Evaluation of the diagnostic accuracy of prototype rapid tests for human African trypanosomiasis

Peer reviewedPublisher PD

Predictive value for cardiovascular events of common carotid intima media thickness and its rate of change in individuals at high cardiovascular risk - Results from the PROG-IMT collaboration.

AIMS: Carotid intima media thickness (CIMT) predicts cardiovascular (CVD) events, but the predictive value of CIMT change is debated. We assessed the relation between CIMT change and events in individuals at high cardiovascular risk. METHODS AND RESULTS: From 31 cohorts with two CIMT scans (total n = 89070) on average 3.6 years apart and clinical follow-up, subcohorts were drawn: (A) individuals with at least 3 cardiovascular risk factors without previous CVD events, (B) individuals with carotid plaques without previous CVD events, and (C) individuals with previous CVD events. Cox regression models were fit to estimate the hazard ratio (HR) of the combined endpoint (myocardial infarction, stroke or vascular death) per standard deviation (SD) of CIMT change, adjusted for CVD risk factors. These HRs were pooled across studies. In groups A, B and C we observed 3483, 2845 and 1165 endpoint events, respectively. Average common CIMT was 0.79mm (SD 0.16mm), and annual common CIMT change was 0.01mm (SD 0.07mm), both in group A. The pooled HR per SD of annual common CIMT change (0.02 to 0.43mm) was 0.99 (95% confidence interval: 0.95-1.02) in group A, 0.98 (0.93-1.04) in group B, and 0.95 (0.89-1.04) in group C. The HR per SD of common CIMT (average of the first and the second CIMT scan, 0.09 to 0.75mm) was 1.15 (1.07-1.23) in group A, 1.13 (1.05-1.22) in group B, and 1.12 (1.05-1.20) in group C. CONCLUSIONS: We confirm that common CIMT is associated with future CVD events in individuals at high risk. CIMT change does not relate to future event risk in high-risk individuals

Emerging Themes from EBV and KSHV microRNA Targets

EBV and KSHV are both gamma-herpesviruses which express multiple viral microRNAs. Various methods have been used to investigate the functions of these microRNAs, largely through identification of microRNA target genes. Surprisingly, these related viruses do not share significant sequence homology in their microRNAs. A number of reports have described functions of EBV and KSHV microRNA targets, however only three experimentally validated target genes have been shown to be targeted by microRNAs from both viruses. More sensitive methods to identify microRNA targets have predicted approximately 60% of host targets could be shared by EBV and KSHV microRNAs, but by targeting different sequences in the host targets. In this review, we explore the similarities of microRNA functions and targets of these related viruses

Regulation of Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Receptor Protein (TWEAKR) Expression by Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Prevents TWEAK-Induced Apoptosis and Inflammatory Cytokine Expression▿ †

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, the second most common AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs) during latency. To identify cellular targets of KSHV miRNAs, we have analyzed a previously reported series of microarrays examining changes in cellular gene expression in the presence of KSHV miRNAs. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor (TWEAKR) was among the most consistently and robustly downregulated genes in the presence of KSHV miR-K12-10a (miR-K10a). Results from luciferase assays with reporter plasmids containing the 3′ untranslated region (UTR) of TWEAKR suggest a targeting of TWEAKR by miR-K10a. The mutation of two predicted miR-K10a recognition sites within the 3′ UTR of TWEAKR completely disrupts inhibition by miR-K10a. The expression of TWEAKR was downregulated in cells transfected with miR-K10a as well as during de novo KSHV infection. In a KS tumor-derived endothelial cell line, the downregulation of TWEAKR by miR-K10a resulted in reduced levels of TWEAK-induced caspase activation. In addition, cells transfected with miR-K10a showed less induction of apoptosis by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Finally, the downregulation of TWEAKR by miR-K10a in primary human endothelial cells resulted in a decrease in levels of expression of the proinflammatory cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to TWEAK. These results identify and validate an important cellular target of KSHV miRNAs. Furthermore, we demonstrate that a viral miRNA protects cells from apoptosis and suppresses a proinflammatory response, which may have significant implications in the complex context of KS lesions

Virus-Mediated Alterations in miRNA Factors and Degradation of Viral miRNAs by MCPIP1

<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi’s sarcoma, encodes 25 mature viral miRNAs. MCP-1-induced protein-1 (MCPIP1), a critical regulator of immune homeostasis, has been shown to suppress miRNA biosynthesis via cleavage of precursor miRNAs through its RNase domain. We demonstrate that MCPIP1 can directly cleave KSHV and EBV precursor miRNAs and that MCPIP1 expression is repressed following de novo KSHV infection. In addition, repression with siRNAs to MCPIP1 in KSHV-infected cells increased IL-6 and KSHV miRNA expression, supporting a role for MCPIP1 in IL-6 and KSHV miRNA regulation. We also provide evidence that KSHV miRNAs repress MCPIP1 expression by targeting the 3’UTR of MCPIP1. Conversely, expression of essential miRNA biogenesis components Dicer and TRBP is increased following latent KSHV infection. We propose that KSHV infection inhibits a negative regulator of miRNA biogenesis (MCPIP1) and up-regulates critical miRNA processing components to evade host mechanisms that inhibit expression of viral miRNAs. KSHV-mediated alterations in miRNA biogenesis represent a novel mechanism by which KSHV interacts with its host and a new mechanism for the regulation of viral miRNA expression.</p></div

MCPIP1 represses mature miRNA expression by cleaving pre-miRNA.

<p>(A) Experimental design schematic for Fig 2B–2G. (B) 293 cells were transfected with a KSHV miRNA expression vector and a MCPIP1 expression plasmid. Expression of mature miRNA was measured using qPCR assays. Results are shown relative to RNU48 and normalized to the cells transfected with KSHV miRNA expression vector and the EGFP control. <i>N</i> = 3. (C) Expression of the KSHV miRNA primary transcript was measured from the same RNA samples as in (B) by qPCR using two different SYBR green primer sets. Results are shown relative to GAPDH, <i>N</i> = 3. (D) RNA from (B) was assayed for expression of KSHV pre-miRNAs using qPCR. Results are normalized to U6 and the EGFP control, <i>N</i> = 3. (E) Expression of hsa-miR-135b was measured from the same RNA samples as in (B) and normalized to the empty vector controls, <i>N</i> = 3. (F) Primary transcript level of hsa-miR-135b was measured using qPCR as in (B). Results are shown relative to β-actin and normalized to empty vector controls, <i>N</i> = 3. (G) 293 cells were transfected with a KSHV miRNA expression vector and an RNase dead, MCPIP1 D141N expression plasmid as in (B). <i>N</i> = 4. (H) BCBL-1 cells were transfected with siRNA to MCPIP1 followed by isolation of newly synthesized labeled RNA. cDNA was obtained and mature KSHV miRNA expression was measured by qPCR. Results are shown relative to RNU48 and normalized to the siNeg control. <i>N</i> = 3–5. (I) In vitro cleavage assay for pre-KSHV-mirs-3, 5, and 6, synthesized with 5’IRD800CWN labels. Pre-miRNAs were incubated with MCPIP1 and assayed for degradation. <i>N</i> = 3. The percent of full-length pre-miRNAs is shown in the accompanying graph. For all graphs, results are shown as mean ± SD (standard deviation). Significance was assessed using a Student’s <i>t</i> test, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01. Numerical data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000998#pbio.2000998.s010" target="_blank">S1 Data</a>.</p

MCPIP1 directly cleaves human and viral pre-miRNAs.

<p>(A) In vitro cleavage assay for pre-hsa-mir-135b synthesized with 5’IRD800CWN labels and incubated with or without recombinant MCPIP1. Samples were resolved on a denaturing urea gel and scanned using an Odyssey scanner (LI-COR). Full-length pre-miRNAs and cleaved RNA products are indicated. (B) In vitro cleavage assay for pre-KSHV-mir-K12-3 and pre-EBV-mir-BART15 in the presence or absence of MCPIP1. (C) In vitro cleavage assay for pre-hsa-mir-135b with WT MCPIP1 or RNase dead, MCPIP1 D141N. (D) In vitro cleavage assays for pre-KSHV-mir-K12-3 and pre-EBV-mir-BART15 with WT or MCPIP1 D141N. Three independent in vitro cleavage assays were performed, and a representative image is shown for each condition.</p

Repression of MCPIP1 increases Dicer expression and TRBP association.

<p>(A) RNA from WT and MCPIP1 -/- MEFs were analyzed for Dicer expression using qPCR. Results are shown relative to β-actin and normalized to the WT control. <i>N</i> = 3. (B) WT and MCPIP1 -/- MEF protein lysates were evaluated for Dicer using western blot analysis. β-actin was used as a loading control. The results are averaged for six replicates, and a representative western blot is shown. (C) WT and MCPIP1 -/- MEFs were transfected with a MCPIP1 expression vector or an EGFP negative control vector, and RNA was isolated 48 hpt. RNA was DNase treated and analyzed for Dicer expression using qPCR. Results are shown relative to β-actin and normalized to the WT control, <i>N</i> = 4. (D) MC116 and KSHV infected MC116.219 cells were immunoprecipitated with anti-Dicer and anti-TRBP antibodies and blotted for Dicer. The results are averaged for four replicates and a representative blot is shown. For all graphs, results are shown as mean ± SD. Significance was assessed using a Student’s <i>t</i> test, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01. Numerical data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000998#pbio.2000998.s010" target="_blank">S1 Data</a>.</p