Influence of chemotherapy on the Plasmodium gametocyte sex ratio of mice and humans

Plasmodium species, the etiologic agents of malaria, are obligatory sexual organisms. Gametocytes, the precursors of gametes, are responsible for parasite transmission from human to mosquito. The sex ratio of gametocytes has been shown to have consequences for the success of this shift from vertebrate host to insect vector. We attempted to document the effect of chemotherapy on the sex ratio of two different Plasmodium species: Plasmodium falciparum in children from endemic area with uncomplicated malaria treated with chloroquine (CQ) or sulfadoxine-pyrimethamine (SP), and P. vinckei petteri in mice treated with CQ or untreated. The studies involved 53 patients without gametocytes at day 0 (13 CQ and 40 SP) followed for 14 days, and 15 mice (10 CQ and 5 controls) followed for five days. During the course of infection, a positive correlation was observed between the time of the length of infection and the proportion of male gametocytes in both Plasmodium species. No effects of treatment (CQ versus SP for P. falciparum or CQ versus controls for P. vinckei petteri) on the gametocyte sex ratio were found for either Plasmodium species. This indicates that parasites do not respond to chemotherapy by altering their sex allocation strategy, even though, in the case of P. falciparum, they apparently increase their overall investment in sexual stages. This suggests that malaria parasite species respond to different environmental cues for their sex differentiation and sex determination.     

Protective immunity to malaria: studies with cloned lines of rodent malaria in CBA/Ca mice. IV. The specificity of mechanisms resulting in crisis and resolution of the primary acute phase parasitaemia of. Plasmodium chabaudi chabaudi and P. yoelii yoelii

Low numbers of parasites from cloned lines of the rodent malaria parasites, Plasmodium chabaudi chabaudi AS and P. yoelii yoelii A, injected into CBA/Ca mice produce acute but usually self-limiting infections. During crisis, i.e. 1-2 days after peak parasitaemia, 'pre-immune' mice experiencing such 'background' infections were reinfected intravenously with homologous parasites or parasites of heterologous strains or species. P. c. chabaudi AS pre-immune mice controlled an AS challenge with essentially the same kinetics as the background infection. Reinfection of AS pre-immune mice with the heterologous (CB and IP-PCI) P. c. chabaudi strains or P. chabaudi adami DS had little effect on the initial growth of these parasites, although eventually the parasitaemia was controlled. In contrast, a partial inhibitory effect on the growth of P. vinckei lentum DS was evident. Challenge with the non-lethal (A) or lethal (YM) variants of P. y. yoelii resulted in an increase in both the growth and virulence of these parasites. P. y. yoelii A pre-immune mice controlled a homologous challenge, but were less effective at controlling the YM variant. In addition, they were unable to clear rapidly a P. c. chabaudi AS or P. v. lentum DS challenge. Both the multiplication and virulence of P. berghei ANKA were enhanced. These findings demonstrate that resolution of the primary acute parasitaemia in P. c. chabaudi AS- and P. y. yoelii A-infected mice is predominantly mediated by species- and strain-specific mechanisms.     

The chemotherapy of rodent malaria, XXIX DNA relationships within the subgenus Plasmodium (Vinckeia).

The relationships between rodent malarias were examined by means of DNA buoyant density determinations and DNA--DNA hybridization data. Current views of the existence in this group of four species: Plasmodium berghei, P. yoelli, P. vinckei and P. chabaudi were confirmed. The identity of two chloroquine-resistant lines derived from P. berghei N strain was established. One of these, the NS line, was found to be a subspecies of P. yoelii; the implications of this finding are discussed.     

Inhibition of Plasmodium yoelii blood-stage malaria by interferon alpha through the inhibition of the production of its target cell, the reticulocyte

The effect of a recombinant hybrid human interferon alpha (IFN-alpha) (which cross-reacts with murine cells) on C57BL/6 mice infected with Plasmodium yoelii sporozoites or parasitized erythrocytes was determined. IFN-alpha did not inhibit the development of the parasite in the liver, but it did reduce the blood parasite load and the hepatosplenomegaly induced by the infection in mice injected with blood-stage parasites. The extent of anemia in IFN-alpha-treated and control mice was similar, despite the lower parasite load in the IFN-alpha-treated mice. The reduced blood parasite load in IFN-alpha-treated mice was associated with reduced erythropoiesis and reticulocytosis. As reticulocytes are the preferred target cells for the strain of P yoelii used (P yoelii yoelii 265 BY), it was postulated that the inhibition of reticulocytosis in IFN-alpha-treated mice was causally related to the observed decreased blood parasite load. This was supported by the finding that IFN-alpha inhibited a different strain of P yoelii (17X clone A), which also displays a tropism for reticulocytes, but not a line of Plasmodium vinckei petteri, which infects only mature red blood cells. As human malaria species also display different tropism for reticulocytes, these findings could be relevant for people coinfected with multiple Plasmodium species or strains or coinfected with Plasmodium and virus. (Blood. 2001;97:3966-3971)     

Heterologous immunity in rodent malaria: comparison of the degree of cross-immunity generated by vaccination with that produced by exposure to live infection

The degree of heterologous immunity occurring between the different rodent malarias was examined. Mice immunized with a killed vaccine consisting of formalin-fixed blood-stage parasites mixed with saponin, were challenged with homologous or heterologous parasites and the degree of protection assessed by monitoring the number of survivors and the duration and severity of parasitaemia. The results indicated that vaccination conferred strong cross-protection between two different Plasmodium yoelii lines and also some cross-protection between different species. The most significant inter-species cross-immunity was in P. berghei-vaccinated mice challenged with P. yoelii (33% survival rate) and in P. vinckei-vaccinated mice challenged with P. chabaudi (50% survival rate). The protection operated only in one direction since mice vaccinated against P. yoelii or P. chabaudi were fully susceptible to P. berghei and P. vinckei respectively, although extension of the survival time did occur in both cases. Significantly greater levels of homologous and heterologous immunity occurred in vaccinated mice which had cleared a primary homologous infection. All recovered animals were fully resistant to homologous secondary challenges. P. berghei-recovered mice had complete resistance to a secondary P. yoelii challenge, with partial resistance also to P. vinckei and P. chabaudi: P. vinckei-recovered mice were completely resistant to P. chabaudi challenge and showed partial resistance to P. berghei (50% survival rate). Conversely, no reduction in mortality occurred in P. chabaudi-recovered mice challenged with any heterologous parasite. Plasmodium yoelii-recovered animals were fully susceptible to P. vinckei but showed some resistance to P. berghei and P. chabaudi (33 and 17% survival respectively). Many lesser instances of cross-resistance were observed in the present study and are discussed in the text but they were not sufficiently powerful to reduce mortality. The present study provides the first reported comprehensive investigation on cross-resistance in rodent malarias generated by a killed vaccine. In addition this is the only complete cross-resistance study available which has concentrated exclusively upon highly lethal strains of each parasite species. Taken with the rechallenge experiments, the results extend, and to a certain point confirm, previous observations on cross-immunity in the rodent malarias. However, several of the present results conflict with previous reports and these are discussed in relation to the relevant literature.     

Inability of Plasmodium vinckei-immune spleen cells to transfer protection to recipient mice exposed to vaccine''vectors''or heterologous species of plasmodium

Mice can be immunized to Plasmodium vinckei by repeated infections followed by cure. Such immunity is dependent on CD4 T cells and an architecturally modified spleen, but has little requirement for antibody. Thus, athymic mice can be exposed to P. vinckei and cured, but do not develop immunity. They are resistant to challenge with parasites, however, if they are then given spleen cells from euthymic immunized animals. Such immune spleen cells, however, cannot transfer resistance to normal mice which have been exposed to BCG, Salmonella typhimurium, or vaccinia virus, and are only partially effective in transferring resistance to mice which have been previously immunized with heterologous plasmodia, P. yoelii, P. chabaudi and P. berghei. Mice exposed to varying numbers of irradiated P. vinckei-pRBC do not develop immunity and nor are such animals protected following adoptive transfer of immune spleen cells. Cellular immunity to malaria may not only be dependent on a population of immune CD4 T cells, but may require a specifically architecturally modified spleen which may not occur following either exposure to candidate vaccine vectors, heterologous plasmodia or non-viable homologous plasmodia.     

Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.     

Anti-Thy-1 treated and irradiated spleen cells from (BALB/cXC57Bl/6) F1 mice infected with Plasmodium chabaudi chabaudi can transfer protection into irradiated hosts

The transfer of spleen cells from (BALB/cXC57Bl/6) F1 mice recovered from a Plasmodium chabaudi chabaudi AS infection into irradiated syngeneic recipients conferred protection. Neither elimination of Thy-1⁺ cells nor in vitro irradiation of immune cells before transfer affected protection while both anti-Thy-1 treatment and irradiation abolished the appearance of anti-P. c. chabaudi antibodies in the recipients. Superinfection of immune spleen cell donors did not improve their capability to transfer protection which was also unaffected by anti-Thy-1 treatment. The serum of mice after one infection was only marginally protective when transferred into irradiated recipients and a second infection improved the protective activity of serum which was not further improved by six infections. The cotransfer of immune serum and immune cells did not result in any synergistic effect. On the other hand, when P. c. chabaudi AS (BALB/cXC57B1/6)F1 infected mice were challenged with a high dose of Plasmodium yoelii 17XL at crisis, the mice were unable to control the heterologous parasite. When mice were challenged with P. yoelii 17XL several weeks after infection with P.c. chabaudi AS, a good degree of cross-protection was observed.     

Improvement of glucose homeostasis in obese diabetic db/db mice given Plasmodium yoelii glycosylphosphatidylinositols

We have previously reported that infection with Plasmodium yoelii, Plasmodium chabaudi, or injection of extracts from malaria-parasitized red blood cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in streptozotocin (STZ)-diabetic mice. P yoelii glycosylphosphatidylinositols (GPIs) were extracted in chloroform:methanol:water (CMW) (10:10:3), purified by high-performance thin layer chromatography (HPTLC) and tested for their insulin-mimetic activities. The effects of P yoelii GPIs on blood glucose were investigated in insulin-resistant C57BL/ks-db/db diabetic mice. A single intravenous injection of GPIs (9 and 30 nmol/mouse) induced a significant dose-related decrease in blood glucose (P < .001), but insignificantly increased plasma insulin concentrations. A single oral dose of 2.7 micromol GPIs per db/db mouse significantly lowered blood glucose (P < .01). P yoelii GPIs in vitro (0.062 to 1 micromol/L) significantly stimulated lipogenesis in rat adipocytes in a dose-dependent manner both in the presence and absence of 10(-8) mol/L insulin (P < .01). P yoelii GPIs stimulated pyruvate dehydrogenase phosphatase (PDH-Pase) and inhibited both cyclic adenosine monophosphate (cAMP)-dependent protein kinase A and glucose-6-phosphatase (G6Pase). P yoelii GPIs had no effect on the activity of the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). This is the first report of the hypoglycemic effect of P yoelii GPIs in murine models of type 2 diabetes. In conclusion, P yoelii GPIs demonstrated acute antidiabetic effects in db/db mice and in vitro. We suggest that P yoelii GPIs, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes.     

Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei.

Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.     

The activity and inhibition of the food vacuole plasmepsin from the rodent malaria parasite Plasmodium chabaudi

The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole plasmepsin-the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this plasmepsin is an important contribution towards understanding its biology.     

Endogenous galectin-3 controls experimental malaria in a species-specific manner

Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses. Galectin-3 has been implicated in several immunological processes as well as in pathogen recognition through specific binding to glycosylated receptors on the surface of host cells or microorganisms. In spite of considerable evidence supporting a role for galectin-3 in host-pathogen interactions, the relevance of this lectin in the regulation of the host defence mechanisms in vivo is poorly understood. In this study, we analysed the impact of galectin-3 deficiency during infection with three distinct species of rodent malaria parasites, Plasmodium yoelii 17XNL, Plasmodium berghei ANKA and Plasmodium chabaudi AS. We found that galectin-3 deficiency showed a marginal effect on the course of parasitaemia during P. chabaudi infection, but did not alter the course of parasitaemia during P. berghei infection. However, lack of galectin-3 significantly reduced P. yoelii parasitaemia. This reduced parasitaemia in Lgals3(-/-) mice was consistent with higher titres of anti-P. yoelii MSP1(19) IgG2b isotype antibodies when compared with their wild-type counterparts. Our results reflect the complexity and singularity of host-pathogen interactions, indicating a species-specific role of endogenous galectin-3 in the control of parasite infections and the modulation of antibody responses.     

An anti-Chitinase malaria transmission-blocking single-chain antibody as an effector molecule for creating a Plasmodium falciparum-refractory mosquito

Indirect evidence has suggested the existence of a second chitinase gene, PgCHT2, in the avian malaria parasite Plasmodium gallinaceum. We have now identified PgCHT2 as the orthologue of the P. falciparum chitinase gene PfCHT1, a malaria transmission-blocking target. Computational phylogenetic evidence and biochemical and cell biological functional data support the hypothesis that an avian-related Plasmodium species was the ancestor of both P. falciparum and P. reichenowi, and this single lineage gave rise to another lineage of malaria parasites, including P. vivax, P. knowlesi, P. berghei, P. yoelii, and P. chabaudi. A recombinant PfCHT1/PgCHT2-neutralizing single-chain antibody significantly reduced P. falciparum and P. gallinaceum parasite transmission to mosquitoes. This single-chain antibody is the first anti-P. falciparum effector molecule to be validated for making a malaria transmission-refractory transgenic Anopheles species mosquito. P. gallinaceum is a relevant animal model that facilitates a mechanistic understanding of P. falciparum invasion of the mosquito midgut.     

约氏疟原虫与伯氏疟原虫侵入期抗原的初步研究

目的　用针对鼠约氏疟原虫(Plasmodiumyoelii)侵入期的8种单克隆抗体,对约氏疟原虫和伯氏疟原虫(P.berghei)侵入期即动合子、裂殖子和子孢子棒状体和表面抗原检测分析。　方法　间接免疫荧光实验(IFA)对各侵入期抗原进行亚细胞结构定位,SDSPAGE及Western印迹对两种鼠疟原虫的不同侵入期进行抗原组分分析。　结果　经上述两种方法检测发现,顶端复合体抗原成分复杂,约氏疟原虫和伯氏疟原虫的棒状体有共同的抗原表位,约氏疟原虫的动合子与其自身的裂殖子有类似成分,也有各自独特的抗原。两种鼠疟原虫动合子抗原有类似成分。约氏疟原虫的子孢子具有与裂殖子、动合子不同的抗原成分。　结论　疟原虫侵入期棒状体和表面抗原在同一虫种的不同侵入期和不同虫种中有共同的抗原表位,也有各自的独特组分。     

Murine malaria decreases hemopoietic stem cells

The causes of anemia and immunosuppression, major outcomes of malaria, are not well established. This study was undertaken to investigate whether erythropoietin (EP) production is adequate and whether the hemopoietic stem cells (CFU-S) were affected during the course of infection. Groups of female Balb/c mice infected with Plasmodium vinckei vinckei, Plasmodium berghei, or Plasmodium chabaudi adami were exposed to five hours of simulated altitude equivalent to 22,000 ft. Plasma samples were collected for EP bioassay and radioimmunoassay (RIA). Using radioiron incorporation as an index of erythropoiesis, differences in response to infection with different species of plasmodia were observed. In general, decreases in erythropoietic activity were observed in bone marrow and spleen as the infection progressed and continued to be depressed after apparent resolution of a nonlethal infection with P. chabaudi adami. Marrow from infected and control femurs were tested for CFU-S content using the spleen colony assay. The cellularity and CFU-S content of the femoral marrow decrease as the parasitemia increases. All three species of plasmodia stimulate EP production during peak parasitemias, indicating that adequate amounts of EP are available to the erythron during malarial infection. Depletion of CFU-S and probable lack of compensatory turnover of CFU-S may contribute to the disease characteristics of malaria.     

Malaria plasmodia in the mouse. Parasitization of mature and immature erythrocytes by Plasmodium berghei, Plasmodium yoelii and Plasmodium chabaudi (author's transl)]

Plasmodium berghei parasites (strain K173) in mice with developing immunity changed to a variant type with increased resistance against antibodies and enhanced invasion of mature erythrocytes; on passages in normal mice this variant type retransformed to the normal type (Kretschmar 1964). On detailed study, parasites of the variant type showed a markedly decreased predilection for polychromatophilic erythrocytes, leading to slowed multiplication during prepatency, increased invasion of mature erythrocytes and rapidly fatal course of the infection. Avoidance of parasitized immature erythrocytes remained unaltered in the variant type. In presence of antibodies many parasites invaded mature erythrocytes even in blood with high concentrations of immature erythrocytes. --Plasmodium yoelii (strain 17X) showed very high predilection for polychromatophilic erythrocytes, only slight lowering of immature erythrocyte concentration and low parasitization of mature erythrocytes. Parasites with altered preference for polychromatophilic erythrocytes were not observed in animals with parasitemia relapsing after spontaneous disappearance of the primary parasitemia or after injection of antiserum. It is suggested that in a variant of Plasmodium yoelii with high invasion of mature erythrocytes (Yoeli et al. 1975), the decisive virulence factor might be a lowered predilection for polychromatophilic erythrocytes. Avoidance of parasitized immature erythrocytes is less marked in Plasmodium yoelii than in Plasmodium berghei. In presence of antibodies more parasites invade mature erythrocytes. --Plasmodium chabaudi showed an appreciable preference for invasion of immature erythrocytes. Moreover, significantly more multiply parasitized mature and immature erythrocytes were found than would have been produced by random invasion of erythrocytes.     

The role of reactive nitrogen intermediates in modulation of gametocyte infectivity of rodent malaria parasites

Direct feeding of Anopheles stephensi mosquitoes on mice infected with Plasmodium vinckei petteri showed that, during the periods of schizogony in the blood, the infectivity of gametocytes was markedly reduced. This could be prevented by prior injection of the L-arginine analogue, Nw-nitro-L-arginine (NwNLA) showing that the altered infectivity was due to reactive nitrogen intermediates (RNI). Similar effects on transmission of P. yoelii nigeriensis were demonstrated in vitro by membrane feeding of the mosquitoes. The in vitro reduction in infectivity could be reversed by injecting the L-arginine analogue either into the infected mouse donor of serum, or into the membrane feeding chamber. Elevated levels of TNF and IL-6 were demonstrated during the course of infection but did not correlate well with nitrogen radical activity. Similarly, direct measurements of NO₂- and NO₃- did not reflect the nitrogen radical activity revealed by addition of the specific L-arginine analogue.     

Aspartic proteases from Plasmodium chabaudi: a rodent model for human malaria

Intraerythrocytic malaria parasites degrade haemoglobin to provide nutrients for their own growth and maturation. Plasmodium aspartic proteases known as plasmepsins play an important role on haemoglobin degradation and are being studied as drug targets for chemotherapy of malaria. The rodent model for human malaria, Plasmodium chabaudi, is an experimentally good model for therapy drug design. The gene encoding an aspartic protease precursor (proplasmepsin) from the rodent malaria parasite P. chabaudi was cloned and sequenced. A theoretical 3D structure model was constructed by comparative homology and used for superimposition with other known models. Analysis of the P. chabaudi and Plasmodium yoelli genomes revealed in both the presence of at least seven plasmepsins and each one has sequence similarity to its plasmepsin counterpart of the human malaria Plasmodium falciparum. The predicted proteins were confirmed as plasmepsins by detection on Blocks Database of three characteristic blocks of the eukaryotic and viral aspartic protease family. Analysis of the proline-rich loop amino acid sequence of these plasmepsins suggests that they constitute characteristic motifs of each plasmepsin group suggesting that these sequence variations are related with different substrate specificities.     

Antimalarial activity of a riboflavin analog against Plasmodium vinckei in vivo and Plasmodium falciparum in vitro

The riboflavin analog 10-(4'-chlorophenyl)-3-methylflavin was found to have significant activity against Plasmodium vinckei vinckei when administered orally and parenterally; it was active against P. falciparum in culture. It inhibited mouse erythrocyte glutathione reductase in a dose-dependent manner. When administered orally, 5-deazariboflavin was not active in vivo although it has been shown to have activity against P. falciparum in vitro.     

Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species.

Merozoite surface antigen 1 (MSA1) of several species of plasmodia has been shown to be a promising candidate for a vaccine directed against the asexual blood stages of malaria. We report the cloning and characterization of the MSA1 gene of the human malaria parasite Plasmodium vivax. This gene, which we call Pv200, encodes a polypeptide of 1726 amino acids and displays features described for MSA1 genes of other species, such as signal peptide and anchoring sequences, conserved cysteine residues, number of potential N-glycosylation sites, and repeats consisting here of 23 glutamine residues in a row. When the nucleotide and deduced amino acid sequences of the MSA1 of P. vivax are compared to those of another human malaria parasite, Plasmodium falciparum, and to those of the rodent parasite Plasmodium yoelii, 10 regions of high amino acid similarity are observed despite the very different dG + dC contents of the corresponding genes. All of the interspecies conserved regions reside within the conserved or semiconserved blocks delimited by the sequences of different alleles of the MSA1 gene of P. falciparum.