Clinical characteristics of patients with cKS at the time of late-EPC culture.

a<p>Mean±standard error.</p>b<p>cKS patients were classified according to our classification that takes into account the prevalent type of lesions, localization, clinical behaviour, evolutive pattern and presence of complications <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-Brambilla1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-DellaBella1" target="_blank">[8]</a>.</p><p>A = slow evolution; B = rapid evolution; rapid denotes an increase in the total number of nodules/plaques or in the total area of plaques in the three months following the last examination.</p

Immunophenotypic characterization of late-EPCs obtained from 15 patients with cKS.

<p>Late-EPCs express high levels of endothelial antigens but lack leukocyte markers. A) Percentage of positive cells for the indicated antigens, data expressed as mean±standard error. B) Representative flow cytometry analysis. Note that binding of UEA-1, uptake of ac-LDL and staining of e-NOS, vWF and Cav-1 were examined by conventional fluorescence-microscopy. UEA-1 = Ulex Europaeus Agglutinin-1; ac-LDL = acetylated-low-density lipoprotein; e-NOS = endothelial nitric oxide synthase; vWF = von Willebrand Factor; Cav-1 = caveolin-1.</p

Flow cytometry analysis showing CD146 expression on different late-EPC populations.

<p>To confirm that late-EPCs with proven endothelial phenotype could support KSHV infection, late-EPCs from one cKS patient underwent cell sorting of CD146+ cells and were cultured for further 2 weeks. Analysis of unsorted late-EPCs stained with isotype control (A) or anti-human CD146 mAb (B); analysis of highly purified CD146+ late-EPCs stained with anti-human CD146 mAb immediately after sorting (C) or after 2 weeks of culture (D).</p

KSHV-DNA in peripheral blood mononuclear cells and in late-EPC cultures from patients with cKS according to their clinical stage and KSHV serology.

a<p>Antibody titers were calculated as the reciprocal of the highest plasma dilution giving positive results.</p>b<p>cKS patients were classified according to our classification that takes into account the prevalent type of lesions, localization, clinical behaviour, evolutive pattern and presence of complications <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-Brambilla1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-DellaBella1" target="_blank">[8]</a>.</p>c<p>In two patients the presence of KSHV-DNA was determined in multiple passages of unstimulated late-EPC cultures.</p>d<p>KSHV-DNA determined in highly pure CD146+ late-EPCs, maintained in culture for further 2 weeks after CD146 sorting.</p><p>KSHV = Kaposi's sarcoma-associated herpesvirus; PBMCs = peripheral blood mononuclear cells; late-EPC = late-endothelial progenitor cell;</p><p>cKS = classic Kaposi's sarcoma; LANA = latency-associated nuclear antigen; GE = genome equivalents.</p

Late-EPCs obtained from patients with cKS support KSHV productive replication.

<p>A) To induce KSHV lytic replication, late-EPCs from cKS patients underwent treatment with <i>n</i>-butyrate (5 patients, solid lines) or TPA (2 patients, hatched lines) for 48 hours. Multiple colonies from each patient were pooled before treatment. B) To confirm that late-EPCs with proven endothelial phenotype could support KSHV lytic replication, late-EPCs from one cKS patient underwent cell sorting of CD146+ cells. Unsorted late-EPCs (solid line) or highly purified CD146+ late-EPCs from the same cKS patient were cultured for further 2 weeks after sorting (hatched line) and underwent treatment with <i>n</i>-butyrate for 48 hours. In any case, KSHV genomes were analyzed by real-time PCR in DNA extracted from late-EPC supernatants. <i>P</i> value was determined using the Wilcoxon signed-rank test.KSHV genomes were analyzed by real-time PCR in DNA extracted from culture supernatants. KSHV = Kaposi's sarcoma-associated herpesvirus; TPA = phorbol 12-myristate 13-acetate.</p