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

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

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

Immunohistochemical detection of the viral interleukin-6 (vIL-6) in placental histocultures.

<p>(A) Immunohistochemical staining showed a strongly positive cytoplasmic staining in both endothelial cells (white arrow) and syncytiotrophoblasts (blue arrow) in HHV-8-infected placental histocultures. (B) Mock-infected placental histocultures. Original magnifications, ×100 (A), ×63 (B).</p

HHV-8 infection of placental histocultures.

<p>HHV-8 load was measured 48 h, 72 h and 7 days after infection in the placental tissues (histograms) and in culture supernatants (dots). Increasing viral loads were detected in tissue fragments and in culture supernatants of infected histocultures, suggesting a productive viral infection. Results are reported as HHV-8 genome equivalents (GE) per 100,000 cells or per ml of supernatant, and are plotted as means±standard errors of three independent experiments performed in duplicate.</p

Immunohistochemistry for detection of apoptotic cells.

<p>Apoptotic nuclei, visualized by immunohistochemistry, were more frequently detected in HHV-8-infected placental histocultures (A) than in mock-infected microexplants (B) 48 h post-infection. Original magnifications, ×40.</p

Immunofluorescence for detection of apoptotic cells.

<p>Immunofluorescence for qualitative detection of apoptotic cells in placental histocultures was performed by using the TUNEL assay. Apoptotic nuclei were more frequently evidenced in HHV-8-infected microexplants 48 h (A) and 72 h (B) after infection than in mock-infected microexplants 48 h (C) and 72 h (D) after mock infection. Original magnifications, ×40.</p

Immunohistochemical detection of the HHV-8 LANA protein in <i>ex vivo</i> placenta tissues.

<p>Specific reactivity was visualized with immunoperoxidase staining using anti-LANA monoclonal antibody with a DAB developer (brown colour) and haematoxylin counterstaining. A term placenta tissue obtained from an HHV-8-seropositive woman, and found to be HHV-8-DNA positive by PCR, showed positive nuclear immunostaining in rare cytotrophoblasts (A) (yellow arrow) and endothelial cells (B) (white arrow). A term placenta tissue from an HHV-8-seropositive woman, found to be HHV-8-DNA negative, showed no specific immunostaining (C). Original magnifications, ×63 (A), ×100 (B), ×20 (C).</p