Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC-MS/MS from Dried Serum Spots.

One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.

Protein Susceptibility to Peroxidation by 4-Hydroxynonenal in Hereditary Hemochromatosis.

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.

Hypothyroidism confers tolerance to cerebral malaria.

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.

Plasmodium falciparum immunodominant IgG epitopes in subclinical malaria.

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-stage Plasmodium falciparum rapidly 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 for P. falciparum antigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients' IgGs. Epitope mapping of them, using adult and children sera samples from an endemic malaria region in Ghana segregated into patients with positive or negative subclinical detection of P. falciparum, revealed binding specificity for two 20-mer immunodominant antigenic regions within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer epitopes 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 humoral response against START and PDI8 antigens may be triggered at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.

Screening for retroviruses and hepatitis viruses using dried blood spots reveals a high prevalence of occult hepatitis B in Ghana.

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.

First homology model of Plasmodium falciparum glucose-6-phosphate dehydrogenase: Discovery of selective substrate analog-based inhibitors as novel antimalarial agents.

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.

Iron supplementation in mouse expands cellular innate defences in spleen and defers lethal malaria infection.

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.

Early and late B cell immune responses in lethal and self-cured rodent malaria.

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.

Differential immune response associated to malaria outcome is detectable in peripheral blood following Plasmodium yoelii 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.

Glutathione peroxidase contributes with heme oxygenase-1 to redox balance in mouse brain during the course of cerebral malaria.

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.

Estandarización de un modelo murino de malaria cerebral en fases clínicas para la evaluación de terapias antimaláricas y de rescate / Standardization of a cerebral malaria murine model in clinical stages for antimalarial and rescue therapies evaluation

Entre las enfermedades infecciosas más devastadoras del SNC se incluye la MC, debido a la alta mortalidad y las graves secuelas que ocasiona. Actualmente, no existe tratamiento farmacológico específico, ni de rescate de lesiones neurocognitivas residuales, y su desarrollo está limitado por la inexistencia de modelos experimentales bien definidos. En este trabajo se caracterizó fenotípicamente la infección en un modelo murino de MC evaluando parámetros clínicos que permitieron establecer cuatro estadios de la enfermedad. Este protocolo proporciona el marco experimental adecuado para estudiar terapias coadyuvantes neuroprotectoras que puedan prevenir y/o eliminar las secuelas neurológicas presentes en los individuos que sobreviven (AU)

Cerebral malaria (CM) is included among the more devastating SNC infectious diseases due to its high mortality and severe sequelae in children. Currently, no specific pharmacological treatment for CM or rescue therapy for neurocognitive residual injury are available, and research on this topic has been hampered due to the lack of well-defined experimental models. In the present study we have characterized the CM murine infection phenotypically, evaluating clinical parameters, which allowed establishing a model encompassing four distinct disease stages. This protocol provides the experimental framework to study adjunctive neuroprotective therapies that may prevent and/or eliminate the neurological sequelae in individuals surviving CM (AU)

Brain-derived neurotrophic factor and the course of experimental cerebral malaria.

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.

Early transcriptional response to chloroquine of the Plasmodium falciparum antioxidant defence in sensitive and resistant clones.

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.

Conjugated linoleic acid affects lipid composition, metabolism, and gene expression in gilthead sea bream (Sparus aurata L).

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.

Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression.

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.