Correlation between <i>HLA-</i> gene expression and the expression of the genes encoding the HLA transcriptional regulators and peptide-loading machinery.

<p>Correlation between <i>HLA-</i> gene expression and the expression of the genes encoding the HLA transcriptional regulators and peptide-loading machinery.</p

Schematic illustration of tumor characteristics and infiltrate.

<p>UM with monosomy 3 attract an infiltrate, producing different cytokines, including Interferon-gamma. The tumor cell (UM cell) responds by increasing HLA class I and II levels, as well as rendering the infiltrating immune cells ineffective (immune suppression) and creating a tumor-favorable environment, with amongst others, stimulation of angiogenesis.</p

Tumor infiltrating immune cells.

<p>Association between a low or high density of tumor-infiltrating CD3+ (A), and CD68+ (B)cells and several HLA and HLA-related genes (Illumina array) in primary UM. CD3 (cells/mm²) and CD68 (pixels x 10³/mm²) scores were dichotomized at the median.</p

Chromosomal aberrations and HLA expression.

<p>Comparison between chromosomal aberrations and the expression of HLA class I and II antigens in a set of 27 primary UM. Tumors are divided according to their chromosome 3 and 6 status (disomy or monosomy of chromosome 3, and disomy of chromosome 6 or gain of 6p). HLA gene expression was determined using an Illumina microarray (A) and protein expression by immunohistochemistry (B) in UM. Additionally, HLA gene expression was determined using qPCR, which served to validate the Illumina findings (C). Four data points of the qPCR that are outside the axis limits (> 11 and < 24) are not shown (HLA-A, D3D6p: 17; HLA-B, D3D6p: 24, and M3D6p: 12; B2M, D3D6p: 13). Only significant p-values are shown, all other comparisons between the groups were not significant (p-values not shown). Error-bars represent the interquartile range. Results were obtained using the Mann-Whitney U tests.</p

Effect of the absence of human leukocytes.

<p>Gene-expression (log 2 intensity values) of HLA-B, HLA-DRA, CD3D (as marker for T-cells), and CD163 (as marker for macrophages), of the original tumors (patient) compared to the xenografts (xeno). MP’s are primary tumors; MM’s are metasisis.</p

Effect of PCAF deficiency on intimal hyperplasia and vascular smooth muscle cell content <i>in vivo</i>.

<p>Representative images of PCAF staining (A), scale bar = 100 μm. Quantification of intimal hyperplasia (B), intima/media ratio (C) and lumenstenosis (D) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. ***<i>P</i><0.001. Representative images of elastin staining (E), scale bar = 50 μm. Quantification intimal (F) and medial (G) smooth muscle cell area (μm²) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 8) mice. **<i>P</i><0.001. (H) Representative images of smooth muscle actin (SMA) staining of cuffed femoral arteries, scale bar = 50 μm. Results are mean±SEM.</p

Effect of garcinol treatment on inflammatory cell recruitment and CCL2 expression <i>in vivo</i>.

<p>(A) Quantification of CD45 positive cells (leukocytes) in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 8). *<i>P</i><0.05, **<i>P</i><0.01. (B) Quantification of Mac3 positive cells (macrophages) in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 6). **<i>P</i><0.01. (C) Quantification of CCL2 positive cells in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 7). **<i>P</i><0.01, ***<i>P</i><0.001. Representative images of CD45, Mac3 and CCL2 staining of cuffed femoral arteries, scale bar = 20 μm. Results are mean±SEM.</p

Effect of PCAF deficiency on inflammatory cytokine expression <i>in vitro</i>.

<p>(A) TNF-alpha production of whole blood derived leukocytes (n = 5) from WT and PCAF KO mice 24 hours after LPS (0–250 ng/ml) stimulation. *<i>P</i><0.05. TNF-alpha(B) and IL-6 (C) production of bone marrow derived macrophages (n = 3) from WT and PCAF KO mice 24 hours after LPS (0–250 ng/ml) stimulation. *<i>P</i><0.05. (D) CCL2 production of vascular smooth muscle cells (n = 3) from WT and PCAF KO mice 24 hours after LPS (0–1 ng/ml) stimulation. *<i>P</i><0.05, ***<i>P</i><0.001. Results are mean±SEM.</p

Effect of PCAF deficiency on macrophage influx and CCL2 expression <i>in vivo</i>.

<p>Quantification of Mac3 positive cells (macrophages) in the intima (A) and media (B) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. Representative images of Mac3 staining (C), scale bar = 50 μm. Quantification of CCL2 positive area in the intima (D) and media (E) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. Representative images of CCL2 staining (F), scale bar = 50 μm. Results are mean±SEM.</p

Effect of garcinol treatment and PCAF downregulation on inflammatory cytokine expression <i>in vitro</i>.

<p>(A) TNF-alpha production of whole blood derived leukocytes (n = 5) after 24 hours stimulation with LPS (100 ng/ml) in combination with garcinol (0–20 μM). *<i>P</i><0.05 compared to vehicle (0 μM garcinol), n.d.: non-detectable. (B) CCL2 production of vascular smooth muscle cells (n = 3) after 24 hours stimulation with LPS (1 ng/ml) in combination with garcinol (0–15 μM). *<i>P</i><0.05 compared to vehicle (0 μM garcinol). (C) Whole blood derived leukocyte viability (n = 4) after 24 hours incubation with garcinol (0–250 μM), expressed as percentage fluorescence intensity. ***<i>P</i><0.001 compared to 0 μM garcinol. (D) Relative Pcaf expression of vascular smooth muscle cells (n = 3) transfected with scrambled or PCAF siRNA and stimulated with 1 ng/ml for 24 hours. **<i>P</i><0.01 compared to scrambled siRNA. (E) CCL2 production of vascular smooth muscle cells (n = 3) transfected with scrambled or PCAF siRNA and stimulated with 1 ng/ml for 24 hours. *<i>P</i><0.05 compared to scrambled siRNA. Results are mean±SEM.</p