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Incomplete non-sterile immunity to malaria is attained in endemic regions after recurrent infections by a large percentage of the adult population, who carry the malaria parasite asymptomatically. Although blood-stagePlasmodium falciparumrapidly elicits IgG responses, the target antigens of partially protective and non-protective IgG antibodies as well as the basis for the acquisition of these antibodies remain largely unknown. We performed IgG-immunomics to screen forP. falciparumantigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients’ IgGs. For further epitope mapping, peptide microarrays designed to cover their sequences were probed with a set of 38 sera samples from adult individuals of an endemic malaria region in Ghana. Eight 20-mer peptides with the highest affinity and frequency of recognition among the population were subsequently validated with 16 sera from the same region, segregated into patients with positive or negative subclinical detection ofP. falciparum. Significant binding specificity for two immunodominant antigenic regions was uncovered within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer peptides challenged with sera samples from children under 5 years old displayed specific IgG binding in those with detectable parasitemia, even at subclinical level. These results suggest that the humoral response against START and PDI8 antigens may be triggered even at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.SignificanceMalaria in Africa is a leading cause of morbidity and mortality. The reservoirs of the malaria parasite are asymptomatic patients who carry it subclinically. Identifying the parasite antigens and its fragments that trigger the most common immunity response by immunoglobulin G that partially protect people can have profound implications for both, development of a malaria vaccine and diagnosis of the subclinical parasite carriers. Antigen discovery and mapping, validated with sera from subclinical carriers, showed that immunoglobulin G responses in children against parasite’s START and PDI8 may eventually be used to differentially diagnose non-infected from subclinical cases. Furthermore, anti-START and anti-PDI8 endemic immunodominance provides association of these antigens with long-term acquired immunity and immune evasion to malaria.

Proteomics is improving malaria research by providing global information on relevant protein sets from the parasite and the host in connection with its cellular structures and specific functions. In the last decade, reports have described biologically significant elements in the proteome of Plasmodium, which are selectively targeted and quantified, allowing for sensitive and high-throughput comparisons. The identification of molecules by which the parasite and the host react during the malaria infection is crucial to the understanding of the underlying pathogenic mechanisms. Hence, proteomics is playing a major role by defining the elements within the pathogenic space between both organisms that change across the parasite life cycle in association with the host transformation and response. Proteomics has identified post-translational modifications in the parasite and the host that are discussed in terms of functional interactions in malaria parasitism. Furthermore, the contribution of proteomics to the investigation of immunogens for potential vaccine candidates is summarized. The malaria-specific technological advances in proteomics are particularly suited now for identifying host-parasite interactions that could lead to promising targets for therapy, diagnosis or prevention. In this review, we examine the knowledge gained on the biology, pathogenesis, immunity and diagnosis of Plasmodium infection from recent proteomic studies. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

The molecular basis for the prevalence of blood group O in regions where malaria is endemic remains unclear. In some genetic backgrounds oxidative modifications have been linked to a reduced susceptibility to severe malaria disease. Through redox proteomics, we detected differences in carbonylated membrane proteins among the different blood groups, both in Plasmodium-infected and uninfected erythrocytes (RBC). Carbonylation profiles of RBC membrane proteins revealed that group O blood shows a reduced protein oxidation pattern compared to groups A, B and AB. Upon infection with Plasmodium falciparum Dd2, erythrocytes of all blood groups showed increased oxidation of membrane proteins. By examining 4-hydroxy-2-nonenal (4-HNE) modified proteins by LC-MS/MS (liquid chromatography/mass spectrometry) we observed that, upon malaria infection, the protein components of lipid rafts and cytoskeleton were the main targets of 4-HNE carbonylation in all blood groups. Ankyrins and protein bands 4.2 and 4.1 were differentially carbonylated in group O as compared to A and B groups. During trophozoite maturation in group O erythrocytes, a steady increase was observed in the number of 4-HNE-modified proteins, suggesting a parasite-driven 4-HNE-carbonylation process. Our findings indicate a possible correlation between the protection against severe malaria in blood group O individuals and a specific pattern of 4-HNE-carbonylation of cytoskeleton proteins.

As the search for an effective human malaria vaccine continues, understanding immune responses to Plasmodium in rodent models is perhaps the key to unlocking new vaccine strategies. The recruitment of parasite-specific antibodies is an important component of natural immunity against infection in blood-stage malaria. Here, we describe the use of sera from naturally surviving ICR mice after infection with lethal doses of Plasmodium yoelii yoelii 17XL to identify highly immunogenic blood-stage antigens. Immobilized protein A/G was used for the affinity-chromatography purification of the IgGs present in pooled sera from surviving mice. These protective IgGs, covalently immobilized on agarose columns, were then used to isolate reactive antigens from whole P. yoelii yoelii 17XL protein extracts obtained from the blood-stage malaria infection. Through proteomics analysis of the recovered parasite antigens, we were able to identify two endoplasmic reticulum lumen proteins: protein disulfide isomerase and a member of the heat shock protein 70 family. Also identified were the digestive protease plasmepsin and the 39 kDa-subunit of eukaryotic translation initiation factor 3, a ribosome associated protein. Of these four proteins, three have not been previously identified as antigenic during blood-stage malaria infection. This procedure of isolating and identifying parasite antigens using serum IgGs from malaria-protected individuals could be a novel strategy for the development of multi-antigen-based vaccine therapies. 2.814 JCR (2012) Q3, 70/137 Immunology

Resistance of Plasmodium falciparum to chloroquine hinders malaria control in endemic areas. Current hypotheses on the action mechanism of chloroquine evoke its ultimate interference with the parasite's oxidative defence systems. Through carbonyl derivatization by 2,4-dinitrophenylhydrazine and proteomics, we compared oxidatively modified proteins across the parasite's intraerythrocytic stages in untreated and transiently IC 50 chloroquine-treated cultures of the chloroquine-resistant P. falciparum strain Dd2. Functional plasmodial protein groups found to be most oxidatively damaged were among those central to the parasite's physiological processes, including protein folding, proteolysis, energy metabolism, signal transduction, and pathogenesis. While an almost constant number of oxidized proteins was detected across the P. falciparum life cycle, chloroquine treatment led to increases in both the extent of protein oxidation and the number of proteins oxidized as the intraerythrocytic cycle progressed to mature stages. Our data provide new insights into early molecular effects produced by chloroquine in the parasite, as well as into the normal protein-oxidation modifications along the parasite cycle. Oxidized proteins involved in the particular parasite drug-response suggest that chloroquine causes specific oxidative stress, sharing common features with eukaryotic cells. Targeting these processes might provide ways of combating chloroquine-resistance and developing new antimalarial drugs.

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

The bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus. This study was designed to examine the effects of RNA-mediated PfG6PD-6PGL gene silencing in cultures of P. falciparum on the expression of parasite antioxidant defense genes at the transcription level. The highest degree of G6PD-6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD-6PGL gene. PfG6PD-6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation. In addition, an immediate transcriptional response was shown by thioredoxin reductase suggesting that P. falciparum G6PD-6PGL plays a physiological role in the specific response of the parasite to intracellullar oxidative stress. P. falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up-regulation of antioxidant genes. Collectively, our findings point to an important role for this enzyme in the parasite's infection cycle. The different characteristics of G6PD-6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies.

Background In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. Methods We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. Results Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the ‘transcription index’ (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. Conclusions The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels. Copyright © 2006 John Wiley & Sons, Ltd.

ICR mice have heterogeneous susceptibility to lethal Plasmodium yoelii yoelii 17XL from the first days of experimental infection as evidenced by the different parasitemia levels and clinical outcomes. This mouse model has revealed specific immune responses on peripheral blood correlating with the infection fate of the animals. To search for immune-markers linked to parasitemia we examined B lymphocytes in organs of the immune system as key effectors of rodent immunity against malaria. To determine changes in immune cellularity fostered by the different prognostic parasitemia we examined B cell subsets in low (15%) and high (50%) parasitized mice during the first days of the infection. In the case of surviving mice, we studied the preservation of memory immune response 500 days after the primary P. yoelii challenge. Correlating with the parasitemia level, it was observed an increase in total cellularity of spleen during the first week of infection which remained after 16 months of the infection in surviving animals. B cell subsets were also modified across the different infection fates. Subpopulation as follicular B cells and B-1 cells proportions behaved differently depending on the parasitemia kinetics. In addition, peritoneal cavity cells proliferated in response to high parasitemia. More significantly, P. yoelii -specific memory B cells remained in the spleen 500 days after the primo-infection. This study demonstrates that B cell kinetics is influenced by the different parasitemia courses which are naturally developed within a same strain of untreated mice. We show that high levels of parasitemia at the beginning of infection promote an extremely fast and exacerbate response of several cell populations in spleen and peritoneal cavity that, in addition, do not follow the kinetics observed in peripheral blood. Furthermore, our results describe the longest persistence of memory B cells long time upon a single malaria infection in mice.

ABSTRACT: Using the published protein sequence from a rabbit microsomal glucose-6-phosphate dehydrogenase G6PD we have isolated and sequenced a cDNA clone coding for its human equivalent, which is also known as hexose-6-phosphate dehydrogenase (H6PD) and glucose dehydrogenase. The corresponding genomic sequence is in the databases enabling its localization to chromosome 1p36. The gene spans 37 kb and consists of 5 exons, the fifth of which codes for more than half of the 89kDa protein. The first intron is a 10kb insertion in the 5′ untranslated sequence. The predicted mRNA has an exceptionally long (6.5kb) 3′ untranslated sequence. The predicted protein shows extensive homology with X-linked G6PD, suggesting the two genes share a common ancestor but no intron positions are conserved between the two genes suggesting the gene duplication was an ancient event. The C-terminal portion of the protein is not homologous with G6PD but shows limited homology with proteins of unknown function found throughout evolution and encoded next to G6PD in various micro-organisms. Intriguingly this C-terminal portion has some homology with the N-terminal sequence of Plasmodium falciparum G6PD.

Malaria infection in humans elicits a wide range of immune responses that can be detected in peripheral blood, but we lack detailed long-term follow-up data on the primary and subsequent infections that lead to naturally acquired immunity. Studies on antimalarial immune responses in mice have been based on models yielding homogenous infection profiles. Here, we present a mouse model in which a heterogeneous course of Plasmodium yoelii lethal malaria infection is produced in a non-congenic ICR strain to allow comparison among different immunological and clinical outcomes. Three different disease courses were observed ranging from a fatal outcome, either early or late, to a self-resolved infection that conferred long-term immunity against re-infection. Qualitative and quantitative changes produced in leukocyte subpopulations and cytokine profiles detected in peripheral blood during the first week of infection revealed that monocytes, dendritic cells and immature B cells were the main cell subsets present in highly-parasitized mice dying in the first week after infection. Besides, CD4(+)CD25(high) T cells expanded at an earlier time point in early deceased mice than in surviving mice and expressed higher levels of intracellular Foxp3 protein. In contrast, survivors showed a limited increase of cytokines release and stable circulating innate cells. From the second week of infection, mice that would die or survive showed similar immune profiles, although CD4(+)CD25(high) T cells number increased earlier in mice with the worst prognosis. In surviving mice the expansion of activated circulating T cell and switched-class B cells with a long-term protective humoral response from the second infection week is remarkable. Our results demonstrate that the follow-up studies of immunological blood parameters during a malaria infection can offer information about the course of the pathological process and the immune response.

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum ( p.f. -ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f. -ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f. -ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f. -ChoK as an accessible drug target against the parasite.

We report a case of an African patient with sickle cell trait who was diagnosed in Spain with B-cell lymphoma. Blood smears were negative for malaria, and no plasmodium antigens were detected in the blood. To treat his lymphoma, the patient underwent chemotherapy and autologous stem cell transplantation. Following a splenectomy due to a worsening condition, he developed clinical malaria with detectable parasitemia. This case suggests that the humoral response and parasite removal by the spleen may afford protection from overt disease and may even help maintain subclinical human reservoirs of the disease.

Multiple glucose-6-phosphate dehydrogenase (G6PD)-deficient alleles have reached polymorphic frequencies because of the protection they confer against malaria infection. A protection mechanism based on enhanced phagocytosis of parasitized G6PD-deficient erythrocytes that are oxidatively damaged is well accepted. Although an association of this phenotype with the impairment of the antioxidant defense in G6PD deficiency has been demonstrated, the dysfunctional pathway leading to membrane damage and modified exposure of the malaria-infected red cell to the host is not known. Thus, in this study, erythrocytes from the common African variant G6PD A- were used to analyze by redox proteomics the major oxidative changes occurring in the host membrane proteins during the intraerythrocytic development of Plasmodium falciparum, the most lethal malaria parasite. Fifteen carbonylated membrane proteins exclusively identified in infected G6PD A- red blood cells revealed selective oxidation of host proteins upon malarial infection. As a result, three pathways in the host erythrocyte were oxidatively damaged in G6PD A-: (1) traffic/assembly of exported parasite proteins in red cell cytoskeleton and surface, (2) oxidative stress defense proteins, and (3) stress response proteins. Additional identification of hemichromes associated with membrane proteins also supports a role for specific oxidative modifications in protection against malaria by G6PD polymorphisms.

Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in proteomic post-transcriptional modification analysis to explain the nature of the specificity underlying oxidative stress mechanisms. For this purpose, a combined protocol of immune-detection, peptide enrichment, mass spectrometry, and de novo protein sequencing has been developed. The methodology was first examined in the model protein bovine serum albumin (BSA), allowing the comparison of matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) performance and sensitivity. Peptide enrichment was optimized by affinity chromatography on HNE-BSA resulting in increased sensitivity. Identification of amino acid residues modified by HNE was finally ascertained by de novo sequencing analysis. The improved methodology was demonstrated on human erythrocyte membrane proteins allowing the identification of HNE-lysine and HNE-histidine Michael adducts in the β-spectrin under physiological conditions.

This protocol describes a method for preparing cultures of Plasmodium falciparum synchronized at any intraerythrocytic stage. Using this method, around 60% parasitized cells may be obtained. On the basis of Trager and Jensen's original continuous culture method, our approach relies on the use of fresh human blood not older than 2 weeks, a low hematocrit between 0.8 and 1.5%, a starting frozen inoculum of 10% ring-stage parasitemia, human serum replaced with AlbuMAX I and alternating sorbitol and Percoll synchronization methods to shorten the cycle window to 4–6 h and reduce sorbitol toxicity. From our synchronized high parasite density cultures, 3–5 ml of infected red blood cells can be obtained in 1 week, corresponding to 1.2 mg of total parasite protein per ml of harvested culture. On the basis of the variables parasitemia and packed cell volume, we provide an equation to accurately calculate the amount of complete medium required every 24 h corrected for the cycle stage and capacity of the culture flask. Ten days suffice to complete the protocol from a frozen stock of parasites.

Background Improvements on malarial diagnostic methods are currently needed for the correct detection in low-density Plasmodium falciparum infections. Microfluorimetric DNA-based assays have been previously used for evaluation of anti-malarial drug efficacy on Plasmodium infected erythrocytes. Several factors affecting the sensitivity of these methods have been evaluated, and tested for the detection and quantification of the parasite in low parasitaemia conditions. Methods Parasitaemia was assessed by measuring SYBRGreen I® (SGI) and PicoGreen® (PG) fluorescence of P. falciparum Dd2 cultures on human red blood cells. Different modifications of standard methods were tested to improve the detection sensitivity. Calculation of IC50 for chloroquine was used to validate the method. Results Removal of haemoglobin from infected red-blood cells culture (IRBC) increased considerably the fluorescent signal obtained from both SGI and PG. Detergents used for cell lysis also showed to have an effect on the fluorescent signal. Upon depletion of haemoglobin and detergents the fluorescence emission of SGI and PG increased, respectively, 10- and 60-fold, extending notably the dynamic range of the assay. Under these conditions, a 20-fold higher PG vs. SGI fluorescent signal was observed. The estimated limits of detection and quantification for the PG haemoglobin/detergent-depleted method were 0.2% and 0.7% parasitaemia, respectively, which allow the detection of ~10 parasites per microliter. The method was validated on whole blood-infected samples, displaying similar results as those obtained using IRBC. Removal of white-blood cells prior to the assay allowed to increase the accuracy of the measurement, by reducing the relative uncertainty at the limit of detection from 0.5 to 0.1. Conclusion The use of PG microassays on detergent-free, haemoglobin-depleted samples appears as the best choice both for the detection of Plasmodium in low-density infections and anti-malarial drugs tests.

The aim of this study was to evaluate the biological correlation and prognostic impact of Gata-1, Gata-2, EKLF, and c-MPL transcript level in a group of 41 acute mieloid leukemia (AML) patients. Gata-1 overexpression was related to advanced age and a low percentage of bone marrow blasts and was associated with the expression of CD34 antigen and lymphoid T markers. The negative impact of Gata-1 expression on the probability of achieving complete remission has been confirmed. Gata-2 overexpression was associated with a low percentage of blasts in BM and males. Expression of c-MPL was associated with CD34+ AML and M2 FAB AML subtype. A higher expression of EKLF was found in secondary AML versus primary AML. Nevertheless, patients expressing EKLF had a longer overall survival and event free survival than those patients that did not express EKLF. Our study has identified expression of EKLF as a factor with a favorable impact on prognosis in AML. Am. J. Hematol, 2009. © 2008 Wiley-Liss, Inc.

Human erythrocyte R-type pyruvate kinase (RPK) deficiency is an autosomal recessive disorder produced by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Survival of patients with severe RPK deficiency has been associated with compensatory expression in red blood cells (RBCs) of M2PK, an isoenzyme showing wide tissue distribution. We describe a novel homozygous null mutation of the PKLR gene found in a girl with a prenatal diagnosis of PK deficiency. The mutant PK gene revealed an 11-nucleotide (nt) duplication at exon 8, causing frameshift of the PKLR transcript, predicting a truncated protein inferred to have no catalytic activity. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detected no M2PK expression in the peripheral blood red cell fraction. The expression of mutant RPK mRNA in the RBCs was almost 6 times higher than that detected in a control patient with hereditary spherocytosis. This molecular phenotypic analysis of the null mutation in the PKLR gene provides evidence for a lack of M2PK in the mature RBCs of this patient and suggests that normal red cell functions and survival are achieved through a population of young erythroid cells released into the circulation in response to anemia. (Blood. 2005;106:1851-1856)