15 research outputs found
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
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
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
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
B cell depletion (BCD) maintenance therapy after short-term depletion (STD) of B and plasma cells with ant-CD20 and bortezomib improves the disease in NZB/W F1 mice.
<p>Mice (n = 4) were treated with anti-CD20 and bortezomib (STD) alone or continuous B cell depletion without bortezomib (BCD, n = 5) or treated as STD followed by BCD maintenance therapy with anti-CD20 (STD+BCD, n = 4). (<b>A)</b> Serum IgM and IgG anti-dsDNA antibody levels in treated and untreated mice (n = 9), as measured by ELISA. (<b>B)</b> Proteinuria in treated and untreated mice. Statistical differences between treated and untreated mice were analyzed using the post-hoc test (ns, non-significant, P>0.05, *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001). (<b>C)</b> Survival curves for treated and untreated NZB/W F1 mice (Kaplan—Meier log-rank test). Abbreviations: STD, Short-term depletion (anti-CD20 and bortezomib); BCD; B cell depletion (anti-CD20).</p
Effects of short-term depletion treatments on the numbers of different B-cell subsets in bone marrow and spleen.
<p>Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (<b>A)</b> Bone marrow B-cell subsets identified by flow cytometry: total B cells (BCs) (CD19<sup>+</sup>), bone marrow pro-B cells (CD93<sup>+</sup>CD117<sup>+</sup>), pre-B cells (CD24<sup>+</sup>IgM<sup>-</sup>IgD<sup>-</sup>), immature B cells (CD24<sup>+</sup>IgM<sup>+</sup>IgD<sup>-</sup>), and mature B cells (CD24<sup>-</sup>IgM<sup>+</sup>IgD<sup>+</sup>). (<b>B)</b> Splenic B-cell subsets identified by flow cytometry: follicular (FO) B cells (CD23<sup>+</sup>CD21<sup>+</sup>IgM<sup>+</sup>), marginal zone (MZ) B cells (CD23<sup>-</sup> CD21<sup>+</sup>IgM<sup>+</sup>), germinal center (GC) B cells (IgD<sup>-</sup>GL7<sup>+</sup>), and B1 B cells (CD23<sup>-</sup>CD21<sup>-</sup>IgM<sup>+</sup>). Values are mean±SEM; ns, non-significant, P>0.05, *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test (<i>n</i> = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.</p
Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.
<p><b>(A)</b> Representative FACS histogram of bone marrow and splenic CD138<sup>+</sup> intracellular κ<sup>+</sup> BrdU<sup>+</sup> short-lived plasma cells (SLPCs), and CD138<sup>+</sup> intracellular κ<sup>+</sup> BrdU<sup>-</sup> long-lived plasma cells (LLPCs) from each treatment group. Percentage of remaining cell numbers relative to the control mean of (<b>B)</b> bone marrow and (<b>C)</b> splenic CD138<sup>+</sup> intracellular κ<sup>+</sup> total plasma cells (PCs), SLPCs, and LLPCs in mice treated with PBS, anti-CD20, anti-CD20 plus integrin-blocking antibodies (Int; anti-LFA1 and anti-VLA4 antibodies), anti-CD20 plus bortezomib (Bz) and anti-CD20 plus Int and Bz. Total PCs, SLPCs and LLPCs were enumerated by flow cytometry 7 days after the start of treatment (<i>n</i> = 5–6 mice per each group). Values are mean±SEM; ns, non-significant; P>0.05, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test. Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; FMO, Fluorescence-minus-one; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.</p
Clinical characteristics of cKS patients.
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
B cells and their non-memory subsets are increased in patients with cKS.
<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