Generation of second messengers in Plasmodium

Signalling in malaria parasites is a field of growing interest as its components may prove to be valuable drug targets, especially when one considers the burden of a disease that is responsible for up to 500 million infections annually. The scope of this review is to discuss external stimuli in the parasite life cycle and the upstream machinery responsible for translating them into intracellular responses, focussing particularly on the calcium signalling pathway. (C) 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Underlying Factors Associated with Anemia in Amazonian Children: A Population-Based, Cross-Sectional Study

Background: Although iron deficiency is considered to be the main cause of anemia in children worldwide, other contributors to childhood anemia remain little studied in developing countries. We estimated the relative contributions of different factors to anemia in a population-based, cross-sectional survey. Methodology: We obtained venous blood samples from 1111 children aged 6 months to 10 years living in the frontier town of Acrelandia, northwest Brazil, to estimate the prevalence of anemia and iron deficiency by measuring hemoglobin, erythrocyte indices, ferritin, soluble transferrin receptor, and C-reactive protein concentrations. Children were simultaneously screened for vitamin A, vitamin B-12, and folate deficiencies; intestinal parasite infections; glucose-6-phosphate dehydrogenase deficiency; and sickle cell trait carriage. Multiple Poisson regression and adjusted prevalence ratios (aPR) were used to describe associations between anemia and the independent variables. Principal Findings: The prevalence of anemia, iron deficiency, and iron-deficiency anemia were 13.6%, 45.4%, and 10.3%, respectively. Children whose families were in the highest income quartile, compared with the lowest, had a lower risk of anemia (aPR, 0.60; 95% CI, 0.37-0.98). Child age (<24 months, 2.90; 2.01-4.20) and maternal parity (>2 pregnancies, 2.01; 1.40-2.87) were positively associated with anemia. Other associated correlates were iron deficiency (2.1; 1.4-3.0), vitamin B-12 (1.4; 1.0-2.2), and folate (2.0; 1.3-3.1) deficiencies, and C-reactive protein concentrations (>5 mg/L, 1.5; 1.1-2.2). Conclusions: Addressing morbidities and multiple nutritional deficiencies in children and mothers and improving the purchasing power of poorer families are potentially important interventions to reduce the burden of anemia.

Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen (1O2). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of 1O2 by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While 1O2 is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of 1O2 we have established a new cytometric method that allows the stage specific quantification of 1O2. Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200600 pixels for rings, 7001,200 pixels for trophozoites and 1,4002,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous 1O2 of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that 1O2 derives predominantly from the digestion of hemoglobin. (c) 2012 International Society for Advancement of Cytometry

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (arg Delta SKL) restored growth but failed to restore infectivity. Further study showed that the ARG Delta SKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.

Burden of maternal bipolar disorder on at-risk offspring: A controlled study on family planning and maternal care

Introduction: Bipolar disorder (BD) is a highly incapacitating disease typically associated with high rates of familial dysfunction. Despite recent literature suggesting that maternal care is an important environmental factor in the development of behavioral disorders, it is unclear how much maternal care is dysfunctional in BD subjects. Objective: The objective of this study was to characterize maternal care in DSM-IV/SCID diagnosed BD type I subjects compared to healthy controls with (PD) and without (NPD) other psychiatric diagnoses. Materials and methods: Thirty-four BD mothers and 106 controls underwent an interview about family planning and maternal care, obstetrical complications, and mother-child interactions. K-SADS-PL questions about violence exposure were used to ascertain domestic violence and physical/sexual abuse. Results: BD mothers were less likely to have stable unions (45.5%; p < 0.01) or to live with the biological father of their children (33.3%; p < 0.01), but had higher educational level and higher rates of social security use/retirement. They also had fewer children and used less contraceptive methods than controls. Children of BD women had higher rates of neonatal anoxia, and reported more physical abuse (16.1%; p = 0.02) than offspring of NPD mothers. Due to BD mothers' symptoms, 33.3% of offspring suffered physical and/or psychological abuse. Limitations: Post hoc analysis, and the use of questions as a surrogate of symptoms as opposed to validated instruments. Conclusion: This is one of few reports confirming that maternal care given by BD women is dysfunctional. BD psychopathology can lead to poor maternal care and both should be considered important environmental risk factors in BD, suggesting that BD psychoeducation should include maternal care orientation. (C) 2012 Elsevier B.V. All rights reserved.

Phylogenetic and host-parasite relationship analysis of Henneguya multiplasmodialis n. sp infecting Pseudoplatystoma spp. in Brazilian Pantanal wetland

A new species of the genus Henneguya (Henneguya multiplasmodialis n. sp.) was found infecting the gills of three of 89 specimens (3.3%) of Pseudoplatystoma corruscans and two of 79 specimens (2.6%) of Pseudoplatystoma reticulatum from rivers in the Pantanal wetland, Brazil. Partial sequencing of the 18S rDNA gene of the spores obtained from one plasmodium from the gills of P. corruscans and other one from the gills of P. reticulatum, respectively, resulted in a total of 1560 and 1147 base pairs. As the spores of H. multiplasmodialis n. sp. resemble those of Henneguya corruscans, which is also a parasite of P. corruscans, sequencing of the 18S rDNA gene of the spores of H. corruscans found on P. corruscans caught in the Brazilian Pantanal wetland was also provided to avoid any taxonomic pendency between these two species, resulting in 1913 base pairs. The sequences of H. multiplasmodialis n. sp. parasite of P. corruscans and P. reticulatum and H. corruscans did not match any of the Myxozoa available in the GenBank. The similarity of H. multiplasmodialis n. sp. obtained from P. corruscans to that from P. reticulatum was of 99.7%. Phylogeny revealed a strong tendency among Henneguya species to form clades based on the order and/or family of the host fish. H. multiplasmodialis n. sp. clustered in a clade with Henneguya eirasi and H. corruscans, which are also parasites of siluriforms of the family Pimelodidae and, together with the clade composed of Henneguya spp. parasites of siluriforms of the family Ictaluridae, formed a monophyletic clade of parasites of siluriform hosts. The histological study revealed that the wall of the plasmodia of H. multiplasmodialis n. sp. were covered with a stratified epithelium rich in club cells and supported by a layer of connective tissue. The interior of the plasmodia had a network of septa that divided the plasmodia into numerous compartments. The septa were composed of connective tissue also covered on both sides with a stratified epithelium rich in club cells. Inflammatory infiltrate was found in the tissue surrounding the plasmodia as well as in the septa. (C) 2011 Elsevier B.V. All rights reserved.

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.

Burden of care and its impact on health-related quality of life of caregivers of individuals with spinal cord injury

Objective: to analyze the impact and burden of care on the Health-Related Quality of Life (HRQOL) of caregivers of individuals with a spinal cord injury (SCI). Method: cross-sectional observational study carried out by reviewing medical records and applying questionnaires. The scale Short Form 36 (SF-36) was used to assess HRQOL and the Caregiver Burden Scale (CBScale) for care burden. Results were analyzed quantitatively. Most patients with SCIs were male, aged 35.4 years old on average, with a predominance of thoracic injuries followed by cervical injuries. Most caregivers were female aged 44.8 years old on average. Results: tetraplegia and secondary complications stand out among the clinical characteristics that contributed to greater care burden and worse HRQOL. Association between care burden with HRQOL revealed that the greater the burden the worse the HRQOL. Conclusion: Preventing care burden through strategies that prepare patients for hospital discharge, integrating the support network, and enabling access to health care services are interventions that could minimize the effects arising from care burden and contribute to improving HRQOL.

Correlation between carotid bifurcation calcium burden on non-enhanced CT and percentage stenosis, as confirmed by digital subtraction angiography

Objectives: Previous evidence supports a direct relationship between the calcium burden (volume) on post-contrast CT with the percent internal carotid artery (ICA) stenosis at the carotid bifurcation. We sought to further investigate this relationship by comparing non-enhanced CT (NECT) and digital subtraction angiography (DSA). Methods: 50 patients (aged 41-82 years) were retrospectively identified who had undergone cervical NECT and DSA. A 64-multidetector array CT (MDCT) scanner was utilised and the images reviewed using preset window widths/levels (30/300) optimised to calcium, with the volumes measured via three-dimensional reconstructive software. Stenosis measurements were performed on DSA and luminal diameter stenoses >40% were considered "significant". Volume thresholds of 0.01, 0.03, 0.06, 0.09 and 0.12 cm(3) were utilised and Pearson's correlation coefficient (r) was calculated to correlate the calcium volume with percent stenosis. Results: Of 100 carotid bifurcations, 88 were available and of these 7 were significantly stenotic. The NECT calcium volume moderately correlated with percent stenosis on DSA r=0.53 (p<0.01). A moderate-strong correlation was found between the square root of calcium volume on NECT with percent stenosis on DSA (r=0.60, p<0.01). Via a receiver operating characteristic curve, 0.06 cm(3) was determined to be the best threshold (sensitivity 100%, specificity 90.1%, negative predictive value 100% and positive predictive value 46.7%) for detecting significant stenoses. Conclusion: This preliminary investigation confirms a correlation between carotid bifurcation calcium volume and percent ICA stenosis and is promising for the optimal threshold for stenosis detection. Future studies could utilise calcium volumes to create a "score" that could predict high grade stenosis.

Chronic pain associated with the Chikungunya Fever: long lasting burden of an acute illness

Abstract Background Chikungunya virus (CHIKV) is responsible for major epidemics worldwide. Autochthonous cases were recently reported in several European countries. Acute infection is thought to be monophasic. However reports on chronic pain related to CHIKV infection have been made. In particular, the fact that many of these patients do not respond well to usual analgesics suggests that the nature of chronic pain may be not only nociceptive but also neuropathic. Neuropathic pain syndromes require specific treatment and the identification of neuropathic characteristics (NC) in a pain syndrome is a major step towards pain control. Methods We carried out a cross-sectional study at the end of the major two-wave outbreak lasting 17 months in Réunion Island. We assessed pain in 106 patients seeking general practitioners with confirmed infection with the CHIK virus, and evaluated its impact on quality of life (QoL). Results The mean intensity of pain on the visual-analogical scale (VAS) was 5.8 ± 2.1, and its mean duration was 89 ± 2 days. Fifty-six patients fulfilled the definition of chronic pain. Pain had NC in 18.9% according to the DN4 questionnaire. Conversely, about two thirds (65%) of patients with NC had chronic pain. The average pain intensity was similar between patients with or without NC (6.0 ± 1.7 vs 6.1 ± 2.0). However, the total score of the Short Form-McGill Pain Questionnaire (SF-MPQ)(15.5 ± 5.2 vs 11.6 ± 5.2; p < 0.01) and both the affective (18.8 ± 6.2 vs 13.4 ± 6.7; p < 0.01) and sensory subscores (34.3 ± 10.7 vs 25.0 ± 9.9; p < 0.01) were significantly higher in patients with NC. The mean pain interference in life activities calculated from the Brief Pain Inventory (BPI) was significantly higher in patients with chronic pain than in patients without it (6.8 ± 1.9 vs 5.9 ± 1.9, p < 0.05). This score was also significantly higher in patients with NC than in those without such a feature (7.2 ± 1.5 vs 6.1 ± 1.9, p < 0.05). Conclusions There exists a specific chronic pain condition associated to CHIKV. Pain with NC seems to be associated with more aggressive clinical picture, more intense impact in QoL and more challenging pharmacological treatment.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

The burden of dengue: Jundiaí, Brazil - January 2010

OBJECTIVE: To study the antibody prevalence against dengue in the municipality of Jundiaí, São Paulo, Brazil, due to the low number of official confirmed autochthonous cases. METHODS: A serological study on dengue infection was conducted during January 2010 and previous reports on dengue and entomological surveillance during that period were reviewed. RESULTS: A prevalence of 7.8% IgG positive (68:876) was found. Furthermore, based on the detection of IgM antibodies in five samples, it was observed that the incidence of dengue in the city at the time of the survey contrasts with the absence of notifications by local health authorities over the same period of time. CONCLUSION: These results highlight the discrepancies between the actual and the detected number of dengue infections, possibly due to significant numbers of asymptomatic infections aggravated by difficulties with dengue clinical diagnosis.