Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria

Abstract Background The development of protective immunity against malaria is slow and to be maintained, it requires exposure to multiple antigenic variants of malaria parasites and age-associated maturation of the immune system. Evidence that the protective immunity is associated with different classes and subclasses of antibodies reveals the importance of considering the quality of the response. In this study, we have evaluated the humoral immune response against Plasmodium falciparum blood stages of individuals naturally exposed to malaria who live in endemic areas of Brazil in order to assess the prevalence of different specific isotypes and their association with different malaria clinical expressions. Methods Different isotypes against P. falciparum blood stages, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA, were determined by ELISA. The results were based on the analysis of different clinical expressions of malaria (complicated, uncomplicated and asymptomatic) and factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks. The occurrence of the H131 polymorphism of the FcγIIA receptor was also investigated in part of the studied population. Results The highest levels of IgG, IgG1, IgG2 and IgG3 antibodies were observed in individuals with asymptomatic and uncomplicated malaria, while highest levels of IgG4, IgE and IgM antibodies were predominant among individuals with complicated malaria. Individuals reporting more than five previous clinical malaria attacks presented a predominance of IgG1, IgG2 and IgG3 antibodies, while IgM, IgA and IgE antibodies predominated among individuals reporting five or less previous clinical malaria attacks. Among individuals with uncomplicated and asymptomatic malaria, there was a predominance of high-avidity IgG, IgG1, IgG2 antibodies and low-avidity IgG3 antibodies. The H131 polymorphism was found in 44.4% of the individuals, and the highest IgG2 levels were observed among asymptomatic individuals with this allele, suggesting the protective role of IgG2 in this population. Conclusion Together, the results suggest a differential regulation in the anti-P. falciparum antibody pattern in different clinical expressions of malaria and showed that even in unstable transmission areas, protective immunity against malaria can be observed, when the appropriated antibodies are produced.

Increased interleukin-10 and interferon-γ levels in Plasmodium vivax malaria suggest a reciprocal regulation which is not altered by IL-10 gene promoter polymorphism

Background In human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed. Methods The serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). Results The levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma. Conclusions This study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Signal transduction in Plasmodium-Red Blood Cells interactions and in cytoadherence

Malaria is responsible for more than 1.5 million deaths each year, especially among children (Snow et al. 2005). Despite of the severity of malaria situation and great effort to the development of new drug targets (Yuan et al. 2011) there is still a relative low investment toward antimalarial drugs. Briefly there are targets classes of antimalarial drugs currently being tested including: kinases, proteases, ion channel of GPCR, nuclear receptor, among others (Gamo et al. 2010). Here we review malaria signal transduction pathways in Red Blood Cells (RBC) as well as infected RBCs and endothelial cells interactions, namely cytoadherence. The last process is thought to play an important role in the pathogenesis of severe malaria. The molecules displayed on the surface of both infected erythrocytes (IE) and vascular endothelial cells (EC) exert themselves as important mediators in cytoadherence, in that they not only induce structural and metabolic changes on both sides, but also trigger multiple signal transduction processes, leading to alteration of gene expression, with the balance between positive and negative regulation determining endothelial pathology during a malaria infection.

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n = 87) and P. falciparum (n = 164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P = 0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P = 0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.

IFN-γ-induced priming maintains long-term strain-transcending immunity against blood-stage Plasmodium chabaudi malaria

The mechanism by which protective immunity to Plasmodium is lost in the absence of continued exposure to this parasite has yet to be fully elucidated. It has been recently shown that IFN-γ produced during human and murine acute malaria primes the immune response to TLR agonists. In this study, we investigated whether IFN-γ-induced priming is important to maintain long-term protective immunity against Plasmodium chabaudi AS malaria. On day 60 postinfection, C57BL/6 mice still had chronic parasitemia and efficiently controlled homologous and heterologous (AJ strain) challenge. The spleens of chronic mice showed augmented numbers of effector/effector memory (TEM) CD4(+) cells, which is associated with increased levels of IFN-γ-induced priming (i.e., high expression of IFN-inducible genes and TLR hyperresponsiveness). After parasite elimination, IFN-γ-induced priming was no longer detected and protective immunity to heterologous challenge was mostly lost with >70% mortality. Spontaneously cured mice had high serum levels of parasite-specific IgG, but effector T/TEM cell numbers, parasite-driven CD4(+) T cell proliferation, and IFN-γ production were similar to noninfected controls. Remarkably, the priming of cured mice with low doses of IFN-γ rescued TLR hyperresponsiveness and the capacity to control heterologous challenge, increasing the TEM cell population and restoring the CD4(+) T cell responses to parasites. Contribution of TLR signaling to the CD4(+) T cell responses in chronic mice was supported by data obtained in mice lacking the MyD88 adaptor. These results indicate that IFN-γ-induced priming is required to maintain protective immunity against P. chabaudi and aid in establishing the molecular basis of strain-transcending immunity in human malaria.

Liver accumulation of Plasmodium chabaudi-infected red blood cells and modulation of regulatory T Cell and dendritic cell responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (PciRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

A study of the anti-plasmodium activity of angiotensin II analogs

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase method on Merrifield's resin using the t-Boc strategy, purified by RP-HPLC and characterized by liquid chromatography/ESI (+) MS (LC-ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti-plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least-square analysis, assessing the position-wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C-terminus, as well as that of hydrophobic amino acids in the N-terminus, suggests that the mechanism underlying the anti-malarial activity of these peptides is attributed to its amphiphilic character.

Antibody responses in Plasmodium falciparum malaria and their relation to protection against the disease

Protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the P. Falciparum infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to P. falciparum infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure. Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an in vitro assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes. The P. falciparum parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated P. falciparum parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition in vitro by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition in vitro, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses. Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area. There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against P. falciparum asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.

Computational investigation of the Plasmodium falciparum fatty acid biosynthetic pathway toward the discovery of novel antimalarials

The structural peculiarities of a protein are related to its biological function. In the fatty acid elongation cycle, one small carrier protein shuttles and delivers the acyl intermediates from one enzyme to the other. The carrier has to recognize several enzymatic counterparts, specifically interact with each of them, and finally transiently deliver the carried substrate to the active site. Carry out such a complex game requires the players to be flexible and efficiently adapt their structure to the interacting protein or substrate. In a drug discovery effort, the structure-function relationships of a target system should be taken into account to optimistically interfere with its biological function. In this doctoral work, the essential role of structural plasticity in key steps of fatty acid biosynthesis in Plasmodium falciparum is investigated by means of molecular simulations. The key steps considered include the delivery of acyl substrates and the structural rearrangements of catalytic pockets upon ligand binding. The ground-level bases for carrier/enzyme recognition and interaction are also put forward. The structural features of the target have driven the selection of proper drug discovery tools, which captured the dynamics of biological processes and could allow the rational design of novel inhibitors. The model may be perspectively used for the identification of novel pathway-based antimalarial compounds.

Charakterisierung der biologischen Funktion der Plasmodium falciparum Calcium-abhängigen Proteinkinase 1

Im Rahmen dieser Arbeit wurden biologische Funktionen einer Proteinkinase des Erregers der Malaria tropica, genauer der „Plasmodium falciparum calcium dependent protein kinase 1“ (PfCDPK1), in parasitären Blutstadien untersucht.rnUm Einblicke in die Funktion der Kinase, die sie in den extrazellulären Kompartimenten des Parasiten übernimmt, zu gewinnen, wurden sechs Proteine untersucht, die dasselbe Translokationsignal wie PfCDPK1 besitzen. Es konnte gezeigt werden, dass fünf der untersuchten Proteine mit der PfCDPK1 im Bereich der parasitophoren Vakuole sowie des tubovesikulären Systems co-lokalisiert sind. Deletionsmutanten, denen das Translokationssignal fehlte, sowie ein Peptid, das lediglich aus diesem bestand, bestätigten, dass die Translokation in die extrazellulären Kompartimente von keinen weiteren Faktoren, außer dem Signalmotiv abhängt. Mit PfCAP und PfRKIP konnten zwei Regulatoren der PfCDPK1 identifiziert werden. PfARM, Pfrab_5b sowie PfGAP45 sind Substrate der PfCDPK1. Mit Hilfe von massenspektrometrischen Messungen wurde der Phosphorylierungsstatus der untersuchten Proteine durch die PfCDPK1 sowie der Autophosphorylierungsstatus der Kinase bestimmt, um Rückschlüsse auf regulatorische Prozesse ziehen zu können.rnDie Phosphorylierung von PfGAP45 durch die PfCDPK1 steht vermutlich mit dem Invasionsprozess des Parasiten in direktem Zusammenhang, da gezeigt wurde, dass eine Hemmung der Kinase mit PP1 einen 90%igen Rückgang an neu infizierten Erythrozyten zur Folge hatte.rnrn

High gradient magnetic separation (HGMS) of erythrocytes infected with plasmodium falciparum

This thesis was undertaken to explore possible applications of high gradient magnetic separation (HGMS) for the separation of RBCs infected with Plasmodium falciparum, with the dual aim of establishing a novel and superior method for isolating late-stage infected cells, and of obtaining synchronized cell cultures.rnThe presented work presents protocols for HGMS of parasitized RBCs that fulfil these aims. Late-stage parasitized cell can be isolated essentially devoid of contamination with non-infected and ring-stage infected cells. Such an easy method for a highly quantitative and qualitative purification has not yet been reported. Synchronous cultures can be obtained both following depletion of late-stage infected cells, and following isolation of the latter. The quality of synchronization cultures matches that of sorbitol lysis, the current standard method for malaria culture synchronization. An advantage of HGMS is the avoidance of osmotic stress for RBCs. The new methods further have the appeal of high reproducibility, cost-effectiveness, and simple protocol.rnIt should be possible to take the methods beyond Plasmodium infected RBCs. Most magnetic separation techniques in the sector of biomedical research employ columns with a hydrophilic polymer-coated matrix. Our procedure employs an optimized buffer system. Polymer coating becomes unnecessary and uncoated columns are available at a fraction of the cost.