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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.

The modulation of the host’s metabolism to protect tissue from damage induces tolerance to infections increasing survival. Here, we examined the role of the thyroid hormones, key metabolic regulators, in the outcome of malaria. Hypothyroidism confers protection to experimental cerebral malaria by a disease tolerance mechanism. Hypothyroid mice display increased survival after infection with Plasmodium berghei ANKA, diminishing intracranial pressure and brain damage, without altering pathogen burden, blood-brain barrier disruption, or immune cell infiltration. This protection is reversed by treatment with a Sirtuin 1 inhibitor, while treatment of euthyroid mice with a Sirtuin 1 activator induces tolerance and reduces intracranial pressure and lethality. This indicates that thyroid hormones and Sirtuin 1 are previously unknown targets for cerebral malaria treatment, a major killer of children in endemic malaria areas., This work was funded by grants SAF2017-83289-R to S.A. and A.A., SAF2017-90604REDT to A.A. supported by the The European Regional Development Fund (FEDER) and BIO2016-77430-R to J.M.B. from the Ministerio de Economía y Competitividad; B2017/BMD-3724 to S.A. and A.A. from the Comunidad de Madrid; and CIBERONC CB/16/00228 to A.A. from the Instituto de Salud Carlos III.

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.

In Plasmodium falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase‒6-phosphogluconolactonase (PfG6PD‒6PGL) is involved in the catalysis of the first reaction of the pentose phosphate pathway. Since this enzyme has a key role in parasite development, its unique structure represents a potential target for the discovery of antimalarial drugs. Here we describe the first 3D structural model of the G6PD domain of PfG6PD‒6PGL. Compared to the human enzyme (hG6PD), the 3D model has enabled the identification of a key difference in the substrate-binding site, which involves the replacement of Arg365 in hG6PD by Asp750 in PfG6PD. In a prospective validation of the model, this critical change has been exploited to rationally design a novel family of substrate analog-based inhibitors that can display the necessary selectivity towards PfG6PD. A series of glucose derivatives featuring an α-methoxy group at the anomeric position and different side chains at position 6 bearing distinct basic functionalities has been synthesized, and their PfG6PD and hG6PD inhibitory activities and their toxicity against parasite and mammalian cells have been assessed. Several compounds displayed micromolar affinity (Ki up to 23 μM), favorable selectivity (up to > 26-fold), and low cytotoxicity. Phenotypic assays with P. falciparum cultures revealed high micromolar IC50 values, likely as a result of poor internalization of the compounds in the parasite cell. Overall, these results endorse confidence to the 3D model of PfG6PD, paving the way for the use of target-based drug design approaches in antimalarial drug discovery studies around this promising target.

Background:Recent advances in antiviral therapy show potential for a cure and/or control of most human infections caused by hepatitis viruses and retroviruses. However, medical success is largely dependent on the identification of the large number of people unaware of these infections, especially in developing countries. Dried blood spots (DBS) have been demonstrated to be a good tool for collecting, storing and transporting clinical specimens from rural areas and limited-resource settings to laboratory facilities, where viral infections can be more reliably diagnosed.Methods:The seroprevalence and virological characterization of hepatitis B virus (HBV) and hepatitis C virus (HCV), as well as human retroviruses (HIV-1, HIV-2, human T-cell leukaemia virus type 1 [HTLV-1] and human T-cell leukaemia virus type 2 [HTLV-2]), were investigated in clinical specimens collected from DBS in Ghana.Results:A total of 305 consecutive DBS were collected. A high prevalence of chronic HBV (8.5%) and occult hepatitis B (14.2%) was found, whereas rates were lower for HIV-1, HTLV-1 and HCV (3.2%, 1.3% and 0.6%, respectively). HIV-2 and HTLV-2 were absent. CRF02_AG was the predominant HIV-1 subtype, whereas genotype E was the most frequent HBV variant.Conclusions:DBS are helpful in the diagnosis and virological characterization of hepatitis and retrovirus infections in resource-limited settings. The high rate of hepatitis B in Ghana, either overt or occult, is noteworthy and confirms recent findings from other sub-Saharan countries. This should encourage close clinical follow up and antiviral treatment assessment in this population, as well as universal HBV vaccine campaigns.

Malaria is a parasitic disease caused by Plasmodium spp., being one of the major causes of death worldwide with two-hundred million new infections and hundreds of thousands of deaths in 2015. Despite the important advances in its prevention and treatment, its resistance to current drug therapies is still a serious risk in its eradication. There is urgency in finding novel targets and drugs operating by novel mechanisms, avoiding cross-resistance to classical antimalarials. In this context, the bifunctional enzyme Glucose-6- phosphate dehydrogenase 6-phosphogluconolactonase appears to be a promising therapeutic target due to its crucial role in regulating the PPP pathway (pentose phosphate pathway), which is the major source of redox potential in Plasmodium falciparum. In the last few years, our group detected a specific mutation between the human and the Plasmodium falciparum form in the binding site of Glucose-6-phosphate (G6P), the endogenous ligand of Glucose-6-phosphate dehydrogenase (G6PD). This mutation involves the substitution of an Arginine (human) by an Aspartate (parasite), which allowed us to create a validated in-house homology model of PfG6PD. Based on this result, the group has focused their efforts, through different molecular modelling techniques, in the discovery of selective scaffolds against PfG6PD. Current efforts address the development of a complete structural model of the bifunctional enzyme, which may offer novel opportunities to develop molecules capable of inhibiting this relevant enzyme.

There currently exists a dire need for safe and inexpensive new antimalarial drugs, which are effective against multiple life cycle stages of Plasmodium falciparum, and act through mechanisms that differ from those of the available drugs to prevent drug resistance. [...]

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.

Oxidative stress has been attributed both a key pathogenic and rescuing role in cerebral malaria (CM). In a Plasmodium berghei ANKA murine model of CM, host redox signaling and functioning were examined during the course of neurological damage. Host antioxidant defenses were early altered at the transcriptional level indicated by the gradually diminished expression of superoxide dismutase-1 (sod-1), sod-2, sod-3 and catalase genes. During severe disease, this led to the dysfunctional activity of superoxide dismutase and catalase enzymes in damaged brain regions. Vitagene associated markers (heat shock protein 70 and thioredoxin-1) also showed a decaying expression pattern that paralleled reduced expression of the transcription factors Parkinson disease 7, Forkhead box O 3 and X-box binding protein 1 with a role in preserving brain redox status. However, the oxidative stress markers reactive oxygen/nitrogen species were not accumulated in the brains of CM mice and redox proteomics and immunohistochemistry failed to detect quantitative or qualitative differences in protein carbonylation. Thus, the loss of antioxidant capacity was compensated for in all cerebral regions by progressive upregulation of heme oxygenase-1, and in specific regions by early glutathione peroxidase-1 induction. This study shows for the first time a scenario of cooperative glutathione peroxidase and heme oxygenase-1 upregulation to suppress superoxide dismutase, catalase, heat shock protein-70 and thioredoxin-1 downregulation effects in experimental CM, counteracting oxidative damage and maintaining redox equilibrium. Our findings reconcile the apparent inconsistency between the lack of oxidative metabolite build up and reported protective effect of antioxidant therapy against CM.

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.

Resistance to chloroquine (CQ) in Plasmodium falciparum has a major impact on malaria control worldwide. To gain insight into early parasite stress response, mRNA expression profiles were determined for a set of 10 antioxidant defence genes in synchronized CQ-sensitive (3D7) and CQ-resistant (Dd2) clones under transient IC50 CQ-exposure (Dd2, 200 nM; 3D7, 14 nM). Upon 2-h CQ challenge, the mRNA upregulation detected was greater in 3D7 (six genes overexpressed at 1/3 of the intraerythrocytic cycle) than in Dd2 clone (three genes responding), providing evidence of an early transcriptional response to CQ-induced oxidative stress which might underlie some of the parasite's metabolic adaptation to the drug.

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

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.

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.

The role of neurotrophic factors on the integrity of the central nervous system (CNS) during cerebral malaria (CM) infection remains obscure, but the long-standing neurocognitive sequelae often observed in rescued children can be attributed in part to the modulation of neuronal survival and synaptic plasticity. To discriminate the contribution of key responses in the time-sequence of the pathogenic events that trigger the development of neurocognitive malaria syndrome we defined four stages (I-IV) of the neurological progression of CM in C57BL/6 mice infected with Plasmodium berghei ANKA. Upregulation of ICAM-1, VCAM-1, e-selectin and p-selectin expression was detected in all cerebral regions before parasitized red blood cells (pRBC) accumulation. As the severity of symptoms increased, BDNF mRNA progressively diminished in several brain regions, earliest in the thalamus-hypothalamus, cerebellum, brainstem and cortex, and correlated with a four-stage disease sequence. Immunohistochemical confocal microscopy revealed changes in the BDNF distribution pattern, suggesting altered axonal transport. During CM progression, molecular markers of neurological infection and inflammation in the parasite and the host, respectively, were accompanied by a switch in the brain constitutive proteasome to the immunoproteasome, which could impede normal protein turnover. In parallel with BDNF downregulation, NCAM expression also diminished with increased CM severity. Together, these data suggest that changes in BDNF availability could be involved in the pathogenesis of CM.

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

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)

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k2nd (M−1s−1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.