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Iron overload caused by hereditary hemochromatosis (HH) increases free reactive oxygen species that, in turn, induce lipid peroxidation. Its 4-hydroxynonenal (HNE) by-product is a well-established marker of lipid peroxidation since it reacts with accessible proteins with deleterious consequences. Indeed, elevated levels of HNE are often detected in a wide variety of human diseases related to oxidative stress. Here, we evaluated HNE-modified proteins in the membrane of erythrocytes from HH patients and in organs of Hfe−/− male and female mice, a mouse model of HH. For this purpose, we used one- and two-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF analysis. We identified cytoskeletal membrane proteins and membrane receptors of erythrocytes bound to HNE exclusively in HH patients. Furthermore, kidney and brain of Hfe−/− mice contained more HNE-adducted protein than healthy controls. Our results identified main HNE-modified proteins suggesting that HH favours preferred protein targets for oxidation by HNE.

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.

The co-endemicity of malnutrition, erythrocytopathies, transmissible diseases and iron-deficiency contribute to the prevalence of chronic anaemia in many populations of the developing world. Although iron dietary supplementation is applied or recommended in at risk populations, its use is controversial due to undesirable outcomes, particularly regarding the response to infections, including highly prevalent malaria. We hypothesized that a boosted oxidative stress due to iron supplementation have a similar impact on malaria to that of hereditary anaemias, enhancing innate response and conditioning tissues to prevent damage during infection. Thus, we have analysed antioxidant and innate responses against lethal Plasmodium yoelii during the first five days of infection in an iron-supplemented mouse. This murine model showed high iron concentration in plasma with upregulated expression of hemoxygenase-1. The sustained homeostasis after this extrinsic iron conditioning, delayed parasitemia growth that, once installed, developed without anaemia. This protection was not conferred by the intrinsic iron overload of hereditary hemochromatosis. Upon iron-supplementation, a large increase of the macrophages/dendritic cells ratio and the antigen presenting cells was observed in the mouse spleen, independently of malaria infection. Complementary, malaria promoted the splenic B and T CD4 cells activation. Our results show that the iron supplementation in mice prepares host tissues for oxidative-stress and induces unspecific cellular immune responses, which could be seen as an advantage to promote early defences against malaria infection.

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.

Background and Purpose Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. Experimental Approach Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. Key Results Borrelidin protected against lethal malaria at 0.25 mg·kg−1·day−1. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. Conclusions and Implications Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Babesiosis is an emerging zoonosis now found in several areas of the world. Using PCR and indirect immunofluorescence assay, we have diagnosed the first case of human babesiosis caused by Babesia microti in Spain. Diagnosis was delayed because of the nonspecific clinical symptoms that occurred in an immunocompetent patient. We thank Cesar Eguiluz for his contribution to this study. This work is funded by the Surveillance Program of the Centro Nacional de Microbiología, the Center for Collaborative Research (RETIC-RICET), and the grants from Ministerio de Economia y Competitividad of Spain (grant numbers AGL2010-21774 and AGL2014-56193-R awarded to E.M. and L.M.G. and grant number BIO2013-44565-R awarded to J.M.B.) and the NIH of the USA (grant numbers HL105694 and HL129215 awarded to C.A.L.).

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

Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethalPlasmodium yoelii yoelii17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses ofP. yoelii yoelii17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

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.

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.