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Bulletin of the World Health... 1977Studies using erythrocyte-free preparations of P. berghei were conducted with a view to improving knowledge of parasite metabolism, particularly nucleotide metabolism.... (Comparative Study)
Comparative Study
Studies using erythrocyte-free preparations of P. berghei were conducted with a view to improving knowledge of parasite metabolism, particularly nucleotide metabolism. The free parasites employed in these studies were prepared by saponin lysis of parasitized mouse erythrocytes in isotonic glucose solutions. A comparative study of post-lytic metabolic activity of free parasites prepared by saponin, ammonium chloride, or osmotic lysis indicated a significantly greater retention of metabolic activity in the saponin-lysis preparations. Separations of nucleoside mono-, di-, and triphosphates extracted from free parasites were performed by means of high pressure liquid chromatography (HPLC), and ATP was additionally measured by luciferin-luciferase assay. Studies designed to differentiate among uptake, phosphorylation, and subsequent incorporation of (3)H-adenosine into nucleic acids of the free parasite strongly suggested that adenosine is metabolized either outside or on the parasite membrane, being first deaminated to inosine and then deribosylated to hypoxanthine. Observations from HPLC and radioisotope precursor studies support a hypothesis in which hypoxanthine may be proposed as being a pivotal substrate for purine salvage by malarial parasites. Some of the key steps in purine salvage and pyrimidine biosynthesis were investigated, using radiolabel uptake studies and HPLC analysis of nucleotides of the free malarial parasite. These studies suggest that hypoxanthine uptake may constitute an important new basis for chemotherapeutic attack on the malarial parasite.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Erythrocytes; Hemolysis; Malaria; Methods; Mice; Nucleotides; Plasmodium berghei; Purine Nucleotides
PubMed: 338182
DOI: No ID Found -
Malaria Journal Jul 2020Malaria is one of the most prevalent infectious disease in the world with 3.2 billion humans at risk. Malaria causes splenomegaly and damage in other organs including...
BACKGROUND
Malaria is one of the most prevalent infectious disease in the world with 3.2 billion humans at risk. Malaria causes splenomegaly and damage in other organs including skeletal muscles. Skeletal muscles comprise nearly 50% of the human body and are largely responsible for the regulation and modulation of overall metabolism. It is essential to understand how malaria damages muscles in order to develop effective preventive measures and/or treatments. Using a pre-clinical animal model, the potential molecular mechanisms of Plasmodium infection affecting skeletal muscles of mice were investigated.
METHODS
Mouse Signal Transduction Pathway Finder PCR Array was used to monitor gene expression changes of 10 essential signalling pathways in skeletal muscles from mice infected with Plasmodium berghei and Plasmodium chabaudi. Then, a new targeted-lipidomic approach using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to profile 158 lipid signalling mediators (LMs), mostly eicosanoids derived from arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was applied. Finally, 16 key LMs directly associated with inflammation, oxidative stress, and tissue healing in skeletal muscles, were quantified.
RESULTS
The results showed that the expression of key genes altered by Plasmodium infection is associated with inflammation, oxidative stress, and atrophy. In support to gene profiling results, lipidomics revealed higher concentrations of LMs in skeletal muscles directly related to inflammatory responses, while on the levels of LMs crucial in resolving inflammation and tissue repair reduced significantly.
CONCLUSION
The results provide new insights into the molecular mechanisms of malaria-induced muscle damage and revealed a potential mechanism modulating inflammation in malarial muscles. These pre-clinical studies should help with future clinical studies in humans aimed at monitoring of disease progression and development of specific interventions for the prevention and mitigation of long-term chronic effects on skeletal muscle function.
Topics: Animals; Malaria; Male; Mice; Muscle, Skeletal; Plasmodium berghei; Plasmodium chabaudi
PubMed: 32664933
DOI: 10.1186/s12936-020-03332-3 -
The Journal of Parasitology Feb 2017Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported...
Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a "pseudoparasite." This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant. Here we improved the original transfection plasmid by incorporating a multicloning site, an enterokinase recognition sequence upstream of GFP, and a His-tag and demonstrate its potential suitability for the heterologous expression of Plasmodium sp. genes relevant to the development of anti-malarial vaccines. Plasmodium berghei HAP2 and MSP8, currently considered candidate genes for a malaria vaccine, were cloned into p[MOE]:GFP, and the constructs were used to transfect P. marinus trophozoites. Within 48 hr of transfection we observed fluorescent cells indicating that the P. berghei genes fused to GFP were expressed. The expression appeared to be transient for both P. berghei genes, as florescence of the transfectants diminished gradually over time. Although this heterologous expression system will require optimization for integration and constitutive expression of Plasmodium genes, our results represent attainment of proof for the "pseudoparasite" concept we previously proposed, as we show that the engineered P. marinus system has the potential to become a surrogate system suitable for expression of Plasmodium spp. genes of interest, which could eventually be used as a malaria vaccine delivery platform. The aim of the present study was to test the ability of marine protozoan parasite P. marinus to express genes of P. berghei .
Topics: Alveolata; Animals; Antigens, Protozoan; Female; Gene Expression Regulation; Genetic Vectors; Malaria; Malaria Vaccines; Male; Plasmids; Plasmodium berghei; Protozoan Proteins; Transfection
PubMed: 27723436
DOI: 10.1645/16-88 -
Malaria Journal Apr 2016The approach of using transgenic rodent malaria parasites to assess the immune system's response to antigenic targets from a human malaria parasite has been shown to be...
Development of a Plasmodium berghei transgenic parasite expressing the full-length Plasmodium vivax circumsporozoite VK247 protein for testing vaccine efficacy in a murine model.
BACKGROUND
The approach of using transgenic rodent malaria parasites to assess the immune system's response to antigenic targets from a human malaria parasite has been shown to be useful for preclinical evaluation of new vaccine formulations. The transgenic Plasmodium berghei parasite line [PvCSP(VK210)/Pb] generated previously expresses the full-length circumsporozoite protein (CSP) VK210 from Plasmodium vivax. The transgenic parasite expresses one of the two most common alleles of CSP, defined by nine amino acids at the central repeat region of this protein. In the present study, a transgenic P. berghei parasite line [PvCSP(VK247)/Pb] expressing the full-length PvCSP(VK247), which is the alternative common allele, was generated and characterized.
METHODS
The P. berghei expressing full-length PvCSP(VK247) was generated and examined its applicability to CSP-based vaccine research by examining its biological characteristics in mosquitoes and mice.
RESULTS
Similar to PvCSP(VK210)/Pb, PvCSP(VK247)/Pb developed normally in mosquitoes and produced infectious sporozoites equipped to generate patent infections in mice. Invasion of HepG2 cells by PvCSP(VK247)/Pb sporozoites was inhibited by an anti-PvCSP(VK247) repeat monoclonal antibody (mAb), but not by an anti-PvCSP(VK210) repeat mAb.
CONCLUSIONS
These two transgenic parasites thus far can be used to evaluate the potential efficacy of PvCSP-based vaccine candidates encompassing the two major genetic variants in preclinical trials.
Topics: Animals; Malaria; Malaria Vaccines; Mice; Organisms, Genetically Modified; Plasmodium berghei; Plasmodium vivax; Protozoan Proteins; Treatment Outcome
PubMed: 27129682
DOI: 10.1186/s12936-016-1297-3 -
Parasites & Vectors Jul 2021Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen,...
BACKGROUND
Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 h of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule.
METHODS
We identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation.
RESULTS
We found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild-type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation.
CONCLUSION
These findings may be useful for the development of a transmission-blocking tool for malaria.
Topics: Animals; Antibodies, Protozoan; Culicidae; Female; Malaria; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; Mosquito Vectors; Oocysts; Plasmodium berghei; Protozoan Proteins
PubMed: 34289894
DOI: 10.1186/s13071-021-04868-2 -
PloS One Oct 2010Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1(19)) and apical...
BACKGROUND
Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1(19)) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic.
METHODOLOGY AND RESULTS
In this study, we assessed the protective efficacies of a series of MSP1(19)- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1(19) induced high titers of PfMSP1(19)-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1(19) in place of native PbMSP1(19). Similarly, neither P. berghei MSP1(19)- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1(19)- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa.
CONCLUSION
This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1(19)- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP1(19) and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.
Topics: Animals; Antigens, Protozoan; Base Sequence; Blotting, Western; DNA Primers; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Malaria Vaccines; Membrane Proteins; Merozoite Surface Protein 1; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Plasmodium berghei; Protozoan Proteins
PubMed: 21060850
DOI: 10.1371/journal.pone.0013727 -
Ethiopian Journal of Health Sciences Sep 2018The emergence and spread of Plasmodium falciparum resistance to antimalarial drugs necessitated the search for new drugs from natural products. Zingiber officinal Roscoe...
BACKGROUND
The emergence and spread of Plasmodium falciparum resistance to antimalarial drugs necessitated the search for new drugs from natural products. Zingiber officinal Roscoe and Echinops Kebericho Mesfin are traditional herbal medicines widely used for the treatment of malaria in Ethiopia. The aim of the study was to assess the toxicity profile and in vivo antiplasmodial activities of 70% methanol crude extracts of both plant materials against Plasmodium berghei.
METHODS
Healthy male Swiss Albino mice of age 4-5 weeks and weight 25-36 g were infected by P. berghei. The extracts were administered orally at doses 5000, 2500 and 1250 mg/kg for acute toxicity of E. kebericho Mesfin. Graded doses at 1000, 500 and 250 mg/kg used for four days suppressive studies. Parasitemia, body weight, packed cell volume (PCV) and survival time were determined. SPSS Version 20 was used for the analysis of data of parasitemia, body weight, PCV, and survival times. Statistical significance was determined by one-way ANOVA. Independent t-test was used to compare results. Results were presented as a mean ± standard error of the mean (M ± SEM). All data were analyzed at a 95% confidence interval (α= 0.05).
RESULTS
At the dose of 5000 mg/kg, E. kebericho Mesfin showed no toxic effects. The LD50 of extract could go beyond the dose used. In vivo antiplasmodial activity of extracts showed excellent chemo suppression at 500 and 1000 mg/kg in a dose dependent manner compared with the negative control. The chemo suppressions of the 1000 mg/kg of both plant extracts were 49.53 ± 1.90% and 32.83 ± 1.03%, respectively. The survival times of P. berghei infected mice were also a dose dependent manner while failed to prevent weight loss.
CONCLUSION
The extracts of both medicinal plants showed antiplasmodial activities against P. berghei. It confirmed the literature findings and their traditional uses.
Topics: Animals; Antimalarials; Asteraceae; Dose-Response Relationship, Drug; Ethiopia; Zingiber officinale; Male; Mice; Mice, Inbred Strains; Parasitemia; Phytotherapy; Plant Extracts; Plasmodium berghei
PubMed: 30607081
DOI: 10.4314/ejhs.v28i5.17 -
African Health Sciences Dec 2020A study on the biochemical indices of albino mice infected with and treated with aqueous and ethanolic extracts was undertaken.
BACKGROUND
A study on the biochemical indices of albino mice infected with and treated with aqueous and ethanolic extracts was undertaken.
METHODS
216 males mice were randomly assigned to six treatment groups each containing six mice for both aqueous and ethanolic extracts experiments. NK-65 was inoculated into the mice intraperitoneally and establishment of infection confirmed. Administration of extracts of was done after phytochemical and acute toxicity tests at varying concentrations, for both suppressive and curative tests. Blood samples collected by ocular puncturing were examined for the biochemical indices; ALT, AST, ALP, creatinine and total protein using the standard procedures.
RESULTS
extracts suppression of in mice was comparable to the standard drug. Significantly higher (p<0.05) recovery of mice treated with A. boonei extracts was observed. The biochemical indices examined all had significantly (p<0.05) increased concentration after 7 days post-infection, except for total protein concentration which had no significant increase or decrease due to extracts administration.
CONCLUSION
The antiplasmodial potentials of leaf and root extracts were dosage and duration-dependent, and have demonstrated satisfactory normalization of altered biochemical indices due to malaria.
Topics: Alstonia; Animals; Antimalarials; Malaria; Mice; Plant Extracts; Plant Leaves; Plant Roots; Plasmodium berghei
PubMed: 34394229
DOI: 10.4314/ahs.v20i4.21 -
Malaria Journal Nov 2017A vaccine that targets multiple developmental stages of malaria parasites would be an effective tool for malaria control and elimination.
BACKGROUND
A vaccine that targets multiple developmental stages of malaria parasites would be an effective tool for malaria control and elimination.
METHODS
A conserved gene in Plasmodium, the Plasmodium berghei gene (PBANKA_020570) encoding a 51 kDa protein (pb51 gene), was identified through search of the PlasmoDB database using a combination of expression and protein localization criteria. A partial domain of the Pb51 protein was expressed in a prokaryotic expression system (rPb51) and used for immunization in mice. The protein expression profile and localization were studied by Western blot and indirect immunofluorescence assay (IFA), respectively. The inhibitory effect of the anti-rPb51 antibodies on parasite proliferation was evaluated in erythrocytes in vivo. The transmission-blocking activity of the immune sera was determined by in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA).
RESULTS
The rPb51 elicited specific antibodies in mice. Western blot confirmed Pb51 expression in schizonts, gametocytes and ookinetes. IFA showed localization of Pb51 on the outer membranes of schizonts, gametocytes, zygotes, retorts, ookinetes and sporozoites of P. berghei. Mice immunized with the rPb51 protein significantly reduced parasite proliferation and gametocyte conversion in vivo. Moreover, the rPb51 antisera also significantly reduced the in vitro ookinete conversion when added into the ookinete culture medium. In DFA, mice immunized with the rPb51 reduced the prevalence of mosquito infection by 21.3% and oocyst density by 54.8%.
CONCLUSIONS
In P. berghei, P51 was expressed in both asexual erythrocytic and sexual stages and localized on the surface of these stages with the exception of the ring stage. The anti-rPb51 antibodies inhibited both P. berghei proliferation in mice and transmission of the parasite to mosquitoes.
Topics: Animals; Blotting, Western; Cell Proliferation; Female; Fluorescent Antibody Technique, Indirect; Malaria; Malaria Vaccines; Mice; Mice, Inbred BALB C; Plasmodium berghei; Protozoan Proteins; Rabbits
PubMed: 29132428
DOI: 10.1186/s12936-017-2107-2 -
MBio Jun 2016Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei...
UNLABELLED
Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity.
IMPORTANCE
Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite's successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite's autophagic pathway may represent a novel approach to control malarial infections.
Topics: Acyl Carrier Protein; Animals; Apicoplasts; Autophagy; Autophagy-Related Protein 8 Family; Hepatocytes; Humans; Liver; Malaria; Membrane Proteins; Merozoites; Mice, Transgenic; Mutation; Organelles; Plasmodium berghei; Protozoan Proteins
PubMed: 27353755
DOI: 10.1128/mBio.00682-16