Diagnosis and Treatment of Kaposi Sarcoma

The full text of this article can be found here: https://link.springer.com/article/10.1007/s40257-017-0270-

WNV-induced miRNAs target processes functionally relevant to WNV pathogenesis.

<p>WNV signature miRNAs were analyzed for functions of predicted targets using Ingenuity Pathway Analysis software (IPA). Predicted targets of these miRNAs were determined and filtered through several criteria to yield functional categories for target genes. (A) The top functional categories are represented and the number of miRNA target genes in each category is denoted. (B) More detailed functions of the WNV-induced miRNA targets. Functions are shown along with statistical significance (P value) and the number of genes that correspond to each function. The p value was calculated in IPA using the right-tailed Fisher Exact Test and compares the number of focus genes that participate in a given functional process and the total number of genes known to be associated with the function in the selected set of miRNA target genes.</p

Toll-Like Receptor-3 Is Dispensable for the Innate MicroRNA Response to West Nile Virus (WNV)

<div><p>The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.</p></div

MiRNA profiling reveals a WNV-induced signature.

<p>Large-scale miRNA profiling was carried out in HEK293-Null and –TLR3 cells using Taqman-based qPCR. Heatmap representation of all raw data points for the experiment. Sample abbreviations are as follows: UN – mock infected cells; WNV – WNV-infected cells; polyIC – cells treated with TLR3 agonist polyI:C; MB – cells exposed to methylene blue-inactivated WNV; Form – cells exposed to formalin-inactivated WNV. Highly abundant miRNAs are indicated by red and non-expressed miRNAs by blue color. Yellow indicates an intermediate expression level. This is also shown underneath the heatmap. The miRNAs were clustered based on expression. The dendrogram is shown on the top and interesting clusters are highlighted in green and indicated by small letters (a high resolution figure is presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104770#pone.0104770.s002" target="_blank">Figure S2</a>).</p

Venn diagram of miRNAs that were induced in HEK293 Null and HEK293 TLR3 cells.

<p>Venn diagram representation of miRNAs induced following WNV infection of HEK293-Null and HEK293-TLR3 cells or polyI:C stimulation of HEK293-TLR3 cells. The number of shared, induced miRNAs are shown for each sample subset. A detailed list of the miRNAs induced for each category represented in the Venn diagram (HEK293-NULL +WNV, HEK293-TLR3 + WNV and HEK293-TLR3 + polyI:C treatment) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104770#pone.0104770.s008" target="_blank">Table S6</a>.</p

WNV infection induces a unique NFκB-responsive gene expression profile.

<p>HEK293-Null and –TLR3 cells under the specified conditions were analyzed for gene expression profiles using a qPCR array consisting of ∼90 NFκB-responsive genes. (A,B) Fold induction of genes was calculated by comparing WNV-infected samples to cells treated with formalin-inactivated virus. WNV-induced gene expression is shown at 0 hours post-infection (A) and 2 hours post-infection (B) with WNV (MOI = 10). Black bars denote TLR3-expressing HEK293 cells while open bars represent HEK293-Nulls. (C) The median CT across all samples was calculated and is shown +/− SEM for each gene analyzed. All genes shown were abundantly expressed (CT<35). (D) Heatmap of NFκB genes profiled. Highly abundant miRNAs are indicated by red and non-expressed miRNAs by blue color. Yellow indicates an intermediate expression level. This is also shown underneath the heatmap. The miRNAs were clustered based on expression.</p

The cellular response to WNV infection is unaffected by TLR3 status.

<p>(A) RT-PCR analysis of TLR3 expression in HEK293-Null and HEK293-TLR3 cells. RT negative reactions were performed as a control. (B) Immunofluorescence images of cells either mock- or WNV-infected are shown. Cells were stained with WNV E antibody (red) and DAPI (blue) and merged, deconvoluted images are shown at 630× magnification. (C–F) Western blots of phosphorylated IRF3 and beta actin in HEK293-Null and HEK293-TLR3 cells at various time points following WNV infection or post-treatment with the TLR3 agonist poly I:C. Mock infected cells and cells treated with inactivated WNV are shown as controls. (G, H) xCelligence analysis of cell viability following infection with serial dilutions of WNV NY99 (MOI = 5 to 0.00005). WNV-induced cytopathogenicity curves are shown for HEK293-Null (G) and HEK293-TLR3 cells (H). Reads were taken every 30 minutes for a period of 100 hours. (I) WNV-induced CPE kinetics were obtained by plotting the time of peak cell index for each sample against the concentration of WNV. HEK293-Nulls are represented by the blue line while HEK293-TLR3s are denoted by the red line. This panel shows a linear regression across 5 log dilutions. Shown are the individual cell indices at each dilution, the calculated robust linear regression lines (R² = 0.968) and in the gray shading the standard error (SE) band across the entire dilution range. (J) Viral load was determined by QPCR analysis for WNV envelope protein using a set of known WNV envelope standards. Panel J shows WNV copy number as determined by real-time qPCR using two biological replicate experiments (1 and 2) conducted one week apart. There was no significant growth difference.</p

Directed Comparison of abundant miRNAs.

<p>All miRNAs with at least one data point had a CT≤28 were selected, normalized to stable small RNAs, and the median CT across the 2, 4, and 8 hr time point was calculated. These were further standardized to yield a Z score, which reflects relative changes. The Z score is shown on the vertical axis and miRNAs on the horizontal axis. The Z scores were ordered by the level of uninfected HEK293-Null (A, B) or HEK293-TLR3 (C, D) cells. Panels (A) and (C) compare the response of WNV to formalin-inactivated virus. Panels (B) and (D) compare the response of WNV infection to polyI:C stimulation. (E) Caliper LabChip analysis of a highly expressed and undetected miRNA. qPCR products of miRNA assays were run on a Caliper LabChip GX and the resulting electropheregram is shown. The sample yields a single peak corresponding to the expressed miRNA and two assay marker bands (UM – upper marker and LM – lower marker). The undetected miRNA electropheregram shows only an upper and lower marker peak with very little to no background signal, confirming the high specificity of this assay.</p

KSHV Latency Locus Cooperates with Myc to Drive Lymphoma in Mice

<div><p>Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma and B-cell malignancies. Mechanisms of KSHV-induced oncogenesis remain elusive, however, in part due to lack of reliable in vivo models. Recently, we showed that transgenic mice expressing the KSHV latent genes, including all viral microRNAs, developed splenic B cell hyperplasia with 100% penetrance, but only a fraction converted to B cell lymphomas, suggesting that cooperative oncogenic events were missing. Myc was chosen as a possible candidate, because Myc is deregulated in many B cell lymphomas. We crossed KSHV latency locus transgenic (latency) mice to Cα Myc transgenic (Myc) mice. By itself these Myc transgenic mice develop lymphomas only rarely. In the double transgenic mice (Myc/latency) we observed plasmacytosis, severe extramedullary hematopoiesis in spleen and liver, and increased proliferation of splenocytes. Myc/latency mice developed frank lymphoma at a higher rate than single transgenic latency or Myc mice. These data indicate that the KSHV latency locus cooperates with the deregulated Myc pathways to further lymphoma progression.</p></div

Lymphoma development.

<p>*: Data were analyzed using Fisher’s exact test. A p value ≤ 0.05 was regarded as significant.</p><p>Lymphoma development.</p