High prevalence of Strongyloides stercoralis in school-aged children in a rural highland of north-western Ethiopia: the role of intensive diagnostic work-up

Background: Soil-transmitted helminthiases (hookworms, Ascaris lumbricoides and Trichuris trichiura) are extremely prevalent in school-aged children living in poor sanitary conditions. Recent epidemiological data suggest that Strongyloides stercoralis is highly unreported. However, accurate data are essential for conducting interventions aimed at introducing control and elimination programmes. Methods: We conducted a cross-sectional survey of 396 randomly selected school-aged children in Amhara region in rural area in north-western Ethiopia, to assess the prevalence of S. stercoralis and other intestinal helminths. We examined stools using three techniques: conventional stool concentration; and two S. stercoralis-specific methods, i.e. the Baermann technique and polymerase chain reaction. The diagnostic accuracy of these three methods was then compared. Results: There was an overall prevalence of helminths of 77.5%, with distribution differing according to school setting. Soil-transmitted helminths were recorded in 69.2%. Prevalence of S. stercoralis and hookworm infection was 20.7 and 54.5%, respectively, and co-infection was detected in 16.3% of cases. Schistosoma mansoni had a prevalence of 15.7%. Prevalence of S. stercoralis was shown 3.5% by the conventional method, 12.1% by the Baermann method, and 13.4% by PCR, which thus proved to be the most sensitive. Conclusions: Our results suggest that S. stercoralis could be overlooked and neglected in Ethiopia, if studies of soil-transmitted helminths rely on conventional diagnostic techniques alone. A combination of molecular and stool microscopy techniques yields a significantly higher prevalence. In view of the fact that current control policies for triggering drug administration are based on parasite prevalence levels, a comprehensive diagnostic approach should instead be applied to ensure comprehensive control of helminth infections.This study was funded by the Mundo Sano Foundation and the Network of Tropical Diseases Research Center (Red de Investigación cooperative de Enfermedades Tropicales-RICET)S

Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin.

Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36

Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

<div><p>Specific oxidized phospholipids (oxPC_CD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPC_CD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPC_CD36 as well as by the strong physiological agonist thrombin. oxPC_CD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPC_CD36. Subsequent <i>ex vivo</i> studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPC_CD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPC_CD36.</p></div

Phosphoproteome network regulating integrin in platelets activated by thrombin.

<p>Proteins and their sites of phosphorylation identified by mass spectrometry were mapped onto an integrin protein- interaction network (reference 32) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084488#pone.0084488.s003" target="_blank">Materials and Methods S1</a>. Graphical representation was done with Cytoscape. Proteins are colored based on the changes in phosphorylation (yellow circle for at least one phosphorylation event induced by thrombin; white circle for no measurable change by thrombin). Phosphorylation sites are colored based on the up-regulation (red diamond), down-regulation (green diamond), or absence of modification (white diamond) by thrombin.</p

Phosphoproteomic analysis of platelets activated by the oxidized phospholipid KODA-PC and thrombin.

<p>Human platelets isolated by gel filtration were incubated with the oxidized phospholipid KODA-PC, or PLPC (as control). Platelets were then lysed and the proteins digested with trypsin. Phospho-tyrosine peptides were enriched from tryptic peptides with 4G10 antibody followed by metal affinity enrichment (IMAC). The supernatant leftover after immunoprecipitation was fractionated by Strong Cation Exchange (SCX) to separate phospho-serine/threonine peptides from unphosphorylated peptides. Fractions collected by SCX were further enriched for phosphopeptides using TiO₂ metal affinity. Phosphopeptide-enriched samples were analyzed by LC-MS/MS for identification and quantitation. A detailed description of the method can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084488#pone.0084488.s003" target="_blank">Materials and Methods S1</a>. Differences in protein phosphorylation between platelets incubated with KODA-PC and PLPC as determined by the phosphoproteomic method were used for analysis at the peptide level (Motif-X), protein level (GO-term, KEGG-pathway, and Kinase enrichment analysis-KEA), and compared to databases relevant to platelet biology. The same methodology was employed to study differences in protein phosphorylation between thrombin-induced and resting platelets.</p

Motifs extracted from significantly (de)phosphorylated sites induced by KODA-PC or thrombin.

<p>¹ Motif extracted using web-based software Motif-X (reference 29). Phosphorylated residues are indicated as “s”, “t”, or “y”. “X” represents any amino acid.</p><p>² Assignment of substrate or binding motif based on PhosphoMotif Finder (reference 30)</p

KODA-PC induces platelet P-selectin expression in a SFK- and SYK-dependent manner.

<p><b>(A-B)</b> Human platelets isolated by gel filtration were incubated with 10 µM SFK inhibitor (PP2), 0.2 µM SYK inhibitor (BAY 61-3606), or controls (DMSO and 10 µM PP3) for 30 min. Then, 30 µM PLPC, 30 µM KODA-PC, 10 µM ADP, or 0.05 U/mL thrombin were added for 30 min. Platelet P-selectin expression was determined by flow cytometry using PE-conjugated antibody to P-selectin. <b>(A)</b> Flow cytometry histograms from representative experiments are shown. <b>(B)</b> Quantitation of flow cytometry data presented as mean ± SD of at least 3 independent experiments. * P<0.05.</p

KODA-PC activates the sequential phosphorylation of SFK, SYK, and PLCγ2 through CD36.

<p><b>(A)</b> Human platelets isolated by gel filtration were incubated with buffer alone (–), CD36 blocking antibody clone FA6-152 (CD36), or a negative control antibody (IgG); then, 50 µM PLPC or 50 µM KODA-PC were added to the platelets for 7 minutes. Equal amount of protein were separated by gel electrophoresis and probed with antibodies raised against phosphorylated SYK (pTyr323) and PLCγ2 (pTyr1217). The blots were reprobed with SYK and PLCγ2 antibodies for normalization. <b>(B)</b> Human platelets isolated by gel filtration were incubated with DMSO, 10 µM SFK inhibitor (PP2), or 0.2 µM SYK inhibitor (BAY61-3606) for 30 min; then, 50 µM PLPC or 50 µM KODA-PC were added to the platelets for 7 minutes. Equal amount of protein were separated by gel electrophoresis and probed with antibodies raised against phosphorylated SYK (pTyr525/526) and PLCγ2 (pTyr759 and pTyr1217). The blots were reprobed with SYK and PLCγ2 antibodies for normalization.</p