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Journal of Global Antimicrobial... Mar 2020The aim of this study was to evaluate the efficacy of pyrimethamine-loaded poloxamer 407 nanomicelles on Plasmodium berghei strain NICD in vivo.
OBJECTIVES
The aim of this study was to evaluate the efficacy of pyrimethamine-loaded poloxamer 407 nanomicelles on Plasmodium berghei strain NICD in vivo.
METHODS
Pyrimethamine-loaded nanomicelles were prepared and their zeta potential, particle size and polydispersity index were measured. For antiplasmodial assessment, 54 mice were randomly divided into six groups. Four groups were infected intraperitoneally with P. berghei, whereas the two remaining groups did not receive the parasite (negative controls). Three of the P. berghei-infected groups received treatment with either pyrimethamine-loaded nanomicelles (2 mg/kg), pyrimethamine (2 mg/kg) or empty nanomicelles (2 mg/kg); the fourth group remained untreated (positive control). The parasitaemia rate, survival rate and histopathological changes in the liver, spleen and kidneys were examined and were compared with the negative and positive control groups.
RESULTS
The mean parasitaemia rate differed significantly between the nanoformulated pyrimethamine group and each of the other groups (P<0.05). Moreover, the survival rate of mice in the nanoformulated pyrimethamine group (7/9; 78%) was significantly higher compared with each of the other groups (P<0.01). The main histopathological changes, including hepatic necrosis in the liver, lymphoid hypoplasia in the spleen, and tubular nephrosis and perivascular and interstitial lymphocytic infiltration in the kidneys, were considerably lower in the nanoformulated pyrimethamine group than in the pyrimethamine and positive control groups.
CONCLUSION
Pyrimethamine-loaded nanomicelles showed potent antimalarial activity and can be considered as a potential candidate for further examination of their suitability as an antimalarial drug.
Topics: Animals; Antimalarials; Disease Models, Animal; Drug Compounding; Liver; Malaria; Male; Mice; Micelles; Nanoparticles; Parasitemia; Particle Size; Plasmodium berghei; Poloxamer; Pyrimethamine; Random Allocation; Spleen; Survival Analysis; Treatment Outcome
PubMed: 31404680
DOI: 10.1016/j.jgar.2019.08.002 -
Advances in Experimental Medicine and... 2016To understand much of the behaviour of microbial pathogens, it is necessary to image living cells, their interactions with each other and with host cells. Species such... (Review)
Review
To understand much of the behaviour of microbial pathogens, it is necessary to image living cells, their interactions with each other and with host cells. Species such as Escherichia coli are difficult subjects to image: they are typically microscopic, colourless and transparent. Traditional cell visualisation techniques such as fluorescent tagging or phase-contrast microscopy give excellent information on cell behaviour in two dimensions, but no information about cells moving in three dimensions. We review the use of digital holographic microscopy for three-dimensional imaging at high speeds, and demonstrate its use for capturing the shape and swimming behaviour of three important model pathogens: E. coli, Plasmodium spp. and Leishmania spp.
Topics: Escherichia coli; Holography; Image Processing, Computer-Assisted; Leishmania mexicana; Microscopy; Movement; Optical Imaging; Plasmodium berghei; Time Factors
PubMed: 27193535
DOI: 10.1007/978-3-319-32189-9_3 -
Malaria Journal Nov 2023Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for...
BACKGROUND
Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays.
METHODS
IC values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 10 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH.
RESULTS
The IC values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification.
CONCLUSIONS
Results from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody IC values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system.
Topics: Mice; Animals; Antibodies, Monoclonal; Plasmodium berghei; Plasmodium falciparum; RNA, Ribosomal, 18S; Protozoan Proteins; Animals, Genetically Modified; Antibodies, Protozoan; Malaria Vaccines
PubMed: 37936181
DOI: 10.1186/s12936-023-04765-2 -
Proceedings of the National Academy of... Feb 2019The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human...
The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite , the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five species and two related apicomplexan parasites. species mainly showed clustering of centromeres, telomeres, and virulence genes. In , the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in and , and absent in In , telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most species is constrained by the colocalization of virulence genes. and , the only two species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.
Topics: Animals; Centromere; Gene Expression Regulation; Genome, Protozoan; Genomics; Heterochromatin; Humans; Malaria, Falciparum; Plasmodium berghei; Plasmodium falciparum; Plasmodium knowlesi; Plasmodium vivax; Telomere; Toxoplasma
PubMed: 30723152
DOI: 10.1073/pnas.1810815116 -
JCI Insight Dec 2019Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing....
Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing. Efforts toward eradication are threatened by the lack of an effective vaccine and a rise in antiparasite drug resistance. Alternative approaches are urgently needed. Repurposing of available, approved drugs with distinct modes of action are being considered as viable and immediate adjuncts to standard antimicrobial treatment. Such strategies may be well suited to the obligatory and clinically silent first phase of Plasmodium infection, where massive parasite replication occurs within hepatocytes in the liver. Here, we report that the widely used antidiabetic drug, metformin, impairs parasite liver stage development of both rodent-infecting Plasmodium berghei and human-infecting P. falciparum parasites. Prophylactic treatment with metformin curtails parasite intracellular growth in vitro. An additional effect was observed in mice with a decrease in the numbers of infected hepatocytes. Moreover, metformin provided in combination with conventional liver- or blood-acting antimalarial drugs further reduced the total burden of P. berghei infection and substantially lessened disease severity in mice. Together, our findings indicate that repurposing of metformin in a prophylactic regimen could be considered for malaria chemoprevention.
Topics: Animals; Antimalarials; Cells, Cultured; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Repositioning; Drug Therapy, Combination; Hepatocytes; Humans; Inhibitory Concentration 50; Liver; Malaria; Male; Mefloquine; Metformin; Mice; Parasite Load; Parasitic Sensitivity Tests; Plasmodium berghei; Plasmodium falciparum; Primaquine; Primary Cell Culture
PubMed: 31852843
DOI: 10.1172/jci.insight.127441 -
Frontiers in Cellular and Infection... 2017Continuous stage conversion and swift changes in the antigenic repertoire in response to acquired immunity are hallmarks of complex eukaryotic pathogens, including... (Review)
Review
Continuous stage conversion and swift changes in the antigenic repertoire in response to acquired immunity are hallmarks of complex eukaryotic pathogens, including species, the causative agents of malaria. Efficient elimination of liver stages prior to blood infection is one of the most promising malaria vaccine strategies. Here, we describe different genetically arrested parasites (GAPs) that have been engineered in and and compare their vaccine potential. A better understanding of the immunological mechanisms of prime and boost by arrested sporozoites and experimental strategies to enhance vaccine efficacy by further engineering existing GAPs into a more immunogenic form hold promise for continuous improvements of GAP-based vaccines. A critical hurdle for vaccines that elicit long-lasting protection against malaria, such as GAPs, is safety and efficacy in vulnerable populations. Vaccine research should focus on solutions toward turning malaria into a vaccine-preventable disease, which would offer an exciting new path of malaria control.
Topics: Animals; CD8-Positive T-Lymphocytes; Gene Deletion; Humans; Liver; Malaria; Malaria Vaccines; Malaria, Falciparum; Plasmodium; Plasmodium berghei; Sporozoites; Vaccination; Vaccines, Attenuated
PubMed: 28620583
DOI: 10.3389/fcimb.2017.00198 -
Infection and Immunity Aug 2018Transmission-blocking vaccines (TBVs) interrupting malaria transmission are an integrated tool for malaria eradication. We characterized a sexual-stage-specific gene...
Transmission-blocking vaccines (TBVs) interrupting malaria transmission are an integrated tool for malaria eradication. We characterized a sexual-stage-specific gene (PBANKA_060330) from and studied its potential for use as a TBV. This gene, referred to as , encodes a protein of 37 kDa with a signal peptide and multiple transmembrane domains and is preferentially expressed in gametocytes. A recombinant Pbg37 (rPbg37) protein targeting the N-terminal 63 amino acids (amino acids 26 to 88) expressed in bacteria elicited strong antibody responses in mice. Western blotting demonstrated Pbg37 expression in gametocytes, zygotes, and, to a lesser extent, ookinetes and its predominant association with the membranes of gametocytes. Indirect immunofluorescence assay showed an abundant surface localization of Pbg37 on gametes and zygotes but reduced amounts on retorts and ookinetes. Knockout of (Δ) led to a considerable reduction in gametocytemia, which translated into a ~92.1% decrease in the oocyst number in mosquitoes. Deletion of had a more substantial influence on the development and maturation of microgametocytes. As a result, the Δ lines exhibited a higher female/male gametocyte ratio, fewer mature male gametocytes, and defects in the exflagellation of mature microgametocytes. To test the transmission-blocking potential of Pbg37, an ookinete assay showed that the major inhibitory effects of anti-Pbg37 antiserum were on the exflagellation and fertilization processes. Direct feeding of mosquitoes on mice immunized with rPbg37 or a control protein showed that rPbg37-immunized and -infected mice had a significant reduction (49.1%) in oocyst density compared to the controls. The conservation of this gene in warrants further investigations in human malaria parasites.
Topics: Animals; Antibodies, Protozoan; Antibody Formation; Blotting, Western; Disease Models, Animal; Disease Transmission, Infectious; Female; Fluorescent Antibody Technique, Indirect; Gene Deletion; Gene Expression Profiling; Malaria; Malaria Vaccines; Male; Membrane Proteins; Mice, Inbred BALB C; Mosquito Vectors; Parasite Load; Parasitemia; Plasmodium berghei; Protozoan Proteins; Vaccines, Synthetic; Virulence
PubMed: 29866905
DOI: 10.1128/IAI.00785-17 -
Molecular Microbiology Mar 2024Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade...
Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.
Topics: Animals; Culicidae; Parasites; Protozoan Proteins; Oocysts; Plasmodium berghei; Sporozoites
PubMed: 37314965
DOI: 10.1111/mmi.15078 -
Frontiers in Cellular and Infection... 2019In the lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively....
In the lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively. The apical structure of merozoites and sporozoites contains rhoptry and microneme secretory organelles, which are conserved with other infective forms of apicomplexan parasites. During merozoite invasion of erythrocytes, some rhoptry proteins are secreted to form a tight junction between the parasite and target cell, while others are discharged to maintain subsequent infection inside the parasitophorous vacuole. It has been questioned whether the invasion mechanisms mediated by rhoptry proteins are also involved in sporozoite invasion of two distinct target cells, mosquito salivary glands and mammalian hepatocytes. Recently we demonstrated that rhoptry neck protein 2 (RON2), which is crucial for tight junction formation in merozoites, is also important for sporozoite invasion of both target cells. With the aim of comprehensively describing the mechanisms of sporozoite invasion, the expression and localization profiles of rhoptry proteins were investigated in sporozoites. Of 12 genes representing merozoite rhoptry molecules, nine are transcribed in oocyst-derived sporozoites at a similar or higher level compared to those in blood-stage schizonts. Immuno-electron microscopy demonstrates that eight proteins, namely RON2, RON4, RON5, ASP/RON1, RALP1, RON3, RAP1, and RAMA, localize to rhoptries in sporozoites. It is noteworthy that most rhoptry neck proteins in merozoites are localized throughout rhoptries in sporozoites. This study demonstrates that most rhoptry proteins, except components of the high-molecular mass rhoptry protein complex, are commonly expressed in merozoites and sporozoites in spp., which suggests that components of the invasion mechanisms are basically conserved between infective forms independently of their target cells. Combined with sporozoite-stage specific gene silencing strategies, the contribution of rhoptry proteins in invasion mechanisms can be described.
Topics: Animals; Anopheles; Blotting, Western; Cells, Cultured; Epithelial Cells; Gene Expression Profiling; Hepatocytes; Mammals; Merozoites; Microscopy, Immunoelectron; Organelles; Plasmodium berghei; Protein Transport; Protozoan Proteins; Real-Time Polymerase Chain Reaction; Sporozoites
PubMed: 31552198
DOI: 10.3389/fcimb.2019.00316 -
Vaccine Jan 2023Antigens expressed during the sexual development of malaria parasites are transmission-blocking vaccine (TBV) targets. Pb22, a protein expressed and localized to the...
Antigens expressed during the sexual development of malaria parasites are transmission-blocking vaccine (TBV) targets. Pb22, a protein expressed and localized to the plasma membrane of gametes and ookinetes in Plasmodium berghei, is an excellent TBV candidate. Here, we evaluated the TB potential of the Plasmodium vivax ortholog Pv22 using a transgenic P. berghei parasite line and P. vivax clinical isolates. The full-length recombinant Pv22 (rPv22) protein was produced and used to immunize mice and rabbits to obtain antibodies. We generated a transgenic P. berghei line (TrPv22Pb) by inserting the pv22 gene into the pb22 locus and showed that Pv22 expression completely rescued the defects in male gametogenesis of the pb22 deletion parasite. Since Pv22 in the transgenic parasite showed similar expression and localization patterns to Pb22, we used the TrPv22Pb parasite as a surrogate to evaluate the TB potential of Pv22. In mosquito feeding assays, mosquitoes feeding on rPv22-immunized mice infected with TrPv22Pb parasites showed a 49.3-53.3 % reduction in the oocyst density compared to the control group. In vitro assays showed that the rPv22 immune sera significantly inhibited exflagellation and ookinete formation of the TrPv22Pb parasites. In a direct membrane feeding assay using three clinical P. vivax isolates, the rabbit anti-rPv22 antibodies also significantly decreased the oocyst density by 53.7, 30.2, and 26.2 %, respectively. This study demonstrated the feasibility of using transgenic P. berghei parasites expressing P. vivax antigens as a potential tool to evaluate TBV candidates. However, the much weaker TB activity of Pv22 obtained from two complementary assays suggest that Pv22 may not be a promising TBV candidate for P. vivax.
Topics: Male; Animals; Mice; Rabbits; Malaria; Plasmodium vivax; Plasmodium berghei; Malaria Vaccines; Protozoan Proteins; Malaria, Vivax; Recombinant Proteins; Antibodies, Protozoan; Culicidae
PubMed: 36503858
DOI: 10.1016/j.vaccine.2022.11.058