An Improved Medium For Plasmodium chabaudi In Vitro Erythrocyte Invasion Assays

SUMMARYErythrocyte invasion assays are described for two species of rodent malaria, namely Plasmodium berghei and P. c. chabaudi. These invasion assays are simple, are carried out using a candle jar and allow a number of assays to be performed simultaneously. Our results demonstrate that both rodent malaria species show an in vitro preference for reticulocytes although the preference of P. c. chabaudi for these cells is not as marked as that of P. berghei. The details of our invasion assays and our results obtained are discussed.

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An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi

Wellcome Open Research ◽

10.12688/wellcomeopenres.15587.2 ◽

2020 ◽

Vol 5 ◽

pp. 71

Author(s):

Edward J Marr ◽

Rachel M Milne ◽

Burcu Anar ◽

Gareth Girling ◽

Frank Schwach ◽

...

Keyword(s):

Marker Genes ◽

Plasmodium Chabaudi ◽

Chronic Infections ◽

Selectable Marker Genes ◽

Erythrocyte Invasion ◽

Infection Dynamics ◽

Marker Free ◽

Parasite Replication

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti-Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and –tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

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An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi

Wellcome Open Research ◽

10.12688/wellcomeopenres.15587.1 ◽

2020 ◽

Vol 5 ◽

pp. 71 ◽

Cited By ~ 3

Author(s):

Edward J Marr ◽

Rachel M Milne ◽

Burcu Anar ◽

Gareth Girling ◽

Frank Schwach ◽

...

Keyword(s):

Marker Genes ◽

Plasmodium Chabaudi ◽

Chronic Infections ◽

Selectable Marker Genes ◽

Erythrocyte Invasion ◽

Infection Dynamics ◽

Marker Free ◽

Parasite Replication

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti-Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and –tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

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Plasmodium falciparum Field Isolates Commonly Use Erythrocyte Invasion Pathways That Are Independent of Sialic Acid Residues of Glycophorin A

Infection and Immunity ◽

10.1128/iai.67.11.5784-5791.1999 ◽

1999 ◽

Vol 67 (11) ◽

pp. 5784-5791 ◽

Cited By ~ 116

Author(s):

Jude Nnaemeka Okoyeh ◽

C. R. Pillai ◽

Chetan E. Chitnis

Keyword(s):

Plasmodium Falciparum ◽

Sialic Acid ◽

Vaccine Development ◽

Glycophorin A ◽

Invasion Pathways ◽

Field Isolates ◽

Erythrocyte Invasion ◽

In Vitro Invasion ◽

Invasion Assays

ABSTRACT Erythrocyte invasion by malaria parasites is mediated by specific molecular interactions. Sialic acid residues of glycophorin A are used as invasion receptors by Plasmodium falciparum. In vitro invasion studies have demonstrated that some cloned P. falciparum lines can use alternate receptors independent of sialic acid residues of glycophorin A. It is not known if invasion by alternate pathways occurs commonly in the field. In this study, we used in vitro growth assays and erythrocyte invasion assays to determine the invasion phenotypes of 15 P. falciparum field isolates. Of the 15 field isolates tested, 5 multiply in both neuraminidase and trypsin-treated erythrocytes, 3 multiply in neuraminidase-treated but not trypsin-treated erythrocytes, and 4 multiply in trypsin-treated but not neuraminidase-treated erythrocytes; 12 of the 15 field isolates tested use alternate invasion pathways that are not dependent on sialic acid residues of glycophorin A. Alternate invasion pathways are thus commonly used by P. falciparum field isolates. Typing based on two polymorphic markers, MSP-1 and MSP-2, and two microsatellite markers suggests that only 1 of the 15 field isolates tested contains multiple parasite genotypes. Individual P. falciparum lines can thus use multiple invasion pathways in the field. These observations have important implications for malaria vaccine development efforts based on EBA-175, the P. falciparumprotein that binds sialic acid residues of glycophorin A during invasion. It may be necessary to target parasite ligands responsible for the alternate invasion pathways in addition to EBA-175 to effectively block erythrocyte invasion by P. falciparum.

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Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

Journal of Experimental Medicine ◽

10.1084/jem.165.2.359 ◽

1987 ◽

Vol 165 (2) ◽

pp. 359-367 ◽

Cited By ~ 31

Author(s):

F W Klotz ◽

D E Hudson ◽

H G Coon ◽

L H Miller

Keyword(s):

Monoclonal Antibodies ◽

Malaria Infection ◽

Rhesus Monkeys ◽

Plasmodium Knowlesi ◽

Rabbit Antiserum ◽

Antigenic Determinants ◽

Partial Protection ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

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Naturally induced humoral response against Plasmodium vivax reticulocyte binding protein 2P1

Malaria Journal ◽

10.1186/s12936-021-03784-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jenni Hietanen ◽

Anongruk Chim-ong ◽

Jetsumon Sattabongkot ◽

Wang Nguitragool

Keyword(s):

Plasmodium Vivax ◽

Vaccine Development ◽

Natural Infection ◽

Humoral Response ◽

Human Antibody ◽

Asymptomatic Carriers ◽

Erythrocyte Invasion ◽

Human Proteins ◽

Human Immune Response

Abstract Background Plasmodium vivax is the most prevalent malaria parasite in many countries. A better understanding of human immunity to this parasite can provide new insights for vaccine development. Plasmodium vivax Reticulocyte Binding Proteins (RBPs) are key parasite proteins that interact with human proteins during erythrocyte invasion and are targets of the human immune response. The aim of this study is to characterize the human antibody response to RBP2P1, the most recently described member of the RBP family. Methods The levels of total IgG and IgM against RBP2P1 were measured using plasmas from 68 P. vivax malaria patients and 525 villagers in a malarious village of western Thailand. The latter group comprises asymptomatic carriers and healthy uninfected individuals. Subsets of plasma samples were evaluated for anti-RBP2P1 IgG subtypes and complement-fixing activity. Results As age increased, it was found that the level of anti-RBP2P1 IgG increased while the level of IgM decreased. The main anti-RBP2P1 IgG subtypes were IgG1 and IgG3. The IgG3-seropositive rate was higher in asymptomatic carriers than in patients. The higher level of IgG3 was correlated with higher in vitro RBP2P1-mediated complement fixing activity. Conclusions In natural infection, the primary IgG response to RBP2P1 was IgG1 and IgG3. The predominance of these cytophilic subtypes and the elevated level of IgG3 correlating with complement fixing activity, suggest a possible role of anti-RBP2P1 antibodies in immunity against P. vivax.

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Plasmodium falciparum Line-Dependent Association ofIn VitroGrowth-Inhibitory Activity and Risk of Malaria

Infection and Immunity ◽

10.1128/iai.06190-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1900-1908 ◽

Cited By ~ 11

Author(s):

Josea Rono ◽

Anna Färnert ◽

Daniel Olsson ◽

Faith Osier ◽

Ingegerd Rooth ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Inhibitory Activity ◽

Content Type ◽

Phenotypic Differences ◽

Erythrocyte Invasion ◽

Growth Inhibitory ◽

Growth Inhibitory Activity ◽

Control Study

ABSTRACTPlasmodium falciparum's ability to invade erythrocytes is essential for its survival within the human host. Immune mechanisms that impair this ability are therefore expected to contribute to immunity against the parasite. Plasma of humans who are naturally exposed to malaria has been shown to have growth-inhibitory activity (GIA)in vitro. However, the importance of GIA in relation to protection from malaria has been unclear. In a case-control study nested within a longitudinally followed population in Tanzania, plasma samples collected at baseline from 171 individuals (55 cases and 116 age-matched controls) were assayed for GIA using threeP. falciparumlines (3D7, K1, and W2mef) chosen based on their erythrocyte invasion phenotypes. Distribution of GIA differed between the lines, with most samples inhibiting the growth of 3D7 and K1 and enhancing the growth of W2mef. GIA to 3D7 was associated with a reduced risk of malaria within 40 weeks of follow-up (odds ratio, 0.45; 95% confidence interval [CI], 0.21 to 0.96;P= 0.04), whereas GIA to K1 and W2mef was not. These results show that GIA, as well as its association with protection from malaria, is dependent on theP. falciparumline and can be explained by differences in erythrocyte invasion phenotypes between parasite lines. Our study contributes knowledge on the biological importance of growth inhibition and the potential influence ofP. falciparumerythrocyte invasion phenotypic differences on its relationship to protective immunity against malaria.

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Η χρήση του πεπτιδίου Ec της ισόμορφης IGF-1Ec στην επισκευή χόνδρινων βλαβών

10.12681/eadd/43322 ◽

2018 ◽

Author(s):

Νικόλαος Αρμακόλας

Keyword(s):

Polymerase Chain Reaction ◽

Trypan Blue ◽

Quantitative Polymerase Chain Reaction ◽

Blue Staining ◽

Alcian Blue Staining ◽

Chain Reaction ◽

Polymerase Chain ◽

Tgf Β1 ◽

Invasion Assays

Το πεπτίδιο Ec (PEc) του IGF-1Ec (IGF-1Ec) επάγει την κινητοποίηση των ανθρωπίνων μεσεγχυματικών βλαστικών κυττάρων (hMSC) και ενεργοποιεί την εξωκυτταρική κινάση 1 και 2 (ERK 1/2) διαφόρων κυττάρων. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της επιδρασης του PEc στην κινητοποίηση και τη διαφοροποίηση των hMSCs, καθώς και η δυνατότητα εφαρμογής του σε συνδυασμό με τον TGF-β1 (TGF-β1) στην επιδιόρθωση του αρθρικού χόνδρου. Τα αποτελέσματα της εξωγενούς χορήγησης του ΡΕc και του ΤGF-β1, ξεχωριστά και σε συνδυασμό, σε hMSCs εκτιμήθηκαν χρησιμοποιώντας trypan blue assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis, Alcian blue staining, wound healing assays και migration/invasion assays. Προσδιορίστηκε ότι το PEc εμπλέκεται στη διαδικασία διαφοροποίησης των hMSCs προς υαλώδη χόνδρο. Η χορήγηση PEc ή / και TGF-β1 σε hMSCs έδειξε συγκρίσιμη εναπόθεση χονδρικής θεμέλειας ουσίας. Ακόμα, η χορήγηση του ΡΕc σε συνδυασμό με τον ΤGF-β1 συσχετίστηκε με μια σημαντική αύξηση στην κινητοποίηση των hMSC σε σύγκριση με την χορήγηση μόνο του TGF-β1 ή του ΡEc (Ρ <0,05). Επομένως, το ΡΕc φαίνεται να διευκολύνει in vitro την κινητοποίηση των hMSC και την διαφοροποίηση τους προς χονδροκύτταρα, ενισχύοντας το ρόλο του ΤGF-β1.

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Acute Plasmodium chabaudi chabaudi Malaria Infection Induces Antibodies Which Bind to the Surfaces of Parasitized Erythrocytes and Promote Their Phagocytosis by Macrophages In Vitro

Infection and Immunity ◽

10.1128/iai.66.9.4080-4086.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4080-4086 ◽

Cited By ~ 52

Author(s):

Maria M. Mota ◽

K. Neil Brown ◽

Anthony A. Holder ◽

William Jarra

Keyword(s):

Parasite Clearance ◽

Enzyme Linked Immunosorbent Assay ◽

Antibody Activity ◽

Parasite Line ◽

Primary Role ◽

Plasmodium Chabaudi ◽

Species Specific ◽

Cellular Immune ◽

Variant Line

ABSTRACT CBA/Ca mice infected with 5 × 104 Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks (∼40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P. c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken fromP. c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P. c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P. c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P. c. chabaudi CB) did not mediate the same effect against P. c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.

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