Sensitivity Analyis: Risk Ratios for viral suppression following different adjustment schema for missing data.

<p>Sensitivity Analyis: Risk Ratios for viral suppression following different adjustment schema for missing data.</p

Antiretroviral treatment by trial participation.

<p>Antiretroviral treatment by trial participation.</p

Baseline sample characteristics for study population, complete cases and comparing trial to non-trial participants restricted to complete cases.

*<p>p value comparing trial to non trial participants restricted to complete cases.</p>1<p>MSM = Men who have sex with Men;</p>2<p>Non Black includes Caucasian, Hispanic and Other race.</p>3<p>Public insurance =  Medicaid/Medicare;</p>4<p>ID =  University of North Carolina Infectious Disease.</p>5<p>HAART Category; NRTI – Nucleoside Reverse Transcriptase Inhibitor; PI/r – Protease Inhibitor/Ritonavir; NNRTI - Non Nucleoside Reverse Transcriptase Inhibitor.</p>6<p>ANC = Absolute Neutrophil Count.</p>7<p>ALT = Alanine amino transferase.</p

Risk Ratios for virologic suppression by trial participation within strata of HAART period.

<p>**adjusted for age, distance traveled to receive care at UNC ID clinic, baseline HIV RNA levels, CD4 cell count, months from HIV diagnosis to HAART initiation, creatinine, type of HAART.</p

Systemically Circulating Viral and Tumor-Derived MicroRNAs in KSHV-Associated Malignancies

<div><p>MicroRNAs (miRNAs) are stable, small non-coding RNAs that modulate many downstream target genes. Recently, circulating miRNAs have been detected in various body fluids and within exosomes, prompting their evaluation as candidate biomarkers of diseases, especially cancer. Kaposi's sarcoma (KS) is the most common AIDS-associated cancer and remains prevalent despite Highly Active Anti-Retroviral Therapy (HAART). KS is caused by KS-associated herpesvirus (KSHV), a gamma herpesvirus also associated with Primary Effusion Lymphoma (PEL). We sought to determine the host and viral circulating miRNAs in plasma, pleural fluid or serum from patients with the KSHV-associated malignancies KS and PEL and from two mouse models of KS. Both KSHV-encoded miRNAs and host miRNAs, including members of the miR-17–92 cluster, were detectable within patient exosomes and circulating miRNA profiles from KSHV mouse models. Further characterization revealed a subset of miRNAs that seemed to be preferentially incorporated into exosomes. Gene ontology analysis of signature exosomal miRNA targets revealed several signaling pathways that are known to be important in KSHV pathogenesis. Functional analysis of endothelial cells exposed to patient-derived exosomes demonstrated enhanced cell migration and IL-6 secretion. This suggests that exosomes derived from KSHV-associated malignancies are functional and contain a distinct subset of miRNAs. These could represent candidate biomarkers of disease and may contribute to the paracrine phenotypes that are a characteristic of KS.</p></div

Linear, multivariate analysis of scratch assays^a.

a<p>Total number of assays n = 94.</p>b<p>Estimates relative effect of variable on fraction of closed area of the scratch after 8 hours relative to mock treatment. A negative coefficient indicates inhibition relative to control.</p>c<p>SEM, standard error of the mean.</p>d<p>Unadjusted p-value of F test for significance (p≤0.05 is considered significant.</p>e<p>Intercept term of the linear model.</p>f<p>n.s., not statistically significant.</p>g<p>Total number of independent experiments n = 9.</p>h<p>CHP, control human plasma.</p>i<p>Human IL6.</p>j<p>SN, supernatant fraction after exo quick kit.</p>k<p>Presence of Annexin V, which prevents exosome fusion.</p

Analysis of oncomiRs and exosomal miRNA subsets.

<p>Oncomirs belonging to the miR-17-92 cluster were analyzed for expression. (A) Box plot showing the distribution of expression scores for ∼150 miRNAs associated with cancer for KS biopsies, exosomes in KS patients, exosomes in normal human plasma and RNase-treated, exosome-depleted supernatant from control plasma. Individual circles represent individual miRNAs and their respective expression levels in the samples. Blue circles represent members of the oncogenic miR-17-92 cluster while other oncogenic miRNAs are denoted by red circles. Box plots of relative expression levels of the miR-17-92 and 106b-25 clusters in control and tumor samples are shown in human (B) and mouse (C) exosome samples. (D) A subset of miRNAs showed exclusive expression in mouse exosomes and not in plasma exosomal supernatants (free miRNAs). Expression levels in transgenic and xenograft mouse exosomes are also higher than control exosomes for these miRNAs. Sample abbreviations: exo, KS exosomes; neg, control exosomes; tumor, tumor biopsies; mock, control human or mouse plasma/serum, plasma, KS patient plasma; pleural, pleural fluid; tg, KSHV latency locus transgenic mice; tum, xenograft tumor mice; cEXO, control exosomes; cSN, control supernatant; tExo, tumor exosomes; tSN, tumor supernatant fraction.</p

Exosome purification and analysis of miRNAs in sample subsets.

<p>Exosomes were purified and miRNAs isolated from exosome samples were analyzed for expression in various subsets. (A) Schematic for profiling of circulating miRNAs from plasma, serum and pleural fluid samples. (B–E) Box plots show the distribution of relative levels (CT) for 12 miRNAs for various conditions (mir-106b, mir-150, mir-16, mir-195, mir-197, mir-205, mir-23a, mir-30c, mir-425-5p, mir-548a, mir-92a, U6 snRNA). We selected those miRNAs, as they were highly expressed and as being representative of the different patterns we see across the experimental controls. Two independent experiments were performed and both replicates are shown. The line represents the median expression of microRNAs for a given sample group while individual microRNAs are denoted by closed circles (n = 24). In some cases, the median of the group is equal to 50 and the line is along the x axis due to >50% of miRNAs with a CT = 50. MiRNA expression following RNase treatment of control human plasma supernatants (B), comparison of human and mouse exosomal miRNA expression in control human plasma and mouse serum (C), differential expression in purified subsets from control human plasma (D) and tissue-specific expression (E) are shown. Asterisks denote previously detected plasma miRs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003484#ppat.1003484-Arroyo1" target="_blank">[35]</a>.</p

Analysis of KSHV miRNA expression and viral DNA in exosome samples.

<p>Box plot representation of miR-K12-11 expression (A) and KSHV load (B) in latent and lytic exosomes purified using the CD63+ Dynabeads method. Sample filtration is noted below each plot. MiRNA expression is shown as fold above background and viral load data is shown as copy number of LANA DNA per reaction. (C) Box plot of viral load for filtered samples purified using either CD63 (left panel) or differential ultracentrifugation (centri, right panel). Viral load is shown for both exosome (exo) and supernatant (sn) fractions. Asterisks denote significance of p≤0.05. (D, E) KSHV viral load from ExoQuick samples was determined by qPCR (D) and products were run on the Caliper LabChip GX (E). (D) Sample groups are as follows: neg (control human and mouse samples negative for KSHV), ntc (no template control), KS or PEL (KS patients and primary PEL fluid), pos (dilutions of oligonucleotide positive controls; high to low concentrations) and tive (xenograft mouse models of KS). (E) Sample abbreviations are as follows: KS = plasma from KS, AMT = AIDS Malignancy, non-KS, PF = pleural fluid, CHP = control human plasma, E = exosome fraction, S = exosome-depleted supernatant fraction. (F) Western blot for the exosomal marker CD9. Samples were enriched for exosomes using CD63+ Dynabeads and were filtered prior to bead purification as noted. Resulting CD63+ and CD63− fractions were treated with RNase as denoted and protein lysates were evaluated. As a positive control, a lysate of pleural fluid-derived exosomes using the ExoQuick (EQ) method were assessed for CD9 expression.</p

Characterization of patient- and mouse model-derived exosomes.

<p>(A) EM images of exosomes prepared from patient and tissue culture samples using Exoquick and ultracentrifugation (UC) methods. PF1, pleural fluid patient 1; PF2, pleural fluid patient 2; BCBL1 – PEL cell line. Scalebar is shown below images. (B–H) Abbreviations are as follows: CHP – Control, KSHV(−) Human Plasma, AMT – patients with non-KS AIDS malignancies, KS – Kaposi's Sarcoma patients, PF – Primary PEL Pleural Fluid, Ctrl – Control Mouse Serum, Tg – KSHV Latency Locus Transgenic Mouse Model, Xeno – TIVE-KSHV Xenograft Mouse Model, (−) KSHV-negative BJAB cell line. The exosomal markers flotillin-2 (B,C), Hsp90 alpha (E,G), Hsp90 beta (F) and CD9 (H) were analyzed by Western blot in human and mouse exosomes (abbreviated E) isolated using the Exoquick method. Exosome-depleted supernatants (abbreviated S) were also analyzed for the presence of Flotillin (B,D) and Hsp90 alpha (E,G). CD9 was detected in mouse exosome samples and exosomes from KS patients (KS), confirming our method of exosome isolation (H). As expected, the exosomal marker was absent in the supernatant fraction and in our negative control BJAB exosome-depleted supernatant fraction. Flotillin was present in exosomes derived from control (Ctrl), transgenic (Tg) and xenograft (Xeno) mouse models but was not present in the supernatant fraction. Hsp90 alpha and beta were expressed in PEL cells (VG1, a KSHV+ PEL cell line) and pleural fluid-derived exosomes (PF) but not in the supernatant. (I) KSHV miR-K2 expression was determined by qPCR and products were run on the Caliper LabChip GX. BCP1-KSHV (+) PEL cell line, Exo – RNA from exosome fraction, Cells – RNA from cell pellet. Exo1,2 and 3 denote three individual TIVE xenograft mice.</p