Plasma levels of non-HDL cholesterol and HDL cholesterol.

<p>Plasma was collected from PLTP^wt/wt→LDLR^−/−, huPLTP^tg/wt→LDLR^−/−, and huPLTP^tg/tg→LDLR^−/− recipient mice at the end of the high fat, high cholesterol diet period (i.e., at time of sacrifice) and separated into two fractions: d<1.063 g/L (non-HDL) and d>1.063 g/L (HDL) by ultracentrifugation. In both fractions, the cholesterol concentration was measured. A: non-HDL cholesterol; B: HDL-cholesterol. N = 11–15 mice per group. ** <i>p</i><0.01, *** <i>p</i><0.001 versus PLTP^wt/wt→LDLR^−/− mice; $$$ <i>p</i><0.001 versus huPLTP^tg/wt→LDLR^−/− mice.</p

Plasma lipid levels.

<p>Plasma levels of cholesterol (A), phospholipids (B) and triglycerides (C) were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002255#s2" target="_blank">Methods</a>. Left panels: Plasma samples were collected from non-transplanted LDLR^−/− and LDLR^−/−/huPLTP^tg/wt mice just before the start of the high fat, high cholesterol diet (0 weeks, white bars) and after 9 weeks of the diet (9 weeks, black bars). *** <i>p</i><0.001 versus 0 weeks (same genotype), ### <i>p</i><0.001 versus LDLR^−/− mice (on the same diet). Right panels: plasma samples were collected from PLTP^wt/wt→LDLR^−/−, huPLTP^tg/wt→LDLR^−/−, and huPLTP^tg/tg→LDLR^−/− recipient mice at 1 week before the start of the transplantation procedure (i.e. at 10 weeks before the start of the diet: −10 weeks, grey bars), just before the start of the diet (0 weeks, white bars) and after 9 weeks of diet (9 weeks, black bars). N = 11–15 mice per group. * <i>p</i><0.05, *** <i>p</i><0.001 versus −10 weeks (same genotype); ### <i>p</i><0.001 versus PLTP^wt/wt→LDLR^−/− mice (on the same diet).</p

Plasma PLTP levels.

<p>A. PLTP activity was measured in plasma samples and expressed in arbitrary units as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002255#pone-0002255-g001" target="_blank">Fig. 1</a>. Left panel: plasma samples were collected from non-transplanted LDLR^−/− and LDLR^−/−/huPLTP^tg/wt mice just before the start of the high fat, high cholesterol diet (0 weeks, white bars) and after 9 weeks of the diet (9 weeks, black bars). *** <i>p</i><0.001 versus 0 weeks (same genotype), ### <i>p</i><0.001 versus LDLR^−/− mice (on the same diet). Right panel: plasma samples were collected from PLTP^wt/wt→LDLR^−/−, huPLTP^tg/wt→LDLR^−/−, and huPLTP^tg/tg→LDLR^−/− recipient mice at 1 week before the start of the transplantation procedure (i.e. at 10 weeks before the start of the diet: −10 weeks, grey bars), just before the start of the diet (0 weeks, white bars) and after 9 weeks of diet (9 weeks, black bars). *** <i>p</i><0.001 versus −10 weeks (same genotype); ### <i>p</i><0.001 versus PLTP^wt/wt→LDLR^−/− mice (on the same diet); ¶¶¶ <i>p</i><0.001 versus 0 weeks (same genotype). B. Mass of human PLTP in plasma from mice was measured by ELISA as described in Methods in the Supplemental Data. C. Specific Activities of PLTP were calculated using the ratio between the activity in AU and the mass in mg/L. ¶¶¶ <i>p</i><0.001 versus 0 weeks (same genotype); $$$ <i>p</i><0.001 versus PLTP^tg/wt→LDLR^−/− mice (on the same diet). N = 11–15 mice per group.</p

Transplantations with β-actin GFP mice→LDLR^−/− mice.

<p>A–D: Early lesions (A,B) and advanced lesions (C,D) with donor cells expressing GFP (green), CD 68 (marker for macrophages) in red (A,C) and α-actin (marker for VSMCs) in red (B,D) and nuclei stained with DAPI (blue). Endothelial cells covering the lesions do not express GFP (nuclei indicated with arrows). Co-localization of GFP and CD68 results in an orange color (arrowheads). The necrotic core in the advanced lesion (located centrally in C and D) is diffusely positive for CD68 but does not show any GFP signal. The media is marked with a double arrow (↔). E,F: Early lesion (E) and advanced lesion (F) with donor cells expressing GFP (green), and CD 31 (marker for endothelial cells) in red (arrows). Representative pictures from N = 6 animals are shown. Original magnifications: 200X.</p

Characteristics of mouse macrophages.

<p>A. Cholesterol efflux from cultured peritoneal macrophages. Cells were loaded with radioactive cholesterol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002255#s2" target="_blank">Methods</a>. Subsequently, culture medium with human HDL as an acceptor was added. Left panel: Radioactivity was measured in aliquots from the cultured medium taken at 0, 1, 2, 4, and 6 hours. Circles: macrophages from C57BL/6J mice, squares: macrophages from huPLTP^tg/tg mice. Differences were not statistically significant. B. PLTP activity measured in the medium of macrophages cultured for 24 hours in the absence (white bars) or presence (black bars) of 100 ng/mL LPS. Cells from 6 mice were used per group. C. PLTP activity measured in plasma samples from mice treated with thioglycolate. N = 6 mice per group. ** <i>p</i><0.005, *** <i>p</i><0.001 versus cells without LPS or thioglycolate (same genotype), ## <i>p</i><0.005, ### <i>p</i><0.001 versus PLPT^wt/wt (same culture medium, with or without LPS or thioglycolate).</p

Atherosclerosis in non-transplanted and transplanted mice.

<p>A. Plaque area was measured in sections from the aortic root (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002255#s2" target="_blank">Methods</a>). Left panel: Plaque area in non-transplanted LDLR^−/− and LDLR^−/−/huPLTP^tg/wt mice. *** <i>p</i><0.001 versus LDLR^−/− mice. Right panel: Plaque area in PLTPwt/wt→LDLR^−/−, huPLTP^tg/wt→LDLR^−/−, and huPLTP^tg/tg→LDLR^−/− recipient mice. N = 11–15 mice per group. *** <i>p</i><0.001 versus PLTP^wt/wt→LDLR^−/− mice; $$$ *** <i>p</i><0.001 versus huPLTP^tg/wt→LDLR^−/− mice. B, C. Immunohistochemistry of atherosclerotic lesions from non-transplanted LDLR^−/−/huPLTP^tg/wt mice (B) and from transplanted huPLTP^tg/tg→LDLR^−/− mice (C), stained for macrophages (MΦ) with anti-CD68 and for PLTP and counterstained with Nuclear-fast red (serial sections). The right panels are magnifications of the boxed parts in the left panels. Original magnifications: 100x (B) and 250x (C).</p

Presentation_1_Oral prednisolone suppresses skin inflammation in a healthy volunteer imiquimod challenge model.pptx

Imiquimod (IMQ) is a topical agent that induces local inflammation via the Toll-like receptor 7 pathway. Recently, an IMQ-driven skin inflammation model was developed in healthy volunteers for proof-of-pharmacology trials. The aim of this study was to profile the cellular, biochemical, and clinical effects of the marketed anti-inflammatory compound prednisolone in an IMQ model. This randomized, double-blind, placebo-controlled study was conducted in 24 healthy volunteers. Oral prednisolone (0.25 mg/kg/dose) or placebo (1:1) was administered twice daily for 6 consecutive days. Two days after treatment initiation with prednisolone or placebo, 5 mg imiquimod (IMQ) once daily for two following days was applied under occlusion on the tape-stripped skin of the back for 48 h in healthy volunteers. Non-invasive (imaging and biophysical) and invasive (skin punch biopsies and blister induction) assessments were performed, as well as IMQ ex vivo stimulation of whole blood. Prednisolone reduced blood perfusion and skin erythema following 48 h of IMQ application (95% CI [−26.4%, −4.3%], p = 0.0111 and 95% CI [−7.96, −2.13], p = 0.0016). Oral prednisolone suppressed the IMQ-elevated total cell count (95% CI [−79.7%, −16.3%], p = 0.0165), NK and dendritic cells (95% CI [−68.7%, −5.2%], p = 0.0333, 95% CI [−76.9%, −13.9%], p = 0.0184), and classical monocytes (95% CI [−76.7%, −26.6%], p = 0.0043) in blister fluid. Notably, TNF, IL-6, IL-8, and Mx-A responses in blister exudate were also reduced by prednisolone compared to placebo. Oral prednisolone suppresses IMQ-induced skin inflammation, which underlines the value of this cutaneous challenge model in clinical pharmacology studies of novel anti-inflammatory compounds. In these studies, prednisolone can be used as a benchmark.</p