonts have begun to rupture at around 44 h postinjection of sporozoites. These differences in the cellular inflammatory response against two different, closely related species of sporozoites are of considerable interest. The data strongly suggest that the BALB/c-P. berghei sporozoite system is a relatively poor biological model for sporozoite immunization studies.     

Antibodies Reactive with the N-Terminal Domain of Plasmodium falciparum Serine Repeat Antigen Inhibit Cell Proliferation by Agglutinating Merozoites and Schizonts

The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum. Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.     

Differential sensitivity in vivo of lethal and nonlethal malarial parasites to endotoxin-induced serum factor.

Sera from mice infected with Plasmodium yoelii or Plasmodium berghei and given endotoxin contained nonspecific mediators which killed both species of parasite and tumor cells in vitro. The sera resembled tumor necrosis sera obtained from mice given macrophage-activating agents such as Propionibacterium acnes (formerly designated Corynebacterium acnes) or Mycobacterium bovis BCG and then endotoxin. Cytotoxicity developed parallel to parasite killing activity and indicated that macrophages were activated. Activation occurred sooner with P. berghei, which is lethal, and serum activity remained on a plateau until the mice died. In nonlethal P. yoelii infections, activation was related to the course of parasitemia. Endotoxin given to mice infected with P. yoelii caused an immediate decrease in parasitemia, presumably through the release of parasite killing factors. The extent of the decrease depended upon the time of administration. No immediate drop in the parasitemia caused by P. berghei was observed at any time. Early administration of endotoxin prolonged survival; late administration accelerated death. Passive transfer of rabbit tumor necrosis serum to infected mice decreased the parasitemia caused by P. yoelii but not that caused by P. berghei. Other components of the immune response appeared to act together with these soluble mediators to eliminate P. yoelii; they may be absent or suppressed in infections with P. berghei.     

Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide.

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.     

Isolation and characterization of a soluble antigen complex of Plasmodium falciparum with pyrogenic properties

A soluble antigen complex, previously designated antigen no. 7 (Ag7) on the basis of the pattern obtained by crossed immunoelectrophoresis of culture supernatants of P. falciparum, was isolated by affinity chromatography. It was shown to be synthesized at the schizont stage of the parasite growth cycle and to be located on the surface of the schizonts. Antibodies to Ag7 did not inhibit the growth of the parasite in vitro. Ag7 is recognized by immune human sera from many parts of the world and it stimulated the production of specific antibody in mice when incorporated into immune-stimulating complex (ISCOM) structures. It also specifically stimulated in vitro proliferation of lymphocytes from clinically immune adults. That it induced the secretion of interleukin 1 by human monocytes and was pyrogenic in rabbits was of particular interest. Thus Ag7 has endotoxin-like properties which make it a possible candidate for an antitoxic malaria vaccine.     

Cyclical transmission of Plasmodium berghei (Coccidiida: plasmodiidae) by Anopheles omorii (Diptera: Culicidae).

Anopheles omorii, a tree-hole breeding anopheline collected in Japan, transmitted a rodent malaria, Plasmodium berghei ANKA strain, in the laboratory. Mice with 0.2-15% parasitemia, 0.01-1.5% gametocytemia, and 0.001-0.5% exflagellations were used as infective hosts. Oocyst numbers ranged from 2 to 171 (mean 44) on the midgut 8-16 d after the blood meal. Several to hundreds of sporozoites were detected in the salivary glands 14 d after feeding. The mosquitoes were infective to mice from 13 to 40 d after feeding. An. omorii occurs naturally at temperatures less than 25 degrees C and therefore is a suitable laboratory vector for the cyclic transmission of P. berghei, a malaria parasite that completes sporogony at 18-21 degrees C.     

Effects of Acquired Resistance on Infection with Eimeria falciformis var. pragensis in Mice

Mice immunized with infections of 500, 5,000, or 20,000 oocysts of E. falciformis var. pragensis were reinfected with 20,000 and 100,000 oocysts at 20 and 38 days, respectively, after the initial infection. After the first challenge infection, none of the immunized mice showed clinical signs of coccidiosis; a few mice passed very low numbers of oocysts, and oocyst discharge seemed to correlate negatively with immunizing dose. None of the mice immunized twice passed oocysts after challenge. Mice immunized with three infections were completely immune to challenge for 4 months. The effect of the immune response on the life cycle of the coccidium was determined by histological examination of the intestines of immune and nonimmune mice infected with the parasite. In both the immune and nonimmune groups, sporozoites penetrated absorptive epithelial cells and migrated to crypt epithelial cells during the first 6 to 24 h postinfection. At 48 to 72 h postinfection, the sporozoites developed into mature first-generation schizonts in the nonimmune mice, whereas the developing first-generation schizonts degenerated within the crypt epithelial cells of the immune mice. In nonimmune mice, third-generation merozoites, inoculated intracecally, developed into mature fourth-generation schizonts, whereas in immune mice the developing fourth-generation schizonts degenerated before maturing. The possibility that a cellmediated immune mechanism is responsible for the arrest in schizogony is discussed.     

PTRAMP; a conserved Plasmodium thrombospondin-related apical merozoite protein

A gene encoding a 352 amino acid protein with a putative signal sequence, transmembrane domain and thrombospondin structural homology repeat was identified in the genome of the human malaria parasite, Plasmodium falciparum and the rodent malaria parasite, Plasmodium berghei. The protein localises in the apical organelles of P. falciparum and P. berghei merozoites within intraerythrocytic schizonts and has, therefore, been termed the Plasmodium thrombospondin-related apical merozoite protein (PTRAMP). PTRAMP co-localises with the Apical Merozoite Antigen-1 (AMA-1) in developing micronemes and subsequently relocates onto the merozoite surface. Although the gene appears to be specific to the Plasmodium genus, orthologues are present in the genomes of all malaria parasite species examined suggesting a conserved function in host-cell invasion. PTRAMP, therefore, has all the features to merit further evaluation as a malaria vaccine candidate.     

Induced immunity against the mosquito Anopheles stephensi (Diptera: Culicidae): effects of cell fraction antigens on survival, fecundity, and plasmodium berghei (Eucoccidiida: Plasmodiidae) transmission

Two subeellular fractions from the midgut of the malaria mosquito Anopheles stephensi (Liston) were used to immunize BALB/c mice. Mice were subsequently infected with the rodent malaria parasite Plasmodium berghei (Vineke & Lips), and the effects of anti-mosquito immunity on mosquito survival and fecundity and on parasite transmission were investigated. Mosquitoes were infected directly from mice (in vivo) or by feeding cultured ookinetes through a membrane (in vitro). Infections were monitored by counting oocysts on the midgut wall. Microvilli extracts induced a strong and partially specific antibody reaction against the midgut, which was manifest as decreased survival in in vivo fed mosquitoes and reduced fecundity in both kinds of feeding. Antisera against microvilli reduced the mean intensity of P. berghei oocysts when fed in vitro, while mosquitoes fed antiserum against basolateral plasma membranes in vivo, showed higher oocyst burdens.     

Effects of interruption of apicoplast function on malaria infection, development, and transmission

A chloroplast-like organelle is present in many species of the Apicomplexa phylum. We have previously demonstrated that the plastid organelle of Plasmodium faciparum is essential to the survival of the blood-stage malaria parasite in culture. One known function of the plastid organelle in another Apicomplexan, Toxoplasma gondii, involves the formation of the parasitophorous vacuole. The effects of interruption of plastid function on sporozoites and sexual-stage parasites have not been investigated. In our previous studies of the effects of thiostrepton, a polypeptide antibiotic from streptococcus spp., on erythrocytic schizongony of the human malaria P. falciparium, we found that this antibiotic appears to interact with the guanosine triphosphatase (GTPase) binding domain of the organellar large subunit ribosomal RNA, as it does in bacteria. We investigate here the effects of this drug on life-cycle stages of the malaria parasite in vivo. Preincubation of mature infective sporozoites with thiostrepton has no observable effect on their infectivity. Sporozoite infection both by mosquito bite and sporozoite injection was prevented by pretreatment of mice with thiostrepton. Thiostrepton eliminates infection with erythrocytic forms of Plasmodium berghei in mice. Clearance of infected red blood cells follows the delayed kinetics associated with drugs that interact with the apicoplast. Thiostrepton treatment of infected mice reduces transmission of parasites by more than ten-fold, indicating that the plastid has a role in sexual development of the parasite. These results indicate that the plastid function is accessible to drug action in vivo and important to the development of both sexual and asexual forms of the parasite.     

γδ+ T Cells Preferentially Respond to Live Rather than Killed Malaria Parasites

We have compared the in vitro responses of peripheral blood T cells from malaria-unexposed donors to live Plasmodium falciparum schizonts, freeze-thawed schizont extracts (P. falciparum schizont extracts [PfSE]), and parasite culture supernatants. We show that the cells responding to PfSE and parasite culture supernatants are predominantly CD4⁺ TCRαβ⁺ while in the presence of live schizonts there is an additional activation of TCRγδ⁺ cells. Activation of TCRγδ⁺ cells in response to PfSE was seen only when irradiated autologous feeder cells or recombinant interleukin-2 (IL-2) was added to the cultures. Live schizonts but not PfSE induced significant IL-2 production in vitro in the first 5 days after stimulation, suggesting that induction of early IL-2 by live parasites may contribute to the marked activation of the TCRγδ⁺ population.     

A simple and efficient method for purifying and quantifying schizonts fromTheileria parva-infected cells

An improved method for the purification ofTheileria parva schizonts from infected bovine cells is described. The technique is simpler and more rapid than previously described methods and gives rise to greater yields of schizonts with negligible contamination by host-cell components. In addition, a fluorescent staining technique was developed whereby live schizonts purified from infected cells can be enumerated and sorted using the flow cytometer. An assessment of the quality of schizonts prepared according to our method as a source of RNA for the construction of parasite cDNA libraries suggests that RNA derived from these preparations is free of host nucleic acids.     

Identification of a Plasmodium berghei antigen sharing common features with P. falciparum and P. chabaudi parasitophorous-vacuole membrane antigens

 On the basis of immunological cross-reactivity, we identified a 43-kDa Plasmodium berghei antigen with homology to the exp-1 antigen from P. falciparium. The P. berghei antigen was recognized by an antibody directed against an epitope on the C-terminus of the P. falciparum exp-1 protein. This antigen is localized on the surface of the parasite and shares peptide sequence homology with the P. chabudi antigen Ag3008. To investigate further the role of the P. berghei antigen, we designed antisense phosphorothioate oligodeoxynucleotides (PS oligos) complementary to sequences of the exp-1 mRNA from P. falciparum. The PS oligos were capable of inhibiting the development of P. falciparum in vitro by 47%. In vivo, experiments in mice showed that the same PS oligos had the potential to extend the life span of mice infected with P. berghei by a factor of 2–4. The immunological cross-reactivity and the antisense inhibition of P. berghei parasite development in vivo indicate that this antigen may be a homologue of exp-1 from P. falciparum that has functional importance for parasite survival. Received: 3 April 1995 / Accepted: 16 June 1995     

Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo.

The effect of induction of an acute-phase response and its mediators on the development of liver schizonts of the rodent malaria parasite Plasmodium berghei was investigated in Brown Norway rats. Subcutaneous injection of turpentine oil 24 h or 5 min before inoculation of sporozoites resulted in 80% and 35% reduction of schizont development, respectively. Turpentine oil induced high plasma levels of interleukin-6 (IL-6). Intraperitoneal administration of IL-1, IL-6 or both, significantly reduced liver schizont development. This reduction was also present if IL-6 had been administered 24 h after sporozoite inoculation. Inhibition induced by IL-1 could be prevented by simultaneous administration of polyclonal anti-IL-6. Administration of polyclonal anti-IL-6 without IL-1 resulted in a 40% increase of liver schizonts compared to control animals. We conclude that induction of an acute-phase response during experimental Plasmodium berghei infections in Brown Norway rats, strongly inhibits liver schizont development and that IL-6 is a key mediator in this process.     

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.     

Crossreactivity with sporozoites, exoerythrocytic forms and blood schizonts of Plasmodium berghei in indirect fluorescent antibody tests with sera of rats immunized with sporozoites or infected blood.

IFA studies are reported using plasmodial antigens from three different stages of the life cycle of Plasmodium berghei: sporozoites (SP); exoerythrocytic schizonts in rat liver (EEF); and parasitized rat erythrocytes (SCH = schizonts). Two series of specific sera were applied: sera from adult rats with a blood-induced infection (series A) and sera from rats immunized against sporozoites by mosquito bites and protected against parasitaemia by chloroquine (series B). In series A antibody titres with all three antigens were seen, but those with SCH were generally the highest. Superinfection with parasitized rat blood did not change the titre. In series B sera, collected from rats after a single exposure to infected mosquitoes, showed only titres with SP from day 3 onwards, but after a second exposure titres to all three antigens developed. Crossreactivity with the heterologous antigens in series B was clearly less than in series A. Anti-P. berghei sporozoite antibodies did not crossreact with P. vivax sporozoites. Rats of series A were resistant to a challenge of parasitized blood and could also inhibit the development of sporozoites. Rats of series B were protected against a challenge of sporozoites but not of infected blood. The results are discussed.     

Gene targeting in the rodent malaria parasite Plasmodium yoelii

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) fused to green fluorescent protein (gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.