Serologically defined variations in malaria endemicity in Pará state, Brazil

BACKGROUND: Measurement of malaria endemicity is typically based on vector or parasite measures. A complementary approach is the detection of parasite specific IgG antibodies. We determined the antibody levels and seroconversion rates to both P. vivax and P. falciparum merozoite antigens in individuals living in areas of varying P. vivax endemicity in Pará state, Brazilian Amazon region. METHODOLOGY/PRINCIPAL FINDINGS: The prevalence of antibodies to recombinant antigens from P. vivax and P. falciparum was determined in 1,330 individuals. Cross sectional surveys were conducted in the north of Brazil in Anajás, Belém, Goianésia do Pará, Jacareacanga, Itaituba, Trairão, all in the Pará state, and Sucuriju, a free-malaria site in the neighboring state Amapá. Seroprevalence to any P. vivax antigens (MSP1 or AMA-1) was 52.5%, whereas 24.7% of the individuals were seropositive to any P. falciparum antigens (MSP1 or AMA-1). For P. vivax antigens, the seroconversion rates (SCR) ranged from 0.005 (Sucuriju) to 0.201 (Goianésia do Pará), and are strongly correlated to the corresponding Annual Parasite Index (API). We detected two sites with distinct characteristics: Goianésia do Pará where seroprevalence curve does not change with age, and Sucuriju where seroprevalence curve is better described by a model with two SCRs compatible with a decrease in force of infection occurred 14 years ago (from 0.069 to 0.005). For P. falciparum antigens, current SCR estimates varied from 0.002 (Belém) to 0.018 (Goianésia do Pará). We also detected a putative decrease in disease transmission occurred ∼29 years ago in Anajás, Goianésia do Pará, Itaituba, Jacareacanga, and Trairão. CONCLUSIONS: We observed heterogeneity of serological indices across study sites with different endemicity levels and temporal changes in the force of infection in some of the sites. Our study provides further evidence that serology can be used to measure and monitor transmission of both major species of malaria parasite

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s)

EqSAPS

Decision-making tree and prediction algorithm for equine patient triag

Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses

The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hard remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material. (C) 2012 Elsevier B.V. All rights reserved.Swedish Foundation for Equine Research, SwedenSwedish Foundation for Equine Research, Swede

Chemical chaperones reverse early suppression of regulatory circuits during Unfolded Protein Response in B cells from Common Variable Immunodeficiency patients

Rare B cell samples from two CVID patients (P1 and P2) and a healthy donor (C) were used in controlled experiments analyzing the early UPR with and without the presence of chemical chaperones. As ER stressors we used Thapsigargin (Tg) and Tunicamycin (Tm). As exogenous chaperones we used DMSO, PBA, and TUDCA. For quantification of specific transcripts of interest we used a high-throughput platform based on TaqMan technology (Applied Biosystems TaqMan OpenArray Real-Time PCR System). Samples containing 3 x 10e5 EBV-B cells that received treatments with ER stressors and/or chemical chaperones for 0, 1, 2, 3, or 4 hr. Purified RNA was quali- and quantified by spectrometry in a Nanodrop and by fluorimetry in a Qubit, respectively. cDNA was generated using 300 ng of total RNA, and amplified in triplicate reactions for quantification of specific transcripts using a QuantStudio 12K Flex Real-Time PCR System. TaqMan OpenArray primers were custom-made by Thermo Fisher Scientific. Genes access numbers can be found in Supplementary Table 2 of the respective publication. Target DCq values were normalized against Gapdh DCq levels. Replicates were averaged and filtered for SE < 0.5 for removal of low-abundance measurements. A ratio of Treated / Untreated was calculated, log2-transformed, and a T-test was applied. Only those ratios whose SE were < 0.5 and that were significantly different (p ≤ 0.05) from untreated controls in at least two time-points were considered relevant. Regulatory circuits were built using BioTapestry 7.1 software. Networks show only those elements assayed in this study. Inputs and outputs of indicated genes are color coded according to their upstream origin (yellow for ATF6, red for PERK, blue for IRE1a, and green for sXBP1). Orange lines indicate those elements whose expression depends on inputs from both ATF6 and PERK pathways. Linkages substantiated by cis-regulatory data are indicated by diamonds colored according to strength of the experimental evidence: blue diamonds for expression studies using gain/loss of function, pink diamonds for binding affinity assays, and orange diamonds for promoter analysis in vivo. A green bar represents post-transcriptional modification of Xbp1 mRNA. A yellow bar represents post-translational modification of ATF6 protein. # (OR) and & (AND) are Boolean rules governing input elements in specific promoters. Bold gene = active expression. Bold gene + thick line = upregulated expression compared to untreated control. Grey gene + grey line = downregulated expression compared to untreated control. For visualization of each patient’s experimental regulatory network, refer to Supplementary Movies (A-G) for average [CVID patients minus healthy control], (H-N) for healthy control, (O-U) for patient P1, and (V-Y) for patient P2

Equine Simplified Acute Physiology Score (EqSAPS): personalized medicine for the equine emergency patient

Scoring models are useful tools that guide the attending clinician in gauging the severity of disease evolution, and in evaluating the efficacy of treatment. There are few tools available with this purpose for the non-human patient, including horses. We aimed (i) to adapt the Simplified Acute Physiology Score 3 (SAPS-3) model for the equine species, reaching a margin of accuracy greater than 75% in the calculation of the probability of death, and (ii) to build a decision tree that helps the attending veterinarian in assessment of the clinical evolution of the equine patient. From an initial pool of 5 568 medical records from University-based Veterinary Hospitals, a final cohort of 1 000 was further mined manually for data extraction. A set of 19 variables were evaluated and tested by five data mining algorithms. The final scoring model, named EqSAPS for Equine Simplified Acute Physiology Score, reached 91.83% of correct estimates for probability of death within 24 hours upon hospitalization. The Area Under Receiver Operating Characteristic Curve (AUROC) for outcome “death” was 0.742, while for “survival” was 0.652. The final decision tree was able to refine prognosis of patients whose EqSAPS score suggested “death”. EqSAPS is an useful tool to gauge the severity of the clinical presentation of the equine patient

p38␣ MAP Kinase Controls IL-17 Synthesis in Vogt-Koyanagi-Harada Syndrome and Experimental Autoimmune Uveitis

PURPOSE. Interleukin (IL)-17, which is responsible for the initial influx of leukocytes into the target tissue, was recently described as the main cytokine involved in autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is a significant cause of noninfectious blindness in the world. Herein the authors aimed at unraveling the involvement of IL-17 in VKH and in experimental autoimmune uveitis, focusing on the signaling pathways involved in IL-17 synthesis. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin. Draining lymph node cells, harvested 21 days after immunization, were cultured in the presence or absence of p38␣ mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and assayed for cytokine production and quantification of CD4 ϩ IL-17 ϩ cells. Mice received intraocular injections of SB203580, and disease severity was evaluated by histologic examination of the enucleated eyes at day 21. CD4 ϩ lymphocytes from MSK-1/2-deficient mice, human CD4 ϩ cells silenced with MSK1 siRNA, or peripheral blood mononuclear cells (PBMCs) from VKH patients were cultured in the presence or absence of p38␣ MAPK inhibitor and then assayed for IL-17, IFN-␥, and IL-4 production. RESULTS. The inhibition of p38␣ MAPK fully blocked the synthesis of IL-17 by PBMCs from VKH patients and lymphocytes from EAU mice. The absence of the msk1/2 gene resulted in failure to produce IL-17 by murine and human lymphocytes. Interestingly, intraocular injections of SB203580 in EAU mice did not suppress development of the disease. CONCLUSIONS. These data show that p38␣ MAPK-MSK1/2 is involved in the control of IL-17 synthesis by CD4 ϩ T cells and that inhibition of p38␣ MAPK in vitro suppresses IL-17 synthesis but that inhibition of this kinase in vivo did not protect from EAU. (Invest Ophthalmol Vis Sci