Evidence for superantigenic activity during murine malaria infection

TCR V beta usage was examined in C57BL/6 mice infected with Plasmodium yoelii. In addition to a polyclonal T cell activation, already described, a superantigenic-like activity was observed during the acute infection. This superantigenic activity induces a preferential deletion without prior expansion of CD4+ and CD8+ T cells bearing the TCR V beta 9 segment. The superantigen could be released by the parasite at different stages of its development since the deletion of V beta 9+ T cells was observed in blood and lymph nodes of mice infected either with sporozoites or with erythrocytic stages. Injection of sporozoite or parasitized erythrocytes to newborn mice led to a deletion and anergy of peripheral V beta 9+ T cells, without affecting thymic T cell populations. These observations suggest that the superantigen is released at very low concentrations during parasite development. The role of such parasite superantigenic activity in infectivity can be underlined by the observation that congenic BALB.D2 Mis1a mice lacking V beta 9 T cells are more susceptible to infection by P. yoelii.      

Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria.

This study was undertaken to explore early differences in cytokine production during nonlethal and lethal blood-stage murine malaria infections. Cytokine analysis of spleens during these infections showed that the principal difference between two nonlethal and two lethal Plasmodium species was the production of gamma interferon 24 h after infection with nonlethal parasites. In contrast, no increases in interleukin-4 production were observed in the first 24 h and tumor necrosis factor alpha levels increased equally in both nonlethal and lethal infections. During the later phase of infection with nonlethal parasites, both gamma interferon and interleukin-4 levels increased markedly a few days before parasite clearance. Early increases in gamma interferon production in nonlethal infections of Plasmodium yoelii and Plasmodium chabaudi were dose related and increased significantly with the size of the inoculum. Studies with the nonlethal P. yoelii suggest that the early gamma interferon response is mediated by T cells and natural killer cells, as it was reduced in athymic mice and in mice depleted of their natural killer cells by treatment with specific antiserum. Infecting mice with increasing numbers of lethal P. yoelii and Plasmodium berghei parasites did not increase the amount of gamma interferon, interleukin-4, and tumor necrosis factor alpha produced in a dose-dependent fashion. We conclude that one consequence of the early production of gamma interferon and tumor necrosis factor-alpha, particularly after nonlethal P. yoelii infection, may be to adjust the balance of T-helper cell subset activation, and probably that of other immune responses, so as to enhance the mechanisms that are essential for elimination of the parasites. This suggests that a successful vaccine should contain antigens capable of inducing such responses.     

Roles of CD4- and CD8-bearing T lymphocytes in the immune response to the erythrocytic stages of Plasmodium chabaudi.

The possible role of CD4- and CD8-bearing T lymphocytes in parasite clearance in vivo was investigated, using Plasmodium chabaudi in C57BL/6 mice as a model. Monoclonal antibodies specific for the CD4 and CD8 molecules were administered in vivo to deplete selectively the appropriate subset of T cells. The efficacy of depletion was ascertained by flow cytometry and functional studies. These mice were then infected with P. chabaudi, and the course of infection was followed. The control groups had maximum parasitemias of approximately 30% 10 days after infection, and the infection was cleared within 27 days. Mice without CD4+ cells had significantly higher parasitemias which they were unable to reduce below 20% for the duration of the experiment. Mice without CD8 cells had slightly higher parasitemias which were cleared after 34 days. Because of the possibility that CD8+ cells alone could not be activated in the absence of growth factors, exogenous interleukin-2 was administered to the mice depleted of CD4 cells. This did not significantly affect parasitemias, and the mice were still unable to clear their infections. The data suggest that CD4+ T cells play a crucial role in the protective immune response to the erythrocytic stages of P. chabaudi.     

Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii

The rodent malaria Plasmodium yoelii is a useful model to study protective immunity to pre-erythrocytic stages of infection, pathogenesis of erythrocytic stages, and vaccine development. However, the utility of the P. yoelii model system has not been fully realized because transfection and genetic manipulation methodologies for this rodent species are less developed than that of another rodent species Plasmodium berghei. Here we report improved transfection efficiency using the AMAXA nucleofector system compared to conventional transfection methodologies. We also show that heterologous promoters from P. berghei can be used to drive expression of a green fluorescent protein (GFP) reporter protein in P. yoelii. In an effort to develop additional selectable markers for this parasite, we also tested positive selectable markers that have been used successfully in P. falciparum and P. berghei. Human dihydrofolate reductase (hdhfr) and Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr-ts) conferred drug resistance to WR99210 and pyrimethamine, respectively, when introduced as episomes. These improvements should make genetic manipulation of P. yoelii more amenable and facilitate further studies of host-parasite interactions using this attractive rodent model.     

Anti-lymphocyte antibodies in lethal mouse malaria. II. Induction of an autoantibody specific suppressor T cell by non-lethal P. yoelii

The anti-lymphocyte autoantibody response to irradiated lethal Plasmodium berghei malaria parasites in normal mice was significantly reduced when recipients were pre-treated with splenic T cells from mice recovered from a non-lethal Plasmodium yoelii infection. Suppression was specific for the autoantibody and did not affect the antibody response to the parasite. Experiments involving sequential P. yoelii-P. berghei infections in situ revealed that recovery from P. berghei was possible when the interval between the two infections was 14 days or more. This ability to recover from P. berghei correlated with a progressive reduction of anti-lymphocyte autoantibody suggesting a useful role for the suppressor cell. The possible link between suppressor cells and anti-lymphocyte autoantibodies in malaria is discussed.     

Glucan-enhanced immunogenicity of killed erythrocyte stages of Plasmodium berghei.

Intravenous injections of glucan simultaneously with Formalin-killed erythrocytic stages of Plasmodium berghei elicited a greater degree of resistance in mice against subsequent infection with viable parasites than injections of killed erythrocytic stages alone. In two experiments with P. berghei strain NK 65, 100% of mice immunized with the glucan-dead parasite preparation survived challenge, whereas only 28.6% of mice receiving dead parasites alone survived. In the third experiment, using P. berghei strain NYU-2, the same proportion of mice survived after immunization with glucan and dead parasites as with dead parasites alone (i.e., 10 of 11 in each group), but mice immunized with the glucan-dead parasite preparation experienced parasitemias of significantly less intensity and shorter duration than mice which received only dead parasites before infection. Inoculation of glucan alone or with normal erythrocytes conferred no protection against challenge.     

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.     

Phagocyte-derived reactive oxygen species do not influence the progression of murine blood-stage malaria infections

Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91(phox-/-) mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoelii and Plasmodium chabaudi K562) or fatal (Plasmodium berghei ANKA, Plasmodium berghei K173 and Plasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections with P. yoelii and P. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91(phox-/-) or wild-type strains of mice infected with malaria. Progression of P. berghei ANKA and P. berghei K173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91(phox-/-) mice and wild-type mice were not significantly different when they were treated with l-N(G)-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.     

A comparison of the stage-specific efficacy of chloroquine, artemether and dioncophylline B against the rodent malaria parasite Plasmodium chabaudi chabaudi in vivo

Chloroquine, artemether and dioncophylline B efficacy against Plasmodium chabaudi was compared. One intraperitoneal injection (10 mg/kg body weight) was given daily over 3 consecutive days to OF1 mice when they were predominantly bearing ring, trophozoite and schizont forms. The parasitaemia was monitored every 2 h during two schizogonic cycles and daily thereafter until parasites were cleared. Chloroquine was more efficient at the trophozoite stage, while artemether was effective against all erythrocytic stages, with a marked efficacy against the trophozoite stage. Chloroquine-treated and artemether-treated parasites displayed a pigment-clumping morphology and lowered the parasitaemia faster than dioncophylline B. Dioncophylline B was effective at trophozoite and schizont stages, but completely ineffective at the ring stage. These results demonstrate that a better timing of drug administration increases the efficacy of common and new antimalarial drugs and provides a model for antimalarial-action monitoring. Drug-induced changes in infected erythrocyte morphology are presented.     

Parasite-specific immunoglobulin isotypes during lethal and non-lethal murine malaria infections

Production of parasite-specific antibodies is an important component of immunity to blood stage malaria infection, as shown by several previous studies in rodent models. However, no study has addressed the induction of humoral immunity by different parasites in a genetically homogeneous host population. Here, levels of parasite-specific immunoglobulin isotypes were measured during primary infections of Plasmodium chabaudi and of Plasmodium yoelii in inbred NIH mice inoculated with cloned lines of either avirulent or virulent erythrocytic parasites. Non-lethal infections were characterized by early and late significant upregulation of IgG2a and IgG1, respectively. In contrast, for lethal infections, a slower, reduced IgG2a response correlated with a rapidly fatal outcome prior to any significant synthesis of IgG1. It is proposed that the sequential upregulated synthesis of parasite-specific IgG2a (cytophilic) and IgG1 (non-cytophilic) is associated with protective immunity to blood stage malaria infections in mice. This may provide an immunological framework for examining humoral immunity to malaria in humans.     

Host defenses in murine malaria: analysis of plasmodial infection-caused defects in macrophage microbicidal capacities.

Macrophage-dependent killing of facultative intracellular bacteria was markedly impaired by overt erythrocytic Plasmodium yoelii or Plasmodium berghei infection of mice. P. yoelii infection was capable of ablating not only the macrophage microbicidal capacity of "normal" animals but also the bactericidal capacities of "activated" macrophages. The uptake by spleen and liver of an intravenous challenge of Listeria monocytogenes was not altered by plasmodial infection, but within hours of injection markedly enhanced bacterial growth was found in tissues of malarious mice. The evidence gives credence to the view that the uptake of bacteria by macrophages of malarious mice was normal but that malarious mice, unlike normal mice, were unable to kill the bacteria. The plasmodial infection-caused defect in macrophage microbicidal capacity could be partially mimicked by the intravenous injection of large numbers of nonreplicating heterologous particles (i.e., killed bacteria, sheep erythrocytes). This result suggests that the uptake of particles generated during overt erythrocytic malaria may be responsible for the malaria-associated defects in macrophage bactericidal capacity.     

Vitamin E-deficient diets enriched with fish oil suppress lethal Plasmodium yoelii infections in athymic and scid/bg mice.

Mice fed vitamin E-deficient diets containing omega-3 fatty acids survive infection with lethal Plasmodium yoelii. The current study sought to determine if antimalarial T- and B-cell responses were required for such dietary-mediated protection. In the first set of experiments, nu/nu mice (which lack alphabeta T-cell-receptor-positive T cells and do not produce antimalarial antibody) and nu/+ mice were fed casein-based diets containing 4% menhaden oil, with or without vitamin E supplementation, for 4 weeks prior to infection with lethal P. yoelii. All mice fed diets containing vitamin E developed fulminating parasitemias and quickly died, whereas both nu/nu and nu/+ mice fed diets deficient in vitamin E controlled their parasitemias for the first 18 days of infection. Thereafter, the nu/nu mice became anemic and died, whereas the nu/+ mice produced antimalarial antibodies and survived. In the second set of experiments, scid/scid.bg/bg mice (which lack B cells and alphabeta and gammadelta T cells and have reduced NK-cell activity) were fed the experimental diet for 6 weeks and then infected with the less virulent 17XNL strain of P. yoelii. Mice fed vitamin E-containing diets quickly died, whereas those fed the vitamin E-deficient diet survived without developing detectable parasitemias. Results from these experiments show that under prooxidant dietary conditions, mice were able to control and even survive malaria in the absence of malaria-primed T cells and antimalarial antibody. These results emphasize the importance of cellular oxidative processes in parasite elimination.     

Cell-mediated immunity in mice vaccinated against malaria.

Mice vaccinated with a formalin-fixed preparation of either Plasmodium berghei or P. yoelli exhibited delayed type hypersensitivity (DTH) to the homologous antigen. This manifested itself in increased delayed thickening of antigen-challenged pinnae of the vaccinated mice as compared to the non-vaccinated controls. DTH was also evident in the vaccinated mice using the homing of radio-labelled bone marrow cells (BMC) to the delayed lesion as a criterion of reactivity. When P. yoelii vaccinated mice were given a live infection P. yoelii, a marked migration of BMC into the spleen occurred, with a peak at 48 hr, and it is suggested that this was a systemic response of DTH. The splenic T-cells of P. yoelii-vaccinated animals transformed in vitro with a soluble extract of the homologous parasite. The potential function of cell-mediated mechanisms in immunity to malarial infections is 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.     

Antimalarial efficacy,cytotoxicity, and genotoxicity of methanolic stem bark extract from Hintonia latiflora in a Plasmodium yoelii yoelii lethal murine malaria model

Traditional medicines have been used to treat malaria for thousands of years and are the source of artemisinin and quinine derivatives. With the increasing levels of drug resistance, the high cost of artemisisnin-based combination therapies, and fake antimalarials drugs, traditional medicine have become an important and sustainable source of malaria treatment. For the benefit of those who use traditional medicine to treat malaria, there is an urgent need to study the efficacy and toxicity of herbal remedies. Hintonia latiflora stem bark infusions are use in Mexican traditional medicine to treat malaria, diabetes, and gastrointestinal diseases. Its efficacy in the treatment of complicated malaria and its ability to generate DNA damage to the host is not fully evaluated. In our search for antimalarial natural products, in the present study, we tested the efficacy of H. latiflora stem bark methanolic extract (HlMeOHe) in CD1 male mice infected with lethal Plasmodium yoelii yoelii and its in vivo cytotoxicity and genotoxicity. To assess the antimalarial activity, the extract was evaluated in a 4-day test scheme in oral doses of 1,200, 600, and 300 mg/kg prior acute toxicity test; oral chloroquine (15 mg/kg) was used as positive control. The ability of 1,200 mg/kg of HlMeOHe to induce cytotoxicity and DNA damage in the peripheral blood of mice was assessed using a fluorochrome-mediated viability test and the micronucleus (MN) assay; N-ethyl-N-nitrosourea (ENU) was used as a positive control. HlMeOHe median acute toxicity (LD₅₀) was 2,783.71 mg/kg and LD₁₀ was 1,293.76 mg/kg (taken as the highest work dose). Plasmodium yoelii yoelii-infected mice in the untreated control group died between 6 and 7 days post-infection (PI) with parasitemia over 70 %. Even though mice treated with 600 and 300 mg/kg showed a chemosuppression percentage of total parasitemia of 99.23 and 23.66, respectively, animals in both groups died 6 to 7 days PI with parasitemia over 45 %. A 4-day dosage of 1,200 mg/kg of the extract showed, in the P. yoelii yoelii-infected mice, a 100 % chemosuppression of total parasitemia on 5 days PI and a 23 days survival time with a mean parasitemia of 23.6 % at the date of death. Only mice treated with chloroquine survived until the end of the experiment. Cell viability was not affected. The average number of micronuclei in the treated mice increased significantly (P?<?0.05) to 4.8 MN when compared with the untreated control group (0.9 MN). The results obtained in this study showed that the infection outcome of P. yoelii yoelii-infected mice is affected by HlMeOHe. Although a concentration of 1,200 mg/kg of HlMeOHe is suitable to use in the treatment of malaria fever, slowed down the parasite replication, retarded the patency time, and increased the infected P. yoelii yoelii mice survival time, its chemical composition should be studied in detail in order to reduce its genotoxic potential.     

Rapid parasite multiplication rate, rather than immunosuppression, causes the death of mice infected with virulent Plasmodium yoelii.

Protective immunity against a lethal malaria challenge infection was passively transferred to naive recipient mice with spleen cells from donor mice bearing a lethal infection with the virulent YM strain of Plasmodium yoelii. Successful transfer of protection was contingent upon the elimination of residual, viable parasites from donor spleen cell suspensions prior to the infusion of cells. Passive transfer experiments failed to detect suppressor cells in the spleens of lethally infected mice because unfractionated spleen cells or T-cell-enriched spleen cells from mice infected with P. yoelii YM did not enhance parasitemias upon infusion into mice infected with cross-reactive nonvirulent P. yoelii 17X. We concluded that a form of protective immunity was generated during the course of virulent infection but that its expression was inconsequential because parasite growth apparently exceeded the capacity of the immune system to clear the infection.     

Synchronization ofPlasmodium yoelii nigeriensis andP. y. killicki infection in the mouse by means of Percoll-glucose gradient stage fractionation: Determination of the duration of the schizogonic cycle

The youngest (rings and young trophozoites) erythrocytic stages ofPlasmodium yoelii nigeriensis andP. yoelii killicki, two rodent malaria subspecies developing very asynchronously in the blood, were separated from the other stages using a discontinuous Percoll-glucose gradient. They were inoculated into mice and the subsequent infection remained synchronous for two generations. The duration of the asexual cycle was found to be 18 h.     

Blackwater fever like in murine malaria

Blackwater fever (BWF) is the term used to designate the occurrence of hemoglobin pigments in the urine of patients infected with malaria parasites. BWF is more often associated with Plasmodium falciparum infection in man. The pathogenesis of BWF has not been explained satisfactorily. In the present study, the clinical and pathological observations made upon CD1 mice infected with Plasmodium yoelii yoelii lethal strain with clinical signs of hemoglobinuria and acute renal failure were evaluated. From the 40 P. yoelii yoelii-infected mice, 14 presented hemoglobinuria. In the observations, it was emphasized that hemoglobinuria occurred in the animals 1–2 days before they die. At 6 days post-infection, infected hemoglobinuric mice (HM) exhibited clinical signs such as dark red urine, apnea, and evident oliguria and hematuria; urine microscopical examination showed very few red blood cells. The entire non hemoglobinuric infected mice had a high parasitemia preceding the time of death, while the HM parasitemia was just detectable. In HM, marked hepatosplenomegaly, anemia, and renal and hepatic dysfunction were observed with the blood chemistry analysis at 6 days post-infection. Severe renal lesions were demonstrated in histopathological and scanning electron microscopy samples. Occlusion and necrosis of convoluted tubules were the main lesions found. The conditions required for the experimental production of hemoglobinuria in CD1 mouse infected by P. yoelii yoelii is still unknown. The clinical picture of a BWF, like in our rodents, was produced exclusively by the interaction between the parasite and its host. Results showed that hemoglobinuria in CD1 mice infected with P. yoelii yoelii and BWF in man infected with P. falciparum are similar in their pathogenesis.     

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.     

Prevention of recrudescent malaria in nude mice by thymic grafting or by treatment with hyperimmune serum.

Nude mice died when infected with the normally avirulent malarial parasite Plasmodium berghei yoelii. Furthermore, malaria recrudesced in Nu/Nu mice after the termination of acute disease by treatment with clindamycin. Recrudescence was not observed in Nu/Nu mice that had been grafted with thymic tissue or treated with hyperimmune serum. Mice mad B cell deficient by treatment with anti-mu-chain serum also died when infected with P. berghei yoelii. The data suggest that a crucial role of the thymus in preventing recrudescent malaria in this model system is to provide a helper function in the production of protective antibody.