11 research outputs found

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

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    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

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

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    <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.

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    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

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

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    <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

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

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    <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

    B cells and their non-memory subsets are increased in patients with cKS.

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    <p>The number of total B cells and CD27<sup>−</sup> B cells was significantly higher in cKS patients (grey bars) than HCs (white bars). All the subsets composing the preimmune/natural effector compartment, namely transitional, pre-naïve, naïve and MZ-like B lymphocytes, were increased in cKS patients, while the subsets composing the antigen-experienced T cell-dependent compartment, namely IgM-only, switched and CD27<sup>−</sup> memory B cells, were unaffected. Data shown as mean ± SE. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples. *<i>P</i><.05; **<i>P</i><.01; ***<i>P</i><.001.</p

    B cells from cKS patients show a low state of activation.

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    <p>B cells from cKS patients (grey bars) expressed lower levels of the costimulatory molecules CD80 and CD86 and higher levels of CD20 than B cells from HCs (white bars). The expression of the indicated markers on total B cells and their CD27<sup>−</sup> and CD27<sup>+</sup> subsets is shown. Data presented as mean ± SE of mean fluorescence intensity (MFI) values. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples. *<i>P</i><.05; **<i>P</i><.01; ***<i>P</i><.001.</p

    Clinical characteristics of cKS patients.

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    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 <sup>(53,54)</sup>.</p><p>A indicates slow evolution; B, rapid evolution; rapid denotes an increase in the total number of nodules/plaques or in the total area of plaques in three months following the last examination.</p

    HHV-8 load in isolated PBMCs, B cells and non-B cells from cKS patients and healthy HHV-8-seropositive (HSP) controls according to their peripheral blood B cells count.

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    a<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.</p>b<p>gEq =  genome Equivalents.</p

    B cells from cKS patients show increased resistance to spontaneous apoptosis and unaffected <i>in vivo</i> turnover.

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    <p>Apoptotic cells were detected by annexin V binding after 24 h culture in unstimulated conditions. <i>A</i>, representative flow cytometric analysis showing annexin V binding gated on total B cells, CD27<sup>−</sup>, MZ-like (CD27<sup>+</sup>IgD<sup>lo</sup>) and switched memory (CD27<sup>+</sup>IgD<sup>−</sup>) B cells, as indicated; comparison between one HC (upper row) and one cKS patient (lower row). B, B cells from cKS patients (grey bars) showed significantly lower annexinV binding than B cells from HCs (white bars); this lower annexin V binding was evident on CD27<sup>−</sup> and MZ-like (CD27<sup>+</sup>IgD<sup>lo</sup>) B cells, roughly composing the preimmune/natural effector compartment, but not on antigen-experienced switched memory (CD27<sup>+</sup>IgD<sup>−</sup>) B cells. Data shown as mean ± SE. <i>C, In vivo</i> cell turnover was estimated by Ki67 staining of freshly isolated PBMCs. Representative flow cytofluorimetric analysis showing Ki67 binding gated on total B cells, CD27<sup>−</sup> and CD27<sup>+</sup> B cells, as indicated; comparison between one HC (upper row) and one cKS patient (lower row). <i>D</i>, The proportion of Ki67<sup>+</sup> cells within total B cells, CD27<sup>−</sup> and CD27<sup>+</sup> B cells did not differ between cKS patients (grey bars) and HCs (white bars). Data shown as mean ± SE. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples.*<i>P</i><.05; **<i>P</i><.01.</p
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