Systemic endothelial abnormalities.

<p>(A) A representative cardiac section stained with H&E, EGFP and CD34 and/or CD31, PROX1 and Ki67 shows numerous atypical spindle-like endothelial cells. Similar cells were found in the skeletal muscle (B) and in brown fat (C). Analysis was done in 2–3 month-old mice, about one month after <i>i.p.</i> injection of tamoxifen. Scale bar, 200 μm. TG/Endo, ROSA26.vFLIP;Cdh5(PAC).creER^T2; TG/B-cells, ROSA26.vFLIP;CD19.cre mice used as control.</p

Perturbation in serum cytokines.

<p>Fourteen serum cytokines were analyzed in ROSA26.vFLIP;Cdh5(PAC).creER^T2 mice by using a quantitative flow cytometry-based assay. Analysis was done in 2–3 month-old mice, about one month after <i>i.p.</i> injection of tamoxifen. Data are representative of at least three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment. <i>P</i>-values derived from two-tailed unpaired Student’s t-test on the means (bars) of WT versus TG mice are shown. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.005</p

Model of KSHV vFLIP-mediated tumorigenesis through endothelial alterations, aberrant myeloid differentiation, and chronic proinflammatory changes in the tumor microenvironment.

<p>Expression of vFLIP in either B-cells or endothelial cells activates several cytokines, both <i>in vivo</i> and <i>in vitro</i>, that lead to aberrant myeloid differentiation with the emergence of myeloid subsets well known to have a role in angiogenesis, tumor immune evasion and tumor progression. <i>(Drawing of myeloid differentiation was modified from Gabrilovich et al)[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004581#ppat.1004581.ref043" target="_blank">43</a>]</i></p

Systemic perineurinal proliferation.

<p>(A) Representative section of the perirenal capsule, diaphram muscle, salivary gland, and pancreas stained with H&E shows atypical proliferation of perineurinal endothelial-like cells (arrows). Analysis was done in 2–3 month-old mice, about one month after <i>i.p.</i> injection of tamoxifen. Scale bar, 200 μm. (B) Serum glucose levels are shown. Data represent one of three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment.</p

Systemic Expression of Kaposi Sarcoma Herpesvirus (KSHV) Vflip in Endothelial Cells Leads to a Profound Proinflammatory Phenotype and Myeloid Lineage Remodeling <i>In Vivo</i>

<div><p>KSHV is the causative agent of Kaposi sarcoma (KS), a spindle-shaped endothelial cell neoplasm accompanied by an inflammatory infiltrate. To evaluate the role of KSHV vFLIP in the pathogenesis of KS, we constructed mice with inducible expression of vFLIP in endothelial cells. Abnormal cells with endothelial marker expression and fusiform appearance were observed in several tissues reminiscent of the spindle cells found in KS. Serum cytokines displayed a profound perturbation similar to that described in KSHV inflammatory cytokine syndrome (KICS), a recently described clinical condition characterized by elevated IL6 and IL10. An increased myeloid component with suppressive immune phenotype was found, which may contribute to functional changes in the microenvironment and cellular heterogeneity as observed in KS. These mice represent the first in vivo demonstration that vFLIP is capable of inducing vascular abnormalities and changes in host microenvironment with important implications for understanding the pathogenesis and treating KSHV-associated diseases.</p></div

Expansion of myeloid cells with PMN-MDSC immunophenotype.

<p>(A) Flow cytometry analysis displayed increase in CD45⁺CD11b⁺ myeloid cells in lung, spleen, liver and heart. (B) Ly6G, Ly6C, Gr1 markers and forward/side scatter parameters were used to define the following myeloid cell subsets: polymorphonuclear myeloid derived cells (PMN-MDSC), monocytic myeloid derived cells (M-MDSC), tumor-associated macrophages (TAM). Analysis was done in 2–3 month-old mice, about one month after <i>i.p.</i> injection of tamoxifen. Data are representative of at least three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment. TG/Endo, ROSA26.vFLIP;Cdh5(PAC).creER^T2; TG/B-cells, ROSA26.vFLIP;CD19.cre mice used as control.</p

Generation of ROSA26.vFLIP;Cdh5(PAC).creER^T2 mice.

<p>(A) The strategy for inducible recombinant activation of vFLIP expression in endothelial cells <i>in vivo</i> is shown with a schematic representation of the ROSA26 locus before (left) and after (right) inducible recombinant activation of vFLIP expression in endothelial cells. ROSA26.vFLIP knock-in mice were bred with Cdh5(PAC).creER^T2 mice to obtain transgene expression in endothelial cells upon tamoxifen injection. (B) Transgene expression was specifically detected, both by RT-PCR (left panel) and anti-FLAG immunoblotting (right panel), in lung, spleen, liver and heart derived from ROSA26.vFLIP;Cdh5(PAC).creER^T2. (C) Quantitative real-time RT-PCR for vFLIP expression. Analysis was done in 2–3 month-old mice, about one month after <i>i.p.</i> injection of tamoxifen. (D) Flow cytometry showing percentage of cardiac endothelial cells and (E) splenic B-cells expressing EGFP. Data represent one of three experiments with similar results; at least three TG and control animals were analyzed in each experiment. Analysis was done in 2–3 month-old mice, about one month after <i>i.p.</i> injection of tamoxifen. TG/Endo are ROSA26.vFLIP;Cdh5(PAC).creER^T2 mice; TG/B-cells represent ROSA26.vFLIP;CD19.cre mice used as control [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004581#ppat.1004581.ref023" target="_blank">23</a>]. </p

Visual comparison of pseudo-biopsy and human biopsy samples.

<p>Cell block pseudo-biopsies with varying KS-positive cells (bottom) were used to imitate human biopsies (top) to validate function of the device. Cell block pseudo-biopsies were embedded in paraffin and stained for latency-associated nuclear antigen (LANA) for comparison with LANA stained human biopsies. KSHV-infected nuclei are brown with dark punctae, while uninfected nuclei are blue. Images of human biopsies were taken from representative sections of KS-positive samples with concentrations similar to the cell block biopsies by visual inspection. The 0% image was taken from an uninfected region of a human sample with low concentration of KS positive cells. Precise analysis of infected cell percentage per sample was performed using HALO image analysis software (Indica Labs), and can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147636#pone.0147636.s002" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147636#pone.0147636.s006" target="_blank">S5</a> Figs. Scale bar applies to all images.</p

Light source comparison: indoors and outdoors.

<p>(A) The KS-Detect system during an experiment using sunlight. Lens must be manually aligned with the sun as the sun moves across the sky. (B) For experiments conducted indoors, the lens and microfluidics are removed from our portable kit, and are fixed in front of a 100 Watt LED array. (C) A typical solar temperature profile is more variable when compared to a typical LED array temperature profile, due to cloud coverage (as seen at about 35 min.) and intermittent realignment of the lens with the sun.</p

The KS-Detect system.

<p>(A) The system contains all components necessary for solar thermal PCR and subsequent analysis, including reagents, tablet, and solar panel. The focusing lens is fixed to the red container on a hinge, allowing rotation (blue arrow) for alignment with the sun. (B) The system is easily carried in one hand, affording easy transportation to patients in remote communities. (C) Microfluidics schematic. Samples are cycled between the warmer center of a PDMS chip (for denaturation of DNA) and the cooler edges (for annealing of primers). A thin, black PDMS layer (not pictured) serves as the bottom of the microfluidic chip, and absorbs solar radiation. (D) Our custom Android application is used to track each temperature zone within the microfluidics and to (E) analyze results via fluorescence levels imaged by a smartphone or tablet.</p